CN107522786A - A kind of molecule, the cell for expressing it and its production and use - Google Patents
A kind of molecule, the cell for expressing it and its production and use Download PDFInfo
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- CN107522786A CN107522786A CN201610443073.7A CN201610443073A CN107522786A CN 107522786 A CN107522786 A CN 107522786A CN 201610443073 A CN201610443073 A CN 201610443073A CN 107522786 A CN107522786 A CN 107522786A
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- cell
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- acceptor molecule
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Classifications
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- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- 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
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- 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
- C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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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
- C12N15/86—Viral vectors
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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
- 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
- C12N5/0636—T lymphocytes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2510/00—Genetically modified cells
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- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
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- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2800/107—Plasmid DNA for vertebrates for mammalian
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Immunology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
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- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Wood Science & Technology (AREA)
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- Pharmacology & Pharmacy (AREA)
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- Developmental Biology & Embryology (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A kind of molecule, the cell for expressing it and its production and use.The present invention relates to a kind of chimeric immunosupress checkpoint acceptor molecule, its nucleic acid is encoded, the cell of construct, expression vector and conversion containing the nucleic acid, and their pharmaceutical applications.The chimeric immunosupress checkpoint acceptor molecule of the present invention includes ectodomain, membrane spaning domain and intracellular domain, the genetic modification of the ectodomain and optionally membrane spaning domain for the corresponding construction domain of immunosupress checkpoint acceptor molecule or based on domain progress, the intracellular domain is one or more immune activation signal domains.There is the cell of conversion of the present invention strong tumor-killing to act on, and can release tumour immunity and suppress microenvironment, strengthen the immunization in tumor microenvironment；Also, the cell of the conversion has self-regulating function, the side effects such as excessive nonspecific immunity effect and cytokine storm can be alleviated.
Description
Technical field
The present invention relates to the cellular immunotherapy technical field of tumour, in particular it relates to a kind of chimeric immunosupress inspection
Acceptor molecule is made an inventory of, encodes its nucleic acid, the cell of construct, expression vector and conversion containing the nucleic acid, and they
Pharmaceutical applications.
Background technology
CAR-T cell tumour current treatment status
In October, 2015, National Cancer Center is in famous cancer professional journals《Cancer Letters》On issue me first
State's resident's cancer now suffers from data.As a result show, China is diagnosed as cancer and still survived case load in 5 years is about 7,490,000 (wherein
The people of male patient 3,680,000, the people of female patient 3,810,000), cancer illness rate is 5,56/,100,000 within overall 5 years, and data reflect on the whole
The active demand that cancer patient is in progress to new Therapy study.
And immunotherapy already turns into mankind's hope new to anticancer, and CAR-T treats neoplastic hematologic disorder particularly B-ALL
The clinical test results of leukaemia, one of tumour immunotherapy most to get most of the attention in recent years is become.But targeting is real
The CAR T immunization therapies research of body knurl still faces huge test：How to break through solid tumor mass barrier, how to resist tumour
How immunosupress, immunocyte in tissue compete existence etc. in micro-environmental hypoxia.
Immunosuppressive action in the problem of oncotherapy, particularly solid tumor and its tumor microenvironment
Tumor microenvironment can lower the number quantity and inflammatory factor of the auxiliary of specific for tumour antigen/toxicity killing cell
Release, up-regulation immunosuppressant cell quantity and suppression function, as regulatory T cells (Regulatory T cells) and medullary system come
The suppression cell (Myeloid-Derived Suppressor Cells) in source, normal immune system is overturned to promote tumour certainly
The superior items of body growth.
The corresponding measure present situation suppressed for tumour immunity
For the immunosuppressive situation of solid tumor, scientist researches and develops blocking outflow and suppresses signal path successively, and it is micro- to lower tumour
The method of inhibitory cells in environment, such as selectively targeted PD-1 (Programmed Death 1), PD-L1
(Programmed Death Ligand 1)、CTLA4(Cytotoxic T-Lymphocyte-associated Antigen-
4) antibody or inhibitor, the immune activation factor etc..
In the prior art, the means that immunosupress related antigen PD-1 and CTLA-4 are applied to immunotherapy of tumors are led to
It is often structure PD-1 antibody (Pembrolizumab, Nivolumab) or CTLA-4 antibody (Ipilimumab), or uses PD-
1 or CTLA-4 inhibitor or signal path blocking agent.However, necessarily restricted be present in PD-1 antibody and CTLA-4 antibody,
That is, antibody, which needs to penetrate into tumor tissues, can just play curative effect, so the clinical practice of antibody has uncertainty.It is reported that
When the antibody of CAR-GD2T cells combined PD -1 carries out immunization therapy, the immunosuppressive action of tumor microenvironment can be resisted, strengthens CAR
T cell tumor-killing.Immunization therapy application for PD-1 and CTLA-4, also reports coexpression target tumor in the prior art
(extracellular antigen is normal cell antigen, and intracellular section is PD-1 or CTLA-4 suppression by related antigen CAR T and inhibition CART
Signal domain), it can prevent that CAR T from missing the target the generation (phenomenon of missing the target of phenomenon：CAR T cells identification killing normal cell).
In addition, coexpression double antibody PD-1 and CD19/Mesothelin/PSCA CART is also reported in the prior art,
Although this expands target spot scope (expression PD-1 or CD19/Mesothelin/PSCA or simultaneously both tumour cells of expression),
But it have ignored the blindly side effect caused by increase killing functions of immunocytes：Cytokine storm, phenomenon of missing the target (normal cell
Express two antigens or one of, cause CAR T kill normal cell, cause normal cell lack body function defect).
