Preparation method of adult T cells and universal adult CAR-T cells induced and differentiated by pluripotent stem cells

Abstract

The invention provides a preparation method of adult T cells and universal adult CAR-T cells induced and differentiated by pluripotent stem cells, and belongs to the technical field of cell differentiation. By intervening the SP140 gene or SP140 protein, advanced hematopoiesis of pluripotent stem cells can be induced, and hematopoietic precursor cells and adult T cells with T gonadotrophy differentiation potential are obtained. The inducible CAR is knocked into the TRAC site of the pluripotent stem cell at a fixed point, so that endogenous regulation of CAR expression is realized, the in-vivo maintenance and anti-tumor capability of CAR-T is improved, and TCR-mediated graft versus host disease is blocked. The invention also provides a separated CAR-T cell, the CAR-T cell aiming at different antigens can be prepared by editing a seed cell of a pluripotent stem cell once, B2M and CIITA genes are further knocked out, HLA-E is overexpressed, and the immunogenicity of the CAR-T cell and the resistance of a host to the CAR-T cell are reduced.

Description

Technical Field

[0001] This invention belongs to the field of cell differentiation technology, specifically relating to a method for preparing adult T cells and universal adult CAR-T cells induced from pluripotent stem cells. Background Technology

[0002] Chimeric Antigen Receptor (CAR-T cell) therapy involves infecting a patient's own or another person's T cells with a CAR virus in vitro, and then reinfusing the quality-controlled CAR-T cells back into the patient to precisely identify and attack tumor cells, thus treating the disease. Currently, CAR-T cell therapy has made groundbreaking progress in the treatment of liquid tumors and solid tumors, bringing new hope to cancer treatment. However, one of the most prominent problems with CAR-T cell therapy is the limitation of the quality and quantity of the patient's own T cells, which directly affects the feasibility and efficacy of the therapy.

[0003] Current in vitro T cell differentiation protocols can only differentiate T cells that are predominantly embryonic in origin. These T cells have three main characteristics: first, they are derived from hematopoietic progenitor cells during embryonic development, exhibiting weak T-lymphocyte differentiation potential; second, they do not express DNTT during T cell differentiation, resulting in limited TCR diversity and poor anti-tumor ability; and third, they are more responsive to stimulation by innate cytokines such as IL12 / IL18, rapidly secreting effector molecules like IFNγ. Therefore, the limited availability of T cells severely restricts the application of CAR-T cell therapy in cancer treatment. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing adult T cells induced from pluripotent stem cells. This method promotes the differentiation of human pluripotent stem cells into adult T cells by inhibiting the expression or function of SP140 at the gene or protein level in conjunction with other cytokines. BCL11A Positive hematopoietic precursor cells differentiate, and then further differentiate to obtain adult-like T cells.

[0005] This invention provides a method for inhibiting SP140 Application of reagents for gene and / or SP140 protein expression in the preparation of at least one cell type from pluripotent stem cells, including hematopoietic progenitor cells, adult T cells, and CAR-T cells.

[0006] Preferably, the pluripotent stem cells include embryonic stem cells and / or induced pluripotent stem cells.

[0007] Preferably, the inhibition SP140 Reagents for gene expression include those that knock down SP140 protein using RNA interference technology orSP140 an agent for knocking out SP140 protein or SP140 an agent for gene expression; The agent for inhibiting the expression of SP140 protein includes an SP140 protein inhibitor.

[0008] The application provides a composition for preparing CAR-T cells based on multifunctional stem cells, which comprises an agent for inhibiting SP140 gene and / or SP140 protein expression, knocking out TRAC gene with an agent, Cre recombinase recombinant virus and conditional knockout agent; The conditional knockout agent includes a first recombinant expression vector or a second recombinant expression vector and a CAR fusion protein. The first recombinant expression vector comprises the following gene elements in series: TRAC gene left homologous arm LoxP - TRAC CDS -first ployA- LoxP- enzyme cutting site - first connecting peptide sequence -CAR -second ployA-TRAC gene right homologous arm The second recombinant expression vector comprises the following gene elements in series: TRAC gene left homologous arm LoxP - TRAC CDS -first ployA- LoxP- enzyme cutting site - connecting peptide sequence - leucine zipper sequence -CAR signaling domain-second ployA-TRAC gene right homologous arm The CAR fusion protein includes an antibody signal peptide, a single-chain antibody and a leucine zipper ligand.

[0009] Preferably, the agent for knocking out TRAC gene includes sgRNA or CRISPR Cas gene editing vector containing the sgRNA targeting TRAC gene; the sgRNA targeting TRAC gene includes sgRNA1 and sgRNA2; the DNA sequence corresponding to the sgRNA1 is shown as SEQ ID NO: 10; the DNA sequence corresponding to the sgRNA2 is shown as SEQ ID NO: 11.

[0010] Preferably, the nucleotide sequence of the exogenous gene contained in the first recombinant expression vector is as shown in SEQ ID NO: 54; The nucleotide sequence of the exogenous gene contained in the second recombinant expression vector is as shown in SEQ ID NO: 55; The amino acid sequence of the CAR fusion protein is as shown in SEQ ID NO: 56; The Cre Recombinant enzyme Recombinant virus Recombinant expression Cre A fusion protein formed by the coding sequence of the recombinase and the mutant ligand binding domain of the estrogen receptor.

[0011] Preferably, it also includes a sgRNA targeting B2M The sgRNA of the sgRNA or the CRISPR Cas gene editing vector containing the sgRNA and the third recombinant vector; The third recombinant vector includes the following gene elements in series: B2M Gene left homologous arm-HLA-E secretion signal peptide-HLA-E CDS-polyA sequence B2M Gene right homologous arm; The targeting B2M The nucleotide sequence of the sgRNA of the gene is as shown in SEQ ID NO: 37.

[0012] Preferably, it also includes a knockout CIITA The knockout CIITA The knockout reagent includes a sgRNA targeting CIITA The sgRNA of the sgRNA or the CRISPR Cas gene editing vector containing the sgRNA; The targeting CIIT The nucleotide sequence of the sgRNA of the gene is as shown in SEQ ID NO: 49.

[0013] The application provides a use of a composition for preparing CAR-T cells based on multifunctional stem cells in the preparation of anti-tumor CAR-T cells or drugs.

[0014] The application provides a method for preparing anti-tumor CAR-T cells based on a composition for preparing CAR-T cells based on multifunctional stem cells, comprising the following steps: The human pluripotent stem cells are treated with the composition, in vitro induced differentiation, and the obtained hematopoietic precursor cells are in vitro differentiated to obtain adult T cells; The adult T cells are treated by tamoxifen induction, and the expression of the gene is knocked out TRAC The CAR, to obtain CAR-T cells.

[0015] The application provides a method for inhibitingSP140 The invention relates to the application of reagents that inhibit the expression of the SP140 gene and / or protein in the preparation of at least one cell type, including hematopoietic progenitor cells, adult T cells, and CAR-T cells, from human pluripotent stem cells. Specifically, this invention describes the in vitro differentiation of hematopoietic progenitor cells, which are similar to postnatal bone marrow hematopoietic stem-progenitor cells (HSPCs), by using reagents that inhibit SP140 expression at the gene or protein level. These cells specifically express the BCL11A gene, a key factor in hematopoietic regulation, and possess the developmental potential to differentiate into adult-like lymphocytes. The reagents facilitate efficient in vitro differentiation into hematopoietic progenitor cells and adult T cells, with CD34 produced on day 2 of hematopoietic differentiation. + The hematopoietic endothelial cells produce CD45 on day 3. + The hematopoietic progenitor cells of the control group (WT) produced CD34 only on day 6. + The hematopoietic endothelial cells produce CD45 on day 7. + The reagent significantly shortens the differentiation time of hematopoietic progenitor cells and yields a large number of hematopoietic progenitor cells and adult-type T cells, meeting the clinical needs for CAR-T cell preparation. Adult-type T cells obtained by further differentiating hematopoietic progenitor cells prepared from human pluripotent stem cells treated with the reagent express adult-type marker genes. DNTT Due to the properties of the (encoding terminal transferase TdT), the adult-type cells exhibit better maintenance and tumor-killing capabilities. Furthermore, CAR-T cells prepared from adult-type T cells treated with the aforementioned reagents demonstrate stronger tumor-killing capabilities compared to control cells (CAR-T cells prepared using the aforementioned reagents are omitted).

[0016] This invention provides a composition for preparing CAR-T cells based on pluripotent stem cells, comprising the inhibitory component used in the above application. SP140 Reagents for gene or SP140 protein expression, knockout reagents Cre Recombinase, recombinant virus, and conditional knockout reagent; wherein the first recombinant expression vector in the conditional knockout reagent comprises the following tandem gene elements: TRAC Left homologous arm of the gene - LoxP - TRAC CDS -First PloyA- LoxP-SA Enzyme cleavage sites - First linker peptide sequence -CAR - The right homologous arm of the second ployA-TRAC gene. The first recombinant expression vector, in conjunction with a knockout reagent, is knocked into human pluripotent stem cells, hematopoietic progenitor cells, or adult T cells at a specific site. TRAC Gene loci, adult T cells obtained by in vitro differentiation induced by the reagents in... CreThe endogenous TCR can be conditionally knocked out under the infection of the recombinase recombinant virus, and the CAR is expressed, to obtain the CAR-T cell with conditionally knocked out TCR, and the CAR is activated due to the regulation of the element, and the CAR-T cell has stronger anti-tumor ability. TRAC The conditional knockout reagent also comprises a second recombinant expression vector and a CAR fusion protein, the CAR is divided into two parts, one part of the CAR structure (CAR signaling domain, denoted as CAR1) is fused with the leucine zipper to replace the CAR in the first recombinant expression vector, to form the second recombinant expression vector, and the other part is fused with the leucine zipper ligand (SYN) to form the CAR fusion protein. The second recombinant expression vector is expressed in the human pluripotent stem cell, the hematopoietic precursor cell or the adult T cell according to the expression method of the first recombinant expression vector, to obtain the CAR-T cell with conditionally knocked out TCR through in-vitro induction and differentiation culture, and then the CAR-T cell is incubated with the CAR fusion protein to obtain the isolated CAR-T cell. The isolated CAR-T cell uses the pairing and binding characteristics of the leucine zipper to modify the CAR structure, one part of the CAR structure is inserted into TRAC the gene to knock out the TCR, and the other part of the CAR structure is fused with the leucine zipper ligand to be expressed, and different single-chain antibodies (scFv) are synthesized according to different antigens. Experiments show that the CAR-T cell with the separated CAR structure has good tumor killing effect, and the cell for knocking out the TCR is constructed with the unchanged CAR1 structure, and then different CAR fusion proteins are introduced according to different requirements, to achieve the purpose of killing different types of tumors, which greatly simplifies the experimental operation and makes the preparation of the anti-tumor drug more flexible.

