CN106047817A

CN106047817A - CAR-T cell as well as preparation method and application thereof

Info

Publication number: CN106047817A
Application number: CN201610602458.3A
Authority: CN
Inventors: 曾宪卓; 张翼
Original assignee: ISTEM REGENERATIVE MEDICINE SCI-TECH Co Ltd
Current assignee: ISTEM REGENERATIVE MEDICINE SCI-TECH Co Ltd
Priority date: 2016-07-28
Filing date: 2016-07-28
Publication date: 2016-10-26

Abstract

The invention discloses a chimeric antigen receptor T (CAR-T) cell as well as a preparation method and application thereof, wherein the CAR of the CAR-T cell comprises a cell membrane exoantigen binding region, a hinge region and an intracellular signal transduction region; the cell membrane exoantigen binding region is chA21scFv for combining with HER2 protein; and the intracellular signal transduction region is CD28-X-ITAM, wherein the X is ICOS, CD137, CD134, CD80 or CD86 costimulatory molecule. The chA21scFv of the CAR-T cell can express anti-HER2 monoclonal antibody, and the HER2 protein is as a target to directly recognize and combine with HER2 protein highly expressed on the surfaces of tumor cells, so that T cells are activated, and cell factors for killing the tumor cells expressing the HER2 protein are secreted.

Description

CAR-T cell and preparation method and application

Technical field

The present invention relates to T lymphocyte technical field, particularly relate to a kind of CAR-T cell and preparation method and application.

Background technology

CAR-T cell (Chimeric antigen receptor T cell, Chimeric antigen receptor T cell) is i.e. to express The T cell that Chimeric antigen receptor is modified, and the Chimeric antigen receptor of CAR-T cell (Chimeric antigen receptor, CAR) include that after birth exoantigen land, hinge region and intracellular signal transduction district three part are constituted.Wherein, after birth exoantigen combines District is one section of single-chain variable domain (single chain Fv domain, scFv), has specific recognition and combines TAA The function of (tumor-associated antigen, tumor related antigen).The composition of scFv is by the weight of monoclonal antibody Chain variable region (the variable region of heavy chain, VH) and variable region of light chain (the variable Region of light chain, VL) with a flexible peptide linker (Linker), VH and VL is coupled together, constitute VH- Linker-VL or VL-Linker-VH.Hinge region is typically to be made up of immunoglobulin superfamily, such as CD8, CD28 and IgG. Intracellular signal transduction district includes costimulatory molecules (costimulatory molecule, CM) and immunity receptor tyrosine activation base Sequence (immunoreceptor tyrosine-based activation motifs, ITAM), wherein, CM is usually CD28, ITAM is usually CD3 ζ or Fc ε RI γ.The T cell expressing CAR can be passed through scFv Direct Recognition and combine tumor cell surface TAA, CAR, by incoming for signal T cell, activate T cell secrete cytokines and include perforin, granzyme, INF-γ, TNF-a Deng, thus play killing tumor cell effect.Therefore, CAR-T cell is that MHC is nonrestrictive.CAR-T cell is by antibody-anti- The killing ability of former specific binding capacity and T cell mediation is incorporated into one, is the important method of immunity antineoplaston. That mosaic antigen receptor method is applied it is crucial that determine a kind of tumor related antigen, at tumor cell surface high expressed, and In the normal tissue without expressing or low expression.

Proto-oncogene people's epithelial growth factor receptor 2 (human epidermal growth factor receptor 2, Her-2/neu) also known as HER2 or c-erbB-2 gene, play a significant role in the morbidity and Clinical course of kinds of tumors, body It is outer that oneself is explicitly shown Her-2/neu gene amplification with zoopery, protein overexpression converts at tumorigenesis and rises in tumor development Pivotal role.Research confirms: in the mankind tumor tissue of more than 30%, such as breast carcinoma, ovarian cancer, carcinoma of endometrium, defeated ovum Pipe cancer, cervical cancer, carcinoma of prostate, gastric cancer, salivary-gland carcinoma, carcinoma of tongue, G. cephalantha, lung cancer in non-cellule type etc., all with The amplification of Her-2/neu gene and the overexpression of p185 albumen, and in normal structure, p185 albumen is negative or trace expression. And in breast carcinoma, patient's process LAN HER2 albumen of about 1/3.Process LAN HER2 albumen makes the aggressive of tumor higher, It it is the independent risk factor of breast cancer patients prognosis mala.Although anti-HER 2 monoclonal antibody (such as bent appropriate strain monoclonal antibody) is in clinic Application make some patients benefit, but it is expensive, largely limit its larger scale clinical application.HER2 albumen Being positioned at cell surface, easily by antibody recognition, therefore, HER2 albumen can be as an ideal of CAR-T cell anti-tumor treatment Target spot.

But, prior art does not provide the CAR-T cell expressing anti-HER 2 monoclonal antibody, the most existing CAR-T Cell can not with HER2 albumen as targeting Direct Recognition combine HER2 albumen, cause the CAR cannot be by incoming for signal T cell In, and the cytokine of the tumor cell killing high expressed HER2 albumen cannot be secreted.

Summary of the invention

Present invention is primarily targeted at a kind of CAR-T cell of offer, it is intended to expression anti-HER 2 monoclonal antibody, and with HER2 albumen is targeting Direct Recognition the HER2 albumen combining tumor cell surface high expressed.

For achieving the above object, the present invention provides a kind of CAR-T cell, and the CAR of described CAR-T cell includes outside after birth anti- Former land, hinge region and intracellular signal transduction district, described after birth exoantigen land is for combining HER2 albumen chA21scFv；Described intracellular signal transduction district is CD28-X-ITAM, wherein, described X be ICOS, CD137, CD134, CD80 or Person's CD86 costimulatory molecules.

Preferably, described X is ICOS costimulatory molecules.

The present invention also provides for the preparation method of a kind of CAR-T cell, comprises the following steps that

Building CAR expression vector, wherein, the CAR of described CAR expression vector includes after birth exoantigen land, hinge region With intracellular signal transduction district, described after birth exoantigen land is the chA21scFv for combining HER2 albumen, and described intracellular is believed Number conducting region is CD28-X-ITAM, and described X is ICOS, CD137, CD134, CD80 or CD86 costimulatory molecules；

Pack described CAR expression vector, and prepare and infect mixture；

Described infection mixture is infected T cell, and prepares CAR-T cell.