The content of the invention
It is an object of the invention to provide a kind of chimeric immunosupress checkpoint acceptor molecule, its nucleic acid is encoded, is contained
There are construct, expression vector and the cell of conversion of the nucleic acid, and their pharmaceutical applications.The expression of the present invention is described chimeric
The cell of conversion of immunosupress checkpoint acceptor molecule can build a kind of tumour immunity and suppress microenvironment, there is the swollen of enhancing
Knurl lethal effect；Also, the cell of the conversion has self-regulating function, excessive nonspecific immunity effect and cell factor can be alleviated
The side effects such as storm.
The present invention is achieved through the following technical solutions above-mentioned purpose：
In a first aspect, the invention provides a kind of chimeric immunosupress checkpoint acceptor molecule, it includes extracellular structure
Domain, membrane spaning domain and intracellular domain, wherein, the ectodomain and optionally membrane spaning domain are immunosupress inspection
The corresponding construction domain of point acceptor molecule or the genetic modification carried out based on the domain, the intracellular domain are one or more
Immune activation signal domain.
Preferably, immunosupress checkpoint acceptor molecule be selected from PD-1, CTLA-4, LAG3, TIM3, A2AR,
B7H3, B7H4, BTLA, IDO, KIR, CD160,2B4 (CD244) and VISTA (C10orf54)；
It is further preferred that the immunologic test point molecule is PD-1, CTLA-4, LAG3 or TIM3.
Preferably, the intracellular domain includes immune costimulatory signal combination and optional signal peptide；
It is further preferred that the immune costimulatory signal combination includes TLR1 and/or TLR2 signal domains；
It is further preferred that the intracellular domain is TLR1 and/or TLR2 and 41BB and/or CD28 signal domains
Combination.
It is further preferred that optional signal peptide is the corresponding signal peptide of immunosupress checkpoint acceptor molecule.
It is further preferred that the intracellular domain also includes CD3 ζ intracellular domain；
In specific preferred embodiment, TLR1 the and/or TLR2 signal domains and optionally 41BB and/or
N-terminal side of the CD28 signal structures configuration of territory in CD3 ζ intracellular domain.
Second aspect, the invention provides the chimeric immunosupress checkpoint acceptor molecule of coding as described in relation to the first aspect
Nucleic acid.
The third aspect, the invention provides a kind of nucleic acid construct, and it includes the nucleic acid as described in second aspect, Yi Jiyu
One or more being operatively connected, can instructing that the chimeric immunosupress checkpoint acceptor molecule expresses in host cell
Individual control sequence.
Fourth aspect, the invention provides a kind of expression vector, and it includes the nucleic acid construct as described in the third aspect；It is excellent
Selection of land, the expression vector are slow virus.
5th aspect, the invention provides a kind of cell of conversion, and it includes the nucleic acid as described in second aspect, Huo Zheqi
The middle nucleic acid construct converted as described in the third aspect or the expression vector as described in fourth aspect；
Preferably, the cell of the conversion is immunocyte, more preferably T cell, B cell or NK cells.
In specific embodiments, the cell of the conversion be Zeus's shield cytotoxic T lymphocyte (S-CTL,
Shielded Cytotoxic T Lymphocyte), it is leaching of the overexpression immunosupress to the checkpoint molecule of activation transition
Bar cell, particularly T lymphocytes.SP-CTL cell of the S-CTL cells including the chimeric PD-1 acceptor molecules of expression, expression are embedding
What the ST-CTL cells of the chimeric TIM3 acceptor molecules of the SC-CTL cells of the CTLA-4 acceptor molecules of conjunction, expression and expression were fitted together to
SL-CTL cells of LAG3 acceptor molecules etc..S-CTL cells in human T cell by transfecting chimeric immunosupress-excitement
The checkpoint molecular gene of transition, acquisition can resist immunosuppressive lymphocyte.S-CTL lymphocytes have the characteristics that：
1) to part (such as PD-L1, B7.1, B7.2, Galectin-9, MHC II of expression immunosupress checkpoint molecule
Deng) tumour cell there is strong cellular lethal effect；
2) expression of natural immunosupress checkpoint molecular receptor (such as PD-1, CTLA-4, TIM3, LAG3) is thin in lymph
Behind the born of the same parents particularly surface, with tumour cell ligand binding such as T cell, NK cells, the activation of immunocyte itself can be suppressed, increased
The performance of function such as grow, identify, killing；
3) S-CTL structures：Extracellular fragment and trans-membrane region are complete immunosupress checkpoint molecular structure, for specificity
Tumor cell surface ligand；Intracellular region is one or more immune activation signal domains, wherein present invention discover that containing TLR1
And/or the intracellular domain in TLR2 signal activations domain, it can further promote Tumor cytotoxicity；
4) self-regulating function, because normal lymphocytes express natural immunosupress checkpoint molecular receptor, conversion
The checkpoint molecule of the chimeric immunosupress of cell expression-excitement transition, and natural immunosupress checkpoint molecular receptor is
Conduction suppresses signal, and chimeric immunosupress checkpoint molecular receptor is conduction activation signal, thus can be adjusted by oneself expression
Section mode slows down the side effect of S-CTL cells, such as miss the target killing, cell factor；
5) SP-CTL cells can promote the propagation of itself.
6th aspect, the invention provides the method for the cell for preparing the conversion as described in terms of the 5th, it includes：To thin
Introduce nucleic acid as described in second aspect in born of the same parents, or nucleic acid construct of the conversion as described in the third aspect or such as fourth aspect institute
The step of expression vector stated.
