Engineered immune killer cell as well as preparation method and application thereof

Abstract

The invention discloses an engineered immune killer cell as well as a preparation method and application thereof. The engineered immune killer cell is an immune killer lymphocyte induced by reprogramming a human T cell, retains a marker and a function of the human T cell derived from the engineered immune killer lymphocyte, and has a marker and a function of an NK cell; cAR molecules for recognizing tumor and virus related antigens or cells for specifically recognizing tumor TCR molecules are transfected and expressed in the obtained immune killer lymphocytes. The engineered immune killer cell not only has a wider-spectrum tumor antigen recognition and killing function and a function of recognizing and removing microorganisms such as viruses and bacteria, but also has an efficient in-vitro amplification capability and an enhanced tumor specific recognition and killing function.

Description

[0001] This application is a divisional application of the invention patent application with application number 202080003111.1, application date September 28, 2020, and invention name “An engineered immune killer cell, its preparation method and application”. Technical Field

[0002] The present application relates to the field of biomedicine technology, and specifically to an engineered immune killer cell, a preparation method thereof, and an application thereof. Background Art

[0003] 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.

[0004] 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.

[0005] 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.

[0006] Cell reprogramming refers to the process in which differentiated cells are reversed under specific conditions and restored to a pluripotent 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 type transformation of immune cells achieved 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. At present, there is no application of cell reprogramming technology in human tumor immunotherapy.

[0007] Chimeric Antigen Receptor (CAR) molecules usually include an extracellular segment, a transmembrane region, and an intracellular segment: the extracellular segment is a single-chain variable region (ScFv) formed by connecting the variable regions of the heavy chain and light chain of an antibody through a peptide segment; the intracellular segment is a chimera of the intracellular segments of various signal transduction molecules, including CD3zeta, CD28, OX-40, 4-1BB, etc.; the transmembrane region comes from the transmembrane region of other molecules (such as CD8, CD4, CD28, and CD3zeta). The gene of the single-chain variable fragment is isolated from, for example, a hybridoma that produces a monoclonal antibody that recognizes a target antigen.

[0008] The structural design of CAR molecules has undergone multiple generations of research and development. The structure of the first-generation CAR molecule contains a single-chain variable fragment (scFv) that recognizes tumor cell surface antigens, a transmembrane domain, and an intracellular domain of the TCR complex CD3δ that activates T cells. Since the intracellular segment of the first-generation CAR only has the CD3δ signaling region and no co-stimulatory signal, the function of the first-generation CART cells is greatly defective, and it shows low levels in terms of proliferation, persistence, and effector function in patients. In order to enhance the ability of the first-generation CAR to activate T cells, the second-generation CAR has been developed. The second-generation CAR has added intracellular molecular signaling domains derived from co-stimulatory molecules (such as CD28, CD134 (OX-40), CD137 (4-1BB), etc.) into the cell. Clinical trials have shown that the second-generation CAR T cells show good proliferation, persistence, and effector function in patients. Most of the clinical trials of the second-generation CAR T cells are for the treatment of B-cell leukemia with anti-CD19 CAR T. Although CAR T cell clinical trials have achieved efficacy, there is still room for further improvement. The third-generation CAR was developed to further improve the efficacy of CAR T cell therapy. The intracellular segment of the third-generation CAR introduces the signal transduction regions of two co-stimulatory molecules. Usually, one co-stimulatory signal is the intracellular region of CD28, and the other is the intracellular signal transduction region of CD134, CD137 or ICOS. Different combinations of co-stimulatory signals may affect the function and efficacy of CAR T cells. Studies have shown that not all third-generation CARs are better than the second generation.

[0009] Immune cells expressing CAR molecules can play an important role in anti-tumor. For example, CAR T cells directly recognize tumor cell surface antigens independently of the expression of major histocompatibility antigen type I on tumor cells, and activate T cells at the same time, so that T cells expressing CAR can effectively kill tumor cells. In short, CAR T cells recognize specific molecules on the surface of tumor cells through antigen-antibody recognition mode, and then activate, proliferate and play a cell-killing function through their intracellular signal transduction. Summary of the invention

[0010] In view of the deficiencies of the prior art and actual needs, the present application provides an engineered immune killer cell, its preparation method and application. The engineered immune killer cell of the present application 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; compared with the mature human T cells from which they are derived, the engineered human immune cells of the present application have enhanced amplification capabilities and better anti-tumor effects; at the same time, because they express CAR molecules or tumor-specific TCR molecules of tumor-associated antigens, they also have enhanced tumor-specific recognition and killing functions.

