CN108220247A - A kind of double CAR-T cells and its preparation method and application - Google Patents

A kind of double CAR-T cells and its preparation method and application Download PDF

Info

Publication number
CN108220247A
CN108220247A CN201810228590.1A CN201810228590A CN108220247A CN 108220247 A CN108220247 A CN 108220247A CN 201810228590 A CN201810228590 A CN 201810228590A CN 108220247 A CN108220247 A CN 108220247A
Authority
CN
China
Prior art keywords
cell
cells
double car
expression
car
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201810228590.1A
Other languages
Chinese (zh)
Inventor
屈彩云
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.)
Hangzhou Shidimu Biological Technology Co Ltd
Original Assignee
Hangzhou Shidimu Biological Technology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hangzhou Shidimu Biological Technology Co Ltd filed Critical Hangzhou Shidimu Biological Technology Co Ltd
Priority to CN201810228590.1A priority Critical patent/CN108220247A/en
Publication of CN108220247A publication Critical patent/CN108220247A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • 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
    • C07K14/70521—CD28, CD152
    • 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
    • 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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • 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
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Landscapes

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

Abstract

The invention discloses a kind of double CAR T cells, it is co-expressed using in tumour cell, but two target antigens not co-expressed in normal cell, signal fused is killed in the antibody variable region of one of target antigen and T cell, it is merged with costimulatory signal in T cell the antibody variable region of another target antigen, two different expression cassettes are formed, build double CAR T cells；Double CAR T cells of structure only identify while express the tumour cell of two target antigens, and the normal cell of one of target antigen is only expressed in nonrecognition.The invention also discloses preparation methods and application.The present invention not only increases killing specificity, and reduces the side effect that the killing to normal cell comes with reducing inflammatory factor storm-zone.

