CN107286246A

CN107286246A - Treat BMDC of Chimeric antigen receptor modification of glioma and preparation method thereof

Info

Publication number: CN107286246A
Application number: CN201611232788.4A
Authority: CN
Inventors: 樊克兴; 高同同
Original assignee: When Biological Technology (beijing) Co Ltd
Current assignee: When Biological Technology (beijing) Co Ltd
Priority date: 2016-12-28
Filing date: 2016-12-28
Publication date: 2017-10-24
Anticipated expiration: 2036-12-28
Also published as: CN107286246B

Abstract

The present invention relates to the BMDC that a kind of Chimeric antigen receptor and the Chimeric antigen receptor are modified, and preparation method thereof, application of the BMDC in treatment glioma immunocyte is further related to, the BMDC is preparing the purposes in being used to treat the medicine of glioma.

Description

Treat BMDC and its preparation of the Chimeric antigen receptor modification of glioma Method

Technical field

The invention belongs to biomedicine field, in particular it relates to be used for a kind of Chimeric antigen receptor (Chimeric Antigen Receptor, CAR) and the Chimeric antigen receptor modification BMDC (CAR-DC), and preparation method thereof, And the BMDC is preparing the purposes in being used to treat the medicine of glioma.

Background technology

BMDC is a class and granulocyte, macrophage and lymphocyte form and work(all different leucocytes, its Cell membrane is protruding, forms the film dendrite similar to nerve cell axons, is named as BMDC (dendritic cells, DC).It is body function most strong professional antigen presenting cell, can efficiently be absorbed, at processing Reason and present antigen, DC are combined by the MHC molecule on surface with its tumour antigen for capturing processing, are formed peptide-MHC molecule and are combined Thing, and submission is to T cell, so as to start the reaction of MHC-I classes Restricted CTL and the restricted CD4+Thl reactions of the classes of MHC- II.Together When, DC also provides T cell activation by the costimulatory molecules (CD80/B7-1, CD86/B7-2, CD40 etc.) of its height expression must The secondary signal of palpus, starts immune response (Stumbles PA.Regulation ofT helper cell differentiation by respiratory tractdendritic cells.Immunology and Cell Biology(1999)77,428–433)。

The mononuclearcell that current DC cell therapies use tumor patient itself breeds, cultivated, inducing life in vitro Into DC, then allow DC to load corresponding tumour antigen, the DC of load tumour antigen is prepared into after strict screening, then by these DC cells are fed back in patient's body, the natural antitumor system in human activin, and DC is in this course by the letter of tumour cell Breath passes to immune system, and guide human body itself antitumor immune system to go to recognize, remove tumour cell (Turnis ME, Rooney CM.Enhancement ofdendritic cells as vaccines for cancer.Immunotherapy 2010；2(6):847–62).Clinical research for many years is contrasted and drawn, the generally applicable clinical various oncotherapy of DC cell therapies, especially It is to malignant mela noma, prostate cancer, kidney, carcinoma of urinary bladder, oophoroma, colon and rectum carcinoma, breast cancer, cervical carcinoma, various The therapeutic effects such as lung cancer, laryngocarcinoma, nasopharyngeal carcinoma, cancer of pancreas, liver cancer, stomach cancer preferably (Constantino J, Gomes C, Falcao A,Cruz MT,Neves BM.Antitumor dendritic cell-based vaccines:lessons from 20years of clinical trials and future perspectives.Transl Res2016；168:74–95). But, the simple curative effect obtained by Activated in Vitro DC, load tumour antigen is limited, therefore in DC cellular immunotherapy tumours Method on still need to further improvement.

EGFRvIII is the final commonly-encountered mutant egf R in human tumour, and thin being referred to as glioblastoma multiforme It is especially prevalent in the brain tumor of born of the same parents' knurl.It is reported that EGFRvIII is present in 56% spongioblastoma, 16% non-small cell (Carol J.Wikstrand, Laura P.Hale, et al.Monoclonal in lung cancer and 78% breast cancer Antibodies against EGFRvIII Are Tumor Specific and React with Breast and Lung Carcinomas and Malignant Gliomas.Cancer Res.1995Jul 15；55(14):3140-8), and do not exist The sequence is found in any normal structure.Therefore, EGFRvIII is potential preferable tumor targets.

