WO2021063285A1 - 一种工程化人体免疫细胞、其制备方法及应用 - Google Patents

一种工程化人体免疫细胞、其制备方法及应用 Download PDF

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WO2021063285A1
WO2021063285A1 PCT/CN2020/118104 CN2020118104W WO2021063285A1 WO 2021063285 A1 WO2021063285 A1 WO 2021063285A1 CN 2020118104 W CN2020118104 W CN 2020118104W WO 2021063285 A1 WO2021063285 A1 WO 2021063285A1
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cells
cell
human
itnk
mature
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PCT/CN2020/118104
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French (fr)
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李鹏
蒋治武
汤朝阳
秦乐
廖芮
郑迪威
崔元彬
林思妙
姚瑶
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昭泰英基生物医药（香港）有限公司
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Priority to US17/639,237 priority Critical patent/US20220325246A1/en
Priority to AU2020359872A priority patent/AU2020359872B2/en
Priority to EP20871608.4A priority patent/EP4039803A4/en
Publication of WO2021063285A1 publication Critical patent/WO2021063285A1/zh

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Definitions

  • This application relates to the field of biomedicine technology, in particular to an engineered human immune cell, its preparation method and application.
  • T cells can be divided into different subgroups according to the differences in their surface markers and functions; for example, according to differences in TCR types, they can be divided into ⁇ T cells and ⁇ T cells.
  • ⁇ T cells account for more than 95% of T cells. They are the main cell group with T cell differentiation markers and perform T cell functions in the body, representing the diversity of T cells.
  • ⁇ T cells are a group of highly heterogeneous cells, and their surface T cell receptors are composed of ⁇ chains and ⁇ chains. They have many subtypes, variable phenotypes, and rich functions. The biological characteristics of each subtype of ⁇ T cells are different. It plays an important role in the occurrence and development of tumors, infections and autoimmune diseases in the body, and is considered to be a bridge between the body's natural immunity and adaptive immunity.
  • NK cells are an indispensable 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 non-restrictive manner. NK cells can exhibit antiviral, anti-GvH, and anticancer effects. Specifically, NK cells directly kill malignant tumors, including sarcoma, myeloma, cancer, lymphoma, and leukemia, or help by inducing dendritic cells ( DC) activity or adaptive immune activation of tumor-specific cytotoxic T lymphocytes (CTL), thereby eliminating abnormal cells, which are tumor cells or cells that are developing into tumor cells.
  • DC dendritic cells
  • CTL tumor-specific cytotoxic T lymphocytes
  • NK cells Although NK cells have potential 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 use natural killer cells as therapeutic agents, activated natural killer cells that can recognize and destroy tumor cells are required. 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. .
  • Cell reprogramming refers to the process by which differentiated cells are reversed under certain conditions to return to a pluripotent state, or form embryonic stem cell lines, or further develop into a new individual.
  • immunotherapy of diseases there have been reports about the transformation of immune cell types through cell reprogramming.
  • pro-inflammatory effector T cells are reprogrammed into anti-inflammatory regulatory T cells;
  • the treatment of immune diseases is of great significance.
  • over-stimulated effector T cells can cause damage to the body. Converting these cells into regulatory T cells can help reduce the overactivity of the immune system. It restores its balance, thereby fundamentally curing the disease.
  • the present application provides a human immune cell engineered through reprogramming, and a preparation method and application thereof.
  • the engineered human immune cells of the present application have part of the markers and functions of T cells and NK cells, and simultaneously express NK cells and T cell antigen recognition killer receptors. Therefore, they have a broader spectrum than NK cells and T cells themselves.
  • the tumor antigen recognizes and kills the function; at the same time, compared with the mature human T cells derived from it, the engineered human immune cells of the present application have enhanced expansion capacity and better anti-tumor effects.
  • this application mentions the immune killer lymphocytes (ITNK) induced by reprogramming human T cells, which retain the markers and functions of the T cells derived from them and have the markers and functions of NK cells.
  • ITNK immune killer lymphocytes
  • the reprogramming of the human T cells involves the deletion of the BCL11B gene.
  • 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 cell populations containing mature human T cells are derived from mature T cells or cell populations obtained by differentiation of pluripotent stem cells, embryonic stem cells or cord blood stem cells.
  • the reprogrammed immune killer lymphocytes express functional TCR, CD3 and NKp30.
  • the reprogrammed immune killer lymphocytes express NK cell markers selected from the group consisting of CD11c, NKG2D and CD161.
  • the reprogrammed immune killer lymphocytes have low or no expression of PD-1, CTLA-4 or FOXP3 immunosuppressive checkpoints.
  • the reprogrammed immune killer lymphocytes have low or no expression of NK-related markers: CD127, CD16, KIRDL2, KIRDL3, NKG2A.
  • the expression of NOTCH of the reprogrammed immune killer lymphocytes is up-regulated compared to the T cells from which it is derived.
  • the expression of LEF1 and TCF7 transcription factors is decreased, and the expression of NOTCH, AP1, mTOR, ID2, TBX21 and NFIL3 is increased in the reprogrammed immune killer lymphocytes.
  • the TCR-mediated signal transduction of the reprogrammed immune killer lymphocytes is enhanced
  • the reprogrammed immune killer lymphocytes have genes CSF2, FOS, MAPK12, MAP3K8, IFN ⁇ , NFKBIA, MAPK11, IL-10 related to TCR-mediated signal transduction And the expression of TEC is up-regulated.
  • 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 up-regulated.
  • the NK-killing toxicity-related signal transduction of the reprogrammed immune killer lymphocytes is enhanced compared to the T cells derived therefrom;
  • the reprogrammed immune killer lymphocytes have genes PRF1, CSF2, ICAM1, CD244, PLCG2, IFNG, FCER1G, GZMB, NCR2 related to signal transduction related to NK killing toxicity
  • PRF1, CSF2, ICAM1, CD244, PLCG2, IFNG, FCER1G, GZMB NCR2 related to signal transduction related to NK killing toxicity
  • NCR1, KIR2DL4 and SYK was up-regulated.
  • the reprogrammed immune killer lymphocytes include CD8+NKp46 hi NKp44+NKp30+, CD4+NKp30+ and ⁇ TCR+NKp46 hi NKp44+NKp30+T cell subsets.
  • the human T cell is a mature human T cell, and reprogramming the human mature T cell includes:
  • step 2) The cells obtained in step 2) are cultured with T cell culture medium.
  • step 1) use anti-human CD3 antibody, anti-human CD28 antibody and anti-human CD2 antibody for activation;
  • magnetic beads of anti-human CD3 antibody, anti-human CD28 antibody and anti-human CD2 antibody are mixed with human mature T cells to incubate the activated T cells at a ratio of 1:2.
  • step 2) use CRISPR/CAS9 technology to knock out the BCL11B gene;
  • the target of the gene knockout is at the second exon and/or the third exon of the BCL11B gene.
  • the T cell culture medium contains IL-2; preferably, it is not co-cultured with OP9-DL1.
  • the present application provides a method for preparing the cell according to the first aspect, which includes:
  • step 3' The cells obtained in step 2') are cultured in T cell culture medium.
  • 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 cell populations containing mature human T cells are derived from mature T cells or cell populations obtained by differentiation of pluripotent stem cells, embryonic stem cells or cord blood stem cells.
  • step 1' use anti-human CD3 antibody, anti-human CD28 antibody, and anti-human CD2 antibody for activation; in a preferred embodiment, use anti-human CD3 antibody, anti-human CD28 antibody Incubate the magnetic beads with anti-human CD2 antibody and human mature T cells at a ratio of 1:2 to activate T cells.
  • the BCL11B gene knockout is performed using CRISPR/CAS9 technology; further preferably, the gene knockout is performed at the second exon and/or the third exon of the BCL11B gene.
  • the T cell culture medium contains IL-2; preferably, it is not co-cultured with OP9-DL1.
  • the application also provides the use of the cells as described in the first aspect in the preparation of drugs for treating diseases selected from the group consisting of tumors, AIDS, and infectious diseases; preferably, the infectious diseases are viruses Infectious diseases.
  • the medicament further contains a pharmaceutically acceptable excipient.
  • This application realizes the reprogramming of human T cells into immune killer lymphocytes for the first time.
  • the reprogrammed cells simultaneously express NK cells and T cell antigen-recognizing killer receptors, especially functional TCRs, and have both T cell and NK cell functions. ; Because it expresses both NK cells and T cell antigens to recognize killer receptors, it can recognize the sensitive antigens of these receptors. Compared with T cells and NK cells, it not only has a broader spectrum of tumor antigen recognition and killing function, but also has Broader spectrum of virus, bacteria and other microorganisms identification and removal function.
  • the reprogrammed cells of the present application have efficient in vitro expansion capabilities.
  • adoptive cell transfer (ACT) therapy both T cells and NK cells are used to treat cancer.
  • the reprogrammed cells of the present application have both the functions of T cells and NK cells.
  • ACT adoptive cell transfer
  • the user can obtain from the patient’s peripheral blood Obtain a large number of T cells to produce the reprogrammed immune killer lymphocytes of the present application, and within 2 to 3 weeks, 200-1248 ⁇ can be prepared from about 100 ⁇ 10 6 peripheral blood mononuclear cells of solid tumor patients.
  • 106 reprogramming immune killer lymphocytes the patient achieve demand reinfusion cells.
  • Figure 1 is a schematic diagram of the PX458-gBCL11B vector constructed in Example 1 of the present application.
  • Figure 2 is a diagram of gene sequencing to detect and verify whether the BCL11B exon of PX458-gBCL11B transduced T cells has been knocked out.
  • the control group is T cells transduced with PX458 empty vector (Mock).
  • Figure 3 is a diagram of Western Blotting detecting and verifying the expression level of BCL11B protein in T cells transduced with PX458-gBCL11B to further confirm whether the BCL11B protein is missing.
  • the control group is T cells transduced with PX458 empty vector (Mock).
  • Figure 4 is a scatter diagram of flow cytometry results, which shows that ITNK cells (ie, PX458-gBCL11B transduced T cells, indicated by PAX458 in the figure) simultaneously highly express T cells compared to Mock T cells and NK cells Markers CD3 and NK cell markers CD56 and NKp46. All samples are derived from the same PBMC sample.
  • ITNK cells ie, PX458-gBCL11B transduced T cells, indicated by PAX458 in the figure
  • NK cells Markers 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 7.2-7.8% CD3-CD56+NKp46+ NK cell population; PX458 is Mock T obtained by PX458-gBCL11B transfection, and Mock-T has the same 7.6-7.8% NK cell subpopulation; NK cells are PBMC obtained by NK cell culture and purification, so there is a small 10.4% CD3+CD56+ NKT or ⁇ T cell population.
