WO2020103777A1 - 白介素21蛋白(il21)突变体及其应用 - Google Patents

白介素21蛋白(il21)突变体及其应用

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WO2020103777A1
WO2020103777A1 PCT/CN2019/119088 CN2019119088W WO2020103777A1 WO 2020103777 A1 WO2020103777 A1 WO 2020103777A1 CN 2019119088 W CN2019119088 W CN 2019119088W WO 2020103777 A1 WO2020103777 A1 WO 2020103777A1
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mutant
herceptin
protein
interleukin
fusion protein
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PCT/CN2019/119088
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French (fr)
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张信
赵云
杭海英
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海珂分子（北京）科技有限责任公司
中国科学院生物物理研究所
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Priority to US17/296,036 priority Critical patent/US20220025006A1/en
Priority to EP19886303.7A priority patent/EP3885357A4/en
Priority to JP2021529120A priority patent/JP7207786B2/ja
Publication of WO2020103777A1 publication Critical patent/WO2020103777A1/zh

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    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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Definitions

  • the invention belongs to the technical field of medicine, and specifically relates to an interleukin 21 protein (IL21) mutant and its application.
  • IL21 interleukin 21 protein
  • cytokines and small molecule inhibitors can play a role in immune regulation. Cytokines and small molecule inhibitors can target inhibitory cell subpopulations, inhibit their number or function, or Anti-tumor effector cells are activated by immunostimulatory molecules to enhance the patient's anti-tumor immune response.
  • IL2 interleukin 2
  • IL2 directly acts on thymus T cells and promotes the differentiation of Treg cells.
  • Inhibiting autoimmunity can also promote the differentiation of initial T cells into effector T cells and memory T cells that can be activated by antigen stimulation, which can help the body resist infection.
  • systemic administration of IL2 can cause strong adverse reactions, which limits the therapeutic dose of IL2 [1-2] , and the plasma half-life is short (about 0.5-2 hours) [3] , which cannot be achieved in the local tumor microenvironment
  • the optimal dose of treatment also limits its clinical application.
  • IL2 In the cytokine family, IL2, IL15 and IL21 all belong to the co-receptor ⁇ chain family, and they all promote the activity of T cells and NK cells and enhance their killing target cells. These characteristics make them useful in the research of tumor immunotherapy. Attractive cytokines [4-5] .
  • IL21 (interleukin 21) is a type I cytokine discovered in 2000. It belongs to the ⁇ c family and consists of 134 amino acids. It is mainly produced by CD4 + T cell subsets, such as TH17 cells and TFH (Tfollicular helper) cells, and NKT cells. It can also secrete higher levels of IL21 [19-20]. It has been reported that the IL21 receptor IL21R is expressed on the surface of T cells, B cells, NK cells, dendritic cells and monocytes / macrophages, and is a cytokine with a wide range of immunomodulatory effects.
  • IL21 is anti-tumor through a variety of mechanisms, including the innate immune system and the acquired immune system [6] .
  • IL21 has been approved to enter human clinical trials for the treatment of melanoma and kidney cancer with good therapeutic effects [7-8] ;
  • IL21 can enhance the response of NK cells to pancreatic cancer cells pre-coated with cetuximab [9] ;
  • the fusion protein constructed by IL21 and monoclonal antibody Rituximab (anti-CD20) has been shown in animal experiments to directly induce apoptosis or kill non-Hodgkin's lymphoma (NHL) through effector effector cells [10] .
  • NHL non-Hodgkin's lymphoma
  • IL21 can greatly improve antibody breast cancer by transforming M2 macrophages (with the effect of increasing tumor resistance and malignancy) into M1 macrophages (with anti-tumor function) Antitumor effect of antibody Herceptin [11] .
  • IL21 has a short plasma half-life of only about 3.09 hours [12] . Even if the molecular weight of the fusion protein constructed by IL21 and monoclonal antibody has reached about 180 kD, the plasma half-life is still short, IL21 and Rituximab (anti-CD20 ) The plasma half-life of the constructed fusion protein in animal experiments is only about 18.94 hours [10] , and the drug-forming property is still not high. Therefore, improving the stability of IL21 is an important condition for prolonging the half-life and improving the drugability.
  • the present invention first relates to an interleukin 21 (IL21) mutant.
  • the mutant is: the 16th ILE and the 70th SER of the amino acid sequence of wild-type IL21 are both mutated to CYS, and the two mutations A disulfide bond is formed between CYS, and the amino acid sequence of the wild-type IL21 is shown in SEQ ID NO.1.
  • the present invention also relates to an interleukin 21 (IL21) mutant, wherein the mutant is: the 16th ILE, the 17th VAL, the 70th SER, and the 112th LYS of the amino acid sequence of the wild-type IL21 It is mutated to CYS and forms two groups of 16-70 and 17-112 disulfide bonds.
  • the amino acid sequence of the wild-type IL21 is shown in SEQ ID NO.1.
  • the invention also relates to a mutant of interleukin 21 and interleukin 4 chimera protein (IL21 / 4).
  • the mutant is: the 16th position of the amino acid sequence of interleukin 21 and interleukin 4 chimera (IL21 / 4) ILE, 70th SER, are mutated to CYS, and form a disulfide bond between the two mutated CYS, the interleukin 21 and interleukin 4 chimeric protein (IL21 / 4) amino acid sequence such as SEQ ID ID NO. 2 shows.
  • the invention also relates to a mutant of interleukin 21 and interleukin 4 chimera protein (IL21 / 4).
  • the mutant is: the 16th position of the amino acid sequence of interleukin 21 and interleukin 4 chimera (IL21 / 4) ILE, 17th VAL, 70th SER, 106th LYS all mutated to CYS, and formed two groups of 16-70, 17-106 disulfide bonds, the interleukin 21 and interleukin 4 chimeric protein (IL21 / 4)
  • the amino acid sequence is shown in SEQ ID NO.2.
  • the invention also relates to the nucleotide sequence encoding the IL21 mutant and IL21 / 4 mutant.
  • the present invention also relates to a fusion protein comprising the IL21 mutant or IL21 / 4 mutant, the fusion protein comprising:
  • the invention also relates to the application of the IL21 mutant or IL21 / 4 mutant in the preparation of a medicament, the medicament is a drug that regulates or activates immunity or an antitumor drug.
  • the invention also relates to the application of the fusion protein containing the IL21 mutant or IL21 / 4 mutant in the preparation of a medicament, the medicament is a drug that regulates or activates immunity or an antitumor drug.
  • the invention also relates to the application of the IL21 mutant or IL21 / 4 mutant in the preparation of preparations that promote B cell differentiation and proliferation, T cell differentiation and proliferation, and NK cell differentiation and proliferation.
  • the invention also relates to the application of the fusion protein containing the IL21 mutant or IL21 / 4 mutant in the preparation of a preparation that promotes B cell differentiation and proliferation, T cell differentiation and proliferation, and NK cell differentiation and proliferation.
  • the present invention also relates to a drug or pharmaceutical composition using the IL21 mutant or IL21 / 4 mutant as an active ingredient, the drug or pharmaceutical composition including a therapeutically effective amount of the IL21 mutant or IL21 / 4 mutant , And the necessary medicinal accessories.
  • the present invention also relates to a drug or pharmaceutical composition using the fusion protein containing the IL21 mutant or IL21 / 4 mutant as an active ingredient, the drug or pharmaceutical composition including a therapeutically effective amount of the fusion protein, and necessary Medicinal accessories.
  • FIG. 1 Recombinant replacement plasmid fragment showing fusion protein of IL21 / 4 and Herceptin antibody
  • FIG. 1 Schematic diagram of replacement of FRT-IL21 / 4-Herceptin plasmid into CHO working cell genome by homologous recombination
  • the left picture is the CHO working cell negative control group
  • the right picture is the experimental group after transfection.
  • the ordinate is the HerceptinHC display rate after combining with the Herceptin heavy chain constant region.
  • the p3 region shows the IL21 / 4-Herceptin display rate after successful replacement, about 0.269%.
  • the left picture shows the CHO working cell negative control group
  • the right picture shows the experimental group after transfection.
  • the abscissa indicates the display rate after binding to the extracellular domain of IL21 receptor IL21R ⁇ ; the ordinate indicates the display rate after binding to the extracellular domain of IL21 receptor ⁇ c, visible
  • the display rate was 17.2% after binding to the extracellular domain of IL21R ⁇ , and ⁇ 0.3% after combining with the extracellular domain of ⁇ c.
  • the left picture shows the CHO working cell negative control group
  • the right picture shows the experimental group after transfection.
  • the abscissa indicates the display rate after binding to the extracellular domain of IL21 receptor ⁇ c
  • the ordinate indicates the HerceptinHC display rate after combining with the Herceptin heavy chain constant region. The results showed that Herceptin display rate after fusion protein binding was 2.54%.
  • CHO cells displaying 16c IL21 / 4-Herceptin, 17c IL21 / 4-Herceptin and 4c IL21 / 4-Herceptin protein are enriched to detect the display rate results.
  • the abscissa indicates the display rate after binding to the extracellular domain of IL21 receptor IL21R ⁇ ; the ordinate The display rate after the combination of the label with the Herceptin heavy chain constant region and the labeled antibody Mouse Anti-human IgG-APC antibody can be seen in the 16c IL21 / 4-Herceptin, 4c IL21 / 4-Herceptin group combined with the Herceptin heavy chain constant region Compared with before enrichment, the display rate of the 17c IL21 / 4-Herceptin group combined with the Herceptin heavy chain constant region is only 1% after enrichment; it can be seen that the 16c IL21 / 4-Herceptin, 4c IL21 / 4-Herceptin group and the recipient The binding rate of the IL21R ⁇ extracellular domain in the body was significantly higher than
  • FIG. 7 Construction of IL21; 16c IL21; 16c IL21 / 4 mutant protein expression plasmids (structure diagram), respectively, each mutant protein coding gene is inserted into PCEP4 vector to construct an expression plasmid, and added His tag.
  • IL21, 16c, IL21 and 16c IL21 / 4 proteins stimulate the proliferation of KOB cells.
  • FIG. 13 Fusion protein 16cIL21 / 4-Herceptin anti-tumor test results; in the figure, Control is the control group injected with PBS; Herceptin is the experimental group injected with Herceptin alone; IL-21 mutant -Herceptin represents 16cIL21 / 4 and Herceptin The experimental group of the fusion protein; IL-21 mutant + Herceptin represents the experimental group of the mixed solution of 16cIL21 / 4 and Herceptin, not the fusion protein.
  • PCR and DNA digestion products are recovered using AxyPrep DNA Extraction Kit (AXYGEN) kit; for small-scale extraction and purification of plasmids, full-scale gold (transgen, China) plasmid small extraction kits are used; for medium-scale extraction and purification of plasmids, Qiagen extraction is used Kits; Tiangen endotoxin-free plasmid large extraction kit is used for mass extraction, and the specific operation is carried out according to the instructions.
  • AxyPrep DNA Extraction Kit AXYGEN
  • AXYGEN AxyPrep DNA Extraction Kit
  • Qiagen extraction is used Kits
  • Tiangen endotoxin-free plasmid large extraction kit is used for mass extraction, and the specific operation is carried out according to the instructions.
  • CHO / dhFr- dihydrofolate reductase deficient Chinese hamster ovary cells
  • 293F cells human embryonic kidney cells, cultured in FreeStyle TM 293 Expression Medium, 37 ° C, 5% CO 2 incubator, shake flask at 125 rpm.
