CN117625531A

CN117625531A - Preparation method of dendritic cell progenitor cells and culture medium thereof

Info

Publication number: CN117625531A
Application number: CN202211049984.3A
Authority: CN
Inventors: 佟曼; 马丽; 向葱; 崔晓燕; 钱其军
Original assignee: Shanghai Cell Therapy Group Co Ltd; Maxirna Shanghai Pharmaceutical Co Ltd; Maxirna Zhejiang Technology Co Ltd
Current assignee: Shanghai Cell Therapy Group Co Ltd; Maxirna Shanghai Pharmaceutical Co Ltd; Maxirna Zhejiang Technology Co Ltd
Priority date: 2022-08-30
Filing date: 2022-08-30
Publication date: 2024-03-01
Also published as: WO2024045890A1

Abstract

The invention relates to a preparation method of dendritic cell progenitor cells and a culture medium thereof, in particular to a method for preparing dendritic cell progenitor cells, which comprises the following steps: cells from peripheral blood are cultured in a medium to obtain dendritic cell progenitors. The method can rapidly and accurately obtain dendritic cell progenitor cells from peripheral blood.

Description

Preparation method of dendritic cell progenitor cells and culture medium thereof

Technical Field

The invention relates to the technical field of cell culture. And more particularly to a method for obtaining a large amount of iGDP (individual general dendritic cell progenitor, dendritic cell group cells) from peripheral blood and application thereof.

Background

The immune system, including innate immunity and adaptive immunity, is an important protective barrier for humans. Dendritic Cells (DCs) are the most powerful professional antigen presenting cells (Antigen presenting cells, APC), which are the bridge between innate and adaptive immunity. Dendritic cells can activate initial T cells and control the in vivo immune response reaction process, and are central links of anti-tumor immune response. Immunotherapy with dendritic cells can induce a tumor-specific, sustained immune response in the patient's body.

Clinical application of dendritic cell vaccines has made great progress. By day 7 and 25 of 2022, clinical trail Clinical data registration platform managed by national institutes of health shows that 1183 Dendritic cell therapeutic Clinical trials are registered with "Dendritic Cells" as a keyword, and 27 Dendritic cell therapeutic Clinical trials in Clinical III/IV stage are mainly indicated by melanoma, renal cancer, prostate cancer and the like, leukemia, HIV infection and the like. The autologous prostate cancer vaccine Sipuleucel-T (profnge) based on dendritic cell vaccine in 2010 was approved by the FDA for market, the first-line dendritic cell therapeutic tumor vaccine worldwide.

Clinical application of dendritic cell vaccines is also facing significant challenges. The clinical efficacy of dendritic cell vaccine products is closely related to the dosage of dendritic cells. In vivo, dendritic cells are derived from intramedullary CD34 ⁺ Hematopoietic stem cells (hematopoietic stem cell, HSCs) develop. In vitro, 90% of the dendritic cell vaccine product was derived from peripheral blood mononuclear cells (monocyte), 10% was derived from cord blood/mobilized peripheral bloodCD34 ⁺ Hematopoietic stem cells, however, are not available in sufficient numbers. For example, using Peripheral Blood Mononuclear Cells (PBMC) to isolate monocyte differentiation to obtain a dendritic cell technology route, the dendritic cell yield is only about 10%: 1.3E8 monocytes (13%) and 1.3E8 monocytes can differentiate to obtain 1E8 dendritic cells (80%) per 1E9 peripheral blood mononuclear cells. Such as using cord blood/mobilizing CD34 in peripheral blood ⁺ The technological route of obtaining dendritic cells by expanding and differentiating hematopoietic stem cells has the dendritic cell yield of only about 15 percent: 1E7 CD34 can be separated from 1E9 cord blood/mobilized peripheral blood mononuclear cells ⁺ Hematopoietic stem cells (1%), 1E7 CD34 ⁺ Hematopoietic stem cells can be expanded in vitro by about 20-fold to 2E8 cells, and 2E8 cells can differentiate to obtain 1.5E8 dendritic cells (75%).

Since dendritic cells have no expansion capacity, how to efficiently obtain a sufficient number of dendritic cell progenitors is of substantial significance for future immunotherapy.

Disclosure of Invention

The first aspect of the present invention provides a method for preparing dendritic cell progenitors comprising the steps of: cells from peripheral blood are cultured in a medium to obtain dendritic cell progenitors.

In one or more embodiments, the medium contains at least two of a growth factor, a Raf/MEK/ERK pathway modulator, an isoxazole derivative. Preferably, the medium contains growth factors, raf/MEK/ERK pathway modulators and isoxazole derivatives.

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 comprises SCF and at least one selected from GM-CSF, G-CSF, IL-3, FLT-3L.

In one or more embodiments, the SCF is present at a concentration of 10 to 150ug/L, preferably 30 to 120ug/L.

In one or more embodiments, the concentration of GM-CSF, when present, is in the range of 0 to 80ug/L, preferably 0 to 50ug/L.

In one or more embodiments, the concentration of G-CSF, when present, is in the range of 0 to 200ug/L, preferably 0 to 150ug/L.

In one or more embodiments, the IL-3 concentration is, when present, from 0 to 80ug/L, preferably from 0 to 50ug/L.

In one or more embodiments, the concentration of FLT-3L is, when present, from 0 to 80ug/L, preferably from 0 to 50ug/L.

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 (PLX 4032), sorafenib (BAY 43-9006), dabrafenib (GSK 2118436), more preferably GDC-0879. In one or more embodiments, the Raf/MEK/ERK pathway modulator is at a concentration of 0.1 to 5. Mu.M, preferably 0.5 to 1.5. Mu.M.

In one or more embodiments, the isoxazole derivative is a compound represented by formula (I).

Wherein R is ₁ Is an unsubstituted or substituted heteroaryl group,

m is an integer from 0 to 4, preferably an integer from 1 to 3,

x is CH ₂ Or NH or O, or a combination of a reactive species,

R ₂ is unsubstituted or substituted heteroaryl.

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 to 15. Mu.M, preferably 0 to 10. Mu.M, more preferably 1 to 10. Mu.M.

In one or more embodiments, the medium further contains a pyrimidoindole derivative, preferably the pyrimidoindole derivative is selected from one or both of UM171 and UM729, more preferably the pyrimidoindole derivative is UM171. In one or more embodiments, the pyrimidoindole derivative is present at a concentration of 0-80nM, preferably 0-50nM.

In one or more embodiments, the medium further comprises a basal medium, such as a basal serum-free medium, including commercial serum-free medium and basal medium supplemented with additives.

In one or more embodiments, the commercial serum-free medium is selected from the group consisting of StemSpan ^TM SFEM culture medium, stemSpan ^TM SFEM II Medium, stemSpan ^TM XF medium, stemSpan ^TM -AOF medium, stemPro ^TM -34 one or more of the SFMs.

In one or more embodiments, the minimal medium is selected from one or more of IMDM medium, DMEM/F-12 medium, neurobasal medium, AIM-V; preferably, the supplement additive is selected from one or more of human serum albumin, insulin, transferrin, sodium selenite, DL-alpha-tocopherol, linoleic acid.

In one or more embodiments, the human serum albumin is present at a concentration of from 1 to 10g/L, preferably from 1g/L to 5g/L.

In one or more embodiments, the insulin concentration is comprised between 1 and 20mg/L, preferably between 3 and 15mg/L.

In one or more embodiments, transferrin is present at a concentration of 1 to 30mg/L, preferably 3 to 25mg/L.

In one or more embodiments, sodium selenite is present at a concentration of 1-20ug/L, preferably 5-15ug/L.

In one or more embodiments, the concentration of DL-alpha-tocopherol is, when present, from 0.5 to 10mg/L, preferably from 0.5 to 8mg/L.

In one or more embodiments, the concentration of linoleic acid is, when present, from 0.5 to 10mg/L, preferably from 0.5 to 5mg/L.

In one or more embodiments, the peripheral blood is peripheral blood that is not mobilized by hematopoietic stem cells.

