Method for enhancing the differentiation efficiency and survival duration of type I classical dendritic cells

Abstract

The present invention discloses a method for enhancing the differentiation efficiency and survival duration of type I classical dendritic cells; during the induction process, by introducing the gene mycn, the present invention achieves a dual improvement in the induction efficiency of cDC1 and the survival of cDC cells. Through a humanized mouse tumor model, the greater effectiveness of the obtained mycn transgenic cDC1 for tumor treatment is verified.

Description

Technical Field

[0001] The present invention belongs to the technical fields of cell therapy, tumor prevention and treatment, and tumor vaccines, and relates to a method for inducing gene-modified primary human cDC1 cells; specifically, it relates to a method for enhancing the differentiation efficiency and survival duration of type I classical dendritic cells. Background Art

[0002] As a main subset of dendritic cells, the conventional type 1 dendritic cell subset (cDC1) plays a key role in maintaining tolerance, anti-tumor immunity, and anti-infection. Compared with other DCs, cDC1 has the most powerful "cross-presentation" ability and can strongly activate CD8+ T and CD4+ T cells. By inducing CD8+ T cells to produce molecules such as IFN-γ, TNFα, perforin, and GZMB, cDC1 plays an anti-tumor role during the internodes. By activating CD4+ T cells, cDC1 can promote the differentiation of memory CD8+ T cells, thus maintaining long-term inhibition of tumors. Activated CDC1 highly expresses CXCL9 / 10 chemokines and can recruit a large number of T cells and NK cells from the periphery and tumor-associated lymph nodes to the tumor site. The IL-12p70 cytokine secreted by CDC1 can also activate NK cells to produce IFN-γ and play an anti-tumor role through NK cells. None of the above advantages are possessed by other dendritic cells. Therefore, cDC1 is the most critical anti-tumor DC subset.

[0003] Although cDC1 has advantages in anti-tumor, and there are also studies attempting to use human cDC1 for anti-tumor treatment, the existing methods still have relatively low induction efficiency, usually around 30%, and it is difficult to obtain a larger number of cDC1 cells, which limits the application of cDC1 in cell therapy. In addition, the cDC1 induced by the existing methods is prone to apoptosis after activation, and the survival time after activation is very short, usually within 24 - 48 hours, which limits the duration of action of cDC1 and thus leads to insufficient anti-tumor ability. Therefore, further improving the differentiation efficiency of primary human cDC1 and the survival duration of cDC1 will help improve the efficacy of cDC1 while reducing the preparation cost of cDC1 cells. Summary of the Invention

[0004] Aiming at the deficiencies of the existing methods such as the still relatively low induction efficiency and the easy apoptosis of the induced cDC1 after activation in the above-mentioned existing technology, the purpose of the present invention is to provide a method for enhancing the differentiation efficiency and survival duration of type I classical dendritic cells; thus, it helps to obtain a larger number and more long-lasting cDC1 cells for tumor treatment.

[0005] In the present invention, by introducing the gene mycn during the induction process, a dual improvement in the induction efficiency of cDC1 and the survival of cDC cells is achieved. Through a humanized mouse tumor model, the greater effectiveness of the obtained mycn transgenic cDC1 for tumor treatment is verified. Specifically, the object of the present invention is achieved through the following technical solutions:

[0006] In a first aspect, the present invention relates to a method for enhancing the differentiation efficiency and survival duration of type I classical dendritic cells, comprising the step of overexpressing the human mycn gene in umbilical cord blood stem cells to induce cDC1 differentiation.

[0007] As an embodiment, the overexpression includes infecting umbilical cord blood stem cells with a lentiviral vector overexpressing the coding region of the human mycn gene.

[0008] As an embodiment, the coding region sequence of the human mycn gene is at least one of the following:

[0009] A. The sequence shown in SEQ ID NO.1;

[0010] B. A sequence having a homology of greater than or equal to 75% with the sequence shown in SEQ ID NO.1;

[0011] C. The sequence shown in SEQ ID NO.2;

[0012] D. A sequence having a homology of greater than or equal to 75% with the sequence shown in SEQ ID NO.2.

