WO2019205867A1 - Polypeptide se liant de manière spécifique à des cellules du cancer du côlon humain - Google Patents

Polypeptide se liant de manière spécifique à des cellules du cancer du côlon humain Download PDF

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Publication number
WO2019205867A1
WO2019205867A1 PCT/CN2019/079772 CN2019079772W WO2019205867A1 WO 2019205867 A1 WO2019205867 A1 WO 2019205867A1 CN 2019079772 W CN2019079772 W CN 2019079772W WO 2019205867 A1 WO2019205867 A1 WO 2019205867A1
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WIPO (PCT)
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polypeptide
colon cancer
cancer cells
human colon
cells
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PCT/CN2019/079772
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English (en)
Chinese (zh)
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于丽凤
魏敏杰
赵琳
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中国医科大学
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Publication of WO2019205867A1 publication Critical patent/WO2019205867A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the field of biomedicine, and particularly relates to a polypeptide which specifically binds to colon cancer cell SW480 and uses thereof.
  • Colon cancer is one of the most common malignant tumors of the digestive tract, and its incidence is second only to gastric cancer and esophageal cancer.
  • the age of good hair is generally over 50 years old, males are more than women, and the ratio of male to female is 2:1.
  • the cause of colon cancer is not clear, mainly environmental factors, genetic factors, precancerous diseases.
  • domestic surveys show that the survival rate of early colon cancer surgery is 90-95%, but in China more than 80% of patients have developed into the middle and late stage of diagnosis, the early diagnosis rate is only 10-15%; and the advanced colon cancer The survival rate is only 5%. Therefore, early diagnosis and early treatment can significantly improve the recovery of colon cancer patients.
  • Targeted therapy is the treatment of established cancer sites at the cellular level.
  • the corresponding therapeutic drug can be designed, and the drug enters the body to specifically select the carcinogenic site to combine and act to cause the tumor cell to specifically die without affecting the normal tissue cells surrounding the tumor, so the molecular targeted therapy is cancer treatment.
  • Another research focus is the treatment of established cancer sites at the cellular level.
  • the corresponding therapeutic drug can be designed, and the drug enters the body to specifically select the carcinogenic site to combine and act to cause the tumor cell to specifically die without affecting the normal tissue cells surrounding the tumor, so the molecular targeted therapy is cancer treatment.
  • Phage display technology is an important technique for screening intermolecular interactions in the field of molecular biology.
  • the main principle of phage display technology is that the gene of interest or the gene encoding the protein and polypeptide is cloned into the appropriate position of the phage surface protein gene by genetic engineering technology, and it is expressed on the surface of the phage with the amplification of phage DNA, due to the foreign gene.
  • the expression product polypeptide or protein of the foreign gene can maintain its original spatial structure and corresponding biological activity.
  • the target cells to perform subtractive screening on the phage, and finally select the phage which can specifically bind to the target cells from the phage polypeptide library, and sequence the DNA to obtain the coding sequence of the corresponding polypeptide.
  • This technology enables the connection between genotypes and phenotypes of proteins or peptides, and has the characteristics of simple operation and high-throughput detection, thus becoming an efficient means for screening tumor cell-specific binding peptides, for early detection of tumors and Targeted vector research in drug therapy offers new directions.
  • the object of the present invention is to provide a polypeptide which can specifically bind to colon cancer cells without affecting normal intestinal epithelial cells, which plays an important role in the early diagnosis of colon cancer and the development of targeted drugs.
  • Human normal intestinal epithelial HIEC cells were used as control.
  • Human phage display peptide library was used for subtractive screening of human phage display peptide library. Blue-white screening test was used to select positive phage which can specifically bind to colon cancer cells. The specificity of phage binding to colon cancer was verified by ELISA.
  • the purified phage is amplified and the DNA is extracted for sequencing, thereby obtaining a coding sequence of a polypeptide capable of specifically binding to colon cancer, and synthesizing a fluorescently labeled positive polypeptide for fluorescent labeling-polypeptide and human
  • the verification of the binding effect of colon cancer cells provides an experimental basis for early diagnosis and targeted therapy of colon cancer.
  • the present invention adopts the following technical scheme: a polypeptide which specifically binds to human colon cancer cells, and the polypeptide is any of the following: (1) the amino acid sequence of the polypeptide is: GLTSMRYHSVIV (SEQ ID No. 1), ( 2) A polypeptide derivative which has undergone deletion, insertion or substitution of one or several amino acids in the polypeptide molecule of (1) and which has the same biological function as the polypeptide molecule of (1).
  • the polypeptide has targeted binding to tumor cells and specifically binds to tumor cells.
  • the tumor cells are colon cancer cells.
  • polypeptide in the preparation of a tumor diagnostic kit comprising the polypeptide or polypeptide conjugate.
  • a polypeptide for specifically binding to human colon cancer cells for use in the preparation of a medicament for treating colon cancer, the medicament comprising the polypeptide and a pharmaceutically active ingredient, or comprising the polypeptide and a delivery vehicle.