  • KOB cells adult T lymphoma cells with high expression of IL21 receptor, kindly donated by the research team of Wang Shengdian, Institute of Biophysics, Chinese Academy of Sciences, using 10% calf serum (Hyclone), 100U / mL double antibody, RPMI1640 medium at 37 °C , Cultured in a 5% CO 2 incubator.
  • Cell preparation one day before the transfection, the cells were planted in a six-well plate with a cell volume of 200,000 / well to achieve 80% fullness, uniform distribution, and good growth during transfection;
  • transfection complex Preparation of transfection complex: mix 5 ⁇ l of Lipo2000 with Opti-MEM serum-free medium 150 ⁇ l; 2 ⁇ g of target plasmid and Opti-MEM serum-free medium 150 ⁇ l; mix at room temperature for 5 minutes and mix to make transfection complex Mix gently and let stand at room temperature for 25 minutes;
  • Cell preparation Inoculate cells at 6-7x10 5 cells / ml the day before transfection, and the cell density should be 1x10 6 cells / ml at the time of transfection;
  • transfection complex take 50 ⁇ g of the target plasmid and dilute it with OptiPROTMSFM (Invitrogen) serum-free medium 2ml, mix well to make a DNA dilution, take 250 ⁇ L of transfection reagent PEI into the DNA dilution to prepare a transfection complex , Mix well and leave at room temperature for 15 minutes;
  • OptiPROTMSFM Invitrogen
  • the transfection complex was added to 293F cell culture medium, 37 ° C., 5% CO 2 incubator, and shake flask culture was continued at 125 rpm for 96 hours.
  • Washing and equilibrating nickel column first clean the nickel column with 50ml of ddH 2 O, then wash the balance nickel column with 50ml of buffer solution containing 5mM imidazole;
  • Sample loading Add the supernatant containing the protein of interest to the nickel column, which can be sampled 2-3 times;
  • Elution After loading the sample, elute with 30 ml of buffer solution containing 5, 30, 60, 90, 120, and 250 mM imidazole, collect the eluent, and select different ultrafiltration tubes according to the molecular weight of the target protein. Concentrate with ultrafiltration tube, the volume concentration is 50-100 times;
  • Protein preservation The final protein was divided into aliquots, quick frozen in liquid nitrogen, and stored at -80 °C.
  • Binding buffer 0.1M Na 3 PO 4 , 0.15M NaCl, PH 7.2;
  • elution buffer 0.1M citric acid, PH 2.7;
  • Sample loading Add the supernatant containing the protein of interest to the chromatography column and load the sample 2-3 times;
  • Segmented recovery collect the eluent, use nanodrop to determine the protein concentration of each tube, discard it below 0.05 mg / ml, select different ultrafiltration tubes according to the molecular weight of the target protein, and concentrate the eluent with the ultrafiltration tube;
  • Protein preservation The final protein is divided into aliquots, quick frozen in liquid nitrogen, and stored at -80 °C.
  • Tm protein melting temperature
  • Example 1 Establishment of a CHO cell line capable of stably displaying the chimera IL21 / 4
  • a chimera IL21 / 4 was constructed by replacing the unstable region in the protein structure of IL21 with the homologous region of IL4. The results showed that the protein conformation of the chimera IL21 / 4 Single and stable, and biological activity has improved.
  • a CHO cell line that efficiently and stably displays the chimeric IL21 / 4
  • a recombinant enzyme-mediated cassette replacement technology recombinase-mediated cassette exchange (RMCE) (specific (See CN 201410803422).
  • a recombinant replacement plasmid fragment showing the fusion protein of IL21 / 4 and Herceptin.
  • the fragments of the fusion protein are: Herceptin light chain with signal peptide, IRES (ribosomal binding site) , Herceptin heavy chain signal peptide, IL21 / 4 is connected to the N-terminus of Herceptin heavy chain by using 3 (G4S) linker, and the transmembrane region (TM) is connected to the C-terminus of Herceptin heavy chain, so that IL21 / 4 can be fused with Herceptin
  • the protein is displayed on the surface of CHO working cells through the anchoring of the transmembrane region.
  • the sequence structure of the fusion protein is shown in Figure 1 (the fusion protein from left to right is: FRT recombination site, Herceptin light chain signal peptide, Herceptin light chain, can IRES (ribosomal binding site) that initiates translation by binding to ribosomes, Herceptin heavy chain signal peptide, IL21 / 4, linker adopts 3 (G4S), Herceptin heavy chain, enables protein molecules to be anchored and displayed on the cell surface of the transmembrane Region (TM), LOXp recombination site.
  • IRES ribosomal binding site
  • Herceptin heavy chain signal peptide Herceptin heavy chain signal peptide
  • IL21 / 4 linker adopts 3
  • Herceptin heavy chain enables protein molecules to be anchored and displayed on the cell surface of the transmembrane Region (TM), LOXp recombination site.
  • the abscissa shows the display rate (17.2%) of IL21 / 4 in the IL21 / 4-Herceptin fusion protein after binding to the extracellular domain of the IL21 receptor IL21R ⁇ (17.2%); the ordinate shows IL21 / In the 4-Herceptin fusion protein, the display rate of IL21 / 4 after binding to the extracellular domain of IL21 receptor ⁇ c ( ⁇ 0.3%).
  • the affinity of IL21 for binding to receptor ⁇ c is much lower than that for binding to receptor IL21R ⁇ . This experiment also confirmed this many times.
  • the abscissa shows that the IL21 / 4 in the IL21 / 4-Herceptin fusion protein binds to the extracellular domain of the IL21 receptor ⁇ c ⁇ 0.3%, and the ordinate shows the Herceptin heavy chain constant region in the fusion protein and the labeled antibody Mouse
  • the display rate of Anti-human IgG-APC antibody after binding was 2.54%; it was improved compared with the pre-enrichment display rate of 0.26%, combined with the result of IL21R ⁇ extracellular domain-linker-GFP fusion protein labeling, it can be considered that the Herceptin heavy chain constant region in the fusion protein
  • the affinity of the labeled antibody Anti-human IgG-APC antibody is lower than that of IL21R ⁇ extracellular domain-linker-GFP.
  • the CHO cells stably displaying the IL21 / 4-Herceptin fusion protein were sorted by flow cytometry FACSAriaIII and named s0 cells.
  • the 16th ILE and the 70th SER are all mutated to CYS, introducing 16-70 disulfide bond; named 16cIL21 / 4 ;
  • the abscissa shows the display rate of different mutant IL21 / 4-Herceptin fusion proteins after binding to the extracellular domain of the IL21 receptor IL21R ⁇ ;
  • the ordinate shows the Herceptin heavy chain constant region of the fusion protein and the labeled antibody Mouse Anti-human IgG -The display rate of APC after antibody binding; it can be seen that the display rate of 16c IL21 / 4-Herceptin; 4c IL21 / 4-Herceptin are significantly higher than that before enrichment, and they bind well to the extracellular domain of receptor IL21R ⁇ ; while the control group 17c IL21 / 4-Herceptin ordinate shows that the display rate after binding to the labeled antibody Mouse Anti-human IgG-APC antibody is only 1%, and there is no obvious binding to the extracellular domain of receptor IL21R ⁇ , indicating that the control group 17c IL21 / 4-Herceptin The mutant is not displayed.
  • CHO cells stably displaying IL21 mutants on the cell membrane surface were digested with 0.02% EDTA-PBS, then eluted with serum-containing medium, centrifuged at 830 g, 3 min, the supernatant was discarded, and the cells were collected;
  • each group binds to the extracellular domain of IL21 receptor IL21R ⁇ , the mutant 16c IL21 / 4-Herceptin display rate is 20%, and the highest binding rate;
  • mutant 16c IL21 / 4-Herceptin group After heating at 49 °C, IL21 / 4-Herceptin group, 4c IL21 / 4-Herceptin group and IL21 receptor IL21R ⁇ extracellular domain are basically lost, mutant 16c IL21 / 4-Herceptin group can also detect IL21 The binding of the extracellular domain of the receptor IL21R ⁇ initially suggests that the mutant 16c IL21 / 4 has improved thermal stability.
  • IL21-Herceptin fusion protein of wild type IL21 and Herceptin;
  • 16c IL21-Herceptin at the same position, the 16th ILE-70th SER, all of which are mutated to CYS, a fusion protein constructed by introducing disulfide bond IL21 and Herceptin;
  • the three proteins IL21, 16c, IL21, 16c and IL21 / 4 were subjected to SDS-PAGE electrophoresis. The results are shown in Figure 8. According to the results, the position of the gel bands of the three proteins IL21, 16c, IL21, 16c and IL21 / 4 is consistent with the theory. From the molecular weight level, these three proteins are the target proteins.
  • the sample protein Under the condition of disulfide bond opening or not, the sample protein will be cut into peptides of different sizes, and there will be different peptide-to-nucleus ratio distributions, according to whether it is predicted by theory The match can determine whether the predicted disulfide bond exists in the sample protein.
  • the amino acid sequences IINVCIK and QLIDCVDQLK that theoretically produce two fragments of disulfide bonds. When digested in the reduced state, the disulfide bond is opened, and C will be alkylated (molecular weight increased by 57Da).
  • the theoretical values of the mass ratios of the two peptides are 859.507 and 1231.635, respectively. As can be seen from FIG. 9A, in the reduced state (plus DTT), 859.587 and 1231.769 have relatively obvious signal peaks, and in the non-reduced state, the signals of these two peaks are very low.
  • disulfide bond is opened when the 16cIL21 / 4 sample protein is added with DTT, and two peptides can be cut out; when the disulfide bond is not opened without DTT, there is no signal from these two peptides, which is the same as the theoretically predicted disulfide bond The structure matches.
  • KOB cells express the IL21 receptor and can be stimulated by IL21 to proliferate.
  • the biological activity of each mutant of IL21 can be determined by stimulating the proliferation ability of KOB cells, the specific method is as follows:
  • KOB cells use 10% calf serum (Hyclone), 100U / mL double antibody, RPMI1640 medium, culture at 37 ° C, 5% CO 2 incubator;
  • 16cIL21 and 16cIL21 / 4 proteins of the two mutants are proteins with improved thermal stability and retaining biological activity.
  • IL21-Herceptin In order to determine whether improving the stability of IL21 can extend the plasma half-life in animals, we selected IL21-Herceptin and the mutated 16c IL21-Herceptin fusion protein and 16c IL21 / 4-Herceptin fusion protein for comparison and detection.
  • the plasma half-life in mice is shown in Table 4 below:
  • IL21 fusion protein stimulates T cell proliferation function determination
  • 16cIL21 / 4-Herceptin fusion protein was tested using the BT474 breast cancer tumor-bearing mouse model. 1.5x10 7 human BT474 breast cancer cells were inoculated on the right ventral back of the mouse, and the drug was administered 35 days later. The proliferation of CD8 + T cells stimulated by Herceptin in mice was tested. The results are shown in Figure 12. It can be seen that the fusion protein of 16cIL21 / 4-Herceptin began to obviously appear to increase in the proportion of CD8 + T cells in the peripheral blood about 53 days after tumor inoculation. At the same time, the Herceptin group showed a trend of slow decline in T cells.
  • BT474 breast cancer tumor-bearing mouse model was used to detect the tumor suppressive activity of 16cIL21 / 4-Herceptin fusion protein.
  • Each mouse was inoculated with 1 ⁇ 10 7 BT474 cells (human breast cancer cells); intraperitoneal injection was given every four days PBS (Control), Herceptin (Herceptin injected alone), 16cIL21 / 4-Herceptin fusion protein (IL-21 mutant -Herceptin), 16cIL21 / 4 and Herceptin mixed solution (IL-21 mutant + Herceptin); each 500ug dose.
  • the results are shown in Figure 13. Compared with the control group, herceptin group and mixed solution group, the 16cIL21 / 4-Herceptin fusion protein group can significantly reduce the tumor volume.

