WO2013053080A1 - Protéine de fusion antitumorale, et gène codant et utilisation associés - Google Patents

Protéine de fusion antitumorale, et gène codant et utilisation associés Download PDF

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Publication number
WO2013053080A1
WO2013053080A1 PCT/CN2011/001711 CN2011001711W WO2013053080A1 WO 2013053080 A1 WO2013053080 A1 WO 2013053080A1 CN 2011001711 W CN2011001711 W CN 2011001711W WO 2013053080 A1 WO2013053080 A1 WO 2013053080A1
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Prior art keywords
cells
melanoma
colon cancer
fusion protein
cancer
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PCT/CN2011/001711
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English (en)
Chinese (zh)
Inventor
刘志敏
赵洪亮
薛冲
杜济良
任敏
夏姗
Original Assignee
中国人民解放军军事医学科学院生物工程研究所
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Priority to PCT/CN2011/001711 priority Critical patent/WO2013053080A1/fr
Publication of WO2013053080A1 publication Critical patent/WO2013053080A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • Anti-tumor fusion protein and coding gene thereof and application thereof are examples of Anti-tumor fusion protein and coding gene thereof and application thereof
  • the present invention relates to an anti-tumor fusion protein and its encoding gene and application.
  • Onconase (One) protein is a ribonuclease extracted from the fertilized eggs of the Leopard frog. Unlike endogenous enzymes (such as the human ribonuclease), the activity of One protein is not inhibited by RNase Inhibitor (RI) in the cytoplasm, so it can be degraded after endocytosis RNA in a variety of cytoplasms causes apoptosis in cells. The unique mechanism and target make One protein possible to become a new type of anti-tumor drug.
  • RI RNase Inhibitor
  • One protein lacks a specific cell surface receptor whose endocytosis is dependent on electrostatic interactions.
  • One protein is positively charged at physiological pH, and all eukaryotic cells are negatively charged under physiological conditions, and they can interact electrostatically with One protein to engulf it. Cell. Invention disclosure
  • the invention provides an anti-tumor fusion protein and a gene encoding the same and application.
  • the fusion protein provided by the present invention is as follows) or (b):
  • the HAS protein consists of sequence 1 of the sequence listing from amino acid residues 144 to 728 of the N-terminus;
  • the 4D5M0CB polypeptide fragment consists of sequence 1 of the sequence listing from amino acid residues 739 to 990 of the N-terminus;
  • a label as shown in Table 1 may be attached to the amino terminus or the carboxy terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing.
  • the protein in (b) above may be artificially synthesized, or may be synthesized by first synthesizing the encoded gene.
  • the gene encoding the protein in (b) above may be deleted by one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 2 in the sequence listing, and/or one or several base pairs may be missed.
  • the mutation, and/or the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end is obtained.
  • the fusion protein can be specifically as shown in SEQ ID NO:1 of the Sequence Listing.
  • HAS protein human serum albumin; human serum albumin
  • 4D5M0CB polypeptide fragments tumor-directed molecules; single-chain antibody fragments that recognize EpCAM promote the uptake of One by tumor cells and tissues.
  • the gene may specifically be a DNA molecule of the following (1) or (2) or (3) or (4):
  • the above stringent conditions may be to hybridize and wash the membrane in a solution of 0.1XSSPE (or 0.1XSSC) and 0.1% SDS at 65 °C.
  • a recombinant expression vector, expression cassette, transgenic cell line or recombinant strain containing the gene is within the scope of the present invention.
  • the recombinant expression vector may specifically insert the gene into the yeast expression vector PPIC9.
  • the recombinant plasmid obtained by cloning the site.
  • the recombinant strain may specifically be a recombinant strain obtained by introducing the recombinant expression vector into yeast.
  • the yeast is preferably Pichia pastoris.
  • the Pichia pastoris may specifically be Pichia pastoris, preferably Pichia pastoris GS 115 hi s4 (Mut + hi s- ) NRRLY-15851.
  • the present invention also contemplates a method of preparing the fusion protein by culturing the recombinant bacterium to obtain the protein.
