CN105949316B - Anti- EGFRvIII Chimeric antigen receptor, encoding gene, recombinant expression carrier and its construction method and application - Google Patents

Abstract

The invention discloses a kind of anti-EGFRvIII Chimeric antigen receptor, encoding gene, recombinant expression carrier and its construction method and applications.The anti-EGFRvIII Chimeric antigen receptor includes the CD8 leader Chimerical receptor signal peptide being sequentially connected in series, EGFRvIII single-chain antibody light chain VL, Optimal Linker C, EGFRvIII single-chain antibody light and weight chain VH, CD8 Hinge Chimerical receptor hinge, CD8 Transmembrane Chimerical receptor transmembrane region, CD137 Chimerical receptor costimulating factor and TCR Chimerical receptor t cell activation domain.In addition, also disclosing encoding gene, recombinant expression carrier and its construction method and the application of the anti-EGFRvIII Chimeric antigen receptor.The method can significantly improve the secretions of cell factor, the killing effect in vitro of CAR-T cell, and clinical therapeutic efficacy is prominent.

Description

Anti- EGFRvIII Chimeric antigen receptor, encoding gene, recombinant expression carrier and its building Methods and applications

Technical field

The invention belongs to immunotherapy of tumors technical fields, and in particular to a kind of anti-EGFRvIII Chimeric antigen receptor is compiled Code gene, recombinant expression carrier (especially a kind of CAR-T transgene carrier based on replication defective recombinant slow virus) and its Construction method and application.

Background technique

The theoretical basis of immunotherapy of tumors is that immune system has identification tumor associated antigen, regulation body attack tumour The ability of cell (cell dissolution of high degree of specificity).This bioprocess is sufficiently complex, at present still in research among.Previous generation It records the nineties, multiple computer MSR Information systems have discovered that tumour antigen (t μm of or antigens), and T lymphocyte can be by main Histocompatibility complex (major histocompatibility complex, MHC) dependence mode identifies these tumours Antigen.

Immunotherapy of tumors is generally divided into two classes, nospecific immunity and specific immunity.Nonspecific immunotherapy for bronchus master It to include interleukin 2 (interle μ kin-2, IL-2), interferon-' alpha ' (interferon α, IFN-α), tumor necrosis factor Sub (t μm of or necrosis factor, TNF-α), the cell factors such as BCG vaccine and toxin, adoptive cellular immunotherapy etc.. Specific active immunotherapy is mainly tumor vaccine.

1.1 tumour Nonspecific immunotherapy for bronchus

Nonspecific immune response be it is inherent, its formation does not need antigenic stimulus, can be widely for more Kind antigen, is the basis of immune response, but specificity is not strong, tends not to generate sufficient intensity to certain specific antigen substance React nonspecific immunization therapy.In the cytokine profiles for entering clinical test, interleukin 2 and interferon are answered With the most extensively [Rosenberg S A, Lotze M T, M μ μ l L M, et al.A progressreport on the treatment of 157patients with advanced cancerμsing lymphokine-activated Killer cells and interle μ kin-2orhigh-dose interle μ kin-2alone [J] .N Engl J Med, 1987,316 (15): 889-897.].

The immunization therapy of 1.2 anti-cancer monoclonal antibodies

Monoclonal antibody is used widely in therapeutic field of tumor over nearly more than 20 years.Antitumor monoclonal antibody medicine generally wraps Include two classes: first is that antitumor monoclonal antibody, second is that antitumor monoclonal antibody conjugate or immune conjugate.Immune conjugate molecule is by list Anti- to form with " bullet " drug two parts, " bullet " mainly includes radionuclide, drug and toxin, is distinguished after connecting with monoclonal antibody Constitute radio-immunity conjugate, chemo-immunity conjugate and immunotoxin.In in November, 1997 and U.S. FDA in October, 1998 point Two monoclonal antibodies for clinical cancer therapy-Rit μ ximab (rit μ xan) and Trast μ z μm ab is not passed through (herceptin)[Dillman R O.Magic bμllets at last！Finally—approval of Amonoclonal antibody for the treatment of cancer [J] .CancerBiother Radiopharm, 1997,12:223-225.].It is now recognized that the mechanism of action of monoclonal antibody has blocking effect, signal transduction effect and targeting Deng three kinds of mechanism of action, without direct lethal effect.The problem of additionally there are in terms of pharmacology, mainly reaches tumour Dose is insufficient.Since conjugate is foreign protein, can be absorbed by reticuloendothelial system, have it is a great deal of will accumulate in liver, spleen and Marrow.Conjugate is macromolecular substances, by capillary endothelium layer and penetrates tumour cell external series gap and is restricted.

The adoptive immunotherapy of 1.3 tumours

The adoptive immunotherapy of tumour, which refers to, is transfused to patient for the self or alloimmune effector cell of Activation In Vitro, with Kill the tumour cell of patient's body.A critical issue in tumour adoptive immunotherapy is to find suitable tumor-killing Cell.Since the eighties in last century, including LAK, cell because induction killing cell (cytokine-ind μ cedkillers, CIK), the cells such as TIL are successively applied to clinic, but since there is expanding, speed is lower, cell origin is difficult, cytotoxicity Not high problems, are restricted in clinical application.The specific for tumour antigen for how improving T cell has important face Bed meaning.T cell mainly passes through T cell receptor (T cell receptor, TCR) identification tumour to the identification of tumour antigen Human leukocyte antigen (h μm of an le μ kocyte antigen, HLA)-peptide complexes of cell surface, therefore, T cell is to swollen The specificity of tumor antigen identification depends on the TCR on T cell surface.It is thin using the means clonal tumours specificity T of molecular biology The TCR of born of the same parents, and by viral vectors of the building containing TCR, TCR is transferred in normal T cell, keeps these T cells swollen because carrying Tumor is specific and becomes specific tumor killing cell [Johnson L A, Morgan R A, D μ dley M E, et al.Gene therapywith hμman and moμse T-cell receptors mediates cancerregression and Targets normal tiss μ es expressing cognate antigen [J] .Blood, 2009,114 (3): 535- 546.]。

1.4 tumor vaccine therapy

Tumor vaccine therapy is to excite the specificity antineoplastic immunity of patient by importing tumour antigen to patient's body Reaction.Due to vaccine therapy have many advantages, such as specificity, in vivo immunological effect hold time it is long, have become at present research heat Point.In recent years polypeptide vaccine, nucleic acid vaccine, holoprotein vaccine, anti-unique antibody vaccine, recombinant viral vaccine, bacterial vaccine, Tumor cell vaccine, Dendritic Cells (dendriticcells, DC) vaccine of gene modification etc. are widely studied and apply [Robbins P F, Morgan R A, Feldman S A, et al.T μm or regressionin patients with metastatic synovial cell sarcoma andmelanomaμsing genetically engineered Lymphocytes reactivewith NY-ESO-1 [J] .J Clin Oncol, 2011,29 (7): 917-924.].Tumour The large-scale application of vaccine therapy is urgently to be resolved the problem of there are three aspects.It is each tumour, every firstly, tumor associated antigen A hypotype, each neoplasm staging, these are different with respect to antigen presentation, so making an accurate selection of antigen, the patient crowd that makes an accurate selection of is to cause It closes important.Second, how to reach tumour antigen efficient absorption and expression in Dendritic Cells? antigen is inhaled by Dendritic Cells Receipts are with surface receptor for mediation.Dendritic Cells has more than ten of receptor, how according to the selection of specific antigen accordingly by Body? third, for the regulation of differentiation of dendritic cells maturation.The differentiation and maturation of Dendritic Cells is an extremely complex mistake Journey, it, which can both move towards activation T cell, can also move towards to inhibit T cell.[targeting DC cell therapeutic type tumor vaccine: bright spot with It challenges and deposits.

http://www.biodiscover.com/news/research/115794.html]

1.5 tumour CAR-T treatment

CAR-T, full name are Chimeric Antigen Receptor T-Cell Imm μ notherapy, chimeric antigen by Body T cell immunotherapy, structure [Eleanor J.Cheadle, et al.CAR T cells:driving the as shown in Figure 1 road fromthe laboratory to the clinic.Immμnological Reviews 2014.Vol.257:91– 106]。

The t cell activation that first generation CAR is mediated is completed by the Tyrosine Activating Motifs on CD3z chain or FceRIg. Letter needed for CD3z chain is capable of providing t cell activation, cracking target cell, adjusts IL-2 secretion and play anti-tumor activity in vivo Number.But the anti-tumor activity of first generation CAR transformation T cell is restricted in vivo, and it is thin that T cell proliferation reduction eventually leads to T The apoptosis of born of the same parents.