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Abstract

本发明提供了一种白介素21蛋白(IL21)突变体及其应用，所述的突变体是将野生型IL21的第16位ILE、第70位SER都突变为CYS，并在两个突变的CYS之间形成二硫键。还提供了包含所述的IL21蛋白突变体的融合蛋白。所述IL21蛋白突变体或融合蛋白能用于调节或激活免疫、抗肿瘤以及促进B细胞、T细胞或NK细胞的分化增殖。

Description

白介素21蛋白(IL21)突变体及其应用 技术领域
本发明属于医药技术领域，具体而言，涉及一种白介素21蛋白(IL21)突变体及其应用。
背景技术
针对肿瘤免疫治疗研究中的免疫抑制问题，细胞因子和小分子抑制剂治疗，能够起到免疫调节的作用，细胞因子和小分子抑制剂能针对抑制性细胞亚群，抑制其数量或功能，或者通过免疫刺激分子活化抗肿瘤效应细胞，增强患者的抗肿瘤免疫应答。IL2(白介素2)是免疫反应中的重要介导分子，也是已知最强有力的抗肿瘤细胞因子之一，具有广泛的生物学活性，IL2直接作用于胸腺T细胞，促进Treg细胞的分化，抑制自身免疫，也能够促进初始T细胞分化成为可被抗原刺激激活的效应T细胞与记忆T细胞，可帮助机体抵抗感染。然而，IL2的全身用药可导致较强的不良反应，使得IL2的治疗剂量受到限制 [1-2]，而且血浆半衰期短(约0.5-2小时) [3]，在肿瘤局部微环境中不能达到治疗的最佳剂量，也限制了其临床应用。
在细胞因子家族中，IL2、IL15和IL21同属于共受体γ链家族，都有促进T细胞、NK细胞活性，增强其杀伤靶细胞的作用，这些特性使它们在研究肿瘤免疫治疗中成为有吸引力的细胞因子 [4-5]。
IL21(白介素21)是2000年发现的Ⅰ型细胞因子，属于γc家族，由134个氨基酸组成，主要由CD4+T细胞亚群，如TH17细胞以及TFH(T follicular helper)细胞分泌产生，NKT细胞也能分泌较高水平的IL21[19-20]。有报道IL21受体IL21R在T细胞、B细胞、NK细胞、树突状细胞和单核/巨噬细胞表面均有表达，是具有广泛免疫调节作用的细胞因子。
许多临床前实验指出，IL21通过多种机制抗肿瘤，包括先天免疫系统和获得性免疫系统 [6]。IL21已被批准进入人体临床实验治疗黑色素瘤、肾癌，获得良好得治疗效果 [7-8]；IL21可以增强NK细胞对西妥昔单抗预先包被的胰腺癌细胞的反应 [9]；IL21与单克隆抗体Rituximab(anti-CD20)所构建融合蛋白在动物实验中显示可以直接诱导凋亡或通过效应效应细胞来杀伤非何杰金氏淋巴瘤(NHL) [10]。
我们的前期研究发现：在乳腺癌小鼠模型中，IL21可以通过将M2巨噬细胞(具有增加肿瘤耐药和恶性的作用)转化为M1巨噬细胞(具有抗肿瘤功能)大幅提升抗体乳腺癌抗体赫赛汀的肿瘤杀伤效果 [11]。
但同时，在多项IL21临床应用研究中显示，虽然IL21毒副作用较相同剂量的IL2或IFN-α小，主要表现为发热、疲劳、头痛、皮疹等较轻症状，也有出现腹痛、血小板减少、低磷血症、肝功能损害等严重表现，但在30μg/kg通常给药量时，各种毒副作用的发生率仍达100％ [12]，这是全身用药时需要慎重对待的问题。事实上，开展IL21临床应用研究多年，目前仍停留在临床前Ⅰ期、Ⅱ期阶段，存在毒副作用发生率高的问题是重要原因。
为解决提高细胞因子在肿瘤局部的有效浓度从而避免全身性毒性反应的问题，有研究者利用抗体靶向性传递细胞因子，使细胞因子的免疫治疗能力与抗体的靶向性抗肿瘤反应结合起来，在肿瘤局部获得高浓度的细胞因子，有效刺激细胞免疫反应，这种将细胞因子与细胞特异性的抗体偶联，将其命名为免疫细胞因子 [13]。
但是，有临床研究发现IL21血浆半衰期较短，仅有约3.09小时 [12]，即使IL21与单克隆抗体构建的融合蛋白分子量已达约180kD，血浆半衰期仍较短，IL21与Rituximab(anti-CD20)所构建融合蛋白在动物实验中血浆半衰期也仅有约18.94小时 [10]，成药性仍然不高。因此，提高IL21稳定性是延长半衰期、提高成药性的重要条件。
结构分析显示，IL21的蛋白存在稳定与不稳定两种构象，这是IL21半衰期短的一个原因；有报道表明通过将IL21的蛋白结构中不稳定区域替换为IL4(白介素4)的同源区域构建了一种嵌合体IL21/4，结果显示嵌合体IL21/4的蛋白构象单一稳定，而且生物学活性有提高 [14]。
在我们的前期工作中，已经建立了实现蛋白质构像体外控制的哺乳动物细胞表面蛋白质稳定展示系统，(参见CN201410803422，CN201810795499)，能够对理性设计蛋白或随机突变蛋白进行筛选及鉴定。在本发明中，我们使用该系统，对白介素21(IL21)的进行了稳定展示，并通过基于蛋白质结构分析的设计，优化并获得了一组突变体IL21蛋白。
参考文献：
1、Roth JA,Cristiano RJ,Gene therapy for cancer:what have we done and where are we going？J.Natl.Cancer Inst.1997；89:21-39.
2、Zatloukal K,Schneeberger A,Berger M et al.Elicitition of a systemic and protective anti-melanoma immune response by an IL-2 based vaccine.Assessment of critical cellular and molecular parameters.J.Immunol.1995；154:3406-3419.
3、Lotze MT,et al.In vivo administration of purified human interleukin 2.I.Half-life and immunologic effects of the Jurkat cell line-derived interleukin 2.[J]Immunol.1985；134(1):157–66.
4、Overwijk,W.W.,Schluns,K.S.Functions of γC cytokines in immune homeostasis:Current and potential clinical applications[J].Clin Immunol.2009；132(2):153-165.
5、Malek TR,Castro I.Interleukin-2 Receptor Signaling:At the Interface between Tolerance and Immunity[J].Immunity.2010；33(2):153–165.
6、Rosanne S,Warren J.L.Interleukin-21:Basic Biology and Implications for Cancer and Autoimmunity[J].Annu.Rev.Immunol.2008.26:57–79.
7、Schmidt H,Brown J,Mouritzen U,et al.Safety and clinical effect of subcutaneous human Interleukin-21 in patients with metastatic melanoma or renal rell carcinoma:a phase I trial[J].Clin Cancer Res.2010；16(21):5312-9.
8、Davis ID,Skrumsager BK,Cebon J,et al.An open-label,two-arm,phase I trial of recombinant human interleukin-21 in patients with metastatic melanoma[J].Clin Cancer Res.2007；13(12):3630-6.
9、McMichael EL,Jaime-Ramirez AC,Guenterberg KD，et al.IL21 Enhances Natural Killer Cell Response to Cetuximab-Coated Pancreatic Tumor Cells[J].Clin Cancer Res.2017；23(2)：489-502.
10、Bhatt S,Parvin S,Zhang Y,et al.Anti-CD20-interleukin-21 fusokine targets malignant B cells viadirect apoptosis and NK-cell-dependentcytotoxicity[J].Blood.2017；129(16):2246-2256.
11、Xu M,Liu M,Du X,et al.Intratumoral Delivery of IL-21 Overcomes Anti-Her2/Neu Resistance through Shifting Tumor-Associated Macrophages from M2to M1Phenotype[J].J Immunol.2015；194(10):4997-5006.
12、Thompson JA,Curti BD,Redman BG,et al.Phase I Study of Recombinant Interleukin-21 in Patients With Metastatic Melanoma and Renal Cell Carcinoma[J].Clin Oncol,2008 Apr 20；26(12):2034-9.
13、重组抗体，沈倍奋，陈志南，刘民培，科学出版社，2005，P373.
14、Kent B,Jens B,Dennis M,et al.The Existence of Multiple Conformers of Interleukin-21Directs Engineering of a Superpotent Analogue[J].THE JOURNAL OF BIOLOGICAL CHEMISTRY,2007,282(32):23326–23336.
发明内容
本发明首先涉及一种白介素21(IL21)突变体，所述的突变体为：将野生型IL21的氨基酸序列的第16位ILE、第70位SER，都突变为CYS，并在两个突变的CYS之间形成二硫键，所述的野生型IL21的 氨基酸序列如SEQ ID NO.1所示。
