Patent Application
20230055186
The present application relates to the technical field of biomedicine and, in particular, to an engineered immune killer cell, a preparation method therefor, and a use thereof.
At present, immunotherapy has become the most concerned and promising “new” idea in the field of cancer treatment. Among the top 10 scientific breakthroughs of the year ranked by Science magazine in 2013, tumor immunotherapy tops the list. Chimeric antigen receptor (CAR) T cells of Novartis and Kite have been approved by the U.S. FDA, and tumor immune cell therapy has made milestone progress in the treatment field of hematological malignancies. However, tumor immune cell therapy still has technical bottlenecks in the clinical treatment. For example, genetically modified immune cells have a single target, causing tumor immune escape and tumor recurrence; a solid tumor lacks a specific marker with high efficiency and low side effects, and current genetically modified immune cell therapy has no effective and safe clinical effect on the solid tumor.
T cells can be divided into different subgroups according to their surface markers and functions. For example, T cells can be divided into γδT cells and αβT cells according to the types of TCRs. αβT cells, which account for more than 95% of T cells, are the main cell population having T cell differentiation markers in vivo and performing T cell functions and represent the diversity of T cells. γδT cells are a group of highly heterogeneous cells. A T-cell receptor on the surface of γδT cells is composed of a γ chain and a 6 chain. γδT cells have many subtypes, variable phenotypes, and rich functions. The subtypes of γδT cells have different biological characteristics, and γδT cells play an important role in the occurrence and development of tumors, infections, and autoimmune diseases in the body and are considered to be a bridge of the body which links innate immunity to adaptive immunity.
Like T cells, natural killer (NK) cells are an indispensable part of a human immune system. NK cells are considered to be lymphoid cells which account for about 10% to 15% of peripheral blood lymphocytes and play a key role in an innate immune response. Different from T cells, NK cells recognize their targets in an MHC-unrestricted manner. NK cells have antiviral, anti-GvH, and anti-cancer effects. Specifically, NK cells directly kill malignant tumors including sarcomas, myelomas, cancers, lymphomas, and leukemia or eliminate abnormal cells by inducing the activity of dendritic cells (DCs) or the adaptive immune activation of tumor-specific cytotoxic T lymphocytes (CTLs), where the abnormal cells are tumor cells or cells that are developing into tumor cells. Although NK cells have the potential to be used as a therapeutic agent against cancer or infectious diseases, most NK cells in a normal human body are present in a dormant state, and NK cells in cancer patients lose their functions due to the immune escape mechanism of cancer cells. To use natural killer cells as the therapeutic agent in practice, 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 in which differentiated cells are reversed under particular conditions and then return to a totipotent 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 transformation of immune cells through cell reprogramming. For example, Dr. Ding Sheng from Gladstone Institutes in the U.S. has reported that pro-inflammatory effector T cells are reprogrammed into anti-inflammatory regulatory T cells by a particular reprogramming method. Such reprogramming is of great significance to the treatment of autoimmune diseases. Specifically, for autoimmune diseases, over-stimulated effector T cells cause damages to the body, and when these cells are transformed into regulatory T cells, the overactivity of the immune system can be reduced and thus the immune system restores its balance, thereby fundamentally treating the diseases. At present, cell reprogramming is still to be applied to human tumor immunotherapy.
A chimeric antigen receptor (CAR) molecule typically includes an extracellular fragment, a transmembrane region, and an intracellular fragment. The extracellular fragment is a single-chain variable fragment (scFv) formed by linking heavy and light chain variable regions of an antibody via a peptide fragment. The intracellular fragment is a chimera of various signal transduction molecules including CD3zeta, CD28, OX-40, 4-1BB, and the like. The transmembrane region is derived from the transmembrane region of another molecule (such as CD8, CD4, CD28, and CD3zeta). The genes of the single-chain variable fragment are isolated from, for example, hybridomas that produce monoclonal antibodies that recognize target antigens.