[0017] The application provides a composition for preparing the CAR-T cell based on the multifunctional stem cell, which further comprises a knockout reagent for targeting B2M the gene and a third recombinant vector for knocking in the HLA-E gene at the gene site on the basis of the two groups of reagents. B2M The composition can be used for preparing the CAR-T cell with conditionally knocked out TCR and expressing the HLA-E molecule. B2M The application prepares the adult T cell by knocking out the HLA molecule (HLA-A, HLA-B and HLA-C) and knocking in the HLA-E, to avoid the activation of the CD8 + T cell and the NK cell and the immune rejection killing caused by the activation, so that the drug efficacy of the cell therapy is ensured. + The experimental results show that, compared with the control group, the CAR-T cell introduced into the body greatly reduces the activation and proliferation quantity of the CD8 + T cell and the NK cell, ensures that a sufficient number of T cells accurately recognize and attack the tumor cells, and greatly achieves the anti-tumor purpose.

[0018] The application provides a composition for preparing CAR-T cells based on multifunctional stem cells, which further comprises a reagent for knocking out CIITA a gene on the basis of the three groups of reagents CIITA Experimental results show that after knocking out CIITA the CAR-T cells differentiated in culture, the proportion of CD4 + T cells in the proliferative activation state is generally significantly reduced. This shows that knocking out the gene by human pluripotent stem cells can reduce immune rejection caused by HLA-I class molecules. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Results of detection of the induction of differentiation of human pluripotent cells into hematopoietic precursor cells by SP140 inhibitors; Figure 2 Results of detection of the induction of differentiation of human pluripotent cells into hematopoietic precursor cells by knocking down SP140 gene expression; + Results of detection of CD45 Figure 3 Results of detection of CD5 + CD7 + cells in T cell differentiation culture (A) and gene expression in cells (B); DNTT Results of detection of the number of T cells in the SP140 inhibitor treatment group and the control group (A) and the secretion of IFNγ by cells after stimulation by IL-12 and IL-18 (B); Figure 4 Results of tumor killing in mice by CAR-T cells, wherein A is the process of the tumor killing experiment in mice, B is the survival period of the mice, and C is the results of tumor killing in vivo by CAR-T cells; Figure 5 Schematic diagram of a fusion gene of CAR-T cells with conditional knockout Figure 6 of gene expression; TRAC Results of detection of hPSCs with conditional knockout Figure 7 of gene expression CAR; wherein A is the detection result of gene editing; and B is the homozygosity detection result; TRAC Results of the influence of tamoxifen (OHT) induction or non-induction on the expression of CAR and Figure 8 in hPSCs; TRAC Results of tumor killing by CAR-T cells with conditional knockout Figure 9 of a gene; wherein A is the killing rate result; and B is the detection result of the secretion of cytokines by CAR-T cells; TRAC Figure 10The design concept and tumor-killing ability assay results of isolated CAT-T cells; Figure 11 In order to be in B2M hPSC detection results for HLA-E expression at specific sites; Figure 12 Inserting the B2M-HLA-E structure into B2M Detection results of the locus (A) and CD8 + Results of T cell activation and proliferation assay (B); Figure 13 Results of NK cell activation and proliferation assay; Figure 14 To knock out CIITA Results of sgRNA knockout of the gene; Figure 15 Results of flow cytometry detection of HLA-DR expression and CD4 expression on the surface of activated T cells (A) + Results of T cell activation and proliferation assay (B). Detailed Implementation

[0020] This invention provides a method for inhibiting SP140 Application of reagents for gene and / or SP140 protein expression in the preparation of at least one cell type from pluripotent stem cells, including hematopoietic progenitor cells, adult T cells, and CAR-T cells.

[0021] In this invention, the inhibition SP140 Preferred agents for gene expression include knocking down SP140 protein using RNA interference technology and / or SP140 Reagents for gene expression levels or using CRISPR / Cas9 to knock out SP140 protein or SP140 Reagents for gene expression. The method utilizes RNA interference technology to knock down SP140 protein or... SP140 The preferred reagents for gene expression level include those targeting... SP140 The gene's siRNA, shRNA, or recombinant vector containing siRNA or shRNA. The SP140 protein is knocked out using the CRISPR / Cas9 method. SP140 Gene expression reagents preferably include targeted agents. SP140 The sgRNA of the gene or a gene editing vector containing the sgRNA. The reagent for inhibiting SP140 protein expression preferably includes an SP140 protein inhibitor. In this embodiment of the invention, GSK761 is used as the SP140 protein inhibitor, and SP140-shRNA-1 (corresponding DNA sequence CCGAGCAGATGTATGAACA) and SP140-shRNA-2 (corresponding DNA sequence CAAGAGTGATGTATTGTGT) are used as interferons. SP140The gene expression reagents were used for the experiment. The preparation method of GSK761 was based on existing technology (Ghiboub, M., Koster, J., Craggs, PD et al. Modulation of macrophage inflammatory function through selective inhibition of the epigenetic reader protein SP140. BMC Biol 20, 182 (2022). https: / / doi.org / 10.1186 / s12915-022-01380-6).

[0022] In this invention, the species of pluripotent stem cells is not particularly limited; common animal and human cell sources are acceptable. Preferably, the pluripotent stem cells include embryonic stem cells and / or induced pluripotent stem cells. Experiments were conducted on embryonic stem cells and induced pluripotent stem cells as initially differentiated cells, respectively. Results showed that the reagent, by interfering with SP140 expression at both the gene and protein levels, promotes the differentiation of pluripotent stem cells into hematopoietic progenitor cells, not only shortening the induction differentiation time but also producing CD34 on day 2. + The hematopoietic endothelial cells produce CD45 on day 3. + The hematopoietic progenitor cells of the control group (WT) produced CD34 only on day 6. + The hematopoietic endothelial cells produce CD45 on day 7. + The differentiation effect of embryonic stem cells and induced pluripotent stem cells was significantly improved, and a large number of hematopoietic progenitor cells could be obtained.

[0023] In this invention, the method for preparing hematopoietic progenitor cells from pluripotent stem cells is preferably divided into two types depending on the type of reagent: when the reagent is an inhibitor... SP140 When using the gene expression reagent, the reagent is introduced into pluripotent stem cells, followed by identification and screening. SP140 Cells with downregulated gene expression were subjected to in vitro induced differentiation culture; when the reagent was an SP140 protein inhibitor, the SP140 protein inhibitor was added to the culture medium for culture.

[0024] In this invention, the culture medium for in vitro induced differentiation culture preferably includes a culture medium for the first day of culture, a culture medium for the second day of culture, and a culture medium for the third to sixth days of culture. The culture medium used for the first day of culture is preferably a basal medium containing 1~200 ng / mL rhBMP4, 1~50 μM SB-431542, 1~50 μM CHIR-99021, 1~200 ng / mL rhVEGF, and 2~100 μM RETA. It can also be a basal medium containing 30~50 ng / mL rhBMP4, 8~12 μM SB-431542, 16~20 μM CHIR-99021, 18~22 ng / mL rhVEGF, and 70~90 nM RETA. Alternatively, it can be a basal medium containing 40 ng / mL rhBMP4, 10 μM SB-431542, 18 μM CHIR-99021, 20 ng / mL rhVEGF, and 80 nM retinyl acetate (RETA). The preferred culture medium for the second day is 1-200 ng / mL rhBMP4, 1-200 ng / mL IGF1, 1-200 ng / mL IGF2, 1-200 ng / mL SCF, and 2-100 μM RETA. Alternatively, it can be a basal medium containing 10-20 ng / mL rhBMP4, 30-50 ng / mL IGF1, 18-22 ng / mL IGF2, 30-50 ng / mL SCF, and 1.8-2.2 μM RETA, or a basal medium containing 15 ng / mL rhBMP4, 40 ng / mL IGF1, 20 ng / mL IGF2, 40 ng / mL SCF, and 2 μM RETA. The culture medium used for the third to sixth days of culture is preferably a basal medium containing 1-200 ng / mL IGF1, 1-200 ng / mL SCF, 2-100 μM RETA, 1-200 ng / mL TPO, 2-100 nM SR1 (StemRegenin1), and 1-200 ng / mL bFGF. It can also be a basal medium containing 20 ng / mL IGF1, 40 ng / mL SCF, 120 nM RETA, 40 ng / mL TPO, 25 nM SR1 (StemRegenin1), and 40 ng / mL bFGF. Alternatively, it can be a basal medium containing 20 ng / mL IGF1, 40 ng / mL SCF, 120 nM RETA, 40 ng / mL TPO, 25 nM SR1 (StemRegenin1), and 40 ng / mL bFGF.The basal culture medium was StemPro containing 1× GlutaMAX, 0.039 µL / mL 1-thioglycerol (Sigma-Aldrich), 50 µg / mL ascorbic acid, and 150 µg / mL transferrin (Sigma). TM -34 SFM (purchased from Gibco, 10639011). When using the SP140 protein inhibitor (GSK761), it is added to the culture medium for the first day of culture and the culture medium for the second day of culture. The working concentration of the SP140 protein inhibitor is preferably 2~1000 nM, but can be 18~22 nM or 20 nM.

