WO2015161832A1 - Long-acting recombinant human interferon α2b-fc fusion protein - Google Patents

Long-acting recombinant human interferon α2b-fc fusion protein Download PDF

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WO2015161832A1
WO2015161832A1 PCT/CN2015/077504 CN2015077504W WO2015161832A1 WO 2015161832 A1 WO2015161832 A1 WO 2015161832A1 CN 2015077504 W CN2015077504 W CN 2015077504W WO 2015161832 A1 WO2015161832 A1 WO 2015161832A1
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fusion protein
long
human interferon
recombinant human
acting recombinant
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WO2015161832A8 (en
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朱文臣
朱成功
朱鹿燕
朱石桥
王晓山
刘宾
任子甲
朱松林
孙乃超
***
苏鸿声
孟庆乐
崔浩
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辅仁药业集团有限公司
河南辅仁医药科技开发有限公司
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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
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • Interferon is a defensive substance produced by human cells. After more than half a century of basic and clinical research, it has proven to be an important broad-spectrum antiviral and anti-tumor therapeutic. Among them, IFN ⁇ 2b is widely used in clinical practice and is the most used interferon. The US FDA approved it for the treatment of chronic hepatitis B, hepatitis C, hairy cell leukemia, Kaposi sarcoma and pigmentoma associated with AIDS. Subacute sclerosing encephalitis and T cell leukemia and other diseases. In China, IFN ⁇ 2b also belongs to the first batch of genetic engineering high-tech drugs officially approved by the state. After more than 20 years of market introduction, it has treated a variety of common diseases and frequently-occurring diseases in China, and has achieved great economic and social benefits.
  • IFN ⁇ 2b is widely used in clinical practice, it has the following disadvantages: due to its relatively small molecular weight, it is easily filtered by glomerulus during circulation in the body; it is unstable in the body and is easily degraded by serum protease; the plasma half-life is short and the treatment period is long. The half-life after intramuscular injection is 2-4 hours, and is almost undetectable in serum after 24 hours. Therefore, frequent administration is required in clinical treatment, and patient compliance is low. When used at high doses, it often leads to such as Red blood cells, white blood cells and thrombocytopenia, a series of side effects such as flu-like symptoms and gastrointestinal disorders directly affect the application of interferon in therapy. Therefore, improving the performance of interferon is still one of the focuses of the medical research.
  • the internationally developed techniques for long-acting interferon mainly include the following: First, chemical modification, mainly by covalently linking a polymer (such as PEG) to interferon, and reducing the kidney by increasing the relative molecular weight of the interferon. Filtration of the pellets prolongs the half-life of the interferon in the body.
  • PEG-Intron a polymer
  • Pegasys a polymer that reduces the kidney by increasing the relative molecular weight of the interferon. Filtration of the pellets prolongs the half-life of the interferon in the body.
  • PEG-Intron FDA-approved PEGylated interferon drugs
  • Pegasys The half-life of PEG-Intron is only extended to 40 hours. Although the half-life of Pegasys is extended to 65 hours, the antiviral activity in vitro is only 7 ⁇ .
  • the second is the site-directed mutagenesis technology, which changes the individual amino acids of interferon to reduce the digestion ability of proteases in the blood.
  • the use of site-directed mutagenesis to alter the original gene sequence of a protein often leads to a large change in the structure of the protein, which may lead to the production of new antigenic determinants and increase the toxic side effects of protein drugs.
  • the third is protein fusion technology, which fuses the drug protein gene with a specific protein gene by genetic recombination, and prolongs the half-life of interferon.
  • Human immunoglobulin G (IgG) is one of the most abundant proteins in human blood, and their half-life in the body is about 20 days.
  • an object of the present invention is to provide a novel long-acting recombinant human interferon ⁇ 2b-Fc fusion protein which passes through a peptide linker of a specific length and an N-terminus of Fc at the C-terminus of human interferon ⁇ 2b
  • the fusion forms a fusion protein, and the expression of the fusion protein is carried out by using a eukaryotic cell expression system, thereby obtaining a high protein expression amount, and the anti-viral activity of the prepared fusion protein is improved, and the half-life of the fusion protein is also prolonged.
  • the peptide linker has SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6.
  • SEQ ID NO: 7 SEQ ID NO: 8 or SEQ ID NO: 9.
  • amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331 that are known to be important for binding can be used as appropriate targets for modification.
  • amino acid substitutions that generally do not alter the activity of the protein or peptide are well known to those skilled in the art (H. Neurath, R. L. Hill, Protein, Academic Press, New York, 1979).
  • the invention also provides a nucleic acid sequence encoding the aforementioned long acting recombinant human interferon alpha 2b-Fc fusion protein.
  • the present invention also provides a cell expression system expressing a long-acting recombinant human interferon ⁇ 2b-Fc fusion protein, wherein the cell expression system is a yeast expression system, an insect cell expression system or a mammalian cell expression system.
  • the IFN ⁇ 2b-Fc fusion protein prepared by the present invention is formed by linking the C-terminus of IFN ⁇ 2b to the N-terminus of Fc via a peptide linker, and can also form a double-headed homodimeric Fc fusion protein, and the obtained fusion protein is similar. IgG but no CH1 region and light chain.
  • the IFN ⁇ 2b-Fc fusion protein exhibits in vivo pharmacokinetic properties comparable to isotype human IgG, resulting in an extended half-life of the IFN ⁇ 2b-Fc fusion protein, thereby reducing the frequency of dosing and enabling patients There is better compliance; and a decrease in fluctuations in the concentration of IFN ⁇ 2b-Fc fusion protein in serum means an improvement in its safety and tolerability.
  • IFN ⁇ 2b fractions on each fusion protein molecule both of which can perform their functions separately, resulting in higher molar activity (herein "molar activity" refers to the specific activity per unit mole of fusion protein. Increased activity of the fusion protein can reduce the dose administered.
  • the present inventors have surprisingly found that the in vivo half-life of the IFN ⁇ 2b-Fc fusion protein can be extended to more than 90 hours, while the in vitro antiviral activity can reach 10 7 -10 8 IU/mg.
  • the eukaryotic cell expression system provided by the invention can increase the stability of the protein and the expression of the protein, can make the fusion protein yield at least greater than 900 mg/L, and simplifies the purification step and reduces the production cost, and has a larger Practical significance and broad application prospects.
  • Figure 4 is a result of HPLC detection of the purity of the purified IFN ⁇ 2b-16L-IgG2Fc fusion protein of the present invention
  • the fusion protein codons containing the peptide linkers in Table 1 were separately synthesized: IFN ⁇ 2b-Fc was optimized according to CHO cell preference codon, and restriction endonuclease sites SpeI and EcoRI were added at the 5' and 3' ends.
  • the EMCV IRES fragment obtained by the artificial synthesis method and the mouse dihydrofolate reductase (DHFR) gene were inserted into the sites of EcoRI (5') and XhoI (3') of the mammalian expression vector PCDNA3 (Invitrogen).
  • the PCDNA3-DHFR vector was obtained.
  • the full-length DNA fragment of the optimized IFN ⁇ 2b-Fc was transferred to the SpeI (5') and EcoRI (3') sites on the PCDNA3-DHFR vector to obtain a PCDNA3-DHFR-IFN ⁇ 2b-Fc expression vector.