Second generation CAR increases a new costimulatory signal intracellular, it is demonstrated experimentally that this original to make to be originated from " signal 1 " of TCR/CD3 complex expands, and many researchs all show the second generation CAR and first generation CAR that are equipped with " signal 2 " It compares, antigentic specificity is constant, and T cell proliferation, cytokine secretion increase, and Anti-apoptotic proteins secretion increases, and cell is dead Die delay.Common costimulatory molecules are CD28, but have research to be replaced CD28 with CD137 (4-1BB) later, except this it Outside, a kind of thinking using NK cell receptor CD244 is also suggested.Although which is better and which is worse actually by different second generation CAR, no Same researcher is not quite similar with result obtained in external research in vivo with different tumours.[depth full version: CAR- The status and future biology paddy .2015-051-15 of T]

In order to further improve the design of CAR, it not only includes " letter that many study groups, which start to be conceived to, which develops third generation CAR, Number 1 ", " signal 2 ", further comprise additional costimulatory signal.Different researchers are opened with different target spot and costimulatory signal The having a certain difference property of comparison result of the obtained second generation CAR and third generation CAR of research of exhibition.Table is reported in some researchs Recombination T cell up to third generation CAR is significantly increased in terms of anti-tumor activity, time to live and cytokine release； Second generation CAR and the third generation CAR recombination T cell of the result of study display targeting M Μ C1 of Wilkie etc. is in antitumor cell poison Property aspect and no significant difference, although expression third generation CAR T cell can secrete a greater amount of IFN-γ (Wilkie S, Picco G,Foster J,et al.Retargeting of hμman T cells to tμmorassociated MΜC1: the evolμtion of a chimeric antigen receptor.J Immμnol 2008；180:4901–4909.). It is worth noting that, above-mentioned difference is only the conclusion obtained in experiment in vitro, not yet compare the second generation and in vivo at present The report of three generations CAR.

Difference between this several generations CAR may be incessantly from signal transduction domain, extracellular antigen binding domain (scFv), again The transfection method (slow virus VS retrovirus) of group T cell, feedback mode (the venous re-transfusion VS peritonaeum VS tumor for recombinating T cell Body) etc. may influence the final antitumous effect of CAR-T cell.

Summary of the invention

It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of anti-EGFRvIII Chimeric antigen receptor, Encoding gene, recombinant expression carrier and its construction method and application.

The purpose of the present invention is achieved through the following technical solutions:

The first object of the present invention is to provide a kind of anti-EGFRvIII Chimeric antigen receptor (two generation CAR), including successively The concatenated CD8leader Chimerical receptor signal peptide as shown in SEQ ID NO.15, as shown in SEQ ID NO.16 EGFRvIII single-chain antibody light chain VL, Optimal Linker C as shown in SEQ ID NO.19, such as SEQ ID NO.20 institute EGFRvIII single-chain antibody heavy chain VH, CD8Hinge Chimerical receptor hinge, such as the SEQ ID as shown in SEQ ID NO.21 shown CD8Transmembrane Chimerical receptor transmembrane region, the CD137 Chimerical receptor as shown in SEQ ID NO.23 shown in NO.22 are total Stimulating factor, and the TCR Chimerical receptor t cell activation domain as shown in SEQ ID NO.24.

Further, the amino acid sequence of the Chimeric antigen receptor is as shown in SEQ ID NO.50.

A kind of anti-EGFRvIII Chimeric antigen receptor (three generations CAR), including being sequentially connected in series as shown in SEQ ID NO.15 CD8leader Chimerical receptor signal peptide, EGFRvIII single-chain antibody light chain VL, such as SEQ ID as shown in SEQ ID NO.16 Optimal Linker C shown in NO.19, EGFRvIII single-chain antibody heavy chain VH, such as SEQ as shown in SEQ ID NO.20 CD8Hinge Chimerical receptor hinge, the CD8Transmembrane as shown in SEQ ID NO.22 shown in ID NO.21 it is chimeric by Body transmembrane region, CD28 Chimerical receptor costimulating factor, the CD137 as shown in SEQ ID NO.23 as shown in SEQ ID NO.25 Chimerical receptor costimulating factor and the TCR Chimerical receptor t cell activation domain as shown in SEQ ID NO.24.

Further, the amino acid sequence of the Chimeric antigen receptor is as shown in SEQ ID NO.51.

The second object of the present invention is to provide a kind of gene for encoding above-mentioned anti-EGFRvIII Chimeric antigen receptor.

Further, the nucleotide sequence of the gene such as SEQ ID NO.52 (two generation CAR) or SEQ ID NO.53 (three For CAR) shown in.

The third object of the present invention is to provide the recombinant expression carrier containing said gene.

Further, the recombinant expression carrier is Lentiviral, retrovirus expression vector, adenovirus table Up to carrier, glandular associated virus expression vector or plasmid.

Further, the Lentiviral includes the gene of above-mentioned anti-EGFRvIII Chimeric antigen receptor.

Further, the Lentiviral includes: the prokaryotic replions pUC Ori sequence for plasmid replication Column, as shown in SEQ ID NO.2；For the sequence of AmpR containing ampicillin resistance gene of purpose bacterial strain massive amplification, such as SEQ Shown in ID NO.1；For enhancing the Viral Replicon SV40Ori sequence of the duplication in eukaryocyte, such as SEQ ID NO.3 institute Show；Slow virus for slow virus packaging packs cis element；ZsGreen1 green for eukaryotic cell expression green fluorescence Fluorescin, as shown in SEQ ID NO.11；For the IRES ribosome binding sequence of common transcriptional expression protein, such as SEQ Shown in ID NO.12；People's EF1 α promoter of eukaryotic transcription for Chimeric antigen receptor gene, as shown in SEQ ID NO.14； For form integrate identification, transmitting, two generation CAR of starting or three generations CAR Chimeric antigen receptor encoding gene, such as Shown in SEQ ID NO.52 or SEQ ID NO.53；The enhanced marmot second of eWPRE for enhancing the expression efficiency of transgenosis Hepatovirus posttranscriptional regulatory element, as shown in SEQ ID NO.13.

Further, the slow virus packaging cis element includes: such as SEQ ID NO.5 using second generation slow virus carrier Shown in slow virus 5terminal LTR, the slow virus 3terminal Self- as shown in SEQ ID NO.6 Inactivating LTR, the Gag cis element as shown in SEQ ID NO.7, the cis- member of RRE as shown in SEQ ID NO.8 Part, the env cis element as shown in SEQ ID NO.9, the cPPT cis element as shown in SEQ ID NO.10.

Further, the slow virus packaging cis element includes: such as SEQ ID NO.5 using third generation slow virus carrier Shown in slow virus 5terminal LTR, the slow virus 3terminal Self- as shown in SEQ ID NO.6 Inactivating LTR, the Gag cis element as shown in SEQ ID NO.7, the cis- member of RRE as shown in SEQ ID NO.8 Part, the env cis element as shown in SEQ ID NO.9, slow virus as described in the cPPT cis element shown in SEQ ID NO.10 Pack cis element, and the RSV promoter as shown in SEQ ID NO.4.

Further, the enhanced marmot hepatitis B posttranscriptional regulatory element of the eWPRE has the enhancing of 6 nucleotide Mutation, specifically: g.396G > A, g.397C > T, g.398T > C, g.399G > A, g.400A > T, g.411A > T.

The fourth object of the present invention is to provide a kind of construction method of above-mentioned Lentiviral, including following step It is rapid:

(1) ampicillin resistance gene AmpR sequence (as shown in SEQ ID NO.1), prokaryotic replions pUC will be contained Ori sequence (as shown in SEQ ID NO.2), Viral Replicon SV40Ori sequence (as shown in SEQ ID NO.3) are used for slow disease The slow virus of poison packaging packs cis element, ZsGreen1 green fluorescent protein (as shown in SEQ ID NO.11), IRES ribose Body binding sequence (as shown in SEQ ID NO.12), enhanced marmot hepatitis B posttranscriptional regulatory element (such as SEQ of eWPRE Shown in ID NO.13) it is stored on slow virus skeleton plasmid；

(2) integrate identification, transmitting, starting by people EF1 α promoter (as shown in SEQ ID NO.14), for forming Two generation CAR or the Chimeric antigen receptor of three generations CAR be combined into two generation CAR or three generations's CAR design scheme, by digestion, connection, Recombining reaction is cloned into slow virus skeleton plasmid, obtains the recombinant slow virus plasmid of two generation CAR or three generations CAR design；

(3) by obtained recombinant slow virus plasmid and slow virus packaging plasmid pPac-GP, pPac-R and memebrane protein plasmid PEnv-G transfects HEK293T/17 cell jointly, after carrying out gene transcript expression in HEK293T/17 cell, packs and successfully weighs Group slow virus carrier can be discharged into cells and supernatant, collect the supernatant for the recombined lentivirus vector for including；

(4) obtained recombinant slow virus supernatant is purified using the ion-exchange method for filtering, adsorbing, eluting, point Recombined lentivirus vector is not obtained.