  • Figure 5 is a scatter plot of flow cytometry results (A), which shows that cord blood-derived ITNK cells (ie, PX458-gBCL11B transduced T cells, indicated by PAX458 in the figure) simultaneously highly express T cell markers compared to mock T cells CD3 and NK cell markers NKp46, CD56, NKp30 and NKp44, and the corresponding cell percentage statistics (B).
  • A cord blood-derived ITNK cells
  • PX458-gBCL11B transduced T cells indicated by PAX458 in the figure
  • B the corresponding cell percentage statistics
  • Figure 6 is a scatter diagram of flow cytometry results (A), which shows that ITNK cells derived from peripheral blood (ie, PX458-gBCL11B transduced T cells, indicated by PAX458 in the figure) simultaneously express high T cell markers compared to mock T cells CD3 and NK cell markers NKp46, CD56, NKp30 and NKp44, and the corresponding cell percentage statistics (B).
  • A flow cytometry results
  • B shows that ITNK cells derived from peripheral blood (ie, PX458-gBCL11B transduced T cells, indicated by PAX458 in the figure) simultaneously express high T cell markers compared to mock T cells CD3 and NK cell markers NKp46, CD56, NKp30 and NKp44, and the corresponding cell percentage statistics (B).
  • Figure 7 is a transmission electron microscope image of Mock T cells, NK cells and ITNK cells (ie, PX458-gBCL11B transduced T cells, indicated by PAX458 in the figure), which shows: compared with T cells, the nucleoplasmic comparison of ITNK cells Low; Among them, 1 indicates the nucleus; 2 indicates the mitochondria; 3 indicates the endoplasmic reticulum; 4 indicates the granule; the ruler, the left picture is 2 ⁇ m, the right picture is 500nm.
  • ITNK cells ie, PX458-gBCL11B transduced T cells, indicated by PAX458 in the figure
  • Figure 8 shows the expression of NK receptors in the CD4+, CD8+, CD3+CD4-CD8-T cell subsets of PX458-gBCL11B-transduced cord blood and peripheral blood-derived T cells (ie, ITNK cells).
  • NKp46 is in CD8.
  • Figure 9 shows the flow cytometry analysis of PX458-gBCL11-transduced T cells.
  • the CD4+, CD8+ and CD4-CD8- subgroups of CD56+ cells were sorted and subjected to DNA sequencing analysis, further confirming the BCL11B in ITNK cells The site is knocked out.
  • FIG. 10 shows TCR ⁇ diversity sequencing. All TCR ⁇ chain sequence trajectory variables have the same diversity in T cells and ITNK cells from the same source.
  • FIG. 11 shows TCR ⁇ diversity sequencing. All TCR ⁇ chain sequence trajectory variables have the same diversity in T cells and ITNK cells from the same source.
  • Figure 12 is a mass spectrometry flow cluster analysis of t-SEN dot density map.
  • A shows CD45+ monocytes derived from cord blood and peripheral blood respectively, which are divided into transduction PX458 empty vector (Mock-T) and transduction The t-SEN point density of the three experimental groups of PX458-gBCL11B (PX458-T) and K562 cells after being transduced with PX458-gBCL11B for 24 hours (activated PX458-T);
  • BC respectively show cord blood (CB) , Adult peripheral blood (PBMC)-derived Mock-T and BCL11B knock-out T cells (BCL11B-KO T), whether activated BCL11B knock-out T cells (BCL11B-KO T) and (Activated BCL11B-KO T) ) Between clustering differences and over-transition conditions (arrows).
  • Figure 13 is a t-SEN color enrichment cluster map of fusion of cord blood and peripheral blood CD45+ cells; ITNK cells are labeled as indicated.
  • Figure 14 is the immunophenotyping analysis of ITNK cells of the present application-t-SEN color enrichment cluster map of fusion of cord blood and peripheral blood CD45+ cells; among them, (A) shows the expression of NKG2D and CD161 markers, (B) shows CD25 , CD127, CD16, KIRDL2, KIRDL 3 and NKG2A marker expression, and (C) shows the expression of immune checkpoint markers PD-1, CTLA-4, FOXP3 and TIM-3.
  • Figure 15 is based on the frequency of umbilical cord blood T cell immune cell subpopulations.
  • ITNK cells can be differentiated from CD4+, CD8+ and ⁇ TCR+ T cells. All ITNK subtypes can be activated by HLA-negative cells (K562).
  • Figure 16 shows the frequency of grouping according to the immune cell subsets of peripheral blood T cells.
  • ITNK cells can be differentiated from CD4+, CD8+ and ⁇ TCR+ T cells. All ITNK subtypes can be activated by HLA-negative cells (K562).
  • Figure 17 shows the results of mass spectrometry flow heat map analysis, which shows the marker expression of each immune cell subpopulation.
  • Figure 18 shows ITNK cell sorting for RNA-Seq. Sort CD3+T, CD3-NKp46+NK, CD3+NKp46+CB-ITNK and PBMC-ITNK in cord blood or adult peripheral blood for RNA sequencing and atac sequencing.
  • Figure 19 is RNA sequencing-principal component analysis of gene expression data of cell subgroups.
  • Figure 20 is the RNA sequencing-KEGG enrichment approach to analyze the gene up-regulation of ITNK cells relative to T cells (cut-off: absolute logarithm 2 times, change ⁇ 1; adjusted P value ⁇ 0.05).
  • Figure 21 is the RNA sequencing-KEGG enrichment approach to analyze the gene up-regulation of ITNK cells relative to NK cells (cut-off: absolute logarithm 2 times, change ⁇ 1; adjusted P value ⁇ 0.05).
  • Figure 22 is an RNA-Seq hierarchical clustering heat map.
  • the left image shows the difference in gene transcription expression of T, ITNK, and NK cells (log2 absolute fold change ⁇ 1; adjusted P value ⁇ 0.05), and the right image is through RNA -Seq analysis of the heat map of the differential expression of the selected genes in T cells, ITNK cells and NK cells; compared with T cells, the expression of NK signaling genes in ITNK cells is up-regulated, and the expression of TCF1 and LEF1 genes is down-regulated (log2 absolute fold change ⁇ 1; P value after adjustment ⁇ 0.05).
  • Figure 23 is a dynamic flow cytometric analysis diagram of the immunophenotype of ITNK cells; flow cytometry is used to analyze the 0, 5, 10, 15, and 20 days after knocking out BCL11B in human T cells to detect CD3+CD4+, CD3
  • the ratio of NKp30 and NKp46 positive subgroups in the +CD8+ subgroup changed; the data represents three experiments; NKp46 was detected on the 5th day after BCL11B knockout, and it was stable on the 10-15th day.
  • Figure 24(A) is a t-SNE dot plot of the results of Sc-RNA seq analysis.
  • the top figure shows the cell distribution of D0-D20, and the bottom figure shows the PX458-gBCL11B gene transduction of T cells after BCL11B knockout, D0(2263), Cluster distribution map of 4948 cells on D5 (1565), D10 (498), D15 (204), D20 (418) days.
  • 4948 cells were clustered into 11 cell subpopulations, ITNK cells were mainly enriched in the subpopulations marked by the (red) circle;
  • B the 4948 cells (11 subpopulations) detected by t-SNE dot plot The expression of NK cell markers;
  • C T cell markers, NK cell markers, immune checkpoints, transcription factors, and apoptotic genes in 4948 cells (11 subgroups) detected by t-SNE dot diagram The expression of related genes.
  • FIG. 25 (A) KEGG signal pathway analysis, which shows that NK cytotoxicity genes are highly expressed in ITNK cells; (B) Violin diagram shows the expression profiles of KAR and KIR genes related to NK cells in different subgroups; (C) Violin diagram Shows the expression profile of genes related to the development of T cells and NK cells.
  • NK signaling genes ID2, TBX21
  • NOTCH related genes MXI1, ZMIZ1, RBPJ
  • AP-1 related genes (FOS, JUN, JUNB, JUND) are up-regulated in ITNK cells.
  • Figure 26(A) is an unsupervised trajectory analysis of single cells from subgroup 5 (CD8+T), subgroup 0 (early CD8+ITNK) and subgroup 1 (late CD8+ITNK) showing CD8+ T cells to CD8 + Progressive transition of ITNK cells;
  • Figure 27 (A) is a bar graph showing the up-regulation of the expression levels of NK-related transcription factors ID2 and TBX21 in ITNK cells; (B) is a Western blot analysis of TBX21 and ID2 in immune cell lysates, where, left The lane is NK cells, the middle lane is ITNK cells, the right lane is T cells, and ⁇ -Tublin is used as an internal reference.
  • Figure 28 is a bar graph showing the amount of IFN ⁇ secreted by T cells and ITNK cells after being stimulated with anti-NKp30, anti-NKp46 and anti-CD3/CD28 antibodies (5ug/ml) through the Elisa experiment; where the data comes from three A sample of two independent donors; data are expressed as mean ⁇ SD; **P ⁇ 0.01, ***P ⁇ 0.001; paired t test.
  • FIG. 29 shows the cytokine secretion of T cells, ITNK cells and NK cells after stimulation by K562 cells; specifically, T, ITNK and NK cells are respectively E (effector): T (target) ratio 1:1 and K562 cells were incubated at 37°C for 18 hours; the supernatant was taken, and the cytokines (CSF2, CCL4, IFN ⁇ , CCL3, IL13, IL2, TNF, CX3CL1, IL8, IL10, IL23, IL7, IL4, IL5, CXCL11, CCL20, IL6, IL17A, IL21, IL12, IL1 ⁇ ) concentration; the value is expressed as the mean ⁇ SD of 3 different donors.
  • cytokines CSF2, CCL4, IFN ⁇ , CCL3, IL13, IL2, TNF, CX3CL1, IL8, IL10, IL23, IL7, IL4, IL5, CXCL11, CCL20, IL6, IL17A
  • Figure 30 shows the percentage of specific cytotoxicity of ITNK cells to HLA-negative K562 cells (A), HLA-positive Hela cells (B), HLA-positive A549 cells (C) and HLA-positive NALM-6 cells (D), where the data Expressed as mean ⁇ standard deviation; **P ⁇ 0.01; unpaired t-test.
  • Figure 31 shows the protein and phosphorylation levels of Fyn, PLC-g2, Syk, Erk1/2 and mTOR in the immune cell lysate measured by western blotting after K562 cell stimulation for 6 hours.