  • SEQ ID No. 10 is the amino acid sequence of IFN ⁇ 2b-16L-IgG2Fc fusion protein (wherein 1-16 amino acids are signal peptides, which are cleaved after secretion), and SEQ ID No. 11 is a fusion protein encoding IFN ⁇ 2b-16L-IgG2Fc a nucleic acid sequence (wherein 1-6 bp in the sequence is a SpeI cleavage site; 7-12 bp is a Kozak sequence; 13-60 bp is a signal peptide sequence; 61-558 bp is an IFN- ⁇ 2b sequence; and 559-606 bp is a peptide linker sequence; 607-1284 bp is the IgG2 Fc sequence); SEQ ID No.
  • SEQ ID No. 12 is the amino acid sequence of the IFN ⁇ 2b-32L-IgG2 Fc fusion protein (wherein 1-16 amino acids are signal peptides, which are cleaved after secretion), and SEQ ID No. 13 is an encoding The nucleic acid sequence of the IFN ⁇ 2b-32L-IgG2Fc fusion protein (wherein 1-6 bp in the sequence is a SpeI cleavage site; 7-12 bp is Kozak) Sequence; 13-60 bp is the signal peptide sequence; 61-558 bp is the IFN- ⁇ 2b sequence; 559-654 bp is the peptide linker sequence; 655-1332 bp is the IgG2 Fc sequence Pro331Ser).
  • the host cells were transfected by electroporation (CHO DG44): a Bio-Rad electrorotator was used, and a 4 mm electric rotor was used for the electrorotation, the voltage was set to 280 V and the shock time was 25 msec. Each shock was 1 ⁇ 10 7 cells, 40 ⁇ g of plasmid, and the total volume was 0.7 ml.
  • electroporation the cells were transferred to shake flasks containing 30 ml of growth medium. After 24 hours of culture, the medium was changed to a screening culture containing 50 nM MTX, and seeded in a 96-well plate at 1000 cells/well.
  • the cells were cultured for about 2 weeks until the clonal confluence rate reached 80% or higher.
  • the expression of the anti-human IFN- ⁇ 2b antibody and the anti-human IgG antibody was analyzed by sandwich ELISA, and the clones with relatively high expression were screened and transferred to 24 Expanded culture in well plates, 6-well plates, T25 cell culture flasks, and cell culture shake flasks.
  • DHFR gene and the fusion protein gene are co-amplified by inhibition of the DHFR gene by MTX.
  • MTX a pressurized method of increasing MTX concentration.
  • clones with high levels of expression of the fusion protein were screened by limiting dilution subcloning. Single cell clones stably and highly expressed were screened by cell proliferation and expression of the target protein of different clones.
  • the culture medium components were optimized, and the selected high-yield cell strains were inoculated at 5 ⁇ 10 5 /ml in a 100 ml volume shake flask, and after 3 to 4 days of culture, the fed stream was cultured for 14 days, and the target protein was obtained after fermentation.
  • Figure 1 shows the results of SDS-PAGE electrophoresis of the fusion protein IFN ⁇ 2b-16L-IgG2Fc and the fusion protein IFN ⁇ 2b-32L-IgG2Fc, wherein the R lane is a reduced fusion protein (ie, monomer) and the NR lane is a non-reduced fusion protein. (ie dimer).
  • the high-yielding cell line expressing the IFN ⁇ 2b-16L-IgG2Fc fusion protein has a viable cell density of up to 1.6 ⁇ 10 7 cells/ml, a cell viability of more than 90%, and a protein yield of greater than 900 mg. /L.
  • the high-yielding cell line expressing the IFN ⁇ 2b-32L-IgG2Fc fusion protein has a viable cell density of up to about 1.5 ⁇ 10 7 cells/ml, a cell viability of more than 90%, and a production of the target protein of which is greater than 1500mg/L.
  • the cell culture medium containing the protein of interest was collected by centrifugation and filtered through a 0.22 ⁇ m nitrocellulose filter. The filtrate was loaded onto a column MabSelect TM protein A (GE Healthcare) phosphate buffered saline (PBS) equilibrated. After the fusion protein was bound to the Protein A column, the effluent fraction was discarded and the column was washed with PBS until the OD value at 280 nm was less than 0.01.
  • PBS phosphate buffered saline
  • the bound fusion protein was then eluted with 20 mM Tris, 20 mM CaCl 2 , 300 mM NaCl, 900 mM arginine, 45% propylene glycol (v/v), 0.05% Tween 80 (v/v), pH 6.8 buffer.
  • the fractions containing the purified protein were pooled and dialyzed against PBS. It was then filtered through a 0.22 ⁇ m nitrocellulose filter and immediately stored at -70 °C. Under reducing conditions, the purified protein migrates to about 40-50 kDa to give a fusion protein.
  • HPLC results of the purified IFN ⁇ 2b-16L-IgG2Fc fusion protein were shown in Figure 4.
  • the HPLC detection conditions were as follows: column: Ferromen S2000; flow rate: 0.5 mL/min; mobile phase: 0.1 M phosphate buffer + 0.1 M NaCl (pH 7.0) solution; detection wavelength: 280 nm.
  • a human amniotic cell line (WISH cells) was prepared into a cell suspension of 2.5 ⁇ 10 5 -3.5 ⁇ 10 5 cells/ml in complete culture medium, and inoculated into a 96-well cell culture plate, 100 ⁇ l per well, and placed in CO 2 . Incubate for 4-6 hours in an incubator. The sample to be tested was diluted 1000 times with the assay medium. 150 ⁇ l of the assay medium was added to each well of a 96-well plate, and then 50 ⁇ l of the diluted sample and the positive control rHuIFN (purchased from Beijing Yuance Pharmaceutical Co., Ltd.) were added to the A row, and each of the three replicate wells was sequentially performed.
  • WISH cells human amniotic cell line
  • rHuIFN commercially produced rHuIFN (purchased from Beijing Yuance Pharmaceutical Co., Ltd.) and purified IFN ⁇ 2b-Fc of the present invention were injected into SD rats (injection dose of 5 ⁇ g/kg body weight) by subcutaneous injection to inject SD rats of the same volume of PBS.
  • injection dose 5 ⁇ g/kg body weight
  • PBS PBS
  • As a control (3 per group) blood samples were collected by tail-cutting blood at different time points (0, 1, 2, 4, 8, 12, 24, 36, 48, 60, 72, 96, 120 hours), and heparin sodium anticoagulation will The collected blood samples were centrifuged at 3000 rpm for 10 min, plasma was collected, and stored at -70 ° C for cryopreservation.
  • the blood sample was tested for the content of the fusion protein having the activity of the IFN ⁇ 2b of the present invention in the serum by using an ELISA kit according to the specification, and a graph reflecting the change in the protein content was plotted based on the result.
  • rHuIFN reached the blood concentration after 4 hours.
  • the peak value was 1 nmol/ml, and the elimination half-life was about 8.07 hours.
  • the IFN ⁇ 2b-16L-IgG2Fc fusion protein reached a peak plasma concentration of 1.15 nmol/ml at 24 hours, and the elimination half-life was 91.15 hours.
  • the IFN ⁇ 2b-32L-IgG2Fc fusion protein reached blood at 24 hours.
  • the drug concentration peaked at 1.1 nmol/ml and the elimination half-life was 93.25 hours.
  • Table 2 shows the results of comparison of the in vitro antiviral activity and elimination half-life of a part of the fusion protein of the present invention with the long-acting interferon reported in the literature.
  • the long-acting recombinant human interferon ⁇ 2b-Fc of the present invention is due to the steric hindrance effect of the fusion protein.
  • the fusion protein although reduced in vitro, has antiviral activity comparable to PEG-Intron and higher than Pegasys; and the in vivo half-life of the long-acting recombinant human interferon ⁇ 2b-Fc fusion protein of the present invention is long-lived with these two commercially available products.