Description

A kind of double CAR-T cells and its preparation method and application
Technical field
The present invention relates to biotechnologies, and in particular to a kind of double CAR-T cells and its preparation method and application.
Background technology
Leukaemia is a kind of candidate stem cell malignant proliferative hematologic disease.The leucocyte side that marrow generates under normal circumstances Body of helping others resists infection, and generates abnormal leukocyte cell in leukaemia people marrow, these pernicious leukocyte cells are a large amount of Existing normal blood cell in vivo is repelled in amplification, causes immunocyte in blood unbalance, influences the normal work(of body blood cell Energy.Leukaemia includes acute myeloid leukaemia, chronic lymphocytic leukemia, hodgkin's leukaemia, the white blood of non-Hodgkins Disease etc..Chronic lymphocytic leukemia is usually happened in mid-aged population, is seldom occurred in population of adolescent.Treatment is white at present The method of blood disease mainly has chemotherapy, radiotherapy, immunization therapy.Chemotherapy cannot effectively remove pernicious leukocyte cell with radiotherapy, because of it Side effect also brings great pain to patient.Using CD19 as the targeting specific immunotherapy method (CAR- of main immunizing antigen T-CD19) go out preferable therapeutic effect in clinical manifestation, can effectively remove leukaemia cell.But CAR-T-CD19 is by costimulation Signal, to together, identifies leukaemia cell, this design method can effectively activate T thin with killing signal fused by CD19 antibody Born of the same parents direct killing tumour target cell, but also containing CD19 antigens in normal structure, therefore, CAR-T-CD19 can also be attacked and be killed Wound expresses the normal structure of these antigens, generates serious side effect.Occur the higher death rate in clinical trial and be called more Stop.Meanwhile the diversity of tumour complexity can also cause antigen to be lost so that CAR-T cells generate undershooting-effect.In addition to this, This Casualty Mode by costimulatory signal and killing signal fused acute activation T cell because its it is strong to tumour cell with just Normal histiocytic lethal effect, causes a series of immune responses to generate inflammatory factor storm, and huge wound is generated to patient body Evil, has also seriously affected immunotherapeutic effects.
Invention content
The object of the present invention is to provide a kind of double CAR-T cells and its preparation method and application, to solve in CAR-T technologies The problem of toxic effect is excessively high.Because in tumour, as a consequence it is hardly possible to it finds a target position and is only expressed in tumour cell completely, It does not express in the normal tissue, so, any single target treatment scheme can not be solved while tumour cell is attacked, Do not attack normal structure, i.e. toxic side effect problem in clinic.But using the targets of two high expression in tumor tissues, Only the two targets just can activate CAR-T and tumour cell is killed when height is expressed simultaneously, and the two targets are almost It does not express simultaneously in the normal tissue, therefore, CAR-T would not attack normal structure, so as to reduce toxic side effect.
The present invention uses following technical scheme：
A kind of double CAR-T cells, co-express using in tumour cell, but two not co-expressed in normal cell target Antigen is marked, kills signal fused in the antibody variable region of one of target antigen and T cell, the antibody of another target antigen Variable region is merged with costimulatory signal in T cell, forms two different expression cassettes, builds double CAR-T cells；Structure Double CAR-T cells only identify while express the tumour cell of two target antigens, and nonrecognition is only expressed one of target and resisted Former normal cell.
Further, a target antigen is CDX, X 19 in described two target antigens；Another target antigen is CDY, Y 160,123 or 23.
Further, signal is killed in the T cell and includes CD3zeta.
Further, costimulatory signal includes CD28,4-1BB, OX40 in the T cell.
The preparation method of above-mentioned double CAR-T cells, includes the following steps：
Step 1, structure expression CDY/XCAR slow virus carriers；
Step 2, slow virus packaging：Obtain expression CDY/XCAR slow virus；
Step 3, slow-virus infection T cell：Human peripheral blood mononuclear cell, culture and amplification T cell are detached, utilizes step 2 Obtained expression CDY/XCAR slow-virus infection T cells obtain the T cell of expression CDY/XCAR：CDY/XCAR-T, i.e., double CAR- T cell.
Further, the preparation of CD19/160CAR-T cells includes the following steps：
Step 1, structure expression CD19/160CAR slow virus carriers：CD160CAR gene orders are cloned into pHAGE2- Cca-loxP carrier NotI sites, by IRES-mcherry fusion gene clonings to CD160CAR sequence downstreams NheI/XbaI Between point, the carrier obtained is named as pHAGE-CD160；CD19CAR sequences are cloned into pHAGE-CD160 carriers BstpDI sites obtain carrier and are named as pHAGE-CD160/19；
Step 2, slow virus packaging：293T cell culture is in the dual anti-DMEM culture mediums of the green chain containing 10%FBS and 1%, carefully Density reaches 50%-60% after born of the same parents are adherent, and cell changes plasma-free DMEM medium into；Plasmid presses pHAGE-CD160/19： pspAX2：PMD2.G=4:3:Mixing in serum-free DMEM is added to after the mixing of 1 ratio, PEI is added to serum-free DMEM mixings, stands DMEM and PEI mixed liquors are added in DMEM and plasmid mixed liquor afterwards and blow and beat mixing, 293T is dropped evenly after being stored at room temperature In cell；After cell constant temperature incubator culture 6 hours, it is changed to the dual anti-DMEM culture mediums of the green chain containing 10%FBS and 1%, 37 DEG C, 5%CO2Incubator is further cultured for 24 hours；Supernatant is collected, 1500rpm room temperatures centrifugation 10min abandons cell precipitation, supernatant mistake 0.22um filter membranes as express CD160/19CAR slow virus, and -80 DEG C save backup；
Step 3, slow-virus infection T cell：Human peripheral blood mononuclear cell, culture and amplification T cell are detached, utilizes step 2 Obtained expression CD160/19CAR slow-virus infection T cells obtain the T cell of expression CD160/19CAR：CD160/19CAR- T, i.e., double CAR-T cells.
Application of the above-mentioned double CAR-T cells in tumor is prepared.
Above-mentioned double CAR-T cells are preparing the application in treating leukemia medicament.
Beneficial effects of the present invention：
Killing signal is building up to two by the present invention with two target antigens (such as CD19 and CD160) respectively with costimulatory signal A different expression cassette, only the two target antigens are expressed simultaneously, and T cell could activate and specific recognition tumour is (as in vain Blood disease) cell, in normal structure, because the two antigens cannot be expressed simultaneously, therefore, normal group of the T cell nonrecognition of activation It knits, if normal B cells low expression CD19 is without expressing CD160, other tissue low expression CD160 are without expressing CD19.It is this Design not only increases killing specificity, and reduces the pair that the killing to normal cell comes with reducing inflammatory factor storm-zone Effect.
Description of the drawings
Fig. 1 is CAR-T ideographs.
Fig. 2 is pHAGE2-cca-loxP carrier structure figures.
Fig. 3 centrifuges each component distribution schematic diagram for Ficol.
Fig. 4 is that T cell is distributed in PBMC.
Fig. 5 expresses streaming figure for CAR.
Fig. 6 is different target fragmentation effect figures.
Fig. 7 is IFN-γ secretion figure.
Fig. 8 is ability of cell proliferation figure.
Fig. 9 is animal interior tumor cell quantity (K562).
Figure 10 is animal interior tumor cell quantity (K562-CD19-CD160).
Specific embodiment
The present invention is done with reference to embodiment and attached drawing and is further explained.The following example is merely to illustrate this hair It is bright, but it is not used to limit the practical range of the present invention.
Embodiment 1
First, experiment material and reagent
1.1st, cell derived：The T cell in normal pbmc source, the T cell in leukaemia human PBMC source, human peripheral blood B Cell, people's acute B _ Lymphoid Leukemic Cells Nalm-6 (CD19 is positive)；People's marrow leukaemia cell K562 (CD19 is negative).
1.2nd, initial medium：PRIM1640 (GIBCO) culture medium, containing 5% heat shock human AB serum, 1% glutamine And 40IU/mL interleukin (IL) -2.
1.3rd, amplification culture medium：PRIM1640 culture mediums, containing 5% heat shock human AB serum, 1% glutamine and 300IU/mL IL-2。
1.4th, complete medium：PRIM1640, containing 10%FBS, 10% green chain is dual anti-.
1.5th, PBMC frozen stock solutions：90%FBS+10%DMSO.
1.6th, experiment reagent：PRIM1640 culture mediums (GIBCO), IL-2 (GIBCO), AB serum (GIBCO), mycillin (GIBCO), Anti-CD3 (OKT3) (BD companies), Anti-CD3-APC (BD companies), Anti-CD8-PE (BD companies), Anti- CD4-FITC (BD companies), anti-CD56-PerCP-Cy5.5 (BD companies), Anti-IgG1-APC (BD companies), Anti- IgG1-PE (BD companies), Anti-IgG1-FITC (BD companies), Anti-his-APC, Anti-Flag-PEcy7 (BD companies), Fetal calf serum (GIBCO), DMSO.
