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

Abstract

The invention discloses a method for enhancing differentiation efficiency and survival time of type I classical dendritic cells. In the induction process, the gene mycn is introduced, so that the induction efficiency of the cDC1 and the survival of the cDC are both improved. Through a humanized mouse tumor model, the obtained mycn transgenic cDC1 is verified to be more effective in tumor treatment.

Description

Technical Field

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

[0002] As a major dendritic cell subset, the conventional type 1 dendritic cell (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, GZMB, etc., cDC1 internodes play an anti-tumor effect. By activating CD4+ T cells, cDC1 can promote the differentiation of memory CD8+ T cells, thereby maintaining long-term suppression of tumors. Activated CDC1 highly expresses CXCL9 / 10 chemokines, which can recruit a large number of T cells and NK cells from peripheral 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 exert anti-tumor effects through NK cells. All of the above advantages are not possessed by other dendritic cells. Therefore, cDC1 is the most critical anti-tumor DC subset.

[0003] Although cDC1 has advantages in anti-tumor, and some studies have attempted to use human cDC1 for anti-tumor treatment, the induction efficiency of existing methods is still low, usually around 30%, making it difficult to obtain a larger number of cDC1 cells, limiting the application of cDC1 for cell therapy. In addition, cDC1 induced by existing methods is prone to apoptosis after activation, and has a short survival time after activation, usually 24-48 hours, which limits the duration of cDC1's effectiveness and leads to insufficient anti-tumor ability. Therefore, further improving the differentiation efficiency of human primary cDC1 and the survival time of cDC1 will help to reduce the cost of cDC1 cell preparation while improving the efficacy of cDC1. Summary of the invention

[0004] In view of the shortcomings of the existing methods in the above-mentioned prior art, such as the low induction efficiency and the induced cDC1 being prone to apoptosis after activation, the object of the present invention is to provide a method for enhancing the differentiation efficiency and survival time of type I classical dendritic cells; thereby helping to obtain more cDC1 cells with more lasting effects for tumor treatment.

[0005] The present invention achieves a dual improvement in cDC1 induction efficiency and cDC cell survival by introducing the mycn gene during the induction process. The humanized mouse tumor model verifies the more effective use of the mycn transgenic cDC1 for tumor treatment. Specifically, the purpose of the present invention is achieved through the following technical solutions: In a first aspect, the present invention relates to a method for enhancing the differentiation efficiency and survival time of type I classical dendritic cells, comprising the steps of overexpressing the human mycn gene in umbilical cord blood stem cells to induce cDC1 differentiation.

[0006] As one embodiment, the overexpression comprises infecting umbilical cord blood stem cells with a lentiviral vector that overexpresses the human mycn gene coding region.

[0007] As an embodiment, the human mycn gene coding region sequence is at least one of the following: A, sequence as shown in SEQ ID NO.1; B. A sequence with a homology of greater than or equal to 75% with the sequence shown in SEQ ID NO.1; C. The sequence shown in SEQ ID NO.2; D. A sequence having a homology of greater than or equal to 75% with the sequence shown in SEQ ID NO.2.

[0008] As an embodiment, the cord blood stem cells are expanded and cultured using a culture medium based on SFEM II, supplemented with penicillin / streptomycin, and a combination of human cytokines SCF, Flt3L, and TPO. Preferably, the culture medium contains 25U / ml-100U / ml penicillin / streptomycin, 20-500ng / ml SCF, 20-500ng / ml Flt3L, and 20-500ng / ml TPO.

[0009] As an embodiment, the cord blood stem cells are CD34+ cord blood stem cells. Preferably, the cord blood is subjected to density gradient centrifugation to obtain mononuclear cells, and the CD34+ cord blood stem cells are separated using a CD34 positive selection kit.

[0010] As an embodiment, the differentiation medium used to induce cDC1 differentiation is SFEM II as the basic medium, supplemented with penicillin / streptomycin, and a combination of human cytokines SCF, Flt3L, GM-CSF, IL4 and IFNγ.

[0011] As an implementation plan, it includes at least one of the following technical features: A, differentiation medium containing penicillin / streptomycin 25U / ml-100U / ml, SCF 10-500ng / ml, Flt3L 10-500ng / ml, GM-CSF 1-200ng / ml, IL4 2.5ng / ml 1-200ng / ml and IFNγ2-100ng / ml; B. The differentiation culture medium was changed every 4 days under the same conditions; cells were preferably collected on the 10th day.

[0012] In the second aspect, the cDC1 cells prepared by the method of the present invention belong to the protection scope of the present invention.