7th aspect, the invention provides chimeric immunosupress checkpoint acceptor molecule as described in relation to the first aspect, such as
Nucleic acid described in second aspect, the nucleic acid construct as described in the third aspect, the expression vector as described in fourth aspect or such as the
Five aspect described in conversion cell prepare with immunosupress checkpoint acceptor molecule, preferably with PD-L1, B7.1, B7.2,
Purposes in the medicine of the related disease of Galectin-9, MHC II expression；
Preferably, the disease be expression immunosupress checkpoint acceptor molecule, preferred expression PD-L1, B7.1, B7.2,
Galectin-9, MHC II tumour；
It is further preferred that expression immunosupress checkpoint acceptor molecule, preferred expression PD-L1, B7.1, B7.2,
Galectin-9, MHC II tumour be selected from lung cancer, leukaemia, breast cancer, prostate cancer, cancer of pancreas, liver cancer, melanoma and
Nonmelanoma skin cancer.
Term defines
In the context of the present invention, " immunologic test point " (Immune Checkpoint) can be raised in immune system
Or the molecule of immunostimulatory signals is lowered, it is divided into immunostimulation checkpoint molecule (Stimulatory Checkpoint) and is immunized
Suppress checkpoint molecule (Inhibitory Checkpoint).
" immunosupress checkpoint " is the molecule that immunostimulatory signals can be lowered in immune system, such as：PD-1、CTLA-
4、TIM3、LAG3、A2AR、B7-H3、B7-H4、BTLA、IDO、KIR、CD160、2B4(CD244)、VISTA(C10orf54)、
TIGIT, LAIR1,2B4 etc..
" immune activation signal "：During lymphocyte activator, T cell needs to obtain two signals fully to activate；The
One signal is antigentic specificity, i.e., by φt cell receptor with antigen presenting cell APC surfaces MHC molecule with reference to caused by；The
Binary signal, i.e. costimulatory signal, it is antigen-non-specific, passes through T cell and the costimulatory signal molecule knot of APC cell surfaces
Caused by conjunction.
The intracellular domain of the chimeric immunosupress checkpoint acceptor molecule of the present invention can be including costimulatory signal point
Sub- CD28, OX40, ICOS (CD278), 4-1BB (CD137), CD40, CD27, CD134, CDS, ICAM-1, LFA-1 (CD11a/
CD18), CD2, BTLA etc. intracellular section, or their any combination.
Beneficial effect
The cell of conversion of the present invention is a kind of new tumor-killing cell, and it suppresses micro-loop to build tumour immunity
Border and express immunosupress checkpoint acceptor molecule, there is low miss rate；Also, the unique design of its intracellular domain can enter one
Step promotes its Tumor cytotoxicity, while can effectively eliminate tumour immunity inhibitory action；And the cell (such as T cell) converted
A certain amount of innate immunity of meeting induced expression suppresses checkpoint acceptor molecule in tumor microenvironment, and it resists with tumour PD-L1
Original, which combines, produces certain suppression signal, there is certain adjustment effect to immunological effect caused by the cell of conversion.It is in view of above-mentioned
Advantage, the cell of the conversion of the expression Chimerical receptor molecule of the invention have good clinical practice in antineoplaston
Prospect.
Brief description of the drawings
Fig. 1 shows the composition sequence collection of illustrative plates in embodiment 1.
Fig. 2 show embodiment 1 in build pWPXLd-S (PD1)-CTL-2A-EGFP plasmids, pWPXLd-S (CTLA-4)-
CTL-2A-EGFP plasmids, pWPXLd-S (TIM3)-CTL-2A-EGFP plasmids and pWPXLd-S (LAG3)-CTL-2A-EGFP matter
Grain collection of illustrative plates.
Fig. 3 shows the virus infection positive rate of flow cytomery SP-CTL cells；Blank groups：Without using CD3 streamings
The non-transfecting T cells that antibody stimulates.
Fig. 4 shows the virus infection positive rate of flow cytomery SC-CTL cells；Normal groups：Non- transfecting T cells.
Fig. 5 shows lethal effect of the SP-CTL cells to people's T-ALL cell lines JURKAT-GL.
Fig. 6 shows lethal effect of the SP-CTL cells to people's lung cancer in non-cellule type H460-GL.
Fig. 7 shows SC-CTL cells to people's B-ALL cell line NALM6-GL lethal effects.
Fig. 8 shows SP-CTL cells to lethal effect inside people's lung cancer in non-cellule type H460-GL.
Embodiment
For ease of understanding the present invention, it is as follows that the present invention enumerates embodiment.Those skilled in the art are it will be clearly understood that the implementation
Example is only to aid in understanding the present invention, is not construed as the concrete restriction to the present invention.
The preparation of embodiment 1, S-CTL cells
Plasmid construction
1) by gene chemical synthesis, " immunosupress checkpoint molecule (signal peptide+extracellular fragment+transmembrane region)-stimulus signal is obtained
1- stimulus signals 2 (generally TLR1 or TLR2)-CD3 ε " DNA sequence dna, the head and the tail of DNA sequence dna respectively comprising PmeI and
Spe1 restriction enzyme sites.Composition sequence collection of illustrative plates is as shown in Figure 1.
Synthesized gene order see SEQ NO.1 (for prepare target PD-1 S-CTL, i.e. SP-CTL, and synthesize
DNA sequence dna) and SEQ NO.2 (to prepare targeting CTLA-4 S-CTL, i.e. SC-CTL, and the DNA sequence dna synthesized) and SEQ
NO.3 (for prepare targeting TIM3 S-CTL, i.e. ST-CTL, and synthesize DNA sequence dna) and SEQ NO.4 (for prepare targeting LAG3
S-CTL, i.e. SL-CTL, and synthesize DNA sequence dna).
S-CTL (Shielded Cytotoxic T Lymphocyte), it is the cytotoxic T lymphocyte of Immune-enhancing effect,
Chimeric immunosupress checkpoint molecule, extracellular fragment and cross-film section are expressed as the corresponding of people's immunosupress checkpoint acceptor molecule
Section, and intracellular section combines for immunostimulatory signals domain, can specific recognition and killing expression immunosupress checkpoint part
Tumour cell and immunosuppressant cell.