In one or more embodiments, the cells from peripheral blood comprise one or more selected from the group consisting of cd45+ leukocytes, cd14+ cells, CD 14-cells, cd123+ cells, CD 123-cells, CD 34-cells.

The invention also provides a culture composition comprising at least two of a growth factor, a Raf/MEK/ERK pathway modulator, an isoxazole derivative, or a medium comprising the culture composition for preparing dendritic cell progenitors from peripheral blood.

In one or more embodiments, the medium contains a growth factor, a Raf/MEK/ERK pathway modulator, and an isoxazole derivative.

In one or more embodiments, the concentration of SCF in the medium is 10-150ug/L, preferably 30-120ug/L.

In one or more embodiments, the concentration of GM-CSF in the medium is from 0 to 80ug/L, preferably from 0 to 50ug/L.

In one or more embodiments, the concentration of G-CSF in the medium is in the range of 0-200ug/L, preferably 0-150ug/L.

In one or more embodiments, the concentration of IL-3 in the medium is from 0 to 80ug/L, preferably from 0 to 50ug/L.

In one or more embodiments, the concentration of FLT-3L in the medium is 0-80ug/L, preferably 0-50ug/L.

Wherein R1 is unsubstituted or substituted heteroaryl,

m 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.

In one or more embodiments, the concentration ratio of each growth factor, raf/MEK/ERK pathway modulator, isoxazole derivative in the culture composition is the same as the concentration ratio of the corresponding components in the culture medium.

In one or more embodiments, the concentration of human serum albumin is 1-10g/L, preferably 1-5 g/L.

In one or more embodiments, the insulin concentration is 1-20mg/L, preferably 3-15mg/L.

In one or more embodiments, the concentration of transferrin is 1-30mg/L, preferably 3-25mg/L.

In one or more embodiments, the concentration of sodium selenite is 1-20ug/L, preferably 5-15ug/L.

In one or more embodiments, the concentration of DL-alpha-tocopherol is from 0.5 to 10mg/L, preferably from 0.5 to 8mg/L.

In one or more embodiments, the concentration of the linoleic acid is between 0.5 and 10mg/L, preferably between 0.5 and 5mg/L.

The invention also provides the use of a culture composition or medium according to any of the embodiments of the invention for the preparation of a reagent comprising dendritic cell progenitors.

The invention also provides a dendritic cell progenitor cell prepared by the method of any of the embodiments herein.

The invention also provides a preparation method of the dendritic cell, which comprises the following steps: the dendritic cell progenitor cells described in any of the embodiments herein are cultured in a dendritic cell differentiation medium to obtain dendritic cells.

The invention also provides a dendritic cell prepared by the method of preparing a dendritic cell described herein.

The invention also provides the use of dendritic cell progenitors prepared by the methods described in any of the embodiments herein in the preparation of a medicament.

In one or more embodiments, the medicament is for treating cancer, an infectious disease, and/or an aging-related disease.

The invention also provides a pharmaceutical composition comprising an effective amount of dendritic cell progenitor cells prepared according to the method of any of the embodiments herein and a pharmaceutically acceptable adjuvant.

The invention also provides a cell cryopreservation formulation comprising dendritic cell progenitors obtained from the methods described in any of the embodiments herein and a cryopreservation solution.

The invention also provides a method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition as described in any of the embodiments herein.

In some embodiments, the disease or disorder is cancer, an infectious disease, and/or an aging-related disease.

The invention has the beneficial effects that: dendritic cell progenitors (iGDP cells) are expanded from peripheral blood, and are readily available, with high accessibility, and high expansion of iGDP, e.g., 83-197 times the number of iGDP cells in the initial peripheral blood mononuclear cell population.

Drawings

FIG. 1 shows cell expansion on days 14 of samples 2-5 cultured using different iGDP expansion media (media 11-27).

FIG. 2 shows cell expansion on day 14 for samples 6 and 7 cultured using different iGDP expansion media (media 15, 23, 24, 28-32).

Fig. 3 shows morphology of iGDP cells expanded from different cell sources, scale bar=50 μm or 100 μm.

Fig. 4 shows flow-through characterization of the induced high-efficiency differentiation of iGDP into DCs for 14 days, differentiation initiating cells as medium 15 to culture the iGDP cells of sample 4 for 14 days, and control as blank for samples without antibody incubation.

Detailed Description

In order to more clearly illustrate the present invention, the present invention will be further described with reference to preferred embodiments and the accompanying drawings. Like parts in the drawings are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.

The inventors found that dendritic cell progenitors can be obtained by culturing cells derived from peripheral blood. Herein, dendritic cell progenitor cells (iGDP, individual general dendritic cell progenitor) refer to precursor cells that can differentiate to form dendritic cells.

Accordingly, the present invention provides a method of preparing dendritic cell progenitors comprising the steps of: cells from peripheral blood are cultured in a medium to obtain dendritic cell progenitors. The invention also provides a culture composition for preparing dendritic cell progenitor cells from peripheral blood or a culture medium comprising the culture medium composition. The culture composition or medium contains at least two of a growth factor, a Raf/MEK/ERK pathway modulator, and an isoxazole derivative. The invention also provides dendritic cell progenitors prepared by the methods.

The collection method of the peripheral blood is not limited, and the peripheral blood may be singly collected or mechanically collected.

In some embodiments, the peripheral blood is peripheral blood that is not mobilized by hematopoietic stem cells. In some embodiments, the cells from peripheral blood are peripheral blood CD45 ⁺ White blood cells. In an exemplary embodiment, the cells from peripheral blood comprise a polypeptide selected from the group consisting of CD45 ⁺ White blood cells, CD14 ⁺ Cell, CD14 ^- Cell, CD123 ⁺ Cell, CD123 ^- Cells, CD34 ^- One or more of the cells.

Thus, some embodiments of the methods of the invention further comprise the step of obtaining one or more of CD45+ leukocytes, CD14+ cells, CD 14-cells, CD123+ cells, CD 123-cells, CD 34-cells from peripheral blood.

As a method for separating cells from peripheral blood, a method conventional in the art, such as hydroxyethyl starch centrifugation, density gradient centrifugation, immunomagnetic bead separation, flow cytometry separation, etc., can be used. Density gradient centrifugation media include Ficoll-Hypaque, percoll, and the like.

The cells from the peripheral blood may be fresh cells or cells recovered after cryopreservation.

The conditions and time for culturing cells from peripheral blood in the medium can be determined by one skilled in the art as desired, e.g., 37 ℃, 5% co2 for at least 1 day, at least 3 days, at least 7 days, at least 14 days, at least 28 days.

"growth factor" refers to a substance that is effective to promote cell growth and that 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 at least one of stem cell growth factor (SCF), granulocyte-macrophage colony-stimulating factor (granulocyte-macrophage colony stimulating factor, GM-CSF), granulocyte colony-stimulating factor (granulocyte colony stimulating factor, G-CSF), interleukin 3 (Intereukin-3, IL-3), FMS-associated tyrosine kinase 3ligand (FMS-related tyrosine kinase 3ligand, FLT-3L). Preferably, the growth factor comprises SCF. The concentration of growth factor is not limited, illustratively: 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, e.g., 0, 5ug/L, 8ug/L, 10ug/L, 15ug/L, 20ug/L, 30ug/L, 40ug/L, 50ug/L; G-CSF is present in a concentration of 0-200ug/L, preferably 0-150ug/L, for example 0, 10ug/L, 20ug/L, 25ug/L, 40ug/L, 50ug/L, 60ug/L, 80ug/L, 100ug/L, 150ug/L; IL-3 concentration 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, for example 0, 10ug/L, 20ug/L, 25ug/L, 30ug/L, 40ug/L, 50ug/L.