  • Interferon PEG-Intron has been greatly improved compared to Pegasys.
  • the prolongation of the half-life means that the administration time can be extended and the number of administrations can be reduced when the same dose is administered.

Abstract

Provided is a human interferon α2b-Fc fusion protein, wherein the C-terminal of the human interferon α2b is linked to the N-terminal of Fc by a peptidic linker. Provided is a nucleic acid sequence encoding the fusion protein and a eukaryotic expression system expressing the fusion protein. Also provided is a use of the fusion protein in the preparation of an antiviral or antitumour drug. The fusion protein has an improved antiviral activity and prolonged in vivo half-life.

Description

长效重组人干扰素α2b-Fc融合蛋白Long-acting recombinant human interferon α2b-Fc fusion protein
本申请要求于2014年4月25日提交中国专利局、申请号为CN201410170133.3发明名称为“长效重组Fc融合人干扰素α2b及其制备方法”的中国专利申请的优先权，其全部内容通过引用并入本申请中。This application claims the priority of the Chinese Patent Application entitled "Long-term Recombinant Fc Fusion Human Interferon α2b and Its Preparation Method" filed on April 25, 2014 by the Chinese Patent Office, Application No. CN201410170133.3, the entire contents of which is hereby incorporated by reference. This application is incorporated by reference.
技术领域Technical field
本发明涉及人干扰素(IFN，interferon)α2b-Fc融合蛋白，尤其涉及含有肽连接体的长效重组人干扰素α2b-Fc融合蛋白，属生物基因工程领域。The present invention relates to a human interferon (IFN) interferon α2b-Fc fusion protein, and more particularly to a long-acting recombinant human interferon α2b-Fc fusion protein comprising a peptide linker, belonging to the field of biological genetic engineering.
背景技术Background technique
干扰素是人体细胞产生的一种防御物质，经过半个多世纪的基础和临床研究，证实其是一种重要的广谱抗病毒、抗肿瘤治疗药物。其中IFNα2b在临床上应用甚广，也是使用量最大的干扰素，美国FDA批准其用于治疗慢性乙型肝炎、丙型肝炎、毛细胞白血病、与AIDS病相关的卡波济肉瘤和色素瘤、亚急性硬化性脑炎和T细胞白血病等疾病。在我国IFNα2b也属于经国家正式批准的第一批基因工程高科技药物。投放市场20多年来，治疗多种我国常见病、多发病，取得了巨大的经济和社会效益。Interferon is a defensive substance produced by human cells. After more than half a century of basic and clinical research, it has proven to be an important broad-spectrum antiviral and anti-tumor therapeutic. Among them, IFNα2b is widely used in clinical practice and is the most used interferon. The US FDA approved it for the treatment of chronic hepatitis B, hepatitis C, hairy cell leukemia, Kaposi sarcoma and pigmentoma associated with AIDS. Subacute sclerosing encephalitis and T cell leukemia and other diseases. In China, IFNα2b also belongs to the first batch of genetic engineering high-tech drugs officially approved by the state. After more than 20 years of market introduction, it has treated a variety of common diseases and frequently-occurring diseases in China, and has achieved great economic and social benefits.
虽然IFNα2b在临床上应用广泛，但是存在以下缺点：由于相对分子量小，在体内循环过程中易被肾小球滤过；体内不稳定，很容易被血清蛋白酶降解；血浆半衰期短而治疗周期长，通过肌肉注射后半衰期为2-4小时，24小时后在血清中几乎检测不到，所以在临床治疗时需要频繁给药，患者依从性低；而在高剂量使用的时候，又往往会导致诸如红细胞、白细胞和血小板减少，出现流感样症状以及肠胃紊乱等一系列副作用，直接影响到干扰素在治疗中的应用。因此改进干扰素的性能，仍是医药界研究的重点之一。Although IFNα2b is widely used in clinical practice, it has the following disadvantages: due to its relatively small molecular weight, it is easily filtered by glomerulus during circulation in the body; it is unstable in the body and is easily degraded by serum protease; the plasma half-life is short and the treatment period is long. The half-life after intramuscular injection is 2-4 hours, and is almost undetectable in serum after 24 hours. Therefore, frequent administration is required in clinical treatment, and patient compliance is low. When used at high doses, it often leads to such as Red blood cells, white blood cells and thrombocytopenia, a series of side effects such as flu-like symptoms and gastrointestinal disorders directly affect the application of interferon in therapy. Therefore, improving the performance of interferon is still one of the focuses of the medical research.
目前国际上研制长效干扰素的技术手段主要有以下几种：一是化学修饰，主要是将多聚物(例如PEG)共价连接到干扰素上，通过增加干扰素的相对分子量，减少肾小球的滤过，延长干扰素在体内的半衰期。目前FDA批准的PEG化的干扰素药物有两种：PEG-Intron和Pegasys，PEG-Intron的半衰期也只延长到40小时，Pegasys的半衰期虽然延长到65小时，但体 外抗病毒活性仅为7×106IU/mg；二是基因的定点突变技术，改变干扰素个别氨基酸从而降低血液中蛋白酶的消化能力。使用定点突变来改变蛋白质的原始基因序列，往往导致蛋白质结构发生很大的改变，很可能导致新的抗原决定簇的产生，而加大蛋白质药物的毒副作用。三是蛋白融合技术，通过基因重组手段将药物蛋白质基因与特定的蛋白基因融合，延长了干扰素的半衰期。人免疫球蛋白G(IgG)是人类血液中最丰富的蛋白质之一，它们在体内的半衰期约为20天。Fc片段是IgG保持较长体内半衰期的主要原因，Fc片段还有助于蛋白的稳定性。这是由于Fc片段可以与新生Fc受体(FcRn)结合，避免IgG进入溶酶体中被降解，Fc还可以增加蛋白的分子量，减少肾小球的滤过。将IgG的Fc片段与活性蛋白质链接构成融合蛋白，已经证实可以延长活性蛋白的半衰期。At present, the internationally developed techniques for long-acting interferon mainly include the following: First, chemical modification, mainly by covalently linking a polymer (such as PEG) to interferon, and reducing the kidney by increasing the relative molecular weight of the interferon. Filtration of the pellets prolongs the half-life of the interferon in the body. There are currently two FDA-approved PEGylated interferon drugs: PEG-Intron and Pegasys. The half-life of PEG-Intron is only extended to 40 hours. Although the half-life of Pegasys is extended to 65 hours, the antiviral activity in vitro is only 7×. 10 6 IU / mg; The second is the site-directed mutagenesis technology, which changes the individual amino acids of interferon to reduce the digestion ability of proteases in the blood. The use of site-directed mutagenesis to alter the original gene sequence of a protein often leads to a large change in the structure of the protein, which may lead to the production of new antigenic determinants and increase the toxic side effects of protein drugs. The third is protein fusion technology, which fuses the drug protein gene with a specific protein gene by genetic recombination, and prolongs the half-life of interferon. Human immunoglobulin G (IgG) is one of the most abundant proteins in human blood, and their half-life in the body is about 20 days. The Fc fragment is the main reason for IgG to maintain a longer half-life in vivo, and the Fc fragment also contributes to protein stability. This is because the Fc fragment can bind to the neonatal Fc receptor (FcRn), preventing IgG from being degraded into the lysosome, and Fc can also increase the molecular weight of the protein and reduce glomerular filtration. Linking the Fc fragment of IgG to the active protein to form a fusion protein has been shown to prolong the half-life of the active protein.
