A method for improving the induction efficiency of type I classical dendritic cell subsets

Abstract

The present invention discloses a method for improving the induction efficiency of type I classical dendritic cell subsets; the method comprises the steps of knocking out / inactivating / inhibiting the transcription factor ZEB2 in umbilical cord blood stem cells to induce cDC1 differentiation. The present invention achieves the goal of improving cDC1 induction efficiency by knocking out the transcription factor ZEB2, which inhibits cDC1 differentiation. The therapeutic efficacy of the obtained cDC1 cells in a humanized mouse tumor model is further evaluated. Compared with existing technologies, the present invention improves the induction efficiency of primary human cDC1 cells and increases the number of cDC1 cells obtained, thereby reducing the cost of cDC1 cell therapy.

Description

Technical Field

[0001] This invention belongs to the field of cell therapy technology and relates to a method for improving the induction efficiency of type I classical dendritic cell subsets. Background Technology

[0002] Dendritic cells (DCs), as an important type of antigen-presenting cell, play a crucial role in various diseases. DCs exert their anti-tumor effects by activating immune cells such as T cells and NK cells. Although DCs have been used in clinical trials for a long time, breakthroughs have been lacking. The most critical reason is that most DCs used for anti-tumor purposes are derived from peripheral blood monocytes through induction, i.e., monocyte-derived DCs (moDCs). However, increasing research indicates that other DC subsets are the ones that truly exert anti-tumor effects in vivo.

[0003] Among the many dendritic cell (DC) subsets, conventional type 1 dendritic cells (cDC1) have been found to play a crucial role in tumor immunity. Compared to moDCs, cDC1 possesses the strongest "cross-presentation" capability, powerfully activating CD8+ T cells. Simultaneously, cDC1 can also activate CD4+ T cells and promote the differentiation of memory CD8+ T cells. Activated cDC1 highly expresses CXCL9 / 10 chemokines, recruiting 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. These advantages are not possessed by moDCs. Therefore, cDC1 is a more suitable anti-tumor DC subset.

[0004] Although cDC1 possesses unique advantages in anti-tumor activity, and some studies have begun utilizing human cDC1 for anti-tumor therapy, current methods still exhibit low induction efficiency, typically only around 30%, limiting the number of cDC1 cells that can be obtained and consequently restricting the application of cDC1 in cell therapy. Therefore, there is an urgent need to further improve the induction efficiency of primary human cDC1, reduce the cost of cDC1 cell preparation, and enhance economic efficiency. Summary of the Invention

[0005] To address the shortcomings of existing technologies, such as the relatively low induction efficiency of cDC1 cells, which limits their application in cell therapy, this invention aims to provide a method for improving the induction efficiency of type I classical dendritic cell subsets, particularly for improving the induction efficiency of human cDC1 cells, thereby facilitating the acquisition of a larger number of cDC1 cells for tumor treatment. This invention employs gene editing to knock out the zeb2 gene, which inhibits cDC1 differentiation, achieving higher cDC1 induction efficiency. Furthermore, the effectiveness of the obtained cDC1 cells for tumor treatment was verified using a humanized mouse tumor model.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] <First Aspect>

[0008] This invention relates to a method for improving the induction efficiency of type I classical dendritic cell subsets, including the steps of knocking out / inactivating / inhibiting the transcription factor zeb2 in umbilical cord blood stem cells to induce cDC1 differentiation.

[0009] As one embodiment of the present invention, the sgRNA targeting transcription factor zeb2 is selected from any of the following:

[0010] Sgzeb2-1 5'-GGCGCAAACAAGCCAATCCC-3' (SEQ ID NO.1),

[0011] Sgzeb2-2 5'-TGTTTGCGCCTCTTGCACCG-3' (SEQ ID NO.2),

[0012] Sgzeb2-3 5'-ATCCAGACCGCAATTAACAA-3' (SEQ ID NO. 3).

[0013] As one embodiment of the present invention, the knockout includes mixing pre-amplified umbilical cord blood stem cells with CAS9 protein and sgRNA, electroporating, and culturing.