  • methanol may be induced for 2-4 days in the process of culturing the recombinant bacteria.
  • the methanol induction time is preferably 72 hours.
  • the concentration of the methanol in the culture system is preferably 0.5% by volume.
  • the use of the fusion protein in the preparation of the following products is also within the scope of the invention: a tumor cell growth inhibitor and/or a cancer therapeutic drug and/or a cancer preventive drug.
  • the tumor cells may be colon cancer cells and/or melanoma cells.
  • the cancer can be colon cancer and/or melanoma.
  • the colon cancer cells are preferably HT29 cells.
  • the melanoma cell is preferably A375 cells.
  • the colon cancer is preferably colon cancer caused by HT29 cells.
  • the melanoma is preferably a melanoma caused by A375 cells.
  • the present invention also protects a product whose active ingredient is the fusion protein; the product is a tumor cell growth inhibitor and/or a cancer therapeutic drug and/or a cancer preventive drug.
  • the tumor cells may be colon cancer cells and/or melanoma cells.
  • the cancer can be colon cancer and/or melanoma.
  • the colon cancer cells are preferably HT29 cells.
  • the melanoma cell is preferably A375 cells.
  • the colon cancer is preferably colon cancer caused by HT29 cells.
  • the melanoma is preferably a melanoma caused by A375 cells.
  • the protein can be used to inhibit tumor cell growth and/or treat cancer and/or prevent cancer.
  • the tumor cells may be colon cancer cells and/or melanoma cells.
  • the cancer can be colon cancer and/or melanoma.
  • the colon cancer cells are preferably HT29 cells.
  • the melanoma cell is preferably A375 cells.
  • the colon cancer is preferably colon cancer caused by HT29 cells.
  • the melanoma is preferably a melanoma caused by A375 cells.
  • the product can be used to inhibit tumor cell growth and/or treat cancer and/or prevent cancer.
  • the cancer can be colon cancer and/or melanoma.
  • the colon cancer cells are preferably HT29 cells.
  • the melanoma cell is preferably A375 cells.
  • the colon cancer is preferably colon cancer caused by HT29 cells.
  • the melanoma is preferably a melanoma caused by A375 cells.
  • One or more pharmaceutically acceptable carriers may also be added to the above products as needed.
  • the carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrating agents, absorption enhancers, surfactants, adsorption carriers and the like in the pharmaceutical field.
  • the product of the present invention can be prepared into various forms such as an injection solution, a tablet, a powder, a granule, a capsule, and an oral solution.
  • the products of the above various dosage forms can be prepared according to a conventional method in the field of pharmacy.
  • the above fusion protein is generally administered in an amount of 500-1000 ⁇ g of fusion protein per adult per administration, once every 14-28 days for a course of 3 to 12 months.
  • Figure 1 shows the expression of the fusion gene in Pichia pastoris; M is the marker; 2 is the supernatant.
  • Figure 2 shows the purified ft7c- ⁇ S4-4Z ⁇ (electropherogram of 9 fusion protein; M is marker; 2 is the purified Onc-HSA-4D5M0CB fusion protein.
  • Figure 3 shows the in vitro antitumor biological activity of the 0nc_HSA-4D5M0CB fusion protein and One protein; A is HT29 cells; B is A375 cells.
  • Figure 4 Toxicity and antitumor activity of 0nc-HSA-4D5M0CB fusion protein and One protein in tumor-bearing nude mice.
  • the following examples are provided to facilitate a better understanding of the invention but are not intended to limit the invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. In the quantitative tests in the following examples, three replicate experiments were set, and the results were averaged.
  • the PCR amplification kit is a product of the Swedish Pharmacia company.
  • the DNA sequence analysis kit is a USB product of the United States.
  • the casein hydrolysate is a product of the German MERK company.
  • Bacto-yeast extract is a product of Di Fco Corporation of the United States.
  • a PCR amplification kit (product of Pharmacia, Sweden) was used.
  • Yeast expression vector PPIC9 purchased from Invi trogen, Catalog no. K1710-01.
  • NRRLY-15851 purchased from Inv i trogen, Catalog no. K1710-01.