Further, in step (1), slow virus packaging cis element using second generation slow virus carrier include: as Slow virus 5terminal LTR shown in SEQ ID NO.5, the slow virus 3terminal Self- as shown in SEQ ID NO.6 Inactivating LTR, the Gag cis element as shown in SEQ ID NO.7, the cis- member of RRE as shown in SEQ ID NO.8 Part, the env cis element as shown in SEQ ID NO.9, the cPPT cis element as shown in SEQ ID NO.10；The slow virus Packaging cis element using third generation slow virus carrier include: slow virus 5terminal LTR as shown in SEQ ID NO.5, Slow virus 3terminal Self-Inactivating LTR as shown in SEQ ID NO.6, as shown in SEQ ID NO.7 Gag cis element, the RRE cis element as shown in SEQ ID NO.8, the env cis element as shown in SEQ ID NO.9, such as Slow virus described in cPPT cis element shown in SEQ ID NO.10 packs cis element, and as shown in SEQ ID NO.4 RSV promoter.

Further, described for forming the chimeric of the two generation CAR for integrating identification, transmitting, starting in step (2) Antigen receptor includes: the CD8leader Chimerical receptor signal peptide as shown in SEQ ID NO.15, as shown in SEQ ID NO.16 EGFRvIII single-chain antibody light chain VL, Optimal Linker C as shown in SEQ ID NO.19, such as SEQ ID NO.20 institute EGFRvIII single-chain antibody heavy chain VH, CD8Hinge Chimerical receptor hinge, such as the SEQ ID as shown in SEQ ID NO.21 shown CD8Transmembrane Chimerical receptor transmembrane region, the CD137 Chimerical receptor as shown in SEQ ID NO.23 shown in NO.22 are total Stimulating factor, the TCR Chimerical receptor t cell activation domain as shown in SEQ ID NO.24；It is described for form collection identification, transmitting, Start in one three generations CAR Chimeric antigen receptor include: the CD8leader Chimerical receptor as shown in SEQ ID NO.15 letter Number peptide, EGFRvIII single-chain antibody light chain VL, the Optimal as shown in SEQ ID NO.19 as shown in SEQ ID NO.16 Linker C, the EGFRvIII single-chain antibody heavy chain VH as shown in SEQ ID NO.20, as shown in SEQ ID NO.21 CD8Hinge Chimerical receptor hinge, CD8Transmembrane Chimerical receptor transmembrane region, such as SEQ as shown in SEQ ID NO.22 CD28 Chimerical receptor costimulating factor, the CD137 Chimerical receptor costimulation as shown in SEQ ID NO.23 shown in ID NO.25 The factor and the TCR Chimerical receptor t cell activation domain as shown in SEQ ID NO.24.

Further, in step (1), the enhanced marmot hepatitis B posttranscriptional regulatory element of eWPRE has 6 cores The enhancing of thuja acid is mutated, specifically: g.396G > A, g.397C > T, g.398T > C, g.399G > A, g.400A > T, g.411A > T.

Further, in step (2), entire CAR gene expression is started by people's EF1 α promoter；CD8leader it is chimeric by Body signal peptide is located at the N-terminal of CAR coded sequence, for guiding CAR albumen to be positioned at cell membrane；EGFRvIII single-chain antibody light chain VL, Optimal Linker C, EGFRvIII single-chain antibody heavy chain VH are combined into the region scfv, and EGFRvIII is anti-for identification It is former；CD8Hinge Chimerical receptor hinge is used to for scfv being anchored on the outside of cell membrane；CD8Transmembrane Chimerical receptor across Film area is used to entire Chimerical receptor being fixed on cell membrane；CD137 Chimerical receptor costimulating factor is for stimulating T cell to be proliferated And cytokine secretion；The Chimerical receptor t cell activation domain TCR is used to activate the expression of downstream signaling pathway；When the region scfv with When EGFRvIII antigen binding, signal is transferred into the cell by Chimerical receptor, thus generate include T cell proliferation, cell because Son secretes increase, Anti-apoptotic proteins secretion increases, cell death delay, a series of biological effects for cracking target cell.

Further, in step (4), the slow virus carrier has the version with fluorescence labels zsGreen1 and without fluorescence Label zsGreen1 version, the version with fluorescence labels are used for experiment in vitro, are used for clinical trial without the version of fluorescence labels.

Further, in step (4), the suction filtration step control supernatant volume is in 200ml~2000ml, vacuum degree control In -0.5MPA~-0.9MPA, prevent due to plug-hole bring carrier loss；The pH value of adsorption step control solution 6~ 8, prevent the variation of PH from carrier being caused to inactivate；The ionic strength of the elution step control eluent is prevented in 0.5M~1.0M The variation of ionic strength causes elution not exclusively or carrier inactivation.

Expression vector of the present invention includes prokaryotic replions (pUC ori) for plasmid replication；Protokaryon screening mark Remember that (AmpR) is used for purpose bacterial strain massive amplification；Viral Replicon (SV40Ori) is used to enhance the duplication in eukaryocyte；Slow disease Poison packaging cis element (RSV, 5terminal LTR, 3terminal Self-Inactivating LTR, Gag, RRE, env, CPPT it) is packed for slow virus；Eukaryon fluorescence labels albumen (ZsGreen1) is used for eukaryotic cell expression green fluorescence；Coexpression Element (IRES) is used for common transcriptional expression protein；Eukaryon of the eukaryotic promoter (EF1 α) for Chimeric antigen receptor gene turns Record；Chimeric antigen receptor (CD8leader, EGFRvIII VL, Common Linker A (SEQ ID NO.17)/Common Linker B(SEQ ID NO.18)/Optimal Linker C(SEQ ID NO.19)、EGFRvIII VH、CD8Hinge、 CD8Transmembrane, CD137, TCR) it is used to form to integrate identification, transmitting, two generations of starting and three generations CAR；Transcription Controlling element (eWPRE) is used to enhance the expression efficiency of transgenosis afterwards.

The fifth object of the present invention is to provide a kind of CAR-T cell, and the CAR-T cell is by above-mentioned anti- The T lymphocyte of EGFRvIII Chimeric antigen receptor modification.

A further object of the present invention is to provide above-mentioned CAR-T cell answering in preparation glioblastoma therapeutic agent With.

The present invention relates to the medical configuration product containing peptide, and in particular to:

One, the sequence of AmpR containing ampicillin resistance gene, prokaryotic replions p Μ C Ori sequence, Viral Replicon SV40Ori sequence, RSV promoter, people EF1 α promoter, slow virus 5terminal LTR, slow virus 3terminal Self- Inactivating LTR, Gag cis element, RRE cis element, env cis element, cPPT cis element, IRES ribosomes The recombinant slow virus load of binding sequence, ZsGreen1 green fluorescent protein, WPRE marmot hepatitis B posttranscriptional regulatory element Body skeleton, this recombined lentivirus vector skeleton can carry different therapeutic genes and be widely used in adoptive cell and control Treatment field.

Two, recombined lentivirus vector skeleton, CD8leader Chimerical receptor signal peptide, EGFRvIII single-chain antibody light chain VL, Single-chain antibody hinge LinkerA, single-chain antibody hinge Linker B, single-chain antibody hinge Linker C, EGFRvIII are single-stranded anti- Weight chain VH, CD8Hinge Chimerical receptor hinge, CD8Transmembrane Chimerical receptor transmembrane region, CD28 Chimerical receptor pierce altogether The sharp factor, CD137 Chimerical receptor costimulating factor, TCR Chimerical receptor t cell activation domain construct to form recombined lentivirus vector, The recombined lentivirus vector that this method obtains may be implemented to express EGFRvIII Chimeric antigen receptor on human T lymphocyte, draw It leads and activated T lymphocytes is to the lethal effect of EGFRvIII positive cell, be used clinically for treatment glioblastoma.

CAR-T technology of the present invention for EGFRvIII is a kind of to combine exempting from for anti-cancer monoclonal antibody The targeted therapy new technology of the adoptive immunotherapy advantage of epidemic disease treatment and tumour.EGFRvIII is the mutant of epidermal growth factor Three, it is the most common EGFR mutant in human tumor.Since reading frame has lacked exon 2 to exon 7 to frame, cause to produce The transcript mutant that a kind of exons 1 and exon 8 adjoin is given birth to.This new exon arrangement mode leads to the born of the same parents of EGFR Foreign section forms a kind of epitope of new tumour-specific.About 30% glioblastoma patient is detected to express EGFRvIII, this can be used as the molecular target of CAR-T treatment entity tumor just.(Laura A.Johnson,et al.Rational development and characterization of humanized anti–EGFR variant III chimeric antigen receptor T cells for glioblastoma.Sci Transl Med.2015February 18；7 (275): 275ra22.), therefore, recent years is at glioblastoma (glioblastoma) Treatment on have significant curative effect, it is considered to be one of the therapeutic modality of most promising glioblastoma.

Chimeric antigen receptor (CAR) is the core component (shown in Fig. 1) of CAR-T, and it is non-dependent to assign T lymphocyte HLA Mode identifies the ability of tumour antigen, this enables by the T cell of CAR transformation compared to nave T cell surface receptor TCR Identify wider target.It include a tumor associated antigen (tumor-associated in the basic engineering of CAR Antigen, TAA) combined area (the scFV section for being typically derived from monoclonal antibody antigen bond area), an extracellular hinge area, One transmembrane region and an intracellular signal transduction area.Specificity, validity and genetic modification of scFV sections of the design for CAR The safety of T cell itself is being crucial determinant.