Raf/MEK/ERK pathway modulators refer to substances capable of blocking or activating the Raf/MEK/ERK pathway. MAPK, mitogen-activated protein kinase, has a signal pathway capable of transducing extracellular signals into cells, and transmits cell signals through three-stage kinase cascade (MAPK, MAPK kinase (MEK or MKK) and MAPK kinase (MEKK or MKKK)) to regulate cell proliferation, differentiation, apoptosis, inflammatory response, vascular development and other biological functions. The Raf/MEK/ERK pathway is 1 of the 4 signal pathways of the MAPK signal pathway network. In some embodiments, the Raf/MEK/ERK pathway modulator contained in the medium is a MEK activator and/or an ERK activator. In some embodiments, the Raf/MEK/ERK pathway modulator contained in the 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 (PLX 4032), sorafenib (BAY 43-9006), dabrafenib (GSK 2118436). The concentration of Raf/MEK/ERK pathway modulator is not limited, and exemplary concentrations are 0.1-5. Mu.M, preferably 0.5-1.5. Mu.M, e.g., 0.5. Mu.M, 0.6. Mu.M, 0.8. Mu.M, 1.0. Mu.M, 1.2. Mu.M, 1.5. Mu.M.

In some embodiments, the isoxazole derivative is a compound of formula (I).

Wherein R is ₁ Is an unsubstituted or substituted heteroaryl group,

m is an integer from 0 to 4, preferably an integer from 1 to 3,

x is CH ₂ Or NH or O, or a combination of a reactive species,

R ₂ is unsubstituted or substituted heteroaryl.

In some embodiments, R ₁ Is an unsubstituted or substituted five-membered heterocyclic ring containing 1 to 2 hetero atoms selected from N, O, S, for example, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, triazolyl and the like, and thienyl, furyl and the like are preferable. Which may be attached to the parent structure through a heteroatom or carbon atom in a five membered heterocyclic ring. When substituted, the number of substituents may be 1 or 2 (mono-or di-substituted), said substituents being selected from alkyl, haloalkyl, hydroxyalkyl, cyano, halogen, ester, amino, preferably said substituents are selected from C1-C4 alkylTrifluoromethyl, C1-C4 alkyl substituted by 1-2 hydroxy, R ₃ OC (=o) -, wherein R ₃ Is a C1-C4 alkyl group.

In some embodiments, R ₂ Is an unsubstituted or substituted five-membered heterocyclic ring containing 1 to 2 hetero atoms selected from N, O, S, for example, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, triazolyl and the like, and is preferably pyrazolyl, oxazolyl, thiazolyl and the like. The heteroaryl group may be attached to the parent structure through a heteroatom or carbon atom in a five membered heterocyclic ring. When substituted, the number of substituents may be 1 or 2 (mono-or di-substituted), the substituents being selected from alkyl, haloalkyl, hydroxyalkyl, cyano, halogen, ester, amino, preferably the substituents are selected from C1-C4 alkyl, trifluoromethyl, C1-C4 alkyl substituted with 1-2 hydroxy, R ₃ OC (=o) -, wherein R ₃ Is a C1-C4 alkyl group. In a preferred embodiment, the substituents are selected from methyl, trifluoromethyl, hydroxypropyl, cyano, halogen, CH ₃ OC(＝O)-。

In some embodiments, the compound of formula (I) has a structure represented by formula (Ia):

wherein A is O or S, m, X and R ₂ Is defined as above.

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

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-carboxilic acid (2-pyrazol-1-yl-ethyl) -amide, compound 21, CAS:943820-93-7, described above.

The concentration of the isoxazole derivative is not limited, and exemplary concentrations are 0 to 15. Mu.M, preferably 0 to 10. Mu.M, more preferably 1 to 10. Mu.M, for example, 0, 1. Mu.M, 2. Mu.M, 4. Mu.M, 5. Mu.M, 6. Mu.M, 10. Mu.M.

The medium may also contain pyrimidoindole derivatives. In some embodiments, the pyrimidoindole derivative is a compound of formula (II):

wherein R is _a Selected from unsubstituted or substituted C1-C4 alkyl, unsubstituted or substituted heteroaryl, R _d OC(＝O)-、R _e NHC (=o) -, wherein R _d Is unsubstituted or substituted C1-C4 alkyl, R _e Is unsubstituted or substituted C1-C4 alkyl;

R _b Selected from-NR _f R _g 、-OR _h Wherein R is _f 、R _g 、R _h Each independently selected from H, C-C4 alkyl, - (CH) ₂ ) _n R _i Wherein n is an integer of 0 to 4, R _i 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 、R _k Each independently selected from unsubstituted or substituted C1-C4 alkyl;

R _c selected from H, - (CH) ₂ ) _i R _p Wherein i is an integer of 0 to 4, R _p 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 、R _k Each independently selected from unsubstituted or substituted C1-C4 alkyl groups.

In some embodiments, R _a Is unsubstituted or substituted heteroaryl, said heteroaryl being a compound containing 1Five-or six-membered heterocyclic ring having up to 2 hetero atoms selected from N, O, S, for example, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl and the like. Which may be attached to the parent structure through a heteroatom or carbon atom in a five-membered or six-membered ring. When substituted, the number of substituents may be 1 or 2 (mono-or di-substituted), said substituents being selected from alkyl, haloalkyl, hydroxyalkyl, cyano, halogen, ester groups, preferably said substituents are selected from C1-C4 alkyl, trifluoromethyl, C1-C4 alkyl substituted with 1-2 hydroxy, R ₃ OC (=o) -, wherein R ₃ Is a C1-C4 alkyl group. In a preferred embodiment, the substituents are selected from methyl, trifluoromethyl, hydroxypropyl, cyano, halogen, CH ₃ OC(＝O)-。

In some embodiments, R _f 、R _g One of them is H or C1-C4 alkyl, and the other is- (CH) ₂ ) _n R _i Wherein n is an integer of 0 to 4, R _i An unsubstituted or substituted C1-C4 alkyl group, an unsubstituted or substituted C3-C6 cycloalkyl group, an unsubstituted or substituted five-or six-membered heterocyclic group (which contains 1 to 2 heteroatoms selected from N, O, S and which may be attached to the parent structure via heteroatoms or carbon atoms on the five-or six-membered ring), an unsubstituted or substituted aryl group, an unsubstituted or substituted five-or six-membered heteroaryl group (which contains 1 to 2 heteroatoms selected from N, O, S and which may be attached to the parent structure via heteroatoms or carbon atoms), -NR _j R _k -CN, wherein R _j 、R _k Each independently selected from unsubstituted or substituted C1-C4 alkyl groups.

In some embodiments, R _f 、R _g One of them is selected from H, C1-C4 alkyl, and the other is- (CH) ₂ ) _n R _i Wherein n is an integer of 0 to 4, and,

R _i selected from: C1-C4 alkyl, C1-C4 alkyl in which one hydrogen atom is substituted by C1-C4 alkoxy, C1-C4 alkyl which is unsubstituted or substituted by C1-C4 alkyl, hydroxy, cyano, nitro, amino, C1-C4 alkoxy, C3-C6 cycloalkyl which is unsubstituted or substituted (e.g. A) A group) containing 1 to 2 hetero atoms selected from N, O, S (e.g., pyrrolidinyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl), C1-C4 alkyl, cyano, nitro, amino, C1-C4 alkoxy-substituted aryl, unsubstituted or C1-C4 alkyl, cyano, nitro, amino, C1-C4 alkoxy-substituted aryl, containing 1 to 2 hetero atoms selected from N, O, S (e.g., pyrrolyl, furanyl, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyridinyl, pyrimidinyl), -NR _j R _k -CN, wherein R _j 、R _k Each independently selected from C1-C4 alkyl.

In some embodiments, R _h Selected from H, - (CH) ₂ ) _n R _i Wherein n is an integer of 0 to 4, and,

R _i selected from: C1-C4 alkyl, C1-C4 alkyl in which one hydrogen atom is substituted by C1-C4 alkoxy, C1-C4 alkyl which is unsubstituted or substituted by C1-C4 alkyl, hydroxy, cyano, nitro, amino, C1-C4 cycloalkyl which is substituted by C1-C4 alkoxy, C1-C4 alkyl which is unsubstituted or substituted by C1-C4 alkyl, hydroxy, cyano, nitro, amino, C1-C4 alkoxy, five-or six-membered heterocyclyl (e.g. pyrrolidinyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl) containing 1 to 2 heteroatoms selected from N, O, S, aryl, unsubstituted or substituted by C1-C4 alkyl, hydroxy, C1-C4 alkyl, cyano, nitro, amino, C1-C4 alkoxy, unsubstituted or substituted by C1-C4 alkyl, hydroxy, five-or six-membered heteroaryl containing 1 to 2 heteroatoms selected from N, O, S (e.g. pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl), -NR _j R _k -CN, wherein R _j 、R _k Each independently selected from C1-C4 alkyl.

