WO2024045890A1 - Preparation method for dendritic cell progenitors and culture medium thereof - Google Patents

Preparation method for dendritic cell progenitors and culture medium thereof Download PDF

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WO2024045890A1
WO2024045890A1 PCT/CN2023/105841 CN2023105841W WO2024045890A1 WO 2024045890 A1 WO2024045890 A1 WO 2024045890A1 CN 2023105841 W CN2023105841 W CN 2023105841W WO 2024045890 A1 WO2024045890 A1 WO 2024045890A1
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cells
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medium
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佟曼
马丽
向葱
崔晓燕
钱其军
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浙江吉量科技有限公司
上海吉量医药工程有限公司
上海细胞治疗集团有限公司
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • the present invention relates to the technical field of cell culture. More specifically, it relates to methods and applications for obtaining large amounts of iGDP (individual general dendritic cell progenitor, dendritic cell group cells) from peripheral blood.
  • iGDP individual general dendritic cell progenitor, dendritic cell group cells
  • the immune system including innate immunity and adaptive immunity, is an important protective barrier for the human body.
  • Dendritic cells are the most powerful professional antigen-presenting cells (APC) and are the bridge between innate immunity and adaptive immunity. Dendritic cells can activate initial T cells and control the immune response process in the body, and are the central link in the anti-tumor immune response. Immunotherapy using dendritic cells can induce the patient's body to produce a tumor-specific and long-lasting immune response.
  • dendritic cell vaccines also faces huge challenges.
  • the clinical efficacy of dendritic cell vaccine products is closely related to the dose of dendritic cells.
  • dendritic cells develop from CD34 + hematopoietic stem cells (HSC) in the bone marrow.
  • HSC hematopoietic stem cells
  • 90% of dendritic cell vaccine products are derived from peripheral blood mononuclear cells (monocytes), and 10% are derived from CD34 + hematopoietic stem cells in cord blood/mobilized peripheral blood, but sufficient numbers of dendritic cells cannot be obtained.
  • PBMC peripheral blood mononuclear cells
  • the yield of dendritic cells is only about 10%: 1.3E8 monocytes can be isolated from every 1E9 peripheral blood mononuclear cells ( 13%), 1.3E8 monocytes can differentiate into 1E8 dendritic cells (80%).
  • the technical route of obtaining dendritic cells through expansion and differentiation of CD34 + hematopoietic stem cells in cord blood/mobilized peripheral blood is used, the dendritic cell yield is only about 15%: 1E7 mononuclear cells can be isolated from every 1E9 cord blood/mobilized peripheral blood mononuclear cells.
  • 1E7 CD34 + hematopoietic stem cells can be expanded approximately 20 times in vitro to 2E8 cells can differentiate into 1.5E8 dendritic cells (75%).
  • dendritic cells have no ability to expand, how to efficiently obtain sufficient numbers of dendritic cell progenitor cells is of substantial significance for future immunotherapy.
  • a first aspect of the present invention provides a method for preparing dendritic cell progenitor cells, including the steps of: culturing cells from peripheral blood in a culture medium to obtain dendritic cell progenitor cells.
  • the culture medium contains at least two of growth factors, Raf/MEK/ERK pathway modulators, and isoxazole derivatives.
  • the culture medium contains growth factors, Raf/MEK/ERK pathway modulators and isoxazole derivatives.
  • the growth factor is selected from at least one of SCF, GM-CSF, G-CSF, IL-3, FLT-3L; preferably, the growth factor contains SCF and is selected from At least one of GM-CSF, G-CSF, IL-3, and FLT-3L.
  • the concentration of SCF when included, is 10-150ug/L, preferably 30-120ug/L.
  • the concentration of GM-CSF when included, is 0-80ug/L, preferably 0-50ug/L.
  • the concentration of G-CSF when included, is 0-200ug/L, preferably 0-150ug/L.
  • IL-3 when included, is present at a concentration of 0-80ug/L, preferably 0-50ug/L.
  • FLT-3L when included, FLT-3L is present at a concentration of 0-80ug/L, preferably 0-50ug/L.
  • the Raf/MEK/ERK pathway modulator is a MEK activator and/or an ERK activator.
  • the Raf/MEK/ERK pathway modulator is selected from one or more of GDC-0879, PLX4720, Vemurafenib (PLX4032), Sorafenib (BAY 43-9006), Dabrafenib (GSK2118436), more preferably GDC -0879.
  • the concentration of the Raf/MEK/ERK pathway modulator is 0.1-5 ⁇ M, preferably 0.5-1.5 ⁇ M.
  • the isoxazole derivative is a compound represented by formula (I).
  • R 1 is unsubstituted or substituted heteroaryl
  • n is an integer from 0 to 4, preferably an integer from 1 to 3,
  • X is CH 2 , NH or O
  • R 2 is unsubstituted or substituted heteroaryl.
  • the isoxazole derivative is a compound represented by any one of Formulas 1-21.
  • the isoxazole derivative is 5-furan-2-yl-isoxazole-3-carboxylic acid (2-pyrazol-1-yl-ethyl)-amide.
  • the concentration of the isoxazole derivative is 0-15 ⁇ M, preferably 0-10 ⁇ M, more preferably 1-10 ⁇ M.
  • the culture medium further contains a pyrimidinoindole derivative.
  • the pyrimidinoindole derivative is selected from one or both of UM171 and UM729. More preferably, The pyrimidinoindole derivative is UM171.
  • the concentration of the pyrimidoindole derivative is 0-80 nM, preferably 0-50 nM.
  • the medium further contains a basal medium, such as a basal serum-free medium, including commercial serum-free medium and basal medium supplemented with additives.
  • a basal medium such as a basal serum-free medium, including commercial serum-free medium and basal medium supplemented with additives.
  • the commercial serum-free medium is selected from StemSpan TM SFEM medium, StemSpan TM SFEM II medium, StemSpan TM -XF medium, StemSpan TM -AOF medium, StemPro TM -34 SFM one or more of them.
  • the basic medium is selected from one or more of IMDM medium, DMEM/F-12 medium, Neurobasal medium, and AIM-V; preferably, the supplement is added
  • the substance is selected from one or more of human albumin, insulin, transferrin, sodium selenite, DL- ⁇ -tocopherol, and linoleic acid.
  • the concentration of human albumin when contained, is 1-10g/L, preferably 1g/L-5g/L.
  • the insulin concentration is 1-20 mg/L, preferably 3-15 mg/L.
  • the concentration of transferrin when included, is 1-30 mg/L, preferably 3-30 mg/L. 25mg/L.
  • the concentration of sodium selenite when included, is 1-20ug/L, preferably 5-15ug/L.
  • DL- ⁇ -tocopherol when included, is present at a concentration of 0.5-10 mg/L, preferably 0.5-8 mg/L.
  • the concentration of linoleic acid when included, is 0.5-10 mg/L, preferably 0.5-5 mg/L.
  • the peripheral blood is peripheral blood without hematopoietic stem cell mobilization.
  • the cells from peripheral blood comprise one or more selected from the group consisting of CD45+ leukocytes, CD14+ cells, CD14- cells, CD123+ cells, CD123- cells, CD34- cells.
  • the present invention also provides a culture composition or a culture medium containing the culture medium composition for preparing dendritic cell progenitor cells from peripheral blood, the culture composition containing growth factors, Raf/MEK/ERK pathway regulators, isoforms At least two of the oxazole derivatives.
  • the culture medium contains growth factors, Raf/MEK/ERK pathway modulators, and isoxazole derivatives.
  • the growth factor is selected from at least one of SCF, GM-CSF, G-CSF, IL-3, FLT-3L; preferably, the growth factor contains SCF and is selected from At least one of GM-CSF, G-CSF, IL-3, and FLT-3L.
  • the concentration of SCF in the culture medium is 10-150ug/L, preferably 30-120ug/L.
  • the concentration of GM-CSF in the culture medium is 0-80ug/L, preferably 0-50ug/L.
  • the concentration of G-CSF in the culture medium is 0-200ug/L, preferably 0-150ug/L.
  • the concentration of IL-3 in the culture medium is 0-80ug/L, preferably 0-50ug/L.
  • the concentration of FLT-3L in the culture medium is 0-80ug/L, preferably 0-50ug/L.
  • the Raf/MEK/ERK pathway modulator is a MEK activator and/or an ERK activator.
  • the Raf/MEK/ERK pathway modulator is selected from one or more of GDC-0879, PLX4720, Vemurafenib (PLX4032), Sorafenib (BAY 43-9006), Dabrafenib (GSK2118436), more preferably GDC -0879.
  • the concentration of the Raf/MEK/ERK pathway modulator is 0.1-5 ⁇ M, preferably 0.5-1.5 ⁇ M.
  • the isoxazole derivative is a compound represented by formula (I).
  • R1 is unsubstituted or substituted heteroaryl
  • n is an integer from 0 to 4, preferably an integer from 1 to 3,
  • X is CH2, NH or O
  • R2 is unsubstituted or substituted heteroaryl.
  • the isoxazole derivative is a compound represented by any one of Formulas 1-21.
  • the isoxazole derivative is 5-furan-2-yl-isoxazole-3-carboxylic acid (2-pyrazol-1-yl-ethyl)-amide.
  • the concentration of the isoxazole derivative is 0-15 ⁇ M, preferably 0-10 ⁇ M, more preferably 1-10 ⁇ M.
  • the culture medium further contains a pyrimidinoindole derivative.
  • the pyrimidinoindole derivative is selected from one or both of UM171 and UM729. More preferably, The pyrimidinoindole derivative is UM171.
  • the concentration of the pyrimidoindole derivative is 0-80 nM, preferably 0-50 nM.
  • the concentration ratio of each growth factor, Raf/MEK/ERK pathway regulator, and isoxazole derivative in the culture composition is the same as the concentration ratio of the corresponding component in the culture medium.
  • the medium further contains a basal medium, such as a basal serum-free medium, including commercial serum-free medium and basal medium supplemented with additives.
  • a basal medium such as a basal serum-free medium, including commercial serum-free medium and basal medium supplemented with additives.
  • the commercial serum-free medium is selected from StemSpan TM SFEM medium, StemSpan TM SFEM II medium, StemSpan TM -XF medium, StemSpan TM -AOF medium, StemPro TM -34 SFM one or more of them.
  • the minimal medium is selected from IMDM medium, DMEM/F-12 One or more of medium, Neurobasal medium, and AIM-V; preferably, the supplement is selected from human albumin, insulin, transferrin, sodium selenite, and DL- ⁇ -tocopherol. , one or more of linoleic acid.
  • the concentration of human albumin is 1-10g/L, preferably 1g/L-5g/L.
  • the insulin concentration is 1-20 mg/L, preferably 3-15 mg/L.
  • the concentration of transferrin is 1-30 mg/L, preferably 3-25 mg/L.
  • the concentration of sodium selenite is 1-20ug/L, preferably 5-15ug/L.
  • the concentration of DL-alpha-tocopherol is 0.5-10 mg/L, preferably 0.5-8 mg/L.
  • the concentration of linoleic acid is 0.5-10 mg/L, preferably 0.5-5 mg/L.
  • the peripheral blood is peripheral blood without hematopoietic stem cell mobilization.
  • the cells from peripheral blood comprise one or more selected from the group consisting of CD45+ leukocytes, CD14+ cells, CD14- cells, CD123+ cells, CD123- cells, CD34- cells.
  • the present invention also provides the use of the culture composition or culture medium according to any embodiment of the present invention in preparing a reagent containing dendritic cell progenitor cells.
  • the invention also provides a dendritic cell progenitor cell prepared by the method described in any embodiment herein.
  • the present invention also provides a method for preparing dendritic cells, which includes: culturing the dendritic cell progenitor cells described in any embodiment of this article in a dendritic cell differentiation medium to obtain dendritic cells.
  • the present invention also provides a dendritic cell prepared by the preparation method of dendritic cells described herein.
  • the present invention also provides use of dendritic cell progenitor cells prepared by the method described in any embodiment herein in the preparation of a medicament.
  • the medicament is used to treat cancer, infectious diseases, and/or aging-related diseases.
  • the present invention also provides a pharmaceutical composition, comprising an effective amount of dendritic cell progenitor cells prepared by the method described in any embodiment of this document and pharmaceutically acceptable excipients.
  • the present invention also provides a cell cryopreservation preparation, including the aforementioned dendritic cell progenitor cells obtained by the method described in any embodiment of this article and a cryopreservation solution.
  • the invention also provides a method of treating or preventing a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition according to any embodiment herein.
  • the disease or disorder is cancer, infectious diseases, and/or aging-related diseases.
  • amplifying dendritic cell progenitor cells (iGDP cells) from peripheral blood is simple and easy to obtain, has high accessibility, and the iGDP amplification multiple is high.
  • the number of iGDP cells is the number of initial peripheral blood mononuclear cells. 83-197 times.
  • Figure 1 shows the cell expansion fold of samples 2-5 on day 14 using different iGDP amplification media (medium 11-27).
  • Figure 2 shows the cell expansion fold of sample 6 and sample 7 on the 14th day of culture using different iGDP amplification media (medium 15, 23, 24, 28-32).
  • Figure 4 shows the flow cytometric characterization of the efficient differentiation of iGDP into DCs for 14 days.
  • the differentiation starting cells were iGDP cells cultured on the 14th day of sample 4 in medium 15.
  • the control group was a blank control in which the sample was not incubated with antibodies.
  • Figure 5 shows the relative cell expansion folds of samples cultured in different iGDP amplification media (Medium 33-48) on days 13 and 14.
  • Figure 6 shows the relative cell numbers of iGDP-induced differentiated dendritic cells obtained with different iGDP amplification media (Medium 33-44).
  • dendritic cell progenitor cells can be obtained by culturing cells from peripheral blood.
  • dendritic cell progenitor cells iGDP, individual general dendritic cell progenitor refers to precursor cells that can differentiate to form dendritic cells.
  • the present invention provides a method for preparing dendritic cell progenitor cells, which includes the steps of: culturing cells from peripheral blood in a culture medium to obtain dendritic cell progenitor cells.
  • the present invention also provides a culture composition for the above preparation of dendritic cell progenitor cells from peripheral blood or a culture medium comprising the culture medium composition.
  • the culture composition or culture medium contains at least two of growth factors, Raf/MEK/ERK pathway regulators, and isoxazole derivatives.
  • the invention also provides dendritic cell progenitor cells prepared by the method.
  • the method of collecting peripheral blood described in this article is not limited, and it can be apheresis blood collection or machine collection peripheral blood, etc.
  • the peripheral blood is peripheral blood that has not been mobilized by hematopoietic stem cells.
  • the cells from peripheral blood are peripheral blood CD45 + leukocytes.
  • the cells from peripheral blood comprise one or more selected from the group consisting of CD45 + leukocytes, CD14 + cells, CD14- cells, CD123 + cells, CD123- cells, and CD34- cells.
  • some embodiments of the method of the present invention further include the step of obtaining one or more of CD45+ leukocytes, CD14+ cells, CD14- cells, CD123+ cells, CD123- cells, CD34- cells from peripheral blood.
  • the method of isolating cells from peripheral blood can adopt conventional methods in this field, such as hydroxyethyl starch centrifugal precipitation method, density gradient centrifugation method, immunomagnetic bead sorting method, flow cytometry sorting method, etc.
  • Density gradient centrifugation media include Ficoll-Hypaque, Percoll, etc.
  • Cells from peripheral blood can be fresh cells or cells that have been cryopreserved and then revived.
  • the conditions and time for culturing cells from peripheral blood in the culture medium can be determined by those skilled in the art as needed, for example, culturing at 37°C, 5% CO2 for at least 1 day, at least 3 days, at least 7 days, at least 14 days, At least 28 days.
  • the growth factor contained in the culture medium is selected from the group consisting of stem cell growth factor (SCF), granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), colony stimulating factor in granulocyte colony stimulating factor (G-CSF), interleukin-3 (IL-3), Fms-related tyrosine kinase 3 ligand (FLT-3L) At least one.
  • the growth factor comprises SCF.
  • the concentration of the growth factor is not limited, for example: the concentration of SCF is 10-150ug/L, preferably 30-120ug/L, such as 30ug/L, 40ug/L, 50ug/L, 60ug/L, 80ug/L , 100ug/L, 120ug/L; the concentration of GM-CSF is 0-80ug/L, preferably 0-50ug/L, for example, 0, 5ug/L, 8ug/L, 10ug/L, 15ug/L, 20ug /L, 30ug/L, 40ug/L, 50ug/L; the concentration of G-CSF is 0-200ug/L, preferably 0-150ug/L, such as 0, 10ug/L, 20ug/L, 25ug/L, 40ug/L, 50ug/L, 60ug/L, 80ug/L, 100ug/L, 150ug/L; the concentration of IL-3 is 0-80u
  • Raf/MEK/ERK pathway modulators refer to substances that can block or activate the Raf/MEK/ERK pathway.
  • MAPK mitogen-activated protein kinase
  • its signaling pathway can transduce extracellular signals into cells through the form of a three-level kinase cascade (MAPK, MAPK kinase (MEK or MKK) and MAPK kinase kinase (MEKK or MKKK))
  • MAPK mitogen-activated protein kinase
  • MEKK or MKKK MAPK kinase kinase
  • MEKK or MKKK MAPK kinase kinase
  • the Raf/MEK/ERK pathway is one of the four signaling pathways in the MAPK signaling pathway network.
  • the Raf/MEK/ERK pathway modulator contained in the culture medium is a MEK activator and/or an ERK activator.
  • the Raf/MEK/ERK pathway modulator contained in the culture medium is a B-Raf kinase inhibitor.
  • the Raf/MEK/ERK pathway modulator is selected from one or more of GDC-0879, PLX4720, Vemurafenib (PLX4032), Sorafenib (BAY 43-9006), Dabrafenib (GSK2118436).
  • the concentration of the Raf/MEK/ERK pathway modulator is not limited, and an exemplary concentration is 0.1-5 ⁇ M, preferably 0.5-1.5 ⁇ M, such as 0.5 ⁇ M, 0.6 ⁇ M, 0.8 ⁇ M, 1.0 ⁇ M, 1.2 ⁇ M, and 1.5 ⁇ M.
  • the Raf/MEK/ERK pathway modulator is 0.5-1.0 ⁇ M GDC-0879 or 1.0 ⁇ M Dabrafenib.
  • the isoxazole derivative is a compound represented by formula (I).
  • R 1 is unsubstituted or substituted heteroaryl
  • n is an integer from 0 to 4, preferably an integer from 1 to 3,
  • X is CH 2 , NH or O
  • R 2 is unsubstituted or substituted heteroaryl.
  • R1 is an unsubstituted or substituted five-membered heterocyclic ring containing 1 to 2 heteroatoms selected from N, O, S, such as pyrrolyl, furyl, thienyl, imidazolyl, pyrazole group, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, triazolyl, etc., preferably thienyl and furyl. It can be connected to the parent structure through heteroatoms or carbon atoms on the five-membered heterocyclic ring.
  • R is an unsubstituted or substituted five-membered heterocyclic ring containing 1 to 2 heteroatoms selected from N, O, S, such as pyrrolyl, furyl, thienyl, imidazolyl, pyrazole group, thiazolyl, isothiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, triazolyl and the like, preferably pyrazolyl, oxazolyl and thiazolyl.
  • the heteroaryl group can be connected to the parent structure through a heteroatom or a carbon atom on the five-membered heterocyclic ring.
  • the number of substituents may be 1 or 2 (mono-substituted or disubstituted), and the substituents are selected from alkyl, haloalkyl, hydroxyalkyl, cyano, halogen, ester, amino,
  • compounds of Formula (I) have the structure of Formula (Ia):
  • A is O or S
  • m, X and R2 are defined as above.
  • Formula (I) is selected from the following compounds:
  • the isoxazole derivative is 5-furan-2-yl-isoxazole-3-carboxylic acid (2-pyrazol-1-yl-ethyl)-amide (5-furan- 2-yl-isoxazole-3-carboxylic acid(2-pyrazol-1-yl-ethyl)-amide, the above-mentioned compound 21, CAS: 943820-93-7).
  • the concentration of the isoxazole derivative is not limited, and an exemplary concentration is 0-15 ⁇ M, preferably 0-10 ⁇ M, more preferably 1-10 ⁇ M, such as 0, 1 ⁇ M, 2 ⁇ M, 4 ⁇ M, 5 ⁇ M, 6 ⁇ M, and 10 ⁇ M.
  • the medium may also contain pyrimidoindole derivatives.
  • the pyrimidoindole derivative is a compound represented by formula (II):
  • R b is selected from -NR f R g , -OR h , where R f , R g , and R h are each independently selected from H, C1-C4 alkyl, -(CH 2 ) n R i , where n is 0 -4 is an integer, R i is selected from unsubstituted or substituted C1-C4 alkyl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclyl, unsubstituted or substituted aryl, unsubstituted or substituted Heteroaryl, -NR j R k , -CN, wherein R j and R k are each independently selected from unsubstituted or substituted C1-C4 alkyl;
  • R c is selected from H, -(CH 2 ) i R p , where i is an integer from 0 to 4, and R p is selected from unsubstituted or substituted C1-C4 alkyl, unsubstituted or substituted cycloalkyl, unsubstituted Substituted or substituted heterocyclyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, -NR j R k , -CN, wherein R j and R k are each independently selected from unsubstituted or substituted C1 -C4 alkyl.
  • Ra is an unsubstituted or substituted heteroaryl group, which is a five- or six-membered heterocyclic ring containing 1 to 2 heteroatoms selected from N, O, and S, such as Pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyridyl, Pyrimidinyl etc. It can be connected to the parent structure through heteroatoms or carbon atoms on the five- or six-membered ring.
