WO2023246001A1 - Car-t和car-m联用在制备抗肿瘤药物中的应用 - Google Patents

Car-t和car-m联用在制备抗肿瘤药物中的应用 Download PDF

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WO2023246001A1
WO2023246001A1 PCT/CN2022/137492 CN2022137492W WO2023246001A1 WO 2023246001 A1 WO2023246001 A1 WO 2023246001A1 CN 2022137492 W CN2022137492 W CN 2022137492W WO 2023246001 A1 WO2023246001 A1 WO 2023246001A1
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car
region
cells
chimeric antigen
nkg2d
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French (fr)
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万晓春
陈有海
刘茂玄
戴昆
刘骏晨
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深圳先进技术研究院
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Definitions

  • the present invention relates to the field of tumor treatment, specifically to cellular immunotherapy of tumors, in particular to chimeric antigen receptor T cell (CAR-T) and chimeric antigen receptor macrophage (CAR-M) cellular immunotherapy, and more specifically to Application of the combination of CAR-T and CAR-M in the preparation of anti-tumor drugs.
  • CAR-T chimeric antigen receptor T cell
  • CAR-M chimeric antigen receptor macrophage
  • chimeric antigen receptor T cells Chomeric Antigen Receptor T cells
  • CAR-T Receptor T-Cell
  • CAR-T Receptor T-Cell
  • the basic principle is to use genetic engineering to modify T lymphocytes so that they express chimeric antigen receptors and use non-major histocompatibility complex (MHC, major histocompatibility complex) to kill tumor cells in a restricted manner.
  • MHC non-major histocompatibility complex
  • CAR T cells have achieved good results in clinical trials and clinical trials, they have also experienced serious side effects, including cytokine release syndrome, neurotoxicity, and off-target effects.
  • CAR-T cell therapy on tumors other than some hematological tumors is not satisfactory in clinical trials and needs to be improved urgently.
  • CAR-T cell therapy has not made a major breakthrough for the following reasons: First, CAR-T cells must transport and penetrate into the tumor, which requires extravasation, chemotaxis, and stromal tissue. The process of infiltration. CAR-T cells must traverse abnormal tumor vessels with reduced adhesion molecules, undergo chemokine/chemokine receptor mismatches, and must migrate through dense cellular and stromal barriers.
  • TME Tumor Microenvironment
  • effector cells encounter adverse conditions such as hypoxic and acidic environments, expression of immune checkpoint ligands, and large numbers of immunosuppressive cells such as tumor-associated macrophages (Tumor Associated Macrophage (TAM), myeloid-derived suppressor cells (Myeloid Derived Suppressor Cell (MDSC) and regulatory T cells (Regulatory Cells, T-regs).
  • TAM Tumor Associated Macrophage
  • MDSC myeloid-derived suppressor cells
  • Regulatory Cells regulatory Cells
  • CAR T cells Even if CAR T cells survive in the TME, solid tumors often have heterogeneous surface antigen expression, which can lead to evasion of CAR T cell detection, incomplete tumor clearance, and eventual growth of antigen-negative tumor cells. This was clearly demonstrated in the treatment of glioblastoma using CAR T cells targeting EGFRvIII, where EGFRvIII expression decreased in 5/7 patients after treatment. Finally, CAR-T cells may recognize tumor-associated antigens in normal tissues and cells, resulting in on-target/off-tumor toxicity. toxicity), which is another obstacle to cell therapy of solid tumors.
  • combining CAR-T with other treatments may be able to make up for the deficiencies of CAR-T in this regard and resist the immunosuppressive influence of the local tumor microenvironment of solid tumors, thereby achieving a synergistic anti-tumor effect.
  • CAR-M CAR-modified macrophages
  • Macrophages mainly phagocytose cell debris and pathogens in body fluids and tissues and present antigens to activate lymphatic or other immune cells to respond to pathogens.
  • macrophages are the innate immune cells with the highest infiltration rate and can interact with almost all cellular components in the TME to stimulate angiogenesis, increase tumor infiltration, and mediate immunosuppression.
  • CAR-M has limited effect in directly killing tumor cells.
  • CAR T cells have achieved good results in clinical trials and clinical trials, they have also experienced serious side effects, including cytokine release syndrome, neurotoxicity, and off-target effects. Moreover, the effectiveness of CAR-T cell therapy on tumors other than some hematological tumors (such as solid tumors) is not satisfactory in clinical trials and needs to be improved urgently. In the treatment of solid tumors, CAR-T cell therapy has not made a major breakthrough for the following reasons: First, CAR-T cells must transport and penetrate into the tumor, which requires extravasation, chemotaxis, and stromal tissue. The process of infiltration.
  • CAR-T cells must traverse abnormal tumor vessels with reduced adhesion molecules, undergo chemokine/chemokine receptor mismatches, and must migrate through dense cellular and stromal barriers.
  • TME Tumor Microenvironment
  • effector cells encounter adverse conditions such as hypoxic and acidic environments, expression of immune checkpoint ligands, and large numbers of immunosuppressive cells such as tumor-associated macrophages (Tumor Associated Macrophage (TAM), myeloid-derived suppressor cells (Myeloid Derived Suppressor Cell (MDSC) and regulatory T cells (Regulatory Cells, T-regs).
  • TAM tumor-associated macrophages
  • MDSC myeloid-derived suppressor cells
  • Regulatory Cells regulatory Cells, T-regs.
  • long-term antigen exposure can lead to T cell exhaustion, thereby reducing the effector function of CAR T cells.
  • CAR T cells Even if CAR T cells survive in the TME, solid tumors often have heterogeneous surface antigen expression, which can lead to evasion of CAR T cell detection, incomplete tumor clearance, and eventual growth of antigen-negative tumor cells. This was clearly demonstrated in the treatment of glioblastoma using CAR T cells targeting EGFRvIII, where EGFRvIII expression decreased in 5/7 patients after treatment. Finally, CAR-T cells may recognize tumor-associated antigens in normal tissues and cells, resulting in on-target/off-tumor toxicity. toxicity), which is another obstacle to cell therapy of solid tumors.
  • combining CAR-T with other treatments may be able to make up for the deficiencies of CAR-T in this regard and resist the immunosuppressive influence of the local tumor microenvironment of solid tumors, thereby achieving a synergistic anti-tumor effect.
  • CAR-T In order to solve the existing technology's (1) shortcomings in CAR-T treatment of solid tumors: including tumor heterogeneity, leading to the escape of CAR target-negative cells; CAR-T is difficult to infiltrate into the tumor and has insufficient killing effect; immunosuppression of solid tumors The influence of the microenvironment leads to CAR-T exhaustion and loss of killing function; CAR-T's own side effects, such as cytokine release syndrome, neurotoxicity and off-target effects; (2) CAR-M cells phagocytose or gnaw tumor cells In order to kill tumors, the efficiency of killing tumor cells is not high.
  • the present invention aims to provide the application of combined use of CAR-T and CAR-M in the preparation of anti-tumor drugs.
  • CAR-M can improve the immune microenvironment of solid tumors; it can serve as an antigen-presenting cell to present antigens after engulfing tumor cells; it is easier to infiltrate into the tumor and cooperate with other immune systems Cells infiltrate tumors, etc.; (2) CAR-T has the ability to directly kill tumor cells.
  • the present invention proposes a plan for tumor treatment using CAR-T and CAR-M in combination. The specific plans are as follows:
  • a first aspect of the present invention provides the application of CAR-T and macrophages in the preparation of anti-tumor drugs.
  • the CAR-T is a T cell modified by a chimeric antigen receptor.
  • the chimeric antigen receptor is a primary The protein structure from the amino terminus to the carboxyl terminus is: signal peptide, NKG2D extracellular region, hinge region, transmembrane region, intracellular costimulatory signal region and intracellular signal region;
  • the NKG2D extracellular domain is the extracellular domain of human NKG2D, and its amino acid sequence is shown in SEQ ID NO. 2.
  • the second aspect of the present invention provides the application of CAR-M and T cells in the preparation of anti-tumor drugs.