本发明还涉及一种白介素21(IL21)突变体，所述的突变体为：将野生型IL21的氨基酸序列的第16位ILE、第17位VAL、第70位SER、第112位LYS的都突变为CYS，并且形成16-70、17-112两组二硫键，所述的野生型IL21的氨基酸序列如SEQ ID NO.1所示。
本发明还涉及一种白介素21和白介素4嵌合体蛋白(IL21/4)的突变体，所述的突变体为：将白介素21和白介素4嵌合体(IL21/4)的氨基酸序列的第16位ILE、第70位SER，都突变为CYS，并在两个突变的CYS之间形成二硫键，所述的白介素21和白介素4嵌合体蛋白(IL21/4)的氨基酸序列如SEQ ID NO.2所示。
本发明还涉及一种白介素21和白介素4嵌合体蛋白(IL21/4)的突变体，所述的突变体为：将白介素21和白介素4嵌合体(IL21/4)的氨基酸序列的第16位ILE、第17位VAL、第70位SER、第106位LYS的都突变为CYS，并且形成16-70、17-106两组二硫键，所述的白介素21和白介素4嵌合体蛋白(IL21/4)的氨基酸序列如SEQ ID NO.2所示。
本发明还涉及编码所述IL21突变体、IL21/4突变体的核苷酸序列。
本发明还涉及包含所述的IL21突变体或IL21/4突变体的融合蛋白，所述的融合蛋白包括：
(1)功能片段1：IL21突变体或IL21/4突变体；
(2)功能片段2：单克隆抗体功能；
及连接不同功能片段的连接结构域。
本发明还涉及所述的IL21突变体或IL21/4突变体在制备药物中的应用，所述的药物是调节或激活免疫的药物或抗肿瘤药物。
本发明还涉及所述包含IL21突变体或IL21/4突变体的融合蛋白在制备药物中的应用，所述的药物是调节或激活免疫的药物或抗肿瘤药物。
本发明还涉及所述的IL21突变体或IL21/4突变体在制备促进B细胞分化增殖，促进T细胞分化增殖，促进NK细胞分化增殖的制剂中的应用。
本发明还涉及所述包含IL21突变体或IL21/4突变体的融合蛋白在制备促进B细胞分化增殖，促进T细胞分化增殖，促进NK细胞分化增殖的制剂中的应用。
本发明还涉及以所述IL21突变体或IL21/4突变体为活性成分的药物或药物组合物，所述的药物或药物组合物包括治疗有效量的所述IL21突变体或IL21/4突变体，以及必要的药用辅料。
本发明还涉及以所述包含IL21突变体或IL21/4突变体的融合蛋白为活性成分的药物或药物组合物，所述的药物或药物组合物包括治疗有效量的所述融合蛋白，以及必要的药用辅料。
附图说明
图1、展示IL21/4与Herceptin抗体的融合蛋白的重组替换质粒片段
图2、通过同源重组将FRT-IL21/4-Herceptin质粒替换入CHO工作细胞基因组示意图
图3、FRT-IL21/4-Herceptin质粒替换入CHO工作细胞后IL21/4-Herceptin展示率
左图为CHO工作细胞阴性对照组，右图为转染后实验组，纵坐标标识为与Herceptin重链恒定区结合后HerceptinHC展示率，p3区显示IL21/4-Herceptin成功替换后展示率，约0.269％。
图4、FRT-IL21/4-Herceptin质粒替换入CHO工作细胞筛选富集后IL21/4-Herceptin展示率
左图为CHO工作细胞阴性对照组，右图为转染后实验组，横坐标标识与IL21受体IL21Rα胞外域结 合后展示率；纵坐标标识与IL21受体γc胞外域结合后展示率，可见实验组与IL21Rα胞外域结合后展示率为17.2％，与γc胞外域结合后展示率<0.3％。
图5、展示IL21/4-Herceptin细胞与Mouse Anti-human IgG-APC antibody结合展示率
左图为CHO工作细胞阴性对照组，右图为转染后实验组，横坐标标识与IL21受体γc胞外域结合后展示率，纵坐标标识为与Herceptin重链恒定区结合后HerceptinHC展示率，结果显示融合蛋白结合后的Herceptin展示率2.54％。
图6、IL21/4-Herceptin通过突变二硫键后检测展示率结果
展示16c IL21/4-Herceptin、17c IL21/4-Herceptin及4c IL21/4-Herceptin蛋白的CHO细胞富集后检测展示率结果，横坐标标识与IL21受体IL21Rα胞外域结合后展示率；纵坐标标识与Herceptin重链恒定区与标记抗体Mouse Anti-human IgG-APC antibody结合后的展示率，可见16c IL21/4-Herceptin、4c IL21/4-Herceptin组与Herceptin重链恒定区结合后的展示率较富集前明显提高；而17c IL21/4-Herceptin组与Herceptin重链恒定区结合后的展示率富集后只有1％；可见16c IL21/4-Herceptin、4c IL21/4-Herceptin组与受体IL21Rα胞外域结合后的展示率较富集前明显提高；而17c IL21/4-Herceptin组与受体IL21Rα胞外域无明显结合，与control组结果相似。
图7、构建IL21；16c IL21；16c IL21/4各突变体蛋白表达质粒(结构图)，分别各突变体蛋白编码基因插入PCEP4载体构建表达质粒，并添加His标签。
图8、IL21及各突变体蛋白SDS-PAGE胶电泳结果图。
图9、16c IL21；16c IL21/4突变体蛋白的酶解后的质谱图。
图10、IL21、16c IL21、16c IL21/4三种蛋白熔解温度(Tm)以明确IL21及各突变体蛋白热稳定性，
图11、IL21、16c IL21与16c IL21/4蛋白刺激KOB细胞增殖结果。
图12、融合蛋白16cIL21/4-Herceptin与Herceptin小鼠体内刺激CD8+T细胞增殖效果。
图13、融合蛋白16cIL21/4-Herceptin体外抗肿瘤试验结果；图中，Control是注射PBS的对照组；Herceptin是单独注射赫赛汀的实验组；IL-21 mutant-Herceptin代表16cIL21/4与Herceptin的融合蛋白的实验组；IL-21 mutant+Herceptin代表16cIL21/4和赫赛汀的混合溶液的实验组，而不是融合蛋白。
具体实施方式
PCR、DNA酶切产物和质粒的回收纯化制备：
PCR和DNA酶切产物回收采用AxyPrep DNA Gel Extraction Kit(AXYGEN)试剂盒回收；质粒的小量提取纯化采用全式金(transgen,China)质粒小提试剂盒；质粒中量提取纯化使用Qiagen中提试剂盒；大量提取采用Tiangen无内毒素质粒大提试剂盒，具体操作按照说明书进行。
细胞培养：
CHO/dhFr-(二氢叶酸还原酶缺陷型中国仓鼠卵巢细胞)：使用含10％小牛血清(Hyclone)、100U/mL双抗、0.1mM次黄嘌呤和0.016mM胸腺嘧啶的IMDM培养基，于37℃，5％的CO 2培养箱中培养。
293F细胞：人胚肾细胞，用FreeStyle TM293 Expression Medium培养基，于37℃，5％的CO 2培养箱，125rpm转速下摇瓶培养。
KOB细胞：高表达IL21受体成人T淋巴瘤细胞，由中科院生物物理研究所王盛典研究员组惠赠，用含10％小牛血清(Hyclone)、100U/mL双抗、RPMI1640培养基，于37℃，5％的CO 2培养箱中培养。
细胞转染：
CHO细胞转染：
细胞准备：转染前一天以20万/孔的细胞量将细胞种于六孔板中，使其在转染时达到80％满度，分布均匀，生长良好；
配制转染复合物：将5μl Lipo2000与Opti-MEM无血清培养基150μl均匀混合；2μg目的质粒与 Opti-MEM无血清培养基150μl均匀混合；室温放置5分钟后将两者混合配制成转染复合物，轻柔混匀，室温放置25分钟；
吸除六孔板中原有培养基，用Opti-MEM无血清培养基清洗三遍，然后将转染复合物滴加至细胞表面，再加入Opti-MEM无血清培养基500μl，4-6小时后换成普通培养基继续培养。
293F细胞转染(以50ml培养液为例)：
细胞准备：在转染前一天按6-7x10 5个细胞/ml接种细胞，待转染时细胞密度应在1x10 6个细胞/ml；
配制转染复合物：取50μg目的质粒用OptiPROTMSFM(Invitrogen)无血清培养基2ml稀释，充分混匀，制成DNA稀释液，取250μL转染试剂PEI加入到DNA稀释液中配制成转染复合物，充分混匀，室温放置15分钟；
将转染复合物加入293F细胞培养液中，37℃，5％的CO 2培养箱，125rpm转速下继续摇瓶培养96小时。
蛋白质的表达与纯化
(1)真核细胞表达与纯化：用含有表达目的基因的质粒转染293F细胞后，摇床继续培养96h，200g离心3分钟弃去细胞沉淀，收取上清培养基，然后再次10000g离心15分钟去掉培养基中的杂质,再用0.45μm滤膜过滤，然后用30kd浓缩管在3800rmp条件下，4℃离心，体积浓缩20倍。
(2)His标签蛋白的纯化：
使用填有硫酸镍(NiSO 4)的Sepharose High Performance(Amersham Bioscience)的层析柱；分别配制分别含有5、30、60、90、120、250mM咪唑的缓冲液：缓冲液其他成分为：20mM Tris-HCL、500mM NaCl、10％甘油；
清洗、平衡镍柱：，先用ddH 2O50ml清洗镍柱，再用含有5mM咪唑的缓冲液50ml清洗平衡镍柱；
上样：将含有目的蛋白的上清滴加入镍柱中，可上样2-3遍；
洗脱：上完样后，用分别含有5、30、60、90、120、250mM咪唑的缓冲液30ml洗脱，收集洗脱液，根据目的蛋白分子量选用不同规格超滤管，将洗脱液用超滤管浓缩，体积浓缩50-100倍；
蛋白保存：将最后得到的蛋白分装，液氮速冻，-80℃保存。
(3)全长抗体及全长抗体融合蛋白的纯化：
使用Protein A SefinoseTM-5ml(Pre-Packed Gravity Column)层析柱，
预先配制：
binding buffer：0.1M Na 3PO 4，0.15M NaCl，PH 7.2；
elution buffer：0.1M citric acid,PH 2.7；
neutralizing buffer：1M Tris-HCl PH 9.0；
清洗平衡；先用ddH 2O50ml冲洗后用elution buffer 50ml清洗层析柱，再用binding buffer 50ml平衡；
上样：将含有目的蛋白的上清滴加入层析柱中，可上样2-3遍；