With the development of tumour immunity theory and clinical technology, Chimeric antigen receptor T cell therapy (Chimeric Antigen receptor T-cell immunotherapy, CAR-T) (June CH, Blazar BR, Riley JL.Engineering lymphocyte subsets:tools,trials and tribulations.NatRev Immunol.2009；9:704-16) turn into one of most promising tumour immunotherapy at present.Chimeric antigen receptor (CAR) by a tumor associated antigen land, extracellular hinge area, trans-membrane region and intracellular signal transduction district's groups into.CAR-T Cell therapy is by foreign gene rotaring dyeing technology, single-chain antibody (the Single chain of identification tumor associated antigen Fragment variable, scFv) and T cell activation sequence expressing fusion protein to T cell surface, make specifically to know The scFv of other tumor associated antigen is coupled by the activation and proliferation signal domain of transmembrane region and T cell intracellular.Express CAR T cell Tumour antigen is combined in the way of antigen is relied on but non-MHC is limited, is started and activation specific killing tumor response.But, CAR-T can produce certain toxicity of missing the target, and influence its cellular immunity curative effect；And importantly, due to the heterogeneity of tumour, Often there is kinds of tumors antigen in same tumour, therefore multiple for the CAR-T offer limited effectiveness of single tumour antigen, and easily Hair.

In view of DC cells are the most important antigen presenting cells of human body, we make DC by the way that CAR structures are transduceed into DC More effectively identification submission tumour antigen, while adding DC activation amplified signals, further promotes DC activation；And it is more important , the CAR-DC of target tumor can be by the effective submission on-effect T cell of a variety of antigens of same tumour, so as to overcome CAR-T single target spot identification lethal effect.In the present invention, we devise a kind of new DC cell therapy tumour strategies, CD40 signal domains Chimeric antigen receptor related to tumour (anti-EGFRvIII scfv) is merged using slow virus system, transduction DC cells, build EGFRvIII CAR-DC.Activation DC is combined with brain glioblastoma cell by the related Chimeric antigen receptor of its tumour Cell, while submission tumour antigen, stimulates T cell activation, so as to play antineoplastic effect.

The content of the invention

In order to overcome the lethal effect of Activated in Vitro DC cells against tumor in the prior art is weaker, specific killing is active to have Defect to be improved, the invention provides following aspect.

Present invention firstly provides a kind of Chimeric antigen receptor (CAR), the Chimeric antigen receptor is by CD8 alpha signals peptide, anti- EGFRvIIIscFv, people CD8 α hinge areas, people CD40 transmembrane region and intracellular signal area are sequentially spliced to form, and are referred to as EGFRvIIICAR。

Further, the amino acid sequence of the single-chain antibody is as shown in SEQ ID NO.1.

Present invention also offers the gene for encoding the Chimeric antigen receptor, its nucleotide sequence such as SEQ ID NO.2 institutes Show.

Present invention also offers expression vector, it is embedding shown in SEQ ID NO.1 comprising simultaneously energy express amino acid sequence Close the encoding gene of antigen receptor.

Further, the expression vector is slow virus carrier pCDH-EGFRvIIICAR.

Present invention also offers a kind of BMDC of engineering, it contains the expression vector.

Further, the expression vector is slow virus carrier pCDH-EGFRvIIICAR.

The purposes in being used to treat the medicine of tumour is being prepared present invention also offers the DC cells of described engineering.

Further, the tumour is selected from：Glioma, non-small cell lung cancer and breast cancer.

Present invention also offers a kind of method for the BMDC for expressing Chimeric antigen receptor, it includes will be described chimeric The expression vector of antigen receptor enters BMDC.

Further, the Chimeric antigen receptor in methods described is the EGFRvIIICAR, it is however preferred to have SEQ ID Amino acid sequence shown in NO.1.

Further, the expression vector in methods described is shown in SEQ ID NO.1 comprising simultaneously energy express amino acid sequence Chimeric antigen receptor encoding gene, preferably slow virus carrier pCDH-EGFRvIIICAR.