  • one of R f and R g is H or C1-C4 alkyl, and the other is -(CH 2 ) n R i , where n is an integer from 0 to 4, and R i is selected from the group consisting of Substituted or substituted C1-C4 alkyl, unsubstituted or substituted C3-C6 cycloalkyl, unsubstituted or substituted five- or six-membered heterocyclyl (which contains 1 to 2 selected from N, O, S heteroatoms, which can be connected to the parent structure through heteroatoms or carbon atoms on the five- or six-membered ring), unsubstituted or substituted aryl groups, unsubstituted or substituted five- or six-membered heteroaryl groups (which contain 1 to 2 heteroatoms selected from N, O, S, which can be connected to the parent structure through heteroatoms or carbon atoms), -NR j R k , -
  • one of R f and R g is selected from H, C1-C4 alkyl, and the other is -(CH 2 ) n R i , where n is an integer from 0 to 4, and,
  • R i is selected from: C1-C4 alkyl, C1-C4 alkyl in which one hydrogen atom is substituted by C1-C4 alkoxy, C1-C4 alkyl that is unsubstituted or substituted by C1-C4 alkyl, hydroxyl, cyano Base, nitro, amino, C1-C4 alkoxy substituted C3-C6 cycloalkyl, unsubstituted or substituted (such as methyl) five-membered cycloalkyl group containing 1 to 2 heteroatoms selected from N, O, S Or six-membered heterocyclyl (such as pyrrolidinyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl), unsubstituted or C1-C4 alkyl, hydroxyl substituted C1-C4 alkyl, cyano Aryl group substituted by base, nitro, amino, C1-C4 alkoxy, unsubsti
  • R h is selected from H, -(CH 2 ) n R i , where n is an integer from 0 to 4, and,
  • R i is selected from: C1-C4 alkyl, C1-C4 alkyl in which one hydrogen atom is substituted by C1-C4 alkoxy, C1-C4 alkyl that is unsubstituted or substituted by C1-C4 alkyl, hydroxyl, cyano base, nitro, amino, C1-C4 alkoxy substituted C3-C6 cycloalkyl, unsubstituted or substituted C1-C4 alkyl, hydroxyl C1-C4 alkyl, cyano, nitro base, amino, C1-C4 alkoxy substituted five- or six-membered heterocyclic group containing 1 to 2 heteroatoms selected from N, O, S (such as pyrrolidinyl, morpholinyl, piperazinyl, Homopiperazinyl, piperidinyl), unsubstituted or C1-C4 alkyl substituted by C1-C4 alkyl, hydroxyl,
  • R c is selected from H, -(CH 2 ) i R p , where i is an integer from 0 to 4, and,
  • R p is selected from: C1-C4 alkyl, unsubstituted or C1-C4 alkyl substituted by C1-C4 alkyl, hydroxyl, cyano, nitro, amino, C1-C4 alkoxy substituted aryl, unsubstituted C1-C4 alkyl, cyano, nitro, amino, C1-C4 alkoxy substituted or substituted by C1-C4 alkyl, hydroxyl containing 1 to 2 heteroatoms selected from N, O, S One- or six-membered heteroaryl (such as pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, triazolyl, tetrazolyl, Pyridyl, pyrimidinyl), -NR j R k , -CN, wherein R j and R k are each
  • the pyrimidoindole derivative is selected from:
  • the pyrimidoindole derivative is selected from UM171 and UM729.
  • the concentration of the pyrimidoindole derivative is not limited, and an exemplary concentration is 0-80 nM, preferably 0-50 nM, such as 0, 5 nM, 10 nM, 20 nM, 25 nM, 40 nM, 50 nM. In some embodiments, the pyrimidinoindole derivative is 5-50 nM UM171 or 500 nM UM729.
  • the basal medium of the culture medium is not limited as long as it is suitable for the growth of dendritic cell progenitor cells.
  • the basal culture medium is a serum-free culture system suitable for the growth of dendritic cell progenitor cells.
  • the serum-free culture system can use commercial serum-free culture medium, such as StemSpan TM SFEM medium, StemSpan TM SFEM II medium, StemSpan TM -XF medium, StemSpan TM -AOF medium, StemPro TM -34SFM; it can also be used containing basic Medium and basal serum-free medium supplemented with supplements.
  • the basic medium is selected from one or more of IMDM medium, DMEM/F-12 medium, Neurobasal medium, and AIM-V.
  • the supplementary additive is selected from one or more of human albumin, insulin, transferrin, sodium selenite, DL- ⁇ -tocopherol, and linoleic acid. The content of supplementary additives is not limited.
  • the concentration of human albumin is 1-10g/L, preferably 1g/L-5g/L, such as 1g/L, 1.5g/L, 2g/L, 2.5g/L, 3g/L, 3.5g/L, 4g/L, 4.5g/L, 5g/L; insulin concentration is 1-20mg/L, preferably 3-15mg/L, such as 3mg/L, 4mg /L, 5mg/L, 6mg/L, 7mg/L, 8mg/L, 9mg/L, 10mg/L, 11mg/L, 12mg/L, 13mg/L, 14mg/L, 15mg/L; transferrin The concentration is 1-30mg/L, preferably 3-25mg/L, such as 3mg/L, 5mg/L, 8mg/L, 12mg/L, 15mg/L, 20mg/L, 21mg/L, 25mg/L; The concentration of sodium sele
  • the culture composition or medium contains a Raf/MEK/ERK pathway modulator and a growth factor, including SCF, and optionally G-CSF and/or IL-3.
  • the present invention also provides a method for preparing dendritic cells, which includes the step of culturing the aforementioned cells obtained by the method described in any embodiment of this article in a dendritic cell differentiation medium to obtain dendritic cells.
  • the method of preparing dendritic cells includes: (1) culturing cells from peripheral blood in the culture medium described herein (e.g., at least 1 day, at least 3 days, at least 7 days, at least 14 days, at least 28 days ), obtain a first cell population, and (2) culture the first cell population in dendritic cell differentiation medium (e.g., for at least 3 days, at least 6 days, at least 9 days, at least 12 days) to obtain dendritic cells.
  • the cells from peripheral blood include one or more selected from the group consisting of CD45+ leukocytes, CD14+ cells, CD14- cells, CD123+ cells, CD123- cells, and CD34- cells.
  • the dendritic cell differentiation medium is not limited, and dendritic cell differentiation medium commonly used in the field can be used.
  • the dendritic cell differentiation medium contains cytokines.
  • the cytokine may be selected from one or more of SCF, GM-CSF, IL-4, FLT-3L, IFN ⁇ , IL-15, etc.
  • the basal medium of the dendritic cell differentiation medium is not limited as long as it is suitable for dendritic cell differentiation.
  • the basal medium is selected from IMDM, RPMI-1640, AIM-V, etc.
  • serum or serum substitutes may be included as supplementary additives, such as FBS, etc.
  • the basal culture medium is a commercial culture medium, such as Immunocult TM -ACF Dendritic Cell Medium, etc.
  • supplementary additives such as Immunocult TM -ACF Dendritic Cell Supplement can also be added.
  • the dendritic cell differentiation medium is an IMDM base medium supplemented with 10% FBS, 100 ng/mL FLT-3L, 20 ng/mL SCF, 20 ng/mL GM-CSF, and 20 ng/mL IL-4.
  • the dendritic cell differentiation medium further contains DC cell maturation factors.
  • the DC cell maturation factor can be a ligand and/or activator of a toll-like receptor (TLR), for example, it can be selected from one or more of poly(I:C), R848, LPS, etc. kind.
  • the invention also provides dendritic cells obtained by the method described in any embodiment herein.
  • the present invention also provides a cell cryopreservation preparation, including the aforementioned dendritic cell progenitor cells and/or dendritic cells obtained by the method described in any embodiment of this article; and a cryopreservation solution.
  • dendritic cell progenitor cells and dendritic cells prepared by the methods described herein can be used to prepare pharmaceutical compositions. Therefore, the present invention also provides a pharmaceutical composition comprising an effective amount of dendritic cell progenitor cells and/or dendritic cells prepared by the method described in any embodiment of this article; and pharmaceutically acceptable excipients.
  • pharmaceutically acceptable excipient refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).
  • Pharmaceutically acceptable excipients include, but are not limited to, diluents, carriers, solubilizers, emulsifiers, preservatives and/or adjuvants. Excipients are preferably non-toxic to the recipient at the doses and concentrations employed.
  • excipients include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • pharmaceutical compositions may contain chemicals used to improve, maintain, or preserve, for example, the pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution, or release rate, absorption or Penetrating substances. These substances are known from the prior art. The optimal pharmaceutical composition will be determined based on the intended route of administration, mode of delivery and desired dosage.
  • compositions for in vivo administration are generally provided in sterile preparations. Sterilization is achieved by filtration through a sterile filter membrane. When the composition is lyophilized, this method can be used for sterilization before or after lyophilization and reconstitution.
  • the pharmaceutical compositions of the present invention may be selected for parenteral delivery.
  • Compositions for parenteral administration may be stored in lyophilized form or in solution. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Parenteral compositions are typically placed in containers with sterile access openings, such as intravenous solution strips or vials with a stopper pierceable by a hypodermic needle.
  • compositions may be selected for inhalation or delivery through the digestive tract (such as orally).
  • the preparation of such pharmaceutically acceptable compositions is within the skill in the art.
  • Other pharmaceutical compositions will be apparent to those skilled in the art. Techniques for formulating a variety of other modes of sustained or controlled delivery, such as liposomal vehicles, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art.
  • kits for producing single dose administration units may each contain a first container with dry protein and a second container with an aqueous formulation.
  • kits are provided containing single and multi-lumen prefilled syringes (eg, liquid syringes and lyophilized syringes).
  • the invention also provides for the treatment of patients (especially dendritic cells or dendritic cell progenitor cell-related diseases, including but not limited to infectious diseases, by administering dendritic cell progenitor cells or pharmaceutical compositions thereof according to any embodiment of the invention) metabolic diseases and/or cancer).
  • patients especially dendritic cells or dendritic cell progenitor cell-related diseases, including but not limited to infectious diseases, by administering dendritic cell progenitor cells or pharmaceutical compositions thereof according to any embodiment of the invention) metabolic diseases and/or cancer.
  • the terms “patient”, “subject”, “individual” and “subject” are used interchangeably herein and include any organism, preferably an animal, more preferably a mammal (e.g. rat, mouse, dog , cats, rabbits, etc.), and most preferably humans.
  • Treatment refers to a subject taking a treatment regimen described herein to achieve at least one positive therapeutic effect (for example, reduction in the number of cancer cells, reduction in tumor volume, reduction in the rate of cancer cell infiltration into surrounding organs, or reduction in tumor metastasis or tumor growth) rate decreases).
  • the treatment regimen that effectively treats a patient will vary based on a variety of factors, such as the patient's disease state, age, weight, and the ability of the therapy to elicit an anti-cancer response in the subject.
  • the therapeutically effective amount of a pharmaceutical composition containing dendritic cell progenitor cells of the invention to be employed will depend, for example, on the extent and goals of the treatment.
  • dosage levels for treatment will depend in part on It varies with the molecule delivered, the indication, the route of administration and the patient's size (body weight, body surface or organ size) and/or condition (age and general health).
  • the clinician can titrate the dosage and vary the route of administration to achieve optimal therapeutic effects. For example, about 10 micrograms/kg of body weight per day - about 50 mg/kg of body weight per day.
  • the frequency of dosing will depend on the pharmacokinetic parameters of the dendritic cell progenitors in the formulation used. Clinicians typically administer the composition until a dose is achieved that achieves the desired effect.
  • the composition may thus be administered as a single dose, or as two or more doses over time (which may or may not contain the same amount of the desired molecule), or as a continuous infusion through an implanted device or catheter.
  • the route of administration of the pharmaceutical composition is according to known methods, such as orally, by intravenous, intraperitoneal, intracerebral (intrabrain parenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional route; Either through a sustained release system or through an implanted device.
  • intravenous, intraperitoneal, intracerebral (intrabrain parenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional route Either through a sustained release system or through an implanted device.
  • StemSpan TM SFEM medium Stemcell Technologies, Cat. No. 09650.
  • StemSpan TM SFEM II medium Stemcell Technologies, Cat. No. 09655.
  • StemSpan TM -XF medium Stemcell Technologies, Cat. No. 100-0073.
  • StemSpan TM -AOF medium Stemcell Technologies, Cat. No. 100-0130.
  • DMEM/F12 medium Thermo Fisher Company, Cat. No. 11330032.
  • Neurobasal medium Thermo Fisher Company, Cat. No. 21103049.
  • Human serum albumin (CAS No. 70024-90-7), Sigma-Aldrich Company, Cat. No. A3782.
  • Linoleic acid (CAS number 60-33-3), Sigma-Aldrich Company, product number L1012.
  • Vemurafenib (PLX4032), Selleck Company, product number S1267.
  • Sorafenib (BAY 43-9006), Selleck Company, product number S7397.
  • CD34 MicroBeads Miltenyi Biotec, Cat. No. 130-100-453. Fixable Viability Stain 780, BD Biosciences, Cat. No. 565388.
  • the Ficoll process for separating peripheral blood cells includes the following steps:
  • the method of obtaining iGDP from peripheral blood cells includes the following steps:
  • the method of obtaining iGDP from CD14 + monocytes and CD14 - non-monocytes includes the following steps:
  • Peripheral blood cells were resuspended in DPBS, centrifuged at 300g at room temperature for 10 minutes, and the parameters were set to increase from 9 to 9;
  • the method of obtaining iGDP from CD123 + and CD123- cells includes the following steps:
  • Peripheral blood cells were resuspended in DPBS, centrifuged at 300g at room temperature for 10 minutes, and the parameters were set to increase from 9 to 9;
  • the method of obtaining iGDP from CD34 - cells includes the following steps:
  • Peripheral blood cells were resuspended in DPBS, centrifuged at 300g at room temperature for 10 minutes, and the parameters were set to increase from 9 to 9;
  • Methods for inducing iGDP differentiation and maturation into dendritic cells including the following steps:
  • IMDM basal medium IMDM basal medium plus 10% FBS, 100ng/mL FLT-3L, 20ng/mL SCF, 20ng/mL GM-CSF, 20ng/mL IL-4) to adjust the density to 5E5 viable cells /mL suspension, and after inoculation, place it in a 37°C, 5% CO 2 incubator for culture.
  • IMDM basal medium 10% FBS, 100ng/mL FLT-3L, 20ng/mL SCF, 20ng/mL GM-CSF, 20ng/mL IL-4
  • IMDM basic medium supplemented with 10% FBS, 100ng/mL FLT-3L, 20ng/mL SCF, 20ng/mL GM-CSF, 20ng/mL IL-4, 200IU/ mL IFN ⁇
  • the flow cytometric detection method of iGDP cells and iGDP-derived dendritic cells includes the following steps:
  • the composition and proportion of iGDP amplification medium 1-48 are shown in Table 1, in which the culture medium StemSpan TM SFEM, StemSpan TM SFEM II, StemSpan TM -XF, StemSpan TM -AOF, IMDM, DMEM/F-12 and Neurobasal
  • the dosage unit is v/v
  • the dosage unit of human albumin is g/L
  • the dosage unit of insulin, transferrin, DL- ⁇ -tocopherol and linoleic acid is mg/L
  • the dosage unit of sodium selenite is ⁇ g/L
  • the dosage unit of SCF, GM-CSF, G-CSF, IL-3 and FLT-3L is ⁇ g/L
  • the dosage unit of GDC-0879 and Dabrafenib is ⁇ M
  • the blood cell samples in the present invention are derived from non-mobilized peripheral blood, and the donors include healthy people aged 18-70 years old, with no gender limit.
  • Non-mobilized peripheral blood CD45+ cells were isolated using the Ficoll process, and fresh cells and cryopreserved and resuscitated cells were used as starting cells for expansion.
  • Samples 8 and 9 are CD14 + monocytes and CD14 - non-monocytes separated by magnetic beads in sample 6.
  • Samples 10 and 11 are CD123 + and CD123 - cells separated by magnetic beads in sample 7.
  • Sample 12 is CD34- cells after peripheral cell magnetic bead sorting.
  • Example 3 iGDP amplification medium formulation research - cell expansion multiples on days 7/14 of sample 1 cultured in different media
  • Example 4 iGDP amplification medium formulation research - cell expansion multiples on the 14th day of samples 2-5 cultured in different media
  • Hand-collected peripheral blood cells separated by the Ficoll process were used as starting cells, iGDP medium 11-27 was used as the test medium, and the number of viable cells on days 0 and 14 of culture was collected.
  • Example 5 iGDP amplification medium formulation study - cell expansion multiples on days 14 of culture samples 6-7 in different media
  • Machine-collected peripheral blood cells isolated by the Ficoll process were used as the starting cells, and iGDP medium 15, 23, 24, and 28-32 were used as the test medium, and the number of viable cells on the 0th and 14th days of culture was collected.
  • Example 6 iGDP long-term amplification ability study - cell expansion times of sample 7 cultured in different media on the 28th day
  • the machine-collected peripheral blood cell sample 7 separated by the Ficoll process was used as the starting cells, and iGDP medium 15, 24, and 30 were used as the test medium.
  • the starting cell inoculation number was 1E6 viable cells, and viable cells were counted on the 28th day of culture. , collecting cell viability and cell diameter data.
  • sample 7 has the best amplification ability in iGDP amplification medium 15, which expanded 197 times after 28 days of culture and maintained cell viability at 85.8%, followed by culture media 30 and 24, which lasted 28 days.
  • the amplifications were 194-fold and 83-fold, respectively, and the cell diameter ranged from 11.8 to 12.1 ⁇ m.
  • Example 7 iGDP amplification starting cell source study - CD45 + cells, CD14 + monocytes, CD14 - non-monocytes, CD123 + cells, CD123 - cells and CD34 - cells starting cell culture
  • CD45 + cells, CD14 + monocytes, CD14 - non-monocytes, CD123 + cells Cells, CD123 - cells and CD34 - cells can all be expanded to obtain iGDP cells.
  • iGDP cells grow in suspension or adherent, and the cells are spherical in shape and relatively uniform in size.
  • Example 8 iGDP-derived DC cell yield and flow cytometric characterization detection
  • iGDP cells as the starting cells for differentiation, directional differentiation and maturation are carried out according to the dendritic cell differentiation method of the present invention. After differentiation and maturity, the yield (number of differentiated cells/number of initial PBMC cells*CD11c + cell proportion) and cell flow Formula characterization detection.
  • the DC cell yields of sample 7 in iGDP amplification media 15, 24 and 30 were 138.05 times, 75.42 times and 132.64 times respectively.
  • iGDP cells obtained from different culture media can efficiently differentiate into DC cells.
  • iGDP cells obtained from culture medium 24 have the highest differentiation efficiency into DC cells, with CD11c + cells accounting for 85.70% and HLA-DR + The proportion of cells was 56.04%, the proportion of CD40 + cells was 79.91%, and the proportion of CD8 + cells was 64.83%.
  • Example 9 relative cell amplification times of samples 13 and 14 cultured in different iGDP amplification media (medium 33-48) on the 14th day
  • Hand-collected peripheral blood cells separated by the Ficoll process were used as starting cells, iGDP amplification media 24 and 33-48 were used as test media, and the number of viable cells on days 0 and 14 of culture was collected, with culture medium 24 as The control group is used to calculate the relative cell expansion multiple of culture medium 33-48 (that is, the cell expansion multiple of culture medium 33-48 divided by the cell expansion multiple of culture medium 24).
  • culture medium 44 has the best effect, and culture medium 37 has the same effect as culture medium 24 and is better than other culture media.
  • Medium 45-48 contains only serum-free medium and growth factors, which results in poorer amplification compared to other media. (Data is the mean and standard deviation of samples 13-14)
  • Example 10 Relative cell numbers of iGDP-induced differentiated dendritic cells obtained from different iGDP amplification media (Medium 33-44)
  • iGDP amplification media 24 and 33-44 were used as test media to obtain iGDP
  • iGDP was induced to differentiate into dendritic cells
  • flow cytometry was used to detect differentiation on the 13th day.
  • Example 11 Characterization and identification of iGDP-derived dendritic cells obtained from different iGDP amplification media (medium 38-44)
  • iGDP cells as the starting cells for differentiation, directional differentiation and maturation are carried out according to the dendritic cell differentiation method of the present invention, and characterization and identification are performed after differentiation and maturity.
  • Table 6 iGDP cells obtained from different media can be efficiently differentiated into DC cells.
  • culture medium 39 replaces the isoxazole derivative compound 21 with SKL2001. Although the two are equally effective in the expansion and differentiation stages, there is a difference in the proportion of CD11c+ cells in the characterization and identification after differentiation and maturity. , the proportion of CD11c+ cells in culture medium 38 was 71.95%, and the proportion of CD11c+ cells in culture medium 39 was 26.07%.

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Abstract

The present invention provides a preparation method for dendritic cell progenitors and a culture medium thereof, and particularly provides a method for preparing dendritic cell progenitors, comprising the step of: culturing cells from peripheral blood in the culture medium to obtain dendritic cell progenitors. By means of the preparation method, dendritic cell progenitors can be quickly and accurately obtained from peripheral blood.

Description

树突细胞祖细胞的制备方法及其培养基Preparation method and culture medium of dendritic cell progenitor cells 技术领域Technical field
本发明涉及细胞培养技术领域。更具体地，涉及从外周血中获得大量iGDP(individual general dendritic cell progenitor，树突细胞组细胞)的方法及其应用。The present invention relates to the technical field of cell culture. More specifically, it relates to methods and applications for obtaining large amounts of iGDP (individual general dendritic cell progenitor, dendritic cell group cells) from peripheral blood.
背景技术Background technique
免疫***，包括固有免疫和适应性免疫，是人体重要的保护屏障。树突细胞(dendritic cell，DC)是功能最强的专职抗原递呈细胞(Antigen presenting cells，APC)，是固有免疫和适应性免疫之间的桥梁。树突细胞能激活初始型T细胞、控制着体内免疫应答反应过程，是抗肿瘤免疫反应的中心环节。利用树突细胞进行免疫治疗能诱导患者机体产生肿瘤特异性、持久的免疫应答。The immune system, including innate immunity and adaptive immunity, is an important protective barrier for the human body. Dendritic cells (DC) are the most powerful professional antigen-presenting cells (APC) and are the bridge between innate immunity and adaptive immunity. Dendritic cells can activate initial T cells and control the immune response process in the body, and are the central link in the anti-tumor immune response. Immunotherapy using dendritic cells can induce the patient's body to produce a tumor-specific and long-lasting immune response.
树突细胞疫苗的临床应用取得了巨大进展。截至2022年7月25日，美国国立卫生研究院管理的“Clinical trail”临床数据登记平台显示，以“Dendritic Cells”为关键词共有1183项树突细胞治疗临床试验登记，处于临床III/IV期的树突细胞治疗临床试验有27项，主要适应症为黑色素瘤、肾癌、***癌等，白血病和HIV感染等。2010年基于树突细胞疫苗的自体***癌疫苗Sipuleucel-T(Provenge)获FDA批准上市，是全球首款树突细胞治疗性肿瘤疫苗。Tremendous progress has been made in the clinical application of dendritic cell vaccines. As of July 25, 2022, the "Clinical trail" clinical data registration platform managed by the National Institutes of Health shows that there are 1,183 dendritic cell therapy clinical trials registered with the keyword "Dendritic Cells", which are in clinical phase III/IV. There are 27 clinical trials of dendritic cell therapy. The main indications are melanoma, kidney cancer, prostate cancer, leukemia and HIV infection. In 2010, the autologous prostate cancer vaccine Sipuleucel-T (Provenge) based on dendritic cell vaccine was approved by the FDA. It is the world's first dendritic cell therapeutic tumor vaccine.