洗脱：上完样后，用binding buffer 50ml清洗，以除去非特异性结合；准备10个15ml离心管，每个15ml离心管中加预先配制的neutralizing buffer 800μl；用elution buffer 30ml将蛋白洗脱下来，用已加入neutralizing buffer准备好的15ml离心管接洗脱液，每3ml换管；
分段回收：收集洗脱液，用nanodrop测定每管蛋白浓度，0.05mg/ml以下的弃用，根据目的蛋白分子量选用不同规格超滤管，将洗脱液用超滤管浓缩；
蛋白保存：将最后得到的蛋白分装，液氮速冻，—80℃保存。
清洗层析柱并保存：用bingding buffer 50ml清洗层析柱，再用ddH 2O 50ml清洗，最后用20％乙醇50ml清洗并封存。
蛋白质熔解温度(Tm)测定
蛋白质熔解温度(Tm)测定原理：蛋白质随周围温度升高，到达熔解温度(Tm)时，蛋白质构象破坏，疏水核心打开，染料可以与疏水区结合发出荧光而被测定。
首先，将待测定蛋白质浓度调整至20-40μM，体积24μl；
染料用
Figure PCTCN2019119088-appb-000001
orange protein(5000x)，用DMSO稀释至25x；
配制25μl检测体系：取96孔上样板，每孔加待测蛋白质浓度(20-40μM)24μl，再加
Figure PCTCN2019119088-appb-000002
orange protein(25x)1μl，混匀，常温避光放置；
使用qPCR仪stepone software 2.1程序检测，按说明书操作并设置参数；
检测并保存实验结果；使用Protein Thermal Shift 3.1程序分析结果。
实施例1、建立能够稳定展示嵌合体IL21/4的CHO细胞株
IL21的蛋白构象存在稳定与不稳定两种构象，通过将IL21的蛋白结构中不稳定区域替换为IL4的同源区域构建了一种嵌合体IL21/4，结果显示嵌合体IL21/4的蛋白构象单一稳定，而且生物学活性有提高。并且考虑到构建IL21突变体分别与赫赛汀(Herceptin)融合蛋白是实验重要目的，选择将嵌合体IL21/4与Herceptin融合蛋白展示在CHO工作细胞表面后，通过对Herceptin重链恒定区的检测也有利于检测嵌合体IL21/4的展示效率，因此我们首先将嵌合体IL21/4与Herceptin融合蛋白展示在CHO工作细胞表面。
为了获得高效、稳定展示嵌合体IL21/4的CHO细胞株，我们利用前期本实验室通过结合重组酶介导的盒式替换技术(recombinase-mediated cassette exchange，RMCE)建立的蛋白质展示系统构建(具体可参见CN 201410803422)。
首先构建展示IL21/4与Herceptin融合蛋白的重组替换质粒片段(FRT-IL21/4-Herceptin质粒)，融合蛋白的片段依次为：带有信号肽的Herceptin轻链，IRES(核糖体结合位点)，Herceptin重链信号肽，IL21/4通过采用3(G4S)linker连接在Herceptin重链的N端，跨膜区(TM)连接在Herceptin重链的C端，这样能够将IL21/4与Herceptin融合蛋白通过跨膜区锚定展示在CHO工作细胞表面，融合蛋白的序列结构如图1所示(融合蛋白从左至右依次为：FRT重组位点、Herceptin轻链信号肽、Herceptin轻链、可以与核糖体结合起始翻译的IRES(核糖体结合位点)、Herceptin重链信号肽、IL21/4、linker采用3(G4S)、Herceptin重链、使蛋白分子锚定展示在细胞表面的跨膜区(TM)、LOXp重组位点。
利用实验室已经建立的CHO工作细胞(具体可参见CN 201410803422)，其基因组内已经插入单拷贝的可重组替换区FRT-puromysin-Loxp，用FRT-IL21/4-Herceptin质粒与实验室前期构建的pCI-2A质粒共同转染，可将FRT-IL21/4-Herceptin质粒中FRT、Loxp位点之间的IL21/4与Herceptin融合蛋白序列以单拷贝方式重组替换入CHO工作细胞基因组内，如图2所示。
转染后按时收集细胞，然后用Mouse Anti-human IgG-APC antibody标记，流式检测IL21/4-Herceptin替换后展示率。结果显示(图3)，展示率为0.269％，转染后能够发生重组替换的概率是很低的，因此这些替换成功检测结果有展示的细胞需要被富集后再次检测，为避免假阳性信号，将上图中左上象限p3区阳性信号较强细胞用流式FACSAriaIII分选出来，继续培养、扩增。
富集后再次检测，这次我们采用预先表达纯化的IL21Rα胞外域-linker-GFP-his融合蛋白，γc胞外域-linker-mRFP-his融合蛋白和Mouse Anti-human IgG-APC antibody标记，以同时检测IL21/4-Herceptin融合蛋白中IL21/4与Herceptin结构域的完整性。结果如图4、5所示，如图4所示，横坐标显示IL21/4-Herceptin融合蛋白中IL21/4与IL21受体IL21Rα胞外域结合后展示率(17.2％)；纵坐标显示IL21/4-Herceptin融合蛋白中IL21/4与IL21受体γc胞外域结合后展示率(<0.3％)，根据文献报道，IL21与受体γc结合的亲和力较与受体IL21Rα结合的亲和力低得多，本实验中也多次证实这一点。
如图5所示，横坐标显示IL21/4-Herceptin融合蛋白中IL21/4与IL21受体γc胞外域结合后展示率<0.3％，纵坐标显示融合蛋白中Herceptin重链恒定区与标记抗体Mouse Anti-human IgG-APC antibody结合后的展 示率2.54％；较富集前展示率0.26％有提高，结合IL21Rα胞外域-linker-GFP融合蛋白标记结果，可以认为融合蛋白中Herceptin重链恒定区与标记抗体Mouse Anti-human IgG-APC antibody亲和力较与IL21Rα胞外域--linker-GFP低。
将上述稳定展示IL21/4-Herceptin融合蛋白的CHO细胞用流式FACSAriaIII分选出来，命名为s0细胞。
上述将嵌合体IL21/4稳定展示在CHO细胞表面的分子生物学实验过程的质粒构建步骤如下：
1、构建pFRT-Herceptin LC-IRES-IL21/4-linker-Herceptin HC-TM-Loxp质粒，将突变体IL21/4稳定展示在CHO细胞表面：
(1)以IL21为模板设计引物，用overlap PCR得到IL21/4片段，
(2)用overlap PCR将IL21/4片段通过linker连接在Herceptin重链HC的N端，再将sp-IL21/4-linker-HerceptinHC片段替换入本实验室已构建的抗体展示质粒pFRT的重链部分，构建pFRT-Herceptin LC-IRES-IL21/4-linker-Herceptin HC-TM-Loxp质粒，该质粒能将突变体IL21/4稳定展示在CHO细胞表面。
2、构建IL21Rα-linker-GFP融合蛋白分泌表达质粒，分泌表达检测IL21功能活性的IL21受体α胞外结构域：
(1)查阅数据库，合成IL21受体IL21Rα链的胞外区，再设计引物以GFP为模板用PCR扩增出GFP片段，
(2)用overlap PCR将IL21Rα链的胞外区通过linker连接在GFP的N端，再将sp-IL21Rα链的胞外区-linker-GFP片段连接入pCEP4，构建IL21Rα-linker-GFP型融合蛋白分泌表达质粒。
3、构建γc-linker-mRFP融合蛋白分泌表达质粒，分泌表达检测IL21功能活性的IL21受体γc胞外结构域：
查阅数据库，合成IL21受体γc链的胞外区；再设计引物以mRFP为模板用PCR扩增出mRFP片段，再次用overlap PCR将γc链的胞外区通过linker连接在mRFP的N端，再将sp-γc链的胞外区-linker-mRFP片段连接入pCEP4，构建γc-linker-mRFP融合蛋白分泌表达质粒。
4、构建IL21突变体及融合蛋白分泌表达质粒
(2)构建IL21不同突变体稳定展示质粒：为将IL21其它突变体稳定展示在CHO细胞表面，我们采用点突变PCR方法构建质粒：以pFRT-Herceptin LC-IRES-IL21/4-linker-Herceptin HC-TM-Loxp质粒为模板，设计引物，构建IL21不同突变体稳定展示质粒。
(3)构建IL21突变体蛋白分泌表达质粒：以多种IL21突变体展示质粒为模板，设计引物，用PCR扩增出IL21突变体片段，再连接入pCEP4，构建IL21突变体蛋白分泌表达质粒。
实施例2、通过蛋白质设计引入不同位点的二硫键进行IL21/4稳定性研究
1、通过蛋白质结构分析和设计，选取IL21/4蛋白上的不同位点进行突变，引入二硫键，获得如下一组突变体。引入的二硫键如下：
(1)第16位ILE、第70位SER，都突变为CYS，引入16-70二硫键；命名为 16cIL21/4；
(2)第17位ILE、第106位SER，都突变为CYS，引入17-106二硫键；命名为 17cIL21/4；
(2)将第16位ILE、第70位SER、第17位VAL、第106位LYS，都突变为CYS，同时引入两组二硫键；命名为 4cIL21/4。
2、获得能稳定展示IL21/4各突变体的CHO细胞
为了获得能稳定展示IL21/4各突变体的CHO细胞系，首先，以FRT-IL21/4-Herceptin为模板，利用点突变技术获得IL21/4各突变体的重组替换展示质粒，命名为：16cIL21/4-Herceptin；17cIL21/4-Herceptin； 4cIL21/4-Herceptin；再次与预先已构建的pci-2A质粒共同转染CHO工作细胞，重组替换成功后富集，以标记抗体Mouse Anti-human IgG-APC antibody结合检测展示率：
如图6所示，横坐标显示不同突变体IL21/4-Herceptin融合蛋白与IL21受体IL21Rα胞外域结合后展示率；纵坐标显示融合蛋白中Herceptin重链恒定区与标记抗体Mouse Anti-human IgG-APC antibody结合后的展示率；可以看到，16c IL21/4-Herceptin；4c IL21/4-Herceptin的展示率都较富集前明显提高，且与受体IL21Rα胞外域结合良好；而对照组17c IL21/4-Herceptin纵坐标显示与标记抗体Mouse Anti-human IgG-APC antibody结合后的展示率只有1％，与受体IL21Rα胞外域无明显结合，说明对照组17c IL21/4-Herceptin这种突变体并无展示。