It is verified through clinical test, carrier of the present invention is applied to clinical treatment bone-marrow-derived lymphocyte leukaemia, B lymthoma, Yi Jiduo Hair property myeloma realizes the fully erased of tumour cell in patient's body, therefore carrier of the present invention is treated in CART Field, which is gathered around, to have broad application prospects.

Compared with prior art, the invention has the following beneficial effects:

The linker for the scFV section that the present invention uses is designed, and is the Linkers pool using Shi Ao company, by albumen The analysis of matter Structure bioinformatics database (https: //www.predictprotein.org/), by protein two The comparison of the protein properties such as level structure, Solvent accessibility, protein flexibility, disulfide bridge, binding site, preferably Out.Detecting test and killing-efficiency test by cell in vitro cytokine secretion proves, can be significant compared with external design Improve the killing effect in vitro of CAR-T cell.Also, it is also better than the effect of external clinical trial in the effect of clinical treatment. The scFV sections of linker design, can be applied equally to third generation CAR design scheme.Third generation CAR design is set with the second generation Meter is compared, and is increased CD28 Chimerical receptor costimulating factor (SEQ ID NO.25), is had stronger signal amplification.

Slow virus carrier column purification system of the present invention is the slow virus large-scale production that the applicant develops Technique.Common supercentrifugation or supercentrifugal process are to separate lentiviral particle using centrifugal sedimentation principle, unavoidably Meeting remain impurity similar in many sedimentation coefficients, adverse effect is brought to subsequent experimental.Also, tubulature process is complicated, operation Cumbersome, multiple conversions container brings more opportunities for contamination.And the slow virus carrier column purification technique of the applicant's exploitation is half Automatic operation, all processes are completed hundred grades of Experimental Areas, avoid manually-operated cumbersome and pollution probability, that is recycled is slow Clinical criteria is fully achieved in the indexs such as endotoxin, mycoplasma in viral vectors.Subsequent follow up develops full-automatic purifying instrument.

The used CAR design scheme of the present invention also can be applied in second generation slow virus carrier structure.The second generation and Difference (as shown in Figure 2 B) of three generations's slow virus carrier in structure, mainly third generation slow virus carrier is second generation carrier 5 ' The region U3 of LTR replaces with RSV promoter, and thus eliminating the need dependences when U3 transcription to Tat albumen, both can be in slow virus Structural gene in remove Tat sequence, also improve lentiviral gene group transcriptional level and transcription duration.The second generation and third It is mainly the difference of subgenomic transcription mode for slow virus carrier, therefore the used CAR design scheme of the present invention can be applied to This two generations slow virus carrier.

The third generation slow virus skeleton plasmid pLenti-3G basic that the present invention uses, with University of Pennsylvania Carl H.June Et al. (Porter DL, Levine BL, Kalos M, BaggA, June CH.Chimeric antigen receptormodifiedT cells in chronic lymphoid leukemia.N Engl J Med 2011；365: Third generation slow virus carrier used by 725-33.) is compared, and bacteriophage f1 replication orgin is eliminated, using eukaryotic viral SV40 Replicon increases copy number of the target gene in eukaryocyte, enhances eukaryotic expression effect.

The third generation slow virus skeleton plasmid pLenti-3G basic that the present invention uses, using enhancedWPRE member Part, with University of Pennsylvania Carl H.June et al. (Porter DL, Levine BL, Kalos M, BaggA, June CH.Chimeric antigen receptormodifiedT cells in chronic lymphoid leukemia.N Engl J Med 2011；WPRE element used by 365:725-33.) is compared, and has the enhancing mutation of 6 nucleotide (g.396G > A, g.397C > T, g.398T > C, g.399G > A, g.400A > T, g.411A > T), the poly gland of primary transcript can be enhanced Glycosidation increases the content of intracellular mRNA, enhances the expression efficiency of transgenosis.

The Lentival packaging system that the present invention uses is four plasmid packaging systems of non-auxiliary virus, passes through four kinds of plasmids Common transfection generates recombined lentivirus vector into HEK293T/17 cell.Slow virus carrier after recombination is replication defect type Exogenous sequences can be integrated into host gene by carrier, disposable, can not be replicated and be proliferated, safety improves a lot.

The slow virus carrier that the present invention uses has the version with fluorescence labels zsGreen1 and without fluorescence labels ZsGreen1 version, the version with fluorescence labels are used for experiment in vitro, are used for clinical trial without the version of fluorescence labels.

Present invention preferably employs third generation slow virus carrier (shown in Fig. 2A), 3 ' SIN LTR eliminate the region U3, eliminate A possibility that slow virus carrier self-replacation, substantially increase safety；CPPT and WPRE element is increased, transduction effect is improved The expression efficiency of rate and transgenosis；Core RNA continues efficiently to turn when ensure that slow virus carrier packaging using RSV promoter Record；Using the EF1 α promoter of people itself, enable CAR gene in human body long lasting for expression.

As it can be seen that recombined lentivirus vector of the present invention will provide reliable turn to the CAR-T of glioblastoma treatment Gene guarantee.

Detailed description of the invention

Fig. 1 is the schematic diagram of CAR of the present invention, wherein Fig. 1 (A) is the basic block diagram of CAR, and Fig. 1 (B) is CAR Generation improve schematic diagram；

Fig. 2 is slow virus carrier structural schematic diagram of the present invention；Wherein, Fig. 2 (A) is the third generation that the present invention uses Slow virus carrier structural schematic diagram, Fig. 2 (B) are that the second generation and third generation slow virus carrier structure compare；

Fig. 3 is the building flow chart that recombined lentivirus vector of the present invention is constructed in the embodiment of the present invention 1, wherein Fig. 3 (A) is the structural schematic diagram of slow virus skeleton plasmid pLenti-3G basic；Fig. 3 (B) is pCAR-EGFRvIII-CLA matter The structural schematic diagram of grain；Fig. 3 (C) figure is the structural schematic diagram of pCAR-EGFRvIII-CLB plasmid；Fig. 3 (D) figure is pCAR- The structural schematic diagram of EGFRvIII-OLC plasmid；Fig. 3 (E) figure is the structural schematic diagram of slow virus packaging plasmid pPac-GP；Fig. 3 (F) figure is the structural schematic diagram of slow virus packaging plasmid pPac-R；Fig. 3 (G) figure is the structural schematic diagram of memebrane protein pEnv-G；

Fig. 4 is the digestion prediction of recombinant slow virus plasmid pCAR-EGFRvIII-CLA and digestion fine jade in the embodiment of the present invention 1 Sepharose electrophoretogram；Wherein, Fig. 4 A is the digestion prediction schematic diagram of recombinant slow virus plasmid pCAR-EGFRvIII-CLA, In, lane1 is 1kb DNA ladder Marker: band is from top to bottom successively are as follows: 10kb, 8Kb, 6kb, 5Kb, 4kb, 3.5Kb,3Kb,2.5kb,2Kb,1.5kb,1Kb,750bp,500bp,250bp；Lane2 is the Pvu of pCAR-EGFRvIII-CLA I digestion prediction: band is from top to bottom successively are as follows: 8616bp, 896bp, 249bp；Fig. 4 B is recombinant slow virus plasmid pCAR- The digestion agarose gel electrophoresis figure of EGFRvIII-CLA；Wherein, lane1 is the electrophoresis knot of 1kb DNA ladder Marker Fruit；Lane2 is the Pvu I restriction enzyme digestion and electrophoresis result of pCAR-EGFRvIII-CLA；

Fig. 5 is the digestion prediction and digestion of recombinant slow virus plasmid pCAR-EGFRvIII--CLB in the embodiment of the present invention 1 Agarose gel electrophoresis figure；Wherein, Fig. 5 A is the digestion prediction schematic diagram of recombinant slow virus plasmid pCAR-EGFRvIII--CLB, Wherein, lane1 is 1kb DNA ladder Marker: band is from top to bottom successively are as follows: 10kb, 8Kb, 6kb, 5Kb, 4kb, 3.5Kb,3Kb,2.5kb,2Kb,1.5kb,1Kb,750bp,500bp,250bp；Lane2 is the Nco of pCAR-EGFRvIII-CLB I digestion prediction: band is from top to bottom successively are as follows: 5867bp, 3329bp, 565bp；Fig. 5 B is recombinant slow virus plasmid pCAR- The digestion agarose gel electrophoresis figure of EGFRvIII--CLB；Wherein, lane1 is the electrophoresis knot of 1kb DNA ladder Marker Fruit；Lane2 is the Nco I restriction enzyme digestion and electrophoresis result of pCAR-EGFRvIII-CLB；

