CN109593786A - The double target spot CAR carriers and its construction method of joint EpCAM and MSLN single-chain antibody and in breast cancer application - Google Patents

The double target spot CAR carriers and its construction method of joint EpCAM and MSLN single-chain antibody and in breast cancer application Download PDF

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CN109593786A
CN109593786A CN201910019779.4A CN201910019779A CN109593786A CN 109593786 A CN109593786 A CN 109593786A CN 201910019779 A CN201910019779 A CN 201910019779A CN 109593786 A CN109593786 A CN 109593786A
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epcam
chain antibody
cell
msln
target spot
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田晓丽
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Shanghai Yi Hao Biotechnology Co Ltd
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Shanghai Yi Hao Biotechnology Co Ltd
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • 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
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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    • 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
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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    • C12N2510/00—Genetically modified cells
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    • 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

Abstract

The invention belongs to field of pharmaceutical biology, and in particular to the double target spot CAR carriers and its construction method and in breast cancer application of joint EpCAM and MSLN single-chain antibody.Double target spot CAR-T therapy vectors pass through joint EpCAM single-chain antibody and MSLN single-chain antibody, specially CD8leader-EpCAM scFv-CD8 α-CD28-CD137-IRES-CD8leader-MSLN scFv-CD8 α-CD3 ζ.It is obtained in a manner of virus infection containing therapy vector CAR-T cell (Chimeric Antigen Receptor T-Cell), it, can targets identification and the high breast cancer cell for expressing two kinds of tumor associated antigens of EpCAM and MSLN of killing by expressing CAR structure.

Description

The double target spot CAR carriers and its construction method of joint EpCAM and MSLN single-chain antibody and In breast cancer application
Technical field
The invention belongs to field of pharmaceutical biology, and in particular to double target spot CAR carriers of joint EpCAM and MSLN single-chain antibody And its construction method and in breast cancer application.
Background technique
Breast cancer is common one of the malignant tumour of female sex organ, and the death rate accounts for the first of female malignant Position, is the primary cause of the death of female cancer patient's death.At present the treatment method of breast cancer have operative treatment, chemotherapy, Chinese traditional treatment, Immunization therapy etc..In recent years, with the continuous development of basic scientific research, immunization therapy is increasingly valued by people.
LAK cell, DC, CIK cell are all once as the immunotherapeutic of tumour, but the above various treatment methods lack Weary specific recognition tumor associated antigen (tumor associated antigen, TAA) and killing specific tumors target cell Ability；And the process of cancer immunoediting can make the major histocompatibility complex (main of target cell surface Histocompatibility complex, MHC) decline in tumor cell surface expression, immunologic escape is formed, tumour cell is made T cell attack can successfully be hidden.
Chimeric antigen receptor (CAR)-T cell therapy is artificially to be overexpressed energy on T cell surface by technique for gene engineering The single-chain variable fragments of specific tumor surface antigen are identified, so that T cell be made to identify specific antigen, killing table Up to the target cell of the antigen.Simultaneously as CAR-T cell is to identify antigen mode using antibody, therefore do not limited by MHC.
Epithelium specific adhesion molecule (EpCAM, Epithelial cell adhesion molecule) is a kind of epithelium Cell transmembrane glycoprotein participates in Wnt signal transduction pathway, regulation target gene transcription, adherency, migration, proliferation, differentiation with cell Etc. related.It is initially to be found with a kind of tumor associated antigen, since EpCAM is in kinds of tumors tissue such as oophoroma, stomach Expression in cancer, Colon and rectum gland cancer, breast cancer etc. is increased and is concerned.The study found that the expression and ovarian neoplasm of EpCAM Progress and prognosis are related, can be used as the target spot of breast cancer targeted therapy.
Mesothelin (mesothelin, MSLN) is a kind of anchored glycoprotein for being present in cell surface, because it is at normal group Knit do not express or mesothelial tissue in low expression, but the high expression in 30% tumour, such as breast cancer, gastric cancer, colon cancer are real Body tumor, and become the tumor cell specific targeting antigen being concerned.
Undershooting-effect is the main source of CAR-T cell therapy side effect, and undershooting-effect, which refers to fail to reach, to be preset Target, have offset phenomenon.The extracellular identification structural domain overwhelming majority of CAR-T cell is tumor associated antigen (Tumor Associated antigen, TAA), and tumor associated antigen and non-tumor cell institute it is peculiar, on health tissues there is also Different degrees of expression.This CAR-T cell can also attack normal tissue, this toxicity, that is, non-while killing tumor cell Cancer target toxicity, poisonous effect caused by the toxic mechanism are known as undershooting-effect.
Therefore, the generation of undershooting-effect how is effectively reduced, enhances killing of the T lymphocyte to tumour cell, to mention The effect of high CAR-T immunotherapy anti-breast cancer becomes a technical problem of CAR-T treatment.
Summary of the invention
The invention aims to solve the above problems, the present invention provides pass through joint EpCAM and MSLN single-chain antibody Double target spot CAR carriers, which is effectively reduced undershooting-effect.It is of the present invention to be directed to EpCAM's and MSLN Undershooting-effect is effectively reduced in CAR-T technology, enhances killing of the T lymphocyte to tumour cell, exempts to improve CAR-T The effect of epidemic disease therapy anti-breast cancer.The design of double target spots can reduce bring risk in CAR-T treatment, meanwhile, parallel-connection structure can Effectively solve different single-chain antibodies affecting one another on space structure.In addition, further including joint EpCAM and MSLN single-chain antibody Double target spot CAR carriers construction method and its application.