  • the CAR-M is a macrophage modified by a chimeric antigen receptor.
  • the chimeric antigen receptor is a primary The protein structure from the amino terminus to the carboxyl terminus is: signal peptide, NKG2D extracellular region, hinge region, transmembrane region and intracellular signal region;
  • the NKG2D extracellular domain is the extracellular domain of human NKG2D, and its amino acid sequence is shown in SEQ ID NO. 2.
  • the third aspect of the present invention provides the application of CAR-T and CAR-M in the preparation of anti-tumor drugs.
  • the CAR-T is a T cell modified by a chimeric antigen receptor.
  • the chimeric antigen receptor is a primary The protein structure from the amino terminus to the carboxyl terminus is: signal peptide, NKG2D extracellular region, hinge region, transmembrane region, intracellular costimulatory signal region and intracellular signal region;
  • the CAR-M is a macrophage modified by a chimeric antigen receptor.
  • the primary protein structure of the chimeric antigen receptor is, from the amino terminus to the carboxyl terminus, in order: signal peptide, NKG2D extracellular region, hinge region, transmembrane region and intracellular signaling region;
  • the extracellular domain of the chimeric antigen receptor NKG2D in the CAR-T and CAR-M is the extracellular domain of human NKG2D, and its amino acid sequence is shown in SEQ ID NO. 2.
  • the signal peptide is selected from CD8 ⁇ signal peptide, CD28 signal peptide, CD4 signal peptide or GM-CSF signal peptide;
  • the hinge region is selected from the group consisting of CD8 ⁇ hinge region or CD28 hinge region;
  • the transmembrane region is selected from the group consisting of CD8 ⁇ transmembrane region or CD28 transmembrane region;
  • the intracellular signal region is selected from CD3 ⁇ or FcR ⁇ .
  • the signal peptide is derived from human CD8 ⁇ , and its amino acid sequence is shown in SEQ ID NO. 1;
  • the hinge region is derived from human CD8 ⁇ , and its amino acid sequence is shown in SEQ ID NO. 3;
  • transmembrane region is derived from human CD8 ⁇ , and its amino acid sequence is shown in SEQ ID NO. 4;
  • the intracellular signal region is CD3 ⁇ , and its amino acid sequence is shown in SEQ ID NO. 5.
  • the intracellular costimulatory signal region is selected from 4-1BB, CD28, CD27, OX40 or ICOS;
  • the intracellular costimulatory signal region is selected from 4-1BB, and its amino acid sequence is shown in SEQ ID NO. 11.
  • the CAR-T construction method is: introducing the nucleic acid or vector of the CAR-T chimeric antigen receptor encoding gene into T cells;
  • the introduction method includes electroporation, transduction or transfection
  • the nucleic acid is located on different viral vectors;
  • the viral vector is a lentiviral vector, an adenoviral vector or a retroviral vector;
  • the vector is a transposon or an mRNA vector
  • the lentiviral vector containing the CAR-T chimeric antigen receptor encoding gene is introduced into T cells by transfection.
  • the CAR-M construction method is: introducing the nucleic acid or vector of the CAR-M chimeric antigen receptor encoding gene into macrophage cells;
  • the introduction method includes electroporation, transduction or transfection
  • the nucleic acid is located on different viral vectors;
  • the viral vector is a lentiviral vector, an adenoviral vector or a retroviral vector;
  • the vector is a transposon or an mRNA vector
  • the adenoviral vector containing the CAR-M chimeric antigen receptor encoding gene is introduced into macrophage cells by transfection.
  • the macrophages are derived from healthy humans or cancer patients;
  • the macrophages are selected from autologous macrophages, allogeneic macrophages or iPSC-induced macrophages;
  • the macrophages are primary macrophages
  • the T cells are derived from healthy humans or cancer patients;
  • the T cells are selected from autologous T cells, allogeneic T cells or iPSC-induced T cells.
  • the tumor is a tumor expressing NKG2D ligand
  • the tumor is a hematological tumor or a solid tumor
  • the tumor is leukemia, multiple myeloma, malignant lymphoma, glioma, liver cancer, lung cancer, gastric cancer, colon cancer, pancreatic cancer or breast cancer.
  • the present invention uses the natural sequence of the extracellular segment of NKG2D as the CAR recognition region, so that the chimeric antigen receptor has the advantages of low immunogenicity, easy expression, etc., and can be expressed normally on macrophages and T cells, and is a preparation for specific killing.
  • CD3 ⁇ is preferably used as the intracellular signal region.
  • the ITAM structure contained in CD3 ⁇ gives CAR-M better phagocytosis/tumor killing effect.
  • the present invention can achieve synergy in anti-tumor and improve the effect of treating tumors through the combined use of CAR-T and CAR-M, the combined use of CAR-T and M cells, or the combined use of CAR-M and T cells.
  • the combination of CAR-T and CAR-M cells for tumor treatment which combines the ability of CAR-M to improve the tumor microenvironment with the ability of CAR-T to directly kill tumor cells, has a synergistic effect and can achieve the goal of killing tumor cells.
  • the powerful and efficient killing of solid tumors greatly improves the efficacy and solves the shortcomings of CAR-T alone in treating solid tumors and the low efficiency of CAR-M alone in killing tumor cells.
  • the combination regimen can reduce the amount of CAR-T cells used, thereby reducing the side effects of CAR-T treatment.
  • Figure 1 Schematic representation of NKG2D CAR in NKG2D CAR-M.
  • Figure 2 Schematic diagram of NKG2D CAR in NKG2D CAR-T.
  • FIG. 3 CAR positive rate of NKG2D CAR-M cells.
  • Figure 4 NKG2D CAR-T cell CAR positivity rate.
  • NKG2D chimeric antigen receptor used in NKG2D CAR-M
  • This embodiment provides the NKG2D chimeric antigen receptor used in NKG2D CAR-M. Its primary protein structure from the amino terminus to the carboxyl terminus is: signal peptide (Signal pep), NKG2D extracellular domain (NKG2D ECD), Hinge region (Hinge), transmembrane region (TM) and intracellular signaling region.
  • the signal peptide can be selected from the signal peptides of human CD8 ⁇ , CD28, CD4, GM-CSF, etc.
  • the hinge region and the transmembrane region can be selected from the hinge region and transmembrane region of human CD8 ⁇ , CD28, etc.
  • the intracellular signal region can be selected from From CD3 ⁇ , FcR ⁇ , etc.
  • the signal peptide is derived from human CD8 ⁇ , and its amino acid sequence is as shown in SEQ ID NO. 1; the NKG2D extracellular region is the extracellular domain of human NKG2D (NM_007360.4), and its amino acid sequence is as follows As shown in SEQ ID NO. 2; the hinge region and transmembrane region are derived from human CD8 ⁇ , the amino acid sequence of the CD8 ⁇ hinge region is shown in SEQ ID NO. 3, and the amino acid sequence of the CD8 ⁇ transmembrane region is shown in SEQ ID NO. 4; intracellular The signal region is CD3 ⁇ , which provides an activation signal, and its amino acid sequence is shown in SEQ ID NO. 5.
  • CD8 ⁇ signal peptide SEQ ID NO. 6
  • NKG2D extracellular region SEQ ID NO. 7
  • CD8 ⁇ hinge region SEQ ID NO. 8
  • CD8 ⁇ transmembrane region SEQ ID NO. 9
  • CD3 ⁇ intracellular signal region SEQ ID NO. 10
  • This embodiment provides the NKG2D chimeric antigen receptor used in NKG2D CAR-T. Its primary protein structure from the amino terminus to the carboxyl terminus is: signal peptide (Signal pep), NKG2D extracellular domain (NKG2D ECD), Hinge region (Hinge), transmembrane region (TM), intracellular costimulatory signal region and intracellular signal region.
  • the signal peptide can be selected from the signal peptides of human CD8 ⁇ , CD28, CD4, GM-CSF, etc.; the hinge region and the transmembrane region can be selected from the hinge region and transmembrane region of human CD8 ⁇ , CD28, etc.; the intracellular costimulatory signal region It can be selected from 41-BB, CD28, CD27, OX40 or ICOS; the intracellular signal region can be selected from CD3 ⁇ , FcR ⁇ , etc.