我们已经获得在CHO工作细胞膜表面稳定展示两种IL21/4突变体的细胞，接下来要对这两种IL21/4突变体做稳定性初步检测。
实施例3、IL21/4各突变体稳定性初步检测
为了检测IL21/4各突变体稳定性，将展示IL21/4-Herceptin的CHO工作细胞与展示突变体16c IL21/4-Herceptin、4c IL21/4-Herceptin的CHO工作细胞做实验组，在4-50℃不同温度梯度下加热，再用IL21受体IL21Rα胞外域-linker-GFP-his结合标记后，流式检测各组在不同温度梯度时与受体结合后展示率，由于随着温度变化，热稳定性差的蛋白会先发生变性，正常构象破坏，丧失与受体结合能力，可以检测各突变体的热稳定性。
具体实验方法如下：
(1)将在细胞膜表面稳定展示IL21突变体的CHO细胞用0.02％EDTA-PBS消化后，用带有血清的培养基洗脱，830g离心，3min，弃去上清，收集细胞；
(2)将细胞用4℃预冷的Opti-MEM无血清培养基1ml重悬，830g离心,3min,弃去上清，收集细胞，再用4℃预冷的Opti-MEM无血清培养基重悬，约5x10 6细胞/50μl；
(3)细胞悬液加入PCR管，用PCR仪设温度梯度加热，至预设温度时加热0.5h，然后4℃，15min，取出PCR管；
(4)按一定稀释比例加入相应的IL21受体胞外域与荧光蛋白融合蛋白混匀，4℃摇床，避光放置1h；
(5)830g离心3min，弃去上清，加新的4℃预冷的Opti-MEM洗涤二遍，分别离心，弃去上清，然后用4℃预冷的Opti-MEM 200μl重悬，标记后的细胞用FACSAriaIII(BD)或FACSCalibur进行检测或分选。
结果显示，
(1)4℃时，除阴性对照组外，各组与IL21受体IL21Rα胞外域均有结合，突变体16c IL21/4-Herceptin展示率为20％，结合率最高；
(2)经过49℃加热后，IL21/4-Herceptin组，4c IL21/4-Herceptin组与IL21受体IL21Rα胞外域结合均基本丧失，突变体16c IL21/4-Herceptin组还能检测到与IL21受体IL21Rα胞外域的结合，初步认为突变体16c IL21/4热稳定性有所提高。
实施例4、构建野生型IL21的突变体并初步检测稳定性
1、流式检测突变体的热稳定性
为了研究在野生型IL21分子中引入同样位置(16、70位)的二硫键对稳定性的影响，我们构建了另外二种结构：
IL21-Herceptin：野生型IL21与Herceptin的融合蛋白；
16c IL21-Herceptin：在同样位置，第16位ILE——第70位SER，将其都突变为CYS，引入二硫键的IL21与Herceptin构建的融合蛋白；
同样通过转染，重组替换，将其展示在CHO工作细胞膜表面，与16c IL21/4-Herceptin及IL21/4-Herceptin比较，在4-55℃不同温度梯度下加热，再用IL21受体IL21Rα胞外域--linker-GFP-his，及标记抗体Mouse Anti-human IgG-APC antibody结合结合标记后，流式检测各组在不同温度梯度时与受体结合后展示率。
结果显示，引入16-70二硫键的16c IL21/4-Herceptin和16c IL21-Herceptin在加热至48℃后，与受体结合仍有部分展示率，说明稳定性较IL21/4-Herceptin和IL21-Herceptin有提高。这四种蛋白是融合蛋白，流式检测只是初筛，需要将每种IL21及突变体蛋白表达，纯化后进一步鉴定热稳定性。
2、表达IL21及各突变体蛋白并检测二硫键结构
为了获得IL21及各突变体蛋白，首先构建质粒，将IL21及各突变体编码基因分别插入PCEP4载体质粒，并添加His标签，构建了三种分别表达IL21；16c IL21；16c IL21/4各突变体蛋白的质粒(如图7)：
将IL21、16c IL21、16c IL21/4三种蛋白进行SDS-PAGE电泳，结果如图8所示，根据结果，IL21、16c IL21、16c IL21/4三种蛋白跑胶条带位置与理论相符，从分子量的层面估计，这三种蛋白是目的蛋白。
为确认16c IL21/4蛋白中引入的目的二硫键是否形成，进一步行质谱检测，将16c IL21/4蛋白送质谱鉴定，跑胶时注意保护二硫键。样品蛋白首先用蛋白酶进行酶解，会切成不同大小肽段。蛋白质中的二硫键会影响酶解结果，在二硫键打开与否的条件下，样品蛋白会切成不同大小的肽段，会有不同的肽段质核比分布，根据是否与理论预测相符可判断样品蛋白是否存在预测二硫键。
本样品16c IL21/4二硫键分析：
理论上会产生二硫键的两个片段的氨基酸序列IINVCIK和QLIDCVDQLK。当在还原状态下酶解时，二硫键被打开，C上面会被烷基化(分子量增加57Da)，两个肽段质核比理论值分别为859.507和1231.635。从图9A中可以看出，在还原状态(加DTT)下，859.587和1231.769有比较明显的信号峰，在非还原状态下，这两个峰信号非常低。说明16cIL21/4样品蛋白加DTT时二硫键被打开，可以切出两个肽段；不加DTT时二硫键未打开，则无这两个肽段信号，这与理论预测的二硫键结构相符。
如果在非还原状态下酶解，这两个肽段之间的二硫键没有被打开，我们应该能检测到一个质核比为两个肽段质核比加和的肽段。在非还原状态下，加和肽段理论质核比为(859.587-57)+(1231.769-57)－2－1＝1974.356。在图9B中，我们可以看到在非还原状态下，有明显的质核比1974.230的峰，且该峰在还原状态下信号很差。说明当样品蛋白不加DTT二硫键未打开时，酶解产物会有一较大肽段信号；加DTT时二硫键被打开，较大肽段被切开，则此信号不存在。
上述结果在16c IL21蛋白的酶解过程中也同样观察到，表明16位C和70位C形成了正确的二硫键。
3、测定IL21及各突变体蛋白熔解温度(Tm)
确认16c IL21/4蛋白中16-70位cys间二硫键的形成后，我们测定了IL21、16c IL21、16c IL21/4三种蛋白熔解温度(Tm)以明确IL21及各突变体蛋白热稳定性，结果如图10所示：
根据结果，IL21、16c IL21、16c IL21/4三种蛋白Tm值比较：16c IL21(65.62℃)>16cIL21/4(56.52℃)>IL21(48.54℃)。
说明引入二硫键的两种突变体16cIL21、16cIL21/4蛋白较野生型IL21蛋白热稳定性均明显提高；可认为引入的二硫键是导致热稳定性提高的原因。
实施例5、测定IL21各突变体生物学活性
1、刺激KOB细胞增殖能力
为了检测IL21、16c IL21、16c IL21/4三种蛋白在热稳定性提高的同时生物学活性是否保留。KOB细胞表达IL21受体，可被IL21刺激而增殖。可通过刺激KOB细胞增殖能力来测定IL21各突变体生物学活性，具体方法如下：
(1)准备KOB细胞：用含10％小牛血清(Hyclone)、100U/mL双抗、RPMI1640培养基，于37℃，5％的CO 2培养箱中培养；
(2)准备IL21及突变体蛋白：构建IL21及突变体蛋白(带His标签)表达质粒，转染293F细胞，常规培养、表达，镍柱纯化；BCA法测定浓度；
(3)生物学活性测定：用96孔板，KOB细胞以2x10 4/每孔接种，200μl培养基，每孔中分别加入IL21或突变体蛋白，设置梯度浓度为0.1ng/ml，1ng/ml，10ng/ml，37℃，5％的CO 2培养箱中继续培养，96h后细胞计数统计。
结果如图11所示：
利用无重复双因素方差分析结果，ctr组与各蛋白组间p<0.05，说明组间存在显著差异；三种蛋白组间p>0.05，说明三种蛋白组间无显著差异；三种蛋白不同浓度组间比较p>0.05，说明三种蛋白不同浓度组间无显著差异；
由本次结果可知，三种蛋白都有刺激KOB细胞增殖的作用，且生物学活性统计学结果无显著差异，在加入蛋白梯度浓度为0.04μg/ml，0.2μg/ml，1μg/ml组间亦无显著差异。
可知，两种突变体16cIL21、16cIL21/4蛋白是具有热稳定性提高并保留生物学活性的蛋白。
2、IL21及各突变体与Herceptin融合蛋白小鼠体内血浆半衰期测试结果
为了明确IL21稳定性提高是否能够延长动物体内血浆半衰期，我们选择IL21-Herceptin以及突变的16c IL21-Herceptin融合蛋白、16c IL21/4-Herceptin融合蛋白进行对比检测，同时加Herceptin组做对照，检测小鼠体内血浆半衰期，结果如下表4所示：
表4：不同类型的IL21/融合蛋白的半衰期检测
Figure PCTCN2019119088-appb-000003
由结果可知，16c IL21-Herceptin、16c IL21/4-Herceptin融合蛋白中包含的高稳定性IL21突变体使小鼠体内血浆半衰期较野生型IL21有了明显延长，以融合蛋白16c IL21/4-Herceptin最好，说明IL21稳定性的提高是其血浆半衰期延长的重要原因。
3、IL21融合蛋白刺激T细胞增殖功能测定
采用BT474乳腺癌荷瘤小鼠模型检测16cIL21/4-Herceptin融合蛋白的功能活性，小鼠右侧腹背部接种1.5x10 7人BT474乳腺癌细胞，35天后给药，对16cIL21/4-Herceptin融合蛋白与Herceptin刺激CD8 +T细胞在小鼠体内增殖情况做了检测，结果见附图12所示。可见16cIL21/4-Herceptin融合蛋白在肿瘤接种后约53天开始明显出现CD8+T细胞在外周血中比例明显升高；同时Herceptin组反而出现T细胞缓慢下降的趋势。
4、IL21融合蛋白肿瘤抑制效果
采用BT474乳腺癌荷瘤小鼠模型检测16cIL21/4-Herceptin融合蛋白的肿瘤抑制活性，每只小鼠接种1×10 7的BT474细胞(人乳腺癌细胞)；采用腹腔注射，每四天注射一次PBS(Control)、Herceptin(单独注射赫赛汀)、16cIL21/4-Herceptin融合蛋白(IL-21 mutant-Herceptin)、16cIL21/4和赫赛汀的混合溶液(IL-21 mutant+Herceptin)；每次注射500ug的剂量。结果如图13所示，与对照组、赫赛汀组以及混合溶液组相比，16cIL21/4-Herceptin融合蛋白组能够显著的减少肿瘤体积。
最后需要说明的是，以上实施例仅用作帮助本领域技术人员理解本发明的实质，并不用做对本发明保护范围的限定。

