CN116410336B - Chimeric antigen receptor encoding nucleotide, CAR-NK cell, construction method and application thereof - Google Patents
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Classifications
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P35/02—Antineoplastic agents specific for leukemia
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/5409—IL-5
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0646—Natural killers cells [NK], NKT cells
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07—ORGANIC CHEMISTRY
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Abstract
The invention relates to the technical field of cell therapy, in particular to construction and application of a chimeric antigen receptor capable of being expressed efficiently and a CAR-NK cell secreting a functional stimulating factor IL-15. The invention is based on the shortcomings and drawbacks of chimeric antigen receptor-NK cell therapies in clinical trials for the treatment of tumors: 1. the source of the adult NK cells is insufficient; 2. the CAR molecules are unevenly transduced in the adult cells, and the functions of the product are unstable; 3. traditional CAR-NK cells have a low function and a short survival after infusion due to effective stimulation defects. In order to solve the key clinical obstacle of NK cell therapy, the invention provides the CAR-CD19-IPS-iNK cell which can efficiently express the CAR molecule and has the function of autocrine stimulation of the IL-15 and is based on the differentiation of induced pluripotent stem cells, which not only solves the problem of insufficient cell sources in immunotherapy, but also can efficiently and stably express the CAR molecule in the NK cell, and the NK cell expressing the CAR molecule can ensure that the cell can maintain sufficient cell activity in the aspect of killing tumors and exert lasting functional effect by secreting the IL-15.
Description
Technical Field
The invention relates to the technical field of cell therapy, in particular to a chimeric antigen receptor coding nucleotide, a CAR-NK cell, a construction method and application thereof.
Background
Cancer is the second leading cause of death worldwide, causing heavy injuries to society. The scientific community has been devoted to the study of cancer attacks, and good success has been achieved in this regard. However, the complexity of tumor therapy has created a bottleneck in the treatment of most tumors with conventional small molecule inhibitors, diabodies and monoclonal antibodies, and thus the development of advanced and effective antitumor drugs is urgently needed to overcome this difficulty. In such a background, T cell immunotherapy expressing Chimeric Antigen Receptor (CAR) has been developed and has brought great promise in clinical treatment of tumors, but CAR-T cells have many disadvantages in treating tumors, such as failure to obtain a sufficient amount of T cells from patients, high manufacturing cost, T cell production cytokine storm, unsatisfactory effectiveness against solid tumors, and serious toxic side effects on patients, severely limiting the effect of CAR-T cells in clinical tumor treatment.
In order to address the above shortcomings of immunotherapy with T cells, the scientific community has put eye light on Natural Killer (NK) cells, and it is desirable to manufacture CAR-NK cells from NK cells to replace CAR-T immunotherapy, and the reasons for considering replacement of T cells with NK cells mainly include:
firstly, NK cells do not first develop graft versus host disease compared to T cells, which avoids the risk of host resistance due to T cells, thereby greatly improving the safety of immunotherapy; second, NK cells do not secrete inflammatory factors such as IL-I, IL-6, etc. that cause cytokine release syndrome, which reduces life threatening adverse events that induce cytokine storm and neurotoxicity in patients. At the same time, however, tumor immunotherapy with NK cells instead of T cells also shows some drawbacks, such as that the metastasis of expanded autologous NK cells does not show positive clinical effects on some solid tumors such as metastatic melanoma, renal cells or advanced gastrointestinal cancers, mainly due to decreased NK cell trafficking and infiltration to tumor sites after adoptive infusion of NK cells by solid tumor patients, decreased tumor microenvironment inhibition signals and altered tumor cell immunogenicity such that tumor cells lack sensitivity to NK cytotoxicity and inhibited immune cells alter NK cell function, and unmodified autologous or allogeneic cells often show weak anti-tumor cytotoxicity without further in vitro or in vivo stimulation in the circulation, and many other factors such as poor NK cell infiltration and activation at tumor sites. Thus, enhancing NK cell tumoricidal activity by increasing NK cell-directed tumor site infiltration is a fundamental approach to increasing NK cell adoptive immunotherapy efficacy.