1.7th, laboratory apparatus：Microscope, cell incubator, Biohazard Safety Equipment, flow cytometer, ultra low temperature freezer, liquid nitrogen Tank, centrifuge.
2nd, CAR designs and its gene order
2.1st, CAR-T ideographs are as shown in Figure 1.
2.2nd, CD160CAR gene orders such as SEQ ID NO:Shown in 1.
2.3rd, CD19CAR gene orders such as SEQ ID NO:Shown in 2.
2.4th, IRES-mcherry fusion gene sequences such as SEQ ID NO:Shown in 3.
2.5th, CD19-CD28-CD3 ζ CAR gene orders such as SEQ ID NO:Shown in 4.
2.6th, Luciferase gene orders such as SEQ ID NO:Shown in 5.
2.7th, the gene order of CD19 such as SEQ ID NO:Shown in 6.
2.8th, the gene order of CD160 such as SEQ ID NO:Shown in 7.
2.9th, CD19-CD160 fusion gene sequences such as SEQ ID NO:Shown in 8.
2.10th, pHAGE2-cca-loxP carrier structures figure
PHAGE2-cca-loxP carrier structures as shown in Fig. 2, show CD160CAR gene orders, CD19CAR gene orders, IRES-mcherry fusions are in the position of carrier.
3rd, series CAR vector constructions
As shown in Fig. 2 pHAGE2-cca-loxP carrier structure figures：CD160CAR gene orders are cloned into pHAGE2- Cca-loxP carrier NotI sites, by IRES-mcherry fusion gene clonings to CD160CAR sequence downstreams NheI/XbaI Between point, the carrier obtained is named as pHAGE-CD160；CD19CAR sequences are cloned into pHAGE2-cca-loxP carriers BstpDI sites, then by IRES-mcherry fusion gene clonings between CD19CAR sequence downstream NheI/XbaI sites, institute The carrier of acquisition is named as pHAGE-CD19；CD19CAR sequences are cloned into the BstpDI sites of pHAGE-CD160 carriers, institute It obtains carrier and is named as pHAGE-CD160/19.The gene order of CD160CAR, CD19CAR, IRES-mcherry are in Shanghai Raw work synthesis.CD19-CD28-CD3 ζ CAR are cloned into pHAGE2-cca-loxP carrier NotI/BstpDI sites, by IRES- For mcherry fusion gene clonings between NheI/XbaI sites, the carrier obtained is named as pHAGE-CD19-CD28-CD3 ζ。
3.1st, the structure of luciferase Lentivirals
Using PGL3-Basical plasmids as template amplification Luciferase, Pcmvie-IRES-puro carriers are inserted into, are ordered Entitled Pcmvie-luc, after sequencing identification is correct, a large amount of extractions be stored in -80 DEG C it is spare.
3.2nd, the structure of CD19-CD28-CD3 ζ Lentivirals
CD19-CD28CD3 ζ sequences in 2.5 are connected to pHAGE2-cca-loxP carrier NotI/BstpDI sites, it will For IRES-mcherry fusion gene clonings between NheI/XbaI sites, the carrier obtained is named as pHAGE-CD19- CD28-CD3ζ。
3.3rd, the structure of CD19 slow virus carriers is expressed
Nalm-6 cell RNAs are extracted, reverse transcription obtains CD19 protein gene sequences into cDNA by template amplification of this cDNA Row, and the sequence of acquisition is inserted into Plvx-cmvie-puro carrier EcoRI/XbaI sites, it is named as Pcmvie-CD19.
3.4th, the structure of CD160 slow virus carriers is expressed
PBMC cell RNAs are extracted, reverse transcription is into cDNA, using this cDNA for template amplification acquisition CD160 protein gene sequences, And the sequence of acquisition is inserted into Plvx-cmvie-puro carrier EcoRI/XbaI sites, it is named as Pcmvie-CD160.
3.5th, the structure of CD160-CD19 slow virus carriers is expressed
2.3 are merged with cloning the CD19 and CD160 of acquisition in 2.2 by chimeric primers, CD19 and CD160 fusions Between be inserted into F2A sequences (such as SEQ ID NO:Shown in 9).CD19-F2A-CD160 sequences are inserted into Plvx- by EcoRI/XbaI Cmvie-puro is named as Pcmvie-CD19-CD160.
4th, slow virus is packed
4.1st, the packaging of the slow virus of expression Luciferase
293T cell culture is in DMEM culture mediums (10%FBS, 1% green chain are dual anti-), and density reaches 50%- after cell is adherent 60%, cell changes plasma-free DMEM medium into.Plasmid presses Pcmvie-luc：psPAX2：PMD2.G=4:3:1 (ug) ratio is mixed Mixing in 250uL serum-frees DMEM is added to after conjunction, 32uLPEI is added to 250uL serum-free DMEM mixings, will after standing 5min 250uLDMEM is added in 250uLDMEM and plasmid mixed liquor with PEI mixed liquors and blows and beats mixing, is stored at room temperature after 15min uniformly It is added drop-wise in 293T cells.After cell constant temperature incubator culture 6 hours, being changed to normal DMEM culture mediums, (10%FBS, 1% is green Chain is dual anti-), 37 DEG C, 5%CO2Incubator is further cultured for 24 hours.All supernatants are collected, 1500rpm room temperatures centrifugation 10min is abandoned thin Born of the same parents are precipitated, and supernatant crosses 0.22um filter membranes, as expresses the slow virus of Luciferase, -80 DEG C save backup.Wherein psPAX2 and PMD2.G is viral packaging plasmid, purchased from addgene companies.
4.2nd, the packaging of CD160CAR slow virus is expressed
Specific method replaces Pcmvie-luc, Qi Tatong referring to 4.1, by pHAGE-CD160 carriers, and the virus obtained is CD160CAR slow virus is expressed, -80 DEG C save backup.
4.3rd, the packaging of CD19CAR slow virus is expressed
Specific method replaces Pcmvie-luc, Qi Tatong referring to 4.1, by pHAGE-CD19 carriers, and the virus obtained is CD19CAR slow virus is expressed, -80 DEG C save backup.
4.4th, the packaging of the slow virus of CD19CAR and CD160CAR is co-expressed
Specific method replaces Pcmvie-luc, Qi Tatong, the disease obtained referring to 4.1, by pHAGE-CD160/19 carriers Poison is expression CD160/19CAR slow virus, and -80 DEG C save backup.
4.5th, the packaging of CD19-CD28-CD3 ζ CAR slow virus
Specific method replaces Pcmvie-luc, Qi Tatong referring to 4.1, by pHAGE-CD19-CD28-CD3 ζ carriers, is obtained The virus obtained is expression CD19-CD28-CD3 ζ slow virus, and -80 DEG C save backup.
4.6th, the packaging of CD19 slow virus is expressed
Specific method replaces Pcmvie-luc, Qi Tatong referring to 4.1, by Pcmvie-CD19 carriers, and the virus obtained is CD19 slow virus is expressed, -80 DEG C save backup.
4.7th, the packaging of CD160 slow virus is expressed
Specific method replaces Pcmvie-luc, Qi Tatong, the virus obtained referring to 4.1, by Pcmvie-CD160 carriers To express CD160 slow virus, -80 DEG C save backup.
4.8th, the packaging of CD19-CD160 slow virus is expressed
Specific method replaces Pcmvie-luc, Qi Tatong referring to 4.1, by Pcmvie CD19-CD160 carriers, is obtained Virus is expression CD19-CD160 slow virus, and -80 DEG C save backup.
5th, for measuring the foundation and screening of the K562 stable cell lines of serial CAR-T activity of cell biology
5.1st, the structure (K562-Luc) of the K562 stable cell lines of expression Luciferase：Cell is trained with complete medium It supports.0.5×105/ mL K562 cell inoculations after constant incubator is incubated 5h, add 1mL to package expression to 6 orifice plates The slow virus of Luciferase gently pats mixing, and constant incubator continues to cultivate 12h, and cell replaces complete medium, continues Cultivate 36h.3ug/mL G418 is added to screen, after normally passing on step sizing 5-7 days, diluting cells to 48 orifice plates make each hole one A cell, normal culture observation.PCR amplification luciferase gene after cell monoclonal amplification, identification Luc positive cell strains are K562-Luc cell lines.
5.2nd, the structure (K562-CD19) of the K562-Luc stable cell lines of expression CD19：Specific method will referring to 5.1. The virus of infection changes the slow virus of expression CD19 into, changes the cell of infection into K562-Luc.Other methods are identical.
5.3rd, the structure (K562-CD160) of the K562-Luc stable cell lines of expression CD160：Specific method is referring to 5.1. The virus of infection is changed into the slow virus of expression CD160, changes the cell of infection into K562-Luc.Other methods are identical.
5.4th, the structure (K562-CD19-CD160) of the K562-Luc stable cell lines of expression CD19-CD160：Specific side The virus of infection is changed into the slow virus of expression CD19-CD160 referring to 5.1. by method, changes the cell of infection into K562-Luc.Its Its method is identical.
6th, the separation of human peripheral blood mononuclear cell (PBMC) is with freezing
6.1st, anticoagulant pre- centrifugation.
6.1.1 normal person and the fresh anticoagulated whole blood 20mL of leukaemia people are taken respectively in 50mL centrifuge tubes, with 2 000r/ Min centrifuges 20min, and upper plasma is abandoned in suction, lower sediment cell about 10~12.5mL is obtained, with Hank ' s liquid or PBS with 1:1 body Long-pending mixing in test tube.
6.1.2 another centrifuge tube is taken, adds in Ficol lymphocyte separation mediums, cell hangs in then taking 1.1 with capillary syring Liquid is slowly added on Ficol lymphocyte separation mediums upper strata, and cell suspension is made to be overlapped on layering liquid.With horizontal centrifuge with 2000r/min centrifuges 20min, after centrifugation, takes out centrifuge tube, it is seen that liquid is layered in pipe.