[0011] In the first aspect, the present application proposes an engineered immune killer cell (hereinafter also referred to as CAR ITNK cell), which is prepared by the following: transfecting CAR molecules or TCR molecules targeting tumor- or virus-associated antigens into human T cells, and simultaneously or thereafter, performing reprogramming involving the deletion or inhibition of the BCL11B gene.

[0012] Preferably, the immune killer cells express CAR molecules or TCR molecules targeting tumor- or virus-associated antigens, retain the markers and functions of the human T cells from which they are derived, and have the markers and functions of NK cells.

[0013] 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 pluripotent stem cells, embryonic stem cells or umbilical cord blood stem cells.

[0014] Preferably, the reprogrammed immune killer lymphocytes express functional TCR, CD3 and NKp30.

[0015] Preferably, the reprogrammed immune killer lymphocytes express NK cell markers selected from the following: CD11c, NKG2D and CD161.

[0016] Preferably, the reprogrammed immune killer lymphocytes lowly express or do not express PD-1, CTLA-4 or FOXP3 immune inhibitory checkpoints.

[0017] Preferably, the reprogrammed immune killer lymphocytes lowly express or do not express NK-related markers: CD127, CD16, KIRDL2, KIRDL3, NKG2A.

[0018] Preferably, the reprogrammed immune killer lymphocytes have upregulated NOTCH expression compared to the T cells from which they are derived.

[0019] 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.

[0020] Preferably, the TCR-mediated signal transduction of the reprogrammed immune killer lymphocytes is enhanced;

[0021] 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.

[0022] 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.

[0023] Preferably, compared to the T cells from which they are derived, the reprogrammed immune killer lymphocytes have enhanced NK killer toxicity-related signal transduction;

[0024] 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.

[0025] 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.

[0026] In a preferred embodiment, the human T cells are mature human T cells, and reprogramming the human mature T cells comprises:

[0027] 1') Activate mature human T cells;

[0028] 2') knocking out the BCL11B gene in the activated mature human T cells obtained in step 1');

[0029] 3') The cells obtained in step 2') are cultured in T cell culture medium.

[0030] Wherein, in step 1'), anti-human CD3 antibody, anti-human CD28 antibody and anti-human CD2 antibody are used for activation;

[0031] 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.

[0032] Wherein, in step 2'), the BCL11B gene is knocked out using CRISPR / CAS9 technology;

[0033] Preferably, the target of gene knockout is at the second exon of the BCL11B gene.

[0034] Preferably, the target of gene knockout is at the third exon of the BCL11B gene.

[0035] Wherein, in step 3'), the T cell culture medium contains IL-2; preferably, it is not co-cultured with OP9-DL1.

[0036] Wherein, the CAR molecule comprises the following domains: a signal peptide, an extracellular antigen recognition domain, a transmembrane region, and an intracellular co-stimulatory domain; in a preferred specific embodiment, the CAR molecule comprises the following domains in sequence from N-terminus to C-terminus: a signal peptide, an extracellular antigen recognition domain, a transmembrane region, and an intracellular co-stimulatory domain.

[0037] Among them, the tumor-associated antigens are tumor surface antigens, cytokines secreted by tumors, surface antigens of cells related to the immunosuppression of the tumor microenvironment and their secreted cytokines, and tumor-associated microbial antigens, preferably CD19, GPC3, Mesothelin, PSCA, and MUC1.

[0038] In a second aspect, the present application provides a method for preparing the cell described in the first aspect, comprising:

[0039] 1”) activates human T cells;

[0040] 2”) Transfecting activated human T cells with CAR molecules or tumor-specific TCR molecule vectors expressing tumor-associated antigens, and simultaneously or subsequently, knocking out the BCL11B gene;

[0041] 3") The cells obtained in step 2") are cultured in T cell culture medium.

[0042] In the above method, preferably, in step 1″), 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 pluripotent stem cells, embryonic stem cells or umbilical cord blood stem cells.

[0043] 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.