  • the medicament is any pharmaceutically therapeutically acceptable dosage form, and the preferred dosage form of the medicament is an injectable preparation.
  • the drug is any pharmacologically acceptable dose.
  • the invention has the advantages that the phage display technology of the invention has simple operation, high-throughput panning, high efficiency, and can screen mimic epitopes, display polypeptides or proteins and genes contained in the phage.
  • Figure 1 ELISA results showing the OD 405 results of the affinity of clone 1-20 positive phage clones and human colon cancer SW480 cells.
  • Figure 2 is a graph showing the results of cellular immunofluorescence experiments on the affinity of No. 8 positive phage clones to human normal intestinal epithelial HIEC cells and human colon cancer SW480 cells.
  • Figure 3 is a graph showing the sequencing results of the positive phage clone No. 8.
  • Figure 4 is a graph showing the results of cellular immunofluorescence of FITC-GV12 and HIEC, SW480 cells.
  • Fig. 5 is a flow chart showing the results of flow cytometry of FITC-GV12 binding to HIEC and SW480 cells.
  • A is the binding ability result of HIEC and PBS;
  • B is the binding ability result of HIEC and the targeting polypeptide FITC-GV12;
  • C is the binding ability result of SW480 and PBS;
  • D is the combination of SW480 and the targeting polypeptide FITC-GV12 Capability results map.
  • Phage 12 peptide library E. coli ER2738, human colon cancer SW480 cells, human normal intestinal epithelial HIEC cells.
  • L-15 medium trypsin, FITG-labeled rabbit anti-mouse, fetal bovine serum, yeast powder, peptone, agar powder, tetracycline stock solution, Tween-20, bovine serum albumin BSA, M13 phage single-strand DNA extraction kit, IPTG, X-gal, PEG-8000, TMB.
  • Preparing cells The 6-well culture plate was pretreated with poly-lysine, and human colon cancer SW480 cells and human normal intestinal epithelial HIEC cells were taken and treated with trypsin and then cultured until the cells were successfully labeled. Screening was performed after the wall was in good growth.
  • Serum-free culture The cell culture medium was aspirated, washed once with PBS, and then added to serum-free medium, and placed in a 37 ° C constant temperature cell incubator with 5% CO 2 for 1 h.
  • Closing the cells Aspirate the cell culture medium, place the plate on a clean paper towel, remove the remaining medium, and block the human colon cancer SW480 cells and human normal intestinal epithelium HIEC with 1% BSA medium. The cells were placed in a 37 ° C thermostatic cell incubator with 5% CO 2 for 1 h.
  • Binding The adsorbed phage supernatant was incubated with human colon cancer SW480 cells for 1 h.
  • Washing Discard the unbound phage, place the microplate on a clean paper towel and pat it vigorously to remove the remaining solution. The plate was washed 5 times with 0.1% TBST as described above.
  • the IPTG/X-gal plate was preheated in an electrothermal incubator at 37 ° C; the appropriate amount of top agar was taken out and heated in a microwave oven, and after it was completely melted, it was taken out, and 3 ml was dispensed in each 10 ml centrifuge tube; After the phage was diluted in a ratio, 10 ⁇ l and 200 ⁇ l of E. coli were mixed and reacted for 5 min, then added to 3 ml of top agar, and then evenly spread on preheated IPTG/X-gal plate, after condensation. The titration results were observed at 37 ° C in an electrothermal incubator overnight.
  • Amplification of phage 20 ml of LB/Tet liquid medium was added to the Erlenmeyer flask, then E. coli bacteria solution and phage to be amplified were added at 1:100, placed at 37 ° C, and shaken vigorously in a constant temperature oscillator 4.5 h, an phage amplification solution is obtained.
  • Blocking Remove the fixed 96-well plate, take the liquid in the dry well, wash it with PBS 3 times, then add 3% hydrogen peroxide, and block it in a constant temperature incubator at 37 °C for 30 min to block Endogenous peroxidase activity.
  • Adding phage sample The blocked 96-well plate was taken out, and the liquid in the dry well was photographed, and the purified positive phage was added and reacted in a constant temperature incubator at 37 ° C for 1 h.
  • Termination The 96-well plate after the reaction was taken out, and the reaction was terminated by adding 2 M sulfuric acid.
  • DAPI staining washing with PBS 3 times, adding DAPI 100 ⁇ l, room temperature, 15 min
  • DAPI staining washing with PBS 3 times, adding DAPI 100 ⁇ l, room temperature, 15 min
  • the green fluorescently-labeled positive polypeptide FITC-GV12 was artificially synthesized, and the targeted binding of the polypeptide to human colon cancer cell SW480 was verified by immunofluorescence staining and flow cytometry.
  • the polypeptide sequence has weak binding ability to human normal intestinal epithelial HIEC cells, and the relative fluorescence intensity value is 1129.92 ⁇ 460.65, while the binding ability to human colon cancer cell SW480 is stronger, and the relative fluorescence intensity value is 36429.45 ⁇ 4173.03, there was a significant difference between the two P ⁇ 0.0001.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Veterinary Medicine (AREA)
  • Genetics & Genomics (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biophysics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne un polypeptide se liant de manière spécifique à des cellules du cancer du côlon et une utilisation associée. Le polypeptide est l'un quelconque parmi les éléments suivants : (1) un polypeptide ayant la séquence d'acides aminés : GLTSMRYHSVIV (SEQ ID No. 1), et (2) un dérivé polypeptidique pour lequel un ou plusieurs acides aminés dans la molécule polypeptidique de (1) ont subi une délétion, une insertion ou un déplacement et qui a la même fonction biologique que celle de la molécule polypeptidique de (1). Le polypeptide selon la présente invention est capable de se lier de manière spécifique à des cellules du cancer du côlon sans action spécifique avec des cellules épithéliales intestinales normales.
PCT/CN2019/079772 2018-04-26 2019-03-27 Polypeptide se liant de manière spécifique à des cellules du cancer du côlon humain WO2019205867A1 (fr)