Based on some defects exhibited by NK cells, the idea of stimulating the amplification and activation of NK cells with interleukin and using interleukin in combination with NK cells for treating tumors is also presented. NK-92 cells have also been stimulated with IL-2 and used to treat advanced lung cancer, producing a better clinical response, but IL-2 induces stimulatory activation of regulatory T cells, suggesting that IL-2 does not act as an optimal stimulatory factor to stimulate NK cell activation and play a role in tumor therapy. IL-15, a member of the chemokine family, is a factor found in recent years and can be produced by activated mononuclear macrophages, epidermal cells, fibroblasts and other cells, and has many similarities to IL-2 in structure, and can use the beta chain and gamma chain of the IL-2 receptor to bind to target cells, exerting biological activity similar to IL-2, and has the biggest characteristic of not inducing activation of regulatory T cells compared with IL-2; in addition, it can induce B cell proliferation and differentiation, stimulate T cell and NK cell proliferation, induce LAK cell activity, and produce IFN-gamma with IL-12 synergistic stimulation NK cell. Because of these characteristics of IL-15, it has been seen in the line of sight of scientists, and studies have been carried out to confirm that IL-15 has a good feasibility in stimulating NK cells and enhancing NK cells in killing tumors, such as treating a variety of symptoms of acute myelogenous leukemia, advanced non-small cell lung cancer, and pediatric refractory solid tumors, by stimulating NK cells with IL-15. While IL-15 has been shown to stimulate NK cells in the hope of tumor therapy, research has been carried out to break this hope, and evidence has been presented that continued use of IL-15 as an exogenous stimulating factor to stimulate NK cells results in functional failure of NK cells, which greatly limits the functional role of NK cells in anti-tumor therapy.
Based on the defects of the prior art, only a method for more effectively promoting the proliferation of NK cells and keeping the NK cells with lasting functional activity or enabling the NK cells to exert larger functional characteristics is found, and the method is urgent for the NK cell immunotherapy to exert an anti-tumor effect.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a chimeric antigen receptor coding nucleotide, a CAR-NK cell, a construction method and application thereof, and provides a CAR-CD19 molecule and an autocrine function stimulating molecule IL-15-based CAR-CD19-IPS-iNK cell which induces differentiation of pluripotent stem cells (IPS), based on the current situation and deficiency of chimeric antigen receptor T cells and NK cell therapies, which not only overcomes the problem of insufficient cell sources in immunotherapy, but also enables the CAR molecule to be stably expressed in NK cells with high efficiency, and the NK cells expressing the CAR-CD19 molecule can enable the cells to maintain sufficient cell viability in killing tumors and exert durable functional effects by secreting IL-15.
In order to achieve the technical effects, the invention adopts the following technical scheme:
first, the present invention provides a chimeric antigen receptor capable of simultaneously and efficiently expressing a target CD19, wherein the chimeric antigen receptor is an amino acid sequence from N-terminus to C-terminus, and the sequence comprises: the promoter region of EF1a, the CD8aSP signal peptide, the extracellular antibody binding domain FMC63scFv, the CD8 hinge region, the CD28TM transmembrane domain, the intracellular co-stimulatory domains CD28 and 4-1BB and CD3 zeta triplets, the P2A self-cleaving structure and the IL-15 original sequence.
Preferably, the signal peptide region is a CD8aSP signal peptide having an optimized amino acid sequence as set forth in SEQ ID NO:1, the coding nucleotide sequence of which is shown as SEQ ID NO: 2.
Preferably, the extracellular antibody binding domain is FMC63scFv and comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:3, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:5, the amino acid sequence of the Linker is shown as SEQ ID NO:7, the light chain variable region coding nucleotide sequence is shown as SEQ ID NO:4 is shown in the figure; the heavy chain variable region codes for a nucleotide sequence such as a gene sequence shown in SEQ ID NO:6 is shown in the figure; the Linker coding nucleotide is shown as SEQ ID NO: shown at 8.
