Method for improving induction efficiency of type I classical dendritic cell subpopulation

Abstract

The invention discloses a method for improving the induction efficiency of a type I classical dendritic cell subset. Comprising the steps of knocking out / inactivating / inhibiting a transcription factor zeb2 in the cord blood stem cells and inducing cDC1 differentiation. According to the invention, a transcription factor zeb2 for inhibiting cDC1 differentiation is knocked out, so that the purpose of improving the cDC1 induction efficiency is achieved. And further evaluating the treatment effect of the obtained cDC1 cells on a tumor model by using a humanized mouse tumor model. Compared with the prior art, the induction efficiency of human primary cDC1 cells is improved, and the number of obtained cDC1 cells is increased, so that the purpose of reducing the cost of cDC1 cell treatment is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell therapy and relates to a method for improving the induction efficiency of type I classical dendritic cell subsets. Background Art

[0002] Dendritic cells (DC), as an important type of antigen-presenting cells, play an important role in many diseases. DC exerts anti-tumor effects by activating immune cells such as T cells and NK cells. Although DC has been used in clinical trials for a long time, there is still a lack of breakthroughs. The most critical reason is that most DC used for anti-tumor is induced from monocytes derived from peripheral blood, that is, monocyte-derived DC (moDC). However, more and more studies have shown that the DC that really plays an anti-tumor role in the body is other DC subsets.

[0003] Among the many DC subsets, the conventional type 1 dendritic cell (cDC1) subset has been found to play a key role in tumor immunity. Compared with moDC, cDC1 has the most powerful "cross-presentation" ability and can strongly activate CD8+ T cells. At the same time, cDC1 can also activate CD4+ T cells and promote the differentiation of memory CD8+ T cells. Activated CDC1 highly expresses CXCL9 / 10 chemokines and can recruit a large number of T cells and NK cells to the tumor site. IL-12 secreted by CDC1 can also activate NK cells and induce CD8+ T cells to produce IFN-γ, exerting an anti-tumor effect. All of the above advantages are not possessed by moDC. Therefore, cDC1 is a more suitable anti-tumor DC subset.

[0004] Although cDC1 has unique advantages in anti-tumor, and some studies have begun to use human cDC1 for anti-tumor treatment, the induction efficiency of existing methods is still low, usually only about 30%, which limits the number of cDC1 cells obtained, and further limits the application of cDC1 for cell therapy. Therefore, it is urgent to further improve the induction efficiency of human primary cDC1, reduce the preparation cost of cDC1 cells, and improve economic benefits. Summary of the invention

[0005] In view of the shortcomings of the existing methods in the above-mentioned prior art, such as the low induction efficiency, which limits the application of cDC1 in cell therapy, the purpose of the present invention is to provide a method for improving the induction efficiency of type I classical dendritic cell subsets; in particular, a method for improving the induction efficiency of human cDC1 cells, thereby helping to obtain a larger number of cDC1 cells for tumor treatment. The present invention uses gene editing to knock out the gene zeb2 that inhibits cDC1 differentiation, thereby achieving a higher efficiency induction of cDC1; further, through a humanized mouse tumor model, the more effective use of the obtained cDC1 for tumor treatment is verified.

[0006] The objective of the present invention is achieved through the following technical solutions: <First aspect> The present invention relates to a method for improving the induction efficiency of type I classical dendritic cell subsets, comprising the steps of knocking out / inactivating / inhibiting transcription factor zeb2 in umbilical cord blood stem cells and inducing cDC1 differentiation.

[0007] As an embodiment of the present invention, the sgRNA targeting the transcription factor zeb2 is selected from any of the following: Sgzeb2-1 5'-GGCGCAAACAAGCCAATCCC-3' (SEQ ID NO.1), Sgzeb2-2 5'-TGTTTGCGCCTCTTGCACCG-3' (SEQ ID NO.2), Sgzeb2-3 5'-ATCCAGACCGCAATTAACAA-3' (SEQ ID NO. 3).