[0014] As one embodiment of the present invention, it includes at least one of the following technical features:

[0015] The umbilical cord blood stem cells are CD34+ umbilical cord blood stem cells; B. The culture medium used for the pre-expansion is based on SFEM II, with the addition of penicillin / streptomycin, and a combination of human cytokines SCF, Flt3L, and TPO.

[0016] C. Pre-expanded umbilical cord blood stem cells at 1×10 5 Cells were resuspended in 5 μl R buffer and then mixed with CAS9 protein and sgRNA;

[0017] D. The electrical discharge conditions are: 1400V, 10ms / pulse, 3 pulses;

[0018] E. The culture medium used for cell culture after electroporation is the same as that used for pre-amplification.

[0019] As one embodiment of the present invention, technical feature A preferably involves density gradient centrifugation of umbilical cord blood to obtain mononuclear cells, and CD34+ umbilical cord blood stem cells are obtained by separation using a CD34 positivity kit.

[0020] As one embodiment of the present invention, technical feature B preferably contains penicillin / streptomycin 25U / ml-100U / ml, SCF 20-500ng / ml, Flt3L 20-500ng / ml, and TPO 20-500ng / ml in the culture medium. More preferably, the amplification culture density is 1×10⁻⁶. 5 / ml.

[0021] As one embodiment of the present invention, in the preferred embodiment 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.

[0022] The electroporation conditions of this invention are: 1400V-1600V, 10-20 ms / pulse, 1-3 pulses. Deviating from these parameters will lead to reduced knockout efficiency or increased cell death, ultimately affecting the induction differentiation efficiency of cDC1. As one embodiment of this invention, the preferred electroporation conditions for 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.

[0023] As one embodiment of the present invention, technical feature E preferably involves culturing for 2 days. More preferably, the culture density is 1×10⁻⁶. 5 / ml.

[0024] As one embodiment of the present invention, the differentiation medium used to induce cDC1 differentiation is based on SFEM II medium, with the addition of penicillin / streptomycin, and a combination of human cytokines SCF, Flt3L, GM-CSF, IL4 and IFNγ.

[0025] As one embodiment of the present invention, it includes at least one of the following technical features:

[0026] A. In the differentiation medium, penicillin / streptomycin 25U / ml-100U / ml, SCF 10-500ng / ml, Flt3L 10-500ng / ml, GM-CSF 1-200ng / ml, IL4 1-200ng / ml, and IFN γ 2-100ng / ml;

[0027] B. Change the medium every 4 days under the same conditions during differentiation culture;

[0028] C. Cells obtained from differentiation culture were sorted using flow cytometry with anti-human CLEC9A PE and anti-human CD141 PECY7 antibodies. The sorted CD141+CLEC9A+ cells were cDC1 cells.

[0029] The above-mentioned technical feature A preferably contains penicillin / streptomycin 50U / ml, SCF 200ng / ml5, Flt3L 200ng / ml, GM-CSF 2.5ng / ml, IL4 2.5ng / ml, and IFN γ 2-100ng / ml.

[0030] The aforementioned technical feature B preferably involves collecting cells on the 10th day.

[0031] <Second aspect>

[0032] The cDC1 cells prepared by the method of this invention are within the scope of protection of this invention.

[0033] <Third aspect>

[0034] The method of this invention or the application of cDC1 cells in the preparation of type I classical dendritic cell tumor vaccines also fall within the scope of protection of this invention.

[0035] <Fourth Aspect>

[0036] The method of this invention or the application of cDC1 cells in the preparation of type I classical dendritic cell antitumor drugs also fall within the scope of protection of this invention.

[0037] The tumors mentioned include various solid tumors and hematologic malignancies; specifically, they include, but are 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's tumor, cervical cancer, testicular cancer, stomach cancer, colon cancer, genitourinary cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell carcinoma, endometrial cancer, and adrenal cortex cancer. 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, piloblastic 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.

