Patent Application
20220325246
The present application relates to the technical field of biomedicine, in particular, engineered human immune cells, a preparation method, and an application thereof.
At present, immunotherapy has become the most concerned and promising “new” idea in the field of cancer treatment. In 2013, in the top ten scientific breakthroughs ranked by Science, tumor immunotherapy topped the list. Chimeric antigen receptor (CAR) T cells produced by Novartis and Kite Pharma have been approved by Food and Drug Administration (FDA), U.S., and the tumor immunotherapy has made milestone progress in the field of hematological malignancies treatment. However, there are still technical bottlenecks in the clinical treatment of tumor immunotherapy, for example, the single target of genetically modified immune cells leads to tumor immune escape and tumor recurrence; there is a lack of specific markers with high efficiency and low side effects for solid tumors, and the current gene-modified immunotherapies show no efficient and safe clinical effect on solid tumors.
T cells can be grouped into different subsets according to their surface markers and functions. For example, according to the difference in TCR types, T cells can be classified into γδ T cells and αβ T cells. αβ T cells account for more than 95% of T cells, are the main cell groups that have T cell differentiation markers and execute T cell functions in vivo, and represent the diversity of T cells. γδ T cells are a group of highly heterogeneous cells, whose surface T cell receptor of γδ T cells is composed of γ chain and δ chain, γδ T cells have features of various subtype, variable phenotype and rich functions, the biological characteristics of γδ T cell subtypes are different, and γδ T cells play an important role in the occurrence and development of tumors, infections and autoimmune diseases and are considered as the bridge spanning innate immunity and adaptive immunity.
Like T cells, natural killer (NK) cells are an indispensable part of the 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 the 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 that are tumor cells or cells developing into tumor cells by inducing the activity of dendritic cells (DCs) or the adaptive immune activation of tumor-specific cytotoxic T lymphocytes (CTLs). Although NK cells have the potential to be used as therapeutic agents against cancers or infectious diseases, most NK cells in the 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. In order to use natural killer cells as therapeutic agents, activated natural killer cells that can recognize and destroy tumor cells are required. Because the number of natural killer cells in vivo is limited, it is very important to obtain a sufficient number of activated natural killer cells.
Cell reprogramming refers to the process that differentiated cells are reversed under specific conditions and then return to the totipotent state, or form embryonic stem cell lines, or further develop into a new individual. In the field of immunotherapy of diseases, there have been reports on the transformation of immune cells by cell reprogramming For example, Dr. Ding Sheng from Gladstone Institutes in the United States reported that pro-inflammatory effector T cells were reprogrammed into anti-inflammatory regulatory T cells by specific reprogramming Such reprogramming is of great significance to the treatment of autoimmune diseases. Specifically, for autoimmune diseases, over-stimulated effector T cells cause damage 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 balance, thereby fundamentally treating the diseases.
At present, there is no application of cell reprogramming in human tumor immunotherapy.
In view of the deficiency of the prior art and the actual demands, the present application provides human immune cells engineered by reprogramming, a preparation method, and an application thereof. The engineered human immune cells of the present application have partial markers and functions of T cells and NK cells and simultaneously express antigen recognition and killing receptors of NK cells and T cells, and thus have a wider spectrum of tumor antigen recognition and killing functions than NK cells and T cells. Meanwhile, compared with human mature T cells from which the engineered human immune cells are derived, the engineered human immune cells of the present application have an enhanced amplification ability and a better anti-tumor effect.
In a first aspect, the present application provides immune killer lymphocytes induced by reprogramming of human T cells (induced T-to-natural killer (ITNK) cells), which retain markers and functions of T cells from which the immune killer lymphocytes are derived and have markers and functions of NK cells, wherein the reprogramming of the human T cells involves deletion of a BCL11B gene.
Preferably, the human T cells are mature human T cells or a cell population including mature human T cells; further preferably, the mature human T cells or the cell population including the mature human T cells are derived from human cord blood or human peripheral blood; and further preferably, the mature human T cells or the cell population including the mature human T cells are derived from mature T cells or cell populations obtained by differentiation of pluripotent stem cells, embryonic stem cells or cord blood stem cells.