Preferably, the intracellular hinge region and the transmembrane domain are CD8 hinge region combination CD28TM, and the optimized amino acid sequence of CD8 is shown in SEQ ID NO:9, the optimized amino acid sequence of the CD28TM is shown as SEQ ID NO:11, the coding nucleotide sequence of the CD8 is shown as SEQ ID NO:10, the coding nucleotide sequence of the CD28TM is shown as SEQ ID NO: shown at 12.
Preferably, the intracellular co-stimulatory domain comprises a CD28 and 4-1BB and a CD3 zeta signaling domain, connected in sequence, wherein the amino acid sequences of the optimized CD28, 4-1BB are set forth in SEQ ID NO:13 and SEQ ID NO: 15; the coding nucleotide sequence is shown as SEQ ID NO:14 and SEQ ID NO: shown at 16; the optimized amino acid sequence of the CD3 zeta signal transduction domain is shown as SEQ ID NO:17, the coding nucleotide sequence of which is shown as SEQ ID NO: shown at 18.
Preferably, the chimeric antigen receptor expresses a functional stimulator IL-15, and the IL-15 coding nucleotide sequence is optimally designed, and the optimized amino acid sequence is shown as SEQ ID NO:19, the coding nucleotide sequence is shown as SEQ ID NO: shown at 20.
Preferably, the chimeric antigen receptor comprises a P2A self-cleaving structure, the optimized amino acid sequence of the P2A self-cleaving structure is set forth in SEQ ID NO:21, the coding nucleotide sequence is shown as SEQ ID NO: shown at 22.
In a second aspect, the present invention also provides a CAR-NK cell secreting a functional stimulating factor IL-15, which is a tumor killer cell CAR-CD19-IPS-iNK integrated with multiple functions, obtained by transducing the chimeric antigen receptor provided in the first aspect described above in an immune cell-targeted differentiated IPS-iNK cell as a target cell.
In a third aspect, the present invention also provides the construction of a CAR-NK cell secreting a functional stimulating factor IL-15 as provided in the second aspect above, namely: transduction of immune cells obtained using the chimeric antigen receptor provided in the first aspect, wherein the immune cells are any one of NK cells, hematopoietic stem cells or iPSC cells, and preferably iPSC cells.
In a fourth aspect, the invention also provides the use of a chimeric antigen receptor provided in the first aspect or a CAR-NK cell secreting a functional stimulating factor IL-15 provided in the second aspect for the preparation of a medicament or cell killing agent for preventing, diagnosing or treating an anti-hematological tumor.
Preferably, the hematological neoplasm comprises any one or a combination of at least two of acute myelogenous leukemia, multiple myeloma, chronic lymphocytic leukemia, acute lymphocytic leukemia, non-hodgkin lymphoma, plasmablasts lymphoma or plasmacytoid dendritic cell neoplasm.
The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description more particularly exemplifies illustrative embodiments. At several points in this application, guidance is provided by listing embodiments that may be used in various combinations. In each case, the list given is used as a representative group only and should not be construed as an exclusive list.
Compared with the prior art, the invention has the beneficial effects that:
defects and deficiencies in clinical trials for treatment of tumors for chimeric antigen receptor-NK cell therapies: 1. the source of the adult NK cells is insufficient; 2. the CAR molecules are unevenly transduced in the adult cells, and the functions of the product are unstable; 3. traditional CAR-NK cells have a low function and a short survival after infusion due to effective stimulation defects. In order to solve the key clinical obstacle of NK cell therapy, the invention provides the CAR molecule and the CAR-CD19-IPS-iNK cell which is the autocrine function stimulating molecule IL-15 and is based on induced differentiation of pluripotent stem cells (IPS), which not only overcomes the problem of insufficient cell sources in immunotherapy, but also enables the CAR molecule to be efficiently and stably expressed in NK cells, and the NK cells expressing the CAR molecule can keep enough cell viability in the aspect of killing tumors and exert lasting functional effects by secreting the IL-15.
