CN108295244A - Polypeptide for treating tumor of breast - Google Patents

Polypeptide for treating tumor of breast Download PDF

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CN108295244A
CN108295244A CN201710026136.3A CN201710026136A CN108295244A CN 108295244 A CN108295244 A CN 108295244A CN 201710026136 A CN201710026136 A CN 201710026136A CN 108295244 A CN108295244 A CN 108295244A
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amino acids
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CN108295244B (en
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刘宏利
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Shanghai Jibei Pharmaceutical Technology Co Ltd
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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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]

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Abstract

The application provides the polypeptide for treating tumor of breast (especially triple negative breast cancer).Specifically, the application of polypeptide of the present invention or its pharmaceutical composition in preparing the drug for treating tumor of breast, the polypeptide is the segment within Endostatin N-terminal 45 amino acid residues of length, and at least contain the 1st 20 amino acids residue of N-terminal, and wherein described the 2nd amino acids residue of Endostatin N-terminal and the 18th amino acids residue, respectively as shown in text, and optionally there are mutation described herein in the 17th, 20-22.

Description

Polypeptide for treating tumor of breast
Technical field
The invention belongs to therapeutic field of tumor, and in particular to the polypeptide for treating tumor of breast.
Background technology
Breast cancer is to be happened at the malignant tumour of mammary gland galandular epithelium tissue, wherein 99% is happened at women.Mammary gland is not The vitals of human life activity, breast cancer in situ are maintained simultaneously to be not fatal；But since breast cancer cell loses normal cell Characteristic, connect between cell loose, be easy to fall off.Cancer cell once falls off, and free cancer cell can be with blood or lymph Liquid sends out whole body, forms transfer, threat to life.Breast cancer has become the kinds of tumor for threatening women physical and mental health at present.The whole world Breast cancer incidence is in rising trend always since the late 1970s.8, U.S. women just has 1 people and suffers from breast in life Gland cancer.China is not the country occurred frequently of breast cancer, but unsuitable optimistic, and the growth rate of China's breast cancer incidence is but higher by recent years National 1~2 percentage point occurred frequently.According to National Cancer Center and prevention and control of diseases office of the Ministry of Public Health breasts in 2009 announced in 2012 Gland cancer morbidity data are shown：National tumour registration area breast cancer incidence occupies the 1st of female malignant.Breast cancer Treatment includes mainly operation, radiotherapy, chemotherapy, estrogen endocrine therapy and the targeted therapy for HER-2.
Three cloudy breast cancer (Triple-Negative Breast Cancer, TNBC) are a kind of specific types of breast cancer, The estrogen receptor (estrogen receptor, ER) of tumour cell, progesterone receptor (progesterone receptor, PR) and human epidermal growth factor receptor 2 (human epidermal growth factor receptor 2, HER-2) is Negative breast cancer type, accounts for the 10%~20% of all breast cancer histological types.Since hormone receptor and HER-2 lack, lead Cause the tumour insensitive to hormone treatment and HER-2 targeted therapies so that three cloudy breast cancer are a kind of most dangerous breasts Gland cancer, invasion is strong, easily shifts, and poor prognosis, the life cycle after patient makes a definite diagnosis is typically not greater than 20 months, and 5 years survival rates are not Foot 15%.
Three cloudy breast cancer carry out conventional therapy currently without clinical therapeutic guideline, generally according to the poor breast cancer of prognosis, Standard treatment is new adjuvant chemotherapy of the postoperative use containing anthracycline or taxanes.Due to the estrogen receptor of TNBC patient, There are a variety of hypotypes, the response level of even same therapeutic scheme, different patients also has very for progesterone receptor or HER2 receptors Big difference.Not yet ratify any targeted drug for treating TNBC at present in the U.S..
Endostatin is that O ' Reilly in 1997 etc. are isolated and purified from mouse endothelial cells tumor (EOMA) supernatant of culture A kind of endogenous angiogenesis inhibitors, be 20kd molecular weight protein matter, from XVIII collagen types hydrolysis produce Object.Experiment shows that Endostatin plays inhibiting effect to blood vessel endothelium and tumour cell.Since the Endostatin of recombination is difficult to again Difficult factors, the EntreMed companies of the U.S. such as property abandon the clinical research of Recombinant Endostatin, there is no method largely to prepare at present Endostatin with higher external activity.
The zinc being made of the 1st, 3,11 three histidines of N-terminal and the 76th asparagicacid residue in Endostatin sequence Ion-binding site, Endostatin are combined most important to its activity with zinc ion.It is reported that from Endostatin N-terminal Polypeptide has activity (the Cancer Res.2005 of certain inhibition vascular endothelial cell and tumour cell；65(9):3656-63, United States Patent (USP) US7524811B2).However above-mentioned experiment is also shown, and contains N-terminal 1-25 amino acids from human endostatin source Polypeptide cannot significantly inhibit the growth for the people source tumour being inoculated on mouse model, and the activity of endostatin derived peptide needs It improves.
Invention content
The application provides application of the polypeptide in preparing the drug for treating tumor of breast, and the polypeptide is Endostatin N-terminal grows the segment within 45 amino acid residues, and at least contains N-terminal 1-20 amino acids residues, and the wherein described endothelium The 2nd amino acids residue of chalone N-terminal and the 18th amino acids residue are respectively selected from the following group：
And optionally, the 17th amino acids of Endostatin N-terminal are that S, A, L, I or T and/or the 20th amino acids are residual Base is S or T, and/or if containing the 21st and/or the 22nd amino acids residue, the 21st amino acids residue is G, A, L, I Or V and/or the 22nd amino acids residue are G, A, L, I or V；
Preferably, the amino acid sequence of the Endostatin such as SEQ ID NO:Shown in 1.
In one or more embodiments, the polypeptide at least contains SEQ ID NO：38 1-22 amino acids are residual Base, and the 2nd and 18 amino acids residues are as mentioned before.
In one or more embodiments, the polypeptide at least contains SEQ ID NO：38 1-25 amino acids are residual Base, and the 2nd and 18 amino acids residues are as mentioned before.
In one or more embodiments, the polypeptide at least contains SEQ ID NO：38 1-22 amino acids are residual Base preferably at least contains SEQ ID NO：38 1-25 amino acids residues, and the 2nd amino acids residue is T, the 18th ammonia Base acid residue is N, G, K, M, F, S or T, and the amino acids of the 17th, 20,21 and 22 are as mentioned before.
In one or more embodiments, the polypeptide at least contains SEQ ID NO：38 1-22 amino acids are residual Base preferably at least contains SEQ ID NO：38 1-25 amino acids residues, and the 18th amino acids residue is N, the 2nd ammonia Base acid residue is T, and the amino acids of the 17th, 20,21 and 22 are as mentioned before.
In one or more embodiments, the polypeptide at least contains SEQ ID NO：38 1-22 amino acids are residual Base preferably at least contains SEQ ID NO：38 1-25 amino acids residues, and the 18th amino acids residue is S, the 2nd ammonia Base acid residue is E, H, L, T, W or V, and the amino acids of the 17th, 20,21 and 22 are as mentioned before.
In one or more embodiments, the amino acid sequence such as SEQ ID NO of the polypeptide:4,5,6,7,27- 30, shown in any in 39,41 and 47-49.
In one or more embodiments, the polypeptide is by SEQ ID NO:38 compositions, wherein the 2nd amino acids are residual Base is T, and the 18th amino acids residue is N or S, and the amino acids of the 17th, 20,21 and 22 are as mentioned before.
In one or more embodiments, the polypeptide is selected from by SEQ ID NO:4 the 1st to the 39th, 38,37,36, 34, the amino acid sequence of 33,32,31,29,28,27 or 26 amino acids residues composition, and by SEQ ID NO:39 the 1st arrive The amino acid sequence of the amino acids residue composition of 39th, 38,37,36,35,34,33,32,31,29,28,27,26 or 25.
In one or more embodiments, the 1st amino acids residue of polypeptide N-terminal is histidine, the histidine quilt Formylated, acetylation, the modification of propionating or Butyrylation, the 1st amino acids of C-terminal can be modified by PEG, cholesterol or amidation.
In one or more embodiments, the polypeptide is selected from：
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；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRG-NH2；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRG；
HTHRDFQPVLHLVALNSSLSGGMRGIRG-NH2；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGA-NH2；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGA；
HTHRDFQPVLHLVALNSSLSGGMRGIRGA-NH2；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADF-NH2；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADF；
HTHRDFQPVLHLVALNSSLSGGMRGIRGADF-NH2；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQ-NH2；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQ；With
HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQ-NH2；
Wherein Ac is acetylation modification, NH2It is modified for amidation.
In one or more embodiments, described pharmaceutical composition also contains pharmaceutically acceptable carrier.
In one or more embodiments, the tumor of breast is selected from HER2, estrogen receptor, progesterone receptor three Triple negative breast cancer, the two for being feminine gender are that negative breast cancer, the breast cancer that one is feminine gender and three are positive Breast cancer.
In one or more embodiments, the tumor of breast is selected from leaf tumour, breast between breast neoplasm, mammary gland Gland fibroepithelial tumour, theloma and male breast carcinoma.
In one or more embodiments, the breast neoplasm is selected from carcinoma microinvasive, wellability mammary gland Cancer, epithelium-myoepithelial tumor, precursor lesion, Intraductal hyperplasia and papillary lesion.
In one or more embodiments, leaf tumour packet is selected from myofibroblastoma, embryonal-cell lipoma between the mammary gland And angiosarcoma.