In some embodiments, R _c Selected from H, - (CH) ₂ ) _i R _p Wherein i is an integer of 0 to 4, and,

R _p selected from: C1-C4 alkyl, C1-C4 alkyl which is unsubstituted or substituted by C1-C4 alkyl, hydroxy, cyano, nitro, amino, aryl which is substituted by C1-C4 alkoxy, C1-C4 alkyl which is unsubstituted or substituted by C1-C4 alkyl, cyano, nitro, amino, C1-C4 alkoxy, five-or six-membered heteroaryl which contains 1 to 2 heteroatoms selected from N, O, S (e.g. pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl), -NR _j R _k -CN, wherein R _j 、R _k Each independently selected from unsubstituted or substituted C1-C4 alkyl groups.

In some embodiments, the pyrimidoindole derivative is selected from the group consisting of:

in a preferred embodiment, the pyrimidoindole derivative is selected from the group consisting of UM171 and UM729.

The concentration of the pyrimidoindole derivative is not limited, and exemplary concentrations are 0 to 80nM, preferably 0 to 50nM, for example 0, 5nM, 10nM, 20nM, 25nM, 40nM, 50nM.

The basal medium of the medium is not limited as long as it is suitable for dendritic cell progenitor cell growth. In a preferred embodiment, the basal medium is a serum-free culture system suitable for the growth of dendritic cell progenitors. Serum-free culture systems can be used with commercial serum-free media, such as StemSpan ^TM SFEM culture medium, stemSpan ^TM SFEM II Medium, stemSpan ^TM XF medium, stemSpan ^TM -AOF medium, stemPro ^TM -34SFM; a basal serum-free medium comprising minimal medium and supplemental additives may also be employed. In exemplary embodiments, the minimal medium is selected from one or more of IMDM medium, DMEM/F-12 medium, neurobasal medium, AIM-V. In exemplary embodiments, the supplemental additive is selected from human serum albuminWhite, insulin, transferrin, sodium selenite, DL-alpha-tocopherol, linoleic acid. The content of the supplemental additive is not limited, and illustratively: the concentration of human serum 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 concentrations of 1-20mg/L, preferably 3-15mg/L, for example 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; the concentration of transferrin is 1-30mg/L, preferably 3-25mg/L, for example 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; the concentration of DL-alpha-tocopherol is 0.5-10mg/L, preferably 0.5-8mg/L, for example 0.5mg/L, 0.8mg/mL, 1mg/L, 1.2mg/mL, 2.5mg/mL, 4mg/mL, 5mg/L; the concentration of the linoleic acid is 0.5-10mg/L, preferably 0.5-5mg/L, for example 0.5mg/L, 0.8mg/mL, 1mg/mL, 1.2mg/mL, 2.5mg/mL, 4mg/mL, 5mg/L.

In some embodiments, the culture composition or medium contains a Raf/MEK/ERK pathway modulator and a growth factor comprising SCF, and optionally G-CSF and/or IL-3.

The invention also provides a preparation method of the dendritic cells, which comprises the following steps: the cells obtained by the method described in any of the embodiments described herein are cultured in a dendritic cell differentiation medium to obtain dendritic cells. Specifically, the method for preparing dendritic cells comprises: (1) Culturing cells from peripheral blood in a medium described herein (e.g., for at least 1 day, at least 3 days, at least 7 days, at least 14 days, at least 28 days) to obtain a first population of cells, and (2) culturing the first population of cells in a 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 comprise one or more selected from the group consisting of CD45+ leukocytes, CD14+ cells, CD 14-cells, CD123+ cells, CD 123-cells, CD 34-cells.

In the present invention, the dendritic cell differentiation medium is not limited, and a dendritic cell differentiation medium commonly used in the art 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, IFN gamma, 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, and the like. Serum or serum substitutes may also be included as a supplemental supplement, such as FBS and the like. In some embodiments, the basal medium is a commercial medium, e.g., immunocult ^TM ACF Dendritic Cell Medium, etc. In addition, supplementary additives such as Immunocult may be added ^TM ACF Dendritic Cell Supplement, etc. In an exemplary embodiment, the dendritic cell differentiation medium is IMDM basal medium supplemented with 10% FBS, 100ng/mL FLT-3L, 20ng/mL SCF, 20ng/mL GM-CSF, 20ng/mL IL-4.

In some embodiments, the dendritic cell differentiation medium further comprises a DC cell maturation factor. The DC cell maturation factor may be a ligand and/or activator of Toll-like receptor (TLR) and the like, which may be selected from one or more of poly (I: C), R848, LPS and the like, for example.

The invention also provides dendritic cells obtained from the foregoing methods described by any of the embodiments herein.

The invention also provides a cell cryopreservation formulation comprising dendritic cell progenitors and/or dendritic cells obtained from the methods described in any of the embodiments herein described; and (5) freezing the stock solution.

In addition, dendritic cell progenitors and dendritic cells prepared by the methods described herein can be used to prepare pharmaceutical compositions. Accordingly, the present invention also provides a pharmaceutical composition comprising an effective amount of dendritic cell progenitors and/or dendritic cells prepared by the methods described in any of the embodiments herein; pharmaceutically acceptable auxiliary materials.

The term "pharmaceutically acceptable excipients" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, as is well known in the art (see, e.g., remington's pharmaceutical sciences. Mediated 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. The adjuvant is preferably non-toxic to the recipient at the dosage and concentration employed. Such excipients include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, the pharmaceutical composition may contain substances for improving, maintaining or retaining, for example, pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or permeation of the composition. These substances are known from the prior art. The optimal pharmaceutical composition can be determined depending on the intended route of administration, the mode of delivery and the dosage required.

Pharmaceutical compositions for in vivo administration are generally provided in the form of sterile formulations. Sterilization is achieved by filtration through sterile filtration membranes. In the case of lyophilization of a composition, this method may be used to sterilize the composition either 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, by using physiological saline or an aqueous solution containing glucose and other auxiliary agents by conventional methods. Parenteral compositions are typically placed in a container having a sterile access port, such as an intravenous solution tape or vial having a stopper pierceable by a hypodermic injection needle. Alternatively, the composition 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 of the art. Other pharmaceutical compositions will be apparent to those skilled in the art. Techniques for formulating a variety of other sustained or controlled delivery means, such as liposome carriers, bioerodible particles or porous beads, and depot injections, are also known to those skilled in the art.

Once formulated, the pharmaceutical compositions are stored in sterile vials as solutions, suspensions, gels, emulsions, solids, crystals, or as dehydrated or lyophilized powders. The formulation may be stored in a ready-to-use form or reconstituted (e.g., lyophilized) prior to administration. The invention also provides kits for producing single dose administration units. Kits of the invention may each contain a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments of the invention, kits are provided that contain single and multi-chamber prefilled syringes (e.g., liquid syringes and lyophilized syringes).

The invention also provides methods of treating a patient (particularly a dendritic cell or dendritic cell progenitor related disease including, but not limited to, infectious disease, metabolic disease, and/or cancer) by administering the dendritic cell progenitor cells of any of the embodiments of the invention or a pharmaceutical composition thereof. The terms "patient," "subject," "individual," "subject" are used interchangeably herein to include any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human. "treating" refers to a subject employing a treatment regimen described herein to achieve at least one positive therapeutic effect (e.g., reduced number of cancer cells, reduced tumor volume, reduced rate of infiltration of cancer cells into peripheral organs, or reduced rate of tumor metastasis or tumor growth). The treatment regimen effective to treat a patient can vary depending on a variety of factors, such as the disease state, age, weight, and ability of the patient to elicit an anti-cancer response in the subject by therapy.

