CN110577605A - Construction method and application of chimeric antigen receptor T (CAR-T) cell targeting multiple myeloma multiple antigens - Google Patents

Construction method and application of chimeric antigen receptor T (CAR-T) cell targeting multiple myeloma multiple antigens Download PDF

Info

Publication number
CN110577605A
CN110577605A CN201810592020.0A CN201810592020A CN110577605A CN 110577605 A CN110577605 A CN 110577605A CN 201810592020 A CN201810592020 A CN 201810592020A CN 110577605 A CN110577605 A CN 110577605A
Authority
CN
China
Prior art keywords
car
cells
multiple myeloma
target
piggybac
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
Application number
CN201810592020.0A
Other languages
Chinese (zh)
Inventor
高基民
魏成
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang Kai Xin Biotechnology Co Ltd
Original Assignee
Zhejiang Kai Xin Biotechnology Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Zhejiang Kai Xin Biotechnology Co Ltd filed Critical Zhejiang Kai Xin Biotechnology Co Ltd
Priority to CN201810592020.0A priority Critical patent/CN110577605A/en
Publication of CN110577605A publication Critical patent/CN110577605A/en
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • 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
    • C07K14/70546—Integrin superfamily
    • 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
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • 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
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Mycology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Hematology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Virology (AREA)
  • Epidemiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a construction method and application of chimeric antigen receptor T cells aiming at multiple myeloma multiple antigens (including BCMA, CS1, CD138, CD38 and Integrin beta 7). The invention utilizes a piggybac transposition system to construct a multi-target CAR expression framework, introduces the multi-target CAR expression framework into T cells by an electrotransfection method and verifies that the multi-target CAR expression framework is inserted into a genome. The invention provides a novel method for constructing CAR-T cells of multi-target multiple myeloma.

