WO2020078079A1 - 能够靶向肿瘤细胞的巨噬细胞及其制备方法 - Google Patents

能够靶向肿瘤细胞的巨噬细胞及其制备方法 Download PDF

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WO2020078079A1
WO2020078079A1 PCT/CN2019/099680 CN2019099680W WO2020078079A1 WO 2020078079 A1 WO2020078079 A1 WO 2020078079A1 CN 2019099680 W CN2019099680 W CN 2019099680W WO 2020078079 A1 WO2020078079 A1 WO 2020078079A1
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cancer
medium
cells
macrophages
pluripotent stem
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张进
张丽
田琳
罗涛
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Zhejiang University
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Zhejiang University
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Priority to ZA2021/02381A priority patent/ZA202102381B/en
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Definitions

  • the present disclosure relates to the field of biotechnology, in particular, to a macrophage capable of targeting tumor cells and a preparation method thereof.
  • adoptive immunotherapy is a method of adoptively reintroducing lymphocytes cultured in vitro to tumor patients to treat tumors.
  • Chimeric antigen receptor (CAR) modified T cells are a new method of adoptive immunotherapy for tumors that has developed rapidly in recent years. The modification of CAR makes T cells have better tumor targeting, stronger killing activity and lasting vitality, and improve the therapeutic effect.
  • the purpose of the present disclosure includes, for example, to provide a macrophage capable of targeting tumor cells to alleviate the poor recognition and killing effect of CAR-T cells on tumor cells, especially solid tumor cells in prior art CAR-T cell therapy Weak technical issues.
  • the object of the present disclosure includes, for example, providing pluripotent stem cells capable of differentiating to obtain the above-mentioned macrophages.
  • the purpose of the present disclosure includes, for example, to provide a method for preparing macrophages capable of targeting tumor cells, so as to alleviate the technical problem of lacking a product that can efficiently target tumor cells in the prior art.
  • the present disclosure provides a macrophage capable of targeting tumor cells, the macrophage including a chimeric antigen receptor.
  • the macrophages are HLA-I deficient macrophages
  • the macrophages are B2M gene-deficient macrophages.
  • the macrophages are derived from the differentiation of pluripotent stem cells, and the pluripotent stem cells contain a gene encoding the chimeric antigen receptor;
  • the pluripotent stem cells are HLA-I deficient pluripotent stem cells
  • the pluripotent stem cells are B2M gene-deficient pluripotent stem cells
  • the pluripotent stem cells include induced pluripotent stem cells and / or embryonic stem cells.
  • the gene encoding the chimeric antigen receptor is located on the vector
  • the vector includes a plasmid vector or a viral vector
  • the viral vector is a retroviral vector, preferably a lentiviral vector
  • the plasmid vector used to construct the B2M gene-deficient type is one of the vectors in a) or b) as follows:
  • the chimeric antigen receptor includes an extracellular antigen binding region, a transmembrane region, a costimulatory domain, and an intracellular signal transduction region;
  • the extracellular antigen binding region includes sc-Fv, Fab, scFab or scIgG antibody fragments;
  • the transmembrane region includes at least one of CD3 ⁇ , CD4, CD8, or CD28;
  • the costimulatory domains include CD27, CD28, CD137, OX40, CD30, CD40, PD-1, LFA-1, CD2, CD7, Lck, DAP10, ICOS, LIGHT, NKG2C, B7-H3 or At least one of the ligands specifically bound by CD3 ⁇ ;
  • the intracellular signal transduction region includes at least one of CD3 ⁇ , Fc ⁇ RI ⁇ , PKC ⁇ , or ZAP70;
  • the chimeric antigen receptor further includes a reporter gene
  • the reporter gene is a fluorescent reporter gene
  • the fluorescent reporter gene is selected from any one of GFP, EGFP, RFP, mCherry, mStrawberry, Luciferase, mApple, mRuby, EosFP.
  • the extracellular antigen binding region specifically binds CD19.
  • a pluripotent stem cell that can differentiate into the above-mentioned macrophages.
  • a method for preparing the above macrophage expresses the gene encoding the chimeric antigen receptor in the macrophage to obtain the macrophage capable of targeting tumor cells.
  • the preparation method further includes the step of preparing macrophages deficient in HLA-I gene;
  • the preparation method further comprises the step of preparing B2M gene-deficient macrophages.
  • the preparation method includes directed differentiation of pluripotent stem cells to obtain macrophages capable of targeting tumor cells, the pluripotent stem cells containing genes encoding chimeric antigen receptors;
  • the pluripotent stem cells are HLA-I deficient pluripotent stem cells
  • the pluripotent stem cells are B2M gene-deficient pluripotent stem cells
  • the pluripotent stem cells include induced pluripotent stem cells and / or embryonic stem cells;
  • the gene encoding the chimeric antigen receptor is recombined on a vector and expressed in macrophages;
  • the reporter gene is recombined with the chimeric antigen receptor and connected to the carrier;
  • the reporter gene is a fluorescent reporter gene
  • the fluorescent reporter gene is selected from any one of GFP, EGFP, RFP, mCherry, mStrawberry, Luciferase, mApple, mRuby, EosFP.
  • the directed differentiation includes the following steps: placing the embryoid body obtained by differentiation induced by pluripotent stem cells in a first medium for first-stage culture, and then using the second medium, the third The culture medium, the fourth culture medium, the fifth culture medium, the sixth culture medium and the seventh culture medium are sequentially cultured in the second stage, the third stage, the fourth stage, the fifth stage, the sixth stage and the seventh stage;
  • the first stage is 0-1 days after inoculation
  • the second stage is 2-7 days after inoculation
  • the third stage is 8-10 days after inoculation
  • the fourth stage is 10-20 days after inoculation.
  • the fifth stage is 20-22 days after inoculation
  • the sixth stage is 22-28 days after inoculation
  • the seventh stage is 29th day after inoculation;
  • the fourth stage, the fifth stage, the sixth stage, and the seventh stage are all required to provide matrigel;
  • the matrigel includes Matrigel or Laminin-521;
  • the step of inducing differentiation of pluripotent stem cells to obtain an embryoid body includes: adding a cell digestion solution Accutase to the pluripotent stem cells and incubating at 36-38 ° C for 10-14 h to obtain an embryoid body ;
  • the pluripotent stem cells are treated with the Rock kinase inhibitor Y27632, and then added to the cell digestion solution Accutase and incubated at 36-38 ° C for 10-14 h to obtain an embryoid body.
  • the first medium includes a first basal medium and a first cytokine, and the first cytokine includes BMP4 and bFGF;
  • the second medium includes a first basal medium and a second cytokine, and the second cytokine includes BMP4, bFGF, VEGF, and SCF;
  • the third medium includes a first basal medium and a third cytokine, and the third cytokine includes bFGF, VEGF, SCF, IGF1, IL-3, M-CSF, and GM-CSF;
  • the fourth medium includes a second basal medium and a third cytokine
  • the fifth medium includes a second basal medium and a fourth cytokine, and the fourth cytokine includes bFGF, VEGF, SCF, IGF1, IL-3, M-CSF, and GM-CSF;
  • the sixth medium includes a second basal medium and a fifth cytokine, and the fifth cytokine includes bFGF, VEGF, SCF, IGF1, M-CSF, and GM-CSF;
  • the seventh medium includes a third basal medium, a sixth cytokine, and FBS, and the sixth cytokine includes M-CSF and GM-CSF;
  • the first basic medium and the second basic medium are serum-free medium
  • the third basic medium is a serum-containing medium
  • the first basal medium is STEMdiff TM APEL TM 2 or mTeSR1;
  • the second basal medium is StemPro TM -34;
  • the third basal medium is RPMI-1640.
  • macrophages capable of targeting tumor cells according to the present disclosure in preventing or treating tumors is also provided.
  • the tumor includes at least one of the following: acute lymphoblastic leukemia, acute myelogenous leukemia, cholangiocarcinoma, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic Myelogenous leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, Hodgkin's lymphoma, lung cancer, medullary thyroid cancer, non-Hodgkin's lymphoma, multiple myeloma , Kidney cancer, ovarian cancer, pancreatic cancer, glioma, melanoma, liver cancer, prostate cancer, or urinary bladder cancer.
  • the tumor includes at least one of the following: acute lymphoblastic leukemia, acute myelogenous leukemia, cholangiocarcinoma, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic Myelogenous leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, Hodgkin's lymphoma, lung cancer, medullary thyroid cancer, non-Hodgkin's lymphoma, multiple myeloma , Kidney cancer, ovarian cancer, pancreatic cancer, glioma, melanoma, liver cancer, prostate cancer, or urinary bladder cancer.
  • the present disclosure provides a macrophage capable of targeting tumor cells, and the macrophage contains a chimeric antigen receptor.
  • CAR-T cell therapy has some technical defects in tumor treatment. Due to the limitation of the microenvironment of solid tumors, it is very difficult for CAR-T cells to enter the tumor. Even if it enters, it will kill the tumor due to the inhibitory effect in the microenvironment. The role of cells will also be weakened.
  • the inventors proposed another idea of tumor immunotherapy, allowing chimeric antigen receptors to be expressed in macrophages.
  • macrophages Compared with T cells, macrophages have the advantage of being easier to enter solid tumors and less likely to be inhibited by other types of cells, so they can better play the role of tumor immunotherapy. Since the expressed chimeric antigen receptor is located on the surface of macrophages, the macrophages can accurately target tumor cells. At the same time, the inventors found through experiments that the chimeric antigen receptors applicable in T cells are also suitable for macrophages, that is, the chimeric antigen receptors in CAR-T cell therapy can be applied to macrophages to achieve chimeric antigen receptors Expression on the surface of macrophages, targeting tumor cells and activating macrophages to engulf tumor cells. Therefore, the discovery of the use of chimeric antigen receptors to modify macrophages provides new ideas and technical means for solid tumor immunotherapy, and is of great significance for tumor immunotherapy.
  • the present disclosure provides a method for preparing macrophages capable of targeting tumor cells, which provides a brand-new idea for immunotumor therapy.
  • 1A is a graph showing the results of flow cytometry detection of the marker CD45 in myeloid cells on day 14 in Example 9 of the present disclosure
  • 1B is a flow chart of flow cytometry detection of the marker CD34 in myeloid cells on day 14 in Example 9 of the present disclosure
  • 1C is a graph showing the results of flow cytometry detection of the marker CD11b in myeloid cells on day 14 in Example 9 of the present disclosure
  • FIG. 1D is a flow chart of flow cytometry detection of the marker CD14 in myeloid cells on day 14 in Example 9 of the present disclosure
  • 1E is a flow chart of flow cytometry detection of the marker CD11b in mature macrophages on day 45 of Example 9 of the present disclosure
  • 1F is a flow chart of flow cytometry detection of the marker CD14 in mature macrophages on day 45 in Example 9 of the present disclosure
  • 1G is a flow chart of flow cytometry detection of the marker CD163 in mature macrophages on day 45 of Example 9 of the present disclosure
  • 1H is a graph showing the results of flow cytometry detection of the marker CD86 in mature macrophages on day 45 in Example 9 of the present disclosure
  • 2A is a flow cytometry technique for detecting expression of a chimeric antigen receptor on the surface of a wild-type iPS cell in Example 10 of the present disclosure
  • 2B is a flow cytometry technique according to Example 10 of the present disclosure for detecting the expression of a chimeric antigen receptor on the surface of an iPS cell stably expressing a chimeric antigen receptor;
  • 2C is a flow cytometry technique in Example 10 of the present disclosure to detect the expression of chimeric antigen receptor on the cell surface after iPS cells stably expressing the chimeric antigen receptor differentiate into macrophages;
  • Example 3 is a graph showing the results of cell immunization after flow cytometry detection of B2M knock-out in Example 11 of the present disclosure
  • 4A is a photograph of a focused microscope photograph of macrophages engulfing Raji cancer cells obtained by iPS differentiation in Example 12 of the present disclosure
  • 4B is a graph showing the statistical results of macrophage phagocytosis of cancer cells by iPS differentiation in Example 12 of the present disclosure.
  • Fig. 5A is a macrophage iMAC differentiated from 2 ⁇ 10 ⁇ 5 iPS or ES cells over-expressing the chimeric antigen receptor CD19 CAR, at 4 ⁇ 10 ⁇ 4 K562 tumor cells expressing CD19 antigen and not expressing CD19 antigen, respectively K562 cells were co-cultured for 24 hours to detect the phagocytosis of iMAC on tumor cells.
  • a macrophage capable of targeting tumor cells includes a chimeric antigen receptor.
  • CAR-T cell therapy has some technical defects in tumor treatment. Due to the limitation of the microenvironment of solid tumors, it is very difficult for CAR-T cells to enter the inside of the tumor. The role of cells will also be weakened.
  • the inventors proposed another idea of tumor immunotherapy, allowing chimeric antigen receptors to be expressed in macrophages. Compared with T cells, macrophages have the advantage of being easier to enter solid tumors and less likely to be inhibited by other types of cells, so they can better play the role of tumor immunotherapy. Since the expressed chimeric antigen receptor is located on the surface of macrophages, the macrophages can accurately target tumor cells.