Brief description of the drawings

Fig. 1 is Chimeric antigen receptor EGFRvIIICAR used in the present invention structural representation, and it is by CD8 alpha signal peptides (CD8 α SP), EGFRvIII antibody light chains variable region (EGFR vIIIscfv-VL), connector area, EGFRvIII heavy chain of antibody are variable Area (EGFRvIIIscfv-VH), CD8 α hinge areas (CD8 α Hinge), CD40 transmembrane regions (CD40TM) and CD40 intracellular signals area Composition.And its contrast structure is designed, its contrast structure is without CD40 intracellular signals area, abbreviation EGFRvIIICAR '.

Fig. 2 is pCDH-EGFRvIIICAR Lentiviral plasmid hum patterns.

In the case of Fig. 3 is external, EGFRvIIICAR-DC cells of the invention and EGFRvIII⁺Tumour cell U87 (EGFRvIII⁺U87 post activation situation) is co-cultured.A：In vitro culture DC cells；B：Slow virus empty carrier transduction in vitro culture DC Cell；C：Slow virus pCDH-EGFRvIIICAR transduction in vitro culture DC cells；D：Slow virus pCDH-EGFRvIIICAR ' transduces In vitro culture DC cells.As illustrated, EGFRvIIICAR-DC cells are in EGFRvIII⁺Under U87 cytositimulations, realize in vitro The activation of higher degree is ripe.

Fig. 4 is T cell activation situation.EGFRvIIICAR-DC cells are incubated altogether with T cell, in EGFRvIII⁺U87 cells Under stimulation, the activation of T cell is promoted.A：Slow virus pCDH-EGFRvIIICAR transduction in vitro culture DC cells (no T cell)； B：Slow virus pCDH empty carriers transduction in vitro culture DC cells (+T cell)；C：Slow virus pCDH-EGFRvIIICAR transductions are external Cultivate DC cells (+T cell)；D：(+T is thin for slow virus pCDH-EGFRvIIICAR ' transduction in vitro culture DC cells Born of the same parents).

Fig. 5 is small mouse tumor volume change in mouse Brain Glioma Model.A：The external training of slow virus EGFRvIIICAR transductions Support DC cells (no T cell)；B：Slow virus empty carrier transduction in vitro culture DC cells (+T cell)；C：Slow virus EGFRvIIICAR ' transduction in vitro culture DC cells (+T cell)；D：Slow virus EGFRvIIICAR transduction in vitro culture DC cells (+T cell).By Tu Ke get, the mouse tumor of injection EGFRvIIICAR-DC cells+T cell group is significantly suppressed, with when Between extend, gross tumor volume be obviously reduced until disappear.

Fig. 6 is small mouse survivorship curve figure in mouse Brain Glioma Model.A：Slow virus EGFRvIIICAR transduction in vitro cultures DC cells (no T cell)；B：Slow virus empty carrier transduction in vitro culture DC cells (+T cell)；C：Slow virus EGFRvIIICAR ' Transduction in vitro culture DC cells (+T cell)；D：Slow virus EGFRvIIICAR transduction in vitro culture DC cells (+T cell).

Embodiment

The embodiment to the present invention is described in detail below.It should be appreciated that described herein specific Embodiment is merely to illustrate and explain the present invention, and is not intended to limit the invention.

Experimental method in following examples, is this area conventional method unless otherwise specified.Institute in following embodiments

Experiment material, is to be commercially available from routine biochemistry reagent shop unless otherwise specified, wherein：

DMEM culture mediums, the culture mediums of X-VIVO 15 are purchased from TaKaRa companies.

T4DNA ligases are purchased from TaKaRa companies.

Restriction endonuclease EcoR I, Xba I are purchased from Thermo Fisher Scientific companies.

Ago-Gel DNA QIAquick Gel Extraction Kits, common DNA product purification kit, the small extraction reagent kit of plasmid are purchased from day Root biochemical technology Co., Ltd.

PCDH-CMV-MCS-EF1-copGFP-T2A-puro (abbreviation pCDH) and its packaging plasmid pLP1, pLP2, pLP/ VSVG is purchased from Addgene companies.

Trans1-T1Phage Resistant Competents cell is purchased from Beijing Quanshijin Biotechnology Co., Ltd.

LipofectamineTM 2000Transfection Reagent transfection reagents are purchased from Invitrogen companies.

