WO2022120934A1 - 一种编码CAR基因的mRNA、组合mRNA、构建方法、CAR-T细胞和方法 - Google Patents

一种编码CAR基因的mRNA、组合mRNA、构建方法、CAR-T细胞和方法 Download PDF

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WO2022120934A1
WO2022120934A1 PCT/CN2020/137996 CN2020137996W WO2022120934A1 WO 2022120934 A1 WO2022120934 A1 WO 2022120934A1 CN 2020137996 W CN2020137996 W CN 2020137996W WO 2022120934 A1 WO2022120934 A1 WO 2022120934A1
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car
mrna
cells
seq
nucleotide sequence
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French (fr)
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胡勇
张苗苗
洪丹
胡迅
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深圳市瑞吉生物科技有限公司
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    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001136—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
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    • C07—ORGANIC CHEMISTRY
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    • C12N2510/00—Genetically modified cells

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  • the invention belongs to the technical field of genetic engineering, and in particular relates to an mRNA encoding a CAR gene, a combined mRNA, a construction method, a CAR-T cell and a method.
  • Biological therapy as a brand new treatment concept that has just emerged, has unique advantages including selectively targeting and killing tumor cells without toxic effects on normal cells.
  • Biological therapy in the treatment of cancer can significantly improve the clinical symptoms of patients, improve the quality of life, and prolong the survival period.
  • Biological therapy in the treatment of cancer can activate the immune function of the body and is expected to eradicate tumor cells. Biological therapy can enhance the success rate of surgery and will be widely accepted by cancer patients.
  • CAR-T therapy is a chimeric antigen receptor-based cellular immunotherapy regimen. It uses in vitro gene transfer technology to transfer the gene sequence encoding chimeric antigen receptor (CAR) into T cells to generate tumor-specific T cells that can bind to target antigens.
  • CAR-T therapy in the treatment of malignant hematological tumors are obvious to all, such as Kymriah for refractory/relapsed acute B lymphoblastic leukemia and refractory/relapsed non-Hodgkin's leukemia.
  • the present invention provides an mRNA encoding a CAR gene, a combined mRNA, a construction method, a CAR-T cell and a method, and a CAR-T cell constructed by using the mRNA encoding the CAR gene provided by the present invention and a combination of mRNAs, the method It is simple, can express CAR gene, and can kill tumor cells.
  • the present invention provides an mRNA encoding a CAR gene, and the nucleotide sequence of the mRNA is selected from any one of SEQ ID No. 1 to 4.
  • the present invention also provides a combined mRNA for preventing and treating tumors, including the mRNA encoding CAR gene and the mRNA encoding cytokines described in the above technical solution; the cytokines are IL-2 cytokine, IL-10 cytokine, IL12A One or more of cytokines, IL12B cytokines and CCL-19 cytokines.
  • the nucleotide sequence encoding the IL-2 cytokine is shown in SEQ ID No. 5
  • the nucleotide sequence encoding the IL-10 cytokine is shown in SEQ ID No. 6
  • the nucleotide sequence encoding the CCL- 19 The nucleotide sequence of cytokine is shown as SEQ ID No.9
  • the nucleotide sequence of IL12A cytokine is shown as SEQ ID No.7
  • the nucleotide sequence of IL12B cytokine is shown as SEQ ID No.8 shown.
  • nucleotide sequence of the mRNA encoding the CAR gene is shown in SEQ ID No.4, and the nucleotide sequence of the mRNA encoding the cytokine is shown in SEQ ID No.5.
  • the nucleotide sequence of the mRNA encoding the CAR gene is shown in SEQ ID No.4, and the nucleotide sequence of the mRNA encoding the cytokine is shown in SEQ ID No.6.
  • the nucleotide sequence of the mRNA encoding the CAR gene is shown in SEQ ID No.4, and the nucleotide sequence of the mRNA encoding the cytokine is selected from the group consisting of SEQ ID No.7 and SEQ ID No.8. kind.
  • nucleotide sequence of the mRNA encoding the CAR gene is shown in SEQ ID No.4, and the nucleotide sequence of the mRNA encoding the cytokine is shown in SEQ ID No.9.
  • the present invention also provides a method for constructing a CAR-T cell, comprising the following steps:
  • the mRNA encoding the CAR gene described in the above technical scheme or the combined mRNA described in the above technical scheme is transfected into cells to obtain CAR-T cells.
  • the present invention also provides a CAR-T cell constructed based on the construction method described in the above technical solution.
  • the present invention also provides the application of the CAR-T cell described in the above technical solution in the preparation of a medicine for preventing and treating tumors.
  • the tumor includes one or more of human Hodgkin's lymphoma, peripheral T-cell lymphoma, diffuse large B-cell lymphoma, lymphoblastic lymphoma and anaplastic large cell lymphoma.
  • the present invention also provides the method for preventing and treating tumors with CAR-T cells according to the above technical solution, comprising the following steps: injecting the CAR-T cells into the spleen of a patient, and the injection dose of the CAR-T cells is 1 mg/50kg Body weight, injected every 2 weeks for a total of 3 injections.
  • the tumor includes one or more of human Hodgkin's lymphoma, peripheral T-cell lymphoma, diffuse large B-cell lymphoma, lymphoblastic lymphoma and anaplastic large cell lymphoma.
  • the present invention provides an mRNA encoding a CAR gene, a combined mRNA, a construction method, a CAR-T cell and an application, wherein the nucleotide sequence of the mRNA is selected from any one of SEQ ID No. 1 to 4.
  • the present invention uses the mRNA or combined mRNA encoding the CAR gene as the target antigen, which is not only convenient for storage and transportation, but also can transform T cells into CAR-T cells in situ. Compared with traditional in vitro CAR-T preparation, the process is greatly simplified. Costs have dropped significantly.
