CN109422815A - Bispecific chimeric antigen receptor c-Met/PD-1 scFv-CAR-T and its construction method and application - Google Patents

Bispecific chimeric antigen receptor c-Met/PD-1 scFv-CAR-T and its construction method and application Download PDF

Info

Publication number
CN109422815A
CN109422815A CN201710759332.1A CN201710759332A CN109422815A CN 109422815 A CN109422815 A CN 109422815A CN 201710759332 A CN201710759332 A CN 201710759332A CN 109422815 A CN109422815 A CN 109422815A
Authority
CN
China
Prior art keywords
antigen receptor
scfv
chimeric antigen
met
cell
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
CN201710759332.1A
Other languages
Chinese (zh)
Inventor
于敏
孙祖俊
王羽雄
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.)
Fudan University
Original Assignee
Fudan University
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 Fudan University filed Critical Fudan University
Priority to CN201710759332.1A priority Critical patent/CN109422815A/en
Publication of CN109422815A publication Critical patent/CN109422815A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • 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
    • 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
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Abstract

The invention belongs to field of biotechnology, it is related to a kind of bispecific chimeric antigen receptor hc-Met scFv-PD-1scFv-CD8 α-CD28-CD137-CD3 ζ and its construction method and application, the Chimeric antigen receptor is by anti-human hepatocyte growth factor receptor (c-Met) monoclonal antibody light chain and heavy chain variable region (c-Met scFv), anti-human 1 receptor of programmed death (PD-1) monoclonal antibody light chain and heavy chain variable region (PD-1 scFv), the extracellular ligand binding domain 1 receptor of people's programmed death (PD-1), people's CD8 α hinge area, people CD28 transmembrane region and intracellular region, people's CD137 and CD3 ζ intracellular region structures in series is constituted.For the Chimeric antigen receptor for modifying T lymphocyte, the T cell (CAR-T cell) after modification is used to prepare entity tumor immunity therapeutic preparation, can especially be used for the oncotherapy preparation of surface c-Met and the PD-L1 positive.