Double target spot CAR carrier specific technical solutions of joint EpCAM and MSLN single-chain antibody provided by the invention are as follows:
Double target spot CAR carriers of joint EpCAM and MSLN single-chain antibody, including Chimeric antigen receptor and carrier, it is described embedding Close antigen receptor connection on the carrier, the Chimeric antigen receptor includes that be respectively as follows: EpCAM single-stranded for two species specificity structures Antibody and MSLN single-chain antibody, for the nucleotide sequence of the EpCAM single-chain antibody as shown in SEQ ID NO.2, the MSLN is mono- For the nucleotide sequence of chain antibody as shown in SEQ ID NO.7, the structure composition of the Chimeric antigen receptor is CD8leader- EpCAM scFv-CD8α-CD28-CD137-IRES-CD8leader-MSLN scFv-CD8α-CD3ζ。
In some embodiments, the CD8leader can express the newly synthesized protein of guidance and carry out transmembrane process Guide peptide, the nucleotide sequence of the CD8leader as shown in SEQ ID NO.1,
The EpCAM scFv indicates EpCAM single-chain antibody,
The CD8 α is transmembrane region, connects extracellular antigen binding domain and intracellular signal domain, and nucleotide sequence such as SEQ ID Shown in NO.3,
The CD28-CD137 is costimulation structural domain, and the CD28 nucleotide sequence is described as shown in SEQ ID NO.4 CD137 nucleotide sequence as shown in SEQ ID NO.5,
The IRES is ribosome recognition site, is to realize that identical carrier efficiently co-expresses the element of two genes, nucleosides Acid sequence as shown in SEQ ID NO.6,
The MSLN scFv indicates MSLN single-chain antibody,
The CD3 ζ is signal transduction domain, and nucleotide sequence is as shown in SEQ ID NO.8.
In some embodiments, the carrier includes PUC19 plasmid, slow virus carrier.
In some embodiments, the slow virus carrier is pCDH-CMV-MCS-EF1-Puro.
The present invention also provides the method for the double target spot CAR carriers for constructing above-mentioned joint EpCAM and MSLN single-chain antibody, Steps are as follows:
(1) Chimeric antigen receptor is stored on the PUC19 plasmid；
(2) double digestion will be carried out containing PUC19 plasmid in step (1) and the slow virus carrier, digestion products will be distinguished It is separated by agarose gel electrophoresis, after the target fragment containing the Chimeric antigen receptor and double digestion after obtaining double digestion Slow virus carrier,
(3) by the slow virus carrier in the target fragment containing the Chimeric antigen receptor and step (2) in step (2) It is attached, connection product is converted, extract plasmid, obtain the recombinant plasmid containing Chimeric antigen receptor structure, it is described Recombinant plasmid is double target spot CAR carriers of joint EpCAM and MSLN single-chain antibody.
In some embodiments, the double digestion in the step (2) is EcoRI and NotI, containing described in (3) The molar ratio that the target fragment of Chimeric antigen receptor is connected with slow virus carrier is 5:1.
The present invention also provides the applications of double target spot CAR carriers of joint EpCAM and MSLN single-chain antibody, by the connection The double target spot CAR carriers for closing EpCAM and MSLN single-chain antibody carry out slow virus packaging, virion are obtained, after centrifugal concentrating The slow virus suspension of high titre is obtained, the slow virus suspension obtains CAR-T cell, the CAR-T is thin for infecting T cell Born of the same parents pass through identification specific proteins EpCAM and MSLN target killing breast cancer cell.
In some embodiments, the slow virus packaging uses three plasmid packaging systems, the three plasmids packaging system Double target spot CAR carriers including PSPAX2 plasmid, pMD2G plasmid and the joint EpCAM and MSLN single-chain antibody, it is described PSPAX2 plasmid, the pMD2G plasmid, the joint EpCAM and MSLN single-chain antibody the ratios of double target spot CAR carriers be 27:3:20, the slow virus packaging use incasing cells of the 293T cell as slow virus.
The invention has the following advantages: the present invention for therapy vector CAR structure include EpCAM single-chain antibody, Two specificity structures of MSLN single-chain antibody；EpCAM single-chain antibody, MSLN single-chain antibody are according to a variety of breast cancer cell surfaces The specificity structure that tumor associated antigen EpCAM, MSLN are determined, Anti-EpCAM, Anti-MSLN of the structure representation can be expressed Two strain specific antibodies are responsible for identifying TAA, on the one hand can targeting killing tumor cell；On the other hand, imparting T cell is new resists Former specificity can effectively avoid tumour cell MHC expression and lower this Immune escaping mechanism.In addition, the design of double target spots can be with The undershooting-effect generated in CAR-T therapeutic process is effectively reduced, CAR-T cell is allow preferably to identify the cream of double positive target spots Adenocarcinoma cell while improving killing-efficiency, reduces bring risk in CAR-T treatment.In addition, parallel-connection structure can be solved effectively Different single-chain antibodies affecting one another on space structure.It is somebody's turn to do when T cell is obtained the carrier and expressed by virus infection mode After CAR structure, T cell can targeting identify and kill breast cancer cell.