Preferably, during described structure CAR expression vector, comprise the following steps that

Obtain the genetic fragment of described chA21scFv, the genetic fragment of described hinge region, the genetic fragment of described CD28, The genetic fragment of described X and the genetic fragment of described ITAM；

Guiding chain is provided, the genetic fragment of guiding chain described in gene fusion and described chA21scFv, and formed guiding chain- chA21scFv；

The genetic fragment of hinge region described in gene fusion, the genetic fragment of described CD28, the genetic fragment of described X and described The genetic fragment of ITAM, and form hinge region-CD28-X-ITAM；

Guiding chain-chA21scFv described in gene fusion and described hinge region-CD28-X-ITAM, and formed guiding chain- ChA21scFv-hinge region-CD28-X-ITAM；

Described guiding chain-chA21scFv-hinge region-CD28-X-ITAM is accessed carrier, and obtains CAR expression vector.

Preferably, the genetic fragment of the described chA21scFv of described acquisition, the genetic fragment of described hinge region, described CD28 Genetic fragment, the genetic fragment of described X and described ITAM genetic fragment during, comprise the following steps that

Obtain and activate PMBCs；

MRNA the reverse transcription of extracting described PMBCs are cDNA；

The primer of described hinge region, the primer of described CD28, the primer of described X and the primer of described ITAM are provided respectively, And all with the cDNA of described PMBCs as template, carry out PCR amplification respectively, and obtain the genetic fragment of described hinge region, described The genetic fragment of CD28, the genetic fragment of described X and the genetic fragment of described ITAM；

Thering is provided template DNA and the primer of described chA21scFv of described chA21scFv, performing PCR of going forward side by side expands, and obtains The genetic fragment of described chA21scFv.

Preferably, described CAR expression vector is that CAR expresses virus particle.

Preferably, during described packaging described CAR expression vector, comprise the following steps that

The packaging plasmid of 12～20 μ g and the described CAR of 12～20 μ g are expressed virus particle be blended by culture medium, and Obtain plasmid mixture；

The liposome of described plasmid mixture and 35～45 μ l is blended by culture medium, and obtains mixed liquor；

By described mixed liquor transfecting eukaryotic cells, and obtain the supernatant of described eukaryotic cell；

Concentrate described supernatant, and prepare and infect mixture.

Preferably, described by during described infection mixture infection T cell, comprise the following steps that

Infection mixture by 0.8～2ml adds (6～10) × 10⁵Individual T cell infects；

Cohesion amine is added in described T cell, makes final concentration of 10～20 μ g/ml of described cohesion amine；

Cultivate and expand metainfective T cell, and obtain CAR-T cell.

Preferably, described X is ICOS costimulatory molecules.

The present invention also provides for the application on the medicine for the treatment of breast carcinoma of a kind of CAR-T cell as above.

Technical solution of the present invention, identifies and combines the HER2 of tumor cell surface high expressed by expressing chA21scFv Albumen, and by intracellular signal transduction district by incoming for signal T cell, thus activating T cell, promote T cell secrete cytokines, This cytokine and then the tumor cell of high expressed HER2 albumen can be killed.And, intracellular signal transduction district incorporates CD28 Costimulatory molecules and X costimulatory molecules, can make T cell continuous activation breed, cytokine continuous release, strengthens and kills tumor Cytosis.

Accompanying drawing explanation

In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing In having technology to describe, the required accompanying drawing used is briefly described, it should be apparent that, the accompanying drawing in describing below is only this Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, it is also possible to Other accompanying drawing is obtained according to the content shown in these accompanying drawings.

Fig. 1 is the gene structure figure of the CAR expression vector of the embodiment of the present invention 1 preparation；

Fig. 2 be ELISA method detection the embodiment of the present invention 1 preparation CAR-T cell and existing NT-T cell respectively with The block diagram of the IFN-γ secretion amount after the co-culturing of HER2+/-tumor cell；

Fig. 3 be ELISA method detection the embodiment of the present invention 1 preparation CAR-T cell and existing NT-T cell respectively with The block diagram of the IL-2 secretory volume after the co-culturing of HER2+/-tumor cell；

Fig. 4 is the specific cartogram of HER2 of the CAR-T cell of the ELISA method detection embodiment of the present invention 1 preparation；

Fig. 5 is⁵¹The CAR-T cell of the Cr release test detection embodiment of the present invention 1 preparation specificity to breast cancer cell The cartogram of lethal effect.

Detailed description of the invention

Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete Describe, it is clear that described embodiment is only a part of embodiment of the present invention rather than whole embodiments wholely.Base Embodiment in the present invention, those of ordinary skill in the art obtained under not making creative work premise all its His embodiment, broadly falls into the scope of protection of the invention.

The present invention provides a kind of CAR-T cell, and wherein, the CAR of this CAR-T cell includes after birth exoantigen land, hinge Sequence and intracellular signal transduction district, after birth exoantigen land is the chA21scFv for combining HER2 albumen；Intracellular signal passes Leading district is CD28-X-ITAM, and wherein, X is ICOS, CD137, CD134, CD80 or CD86 costimulatory molecules.

CAR-T cell of the present invention identifies and combines the HER2 of tumor cell surface high expressed by expressing chA21scFv Albumen, and by intracellular signal transduction district by incoming for signal T cell, thus activating T cell, promote T cell secrete cytokines, This cytokine and then the tumor cell of high expressed HER2 albumen can be killed.And, intracellular signal transduction district incorporates CD28 Costimulatory molecules and X costimulatory molecules, can make T cell continuous activation breed, cytokine continuous release, strengthens and kills tumor Cytosis.

It should be noted that in the structure of this CAR, chA21 is for using surface investment (surface epitope Masking method, SEM) the Humanized monoclonal antibodies A21 that produces, it is a kind of anti-ErbB 2 antibodies, it has the strongest knot Close specificity and antitumor activity, chA21 can the I domain of specific recognition HER2 extracellular region C-end, away from HER2 receptor two Dimerization functional position, therefore, the present invention builds CAR-T cell based on the chA21 of anti-HER2；ChA21scFv is by chA21 Dan Ke The heavy-chain variable domains of grand antibody and light variable domains connect formation by peptide linker, due to many suppliers or section Grind mechanism and the most successfully synthesize chA21scFv, and synthetic method is also prior art, therefore this chA21scFv can be directly by supplying Business or scientific research institution is answered to provide.Hinge region can be CD3, CD4, CD8 or CD28, concrete depending on practical situation.Intracellular is believed The costimulatory molecules X of number conducting region can be ICOS, CD137, CD134, CD80 or CD86 costimulatory molecules, all can play conduction The effect of signal, it is achieved T cell continuous activation is bred, cytokine continuous release, strengthens the purpose of killing tumor cell；Intracellular The ITAM of signal conducting region is usually CD3 ζ or Fc ε RI γ.