The structural design of the CAR molecule has undergone many generations of research and development. The structure of the first-generation CAR molecule includes a single-chain variable fragment (scFv) that recognizes a tumor cell surface antigen, a transmembrane domain, and an intracellular domain that activates the TCR complex CD3 of T cells. Since the intracellular fragment of the first-generation CAR includes only a CD3 signal transduction region and no costimulatory signal, the first-generation CAR-T cells have severely deficient functions and show low levels in terms of the proliferation, persistence and effector functions in patients. For the purpose of enhancing the ability of the first-generation CAR to activate T cells, the second-generation CAR has been developed. The second-generation CAR adds an intracellular signal transduction domain derived from costimulatory molecules (such as CD28, CD134(OX-40), and CD137(4-1BB)). Clinical trials show that the second-generation CAR-T cells have relatively good proliferation, persistence, and effector functions in patients. The clinical trials of the second-generation CAR-T cells are mostly the treatment of B-cell leukemia with anti-CD19 CAR-T cells. Although CAR-T cells have achieved efficacy in clinical trials, the CAR-T cells are to be further improved. The third-generation CAR is developed to further improve the efficacy of CAR-T cell therapy. The signal transduction regions of two costimulatory molecules are introduced into the intracellular fragment of the third-generation CAR. Typically, one costimulatory signal is the intracellular region of CD28, and the other costimulatory signal is the intracellular signal transduction region of CD134, CD137, ICOS or the like. Different combinations of costimulatory signals may affect the function and efficacy of CAR-T cells. Studies show that not all third-generation CARs are better than second-generation CARs.
Immune cells expressing CAR molecules can have an important anti-tumor effect. For example, CAR-T cells are independent of the expression of major histocompatibility antigens of type I on tumor cells, directly recognize tumor cell surface antigens, and simultaneously activate T cells so that the T cells expressing the CAR can effectively kill tumor cells. In short, CAR-T cells recognize specific molecules on the surface of tumor cells through an antigen-antibody recognition pattern and then are activated, proliferate, and kill cells through intracellular signaling.
In view of the defects in the existing art and practical requirements, the present application provides an engineered immune killer cell, a preparation method therefor, and a use thereof. The engineered immune killer cell of the present application has some markers and functions of both T cells and NK cells and simultaneously expresses antigen recognition and killing receptors of NK cells and T cells so that the engineered immune killer cell has more extensive tumor antigen recognition and killing functions than NK cells and T cells. Compared with a mature human T cell from which the engineered human immune cell is derived, the engineered human immune cell of the present application has an enhanced proliferation ability and a better anti-tumor effect. At the same time, the engineered human immune cell also has enhanced tumor-specific recognition and killing functions due to its CAR molecule expressing a tumor-associated antigen or its tumor-specific TCR molecule.
In a first aspect, the present application provides an engineered immune killer cell (hereinafter referred to as a CAR ITNK cell) prepared by transfecting a human T cell with a CAR molecule or a TCR molecule targeting a tumor-associated antigen or a virus-associated antigen along with or followed by reprogramming involving deletion or inhibition of a BCL11B gene.
Preferably, the immune killer cell expresses the CAR molecule or the TCR molecule targeting the tumor-associated antigen or the virus-associated antigen, retains a marker and a function of the human T cell from which the immune killer cell is derived, and has a marker and a function of NK cells.
Preferably, the human T cell is a mature human T cell or a cell population containing mature human T cells. Further preferably, the mature human T cell or the cell population containing mature human T cells is derived from cord blood or peripheral blood of a human body. Further preferably, the mature human T cell or the cell population containing mature human T cells is derived from a mature T cell or a cell population obtained through differentiation of pluripotent stem cells, embryonic stem cells, or cord blood stem cells.
Preferably, the reprogrammed immune killer lymphocyte expresses functional TCR, CD3, and NKp30.
Preferably, the reprogrammed immune killer lymphocyte expresses the following marker of NK cells: CD11c, NKG2D, and CD161.
Preferably, the reprogrammed immune killer lymphocyte performs low expression or no expression of an immunosuppression checkpoint PD-1, CTLA-4, or FOXP3.
Preferably, the reprogrammed immune killer lymphocyte performs low expression or no expression of an NK-associated marker CD127, CD16, KIRDL2, KIRDL3, NKG2A.
Preferably, the reprogrammed immune killer lymphocyte upregulates expression of NOTCH compared with the T cell from which the reprogrammed immune killer lymphocyte is derived.
Preferably, the reprogrammed immune killer lymphocyte downregulates expression of transcription factors LEF1 and TCF7 and upregulates expression of NOTCH, AP1, mTOR, ID2, TBX21, and NFIL3 compared with the T cell from which the reprogrammed immune killer lymphocyte is derived.
Preferably, TCR-mediated signal transduction of the reprogrammed immune killer lymphocyte is enhanced.
Preferably, compared with the T cell from which the reprogrammed immune killer lymphocyte is derived, the reprogrammed immune killer lymphocyte upregulates expression of genes CSF2, FOS, MAPK12, MAP3K8, IFNγ, NFKBIA, MAPK11, IL-10, and TEC which are associated with the TCR-mediated signal transduction.