专利申请CN1361793公开了一种Fc-IFNα融合蛋白，其半衰期仅为19.3小时；美国专利US5723125公开了一种包含肽连接体的IFN-Fc融合蛋白，所述肽连接体是Gly-Ser重复单位，该融合蛋白的半衰期虽然延长，但抗病毒活性仅为2.2×108IU/μmol(换算之后约5×106IU/mg)。综上所述，较小幅度地延长半衰期并不能有效减少给药频率，而以牺牲抗病毒活性来一味延长半衰期，又达不到相应的治疗效果，因此，近些年人们在长效干扰素的研究中不断努力在活性和长效性之间寻找平衡点，希望活性降低在可以接受的范围之内，同时尽可能的延长其在体内的半衰期。Patent application CN1361793 discloses an Fc-IFNα fusion protein having a half-life of only 19.3 hours; US Pat. No. 5,723,125 discloses an IFN-Fc fusion protein comprising a peptide linker which is a Gly-Ser repeat unit, Although the half-life of the fusion protein was prolonged, the antiviral activity was only 2.2 × 10 8 IU/μmol (about 5 × 10 6 IU/mg after conversion). In summary, a small extension of the half-life does not effectively reduce the frequency of dosing, but at the expense of antiviral activity to extend the half-life, and the corresponding therapeutic effect is not achieved. Therefore, in recent years, people have long-term interferon Efforts have been made to find a balance between activity and long-term efficacy, and it is hoped that the activity will be reduced within an acceptable range while maximizing its half-life in vivo.
目前市售的长效干扰素均是在大肠杆菌表达***中表达的，然而原核表达***具有包涵体蛋白不易纯化、蛋白修饰不完整等缺陷。相反地，真核表达***能够克服上述问题，真核表达***表达产物大多能够分泌，易于获取和纯化；表达产物能够糖基化，更接近于天然蛋白；表达***的稳定性好，表达量可以调控。常见的真核表达***有酵母表达***、昆虫细胞表达***和哺乳动物细胞表达***。其中哺乳动物细胞表达***，特别是二氢叶酸还原酶(DHFR)双倍体缺陷的中国仓鼠卵巢细胞(Chinese hamster ovary，CHO)表达***，已经有越来越多的药用蛋白在CHO细胞中获得高效表达，其中部分药物已经投放市场，例如EPO、GCSF等。CHO表达***具有许多优点：1)有准确的转录后修饰功能，表达的糖基化的药物蛋白分子在分子 结构、理化特性和生物学功能方面最接近于天然蛋白分子；2)CHO具有胞外分泌功能，又很少分泌自身的内源性蛋白，便于下游产物的分离纯化；3)可以进行无血清悬浮培养，表达水平高。但目前未见IFNα2b-Fc融合蛋白采用真核细胞表达的相关报道。因此，亟需采用真核表达***来克服原核表达***的各种问题。The long-acting interferons currently available in the market are expressed in the E. coli expression system. However, the prokaryotic expression system has defects such as incompatibility of the inclusion body protein and incomplete protein modification. Conversely, eukaryotic expression systems can overcome the above problems. Most of the expression products of eukaryotic expression systems can be secreted, easily obtained and purified; the expression products can be glycosylated, closer to the natural protein; the stability of the expression system is good, and the expression can be Regulation. Common eukaryotic expression systems are yeast expression systems, insect cell expression systems, and mammalian cell expression systems. Among them, mammalian cell expression systems, especially dihydrofolate reductase (DHFR) diploid-deficient Chinese hamster ovary (CHO) expression system, have more and more pharmaceutical proteins in CHO cells. Highly expressed, some of which have been placed on the market, such as EPO, GCSF, etc. The CHO expression system has many advantages: 1) it has an accurate post-transcriptional modification function, and the expressed glycosylated drug protein molecule is in the molecule. The structure, physical and chemical properties and biological functions are closest to the natural protein molecules; 2) CHO has extracellular secretory function, and rarely secretes its own endogenous protein, which facilitates the separation and purification of downstream products; 3) serum-free suspension culture , the expression level is high. However, there is no report on the expression of IFNα2b-Fc fusion protein in eukaryotic cells. Therefore, there is an urgent need to adopt eukaryotic expression systems to overcome various problems of prokaryotic expression systems.
发明内容Summary of the invention
本发明提供了基于上述问题，本发明目的在于提供一种新的长效重组人干扰素α2b-Fc融合蛋白，通过在人干扰素α2b的C端通过特定长度的肽连接体与Fc的N端连接形成融合蛋白，并采用真核细胞表达***进行融合蛋白表达，获得了较高的蛋白表达量，并使得制备出的融合蛋白抗病毒活性提高，同时还大大延长了其在体内的半衰期。The present invention provides, based on the above problems, an object of the present invention is to provide a novel long-acting recombinant human interferon α2b-Fc fusion protein which passes through a peptide linker of a specific length and an N-terminus of Fc at the C-terminus of human interferon α2b The fusion forms a fusion protein, and the expression of the fusion protein is carried out by using a eukaryotic cell expression system, thereby obtaining a high protein expression amount, and the anti-viral activity of the prepared fusion protein is improved, and the half-life of the fusion protein is also prolonged.
本发明提供了一种长效重组人干扰素α2b-Fc融合蛋白，其结构为人干扰素α2b-肽连接体-Fc，其中，所述肽连接体具有以下结构：X-(GGGS)p-(GGGGS)n-(GGGS)q，其中X为GS或GGS，p＝0-2，q＝0-2，n＝1-8。The present invention provides a long-acting recombinant human interferon α2b-Fc fusion protein having the structure of human interferon α2b-peptide linker-Fc, wherein the peptide linker has the following structure: X-(GGGS)p-( GGGGS)n-(GGGS)q, where X is GS or GGS, p=0-2, q=0-2, n=1-8.
在本发明的一个优选实施方式中，所述肽连接体具有以下结构：X-(GGGS)p-(GGGGS)n-(GGGS)q，其中X为GS或GGS，p＝0-1，q＝0-1，n＝1-6。In a preferred embodiment of the invention, the peptide linker has the structure: X-(GGGS)p-(GGGGS)n-(GGGS)q, wherein X is GS or GGS, p=0-1, q =0-1, n=1-6.
在本发明的更优选实施方式中，所述肽连接体具有SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8或SEQ ID NO：9的结构。In a more preferred embodiment of the invention, the peptide linker has SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6. The structure of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
本发明的Fc片段包括人IgG1、IgG2、IgG3和IgG4亚型的Fc片段，优选IgG2和IgG4亚型的Fc，最优选IgG2亚型的Fc。本发明的Fc片段包括天然氨基酸序列和其序列衍生物(突变体)。氨基酸序列衍生物是由于一个或多个氨基酸残基的缺失、***、非保守性或保守性取代或它们的联合而不同于天然氨基酸序列的序列。例如，在IgG Fc中，已知对于结合重要的、位于位点214至238、297至299、318至322或327至331处的氨基酸残基可用作修饰的适当靶目标。在蛋白质或多肽中，通常不改变该蛋白质或肽活性的氨基酸替换是本领域技术人员所熟知的(H.Neurath，R.L.Hill，蛋白质， Academic Press，纽约，1979)。The Fc fragments of the invention include Fc fragments of human IgGl, IgG2, IgG3 and IgG4 subtypes, preferably Fc of IgG2 and IgG4 subtypes, most preferably Fc of the IgG2 subtype. The Fc fragment of the present invention includes a natural amino acid sequence and a sequence derivative (mutant) thereof. Amino acid sequence derivatives are sequences that differ from the native amino acid sequence due to deletions, insertions, non-conservative or conservative substitutions of one or more amino acid residues, or combinations thereof. For example, in IgG Fc, amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331 that are known to be important for binding can be used as appropriate targets for modification. In proteins or polypeptides, amino acid substitutions that generally do not alter the activity of the protein or peptide are well known to those skilled in the art (H. Neurath, R. L. Hill, Protein, Academic Press, New York, 1979).
