CN109694884A - The preparation of CAR-T carrier for being applied in treatment of colon cancer and its construction method - Google Patents
The preparation of CAR-T carrier for being applied in treatment of colon cancer and its construction method Download PDFInfo
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Abstract
The preparation for the CAR-T carrier that the present invention provides a kind of for applying in treatment of colon cancer and its construction method.Therapy vector of the invention is made of Lentiviral pCDH-CMV-MCS-EF1-Puro and CAR expression structure two parts.CAR structure of the invention is specially CD8leader-EpCAMscfv-CD8 α-CD28-CD137-CD3 ζ-IRES-PD-1mAb.Take three plasmid packaging systems according to (PSPAX2:pMD2G=8:1) therapy vector and packaging plasmid PSPAX2 and pMD2G of the invention: vector plasmid=3:2 infection 293T cell obtains CAR-T cell.CAR-T cell of the invention can targets identification and kill EpCAM positive expression colon cancer cell.
Description
Technical field
The present invention relates to field of biomedicine technology, and in particular to a kind of CAR-T for applying in treatment of colon cancer
The preparation of carrier and its construction method.
Background technique
Colon cancer is the malignant change that mucous membrane of colon epithelium occurs under a variety of carcinogenic factor effects such as environment or heredity, is
One of common malignant tumour.In recent years, disease incidence and lethality of the colon cancer in China are on the rise.Currently, main
Wanting therapeutic modality includes operation, chemotherapy, radiotherapy, Chinese medicine treatment.But it is limited with upper type therapeutic effect, and toxic side effect is larger.
Although the colon cancer multidisciplinary synthesis based on surgical operation treats cooperative groups (m μ ltidisciplinary team, MDT)
Diagnosis and treatment mode and immunization therapy gradually play an increasingly important role, but cannot reach satisfactory effect, such as exempt from
There is lack specific recognition tumor associated antigen (tumor associated antigen, TAA) and killing spy for epidemic disease treatment
Determine the ability and tumor cell surface major histocompatibility complex (main of tumour target cell
Histocompatibility complex, MHC) expression decline lead to the problem of immunologic escape.
However, Chimeric antigen receptor (CAR)-T cell therapy obtains weight in the therapeutic process of anti-malignant tumor in recent years
It is in progress.CAR-T technology is transformed by extracting the T lymphocyte of patient using gene engineering method, so that after transformation
T lymphocyte can secrete CAR to have a specific recognition and binding ability to tumour antigen, and by improved T
Lymphocyte feeds back to patient's body after amplification in vitro, to reach the specific killing to tumour cell.It is treated with traditional immunization
Method is compared, CAR-T therapy in vitro with better targeting and lethal has been shown in clinical test.CAR-T therapy
For effect in multinomial oncotherapy research there are still difference, CAR-T depends on its CAR to swollen in the effect for the treatment of malignant tumour
The targeting and CAR-T cell of tumor are in people's intracorporal time-to-live and activity.Therefore, CAR-T prepares carrier to using CAR-T
The treatment that technology carries out malignant tumour has great influence.
T lymphocyte is PD-1 (the Programmed cell death by its surface when playing antitumor action
Protein 1) apoptosis receptor 1 identified and killed to tumour cell.However, the study found that kinds of tumors
PD-L1 in cell has up-regulated expression.Tumour cell passes through secretion PD-L1 (programmed death ligand1) program
Property cell death receptor ligand 1, and in conjunction with the PD-1 receptor of T cell, so that T cell is caused to inactivate, it is but thinless to tumour
Born of the same parents cause to kill, in conjunction with the PD-1 in T cell after, can inhibit T cell proliferation and activation, T cell made to be in inactivated state,
Therefore tumor immune escape is caused.
It is being carried out in antitumor therapeutic process using CAR-T, the most key is the building of carrier, although to CAR-T
The constructing technology for preparing carrier has had more report, still, there is no PD-L1 pairs for can effectively antagonizing colon cancer cell at present
The interference of T cell and the CAR-T therapy vector and correlative study to colon cancer cell with good discernment.
Summary of the invention
The CAR-T carrier and its construction method that the purpose of the present invention is to provide a kind of for applying in treatment of colon cancer
With the application in treatment of colon cancer.
