An engineered human immune cell, preparation method and application thereof

Abstract

The present invention discloses an engineered human immune cell, a preparation method and application thereof. The engineered human immune cell of the present invention is an immune killer lymphocyte induced by reprogramming human T cells, which retains the markers and functions of the human T cells from which it is derived and has the markers and functions of NK cells, wherein the reprogramming of the human T cells involves the deletion of the BCL11B gene. Compared with T cells and NK cells, the engineered human immune cell not only has a broader spectrum of tumor antigen recognition and killing functions, but also has a broader spectrum of microbial recognition and elimination functions such as viruses and bacteria, and has efficient in vitro amplification capabilities.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an engineered human immune cell, a preparation method and application thereof. Background Art

[0002] Currently, immunotherapy has become the most popular and promising "new" idea in the field of cancer treatment. In 2013, Science magazine ranked the top ten scientific breakthroughs of the year, and tumor immunotherapy topped the list. Novartis and Kite's chimeric antigen receptor (CAR) T cells were approved for marketing by the US FDA, and tumor immune cell therapy has made a milestone in the treatment of hematological malignancies. However, tumor immune cell therapy still has technical bottlenecks in clinical treatment, such as the single target of gene-modified immune cells, which leads to tumor immune escape and tumor recurrence; solid tumors lack specific markers with high efficiency and low side effects, and current gene-modified immune cell therapy has not shown efficient and safe clinical efficacy for solid tumors.

[0003] T cells can be divided into different subgroups based on their surface markers and functions; for example, they can be divided into γδT cells and αβT cells based on TCR type differences. αβT cells account for more than 95% of T cells and are the main cell group in the body that has T cell differentiation markers and performs T cell functions, representing the diversity of T cells. γδT cells are a group of highly heterogeneous cells, whose surface T cell receptors are composed of γ chains and δ chains. There are many subtypes, variable phenotypes, and rich functions. The biological characteristics of each subtype of γδT cells are different. They play an important role in the occurrence and development of tumors, infections, and autoimmune diseases in the body, and are considered to be the bridge connecting the body's natural immunity and adaptive immunity.

[0004] Like T cells, natural killer (NK) cells are an integral part of the human immune system. NK cells are considered to be lymphoid cells that account for about 10-15% of peripheral blood lymphocytes and play a key role in the natural immune response. Unlike T cells, NK cells recognize their targets in an MHC-unrestricted manner. NK cells can exhibit antiviral, anti-GvH and anti-cancer effects. Specifically, NK cells directly kill malignant tumors, including sarcomas, myelomas, carcinomas, lymphomas and leukemias, or help to induce adaptive immune activation of dendritic cells (DC) activity or tumor-specific cytotoxic T lymphocytes (CTLs), thereby eliminating abnormal cells, which are tumor cells or cells that are developing into tumor cells. Although NK cells have the potential to be used as therapeutic agents against cancer or infectious diseases, most NK cells in normal humans exist in a dormant state, and NK cells in cancer patients lack their functions due to the immune escape mechanism of cancer cells. In order to actually use natural killer cells as therapeutic agents, activated natural killer cells that can recognize and destroy tumor cells are required, and since the number of natural killer cells in the body is limited, it is very important to obtain a sufficient number of activated natural killer cells.

[0005] Cell reprogramming refers to the process in which differentiated cells are reversed under specific conditions and restored to a omnipotent state, or form an embryonic stem cell line, or further develop into a new individual. In the field of immunotherapy of diseases, there have been reports on the conversion of immune cell types through cell reprogramming. For example, Dr. Ding Sheng of the Gladstone Institutes in the United States reported that pro-inflammatory effector T cells were reprogrammed into anti-inflammatory regulatory T cells through a specific reprogramming method; this reprogramming is of great significance for the treatment of autoimmune diseases. Specifically, in autoimmune diseases, over-stimulated effector T cells can cause damage to the body. Converting these cells into regulatory T cells helps reduce the overactivity of the immune system and restore its balance, thereby fundamentally treating the disease.

[0006] At present, there is no application of cell reprogramming technology in human tumor immunotherapy. Summary of the invention

[0007] In view of the deficiencies of the prior art and actual needs, the present invention provides a human immune cell engineered by reprogramming, a preparation method and application thereof. The engineered human immune cell of the present invention has some markers and functions of T cells and NK cells, and expresses antigen recognition and killing receptors of NK cells and T cells at the same time, thus having a broader spectrum of tumor antigen recognition and killing functions than NK cells and T cells themselves; at the same time, compared with the mature human T cells from which they are derived, the engineered human immune cell of the present invention has enhanced expansion ability and better anti-tumor effect.

[0008] In a first aspect, the present invention provides immune killer lymphocytes (ITNK) induced by reprogramming human T cells, which retain the markers and functions of the T cells from which they are derived and have the markers and functions of NK cells, wherein the reprogramming of the human T cells involves the deletion of the BCL11B gene.

[0009] Preferably, the human T cells are mature human T cells or a cell population containing mature human T cells; further preferably, the mature human T cells or a cell population containing mature human T cells are derived from human umbilical cord blood or peripheral blood; further preferably, the mature human T cells or a cell population containing mature human T cells are derived from or obtained by differentiation of umbilical cord blood stem cells.

[0010] Preferably, the reprogrammed immune killer lymphocytes express functional TCR, CD3 and NKp30.

[0011] Preferably, the reprogrammed immune killer lymphocytes express NK cell markers selected from the following: CD11c, NKG2D and CD161.

[0012] Preferably, the reprogrammed immune killer lymphocytes lowly express or do not express PD-1, CTLA-4 or FOXP3 immune inhibitory checkpoints.

[0013] Preferably, the reprogrammed immune killer lymphocytes lowly express or do not express NK-related markers: CD127, CD16, KIRDL2, KIRDL3, NKG2A.

[0014] Preferably, the reprogrammed immune killer lymphocytes have upregulated NOTCH expression compared to the T cells from which they are derived.

[0015] Preferably, compared to the T cells from which they are derived, in the reprogrammed immune killer lymphocytes, the expression of LEF1 and TCF7 transcription factors is decreased, and the expression of NOTCH, AP1, mTOR, ID2, TBX21 and NFIL3 is increased.

[0016] Preferably, the TCR-mediated signal transduction of the reprogrammed immune killer lymphocytes is enhanced;

[0017] Preferably, compared with the T cells from which they are derived, the reprogrammed immune killer lymphocytes have upregulated expression of genes CSF2, FOS, MAPK12, MAP3K8, IFNγ, NFKBIA, MAPK11, IL-10 and TEC related to TCR-mediated signal transduction.