树突细胞疫苗的临床应用同样面临巨大挑战。树突细胞疫苗产品的临床疗效与树突细胞剂量息息相关。在体内，树突细胞由骨髓内CD34+造血干细胞(hematopoietic stem cell，HSC)发育而来。在体外，90％树突细胞疫苗产品来源于外周血单核细胞(monocyte)，10％来源于脐血/动员外周血中CD34+造血干细胞，但均无法获得数量充足的树突细胞。如使用外周血单个核细胞(PBMC)分离单核细胞分化获得树突细胞技术路线，树突细胞得率仅约10％：每1E9个外周血单个核细胞可分离出1.3E8个单核细胞(13％)，1.3E8个单核细胞可分化获得1E8个树突细胞(80％)。如使用脐血/动员外周血中CD34+造血干细胞扩增分化获得树突细胞技术路线，树突细胞得率仅约15％：每1E9个脐血/动员外周血单个核细胞可分离出1E7个CD34+造血干细胞(1％)，1E7个CD34+造血干细胞可在体外扩增约20倍至 2E8个细胞，2E8个细胞可分化获得1.5E8个树突细胞(75％)。The clinical application of dendritic cell vaccines also faces huge challenges. The clinical efficacy of dendritic cell vaccine products is closely related to the dose of dendritic cells. In the body, dendritic cells develop from CD34 + hematopoietic stem cells (HSC) in the bone marrow. In vitro, 90% of dendritic cell vaccine products are derived from peripheral blood mononuclear cells (monocytes), and 10% are derived from CD34 + hematopoietic stem cells in cord blood/mobilized peripheral blood, but sufficient numbers of dendritic cells cannot be obtained. If peripheral blood mononuclear cells (PBMC) are used to isolate monocyte differentiation and obtain dendritic cells, the yield of dendritic cells is only about 10%: 1.3E8 monocytes can be isolated from every 1E9 peripheral blood mononuclear cells ( 13%), 1.3E8 monocytes can differentiate into 1E8 dendritic cells (80%). If the technical route of obtaining dendritic cells through expansion and differentiation of CD34 + hematopoietic stem cells in cord blood/mobilized peripheral blood is used, the dendritic cell yield is only about 15%: 1E7 mononuclear cells can be isolated from every 1E9 cord blood/mobilized peripheral blood mononuclear cells. CD34 + hematopoietic stem cells (1%), 1E7 CD34 + hematopoietic stem cells can be expanded approximately 20 times in vitro to 2E8 cells can differentiate into 1.5E8 dendritic cells (75%).
由于树突细胞无扩增能力，因此，如何高效获得数量充足的树突细胞祖细胞对于未来的免疫治疗具有实质性意义。Since dendritic cells have no ability to expand, how to efficiently obtain sufficient numbers of dendritic cell progenitor cells is of substantial significance for future immunotherapy.
发明内容Contents of the invention
本发明第一方面提供制备树突细胞祖细胞的方法，包括步骤：将来自外周血的细胞在培养基中培养，获得树突细胞祖细胞。A first aspect of the present invention provides a method for preparing dendritic cell progenitor cells, including the steps of: culturing cells from peripheral blood in a culture medium to obtain dendritic cell progenitor cells.
在一个或多个实施方案中，所述培养基含有生长因子、Raf/MEK/ERK通路调节剂、异恶唑衍生物中的至少两种。优选地，所述培养基含有生长因子、Raf/MEK/ERK通路调节剂和异恶唑衍生物。In one or more embodiments, the culture medium contains at least two of growth factors, Raf/MEK/ERK pathway modulators, and isoxazole derivatives. Preferably, the culture medium contains growth factors, Raf/MEK/ERK pathway modulators and isoxazole derivatives.
在一个或多个实施方案中，所述生长因子选自SCF、GM-CSF、G-CSF、IL-3、FLT-3L中的至少一种；优选地，所述生长因子含有SCF以及选自GM-CSF、G-CSF、IL-3、FLT-3L中的至少一种。In one or more embodiments, the growth factor is selected from at least one of SCF, GM-CSF, G-CSF, IL-3, FLT-3L; preferably, the growth factor contains SCF and is selected from At least one of GM-CSF, G-CSF, IL-3, and FLT-3L.
在一个或多个实施方案中，含有时，SCF的浓度为10-150ug/L，优选为30-120ug/L。In one or more embodiments, when included, the concentration of SCF is 10-150ug/L, preferably 30-120ug/L.
在一个或多个实施方案中，含有时，GM-CSF的浓度为0-80ug/L，优选为0-50ug/L。In one or more embodiments, when included, the concentration of GM-CSF is 0-80ug/L, preferably 0-50ug/L.
在一个或多个实施方案中，含有时，G-CSF的浓度为0-200ug/L，优选为0-150ug/L。In one or more embodiments, when included, the concentration of G-CSF is 0-200ug/L, preferably 0-150ug/L.
在一个或多个实施方案中，含有时，IL-3的浓度为0-80ug/L，优选为0-50ug/L。In one or more embodiments, when included, IL-3 is present at a concentration of 0-80ug/L, preferably 0-50ug/L.
在一个或多个实施方案中，含有时，FLT-3L的浓度为0-80ug/L，优选为0-50ug/L。In one or more embodiments, when included, FLT-3L is present at a concentration of 0-80ug/L, preferably 0-50ug/L.
在一个或多个实施方案中，所述Raf/MEK/ERK通路调节剂为MEK激活剂和/或ERK激活剂。优选地，所述Raf/MEK/ERK通路调节剂选自GDC-0879、PLX4720、Vemurafenib(PLX4032)、Sorafenib(BAY 43-9006)、Dabrafenib(GSK2118436)中的一种或几种，更优选为GDC-0879。在一个或多个实施方案中，所述Raf/MEK/ERK通路调节剂的浓度为0.1-5μM，优选为0.5-1.5μM。In one or more embodiments, the Raf/MEK/ERK pathway modulator is a MEK activator and/or an ERK activator. Preferably, the Raf/MEK/ERK pathway modulator is selected from one or more of GDC-0879, PLX4720, Vemurafenib (PLX4032), Sorafenib (BAY 43-9006), Dabrafenib (GSK2118436), more preferably GDC -0879. In one or more embodiments, the concentration of the Raf/MEK/ERK pathway modulator is 0.1-5 μM, preferably 0.5-1.5 μM.
在一个或多个实施方案中，所述异恶唑衍生物为式(I)所示的化合物。
In one or more embodiments, the isoxazole derivative is a compound represented by formula (I).
其中，R1为未取代或取代的杂芳基，Wherein, R 1 is unsubstituted or substituted heteroaryl,
m为0-4的整数，优选为l-3的整数，m is an integer from 0 to 4, preferably an integer from 1 to 3,
X为CH2、NH或O，X is CH 2 , NH or O,
R2为未取代或取代的杂芳基。R 2 is unsubstituted or substituted heteroaryl.
在一个或多个实施方案中，所述异恶唑衍生物为式1-21中任一所示的化合物。优选地，所述异恶唑衍生物为5-呋喃-2-基-异恶唑-3-羧酸(2-吡唑-1-基-乙基)-酰胺。在一个或多个实施方案中，所述异恶唑衍生物的浓度为0-15μM，优选为0-10μM，更优选为1-10μM。In one or more embodiments, the isoxazole derivative is a compound represented by any one of Formulas 1-21. Preferably, the isoxazole derivative is 5-furan-2-yl-isoxazole-3-carboxylic acid (2-pyrazol-1-yl-ethyl)-amide. In one or more embodiments, the concentration of the isoxazole derivative is 0-15 μM, preferably 0-10 μM, more preferably 1-10 μM.
在一个或多个实施方案中，所述培养基还含有嘧啶并吲哚衍生物，优选地，所述嘧啶并吲哚衍生物选自UM171和UM729中的一种或两种，更优选地，所述嘧啶并吲哚衍生物为UM171。在一个或多个实施方案中，所述嘧啶并吲哚衍生物的浓度为0-80nM，优选为0-50nM。In one or more embodiments, the culture medium further contains a pyrimidinoindole derivative. Preferably, the pyrimidinoindole derivative is selected from one or both of UM171 and UM729. More preferably, The pyrimidinoindole derivative is UM171. In one or more embodiments, the concentration of the pyrimidoindole derivative is 0-80 nM, preferably 0-50 nM.
在一个或多个实施方案中，所述培养基还含有基础培养基，例如基础无血清培养基，包括商业无血清培养基和补充有添加物的基本培养基。In one or more embodiments, the medium further contains a basal medium, such as a basal serum-free medium, including commercial serum-free medium and basal medium supplemented with additives.
在一个或多个实施方案中，所述商业无血清培养基选自StemSpanTM SFEM培养基、StemSpanTM SFEM II培养基、StemSpanTM-XF培养基、StemSpanTM-AOF培养基、StemProTM-34 SFM中的一种或多种。In one or more embodiments, the commercial serum-free medium is selected from StemSpan ™ SFEM medium, StemSpan ™ SFEM II medium, StemSpan ™ -XF medium, StemSpan ™ -AOF medium, StemPro ™ -34 SFM one or more of them.
在一个或多个实施方案中，所述基本培养基选自IMDM培养基、DMEM/F-12培养基、Neurobasal培养基、AIM-V中的一种或多种；优选地，所述补充添加物选自人血白蛋白、胰岛素、转铁蛋白、***钠、DL-α-生育酚、亚油酸中的一种或多种。In one or more embodiments, the basic medium is selected from one or more of IMDM medium, DMEM/F-12 medium, Neurobasal medium, and AIM-V; preferably, the supplement is added The substance is selected from one or more of human albumin, insulin, transferrin, sodium selenite, DL-α-tocopherol, and linoleic acid.
在一个或多个实施方案中，含有时，人血白蛋白的浓度为1-10g/L，优选为1g/L-5g/L。In one or more embodiments, when contained, the concentration of human albumin is 1-10g/L, preferably 1g/L-5g/L.
在一个或多个实施方案中，含有时，胰岛素浓度为1-20mg/L，优选为3-15mg/L。In one or more embodiments, when included, the insulin concentration is 1-20 mg/L, preferably 3-15 mg/L.
在一个或多个实施方案中，含有时，转铁蛋白的浓度为1-30mg/L，优选为3- 25mg/L。In one or more embodiments, when included, the concentration of transferrin is 1-30 mg/L, preferably 3-30 mg/L. 25mg/L.
在一个或多个实施方案中，含有时，***钠的浓度为1-20ug/L，优选为5-15ug/L。In one or more embodiments, when included, the concentration of sodium selenite is 1-20ug/L, preferably 5-15ug/L.
在一个或多个实施方案中，含有时，DL-α-生育酚的浓度为0.5-10mg/L，优选为0.5-8mg/L。In one or more embodiments, when included, DL-α-tocopherol is present at a concentration of 0.5-10 mg/L, preferably 0.5-8 mg/L.
在一个或多个实施方案中，含有时，亚油酸的浓度为0.5-10mg/L，优选为0.5-5mg/L。In one or more embodiments, when included, the concentration of linoleic acid is 0.5-10 mg/L, preferably 0.5-5 mg/L.
在一个或多个实施方案中，所述外周血为未经造血干细胞动员的外周血。In one or more embodiments, the peripheral blood is peripheral blood without hematopoietic stem cell mobilization.
在一个或多个实施方案中，所述来自外周血的细胞包含选自CD45+白细胞、CD14+细胞、CD14-细胞、CD123+细胞、CD123-细胞、CD34-细胞中的一种或多种。In one or more embodiments, the cells from peripheral blood comprise one or more selected from the group consisting of CD45+ leukocytes, CD14+ cells, CD14- cells, CD123+ cells, CD123- cells, CD34- cells.
本发明还提供培养组合物或包含所述培养基组合物的培养基，用于从外周血制备树突细胞祖细胞，所述培养组合物含有生长因子、Raf/MEK/ERK通路调节剂、异恶唑衍生物中的至少两种。The present invention also provides a culture composition or a culture medium containing the culture medium composition for preparing dendritic cell progenitor cells from peripheral blood, the culture composition containing growth factors, Raf/MEK/ERK pathway regulators, isoforms At least two of the oxazole derivatives.
在一个或多个实施方案中，所述培养基含有生长因子、Raf/MEK/ERK通路调节剂和异恶唑衍生物。In one or more embodiments, the culture medium contains growth factors, Raf/MEK/ERK pathway modulators, and isoxazole derivatives.
在一个或多个实施方案中，所述生长因子选自SCF、GM-CSF、G-CSF、IL-3、FLT-3L中的至少一种；优选地，所述生长因子含有SCF以及选自GM-CSF、G-CSF、IL-3、FLT-3L中的至少一种。In one or more embodiments, the growth factor is selected from at least one of SCF, GM-CSF, G-CSF, IL-3, FLT-3L; preferably, the growth factor contains SCF and is selected from At least one of GM-CSF, G-CSF, IL-3, and FLT-3L.
在一个或多个实施方案中，所述培养基中，SCF的浓度为10-150ug/L，优选为30-120ug/L。In one or more embodiments, the concentration of SCF in the culture medium is 10-150ug/L, preferably 30-120ug/L.
在一个或多个实施方案中，所述培养基中，GM-CSF的浓度为0-80ug/L，优选为0-50ug/L。In one or more embodiments, the concentration of GM-CSF in the culture medium is 0-80ug/L, preferably 0-50ug/L.
在一个或多个实施方案中，所述培养基中，G-CSF的浓度为0-200ug/L，优选为0-150ug/L。In one or more embodiments, the concentration of G-CSF in the culture medium is 0-200ug/L, preferably 0-150ug/L.
在一个或多个实施方案中，所述培养基中，IL-3的浓度为0-80ug/L，优选为0-50ug/L。In one or more embodiments, the concentration of IL-3 in the culture medium is 0-80ug/L, preferably 0-50ug/L.
在一个或多个实施方案中，所述培养基中，FLT-3L的浓度为0-80ug/L，优选为0-50ug/L。 In one or more embodiments, the concentration of FLT-3L in the culture medium is 0-80ug/L, preferably 0-50ug/L.
在一个或多个实施方案中，所述Raf/MEK/ERK通路调节剂为MEK激活剂和/或ERK激活剂。优选地，所述Raf/MEK/ERK通路调节剂选自GDC-0879、PLX4720、Vemurafenib(PLX4032)、Sorafenib(BAY 43-9006)、Dabrafenib(GSK2118436)中的一种或几种，更优选为GDC-0879。在一个或多个实施方案中，所述Raf/MEK/ERK通路调节剂的浓度为0.1-5μM，优选为0.5-1.5μM。In one or more embodiments, the Raf/MEK/ERK pathway modulator is a MEK activator and/or an ERK activator. Preferably, the Raf/MEK/ERK pathway modulator is selected from one or more of GDC-0879, PLX4720, Vemurafenib (PLX4032), Sorafenib (BAY 43-9006), Dabrafenib (GSK2118436), more preferably GDC -0879. In one or more embodiments, the concentration of the Raf/MEK/ERK pathway modulator is 0.1-5 μM, preferably 0.5-1.5 μM.
在一个或多个实施方案中，所述异恶唑衍生物为式(I)所示的化合物。
In one or more embodiments, the isoxazole derivative is a compound represented by formula (I).
其中，R1为未取代或取代的杂芳基，Wherein, R1 is unsubstituted or substituted heteroaryl,
m为0-4的整数，优选为1-3的整数，m is an integer from 0 to 4, preferably an integer from 1 to 3,
X为CH2、NH或O，X is CH2, NH or O,
R2为未取代或取代的杂芳基。R2 is unsubstituted or substituted heteroaryl.
在一个或多个实施方案中，所述异恶唑衍生物为式1-21中任一所示的化合物。优选地，所述异恶唑衍生物为5-呋喃-2-基-异恶唑-3-羧酸(2-吡唑-1-基-乙基)-酰胺。在一个或多个实施方案中，所述异恶唑衍生物的浓度为0-15μM，优选为0-10μM，更优选为1-10μM。In one or more embodiments, the isoxazole derivative is a compound represented by any one of Formulas 1-21. Preferably, the isoxazole derivative is 5-furan-2-yl-isoxazole-3-carboxylic acid (2-pyrazol-1-yl-ethyl)-amide. In one or more embodiments, the concentration of the isoxazole derivative is 0-15 μM, preferably 0-10 μM, more preferably 1-10 μM.
在一个或多个实施方案中，所述培养基还含有嘧啶并吲哚衍生物，优选地，所述嘧啶并吲哚衍生物选自UM171和UM729中的一种或两种，更优选地，所述嘧啶并吲哚衍生物为UM171。在一个或多个实施方案中，所述嘧啶并吲哚衍生物的浓度为0-80nM，优选为0-50nM。In one or more embodiments, the culture medium further contains a pyrimidinoindole derivative. Preferably, the pyrimidinoindole derivative is selected from one or both of UM171 and UM729. More preferably, The pyrimidinoindole derivative is UM171. In one or more embodiments, the concentration of the pyrimidoindole derivative is 0-80 nM, preferably 0-50 nM.
在一个或多个实施方案中，所述培养组合物中各生长因子、Raf/MEK/ERK通路调节剂、异恶唑衍生物的浓度比与培养基中相应组分的浓度比相同。In one or more embodiments, the concentration ratio of each growth factor, Raf/MEK/ERK pathway regulator, and isoxazole derivative in the culture composition is the same as the concentration ratio of the corresponding component in the culture medium.
在一个或多个实施方案中，所述培养基还含有基础培养基，例如基础无血清培养基，包括商业无血清培养基和补充有添加物的基本培养基。In one or more embodiments, the medium further contains a basal medium, such as a basal serum-free medium, including commercial serum-free medium and basal medium supplemented with additives.
在一个或多个实施方案中，所述商业无血清培养基选自StemSpanTM SFEM培养基、StemSpanTM SFEM II培养基、StemSpanTM-XF培养基、StemSpanTM-AOF培养基、StemProTM-34 SFM中的一种或多种。In one or more embodiments, the commercial serum-free medium is selected from StemSpan ™ SFEM medium, StemSpan ™ SFEM II medium, StemSpan ™ -XF medium, StemSpan ™ -AOF medium, StemPro ™ -34 SFM one or more of them.
在一个或多个实施方案中，所述基本培养基选自IMDM培养基、DMEM/F-12 培养基、Neurobasal培养基、AIM-V中的一种或多种；优选地，所述补充添加物选自人血白蛋白、胰岛素、转铁蛋白、***钠、DL-α-生育酚、亚油酸中的一种或多种。In one or more embodiments, the minimal medium is selected from IMDM medium, DMEM/F-12 One or more of medium, Neurobasal medium, and AIM-V; preferably, the supplement is selected from human albumin, insulin, transferrin, sodium selenite, and DL-α-tocopherol. , one or more of linoleic acid.
在一个或多个实施方案中，人血白蛋白的浓度为1-10g/L，优选为1g/L-5g/L。In one or more embodiments, the concentration of human albumin is 1-10g/L, preferably 1g/L-5g/L.
在一个或多个实施方案中，胰岛素浓度为1-20mg/L，优选为3-15mg/L。In one or more embodiments, the insulin concentration is 1-20 mg/L, preferably 3-15 mg/L.
在一个或多个实施方案中，转铁蛋白的浓度为1-30mg/L，优选为3-25mg/L。In one or more embodiments, the concentration of transferrin is 1-30 mg/L, preferably 3-25 mg/L.
在一个或多个实施方案中，***钠的浓度为1-20ug/L，优选为5-15ug/L。In one or more embodiments, the concentration of sodium selenite is 1-20ug/L, preferably 5-15ug/L.
在一个或多个实施方案中，DL-α-生育酚的浓度为0.5-10mg/L，优选为0.5-8mg/L。In one or more embodiments, the concentration of DL-alpha-tocopherol is 0.5-10 mg/L, preferably 0.5-8 mg/L.
在一个或多个实施方案中，亚油酸的浓度为0.5-10mg/L，优选为0.5-5mg/L。In one or more embodiments, the concentration of linoleic acid is 0.5-10 mg/L, preferably 0.5-5 mg/L.
在一个或多个实施方案中，所述外周血为未经造血干细胞动员的外周血。In one or more embodiments, the peripheral blood is peripheral blood without hematopoietic stem cell mobilization.
在一个或多个实施方案中，所述来自外周血的细胞包含选自CD45+白细胞、CD14+细胞、CD14-细胞、CD123+细胞、CD123-细胞、CD34-细胞中的一种或多种。In one or more embodiments, the cells from peripheral blood comprise one or more selected from the group consisting of CD45+ leukocytes, CD14+ cells, CD14- cells, CD123+ cells, CD123- cells, CD34- cells.
本发明还提供本发明任一实施方案所述的培养组合物或培养基在制备含有树突细胞祖细胞的试剂中的应用。The present invention also provides the use of the culture composition or culture medium according to any embodiment of the present invention in preparing a reagent containing dendritic cell progenitor cells.
本发明还提供一种树突细胞祖细胞，其由本文任一实施方案所述的方法制备。The invention also provides a dendritic cell progenitor cell prepared by the method described in any embodiment herein.
本发明还提供一种树突细胞的制备方法，包括：将本文任一实施方案所述的树突细胞祖细胞在树突细胞分化培养基中培养，获得树突细胞。The present invention also provides a method for preparing dendritic cells, which includes: culturing the dendritic cell progenitor cells described in any embodiment of this article in a dendritic cell differentiation medium to obtain dendritic cells.
本发明还提供一种由本文所述树突细胞的制备方法制备的树突细胞。The present invention also provides a dendritic cell prepared by the preparation method of dendritic cells described herein.
本发明还提供由本文任一实施方案所述的方法制备的树突细胞祖细胞在制备药物中的用途。The present invention also provides use of dendritic cell progenitor cells prepared by the method described in any embodiment herein in the preparation of a medicament.
在一个或多个实施方案中，所述药物用于治疗癌症、感染疾病和/或衰老相关疾病。 In one or more embodiments, the medicament is used to treat cancer, infectious diseases, and/or aging-related diseases.
本发明还提供一种药物组合物，包含有效量的本文任一实施方案所述的方法制备的树突细胞祖细胞和药学上可接受的辅料。The present invention also provides a pharmaceutical composition, comprising an effective amount of dendritic cell progenitor cells prepared by the method described in any embodiment of this document and pharmaceutically acceptable excipients.
本发明还提供一种细胞冻存制剂，包括前述由本文任一实施方案所述的方法获得的树突细胞祖细胞和冻存液。The present invention also provides a cell cryopreservation preparation, including the aforementioned dendritic cell progenitor cells obtained by the method described in any embodiment of this article and a cryopreservation solution.
本发明还提供一种治疗或预防有需要的受试者的疾病或病症的方法，包含向所述受试者施用有效量的本文任一实施方案所述的药物组合物。The invention also provides a method of treating or preventing a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition according to any embodiment herein.
一些实施方案中，所述疾病或病症是癌症、感染疾病和/或衰老相关疾病。In some embodiments, the disease or disorder is cancer, infectious diseases, and/or aging-related diseases.
本发明的有益效果：从外周血中扩增树突细胞祖细胞(iGDP细胞)，简便易得，可及性高，而且iGDP扩增倍数高，例如iGDP细胞数量是初始外周血单个核细胞数量的83-197倍。The beneficial effects of the present invention: amplifying dendritic cell progenitor cells (iGDP cells) from peripheral blood is simple and easy to obtain, has high accessibility, and the iGDP amplification multiple is high. For example, the number of iGDP cells is the number of initial peripheral blood mononuclear cells. 83-197 times.