  • the three lanes on the left are NK cells and the three in the middle.
  • the lanes are ITNK cells, the three lanes on the right are T cells, BCL11B is used as a gene editing control, and GAPDH is used as a loading control.
  • FIG 32 (A) is a schematic diagram of the test for detecting ITNK cells killing tumor cells in vivo;
  • (BC) shows the use of in vivo bioluminescence imaging technology to quantitatively analyze the total flux of experimental mouse luciferase activity at a specific time point (each group 5 mice), the result is the mean ⁇ SD, **P ⁇ 0.01, unpaired t test;
  • (E) shows that Hela tumor-bearing mice were treated with PBS, Mock T, ITNK and NK cells on day 7 and day 10. The tumor size was detected at the designated time point (5 mice per group), and the data were expressed as mean ⁇ standard deviation; ***P ⁇ 0.001; unpaired t-test.
  • PB peripheral blood
  • BM bone marrow
  • spleen liver, and lung
  • GN19NGG the target site
  • N is preferably G
  • the target site can be on the antisense strand
  • F forward
  • R reverse
  • gRNA guideRNA
  • F forward
  • R reverse
  • gRNA guideRNA
  • the gRNA gene knockout plasmid vector that knocked out the second and third exons was selected for the next experiment.
  • This 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, and the highest knockout efficiency
  • the third pair of gRNAs, the third pair of gRNAs with the lowest knockout efficiency of the third exon, the gene knockout plasmids corresponding to the third pair of gRNAs, and their mixture can reprogram T cells to obtain the immune killer lymphocytes of the present application.
  • the gRNAs of SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 50 and SEQ ID NO: 51 were used to construct BCL11B gene knockout plasmids and mix them for the next experiment.
  • T cells from all sources use T cell activation kit (Miltenyi Biotec), using magnetic beads coated with anti-human CD3, anti-human CD28 antibody, and anti-human CD2 to incubate with T cells at a ratio of 1:2 Activation (cell density: 2.5 ⁇ 10 6 cells/ml, medium: T551-H3 (Takara, Japan) medium, containing 5% autologous plasma, hIL2 (100IU/ml), gentamicin sulfate (20 ⁇ g/ml) ), 10mm HEPES, 2mm glutamine and 1% penicillin/streptomycin), 24 to 48 hours after activation, the T cells are eluted from the avidin MACS iBead TM particles for use.
  • T cell activation kit Miltenyi Biotec
  • T cell markers such as CD3 and NK cell markers such as NKp46, CD56, NKp30 and NKp44, and it is determined that the human ITNK cells of this application have been obtained .
  • NK cells only express NK cell markers such as NKp46 and CD56, but not T cell markers such as CD3.
  • the T cells electroporated to the empty vector express T cell markers such as CD3, but not NK cell markers.
  • the expression of each cell marker of T cells, NK cells and ITNK cells is shown in Figure 4-6, and their phenotypic differences are also summarized in Table 2 below.
  • ITNK cells reprogrammed from T cells have a cell morphology that is different from T cells, and their morphology is similar to NK cells, with a small nucleus (compared to T cell nucleus occupying the entire cell volume).
  • NK cells a small nucleus (compared to T cell nucleus occupying the entire cell volume).
  • the transmission electron micrographs of T cells, NK cells and ITNK cells are shown in Figure 7.
  • the inventors also compared the expression profiles of these NK markers in BCL11B-deficient T cell subsets derived from cord blood and peripheral blood, and found that the percentage of CD8+NKp46+ and CD8+CD56+ cells was significantly higher than that of CD4+NKp46+ and CD4+CD56+ cells indicate that NKp46+CD3+ITNK is mainly derived from CD8+ T cells (see Figure 8).
  • CD4+ T cells express NKp30 but not NKp46 after losing BCL11B (see Figure 8B).
  • the CD4-CD8-NKp46+ subgroup expresses "TCR ⁇ ", which are ⁇ TCR+ITNK cells (see Figure 9).
  • TCR ⁇ which are ⁇ TCR+ITNK cells
  • the BCL11B deletion in CD4+, CD8+ and ⁇ TCR+ T cells was further verified by DNA sequencing (see Figure 9).
  • TCR ⁇ sequencing Using a human TCR ⁇ analysis kit, T cells from the same donor and ITNK cells obtained in Example 1 were subjected to RNA extraction and CDR3 region targeted amplification to obtain TCR RNA. Use the Hiseq4000 platform to sequence the TCR RNA to obtain the TCR library. Use MiXCR(ref) to perform cluster combination analysis. Export instructions through MiXCR clone and export TCR ⁇ clone type with the "-chain" parameter.
  • TCR sequencing comparing the diversity of TCR clones of T cells and ITNK cells from the same donor, it was found that the diversity of TCR clones was the same (see Figures 10 and 11), which confirmed that the ITNK cells obtained were reprogrammed after T cells lacked BCL11B. It comes, and maintains the TCR diversity of T cells, rather than the expansion of a special, unknown small subpopulation of human T cells.
  • the ITNK cells obtained in Example 1 were subjected to single-cell immunophenotype analysis by Mass Cytometry (CyTOF) technology, and the control group was T cells transduced with an empty vector.
  • Preparation and pretreatment of mass spectrometer samples Centrifuge the cells from the culture suspension, resuspend in PBS containing 0.5% BSA and 0.02% NaN 3, and incubate with anti-human CD16/32 monoclonal antibody for 10 minutes at room temperature To block Fc receptors. Subsequently, a mixture of metal-labeled antibodies against cell surface molecules was added and incubated on ice for a further 20 minutes. Antibodies are pre-coupled antibodies (Fluidigm) or use mass spectrometry flow coupling kit (Fluidigm) and internally coupled according to the instructions. 5mM cisplatin was added to the cells, and the staining was incubated in FBS (Fluidigm) for 1 minute on ice.
  • FBS Fludigm
  • the cells 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 191/193Ir DNA intercalator (Fluidigm) diluted 1:4000 (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 ddH 2 O, and then resuspended and diluted with ultrapure water (ddH 2 O) to approximately 10 6 cells/ Ml. Subsequently, CyTOF2 equipment (Fluidigm) was used to detect and collect cell sample data at an event rate of ⁇ 400 events/sec.
  • Fluidigm 191/193Ir DNA intercalator diluted 1:4000 (the intercalator was diluted with PBS containing 1.6% para
  • PhenoGraph clustering algorithm cluster analysis is performed according to the cellular immune phenotype differences of 40 markers, and ITNK cells derived from cord blood (hereinafter also referred to as CB-ITNK) and ITNK cells derived from peripheral blood (hereinafter also referred to as PBMC -ITNK) and Mock-T cells are integrated and classified into 39 subgroups, as shown in Figures 12, 13, 14, 15, 16 and 17. It can be seen from Figure 12 that PX458T and Mock-T separated before and after K562 cell stimulation, and there was almost no overlap, indicating that there was a significant difference between PX458 T and Mock-T; while there was a significant difference between PX458T cells before and after K562 cell stimulation. Overlap, and the activated PX458-T cells derive more new subpopulations.
  • the ITNK cells of the present application include the CD3 negative cell subpopulation of No. 33, the CD4+ cell subpopulation of No. 5-10, and the No. 20-22 and 26-28 TCR ⁇ + cell subsets of No. 23-24 and TCR ⁇ + cells of NO.23-24, and both express NK-related markers such as CD56, NKp30, NKp44, NKp46 or CD11C, etc.
  • the ITNK cells of the present application are different from conventional NK cells because the ITNK cells of the present application do not express CD127, CD16, NKG2A and KIR2DL2 (as shown in Figure 14B). Moreover, the ITNK cells of the present application have low expression of immunosuppressive checkpoints PD-1, CTLA-4, and FOXP3 (as shown in Figure 14C). The high expression of immunosuppressive checkpoints can induce immune cells to form immunosuppressive states such as low function and exhaustion. This suggests that the ITNK cells of the present application have strong immune effects and are not easily inhibited by tumors and other immunosuppressive microenvironments.
  • the histogram in Figure 15 clearly shows the percentage of various ITNK cells in CD45+ hematopoietic cells, and the dynamic transition from resting ITNK cells to effector cells after stimulation.
  • the dynamic transition of ITNK cells derived from adult peripheral blood from a resting state to an effective state after stimulation is the same as that of ITNK cells derived from umbilical cord blood.
  • the mass spectrum heatmap shown in Figure 17 shows the dynamic changes of the immunophenotype of ITNK cells before and after activation, and the expression of CD25 increases after activation.
  • ITNK is activated, the expression of NK cell activation receptors and T cell markers is still retained.
  • T and ITNK cells were analyzed by RNA sequencing.
  • the sorting operation is as follows: Flow cytometric analysis or sorting is performed by the flow cytometer Canto, FACS Fortessa (BD), FACSAriaII, etc.
  • PCA Principal component analysis
  • scRNA-seq detects cell samples at different time points. On average, each cell detects 2000-4000 genes, and a total of 20,000 human genes are detected in all cells.
  • t-SNE t-distributed random neighbor-embedded
  • ITNK cells are mainly concentrated in subgroup 6 (CD4+ITNK), subgroup 1 (CD8+ITNK) and subgroup 10 ( ⁇ TCR+ITNK) (Figure 24) , And these subpopulations completely overlap with the BCL11B deletion expression subpopulation, further indicating that ITNK cells are reprogrammed by knocking out BCL11B in T cells.
  • KEGG enrichment analysis showed that ITNK cells specifically expressed NK marker genes and related genes (Figure 25).
  • NOTCH1, NOTCH2, ZMIZ1 NOTCH1 cofactor
  • RBPJ NOTCH downstream transcription factor
  • the subunits of FOS, JUN, and JUNB, the three AP-1 transcription factors are lowly expressed in the early stage of ITNK reprogramming, and gradually up-regulated in the late stage of reprogramming (Figure 25C). It can be seen that NOTCH signal and AP-1 signal are up-regulated after ITNK reprogramming.
  • T cells and ITNK cells are tightly clustered in the t-SNE dot plot, indicating that the transition from T cells to NK cells is almost synchronous.
  • NK marker genes of CD8+T subgroup 5
  • early CD8+ITNK subgroup 0
  • late CD8+ITNK subgroup 1
  • T cells began to reprogram into ITNK cells on the fifth day after BCL11B was knocked out.
  • transcription factor genes TBX2, ID2, etc.