  • the inventors found through experiments that the chimeric antigen receptors applicable in T cells are also suitable for macrophages, that is, the chimeric antigen receptors in CAR-T cell therapy can be applied to macrophages to achieve chimeric antigen receptors Expression on the surface of macrophages, targeting tumor cells and activating macrophages to engulf tumor cells. Therefore, the discovery of the use of chimeric antigen receptors to modify macrophages provides new ideas and technical means for solid tumor immunotherapy, and is of great significance for tumor immunotherapy.
  • the macrophages are HLA-I (human lymphocyte antigen I) -deficient macrophages. Allowing macrophages to express chimeric antigen receptors allows macrophages to efficiently target tumor cells and activate themselves to engulf tumor cells, but due to the specific recognition of MHC (major histocompatibility complex, the major histocompatibility complex) Function, leading to immune rejection of allogeneic cell transplantation and anti-host reaction of the graft, therefore, the versatility of macrophages that can target tumor cells needs to be improved.
  • HLA-I human lymphocyte antigen I
  • the HLA-I gene is constructed Defective type can avoid allogeneic rejection, realize the versatility of macrophages that can target tumor cells, and further reduce the cost of immune tumor treatment.
  • HLA-I is a highly polymorphic alloantigen and has a significant relationship with organ transplantation and immune rejection. HLA-I of macrophages can be knocked out, thereby reducing the problem of allogeneic immune rejection.
  • the wild-type CAR-T cells used for immune cell therapy have a wider and universal application range.
  • the method used is to directly knock out the B2M gene in the HLA-I complex, so as to reduce the immunogenicity of the cells, so as to avoid rejection of the transplanted cells by the host after differentiation into immune cells, and thus to achieve allogeneic transplantation.
  • the macrophages are B2M gene-deficient macrophages.
  • B2M or ⁇ 2 microglobulin, is a member of MHC class I molecules. It is present in all nucleated cells, but does not include red blood cells. B2M is necessary for the expression of MHC class I protein cell surface and the stability of the peptide binding region. In fact, in the absence of B2M, MHC class I proteins are rarely detected on the cell surface. The construction of B2M gene-deficient macrophages can effectively reduce the host's immune rejection response to transplanted cells.
  • the macrophages are derived from the pluripotent stem cells directed differentiation, wherein the pluripotent stem cells contain a gene encoding a chimeric antigen receptor.
  • Both T cells and macrophages are mature cells with limited expansion capacity.
  • the number of correctly edited cells obtained is very limited, and may not be able to meet clinical requirements.
  • the cell dose required, the product cost is too high. Therefore, to solve this problem, pluripotent stem cells can be applied to differentiate into macrophages to solve the problem, and the pluripotent stem cells can be genetically modified to express the gene encoding the chimeric antigen receptor and / or become B2M-deficient.
  • the pluripotent stem cells have the ability to proliferate and differentiate into immune cells indefinitely, and after gene editing of the pluripotent stem cells can select the monoclonals that are edited correctly and do not have off-target effects.
  • the pluripotent stem cells are HLA-I deficient pluripotent stem cells.
  • the pluripotent stem cells were modified with HLA-I deficiency to obtain macrophages with strong versatility, allogeneic suppression and no immune rejection, which can target tumor cells.
  • the pluripotent stem cells are B2M gene-deficient pluripotent stem cells.
  • the pluripotent stem cells include induced pluripotent stem cells and / or embryonic stem cells.
  • the gene encoding the chimeric antigen receptor is located on the vector.
  • the vector includes a plasmid vector or a viral vector.
  • the viral vector is a retroviral vector, preferably a lentiviral vector.
  • the plasmid vector used to construct the B2M gene-deficient type is one of the vectors in a) or b) as follows:
  • the chimeric antigen receptor includes an extracellular antigen binding region, a transmembrane region, a costimulatory domain, and an intracellular signal transduction region. It should be noted that a chimeric antigen receptor suitable for T cells can be used as a chimeric antigen receptor for macrophages.
  • the extracellular antigen binding region includes sc-Fv, Fab, scFab, or scIgG antibody fragments.
  • the antigen binding region that recognizes the tumor recognizes any one of the group consisting of CD19, CD20, CD22, CD30, GD2, HER2, CAIX, CD171, Mesothelin, LMP1, EGFR , Muc1, GPC3, EphA2, EpCAM, MG7, CSR, ⁇ -fetoprotein (AFP), ⁇ -actinin-4, A3, antigens specific for A33 antibodies, ART-4, B7, Ba733 , BAGE, BrE3 antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8 / m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16 , CD18, CD21, CD23, CD25, CD29, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55
  • the extracellular antigen binding region specifically binds CD19.
  • the transmembrane region includes at least one of CD3 ⁇ , CD4, CD8, or CD28.
  • the costimulatory domains include CD27, CD28, CD137, OX40, CD30, CD40, PD-1, LFA-1, CD2, CD7, Lck, DAP10, ICOS, LIGHT, NKG2C, At least one of ligands that B7-H3 or CD3 ⁇ specifically binds.
  • the intracellular signal transduction region includes at least one of CD3 ⁇ , Fc ⁇ RI ⁇ , PKC ⁇ , or ZAP70.
  • the chimeric antigen receptor also includes a reporter gene.
  • the reporter gene is a fluorescent reporter gene.
  • the fluorescent reporter gene is selected from any one of GFP, EGFP, RFP, mCherry, mStrawberry, Luciferase, mApple, mRuby, EosFP.
  • the macrophage capable of targeting tumor cells or the therapy capable of differentiating into the macrophage is suitable for treating cancer. It is expected that any type of tumor and any type of tumor antigen can be targeted. Exemplary types of cancer that can be targeted include acute lymphoblastic leukemia, acute myelogenous leukemia, cholangiocarcinoma, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, Endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, Hodgkin's lymphoma, lung cancer, medullary thyroid cancer, non-Hodgkin's lymphoma, multiple myeloma, kidney cancer, ovarian cancer, pancreatic cancer , Glioma, melanoma, liver cancer, prostate cancer and urinary bladder cancer. However, it should be noted that those skilled in the art should recognize that tumor-associated antigens of virtually any type of cancer are
  • a method for preparing macrophages capable of targeting tumor cells The gene encoding the chimeric antigen receptor is expressed in macrophages to obtain macrophages capable of targeting tumor cells. This method provides a new way of thinking for the treatment of immune tumors.
  • the preparation method further includes the step of preparing macrophages deficient in the HLA-I gene.
  • the preparation method further includes the step of preparing B2M gene-deficient macrophages.
  • the preparation method includes directed differentiation of pluripotent stem cells to obtain macrophages capable of targeting tumor cells.
  • the pluripotent stem cells contain genes encoding chimeric antigen receptors.
  • the pluripotent stem cells are HLA-I deficient pluripotent stem cells.
  • the pluripotent stem cells are B2M gene-deficient pluripotent stem cells.
  • the pluripotent stem cells include induced pluripotent stem cells and / or embryonic stem cells.
  • the gene encoding the chimeric antigen receptor is recombined on a vector and expressed in macrophages.
  • the reporter gene is recombined with the chimeric antigen receptor and then ligated to the vector.
  • the reporter gene is a fluorescent reporter gene.
  • the fluorescent reporter gene is selected from any one of GFP, EGFP, RFP, mCherry, mStrawberry, Luciferase, mApple, mRuby, EosFP.
  • the directed differentiation includes the following steps: placing the embryoid body obtained from the differentiation induced by the pluripotent stem cells in the first medium for the first-stage culture, and then using the second medium and the third medium ,
  • the fourth culture medium, the fifth culture medium, the sixth culture medium and the seventh culture medium are successively cultured in the second stage, the third stage, the fourth stage, the fifth stage, the sixth stage and the seventh stage, among which
  • the first stage is 0-1 days after inoculation
  • the second stage is 2-7 days after inoculation
  • the third stage is 8-10 days after inoculation
  • the fourth stage is 10-20 days after inoculation
  • the fifth stage is 20 after inoculation -22 days
  • the sixth stage is 22-28 days after vaccination
  • the seventh stage is the 29th day after vaccination.
  • the above-mentioned cell induction culture method is to first cultivate pluripotent stem cells with a gene encoding a chimeric antigen receptor to form embryoid bodies, and then culture them in a cell induction medium, and finally a large amount of macrophages that can target tumor cells can be obtained cell.
  • mesodermal cells are obtained in the first stage
  • hematopoietic cells are obtained in the second stage
  • myeloid cells are obtained in the third stage
  • mature macrophages are obtained in the fourth stage.
  • a new second medium needs to be replaced every other day during the second-stage cultivation, and a new third medium needs to be replaced every other day during the third-stage cultivation.
  • the cells are suspension cells obtained after the fourth stage of culture.
  • the process by which pluripotent stem cells form an embryoid body (EB) is as follows:
  • mTeSR1, DMEM / F12 and Versene are preheated to 15-25 ° C for cell passage.
  • Y27632 is a Rock kinase inhibitor with a concentration of 3 ⁇ M.
  • the number of cells when cells are seeded on day 10, is 20-25 cells / ml.
  • the culture medium in the solution of the present application, can be replaced in any one of the following ways 1) -3):
  • the volume of the medium of different types of plates is as follows: 6-well plates are 2.0 mL / well; 24-well plates are 0.5 mL; 96-well plates are 150 ⁇ L / well.
  • matrigel needs to be provided during the fourth stage, the fifth stage, the sixth stage, and the seventh stage.
  • Matrigel includes Matrigel or Laminin-521.
  • the step of inducing differentiation of pluripotent stem cells to obtain an embryoid body includes: the pluripotent stem cells are treated with Rock kinase inhibitor Y27632, and then added to the cell digestion solution Accutase and incubated at 36-38 °C 10 -14h to get the embryoid body.
  • the first medium includes a first basal medium and a first cytokine, and the first cytokine includes BMP4 and bFGF;
  • the second medium includes a first basal medium and a second cytokine, and the second cytokine includes BMP4, bFGF, VEGF, and SCF;
  • the third medium includes a first basal medium and a third cytokine, and the third cytokine includes bFGF, VEGF, SCF, IGF1, IL-3, M-CSF, and GM-CSF;
  • the fourth medium includes a second basal medium and a third cytokine
  • the fifth medium includes a second basal medium and a fourth cytokine, and the fourth cytokine includes bFGF, VEGF, SCF, IGF1, IL-3, M-CSF and GM-CSF;
  • the sixth medium includes a second basal medium and a fifth cytokine, and the fifth cytokine includes bFGF, VEGF, SCF, IGF1, M-CSF and GM-CSF;
  • the seventh medium includes the third basal medium, the sixth cytokine and FBS, and the sixth cytokine includes M-CSF and GM-CSF;
  • the first basic medium and the second basic medium are serum-free medium
  • the third basic medium is serum-containing medium.
  • the continuous use of the above cell induction medium combination can achieve rapid and large-scale differentiation of embryoid somatic cells into macrophages. Since the embryoid body is derived from the differentiation of pluripotent stem cells, the pluripotent stem cells can stably express the chimeric antigen receptor, so The resulting macrophages can express chimeric antigen receptors and have the ability to engulf tumor cells.
  • the first six media are serum-free media, which can provide basic nutrients for cell growth, proliferation and differentiation at all stages while reducing the risk of contamination.
  • the seventh medium contains serum and FBS, which can maintain the growth of macrophages. Since each of the above-mentioned culture media contains specific multiple cytokines, it can promote the directional differentiation of cells, and finally obtain a large number of macrophages with stable performance and high quality.
  • BMP4 (bone morphogenetic protein 4, bone morphogenetic protein 4) belongs to the TGF- ⁇ superfamily and plays an important role in the development and regeneration of bone embryos. BMP4 participates in the biological processes of regulating cell proliferation, differentiation and apoptosis, and plays an important role in embryonic development, stable internal environment of various tissues and organs after birth, and the occurrence of various tumors.
  • bFGF is one of fibroblast growth factors. It is a basic fibroblast growth factor. It is an inducing factor for cell morphogenesis and differentiation. It can induce and promote the proliferation and differentiation of various cells.
  • VEGF vascular endothelial growth factor, vascular endothelial growth factor
  • vascular endothelial growth factor is a highly specific vascular endothelial growth factor, with increased vascular permeability, promote vascular endothelial cell migration and extracellular matrix degeneration, promote cell proliferation and vascularization The role.
  • SCF Stem Cell Factor
  • IGF1 is an insulin-like growth factor (insulin-like growth factors) that promotes cell growth and differentiation.
  • IL-3 (Interleukin-3) is a cytokine of the chemokine family, which can regulate hematopoiesis and immunity.
  • M-CSF macrophage CSF
  • GM-CSF granulocyte and macrophage CSF
  • CSF colony stimulating factors
  • FBS is fetal bovine serum. It is a trait with a pale yellow clear appearance, no hemolysis, and no sticky liquid. Antibodies and complements contained in FBS are the least harmful to cells and rich in nutrients necessary for cell growth.
  • the first basal medium is STEMdiff TM APEL TM 2 or mTeSR1.
  • the second basal medium is StemPro TM -34.
  • the third basal medium is RPMI-1640.
  • the final concentrations of BMP4 and bFGF in the first medium are 8-12 ng / ml and 3-7 ng / ml in sequence.
  • the typical but non-limiting BMP4 concentration is 8 ng / ml, 10 ng / ml or 12 ng / ml; the typical but non-limiting bFGF concentration is 3 ng / ml, 5 ng / ml or 7 ng / ml.
  • the final concentrations of BMP4, bFGF, VEGF and SCF in the second medium are 8-12ng / ml, 3-7ng / ml, 48-52ng / ml and 95-105ng / ml.