  • Figure 2 the signal peptide is derived from human CD8 ⁇ , and its amino acid sequence is as shown in SEQ ID NO.
  • NKG2D extracellular domain is the extracellular domain of human NKG2D (NM_007360.4), and its amino acid sequence is as follows As shown in SEQ ID NO. 2; the hinge region and transmembrane region are derived from human CD8 ⁇ , the amino acid sequence of the CD8 ⁇ hinge region is shown in SEQ ID NO. 3, and the amino acid sequence of the CD8 ⁇ transmembrane region is shown in SEQ ID NO. 4; intracellular The co-stimulatory signal region is selected from 41BB, and its amino acid sequence is shown in SEQ ID NO. 11; the intracellular signal region is CD3 ⁇ , which provides an activation signal, and its amino acid sequence is shown in SEQ ID NO. 5.
  • CD8 ⁇ signal peptide SEQ ID NO. 6
  • NKG2D extracellular region SEQ ID NO. 7
  • CD8 ⁇ hinge region SEQ ID NO. 8
  • CD8 ⁇ transmembrane region SEQ ID NO. 9
  • 41BB intracellular costimulatory signal region SEQ ID NO. 12
  • CD3 ⁇ intracellular signal region SEQ ID NO. 10
  • CD8 ⁇ signal peptide amino acid sequence (SEQ ID NO. 1):
  • NKG2D extracellular domain amino acid sequence (SEQ ID NO. 2):
  • CD8 ⁇ hinge region amino acid sequence (SEQ ID NO. 3):
  • CD8 ⁇ transmembrane region amino acid sequence (SEQ ID NO. 4):
  • CD8 ⁇ signal peptide nucleotide sequence (SEQ ID NO. 6):
  • NKG2D extracellular domain nucleotide sequence (SEQ ID NO. 7):
  • CD8 ⁇ hinge region nucleotide sequence (SEQ ID NO. 8):
  • CD8 ⁇ transmembrane region nucleotide sequence (SEQ ID NO. 9):
  • CD3 ⁇ intracellular signal region nucleotide sequence (SEQ ID NO. 10):
  • NKG2D CAR adenovirus is packaged using the pAdEasy system recombinant adenovirus packaging system.
  • the backbone vector used for Ad5F35 adenovirus packaging is Ad5F35 Helper.
  • centrifuge After freezing and thawing is completed, centrifuge at 12000g for 2 minutes and collect the supernatant. The collected virus supernatant was evenly dropped into 10 10 cm petri dishes, mixed and placed in a CO 2 incubator for culture. After 2-3 days, centrifuge to collect the supernatant and cells. Add PEG8000 and NaCl to the virus supernatant. Mix well and place it upright at 4°C overnight. Centrifuge the next day to collect the supernatant. Store the cell pellet at -80°C, and then add adenovirus for cryopreservation. The solution was resuspended and frozen and thawed 4 times, and the supernatant was collected after high-speed centrifugation.
  • the remaining cells were disrupted by sonication, and the sample supernatant was collected after centrifugation. After mixing all the collected samples, the virus is purified using iodixanol density gradient centrifugation. Finally, the purified virus solution is filtered through a 0.22 micron filter membrane and aliquoted for storage at low temperature.
  • PBMC peripheral blood mononuclear cells
  • the above-mentioned CAR-NKG2D coding gene was inserted into the pWPXLD vector between the BamHI and EcoRI enzyme cutting sites, and located behind the elongation factor 1 ⁇ (EF1 ⁇ ) of the pWPXLD vector, with EF1 ⁇ as the promoter.
  • a start codon such as ATG
  • a stop codon such as TAA
  • the pWPXLd-CAR-NKG2D recombinant plasmid obtained above, the packaging plasmid psPAX2, and the envelope plasmid pMD2G were co-transfected into cultured HEK293T cells using lipofectamine transfection reagent Lipofectamine3000.
  • the virus-containing supernatant was harvested at 48 h, filtered through a 0.45 ⁇ m filter, and stored in a -80°C ultra-low temperature refrigerator; the virus-containing supernatant was harvested a second time at 72 h, filtered through a 0.45 ⁇ m filter, and stored with the virus-containing supernatant at 72 h.
  • the virus supernatants were combined and added to the ultracentrifuge tube together, and placed one by one into the Beckman ultracentrifuge. Set the centrifugation parameters to 25000 rpm, the centrifugation time to 2 h, and the centrifugation temperature to 4°C; after centrifugation, discard the supernatant. , try to remove the liquid remaining on the tube wall, add the virus preservation solution, gently pipette and resuspend; after being fully dissolved, centrifuge at 10,000 rpm at high speed, centrifuge for 5 minutes, take the supernatant and measure the titer using the fluorescence method. ⁇ l, 2 ⁇ 10 8 pieces/mL, aliquot and store in -80°C ultra-low temperature refrigerator to obtain recombinant lentivirus with CAR-NKG2D encoding gene.
  • PBMC Peripheral Blood Mononuclear Cells
  • PBMC are derived from autologous venous blood, autologous bone marrow, umbilical cord blood, placental blood, etc. It is best to come from fresh peripheral blood or bone marrow collected from cancer patients one month after surgery and one month after radiotherapy and chemotherapy.
  • the patient's blood is extracted and the sample is sent to the blood separation chamber; peripheral blood mononuclear cells are collected, centrifuged by Ficoll and the middle layer cells are taken; after washing with PBS, PBMC are obtained.
  • PBMC blood pressure
  • CD3/CD28 immunomagnetic beads at a ratio of magnetic beads to cells of 3:1, and incubate at room temperature for 1-2 hours; use a pair of magnets to incubate. Cells with good magnetic beads were screened; after washing with PBS and removing the immunomagnetic beads, CD3-positive T lymphocytes were obtained.
  • CD3-positive T lymphocytes obtained by the immunomagnetic bead separation method in b) Take the CD3-positive T lymphocytes obtained by the immunomagnetic bead separation method in b), add the recombinant lentivirus described in 2.2 with a virus titer corresponding to the number of CD3-positive cells, and culture it.
  • the experiment is divided into 4 groups: Control group; CAR-M group; CAR-T group; CAR-T and CAR-M combination group; 50% CAR-T and 50% CAR-M group.
  • the SPECTROstar Omega microplate reader was used to collect and analyze the fluorescence intensity for cell killing experiments.
  • NKG2D CAR-M cells at 2 ⁇ 10 4 cells/well (CAR-M group; CAR-T and CAR-M combination group) or 1 ⁇ 10 4 cells/well (50% CAR-T and 50% CAR -M group) were inoculated into an opaque 96-well cell culture plate, with 10 multiple wells in each group, and allowed to stand for 24 hours.
  • % cell lysis (Lysis%) [1 - (fluorescence signal of co-cultured cells - background fluorescence signal) /
  • the killing results of PC-3 cells are shown in Figure 5: the CAR-T group is better than the CAR-M group in killing, the 50% CAR-T and 50% CAR-M groups are significantly better than the CAR-T group, and the CAR-T+ CAR-M has the strongest killing effect.
  • the total number of cells in the 50% CAR-T and 50% CAR-M group is the same as that in the CAR-T group, which contains 50% CAR-T and 50% CAR-M cells, but the killing effect of the 50% CAR-T and 50% CAR-M group was significantly better than that of the CAR-T group, which shows that CAR-T and CAR-M have a synergistic effect, 1+1 ⁇ 2.
  • the experiment was divided into 4 groups: Control group; non-CAR-transduced macrophage group (M group); CAR-T group; CAR-T and M combination group; 50% CAR-T and 50% M group.
  • the SPECTROstar Omega microplate reader was used to collect and analyze the fluorescence intensity for cell killing experiments.
  • macrophage (M) cells were added at 2 ⁇ 10 4 cells/well (M group; CAR-T and M combined group) or 1 ⁇ 10 4 cells/well (50% CAR-T and 50% M group). Inoculate into a light-proof 96-well cell culture plate, with 10 multiple wells in each group, and let stand for 24 hours.