Preferably, compared with NK cells, the reprogrammed immune killer lymphocyte has enhanced T cell recognition and TCR signal transduction; preferably, the reprogrammed immune killer lymphocyte upregulates expression of CD3, CD4, CD8, and CD40LG.
Preferably, compared with the T cell from which the reprogrammed immune killer lymphocyte is derived, the reprogrammed immune killer lymphocyte has enhanced NK killing toxicity-associated signal transduction.
Preferably, compared with the T cell from which the reprogrammed immune killer lymphocyte is derived, the reprogrammed immune killer lymphocyte upregulates expression of genes PRF1, CSF2, ICAM1, CD244, PLCG2, IFNG, FCER1G, GZMB, NCR2, NCR1, KIR2DL4, and SYK which are associated with the NK killing toxicity-associated signal transduction.
In a preferred specific embodiment, the reprogrammed immune killer lymphocyte includes CD8+NKp46hiNKp44+NKp30+, CD4+NKp30+, and γδTCR+NKp46hiNKp44+NKp30+ T cell subgroups.
In a preferred specific embodiment, the human T cell is a mature human T cell, and reprogramming the mature human T cell includes:
(1′) activating the mature human T cell;
(2′) performing BCL11B gene knockout on the activated mature human T cell obtained in step (1′); and
(3′) culturing the cell obtained in step (2′) in a T cell culture medium.
In step (1′), the mature human T cell is activated using an anti-human CD3 antibody, an anti-human CD28 antibody, and an anti-human CD2 antibody.
Preferably, the T cell is activated through incubation of magnetic beads of the anti-human CD3 antibody, the anti-human CD28 antibody, and the anti-human CD2 antibody mixed with the mature human T cell at a ratio of 1:2.
In step (2′), the BCL11B gene knockout is performed using CRISPR/CAS9 technology.
Preferably, a target of the gene knockout is at a second exon of the BCL11B gene.
Preferably, the target of the gene knockout is at a third exon of the BCL11B gene.
In step (3′), the T cell culture medium includes IL-2; preferably, the cell obtained in step (2′) is not co-cultured with OP9-DL1.
The CAR molecule includes the following domains: a signal peptide, an extracellular antigen recognition domain, a transmembrane region, and an intracellular costimulatory domain. In a preferred specific embodiment, the CAR molecule includes the signal peptide, the extracellular antigen recognition domain, the transmembrane region, and the intracellular costimulatory domain in sequence from an N-terminal to a C-terminal.
The tumor-associated antigen is a tumor surface antigen, a cytokine secreted by a tumor, a surface antigen of a cell associated with immunosuppression of a tumor microenvironment and a cytokine secreted by the cell, or a tumor-associated microbial antigen, preferably CD19, GPC3, Mesothelin, PSCA, or MUC1.
In a second aspect, the present application provides a method for preparing the cell according to the first aspect. The method includes:
(1″) activating a human T cell;
(2″) transfecting the activated human T cell with a CAR molecule expressing a tumor-associated antigen or a tumor-specific TCR molecule along with or followed by performing BCL11B gene knockout; and
(3″) culturing the cell obtained in step (2″) in a T cell culture medium.
In the preceding method, preferably, in step (1″), the human T cell is a mature human T cell or a cell population containing mature human T cells; further preferably, the mature human T cell or the cell population containing mature human T cells is derived from cord blood or peripheral blood of a human body; further preferably, the mature human T cell or the cell population containing mature human T cells is derived from a mature T cell or a cell population obtained through differentiation of pluripotent stem cells, embryonic stem cells, or cord blood stem cells.
In the preceding method, preferably, in step (1″), the human T cell is activated using an anti-human CD3 antibody, an anti-human CD28 antibody, and an anti-human CD2 antibody. In a preferred specific embodiment, the T cell is activated through incubation of magnetic beads of the anti-human CD3 antibody, the anti-human CD28 antibody, and the anti-human CD2 antibody mixed with the mature human T cell at a ratio of 1:2.
Preferably, in step (2″), the CAR molecule includes the following domains: a signal peptide, an extracellular antigen recognition domain, a transmembrane region, and an intracellular costimulatory domain. In a preferred specific embodiment, the CAR molecule includes the signal peptide, the extracellular antigen recognition domain, the transmembrane region, and the intracellular costimulatory domain in sequence from an N-terminal to a C-terminal. Preferably, the antigen is the tumor-associated antigen and/or an antigen associated with a microorganism such as a virus or a bacterium. Further preferably, the tumor-associated antigen is a tumor surface antigen, a cytokine secreted by a tumor, a surface antigen of a cell associated with immunosuppression of a tumor microenvironment and a cytokine secreted by the cell, or a tumor-associated microbial antigen, more preferably the tumor surface antigen, even more preferably CD19, GPC3, Mesothelin, PSCA, or MUC1.