Preferably, the reprogrammed immune killer lymphocytes express functional TCR, CD3 and NKp30.
Preferably, the reprogrammed immune killer lymphocytes express a marker of NK cells selected from the following group consisting of CD11c, NKG2D and CD161.
Preferably, immunosuppression checkpoints PD-1, CTLA-4 and FOXP3 are of low expression or no expression in the reprogrammed immune killer lymphocytes.
Preferably, NK-related markers CD127, CD16, KIRDL2, KIRDL3 and NKG2A are of low expression or no expression in the reprogrammed immune killer lymphocytes.
Preferably, compared with the T cells from which the immune killer lymphocytes are derived, expression of NOTCH on the reprogrammed immune killer lymphocytes is up-regulated.
Preferably, compared with the T cells from which the immune killer lymphocytes are derived, expression of transcription factors LEF1 and TCF7 is down-regulated, and expression of NOTCH, AP1, mTOR, ID2, TBX21 and NFIL3 is up-regulated.
Preferably, TCR-mediated signal transduction of the reprogrammed immune killer lymphocytes is enhanced.
Preferably, compared with the T cells from which the immune killer lymphocytes are derived, expression of genes of the reprogrammed immune killer lymphocytes, which are related to the TCR-mediated signal transduction and include CSF2, FOS, MAPK12, MAP3K8, IFNγ, NFKBIA, MAPK11, IL-10 and TEC, is up-regulated.
Preferably, compared with NK cells, T cell recognition and TCR signal transduction of the reprogrammed immune killer lymphocytes are enhanced; and preferably, expression of CD3, CD4, CD8 and CD40LG is up-regulated.
Preferably, compared with the T cells from which the immune killer lymphocytes are derived, NK killing toxicity-related signal transduction of the reprogrammed immune killer lymphocytes is enhanced.
Preferably, compared with the T cells from which the immune killer lymphocytes are derived, expression of genes of the reprogrammed immune killer lymphocytes, which are related to the NK killing toxicity-related signal transduction and include PRF1, CSF2, ICAM1, CD244, PLCG2, IFNG, FCER1G, GZMB, NCR2, NCR1, KIR2DL4 and SYK, is up-regulated.
In a preferred specific embodiment, the reprogrammed immune killer lymphocytes include cell subsets CD8+NKp46hiNKp44+NKp30+, CD4+NKp30+, and γδTCR+NKp46hiNKp44+NKp30+T.
In a preferred specific embodiment, the human T cells are mature human T cells, and the reprogramming of the human T cells includes:
1) activating mature human T cells;
2) performing BCL11B gene knockout on the activated mature human T cells obtained in step 1); and
3) culturing the cells obtained in step 2) with a T cell culture medium.
Wherein, in step 1), the activation is performed using an anti-human CD3 antibody, an anti-human CD28 antibody, and an anti-human CD2 antibody.
Preferably, magnetic beads of anti-human CD3 antibody, anti-human CD28 antibody and anti-human CD2 antibody are mixed with the mature human T cells in a ratio of 1:2 and incubated to activate the T cells.
In step 3), the T cell medium includes IL-2; and preferably, the cells obtained in step 2) are not co-cultured with OP9-DLE
In a second aspect, the present application provides a method for preparing the cells described in the first aspect, including:
1′) activating human T cells;
2′) performing BCL11B gene knockout on the activated human T cells obtained in step 1′); and
3′) culturing the cells obtained in step 2′) with a T cell culture medium.
In the above-mentioned method, preferably, the human T cells are mature human T cells or a cell population including mature human T cells; further preferably, the mature human T cells or the cell population including the mature human T cells are derived from human cord blood or human peripheral blood; and further preferably, the mature human T cells or the cell population including the mature human T cells are derived from mature T cells or cell populations obtained by differentiation of pluripotent stem cells, embryonic stem cells or cord blood stem cells.