在本发明的一个优选实施方案中，长效重组人干扰素α2b-Fc融合蛋白可以以同源二聚体的形式存在。In a preferred embodiment of the invention, the long acting recombinant human interferon alpha 2b-Fc fusion protein may be present as a homodimer.
本发明还提供了一种编码前述长效重组人干扰素α2b-Fc融合蛋白的核酸序列。The invention also provides a nucleic acid sequence encoding the aforementioned long acting recombinant human interferon alpha 2b-Fc fusion protein.
本发明还提供了一种表达长效重组人干扰素α2b-Fc融合蛋白的细胞表达***，其中，所述细胞表达***为酵母表达***、昆虫细胞表达***或哺乳动物细胞表达***。The present invention also provides a cell expression system expressing a long-acting recombinant human interferon α2b-Fc fusion protein, wherein the cell expression system is a yeast expression system, an insect cell expression system or a mammalian cell expression system.
在本发明的一个优选实施方案中，利用二氢叶酸还原酶(DHFR)双倍体缺陷的CHO细胞表达***来表达本发明的融合蛋白。In a preferred embodiment of the invention, the dihydrofolate reductase (DHFR) diploid deficient CHO cell expression system is utilized to express the fusion protein of the invention.
本发明还提供了一种前述长效重组人干扰素α2b-Fc融合蛋白在制备抗病毒或抗肿瘤药物中的用途。The present invention also provides the use of the aforementioned long-acting recombinant human interferon α2b-Fc fusion protein for the preparation of an antiviral or antitumor drug.
此外，本发明制备的IFNα2b-Fc融合蛋白是通过一段肽连接体将IFNα2b的C端与Fc的N端连接形成，也可以形成双头同源二聚体Fc融合蛋白，得到的融合蛋白类似于IgG但无CH1区域和轻链。由于结构上的同源性，IFNα2b-Fc融合蛋白表现出与同种型人IgG相当的体内药代动力学特性，使得IFNα2b-Fc融合蛋白的半衰期延长，从而能够降低给药的频率，使患者有更好的依从性；并且血清中IFNα2b-Fc融合蛋白浓度波动的减少意味着其安全性和耐受性的改善。另外，每个融合蛋白分子上有两个IFNα2b部分，这两个部分都能分别行使其功能，得到更高的摩尔比活(此处“摩尔比活”是指每单位摩尔融合蛋白的比活)，融合蛋白活性的增高能够降低给药剂量。Furthermore, the IFNα2b-Fc fusion protein prepared by the present invention is formed by linking the C-terminus of IFNα2b to the N-terminus of Fc via a peptide linker, and can also form a double-headed homodimeric Fc fusion protein, and the obtained fusion protein is similar. IgG but no CH1 region and light chain. Due to structural homology, the IFNα2b-Fc fusion protein exhibits in vivo pharmacokinetic properties comparable to isotype human IgG, resulting in an extended half-life of the IFNα2b-Fc fusion protein, thereby reducing the frequency of dosing and enabling patients There is better compliance; and a decrease in fluctuations in the concentration of IFNα2b-Fc fusion protein in serum means an improvement in its safety and tolerability. In addition, there are two IFNα2b fractions on each fusion protein molecule, both of which can perform their functions separately, resulting in higher molar activity (herein "molar activity" refers to the specific activity per unit mole of fusion protein. Increased activity of the fusion protein can reduce the dose administered.
不受任何理论的限制，本发明出人意料地发现IFNα2b-Fc融合蛋白的体内半衰期可延长至90小时以上，同时体外抗病毒活性能够达到107-108IU/mg。另外，采用本发明提供的真核细胞表达***可以提高蛋白的稳定性和蛋白的表达量，可以使得融合蛋白产量至少大于900mg/L，并且简化了纯化步骤和降低了生产成本，具有较大的实际意义和广阔的应用前景。 Without being bound by any theory, the present inventors have surprisingly found that the in vivo half-life of the IFNα2b-Fc fusion protein can be extended to more than 90 hours, while the in vitro antiviral activity can reach 10 7 -10 8 IU/mg. In addition, the eukaryotic cell expression system provided by the invention can increase the stability of the protein and the expression of the protein, can make the fusion protein yield at least greater than 900 mg/L, and simplifies the purification step and reduces the production cost, and has a larger Practical significance and broad application prospects.
附图说明DRAWINGS
为了更清楚地说明本发明实施例和现有技术的技术方案，下面对实施例和现有技术中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the embodiments of the present invention and the prior art, the following description of the embodiments and the drawings used in the prior art will be briefly described. It is obvious that the drawings in the following description are only Some embodiments of the invention may also be used to obtain other figures from these figures without departing from the art.
图1为SDS-PAGE电泳鉴定本发明IFNα2b-16L-IgG2Fc融合蛋白和IFNα2b-32L-IgG2Fc融合蛋白的电泳图；泳道R点的样品是经还原的融合蛋白(即二聚体打开)，泳道NR点的样品是非还原的融合蛋白；1 is an electrophoresis pattern of the IFNα2b-16L-IgG2Fc fusion protein and the IFNα2b-32L-IgG2Fc fusion protein of the present invention by SDS-PAGE electrophoresis; the sample of the R point of the lane is a reduced fusion protein (ie, dimer opening), lane NR The sample at the point is a non-reduced fusion protein;
图2为细胞摇瓶培养筛选出的高表达细胞株的活细胞生长密度、细胞活力和分泌的IFNα2b-16L-IgG2Fc融合蛋白的浓度趋势曲线；2 is a graph showing the growth cell density, cell viability, and concentration trend of secreted IFNα2b-16L-IgG2Fc fusion protein in a highly expressed cell line selected from cell shake flask culture;
图3为细胞摇瓶培养筛选出的高表达细胞株的活细胞生长密度、细胞活力和分泌的IFNα2b-32L-IgG2Fc融合蛋白的浓度趋势曲线；Figure 3 is a graph showing the growth cell density, cell viability, and concentration trend of secreted IFNα2b-32L-IgG2Fc fusion protein in a highly expressed cell line selected from cell shake flask culture;
图4为纯化后的本发明的IFNα2b-16L-IgG2Fc融合蛋白纯度的HPLC检测结果；Figure 4 is a result of HPLC detection of the purity of the purified IFNα2b-16L-IgG2Fc fusion protein of the present invention;
图5为本发明IFNα2b-16L-IgG2Fc融合蛋白和IFNα2b-32L-IgG2Fc融合蛋白在大鼠体内的药代动力学曲线。Figure 5 is a pharmacokinetic curve of the IFNα2b-16L-IgG2Fc fusion protein and IFNα2b-32L-IgG2Fc fusion protein of the present invention in rats.
具体实施方式detailed description
为使本发明的目的、技术方案、及优点更加清楚明白，以下参照附图并举实施例，对本发明进一步详细说明。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The present invention will be further described in detail below with reference to the accompanying drawings. It is apparent that the described embodiments are only a part of the embodiments of the invention, and not all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts are within the scope of the present invention.