  • the T cells After being transferred into the patient's T cells, the T cells are activated, and the positioning and navigation device CAR (tumor chimeric antigen receptor) is installed to transform the ordinary "warrior" of T cells.
  • CAR tumor chimeric antigen receptor
  • the results of the examples of the present invention show that the CAR-T cells constructed by using the mRNA encoding the CAR gene and the combined mRNA provided by the present invention can express the CAR gene and have a killing effect on tumors.
  • Figure 1 shows the expression rate of CAR in CAR-T cells
  • Figure 2 shows the results of CAR-T cells killing tumor cells
  • FIG. 3 shows the results of exocytosis of IFN ⁇ after CAR-T cells are activated by target cells
  • Figure 4 shows the results of IFN ⁇ secretion by CD4 and CD8 cells after CAR-T cells were activated by target cells.
  • Figure 5 shows the results of IFN ⁇ secretion by CD4 and CD8 cells after CAR-T cells are activated by target cells.
  • Figure 6 shows the results of IFN ⁇ secretion by CD4 and CD8 cells after CAR-T cells were activated by target cells.
  • Figure 7 shows the results of in situ construction of CAR-T cells after injection of CAR gene mRNA.
  • Figure 8 shows the results of CAR-T cells killing target cells in vivo.
  • the present invention provides an mRNA encoding a CAR gene, and the nucleotide sequence of the mRNA is selected from any one of SEQ ID No. 1 to 4.
  • the present invention also provides a combined mRNA for preventing and treating tumors, including the mRNA encoding CAR gene and the mRNA encoding cytokines described in the above technical solution; the cytokines are IL-2 cytokine, IL-10 cytokine, IL12A One or more of cytokines, IL12B cytokines and CCL-19 cytokines.
  • the mRNA whose nucleotide sequence is shown in SEQ ID No.5 encodes IL-2 cytokine
  • the mRNA whose nucleotide sequence is shown in SEQ ID No.6 encodes IL-10 cytokine
  • the nucleotide sequence is shown in SEQ ID No.6.
  • the mRNA whose sequence is shown in SEQ ID No.7 encodes IL12A cytokine
  • the mRNA whose nucleotide sequence is shown in SEQ ID No.8 encodes IL12B cytokine
  • the mRNA whose nucleotide sequence is shown in SEQ ID No.9 encodes CCL-19 cytokine.
  • the mRNA combination comprises the mRNA encoding the CAR gene and the mRNA encoding the cytokine, and the nucleotide sequence of the mRNA encoding the CAR gene is preferably as shown in SEQ ID No. 4, and the cytokine encoding The nucleotide sequence of the mRNA is preferably as shown in SEQ ID No.5.
  • the mRNA combination comprises the mRNA encoding the CAR gene and the mRNA encoding the cytokine, and the nucleotide sequence of the mRNA encoding the CAR gene is preferably as shown in SEQ ID No. 4, and the cytokine encoding The nucleotide sequence of the mRNA is preferably as shown in SEQ ID No.6.
  • the mRNA combination comprises the mRNA encoding the CAR gene and the mRNA encoding the cytokine
  • the nucleotide sequence of the mRNA encoding the CAR gene is preferably as shown in SEQ ID No. 4, and the cytokine encoding
  • the nucleotide sequence of the mRNA is preferably selected from SEQ ID No.7 and SEQ ID No.8.
  • SEQ ID No. 7 and SEQ ID No. 8 encode the A chain and B chain of IL12, respectively, and they can only work together when they are combined, so they are essentially a combination of a CAR gene and a cytokine gene.
  • the mRNA combination comprises the mRNA encoding the CAR gene and the mRNA encoding the cytokine, and the nucleotide sequence of the mRNA encoding the CAR gene is preferably as shown in SEQ ID No. 4, and the cytokine encoding The nucleotide sequence of the mRNA is preferably as shown in SEQ ID No.9.
  • the cytokine mRNA provided by the present invention is expressed after transfecting cells.
  • CAR-T cells can enhance the proliferation of T cells, which can generate unrestricted activation of MHC and further induce the secretion of macrophages.
  • the secretion of cytokines also improves the immune microenvironment and upregulates the antigen-presenting ability of DC cells and macrophages to improve local tumor immunity.
  • the present invention also provides a method for constructing CAR-T cells, comprising the following steps: transfecting the mRNA encoding the CAR gene described in the above technical solution or the mRNA combination described in the above technical solution into T cells to obtain CAR-T cells .
  • the cells are preferably peripheral blood mononuclear cells, H9 (human T lymphocyte line) or jurkaT cells.
  • the present invention also provides a CAR-T cell constructed based on the construction method described in the above technical solution.
  • the present invention also provides the application of the CAR-T cell described in the above technical solution in the preparation of a medicine for preventing and treating tumors.
  • the present invention does not specifically limit the dosage form of the drug, and a medically acceptable dosage form of CAR-T cells may be used.
  • the tumor preferably includes one or more of human Hodgkin's lymphoma, peripheral T-cell lymphoma, diffuse large B-cell lymphoma, lymphoblastic lymphoma and anaplastic large cell lymphoma
  • the injection method is preferably saline solution, intravenous injection.
  • the present invention also provides the method for preventing and treating tumors with CAR-T cells according to the above technical solution, comprising the following steps: injecting the CAR-T cells into the spleen of a patient, and the injection dose of the CAR-T cells is 1 mg.
  • the tumor preferably includes one or more of human Hodgkin's lymphoma, peripheral T-cell lymphoma, diffuse large B-cell lymphoma, lymphoblastic lymphoma and anaplastic large cell lymphoma .
  • PBMC cells were isolated from the peripheral blood of healthy donors and cultured in AIM-V medium supplemented with IL2 (50U/ML). 5. seq4+6, seq4+7+8 and seq4+9 were respectively transfected into cells. After the cell culture was completed, the samples were taken and dispensed into sampling tubes for quality control testing.