SP-CTL, expresses the S-CTL cells of chimeric PD-1 acceptors, and specific recognition killing expression PD-L1 tumour is thin
Born of the same parents and tumor-related cell plastid or immunosuppressant cell.
SC-CTL, expresses the S-CTL cells of chimeric CTLA-4 acceptors, specific recognition killing expression B7.1 and/or
B7.2 tumour cell and tumor-related cell plastid or immunosuppressant cell.
ST-CTL, the S-CTL cells of chimeric TIM3 acceptors are expressed, specific recognition killing expression Galectin-9's
Tumour cell and tumor-related cell plastid or immunosuppressant cell.
SL-CTL, expresses the S-CTL cells of chimeric LAG3 acceptors, and specific recognition killing expression MHC II tumour is thin
Born of the same parents and tumor-related cell plastid or immunosuppressant cell.
2) Thermo restriction enzyme Pme1 and Spe1 are used, obtains the S-CTL DNA of synthesis by double digestion respectively
The linearisation DNA of fragment (SEQ NO.1, SEQ NO.2, SEQ NO.3, SEQ NO.4) and pWPXLd-EGFP carriers is (containing viscosity
End).
3) reclaimed by agargel electrophoresis, obtain the S-CTL (SEQ NO.1, the SEQ that are linearly combined to cohesive end
NO.2, SEQ NO.3, SEQ NO.4) and pWPXLd-EGFP vector DNA fragments.
4) by T4DNA ligases (coming from Invitrogent companies), connect respectively linearisation SEQ NO.1 and
PWPXLd-2A-EGFP, the SEQ NO.2 and pWPXLd-2A-EGFP of linearisation, obtain pWPXLd-S (PD1/CTLA-4/TIM3/
LAG3)-CTL-2A-EGFP plasmids, its plasmid map is referring to accompanying drawing 2.
S-CTL slow virus is packed
1) culture 293T cells to density reaches 80-90%, and culture medium is replaced by：DMEM high glucose mediums+1%FBS+
1% is dual anti-；
2) after 2-6 hours, with PEI respectively by pWPXLd-S (PD1/CTLA-4/TIM3/LAG3)-CTL-2A-EGFP plasmids
293T cells are transfected into jointly with slow virus helper plasmid (pMD2.G, psPAX2), with empty carrier pWPXLd-EGFP and auxiliary matter
Grain cotransfection is as a control group；
3) 24,48 and 72 hours after transfection, collection culture medium supernatant, i.e. S (PD1/CTLA-4/TIM3/LAG3)-
CTL viral supernatants, freeze standby in -80 DEG C.
T cell activation and slow-virus infection
1) monocyte in blood is isolated by Ficoll density gradient methods or cells by red blood cell lysis method；
2) T cell is gone out by MACS Pan-T magnetic bead sortings, (AIM-V adds 5%FBS, penicillin with T cell culture medium
100U/ml and streptomysin 0.1mg/ml) it is diluted to concentration 2.5 × 106Individual/ml is stand-by；
3) T cell is stimulated by being coated with the magnetic bead (U.S. day Ni) of CD2, CD3, CD28 antibody, in 37 DEG C, 5%CO2Under the conditions of
Incubator in culture stimulate 48h；
4) magnetic bead in T cell is removed, supernatant is removed in centrifugation, is resuspended；
5) add the μ g/ml of S (PD1/CTLA-4/TIM3/LAG3)-CTL slow virus supernatant 8 polybrene and
300IU/ml IL-2, viral addition are MOI=10, in 37 DEG C, 5%CO2Under the conditions of incubator in cultivate；
6) after 24h, 300g centrifugation 5min, supernatant is removed, is resuspended with the fresh T cells culture medium of the IL-2 containing 300IU/ml, in
37 DEG C, 5%CO2Under the conditions of incubator in cultivate, half amount was carried out per 2-3 days and changes liquid；
7) by Flow Cytometry, GFP positive T cells S-CTL (SC-CTL or ST-CTL) ratio is detected, as a result as schemed
3rd, shown in Fig. 4.
Embodiment 2, SP-CTL cells are to human leukemia cell line JURKAT Cytotoxicity in vitro effect
1) the transduction expressing luciferase (Luciferace) in Leukemia Cell Lines JURKAT, structure JURKAT-GL reports
Accuse cell line；
2) respectively by SC-CTL GFP positive cells and JURKAT-GL cells with cell number 1：It is (right that the mixing of 2 ratios co-cultures
According to group to add GFP T cells, blank group is to be not added with T cell, three multiple holes of every group of setting)；
3) after 18 hours, the culture medium supernatant of half volume is gently absorbed with pipettor, adds isometric luciferase
Substrate (concentration：300 μ g/ml), mix；
4) RLU (relative light unit) is determined by multi-function microplate reader, minute is set to 1 second.
5) ratio calculation formula is killed：100% × (blank well reading-experimental port reading)/blank well reading, SP-CTL are thin
Born of the same parents are as shown in Figure 5 to human leukemia cell line JURKAT Cytotoxicity in vitro ratio.
Embodiment 3, SP-CTL cells are to lung cancer in non-cellule type H460 Cytotoxicity in vitro effect
1) the transduction expressing luciferase (Luciferace) in lung cancer in non-cellule type H460, structure H460-GL reports
Cell line；
2) respectively by SP-CTL GFP positive cells and H460-GL cells with cell number 1：It is (right that the mixing of 2 ratios co-cultures
According to group to add GFP T cells, blank group is to be not added with T cell, three multiple holes of every group of setting)；
3) after 18 hours, the culture medium supernatant of half volume is gently absorbed with pipettor, adds isometric luciferase
Substrate (concentration：300 μ g/ml), mix；
4) RLU (relative light unit) is determined by multi-function microplate reader, minute is set to 1 second.