In one or more embodiments, the mammary gland fibroepithelial tumour is phyllodes tumor or hamartoma.
The application also provides the application polypeptide or its pharmaceutical composition in preparing the drug for improving chemotherapeutical medicine curative effect Application.In one or more embodiments, the chemotherapeutics is cis-platinum, carboplatin, oxaliplatin, cyclophosphamide, first ammonia In petrin, fluorouracil, adriamycin, taxol, docetaxel, mitoxantrone, Xeloda, gemzar, Epi-ADM, gemcitabine etc. It is one or more.
The application also provides the application polypeptide or its pharmaceutical composition and is preparing raising endocrine therapeutic agents curative effect Application in drug.In one or more embodiments, the endocrine therapeutic agents are megestrol acetate, medroxyprogesterone acetate, Ai Luo It is one or more in U.S. new, tamoxifen, Arimidex, Letrozole, Zoladex library etc..
The application also provides the application polypeptide or its pharmaceutical composition and is preparing the medicine for improving target therapeutic agent curative effect Application in object.In one or more embodiments, the target therapeutic agent be antibody and tyrosine kinase inhibitor, Middle antibody is Trastuzumab, handkerchief trastuzumab, bevacizumab, keytruda monoclonal antibodies, durvalumab monoclonal antibodies etc.；Tyrosine kinase presses down Preparation is Lapatinib, Nola for one or more in Buddhist nun, Sutent etc..
The application also provides the application polypeptide or its pharmaceutical composition and is preparing the medicine for improving immunotherapy medicaments curative effect Application in object.In one or more embodiments, the immunotherapy medicaments be anti-PD-1 antibody, anti-PD-L1 antibody, It is one or more in CAR-T cells etc..
Description of the drawings
Fig. 1 a and 1b show HPLC the and MASS collection of illustrative plates of polypeptide P1 respectively.
Fig. 1 c and 1d show HPLC the and MASS collection of illustrative plates of polypeptide P2 respectively.
Fig. 1 e and 1f show HPLC the and MASS collection of illustrative plates of polypeptide P2T2S18 respectively.
Fig. 1 g and 1h show HPLC the and MASS collection of illustrative plates of polypeptide P2T2N18 respectively.
Fig. 2 shows the biological activity that P1, P2, P3, P4 polypeptide, endostatin, endostar inhibit HUVEC.
Fig. 3 a and 3b show HPLC the and MASS collection of illustrative plates of polypeptide P2T2S18 Δs 1 respectively.
Fig. 3 c and 3d show HPLC the and MASS collection of illustrative plates of polypeptide P2T2S18 Δs 2 respectively.
Fig. 3 e and 3f show HPLC the and MASS collection of illustrative plates of polypeptide P2T2S18 Δs 3 respectively.
The biological activity that Fig. 4 display portions polypeptide inhibits HUVEC.
Fig. 5 a and 5b show inhibiting effect of several polypeptides to BT-483.
Fig. 6 a show inhibiting effect of several polypeptides to BT-483, MDA-MB-453 and MDA-MB-468.
Fig. 6 b show the microscope photo of the P2 and P2T2S18 of each concentration to the inhibiting effect of MDA-MB-453 cells.
Fig. 7 shows inhibiting effect of the P2 and P2T2S18-29 to SK-BR-3, T-47D and BT-474.
Fig. 8 a and 8b show HPLC the and MASS collection of illustrative plates of polypeptide P2T2S18-20 respectively.
Fig. 8 c and 8d show HPLC the and MASS collection of illustrative plates of polypeptide P2T2S18-25 respectively.
Fig. 8 e and 8f show HPLC the and MASS collection of illustrative plates of polypeptide P2T2N18-35 respectively.
Fig. 8 g and 8h show HPLC the and MASS collection of illustrative plates of polypeptide P2T2N18-40 respectively.
Fig. 8 i and 8j show HPLC the and MASS collection of illustrative plates of polypeptide P2T2N18-45 respectively.
Fig. 9 shows inhibition of the polypeptide to HUVEC growth in vitro.In figure, in terms of the cell survival rate at 180 μM of concentration, from Each curve of top to bottm represent successively P2T2-15, P2T2S18-45, P2T2S18-40, P2T2S18-20, P2T2S18-25, The cell survival rate of P2T2S18-35 and P2T2S18.
Figure 10 shows inhibition of the polypeptide to HUVEC growth in vitro.
Figure 11 a and 11b show HPLC the and MASS collection of illustrative plates of polypeptide P2T2S18-29 respectively.
Figure 12 shows inhibiting effect of the polypeptide P2T2S18-29 to HUVEC.In terms of the cell survival rate at 2.5mg/ml, from Top to bottm curve represents Endostar, Endostatin, P2, P2T2S18-29 and P2T2S18 successively.
Specific implementation mode
This application involves the treatments of tumor of breast, and in particular to uses polypeptide therapeutic tumor of breast as described below：Endothelium The N-terminal of chalone grows the segment of amino acid residue within 45, at least contains Endostatin N-terminal 1-20 amino acids residues, and Wherein：
(1) correspond to the 2nd amino acids of Endostatin N-terminal residue be A, R, N, D, Q, E, H, I, L, K, M, F, P, T, W, Y or V；With
(2) correspond to the 18th amino acids of Endostatin N-terminal residue be A, R, N, D, C, E, G, H, I, L, K, M, F, S, T, W, Y or V；
And the polypeptide is compared with its corresponding sequence without mutation, it is high to the inhibiting rate of HUVEC under the conditions of same concentrations At least 15%, it is preferably at least high by 20%；Or the polypeptide, compared with its corresponding sequence without mutation, the former is IC50A concentration of the latter IC50The half of concentration, preferably the former IC50A concentration of the latter IC50/ 5th of concentration, further preferably preferably The former IC of ground50A concentration of the latter IC50/ 10th of concentration.
Endostatin is preferably human endostatin.SEQ ID NO:1 shows an example of Research of Recombinant Human Endostatin Son.Preferably, the amino acid sequence of the application at least contains SEQ ID NO:Endostatin N-terminal 1-20 bit aminos described in 1 Sour residue, and the 2nd and the 18th amino acids it is as described herein.
Preferably, the residue that the polypeptide corresponds to the 2nd amino acids of Endostatin N-terminal is D, L, T, W or Y.Preferably, The residue that the polypeptide corresponds to the 18th amino acids of Endostatin N-terminal is N, E, K, M, S, T or V.It is furthermore preferred that the polypeptide Residue corresponding to the 2nd amino acids of Endostatin N-terminal is D, T, W or Y.It is highly preferred that the polypeptide corresponds to Endostatin The residue of 18th amino acids is N, S or V.
In certain embodiments, the polypeptide corresponds to the 2nd amino acid residue of Endostatin and the 18th amino Sour residue is respectively following combination：
2nd amino acids 18th amino acids
A M
R I
N K
D E, M, T or Y
Q A or H
E S or V
H A or S
L R, E or S
K V
M L or W
F T
P C or V
T N, G, K, M, F, S or T
W C, E, I, K, S or Y
Y R, H, W or V
V D or S
Alternatively, in certain embodiments, the polypeptide correspond to Endostatin the 2nd amino acid residue and the 18th Amino acid residue is respectively following combination：
18th amino acids 2nd amino acids
A Q or H
R L or Y
N T
D V
C P or W
E D, L or W
G T
H Q or Y
I R or W
L M
K N, T or W
M A, D or T
F T
S E, H, L, T, W or V
T D, F or T
W M or Y
Y D or W
V E, K, P or Y
It should be understood that " segment " refers to a part of continuous sequence of full length sequence.For example, the polypeptide of this paper is preferably by endothelium The 1st amino acids of chalone N-terminal to the 20th, 21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37, 38,39,40,41,42,43,44 or 45 amino acids residues form and are amino acid residue described herein at the 2nd and 18 Sequence.In other words, the application polypeptide length is 20-45 amino acid residue, from the 1st amino acid residue of Endostatin N-terminal It calculates.It is highly preferred that the polypeptide length of the application is 25-40 amino acid residue, started from the 1st amino acid residue of N-terminal.
In certain embodiments, the segment is in addition to being amino acid residue described herein at the 2nd and 18, also optionally Any bit, Ren Erwei, Ren Sanwei or whole four in the 17th, 20,21 and 22 are following residue：
17th amino acids residue：S, A, L, I, V or T；
20th amino acids residue：S or T；
21st amino acids residue：G, A, L, I or V；
22nd amino acids residue：G, A, L, I or V.
Therefore, in certain embodiments, the application of the segment within 45 amino acid residues is grown as Endostatin N-terminal Polypeptide at least contains SEQ ID NO：38 1-22 amino acids residues preferably at least contain SEQ ID NO：38 1-25 Amino acid residue, and the 2nd and 18 is amino acid residue as described herein, and any bit in the 17th, 20-22, Ren Erwei, appoint Three or all four are residue described above.Further, the long 25-40 amino acid residue of this kind of polypeptide.
In certain embodiments, the application polypeptide of the segment within 45 amino acid residues is grown as Endostatin N-terminal At least contain SEQ ID NO：38 1-22 amino acids residues preferably at least contain SEQ ID NO：38 1-25 bit aminos Sour residue, and the 2nd amino acids residue is T, the 18th amino acids residue is N, G, K, M, F, S or T (more preferably N or S), is appointed Selection of land, the amino acid of the 17th, 20,21 and 22 are as mentioned before.Further, the long 25-40 amino acid residue of this kind of polypeptide.