  • % cell lysis (Lysis%) [1 - (fluorescence signal of co-cultured cells - background fluorescence signal) /
  • the killing results of PC-3 cells are shown in Figure 7:
  • the 50% T and 50% CAR-M groups were significantly better than the T group and CAR-M group, and T+CAR-M had the strongest killing effect.
  • the total cell number is the same as the T group and CAR-M group, which contains 50% T and 50% CAR-M cells, but 50% T and 50% CAR
  • the killing effect of the -M group was significantly better than that of the T group and CAR-M, which shows that CAR-T and CAR-M have a synergistic effect, 1+1 ⁇ 2.

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Abstract

本发明公开了CAR-T和CAR-M联用在制备抗肿瘤药物中的应用，CAR-T为经嵌合抗原受体修饰的T细胞，嵌合抗原受体一级蛋白结构从氨基端到羧基端顺次为：信号肽、NKG2D胞外区、铰链区、跨膜区、胞内共刺激信号区和胞内信号区；CAR-M为经嵌合抗原受体修饰的巨噬细胞，嵌合抗原受体一级蛋白结构从氨基端到羧基端顺次为：信号肽、NKG2D胞外区、铰链区、跨膜区和胞内信号区；NKG2D胞外区为人NKG2D胞外域，氨基酸序列如SEQ ID NO.2所示。CAR-T和CAR-M联用将CAR-M改善肿瘤微环境的能力与CAR-T直接杀伤肿瘤细胞的能力相结合，可以实现对肿瘤细胞的杀伤。

Description

CAR-T和CAR-M联用在制备抗肿瘤药物中的应用 技术领域
本发明涉及肿瘤治疗领域，具体涉及细胞免疫***，尤其涉及嵌合抗原受体T细胞（CAR-T）和嵌合抗原受体巨噬细胞（CAR-M）细胞免疫疗法，更具体地涉及CAR-T和CAR-M联用在制备抗肿瘤药物中的应用。
 背景技术
肿瘤严重威胁着我国国民健康，并为社会经济造成巨大的负担。尽管随着医药科技的飞速发展，越来越多的方法手段被用于肿瘤的治疗，但是远远还不够，仍面临着巨大的未满足的临床需求。因此，积蓄开发新的肿瘤治疗的方法。
一直以来，恶性肿瘤的治疗疗效不足、治愈率低，使之成为严重威胁着人类健康的重大疾病。传统的放化疗法旨在直接消灭肿瘤，但肿瘤常复发转移，又会杀伤正常组织细胞。肿瘤的细胞免疫治疗的目的则是促进或修饰免疫细胞攻击癌细胞，同时保持正常细胞的完整性。与传统的放化疗相比，细胞的过继治疗具有良好的特异性、安全性、有效性和持久性，在2013年十大科技进步中排名第一。
近年来，以DCs、T细胞、NK细胞等为基础的过继性细胞治疗在肿瘤治疗中取得了较好的效果。尤其是以嵌合抗原受体T细胞（Chimeric Antigen Receptor T-Cell, CAR-T）为代表的免疫细胞治疗技术在血液肿瘤的治疗上展现出了强大的疗效和巨大的发展潜力，已然成为人类对抗癌症的新希望。嵌合抗原受体（CAR）T细胞免疫疗法是一种肿瘤过继细胞免疫疗法，基本原理是利用基因工程修饰T淋巴细胞，使其表达嵌合抗原受体，以非主要组织相容性复合体（MHC，major histocompatibility complex）限制性的方式杀伤肿瘤细胞。虽然CAR T细胞在临床及临床试验中取得了较好的效果，但同时也出现了较为严重的副作用，包括细胞因子释放综合征、神经毒性和脱靶效应等。而且，CAR-T细胞疗法对除一些血液***肿瘤之外的其他肿瘤（如实体瘤）的有效性在临床试验中也不令人满意，亟需提高。在实体瘤的治疗中，CAR-T细胞治疗未取得较大突破有以下几个原因：首先，CAR-T细胞必须运输并渗透到肿瘤中，这是一个需要外渗，趋化性和基质组织渗透的过程。CAR-T细胞必须穿过粘附分子减少的异常肿瘤血管，经历趋化因子/趋化因子受体失配，并且必须通过密集的细胞和基质屏障迁移。进入肿瘤微环境（Tumor Microenvironment, TME）后，效应细胞会遇到不利条件，例如低氧和酸性环境、免疫检查点配体的表达以及大量免疫抑制细胞，如肿瘤相关巨噬细胞（Tumor Associated Macrophage, TAM）、髓源性抑制细胞（Myeloid Derived Suppressor Cell, MDSC）和调节性T细胞（Regulatory Cells, T-regs）。此外，长期的抗原接触会导致T细胞衰竭，从而降低CAR T细胞的效应子功能。即使CAR-T细胞在TME中存活，实体瘤也经常具有异质的表面抗原表达，这可能导致逃避CAR T细胞检测，肿瘤清除不完全以及抗原阴性肿瘤细胞的最终生长。这在使用靶向EGFRvIII的CAR T细胞治疗胶质母细胞瘤中得到了明确的证明，在治疗后5/7的患者中EGFRvIII的表达下降了。最后，CAR-T细胞可能会识别正常组织和细胞中的肿瘤相关抗原，从而导致在靶/脱肿瘤毒性(on-target/off tumor toxicity)，这也是实体肿瘤细胞治疗的另一个障碍。因而，将CAR-T与其他治疗手段联合，有可能能弥补CAR-T在这方面的不足，抵抗实体瘤局部肿瘤微环境免疫抑制性的影响，从而起到协同抗肿瘤的效应。
近年来，巨噬细胞杀伤肿瘤的潜能逐渐受到关注。CAR修饰的巨噬细胞(CAR-M)被认为是一种有前途的细胞类型。巨噬细胞主要通过吞噬体液和组织中的细胞残片及病原体并呈递抗原以激活淋巴或其他免疫细胞，令其对病原体做出反应。在肿瘤微环境中，巨噬细胞是具有最高浸润率的先天免疫细胞并可与TME中几乎所有细胞成分相互作用，刺激血管生成，增加肿瘤浸润，并介导免疫抑制。但是CAR-M直接杀伤肿瘤细胞的作用有限。
 技术问题
虽然CAR T细胞在临床及临床试验中取得了较好的效果，但同时也出现了较为严重的副作用，包括细胞因子释放综合征、神经毒性和脱靶效应等。而且，CAR-T细胞疗法对除一些血液***肿瘤之外的其他肿瘤（如实体瘤）的有效性在临床试验中也不令人满意，亟需提高。在实体瘤的治疗中，CAR-T细胞治疗未取得较大突破有以下几个原因：首先，CAR-T细胞必须运输并渗透到肿瘤中，这是一个需要外渗，趋化性和基质组织渗透的过程。CAR-T细胞必须穿过粘附分子减少的异常肿瘤血管，经历趋化因子/趋化因子受体失配，并且必须通过密集的细胞和基质屏障迁移。进入肿瘤微环境（Tumor Microenvironment, TME）后，效应细胞会遇到不利条件，例如低氧和酸性环境、免疫检查点配体的表达以及大量免疫抑制细胞，如肿瘤相关巨噬细胞（Tumor Associated Macrophage, TAM）、髓源性抑制细胞（Myeloid Derived Suppressor Cell, MDSC）和调节性T细胞（Regulatory Cells, T-regs）。此外，长期的抗原接触会导致T细胞衰竭，从而降低CAR T细胞的效应子功能。即使CAR-T细胞在TME中存活，实体瘤也经常具有异质的表面抗原表达，这可能导致逃避CAR T细胞检测，肿瘤清除不完全以及抗原阴性肿瘤细胞的最终生长。这在使用靶向EGFRvIII的CAR T细胞治疗胶质母细胞瘤中得到了明确的证明，在治疗后5/7的患者中EGFRvIII的表达下降了。最后，CAR-T细胞可能会识别正常组织和细胞中的肿瘤相关抗原，从而导致在靶/脱肿瘤毒性(on-target/off tumor toxicity)，这也是实体肿瘤细胞治疗的另一个障碍。因而，将CAR-T与其他治疗手段联合，有可能能弥补CAR-T在这方面的不足，抵抗实体瘤局部肿瘤微环境免疫抑制性的影响，从而起到协同抗肿瘤的效应。