实施例1制备人干扰素α2b-肽连接体-Fc融合蛋白Example 1 Preparation of Human Interferon α2b-peptide Linker-Fc Fusion Protein
1)表达载体的构建1) Construction of expression vector
分别人工合成含有表1中的肽连接体的融合蛋白密码子：根据CHO细胞偏爱密码子优化IFNα2b-Fc，并在5’和3’端添加限制性内切酶内切位点SpeI和EcoRI。 The fusion protein codons containing the peptide linkers in Table 1 were separately synthesized: IFNα2b-Fc was optimized according to CHO cell preference codon, and restriction endonuclease sites SpeI and EcoRI were added at the 5' and 3' ends.
表1Table 1
Figure PCTCN2015077504-appb-000001
Figure PCTCN2015077504-appb-000001
将用人工合成的方法获得的EMCV IRES片段和小鼠的二氢叶酸还原酶(DHFR)基因***到哺乳动物表达载体PCDNA3(Invitrogen)的EcoRI(5’)和XhoI(3’)的位点，得到PCDNA3-DHFR载体。将优化的IFNα2b-Fc的全长DNA片段转移到PCDNA3-DHFR载体上的SpeI(5’)和EcoRI(3’)位点，得到PCDNA3-DHFR-IFNα2b-Fc表达载体。The EMCV IRES fragment obtained by the artificial synthesis method and the mouse dihydrofolate reductase (DHFR) gene were inserted into the sites of EcoRI (5') and XhoI (3') of the mammalian expression vector PCDNA3 (Invitrogen). The PCDNA3-DHFR vector was obtained. The full-length DNA fragment of the optimized IFNα2b-Fc was transferred to the SpeI (5') and EcoRI (3') sites on the PCDNA3-DHFR vector to obtain a PCDNA3-DHFR-IFNα2b-Fc expression vector.
SEQ ID No.10为IFNα2b-16L-IgG2Fc融合蛋白的氨基酸序列(其中，1-16个氨基酸为信号肽，分泌之后被切掉)，SEQ ID No.11为编码IFNα2b-16L-IgG2Fc融合蛋白的核酸序列(其中，序列中1-6bp为SpeI酶切位点；7-12bp为Kozak序列；13-60bp为信号肽序列；61-558bp为IFN-α2b序列；559-606bp为肽连接体序列；607-1284bp为IgG2Fc序列)；SEQ ID No.12为IFNα2b-32L-IgG2Fc融合蛋白的氨基酸序列(其中，1-16个氨基酸为信号肽，分泌之后被切掉)，SEQ ID No.13为编码IFNα2b-32L-IgG2Fc融合蛋白的核酸序列(其中，序列中1-6bp为SpeI酶切位点；7-12bp为Kozak 序列；13-60bp为信号肽序列；61-558bp为IFN-α2b序列；559-654bp为肽连接体序列；655-1332bp为IgG2Fc序列Pro331Ser)。SEQ ID No. 10 is the amino acid sequence of IFNα2b-16L-IgG2Fc fusion protein (wherein 1-16 amino acids are signal peptides, which are cleaved after secretion), and SEQ ID No. 11 is a fusion protein encoding IFNα2b-16L-IgG2Fc a nucleic acid sequence (wherein 1-6 bp in the sequence is a SpeI cleavage site; 7-12 bp is a Kozak sequence; 13-60 bp is a signal peptide sequence; 61-558 bp is an IFN-α2b sequence; and 559-606 bp is a peptide linker sequence; 607-1284 bp is the IgG2 Fc sequence); SEQ ID No. 12 is the amino acid sequence of the IFNα2b-32L-IgG2 Fc fusion protein (wherein 1-16 amino acids are signal peptides, which are cleaved after secretion), and SEQ ID No. 13 is an encoding The nucleic acid sequence of the IFNα2b-32L-IgG2Fc fusion protein (wherein 1-6 bp in the sequence is a SpeI cleavage site; 7-12 bp is Kozak) Sequence; 13-60 bp is the signal peptide sequence; 61-558 bp is the IFN-α2b sequence; 559-654 bp is the peptide linker sequence; 655-1332 bp is the IgG2 Fc sequence Pro331Ser).
2)稳定表达细胞株的转染和高表达细胞株的筛选2) Screening of transfected and highly expressed cell lines stably expressing cell lines
采用电穿孔的方法转染宿主细胞(CHO DG44)：使用Bio-Rad电转仪，电转时选用4mm的电转杯，设置电压为280V和电击时间为25毫秒。每次电击1×107个细胞，质粒40μg，总体积为0.7ml。电转后细胞转入含有30ml生长培养基的摇瓶中培养。培养24小时后，将培养基换成含50nM MTX的筛选培养中，并以1000个细胞/孔接种于96孔板中。细胞培养2周左右，直到克隆汇合率达到80％或以上，用抗人IFN-α2b抗体和抗人IgG抗体，采用夹心ELISA方法分析表达量，筛选出表达量相对高的克隆，依次转入24孔板、6孔板、T25细胞培养瓶和细胞培养摇瓶中扩大培养。The host cells were transfected by electroporation (CHO DG44): a Bio-Rad electrorotator was used, and a 4 mm electric rotor was used for the electrorotation, the voltage was set to 280 V and the shock time was 25 msec. Each shock was 1 × 10 7 cells, 40 μg of plasmid, and the total volume was 0.7 ml. After electroporation, the cells were transferred to shake flasks containing 30 ml of growth medium. After 24 hours of culture, the medium was changed to a screening culture containing 50 nM MTX, and seeded in a 96-well plate at 1000 cells/well. The cells were cultured for about 2 weeks until the clonal confluence rate reached 80% or higher. The expression of the anti-human IFN-α2b antibody and the anti-human IgG antibody was analyzed by sandwich ELISA, and the clones with relatively high expression were screened and transferred to 24 Expanded culture in well plates, 6-well plates, T25 cell culture flasks, and cell culture shake flasks.
为了提高融合蛋白产量，采用递增MTX浓度的加压方法培养细胞。这样通过MTX对DHFR基因的抑制，实现DHFR基因和融合蛋白基因的共扩增。当细胞适应2μM MTX时，采用有限稀释法亚克隆筛选出高水平表达融合蛋白的克隆。通过对不同克隆的细胞增殖和目的蛋白表达情况筛选出稳定高表达的单细胞克隆。To increase fusion protein production, cells were cultured using a pressurized method of increasing MTX concentration. Thus, the DHFR gene and the fusion protein gene are co-amplified by inhibition of the DHFR gene by MTX. When the cells were adapted to 2 μM MTX, clones with high levels of expression of the fusion protein were screened by limiting dilution subcloning. Single cell clones stably and highly expressed were screened by cell proliferation and expression of the target protein of different clones.
3)生产融合蛋白3) Production of fusion protein
将培养基成分优化，将筛选出的高产细胞株在100ml体积的摇瓶中以5×105个/ml接种，培养3到4天后，补料流加培养14天，发酵后得到目的蛋白。The culture medium components were optimized, and the selected high-yield cell strains were inoculated at 5×10 5 /ml in a 100 ml volume shake flask, and after 3 to 4 days of culture, the fed stream was cultured for 14 days, and the target protein was obtained after fermentation.
图1显示了融合蛋白IFNα2b-16L-IgG2Fc与融合蛋白IFNα2b-32L-IgG2Fc的SDS-PAGE电泳的鉴定结果，其中R泳道为还原的融合蛋白(即单体)，NR泳道为非还原的融合蛋白(即二聚体)。Figure 1 shows the results of SDS-PAGE electrophoresis of the fusion protein IFNα2b-16L-IgG2Fc and the fusion protein IFNα2b-32L-IgG2Fc, wherein the R lane is a reduced fusion protein (ie, monomer) and the NR lane is a non-reduced fusion protein. (ie dimer).