Preferably, in step (2″), the BCL11B gene knockout is performed using CRISPR/CAS9 technology; further preferably, the gene knockout is performed at a second exon of a BCL11B gene; or the gene knockout is performed at a third exon of the BCL11B gene.
Preferably, in step (3″), the T cell culture medium includes IL-2; preferably, the cell obtained in step (2″) is not co-cultured with OP9-DL1.
In a third aspect, the present application further provides a use of the cell according to the first aspect for preparing a drug for treatment of a disease selected from the group consisting of a tumor, AIDS, and an infectious disease; preferably, the infectious disease is a viral infectious disease.
Preferably, the drug further includes a pharmaceutically acceptable excipient.
In the present application, the human T cell is reprogrammed into an immune killer lymphocyte and the obtained immune killer lymphocyte is transfected with the CAR molecule expressing the tumor-associated antigen or the tumor-specific TCR molecule, achieving a better tumor killing effect. The reasons are as follows: the reprogrammed cell simultaneously expresses antigen recognition and killing receptors of NK cells and T cells, especially functional TCRs and have the functions of both T cells and NK cells; since the reprogrammed cell simultaneously expresses the antigen recognition and killing receptors of NK cells and T cells, the reprogrammed cell can recognize antigens sensitive to these receptors. Compared with T cells and NK cells, the reprogrammed cell not only has more extensive tumor antigen recognition and killing functions but also has more extensive functions of recognition and elimination of microorganisms such as viruses and bacteria. At the same time, the reprogrammed cell also has enhanced tumor-specific recognition and killing functions due to its CAR molecule expressing the tumor-associated antigen or its tumor-specific TCR molecule.
In addition, the engineered immune killer cell of the present application has an efficient in vitro proliferation ability. In adoptive cell transfer (ACT) therapy, both T cells and NK cells are used for cancer treatment. The engineered immune killer cell of the present application has the functions of both T cells and NK cells. Compared with NK cells which have limited availability and proliferation ability when applied to adoptive immunotherapy (ACT), the reprogrammed immune killer lymphocyte of the present application can be generated from a large number of T cells obtained by a user from the peripheral blood of a patient, and within 2-3 weeks, 200×106 to 1248×106 reprogrammed immune killer lymphocytes can be prepared and acquired from about 100×106 peripheral blood mononuclear cells (PBMC) of a solid tumor patient, thereby meeting the demand of the patient for cell reinfusion.
To further elaborate on the technical means adopted and the effects achieved in the present application, the technical solutions of the present application are further described below through specific examples in conjunction with drawings. However, the present application is not limited to the scope of the examples.
Unless otherwise stated, the present application is not limited to the relative arrangement, numeric expressions and numerical values of the components and steps set forth in these examples. The techniques, methods, and devices known to those of ordinary skill in the art may not be discussed in detail, but in appropriate circumstances, the techniques, methods, and devices should be regarded as part of the specification.
According to the selection rule of a CRISP/CAS9 target site: GN19NGG, where GN19 was a target site, N was better G, and the target site can be on an antisense strand (that is, the sequence on a sense strand is CCN N19C), the following target sequences were selected and forward (F) and reverse (R) primers were designed separately as guideRNA (gRNA). The gRNA was annealed and ligated into the digested PX458 vector to construct a PX458-gBCL11B vector (as shown in
According to the knockout efficiency in Table 1, the gRNA gene knockout plasmid vectors with knockout at the second exon and the third exon were selected for the next step. In the present application, BCL11B gene knockout was preferably performed at the second exon and the third exon, and gene knockout plasmids corresponding to a mixture of a first pair of gRNA and a second pair of gRNA with the lowest knockout efficiency at the second exon, a third pair of gRNA with the highest knockout efficiency at the second exon, and a third pair of gRNA with the lowest knockout efficiency at the third exon and a mixture thereof can all reprogram T cells into immune killer lymphocytes of the present application. In this example, BCL11B gene knockout plasmids were constructed by using gRNA of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 50 and SEQ ID NO: 51, respectively and mixed for the next step.