The newborn hamster kidney fibroblast of 293T incasing cells, BHK21 and U87 glioma cells are purchased from U.S. ATCC.

Hyclone is purchased from Gibco companies.

All primers and gene chemical synthesis are synthesized by Shanghai Sheng Gong bio tech ltd.

PEG6000 and NaCl are purchased from Shanghai Suo Laibao bio tech ltd.

Human GM-CSF, IL-4, rhIL-2, CD3/CD28 antibody etc. are purchased from ebioscience companies.

The streaming such as anti-MHC-I, MHC-II, CD80, CD86, CD3, CD25 antibody is purchased from BD companies.

People's DC cell sortings kit, T cell sorting kit are purchased from STEM CELL companies.

NSG mousePurchased from Beijing Weitongda Biotechnology Co., Ltd..

Embodiment 1

First, a kind of determination of EGFRvIIICAR gene orders

1.1 from U.S. national library of medicine website (http://www.ncbi.nlm.nih.gov/entrez) GenBank databases obtain people's CD8 alpha signals peptide gene (SEQ ID NO.3), people's CD8 α hinge areas gene (SEQ ID NO.7), People CD40 transmembrane regions (SEQ ID NO.8) and people's CD40 signals domain gene (SEQ ID NO.9) sequence.By people's CD8 alpha signal peptidyls Because of (SEQ ID NO.3), EGFRvIIIscFv light chain genes (SEQ ID NO.4), connector area (Gly4-Ser)₃(SEQ ID NO.5), EGFRvIIIscFv heavy chain genes (SEQ ID NO.6), people's CD8 α hinge areas gene (SEQ ID NO.7), people CD40 Transmembrane region (SEQ ID NO.8) and people's CD40 signals domain gene (SEQ ID NO.9) sequence are attached, and form final complete EGFRvIIICAR gene orders (SEQ ID NO.2) information.

2nd, the structure of EGFRvIIICAR expression plasmids

2.1 full genomes are synthesized：

Synthesize complete EGFRvIIICAR sequences (SEQ ID NO.2) by Shanghai Sheng Gong bio tech ltd, and Its 5 ' end addition Xba I restriction enzyme site, 3 ' end addition EcoR I restriction enzyme sites.

2.2 expand the EGFRvIIICAR ' areas not comprising someone's CD40 intracellular signals area, 5 ' end addition Xba I by PCR Restriction enzyme site, 3 ' end addition EcoR I restriction enzyme sites.F：GCTCTAGAATGGCCCTGCCTGTG(SEQ ID NO.10)；R： GGAATTCTCAGGTATCAGAAACCCCTGTAG(SEQ ID NO.11).1005bp fragments are reclaimed in amplification.

2.3 are cloned into EGFRvIIICAR and EGFRvIIICAR ' in pCDH Lentivirals respectively, build

PCDH-EGFRvIIICAR and pCDH-EGFRvIIICAR ' plasmids.It is specific as follows：By pCDH carriers, EGFRvIIICAR fragments, EGFRvIIICAR ' fragment Xba I/EcoR I digestions, are separately recovered 8192bp, 1186bp, 1000bp Fragment.Respectively using the pCDH carriers after the connection digestion of T4DNA ligases and EGFRvIIICAR, pCDH carrier with EGFRvIIICAR ', Trans1-T1 competence incubated overnights are converted with connection product, and picking Colony Culture extracts plasmid and surveyed Sequence, obtains pCDH-EGFRvIIICAR and pCDH-EGFRvIII plasmids.EGFRvIIICAR and EGFRvIIICAR ' structures are shown in figure 1.The information of pCDH-EGFRvIIICAR Lentivirals is shown in Fig. 2.EGFRvIIICAR ' gene orders and amino acid sequence See sequence table SEQ ID NO.12 and SEQ ID NO.13.

3rd, the packaging of slow virus, concentration and titer determination

The packaging of 3.1 slow virus：

3.1.1 cell is handled：Transfection collects the 3-10 in exponential phase for first 24 hours for 293T cells, by 293T Cell is inoculated in six porocyte culture plates, and inoculum concentration is 5 × 10^5/ holes, and cell is cultivated in the DMEM containing 2ml 10%FBS Grown in base, be placed in 37 DEG C of 5%CO₂Cell culture incubator culture 18 hours, cell density reaches that 60-80% can be transfected.