S1, build CAR expression vector, wherein, the CAR of CAR expression vector include after birth exoantigen land, hinge region and Intracellular signal transduction district, after birth exoantigen land is the chA21scFv for combining HER2 albumen, and intracellular signal transduction district is CD28-X-ITAM, X are ICOS, CD137, CD134, CD80 or CD86 costimulatory molecules；

S2, packaging CAR expression vector, and prepare and infect mixture；

S3, general infect mixture and infect T cell, and prepare CAR-T cell.

The preparation method of CAR-T cell of the present invention is by building CAR expression vector, and incites somebody to action by the way of packaging, transfection CAR expression vector accesses in T cell, owing to this CAR expression vector expresses anti-HER 2 monoclonal chA21, such that it is able to identify and In conjunction with the HER2 albumen of tumor cell surface high expressed, and by intracellular signal transduction district by incoming for signal T cell, thus T is thin Born of the same parents activate, and promote T cell secrete cytokines, this cytokine and then can kill the tumor cell of high expressed HER2 albumen. This preparation method is efficient, simple, low cost.

Further, in step sl, during building CAR expression vector, step is specifically included as follows:

S10, the acquisition genetic fragment of chA21scFv, the genetic fragment of hinge region, the genetic fragment of CD28, the gene sheet of X Section and the genetic fragment of ITAM；

S11, provide guiding chain, the genetic fragment of gene fusion guiding chain and chA21scFv, and formed guiding chain- chA21scFv；

The gene sheet of S12, the genetic fragment of gene fusion hinge region, the genetic fragment of CD28, the genetic fragment of X and ITAM Section, and form hinge region-CD28-X-ITAM；

S13, gene fusion guiding chain-chA21scFv and hinge region-CD28-X-ITAM, and formed guiding chain- ChA21scFv-hinge region-CD28-X-ITAM；

S14, guiding chain-chA21scFv-hinge region-CD28-X-ITAM is accessed carrier, and obtain CAR expression vector.

By PCR amplification technique and overlapping pcr, by the genetic fragment of chA21scFv, the genetic fragment of hinge region, The genetic fragment of the genetic fragment of CD28, the genetic fragment of X and ITAM carries out gene fusion, and builds CAR expression vector, its Building mode is efficiently, simply.It should be noted that this guiding chain is preferably people's CD8 α guiding chain, people's CD8 α guiding chain can be from People's CD8 α cell extracts, it is also possible to obtain from corresponding supplier, and the primer of people's CD8 α guiding chain can be by supplying accordingly Business is answered to provide.

Further, to specifically include step as follows for step S10:

S100, (peripheral blood mononuclear cells, peripheral blood mononuclear is thin to obtain and activate PMBCs Born of the same parents)；

S101, the mRNA extracting PMBCs reverse transcription are cDNA；

S102, provide the primer of hinge region, the primer of CD28, the primer of X and the primer of ITAM respectively, and all with PMBCs CDNA be template, carry out PCR amplification respectively, and obtain the gene sheet of the genetic fragment of hinge region, the genetic fragment of CD28, X Section and the genetic fragment of ITAM；

S103, the template DNA providing chA21scFv and the primer of chA21scFv, performing PCR of going forward side by side expands, and obtains The genetic fragment of chA21scFv.

Obtained the mRNA of T cell by PMBCs, be then cDNA by mRNA reverse transcription, thus be different types of T cell The amplification of genetic fragment provide template, simultaneously by template DNA and the primer of chA21scFv of existing chA21scFv ChA21scFv is carried out PCR amplification, it is clear that above-mentioned PCR amplification is efficient, easy, is conducive to provide the structure of CAR expression vector.

Further, CAR expression vector be CAR express virus particle, in other words, in step S14, by guiding chain- ChA21scFv-hinge region-CD28-X-ITAM has accessed viral vector, and defines CAR and express virus particle, wherein, this disease Poisonous carrier can be retrovirus or slow virus, and by presented in plasmid.

Further, in step s 2, during packaging CAR expression vector, step is specifically included as follows:

S20, the CAR of the packaging plasmid of 12～20 μ g and 12～20 μ g is expressed virus particle it is blended by culture medium, and Obtain plasmid mixture；

S21, the liposome of plasmid mixture and 35～45 μ l is blended by culture medium, and obtains mixed liquor；

S22, by mixed liquor transfecting eukaryotic cells, and obtain the supernatant of eukaryotic cell；

S23, concentrated supernatant, and prepare and infect mixture.

By the packaging to CAR expression vector, CAR can be risen to and express the efficiency of infection of virus particle, thus improve The preparation efficiency of CAR-T cell.

Further, in step S3, during mixture infection T cell will be infected, comprise the following steps that

S30, by 0.8～2ml infection mixture add (6～10) × 10⁵Individual T cell infects；

S31, general's cohesion amine add in T cell, make final concentration of 10～20 μ g/ml of cohesion amine；

S32, cultivation expand metainfective T cell, and obtain CAR-T cell.

This cohesion amine transfection agents can improve the efficiency of infection of T cell, it is achieved the modification to T cell.

Owing to CAR-T cell identifies and combine the HER2 egg of tumor cell surface high expressed by expressing chA21scFv In vain, and by intracellular signal transduction district by incoming for signal T cell, thus activating T cell, promote T cell secrete cytokines, should Cytokine and then can kill the tumor cell of high expressed HER2 albumen, therefore, this CAR-T cell is applied to treat breast carcinoma Time, owing to the apparent height of breast cancer cell expresses HER2 albumen, can be with Efficient killing effect breast cancer cell.

Now by embodiment 1, CAR-T cell of the present invention and preparation method and application is further explained and illustrates, In this CAR-T cell, the X costimulatory molecules in the intracellular signal transduction district of CAR is preferably ICOS costimulatory molecules, CAR-T cell Concrete building mode sees content as detailed below.In like manner, according to following building mode, can build X costimulatory molecules is CAR-T cell during other costimulatory moleculeses, the primer sequence as X costimulatory molecules can be looked into from the GenBank of NCBI Looking for acquisition, the primer of X costimulatory molecules can be bought in businessman accordingly and obtain, it is also possible to designed, designed.

Embodiment 1

One, the material used in the embodiment of the present invention and reagent

1, human peripheral blood mononuclear cell

The peripheral blood lymphocytes of three healthy volunteers derives from carcinoma intervention section of BeiJing University ShenZhen Hospital.

2, cell line

Breast cancer lines SKBR3, T47D, MCF-7, MDA-MB-231 and Ovarian Cancer Cells SKOV3,0VCAR3, A1847 and A2780, and the tumor cell line MDA-MB-468 (buying in ATCC) that HER2 is negative.TC-1 is HPV-16 transfection Murine lung cancer cell, be used as people's HER2 feminine gender express comparison, also buy in ATCC.293T cell is bought in ATCC.