从图2可以看出，表达IFNα2b-16L-IgG2Fc融合蛋白的高产细胞株的活细胞密度最高可达到1.6×107个/ml，细胞活力大于90％以上，其表达的目的蛋白的产量大于900mg/L。It can be seen from Fig. 2 that the high-yielding cell line expressing the IFNα2b-16L-IgG2Fc fusion protein has a viable cell density of up to 1.6×10 7 cells/ml, a cell viability of more than 90%, and a protein yield of greater than 900 mg. /L.
从图3可以看出，表达IFNα2b-32L-IgG2Fc融合蛋白的高产细胞株的活细胞密度最高可达约1.5×107个/ml，细胞活力大于90％以上，其表达的 目的蛋白的产量大于1500mg/L。As can be seen from Fig. 3, the high-yielding cell line expressing the IFNα2b-32L-IgG2Fc fusion protein has a viable cell density of up to about 1.5×10 7 cells/ml, a cell viability of more than 90%, and a production of the target protein of which is greater than 1500mg/L.
4)收集上清液并纯化融合蛋白4) Collect the supernatant and purify the fusion protein
离心收集含有目的蛋白的细胞培养基，用0.22μm的硝酸纤维素过滤器过滤。将滤液加样到磷酸盐缓冲液(PBS)平衡的MabSelectTMprotein A柱(GE Healthcare)上。待融合蛋白结合于ProteinA柱后，弃去流出的组分，用PBS洗涤该柱，直到280nm处的OD值低于0.01。然后用20mM Tris，20mM CaCl2，300mM NaCl，900mM精氨酸，45％丙二醇(v/v)，0.05％吐温80(v/v)，PH6.8的缓冲液洗脱结合的融合蛋白。合并含有纯化蛋白的组分，并用PBS透析。然后用0.22μm的硝酸纤维素过滤器过滤，并立即储存在-70℃。在还原条件下，纯化的蛋白迁移至约40-50kDa，得到融合蛋白。The cell culture medium containing the protein of interest was collected by centrifugation and filtered through a 0.22 μm nitrocellulose filter. The filtrate was loaded onto a column MabSelect TM protein A (GE Healthcare) phosphate buffered saline (PBS) equilibrated. After the fusion protein was bound to the Protein A column, the effluent fraction was discarded and the column was washed with PBS until the OD value at 280 nm was less than 0.01. The bound fusion protein was then eluted with 20 mM Tris, 20 mM CaCl 2 , 300 mM NaCl, 900 mM arginine, 45% propylene glycol (v/v), 0.05% Tween 80 (v/v), pH 6.8 buffer. The fractions containing the purified protein were pooled and dialyzed against PBS. It was then filtered through a 0.22 μm nitrocellulose filter and immediately stored at -70 °C. Under reducing conditions, the purified protein migrates to about 40-50 kDa to give a fusion protein.
纯化后的IFNα2b-16L-IgG2Fc融合蛋白纯度的HPLC检测结果见图4。HPLC检测条件为：色谱柱：费罗门S2000；流速：0.5mL/分钟；流动相：0.1M磷酸盐缓冲液+0.1M NaCl(pH7.0)溶液；检测波长：280nm。The HPLC results of the purified IFNα2b-16L-IgG2Fc fusion protein were shown in Figure 4. The HPLC detection conditions were as follows: column: Ferromen S2000; flow rate: 0.5 mL/min; mobile phase: 0.1 M phosphate buffer + 0.1 M NaCl (pH 7.0) solution; detection wavelength: 280 nm.
实施例2融合蛋白体外抗病毒活性的测定Example 2 Determination of in vitro antiviral activity of fusion protein
将人羊膜细胞株(WISH细胞)用完全培养液配成2.5×105-3.5×105个细胞/ml的细胞悬液，接种于96孔细胞培养板中，每孔100μl，放入CO2培养箱中培养4-6小时。将待检样品用测定培养液稀释1000倍。于96孔板中每孔加入150μl测定培养液，然后在A排中分别加入稀释好的样品和阳性对照品rHuIFN(购自北京远策药业)50μl，各做3个复孔，然后依次进行4倍梯度稀释，共8个稀释度。取接种的细胞培养板。将稀释好的样品和阳性对照品溶液依次移入该细胞培养板，每孔100μl，放入CO2培养箱中培养18-24小时。取出细胞培养板，弃去培养液，加入稀释好的病毒液，每孔100μl。放入CO2培养箱中培养约24小时。镜检阳性对照品的50％病变点在D或E排，弃去上清，每孔加入50μl染色液，室温放置30分钟。小心冲去染色液，吸干残留水分。每孔加入100μl脱色液，室温放置3-5分钟。在酶标仪上比色，测定波长570nm，参比波长630nm，然后采用计算机程序(四参数回归计算法)进行结果处理。几种融合蛋白的抗病毒活性结果见表2。 A human amniotic cell line (WISH cells) was prepared into a cell suspension of 2.5×10 5 -3.5×10 5 cells/ml in complete culture medium, and inoculated into a 96-well cell culture plate, 100 μl per well, and placed in CO 2 . Incubate for 4-6 hours in an incubator. The sample to be tested was diluted 1000 times with the assay medium. 150 μl of the assay medium was added to each well of a 96-well plate, and then 50 μl of the diluted sample and the positive control rHuIFN (purchased from Beijing Yuance Pharmaceutical Co., Ltd.) were added to the A row, and each of the three replicate wells was sequentially performed. 4x gradient dilution for a total of 8 dilutions. Inoculate the inoculated cell culture plate. The diluted sample and the positive control solution were sequentially transferred into the cell culture plate, 100 μl per well, and cultured in a CO 2 incubator for 18-24 hours. The cell culture plate was taken out, the culture solution was discarded, and the diluted virus solution was added to 100 μl per well. It was placed in a CO 2 incubator for about 24 hours. The 50% lesions of the microscopic positive control were in the D or E row, the supernatant was discarded, 50 μl of staining solution was added to each well, and left at room temperature for 30 minutes. Carefully wash away the staining solution and blot the residual moisture. 100 μl of decolorizing solution was added to each well and allowed to stand at room temperature for 3-5 minutes. Colorimetric on a microplate reader, the measurement wavelength was 570 nm, the reference wavelength was 630 nm, and then the result was processed by a computer program (four-parameter regression calculation method). The antiviral activity results of several fusion proteins are shown in Table 2.