[0013] As an embodiment, the umbilical cord blood stem cells are subjected to amplification culture, and the medium used is based on SFEM II and supplemented with penicillin / streptomycin, as well as a combination of human cytokines SCF, Flt3L, and TPO. Preferably, in the medium, penicillin / streptomycin is 25U / ml - 100U / ml, SCF is 20 - 500ng / ml, Flt3L is 20 - 500ng / ml, and TPO is 20 - 500ng / ml.

[0014] As an embodiment, the umbilical cord blood stem cells are CD34+ umbilical cord blood stem cells. Preferably, mononuclear cells are obtained by density gradient centrifugation of umbilical cord blood, and the CD34+ umbilical cord blood stem cells are isolated using a CD34 positive selection kit.

[0015] As an embodiment, the differentiation medium used to induce cDC1 differentiation is based on SFEM II and supplemented with penicillin / streptomycin, as well as a combination of human cytokines SCF, Flt3L, GM-CSF, IL4, and IFN γ.

[0016] As an embodiment, it includes at least one of the following technical features:

[0017] A. In the differentiation medium, penicillin / streptomycin is 25 U / ml - 100 U / ml, SCF is 10 - 500 ng / ml, Flt3L is 10 - 500 ng / ml, GM-CSF is 1 - 200 ng / ml, IL4 is 2.5 ng / ml - 1 - 200 ng / ml, and IFN γ is 2 - 100 ng / ml;

[0018] B. During the differentiation culture, the culture medium is changed every 4 days under the same conditions; preferably, the cells are collected on the 10th day.

[0019] In a second aspect, the cDC1 cells prepared by the method of the present invention are within the scope of protection of the present invention.

[0020] In a third aspect, the use of the cDC1 cells of the present invention in preparing a type I classical dendritic cell tumor vaccine or a type I classical dendritic cell anti-tumor drug is also within the scope of protection of the present invention.

[0021] The tumors include various solid tumors and hematological tumors; specifically, including but not limited to breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain cancer, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck cancer, lung cancer, small cell lung cancer, Wilms tumor, cervical cancer, testicular cancer, gastric cancer, genitourinary cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell cancer, endometrial cancer, adrenocortical cancer, malignant pancreatic insulinoma, malignant carcinoid, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocythemia, Hodgkin's disease, non-Hodgkin lymphoma, soft tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] Improve the induction efficiency of human primary cDC1 cells and the survival duration of cDC1 cells, so as to achieve the purpose of reducing the treatment cost of cDC1 cells and enhancing the efficacy of cDC1. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] By reading the detailed description of the non-limiting embodiments with reference to the following drawings, other features, objects, and advantages of the present invention will become more apparent:

[0025] Figure 1 Schematic flowchart of the method for enhancing the induction efficiency and survival duration of cDC1 in the present invention;

[0026] Figure 2 Flow cytometry intracellular staining for detecting the overexpression effect of MYCN;

[0027] Figure 3 Overexpression of the mycn gene to enhance the induction efficiency of cDC1;

[0028] Figure 4 For the cDC1 cells separated and differentiated, after stimulation with PolyIC and PAM, the apoptosis of the cells was dynamically detected by flow cytometry;

[0029] Figure 5 Therapeutic effect of cDC1 cells on the tumor model; *p<0.05, ***p<0.001;

[0030] Figure 6 Plasmid map of pLenti-EF1α-MCS-CMV-EGFP-P2A-Puromycin. Detailed implementation manners

[0031] The present invention will be described in detail below in conjunction with the embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, several adjustments and improvements can be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0032] Example 1

[0033] This example provides a method for enhancing the differentiation efficiency and survival duration of type I classical dendritic cells; as Figure 1 shown.

[0034] 1. Amplification of umbilical cord blood stem cells

[0035] Umbilical cord blood was subjected to density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare) to obtain mononuclear cells, and then CD34+ umbilical cord blood stem cells were isolated using a CD34 positive selection kit (Miltenyi Biotec).