3, reagent

(1) RPMI-1640 culture medium (Invitrogen company)

(2) inactivated fetal bovine serum (Invitrogen company)

(3) a blue or green streptomycin 10000U/ml 10000IU/ml (Invitrogen company)

(4) AntiCD3 McAb/anti-CD28 magnetic bead (Invitrogen company)

(5) RhIL-2 (IL-2) (PeproTech company)

(6) DMSO (dimethyl sulfoxide) (Invitrogen company)

(7) 0.25% pancreatin (Invitrogen company)

(8) LTS1077 lymphocytes separating solution (Yan Jin bio tech ltd, Shanghai)

(9) mRNA extracts test kit (Invitrogen company)

(10) Reverse Transcription box (Invitrogen company)

(11) PCR kit (Invitrogen company)

(12) PCR primer purification kit (Qiagen company)

(13) the plasmid PEE14-chA21 (Chinese University of Science and Technology's offer) of the chA21scFv containing Humanized anti-HER 2

(14) people CD8 α guiding chain (CD8 α leader chain) (Takara company)

(15) the forward primer AF (Takara company) of people CD8 α guiding chain

(16) the forward primer CF and reverse primer CR (Takara company) of CD8 α hinge region (CD8 α hinge)

(17) the forward primer DF and reverse primer DR (Takara company) of CD28

(18) the forward primer GF and reverse primer GR (Takara company) of ICOS

(19) the forward primer EF and reverse primer ER (Takara company) of CD3 ζ

(20) the forward primer BF and reverse primer BR (Takara company) of chA21scFv

(21) pMD.G plasmid, pMDLg/p plasmid, Rev expression plasmid (Qiagen company)

(22) the mountain sheep anti mouse F (ab ') of FITC labelling₂Antibody (Jackson ImmunoResearch company)

(23) anti-human-CD4 of the anti-human-CD4 of anti-human-CD45, FITC, PE of anti-human-CD3, the PE labelling of APC CY7 labelling, Anti-human-the CD8 of APC, PE anti-human CD45RO, APC anti-human CD62L antibody (Biolegend company)

(24) IFN-γ ELISA kit (Biolegend company)

(25) IL-2ELISA test kit (Biolegend company)

(26) HER2-Fc chimeric protein (R&D Systems company)

(27) CD19-Fc chimeric protein (SPEEDBioSystems company)

(28) sodium chromate (Na₂ ⁵¹CrO₄) (DuPont NEN company)

(29) FITC rabbit anti-human HER2 antibody (Clone24D2, Biolegend company)

(30) liposome 2000 (Invitrogen company)

(31) cohesion amine (Sigma company)

4, primer sequence table

Table one

Two, the technical scheme of the present embodiment

(1), the separation of peripheral blood lymphocytes and activation

1, the separation of human peripheral blood mononuclear cell (peripheral blood mononuclear cell, PBMC)

(1) the peripheral blood 15ml of anticoagulant blood vessel extraction healthy volunteer；

(2) in this anticoagulant blood vessel, add isopyknic PBS, blow and beat into cell suspension 30ml gently；

(3) separately take two 50ml centrifuge tubes, add the LTS1077 lymphocytes separating solution of 15ml to a centrifuge tube；Then, Draw the cell suspension of 15ml with suction pipe, above distance LTS1077 lymphocytes separating solution at 1cm by cell suspension carefully and Adding slowly, make cell suspension be overlapped on LTS1077 lymphocytes separating solution, 2000rpm is centrifuged 20min；

(4) take out centrifugal after centrifuge tube, pipet sucks the blood plasma of the superiors, and it is single that liquid-transfering gun is drawn under plasma layer Nucleus is inserted in another centrifuge tube；Then, the PBS adding l5ml washs, gently recentrifuge after piping and druming uniformly, 1500rpm, L0min, removes supernatant；Washing 3 times altogether；

(5) add in the centrifuge tube after removing supernatant containing 10% inactivated fetal bovine serum, the blue or green streptomycin of 100U/ml, The RPMI-1640 culture medium of 100U/ml IL-2, is positioned over 37 DEG C, 5%CO₂Cell culture incubator in cultivate, and separate acquisition PBMC cell preparation.

2, the activation of human peripheral blood mononuclear cell

(1) take the PBMC cell preparation that step 1 obtains, in 24 orifice plates, plant 1x10⁶Individual PBMC cell (1 × 10⁶/ml)；

(2) according to the method for description of immunological magnetic bead sorting test kit, with PBS, the magnetic bead being coated AntiCD3 McAb/CD28 is washed 3 times；

(3) magnetic bead is added in PBMC cell in 3:1 (magnetic bead: cell) ratio, place 37 DEG C, 5%CO₂Incubator is trained Support overnight；

(4) cell-stimulating 12～after 24 hours, centrifugal, collect the human peripheral blood mononuclear cell being activated, be used as to carry mRNA And reverse transcription is cDNA.

(2) mRNA the reverse transcription of, extracting the peripheral blood lymphocytes activated are cDNA

1, the extraction of the mRNA of peripheral blood lymphocytes

(1) human peripheral blood mononuclear cell 1.5 × 10 after being activated is taken out⁶Individual, rinse 2 times with PBS；

(2), after removing supernatant, in cell precipitates, add the Trizol of 1.5ml, blow and beat uniformly cell suspending liquid；

(3) use 1ml syringe suction of cells suspension back and forth, to shear genomic DNA, then directly will with syringe Sample is transferred in the EP pipe of a new 1.5ml；

(4) in EP pipe, add the chloroform of 250 μ l, acutely shake 30sec, 12000rpm and be centrifuged 5min；

(5) supernatant produced after being centrifuged moves in the Ep pipe of another new 1.5ml, and adds isopyknic isopropanol, Room temperature places 5min；

(6) 12000rpm is centrifuged 5min, siphons away supernatant；

(7) adding the ethanol 750 μ l of 70% in the Ep pipe after siphoning away supernatant, be not required to piping and druming, 12000rpm is centrifuged 2min；

(8) the most thoroughly siphon away centrifugal after the supernatant that produces, drying at room temperature makes ethanol volatilize, and forms precipitation；

(9) DEPC (diethyl pyrocarbonate, the pyrocarbonic acid diethyl ester) water of 60 μ l is added to this precipitation, and molten Solve this precipitation, the mRNA of instant solution peripheral blood lymphocytes；

(10) take the mRNA 3 μ l of dissolving, add in the DEPC water of 1ml, use ultraviolet spectrophotometer measure 260nm and The absorbance of 280nm: OD260/280=1.9～2.0；The productivity of mRNA, OD260/280 is calculated by the mRNA of 1OD=40 μ g The mRNA purity being considered as in 1.9～2.0 extracting is the highest；

(11) next step RT reaction is carried out with the RNA product extracted.