  • the mRNA encoding the CAR gene the nucleotide sequence is shown in SEQ ID No.1, referred to as seq1;
  • the mRNA encoding the CAR gene the nucleotide sequence is shown in SEQ ID No.2, referred to as seq2KY;
  • the mRNA encoding the CAR gene the nucleotide sequence is shown in SEQ ID No.3, abbreviated as seq3YE;
  • the mRNA encoding the CAR gene the nucleotide sequence is shown in SEQ ID No.4, referred to as seq4;
  • nucleotide sequence of mRNA encoding CAR gene is shown in SEQ ID No. 4, and the nucleotide sequence of mRNA encoding cytokine is shown in SEQ ID No.5, referred to as seq4+5;
  • nucleotide sequence of mRNA encoding CAR gene is shown in SEQ ID No. 4
  • nucleotide sequence of mRNA encoding cytokine is shown in SEQ ID No.6, hereinafter referred to as 4+6;
  • nucleotide sequence of mRNA encoding CAR gene is shown in SEQ ID No. 4, and the nucleotide sequence of mRNA encoding cytokine is selected from SEQ ID No. 4 ID No.7 and 8, hereinafter referred to as 4+7+8;
  • nucleotide sequence of mRNA encoding CAR gene is shown in SEQ ID No. 4
  • nucleotide sequence of mRNA encoding cytokine is shown in SEQ ID No. 9, hereinafter referred to as 4+9.
  • PBMC cells Preparation of PBMC cells: Determine the passage density and volume according to the use time, and pass them in a cell culture flask to ensure that the cells are in the logarithmic growth phase when used.
  • Counting of cell digestion take the cells in good growth state, remove the medium, wash the cells with 10ml PBS, add an appropriate amount of 0.25% trypsin (1ml 0.25% trypsin for T75 flask, 3ml 0.25% trypsin for T175 flask) to digest for 5 minutes , then add DMEM medium containing 10% FBS (9ml medium for T75 bottle, 17ml medium for T175 bottle) to neutralize trypsin, pipette cells and transfer to 50ml centrifuge tube, mix by pipetting repeatedly, and then take about 0.3ml The cell suspension was appropriately diluted and counted.
  • Cell dilution Take an appropriate amount of cell suspension, dilute to 5 ⁇ 10 5 cells/ml with AIM-V medium containing 10% FBS, and mix by pipetting.
  • Cell seeding Take 2ml of cell suspension and add it to a 6-well plate. Each sample needs to prepare 2 wells of parallel cells, the control sample needs to prepare 1 well of cells, and the blank control needs to prepare 1 well. Place the 6-well plate in a 37 ⁇ 1°C, 5 ⁇ 0.5% CO 2 incubator overnight.
  • Preparation of single cell suspension take the cells in good growth state, remove the medium, wash the cells with 10ml PBS, add an appropriate amount of 0.25% trypsin for 5 minutes, and then add DMEM medium containing 10% FBS to neutralize the trypsin , the cells were pipetted and transferred to a 50ml centrifuge tube, mixed by repeated pipetting, and then the cell suspension was diluted and counted.
  • Fixation Cells were collected by centrifugation and the supernatant was removed by aspiration. Resuspend cells in 0.5-1 ml of 1x PBS. Formaldehyde was added to a final concentration of 4%. Fix for 15 min at room temperature. Wash with enough 1x PBS by centrifugation. Discard the supernatant in a suitable waste container. Resuspend cells in 0.5-1 ml of 1x PBS.
  • Immunostaining Resuspend cells in 100 ⁇ l of diluted primary antibody (1:2000). Incubate for 1 hour at room temperature. Wash with incubation buffer by centrifugation. Discard the supernatant. Resuspend cells in 100 ⁇ l of diluted fluorochrome-conjugated secondary antibody (1:5000). Incubate for 30 minutes at room temperature. On-board detection.
  • the prepared CAR-T cells were tested for CAR expression rate by flow cytometry, and the results are shown in Figure 1 and Table 1.
  • the first step CAR-T cell preparation
  • the human lymphoma cell line Daudi was resuspended in complete medium to a density of 1 ⁇ 10 6 /mL;
  • T cells After transfection of CAR gene, T cells released immune factors under the stimulation of target cells.
  • the CAR-T cells prepared in Example 1 were co-cultured with the human lymphoma cell line Daudi, and the supernatant and cell pellet were collected 24 hours later.
  • the human IFN ⁇ detection kit (BD) was used in the supernatant to perform ELISA assay to detect the content of IFN ⁇ released by CAR-T cells after stimulation of target cells. Cell pellets were detected by flow cytometry for endogenous cellular immune factors.
  • the antibodies used were allophycocyanin(APC)-Cy7-conjugated mAb to human CD8, PerCP-Cy5.5-conjugated mAb to human CD4, V450-conjugated mAb to human IFNg , PE-Cy7–conjugated antitumor necrosis factor (TNF) mAb, and flfluorescein isothiocyanate (FITC)-conjugated mAb to human IL2 (BD Biosciences). The results are shown in FIGS. 3 to 6 and Tables 3, 4, and 5.
  • Table 3 The content of immune factor IFN ⁇ excreted by CAR-T cells after target cell stimulation
  • mRNA-mediated CAR-T cells can secrete immune factors IFN ⁇ , TNF ⁇ and IL2 under the stimulation of target cells to mediate cellular immunity.
  • Cell line human lymphoma cell line Daudi;
  • Daudi cells are a human lymphoma cell line, and a mouse model of human lymphocytic leukemia leukemia can be constructed by intravenous injection. Its CD19 expression is positive, and it can be used as the target cell of the CAR-T cell of the present invention.
  • the Daudi cells were resuspended in physiological saline, and the viable cell concentration was adjusted to 3 ⁇ 10 8 cells/ml. Vaccination was performed by tail vein injection.