  • HT29 cells human colon cancer cells, EpCAM positive
  • A375 cells human melanoma cells, EpCAM negative
  • ATCC catalog number CRL-1619.
  • Balb/c nude mouse purchased from the Experimental Animal Center of the Academy of Military Medical Sciences.
  • 1 M PBS buffer was obtained by mixing 132 ml of 1 M K 2 HP0 4 buffer and 868 ml of 1 M KH 2 P0 4 buffer; pH 6.0.
  • nucleotide sequences of the primers in the examples are as follows (5 ' ⁇ 3 '):
  • the gene encoding the 4D5M0CB polypeptide fragment shown by nucleotides 2215 to 2970 at the 5' end is i 4D5M0CB ⁇ 576bp).
  • PCR amplification was performed under the guidance of the primer OncF and the primer OncR to obtain a PCR amplification product.
  • PCR amplification was carried out under the guidance of the primer HSAF and the primer HSAR to obtain a PCR amplification product.
  • PCR amplification reaction system 50 ⁇ : PCR amplification product 1 ⁇ 1 of step 2, PCR amplification product 1 ⁇ 1 of step 3, primer OncF 2 ⁇ 1 , primer HSAR 2 ⁇ 1 , dNTP 1 ⁇ 1 , 10x pfu enzyme buffer 5 ⁇ l of liquid, 1 ⁇ l of pfu enzyme, and 37 ⁇ l of distilled water.
  • reaction conditions for PCR amplification were as follows: 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 4 minutes for a total of 3Q cycles.
  • PCR amplification was carried out under the guidance of primer OncF and primer HSAR to obtain a PCR amplification product.
  • PCR amplification was carried out under the guidance of primer 4D5F and primer 4D5R to obtain a PCR amplification product.
  • step 7 the PCR amplification product of step 5 and the PCR amplification product of step 6 are used as a template, and PCR amplification is carried out under the guidance of OncF and primer 4D5R to obtain a PCR amplification product.
  • PCR amplification reaction system 50 ⁇ ⁇ : PCR amplification product 1 ⁇ 1 of step 5, PCR amplification product 1 ⁇ 1 of step 6, primer OncF 2 ⁇ 1 , primer 4D5R 2 ⁇ 1 , dNTP 1 ⁇ 1 , 10 ⁇ pfu enzyme buffer 5 ⁇ l of liquid, 1 ⁇ l of pfu enzyme, and 37 ⁇ l of distilled water.
  • reaction conditions for PCR amplification were as follows: 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 5 minutes for a total of 3Q cycles.
  • step 7 The PCR amplification product of step 7 was digested with restriction endonucleases BamHI and EcoRI to obtain a digested product.
  • the yeast expression vector pPIC9 was digested with restriction endonuclease BamHI and EcoRI, and the vector backbone (about 7700 bp) was recovered.
  • step 8 The restriction enzyme product of step 8 is ligated with the vector backbone of step 9, to obtain a recombinant plasmid 0nc-HSA-4D5M0CB/pPIC9 o DNA sequence analysis kit (USB product of the United States). Recombinant plasmid 0nc-HSA-4D5M0CB/pPIC9 Sequencing was performed. The sequencing results showed that the 0nc-HSA-4D5M0CB fusion gene shown in SEQ ID NO: 2 of the sequence listing was inserted between the BamHI and EcoRI cleavage sites of the yeast expression vector pPIC9.
  • nucleotides 1 to 57 from the 5' end are the coding genes of the signal peptide
  • nucleotides 58 to 369 are the i3 ⁇ 4c gene
  • nucleotides 430 to 2184 are the nucleotides 2215 to 2970.
  • Sequence 1 of the fusion gene coding sequence shown in Sequence 2 of the Sequence Listing The 0nc-HSA-4D5M0CB fusion protein is shown.
  • amino acid residues from positions 1 to 19 of the N-terminus are the signal peptide
  • amino acid residues at positions 20 to 123 are the One protein
  • amino acid residues 144 to 728 are The HAS protein
  • amino acid residues 739 to 990 are 4D5M0CB polypeptide fragments.