附图说明Description of drawings
图1示出使用不同iGDP扩增培养基(培养基11-27)培养样品2-5第14天的细胞扩增倍数。Figure 1 shows the cell expansion fold of samples 2-5 on day 14 using different iGDP amplification media (medium 11-27).
图2示出使用不同iGDP扩增培养基(培养基15、23、24、28-32)培养样品6和样品7第14天的细胞扩增倍数。Figure 2 shows the cell expansion fold of sample 6 and sample 7 on the 14th day of culture using different iGDP amplification media (medium 15, 23, 24, 28-32).
图3示出不同细胞来源扩增出的iGDP细胞形态图，Scale bar＝50μm或100μm。Figure 3 shows the morphological diagram of iGDP cells amplified from different cell sources, Scale bar=50μm or 100μm.
图4示出诱导iGDP向DC高效分化14天的流式表征，分化起始细胞为培养基15培养样品4第14天的iGDP细胞，对照组为样本未进行抗体孵育的空白对照。Figure 4 shows the flow cytometric characterization of the efficient differentiation of iGDP into DCs for 14 days. The differentiation starting cells were iGDP cells cultured on the 14th day of sample 4 in medium 15. The control group was a blank control in which the sample was not incubated with antibodies.
图5示出不同iGDP扩增培养基(培养基33-48)培养样品13、14第14天的相对细胞扩增倍数。Figure 5 shows the relative cell expansion folds of samples cultured in different iGDP amplification media (Medium 33-48) on days 13 and 14.
图6示出不同iGDP扩增培养基(培养基33-44)获得的iGDP诱导分化树突细胞的相对细胞数量。Figure 6 shows the relative cell numbers of iGDP-induced differentiated dendritic cells obtained with different iGDP amplification media (Medium 33-44).
具体实施方式 Detailed ways
为了更清楚地说明本发明，下面结合优选实施例和附图对本发明做进一步的说明。附图中相似的部件以相同的附图标记进行表示。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应以此限制本发明的保护范围。In order to illustrate the present invention more clearly, the present invention will be further described below with reference to preferred embodiments and drawings. Similar parts are designated with the same reference numerals in the drawings. Those skilled in the art should understand that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of the present invention.
发明人发现，通过培养来自外周血的细胞，可以获得树突细胞祖细胞。本文中，树突细胞祖细胞(iGDP，individual general dendritic cell progenitor)是指可分化形成树突细胞的前体细胞。The inventors discovered that dendritic cell progenitor cells can be obtained by culturing cells from peripheral blood. In this article, dendritic cell progenitor cells (iGDP, individual general dendritic cell progenitor) refers to precursor cells that can differentiate to form dendritic cells.
因此，本发明提供制备树突细胞祖细胞的方法，包括步骤：将来自外周血的细胞在培养基中培养，获得树突细胞祖细胞。本发明还提供用于上述从外周血制备树突细胞祖细胞的培养组合物或包含所述培养基组合物的培养基。所述培养组合物或培养基含有生长因子、Raf/MEK/ERK通路调节剂、异恶唑衍生物中的至少两种。本发明还提供由所述的方法制备的树突细胞祖细胞。Therefore, the present invention provides a method for preparing dendritic cell progenitor cells, which includes the steps of: culturing cells from peripheral blood in a culture medium to obtain dendritic cell progenitor cells. The present invention also provides a culture composition for the above preparation of dendritic cell progenitor cells from peripheral blood or a culture medium comprising the culture medium composition. The culture composition or culture medium contains at least two of growth factors, Raf/MEK/ERK pathway regulators, and isoxazole derivatives. The invention also provides dendritic cell progenitor cells prepared by the method.
本文所述的外周血的采集方式不限，可以是单采外周血或机采外周血等。The method of collecting peripheral blood described in this article is not limited, and it can be apheresis blood collection or machine collection peripheral blood, etc.
在一些实施方案中，所述外周血为未经造血干细胞动员的外周血。在一些实施方案中，来自外周血的细胞为外周血CD45+白细胞。在示例性实施方案中，所述来自外周血的细胞包含选自CD45+白细胞、CD14+细胞、CD14-细胞、CD123+细胞、CD123-细胞、CD34-细胞中的一种或多种。In some embodiments, the peripheral blood is peripheral blood that has not been mobilized by hematopoietic stem cells. In some embodiments, the cells from peripheral blood are peripheral blood CD45 + leukocytes. In an exemplary embodiment, the cells from peripheral blood comprise one or more selected from the group consisting of CD45 + leukocytes, CD14 + cells, CD14- cells, CD123 + cells, CD123- cells, and CD34- cells.
因此，本发明方法的一些实施方案还包括由外周血获取CD45+白细胞、CD14+细胞、CD14-细胞、CD123+细胞、CD123-细胞、CD34-细胞中的一种或多种的步骤。Therefore, some embodiments of the method of the present invention further include the step of obtaining one or more of CD45+ leukocytes, CD14+ cells, CD14- cells, CD123+ cells, CD123- cells, CD34- cells from peripheral blood.
从外周血分离细胞的方法可采用本领域常规方法，例如羟乙基淀粉离心沉淀法、密度梯度离心法、免疫磁珠分选法、流式细胞仪分选法等。密度梯度离心介质包括Ficoll-Hypaque、Percoll等。The method of isolating cells from peripheral blood can adopt conventional methods in this field, such as hydroxyethyl starch centrifugal precipitation method, density gradient centrifugation method, immunomagnetic bead sorting method, flow cytometry sorting method, etc. Density gradient centrifugation media include Ficoll-Hypaque, Percoll, etc.
来自外周血的细胞可以是新鲜细胞，也可以是冻存后复苏的细胞。Cells from peripheral blood can be fresh cells or cells that have been cryopreserved and then revived.
将来自外周血的细胞在所述培养基中培养的条件和时间可由本领域技术人员根据需要确定，例如37℃、5％C02培养至少1天、至少3天、至少7天、至少14天、至少28天。The conditions and time for culturing cells from peripheral blood in the culture medium can be determined by those skilled in the art as needed, for example, culturing at 37°C, 5% CO2 for at least 1 day, at least 3 days, at least 7 days, at least 14 days, At least 28 days.
“生长因子”是指有效促进细胞生长且除非作为补充剂添加到培养基中否则它不是基础培养基的组分的物质。在一些实施方案中，培养基中所含的生长因子选自干细胞生长因子(stem cell factor，SCF)、粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating factor，GM-CSF)、粒细胞集落刺激因 子(granulocyte colony stimulating factor，G-CSF)、白细胞介素3(Interleukin-3，IL-3)、FMS相关酪氨酸激酶3配体(Fms-related tyrosine kinase 3 ligand，FLT-3L)中的至少一种。优选地，所述生长因子包含SCF。生长因子的浓度不受限制，示例性地：SCF的浓度为10-150ug/L，优选为30-120ug/L，例如30ug/L、40ug/L、50ug/L、60ug/L、80ug/L、100ug/L、120ug/L；GM-CSF的浓度为0-80ug/L，优选为0-50ug/L，例如，0、5ug/L、8ug/L、10ug/L、15ug/L、20ug/L、30ug/L、40ug/L、50ug/L；G-CSF的浓度为0-200ug/L，优选为0-150ug/L，例如0、10ug/L、20ug/L、25ug/L、40ug/L、50ug/L、60ug/L、80ug/L、100ug/L、150ug/L；IL-3的浓度为0-80ug/L，优选为0-50ug/L，例如0、10ug/L、20ug/L、25ug/L、30ug/L、40ug/L、50ug/L；FLT-3L的浓度为0-80ug/L，优选为0-50ug/L，例如0、10ug/L、20ug/L、25ug/L、30ug/L、40ug/L、50ug/L。"Growth factor" refers to a substance that effectively promotes cell growth and is not a component of the basal medium unless added to the medium as a supplement. In some embodiments, the growth factor contained in the culture medium is selected from the group consisting of stem cell growth factor (SCF), granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), colony stimulating factor in granulocyte colony stimulating factor (G-CSF), interleukin-3 (IL-3), Fms-related tyrosine kinase 3 ligand (FLT-3L) At least one. Preferably, the growth factor comprises SCF. The concentration of the growth factor is not limited, for example: the concentration of SCF is 10-150ug/L, preferably 30-120ug/L, such as 30ug/L, 40ug/L, 50ug/L, 60ug/L, 80ug/L , 100ug/L, 120ug/L; the concentration of GM-CSF is 0-80ug/L, preferably 0-50ug/L, for example, 0, 5ug/L, 8ug/L, 10ug/L, 15ug/L, 20ug /L, 30ug/L, 40ug/L, 50ug/L; the concentration of G-CSF is 0-200ug/L, preferably 0-150ug/L, such as 0, 10ug/L, 20ug/L, 25ug/L, 40ug/L, 50ug/L, 60ug/L, 80ug/L, 100ug/L, 150ug/L; the concentration of IL-3 is 0-80ug/L, preferably 0-50ug/L, such as 0, 10ug/L , 20ug/L, 25ug/L, 30ug/L, 40ug/L, 50ug/L; the concentration of FLT-3L is 0-80ug/L, preferably 0-50ug/L, such as 0, 10ug/L, 20ug/ L, 25ug/L, 30ug/L, 40ug/L, 50ug/L.
Raf/MEK/ERK通路调节剂是指能够阻断或激活Raf/MEK/ERK通路的物质。MAPK，即丝裂原活化蛋白激酶，其信号通路可以将细胞外信号转导至细胞内，通过三级激酶级联的形式(MAPK，MAPK激酶(MEK或MKK)以及MAPK激酶的激酶(MEKK或MKKK))传导细胞信号，从而调控细胞的增殖、分化、凋亡、炎症反应以及血管发育等生物学功能。Raf/MEK/ERK通路是MAPK信号通路网络4条信号通路中的1条。在一些实施方案中，培养基中所含的Raf/MEK/ERK通路调节剂为MEK激活剂和/或ERK激活剂。在一些实施方案中，培养基中所含的Raf/MEK/ERK通路调节剂为B-Raf激酶抑制剂。优选地，所述Raf/MEK/ERK通路调节剂选自GDC-0879、PLX4720、Vemurafenib(PLX4032)、Sorafenib(BAY 43-9006)、Dabrafenib(GSK2118436)中的一种或几种。Raf/MEK/ERK通路调节剂的浓度不受限制，示例性的浓度为0.1-5μM，优选为0.5-1.5μM，例如0.5μM、0.6μM、0.8μM、1.0μM、1.2μM、1.5μM。在一些实施方案中，Raf/MEK/ERK通 路调节剂为0.5-1.0μM的GDC-0879或1.0μM的Dabrafenib。 Raf/MEK/ERK pathway modulators refer to substances that can block or activate the Raf/MEK/ERK pathway. MAPK, mitogen-activated protein kinase, its signaling pathway can transduce extracellular signals into cells through the form of a three-level kinase cascade (MAPK, MAPK kinase (MEK or MKK) and MAPK kinase kinase (MEKK or MKKK)) conducts cell signals, thereby regulating biological functions such as cell proliferation, differentiation, apoptosis, inflammatory response, and vascular development. The Raf/MEK/ERK pathway is one of the four signaling pathways in the MAPK signaling pathway network. In some embodiments, the Raf/MEK/ERK pathway modulator contained in the culture medium is a MEK activator and/or an ERK activator. In some embodiments, the Raf/MEK/ERK pathway modulator contained in the culture medium is a B-Raf kinase inhibitor. Preferably, the Raf/MEK/ERK pathway modulator is selected from one or more of GDC-0879, PLX4720, Vemurafenib (PLX4032), Sorafenib (BAY 43-9006), Dabrafenib (GSK2118436). The concentration of the Raf/MEK/ERK pathway modulator is not limited, and an exemplary concentration is 0.1-5 μM, preferably 0.5-1.5 μM, such as 0.5 μM, 0.6 μM, 0.8 μM, 1.0 μM, 1.2 μM, and 1.5 μM. In some embodiments, the Raf/MEK/ERK pathway modulator is 0.5-1.0 μM GDC-0879 or 1.0 μM Dabrafenib.
在一些实施方案中，异恶唑衍生物为式(I)所示化合物。
In some embodiments, the isoxazole derivative is a compound represented by formula (I).
其中，R1为未取代或取代的杂芳基，Wherein, R 1 is unsubstituted or substituted heteroaryl,
m为0-4的整数，优选为1-3的整数，m is an integer from 0 to 4, preferably an integer from 1 to 3,
X为CH2、NH或O，X is CH 2 , NH or O,
R2为未取代或取代的杂芳基。R 2 is unsubstituted or substituted heteroaryl.
在一些实施方案中，R1为未取代或取代的含有1至2个选自N、O、S的杂原子的五元杂环，例如吡咯基、呋喃基、噻吩基、咪唑基、吡唑基、噻唑基、恶唑基、恶二唑基、异恶唑基、***基等，优选噻吩基、呋喃基。其可通过五元杂环上的杂原子或碳原子与母体结构相连。当被取代时，取代基的数量可为1个或2个(单取代或双取代)，所述取代基选自烷基、卤代烷基、羟基烷基、氰基、卤素、酯基、氨基，优选地，所述取代基选自C1-C4烷基、三氟甲基、被1-2个羟基取代的C1-C4烷基、R3OC(＝O)-，其中R3为C1-C4烷基。In some embodiments, R1 is an unsubstituted or substituted five-membered heterocyclic ring containing 1 to 2 heteroatoms selected from N, O, S, such as pyrrolyl, furyl, thienyl, imidazolyl, pyrazole group, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, triazolyl, etc., preferably thienyl and furyl. It can be connected to the parent structure through heteroatoms or carbon atoms on the five-membered heterocyclic ring. When substituted, the number of substituents may be 1 or 2 (mono-substituted or disubstituted), and the substituents are selected from alkyl, haloalkyl, hydroxyalkyl, cyano, halogen, ester, amino, Preferably, the substituent is selected from C1-C4 alkyl, trifluoromethyl, C1-C4 alkyl substituted by 1-2 hydroxyl groups, R 3 OC(=O)-, wherein R 3 is C1-C4 alkyl.
在一些实施方案中，R2为未取代或取代的含有1至2个选自N、O、S的杂原子的五元杂环，例如吡咯基、呋喃基、噻吩基、咪唑基、吡唑基、噻唑基、异噻唑基、恶唑基、恶二唑基、异恶唑基、***基等，优选为吡唑基、恶唑基、噻唑基。该杂芳基可通过五元杂环上的杂原子或碳原子与母体结构相连。当被取代时，取代基的数量可为1个或2个(单取代或双取代)，所述取代基选自烷基、卤代烷基、羟基烷基、氰基、卤素、酯基、氨基，优选地，所述取代基选自C1-C4烷基、三氟甲基、被1-2个羟基取代的C1-C4烷基、R3OC(＝O)-，其中R3为C1-C4烷基。优选实施方案中，所述取代基选自甲基、三氟甲基、羟丙基、氰基、卤素、CH3OC(＝O)-。In some embodiments, R is an unsubstituted or substituted five-membered heterocyclic ring containing 1 to 2 heteroatoms selected from N, O, S, such as pyrrolyl, furyl, thienyl, imidazolyl, pyrazole group, thiazolyl, isothiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, triazolyl and the like, preferably pyrazolyl, oxazolyl and thiazolyl. The heteroaryl group can be connected to the parent structure through a heteroatom or a carbon atom on the five-membered heterocyclic ring. When substituted, the number of substituents may be 1 or 2 (mono-substituted or disubstituted), and the substituents are selected from alkyl, haloalkyl, hydroxyalkyl, cyano, halogen, ester, amino, Preferably, the substituent is selected from C1-C4 alkyl, trifluoromethyl, C1-C4 alkyl substituted by 1-2 hydroxyl groups, R 3 OC(=O)-, wherein R 3 is C1-C4 alkyl. In a preferred embodiment, the substituent is selected from methyl, trifluoromethyl, hydroxypropyl, cyano, halogen, CH 3 OC(=O)-.
在一些实施方案中，式(I)化合物具有式(Ia)所示的结构：
In some embodiments, compounds of Formula (I) have the structure of Formula (Ia):
其中，A为O或S，m、X和R2的定义如上所述。Where, A is O or S, m, X and R2 are defined as above.
在一些实施方案中，式(I)选自如下化合物：


In some embodiments, Formula (I) is selected from the following compounds:


在一些实施方案中，所述异恶唑衍生物为5-呋喃-2-基-异恶唑-3-羧酸(2-吡唑-1-基-乙基)-酰胺(5-furan-2-yl-isoxazole-3-carboxylic acid(2-pyrazol-1-yl-ethyl)-amide，即上述化合物21，CAS：943820-93-7)。In some embodiments, the isoxazole derivative is 5-furan-2-yl-isoxazole-3-carboxylic acid (2-pyrazol-1-yl-ethyl)-amide (5-furan- 2-yl-isoxazole-3-carboxylic acid(2-pyrazol-1-yl-ethyl)-amide, the above-mentioned compound 21, CAS: 943820-93-7).
所述异恶唑衍生物的浓度不受限制，示例性的浓度为0-15μM，优选为0-10μM，更优选为1-10μM，例如0、1μM、2μM、4μM、5μM、6μM、10μM。The concentration of the isoxazole derivative is not limited, and an exemplary concentration is 0-15 μM, preferably 0-10 μM, more preferably 1-10 μM, such as 0, 1 μM, 2 μM, 4 μM, 5 μM, 6 μM, and 10 μM.
所述培养基还可含有嘧啶并吲哚衍生物。一些实施方案中，嘧啶并吲哚衍生物为式(II)所示化合物：
The medium may also contain pyrimidoindole derivatives. In some embodiments, the pyrimidoindole derivative is a compound represented by formula (II):
其中，Ra选自未取代或取代的C1-C4烷基、未取代或取代的杂芳基、RdOC(＝O)-、ReNHC(＝O)-，其中Rd为未取代或取代的C1-C4烷基，Re为未取代或取代的C1-C4烷基；Wherein, R a is selected from unsubstituted or substituted C1-C4 alkyl, unsubstituted or substituted heteroaryl, R d OC(=O)-, R e NHC(=O)-, where Rd is unsubstituted or Substituted C1-C4 alkyl, R e is unsubstituted or substituted C1-C4 alkyl;
Rb选自-NRfRg、-ORh，其中，Rf、Rg、Rh各自独立选自H、C1-C4烷基、-(CH2)nRi，其中，n为0-4的整数，Ri选自未取代或取代的C1-C4烷基、未取代或取代的环烷基、未取代或取代的杂环基、未取代或取代的芳基、未取代或取代的杂芳基、-NRjRk、-CN，其中Rj、Rk各自独立选自未取代或取代的C1-C4烷基；R b is selected from -NR f R g , -OR h , where R f , R g , and R h are each independently selected from H, C1-C4 alkyl, -(CH 2 ) n R i , where n is 0 -4 is an integer, R i is selected from unsubstituted or substituted C1-C4 alkyl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclyl, unsubstituted or substituted aryl, unsubstituted or substituted Heteroaryl, -NR j R k , -CN, wherein R j and R k are each independently selected from unsubstituted or substituted C1-C4 alkyl;
Rc选自H、-(CH2)iRp，其中，i为0-4的整数，Rp选自未取代或取代的C1-C4烷基、未取代或取代的环烷基、未取代或取代的杂环基、未取代或取代的芳基、未取代或取代的杂芳基、-NRjRk、-CN，其中Rj、Rk各自独立选自未取代或取代的C1-C4烷基。R c is selected from H, -(CH 2 ) i R p , where i is an integer from 0 to 4, and R p is selected from unsubstituted or substituted C1-C4 alkyl, unsubstituted or substituted cycloalkyl, unsubstituted Substituted or substituted heterocyclyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, -NR j R k , -CN, wherein R j and R k are each independently selected from unsubstituted or substituted C1 -C4 alkyl.
一些实施方案中，Ra为未取代或取代的杂芳基，所述杂芳基为含有1至2个选自N、O、S的杂原子的五元杂环或六元杂环，例如吡咯基、呋喃基、噻吩基、咪唑基、吡唑基、噻唑基、恶唑基、恶二唑基、异恶唑基、***基、四唑基、吡啶基、 嘧啶基等。其可通过五元环或六元环上的杂原子或碳原子与母体结构相连。当被取代时，取代基的数量可为1个或2个(单取代或双取代)，所述取代基选自烷基、卤代烷基、羟基烷基、氰基、卤素、酯基，优选地，所述取代基选自C1-C4烷基、三氟甲基、被1-2个羟基取代的C1-C4烷基、R3OC(＝O)-，其中R3为C1-C4烷基。优选实施方案中，所述取代基选自甲基、三氟甲基、羟丙基、氰基、卤素、CH3OC(＝O)-。In some embodiments, Ra is an unsubstituted or substituted heteroaryl group, which is a five- or six-membered heterocyclic ring containing 1 to 2 heteroatoms selected from N, O, and S, such as Pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyridyl, Pyrimidinyl etc. It can be connected to the parent structure through heteroatoms or carbon atoms on the five- or six-membered ring. When substituted, the number of substituents may be 1 or 2 (mono-substituted or disubstituted), and the substituents are selected from alkyl, haloalkyl, hydroxyalkyl, cyano, halogen, ester, preferably , the substituent is selected from C1-C4 alkyl, trifluoromethyl, C1-C4 alkyl substituted by 1-2 hydroxyl groups, R 3 OC(=O)-, where R 3 is C1-C4 alkyl . In a preferred embodiment, the substituent is selected from methyl, trifluoromethyl, hydroxypropyl, cyano, halogen, CH 3 OC(=O)-.
一些实施方案中，Rf、Rg中一者为H或C1-C4烷基、另一者为-(CH2)nRi，其中，n为0-4的整数，Ri选自未取代或取代的C1-C4烷基、未取代或取代的的C3-C6环烷基、未取代或取代的五元或六元杂环基(其含有1至2个选自N、O、S的杂原子，可通过五元或六元环上的杂原子或碳原子与母体结构相连)、未取代或取代的芳基、未取代或取代的五元或六元杂芳基(其含有1至2个选自N、O、S的杂原子，可通过杂原子或碳原子与母体结构相连)、-NRjRk、-CN，其中Rj、Rk各自独立选自未取代或取代的C1-C4烷基。In some embodiments, one of R f and R g is H or C1-C4 alkyl, and the other is -(CH 2 ) n R i , where n is an integer from 0 to 4, and R i is selected from the group consisting of Substituted or substituted C1-C4 alkyl, unsubstituted or substituted C3-C6 cycloalkyl, unsubstituted or substituted five- or six-membered heterocyclyl (which contains 1 to 2 selected from N, O, S heteroatoms, which can be connected to the parent structure through heteroatoms or carbon atoms on the five- or six-membered ring), unsubstituted or substituted aryl groups, unsubstituted or substituted five- or six-membered heteroaryl groups (which contain 1 to 2 heteroatoms selected from N, O, S, which can be connected to the parent structure through heteroatoms or carbon atoms), -NR j R k , -CN, where R j and R k are each independently selected from unsubstituted or substituted C1-C4 alkyl.