[0008] As one embodiment of the present invention, the knockout includes mixing the pre-amplified umbilical cord blood stem cells with CAS9 protein and sgRNA, electroporating, and culturing.

[0009] As an embodiment of the present invention, it includes at least one of the following technical features: The cord blood stem cells are CD34+ cord blood stem cells; B. The culture medium used for the pre-amplification is SFEM II as the basic culture medium, and penicillin / streptomycin, and a combination of human cytokines SCF, Flt3L, and TPO are added; C. Pre-amplified cord blood stem cells were 1×10 5 The cells were resuspended in 5 μl R buffer and mixed with CAS9 protein and sgRNA; D. Electroporation conditions: 1400v, 10ms / pulse, 3 pulses; E. The culture medium used for cell culture after electroporation is the same as the pre-amplification culture medium.

[0010] As an embodiment of the present invention, technical feature A preferably performs density gradient centrifugation on umbilical cord blood to obtain mononuclear cells, and uses a CD34 positive selection kit to separate and obtain CD34+ umbilical cord blood stem cells.

[0011] As an embodiment of the present invention, the technical feature B preferably contains 25U / ml-100U / ml of penicillin / streptomycin, 20-500ng / ml of SCF, 20-500ng / ml of Flt3L, and 20-500ng / ml of TPO in the culture medium. More preferably, the expansion culture density is 1×10 5 / ml.

[0012] As an embodiment of the present invention, in the preferred mixed system of technical feature C, 0.5-5 μg / 6 μl of CAS9 protein and 100 ng / 6 μl-1 μg / 6 μl of sgRNA are mixed in R buffer. More preferably, the mixture is incubated at room temperature for 5-20 min.

[0013] The electroporation conditions of the present invention are: 1400v-1600v, 10-20 ms / pulse, 1-3 pulses; if it is not within the condition parameters, it will lead to reduced knockout efficiency or more cell death, which will ultimately affect the induction and differentiation efficiency of cDC1. As an embodiment of the present invention, the preferred electroporation conditions of technical feature D are: 1) 1400v, 10ms / pulse, 3 pulses; 2) 1600v, 20ms / pulse, 1 pulse; or 3) 1500v, 10ms / pulse, 3 pulses. The most preferred electroporation conditions are: 1400v, 10ms / pulse, 3 pulses.

[0014] As an embodiment of the present invention, the technical feature E is preferably cultured for 2 days. More preferably, the culture density is 1×10 5 / ml.

[0015] As one embodiment of the present invention, 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.

[0016] As an embodiment of the present invention, 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 1-200ng / ml, and IFNγ 2-100ng / ml; B. Differentiation culture medium was changed every 4 days under the same conditions; C. The cells obtained from the differentiation culture were sorted using flow cytometry antibodies anti-human CLEC9A PE and anti-human CD141PECY7, and the sorted CD141+CLEC9A+ cells were cDC1 cells.

[0017] The above technical feature A preferably comprises penicillin / streptomycin 50 U / ml, SCF 200 ng / ml5, Flt3L 200 ng / ml, GM-CSF 2.5 ng / ml, IL4 2.5 ng / ml, and IFNγ 2-100 ng / ml.

[0018] The above technical feature B preferably involves collecting cells on the 10th day.

[0019] <Second Aspect> The cDC1 cells prepared by the method of the present invention belong to the protection scope of the present invention.

[0020] <Third Aspect> The use of the method of the present invention or cDC1 cells in the preparation of type I classical dendritic cell tumor vaccines also falls within the protection scope of the present invention.

[0021] <Fourth Aspect> The use of the method of the present invention or cDC1 cells in the preparation of type I classical dendritic cell anti-tumor drugs also falls within the protection scope of the present invention.

[0022] 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, colon cancer, genital urinary cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell carcinoma, endometrial cancer, adrenal cortex cancer, ovarian cancer, cervical cancer, testicular cancer, gastric cancer, colon cancer, genital urinary cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell carcinoma, endometrial cancer, adrenal cortex cancer, uterine cancer, cervical ...uterine cancer, cervical cancer, uterine cancer, uterine cancer, cervical cancer, uterine cancer, uterine cancer, uterine cancer, uterine 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.