In certain embodiments, the application polypeptide of the segment within 45 amino acid residues is grown as Endostatin N-terminal At least contain SEQ ID NO：38 1-22 amino acids residues preferably at least contain SEQ ID NO：38 1-25 bit aminos Sour residue, and the 18th amino acids residue is N, the 2nd amino acids residue is T, and optionally, the amino acid of the 17th, 20,21 and 22 is such as It is described previously.Further, the long 25-40 amino acid residue of this kind of polypeptide.
In certain embodiments, the application polypeptide of the segment within 45 amino acid residues is grown as Endostatin N-terminal At least contain SEQ ID NO：38 1-22 amino acids residues preferably at least contain SEQ ID NO：38 1-25 bit aminos Sour residue, and the 18th amino acids residue is S, the 2nd amino acids residue is E, H, L, T, W or V, optionally, the 17th, 20,21 It is as mentioned before with 22 amino acids.Further, the long 25-40 amino acid residue of this kind of polypeptide.
The amino acid sequence of the preferred polypeptide of the application such as SEQ ID NO:4,5,6,7,27-30,39,41 and 47-49 In it is any shown in.The application polypeptide further includes by SEQ ID NO:4 the 1st to the 39th, 38,37,36,34,33,32,31,29,28, The amino acid sequence of 27 or 26 amino acids residues composition, and by SEQ ID NO:39 the 1st to the 39th, 38,37,36,35, 34, the amino acid sequence of 33,32,31,29,28,27,26 or 25 amino acids residues composition.
The 1st amino acids residue of polypeptide N-terminal of the application is histidine, which can be by formylated, acetylation, propionyl Change or Butyrylation modification, the 1st amino acids of C-terminal can be modified by PEG, cholesterol or amidation.
Preferably, the 1st amino acids residue histidine of the application polypeptide N-terminal is acetylation modification, the 1st bit amino of C-terminal Acid is amidated modification.
It should be understood that in gene cloning operation, it is often necessary to design suitable restriction enzyme site, this certainly will be in expressed ammonia Base acid sequence end introduces one or more incoherent residues, and this has no effect on the activity of aim sequence.For another example in order to Construction of fusion protein, the expression for promoting recombinant protein obtain the recombinant protein being secreted into automatically outside host cell or conducive to recombination The purifying of albumen, it is often necessary to be added to some amino acid other in the ends N-, the ends C- or the albumen of recombinant protein In appropriate area, it may for example comprise but be not limited to, suitable joint peptide, signal peptide, leader peptide, end extend etc..The application amino The aminoterminal or c-terminus of acid sequence can also contain one or more polypeptide fragments, as protein tag.Any suitable label It may be used to the application.For example, the label can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7,4A6, ε, B, gE and Ty1.These labels can be used for purifying albumen.The mark used It includes Poly-Arg to sign example, such as RRRRR (SEQ ID NO:42)；Poly-His 2-10 (usual 6), such as HHHHHH (SEQ ID NO:43)；FLAG, i.e. DYKDDDDK (SEQ ID NO:44)；Strep-TagII, i.e. WSHPQFEK (SEQ ID NO:45)；And C-myc, i.e. WQKLISEEDL (SEQ ID NO:46).
Therefore, the application also include constitute containing the sequence label or by the sequence label and foregoing segments it is more Peptide.
The amino acid sequence of the application can be chemical synthesis product, or use recombinant technique from protokaryon or eukaryon place The recombinant polypeptide generated in main (for example, bacterium, yeast, filamentous fungi, higher plant, insect and mammalian cell).According to The polypeptide of host used in recombinant production scheme, the application can be glycosylated, or can be nonglycosylated.
For example, chemiluminescent polypeptide synthetic method well known in the art can be used to synthesize the amino acid sequence of the application.Polypeptide It includes solid-phase synthesis and liquid phase synthesizing method to learn synthetic method, wherein common with solid-phase synthesis.Solid phase synthesis process include but It is not limited to two kinds of common methods of Fmoc and tBoc.In general, using resin as insoluble solid phase carrier, usually from C-terminal (carboxyl End) amino acid is connected on peptide chain one by one to N-terminal (aminoterminal), each amino acid connection cycle is by following three-step reaction structure At：1) it is deprotected：Protected amino acid must remove the blocking group of amino with a kind of deprotection solvent；2) it activates：The company of waiting for The carboxyl of the amino acid connect is activated agent and is activated；With 3) coupling：The carboxyl of the activation amino exposed with previous amino acid is anti- It answers, forms peptide bond.Iterative cycles can be completed when peptide chain extends to required length.Finally with cutting liquid cutting peptide chain and admittedly Connection between phase carrier, so that it may obtain required amino acid sequence.It can be on the automation Peptide synthesizer that program controls Above-mentioned chemical synthesis is carried out, this quasi-instrument includes but not limited to the Tribute of Protein Technologies companies release bis- The Focus XC tri- that channel polypeptide synthesizer, the UV Online Monitor systems of C S Bio companies, Aapptec companies release Channel synthesizer etc..
The application also includes the polynucleotides of coding the application polypeptide.For example, SEQ ID NO:30 show SEQ ID NO:1 coded sequence；SEQ ID NO:31 show SEQ ID NO:3 coded sequence；SEQ ID NO:32 show SEQ ID NO:4 coded sequence；SEQ ID NO:33 show SEQ ID NO:5 coded sequence；SEQ ID NO:34 show SEQ ID NO:6 coded sequence；SEQ ID NO:35 show SEQ ID NO:7 coded sequence；SEQ ID NO:36 is aobvious SEQ ID NO are shown:8 coded sequence；SEQ ID NO:37 show SEQ ID NO:9 coded sequence；SEQ ID NO: 40 show SEQ ID NO:39 coded sequence.
In certain embodiments, the polynucleotide sequence is selected from by SEQ ID NO:32 the 1st to the 117th, 114, 111, the polynucleotide sequence of 108,102,99,96,93,87,84,81 or 78 bit bases composition.In certain embodiments, institute Polynucleotide sequence is stated to be selected from by SEQ ID NO:40 the 1st to the 117th, 114,111,108,105,102,99,96,93,87, 84, the polynucleotide sequence of 81,78 or 75 bit bases composition.
The polynucleotides of the application can be DNA form or rna form.DNA form includes cDNA, genomic DNA or people The DNA of work synthesis.DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand.Encoding mature polypeptide Coding region sequence can variant identical as above-mentioned DNA sequence dna or degeneracy.As used herein, " variant of degeneracy " In this application refer to encode the application amino acid sequence, but with such as SEQ ID NO:The differentiated core of sequence shown in 31 etc. Acid sequence.
Term " polynucleotides of coding polypeptide " can be the polynucleotides for including this polypeptide of coding, can also be to further include The polynucleotides of additional code and/or non-coding sequence.
Polypeptide and polynucleotides in the application preferably provide in a separate form, are more preferably purified to homogeneous.
The nucleotide sequence of the application can usually use PCR amplification method, recombination method or artificial synthesized method to obtain.For PCR amplification method can carry out design primer according to related nucleotide sequence, especially open reading frame sequence disclosed in the present application, The commercially available libraries cDNA are used in combination or by the libraries cDNA prepared by conventional method well known by persons skilled in the art as template, amplification and Obtain related sequence.When sequence is longer, it is often necessary to carry out twice or multiple PCR amplification, the piece for then again amplifying each time Section is stitched together by proper order.
Once obtaining related sequence, so that it may to obtain related sequence in large quantity with recombination method.This is typically will It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.
In addition, related sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.In general, logical After first synthesizing multiple small fragments, it is then attached the very long segment of available sequence again.
At present, it is already possible to obtain the DNA sequence dna of coding the application amino acid sequence by chemical synthesis completely.Then The DNA sequence dna can be introduced into various existing DNA moleculars (or such as carrier) as known in the art and cell.
The application also relates to the carriers for including the application polynucleotides, and are generated through genetic engineering with the carrier of the application Host cell, and the method that generates herein described polypeptide through recombinant technique.Preferably, the carrier of the application is that expression carries Body.
By the recombinant dna technology of routine, it can express or produce the application's using the application polynucleotide sequence Polypeptide.In general there are following steps：
(1) polynucleotides of the application or the variant of its degeneracy are used, or is carried with containing the polynucleotide recombinant expression Body converts or suitable host cell of transduceing；
(2) host cell cultivated in suitable culture medium；
(3) it is separated from culture medium or cell, protein purification.
The polynucleotide sequence of the application can be inserted into recombinant expression carrier.Term " recombinant expression carrier " refers to ability Bacterial plasmid, bacteriophage, yeast plasmid known to domain, plant cell virus, mammalian cell virus such as adenovirus, reverse transcription Viral or other carriers.As long as can replicate and stablize in host, any plasmid and carrier can be used.The one of expression vector A important feature is to usually contain replication orgin, promoter, marker gene and translation control element.Expression vector further includes translation The ribosome bind site and transcription terminator of starting.
Method well-known to those having ordinary skill in the art can be used to build nucleic acid sequence containing the application and suitable transcription/translation Control the expression vector of signal.These methods include recombinant DNA technology in vi, DNA synthetic technologys, In vivo recombination technology etc..Institute The nucleic acid sequence stated can be effectively connected in the appropriate promoter in expression vector, to instruct mRNA to synthesize.These promoters Representative example has：Lac the or trp promoters of Escherichia coli；Bacteriophage lambda PL promoters；Eukaryotic promoter includes CMV early immediately Phase promoter, HSV thymidine kinase promoters, early and late SV40 promoters, retrovirus LTRs and some other The promoter that the controllable gene known is expressed in protokaryon or eukaryotic or its virus.