一些实施方案中，Rf、Rg中一者选自H、C1-C4烷基，另一者为-(CH2)nRi，其中，n为0-4的整数，并且，In some embodiments, one of R f and R g is selected from H, C1-C4 alkyl, and the other is -(CH 2 ) n R i , where n is an integer from 0 to 4, and,
Ri选自：C1-C4烷基，其中一个氢原子被C1-C4烷氧基取代的C1-C4烷基，未取代或被C1-C4烷基、羟基取代的C1-C4烷基、氰基、硝基、氨基、C1-C4烷氧基取代的C3-C6环烷基，未取代或取代(例如甲基)的含有1至2个选自N、O、S的杂原子的五元或六元杂环基(例如吡咯烷基、吗啉基、哌嗪基、高哌嗪基、哌啶基)，未取代或被C1-C4烷基、羟基取代的C1-C4烷基、氰基、硝基、氨基、C1-C4烷氧基取代的芳基，未取代或被C1-C4烷基、羟基取代的C1-C4烷基、氰基、硝基、氨基、C1-C4烷氧基取代的含有1至2个选自N、O、S的杂原子五元或六元杂芳基(例如吡咯基、呋喃基、噻吩基、咪唑基、吡唑基、噻唑基、恶唑基、恶二唑基、异恶唑基、***基、四唑基、吡啶基、嘧啶基)，-NRjRk，-CN，其中Rj、Rk各自独立选自C1-C4烷基。R i is selected from: C1-C4 alkyl, C1-C4 alkyl in which one hydrogen atom is substituted by C1-C4 alkoxy, C1-C4 alkyl that is unsubstituted or substituted by C1-C4 alkyl, hydroxyl, cyano Base, nitro, amino, C1-C4 alkoxy substituted C3-C6 cycloalkyl, unsubstituted or substituted (such as methyl) five-membered cycloalkyl group containing 1 to 2 heteroatoms selected from N, O, S Or six-membered heterocyclyl (such as pyrrolidinyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl), unsubstituted or C1-C4 alkyl, hydroxyl substituted C1-C4 alkyl, cyano Aryl group substituted by base, nitro, amino, C1-C4 alkoxy, unsubstituted or substituted by C1-C4 alkyl, hydroxyl C1-C4 alkyl, cyano, nitro, amino, C1-C4 alkoxy A five-membered or six-membered heteroaryl group substituted with 1 to 2 heteroatoms selected from N, O, and S (such as pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl , oxadiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl), -NR j R k , -CN, where R j and R k are each independently selected from C1-C4 alkyl .
一些实施方案中，Rh选自H、-(CH2)nRi，其中，n为0-4的整数，并且，In some embodiments, R h is selected from H, -(CH 2 ) n R i , where n is an integer from 0 to 4, and,
Ri选自：C1-C4烷基，其中一个氢原子被C1-C4烷氧基取代的C1-C4烷基，未取代或被C1-C4烷基、羟基取代的C1-C4烷基、氰基、硝基、氨基、C1-C4烷氧基取代的C3-C6环烷基，未取代或被C1-C4烷基、羟基取代的C1-C4烷基、氰基、硝 基、氨基、C1-C4烷氧基取代的含有1至2个选自N、O、S的杂原子的五元或六元杂环基(例如吡咯烷基、吗啉基、哌嗪基、高哌嗪基、哌啶基)，未取代或被C1-C4烷基、羟基取代的C1-C4烷基、氰基、硝基、氨基、C1-C4烷氧基取代的芳基，未取代或被C1-C4烷基、羟基取代的C1-C4烷基、氰基、硝基、氨基、C1-C4烷氧基取代的含有1至2个选自N、O、S的杂原子五元或六元杂芳基(例如吡咯基、呋喃基、噻吩基、咪唑基、吡唑基、噻唑基、恶唑基、恶二唑基、异恶唑基、***基、四唑基、吡啶基、嘧啶基)，-NRjRk，-CN，其中Rj、Rk各自独立选自C1-C4烷基。R i is selected from: C1-C4 alkyl, C1-C4 alkyl in which one hydrogen atom is substituted by C1-C4 alkoxy, C1-C4 alkyl that is unsubstituted or substituted by C1-C4 alkyl, hydroxyl, cyano base, nitro, amino, C1-C4 alkoxy substituted C3-C6 cycloalkyl, unsubstituted or substituted C1-C4 alkyl, hydroxyl C1-C4 alkyl, cyano, nitro base, amino, C1-C4 alkoxy substituted five- or six-membered heterocyclic group containing 1 to 2 heteroatoms selected from N, O, S (such as pyrrolidinyl, morpholinyl, piperazinyl, Homopiperazinyl, piperidinyl), unsubstituted or C1-C4 alkyl substituted by C1-C4 alkyl, hydroxyl, cyano, nitro, amino, C1-C4 alkoxy substituted aryl, unsubstituted Or C1-C4 alkyl, cyano, nitro, amino, C1-C4 alkoxy substituted by C1-C4 alkyl, hydroxyl, containing 1 to 2 heteroatoms selected from N, O, S. or six-membered heteroaryl (such as pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyridine group, pyrimidinyl), -NR j R k , -CN, where R j and R k are each independently selected from C1-C4 alkyl.
一些实施方案中，Rc选自H、-(CH2)iRp，其中，i为0-4的整数，并且，In some embodiments, R c is selected from H, -(CH 2 ) i R p , where i is an integer from 0 to 4, and,
Rp选自：C1-C4烷基，未取代或被C1-C4烷基、羟基取代的C1-C4烷基、氰基、硝基、氨基、C1-C4烷氧基取代的芳基，未取代或被C1-C4烷基、羟基取代的C1-C4烷基、氰基、硝基、氨基、C1-C4烷氧基取代的含有1至2个选自N、O、S的杂原子五元或六元杂芳基(例如吡咯基、呋喃基、噻吩基、咪唑基、吡唑基、噻唑基、恶唑基、恶二唑基、异恶唑基、***基、四唑基、吡啶基、嘧啶基)，-NRjRk，-CN，其中Rj、Rk各自独立选自未取代或取代的C1-C4烷基。R p is selected from: C1-C4 alkyl, unsubstituted or C1-C4 alkyl substituted by C1-C4 alkyl, hydroxyl, cyano, nitro, amino, C1-C4 alkoxy substituted aryl, unsubstituted C1-C4 alkyl, cyano, nitro, amino, C1-C4 alkoxy substituted or substituted by C1-C4 alkyl, hydroxyl containing 1 to 2 heteroatoms selected from N, O, S One- or six-membered heteroaryl (such as pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, triazolyl, tetrazolyl, Pyridyl, pyrimidinyl), -NR j R k , -CN, wherein R j and R k are each independently selected from unsubstituted or substituted C1-C4 alkyl.
一些实施方案中，嘧啶并吲哚衍生物选自：
In some embodiments, the pyrimidoindole derivative is selected from:
优选实施方案中，嘧啶并吲哚衍生物选自UM171和UM729。 In a preferred embodiment, the pyrimidoindole derivative is selected from UM171 and UM729.
嘧啶并吲哚衍生物的浓度不受限制，示例性的浓度为0-80nM，优选为0-50nM，例如0、5nM、10nM、20nM、25nM、40nM、50nM。在一些实施方案中，嘧啶 并吲哚衍生物是5-50nM的UM171或500nM的UM729。 The concentration of the pyrimidoindole derivative is not limited, and an exemplary concentration is 0-80 nM, preferably 0-50 nM, such as 0, 5 nM, 10 nM, 20 nM, 25 nM, 40 nM, 50 nM. In some embodiments, the pyrimidinoindole derivative is 5-50 nM UM171 or 500 nM UM729.
所述培养基的基础培养基不受限制，只要其适合树突细胞祖细胞生长即可。优选实施方案中，基础培养基是适合树突细胞祖细胞生长的无血清培养体系。无血清培养体系可采用商业无血清培养基，如StemSpanTM SFEM培养基、StemSpanTMSFEM II培养基、StemSpanTM-XF培养基、StemSpanTM-AOF培养基、StemProTM-34SFM；也可以采用包含基本培养基和补充添加物的基础无血清培养基。示例性实施方案中，所述基本培养基选自IMDM培养基、DMEM/F-12培养基、Neurobasal培养基、AIM-V中的一种或多种。示例性实施方案中，所述补充添加物选自人血白蛋白、胰岛素、转铁蛋白、***钠、DL-α-生育酚、亚油酸中的一种或多种。补充添加物的含量不受限制，示例性地：人血白蛋白的浓度为1-10g/L，优选为1g/L-5g/L，例如1g/L、1.5g/L、2g/L、2.5g/L、3g/L、3.5g/L、4g/L、4.5g/L、5g/L；胰岛素浓度为1-20mg/L，优选为3-15mg/L，例如3mg/L、4mg/L、5mg/L、6mg/L、7mg/L、8mg/L、9mg/L、10mg/L、11mg/L、12mg/L、13mg/L、14mg/L、15mg/L；转铁蛋白的浓度为1-30mg/L，优选为3-25mg/L，例如3mg/L、5mg/L、8mg/L、12mg/L、15mg/L、20mg/L、21mg/L、25mg/L；***钠的浓度为1-20ug/L，优选为5-15ug/L，例如5ug/L、8ug/L、10ug/L、12.5ug/L、15ug/L；DL-α-生育酚的浓度为0.5-10mg/L，优选为0.5-8mg/L，例如0.5mg/L、0.8mg/mL、1mg/L、1.2mg/mL、2.5mg/mL、4mg/mL、5mg/L；亚油酸的浓度为0.5-10mg/L，优选为0.5-5mg/L，例如0.5mg/L、0.8mg/mL、1mg/mL、1.2mg/mL、2.5mg/mL、4mg/mL、5mg/L。The basal medium of the culture medium is not limited as long as it is suitable for the growth of dendritic cell progenitor cells. In a preferred embodiment, the basal culture medium is a serum-free culture system suitable for the growth of dendritic cell progenitor cells. The serum-free culture system can use commercial serum-free culture medium, such as StemSpan TM SFEM medium, StemSpan TM SFEM II medium, StemSpan TM -XF medium, StemSpan TM -AOF medium, StemPro TM -34SFM; it can also be used containing basic Medium and basal serum-free medium supplemented with supplements. In an exemplary embodiment, the basic medium is selected from one or more of IMDM medium, DMEM/F-12 medium, Neurobasal medium, and AIM-V. In an exemplary embodiment, the supplementary additive is selected from one or more of human albumin, insulin, transferrin, sodium selenite, DL-α-tocopherol, and linoleic acid. The content of supplementary additives is not limited. For example, the concentration of human albumin is 1-10g/L, preferably 1g/L-5g/L, such as 1g/L, 1.5g/L, 2g/L, 2.5g/L, 3g/L, 3.5g/L, 4g/L, 4.5g/L, 5g/L; insulin concentration is 1-20mg/L, preferably 3-15mg/L, such as 3mg/L, 4mg /L, 5mg/L, 6mg/L, 7mg/L, 8mg/L, 9mg/L, 10mg/L, 11mg/L, 12mg/L, 13mg/L, 14mg/L, 15mg/L; transferrin The concentration is 1-30mg/L, preferably 3-25mg/L, such as 3mg/L, 5mg/L, 8mg/L, 12mg/L, 15mg/L, 20mg/L, 21mg/L, 25mg/L; The concentration of sodium selenite is 1-20ug/L, preferably 5-15ug/L, such as 5ug/L, 8ug/L, 10ug/L, 12.5ug/L, 15ug/L; DL-α-tocopherol The concentration is 0.5-10mg/L, preferably 0.5-8mg/L, such as 0.5mg/L, 0.8mg/mL, 1mg/L, 1.2mg/mL, 2.5mg/mL, 4mg/mL, 5mg/L; The concentration of oleic acid is 0.5-10mg/L, preferably 0.5-5mg/L, such as 0.5mg/L, 0.8mg/mL, 1mg/mL, 1.2mg/mL, 2.5mg/mL, 4mg/mL, 5mg/ L.
一些实施方案中，所述培养组合物或培养基含有Raf/MEK/ERK通路调节剂和生长因子，所述生长因子包括SCF、以及任选的G-CSF和/或IL-3。In some embodiments, the culture composition or medium contains a Raf/MEK/ERK pathway modulator and a growth factor, including SCF, and optionally G-CSF and/or IL-3.
本发明还提供树突细胞的制备方法，包括步骤：将前述由本文任一实施方案所述的方法获得的细胞在树突细胞分化培养基中培养，获得树突细胞。具体地，制备树突细胞的方法包括：(1)将来自外周血的细胞在本文所述的培养基中培养(例如至少1天、至少3天、至少7天、至少14天、至少28天)，获得第一细胞群，和(2)将第一细胞群在树突细胞分化培养基中培养(例如至少3天、至少6天、至少 9天、至少12天)，获得树突细胞。所述来自外周血的细胞包含选自CD45+白细胞、CD14+细胞、CD14-细胞、CD123+细胞、CD123-细胞、CD34-细胞中的一种或多种。The present invention also provides a method for preparing dendritic cells, which includes the step of culturing the aforementioned cells obtained by the method described in any embodiment of this article in a dendritic cell differentiation medium to obtain dendritic cells. Specifically, the method of preparing dendritic cells includes: (1) culturing cells from peripheral blood in the culture medium described herein (e.g., at least 1 day, at least 3 days, at least 7 days, at least 14 days, at least 28 days ), obtain a first cell population, and (2) culture the first cell population in dendritic cell differentiation medium (e.g., for at least 3 days, at least 6 days, at least 9 days, at least 12 days) to obtain dendritic cells. The cells from peripheral blood include one or more selected from the group consisting of CD45+ leukocytes, CD14+ cells, CD14- cells, CD123+ cells, CD123- cells, and CD34- cells.
本发明中，树突细胞分化培养基不受限制，可以使用本领域常用的树突细胞分化培养基。一些实施方案中，树突细胞分化培养基含有细胞因子。所述细胞因子可选自SCF、GM-CSF、IL-4、FLT-3L、IFNγ、IL-15等中的一种或多种。树突细胞分化培养基的基础培养基不受限制，只要适于树突细胞分化即可。一些实施方案中，基础培养基选自IMDM、RPMI-1640，AIM-V等。另外还可含有血清或血清替代物作为补充添加物，例如FBS等。一些实施方案中，基础培养基为商用培养基，例如ImmunocultTM-ACF Dendritic Cell Medium等。另外还可加入有补充添加物例如ImmunocultTM-ACF Dendritic Cell Supplement等。示例性实施方案中，树突细胞分化培养基是添加有10％FBS、100ng/mL FLT-3L、20ng/mL SCF、20ng/mL GM-CSF、20ng/mL IL-4的IMDM基础培养基。In the present invention, the dendritic cell differentiation medium is not limited, and dendritic cell differentiation medium commonly used in the field can be used. In some embodiments, the dendritic cell differentiation medium contains cytokines. The cytokine may be selected from one or more of SCF, GM-CSF, IL-4, FLT-3L, IFNγ, IL-15, etc. The basal medium of the dendritic cell differentiation medium is not limited as long as it is suitable for dendritic cell differentiation. In some embodiments, the basal medium is selected from IMDM, RPMI-1640, AIM-V, etc. In addition, serum or serum substitutes may be included as supplementary additives, such as FBS, etc. In some embodiments, the basal culture medium is a commercial culture medium, such as Immunocult ™ -ACF Dendritic Cell Medium, etc. In addition, supplementary additives such as Immunocult TM -ACF Dendritic Cell Supplement can also be added. In an exemplary embodiment, the dendritic cell differentiation medium is an IMDM base medium supplemented with 10% FBS, 100 ng/mL FLT-3L, 20 ng/mL SCF, 20 ng/mL GM-CSF, and 20 ng/mL IL-4.
一些实施方案中，树突细胞分化培养基还含有DC细胞成熟因子。DC细胞成熟因子可为toll样受体(Toll-like receptor，TLR)的配体和/或激活剂等，例如其可选自poly(I：C)、R848、LPS等中的一种或多种。In some embodiments, the dendritic cell differentiation medium further contains DC cell maturation factors. The DC cell maturation factor can be a ligand and/or activator of a toll-like receptor (TLR), for example, it can be selected from one or more of poly(I:C), R848, LPS, etc. kind.
本发明还提供由前述由本文任一实施方案所述的方法获得的树突细胞。The invention also provides dendritic cells obtained by the method described in any embodiment herein.
本发明还提供一种细胞冻存制剂，包括前述由本文任一实施方案所述的方法获得的树突细胞祖细胞和/或树突细胞；以及冻存液。The present invention also provides a cell cryopreservation preparation, including the aforementioned dendritic cell progenitor cells and/or dendritic cells obtained by the method described in any embodiment of this article; and a cryopreservation solution.
此外，本文所述的方法制备的树突细胞祖细胞和树突细胞可以用于制备药物组合物。因此，本发明还提供一种药物组合物，包含有效量的本文任一实施方案所述的方法制备的树突细胞祖细胞和/或树突细胞；以及药学上可接受的辅料。In addition, dendritic cell progenitor cells and dendritic cells prepared by the methods described herein can be used to prepare pharmaceutical compositions. Therefore, the present invention also provides a pharmaceutical composition comprising an effective amount of dendritic cell progenitor cells and/or dendritic cells prepared by the method described in any embodiment of this article; and pharmaceutically acceptable excipients.
术语“药学上可接受的辅料”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington′s Pharmaceutical Sciences.Edited by Gennaro AR，19th ed.Pennsylvania：Mack Publishing Company，1995)。药学上可接受的辅料包括但不限于稀释剂、载剂、增溶剂、乳化剂、防腐剂和/或佐剂。辅料优选地在所采用的剂量和浓度下对接受者无毒。这类辅料包括但并不限于：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。在某些实施方案中，药物组合物可含有用于改善、维持或保留例如组合物的pH、渗透性、粘度、澄清度、颜色、等渗性、气味、无菌性、稳定性、溶解或释放速率、吸收或 渗透的物质。这些物质为现有技术已知。可视预期的施用途径、递送方式和所需的剂量来确定最佳的药物组合物。The term "pharmaceutically acceptable excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Pharmaceutically acceptable excipients include, but are not limited to, diluents, carriers, solubilizers, emulsifiers, preservatives and/or adjuvants. Excipients are preferably non-toxic to the recipient at the doses and concentrations employed. Such excipients include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, pharmaceutical compositions may contain chemicals used to improve, maintain, or preserve, for example, the pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution, or release rate, absorption or Penetrating substances. These substances are known from the prior art. The optimal pharmaceutical composition will be determined based on the intended route of administration, mode of delivery and desired dosage.
用于体内施用的药物组合物通常以无菌制剂的形式提供。通过经无菌过滤膜过滤来实现灭菌。在组合物冻干时，可在冻干和复水之前或之后使用此方法进行灭菌。可选择本发明的药物组合物用于肠胃外递送。用于肠胃外施用的组合物可以冻干形式或在溶液中储存。例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。肠胃外组合物通常放在具有无菌进入孔的容器中，例如具有皮下注射针可刺穿的塞子的静脉内溶液带或小瓶。或者，可选择组合物用于吸入或通过消化道(诸如经口)递送。所述药学上可接受的组合物的制备在本领域的技术内。其它药物组合物将为本领域技术人员显而易见。用于配制多种其它持续或可控传递方式的技术(诸如脂质体载剂、生物易蚀微粒或多孔珠粒和积存注射)也为本领域技术人员所知。Pharmaceutical compositions for in vivo administration are generally provided in sterile preparations. Sterilization is achieved by filtration through a sterile filter membrane. When the composition is lyophilized, this method can be used for sterilization before or after lyophilization and reconstitution. The pharmaceutical compositions of the present invention may be selected for parenteral delivery. Compositions for parenteral administration may be stored in lyophilized form or in solution. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Parenteral compositions are typically placed in containers with sterile access openings, such as intravenous solution strips or vials with a stopper pierceable by a hypodermic needle. Alternatively, the compositions may be selected for inhalation or delivery through the digestive tract (such as orally). The preparation of such pharmaceutically acceptable compositions is within the skill in the art. Other pharmaceutical compositions will be apparent to those skilled in the art. Techniques for formulating a variety of other modes of sustained or controlled delivery, such as liposomal vehicles, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art.
药物组合物一经配制，就以溶液、悬浮液、凝胶、乳液、固体、晶体或以脱水或冻干粉末的形式储存在无菌小瓶中。所述配制物可储存成即用形式或在施用前复水的形式(例如，冻干)。本发明还提供用于产生单剂量施用单位的试剂盒。本发明的试剂盒可各自含有具有干燥蛋白的第一容器和具有含水配制物的第二容器。在本发明的某些实施方案中，提供含有单腔和多腔预填充注射器(例如，液体注射器和冻干注射器)的试剂盒。Once formulated, pharmaceutical compositions are stored in sterile vials as solutions, suspensions, gels, emulsions, solids, crystals, or as dehydrated or lyophilized powders. The formulations can be stored in ready-to-use form or reconstituted prior to administration (eg, lyophilized). The present invention also provides kits for producing single dose administration units. The kit of the invention may each contain a first container with dry protein and a second container with an aqueous formulation. In certain embodiments of the invention, kits are provided containing single and multi-lumen prefilled syringes (eg, liquid syringes and lyophilized syringes).
本发明也提供通过施用本发明任一实施方案所述的树突细胞祖细胞或其药物组合物来治疗患者(尤其是树突细胞或树突细胞祖细胞相关疾病，包括但不限于感染疾病、代谢疾病和/或癌症)的方法。本文中，术语“患者”、“受试者”、“个体”、“对象”在本文中可互换使用，包括任何生物体，优选动物，更优选哺乳动物(例如大鼠、小鼠、狗、猫、兔等)，且最优选的是人。“治疗”指向受试者采用本文所述治疗方案以达到至少一种阳性治疗效果(比如，癌症细胞数目减少、肿瘤体积减小、癌细胞浸润至周边器官的速率降低或肿瘤转移或肿瘤生长的速率降低)。有效治疗患者的治疗方案可根据多种因素(比如患者的疾病状态、年龄、体重及疗法激发受试者的抗癌反应的能力)而变。The invention also provides for the treatment of patients (especially dendritic cells or dendritic cell progenitor cell-related diseases, including but not limited to infectious diseases, by administering dendritic cell progenitor cells or pharmaceutical compositions thereof according to any embodiment of the invention) metabolic diseases and/or cancer). As used herein, the terms "patient", "subject", "individual" and "subject" are used interchangeably herein and include any organism, preferably an animal, more preferably a mammal (e.g. rat, mouse, dog , cats, rabbits, etc.), and most preferably humans. "Treatment" refers to a subject taking a treatment regimen described herein to achieve at least one positive therapeutic effect (for example, reduction in the number of cancer cells, reduction in tumor volume, reduction in the rate of cancer cell infiltration into surrounding organs, or reduction in tumor metastasis or tumor growth) rate decreases). The treatment regimen that effectively treats a patient will vary based on a variety of factors, such as the patient's disease state, age, weight, and the ability of the therapy to elicit an anti-cancer response in the subject.
