CN111454372A - Construction and application of NKG2D-ACE2CAR-NK cell secreting super I L15 - Google Patents
Construction and application of NKG2D-ACE2CAR-NK cell secreting super I L15 Download PDFInfo
- Publication number
- CN111454372A CN111454372A CN202010390688.4A CN202010390688A CN111454372A CN 111454372 A CN111454372 A CN 111454372A CN 202010390688 A CN202010390688 A CN 202010390688A CN 111454372 A CN111454372 A CN 111454372A
- Authority
- CN
- China
- Prior art keywords
- cells
- nkg2d
- virus
- sars
- car
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- 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]
- C07K14/5443—IL-15
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/7056—Lectin superfamily, e.g. CD23, CD72
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- 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
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/485—Exopeptidases (3.4.11-3.4.19)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/17—Metallocarboxypeptidases (3.4.17)
- C12Y304/17023—Angiotensin-converting enzyme 2 (3.4.17.23)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
-
- 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
- C12N2510/00—Genetically modified cells
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Vascular Medicine (AREA)
- Hematology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a construction method of NKG2D-ACE2CAR-NK cells secreting super I L and application thereof.A expression vector mainly comprises NKG2D extracellular segment, ACE2 extracellular segment, 4-1BB, CD3 zeta and I L R α -I L fusion protein, NKG2D is an activating receptor of the NK cells and a ligand (MICA, MICB, U L BP1-6) for recognizing surfaces of virus infected cells or tumor cells, ACE2 is a receptor of SARS-CoV-2 and is combined with S protein of a virus envelope, the NK cells modified by CAR are proved to break through the limitation of inhibitory receptors to activate the NK cells so as to enhance the specific killing effect of the NK cells on target cells without serious toxic and side effects, an I L super agonist is increased by 20 times compared with the activity of natural I L in vivo, the pharmacokinetics characteristic is improved, the ACE-NK cells have enhanced durable CAR and ACE activity, the antibody target cells are enhanced by the synergistic effect of SARS-CD-19 virus-CD 2V-19 virus-eliminating protein and the antibody of SARS-CD 2 virus-CD 462 cell surface is enhanced by the synergistic effect of SARS-CD 2.
Description
Technical Field
The invention belongs to the field of biotechnology engineering, and particularly relates to construction of NKG2D-ACE2CAR NK cells and application of the NK cells in preventing and treating COVID-19.
Background
At present, no medicine/therapy for specifically clearing SARS-CoV-2 virus exists, and particularly no specific cure method for severe COVID-19 exists. In view of the clinical observation that there is a clear correlation between leucopenia and lymphopenia and the disease deterioration and even death, some immunopotentiators are emphasized in the clinical treatment scheme for patients with severe or critical illness so as to improve immunity and achieve the purpose of effectively controlling the replication of SARS-CoV-2 virus.
CAR-NK cells, also called upgraded NK cells, are modified by genetic engineering means to express Chimeric Antigen Receptors (CAR), and antibodies (or receptors) recognizing surface antigens of target cells (such as virus-infected cells and cancer cells) are connected with signal molecules required for activating immune cells, so that the limitation of inhibitory receptors can be broken through to activate NK cells, and thus the specific killing of the NK cells on the target cells is enhanced. More and more clinical trial researches show that the CAR-NK has good anti-tumor activity and high safety (no serious toxic and side effects such as cytokine storm and the like), can be produced in batches (being a universal and ready-to-use product), and has extremely wide application prospect.
NKG2D is the activating receptor of NK cell, can recognize MHC-I molecule, and is important for natural immunity, NKG2D can recognize 8 different ligands expressed on the surface of target cell, so it can target several different target cells at the same time, NKG2D ligand (NKG2D L) is not expressed or its expression is very little in normal cell, but when the cell is infected or cancerated, the expression of these ligands will be greatly increased, thus it becomes the ideal target of immunotherapy.
The angiotensin converting enzyme 2(ACE2) gene maps to the X chromosome and includes 18 exons and encodes a type I transmembrane glycoprotein consisting of 805 amino acid residues. Expressed in lung, kidney, stomach, intestine, heart, bone marrow, spleen, liver, retina, placenta, ovary, brain tissue, testis, coronary artery, vein, fat tissue, endothelial cell, macrophage and other tissue cells. ACE2 was localized to the apical end of the cell, and no expression of ACE2 was detected on the lateral and basal sides. ACE2 is a receptor for SARS-CoV-2 virus to enter cells by specific binding of S protein, and has relieving effect on acute respiratory distress syndrome.