[0044] Preferably, in step 2"), the CAR molecule comprises the following domains: a signal peptide, an extracellular antigen recognition domain, a transmembrane region, and an intracellular co-stimulatory domain; in a preferred specific embodiment, the CAR molecule comprises the following domains from N-terminus to C-terminus: a signal peptide, an extracellular antigen recognition domain, a transmembrane region, and an intracellular co-stimulatory domain; preferably, the antigen is a tumor-associated antigen and / or a microbial-associated antigen such as a virus or bacteria; further preferably, the tumor-associated antigen is a tumor surface antigen, a cytokine secreted by a tumor, a surface antigen of a cell related to the immunosuppression of the tumor microenvironment and its secreted cytokines, a tumor-associated microbial antigen, more preferably a tumor surface antigen, and even more preferably CD19, GPC3, Mesothelin, PSCA, MUC1.

[0045] 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 of the BCL11B gene; or, the gene is knocked out at the third exon of the BCL11B gene.

[0046] Preferably, in step 3″), the T cell culture medium contains IL-2; preferably, the T cell culture medium is not co-cultured with OP9-DL1.

[0047] In a third aspect, the present application 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.

[0048] Preferably, the medicament further comprises a pharmaceutically acceptable excipient.

[0049] The present application achieves a stronger tumor killing effect by reprogramming human T cells into immune killer lymphocytes, and transfecting and expressing CAR molecules or tumor-specific TCR molecules of tumor-associated antigens in the obtained immune killer lymphocytes. The reason is that the reprogrammed cells simultaneously express antigen recognition and killing receptors of NK cells and T cells, especially functional TCRs, and have both T cell and NK cell functions; because they simultaneously express antigen recognition and killing receptors of NK cells and T cells, they can recognize antigens that are sensitive to these receptors. 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 of microorganisms such as viruses and bacteria; at the same time, because they express CAR molecules or tumor-specific TCR molecules of tumor-associated antigens, they also have enhanced tumor-specific recognition and killing functions.

[0050] In addition, the engineered immune killer cells of the present application have efficient in vitro expansion capabilities. In adoptive cell transfer (ACT) therapy, both T cells and NK cells are used to treat cancer. The engineered immune killer 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 Peripheral blood mononuclear cells were prepared to obtain 200-1248×10 6 Reprogrammed immune killer lymphocytes to meet the patient's demand for transfused cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It is a schematic diagram of the PX458-gBCL11B vector constructed in Example 1 of the present application.

[0052] Figure 2 It is a diagram showing whether the BCL11B exon of T cells transduced with PX458-gBCL11B is knocked out by gene sequencing detection and verification. The control group is T cells transduced with PX458 empty vector (Mock).

[0053] 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).

[0054] Figure 4It is a scatter plot of flow cytometry results, which shows that relative to Mock T cells and NK cells, the ITNK cells of the present application (i.e., T cells transduced by PX458-gBCL11B, indicated by PAX458 in the figure) 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 MockT with PX458-gBCL11B, and has 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.

[0055] FIG5 is a scatter plot of flow cytometry results (A), which shows that umbilical cord blood-derived ITNK cells (i.e., PX458-gBCL11B-transduced T cells, indicated by PAX458 in the figure) highly express T cell marker CD3 and NK cell markers NKp46, CD56, NKp30 and NKp44 relative to Mock T cells, as well as the corresponding cell percentage statistics (B).

[0056] Figure 6 is a scatter plot of flow cytometry results (A), which shows that peripheral blood-derived ITNK cells (i.e., T cells transduced with PX458-gBCL11B, indicated by PAX458 in the figure) highly express T cell marker CD3 and NK cell markers NKp46, CD56, NKp30 and NKp44 relative to Mock T cells, as well as the corresponding cell percentage statistics (B).

[0057] 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.

[0058] Figure 8 The 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.

[0059] 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.

[0060] 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 source.

[0061] 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.

[0062] 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-KOT) and (Activated BCL11B-KO T) derived from or not.

[0063] 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.

[0064] Fig.14 This is the immunophenotypic analysis of ITNK cells of the present application - 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.

[0065] 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).

[0066] 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).

[0067] Fig.17 Shown are mass cytometry heat map analysis results, which display the marker expression of each immune cell subset.

[0068] 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.

[0069] Fig.19 It is principal component analysis of RNA sequencing-cell subpopulation gene expression data.

[0070] 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).

[0071] 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).