  • Example 6 The ITNK cells of this application can recognize and kill MHC I positive/negative tumor cells in vitro
  • NCR and T cell receptor (TCR) expressed on the ITNK cells of this application are functional.
  • TCR T cell receptor
  • anti-NKp30, anti-NKp46 and anti-CD3/CD28 monoclonal antibodies to stimulate ITNK cells. It was found that after stimulation with anti-NKp30 and anti-NKp46 antibodies, the secretion of interferon (IFN) in ITNK cells increased, but the T cells in the control group did not increase ( Figure 28); after stimulation with anti-CD3/CD28 antibodies, the interferon of ITNK cells increased The secretion increased, while the control group T cells did not increase (Figure 28). This indicates that NCR and TCR in ITNK cells are functional.
  • IFN interferon
  • the ITNK cells of the present application can secrete various cytokines, including GM-CSF, IFN, and TNF (as shown in Figure 29), and can recognize and kill MHC-I negative K562 cells (as shown in Figure 30A).
  • ITNK cells can effectively kill Hela and A549 cells (as shown in Figure 30B-C). Both of these cells are highly expressing ligands of NK-activated receptors and MHC I is positive; as shown in Table 2, due to low ITNK cells It expresses NK cell KIR receptors (KIR2DL1, KIR2DL3, KIR3DL1 and KIR3DL2) that mediate immunosuppression, so ITNK cells have a better killing effect on tumors than NK cells.
  • NALM-6 does not express NCR ligands and highly expresses MHC-I molecules.
  • ITNK cells and NK cells do not significantly kill NALM-6 (as shown in Figure 30D).
  • the inventors used K562 cells to stimulate ITNK, NK and T cells, and found that the expression levels of phosphorylated Fyn, PLC- ⁇ 2, Syk, Erk and m-TOR in ITNK and NK cells were similar, but higher than those of T cells (such as Figure 31).
  • Example 7 The ITNK cells of the present application can inhibit tumor growth in vivo
  • the inventors also assessed whether the ITNK cells of the present application 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 (Figure 32A). At a specific time point, the survival status of K562 in mice was detected by in vivo imaging equipment. Compared with the T cell (negative control) and PBS (blank control) group, the experimental mice treated with ITNK cells and NK cells significantly reduced the tumor burden of K562 after 28 days of infusion ( Figures 32B and 32C) and survived longer ( Figure 32D).
  • the inventors also 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 NK, T cell or PBS treatment group ( Figure 32E). It can be seen that the ITNK cells of the present application are effective killers of tumor cells in the body and can prevent tumor progression.
  • ITNK cells In order to verify the distribution and maintenance of ITNK cells in the body.
  • ITNK cells were transplanted ITNK cells into NSI strains of immunodeficient mice lacking T, B, and NK cells, and tested after transplantation 1, 7, 14, 21 and 180.
  • Peripheral blood was measured with peripheral blood (PB) and spleen (SP).
  • PB peripheral blood
  • SP spleen
  • the percentage of ITNK cells in bone marrow (BM), liver (liver) and lung (lung) Figure 33A-B.
  • the proportion of ITNK cells reached a peak 21 days after transplantation, then gradually decreased, and could not be detected after 6 months (Figure 33B). It can be seen from Figure 33B that the maintenance ability of ITNK cells in vivo is better than that of T cells. We have never observed ITNK cells attacking the host or unrestricted expansion.

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Abstract

提供了一种工程化人体免疫细胞、其制备方法及应用。该工程化人体免疫细胞是通过缺失BCL11B基因重编程人T细胞诱导而成的免疫杀伤淋巴细胞，其保留了衍生其的T细胞的标志物和功能并具有NK细胞的标志物和功能。重编程的工程化人体免疫细胞可用于制备治疗肿瘤和感染性疾病的药物。

Description

一种工程化人体免疫细胞、其制备方法及应用 技术领域
本申请涉及生物医药技术领域，具体涉及一种工程化人体免疫细胞、其制备方法及应用。
背景技术
目前，免疫疗法已成为癌症治疗领域中最受关注和最有希望的“新”想法。2013年《科学》杂志评出的年度十大科学突破排行榜中，肿瘤免疫治疗高居榜首。诺华和凯特公司的嵌合抗原受体(CAR,Chimeric Antigen Receptor)T细胞被美国FDA批准上市，肿瘤免疫细胞治疗在血液恶性癌症治疗领域取得了里程碑式进展。然而，肿瘤免疫细胞治疗仍然存在临床治疗的技术瓶颈，如基因修饰免疫细胞的靶点单一等原因导致肿瘤免疫逃逸，肿瘤复发；如实体肿瘤缺乏高效、低副作用的特异性标志物，目前的基因修饰免疫细胞治疗对实体肿瘤均未见高效、安全的临床疗效。
T细胞根据其表面标志和功能的差异，可分为不同的亚群；如根据TCR类型差异可分为γδT细胞和αβT细胞。αβT细胞占T细胞95％以上，是体内具有T细胞分化标记、执行T细胞功能主要的细胞群，代表了T细胞的多样性。而γδT细胞是一群高度异质性细胞，其表面T细胞受体由γ链和δ链组成，其亚类种类多、表型多变、功能丰富，γδT细胞各亚类间生物学特性各异，在机体肿瘤、感染及自身免疫性疾病等的发生、发展中均有重要作用，被认为是机体连接天然免疫与适应性免疫功能的桥梁。
像T细胞一样，自然杀伤(NK)细胞是人体免疫系统中不可或缺的一部分。NK细胞被认为是占外周血淋巴细胞约10-15％且在天然免疫响应中起关键作用的淋巴样细胞。与T细胞不同的是，NK细胞以MHC非限制性方式识别其靶标。NK细胞可呈现抗病毒作用、抗GvH作用和抗癌作用，具体地，NK细胞直接杀伤恶性肿瘤，包括肉瘤、骨髓瘤、癌、淋巴瘤和白血病，或有助于通过诱导树突状细胞(DC)活性或肿瘤特异性细胞毒性T淋巴细胞(CTL)的适应性免疫活化，由此消除异常细胞，所述异常细胞为肿瘤细胞或正发展成为肿瘤细胞的细胞。尽管NK细胞具有作为对抗癌症或感染性疾病的治疗剂的潜力，但是正常人体内大多的NK细胞以休眠状态存在，且癌症患者内的NK细胞由于癌细胞的免疫逃逸机制缺少其功能。为将自然杀伤细胞实际应用为治疗剂，需要能够识别并破坏肿瘤细胞的活化的自然杀伤细胞，且由于体内的自然杀伤细胞数目有限，因此，获得足够数量的活化的自然杀伤细胞是非常重要的。
