WO2022007796A1 - 共表达IL-21和hrCD16嵌合受体的免疫细胞及其应用 - Google Patents

共表达IL-21和hrCD16嵌合受体的免疫细胞及其应用 Download PDF

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WO2022007796A1
WO2022007796A1 PCT/CN2021/104765 CN2021104765W WO2022007796A1 WO 2022007796 A1 WO2022007796 A1 WO 2022007796A1 CN 2021104765 W CN2021104765 W CN 2021104765W WO 2022007796 A1 WO2022007796 A1 WO 2022007796A1
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cells
hrcd16
immune cell
cancer
region
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French (fr)
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徐建青
张晓燕
廖启彬
丁相卿
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上海鑫湾生物科技有限公司
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Definitions

  • the invention belongs to the field of biological therapy, and relates to an immune cell co-expressing IL-21 and hrCD16 chimeric receptors and its application in preparing medicines for treating cancer or viral infectious diseases.
  • Surgical resection can only be applied to primary tumor lesions and is helpless for multiple metastatic lesions; radiotherapy is mostly not suitable for advanced and radiotherapy. Insensitive tumor patients; chemotherapy has poor tumor specificity, often accompanied by a variety of adverse side effects, and is not suitable for chemotherapy-resistant tumor patients.
  • a new type of tumor immunotherapy method has gradually entered people's field of vision, namely chimeric antigen receptor T cell therapy (Chimeric antigen receptor T cells, CAR-T cells).
  • CAR-T cell therapy is effective in the treatment of hematological tumors, so two CAR-T cell drugs for hematological tumors have been born, namely Kymriah of Novartis and Yescarta of Kite.
  • Solid tumors have severely restricted the clinical efficacy of CAR-T cells due to their complex tissue structure, immunosuppressive microenvironment, and lack of tumor-specific targets. Therefore, there is an urgent need to develop new and effective genetically engineered immune cell technologies to improve the therapeutic effect of solid tumors.
  • the tumor antigens currently used are mostly tumor-associated antigens, which are highly expressed in tumor cells, but are also expressed in small amounts in normal tissue cells, resulting in the "on-target off effect of targeting non-tumor cells" in CAR-T cell therapy. -tumor), limiting its clinical application in the treatment of solid tumors.
  • CAIX carbonic anhydrase IX
  • Cytokines as the third signal of T cell activation, contribute to T cell proliferation, survival and function.
  • Cytokine IL-21 a member of the ⁇ chain family of cytokines, is produced and secreted by activated CD4 + T cells and NKT cells. Its receptors are widely distributed and have immunomodulatory effects on the proliferation, differentiation and function of T cells.
  • IL-21 cooperates with IL-15 to promote the proliferation and maturation of NK cells
  • IL-21 cooperates with IL-15 and IL-18 to promote the production of IFN- ⁇ in NK and T cells, and enhance the anti-infection/tumor activity.
  • IL-21 induces B cells to produce anti-infection/tumor-targeted antibodies.
  • the purpose of the present invention is to provide an immune cell that co-expresses IL-21 and hrCD16 chimeric receptors.
  • the hrCD16 chimeric receptor provided by the present invention not only has high Fc affinity and cellular protease resistance, but also can be introduced into a variety of killer immune cells, such as T cells, NK, and IL-21 by viral/non-viral delivery. cells or NKT cells and other immune cells, and stably expressed on them.
  • the immune cells co-expressing IL-21 and hrCD16 chimeric receptors of the present invention can broadly kill different types of tumor cells or virus-infected cells, And have better anti-tumor or virus efficacy.
  • the immune cells co-expressing IL-21 and hrCD16 chimeric receptors of the present application use tumor antigen-targeting antibodies or viral antigen-targeting antibodies in combination, which can not only efficiently kill different types of tumor cells or virus-infected cells, but also act on In paralogous immune cells, endogenous immune response is initiated at the same time.
  • the present invention provides an immune cell, wherein the immune cell co-expresses IL-21 and hrCD16 chimeric receptor;
  • the hrCD16 chimeric receptor comprises:
  • the chimeric receptor further comprises
  • the extracellular recognition domain is a mutant hrCD16 extracellular recognition domain that specifically binds to the Fc fragment of an antibody, which comprises the amino acid sequence shown in SEQ ID NO: 1.
  • the immune cell according to the present invention is characterized in that the immune cell is selected from T cells, natural killer cells (NK), innate lymphocytes (Innate lymphoid cells, ILC), hematopoietic stem cells, embryonic Stem cells and pluripotent stem cells;
  • NK natural killer cells
  • ILC innate lymphocytes
  • hematopoietic stem cells embryonic Stem cells and pluripotent stem cells
  • the T cells are selected from the group consisting of unsorted and purified T cells, sorted and purified T cells, sorted and purified PD-1 + T cells, sorted and purified CD137 + T cells, sorted and purified CD160 cells + T cells, sorted and purified naive T cells (T naive ), sorted and purified central memory T cells (T CM ), sorted and purified effector memory T cells ( TEM ), sorted and purified effector T cells cells ( TEMRA ), sorted and purified transitional memory T cells (Transitional Memory T cells, TTM ), sorted and purified tissue memory T cells (Tissue residential memory T cells, TRM ) and natural killer T cells ( Natural killer T cells, NKT).
  • T naive sorted and purified central memory T cells
  • T CM sorted and purified effector memory T cells
  • TEMRA sorted and purified effector T cells
  • TTM Transitional Memory T cells
  • tissue memory T cells Tissue residential memory T cells, TRM
  • immunoglobulin Fc receptors Fc ⁇ RI (CD64), Fc ⁇ RII (CD32), Fc ⁇ RIII (CD16), Fc ⁇ R (CD89), Fc ⁇ RI, Fc ⁇ RII (CD23);
  • costimulatory molecules CD28 hinge region, CD137 hinge region, CD8 ⁇ hinge region, CD4 hinge region, PD-1 hinge region and CTLA-4 hinge region;
  • the immune cell according to the present invention is characterized in that the extracellular spacer is a CD8 hinge region; preferably, the extracellular spacer comprises the amino acid sequence shown in SEQ ID NO:2.
  • the immune cell according to the present invention is characterized in that the transmembrane region includes but is not limited to any one or more of the following: the transmembrane region of the CD3 ⁇ chain of the T cell receptor complex, the CD28 transmembrane region, the Globulin Fc receptor transmembrane domain, CD4 transmembrane domain, CD8 transmembrane domain, CD16 transmembrane domain, CD137 transmembrane domain, CTLA-4 transmembrane domain, PD-1 transmembrane domain, LAG-3 transmembrane domain, VISTA transmembrane region and combinations thereof;
  • the transmembrane region is a CD8 transmembrane region; more preferably, the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:3.
  • the costimulatory signaling domains include but are not limited to any one or more of the following signaling domains: CD2, CD27, CD28, CD30, CD40, CD40L, CD137(4-1BB), CD134(OX40), CD278(ICOS), GITR, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, Dap10, ICAM-1, LFA-1 , Lck, TNFRI, TNFRII, TIM-1, TIM-2, TIM-3, TIM-4, and combinations thereof.
  • the costimulatory signaling domain is a CD137 signaling domain; more preferably, the costimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO:4.
  • the immune cell according to the present invention is characterized in that the cytokine receptor signaling domains include but are not limited to any one or more of the following signaling domains: IL-2R, IL-7R, IL-15R , IL-18R, IL-21R, IL-23R, and combinations thereof.
  • the immune cell according to the present invention is characterized in that the intracellular signaling domain CD3 ⁇ chain, Fc ⁇ RIII, Fc ⁇ RI, the intracellular signaling domain of Fc receptors, carrying immunoreceptor tyrosine activation motif (ITAM) ) of the signaling domain and combinations thereof; more preferably, its amino acid sequence is shown in SEQ ID NO:5.