  • Typical but non-limiting BMP4 concentrations are 8ng / ml, 10ng / ml or 12ng / ml; typical but non-limiting bFGF concentrations are 3ng / ml, 5ng / ml or 7ng / ml; VEGF concentrations are typical but non-limiting 48ng / ml, 50ng / ml, or 52ng / ml; typical but non-limiting SCF concentrations are 95ng / ml, 99ng / ml, 100ng / ml, 104ng / ml, or 105ng / ml.
  • the final concentrations of bFGF, VEGF, SCF, IGF1, IL-3, M-CSF, and GM-CSF are 8-12ng / ml, 48-52ng / ml, 48-52ng / ml, 8-12ng / ml, 23-27ng / ml, 48-52ng / ml and 48-52ng / ml.
  • Typical but non-limiting bFGF concentrations are 8 ng / ml, 10 ng / ml or 12 ng / ml; typical but non-limiting VEGF concentrations are 48 ng / ml, 50 ng / ml or 52 ng / ml; SCF concentrations are typical but non-limiting 48ng / ml, 50ng / ml or 52ng / ml; typical but non-limiting IGF1 concentration is 8ng / ml, 10ng / ml or 12ng / ml; typical but non-limiting IL-3 concentration is 23ng / ml and 25ng / ml or 27ng / ml; typical but non-limiting M-CSF concentration is 48ng / ml, 50ng / ml or 52ng / ml; typical but non-limiting GM-CSF concentration is 48ng / ml, 50ng / ml or 52ng / ml.
  • the final concentrations of bFGF, VEGF, SCF, IGF1, IL-3, M-CSF and GM-CSF are 8-12ng / ml, 48-52ng / ml, 48-52ng / ml, 8-12ng / ml, 23-27ng / ml, 95-105ng / ml and 95-105ng / ml.
  • Typical but non-limiting bFGF concentrations are 8 ng / ml, 10 ng / ml or 12 ng / ml; typical but non-limiting VEGF concentrations are 48 ng / ml, 50 ng / ml or 52 ng / ml; SCF concentrations are typical but non-limiting 48ng / ml, 50ng / ml or 52ng / ml; typical but non-limiting IGF1 concentration is 8ng / ml, 10ng / ml or 12ng / ml; typical but non-limiting IL-3 concentration is 23ng / ml and 25ng / ml or 27ng / ml; M-CSF concentration is typical but non-limiting 95ng / ml, 99ng / ml, 102ng / ml, 104ng / ml or 105ng / ml; GM-CSF concentration is typical but non-limiting 95ng / ml,
  • the final concentrations of bFGF, VEGF, SCF, IGF1, M-CSF and GM-CSF are 8-12ng / ml, 48-52ng / ml, 48- 52ng / ml, 8-12ng / ml, 95-105ng / ml and 95-105ng / ml.
  • Typical but non-limiting bFGF concentrations are 8 ng / ml, 10 ng / ml or 12 ng / ml; typical but non-limiting VEGF concentrations are 48 ng / ml, 50 ng / ml or 52 ng / ml; SCF concentrations are typical but non-limiting 48ng / ml, 50ng / ml or 52ng / ml; typical but non-limiting IGF1 concentration is 8ng / ml, 10ng / ml or 12ng / ml; typical but non-limiting M-CSF concentration is 95ng / ml and 99ng / ml, 102ng / ml, 104ng / ml or 105ng / ml; GM-CSF concentration is typically but not limited to 95ng / ml, 99ng / ml, 100ng / ml, 104ng / ml or 105ng / ml.
  • the final concentrations of FBS, M-CSF, and GM-CSF are 8-12% by mass, 95-105 ng / ml, and 95-105 ng / ml, respectively.
  • Typical but non-limiting mass fraction of FBS is 8%, 10% or 12%;
  • typical but non-limiting M-CSF concentration is 95ng / ml, 99ng / ml, 100ng / ml, 104ng / ml or 105ng / ml ;
  • the GM-CSF concentration is typically but not limited to 95ng / ml, 97ng / ml, 100ng / ml, 104ng / ml or 105ng / ml.
  • FBS is subjected to inactivation treatment in the seventh medium.
  • the present disclosure also relates to a pluripotent stem cell capable of differentiating into the above-mentioned macrophage capable of targeting tumor cells.
  • the pluripotent stem cells can be modified by gene editing, and at the same time, they can be differentiated by specific culture conditions to obtain the above-mentioned macrophages.
  • peripheral blood was drawn from patients or volunteers, and PBMC (peripheral blood mononuclear cells) were isolated with lymphocyte separation fluid, cultured with H3000 + CC100, and MEF cells (fibroblasts) were recovered.
  • PBMC peripheral blood mononuclear cells
  • iPS cells induced pluripotent stem cells
  • Resuscitation Remove the frozen cells from the liquid nitrogen tank and quickly put them in a 37 ° C water bath, shake them quickly to melt them. Prepare a 15ml centrifuge tube in the ultra-clean table, add 5ml complete medium and cells in the cryopreservation tube, mix well, and centrifuge at 250rcf / min for 5min. Discard the supernatant, resuspend in 5ml of complete medium and transfer to a T25 culture flask, and culture at 37 ° C in a 5% CO 2 incubator. Observe the cell survival rate the next day, discard the old medium and add 5ml of fresh medium.
  • Subculture When cells grow to 80% -90%, subculture. Discard the supernatant, add 5ml PBS and shake gently. Discard PBS, add 1ml 0.25% trypsin, digest for 10s to 20s until the cells become round, the cell gap becomes larger, add 3ml complete medium, mix and move to a 15ml centrifuge tube, centrifuge 250rcf / min, 5min. Discard the supernatant, resuspend in 2ml complete medium and transfer to T75 flask reserved for 13ml complete medium, and then cultivate as before.
  • Lenti-EF1a-CD19-T2A-EGFP-Puro contains scFv that specifically binds to CD19 antigen, transmembrane domain from CD8, costimulatory domain from 4-1BB, and intracellular domain from CD3zeta There are fluorescent gene EGFP and screening gene puromycin.
  • the vector was identified by the enzymes EcoRI and XbaI, and the band size was correct.
  • lentiviral expression vector, packaging vector and envelope vector are transfected with lip2000 in a 10cm cell culture plate at a ratio of 4: 3: 1, and the medium is changed after 6h, respectively after 24h and 48h Collect the supernatant.
  • After filtering the collected supernatant through a 0.22um filter membrane add 1/2 volume of 25% PEG at 4 ° C overnight. The next day, centrifuge at 4000C for 4min at 4 ° C. Discard the supernatant and resuspend in 500ul PBS Precipitate, 50ul per tube, and place at -80 °C.
  • the virus was infected with iPS according to the MOI of 20, and the cells were screened by adding 0.25ug / ml of puromycin on the third day after infection for 3 days to obtain a stable expression cell line, which can be used later for the differentiation of macrophages.
  • the B2M gene is located on chromosome 15q21-22.2.
  • the B2M gene encodes an endogenous low molecular weight serum protein ⁇ 2 microglobulin, which is related to the MHC-I ⁇ 2 chain on the surface of almost all nucleated cells.
  • the medium containing puromycin is selected.
  • the screened cells were divided into two groups, one group was cultured normally, and the other group was cultured normally while adding 50ng / ul IFN- ⁇ treatment for 48h.
  • the iPS cells stably expressed by CAR in wild-type Example 6 were also divided into two groups, one group was cultured normally, and the other group was cultured normally while adding 50ng / ul IFN- ⁇ treatment for 48h. Then incubate these 4 groups of cells with B2M flow cytometry antibody, and test the knockout effect of B2M on the computer. The results showed that compared with the iPS stably expressed by the CAR in wild-type Example 6, cells after B2M knockout were unable to induce B2M after being treated with IFN- ⁇ for 48 hours, indicating that the B2M gene of the cells had been knocked out.
  • CAR stably expressed iPS cells induce the formation of embryoid bodies (EB)
  • mTeSR1, DMEM / F12 and Versene are preheated to 15-25 ° C for cell passage.
  • Y27632 is a Rock kinase inhibitor with a concentration of 3 ⁇ M. Induction of the cells in Example 7:
  • EB Embryoid body
  • Step a) is removed mTeSR1 embryoid body culture medium 1) above, f), on day 1 a first incubated with medium (STEMdiff TM APEL TM 2,10ng / ml BMP4,5ng / mlbFGF) cultured 24h, embryoid Somatic differentiation into mesoderm cells;
  • medium STEMdiff TM APEL TM 2,10ng / ml BMP4,5ng / mlbFGF
  • Step b) Remove the first medium in step a), and use the second medium (STEMdiff TM APEL TM 2, 10ng / ml BMP4, 5ng / ml bFGF, 50ng / ml VEGF and 2nd day after inoculation ) 100ng / ml SCF) Incubation and culture of mesoderm cells, during which a new second medium is replaced every other day to obtain hematopoietic cells;
  • Step c) removing the second medium in step b), and with a third medium (STEMdiff TM APEL TM 2,10ng / ml bFGF, 50ng / ml VEGF, 50ng / ml SCF during 8-10 days after inoculation, 10ng / ml IGF1, 25ng / ml IL-3, 50ng / ml M-CSF and 50ng / ml GM-CSF) incubate and culture hematopoietic cells, and change the new third medium every other day during the period;
  • a third medium STEMdiff TM APEL TM 2,10ng / ml bFGF, 50ng / ml VEGF, 50ng / ml SCF during 8-10 days after inoculation, 10ng / ml IGF1, 25ng / ml IL-3, 50ng / ml M-CSF and 50ng / ml GM-CSF
  • Step d) Remove the third medium in step c), and during the 11-20 days after inoculation, inoculate the cells at a concentration of 20-25 cells / ml into the culture pre-coated with Matrigel (1 mg / ml) In the dish, use the fourth medium (StemPro TM- 34, 10ng / ml bFGF, 50ng / ml VEGF, 50ng / ml SCF, 10ng / ml IGF1, 25ng / ml IL-3, 50ng / ml M-CSF and 50ng / ml GM-CSF) Incubate the cells in step c) to obtain myeloid cells;
  • Step e) Starting on days 21-22 after inoculation, collect the myeloid cells suspended in step d) and re-plate into a petri dish pre-coated with Matrigel, using the fifth medium (StemPro TM- 34, 10ng / ml bFGF, 50 ng / ml VEGF, 50 ng / ml SCF, 10 ng / ml IGF1, 25 ng / ml IL-3, 100 ng / ml M-CSF and 100 ng / ml GM-CSF) were incubated and cultured, and macrophages were differentiated;
  • Step f) Remove the fifth medium in step e), and use the sixth medium (StemPro TM -34, 10ng / ml bFGF, 50ng / ml VEGF, 50ng / ml SCF, 10ng during the 23-28 days after inoculation / ml IGF1, 100ng / ml M-CSF and 100ng / ml GM-CSF) macrophages;
  • Step g) Remove the sixth medium in step f) and start with the seventh medium (RPMI-1640, 10% w / w FBS, 100ng / ml M-CSF, 100ng / ml GM-CSF on the 29th day of inoculation ) Maintain mature macrophages or perform cryopreservation.
  • the seventh medium RPMI-1640, 10% w / w FBS, 100ng / ml M-CSF, 100ng / ml GM-CSF on the 29th day of inoculation
  • Example 8 The cells of each stage obtained in Example 8 were subjected to flow cytometry to detect markers of related cells to evaluate the effect of directed differentiation. The results are shown in FIGS. 1A-1H.
  • 1 indicates iPS cells
  • 2 indicates myeloid cells on day 14
  • 3 indicates mature macrophages on day 45.
  • Figure 1A is the detection result of blood cell marker CD45 in myeloid cells on day 14
  • Figure 1B is the detection result of CD34 marker of hematopoietic stem cells in myeloid cells on day 14
  • FIG. 1C is myeloid cells in day 14
  • the detection result of the macrophage marker CD11b is the detection result of the macrophage marker CD14 in myeloid cells on day 14
  • FIG. 1E is the macrophage marker CD11b in mature macrophages on day 45.
  • Detection results Figure 1F is the detection result of the macrophage marker CD14 in the mature macrophage on day 45
  • Figure 1G is the detection result of the macrophage marker CD163 in the mature macrophage on day 45
  • FIG. 1H The detection result of CD86, a marker of macrophages in 45-day-old mature macrophages.
  • Flow cytometry was used to identify whether the chimeric antigen receptor was expressed on the surface of iPS cells and differentiated macrophages.
  • the HLA-I (B2M) -deficient pluripotent stem cells in Example 7 were divided into two groups, one group was cultured normally, and the other group was treated with 50 ng / ul IFN- ⁇ for 48 hours.
  • the iPS cells with stable expression of CAR in wild-type Example 6 were also divided into two groups, one group was cultured normally, and the other group was treated with 50 ng / ul IFN- ⁇ for 48 hours. Then these four groups of cells were respectively incubated with B2M flow cytometry antibody, and flow cytometry was used to detect the knockout effect of B2M.
  • FIG. 3 shows that compared with the iPS cells with stable CAR expression in Example 6, cells after B2M knockout were unable to induce B2M after IFN- ⁇ treatment for 48 hours, indicating that the B2M gene of the cells had been knocked out.
  • K562 is an acute myeloid leukemia cell line that does not express CD19 antigen on the surface.