[0025] In this invention, the initial cell density during the in vitro induced differentiation culture is preferably 1×10⁻⁶. 5 Above / ml, it can be 5×10 5 / ml~10×10 5 / ml. For in vitro differentiation induction, it is preferably performed in a 96-well U-bottom culture plate. The preferred parameters for in vitro differentiation induction culture are a hypoxic incubator at 37℃, 5% CO2, and 5% O2. During the in vitro differentiation induction culture, it is preferable to take samples daily and digest them with type II collagenase for 4-5 minutes to prepare single-cell suspensions for cell marker detection. Detection results show that CD34 is produced on day 2 of differentiation. + The hematopoietic endothelial cells produce CD45 on day 3. + The control group (WT, omitting SP140 protein inhibitor treatment) produced hematopoietic progenitor cells, while the control group (WT, omitting SP140 protein inhibitor treatment) under the same conditions did not produce CD34 until day 6. + The hematopoietic endothelial cells produce CD45 on day 7. + Hematopoietic precursor cells. This indicates that the use of inhibition... SP140 Reagents expressing the gene or SP140 protein, when used in differentiation culture, can not only promote the differentiation of pluripotent stem cells into hematopoietic progenitor cells, but also shorten the differentiation cycle, achieve efficient differentiation, and obtain a large number of hematopoietic progenitor cells. The hematopoietic progenitor cells are CD45 cells. + CD34 + The cells also specifically express BCL11A, a key factor in hematopoietic regulation. The prepared hematopoietic progenitor cells are similar to bone marrow hematopoietic stem and progenitor cells (HSPCs) and possess lymphocyte developmental potential.

[0026] In this invention, the method for preparing adult T cells from pluripotent stem cells preferably involves preparing hematopoietic progenitor cells from pluripotent stem cells according to the above method, followed by differentiation culture to obtain adult T cells. The differentiation culture medium preferably includes a culture medium for the first week, a culture medium for the second week, and a culture medium for the third to sixth weeks. The culture medium for the first week is preferably IMDM or StemSpan containing IL-3 0.001~0.2 μg / ml, SCF 0.001~0.2 μg / ml, Flt3L 0.001~0.2 μg / ml, IL-7 0.001~0.2 μg / ml, and TPO 0.001~0.2 μg / ml. TM SFEM medium can be IMDM or StemSpan containing IL-3 0.08~0.012 μg / ml, SCF 0.015~0.025 μg / ml, Flt3L 0.015~0.025 μg / ml, IL-7 0.009~0.011 μg / ml, and TPO 0.015~0.025 μg / ml. TM SFEM medium can also be prepared using IMDM or StemSpan containing IL-3 0.01 μg / ml, SCF 0.02 μg / ml, Flt3L 0.02 μg / ml, IL-7 0.01 μg / ml and TPO 0.02 μg / ml. TM SFEM medium. The preferred medium for the second week of culture includes StemSpan containing SCF 0.001–0.2 μg / ml, Flt3L 0.001–0.2 μg / ml, IL-7 0.001–0.2 μg / ml, and TPO 0.001–0.2 μg / ml. TM SFEM medium can be StemSpan containing SCF 0.015~0.025μg / ml, Flt3L 0.015~0.025μg / ml, IL-7 0.009~0.011μg / ml, and TPO 0.015~0.025μg / ml. TM SFEM medium can also be prepared using StemSpan containing SCF 0.02 μg / ml, Flt3L 0.02 μg / ml, IL-7 0.01 μg / ml, and TPO 0.02 μg / ml. TM SFEM medium. The preferred culture medium for the third to sixth weeks of culture includes StemSpan containing 0.001–0.2 μg / ml Flt3L, 0.001–0.2 μg / ml IL-7, and 0.001–0.2 μg / ml TPO. TMSFEM medium can be StemSpan containing Flt3L 0.015~0.025μg / ml, IL-7 0.009~0.011μg / ml, and TPO 0.015~0.025μg / ml. TM SFEM medium can also be used for StemSpan containing Flt3L 0.02 μg / ml, IL-7 0.01 μg / ml and TPO 0.02 μg / ml. TM SFEM culture medium. The preferred density of the hematopoietic progenitor cells is 1×10⁻⁶. 5 / well. During the differentiation culture, the culture medium should be changed to fresh medium every 2-3 weeks. The differentiation culture is preferably carried out on culture plates coated with human DLL4-Fc protein at concentrations of 1-100 μg / ml and VCAM-1 protein at concentrations of 1-100 μg / ml. The DLL4-Fc and VCAM-1 protein coating plates should be changed every week. The preferred differentiation culture time is 6 weeks.

[0027] In this invention, suppression is utilized. SP140 T cells were prepared by treating pluripotent stem cells with reagents that expressed the gene and / or SP140 protein, indicating the inhibition of T cells. SP140 The reagents used to express the gene and / or SP140 protein do not affect the differentiation of pluripotent stem cells into T cells. The differentiated T cells express a gene encoding the terminal transferase TdT. DNTT This gene is one of the characteristics of adult T cells, giving them abundant TCRs. Furthermore, the T cells prepared after stimulation with the combined IL-12 and IL-18 cytokines secreted a lower level of the effector IFNγ compared to the control group; low IFNγ secretion is another characteristic of adult T cells. This indicates that the hematopoietic stem precursor cells produced by the reagent treatment possess adult T cell differentiation potential, while the untreated control group produced more fetal-like T cells. The hematopoietic precursor cells produced by the control group that were not treated with SP140 inhibitors during the hematopoietic stage do not possess adult T cell differentiation potential.

[0028] In this invention, the inhibition is utilized SP140A method for preparing CAR-T cells by treating pluripotent stem cells with a reagent that expresses the SP140 protein includes gene modification of pluripotent stem cells to express CAR, gene modification of hematopoietic progenitor cells, and gene modification of adult T cells. Preferred is gene modification of pluripotent stem cells to express CAR, which is beneficial for improving the tumor-killing ability of CAR-T cells. Simultaneously, the conditional expression model of CAR provides a theoretical and practical basis for the construction of universal off-the-shelf CAR-T cells. Preferably, the method involves preparing hematopoietic progenitor cells using the above method, then differentiating and culturing them to obtain adult T cells, and infecting the cells with a recombinant virus expressing CAR to obtain CAR-expressing adult T cells. The antitumor activity of CAR-expressing adult T cells and CAR-expressing T cells was verified using a mouse tumor model. Results showed that CD19-CAR-T cells induced by differentiation after treatment with SP140 protein inhibitors exhibited stronger antitumor activity. Compared with the untreated group (WT) and the control group (DPBS), the SP140 inhibitor group showed significantly reduced fluorescence signal, significantly decreased tumor burden, and significantly improved mouse survival rate. These results indicate that SP140 inhibition can enhance the in vivo killing ability of CAR-T cells derived from hematopoietic progenitor cells.

[0029] This invention provides a composition for preparing CAR-T cells based on pluripotent stem cells, comprising the inhibitory component used in the above application. SP140 Reagents for gene or SP140 protein expression, knockout TRAC Gene reagents Cre Recombinase, recombinant virus, and conditional knockout reagent.

[0030] In this invention, different conditional knockout reagents are selected according to the type of CAR-T cells prepared. The CAR-T cells preferably include conditional knockout CAR-T cells and isolated CAR-T cells. When preparing conditional knockout CAR-T cells, the conditional knockout reagent includes a first recombinant expression vector; the first recombinant expression vector contains the following tandem gene elements: TRAC Left homologous arm of the gene - LoxP - TRAC CDS -First PloyA- LoxP- Enzyme cleavage sites - First linker peptide sequence -CAR -Second PloyA- TRAC The right homologous arm of the gene. The restriction enzyme site is preferably an SA restriction enzyme site. The first polyA is preferably bGH polyA. The second polyA is preferably an SV40 polyA sequence. TRACThe nucleotide sequence of the left homologous arm of the gene is shown in SEQ ID NO: 1; the nucleotide sequence of the right homologous arm of the TRAC gene is shown in SEQ ID NO: 2; and the nucleotide sequence of the SA cleavage site is shown in SEQ ID NO: 3. The linker peptide is preferably the T2A linker peptide, and its corresponding nucleotide sequence is shown in SEQ ID NO: 4. LoxP The nucleotide sequence of the sequence is shown in SEQ ID NO: 5. Preferably, the CAR is 1928ζCAR, and the nucleotide sequence of the full-length 1928ζCAR is shown in SEQ ID NO: 6; the nucleotide sequence of SV40 polyA is shown in SEQ ID NO: 7; and the nucleotide sequence of bGH polyA is shown in SEQ ID NO: 8. TRAC The nucleotide sequence of the CDS sequence of the gene is shown in SEQ ID NO: 9. The nucleotide sequence of the exogenous gene contained in the first recombinant expression vector is shown in SEQ ID NO: 54. The first recombinant expression vector will carry... TRAC Genes and coding CAR The fusion gene fragment of the sequence was cloned into the TRAC gene, and the TRAC gene in the fusion gene was flanked by clones. LoxP Site, for easy Cre Recombinase digestion enables conditional knockout of the TRAC gene, reducing the incidence of transplant resistance in the host. CAR The CAR-T cells prepared by the TRAC element have stronger anti-tumor capabilities.