Description

Bispecific chimeric antigen receptor c-Met/PD-1 scFv-CAR-T and its building side Method and application
Technical field
The invention belongs to field of biotechnology, are related to antigen receptor c-Met/PD-1 scFv-CAR-T and its construction method, tool Body is related to bispecific chimeric antigen receptor c-Met/PD-1 scFv-CAR-T and its construction method and application.The present invention utilizes Technique for gene engineering obtains the fusion of the recombination Chimeric antigen receptor containing bispecific, makes T cell expression should after infecting T cell Bispecific chimeric antigen receptor.The bispecific chimeric antigen receptor T cell be capable of tumor cell surface c-Met and The Inhibitory receptor PD-1 for blocking activation T cell surface avoids activation T cell from being induced apoptosis, keeps sustained activation ability, from And the tumour cell of the specific killing c-Met positive, it can be applied to the immunization therapy of entity tumor.
Background technique
Prior art discloses in normal immunoreaction process, non-sensitized T cell at least needs the stimulation of two kinds of signals Specific immune response can be activated to generate；Wherein first signal is that T cell is identified by T cell receptor (TCR) Antigen in conjunction with MHC-I or II class molecule transmits antigen recognizing signal by TCR/CD3 complex；Second signal be with The costimulatory molecules of T cell surface receptor molecule identification antigen presenting cell (APC) or other cell surfaces based on CD28, pass Pass costimulatory signal.Studies have shown that tumor specific T cells play mainly in the immune response that body removes tumour cell Effect, while tumour cell is by inhibiting to participate in T cell identification and antigen reactive developed by molecule, reducing the approach such as immunogenicity Generate immunologic escape, thus prevent the immune system of body from play remove tumour cell effect.
Studies have found that replacing α, β chain of TCR variable with the single-stranded variable region (scFv) of tumor specific monoclonal antibodies Area, and scFv is directly connected with costimulatory molecules (such as CD28, CD137) and T cell signal transduction region CD3 ζ, it is formed Chimeric antigen receptor (CAR) is expressed in T cell surface, and tumour specific antigen can be identified by scFv, and directly generation T cell is living The first, second signal changed promotes T cell activation, proliferation, thus specific killing tumour cell.The process relies primarily on The scFv of CAR-T cell surface is to the specific recognition of tumour antigen, the specificity of immune response and lethal relatively strong.
Currently, CAR-T cell obtains significant curative effect in Malignancy treatment, but answered in the clinic of solid tumor With being but faced with lot of challenges.Major obstacle first is that due to entity tumor immunosupress microenvironment, i.e., by up-regulation suppression Property receptor processed inhibits tumor infiltrating lymphocyte (TIL) and the genetic engineering to modify the function of T cell, especially immunosupress The up-regulation of property receptor PD-1.PD-1 is the most inhibitive ability of immunity receptors studied at present, is expressed in T cell and precursor B cell surface, a member of contactin.PD-L1 is the PD-1 major ligand having now been found that, research is found PD-L1 is increased in the expression of kinds of tumor cells film surface, and tumour cell dexterously goes to combine T cell table using PD-L1 The PD-1 receptor in face transmits inhibition signal, so as to cause immunologic escape.
There are preclinical study and clinical test to point out, enhances cancer patient's using PD-L1 or PD-1 blocking antibody Anti-tumor immune response can be used as a kind of novel immunotherapy of tumors means, and can obtain the clinical response of duration. In addition, there is research to prompt, CAR-T cell therapy, which combines anti-PD-1 antibody, can greatly enhance the anti-tumor effect of CAR-T cell, PD-1-scFv-CAR fusion receptors so are expressed in CAR-T cell membrane surface, i.e., CAR-T cell table are closed using PD-1 scFv Does the PD-1 receptor in face enhance the effectiveness of CAR-T cell therapy to avoid the inhibition signal of PD-L/PD-1? utilize anti-c-Met The bispecific chimeric antigen receptor hc-Met scFv-Linker-PD-1 scFv-CD8 α-of scFv and anti-PD-1 scFv building CD28-CD137-CD3 ζ is expressed in T cell surface, can make the tumour cell of the T cell specific killing c-Met positive, seal simultaneously The Inhibitory receptor PD-1 for closing T cell surface makes T cell sustained activation, therefore the T of bispecific chimeric antigen receptor modification is thin Born of the same parents, which are expected to break to adopt, transmits the immunosupress signal shaft of the PD-L/PD-1 in T cell treatment, leads in entity tumor immunization therapy Domain has broad application prospects.
Status based on the prior art, present inventor is quasi- to provide bispecific chimeric antigen receptor c-Met/PD-1 ScFv-CAR-T and its construction method and for the purposes in the immunization therapy of entity tumor.
Summary of the invention
The invention aims to solve technical problem of the existing technology, provide a kind of bispecific chimeric antigen by Body c-Met/PD-1 scFv-CAR-T and its construction method and for the purposes in the immunization therapy of entity tumor.
The present invention constructs the anti-receptor of chimeric antigen (the hc-Met scFv- of anti-hc-Met scFv-PD-1scFv a kind of Linker-PD-1 scFv-CD8 α-CD28-CD137-CD3 ζ), the T cell for expressing the Chimeric antigen receptor has its uniqueness, It is verified by experiments to tumor cell specific killing ability, with the application prospect in clinical treatment.
The present invention is obtained a kind of containing bispecific recombination Chimeric antigen receptor hc-Met scFv- using technique for gene engineering The genetic fragment is inserted into Lentiviral system by the fusion of PD-1 scFv-CD8 α-CD28-CD137-CD3 ζ, The supernatant containing virus is obtained by carrying out packaging to slow virus, T cell is infected, T cell is made to express the bispecific chimeric antigen Receptor.This bispecific chimeric antigen receptor T cell (CAR-T), which is capable of the c-Met on tumor cell surface and blocks, swashs The Inhibitory receptor PD-1 on T cell surface living avoids activation T cell from being induced apoptosis, sustained activation ability is kept, thus specifically Property killing the c-Met positive tumour cell, can be applied to the immunization therapy of entity tumor.
More specifically, the technical solution adopted by the present invention is that: bispecific chimeric antigen receptor hc-Met scFv- Linker-PD-1 scFv-CD8 α-CD28-CD137-CD3 ζ, the Chimeric antigen receptor is by anti-human c-Met and PD-1 monoclonal Antibody light chain and heavy chain variable region hc-Met scFv, PD-1 scFv, people CD8 α hinge area, people CD28 transmembrane region and intracellular region, People's CD137 and CD3 ζ intracellular region structures in series is constituted；The amino acid sequence such as sequence table of the bispecific chimeric antigen receptor Shown in middle SEQ ID NO.1；
The nucleic acid sequence of the bispecific chimeric antigen receptor is as shown in SQE ID NO.2；