3.1.2 cotransfection slow virus carrier plasmid (pCDH or the pCDH-EGFRvIIICAR or pCDH- into 293T cells EGFRvIIICAR ') and its packaging plasmid (pLP1, pLP2, pLP/VSVG)；

3.1.3 24 hours after transfecting, in GFP luciferase expression situations after fluorescence microscopy Microscopic observation 293T cell transfectings.Point Not in 48 hours after transfection, 72 hours collect 293T culture supernatants in, 3000rpm centrifuge 15 minutes, respectively collect cell and on Clearly, multigelation three breakup cell, is collected by centrifugation supernatant；

3.1.4 with 0.45 μm of filter filter virus supernatant, the unloaded precursor virus of pCDH, pCDH- are obtained respectively EGFRvIIICAR ' viruses, pCDH-EGFRvIIICAR viral solutions.

The concentration of 3.2 slow virus and virus titer are determined

3.2.1 cell and culture supernatant are collected respectively, and using PEG6000 concentrating virus, 1/4 body is added into viral solution Long-pending PEG6000/NaCl solution (25%PEG6000+4.4%NaCl), is mixed, and 4 DEG C stand 1 hour；

3.2.24 DEG C, 5000rpm is centrifuged 30 minutes；

3.2.3 supernatant is abandoned, 2ml virolysises liquid (10mM Tris-HCl (pH8.0), 2mM MgCl is added₂, 5% sucrose) Slow virus precipitation is dissolved, and in -80 DEG C of storages.

3.2.4 virus titer is determined.Inoculation BHK21 cells are to 24 orifice plates in advance, and 10^5/ holes are placed in 37 DEG C of 5%CO2 thin Born of the same parents' incubator culture 18 hours；

3.2.5 lytic virus, prepares from 10^-2To 10^-710 times dilution viral samples；

3.2.6 cell culture fluid is removed, the cell culture fluid containing different virus amount is added, and added in nutrient solution liquid 6ug/ml polybrene (polybrene), is placed in 37 DEG C of 5%CO2 cell culture incubators cultures 2 hours；

3.2.7 cell culture fluid is removed, 1%FBS DMEM nutrient solutions is added, is placed in 37 DEG C of 5%CO₂Cell culture incubator is trained Support 48 hours；

3.2.8 cell culture fluid is removed, puromycin containing 2ug/ml, 1%FBS DMEM nutrient solutions is added, is placed in 37 DEG C 5%CO2 cell culture incubators culture 72 hours；

3.2.9 cell culture fluid is removed, violet staining liquid is added, counting is colored clone's number, and calculates virus titer.

2.10 by viral dilution to 10^7TU/ml, in -80 DEG C of storages.

4th, the in vitro culture of BMDC, infection and amplification

4.1 is thin from peripheral blood mononuclear by CD14+ cells with BMDC sorting kit (being purchased from STEM CELL companies) Sorted out in born of the same parents (PBMC).

4.2 are resuspended cell with the culture mediums of X-VIVO 15, add 200U/ml recombinant human granulocyte-macrophage colony thorn Swash the factor (GM-CSF) solution, 500U/ml IL-4 solution and 5% autologous plasma, induce CD14+ cells to BMDC Differentiation.

4.3 are separately added into pCDH or pCDH-EGFRvIIICAR ' or pCDH- into the BMDC of culture 6 days EGFRvIIICAR viral concentrations liquid is mixed, put to viral infection multiplicity MOI=10, and final concentration of 6 μ g/ml polybrene In 37 DEG C of 5%CO₂Cell culture incubator culture, liquid is not changed in 24 hours；Carry out the second subinfection within 24 hours after infection；

96 hours after 4.4 infection, with the expression of the green fluorescence of fluorescence microscope BMDC.

4.5 are collected after cell, and 1000rpm is centrifuged 10 minutes, and PBS is washed 1 time, and cell, which is resuspended, with 200ul PBS is placed in stream In formula pipe, with flow cytomery GFP positive rates.Positive cell is the EGFRvIIICAR- BMDCs of gained.