Detailed description of the invention
Fig. 1 is Lentiviral pCDH-CMV-MCS-EF1-Puro structural schematic diagram of the invention；
Fig. 2 is CAR structural schematic diagram of the invention；
Fig. 3 is that Lentiviral pCDH-CMV-MCS-EF1-Puro EcoRI and NotI double digestion of the invention are produced Object agarose gel electrophoresis figure；
Fig. 4 is the double target spot CAR carrier EcoRI and NotI double digestion of joint EpCAM and MSLN single-chain antibody of the invention Product agarose gel electrophoresis figure；
Fig. 5 is the FCM analysis figure of CAR-T cell of the invention；
Fig. 6 is the killing-efficiency curve graph of RTCA detection CAR-T cell of the invention to SK-BR-3；
Fig. 7 is that CAR-T cell of the invention detects column to the ELISA of the IFNgamma factor after SK-BR-3 killing for 24 hours Figure.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, and reference Attached drawing, the present invention is described in more detail.
The present invention provides double target spot CAR carriers of joint EpCAM and MSLN single-chain antibody, concrete scheme is as follows: including Chimeric antigen receptor and carrier, Chimeric antigen receptor are connected on carrier, and the Chimeric antigen receptor includes two species specificity knots Structure is respectively as follows: EpCAM single-chain antibody and MSLN single-chain antibody, the nucleotide sequence of the EpCAM single-chain antibody such as SEQ ID Shown in NO.2, the nucleotide sequence of the MSLN single-chain antibody is as shown in SEQ ID NO.7.The structure composition of Chimeric antigen receptor For CD8leader-EpCAM scFv-CD8 α-CD28-CD137-IRES-CD8leader-MSLN scFv-CD8 α-CD3 ζ. CD8leader can express the guidance peptide for guiding newly synthesized protein to carry out transmembrane process, the nucleotides sequence of the CD8leader Column are as shown in SEQ ID NO.1；EpCAM scFv indicate EpCAM single-chain antibody, nucleotide sequence as shown in SEQ ID NO.2, CD8 α is transmembrane region, connects extracellular antigen binding domain and intracellular signal domain, by CAR Structure anchor in the transmembrane region on T cell film, And nucleotide sequence is as shown in SEQ ID NO.3；CD28-CD137 is costimulation structural domain, transduction proliferation signal and inducing cell The factor generates, stimulates T cell activation, and CD28 nucleotide sequence is as shown in SEQ ID NO.4, CD137 nucleotide sequence such as SEQ Shown in ID NO.5；IRES is ribosome recognition site, can recruit ribosomes and translate to mRNA, as IRES and external source cDNA When fusion, IRES can independently initiation of translation, nucleotide sequence is as shown in SEQ ID NO.6；MSLN scFv indicates that MSLN is single-stranded Antibody can express the single-chain antibody structure of antibody A nti-MSLN, and nucleotide sequence is as shown in SEQ ID NO.7；CD3 ζ is signal , to conduction TCR sample signal intracellular, T cell, nucleotide sequence such as SEQ are activated when extracellular region and target antigen combination in transduction domain Shown in ID NO.8.Expression for breast cancer cell surface tumor associated antigen EpCAM, MSLN antibody A nti-EpCAM, Anti-MSLN, so as to more accurately identify and kill and meanwhile express above two tumor associated antigen breast cancer it is thin Born of the same parents reduce undershooting-effect.CAR structure mentioned in the present invention imparts the stronger proliferative of T cell, and lasting vitality makes It shows stronger tumor cell killing potential.
Carrier includes PUC19 plasmid, slow virus carrier.Slow virus carrier is pCDH-CMV-MCS-EF1-Puro.Pass through Snap Gene software analyzes the carrier and searches pertinent literature it is found that pCDH-CMV-MCS-EF1-Puro EcoRI and NotI Double digestion Insert Fragment.It is mammalian cell specificity promoter that the expression vector, which includes: CMV promoter-, driving capability compared with By force；Multiple cloning sites (MCS)-include multiple restriction enzyme sites (restriction site), are that foreign gene is inserted into Position；The polyA tailing efficiency of mRNA can be improved in WPRE element-, improves the expression efficiency of metastatic gene；SV40polyA sequence- Transcription can effectively be terminated and add PolyA tail for the mRNA of transcription；Hybrid RSV/5 ' LTR- contains promoter and enhancer Equal controlling elements, make its high-caliber expression overall length virus transcription object in 293T cell；Genetic element (cPPT, gag, env, LTRs)-for packing, transduceing and steadily will be in the genomic DNA of expressing viral structural integrity to host；SV40origin- Plasmid is set to stablize proliferation in incasing cells.
The Lentiviral, which can be used as, makes target gene in nearly all mammalian cell include non-dividing cell With the most effective carrier expressed in dividing cell, can hold carry exogenous genetic fragment it is big, transfection efficiency is higher, also can to T cell Reach satisfied transfection.