5) ratio calculation formula is killed：100% × (blank well reading-experimental port reading)/blank well reading (such as Fig. 4),
SP-CTL cells are as shown in Figure 6 to lung cancer in non-cellule type H460 Cytotoxicity in vitro cell proportion.
Embodiment 4, SC-CTL cells are to human leukemia cell line NALM6 Cytotoxicity in vitro effect
1) the transduction expressing luciferase (Luciferace) in Leukemia Cell Lines NALM6, structure NALM6-GL reports
Cell line；
2) respectively by GFP positive cell numbers be 3.5 × 104、1.75×104、8.6×103、4.4×103、2.2×103、
1.1×103SC-CTL virus infection cell mixing and 1 × 104(control group is thin to add GFP T for individual NALM6-GL cells co-cultivation
Born of the same parents, blank group are to be not added with T cell, three multiple holes of every group of setting)；
3) after 18 hours, the culture medium supernatant of half volume is gently absorbed with pipettor, adds isometric luciferase
Substrate (concentration：300 μ g/ml), mix；
4) RLU (relative light unit) is determined by multi-function microplate reader, minute is set to 1 second.
5) ratio calculation formula is killed：100% × (blank well reading-experimental port reading)/blank well reading, SC-CTL are thin
Born of the same parents are as shown in Figure 7 to human leukemia cell line NALM6 Cytotoxicity in vitro ratio.
Embodiment 5, SP-CTL cells are to lethal effect inside people's lung cancer in non-cellule type H460
1) by 1 × 105Be implanted under individual H460-GL cell skins immunodeficient mouse NOD/SCID IL2rg-/- in vivo；
2) after 10 days, it is 1 × 10 that SP-CTL GFP positive cell numbers are injected in H460-GL tumor-bearing mices5SP-CTL
Virus infection cell mixing, for control group to inject the tumor-bearing mice of GFP T cells, each experimental group sets three repetitions；
3) after tumour transplatation, H460 volume of tumor mass is measured within the 10th, 14,17,20,23,26,29,32,35 day, as a result
As shown in Figure 8.
By above testing result, the various tumours of S-CTL cells of the invention to expression respective ligand
With significantly inside, outer lethal effect.
Applicant states that the present invention illustrates product, purposes and its occupation mode of the present invention by above-described embodiment, but
The invention is not limited in above-mentioned detailed use of commodity and occupation mode, that is, do not mean that the present invention have to rely on above-mentioned detailed use of commodity and
Occupation mode could be implemented.Person of ordinary skill in the field produces it will be clearly understood that any improvement in the present invention to the present invention
The equivalence replacement of each raw material of product and the addition of auxiliary element, the selection of concrete mode etc., all fall within protection scope of the present invention and
Within the scope of open.
The list of gene order involved by this paper
SEQ ID NO.1
gtttAAACATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGG
TTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAA
CGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACC
AGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTG
CCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCAT
CTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGC
CCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTG
CTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCTTCGAAaaacggggcagaaagaaactcctgtatat
attcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaag
aagaaggaggatgtgaactgGAATTCCAGGCCAAAAGGAAGCCCAGGAAAGCTCCCAGCAGGAACATCTGCTATGAT
GCATTTGTTTCTTACAGTGAGCGGGATGCCTACTGGGTGGAGAACCTTATGGTCCAGGAGCTGGAGAACTTCAATCC
CCCCTTCAAGTTGTGTCTTCATAAGCGGGACTTCATTCCTGGCAAGTGGATCATTGACAATATCATTGACTCCATTG
AAAAGAGCCACAAAACTGTCTTTGTGCTTTCTGAAAACTTTGTGAAGAGTGAGTGGTGCAAGTATGAACTGGACTTC
TCCCATTTCCGTCTTTTTGATGAGAACAATGATGCTGCCATTCTCATTCTTCTGGAGCCCATTGAGAAAAAAGCCAT
TCCCCAGCGCTTCTGCAAGCTGCGGAAGATAATGAACACCAAGACCTACCTGGAGTGGCCCATGGACGAGGCTCAGC
GGGAAGGATTTTGGGTAAATCTGAGAGCTGCGATAAAGTCCGCGATCGCAagagtgaagttcagcaggagcgcagac
gcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgtttt
ggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatg
aactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcac
gatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcg
cACTAGT
SEQ ID NO.2
GTTTAAACATGGCTTGCCTTGGATTTCAGCGGCACAAGGCTCAGCTGAACCTGGCTACCAGGACCTGGCCCTGCACT
CTCCTGTTTTTTCTTCTCTTCATCCCTGTCTTCTGCAAAGCAATGCACGTGGCCCAGCCTGCTGTGGTACTGGCCAG
CAGCCGAGGCATCGCCAGCTTTGTGTGTGAGTATGCATCTCCAGGCAAAGCCACTGAGGTCCGGGTGACAGTGCTTC
GGCAGGCTGACAGCCAGGTGACTGAAGTCTGTGCGGCAACCTACATGATGGGGAATGAGTTGACCTTCCTAGATGAT
TCCATCTGCACGGGCACCTCCAGTGGAAATCAAGTGAACCTCACTATCCAAGGACTGAGGGCCATGGACACGGGACT
CTACATCTGCAAGGTGGAGCTCATGTACCCACCGCCATACTACCTGGGCATAGGCAACGGAACCCAGATTTATGTAA
TTGATCCAGAACCGTGCCCAGATTCTGACTTCCTCCTCTGGATCCTTGCAGCAGTTAGTTCGGGGTTGTTTTTTTAT
AGCTTTCTCCTCACATTCGAAaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccg
ccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccGAATTCAACA
TACCCTTAGAAGAACTCCAAAGAAATCTCCAGTTTCATGCATTTATTTCATATAGTGGGCACGATTCTTTCTGGGTG
AAGAATGAATTATTGCCAAACCTAGAGAAAGAAGGTATGCAGATTTGCCTTCATGAGAGAAACTTTGTTCCTGGCAA
GAGCATTGTGGAAAATATCATCACCTGCATTGAGAAGAGTTACAAGTCCATCTTTGTTTTGTCTCCCAACTTTGTCC
AGAGTGAATGGTGCCATTATGAACTCTACTTTGCCCATCACAATCTCTTTCATGAAGGATCTAATAGCTTAATCCTG
ATCTTGCTGGAACCCATTCCGCAGTACTCCATTCCTAGCAGTTATCACAAGCTCAAAAGTCTCATGGCCAGGAGGAC
TTATTTGGAATGGCCCAAGGAAAAGAGCAAACGTGGCCTTTTTTGGGCTAACTTAAGGGCAGCCATTAATATTAAGC
TGACAGAGCAAGCAAAGAAAGCGATCGCAagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggc
cagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccggga
ccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatgg
cggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctc
agtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcACTAGT
SEQ ID NO.3
gtttaaacatgttttcacatcttccctttgactgtgtcctgctgctgctgctgctactacttacaaggtcctcagaa
gtggaatacagagcggaggtcggtcagaatgcctatctgccctgcttctacaccccagccgccccagggaacctcgt
gcccgtctgctggggcaaaggagcctgtcctgtgtttgaatgtggcaacgtggtgctcaggactgatgaaagggatg
tgaattattggacatccagatactggctaaatggggatttccgcaaaggagatgtgtccctgaccatagagaatgtg
actctagcagacagtgggatctactgctgccggatccaaatcccaggcataatgaatgatgaaaaatttaacctgaa
gttggtcatcaaaccagccaaggtcacccctgcaccgactcggcagagagacttcactgcagcctttccaaggatgc
ttaccaccaggggacatggcccagcagagacacagacactggggagcctccctgatataaatctaacacaaatatcc
acattggccaatgagttacgggactctagattggccaatgacttacgggactctggagcaaccatcagaataggcat
ctacatcggagcagggatctgtgctgggctggctctggctcttatcTTCGAAaaacggggcagaaagaaactcctgt
atatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaa
gaagaagaaggaggatgtgaactgGAATTCCAGGCCAAAAGGAAGCCCAGGAAAGCTCCCAGCAGGAACATCTGCTA
TGATGCATTTGTTTCTTACAGTGAGCGGGATGCCTACTGGGTGGAGAACCTTATGGTCCAGGAGCTGGAGAACTTCA
ATCCCCCCTTCAAGTTGTGTCTTCATAAGCGGGACTTCATTCCTGGCAAGTGGATCATTGACAATATCATTGACTCC
ATTGAAAAGAGCCACAAAACTGTCTTTGTGCTTTCTGAAAACTTTGTGAAGAGTGAGTGGTGCAAGTATGAACTGGA
CTTCTCCCATTTCCGTCTTTTTGATGAGAACAATGATGCTGCCATTCTCATTCTTCTGGAGCCCATTGAGAAAAAAG
CCATTCCCCAGCGCTTCTGCAAGCTGCGGAAGATAATGAACACCAAGACCTACCTGGAGTGGCCCATG
GACGAGGCTCAGCGGGAAGGATTTTGGGTAAATCTGAGAGCTGCGATAAAGTCCGCGATCGCAagagtgaagttcag
caggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagagg
agtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaa
ggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggag
gggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcagg
ccctgccccctcgcACTAGT
SEQ ID NO.4
gtttaaacatgtgggaggctcagttcctgggcttgctgtttctgcagccgctttgggtggctccagtgaagcctctc
cagccaggggctgaggtcccggtggtgtgggcccaggagggggctcctgcccagctcccctgcagccccacaatccc
cctccaggatctcagccttctgcgaagagcaggggtcacttggcagcatcagccagacagtggcccgcccgctgccg
cccccggccatcccctggcccccggccctcacccggcggcgccctcctcctgggggcccaggccccgccgctacacg
gtgctgagcgtgggtcccggaggcctgcgcagcgggaggctgcccctgcagccccgcgtccagctggatgagcgcgg
ccggcagcgcggggacttctcgctatggctgcgcccagcccggcgcgcggacgccggcgagtaccgcgccgcggtgc
acctcagggaccgcgccctctcctgccgcctccgtctgcgcctgggccaggcctcgatgactgccagccccccagga
tctctcagagcctccgactgggtcattttgaactgctccttcagccgccctgaccgcccagcctctgtgcattggtt
ccggaaccggggccagggccgagtccctgtccgggagtccccccatcaccacttagcggaaagcttcctcttcctgc
cccaagtcagccccatggactctgggccctggggctgcatcctcacctacagagatggcttcaacgtctccatcatg
tataacctcactgttctgggtctggagcccccaactcccttgacagtgtacgctggagcaggttccagggtggggct
gccctgccgcctgcctgctggtgtggggacccggtctttcctcactgccaagtggactcctcctgggggaggccctg