Description

Construction method and application of chimeric antigen receptor T (CAR-T) cell targeting multiple myeloma multiple antigens
One, the technical field
the patent belongs to the field of biotechnology engineering, and particularly relates to construction and application of chimeric antigen receptor T cells aiming at Multiple Myeloma (MM) multiple targets (including BCMA, CS1, CD138, CD38 and Integrin beta 7).
Secondly, the technical background is as follows:
Multiple Myeloma (MM) is a malignant tumor characterized by massive proliferation of clonal plasma cells, and is currently mainly treated with chemotherapy and targeted drugs, but the disease is currently alleviated and cannot be cured. The incidence rate of MM in China is about one ten-thousandth to two hundred-thousandth, and exceeds the incidence rate of acute leukemia, which is the second place of the incidence rate of malignant tumor in the blood system. However, the cognitive level of MM is generally low, and only 2 chemotherapeutic drugs are available to relieve the pain but not cure it worldwide. More critically, many patients develop resistance to chemotherapeutic drugs, resulting in their survival of only nine months. Although some monoclonal antibodies have shown promise in preclinical studies and early clinical trials for treatment of MM, there is no consistent acceptance. Clearly, the development of new therapeutic approaches to MM is urgently needed.
CAR-T therapy is a cellular immunotherapy based on chimeric antigen receptors. Which is generated by the transfer of a gene sequence encoding a Chimeric Antigen Receptor (CAR) into T cells by in vitro gene transfer techniques to generate tumor-specific T cells that can bind to a target antigen. Relevant antigens for multiple myeloma include mainly: BCMA, CS1, CD138, CD38, and Integrin β 7. CS1 is a cell surface glycoprotein that is highly expressed in normal plasma cells and in more than 95% of MM patients, and there are reports in the literature that there is also expression of CS1 on the cell surface of relapsed MM patients; CD138, also known as syndecan-1, acts as an adhesion protein in the extracellular matrix, binding collagen and fibronectin molecules, and CD138 is almost exclusively expressed on the surface of MM tumor cells, so both molecules may be very attractive targets for the treatment of MM. Although CD138 is expressed in epithelial cells, in 2016, a report from the general hospital of the chinese people's liberation force showed that none of several chemotherapy-refractory MM patients treated with anti-CD138 CAR-T developed epithelial cytotoxicity, which indirectly provided a guarantee of patient life safety; b Cell Maturation Antigen (BCMA) is a receptor protein specifically expressed on MM cells, and BCMA protein is not detected on normal tissues and CD34+ hematopoietic cells except plasma cells. BCMA belongs to the TNF receptor superfamily, which can bind B cell activating factors and proliferation-inducing ligands to promote MM cell growth and adhesion of bone marrow stromal cells, BCMA antibodies have killing effects on both MM cell lines and primary MM cells, and studies have demonstrated the effectiveness of anti-BCMA CAR-T cells in treating MM; CD38 is a single-chain type II transmembrane glycoprotein, and several studies have shown that CD38 is almost expressed on the surface of myeloma cells, and the united states Food and Drug Administration (FDA) has approved the use of the monoclonal antibody drug daRatumumab (CD38 mab) to treat MM, which also supports the notion that CD38 is a suitable target, and suggests that CD38 may be a potential target antigen for CAR-T immunotherapy of MM.
The Piggybab transposition system is a DNA transposon derived from the lepidopteran insect Trichoplusia ni., originally isolated for the first time when Baculovirus (Baculovirus) infects Trichoplusia ni TN-368 cell line. The PiggyBac transposon can be inserted into a TTAA site of a genome, and the PiggyBac transposon is proved to be capable of being transposed efficiently in genomes of mice, pigs and people at present, and has no limit on the size of a carried exogenous gene when being inserted into a target genome.
Thirdly, the invention content:
The invention mainly provides a construction method of a multi-targeting chimeric antigen receptor T cell aiming at MM, aiming at the problems that a patient who treats MM by using a targeted drug is easy to resist drugs and relapse, and antigen escape possibly exists in single-targeting CAR-T.
Constructing piggybac transposition system vectors such as anti-CS1& CD138& BCMA, anti-CS1& CD138 and the like by a genetic engineering technology, transfecting the piggybac transposition system into a T cell by using an electric rotation method, and detecting the expression of CAR by flow after culturing for two days; and extracting T cell genome DNA, and verifying that the CAR molecules are inserted into the genome by Splinkerette PCR.
Compared with the prior art, the invention relates to the following advantages:
The invention relates to a CAR molecule design scheme, which is characterized in that a piggybac transposition system is applied, and the piggybac transposition system is different from a conventional two-generation or three-generation lentivirus vector in that the piggybac transposition system can be used for constructing CAR-T cells in a non-viral form, so that the safety is improved.
Secondly, the transposable gene size of the piggybac transposition system related by the invention is not limited by the virus packaging capacity any more, so that the transposon gene size is not limited to a single target any more, and the multi-target CAR-T cell design can be completed.
Thirdly, the invention adopts multi-target CAR-T to reduce the antigen escape phenomenon, although CD138 is easy to shed on tumor cells, anti-CS1/anti-BCMA/anti-CD38/anti-integrin beta 7 CAR can also recognize the tumor cells and finally eliminate the tumor cells.
Fourthly, explanation of the attached drawings:
FIG. 1 is a schematic diagram of a piggybac transposing system enzyme helper plasmid vector (FIG. 1a) and a piggybac transposing system donor vector (FIG. 1b) involved in the present invention.
FIG. 2 is a diagram of agarose gel electrophoresis obtained by transforming PB vector into PB-anti-CD138-P2A-anti-CS1 in example 1 of the present invention. Wherein, FIG. 2(a) is an agarose electrophoresis picture after cutting PB vector by SmaI, MluI-HF restriction endonuclease, Lane1 is an agarose electrophoresis picture after cutting PB vector, and Lane2 is a marker agarose electrophoresis distribution picture. FIG. 2(b) shows, on the left, DL5000 DNA Ladder, which has the following sizes: 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp, wherein the right side shows agarose gel electrophoresis results after PCR amplification is respectively carried out on anti-CS1CAR and anti-CD138CAR, lane1 shows a CS1 amplification result, lane2 shows a maker electrophoresis distribution result, and lane3 shows a CD138 amplification result. FIG. 2(c) is an agarose electrophoresis picture of anti-CS1CAR and anti-CD138CAR after overlap PCR. Lane1 is the marker electrophoretic distribution result, and Lane2-5 is the anti-CS1 CAR-P2A-anti-CD138 CAR nucleic acid agarose gel electrophoresis result. FIG. 2(d) shows the results of double-restriction agarose electrophoresis with SmaI and MluI restriction enzymes. Lane1 is marker electrophoretic distribution, and Lane6 is PB vector cleavage result.
Figure 3 is a flow cytometry assay of the expression of multi-targeted CARs on T cells as in example 3.
Figure 4 is a validation of CAR molecule insertion into the T cell genome by Splinkerette PCR.
Fifth, detailed description of the invention
The equipment used below was as follows:
The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental method of the present example does not specify the specific conditions, and the general conditions are generally followed.
Example 1: construction of recombinant vector (exemplified by PB-anti-CD138-P2A-anti-CS 1)
The main experimental materials:
The PB vector plasmid and the PB helper plasmid were provided by Pronobio Ltd.
SmaI, MluI-HF, XhoI, MfeI, ClaI restriction enzymes were all purchased from NEB.
The seamless cloning enzymes were purchased from Heyuanzhi.
The high fidelity Prime GXL STAR enzyme was purchased from TAKARA.
Both Trans 5 α and TransStbl3 competent cells were purchased from holo-gold Biotechnology, Inc.
For large-scale extraction, medium-scale particle extraction kit was purchased from QIAGEN.
Primer: the primers are designed according to the primer design principle and are synthesized by Jiangsu Jinzhi Biotechnology GmbH. The primers used are as in table (1) below:
Watch (1)
the amino acid sequence of SEQ ID NO.14 is derived from EP29920202A1, and the amino acid sequence of SEQ ID NO.15 is shown in US20180022815A1, and the nucleotide sequences are synthesized by Suzhou Jinzhi Biotech limited and finally stored in the form of plasmid dry powder.
Sanger sequencing services were all accomplished by Shanghai Sangni biology, Inc.
NaCl, yeast powder, peptone, EDTA, NaOH and other chemical reagents are purchased from Shanghai Producers.
The construction method of the plasmid recombinant vector (taking PB anti-CS1& CD138CAR as an example):
Primers CS1F, CS 1R and CD138F, CD 138R were used to amplify a synthetic fragment (synthetic gene) anti-CS1CAR (hCD8 leader-VL1-Linker-VH1-CD8 hige-CD 8TM-41BB-CD3 ζ) (SEQ ID No.13) and an anti-CD138CAR (P2A-hCD8 leader-VL2-Linker-VH2-CD8 hige-CD 8TM-41BB-CD3 ζ) (SEQ ID No.12) (SEQ ID No.15), respectively, under the conditions of (98 ℃: 10s, 60 ℃: 15s, 68 ℃: 1kb/min) 35 cycles, and the amplification system is shown in Table 2. The target fragment was obtained on the right side of FIG. 5(b) using 1% agarose gel, and the target band was 1500bp, 1545bp, and then fragments of anti-CS1CAR and P2A-anti-CD138CAR were recovered using XYGENE gel recovery kit (see Table 3 below), respectively, and the concentration and purity were determined. Mu.l of each recovered product of the two-fragment gel was taken as a template, CS1F and CD 138R were used as primers to amplify the target fragment hCD8 leader-VL1-Linker-VH1-CD8hinge-CD8TM-41BB-CD3 zeta-P2A-hCD 8 leader-VL2-Linker-VH2-CD8hinge-CD8TM-41BB-CD3 