Drawings
FIG. 1 is a schematic diagram of a chimeric antigen receptor and major components provided in example 1 of the present invention;
FIG. 2 is a flow chart of constructing a CAR-NK cell highly expressing IL-15 according to example 2 of the present invention;
FIG. 3 is a graph showing the results of a pluripotency identification experiment on CARCD19-iPSC cells provided in example 2 of the present invention;
FIG. 4 is a test result of inducing the directional differentiation of CARCD19-iPSC cells into hematopoietic progenitor cells provided in example 2 of the present invention;
FIG. 5 is a graph showing the results of the measurement of the committed differentiation of hematopoietic progenitor cells into CARCD19-NK cells provided in example 2 of the present invention;
FIG. 6 shows the results of measurement of the expression of CARCD19 in NK cells provided in example 3 of the present invention;
FIG. 7 is a graph showing the measurement of IL-15 secretion amount of CarCD19-NK cells according to example 3 of the present invention;
FIG. 8 shows the result of detection of the killing activity of CARCD19-NK cells against K562 tumor cells.
Detailed Description
Embodiments of the technical scheme of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only for more clearly illustrating the technical aspects of the present invention, and thus are merely examples, and are not intended to limit the scope of the present invention.
The specific embodiments of the present invention are listed only as examples of the present invention, and the present invention is not limited to the specific embodiments described below. Any equivalent modifications and substitutions of the embodiments described below will be apparent to those skilled in the art, and are intended to be within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or equipment were commercially available as conventional products without the manufacturer's attention. Numerous specific details are set forth in the following description in order to provide a better understanding of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other embodiments, methods, means, apparatus and steps well known to those skilled in the art have not been described in detail in order to not obscure the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise indicated, all units used in this specification are units of international standard, and the numerical values and numerical ranges appearing in the present invention are understood to include systematic errors unavoidable in industrial production.
Example 1
Referring to FIG. 1, the present embodiment provides a chimeric antigen receptor CAR-CD19 molecule easy to express in immune cells and a method for preparing the same.
Fig. 1 is a schematic diagram of main components of an engineered chimeric antigen receptor CAR-CD19 according to the present invention, and more specifically, a chimeric antigen receptor according to the present embodiment includes, in order from N-terminal to C-terminal:
the promoter region of EF1a, the CD8aSP signal peptide, the extracellular antibody binding domain FMC63scFv, the CD8 hinge region, the CD28TM transmembrane domain, the intracellular co-stimulatory domains CD28 and 4-1BB and CD3 zeta triplets, the P2A self-cleaving structure and the IL-15 element, specifically the sequences of the elements that make up the chimeric antigen receptor are as follows:
the sequence of the signal peptide region of CD8aSP is set forth in SEQ ID NO:1, the coding nucleotide sequence is shown as SEQ ID NO:2 is shown in the figure;
extracellular antibody binding domain Fmc VL comprises a light chain variable region having an amino acid sequence set forth in SEQ ID NO:3, the coding nucleotide sequence is shown as SEQ ID NO:4 is shown in the figure; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:5, the coding nucleotide sequence is shown as SEQ ID NO:6 is shown in the figure; the amino acid sequence of the Linker is shown as SEQ ID NO:7, the coding nucleotide sequence is shown as SEQ ID NO:8.
the hinge region is CD8 walk, and the amino acid sequence is shown in SEQ ID NO:9, the coding nucleotide sequence is shown as SEQ ID NO:10 is shown in the figure;
the transmembrane domain is CD28TM, and the amino acid sequence of the transmembrane domain is shown in SEQ ID NO:11, the coding nucleotide sequence is shown as SEQ ID NO: shown at 12;
the intracellular co-stimulatory domain comprises CD28 and 4-1BB connected in sequence, wherein the amino acid sequences of CD28 and 4-1BB are respectively shown in SEQ ID NO:13 and SEQ ID NO:15, the coding nucleotide sequence is shown as SEQ ID NO:14 and SEQ ID NO: shown at 16;
the amino acid sequence of the CD3 zeta signal transduction domain is shown in SEQ ID NO:17, the coding nucleotide sequence is shown as SEQ ID NO: shown at 18;
the amino acid sequence of the P2A self-shearing structure is shown in SEQ ID NO:21, the coding nucleotide sequence is shown as SEQ ID NO: shown at 22;
the amino acid sequence of the IL-15 element is shown in SEQ ID NO:19, the coding nucleotide sequence is shown as SEQ ID NO: shown at 20.