 技术解决方案
为了解决现有技术中（1）CAR-T治疗实体瘤存在的缺陷：包括肿瘤异质性，导致CAR靶点阴性细胞逃逸；CAR-T难以浸润到肿瘤内部，杀伤效果不足；实体瘤免疫抑制微环境的影响，导致CAR-T耗竭，杀伤功能丧失；CAR-T自身的副作用，如细胞因子释放综合征、神经毒性和脱靶效应等；（2）CAR-M细胞通过吞噬或胞啃肿瘤细胞杀伤肿瘤，其杀伤肿瘤细胞的效率不高的问题，本发明旨在提供CAR-T和CAR-M联用在制备抗肿瘤药物中的应用。
基于CAR-M与CAR-T的比较优势：（1）CAR-M可以改善实体瘤肿瘤免疫微环境；在吞噬肿瘤细胞后可以作为抗原呈递细胞呈递抗原；更容易浸润到肿瘤内部，协同其他免疫细胞浸润肿瘤等；（2）CAR-T直接杀伤肿瘤细胞的能力，本发明提出CAR-T和CAR-M联合使用的方式进行肿瘤治疗的方案。具体方案如下：
本发明第一方面提供CAR-T和巨噬细胞联用在制备抗肿瘤药物中的应用，所述CAR-T为经嵌合抗原受体修饰的T细胞，所述嵌合抗原受体一级蛋白结构从氨基端到羧基端顺次为：信号肽、NKG2D胞外区、铰链区、跨膜区、胞内共刺激信号区和胞内信号区；
所述NKG2D胞外区为人NKG2D的胞外域，其氨基酸序列如SEQ ID NO. 2所示。
本发明第二方面提供CAR-M和T细胞联用在制备抗肿瘤药物中的应用，所述CAR-M为经嵌合抗原受体修饰的巨噬细胞，所述嵌合抗原受体一级蛋白结构从氨基端到羧基端顺次为：信号肽、NKG2D胞外区、铰链区、跨膜区和胞内信号区；
所述NKG2D胞外区为人NKG2D的胞外域，其氨基酸序列如SEQ ID NO. 2所示。
本发明第三方面提供CAR-T和CAR-M联用在制备抗肿瘤药物中的应用，所述CAR-T为经嵌合抗原受体修饰的T细胞，所述嵌合抗原受体一级蛋白结构从氨基端到羧基端顺次为：信号肽、NKG2D胞外区、铰链区、跨膜区、胞内共刺激信号区和胞内信号区；
所述CAR-M为经嵌合抗原受体修饰的巨噬细胞，所述嵌合抗原受体一级蛋白结构从氨基端到羧基端顺次为：信号肽、NKG2D胞外区、铰链区、跨膜区和胞内信号区；
所述CAR-T和CAR-M中嵌合抗原受体NKG2D胞外区为人NKG2D的胞外域，其氨基酸序列如SEQ ID NO. 2所示。
上述应用中，所述信号肽选自CD8α信号肽、CD28信号肽、CD4信号肽或GM-CSF信号肽；
所述铰链区选自CD8α铰链区或CD28铰链区；
所述跨膜区选自CD8α跨膜区或CD28跨膜区；
所述胞内信号区选自CD3ζ或FcRγ。
上述应用中，所述信号肽源于人CD8α，其氨基酸序列如SEQ ID NO. 1所示；
所述铰链区源于人CD8α，其氨基酸序列如SEQ ID NO. 3所示；
所述跨膜区源于人CD8α，其氨基酸序列如SEQ ID NO. 4所示；
所述胞内信号区为CD3ζ，其氨基酸序列如SEQ ID NO. 5所示。
上述应用中，所述胞内共刺激信号区选自4-1BB、CD28、CD27、OX40或ICOS；
优选地，所述胞内共刺激信号区选自4-1BB，其氨基酸序列如SEQ ID NO. 11所示。
上述应用中，所述CAR-T构建方法为：将CAR-T嵌合抗原受体编码基因的核酸或载体导入T细胞中；
所述导入的方式包括电穿孔、转导或转染；
优选地，所述核酸位于不同的病毒载体上；所述病毒载体为慢病毒载体、腺病毒载体或逆转录病毒载体；
优选地，所述载体为转座子或mRNA载体；
优选地，将包含CAR-T嵌合抗原受体编码基因的慢病毒载体通过转染导入T细胞中。
上述应用中，所述CAR-M构建方法为：将CAR-M嵌合抗原受体编码基因的核酸或载体导入巨噬细胞细胞中；
所述导入的方式包括电穿孔、转导或转染；
优选地，所述核酸位于不同的病毒载体上；所述病毒载体为慢病毒载体、腺病毒载体或逆转录病毒载体；
优选地，所述载体为转座子或mRNA载体；
优选地，将包含CAR-M嵌合抗原受体编码基因的腺病毒载体通过转染导入巨噬细胞细胞中。
上述应用中，所述巨噬细胞源自健康人体或癌症患者；
所述巨噬细胞选自自体巨噬细胞、异体巨噬细胞或iPSC诱导的巨噬细胞；
优选地，所述巨噬细胞为原代巨噬细胞；
所述T细胞源自健康人体或癌症患者；
所述T细胞选自自体T细胞、异体T细胞或iPSC诱导的T细胞。
上述应用中，所述肿瘤为NKG2D配体表达的肿瘤；
所述肿瘤为血液瘤或实体瘤；
优选地，所述肿瘤为白血病、多发性骨髓瘤、恶性淋巴瘤、脑胶质瘤、肝癌、肺癌、胃癌、结肠癌、胰腺癌或乳腺癌。
 有益效果
本发明的有益效果为：
1、本发明以NKG2D胞外段天然序列作为CAR识别区，使嵌合抗原受体具有低免疫原性、易表达等优势，可以在巨噬细胞和T细胞上正常表达，为制备特异性杀伤/吞噬肿瘤细胞的嵌合抗原受体巨噬细胞（CAR-M）和嵌合抗原受体T细胞（CAR-T）提供基础。优选地以CD3ζ作为胞内信号区，CD3ζ含有的ITAM结构赋予CAR-M更好的吞噬/杀伤肿瘤效果。
2、本发明通过CAR-T和CAR-M联用，CAR-T和M细胞联用或者CAR-M和T细胞联用，可以实现在抗肿瘤方面的协同增效，提高***的效果。尤其是联合CAR-T和CAR-M细胞进行肿瘤治疗，其将CAR-M改善肿瘤微环境的能力与CAR-T直接杀伤肿瘤细胞的能力相结合，具有协同增效作用，可以实现对肿瘤细胞（尤其是实体瘤）的强力高效杀伤，极大提高疗效，解决CAR-T单独治疗实体瘤时存在的缺陷以及CAR-M单独杀伤肿瘤细胞效率不高的问题。此外，联用方案可以降低CAR-T细胞的使用量，从而降低CAR-T治疗副作用。
 附图说明
图1：NKG2D CAR-M中NKG2D CAR的示意图。
图2：NKG2D CAR-T中NKG2D CAR的示意图。
图3：NKG2D CAR-M细胞CAR阳性率。
图4：NKG2D CAR-T细胞CAR阳性率。
图5：人原代CAR-M和CAR-T细胞对肿瘤细胞的杀伤作用实验，杀伤48小时后各组的杀伤率统计（效应细胞：靶细胞=1）。
图6：人原代巨噬细胞（M）和CAR-T细胞对肿瘤细胞的杀伤作用实验，杀伤48小时后各组的杀伤率统计（效应细胞：靶细胞=1）。
图7：人原代CAR-M和原代T细胞对肿瘤细胞的杀伤作用实验，杀伤48小时后各组的杀伤率统计（效应细胞：靶细胞=1）。
 本发明的实施方式
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例1
1. NKG2D CAR-M中的所用的NKG2D嵌合抗原受体