5th, co culture system in vitro determines the activation and activation T cell effect of CAR-DC cells

5.1 analysis activated dendritic cells

5.1.1 cell is handled：Respectively to EGFRvIIICAR-DC cells and EGFRvIII⁺U87 cells (abbreviation U87vIII) Counted；

5.1.2 by EGFRvIIICAR-DC cells and EGFRvIII⁺U87 cells press 3：1 ratio is mixed, and is placed in 37 DEG C 5% CO₂Cell culture incubator culture 48 hours；

5.1.3 collect after cell, 1000rpm centrifugations 10min, PBS is washed 1 time, with PBS resuspension cells, respectively using anti- People's MHC-I/MHC-II/CD86/CD80 antibody stainings, detect the Activation markers MHC-I/MHC-II/CD86/ of DC cell surfaces CD80 expression.As a result Fig. 3 is seen, the activation degree of EGFRvIIICAR-DC cells is significantly raised.

5.2 analysis t cell activations

5.2.1T cell is separately cultured, and kit (being purchased from STEM CELL companies) is sorted by T cell from outer with T cell Sorted out in all blood monocytes (PBMC)；

5.2.2 cell concentration is adjusted to 1 × 10^6/ml with the culture mediums of RPMI 1640 containing 10%FBS, cell is inoculated with To with the anti-human coated Tissue Culture Flask of CD3 and CD28 antibody, adding 200IU/ml recombinated interleukin-2s in advance (rhIL-2) 37 DEG C of 5%CO, are placed in₂Cell culture incubator culture；

5.2.3 by EGFRvIIICAR-DC cells, EGFRvIII⁺U87 cells and T cell press 3：1：3 ratio mixing, puts In 37 DEG C of 5%CO₂Cell culture incubator culture 48 hours；

5.2.4 collect after cell, 1000rpm is centrifuged 10 minutes, PBS is washed 1 time, cell is resuspended with PBS, respectively using anti- People's CD3/CD25 antibody stainings, the Activation markers CD25 on detection T cell surface expression.As a result Fig. 4 is seen, only EGFRvIIICAR-DC cell effective activation T cells.

6th, the antitumor action of internal CAR-DC cells

The NSG mouse of 20 6-8 week old are being subcutaneously implanted 5x10^5EGFRvIII⁺U87 cells, observation and measurement tumour Growing state, up to tumour growth to 200mm³, mouse is randomly divided into 4 groups, passes through tail vein injection 10^ respectively 6EGFRvIIICAR-DC cells, pCDH empty carriers-DC cells+T cell, pCDH-EGFRvIIICAR '-DC cells+T cell, PCDH-EGFRvIIICAR-DC cells+T cell (T cell number is 10^6/).

CAR-DC cell therapy glioma mouse model effect measurings：After with CAR-DC cell therapies, observation and survey The growth of tumour is measured, and records mouse survival rate.The measurement result of gross tumor volume is referring to Fig. 5, and pCDH-EGFRvIIICAR-DC is thin In the experimental group of born of the same parents+T cell, gross tumor volume increasess slowly, and is gradually reduced after a period of time until disappearing.Survival rate is referring to figure 28 days survival rates are 100% after the tumor-bearing mice treatment of 6, EGFRvIIICAR-DC cells+T cell group, only use DC cell therapies And empty carrier DC groups of cells mouse is all dead, 28 days parts are deposited after EGFRvIIICAR '-DC cells+T cell group mouse treatment Living, tumor growth rate is relative to be slowed down.Confirm that CD40 costimulatory moleculeses, in DC cell-stimulating T cells, start in immune response Key effect.