  • the lentiviral vector expressing CD19 was transformed into K562 cells to construct a cell line expressing CD19 on the cell surface.
  • K562 cells, K562 cells stably expressing CD19, and Raji cells infected with mcherry virus were flow-sorted 4-5 days later, and cultured and expanded mcherry positive stable transfected cell lines.
  • Example 8 After the macrophages differentiated in Example 8 were cultured with the above three mcherry stable cell lines for 4 hours, they were photographed by a confocal microscope and the macrophages expressing mcherry cancer cells were statistically phagocytosed. The results are shown in Figures 4A and 4B. The test results show that the macrophages provided by the present disclosure have the ability to engulf cancer cells, and at the same time, they can be applied in large-scale heterogeneous production.
  • Fig. 5A is a macrophage iMAC differentiated from 2 ⁇ 10 ⁇ 5 iPS or ES cells over-expressing the chimeric antigen receptor CD19 CAR, at 4 ⁇ 10 ⁇ 4 K562 tumor cells expressing CD19 antigen and not expressing CD19 antigen, respectively K562 cells were co-cultured for 24 hours to detect the phagocytosis of iMAC on tumor cells.
  • the iMAC and K562 cells are labeled with fluorescent dyes of different colors, and the double-labeled cells represent iMAC cells that can engulf tumor cells.
  • the results show that CD19CAR iMAC has stronger phagocytic capacity for K562 cells expressing CD19 antigen.
  • the present disclosure provides a macrophage capable of targeting tumor cells, and the macrophage contains a chimeric antigen receptor.
  • CAR-T cell therapy has some technical defects in tumor treatment. Due to the limitation of the microenvironment of solid tumors, it is very difficult for CAR-T cells to enter the inside of the tumor. The role of cells will also be weakened.
  • the inventors proposed another idea of tumor immunotherapy, allowing chimeric antigen receptors to be expressed in macrophages. Compared with T cells, macrophages have the advantage of being easier to enter solid tumors and less likely to be inhibited by other types of cells, so they can better play the role of tumor immunotherapy.
  • the macrophages can accurately target tumor cells.
  • the inventors found through experiments that the chimeric antigen receptors applicable in T cells are also suitable for macrophages, that is, the chimeric antigen receptors in CAR-T cell therapy can be applied to macrophages to achieve chimeric antigen receptors Expression on the surface of macrophages, targeting tumor cells and activating macrophages to engulf tumor cells. Therefore, the discovery of the use of chimeric antigen receptors to modify macrophages provides new ideas and technical means for solid tumor immunotherapy, and is of great significance for tumor immunotherapy.
  • the present disclosure provides a method for preparing macrophages capable of targeting tumor cells, which provides a brand-new idea for immunotumor therapy.

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Abstract

一种能够靶向肿瘤细胞的巨噬细胞及其制备方法。巨噬细胞中含有嵌合抗原受体。由于实体肿瘤的微环境的限制，CAR-T细胞进入肿瘤内部十分困难，即便进入，又由于微环境中的抑制作用，杀伤肿瘤细胞的作用也会减弱。针对上述技术缺陷，提出了另一种肿瘤免疫治疗的思路，让嵌合抗原受体在巨噬细胞中表达。CAR-T细胞疗法中的嵌合抗原受体应用于巨噬细胞可以实现嵌合抗原受体在巨噬细胞表面的表达、靶向肿瘤细胞并且激活巨噬细胞将肿瘤细胞吞噬。为肿瘤免疫治疗提供了新的思路和技术手段。

Description

能够靶向肿瘤细胞的巨噬细胞及其制备方法
相关申请的交叉引用
本申请要求于2018年10月18日提交中国专利局的申请号为201811218443.2、名称为“能够靶向肿瘤细胞的巨噬细胞及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开涉及生物技术领域，具体而言，涉及一种能够靶向肿瘤细胞的巨噬细胞及其制备方法。
背景技术
随着免疫学、基因组编辑和合成生物学的发展，肿瘤免疫治疗的研究突飞猛进，尤其以过继性免疫疗法具有广泛的前景。过继性免疫疗法是将体外刺激培养的淋巴细胞过继回输到肿瘤病人体内从而治疗肿瘤的方法。嵌合抗原受体(chimeric antigen receptor，CAR)修饰T细胞是近年来发展迅猛的肿瘤过继免疫治疗新手段。CAR的修饰使得T细胞具有更好的肿瘤靶向性以及更强的杀伤活性和持久的生命力，提升治疗的效果。
然而，一方面，在CAR-T细胞中进行工程化改造都面临着载体的转化效率和基因编辑的效率低的问题，改造后的细胞的扩增能力也未必能够满足临床上所需要的细胞剂量。所以，CAR-T细胞疗法推广的一个巨大挑战是成本极高，美国最早批准的Norvatis的治疗难治复发性B细胞白血病的CAR-T产品Kymriah的价格是47.5万美元，反映出这种同种异体的个体化细胞产品从细胞收集到病毒/CAR-T细胞制备到回输的高额成本。同时，由于CAR-T细胞疗法中细胞生产数量有限，而且一种CAR-T细胞无法用于多名患者的治疗。如果通过对异体T细胞进行基因编辑和体外扩增后，不仅获得的正确编辑的细胞数量有限，同时免疫排斥也是一个需要解决的技术难题。另一方面，由于肿瘤特别是实体肿瘤内部具有复杂的微环境，不仅包括肿瘤细胞本身和T细胞，还包括巨噬细胞、成纤维细胞等等。复杂的实体肿瘤微环境限制了CAR-T细胞与肿瘤细胞的接触，即便进入了实体肿瘤内部，多种类型的细胞会起到抑制CAR-T细胞杀伤肿瘤细胞的作用，进而促进肿瘤的发生与发展，减弱CAR-T细胞的杀伤作用。因此，一种通用型的、能够用于异体、在低成本的情况下得到大量可以高效靶向肿瘤细胞的成品的的产品十分必要。有鉴于此，特提出本公开。
发明内容
本公开的目的包括例如提供一种能够靶向肿瘤细胞的巨噬细胞，以缓解现有技术CAR-T细胞疗法中CAR-T细胞对肿瘤细胞，特别是实体肿瘤细胞的识别能力差和杀伤作用较弱的技术问题。
本公开的目的包括例如提供能够分化得到上述巨噬细胞的多能干细胞。
本公开的目的包括例如提供一种能够靶向肿瘤细胞的巨噬细胞的制备方法，以缓解现 有技术中缺少一种可以高效靶向肿瘤细胞产品的技术问题。
本公开提供一种能够靶向肿瘤细胞的巨噬细胞，所述巨噬细胞包括嵌合抗原受体。
在一种或多种实施方式中，所述巨噬细胞为HLA-I缺陷型巨噬细胞；
在一种或多种实施方式中，所述巨噬细胞为B2M基因缺陷型巨噬细胞。
在一种或多种实施方式中，所述巨噬细胞由多能干细胞定向分化得到，所述多能干细胞含有编码所述嵌合抗原受体的基因；
在一种或多种实施方式中，所述多能干细胞为HLA-I缺陷型多能干细胞；
在一种或多种实施方式中，所述多能干细胞为B2M基因缺陷型多能干细胞；
在一种或多种实施方式中，所述多能干细胞包括诱导性多能干细胞和/或胚胎干细胞。
在一种或多种实施方式中，编码所述嵌合抗原受体的基因位于载体上；
在一种或多种实施方式中，所述载体包括质粒载体或病毒载体；
在一种或多种实施方式中，所述病毒载体为逆转录病毒载体，优选为慢病毒载体；
在一种或多种实施方式中，用于构建B2M基因缺陷型的质粒载体为如下a)或b)中载体中的一种：
a)能够表达gRNA和Cas9蛋白；
b)能够表达gRNA和Cpf1蛋白；
在一种或多种实施方式中，所述嵌合抗原受体包括胞外抗原结合区、跨膜区、共刺激结构域和胞内信号转导区；
在一种或多种实施方式中，所述胞外抗原结合区包括sc-Fv、Fab、scFab或scIgG抗体片段；
和/或，所述跨膜区包括CD3ζ、CD4、CD8或CD28中的至少一种；
和/或，所述共刺激结构域包括与CD27、CD28、CD137、OX40、CD30、CD40、PD-1、LFA-1、CD2、CD7、Lck、DAP10、ICOS、LIGHT、NKG2C、B7-H3或CD3ζ特异性结合的配体中的至少一种；
和/或，所述胞内信号转导区包括CD3ζ、FcεRIγ、PKCθ或ZAP70中的至少一种；
在一种或多种实施方式中，所述嵌合抗原受体还包括报告基因；
在一种或多种实施方式中，所述报告基因为荧光报告基因；
在一种或多种实施方式中，所述荧光报告基因选自GFP、EGFP、RFP、mCherry、mStrawberry、Luciferase、mApple、mRuby、EosFP中的任一种。
在一种或多种实施方式中，所述胞外抗原结合区特异性结合CD19。
一种能够分化得到上述巨噬细胞的多能干细胞。
一种上述巨噬细胞的制备方法，将编码嵌合抗原受体的基因在巨噬细胞中表达，得到所述能够靶向肿瘤细胞的巨噬细胞。
在一种或多种实施方式中，所述制备方法还包括制备HLA-I基因缺陷的巨噬细胞的步骤；
优选地，所述制备方法还包括制备B2M基因缺陷的巨噬细胞的步骤。
在一种或多种实施方式中，所述制备方法包括将多能干细胞定向分化得到能够靶向肿瘤细胞的巨噬细胞，所述多能干细胞含有编码嵌合抗原受体的基因；