[0031] In this invention, when preparing isolated CAR-T cells, the conditional knockout reagent includes a second recombinant expression vector and a CAR fusion protein. When preparing isolated CAR-T cells, the CAR is divided into two parts; one part replaces the CAR portion in the first recombinant expression vector with a leucine zipper sequence, achieving conditional knockout. TRAC The gene simultaneously expresses part of the CAR structure; the other part of the CAR structure is fused with a leucine zipper ligand to form a CAR fusion protein, and the two bind via a leucine zipper and a leucine zipper ligand. The second recombinant expression vector preferably contains the following tandem gene elements: TRAC Left homologous arm of the gene - LoxP - TRAC CDS -First PloyA- LoxP- Enzyme cleavage sites - Linking peptide sequence - Leucine zipper sequence -CAR Signal transduction domain - the second polyA-TRAC gene right homologous arm. TRAC Left homologous arm of the gene LoxP -、 TRACCDS First polyA, restriction enzyme site 、 Linking peptide sequence, second polyA, TRAC The right homologous arm of the gene is the same as that of the first recombinant expression vector, and will not be described further here. The nucleotide sequence of the leucine zipper sequence is shown in SEQ ID NO:15. CAR The signal transduction domain is 1928ζCAR. CAR The signal transduction domain, with a nucleotide sequence as shown in SEQ ID NO:16. The nucleotide sequence of the exogenous gene contained in the second recombinant expression vector, as shown in SEQ ID NO:55.

[0032] In this invention, the CAR fusion protein preferably comprises an antibody signal peptide, a single-chain antibody, and a leucine zipper ligand, and more preferably, a second linker peptide is used to connect the single-chain antibody and the leucine zipper ligand. The antibody signal peptide preferably adapts to different antibody types. The nucleotide sequence of the signal peptide of the anti-CD19 antibody is shown in SEQ ID NO:57 (MLLLVTSLLLCELPHPAFLLIP). The nucleotide sequence of the single-chain antibody against CD19 is shown in SEQ ID NO:14: DIQMTQTTSSLSASLG DRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQE SGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS. The amino acid sequence of the anti-CD20 scFV sequence is shown in SEQ ID NO: 58: MLLLVTSLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMS CKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIK. The amino acid sequence of the anti-CD22 scFV sequence is shown in SEQ ID NO:59: MELGLSWIFLLAILKGVQCQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSN SAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQSPSSSLSASVGDRVTITC RASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS. The nucleotide sequence of SYN22 of the anti-CD19 scFV is shown in SEQ ID NO: 60: KRIAYLRKKIAALKKDNANLEKDIANLENEIERLIKEIKTLENEVASHEQ. The nucleotide sequence of SYN2 targeting anti-CD20 scFV is shown in SEQ ID NO:61: ARNAYLRKKIARLKKDNLQLERDEQNLEKIIANLRDEIARLENEVASHEQ. The nucleotide sequence of SYN9 targeting anti-CD22 scFV is shown in SEQ ID NO:62: QKVESLKQKIEELKQRKAQLKNDIANLEKEIAYAET. The second linker peptide is preferably GGGSGGGGSGGGGS (SEQ ID NO:63). The amino acid sequence of the CAR fusion protein is shown in SEQ ID NO:56. The CAR fusion protein is preferably obtained by biosynthesis, specifically by cloning the fusion gene encoding the CAR fusion protein into a backbone vector, introducing the resulting recombinant expression vector into a host for expression, and then isolating and purifying to obtain the CAR fusion protein. The design of the second recombinant expression vector and the CAR fusion protein can be flexibly selected according to the tumor type to improve the anti-tumor range of the prepared CAR-T cells.

[0033] In this invention, the knockout TRAC Genetic reagents preferably include targeted reagents. TRAC The sgRNA of the gene or a CRISPR-Cas gene editing vector containing said sgRNA; the targeted TRAC The gene's sgRNA includes sgRNA1 with the nucleotide sequence shown in SEQ ID NO:10 and sgRNA2 with the nucleotide sequence shown in SEQ ID NO:11. The knockout... TRAC Genetic reagents are used to clone exogenous gene fragments from a first or second recombinant expression vector into... TRAC In genes.

[0034] In this invention, the Cre Recombinase, Recombinant Virus, Recombinant Expression Cre A fusion protein formed by a recombinase-encoding sequence and a mutant ligand-binding domain of the estrogen receptor. Cre Recombinase is expressed in cells after the recombinant virus infects the cells. Cre A fusion protein formed by a recombinase and a mutant ligand-binding domain of the estrogen receptor, induced by tamoxifen. Cre The recombinase activity increases the recognition of two LoxP sites, thereby cleaving the LoxP sites and their contents. TRAC Gene sequence, to achieve knockout under certain conditions.

[0035] In this invention, the composition preferably further includes a targeting mechanism. B2M The gene's sgRNA or a CRISPR-Cas gene editing vector containing said sgRNA and a third recombinant vector. The third recombinant vector preferably comprises the following tandem gene elements: the left homologous arm of the B2M gene - HLA-E secretion signal peptide - HLA-E CDS-polyA sequence - the right homologous arm of the B2M gene. The targeting... B2M The nucleotide sequence of the gene's sgRNA is shown in SEQ ID NO:37. The CRISPR-Cas gene editing vector containing the sgRNA, combined with the third recombinant vector, knocks the HLA-E gene into the B2M gene, achieving simultaneous expression of the HLA-E molecule while knocking out the B2M gene, thus avoiding CD8... + The activation of T cells and NK cells and the resulting immune rejection killing reduce the incidence of host transplantation disease.

[0036] In this invention, the composition preferably further includes knockout. CIITA Gene knockout reagents. CIITA Gene reagents preferably include targeted agents. CIITA The sgRNA of the gene or a CRISPR-Cas gene editing vector containing the sgRNA. The targeted gene... CIITA The nucleotide sequence of the gene's sgRNA is shown in SEQ ID NO:49. CIITA Gene knockout further helps to avoid CD8 + The activation of T cells and NK cells and the resulting immune rejection killing can reduce the incidence of host-associated transplant disease.

[0037] This invention provides the application of the composition for preparing CAR-T cells based on pluripotent stem cells in the preparation of anti-tumor CAR-T cells or drugs.

[0038] This invention provides a method for preparing anti-tumor CAR-T cells based on the aforementioned pluripotent stem cell preparation composition, comprising the following steps: Human pluripotent stem cells are treated with the composition and induced to differentiate in vitro. The resulting hematopoietic progenitor cells are then differentiated in vitro to obtain adult T cells. The adult T cells were induced with tamoxifen to knock out the TRAC gene and express CAR, thus obtaining CAR-T cells.

[0039] In this invention, the induction concentration of tamoxifen is preferably 0.08~0.12μM, and can be 0.1μM.

[0040] In this invention, the CAR-T cells include conditional knockout CAR-T cells, isolated CAR-T cells, and universal CAR-T cells. The conditional knockout CAR-T cells are preferably adult T cells expressing CAR with the TRAC gene conditionally knocked out; the adult T cells are inhibitory... SP140 Pluripotent stem cells were obtained by differentiation culture after treatment with a reagent that expressed the gene or SP140 protein; the pluripotent stem cells were transformed with a first recombinant expression vector and knocked out. TRAC Gene reagent was used to infect simultaneously Cre Pluripotent stem cells derived from recombinase and recombinant viruses. The isolated CAR-T cells are preferably adult T cells expressing CAR with the TRAC gene conditionally knocked out; wherein the CAR signaling domain in the CAR is linked to single-chain antibodies against different antigens via leucine zippers and leucine zipper ligands. The universal CAR-T cells are preferably knockout... B2M Gene or simultaneous knockout B2M Genes and CIITA Conditional knockout CAR-T cells or isolated CAR-T cells expressing the HLA-E gene. These universal CAR-T cells possess strong tumor-killing capabilities while exhibiting a low risk of host suppressor disease, significantly enhancing the efficacy of cell therapy.

[0041] The following detailed description, in conjunction with embodiments, illustrates a method for preparing adult T cells and universal adult CAR-T cells induced from pluripotent stem cells according to the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1 In vitro culture method for differentiation of human pluripotent stem cells into hematopoietic progenitor cells The basal medium was StemPro containing 1× GlutaMAX, 0.039 µL / mL 1-thioglycerol (Sigma-Aldrich), 50 µg / mL ascorbic acid, and 150 µg / mL transferrin (Sigma). TM -34 SFM (purchased from Gibco, 10639011).

[0043] On day 0, ES cells (H1) or iPS cells (DF-ZB11AL-D) were cultured in vitro using basal medium supplemented with 40 ng / mL rhBMP4 and 10 µM ROCK inhibitor. Specifically, 1×10 6 3 x 10 6 Cells were resuspended in 2 mL of culture medium in each well and placed in AggreWell 400 24-well plates (STEMCELL Technologies) and aggregated by centrifugation at 200g for 5 min. After 24 hours of culture, cells were cultured on day 1 using basal medium supplemented with 40 ng / mL rhBMP4, 10 μM SB-431542, 18 μM CHIR-99021, 20 ng / mL rhVEGF, 80 nM RETA, and 20 nM SP140 inhibitor. On day 2, cells were cultured on basal medium supplemented with 15 ng / mL rhBMP4, 40 ng / mL IGF1, 20 ng / mL IGF2, 40 ng / mL SCF, 2 μM RETA, and 20 nM SP140 inhibitor. Cells were cultured on basal medium supplemented with 20 ng / mL IGF1, 40 ng / mL SCF, 120 nM RETA, 40 ng / mL TPO, 25 nM SR1 (StemRegenin1) and 40 ng / mL bFGF on days 3–6.