zeta, the amplification system is shown in Table (4), (98 ℃: 10s, 60 ℃: 15s, 68 ℃: 3min) and 35cycle, and the target fragment obtained by using 1% agarose gel has the target band size of 3045bp as shown in FIG. 4 (c). Carrying out double digestion on the vector PB vector by SmaI and Mlul-HF restriction endonuclease under the reaction conditions of 25 ℃ for 3h, 37 ℃ for 3h and 65 ℃ for 20min, wherein the digestion system is shown in table (5), the digestion product is subjected to 1% agarose gel to obtain a vector fragment shown in figure 4(a), the vector and the target fragment are connected by seamless cloning, then plasmid transformation is carried out (after the seamless cloning product is placed on ice for 5min, the seamless cloning product is transferred into 100 microliter Transbl 3 competent state, placed on ice for 30min and 42 ℃ for 45s, then placed on ice for 5min, 500 microliter LB is added, the culture is activated in a shaker at 37 ℃ and 225rpm for 1h, then centrifuged at 5000rpm/min for 20min for 5min, the supernatant is discarded, the residual bacterial liquid is mixed uniformly and then plated, the plate is cultured at 37 ℃ for 12-14 h), the single cloning colony is selected, bacterial liquid amplification is carried out at 37 ℃ and 250rpm/min, 12-14 h, plasmid extraction is finally carried out by Sma, the restriction enzyme digestion of Mlul-HF restriction enzyme is identified, the digestion system is shown in Table 6, the size of the target fragment is observed by using 1% agarose gel, and the size of the band is as follows in sequence as shown in FIG. 4 (d): 7.3kb, 3 kb. Finally Sanger sequencing was performed.
Table (2): PCR system
Table (3): glue recovery procedure
Table (4): overlapping PCR system
Table (5): seamless cloning system
table (6): restriction enzyme system
Example 2: separating Peripheral Blood Mononuclear Cells (PBMC) and PB-CAR of healthy people by density gradient centrifugation, co-transfecting T cells by using PB helper plasmids, and detecting the expression condition of CAR on the surfaces of the T cells. (take anti-CS1& CD138& BCMA CAR as an example)
(1) Taking 10ml of peripheral blood of a healthy person to an EDTA-Na2 anticoagulation tube, and uniformly mixing the anticoagulation tube with DPBS according to the ratio of 1: 1.
(2) And respectively adding 5ml of lymphocyte separating medium into four 15ml sterile centrifuge tubes, and slowly adding the mixed solution of peripheral blood and SPNS onto the lymphocyte separating medium without damaging the liquid level.
(3) The mixture is horizontally centrifuged at 800g for 20min at 25 ℃, and the acceleration and deceleration are adjusted to 0.
(4) After centrifugation, the white flocculent layer, i.e., the PBMC layer, in the centrifuge tube is sucked out by a Pasteur pipette, placed in a new sterile centrifuge tube, PBS is added, and the PBMC is washed twice by centrifugation.
(5) Horizontal centrifugation at 1500rpm/min for 5 min. The supernatant was discarded and 1ml of complete medium was added and counted in a resuspension and was ready for use.
(6) The proportion of CD3 positive cells in PBMC was determined by flow cytometry. CD3/CD28 beads (10) were added to the cell suspension at a ratio of 3: 1 dynabeads CD3/CD28 to CD3 positive cells6CD3 positive cells added with 30ul beads), and the magnetic column is fully contacted and combined with the cells by rotary shaking at the speed of 1rpm for 30min at 4 ℃.
(7) after 30 minutes, add enough (greater than 1ml) buffer 1(DPBS contains 5% FBS) to the tube, then place the tube in the magnetic rack for 1-2 minutes, and aspirate the supernatant.
(8) The cells and magnetic beads were resuspended in complete medium AIM-V and counted.
(9) Adding culture medium to make CD3 positive cell concentration be 0.5-1x106Between/ml. The concentration of plated cells is 0.5-1.0 × 106And/ml, placing in an incubator at 37 ℃ for culture.
(10) and (3) cell preparation: 3x107m/ml T cells, 1640 Medium
(11) Setting the electrotransfer parameters:
Selecting a mode: LV
voltage: 200V
pulse length: 65ms
Pulse number: 1
Turning the cup by points: BTX dispersible packages Model 640
(12) the process is as follows: sample volume: 200ul, DNA content 15ug, RT, T cell 10M.
(13) T cell culture 5% CO2，37℃.
(14) After 48h of T cell culture, the expression of three CARs in T cells was flow analyzed.
Example 3: splinkerette PCR verification of foreign gene insertion into T cell genome
(1) And extracting T cell genome DNA according to a GX kit.
(2) The T cell genome was digested with BglII restriction enzyme.
(3) Splinkerette linkers were annealed. (SEQ. ID. NO.5, SEQ. ID. NO.6),
(4) The digested T cell genome was ligated to a Splinkerette linker. The reaction system is shown in the following Table (10). 16 ℃ and 16h.
(5) First round Splinkerette PCR, the reaction system is as follows (11). Reaction procedure: 98 ℃ C: 5min, (98 ℃: 10s, 60 ℃: 15s, 68 ℃: 2min) 35cycle, 68 ℃: and 5 min.
(6) Second round Splinkerette PCR, the reaction system is as follows (12). Reaction procedure: 98 ℃ C: 5min, (98 ℃: 10s, 60 ℃: 15s, 68 ℃: 2min) 35cycle, 68 ℃: and 5 min.
watch 10
TABLE 11
TABLE 12