2, RT (Reverse Transcription, reverse transcription) reaction

(1) reaction system following with other reagent composition for the mRNA that step 1 is extracted:

Reagent	Volume
		mRNA	1.5μl
OligodT-Adaptor Primer (primer)	1μl
		DNTP Mixture (each 10nM)	2μl
AMV Reverse Transcriptase	1μl
		10×RT Buffer	1.5μl
RNase Free dH₂O	3.75μl
		MgCl₂	2μl
RNase Inhibitor	0.25μl
		Total	13μl

(2) RT reaction condition: room temperature, 10min；42 DEG C, 1h；99 DEG C, 5min inactivates AMV Reverse Transcriptase；And then reverse transcription obtains the cDNA of PMBCs；

(3) cDNA of PMBCs reverse transcription obtained is placed in-80 DEG C of Refrigerator stores, standby.

(3), the structure of mosaic antigen receptor (CAR) carrier of targeting HER2

1, PCR expands chA21scFv, CD8 α hinge, CD28, ICOS and CD3 ζ

1.1, PCR expands chA21scFv

A, the DNA profiling that chA21scFv is provided and the primer of chA21scFv:

The plasmid PEE14-chA21 that the DNA profiling of chA21scFv is provided by Chinese University of Science and Technology obtains；ChA21scFv's Forward primer BF and reverse primer BR is provided by Takara company, and its primer sets specifically sees the above table one)；

B, obtain PCR primer according to following PCR reaction system and PCR reaction condition；

PCR reaction system:

Reagent	Volume
		DNA profiling (chA21scFv)	1.5μl(75ng)
10×PCR buffer	7.5μl
		10mM dNTP	1.5μl
10um forward primer BF	3μl
		10um reverse primer BR	3μl
50mM MgSO₄	3μl
		Taq archaeal dna polymerase	1 μ l (5 unit)
Add tri-distilled water extremely	80μl

PCR reaction condition:

C, electrophoresis

Preparation agarose gel (20g/L): weigh the agarose gel powder of 0.6g, adds the TAE electrophoretic buffer of 30ml, Putting into heating 45s in microwave oven after mixing, add the EB liquid of 1ul, irrigate after mixing, room temperature cools down；Add suitable in electrophoresis tank Amount TAE electrophoretic buffer, with DL2000 as DNA Marker, adds the above-mentioned PCR primer of 5u1, regulation electricity in sample aperture Swimming instrument voltage is 90mV, electrophoresis half an hour；

D, result are observed

Utilize digital gel images collection and analysis system to be scanned analysis to understand: above-mentioned PCR primer is chA21scFv Genetic fragment；

E, PCR primer reclaim

(1) PCR primer remaining after electrophoresis is transferred in the EP pipe of 2ml, adds the PB buffer of 300ul, the most mixed Even；

(2) absorption: the EP pipe of QIAquick pillar link 2ml, is slowly added to the PB buffer containing PCR primer QIAquick pillar, 13000 revs/min are centrifuged 30 seconds；

(3) washing: discard the liquid in EP pipe, add the PE buffer of 800ul in QIAquick pillar, 13000 turns/ Separate the heart 30 seconds；

(4) discarding the liquid in EP pipe, 13000 revs/min are centrifuged 1 minute；

(5) eluting: be placed in the EP pipe of new 1.5ml by QIAquick pillar, adds the EB buffering of 60ul in pillar Liquid, 13000 revs/min are centrifuged 1 minute, discard the liquid in EP pipe and reclaim PCR primer；

(6) PCR primer after reclaiming is placed in-20 DEG C of Refrigerator stores.

1.2, PCR expands CD8 α hinge, CD28 and CD3 ζ

The cDNA of the PBMCs that a, offer primer and step (two) prepare:

The forward primer CF and reverse primer CR of CD8 α hinge are provided by Takara company, and its primer sets sees the above table one；

The forward primer DF and reverse primer DR of CD28 are provided by Takara company, and its primer sets sees the above table one；

The forward primer DF and reverse primer DR of ICOS are provided by Takara company, and its primer sets sees the above table one；

The forward primer EF and reverse primer ER of CD3 ζ are provided by Takara company, and its primer sets sees the above table one；

PCR reaction system:

PCR reaction condition: identical with the PCR reaction condition of PCR amplification chA21scFv；

C, PCR primer reclaim (identical with the step that the PCR primer of PCR amplification chA21scFv reclaims), and then obtain CD8 α The PCR primer of hinge/CD28/ICOS/CD3 ζ.

2, over-lap PCR and carry out gene fusion

2.1, CD8 α leader chain (CD8 α guiding chain) is provided, the chA21scFv after being expanded by CD8 α leader chain with PCR Carry out gene fusion, and form CD8 α leader-chA21scFv:

A, first step PCR are reacted, and its reaction system and response time are as follows；

First step PCR reaction system:

Reagent	Volume
		CD8 α leader chain	～50ng
ChA21scFv after amplification	～50ng
		10×PCR buffer	5μl
10mM dNTP	1μl
		50mM MgSO₄	2μl
Taq archaeal dna polymerase	0.2μl
		Add water to	50μl

First step PCR reaction condition:

B, second step PCR react, and its reaction system and response time are as follows；

Second step PCR reaction system: add the forward of CD8 α leader chain in the system after first step PCR has been reacted The reverse primer BR (its primer sequence is as shown in Table 1) of primer AF (its primer sets is as shown in Table 1) and chA21scFv is each 2ul；

Second step PCR reaction condition:

E, the PCR primer that second step PCR reacts being carried out electrophoresis, digital gel images collection is scanned with analysis system Analyze, and prove that the PCR primer that second step PCR reacts is CD8 α leader-chA21scFv；

The PCR primer of f, second step PCR reaction reclaims (the step phase reclaimed with the PCR primer of PCR amplification chA21scFv With), and obtain CD8 α leader-chA21scFv；

2.2, the CD8 α hinge after amplification and the CD28 after amplification is carried out gene fusion, it is thus achieved that CD8 α hinge-CD28；

ICOS after CD8 α hinge-CD28 and amplification is carried out gene fusion, it is thus achieved that CD8 α hinge-CD28-ICOS；

CD3 ζ after CD8 α hinge-CD28-ICOS and amplification is carried out gene fusion, it is thus achieved that CD8 α hinge-CD28- ICOS-CD3ζ；

CD8 α leader-chA21scFv and CD8 α hinge-CD28-ICOS-CD3 ζ is carried out gene fusion, it is thus achieved that CD8 α Leader-chA21scFv-CD8 α hinge-CD28-ICOS-CD3 ζ, its gene structure is as shown in Figure 1；

Ibid, wherein, the primer added in CD8 α hinge and CD28 fusion process is CF, DR to concrete grammar；CD8α The primer added in hinge-CD28 Yu ICOS fusion process is CF, GR；CD8 α hinge-CD28-ICOS and CD3 ζ merged The primer added in journey is CF, ER；CD8 α leader-chA21scFv and CD8 α hinge-CD28-ICOS-CD3 ζ merged The primer added in journey is AF, ER (primer sequence is as shown in Table 1).PCR primer carries out electrophoresis, and digital gel images is adopted Collection and analysis system are scanned analyzing, final recovery PCR primer CD8 α leader-chA21scFv-CD8 α hinge-CD28- ICOS-CD3ζ。

3, CD8 α leader-chA21scFv-CD8 α hinge-CD28-ICOS-CD3 ζ transfer to U.S.'s Cambridge gene mentation public Department (Addgene, Cambridge, MA, USA) accesses pSin (slow virus) skeleton and forms pSin-chA21-28 ζ plasmid, i.e. CAR Expression vector.