Sequence table

<110>When power biotechnology（Beijing）Co., Ltd

<120>Treat BMDC of Chimeric antigen receptor modification of glioma and preparation method thereof

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<170> PatentIn version 3.3

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<213>Artificial sequence

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<223>EGFRvIIICAR amino acid sequence

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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Gln Leu Val Gln Ser Gly Ala Glu Val

20 25 30

Lys Lys Pro Gly Glu Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Phe

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Asn Ile Glu Asp Tyr Tyr Ile His Trp Val Arg Gln Met Pro Gly Lys

50 55 60

Gly Leu Glu Trp Met Gly Arg Ile Asp Pro Glu Asn Asp Glu Thr Lys

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Tyr Gly Pro Ile Phe Gln Gly His Val Thr Ile Ser Ala Asp Thr Ser

85 90 95

Ile Asn Thr Val Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr

100 105 110

Ala Met Tyr Tyr Cys Ala Phe Arg Gly Gly Val Tyr Trp Gly Gln Gly

115 120 125

Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser Pro Asp Ser

145 150 155 160

Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser

165 170 175

Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln

180 185 190

Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu Ile Ser Leu Val Ser Lys

195 200 205

Leu Asp Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

210 215 220

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val

225 230 235 240

Tyr Tyr Cys Trp Gln Gly Thr His Phe Pro Gly Thr Phe Gly Gly Gly

245 250 255

Thr Lys Val Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

260 265 270

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

275 280 285

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

290 295 300

Ala Cys Asp Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys

305 310 315 320

Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val

325 330 335

Gly Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp Thr

340 345 350

Arg Ser Pro Gly Ser Ala Glu Ser Pro Gly Gly Asp Pro His His His

355 360 365

Leu Arg Asp Pro Val Cys His Pro Leu Gly Ala Gly Leu Ser Gln Lys

370 375 380

Gly Gly Gln Glu Ala Asn Gln

385 390

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atggccctgc ctgtgacagc cctgctgctg cctctggctc tgctgctgca tgccgctaga 60

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tatctgcaat ggtcctcatt gaaagcctca gataccgcga tgtactactg tgctttcaga 360

ggaggggtct actggggaca gggaactacc gtgactgtct cgtccggcgg aggcgggtca 420

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<223>The nucleotide sequence of EGFRvIIIscFv light chains

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ccggggaagg gactcgaatg gatgggacgg atcgacccag aaaacgacga aactaagtac 180

ggtccgatct tccaaggcca tgtgactatt agcgccgata cttcaatcaa taccgtgtat 240

ctgcaatggt cctcattgaa agcctcagat accgcgatgt actactgtgc tttcagagga 300

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ctgcagcaaa agcctggcca gccaccgaag cgccttatct cactggtgtc gaagctggat 180