One, experimental material
1. slow virus expression plasmid pCDH-CMV-MCS-EF1-Puro, slow virus packaging plasmid pMD2G, vector plasmid PSPAX2 is purchased from SBI；Slow virus expression plasmid pCDH-CMV-MCS-EF1-Puro structure is as shown in Figure 1；
2.CAR structure sequence is designed by Shanghai Yi Hao Biotechnology Co., Ltd, You Shenggong bioengineering (Shanghai) share Co., Ltd's synthesis, with the preservation of PUC19 plasmid form；
3. restriction endonuclease EcoRI and NotI are purchased from NEB；
4.T4 DNA ligase、Free H2O is purchased from precious biology；
5. competent cell is purchased from Trans；
6. plastic recovery kit, the small extraction reagent kit of plasmid is purchased from Tiangeng biochemical technology Co., Ltd；
7.293T cell, SK-BR-3 cell are purchased from Chinese Academy of Sciences's cell bank；
8.FBS, DMEM, 1640 culture mediums, PBS, Opti-MEM, lipofectamine 2000 are purchased from Gibco；
9.CD3 monoclonal antibody, CD28 monoclonal antibody, CH38 albumen, IL-2 are purchased from Shanghai offshore protein Science and Technology Ltd.；
10.Multiskan GO microplate reader+uDrop ultra micro template, flow cytometer are purchased from ThermoFisher；
11.HE120 Horizontal electrophoresis tank, Tanon gel imager are purchased from Tanon；
12. water isolation type constant incubator, constant-temperature shaking incubator are purchased from the permanent Science and Technology Ltd. in Shanghai one；
The 13.Bio-Rad small-sized electrophoresis system of Mini-PROTEAN Tetra Cell is purchased from Bio-Rad；
14. Olympus microscope is purchased from Olympus；
15. oese, spreading rod are purchased from Jie Te biofiltration limited liability company；
16. syringe, the culture dish of 0.45 μm of filter membrane, each specification, culture bottle, porous culture plate, various specifications centrifuge tube Purchased from Corning.
Two, combine the construction method of double target spot CAR carriers of EpCAM and MSLN single-chain antibody
(1) plasmid extracts
The preparation method of LB liquid medium: electronic balance weighs 5g fluid nutrient medium dry powder in 500mL conical flask, adds 100mL ultrapure water sterilizes in high-pressure steam sterilizing pan after masking foil sealing, when being cooled to 40 DEG C -50 DEG C, with 1000: 1 is added 0.2% ampicillin (AMP), it is careful mix after, be transferred to spare in clean 500mL reagent bottle, condition of storage is 4℃。
The preparation method of LB solid medium: electronic balance weighs 5g solid medium dry powder in 500mL conical flask, adds 100mL ultrapure water sterilizes in high-pressure steam sterilizing pan after masking foil sealing, when being cooled to 40 DEG C -50 DEG C, with 1000: 1 is added 0.2% ampicillin, and after careful mixing, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices after solidification, seals membrana oralis and is stored in 4 DEG C.
It is taken out respectively from -80 DEG C of refrigerators and contains slow virus expression plasmid pCDH-CMV-MCS-EF1-Puro and PUC19 plasmid Glycerol stock, take 5 μ L to be inoculated in 5mL LB liquid medium (AMP resistance) respectively, in constant-temperature table shaken cultivation 12-16h, Condition is 37 DEG C, 250rmp.
According to purchased from Tiangeng biochemical technology Co., Ltd the small extraction reagent kit of plasmid (article No.: DP103-03) specification into Row plasmid extracts, and obtains slow virus expression plasmid pCDH-CMV-MCS-EF1-Puro and containing double target spot (EpCAM single-chain antibodies With MSLN single-chain antibody) the PUC19 plasmid of CAR structure.
(2) digestion, connection, conversion
By extracted slow virus expression plasmid pCDH-CMV-MCS-EF1-Puro and containing double target spots, (EpCAM is single-stranded Antibody and MSLN single-chain antibody) CAR structure PUC19 plasmid simultaneously carry out EcoRI, NotI double digestion, digestion products are distinguished Agarose gel electrophoresis is carried out, is observed and recorded with gel imager as a result, testing result is shown in attached drawing 3.
Purpose band recycling is carried out with Tiangeng Ago-Gel QIAquick Gel Extraction Kit (article No.: DP209-02) specification, will be returned The section that takes up is by CAR structure: the molar ratio of pCDH-CMV-MCS-EF1-Puro=5:1 is attached, and is converted to competence Competent cell after conversion is dripped on the solid medium of preheating, is marked by cell, is stayed overnight in 37 DEG C of incubators.
(3) upgrading grain, digestion verification, sequencing
Picking part bacterium colony carries out Zengjing Granule in constant-temperature table and goes forward side by side in 5mL LB liquid medium (AMP resistance) Row plasmid extraction, plasmid extraction take out 500 μ L in 1.5mL Ep pipe before, are used as fungi preservation, are stored in -80 DEG C；It will take out It mentions product to verify using EcoRI, NotI double digestion, the correct 1 μ g of plasmid of band is taken to send raw work bioengineering (Shanghai) limited public affairs (as shown in figure 4, target fragment is CAR structure in figure) is sequenced in department, and the plasmid of band exception and the bacterium solution of retention are lost It abandons.The correct plasmid of sequencing result is stripped, the plasmid and its bacterium solution of sequencing result mistake abandon.
Three, combine application of the double target spot CAR carriers of EpCAM and MSLN single-chain antibody in breast cancer
(1) preparation of concentrating virus liquid
Slow virus packaging is carried out using three plasmid packaging systems.Three plasmids are respectively the slow virus expression containing CAR structure Plasmid pCDH-CMV-MCS-EF1-Puro, slow virus packaging plasmid pMD2G, vector plasmid PSPAX2.Cell is 293T cell.