The therapeutically effective amount of the pharmaceutical composition comprising dendritic cell progenitors of the present invention to be employed will depend, for example, on the degree of treatment and the goal. Those skilled in the art will appreciate that the appropriate dosage level for treatment will vary depending in part on the molecule delivered, the indication, the route of administration, and the size (body weight, body surface or organ size) and/or condition (age and general health) of the patient. In certain embodiments, the clinician may titrate the dose and alter the route of administration to obtain the optimal therapeutic effect. Such as from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day.

The frequency of administration will depend on the pharmacokinetic parameters of the dendritic cell progenitors in the formulation used. The clinician typically administers the composition until a dose is reached that achieves the desired effect. The composition may thus be administered as a single dose, or over time as two or more doses (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, for example, by oral, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal or intralesional route injection; either by a sustained release system or by an implanted device.

Embodiments of the present invention will be described in detail below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not noted in the examples, and are carried out according to techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, ind. Molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Examples

Reagents and materials:

the sources of antibodies and reagents used in the examples are as follows:

StemSpan ^TM SFEM medium, stemcell Technologies company, cat# 09650.

StemSpan ^TM SFEM II medium, stemcell Technologies, cat# 09655.

StemSpan ^TM XF Medium, stemcell Technologies company, cat# 100-0073.

StemSpan ^TM AOF medium, company Stemcell Technologies, cat# 100-0130.

IMDM medium, thermo Fisher company, cat No. 12440053.

DMEM/F12 medium, thermo Fisher, cat# 11330032.

Neurobasal medium, thermo Fisher, cat# 21103049.

Human serum albumin (CAS number 70024-90-7), sigma-Aldrich, cat# A3782.

Human insollin (CAS number 11061-68-0), sigma-Aldrich, inc., cat number 91077C.

Transferrin (CAS number 11096-37-0), sigma-Aldrich, cat# T0665.

Sodium selenite (CAS number 10102-18-8), sigma-Aldrich, cat# S9133.

DL-alpha-tocopherol (CAS number 10191-41-0), sigma-Aldrich, inc., cat number T3251.

Linoleic acid (CAS number 60-33-3), sigma-Aldrich, inc., cat# L1012.

Human SCF, peproTech company, cat# AF-300-07.

Animal-Free Recombinant Human GM-CSF, peproTech company, cat# 300-03.

Animal-Free Recombinant Human G-CSF, peproTech company, cat# AF-300-23.

Recombinant Human IL-3, peproTech company, cat# GMP200-03.

Human Flt3-Ligand, peproTech company, cat# 300-19-10.

GDC-0879, selleck, inc., cat# S1104.

PLX-4720, selleck Inc., cat# S1152.

Vemurafenib (PLX 4032), selleck company, cat# S1267.

Sorafenib (BAY 43-9006), selleck company, cat# S7397.

Dabrafienib, selleck, cat# S2807.

Compounds 1-21, 24-44, shanghai Ji Zao medical engineering Co., ltd.

UM171, apexBio Technology company, cat No. a8950.

UM729, apexBio Technology company, cat No. a8952.

Ficoll-Paque PREMUM, GE Healthcare, inc., cat# 17-5442-03.

Dulbecco's phosphate buffer, cat# 14190250.

ViaStain ^TM AO/PI Staining Solution, nexcelom Inc., cat# CS2-0106.

Pan Monocyte Isolation Kit, miltenyi Biotec, cat# 130-096-537.

CD123 microblades, miltenyi Biotec, cat# 130-094-432.

CD34 microblades, miltenyi Biotec, cat# 130-100-453.Fixable Viability Stain 780,BD Biosciences company, cat No. 565388.

FITC Mouse Anti-Human CD11c, BD Biosciences, inc., cat# 561355.

PE Mouse Anti-Human HLA-DR, BD Biosciences, inc., cat# 555812.

BV605 Mouse Anti-Human CD40, BD Biosciences, inc., cat# 740410.

BV786 Mouse Anti-Human CD80, BD Biosciences, cat# 564159.

The experimental method comprises the following steps:

1. the method for separating peripheral blood cells by Ficoll process comprises the following steps:

1) Drawing the blood (containing anticoagulant) in the blood bag out by using a sterile syringe and averagely split charging the blood into a 50mL centrifuge tube;

2) Centrifuging 2500g of the mixture for 10min by using a Thermo Fisher centrifuge, and setting the parameter to rise and fall by 7;

3) The middle white membrane layer is drawn out and transferred to a new 50mL centrifuge tube, and each tube is 20mL;

4) Adding 30mL of normal-temperature DPBS to dilute the blood sample, and gently and fully mixing;

5) According to the sample size, preparing a plurality of 50mL centrifuge tubes (30 mL diluted blood sample/tube), adding 15mL Ficoll for each tube (without touching the upper part of the tube wall), and standing at room temperature;

6) 30mL of diluted blood sample was slowly added to Ficoll (per Ficoll solution: diluted blood was 1: 2) And (3) centrifuging for 20min at room temperature by 800g, and setting the parameter to be increased by 1 and decreased by 0.

7) After centrifugation, the mixture was separated into a red blood cell layer, a Ficoll layer, a white membrane layer and a plasma layer from bottom to top. Discarding the plasma layer to a 5mL scale above the buffy coat, slowly sucking the buffy coat of the cells into a new 50mL centrifuge tube by using a 1mL gun head, and adding DPBS (differential pressure buffer) with the volume of at least 3 times to 50mL; 400g at room temperature, centrifuging for 10min, and setting the parameter to rise and fall to 9;

8) Discarding the supernatant (without dumping the supernatant), re-suspending cells with 1mL of DPBS for each tube, adding DPBS to 50mL, centrifuging at room temperature of 400g for 10min, and setting the parameter to rise and fall to 9;

9) Discarding supernatant, re-suspending with culture medium, inoculating, and adding 5% CO at 37deg.C ₂ Culturing in an incubator.

2. A method of obtaining iGDP from peripheral blood cells comprising the steps of:

1) The separated fresh peripheral blood cells are resuspended by DPBS, 300g is carried out at room temperature, the centrifugation is carried out for 10min, the parameter is set to rise 9 and fall 9 (the frozen peripheral blood cells are taken out from a liquid nitrogen tank, quickly melted in a water bath at 37 ℃ and transferred into 9mL of fresh culture medium, and the mixture is gently blown and evenly mixed, and the centrifugation is carried out with the same parameter);

2) The supernatant was discarded, resuspended in 1mL of the corresponding medium, and 10. Mu.L of the cell suspension was taken with 10. Mu.L of LViaStain ^TM Slightly blowing and uniformly mixing AO/PI Staining Solution;

3) Transferring to a cell counting plate, inserting into a Cellometer K2 Fluorescent Cell ViabilityCounter, and selecting a corresponding program to perform AO/PI cell activity detection;

4) Adding appropriate amount of culture medium to adjust the density to 1.4E6 living cells/mL, inoculating into Tissue-treated culture plate, placing into 37deg.C, 5% CO ₂ Culturing in an incubator.

5) Transferring the cell suspension into a centrifuge tube when the cell confluency reaches 70-80%, and taking 10 mu L of the cell suspension and 10 mu L of ViaStain ^TM Slightly blowing and uniformly mixing AO/PI Staining Solution;

6) Transferring to a cell counting plate, inserting into a Cellometer K2 Fluorescent Cell Viability Counter, and selecting a corresponding program to perform AO/PI cell activity detection;

7) Adding fresh culture medium according to counting result to adjust density to 1E5 living cells/mL culture medium, inoculating, adding 37 deg.C 5% CO ₂ Culturing in an incubator.