T cells were sorted and activated by the following method:
(1) peripheral blood and cord blood including mature human T cells were centrifuged at 300×g for 10 minutes, separately, and plasma was collected and thermally inactivated at 56° C. for 30 minutes;
(2) the precipitated granular blood cells were suspended with 0.9% NaCl, and peripheral blood mononuclear cells (PBMCs) were separated through Ficoll density gradient centrifugation; and
(3) negative sorting was performed with an MACS Pan T separation kit (produced by Miltenyi Biotec in Bergisch Gladbach, Germany) to enrich all T cells (Pan T) from the blood such as peripheral blood and cord blood.
The above (1) to (3) were the steps of isolating mature human T cells from peripheral blood and cord blood. It is to be noted that other T cell sources are also acceptable, such as the committed differentiation of pluripotent stem cells and hematopoietic stem cells. T cells from all sources were activated with a T cell activation kit (produced by Miltenyi Biotec) through the incubation of magnetic beads coated with anti-human CD3, anti-human CD28, and anti-human CD2 antibodies mixed with T cells at a ratio of 1:2 (cell density: 2.5×106 cells/mL, and culture medium:T551-H3 (produced by Takara, Japan) containing 5% autologous plasma, hIL2 (100 IU/mL), gentamicin sulfate (20 μg/mL), 10 mm of HEPES, 2 mm of glutamine, and 1% penicillin/streptomycin). Having been activated for 24-48 hours, T cells were eluted from antibiotin MACS iBead™ granules for later use.
(1) CRISP/CAS9 knockout vectors PX458-gBCL11B were transduced into the above-mentioned activated T cells by an electrotransfer (T-023, LONZA Amaxa Nucleofector, Lonza);
(2) after 12 hours, T cells transduced with PX458-gBCL11B (such cells were simply referred to as PX458-T) were centrifuged and cultured in a T551-H3 (produced by Takara, Japan) medium (containing 5% autologous plasma or fetal bovine serum (FBS), 500 IU/mL hIL2 and gentamicin sulfate (20 μg/mL));
(3) a fresh medium was changed every three days and the cell density was kept within a range of 0.5×106 cells/mL to 1×106 cells/mL until electroporation was performed for 14 days;
(4) whether the second exon or the third exon of BCL11B of the T cells transduced with PX458-gBCL11B was subjected to knockout, such as induced insertion or deletion of sites, was detected and verified through gene sequencing; where the control group was T cells transduced with PX458 empty vectors (Mock); and
(5) the expression level of BCL11B proteins in the T cells transduced with PX458-gBCL11B was detected and verified through Western Blotting to further confirm the deletion of BCL11B proteins, where the control group was the T cells transduced with PX458 empty vectors (Mock). The results of Western Blotting are shown in
As described above, after T cells were subjected to electroporation for 14 days, 19.5% to 68.7% of the resulting cells expressed both T cell markers such as CD3 and NK cell markers such as NKp46, CD56, NKp30, and NKp44, and then it was determined that the human ITNK cells of the present application were obtained. NK cells expressed only NK cell markers such as NKp46 and CD56 and did not express T cell markers such as CD3. The T cells electroporated with empty vectors expressed T cell markers such as CD3 and did not express NK cell markers. The expression of cell markers of T cells, NK cells, and ITNK cells is shown in
In addition, it shows through the observation with a confocal microscope that the cell morphology of an ITNK cell reprogrammed from the T cell is different from that of the T cell and similar to that of an NK cell, and the reprogrammed ITNK cell has a relatively small nucleus (relative to a volume of the nucleus of the T cell which occupies the whole cell), a relatively plentiful intracellular matrix, a larger granule, abundant endoplasmic reticulum, and high protein synthesis activity, indicating that the reprogrammed ITNK cell is an immune killer lymphocyte. The transmission electron microscopic images of the T cell, the NK cell, and the ITNK cell are shown in
In addition, the inventors also compared the expression profiles of these NK markers in BCL11B-deficient T cell subgroups derived from cord blood and peripheral blood and found that the percentages of CD8+NKp46+ cells and CD8+CD56+ cells were significantly higher than the percentages of CD4+NKp46+ cells and CD4+CD56+ cells, indicating that NKp46+CD3+ ITNK cells were mainly derived from CD8+ T cells (see
The CD4-CD8-NKp46+ subgroup expressed “TCRΔδ” and was γδTCR+ ITNK cells (see
TCRαβ sequencing: T cells and ITNK cells obtained in Example 1, both of which were derived from the same donor, were subjected to RNA extraction and CDR3 region targeted proliferation through a human TCRαβ analysis kit to obtain TCR RNA. TCR RNA was sequenced on Hiseq 4000 platform to obtain a TCR library. A clustering combination analysis was performed with MiXCR(ref). The types of TCRαβ clones were derived with the parameter of “—chain” through MiXCR clone derivation instructions. The diversity of TCR clones of T cells and ITNK cells which were both derived from the same donor was compared through TCR sequencing. It was found that the diversity of TCR clones was consistent (see
The ITNK cells obtained in Example 1 were subjected to a single-cell immunophenotyping analysis through mass cytometry (CyTOF), separately. The control group was T cells transduced with empty vectors.