  • CAR-T cells were constructed in situ in vivo, and the synthesized CAR gene mRNA and cytokine mRNA were combined according to the following combinations seq1, seq2KY, seq3 YE, seq4, seq4+5 (mass ratio 1:1), seq4+6 (Mass ratio 1:1), seq4+7+8 (Mass ratio 1:1:1) and seq4+9 (Mass ratio 1:1) were injected into the spleen of mice at 1 mg each, and flow cytometry was performed at different time points The number of CAR-T cells and cancer cells in the blood of mice were detected, see Table 6 and Table 7. The results are shown in Figures 7 and 8.
  • Seq2 11.3% Seq4+7+8 25.75 Seq3 13.9% Seq4+9 20.9% Seq4 14.45%

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Abstract

提供了一种编码CAR基因的mRNA、组合mRNA、构建方法、CAR-T细胞和方法，mRNA的核苷酸序列选自SEQ ID NOs.1～4中的任一种。采用编码CAR基因的mRNA、组合mRNA构建得到的CAR-T细胞能够表达CAR基因，对肿瘤具有杀伤作用。

Description

一种编码CAR基因的mRNA、组合mRNA、构建方法、CAR-T细胞和方法 技术领域
本发明属于基因工程技术领域，具体涉及一种编码CAR基因的mRNA、组合mRNA、构建方法、CAR-T细胞和方法。
背景技术
随着全球环境因素的不断恶化，无论发达国家还是发展中国家，恶性肿瘤的发病率和死亡率均呈上升趋势，恶性肿瘤已成为人类生命健康的最大危害。由于传统治疗方法的固有缺陷以及全球生物高新技术的火热发展，人们对新疗法的出现期待已久。而生物治疗作为刚崭露头角的全新治疗理念，其特有优点包括选择性靶向杀伤肿瘤细胞，对正常细胞无毒性作用。生物疗法治疗癌症能够明显改善患者临床症状，提高生活质量，延长生存期。生物疗法治疗癌症能激活机体免疫功能，有望根除肿瘤细胞，生物治疗能够增强手术成功率，必将为癌症患者所广泛接受。
CAR-T疗法是一种以嵌合型抗原受体为基础的细胞免疫治疗方案。其通过体外基因转移技术，将编码嵌合抗原受体(CAR)的基因序列转导入T细胞中，生成可以结合靶抗原的肿瘤特异性T细胞。近几年，CAR-T疗法在治疗恶性血液肿瘤中取得的成果是有目共睹的，如针对难治性/复发性急性B淋巴母细胞白血病的Kymriah和针对难治性/复发性非何杰金氏淋巴瘤的Yescarta去年已在美国上市，但会有至少50％以上的复发率，且CAR-T细胞治疗实体瘤效果不佳，主要原因是缺乏合适的靶抗原、CAR-T细胞在体内持续时间短、免疫逃逸、免疫抑制肿瘤微环境等。因此，目标抗原的选择对于CAR的特异性、有效性以及基因改造的T细胞自身安全性来讲都至关重要。现有技术中，采用传统Car-T的体外构建Car-T细胞的方法复杂。
发明内容
基于上述原因，本发明提供一种编码CAR基因的mRNA、组合mRNA、构建方法、CAR-T细胞和方法，采用本发明提供的编码CAR基 因的mRNA和mRNA组合构建得到的CAR-T细胞，方法简单，能够表达CAR基因，能够杀伤肿瘤细胞。
本发明通过下述技术方案实现的：
本发明提供了一种编码CAR基因的mRNA，所述mRNA的核苷酸序列选自SEQ ID No.1～4中的任一种。
本发明还提供了一种防治肿瘤的组合mRNA，包括上述技术方案所述的编码CAR基因的mRNA和编码细胞因子的mRNA；所述细胞因子为IL-2细胞因子、IL-10细胞因子、IL12A细胞因子、IL12B细胞因子和CCL-19细胞因子中的一种或几种。
优选的，编码所述IL-2细胞因子的核苷酸序列如SEQ ID No.5所示，编码所述IL-10细胞因子的核苷酸序列如SEQ ID No.6所示，编码CCL-19细胞因子的核苷酸序列如SEQ ID No.9所示，编码IL12A细胞因子的核苷酸序列如SEQ ID No.7所示，编码IL12B细胞因子的核苷酸序列如SEQ ID No.8所示。
优选的，所述编码CAR基因的mRNA的核苷酸序列如SEQ ID No.4所示，所述编码细胞因子的mRNA的核苷酸序列如SEQ ID No.5所示。
优选的，所述编码CAR基因的mRNA的核苷酸序列如SEQ ID No.4所示，所述编码细胞因子的mRNA的核苷酸序列如SEQ ID No.6所示。
优选的，所述编码CAR基因的mRNA的核苷酸序列如SEQ ID No.4所示，所述编码细胞因子的mRNA的核苷酸序列选自SEQ ID No.7和SEQ ID No.8两种。
优选的，所述编码CAR基因的mRNA的核苷酸序列如SEQ ID No.4所示，所述编码细胞因子的mRNA的核苷酸序列如SEQ ID No.9所示。
本发明还提供了一种CAR-T细胞的构建方法，包括以下步骤：