[0023] Differentiation from cord blood stem cells to cDC1 is regulated by a variety of transcription factor proteins. Some transcription factors promote the differentiation of cDC1 cells (including IRF8, BATF3, etc.), while other transcription factors inhibit the differentiation of cDC1 cells (including ZEB2, IRF4, etc.). The present invention achieves the purpose of improving the induction efficiency of cDC1 by knocking out the transcription factor zeb2 that inhibits cDC1 differentiation. The humanized mouse tumor model is further used to evaluate the therapeutic effect of the obtained cDC1 cells on the tumor model. Compared with the prior art, the present invention improves the induction efficiency of human primary cDC1 cells and increases the number of cDC1 cells obtained, thereby achieving the purpose of reducing the cost of cDC1 cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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 This is a schematic diagram of the process of improving the cDC1 induction efficiency of the present invention; Figure 2 Schematic diagram of flow cytometry intracellular staining to detect the knockout effects of three zeb2 sgRNAs; Figure 3 A comparison chart showing the improvement of cDC1 induction efficiency by knocking out the zeb2 gene; sgZEB2-1, 2, and 3 are different sgRNAs targeting the human zeb2 gene, and sgIRF4 is a sgRNA targeting the human IRF4 gene; Figure 4 To isolate and differentiate cDC1, PolyIC and PAM were stimulated, and the expression of cytokines IL12p70 and CXCL10 was shown; Figure 5 The zeb2-knockout cDC1 was loaded with antigens and then co-cultured with CD8+ T cells that recognized the antigens to detect the proliferation of CD8+ T cells; ns, no significant; **p<0.01; ***p<0.001, ****p<0.0001; Figure 6 Schematic diagram of the therapeutic effect of cDC1 cells on tumor models; *p<0.05, ***p<0.001. DETAILED DESCRIPTION

[0025] 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.

[0026] Example 1 This embodiment provides a method for improving the induction efficiency of human cDC1 cells; the process is as follows Figure 1 As shown: 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).

[0027] The following culture conditions were used for culture: SFEM II medium (STEMCELL Technologiesl), penicillin / streptomycin (final concentration of 50 U / ml, Gibco, range 25 U / ml-100 U / ml), 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) were added, and the cells were expanded for 2 days (range 1-2 days). The culture density was 1×10 5 / ml.

[0028] 2. Gene editing of cord blood stem cells The Invitrogen™ Neon transfection system (Invitrogen) was used to electroporate a mixture of CAS9 protein and humanzeb2 gene sgRNA for gene editing of umbilical cord blood stem cells (to improve the induction efficiency of cDC1 by knocking out zeb2). CAS9 protein was 1 μg (range 0.5-5 μg), sgRNA was 250 ng (range 100 ng-1 μg), mixed in Rbuffer (total volume 6 μl), and incubated at room temperature for 5-20 min.

[0029] All three zeb2 sgRNAs can be used alone, and the sequences are as follows: Sgzeb2-1 5'-GGCGCAAACAAGCCAATCCC-3' (SEQ ID NO.1), Sgzeb2-2 5'-TGTTTGCGCCTCTTGCACCG-3' (SEQ ID NO.2), Sgzeb2-3 5'-ATCCAGACCGCAATTAACAA-3' (SEQ ID NO. 3).

[0030] Collect the pre-amplified cord blood stem cells obtained in step 1, 1×10 5 The cells were resuspended in 5 μl R buffer, then mixed with CAS9 protein and sgRNA and electroporated. The electroporation conditions were: 1400v, 10ms / pulse, 3 pulses. Other conditions were: 1) 1600v, 20ms / pulse, 1 pulse; 2) 1500v, 10ms / pulse, 3 pulses. The electroporated cells were then placed in the umbilical cord blood stem cell culture medium in 1 and cultured for 2 days at a culture density of 1×10 5 / ml.