Specific implementation step is as follows:
(1) interior for 24 hours before transfection to carry out bed board: to be typically chosen cell of the passage number within 3 generations, grown according to cell close Degree and state adjust cell density, and the 293T cell that stand density reaches 80% carries out bed board；
(2) density to be grown reaches 60-90%, and cell state is good, can carry out viral packaging；
(3) according to PSPAX2 plasmid, pMD2G plasmid, the double target spot CAR carriers (weight for combining EpCAM and MSLN single-chain antibody Group plasmid) ratio be that 27:3:20 carries out viral packaging, by taking 6cm ware as an example, required three plasmid mixed liquors proportion is as follows: pressing Plasmid additional amount is determined according to each plasmid concentration.
3 μ g of recombinant plasmid
pMD2G 0.5μg
PSPAX2 4μg
(4) transfection reagent selection lipofectamine 2000 (4 DEG C of preservations), additional amount is 2 μ L/ μ g plasmids；
(5) transfection reagent mixtures in plasmid mixture in (3) and (4) are mixed in a pipe, after the static 20min of room temperature, Addition is changed in liquid cell, continues to cultivate；
(6) respectively collect 48h, 72h after culture supernatant, pass through 0.45 μm of membrane filtration；
(7) virus liquid of collection is concentrated using PEG8000 concentration method, and measures virus titer, be stored in 80 DEG C of ﹣ It is spare.
(2) virus liquid infects T cell
1, PBMC is separated
1) about 6mL people periphery new blood is collected with the vacuum blood collection tube containing heparin；
2) it dilutes: isometric PBS being added at room temperature, gently piping and druming mixes；
3) be loaded: taking 50mL centrifuge tube, draw 6mL Ficoll (lymphocyte separation medium) in centrifuge tube (Ficoll with The volume ratio of blood is 1:1 before diluting), centrifuge tube tilts 45 °, by the blood after dilution at Ficoll ullage about 1cm It is added slowly to above Ficoll along tube wall；
4) be centrifuged: 18-20 DEG C, 2000rpm, 30min, points four layers from tube bottom to liquid level after centrifugation, be followed successively by red blood cell and Granulocyte layer, layering liquid layer, mononuclearcell layer, plasma layer；
5) it recycles: pipette is inserted directly into cloud and mist layer (or the blood plasma for first sucking upper layer), cloud and mist layer is gently sucked out, puts Enter in new centrifuge tube；
6) it washs: the PBS of addition to 3 times of volume less than PBMC (peripheral blood mononuclear cells), 18-20 DEG C, 2000rpm, 10min, twice；
7) cell count: abandoning supernatant, adds 1mL lymphocytes culture medium, and piping and druming mixes, and is prepared into PBMC cell suspension.It adopts It is counted with blood cell counting plate: being added in blood cell counting plate after taking a drop PBMC suspension and a 2% trypan blue dye liquor of drop to mix, 4 big lattice inner cell sums are counted under the microscope.Cell number/mL=4 block plaid total number of cells/4 × 104× 2 (dilutions times Number).
2, the activation and slow-virus infection of T cell
(1) prepare before experiment:
1) Anti-CD3 monoclonal antibody liquid is prepared: 50 μ g of CD3 monoclonal antibody is dissolved in 5mLPBS solution, and being configured to concentration is 10 μ g/mL Solution, dispensed after dissolution according to every 400 μ L of EP pipe, be placed in -80 DEG C of refrigerators and save.(under this concentration in every 24 orifice plate 175 μ L antibody-solutions are added)；
2) Anti-CD28 monoclonal antibody liquid is prepared: 50 μ g of CD28 monoclonal antibody is dissolved in 5mLPBS solution, and being configured to concentration is 10 μ g/ The solution of mL is dispensed after dissolution according to every 400 μ L of EP pipe, is placed in -80 DEG C of refrigerators and is saved.(every 24 orifice plate under this concentration 175 μ L antibody-solutions of interior addition)；
3) CH-38 protein liquid is prepared: 500 μ g of CH38 albumen is dissolved in 10mL PBS solution, is configured to 50 μ g/mL solution, It is dispensed after dissolution according to every 400 μ L of EP pipe, is placed in -80 DEG C of refrigerators and saves.(175 μ are added under this concentration in every 24 orifice plate L antibody-solutions)；
4) the IL-2 factor is prepared: IL-2 protein solid presses 1 × 107U/mg is prepared, and every 50 μ g IL-2 albumen is added The PBS solution of 500 μ L, is configured to 103The concentration of U/ μ L is dispensed according to 32 μ L are added in every EP after preparation, is placed in -80 DEG C refrigerator saves；
5) lymphocytes culture medium is prepared: the every 50mL of Takara-551h3 lymphocytes culture medium is added what 30 μ L had been dispensed IL-2 solution, 0.5mL is dual anti-, 250 μ L autoserums.