  • ITAM immunoreceptor tyrosine activation motif
  • the immune cell according to the present invention is characterized in that, the hrCD16 chimeric receptor is composed of a mutant CD16 extracellular domain, a human CD8 hinge region, and a human CD8 transmembrane region comprising the amino acid sequence shown in SEQ ID NO: 1 , composed of human CD137 costimulatory signaling domain and CD3 ⁇ chain signaling domain;
  • amino acid sequence of the hrCD16 chimeric receptor is shown in SEQ ID NO: 6
  • the immune cell according to the present invention is characterized in that, the IL-21 is selected from wild-type IL-21, truncated IL-21, mutant IL-21 and active fragments thereof; preferably, the IL-21 21 is secreted or membrane-type IL-21; preferably, the IL-21 is constitutively expressed or inducibly expressed; more preferably, the IL-21 has the amino acid sequence shown in SEQ ID NO:7.
  • the present invention also provides a polynucleotide, characterized in that, the polynucleotide encodes the IL-21 and hrCD16 chimeric receptors in the immune cells;
  • polynucleotide sequence is shown in SEQ ID NO:8.
  • the present invention also provides a vector comprising the polynucleotide.
  • the vector is selected from plasmids, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, poxvirus vectors, herpes virus vectors, transposons and combinations thereof; more preferably, the vector is a lentiviral vector;
  • the present invention provides a combination comprising the immune cell and a tumor antigen-targeting antibody.
  • the tumor antigens include but are not limited to one or more of the following: CD19, BCMA, CD20, CD22, CD30, CD33, CD38, CD47, CD70, CD117, CD123, CD133, CD138, CD147, CD171, NKG2DL, HER2, MUC1, MUC16, CEA, EpCAM, IL-13R ⁇ 2, EGFR, EGFRvIII, GD2, DR5, EphA2, FR ⁇ , PSCA, PSMA, TARP, cMet, VEGFR2, BCMA, CTLA-4, PD-L1, AFP, GPC3, AXL, ROR1, ROR2, FAP, Mesothelin, DLL3, CLDN18.
  • the tumor antigens include, but are not limited to, one or more of the following: HER2, EGFR, CD47, GPC3, AXL and FAP.
  • the tumor antigen targeting antibody is a GPC3 tumor antigen targeting antibody.
  • the present invention provides a combination comprising the immune cell and viral antigen targeting antibodies.
  • the viral antigen is selected from one or more of the following: gp120 of human acquired immunodeficiency virus HIV-1, surface antigen of hepatitis B virus HBV, hemagglutinin or neuraminidase of influenza virus, Ebola virus spike protein, severe acute respiratory syndrome coronavirus SARS-CoV surface spike protein, Middle East respiratory syndrome coronavirus MERS-CoV surface spike protein and novel coronavirus SARS-CoV-2 surface spike protein;
  • the viral surface antigen is selected from the surface spike protein of the novel coronavirus SARS-CoV-2.
  • the present invention provides the use of the combination of immune cells, immune cells and tumor antigen-targeting antibodies or viral antigen-targeting antibodies of the present invention in the preparation of a medicament for the treatment of tumors or viral infectious diseases;
  • the tumor is selected from one or more of the following: lymphoma, neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, renal cancer, pancreatic cancer, nasopharyngeal cancer cancer, small bowel, large bowel, colorectal, bladder, bone, prostate, thyroid, brain, rhabdoid and leiomyoma.
  • the viral infectious disease is selected from one or more of the following: Human Acquired Immunodeficiency Syndrome, Hepatitis B, Influenza, Ebola Virus Disease, Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and Novel Coronavirus Pneumonia.
  • Human Acquired Immunodeficiency Syndrome Hepatitis B
  • Influenza Influenza
  • Ebola Virus Disease Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and Novel Coronavirus Pneumonia.
  • SARS Severe Acute Respiratory Syndrome
  • MERS Middle East Respiratory Syndrome
  • Novel Coronavirus Pneumonia Novel Coronavirus Pneumonia
  • the present invention provides a method of treating a tumor, the method comprising administering to a subject a therapeutically effective amount of an immune cell, a combination of an immune cell and a tumor antigen-targeting antibody;
  • the tumor is selected from one or more of the following: lymphoma, neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, renal cancer, pancreatic cancer, nasopharyngeal cancer cancer, small bowel, large bowel, colorectal, bladder, bone, prostate, thyroid, brain, rhabdoid and leiomyoma.
  • the present invention provides a method of treating a GPC3+ tumor comprising administering to a patient a combination of an immune cell of the present invention and a GPC3-targeting antibody.
  • the present invention provides a method of treating a viral infectious disease, the method comprising administering to a subject a therapeutically effective amount of an immune cell, a combination of an immune cell and a viral antigen targeting antibody;
  • the viral infectious disease is selected from one or more of the following: Human Acquired Immune Deficiency Syndrome, Hepatitis B, Influenza, Ebola Virus Disease, Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and Novel Coronavirus Pneumonia.
  • the present invention provides a method of treating novel coronavirus pneumonia, the method comprising administering to a patient a combination of the immune cells of the present invention and a SARS-CoV-2 surface spike protein targeting antibody.
  • the present invention provides a method for preparing immune cells of the present invention, comprising the steps of:
  • the immune cells are T cells.
  • the present invention also provides a method for expanding a large number of T cells co-expressing IL-21 and hrCD16 chimeric receptors, the method comprising: transfecting with the vector or infecting T with the virus as described cells; and additionally add anti-human CD3 stimulatory antibodies and anti-human CD28 stimulatory antibodies, tumor antigen-expressing cells or recombinant tumor antigens, and tumor antigen-targeting antibodies to stimulate said T cells to proliferate to produce large amounts of co-expressed IL-21 and high-affinity, cleavage-resistant hrCD16 chimeric receptor T cells.
  • the present invention has the following advantages:
  • the present invention provides an immune cell that co-expresses IL-21 and hrCD16 chimeric receptor, the hrCD16 chimeric receptor of the present invention has high Fc affinity and cellular protease resistance, and it has higher antibody Fc fragment affinity, combined with tumor antigen-targeting antibodies or viral antigen-targeting antibodies, can significantly enhance the effect of ADCC.
  • the hrCD16 chimeric receptor of the present invention also has the ability to resist the cleavage of metalloproteinases, to ensure that hrCD16 is highly expressed on killer cells, and to avoid functional damage caused by down-regulation of CD16.
  • the hrCD16 chimeric receptor of the present invention is different from the traditional single-target CAR strategy, and can flexibly switch the targeting antibodies of different tumor antigens or viral antigens, thereby realizing the efficacy of broad-spectrum immunotherapy of multiple cancer species.
  • the immune cells of the present invention co-express IL-21, which can not only effectively kill different types of tumor cells or virus-infected cells, but also act on paralogous immune cells and initiate endogenous immune responses at the same time.
  • Figure 1 is a map of recombinant lentivirus expression plasmids.
  • FIG. 2 shows that T cells co-expressing IL-21 and hrCD16 chimeric receptors according to the present invention express high levels of hrCD16 and IL-21.
  • Figure 3 shows that T cells co-expressing IL-21 and hrCD16 chimeric receptors and T cells modified with hrCD16 chimeric receptors that do not express IL-21 and wild-type CD16 chimeric receptors secrete anti-tumor factor IFN according to the present invention - ⁇ results, indicating that co-expression of IL-21 can enhance the ability of hrCD16 chimeric receptor-modified T cells to secrete anti-tumor factor IFN- ⁇ .
  • Figure 4 shows T cells co-expressing IL-21 and hrCD16 chimeric receptors and T cells modified with hrCD16 chimeric receptors that do not express IL-21 and wild-type CD16 chimeric receptors in combination with GPC3 targeting antibodies according to the present invention
  • the results of killing liver cancer cells indicated that co-expression of IL-21 could enhance the killing activity of hrCD16 chimeric receptor-modified T cells.
  • DMEM medium and RPMI1640 medium were purchased from Corning Company, and lymphocyte medium X-VIVO 15 was purchased from Lonza Company.
  • T cell growth medium consists of basal medium and cytokines.