[0018] Preferably, relative to NK cells, the T cell recognition and TCR signal transduction of the reprogrammed immune killer lymphocytes are enhanced, and preferably, the expression of CD3, CD4, CD8, and CD40LG is upregulated.

[0019] Preferably, compared to the T cells from which they are derived, the reprogrammed immune killer lymphocytes have enhanced NK killer toxicity-related signal transduction;

[0020] Preferably, compared with the T cells from which they are derived, the reprogrammed immune killer lymphocytes have upregulated expression of genes PRF1, CSF2, ICAM1, CD244, PLCG2, IFNG, FCER1G, GZMB, NCR2, NCR1, KIR2DL4 and SYK related to NK killer toxicity-related signal transduction.

[0021] In a preferred embodiment, the reprogrammed immune killer lymphocytes include CD8+NKp46 hi NKp44+NKp30+, CD4+NKp30+ and γδTCR+NKp46 hi NKp44+NKp30+T cell subsets.

[0022] In a preferred embodiment, the human T cells are mature human T cells, and reprogramming the human mature T cells comprises:

[0023] 1) Activate mature human T cells;

[0024] 2) knocking out the BCL11B gene in the activated mature human T cells obtained in step 1);

[0025] 3) Cultivating the cells obtained in step 2) using T cell culture medium.

[0026] Wherein, in step 1), anti-human CD3 antibody, anti-human CD28 antibody and anti-human CD2 antibody are used for activation;

[0027] Preferably, magnetic beads containing anti-human CD3 antibody, anti-human CD28 antibody and anti-human CD2 antibody are mixed with human mature T cells at a ratio of 1:2 to activate T cells.

[0028] Wherein, in step 2), the BCL11B gene is knocked out using CRISPR / CAS9 technology;

[0029] Preferably, the target of gene knockout is at the second exon and / or the third exon of the BCL11B gene.

[0030] Wherein, in step 3), the T cell culture medium contains IL-2; preferably, it is not co-cultured with OP9-DL1.

[0031] In a second aspect, the present invention provides a method for preparing the cell described in the first aspect, comprising:

[0032] 1') Activate human T cells;

[0033] 2') knocking out the BCL11B gene in the activated human T cells obtained in step 1');

[0034] 3') The cells obtained in step 2') are cultured in T cell culture medium.

[0035] In the above method, preferably, the human T cells are mature human T cells or a cell population containing mature human T cells; further preferably, the mature human T cells or a cell population containing mature human T cells are derived from human umbilical cord blood or peripheral blood; further preferably, the mature human T cells or a cell population containing mature human T cells are derived from mature T cells or cell populations obtained by differentiation of umbilical cord blood stem cells.

[0036] In the above method, preferably, in step 1'), anti-human CD3 antibody, anti-human CD28 antibody and anti-human CD2 antibody are used for activation; in a preferred specific embodiment, magnetic beads containing anti-human CD3 antibody, anti-human CD28 antibody and anti-human CD2 antibody are mixed with human mature T cells in a ratio of 1:2 and incubated to activate T cells.

[0037] Preferably, in step 2'), the BCL11B gene is knocked out using CRISPR / CAS9 technology; further preferably, the gene is knocked out at the second exon and / or the third exon of the BCL11B gene.

[0038] Preferably, in step 3'), the T cell culture medium contains IL-2; preferably, the T cell is not co-cultured with OP9-DL1.

[0039] In a third aspect, the present invention also provides use of the cell as described in the first aspect above in the preparation of a drug for treating a disease selected from the following: tumors, AIDS and infectious diseases; preferably, the infectious disease is a viral infectious disease.

[0040] Preferably, the medicament further comprises a pharmaceutically acceptable excipient.

[0041] This application realizes for the first time the reprogramming of human T cells into immune killer lymphocytes. The reprogrammed cells simultaneously express the antigen recognition and killing receptors of NK cells and T cells, especially functional TCRs, and have both T cell and NK cell functions. Since they simultaneously express the antigen recognition and killing receptors of NK cells and T cells, they can recognize antigens to which these receptors are sensitive. Compared with T cells and NK cells, they not only have a broader spectrum of tumor antigen recognition and killing functions, but also have a broader spectrum of recognition and elimination functions for viruses, bacteria and other microorganisms.

[0042] In addition, the reprogrammed cells of the present application have efficient in vitro expansion ability. In adoptive cell transfer (ACT) therapy, both T cells and NK cells are used to treat cancer. The reprogrammed cells of the present application have the functions of both T cells and NK cells. Compared with the availability and expansion capacity limitations of NK cells when used in adoptive immunotherapy (ACT), users can obtain a large number of T cells from the patient's peripheral blood to produce the reprogrammed immune killer lymphocytes of the present application, and within 2 to 3 weeks, about 100×10 6 200-1248×10 6 Reprogrammed immune killer lymphocytes can meet the patient's demand for cell transfusion. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a schematic diagram of the PX458-gBCL11B vector constructed in Example 1 of the present invention.

[0044] Figure 2 It is a diagram showing whether the BCL11B exon of T cells transduced with PX458-gBCL11B is knocked out by gene sequencing. The control group is T cells transduced with PX458 empty vector (Mock).

[0045] Figure 3 This is a diagram showing the detection and verification of the BCL11B protein expression level in T cells transduced with PX458-gBCL11B by Western Blotting to further confirm whether the BCL11B protein is missing. The control group is T cells transduced with the PX458 empty vector (Mock).

[0046] Figure 4It is a scatter plot of flow cytometry results, which shows that relative to Mock T cells and NK cells, ITNK cells of the present invention (i.e., T cells transduced by PX458-gBCL11B, indicated by PAX458 in the figure) simultaneously highly express T cell marker CD3 and NK cell markers CD56 and NKp46. All samples are derived from the same PBMC sample, Mock T is PBMC sorted by Pan-T, so there is a 7.2-7.8% CD3-CD56+NKp46+ NK cell heterogeneity; PX458 is obtained by transfecting Mock T with PX458-gBCL11B, and there is the same 7.6-7.8% NK cell subpopulation as Mock-T; NK cells are obtained by culturing and purifying PBMC in NK cell culture medium, so there is a small amount of 10.4% CD3+CD56+ NKT or γδT cell heterogeneity.