In addition, expression vector preferably includes one or more selected markers, to provide for selecting conversion The phenotypic character of host cell, such as the dihyrofolate reductase of eukaryotic culture, neomycin resistance and green fluorescence egg (GFP) in vain, or tetracycline or amicillin resistance for Escherichia coli.
The carrier for including above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence can be used for converting suitable When host cell, allow it to expression protein.
Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；It is Filamentous true Bacterium cell or higher eucaryotic cells, such as mammalian cell.Representative example has：Escherichia coli, streptomyces；Mouse typhus The bacterial cell of salmonella；Fungal cell such as yeast, filamentous fungi, plant cell；The insect cell of drosophila S2 or Sf9； The zooblast etc. of CHO, COS, 293 cells or Bowes melanoma cells.
When the polynucleotides of the application are expressed in higher eucaryotic cells, if will when being inserted into enhancer sequence in the carrier Transcription can be made to be enhanced.Enhancer is the cis-acting factors of DNA, generally about has 10 to 300 base-pairs, acts on and open Mover is to enhance the transcription of gene.
Persons skilled in the art are aware that how to select carrier, promoter, enhancer and host cell appropriate.
It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.When host is original When core biology such as Escherichia coli, can absorb the competent cell of DNA can harvest after exponential phase of growth, use CaCl2Method processing, institute With the step of it is generally well-known in the art.Another method is to use MgCl2.If desired, conversion can also use the side of electroporation Method carries out.When host is eucaryote, following DNA transfection methods can be selected：Calcium phosphate precipitation, conventional mechanical methods are such as Microinjection, electroporation, liposome packaging etc..
The transformant of acquisition can use conventional method culture, express the polypeptide of the coded by said gene of the application.According to used Host cell, culture medium used in culture can be selected from various conventional mediums.Under conditions of suitable for host cell growth It is cultivated.After host cell growth is to cell density appropriate, with suitable method (such as temperature transition or chemical induction) Cell is further cultured for a period of time by the promoter for inducing selection.
Recombinant polypeptide in the above methods can be expressed in cells, or on the cell membrane, or secreted outside the cell.Such as Fruit needs, its physics, chemical and other characteristics can be utilized to be separated by various separation methods and purify the albumen of recombination.This A little methods are well-known to those skilled in the art.The example of these methods includes but is not limited to：The renaturation process of routine is used Protein precipitant handles (salting-out method), centrifugation, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel filtration), inhales The combination of attached chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and various other liquid chromatography technologies and these methods. The various methods that polypeptide is prepared by recombinant technique known in the art.
The application also provides a kind of pharmaceutical composition, which contains the polypeptide of the application and pharmaceutically acceptable Carrier.
It can contain in pharmaceutical composition and treat or prevent a effective amount of the application polypeptide." effective quantity " refers to the dosage of certain ingredient It is enough to generate desired reaction.Specific effective quantity depends on many factors, the body of the particular condition, patient such as to be treated Concrete conditions in the establishment of a specific crime (such as patient's weight, age or gender), duration for the treatment of, the therapy (if any) granted jointly and used Specific formula." effective quantity " also refers under the dosage, and the toxicity or counter productive of the application polypeptide are not as good as caused by it Positive curative effect.
Pharmaceutically acceptable carrier is typically safe and nontoxic, and broadly may include being used to prepare in pharmaceutical industries Any known substance of pharmaceutical composition, as filler, diluent, coagulating agent, binder, lubricant, glidant, stabilizer, Colorant, wetting agent, disintegrant etc..When selection is suitable for delivering the excipient of synthetic peptide, this pharmaceutical composition need to be mainly considered The administering mode of object, the known technique of those skilled in the art.
The content of polypeptide described in the application pharmaceutical composition is about 0.01-1000 μM.
Aforementioned pharmaceutical compositions can be prepared according to known pharmaceutical procedures, for example《Remington's Pharmaceutical Science》 (Remington ' s Pharmaceutical Sciences) (the 17th edition, Alfonoso R.Gennaro are compiled, and Mike publishes public Take charge of (Mack Publishing Company), Easton, Pennsylvania (1985)) it is documented in detail in a book.
The pharmaceutical composition of the application can be various suitable dosage forms, including but not limited to tablet, capsule, injection Deng.
Also contain other known chemotherapeutics in the pharmaceutical composition of the application, it is especially known to be used for treating mammary gland The chemotherapeutics of cancer, including but not limited to cis-platinum, carboplatin, oxaliplatin, cyclophosphamide, methopterin, fluorouracil, Ah mould Element, taxol, docetaxel, mitoxantrone, Xeloda, gemzar, Epi-ADM, gemcitabine etc..
Also contain other known endocrine therapeutic agents in the pharmaceutical composition of the application, it is especially known to be used for controlling Treat the endocrine therapeutic agents of breast cancer, including but not limited to megestrol acetate, medroxyprogesterone acetate, Ai Luomeixin, tamoxifen, auspicious peaceful , Letrozole, Zoladex library etc..
Also contain other known target therapeutic agent in the pharmaceutical composition of the application, it is especially known to be used for treating The target therapeutic agent of breast cancer, including but not limited to antibody and tyrosine kinase inhibitor, wherein antibody include but not limited to Trastuzumab, handkerchief trastuzumab, bevacizumab, keytruda monoclonal antibodies, durvalumab monoclonal antibodies etc.；Tyrosine kinase inhibitor includes But Lapatinib, Nola are not limited to for Buddhist nun, Sutent etc..
Also contain other known immunotherapy medicaments in the pharmaceutical composition of the application, it is especially known to be used for treating The immunotherapy medicaments of breast cancer, including but not limited to anti-PD-1 antibody, anti-PD-L1 antibody, CAR-T cells etc..
The polypeptide and pharmaceutical composition of the application can be used to treat or prevent known Endostatin can treat or prevent it is each Kind disease mitigates or mitigates the various symptoms that known Endostatin can mitigate or mitigate.
For example, can the application polypeptide or its pharmaceutical composition give the objects of needs, for treating tumor of breast.Object Can be mammal, especially people.
Tumor of breast includes but not limited to that leaf tumour between breast neoplasm, mammary gland, fibroepithelial tumour, nipple are swollen Tumor, male breast carcinoma.In certain embodiments, tumor of breast is malignant tumour, i.e. breast cancer.Breast neoplasm includes But be not limited to carcinoma microinvasive, infiltrative breast carcinoma, epithelium-myoepithelial tumor, precursor lesion, Intraductal hyperplasia, Papillary lesion etc..Leaf tumour includes but not limited to myofibroblastoma, embryonal-cell lipoma, angiosarcoma etc. between mammary gland.Mammary gland Fibroepithelial tumour is phyllodes tumor or hamartoma etc..In certain embodiments, breast cancer is three cloudy breast cancer.
Therefore, the application also provides a kind of therapy of tumor of breast especially breast cancer, and this method includes giving to need The polypeptide or its pharmaceutical composition of the object the application wanted.
Chemotherapeutics also can be improved (such as treatment breast cancer, especially three cloudy breasts in the polypeptide of the application or its pharmaceutical composition The chemotherapeutics of gland cancer), endocrine therapeutic agents, molecular targeted agents and the effect of immunization therapy.Therefore, the application also provides A method of the effect of improving chemotherapeutics, endocrine therapeutic agents, molecular targeted agents or immunotherapy medicaments, this method Including before giving object chemotherapeutics, endocrine therapeutic agents, molecular targeted agents or the immunotherapy medicaments of needs, simultaneously Or the polypeptide or its pharmaceutical composition of the application is given later.
In methods and applications described herein, the amount of polypeptide contained in polypeptide to be administered or pharmaceutical composition should be Therapeutically effective amount.Tumor of breast can be any one previously described tumor of breast.The method of administration is had no it is specifically limited, Such as can be take orally, subcutaneous or intravenous etc..Chemotherapeutics, endocrine therapeutic agents, molecular targeted agents and immunization therapy Including treatment the tumour especially various chemotherapeutics, endocrine therapeutic agents, molecular targeted agents of breast cancer and various immune Medicine, including but not limited to previously described chemotherapy, molecular targeted and immunotherapy medicaments.
The application also provides the application polypeptide or pharmaceutical composition and is preparing for improving chemotherapeutics (such as treatment mammary gland Cancer, the chemotherapeutics of especially three cloudy breast cancer), the treatments of endocrine therapeutic agents, molecular targeted agents or immunotherapy medicaments Application in the drug of effect.
The application also provides the polypeptide as drug, the polypeptide such as the application various aspects or each embodiment institute above It states.The application is also provided for treating various tumors of breast described previously (being further three cloudy breast cancer) or being used for raisingization Treat drug (such as treatment breast cancer, the chemotherapeutics of especially three cloudy breast cancer), endocrine therapeutic agents, target therapeutic agent or The polypeptide of immunotherapy medicaments curative effect, the polypeptide is as described in the application above various aspects or each embodiment.
It should be understood that each feature in various aspects described herein, each embodiment and following each specific embodiments can Arbitrary combination, constitutes preferred embodiment.For example, each polypeptide as described herein is used equally for treating in listed disease Any one.