[0072] Fig. 22 It 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).

[0073] 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.

[0074] Fig.24 (A) is a t-SNE dot plot of the Sc-RNAseq 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.

[0075] Figure 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.

[0076] Figure 26 (A) shows the gradual transition of CD8+T cells to CD8+ITNK cells by unsupervised trajectory analysis of single cells from subpopulation 5 (CD8+T), subpopulation 0 (early CD8+ITNK) and subpopulation 1 (late CD8+ITNK); (B) shows the gradual transition of CD4+T cells to CD4+ITNK cells by unsupervised trajectory analysis of single cells from subpopulation 4 (CD4+T), subpopulation 5 (early CD4+ITNK) and subpopulation 7 (late CD4+ITNK).

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] Fig.34 The PI labeling and GFP expression of transduced cells were detected by flow cytometry to determine the survival and transduction efficiency of transduced cells (such as Fig.34 A), and the reprogramming of ITNK cells (B).

[0085] Fig.35 The target tumor cell lysis rates when each effector cell group (T, ITNK, 19t2, 19t2 / ITNK) was co-cultured with target cells (A) K562-CD19 cells and (B) NALM6 cells at different effector cell to target cell ratios (E:T ratio) are shown as mean ± SD. **P ≤ 0.01; unpaired t test.

[0086] FIG36 (A) is a schematic diagram showing an experiment in which NSI mice were intravenously injected with CD19+K562-GL cells (5×105). According to the figure, T cells, ITNK cells, and NK cells (2.5×105) were injected into the mice on day 2. 6), and bioluminescence imaging was performed on days 2, 7, 14, 21, and 28, respectively; 5 mice per group. (BC) Total flux statistics (B) and live imaging (C) of in vivo bioluminescence imaging quantitative analysis of luciferase activity, 5 mice per group. Results are shown as mean ± SD. *P ≤ 0.05, unpaired Student's t-test, for the indicated time points. DETAILED DESCRIPTION

[0087] In order to further explain the technical means and effects adopted by the present application, the technical solution of the present application is further explained below in conjunction with the drawings and through specific implementation methods, but the present application is not limited to the scope of the embodiments.

[0088] Unless otherwise specified, the relative arrangement of the parts and steps, numerical expressions and numerical values ​​described in these embodiments do not constitute a limitation of the present application. 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.

[0089] Example 1 Preparation of reprogrammed natural killer (ITNK) cells of the present application

[0090] Construction of gene knockout plasmid vector

[0091] 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 knockout conditions of BCL11B knockout target sites such as base deletion and misalignment by gene sequencing (see Figure 2 ), and the knockout efficiency was statistically calculated as shown in Table 1.

[0092] Table 1. List of BCL11B target sequences and their corresponding gRNAs

[0093]

[0094]

[0095]

[0096] 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 application. 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.

[0097] T cell sorting and activation

[0098] T cell isolation and activation were performed using the following methods:

[0099] (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;

[0100] (2) suspending the precipitated granular blood cells with 0.9% NaCl and isolating peripheral blood mononuclear cells (PBMCs) by Ficoll density gradient centrifugation;

[0101] (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)

[0102] The above (1)-(3) are the steps for isolating human mature T cells from peripheral blood and umbilical cord blood. It should be noted that other T cell sources can also be used, such as pluripotent stem cells, hematopoietic stem cells, etc. T cells from all sources were activated using a T cell activation kit (Miltenyi Biotec) using magnetic beads coated with anti-human CD3, anti-human CD28 antibodies, and anti-human CD2 and incubated with T cells in a ratio of 1:2 (cell density: 2.5×10 6cells / 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.

[0103] Induced reprogramming

[0104] (1) The CRISP / CAS9 knockout vector PX458-gBCL11B was transduced into the activated T cells by electroporation (T-023, LONZA Amata Nucleofector, Lonza);

[0105] (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));

[0106] (3) Fresh culture medium was then replaced every 3 days to maintain the cell density at 0.5-1×10 6 cells / ml until day 14 after electroporation.

[0107] (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);

[0108] (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 .

[0109] Phenotypic characterization of reprogrammed cells

[0110] 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 application 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, and their phenotypic differences are also summarized in the following Table 2.

[0111] Table 2 Phenotypic differences among ITNK cells, T cells, and NK cells

[0112]

[0113] 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.

[0114] 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).