The CAR-T carrier that one of the objects of the present invention is to provide a kind of for applying in treatment of colon cancer comprising
Lentiviral and the CAR gene order for accessing the Lentiviral, the CAR gene order includes EpCAM
Single-chain antibody gene sequence and PD-1 monoclonal antibody gene sequence, the EpCAM single-chain antibody nucleotide sequence such as SEQ ID
Shown in NO.2, the nucleotide sequence of PD-1 monoclonal antibody is as shown in SEQ ID NO.8.By being inserted into CAR gene order
The gene order of PD-1 monoclonal antibody so that T cell expression PD-1 monoclonal antibody and with the PD-1 on T cell surface specificity
In conjunction with the combination to reduce the PD-L1 and PD-1 of cancer cell, and then the lethality of CAR-T cells against tumor cells is improved, in addition,
It can be improved the identification of CAR-T cells on cancer cells by being inserted into EpCAM single-chain antibody gene sequence in CAR gene order
Power.
Further, the structure composition of the CAR is CD8leader-EpCAMscfv-CD8 α-CD28-CD137-CD3 ζ-
The structure composition schematic diagram of IRES-PD-1mAb, CAR are as shown in Figure 1, in which: CD8leader is the guidance peptide portion of CD8 albumen
Point, albumen can be positioned at extracellularly, the nucleotide sequence of CD8leader is as shown in SEQ ID NO.1；EpCAMscfv is
The specific antigen binding domain of EpCAM design, the nucleotide sequence of EpCAMscfv such as SEQ ID are expressed according to colon cancer tissue height
Shown in NO.2；CD8 α is transmembrane region, connects extracellular antigen binding domain and intracellular signal domain, can be by CAR Structure anchor in T cell film
On, the nucleotide sequence of CD8 α is as shown in SEQ ID NO.3；CD28-CD137 is costimulation structural domain, and transduction proliferation signal is simultaneously
The generation of inducing cytokine stimulates T cell activation, the nucleotide sequence of CD28-CD137 such as SEQ ID NO.4 and SEQ ID
Shown in NO.5；CD3 ζ is signal transduction domain, when extracellular region and target antigen combination, will be swashed to conduction TCR sample signal intracellular
T cell living plays the effect of targeting killing colon cancer cell, and the nucleotide sequence of CD3 ζ is as shown in SEQ ID NO.6；IRES
For ribosome recognition site, ribosomes can be recruited, mRNA is translated, when IRES is merged with external source cDNA, IRES can be independent
Ground initiation of translation, the nucleotide sequence of IRES is as shown in SEQ ID NO.7；PD-1mAb can be expressed to be tied with the PD-1 on T cell surface
It closes, blocks the combination of PD-1 and PD-L1, to raise the growth and proliferation of T cell, enhance knowledge of the T cell to colon cancer cell
Not, the PD-1mAb of its attack and killing ability is activated, the nucleotide sequence of PD-1mAb is as shown in SEQ ID NO.8.
Further, the Lentiviral is pCDH-CMV-MCS-EF1-Puro, structural schematic diagram such as Fig. 2
It is shown.PCDH-CMV-MCS-EF1-Puro can pass through EcoRI and NotI double digestion Insert Fragment.The expression vector includes:
CMV promoter-is mammalian cell specificity promoter, and driving capability is stronger；Multiple cloning sites (MCS)-include multiple limits
Property restriction enzyme site (restriction site) processed is the position of foreign gene insertion；WPRE element-can be improved mRNA's
PolyA tailing efficiency, improves the expression efficiency of metastatic gene；SV40polyA sequence-can terminate transcription and effectively for transcription
MRNA adds PolyA tail；Hybrid RSV/5 ' LTR- contains the controlling elements such as promoter and enhancer, makes it in 293T cell
High-caliber expression overall length virus transcription object；Genetic element (cPPT, gag, env, LTRs)-is for packing, transduceing and steadily
It will be in the genomic DNA of expressing viral structural integrity to host；SV40origin- makes plasmid stablize proliferation in incasing cells.
The Lentiviral, which can hold, carries that exogenous genetic fragment is big, and transfection efficiency is higher, and satisfied transfection effect can be also reached to T cell
Fruit.
Further, the Lentiviral pCDH-CMV-MCS-EF1-Puro passes through EcoRI and NotI double digestion
It is inserted into CAR gene order.
The second object of the present invention is to provide a kind of building side of CAR-T carrier for applying in treatment of colon cancer
Method comprising following steps:
1) it will be stored on PUC19 plasmid after the synthesis of CAR gene order；
2) bacterial strain containing Lentiviral and the glycerol stock containing the obtained PUC19 plasmid of step 1) are carried out
After Zengjing Granule, plasmid is extracted, and be utilized respectively EcoRI and NotI double digestion；
3) the obtained digestion products of step 2) are passed through into agarose gel electrophoresis separation respectively, and recycle target fragment；
4) by step 3) the obtained Lentiviral segment of recycling and CAR target fragment according to molar ratio be 1:5 into
Row connection conversion, plasmid extract, and obtain CAR-T therapy vector.