[0036] Culture was carried out under the following culture conditions: using SFEM II medium (STEMCELL Technologiesl), adding penicillin / streptomycin (Gibco, penicillin 500 U / ml, range 25 U / ml - 100 U / ml), and simultaneously adding human cytokines human SCF (200 ng / ml, range 20 - 500 ng / ml), human Flt3L (200 ng / ml, range 20 - 500 ng / ml), human TPO (200 ng / ml, range 20 - 500 ng / ml), and amplifying for 2 days (range 1 - 2 days). The culture density was 1×105 / ml.

[0037] 2. Perform genetic modification on umbilical cord blood stem cells

[0038] Overexpress the coding region of the human mycn gene using a lentiviral vector. The SFFV promoter is used to control the expression of the mycn gene. Specifically, the coding region of human mycn is inserted into the empty lentiviral vector pLenti-EF1α-MCS-CMV-EGFP-P2A-Puromycin to obtain the pLenti-EF1α-MYCN-CMV-EGFP-P2A-Puromycin overexpression vector. The construction of the vector and virus packaging were both completed by GeneChem. The insertion site is between the two restriction enzyme cleavage sites XbaI and BamH1 in the MCS region. The plasmid map is as Figure 6 shown.

[0039] The MOI of virus infection is 100, that is, 100 active virus particles infect 1 umbilical cord blood stem cell. The virus is added to the umbilical cord blood stem cell medium, and 100 μl of the medium contains 1×10 7 active virus particles and 1×10 5 power of umbilical cord blood stem cells. After overnight infection in a 37-degree incubator, the medium is replaced with fresh above-mentioned umbilical cord blood stem cell amplification medium. The culture density is 1×10 5 / ml.

[0040] Umbilical cord blood stem cell medium: SFEM II medium (STEMCELL Technologiesl), and simultaneously adding human cytokines human SCF (200 ng / ml, range 20 - 500 ng / ml), human Flt3L (200 ng / ml, range 20 - 500 ng / ml), human TPO (200 ng / ml, range 20 - 500 ng / ml).

[0041] The overexpressed Human mycn gene sequence is: Note that Sequence 1 and Sequence 2 are different splice variants of the mycn gene.

[0042] Sequence 1

[0043] Sequence source: >hg38_refGene_NM_005378 range=chr2:15942065-15946097 5'pad=0 3'pad=0 strand=+ repeatMasking=none. The specific sequence is SEQ ID NO.1.

[0044] Sequence 2

[0045] Sequence source: >hg38_refGene_NM_001293231 range=chr2:15940587-15946097 5'pad=0 3'pad=0 strand=+ repeatMasking=none. The specific sequence is SEQ ID NO.2.

[0046] 3. Differentiation of cDC1 cells

[0047] Next, induce the differentiation of cDC1. The differentiation medium is: SFEM II medium (STEMCELL Technologies l), supplemented with penicillin / streptomycin (Gibco, penicillin 500 U / ml, range 25 U / ml - 100 U / ml), and at the same time supplemented with human cytokines human SCF (200 ng / ml), human Flt3L (200 ng / ml), human GM-CSF (2.5 ng / ml), human IL4 (2.5 ng / ml), and human IFN γ (5 ng / ml). Change the medium every 4 days under the same conditions. Collect the cells on the 10th day.

[0048] The collected cells were sequentially stained with the flow cytometry antibodies Anti-n-Myc / MYCN antibody [NCM II 100](ab16898) and DyLight® 488 goat anti-mouse IgG (H+L) (ab96879), as Figure 2 shown, and it was detected that MYCN achieved a high level of overexpression.

[0049] The collected cells were stained with the flow cytometry antibodies anti-human CD141 PECY7 and anti-human CD1C BV650 ( Figure 3 ). CDC1 is CD141 high , and cDC2 is CD141 low / - CD1C + The results showed that overexpression of mync significantly improved the induction efficiency of cDC1, and the induction efficiency of cDC1 increased from 28.7% to 50.8%.

[0050] Next, the sorted CD141 high cells were cDC1 cells. Then, they were stimulated with 10 μg / ml PolyI:C (1 - 10 μg / ml) and 10 μg / ml R848 (1 - 10 μg / ml) for 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours. Flow cytometry was used to detect cell viability. It was found that the overexpression of the mycn gene promoted the survival of cDC1 cells in vitro ( Figure 4 ).