[0047] Figure 5 The flow cytometry results are scatter plots (A), which show that umbilical cord blood-derived ITNK cells (i.e., T cells transduced with PX458-gBCL11B, indicated by PAX458 in the figure) highly express both the T cell marker CD3 and the NK cell markers NKp46, CD56, NKp30, and NKp44 relative to Mock T cells, as well as the corresponding cell percentage statistics (B).

[0048] Figure 6 The scatter plot of flow cytometry results (A) shows that peripheral blood-derived ITNK cells (i.e., T cells transduced with PX458-gBCL11B, indicated by PAX458 in the figure) highly express both the T cell marker CD3 and the NK cell markers NKp46, CD56, NKp30 and NKp44 relative to Mock T cells, as well as the corresponding cell percentage statistical graph (B).

[0049] Figure 7 It is a transmission electron micrograph of Mock T cells, NK cells and ITNK cells (i.e., T cells transduced with PX458-gBCL11B, indicated by PAX458 in the figure), which shows that: compared with T cells, the nuclear-cytoplasmic ratio of ITNK cells is relatively low; wherein, 1 indicates nucleus; 2 indicates mitochondria; 3 indicates endoplasmic reticulum; 4 indicates granules; scale, the left picture is 2 μm, and the right picture is 500 nm.

[0050] Figure 8The expression of NK receptors in CD4+, CD8+, and CD3+CD4-CD8-T cell subsets of T cells derived from umbilical cord blood and peripheral blood (i.e., ITNK cells) transduced with PX458-gBCL11B, among which NKp46 expression in CD8+T cells was significantly higher than that in CD4+T cells; n=6, indicating that the experiment was repeated in 6 independent healthy donors; ***P≤0.001, paired t-test.

[0051] Fig. 9 Flow cytometric analysis of PX458-gBCL11-transduced T cells is shown, and CD56+ cells were sorted into CD4+, CD8+, and CD4-CD8- subsets and subjected to DNA sequencing analysis, further confirming that the BCL11B locus was knocked out in ITNK cells.

[0052] Fig.10 TCRβ diversity sequencing shows that all TCRβ chain sequence locus variants have the same diversity in T cells and iTNK cells of the same origin.

[0053] Fig.11 TCRα diversity sequencing shows that all TCRα chain sequence locus variants have the same diversity in T cells and ITNK cells of the same source.

[0054] Fig.12 The t-SEN dot density map of mass spectrometry clustering analysis, (A) shows the t-SEN dot density of CD45+ monocytes derived from umbilical cord blood and peripheral blood, which are divided into three experimental groups: transduced with PX458 empty vector (Mock-T), transduced with PX458-gBCL11B (PX458-T), and transduced with PX458-gBCL11B and activated by K562 cells for 24 hours (activated PX458-T); (BC) respectively show the clustering differences and over-conversion (arrows) between Mock-T and BCL11B knockout T cells (BCL11B-KO T) derived from umbilical cord blood (CB), adult peripheral blood (PBMC), BCL11B knockout T cells (BCL11B-KO T) and activated BCL11B knockout T cells (BCL11B-KO T) and (ActivatedBCL11B-KO T) derived from umbilical cord blood (CB), adult peripheral blood (PBMC), respectively.

[0055] Fig.13 Figure 2 is a t-SEN color enrichment cluster image of a fusion of cord blood and peripheral blood CD45+ cells; ITNK cells are labeled as indicated. Cells are colored by normalized expression of markers CD3, CD4, CD8, γδTCR, CD56, NKp46, NKp30, NKp44, and CD11c on the t-SEN image.

[0056] Fig.14It is an immunophenotypic analysis of ITNK cells of the present invention - a t-SEN color enrichment cluster diagram of umbilical cord blood and peripheral blood CD45+ cell fusion; wherein (A) shows the expression levels of NKG2D and CD161 markers, (B) shows the expression levels of CD25, CD127, CD16, KIRDL2, KIRDL 3 and NKG2A markers, and (C) shows the expression levels of immune checkpoint markers PD-1, CTLA-4, FOXP3 and TIM-3.

[0057] Fig.15 It is based on the frequency of immune cell subset grouping of umbilical cord blood T cells. ITNK cells can be differentiated from CD4+, CD8+ and γδTCR+ T cells, and all ITNK subtypes can be activated by HLA-negative cells (K562).

[0058] Fig.16 It is based on the frequency of immune cell subset grouping of peripheral blood T cells. ITNK cells can be differentiated from CD4+, CD8+ and γδTCR+ T cells, and all ITNK subtypes can be activated by HLA-negative cells (K562).

[0059] Fig.17 Shown are mass cytometry heat map analysis results, which display the marker expression of each immune cell subset.

[0060] Fig.18 Shows ITNK cell sorting for RNA-Seq. CD3+T, CD3-NKp46+NK, CD3+NKp46+CB-ITNK, and PBMC-ITNK in cord blood or adult peripheral blood were sorted for RNA sequencing and atac sequencing. The sorted cell purities of Mock-T (n=6), ITNK (n=6), and NK cells (n=4) were 95.3±3.61%, 92.41±2.6%, and 92.88±2.06%, respectively.

[0061] Fig.19 It is principal component analysis of RNA sequencing-cell subpopulation gene expression data.

[0062] Fig. 20 RNA sequencing-KEGG enrichment pathway analysis of genes upregulated in ITNK cells relative to T cells (cutoff: absolute log 2-fold change ≥1; adjusted P value ≤0.05).

[0063] Fig.21 RNA sequencing-KEGG enrichment pathway analysis of genes upregulated in ITNK cells relative to NK cells (cutoff: absolute log 2-fold change ≥1; adjusted P value ≤0.05).

[0064] Fig. 22It is a heat map of RNA-Seq hierarchical clustering, in which the left figure shows the differences in gene transcription expression among T, ITNK, and NK cells (log2 absolute fold change ≥1; adjusted P value ≤0.05), and the right figure is a heat map of differential expression of genes in T cells, ITNK cells, and NK cells screened by RNA-seq analysis; compared with T cells, the expression of NK signaling genes in ITNK cells is upregulated, and the expression of TCF1 and LEF1 genes is downregulated (log2 absolute fold change ≥1; adjusted P value ≤0.05).

[0065] Fig.23 It is a dynamic flow cytometric analysis of the immunophenotype of ITNK cells. Flow cytometry was used to analyze the changes in the proportion of NKp30 and NKp46 positive subpopulations in CD3+CD4+ and CD3+CD8+ subpopulations on days 0, 5, 10, 15, and 20 after knocking out BCL11B in human T cells. The data represent three experiments. NKp46 was detected starting on day 5 after BCL11B knockout and was stable from day 10 to day 15.