Preparation and pretreatment of mass spectrometer samples: Cells from a culture suspension were centrifuged, re-suspended with PBS containing 0.5% BSA and 0.02% NaN3, and incubated with an anti-human CD16/32 monoclonal antibody at room temperature for 10 minutes to block an Fc receptor. Then, 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 (produced by Fluidigm) or were internally coupled using a mass spectrometry flow coupling kit (produced by Fluidigm) according to the instructions. 5 mM of cisplatin was added to the cells, and the cells were incubated and stained on ice in FBS (produced by Fluidigm) for 1 minute. After the cells were treated with a fixation/permeabilization buffer (produced by Thermo Fisher), the cells were mixed with the metal-labeled antibodies and incubated to label intracellular proteins. After the cells were cleaned, the cells were stained with 1 mL of 191/1931r DNA intercalator (produced by Fluidigm) that was diluted at a ratio of 1:4000 (the intercalator was diluted with PBS containing 1.6% paraformaldehyde (produced by EMS)) and then stored at 4° C. Before an assay, the cells were washed once with PBS containing 0.5% BSA and 0.02% NaN3, washed once with ddH2O, and re-suspended and diluted to about 106 cells/mL with ultrapure water (ddH2O). Then, cell sample data was detected and collected using CyTOF2 (produced by Fluidigm) at an event rate of ≤400 events/sec.
According to the cellular immunophenotypic differences of 40 markers, a clustering analysis was performed through PhenoGraph clustering algorithm. ITNK cells derived from cord blood (hereinafter referred to as CB-ITNK), ITNK cells derived from peripheral blood (hereinafter referred to as PBMC-ITNK), and Mock-T cells were integrated and classified into 39 subgroups, as shown in
According to the results of a cell marker expression heterogeneity analysis through mass cytometry, the ITNK cells of the present application include a CD3-negative cell subgroup of NO. 33, CD4+ cell subgroups of Nos. 5 to 10, CD8+ cell subgroups of NOs. 20 to 22 and 26 to 28, and TCRγδ+cell subgroups of NOs. 23 and 24, and all these ITNK cells express NK-associated markers such as CD56, NKp30, NKp44, NKp46, and CD11C; and compared with γδT cells, TCRγδ+ ITNK cells perform high expression of three markers NKp46, NKp30, and NKp44, that is, (NKp46high NKp30high NKp44high) (as shown in
In addition, as for ITNK cells derived from cord blood, the histogram shown in
To study the entire gene expression profile of ITNK cells, the inventors performed RNA sequencing and analysis on T cells derived from 4 cord blood samples and 3 adult peripheral blood samples, ITNK cells derived from 4 cord blood samples and 3 adult peripheral blood samples, and NK cells derived from 2 cord blood samples and 2 adult peripheral blood samples. The sorting operation was as follows: flow cytometry analysis or sorting was performed by flow cytometers Canto, FACS Fortessa (BD), FACSAriall, etc. Cell surface receptors were labeled as follows: cells and antibodies were mixed in 50 μL of flow buffer (PBS solution containing 2% FBS) and incubated at 4° C. for 30 minutes in the dark. Intracellular labeling: the cells were subjected to permeable treatment with Foxp3/transcription factor staining buffer (produced by eBioscience), and after the buffer was eluted, the cells were blocked with mouse serum or rabbit serum, incubated with antibodies at 4° C. for 30 minutes in the dark, washed once with the flow buffer, and then re-suspended for subsequent flow cytometry analysis or sorting. A cell sorting strategy and the verification of sorting purity were shown in
A principal component analysis (PCA) was performed for similarity evaluation on the RNA sequencing results of 18 samples. It was found that ITNK cells were different from T cells and NK cells according to a transcriptome analysis (as shown in
Flow cytometry shows that CD8+CD3+NKp46+ ITNK and CD4+CD3+NKp30+ ITNK appear on day 5 after BCL11B knockout (as shown in
About 5000 cells were detected and analyzed in all experimental groups. Groups of cell samples at different time points were detected through scRNA-seq, an average of 2000-4000 genes were detected per cell, and a total of 20000 human genes were detected in all cells. In the t-distributed random neighbor-embedded (t-SNE) analysis of transcription profiles, the cells were projected to two dimensions, which provided the visual representation of the cell fate transition in the reprogramming process of ITNK cells. The results of the unbiased t-SEN analysis show that the cells from day 0 to day 20 after knockout can be clustered into 11 subgroups (as shown in