[0013] In a third aspect, the use of the cDC1 cells of the present invention in the preparation of type I classical dendritic cell tumor vaccines or type I classical dendritic cell anti-tumor drugs also falls within the protection scope of the present invention.

[0014] The tumor includes various solid tumors and blood 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, Wilman tumor, cervical cancer, testicular cancer, gastric cancer, genital urinary cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell carcinoma, endometrial cancer, adrenal cortical carcinoma, Malignant pancreatic insulinoma, malignant carcinoid, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic myeloid leukemia, acute myeloid leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocythemia, Hodgkin's disease, non-Hodgkin's lymphoma, soft tissue sarcoma, osteoblastic sarcoma, primary macroglobulinemia and retinoblastoma.

[0015] Compared with the prior art, the present invention has the following beneficial effects: Improve the induction efficiency of human primary cDC1 cells and increase the survival time of cDC1 cells, thereby reducing the cost of cDC1 cell therapy and enhancing the efficacy of cDC1. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 A schematic diagram of a method for improving cDC1 induction efficiency and survival time of the present invention; Figure 2 Flow cytometry intracellular staining was used to detect the overexpression effect of MYCN; Figure 3 To overexpress the mycn gene to improve the induction efficiency of cDC1; Figure 4 To isolate the differentiated cDC1, after stimulation with PolyIC and PAM, flow cytometry was used to dynamically detect cell apoptosis. Figure 5 The therapeutic effect of cDC1 cells on tumor models; *p<0.05, ***p<0.001; Figure 6 This is the plasmid map of pLenti-EF1α-MCS-CMV-EGFP-P2A-Puromycin. DETAILED DESCRIPTION

[0017] The present invention is described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some adjustments and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0018] Example 1 This embodiment provides a method for enhancing the differentiation efficiency and survival time of type I classical dendritic cells; Figure 1 shown.

[0019] 1. Expansion of Umbilical Cord Blood Stem Cells 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).

[0020] The following culture conditions were used for culture: SFEM II medium (STEMCELL Technologiesl), penicillin / streptomycin (Gibco, penicillin 500U / ml, range 25U / ml-100U / ml), human cytokines human SCF (200ng / ml, range 20-500ng / ml), human Flt3L (200ng / ml, range 20-500ng / ml), human TPO (200ng / ml, range 20-500ng / ml) were added, and the cells were expanded for 2 days (range 1-2 days). The culture density was 1×105 / ml.

[0021] 2. Genetically engineered cord blood stem cells The human mycn gene coding region was overexpressed using a lentiviral vector. The SFFV promoter was used to control the expression of the mycn gene. Specifically, the human mycn coding region was 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 vector construction and virus packaging were completed by GeneCare. The insertion site was between the two restriction sites XbaI and BamH1 in the MCS region. The plasmid map is shown in Figure 6 shown.

[0022] The MOI of virus infection is 100, that is, 100 active virus particles infect 1 cord blood stem cell. The virus is added to the cord blood stem cell culture medium. 100 μl of culture medium contains 1×10 7 Active virus particles and 1×10 5 After infection overnight in a 37-degree incubator, the culture medium was replaced with fresh culture medium for the above-mentioned umbilical cord blood stem cell expansion. The culture density was 1×10 5 / ml.

[0023] Umbilical cord blood stem cell culture medium: SFEM II culture medium (STEMCELL Technologiesl), supplemented with human cytokines human SCF (200ng / ml, range 20-500ng / ml), human Flt3L (200ng / ml, range 20-500ng / ml), and human TPO (200ng / ml, range 20-500ng / ml).

[0024] The sequence of the overexpressed Human mycn gene is: Note that sequence 1 and sequence 2 are different splicing forms of the mycn gene.

[0025] Sequence 1 Sequence source: >hg38_refGene_NM_005378 range=chr2:15942065-15946097 5'pad=03'pad=0 strand=+ repeatMasking=none. The specific sequence is SEQ ID NO.1.

[0026] Sequence 2 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.

[0027] 3. Differentiation of cDC1 Cells Next, cDC1 differentiation was induced with SFEM II medium (STEMCELL Technologiesl) supplemented with penicillin / streptomycin (Gibco, penicillin 500U / ml, range 25U / ml-100U / ml), human cytokines human SCF (200ng / ml), human Flt3L (200ng / ml), human GM-CSF (2.5ng / ml), human IL4 (2.5ng / ml) and human IFNγ (5ng / ml), and the medium was changed every 4 days under the same conditions. Cells were collected on day 10.

[0028] The collected cells were sequentially stained with flow cytometry antibodies Anti-n-Myc / MYCN antibody [NCM II 100] (ab16898) and DyLight® 488 goat anti-mouse IgG (H+L) (ab96879), as shown in Figure 2. Figure 2 As shown, MYCN was detected to achieve high levels of overexpression.