本实施例提供NKG2D CAR-M中的所用的NKG2D嵌合抗原受体，其一级蛋白结构从氨基端到羧基端顺次为：信号肽（Signal pep）、NKG2D胞外区（NKG2D ECD）、铰链区（Hinge）、跨膜区（TM）和胞内信号区。其中，信号肽可以选自人CD8α、CD28、CD4、GM-CSF等的信号肽；铰链区和跨膜区可以选自人CD8α、CD28等的铰链区和跨膜区；胞内信号区可以选自CD3ζ、FcRγ等。在一个具体的实施方式中（图1），信号肽源于人CD8α，其氨基酸序列如SEQ ID NO. 1所示；NKG2D胞外区为人NKG2D（NM_007360.4）的胞外域，其氨基酸序列如SEQ ID NO. 2所示；铰链区和跨膜区源于人CD8α，CD8α铰链区氨基酸序列如SEQ ID NO. 3所示，CD8α跨膜区氨基酸序列如SEQ ID NO. 4所示；胞内信号区为CD3ζ，CD3ζ提供激活信号，其氨基酸序列如SEQ ID NO. 5所示。具体核苷酸序列：CD8α信号肽（SEQ ID NO. 6）、NKG2D胞外区（SEQ ID NO. 7）、CD8α铰链区（SEQ ID NO. 8）、CD8α跨膜区（SEQ ID NO. 9）、CD3ζ胞内信号区（SEQ ID NO. 10）通过人工合成，得到NKG2D CAR-M中的所用的NKG2D嵌合抗原受体序列。
2. NKG2D CAR-T中的所用的NKG2D嵌合抗原受体
本实施例提供NKG2D CAR-T中的所用的NKG2D嵌合抗原受体，其一级蛋白结构从氨基端到羧基端顺次为：信号肽（Signal pep）、NKG2D胞外区（NKG2D ECD）、铰链区（Hinge）、跨膜区（TM）、胞内共刺激信号区和胞内信号区。其中，信号肽可以选自人CD8α、CD28、CD4、GM-CSF等的信号肽；铰链区和跨膜区可以选自人CD8α、CD28等的铰链区和跨膜区；胞内共刺激信号区可以选自41-BB、CD28、CD27、OX40或ICOS；胞内信号区可以选自CD3ζ、FcRγ等。在一个具体的实施方式中（图2），信号肽源于人CD8α，其氨基酸序列如SEQ ID NO. 1所示；NKG2D胞外区为人NKG2D（NM_007360.4）的胞外域，其氨基酸序列如SEQ ID NO. 2所示；铰链区和跨膜区源于人CD8α，CD8α铰链区氨基酸序列如SEQ ID NO. 3所示，CD8α跨膜区氨基酸序列如SEQ ID NO. 4所示；胞内共刺激信号区选自41BB，其氨基酸序列如SEQ ID NO. 11所示；胞内信号区为CD3ζ，CD3ζ提供激活信号，其氨基酸序列如SEQ ID NO. 5所示。具体核苷酸序列：CD8α信号肽（SEQ ID NO. 6）、NKG2D胞外区（SEQ ID NO. 7）、CD8α铰链区（SEQ ID NO. 8）、CD8α跨膜区（SEQ ID NO. 9）、41BB胞内共刺激信号区（SEQ ID NO. 12）、CD3ζ胞内信号区（SEQ ID NO. 10）通过人工合成，得到NKG2D CAR-M中的所用的NKG2D嵌合抗原受体序列。
3. 本实施例中氨基酸和核苷酸序列
CD8α信号肽氨基酸序列（SEQ ID NO. 1）：
MALPVTALLLPLALLLHAARP
NKG2D胞外区氨基酸序列（SEQ ID NO. 2）：
IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV
CD8α铰链区氨基酸序列（SEQ ID NO. 3）：
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
CD8α跨膜区氨基酸序列（SEQ ID NO. 4）：
IYIWAPLAGTCGVLLLSLVITLYC
CD3ζ胞内信号区氨基酸序列（SEQ ID NO. 5）：
RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
CD8α信号肽核苷酸序列（SEQ ID NO. 6）：
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG
NKG2D胞外区核苷酸序列（SEQ ID NO. 7）：
ATATGGAGTGCTGTATTCCTAAACTCATTATTCAACCAAGAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACGTACATCTGCATGCAAAGGACTGTG
CD8α铰链区核苷酸序列（SEQ ID NO. 8）：
ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT
CD8α跨膜区核苷酸序列（SEQ ID NO. 9）：
ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC
CD3ζ胞内信号区核苷酸序列（SEQ ID NO. 10）：
AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC
41BB胞内共刺激信号区氨基酸序列（SEQ ID NO. 11）：
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
4-1BB胞内共刺激信号区核苷酸序列（SEQ ID NO. 12）：
AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAA
实施例2
1. NKG2D CAR-M的制备
1.1. 重组腺病毒构建
NKG2D CAR腺病毒采用pAdEasy system重组腺病毒的包装体系进行包装，Ad5F35腺病毒包装所用的骨架载体是Ad5F35 Helper。使用PEI对Ad5F35相关包装质粒在HEK 293细胞中进行转染，待混匀后置于CO 2培养箱中培养。培养14天后3500 g离心收集细胞，加入腺病毒冻存液后重悬沉淀，将悬液在-80℃及37℃反复冻融四次，冻融完成后，12000g离心2 min，收集上清。将收集起来的病毒上清平均滴加到10个10 cm培养皿中，混匀后置于CO 2培养箱中培养。2-3天后离心收集上清和细胞，病毒上清加入PEG8000和NaCl，混匀后4℃直立放置过夜，第二天离心收集上清；细胞沉淀置于-80℃保存，后续加入腺病毒冻存液后重悬后反复冻融4次，高速离心后收集上清。剩余的细胞行超声破碎，离心后收集样本上清。将所有收集的样本混合后，用碘克沙醇密度梯度离心法对病毒进行纯化，最后将纯化好的的病毒溶液通过0.22微米滤膜过滤后分装低温保存。
1.2 靶向NKG2D的嵌合抗原受体M细胞的制备
a）全血分离人外周血单个核细胞（PBMC）
在50 mL 离心管中加入全血25 mL，再加入25 mL PBS 与其混匀；往50 mL 离心管中加入15 mL FICOLL 试剂，然后沿壁缓慢加入30 mL 上述稀释好的血；离心机20℃，2110 r/min，升2降2，离心30 min；离心之后液体分四层，将最上层黄色液体去掉，转圈吸取第二层白膜层，所得白膜层液体均分成两管，加PBS至40-45 mL 混匀，离心机20℃，1800 r/min，升9降9，离心8 min；去上清，加40 mL PBS 把两管重悬成一管，离心机20℃，1200 r/min，升9降9，离心8 min；去上清，加40 mL PBS 重悬计数，剩下的细胞20℃，1200 r/min，升9降9，离心8min；去上清，用冻存液冻存备用。
b）分离人CD14 +细胞