Embodiment
With reference to specific embodiment, the application is expanded on further.The implementation of the application unless otherwise stated, will use this Chemistry, biochemistry, recombinant DNA technology and immunologic conventional method known to field technology personnel.These technologies are in document In have complete explanation.Referring to such as《Peptide：Chemistry and biology》, Science Press, N. Xiu Ede, H.D. Jia Kubuke work, Liu Gram good, what army woods etc. is translated；《Basic immunology》(Fundamental Virology), the second edition, I and II volumes (B.N.Fields and D.M.Knipe are compiled)；《Experiment immunization learns to do volume》(Handbook of Experimental Immunology), (D.M.Weir and C.C.Blackwell are compiled I-IV volumes, Blackwell Scientific Publications)；T.E.Creighton,《Protein：Structure and molecular characterization》(Proteins:Structures and Molecular properties)(W.H.Freeman and Company,1993)；A.L.Lehninger,《Biochemistry》 (Biochemistry) (Worth Publishers, Inc. latest edition)；Sambrook etc.,《Molecular cloning：Laboratory manual》 (Molecular Cloning:A Laboratory Manual), the second edition, 1989；《Enzymology method》(Methods in Engymology) (S.Colowick and N.Kaplan are compiled, Academic Press, Inc.).Furthermore, it is to be understood that in the application " containing " also include " by ... form ".Used herein amino acid sequence number, i.e. " SEQ ID NO:1-29,38,39,41 And 47-49 ", only refer to amino acid sequence itself, does not include N-terminal modification and C-terminal modification.
Embodiment one：The preparation and modification of polypeptide
According to Peptide systhesis standard Fmoc schemes, originated with 0.25mM resins, according to following table sequence from c-terminus to amino Residue extends synthesis one by one at end, can finally add N-terminal modification.After peptide synthesis, cleaved liquid cutting, G6 glass sand hourglasses filter out Resin, filter vacuum are drained, and the C-terminal of polypeptide can further amidation.Deionized water dissolves polypeptide product, 100 type medium pressure liguid chromatograph C18 column purifications of explorer, Fraction collection main peak.Target peak collects sample with Agilent 1100 Type reverse-phase HPLC Phenomenex C18 analytical column Purities, LCQ Advantage type mass spectrograph molecular weight mirror It is fixed.Medium pressure liquid chromatography purifying gained collection liquid freeze-drying, be dissolved in PBS formed polypeptide storing liquid, 0.20 μM of filtration sterilization, -80 It DEG C freezes.Fig. 1 is shown in HPLC Purities and the identification of MASS mass spectroscopy molecular amounts.
Polypeptide is numbered Sequence number Sequence (from N-terminal to C-terminal)
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
Embodiment two：The separation and culture of Human umbilical vein endothelial cells (HUVEC)
Prepare umbilical cord preserving fluid：Dual anti-(blueness/chain) of 150ml PBS+3 times working concentrations；Prepare complete medium：80ml The glutamy of dual anti-+ 1ml heparin solutions (the 0.5%W/V)+1ml 200mM of M199+20ml FBS+1ml ECGS+1ml 100X Amine；Prepare separation instrument：Operation kidney basin 1, vessel forceps 4-5, operating scissors 2, the glass culture dish of diameter 10cm or so；I The configuration of Collagenase Type：It is configured to 1% (W/V).
The nearly fetus end umbilical cords of 20cm are taken, are rinsed well, both ends ligation is placed in 150ml umbilical cord preserving fluids；It is placed in 4 degree of ice Case preserves, and 6 hours digested；Check umbilical cord, remove undamaged portion, umbilical vein is fully rinsed well, after by 10ml clostridiopetidase As Solution injects, and is transferred to 37 DEG C of incubators and digests 15 minutes；Umbilical cord is taken out, digestive juice is collected, is used in combination PBS to clean, is resuspended after centrifugation Culture, changes liquid after 24 hours, removing is unable to attached cell.
Embodiment three：Inhibition of the polypeptide to Human umbilical vein endothelial cells (HUVEC) and tumour cell
Using mtt assay detection to the growth inhibition effect of cell.Its principle is the succinic acid dehydrogenation in living cells mitochondria Enzyme can make exogenous MTT (3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromides) be reduced to the indigo plant of water-insoluble Purple crystal first a ceremonial jade-ladle, used in libation (Formazan) is simultaneously deposited in cell, and dead cell is without this function.Dimethyl sulfoxide (DMSO) (DMSO) can dissolve First a ceremonial jade-ladle, used in libation in cell measures its absorbance value with enzyme-linked immunosorbent assay instrument at 490/570nm wavelength, can reflect living cells indirectly Quantity.Within the scope of certain cell number, it is directly proportional to cell number that MTT crystallizes the amount to be formed.
The HUVEC or tumour cell of exponential phase abandon culture supernatant, and PBS is washed 1 time, and 0.25% pancreatin of 1ml is added (4 DEG C), 37 DEG C of digestion 2min, are added culture supernatant and neutralize, and after piping and druming cell is at suspension, 1000 leave heart 3min.Supernatant is abandoned, is added Enter the resuspension of 5ml culture mediums.By 3 × 104/ ml is inoculated with 48 orifice plates, the holes 500ul/.5%CO2,37 DEG C are cultivated 24 hours.Culture Cell abandons supernatant, and the culture medium containing polypeptide is added and (contains Zn in culture medium2+A concentration of 17.39 μm of ol/L), continue culture 48 Hour.Supernatant, the holes 450ul/ PBS softly rinsing 1 time are carefully abandoned in each hole.450ul MTT culture mediums are added per hole, continue to cultivate 4h.It is careful to abandon culture supernatant, the holes dimethyl sulfoxide (DMSO) 450ul/ are added, sets and is protected from light low-speed oscillation 10min on shaking table.It will be on 150ul 96 hole elisa plates are transferred to clearly, the light absorption value in each hole is measured in enzyme-linked immunosorbent assay instrument OD490nm, 570nm.
Example IV：Polypeptide N-terminal is modified and C-terminal modification is on its active influence
Sequence such as SEQ ID NO shown in the method synthesis following table as shown in embodiment one:2 polypeptide, with or without N-terminal And/or C-terminal modification, wherein Ac are acetylation modification, NH2It is modified for amidation.HPLC Purities and MASS matter are carried out to it Compose molecular weight identification.
Polypeptide is numbered Sequence (from N-terminal to C-terminal)
P1 HSHRDFQPVLHLVALNSPLSGGMRGIRGAD
P2 Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH2
P3 Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD
P4 HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH2
Research of Recombinant Human Endostatin (endostatin, SEQ ID NO:1) can commercially obtain (such as Genetex article No. GTX65524, BioVision product article No.s 4799-1000, the Shanghai bio tech ltd Bo Sheng article No. E2296-05, Wuhan Boster Biological Technology Co., Ltd. article No. BP4153 etc.).The Research of Recombinant Human Endostatin medicine listed Object rhEndostatin (endostar, SEQ ID NO:10) it is bought from medical institutions.
It is (more in peptide concentration 1mg/ml, Research of Recombinant Human Endostatin concentration 5mg/ml according to method shown in embodiment three Peptide and Endostatin are close to equimolar concentration) experimental condition under to P1, P2, P3, P4 polypeptide, endostatin, endostar The HUVEC biological activities inhibited are measured, measurement result such as Fig. 2.
Embodiment five：The structure activity study of polypeptide
Such as the polypeptide of the method composition sequence such as following table of embodiment one, and measure its HPLC Purity and MASS mass spectrums point Son amount.According to method shown in embodiment three, the biological activity that aforementioned polypeptides inhibit HUVEC under 1mg/ml concentration conditions It is measured, measurement result such as Fig. 4.
With document (EMBO J.1998Mar 16；17(6):1656-1664) endostatin research structure (PDB numbers disclosed in According to library structure number 1BNL) based on, Blast search is carried out to aforementioned polypeptides using INSIGHT II softwares, obtains P2 polypeptides Preferential conformation.Use full activation space self-consistent field (CASSCF) to highest occupied molecular orbital (HOMO) energy of P2 polypeptides and minimum again Unoccupied orbital (LUMO) energy is calculated.On this basis, the dimension collaboration iteration of algorithm-polypeptide amino acid 2 created using us Space potential field algorithm (2-D synergistic iterative algorithms in spacial point field, 2-D SIASPF), amino acid carries out combination of two simulative iteration replacement in P2 polypeptides, calculates and replaces to polypeptide Zn ions binding activity domain Electron density variance caused by (1H, 3H, 11H) is cumulative, and combines the corresponding bioactivity of actual test combination of amino acids, to P2 Bioactivity conspiracy relation scores between two site of each amino acid, the results show that the 2nd and the 18th amino acids in polypeptide Highest is influenced on the concertedness of bioactivity.
Embodiment six：Polypeptide amino acid is replaced on its active influence
Build the P2 peptide combined peptides library of the 2nd and the 18th amino acids.It is automatically high using AAPPTEC companies Apex396 The polypeptide of flux Peptide synthesizer composition sequence such as following table, wherein X1And X3For any one natural amino acid (see the table below), X2With X4For S, X5And X6For G.
Polypeptide is numbered Sequence (from N-terminal to C-terminal)
P2X2X18 Ac-HX1HRDFQPVLHLVALNX2X3LX4X5X6MRGIRGAD-NH2(SEQ ID NO:38)
According to method shown in embodiment three, the biology that aforementioned polypeptides inhibit HUVEC under 1mg/ml concentration conditions Activity is measured, and measurement result see the table below.
In table, a, b, c, d, e, f, g, h, i and j of small letter are indicated respectively：
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%.