在一种或多种实施方式中，所述多能干细胞为HLA-I缺陷型多能干细胞；
在一种或多种实施方式中，所述多能干细胞为B2M基因缺陷型多能干细胞；
在一种或多种实施方式中，所述多能干细胞包括诱导性多能干细胞和/或胚胎干细胞；
在一种或多种实施方式中，将所述编码嵌合抗原受体的基因重组在载体上，在巨噬细胞中进行表达；
在一种或多种实施方式中，将报告基因与所述嵌合抗原受体进行重组后与载体进行连接；
在一种或多种实施方式中，所述报告基因为荧光报告基因；
在一种或多种实施方式中，所述荧光报告基因选自GFP、EGFP、RFP、mCherry、mStrawberry、Luciferase、mApple、mRuby、EosFP中的任一种。
在一种或多种实施方式中，所述定向分化包括以下步骤：将由多能干细胞诱导分化得到拟胚体放置在第一培养基中进行第一阶段培养，然后用第二培养基、第三培养基、第四培养基、第五培养基、第六培养基和第七培养基依次进行第二阶段、第三阶段、第四阶段、第五阶段、第六阶段和第七阶段培养；
所述第一阶段为接种后0-1天，所述第二阶段为接种后2-7天，所述第三阶段为接种后8-10天，第四阶段为接种后10-20天，第五阶段为接种后20-22天，第六阶段为接种后22-28天，第七阶段为接种后第29天；
在一种或多种实施方式中，所述第四阶段、第五阶段、第六阶段和第七阶段进行培养时均需要提供基质胶；
在一种或多种实施方式中，所述基质胶包括Matrigel或Laminin-521；
在一种或多种实施方式中，所述多能干细胞诱导分化得到拟胚体的步骤包括：多能干细胞中加入细胞消化液Accutase并在36-38℃条件下孵育10-14h得到拟胚体；
在一种或多种实施方式中，多能干细胞经Rock激酶抑制剂Y27632处理后再加入细胞消化液Accutase并在36-38℃条件下孵育10-14h得到拟胚体。
在一种或多种实施方式中，所述第一培养基包括第一基础培养基和第一细胞因子，第一细胞因子包括BMP4和bFGF；
所述第二培养基包括第一基础培养基和第二细胞因子，第二细胞因子包括BMP4，bFGF，VEGF和SCF；
所述第三培养基包括第一基础培养基和第三细胞因子，第三细胞因子包括bFGF，VEGF，SCF，IGF1，IL-3，M-CSF和GM-CSF；
所述第四培养基包括第二基础培养基和第三细胞因子；
所述第五培养基包括第二基础培养基和第四细胞因子，第四细胞因子包括bFGF，VEGF，SCF，IGF1，IL-3，M-CSF和GM-CSF；
所述第六培养基包括第二基础培养基和第五细胞因子，第五细胞因子包括bFGF，VEGF，SCF，IGF1，M-CSF和GM-CSF；
所述第七培养基包括第三基础培养基、第六细胞因子和FBS，第六细胞因子包括M-CSF和GM-CSF；
其中，所述第一基础培养基和第二基础培养基为无血清培养基；
所述第三基础培养基为含血清培养基；
在一种或多种实施方式中，第一基础培养基为STEMdiff TMAPEL TM2或mTeSR1；
在一种或多种实施方式中，第二基础培养基为StemPro TM-34；
在一种或多种实施方式中，第三基础培养基为RPMI-1640。
还提供根据本公开所述的能够靶向肿瘤细胞的巨噬细胞在预防或治疗肿瘤中的用途。
在一种或多种实施方式中，所述肿瘤包括以下中至少一种：急性淋巴母细胞性白血病、急性骨髓源性白血病、胆管癌、乳腺癌、子宫颈癌、慢性淋巴细胞性白血病、慢性骨髓源性白血病、结肠直肠癌、子宫内膜癌、食道癌、胃癌、头颈部癌、霍奇金氏淋巴瘤、肺癌、甲状腺髓样癌、非霍奇金氏淋巴瘤、多发性骨髓瘤、肾癌、卵巢癌、胰腺癌、神经胶质瘤、黑素瘤、肝癌、前列腺癌或尿路膀胱癌。
还提供一种预防或治疗肿瘤的方法，包括向有需要的受试者给予本公开所述的能够靶向肿瘤细胞的巨噬细胞。
在一种或多种实施方式中，所述肿瘤包括以下中至少一种：急性淋巴母细胞性白血病、急性骨髓源性白血病、胆管癌、乳腺癌、子宫颈癌、慢性淋巴细胞性白血病、慢性骨髓源性白血病、结肠直肠癌、子宫内膜癌、食道癌、胃癌、头颈部癌、霍奇金氏淋巴瘤、肺癌、甲状腺髓样癌、非霍奇金氏淋巴瘤、多发性骨髓瘤、肾癌、卵巢癌、胰腺癌、神经胶质瘤、黑素瘤、肝癌、前列腺癌或尿路膀胱癌。
与现有技术相比，本公开的有益效果为：
本公开提供一种能够靶向肿瘤细胞的巨噬细胞，该巨噬细胞中含有嵌合抗原受体。发明人发现CAR-T细胞疗法在肿瘤治疗中存在一些技术缺陷，由于实体肿瘤的微环境的限制，CAR-T细胞进入肿瘤内部十分困难，即便进入，又由于微环境中的抑制作用，杀伤肿瘤细胞的作用也会减弱。发明人针对上述技术缺陷，提出了另一种肿瘤免疫治疗的思路， 让嵌合抗原受体在巨噬细胞中表达。巨噬细胞相较于T细胞具有更易进入实体肿瘤内部和不易受到其他类型细胞抑制的优势，所以可以更好地发挥肿瘤免疫治疗的作用。由于表达的嵌合抗原受体位于巨噬细胞的表面，所以该巨噬细胞可以准确的靶向肿瘤细胞。同时，发明人通过试验发现，T细胞中适用的嵌合抗原受体同样适用于巨噬细胞，即CAR-T细胞疗法中的嵌合抗原受体应用于巨噬细胞可以实现嵌合抗原受体在巨噬细胞表面的表达、靶向肿瘤细胞并且激活巨噬细胞将肿瘤细胞吞噬。所以，应用嵌合抗原受体修饰巨噬细胞这一发现为实体肿瘤免疫治疗提供了新的思路和技术手段，对于肿瘤免疫治疗具有重要的意义。
本公开提供了一种能够靶向肿瘤细胞的巨噬细胞的制备方法，该方法为免疫肿瘤治疗提供一个全新的思路。
附图说明
图1A为本公开实施例9中第14天髓系细胞中标志物CD45流式细胞技术检测结果图；
图1B为本公开实施例9中第14天髓系细胞中标志物CD34流式细胞技术检测结果图；
图1C为本公开实施例9中第14天髓系细胞中标志物CD11b流式细胞技术检测结果图；
图1D为本公开实施例9中第14天髓系细胞中标志物CD14流式细胞技术检测结果图；
图1E为本公开实施例9中第45天的成熟巨噬细胞中标志物CD11b流式细胞技术检测结果图；
图1F为本公开实施例9中第45天的成熟巨噬细胞中标志物CD14流式细胞技术检测结果图；
图1G为本公开实施例9中第45天的成熟巨噬细胞中标志物CD163流式细胞技术检测结果图；
图1H为本公开实施例9中第45天的成熟巨噬细胞中标志物CD86流式细胞技术检测结果图；
图2A为本公开实施例10中流式细胞技术检测野生型iPS细胞表面嵌合抗原受体表达；
图2B为本公开实施例10中流式细胞技术检测稳定表达嵌合抗原受体的iPS细胞表面的嵌合抗原受体表达；
图2C为本公开实施例10中流式细胞技术检测稳定表达嵌合抗原受体的iPS细胞分化成巨噬细胞后的细胞表面的嵌合抗原受体表达；
图3为本公开实施例11中流式细胞技术检测B2M敲除后的细胞免疫结果图；
图4A为本公开实施例12中iPS分化得到的巨噬细胞吞噬Raji癌细胞的聚焦显微镜拍照图；
图4B为本公开实施例12中iPS分化得到的巨噬细胞吞噬癌细胞的统计结果图。
图5A为将2×10^5过表达嵌合抗原受体CD19 CAR的iPS或ES细胞分化得到的巨噬 细胞iMAC，分别于4×10^4表达CD19抗原的K562肿瘤细胞和不表达CD19抗原的K562细胞共培养24小时，检测iMAC对肿瘤细胞的吞噬作用。
图5B，如同图A的实验，从与肿瘤细胞共培养后的巨噬细胞中提取RNA，测量细胞因子(TNF、IL-6和IL-1 beta)的表达。
具体实施方式
下面将结合实施例对本公开的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本公开，而不应视为限制本公开的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。
一种能够靶向肿瘤细胞的巨噬细胞，该巨噬细胞包括嵌合抗原受体。
发明人发现CAR-T细胞疗法在肿瘤治疗中存在一些技术缺陷，由于实体肿瘤的微环境的限制，CAR-T细胞进入肿瘤内部十分困难，即便进入，又由于微环境中的抑制作用，杀伤肿瘤细胞的作用也会减弱。发明人针对上述技术缺陷，提出了另一种肿瘤免疫治疗的思路，让嵌合抗原受体在巨噬细胞中表达。巨噬细胞相较于T细胞具有更易进入实体肿瘤内部和不易受到其他类型细胞抑制的优势，所以可以更好地发挥肿瘤免疫治疗的作用。由于表达的嵌合抗原受体位于巨噬细胞的表面，所以该巨噬细胞可以准确的靶向肿瘤细胞。同时，发明人通过试验发现，T细胞中适用的嵌合抗原受体同样适用于巨噬细胞，即CAR-T细胞疗法中的嵌合抗原受体应用于巨噬细胞可以实现嵌合抗原受体在巨噬细胞表面的表达、靶向肿瘤细胞并且激活巨噬细胞将肿瘤细胞吞噬。所以，应用嵌合抗原受体修饰巨噬细胞这一发现为实体肿瘤免疫治疗提供了新的思路和技术手段，对于肿瘤免疫治疗具有重要的意义。
在一种或多种实施方式中，巨噬细胞为HLA-I(human lymphocyte antigen I，人类淋巴细胞抗原)缺陷型巨噬细胞。让巨噬细胞表达嵌合抗原受体可以使巨噬细胞高效地靶向肿瘤细胞并激活自身进行肿瘤细胞的吞噬，但是由于MHC(major histocompatibility complex，主要组织相容体复合物)的特异性识别作用，导致异体细胞移植的免疫排斥反应和移植物的抗宿主反应，因此，能够靶向肿瘤细胞的巨噬细胞的通用性有待提高，通过对巨噬细胞的MHC进行改造，构建HLA-I基因缺陷型，可以避免异体排斥反应，实现能够靶向肿瘤细胞的巨噬细胞的通用性，进一步降低免疫肿瘤治疗的成本。HLA-I是具有高度多态性的同种异体抗原，与器官移植、免疫排斥等有重大的关系，巨噬细胞的HLA-I可以被敲除，从而减少异体的免疫排斥问题，相比目前的用于免疫细胞治疗的野生型CAR-T细胞等具有更广泛、通用的应用范围。采用的方法是直接敲除HLA-I复合体中的B2M基因，从而实现降低细胞的免疫原性，以避免分化成免疫细胞后宿主对移植细胞产生排斥反应，从而实现异体移植。
在一种或多种实施方式中，巨噬细胞为B2M基因缺陷型巨噬细胞。B2M即β2微球蛋白，是MHCⅠ类分子中的一员，它存在于所有的有核细胞中，但不包括红血细胞。B2M对于MHCⅠ类蛋白细胞表面的表达和肽结合区域的稳定性是必要的。事实上，在缺少B2M的情况下，在细胞表面几乎很少能检测到MHCⅠ类蛋白。构建B2M基因缺陷型巨噬细胞可以有效降低宿主对移植细胞的免疫排斥反应。
在一种或多种实施方式中，巨噬细胞由多能干细胞定向分化得到，其中，多能干细胞含有编码嵌合抗原受体的基因。无论是T细胞还是巨噬细胞，都是成熟细胞，细胞的扩增能力有限，同时，由于载体的转化效率和基因编辑效率的影响，得到的正确编辑的细胞数量十分有限，未必能够满足临床上需要的细胞剂量，产品成本太高。所以针对这一问题，可以应用多能干细胞定向分化成巨噬细胞来解决，并且对多能干细胞进行基因改造，使其可以表达编码嵌合抗原受体的基因和/或成为B2M缺陷型，再将该多能干细胞定向分化，得到大量的能够靶向肿瘤细胞的巨噬细胞。多能干细胞具有无限增殖和分化成免疫细胞的能力，并且在对多能干细胞进行基因编辑后可以选择编辑正确并且不存在脱靶效应的单克隆。
在一种或多种实施方式中，多能干细胞为HLA-I缺陷型多能干细胞。对多能干细胞进行HLA-I缺陷改造，得到通用性强的，异体抑制没有免疫排斥反应的，能够靶向肿瘤细胞的巨噬细胞。
在一种或多种实施方式中，多能干细胞为B2M基因缺陷型多能干细胞。
在一种或多种实施方式中，多能干细胞包括诱导性多能干细胞和/或胚胎干细胞。
在一种或多种实施方式中，编码嵌合抗原受体的基因位于载体上。
在一种或多种实施方式中，载体包括质粒载体或病毒载体。
在一种或多种实施方式中，病毒载体为逆转录病毒载体，优选为慢病毒载体。
在一种或多种实施方式中，用于构建B2M基因缺陷型的质粒载体为如下a)或b)中载体中的一种：
a)能够表达gRNA和Cas9蛋白；
b)能够表达gRNA和Cpf1蛋白。
在一种或多种实施方式中，嵌合抗原受体包括胞外抗原结合区、跨膜区、共刺激结构域和胞内信号转导区。需要说明的是，适用于T细胞的嵌合抗原受体可以作为巨噬细胞的嵌合抗原受体。
在一种或多种实施方式中，胞外抗原结合区包括sc-Fv、Fab、scFab或scIgG抗体片段。