To determine whether an NK-cell receptor (NCR) and a T-cell receptor (TCR) expressed by ITNK cells of the present application are functional, the ITNK cells were stimulated with an anti-NKp30 monoclonal antibody, an anti-NKp46 monoclonal antibody, and an anti-CD3/CD28 monoclonal antibody, separately. It is found that after stimulated with the anti-NKp30 antibody and the anti-NKp46 antibody, the ITNK cells secrete more interferons (IFNs) while T cells in the control group secrete the same IFNs (as shown in
Similar to NK cells, the ITNK cells of the present application can secrete a variety of cytokines including GM-CSF, IFN and TNF (as shown in
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 K562 tumor-bearing mouse models, and then ITNK cells, NK cells, or T cells were injected for a single time (
To verify the in vivo distribution and maintenance ability of the ITNK cells, the ITNK cells were transplanted into NSI-strain immunodeficient mice lacking T cells, B cells, and NK cells, and the percentages of ITNK cells in peripheral blood (PB), spleen (SP), bone marrow (BM), liver, and lung were measured on day 1, day 7, day 14, day 21, and day 180 after transplantation (
To evaluate the possible off-target mutation induced by PX458-gBCL11B, T cells electroporated with PX458-gBCL11B were subjected to whole genome sequencing of high coverage. Compared with wild-type T cells, it is found from two independent experiments that there are very few off-target mutations caused by nuclease in the T cells edited by PX458-gBCL11B.
Although ITNK cells have TCR and NCR functions, the ITNK cells cannot recognize particular tumor antigens. For this purpose, the inventors transduced PB-CAR molecular vectors (the structure of a CAR molecule: an extracellular domain is the extracellular fragment of a receptor of an antigen such as CD19, GPC3, MUC1, or Mesothelin or the scFv sequence of the corresponding antibody, a transmembrane region is one or two of transmembrane regions of receptors CD28, NKG2D, NKp44, and NKp46, and an intracellular costimulatory domain is an intracellular costimulatory domain of CD28, TLR2, 2B4, DAP10, or DAP12, and CD3) and BCL11B knockout vectors PX458-gBCL11B into human T cells successively or simultaneously to obtain the ITNK cells expressing anti-CD19 chimeric antigen receptor (CAR) molecules. The expression of PI labels and GFP in the cells after transduction was detected through flow cytometry to determine the survival and transduction efficiency of the cells after transduction (as shown in
To evaluate the anti-tumor effect of CAR19 (FMC63 scFv fragment-CD28 transmembrane region-CD28 and TLR2 intracellular domain-CD3 signal domain)-ITNK cells, human chronic myeloid leukemia cell line K562 cells (K562-CD19) expressing human CD19 and luciferase and B acute lymphocytic 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:target cell) ratios for 24 h, respectively. Luciferase substrates were added and the killing situation of tumor cells was detected by a microplate reader.
The in vitro killing experiment on K562-CD19 cells shows that the CAR19-ITNK cells of the present application more effectively recognize and kill K562-CD19 cells than CAR19-T cells and ITNK cells (as shown in
To detect the in vivo anti-tumor activity of CAR19-ITNK cells of the present application, the applicant injected the K562-CD19 cells constructed in Example 10 into NSI mice through veins (5×105 cells per mouse), and then CAR19-ITNK cells, ITNK cells, CAR19-T cells, or T cells were injected (2.5×105 cells per mouse), which were respectively referred to as the CAR19-ITNK group, the ITNK group, the CAR19-T group, or the T cell group. The experimental process is shown in
The experimental results show that mice in the CAR19-ITNK group have lighter tumor loads than the other groups as shown in
CAR-ITNK cells that recognize phosphatidylinositol GPC3 were constructed in the present application, where the structure of the CAR molecule includes an anti-GPC3 scFv extracellular fragment, an NKG2D transmembrane region, a 2B4 intracellular costimulatory domain, and CD3ζ. Four experimental groups, including CAR-ITNK cells, CAR-T cells, ITNK cells, and T cells, were set up in a 96-well plate, and three duplicate wells were set up for each group. Each well was added with 10000 tumor cells (GPC3-positive tumor cell lines Huh?-GL and HepG2-GL, where GL was a luciferase gene marker) as target cells. Effector cells were added into the plate at an E:T ratio of 4:1, 2:1, 1:1, 1:2, 1:4, separately. After the effector cells were incubated with the tumor cells for 24 hours, luciferase substrates were added and the killing ratio of tumor cells was detected by a quantitative spectrophotometer. It is found through the analysis of the experimental results that CAR-ITNK cells have a better tumor killing effect than ITNK cells, CAR-T cells, and T cells (which is not shown by data).