The third object of the present invention is to provide a kind of CAR-T carrier for applying in treatment of colon cancer and is preparing
Application in CAR-T: by the CAR-T therapy vector and packaging plasmid PSPAX2 and pMD2G take three plasmid packaging systems into
Row virus packaging, uses 293T cell as the incasing cells of slow virus, and collection virus liquid is simultaneously concentrated postoperative infection and has expanded activation
T cell, can be obtained CAR-T cell.The CD3 positive rate of CAR-T cell of the invention is high, and the T cell of the CAR positive occupies
44% or more of the T cell total amount of the CD3 positive.
Further, the ratio of the packaging plasmid and the vector plasmid in system be packaging plasmid (PSPAX2:
PMD2G=8:1): vector plasmid=3:2.
The fourth object of the present invention is to provide a kind of CAR-T carrier for applying in treatment of colon cancer in colon cancer
Application in treating.The CAR-T obtained using CAR-T therapy vector of the invention imitates colon cancer cell with good killing
Fruit can kill completely colon cancer cell T84 after CAR-T cell about 60h is added when imitating target ratio is 2.5:1 and 5:1.
The invention has the advantages that since recombination T cell can be expressed the highly expressed EpCAM's in colon cancer cell surface
Antibody A nti-EpCAM specific recognition and can kill colon cancer cell；Antibody A nti-PD-1 can be with T cell surface
PD-1 combine, block the combination of PD-1 and PD-L1, to lower influence of the PD-1 to T cell, enhance T cell to tumour
The lethal effect of cell, meanwhile, the immune function of enhancing human body itself realizes antitumor action.In addition, the CAR in the present invention is tied
Structure imparts the stronger proliferative of T cell, after T cell obtains the carrier by virus infection mode and expresses the CAR structure,
T cell can targeting identify and kill colon cancer cell.
Detailed description of the invention
Fig. 1 is CAR structural schematic diagram；
Fig. 2 is Lentiviral pCDH-CMV-MCS-EF1-Puro structural schematic diagram；
Fig. 3 is EcoRI and NotI the double enzyme digestion product agarose of Lentiviral pCDH-CMV-MCS-EF1-Puro
Gel electrophoresis figure；
Fig. 4 is EcoRI and NotI the double enzyme digestion product agarose gel electrophoresis figure of CAR-T therapy vector；
Fig. 5 is the T cell testing result of the CD3 positive；
Fig. 6 is the T cell testing result of the CD4 positive and the CD8 positive in the T cell of the CD3 positive；
Fig. 7 is the CAR positive cell testing result of the T cell of the CD3 positive；
Fig. 8 is that RTCA detects CART cell to the killing-efficiency of T84；
Fig. 9 is the secretion result of IFN-γ after CAR-T cell and T84 co-cultivation.
Specific embodiment
CAR structure composition of the invention is CD8leader-EpCAM scfv-CD8 α-CD28-CD137-CD3-IRES-
PD-1mAb.Wherein, the nucleotide sequence of CD8leader is as shown in SEQ ID NO.1；The nucleotide sequence of EpCAMscfv is such as
Shown in SEQ ID NO.2, epithelium specific adhesion molecule (EpCAM, Epithelial cell adhesion moleculer)
It is a kind of epithelial cell transmembrane glycoprotein, participates in Wnt signal transduction pathway, regulation target gene transcription, adherency with cell is moved
Shifting, proliferation, differentiation etc. are related.It is initially to be found with a kind of tumor associated antigen, since EpCAM is in kinds of tumors tissue
Such as Colon and rectum gland cancer, gastric cancer, oophoroma, the expression in breast cancer are increased and are concerned.The study found that the expression of EpCAM
It is related to the progress of tumour and prognosis, it can be used as the target spot of neoplasm targeted therapy.The nucleotide sequence of CD8 α such as SEQ ID NO.3
It is shown；The nucleotide sequence of CD28-CD137 is as shown in SEQ ID NO.4 and SEQ ID NO.5；The nucleotide sequence of CD3 ζ is such as
Shown in SEQ ID NO.6；The nucleotide sequence of IRES is as shown in SEQ ID NO.7；The nucleotide sequence of PD-1mAb such as SEQ ID
Shown in NO.8, Anti-PD-1 (PD-1 antibody) can block the combination of PD-1 and PD-L1 in conjunction with the PD-1 on T cell surface, from
And the growth and proliferation of T cell are raised, enhance identification of the T cell to tumour cell, activate its attack and killing ability, passes through tune
The immune function of moving body itself realizes antitumor action.