  • the basal medium is lymphocyte medium X-VIVO 15, and the cytokines are IL-7 at a final concentration of 5ng/mL, IL-15 at 10ng/mL and 30ng/mL mL of IL-21.
  • cytokines IL-7 and IL-15 were purchased from R&D Company, and IL-21 was purchased from Nearshore Protein Technology Co., Ltd. .
  • Fetal bovine serum was purchased from BI Company.
  • Lenti-X lentiviral concentration reagent was purchased from Takara Company.
  • Tumor-targeting antibody is prepared and provided by Shanghai Xinwan Biotechnology Co., Ltd., including GPC3-targeting antibody (XW-GPC3-01) and highly lethal GPC3-targeting antibody (XW-GPC3-02); Sexual GPC3 targeting antibody (XW-GPC3-02) was produced and prepared by the GMP workshop of Beijing Yiqiao Shenzhou Technology Co., Ltd.
  • the synthetic gene was purchased from Shanghai Jierui Biological Engineering Co., Ltd.
  • the lentiviral expression plasmid pXW-EF1 ⁇ -MCS-P2A-EGFP was provided by Shanghai Xinwan Biotechnology Co., Ltd., and the packaging plasmid psPAX2 and the envelope plasmid PMD2.G were purchased from Addgene Company.
  • Stable 3 chemically competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.
  • endotoxin-free plasmid mini-prep kit and endotoxin-free plasmid mid-prep kit were purchased from OMEGA Company and Macherey Nagel Company, respectively.
  • the luciferase substrate was purchased from Promega Biotechnology Co., Ltd.
  • HEK293T cells and HepG2 liver cancer cells were purchased from ATCC in the United States.
  • HepG2-luc liver cancer cells were purchased from Shanghai Xinwan Biotechnology Co., Ltd.
  • the microplate luminescence detector was purchased from Promega Biotechnology Co., Ltd.
  • IL-21 ELISA kit and IFN- ⁇ ELISA kit were purchased from Shanghai Dakewe Biotechnology Co., Ltd. and Shanghai Unionway Biotechnology Co., Ltd., respectively.
  • CD16.BBz (SEQ ID NO: 9) hrCD16.BBz (SEQ ID NO: 10) and hrCD16.BBz-P2A-IL-21 genes (SEQ ID NO: 8) were synthesized by Shanghai Jierui Bioengineering Co., Ltd., and cloned To the blank lentivirus expression plasmid (pXW-EF1 ⁇ -MCS-P2A-EGFP) to obtain pXW-EF1 ⁇ -CD16.BBz (abbreviated as pXW-CD16.BBz), pXW-EF1 ⁇ -hrCD16.BBz (abbreviated as pXW-hrCD16.BBz ) and pXW-EF1 ⁇ -hrCD16.BBz-P2A-IL-21 (abbreviated as pXW-hrCD16.BBz-IL-21) recombinant lentivirus expression plasmid, the plasmid map is shown in Figure 1.
  • Example 2 Packaging, concentration and titer determination of lentivirus
  • HEK293T cell treatment 24 hours before transfection, HEK293T cells in logarithmic growth phase were collected and seeded in 10 cm cell culture dishes (6-8 ⁇ 10 6 cells) in complete DMEM medium containing 10 mL growth, placed in 37 °C, 5% CO 2 cell culture incubator for 18 to 24 hours, the cell density reached to more than 70 to 90% for transfection.
  • the viral supernatant collected by centrifugation was filtered with a 0.45 ⁇ m filter, 1/3 of the viral supernatant volume was added with Lenti-X lentivirus concentration reagent, inverted and mixed several times, incubated overnight at 4°C, centrifuged at 2000 ⁇ g for 45 min at 4°C, and centrifuged. A white precipitate can be seen at the bottom of the tube, which is the virus. Carefully discard the supernatant, resuspend the white pellet with 1/50-1/100 volume of the original virus supernatant in blank RPMI1640 medium, aliquot and store at -80°C for later use.
  • Jurkat T cells were seeded on a 96-well U-bottom plate at 1 ⁇ 10 5 cells/well, and the collected lentivirus concentrate was diluted 10-fold. Add 100 ⁇ L of virus diluent to the corresponding wells, add protamine sulfate, a pro-infection reagent, and adjust the concentration to 10 ⁇ g/mL, 1000 ⁇ g, centrifuge at 32 °C for 90 min, and replace the culture medium after overnight culture, and continue to culture for 48 hours.
  • the pro-infection reagent protamine sulfate and adjust the working concentration to 10 ⁇ g/mL, 1000 ⁇ g, centrifuge at 32 °C for 90 min, and after overnight culture, replace with fresh T cell growth medium continued to culture.
  • Example 4 The hrCD16 chimeric receptor is efficiently expressed on the surface of primary T cells
  • Example 3 The method described in Example 3 was used to prepare hrCD16 chimeric receptor (pXW-hCD16.BBz) modified T cells and hrCD16 chimeric receptor (pXW-hCD16.BBz-IL-21) modified cells that co-express IL-21 T cells.
  • Example 5 hrCD16 chimeric receptor-modified T cells co-expressing IL-21 secrete high levels of IL-21
  • hrCD16 chimeric receptor-modified T cells and hrCD16 chimeric receptor-modified T cells co-expressing IL-21 were taken and placed in a 12-well plate, a total of 1 mL of TGM medium was placed at 37°C, Culture in a 5% CO 2 cell incubator for 48 hours and collect the supernatant, cryopreserved at -80°C for later use or immediately detect the expression level of IL-21 in the supernatant.
  • HepG2 human hepatoma cells
  • the supernatant was collected and frozen at -80°C for future use or immediate detection. The expression level of IFN- ⁇ in the supernatant.
  • Example 7 Combination of hrCD16 chimeric receptor-modified T cells co-expressing or not expressing IL-21 Antibody targeting GPC3 kills liver cancer cells
  • Tumor cell killing efficiency was assessed by a Luciferase-based cytotoxicity assay.
  • 1 ⁇ 10 4 HepG2-Luc luciferase gene-modified human hepatoma cells
  • 100 ⁇ L of medium per well were seeded on a 96-well flat-bottom black plate with 100 ⁇ L of medium per well and cultured in a 37°C, 5% CO 2 cell incubator 18 to 20 hours.
  • the formula for calculating the cell killing rate is as follows,
  • Cell killing rate (%) (luciferase activity value of untransduced T cell group-luciferase activity value of experimental group)/luciferase activity value of untransduced T cell group ⁇ 100
  • T cells modified with hrCD16 chimeric receptor co-expressing or not expressing IL-21 specifically killed liver cancer cells under the GPC3-targeting antibody.
  • the efficiency of hrCD16 chimeric receptor-modified T cells co-expressing IL-21 in killing tumor cells (0.5 ⁇ g/mL and 2 ⁇ g/mL liver cancer killing rates were 60.5% and 62.7%, respectively) was superior.
  • Liver cancer killing rates were 37% and 40.5% at hrCD16 chimeric receptor-modified T cells alone (0.5 ⁇ g/mL and 2 ⁇ g/mL, respectively) and wild-type CD16 chimeric receptor-modified T cells (0.5 ⁇ g/mL and 2 ⁇ g/mL, respectively).
  • Hepatocellular carcinoma killing rates under /mL were 23% and 25%, respectively), and the difference was statistically significant; when combined with a highly lethal GPC3-targeting antibody, hrCD16 chimeric receptor-modified T cells co-expressing IL-21 killed tumors
  • the cell efficiency (68% and 83.8% liver cancer killing rates at 0.5 ⁇ g/mL and 2 ⁇ g/mL, respectively) was better than that of hrCD16 chimeric receptor-modified T cells (0.5 ⁇ g/mL and 2 ⁇ g/mL liver cancer killing rates, respectively). 52.8% and 66.8%) and wild-type CD16 chimeric receptor-modified T cells (27% and 35% liver cancer killing rates at 0.5 ⁇ g/mL and 2 ⁇ g/mL, respectively), and the difference was statistically significant.