The bispecific chimeric antigen receptor contains the single-chain antibody hc-Met scFv for people's c-Met antigen, ammonia Base acid sequence is connected between the heavy chain and light chain of single-chain antibody with the connection small peptide of 15 amino acid as shown in SEQ ID NO.3；
The Chimeric antigen receptor contains the single-chain antibody hc-MetscFv for people's c-Met antigen, and nucleic acid sequence is such as Shown in SEQ ID NO.4；
The Chimeric antigen receptor contains the extracellular combined area receptor PD-1 for human PD-L 1, amino acid sequence such as SEQ Shown in ID NO.5；
The Chimeric antigen receptor contains the extracellular combined area receptor PD-1 for human PD-L 1, nucleic acid sequence such as SEQ Shown in ID NO.6；
The amino terminal of the Chimeric antigen receptor contains a CD8 alpha signal peptide, in amino acid sequence such as sequence table Shown in SEQ ID NO.7；
The amino terminal of the Chimeric antigen receptor contains a CD8 alpha signal peptide, in nucleic acid sequence such as sequence table Shown in SEQ ID NO.8.
Invention further provides the bispecific antigen receptor hc-Met scFv-Linker-PD-1 scFv- CD8 α-CD28-CD137-CD3 ζ is in preparation Chimeric antigen receptor T cell and its application in immunotherapy of tumors.
The present invention uses anti-human c-Met scFv and PD-1 scFv amino acid sequence, is carried out codon optimization, from People's CD8 alpha signal peptide gene, people's CD8 α hinge area gene, people CD28 transmembrane region and born of the same parents are searched in NCBI GenBank database Inner region gene, CD137 and CD3 ζ intracellular region gene sequence information, full genome synthesize CD8 alpha signal peptide, CD8 α-CD28- Then CD137-CD3 ζ genetic fragment connects into bispecific chimeric antigen receptor hc-Met scfv-PD-1 by recombinase ScFv-CD8 α-CD28-CD137-CD3 ζ is inserted into Lentiviral pKC-EF1 α-DS-Red (pKC- empty carrier), Construct anti-human c-Met/PD-1 scFv-CAR expression vector (as shown in Figure 1)；It sends sequencing outside and complies fully with design (such as Fig. 2 institute Show)；The packaging virus in 293T cell using the plasmid and the packaging plasmid VSVG and r-8.91 of slow virus, infection Jurkat are thin Born of the same parents (as shown in Figure 3)；Jurkat cell is set to express the Chimeric antigen receptor.The CAR-Jurkat cell of acquisition in vitro with c- The Met and tumour cell MKN45 of the PD-L1 positive is co-cultured, and ELISA method detection co-cultures the cytokine levels in supernatant (as shown in Figure 4)；Radioactivity cytotoxicity method detects CAR-Jurkat cell to the killing activity of MKN45 cell (as shown in Figure 5)；Experiment in vivo detects its influence to tumour growth, it was confirmed that the T cell pair of Chimeric antigen receptor modification The specific killing action of tumour cell.Meanwhile it is single that targeting c-Met scFv-CAR and PD-1-CAR are constructed in the present invention The Chimeric antigen receptor of target spot, as comparing superiority of the bispecific chimeric antigen receptor in terms of inhibiting tumour function.
Chimeric antigen receptor of the invention is for modifying T lymphocyte, and the T cell (CAR-T cell) after modification is for making Standby entity tumor immunity therapeutic preparation, can especially be used for the oncotherapy preparation of surface c-Met and the PD-L1 positive；Described Bispecific chimeric antigen receptor hc-Met scFv-PD-1 scFv-CD8 α-CD28-CD137-CD3 ζ.Immune in tumour is controlled It has broad application prospects in treatment,
Detailed description of the invention
Fig. 1 is pKC-CAR Lentiviral schematic diagram.
Fig. 2 is that peak value figure is sequenced in pKC-CAR Lentiviral part.
The red fluorescence (Red) of Jurkat T cell expresses feelings after Fig. 3 is fluorescence microscopy microscopic observation slow-virus infection 48h Condition.
Fig. 4 is CAR-T experimental group, IL-2 concentration compares in cellular control unit supernatant.
Fig. 5 is under different effect target ratio situations, and CAR-T experimental group is compared with cellular control unit toxicity.
Fig. 6 is the influence that CAR-T cell grows transplanted tumor in nude mice.
Specific embodiment
Invention is further described in detail with reference to the accompanying drawings and detailed description:
The determination of embodiment 1:hc-MetscFv-PD-1scFv-CD8 α-CD28-CD137-CD3 ζ gene order
People's PD-1 extracellular fragment gene, people's CD8 alpha signal peptidyl are searched from U.S. national library of medicine site databases Cause, people's CD8 α hinge area gene, people CD28 transmembrane region and intracellular region gene, CD137 and CD3 ζ intracellular region gene order；Anti- c- The monoclonal antibody sequence that Met and PD-1 single-chain antibody (anti-c-Met scFv and PD-1 scFv) gene order constructs in fact from this Column, are carried out codon optimization, to guarantee to be more suitable for the expression of T cell in the case where encoding amino acid sequence is constant, respectively Shown in gene sequence information such as SEQUENCE LISTING (sequence table SEQ ID NO.1-8)；
Said gene sequence is successively pressed into people's CD8 alpha signal peptide gene, anti-human c-Met scFv, anti-human PD-1 scFv, CAR (including people's CD8 α hinge area gene, people CD28 transmembrane region and intracellular region gene, people's CD137 and CD3 ζ intracellular region gene order) into Row connection, introduces different restriction enzyme sites in each sequence junction, ultimately forms complete hc-Met scFv-PD-1 scFv- CAR gene sequence information.
Embodiment 2
Construct hc-Met scFv-PD-1 scFv-CD8 α-CD28-CD137-CD3 ζ expression plasmid
It overlaps PCR and synthesizes genetic fragment:
C-Met scFv, PD-1 scFv, CAR sequence are synthesized using overlapping PCR, is introduced in each sequence junction different Restriction enzyme site is linked to be complete c-Met scFv-PD-1 scFv-CAR sequence using recombinase, is cloned into pKC-EF1 α-DS- In RED Lentiviral (pKC- empty carrier), anti-human c-Met scfv-PD-1scFv-CAR expression plasmid (pKC- is obtained CAR plasmid) and DH5 α bacterium solution containing the plasmid, plasmid information it is as shown in Figure 1；
Recombinant plasmid sequencing:
Send Science and Technology Ltd., Shanghai Major Biological Medical Technology Co., Ltd. to be sequenced the recombinant plasmid, by sequencing result be fitted to Hc-Met scFv-PD-1 scFv-CAR sequence alignment is to confirm that sequence is correct, on sequencing primer pKC-EF1 α-DS-Red carrier Universal sequencing primer object:
Sense:5 '-GACTCAGCCGGCTCT-3 '
Anti-sense:5 '-CTCGCCCTCGATCTC-3 '
Part sequencing result is as shown in Figure 2.
Embodiment 3: a large amount of extractings of purpose plasmid and packaging plasmid
By the bacterial strain of pKC-CAR plasmid, pKC- empty carrier plasmid and VSVG and r-8.91 packaging plasmid in LB culture solution Middle mass propgation, with alkaline lysis large quantity extracting plasmid, (Beijing Tiangeng biochemical technology Co., Ltd endotoxin-free plasmid extracts examination Agent box), in case transfection.
1) bacterium solution is added in 100ml LB culture solution with ampicillin in 1: 1000 ratio, 37 DEG C of constant temperature incubators 220rpm shakes 12-16h；
2) a small amount of bacterium solution is taken to survey A600Absorbance stops shaking up to 0.4 or so；