在一种或多种实施方式中，该识别肿瘤的抗原结合区识别以下抗原组成的组中的任一种：CD19、CD20、CD22、CD30、GD2、HER2、CAIX、CD171、Mesothelin、LMP1、EGFR、Muc1、GPC3、EphA2、EpCAM、MG7、CSR、α-甲胎蛋白(AFP)、α-辅肌动蛋白-4、A3、对A33抗体 具有特异性的抗原、ART-4、B7、Ba 733、BAGE、BrE3抗原、CA125、CAMEL、CAP-1、碳酸酐酶IX、CASP-8/m、CCL19、CCL21、CD1、CD1a、CD2、CD3、CD4、CD5、CD8、CD11A、CD14、CD15、CD16、CD18、、CD21、CD23、CD25、CD29、CD32b、CD33、CD37、CD38、CD40、CD40L、CD44、CD45、CD46、CD52、CD54、CD55、CD59、CD64、CD66a-e、CD67、CD70、CD70L、CD74、CD79a、CD79b、CD80、CD83、CD95、CD126、CD132、CD133、CD138、CD147、CD154、CDC27、CDK-4/m、CDKN2A、CTLA4、CXCR4、CXCR7、CXCL12、HIF-1α、结肠特异性抗原p(CSAp)、CEA(CEACAM-5)、CEACAM-6、c-Met、DAM、EGFRvIII、EGP-1(TROP-2)、EGP-2、ELF2-M、Ep-CAM、纤维母细胞生长因子(FGF)、Flt-1、Flt-3、叶酸受体、G250抗原、GAGE、gp100、GRO-β、HLA-DR、HM1.24、人绒毛膜促性腺激素(HCG)和它的亚单位、HMGB-1、缺氧诱导性因子(HIF-1)、HSP70-2M、HST-2、Ia、IGF-1R、IFN-γ、IFN-α、IFN-β、IFN-λ、IL-4R、IL-6R、IL-13R、IL-15R、IL-17R、IL-18R、IL-2、IL-6、IL-8、IL-12、IL-15、IL-17、IL-18、IL-23、IL-25、胰岛素样生长因子1(IGF-1)、KC4抗原、KS-1抗原、KS1-4、Le-Y、LDR/FUT、巨噬细胞迁移抑制因子(MIF)、MAGE、MAGE-3、MART1、MART-2、NY-ESO-1、TRAG-3、mCRP、MCP-1、MIP-1A、MIP-1B、MIF、MUC2、MUC3、MUC4、MUC5ac、MUC13、MUC16、MUM-1/2、MUM-3、NCA66、NCA95、NCA90、胰腺癌粘蛋白、PD1受体、胎盘生长因子、p53、PLAGL2、前列腺酸性磷酸酶、PSA、PRAME、PSMA、PlGF、ILGF、ILGF-1R、IL-6、IL-25、RS5、RANTES、T101、SAGE、S100、存活素、存活素-2B、TAC、TAG-72、腱生蛋白、TRAIL受体、TNF-α、Tn抗原、汤姆逊-弗雷登里希抗原、肿瘤坏死抗原、VEGFR、ED-B纤连蛋白、WT-1、17-1A抗原、补体因子C3、C3a、C3b、C5a、C5、血管生成标志物、bc1-2、bc1-6、Kras、致癌基因标志物和致癌基因产物。
在一种或多种实施方式中，所述胞外抗原结合区特异性结合CD19。
在一种或多种实施方式中，跨膜区包括CD3ζ、CD4、CD8或CD28中的至少一种。
在一种或多种实施方式中，共刺激结构域包括与CD27、CD28、CD137、OX40、CD30、CD40、PD-1、LFA-1、CD2、CD7、Lck、DAP10、ICOS、LIGHT、NKG2C、B7-H3或CD3ζ特异性结合的配体中的至少一种。
在一种或多种实施方式中，胞内信号转导区包括CD3ζ、FcεRIγ、PKCθ或ZAP70中的至少一种。
在一种或多种实施方式中，嵌合抗原受体还包括报告基因。
在一种或多种实施方式中，报告基因为荧光报告基因。
在一种或多种实施方式中，荧光报告基因选自GFP、EGFP、RFP、mCherry、mStrawberry、Luciferase、mApple、mRuby、EosFP中的任一种。
在一种或多种实施方式中，该能够靶向肿瘤细胞的巨噬细胞或能够分化成该巨噬细胞的疗法适用于治疗癌症。预期任何类型的肿瘤和任何类型的肿瘤抗原都可被靶向。可被靶 向的示例性类型的癌症包括急性淋巴母细胞性白血病、急性骨髓源性白血病、胆管癌、乳腺癌、子宫颈癌、慢性淋巴细胞性白血病、慢性骨髓源性白血病、结肠直肠癌、子宫内膜癌、食道癌、胃癌、头颈部癌、霍奇金氏淋巴瘤、肺癌、甲状腺髓样癌、非霍奇金氏淋巴瘤、多发性骨髓瘤、肾癌、卵巢癌、胰腺癌、神经胶质瘤、黑素瘤、肝癌、前列腺癌和尿路膀胱癌等。然而，需要说明的是，本领域技术人员应该认识到实际上任何类型的癌的肿瘤相关抗原都是已知的。
一种能够靶向肿瘤细胞的巨噬细胞的制备方法，将编码嵌合抗原受体的基因在巨噬细胞中表达，得到能够靶向肿瘤细胞的巨噬细胞。该方法为免疫肿瘤治疗提供一个全新的思路。
在一种或多种实施方式中，制备方法还包括制备HLA-I基因缺陷的巨噬细胞的步骤。
在一种或多种实施方式中，制备方法还包括制备B2M基因缺陷的巨噬细胞的步骤。
在一种或多种实施方式中，制备方法包括将多能干细胞定向分化得到能够靶向肿瘤细胞的巨噬细胞，多能干细胞含有编码嵌合抗原受体的基因。
在一种或多种实施方式中，多能干细胞为HLA-I缺陷型多能干细胞。
在一种或多种实施方式中，多能干细胞为B2M基因缺陷型多能干细胞。
在一种或多种实施方式中，多能干细胞包括诱导性多能干细胞和/或胚胎干细胞。
在一种或多种实施方式中，将编码嵌合抗原受体的基因重组在载体上，在巨噬细胞中进行表达。
在一种或多种实施方式中，将报告基因与所述嵌合抗原受体进行重组后与载体进行连接。
在一种或多种实施方式中，报告基因为荧光报告基因。
在一种或多种实施方式中，荧光报告基因选自GFP、EGFP、RFP、mCherry、mStrawberry、Luciferase、mApple、mRuby、EosFP中的任一种。
在一种或多种实施方式中，定向分化包括以下步骤：将由多能干细胞诱导分化得到拟胚体放置在第一培养基中进行第一阶段培养，然后用第二培养基、第三培养基、第四培养基、第五培养基、第六培养基和第七培养基依次进行第二阶段、第三阶段、第四阶段、第五阶段、第六阶段和第七阶段培养，其中，第一阶段为接种后0-1天，第二阶段为接种后2-7天，第三阶段为接种后8-10天，第四阶段为接种后10-20天，第五阶段为接种后20-22天，第六阶段为接种后22-28天，第七阶段为接种后第29天。
上述的细胞诱导培养方法是先将带有编码嵌合抗原受体基因的多能干细胞培养形成拟胚体，再经过细胞诱导培养基的培养，最后可以得到大量的能够靶向肿瘤细胞的巨噬细胞。
需要说明的是，第一阶段得到的为中胚层细胞，第二阶段得到的为造血细胞，第三阶段得到的为髓系细胞，第四阶段得到的为成熟的巨噬细胞。
在一种或多种实施方式中，第二阶段培养时需要每隔一天更换新的第二培养基，第三阶段培养时需要每隔一天更换新的第三培养基，第五阶段进行培养的细胞为第四阶段培养后得到的悬浮细胞。
在一种或多种实施方式中，多能干细胞形成拟胚体(EB)的过程如下：
mTeSR1，DMEM/F12和Versene预热到15-25℃用于进行细胞传代。Y27632为Rock激酶抑制剂，使用浓度为3μM。
a)用1ml不含DPBS洗原孔；
b)吸去DPBS，加入1ml含有Y27632的Versene，37℃孵育4min；
c)使用移液器吹打1-2次并移出细胞(通常细胞仍处于较大的团块中会形成更好的EB)；
d)立即将细胞转移到含有DMEM/F12的离心管中以1:5-9的比例稀释Versene；用1ml DMEM/F12洗一次原孔，收集剩余的细胞并转移到试管中，300×g离心5min；
e)含有Y27632的mTeSR1培养基重悬细胞并将细胞放入超低附板上，分离比为1-2:1(每孔有90％的多能干细胞)。
在一种或多种实施方式中，第10天接种细胞时，细胞数量为20-25个/ml。
在一种或多种实施方式中，在本申请的方案中，可以按下列1)-3)中的任意一种方式更换培养基：
1)在管中放置细胞5min(管中用DPBS中的0.1％BSA包被)；
2)300rpm/min离心3min；
3)过滤器过滤更换。
在一种或多种实施方式中，细胞诱导培养过程中，不同类型平板的培养基体积如下：6孔板为2.0mL/孔；24孔板为0.5mL；96孔板为150μL/孔。
在一种或多种实施方式中，第四阶段、第五阶段、第六阶段和第七阶段进行培养时均需要提供基质胶。
在一种或多种实施方式中，基质胶包括Matrigel或Laminin-521。
在一种或多种实施方式中，多能干细胞诱导分化得到拟胚体的步骤包括：多能干细胞经Rock激酶抑制剂Y27632处理后再加入细胞消化液Accutase并在36-38℃条件下孵育10-14h得到拟胚体。
在一种或多种实施方式中，第一培养基包括第一基础培养基和第一细胞因子，第一细胞因子包括BMP4和bFGF；
第二培养基包括第一基础培养基和第二细胞因子，第二细胞因子包括BMP4，bFGF，VEGF和SCF；
第三培养基包括第一基础培养基和第三细胞因子，第三细胞因子包括bFGF，VEGF，SCF， IGF1，IL-3，M-CSF和GM-CSF；
第四培养基包括第二基础培养基和第三细胞因子；
第五培养基包括第二基础培养基和第四细胞因子，第四细胞因子包括bFGF，VEGF，SCF，IGF1，IL-3，M-CSF和GM-CSF；
第六培养基包括第二基础培养基和第五细胞因子，第五细胞因子包括bFGF，VEGF，SCF，IGF1，M-CSF和GM-CSF；
第七培养基包括第三基础培养基、第六细胞因子和FBS，第六细胞因子包括M-CSF和GM-CSF；
其中，第一基础培养基和第二基础培养基为无血清培养基；
第三基础培养基为含血清培养基。
上述的细胞诱导培养基组合连续使用，可以实现拟胚体细胞快速大量地诱导分化成巨噬细胞，由于拟胚体由多能干细胞分化得到，多能干细胞可以稳定表达嵌合抗原受体，所以，得到的巨噬细胞可以表达嵌合抗原受体，具有肿瘤细胞吞噬能力。其中，前六种培养基为无血清培养基，可以提供细胞各个阶段生长、增殖和分化所用的基础营养物质的同时降低污染风险。此外，第七培养基中含有血清和FBS，可以很好的维持巨噬细胞的生长。由于上述每种培养基中各自含有特定的多种细胞因子，所以能够促进细胞的定向分化，最终得到大量的性能稳定高质量的巨噬细胞。
BMP4(bone morphogenetic protein 4，骨形态发生蛋白4)属于TGF-β超家族，对骨骼的胚胎发育和再生修复起重要作用。BMP4参与调节细胞的增殖、分化和凋亡的等生物学过程，在胚胎发育、出生后各组织器官内环境稳定及多种肿瘤的发生中都有重要作用。
bFGF是成纤维细胞生长因子中的一种，为碱性成纤维细胞生长因子，是细胞形态发生和分化的诱导因子，可诱导促进多种细胞的增殖和分化。
VEGF(vascular endothelial growth factor，血管内皮生长因子)是一种高度特异性的促血管内皮细胞生长因子，具有增加血管通透性、促进血管内皮细胞迁移和细胞外基质变性、促进细胞增殖和血管形成的作用。
SCF(Stem cell factor，干细胞因子)是由骨髓微环境中的基质细胞产生的一种酸性糖蛋白。
IGF1为类胰岛素生长因子(insulin-like growth factors)中的一种，促进细胞生长和分化。
IL-3(Interleukin-3，白细胞介素-3)是趋化因子家族的一种细胞因子，可以调节造血与免疫。
M-CSF(巨噬细胞CSF)和GM-CSF(粒细胞和巨噬细胞CSF)均为集落刺激因子(CSF)中的一种，M-CSF具有刺激巨噬细胞集落、刺激粒细胞功能，降低血胆固醇。GM-CSF可以 刺激粒细胞，巨噬细胞集落形成，刺激粒细胞功能。
FBS为胎牛血清，是一种性状、外观浅黄色澄清、无溶血、无异物稍粘稠液体。FBS中所含的抗体、补体等对细胞有害的成分最少，含有丰富的细胞生长必须的营养成份。
在一种或多种实施方式中，第一基础培养基为STEMdiff TMAPEL TM2或mTeSR1。
在一种或多种实施方式中，第二基础培养基为StemPro TM-34。
在一种或多种实施方式中，第三基础培养基为RPMI-1640。
在一种或多种实施方式中，第一培养基中，BMP4和bFGF的终浓度依次为8-12ng/ml和3-7ng/ml。BMP4浓度典型但非限制性的为8ng/ml、10ng/ml或12ng/ml；bFGF浓度典型但非限制性的为3ng/ml、5ng/ml或7ng/ml。
在一种或多种实施方式中，第二培养基中，BMP4，bFGF，VEGF和SCF的终浓度依次为8-12ng/ml、3-7ng/ml、48-52ng/ml和95-105ng/ml。BMP4浓度典型但非限制性的为8ng/ml、10ng/ml或12ng/ml；bFGF浓度典型但非限制性的为3ng/ml、5ng/ml或7ng/ml；VEGF浓度典型但非限制性的为48ng/ml、50ng/ml或52ng/ml；SCF浓度典型但非限制性的为95ng/ml、99ng/ml、100ng/ml、104ng/ml或105ng/ml。
在一种或多种实施方式中，第三培养基中，bFGF，VEGF，SCF，IGF1，IL-3，M-CSF和GM-CSF的终浓度依次为8-12ng/ml、48-52ng/ml、48-52ng/ml、8-12ng/ml、23-27ng/ml、48-52ng/ml和48-52ng/ml。bFGF浓度典型但非限制性的为8ng/ml、10ng/ml或12ng/ml；VEGF浓度典型但非限制性的为48ng/ml、50ng/ml或52ng/ml；SCF浓度典型但非限制性的为48ng/ml、50ng/ml或52ng/ml；IGF1浓度典型但非限制性的为8ng/ml、10ng/ml或12ng/ml；IL-3浓度典型但非限制性的为23ng/ml、25ng/ml或27ng/ml；M-CSF浓度典型但非限制性的为48ng/ml、50ng/ml或52ng/ml；GM-CSF浓度典型但非限制性的为48ng/ml、50ng/ml或52ng/ml。