tcgggagtgc ccgatcgctt ctccggctcg ggatcgggta ctgacttcac cctcactatc 240

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<213>Artificial sequence

<220>

<223>The nucleotide sequence of people's CD8 α hinge areas

<400> 7

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 8

<211> 66

<212> DNA

<213>Artificial sequence

<220>

<223>People's CD40 transmembrane regions

<400> 8

gtcctgcacc gctcatgctc gcccggcttt ggggtcaagc agattgctac aggggtttct 60

gatacc 66

<210> 9

<211> 186

<212> DNA

<213>Artificial sequence

<220>

<223>The nucleotide sequence in people's CD40 intracellular signals area

<400> 9

atctgcgagc cctgcccagt cggcttctcc aatgtgtcat ctgctttcga aaaatgtcac 60

ccttggacaa ggtccccagg atcggctgag agccctggtg gtgatcccca tcatcatctt 120

cgggatcctg tttgccatcc tcttggtgct ggtctttctc aaaaaggtgg ccaagaagcc 180

aaccaa 186

<210> 10

<211> 23

<212> DNA

<213>Artificial sequence

<220>

<223>PCR it is positive because

<400> 10

gctctagaat ggccctgcct gtg 23

<210> 11

<211> 30

<212> DNA

<213>Artificial sequence

<220>

<223>PCR reverse primers

<400> 11

ggaattctca ggtatcagaa acccctgtag 30

<210> 12

<211> 987

<212> DNA

<213>Artificial sequence

<220>

<223>EGFRvIIICAR ' nucleotide sequence

<400> 12

atggccctgc ctgtgacagc cctgctgctg cctctggctc tgctgctgca tgccgctaga 60

cccgagattc agctcgtgca atcgggagcg gaagtcaaga agccaggaga gtccttgcgg 120

atctcatgca agggtagcgg ctttaacatc gaggattact acatccactg ggtgaggcag 180

atgccgggga agggactcga atggatggga cggatcgacc cagaaaacga cgaaactaag 240

tacggtccga tcttccaagg ccatgtgact attagcgccg atacttcaat caataccgtg 300

tatctgcaat ggtcctcatt gaaagcctca gataccgcga tgtactactg tgctttcaga 360

ggaggggtct actggggaca gggaactacc gtgactgtct cgtccggcgg aggcgggtca 420

ggaggtggcg gcagcggagg aggagggtcc gacgtcgtga tgacccagag ccctgacagc 480

ctggcagtga gcctgggcga aagagctacc attaactgca aatcgtcgca gagcctgctg 540

gactcggacg gaaaaacgta cctcaattgg ctgcagcaaa agcctggcca gccaccgaag 600

cgccttatct cactggtgtc gaagctggat tcgggagtgc ccgatcgctt ctccggctcg 660

ggatcgggta ctgacttcac cctcactatc tcctcgcttc aagcagagga cgtggccgtc 720

tactactgct ggcagggaac ccactttccg ggaaccttcg gcggagggac gaaagtggag 780

atcaagacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 840

cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 900

gggctggact tcgcctgtga tgtcctgcac cgctcatgct cgcccggctt tggggtcaag 960

cagattgcta caggggtttc tgatacc 987

<210> 13

<211> 329

<212> PRT

<213>Artificial sequence

<220>

<223>EGFRvIIICAR ' amino acid sequence

<400> 13

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Gln Leu Val Gln Ser Gly Ala Glu Val

20 25 30

Lys Lys Pro Gly Glu Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Phe

35 40 45

Asn Ile Glu Asp Tyr Tyr Ile His Trp Val Arg Gln Met Pro Gly Lys

50 55 60

Gly Leu Glu Trp Met Gly Arg Ile Asp Pro Glu Asn Asp Glu Thr Lys

65 70 75 80

Tyr Gly Pro Ile Phe Gln Gly His Val Thr Ile Ser Ala Asp Thr Ser

85 90 95

Ile Asn Thr Val Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr

100 105 110

Ala Met Tyr Tyr Cys Ala Phe Arg Gly Gly Val Tyr Trp Gly Gln Gly

115 120 125

Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser Pro Asp Ser

145 150 155 160

Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser

165 170 175

Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln

180 185 190

Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu Ile Ser Leu Val Ser Lys

195 200 205

Leu Asp Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

210 215 220

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val

225 230 235 240

Tyr Tyr Cys Trp Gln Gly Thr His Phe Pro Gly Thr Phe Gly Gly Gly

245 250 255

Thr Lys Val Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

260 265 270

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

275 280 285

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

290 295 300

Ala Cys Asp Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys

305 310 315 320

Gln Ile Ala Thr Gly Val Ser Asp Thr

325

Claims

1. a kind of Chimeric antigen receptor, it is characterised in that the Chimeric antigen receptor is by CD8 alpha signals peptide, anti-EGFRvIII list Chain antibody (EGFRvIIIscFv), people CD8 α hinge areas, people CD40 transmembrane region and intracellular signal area are sequentially spliced to form, and are referred to as EGFRvIIICAR。

2. Chimeric antigen receptor according to claim 1, wherein, the amino acid sequence such as SEQ of the Chimeric antigen receptor Shown in ID NO.1.

3. encoding the gene of the Chimeric antigen receptor described in claim 2, its nucleotide sequence is as shown in SEQ ID NO.2.

4. expression vector, it contains and energy express amino acid sequence is the volume of the Chimeric antigen receptor shown in SEQ ID NO.1 Code gene.

5. expression vector according to claim 4, wherein, the expression vector is Lentiviral

pCDH-EGFRvIIICAR。

6. a kind of BMDC of engineering, it contains the expression vector described in claim 4.

7. the DC cells of the engineering described in claim 6 are preparing the purposes in being used to treat the medicine of tumour.

8. the purposes described in claim 7, wherein the tumour is selected from：Glioma, non-small cell lung cancer and breast cancer.

9. prepare the method for the BMDC of expression Chimeric antigen receptor, it is characterised in that by the table of the Chimeric antigen receptor Enter BMDC up to carrier.

10. the method described in claim 8, wherein the Chimeric antigen receptor be chimeric antigen described in claim 1 or 2 by Body.

11. the method described in claim 9, wherein the expression vector is the expression vector described in claim 4 or 5.