[0031] In this example, cells were collected 2 days after electroporation, and the knockout effects of three sgRNAs on zeb2 were detected by intracellular flow cytometry. It was found that sgZEB2-2 had the highest knockout efficiency; Figure 2 As shown, the knockout effects are sgzeb2-2, sgzeb2-1, and sgzeb2-3, respectively.

[0032] 3. Differentiation of Cdc1 Cells Next, cDC1 differentiation was induced with SFEM II medium (STEMCELL Technologiesl) supplemented with penicillin / streptomycin (final concentration of 50 U / ml, Gibco, range 25 U / ml-100 U / ml), human cytokines human SCF (200 ng / ml) (range: 10-500 ng / ml), Flt3L 200 ng / ml (range: 10-500 ng / ml), GM-CSF 2.5 ng / ml (range: 1-200 ng / ml), IL4 2.5 ng / ml (range: 1-200 ng / ml), and IFNγ 5 ng / ml (range: 2-100 ng / ml). The medium was changed every 4 days under the same conditions. Cells were collected on day 10.

[0033] The collected cells were stained with flow cytometry antibodies anti-human CLEC9A PE, anti-human CD141 PECY7, and anti-human CD1C BV650 ( Figure 3 ). The results showed that knocking out the zeb2 gene significantly increased the induction efficiency of cDC1. However, knocking out another transcription factor IRF4 that inhibits cDC1 differentiation had no effect (sgIRF4 sequence: 5'-CTTTAAACAGTGCCCAAGCC-3', SEQ ID NO.4). The efficiency of improvement was: sgzeb2-2, sgzeb2-1, sgzeb2-3; the highest efficiency was sgZEB2-2.

[0034] The sorted CD141+CLEC9A+ cells were cDC1 cells. They were then stimulated with 10 μg / ml PolyI:C (1-10 μg / ml) and 10 μg / ml R848 (1-10 μg / ml) for 24 hours ( Figure 4 The results showed that knocking out the zeb2 gene did not affect the production of cytokines in cDC1. After knocking out zeb2, cDC1 still highly expressed cytokines IL12p70 and CXCL10.

[0035] Then, cDC1 cells were co-incubated with tumor cell lysate antigens for 12 hours, and then cDC1 cells were co-incubated with CD8+ T cells for 72 hours (DC to T cell ratio was 1:1), and the proliferation of CD8+ T cells was detected. The results showed that knockout of the zeb2 gene promoted the activation of CD8+ T by cDC1 ( Figure 5 sgzeb2-2 showed the most significant effect. Given that sgzeb2-2 showed the most significant effect in vitro, we selected cDC1 knockout with sgzeb2-2 to test its anti-tumor effect in tumor models.

[0036] Among them, tumor cell lysate antigen can be prepared by the following steps: 1) Resuspend 1X10^6 / ml tumor cell line in DMEM medium supplemented with hypochlorous acid at a final concentration of 60uM, and culture at 37 degrees Celsius for 1h; 2) Then wash twice with PBS, centrifuge at 500g, 4 degrees, 5min. 3) Resuspend the tumor cell pellet with PBS to 1X10^7 / ml, then freeze at -80 degrees for 1h, take out and thaw at room temperature. Repeat freezing and thawing for more than 6 times. 4) Finally, the tumor lysate is co-incubated with DC, with the ratio of the lysate obtained from the same number of tumor cells and the same number of cDC1 co-incubated.

[0037] 4. Humanized Mouse Tumor Model HLA0201 genotype umbilical cord blood stem cells from umbilical cord blood bank were injected into NCG-X mice (Jicui Yaokang) through tail vein. After 20 weeks, mice with human immune system were obtained. Then, 1×10 6 A375 tumor cell line was used to construct a melanoma model.