Claims (4)

1. A method for constructing Chimeric Antigen Receptor (CAR) T cells targeting multiple antigens of multiple myeloma, including BCMA, CS1, CD138, CD38 and Integrin beta 7, and application thereof are provided.
2. The multiple myeloma multi-target (such as BAMA, CS1, CD138, CD38, Integrin beta 7 and the like) piggybac transposition system electrotransfection method is introduced into T cells, and expresses multi-target CAR molecules.
3. The piggybac transposition system is characterized in that: the piggybac transposable system is divided into two parts, one part is that the donor plasmid mainly expresses CAR molecules, and the other part is that the helper plasmid expresses the piggybac transposase.
4. The CAR-T cell construction method is characterized in that: a method of plasmid electrotransfection by piggybac transposition system.
CN201810592020.0A 2018-06-11 2018-06-11 Construction method and application of chimeric antigen receptor T (CAR-T) cell targeting multiple myeloma multiple antigens Pending CN110577605A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810592020.0A CN110577605A (en) 2018-06-11 2018-06-11 Construction method and application of chimeric antigen receptor T (CAR-T) cell targeting multiple myeloma multiple antigens

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810592020.0A CN110577605A (en) 2018-06-11 2018-06-11 Construction method and application of chimeric antigen receptor T (CAR-T) cell targeting multiple myeloma multiple antigens

Publications (1)

Publication Number Publication Date
CN110577605A true CN110577605A (en) 2019-12-17

Family

ID=68810239

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810592020.0A Pending CN110577605A (en) 2018-06-11 2018-06-11 Construction method and application of chimeric antigen receptor T (CAR-T) cell targeting multiple myeloma multiple antigens

Country Status (1)

Country Link
CN (1) CN110577605A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113663061A (en) * 2021-08-04 2021-11-19 上海优卡迪生物医药科技有限公司 Application of CD38 in preparation of CAR-T medicine
WO2023217062A1 (en) * 2022-05-10 2023-11-16 星尘生物科技(上海)有限公司 Chimeric antigen receptor and use thereof

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105713881A (en) * 2014-12-04 2016-06-29 科济生物医药（上海）有限公司 Dual-targeting genetically modified immunologic effector cell aiming at GPC3 (Glypican-3) and ASGPR1 (asialoglycoprotein receptor 1) and applications of dual-targeting genetically modified immunologic effector cell
CN106414748A (en) * 2014-02-14 2017-02-15 得克萨斯州大学***董事会 Chimeric antigen receptors and methods of making
CN107325185A (en) * 2017-06-06 2017-11-07 上海优卡迪生物医药科技有限公司 The double targeting Chimeric antigen receptors of anti-PSCA and PDL1 based on OCTS CAR, encoding gene and expression vector
CN107529550A (en) * 2017-08-28 2018-01-02 马晓冬 A kind of CAR T system constituting methods of CD 19 based on PiggyBac carriers
CN107849112A (en) * 2015-06-25 2018-03-27 美商生物细胞基因治疗有限公司 Chimeric antigen receptor (CAR), composition and its application method
CN107922938A (en) * 2015-08-11 2018-04-17 国立大学法人大阪大学 Antibody