Embodiment seven：Polypeptide inhibits the growth in vitro of people's triple negative breast cancer cell BT-483
According to three the method for embodiment, in equimolar concentration, (P2T2S18 polypeptide 150uM concentration is approximately equivalent to each polypeptide It tests polypeptide shown in following table under the conditions of 0.5mg/ml) growth in vitro of people's triple negative breast cancer cell BT-483 cells is inhibited to make With test result is shown in Fig. 5 a and following table.
The results show that under 150uM concentration conditions rhEndostatin and P2 to BT-483 cells almost without effect.And P2T2S18, P2T2N18, P2T2S18-29 are apparent to BT-483 cyto-inhibitions, and activity of tumor cells is less than 15%, almost kills. The tumor suppression of P2T2S18-20, P2T2S18-25, P2T2S18-35, P2T2S18-40, P2T2S18-45 showed different Effect.
Polypeptide is numbered Sequence number Cell survival rate (%) when drug concentration is 150uM
P2T2S18-45 SEQ ID NO:3 44
P2T2S18-40 SEQ ID NO:4 39
P2T2S18-35 SEQ ID NO:5 26
P2T2S18 SEQ ID NO:6 14
P2T2S18-29 SEQ ID NO:41 9
P2T2S18-25 SEQ ID NO:7 17
P2T2S18-20 SEQ ID NO:8 28
P2T2-15 SEQ ID NO:24 86
P2T2N18 SEQ ID NO:9 20
P2 SEQ ID NO:2 99
endostar SEQ ID NO:10 93
Embodiment eight：Polypeptide inhibits the growth in vitro of people's triple negative breast cancer cell BT-483
Such as the method composition sequence following table polypeptide of embodiment one, figure is shown in HPLC Purities and the identification of MASS mass spectroscopy molecular amounts 1 and Fig. 3 a-3f.According to the test of three the method for embodiment to the inhibiting effect of BT-483, measurement result is shown in Fig. 5 b.
Embodiment nine：Polypeptide inhibits the growth in vitro of a variety of people's triple negative breast cancer cells
According to three the method for embodiment, each polypeptide tests P2 (SEQ ID NO under constant gradient concentration conditions:2)、 P2T2S18(SEQ ID NO:6)、P2T2N18(SEQ ID NO:And P2T2S18-29 (SEQ ID NO 9):41) polypeptide is to people three The growth in vitro inhibitory action of negative breast cancer cells BT-483, MDA-MB-453, MDA-MB-468, test result see Fig. 6 a, 6b and following table.
The results show that in 0.5mg/ml, P2 is to a variety of people's triple negative breast cancer cells almost without inhibiting effect, cell Survival rate is more than 90%；And P2T2S18, P2T2N18 and P2T2S18-29 can play multiple people's triple negative breast cancer cells Obvious inhibiting effect.P2T2S18, P2T2N18 and P2T2S18-29 in 2.5mg/ml to people's triple negative breast cancer cell almost It kills completely, Partial tumors cell survival rate is less than 10%.Correspondingly, P2 is bright to the IC50 concentration of people's triple negative breast cancer cell The aobvious IC50 concentration higher than P2T2S18, P2T2N18 and P2T2S18-29, even as high as 17 times.Microscope photo shows (figure 6b), each concentration P2 does not show effect to MDA-MB-453 cells.And P2T2S18 polypeptides cause apparent cellular morphology to change, Most cells are in dead state when 0.7mg/ml concentration；With the increase of peptide concentration, MDA-MB-453 mutually merges shape At agglomerate.As it can be seen that there are essential differences for effects of the P2 and P2T2S18 to MDA-MB-453 cells.
Embodiment ten：Polypeptide inhibits the growth in vitro of a variety of human breast cancer cells
According to three the method for embodiment, each polypeptide tests P2 (SEQ ID NO under constant gradient concentration conditions:2) and P2T2S18-29(SEQ ID NO:41) growth in vitro of human breast cancer cell SK-BR-3, T-47D and BT-474 are inhibited to make With the expression of each cell surface receptor and test result are shown in Fig. 7 and following table.
The results show that for P2 to human breast cancer cell almost without inhibiting effect, cell survival rate is big in 0.5mg/ml In 80%；And P2T2S18-29 can also play obvious inhibiting effect to non-triple negative breast cancer cell, in 0.5mg/ml to non- The cell survival rate of triple negative breast cancer is respectively less than 50%, and the expression with surface receptor does not show obvious relation between persistence.Correspondingly, P2 pairs The IC50 concentration of people's triple negative breast cancer cell is apparently higher than the IC50 concentration of P2T2S18-29, even as high as 17 times.
Embodiment 11：Foundation of the breast cancer in body tumor model
The human breast cancer cells for the exponential phase for taking in vitro culture in good condition, nude mice by subcutaneous inoculation contain 5 × 106 The culture cell suspension 100ul of oncocyte.Well-grown solid tumor is taken after 15 days, and about 3mm sizes are cut under aseptic condition Uniform fritter, inoculate one piece with the right armpit of every nude mice of trochar.Divided again according to tumor size within 10-14 days after inoculation Group, eliminates the excessive and too small animal of tumour, and every group of tumor average volume is almost the same.Each group is given according to testing program and is tested Drug.Major diameter (a), the minor axis (b) of tumor mass are measured 2 times a week.Animal is put to death after the test, and solution takes tumor mass, claims knurl weight, claps According to.Gross tumor volume TV=1/2 × a × b2；Tumour relative volume RTV=Vt/Vo, Vo are (to be administered first 1 day) to measure when dividing cage Gained gross tumor volume, Vt are gross tumor volume when measuring each time.Tumour inhibiting rate (%)=(1-T/C) * 100%, wherein T are treatment Group mean tumour volume, C are negative control group mean tumour volume.
Embodiment 12：Inhibition of the polypeptide to human breast cancer cell tumor growth
Such as the polypeptide of the method composition sequence such as following table of embodiment one：
The method as shown in embodiment 11 establishes human breast carcinoma in body tumor model, and experiment sets following 6 groups, except negative right Outside according to 9 animals of group, every group of 6 animals of other test groups, the wherein dosage of polypeptide and Endostatin close to equimolar, Endostar groups use marketed drug rhEndostatin (SEQ ID NO:10)：
1) negative control group (physiological saline, be subcutaneously injected sc, 2 times/day, successive administration 21 days)；
2) cyclophosphamide CTX (30mg/kg, be injected intraperitoneally ip, 1 times/day, successive administration 7 days)；
3) P2 groups (15mg/kg/ times, sc, 2 times/day, successive administration 21 days)；
4) P2T2S18 groups (15mg/kg/ times, sc, 2 times/day, successive administration 21 days)；
5) P2T2S18-29 groups (15mg/kg/ times, sc, 2 times/day, successive administration 21 days)；
6) endostar (50mg/kg/ times, sc, 2 times/day, successive administration 21 days).
Embodiment 13：Inhibition of the polypeptide to HUVEC growth in vitro
The polypeptide of method composition sequence such as following table as shown in embodiment one, and HPLC Purities and MASS are carried out to it Mass spectroscopy molecular amount is identified.
Polypeptide is numbered Sequence number Sequence (from N-terminal to C-terminal)
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
The sequence of recombinant vascular endothelial chalone (endostatin) such as SEQ ID NO:Shown in 1, marketed drug rhEndostatin (endostar) such as SEQ ID NO:Shown in 10.
According to the test of three the method for embodiment to the inhibiting effect of HUVEC, measurement result see the table below.As a result it shows P2T2S18 and P2T2N18 bioactivity is apparently higher than P2, IC50Concentration reduces about 10 times compared with P2.And carry the 2nd amino acids The polypeptide P2T2 of the simple point mutation and P2N18 and P2S18 for carrying the 18th amino acids simple point mutation, bioactivity are compared with P2 It is low, therefore P2T2S18 and P2T2N18 high bioactivities are difficult to caused by 2 amino acids and 18 amino acids common mutations The synergistic effect of expectation generates, it is seen that by conventional simple point mutation scan method be difficult to obtain this application involves P2T2S18 With P2T2N18 high bioactivity structures.
Embodiment 14：Inhibition of the polypeptide to HUVEC growth in vitro
The polypeptide of method composition sequence such as following table as shown in embodiment one, HPLC Purities and MASS mass spectroscopy molecular amounts Fig. 1 and Fig. 8 a-8j are shown in identification.
According to method test polypeptide shown in embodiment three to the inhibitory activity of HUVEC.Each polypeptide is in equimolar concentration item It is tested under part, wherein P2T2S18 polypeptides 300uM concentration is approximately equivalent to 1mg/ml.As a result see Fig. 9 and following table, show P2T2S18's C-terminal still has bioactivity after shortening or extend in a certain range.
Polypeptide is numbered Sequence number Cell survival rate (%) when peptide concentration is 120uM
P2T2S18-45 SEQ ID NO:3 80
P2T2S18-40 SEQ ID NO:4 49
P2T2S18-35 SEQ ID NO:5 15
P2T2S18 SEQ ID NO:6 2
P2T2S18-25 SEQ ID NO:7 31
P2T2S18-20 SEQ ID NO:8 52
P2T2-15 SEQ ID NO:24 95
Embodiment 15：Inhibition of the polypeptide to tumour cell and HUVEC growth in vitro
Such as the polypeptide of the method composition sequence such as following table of embodiment one, according to method shown in embodiment three in peptide concentration It is the inhibiting effect tested under the conditions of 0.1mg/ml to HUVEC, measurement result is shown in Figure 10.