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Abstract

一种共表达IL-21和hrCD16嵌合受体的免疫细胞，其中，所述hrCD16嵌合受体包含：(1)胞外识别域；(2)胞外间隔区；(3)跨膜区；(4)胞内信号传导结构域；任选地，所述嵌合受体还包含(5)一个或多个共刺激信号传导结构域；和/或(6)一个或多个细胞因子受体信号传导结构域。提供了包含所述免疫细胞和肿瘤抗原靶向抗体或病毒抗原靶向抗体的组合；所提供的免疫细胞共表达IL-21，既可以高效杀伤不同类型的肿瘤细胞/病毒感染细胞，又可以作用于旁系免疫细胞，同时启动内源性抗肿瘤/病毒免疫应答。

Description

共表达IL-21和hrCD16嵌合受体的免疫细胞及其应用 技术领域
本发明属于生物治疗领域，涉及一种共表达IL-21和hrCD16嵌合受体的免疫细胞及其在制备用于治疗癌症或病毒感染性疾病的药物中的应用。
背景技术
目前癌症经典的治疗方案如手术、放疗和化疗等在临床应用中还存在着诸多局限性，手术切除只能应用于原发肿瘤病灶而对于多发转移癌灶束手无策；放射治疗多不适用晚期以及放疗不敏感的肿瘤患者；化学药物治疗的肿瘤特异性较差，常伴随多种不良副反应且不适用于化疗耐受肿瘤患者。近年来，一种新型的肿瘤免疫治疗方法逐渐进入人们的视野，即嵌合抗原受体T细胞疗法(Chimeric antigen receptor T cells，CAR-T cells)。多项临床前和临床研究证实CAR-T细胞疗法在血液肿瘤的治疗中效果显著，因此催生了两款针对血液瘤的CAR-T细胞药物，分别是Novartis公司的Kymriah和Kite公司的Yescarta。实体瘤由于其组织结构复杂、免疫抑制性微环境、肿瘤特异靶标缺乏等诸多因素，严重制约了CAR-T细胞的临床疗效。因此，迫切需要开发新型有效的基因工程免疫细胞技术以提升实体瘤对治疗效果。
目前使用的肿瘤抗原多为肿瘤相关抗原，在肿瘤细胞高表达，但在正常组织细胞亦有少量表达，导致CAR-T细胞疗法出现“靶向非肿瘤细胞的毒副效应”(On-target off-tumor)，限制了其在实体瘤治疗的临床应用。一项靶向碳酸酐酶IX(CAIX)的CAR-T临床研究结果显示，CAIX CAR-T细胞治疗不仅不能控制患者肿瘤生长，反而导致患者肝功能异常和胆管炎的发生(Lamers CH,Sleijfer S,Vulto AG,et al.Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX:first clinical experience[J].J Clin Oncol.2006,24(13):e20-e22)。另外一项靶向酪氨酸激酶受体2(ERBB2)的CAR-T细胞治疗更是引起致死性的炎症因子风暴导致患者死亡(Morgan RA,Yang JC,Kitano M,et al.Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2[J].Mol Ther.2010,18:843-851)。因此，有必要开发一种更为安全的免疫细胞治疗技术。
细胞因子作为T细胞活化的第三信号，有助于T细胞增殖、存活和功能的发挥。细胞因子IL-21作为γ链家族的细胞因子成员，由活化的CD4 +T细胞和NKT细胞产生分泌，其受体分布广泛，对于T细胞的增殖、分化和功能具有免疫调节作用。其中，IL-21协同IL-15促进NK细胞的增殖和成熟，IL-21协同IL-15和IL-18可促进NK和T细胞IFN-γ的产生，增强抗感染/肿瘤的活性。此外，IL-21可诱导B细胞产生抗感染/肿瘤的靶向抗体。
然而，现有技术中，对于包含IL-21的CD16嵌合受体并没有详细的研究。因此，当前对包含IL-21的Fc高亲和性、细胞蛋白酶耐受型CD16(High-affinity,protease-resistant CD16,hrCD16)嵌合受体存在需求。
发明简述
针对现有技术的不足，本发明的目的是提供一种共表达IL-21和hrCD16嵌合受体的免疫细胞。本发明提供的hrCD16嵌合受体不仅具有Fc高亲和性和细胞蛋白酶耐受性，而且可以与IL-21一起通过病毒/非病毒递送方式导入多种杀伤性免疫细胞，如T细胞、NK细胞或NKT细胞等免疫细胞，并稳定表达于其上。使用本发明的共表达IL-21和hrCD16嵌合受体的免疫细胞，联合多种肿瘤抗原靶向抗体或病毒抗原靶向抗体，能够广谱杀伤不同类型的肿瘤细胞或经病毒感染的细胞，并且具有更好的抗肿瘤或病毒疗效。本申请的共表达IL-21和hrCD16嵌合受体的免疫细胞联合使用肿瘤抗原靶向抗体或病毒抗原靶向抗体，既可以高效杀伤不同类型的肿瘤细胞或经病毒感染的细胞，又可以作用于旁系免疫细胞，同时启动内源性免疫应答。
本发明通过如下技术方案以实现所述目的：
一方面，本发明提供了一种免疫细胞，其特征所述，所述免疫细胞共表达IL-21和hrCD16嵌合受体；
其中，所述hrCD16嵌合受体包含：
(1)胞外识别域；
(2)胞外间隔区；
(3)跨膜区；和
(4)胞内信号传导结构域；
任选地，所述嵌合受体还包含
(5)一个或多个共刺激信号传导结构域；和/或
(6)一个或多个细胞因子受体信号传导结构域；
其中，所述胞外识别域为特异性结合抗体Fc片段的突变型hrCD16胞外识别域，其包含如SEQ ID NO:1所示的氨基酸序列。
根据本发明所述的免疫细胞，其特征在于，所述的免疫细胞选自T细胞、天然杀伤细胞(Natural killer cells，NK)、固有淋巴细胞((Innate lymphoid cells,ILC)、造血干细胞、胚胎干细胞和多能干细胞；
优选地，所述T细胞选自未经分选纯化的T细胞、分选纯化的T细胞、分选纯化的PD-1 +T细胞、分选纯化的CD137 +T细胞、分选纯化的CD160 +T细胞、分选纯化的纯真T细胞(T naive)、分选纯化的中央记忆型T细胞(T CM)、分选纯化的效应记忆型T细胞(T EM)、分选纯化的效应T细胞(T EMRA)、分选纯化的过渡记忆型T细胞(Transitional Memory T cells，T TM)、分选纯化的组织记忆型T细胞(Tissue residential memory T cells，T RM)和天然杀伤T细胞(Natural killer T cells，NKT)。
根据本发明所述的免疫细胞，其特征在于，所述胞外间隔区包括但不限于以下任何一种或多种：
(1)抗体IgG4的铰链区及其突变体；
(2)抗体IgG4的铰链区及其突变体和CH2区；
(3)抗体IgG4的铰链区及其突变体、CH2区和CH3区；
(4)抗体IgG1的铰链区及其突变体；
(5)抗体IgG1的铰链区及其突变体和CH2区；
(6)抗体IgG1的铰链区及其突变体、CH2区和CH3区；
(7)免疫球蛋白Fc受体的铰链区：FcγRI(CD64)、FcγRII(CD32)、FcγRIII(CD16)、FcαR(CD89)、FcεRI、FcεRII(CD23)；
(8)共刺激分子CD28铰链区、CD137铰链区、CD8α铰链区、CD4铰链区、PD-1铰链区和CTLA-4铰链区；和
(9)以上的任何一种组合。
根据本发明所述的免疫细胞，其特征在于，所述胞外间隔区为CD8铰链区；优选地，所述胞外间隔区包含如SEQ ID NO:2所示的氨基酸序列。