In the above-mentioned method, preferably, in step 1′), the activation is performed using an anti-human CD3 antibody, an anti-human CD28 antibody, and an anti-human CD2 antibody; and in a preferred specific embodiment, magnetic beads of anti-human CD3 antibody, anti-human CD28 antibody and anti-human CD2 antibody are mixed with the mature human T cells in a ratio of 1:2 and incubated to activate the T cells.
Preferably, in step 2′), the BCL11B gene knockout is performed by CRISPR/CAS9; and further preferably, the gene knockout is performed at a second exon and/or a third exon of a BCL11B gene.
Preferably, in step 3′), the T cell medium includes IL-2; and preferably, the cells obtained in step 2′) are not co-cultured with OP9-DLE
In a third aspect, the present application provides an application of the cells described in the first aspect in the preparation of a medicament for the treatment of a disease selected from the group consisting of tumors, AIDS and infectious diseases; and preferably, the infectious diseases are viral infectious diseases.
Preferably, the medicament further includes a pharmaceutically acceptable excipient.
The present application is the first to implement the reprogramming of human T cells into immune killer lymphocytes. The reprogrammed cells simultaneously express antigen recognition killer receptors of NK cells and T cells, especially functional TCRs, and have the functions of T cells and NK cells; and since the reprogrammed cells simultaneously express antigen recognition and killing receptors of NK cells and T cells, the reprogrammed cells can recognize antigens sensitive to these receptors. Therefore, compared with T cells and NK cells, the reprogrammed cells not only have a wider spectrum of tumor antigen recognition and killing functions, but also have a wider spectrum of microbial recognition and removal functions on microorganisms such as viruses and bacteria.
In addition, the reprogrammed cells of the present application have an efficient in vitro amplification ability. In adoptive cell transfer (ACT) therapy, both T cells and NK cells are used to treat cancers. The reprogrammed cells of the present application have the functions of both T cells and NK cells, and free from the limitation of availability and amplification ability of NK cells in the adoptive immunotherapy (ACT), the user can obtain a large number of T cells from the peripheral blood of a patient to generate the reprogrammed immune killer lymphocytes of the present application, and within 2 to 3 weeks, the user can obtain 200×106 to 1248×106 reprogrammed immune killer lymphocytes 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 solutions of the present application are further described below through specific examples in conjunction with drawings, but the present application is not limited to the scope of the examples.
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, unless otherwise noted. Techniques, methods and devices known to those of ordinary skill in the art may not be discussed in detail, but in appropriate circumstances, techniques, methods and devices should be regarded as part of the specification.
Construction of Gene Knockout Plasmid Vector
According to the selection rule of CRISP/CAS9 target sites: GN19NGG, where GN19 was a target site, N was still better 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 guideRNA (gRNA), respectively. The gRNA was annealed and ligated into the digested PX458 vectors to construct PX458-gBCL11B vectors (as shown in
According to the knockout efficiency in Table 1, the gRNA gene knockout plasmid vectors at the second exon and the third exon were selected to be knocked out for the next experiment. In the present application, preferably, BCL11B gene knockout was performed in the second exon and the third exon, the third pair of gRNA with the highest knockout efficiency was knocked out by using a mixture of the first pair of gRNA with the lowest knockout efficiency and the second pair of gRNA with the lowest knockout efficiency at the second exon, and the gene knockout plasmids corresponding to the third pair of gRNA with the lowest knockout efficiency at the third exon and the mixture thereof could reprogram T cells to obtain the immune killing 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 experiment.
Sorting and Activation of T Cells
T Cells were sorted and activated using the following method.
(1) Peripheral blood including human mature T cells and cord blood including human mature T cells were centrifuged at 300×g for 10 minutes, 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 (PBMC) were separated by Ficoll density gradient centrifugation.
(3) Negative sorting was performed with MACS Pan T Cell Isolation 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) are the steps of isolating human mature T cells from peripheral blood and cord blood, and it should be noted that other T cell sources are also acceptable, such as the directed 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). Magnetic beads coated with anti-human CD3, anti-human CD28 antibody and anti-human CD2 were mixed and incubated with T cells in a ratio of 1:2 (cell density: 2.5×106 cells/ml, and the medium: T551-H3 medium (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) and then activated. After activation for 24 hours to 48 hours, T cells were eluted from antibiotin MACS iBead™ granules for later use.