Polypeptide is numbered Sequence number Sequence (from N-terminal to C-terminal)
P2T2S18 SEQ ID NO:6 Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH2
P2T2A17S18 SEQ ID NO:27 Ac-HTHRDFQPVLHLVALNASLSGGMRGIRGAD-NH2
P2T2S18T20 SEQ ID NO:28 Ac-HTHRDFQPVLHLVALNSSLTGGMRGIRGAD-NH2
P2T2A17S18T20 SEQ ID NO:29 Ac-HTHRDFQPVLHLVALNASLTGGMRGIRGAD-NH2
Embodiment 16：Inhibition of the polypeptide to HUVEC growth in vitro
Such as the polypeptide of the method composition sequence such as following table of embodiment one, HPLC Purities and the identification of MASS mass spectroscopy molecular amounts See Figure 11 a and 11b.According to method test shown in embodiment three to the inhibiting effect of HUVEC.Measurement result is shown in Figure 12.As a result Show that P2T2S18 with P2T2S18-29 bioactivity is similar, obviously higher than P2.
Polypeptide is numbered Sequence number Sequence (from N-terminal to C-terminal)
P2T2S18-29 SEQ ID NO:41 Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGA-NH2
Above-mentioned specific embodiment be only it is illustrative, and not restrictive.The protection domain of the application will be wanted by right It asks to limit.It will be understood by those skilled in the art that without departing from spirit and scope, it can be to the application's Various modification can be adapted and changes for technical solution, these modifications and variation include still within the scope of the present application.
Sequence table
<110>Shanghai Ji Bei Pharmaceutical Technology Co., Ltd
<120>Polypeptide for treating tumor of breast
<130> 170293
<160> 49
<170> PatentIn version 3.3
<210> 1
<211> 183
<212> PRT
<213>Homo sapiens（Homo sapiens）
<400> 1
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
20 25 30
Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ala Gly Thr Phe Arg Ala
35 40 45
Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile Val Arg Arg Ala
50 55 60
Asp Arg Ala Ala Val Pro Ile Val Asn Leu Lys Asp Glu Leu Leu Phe
65 70 75 80
Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu Gly Pro Leu Lys Pro
85 90 95
Gly Ala Arg Ile Phe Ser Phe Asp Gly Lys Asp Val Leu Arg His Pro
100 105 110
Thr Trp Pro Gln Lys Ser Val Trp His Gly Ser Asp Pro Asn Gly Arg
115 120 125
Arg Leu Thr Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Ala Pro Ser
130 135 140
Ala Thr Gly Gln Ala Ser Ser Leu Leu Gly Gly Arg Leu Leu Gly Gln
145 150 155 160
Ser Ala Ala Ser Cys His His Ala Tyr Ile Val Leu Cys Ile Glu Asn
165 170 175
Ser Phe Met Thr Ala Ser Lys
180
<210> 2
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The segment of human endostatin
<400> 2
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 3
<211> 45
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 3
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
20 25 30
Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ala Gly Thr
35 40 45
<210> 4
<211> 40
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 4
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
20 25 30
Cys Phe Gln Gln Ala Arg Ala Val
35 40
<210> 5
<211> 35
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 5
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
20 25 30
Cys Phe Gln
35
<210> 6
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 6
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 7
<211> 25
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 7
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser Gly Gly Met Arg Gly
20 25
<210> 8
<211> 20
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 8
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser
20
<210> 9
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 9
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Asn Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 10
<211> 192
<212> PRT
<213>Artificial sequence
<220>
<223>Research of Recombinant Human Endostatin drug rhEndostatin
<400> 10
Met Gly Gly Ser His His His His His His Ser His Arg Asp Phe Gln
1 5 10 15
Pro Val Leu His Leu Val Ala Leu Asn Ala Pro Leu Ser Gly Gly Met
20 25 30
Arg Gly Ile Arg Gly Ala Asp Phe Gln Cys Phe Gln Gln Ala Arg Ala
35 40 45
Val Gly Leu Ala Gly Thr Phe Arg Ala Phe Leu Ser Ser Arg Leu Gln
50 55 60
Asp Leu Tyr Ser Ile Val Arg Arg Ala Asp Arg Ala Ala Val Pro Ile
65 70 75 80
Val Asn Leu Lys Asp Glu Leu Leu Phe Pro Ser Trp Glu Ala Leu Phe
85 90 95
Ser Gly Ser Glu Gly Pro Leu Lys Pro Gly Ala Arg Ile Phe Ser Phe
100 105 110
Asp Gly Lys Asp Val Leu Arg His Pro Thr Trp Pro Gln Lys Ser Val
115 120 125
Trp His Gly Ser Asp Pro Asn Gly Arg Arg Leu Thr Glu Ser Tyr Cys
130 135 140
Glu Thr Trp Arg Thr Glu Ala Pro Ser Ala Thr Gly Gln Ala Ser Ser
145 150 155 160
Leu Leu Gly Gly Arg Leu Leu Gly Gln Ser Ala Ala Ser Cys His His
165 170 175
Ala Tyr Ile Val Leu Cys Ile Glu Asn Ser Phe Met Thr Ala Ser Lys
180 185 190
<210> 11
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 11
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 12
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 12
His Ala His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 13
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 13
His Glu His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 14
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 14
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Ala Asn
1 5 10 15
Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 15
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 15
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Ala
1 5 10 15
Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 16
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 16
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Thr Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 17
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 17
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ala Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 18
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 18
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ala Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 19
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 19
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Ala Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 20
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 20
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Leu Ala Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 21
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 21
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Leu Ser Ala Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 22
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 22
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Leu Ser Gly Ala Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 23
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 23
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Leu Ser Gly Gly Met Arg Gly Asp Arg Gly Ala Asp
20 25 30
<210> 24
<211> 17
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 24
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser
<210> 25
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 25
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 26
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 26
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Asn Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 27
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 27
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ala Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 28
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 28
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Thr Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 29
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of the segment of human endostatin
<400> 29
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ala Ser Leu Thr Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 30
<211> 549
<212> DNA
<213>Homo sapiens（Homo sapiens）
<400> 30
cacagccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag ccccctgtca 60
ggcggcatgc ggggcatccg cggggccgac ttccagtgct tccagcaggc gcgggccgtg 120
gggctggcgg gcaccttccg cgccttcctg tcctcgcgcc tgcaggacct gtacagcatc 180
gtgcgccgtg ccgaccgcgc agccgtgccc atcgtcaacc tcaaggacga gctgctgttt 240
cccagctggg aggctctgtt ctcaggctct gagggtccgc tgaagcccgg ggcacgcatc 300
ttctcctttg acggcaagga cgtcctgagg caccccacct ggccccagaa gagcgtgtgg 360
catggctcgg accccaacgg gcgcaggctg accgagagct actgtgagac gtggcggacg 420
gaggctccct cggccacggg ccaggcctcc tcgctgctgg ggggcaggct cctggggcag 480
agtgccgcga gctgccatca cgcctacatc gtgctctgca ttgagaacag cttcatgact 540
gcctccaag 549
<210> 31
<211> 135
<212> DNA
<213>Artificial sequence
<220>
<223>The coded sequence of the mutant of human endostatin segment
<400> 31
cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag cagcctgtca 60
ggcggcatgc ggggcatccg cggggccgac ttccagtgct tccagcaggc gcgggccgtg 120
gggctggcgg gcacc 135
<210> 32
<211> 120
<212> DNA
<213>Artificial sequence
<220>
<223>The coded sequence of the mutant of human endostatin segment
<400> 32
cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag cagcctgtca 60
ggcggcatgc ggggcatccg cggggccgac ttccagtgct tccagcaggc gcgggccgtg 120
<210> 33
<211> 105
<212> DNA
<213>Artificial sequence
<220>
<223>The coded sequence of the mutant of human endostatin segment
<400> 33
cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag cagcctgtca 60
ggcggcatgc ggggcatccg cggggccgac ttccagtgct tccag 105
<210> 34
<211> 90
<212> DNA
<213>Artificial sequence
<220>
<223>The coded sequence of the mutant of human endostatin segment
<400> 34
cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag cagcctgtca 60
ggcggcatgc ggggcatccg cggggccgac 90
<210> 35
<211> 75
<212> DNA
<213>Artificial sequence
<220>
<223>The coded sequence of the mutant of human endostatin segment
<400> 35
cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag cagcctgtca 60
ggcggcatgc ggggc 75
<210> 36
<211> 60
<212> DNA
<213>Artificial sequence
<220>
<223>The coded sequence of the mutant of human endostatin segment
<400> 36
cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag cagcctgtca 60
<210> 37
<211> 90
<212> DNA
<213>Artificial sequence
<220>
<223>The coded sequence of the mutant of human endostatin segment
<400> 37
cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag caacctgtca 60