[0115] 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 ).

[0116] Example 2 Identification of the Source of ITNK Cells in the Present Application

[0117] TCRαβ sequencing: RNA was extracted and CDR3 region targeted amplification was performed on T cells from the same donor and ITNK cells obtained in Example 1 using the human TCRαβ analysis kit to obtain TCR RNA. TCR RNA was sequenced using the HiSeq4000 platform to obtain a TCR library. Cluster combination analysis was performed using MiXCR (ref). TCRαβ clone types were exported using the MiXCR clone export command with the "one chain" parameter. TCR clone diversity of T cells and ITNK cells from the same donor was compared using TCR sequencing, and it was found that TCR clone diversity 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.

[0118] Example 3 Single cell immunophenotyping of ITNK cells of the present application

[0119] 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.

[0120] 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.

[0121] 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.

[0122] According to the results of mass spectrometry flow cytometry marker expression heterogeneity analysis, the ITNK cells of the present application include CD3-negative cell subsets of NO.33, CD4+ cell subsets of NO.5-10, CD8+ cell subsets of NO.20-22 and 26-28, and TCRγδ+ cell subsets of 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 application are different from conventional NK cells because the ITNK cells of the present application do not express CD127, CD16, NKG2A and KIR2DL2 (e.g. Fig.14 B). Moreover, the ITNK cells of the present application lowly express 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 suggests that the ITNK cells of the present application have strong immune effects and are not easily inhibited by immunosuppressive microenvironments such as tumors.

[0123] 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.16As 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.

[0124] Example 4 RNA-Seq transcriptional profiling analysis of ITNK cells in this application

[0125] 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 ).

[0126] 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 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).

[0127] Table 3. Gene expression of ITNK cells relative to NK cells

[0128]

[0129] Table 4. Gene expression of ITNK cells relative to NK cells

[0130]

[0131] Example 5 Single-cell transcriptome sequencing analysis of ITNK cells in this application

[0132] 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.

[0133] 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 (as shown in Figure 25). Fig.24 C. Fig.25C It can be seen that NOTCH1, NOTCH2, ZMIZ1 (NOTCH1 auxiliary factor), and RBPJ (NOTCH downstream transcription factor) were 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, were lowly expressed in the early stage of ITNK reprogramming, and gradually upregulated in the late stage of reprogramming (e.g. Fig.25C ). It can be seen that NOTCH signal and AP-1 signal 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. By analyzing the trajectory of NK marker genes of CD8+T (subgroup 5), early CD8+ITNK (subgroup 0) and late CD8+ITNK (subgroup 1), it can be seen that CD8+T cells gradually transition to CD8+ITNK (e.g. Fig.26A ). 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.26B ).and( 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 ).

[0134] Example 6 ITNK cells of the present application can recognize and kill MHCI positive / negative tumor cells in vitro

[0135] In order to determine whether the NK cell receptor (NCR) and T cell receptor (TCR) expressed on the ITNK cells of the present application 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 the control group T cells 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.

[0136] Similar to NK cells, the ITNK cells of the present application 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 of which highly express NK activating receptor ligands and are MHCI 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.

[0137] Example 7 ITNK cells of the present application can inhibit tumor growth in vivo

[0138] The inventors also evaluated 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 ( Fig.32 A). 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 application are effective killers of tumor cells in vivo and can prevent tumor progression.

[0139] Example 8 In vivo safety assessment of ITNK cells of the present application

[0140] 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.

[0141] 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.

[0142] Example 9 Construction of CAR ITNK cells of the present application

[0143] Although the above-mentioned ITNK cells have TCR (T cell receptor) and NCR (NK cell receptor) functions, they cannot recognize specific tumor antigens. To this end, the inventors transduced the PB-CAR molecular vector (CAR molecular structure: the extracellular domain is the extracellular segment of the receptor of antigens such as CD19, GPC3, MUC1 or Mesothelin or the corresponding antibody scFv sequence, the transmembrane region is one or two of the transmembrane regions of receptors such as CD28, NKG2D, NKp44 or NKp46, and the intracellular co-stimulatory domain is CD28, TLR2, 2B4, DAP10 or DAP12 and other intracellular co-stimulatory domains, as well as CD3δ) and the BCL11B knockout vector PX458-gBCL11B into human T cells one after another or simultaneously, and obtained ITNK cells expressing anti-CD19 chimeric antigen receptor (CAR) molecules. The PI labeling and GFP expression of the transduced cells were detected by flow cytometry to determine the survival and transduction efficiency of the transduced cells (such as Fig.34 A) and the reprogramming of ITNK cells (as shown in Fig.34 Specifically, CAR19-ITNK cells were detected on days 10 to 14 (as shown in FIG. Fig.34 ), which showed the percentage of GFP+CD4+NKp30+, CD4-CD8-γδITNK subsets and GFP+CD8+NKp46+ subsets.