将上述技术方案所述的编码CAR基因的mRNA或上述技术方案所述的组合mRNA转染细胞，得到CAR-T细胞。
本发明还提供了一种基于上述技术方案所述的构建方法构建得到的CAR-T细胞。
本发明还提供了上述技术方案所述的CAR-T细胞在制备防治肿瘤的药物中的应用。
优选的，所述肿瘤包括人霍奇金淋巴瘤、外周T细胞淋巴瘤、弥漫性大B细胞淋巴瘤、淋巴母细胞淋巴瘤和间变性大细胞淋巴瘤中的一种或几种。
本发明还提供了上述技术方案所述的CAR-T细胞防治肿瘤的方法，包括以下步骤：将所述CAR-T细胞注射到患者的脾脏，所述CAR-T细胞的注射剂量为1mg/50kg体重，每2周注射一次，共注射3次。
优选的，所述肿瘤包括人霍奇金淋巴瘤、外周T细胞淋巴瘤、弥漫性大B细胞淋巴瘤、淋巴母细胞淋巴瘤和间变性大细胞淋巴瘤中的一种或几种。
本发明提供了一种编码CAR基因的mRNA、组合mRNA、构建方法、CAR-T细胞和应用，所述mRNA的核苷酸序列选自SEQ ID No.1～4中的任一种。本发明以编码CAR基因的mRNA或组合mRNA为靶抗原，不仅便于保存和运输，还可以在原位将T细胞转化成为CAR-T细胞，相比传统体外CAR-T制备在流程上大大简化，成本大幅下降。以信使RNA作为携带嵌合抗原受体的载体，转入患者T细胞后，将T细胞激活，并装上定位导航装置CAR(肿瘤嵌合抗原受体)，将T细胞这个普通"战士"改造成"超级战士"，即CAR-T细胞，他利用其"定位导航装置"-CAR，专门识别体内肿瘤细胞，并通过免疫作用释放大量的多种效应因子，它们能高效地杀灭肿瘤细胞，从而达到治疗恶性肿瘤的目的。
本发明实施例的结果显示：采用本发明提供的编码CAR基因的mRNA、组合mRNA构建得到的CAR-T细胞能够表达CAR基因，对肿瘤具有杀伤作用。
说明书附图
图1为CAR-T细胞中CAR的表达率；
图2为CAR-T细胞杀伤肿瘤细胞结果；
图3为CAR-T细胞被靶细胞激活后外泌IFNγ的结果；
图4为CAR-T细胞被靶细胞激活后CD4和CD8细胞分泌IFNγ的结果。
图5为CAR-T细胞被靶细胞激活后CD4和CD8细胞分泌IFNγ的 结果。
图6为CAR-T细胞被靶细胞激活后CD4和CD8细胞分泌IFNγ的结果。
图7为注射CAR基因mRNA后原位构建CAR-T细胞的结果。
图8为CAR-T细胞体内杀伤靶细胞的结果。
具体实施方式
本发明提供了一种编码CAR基因的mRNA，所述mRNA的核苷酸序列选自SEQ ID No.1～4中的任一种。
本发明还提供了一种防治肿瘤的组合mRNA，包括上述技术方案所述的编码CAR基因的mRNA和编码细胞因子的mRNA；所述细胞因子为IL-2细胞因子、IL-10细胞因子、IL12A细胞因子、IL12B细胞因子和CCL-19细胞因子中的一种或几种。在本发明中，核苷酸序列如SEQ ID No.5所示的mRNA编码IL-2细胞因子，核苷酸序列如SEQ ID No.6所示的mRNA编码IL-10细胞因子，核苷酸序列如SEQ ID No.7所示的mRNA编码IL12A细胞因子，核苷酸序列如SEQ ID No.8所示的mRNA编码IL12B细胞因子，核苷酸序列如SEQ ID No.9所示的mRNA编码CCL-19细胞因子。
在本发明中，所述的mRNA组合包括编码CAR基因的mRNA和编码细胞因子的mRNA，所述编码CAR基因的mRNA的核苷酸序列优选如SEQ ID No.4所示，所述编码细胞因子的mRNA的核苷酸序列优选如SEQ ID No.5所示。
在本发明中，所述的mRNA组合包括编码CAR基因的mRNA和编码细胞因子的mRNA，所述编码CAR基因的mRNA的核苷酸序列优选如SEQ ID No.4所示，所述编码细胞因子的mRNA的核苷酸序列优选如SEQ ID No.6所示。
在本发明中，所述的mRNA组合包括编码CAR基因的mRNA和编码细胞因子的mRNA，所述编码CAR基因的mRNA的核苷酸序列优选如SEQ ID No.4所示，所述编码细胞因子的mRNA的核苷酸序列优选选 自SEQ ID No.7和SEQ ID No.8两种。SEQ ID No.7和SEQ ID No.8分别编码IL12的A链和B链，二者结合在一起才能发挥作用，因此本质上还是一个CAR基因和一个细胞因子基因组合。
在本发明中，所述的mRNA组合包括编码CAR基因的mRNA和编码细胞因子的mRNA，所述编码CAR基因的mRNA的核苷酸序列优选如SEQ ID No.4所示，所述编码细胞因子的mRNA的核苷酸序列优选如SEQ ID No.9所示。
本发明提供的细胞因子mRNA转染细胞后进行表达，CAR-T细胞在细胞因子和肿瘤表面抗原的共刺激下，增强T细胞增殖，可产生MHC非限制性的活化，进一步诱导巨噬细胞分泌细胞因子，增强抗肿瘤反应。细胞因子的分泌同时也改善了免疫微环境，上调了DC细胞和巨噬细胞的抗原呈递能力从而改善局部的肿瘤免疫力。
本发明还提供了一种CAR-T细胞的构建方法，包括以下步骤：将上述技术方案所述的编码CAR基因的mRNA或上述技术方案所述的mRNA组合转染T细胞，得到CAR-T细胞。在本发明中，所述细胞优选为外周血单个核细胞、H9(人T淋巴细胞系)或jurkaT细胞。
本发明还提供了一种基于上述技术方案所述的构建方法构建得到的CAR-T细胞。
本发明还提供了上述技术方案所述的CAR-T细胞在制备防治肿瘤的药物中的应用。本发明对所述药物的剂型没有特殊限定，采用CAR-T细胞在医学上可接受的剂型即可。在本发明中，所述肿瘤优选包括人霍奇金淋巴瘤、外周T细胞淋巴瘤、弥漫性大B细胞淋巴瘤、淋巴母细胞淋巴瘤和间变性大细胞淋巴瘤中的一种或几种，注射方式优选为生理盐水溶解，静脉注射。