将复苏的PBMC细胞置于离心机中300 g 离心5 min，丢弃上清；4-5 mL 左右的MojoSort™缓冲液重悬。用70 μM 的细胞筛网过滤细胞，离心机300 g 离心5 min 后去上清，然后用适当体积的MojoSort™Buffer 重悬并调整细胞浓度至1×10 8个/mL。往100μL 细胞悬液（10 7个细胞）中加入5 μL Human TruStain FcX™（Fc受体封闭溶液），混合均匀后室温下孵育10 min。如果分离更多的细胞，则相应比例增加试剂用量。加入10 μL Biotin-Antibody Cocktail，混合均匀后冰上孵育15 min。如果分离更多的细胞，则相应比例增加试剂用量。加入10μL 通过最大速度涡旋重悬的Streptavidin Nanobeads，混合均匀后冰上孵育15 min。如果分离更多的细胞，则相应比例增加试剂用量。加入4 mL 的MojoSort™缓冲液以洗涤细胞，置于入离心机300 g 离心5 min 后丢弃上清。加入2.5 mL MojoSort™缓冲液重悬细胞于干净的流式管中，并将其放在磁力架中静置5 min。倒出并收集液体于一新的15 mL 离心管中备用。然后用2.5 mL MojoSort™缓冲液重悬剩下的沉淀。将收集好的液体合并，置于离心机300 g 离心5 min后用制备好的人原代巨噬细胞培养基(RPMI-1640 培养基中加入10%的FBS、1%的PS 和终浓度为20 ng/mL 的人GM-CSF)重悬并计数，接种于非TC 处理的6 孔板中培养。可以另取一小部分细胞用作于流式染色，以检测其纯度。
c）人原代巨噬细胞的培养
用配制好的人原代巨噬细胞培养基培养分离好的人CD14 +细胞于非TC 处理的6 孔板中（3-8×10 5个/mL），37℃，5%二氧化碳培养箱中培养。于第三天轻轻吸取上清半量换液，第五天全量换液后获得的贴壁细胞则为人原代巨噬细胞。
d）腺病毒感染人原代巨噬细胞
分离好的人CD14 +细胞计数后培养于非TC 处理的6 孔板中（3-8 ×10 5个/mL）；5 天后按照MOI=200-1000的病毒滴度加入腺病毒（对照病毒及NKG2D CAR腺病毒）；24-48 h后换成不带病毒的培养基；48 h 后用检测CAR-M的阳性率：将消化下来的CAR-M进行流式染色检测CAR的表达。
流式结果显示（附图3）：NKG2D CAR-M的阳性率>50%。表明成功制得得到靶向NKG2D的人原代CAR-M细胞（人原代CAR-M细胞）。
2. NKG2D CAR-T的制备
2.1. 构建pWPXLd-CAR-NKG2D重组质粒
将上述CAR-NKG2D的编码基因***到pWPXLD载体的BamHⅠ和EcoRⅠ酶切位点之间，且位于pWPXLD载体的延伸因子1α（EF1α）之后，以EF1α为启动子。所述CAR-NKG2D的编码基因***到pWPXLD载体时，在所述CAR-NKG2D的编码基因的5’端可加入起始密码子（如ATG）与pWPXLD载体中BamHⅠ酶切位点相连，3’端可加入终止密码子（如TAA）与pWPXLD载体中EcoRⅠ酶切位点相连。然后转入大肠杆菌感受态细胞DH5α，进行阳性克隆PCR鉴定和测序鉴定。经过PCR产物凝胶电泳检测和测序鉴定符合目的片段大小和序列，获得pWPXLd-CAR-NKG2D重组质粒。
2.2. 重组慢病毒构建
将上述获得的pWPXLd-CAR-NKG2D重组质粒与包装质粒psPAX2、包膜质粒pMD2G三者通过脂质体转染试剂Lipofectamine3000共转染培养好的HEK293T细胞。第48 h收获含病毒的上清，经0.45 μm滤膜过滤，于-80℃超低温冰箱中保存；第72 h二次收获含病毒的上清，0.45μm滤膜过滤，与第48 h收获的病毒上清合并后一起加入超速离心管中，逐一放入至Beckman超速离心机内，设置离心参数为25000 rpm，离心时间为2 h，离心温度控制在4℃；离心结束后，弃去上清，尽量去除残留在管壁上的液体，加入病毒保存液，轻轻反复吹打重悬；经充分溶解后，高速离心10000 rpm，离心5 min后，取上清荧光法测定滴度，病毒按照100 μl，2×10 8个/mL分装，保存于-80℃超低温冰箱，得到带CAR-NKG2D编码基因的重组慢病毒。
2.3 靶向NKG2D的嵌合抗原受体T细胞的制备
a）PBMC（外周血单个核细胞）的分离
PBMC来源于自体静脉血、自体骨髓、脐带血和胎盘血等。最好是来源于癌症患者手术一个月后、放化疗一个月后采集的新鲜外周血或骨髓。
抽取病人血液，送样至血液分离室；采集外周血单个核细胞，Ficoll离心分离后取中间层细胞；经PBS洗涤后，得到PBMC。
b）免疫磁珠法分离抗原特异性T淋巴细胞
取上述PBMC，加入不含血清的基础培养基，配成细胞悬液；按磁珠与细胞的比例为3:1，加入CD3/CD28免疫磁珠，室温孵1-2 h；采用磁铁对孵育好磁珠的细胞进行筛选；PBS洗涤，去除免疫磁珠后，得到CD3阳性T淋巴细胞。
c）病毒转染法制备抗原特异性T淋巴细胞
取b）中经免疫磁珠分离法得到的CD3阳性T淋巴细胞，加入与CD3阳性细胞数相应的病毒滴度的2.2中所述重组慢病毒进行培养。
培养的第3天，进行细胞计数和换液，调整细胞浓度为1×10 6个/mL，接种，培养；培养的第5天，观察细胞状态，如果细胞密度增大，则稀释细胞浓度为1×10 6个/mL，检测细胞活性，继续培养。扩增培养到第9-11天，收集细胞，同时经过流式细胞仪检测靶向NKG2D的嵌合抗原受体CAR-NKG2D的表达，结果如图4所示。经检测，经上述重组慢病毒感染的T细胞中，CAR-NKG2D的阳性率约为70%，这表明成功制得得到靶向NKG2D的CAR-T细胞（CAR-T细胞）。
3. 人原代CAR-M和CAR-T细胞对肿瘤细胞的杀伤作用
实验共分为4组：Control组；CAR-M组；CAR-T组；CAR-T和CAR-M联用组；50% CAR-T和50% CAR-M组。采用SPECTROstar Omega酶标仪对荧光强度进行采集分析进行细胞杀伤实验。首先把NKG2D CAR-M细胞以2×10 4个/孔（CAR-M组；CAR-T和CAR-M联用组）或1×10 4个/孔（50% CAR-T和50% CAR-M组）接种至不透光的96孔细胞培养板中，每组10个复孔，静置24 h。24 h后按照效应细胞：靶细胞=1：1的比例加入稳定表达luciferase的PC-3细胞，以及2×10 4个/孔（CAR-T组；CAR-T和CAR-M联用组）或1×10 4个/孔（50% CAR-T和50% CAR-M组）的CAR-T数量。
共培养48 h后，37℃，5%二氧化碳培养箱中培养48 h后，向不透光96孔板中加入浓度为100-200 μg/mL的D-luciferin，potassium salt底物， 37℃避光孵育10 min后，使用SPECTROstar Omega酶标仪对荧光强度进行采集分析，并通过以下公式各免疫细胞组对PC-3靶细胞的杀伤进行计算：
%细胞溶解(Lysis%) = 【1 -（共培养细胞的荧光信号-背景荧光信号）/
（单独培养PC-3细胞荧光信号-背景荧光信号）】*100
PC-3细胞的杀伤结果如附图5所示：CAR-T组杀伤优于CAR-M组，50% CAR-T和50% CAR-M组显著优于CAR-T组，CAR-T+CAR-M杀伤作用最强。鉴于CAR-T细胞杀伤效果强于CAR-M细胞，在50% CAR-T和50% CAR-M组中，其总细胞数和CAR-T组一样，其中含50%CAR-T和50%CAR-M细胞，但是50% CAR-T和50% CAR-M组杀伤效果显著优于CAR-T组，这表明CAR-T和CAR-M具有协同增效作用，1+1》2。
4. 人原代巨噬细胞（M）和CAR-T细胞对肿瘤细胞的杀伤作用
实验共分为4组：Control组；未转CAR的巨噬细胞组（ M组）；CAR-T组；CAR-T和M联用组；50% CAR-T和50% M组。采用SPECTROstar Omega酶标仪对荧光强度进行采集分析进行细胞杀伤实验。首先把巨噬细胞（M）细胞以2×10 4个/孔（M组；CAR-T和M联用组）或1×10 4个/孔（50% CAR-T和50% M组）接种至不透光的96孔细胞培养板中，每组10个复孔，静置24 h。24 h后按照效应细胞：靶细胞=1：1的比例加入稳定表达luciferase的PC-3细胞，以及2×10 4个/孔（CAR-T组；CAR-T和M联用组）或1×10 4个/孔（50% CAR-T和50% M组）的CAR-T数量。