[0144] Example 10 CAR ITNK cells of the present application kill CD19+CML and BALL in vitro

[0145] In order to evaluate the anti-tumor effect of CAR19 (FMC63 scFv segment-CD28 transmembrane region-CD28 and TLR2 intracellular domain-CD3δ signaling domain)-ITNKs, human chronic myeloid leukemia cell line K562 cells (K562-CD19) expressing human CD19 and luciferase and acute B lymphoblastic leukemia NALM-6 cells expressing luciferase were constructed. CAR19-ITNK cells, CAR19-T cells, NK cells, and T cells were mixed with the two leukemia cell lines at different E:T effector cell and target cell ratios for 24 hours, and luciferase substrate was added. The killing of tumor cells was detected by microplate reader.

[0146] The above in vitro killing experiment on K562-CD19 cells showed that the CAR19-ITNK cells of the present application are more effective in recognizing and killing K562-CD19 cells than CAR19 T cells and ITNK cells (such as Fig.35 However, when eliminating NALM-6 cells, the killing ability of CAR19-ITNK cells and CAR19 T cells was similar (as shown in Figure 2A). Fig.35 B), which may be due to HLA-I positivity or the lack of NCR ligands in NALM-6. ITNK cells never cleaved NALM-6 (as shown in Fig.35 These results suggest that CAR and NCR signaling are compatible and can work synergistically.

[0147] Example 11 In vivo killing of CD19+CML by CAR19 ITNK cells of the present application

[0148] In order to detect the in vivo anti-tumor activity of the CAR19-ITNK cells of the present application, the applicant injected the K562-CD19 cells constructed in Example 10 into NSI mice via intravenous injection (the injection dose for each mouse was 5×10 5 cells), followed by injection of CAR19-ITNK cells, ITNK cells, CAR19-T or T cells (the injection dose per mouse was 2.5×10 5 The experimental process is as follows: Fig.36A As shown, on days 2, 7, 14, 21, and 28 after K562-CD19 transplantation, the leukemia mice were treated with luciferase substrate (which can react with luciferase-expressing cells, and cells expressing luciferase can be detected by a live imaging device), and the survival or killing of luciferase-labeled K562-CD19 cells in the leukemia mice was detected by live imaging technology. The live imaging device can detect the distribution of luciferase-labeled cells and determine the number of labeled cells by fluorescence intensity.

[0149] The experimental results showed that the tumor burden of mice in the CAR19-ITNK group was lighter than that in other groups. Fig.36B -C; The experimental results also showed that CAR19-ITNK cells inhibited the growth of NALM-6 more significantly than other cells such as CAR19-T cells and ITNK cells. Fig.36B It is worth noting that although CAR19-ITNK and CAR19 T cells showed similar killing ability against NALM-6 in vitro (as shown in Fig.35 B), but CAR19-ITNK cells outperformed CAR19 T cells in clearing NALM-6 in vivo. Taken together, these results suggest that CAR enhances the cytotoxicity of ITNK cells against tumors, especially in vivo.

[0150] Example 12 Anti-GPC3 CAR-ITNK cells of the present application kill liver cancer cells

[0151] This application constructs CAR-ITNK cells that recognize phosphatidylinositol GPC3. The molecular structure of CAR is the extracellular segment of anti-GPC3 scFv, NKG2D transmembrane region, 2B4 intracellular co-stimulatory domain and CD3δ. Four experimental groups were set up in a 96-well plate, including CAR-ITNK cells, CAR-T, ITNK cells, and T cells, with 3 duplicate wells in each group. 10,000 tumor cells (GPC3-positive tumor cell lines Huh7-GL and HepG2-GL, GL is a luciferase gene marker) were added to each well as target cells, and effector cells were added to the well plate according to E:T of 4:1, 2:1, 1:1, 1:2, and 1:4, respectively. After the effector cells and tumor cells were co-incubated for 24 hours, luciferase substrate was added, and the killing ratio of tumor cells was detected by quantitative spectrophotometer. The experimental results showed that the tumor killing effect of CAR-ITNK cells was better than that of ITNK, CAR T and T cells (data not shown).