Claims (7)

  1. 一种白介素21(IL21)突变体，所述的突变体为：
    (1)将野生型IL21的氨基酸序列的第16位ILE、第70位SER，都突变为CYS，并在两个突变的CYS之间形成二硫键，所述的野生型IL21的氨基酸序列如SEQ ID NO.1所示；
    或(2)将野生型IL21的氨基酸序列的第16位ILE、第17位VAL、第70位SER、第112位LYS的都突变为CYS，并且形成16-70、17-112两组二硫键；
    所述的野生型IL21的氨基酸序列如SEQ ID NO.1所示。
  2. 一种白介素21和白介素4嵌合体蛋白(IL21/4)的突变体，所述的突变体为：
    (1)将白介素21和白介素4嵌合体(IL21/4)的氨基酸序列的第16位ILE、第70位SER，都突变为CYS，并在两个突变的CYS之间形成二硫键；
    或(2)将白介素21和白介素4嵌合体(IL21/4)的氨基酸序列的第16位ILE、第17位VAL、第70位SER、第106位LYS的都突变为CYS，并且形成16-70、17-106两组二硫键；
    所述的白介素21和白介素4嵌合体蛋白(IL21/4)的氨基酸序列如SEQ ID NO.2所示。
  3. 编码权利要求1所述的IL21突变体或权利要求2所述的IL21/4突变体的核苷酸序列。
  4. 包含权利要求1所述的IL21突变体或权利要求2所述的IL21/4突变体的融合蛋白，所述的融合蛋白包括：
    (1)功能片段1：IL21突变体或IL21/4突变体；
    (2)功能片段2：单克隆抗体功能；
    及连接不同功能片段的连接结构域。
  5. 权利要求1所述的IL21突变体、权利要求2所述的IL21/4突变体或权利要求4所述的融合蛋白在制备药物中的应用，所述的药物是调节或激活免疫的药物或抗肿瘤药物。
  6. 权利要求1所述的IL21突变体、权利要求2所述的IL21/4突变体或权利要求4所述的融合蛋白在制备促进B细胞分化增殖，促进T细胞分化增殖，促进NK细胞分化增殖的制剂中的应用。
  7. 以权利要求1所述的IL21突变体、权利要求2所述的IL21/4突变体或权利要求4所述的融合蛋白为活性成分的药物或药物组合物，所述的药物或药物组合物包括治疗有效量的所述IL21突变体、所述IL21/4突变体或所述融合蛋白，以及必要的药用辅料。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11066644B2 (en) 2018-02-01 2021-07-20 Nkmax Co., Ltd. Method of producing natural killer cells and composition for treating cancer