  • sequence 2 of the sequence listing was inserted between the BamHI and EcoRI cleavage sites of the yeast expression vector PPIC9 from the nucleotides 1 to 369 of the 5' end to obtain the recombinant plasmid 0nc/pPIC9 (control plasmid). ).
  • the recombinant plasmid 0nc-HSA-4D5M0CB/pPIC9 was transformed into Pichia pastoris GS115 hi s4 (Mut + hi s- ) LY-15851 by yeast protoplast transformation method to obtain recombinant bacteria.
  • step 3 Take 1ml of the bacterial solution from step 2 and transfer it to 50ml liquid BMGY medium. Continue to shake.
  • step 4 The precipitate of step 4 was treated with 200 ml of liquid BMMY medium (1% yeast extract, 2% tryptone, 1. 34% YNB, 4 X 10 - 5 % biotin, 0.5% methanol, 100 mM pH 6. 5%) The amount of methanol in the medium is 0.5% by volume. The suspension is incubated at 30 ° C for 1 hr. .
  • liquid BMMY medium 1% yeast extract, 2% tryptone, 1. 34% YNB, 4 X 10 - 5 % biotin, 0.5% methanol, 100 mM pH 6. 5%
  • step 6 The supernatant collected in step 6 was subjected to non-reducing SDS-PAGE electrophoresis, and the results are shown in Fig. 1. The results indicate that the fusion gene can be efficiently secreted and expressed by Pichia pastoris.
  • the equilibration buffer used for the cation exchange chromatography was 20 mM phosphate buffer of pH 6.0, and the elution buffer was 20 mM pH 6.0 buffer containing 1 M NaCl.
  • the XK50/30 column was used with a column volume of 500 ml.
  • Elution process Equilibration buffer was used at the initial time, elution buffer was used at the end of the elution, and a mixture of equilibration buffer and elution buffer was used during the elution ( During the elution process, the volume ratio of the elution buffer in the mixture is linearly increased, and the volume ratio of the equilibration buffer in the mixture is linearly reduced. A total of 10 columns are eluted from the initial time to the end time. volume.
  • the post-column solution in the range of 60-70% (volume ratio) of the elution buffer was collected.
  • step 2 The post-column solution collected in step 1 was purified by Phenyl Sepharose HP hydrophobic chromatography.
  • the equilibration buffer used for hydrophobic chromatography was 20 mM pH 6.0 buffer containing 1 M (NH 4 ) 2 S0 4 , and the elution buffer was 20 mM P H6.0 phosphate buffer.
  • the XK50/30 column was used with a column volume of 500 ml.
  • Elution process Equilibration buffer was used at the initial time, elution buffer was used at the end of the elution, and a mixture of equilibration buffer and elution buffer was used during the elution ( During the elution process, the volume ratio of the elution buffer in the mixture is linearly increased, and the volume ratio of the equilibration buffer in the mixture is linearly reduced. A total of 10 columns are eluted from the initial time to the end time. volume.
  • the post-column solution in the range of 30-40% (volume ratio) of the elution buffer was collected.
  • step 3 The post-column solution collected in step 2 was further purified by Superde X 200 molecular sieve to obtain a final product.
  • the buffer used for the molecular sieve was 20 mM phosphate buffer pH 6.0.
  • the XK50/950 column was used and the volume of the column was 1500 ml.
  • the post-column solution from 700 mL to 800 mL was collected starting from the addition of buffer.
  • the supernatant obtained in step 6 of step 2 can be purified to obtain about 100 mg of the 0nc-HSA-4D5M0CB fusion protein.
  • the recombinant plasmid 0nc/pPIC9 was used instead of the recombinant plasmid 0nc-HSA-4D5M0CB/pPIC9 to carry out the steps 2 and 3, respectively, to obtain a purified One protein.
  • Example 2 In vitro antitumor activity of 0nc-HSA-4D5M0CB fusion protein The in vitro antitumor activity of the 0nc-HSA-4D5M0CB fusion protein was determined by the proliferation inhibition method.