acctcctggtgactggagacaatggcgactttacccttcgactagaggatgtgagccaggcccaggctgggacctac
acctgccatatccatctgcaggaacagcagctcaatgccactgtcacattggcaatcatcacagtgactcccaaatc
ctttgggtcacctggatccctggggaagctgctttgtgaggtgactccagtatctggacaagaacgctttgtgtgga
gctctctggacaccccatcccagaggagtttctcaggaccttggctggaggcacaggaggcccagctcctttcccag
ccttggcaatgccagctgtaccagggggagaggcttcttggagcagcagtgtacttcacagagctgtctagcccagg
tgcccaacgctctgggagagccccaggtgccctcccagcaggccacctcctgctgtttctcatccttggtgtccttt
ctctgctccttttggtgactggagcctttggctttTTCGAAaaacggggcagaaagaaactcctgtatatattcaaa
caaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaagg
aggatgtgaactgGAATTCCAGGCCAAAAGGAAGCCCAGGAAAGCTCCCAGCAGGAACATCTGCTATGATGCATTTG
TTTCTTACAGTGAGCGGGATGCCTACTGGGTGGAGAACCTTATGGTCCAGGAGCTGGAGAACTTCAATCCCCCCTTC
AAGTTGTGTCTTCATAAGCGGGACTTCATTCCTGGCAAGTGGATCATTGACAATATCATTGACTCCATTGAAAAGAG
CCACAAAACTGTCTTTGTGCTTTCTGAAAACTTTGTGAAGAGTGAGTGGTGCAAGTATGAACTGGACTTCTCCCATT
TCCGTCTTTTTGATGAGAACAATGATGCTGCCATTCTCATTCTTCTGGAGCCCATTGAGAAAAAAGCCATTCCCCAG
CGCTTCTGCAAGCTGCGGAAGATAATGAACACCAAGACCTACCTGGAGTGGCCCATGGACGAGGCTCAGCGGGAAGG
ATTTTGGGTAAATCTGAGAGCTGCGATAAAGTCCGCGATCGCAagagtgaagttcagcaggagcgcagacgcccccg
cgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaag
agacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgca
gaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcc
tttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcACTAGT
Claims (10)
1. a kind of chimeric immunosupress checkpoint acceptor molecule, it includes ectodomain, membrane spaning domain and intracellular structure
Domain, wherein, the ectodomain and optionally membrane spaning domain are the corresponding construction domain of immunosupress checkpoint acceptor molecule
Or the genetic modification carried out based on the domain, the intracellular domain is one or more immune activation signal domains.
2. immunosupress checkpoint according to claim 1 acceptor molecule, it is characterised in that the immunosupress checkpoint
Acceptor molecule is selected from PD-1, CTLA-4, LAG3, TIM3, A2AR, B7H3, B7H4, BTLA, IDO, KIR, CD160,2B4
And VISTA (C10orf54) (CD244)；
Preferably, the immunologic test point molecule is PD-1, CTLA-4, LAG3 or TIM3.
3. immunosupress checkpoint according to claim 1 or 2 acceptor molecule, it is characterised in that the intracellular domain
Include the combination of immune costimulatory signal and optional signal peptide；
Preferably, the immune costimulatory signal combination includes TLR1 and/or TLR2 signal domains；
It is further preferred that the intracellular domain is TLR1 and/or TLR2 and the group of 41BB and/or CD28 signal domains
Close；
Preferably, optional signal peptide is the corresponding signal peptide of immunosupress checkpoint acceptor molecule.
4. immunosupress checkpoint according to claim 3 acceptor molecule, it is characterised in that the intracellular domain is also wrapped
Include CD3 ζ intracellular domain；
Preferably, TLR1 and/or TLR2 signal domains and optionally 41BB and/or CD28 signal structures configuration of territory exists
The N-terminal side of CD3 ζ intracellular domain.
5. encode the nucleic acid of the chimeric immunosupress checkpoint acceptor molecule as described in claim any one of 1-4.
6. a kind of nucleic acid construct, it includes nucleic acid as claimed in claim 5, and is operatively connected therewith, can instruct institute
State one or more control sequences that chimeric immunosupress checkpoint acceptor molecule is expressed in host cell.
7. a kind of expression vector, it includes nucleic acid construct as claimed in claim 6；Preferably, the expression vector is slow
Virus.
8. a kind of cell of conversion, it includes nucleic acid as claimed in claim 5, or has wherein converted such as claim 6 institute
The nucleic acid construct or expression vector as claimed in claim 7 stated；
Preferably, the cell of the conversion is immunocyte, preferably T cell, B cell or NK cells.
9. preparing the method for the cell converted as claimed in claim 8, it includes：Introducing right such as into cellular genome will
The nucleic acid described in 5 is sought, or converts nucleic acid construct as claimed in claim 6 or expression vector as claimed in claim 7
The step of.