[0066] Fig.24 (A) is a t-SNE dot plot of the Sc-RNA seq analysis results. The upper figure shows the cell distribution map from D0 to D20, and the lower figure shows the cluster distribution map of 4948 cells on D0 (2263), D5 (1565), D10 (498), D15 (204), and D20 (418) days after PX458-gBCL11B gene transduction of T cells to knock out BCL11B. The 4948 cells were clustered into 11 cell subsets, and ITNK cells were mainly enriched in the subpopulations marked by (red) circles; (B) The expression of NK cell markers in the 4948 cells (11 subsets) detected was shown by t-SNE dot plots; (C) The expression of T cell markers, NK cell markers, immune checkpoints, transcription factors, and apoptosis-related genes in the 4948 cells (11 subsets) detected was shown by t-SNE dot plots.

[0067] Fig.25 (A) KEGG signaling pathway analysis, which shows that NK cytotoxicity genes are highly expressed in ITNK cells; (B) Violin plots show the expression profiles of NK cell-related KAR and KIR genes in different subsets; (C) Violin plots show the expression profiles of genes related to T cell and NK cell development. NK signaling genes (ID2, TBX21), NOTCH-related genes (MXI1, ZMIZ1, RBPJ), and AP-1-related genes (FOS, JUN, JUNB, JUND) are upregulated in ITNK cells.

[0068] Fig.26(A) Unsupervised trajectory analysis of single cells from subset 5 (CD8+T), subset 0 (early CD8+ITNK), and subset 1 (late CD8+ITNK) shows the gradual transition of CD8+T cells to CD8+ITNK cells; (B) Unsupervised trajectory analysis of single cells from subset 4 (CD4+T), subset 5 (early CD4+ITNK), and subset 7 (late CD4+ITNK) shows the gradual transition of CD4+T cells to CD4+ITNK cells.

[0069] Fig. 27 (A) is a bar graph showing that the expression levels of NK-related transcription factors ID2 and TBX21 are upregulated in ITNK cells; (B) is a Western Blot analysis of TBX21 and ID2 in immune cell lysates, where the left lane is NK cells, the middle lane is ITNK cells, the right lane is T cells, and β-Tublin is used as an internal reference.

[0070] Fig.28 A bar graph showing the amount of IFNγ secretion by T cells and ITNK cells after stimulation with anti-NKp30, anti-NKp46 and anti-CD3 / CD28 antibodies (5ug / ml) by Elisa experiments; data were from samples of three independent donors; data were expressed as mean ± SD; **P ≤ 0.01, ***P ≤ 0.001; paired t test.

[0071] Fig.29 The cytokine secretion of T cells, ITNK cells and NK cells after stimulation with K562 cells is shown; specifically, T, ITNK and NK cells were incubated with K562 cells at an E (effector): T (target) ratio of 1:1 at 37°C for 18 hours; the supernatant was taken and the cytokine (CSF2, CCL4, IFNγ, CCL3, IL13, IL2, TNF, CX3CL1, IL8, IL10, IL23, IL7, IL4, IL5, CXCL11, CCL20, IL6, IL17A, IL21, IL12, IL1β) concentrations were determined by multiplex immunoassay; the values ​​are expressed as the mean ± SD of 3 different donors.

[0072] Fig.30 The specific cytotoxicity percentages of ITNK cells against HLA-negative K562 cells (A), HLA-positive Hela cells (B), HLA-positive A549 cells (C), and HLA-positive NALM-6 cells (D) are shown, where the data are expressed as mean ± standard deviation; **P ≤ 0.01; unpaired t-test.

[0073] Fig.31 The protein and phosphorylation levels of Fyn, PLC-g2, Syk, Erk1 / 2 and mTOR in the immune cell lysate were measured by immunoblotting after 6 hours of stimulation of K562 cells. The three lanes on the left are NK cells, the three lanes in the middle are ITNK cells, and the three lanes on the right are T cells. BCL11B was used as a gene editing control and GAPDH was used as a loading control.

[0074] Fig.32 (A) is a schematic diagram of the in vivo tumor cell killing test of ITNK cells; (BC) shows the quantitative analysis of the total flux of luciferase activity of experimental mice at specific time points using in vivo bioluminescence imaging technology (5 mice in each group), and the results are mean ± SD, **P ≤ 0.01, unpaired t test; (D) is a statistical chart of the survival time of K562 tumor-bearing mice treated with PBS (n = 10), Mock T (n = 15), ITNK (n = 15), and NK cells (n = 5); (E) shows that Hela tumor-bearing mice were treated with PBS, Mock T, ITNK and NK cells on the 7th and 10th days, respectively, and the tumor size was detected at the specified time points (5 mice in each group), and the data are expressed as mean ± standard deviation; ***P ≤ 0.001; unpaired t test.

[0075] Fig.33 The distribution and maintenance of ITNK cells in mice after transplantation into the NSI strain of immunodeficient mice lacking T, B, and NK cells; wherein, (A top) is a schematic diagram of ITNK cell injection into NSI mice and detection experiments, which shows that ITNK was analyzed in the short term and long term on the 1st day, 7th day, 14th day, 21st day and 6th month, respectively (n=3 in each group); (A bottom) shows the percentage of ITNK cells in CD3+T cells analyzed by representative flow cytometry; (B) is a dynamic distribution diagram of ITNK cells in mouse peripheral blood (PB), bone marrow (BM), spleen, liver and lungs, which shows that no T cells and ITNK cells can be detected 6 months after injection of ITNK cells. DETAILED DESCRIPTION

[0076] To further explain the technical means and effects of the present invention, the technical solution of the present invention is further described below with reference to the accompanying drawings and through specific implementation methods, but the present invention is not limited to the scope of the embodiments.

[0077] Unless otherwise specified, the relative arrangement of the parts and steps, the numerical expressions and the numerical values ​​set forth in these embodiments do not constitute limitations of the present invention. Technologies, methods and equipment known to those of ordinary skill in the art may not be discussed in detail, but in appropriate cases, technologies, methods and equipment should be considered as part of this description.