Lentiviral specific structure mentioned in the present invention is pCDH-CMV-MCS-EF1-Puro, structural representation
Figure is as shown in Fig. 2.The carrier is analyzed by Snap Gene software and searches pertinent literature it is found that pCDH-CMV-MCS-EF1-
Puro EcoRI and NotI double digestion Insert Fragment.The expression vector includes: that CMV promoter-is mammalian cell specificity
Promoter, driving capability are stronger；Multiple cloning sites (MCS)-include multiple restriction enzyme sites (restriction site),
It is the position of foreign gene insertion；The polyA tailing efficiency of mRNA can be improved in WPRE element-, improves the expression effect of metastatic gene
Rate；SV40polyA sequence-can effectively terminate transcription and add PolyA tail for the mRNA of transcription；Hybrid RSV/5 ' LTR- contains
There are the controlling elements such as promoter and enhancer, makes its high-caliber expression overall length virus transcription object in 293T cell；Heredity is wanted
Plain (cPPT, gag, env, LTRs)-is for packing, transduceing and steadily by the genome of expressing viral structural integrity to host
In DNA；SV40origin- makes plasmid stablize proliferation in incasing cells.
The construction method of CAR-T carrier for being applied in treatment of colon cancer of the invention are as follows: close CAR gene order
It is present on PUC19 plasmid vector after；PUC19 plasmid vector and Lentiviral are all made of I pair of ECoR I, Not enzyme
It cuts, digestion products is separated by agarose gel electrophoresis, purpose band is recycled, obtains the concentration of carrier and target fragment, it will
The two is attached after conversion for 1:5 according to molar ratio and carries out plasmid extraction, and the treatment that final acquisition contains specific CAR structure carries
Body.
The application of CAR-T carrier for applying in treatment of colon cancer of the invention: therapy vector uses three plasmid packets
Dress system, group become PSPAX2, and pMD2G, therapy vector, this ratio of three kinds of plasmids in system is packaging plasmid
(PSPAX2:pMD2G=8:1): vector plasmid=3:2 uses 293T cell as the incasing cells of slow virus, collects training respectively
Virus liquid after supporting 48h, 72h, this virus liquid is concentrated, and survey virus titer, has finally been expanded with MOI=30 infection sharp
T cell living can be obtained CAR-T cell, and using the cell typing of Flow cytometry CAR-T cell and CAR knot
Structure the positive expression rate, in cellular level, using RTCA technology detection CAR-T cell to the killing-efficiency of colon cancer tumours cell,
Lethal cell factor IFN-γ is detected using ELISA method simultaneously.
The content of present invention is illustrated combined with specific embodiments below:
Main material source used in each embodiment of the present invention is described as follows:
1. slow virus expression plasmid pCDH-CMV-MCS-EF1-Puro, slow virus packaging plasmid pMD2G, vector plasmid
PSPAX2 is purchased from SBI；
2.CAR gene order is designed by Shanghai Beautiful medical science and technology Co., Ltd, send raw work bioengineering (Shanghai)
Limited liability company's synthesis, with the preservation of PUC19 plasmid form；
3. restriction endonuclease EcoRI, Not I is purchased from NEB；
4. Ago-Gel QIAquick Gel Extraction Kit (article No.: DP209-02), the small extraction reagent kit of plasmid (article No.: DP103-03) purchase
From Tiangeng biochemical technology Co., Ltd；
5.293T cell, T84 cell are purchased from Chinese Academy of Sciences's cell bank；
6.lipofectamine 2000 is purchased from Gibco；
7.CD3 monoclonal antibody (model GMP-A018), CD28 monoclonal antibody (model GMP-A063), CH38 albumen (model GMP-
CH38), IL-2 (model GMP-CD66) is purchased from offshore protein Science and Technology Ltd.；
8. flow cytometer is purchased from purchased from ThermoFisher, ST16R tabletop refrigerated centrifuge, Fresco microcentrifuge
ThermoFisher, HE120 Horizontal electrophoresis tank, Tanon gel imager are purchased from Tanon.
Embodiment 1: the building of the therapy vector for CAR-T preparation
(1) plasmid extracts:
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 are 37 DEG C, 250rpm.
Plasmid extraction is carried out according to the specification of the small extraction reagent kit of the plasmid of Tiangeng.