细胞重编程(cell reprogramming)指的是分化的细胞在特定的条件下被逆转后恢复到全能性状态，或者形成胚胎干细胞系，或者进一步发育成一个新的个体的过程。在疾病的免疫治疗领域，已有通过细胞重编程来实现免疫细胞的类型转化的相关报道。例如，美国格拉斯通研究所(Gladstone Institutes)的丁胜博士曾报道，通过特定的重编程方法，将促炎的效应T细胞重编程为抗炎的调节性T细胞；这种重编程对于自身免疫性疾病的治疗意义重大，具体地，在自身免疫性疾病中，被过度激发的效应T细胞会对机体造成损害，将这些细胞转化为调节性T细胞有助于减少免疫系统的过度活跃，使其恢复平衡，从而根本上治疗疾病。
目前，尚未见细胞重编程技术在人类肿瘤免疫治疗中的应用。
发明内容
针对现有技术的不足及实际的需求，本申请提供了一种通过重编程工程化的人体免疫细胞、其制备方法及应用。本申请的工程化人体免疫细胞兼具T细胞和NK细胞的部分标志物和功能，同时表达NK细胞和T细胞的抗原识别杀伤受体，因而，比NK细胞和T细胞本身具有更广谱的肿瘤抗原识别杀伤功能；同时，相较于衍生其的人成熟T细胞，本申请的工程化人体免疫细胞扩增能力增强，抗肿瘤效果更好。
在第一方面，本申请提过了通过重编程人T细胞诱导而成的免疫杀伤淋巴细胞(ITNK)，其保留衍生其的T细胞的标志物和功能并具有NK细胞的标志物和功能，其中，所述人T细胞的重编程涉及BCL11B基因的缺失。
优选地，所述人T细胞为成熟的人T细胞或含有成熟人T细胞的细胞群；进一步优选地，所述成熟人T细胞或含有成熟人T细胞的细胞群来源于人体脐带血或外周血；进一步优选地，所述成熟人T细胞或含有成熟人T细胞的细胞群来源于多能干细胞、胚胎干细胞或脐带血干细胞分化获得的成熟T细胞或细胞群。
优选地，所述重编程的免疫杀伤淋巴细胞表达功能性的TCR、CD3和NKp30。
优选地，所述重编程的免疫杀伤淋巴细胞表达选自以下的NK细胞的标志物：CD11c、NKG2D和CD161。
优选地，所述重编程的免疫杀伤淋巴细胞低表达或不表达PD-1、CTLA-4或FOXP3免疫抑制检查点。
优选地，所述重编程的免疫杀伤淋巴细胞低表达或不表达NK相关标志物：CD127、CD16、KIRDL2、KIRDL3、NKG2A。
优选地，相较于衍生其的T细胞，所述重编程的免疫杀伤淋巴细胞的NOTCH表达上调。
优选地，相较于衍生其的T细胞，所述重编程的免疫杀伤淋巴细胞中，LEF1和TCF7转录因子表达下降，NOTCH、AP1、mTOR、ID2、TBX21和NFIL3表达上升。
优选地，所述重编程的免疫杀伤淋巴细胞的TCR介导的信号转导增强；
优选地，相较于衍生其的T细胞，所述重编程的免疫杀伤淋巴细胞的与TCR介导的信号转导相关的基因CSF2、FOS、MAPK12、MAP3K8、IFNγ、NFKBIA、MAPK11、IL-10和TEC的表达上调。
优选地，相对于NK细胞，所述重编程的免疫杀伤淋巴细胞的T细胞识别和TCR信号转导增强，优选地，CD3、CD4、CD8、CD40LG的表达上调。
优选地，相较于衍生其的T细胞，所述重编程的免疫杀伤淋巴细胞的NK杀伤毒性相关信号转导增强；
优选地，相较于衍生其的T细胞，所述重编程的免疫杀伤淋巴细胞的与NK杀伤毒性相关信号转导相关的基因PRF1、CSF2、ICAM1、CD244、PLCG2、IFNG、FCER1G、GZMB、NCR2、NCR1、KIR2DL4和SYK的表达上调。
在一个优选的具体实施方案中，所述重编程的免疫杀伤淋巴细胞包括CD8+NKp46 hiNKp44+NKp30+、CD4+NKp30+和γδTCR+NKp46 hiNKp44+NKp30+T细胞亚群。
在一个优选的具体实施方案中，所述人T细胞为成熟人T细胞，并且重编程所述人成熟T细胞包括：
1)激活成熟人T细胞；
2)对步骤1)所得激活的成熟人T细胞实施BCL11B基因敲除；
3)将步骤2)所得细胞用T细胞培养基进行培养。
其中，步骤1)中，使用抗人CD3抗体、抗人CD28抗体和抗人CD2抗体进行激活；
优选地，使用抗人CD3抗体、抗人CD28抗体和抗人CD2抗体的磁珠与人成熟T细胞以1:2混合孵育激活T细胞。
其中，步骤2)中，使用CRISPR/CAS9技术进行BCL11B基因敲除；
优选地，所述基因敲除的靶点在BCL11B基因的第二外显子和/或第三外显子处。
其中，步骤3)中，所述T细胞培养基包含IL-2；优选地，不与OP9-DL1进行共培养。
第二方面，本申请提供了一种制备上述第一方面所述的细胞的方法，其包括：
1’)激活人T细胞；
2’)对步骤1’)所得激活的人T细胞实施BCL11B基因敲除；
3’)将步骤2’)所得细胞用T细胞培养基进行培养。
上述方法中，优选地，所述人T细胞为成熟人T细胞或含有成熟人T细胞的细胞群；进一步优选地，所述成熟人T细胞或含有成熟人T细胞的细胞群来源于人体脐带血或外周血；进一步优选地，所述成熟人T细胞或含有成熟人T细胞的细胞群来源于多能干细胞、胚胎干细胞或脐带血干细胞分化获得的成熟T细胞或细胞群。
上述方法中，优选地，步骤1’)中，使用抗人CD3抗体、抗人CD28抗体和抗人CD2抗体进行激活；在一个优选的具体实施方案中，使用抗人CD3抗体、抗人CD28抗体和抗人CD2抗体的磁珠与人成熟T细胞以1:2混合孵育激活T细胞。
优选地，步骤2’)中，使用CRISPR/CAS9技术进行BCL11B基因敲除；进一步优选地，在BCL11B基因的第二外显子和/或第三外显子处进行基因敲除。
优选地，步骤3’)中，所述T细胞培养基包含IL-2；优选地，不与OP9-DL1进行共培养。
第三方面，本申请还提供了如上述第一方面所述的细胞在制备治疗选自以下的疾病的药物中的用途：肿瘤、艾滋病和感染性疾病；优选地，所述感染性疾病为病毒感染性疾病。
优选地，所述药物还包含药学上可接受的赋形剂。
本申请首次实现了将人T细胞重编程为免疫杀伤淋巴细胞，重编程后的细胞同时表达NK细胞和T细胞的抗原识别杀伤受体，特别是功能性TCR，同时具备T细胞和NK细胞功能；由于其同时表达NK细胞和T细胞的抗原识别杀伤受体，所以可识别这些受体敏感的抗原，相对于T细胞和NK细胞，不仅具有更广谱的肿瘤抗原识别杀伤功能，而且还具有更广谱的病毒、细菌等微生物识别清除功能。
此外，本申请的重编程后的细胞具有高效的体外扩增能力。在过继细胞转移(ACT)治疗中，T细胞和NK细胞都被用于治疗癌症。本申请的重编程后的细胞兼具T细胞和NK细胞的功能，相较于NK细胞应用于过继性免疫治疗(ACT)时的可用性和扩增能力限制，使用者可从患者的外周血中获得大量的T细胞来产生本申请的重编程的免疫杀伤淋巴细胞，并在2～3周内，即可从实体瘤患者约100×10 6个外周血单核细胞中制备获取200-1248×10 6个重编程的免疫杀伤淋巴细胞，达到患者回输细胞的需求量。
附图说明
图1是本申请实施例1中所构建的PX458-gBCL11B载体的示意图。
图2是基因测序检测并验证转导了PX458-gBCL11B的T细胞的BCL11B外显子是否被敲除的图示，对照组为转导了PX458空载体(Mock)的T细胞。
图3是Western Blotting检测并验证转导了PX458-gBCL11B的T细胞中BCL11B蛋白表达水平，以进一步确认BCL11B蛋白是否缺失的图示，对照组为转导了PX458空载体(Mock)的T细胞。
图4为流式细胞结果散点图，其显示相对于Mock T细胞和NK细胞，本申请的ITNK细胞(即，PX458-gBCL11B转导的T细胞，图中以PAX458指示)同时高表达T细胞标志物CD3和NK细胞标志物CD56和NKp46。所有样本来源于同一PBMC样本，Mock T为PBMC通过Pan-T分选，所以存在7.2-7.8％的CD3-CD56+NKp46+的NK细胞杂群；PX458为Mock T经过PX458-gBCL11B转染获得，与Mock-T存在相同的7.6-7.8％的NK细胞亚群；NK细胞为PBMC通过NK细胞培养基培养纯化获得，所以存在少量10.4％的CD3+CD56+的NKT或γδT细胞杂群。
图5为流式细胞结果散点图(A)其显示脐带血来源的ITNK细胞(即，PX458-gBCL11B转导的T细胞，图中以PAX458指示)相对于Mock T细胞同时高表达T细胞标志物CD3和NK细胞标志物NKp46、CD56、NKp30和NKp44，以及相应的细胞百分比统计图(B)。
图6为流式细胞结果散点图(A)其显示外周血来源的ITNK细胞(即，PX458-gBCL11B转导的T细胞，图中以PAX458指示)相对于Mock T细胞同时高表达T细胞标志物CD3和 NK细胞标志物NKp46、CD56、NKp30和NKp44，以及相应的细胞百分比统计图(B)。
图7是Mock T细胞、NK细胞和ITNK细胞(即，PX458-gBCL11B转导的T细胞，图中以PAX458指示)的透射电镜图，其显示：与T细胞相比，ITNK细胞的核质比较低；其中，1指示核；2指示线粒体；3指示内质网；4指示粒；标尺，左图为2μm，右图为500nm。
图8显示PX458-gBCL11B转导的脐带血、外周血来源的T细胞(即，ITNK细胞)的CD4+、CD8+、CD3+CD4-CD8-T细胞亚群中NK受体表达，其中，NKp46在CD8+T细胞中表达明显高于CD4+T细胞；n＝6，表示试验为6个独立健康供体的重复；***P≤0.001，配对t检验。
图9显示PX458-gBCL11转导的T细胞的流式细胞术分析，并将CD4+、CD8+和CD4-CD8-亚群中的CD56+细胞分选并进行DNA测序分析，进一步证实了ITNK细胞中的BCL11B位点被敲除。
图10显示TCRβ多样性测序，所有TCRβ链序列轨迹变量在同一来源的T细胞和ITNK细胞中具有相同的多样性。
图11显示TCRα多样性测序，所有TCRα链序列轨迹变量在同一来源的T细胞和ITNK细胞中具有相同的多样性。
图12是质谱流式聚类分析t-SEN点密度图，(A)显示分别来源于脐带血和外周血的CD45+单核细胞，分为进行转导PX458空载体(Mock-T)、转导PX458-gBCL11B(PX458-T)和转导了PX458-gBCL11B后经过K562细胞24小时活化(激活的PX458-T)三个实验组的t-SEN点密度；(B-C)分别展示脐带血(CB)、成体外周血(PBMC)来源的Mock-T和敲除BCL11B的T细胞(BCL11B-KO T)、是否经过激活的敲除BCL11B的T细胞(BCL11B-KO T)和(Activated BCL11B-KO T)之间的分群差异和过度转变情况(箭头)。