CAR-ITNK cells that recognize cytokine TGFβ were constructed in the present application, where the structure of the CAR molecule includes an anti-TGFβ scFv extracellular fragment, a CD28 intracellular costimulatory domain, a TLR2 intracellular costimulatory domain, and CD3. Four experimental groups, including CAR-ITNK cells, CAR-ITNK+TGFβ, ITNK cells, and ITNK cells+TGFβ, were set up in a 96-well plate, and five duplicate wells were set up for each group, with 105 cells per well. 6, 24, 48, 72, and 96 hours after TGFβ (3 ng/mL) was added, the absolute number of cells in each well was recorded through cell counting, and the secretion of related immune effector cytokines in different experimental groups was detected by ELISA. Through the comparison and data analysis of statistical results, it is found that TGFβ inhibits the proliferation of ITNK cells and the secretion of immune effector cytokines, while the anti-TGFβ CAR-ITNK cells exhibit enhanced cell proliferation and secretion of immune effector cytokines in the presence of TGFβ.
To evaluate the killing effect of the anti-TGFβ CAR-ITNK cells on tumor cells, four experimental groups, including CAR-ITNK cells, CAR-ITNK+TGFβ, ITNK cells, and ITNK cells+TGFβ, were set up in a 24-well plate, and three duplicate wells were set up for each group. Each well had 2×105 effector cells and was added with 105 tumor cells (tumor cell line HepG2 with a luciferase gene marker) as target cells. 24 hours after the TGFβ cytokine was added, luciferase substrates were added and the killing ratio of tumor cells was detected by a fluorometer. Through the analysis of the experimental results, it is found that TGFβ inhibits the tumor killing effect of ITNK cells, while the presence of TGFβ relatively enhances the killing effect of the anti-TGFβ CAR ITNK cells on tumor cells (which is not shown by data).
CAR-ITNK cells that recognize Mesothelin were constructed in the present application, where the structure of the CAR molecule includes an anti-Mesothelin scFv extracellular fragment, a CD28 transmembrane region, a DAP10/DAP12 sequence, and CD3. Four experimental groups, including CAR-ITNK cells, CAR-T cells, ITNK cells, and T cells, were set up in a 96-well plate, and three duplicate wells were set up for each group. Each well was added with 10000 tumor cells (Mesothelin-positive tumor cell lines BGC-823-GL and MKN-28-GL, where GL was a luciferase gene marker) as target cells. Effector cells were added into the plate at an E:T ratio of 4:1, 2:1, 1:1, 1:2, 1:4, separately. After the effector cells were incubated with the tumor cells for 24 hours, luciferase substrates were added and the killing ratio of tumor cells was detected by a quantitative spectrophotometer. These experimental results are similar to the results in the preceding example and show that CAR-ITNK cells have a better tumor killing effect than ITNK cells, CAR-T cells, and T cells (which is not shown by data).
Tumor and virus recognition and killing activation pathways of the CAR ITNK cells of the present application do not interfere with each other and have a mutual synergistic effect. The CAR ITNK cells of the present application can not only activate and recognize tumor- or virus-associated antigens through CAR molecules but also recognize tumor- or virus-associated antigens through the pathway of the NK-cell receptor and the TCR in ITNK cells. The CAR ITNK cells not only have an efficient specific killing effect on particular tumors and viruses so as to rapidly control tumor progression and virus deterioration but also have broad-spectrum anti-tumor and anti-virus effects so as to prevent the escape and recurrence of tumors and viruses. The CAR-ITNK technology of the present application solves the problems in the existing art of tumor antigen escape, recurrence, and low efficiency in the CAR T and CAR NK treatment.
The applicant has stated that although the detailed method of the present application is described through the examples described above, the present application is not limited to the detailed method described above, which means that implementation of the present application does not necessarily depend on the detailed method described above. It should be apparent to those skilled in the art that any improvements made to the present application, equivalent replacements of raw materials of the product of the present application, additions of adjuvant ingredients to the product of the present application, and selections of specific manners, etc., all fall within the protection scope and the disclosure scope of the present application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201911382947.2 | Dec 2019 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/118265 | 9/28/2020 | WO |