(2) experiment flow:
Day ﹣ 1:24 orifice plate coating: 24 orifice plate of Corning is taken, by taking 2 holes as an example, CD3 monoclonal antibody liquid 175 is respectively added in 2 holes 175 μ L, CH-38 protein liquid of μ L, CD28 monoclonal antibody liquid, 175 μ L.After gently concussion mixes after addition, orifice plate is closed with sealed membrane, is put Enter 4 DEG C of fridge overnights.Cell recovery: taking the PBMC cell in liquid nitrogen, recovery；
Day0: cleaning coating plate: taking out coated 24 orifice plate yesterday, discard supernatant, and after PBS is cleaned 2 times, PBS is added and waits for With；
PBMC bed board: collecting PBMC cell, counts and by concentration final adjustment to 0.7 × 106Cells/mL, every Kong Zhongjia Enter 400 μ L cell suspensions, i.e., is added 2.8 × 10 in every hole5A cells；
Virus infection: being infected with MOI=30, prepare 1mL Virus culture base suspension, be added 24 orifice plates in, 1000g from Centrifuge temperature is adjusted to 32 DEG C by heart 30min；
Day1-Day2: observation cell state；
Day3: all cells in 24 orifice plates are transferred to the 25cm that 10mL culture medium is added2In culture bottle, cell is observed State；
Day4-Day7: observation cell state and cell quantity, if cell starts obviously to expand, and regional area cell Density is more, and 10mL culture medium is added；
Day8: 25cm at this time2Cell in culture bottle has covered with, and is transferred to the 75cm that 20mL culture medium is added2Culture bottle In continue to cultivate；
Day9-Day10: observation cell state, when cell is in 75cm2When being in full state in culture bottle, stop continuing Growth, enrichment of cell simultaneously calculate amplification ratio, and flow cytometer detection detects cell typing, carry out cell killing detection or cell cryopreservation etc. Subsequent experimental.
Four, the identification and detection of CAR-T cell
(1) Flow cytometry CAR structure positive expression rate
1, cell CD3, CD4, CD8 positive rate are detected
1) the NC group cell (not carrying out virus infection) of acquirement and sample group cell (having carried out virus infection) are used into PBS + 2%BSA is mildly washed 2 times, centrifugal condition 1500rpm, 3min；
2) 1000 μ LPBS+2%BSA are added in NC pipe, are distributed into 5 pipes, respectively NC, NC-CD3, NC-CD4, NC-CD8, 200 μ LPBS are added in sample tube in NC-CD3/4/8, are labeled as sample-CD3/4/8, are separately added into 5 μ L/20 μ of CD3/4/8 antibody L/20 μ L is mild to mix；
3) after room temperature is protected from light incubation 30min, 1500rpm, 3min discard waste liquid；
4) 200 μ LPBS+2%BSA are added, mild mix is resuspended, and 1500rpm/3min discards waste liquid；
5) 100 μ LPBS+2%BSA are added, mild mixing can go up machine testing after being resuspended.
As shown in figure 5, CD3+Cell accounts for 95.03%, CD4 of detected cell total amount-CD8+Cell accounts for CD3+Cell total amount 57.36%, CD4+CD8-Cell accounts for CD3+The 34.90% of cell total amount meets the phenotype of T cell.
2, CAR structure positive expression rate is detected
1) it by the NC group cell (not carrying out virus infection) of acquirement and sample group cell (having carried out virus infection), uses PBS+2%BSA is mildly washed 2 times, and 1500rpm/6min discards waste liquid；
2) 200 μ LPBS are added in NC pipe, are resuspended；100 μ LPBS are added in sample tube, is resuspended, adds 100 μ L primary antibody works Make liquid (3 μ g/mL), mixes；
3) it is incubated at room temperature 1h, 1500rpm/3min, discards waste liquid；
4) 200 μ LPBS are added, mild mix is resuspended, and 1500rpm/3min discards waste liquid；
5) 200 μ LPBS are added in NC pipe, are resuspended；200 μ LPBS are added in sample tube, are resuspended, add the work of 5 μ L secondary antibodies Liquid mixes；
6) after room temperature is protected from light incubation 1h, 1500rpm/3min discards waste liquid, is mildly washed using PBS+2%BSA 3 times, from Heart condition is 1500rpm, 3min, discards waste liquid；
7) 100 μ LPBS, mild upper machine testing after mixing resuspension is added.
As shown in figure 5, CAR-T cell accounts for the 51.90% of T cell total amount.
(2) RTCA real-time cell killing detection
1) by taking breast cancer cell SK-BR-3 as an example, cell suspension is prepared into after digestion, piping and druming carries out cytometer after mixing Number；
2) cell suspension is diluted to 5 × 104Cells/mL concentration is put on ice for spare；
3) RTCA detection plate is taken out, 50 μ L culture mediums is added；
4) test procedure of this test is compiled in RTCA detector program；
5) RTCA detection plate is placed in detector, observe in program Messege it is whether normal, starting is in fact after normal Test program；
6) program 1 takes out detection plate after running, and 100 μ L of tumor cell suspension is added in corresponding aperture, before addition Mix every solencyte suspension；
7) after cell suspension is added, it will test in plate merging incubator and stand 30min, make cell natural subsidence；
8) after 30min, it will test plate and be put into detector, run program 2；
9) it observes cell growth curve afterwards for 24 hours, when cell is in logarithmic growth phase, prepares that effector T cell is added；
10) effector T cell is taken out in culture bottle, is centrifuged, is cleaned, and is counted, it is denseer than preparing effect group cell by different effect targets Degree；
11) time out program, takes out detection plate, and corresponding position is added 50 μ L of effector cell, puts back in detector, after onward Sequence is observed daily.
Fig. 6 is that double target spot (EpCAM single-chain antibody and MSLN single-chain antibody) the CAR-T cells of RTCA detection are thin to SK-BR-3 The killing-efficiency of born of the same parents, 5:1 is indicated in figure: effector cell: target cell=5:1；2.5:1 is indicated: effector cell: target cell=2.5: 1；1.25:1 is indicated: effector cell: target cell=1.25:1；NCT is indicated: effector cell is the T cell being uninfected by, and effect is thin Born of the same parents: target cell=1.25:1.As shown, the fragmentation effect of CAR-T cell is significantly stronger than the T cell being uninfected by, and it is killed Effect has concentration dependent, and effector cell's ratio is higher, and fragmentation effect is better.