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116496408A (zh) * 2022-01-21 2023-07-28 广东菲鹏制药股份有限公司 白细胞介素21及其受体复合物

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101553501A (zh) * 2006-10-26 2009-10-07 诺沃-诺迪斯克有限公司 Il-21变种
WO2010076339A1 (en) * 2009-01-05 2010-07-08 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Means and method for the treatment of antibody deficiency diseases based on il-21 and il-21 variants

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003087320A2 (en) * 2002-04-09 2003-10-23 Beth Israel Deaconess Medical Center, Inc. Antagonists of il-21 and modulation of il-21-mediated t cell responses
RU2412199C2 (ru) * 2005-04-18 2011-02-20 Ново Нордиск А/С Варианты ил-21
CN100425697C (zh) * 2006-05-31 2008-10-15 中国药科大学 一种人白细胞介素21的核苷酸序列及生产成熟的人白细胞介素21的方法和应用
WO2010103038A1 (en) * 2009-03-11 2010-09-16 Novo Nordisk A/S Interleukin-21 variants having antagonistic binding to the il-21 receptor
CN108686202A (zh) * 2017-04-06 2018-10-23 张晋宇 肿瘤免疫疗法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101553501A (zh) * 2006-10-26 2009-10-07 诺沃-诺迪斯克有限公司 Il-21变种
WO2010076339A1 (en) * 2009-01-05 2010-07-08 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Means and method for the treatment of antibody deficiency diseases based on il-21 and il-21 variants

Non-Patent Citations (18)

* Cited by examiner, † Cited by third party
Title
BHATT SPARVIN SZHANG Y ET AL.: "Anti-CD20-interleukin-21 fusokine targets malignant B cells via direct apoptosis and NK-cell-dependent cytotoxicity [J", BLOOD, vol. 129, no. 16, 2017, pages 2246 - 2256
DAVIS IDSKRUMSAGER BKCEBON J ET AL.: "An open-label, two-arm, phase I trial of recombinant human interleukin-21 in patients with metastatic melanoma [J", CLIN CANCER RES., vol. 13, no. 12, 2007, pages 3630 - 6, XP055601201, DOI: 10.1158/1078-0432.CCR-07-0410
KENT BJENS BDENNIS M ET AL.: "The Existence of Multiple Conformers of Interleukin-21 Directs Engineering of a Superpotent Analogue[J", THE JOURNALOF BIOLOGICAL CHEMISTRY, vol. 282, no. 32, 2007, pages 23326 - 23336
KENT BONDENSGAARD ET AL: "The Existence of Multiple Conformers of Interleu- kin-21", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 282, no. 32, 10 August 2007 (2007-08-10), pages 23326 - 23336, XP002470498, ISSN: 0021-9258, DOI: 10.1074/jbc.M701313200 *
LOTZE MT ET AL.: "In vivo administration of purified human interleukin 2.1. Half-life and immunologic effects of the Jurkat cell line-derived interleukin 2", J] LMMUNOL., vol. 134, no. l, 1985, pages 157 - 66
MALEK TRASTRO I: "Interleukin-2 Receptor Signaling: At the Interface between Tolerance and mmunity[J", MMUNITY, vol. 33, no. 2, 2010, pages 153 - 165, XP055226773, DOI: 10.1016/j.immuni.2010.08.004
MCMICHAEL ELJAIME-RAMIREZ ACGUENTERBERG KD ET AL.: "IL21 Enhances Natural Killer Cell Response to Cetuximab-Coated Pancreatic Tumor Cells [J", CLIN CANCER RES., vol. 23, no. 2, 2017, pages 489 - 502
OVERWIJK,W.W.SCHLUNS,K.S.: "Functions of yc cytokines in immune homeostasis: Current and potential clinical applications[J", CLIN IMMUNOL., vol. 132, no. 2, 2009, pages 153 - 165, XP026301929, DOI: 10.1016/j.clim.2009.03.512
ROSANNE SWARREN J.L: "Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity[J", ANNU.REV.LMMUNOL., vol. 26, 2008, pages 57 - 79
ROTH JACRISTIANO RJ: "Gene therapy for cancer: what have we done and where are we going?", J.NATL.CANCER INST., vol. 89, 1997, pages 21 - 39
SCHMIDT HBROWN JMOURITZEN U ET AL.: "Safety and clinical effect of subcutaneous human Interleukin-21 in patients with metastatic melanoma or renal cell carcinoma: a phase I trial[J", CLIN CANCER RES., vol. 16, no. 21, 2010, pages 5312 - 9.8, XP055624568, DOI: 10.1158/1078-0432.CCR-10-1809
See also references of EP3885357A4
SHEN BEIFENCHEN ZHINANLIU MINPEI: "Recombinant Antibody", 2005, SCIENCE PRESS, pages: 373
THOMPSON JACURTI BDREDMAN BG ET AL.: "Phase I Study of Recombinant Interleukin-21 in Patients With Metastatic Melanoma and Renal Cell Carcinoma [J", CLIN ONCOL, vol. 26, no. 12, 20 April 2008 (2008-04-20), pages 2034 - 9
XU MLIU MDU X ET AL.: "Intratumoral Delivery of IL-21 Overcomes Anti-Her2/Neu Resistance through Shifting Tumor-Associated Macrophages from M2 to M1 Phenotype [J", J IMMUNOL., vol. 194, no. 10, 2015, pages 4997 - 5006, XP055376961, DOI: 10.4049/jimmunol.1402603
ZATLOUKAL KSCHNEEBERGER ABERGER M ET AL.: "Elicitation of a systemic and protective anti-melanoma immune response by an IL-2 based vaccine. Assessment of critical cellular and molecular parameters", J. IMMUNOL., vol. 154, 1995, pages 3406 - 3419
ZHANG, NIJIA: "Building Highly Active Cytokine Interleukin IL-21 Based on the Molecular Conformation Design", MASTER THESIS, 15 September 2014 (2014-09-15), pages 1 - 58, XP009521345 *
ZHOU, YUJUN: "In Silico Study of the Increase in Potency about Cytokine Interleukin-21", MASTER THESIS, 15 September 2014 (2014-09-15), pages 1 - 56, XP009521346 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11066644B2 (en) 2018-02-01 2021-07-20 Nkmax Co., Ltd. Method of producing natural killer cells and composition for treating cancer

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