(2) digestion, connection, conversion
By extracted slow virus expression plasmid pCDH-CMV-MCS-EF1-Puro, PUC19 plasmid simultaneously carry out EcoRI,
Digestion products are carried out agarose gel electrophoresis respectively, are observed and recorded with gel imager as a result, pCDH- by NotI double digestion
CMV-MCS-EF1-Puro agarose gel electrophoresis results are as shown in Figure 3.
Purpose band recycling is carried out with Ago-Gel QIAquick Gel Extraction Kit specification, recycling segment is pressed into CAR structure:
The molar ratio of pCDH-CMV-MCS-EF1-Puro=5:1 is attached, and is converted.Conversion reaction conditions be 16 DEG C, 4h in
It is connected in ProFlex PCR instrument, conversion reaction system is as follows:
Linked system is as follows:
Specific conversion process is as follows:
1. taking out competent cell from -80 DEG C of refrigerators to melt on ice, 37 DEG C of incubators are based on from 4 DEG C of taking-up solid cultures
Preheating；
2. connection product is transferred in 1.5mL centrifuge tube；
3. being slowly added to 50 μ L competent cells thereto, agitate by adding, mixes the two；
4. after the centrifuge tube in 3 is put in 30min on ice, the heat shock 90s in 37 DEG C of water-baths；
5. after heat shock, taking out be placed in 2min on ice immediately；
6. 950 μ L fluid nutrient mediums are added thereto, in constant-temperature table 40~60min of culture, condition is 37 DEG C,
200rpm；
7. 6000rpm is centrifuged 2min, discards most of supernatant, stay 40~60 μ L；
8. blowing and beating resuspended bacterium solution with pipette tips, bacterium solution is dripped on the solid medium of preheating, is marked, 37 DEG C of incubator mistakes
Night.
(3) conversion results sequence verification extracts therapy vector
The part bacterium colony of picking step (2) culture carries out in 5mL LB liquid medium (AMP resistance) in constant-temperature table
Zengjing Granule simultaneously carries out plasmid extraction using the small extraction reagent kit of the plasmid of Tiangeng, and 500 μ L are taken out before plasmid extraction in 1.5mL
In Ep pipe, it is used as fungi preservation, is stored in -80 DEG C；By extract product using after EcoRI, NotI double digestion through Ago-Gel
Electrophoresis is verified, as a result as shown in Figure 4.Take the correct 1 μ g of plasmid of band send Sangon Biotech (Shanghai) Co., Ltd. into
Row sequencing, the plasmid of band exception and the bacterium solution of retention abandon.The correct plasmid of sequencing result is stripped to get arriving
Therapy vector plasmid.
Embodiment 2: the preparation of concentrating virus liquid
Slow virus packaging is carried out using three plasmid packaging systems.Three plasmids are respectively therapy vector obtained in embodiment 1
Plasmid, slow virus packaging plasmid pMD2G and vector plasmid PSPAX2, cell are 293T cell.
Specific implementation step is as follows:
(1) before transfection for 24 hours in carry out bed board: cell of the selection passage number within 3 generations, according to cell density and
State adjusts cell density, selects stand density to reach 80% 293T cell progress bed board, selects 6cm ware in the present embodiment；
(2) 60-90% is reached to cell density, cell state is good, viral packaging can be carried out, according to slow virus packet
It fills mixture and vector plasmid=3:2 carries out viral packaging, required three plasmid mixed liquors proportion is as follows:
3 μ g of vector plasmid
pMD2G 0.5μg
PSPAX2 4μg
(3) plasmid mixture and 15 μ L transfection reagent lipofectamine 2000 are mixed in a pipe, are stored at room temperature
It is slowly adherent to be added in the 293T cell for having replaced culture medium after 20min, continue to cultivate；
(4) 48h, 72h virus liquid being collected respectively, 80 DEG C of ﹣ storages are set by 0.45 μm of membrane filtration；
(5) virus liquid of collection is concentrated using PEG8000 concentration method, and measures virus titer, be stored in 80 DEG C of ﹣
It is spare.Virus titer is measured with the following method:
1. taking the good 293T cell of growth conditions, counted after digestion, by 2 × 105Cells/well uniformly spreads cell to 24
Porocyte culture plates, 6~10h of culture are adherent to cell；
2. the concentrating virus liquid of various concentration is added in cells and supernatant into 24 orifice plates, 24 hole edges of boards are gently patted
Edge is uniformly mixed virus liquid with culture solution, and 48h is infected in incubator；
3. cell is digested and collected after infecting 48h, Flow cytometry positive cell percentage, and according to following public affairs
Formula calculates virus titer, unit TU/mL:
T=(cell number × positive cell percentage × 1000 when bed board)/addition virus liquid volume (μ L).