(4) packaging CAR expression vector (packaging pSin-chA21-28 ζ slow virus plasmid), and prepare and infect mixture

1,293T passage is cultivated and prepares

A, 293T cell recovery and cultivation

(1) cell culture medium: configuration is cultivated containing the RPMI-1640 of 10% inactivated fetal bovine serum, the blue or green streptomycin of 100U/ml Base；

(2) taking out frozen 293T cell from liquid nitrogen container, put into rapidly 37 DEG C of water-baths, frozen 293T cell melts After, in super-clean bench, cell suspension is moved in centrifuge tube, adds the RPMI-1640 culture medium of 4ml, put in centrifuge, 1000 revs/min are centrifuged 3～4 minutes；

(3) abandoning supernatant, the RPMI-1640 culture medium adding 1ml preparation is blown and beaten into cell suspension gently, is moved into 75cm²In Tissue Culture Flask, addition RPMI-1640 culture medium, to 10ml, puts into 37 DEG C, 5%CO₂Cell culture incubator in cultivate, Liquid is changed after 24 hours；

B, 293T passage

Cell in culture dish passes on when being covered with about 80～90%.Old culture medium is siphoned away, adds in culture dish Enter 2～3ml PBS rinse 1 time, siphon away PBS, in culture dish add 0.25% trypsin 2ml, digest 3～5min, note Being intended to the change of observation of cell form under inverted microscope, when seeing cell retraction, form becomes round, and Cell tracking gap increases Time, the RPMI-1640 culture medium adding 5ml in culture dish terminates digestion, and suction pipe is repeatedly blown and beaten and made cell detachment, makes cell and hangs After liquid, in about 1:3 ratio Secondary Culture；

C, 293T cell cryopreservation

(1) cell that growth conditions is good, can be frozen when being covered with culture bottle about 80～90%.Cell dissociation after-blow breaks into Cell suspension, moves in 15ml centrifuge tube, puts in centrifuge, and 1000 revs/min are centrifuged 3～4 minutes；

(2) abandoning supernatant, adds the inactivated fetal bovine serum 1ml containing 10% dimethyl sulfoxide, blows and beats into cell gently and hangs Liquid, moves in cell cryopreservation tube, carries out labelling, insert on ice, and puts into rapidly preservation in liquid nitrogen container.

2, the packaging of slow virus plasmid

(1) take out frozen 293T cell, be placed in 75cm²Tissue Culture Flask cultivate；Choose degrees of fusion 60～70% 293T cell, standby after three hours after changing liquid；

(2) by 3 kinds of packaging plasmid pMD.G, pMDLg/p, Rev and pSin-chA21-28z plasmid according to the form below ratios in 1ml Serum-free is without mix homogeneously, incubated at room 5 minutes in the RPMI-1640 of 37 DEG C of dual anti-preheatings, and obtains plasmid mixture；

pMDLg/p	5.5μg
		Rev	3μg
pMD.G	4.5μg
		pSin-chA21-28z	13μg
Amount to	26μg

(3) by the above-mentioned plasmid mixture of 26 μ g and 39 μ l liposomees 2000, (plasmid is 1 μ with the ratio of liposome 2000 G:1.5 μ l) it is added dropwise in the RPMI-1640 of 1ml serum-free antibiotic-free being gently mixed uniformly, incubated at room 5 minutes, and obtain Obtain mixed liquor；

(4) mixed liquor of final liposome 2000 and plasmid blend is added dropwise in 293T Tissue Culture Dish, gently Light mix homogeneously, 37 DEG C, 5%CO₂Incubator is cultivated；

(5) the transfection 293T cell supernatant of 24 hours and 48 hours is collected；

(6) get the 293T cell supernatant about 35ml of collection, be collected in 50ml centrifuge tube, 2000 turns, 10 minutes, remove 293T cell debris, and obtain the supernatant after removing fragment；

(7) being placed on ice by the supernatant that step (6) obtains, connecting 0.45 μm syringe needle filter with 60ml syringe will be centrifugal After supernatant liquid filtering in 35ml ultracentrifugation pipe, 25000 turns, 3 hours；

(8) centrifuge tube is taken out gently, be placed on ice；

(9) in viral special super-clean bench, open metal centrifuge tube, suck the viral supernatants after part centrifugal, use tweezers Take out ultracentrifugation pipe, suck major part supernatant, remain about 4ml；

(10) repeatedly blowing and beating remaining 4ml viral supernatants about 10 times with 5ml pipet, 0.75ml/ pipe carries out subpackage, puts Enter in-80 DEG C of refrigerators and save backup, and obtain lentiviral particle, i.e. infect mixture.

(5), transfection peripheral blood lymphocytes

1, the separation of human peripheral blood mononuclear cell and activation

Identical with step ().

2, the transfection of human peripheral blood mononuclear cell

(1) after human peripheral blood mononuclear cell activates 12～24 hours in 24 orifice plates, centrifugal 24 orifice plates, siphon away on 800 μ l Clear culture fluid, adds new culture fluid, adjusts every porocyte number about 0.5 × 10⁶/200μl；

(2) sub-elect T cell from human peripheral blood mononuclear cell and activate, taking (7～9) × 10⁵The T cell activated, uses CD45RO, CD62L antibody staining, (method is same for the typing of the human peripheral blood mononuclear cell activated before flow cytomery transfection On)；

(3) at room temperature frozen lentiviral particle is melted, mix gently, the lentiviral particle of 1ml is added T cell In, in 24 orifice plates add cohesion amine, adjust to final concentration of 12 μ g/ml by RPMI1640 culture medium, 24 orifice plates are put into from Scheming is centrifuged, 2500r/min, centrifugal 1.5 hours；After Li Xin, 24 orifice plates are put in 37 DEG C, 5%CO₂Overnight incubation in incubator；

(4) 24 orifice plates that centrifuged overnight is cultivated, remove the major part supernatant culture fluid containing lentiviral particle, add fresh RPMI-1640 culture fluid, expands T cell；

(5) every 2 days counting T cell, add IL-250IU/ml in RPMI-1640 culture medium, and maintenance T cell density is (0.5～1) × 10⁶/ml；

After (6) 2 weeks, use anti-human CD3, CD45, CD4, CD8, CD45RO, CD62L antibody and the goat-anti of FITC labelling Mus F (ab') 2 antibody, carries out flow cytometry analysis (method is ibid)；

(7) successful chA21-28-ICOS ζ CAR-T cell is transfected standby.