It is visible in pipe to be divided into 4 layers：Top layer is blood plasma, blood platelet containing part：The second layer is very thin tunica albuginea layer, mainly Mononuclearcell is also contaminated with a small amount of blood platelet；Third layer is separation liquid layer；4th layer be granulocyte and red blood cell, red blood cell Tube bottom is sunken to, and it is in one layer of very thin tunica albuginea (Fig. 3) that granulocyte, which is then tightly attached on packed red cells,.
6.2nd, extraction, washing and the suspension of PBMC
The blood plasma of top layer is sucked, collects the PBMC of plasma layer and lymphocyte separation medium interface, is all sucked out as possible PBMC.Add 3~4 times with upper volume Hanks or PBS liquid in the PBMC of gained, gently blown and beaten with capillary syring uniformly, avoid producing Minute bubbles, liquid-column height are no more than the 2/3 of centrifuge tube.1500r/min centrifugation 10min are centrifuged after mixing, low-speed centrifugal is conducive to The blood platelet retained in removal cell suspension, removes supernatant.Cell is washed with phosphate buffer 2 times, 1500r/min centrifugations again 10min washes away remaining lymphocyte separation medium.Again with RPMI-1640 culture mediums centrifuge washing 1 time, supernatant is exhausted, with abundant Remove the impurity such as blood platelet.
6.3rd, the identification of the purity of monocyte and cell viability
200uLPBMC is taken to be resuspended to 1mLPBS, is assigned in four streaming glass tubes, number 1,2,3,4.Wherein No. 1 For pipe as negative control, No. 2 pipes are incubated anti-CD3-APC, anti-CD8-PE, anti-CD4-FITC, and No. 3 pipes are incubated anti- CD3-APC, anti-CD56-PerCP-Cy5.5,4 DEG C are protected from light and are incubated 30min, and anti-IgG control equally handle.Streaming Cell instrument detects PBMC cell colonys.T cell is distributed in PBMC as shown in Figure 4, and it is the mono- positive cells of CD3 to scheme Q1 in A, represents that T is thin Born of the same parents；Q4 is the mono- positive cells of CD56, represents NK cells；Q2 is the bis- positive cells of CD3 and CD56, represents NKT cells.Q1 cells continue point Analyse CD4-T and CD8-T cell subsets as shown in panelb, Q1 represents CD4-T cells, and Q4 represents CD8-T cells.
Separately 1 drop cell suspension is taken to be placed in blood cell counting plate inside counting.Cell viability is detected with trypan exclusion stain.Take 1 Drop cell suspension adds 1 drop, 2% trypan blue dye liquor mixing, is capped piece, microscope high power microscopy, living cells is not colored, and refractive power is strong；Extremely Cell is dyed to blue, and volume is slightly expanded.
6.4th, PBMC freezes
1×105PBMC cells are resuspended in 1mL frozen stock solutions, and re-suspension liquid is transferred to cryopreservation tube, and cryopreservation tube is put into procedural drop In warm box, Programmed cryopreservation box is transferred to -80 DEG C of ultra low temperature freezers and is stayed overnight, liquid nitrogen is moved within second day and preserves for a long time.
7th, the PBMC that the culture of T cell freezes from liquid nitrogen taking-up is immediately placed in 37 DEG C of water-bath fast melts, and T cell is initial Culture medium washed once.PBMC presses 1 × 106Cell/mL is resuspended in T cell initial medium, while adds 50ng/mL Anti- CD3(OKT3).1mL cell re-suspension liquids are added to 24 orifice plates (corning), in 37 DEG C, 5%CO2Constant incubator culture.
The culture of leukaemia human T-cell：Due to containing a large amount of CD19 leukaemia cells in leukaemia human PBMC, first The leukaemia cell of expression CD19 surface antigens is removed with Anti-CD19Beads (Miltenyi, Auburn, CA).Normal control PBMC after leucocyte of the PBMC cells with detaching removal expression CD19 surface antigens is by 1 × 106Cell/mL is resuspended in T cell Amplification culture medium, 24 orifice plates for being inoculated into OKT3 pretreatments carry out culture 4 days, intermediate fresh according to cell amplification situation addition Amplification culture medium is collected by centrifugation cell on the 4th day, is resuspended in commercialized 1640 culture medium, 1mL cell suspensions are made.Orifice plate Processing method：1mL is added to 24 orifice plates containing 10 μ g/mL OKT3PBS solution, and PBS is washed twice after being placed at room temperature for 4h.
8th, slow-virus infection T cell 2mL is incubated 6 orifice plate 2h containing 15 μ g/mL RetroNectin PBS room temperature, abandons aggegation Liquid.Pay attention to keeping moistening, it is impossible to kill.
Add 2mL confining liquids (2% seralbumin (BSA) is dissolved in HBSS), RT is incubated 30min, abandons confining liquid.
It is cleaned three times with washing buffer (HBSS containing 2.5%HEPES).
Virus liquid (expression CD160CAR slow virus, expression CD19CAR slow virus, expression CD160/19CAR slow virus, table Up to CD19-CD28-CD3 ζ slow virus) it is rapid thaw after respectively by cell suspension=1 collected in virus liquid and step 7:1 is mixed Even, 2mL mixed liquors are added in 6 orifice plates of above-mentioned preparation.2h is centrifuged in 32 DEG C of 2000g.
Abandon supernatant, 0.5 × 106It is resuspended in the T cell amplification culture medium of the IL-2 containing 300IU/mL, is uniformly laid on 6 holes Plate, orifice plate centrifuge 2h in 32 DEG C of 2000g, then put cultivate 24 hours in 37 degree of incubators after collect part cell, flow cytometer detection is sick The expression of malicious transfection efficiency and CAR.Expression CD160CAR, CD19CAR, CD160/19CAR and the CD19- respectively obtained The T cell of CD28-CD3 ζ be respectively designated as CD160CAR-T, CD19CAR-T, CD160/19CAR-T, CD19-CD28-CD3 ζ- T。
9th, CD160CAR in CD160CAR-T, CD19CAR-T, CD160/19CAR-T, CD19-CD28-CD3 ζ-T, The expression identification of CD19CAR, CD160/19CAR and CD19-CD28-CD3 ζ
The T cell of transfection and the T cell (2 × 10 of untransfected5/ tube) respectively with anti-CD160-His, anti- CD19-Flag.It is incubated 30min on ice.After the PBS washings of precooling, 1 μ l anti-His-APC, 1 μ lanti-Flag-PE-Cy7 It is added in sample, ice bath.10 μ l anti-CD4-FITC, 10 μ l anti-CD8-PE additions, are incubated 15min on ice.anti- IgG control are equally handled.Flow cytomery.As shown in figure 5, A：The T cell normally cultivated；B：T cell transfects CD160CAR slow virus, positive CD160CAR-T cells accounting 52%；C：T cell transfects CD19CAR slow virus, positive CD19CAR-T cells accounting 50.1%；D：T cell transfects CD160/CD19CAR-T slow virus, positive CD160/CD19CAR-T Cell accounting 62%；E：T cell transfects CD19-CD28-CD3 ζ CAR slow virus, and positive CD19-CD28-CD3 ζ CAR-T cells account for Than 53%.
Tenth, CD160CAR-T, CD19CAR-T, CD160/19CAR-T, CD19-CD28-CD3 ζ-T toxicity detections
Experimental principle：In the several cell lines of K562-luc, K562-CD19, K562-CD160, K562-CD19-CD160 Constitutive expression luciferase since luciferase cannot be secreted extracellular, theoretically cultivates fluorescein in the culture medium of cell Enzymatic activity is very low or does not have, when with CD19CAR-T, CD19-CD28-CD3 ζ-T, CD160CAR-T, CD160/19CAR-T, normal When T cell handles K562-luc, K562-CD19, K562-CD160, K562-CD19-CD160, if the CD19CAR-T of processing, CD19-CD28-CD3 ζ-T, CD160CAR-T, CD160/19CAR-T, normal T-cell are to K562-luc, K562-CD19, K562- When CD160, K562-CD19-CD160 have lethal effect, K562-luc, K562-CD19, K562-CD160, K562- for being killed CD19-CD160 will be cracked, and luciferase is discharged into extracellular, therefore, passed through and measured after killing luciferase in cell conditioned medium Activity, can be with the killing activity of the various T cells of indirect determination.
Experimental method：It is pressed after the T cell that the 12-14 days collect activation, PBS washings per hole 2.5/1.25/0.25/0.05 × 105T cells, 5 × 103Target cell (E/T=50:1,25:1,5:1,1:1) 96 orifice plate of round bottom is inoculated into, 37 DEG C are incubated 4h, double Multiple holes.Supernatant is abandoned in centrifugation, and PBS is washed three times, adds 20uL lysates per hole, after lysis at room temperature 5min, adds 100uL luciferases anti- Substrate is answered, in microplate reader, procedure selection chemiluminescence, quick read plate.
Wherein Maxluc refers to the enzyme activity of the luciferase measured by cracking K562-luc cells；Expluc is respectively referred to CD19CAR-T, CD19-CD28-CD3 ζ-T, CD160CAR-T, CD160/19CAR-T, normal T-cell processing are a variety of different Fluorescein in the supernatant measured after K562 (K562-Luc, K562-CD19, K562-CD160, K562-CD19-CD160) cell line Enzyme enzyme activity.Experiment packet is as shown in table 1.
Table 1
Experimental result is as shown in Figure 6, the results showed that CD160/19CAR-T energy selective killings K562-CD19-CD160 is thin Born of the same parents without killing K562-Luc, K562-CD19, K562-CD160, show apparent selective killing；CD19-CD28-CD3 ζ-T have lethal effect to K562-CD19 and K562-CD19-CD160, without selectivity.
11, ELISA detects IFN-γ