3. From CD14 ⁺ Monocytes and CD14 ^- A method for obtaining iGDP in non-monocytes comprising the steps of:

1) The peripheral blood cells are resuspended by DPBS, centrifuged for 10min at room temperature of 300g, and the parameters are set to rise and fall by 9;

2) Preparing and sorting buffer: MACS BSA Stock Solution and autoMACS Rinsing Solution are combined as 1: mixing at a ratio of 20, and placing on ice;

3) Each 1E7 cell was resuspended in 30. Mu.L of sorting buffer, mixed well with 10. Mu.L of reagent L FcR Blocking Solution and 10. Mu.L of Biotin-Antibody Cocktail, and incubated at 2-8deg.C for 5min;

4) Separating buffer with 30 μl of each 1E7 cell, adding 20 μl of Anti-Biotin MicroBeads, mixing, and incubating at 2-8deg.C for 5min;

5) Fixing LS column by using MACS Separator, adding 3mL buffer to infiltrate LS column;

6) Dropping cell suspension, collecting unlabeled high purity CD14 ⁺ The mononuclear cell suspension is then washed by adding 3mL buffer, the unlabeled cell suspension is collected, and the cell suspension is mixed twice;

7) LS column was removed from MACS Separator and placed on collection tube, 5mL buffer was added, plug was pushed into column, magnetically labeled cells were immediately washed out, and CD14 was collected ^- A non-monocyte suspension;

8) Respectively mix CD14 ⁺ Monocytes and CD14 ^- Non-monocyte suspensions, 10. Mu.L of each cell suspension were placed in a centrifuge tube and 10. Mu.L of ViaStain was added ^TM The AO/PI Staining Solution is gently blown and evenly mixed, and added into a counting plate, a Cellometer K2 Fluorescent Cell Viability Counter is selected to carry out AO/PI cell activity detection by a corresponding program;

9)CD14 ⁺ monocytes and CD14 ^- Centrifuging non-monocyte suspension respectively, centrifuging for 10min at 300g, setting the parameter to rise 9 and fall 9, and gently sucking the supernatant;

10 Resuspension with a proper volume of culture medium, adjusting cell density to 1E5 living cells/mL of culture medium, inoculating, and adding 5% CO at 37deg.C ₂ Culturing in an incubator.

4. From CD123 ⁺ And CD123 ^- A method for obtaining iGDP in a cell, comprising the steps of:

3) Each 1E8 cell was resuspended with 400. Mu.L of sorting buffer, 100. Mu.L of CD123 microblades were added and mixed well and incubated at 2-8deg.C for 15min;

4) Each 1E8 cell is resuspended by using 10mL sorting buffer, 300g is centrifuged for 10min, the parameters are set to rise 9 and fall 9, and the supernatant is gently sucked;

5) Each 1E8 cell was resuspended with 500. Mu.L of sorting buffer;

6) Fixing LS column by using MACS Separator, adding 3mL buffer to infiltrate LS column;

7) Dropwise adding cell suspension, and collecting unlabeled CD123 ^- The cell suspension is then washed by adding 3mL buffer, unlabeled cell suspension is collected, and the cell suspension is mixed twice;

8) LS column was removed from MACS Separator and placed on collection tubes, 5mL buffer was added, plugs were pushed into the column, and magnetically labeled cells were immediately washed out (to increase CD 123) ⁺ Cell purity, the washed cell suspension can be subjected to secondary sorting with a new LS column);

9) Respectively mix CD123 ⁺ And CD123 ^- Cell suspensions, 10. Mu.L of each cell suspension was placed in a centrifuge tube, and 10. Mu.L of ViaStain was added ^TM The AO/PI Staining Solution is gently blown and evenly mixed, and added into a counting plate, a Cellometer K2 Fluorescent Cell Viability Counter is selected to carry out AO/PI cell activity detection by a corresponding program;

10)CD123 ⁺ and CD123 ^- The cell suspensions were centrifuged separately, 300g, for 10min, and the supernatant gently aspirated;

11 Resuspension with corresponding volume of corresponding culture medium, adjusting density to 1E5 living cells/mL culture medium, inoculating, and adding 5% CO at 37deg.C ₂ Culturing in an incubator.

5. From CD34 ^- A method for obtaining iGDP in a cell, comprising the steps of:

2) Preparing and sorting buffer: MACS BSA Stock Solution and autoMACS RinsingSolution are combined as 1: mixing at a ratio of 20, and placing on ice;

6) Dropping cell suspension, collecting unlabeled high purity CD34 ^- The cell suspension is then washed by adding 3mL buffer, unlabeled cell suspension is collected, and the cell suspension is mixed twice;

7) Blending CD34 ^- Cell suspensions, 10. Mu.L each were placed in a centrifuge tube and 10. Mu.L of LViaStain was added ^TM The AO/PI Staining Solution is gently blown and evenly mixed, and added into a counting plate, a Cellometer K2Fluorescent Cell Viability Counter is selected to carry out AO/PI cell activity detection by a corresponding program;

9)CD34 ^- respectively centrifuging the cell suspensions, 300g, centrifuging for 10min, setting the parameters to rise 9 and fall 9, and gently sucking the supernatant;

6. A method of inducing differentiation and maturation of iGDP into dendritic cells comprising the steps of:

1) iGDP cells were resuspended using DPBS, centrifuged for 10min at 300g at room temperature, and the parameters were set to 9 drops;

2) The supernatant was discarded, and 10. Mu.L of the cell suspension was gently mixed with 10. Mu.L of ViaStain AO/PI Staining Solution using 1mL of medium;

3) Transferring to a cell counting plate, inserting into a Cellometer K2Fluorescent Cell Viability Counter, and selecting a corresponding program to perform AO/PI cell activity detection;

4) Adding adaptationsQuantitative differentiation Medium (IMDM basal Medium supplemented with 10% FBS, 100ng/mL FLT-3L, 20ng/mL SCF, 20ng/mL GM-CSF, 20ng/mL IL-4) was adjusted to a density of 5E5 viable cells/mL suspension, inoculated and placed at 37℃in 5% CO ₂ Culturing in an incubator.

5) Half liquid exchange every 3 days;

6) Culturing on 12 th day, collecting half volume of cell suspension, centrifuging at room temperature of 300g for 10min, and setting 9 to rise and fall parameters;

7) The supernatant was discarded, and the cells were resuspended in an appropriate amount of maturation medium (IMDM basal medium supplemented with 10% FBS, 100ng/mL FLT-3L, 20ng/mL SCF, 20ng/mL GM-CSF, 20ng/mL IL-4, 200IU/mL IFNγ, 60. Mu.g/mL poly (I: C), 10. Mu.g/mL R848, 2. Mu.g/mL LPS), and inoculated into primary Kong Zhongji for further culture for 24 hours.

7. The flow detection method of the iGDP cells and the iGDP derived dendritic cells comprises the following steps:

1) Taking known number of iGDP/iGDP derived dendritic cells, standing at room temperature for 3-5min, centrifuging for 5min, and gently sucking off the supernatant;

2) Adjusting the cell density to 1E7 living cells/mL DPBS, adding FVS780 according to the proportion of 1.5 mu L for each 1E7, fully mixing the sample and the dye, and incubating for 10min at room temperature in a dark place;

3) Taking 4mL of DPBS, cleaning twice, centrifuging to 400g after each cleaning, centrifuging for 10min, and gently sucking the supernatant;

4) Adding corresponding antibodies (FITC Mouse Anti-Human CD11c, PE Mouse Anti-Human HLA-DR, BV605 Mouse Anti-Human CD40, BV786 Mouse Anti-Human CD 80) in a 1.5mL centrifuge tube according to a proportion, and completely mixing the antibodies;

5) The cell density after uniform mixing is adjusted to 2E6 living cells/100 mu L suspension, 100 mu L of cell suspension is added into a sample tube, and the mixture is incubated for 15min at room temperature and in a dark place;

6) 1mL of DPBS is added into a sample tube, and the mixture is centrifuged by a table centrifuge, 400g of the mixture is centrifuged for 5min, and the supernatant is gently sucked;

7) Repeating the step 6 for cleaning once;

8) Adding 300 mu L of DPBS into a sample tube, and carefully blowing and mixing by using a gun head;

9) And (5) detecting on the machine.