(6) detection of the CAR-T cells in vitro function of anti-HER2, is expressed

Before detection, the tumor cell line of the CAR-T cell first embodiment 1 prepared and expression HER2 is (involved by embodiment 1 And material in cell line) co-culture as follows, and obtain co-culture media:

(1) with after 0.25% trypsinization tumor cell, add in RPMI1640 culture medium and pancreatin, adjust thin after being centrifuged Born of the same parents' density is 1x10⁶/ ml, takes 100 μ l (1x10⁵) tumor cell inoculation in U-shaped 96 orifice plates, 3 multiple holes are set；

(2) the chA21-28-ICOS ζ CAR-T cell injuring model expressing anti-HER2 expanded after 14 days, centrifugal remove anti- CD3/CD28 magnetic bead, overnight incubation in without the RPMI1640 culture fluid of IL-2, make T lymphocyte tranquillization；

(3) collect the T lymphocyte of tranquillization, be centrifuged and remove supernatant, cell is resuspended in RPMI1640 culture fluid, meter Number, adjustment cell density is 1x10⁶/ ml, takes 100 μ l (1x10⁵) T cell vaccination inoculated tumour cell in U-shaped 96 orifice plates Corresponding hole in, 96 orifice plates are positioned over 37 DEG C, 5%CO₂Overnight incubation in incubator.Only the hole of T cell is as feminine gender Control wells.

1, the content of IFN-γ in ELISA method detection co-culture media

(1) it is coated: by specification instructs, and Capture antibody is diluted in 1X and is coated in buffer, provides to test kit Respectively adding 0.1ml in each reacting hole in microwell plate, plastics paster covers microwell plate, places 4 DEG C overnight；

(2) close: the automatic ELISA of the microwell plate after overnight is washed after trigger washes paint 4 times, dry, analyze buffering with IX Liquid 100 μ l closes 1 hour；

(3) sample-adding: microwell plate is washed again after painting 4 times, dry, add T cell to be detected and tumor to each reacting hole The supernatant 100 μ l of co-culture of cells, the supernatant of corresponding extension rate and IFN-γ standard substance (1000pg/ml, doubling dilution To 15.6pg/ml), incubated at room 2 hours；

(4) adding detection antibody: after microwell plate washs 4 times, dry, by specification is specified and is divided by Detection antibody 1X Analysis buffer dilution, takes 100 μ l and adds in each reacting hole, incubated at room 1 hour；

(5) add the two of horseradish peroxidase-labeled to resist: microwell plate is washed after painting 4 times, dries, and is used by the antibody of HRP labelling 1X analysis buffer dilutes, and respectively adds 100 μ l, incubated at room 0.5 hour in each reacting hole；

(6), after microwell plate is washed and painted 4 times, dry, in each reacting hole, add freshly prepared tmb substrate solution 100 μ l, keep away Light hatches 15 minutes；

(7) in each reacting hole, add 1M sulphuric acid 100 μ l and terminate reaction；

(8) result judges: on ELISA detector, returns to zero with blank control wells, detects each hole 450nm light absorption value (OD Value), calculate IFN-γ concentration value.Testing result is as shown in Figure 2.

According to Fig. 2, chA21-28-ICOS ζ CAR-T cell can the tumor of all HER2+ of specific identification thin Born of the same parents a large amount of secretion of gamma-IFN, the IFN-γ secreted for breast cancer cell (SKBR3) is up to more than 19000pg/ml, but will When chA21-28-ICOS ζ CAR-T cell and MDA-MB-468 and the TC-1 co-culture of cells that HER2 is negative, on cell culture fluid Very low dose of IFN-γ it is only able to detect in Qing.And NT-T cell and HER2 positive tumor cell is when co-culturing, cell Culture fluid supernatant can't detect IFN-γ.Therefore, result shows: the specific T cell of HER2-can specific identification also Kill the positive tumor cell of HER2.

2, the content of IL-2 in ELISA method detection co-culture media

Detecting step is identical with the step of IFN-γ, and testing result is as shown in Figure 3.

According to Fig. 3, chA21-28-ICOS ζ CAR-T cell can the tumor of all HER2+ of specific identification thin Born of the same parents also secrete IL-2 in a large number, and the IL-2 secreted for breast cancer cell (SKBR3) is up to more than 2800pg/ml.But by chA21- When 28-ICOS ζ CAR-T cell and MDA-MB-468 and the TC-1 co-culture of cells that HER2 is negative, in cell culture supernatant only Very low dose of IL-2 can be detected.And NT-T cell and HER2 positive tumor cell is when co-culturing, on cell culture fluid IL-2 is can't detect in Qing.Therefore, result shows: the specific T cell of HER2-can specific identification and kill HER2 sun The tumor cell of property.

3, the antigenic specificity evaluation test of CAR-T cell

(1) it is coated: be coated 96 holes with 200 μ l 5 μ g/ml HER2-Fc chimeric proteins or CD19-Fc chimeric protein Plate, covers 96 orifice plates with plastics paster, and 4 DEG C overnight；

(2) add the T cell of activation: next day, after PBS washs 3 times, dry, in 96 orifice plates, add 1 × 10⁵Individual ChA21-28-ICOS ζ CAR-T cell or NTT cell, 37 DEG C of overnight incubation；

(3) secretion of euzymelinked immunosorbent assay (ELISA) detection IFN-γ, testing result is as shown in Figure 4.

According to Fig. 4, NT-T cell to HER2-FC chimeric protein and CD19-FC chimeric protein all without specific response, ChA21-28-ICOS ζ CAR-T cell to CD19-Fc chimeric protein also without specific response, but HER2-FC chimeric protein seal In 96 orifice plates closed, chA21-28-ICOS ζ CAR-T cell releases substantial amounts of INF-γ, and up to more than 19000pg/ml.