The acquisition of embodiment 3:CAR-T cell
1.PBMC (peripheral blood mononuclear cells) separation
(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) it is loaded: taking 50mL centrifuge tube, draw 6mLFicoll (lymphocyte separation medium) in centrifuge tube, (Ficoll
With dilute before blood volume ratio be 1:1), pipe tilt 45 °, by the blood after dilution at Ficoll ullage about 1cm edge
Tube wall is added slowly to above Ficoll；
(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,
It is put into new centrifuge tube；
(6) it washs: the PBS that no less than 3 times of PBMC volume of addition, 18-20 DEG C, 2000rpm, 10min, twice；
(7) cell count: abandoning supernatant, adds 1mL RPMI-1640 culture medium (containing 10% fetal calf serum), piping and druming mixes, system
For at PBMC cell suspension.It is counted using blood cell counting plate: after taking a drop PBMC suspension and a 2% trypan blue dye liquor of drop to mix
It is added in blood cell counting plate, counts 4 big lattice inner cell sums under the microscope.(4 block plaid cells are total for cell number/mL=
Number/4) × 104× 2 (extension rates).
The activation and slow-virus infection of 2.T cell obtain CAR-T cell
(1) related reagent is prepared:
1. Anti-CD3 monoclonal antibody liquid is prepared: 50 μ g of CD3 monoclonal antibody is dissolved in 5ml PBS 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 5ml PBS 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 dual anti-(mycillin mixed liquor (100X)), 250 μ l autoserums.
(2) operating procedure:
1. Day ﹣ 1:24 orifice plate is coated with: taking 24 orifice plate of Corning, by taking 2 holes as an example, CD3 monoclonal antibody liquid is respectively added in 2 holes
175 μ l, CD28 monoclonal antibody liquid, 175 μ l, CH-38 protein liquid, 175 μ l.After gently concussion mixes after addition, orifice plate is closed with sealed membrane,
It is put into 4 DEG C of fridge overnights.Cell recovery: taking the PBMC cell in liquid nitrogen, recovery；
2. Day0: cleaning coating plate: taking out coated 24 orifice plate of Day ﹣ 1, discard supernatant, and after PBS is cleaned 2 times, PBS is added
For use；
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 cell；
Virus infection: the viral concentration liquid in embodiment 2 infects PMBC cell with MOI=30, prepares 1ml disease
Malicious culture medium suspension is added into 24 orifice plates, and orifice plate is placed in a centrifuge, and 1000g is centrifuged 30min, by centrifuge temperature tune
Section is to 32 DEG C；
3. Day1-Day2: observation cell state；
4. Day3: all cells in 24 orifice plates are transferred to the 25cm that 10ml culture medium is added2In culture bottle, observation is thin
Born of the same parents' state；
5. Day4-Day7: observation cell state and cell quantity, if cell starts obviously to expand, and regional area is thin
Born of the same parents' density is more, and 10ml culture medium is added；
6. Day8: 25cm at this time2Cell in culture bottle has covered with, and is transferred to the 75cm that 20ml culture medium is added2Culture
Continue to cultivate in bottle；
7. Day9-Day10: observation cell state, when cell is in 75cm2In culture bottle be in full state when, stop after
Continuous growth, enrichment of cell simultaneously calculate amplification ratio, flow cytometer detection cell typing, after carrying out cell killing detection or cell cryopreservation etc.
Continuous experiment.
Embodiment 4: Flow cytometry CAR structure positive expression rate
1. detecting cell CD3, CD4, CD8 positive rate
1. the NC group PBMC cell (not carrying out virus infection) of acquirement and sample group cell (having carried out virus infection) are made
It is mildly washed with PBS+2%BSA 2 times, centrifugal condition 1500rpm, 3min；
2. 1000 μ lPBS+2%BSA are added in NC pipe, be distributed into 5 pipes, respectively NC, NC-CD3, NC-CD4, NC-CD8,
200 μ l PBS 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 of CD3/4/8 antibody
μ l/20 μ l is mild to mix；
3. 1500rpm, 3min discard waste liquid after room temperature is protected from light incubation 30min；
4. 200 μ l PBS+2%BSA are added, mild mix is resuspended, and 1500rpm, 3min discard waste liquid；
5. 100 μ l PBS+2%BSA are added, mild upper machine testing after mixing resuspension.