图13是脐带血和外周血CD45+细胞融合的t-SEN彩色富集簇图；ITNK细胞按指示标记。t-SEN图上标记物CD3、CD4、CD8、γδTCR、CD56、NKp46、NKp30、NKp44和CD11c的标准化表达使细胞着色。
图14是本申请ITNK细胞的免疫表型分析-脐带血和外周血CD45+细胞融合的t-SEN彩色富集簇图；其中，(A)显示NKG2D和CD161标记物表达量，(B)显示CD25、CD127、CD16、KIRDL2、KIRDL 3和NKG2A标志物表达量，以及(C)显示免疫检查点标记物PD-1、CTLA-4、FOXP3和TIM-3的表达量。
图15是根据脐带血T细胞的免疫细胞亚群分组的频率，ITNK细胞可由CD4+、CD8+和γδTCR+T细胞分化而来，所有ITNK亚型均可被HLA阴性细胞(K562)激活。
图16是根据外周血T细胞的免疫细胞亚群分组的频率，ITNK细胞可由CD4+、CD8+和γδTCR+T细胞分化而来，所有ITNK亚型均可被HLA阴性细胞(K562)激活。
图17显示质谱流式热图分析结果，其显示了每个免疫细胞亚群的标志物表达情况。
图18显示用于RNA-Seq的ITNK细胞分选。分选脐带血或成体外周血中CD3+T、CD3-NKp46+NK、CD3+NKp46+CB-ITNK和PBMC-ITNK用于RNA测序和atac测序。Mock-T(n＝6)、ITNK(n＝6)和NK细胞(n＝4)的分选细胞纯度分别为95.3±3.61％、92.41±2.6％和92.88±2.06％。
图19是RNA测序-细胞亚群基因表达数据的主成分分析。
图20是RNA测序-KEGG富集途径分析ITNK细胞相对于T细胞的基因上调情况(截止:绝对对数2倍，变化≥1；调整P值≤0.05)。
图21是RNA测序-KEGG富集途径分析ITNK细胞相对于NK细胞的基因上调情况(截止:绝对对数2倍，变化≥1；调整P值≤0.05)。
图22是RNA-Seq分层聚类热图，其中，左图显示T、ITNK、NK细胞基因转录表达的差异(log2绝对倍数变化≥1；调整后P值≤0.05)，右图是通过RNA-seq分析筛选出的基因在T细胞、ITNK细胞和NK细胞中差异表达的热点图；与T细胞相比，ITNK细胞中NK信号基 因表达上调，TCF1和LEF1基因表达下调(log2绝对倍数变化≥1；调整后P值≤0.05)。
图23是ITNK细胞的免疫表型动态流式分析图；通过流式细胞技术，分析在人T细胞中敲除BCL11B后的第0、5、10、15、20天，检测CD3+CD4+、CD3+CD8+亚群中NKp30和NKp46阳性亚群的比例变化；数据代表了三个实验；BCL11B敲除后的第5天开始检测到NKp46，第10-15天稳定。
图24(A)是Sc-RNA seq分析结果的t-SNE点图，上图表示D0-D20的细胞分布图，下图表示PX458-gBCL11B基因转导T细胞敲除BCL11B后D0(2263)、D5(1565)、D10(498)、D15(204)、D20(418)天的4948个细胞聚类分布图。4948个细胞通过聚类为11个细胞亚群，ITNK细胞主要富集在(红色)圆圈标记的亚群；(B)通过t-SNE点图表示检测的4948个细胞(11个亚群)中NK细胞标志物表达的情况；(C)通过t-SNE点图表示检测的4948个细胞(11个亚群)中T细胞标志物、NK细胞标志物、免疫检查点、转录因子、凋亡基因相关基因的表达情况。
图25(A)KEGG信号通路分析，其显示NK细胞毒性基因在ITNK细胞中高表达；(B)小提琴图显示了不同亚群中NK细胞相关的KAR和KIR基因的表达谱；(C)小提琴图显示了与T细胞和NK细胞发育相关基因的表达谱。NK信号基因(ID2,TBX21)、NOTCH相关基因(MXI1,ZMIZ1,RBPJ)、AP-1相关基因(FOS,JUN,JUNB,JUND)在ITNK细胞中表达上调。
图26(A)为从亚群5(CD8+T)、亚群0(早期CD8+ITNK)和亚群1(晚期CD8+ITNK)的单个细胞的无监督轨迹分析显示CD8+T细胞向CD8+ITNK细胞的逐步过渡；(B)从亚群4(CD4+T)、亚群5(早期CD4+ITNK)和亚群7(晚期CD4+ITNK)的单个细胞的无监督轨迹分析显示CD4+T细胞向CD4+ITNK细胞的逐步过渡。
图27(A)为显示NK相关转录因子ID2和TBX21在ITNK细胞中表达水平上调的条形图；(B)为免疫细胞裂解液中TBX21和ID2的免疫印迹(Western Blot)分析，其中，左泳道为NK细胞，中泳道为ITNK细胞，右泳道为T细胞，并且β-Tublin作为内部参考。
图28为显示通过Elisa实验，检测T细胞和ITNK细胞分别经过anti-NKp30、anti-NKp46和anti-CD3/CD28抗体(5ug/ml)刺激后的IFNγ分泌量的柱状图；其中，数据来自三个独立供体的样本；数据以平均值±SD表示；**P≤0.01，***P≤0.001；配对t测试。
图29显示经过K562细胞刺激后，T细胞、ITNK细胞和NK细胞的细胞因子分泌情况；具体地，将T、ITNK和NK细胞分别以E(效应子):T(靶标)比1:1和K562细胞，在37℃下孵育18小时；取上清液，用多重免疫法(multiplex immunoassay)测定细胞因子(CSF2、CCL4、IFNγ、CCL3、IL13、IL2、TNF、CX3CL1、IL8、IL10、IL23、IL7、IL4、IL5、CXCL11、CCL20、IL6、IL17A、IL21、IL12、IL1β)浓度；值表示为3个不同供体的平均值±SD。
图30显示ITNK细胞对HLA阴性K562细胞(A)、HLA阳性Hela细胞(B)、HLA阳性A549细胞(C)和HLA阳性的NALM-6细胞(D)的特异性细胞毒性百分比，其中，数据以均数±标准差表示；**P≤0.01；非配对t检验。
图31显示免疫印迹法测定K562细胞刺激6小时后，免疫细胞裂解液中Fyn、PLC-g2、Syk、Erk1/2和mTOR的蛋白和磷酸化水平，其中，左边三泳道为NK细胞，中间三泳道为ITNK细胞，右边三泳道为T细胞，以BCL11B作为基因编辑对照，以GAPDH作为上样对照。
图32(A)为检测ITNK细胞体内杀伤肿瘤细胞试验的示意图；(B-C)显示利用体内生物荧光成像技术，对特定时间点的实验小鼠荧光素酶活性的总通量进行定量分析(每组5只小鼠)，结果为平均值±SD，**P≤0.01，未配对t检验；(D)为PBS(n＝10)、Mock T(n＝15)、ITNK(n＝15)、NK细胞(n＝5)处理K562荷瘤小鼠的生存时间统计图；(E)显示第7天和第10天对Hela荷瘤小鼠分别进行PBS、Mock T、ITNK和NK细胞治疗，并于指定的时间点检测肿瘤大小(每组5只小鼠)，数据以均数±标准差表示；***P≤0.001；非配对t检验。
图33显示ITNK细胞移植到缺失T、B、NK细胞的免疫缺陷小鼠NSI品系后其在小鼠体内的分布和维持情况；其中，(A上)为向NSI小鼠注射ITNK细胞和检测实验示意图，其显示ITNK在第1天、第7天、第14天、第21天和第6个月分别进行短期和长期的分析(每组n＝3)；(A下)显示了代表性的流式细胞术分析的CD3+T细胞中ITNK细胞的百分比；(B)为小鼠外周血(PB)、骨髓(BM)、脾脏、肝脏和肺中ITNK细胞的动态分布图，其显示在注射ITNK细胞6个月后检测不到T细胞和ITNK细胞。
具体实施方式
为更进一步阐述本申请所采取的技术手段及其效果，以下结合附图并通过具体实施方式来进一步说明本申请的技术方案，但本申请并非局限在实施例范围内。
除非另有说明，否则在这些实施例中阐述的部件和步骤的相对布置、数字表达式和数值不构成对本申请的限制。对于本领域普通技术人员已知的技术、方法和设备可能不作详细讨论，但在适当情况下，技术、方法和设备应当被视为本说明的一部分。
实施例1 本申请的重编程的自然杀伤(ITNK)细胞的制备
基因敲除质粒载体构建
根据CRISP/CAS9靶位点的选择规则：GN19NGG，GN19是靶位点，N最好仍是G，靶位点可以在反义链上(即，在正义链上的序列顺序为：CCN N19C)，选择了以下靶点序列并分别设计了正向(F)、反向(R)引物作为guideRNA(gRNA)，将gRNA退火、连接入酶切好的PX458载体，构建了PX458-gBCL11B载体(如图1)。在293T细胞系中转染PX458-gBCL11B，并挑取单克隆，通过基因测序检测其中BCL11B敲除靶点的碱基缺失、错位等敲除情况(见图2)，并统计计算敲除效率，如下表1。
表1、BCL11B靶点序列及其对应的gRNA列表
Figure PCTCN2020118104-appb-000001
Figure PCTCN2020118104-appb-000002
Figure PCTCN2020118104-appb-000003
根据上述表1的敲除效率情况，选择敲除第二、第三外显子的gRNA基因敲除质粒载体，用于下一步实验。本申请优选在第二外显子、第三外显子中进行BCL11B基因敲除，利用第二外显子敲除效率最低的第一对gRNA和第二对gRNA的混合物，敲除效率最高的第三对gRNA，第三外显子敲除效率最低的第三对gRNA对应的基因敲除质粒以及它们的混合物均能将T细胞重编程获得了本申请的免疫杀伤淋巴细胞。在本实施例中，采用SEQ ID NO:5和SEQ ID NO:6、SEQ ID NO:50和SEQ ID NO:51的gRNA分别构建BCL11B基因敲除质粒并将其混合，用于下一步实验。
T细胞的分选和激活
采用以下方法进行T细胞的分选和激活：
(1)分别将包含人成熟T细胞的外周血和脐带血血液以300×g离心10分钟，收集血浆，在56℃下热灭活30分钟；
(2)将沉淀的颗粒状血细胞用0.9％NaCl悬浮，通过Ficoll密度梯度离心分离外周血单核细胞(PBMC)；
(3)通过MACS Pan T分离试剂盒(Miltenyi Biotec,Bergisch Gladbach,Germany)阴性分选，从血液如外周血、脐带血等富集全部T细胞(Pan T)
以上(1)-(3)为从外周血和脐带血血液中分离人成熟T细胞的步骤，需要说明的是，其它T细胞来源亦可，如多能干细胞、造血干细胞定向诱导分化等。所有来源的T细胞均利用T细胞活化试剂盒(T cell activation kit(Miltenyi Biotec))，使用涂有抗人CD3、抗人CD28抗体和抗人CD2的磁珠与T细胞以1：2混合孵育激活(细胞密度：2.5×10 6个细胞/ml，培养基：T551-H3(Takara,Japan)培养基，含有5％自体血浆，hIL2(100IU/ml)，硫酸庆大霉素(20μg/ml)，10mm HEPES，2mm谷氨酰胺和1％青霉素/链霉素)，活化24-48小时后，将T细胞从抗生物素MACS iBead TM颗粒中洗脱，备用。