Induced Reprogramming
(1) CRISP/CAS 9 knockout vectors PX458-gBCL11B were transduced into the above-mentioned activated T cells by electrotransfer (T-023, LONZA Amaxa Nucleofector, Lonza).
(2) After 12 hours, T cells transduced with PX458-gBCL11B (such cells were referred to PX458-T) were centrifuged and cultured with 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) The fresh medium was changed every 3 days, and the cell density was kept in the range of 0.5×106 cells/ml to 1×106 cells/ml until day 14 after electrotransfer.
(4) Whether the second exon and the third exon of BCL11B of T cells transduced with PX458-gBCL11B were subjected to knockout such as site induced insertion or deletion, was detected and verified by gene sequencing. The control group was T cells transduced with PX458 empty vectors (Mock).
(5) The expression level of BCL11B proteins in T cells transduced with PX458-gBCL11B was detected and verified by Western Blotting to further confirm the deletion of BCL11B proteins, and the control group was T cells transduced with PX458 empty vectors (Mock). The results of Western Blotting are shown in
Phenotype Identification of Reprogrammed Cells
As described above, after 14 days of electrotransfer of T cells, 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 thus it was determined that the human ITNK cells of the present application were obtained. NK cells only expressed NK cell markers such as NKp46 and CD56, but did not express T cell markers such as CD3. T cells subjected to electrotransfer of empty vectors expressed T cell markers such as CD3, but did not express NK cell markers. The expression of cell markers of T cells, NK cells and ITNK cells is shown in
In addition, according to the observation from the confocal microscope, the cell morphology of ITNK cells reprogrammed from T cells was different from the cell morphology of T cells and similar to the cell morphology of NK cells, and the reprogrammed ITNK cells had small nucleus (the volume of T cell nucleus relative to the whole cell), plentiful intracellular matrix, large granules, abundant endoplasmic reticulum and high protein synthesis activity, indicating that reprogrammed ITNK cells were immune killer lymphocytes. The transmission electron microscopic images of T cells, NK cells and ITNK cells are shown in
In addition, the inventors also compared the expression profiles of these NK markers in the subsets of BCL11B-deficient T cells 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 mainly derived from CD8+ T cells (see
CD4−CD8−NKp46+ subset expressed “TCRγδ” and was γδTCR+ ITNK cells (see
TCRαβ sequencing: T cells derived from the same donor and ITNK cells obtained in Example 1 were subjected to RNA extraction and CDR3 region targeted amplification through human TCRαβ analysis kit to obtain TCR RNA. TCR RNA was subjected to sequencing on Hiseq 4000 platform to obtain a TCR library. Clustering combination analysis was performed with MiXCR(ref). The type of TCRαβ clone was derived with the parameter of “-chain” through the MiXCR clone derivation instruction. The diversity of TCR clones of T cells derived from the same donor and ITNK cells was compared by TCR sequencing, and it was found that the diversity of TCR clones was consistent (see
ITNK cells obtained in Example 1 were subjected to single-cell immunophenotype analysis by mass cytometry (CyTOF) respectively, and the control group was T cells transduced with empty vectors.
Preparation and pretreatment of mass spectrometer samples: Cells from culture suspension were centrifuged, re-suspended with PBS containing 0.5% BSA and 0.02% NaN3, and incubated with anti-human CD16/32 monoclonal antibody at room temperature for 10 minutes to block the Fc receptor. Then, a mixture of metal-labeled antibodies against cell surface molecules was added and incubated on ice for further 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 and incubated with the metal-labeled antibodies to label intracellular proteins. After the cells were cleaned, the cells were stained with 1 mL of 191/193Ir 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 detection, the cells were washed once with PBS containing 0.5% BSA and 0.02% NaN3 and once with ddH2O, then re-suspended and diluted to about 106 cells/ml with ultrapure water (ddH2O). Subsequently, cell sample data were detected and collected using CyTOF2 device (produced by Fluidigm) at an event rate of <400 events/sec.