[0078] Example 1 Preparation of reprogrammed natural killer (ITNK) cells of the present invention

[0079] Construction of gene knockout plasmid vector

[0080] According to the selection rule of CRISP / CAS9 target site: GN19NGG, GN19 is the target site, N is preferably still G, and the target site can be on the antisense strand (i.e., the sequence order on the sense strand is: CCN N19C), the following target sequences were selected and forward (F) and reverse (R) primers were designed as guide RNA (gRNA), the gRNA was annealed and ligated into the PX458 vector that had been digested with enzymes, and the PX458-gBCL11B vector was constructed (such as Figure 1 ). PX458-gBCL11B was transfected into 293T cell lines, and single clones were picked to detect the base deletion, mislocation and other knockout conditions of the BCL11B knockout target by gene sequencing (see Figure 2 ), and the knockout efficiency was statistically calculated as shown in Table 1.

[0081] Table 1. List of BCL11B target sequences and their corresponding gRNAs

[0082]

[0083]

[0084]

[0085] According to the knockout efficiency of Table 1 above, the gRNA gene knockout plasmid vector for knocking out the second and third exons is selected for the next experiment. The present application preferably performs BCL11B gene knockout in the second exon and the third exon, using the mixture of the first pair of gRNA and the second pair of gRNA with the lowest knockout efficiency of the second exon, the third pair of gRNA with the highest knockout efficiency, the third pair of gRNA with the lowest knockout efficiency of the third exon, and the gene knockout plasmid corresponding to the third pair of gRNA and their mixture can reprogram T cells to obtain the immune killer lymphocytes of the present invention. In the present embodiment, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:50 and SEQ ID NO:51 gRNA are used to construct BCL11B gene knockout plasmids respectively and mix them for the next experiment.

[0086] T cell sorting and activation

[0087] T cell isolation and activation were performed using the following methods:

[0088] (1) Peripheral blood and umbilical cord blood containing mature human T cells were centrifuged at 300 × g for 10 minutes, and plasma was collected and heat-inactivated at 56°C for 30 minutes;

[0089] (2) suspending the precipitated granular blood cells with 0.9% NaCl and isolating peripheral blood mononuclear cells (PBMCs) by Ficoll density gradient centrifugation;

[0090] (3) Enrichment of all T cells (Pan T) from blood such as peripheral blood and umbilical cord blood by negative sorting using the MACS Pan T isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany)

[0091] The above (1)-(3) are steps for isolating mature human T cells from peripheral blood and umbilical cord blood. It should be noted that other T cell sources are also acceptable, such as hematopoietic stem cell directed differentiation. T cells from all sources were activated using a T cell activation kit (Miltenyi Biotec) by mixing magnetic beads coated with anti-human CD3, anti-human CD28 antibodies and anti-human CD2 with T cells in a ratio of 1:2 (cell density: 2.5×10 6 cells / ml, culture medium: T551-H3 (Takara, Japan) medium, containing 5% autologous plasma, hIL2 (100 IU / ml), gentamicin sulfate (20 μg / ml), 10 mm HEPES, 2 mm glutamine and 1% penicillin / streptomycin). After activation for 24-48 hours, T cells were eluted from the anti-biotin MACS iBead TM particles and set aside.

[0092] Induced reprogramming

[0093] (1) The CRISP / CAS9 knockout vector PX458-gBCL11B was transduced into the activated T cells by electroporation (T-023, LONZA Amata Nucleofector, Lonza);

[0094] (2) 12 h later, PX458-gBCL11B-transduced T cells (referred to as PX458-T) were centrifuged and cultured with T551-H3 (Takara, Japan) medium (containing 5% autologous plasma or fetal bovine serum (FBS), 500 IU / ml hIL2, and gentamicin sulfate (20 μg / ml));

[0095] (3) Fresh culture medium was then replaced every 3 days to maintain the cell density at 0.5-1×10 6cells / ml until day 14 after electroporation.

[0096] (4) Using gene sequencing, detect and verify whether the second exon and third exon of BCL11B in T cells transduced with PX458-gBCL11B are knocked out, such as site-induced insertion or deletion, etc. The control group is T cells transduced with the PX458 empty vector (Mock);

[0097] (5) Western Blotting was used to detect and verify the expression level of BCL11B protein in T cells transduced with PX458-gBCL11B to further confirm the absence of BCL11B protein. The control group was T cells transduced with the PX458 empty vector (Mock). The results of Western Blotting are shown in Figure 3 .

[0098] Phenotypic characterization of reprogrammed cells

[0099] As mentioned above, 14 days after electroporation of T cells, 19.5-68.7% of the obtained cells expressed both T cell markers such as CD3 and NK cell markers such as NKp46, CD56, NKp30 and NKp44, and it was determined that the human ITNK cells of the present invention were obtained. NK cells only express NK cell markers such as NKp46 and CD56, but do not express T cell markers such as CD3. T cells electroporated with empty vectors express T cell markers such as CD3, but do not express NK cell markers. The expression of each cell marker of T cells, NK cells and ITNK cells is shown in the figure. Figure 4-6 Their phenotypic differences are also summarized in Table 2 below.

[0100] Table 2 Phenotypic differences among ITNK cells, T cells, and NK cells

[0101]

[0102]

[0103] In addition, observations under a confocal microscope showed that ITNK cells reprogrammed from T cells have a cell morphology that is different from that of T cells. Their morphology is similar to that of NK cells, with smaller nuclei (relative to the T cell nucleus occupying the entire cell volume), more intracellular matrix, larger particles, rich endoplasmic reticulum, and high protein synthesis activity, indicating that the reprogrammed ITNK cells are immune killer lymphocytes. Transmission electron microscopy images of T cells, NK cells, and ITNK cells are shown in Figure 2. Figure 7 shown.

[0104] In addition, the inventors also compared the expression profiles of these NK markers in BCL11B-deficient T cell subsets derived from umbilical cord blood and peripheral blood, and found that the percentages of CD8+NKp46+ and CD8+CD56+ cells were significantly higher than those of CD4+NKp46+ and CD4+CD56+ cells, indicating that NKp46+CD3+ITNK mainly originated from CD8+T cells (see Figure 8 Unlike CD8+ T cells, CD4+ T cells express NKp30 but not NKp46 after losing BCL11B (see Figure 8 B).

[0105] The CD4-CD8-NKp46+ subset expressed "TCRγδ", which was γδTCR+ITNK cells (see Fig. 9 ). BCL11B deletion in CD4+, CD8+, and γδTCR+ T cells was further verified by DNA sequencing (see Fig. 9 ).