After adoptive transfusion into the body, the NK cells have very short survival time without continuous support of cytokines such as I L-15, and compared with natural non-compound I L-15 in vivo, the activity of super I L-15 (sI L-15/I L-15R α sushi domain fusion protein) is increased by nearly 20 times, and the NK cells have improved pharmacokinetic properties, longer persistence in lymphatic tissues and enhanced target cell killing activity.
Disclosure of Invention
The NKG2D-ACE2CAR-NK cell (secreting super I L) from umbilical cord blood is prepared and utilized, S protein of SARS-COV-2 virus and NKG2D L on the surface of an infected cell are respectively targeted by ACE2 and NKG2D, and the strong synergistic effect of super I L is cooperated to specifically remove SARS-COV-2 virus particles and the infected cell, so that a safe and special-effect cell therapy is provided for COVID-19.
The method comprises the steps of constructing a plenti-NKG2D-ACE 2CAR (super I L15 secreted) lentiviral vector by a genetic engineering technology, preparing and utilizing high-titer and high-purity lentiviruses to transduce NK cells, respectively detecting the expression efficiency of NKG2D and ACE2 by flow type after five days of culture, and verifying the specific killing effect of NKG2D-ACE2CAR-NK cells on target cells expressing SARS-CoV-2-S or/and NKG2D L, wherein the plenti-NKG2D-ACE 2CAR lentiviral vector has the following characteristics:
CAR-NK therapy is safe: severe toxic and side effects such as cytokine storm and the like generally do not occur.
2. The double-target combined therapy is that S protein of SARS-CoV-2 virus and NKG2D L-NK cells on the surface of infected cells can be respectively targeted by ACE2 and NKG2D, and SARS-CoV-2 virus particles and infected cells can be effectively eliminated.
The CAR-NK cell has strong and durable activity, and the CAR-NK cell can overcome the immunosuppressive microenvironment in the body to effectively expand by virtue of the strong synergistic effect of super I L15.
Decoy effect of CAR-NK cells: the ACE2 competitively inhibits SARS-COV-2 virus particles from infecting important organ tissue cells such as II type alveolar epithelial cells, and causes SARS-COV-2 virus entering CAR-NK cells to have frustrating infection, thereby infectious virus particles cannot be produced.
The regulatory effect of CAR-NK cells on adaptive immunity regulates the function of B cells, NK-T and CD 8T by overexpressing membrane receptors such as CD16 and secreting cytokines (e.g., super I L15).
CAR-NK cells have versatility: is a ready-to-use cell therapy product, is suitable for different individuals, and can be frozen for standby use for a long time.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is within the scope of the present invention for those skilled in the art to obtain other drawings based on the drawings without inventive exercise.
FIG. 1 is a schematic structural diagram of NKG2D-ACE2 dual target CAR;
fig. 2 is a slow virus packaging system used for preparing NKG2D-ACE2CAR virus particles, NK cells are infected with MOI 40, and the expression of NKG2D and ACE2 on the surface of NK cells is detected by flow cytometry;
FIG. 3 is a schematic diagram of a membrane anchoring S1-luc-GFP structure and a flow detection method for expression of Raji-S1-luc-GFP cell surface S1 protein and a GFP fluorescent reporter gene, which indicates that the construction of a Raji-S1-luc-GFP cell strain is successful;
FIG. 4 shows that the in vitro killing activity of NKG2D CAR-NK cells on human lung cancer cell line A549 expressing luciferase under different effective target ratio conditions is verified by a luciferase method.
FIG. 5 shows that the in vitro killing activity of NKG2D-ACE2CAR-NK cells on Raji-S1-luc-GFP cells expressing luciferase is verified by a luciferase method under different effective target ratio conditions, and the specific recognition of S1 protein is proved.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.