根据本发明所述的免疫细胞，其特征在于，所述跨膜区包括但不限于以下任何一种或多种：T细胞受体复合物的CD3ξ链的跨膜区、CD28跨膜区、免疫球蛋白Fc受体跨膜区、CD4跨膜区、CD8跨膜区、CD16跨膜区、CD137跨膜区、CTLA-4跨膜区、PD-1跨膜区、LAG-3跨膜区、VISTA跨膜区及其组合；
优选地，所述跨膜区为CD8跨膜区；更优选地，所述跨膜区包含如SEQ ID NO:3所示的氨基酸序列。
根据本发明所述的免疫细胞，其特征在于，所述共刺激信号传导结构域 包括但不限于以下任何一种或多种的信号传导结构域：CD2、CD27、CD28、CD30、CD40、CD40L、CD137(4-1BB)、CD134(OX40)、CD278(ICOS)、GITR、TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、TLR11、Dap10、ICAM-1、LFA-1、Lck、TNFRI、TNFRII、TIM-1、TIM-2、TIM-3、TIM-4及其组合。
优选地，所述共刺激信号传导结构域为CD137信号传导结构域；更优选地，所述共刺激信号传导结构域包含如SEQ ID NO:4所示的氨基酸序列。
根据本发明所述的免疫细胞，其特征在于，所述细胞因子受体信号传导结构域包括但不限于以下任何一种或多种信号传导结构域：IL-2R、IL-7R、IL-15R、IL-18R、IL-21R、IL-23R及其组合。
根据本发明所述的免疫细胞，其特征在于，所述胞内信号传导结构域CD3ξ链、FcγRIII、FcεRI、Fc受体的胞内信号传导域、携带免疫受体酪氨酸活化基序(ITAM)的信号传导结构域及其组合；更优选地，其氨基酸序列如SEQ ID NO:5所示。
根据本发明所述的免疫细胞，其特征在于，所述hrCD16嵌合受体由包含如SEQ ID NO:1所示的氨基酸序列的突变型CD16胞外域、人CD8铰链区、人CD8跨膜区、人CD137共刺激信号传导结构域和CD3ξ链信号传导结构域组成；
优选地，所述hrCD16嵌合受体的氨基酸序列如SEQ ID NO:6所示
根据本发明所述的免疫细胞，其特征在于，所述IL-21选自野生型IL-21、截短型IL-21和突变型IL-21及其活性片段；优选地，所述IL-21是分泌型或膜型IL-21；优选地，所述IL-21是组成性表达或诱导性表达；更优选地，所述IL-21具有如SEQ ID NO:7所示的氨基酸序列。
本发明还提供了一种多核苷酸，其特征在于，所述多核苷酸编码所述的免疫细胞中的IL-21和hrCD16嵌合受体；
优选地，所述多核苷酸序列如SEQ ID NO:8所示。
本发明还提供了一种载体，所述载体包含所述多核苷酸。
优选地，所述载体选自质粒、慢病毒载体、腺病毒载体、腺相关病毒载体、逆转录病毒载体、痘病毒载体、疱疹病毒载体、转座子及其组合；更优选地，所述载体为慢病毒载体；
再一方面，本发明提供了包含所述免疫细胞和肿瘤抗原靶向抗体的组合。
优选地，所述肿瘤抗原包括但不限于以下一种或多种：CD19、BCMA、CD20、CD22、CD30、CD33、CD38、CD47、CD70、CD117、CD123、CD133、 CD138、CD147、CD171、NKG2DL、HER2、MUC1、MUC16、CEA、EpCAM、IL-13Rα2、EGFR、EGFRvIII、GD2、DR5、EphA2、FRα、PSCA、PSMA、TARP、cMet、VEGFR2、BCMA、CTLA-4、PD-L1、AFP、GPC3、AXL、ROR1、ROR2、FAP、Mesothelin、DLL3、CLDN18。
更优选地，所述肿瘤抗原包括但不限于以下一种或多种：HER2、EGFR、CD47、GPC3、AXL和FAP。
进一步优选地，所述肿瘤抗原靶向抗体为GPC3肿瘤抗原靶向抗体。
再一方面，本发明提供了包含所述免疫细胞和病毒抗原靶向抗体的组合。
优选地，所述病毒抗原选自以下的一种或多种：人类获得性免疫缺陷病毒HIV-1的gp120、乙型肝炎病毒HBV的表面抗原、流感病毒的血凝素或神经氨酸酶、埃博拉病毒的刺突蛋白、严重急性呼吸系统综合征冠状病毒SARS-CoV的表面刺突蛋白，中东呼吸综合征冠状病毒MERS-CoV的表面刺突蛋白和新型冠状病毒SARS-CoV-2的表面刺突蛋白；
更优选地，所述病毒表面抗原选自新型冠状病毒SARS-CoV-2的表面刺突蛋白。
再另一方面，本发明提供了本发明所述的免疫细胞、免疫细胞和肿瘤抗原靶向抗体或病毒抗原靶向抗体的组合在制备用于治疗肿瘤或病毒感染性疾病的药物中的用途；
优选地，所述肿瘤选自以下一种或多种：淋巴瘤、神经母细胞瘤、肺癌、乳腺癌、食管癌、胃癌、肝癌、子宫颈癌、卵巢癌、肾癌、胰腺癌、鼻咽癌、小肠癌、大肠癌、结直肠癌、膀胱癌、骨癌、前列腺癌、甲状腺癌、脑癌、横纹肌瘤和平滑肌瘤。
优选地，所述病毒感染性疾病选自以下的一种或多种：人类获得性免疫缺陷综合征、乙型肝炎、流感、埃博拉病毒病、严重急性呼吸系统综合征(SARS)、中东呼吸综合征(MERS)和新型冠状病毒肺炎。
再另一方面，本发明提供了治疗肿瘤的方法，所述方法包括给予受试者治疗有效量的免疫细胞，免疫细胞和肿瘤抗原靶向抗体的组合；
优选地，所述肿瘤选自以下一种或多种：淋巴瘤、神经母细胞瘤、肺癌、乳腺癌、食管癌、胃癌、肝癌、子宫颈癌、卵巢癌、肾癌、胰腺癌、鼻咽癌、小肠癌、大肠癌、结直肠癌、膀胱癌、骨癌、前列腺癌、甲状腺癌、脑癌、横纹肌瘤和平滑肌瘤。在优选的实施方案中，本发明提供了治疗GPC3+肿瘤的方法，所述方法包括给予患者本发明所述的免疫细胞和GPC3靶向抗 体的组合。
再另一方面，本发明提供了治疗病毒感染性疾病的方法，所述方法包括给予受试者治疗有效量的免疫细胞，免疫细胞和病毒抗原靶向抗体的组合；
所述病毒感染性疾病选自以下的一种或多种：人类获得性免疫缺陷综合征、乙型肝炎、流感、埃博拉病毒病、严重急性呼吸系统综合征(SARS)、中东呼吸综合征(MERS)和新型冠状病毒肺炎。
在优选的实施方案中，本发明提供了治疗新型冠状病毒肺炎的方法，所述方法包括给予患者本发明所述的免疫细胞和SARS-CoV-2表面刺突蛋白靶向抗体的组合。
再另一方面，本发明提供了本发明的免疫细胞的制备方法，其包括如下步骤：
1)获取免疫细胞中的IL-21和hrCD16嵌合受体的核酸序列；
2)将核酸序列克隆至慢病毒表达载体中，获得hrCD16-IL-21慢病毒表达质粒(pXW-hrCD16.BBz-IL-21)；
3)将表达质粒、骨架质粒和包膜质粒共转染至HEK293T细胞，包装并获得慢病毒颗粒，经离心浓缩后获得慢病毒浓缩液；
4)将慢病毒转导免疫细胞，从而获得共表达IL-21和hrCD16嵌合受体的免疫细胞；
优选地，所述免疫细胞为T细胞。
本发明还提供了一种用于扩增大量共表达IL-21和hrCD16嵌合受体的T细胞的方法，所述方法包括：用所述的载体转染或用如所述的病毒感染T细胞；及额外添加抗人CD3刺激抗体和抗人CD28刺激抗体、表达肿瘤抗原的细胞或重组肿瘤抗原以及肿瘤抗原靶向抗体以刺激所述T细胞，大量增殖以产生大量的共表达IL-21和高亲和、酶切抵抗型hrCD16嵌合受体的T细胞。
与现有技术相比，本发明具有以下优点：