(3) ELISA detects cytokine secretion
1) ddH is used2O dilutes 10 × coating buffer buffer, prepares 250 × coating protein, such as 2mL coating buffer in proportion 8 μ 250 × coating proteins of L are added in buffer；
2) 100 holes μ L/ 1 are added into affine 96 orifice plate of Corning 9018Elisa high) in prepare complete coating buffer, Sealing is put into 4 DEG C of refrigerators, overnight；
3) coated 96 orifice plate is cleaned using PBST (0.05%Tween 20), 3 times；
4) ddH is used2O prepares 5 × confining liquid, and 200 holes μ L/ are added, and closes 1h at room temperature；
5) 1 × confining liquid is added by bottled standard items requirement to be prepared, carries out 7 doubling dilutions, while (taking to sample CAR-T cell in RTCA test experience is to the supernatant after SK-BR-3 killing for 24 hours) carry out 5 times of dilutions；
6) plank 5 times after PBST cleaning closing, the sample liquid after standard items and dilution is added are incubated at room temperature 2h or 4 DEG C Overnight；
7) PBST is cleaned 4 times；
8) 250 × detection antibody is diluted using 1 × confining liquid, is added in 100 holes μ L/, is incubated at room temperature 1h；
9) PBST is cleaned 4 times, is diluted 250 × HRP using 1 × confining liquid, is added in 100 holes μ L/, is incubated at room temperature 30min；
10) PBST is cleaned 5 times, and 1 × TMB reagent, 100 μ L is added in every hole, is incubated at room temperature 15min；
11) 50 hole μ L/ terminate liquid color development stoppings are added；
12) microplate reader 450nm detects OD value.
Fig. 7 is that the ELISA of the IFNgamma factor after CAR-T cell kills for 24 hours SK-BR-3 is detected, and 5:1 is indicated in figure: Effector cell: target cell=5:1；2.5:1 is indicated: effector cell: target cell=2.5:1；1.25:1 is indicated: effector cell: target Cell=1.25:1.After CAR-T cell and SK-BR-3 cell co-culture for 24 hours, there is apparent cell factor IFNgamma to discharge, Effector cell: when target cell is 5:1, cell factor IFNgamma release is most.
In conclusion double target spot CAR carriers of joint EpCAM and MSLN single-chain antibody provided by the invention are applied to infection T cell, the antibody of tumor associated antigen EpCAM, MSLN for breast cancer cell surface can be expressed simultaneously by obtaining CAR-T cell Anti-EpCAM, Anti-MSLN, so as to more accurately identify and kill while expressing above two tumor associated antigen Breast cancer cell, considerably increase the targeting of CAR-T cell, reduce undershooting-effect；In addition, CAR mentioned in the present invention Structure imparts the stronger proliferative of T cell, and lasting vitality makes it show stronger tumor cell killing potential.
Above-mentioned preferable possible embodiments only of the invention, are not limitations of the present invention, the present invention is also not limited to above-mentioned Citing, those skilled in the art, within the essential scope of the present invention, made variations, modifications, additions or substitutions, Also it should belong to protection scope of the present invention.

Claims (8)

1. combining double target spot CAR carriers of EpCAM and MSLN single-chain antibody, which is characterized in that including Chimeric antigen receptor and load Body, the Chimeric antigen receptor connect on the carrier,
The Chimeric antigen receptor includes that two species specificity structures are respectively as follows: EpCAM single-chain antibody and MSLN single-chain antibody, described The nucleotide sequence of EpCAM single-chain antibody is as shown in SEQ ID NO.2, the nucleotide sequence such as SEQ of the MSLN single-chain antibody Shown in ID NO.7,
The structure composition of the Chimeric antigen receptor is CD8leader-EpCAM scFv-CD8 α-CD28-CD137-IRES- CD8leader-MSLN scFv-CD8α-CD3ζ。
2. double target spot CAR carriers of joint EpCAM and MSLN single-chain antibody according to claim 1, which is characterized in that institute The guidance peptide for guiding newly synthesized protein to carry out transmembrane process, the nucleotide of the CD8leader can be expressed by stating CD8leader Sequence as shown in SEQ ID NO.1,
The EpCAM scFv indicates EpCAM single-chain antibody,
The CD8 α is transmembrane region, connects extracellular antigen binding domain and intracellular signal domain, and nucleotide sequence such as SEQ ID NO.3 It is shown,
The CD28-CD137 is costimulation structural domain, and the CD28 nucleotide sequence is described as shown in SEQ ID NO.4 CD137 nucleotide sequence as shown in SEQ ID NO.5,
The IRES is ribosome recognition site, is to realize that identical carrier efficiently co-expresses the element of two genes, nucleotides sequence It arranges as shown in SEQ ID NO.6,
The MSLN scFv indicates MSLN single-chain antibody,
The CD3 ζ is signal transduction domain, and nucleotide sequence is as shown in SEQ ID NO.8.
3. double target spot CAR carriers of joint EpCAM and MSLN single-chain antibody according to claim 1, which is characterized in that institute Stating carrier includes PUC19 plasmid, slow virus carrier.
4. double target spot CAR carriers of joint EpCAM and MSLN single-chain antibody according to claim 3, which is characterized in that institute Stating slow virus carrier is pCDH-CMV-MCS-EF1-Puro.