[0038] Differentiation from umbilical cord blood stem cells to cDC1 cells is regulated by various transcription factors. Some transcription factors promote cDC1 cell differentiation (including IRF8, BATF3, etc.), while others inhibit it (including ZEB2, IRF4, etc.). This invention improves cDC1 induction efficiency by knocking out the transcription factor zeb2, which inhibits cDC1 differentiation. Furthermore, the therapeutic effect of the obtained cDC1 cells on the tumor model was evaluated using a humanized mouse tumor model. Compared with existing technologies, this invention improves the induction efficiency of primary human cDC1 cells and increases the number of cDC1 cells obtained, thereby reducing the cost of cDC1 cell therapy. Attached Figure Description

[0039] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0040] Figure 1 This is a flowchart illustrating the method for improving cDC1 induction efficiency according to the present invention.

[0041] Figure 2 A schematic diagram illustrating the effect of knockout of three zeb2 sgRNAs detected by flow cytometry intracellular staining.

[0042] Figure 3 A comparison of the induction efficiency of cDC1 by knocking out the zeb2 gene; where sgZEB2-1, 2, and 3 are different sgRNAs targeting the human zeb2 gene, and sgIRF4 is the sgRNA targeting the human IRF4 gene.

[0043] Figure 4 A schematic diagram showing the expression of cytokines IL12p70 and CXCL10 by stimulating cDC1 cells obtained from isolation and differentiation with PolyIC and PAM.

[0044] Figure 5 To load zeb2 knockout cDC1 cells with antigen, and then co-incubate them with CD8+ T cells that recognize the antigen, the proliferation of CD8+ T cells was detected; where, ns, no significant; **p<0.01; ***p<0.001, ****p<0.0001;

[0045] Figure 6 This is a schematic diagram illustrating the therapeutic effect of cDC1 cells on a tumor model; where *p<0.05, ***p<0.001. Detailed Implementation

[0046] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0047] Example 1

[0048] This embodiment provides a method for improving the induction efficiency of human cDC1 cells; the process is as follows: Figure 1 As shown:

[0049] 1. Expansion of umbilical cord blood stem cells

[0050] Uninuclear cells were obtained by density gradient centrifugation of umbilical cord blood using Ficoll-Paque Plus (GE Healthcare), and CD34+ umbilical cord blood stem cells were then isolated using the CD34 positive selection kit (Miltenyi Biotec).

[0051] Cultured under the following conditions: SFEM II medium (STEMCELL Technologies), supplemented with penicillin / streptomycin (final concentration 50 U / ml, Gibco, range 25 U / ml-100 U / ml), and human cytokines: human SCF (200 ng / ml, range 20-500 ng / ml), human Flt3L (200 ng / ml, range 20-500 ng / ml), and human TPO (200 ng / ml, range 20-500 ng / ml). Amplification was performed for 2 days (range 1-2 days). The culture density was 1×10⁶. 5 / ml.

[0052] 2. Gene editing of umbilical cord blood stem cells

[0053] Gene editing of umbilical cord blood stem cells was performed by electroporation of a mixture of CAS9 protein and humanzeb2 gene sgRNA using the Invitrogen™ Neon transfection system (Invitrogen). (The induction efficiency of cDC1 was improved by knocking out zeb2.) The CAS9 protein was 1 μg (range 0.5-5 μg), and the 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.

[0054] All three zeb2 sgRNAs can be used individually. The sgRNA sequences are as follows:

[0055] Sgzeb2-1 5'-GGCGCAAACAAGCCAATCCC-3' (SEQ ID NO.1),

[0056] Sgzeb2-2 5'-TGTTTGCGCCTCTTGCACCG-3' (SEQ ID NO.2),

[0057] Sgzeb2-3 5'-ATCCAGACCGCAATTAACAA-3' (SEQ ID NO. 3).