1)本发明提供了一种共表达IL-21和hrCD16嵌合受体的免疫细胞，本发明的hrCD16嵌合受体具有Fc高亲和性和细胞蛋白酶耐受型，其具备更高的抗体Fc片段亲和力，联合肿瘤抗原靶向抗体或病毒抗原靶向抗体使用，可显著增强ADCC作用。
2)本发明的hrCD16嵌合受体同时具备抵抗金属蛋白酶的切割，保证hrCD16高效表达于杀伤细胞上，避免因CD16下调而导致的功能受损。
3)本发明的hrCD16嵌合受体区别于传统的单靶点CAR策略，可灵活切换不同肿瘤抗原或病毒抗原的靶向抗体，从而实现多癌种的广谱免疫治疗 的功效。
4)本发明的的免疫细胞共表达IL-21，既可以高效杀伤不同类型的肿瘤细胞或经病毒感染的细胞，又可以作用于旁系免疫细胞，同时启动内源性免疫应答。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1为重组慢病毒表达质粒图谱。
图2为根据本发明的共表达IL-21和hrCD16嵌合受体的T细胞表达高水平的hrCD16和IL-21。
图3为根据本发明的共表达IL-21和hrCD16嵌合受体的T细胞和不表达IL-21的hrCD16嵌合受体及野生型CD16嵌合受体修饰的T细胞分泌抗肿瘤因子IFN-γ的结果，表明共表达IL-21可以增强hrCD16嵌合受体修饰T细胞分泌抗肿瘤因子IFN-γ能力。
图4为根据本发明的共表达IL-21和hrCD16嵌合受体的T细胞和不表达IL-21的hrCD16嵌合受体及野生型CD16嵌合受体修饰的T细胞联合GPC3靶向抗体杀伤肝癌细胞的结果，表明共表达IL-21可以增强hrCD16嵌合受体修饰T细胞的杀伤活性。
实施发明的最佳方式
以下实施例仅用于说明本发明，但不用来限制本发明的范围。
以下实施例的实验方法，如无特殊说明，均为本领域的常规实验方法。以下实施例中所使用的实验材料，若无特殊说明，均为自常规生化试剂销售公司购买所得，其中：
DMEM培养基、RPMI1640培养基均购自Corning公司，淋巴细胞培养基X-VIVO 15购自Lonza公司。
T细胞生长培养基由基础培养基和细胞因子组成，基础培养基为淋巴细胞培养基X-VIVO 15，细胞因子为终浓度5ng/mL的IL-7，10ng/mL的IL-15和30ng/mL的IL-21。其中，细胞因子IL-7和IL-15购自R&D公司，IL-21则购自近岸蛋白质科技有限公司。。
胎牛血清购自BI公司。
TurboFect转染试剂盒购自Thermo Fisher Scientific公司。
Lenti-X慢病毒浓缩试剂购自Takara公司。
肿瘤靶向抗体肿瘤靶向抗体由上海鑫湾生物科技有限公司制备并提供，包括GPC3靶向抗体(XW-GPC3-01)和高杀伤性GPC3靶向抗体 (XW-GPC3-02)；高杀伤性GPC3靶向抗体(XW-GPC3-02)由北京义翘神州科技有限公司的GMP车间生产制备。
合成基因购自上海捷瑞生物工程有限公司。
慢病毒表达质粒pXW-EF1α-MCS-P2A-EGFP由上海鑫湾生物科技有限公司提供，包装质粒psPAX2和包膜质粒PMD2.G购自Addgene公司。
Stable 3化学感受态细胞购自上海唯地生物技术有限公司。
无内毒素质粒小提试剂盒和无内毒素质粒中提试剂盒分别购自OMEGA公司和Macherey Nagel公司。
荧光素酶底物购自普洛麦格生物技术有限公司。
HEK293T细胞、HepG2肝癌细胞购自美国ATCC。HepG2-luc肝癌细胞购自上海鑫湾生物科技有限公司。
Figure PCTCN2021104765-appb-000001
微孔板发光检测仪购自普洛麦格生物技术有限公司。
IL-21 ELISA kit和IFN-γELISA kit分别购自上海达科为生物技术有限公司和上海优宁维生物科技有限公司。
实施例1慢病毒表达质粒的构建
由上海捷瑞生物工程有限公司合成CD16.BBz(SEQ ID NO:9)hrCD16.BBz(SEQ ID NO:10)和hrCD16.BBz-P2A-IL-21基因(SEQ ID NO:8)，并克隆至空白慢病毒表达质粒(pXW-EF1α-MCS-P2A-EGFP)获得pXW-EF1α-CD16.BBz(简写为pXW-CD16.BBz)、pXW-EF1α-hrCD16.BBz(简写为pXW-hrCD16.BBz)和pXW-EF1α-hrCD16.BBz-P2A-IL-21(简写为pXW-hrCD16.BBz-IL-21)重组慢病毒表达质粒，质粒图谱如图1所示。
实施例2慢病毒的包装、浓缩和滴度测定
1.1慢病毒的包装
HEK293T细胞处理：转染前24小时，收集处于对数生长期的HEK293T细胞，将其接种于10cm细胞培养皿中(6～8×10 6个细胞)，细胞在含有10mL的完全DMEM培养基中生长，置于37℃，5％CO 2细胞培养箱中培养18～24小时，细胞密度达到70～90％以上即可进行转染。
HEK293T细胞转染：在15mL离心管中加入1mL基础DMEM培养基，按照质量比为慢病毒表达质粒慢病毒表达质粒(pXW-CD16.BBz、pXW-hrCD16.BBz或pXW-hrCD16.BBz-IL-21):包装质粒psPAX2:包膜质粒PMD2.G＝1:3:1配制转染混合液，质粒总量合计15μg/皿。以质粒量(μg)：转染试剂(μL)＝1：2的比例加入TurboFect转染试剂30μL，室温孵育15～20min后加至细胞培养皿中，置于37℃，5％CO 2细胞培养箱中培养48小时 并收集病毒上清，1000×g，4℃离心10min，收集上清病毒。
1.2慢病毒的浓缩
离心收集的病毒上清采用0.45μm滤器过滤，加入1/3病毒上清体积的Lenti-X慢病毒浓缩试剂，颠倒混匀数次，4℃孵育过夜，2000×g，4℃离心45min，离心管底部可见白色沉淀，即为病毒。小心弃除上清，以原病毒上清的1/50～1/100体积的空白RPMI1640培养基重悬白色沉淀，分装并于-80℃冻存备用。
1.3慢病毒滴度测定
将Jurkat T细胞按照1×10 5个/孔接种于96孔U底板上，将所收集的慢病毒浓缩液按10倍递增稀释。将100μL的病毒稀释液加入到相应孔中，加入促感染试剂硫酸鱼精蛋白并调整浓度至10μg/mL，1000×g，32℃离心感染90min，过夜培养后更换培养液，继续培养48小时，流式细胞仪检测荧光阳性细胞比例，采用下面的公式计算病毒滴度：病毒滴度(TU/mL)＝1×10 5×荧光阳性细胞比例/100×1000×相应的稀释倍数。
实施例3 T细胞的感染和扩增
分别将实施例2包装、浓缩获得的慢病毒载体(LV-XW-hrCD16.BBz或LV-XW-hrCD16.BBz-IL-21)(MOI＝5～10)加至铺有1×10 6个预先活化的外周血单个核细胞的48孔平底板中，添加促感染试剂硫酸鱼精蛋白并将工作浓度调至10μg/mL，1000×g，32℃离心感染90min，过夜培养后，更换新鲜的T细胞生长培养基继续培养。每2～3天添加新鲜的T细胞生长培养基，并调整细胞密度至0.5～2×10 6个细胞。自感染后6～7天，移除活化T细胞的免疫磁珠，继续培养扩增hrCD16嵌合受体和/或IL-21修饰的T细胞，待细胞静息后方可进行后续的功能实验。
实施例4 hrCD16嵌合受体有效地表达在原代T细胞表面