5. combining the construction method of double target spot CAR carriers of EpCAM and MSLN single-chain antibody, for constructing such as claim 1-4 Double target spot CAR carriers of described in any item joint EpCAM and MSLN single-chain antibodies, which is characterized in that the construction method Steps are as follows:
(1) Chimeric antigen receptor is stored on the PUC19 plasmid；
(2) double digestion will be carried out containing PUC19 plasmid in step (1) and the slow virus carrier, digestion products will be passed through respectively Agarose gel electrophoresis separates, slow disease after the target fragment containing the Chimeric antigen receptor and double digestion after obtaining double digestion Poisonous carrier,
(3) slow virus carrier in the target fragment containing the Chimeric antigen receptor and step (2) in step (2) is carried out Connection, connection product is converted, and extracts plasmid, obtains the recombinant plasmid containing Chimeric antigen receptor structure, the recombination Plasmid is double target spot CAR carriers of joint EpCAM and MSLN single-chain antibody.
6. the construction method of double target spot CAR carriers of joint EpCAM and MSLN single-chain antibody according to claim 5, It is characterized in that, the double digestion in the step (2) is EcoRI and NotI, and the mesh of the Chimeric antigen receptor is contained in (3) The molar ratio that is connected with slow virus carrier of segment be 5:1.
7. combining the application of double target spot CAR carriers of EpCAM and MSLN single-chain antibody, based on described in any one of claim 1-4 Joint EpCAM and MSLN single-chain antibody double target spot CAR carriers, which is characterized in that the joint EpCAM and MSLN is mono- Double target spot CAR carriers of chain antibody carry out slow virus packaging, obtain virion, and the slow disease of high titre is obtained after centrifugal concentrating Malicious suspension, the slow virus suspension obtain CAR-T cell, the CAR-T cell passes through identification specificity for infecting T cell Albumen EpCAM and MSLN target killing breast cancer cell.
8. the application of double target spot CAR carriers of joint EpCAM and MSLN single-chain antibody according to claim 7, feature It is, the slow virus packaging uses three plasmid packaging systems, and the three plasmids packaging system includes PSPAX2 plasmid, pMD2G Double target spot CAR carriers of plasmid and the joint EpCAM and MSLN single-chain antibody, the PSPAX2 plasmid, the pMD2G matter Grain, the joint EpCAM and MSLN single-chain antibody the ratios of double target spot CAR carriers be 27:3:20, the slow virus packs and adopts Use 293T cell as the incasing cells of slow virus.
CN201910019779.4A 2019-01-09 2019-01-09 The double target spot CAR carriers and its construction method of joint EpCAM and MSLN single-chain antibody and in breast cancer application Withdrawn CN109593786A (en)

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US10927180B2 (en) 2017-10-13 2021-02-23 Harpoon Therapeutics, Inc. B cell maturation antigen binding proteins
US10954311B2 (en) 2015-05-21 2021-03-23 Harpoon Therapeutics, Inc. Trispecific binding proteins and methods of use
US11136403B2 (en) 2017-10-13 2021-10-05 Harpoon Therapeutics, Inc. Trispecific proteins and methods of use
US11180563B2 (en) 2020-02-21 2021-11-23 Harpoon Therapeutics, Inc. FLT3 binding proteins and methods of use
US11453716B2 (en) 2016-05-20 2022-09-27 Harpoon Therapeutics, Inc. Single domain serum albumin binding protein
US11535668B2 (en) 2017-02-28 2022-12-27 Harpoon Therapeutics, Inc. Inducible monovalent antigen binding protein
US11607453B2 (en) 2017-05-12 2023-03-21 Harpoon Therapeutics, Inc. Mesothelin binding proteins
US11623958B2 (en) 2016-05-20 2023-04-11 Harpoon Therapeutics, Inc. Single chain variable fragment CD3 binding proteins
US11807692B2 (en) 2018-09-25 2023-11-07 Harpoon Therapeutics, Inc. DLL3 binding proteins and methods of use

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US10954311B2 (en) 2015-05-21 2021-03-23 Harpoon Therapeutics, Inc. Trispecific binding proteins and methods of use
US11453716B2 (en) 2016-05-20 2022-09-27 Harpoon Therapeutics, Inc. Single domain serum albumin binding protein
US11623958B2 (en) 2016-05-20 2023-04-11 Harpoon Therapeutics, Inc. Single chain variable fragment CD3 binding proteins
US10844134B2 (en) 2016-11-23 2020-11-24 Harpoon Therapeutics, Inc. PSMA targeting trispecific proteins and methods of use
US11535668B2 (en) 2017-02-28 2022-12-27 Harpoon Therapeutics, Inc. Inducible monovalent antigen binding protein
US11607453B2 (en) 2017-05-12 2023-03-21 Harpoon Therapeutics, Inc. Mesothelin binding proteins
US10927180B2 (en) 2017-10-13 2021-02-23 Harpoon Therapeutics, Inc. B cell maturation antigen binding proteins
US11136403B2 (en) 2017-10-13 2021-10-05 Harpoon Therapeutics, Inc. Trispecific proteins and methods of use
US11807692B2 (en) 2018-09-25 2023-11-07 Harpoon Therapeutics, Inc. DLL3 binding proteins and methods of use
US11180563B2 (en) 2020-02-21 2021-11-23 Harpoon Therapeutics, Inc. FLT3 binding proteins and methods of use

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