[0044] On day 7, aggregates were collected, centrifuged at 200g for 5 min, the supernatant was discarded, and the cells were digested with type II collagenase for 4-5 min to prepare a single-cell suspension. The cells were washed and counted. CD34 was enriched using the Miltenyi Biotec CD34 positive selection kit. + Hematopoietic precursor cells.

[0045] Comparative Example 1 Human pluripotent stem cells were induced to differentiate into hematopoietic progenitor cells in vitro according to the method of Example 1, the only difference being that the SP140 inhibitor was omitted from the culture medium used on the second and third days.

[0046] Flow cytometry was used to monitor the culture of ES cells or iPS cells in Example 1 and Comparative Example 1 from day 1 to day 7. The results are shown in the figure. Figure 1 In Example 1, the hematopoietic progenitor cells were designated as the inhibitor group, and Comparative Example 1 was designated as the control group. Treatment with the SP140 inhibitor induced ES cells or iPS cells to differentiate prematurely and produce CD45. + CD34 +Hematopoietic progenitor cells: CD34 is produced on day 2 of hematopoietic differentiation culture. + The hematopoietic endothelial cells produce CD45 on day 3. + The blood cells produced in the control group (e.g., WT) were only produced on day 6, while in the control group (WT) under the same conditions, CD34 was not produced until day 6. + The hematopoietic endothelial cells produce CD45 on day 7. + Hematopoietic precursor cells.

[0047] Example 2 In vitro culture method for differentiation of human pluripotent stem cells into hematopoietic progenitor cells 1. Targeted SP140 Construction of gene shRNA and its recombinant vector A knockdown SP140 Methods for constructing human embryonic stem cells and human induced pluripotent stem cells with gene expression 1. Materials Human embryonic stem cells are H1 cells, sourced from the cell bank of the Institute of Cell Biology, Chinese Academy of Sciences. The human multipotent induced cellular stem cells were DF-ZB11AG-F cells, which were derived from the stem cell bank of Dongfang Hospital.

[0048] 2. Construction of shRNA recombinant vector 2.1 shRNA Design shRNAs were designed using conventional software targeting the SP140 gene of human embryonic stem cells and human pluripotent induced stem cells, resulting in two shRNA sequences: the DNA sequence corresponding to SP140-shRNA-1 is CCGAGCAGATGTATGAACA (SEQ IN NO:17); and the DNA sequence corresponding to SP140-shRNA-2 is CAAGAGTGATGTATTGTGT (SEQ IN NO:18).

[0049] Palindromic sequences were designed based on two shRNA sequences, resulting in two forward and reverse primers, as follows: SP140-shRNA-1: Oligo F: 5'- AATTCCGGAACCGAGCAGATGTATGAACACTCGAGCCGAG CAGATGTATGAACATTTTTTTTTAT-3' (SEQ IN NO:19); Oligo R: 5'-AAAAAAAAAACCGAGCAGATGTATGAACACTCGAGCCGAG CAGATGTATGAACATTGGGG-3' (SEQ IN NO:20).

[0050] SP140-shRNA-2: Oligo F: 5'- AATTCCGGAACAAGAGTGATCTATTGTGTCTCGAGCAAGA GTGATCTATTGTGTTTTTTTTTAT -3' (SEQ IN NO:21); Oligo R: 5'-AAAAAAAAACAAGAGTGATCTATTGTGTCTCGAGCAAGAG TGATCTATTGTGTTTGGGG-3' (SEQ IN NO:22).

[0051] 2.2 Phosphorylation and Annealing Configure the phosphorylation reaction system according to Table 1. After incubating in a water bath at 37°C for 30 minutes, place the annealed product directly into boiling hot water and wait for it to cool completely to room temperature before use.

[0052] Table 1 Reaction System

[0053] 2.3 Linearization of the carrier Using enzyme cleavage sites EcoR I and Pac Linearized shRNA backbone plasmid PLKO.3G was prepared according to the enzyme digestion system in Table 2, incubated in a water bath at 37°C for 1 hour, and then subjected to agarose gel electrophoresis (1.2%). The purified linearized vector was obtained by gel excision and recovery.

[0054] Table 2 Enzyme digestion reaction system

[0055] 2.4 Connection Reaction Configure the enzyme ligation reaction system according to Table 3, ligate at room temperature (25℃) for 30 min, and obtain the ligation product.

[0056] Table 3 Enzyme ligation reaction system

[0057] 2.5 Conversion and Thermal Shock Add 20 μl of competent cells thawed on ice to 3 μl of ligation product, incubate on ice for 30 min, then place in 42℃ water for 30 s, then place on ice to recover for 2 min, add 500 μl of LB medium, and incubate on a shaker at 220 rpm and 37℃ for 1 hour. Take an appropriate amount and spread it on a plate, incubate upright for 15 min, then invert the plate overnight. Single clones can be selected the next day.

[0058] The PLKO.3G plasmid containing SP140 shRNA was obtained by Sanger sequencing, indicating that the recombinant expression vector containing SP140 shRNA was successfully prepared.

[0059] 2.6 Gene editing of cells using liposome transfection Plasmid extraction of the recombinant expression vector was performed to ensure endotoxin-free processing. H1 or DF-ZB11AG-F cells were seeded in 6-well plates two days in advance to achieve a cell density of 20%–30% after two days. Liposome transfection was performed two days later using Lipofectamine® LTX&PLUS™ (Invitrogen) reagent. RT-qPCR was used to detect cell mutations. SP140 Gene expression levels, compared to wild-type cells, SP140 Downregulation of gene expression levels indicates successful knockdown construction SP140 Human embryonic stem cells (hESC-SP140-shRNA cells) expressing genes and knockdown SP140 Human polymorphic stem cells (hiPSC-SP140-shRNA cells) with gene expression.

[0060] Example 3 An in vitro culture method for differentiating human stem cells into hematopoietic stem cells 1. Single-cell preparation Cells containing hESC-SP140-shRNA and hiPSC-SP140-shRNA prepared in Example 2 were cultured to 90% confluence and then dissociated into single-cell suspensions by incubation at 37°C for 5 min using a cell digestion solution (accutase). The dissociated cells were collected, counted, and centrifuged at 200g for 3 min. After removing the supernatant, the cells were resuspended.

[0061] 2. In vitro differentiation culture The basal medium was StemPro supplemented with 1× GlutaMAX, 0.039 µL / mL 1-thioglycerol (Sigma-Aldrich), 50 µg / mL ascorbic acid, and 150 µg / mL transferrin (Sigma). TM -34 SFM (purchased from Gibco).

[0062] On day 0, hESC-SP140-shRNA and IPSC-SP140-shRNA cells were cultured in basal medium supplemented with 20 ng / mL rhBMP4 and 40 µM ROCK inhibitor. 1×10⁶ cells were used for the culture. 6Cells were resuspended in 2 mL of cell culture medium in each well and placed in AggreWell 400 24-well plates (STEMCELL Technologies) and aggregated by centrifugation at 200 g for 5 min. After 24 hours of culture, cells were cultured on day 1 using basal medium supplemented with 40 ng / mL rhBMP4, 10 μM SB-431542, 18 μM CHIR-99021, 20 ng / mL rhVEGF, and 80 nM RETA. On day 2, cells were cultured on basal medium supplemented with 15 ng / mL rhBMP4, 40 ng / mL IGF1, 20 ng / mL IGF2, 40 ng / mL SCF, and 2 μM RETA. Cells were cultured on basal medium supplemented with 20 ng / mL IGF1, 40 ng / mL SCF, 120 nM RETA, 40 ng / mL TPO, 25 nM SR1 (StemRegenin1) and 40 ng / mL bFGF on days 3–6.

[0063] On day 7, aggregates were collected, centrifuged at 200g for 5 min, the supernatant was discarded, and the cells were digested with type II collagenase for 4-5 min to prepare a single-cell suspension. The cells were washed and counted. CD34 was enriched using the Miltenyi Biotec CD34 positive selection kit. + Hematopoietic precursor cells.

[0064] Flow cytometry was used to monitor the levels of CD34, CD45, CD38, CD90, CD49f, CD133, and BCL11A cell markers in cells cultured on days 3, 5, and 7 of Example 3 and Comparative Example 1. Results are as follows: Figure 2 As shown in Figure A, a small amount of CD45 could only be detected in the comparative group (WT group) on day 7 of hematopoietic differentiation. + hematopoietic stem cells, and SP140 Example 3, which involved knockout treatment, showed that a large number of CD45 molecules could be detected as early as the third day of hematopoietic differentiation. + The cells were identified as hematopoietic progenitor cells, and flow cytometry analysis confirmed the expression of BCL11A, a key factor in hematopoietic regulation. This indicates that SP140 inhibitor treatment promotes the early expression of the hematopoietic stem cell marker Lin-CD34. + CD38 - CD90 + CD45RA - CD49f + EPCR + .

[0065] In summary, this invention inhibits the activity of human pluripotent stem cells. SP140Gene expression or SP140 protein function promotes the induced differentiation of human pluripotent stem cells into hematopoietic progenitor cells and greatly shortens the differentiation time by 4-5 days compared with the control; at the same time, it also increases the number of hematopoietic progenitor cells, achieving the goal of highly efficient in vitro induced differentiation.