采用实施例3所述的方法制备hrCD16嵌合受体(pXW-hCD16.BBz)修饰的T细胞和共表达IL-21的hrCD16嵌合受体(pXW-hCD16.BBz-IL-21)修饰的T细胞。分别取1×10 5个下列三种细胞置于1.5mL EP管中：未转导T细胞、hrCD16嵌合受体修饰的T细胞、以及hrCD16-IL-21修饰的T细胞，加入0.5μL流式抗体PerCP/Cy5.5-anti-human CD16，室温避光孵育20min，结束后用FACS缓冲液(含有2％FBS的1×PBS)洗脱2遍，200～300μL FACS缓冲液重悬后使用流式细胞仪检测。
结果如图2A所示：流式分析hrCD16嵌合受体在原代T细胞上表达，两人份的hrCD16嵌合受体和/或共表达IL-21修饰的T细胞均可以正常表达 hrCD16，两者阳性率相当，且CD4 +T细胞亚群比例相似。另外，未转导T细胞不表达hrCD16嵌合受体。
实施例5共表达IL-21的hrCD16嵌合受体修饰T细胞分泌高水平的 IL-21
分别取1×10 6个hrCD16嵌合受体修饰的T细胞与共表达IL-21的hrCD16嵌合受体修饰的T细胞置于12孔板中，合计1mL的TGM培养基，置于37℃，5％CO 2细胞培养箱中培养48小时并收集上清，-80℃冻存备用或即时检测上清IL-21的表达水平。
结果如图2B所示，共表达IL-21的hrCD16嵌合受体修饰T细胞可分泌高水平的IL-21，而hrCD16嵌合受体及野生型CD16嵌合受体修饰T细胞培养上清未检测到IL-21的分泌。
实施例6共表达或未表达IL-21的hrCD16嵌合受体修饰的T细胞的活 化
首先，将3×10 4个HepG2(人肝癌细胞)接种于96孔平底板上，每孔100μL培养基，置于37℃，5％CO 2细胞培养箱中培养18～20小时。第二天，以效应细胞：靶细胞＝3:1的比例加入共表达或为表达IL-21的hrCD16嵌合受体修饰的T细胞至含有靶细胞的孔中，并加入GPC3靶向抗体，抗体工作浓度调整为0.5、1和2μg/mL，置于37℃，5％CO 2细胞培养箱中继续培养18～20小时，共培养结束后收集上清，-80℃冻存备用或即时检测上清IFN-γ的表达水平。
结果图3所示，三种工程化T细胞均在GPC3靶向抗体的下被肝癌细胞活化并产生抗肿瘤因子IFN-γ。其中，共表达IL-21的hrCD16嵌合受体修饰T细胞可以分泌更高水平的IFN-γ，并且差异具有统计学意义。
实施例7共表达或未表达IL-21的hrCD16嵌合受体修饰的T细胞联合 GPC3靶向抗体杀伤肝癌细胞
肿瘤细胞杀伤效率由基于荧光素酶的细胞杀伤检测方法(Luciferase-based cytotoxicity assay)进行评估。首先，将1×10 4个HepG2-Luc(荧光素酶基因修饰的人肝癌细胞)接种于96孔平底黑板上，每孔100μL培养基，置于37℃，5％CO 2细胞培养箱中培养18～20小时。第二天，以效应细胞：靶细胞＝3:1的比例加入hrCD16嵌合受体修饰T细胞至含有靶细胞的孔中，并加入GPC3靶向抗体，抗体工作浓度调整为0.5和2μg/mL，置于37℃，5％CO 2细胞培养箱中继续培养18～20小时，共培养结束后使用
Figure PCTCN2021104765-appb-000002
微孔板发光检测仪检测靶细胞的荧光素酶活力值。
细胞杀伤率的计算公式如下所示，
细胞杀伤率(％)＝(未转导T细胞组荧光素酶活力值-实验组荧光素酶活力值)/未转导T细胞组荧光素酶活力值×100
结果如图4所示，共表达或未表达IL-21的hrCD16嵌合受体修饰的T细胞均在GPC3靶向抗体下特异性杀伤肝癌细胞。联用GPC3靶向抗体下，共表达IL-21的hrCD16嵌合受体修饰T细胞杀伤肿瘤细胞的效率(0.5μg/mL和2μg/mL下的肝癌杀伤率分别为60.5％和62.7％)优于单独hrCD16嵌合受体修饰T细胞(0.5μg/mL和2μg/mL下的肝癌杀伤率分别为37％和40.5％)及野生型CD16嵌合受体修饰T细胞(0.5μg/mL和2μg/mL下的肝癌杀伤率分别为23％和25％)，并且差异具有统计学意义；联用高杀性GPC3靶向抗体下，共表达IL-21的hrCD16嵌合受体修饰T细胞杀伤肿瘤细胞的效率(0.5μg/mL和2μg/mL下的肝癌杀伤率分别为68％和83.8％)优于hrCD16嵌合受体修饰T细胞(0.5μg/mL和2μg/mL下的肝癌杀伤率分别为52.8％和66.8％)及野生型CD16嵌合受体修饰T细胞(0.5μg/mL和2μg/mL下的肝癌杀伤率分别为27％和35％)，并且差异具有统计学意义。
虽然以上仅描述了本发明的具体实施方式的范例，但本领域的技术人员应当理解，以上这些仅为举例说明，本发明的保护范围是由所附权利要求书限定的。本领域的技术人员在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改，但这些变更或修改均应落入本发明的保护范围。

Claims (11)

  1. 一种免疫细胞，其特征在于，所述免疫细胞共表达IL-21和hrCD16嵌合受体；
    其中，所述hrCD16嵌合受体包含：
    (1)胞外识别域；
    (2)胞外间隔区；
    (3)跨膜区；和
    (4)胞内信号传导结构域；
    任选地，所述嵌合受体还包含
    (5)一个或多个共刺激信号传导结构域；和/或
    (6)一个或多个细胞因子受体信号传导结构域；
    其中，所述胞外识别域为特异性结合抗体Fc片段的突变型hrCD16胞外识别域，其包含如SEQ ID NO:1所示的氨基酸序列。
  2. 如权利要求1所述的免疫细胞，其特征在于，所述的免疫细胞选自T细胞、天然杀伤细胞(Natural killer cells，NK)、固有淋巴细胞((Innate lymphoid cells,ILC)、造血干细胞、胚胎干细胞和多能干细胞；
    优选地，所述T细胞选自未经分选纯化的T细胞、分选纯化的T细胞、分选纯化的PD-1 +T细胞、分选纯化的CD137 +T细胞、分选纯化的CD160 +T细胞、分选纯化的纯真T细胞(T naive)、分选纯化的中央记忆型T细胞(T CM)、分选纯化的效应记忆型T细胞(T EM)、分选纯化的效应T细胞(T EMRA)、分选纯化的过渡记忆型T细胞(Transitional Memory T cells，T TM)、分选纯化的组织记忆型T细胞(Tissue residential memory T cells，T RM)和天然杀伤T细胞(Natural killer T cells，NKT)。
  3. 如权利要求1所述的免疫细胞，其特征在于，所述胞外间隔区选自以下任何一种或多种：
    (1)抗体IgG4的铰链区及其突变体；
    (2)抗体IgG4的铰链区及其突变体和CH2区；
    (3)抗体IgG4的铰链区及其突变体、CH2区和CH3区；
    (4)抗体IgG1的铰链区及其突变体；
    (5)抗体IgG1的铰链区及其突变体和CH2区；
    (6)抗体IgG1的铰链区及其突变体、CH2区和CH3区；
    (7)免疫球蛋白Fc受体的铰链区：FcγRI(CD64)、FcγRII(CD32)、 FcγRIII(CD16)、FcαR(CD89)、FcεRI、FcεRII(CD23)；