Fig. 6 is the digestion prediction of recombinant slow virus plasmid pCAR-EGFRvIII-OLC and digestion fine jade in the embodiment of the present invention 1 Sepharose electrophoretogram；Wherein, Fig. 6 A is the digestion prediction schematic diagram of recombinant slow virus plasmid pCAR-EGFRvIII-OLC, In, lane1 is 1kb DNA ladder Marker: band is from top to bottom successively are as follows: 10kb, 8Kb, 6kb, 5Kb, 4kb, 3.5Kb,3Kb,2.5kb,2Kb,1.5kb,1Kb,750bp,500bp,250bp；Lane2 is the Kpn of pCAR-EGFRvIII-OLC I digestion prediction: band is from top to bottom successively are as follows: 8335bp, 1426bp；Fig. 6 B is recombinant slow virus plasmid pCAR-EGFRvIII- The digestion agarose gel electrophoresis figure of OLC, wherein lane1 is the electrophoresis result of 1kb DNA ladder Marker；Lane2 is The Kpn I restriction enzyme digestion and electrophoresis result of pCAR-EGFRvIII-OLC；

Fig. 7 is the flow chart of 2 intermediate ion exchange chromatography of embodiment of the present invention purifying recombined lentivirus vector；

Fig. 8 is the titre testing result schematic diagram of the different way of purification of recombined lentivirus vector in the embodiment of the present invention 2；

Fig. 9 is the detection of mycoplasma result signal of the different way of purification of recombined lentivirus vector in the embodiment of the present invention 2 Figure；Wherein, lane1 DL2000marker, counterband tape is from top to bottom successively from top to bottom are as follows: 2kb, 1kb, 750bp, 500bp,250bp,100bp；Lane2 is positive control；Lane3 is negative control；Lane4 is PBS；Lane5 is water；lane6 For lysate；

Lane7 is the slow virus of ultracentrifugation purifying；Lane8 is the slow virus of high speed centrifugation purifying；Lane9 is ion friendship Change the slow virus of chromatogram purification；Lane10 is ghost；

Figure 10 is the histogram of mRNA relative expression quantity in the embodiment of the present invention 3, RT-QPCR the result shows that CAR in PBMC Intracellular efficient transcription；

Figure 11 is the WB detection figure of CAR expressing quantity in the embodiment of the present invention 3, the results showed that CAR albumen is thin in PBMC High efficient expression intracellular；Wherein, in Figure 11 A, lane1 is PBMC ghost, and lane2 is control virus MOCK, lane3 lvCAR- EGFRvIII-CLA, lane4 lvCAR-EGFRvIII-CLB, lane5 lvCAR-EGFRvIII-OLC；Figure 11 B is beta- Actin internal reference band；

Figure 12 is the killing-efficiency that LDH detects under the conditions of different effect target ratios in the embodiment of the present invention 3, and E is effector cell, T For target cell；

Figure 13 is that qPCR detects Cytokine Expression Level schematic diagram under the conditions of different effect target ratios, E in the embodiment of the present invention 3 For effector cell, T is target cell；Wherein, Figure 13 A indicates the mRNA transcriptional level of IL-2；Figure 13 B indicates that the mRNA of IFN-γ turns Record is horizontal；

Figure 14 is tumour body in the embodiment of the present invention 3 after CAR-EGFRvIII-T cells i injection encephalic tumor-bearing mice Long-pending and T cell quantity situation of change；PBS indicates the blank group of intravenous injection PBS, and UTD indicates to be injected intravenously the CAR- that do not transduce The experimental group of the human T-cell of EGFRvIII, 2173 indicate transduceed CAR-EGFRvIII human T-cell experimental group；Figure 14 A table Show after injecting the 11st day, the nuclear magnetic resonance result of different experiments group intracranial tumors volume；Figure 14 B is indicated after injection the 18th day, is flowed Formula cell art detect different tissues in CD4+, CD8+ cell there are situations；

Figure 15 is the titre of the second generation of continuous 3 batches and third generation recombined lentivirus vector in the embodiment of the present invention 4 As a result compare；

Figure 16 is the killing-efficiency that LDH detects under the conditions of different effect target ratios in the embodiment of the present invention 5, wherein E is that effect is thin Born of the same parents, T are target cell.

Specific embodiment

Following embodiment is only illustrative of the invention and is not intended to limit the scope of the invention.It is not specified in embodiment specific The experimental method of condition, usually according to normal condition, or according to the normal condition proposed by manufacturer.

Embodiment 1 constructs recombined lentivirus vector

One, material

1, slow virus skeleton plasmid pLenti-3G basic, slow virus packaging plasmid pPac-GP, pPac-R and film egg White matter grain pEnv-G, HEK293T/17 cell, homologous recombination enzyme by generation take wing (Shanghai) biological medicine Science and Technology Ltd. provide；

2, primer: primer needed for designing amplification of DNA fragments and target site according to design of primers principle, the primer is by Shanghai Biotech firm's synthesis, specifically:

EF1 α-F:5 '-ATTCAAAATTTTATCGATGCTCCGGTGCCCGTCAGT-3 ' (SEQ ID NO.26)

EF1 α-R:5 '-TCACGACACCTGAAATGGAAGA-3 ' (SEQ ID NO.27)

CD8leader-F:5 '-GGTGTCGTGAGGATCCGCCACCATGGCCTTACCAGTGACCGC-3 '

(SEQ ID NO.28)

CD8leader-R:5 '-GTGTCATCTGGATGTCCGGCCTGGCGGCGTG-3 ' (SEQ ID NO.29)

VL-F:5 '-CCACGCCGCCAGGCCGGATGTTGTGATGACCCAGACTCC-3 ' (SEQ ID NO.30)

VL-R:5 '-TTTGATTTCCAGCTTGGTGCCT-3 ' (SEQ ID NO.31)

CLA-VH-F:5 '-CAAGCTGGAAATCAAAGGCGGTGGCTCGGGTGGTGGGTCGGGCGGC

GGATCTGGGGGAGGTTCTGAGGTCCAGCTGCAACAGTCT-3’(SEQ ID NO.32)

CLB-VH-F:5 '-CAAGCTGGAAATCAAAGGATCCACCTCCGGATCCGGAAAACCCGGAT

CCGGAGAAGGATCCACCAAAGGAGAGGTCCAGCTGCAACAGTCT-3’(SEQ ID NO.33)

OLC-VH-F:5 '-CAAGCTGGAAATCAAAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCG

GGTGGCGGCGGATCTGAGGTCCAGCTGCAACAGTCT-3’(SEQ ID NO.34)

VH-R:5 '-GGAGGAGACGGTGACTGAGGT-3 ' (SEQ ID NO.35)

CD8Hinge-F:5 '-GTCACCGTCTCCTCCACCACGACGCCAGCGCC-3 ' (SEQ ID NO.36)

CD8Hinge-R:5 '-GTAGATATCACAGGCGAAGTCCA-3 ' (SEQ ID NO.37)

CD8Transmembrane-F:5 '-CGCCTGTGATATCTACATCTGGGCGCCCTTGGC-3 ' (SEQ ID NO.38)

CD8Transmembrane-R:5 '-TCTTTCTGCCCCGTTTGCAGTAAAGGGTGATAACCAGTG-3 ' (SEQ ID NO.39)

CD137-F:5 '-AAACGGGGCAGAAAGAAACTC-3 ' (SEQ ID NO.40)

CD137-R:5 '-TGCTGAACTTCACTCTCAGTTCACATCCTCCTTCTTCTTC-3 ' (SEQ ID NO.41)

TCR-F:5 '-AGAGTGAAGTTCAGCAGGAGCG-3 ' (SEQ ID NO.42)

TCR-R:5 '-GGAGAGGGGCGTCGACTTAGCGAGGGGGCAGGGC-3 ' (SEQ ID NO.43)

WPRE-QPCR-F:5 '-CCTTTCCGGGACTTTCGCTTT-3 ' (SEQ ID NO.44)

WPRE-QPCR-R:5 '-GCAGAATCCAGGTGGCAACA-3 ' (SEQ ID NO.45)

Actin-QPCR-F:5 '-CATGTACGTTGCTATCCAGGC-3 ' (SEQ ID NO.46)

Actin-QPCR-R:5 '-CTCCTTAATGTCACGCACGAT-3 ' (SEQ ID NO.47)

CAR-QPCR-F:5 '-GACTTGTGGGGTCCTTCTCCT-3 ' (SEQ ID NO.48)

CAR-QPCR-R:5 '-GCAGCTACAGCCATCTTCCTC-3 ' (SEQ ID NO.49)

3、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、 SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31、、SEQ ID NO.32、SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35、SEQ ID NO.36、SEQ ID NO.37、SEQ ID NO.38、SEQ ID NO.39、SEQ ID NO.40、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.45、SEQ ID NO.46、SEQ ID NO.47、SEQ DNA sequence dna shown in ID NO.48, SEQ ID NO.49 is synthesized by Shanghai Jierui Biology Engineering Co., Ltd, and with oligonucleotides Dry powder or plasmid form save；SEQ ID NO.52, SEQ ID NO.53 are synthesized by Changzhou Ji Yu Biotechnology Co., Ltd.