[0152] Example 13 Cell expansion and killing of solid tumor cells by anti-TGFβ CAR-ITNK cells of the present application

[0153] This application constructs CAR-ITNK cells that recognize cytokine TGFβ, and the CAR molecular structure is an anti-TGFβ scFv extracellular segment, CD28 intracellular co-stimulatory domain, TLR2 intracellular co-stimulatory domain and CD3δ. Four experimental groups were set up in a 96-well plate, including CAR-ITNK cells, CAR-ITNK+TGFβ, ITNK cells, ITNK cells+TGFβ, 5 duplicate wells in each group, 105 cells per well, and 6, 24, 48, 72, and 96 hours after adding TGFβ (3ng / ml) cytokines, by cell counting, the absolute value of cells in each well was recorded, and the secretion of related immune effector cytokines in different experimental groups was detected by ELISA detection method. Statistical results and comparative data analysis will show that TGFβ has an inhibitory effect on the proliferation of ITNK cells and the secretion of immune effector cytokines, while the anti-TGFβ CAR-ITNK cells have relatively enhanced cell proliferation and immune effector cytokine secretion under the condition of TGFβ.

[0154] To evaluate the killing effect of anti-TGFβ CAR-ITNK cells on tumor cells, four experimental groups were set up in a 24-well plate, including CAR-ITNK cells, CAR-ITNK+TGFβ, ITNK cells, and ITNK cells+TGFβ, with 3 replicates in each group and 2X105 effector cells in each well. 105 tumor cells (tumor cell line HepG2, luciferase gene marker) were added as target cells. Luciferase substrate was added 24 hours after the addition of TGFβ cytokine, and the killing ratio of tumor cells was detected by fluorescence illuminometer. The experimental results showed that TGFβ inhibited the tumor killing effect of ITNK cells, while the presence of TGFβ relatively promoted the killing effect of anti-TGFβ CAR ITNK cells on tumor cells (data not shown).

[0155] Example 14 Killing of solid tumor cells by the anti-Mesothelin CAR-ITNK cells of the present application

[0156] This application constructs CAR-ITNK cells that recognize mesothelin, and the CAR molecular structure is an anti-mesothelin scFv extracellular segment, a CD28 transmembrane region, a DAP10 or DAP12 sequence, and CD3δ. Four experimental groups were set up in a 96-well plate, including CAR-ITNK cells, CAR-T, ITNK cells, and T cells, with 3 duplicate wells in each group. 10,000 tumor cells (Mesothelin-positive tumor cell lines BGC-823-GL and MKN-28-GL, GL is a luciferase gene marker) were added to each well as target cells, and effector cells were added to the well plate according to E:T of 4:1, 2:1, 1:1, 1:2, and 1:4, respectively. After the effector cells and tumor cells were co-incubated for 24 hours, luciferase substrate was added, and the killing ratio of tumor cells was detected by a quantitative spectrophotometer. These experimental results are similar to those of the above examples, and all show that the tumor killing effect of CAR-ITNK cells is better than that of ITNK, CAR T and T cells (data not shown).

[0157] The various tumor and virus recognition and killing activation pathways in the CAR ITNK cells of the present application do not interfere with each other and have mutual synergy. The CAR ITNK of the present application can not only activate and recognize tumor or virus-related antigens through CAR molecules, but also recognize tumor or virus-related antigens through NK cell receptors and TCR receptor pathways in ITNK cells. It not only has a specific and efficient killing effect on specific tumors and viruses, thereby quickly controlling tumor progression and viral deterioration, but also has a broad spectrum of anti-tumor and viral effects, thereby preventing the escape and recurrence of tumors and viruses. The CAR-ITNK technology of the present application solves the problems of tumor antigen escape, treatment recurrence and low efficiency in CAR T and CAR NK treatment in the prior art.