3) the equilibrium liquid BL, 8000rpm that 2.5ml is added into adsorption column CP6 are centrifuged 2min, outwell useless in collecting pipe Liquid places back in adsorption column in collecting pipe；
4) bacterium solution is centrifuged 3min in 4 DEG C of 8000rpm, as far as possible absorption supernatant, collects bacterial precipitation；
5) 8ml solution P1 (RNaseA has been added in determination), thorough suspended bacterial cell precipitation is added；
6) 8ml solution P2 is added, leniently turns upside down 6-8 times immediately, cracks thallus sufficiently, be placed at room temperature for 5min；
7) 8ml solution P4 is added, leniently turns upside down 6-8 times, mixes well immediately, until white dispersion wadding occurs in solution Shape precipitating, is placed at room temperature for 10min, and 8000rpm is centrifuged 10min；Make white precipitate to tube bottom, complete soln is carefully poured into In filter CS1, push handle filtering is slowly pushed, filtrate is collected in the centrifuge tube of clean 50ml；
8) isopropanol of 0.3 times of filtrate volume is added into filtrate, is transferred to after mixing of turning upside down in absorption note CP6；
9) room temperature 8000rpm is centrifuged 2min, outwells the waste liquid in collecting pipe, adsorption column CP6 is placed back in collecting pipe；
10) 10ml rinsing liquid PW (please first check whether and dehydrated alcohol has been added) is added into adsorption column CP6,8000rpm It is centrifuged 2min, the waste liquid in collecting pipe is discarded, adsorption column is placed back in collecting pipe；
11) repetitive operation step 10；
12) 3ml dehydrated alcohol is added into adsorption column CP6, room temperature 8000rpm is centrifuged 2min, outwells waste liquid；
13) adsorption column CP6 is placed back in collecting pipe, 8000rpm is centrifuged 5min, by rinsing liquid remaining in adsorption column Removal；
14) it is sterile it is ultra-clean it is interior adsorption column CP6 is placed in a new 50ml collecting pipe, phase adsorbed film middle part position is outstanding 2ml elution buffer TB is added dropwise in sky, is placed at room temperature for 5min, and then 8000rpm is centrifuged 2min, by the eluent in 50ml centrifuge tube 2 clean 1.5ml centrifuge tubes all are distributed into, -20 DEG C save backup；
15) 2 μ l DNA solution ultraviolet spectrophotometries will be taken to survey plasmid DNA concentration and A260/A280, and carry out fine jade simultaneously The quality of sepharose electrophoretic analysis plasmid.
Embodiment 4: the packaging of slow virus
Cell processing: being digested for 24 hours with pancreatin before transfection, collect the 4-12 band 293T cell in logarithmic growth phase, will Cell is transferred in 10cm culture dish, and cell is grown in the DMEM culture medium that 10ml contains 10%FBS, 1%PS, sets 37 DEG C 5%CO2Incubator is cultivated for 24 hours, is transfected when converging rate up to 60-70%；
Rotaring redyeing system configuration:
1) in 50 μ l serum-free Opti-MEM culture mediums are added in 1.5ml sterilizing EP pipe, 10 μ g plasmid (pKC- are added The plasmid or pKC- empty plasmid of CAR mesh: VSVG: r-8.91=4: 3: 3), mixing well, be stored at room temperature 5min；Another 1.5 50 μ l serum-free Opti-MEM culture mediums and 30 μ l transfection reagent lipofectamine 2000 are added in sterile centrifugation tube (Lipo2000: total DNA (μ g)=2: 1), mixes well, is stored at room temperature 5min, then mixes solution in two pipes, is stored at room temperature 20min；
2) culture solution in former culture dish is carefully sucked out, 8ml serum-free Opti-MEM culture medium is added；
3) above-mentioned plasmid-Lipo2000 mixed liquor is added in 10cm culture dish, it is careful to mix；
4) culture dish is put into 37 DEG C of 5%CO212-16h is cultivated, 2ml FBS is added, continues to cultivate；
5) transfection for 24 hours after after fluorescence microscopy microscopic observation 293T cell transfecting Red luciferase expression situation, respectively at transfection 48h, 2h collect cells and supernatant afterwards, and room temperature 3000rpm is centrifuged 15min, collect supernatant；
6) with 0.45 μm of filter filter virus supernatant, pKC- zero load and pKC-CAR virus stock solution used are obtained respectively.
Culture, infection and the amplification of embodiment 5Jurkat T cell
1) the JurkatT cell in the T cell source that this room of Soviet Union freezes is incubated, 4 generations of biography are seeded to 24 orifice plates later, to culture The polybrene pretreatment 4h of final concentration of 4 μ g/ml is added in every hole in Jurkat cell for 24 hours, and pKC- empty carrier is then added Or 200 μ l of pKC-CAR viral concentration liquid, it is placed in incubator culture, it is interior for 24 hours not change liquid；
2) infection carries out infecting for second afterwards for 24 hours, and cell 1000rpm is centrifuged 10min, carefully sucks supernatant, is added new The fresh RPMI 1640 culture medium containing 10%FBS adjusts cell concentration to 1 × 10 after being resuspended6/ ml pre-processes 4h with polybrene After 200 μ l pKC-CAR viral concentration liquid be added continue to cultivate；
3) continue after cultivating 96h, with the RED red fluorescence expression of fluorescence microscope JurkatT cell and shine Phase.Jurkat infection rate is up to 50% or so (as shown in Figure 3) as the result is shown.
Embodiment 6
Co culture system in vitro measures CAR-Jurkat T cell secrete cytokines and tests to tumor cell killing potential
ELISA method measures cytokine levels:
ELISA kit (is purchased from R&D company), operates by kit specification.
1) it will be taken out from the supernatant of C, D, E group culture 36h from -20 DEG C, room temperature is melted；
2) concentrated cleaning solution, dilution, standard items, sealing lath are taken out into room temperature pre-temperature from refrigerator；
3) concentrated cleaning solution and dilution are diluted to working concentration with distilled water respectively；
4) standard items are prepared: 500ml dilution is added into freeze-drying standard items, dissolves at least 20min naturally, mixes, it is dilute It is interpreted into 2000,1000,500,250,125,62.5,31.25,15.6 and 0pg/ml；
5) standard items of sample and various concentration being added in corresponding aperture, 100 holes μ l/ seal reacting hole with sealing plate gummed paper, 37 DEG C of incubation 2h；
6) board-washing 5 times dry liquid in plate；
7) corresponding antibodies are balanced to room temperature, 100 holes μ l/ is added, seal reacting hole, 37 DEG C of incubation 1h with sealing plate gummed paper；
8) board-washing 5 times dry liquid in plate；
9) chromogenic substrate is balanced to room temperature, 100 μ l are added in every hole, and 37 DEG C are protected from light incubation 30min；
10) terminate liquid is balanced to room temperature, 50 hole μ l/ of terminate liquid is added, microplate reader 450nm (detection in 30min after mixing Wavelength) and 570nm (tuning wavelength) wavelength measurement absorbance value；
11) 4 parameters (4-PL) linear standard curve is drawn, its concentration is found by the OD value of sample.Double spies as the result is shown The Jurkat cell of anisotropic Chimeric antigen receptor modification can be obviously promoted the secretion (as shown in Figure 4) of IL-2 and IFN-γ.
It is living to the killing of MKN45 cell that 96 non-radioactive cell toxicity method of CytoTox detects CAR-Jurkat T cell Property:
Kit is purchased from Promega company, and by specification requires operation.
1) prepared by target cell: 800rpm is centrifuged 5min and collects logarithmic growth phase MKN45 cell, with the RPMI containing 5%FBS 1640 adjust cell concentrations to 2 × 106/ml；
2) prepared by effector cell: 1000rpm is centrifuged 10min and collects the CAR-Jurkat for infecting latter week, with containing 5%FBS RPMI 1640 adjust cell concentration to 2 × 106/ml。
3) following control group and experimental group (three wells) are set in 96 orifice plate of the bottom U:
4) in different effects: target ratio (2: 1,1: 2,1: 4,1: 8 and 1: 16) adding effector cell and target cell, with containing 5% It is 200 holes μ l/ that the RPMI 1640 of FBS, which supplies volume,；
5) 200g is centrifuged 96 hole plate 4min, sets 37 DEG C, 5%CO2, saturated humidity incubator culture 7h, 45min exists in advance Target cell maximum relief hole adds 10 μ l cell pyrolysis liquids (10X), and 200g is centrifuged plate 4min；
6) LDH activity measures；
7) CTL activity calculates: calculating each group (3 multiple holes) mean light absorbency (A490), calculates CTL by operating procedure and crack target The percentage of cell, the Jurkat cell killing target cell activity of bispecific chimeric antigen receptor modification is obvious strong as the result is shown In control group (as shown in Figure 5).
CAR-T cell inhibits the capacity experimental of tumour growth in 7 body of embodiment
Using CD3+T cell in immuno magnetic cell separation human peripheral
1) healthy volunteer is chosen, 20mL periphery whole blood is extracted from ulnar vein, isolates PBMC first；
2) RPMI 1640 culture medium of the isolated PBMC containing 10%FBS is resuspended, and counts cell concentration；
3) to wash PBMC with MACS buffer primary, and 1000rpm is centrifuged 10min sedimentation cell, abandons supernatant；
4) suitable anti-human-CD3 immunomagnetic beads (20uL/10 is added7A PBMC)) it mixes, 4 DEG C of incubation 15min；
5) appropriate MACS buffer is added, 1000rpm is centrifuged 10min, and sedimentation cell is resuspended with MACS buffer；
6) MiniMACS separator is taken out, MS splitter is put into separator, and wash MS with appropriate MACS buffer Splitter；
7) cell suspension in step (5) is slowly added in MS splitter, carries out cell sorting, is not to be immunized by CD3 The negative cells of marked by magnetic bead flow out first；
8) splitter is washed three times with appropriate MACS buffer, every time flow to end the buffer in pillar；
9) prepare a new 15mL centrifuge tube on, MS splitter is removed from MiniMACS separator, place it is new from On heart pipe；
10) 1mL MACS buffer is added on splitter, the cell of delay is eluted completely quickly, repeats three Secondary, the positive cell of CD3 immunomagnetic beads label finally all elutes；
11) supernatant is removed after the cell centrifugation collected, cell, cytometer is resuspended with 1640 culture medium of RPMI containing 10%FBS Number is used for subsequent experimental.
Activation, amplification and the infection experiment of T lymphocyte
CD3+T lymphocyte is needed to activate and be expanded before being infected, by CD3/CD28 immune activation magnetic bead specification It carries out, specific steps are as follows:
1) by above-mentioned isolated CD3+T cell count, adjusting concentration is 2 × 106/ mL, every hole total volume are 500 μ L It plants in 24 orifice plates；
2) 25 μ L immunomagnetic beads are added in every hole, to obtain magnetic bead and cell ratio as 1: 1；
3) recombinant human il-2 is added, makes its final concentration of 30U/mL；
4) cell checks daily, observes the upgrowth situation of cell.Every 2-3 days replacement culture mediums, and rejoin recombined human IL-2；
5) when finding that cell density is larger, cell centrifugation is collected, count after resuspension and density is adjusted to 1 × 106/ mL is passaged to and continues to cultivate in 6 orifice plates；
6) cell is collected after cultivating 1 week, 1640 culture medium of RPMI that serum-free is added is resuspended, and is added in 6 orifice plates, overall Product is 2mL, with 8 μ g/mL polybrene pretreatment cell 4h；
7) appropriate concentration slow virus suspension is slowly homogeneously added into 6 orifice plates after 4h, replaces culture medium afterwards for 24 hours；
8) fluorescence microscope T cell state is used daily, is taken pictures under the same visual field with red fluorescence and white light, is united Cell infection efficiency is counted, is infecting the 5th day collection each group T cell in centrifuge tube, 1000rpm is centrifuged 5min；
9) it is resuspended, is cleaned one time with sterile PBS after abandoning supernatant, cell activity identification is placed under microscope through Trypan Blue Observation counts；
10) 1 × 10 finally is made with PBS8The suspension of/mL is for being transfused nude mice.
Zoopery:
Establish Nude Mouse Model
1) Male nude mice of 6-8 week old, which is raised, at least shifts to an earlier date 1 week before University Animal room SPF receptacle, transplantation tumor It is purchased from Shanghai Slac Experimental Animal Co., Ltd.；
2) MKN45 cell DMEM in high glucose culture medium (10%FBS, 1%PS), 37 DEG C, 5%CO2It is cultivated in incubator；
3) 0.25% pancreatin of MKN45 cell in logarithmic growth phase is digested, neutralized, be centrifuged, abandoned on centrifuge tube Clearly；
4) cell is resuspended with sterile PBS, 1000rpm is centrifuged 5min, abandons supernatant, sterile PBS gravity treatment cell is added and counts Number, adjustment concentration are 5 × 107The suspension of/mL；
5) with skin of back on the right side of iodophor disinfection nude mice, then with 75% ethanol back, hanging on allows alcohol Volatilization, the cell suspension of 100 μ L of dorsal sc injection on the right side of every nude mice, gently oppressing pin hole prevents cell from overflowing, then It puts back to and continues to cultivate in cage；
6) it observes mouse 2 times weekly, weighs, surveys lump length and width.Calculating gross tumor volume V=1/2 × length × wide Square.
The infusion of CAR-T cell:
1) CAR-T cell is calculated and prepared in advance, measures and calculate transplanted tumor in nude mice volume, when growth reaches 50mm3It is left When right (1 week after about injecting), nude mouse is randomly divided into 3 groups, every group 5；
2) CAR-T cell is resuspended in sterile PBS, is transfused in Mice Body by tail vein.First group is control group input 1 ×107T cell；Second group is the input of experimental group 11 × 107PKC-c-Met-CAR-T cell；Third group be experimental group 2 input 1 × 107PKC-c-Met/PD-1-CAR-T cell；
3) it then proceedes to observe 2 nude mice diet, defecation and the state of mind weekly, weigh, survey length of tumor and width, Totally 6 weeks；
4) chloral hydrate anesthesia nude mice removes tumor tissues and dissects nude mice, and statistics each group of data is analyzed, and as a result shows Show that the T cell of bispecific chimeric antigen receptor modification can significantly press down the growth (as shown in Figure 6) of in-vivo tumour.
In conclusion the contents of the present invention are not limited in the above embodiments, the knowledgeable people of same area can be with It is proposed other embodiments easily within technological guidance's thought of the invention, but this embodiment is included in model of the invention Within enclosing.