4, toolenzyme Pvu I, Nco I, Kpn I, ApaL I, Sac I, Cla I, Sal I, T4DNA ligase are purchased from NEB company；

5, high fidelity enzyme PrimeSTAR, RN is purchased from Takara company；

6,0.22 μm of -0.8 μm of PES filter is purchased from millipore company；

7, plasmid extraction kit, Ago-Gel QIAquick Gel Extraction Kit are purchased from MN company；

8, competent cell TOP10 is purchased from tiangen company；

9、NaCl、KCl、Na₂HPO₄.12H₂O、KH₂PO₄、Trypsin、EDTA、CaCl₂, NaOH, PEG6000 be purchased from Hai Shenggong；

10, Opti-MEM, FBS, DMEM, 1640, Hepes, be purchased from invitrogen company；

11, Biotinylated protein L is purchased from GeneScript company；

12, the secondary antibody of horseradish peroxidase-labeled, DAB working solution are purchased from Beijing Zhong Shan Golden Bridge；

13, ECL+plusTM Western blotting system is purchased from Amersham company；

14, DNeasy kit is purchased from Shanghai JaRa company；

15, it is company that lymphocyte separation medium, which reaches section purchased from Shenzhen,；

16, phycoerythrin (PE)-conjugated streptavidin is purchased from BD Bioscience company；

17, SA-HRP is purchased from Shanghai Yi Sheng company；

18, mycoplasma test reagent box, endotoxin detection kit, EGFRvIII⁺K562 cell takes wing (Shanghai) purchased from generation Company；

19, LDH detection kit is purchased from promega company；

Two, recombined lentivirus vector lvCAR-EGFRvIII-CLA, lvCAR-EGFRvIII-CLB, lvCAR- The construction method of EGFRvIII-OLC.

Referring to Fig. 3, the construction method of recombined lentivirus vector of the present invention is as follows:

1, by people EF1 α promoter, CD8leader Chimerical receptor signal peptide, EGFRvIII single-chain antibody light chain VL, Common Linker A, Common Linker B, Optimal Linker C, EGFRvIII single-chain antibody heavy chain VH, CD8Hinge Chimerical receptor hinge, CD8Transmembrane Chimerical receptor transmembrane region, CD137 Chimerical receptor costimulating factor, TCR Chimerical receptor t cell activation domain segment is cloned into slow virus skeleton plasmid pLenti-3G basic, respectively obtains recombinant lentiviral Virus particle pCAR-EGFRvIII-CLA, pCAR-EGFRvIII-CLB, pCAR-EGFRvIII-OLC.

(1) slow virus skeleton plasmid pLenti-3G basic is carried out using Cla I and Sal I restriction enzyme double Digestion, product pass through 1.5% agarose gel electrophoresis, confirm the segment V1 (shown in Fig. 4) of 8303bp, and be tapped and recovered and be placed in In Eppendorf pipe, corresponding segment (being shown in Table 1) is recycled with the Ago-Gel QIAquick Gel Extraction Kit of MN company, and measure product Purity and concentration；

1, colloidal sol	Sol solutions are added in 200 μ l NTI/100mg gel ratios, 50 DEG C of water-baths are placed 5-10 minutes.
		2, in conjunction with DNA	11000g is centrifuged 30 seconds, discards filtrate.
3, film is washed	700 μ l NT3,11000g is added centrifugation 30 seconds, discards filtrate.
		4, film is washed	It is primary to repeat third step
5, it dries	11000g is centrifuged 1 minute, and the collecting pipe renewed is placed at room temperature for 1 minute.
		6, eluted dna	15-30 μ l NE is added, is placed at room temperature for 1 minute, 11000g is centrifuged 1 minute, collects filtrate.

1 Ago-Gel recycling step of table

(2) system in table 2, PCR are used using the SEQ ID NO.14 synthesized as template with primer EF1 α-F and EF1 α-R Cycling condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 2min) * 35cycle, 72 DEG C of 10min.Product passes through 1.5% agarose gel electrophoresis, confirms the segment a of 1208bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company Ago-Gel QIAquick Gel Extraction Kit recycle corresponding segment (being shown in Table 1), and measure the purity and concentration of product；

Reagent	Volume (μ l)
		H2O	32.5
5×Buffer(with Mg2+)	10
		DNTP (each 2.5mM)	4
Primer1(+)(10μM)	1
		Primer2 (-) (10 μM)	1
Template	1
		PrimeSTAR	0.5

2 50 μ l PCR reaction system of table

(3) with primer CD8leader-F and CD8leader-R using the SEQ ID NO.15 synthesized as template, using in table 2 System, PCR cycle condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C 5min.Product passes through 1.5% agarose gel electrophoresis, confirms the segment b of 101bp, and is tapped and recovered and is placed in Eppendorf pipe It is interior, corresponding segment (being shown in Table 1) is recycled with the Ago-Gel QIAquick Gel Extraction Kit of MN company, and measure the purity of product and dense Degree；

(4) system in table 2, PCR cycle are used using the SEQ ID NO.16 synthesized as template with primer VL-F and VL-R Condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through 1.5% agarose gel electrophoresis, confirms the segment c of 336bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company Ago-Gel QIAquick Gel Extraction Kit recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；

(5) system in table 2, PCR are used using the SEQ ID NO.20 synthesized as template with primer CLA-VH-F and VH-R Cycling condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through 1.5% agarose gel electrophoresis, confirms the segment d of 424bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company Ago-Gel QIAquick Gel Extraction Kit recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；

(6) system in table 2, PCR are used using the SEQ ID NO.20 synthesized as template with primer CLB-VH-F and VH-R Cycling condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through 1.5% agarose gel electrophoresis, confirms the segment e of 430bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company Ago-Gel QIAquick Gel Extraction Kit recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；

(7) system in table 2, PCR are used using the SEQ ID NO.20 synthesized as template with primer OLC-VH-F and VH-R Cycling condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through 1.5% agarose gel electrophoresis, confirms the segment f of 421bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company Ago-Gel QIAquick Gel Extraction Kit recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；

(8) with primer CD8Hinge-F and CD8Hinge-R using the SEQ ID NO.21 synthesized as template, using in table 2 System, PCR cycle condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min. Product passes through 1.5% agarose gel electrophoresis, confirms the segment g of 147bp, and be tapped and recovered and be placed in Eppendorf pipe, uses The Ago-Gel QIAquick Gel Extraction Kit of MN company recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；

(9) it is with the SEQ ID NO.22 synthesized with primer CD8Transmembrane-F and CD8Transmembrane-R Template uses the system in table 2, PCR cycle condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through 1.5% agarose gel electrophoresis, confirms the segment h of 100bp, and be tapped and recovered and set In in Eppendorf pipe, corresponding segment (being shown in Table 1) is recycled with the Ago-Gel QIAquick Gel Extraction Kit of MN company, and measure production The purity and concentration of object；

(10) with primer CD137-F and CD137-R using the SEQ ID NO.23 synthesized as template, using the system in table 2, PCR cycle condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product By 1.5% agarose gel electrophoresis, the segment i of 142bp is confirmed, and be tapped and recovered and be placed in Eppendorf pipe, with MN public affairs The Ago-Gel QIAquick Gel Extraction Kit of department recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；

(11) system in table 2, PCR are used using the SEQ ID NO.24 synthesized as template with primer TCR-F and TCR-R Cycling condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through 1.5% agarose gel electrophoresis, confirms the segment j of 355bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company Ago-Gel QIAquick Gel Extraction Kit recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；

(12) Eppendorf is added in addition to primer using the system in table 3 using each 1 μ l of DNA fragmentation b, c, d as template In pipe, PCR cycle condition are as follows: primer is added in 98 DEG C of 3min, (98 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 30sec) * 6cycle CD8leader-F/VL-R, (98 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 40sec) * 24cycle, 72 DEG C of 5min.Product passes through 1.5% agarose gel electrophoresis, confirms the segment k of 814bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company Ago-Gel QIAquick Gel Extraction Kit recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；

Reagent	Volume (μ l)
		H2O	33.5-1* template number
5×Buffer(with Mg2+)	10
		DNTP (each 2.5mM)	4
Primer1(+)(10μM)	1
		Primer2 (-) (10 μM)	1
Template	1* template number
		PrimeSTAR	0.5

3 50 μ l over-lap PCR reaction system of table

(13) Eppendorf is added in addition to primer using the system in table 3 using each 1 μ l of DNA fragmentation b, c, e as template In pipe, PCR cycle condition are as follows: primer is added in 98 DEG C of 3min, (98 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 30sec) * 6cycle CD8leader-F/VL-R, (98 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 40sec) * 24cycle, 72 DEG C of 5min.Product passes through 1.5% agarose gel electrophoresis, confirms the segment l of 820bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company Ago-Gel QIAquick Gel Extraction Kit recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；

(14) Eppendorf is added in addition to primer using the system in table 3 using each 1 μ l of DNA fragmentation b, c, f as template In pipe, PCR cycle condition are as follows: primer is added in 98 DEG C of 3min, (98 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 30sec) * 6cycle CD8leader-F/VL-R, (98 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 40sec) * 24cycle, 72 DEG C of 5min.Product passes through 1.5% agarose gel electrophoresis, confirms the segment m of 811bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company Ago-Gel QIAquick Gel Extraction Kit recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；