[0158] The applicant declares that the present application uses the above-mentioned embodiments to illustrate the detailed methods of the present application, but the present application is not limited to the above-mentioned detailed methods, that is, it does not mean that the present application must rely on the above-mentioned detailed methods to be implemented. The technicians in the relevant technical field should understand that any improvement to the present application, the equivalent replacement of the raw materials of the product of the present application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present application.

Claims

1. An engineered immune killer cell prepared by: transfecting a CAR molecule targeting a tumor or virus-associated antigen in a human T cell, and simultaneously or subsequently, performing reprogramming involving the deletion or inhibition of the BCL11B gene; The immune killer cells express CAR molecules targeting tumor or virus-associated antigens, retain the markers and functions of the human T cells from which they are derived, and have the markers and functions of NK cells; It expresses functional TCR, CD3, and NKp30; It expresses a marker of NK cells selected from the group consisting of: CD11c, NKG2D, and CD161; The human T cells are mature human T cells, and reprogramming the human mature T cells comprises: 1') Activate mature human T cells; 2') knocking out the BCL11B gene in the activated mature human T cells obtained in step 1'); 3') The cells obtained in step 2') are cultured in T cell culture medium.

2. The cell according to claim 1, wherein The mature human T cells are derived from human umbilical cord blood or peripheral blood; Preferably, the mature human T cells are derived from mature T cells or cell populations differentiated from pluripotent stem cells, embryonic stem cells or umbilical cord blood stem cells; Preferably, it has low or no expression of PD-1, CTLA-4, or FOXP3 immune inhibitory checkpoints; Preferably, it lowly expresses or does not express NK-related markers: CD127, CD16, KIRDL2, KIRDL3, NKG2A. Preferably, NOTCH expression is upregulated compared to the T cell from which it is derived; Preferably, 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.

3. The cell according to any one of claims 1 to 2, wherein Its TCR-mediated signal transduction is enhanced; Preferably, compared to the T cells from which it is derived, the expression of genes CSF2, FOS, MAPK12, MAP3K8, IFNγ, NFKBIA, MAPK11, IL-10 and TEC related to TCR-mediated signal transduction is upregulated; Preferably, relative to NK cells, T cell recognition and TCR signal transduction are enhanced; preferably, the expression of CD3, CD4, CD8, and CD40LG is upregulated.

4. The cell according to any one of claims 1 to 3, wherein Compared with the T cells from which they are derived, their NK cell cytotoxicity-related signal transduction is enhanced; Preferably, compared with the T cells from which it is 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 is upregulated.

5. The cell according to any one of claims 1-4, comprising CD8+NKp46+NKp44+NKp30+, CD4+NKp30+ and γδTCR+NKp46+NKp44+NKp30+ T cell subsets.

6. The cell according to any one of claims 1 to 5, wherein In step 1'), anti-human CD3 antibody, anti-human CD28 antibody and anti-human CD2 antibody are used for activation; 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; Preferably, in step 2'), CRISPR / CAS9 technology is used to knock out the BCL11B gene; Preferably, the target of gene knockout is at the second exon of the BCL11B gene; Preferably, the target of gene knockout is at the third exon of the BCL11B gene; Preferably, in step 3'), the T cell culture medium contains IL-2; preferably, the T cell is not co-cultured with OP9-DL1.

7. The cell according to any one of claims 1 to 6, which is a CAR-ITNK cell that recognizes phosphatidylinositol GPC3, wherein: The CAR molecular structure is an anti-GPC3 scFv extracellular segment, an NKG2D transmembrane region, a 2B4 intracellular co-stimulatory domain and CD3ζ.

8. The cell according to any one of claims 1 to 6, which is a CAR-ITNK cell that recognizes the cytokine TGFβ, wherein The CAR molecular structure is an anti-TGFβ scFv extracellular segment, a CD28 intracellular co-stimulatory domain, a TLR2 intracellular co-stimulatory domain and CD3ζ.

9. The cell according to any one of claims 1 to 6, which is a CAR-ITNK cell that recognizes mesothelin, wherein: The CAR molecular structure is an anti-Mesothelin scFv extracellular segment, a CD28 transmembrane region, a DAP10 or DAP12 sequence and CD3ζ.

10. Use of the cell according to any one of claims 1 to 9 in the preparation of a medicament for treating a disease selected from the group consisting of tumors, AIDS and infectious diseases; Preferably, the tumor is a solid tumor; More preferably, the tumor is liver cancer and / or gastric cancer.

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