在一种或多种实施方式中，第五培养基中，bFGF，VEGF，SCF，IGF1，IL-3，M-CSF和GM-CSF的终浓度依次为8-12ng/ml、48-52ng/ml、48-52ng/ml、8-12ng/ml、23-27ng/ml、95-105ng/ml和95-105ng/ml。bFGF浓度典型但非限制性的为8ng/ml、10ng/ml或12ng/ml；VEGF浓度典型但非限制性的为48ng/ml、50ng/ml或52ng/ml；SCF浓度典型但非限制性的为48ng/ml、50ng/ml或52ng/ml；IGF1浓度典型但非限制性的为8ng/ml、10ng/ml或12ng/ml；IL-3浓度典型但非限制性的为23ng/ml、25ng/ml或27ng/ml；M-CSF浓度典型但非限制性的为95ng/ml、99ng/ml、102ng/ml、104ng/ml或105ng/ml；GM-CSF浓度典型但非限制性的为95ng/ml、99ng/ml、102ng/ml、104ng/ml或105ng/ml。
在一种或多种实施方式中，第六培养基中，bFGF，VEGF，SCF，IGF1，M-CSF和GM-CSF的终浓度依次为8-12ng/ml、48-52ng/ml、48-52ng/ml、8-12ng/ml、95-105ng/ml和95-105ng/ml。bFGF浓度典型但非限制性的为8ng/ml、10ng/ml或12ng/ml；VEGF浓度典 型但非限制性的为48ng/ml、50ng/ml或52ng/ml；SCF浓度典型但非限制性的为48ng/ml、50ng/ml或52ng/ml；IGF1浓度典型但非限制性的为8ng/ml、10ng/ml或12ng/ml；M-CSF浓度典型但非限制性的为95ng/ml、99ng/ml、102ng/ml、104ng/ml或105ng/ml；GM-CSF浓度典型但非限制性的为95ng/ml、99ng/ml、100ng/ml、104ng/ml或105ng/ml。
在一些实施方式中，第七培养基中，FBS，M-CSF和GM-CSF的终浓度依次为质量分数8-12％、95-105ng/ml和95-105ng/ml。FBS的质量分数典型但非限制性的为8％、10％或12％；M-CSF浓度典型但非限制性的为95ng/ml、99ng/ml、100ng/ml、104ng/ml或105ng/ml；GM-CSF浓度典型但非限制性的为95ng/ml、97ng/ml、100ng/ml、104ng/ml或105ng/ml。
在一种或多种实施方式中，第七培养基中，FBS经过灭活处理。
在一种或多种实施方式中，本公开还涉及一种能够分化得到上述能够靶向肿瘤细胞的巨噬细胞的多能干细胞。该多能干细胞通过基因编辑改造，同时通过特定的培养条件可以定向分化得到上述巨噬细胞。
下面通过具体的实施例进一步说明本公开，但是，应当理解为，这些实施例仅仅是用于更详细地说明之用，而不应理解为用于以任何形式限制本公开。
实施例1诱导多能干细胞的制备
-1天，从病人或志愿者身上抽取10ml外周血，用淋巴细胞分离液分离得到PBMC(外周血单核细胞)，H3000+CC100培养，复苏MEF细胞(成纤维细胞)。
0天，取1-2million的PBMC，电转含重编程因子OCT4、SOX2、KLF4、LIN28、L-MYC的质粒，电转后细胞放入H3000+CC100的培养基中培养，4h后250rcf离心5min，弃上清，用H3000+CC100的培养基重悬，放入MEF细胞板中培养。
2天，复苏MEF细胞。
3天，取细胞上清到15ml离心管中，贴壁细胞加200ul Tryple消化5min，1ml H3000终止消化，吹打转移到相对应的离心管中，250rcf离心5min，弃上清，用H3000+CC100的培养基重悬，放入新的MEF细胞板中培养。
4天，加200ul E8培养基。
6天，8天，10天，取1ml培养基，250rcf离心5min,弃上清，用1.2ml E8培养基重悬，放入原来的细胞板中培养。
11-20天，吸掉上清，换成E8培养基。
约15天左右出现克隆，待其生长至一定的程度，挑单克隆细胞至含Matrigel的96孔板中，继续培养传代，得到iPS细胞(诱导性多能干细胞)。
实施例2 293T细胞的复苏、培养传代
(1)复苏：从液氮罐中取出冻存的细胞迅速放到37℃水浴锅中，快速晃动使之融化。在超净台中准备一支15ml离心管，加入5ml完全培养基和冻存管中的细胞，混匀，离心 250rcf/min，5min。弃上清，用5ml完全培养基重悬转入T25培养瓶中，37℃，5％CO 2培养箱中培养。第二天观察细胞存活率，弃旧培养基，加入5ml新鲜培养基。
(2)培养传代：当细胞生长到80％-90％时传代培养。弃上清，加入5ml PBS轻轻振荡，弃PBS，加入1ml 0.25％胰酶，消化10s到20s至细胞变圆，细胞间隙变大，加入3ml完全培养基混匀移至15ml离心管中，离心250rcf/min，5min。弃上清，用2ml完全培养基重悬转入预留13ml完全培养基的T75培养瓶中，后同前培养。
实施例3慢病毒载体的构建
Lenti-EF1a-CD19-T2A-EGFP-Puro，包含特异性结合CD19抗原的scFv,来自CD8的跨膜结构域、来自4-1BB的共同刺激结构域，以及来自CD3zeta的胞内结构域，同时带有荧光基因EGFP和筛选基因puromycin。
实施例4慢病毒载体的鉴定
载体经内切酶EcoRI和XbaI酶切鉴定，酶切条带大小正确。
实施例5慢病毒的制备
当293T细胞长至60-70％时，慢病毒表达载体、包装载体和信封载体按照4:3:1的比例在10cm细胞培养板中用lip2000转染，6h后换液，24h和48h后分别收集上清，收集的上清经0.22um滤膜过滤后，加1/2体积的25％PEG，4℃过夜，第二天，4℃4000rcf离心20min，弃上清，用500ul的PBS重悬沉淀，每管50ul分装，放于-80℃。
实施例6 CAR稳定表达的iPS细胞的构建
测定病毒的滴度后，按照MOI为20将病毒感染iPS，感染后第3天加0.25ug/ml的puromycin筛选细胞3天，可获得稳定表达细胞系，后面可用于巨噬细胞的分化。
实施例7 HLA-I改造
B2M基因位于染色体15q21-22.2。B2M基因编码一种内源性低分子量血清蛋白质β2微球蛋白，β2微球蛋白与几乎所有有核细胞表面的MHC-Iβ2链有关。我们在B2M基因的第一个外显子设计了三条gRNA，分别连接到含有Cas9蛋白的PX458的载体上，再通过电转的方法将载体导入实施例6中的CAR稳定表达的iPS细胞内，用含有puromycin的培养基进行筛选。筛选后的细胞分成两组，一组正常培养，另一组正常培养的同时加50ng/ul的IFN-γ处理48h。野生型实施例6中的CAR稳定表达的iPS细胞也分成两组，一组正常培养，另一组正常培养的同时加50ng/ul的IFN-γ处理48h。然后再用B2M的流式抗体分别对这4组细胞进行孵育，上机检测B2M的敲除效果。结果显示与野生型实施例6中的CAR稳定表达的iPS相比，B2M敲除后的细胞经IFN-γ处理48h后无法诱导B2M，表明细胞的B2M基因已经敲除。
实施例8能够靶向肿瘤细胞的巨噬细胞的制备
1)CAR稳定表达的iPS细胞诱导形成拟胚体(EB)
mTeSR1，DMEM/F12和Versene预热到15-25℃用于进行细胞传代。Y27632为Rock激酶抑制剂，使用浓度为3μM。将实施例7中的细胞进行诱导：
a)用1ml DPBS洗原孔；
b)吸去DPBS，加入1ml含有Y27632的Versene，37℃孵育4min；
c)使用移液器吹打1-2次并移出细胞(通常细胞仍处于较大的团块中会形成更好的EB)；
d)立即将细胞转移到含有DMEM/F12的离心管中以1:5-9的比例稀释Versene；用1ml DMEM/F12洗一次原孔，收集剩余的细胞并转移到试管中，300×g离心5min；
e)含有Y27632的mTeSR1培养基重悬细胞并将细胞放入超低附板上，分离比为1-2:1(每孔有90％的诱导性多能干细胞)。
2)拟胚体(EB)诱导分化成巨噬细胞
步骤a)去除上述1)的f)中拟胚体的mTeSR1培养基，在第1天用第一培养基(STEMdiff TMAPEL TM2，10ng/ml BMP4，5ng/mlbFGF)孵育培养24h，拟胚体分化成中胚层细胞；
步骤b)去除步骤a)中的第一培养基，接种后的第2-7天期间用第二培养基(STEMdiff TM APEL TM2，10ng/ml BMP4，5ng/ml bFGF，50ng/ml VEGF和100ng/ml SCF)孵育培养中胚层细胞，期间每隔一天更换一次新的第二培养基，得到造血细胞；
步骤c)去除步骤b)中的第二培养基，接种后的第8-10天期间用第三培养基(STEMdiff TM APEL TM2，10ng/ml bFGF，50ng/ml VEGF，50ng/ml SCF，10ng/ml IGF1，25ng/ml IL-3，50ng/ml M-CSF和50ng/ml GM-CSF)孵育培养造血细胞，期间每隔一天更换一次新的第三培养基；
步骤d)去除步骤c)中的第三培养基，接种后的第11-20天期间，将细胞按照20-25个/ml的浓度接种到预先涂有基质胶Matrigel(1mg/ml)的培养皿中，用第四培养基(StemPro TM-34，10ng/ml bFGF，50ng/ml VEGF，50ng/ml SCF，10ng/ml IGF1，25ng/ml IL-3，50ng/ml M-CSF和50ng/ml GM-CSF)孵育培养步骤c)中的细胞，得到髓系细胞；
步骤e)接种后的第21-22天开始，收集步骤d)中悬浮的髓系细胞并重新铺板到预先涂有基质胶的培养皿中，用第五培养基(StemPro TM-34，10ng/ml bFGF，50ng/ml VEGF，50ng/ml SCF，10ng/ml IGF1，25ng/ml IL-3，100ng/ml M-CSF和100ng/ml GM-CSF)孵育培养，分化得到巨噬细胞；
步骤f)去除步骤e)中的第五培养基，接种后的第23-28天期间用第六培养基(StemPro TM-34，10ng/ml bFGF，50ng/ml VEGF，50ng/ml SCF，10ng/ml IGF1，100ng/ml M-CSF和100ng/ml GM-CSF)孵育巨噬细胞；
步骤g)去除步骤f)中的第六培养基，接种的第29天开始用第七培养基(RPMI-1640， 10％w/w FBS，100ng/ml M-CSF，100ng/ml GM-CSF)维持成熟的巨噬细胞或进行细胞冻存。
通过方法得到大量高质量高纯度的能够靶向肿瘤细胞的成熟巨噬细胞。
实施例9流式细胞检测
将实施例8中得到的各阶段细胞进行流式细胞检测，检测相关细胞的标记物，以评价定向分化的效果。结果如图1A-1H所示，需要说明的是，图1A-图1H中，1表示iPS细胞，2表示第14天的髓系细胞，3表示第45天的成熟巨噬细胞。
图1A为第14天的髓系细胞中血细胞的标志物CD45检测结果，图1B为第14天的髓系细胞中造血干细胞的标志物CD34检测结果，图1C为第14天的髓系细胞中巨噬细胞的标志物CD11b检测结果，图1D为第14天的髓系细胞中巨噬细胞的标志物CD14检测结果，图1E为第45天的成熟巨噬细胞中巨噬细胞的标志物CD11b检测结果，图1F为第45天的成熟巨噬细胞中巨噬细胞的标志物CD14检测结果，图1G为第45天的成熟巨噬细胞中巨噬细胞的标志物CD163检测结果，图1H第45天的成熟巨噬细胞中巨噬细胞的标志物CD86检测结果。
结果显示第14天出现巨噬细胞的标志物CD11b和CD14，第45天CD14的表达量上升，另外出现巨噬细胞新的标志物CD86和CD163，说明多能干细胞成功定向分化成成熟巨噬细胞。
实施例10嵌合抗原受体在巨噬细胞表面的表达
采用流式分析鉴定嵌合抗原受体在iPS细胞和分化的巨噬细胞表面是否表达。取野生型iPS细胞及实施例6中稳定表达嵌合抗原受体的iPS细胞，以及其分化成为的巨噬细胞(实施例8中的巨噬细胞)。300rcf离心5min后,去上清，PBS洗一次，重复离心，CAR的流式抗体孵育15min，300rcf离心5min，去上清，PBS洗一次，二抗孵育10min，离心5min，去上清，PBS洗一次，用含0.1％BSA的PBS重悬细胞，流式上机检测，结果如图2A-图2C所示，发现CAR能够表达在巨噬细胞的表面。
实施例11 HLA-I缺陷的免疫检测
取实施例7中的HLA-I(B2M)缺陷型的多能干细胞分为两组，一组正常培养，另一组加50ng/ul的IFN-γ处理48h。野生型实施例6中的CAR稳定表达的iPS细胞也分成两组，一组正常培养，另一组加50ng/ul的IFN-γ处理48h。然后再用B2M的流式抗体分别对这4组细胞进行孵育，流式分析检测B2M的敲除效果。结果如图3所示，说明与实施例6中的CAR稳定表达的iPS细胞相比，B2M敲除后的细胞经IFN-γ处理48h后无法诱导B2M，表明细胞的B2M基因已经敲除。