4、⁵¹Cr release test detection chA21-28-ICOS ζ CAR-T cell killing breast cancer cell ability

(1) results are in the breast cancer cell (SKBR3) of exponential phase, and adjusting cell concentration is 2 × 10⁶/ml；

(2) 1 × 10 is taken⁶Cell (0.5ml), adds Na₂ ⁵¹CrO₄100uCi, hatches 2～3h for 37 degree, and every 15min shakes gently Once；

(3) wash 3 times with 10%FCS RPMI 1640, each 800rpm, 5min.

(4) re-suspended cell concentration 1 × 10⁵/ml；

In (5) the 96 U-shaped culture plates in hole, every hole adds 100 μ l (1x10⁴Cell), if 3 multiple holes；

(6) being the ratio of 1:1,3:1 and 10:1 according to effector T cell and target cell, it is dense that every hole adds 100 μ l difference cells The CAR-T cell of embodiment 1 preparation of degree；Maximum release group is set, adds 100 μ l 1%Triton X-to breast cancer cell 100；Spontaneous release group is set, in breast cancer cell, adds 100 μ l RPMI1640 culture medium；

(7) 200g is centrifuged 1min, puts into 37 DEG C, 5%CO₂Incubator cultivates 4h.

(8) 200g is centrifuged 1min, and 100 μ supernatants are taken out in every hole, with Wizard2gamma counting (PerkinElmer) detection；

(9) calculate: specific killing rate (%)=(experimental group cpm-Spontaneous release cpm)/(maximum release group-naturally release Put cpm), shown in result of calculation Fig. 5.

SKBR3 tumor positive for cracking HER2-according to Fig. 5, chA21-28-ICOS ζ CAR-T cell-specific is thin Born of the same parents, and NTT cell does not all have splitting action for SKBR3 tumor cell positive or negative for HER2.Meanwhile, by NT-T cell With chA21-28-ICOS ζ CAR-T cell respectively with express the SK0V3 co-culture of cells 24 hours of HER2 after, it can be seen that NT-T The growth of SK0V3 cell is not affected by cell, and add in the culture dish of chA21-28-ICOS ζ CAR-T cell it can be seen that The formation of T cell colony, and SKBR3 tumor cell significantly reduces.

5, statistical analysis

All data areRepresent.Utilize GraphPad Prism5.0 (GraphPad Software, Inc., San Diego, California USA) software, T method of inspection analyzes difference and the difference of Specific cell lysis of cytokine secretion Different.P ＜ 0.05 thinks statistically significant.

Shown in sum up, CAR-T cell of the present invention has good fragmentation effect, and CAR-to the tumor cell expressing HER2 When T cell and breast cancer cell (SKBR3) co-culture, the INF-γ of secretion is up to more than 19000pg/ml, IL-2 up to More than 2800pg/ml, far above INF-γ and IL-2 of other third generations CAR-T emiocytosis.

The foregoing is only the preferred embodiments of the present invention, not thereby limit the scope of the claims of the present invention, every at this Under the inventive concept of invention, utilize the equivalent transformation that description of the invention and accompanying drawing content are made, or directly/indirectly it is used in it The technical field that he is correlated with is included in the scope of patent protection of the present invention.

Claims

1. a CAR-T cell, the CAR of described CAR-T cell includes that after birth exoantigen land, hinge region and intracellular signal pass Lead district, it is characterised in that

Described after birth exoantigen land is the chA21 scFv for combining HER2 albumen；

Described intracellular signal transduction district is CD28-X-ITAM, and wherein, described X is ICOS, CD137, CD134, CD80 or CD86 Costimulatory molecules.

2. Chimeric antigen receptor T cell as claimed in claim 1, it is characterised in that described X is ICOS costimulatory molecules.

3. the preparation method of a CAR-T cell, it is characterised in that comprise the following steps that

Building CAR expression vector, wherein, the CAR of described CAR expression vector includes after birth exoantigen land, hinge region and born of the same parents Interior signal conducting region, described after birth exoantigen land is the chA21 scFv for combining HER2 albumen, described intracellular signal Conducting region is CD28-X-ITAM, and described X is ICOS, CD137, CD134, CD80 or CD86 costimulatory molecules；

Pack described CAR expression vector, and prepare and infect mixture；

Described infection mixture is infected T cell, and prepares CAR-T cell.

4. the preparation method of CAR-T cell as claimed in claim 3, it is characterised in that the mistake of described structure CAR expression vector Cheng Zhong, comprises the following steps that

Obtain the genetic fragment of described chA21 scFv, the genetic fragment of described hinge region, the genetic fragment of described CD28, described The genetic fragment of X and the genetic fragment of described ITAM；

Guiding chain is provided, the genetic fragment of guiding chain described in gene fusion and described chA21 scFv, and formed guiding chain- chA21 scFv；

The genetic fragment of hinge region described in gene fusion, the genetic fragment of described CD28, the genetic fragment of described X and described ITAM Genetic fragment, and form hinge region-CD28-X-ITAM；

Guiding chain-chA21 scFv described in gene fusion and described hinge region-CD28-X-ITAM, and form guiding chain-chA21 ScFv-hinge region-CD28-X-ITAM；

Described guiding chain-chA21 scFv-hinge region-CD28-X-ITAM is accessed carrier, and obtains CAR expression vector.

5. the preparation method of CAR-T cell as claimed in claim 4, it is characterised in that obtain the base of described chA21 scFv Because of fragment, the genetic fragment of described hinge region, the genetic fragment of described CD28, the genetic fragment of described X and described ITAM During genetic fragment, comprise the following steps that

Obtain and activate PMBCs；

MRNA the reverse transcription of extracting described PMBCs are cDNA；

The primer of described hinge region, the primer of described CD28, the primer of described X and the primer of described ITAM be provided respectively, and all With the cDNA of described PMBCs as template, carry out PCR amplification respectively, and obtain the genetic fragment of described hinge region, described CD28 Genetic fragment, the genetic fragment of described X and the genetic fragment of described ITAM；

Thering is provided template DNA and the primer of described chA21 scFv of described chA21 scFv, performing PCR of going forward side by side expands, and obtains institute State the genetic fragment of chA21 scFv.

6. the preparation method of CAR-T cell as claimed in claim 3, it is characterised in that described CAR expression vector is CAR table Da virus plasmid.

7. the preparation method of CAR-T cell as claimed in claim 6, it is characterised in that described packaging described CAR expression vector During, comprise the following steps that

Concentrate described supernatant, and prepare and infect mixture.

8. the preparation method of CAR-T cell as claimed in claim 3, it is characterised in that described by described infection mixture sense During dye T cell, comprise the following steps that

Cultivate and expand metainfective T cell, and obtain CAR-T cell.

9. the preparation method of the CAR-T cell as described in claim 3 to 8 any one, it is characterised in that described X is ICOS Costimulatory molecules.

10. a CAR-T cell as claimed in claim 1 or 2 application on the medicine for the treatment of breast carcinoma.