After target cell K562-Luc, K562-CD19, K562-CD160, K562-CD160-CD19 are washed respectively with PBS, press 1×106/ mL is resuspended in the T cell amplification culture medium without IL-2.1×105K562-Luc、K562-CD19、K562- CD160, K562-CD160-CD19 target cell are inoculated into 96 orifice plate of round bottom, and each target cell sets two multiple holes.Normal T-cell (Control), CD19CAR-T, CD19-CD28-CD3 ζ-T, the difference PBS washings of CD160CAR-T, CD160/19CAR-T cell 1 × 10 is pressed afterwards6/ mL cell densities are diluted in the T cell amplification culture medium without IL-2, and 1 × 105T cell vaccination is thin to target is contained In the culture hole of born of the same parents.37 DEG C, 5%CO2After incubator culture 18-20h, by IFN-γ ELISA kit (Pierce, Rockford) operating instruction detection IFN γ secreting, expressing.As shown in Figure 7, the results showed that：CD19-CD28-CD3 ζ-T processing K562-CD19 and K562-CD19-CD160, CD160CAR-T processing K562-CD160 and K562-CD19-CD160, CD160/ 19CAR-T processing K562-CD19, K562-CD160, K562-CD19-CD160 have IFN-γ secretion, show CD160/ 19CAR-T can equally generate bystander effect, and have same efficiency with traditional CAR-T.
12, proliferative capacity detects
1×105Normal T-cell (Control) respectively with after irradiation K562-Luc, K562-CD19, K562-CD160, 562-CD19/CD160 cells, CD19CAR-T respectively with K562-Luc, K562-CD19, K562-CD160,562- after irradiation CD19/CD160 cells, CD19-CD28-CD3 ζ-T respectively with after irradiation K562-Luc, K562-CD19, K562-CD160, 562-CD19/CD160 cells, CD160CAR-T respectively with K562-Luc, K562-CD19, K562-CD160,562- after irradiation CD19/CD160 cells, CD160/19CAR-T cells respectively with after irradiation K562-Luc, K562-CD19, K562-CD160, 562-CD19/CD160 cells, by 1:1 co-cultures, in the new irradiated cells of addition in 7,14,21,31 days, with blood counting chamber meter Number statistics cell quantity.Experimental result is as shown in figure 8, CD19-CD28-CD3 ζ-T and K562-CD19 or 562-CD19-CD160 Co-cultivation, CD160CAR-T and K562-CD160 or K562-CD19-CD160 co-cultivations, CD160/19CAR-T and K562- CD19, K562-CD160 or K562-CD19-CD160 co-cultivation can stimulate CD160CAR-T, CD19-CD28-CD3 ζ-T, CD160/19CAR-T cell Proliferations, but CD160/19CAR-T holds and is better than CD160CAR-T, CD19-CD28- after proliferative capacity CD3 ζ-T, and without apoptosis phenomenon after activation, show may there is longer Effect time in vivo.
13, zoopery
Mouse：Nonobese diabetic severe/combined immunodeficient(NOD.SCID) Il2rg-/-(NSG)mice.It is 1,2,3,4,5 that 30 mouse are divided into five big group # by table 2.It is divided into per 6 mouse of group greatly Two groups, per each three mouse of group.K562, K562-CD19-CD160 each 1 × 105Cell is small by 2 tail vein injection of table respectively Mouse.After 4 days, tail vein injects 1 × 10 by table two respectively6Tcell, CD19CAR-T, CD19-CD28-CD3 ζ CAR-T, CD160CAR-T, CD160/CD19CAR-T.In 0 day, 7 days, 14 days, 21 days, 31 days tail veins took blood, analyzed K562 in blood, K562-CD19-CD160 cell quantities.
Table 2
As shown in Figure 9 and Figure 10, it is all injection K562 mouse because tumor cell proliferation rapidly the 21st day all it is peaceful and comfortable Extremely.The result shows that CD19-CD28-CD3 ζ CAR-T can kill K562-CD160-CD19 in early period, in later stage K562-CD160- CD19 cells expand rapidly, and fragmentation effect is poor；And CD160/CD19CAR-T energy continuous and effective killings K562-CD160-CD19 swells Oncocyte, the interior K562-CD160-CD19 survived of the 31st day Mice Body are almost nil.Show CD160/CD19CAR-T cell energy The permanently effective killing bis- target tumour cells of K562-CD160-CD19.
During tumour immunity killing, after tumour antigen contact immunocyte, first by TCR activation CD3zeta killings Signal, this is the first signal of immune activation.The first signal of immune cell activated can cause the activation-inducing of immunocyte to be exempted from Epidemic disease apoptosis.The activation of costimulatory signal CD28,4-1BB, OX40 can be with acute activation immune cell expansion and cytotoxicity, the factors Secretion.Killing signal activates simultaneously with costimulatory signal just plays the role of immunologic cytotoxicity, and traditional CAR-T is pierced together by signal is killed The advantages of energizing signal is building up to same expression cassette, translates and forms a fusion protein being covalently attached, this design is people To increase the interaction of killing signal and costimulatory signal, the intensity of killing signal is greatly strengthened, but can cause to lack as follows It falls into：
Single antigen target, which easily causes, to miss the target.Used single antigen also has expression, this list in the normal tissue No matter normally and tumor tissues one antigen is designed with extremely strong killing activity, can kill, lack simply by the presence of this antigen Selectivity.Such as CD19 both high expression in leukaemia human B cell, also expressed in normal B cells, while leukaemia is killed Also normal B cells can be removed and cause serious factor storm.
In the present invention, killing signal and costimulatory signal are building up to two targets respectively, kill signal and costimulation Signal target antigen is generally peculiar in tumour or high expression, as long as there are one do not express this antigen combination in the normal tissue Or low expression killing signal cannot activate, normal cell is protected, and is realized targeting, the high efficiency of immunization therapy, is carried The high safety for the treatment of.Such as CD19 and CD160, CD19 is expressed, while CD160 is white in chronic granulocytic in leukemia patient height Blood patient in hair cell lymphocytic leukemia people with expressing, and using this feature, the present invention transmits killing signal with CD19, uses CD160 transmits costimulatory signal, successfully constructs a kind of double CAR-T cells, can be applied to double target-specific treatment leukaemia.This The cell that kind CD19 and CAR-T specific killing CD19 of CD160 bispecifics target and CD160 is expressed simultaneously, effectively improves The safety of immunization therapy.
Sequence table
<110>Hangzhou Shi Dimu bio tech ltd
<120>A kind of double CAR-T cells and its preparation method and application
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1254
<212> DNA
<213>Artificial sequence (CD160CAR genes)
<400> 1
atgctgctcc tggtgacaag cctgctgctc tgtgagctgc cacacccagc cttcctcctg 60
atcccagata tcgtgctgac ccagagccca gccaccctga gcgtgacccc aggcaacagc 120
gtcagcctgt cctgcagggc cagccagagc atcagcaacc acctgcactg gtaccagcag 180
aagagccacg agagcccaag gctgctcatc aagtacgctt cccagtccat cagcggcatc 240
ccctccaggt tcagcggcag cggcagcggg acagatttca ccctcagcat caacagcgtg 300
gagaccgagg attttggcat gtacttctgt cagcagagca acagctggcc gctcacgttc 360
ggcgctggga ccaagctgga gctgaagcgg ggctccacca gcggctccgg caagcccggc 420
agcggcgagg gctccaccaa gggccaggtg cacctccagc agagcggggc tgagctggcc 480
cgccctgggg ctagcgtgaa gctgtcctgc aaggctagcg gctacacctt taccgactac 540
tggatgcagt ggatcaagca gaggcctggc cagggcctgg agtggatcgg gagcatctac 600
cctggcgatg atgatgctag gtacacccag aagttcaggg gcaaggccac actgaccgcc 660
gataagtcct ccagcacagc ctacatgcag ctcagcagcc tggccagcga ggacagcgcg 720
gtctactact gtgcccgcag gggcatcgct gcggtggtgg gcggctttga ctactggggc 780
cagggcacca ccctcacagt ctccagcatc gaggtgatgt accctcctcc ttacctggac 840
aacgagaaga gcaacggcac catcatccac gtgaagggga agcacctgtg tccaagcccc 900
ctgtttcccg gccctagcaa gcccttttgg gtgctggtgg tggtgggcgg cgtcctggct 960
tgctacagcc tgctggtgac agtggccttt atcatcttct gggtgaggag caagaggagc 1020
aggctcctgc acagcgacta catgaacatg accccccgcc gccccgggcc cacccgcaag 1080
cactaccagc cctacgcccc accacgcgac ttcgccgcct accgctccaa gcggggccgc 1140
aagaagctcc tgtacatctt caagcagcca tttatgcgcc cagtgcagac cacccaggag 1200
gaggatggct gtagctgccg ctttccagag gaggaggagg gcggctgtga gctg 1254
<210> 2
<211> 1377
<212> DNA
<213>Artificial sequence (CD19CAR genes)
<400> 2
atgctgctcc tggtgacaag cctgctgctc tgtgagctgc cacacccagc cttcctcctg 60
atcccagaca tccagatgac acagaccaca tcctccctga gcgccagcct gggcgaccgc 120
gtcaccatca gctgcagggc cagccaggac atcagcaagt acctgaactg gtaccagcag 180
aagccagatg gcaccgtgaa gctcctgatc taccacacaa gccgcctgca cagcggcgtc 240
ccaagcaggt tcagcggcag cgggagcggc acagattaca gcctcaccat cagcaacctg 300
gagcaggagg atatcgccac ctacttttgc cagcagggca acacgctgcc gtacacgttc 360
ggcgggggga ccaagctgga gatcacaggc tccaccagcg gctccggcaa gcccggcagc 420
ggcgagggct ccaccaaggg cgaggtgaag ctgcaggaga gcggccctgg cctggtggcg 480
cccagccaga gcctgtccgt cacatgcacc gtcagcgggg tcagcctgcc cgactacggc 540
gtgagctgga tccgccagcc tccacgcaag ggcctggagt ggctgggcgt gatctggggc 600
agcgagacca catactacaa cagcgctctc aagtcccgcc tgaccatcat caaggacaac 660
tccaagagcc aggtgttcct gaagatgaac agcctgcaga ccgatgacac agccatctac 720
tactgtgcca agcactacta ctacggcggc agctacgcta tggactactg gggccagggc 780
accagcgtca ccgtctccag cgcggccgcc ttcgtgccgg tcttcctgcc agcgaagccc 840
accacgacgc cagcgccgcg cccaccaaca ccggcgccca ccatcgcgtc gcagcccctg 900
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg ccgtgcacac gagggggctg 960
gacttcgcct gtgatatcta catctgggcg cccctggccg ggacctgtgg ggtcctgctc 1020
ctgagcctgg tgatcacccg cgtgaagttc agcaggagcg ccgacgcccc cgcgtaccag 1080
cagggccaga accagctcta caacgagctc aacctgggcc gccgcgagga gtacgatgtg 1140
ctggacaagc gccgcggccg ggaccctgag atggggggca agccgcagcg caggaagaac 1200
cctcaggagg gcctgtacaa cgagctgcag aaggataaga tggcggaggc ctacagcgag 1260
atcgggatga agggcgagcg ccggaggggc aaggggcacg atggcctgta ccagggcctc 1320
agcacagcca ccaaggacac ctacgacgcc ctgcacatgc aggccctgcc ccctcgc 1377
<210> 3
<211> 1302
<212> DNA
<213>Artificial sequence (IRES-mcherry fusions)