In addition, the present example further predicts the DNA secondary structure of the modified CAR-CD19 molecule of the present invention and the conventional CAR molecule through a RNA folder web serverRNAfold web server (univie. Ac. At) website, and it is known from the secondary structure prediction and analysis results that the modified CAR-CD19 molecule of the present invention is significantly different from the conventional CAR molecule, and it is known that the CARCD19 molecule of the present invention is more easily expressed in cells after website analysis.
Example 2
The embodiment provides a CAR-NK cell capable of highly expressing IL-15, please refer to FIG. 2, the construction flow is as follows:
s1: vector construction
Transferring the chimeric antigen receptor synthesized in the example 1 into a piggyBac transposon vector according to the manufacturer's proposal to construct a CAR CD19-piggyBac vector, wherein the constructed CAR CD19-piggyBac vector is used for subsequent transduction of IPSC cells;
s2: iPSC cell transduction
iPSC cells were plated in 6-well plates for culturing when the iPSC cell density reached 80% for transduction.
And (3) transfecting the CAR CD19-piggyBac vector into the iPSC cells by using a Lipofectamine2000 transfection mode, screening the transfected iPSC cells by using puromycin after 72 hours, and obtaining CARCD19-iPSC cells.
S3, sequencing and identifying monoclonal cells
Selecting monoclonal cells for sequencing identification, sequencing by the engine family organism limited company, generating a sequencing result in the sequencing process, and sequencing identification results of the CARCD19 chimeric antigen receptor in the CARCD19-iPSC cells are shown as SEQ ID NO: 23.
After BLASTA comparison and analysis, the sequencing result shows that the sequence is consistent with the pre-designed CARCD19 sequence, so that the CARCD19 can be successfully transduced into iPSC cells, and the construction of the CARCD19-iPSC cells is successful and can be continuously used for subsequent differentiation research.
S4: immunofluorescence staining and karyotype analysis for identifying pluripotency of CARCD19-iPS
S41: immunofluorescence analysis
Cells were seeded at 30 ten thousand per well in 6-well plates with Metragel matrigel and placed in 5% CO 2 And 37 ℃ in an incubator, during which the liquid is changed every day. When the cell fusion reached 80%, the cells were washed 3 times with PBS, followed by fixing the cells 10-fold with 1ml of 4% paraformaldehyde at room temperature20 After the completion of the permeation, the cells were washed 3 times with 1ml of PBS for 3-5min each, blocked with BSA at room temperature for 1h, and incubated overnight at 4℃with the addition of OCT4 primary antibody. After the incubation, the cells were washed 3 times with PBS for 5min each. The secondary antibody and 1ul of DAPI were added and incubated at room temperature in the dark for 1h, after the incubation was completed, the cells were washed 3 times with PBS, photographed with a microscope and analyzed for OCT4 expression, and the experimental results are shown in FIG. 3.
The results of the above-described fig. 3 show that: OCT4 is stably expressed in iPSC cell nuclei, the cell nuclei are dyed green under fluorescent irradiation, and the cell nuclei can be seen to be dyed blue at the same position of green fluorescence after being counterstained by DAPI, so that the construction of CARCD19-iPSC cells is successful, and the iPSC cells can maintain the pluripotency.
S42: genetic analysis
Cells 1h were treated with 0.1 μg/mL Colcemid at 37 ℃, cells at the exponential phase were captured in the metaphase and harvested according to standard methods. Hypotonic treatment was performed with 0.075M KCl solution for 20 minutes at room temperature. The cell-fixed slides were subjected to trypsin-giemsa banding to identify single metaphase chromosomes. Representative genomic images were obtained for karyotyping.