Examples
The main experimental materials:
NdeI-HF, MluI-HF (NEB), seamless clonase (and Yuan biol.), high fidelity Prime GX L STAR enzyme (TAKARA), TransStbl3 competent cells (all-grass Biotech Co., Ltd.), Plasmid Mini Kit I (OMEGA),
plasmid Maxi Kit (QIAGEN), DMEM, RPMI-1640, Opti-MEM medium, Gibco FBS (Thermo Fisher Scientific), Sanger sequencing (Shanghai Sangnie Biotech Co., Ltd.), yeast powder, peptone, EDTA, NaOH (Shanghai Biotech Co., Ltd.), primers (Jiangsu jin Zhi Biotech Co., Ltd.).
Construction of recombinant plasmid
① construction Plenti-NKG2D-ACE2-CAR was synthesized by Sovizhou Jinzhi Biotechnology Ltd, using NdeI-HF and MluI-HF double digestion Plenti-EF1a- -MCS vector, and synthetic NKG2D-ACE2CAR gene, both carrying NdeI and MluI cohesive ends, with the reaction conditions of 37 ℃ for 3h and 65 ℃ for 20min, and the digestion system as in Table 3. the digestion product was subjected to 1% agarose gel electrophoresis to obtain vector fragments, then Plynti vector fragments and NKG2D-ACE2CAR were recovered with XYGENE gel recovery kit (see Table 1), the concentration and purity were determined, the vector fragments and the target fragments were cloned by T4 cloning system (Table 2), 16-24 min at 16 ℃, 10min at 65 ℃, then transformed (after placing the reaction product on ice for 5min, they were transferred to St bl3, placed on ice 30min, then 5min, 5min at 5000 ℃ for 10min, removed from 65 ℃ for 10min, after centrifugation, the remaining reaction product was placed on ice for 5rpm, after centrifugation, the strain was added to 5rpm, the strain was cultured for 5rpm, the strain was added to 5rpm, and the strain was added to the strain, and the strain was added to the.
TABLE 1 gum recovery
TABLE 2 cloning System of T4
TABLE 3 restriction enzyme cleavage System
II, transducing 293T cells by utilizing Plenti vector plasmid and helper plasmid to package lentivirus, and transfecting the packaged lentivirus into Jurkat cells to calculate virus titer
(1) 293T cells were cultured in 15cm cell dishes and 60. mu.g PEI was resuspended in 1.5ml PBS when 293T cells reached 70% of full field, and 1.5ml PBS was resuspended with 20. mu.g total mass of Plenti vector plasmid and helper plasmid;
(2) standing at room temperature for 5min, adding the PBS-PEI mixed solution into the PBS-DNA mixed solution, and standing at room temperature for 20 min;
(3) preparing OPTI-DMEM full culture in an incubator at 37 ℃ for rewarming, sucking out a DMEM original culture medium in 293T cells, and adding the OPTI-DMEM into the 293T cells along the dish wall;
(4) adding the PEI-DNA-PBS mixed solution into a culture dish, and culturing for 48h at 37 ℃;
(5) collecting the lentivirus in the supernatant in a 50ml centrifuge tube, adding 20ml of culture medium, and incubating for 24h to collect the virus within 72 h;
(6) centrifuging at 1500rpm for 5min to remove cell debris, or filtering with 0.45um filter, centrifuging for 12-14 hr at 4 deg.C to concentrate virus;
(7) removing supernatant, adding VIVO or AIM-V (preferably 1% HEPES) at ratio of 1: 200-1: 400, and resuspending virus;
(8) the virus is subpackaged in 1.5ml Ep tubes, the Ep tubes are preserved at the temperature of-80 ℃, repeated freeze thawing is avoided (the titer is reduced by one order of magnitude by freeze thawing), and a little virus is used for the next virus titer detection experiment;
(9) centrifuging Jurkat cells at 1500rpm for 5min, discarding the supernatant, resuspending the cells in 1ml 1640 medium, and counting;
(10) add 0.5 × 10 to 96-well plates6Jurkat cells, adding viruses in a gradient proportion of 1: 50, 1: 500, 1: 1000, 1: 2000 and the like, and supplementing a culture medium until the total volume of each hole is 200 ul;
(11) 0.1ul polybrene B protein per well for promoting transduction (0.1ul/200ul system);
(12) centrifuging a 96-well plate at the temperature of 32 ℃ for 90min at 1200g, and incubating the plate in an incubator at the temperature of 37 ℃ for 4 h;
(13) the Jurkat cell suspension of each well of a 96-well plate is blown, evenly mixed, transferred to a 1.5ml Ep tube, centrifuged at 1500rpm for 5min, the supernatant is discarded, resuspended by 1ml 1640 full culture and transferred to a 24-well plate, and then expanded and cultured for 48h at 37 ℃.