<400> 3
gctagccctc ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg 60
cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga 120
aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa 180
tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa 240
caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct 300
gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg 360
ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg 420
ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca 480
catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg 540
acgtggtttt cctttgaaaa acacgatgat aatatggcca cacatatggt gagcaagggc 600
gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc 660
tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc 720
acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc 780
ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc 840
gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag 900
gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac 960
aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc 1020
atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag 1080
atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc 1140
tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac 1200
atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc 1260
cactccaccg gcggcatgga cgagctgtac aagtgatcta ga 1302
<210> 4
<211> 1470
<212> DNA
<213>Artificial sequence (CD19-CD28-CD3 ζ CAR genes)
<400> 4
atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60
atcccagaca tccagatgac acagactaca tcctccctgt ctgcctctct gggagacaga 120
gtcaccatca gttgcagggc aagtcaggac attagtaaat atttaaattg gtatcagcag 180
aaaccagatg gaactgttaa actcctgatc taccatacat caagattaca ctcaggagtc 240
ccatcaaggt tcagtggcag tgggtctgga acagattatt ctctcaccat tagcaacctg 300
gagcaagaag atattgccac ttacttttgc caacagggta atacgcttcc gtacacgttc 360
ggagggggga ctaagttgga aataacaggc tccacctctg gatccggcaa gcccggatct 420
ggcgagggat ccaccaaggg cgaggtgaaa ctgcaggagt caggacctgg cctggtggcg 480
ccctcacaga gcctgtccgt cacatgcact gtctcagggg tctcattacc cgactatggt 540
gtaagctgga ttcgccagcc tccacgaaag ggtctggagt ggctgggagt aatatggggt 600
agtgaaacca catactataa ttcagctctc aaatccagac tgaccatcat caaggacaac 660
tccaagagcc aagttttctt aaaaatgaac agtctgcaaa ctgatgacac agccatttac 720
tactgtgcca aacattatta ctacggtggt agctatgcta tggactactg gggtcaagga 780
acctcagtca ccgtctcctc agcggccgca attgaagtta tgtatcctcc tccttaccta 840
gacaatgaga agagcaatgg aaccattatc catgtgaaag ggaaacacct ttgtccaagt 900
cccctatttc ccggaccttc taagcccttt tgggtgctgg tggtggttgg gggagtcctg 960
gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1020
agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1080
aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cagagtgaag 1140
ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 1200
ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1260
gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1320
aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380
aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1440
cttcacatgc aggccctgcc ccctcgctaa 1470
<210> 5
<211> 1470
<212> DNA
<213>Artificial sequence (Luciferase genes)
<400> 5
atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60
atcccagaca tccagatgac acagactaca tcctccctgt ctgcctctct gggagacaga 120
gtcaccatca gttgcagggc aagtcaggac attagtaaat atttaaattg gtatcagcag 180
aaaccagatg gaactgttaa actcctgatc taccatacat caagattaca ctcaggagtc 240
ccatcaaggt tcagtggcag tgggtctgga acagattatt ctctcaccat tagcaacctg 300
gagcaagaag atattgccac ttacttttgc caacagggta atacgcttcc gtacacgttc 360
ggagggggga ctaagttgga aataacaggc tccacctctg gatccggcaa gcccggatct 420
ggcgagggat ccaccaaggg cgaggtgaaa ctgcaggagt caggacctgg cctggtggcg 480
ccctcacaga gcctgtccgt cacatgcact gtctcagggg tctcattacc cgactatggt 540
gtaagctgga ttcgccagcc tccacgaaag ggtctggagt ggctgggagt aatatggggt 600
agtgaaacca catactataa ttcagctctc aaatccagac tgaccatcat caaggacaac 660
tccaagagcc aagttttctt aaaaatgaac agtctgcaaa ctgatgacac agccatttac 720
tactgtgcca aacattatta ctacggtggt agctatgcta tggactactg gggtcaagga 780
acctcagtca ccgtctcctc agcggccgca attgaagtta tgtatcctcc tccttaccta 840
gacaatgaga agagcaatgg aaccattatc catgtgaaag ggaaacacct ttgtccaagt 900
cccctatttc ccggaccttc taagcccttt tgggtgctgg tggtggttgg gggagtcctg 960
gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1020
agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1080
aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cagagtgaag 1140
ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 1200
ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1260
gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1320
aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380
aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1440
cttcacatgc aggccctgcc ccctcgctaa 1470
<210> 6
<211> 943
<212> DNA
<213>Artificial sequence (gene of CD19)
<400> 6
atgccacctc ctcgcctcct cttcttcctc ctcttcctca cccccatgga agtcaggccc 60
gaggaacctc tagtggtgaa ggtggaagag ggagataacg ctgtgctgca gtgcctcaag 120
gggacctcag atggccccac tcagcagctg acctggtctc gggagtcccc gcttaaaccc 180
ttcttaaaac tcagcctggg gctgccaggc ctgggaatcc acatgaggcc cctggccatc 240
tggcttttca tcttcaacgt ctctcaacag atggggggct tctacctgtg ccagccgggg 300
cccccctctg agaaggcctg gcagcctggc tggacagtca atgtggaggg cagcggggag 360
ctgttccggt ggaatgtttc ggacctaggt ggcctgggct gtggcctgaa gaacaggtcc 420
tcagagggcc ccagctcccc ttccgggaag ctcatgagcc ccaagctgta tgtgtgggcc 480
aaagaccgcc ctgagatctg ggagggagag cctccgtgtc tcccaccgag ggacagcctg 540
aaccagagcc tcagccagga cctcaccatg gcccctggct ccacactctg gctgtcctgt 600
ggggtacccc ctgactctgt gtccaggggc cccctctcct ggacccatgt gcaccccaag 660
gggcctaagt cattgctgag cctagagctg aaggacgatc gcccggccag agatatgtgg 720
gtaatggaga cgggtctgtt gttgccccgg gccacagctc aagacgctgg aaagtattat 780
tgtcaccgtg gcaacctgac catgtcattc cacctggaga tcactgctcg gccagtacta 840
tggcactggc tgctgaggac tggtggctgg aaggtctcag ctgtgacttt ggcttatctg 900
atcttctgcc tgtgttccct tgtgggcatt cttcatcttc aaa 943
<210> 7
<211> 480
<212> DNA
<213>Artificial sequence (gene of CD160)
<400> 7
atgctgttgg aacccggcag aggctgctgt gccctggcca tcctgctggc aattgtggac 60
atccagtctg gtggatgcat taacatcacc agctcagctt cccaggaagg aacgcgacta 120
aacttaatct gtactgtatg gcataagaaa gaagaggctg aggggtttgt agtgtttttg 180
tgcaaggaca ggtctggaga ctgttctcct gagaccagtt taaaacagct gagacttaaa 240
agggatcctg ggatagatgg tgttggtgaa atatcatctc agttgatgtt caccataagc 300
caagtcacac cgttgcacag tgggacctac cagtgttgtg ccagaagcca gaagtcaggt 360
atccgccttc agggccattt tttctccatt ctattcacag agacagggaa ctacacagtg 420
acgggattga aacaaagaca acaccttgag ttcagccata atgaaggcac tctcagttaa 480
<210> 8
<211> 2151
<212> DNA
<213>Artificial sequence (CD19-CD160 fusions)
<400> 8
atgccacctc ctcgcctcct cttcttcctc ctcttcctca cccccatgga agtcaggccc 60
gaggaacctc tagtggtgaa ggtggaagag ggagataacg ctgtgctgca gtgcctcaag 120
gggacctcag atggccccac tcagcagctg acctggtctc gggagtcccc gcttaaaccc 180
ttcttaaaac tcagcctggg gctgccaggc ctgggaatcc acatgaggcc cctggccatc 240
tggcttttca tcttcaacgt ctctcaacag atggggggct tctacctgtg ccagccgggg 300
cccccctctg agaaggcctg gcagcctggc tggacagtca atgtggaggg cagcggggag 360
ctgttccggt ggaatgtttc ggacctaggt ggcctgggct gtggcctgaa gaacaggtcc 420
tcagagggcc ccagctcccc ttccgggaag ctcatgagcc ccaagctgta tgtgtgggcc 480
aaagaccgcc ctgagatctg ggagggagag cctccgtgtc tcccaccgag ggacagcctg 540
aaccagagcc tcagccagga cctcaccatg gcccctggct ccacactctg gctgtcctgt 600
ggggtacccc ctgactctgt gtccaggggc cccctctcct ggacccatgt gcaccccaag 660
gggcctaagt cattgctgag cctagagctg aaggacgatc gcccggccag agatatgtgg 720
gtaatggaga cgggtctgtt gttgccccgg gccacagctc aagacgctgg aaagtattat 780
tgtcaccgtg gcaacctgac catgtcattc cacctggaga tcactgctcg gccagtacta 840
tggcactggc tgctgaggac tggtggctgg aaggtctcag ctgtgacttt ggcttatctg 900
atcttctgcc tgtgttccct tgtgggcatt cttcatcttc aaagagccct ggtcctgagg 960
aggaaaagaa agcgaatgac tgaccccacc aggagattct tcaaagtgac gcctccccca 1020
ggaagcgggc cccagaacca gtacgggaac gtgctgtctc tccccacacc cacctcaggc 1080
ctcggacgcg cccagcgttg ggccgcaggc ctggggggca ctgccccgtc ttatggaaac 1140
ccgagcagcg acgtccaggc ggatggagcc ttggggtccc ggagcccgcc gggagtgggc 1200
ccagaagaag aggaagggga gggctatgag gaacctgaca gtgaggagga ctccgagttc 1260
tatgagaacg actccaacct tgggcaggac cagctctccc aggatggcag cggctacgag 1320
aaccctgagg atgagcccct gggtcctgag gatgaagact ccttctccaa cgctgagtct 1380
tatgagaacg aggatgaaga gctgacccag ccggtcgcca ggacaatgga cttcctgagc 1440
cctcatgggt cagcctggga ccccagccgg gaagcaacct ccctggcagg gtcccagtcc 1500
tatgaggata tgagaggaat cctgtatgca gccccccagc tccgctccat tcggggccag 1560
cctggaccca atcatgagga agatgcagac tcttatgaga acatggataa tcccgatggg 1620
ccagacccag cctggggagg agggggccgc atgggcacct ggagcaccag gatgctgttg 1680
gaacccggca gaggctgctg tgccctggcc atcctgctgg caattgtgga catccagtct 1740
ggtggatgca ttaacatcac cagctcagct tcccaggaag gaacgcgact aaacttaatc 1800
tgtactgtat ggcataagaa agaagaggct gaggggtttg tagtgttttt gtgcaaggac 1860
aggtctggag actgttctcc tgagaccagt ttaaaacagc tgagacttaa aagggatcct 1920
gggatagatg gtgttggtga aatatcatct cagttgatgt tcaccataag ccaagtcaca 1980
ccgttgcaca gtgggaccta ccagtgttgt gccagaagcc agaagtcagg tatccgcctt 2040
cagggccatt ttttctccat tctattcaca gagacaggga actacacagt gacgggattg 2100
aaacaaagac aacaccttga gttcagccat aatgaaggca ctctcagtta a 2151
<210> 9
<211> 66
<212> DNA
<213>Artificial sequence (F2A)
<400> 9
gtgaaacaga ctttgaattt tgaccttctc aagttggcgg gagacgtgga gtccaaccca 60
gggccc 66