共培养48 h后，37℃，5%二氧化碳培养箱中培养48 h后，向不透光96孔板中加入浓度为100-200 μg/mL的D-luciferin，potassium salt底物， 37℃避光孵育10 min后，使用SPECTROstar Omega酶标仪对荧光强度进行采集分析，并通过以下公式各免疫细胞组对PC-3靶细胞的杀伤进行计算：
%细胞溶解(Lysis%) = 【1 -（共培养细胞的荧光信号-背景荧光信号）/
（单独培养PC-3细胞荧光信号-背景荧光信号）】*100
PC-3细胞的杀伤结果如附图6所示：CAR-T组杀伤优于M组，50% CAR-T和50% M组显著优于CAR-T组，CAR-T+M杀伤作用最强。鉴于CAR-T细胞杀伤效果强于M细胞，在50% CAR-T和50% M组中，其总细胞数和CAR-T组一样，其中含50% CAR-T和50% M细胞，但是50% CAR-T和50% M组杀伤效果显著优于CAR-T组，这表明CAR-T和M具有协同增效作用，1+1》2。
5. 人原代CAR-M和原代T细胞对肿瘤细胞的杀伤作用
实验共分为4组：Control组；CAR-M组；T细胞组；T细胞和CAR-M联用组；50% T和50% CAR-M组。采用SPECTROstar Omega酶标仪对荧光强度进行采集分析进行细胞杀伤实验。首先把NKG2D CAR-M细胞以2×10 4个/孔（CAR-M组；T和CAR-M联用组）或1×10 4个/孔（50% T和50% CAR-M组）接种至不透光的96孔细胞培养板中，每组10个复孔，静置24 h。24h后按照效应细胞：靶细胞=1：1的比例加入稳定表达luciferase的PC-3细胞，以及2×10 4个/孔（T组； T和CAR-M联用组）或1×10 4个/孔（50% T和50% CAR-M组）的T数量。
共培养48 h后，37℃，5%二氧化碳培养箱中培养48 h后，向不透光96孔板中加入浓度为100-200 μg/mL的D-luciferin，potassium salt底物， 37℃避光孵育10 min后，使用SPECTROstar Omega酶标仪对荧光强度进行采集分析，并通过以下公式各免疫细胞组对PC-3靶细胞的杀伤进行计算：
%细胞溶解(Lysis%) = 【1 -（共培养细胞的荧光信号-背景荧光信号）/
（单独培养PC-3细胞荧光信号-背景荧光信号）】*100
PC-3细胞的杀伤结果如附图7所示： 50% T和50% CAR-M组显著优于T组和CAR-M组，T+CAR-M杀伤作用最强。在50% T和50% CAR-M联用组中，其总细胞数和T组、CAR-M组一样，其中含50% T和50%CAR-M细胞，但是50% T和50% CAR-M组杀伤效果显著优于 T组和CAR-M，这表明CAR-T和CAR-M具有协同增效作用，1+1》2。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (10)

  1. CAR-T和巨噬细胞联用在制备抗肿瘤药物中的应用，其特征在于，所述CAR-T为经嵌合抗原受体修饰的T细胞，所述嵌合抗原受体一级蛋白结构从氨基端到羧基端顺次为：信号肽、NKG2D胞外区、铰链区、跨膜区、胞内共刺激信号区和胞内信号区；
    所述NKG2D胞外区为人NKG2D的胞外域，其氨基酸序列如SEQ ID NO. 2所示。
  2. CAR-M和T细胞联用在制备抗肿瘤药物中的应用，其特征在于，所述CAR-M为经嵌合抗原受体修饰的巨噬细胞，所述嵌合抗原受体一级蛋白结构从氨基端到羧基端顺次为：信号肽、NKG2D胞外区、铰链区、跨膜区和胞内信号区；
    所述NKG2D胞外区为人NKG2D的胞外域，其氨基酸序列如SEQ ID NO. 2所示。
  3. CAR-T和CAR-M联用在制备抗肿瘤药物中的应用，其特征在于，所述CAR-T为经嵌合抗原受体修饰的T细胞，所述嵌合抗原受体一级蛋白结构从氨基端到羧基端顺次为：信号肽、NKG2D胞外区、铰链区、跨膜区、胞内共刺激信号区和胞内信号区；
    所述CAR-M为经嵌合抗原受体修饰的巨噬细胞，所述嵌合抗原受体一级蛋白结构从氨基端到羧基端顺次为：信号肽、NKG2D胞外区、铰链区、跨膜区和胞内信号区；
    所述CAR-T和CAR-M中嵌合抗原受体NKG2D胞外区为人NKG2D的胞外域，其氨基酸序列如SEQ ID NO. 2所示。
  4. 根据权利要求1-3任一项所述的应用，其特征在于，所述信号肽选自CD8α信号肽、CD28信号肽、CD4信号肽或GM-CSF信号肽；
    所述铰链区选自CD8α铰链区或CD28铰链区；
    所述跨膜区选自CD8α跨膜区或CD28跨膜区；
    所述胞内信号区选自CD3ζ或FcRγ。
  5. 根据权利要求1-3任一项所述的应用，其特征在于，所述信号肽源于人CD8α，其氨基酸序列如SEQ ID NO. 1所示；
    所述铰链区源于人CD8α，其氨基酸序列如SEQ ID NO. 3所示；
    所述跨膜区源于人CD8α，其氨基酸序列如SEQ ID NO. 4所示；
    所述胞内信号区为CD3ζ，其氨基酸序列如SEQ ID NO. 5所示。
  6. 根据权利要求1或3所述的应用，其特征在于，所述胞内共刺激信号区选自4-1BB、CD28、CD27、OX40或ICOS；
    优选地，所述胞内共刺激信号区选自4-1BB，其氨基酸序列如SEQ ID NO. 11所示。
  7. 根据权利要求1或3所述的应用，其特征在于，所述CAR-T构建方法为：将CAR-T嵌合抗原受体编码基因的核酸或载体导入T细胞中；
    所述导入的方式包括电穿孔、转导或转染；
    优选地，所述核酸位于不同的病毒载体上；所述病毒载体为慢病毒载体、腺病毒载体或逆转录病毒载体；
    优选地，所述载体为转座子或mRNA载体；
    优选地，将包含CAR-T嵌合抗原受体编码基因的慢病毒载体通过转染导入T细胞中。
  8. 根据权利要求2或3所述的应用，其特征在于，所述CAR-M构建方法为：将CAR-M嵌合抗原受体编码基因的核酸或载体导入巨噬细胞细胞中；
    所述导入的方式包括电穿孔、转导或转染；
    优选地，所述核酸位于不同的病毒载体上；所述病毒载体为慢病毒载体、腺病毒载体或逆转录病毒载体；
    优选地，所述载体为转座子或mRNA载体；
    优选地，将包含CAR-M嵌合抗原受体编码基因的腺病毒载体通过转染导入巨噬细胞细胞中。
  9. 根据权利要求1-3任一项所述的应用，其特征在于，所述巨噬细胞源自健康人体或癌症患者；
    所述巨噬细胞选自自体巨噬细胞、异体巨噬细胞或iPSC诱导的巨噬细胞；
    优选地，所述巨噬细胞为原代巨噬细胞；
    所述T细胞源自健康人体或癌症患者；
    所述T细胞选自自体T细胞、异体T细胞或iPSC诱导的T细胞。
  10. 根据权利要求1-3任一项所述的应用，其特征在于，所述肿瘤为NKG2D配体表达的肿瘤；
    所述肿瘤为血液瘤或实体瘤；
    优选地，所述肿瘤为白血病、多发性骨髓瘤、恶性淋巴瘤、脑胶质瘤、肝癌、肺癌、胃癌、结肠癌、胰腺癌或乳腺癌。
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