本发明还提供了上述技术方案所述的CAR-T细胞防治肿瘤的方法，包括以下步骤：将所述CAR-T细胞注射到患者的脾脏，所述CAR-T细胞的注射剂量为1mg。在本发明中，所述肿瘤优选包括人霍奇金淋巴瘤、外周T细胞淋巴瘤、弥漫性大B细胞淋巴瘤、淋巴母细胞淋巴瘤和间变性大细胞淋巴瘤中的一种或几种。
下面结合实施例，对本发明提供的一种编码CAR基因的mRNA、组合mRNA、构建方法、CAR-T细胞和方法进行详细的描述，但不能将它们认为是对本发明保护范围的限制。
实施例1
制备CAR-T细胞
从健康捐献者的外周血中分离出PBMC细胞，在加入IL2(50U/ML)的AIM-V培养基中培养，将CAR基因mRNA和细胞因子mRNA按照seq1、seq2KY、seq3 YE、seq4、seq4+5、seq4+6、seq4+7+8和seq4+9分别转染细胞，在细胞培养结束后，取样后分装至取样管中，进行质控检测。
编码CAR基因的mRNA，核苷酸序列如SEQ ID No.1所示，简称seq1；
编码CAR基因的mRNA，核苷酸序列如SEQ ID No.2所示，简称seq2KY；
编码CAR基因的mRNA，核苷酸序列如SEQ ID No.3所示，简称seq3YE；
编码CAR基因的mRNA，核苷酸序列如SEQ ID No.4所示，简称seq4；
防治肿瘤的mRNA组合，包括编码CAR基因的mRNA和编码细胞因子的mRNA，编码CAR基因的mRNA的核苷酸序列如SEQ ID No.4所示，编码细胞因子的mRNA核苷酸序列如SEQ ID No.5所示，简称seq4+5；
防治肿瘤的mRNA组合，包括编码CAR基因的mRNA和编码细胞因子的mRNA，编码CAR基因的mRNA的核苷酸序列如SEQ ID No.4所示，编码细胞因子的mRNA核苷酸序列如SEQ ID No.6所示，以下简称4+6；
防治肿瘤的mRNA组合，包括编码CAR基因的mRNA和编码细胞因子的mRNA，编码CAR基因的mRNA的核苷酸序列如SEQ ID No.4所示，编码细胞因子的mRNA核苷酸序列选自SEQ ID No.7和8，以下简称4+7+8；
防治肿瘤的mRNA组合，包括编码CAR基因的mRNA和编码细胞因子的mRNA，编码CAR基因的mRNA的核苷酸序列如SEQ ID No.4所示，编码细胞因子的mRNA核苷酸序列如SEQ ID No.9所示，以下简 称4+9。
1、PBMC细胞准备：根据使用时间确定传代密度和体积，传代于细胞培养瓶内，保证使用时细胞处于对数生长期。
细胞消化计数：取生长状态良好的细胞，去掉培养基，以10ml PBS清洗细胞后，加入适量0.25％胰酶(T75瓶加1ml 0.25％胰酶，T175瓶加3ml 0.25％胰酶)消化5分钟，然后加入含10％FBS的DMEM培养基(T75瓶用9ml培养基，T175瓶用17ml培养基)中和胰酶，吹打细胞并转至50ml离心管，反复吹打混匀，然后取约0.3ml的细胞悬液，适当稀释后计数。
2、细胞稀释：取适量细胞悬液，用含10％FBS的AIM-V培养基稀释到5×10 5个/ml，吹打混匀。
3、细胞接种：取2ml细胞悬液加到6孔板内。每个样品需要准备2孔平行细胞，对照组样品需要准备1孔细胞，空白对照1孔。将6孔板放入37±1℃、5±0.5％CO 2培养箱培养过夜。
4、转染：
4.1接种完细胞后约24小时，观察6孔板内的细胞状态，汇合度在90％。在生物安全柜内，配制所需体积的90％AIM-V+10％FBS培养基。转染前30min弃掉孔板的培养基，每孔加入1ml新鲜培养基(90％AIM-V+10％FBS)。
4.2配制转染体系：取200μl opti-MEM，加入10μg seq1、seq2KY、seq3 YE、seq4、seq4+5(质量比1:1)、seq4+6(质量比1:1)、seq4+7+8(质量比1:1:1)和seq4+9(质量比1:1)或阴性对照GFP-mRNA，用枪头轻轻吹打混匀，再加入60μl PEI(浓度1mg/ml)，立即置于漩涡振荡器上振荡10次，每次1s，充分混匀，静置10min。
4.3将配制好的转染体系，直接均匀滴加进入培养的细胞中，再前后左右摇匀，使得转染体系均匀分布于细胞上。
流式细胞术
1、单细胞悬液制备：取生长状态良好的细胞，去掉培养基，以10ml PBS清洗细胞后，加入适量0.25％胰酶消化5分钟，然后加入含10％FBS的DMEM培养基中和胰酶，吹打细胞并转至50ml离心管，反复吹打混 匀，然后将细胞悬液稀释后计数。
2、固定：通过离心收集细胞并吸除上清液。用0.5～1ml 1×PBS中重悬细胞。添加甲醛并使其终浓度为4％。在室温下固定15分钟。用足够的1×PBS离心洗涤。将上清液丢弃在合适的废液缸中。在0.5～1ml1×PBS中重悬细胞。
4、免疫染色：在100μl稀释一抗(1：2000)中重悬细胞。在室温下孵育1小时。通过离心分离，用孵育缓冲液洗涤。丢弃上清液。在100μl稀释的荧光染料标记的二抗(1：5000)中重悬细胞。在室温下孵育30分钟。上机检测。
将制得的CAR-T细胞通过流式细胞术进行CAR表达率的检测，结果如图1和表1所示。
表1制得的CAR-T细胞中CAR的表达率
细胞名称 表达率
Seq1 35.29％
Seq2 KY 38.02％
Seq3 YE 37.00％
Seq4 41.78％
Seq4+5 38.08％
Seq4+6 44.53％
Seq4+7+8 45.31％
Seq4+9 46.54