Claims (8)

1. a kind of double CAR-T cells, which is characterized in that co-expressed using in tumour cell, but table is not total in normal cell Two target antigens reached kill signal fused in the antibody variable region of one of target antigen and T cell, another target The antibody variable region of antigen is merged with costimulatory signal in T cell, forms two different expression cassettes, builds double CAR-T Cell；Double CAR-T cells of structure only identify while express the tumour cell of two target antigens, and nonrecognition is only expressed wherein The normal cell of one target antigen.
2. double CAR-T cells according to claim 1 a, which is characterized in that target resists in described two target antigens Originally it was CDX, X 19；Another target antigen is CDY, Y 160,123 or 23.
3. double CAR-T cells according to claim 1, which is characterized in that signal is killed in the T cell and is included CD3zeta。
4. double CAR-T cells according to claim 1, which is characterized in that costimulatory signal includes in the T cell CD28、4-1BB、OX40。
5. the preparation method of double CAR-T cells described in claim 1-4 any claims, which is characterized in that including as follows Step：
Step 1, structure expression CDY/XCAR slow virus carriers；
Step 2, slow virus packaging：Obtain expression CDY/XCAR slow virus；
Step 3, slow-virus infection T cell：Human peripheral blood mononuclear cell, culture and amplification T cell are detached, is obtained using step 2 Expression CDY/XCAR slow-virus infection T cells, obtain expression CDY/XCAR T cell：CDY/XCAR-T, i.e., double CAR-T are thin Born of the same parents.
6. the preparation method of double CAR-T cells according to claim 5, which is characterized in that CD19/160CAR-T cells Preparation includes the following steps：
Step 1, structure expression CD19/160CAR slow virus carriers：CD160CAR gene orders are cloned into pHAGE2-cca- LoxP carrier NotI sites, by IRES-mcherry fusion gene clonings to CD160CAR sequence downstream NheI/XbaI sites it Between, the carrier obtained is named as pHAGE-CD160；CD19CAR sequences are cloned into the BstpDI positions of pHAGE-CD160 carriers Point obtains carrier and is named as pHAGE-CD160/19；
Step 2, slow virus packaging：293T cell culture is in the dual anti-DMEM culture mediums of the green chain containing 10%FBS and 1%, cell patch Density reaches 50%-60% after wall, and cell changes plasma-free DMEM medium into；Plasmid presses pHAGE-CD160/19：pspAX2： PMD2.G=4:3:Mixing in serum-free DMEM is added to after the mixing of 1 ratio, PEI is added to serum-free DMEM mixings, will after standing DMEM is added in DMEM and plasmid mixed liquor with PEI mixed liquors and blows and beats mixing, and 293T cells are dropped evenly after being stored at room temperature In；After cell constant temperature incubator culture 6 hours, it is changed to the dual anti-DMEM culture mediums of the green chain containing 10%FBS and 1%, 37 DEG C, 5%CO2Incubator is further cultured for 24 hours；Supernatant is collected, 1500rpm room temperatures centrifugation 10min abandons cell precipitation, supernatant mistake 0.22um filter membranes as express CD160/19CAR slow virus, and -80 DEG C save backup；
Step 3, slow-virus infection T cell：Human peripheral blood mononuclear cell, culture and amplification T cell are detached, is obtained using step 2 Expression CD160/19CAR slow-virus infection T cells, obtain expression CD160/19CAR T cell：CD160/19CAR-T, i.e., Double CAR-T cells.
7. application of double CAR-T cells in tumor is prepared described in claim 1-4 any claims.
8. double CAR-T cells described in claim 1-4 any claims are preparing the application in treating leukemia medicament.
CN201810228590.1A 2018-03-20 2018-03-20 A kind of double CAR-T cells and its preparation method and application Pending CN108220247A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810228590.1A CN108220247A (en) 2018-03-20 2018-03-20 A kind of double CAR-T cells and its preparation method and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810228590.1A CN108220247A (en) 2018-03-20 2018-03-20 A kind of double CAR-T cells and its preparation method and application