And thirdly, establishing a Raji-S1-luc-GFP cell line.
Raji-S1-luc-GFP cells respectively transduce lentiviruses expressing S1-luc-GFP, observe the GFP green fluorescence expression condition the next day, sort GFP positive cells by flow cytometry and further amplify, and finally flow analyze the S1 protein on the cell strain surface and the expression of GFP, and the result is shown in figure 3.
Fifth, detecting the killing effect of CAR-NK cells on target cells by a luciferase method
(1) Culturing A549-L uc-GFP/Raji-S1-luc-GFP cells to logarithmic growth state, taking a certain number of cells, centrifuging and precipitating, and counting;
(2) adding 1 × 10 into 96-hole flat-bottom opaque white board4A549-L uc-GFP/Raji-S1-luc-GFP cells, the culture medium is supplemented to 100u L;
(3) NKG2D-ACE2-CAR NK cells and A549-L uc-GFP/Raji-S1-luc-GFP cells are set to have different effective target ratios, and corresponding CAR-NK cells are added into each well for mixed culture;
(4) setting a Mock cell group, and adding the NK cells which are not transduced with the virus, wherein the number of the NK cells is the same as that of the CAR-NK cells in the step (3);
(5) two controls are set simultaneously, the negative control is A549-L uc-GFP/Raji-S1-luc-GFP cells are cultured in a culture medium, the positive control is that 2.5 percent of Triton-X100 is added into the culture medium, both the positive control and the negative control are not added with Mock cells or CAR-NK cells, and the Mock cells or the CAR-NK cells are used as the minimum and maximum background values of cell killing, namely Kmin and Kmax.
(6) After 24 hours of culture, centrifuging the cells for 5min at 1500rpm of a 96-well plate, discarding supernatant, washing the cells once with a culture medium, and then resuspending the cells;
(7) adding 0.5mM D-fluorescein into each hole, standing for 10min in a dark place, and detecting the fluorescence intensity by using a chemiluminescence mode (L fluorometric Measurement) on an enzyme labeling instrument, wherein the detection time of each hole is 1000 ms;
(8) the fluorescence intensity value K of each well is counted, and the killing efficiency% of the CAR-NK cells and the Mock NK cells to A549-L uc-GFP/Raji-S1-luc-GFP cells is compared, wherein the formula is that the killing efficiency% is (Kmin-K)/(Kmin-Kmax) × 100%, and the result is shown in FIG. 4 and FIG. 5.
The above disclosure is only a preferred embodiment of the present invention, and certainly should not be taken as limiting the scope of the invention, which is defined by the claims and their equivalents.
Claims (3)
1. The coded chimeric antigen receptor is characterized by comprising NKG2D extracellular segment, ACE2 extracellular segment, 4-1BB, CD3 zeta and I L15R α -I L15 thaws protein, and the structure of the chimeric antigen receptor is coded by an amino acid sequence SEQ ID NO. 1.
NKG2D-ACE2CAR-NK cells characterized by: which expresses a chimeric antigen receptor according to claim 1.