[0106] Example 2 Identification of the Source of ITNK Cells of the Present Invention

[0107] TCRαβ sequencing: Using the human TCRαβ analysis kit, RNA was extracted and CDR3 region targeted amplification was performed on T cells from the same donor and ITNK cells obtained in Example 1 to obtain TCR RNA. Using the HiSeq4000 platform, TCR RNA was sequenced to obtain a TCR library. Cluster combination analysis was performed using MiXCR (ref). Using the MiXCR clone export command, the TCRαβ clone type was exported with the "one chain" parameter. By TCR sequencing, the diversity of TCR clones of T cells and ITNK cells from the same donor was compared, and it was found that the diversity of TCR clones was consistent (see Fig.10 and 11 ), which determined that the ITNK cells obtained were reprogrammed after T cells lost BCL11B and maintained the TCR diversity of T cells, rather than being expanded from a special, unknown small subset of human T cells.

[0108] Example 3 Single cell immunophenotyping of ITNK cells of the present invention

[0109] The ITNK cells obtained in Example 1 were subjected to single cell immunophenotype analysis by mass cytometry (CyTOF), and the control group was T cells transduced with an empty vector.

[0110] Preparation and pretreatment of mass spectrometer samples: Cells from the culture suspension were centrifuged, resuspended in PBS containing 0.5% BSA and 0.02% NaN 3, and incubated with a monoclonal antibody against human CD16 / 32 for 10 minutes at room temperature to block Fc receptors. Subsequently, a mixture of metal-labeled antibodies against cell surface molecules was added and further incubated on ice for 20 minutes. The antibodies were pre-coupled antibodies (Fluidigm) or internally coupled using a mass spectrometry flow coupling kit (Fluidigm) according to the instructions. 5mM cisplatin was added to the cells and incubated on ice for 1 minute in FBS (Fluidigm). After treatment with fixation / permeabilization buffer (Thermo Fisher), the cells were mixed and incubated with metal-labeled antibodies to label intracellular proteins. After washing the cells, they were stained with 1 mL of 1:4000 diluted 191 / 193Ir DNA intercalator (Fluidigm) (the intercalator was diluted with PBS containing 1.6% paraformaldehyde (EMS)) and stored at 4°C. Before detection, the cells were washed once with PBS containing 0.5% BSA and 0.02% NaN 3, washed once with ddH2O, and then resuspended and diluted to about 10 with ultrapure water (ddH2O). 6 Subsequently, the cell sample data were detected and collected using a CyTOF2 instrument (Fluidigm) at an event rate of <400 events / second.

[0111] Using the PhenoGraph clustering algorithm, cluster analysis was performed based on the differences in cell immune phenotypes of 40 markers, and cord blood-derived ITNK cells (hereinafter also referred to as CB-ITNK), peripheral blood-derived ITNK cells (hereinafter also referred to as PBMC-ITNK), and Mock-T cells were integrated and classified into 39 subgroups, such as Fig.12 , 13 , 14, 15, 16 and 17. Depend on Fig.12 It can be seen that PX458 T and Mock-T were separated before and after K562 cell stimulation, with almost no overlap, indicating that there was a significant difference between PX458T and Mock-T; there was an obvious overlap between PX458 T cells before and after K562 cell stimulation, and the activated PX458-T cells derived more new subpopulations.

[0112] According to the results of mass spectrometry flow cytometry marker expression heterogeneity analysis, the ITNK cells of the present invention include CD3 negative cell subpopulation NO.33, CD4+ cell subpopulation NO.5-10, CD8+ cell subpopulation NO.20-22 and 26-28, and TCRγδ+ cell subpopulation NO.23-24, and all express NK-related markers such as CD56, NKp30, NKp44, NKp46 or CD11C, etc. Compared with γδT cells, the three markers NKp46, NKp30 and NKp44 of TCRγδ+ITNK are all highly expressed, that is, (NKp46 high NKp30 high NKp44 high )(like Fig.13 and 14 A). The ITNK cells of the present invention are distinguished from conventional NK cells because the ITNK cells of the present invention do not express CD127, CD16, NKG2A and KIR2DL2 (e.g. Fig.14 B). Moreover, the ITNK cells of the present invention underexpress the immunosuppressive checkpoints PD-1, CTLA-4, and FOXP3 (e.g. Fig.14 C), high expression of immunosuppressive checkpoints will induce immune cells to form immunosuppressive states such as low function and exhaustion, which indicates that the ITNK cells of the present invention have strong immune effects and are not easily inhibited by immunosuppressive microenvironments such as tumors.

[0113] In addition, in cord blood-derived ITNK cells, e.g. Fig.15 The bar graph clearly shows the percentage of various ITNK cells in CD45+ hematopoietic cells, as well as the dynamic transformation from quiescent ITNK cells to effector cells after stimulation. Fig.16 As shown in Figure 2, the dynamic transition of adult peripheral blood-derived ITNK cells from a resting state to an effector state after stimulation is also the same as that of cord blood-derived ITNK cells. Fig.17 The mass spectrometry heatmap shows the dynamic changes of the immune phenotype of ITNK cells before and after activation, and the expression of CD25 increases after activation. In summary, after ITNK activation, the expression of NK cell activation receptors and T cell markers is still retained.

[0114] Example 4 RNA-Seq transcriptional profiling analysis of ITNK cells of the present invention

[0115] In order to study the full gene expression profile of ITNK cells, the inventors performed RNA sequencing analysis on T and ITNK cells from 4 umbilical cord blood samples and 3 adult peripheral blood samples, as well as NK cells from 2 umbilical cord blood samples and 2 adult peripheral blood samples. The sorting operation is as follows: flow cytometric analysis or sorting is performed using flow cytometers such as Canto, FACS Fortessa (BD), and FACSAriaII. For cell surface receptor labeling, the cells are mixed with antibodies with 50 μl of flow buffer (PBS solution containing 2% FBS) and incubated at 4 degrees in the dark for 30 minutes; for intracellular labeling, the cells are permeabilized using Foxp3 / transcription factor staining buffer (eBioscience), and after elution of the buffer, they are blocked with mouse or rabbit serum, incubated with antibodies at 4 degrees in the dark for 30 minutes, rinsed once with flow buffer and resuspended for use in flow cytometric analysis or sorting. Cell sorting strategy and sorting purity verification (such as Fig.18 ).