实施例12特异性吞噬癌症细胞检测
K562是表面不表达CD19抗原的急性髓系白血病细胞系。将表达CD19的慢病毒载体转化入K562细胞构建细胞表面表达CD19的细胞株。Raji细胞表面表达CD19抗原的来自B 细胞淋巴瘤的细胞系。将K562细胞、稳定表达CD19的K562细胞，以及Raji细胞感染mcherry的病毒，4-5天后流式分选，培养扩增mcherry阳性的稳转细胞系。
将实施例8中分化得到的巨噬细胞分别与以上三种mcherry的稳转细胞系培养4h后，共聚焦显微镜拍照，统计吞噬了表达mcherry癌症细胞的巨噬细胞。结果如图4A和图4B所示。试验结果表明本公开提供的巨噬细胞具有吞噬癌症细胞的能力，同时可以异源大规模的生产应用。
图5A为将2×10^5过表达嵌合抗原受体CD19 CAR的iPS或ES细胞分化得到的巨噬细胞iMAC，分别于4×10^4表达CD19抗原的K562肿瘤细胞和不表达CD19抗原的K562细胞共培养24小时，检测iMAC对肿瘤细胞的吞噬作用。iMAC与K562细胞分别用不同颜色的荧光染料标记，被双标的细胞代表能够吞噬肿瘤细胞的iMAC细胞。结果显示CD19CAR iMAC对表达CD19抗原的K562细胞的吞噬能力更强。图5B，如同图A的实验，从与肿瘤细胞共培养后的巨噬细胞中提取RNA，测量细胞因子(TNF、IL-6和IL-1β)的表达。与表达CD19抗原的K562细胞共培养过的巨噬细胞表达TNF、IL-6和IL-1β细胞因子的水平显著提高。
尽管已用具体实施例来说明和描述了本公开，然而应意识到，在不背离本公开的精神和范围的情况下可以作出许多其它的更改和修改。因此，这意味着在所附权利要求中包括属于本公开范围内的所有这些变化和修改。
工业实用性
本公开提供一种能够靶向肿瘤细胞的巨噬细胞，该巨噬细胞中含有嵌合抗原受体。发明人发现CAR-T细胞疗法在肿瘤治疗中存在一些技术缺陷，由于实体肿瘤的微环境的限制，CAR-T细胞进入肿瘤内部十分困难，即便进入，又由于微环境中的抑制作用，杀伤肿瘤细胞的作用也会减弱。发明人针对上述技术缺陷，提出了另一种肿瘤免疫治疗的思路，让嵌合抗原受体在巨噬细胞中表达。巨噬细胞相较于T细胞具有更易进入实体肿瘤内部和不易受到其他类型细胞抑制的优势，所以可以更好地发挥肿瘤免疫治疗的作用。由于表达的嵌合抗原受体位于巨噬细胞的表面，所以该巨噬细胞可以准确的靶向肿瘤细胞。同时，发明人通过试验发现，T细胞中适用的嵌合抗原受体同样适用于巨噬细胞，即CAR-T细胞疗法中的嵌合抗原受体应用于巨噬细胞可以实现嵌合抗原受体在巨噬细胞表面的表达、靶向肿瘤细胞并且激活巨噬细胞将肿瘤细胞吞噬。所以，应用嵌合抗原受体修饰巨噬细胞这一发现为实体肿瘤免疫治疗提供了新的思路和技术手段，对于肿瘤免疫治疗具有重要的意义。
本公开提供了一种能够靶向肿瘤细胞的巨噬细胞的制备方法，该方法为免疫肿瘤治疗提供一个全新的思路。

Claims (16)

  1. 一种能够靶向肿瘤细胞的巨噬细胞，其中，所述巨噬细胞包括嵌合抗原受体。
  2. 根据权利要求1所述的巨噬细胞，其中，所述巨噬细胞为HLA-I缺陷型巨噬细胞；
    优选地，所述巨噬细胞为B2M基因缺陷型巨噬细胞。
  3. 根据权利要求1或2所述的巨噬细胞，其中，所述巨噬细胞由多能干细胞定向分化得到，所述多能干细胞含有编码所述嵌合抗原受体的基因；
    优选地，所述多能干细胞为HLA-I缺陷型多能干细胞；
    优选地，所述多能干细胞为B2M基因缺陷型多能干细胞；
    优选地，所述多能干细胞包括诱导性多能干细胞和/或胚胎干细胞。
  4. 根据权利要求1-3任一项所述的巨噬细胞，其中，编码所述嵌合抗原受体的基因位于载体上；
    优选地，所述载体包括质粒载体或病毒载体；
    优选地，所述病毒载体为逆转录病毒载体，优选为慢病毒载体；
    优选地，用于构建B2M基因缺陷型的质粒载体为如下a)或b)中载体中的一种：
    a)能够表达gRNA和Cas9蛋白；
    b)能够表达gRNA和Cpf1蛋白；
    优选地，所述嵌合抗原受体包括胞外抗原结合区、跨膜区、共刺激结构域和胞内信号转导区；
    优选地，所述胞外抗原结合区包括sc-Fv、Fab、scFab或scIgG抗体片段；
    和/或，所述跨膜区包括CD3ζ、CD4、CD8或CD28中的至少一种；
    和/或，所述共刺激结构域包括与CD27、CD28、CD137、OX40、CD30、CD40、PD-1、LFA-1、CD2、CD7、Lck、DAP10、ICOS、LIGHT、NKG2C、B7-H3或CD3ζ特异性结合的配体中的至少一种；
    和/或，所述胞内信号转导区包括CD3ζ、FcεRIγ、PKCθ或ZAP70中的至少一种；
    优选地，所述嵌合抗原受体还包括报告基因；
    优选地，所述报告基因为荧光报告基因；
    优选地，所述荧光报告基因选自GFP、EGFP、RFP、mCherry、mStrawberry、Luciferase、mApple、mRuby、EosFP中的任一种。
  5. 根据权利要求1-4任一项所述的巨噬细胞，其中，所述胞外抗原结合区特异性 结合以下中的至少一种：CD19、CD20、CD22、CD30、GD2、HER2、CAIX、CD171、Mesothelin、LMP1、EGFR、Muc1、GPC3、EphA2、EpCAM、MG7、CSR、α-甲胎蛋白、α-辅肌动蛋白-4、A3、对A33抗体具有特异性的抗原、ART-4、B7、Ba 733、BAGE、BrE3抗原、CA125、CAMEL、CAP-1、碳酸酐酶IX、CASP-8/m、CCL19、CCL21、CD1、CD1a、CD2、CD3、CD4、CD5、CD8、CD11A、CD14、CD15、CD16、CD18、、CD21、CD23、CD25、CD29、CD32b、CD33、CD37、CD38、CD40、CD40L、CD44、CD45、CD46、CD52、CD54、CD55、CD59、CD64、CD66a-e、CD67、CD70、CD70L、CD74、CD79a、CD79b、CD80、CD83、CD95、CD126、CD132、CD133、CD138、CD147、CD154、CDC27、CDK-4/m、CDKN2A、CTLA4、CXCR4、CXCR7、CXCL12、HIF-1α、结肠特异性抗原p、CEACAM-5、CEACAM-6、c-Met、DAM、EGFRvIII、EGP-1、EGP-2、ELF2-M、Ep-CAM、纤维母细胞生长因子、Flt-1、Flt-3、叶酸受体、G250抗原、GAGE、gp100、GRO-β、HLA-DR、HM1.24、人绒毛膜促性腺激素和它的亚单位、HMGB-1、缺氧诱导性因子、HSP70-2M、HST-2、Ia、IGF-1R、IFN-γ、IFN-α、IFN-β、IFN-λ、IL-4R、IL-6R、IL-13R、IL-15R、IL-17R、IL-18R、IL-2、IL-6、IL-8、IL-12、IL-15、IL-17、IL-18、IL-23、IL-25、胰岛素样生长因子1、KC4抗原、KS-1抗原、KS1-4、Le-Y、LDR/FUT、巨噬细胞迁移抑制因子、MAGE、MAGE-3、MART1、MART-2、NY-ESO-1、TRAG-3、mCRP、MCP-1、MIP-1A、MIP-1B、MIF、MUC2、MUC3、MUC4、MUC5ac、MUC13、MUC16、MUM-1/2、MUM-3、NCA66、NCA95、NCA90、胰腺癌粘蛋白、PD1受体、胎盘生长因子、p53、PLAGL2、前列腺酸性磷酸酶、PSA、PRAME、PSMA、PlGF、ILGF、ILGF-1R、IL-6、IL-25、RS5、RANTES、T101、SAGE、S100、存活素、存活素-2B、TAC、TAG-72、腱生蛋白、TRAIL受体、TNF-α、Tn抗原、汤姆逊-弗雷登里希抗原、肿瘤坏死抗原、VEGFR、ED-B纤连蛋白、WT-1、17-1A抗原、补体因子C3、C3a、C3b、C5a、C5、血管生成标志物、bc1-2、bc1-6或Kras。
  6. 根据权利要求1-4任一项所述的巨噬细胞，其中，所述胞外抗原结合区特异性结合CD19。
  7. 一种能够分化得到权利要求1-6任一项所述巨噬细胞的多能干细胞。
  8. 一种权利要求1-6任一项所述的巨噬细胞的制备方法，其中，将编码嵌合抗原受体的基因在巨噬细胞中表达，得到所述能够靶向肿瘤细胞的巨噬细胞。
  9. 根据权利要求8所述的制备方法，其中，所述制备方法还包括制备HLA-I基因缺陷的巨噬细胞的步骤；
    优选地，所述制备方法还包括制备B2M基因缺陷的巨噬细胞的步骤。
  10. 根据权利要求8或9所述的制备方法，其中，所述制备方法包括将多能干细胞 定向分化得到能够靶向肿瘤细胞的巨噬细胞，所述多能干细胞含有编码嵌合抗原受体的基因；
    优选地，所述多能干细胞为HLA-I缺陷型多能干细胞；
    优选地，所述多能干细胞为B2M基因缺陷型多能干细胞；
    优选地，所述多能干细胞包括诱导性多能干细胞和/或胚胎干细胞；
    优选地，将所述编码嵌合抗原受体的基因重组在载体上，在巨噬细胞中进行表达；
    优选地，将报告基因与所述嵌合抗原受体进行重组后与载体进行连接；
    优选地，所述报告基因为荧光报告基因；
    优选地，所述荧光报告基因选自GFP、EGFP、RFP、mCherry、mStrawberry、Luciferase、mApple、mRuby、EosFP中的任一种。
  11. 根据权利要求10所述的制备方法，其中，所述定向分化包括以下步骤：将由多能干细胞诱导分化得到拟胚体放置在第一培养基中进行第一阶段培养，然后用第二培养基、第三培养基、第四培养基、第五培养基、第六培养基和第七培养基依次进行第二阶段、第三阶段、第四阶段、第五阶段、第六阶段和第七阶段培养；
    所述第一阶段为接种后0-1天，所述第二阶段为接种后2-7天，所述第三阶段为接种后8-10天，第四阶段为接种后10-20天，第五阶段为接种后20-22天，第六阶段为接种后22-28天，第七阶段为接种后第29天；
    优选地，所述第四阶段、第五阶段、第六阶段和第七阶段进行培养时均需要提供基质胶；
    优选地，所述基质胶包括Matrigel或Laminin-521。
  12. 根据权利要求11所述的制备方法，其中，所述第一培养基包括第一基础培养基和第一细胞因子，第一细胞因子包括BMP4和bFGF；
    所述第二培养基包括第一基础培养基和第二细胞因子，第二细胞因子包括BMP4，bFGF，VEGF和SCF；
    所述第三培养基包括第一基础培养基和第三细胞因子，第三细胞因子包括bFGF，VEGF，SCF，IGF1，IL-3，M-CSF和GM-CSF；
    所述第四培养基包括第二基础培养基和第三细胞因子；
    所述第五培养基包括第二基础培养基和第四细胞因子，第四细胞因子包括bFGF，VEGF，SCF，IGF1，IL-3，M-CSF和GM-CSF；
    所述第六培养基包括第二基础培养基和第五细胞因子，第五细胞因子包括bFGF，VEGF，SCF，IGF1，M-CSF和GM-CSF；
    所述第七培养基包括第三基础培养基、第六细胞因子和FBS，第六细胞因子包括 M-CSF和GM-CSF；
    其中，所述第一基础培养基和第二基础培养基为无血清培养基；
    所述第三基础培养基为含血清培养基；
    优选地，第一基础培养基为STEMdiff TMAPEL TM2或mTeSR1；
    优选地，第二基础培养基为StemPro TM-34；
    优选地，第三基础培养基为RPMI-1640。
  13. 根据权利要求1-6任一项所述的能够靶向肿瘤细胞的巨噬细胞在预防或治疗肿瘤中的用途。
  14. 根据权利要求13所述的用途，其中所述肿瘤包括以下中至少一种：急性淋巴母细胞性白血病、急性骨髓源性白血病、胆管癌、乳腺癌、子宫颈癌、慢性淋巴细胞性白血病、慢性骨髓源性白血病、结肠直肠癌、子宫内膜癌、食道癌、胃癌、头颈部癌、霍奇金氏淋巴瘤、肺癌、甲状腺髓样癌、非霍奇金氏淋巴瘤、多发性骨髓瘤、肾癌、卵巢癌、胰腺癌、神经胶质瘤、黑素瘤、肝癌、前列腺癌或尿路膀胱癌。
  15. 预防或治疗肿瘤的方法，包括向有需要的受试者给予根据权利要求1-6任一项所述的能够靶向肿瘤细胞的巨噬细胞。
  16. 根据权利要求15所述的方法，其中所述肿瘤包括以下中至少一种：急性淋巴母细胞性白血病、急性骨髓源性白血病、胆管癌、乳腺癌、子宫颈癌、慢性淋巴细胞性白血病、慢性骨髓源性白血病、结肠直肠癌、子宫内膜癌、食道癌、胃癌、头颈部癌、霍奇金氏淋巴瘤、肺癌、甲状腺髓样癌、非霍奇金氏淋巴瘤、多发性骨髓瘤、肾癌、卵巢癌、胰腺癌、神经胶质瘤、黑素瘤、肝癌、前列腺癌或尿路膀胱癌。
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