    (8)共刺激分子CD28铰链区、CD137铰链区、CD8α铰链区、CD4铰链区、PD-1铰链区和CTLA-4铰链区；和
    (9)以上任何的一种组合；
    优选地，所述胞外间隔区为CD8铰链区；更优选地，所述胞外间隔区包含如SEQ ID NO:2所示的氨基酸序列；
    优选地，所述跨膜区选自以下任何一种或多种：T细胞受体复合物的CD3ξ链的跨膜区、CD28跨膜区、免疫球蛋白Fc受体跨膜区、CD4跨膜区、CD8跨膜区、CD16跨膜区、CD137跨膜区、CTLA-4跨膜区、PD-1跨膜区、LAG-3跨膜区、VISTA跨膜区及其组合；更优选地，所述跨膜区为CD8跨膜区；进一步优选地，所述跨膜区包含如SEQ ID NO:3所示的氨基酸序列；
    优选地，所述共刺激信号传导结构域选自以下任何一种或多种的信号传导结构域：CD2、CD27、CD28、CD30、CD40、CD40L、CD137(4-1BB)、CD134(OX40)、CD278(ICOS)、GITR、TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、TLR11、Dap10、ICAM-1、LFA-1、Lck、TNFRI、TNFRII、TIM-1、TIM-2、TIM-3、TIM-4及其组合；更优选地，所述共刺激信号传导结构域为CD137信号传导结构域；进一步优选地，所述共刺激信号传导结构域包含如SEQ ID NO:4所示的氨基酸序列；
    优选地，所述细胞因子受体信号传导结构域选自以下任何一种或多种信号传导结构域：IL-2R、IL-7R、IL-15R、IL-18R、IL-21R、IL-23R及其组合；
    优选地，所述胞内信号传导结构域选自CD3ξ链、FcγRIII、FcεRI、Fc受体的胞内信号传导域、携带免疫受体酪氨酸活化基序(ITAM)的信号传导结构域及其组合；更优选地，其氨基酸序列如SEQ ID NO:5所示；
    优选地，所述hrCD16嵌合受体由包含如SEQ ID NO:1所示的氨基酸序列的突变型CD16胞外域、人CD8铰链区、人CD8跨膜区、人CD137共刺激信号传导结构域和CD3ξ链信号传导结构域组成；
    优选地，所述hrCD16嵌合受体的氨基酸序列如SEQ ID NO:6所示。
  4. 如权利要求1所述的免疫细胞，其特征在于，所述IL-21选自野生型IL-21、截短型IL-21和突变型IL-21及其活性片段；
    优选地，所述IL-21是分泌型或膜型IL-21；
    优选地，所述IL-21是组成性表达或诱导性表达；
    更优选地，所述IL-21具有如SEQ ID NO:7所示的氨基酸序列。
  5. 一种多核苷酸，其特征在于，所述多核苷酸编码如权利要求1至4中任一项所述的免疫细胞中的IL-21和hrCD16嵌合受体；
    优选地，所述多核苷酸包含如SEQ ID NO:8所示的核苷酸序列。
  6. 一种载体，所述载体包含如权利要求5所述的多核苷酸；
    优选地，所述载体选自质粒、慢病毒载体、腺病毒载体、腺相关病毒载体、逆转录病毒载体、痘病毒载体、疱疹病毒载体、转座子及其组合；
    更优选地，所述载体为慢病毒载体。
  7. 一种组合，所述组合包含如权利要求1-4中任一项所述的免疫细胞和肿瘤抗原靶向抗体或病毒抗原靶向抗体；
    优选地，所述肿瘤抗原选自以下一种或多种：CD19、BCMA、CD20、CD22、CD30、CD33、CD38、CD47、CD70、CD117、CD123、CD133、CD138、CD147、CD171、NKG2DL、HER2、MUC1、MUC16、CEA、EpCAM、IL-13Rα2、EGFR、EGFRvIII、GD2、DR5、EphA2、FRα、PSCA、PSMA、TARP、cMet、VEGFR2、BCMA、CTLA-4、PD-L1、AFP、GPC3、AXL、ROR1、ROR2、FAP、Mesothelin、DLL3和CLDN18；
    更优选地，所述肿瘤抗原选自以下一种或多种：HER2、EGFR、CD47、GPC3、AXL和FAP；
    进一步优选地，所述肿瘤抗原靶向抗体为GPC3肿瘤抗原靶向抗体；
    优选地，所述病毒抗原选自以下的一种或多种：人类获得性免疫缺陷病毒HIV-1的gp120、乙型肝炎病毒HBV的表面抗原、流感病毒的血凝素或神经氨酸酶、埃博拉病毒的刺突蛋白、严重急性呼吸系统综合征冠状病毒SARS-CoV的表面刺突蛋白，中东呼吸综合征冠状病毒MERS-CoV的表面刺突蛋白和新型冠状病毒SARS-CoV-2的表面刺突蛋白；
    更优选地，所述病毒抗原选自新型冠状病毒SARS-CoV-2的表面刺突蛋白。
  8. 如权利要求1-4中任一项所述的免疫细胞，如权利要求7所述的免疫细胞和肿瘤抗原靶向抗体或病毒抗原靶向抗体的组合在制备用于治疗肿瘤或病毒感染性疾病的药物中的用途；
    优选地，所述肿瘤选自以下一种或多种：淋巴瘤、神经母细胞瘤、肺癌、乳腺癌、食管癌、胃癌、肝癌、子宫颈癌、卵巢癌、肾癌、胰腺癌、鼻咽癌、小肠癌、大肠癌、结直肠癌、膀胱癌、骨癌、前列腺癌、甲状腺癌、脑癌、横纹肌瘤和平滑肌瘤；
    优选地，所述病毒感染性疾病选自以下的一种或多种：人类获得性免疫缺陷综合征、乙型肝炎、流感、埃博拉病毒病、严重急性呼吸系统综合征(SARS)、中东呼吸综合征(MERS)和新型冠状病毒肺炎。
  9. 一种治疗肿瘤的方法，所述方法包括给予受试者治疗有效量的如权利要求1-4中任一项所述的免疫细胞，如权利要求7所述的免疫细胞和肿瘤抗原靶向抗体的组合；
    优选地，所述肿瘤选自以下一种或多种：淋巴瘤、神经母细胞瘤、肺癌、乳腺癌、食管癌、胃癌、肝癌、子宫颈癌、卵巢癌、肾癌、胰腺癌、鼻咽癌、小肠癌、大肠癌、结直肠癌、膀胱癌、骨癌、前列腺癌、甲状腺癌、脑癌、横纹肌瘤和平滑肌瘤；
    优选地，所述肿瘤为GPC3+肿瘤，所述方法包括给予患者如权利要求1-4中任一项所述的免疫细胞和GPC3靶向抗体的组合。
  10. 一种治疗病毒感染性疾病的方法，所述方法包括给予受试者治疗有效量的如权利要求1-4中任一项所述的免疫细胞，如权利要求7所述的免疫细胞和病毒抗原靶向抗体的组合；
    所述病毒感染性疾病选自以下的一种或多种：人类获得性免疫缺陷综合征、乙型肝炎、流感、埃博拉病毒病、严重急性呼吸系统综合征、中东呼吸综合征和新型冠状病毒肺炎；
    优选地，所述疾病为新型冠状病毒肺炎，所述方法包括给予患者如权利要求1-4中任一项所述的免疫细胞和SARS-CoV-2表面刺突蛋白靶向抗体的组合。
  11. 如权利要求1-4中任一项所述的免疫细胞的制备方法，其包括如下步骤：
    1)获取如权利要求1-4中任一项所述的免疫细胞中的IL-21和hrCD16嵌合受体的核酸序列；
    2)将IL-21和hrCD16嵌合受体的核酸序列克隆至慢病毒表达载体中，获得hrCD16-IL-21慢病毒表达质粒；
    3)将表达质粒、骨架质粒和包膜质粒共转染至HEK293T细胞，包装并获得慢病毒颗粒，经离心浓缩后获得慢病毒浓缩液；
    4)将慢病毒转导免疫细胞，从而获得共表达IL-21和hrCD16嵌合受体的免疫细胞；优选地，所述免疫细胞为T细胞。
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