[0051] 4. Humanized mouse tumor model

[0052] Umbilical cord blood-derived HLA0201-genotype umbilical cord blood stem cells were injected into the tail vein of NCG-X mice (Jicuiyaokang). After 20 weeks, mice with a human immune system were obtained. Then, 1×10 6 A375 tumor cell lines were subcutaneously injected into the mice to construct a melanoma model.

[0053] 5. cDC1 subset cells were used for tumor model treatment

[0054] After the mice developed tumors, HLA0201-genotype human cDC1 cells activated in vitro (range: 5×10 6 -1×10 9 cells / kg body weight / time, once a week) were injected into the tumor model mice intratumorally. Tumor changes were dynamically monitored. Figure 5 The therapeutic effects of mycn-overexpressing cDC1 cells (mycn-cDC1) and control cDC1 cells (Control cDC1) on melanoma tumors were dynamically compared; it was found that the overexpression of the mycn gene made cDC1 have stronger anti-tumor ability.

[0055] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which does not affect the essence of the present invention.

Claims

1. A method for enhancing the differentiation efficiency and survival duration of classical type I dendritic cells in vitro, characterized in that, The step of inducing the differentiation of cDC1 by overexpressing the human mycn gene in umbilical cord blood stem cells; The coding region sequence of the human mycn gene is as follows at least one of the following: A. The sequence shown in SEQ ID NO.1; B. The sequence shown in SEQ ID NO.

2.

2. The method for enhancing the differentiation efficiency and survival duration of type I classical dendritic cells in vitro according to claim 1, wherein The overexpression includes: infecting umbilical cord blood stem cells with a lentiviral vector overexpressing the coding region of the human mycn gene.

3. The method for enhancing the differentiation efficiency and survival duration of type I classical dendritic cells in vitro according to claim 1, characterized in that The umbilical cord blood stem cells are amplified and cultured, and the medium used is based on SFEM II as the basal medium, and penicillin / streptomycin, and a combination of human cytokines SCF, Flt3L, and TPO are added; the penicillin / streptomycin in the medium is 25 U / ml - 100 U / ml, SCF is 20 - 500 ng / ml, Flt3L is 20 - 500 ng / ml, and TPO is 20 - 500 ng / ml.

4. The method for enhancing the differentiation efficiency and survival duration of type I classical dendritic cells in vitro according to claim 1 or 3, characterized in that The umbilical cord blood stem cells are CD34+ umbilical cord blood stem cells.

5. The method for enhancing the differentiation efficiency and survival duration of type I classical dendritic cells in vitro according to claim 4, wherein The umbilical cord blood is subjected to density gradient centrifugation to obtain mononuclear cells, and the CD34+ umbilical cord blood stem cells are isolated using a CD34 positive selection kit.

6. The method for enhancing the differentiation efficiency and survival duration of type I classical dendritic cells in vitro according to claim 1, wherein The differentiation medium used to induce the differentiation of cDC1 is based on SFEM II as the basal medium, and penicillin / streptomycin, and a combination of human cytokines SCF, Flt3L, GM-CSF, IL4, and IFN γ are added.

7. The method for enhancing the differentiation efficiency and survival duration of type I classical dendritic cells in vitro according to claim 6, wherein At least includes one of the following technical features: A. In the differentiation medium, penicillin / streptomycin is 25 U / ml - 100 U / ml, SCF is 10 - 500 ng / ml, Flt3L is 10 - 500 ng / ml, GM-CSF is 1 - 200 ng / ml, IL4 is 2.5 ng / ml - 1 - 200 ng / ml, and IFN γ is 2 - 100 ng / ml; B. The differentiation culture is changed once every 4 days under the same conditions.

8. A cDC1 cell prepared by the method according to any one of claims 1 - 7.

9. An application of the cDC1 cell according to claim 8 in the preparation of an anti-tumor drug for classical type I dendritic cells; the tumor is melanoma.

10. The application according to claim 9, characterized in that, The anti-tumor drug for classical type I dendritic cells is a classical type I dendritic cell tumor vaccine.

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