The testing result of the T cell of the CD3 positive is as shown in figure 5, the accounting of the T cell of the CD3 positive reaches 94.85%.CD4
Positive and the CD8 positive the T cell accounting is as shown in fig. 6, the T cell of the CD4 feminine gender CD8 positive accounts for the T cell total amount of the CD3 positive
The T that the T cell of 51.20%, the CD4 positive CD8 positive accounts for 1.08%, the CD4 positive CD8 feminine gender of the T cell total amount of the CD3 positive is thin
Born of the same parents account for the 37.44% of the T cell total amount of the CD3 positive.
2. detecting CAR structure positive expression rate
1. the NC group cell (not carrying out virus infection) and sample group cell (having carried out virus infection) of acquirement are used
PBS+2%BSA is mildly washed 2 times, and centrifugal condition 1500rpm, 6min discard waste liquid；
2. 200 μ lPBS are added in NC pipe, it is 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. being incubated at room temperature 1h, centrifugal condition 1500rpm, 3min discard 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, it is resuspended；200 μ lPBS are added in sample tube, are resuspended, add the work of 5 μ l secondary antibodies
Liquid mixes；
6. centrifugal condition 1500rpm, 3min discard waste liquid, mild using PBS+2%BSA after room temperature is protected from light incubation 1h
Washing 3 times, centrifugal condition 1500rpm, 3min discard waste liquid；
7. 100 μ lPBS are added, mild upper machine testing after mixing resuspension.
As a result as shown in fig. 7, the T cell of the CAR positive occupies the 44.10% of the T cell total amount of the CD3 positive.3.RTCA is real
When Cell killing efficacy detect
1. by cell suspension is prepared into after colon cancer cell T84 digestion, piping and druming carries out RTCA cell count after mixing, will be thin
Born of the same parents' suspension is diluted to 5 × 104Cells/ml concentration is put on ice for spare；
2. RTCA detection plate is taken out, 50 μ l culture mediums are added, the examination of this test is compiled in RTCA detector program
Test program；
3. RTCA detection plate is placed in detector, observe in program Messege it is whether normal, starting is in fact after normal
Test program 1；
4. program takes out detection plate after running, 100 μ l of colon cancer tumours cell suspension is added in corresponding aperture, is added
Every solencyte suspension is mixed before；
5. will test in plate merging incubator and stand 30min, make cell natural subsidence；
6. after 30min, will test plate and be put into detector, program 2 is run；
7. observing cell growth curve afterwards for 24 hours, when cell is in logarithmic growth phase, prepare to obtain in addition embodiment 3
CAR-T cell:
Effector T cell is taken out in culture bottle, is centrifuged, is cleaned, is counted, by effect target ratio (CAR-T cell: colon cancer cell
T84 it is respectively) that 5:1,2.5:1 and 1:1 prepare effect group cell concentration, control group is with the T cell without infection according to T cell:
Colon cancer cell T84=1.25:1 is prepared.
8. time out program takes out detection plate, corresponding position is added 50 μ l of effector cell, puts back in detector, continues program,
Observation daily.
Experimental results are shown in figure 8, can be seen that by result, can be to knot after CAR-T cell about 30h of the invention is added
Colon-cancer cell T84 has apparent killing effect, and the higher killing to colon cancer cell T84 of the adding proportion of CAR-T cell is imitated
Fruit is better, when imitating target ratio is 2.5:1 and 5:1, can kill completely colon cancer cell T84 after CAR-T cell about 60h is added
It goes out, and the T lymphocyte of control group then has no killing effect to colon cancer cell T84.
4.ELISA detects cytokine secretion
1. using ddH2O dilution 10 × coating buffer, by 8 μ 250 × coating proteins of l are added in 2ml coating buffer buffer
Ratio is prepared；
2. the step 1. middle packet for preparing completion is added by 100 holes μ l/ into affine 96 orifice plate of Corning 9018Elisa high
By liquid, sealing is put into 4 DEG C of refrigerators, overnight；
3. carrying out 3 cleanings to coated 96 orifice plate using PBST (0.05%Tween 20)；
4. using ddH2O prepares 5 × confining liquid, is added by 200 holes μ l/, closes 1h at room temperature；
5. 1 × confining liquid is added by bottled standard items requirement to be prepared, carry out 7 doubling dilutions, at the same to sample into
5 times of row dilutions；
6. plank 5 times after PBST cleaning closing, the sample liquid after standard items and dilution is added is incubated at room temperature after 2h 4 DEG C
Overnight；
7. PBST is cleaned 4 times；
8. being added in 100 holes μ l/ using 1 × confining liquid dilution 250 × detection antibody, being incubated at room temperature 1h；
9. PBST is cleaned 4 times, 250 × HRP is diluted using 1 × confining liquid, is added in 100 holes μ l/, is incubated at room temperature 30min；
10. PBST is cleaned 5 times, 1 × TMB reagent, 100 μ l is added in every hole, is incubated at room temperature 15min；
50 hole μ l/ terminate liquid color development stoppings are added；
Microplate reader 450nm detects OD value.