将采用的含有本发明树突细胞祖细胞的药物组合物的治疗有效量将取决于例如治疗程度和目标。本领域技术人员将了解，用于治疗的适当剂量水平将部分取决于 所递送的分子、适应症、施用途径和患者的大小(体重、体表或器官大小)和/或状况(年龄和一般健康状况)而变化。在某些实施方案中，临床医生可滴定剂量并改变施用途径来获得最佳的治疗效果。例如每天约10微克/千克体重-约50毫克/千克体重。The therapeutically effective amount of a pharmaceutical composition containing dendritic cell progenitor cells of the invention to be employed will depend, for example, on the extent and goals of the treatment. One skilled in the art will appreciate that appropriate dosage levels for treatment will depend in part on It varies with the molecule delivered, the indication, the route of administration and the patient's size (body weight, body surface or organ size) and/or condition (age and general health). In certain embodiments, the clinician can titrate the dosage and vary the route of administration to achieve optimal therapeutic effects. For example, about 10 micrograms/kg of body weight per day - about 50 mg/kg of body weight per day.
给药频率将取决于所用配制物中树突细胞祖细胞的药物动力学参数。临床医生典型地施用组合物直到达到实现所需效果的剂量。组合物因此可作为单次剂量施用，或随时间以作为两次或多次剂量(可含有或不含有相同量的所需分子)施用，或通过植入装置或导管以连续输液的方式施用。The frequency of dosing will depend on the pharmacokinetic parameters of the dendritic cell progenitors in the formulation used. Clinicians typically administer the composition until a dose is achieved that achieves the desired effect. The composition may thus be administered as a single dose, or as two or more doses over time (which may or may not contain the same amount of the desired molecule), or as a continuous infusion through an implanted device or catheter.
药物组合物的施用途径是根据已知方法，例如经口、通过静脉内、腹膜内、脑内(脑实质内)、脑室内、肌肉内、眼内、动脉内、门静脉或病灶内途径注射；通过持续释放***或通过植入装置。The route of administration of the pharmaceutical composition is according to known methods, such as orally, by intravenous, intraperitoneal, intracerebral (intrabrain parenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional route; Either through a sustained release system or through an implanted device.
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市场购买获得的常规产品。The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field shall be followed (for example, refer to "Molecular Cloning Experimental Guide" translated by J. Sambrook et al., Huang Peitang et al., third edition, Science Press) or follow the product instructions. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased in the market.
实施例Example
试剂和材料：Reagents and materials:
实施例中所使用的抗体、试剂来源如下：The sources of antibodies and reagents used in the examples are as follows:
StemSpanTM SFEM培养基，Stemcell Technologies公司，货号09650。StemSpan TM SFEM medium, Stemcell Technologies, Cat. No. 09650.
StemSpanTM SFEM II培养基，Stemcell Technologies公司，货号09655。StemSpan ™ SFEM II medium, Stemcell Technologies, Cat. No. 09655.
StemSpanTM-XF培养基，Stemcell Technologies公司，货号100-0073。StemSpan ™ -XF medium, Stemcell Technologies, Cat. No. 100-0073.
StemSpanTM-AOF培养基，Stemcell Technologies公司，货号100-0130。StemSpan TM -AOF medium, Stemcell Technologies, Cat. No. 100-0130.
IMDM培养基，Thermo Fisher公司，货号12440053。IMDM medium, Thermo Fisher Company, Cat. No. 12440053.
DMEM/F12培养基，Thermo Fisher公司，货号11330032。DMEM/F12 medium, Thermo Fisher Company, Cat. No. 11330032.
Neurobasal培养基，Thermo Fisher公司，货号21103049。Neurobasal medium, Thermo Fisher Company, Cat. No. 21103049.
人血清白蛋白(CAS号70024-90-7)，Sigma-Aldrich公司，货号A3782。Human serum albumin (CAS No. 70024-90-7), Sigma-Aldrich Company, Cat. No. A3782.
Human insulin(CAS号11061-68-0)，Sigma-Aldrich公司，货号91077C。Human insulin (CAS No. 11061-68-0), Sigma-Aldrich Company, Cat. No. 91077C.
转铁蛋白(CAS号11096-37-0)，Sigma-Aldrich公司，货号T0665。 Transferrin (CAS No. 11096-37-0), Sigma-Aldrich Company, Cat. No. T0665.
***钠(CAS号10102-18-8)，Sigma-Aldrich公司，货号S9133。Sodium selenite (CAS No. 10102-18-8), Sigma-Aldrich Company, Cat. No. S9133.
DL-α-生育酚(CAS号10191-41-0)，Sigma-Aldrich公司，货号T3251。DL-α-Tocopherol (CAS No. 10191-41-0), Sigma-Aldrich Company, Cat. No. T3251.
亚油酸(CAS号60-33-3)，Sigma-Aldrich公司，货号L1012。Linoleic acid (CAS number 60-33-3), Sigma-Aldrich Company, product number L1012.
Human SCF，PeproTech公司，货号AF-300-07。Human SCF, PeproTech, Cat. No. AF-300-07.
Animal-Free Recombinant Human GM-CSF，PeproTech公司，货号300-03。Animal-Free Recombinant Human GM-CSF, PeproTech, Cat. No. 300-03.
Animal-Free Recombinant Human G-CSF，PeproTech公司，货号AF-300-23。Animal-Free Recombinant Human G-CSF, PeproTech, Cat. No. AF-300-23.
Recombinant Human IL-3，PeproTech公司，货号GMP200-03。 Recombinant Human IL-3, PeproTech, Cat. No. GMP200-03.
Human Flt3-Ligand，PeproTech公司，货号300-19-10。Human Flt3-Ligand, PeproTech, Cat. No. 300-19-10.
GDC-0879，Selleck公司，货号S1104。GDC-0879, Selleck Company, Cat. No. S1104.
PLX-4720，Selleck公司，货号S1152。PLX-4720, Selleck Company, Cat. No. S1152.
Vemurafenib(PLX4032)，Selleck公司，货号S1267。Vemurafenib (PLX4032), Selleck Company, product number S1267.
Sorafenib(BAY 43-9006)，Selleck公司，货号S7397。Sorafenib (BAY 43-9006), Selleck Company, product number S7397.
Dabrafenib，Selleck公司，货号S2807。Dabrafenib, Selleck, Cat. No. S2807.
化合物1-21、24-44，上海吉量医药工程有限公司。Compounds 1-21, 24-44, Shanghai Jiliang Pharmaceutical Engineering Co., Ltd.
UM171，ApexBio Technology公司，货号A8950。UM171, ApexBio Technology, Cat. No. A8950.
UM729，ApexBio Technology公司，货号A8952。UM729, ApexBio Technology, Cat. No. A8952.
Ficoll-Paque PREMIUM，GE Healthcare公司，货号17-5442-03。Ficoll-Paque PREMIUM, GE Healthcare, Cat. No. 17-5442-03.
Dulbecco′s磷酸盐缓冲液，货号14190250。Dulbecco's Phosphate Buffered Saline, Cat. No. 14190250.
ViaStainTM AO/PI Staining Solution，Nexcelom公司，货号CS2-0106。ViaStain TM AO/PI Staining Solution, Nexcelom Company, Cat. No. CS2-0106.
Pan Monocyte Isolation Kit，Miltenyi Biotec公司，货号130-096-537。Pan Monocyte Isolation Kit, Miltenyi Biotec, Cat. No. 130-096-537.
CD123 MicroBeads，Miltenyi Biotec公司，货号130-094-432。CD123 MicroBeads, Miltenyi Biotec, Cat. No. 130-094-432.
CD34 MicroBeads，Miltenyi Biotec公司，货号130-100-453。Fixable Viability Stain 780，BD Biosciences公司，货号565388。CD34 MicroBeads, Miltenyi Biotec, Cat. No. 130-100-453. Fixable Viability Stain 780, BD Biosciences, Cat. No. 565388.
FITC Mouse Anti-Human CD11c，BD Biosciences公司，货号561355。FITC Mouse Anti-Human CD11c, BD Biosciences, Cat. No. 561355.
PE Mouse Anti-Human HLA-DR，BD Biosciences公司，货号555812。PE Mouse Anti-Human HLA-DR, BD Biosciences, Cat. No. 555812.
BV605 Mouse Anti-Human CD40，BD Biosciences公司，货号740410。BV605 Mouse Anti-Human CD40, BD Biosciences, Cat. No. 740410.
BV786 Mouse Anti-Human CD80，BD Biosciences公司，货号564159。BV786 Mouse Anti-Human CD80, BD Biosciences, Cat. No. 564159.
实验方法： experimental method:
1、Ficoll工艺分离外周血细胞的方法，包括以下步骤：1. The Ficoll process for separating peripheral blood cells includes the following steps:
1)将血袋中的血(含抗凝剂)使用无菌注射器抽出平均分装到50mL离心管中；1) Use a sterile syringe to draw the blood (containing anticoagulant) from the blood bag and distribute it evenly into 50mL centrifuge tubes;
2)使用Thermo Fisher的离心机2500g离心10min，设置参数升7降7；2) Use a Thermo Fisher centrifuge to centrifuge at 2500g for 10 minutes, and set the parameters to increase from 7 to 7;
3)抽出中间白膜层转移至新的50mL离心管中，每管20mL；3) Pull out the middle white film layer and transfer it to a new 50mL centrifuge tube, 20mL in each tube;
4)加入30mL常温DPBS稀释血样，轻柔充分混匀；4) Add 30mL of room temperature DPBS to dilute the blood sample and mix gently and thoroughly;
5)根据样本量，准备若干50mL离心管(30mL稀释血样/管)，每管加入15mL Ficoll备用(不要碰到管壁上部)，室温放置；5) According to the sample volume, prepare several 50mL centrifuge tubes (30mL diluted blood sample/tube), add 15mL Ficoll to each tube for later use (do not touch the upper part of the tube wall), and place at room temperature;
6)将30mL稀释血样缓慢加到Ficoll上(按Ficoll溶液：稀释好的血液为1∶2)，室温，800g，离心20min，设置参数升1降0。6) Slowly add 30 mL of diluted blood sample to Ficoll (according to Ficoll solution: the diluted blood is 1:2), centrifuge at room temperature, 800g, for 20 minutes, and set the parameters to increase by 1 and decrease by 0.
7)离心结束后，从下往上分为红细胞层、Ficoll层、白膜层、血浆层。弃血浆层至白膜层上方5mL刻度处，用1mL枪头缓慢吸取细胞白膜层至新50mL离心管中，加至少3倍体积DPBS至50mL；室温400g，离心10min，设置参数升9降9；7) After centrifugation, it is divided into red blood cell layer, Ficoll layer, buffy coat layer and plasma layer from bottom to top. Discard the plasma layer to the 5mL mark above the buffy coat layer, use a 1mL pipette tip to slowly absorb the cell buffy coat layer into a new 50mL centrifuge tube, add at least 3 times the volume of DPBS to 50mL; centrifuge at 400g at room temperature for 10 minutes, and set the parameters to increase from 9 to decrease by 9 ;
8)弃去上清(不要倒尽上清)，每管用1mL DPBS重悬细胞，加DPBS至50mL，室温400g，离心10min，设置参数升9降9；8) Discard the supernatant (do not pour out all the supernatant), resuspend the cells in 1mL DPBS for each tube, add DPBS to 50mL, 400g at room temperature, centrifuge for 10min, set the parameters to increase from 9 to 9;
9)弃去上清，用培养基重悬接种，放入37℃5％CO2培养箱培养。9) Discard the supernatant, resuspend the inoculation in culture medium, and place it in a 37°C 5% CO2 incubator for culture.
2、从外周血细胞中获得iGDP的方法，包括以下步骤：2. The method of obtaining iGDP from peripheral blood cells includes the following steps:
1)分离的新鲜外周血细胞使用DPBS重悬，室温300g，离心10min，设置参数升9降9(冻存外周血细胞从液氮罐取出，37℃水浴锅内快速融化，转移至9mL新鲜培养基中，轻轻吹打混匀，同样参数进行离心)；1) Resuspend the separated fresh peripheral blood cells in DPBS, centrifuge at 300g at room temperature for 10 minutes, and set the parameters to increase from 9 to decrease by 9 (the frozen peripheral blood cells are taken out of the liquid nitrogen tank, thawed quickly in a 37°C water bath, and transferred to 9 mL of fresh culture medium , mix gently by pipetting, and centrifuge with the same parameters);
2)弃去上清，使用1mL相应培养基重悬，取10μL细胞悬液与10μLViaStainTM AO/PI Staining Solution轻轻吹打混匀；2) Discard the supernatant and resuspend in 1 mL of the corresponding culture medium. Take 10 μL of cell suspension and 10 μL of ViaStain TM AO/PI Staining Solution and mix gently by pipetting;
3)转移至细胞计数板，***Cellometer K2 Fluorescent Cell Viability Counter，选择相应程序进行AO/PI细胞活性检测；3) Transfer to the cell counting board, insert the Cellometer K2 Fluorescent Cell Viability Counter, and select the corresponding program for AO/PI cell viability detection;
4)加入适量培养基调整密度为1.4E6个活细胞/mL培养基，接种至Tissue-treated培养板内，放入37℃，5％CO2培养箱培养。4) Add an appropriate amount of culture medium to adjust the density to 1.4E6 viable cells/mL culture medium, inoculate it into a Tissue-treated culture plate, and place it in a 37°C, 5% CO 2 incubator for culture.
5)细胞汇合度达70-80％时，转移细胞悬液于离心管内，取10μL细胞悬 液与10μLViaStainTM AO/PI Staining Solution轻轻吹打混匀；5) When the cell confluence reaches 70-80%, transfer the cell suspension into a centrifuge tube and take 10 μL of cell suspension. The solution was mixed with 10 μL ViaStain TM AO/PI Staining Solution by gently pipetting;
6)转移至细胞计数板，***Cellometer K2 Fluorescent Cell Viability Counter，选择相应程序进行AO/PI细胞活性检测；6) Transfer to the cell counting board, insert the Cellometer K2 Fluorescent Cell Viability Counter, and select the corresponding program for AO/PI cell viability detection;
7)根据计数结果补加新鲜培养基调整密度为1E5个活细胞/mL培养基，接种后放入37℃5％CO2培养箱培养。7) Add fresh culture medium according to the counting results to adjust the density to 1E5 viable cells/mL culture medium. After inoculation, place it in a 37°C 5% CO 2 incubator for culture.
3、从CD14+单核细胞和CD14-非单核细胞中获得iGDP的方法，包括以下步骤：3. The method of obtaining iGDP from CD14 + monocytes and CD14 - non-monocytes includes the following steps:
1)外周血细胞使用DPBS重悬，室温300g，离心10min，设置参数升9降9；1) Peripheral blood cells were resuspended in DPBS, centrifuged at 300g at room temperature for 10 minutes, and the parameters were set to increase from 9 to 9;
2)配制分选buffer：将MACS BSA Stock Solution和autoMACS Rinsing Solution按1∶20比例混合，置于冰上；2) Prepare sorting buffer: Mix MACS BSA Stock Solution and autoMACS Rinsing Solution in a ratio of 1:20 and place on ice;
3)每1E7细胞用30μL分选buffer重悬，加入10μL FcRBlocking Solution试剂和10μL Biotin-Antibody Cocktail混匀，2-8℃孵育5min；3) Resuspend each 1E7 cell with 30 μL of sorting buffer, add 10 μL of FcRBlocking Solution reagent and 10 μL of Biotin-Antibody Cocktail, mix well, and incubate at 2-8°C for 5 minutes;
4)每1E7细胞用30μL分选buffer，加入20μL Anti-Biotin MicroBeads混匀，2-8℃孵育5min；4) Use 30μL sorting buffer for each 1E7 cell, add 20μL Anti-Biotin MicroBeads, mix well, and incubate at 2-8°C for 5 minutes;
5)使用MACS Separator固定LS column，加入3mL buffer浸润LS column；5) Use MACS Separator to fix the LS column, and add 3mL buffer to infiltrate the LS column;
6)滴加细胞悬液，收集未标记即高纯CD14+单核细胞悬液，随后加入3mL buffer清洗，收集未标记细胞悬液，混合两次细胞悬液；6) Drop the cell suspension, collect the unlabeled, high-purity CD14 + monocyte suspension, then add 3mL buffer for washing, collect the unlabeled cell suspension, and mix the cell suspension twice;
7)从MACS Separator上取下LS column至于收集管上，加入5mL buffer，将塞子推入柱内，立即将磁性标记的细胞冲洗出来，收集CD14-非单核细胞悬液；7) Remove the LS column from the MACS Separator and place it on the collection tube, add 5mL buffer, push the plug into the column, immediately flush out the magnetically labeled cells, and collect the CD14 - non-mononuclear cell suspension;
8)分别混匀CD14+单核细胞和CD14-非单核细胞悬液，各取10μL细胞悬液置于离心管中，加入10μLViaStainTM AO/PI Staining Solution轻轻吹打混匀加入计数板，Cellometer K2 Fluorescent Cell Viability Counter，选择相应程序进行AO/PI细胞活性检测；8) Mix the CD14 + monocyte and CD14 - non-monocyte suspensions separately, take 10 μL of each cell suspension into a centrifuge tube, add 10 μL ViaStain TM AO/PI Staining Solution, gently pipet and mix, and add to the counting plate, Cellometer K2 Fluorescent Cell Viability Counter, select the corresponding program to detect AO/PI cell viability;
9)CD14+单核细胞和CD14-非单核细胞悬液分别离心，300g，离心10min，设置参数升9降9，轻柔吸弃上清；9) Centrifuge the CD14 + monocyte and CD14 - non-monocyte suspensions respectively at 300g for 10 minutes, set the parameters up to 9 and down to 9, and gently aspirate and discard the supernatant;
10)使用适当体积培养基重悬，调整细胞密度为1E5个活细胞/mL培养基，接种后放入37℃5％CO2培养箱培养。 10) Use an appropriate volume of culture medium to resuspend, adjust the cell density to 1E5 viable cells/mL culture medium, and place it in a 37°C 5% CO 2 incubator after inoculation.
4、从CD123+和CD123-细胞中获得iGDP的方法，包括以下步骤：4. The method of obtaining iGDP from CD123 + and CD123- cells includes the following steps:
1)外周血细胞使用DPBS重悬，室温300g，离心10min，设置参数升9降9；1) Peripheral blood cells were resuspended in DPBS, centrifuged at 300g at room temperature for 10 minutes, and the parameters were set to increase from 9 to 9;
2)配制分选buffer：将MACS BSA Stock Solution和autoMACS Rinsing Solution按1∶20比例混合，置于冰上；2) Prepare sorting buffer: Mix MACS BSA Stock Solution and autoMACS Rinsing Solution in a ratio of 1:20 and place on ice;
3)每1E8细胞用400μL分选buffer重悬，加入100μL CD123 MicroBeads混匀，2-8℃孵育15min；3) Resuspend each 1E8 cell with 400μL sorting buffer, add 100μL CD123 MicroBeads, mix well, and incubate at 2-8°C for 15 minutes;
4)每1E8细胞用10mL分选buffer重悬，300g，离心10min，设置参数升9降9，轻柔吸弃上清；4) Resuspend each 1E8 cell in 10mL of sorting buffer, centrifuge at 300g for 10 minutes, set the parameters from 9 to 9, and gently aspirate and discard the supernatant;
5)每1E8细胞用500μL分选buffer重悬；5) Resuspend each 1E8 cell in 500 μL of sorting buffer;
6)使用MACS Separator固定LS column，加入3mL buffer浸润LS column；6) Use MACS Separator to fix the LS column, and add 3mL buffer to infiltrate the LS column;
7)滴加细胞悬液，收集未标记即CD123-细胞悬液，随后加入3mL buffer清洗，收集未标记细胞悬液，混合两次细胞悬液；7) Drop the cell suspension, collect the unlabeled CD123-cell suspension, then add 3mL buffer for washing, collect the unlabeled cell suspension, and mix the cell suspension twice;
8)从MACS Separator上取下LS column至于收集管上，加入5mL buffer，将塞子推入柱内，立即将磁性标记的细胞冲洗出来(为提高CD123+细胞纯度，可以将冲洗的细胞悬液用一根新的LS column进行二次分选)；8) Remove the LS column from the MACS Separator and place it on the collection tube, add 5mL buffer, push the plug into the column, and immediately rinse out the magnetically labeled cells (to improve the purity of CD123 + cells, the washed cell suspension can be used A new LS column is used for secondary sorting);
9)分别混匀CD123+和CD123-细胞悬液，各取10μL细胞悬液置于离心管中，加入10μLViaStainTM AO/PI Staining Solution轻轻吹打混匀加入计数板，Cellometer K2 Fluorescent Cell Viability Counter，选择相应程序进行AO/PI细胞活性检测；9) Mix the CD123 + and CD123 - cell suspensions respectively, place 10 μL of each cell suspension in a centrifuge tube, add 10 μL of ViaStain TM AO/PI Staining Solution, mix gently by pipetting, and add to the counting plate, Cellometer K2 Fluorescent Cell Viability Counter. Select the corresponding program for AO/PI cell activity detection;
10)CD123+和CD123-细胞悬液分别离心，300g，离心10min，轻柔吸弃上清；10) Centrifuge the CD123 + and CD123 - cell suspensions respectively at 300g for 10 minutes, and gently aspirate and discard the supernatant;
11)使用相应体积相应培养基重悬，调整密度为1E5个活细胞/mL培养基，接种后放入37℃5％CO2培养箱培养。11) Resuspend in the corresponding volume of the corresponding medium, adjust the density to 1E5 viable cells/mL medium, and place it in a 37°C 5% CO 2 incubator after inoculation.