According to the cellular immunophenotypic differences of 40 markers, 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 subsets, as shown in
According to results of cell marker expression heterogeneity analysis by mass cytometry, the ITNK cells of the present application included CD3-negative cell subset of NO. 33, CD4+ cell subsets of NOs. 5 to 10, CD8+ cell subsets of NOs. 20 to 22 and 26 to 28, and TCRγδ+ cell subsets of NOs. 23 to 24, and all expressed NK-related markers such as CD56, NKp30, NKp44, NKp46 or CD11C, and compared with γδT cells, NKp46, NKp30 and NKp44 markers were of high expression in TCRγδ+ ITNK cells, i.e., (NKp46high NKp30high NKp44high) (as shown in
In addition, in ITNK cells derived from cord blood, the histogram as shown in
In order to study the entire gene expression profile of ITNK cells, the inventors performed RNA sequencing 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 is as follows: flow cytometry analysis or sorting was performed by flow cytometers Canto, FACS Fortessa (BD), FACSAriaII, etc. Cell-surface receptor labeling: the cells and antibody were mixed in 50 μl of flow buffer (PBS solution containing 2% FBS) and incubated at 4° C. for 30 minutes in the dark. Cell intracellular labeling: the cells were subjected to permeable treatment with Foxp3/transcription factor staining buffer (produced by eBioscience), after the buffer was eluted, blocked with mouse serum or rabbit serum, incubated with antibodies at 4° C. for 30 minutes in the dark, washed with flow buffer and then suspended for subsequent flow cytometry analysis or sorting. Cell sorting strategy and sorting purity were verified (as shown in
Principal component analysis (PCA) was performed on RNA sequencing results of 18 samples for similarity evaluation, and it was found that ITNK cells were different from T cells and NK cells according to transcriptome analysis (as shown in
Flow cytometry shows that CD8+CD3+NKp46+ ITNK cells and CD4+CD3+NKp30+ ITNK cells appeared on day 5 after BCL11B was knocked out (as shown in
About 5000 cells were detected and analyzed in all experimental groups. Different time-point groups of cell samples were detected by scRNA-seq, 2000 to 4000 genes per cell were detected, and a total of 20000 human genes were detected among all cells. In the t-distributed random neighbor-embedded (t-SNE) analysis of transcription profiles, the cells were projected into two dimensions, which provided a visual representation of the cell fate transition in the process of ITNK cell reprogramming. The results of the unbiased t-SEN analysis show that the cells from day 0 to day 20 after knockout could be clustered into 11 subsets (as shown in
In order to determine whether the NK cell receptor (NCR) and T cell receptor (TCR) expressed on ITNK cells of the present application are functional, the ITNK cells were stimulated with anti-NKp30 monoclonal antibody, anti-NKp46 monoclonal antibody and anti-CD3/CD28 monoclonal antibody, respectively. It is found that the secretion of interferon (IFN) in ITNK cells increased after the ITNK cells were stimulated with anti-NKp30 antibody and anti-NKp46 antibody, while the secretion of IFN in T cells of the control group did not increase (as shown in
Similar to NK cells, ITNK cells of the present application could 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 could 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 in a single time (
In order to verify the distribution and maintenance ability of ITNK cells in vivo, ITNK cells were transplanted into NSI-strain immunodeficient mice lacking T cells, B cells and NK cells, and the percentage of ITNK cells in peripheral blood (PB), spleen (SP), bone marrow (BM), liver, and lung was measured on day 1, day 7, day 14, day 21 and day 180 after transplantation (
In order to evaluate the possible off-target mutation induced by PX458-gBCL11B, T cells electroporated with PX458-gBCL11B were subjected to the whole genome sequencing of high coverage. Compared with wild-type T cells, it is found from two independent experiments that there were very few off-target mutations caused by nuclease in T cells edited by PX458-gBCL11B.
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 disclosed scope of the present application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201910945529.3 | Sep 2019 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/118104 | 9/27/2020 | WO |