(15) it using each 1 μ l of DNA fragmentation g, h, i, j as template, using the system in table 3, is added in addition to primer In Eppendorf pipe, PCR cycle condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 30sec) * 6cycle, Primer CD8Hinge-F/TCR-R, (98 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 30sec) * 24cycle, 72 DEG C of 5min is added.It produces Object passes through 1.5% agarose gel electrophoresis, confirms the segment n of 704bp, and be tapped and recovered and be placed in Eppendorf pipe, uses MN The Ago-Gel QIAquick Gel Extraction Kit of company recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；

(16) Eppendorf pipe is added with the ratio of 5 μ l total volumes and molar ratio 1:1:1:1 in DNA fragmentation V1, a, k, n It is interior, 15 μ l of homologous recombination enzyme reaction solution is added, is incubated for 30 minutes after mixing at 42 DEG C, is transferred to and places 2-3 minutes on ice, it will be anti- It answers liquid to be added in 50 μ l TOP10, gently rotates to mix content, placed 30 minutes in ice, pipe is put into pre-heating to 42 DEG C thermostat water bath in heat shock 90 seconds, quickly pipe is transferred in ice bath, makes cell cooling 2-3 minutes, every pipe adds 900 μ l Then pipe is transferred on 37 DEG C of shaking tables by LB culture solution, incubating 1 hour makes bacteria resuscitation, and the transformed bacteria solution of 100 μ l is taken to be coated on On Amp LB agar plate, it is inverted plate, 37 DEG C of cultures in constant incubator, 16 hours.

Picked clones carry out bacterium colony PCR identification, identify that correctly clone is recombinant slow virus plasmid pCAR- EGFRvIII-CLA carries out digestion identification to correct clone (see Fig. 4)；

(17) Eppendorf pipe is added with the ratio of 5 μ l total volumes and molar ratio 1:1:1:1 in DNA fragmentation V1, a, l, n It is interior, 15 μ l of homologous recombination enzyme reaction solution is added, is incubated for 30 minutes after mixing at 42 DEG C, is transferred to and places 2-3 minutes on ice, it will be anti- It answers liquid to be added in 50 μ l TOP10, gently rotates to mix content, placed 30 minutes in ice, pipe is put into pre-heating to 42 DEG C thermostat water bath in heat shock 90 seconds, quickly pipe is transferred in ice bath, makes cell cooling 2-3 minutes, every pipe adds 900 μ l Then pipe is transferred on 37 DEG C of shaking tables by LB culture solution, incubating 1 hour makes bacteria resuscitation, and the transformed bacteria solution of 100 μ l is taken to be coated on On Amp LB agar plate, it is inverted plate, 37 DEG C of cultures in constant incubator, 16 hours.

Picked clones carry out bacterium colony PCR identification, identify that correctly clone is recombinant slow virus plasmid pCAR- EGFRvIII-CLB carries out digestion identification to correct clone (see Fig. 5)；

(18) Eppendorf pipe is added with the ratio of 5 μ l total volumes and molar ratio 1:1:1:1 in DNA fragmentation V1, a, m, n It is interior, 15 μ l of homologous recombination enzyme reaction solution is added, is incubated for 30 minutes after mixing at 42 DEG C, is transferred to and places 2-3 minutes on ice, it will be anti- It answers liquid to be added in 50 μ l TOP10, gently rotates to mix content, placed 30 minutes in ice, pipe is put into pre-heating to 42 DEG C thermostat water bath in heat shock 90 seconds, quickly pipe is transferred in ice bath, makes cell cooling 2-3 minutes, every pipe adds 900 μ l Then pipe is transferred on 37 DEG C of shaking tables by LB culture solution, incubating 1 hour makes bacteria resuscitation, and the transformed bacteria solution of 100 μ l is taken to be coated on On Amp LB agar plate, it is inverted plate, 37 DEG C of cultures in constant incubator, 16 hours.Picked clones carry out bacterium colony PCR mirror It is fixed, it identifies that correctly clone is recombinant slow virus plasmid pCAR-EGFRvIII-OLC, digestion identification is carried out to correct clone (see Fig. 6).

2, recombined lentivirus vector lvCAR-EGFRvIII-CLA, lvCAR-EGFRvIII-CLB, lvCAR-EGFRvIII- The packaging of OLC.

(1) complete medium: taking out preheated fresh culture, and 10%FBS+5ml Pen-Srep is added, runs up and down Mix；(2) NaCl 8g, KCl 0.2, Na 1XPBS solution: are weighed₂HPO₄.12H₂O 3.58g, KH₂PO4 0.24g

It is placed in 1000ml beaker, the dissolution of 900ml Milli-Q grade ultrapure water is added, after the completion of dissolution, use 1000ml graduated cylinder is settled to 1000ml, 121 DEG C of high-temperature heat sterilization 20min；

(3) 0.25%Trypsin solution: weighing Trypsin 2.5g, and EDTA 0.19729g is placed in 1000ml beaker, 900ml1XPBS dissolution is added, after the completion of dissolution, is settled to 1000ml using 1000ml graduated cylinder, 0.22 μM of filtration sterilization, for a long time Using can be reserved for -20 DEG C of refrigerators；

(4) 0.5M CaCl2 solution: 36.75g CaCl is weighed₂It is dissolved with 400ml Milli-Q grade ultrapure water；With Total volume is settled to 500ml by Milli-Q grade ultrapure water, is mixed；0.22 μm of filtration sterilization, packing are saved in 50ml centrifugation Guan Zhong, every pipe 45ml or so, 4 DEG C of preservations.

(5) 4.09g NaCl, 0.269g Na 2XHBS solution: are weighed₂HPO4,5.96g Hepes, with 400ml Milli- The dissolution of Q grade ultrapure water；After calibrating pH instrument, the pH of HBS solution is transferred to 7.05 with 2M NaOH solution.Adjust every bottle of HBS's It is 3ml or so that PH, which consumes 2M NaOH,；

(6) the HEK293T/17 cell frozen is taken out from liquid nitrogen container, is quickly transferred in 37 DEG C of water-baths, after 1~2min It is transferred in super-clean bench, the liquid in cryopreservation tube is fully transferred to 10cm by sterile working²In culture dish, supply containing 10%FBS DMEM to 8mL/10cm²Dish, rear micro- sem observation cell, the degree of cell confluency are greater than 80% and are passed on for 24 hours；

(7) selection cell state is good, free of contamination HEK293T/17 cell, and every 2-6 culture dish is one group, by cell After pancreatin digestion, 4-12ml complete medium is drawn with electric pipettor, 2ml is added into each postdigestive culture dish, is avoided Culture dish dries out；All cells are blown and beaten into single cell suspension using 1ml pipettor, are transferred in medium bottle；

(8) remaining cell in above-mentioned 2-6 culture dish is transferred in medium bottle, and rinsed again with culture medium once Culture dish；

(9) culture medium bottle cap is covered tightly, turns upside down 10 times or so and mixes well cell suspension, cell is passed to 8-24 10cm²In culture dish, the cell density of every ware should about 4 × 10⁶A/10ml complete medium or so.If cell density and pre- The difference of phase is larger, then needs to count cell, then according to 4 × 10⁶The amount of a/ware is inoculated with；

(10) every 6 culture dishes arrange piles up for one, pays attention to keeping the cooperation between ware up and down.By culture dish or so, front and back It shakes for several times, spreads out cell sufficiently, be then placed in 5%CO₂Incubator.Remaining cell does same processing；

(11) checking that institute's passage cell, cell confluency degree should be 70-80%, profile is full, and it is adherent good, it is trained in cell It supports and is uniformly distributed in ware；

(12) liquid is changed for cell, culture medium is replaced with into fresh complete medium, every ware 9ml, and by the CO of incubator₂It is dense Degree setting value is increased to 8%；

(13) match DNA/CaCl according to N+0.5₂Solution.Every ware HEK293T/17 cell transfecting plasmid amount is according to following ratio It uses: recombinant slow virus plasmid (20 μ g), pPac-GP (15 μ g), pPac-R (10 μ g), pEnv-G (7.5 μ g).Take one it is new 0.5M CaCl2:0.25ml, 20 μ g:pPac-GP of recombinant slow virus plasmid, 15 μ g:pPac-R, 10 μ g is added in 5ml centrifuge tube: 7.5 μ g of pEnv-G, supplement ultrapure water close the lid to 0.5ml, mix well；

(14) a 5ml centrifuge tube is separately taken, 0.5ml DNA/CaCl2 solution is added.Turbula shaker is opened, a hand is taken The firmly upper end of 5ml centrifuge tube makes tube bottom contact oscillating end, and liquid is made to scatter on tube wall flowing, and another hand moves 1mL by one Liquid rifle is drawn 2 × HBS of 0.5mL solution, is slowly added dropwise into centrifuge tube, coutroi velocity, being dripped off with half a minute is advisable.2×HBS It after addition, after persistent oscillation 5 seconds, stops oscillation, can be directly added into the cell for needing to transfect；

(15) take a ware cell, the 1mL calcium in centrifuge tube turned into drop and is added, make as far as possible calcium turn reagent be distributed to it is whole In a culture dish；