Claims (10)

1. bispecific chimeric antigen receptor hc-Met scFv-PD-1 scFv-CD8 α-CD28-CD137-CD3 ζ, feature exist In the Chimeric antigen receptor is by anti-human c-Met monoclonal antibody light chain and heavy chain variable region hc-Met scFv, anti-human PD-1 Monoclonal antibody light chain and heavy chain variable region hPD-1 scFv, people CD8 α hinge area, people CD28 transmembrane region and intracellular region, people CD137 and CD3 ζ intracellular region structures in series is constituted；The amino acid sequence of the Chimeric antigen receptor such as sequence SEQ ID NO.1 institute Show.
2. Chimeric antigen receptor hc-Met scFv-PD-1 scFv-CD8 α-CD28-CD137- according to claim 1 CD3 ζ, which is characterized in that the Chimeric antigen receptor is by anti-human c-Met monoclonal antibody light chain and heavy chain variable region hc-Met ScFv, anti-human PD-1 monoclonal antibody light chain and heavy chain variable region hPD-1 scFv, people CD8 α hinge area, people CD28 transmembrane region and Intracellular region, people's CD137 and CD3 ζ intracellular region structures in series are constituted, the nucleic acid sequence of the Chimeric antigen receptor such as SEQ ID Shown in NO.2.
3. Chimeric antigen receptor hc-Met scFv--CD8 α-CD28-CD137-CD3 ζ according to claim 1, feature It is, the Chimeric antigen receptor contains the single-chain antibody hc-Met scFv for people's c-Met antigen, and amino acid sequence is such as SEQ ID NO.3, with connection small peptide Gly4Ser connection between the light chain and heavy chain of single-chain antibody.
4. Chimeric antigen receptor hc-Met scFv-CD8 α-CD28-CD137-CD3 ζ according to claim 3, feature It is, the Chimeric antigen receptor contains the single-chain antibody hc-Met scFv for people's c-Met antigen, nucleic acid sequence such as SEQ Shown in ID NO.4.
5. Chimeric antigen receptor PD-1-CD8 α-CD28-CD137-CD3 ζ according to claim 1, which is characterized in that institute Chimeric antigen receptor is stated to contain for human PD-L 1 and PD-1 extracellular fragment combined area, it can be with PD-L1 ligand binding, amino acid Sequence is as shown in sequence SEQ ID NO.5.
6. Chimeric antigen receptor PD-1-CD8 α-CD28-CD137-CD3 ζ according to claim 5, which is characterized in that institute Chimeric antigen receptor is stated to contain for human PD-L 1 and PD-1 extracellular fragment combined area, it can be with PD-L1 ligand binding, nucleic acid sequence Column are as shown in SEQ ID NO.6.
7. Chimeric antigen receptor hc-Met scFv-PD-1 scFv-CD8 α-CD28-CD137- according to claim 1 CD3 ζ, which is characterized in that the amino terminal of the antigen receptor contains a CD8 alpha signal peptide, amino acid sequence such as sequence Shown in SEQ ID NO.7.
8. Chimeric antigen receptor hc-Met scFv-PD-1 scFv-CD8 α-CD28-CD137- according to claim 7 CD3 ζ, which is characterized in that the amino terminal of the antigen receptor contains a CD8 alpha signal peptide, nucleic acid sequence such as SEQ ID Shown in NO.8.
9. bispecific chimeric antigen receptor hc-Met scFv-PD-1 scFv-CD8 α-CD28- as described in claim 1 Purposes of the CD137-CD3 ζ in preparation Chimeric antigen receptor T cell.
10. purposes as claimed in claim 9, which is characterized in that it is swollen that the Chimeric antigen receptor T cell is used to prepare entity Tumor immunity therapeutic preparation.
CN201710759332.1A 2017-08-28 2017-08-28 Bispecific chimeric antigen receptor c-Met/PD-1 scFv-CAR-T and its construction method and application Pending CN109422815A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710759332.1A CN109422815A (en) 2017-08-28 2017-08-28 Bispecific chimeric antigen receptor c-Met/PD-1 scFv-CAR-T and its construction method and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710759332.1A CN109422815A (en) 2017-08-28 2017-08-28 Bispecific chimeric antigen receptor c-Met/PD-1 scFv-CAR-T and its construction method and application