EXAMPLE 1 preparation of iGDP amplification Medium

The composition and ratio of iGDP amplification medium 1-32 is shown in Table 1, wherein the medium is StemSpan ^TM SFEM、StemSpan ^TM SFEM II、StemSpan ^TM -XF、StemSpan ^TM AOF, IMDM, DMEM/F-12 and Neurobasal in v/v, human serum albumin in G/L, insulin, transferrin, DL-alpha-tocopherol and linoleic acid in mg/L, sodium selenite in μg/L, SCF, GM-CSF, G-CSF, IL-3 and FLT-3L in μg/L, GDC-0879 in μM, 5-furan-2-yl-isoxazole-3-carboxilic acid (compound 21) in μM, and pyrimidoindol derivative UM171 in nM.

Details are given in table 1 below.

The unwritten amount in Table 1 was 0.

Furthermore, the inventors replaced GDC-0879 in the above media with PLX-4720, vemurafenib (PLX 4032), sorafenib (BAY 43-9006), or Dabrafenib, obtaining media 33-61 (PLX-4720), 62-90 (PLX 4032), 91-119 (Sorafenib), 120-148 (Dabrafenib) corresponding to media 2, 3, 5-9, 11-32.

In addition, the inventors replaced compound 21 in the above-mentioned media 3-10, 12-22, 24-30, 32 with compound 1-20 to obtain other various media corresponding to media 3-10, 12-22, 24-30, 32.

In addition, the inventors replaced UM171 in the above-described media 14-20 with UM729 and compound 24-44 to obtain other various media corresponding to media 14-20.

Example 2 collection of blood cell samples

The blood cell sample in the invention is derived from non-mobilized peripheral blood, and the donor comprises healthy people aged 18-70 years, and has unlimited sexuality. Separation of non-mobilized peripheral blood C using Ficoll processD45 ⁺ Cells, cells were expanded with fresh cells and cryopreserved resuscitated cells as starting cells. Samples 8 and 9 are CD14 after magnetic bead sorting of sample 6 ⁺ Monocytes and CD14 ^- Non-mononuclear cells, samples 10 and 11 were CD123 after magnetic bead sorting for sample 7 ⁺ And CD123 ^- Cells, sample 12 was CD34 after magnetic bead sorting of peripheral cells ^- And (3) cells.

See table 2 below for details.

EXAMPLE 3 iGDP amplification Medium formulation study-cell expansion fold at day 7/14 of different Medium culture sample 1

The machine-collected peripheral blood cell sample 1 separated by Ficoll process is used as a starting cell, and the iGDP culture media 1-9 are used as test culture media, so that the number of living cells on day 0 and day 7/14 of culture is collected.

Example 4 iGDP amplification Medium formulation study-cell expansion fold at day 14 of different Medium culture samples 2.5

The viable cell count on day 0 and day 14 of culture was collected using hand-harvested peripheral blood cells isolated by Ficoll process as starting cells and iGDP medium 11-27 as test medium.

As can be seen from FIG. 1, after 14 days of culture using iGDP amplification medium 11-27, the cell expansion fold was 0.32.+ -. 0.15 to 4.94.+ -. 1.19 (data are mean and standard deviation of samples 2-5).

Example 5 iGDP amplification Medium formulation study-cell expansion fold on day 14 of different Medium culture samples 6.7

The machine-harvested peripheral blood cells separated by Ficoll process are used as initial cells, and iGDP culture media 15, 23, 24 and 28-32 are used as test culture media, so that the number of living cells on day 0 and day 14 of culture is collected.

As can be seen from FIG. 2, after 14 days of culture using iGDP amplification medium 15, 23, 24, 28-32, the cell expansion factors were 11.83.+ -. 1.36 to 43.23.+ -. 5.34 (data are mean and standard deviation of samples 6-7).

EXAMPLE 6 iGDP Long-term amplification Capacity study-cell expansion fold at day 28 of different Medium culture samples 7

Taking a machine-collected peripheral blood cell sample 7 separated by a Ficoll process as initial cells, taking iGDP culture media 15, 24 and 30 as test culture media, inoculating 1E6 living cells with initial cells, culturing for counting living cells on the 28 th day, and collecting cell viability and cell diameter data.

As can be seen from Table 3, sample 7 was optimal in terms of amplification capacity in iGDP amplification medium 15, and had been cultured for 28 days to amplify 197-fold and maintained at a cell viability of 85.8%, followed by medium 30 and 24, and cultured for 28 days to amplify 194-fold and 83-fold, respectively, with a cell diameter in the range of 11.8-12.1. Mu.m.

See table 3 below for details.

TABLE 3 Table 3

EXAMPLE 7 iGDP amplification initiation cell Source study-CD 45 ⁺ Cell, CD14 ⁺ Monocytes, CD14 ^- Non-monocytes, CD123 ⁺ Cell, CD123 ^- Cells and CD34 ^- Cell-initiated cell culture

Machine-collected peripheral blood CD45 separated by Ficoll process ⁺ Cell (sample 7), ficoll process separation combined with magnetic bead separation to obtain CD14 ⁺ Monocytes (sample 8), CD14 ^- Non-monocytes (sample 9), CD123 ⁺ Cell (sample 10), CD123 ^- Cells (sample 11) and CD34 ^- Cells (sample 12) were used as starting cells, inoculated with iGDP medium 15, and iGDP cytophotograph taken on day 14 of culture.

As can be seen from fig. 4, CD45 ⁺ Cell, CD14 ⁺ Monocytes, CD14 ^- Non-monocytes, CD123 ⁺ Cell, CD123 ^- Both cells and CD 34-cells can be expanded to obtain iGDP cells, and the iGDP cells are grown in a suspended or adherent manner, and the cells are spherical and uniform in size.

Example 8 iGDP derived DC cell yield and flow characterization assays

The differentiation and maturation of dendritic cells according to the present invention was performed using iGDP cells as differentiation initiating cells, and the yield (number of differentiated cells/number of initial PBMC cells. CD11 c) ⁺ Cell fraction) and cell flow characterization detection.

As can be seen from table 4, the DC cell yields in iGDP expansion media 15, 24 and 30 for sample 7 were 138.05-fold, 75.42-fold and 132.64-fold, respectively.

Details are given in table 4 below.

As can be seen from Table 5, iGDP cells obtained from different media can be efficiently differentiated into DC cells, wherein the iGDP cells obtained from the medium 24 have the highest differentiation efficiency into DC cells, CD11c ⁺ Cell ratio is 85.70%, HLA-DR ⁺ Cell ratio is 56.04%, CD40 ⁺ Cell ratio is 79.91%, CD8 ⁺ The cell fraction was 64.83%.

See table 5 below for details.

In addition, the flow characterization detection result of the iGDP-derived DC cells obtained in the culture medium 15 is shown in FIG. 4, and the CD11c of the iGDP-derived DC cells ⁺ The ratio of HLA-DR is 75.77% ⁺ With a 30.29% ratio, CD40 ⁺ Has a ratio of 72.12% and CD80 ⁺ The duty cycle was 75.28%, indicating that iGDP has DC differentiation potential.

Claims

1. A method for preparing dendritic cell progenitors, characterized in that cells from peripheral blood are cultured in a medium to obtain dendritic cell progenitors.

2. The method of claim 1, wherein the medium comprises at least two of a growth factor, a Raf/MEK/ERK pathway modulator, an isoxazole derivative.

3. The method of claim 2, wherein the growth factor is selected from at least one of SCF, GM-CSF, G-CSF, IL-3, FLT-3L, more preferably the growth factor comprises SCF and at least one of GM-CSF, G-CSF, IL-3, FLT-3L.

4. The method of claim 3, wherein the step of,

the concentration of SCF is 10-150ug/L, preferably 30-120ug/L;

The concentration of GM-CSF is 0-80ug/L, preferably 0-50ug/L;

G-CSF is present at a concentration of 0-200ug/L, preferably 0-150ug/L;

IL-3 concentration is 0-80ug/L, preferably 0-50ug/L; and/or

The concentration of FLT-3L is 0-80ug/L, preferably 0-50ug/L.