3. A gene for expressing a fourth generation chimeric antigen receptor forming NKG2D-ACE2-4-1BB-CD3 zeta-P2A-I L15R α -I L15 is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010390688.4A CN111454372A (en) | 2020-05-08 | 2020-05-08 | Construction and application of NKG2D-ACE2CAR-NK cell secreting super I L15 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010390688.4A CN111454372A (en) | 2020-05-08 | 2020-05-08 | Construction and application of NKG2D-ACE2CAR-NK cell secreting super I L15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111454372A true CN111454372A (en) | 2020-07-28 |
Family
ID=71674691
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010390688.4A Pending CN111454372A (en) | 2020-05-08 | 2020-05-08 | Construction and application of NKG2D-ACE2CAR-NK cell secreting super I L15 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111454372A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112098660A (en) * | 2020-11-03 | 2020-12-18 | 北京百普赛斯生物科技股份有限公司 | Novel coronavirus neutralizing antibody detection kit |
| WO2021201679A1 (en) * | 2020-04-01 | 2021-10-07 | Kiadis Pharma Intellectual Property B.V. | Compositions and methods targeting coronaviruses |
| WO2022035998A1 (en) * | 2020-08-11 | 2022-02-17 | City Of Hope | Compositions and uses of sars-cov-2 targeted chimeric antigen receptor modified nk cells |
| WO2022114798A1 (en) * | 2020-11-27 | 2022-06-02 | 한국생명공학연구원 | Composition for increasing natural killer cell activity and preventing and treating infectious diseases and cancer using peptide derived from sars-cov-2 s protein that binds to nkg2d receptor on natural killer cells |
| CN114569708A (en) * | 2020-12-02 | 2022-06-03 | 四川大学华西医院 | Application of NKG2D CAR-immune cell in anti-aging |
| CN115873803A (en) * | 2022-11-28 | 2023-03-31 | 上海恩凯细胞技术有限公司 | Method and application of improving NK cell survival and anti-tumor activity |
| CN115947869A (en) * | 2022-11-28 | 2023-04-11 | 广州佰芮慷生物科技有限公司 | Chimeric antigen receptor targeting human cytomegalovirus, CAR-NK cell and application |
| WO2024099265A1 (en) * | 2022-11-07 | 2024-05-16 | 上海先博生物科技有限公司 | Engineered chimeric antigen receptor immune cell and use thereof |
| CN119265201A (en) * | 2024-08-23 | 2025-01-07 | 中国人民解放军海军军医大学第一附属医院 | NKG2DL-FAP dual-target CAR-NK cells encoding genes, proteins, and expressing IL-15, and construction methods and uses thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105647873A (en) * | 2016-03-14 | 2016-06-08 | 紫程瑞生会(北京)生物技术发展有限公司 | Preparation method and kit of bispecific chimeric antigen receptor gene modified natural killer cells |
| CN110922490A (en) * | 2019-12-05 | 2020-03-27 | 浙江启新生物技术有限公司 | CAR expression vector secreting interleukin 7 and chemokine 21 and application thereof |
-
2020
- 2020-05-08 CN CN202010390688.4A patent/CN111454372A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105647873A (en) * | 2016-03-14 | 2016-06-08 | 紫程瑞生会(北京)生物技术发展有限公司 | Preparation method and kit of bispecific chimeric antigen receptor gene modified natural killer cells |
| CN110922490A (en) * | 2019-12-05 | 2020-03-27 | 浙江启新生物技术有限公司 | CAR expression vector secreting interleukin 7 and chemokine 21 and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| JIMIN GAO: "A Phase I/II Study of Universal Off-the-shelf NKG2D-ACE2 CAR-NK Cells for Therapy of COVID-19" * |
| KAI-PING HAN ET AL: "IL-15:IL-15 receptor alpha superagonist complex: High-level co-expression in recombinant mammalian cells, purification and characterization" * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021201679A1 (en) * | 2020-04-01 | 2021-10-07 | Kiadis Pharma Intellectual Property B.V. | Compositions and methods targeting coronaviruses |
| WO2022035998A1 (en) * | 2020-08-11 | 2022-02-17 | City Of Hope | Compositions and uses of sars-cov-2 targeted chimeric antigen receptor modified nk cells |
| CN112098660A (en) * | 2020-11-03 | 2020-12-18 | 北京百普赛斯生物科技股份有限公司 | Novel coronavirus neutralizing antibody detection kit |
| CN112098660B (en) * | 2020-11-03 | 2021-02-09 | 北京百普赛斯生物科技股份有限公司 | Novel coronavirus neutralizing antibody detection kit |
| WO2022114798A1 (en) * | 2020-11-27 | 2022-06-02 | 한국생명공학연구원 | Composition for increasing natural killer cell activity and preventing and treating infectious diseases and cancer using peptide derived from sars-cov-2 s protein that binds to nkg2d receptor on natural killer cells |