实施例3融合蛋白大鼠体内药代动力学实验Example 3 Pharmacokinetics experiment of fusion protein in rats
通过皮下注射，将商品化的rHuIFN(购自北京远策药业)和本发明纯化后的IFNα2b-Fc分别注入SD大鼠(注射剂量5μg/kg体重)，以注射相同体积PBS的SD大鼠作为对照(每组3只)。注射后，在不同时间点(0、1、2、4、8、12、24、36、48、60、72、96、120小时)通过剪尾取血法收集血样，肝素钠抗凝，将采集的血样3000rpm离心10min，收集血浆，-70℃冷冻保藏。将血样用ELISA试剂盒参照说明书检测血清中具有本发明IFNα2b活性的融合蛋白的含量，并根据结果绘制出反映蛋白含量变化的曲线图，如图4所示，rHuIFN在4小时后达到血药浓度峰值1nmol/ml，消除半衰期约为8.07小时；IFNα2b-16L-IgG2Fc融合蛋白在24小时达到血药浓度峰值1.15nmol/ml，消除半衰期为91.15小时；IFNα2b-32L-IgG2Fc融合蛋白在24小时达到血药浓度峰值1.1nmol/ml，消除半衰期为93.25小时。Commercially produced rHuIFN (purchased from Beijing Yuance Pharmaceutical Co., Ltd.) and purified IFNα2b-Fc of the present invention were injected into SD rats (injection dose of 5 μg/kg body weight) by subcutaneous injection to inject SD rats of the same volume of PBS. As a control (3 per group). After injection, blood samples were collected by tail-cutting blood at different time points (0, 1, 2, 4, 8, 12, 24, 36, 48, 60, 72, 96, 120 hours), and heparin sodium anticoagulation will The collected blood samples were centrifuged at 3000 rpm for 10 min, plasma was collected, and stored at -70 ° C for cryopreservation. The blood sample was tested for the content of the fusion protein having the activity of the IFNα2b of the present invention in the serum by using an ELISA kit according to the specification, and a graph reflecting the change in the protein content was plotted based on the result. As shown in Fig. 4, rHuIFN reached the blood concentration after 4 hours. The peak value was 1 nmol/ml, and the elimination half-life was about 8.07 hours. The IFNα2b-16L-IgG2Fc fusion protein reached a peak plasma concentration of 1.15 nmol/ml at 24 hours, and the elimination half-life was 91.15 hours. The IFNα2b-32L-IgG2Fc fusion protein reached blood at 24 hours. The drug concentration peaked at 1.1 nmol/ml and the elimination half-life was 93.25 hours.
表2显示了部分本发明的融合蛋白与文献中报道的长效干扰素的体外抗病毒活性和消除半衰期的比较结果。Table 2 shows the results of comparison of the in vitro antiviral activity and elimination half-life of a part of the fusion protein of the present invention with the long-acting interferon reported in the literature.
表2Table 2
Figure PCTCN2015077504-appb-000002
Figure PCTCN2015077504-appb-000002
可见，由于融合蛋白空间位阻效应，本发明的长效重组人干扰素α2b-Fc 融合蛋白，虽然体外活性有所降低，但是抗病毒活性与PEG-Intron相当并高于Pegasys；且本发明的长效重组人干扰素α2b-Fc融合蛋白体内半衰期与这两种市售的长效干扰素PEG-Intron与Pegasys相比都有很大的提高。而半衰期的延长意味着同样剂量给药时可以延长给药时间，降低给药次数。It can be seen that the long-acting recombinant human interferon α2b-Fc of the present invention is due to the steric hindrance effect of the fusion protein. The fusion protein, although reduced in vitro, has antiviral activity comparable to PEG-Intron and higher than Pegasys; and the in vivo half-life of the long-acting recombinant human interferon α2b-Fc fusion protein of the present invention is long-lived with these two commercially available products. Interferon PEG-Intron has been greatly improved compared to Pegasys. The prolongation of the half-life means that the administration time can be extended and the number of administrations can be reduced when the same dose is administered.
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明保护的范围之内。 The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalents, improvements, etc., which are made within the spirit and principles of the present invention, should be included in the present invention. Within the scope of protection.

Claims (11)

  1. 一种长效重组人干扰素α2b-Fc融合蛋白，其结构为人干扰素α2b-肽连接体-Fc，其中，所述肽连接体具有以下结构：X-(GGGS)p-(GGGGS)n-(GGGS)q，其中X为GS或GGS，p＝0-2，q＝0-2，n＝1-8。A long-acting recombinant human interferon α2b-Fc fusion protein having the structure of human interferon α2b-peptide linker-Fc, wherein the peptide linker has the following structure: X-(GGGS)p-(GGGGS)n- (GGGS)q, where X is GS or GGS, p=0-2, q=0-2, n=1-8.
  2. 根据权利要求1所述的长效重组人干扰素α2b-Fc融合蛋白，其中，Fc为人IgG2 Fc或人IgG2 Fc的突变体。The long-acting recombinant human interferon α2b-Fc fusion protein according to claim 1, wherein the Fc is a mutant of human IgG2 Fc or human IgG2 Fc.
  3. 根据权利要求2所述的长效重组人干扰素α2b-Fc融合蛋白，其中，人IgG2 Fc的突变体包含Pro331Ser突变。The long-acting recombinant human interferon α2b-Fc fusion protein according to claim 2, wherein the mutant of human IgG2 Fc comprises a Pro331Ser mutation.
  4. 根据前述权利要求任一项所述的长效重组人干扰素α2b-Fc融合蛋白，其中，所述肽连接体具有以下结构：X-(GGGS)p-(GGGGS)n-(GGGS)q，其中X为GS或GGS，p＝0-1，q＝0-1，n＝1-6。The long-acting recombinant human interferon α2b-Fc fusion protein according to any of the preceding claims, wherein the peptide linker has the following structure: X-(GGGS)p-(GGGGS)n-(GGGS)q, Wherein X is GS or GGS, p=0-1, q=0-1, n=1-6.
  5. 根据权利要求4所述的长效重组人干扰素α2b-Fc融合蛋白，其中，所述肽连接体具有以下结构：X-(GGGS)p-(GGGGS)n-(GGGS)q，其中X为GGS，p＝1，q＝1，n＝1。The long-acting recombinant human interferon α2b-Fc fusion protein according to claim 4, wherein the peptide linker has the following structure: X-(GGGS)p-(GGGGS)n-(GGGS)q, wherein X is GGS, p=1, q=1, n=1.
  6. 根据权利要求4所述的长效重组人干扰素α2b-Fc融合蛋白，其中，所述肽连接体具有以下结构：X-(GGGS)p-(GGGGS)n-(GGGS)q，其中X为GS，p＝0，q＝0，n＝6。The long-acting recombinant human interferon α2b-Fc fusion protein according to claim 4, wherein the peptide linker has the following structure: X-(GGGS)p-(GGGGS)n-(GGGS)q, wherein X is GS, p=0, q=0, n=6.
  7. 根据前述权利要求任一项所述的长效重组人干扰素α2b-Fc融合蛋白，可以以同源二聚体的形式存在。The long-acting recombinant human interferon α2b-Fc fusion protein according to any of the preceding claims, which may exist in the form of a homodimer.
  8. 一种编码前述权利要求任一项所述的长效重组人干扰素α2b-Fc融合蛋白的核酸序列。A nucleic acid sequence encoding the long acting recombinant human interferon alpha 2b-Fc fusion protein of any of the preceding claims.
  9. 一种表达权利要求1-7任一项所述的长效重组人干扰素α2b-Fc融合蛋白的细胞表达***，其中，所述细胞表达***为酵母表达***、昆虫细胞表达***或哺乳动物细胞表达***。A cell expression system for expressing the long-acting recombinant human interferon α2b-Fc fusion protein according to any one of claims 1 to 7, wherein the cell expression system is a yeast expression system, an insect cell expression system or a mammalian cell Expression system.
  10. 根据权利要求9所述的表达长效重组人干扰素α2b-Fc融合蛋白的细胞表达***，其中，所述细胞表达***为二氢叶酸还原酶(DHFR)双倍体缺 陷的CHO细胞表达***。The cell expression system for expressing a long-acting recombinant human interferon α2b-Fc fusion protein according to claim 9, wherein the cell expression system is dihydrofolate reductase (DHFR) diploid deficiency A trapped CHO cell expression system.
  11. 权利要求1-7任一项所述的长效重组人干扰素α2b-Fc融合蛋白在制备抗病毒或抗肿瘤药物中的用途。 Use of the long acting recombinant human interferon alpha 2b-Fc fusion protein of any of claims 1-7 for the preparation of an antiviral or antitumor drug.
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