[0066] Example 4 CD34 + Evaluation of transplantation efficiency of hematopoietic progenitor cells.

[0067] Six- to eight-week-old NSG mice were used as recipient mice for the artificial blood cell chimerism experiment. Within 24 hours before transplantation, the mice were irradiated with 2–4 Gy and given water containing antibiotics (1% enrofloxacin, Bayer) for 14 days after irradiation.

[0068] For CD34 originating from EB + The initial cell transplantation was performed via intrafemoral injection. Mice were temporarily anesthetized with isoflurane prior to injection. The knee was disinfected with 75% alcohol before injection, followed by an injection of 4 × 10⁻⁶ cells. 5 CD34 + Human cells. The injection site was disinfected again with 75% alcohol after injection, and then sealed with tissue glue (3M, Vetbond). The mice's condition and recovery were monitored daily post-surgery.

[0069] Following human cell transplantation, peripheral blood tests were performed every 4 weeks until week 16 to assess human CD45 levels. + The chimerism level of cells, WT-D7-CD34, SP140 inhibition-D7-CD34, and cord blood-CD34 were compared. CD45 is generally considered... + A cell chimerism rate of 0.1% was considered to have achieved effective human hematopoietic cell chimerism in NSG mice. At the end of the primary transplantation experiment, the chimerism of human hematopoietic cells in the bone marrow and spleen of NSG mice was analyzed by flow cytometry.

[0070] See results Figure 2 In the SP140 inhibitor treatment group, the cell transplantation efficiency was similar to that of umbilical cord blood transplantation, but significantly higher than that of the control group. Therefore, hematopoietic stem cells obtained after SP140 inhibitor treatment can achieve multi-lineage human hematopoietic reconstitution in vivo, demonstrating their potential for functional transplantation.

[0071] Example 5 A CD34-based + In vitro culture methods for differentiating hematopoietic progenitor cells into T cells Two hours prior to application, 24-well plates were coated with 10 μg / ml human DLL4-Fc protein and 2 mg / ml VCAM-1 protein to obtain pretreated 24-well plates. Subsequently, CD34 enriched in Example 1 was... + Hematopoietic progenitor cells were seeded in pretreated 24-well plates at a seeding density of 1 × 10⁻⁶ cells / well. 5 Cells were resuspended in SFEM medium in each well, and then the medium was changed weekly: Week 1: SFEM medium containing IL-3 0.01 μg / ml, SCF 0.02 μg / ml, Flt3L 0.02 μg / ml, IL-7 0.01 μg / ml, and TPO 0.02 μg / ml; Week 2: SFEM medium containing SCF 0.02 μg / ml, Flt3L 0.02 μg / ml, IL-7 0.01 μg / ml, and TPO 0.02 μg / ml; Weeks 3-6: SFEM medium containing Flt3L 0.02 μg / ml, IL-7 0.01 μg / ml, and TPO 0.02 μg / ml. The medium was changed every 2-3 days during T cell differentiation. The culture plates coated with DLL4-Fc and VCAM-1 proteins should be replaced every week, and T cells can be collected around week 6 of differentiation.

[0072] Comparative Example 2 CD34 prepared in Comparative Example 1 was cultured using the method described in Example 5. + Hematopoietic precursor cells, collect T cells.

[0073] During T cell differentiation, CD5 and CD7 cell markers were detected by flow cytometry. The results are shown in [Figure number missing]. Figure 3 In Example A, the T cells prepared in Example 5 were designated as the inhibitor group, and the T cells prepared in Comparative Example 2 were designated as the control group. The results showed that T cells could be successfully differentiated using the SP140 inhibitor, and the surface molecule expression of the T cells differentiated from the control group (WT) was similar, indicating that the SP140 inhibitor treatment did not affect T cell differentiation.

[0074] When treating differentiated cells with SP140 inhibitor for T cell differentiation culture, flow cytometry was used to detect proT cells (CD5+). + CD7 + The expression of the DNTT gene at the proT stage was investigated. The results showed that proT cells expressed the gene DNTT, which encodes the terminal transferase TdT, while the control group (WT) did not express DNTT at all.

[0075] The average number of T cells differentiated from each ES cell in the two groups was counted separately. The results showed that the SP140 inhibitor group was able to differentiate more T cells. Figure 4(A)

[0076] Differentiated T cells were subjected to combined IL-12 and IL-18 stimulation experiments: 96-well plates with a U-bottom base were used, with 0.2 × 10⁶ cells per well. 6 Differentiated T cells were resuspended in RPMI medium containing 10 ng / ml of the cytokines human IL-2, human IL-12, and human IL-18. After 20 h of stimulation, IFNγ was measured. When using flow cytometry, 1.5 μg / ml of Brefeldin A was added 4 hours before the end of stimulation.

[0077] The effector cytokine IFNγ secreted by differentiated T cells after combined stimulation with IL12 and IL18 was detected. The specific experimental procedure is as follows: A 96-well plate with a U-bottom was used for the experiment, and 0.2 × 10⁻⁶ cells were collected from each well. 6 Cells were resuspended in RPMI medium containing 10 ng / ml of the cytokines human IL-2, human IL-12, and human IL-18. The experiment could be performed 18-22 hours after stimulation. If flow cytometry was to be performed, 1.5 ug / ml of Brefeldin A should be added 4 hours before the end of stimulation.

[0078] Fewer than the control group ( Figure 4 In contrast, the control group (WT) secreted more of the effector IFNγ.

[0079] Gene DNTT Encoding the terminal transferase TdT is one of the characteristics of adult T cells, contributing to the rich diversity of TCRs. The secreted effector IFNγ is another characteristic of adult T cells. The adult T cells prepared in this invention not only express the DNTT gene but also respond more readily to stimulation by innate cytokines IL12 and IL18, rapidly secreting effector molecules like IFNγ, ensuring good maintenance and tumor-killing capabilities of T cells. It is evident that the T cells obtained in the SP140 inhibitor group possess the characteristics of adult T cells. Since T cells are derived from hematopoietic stem and progenitor cells through directed differentiation, this also demonstrates that the hematopoietic stem progenitor cells produced by SP140 inhibitor treatment have the differentiation potential of adult T cells. In contrast, the untreated control group produced more fetal-like T cells, and the hematopoietic progenitor cells produced in the control group not treated with SP140 inhibitors during the hematopoietic stage did not possess the differentiation potential of adult T cells.

[0080] Example 6 A study on the construction of CAR-T cells based on adult T cells and in vivo tumor killing experiments. 1. Construction of CAR-T cells 1.1 CAR Lentiviral Infection of Adult T Cells The T cells prepared in Example 5 were activated with CD3 / CD28 beads for 3 days. On the 4th day, clinical-grade CAR lentiviral particles (GV400) were added to infect and cultured for 4 days to obtain CAR-T cells.

[0081] 1.2 In vivo killing experiment of CAR-T cells Three days in advance, administer 1×10⁻⁶ mmol / L via tail vein injection to NSG mice. 5 5 × 10⁵ NALM6-Luciferase tumor cells, designated Day-3, were used to... 6 CAR-T cells were injected into mice via tail vein on Day 0, Day 3, and Day 7, respectively. For the first three weeks after CAR-T cell administration, mice were intraperitoneally administered human cytokines IL-2 (100 KU per mouse) and IL-15 (100 KU per mouse) three times a week. Tumor growth was monitored using an in vivo imaging system, Lumina3 (IVIS LuminaXRMS), starting on Day 0 and photographed weekly for four consecutive weeks. Two control groups were included: the untreated group (WT) and the control group (DPBS). The untreated group (WT), representing the tumor mouse model, received CAR-T cells derived from T cells that were not induced to differentiate using SP140 inhibitors; the control group (DPBS), representing the tumor mouse model, received only the solvent DPBS.

[0082] See results Figure 5 The results showed that T cells differentiated from the SP140 inhibitor group exhibited better tumor-killing ability after treatment at the hematopoietic differentiation stage. In the Nalm6-Luc mouse model, CD19-CAR-T cells induced by differentiation with SP140 inhibitor treatment showed stronger anti-tumor activity. Compared with the untreated group (WT) and the control group (DPBS), mice in the SP140 inhibitor group showed significantly reduced fluorescence signal, significantly decreased tumor burden, and significantly improved survival rate. P = 0.0006). These results indicate that SP140 inhibition can enhance the in vivo killing ability of CAR-T cells derived from hematopoietic progenitor cells.

[0083] Example 7 A method for constructing CAR-T cells with conditional TCR knockout Using gene editing technologies (such as CRISPR / Cas9) to insert DNA fragments (cassetes) into the "LA" -LoxP - TRAC -cDNA-PA1- LoxP-SA-T2A-CAR-PA2-RA" was knocked into the TRAC site, and homozygotes were screened to obtain individuals with the "SA-T2A-CAR-PA2-RA" gene. LoxP - TRAC -cDNA-PA1- LoxP -SA-T2A- CAR hPSC "seed" cells with the "-PA2" sequence were cultured in vitro to obtain CAR-T cells.

[0084] 1. Design of CARs for Conditional Expression A CAR gene was inserted into the TRAC gene site in CAR-T cells to design a conditionally knockout fusion gene for the TRAC gene. Specifically, this fusion gene includes the following tandem gene elements: TRAC gene 5' homologous arm - LoxP sequence - TRAC CDS sequence - bGHployA - LoxP sequence - SA splice site - T2A linker peptide sequence - 1928ζ CAR full sequence - SV40 polyA - TRAC gene 3' homologous arm. See details... Figure 6 .