  • Example 2000 cells (HT29 cells or A375 cells) were added to each well of a 96-well plate, and then different concentrations of the 0nc-HSA-4D5M0CB fusion protein prepared in Example 1 (or the One protein prepared in Example 1) were added at 37 ° C. After 4 days of culture, viable cells were detected by MTT staining. Three replicates were performed, and five replicate wells were set for each concentration in each test, and the results were averaged.
  • Group 1 Each nude mouse was intraperitoneally injected with 50 One protein per day, each injection volume was 0.3 ml (adjusted volume with 1 M PBS buffer); continuous injection for 4 days; second group (0nc -HSA-4D5M0CB group): Each nude mouse was intraperitoneally injected 300 ⁇ g per day.
  • 0nc-HSA-4D5M0CB fusion protein 300 ⁇ g 0nc-HSA-4D5M0CB fusion protein, the content of One protein is about 30 ⁇ g), each injection volume is 0.3 ml (volume adjusted with 1M PBS buffer); continuous Injection for 4 days.
  • the third group (PBS group): Each nude mouse was intraperitoneally injected with 1 M PBS buffer per day, each injection volume was 0.3 ml; continuous injection for 4 days;
  • the body weight change (mean) of each group of nude mice from the first day of administration is shown in Figure 4A and Table 4.
  • the tumor volume (mean) of each group of nude mice from the first day of administration is shown in Figure 4B and Table 5.
  • Table 5 Mean tumor volume (cubic mm) of each group of nude mice from the first day of dosing
  • the 0nc-HSA-4D5M0CB fusion protein had significant tumor suppressor activity, and the tumor growth inhibition rate was 67% on day 18 (after 2 weeks of the last treatment).
  • the toxicity of the 0nc-HSA-4D5M0CB fusion protein and One protein in mice was based on the decrease in body weight of mice.
  • the maximum dose at which the weight loss does not exceed 25% is defined as the maximum tolerated dose (MTD).
  • the maximal tolerated dose (MTD) of the 0nc-HSA_4D5M0CB fusion protein was significantly increased; the MTD of One protein was 13.2 mg/kg, and the MTD of the 0nc-HSA-4D5M0CB fusion protein was 80 mg/kg.
  • Onconase showed no anti-tumor activity, while the 0nc_HSA-4D5M0CB fusion protein had significant anti-tumor activity.
  • the present invention fuses the existing One protein with the HAS protein and the 4D5M0CB polypeptide fragment, and the HAS protein functions to increase the molecular weight, improve the pharmacokinetics and biodistribution, and the 4D5M0CB polypeptide fragment increases the specificity and selectivity for the tumor cells. .
  • the pharmacokinetic and biological distribution of the 0nc-HSA-4D5M0CB fusion protein provided by the present invention is remarkably improved, and tumor targeting is significantly increased.
  • the antitumor activity of tumor-bearing mice at the same toxic dose was significantly improved, showing a good application prospect.
  • the fusion protein of the invention has the advantages of high safety and good curative effect, high expression, easy purification, short production cycle, large scale of production and low cost, and has important application prospects and practical significance.

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Abstract

La présente invention concerne une protéine de fusion antitumorale, la protéine de fusion comprenant une protéine Onc constituée de 20 à 123 résidus d'acides aminés de SEQ ID ΝΟ : 1, une protéine HSA constituée de 144 à 728 résidus d'acides aminés de SEQ ID ΝΟ : 1 et un fragment de polypeptide 4D5MOCB constitué de 739 à 990 résidus d'acides amines de SEQ ID ΝΟ : 1. La présente invention concerne également le gène codant et l'utilisation de la protéine de fusion.
PCT/CN2011/001711 2011-10-12 2011-10-12 Protéine de fusion antitumorale, et gène codant et utilisation associés WO2013053080A1 (fr)

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Cited By (1)

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CN112386686A (zh) * 2019-08-14 2021-02-23 深圳市中科艾深医药有限公司 人sDR5-Fc重组融合蛋白在制备防治急性肾损伤药物中的应用

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112386686A (zh) * 2019-08-14 2021-02-23 深圳市中科艾深医药有限公司 人sDR5-Fc重组融合蛋白在制备防治急性肾损伤药物中的应用

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