According to the karyotype analysis of the iPSC cells, the iPSC cells transduced with CARCD19 still show normal chromosome karyotype, are in a diploid karyotype state, and have no chromosome abnormal condition. From this, it was confirmed that transduction of CARCD19 had no effect on the karyotype of iPSC cells.
Immunofluorescence and karyotype analysis experiments prove that the transduction of CARCD19 does not affect the pluripotency and karyotype change of the iPSC cells, and the functions of the iPSC cells can be reserved.
S5: CARCD19-iPSC cell induced differentiation
The CARCD19-iPSC cells obtained are subjected to expansion culture, and then induced to differentiate into hematopoietic progenitor cells, and then subjected to directed differentiation into CARCD19-NK cells.
S51 inducing directed differentiation of CARCD19-iPSC cells into hematopoietic progenitor cells
The CARCD19-iPSC cells were seeded at a rate of 20 ten thousand per well. After the cells were grown to 80% confluence, the medium was aspirated, the cells were washed once with 1ml of PBS, then incubated for 5 minutes at 37 ℃ with 0.5ml of digest, the digestion was stopped with 0.5ml of complete medium, then the cell aggregates were carefully separated into individual cells using a 1mL pipette, the cells were transferred to a 15ml conical tube with micropipettes, additional 5mL of complete medium was added, and the cell aggregates were removed by a 70 μm screen. 300g, 5min, removing the supernatant, washing the cells once with 5mL PBS, centrifuging the cells again 300g, 5min, removing the supernatant, and re-suspending the cells using 1mL APEL medium.
After cell counting, cells were resuspended to 80000 cells/mL using APEL medium containing cytokines (SCF: 40 ng/mL, BMP4: 20ng/mL, VEGF:20 ng/mL; 10. Mu.M ROCKi (Y-27632), followed by seeding the cells into round bottom 96 well plates, 100. Mu.L (8000 cells) per well 300g, 5min centrifugation 96 well plates, and then transferring the 96 well plates to 37℃5% CO 2 Culturing in an incubator for 6 days. After 6 days, the plates were removed, and a portion of the cells were collected for CD34, CD43 flow assay, the results of which are shown in FIG. 4, and the remaining EBs were used in the subsequent NK differentiation experiments to prepare CARCD19-NK cells.
S52 directed differentiation of hematopoietic progenitor cells into CARCD19-NK cells
The plates were plated with 6-well plates using 0.1% gelatin and placed in an incubator for 4 hours. The induced differentiation medium was prepared according to the following ingredients and addition ratios:
56.6% DMEM+Glutamax-1,28.3% F12+Glutamax-1,15% human AB serum, 1%p/s diab, 2mM L-gluta-mine L-glutamine, 1uM P-mercaptoethanol P-mercaptoethanol, 5ng/ml sodium selenite sodium selenite, 50uM ethane (MP) aminoethanol, 2mg/L ascoribe acid ascorbic acid, 5ng/ml IL-3,20ng/ml SCF,20ng/ml IL-7,10ng/ml IL-15,10ng/ml FLT3L. After the culture medium is prepared, EB cells in 96 holes are picked into a hole without gelatin, and the cells are washed twice with 3-5ml of prepared induced differentiation culture medium. Gelatin is sucked and removed, 2ml of induction differentiation medium is added, and the cleaned EB cells are picked into a 6-hole plate one by one and then placed into an incubator for culture. After the cells were cultured for 7 days, IL-3 was withdrawn and changed, followed by changing the liquid every 3-5 days, during which the state of the differentiated cells was photographed and preserved, and the experimental results are shown in FIG. 5. In addition, when the cells were cultured until 32 days, part of the cells were collected for CD56 flow assay while changing the liquid, and after the cells were harvested and differentiated after continuing to culture until 39 days for CD56 flow assay, the experimental result showed that CARCD19-NK preparation was successful.
Example 3
This example further examined CARCD19-NK cells obtained in example 2 above, including the expression of CARCD19 in NK cells and the secretion of IL-15 by CARCD19-NK cells.