[0085] in, TRAC The nucleotide sequence of the 5' homologous arm of the gene is shown in SEQ ID NO: 1; TRAC The nucleotide sequence of the 3' homologous arm of the gene is shown in SEQ ID NO: 2; The nucleotide sequence of the SA cleavage site is shown in SEQ ID NO: 3; The nucleotide sequence of the T2A linker peptide is shown in SEQ ID NO: 4; LoxP The nucleotide sequence of the sequence is shown in SEQ ID NO: 5; The nucleotide sequence of the full 1928ζCAR is shown in SEQ ID NO: 6; The nucleotide sequence of SV40 polyA is shown in SEQ ID NO: 7; The nucleotide sequence of bGH ployA is shown in SEQ ID NO: 8; TRAC The nucleotide sequence of the gene's CDS sequence is shown in SEQ ID NO: 9.

[0086] The above-mentioned fusion gene was artificially synthesized and cloned into the gene editing vector cDNA3.1(+) (addgene, V790-20) to obtain the CAR recombinant gene editing vector.

[0087] 2. Construction of recombinant gene editing vectors CRISPR Cas9 technology is used to knock out the target gene. Two sgRNAs are designed targeting the flanking sequences of the TRAC gene, namely: Targeted TRAC DNA sequence of sgRNA at the 5' homologous arm of the gene: tagaaaccagctgctcgtga (SEQ ID NO: 10); targeting TRAC DNA sequence of sgRNA at the 3' end homologous arm of the gene: gagctaagagagccgtactg (SEQ ID NO: 11).

[0088] The sgRNA was constructed into a Cas9 co-expression vector (addgene: PX458) to obtain a recombinant gene editing vector.

[0089] 3. Expression of recombinase Cre Construction of recombinant viruses The specific steps for lentivirus packaging are as follows: Pack 10×10 6 A number of HEK293FT cells were co-transfected with 5.4 µg pCAG-ERT2CreERT2 (purchased from Addgene, catalog number: #13777), 5.4 µg psPAX2, and 1.2 µg pMD2.G. The transfection system consisted of 12 µL PLUS Reagent and 3 mL Opti-MEM (Gibco), incubated at room temperature for 5 min, followed by the addition of 36 µL Lipofectamine LTX (Invitrogen) and incubation at room temperature for 30 min, then 5 mL Opti-MEM was added. After 8 h, the solution was replaced with DMEM (HyClone) containing 10% FBS and 1% PS. After 72 h, the viral supernatant was collected, filtered through a 0.45 µm filter, and stored at -80°C.

[0090] 4. Methods for constructing recombinant human pluripotent stem cells (hPSCs) containing CAR sequences Recombinant lentivirus and CAR recombinant gene editing vector were simultaneously introduced into embryonic stem cells or human induced pluripotent stem cells. After 6 days of culture, once the clones reached a visible size, they were picked using a pipette tip. The picked clones were seeded into 48-well plates containing mTesr1 (STEMCELL, 85850) medium. The medium did not need to be changed for the first three days. On the fourth day, the medium was changed, and dead cells in the supernatant were lysed. Preliminary PCR identification of the clones was performed using the primers and amplification conditions shown in Tables 4-6, and positive cells containing the fusion gene were screened. Clones obtained from the preliminary identification were labeled, and half of the cells were lysed and confirmed during passage. Homozygosity testing of the clones was also performed.

[0091] Table 4. Identification primers for TRAC-1928ζ targeting clones

[0092] Table 5 Amplification reaction system

[0093] Table 6 PCR Amplification Procedure

[0094] See results Figure 7 Gene editing was performed using transient transfection with sgRNA and a gene-editing vector. PCR identification at multiple sites showed that the CAR gene was successfully knocked into the TRAC site. Homozygosity test results are as follows... Figure 7 As shown in Figure B, the obtained clone was found to be a homozygous knock-in clone, which was named hPSC-CAR.

[0095] 5. Methods for preparing T cells that conditionally knock out endogenous TCRs and simultaneously express CAR The hPSC-CAR cells were cultured in vitro according to the methods in Examples 1 and 5. The resulting adult T cells were induced with 0.1 μM tamoxifen (OHT) to knock out the TRAC gene. Simultaneously, adult T cells were not induced with tamoxifen (OHT) to obtain control CAR T cells.

[0096] See results Figure 8 After the cloned hPSC-CAR was directed to differentiate into T cells, the CAR was only expressed when OHT was added, which at the same time destroyed the expression of TRAC, thus achieving the knockout of TCR while expressing CAR.

[0097] 6. Tumor killing ability test CAR-T cell in vitro killing experiment 1. Effector cells (CAR-T cells) and target cells (tumor cells) were mixed in the corresponding ratio and placed in a 96-well U-shaped culture dish to a culture volume of 200 μl. A control group without effector cells and a control group without tumor cells were set up. 250 μl of PBS was placed in the empty plates around the culture dish to prevent evaporation.

[0098] 2. After co-culturing for a certain period of time, cell counting is performed to obtain the total number of mixed cells. At the same time, the proportion of tumor cells in the flow cytometry analysis is used to calculate the total number of remaining tumor cells.

[0099] 3. Cell killing rate is calculated according to formula I.

[0100] Cell killing rate = (1 - number of remaining tumor cells / number of original tumor cells) × 100% Formula I.

[0101] See resultsFigure 9 OHT-treated CAR-T cells have good tumor-killing ability.

[0102] Example 8 A method for constructing isolated CAR-T cells Will have "left homologous arm LA- LoxP-TRAC -cDNA-PA- LoxP -SA-T2A-FOS-CAR1-PA- The right homologous arm of RA was knocked in at a specific site using gene editing technology (such as CRISPR / Cas9). TRAC Locus, screen for homozygotes, obtain those with " LoxP-TRAC -cDNA-PA- LoxP -SA-T2A-FOS- CAR1 CAR-T cells were obtained by culturing hPSC seed cells with "-PA" in vitro.

[0103] 1. Design of CARs for Conditional Expression The CAR is divided into two parts: a 28ζCAR sequence and a single-chain antibody sequence. The 28ζCAR sequence is fused with a leucine zipper and inserted into the TRAC gene site, while the single-chain antibody sequence forms a CAR fusion protein with a signal peptide, a linker peptide sequence, and a leucine zipper ligand (LZ1).

[0104] A fusion gene capable of conditionally knocking out the TRAC gene was designed by inserting leucine zipper (FOS) and CAR into the TRAC gene site. Specifically, it includes the following tandem gene elements: 5' homologous arm - LoxP sequence - TRAC CDS sequence - bGHployA - LoxP sequence - SA splice site - T2A linker peptide sequence - FOS - 28ζCAR sequence - SV40 polyA - 3' homologous arm. See details... Figure 10 In section A, the nucleotide sequence of the leucine zipper (FOS) is shown in SEQ ID NO:12. The fusion gene was constructed into a recombinant gene editing vector according to the method in Example 7.

[0105] The CAR fusion protein is a protein fragment prepared by recombinant expression in E. coli, containing signal peptides (SP) of different antibodies, expression sequences (scFV) of different antibodies, linker sequences, and leucine zipper ligands (SYN22).

[0106] The linker sequence is GGGGSGGGGSGGGGS (SEQ ID NO:63). The nucleotide sequence of the SYN22 sequence is shown in SEQ ID NO:13: KRIAYLRKKIAALKKDNANLEKDIANLENEIERLIKEIKTLENEVASHEQ.

[0107] The amino acid sequence of anti-CD19 scFV sequence is as SEQ ID NO:14 shows: DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSG EGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS.

[0108] The amino acid sequence of FOS is shown in SEQ ID NO:15: ELTDTLQAETDQLEDEKSALQTEIANLLKEKEKLEFILAAHR.

[0109] The amino acid sequence of 28ζCAR sequence is as SEQ ID NO:16 shows: AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA YRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0110] 2. Methods for constructing recombinant human pluripotent stem cells (hPSCs) containing separable CAR sequences Recombinant human pluripotent stem cells with separable CAR sequences were constructed according to steps 2-4 of Example 7. Positive clones were identified using the primers in Table 4 of Example 7, and positive cells were obtained.

[0111] 3. Methods for preparing isolated CAR T cells The positive cells were induced to differentiate in vitro according to the methods in Examples 1 and 5. The resulting adult T cells were then induced with 0.1 μM tamoxifen (OHT) to knock out the TRAC gene, resulting in isolated CAR T cells. Simultaneously, adult T cells were not induced with tamoxifen (OHT) to obtain control CAR T cells.

[0112] 4. Tumor killing experiment T cell in vitro killing experiment 1. Effector cells (T cells) and target cells (tumor cells) were mixed in the corresponding ratio and placed in a 96-well U-shaped culture dish to a culture volume of 200 μL. Simultaneously, the culture medium was supplemented with the corresponding concentration of in vitro synthesized leucine zipper-antibody sequence protein (the control group received the same volume of DPBS). Groups without effector cells and without tumor cells were set up as controls. 250 μL of PBS was placed in the empty plates around the culture dish to prevent evaporation.

[0113] 2. After co-culturing for a certain period of time, cell counting is performed to obtain the total number of mixed cells. At the same time, the proportion of tumor cells in the flow cytometry analysis is used to calculate the total number of remaining tumor cells.

[0114] 3. Cell killing rate is calculated according to formula I.

[0115] Cell killing rate = (1 - number of remaining tumor cells / number of original tumor cells) × 100% Formula I.

[0116] See results Figure 10 In Figure B, universal CAR-T cells, after binding to an in vitro synthesized CAR2 fusion protein via a leucine zipper, effectively killed NALM6 tumors both in vitro and in NSG mice. This indicates that the isolated CAR-T cells have a good tumor-killing effect.