Publications (1)

Publication Number Publication Date
CN108220247A true CN108220247A (en) 2018-06-29

Family

ID=62659746

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810228590.1A Pending CN108220247A (en) 2018-03-20 2018-03-20 A kind of double CAR-T cells and its preparation method and application

Country Status (1)

Country Link
CN (1) CN108220247A (en)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109504660A (en) * 2018-11-02 2019-03-22 温州启星生物技术有限公司 A kind of forth generation CAR-T cell and its construction method and application
CN109735558A (en) * 2018-12-12 2019-05-10 中南大学 A kind of recombinant C AR19-IL24 gene, slow virus carrier, CAR19-IL24-T cell and application
CN109868260A (en) * 2019-04-16 2019-06-11 上海汉尼生物细胞技术有限公司 A kind of preparation method of CAR-T cell
CN110951694A (en) * 2019-12-30 2020-04-03 北京鼎成肽源生物技术有限公司 Preparation method of autologous trophoblast and culture method of SNK cells
CN111041064A (en) * 2019-07-22 2020-04-21 徐州医科大学 Method for evaluating CAR-T killing activity in vitro
WO2021038036A1 (en) * 2019-08-28 2021-03-04 King's College London B CELL TARGETED PARALLEL CAR (pCAR) THERAPEUTIC AGENTS
CN113382745A (en) * 2016-09-02 2021-09-10 莱蒂恩技术公司 Compositions and methods for treating cancer with DuoCAR
WO2022089656A1 (en) * 2020-11-02 2022-05-05 上海医药集团生物治疗技术有限公司 Vector genetically engineered with chimeric antigen receptor and against two or more targets and application thereof
WO2022195241A1 (en) * 2021-03-17 2022-09-22 Ucl Business Ltd Cd160 binding domain

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105384825A (en) * 2015-08-11 2016-03-09 南京传奇生物科技有限公司 Bispecific chimeric antigen receptor based on variable domains of heavy chain of heavy-chain antibody and application thereof
CN106822184A (en) * 2016-12-15 2017-06-13 梁爱斌 The united CAR T cells preparation of Mutiple Targets and preparation method
WO2017175072A1 (en) * 2016-04-08 2017-10-12 Feldan Bio Inc. Peptide shuttle based gene disruption
CN107326014A (en) * 2017-07-31 2017-11-07 时力生物科技（北京）有限公司 A kind of T lymphocytes of bispecific chimeric antigen receptor modification and its preparation method and application

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105384825A (en) * 2015-08-11 2016-03-09 南京传奇生物科技有限公司 Bispecific chimeric antigen receptor based on variable domains of heavy chain of heavy-chain antibody and application thereof
WO2017175072A1 (en) * 2016-04-08 2017-10-12 Feldan Bio Inc. Peptide shuttle based gene disruption
CN106822184A (en) * 2016-12-15 2017-06-13 梁爱斌 The united CAR T cells preparation of Mutiple Targets and preparation method
CN107326014A (en) * 2017-07-31 2017-11-07 时力生物科技（北京）有限公司 A kind of T lymphocytes of bispecific chimeric antigen receptor modification and its preparation method and application

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
丁润生等: ""CD160 在慢性淋巴细胞白血病中的表达及临床意义"", 《实用医学杂志》 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113382745B (en) * 2016-09-02 2024-04-19 莱蒂恩技术公司 Compositions and methods for treating cancer with DuoCAR
CN113382745A (en) * 2016-09-02 2021-09-10 莱蒂恩技术公司 Compositions and methods for treating cancer with DuoCAR
CN109504660A (en) * 2018-11-02 2019-03-22 温州启星生物技术有限公司 A kind of forth generation CAR-T cell and its construction method and application
CN109504660B (en) * 2018-11-02 2021-08-06 温州启星生物技术有限公司 Fourth-generation CAR-T cell and construction method and application thereof
CN109735558A (en) * 2018-12-12 2019-05-10 中南大学 A kind of recombinant C AR19-IL24 gene, slow virus carrier, CAR19-IL24-T cell and application
CN109735558B (en) * 2018-12-12 2022-04-15 中南大学 Recombinant CAR19-IL24 gene, lentiviral vector, CAR19-IL24-T cell and application
CN109868260A (en) * 2019-04-16 2019-06-11 上海汉尼生物细胞技术有限公司 A kind of preparation method of CAR-T cell
CN111041064A (en) * 2019-07-22 2020-04-21 徐州医科大学 Method for evaluating CAR-T killing activity in vitro
WO2021038036A1 (en) * 2019-08-28 2021-03-04 King's College London B CELL TARGETED PARALLEL CAR (pCAR) THERAPEUTIC AGENTS
CN110951694A (en) * 2019-12-30 2020-04-03 北京鼎成肽源生物技术有限公司 Preparation method of autologous trophoblast and culture method of SNK cells
CN115380112A (en) * 2020-11-02 2022-11-22 上海医药集团生物治疗技术有限公司 Two or more target point chimeric antigen receptor gene engineering carrier and its application
CN115380112B (en) * 2020-11-02 2024-03-29 上海医药集团生物治疗技术有限公司 Two or more target chimeric antigen receptor gene engineering vector and application thereof
WO2022089656A1 (en) * 2020-11-02 2022-05-05 上海医药集团生物治疗技术有限公司 Vector genetically engineered with chimeric antigen receptor and against two or more targets and application thereof
WO2022195241A1 (en) * 2021-03-17 2022-09-22 Ucl Business Ltd Cd160 binding domain

Similar Documents

Publication Publication Date Title
CN108220247A (en) A kind of double CAR-T cells and its preparation method and application
US11932872B2 (en) Dual chimeric antigen receptor-t cell which can be regulated, construction method therefor and use thereof
CN108373504A (en) CD24 specific antibodies and anti-CD24-CAR-T cells
CN108752482B (en) Carry the Chimeric antigen receptor and its application of truncation or not truncated myeloid cell triggering property receptor signal structure
CN109678965A (en) The T cell and its application of Chimeric antigen receptor and its gene and recombinant expression carrier, the bis- targetings of CD22-CD19
CN108409840A (en) The Chimeric antigen receptor of anti-CD123 single-chain antibodies and combinations thereof and application
KR20220009966A (en) Engineered Immune Cells Containing Recognition Molecules
CN109503716A (en) A kind of bispecific chimeric antigen receptor molecule and its application in oncotherapy
CN107033248A (en) Recognize Chimeric antigen receptor and its application of carcinomebryonic antigen
CN107365798A (en) A kind of CD19 CAR T cells of carrying iCasp9 suicide genes and its application
CN106317228A (en) Chimeric antigen receptor molecule and application thereof
CN108641000A (en) The double target spot CAR-T therapy vectors and its construction method of liver cancer and application
CN106279432B (en) A kind of VC-CAR molecule and the application in removing HIV-1 infection cell
CN108822216B (en) Carry the Chimeric antigen receptor and its application of truncation or not truncated nature cell toxin receptor signal structure
CN108341881A (en) Chimeric antigen receptor and its expressing gene with safety switch, the NK cells of its modification and application
CN110423767A (en) Express Chimeric antigen receptor CAR gene and the application of solubility PD-1
CN116144599A (en) Immune cell for killing escape variant NK cells and application thereof
CN105132445B (en) A kind of receptor protein of specially recognizing tumor cells, T lymphocytes and NK cells
CN107541499B (en) Preparation and application of CIK of TNFR2 in targeted immunodetection point
JP2021519063A (en) Lymphocytes expressing heterologous targeting constructs
CN107058234B (en) CAR.IL-33-T and its preparation and application
CN109265563A (en) It is a kind of for treating the source of people Chimeric antigen receptor and its application of neoplastic hematologic disorder
CN108864289A (en) The double target spot CAR-T therapy vectors and its construction method of gastric cancer and application
CN111704674A (en) Chimeric antigen receptor targeting c-Met and autocrine PD-L1scFv and application thereof
CN108699163A (en) A kind of polygenes recombination Chimeric antigen receptor molecule and its application

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20180629