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106414748A (en) * 2014-02-14 2017-02-15 得克萨斯州大学***董事会 Chimeric antigen receptors and methods of making
CN105713881A (en) * 2014-12-04 2016-06-29 科济生物医药（上海）有限公司 Dual-targeting genetically modified immunologic effector cell aiming at GPC3 (Glypican-3) and ASGPR1 (asialoglycoprotein receptor 1) and applications of dual-targeting genetically modified immunologic effector cell
CN107849112A (en) * 2015-06-25 2018-03-27 美商生物细胞基因治疗有限公司 Chimeric antigen receptor (CAR), composition and its application method
CN107922938A (en) * 2015-08-11 2018-04-17 国立大学法人大阪大学 Antibody
CN107325185A (en) * 2017-06-06 2017-11-07 上海优卡迪生物医药科技有限公司 The double targeting Chimeric antigen receptors of anti-PSCA and PDL1 based on OCTS CAR, encoding gene and expression vector
CN107529550A (en) * 2017-08-28 2018-01-02 马晓冬 A kind of CAR T system constituting methods of CD 19 based on PiggyBac carriers

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DAISUKE MORITA等: "Enhanced Expression of Anti-CD19 Chimeric Antigen Receptor in piggyback Transposon-Engineered T Cells" *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113663061A (en) * 2021-08-04 2021-11-19 上海优卡迪生物医药科技有限公司 Application of CD38 in preparation of CAR-T medicine
WO2023217062A1 (en) * 2022-05-10 2023-11-16 星尘生物科技(上海)有限公司 Chimeric antigen receptor and use thereof

Similar Documents

Publication Publication Date Title
US20240011051A1 (en) Methods for transduction and cell processing
CN106279434B (en) Engineered CD20 targeted NKT cell and preparation method and application thereof
US11090335B2 (en) Chimeric antigen receptor targeting human NKG2DL and methods of preparing said receptor and pharmaceutical composition
CN107429260A (en) The hepatitis posttranscriptional regulatory element of modification
CN110818803B (en) Chimeric antigen receptor-T cell for secreting and expressing IL15RA-IL15 fusion protein and CCL21 chemokine and application
CN109504660B (en) Fourth-generation CAR-T cell and construction method and application thereof
CN109844125A (en) The preparation method and application of the CLDN18.2 specific chimeric antigen receptor T cell of people's PD-1 gene knockout
CN111304255B (en) Nourishing cell, preparation method thereof and application of nourishing cell in efficient amplification of NK cells
WO2018041220A1 (en) Human-cd123-targeting chimeric receptor ligand and application thereof
CN109652380B (en) CAR-T cell based on base editing targeting Lewis Y and preparation method and application thereof
WO2018218879A1 (en) Octs technology-based pancreatic cancer and malignant mesothelioma car-t therapeutic vector, construction method therefor, and use thereof
CN111848820B (en) CD19 and BCMA double-target chimeric antigen receptor and application thereof
CN114901808A (en) Method for producing CAR-T cells
CN110684790A (en) Encoding gene of anti-B7-H3 chimeric antigen receptor, preparation method, plasmid with encoding gene, immune cell and application of immune cell
CN114317607A (en) Double-target universal CAR-T cell fusing primary targeting CD7CAR and secondary targeting BCMA and preparation method thereof
CN112210539A (en) Fourth-generation CAR-T cell and application thereof
CN110577605A (en) Construction method and application of chimeric antigen receptor T (CAR-T) cell targeting multiple myeloma multiple antigens
CN111875711A (en) Enhanced immune cell and application thereof
US20230398148A1 (en) Cells expressing a chimeric receptor from a modified invariant cd3 immunoglobulin superfamily chain locus and related polynucleotides and methods
CN112552404A (en) Single-chain antibody targeting c-Met, chimeric antigen receptor, recombinant vector, CAR-T cell and application
WO2023123195A1 (en) Engineered immune cell target gene of which can be regulated, preparation method therefor, and use thereof
CN110819596A (en) Modified cells with enhanced migratory capacity
CN107098981B (en) Chimeric antigen receptor modified T lymphocyte targeting CD19
CN112626028B (en) Engineering cell for activating NK-like cells and preparation method and application thereof
CN110951695A (en) Universal CAR-T cell, preparation method 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

Application publication date: 20191217

WD01 Invention patent application deemed withdrawn after publication