Publications (1)

Publication Number Publication Date
CN109422815A true CN109422815A (en) 2019-03-05

Family

ID=65503676

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710759332.1A Pending CN109422815A (en) 2017-08-28 2017-08-28 Bispecific chimeric antigen receptor c-Met/PD-1 scFv-CAR-T and its construction method and application

Country Status (1)

Country Link
CN (1) CN109422815A (en)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106350533A (en) * 2015-10-09 2017-01-25 上海宇研生物技术有限公司 Anti-PD-L1-CAR-T, and preparation method and application thereof
CN111704674A (en) * 2020-07-07 2020-09-25 南京医科大学 Chimeric antigen receptor targeting c-Met and autocrine PD-L1scFv and application thereof
CN111763262A (en) * 2020-07-07 2020-10-13 南京医科大学第二附属医院 Bispecific chimeric antigen receptor targeting c-Met and PD-L1 and application thereof
CN112239506A (en) * 2019-07-16 2021-01-19 复旦大学 Micromolecular bispecific antibody Diabody for resisting PD-1 and c-Met antigens
CN111848797B (en) * 2020-07-20 2021-02-09 北京鼎成肽源生物技术有限公司 Single-chain antibody targeting c-Met, chimeric antigen receptor, recombinant vector, CAR-T cell and application
CN114450407A (en) * 2019-09-26 2022-05-06 赫利世弥斯株式会社 Chimeric antigen receptor comprising anti-mesenchyma epidermal transforming factor antibody or antigen binding fragment thereof and use thereof
WO2023200267A1 (en) * 2022-04-14 2023-10-19 주식회사 셀랩메드 Combination therapy of chimeric antigen receptor and hgf binding inhibitor substance

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140242077A1 (en) * 2013-01-23 2014-08-28 Abbvie, Inc. Methods and compositions for modulating an immune response
CN104583230A (en) * 2012-07-13 2015-04-29 宾夕法尼亚大学董事会 Enhancing activity of CAR T cells by co-introducing a bispecific antibody
CN105085680A (en) * 2014-05-23 2015-11-25 复旦大学 Humanized anti-PD-1 and c-MET bispecific antibody, and preparation method and application thereof
CN106008721A (en) * 2016-08-09 2016-10-12 安徽未名细胞治疗有限公司 C-Met specific chimeric antigen receptor and application thereof
WO2017055388A2 (en) * 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Bispecific t cell activating antigen binding molecules
CN107002119A (en) * 2014-03-24 2017-08-01 豪夫迈·罗氏有限公司 Treatment of cancer and the former and associating that HGF is expressed using C MET antagonists
CN107082813A (en) * 2017-05-17 2017-08-22 董克海 Target PD 1 Chimeric antigen receptor molecule and its application

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104583230A (en) * 2012-07-13 2015-04-29 宾夕法尼亚大学董事会 Enhancing activity of CAR T cells by co-introducing a bispecific antibody
US20140242077A1 (en) * 2013-01-23 2014-08-28 Abbvie, Inc. Methods and compositions for modulating an immune response
CN107002119A (en) * 2014-03-24 2017-08-01 豪夫迈·罗氏有限公司 Treatment of cancer and the former and associating that HGF is expressed using C MET antagonists
CN105085680A (en) * 2014-05-23 2015-11-25 复旦大学 Humanized anti-PD-1 and c-MET bispecific antibody, and preparation method and application thereof
WO2017055388A2 (en) * 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Bispecific t cell activating antigen binding molecules
CN106008721A (en) * 2016-08-09 2016-10-12 安徽未名细胞治疗有限公司 C-Met specific chimeric antigen receptor and application thereof
CN107082813A (en) * 2017-05-17 2017-08-22 董克海 Target PD 1 Chimeric antigen receptor molecule and its application