[0038] 5.cDC1 subpopulation cells are used for tumor model treatment After the mice were tumor-bearing, human cDC1 cells with HLA0201 genotype induced and activated in vitro (in vitro induction and activation were the same as steps 1-3 above) (range: 5×10 6 -1×10 9The results showed that zeb2 knockout cDC1 had stronger anti-tumor ability (see Figure 6 ). It should be noted that cDC1 subpopulation cells can be used for the treatment of various tumors, including various solid tumors and hematological tumors; they are not limited to melanoma in this embodiment. The inhibitory effect on various tumors was tested, and the operation steps were the same. Among them, A375: human melanoma cell line (Chinese Academy of Sciences Cell Bank: SCSP-533); Siha: human cervical cancer cell line (Chinese Academy of Sciences Cell Bank: SCSP-5058); SKOV3: human ovarian cancer cell line (Chinese Academy of Sciences Cell Bank: SCSP-5214); PANC-1: human pancreatic cancer cell line (Chinese Academy of Sciences Cell Bank: SCSP-535); T98G: human brain glioma cell line (Chinese Academy of Sciences Cell Bank: SCSP-SCSP-5274); HT-29: human colorectal cancer cell line (Chinese Academy of Sciences Cell Bank: SCSP-5032). Since cDC1 can directly take up a variety of tumor antigens and present them to T cells after being introduced into the body, the anti-tumor ability of cDC1 is not limited by individual and tumor type differences. It has a wide range of anti-tumor potential and is suitable for the treatment of various solid tumors and hematological tumors.

[0039] 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 improving the induction efficiency of type I classical dendritic cell subsets, characterized in that: The method comprises the steps of knocking out / inactivating / inhibiting the transcription factor zeb2 in umbilical cord blood stem cells and inducing cDC1 differentiation; the sgRNA targeting the transcription factor zeb2 is selected from any one of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.

3.

2. The method for improving the induction efficiency of type I classical dendritic cell subsets according to claim 1, characterized in that: The knockout comprises mixing the pre-amplified umbilical cord blood stem cells with CAS9 protein and sgRNA, electroporating, and culturing.

3. The method for improving the induction efficiency of type I classical dendritic cell subsets according to claim 2, characterized in that: At least one of the following technical features: A. The cord blood stem cells are CD34+ cord blood stem cells; the cord blood is subjected to density gradient centrifugation to obtain mononuclear cells, and CD34+ cord blood stem cells are separated using a CD34 positive selection kit; B. The culture medium used for the pre-amplification is SFEM II as the basic culture medium, and penicillin / streptomycin, and a combination of human cytokines SCF, Flt3L, and TPO are added; the culture medium contains SCF 20-500ng / ml, Flt3L 20-500ng / ml, and TPO 20-500ng / ml; C. Pre-amplified cord blood stem cells were 1×10 5 After the cells were resuspended in 5 μl R buffer, they were mixed with CAS9 protein and sgRNA; in the mixed system, CAS9 protein 0.5-5 μg / 6 μl, sgRNA 100 ng / 6 μl-1 μg / 6 μl, mixed in R buffer; incubated at room temperature for 5-20 min; D. Electroporation conditions: 1400v-1600v, 10-20 ms / pulse, 1-3 pulse; E. The culture medium used for cell culture after electroporation is the same as the pre-amplification culture medium.

4. The method for improving the induction efficiency of type I classical dendritic cell subsets 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.

5. The method for improving the induction efficiency of type I classical dendritic cell subsets according to claim 4, characterized in that: At least one of the following technical features: A, differentiation medium contains SCF 10-500ng / ml, Flt3L 10-500ng / ml, GM-CSF 1-200ng / ml, IL4 2.5ng / ml-200ng / ml, and IFNγ 2-100ng / ml; B. Differentiation culture medium was changed every 4 days under the same conditions; C. The cells obtained from the differentiation culture were sorted using flow cytometry antibodies anti-human CLEC9A PE and anti-human CD141 PECY7, and the sorted CD141+CLEC9A+ cells were cDC1 cells.

6. Use of the method according to any one of claims 1 to 5 in the preparation of type I classical dendritic cell tumor vaccine.

7. Use of the method according to any one of claims 1 to 5 in preparing type I classical dendritic cell anti-tumor drugs.

8. The use according to claim 7, characterized in that: The tumors include various solid tumors and blood tumors.

9. The use according to claim 8, characterized in that: The tumors include 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's 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.

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