[0116] Principal component analysis (PCA) was used to evaluate the similarity of RNA sequencing results of 18 samples, and it was found that from the transcriptome analysis, ITNK was different from T cells and NK cells (such as Fig.19 Compared with T cells, 776 genes were increased (data not shown), including NK-specific transcription factors (such as ID2, TBX21, NFIL3, IRF8), activating and inhibitory NK cell receptors / proteins (such as IL2RB, IFNG, PRF1, GZMB, TNFRSF4, NCR1, NCR2, PLCG2), histone genes (HIST1H1D, HIST1H2AC / D / E / G / M, HIST2H2A3 / 4) (such as Fig. 22 and 20 ); In contrast, compared with T cells, 592 genes such as TCF-1 / TCF-7, LEF-1, IL-7R, MYC, PD-L1, FOXP3, etc. were downregulated in ITNK cells (Table 3). Compared with NK cells, 666 genes were increased in ITNK (data not shown), most of which were enriched in T cell recognition and TCR signal transduction (CD3, CD4, CD8, CD40LG) ( Fig.21). Interestingly, compared with NK cells, the expression of KIR genes KIR2DL1, KIR2DL3, KIR3DL1 and KIR3DL2 in ITNK cells was downregulated (as shown in Table 4), while KIR2DL1, KIR2DL3, KIR3DL1 and KIR3DL2 are NK cell inhibitory receptors that mediate the inhibitory effect of immune cells, and their low expression indicates that ITNK cells have resistance to the immunosuppressive microenvironment. The results of whole transcriptome sequencing analysis showed that NK cell signature genes IL2RB, ID2, NFIL3, etc. were upregulated in ITNK cells ( Fig. 22 right).

[0117] Table 3. Gene expression of ITNK cells relative to NK cells

[0118]

[0119]

[0120] Table 4. Gene expression of ITNK cells relative to NK cells

[0121]

[0122] Example 5 Single-cell transcriptome sequencing analysis of ITNK cells of the present invention

[0123] Flow cytometry showed that CD8+CD3+NKp46+ITNK and CD4+CD3+NKp30+ITNK appeared 5 days after knocking out BCL11B (e.g. Fig.23 To further explain the cell fate transition during T cell to ITNK reprogramming, we used microwell-based single-cell transcriptome sequencing (scRNA-seq) technology to investigate the gene expression profiles of T and ITNK cells at the single-cell level in CD3+ umbilical cord blood samples at different time points after knockout of BCL11B.

[0124] A total of about 5,000 cells were detected and analyzed in all experimental groups. scRNA-seq detected cell samples at different time points, with an average of 2,000-4,000 genes detected per cell, and a total of 20,000 human genes detected in all cells. In the t-SNE (t-distributed random neighbor-embedded) analysis of the transcriptional profile, the cells were projected onto two dimensions, providing a visual representation of the cell fate transition during ITNK cell reprogramming. The unbiased t-SNE analysis results showed that cells from day 0 to day 20 after knockout could be clustered into 11 subgroups (such as Fig.24). According to the expression of marker genes of T cells and NK cells, it was determined that ITNK cells were mainly concentrated in subgroup 6 (CD4+ITNK), subgroup 1 (CD8+ITNK) and subgroup 10 (γδTCR+ITNK) (such as Fig.24 ), and these subgroups completely overlap with the BCL11B-deficient subgroup, further indicating that ITNK cells are reprogrammed by knocking out BCL11B in T cells. KEGG enrichment analysis showed that ITNK cells specifically highly expressed NK marker genes and their related genes (such as Fig.25 ).Depend on Fig.24 C. Fig.25 C shows that NOTCH1, NOTCH2, ZMIZ1 (NOTCH1 auxiliary factor), and RBPJ (NOTCH downstream transcription factor) are all upregulated in human ITNK cells, suggesting that the NOTCH signaling pathway plays a role in ITNK cell reprogramming. FOS, JUN, and JUNB, three subunits of AP-1 transcription factors, are lowly expressed in the early stage of ITNK reprogramming, and gradually upregulated in the late stage of reprogramming (e.g. Fig.25 C). It can be seen that NOTCH signaling and AP-1 signaling are upregulated after ITNK reprogramming. T cells and ITNK cells are closely clustered in the t-SNE dot plot, indicating that the transformation of T cells to NK cells is almost synchronous.

[0125] Trajectory analysis of NK signature genes in late CD8+ITNK (subgroup 1) revealed a gradual transition of CD8+T cells to CD8+ITNK (e.g. Fig.26 A). Similarly, trajectory analysis of NK signature genes in CD4+ T cells and ITNK cells also showed a gradual transition of T cells to ITNK cells ( Fig.26 B). Fig.23 ) The results of dynamic flow cytometry analysis of the immunophenotype of ITNK cells were consistent. T cells began to reprogram into ITNK cells on the fifth day after BCL11B knockout. We found that the expression of transcription factor genes (TBX2, ID2, etc.) was significantly upregulated during the reprogramming of T cells to ITNK cells, and this was further verified by immunoblotting (e.g. Fig. 27 ).

[0126] Example 6 ITNK cells of the present invention can recognize and kill MHCI positive / negative tumor cells in vitro

[0127] In order to determine whether the NK cell receptor (NCR) and T cell receptor (TCR) expressed on the ITNK cells of the present invention are functional, we stimulated ITNK cells with anti-NKp30, anti-NKp46 and anti-CD3 / CD28 monoclonal antibodies, respectively, and found that after stimulation with anti-NKp30 and anti-NKp46 antibodies, the interferon (IFN) secretion of ITNK cells increased, while that of T cells in the control group did not increase (e.g. Fig.28 ); after stimulation with anti-CD3 / CD28 antibodies, the secretion of interferon by ITNK cells increased, while that of control T cells did not increase (e.g. Fig.28 ). This suggests that the NCR and TCR in ITNK cells are functional.

[0128] Similar to NK cells, ITNK cells of the present invention can secrete various cytokines, including GM-CSF, IFN and TNF (e.g. Fig.29 ), and can recognize and kill MHC-I negative K562 cells (such as Fig.30 A). In addition, ITNK cells can effectively kill Hela and A549 cells (such as Fig.30 BC), both cells highly express NK activating receptor ligands and are MHC I positive; as shown in Table 2, since ITNK cells lowly express NK cell KIR receptors (KIR2DL1, KIR2DL3, KIR3DL1 and KIR3DL2) that mediate immunosuppression, ITNK cells have a better killing effect on tumors than NK cells. However, NALM-6 does not express NCR ligands and highly expresses MHC-I molecules, and ITNK cells and NK cells have no significant killing effect on NALM-6 (such as Fig.30 D). Then, the inventors used K562 cells to stimulate ITNK, NK and T cells respectively and found that the expression levels of phosphorylated Fyn, PLC-γ2, Syk, Erk and m-TOR in ITNK and NK cells were similar, but higher than those in T cells (e.g. Fig.31 ). These results suggest that ITNK cells have similar NK cell functions in terms of NCR activation, cytokine secretion, cytotoxicity, and signaling pathways.