[0058] Collect 1×102 pre-expanded umbilical cord blood stem cells obtained in step 1. 5 Cells were resuspended in 5 μl of R buffer, then mixed with CAS9 protein and sgRNA, and electroporated. Electroporation conditions were: 1400 V, 10 ms / pulse, 3 pulses. Other conditions were: 1) 1600 V, 20 ms / pulse, 1 pulse; 2) 1500 V, 10 ms / pulse, 3 pulses. The electroporated cells were then cultured in the umbilical cord blood stem cell culture medium described in step 1 for 2 days at a density of 1 × 10⁶ cells / day. 5 / ml.

[0059] In this embodiment, cells were first collected two days after electroporation, and the knockout effect of the three sgRNAs on zeb2 was detected by intracellular flow cytometry. It was found that sgZEB2-2 had the highest knockout efficiency; as shown... Figure 2 As shown, the knockout effects are sgzeb2-2, sgzeb2-1, and sgzeb2-3 in sequence.

[0060] 3. Differentiation of Cdc1 cells

[0061] Next, cDC1 differentiation was induced using SFEM II medium (STEMCELL Technologies, p. 1), supplemented with penicillin / streptomycin (final concentration 50 U / ml, Gibco, range 25 U / ml-100 U / ml). Human cytokines were also added: 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 IFN-γ (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.

[0062] The collected cells were stained using flow cytometry with anti-human CLEC9A PE, anti-human CD141 PECY7, and anti-human CD1C BV650 antibodies. Figure 3 The results showed that knocking out the zeb2 gene significantly improved the induction efficiency of cDC1. However, knocking out another transcription factor that inhibits cDC1 differentiation, IRF4, had no effect (sgIRF4 sequence: 5'-CTTTAAACAGTGCCCAAGCC-3', SEQ ID NO.4). The efficiency improvements were as follows: sgzeb2-2, sgzeb2-1, sgzeb2-3; the highest efficiency improvement was observed in sgZEB2-2.

[0063] The sorted CD141+CLEC9A+ cells were designated as cDC1 cells. They were then stimulated for 24 hours with 10 μg / ml PolyI:C (1-10 μg / ml) and 10 μg / ml R848 (1-10 μg / ml). Figure 4 The results showed that knockout of the zeb2 gene did not affect the production of cDC1 cytokines. Even after zeb2 knockout, cDC1 still highly expressed the cytokines IL12p70 and CXCL10.

[0064] Next, cDC1 cells were co-incubated with tumor cell lysate antigen for 12 hours, and then co-incubated with CD8+ T cells for 72 hours (DC to T cell ratio of 1:1). CD8+ T cell proliferation was then assessed. The results showed that knockout of the zeb2 gene promoted cDC1 activation of CD8+ T cells. Figure 5 Moreover, sgzeb2-2 showed the most significant effect. Given that sgzeb2-2 had the most obvious in vitro effect, the antitumor effect of cDC1 knocked out with sgzeb2-2 was tested in a tumor model.

[0065] The tumor cell lysate antigen can be prepared through the following steps: 1) Resuspend a 1×10^6 / ml tumor cell line in DMEM medium supplemented with hypochlorous acid to a final concentration of 60 μM, and incubate at 37°C for 1 h; 2) Wash twice with PBS, centrifuge at 500 g, 4°C, for 5 min; 3) Resuspend the tumor cell pellet in PBS to a concentration of 1×10^7 / ml, then freeze at -80°C for 1 h, and thaw at room temperature. Repeat the freeze-thaw cycle at least 6 times; 4) Finally, co-incubate the tumor lysate with DCs in a ratio of equal amounts of lysate obtained from tumor cells and equal amounts of cDC1.

[0066] 4. Humanized mouse tumor model

[0067] HLA0201 genotype umbilical cord blood stem cells from the umbilical cord blood bank were injected via the tail vein into NCG-X mice (Jicui Yaokang). After 20 weeks, mice with human immune systems were obtained. Next, the mice were subcutaneously injected with 1×10 6 A melanoma model was constructed using an A375 tumor cell line.