诱导重编程
(1)通过电转(T-023,LONZA Amaxa Nucleofector,Lonza)，将CRISP/CAS9敲除载体PX458-gBCL11B转导入上述激活后的T细胞；
(2)12小时后，将PX458-gBCL11B转导的T细胞(简称PX458-T)离心，并用T551-H3(Takara,Japan)培养基(含有5％自体血浆或胎牛血清(FBS)，500IU/ml hIL2和硫酸庆大霉素(20μg/ml))培养；
(3)随后每3天更换新鲜培养基，保持细胞密度在0.5-1×10 6个细胞/ml范围内，直至电转后第14天。
(4)通过基因测序，检测并验证转导了PX458-gBCL11B的T细胞的BCL11B的第二外显子、第三外显子是否被敲除，如位点诱导插入或缺失等，对照组为转导了PX458空载体(Mock)的T细胞；
(5)通过Western Blotting，检测并验证转导了PX458-gBCL11B的T细胞中BCL11B蛋白表达水平，以进一步确认BCL11B蛋白的缺失，对照组为转导了PX458空载体(Mock)的T细胞，Western Blotting结果见图3。
重编程细胞的表型鉴定
如上，电转T细胞14天后，所得细胞中有19.5-68.7％既表达T细胞标志物如CD3，也表达NK细胞标志物如NKp46、CD56、NKp30和NKp44，则确定获得了本申请的人ITNK细胞。NK细胞只表达NK细胞标志物如NKp46、CD56，但不表达T细胞标志物如CD3。电转空载体的T细胞表达T细胞标志物如CD3，但不表达NK细胞标志物。T细胞、NK细胞和ITNK细胞的各细胞标志物的表达情况如图4-6所示，它们的表型差异也归纳于以下表2。
表2、ITNK细胞与T细胞、NK细胞的表型差异
Figure PCTCN2020118104-appb-000004
此外，通过共聚焦显微镜观察显示，由T细胞重编程而来的ITNK细胞具有区别于T细胞的细胞形态，其形态与NK细胞类似，细胞核(相对于T细胞细胞核占据细胞整体体积)较小，细胞内基质较多，颗粒增大，内质网丰富，高蛋白合成活性，表明重编程后的ITNK细胞为免疫杀伤淋巴细胞。T细胞、NK细胞和ITNK细胞的透射电镜图如图7所示。
此外，发明人还比较了脐带血来源和外周血来源的BCL11B缺失的T细胞亚群中这些NK标志物的表达谱，发现CD8+NKp46+和CD8+CD56+细胞的百分比显着高于CD4+NKp46+和CD4+CD56+细胞，表明NKp46+CD3+ITNK主要来源于CD8+T细胞(见图8)。与CD8+T细胞不同，CD4+T细胞在失去BCL11B后表达NKp30但不表达NKp46(见图8B)。
CD4-CD8-NKp46+亚群表达了“TCRγδ”，为γδTCR+ITNK细胞(见图9)。通过DNA测序进一步验证CD4+、CD8+和γδTCR+T细胞中的BCL11B缺失(见图9)。
实施例2 本申请ITNK细胞的来源鉴定
TCRαβ测序：通过人TCRαβ分析试剂盒，将同一供者来源的T细胞和实施例1所得ITNK细胞进行RNA提取和CDR3区域靶向扩增，获得TCR RNA。利用Hiseq4000平台，对TCR RNA进行测序，获得TCR文库。使用MiXCR(ref)进行聚类组合分析。通过MiXCR克隆导出指令，以“—链”参数导出TCRαβ克隆类型。通过TCR测序，比较同一供者来源的T细胞和ITNK细胞的TCR克隆的多样性，发现TCR克隆多样性一致(见图10和11)，由此确定所得ITNK细胞是T细胞缺失BCL11B后重编程而来，并保持了T细胞的TCR多样性，而不是人T细胞中的特殊、未知小亚群扩增而来的。
实施例3 本申请ITNK细胞的单细胞免疫表型鉴定
通过质谱流式细胞技术(Mass Cytometry，CyTOF)，分别对实施例1所得ITNK细胞进行单细胞免疫表型分析，对照组为转导了空载体的T细胞。
质谱仪样品的制备和预处理：将来自培养悬浮液的细胞离心，用含有0.5％BSA和0.02％NaN 3的PBS重悬，并在室温下用抗人CD16/32的单克隆抗体孵育10分钟以阻断Fc受体。随后，加入针对细胞表面分子的金属标记的抗体混合物，并在冰上进一步孵育20分钟。抗体为预耦合的抗体(Fluidigm)或使用质谱流式耦合试剂盒(Fluidigm)并根据说明书进行内部耦合。向细胞中加入5mM顺铂(cisplatin)，在FBS(Fluidigm)中在冰上孵育染色1分钟。在用固定/透化缓冲液(Thermo Fisher)处理后，将细胞与金属标记抗体混合孵育以标记细胞内蛋白质。清洗细胞后，用1mL的1：4000稀释的191/193Ir DNA嵌入剂(Fluidigm)(嵌入剂用含有1.6％多聚甲醛(EMS)的PBS稀释)染色，并储存在4℃。检测前，将细胞用含有0.5％BSA和0.02％NaN 3的PBS洗涤一次，用ddH 2O洗涤一次，然后用超纯水(ddH 2O)将细胞重悬和稀释至约10 6个细胞/毫升。随后，利用CyTOF2设备(Fluidigm)以<400事件/秒的事件速率检测和收集细胞样品数据。
通过PhenoGraph聚类算法，根据40个标记物的细胞免疫表型差异进行聚类分析，将脐带血来源的ITNK细胞(下文也称CB-ITNK)、外周血来源的ITNK细胞(下文也称为PBMC-ITNK)和Mock-T细胞整合并分类为39个亚群，如图12、13、14、15、16和17。由图12可知，K562细胞刺激前后的PX458T与Mock-T之间分离，几乎不存在重叠，表明PX458  T与Mock-T之间差异显著；而经过K562细胞刺激前后的PX458T细胞之间存在明显的重叠，且激活后的PX458-T细胞衍生了更多新的亚群。
根据质谱流式细胞标记物表达异质性分析结果，本申请的ITNK细胞包括NO.33的CD3阴性细胞亚群、NO.5-10的CD4+细胞亚群、NO.20-22和26-28的CD8+细胞亚群以及NO.23-24的TCRγδ+细胞亚群，且均表达NK相关标志物如CD56、NKp30、NKp44、NKp46或CD11C等，TCRγδ+ITNK相对于γδT细胞，NKp46、NKp30和NKp44三个标志物均为高表达，即(NKp46 highNKp30 highNKp44 high)(如图13和14A)。本申请的ITNK细胞区别于常规的NK细胞，因为本申请的ITNK细胞不表达CD127、CD16、NKG2A和KIR2DL2(如图14B)。而且，本申请的ITNK细胞低表达免疫抑制检查点PD-1、CTLA-4、FOXP3(如图14C)，免疫抑制检查点的高表达会诱导免疫细胞形成低功能、衰竭等免疫抑制状态，由此提示本申请的ITNK细胞具有强免疫效应，且不容易被肿瘤等免疫抑制微环境所抑制。
此外，在脐带血来源的ITNK细胞中，如图15的柱状图清晰地显示了各种ITNK细胞在CD45+造血细胞中所占的百分比，以及刺激后从静止ITNK细胞向效应细胞的动态转变。如图16所示，成年人外周血来源的ITNK细胞经刺激后从静息状态向效应状态的动态转变也与脐带血来源的ITNK相同。如图17的质谱流式热图(heatmap)显示了激活前后ITNK细胞的免疫表型动态变化，激活后CD25表达升高。综上所述，ITNK激活后，仍然保留NK细胞激活受体和T细胞标志物的表达。
实施例4 本申请ITNK细胞的RNA-Seq转录谱分析
为了研究ITNK细胞的全部基因表达谱，发明人对来自于4个脐带血样品和3个成体外周血样品的T和ITNK细胞，以及来自于2个脐带血样品和2个成体外周血样品的NK细胞进行了RNA测序分析。分选操作如下：通过流式细胞仪Canto、FACS Fortessa(BD)、FACSAriaII等进行流式细胞分析或分选。细胞表面受体标记，将细胞与抗体用50μl流式缓冲液(含2％FBS的PBS溶液)混合，4度避光孵育30分钟；细胞胞内标记，细胞利用Foxp3/转录因子染色缓冲液(eBioscience)进行通透性处理，洗脱缓冲液后，用鼠或兔血清阻断，与抗体4度避光孵育30分钟，利用流式缓冲液冲洗一遍后重悬，备用进行流式细胞分析或分选。细胞分选策略和分选纯度验证(如图18)。
采用主成分分析(PCA)对18个样本的RNA测序结果进行相似性评价，发现从转录组分析，ITNK不同于T细胞和NK细胞(如图19)。与T细胞相比，增加776个基因(数据未显示)，包括NK特异性转录因子(如ID2、TBX21、NFIL3、IRF8)、激活和抑制NK细胞受体/蛋白(如IL2RB、IFNG、PRF1、GZMB、TNFRSF4、NCR1、NCR2、PLCG2)、组蛋白基因(HIST1H1D、HIST1H2AC/D/E/G/M、HIST2H2A3/4)(如图22和20)；相反，与T细胞相比，ITNK细胞中有592个基因如TCF-1/TCF-7、LEF-1、IL-7R、MYC、PD-L1、FOXP3等被下调(如表3)。与NK细胞相比，ITNK中666个基因表达增加(数据未显示)，其中大多数基因在T细胞识别和TCR信号转导(CD3、CD4、CD8、CD40LG)中富集(图21)。有趣的是，与NK细胞相比，ITNK中KIR基因KIR2DL1、KIR2DL3、KIR3DL1和KIR3DL2表达下调(如表4)，而KIR2DL1、KIR2DL3、KIR3DL1和KIR3DL2为NK细胞抑制性受体，介导免疫细胞的抑制作用，而其低表达则表示ITNK细胞具有对免疫抑制微环境的抵抗作用。全转录组测序分析结果显示，NK细胞标志性基因IL2RB、ID2、NFIL3等在ITNK细胞中上调表达(图22右)。
表3、ITNK细胞相对于NK细胞的相关基因表达
Figure PCTCN2020118104-appb-000005
表4、ITNK细胞相对于NK细胞的相关基因表达
Figure PCTCN2020118104-appb-000006
实施例5 本申请ITNK细胞的单细胞转录组测序分析
流式细胞术显示CD8+CD3+NKp46+ITNK和CD4+CD3+NKp30+ITNK在敲除BCL11B后5天即出现(如图23)。为了进一步解释T细胞向ITNK重编程过程中的细胞命运转变，我们使用基于微孔的单细胞转录组测序(scRNA-seq)技术，研究了敲除BCL11B后不同时间点CD3+脐带血样品中T和ITNK细胞在单细胞水平上的基因表达谱。