3.1 Detection of expression of CARCD19 in NK cells
Post-differentiation CARCD19-NK cells expression of CARCD19 was examined by flow cytometry: the cells were collected and washed, then the differentiated cells were prepared into cell suspensions, 100. Mu.l of the cell suspensions were added to each 15mL centrifuge tube, followed by 0.1-10. Mu.g/mL of CD19 antibody, incubated at room temperature for 30 minutes in the absence of light, followed by washing the cells three times with PBS and centrifuging at 400 g for 5 minutes, finally the cells were resuspended with 1mL of PBS, and the expression level of CD19 was detected in a flow cytometer, and the experimental results are shown in FIG. 6.
The experimental results show that the CD19 is highly expressed in the CARCD19-NK cells, which ensures that the differentiated CARCD19-NK cells can target tumor cells taking the CD19 as an antigen.
3.2 CARCD19-NK cell IL-15 expression level detection
Cell samples were collected, centrifuged to obtain supernatant for IL-15 detection, treated samples were prepared according to the kit instructions, and finally absorbance OD values were measured at 450nm wavelength, and the expression level of IL-15 was calculated, and the experimental results were shown in FIG. 7.
The above experimental results show that the amount of IL-15 secreted by the CARCD19-NK of the first generation is equivalent to that of IL-15 secreted by NK cells which do not carry out CARCD19, and the secretion amount is small, the IL-15 secreted by the CARCD19-NK of the second generation is obviously increased compared with that of normal group cells, and the IL-15 secreted by the CARCD19-NK of the third generation is obviously increased compared with that of the CARCD19-NK of the second generation.
Example 4
1x10 in 96 well plates ^4 K562 cells with fluorescent label were placed in a medium containing 5% CO 2 K562 cells were allowed to adhere to the wall in an incubator at 37℃for 4 hours. The CARCD19-NK cells were then accessed according to different potency target ratios, according to 1: 1. 5: 1. 10: 1. 20:1 by 1 to be added respectively ^4 、5x10 ^4 、10x10 ^4 、20x10 ^4 And CARCD19-NK cells. K562 cells with fluorescence were counted 24h after the addition of CARCD19-NK cells using a cytometer.
The experimental results are shown in fig. 8, and statistical analysis is performed based on the count results.
The counting result shows that the CARCD19-NK cells have remarkable killing effect on K562 cells, and the better the killing effect is along with the increase of the effective target ratio, the best killing effect is achieved when the effective target ratio is 10:1 and 20:1, and the killing effect of the two effective target ratios is equivalent.
The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention. The technology, shape, and construction parts of the present invention, which are not described in detail, are known in the art.
Claims (4)
1. A chimeric antigen receptor encoding nucleotide sequence comprising, in order: the promoter region of EF1a, the CD8aSP signal peptide, the extracellular antibody binding domain FMC63scFv, the CD8 hinge region, the CD28TM transmembrane domain, the intracellular co-stimulatory domains CD28 and 4-1BB and CD3 zeta triplets, the P2A self-cleaving structure and the IL-15 element sequence, and the nucleic acid sequence of said chimeric antigen receptor is as set forth in SEQ ID NO: 23.
2. A CAR-NK cell that secretes the functional stimulatory factor IL-15, characterized in that: an immune cell-oriented differentiated IPS-iNK cell is taken as a target cell, and the coding nucleotide of the chimeric antigen receptor as claimed in claim 1 is transduced in the target cell, so that a tumor killing cell CAR-CD19-IPS-iNK is obtained.
3. The method of constructing CAR-NK cells secreting the functional stimulating factor IL-15 according to claim 2, wherein said cells are obtained by transducing immune cells, which are any one of NK cells, hematopoietic stem cells or iPSC cells, with the nucleotide encoding the chimeric antigen receptor according to claim 1.
4. Use of a chimeric antigen receptor-encoding nucleotide according to claim 1, or a CAR-NK cell secreting a functional stimulatory factor IL-15 according to claim 2, for the preparation of a medicament for the prevention, diagnosis or treatment of CD19 expressing hematological tumors or a killer of CD19 expressing cells.
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