5、从CD34-细胞中获得iGDP的方法，包括以下步骤：5. The method of obtaining iGDP from CD34 - cells includes the following steps:
1)外周血细胞使用DPBS重悬，室温300g，离心10min，设置参数升9降9；1) Peripheral blood cells were resuspended in DPBS, centrifuged at 300g at room temperature for 10 minutes, and the parameters were set to increase from 9 to 9;
2)配制分选buffer：将MACS BSA Stock Solution和autoMACS Rinsing Solution按1∶20比例混合，置于冰上；2) Prepare sorting buffer: Mix MACS BSA Stock Solution and autoMACS Rinsing Solution in a ratio of 1:20 and place on ice;
3)每1E7细胞用30μL分选buffer重悬，加入10μL FcR Blocking Solution 试剂和10μL Biotin-Antibody Cocktail混匀，2-8℃孵育5min；3) Resuspend each 1E7 cell in 30 μL of sorting buffer and add 10 μL of FcR Blocking Solution. Mix the reagent and 10 μL Biotin-Antibody Cocktail and incubate at 2-8°C for 5 minutes;
4)每1E7细胞用30μL分选buffer，加入20μL Anti-Biotin MicroBeads混匀，2-8℃孵育5min；4) Use 30μL sorting buffer for each 1E7 cell, add 20μL Anti-Biotin MicroBeads, mix well, and incubate at 2-8°C for 5 minutes;
5)使用MACS Separator固定LS column，加入3mL buffer浸润LS column；5) Use MACS Separator to fix the LS column, and add 3mL buffer to infiltrate the LS column;
6)滴加细胞悬液，收集未标记即高纯CD34-细胞悬液，随后加入3mLbuffer清洗，收集未标记细胞悬液，混合两次细胞悬液；6) Drop the cell suspension, collect the unlabeled or high-purity CD34-cell suspension, then add 3mL buffer for washing, collect the unlabeled cell suspension, and mix the cell suspension twice;
7)混匀CD34-细胞悬液，各取10μL细胞悬液置于离心管中，加入10μLViaStainTM AO/PI Staining Solution轻轻吹打混匀加入计数板，Cellometer K2 Fluorescent Cell Viability Counter，选择相应程序进行AO/PI细胞活性检测；7) Mix the CD34-cell suspension, place 10 μL of each cell suspension in a centrifuge tube, add 10 μL ViaStain TM AO/PI Staining Solution, mix gently by pipetting, add to the counting plate, Cellometer K2 Fluorescent Cell Viability Counter, and select the corresponding program. AO/PI cell activity detection;
9)CD34-细胞悬液分别离心，300g，离心10min，设置参数升9降9，轻柔吸弃上清；9) Centrifuge the CD34-cell suspension separately at 300g for 10 minutes, set the parameters up to 9 and down to 9, and gently aspirate and discard the supernatant;
10)使用适当体积培养基重悬，调整细胞密度为1E5个活细胞/mL培养基，接种后放入37℃5％CO2培养箱培养。10) Use an appropriate volume of culture medium to resuspend, adjust the cell density to 1E5 viable cells/mL culture medium, and place it in a 37°C 5% CO 2 incubator after inoculation.
6、诱导iGDP向树突细胞分化和成熟的方法，包括以下步骤：6. Methods for inducing iGDP differentiation and maturation into dendritic cells, including the following steps:
1)iGDP细胞使用DPBS重悬，室温300g，离心10min，设置参数升9降9；1) Resuspend iGDP cells in DPBS, centrifuge at 300g at room temperature for 10 minutes, and set the parameters from 9 to 9;
2)弃去上清，使用1mL培养基，取10μL细胞悬液与10μL ViaStain AO/PI Staining Solution轻轻吹打混匀；2) Discard the supernatant, use 1mL of culture medium, take 10μL of cell suspension and 10μL of ViaStain AO/PI Staining Solution and mix gently by pipetting;
3)转移至细胞计数板，***Cellometer K2 Fluorescent Cell Viability Counter，选择相应程序进行AO/PI细胞活性检测；3) Transfer to the cell counting board, insert the Cellometer K2 Fluorescent Cell Viability Counter, and select the corresponding program for AO/PI cell viability detection;
4)加入适量分化培养基(IMDM基础培养基添加10％FBS、100ng/mL FLT-3L、20ng/mL SCF、20ng/mL GM-CSF、20ng/mL IL-4)调整密度为5E5个活细胞/mL悬液，接种后放入37℃，5％CO2培养箱培养。4) Add an appropriate amount of differentiation medium (IMDM basal medium plus 10% FBS, 100ng/mL FLT-3L, 20ng/mL SCF, 20ng/mL GM-CSF, 20ng/mL IL-4) to adjust the density to 5E5 viable cells /mL suspension, and after inoculation, place it in a 37°C, 5% CO 2 incubator for culture.
5)每隔3天半换液；5) Change the medium every 3 and a half days;
6)培养第12天，收集一半体积细胞悬液，室温300g，离心10min，设置参数升9降9；6) On the 12th day of culture, collect half the volume of cell suspension, centrifuge at 300g at room temperature for 10 minutes, and set the parameters to increase from 9 to 9;
7)弃去上清，使用适量成熟培养基(IMDM基础培养基添加10％FBS、100ng/mL FLT-3L、20ng/mL SCF、20ng/mL GM-CSF、20ng/mL IL-4、200IU/mL IFNγ、 60μg/mL poly(I：C)、10μg/mL R848、2μg/mL LPS)重悬细胞，接种至原孔中继续培养24h。7) Discard the supernatant and use an appropriate amount of mature medium (IMDM basic medium supplemented with 10% FBS, 100ng/mL FLT-3L, 20ng/mL SCF, 20ng/mL GM-CSF, 20ng/mL IL-4, 200IU/ mL IFNγ, Resuspend the cells in 60 μg/mL poly(I:C), 10 μg/mL R848, 2 μg/mL LPS), inoculate them into the original wells and continue culturing for 24 h.
7、iGDP细胞和iGDP衍生树突细胞流式检测方法，包括以下步骤：7. The flow cytometric detection method of iGDP cells and iGDP-derived dendritic cells includes the following steps:
1)取已知数量的iGDP/iGDP衍生树突细胞，室温静置3-5min，400g，离心5min，轻柔吸弃上清；1) Take a known number of iGDP/iGDP-derived dendritic cells, let stand at room temperature for 3-5 minutes, centrifuge at 400g for 5 minutes, and gently aspirate and discard the supernatant;
2)调整细胞密度为1E7个活细胞/mL DPBS，按每1E7加1.5μL比例加入FVS780，将样品与染料充分混匀，室温避光孵育10min；2) Adjust the cell density to 1E7 viable cells/mL DPBS, add FVS780 at a ratio of 1.5 μL per 1E7, mix the sample and dye thoroughly, and incubate at room temperature in the dark for 10 minutes;
3)再取4mL DPBS清洗两次，每次清洗后离心，400g，离心10min，轻柔吸弃上清；3) Take another 4mL of DPBS and wash it twice. After each wash, centrifuge at 400g for 10 minutes. Gently aspirate and discard the supernatant;
4)按比例加入对应抗体(FITC Mouse Anti-Human CD11c，PE Mouse Anti-Human HLA-DR，BV605 Mouse Anti-Human CD40，BV786 Mouse Anti-Human CD80)于1.5mL离心管中，将抗体完全混匀；4) Add the corresponding antibodies (FITC Mouse Anti-Human CD11c, PE Mouse Anti-Human HLA-DR, BV605 Mouse Anti-Human CD40, BV786 Mouse Anti-Human CD80) in proportion to the 1.5mL centrifuge tube, and mix the antibodies completely ;
5)将混匀后的细胞密度调整至2E6个活细胞/100μL悬液，样品管中加入100μL细胞悬液，室温避光孵育15min；5) Adjust the mixed cell density to 2E6 viable cells/100 μL suspension, add 100 μL cell suspension to the sample tube, and incubate at room temperature in the dark for 15 minutes;
6)样品管中加入1mL DPBS，使用台式离心机离心，400g离心5min，轻柔吸弃上清；6) Add 1mL DPBS to the sample tube, centrifuge using a desktop centrifuge at 400g for 5 minutes, and gently aspirate and discard the supernatant;
7)重复步骤6清洗一次；7) Repeat step 6 for cleaning once;
8)样品管中加入300μL DPBS，使用枪头小心吹打混匀；8) Add 300μL DPBS to the sample tube, use the pipette tip to carefully mix by pipetting;
9)上机检测。9) On-machine testing.
实施例1，iGDP扩增培养基的配制Example 1, Preparation of iGDP amplification medium
iGDP扩增培养基1-48的组成及比例如表1所示，其中培养基StemSpanTM SFEM、StemSpanTM SFEM II、StemSpanTM-XF、StemSpanTM-AOF、IMDM、DMEM/F-12和Neurobasal的用量单位为v/v，人血白蛋白用量单位为g/L，胰岛素、转铁蛋白、DL-α-生育酚和亚油酸的用量单位为mg/L，***钠的用量单位为μg/L，SCF、GM-CSF、G-CSF、IL-3和FLT-3L的用量单位为μg/L，GDC-0879、Dabrafenib的用量单位为μM，5-furan-2-yl-isoxazole-3-carboxylic acid(2-pyrazol-1-yl-ethyl)-amide(化合物21)、SKL2001的用量单位为μM，嘧啶并吲哚衍生物UM171、UM729的用量单位 为nM。The composition and proportion of iGDP amplification medium 1-48 are shown in Table 1, in which the culture medium StemSpan TM SFEM, StemSpan TM SFEM II, StemSpan TM -XF, StemSpan TM -AOF, IMDM, DMEM/F-12 and Neurobasal The dosage unit is v/v, the dosage unit of human albumin is g/L, the dosage unit of insulin, transferrin, DL-α-tocopherol and linoleic acid is mg/L, the dosage unit of sodium selenite is μg/L, the dosage unit of SCF, GM-CSF, G-CSF, IL-3 and FLT-3L is μg/L, the dosage unit of GDC-0879 and Dabrafenib is μM, 5-furan-2-yl-isoxazole- The dosage units of 3-carboxylic acid(2-pyrazol-1-yl-ethyl)-amide (compound 21) and SKL2001 are μM, and the dosage units of pyrimidinoindole derivatives UM171 and UM729 are is nM.
详见下表1。

See Table 1 below for details.

表1中未写用量的即用量为0。The ready-to-use quantity without written quantity in Table 1 is 0.
实施例2，血液细胞样本的采集Example 2, collection of blood cell samples
本发明中血液细胞样品来源于非动员外周血，供者包含18-70岁健康人群，性别不限。采用Ficoll工艺分离非动员外周血CD45+细胞，以新鲜细胞和冻存再复苏细胞为起始细胞进行扩增。样本8和9为样本6磁珠分选后的CD14+单核细胞和CD14-非单核细胞，样本10和11为样本7为磁珠分选后的CD123+和CD123-细胞，样本12为外周细胞磁珠分选后的CD34-细胞。The blood cell samples in the present invention are derived from non-mobilized peripheral blood, and the donors include healthy people aged 18-70 years old, with no gender limit. Non-mobilized peripheral blood CD45+ cells were isolated using the Ficoll process, and fresh cells and cryopreserved and resuscitated cells were used as starting cells for expansion. Samples 8 and 9 are CD14 + monocytes and CD14 - non-monocytes separated by magnetic beads in sample 6. Samples 10 and 11 are CD123 + and CD123 - cells separated by magnetic beads in sample 7. Sample 12 is CD34- cells after peripheral cell magnetic bead sorting.
详见下表2。
See Table 2 below for details.
实施例3，iGDP扩增培养基配方研究-不同培养基培养样品1第7/14天的细胞扩增倍数Example 3, iGDP amplification medium formulation research - cell expansion multiples on days 7/14 of sample 1 cultured in different media
以Ficoll工艺分离的机采外周血细胞样本1为起始细胞，使用iGDP培养基1-9为受试培养基，采集培养第0天和第7/14天的活细胞数。Use machine-collected peripheral blood cell sample 1 separated by the Ficoll process as the starting cells, use iGDP medium 1-9 as the test medium, and collect the number of viable cells on day 0 and day 7/14 of culture.
实施例4，iGDP扩增培养基配方研究-不同培养基培养样品2-5第14天的细胞扩增倍数Example 4, iGDP amplification medium formulation research - cell expansion multiples on the 14th day of samples 2-5 cultured in different media
以Ficoll工艺分离的手采外周血细胞为起始细胞，使用iGDP培养基11-27为受试培养基，采集培养第0天和第14天的活细胞数。Hand-collected peripheral blood cells separated by the Ficoll process were used as starting cells, iGDP medium 11-27 was used as the test medium, and the number of viable cells on days 0 and 14 of culture was collected.
由图1以看出，使用iGDP扩增培养基11-27培养14天后，细胞扩增倍数为 0.32±0.15至4.94±1.19(数据为样本2-5的平均值和标准差)。As can be seen from Figure 1, after 14 days of culture using iGDP amplification medium 11-27, the cell expansion fold is 0.32±0.15 to 4.94±1.19 (data are the mean and standard deviation of samples 2-5).
实施例5，iGDP扩增培养基配方研究-不同培养基培养样品6-7第14天的细胞扩增倍数Example 5, iGDP amplification medium formulation study - cell expansion multiples on days 14 of culture samples 6-7 in different media
以Ficoll工艺分离的机采外周血细胞为起始细胞，使用iGDP培养基15、23、24、28-32为受试培养基，采集培养第0天和第14天的活细胞数。Machine-collected peripheral blood cells isolated by the Ficoll process were used as the starting cells, and iGDP medium 15, 23, 24, and 28-32 were used as the test medium, and the number of viable cells on the 0th and 14th days of culture was collected.
由图2可以看出，使用iGDP扩增培养基15、23、24、28-32培养14天后，细胞扩增倍数为11.83±1.36至43.23±5.34(数据为样本6-7的平均值和标准差)。As can be seen from Figure 2, after 14 days of culture using iGDP amplification medium 15, 23, 24, 28-32, the cell expansion fold ranged from 11.83±1.36 to 43.23±5.34 (the data are the average and standard values of samples 6-7 Difference).
实施例6，iGDP长期扩增能力研究-不同培养基培养样品7第28天的细胞扩增倍数Example 6, iGDP long-term amplification ability study - cell expansion times of sample 7 cultured in different media on the 28th day
以Ficoll工艺分离的机采外周血细胞样本7为起始细胞，使用iGDP培养基15、24、30为受试培养基，起始细胞接种数量为1E6个活细胞，培养第28天进行活细胞计数，收集细胞活率和细胞直径数据。The machine-collected peripheral blood cell sample 7 separated by the Ficoll process was used as the starting cells, and iGDP medium 15, 24, and 30 were used as the test medium. The starting cell inoculation number was 1E6 viable cells, and viable cells were counted on the 28th day of culture. , collecting cell viability and cell diameter data.
由表3可以看出，样本7在iGDP扩增培养基15中扩增能力最优，培养28天扩增197倍且细胞活率维持在85.8％，其次是培养基30和24，培养28天扩增分别扩增194倍和83倍，细胞直径范围为11.8-12.1μm。As can be seen from Table 3, sample 7 has the best amplification ability in iGDP amplification medium 15, which expanded 197 times after 28 days of culture and maintained cell viability at 85.8%, followed by culture media 30 and 24, which lasted 28 days. The amplifications were 194-fold and 83-fold, respectively, and the cell diameter ranged from 11.8 to 12.1 μm.
详见下表3。See Table 3 below for details.
表3
table 3
实施例7，iGDP扩增起始细胞来源研究-CD45+细胞、CD14+单核细胞、CD14-非单核细胞、CD123+细胞、CD123-细胞和CD34-细胞起始细胞培养Example 7, iGDP amplification starting cell source study - CD45 + cells, CD14 + monocytes, CD14 - non-monocytes, CD123 + cells, CD123 - cells and CD34 - cells starting cell culture
分别以Ficoll工艺分离的机采外周血CD45+细胞(样品7)、Ficoll工艺分离结合磁珠分选获得的CD14+单核细胞(样品8)、CD14-非单核细胞(样品9)、CD123+细胞(样品10)、CD123-细胞(样品11)和CD34-细胞(样品12)为起始细胞，使用iGDP培养基15接种，采集培养第14天的iGDP细胞光镜图片。Machine-collected peripheral blood CD45 + cells separated by the Ficoll process (sample 7), CD14 + monocytes separated by the Ficoll process combined with magnetic bead sorting (sample 8), CD14 - non-monocytes (sample 9), CD123 + cells (sample 10), CD123 - cells (sample 11) and CD34 - cells (sample 12) were used as starting cells, inoculated using iGDP medium 15, and light microscope pictures of iGDP cells on the 14th day of culture were collected.
由图4可以看出，CD45+细胞、CD14+单核细胞、CD14-非单核细胞、CD123+细 胞、CD123-细胞和CD34-细胞均可扩增获得iGDP细胞，iGDP细胞悬浮或贴壁生长、细胞呈圆球形、大小较均一。As can be seen from Figure 4, CD45 + cells, CD14 + monocytes, CD14 - non-monocytes, CD123 + cells Cells, CD123 - cells and CD34 - cells can all be expanded to obtain iGDP cells. iGDP cells grow in suspension or adherent, and the cells are spherical in shape and relatively uniform in size.
实施例8，iGDP衍生DC细胞得率和流式表征检测Example 8, iGDP-derived DC cell yield and flow cytometric characterization detection
以iGDP细胞为分化起始细胞，根据本发明中树突状细胞分化方法进行定向分化和成熟，分化成熟后进行得率(分化细胞数/初始PBMC细胞数*CD11c+细胞占比)和细胞流式表征检测。Using iGDP cells as the starting cells for differentiation, directional differentiation and maturation are carried out according to the dendritic cell differentiation method of the present invention. After differentiation and maturity, the yield (number of differentiated cells/number of initial PBMC cells*CD11c + cell proportion) and cell flow Formula characterization detection.
由表4可以看出，样本7在iGDP扩增培养基15、24和30中的DC细胞得率分别为138.05倍、75.42倍和132.64倍。As can be seen from Table 4, the DC cell yields of sample 7 in iGDP amplification media 15, 24 and 30 were 138.05 times, 75.42 times and 132.64 times respectively.
详见下表4。
See Table 4 below for details.
由表5可以看出，由不同培养基获得的iGDP细胞可高效分化为DC细胞，其中培养基24获得的iGDP细胞向DC细胞分化效率最高，CD11c+细胞占比为85.70％、HLA-DR+细胞占比为56.04％、CD40+细胞占比为79.91％、CD8+细胞占比为64.83％。As can be seen from Table 5, iGDP cells obtained from different culture media can efficiently differentiate into DC cells. Among them, iGDP cells obtained from culture medium 24 have the highest differentiation efficiency into DC cells, with CD11c + cells accounting for 85.70% and HLA-DR + The proportion of cells was 56.04%, the proportion of CD40 + cells was 79.91%, and the proportion of CD8 + cells was 64.83%.
详见下表5。
See Table 5 below for details.
另，培养基15获得的iGDP衍生DC细胞流式表征检测结果如图4所示，iGDP衍生DC细胞CD11c+占比为75.77％，HLA-DR+占比为30.29％，CD40+占比为72.12％，CD80+占比为75.28％，表明iGDP具有DC分化潜能。 In addition, the flow cytometric characterization test results of iGDP-derived DC cells obtained in culture medium 15 are shown in Figure 4. The CD11c + proportion of iGDP-derived DC cells was 75.77%, the HLA-DR + proportion was 30.29%, and the CD40 + proportion was 72.12 %, CD80 + accounted for 75.28%, indicating that iGDP has DC differentiation potential.
实施例9，不同iGDP扩增培养基(培养基33-48)培养样品13、14第14天的相对细胞扩增倍数Example 9, relative cell amplification times of samples 13 and 14 cultured in different iGDP amplification media (medium 33-48) on the 14th day
以Ficoll工艺分离的手采外周血细胞为起始细胞，使用iGDP扩增培养基24，33-48为受试培养基，采集培养第0天和第14天的活细胞数，其中培养基24作为对照组，用以计算培养基33-48的相对细胞扩增倍数(即培养基33-48的细胞扩增倍数除以培养基24的细胞扩增倍数)。Hand-collected peripheral blood cells separated by the Ficoll process were used as starting cells, iGDP amplification media 24 and 33-48 were used as test media, and the number of viable cells on days 0 and 14 of culture was collected, with culture medium 24 as The control group is used to calculate the relative cell expansion multiple of culture medium 33-48 (that is, the cell expansion multiple of culture medium 33-48 divided by the cell expansion multiple of culture medium 24).
由图5可以看出，培养基44效果最优，培养基37与培养基24的效果相当，优于其他培养基。培养基45-48仅含有无血清培养基和生长因子，与其他培养基相比，扩增效果较差。(数据为样品13-14的平均值和标准差)It can be seen from Figure 5 that culture medium 44 has the best effect, and culture medium 37 has the same effect as culture medium 24 and is better than other culture media. Medium 45-48 contains only serum-free medium and growth factors, which results in poorer amplification compared to other media. (Data is the mean and standard deviation of samples 13-14)
实施例10，不同iGDP扩增培养基(培养基33-44)获得的iGDP诱导分化树突细胞的相对细胞数量Example 10, Relative cell numbers of iGDP-induced differentiated dendritic cells obtained from different iGDP amplification media (Medium 33-44)
以Ficoll工艺分离的手采外周血细胞为起始细胞，使用iGDP扩增培养基24，33-44为受试培养基获得iGDP，诱导iGDP向树突细胞分化，使用流式检测分化第13天的树突细胞数量，其中培养基24作为对照组，用以计算培养基33-44的相对细胞数量(即培养基33-44的树突细胞数量除以培养基24的树突细胞数量)。Hand-collected peripheral blood cells isolated by the Ficoll process were used as starting cells, iGDP amplification media 24 and 33-44 were used as test media to obtain iGDP, iGDP was induced to differentiate into dendritic cells, and flow cytometry was used to detect differentiation on the 13th day. The number of dendritic cells, with medium 24 as the control group, was used to calculate the relative cell number of medium 33-44 (ie, the number of dendritic cells in medium 33-44 divided by the number of dendritic cells in medium 24).
由图6可以看出，iGDP扩增培养基33-44获得的iGDP，经过诱导分化后，均可获得较多数量的DC细胞。(数据来源为样品14)As can be seen from Figure 6, a larger number of DC cells can be obtained from iGDP obtained from iGDP amplification medium 33-44 after induction of differentiation. (Data source is sample 14)
实施例11，不同iGDP扩增培养基(培养基38-44)获得的iGDP衍生树突细胞表征鉴定Example 11, Characterization and identification of iGDP-derived dendritic cells obtained from different iGDP amplification media (medium 38-44)
以iGDP细胞为分化起始细胞，根据本发明中树突状细胞分化方法进行定向分化和成熟，分化成熟后进行表征鉴定。由表6可以看出，不同培养基获得的iGDP细胞均可高效分化为DC细胞。Using iGDP cells as the starting cells for differentiation, directional differentiation and maturation are carried out according to the dendritic cell differentiation method of the present invention, and characterization and identification are performed after differentiation and maturity. As can be seen from Table 6, iGDP cells obtained from different media can be efficiently differentiated into DC cells.
与培养基38相比，培养基39将异恶唑衍生物化合物21替换为了SKL2001，虽然两者在扩增和分化阶段效果相当，但在分化成熟后的表征鉴定中，CD11c+细胞占比存在区别，培养基38的CD11c+细胞占比为71.95％，培养基39的CD11c+细胞占比为26.07％. Compared with culture medium 38, culture medium 39 replaces the isoxazole derivative compound 21 with SKL2001. Although the two are equally effective in the expansion and differentiation stages, there is a difference in the proportion of CD11c+ cells in the characterization and identification after differentiation and maturity. , the proportion of CD11c+ cells in culture medium 38 was 71.95%, and the proportion of CD11c+ cells in culture medium 39 was 26.07%.
详见下表6。
See Table 6 below for details.

Claims (34)

  1. 一种制备树突细胞祖细胞的方法，其特征在于，将来自外周血的细胞在培养基中培养，获得树突细胞祖细胞。A method for preparing dendritic cell progenitor cells, characterized by culturing cells from peripheral blood in a culture medium to obtain dendritic cell progenitor cells.