所有实验组共计检测分析约5000个细胞。scRNA-seq检测不同时间点组细胞样本，平均每个细胞检测2000-4000个基因，在所有细胞中共检测到20000个人类基因。在转录谱的t-SNE(t-distributed random neighbor-embedded)分析中，将细胞投射到二维上，为ITNK细胞重编程过程中的细胞命运转变提供了可视化的表现方式。无偏t-SEN分析结果显示，从敲除后第0天到第20天的细胞可聚类为11个亚群(如图24)。根据T细胞和NK细胞的标志性基因表达情况，确定ITNK细胞主要集中在亚群6(CD4+ITNK)、亚群1(CD8+ITNK)和亚群10(γδTCR+ITNK)(如图24)，而这些亚群与BCL11B缺失表达亚群完全重合，进一步说明ITNK细胞是由T细胞中敲除BCL11B重编程而来。KEGG富集分析表明，ITNK细胞特异性高表达NK标记基因及其相关基因(如图25)。由图24C、图25C可知，人ITNK细胞中NOTCH1、NOTCH2、ZMIZ1(NOTCH1辅助因子)、RBPJ(NOTCH下游转录因子)均上调，提示NOTCH信号通路在ITNK细胞重编程中的发挥作用。FOS、JUN、JUNB，三个AP-1转录因子的亚基在ITNK重编程早期低表达，而重编程晚期逐渐上调表达(如图25C)。可知NOTCH信号和AP-1信号在ITNK重编后上调。T细胞和ITNK细胞在t-SNE点图中紧密聚集，说明T细胞向NK细胞的转变几乎是同步的。通过对CD8+T(亚群5)、早期CD8+ITNK(亚群0)和晚期CD8+ITNK(亚群1)的NK标志性基因的轨迹分析，可以看出CD8+T细胞向CD8+ITNK的逐步过渡(如图26A)。同样，CD4+T细胞和ITNK细胞NK标志性基因的轨迹分析也显 示了T细胞向ITNK细胞的逐步过渡(图26B)。与(图23)ITNK细胞的免疫表型动态流式分析结果一致，T细胞敲除BCL11B后第五天开始重编程为ITNK细胞。我们发现转录因子基因(TBX2、ID2等)在T细胞向ITNK细胞重编程过程中表达明显上调，并通过免疫印迹进一步进行了验证(如图27)。
实施例6 本申请ITNK细胞可体外识别和杀伤MHCI阳性/阴性的肿瘤细胞
为了确定本申请的ITNK细胞上表达的NK细胞受体(NCR)和T细胞受体(TCR)是否有功能，我们分别利用抗NKp30、抗NKp46和抗CD3/CD28的单克隆抗体刺激ITNK细胞，发现：抗NKp30、抗NKp46抗体刺激后，ITNK细胞的干扰素(IFN)分泌增加，而对照组T细胞的不增加(如图28)；用抗CD3/CD28抗体刺激后，ITNK细胞的干扰素分泌增加，而对照组T细胞的不增加(如图28)。这表明，ITNK细胞中的NCR和TCR是功能性的。
与NK细胞类似，本申请的ITNK细胞可分泌各种细胞因子，包括GM-CSF、IFN和TNF(如图29)，且可识别杀伤MHC-I阴性的K562细胞(如图30A)。此外，ITNK细胞能有效杀死Hela和A549细胞(如图30B-C)，这两种细胞都是高表达NK激活型受体的配体且MHC I为阳性；如表2，由于ITNK细胞低表达介导免疫抑制作用的NK细胞KIR受体(KIR2DL1、KIR2DL3、KIR3DL1和KIR3DL2)，所以ITNK细胞对肿瘤的杀伤效果比NK细胞更好。但是，NALM-6不表达NCR配体，且高表达MHC-I分子，ITNK细胞与NK细胞对NALM-6无显著杀伤(如图30D)。然后，发明人利用K562细胞分别刺激ITNK、NK和T细胞，发现：ITNK和NK细胞中的磷酸化Fyn、PLC-γ2、Syk、Erk和m-TOR表达水平相似，但高于T细胞(如图31)。这些结果表明，ITNK细胞在NCR活化、细胞因子分泌、细胞毒性和信号通路等方面具有相似的NK细胞功能。
实施例7 本申请的ITNK细胞可体内抑制肿瘤生长
发明人还评估了本申请的ITNK细胞是否能够抑制异种移植瘤的生长。具体地，将标记有荧光素酶的K562细胞植入NSI小鼠，构建K562荷瘤小鼠模型，然后单次注入ITNK、NK或T细胞(图32A)。于特定时间点，通过活体成像设备检测K562在小鼠体内的存活状况。与注射T细胞(阴性对照)、PBS(空白对照)组相比，ITNK细胞和NK细胞处理的实验小鼠在输注28天后K562肿瘤负荷显著减少(图32B和32C)，存活时间更长(图32D)。此外，发明人还用Hela细胞移植入NSI小鼠，并分别用ITNK、NK或T细胞治疗Hela异种移植小鼠，结果显示，用ITNK细胞治疗的荷瘤小鼠的Hela肿瘤生长速度显著慢于NK、T细胞或PBS处理组(图32E)。由此可见，本申请的ITNK细胞是体内肿瘤细胞的有效杀手，可以阻止肿瘤进展。
实施例8 本申请的ITNK细胞体内安全性评估
为了验证ITNK细胞在体内的分布和维持能力。我们将ITNK细胞移植到缺失T、B、NK细胞的免疫缺陷小鼠NSI品系体内，并检测移植1、7、14、21和180后，外周血测量外周血与(PB)、脾脏(SP)、骨髓(BM)、肝脏(liver)和肺(lung)中ITNK细胞的百分比(图33A-B)。ITNK细胞比例在移植后21天达到最高点，其后逐渐减少，6个月后无法检测到(图33B)。由图33B可知，ITNK细胞体内维持能力相对于T细胞更好。我们始终没有观察到ITNK细胞攻击宿主或无限制扩增的情况。
为了评估PX458-gBCL11B可能引起的靶外突变，我们对PX458-gBCL11B电穿孔T细胞进行了高覆盖率的全基因组测序。与野生型T细胞相比，我们从两个独立的实验中发现，PX458-gBCL11B编辑的T细胞中，由核酸酶引起的脱靶突变非常少。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (19)

  1. 一种通过重编程人T细胞诱导而成的免疫杀伤淋巴细胞，其保留衍生其的人T细胞的标志物和功能并具有NK细胞的标志物和功能，其中，所述人T细胞的重编程涉及BCL11B基因的缺失。
  2. 根据权利要求1所述的细胞，其中，所述人T细胞为成熟人T细胞或含有成熟人T细胞的细胞群；
    优选地，所述成熟人T细胞或含有成熟人T细胞的细胞群来源于人体脐带血或外周血；
    优选地，所述成熟人T细胞或含有成熟人T细胞的细胞群来源于多能干细胞、胚胎干细胞或脐带血干细胞分化获得的成熟T细胞或细胞群。
  3. 根据权利要求1或2所述的细胞，其表达功能性的TCR、CD3和NKp30。
  4. 根据权利要求1-3任一项所述的细胞，其表达选自以下的NK细胞的标志物：CD11c、NKG2D和CD161；
    优选地，其低表达或不表达PD-1、CTLA-4或FOXP3免疫抑制检查点；
    优选地，其低表达或不表达NK相关标志物：CD127、CD16、KIRDL2、KIRDL3、NKG2A。
  5. 根据权利要求1-4任一项所述的新型免疫杀伤淋巴细胞，其中，相较于衍生其的T细胞，NOTCH表达上调。
  6. 根据权利要求1-5任一项所述的细胞，其中，相较于衍生其的T细胞，LEF1和TCF7转录因子表达下降，NOTCH、AP1、ID2、TBX21和NFIL3表达上升。
  7. 根据权利要求1-6任一项所述的细胞，其中，其TCR介导的信号转导增强；
    优选地，相较于衍生其的T细胞，其与TCR介导的信号转导相关的基因CSF2、FOS、MAPK12、MAP3K8、IFNγ、NFKBIA、MAPK11、IL-10和TEC的表达上调；
    优选地，相对于NK细胞，其T细胞识别和TCR信号转导增强；优选地，CD3、CD4、CD8、CD40LG的表达上调。
  8. 根据权利要求1-7任一项所述的细胞，其中，相较于衍生其的T细胞，其NK杀伤毒性相关信号转导增强；
    优选地，相较于衍生其的T细胞，其与NK杀伤毒性相关信号转导相关的基因PRF1、CSF2、ICAM1、CD244、PLCG2、IFNG、FCER1G、GZMB、NCR2、NCR1、KIR2DL4和SYK的表达上调。
  9. 根据权利要求1-8任一项所述的细胞，其包括CD8+NKp46+NKp44+NKp30+、CD4+NKp30+和γδTCR+NKp46+NKp44+NKp30+T细胞亚群。
  10. 根据权利要求1-9任一项所述的细胞，其中，所述人T细胞为成熟人T细胞，并且重编程所述人成熟T细胞包括：
    1)激活成熟人T细胞；
    2)对步骤1)所得激活的成熟人T细胞实施BCL11B基因敲除；
    3)将步骤2)所得细胞用T细胞培养基进行培养。
  11. 根据权利要求10所述的细胞，其中，步骤1)中，使用抗人CD3抗体、抗人CD28抗体和抗人CD2抗体进行激活；
    优选地，使用抗人CD3抗体、抗人CD28抗体和抗人CD2抗体的磁珠与人成熟T细胞以1:2混合孵育激活T细胞。
  12. 根据权利要求10或11所述的细胞，其中，步骤2)中，使用CRISPR/CAS9技术进行BCL11B基因敲除；
    优选地，所述基因敲除的靶点在BCL11B基因的第二外显子处；
    优选地，所述基因敲除的靶点在BCL11B基因的第三外显子处。
  13. 根据权利要求10-12任一项所述的细胞，其中，步骤3)中，所述T细胞培养基包含IL-2；优选地，不与OP9-DL1进行共培养。
  14. 一种制备权利要求1-13任一项所述的细胞的方法，其包括：
    1’)激活人T细胞；
    2’)对步骤1’)所得激活的人T细胞实施BCL11B基因敲除；
    3’)将步骤2’)所得细胞用T细胞培养基进行培养。
  15. 根据权利要求14所述的方法，其中，所述人T细胞为成熟人T细胞或含有成熟人T细胞的细胞群；
    优选地，所述成熟人T细胞或含有成熟人T细胞的细胞群来源于人体脐带血或外周血；
    优选地，所述成熟人T细胞或含有成熟人T细胞的细胞群来源于多能干细胞、胚胎干细胞或脐带血干细胞分化获得的成熟T细胞或细胞群。
  16. 根据权利要求14或15所述的方法，其中，步骤1’)中，使用抗人CD3抗体、抗人CD28抗体和抗人CD2抗体进行激活；
    优选地，使用抗人CD3抗体、抗人CD28抗体和抗人CD2抗体的磁珠与人成熟T细胞以1:2混合孵育激活T细胞。
  17. 根据权利要求14-16任一项所述的方法，其中，步骤2’)中，使用CRISPR/CAS9技术进行BCL11B基因敲除；
    优选地，在BCL11B基因的第二外显子处进行基因敲除；
    优选地，在BCL11B基因的第三外显子处进行基因敲除。
  18. 根据权利要求14-17任一项所述的方法，其中，步骤3’)中，所述T细胞培养基包含IL-2；优选地，不与OP9-DL1进行共培养。
  19. 权利要求1-13任一项所述的细胞在制备治疗选自以下的疾病的药物中的用途：肿瘤、艾滋病和感染性疾病；优选地，所述感染性疾病为病毒感染性疾病。
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