[0129] Example 8 ITNK cells of the present invention can inhibit tumor growth in vivo

[0130] The inventors also evaluated whether the ITNK cells of the present invention can inhibit the growth of xenograft tumors. Specifically, K562 cells labeled with luciferase were implanted into NSI mice to construct a K562 tumor-bearing mouse model, and then a single injection of ITNK, NK or T cells ( Fig.32A). At specific time points, the survival of K562 in mice was detected by in vivo imaging equipment. Compared with the T cell injection (negative control) and PBS (blank control) groups, the K562 tumor burden of experimental mice treated with ITNK cells and NK cells was significantly reduced 28 days after infusion ( Fig.32 B and 32C), survive longer ( Fig.32 D). In addition, the inventors transplanted Hela cells into NSI mice and treated Hela xenograft mice with ITNK, NK or T cells respectively. The results showed that the growth rate of Hela tumors in tumor-bearing mice treated with ITNK cells was significantly slower than that in the NK, T cell or PBS treatment groups ( Fig.32 E). It can be seen that the ITNK cells of the present invention are effective killers of tumor cells in vivo and can prevent tumor progression.

[0131] Example 9 In vivo safety assessment of ITNK cells of the present invention

[0132] In order to verify the distribution and maintenance capacity of ITNK cells in vivo, we transplanted ITNK cells into the immunodeficient mouse NSI strain lacking T, B, and NK cells, and measured the percentage of ITNK cells in peripheral blood (PB), spleen (SP), bone marrow (BM), liver (liver), and lung (lung) 1, 7, 14, 21, and 180 days after transplantation. Fig.33 AB). The proportion of ITNK cells reached the highest point 21 days after transplantation, then gradually decreased and could not be detected after 6 months ( Fig.33 B) By Fig.33 B shows that ITNK cells have better in vivo maintenance ability than T cells. We have never observed ITNK cells attacking the host or expanding without limit.

[0133] To assess the potential off-target mutations caused by PX458-gBCL11B, we performed high-coverage whole-genome sequencing of PX458-gBCL11B electroporated T cells. Compared with wild-type T cells, we found from two independent experiments that PX458-gBCL11B-edited T cells had very few off-target mutations caused by the nuclease.

[0134] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An immune killer lymphocyte induced by reprogramming human mature T cells, which retains the markers and functions of the human mature T cells from which it is derived and has the markers and functions of NK cells, characterized in that: The reprogramming of the human mature T cells involves the deletion of the BCL11B gene; the immune killer lymphocytes have upregulated NOTCH expression compared to the T cells from which they are derived; the expression of LEF1 and TCF7 transcription factors is decreased, and the expression of NOTCH, AP1, ID2, TBX21 and NFIL3 is increased; its TCR-mediated signal transduction is enhanced; compared to the T cells from which they are derived, the expression of genes CSF2, FOS, MAPK12, MAP3K8, IFNγ, NFKBIA, MAPK11, IL-10 and TEC related to TCR-mediated signal transduction is upregulated; The method for reprogramming the human mature T cells comprises: 1) Activate T cells by incubating magnetic beads containing anti-human CD3 antibody, anti-human CD28 antibody and anti-human CD2 antibody with mature human T cells or cell populations containing mature human T cells at a ratio of 1:2; 2) knocking out the BCL11B gene in the activated mature human T cells obtained in step 1); knocking out the second exon and the third exon; 3) culturing the cells obtained in step 2) using T cell culture medium; The mature human T cells or cell populations containing mature human T cells are derived from mature T cells or cell populations differentiated from umbilical cord blood stem cells; The immune killer lymphocytes express NK cell markers selected from the following: CD11c, NKG2D and CD161; The immune killer lymphocytes lowly express or do not express PD-1, CTLA-4 or FOXP3 immune inhibitory checkpoints; Compared with NK cells, their T cell recognition and TCR signal transduction are enhanced; the expression of CD3, CD4, CD8, and CD40LG is upregulated.

2. The cell according to claim 1, which expresses functional TCR, CD3 and NKp30.

3. The cell according to claim 1, characterized in that It lowly expresses or does not express NK-related markers: CD127, CD16, KIRDL2, KIRDL3, and NKG2A.

4. The cell according to claim 1, characterized in that Compared with the T cells from which they were derived, the expression of genes PRF1, CSF2, ICAM1, CD244, PLCG2, IFNG, FCER1G, GZMB, NCR2, NCR1, KIR2DL4 and SYK related to NK cytotoxicity-related signal transduction was upregulated.

5. The cell according to claim 1, characterized in that It includes CD8+NKp46+NKp44+NKp30+, CD4+NKp30+ and γδTCR+NKp46+NKp44+NKp30+ T cell subsets.

6. The cell according to claim 1, characterized in that In step 2), the BCL11B gene was knocked out using CRISPR / CAS9 technology.

7. The cell according to claim 1, characterized in that, in step 3), the T cell culture medium contains IL-2.

8. The cell according to claim 1, characterized in that In step 3), co-culture with OP9-DL1 was not performed.

9. A method for preparing the human Mature A method for inducing immune killing lymphocytes by T cell induction, characterized in that: It includes: 1') using magnetic beads containing anti-human CD3 antibody, anti-human CD28 antibody and anti-human CD2 antibody to incubate with mature human T cells or a cell population containing mature human T cells at a ratio of 1:2 to activate T cells; 2') knocking out the BCL11B gene in the activated mature human T cells obtained in step 1'); knocking out the second exon and the third exon; 3') culturing the cells obtained in step 2') using T cell culture medium; The mature human T cells or the cell population containing mature human T cells are derived from human umbilical cord blood or peripheral blood.

10. The method according to claim 9, characterized in that The mature human T cells or the cell population containing mature human T cells are derived from the mature T cells or cell population obtained by differentiation of umbilical cord blood stem cells.

11. The method according to claim 9, characterized in that In step 2'), the BCL11B gene was knocked out using CRISPR / CAS9 technology.

12. The method according to claim 9, characterized in that In step 3'), the T cell culture medium contains IL-2.

13. The method according to claim 9, characterized in that In step 3'), co-culture with OP9-DL1 was not performed.

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