[0117] Example 9 A method for preparing universal CAR-T cells The hPSC "seed" cells 2 obtained in Example 8, carrying the gene "LoxP-TRAC-cDNA-PA-LoxP-SA-T2A-FOS-CAR1-PA", were subjected to B2M gene knockout using gene editing technology (such as CRISPR / Cas9) and HLA-E expression to obtain hPSC "seed" cells 3 with conditional TCR knockout / CAR1 expression. After in vitro induction and differentiation culture, adult T cells were obtained. After conditional knockout and expression, universal CAR-T cells 1 were obtained.

[0118] 1. In B2M Design of HLA-E fusion gene knock-in at gene locus exist B2MsgRNA was designed into the gene, with the specific DNA sequence as follows: actcacgctggatagcctcc (SEQ ID NO:37). The sgRNA was cloned into a Cas9 co-expression vector (addgene: PX458) to obtain a recombinant gene editing vector containing sgRNA.

[0119] The fusion gene comprises the following tandem gene elements: the left homologous arm of the B2M gene, the HLA-E secretory signal peptide sp, the CDS-SV40 polyA sequence of HLA-E, and the right homologous arm of the B2M gene.

[0120] The nucleotide sequence of the HLA-E secretion signal peptide sp is shown in SEQ ID NO:38: atgtctcgctccgtggccttagctgtgctcgcgctactctctctctttctggcctggaggct.

[0121]

[0122] The nucleotide sequence of the SV40 polyA sequence is shown in SEQ ID NO:40: aacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcattttttttcactgc;

[0123] The fusion gene designed above was cloned into the pcDNA3.1(+) (addgene, V790-20) vector to obtain the recombinant vector.

[0124] 2. hPSCs expressing HLA-E Targeted B2M The recombinant gene editing vector containing the sgRNA and the recombinant vector were electroporated into the hPSC "seed" cells 1. Once the clones reached a visible size, they were picked using a pipette tip. The picked clones were generally placed in 48-well plates. No medium change was needed for the first three days. On the fourth day, the medium was changed, and dead cells in the supernatant were lysed for preliminary identification. Clones obtained from the preliminary identification were labeled. Simultaneously, half of the cells were lysed and used for confirmation identification during passage. The primer sequences are shown in Table 7.

[0125] Table 7 Primers for identification

[0126] After the hPSC "seed" cell clones were identified as positive, they were induced to differentiate into adult-type T cells in vitro according to the methods in Examples 1 and 5. The expression of HLA-A / B / C and HLA-E was detected by flow cytometry, and CD8 expression from different human sources was also detected. + The immune killing effect of T cells and NK cells on differentiated T cells.

[0127] See results Figure 11 B2M site gene editing in hPSCs can simultaneously knock out HLA-E molecules and eliminate a class of HLA molecules, potentially avoiding the influence of CD8+. + T-cell and NK-cell-induced immune rejection. HLA-A / B / C are classic HLA class molecules; this gene editing design can achieve the simultaneous expression of HLA-E while knocking out HLA class molecules.

[0128] CD8 was measured by flow cytometry. + The activation and proliferation of T cells and NK cells were observed. Results are shown below. Figure 12 and Figure 13 Experimental results show that after the B2M-HLA-E structure is inserted into the B2M site, CD8... + The activation and proliferation of T cells and NK cells were significantly reduced, and similar results were observed in different individuals.

[0129] Example 10 A method for preparing isolated universal CAR-T cells 2 Gene editing technologies (such as CRISPR / Cas9) are used to target genes containing " LoxP-TRAC-cDNA-PA- LoxP -SA-T2A- CAR hPSC cells 3 of "-PA" were subjected to CIITA Gene knockout yielded hPSC "seed" cells 4 with conditionally knocked-out TCR / CAR1 expression. These cells were then induced to differentiate and culture in vitro to obtain adult-type T cells. After conditional knockout and expression, universal CAR-T cells 2 were obtained.

[0130] 1. CIITA Knockout using CRISPR Cas9 technology, targeting CIITA The specific DNA sequence of the gene's sgRNA is as follows: GCTGAACTGGTCGCAGTTGA (SEQ ID NO:49).

[0131] Target CIITA The sgRNA was cloned into a Cas9 co-expression vector (addgene: PX458) and electroporated into conditionally knocked-out TCR / CAR1-expressing hPSC "seed" cells 3. After 5-7 days of electroporation, once the clones reached a visible size, they were picked using a pipette tip. The picked clones were generally placed in 48-well plates. No medium change was needed for the first 3 days. On the fourth day, the medium was changed, and dead cells in the supernatant were lysed for preliminary identification. Clones obtained from the preliminary identification were labeled, and half of the cells were lysed and confirmed during passage. The primer sequences for identification are shown in Table 8. Results are shown below. Figure 14 hPSC "seed" cells 3 were successfully knocked out. CIITA Gene.

[0132] Table 8 CIITA Primers for gene knockout identification

[0133] After identifying positive clones, they were cultured in vitro using the methods described in Examples 1 and 5 to obtain adult-type T cells. Flow cytometry was used to detect the expression of HLA-DR on the surface of activated T cells, and CD4+ from different human sources was also detected. + The immune killing effect of T cells on differentiated T cells.

[0134] After identifying positive clones, they were cultured in vitro using the methods described in Examples 1 and 5 to obtain adult-type T cells. These cells were then inoculated with CD4+. + T cell co-culture revealed that knockout CIITA After the gene, CD4 is in a proliferating and activated state. + The proportion of T cells decreased significantly. Figure 15 In group A), we obtained similar results among several different people. Figure 15(B). The above results indicate that knocking out during the ES stage... CIITA Genes can reduce immune rejection caused by HLA-I molecules.

[0135] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A kind of inhibition SP140 Application of reagents for gene and / or SP140 protein expression in the preparation of at least one cell type from pluripotent stem cells, including hematopoietic progenitor cells, adult T cells, and CAR-T cells.

2. The application according to claim 1, characterized in that, The pluripotent stem cells include embryonic stem cells and / or induced pluripotent stem cells.

3. The application according to claim 1, characterized in that, The inhibition SP140 Reagents for gene expression include those that knock down SP140 protein using RNA interference technology or SP140 Reagents for gene expression levels or using CRISPR / Cas9 to knock out SP140 protein or SP140 Reagents for gene expression; The reagents used to inhibit SP140 protein expression include SP140 protein inhibitors.

4. A composition for preparing CAR-T cells based on pluripotent stem cells, characterized in that, Including the suppression in any one of claims 1 to 3 SP140 Reagents for gene and / or SP140 protein expression, knockout TRAC Gene reagents Cre Recombinase, recombinant virus, and conditional knockout reagent; The conditional knockout reagent includes a first recombinant expression vector or a second recombinant expression vector and a CAR fusion protein; The first recombinant expression vector contains the following tandem gene elements: TRAC Left homologous arm of the gene - LoxP - TRAC CDS -First PloyA- LoxP- Enzyme cleavage sites - First linker peptide sequence -CAR - The right homologous arm of the second ployA-TRAC gene; The second recombinant expression vector contains the following tandem gene elements: TRAC Left homologous arm of the gene - LoxP - TRAC CDS -First PloyA- LoxP- Enzyme cleavage sites - Linking peptide sequence - Leucine zipper sequence -CAR Signal transduction domain - the right homologous arm of the second polyA-TRAC gene; The CAR fusion protein includes an antibody signal peptide, a single-chain antibody, and a leucine zipper ligand.

5. The composition according to claim 4, characterized in that, The knockout TRAC Genetic reagents include targeted TRAC The sgRNA of the gene or a CRISPR-Cas gene editing vector containing the sgRNA; The target TRAC The sgRNAs of a gene include sgRNA1 and sgRNA2; The DNA sequence corresponding to sgRNA1 is shown in SEQ ID NO:10; The DNA sequence corresponding to sgRNA2 is shown in SEQ ID NO:

11.

6. The composition according to claim 4, characterized in that, The nucleotide sequence of the exogenous gene contained in the first recombinant expression vector is shown in SEQ ID NO:54; The nucleotide sequence of the exogenous gene contained in the second recombinant expression vector is shown in SEQ ID NO:55; The amino acid sequence of the CAR fusion protein is shown in SEQ ID NO:56; The Cre Recombinase, Recombinant Virus, Recombinant Expression Cre A fusion protein formed by a recombinase and a mutant ligand-binding domain of the estrogen receptor.

7. The composition according to claim 4, characterized in that, Also includes targeted B2M The sgRNA of the gene or a CRISPR-Cas gene editing vector containing the sgRNA and a third recombinant vector; The third recombinant vector comprises the following tandem gene elements: B2M Left homologous arm of the gene - HLA-E secretory signal peptide - HLA-E CDS - polyA-B2M gene right homologous arm; The target B2M The nucleotide sequence of the gene's sgRNA is shown in SEQ ID NO:

37.

8. The composition according to any one of claims 4 to 7, characterized in that, Also includes knockout CIITA Gene knockout reagent CIITA Gene reagents include targeted CIITA The sgRNA of the gene or a CRISPR-Cas gene editing vector containing the sgRNA; The target CIITA The nucleotide sequence of the gene's sgRNA is shown in SEQ ID NO:

49.

9. The use of the composition according to any one of claims 4 to 8 in the preparation of antitumor CAR-T cells or drugs.

10. A method for preparing anti-tumor CAR-T cells from the composition according to any one of claims 4 to 8, characterized in that, Includes the following steps: Pluripotent stem cells are treated with the composition and induced to differentiate in vitro. The resulting hematopoietic progenitor cells are then differentiated in vitro to obtain adult T cells. The adult T cells were induced with tamoxifen and then knocked out. TRAC Simultaneous expression of the CAR gene yields CAR-T cells.

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