5. The method of any one of claims 2-4, wherein the Raf/MEK/ERK pathway modulator is a MEK activator and/or an ERK activator.

6. The method according to claim 5, wherein the Raf/MEK/ERK pathway modulator is selected from one or more of GDC-0879, PLX4720, vemurafenib, sorafenib, dabrafenib, preferably GDC-0879.

7. The method according to any one of claims 2 to 6, wherein the Raf/MEK/ERK pathway modulator is at a concentration of 0.1 to 5 μm, preferably 0.5 to 1.5 μm.

8. The method according to any one of claims 2 to 7, wherein the isoxazole derivative is a compound of formula (I):

wherein R is ₁ Is an unsubstituted or substituted heteroaryl group,

m is an integer from 0 to 4, preferably an integer from 1 to 3,

x is CH ₂ Or NH or O, or a combination of a reactive species,

R ₂ is unsubstituted or substituted heteroaryl;

preferably, the isoxazole derivative is a compound represented by any one of formulas 1 to 21;

More preferably, the isoxazole derivative is 5-furan-2-yl-isoxazole-3-carboxylic acid (2-pyrazol-1-yl-ethyl) -amide.

9. The method according to any one of claims 2 to 8, wherein the concentration of the isoxazole derivative is 0 to 15 μΜ, preferably 0 to 10 μΜ, more preferably 1 to 10 μΜ, even more preferably 4 to 6 μΜ.

10. The method according to any one of claims 2 to 9, wherein the medium further comprises a pyrimidoindole derivative, preferably the pyrimidoindole derivative is represented by formula (II),

R _b selected from-NR _f R _g 、-OR _h Wherein R is _f 、R _g 、R _h Each independently selected from H, C-C4 alkyl, - (CH) ₂ ) _n R _i Wherein n is the integer of 0 to 4Number, R _i 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 、R _k Each independently selected from unsubstituted or substituted C1-C4 alkyl;

R _c selected from H, - (CH) ₂ ) _i R _p Wherein i is an integer of 0 to 4, R _p 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 、R _k Each independently selected from unsubstituted or substituted C1-C4 alkyl;

more preferably, the pyrimidoindole derivative is a compound represented by any one of formulas 22 to 44;

further preferably, the pyrimidoindole derivative is selected from one or both of UM171 and UM729, and still further preferably, the pyrimidoindole derivative is UM171.

11. The method according to claim 10, characterized in that the pyrimidoindole derivative is present in a concentration of 0-80nM, preferably 0-50nM.

12. The method according to any one of claims 2-11, wherein the medium further comprises a basal medium, such as a basal serum-free medium; preferably, the basal medium comprises a commercial serum-free medium or basal medium supplemented with additives.

13. The method according to claim 12, wherein,

the commercial serum-free medium is selected from the group consisting of StemSpan ^TM SFEM culture medium, stemSpan ^TM SFEM II Medium, stemSpan ^TM XF medium, stemSpan ^TM -AOF medium, stemPro ^TM One or more of-34 SFM,

the basic culture medium is selected from one or more of IMDM culture medium, DMEM/F-12 culture medium, neurobasal culture medium and AIM-V,

the supplement additive is selected from one or more of human serum albumin, insulin, transferrin, sodium selenite, DL-alpha-tocopherol and linoleic acid.

14. The method of claim 13, wherein the step of determining the position of the probe is performed,

the concentration of the human serum albumin is 1-10g/L, preferably 1-5 g/L;

insulin concentration is 1-20mg/L, preferably 3-15mg/L;

the concentration of transferrin is 1-30mg/L, preferably 3-25mg/L;

the concentration of sodium selenite is 1-20ug/L, preferably 5-15ug/L;

the concentration of DL-alpha-tocopherol is 0.5-10mg/L, preferably 0.5-8mg/L; and/or

The concentration of the linoleic acid is 0.5-10mg/L, preferably 0.5-5mg/L.

15. The method of any one of claims 1-14, wherein the peripheral blood is peripheral blood that has not been mobilized by hematopoietic stem cells.

16. The method of any one of claims 1-15, wherein the cells are selected from CD45 ⁺ White blood cells, CD14 ⁺ Cell, CD14 ^- Cell, CD123 ⁺ Cell, CD123 ^- Cells, CD34 ^- One or more of the cells.

17. A culture medium for the preparation of dendritic cell progenitors from peripheral blood, the culture medium comprising at least two of growth factors, raf/MEK/ERK pathway modulators, isoxazole derivatives.

18. The medium of claim 17, wherein the growth factor is selected from at least one of SCF, GM-CSF, G-CSF, IL-3, FLT-3L, more preferably wherein the growth factor comprises SCF and at least one of GM-CSF, G-CSF, IL-3, FLT-3L.

19. The medium according to claim 18, wherein,

the concentration of SCF is 10-150ug/L, preferably 30-120ug/L;

the concentration of GM-CSF is 0-80ug/L, preferably 0-50ug/L;

G-CSF is present at a concentration of 0-200ug/L, preferably 0-150ug/L;

IL-3 concentration is 0-80ug/L, preferably 0-50ug/L; and/or

The concentration of FLT-3L is 0-80ug/L, preferably 0-50ug/L.

20. The medium of any one of claims 17-19, wherein the Raf/MEK/ERK pathway modulator is a MEK activator and/or an ERK activator.

21. The culture medium according to claim 20, wherein the Raf/MEK/ERK pathway modulator is selected from one or more of GDC-0879, PLX4720, vemurafenib (PLX 4032), sorafenib (BAY 43-9006), dabrafenib (GSK 2118436), preferably GDC-0879.

22. The medium according to any one of claims 17 to 21, wherein the Raf/MEK/ERK pathway modulator is at a concentration of 0.1 to 5 μm, preferably 0.5 to 1.5 μm.

23. The medium according to any one of claim 17 to 22,

the isoxazole derivative is a compound shown in a formula (I):

wherein R is ₁ Is an unsubstituted or substituted heteroaryl group,

m is an integer from 0 to 4, preferably an integer from 1 to 3,

x is CH ₂ Or NH or O, or a combination of a reactive species,

R ₂ is unsubstituted or substituted heteroaryl;

24. The medium according to any one of claims 17-23, wherein the concentration of the isoxazole derivative is 0-15 μm, preferably 0-10 μm, more preferably 1-10 μm, even more preferably 4-6 μm.

25. The medium according to any one of claim 17 to 24,

the medium also contains a pyrimidoindole derivative, preferably, the pyrimidoindole derivative is shown as a formula (II),

26. The medium according to claim 25, characterized in that the pyrimidoindole derivative is present in a concentration of 0-80nM, preferably 0-50nM.

27. The medium according to any one of claims 17-25, further comprising a basal medium, such as basal serum-free medium; preferably, the basal medium comprises a commercial serum-free medium or basal medium supplemented with additives.

28. The medium according to claim 27, wherein,

29. The medium according to claim 28, wherein,

the concentration of the human serum albumin is 1-10g/L, preferably 1-5 g/L;

insulin concentration is 1-20mg/L, preferably 3-15mg/L;

the concentration of transferrin is 1-30mg/L, preferably 3-25mg/L;

the concentration of sodium selenite is 1-20ug/L, preferably 5-15ug/L;

The concentration of the linoleic acid is 0.5-10mg/L, preferably 0.5-5mg/L.

30. A dendritic cell progenitor cell prepared by the method of any one of claims 1-16.

31. A method for producing a dendritic cell, wherein the dendritic cell progenitor cell according to claim 30 is cultured in a dendritic cell differentiation medium to obtain a dendritic cell.

32. A dendritic cell prepared by the method of preparation of claim 31.

33. Use of the dendritic cell progenitor cell of claim 30 or the dendritic cell of claim 32 in the manufacture of a medicament, preferably for the treatment of cancer, an infectious disease and/or an aging-related disease.

34. A pharmaceutical composition comprising an effective amount of the dendritic cell progenitor cell of claim 30 and/or the dendritic cell of claim 32, and a pharmaceutically acceptable adjuvant.