| CN114569708A (en) * | 2020-12-02 | 2022-06-03 | 四川大学华西医院 | Application of NKG2D CAR-immune cell in anti-aging |
| WO2024099265A1 (en) * | 2022-11-07 | 2024-05-16 | 上海先博生物科技有限公司 | Engineered chimeric antigen receptor immune cell and use thereof |
| CN115873803A (en) * | 2022-11-28 | 2023-03-31 | 上海恩凯细胞技术有限公司 | Method and application of improving NK cell survival and anti-tumor activity |
| CN115947869A (en) * | 2022-11-28 | 2023-04-11 | 广州佰芮慷生物科技有限公司 | Chimeric antigen receptor targeting human cytomegalovirus, CAR-NK cell and application |
| CN115947869B (en) * | 2022-11-28 | 2023-12-12 | 广州佰芮慷生物科技有限公司 | Chimeric antigen receptor targeting human cytomegalovirus, CAR-NK cell and application |
| WO2024113649A1 (en) * | 2022-11-28 | 2024-06-06 | 上海恩凯细胞技术有限公司 | Method for improving viability and anti-tumor activity of nk cells and use thereof |
| CN119265201A (en) * | 2024-08-23 | 2025-01-07 | 中国人民解放军海军军医大学第一附属医院 | NKG2DL-FAP dual-target CAR-NK cells encoding genes, proteins, and expressing IL-15, and construction methods and uses thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111454372A (en) | Construction and application of NKG2D-ACE2CAR-NK cell secreting super I L15 | |
| CN106279434B (en) | Engineered CD20 targeted NKT cell and preparation method and application thereof | |
| CN107034237B (en) | A kind of CAR-NK cells and the preparation method and application thereof | |
| CN110684730B (en) | Preparation method for efficiently amplifying NK cells by using trophoblasts | |
| CN105368859B (en) | A kind of chimeric antigen receptor hCD87-CAR, lentivirus and plasmid carrying hCD87-CAR gene structure and application thereof | |
| CN112391414A (en) | HER 2-targeted CAR-T expression vector and construction and application thereof | |
| CN106978442B (en) | Preparation method of chimeric antigen receptor T cell | |
| CN105924528B (en) | Chimeric antigen receptor and its gene and recombinant expression vector, CARMSLN-NKT cell and its preparation method and application | |
| CN110760480A (en) | Anti-tumor NK (Natural killer) cell and preparation method thereof | |
| CN107793483B (en) | Chimeric antigen receptor and its gene and recombinant expression carrier, CARMSLN-NKT cell and its preparation method and application | |
| CN111484559B (en) | Construction and application of third generation NKG2D chimeric antigen receptor T or NK cell | |
| WO2021093250A1 (en) | Fgfr4-targeted single-chain antibody, chimeric antigen receptor, chimeric antigen receptor t cell, preparation method therefor and application thereof | |
| CN105384822A (en) | Chimeric antigen receptor and gene and recombinant expression vector thereof, engineered CD138 targeting NKT cell and application thereof | |
| CN111139222A (en) | A kind of recombinant mesenchymal stem cell and its preparation method and use | |
| US20240342266A1 (en) | Method for treating tumor with combination of exogenous antigen and therapeutic agent | |
| CN105384823A (en) | Chimeric antigen receptor and gene and recombinant expression vector thereof, engineered CD33 targeting NKT cell and application thereof | |
| WO2021036245A1 (en) | Chimeric antigen receptor t cell carrying safety switch and targeting egfrvⅲ, and preparation method therefor and use thereof | |
| CN103820483B (en) | Ovarian cancer stem cell vaccine of express alpha-gal epi-position and preparation method thereof | |
| CN105920592B (en) | Application of the CARHER1-NKT cell in the preparation that preparation is used for treatment of advanced HER1 positive lung cancer | |
| CN102533657B (en) | Human 41 type adenovirus (Ad41) packaging cell line and application thereof | |
| WO2021036246A1 (en) | Chimeric antigen receptor t cell targeting egfrviii and interfering with il-6 expression, preparation method therefor and application thereof | |
| WO2021036247A1 (en) | Chimeric antigen receptor t cell targeting her2 and interfering with il-6 expression, preparation method therefor and use thereof | |
| CN114540422A (en) | Preparation and application of mesenchymal stem cell exosome for delivering RNA (ribonucleic acid) medicament to damaged part in targeted manner | |
| CN107245501A (en) | PGC-1 alpha gene overexpression lentiviral vector, PGC-1 alpha lentivirus, construction method and application | |
| CN113214366A (en) | Recombinant Newcastle disease virus matrix protein and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20200728 |