As a result as shown in figure 9, using in " detection of RTCA real-time cell fragmentation effect " of the present embodiment CAR-T cell with
After colon cancer cell T84 is co-cultured 24 hours, it is able to detect that apparent cell factor IFN-γ release, effect CAR-T is thin
Born of the same parents' adding proportion is higher, and IFN-γ release is more.
The technical concepts and features of embodiment of above only to illustrate the invention, its object is to allow be familiar with technique
People understands the contents of the present invention and is implemented, and it is not intended to limit the scope of the present invention, all spiritual according to the present invention
The equivalent change or modification that essence is done, should be covered by the scope of protection of the present invention.
Claims (10)
1. the CAR-T carrier for being applied in treatment of colon cancer, which is characterized in that including Lentiviral and access institute
The CAR gene order of Lentiviral is stated, the CAR gene order includes EpCAM single-chain antibody gene sequence and PD-1
Monoclonal antibody gene sequence, for the EpCAM single-chain antibody nucleotide sequence as shown in SEQ ID NO.2, PD-1 monoclonal is anti-
The nucleotide sequence of body is as shown in SEQ ID NO.8.
2. the CAR-T carrier according to claim 1 for being applied in treatment of colon cancer, which is characterized in that the CAR
Structure composition be CD8leader-EpCAMscfv-CD8 α-CD28-CD137-CD3 ζ-IRES-PD-1mAb.
3. the CAR-T carrier according to claim 2 for being applied in treatment of colon cancer, which is characterized in that the CAR
Gene order are as follows:
The nucleotide sequence of CD8leader is as shown in SEQ ID NO.1；
The nucleotide sequence of EpCAMscfv is as shown in SEQ ID NO.2；
The nucleotide sequence of CD8 α is as shown in SEQ ID NO.3；
The nucleotide sequence of CD28-CD137 is as shown in SEQ ID NO.4 and SEQ ID NO.5；
The nucleotide sequence of CD3 ζ is as shown in SEQ ID NO.6；
The nucleotide sequence of IRES is as shown in SEQ ID NO.7；
The nucleotide sequence of PD-1mAb is as shown in SEQ ID NO.8.
4. the CAR-T carrier according to claim 1 for being applied in treatment of colon cancer, which is characterized in that described slow
Virus expression carrier is pCDH-CMV-MCS-EF1-Puro.
5. the CAR-T carrier according to claim 4 for being applied in treatment of colon cancer, which is characterized in that described slow
Virus expression carrier pCDH-CMV-MCS-EF1-Puro is inserted into CAR gene order by EcoRI and NotI double digestion.
6. the building side of the CAR-T carrier according to any one of claims 1-5 for being applied in treatment of colon cancer
Method, which comprises the following steps:
1) it will be stored on PUC19 plasmid after the synthesis of CAR gene order；
2) bacterial strain containing Lentiviral and the glycerol stock containing the obtained PUC19 plasmid of step 1) are subjected to increasing bacterium
After culture, plasmid is extracted, and be utilized respectively EcoRI and NotI double digestion；
3) the obtained digestion products of step 2) are passed through into agarose gel electrophoresis separation respectively, and recycle target fragment；
4) Lentiviral segment and CAR target fragment that step 3) recycling obtains are connected according to molar ratio for 1:5
Switching through, plasmid extract, and obtain CAR-T therapy vector.
7. prepared by the CAR-T carrier according to any one of claims 1-5 for being applied in treatment of colon cancer
Application in CAR-T: by the CAR-T therapy vector and packaging plasmid PSPAX2 and pMD2G take three plasmid packaging systems into
Row virus packaging, uses 293T cell as the incasing cells of slow virus, and collection virus liquid is simultaneously concentrated postoperative infection and has expanded activation
T cell, can be obtained CAR-T cell.
8. the CAR-T carrier according to claim 7 for applying in treatment of colon cancer is preparing answering in CAR-T
With, which is characterized in that the ratio of the packaging plasmid and the vector plasmid in system is packaging plasmid (PSPAX2:pMD2G
=8:1): vector plasmid=3:2.
9. a kind of CAR-T cell, which is characterized in that according to claim 7 for being applied in treatment of colon cancer
CAR-T carrier is obtained in the application method prepared in CAR-T.
10. the CAR-T carrier according to any one of claims 1-5 for applying in treatment of colon cancer is in colon cancer
Application in treating.
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