[0029] The collected cells were stained with flow cytometry antibodies anti-human CD141 PECY7 and anti-human CD1C BV650 ( Figure 3 CDC1 is CD141 high , cDC2 is CD141 low / - CD1C + The results showed that overexpression of mync significantly increased the induction efficiency of cDC1, from 28.7% to 50.8%.

[0030] Next, the sorted CD141 high The cells were cDC1 cells. Then they were stimulated with 10ug / ml PolyI:C (1-10ug / ml) and 10ug / ml R848 (1-10ug / ml) for 0 hours, 24 hours, 48 ​​hours, 72 hours, and 96 hours. Flow cytometry was used to detect cell survival. The results showed that overexpression of the mycn gene promoted the survival of cDC1 cells in vitro ( Figure 4 ).

[0031] 4. Humanized Mouse Tumor Model The HLA0201 genotype umbilical cord blood stem cells derived from umbilical cord blood were injected into the tail vein of NCG-X mice (Jicui Yaokang). After 20 weeks, mice with human immune system were obtained. Then, 1×10 6A375 tumor cell line was used to construct a melanoma model.

[0032] 5.cDC1 subpopulation cells are used for tumor model treatment After the mice were tumor-bearing, human cDC1 cells (range: 5×10 6 -1×10 9 The cells were injected intratumorally into tumor model mice (10 cells / kg body weight / time, once a week) to dynamically monitor tumor changes. Figure 5 The therapeutic effects of mycn-overexpressing cDC1 cells (mycn-cDC1) and control cDC1 cells (Control cDC1) on melanoma tumors were dynamically compared; the results showed that overexpression of the mycn gene gave cDC1 a stronger anti-tumor ability.

[0033] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for enhancing the differentiation efficiency and survival time of type I classical dendritic cells, characterized in that: The method comprises the steps of overexpressing the human mycn gene in umbilical cord blood stem cells to induce cDC1 differentiation.

2. The method for enhancing the differentiation efficiency and survival time of type I classical dendritic cells according to claim 1, characterized in that: The overexpression comprises: infecting umbilical cord blood stem cells with a lentiviral vector that overexpresses the human mycn gene coding region.

3. The method for enhancing the differentiation efficiency and survival time of type I classical dendritic cells according to claim 2, characterized in that: The human mycn gene coding region sequence is at least one of the following: A, sequence as shown in SEQ ID NO.1; B. A sequence with a homology of greater than or equal to 75% with the sequence shown in SEQ ID NO.1; C. The sequence shown in SEQ ID NO.2; D. A sequence having a homology of greater than or equal to 75% with the sequence shown in SEQ ID NO.

2.

4. The method for enhancing the differentiation efficiency and survival time of type I classical dendritic cells according to claim 1, characterized in that: The umbilical cord blood stem cells are expanded and cultured using a culture medium based on SFEM II, supplemented with penicillin / streptomycin, and a combination of human cytokines SCF, Flt3L, and TPO; the culture medium contains 25U / ml-100U / ml of penicillin / streptomycin, 20-500ng / ml of SCF, 20-500ng / ml of Flt3L, and 20-500ng / ml of TPO.

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

6. The method for enhancing the differentiation efficiency and survival time of type I classical dendritic cells according to claim 5, characterized in that: The umbilical cord blood is subjected to density gradient centrifugation to obtain mononuclear cells, and the CD34+ umbilical cord blood stem cells are separated using a CD34 positive selection kit.

7. The method for enhancing the differentiation efficiency and survival time of type I classical dendritic cells according to claim 1, characterized in that: The differentiation medium used to induce cDC1 differentiation was SFEM II as the basal medium, supplemented with penicillin / streptomycin, and a combination of human cytokines SCF, Flt3L, GM-CSF, IL4, and IFNγ.

8. The method for enhancing the differentiation efficiency and survival time of type I classical dendritic cells according to claim 7, characterized in that: At least one of the following technical features: A, differentiation medium containing penicillin / streptomycin 25U / ml-100U / ml, SCF 10-500ng / ml, Flt3L 10-500ng / ml, GM-CSF 1-200ng / ml, IL4 2.5ng / ml 1-200ng / ml and IFNγ2-100ng / ml; B. In differentiation culture, the medium was changed every 4 days under the same conditions.

9. A cDC1 cell prepared according to the method according to any one of claims 1 to 8.

10. Use of the cDC1 cell according to claim 9 in preparing a type I classical dendritic cell tumor vaccine or a type I classical dendritic cell anti-tumor drug.

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