从图1和表1中可以得出，本发明提供的mRNA剂型能够有效地对T细胞进行转染，使其成为CAR-T细胞。
实施例2
对外周血制成的CAR-T细胞进行体外杀实验，具体实验步骤如下：
第一步：CAR-T细胞制备
如实施例1。
第二步：Calcein-AM标记靶细胞
1)将Calcein-AM用DMSO稀释成lmg/mL；
2)将人淋巴瘤细胞系Daudi用全培养基重悬成1×10 6/mL的密度；
3)加入15μl的Calcein-AM,37℃、5％CO 2培养30min，每l0min 轻轻混匀；
4)1500rpm离心，去上清，用培养基重悬，重复两遍；
第三步：杀伤
1)将标记好的人淋巴瘤细胞系Daudi按照5000-50000个/mL的密度重悬，取100μl加入到96孔板中；
2)按照适当的比例加入CAR-T细胞100μl，使CAR-T细胞与癌细胞的数目比分别为50:1、25:1、12.5:1、6:1和3:1，每组3个平行；同时，有单独的A组6个平行，只有靶细胞(阴性对照)：有单独的B组6个平行，只有靶细胞+2％TritonX-100(阳性对照)；
第四步：
1)37℃、5％CO 2培养4小时后，离心，取75μl上清，转移到一个新的培养板上；
2)通过分光光度计检测样品，并记录AFU数据；
3)计算细胞裂解的百分比：[(实验组吸光度-阴性对照吸光度)/(阳性对照组吸光度-阴性对照组吸光度)]×l00％。得到的结果如图2、表2所示。
表2本发明制备的CAR-T细胞体外杀伤癌细胞结果统计(细胞裂解百分比)
Figure PCTCN2020137996-appb-000001
从图2可以看出，制备的CAR-T细胞具有人淋巴瘤细胞杀伤能力。
实施例3
检测转染CAR基因后，在靶细胞的刺激下T细胞释放免疫因子情况。
将实施例1制备的CAR-T细胞和人淋巴瘤细胞系Daudi共培养，24小时后收取上清和细胞沉淀。
上清使用人IFNγ检测试剂盒(BD)进行elisa实验检测CAR-T细胞在靶细胞刺激后释放的IFNγ含量。细胞沉淀采用流式细胞术进行内源细胞免疫因子检测，所用抗体为allophycocyanin(APC)-Cy7-conjugated mAb to human CD8,PerCP-Cy5.5–conjugated mAb to human CD4,V450-conjugated mAb to human IFNg,PE-Cy7–conjugated antitumor necrosis factor(TNF)mAb,and flfluorescein isothiocyanate(FITC)-conjugated mAb to human IL2(BD Biosciences)。结果如图3～6及表3、4、5所示。
表3 CAR-T细胞在靶细胞刺激后外泌的免疫因子IFNγ含量
分组 IFNγ(pg/ml) 分组 IFNγ(pg/ml)
对照 105 Seq4+5 411
Seq1 310 Seq4+6 422
Seq2 336 Seq4+7+8 440
Seq3 391 Seq4+9 400
Seq4 382 —— ——
表4受靶细胞激活后CD4细胞中表达IFNγ、TNFα和IL2的阳性细胞比例(％)
  对照 Seq1 Seq2 Seq3 Seq4 Seq4+5 Seq4+6 Seq4+7+8 Seq4+9
INFγ 0.65 27.55 30 25.5 32 34.6 37.1 37.5 42.5
TNFα 0.35 1.5 1.3 1.15 1.5 1.6 1.35 1.4 1.55
IL2 3.65 71.5 77 79.2 78.8 78.5 81 81.3 86.4
表5受靶细胞激活后CD8细胞中表达IFNγ、TNFα和IL2的阳性细胞比例(％)
  对照 Seq1 Seq2 Seq3 Seq4 Seq4+5 Seq4+6 Seq4+7+8 Seq4+9
INFγ 0.5 2.6 3.05 1.4 2025 2.3 2.4 1.8 1.915
TNFα 0.65 15 12.5 12 12.25 14 13.15 14.4 14.35
IL2 0.75 20.5 23.5 19 18.3 15.2 16 11.5 13
由图3～6可知，mRNA介导的CAR-T细胞在靶细胞的刺激下，可分泌免疫因子IFNγ、TNFα和IL2，介导细胞免疫。
实施例4
对实施例1制备得到的CAR-T细胞验证其安全性和有效性。
首先利用肿瘤模型小鼠验证实施例1构建的CAR-T细胞的有效性，具体步骤如下：
一、淋巴瘤小鼠模型的构建：
1.细胞系：人淋巴瘤细胞系Daudi；
Daudi细胞是人淋巴瘤细胞系，可以通过静脉注射的方式构建小鼠的人淋巴细胞性白血病白血病模型。其CD19表达为阳性，可以作为本发明所述CAR-T细胞的靶细胞。
2.细胞培养
Daudi细胞培养(1640+20％FBS)
计数并测定活率，离心后用生理盐水重悬，调整其活细胞浓度为3×10 8个/ml，总数达1.8×10 9个。
3.细胞系接种
用生理盐水重悬Daudi细胞，调整其活细胞浓度为3×10 8个/ml。通过尾静脉注射的方式进行接种。
4.一周后，体内原位构建CAR-T细胞，将合成的CAR基因mRNA和细胞因子mRNA按照以下组合seq1、seq2KY、seq3 YE、seq4、seq4+5(质量比1:1)、seq4+6(质量比1:1)、seq4+7+8(质量比1:1:1)和seq4+9(质量比1:1)各1mg对小鼠进行脾脏注射，不同时间点通过流式细胞术检测小鼠血液中的CAR-T细胞数目和癌症细胞数目，见表6和表7。结果如图7和图8所示。