[0068] 5. cDC1 subset cells used for tumor model therapy

[0069] After tumor formation in mice, human cDC1 cells with the HLA0201 genotype were in vitro activated (in vitro activation was performed according to steps 1-3 above) (range: 5 × 10⁻⁶). 6 -1×10 9 (1 cell / kg body weight / dose, once a week) was injected intratumorally into tumor model mice. Tumor changes were dynamically monitored. The results showed that zeb2 knockout of cDC1 had a stronger anti-tumor effect (see...). Figure 6It should be noted that the cDC1 subset of cells can be used for the treatment of various tumors, including various solid tumors and hematologic malignancies; it is not limited to melanoma as described in this example. The inhibitory effects on various tumors were tested, and the procedures 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 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). Given that 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 broad anti-tumor potential and is suitable for the treatment of various solid tumors and hematological malignancies.

[0070] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can 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 preparing a type I classical dendritic cell anti-tumor drug or a type I classical dendritic cell tumor vaccine by inducing differentiation of cDC1 cells obtained by knocking out the transcription factor ZEB2 in umbilical cord blood stem cells; the cDC1 cells are induced to differentiate by knocking out the transcription factor ZEB2 in umbilical cord blood stem cells, and the sgRNA for targeting the knockout of the transcription factor ZEB2 in umbilical cord blood stem cells is selected from SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The tumors include various solid tumors and hematological tumors.

3. The use according to claim 2, characterized in that The tumors include breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, ovarian cancer, brain cancer, head and neck cancer, liver cancer, bladder cancer, lung cancer, Wilm's tumor, cervical cancer, stomach cancer, thyroid cancer, esophageal cancer, myeloma, adrenal cancer, renal cell carcinoma, endometrial cancer, adrenocortical carcinoma, malignant pancreatic insulinoma, malignant carcinoid, choriocarcinoma, leukemia, neuroblastoma, sarcoma, germ cell tumor, polycythemia vera, essential thrombocythemia and retinoblastoma.

4. The use according to claim 3, characterized in that The lymphoma includes Hodgkin's disease or non-Hodgkin's lymphoma; The leukemia includes acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia or chronic myeloid leukemia; The sarcoma includes Kaposi's sarcoma, rhabdomyosarcoma, soft tissue sarcoma or osteogenic sarcoma; The lung cancer includes non-small cell lung cancer or small cell lung cancer; The brain cancer includes glioma or glioblastoma; The germ cell tumors include testicular tumors.

5. The use according to claim 1, characterized in that The knockout comprises mixing pre-amplified umbilical cord blood stem cells with CAS9 protein and sgRNA, electroporating, and culturing.

6. The use according to claim 5, 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 isolated using a CD34 positive selection kit; B. The culture medium used for the pre-amplification is SFEM II as the basal medium, supplemented with penicillin / streptomycin, and a combination of human cytokines SCF, Flt3L, and TPO; the concentrations of SCF in the culture medium are 20-500 ng / ml, Flt3L 20-500 ng / ml, and TPO 20-500 ng / ml; C. Pre-amplified umbilical cord blood stem cells were 1×10 5 Resuspend the cells in 5 μl R buffer and mix with CAS9 protein and sgRNA; in the mixed system, 0.5-5 μg CAS9 protein / 6 μl and 100 ng / 6 μl-1 μg sgRNA are mixed in R buffer; incubate at room temperature for 5-20 minutes; D. Electroporation conditions: 1400v-1600v, 10-20 ms / pulse, 1-3 pulses; E. The culture medium used for cell culture after electroporation is the same as the pre-amplification medium.

7. The use according to claim 1, characterized in that The differentiation medium used to induce cDC1 differentiation was SFEMII basal medium supplemented with penicillin / streptomycin and a combination of human cytokines SCF, Flt3L, GM-CSF, IL4, and IFNγ.

8. The use according to claim 7, characterized in that Include at least one of the following technical features: A, Differentiation medium containing SCF 10-500 ng / ml, Flt3L 10-500 ng / ml, GM-CSF 1-200 ng / ml, IL4 2.5 ng / ml-200 ng / ml, and IFNγ 2-100 ng / 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. The sorted CD141+CLEC9A+ cells were cDC1 cells.

Images