10. chimeric immunosupress checkpoint acceptor molecule as described in claim any one of 1-4, as claimed in claim 5
Nucleic acid, nucleic acid construct as claimed in claim 6, expression vector as claimed in claim 7 or as claimed in claim 8
Conversion cell prepare with immunosupress checkpoint acceptor molecule, preferably with PD-L1, B7.1, B7.2, Galectin-9,
Purposes in the medicine of the disease of MHC II expression correlation；
Preferably, the disease be expression immunosupress checkpoint acceptor molecule, preferred expression PD-L1, B7.1, B7.2,
Galectin-9, MHC II tumour；
It is further preferred that expression immunosupress checkpoint acceptor molecule, preferred expression PD-L1, B7.1, B7.2,
Galectin-9, MHC II tumour be selected from lung cancer, leukaemia, breast cancer, prostate cancer, cancer of pancreas, liver cancer, melanoma and
Nonmelanoma skin cancer.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610443073.7A CN107522786A (en) | 2016-06-20 | 2016-06-20 | A kind of molecule, the cell for expressing it and its production and use |
| PCT/CN2017/088594 WO2017219916A1 (en) | 2016-06-20 | 2017-06-16 | Molecule, cell expressing the same, and preparation method and use thereof |
| CN202110620458.7A CN113512126B (en) | 2016-06-20 | 2017-06-16 | Molecule, cell expressing same and preparation method and application thereof |
| CN201780000466.3A CN107624119B (en) | 2016-06-20 | 2017-06-16 | Molecules, cells expressing the same, and preparation methods and uses thereof |
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|---|---|---|---|
| CN201610443073.7A CN107522786A (en) | 2016-06-20 | 2016-06-20 | A kind of molecule, the cell for expressing it and its production and use |
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| CN107522786A true CN107522786A (en) | 2017-12-29 |
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| CN201610443073.7A Pending CN107522786A (en) | 2016-06-20 | 2016-06-20 | A kind of molecule, the cell for expressing it and its production and use |
| CN201780000466.3A Active CN107624119B (en) | 2016-06-20 | 2017-06-16 | Molecules, cells expressing the same, and preparation methods and uses thereof |
| CN202110620458.7A Active CN113512126B (en) | 2016-06-20 | 2017-06-16 | Molecule, cell expressing same and preparation method and application thereof |
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| CN202110620458.7A Active CN113512126B (en) | 2016-06-20 | 2017-06-16 | Molecule, cell expressing same and preparation method and application thereof |
Country Status (2)
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| CN (3) | CN107522786A (en) |
| WO (1) | WO2017219916A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112226412A (en) * | 2020-10-12 | 2021-01-15 | 广东昭泰体内生物医药科技有限公司 | A T cell expressing immunosuppressive checkpoint receptor molecule and its application |
| CN114081941A (en) * | 2021-11-19 | 2022-02-25 | 上海奢旭企业管理中心(有限合伙) | The preparation method of B7-H4 protein and its application in the preparation of anti-excessive immune response or anti-cytokine storm drugs |
| CN116731205A (en) * | 2023-05-19 | 2023-09-12 | 皖南医学院第一附属医院（皖南医学院弋矶山医院） | Immunopotentiating fusion protein for expressing TGF beta RII and TIM3 extracellular region and application thereof |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2018341244A1 (en) | 2017-09-26 | 2020-03-05 | Cero Therapeutics, Inc. | Chimeric engulfment receptor molecules and methods of use |
| WO2019191340A1 (en) | 2018-03-28 | 2019-10-03 | Cero Therapeutics, Inc. | Cellular immunotherapy compositions and uses thereof |
| WO2019191334A1 (en) | 2018-03-28 | 2019-10-03 | Cero Therapeutics, Inc. | Chimeric tim4 receptors and uses thereof |
| CN117402262A (en) * | 2022-10-19 | 2024-01-16 | 上海君赛生物科技有限公司 | LAG3-based chimeric immune cell coreceptors and their uses |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2842102T3 (en) * | 2012-08-20 | 2021-07-12 | Hutchinson Fred Cancer Res | Method and compositions for cellular immunotherapy |
| CN103965361B (en) * | 2013-02-06 | 2018-10-30 | 上海细胞治疗工程技术研究中心集团有限公司 | A kind of chimeric molecule converter of T cell signal and application thereof |
| ES2837856T3 (en) * | 2013-12-20 | 2021-07-01 | Hutchinson Fred Cancer Res | Labeled Chimeric Effector Molecules and Receptors |
| EP4303229A3 (en) * | 2014-01-21 | 2024-04-17 | Novartis AG | Enhanced antigen presenting ability of car t cells by co-introduction of costimulatory molecules |
| US20170258835A1 (en) * | 2014-10-31 | 2017-09-14 | The Trustees Of The University Of Pennsylvania | Methods and compositions for modified t cells |
| CN104673897B (en) * | 2015-01-26 | 2017-09-29 | 江苏先声药业有限公司 | A kind of method of the biological activity of the measure pathway inhibitors of PD 1 |
| CN104829733B (en) * | 2015-05-25 | 2018-06-05 | 广州百暨基因科技有限公司 | The Chimeric antigen receptor and preparation method and application that antigen-binding unit is stablized |
| CN105153315B (en) * | 2015-10-09 | 2019-04-02 | 重庆精准生物技术有限公司 | Immunosupress receptor combination tumor antigen Chimerical receptor and its application |
| CN105331585A (en) * | 2015-11-13 | 2016-02-17 | 科济生物医药（上海）有限公司 | Chimeric antigen receptor-modified immunologic effector cell with PD-L1 blocking agent |
| CN105601752B (en) * | 2016-02-01 | 2019-08-23 | 海门郝怡雅医药科技有限公司 | A kind of multigene recombinant chimeric antigen receptor molecule |
-
2016
- 2016-06-20 CN CN201610443073.7A patent/CN107522786A/en active Pending
-
2017
- 2017-06-16 CN CN201780000466.3A patent/CN107624119B/en active Active
- 2017-06-16 WO PCT/CN2017/088594 patent/WO2017219916A1/en not_active Ceased
- 2017-06-16 CN CN202110620458.7A patent/CN113512126B/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112226412A (en) * | 2020-10-12 | 2021-01-15 | 广东昭泰体内生物医药科技有限公司 | A T cell expressing immunosuppressive checkpoint receptor molecule and its application |
| CN114081941A (en) * | 2021-11-19 | 2022-02-25 | 上海奢旭企业管理中心(有限合伙) | The preparation method of B7-H4 protein and its application in the preparation of anti-excessive immune response or anti-cytokine storm drugs |
| CN116731205A (en) * | 2023-05-19 | 2023-09-12 | 皖南医学院第一附属医院（皖南医学院弋矶山医院） | Immunopotentiating fusion protein for expressing TGF beta RII and TIM3 extracellular region and application thereof |
| CN116731205B (en) * | 2023-05-19 | 2024-02-27 | 皖南医学院第一附属医院（皖南医学院弋矶山医院） | Immunopotentiating fusion protein for expressing TGF beta RII and TIM3 extracellular region and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107624119A (en) | 2018-01-23 |
| CN113512126A (en) | 2021-10-19 |
| CN113512126B (en) | 2023-05-05 |
| CN107624119B (en) | 2021-07-09 |
| WO2017219916A1 (en) | 2017-12-28 |
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Application publication date: 20171229 |