表6注射mRNA原位构建CAR-T细胞结果
分组 CAR-T比例 分组 CAR-T比例
对照 0.4％ Seq4+5 19.1％
Seq1 8.6％ Seq4+6 21.1％
Seq2 11.3％ Seq4+7+8 25.75
Seq3 13.9％ Seq4+9 20.9％
Seq4 14.45％    
表7接受mRNA介导的CAR-T治疗的小鼠体内癌细胞数目变化(Log 10/μl)
  对照 Seq1 Seq2 Seq3 Seq4 Seq4+5 Seq4+6 Seq4+7+8 Seq4+9
7d 1.4 1.1 1.25 1.12 1.21 1.33 1.25 1.52 1.28
14d 2.2 1.0 0.72 0.56 0.36 0.42 0.5 0.21 0.33
21d 2.3 0.9 0.63 0.46 0.34 0.4 0.48 0.23 0.25
28d 2.6 0.85 0.56 0.36 0.35 0.38 0.45 0.2 0.21
35d 2.7 0.8 0.45 0.34 0.31 0.36 0.42 0.16 0.18
42d 2.9 0.74 0.44 0.31 0.33 0.32 0.35 0.15 0.16
49d 3.1 0.68 0.42 0.26 0.25 0.3 0.33 0.17 0.15
56d 3.2 0.62 0.32 0.24 0.21 0.21 0.31 0.14 0.12
63d 3.4 0.56 0.21 0.21 0.16 0.18 0.26 0.12 0.08
70d 2.9 0.49 0.21 0.19 0.12 0.16 0.25 0.11 0.06
77d 3.3 0.38 0.14 0.15 0.11 0.15 0.25 0.06 0.06
84d 3.1 0.36 0.1 0.11 0.1 0.11 0.16 0.05 0.04
91d 3.4 0.2 0.07 0.05 0.06 0.09 0.1 0.01 0.02
由图7～图8可知，注射CAR-T mRNA能有效地进行原位CAR-T细胞构建，同时对血液中地淋巴瘤细胞进行有效的杀灭。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (13)

  1. 一种编码CAR基因的mRNA，其特征在于，所述mRNA的核苷酸序列选自SEQ ID No.1～4中的任一种。
  2. 一种防治肿瘤的组合mRNA，其特征在于，包括权利要求1所述的编码CAR基因的mRNA和编码细胞因子的mRNA；所述细胞因子为IL-2细胞因子、IL-10细胞因子、IL12A细胞因子、IL12B细胞因子和CCL-19细胞因子中的一种或几种。
  3. 根据权利要求2所述的组合mRNA，其特征在于，编码所述IL-2细胞因子的核苷酸序列如SEQ ID No.5所示，编码所述IL-10细胞因子的核苷酸序列如SEQ ID No.6所示，编码CCL-19细胞因子的核苷酸序列如SEQ ID No.9所示，编码IL12A细胞因子的核苷酸序列如SEQ ID No.7所示，编码IL12B细胞因子的核苷酸序列如SEQ ID No.8所示。
  4. 根据权利要求2所述的组合mRNA，其特征在于，所述编码CAR基因的mRNA的核苷酸序列如SEQ ID No.4所示，所述编码细胞因子的mRNA的核苷酸序列如SEQ ID No.5所示。
  5. 根据权利要求2所述的组合mRNA，其特征在于，所述编码CAR基因的mRNA的核苷酸序列如SEQ ID No.4所示，所述编码细胞因子的mRNA的核苷酸序列如SEQ ID No.6所示。
  6. 根据权利要求2所述的组合mRNA，其特征在于，所述编码CAR基因的mRNA的核苷酸序列如SEQ ID No.4所示，所述编码细胞因子的mRNA的核苷酸序列选自SEQ ID No.7和SEQ ID No.8两种。
  7. 根据权利要求2所述的组合mRNA，其特征在于，所述编码CAR基因的mRNA的核苷酸序列如SEQ ID No.4所示，所述编码细胞因子的mRNA的核苷酸序列如SEQ ID No.9所示。
  8. 一种CAR-T细胞的构建方法，其特征在于，包括以下步骤：
    将权利要求1所述的编码CAR基因的mRNA或权利要求2～6任一项所述的组合mRNA转染T细胞，得到CAR-T细胞。
  9. 一种基于权利要求8所述的构建方法构建得到的CAR-T细胞。
  10. 权利要求9所述的CAR-T细胞在制备防治肿瘤的药物中的应用。
  11. 根据权利要求10所述的应用，其特征在于，所述肿瘤包括人霍奇金淋巴瘤、外周T细胞淋巴瘤、弥漫性大B细胞淋巴瘤、淋巴母细胞淋巴瘤和间变性大细胞淋巴瘤中的一种或几种。
  12. 权利要求9所述的CAR-T细胞防治肿瘤的方法，包括以下步骤：将所述CAR-T细胞注射到患者的脾脏，所述CAR-T细胞的注射剂量为1mg/50kg体重，每2周注射一次，共注射3次。
  13. 根据权利要求12所述的方法，其特征在于，所述肿瘤包括人霍奇金淋巴瘤、外周T细胞淋巴瘤、弥漫性大B细胞淋巴瘤、淋巴母细胞淋巴瘤和间变性大细胞淋巴瘤中的一种或几种。
PCT/CN2020/137996 2020-12-07 2020-12-21 一种编码CAR基因的mRNA、组合mRNA、构建方法、CAR-T细胞和方法 WO2022120934A1 (zh)

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