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CN108640976B (zh) * 2018-04-26 2021-04-09 中国医科大学 一种与人结肠癌细胞特异性结合的多肽
CN113527431B (zh) * 2020-04-15 2024-04-19 辽宁中健医药科技有限公司 特异靶向人结直肠癌细胞的多肽及其用途
CN111518171B (zh) * 2020-05-06 2022-05-13 中国医科大学 一种靶向人肝癌细胞的多肽及其用途
CN113925973A (zh) * 2020-07-14 2022-01-14 辽宁中健医药科技有限公司 一种多肽偶联药物及其制备方法和应用

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CN102127153A (zh) * 2010-12-16 2011-07-20 陕西师范大学 Caco-2细胞表面特异性结合的多肽及其筛选方法
CN102477083A (zh) * 2010-11-22 2012-05-30 大连创达技术交易市场有限公司 具有导向治疗低毒副作用的癌胚抗原特异性结合肽
CN106967154A (zh) * 2017-03-24 2017-07-21 孟祥军 人结肠癌细胞特异性结合多肽的筛选方法及其应用
CN108640976A (zh) * 2018-04-26 2018-10-12 中国医科大学 一种与人结肠癌细胞特异性结合的多肽

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CN104086627B (zh) * 2014-05-29 2016-06-08 郑州大学 具有抗肿瘤活性的PD-L1 IgV亲和肽S10
CN104650190B (zh) * 2015-01-21 2018-02-13 陕西师范大学 肝癌细胞表面特异性结合的多肽
EP3117824A1 (fr) * 2015-07-17 2017-01-18 BE Pharbel Manufacturing Microparticules multicouche à libération de composé pharmaceutiquement actif sous forme posologique liquide

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CN102477083A (zh) * 2010-11-22 2012-05-30 大连创达技术交易市场有限公司 具有导向治疗低毒副作用的癌胚抗原特异性结合肽
CN102127153A (zh) * 2010-12-16 2011-07-20 陕西师范大学 Caco-2细胞表面特异性结合的多肽及其筛选方法
CN106967154A (zh) * 2017-03-24 2017-07-21 孟祥军 人结肠癌细胞特异性结合多肽的筛选方法及其应用
CN108640976A (zh) * 2018-04-26 2018-10-12 中国医科大学 一种与人结肠癌细胞特异性结合的多肽

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