  2. 根据权利要求1所述的方法，其特征在于，所述培养基含有生长因子、Raf/MEK/ERK通路调节剂、异恶唑衍生物中的至少两种。The method of claim 1, wherein the culture medium contains at least two of growth factors, Raf/MEK/ERK pathway regulators, and isoxazole derivatives.
  3. 根据权利要求2所述的方法，其特征在于，所述生长因子选自SCF、GM-CSF、G-CSF、IL-3、FLT-3L中的至少一种，更优选地，所述生长因子含有SCF以及选自GM-CSF、G-CSF、IL-3、FLT-3L中的至少一种。The method according to claim 2, wherein the growth factor is selected from at least one of SCF, GM-CSF, G-CSF, IL-3, and FLT-3L. More preferably, the growth factor Contains SCF and at least one selected from GM-CSF, G-CSF, IL-3, and FLT-3L.
  4. 根据权利要求3所述的方法，其特征在于，The method according to claim 3, characterized in that:
    SCF的浓度为10-150ug/L，优选为30-120ug/L；The concentration of SCF is 10-150ug/L, preferably 30-120ug/L;
    GM-CSF的浓度为0-80ug/L，优选为0-50ug/L；The concentration of GM-CSF is 0-80ug/L, preferably 0-50ug/L;
    G-CSF的浓度为0-200ug/L，优选为0-150ug/L；The concentration of G-CSF is 0-200ug/L, preferably 0-150ug/L;
    IL-3的浓度为0-80ug/L，优选为0-50ug/L；和/或The concentration of IL-3 is 0-80ug/L, preferably 0-50ug/L; and/or
    FLT-3L的浓度为0-80ug/L，优选为0-50ug/L。The concentration of FLT-3L is 0-80ug/L, preferably 0-50ug/L.
  5. 根据权利要求2-4中任一项所述的方法，其特征在于，所述Raf/MEK/ERK通路调节剂为MEK激活剂和/或ERK激活剂。The method according to any one of claims 2 to 4, characterized in that the Raf/MEK/ERK pathway modulator is a MEK activator and/or an ERK activator.
  6. 根据权利要求5所述的方法，其特征在于，所述Raf/MEK/ERK通路调节剂选自GDC-0879、PLX4720、Vemurafenib、Sorafenib、Dabrafenib中的一种或几种，优选为GDC-0879。The method according to claim 5, characterized in that the Raf/MEK/ERK pathway modulator is selected from one or more of GDC-0879, PLX4720, Vemurafenib, Sorafenib, and Dabrafenib, preferably GDC-0879.
  7. 根据权利要求2-6中任一项所述的方法，其特征在于，所述Raf/MEK/ERK通路调节剂的浓度为0.1-5μM，优选为0.5-1.5μM。The method according to any one of claims 2-6, characterized in that the concentration of the Raf/MEK/ERK pathway modulator is 0.1-5 μM, preferably 0.5-1.5 μM.
  8. 根据权利要求2-7中任一项所述的方法，其特征在于，所述异恶唑衍生物为式(I)所示的化合物：
    The method according to any one of claims 2-7, characterized in that the isoxazole derivative is a compound represented by formula (I):
    其中，R1为未取代或取代的杂芳基，Wherein, R 1 is unsubstituted or substituted heteroaryl,
    m为0-4的整数，优选为1-3的整数， m is an integer from 0 to 4, preferably an integer from 1 to 3,
    X为CH2、NH或O，X is CH 2 , NH or O,
    R2为未取代或取代的杂芳基；R 2 is unsubstituted or substituted heteroaryl;
    优选地，所述异恶唑衍生物为式1-21中任一所示的化合物；Preferably, the isoxazole derivative is a compound represented by any one of formulas 1-21;
    更优选地，所述异恶唑衍生物为5-呋喃-2-基-异恶唑-3-羧酸(2-吡唑-1-基-乙基)-酰胺。More preferably, the isoxazole derivative is 5-furan-2-yl-isoxazole-3-carboxylic acid (2-pyrazol-1-yl-ethyl)-amide.
  9. 根据权利要求2-8中任一项所述的方法，其特征在于，所述异恶唑衍生物的浓度为0-15μM，优选为0-10μM，更优选为1-10μM，进一步优选为4-6μM。The method according to any one of claims 2 to 8, characterized in that the concentration of the isoxazole derivative is 0-15 μM, preferably 0-10 μM, more preferably 1-10 μM, further preferably 4 -6μM.
  10. 根据权利要求2-9中任一项所述的方法，其特征在于，所述培养基还含有嘧啶并吲哚衍生物，优选地，所述嘧啶并吲哚衍生物如式(II)所示，
    The method according to any one of claims 2 to 9, characterized in that the culture medium further contains a pyrimidinoindole derivative. Preferably, the pyrimidinoindole derivative is represented by formula (II) ,
    其中，Ra选自未取代或取代的C1-C4烷基、未取代或取代的杂芳基、RdOC(＝O)-、ReNHC(＝O)-，其中Rd为未取代或取代的C1-C4烷基，Re为未取代或取代的C1-C4烷基；Wherein, R a is selected from unsubstituted or substituted C1-C4 alkyl, unsubstituted or substituted heteroaryl, R d OC(=O)-, R e NHC(=O)-, where R d is unsubstituted Or substituted C1-C4 alkyl, R e is unsubstituted or substituted C1-C4 alkyl;
    Rb选自-NRfRg、-ORh，其中，Rf、Rg、Rh各自独立选自H、C1-C4烷基、-(CH2)nRi，其中，n为0-4的整数，Ri选自未取代或取代的C1-C4烷基、未取代或取代的环烷基、未取代或取代的杂环基、未取代或取代的芳基、未取代或取代的杂芳基、-NRjRk、-CN，其中Rj、Rk各自独立选自未取代或取代的C1-C4烷基；R b is selected from -NR f R g , -OR h , where R f , R g , and R h are each independently selected from H, C1-C4 alkyl, -(CH 2 ) n R i , where n is 0 -4 is an integer, R i is selected from unsubstituted or substituted C1-C4 alkyl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclyl, unsubstituted or substituted aryl, unsubstituted or substituted Heteroaryl, -NR j R k , -CN, wherein R j and R k are each independently selected from unsubstituted or substituted C1-C4 alkyl;
    Rc选自H、-(CH2)iRp，其中，i为0-4的整数，Rp选自未取代或取代的C1-C4烷基、未取代或取代的环烷基、未取代或取代的杂环基、未取代或取代的芳基、未取代或取代的杂芳基、-NRjRk、-CN，其中Rj、Rk各自独立选自未取代或取代的C1-C4烷基；R c is selected from H, -(CH 2 ) i R p , where i is an integer from 0 to 4, and R p is selected from unsubstituted or substituted C1-C4 alkyl, unsubstituted or substituted cycloalkyl, unsubstituted Substituted or substituted heterocyclyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, -NR j R k , -CN, wherein R j and R k are each independently selected from unsubstituted or substituted C1 -C4 alkyl;
    更优选地，所述嘧啶并吲哚衍生物为式22-44中任一所示的化合物；More preferably, the pyrimidinoindole derivative is a compound represented by any one of formulas 22-44;
    进一步优选地，所述嘧啶并吲哚衍生物选自UM171和UM729中的一种或两种，更进一步优选地，所述嘧啶并吲哚衍生物为UM171。 It is further preferred that the pyrimidinoindole derivative is selected from one or both of UM171 and UM729. It is further preferred that the pyrimidoindole derivative is UM171.
  11. 根据权利要求10所述的方法，其特征在于，所述嘧啶并吲哚衍生物的浓度为0-80nM，优选为0-50nM。The method according to claim 10, characterized in that the concentration of the pyrimidoindole derivative is 0-80 nM, preferably 0-50 nM.
  12. 根据权利要求2-11中任一项所述的方法，其特征在于，所述培养基还含有基础培养基，例如基础无血清培养基；优选地，所述基础培养基包括商业无血清培养基或补充有添加物的基本培养基。The method according to any one of claims 2 to 11, characterized in that the culture medium also contains a basal culture medium, such as a basal serum-free culture medium; preferably, the basal culture medium includes a commercial serum-free culture medium or minimal medium supplemented with additives.
  13. 根据权利要求12中任一项所述的方法，其特征在于，The method according to any one of claims 12, characterized in that:
    所述商业无血清培养基选自StemSpanTMSFEM培养基、StemSpanTMSFEM II培养基、StemSpanTM-XF培养基、StemSpanTM-AOF培养基、StemProTM-34 SFM中的一种或多种，The commercial serum-free medium is selected from one or more of StemSpan TM SFEM medium, StemSpan TM SFEM II medium, StemSpan TM -XF medium, StemSpan TM -AOF medium, and StemPro TM -34 SFM,
    所述基本培养基选自IMDM培养基、DMEM/F-12培养基、Neurobasal培养基、AIM-V中的一种或多种，The basic medium is selected from one or more of IMDM medium, DMEM/F-12 medium, Neurobasal medium, and AIM-V,
    所述补充添加物选自人血白蛋白、胰岛素、转铁蛋白、***钠、DL-α-生育酚、亚油酸中的一种或多种。The supplementary additive is selected from one or more of human albumin, insulin, transferrin, sodium selenite, DL-α-tocopherol, and linoleic acid.
  14. 根据权利要求13所述的方法，其特征在于，The method according to claim 13, characterized in that:
    人血白蛋白的浓度为1-10g/L，优选为1g/L-5g/L；The concentration of human serum albumin is 1-10g/L, preferably 1g/L-5g/L;
    胰岛素浓度为1-20mg/L，优选为3-15mg/L；The insulin concentration is 1-20 mg/L, preferably 3-15 mg/L;
    转铁蛋白的浓度为1-30mg/L，优选为3-25mg/L；The concentration of transferrin is 1-30 mg/L, preferably 3-25 mg/L;
    ***钠的浓度为1-20ug/L，优选为5-15ug/L；The concentration of sodium selenite is 1-20ug/L, preferably 5-15ug/L;
    DL-α-生育酚的浓度为0.5-10mg/L，优选为0.5-8mg/L；和/或The concentration of DL-α-tocopherol is 0.5-10 mg/L, preferably 0.5-8 mg/L; and/or
    亚油酸的浓度为0.5-10mg/L，优选为0.5-5mg/L。The concentration of linoleic acid is 0.5-10 mg/L, preferably 0.5-5 mg/L.
  15. 根据权利要求1-14中任一项所述的方法，所述外周血为未经造血干细胞动员的外周血。The method according to any one of claims 1-14, wherein the peripheral blood is peripheral blood without hematopoietic stem cell mobilization.
  16. 根据权利要求1-15中任一项所述的方法，其特征在于，所述细胞选自CD45+白细胞、CD14+细胞、CD14-细胞、CD123+细胞、CD123-细胞、CD34-细胞中的一种或多种。The method according to any one of claims 1-15, wherein the cells are selected from one of CD45 + leukocytes, CD14 + cells, CD14- cells, CD123 + cells, CD123- cells, and CD34- cells. Kind or variety.
  17. 一种培养基，其特征在于，用于从外周血制备树突细胞祖细胞，所述培养基含有生长因子、Raf/MEK/ERK通路调节剂、异恶唑衍生物中的至少两种。A culture medium, characterized in that it is used to prepare dendritic cell progenitor cells from peripheral blood, and the culture medium contains at least two of growth factors, Raf/MEK/ERK pathway regulators, and isoxazole derivatives.
  18. 根据权利要求17所述的培养基，其特征在于，所述生长因子选自 SCF、GM-CSF、G-CSF、IL-3、FLT-3L中的至少一种，更优选地，所述生长因子含有SCF以及选自GM-CSF、G-CSF、IL-3、FLT-3L中的至少一种。The culture medium according to claim 17, wherein the growth factor is selected from the group consisting of At least one of SCF, GM-CSF, G-CSF, IL-3, and FLT-3L. More preferably, the growth factor contains SCF and is selected from the group consisting of GM-CSF, G-CSF, IL-3, and FLT-3L. At least one of 3L.
  19. 根据权利要求18所述的培养基，其特征在于，The culture medium according to claim 18, characterized in that,
    SCF的浓度为10-150ug/L，优选为30-120ug/L；The concentration of SCF is 10-150ug/L, preferably 30-120ug/L;
    GM-CSF的浓度为0-80ug/L，优选为0-50ug/L；The concentration of GM-CSF is 0-80ug/L, preferably 0-50ug/L;
    G-CSF的浓度为0-200ug/L，优选为0-150ug/L；The concentration of G-CSF is 0-200ug/L, preferably 0-150ug/L;
    IL-3的浓度为0-80ug/L，优选为0-50ug/L；和/或The concentration of IL-3 is 0-80ug/L, preferably 0-50ug/L; and/or
    FLT-3L的浓度为0-80ug/L，优选为0-50ug/L。The concentration of FLT-3L is 0-80ug/L, preferably 0-50ug/L.
  20. 根据权利要求17-19中任一项所述的培养基，其特征在于，所述Raf/MEK/ERK通路调节剂为MEK激活剂和/或ERK激活剂。The culture medium according to any one of claims 17-19, wherein the Raf/MEK/ERK pathway regulator is a MEK activator and/or an ERK activator.
  21. 根据权利要求20所述的培养基，其特征在于，所述Raf/MEK/ERK通路调节剂选自GDC-0879、PLX4720、Vemurafenib(PLX4032)、Sorafenib(BAY 43-9006)、Dabrafenib(GSK2118436)中的一种或几种，优选为GDC-0879。The culture medium according to claim 20, characterized in that the Raf/MEK/ERK pathway regulator is selected from GDC-0879, PLX4720, Vemurafenib (PLX4032), Sorafenib (BAY 43-9006), Dabrafenib (GSK2118436) One or more of them, preferably GDC-0879.
  22. 根据权利要求17-21中任一项所述的培养基，其特征在于，所述Raf/MEK/ERK通路调节剂的浓度为0.1-5μM，优选为0.5-1.5μM。The culture medium according to any one of claims 17-21, wherein the concentration of the Raf/MEK/ERK pathway modulator is 0.1-5 μM, preferably 0.5-1.5 μM.
  23. 根据权利要求17-22中任一项所述的培养基，其特征在于，The culture medium according to any one of claims 17-22, characterized in that,
    所述异恶唑衍生物为式(I)所示的化合物：
    The isoxazole derivative is a compound represented by formula (I):
    其中，R1为未取代或取代的杂芳基，Wherein, R 1 is unsubstituted or substituted heteroaryl,
    m为0-4的整数，优选为1-3的整数，m is an integer from 0 to 4, preferably an integer from 1 to 3,
    X为CH2、NH或O，X is CH 2 , NH or O,
    R2为未取代或取代的杂芳基；R 2 is unsubstituted or substituted heteroaryl;
    优选地，所述异恶唑衍生物为式1-21中任一所示的化合物；Preferably, the isoxazole derivative is a compound represented by any one of formulas 1-21;
    更优选地，所述异恶唑衍生物为5-呋喃-2-基-异恶唑-3-羧酸(2-吡唑-1-基-乙基)-酰胺。More preferably, the isoxazole derivative is 5-furan-2-yl-isoxazole-3-carboxylic acid (2-pyrazol-1-yl-ethyl)-amide.
  24. 根据权利要求17-23中任一项所述的培养基，其特征在于，所述异 恶唑衍生物的浓度为0-15μM，优选为0-10μM，更优选为1-10μM，进一步优选为4-6μM。The culture medium according to any one of claims 17-23, characterized in that the heterogeneous The concentration of the oxazole derivative is 0-15 μM, preferably 0-10 μM, more preferably 1-10 μM, further preferably 4-6 μM.
  25. 根据权利要求17-24中任一项所述的培养基，其特征在于，The culture medium according to any one of claims 17-24, characterized in that,
    所述培养基还含有嘧啶并吲哚衍生物，优选地，所述嘧啶并吲哚衍生物如式(II)所示，
    The culture medium also contains a pyrimidinoindole derivative. Preferably, the pyrimidinoindole derivative is represented by formula (II),
    其中，Ra选自未取代或取代的C1-C4烷基、未取代或取代的杂芳基、RdOC(＝O)-、ReNHC(＝O)-，其中Rd为未取代或取代的C1-C4烷基，Re为未取代或取代的C1-C4烷基；Wherein, R a is selected from unsubstituted or substituted C1-C4 alkyl, unsubstituted or substituted heteroaryl, R d OC(=O)-, R e NHC(=O)-, where R d is unsubstituted Or substituted C1-C4 alkyl, R e is unsubstituted or substituted C1-C4 alkyl;
    Rb选自-NRfRg、-ORh，其中，Rf、Rg、Rh各自独立选自H、C1-C4烷基、-(CH2)nRi，其中，n为0-4的整数，Ri选自未取代或取代的C1-C4烷基、未取代或取代的环烷基、未取代或取代的杂环基、未取代或取代的芳基、未取代或取代的杂芳基、-NRjRk、-CN，其中Rj、Rk各自独立选自未取代或取代的C1-C4烷基；R b is selected from -NR f R g , -OR h , where R f , R g , and R h are each independently selected from H, C1-C4 alkyl, -(CH 2 ) n R i , where n is 0 -4 is an integer, R i is selected from unsubstituted or substituted C1-C4 alkyl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclyl, unsubstituted or substituted aryl, unsubstituted or substituted Heteroaryl, -NR j R k , -CN, wherein R j and R k are each independently selected from unsubstituted or substituted C1-C4 alkyl;
    Rc选自H、-(CH2)iRp，其中，i为0-4的整数，Rp选自未取代或取代的C1-C4烷基、未取代或取代的环烷基、未取代或取代的杂环基、未取代或取代的芳基、未取代或取代的杂芳基、-NRjRk、-CN，其中Rj、Rk各自独立选自未取代或取代的C1-C4烷基；R c is selected from H, -(CH 2 ) i R p , where i is an integer from 0 to 4, and R p is selected from unsubstituted or substituted C1-C4 alkyl, unsubstituted or substituted cycloalkyl, unsubstituted Substituted or substituted heterocyclyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, -NR j R k , -CN, wherein R j and R k are each independently selected from unsubstituted or substituted C1 -C4 alkyl;
    更优选地，所述嘧啶并吲哚衍生物为式22-44中任一所示的化合物；More preferably, the pyrimidinoindole derivative is a compound represented by any one of formulas 22-44;
    进一步优选地，所述嘧啶并吲哚衍生物选自UM171和UM729中的一种或两种，更进一步优选地，所述嘧啶并吲哚衍生物为UM171。It is further preferred that the pyrimidinoindole derivative is selected from one or both of UM171 and UM729. It is further preferred that the pyrimidoindole derivative is UM171.
  26. 根据权利要求25所述的培养基，其特征在于，所述嘧啶并吲哚衍生物的浓度为0-80nM，优选为0-50nM。The culture medium according to claim 25, characterized in that the concentration of the pyrimidoindole derivative is 0-80 nM, preferably 0-50 nM.
  27. 根据权利要求17-25中任一项所述的培养基，其特征在于，所述培养基还含有基础培养基，例如基础无血清培养基；优选地，所述基础培养基包括商业无血清培养基或补充有添加物的基本培养基。 The culture medium according to any one of claims 17-25, characterized in that the culture medium also contains a basal culture medium, such as a basal serum-free culture medium; preferably, the basal culture medium includes commercial serum-free culture medium Base or minimal medium supplemented with additives.
  28. 根据权利要求27所述的培养基，其特征在于，The culture medium according to claim 27, characterized in that,
    所述商业无血清培养基选自StemSpanTMSFEM培养基、StemSpanTMSFEM II培养基、StemSpanTM-XF培养基、StemSpanTM-AOF培养基、StemProTM-34 SFM中的一种或多种，The commercial serum-free medium is selected from one or more of StemSpan TM SFEM medium, StemSpan TM SFEM II medium, StemSpan TM -XF medium, StemSpan TM -AOF medium, and StemPro TM -34 SFM,
    所述基本培养基选自IMDM培养基、DMEM/F-12培养基、Neurobasal培养基、AIM-V中的一种或多种，The basic medium is selected from one or more of IMDM medium, DMEM/F-12 medium, Neurobasal medium, and AIM-V,
    所述补充添加物选自人血白蛋白、胰岛素、转铁蛋白、***钠、DL-α-生育酚、亚油酸中的一种或多种。The supplementary additive is selected from one or more of human albumin, insulin, transferrin, sodium selenite, DL-α-tocopherol, and linoleic acid.
  29. 根据权利要求28所述的培养基，其特征在于，The culture medium according to claim 28, characterized in that,
    人血白蛋白的浓度为1-10g/L，优选为1g/L-5g/L；The concentration of human albumin is 1-10g/L, preferably 1g/L-5g/L;
    胰岛素浓度为1-20mg/L，优选为3-15mg/L；The insulin concentration is 1-20 mg/L, preferably 3-15 mg/L;
    转铁蛋白的浓度为1-30mg/L，优选为3-25mg/L；The concentration of transferrin is 1-30 mg/L, preferably 3-25 mg/L;
    ***钠的浓度为1-20ug/L，优选为5-15ug/L；The concentration of sodium selenite is 1-20ug/L, preferably 5-15ug/L;
    DL-α-生育酚的浓度为0.5-10mg/L，优选为0.5-8mg/L；和/或The concentration of DL-α-tocopherol is 0.5-10 mg/L, preferably 0.5-8 mg/L; and/or
    亚油酸的浓度为0.5-10mg/L，优选为0.5-5mg/L。The concentration of linoleic acid is 0.5-10 mg/L, preferably 0.5-5 mg/L.
  30. 一种由权利要求1-16中任一项所述的方法制备的树突细胞祖细胞。A dendritic cell progenitor cell prepared by the method of any one of claims 1-16.
  31. 一种树突细胞的制备方法，其特征在于，将权利要求30所述的树突细胞祖细胞在树突细胞分化培养基中培养，获得树突细胞。A method for preparing dendritic cells, characterized in that the dendritic cell progenitor cells described in claim 30 are cultured in a dendritic cell differentiation medium to obtain dendritic cells.
  32. 一种由权利要求31所述的制备方法制备的树突细胞。A dendritic cell prepared by the preparation method of claim 31.
  33. 权利要求30所述的树突细胞祖细胞或权利要求32所述的树突细胞在制备用于药物中的用途，优选地，所述药物用于治疗癌症、感染疾病和/或衰老相关疾病。The use of the dendritic cell progenitor cells according to claim 30 or the dendritic cells according to claim 32 in the preparation of medicines. Preferably, the medicines are used to treat cancer, infectious diseases and/or aging-related diseases.
  34. 一种药物组合物，包含有效量的权利要求30所述的树突细胞祖细胞和/或权利要求32所述的树突细胞，以及药学上可接受的辅料。 A pharmaceutical composition comprising an effective amount of the dendritic cell progenitor cells of claim 30 and/or the dendritic cells of claim 32, and pharmaceutically acceptable excipients.
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