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
MARK MERCHANT ET AL.: "Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent", 《PNAS》 *
XINGXING YUAN ET AL.: "Dual-function chimeric antigen receptor T cells targeting c-Met and PD-1 exhibit potent anti-tumor efficacy in solid tumors", 《INVESTIGATIONAL NEW DRUGS》 *
ZU-JUN SUN ET AL.: "A novel bispecific c-MET/PD-1 antibody with therapeutic potential in solid cancer", 《ONCOTARGET》 *
周荧等: "靶向 c-Met 嵌合抗原受体 T 细胞的制备及其对鼻咽癌细胞作用的研究", 《山东大学耳鼻喉眼学报》 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106350533A (en) * 2015-10-09 2017-01-25 上海宇研生物技术有限公司 Anti-PD-L1-CAR-T, and preparation method and application thereof
CN106350533B (en) * 2015-10-09 2020-07-17 上海宇研生物技术有限公司 Anti-PD-L1-CAR-T and preparation method and application thereof
CN112239506A (en) * 2019-07-16 2021-01-19 复旦大学 Micromolecular bispecific antibody Diabody for resisting PD-1 and c-Met antigens
CN112239506B (en) * 2019-07-16 2023-09-26 复旦大学 Small molecule bispecific antibody diabetes of anti-PD-1 and c-Met antigen
CN114450407A (en) * 2019-09-26 2022-05-06 赫利世弥斯株式会社 Chimeric antigen receptor comprising anti-mesenchyma epidermal transforming factor antibody or antigen binding fragment thereof and use thereof
CN111704674A (en) * 2020-07-07 2020-09-25 南京医科大学 Chimeric antigen receptor targeting c-Met and autocrine PD-L1scFv and application thereof
CN111763262A (en) * 2020-07-07 2020-10-13 南京医科大学第二附属医院 Bispecific chimeric antigen receptor targeting c-Met and PD-L1 and application thereof
CN111763262B (en) * 2020-07-07 2021-10-26 南京医科大学第二附属医院 Bispecific chimeric antigen receptor targeting c-Met and PD-L1 and application thereof
CN111704674B (en) * 2020-07-07 2022-05-03 南京医科大学 Chimeric antigen receptor targeting c-Met and autocrine PD-L1 scFv and application thereof
CN111848797B (en) * 2020-07-20 2021-02-09 北京鼎成肽源生物技术有限公司 Single-chain antibody targeting c-Met, chimeric antigen receptor, recombinant vector, CAR-T cell and application
WO2023200267A1 (en) * 2022-04-14 2023-10-19 주식회사 셀랩메드 Combination therapy of chimeric antigen receptor and hgf binding inhibitor substance

Similar Documents

Publication Publication Date Title
CN109422815A (en) Bispecific chimeric antigen receptor c-Met/PD-1 scFv-CAR-T and its construction method and application
CN107312091B (en) Target the Humanized monoclonal antibodies of people CD19 antigen
CN105907719B (en) Anti ROBO1 CAR-T cell and its preparation and application
CN106399255B (en) PD-1 CAR-T cell and its preparation method and application
CN105949325B (en) Chimeric antigen receptor, slow virus carrier and its application comprising CD27 intracellular domain
CN105296431B (en) The α β T cells and its suppression cancer purposes of tumor combination specificity gamma delta T CR genetic modifications
CN107249602A (en) Target the Chimeric antigen receptor of hematologic malignancies（CAR）, its composition and application method
CN107810267A (en) γ delta cells of modification and application thereof
CN103502438A (en) Method and compositions for cellular immunotherapy
CN109652378B (en) Function-enhanced universal CAR-T cell and preparation method and application thereof
CN105837693A (en) BCMA-based (B cell maturation antigen-based) chimeric antigen receptor and preparation method and application thereof
CN108315305A (en) Carry the preparation method and applications of the immunocyte excretion body of Chimeric antigen receptor
CN106661129A (en) Chimeric antigen receptor specific for SSEA4 antigen
CN109503716A (en) A kind of bispecific chimeric antigen receptor molecule and its application in oncotherapy
CN109385403A (en) Target the CAR NK cell of GPC3
CN109721659A (en) It is a kind of target CD19 Novel chimeric antigen receptor (CAR) and its application
CN108948211A (en) A kind of Chimeric antigen receptor and its application based on GD2
CN107236762A (en) A kind of method that minicircle dna transfecting T cells prepare clinical grade CAR T cell preparations
CN109553686A (en) The novel regulatable double Chimeric antigen receptor T cells of one kind and its construction method and application
CN109722437A (en) A kind of universal CAR-T cell and its preparation method and application
CN107287163B (en) Express the dendritic cells and application thereof of Chimeric antigen receptor
CN109609465A (en) A kind of gamma delta T cells using derived from cord blood prepare the method and the CAR-T cell and application of CAR-T cell
CN106317228A (en) Chimeric antigen receptor molecule and application thereof
CN105316362A (en) Dual-RMCE-mediated (dual-recombinase mediated cassette exchange-mediated) TCR (T cell receptor) gene replacement system and method
CN110305906A (en) A kind of slow virus carrier and PDL1-CAR-T cell of the CAR Chimerical receptor targeting PDL1

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
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20190305