ggcggcatgc ggggcatccg cggggccgac 90
<210> 38
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223>Xaa is arbitrary amino acid
<220>
<221> MISC_FEATURE
<222> (17)..(17)
<223>Xaa is S, A, L, I, V or T
<220>
<221> MISC_FEATURE
<222> (18)..(18)
<223>Xaa is arbitrary amino acid
<220>
<221> MISC_FEATURE
<222> (20)..(20)
<223>Xaa is S or T
<220>
<221> MISC_FEATURE
<222> (21)..(21)
<223>Xaa is G, A, L, I or V
<220>
<221> MISC_FEATURE
<222> (22)..(22)
<223>Xaa is G, A, L, I or V
<400> 38
His Xaa His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Xaa Xaa Leu Xaa Xaa Xaa Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 39
<211> 40
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of human endostatin segment
<400> 39
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Asn Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
20 25 30
Cys Phe Gln Gln Ala Arg Ala Val
35 40
<210> 40
<211> 120
<212> DNA
<213>Artificial sequence
<220>
<223>The coded sequence of the mutant of human endostatin segment
<400> 40
cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag caacctgtca 60
ggcggcatgc ggggcatccg cggggccgac ttccagtgct tccagcaggc gcgggccgtg 120
<210> 41
<211> 29
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of human endostatin segment
<400> 41
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala
20 25
<210> 42
<211> 5
<212> PRT
<213>Artificial sequence
<220>
<223> Tag
<400> 42
Arg Arg Arg Arg Arg
1 5
<210> 43
<211> 6
<212> PRT
<213>Artificial sequence
<220>
<223> Tag
<400> 43
His His His His His His
1 5
<210> 44
<211> 8
<212> PRT
<213>Artificial sequence
<220>
<223> Tag
<400> 44
Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
<210> 45
<211> 8
<212> PRT
<213>Artificial sequence
<220>
<223> Tag
<400> 45
Trp Ser His Pro Gln Phe Glu Lys
1 5
<210> 46
<211> 10
<212> PRT
<213>Artificial sequence
<220>
<223> Tag
<400> 46
Trp Gln Lys Leu Ile Ser Glu Glu Asp Leu
1 5 10
<210> 47
<211> 28
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of human endostatin segment
<400> 47
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly
20 25
<210> 48
<211> 31
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of human endostatin segment
<400> 48
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe
20 25 30
<210> 49
<211> 32
<212> PRT
<213>Artificial sequence
<220>
<223>The mutant of human endostatin segment
<400> 49
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
20 25 30

Claims (10)

1. the application of polypeptide or its pharmaceutical composition in preparing the drug for treating tumor of breast, wherein the polypeptide is Endostatin N-terminal grows the segment within 45 amino acid residues, and at least contains N-terminal 1-20 amino acids residues, and wherein The 2nd amino acids residue of Endostatin N-terminal and the 18th amino acids residue are respectively selected from following combination：
2nd amino acids 18th amino acids A M R I N K D E, M, T or Y Q A or H E S or V H A or S L R, E or S K V M L or W F T P C or V T N, G, K, M, F, S or T W C, E, I, K, S or Y Y R, H, W or V V D or S
And optionally, the 17th amino acids of Endostatin N-terminal are S, A, L, I or T；And/or the 20th amino acids residue be S or T；And/or if contain the 21st and/or the 22nd amino acids residue, the 21st amino acids residue be G, A, L, I or V and/or the 22nd amino acids residue are G, A, L, I or V；
Preferably, the amino acid sequence of the Endostatin such as SEQ ID NO:Shown in 1.
2. application as described in claim 1, which is characterized in that the polypeptide at least contains SEQ ID NO：38 1-22 Amino acid residue preferably at least contains SEQ ID NO：38 1-25 amino acids residues, and the 2nd and 18 amino acids residues As described in claim 1.
3. application as claimed in claim 1 or 2, which is characterized in that
The polypeptide at least contains SEQ ID NO：38 1-22 amino acids residues preferably at least contain SEQ ID NO：38 1-25 amino acids residues, and the 2nd amino acids residue be T, the 18th amino acids residue be N, G, K, M, F, S or T, and The amino acids of 17th, 20,21 and 22 are as described in claim 1；Or
The polypeptide at least contains SEQ ID NO：38 1-22 amino acids residues preferably at least contain SEQ ID NO：38 1-25 amino acids residues, and the 18th amino acids residue is N, the 2nd amino acids residue is T, and the 17th, 20,21 and 22 Amino acids are as described in claim 1；Or
The polypeptide at least contains SEQ ID NO：38 1-22 amino acids residues preferably at least contain SEQ ID NO：38 1-25 amino acids residues, and the 18th amino acids residue is S, the 2nd amino acids residue are E, H, L, T, W or V, and the 17,20,21 and 22 amino acids are as described in claim 1；Or
The amino acid sequence of the polypeptide such as SEQ ID NO:4, shown in any in 5,6,7,27-30,39,41 and 47-49；Or
The polypeptide is by SEQ ID NO:38 composition, wherein the 2nd amino acids residue be T, the 18th amino acids residue be N or S, and the amino acids of the 17th, 20,21 and 22 are as described in claim 1；Or
The polypeptide is selected from by SEQ ID NO:4 the 1st to the 39th, 38,37,36,34,33,32,31,29,28,27 or 26 ammonia The amino acid sequence of base acid residue composition, and by SEQ ID NO:39 the 1st to the 39th, 38,37,36,35,34,33,32,31, 29, the amino acid sequence of 28,27,26 or 25 amino acids residues composition；And/or
The 1st amino acids residue of polypeptide N-terminal is histidine, and the histidine is by formylated, acetylation, propionating or Butyrylation Modification, the 1st amino acids of C-terminal can be modified by PEG, cholesterol or amidation.
4. application as described in claim 1, which is characterized in that the polypeptide is selected from：
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；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRG-NH2；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRG；
HTHRDFQPVLHLVALNSSLSGGMRGIRG-NH2；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGA-NH2；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGA；
HTHRDFQPVLHLVALNSSLSGGMRGIRGA-NH2；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADF-NH2；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADF；
HTHRDFQPVLHLVALNSSLSGGMRGIRGADF-NH2；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQ-NH2；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQ；With
HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQ-NH2；
Wherein Ac is acetylation modification, NH2It is modified for amidation.
5. the application as described in any one of claim 1-4, which is characterized in that described pharmaceutical composition also contains pharmaceutically Acceptable carrier.
6. the application as described in any one of claim 1-5, which is characterized in that the tumor of breast is selected from HER2, estrogen Receptor, progesterone receptor three be negative triple negative breast cancer, both be negative breast cancer, the mammary gland that one is feminine gender Cancer and three are the breast cancer of the positive.
7. the application as described in any one of claim 1-5, which is characterized in that the tumor of breast is selected from breast epithelium Leaf tumour, mammary gland fibroepithelial tumour, theloma and male breast carcinoma between tumour, mammary gland.
8. the use as claimed in claim 7, which is characterized in that
The breast neoplasm be selected from carcinoma microinvasive, infiltrative breast carcinoma, epithelium-myoepithelial tumor, precursor lesion, Intraductal hyperplasia and papillary lesion；
Leaf tumour packet is selected from myofibroblastoma, embryonal-cell lipoma and angiosarcoma between the mammary gland；
The mammary gland fibroepithelial tumour is phyllodes tumor or hamartoma.
9. polypeptide or its pharmaceutical composition prepare for improve chemotherapeutics, endocrine therapeutic agents, molecular targeted agents or Application in the drug of the effect of immunotherapy medicaments, wherein the polypeptide is as described in any one of claim 1-4.
10. application as claimed in claim 9, which is characterized in that
The chemotherapeutics be cis-platinum, carboplatin, oxaliplatin, cyclophosphamide, methopterin, fluorouracil, adriamycin, taxol, It is one or more in docetaxel, mitoxantrone, Xeloda, gemzar, Epi-ADM or gemcitabine；
The endocrine therapeutic agents are megestrol acetate, medroxyprogesterone acetate, Ai Luomeixin, tamoxifen, Arimidex, Letrozole and Nuo Lei It is one or more in moral library；
The target therapeutic agent is antibody and tyrosine kinase inhibitor, and wherein antibody is that Trastuzumab, handkerchief trastuzumab, shellfish are cut down Monoclonal antibody, keytruda monoclonal antibodies or durvalumab monoclonal antibodies；Tyrosine kinase inhibitor is Lapatinib, Nola replaces for Buddhist nun and Shu Ni It is one or more in Buddhist nun；
The immunotherapy medicaments are one or more in anti-PD-1 antibody, anti-PD-L1 antibody or CAR-T cells.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112813042A (en) * 2021-02-08 2021-05-18 南京市妇幼保健院 Mammary gland adipose tissue-derived polypeptide and anti-tumor application thereof
CN114931634A (en) * 2022-03-18 2022-08-23 广州达博生物制品有限公司 Combined treatment method and pharmaceutical application of E10A and PD1 monoclonal antibody to tumors

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1875104A (en) * 2003-08-29 2006-12-06 儿童医学中心公司 Anti-angiogenic peptides from the N-terminus of endostatin
CN102924578A (en) * 2011-08-09 2013-02-13 哈药集团技术中心 Anti-tumor polypeptide, preparation method and anti-tumor applications thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1875104A (en) * 2003-08-29 2006-12-06 儿童医学中心公司 Anti-angiogenic peptides from the N-terminus of endostatin
CN102924578A (en) * 2011-08-09 2013-02-13 哈药集团技术中心 Anti-tumor polypeptide, preparation method and anti-tumor applications thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112813042A (en) * 2021-02-08 2021-05-18 南京市妇幼保健院 Mammary gland adipose tissue-derived polypeptide and anti-tumor application thereof
CN112813042B (en) * 2021-02-08 2021-08-27 南京市妇幼保健院 Mammary gland adipose tissue-derived polypeptide and anti-tumor application thereof
CN114931634A (en) * 2022-03-18 2022-08-23 广州达博生物制品有限公司 Combined treatment method and pharmaceutical application of E10A and PD1 monoclonal antibody to tumors

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