(16) it after calcium turns liquid addition, is marked in ware lid, culture dish is released to another 5%CO₂In incubator. Ensure that culture dish is horizontal positioned, every pile culture dish does not exceed 6.In 5%CO₂(6-8h) is placed in incubator；

(17) by the CO of first incubator₂Concentration set point adjusts back to 5%；

After (18) 24 hours, cell state is checked.Cell confluency degree should be 80-85% or so, in good condition.It will culture Base siphons away, replacement 10ml fresh DMEM complete medium；

After (19) 48 hours, transfection efficiency is observed.Most cells are still adherent.It can be seen that thin more than 95% Born of the same parents can have green fluorescence.By the same virus packaging supernatant collection to together, and continue addition 10mL into culture dish Fresh culture；

After (20) 72 hours, the same vial supernatant is collected into the virus together, collected twice again to be placed on Together, culture dish is abandoned；Recombined lentivirus vector lvCAR-EGFRvIII-CLA, lvCAR- are contained in the supernatant collected at this time EGFRvIII-CLB、lvCAR-EGFRvIII-OLC。

The concentration and detection of 2 recombined lentivirus vector of embodiment

One, supercentrifugation purifies recombined lentivirus vector；

(1) supernatant of collection is dispensed into 50ml centrifuge tube, 500g room temperature is centrifuged 10min, removes cell and big Fragment；

(2) with 0.22 μm of -0.8 μm of filter filtering supernatant；

(3) 6 Hitachi 40PA ultracentrifugation pipes are taken, 70% ethanol disinfection is sprayed on surface, is placed on super-clean bench with ultraviolet Light irradiation is sterilized 30 minutes.High-temperature heat sterilization can also be passed through；

(4) the cell conditioned medium sample packing 32ml handled step 2 well is into centrifuge tube；

(5) metal cover is covered, by centrifuge tube together with metal cover trim, makes deviation of weight in 0.02g with 1XPBS adjustment In range；

(6) centrifuge tube of trim is symmetrically placed in ultracentrifugation rotor P50AT2.Centrifugal rotational speed 100,000g is set, 4 DEG C are centrifuged 2 hours；

(7) after being centrifuged, carefully centrifuge tube is taken out from rotor, it can be seen that have a small group heavy at centrifuge tube bottom It forms sediment, is made marks on outer tube wall with Marker, outwell supernatant.Centrifuge tube is tipped upside down on the paper handkerchief completed in advance, remnants are made Liquid pours off.The drop hung on wall can be siphoned away with liquid-transfering gun；

(8) Opti-MEM of 200 μ l is added into each centrifuge tube, dissolves precipitating with the piping and druming of 200 μ l pipettors, as far as possible Reduce the generation of foam；

(9) ultracentrifugation pipe is inserted into 50ml centrifuge tube, closes the lid, is put into 4 DEG C of refrigerator overnights；

(10) 500g, room temperature are centrifuged 1min, virus liquid are made to focus on tube bottom；

(11) all identical viral concentration liquid are brought together, are filtered with 0.22 μm -0.8 μm of PES filter；It will be sick Poison is divided into 25 to 50 μ l mono- pipe, freezes -80 DEG C of refrigerators, carries out long-term preservation；

Two, supercentrifugal process purifies recombined lentivirus vector；

(1) the supernatant 204ml of collection is filtered using 0.22 μm -0.8 μm of PES filter；

(2) 6000 solution of PEG of 51ml 50% is added；

(3) 21.7ml 4M NaCl solution is added；

(4) be added 23.3ml PBS solution, at this moment overall solution volume be 300ml.PEG 6000 final concentration 8.5%, NaCl final concentration 0.3M；

(5) solution is dispensed into 250ml wide-mouth bottle, every part of 150ml；

(6) 4 DEG C are placed 1.5 hours, and every 20-30 minutes mixes once；

(7) 4 DEG C, 7000g centrifugation 10min；

(8) it can see that tube bottom has white precipitate after being centrifuged；

(9) liquid is carefully discarded supernatant, it is heavy acutely to rock resuspension for every bottle of addition 1.2ml 50mM Tris-HCl (pH 7.4) It forms sediment；

(10) it is vortexed concussion 20-30 seconds and precipitating is further resuspended；

(11) virus is divided into 25 to 50 μ l mono- to manage, freezes -80 DEG C of refrigerators, carry out long-term preservation；

Three, ion exchange chromatography recombined lentivirus vector (as shown in Figure 7)；

(1) supernatant of collection is used into Thermo vacuum pump, is filtered through 0.22 μm -0.8 μm of PES filter, remove impurity elimination Matter；

(2) 1.5M NaCl 250mM Tris-HCl (pH 6-8) is added into supernatant in the ratio of 1:1~1:10；

(3) 2 ion exchange columns are placed in series, with 4ml 1M NaOH, 4ml 1M NaCl, 5ml 0.15M NaCl 25mM Tris-HCl (pH 6-8) solution successively crosses column；

(4) solution obtained in step 2 is given to ion exchange column loading with the speed of 1-10ml/min by peristaltic pump；

(5) it after whole supernatants cross column, is cleaned using 10ml 0.15M NaCl 25mM Tris-HCl (pH 6-8) solution One time；

(6) it is eluted according to applied sample amount using 1-5ml 1.5M NaCl 25mM Tris-HCl (pH 6-8), collection is washed De- liquid；

(7) eluent is divided into 25 to 50 μ l mono- to manage, freezes -80 DEG C of refrigerators, carry out long-term preservation；

Four, titer determination and compare；

(1) 24 orifice plates is taken to be inoculated with 293T cell.Every hole cell is 5 × 10⁴A, added culture volume is 500ul, different Difference, cell confluency when carrying out virus infection are 40%-60% to the vitro growth rates of type；

(2) prepare 3 sterile EP tubes, the fresh complete medium (DMEM in high glucose+10% of 90ul is added in each pipe FBS) after inoculating cell 24 hours, the cell in two holes is taken to be counted with blood counting chamber, the actual number of cell when determining infection, It is denoted as N；

(3) it takes virus stock solution used 10ul to be determined to be added in first pipe, after mixing gently, 10ul is taken to be added to second In a pipe, a to the last pipe is then successively operated；410ul complete medium (DMEM in high glucose+10% is added in every pipe ), FBS final volume 500ul；

(4) 20 hours after infection starts, culture supernatant is removed, 500 μ l complete medium (DMEM in high glucose+10% are changed to FBS), 5%CO₂Continue culture 48 hours；

After (5) 72 hours, luciferase expression situation is observed, under normal circumstances, fluorecyte number increases and phase with extension rate It should reduce, and take pictures；

(6) 0.25% pancreas enzyme -EDTA solution digestion cell of 0.2ml is used, is placed 1 minute at 37 DEG C.It is purged with culture medium whole A cell face, is collected by centrifugation cell.Illustrate extracting genomic DNA according to DNeasy kit.200 are added in each sample cell μ l eluent is washed lower DNA and is quantified；

(7) preparing target DNA detection qPCRmix general pipeline I, (QPCR primer sequence is SEQ ID NO.44---SEQ ID NO.45):

N=number of reactions. is for example: overall reaction number is 40, by 2 × TaqMan of 1ml Universal PCR Master Mix, 4 μ l

Forward primer, 4 μ l reverse primer, 4 μ l probe and 788 μ l H2O mixing.It is placed on after concussion On ice；

(8) preparing internal reference DNA detection qPCRmix pipe II, (QPCR primer sequence is SEQ ID NO.46---SEQ ID NO.47):

2×TaqMan Master Mix 25μl×n

10×RNaseP primer/probe mix 2.5μl×n

H₂O 17.5μl×n

N=number of reactions. is for example: overall reaction number is 40, by 2 × TaqMan of 1ml Universal PCR Master Mix, 100 μ l10 × RNaseP primer/probe mix and 700 μ l H₂O is mixed.Ice is placed on after concussion On；

(9) PCR system is completed in 96 hole PCR plates of pre-cooling to establish.45 μ l are respectively taken to be added to each row of A-D from general pipeline I Hole in, from respectively taking 45 μ l to be added in the hole of each row of E-G in general pipeline II.

(10) 5 μ l plasmid standards and sample to be tested genomic DNA are taken to be added in A-D row respectively, each sample repeats 1 It is secondary.Separately stay 1 hole that the water of 5 μ l is added as no template control (no-template control).

(11) 5 μ l genome standard items and sample to be tested genomic DNA are taken to be added in E-G row respectively, each sample weight It is 1 time multiple.Separately stay 1 hole that the water of 5 μ l is added as no template control (no-template control).

(12) used quantitative PCR apparatus is 7500 quantitative system of ABI PRISM.Cycling condition setting are as follows: 50 DEG C 2 minutes, 95 DEG C 10 minutes, followed by 95 DEG C 15 seconds, 60 DEG C of 40 of 1 minute circulations.

Data analysis: the slow virus carrier copy number integrated in the DNA sample measured is demarcated with genome number, is obtained The viral copy number of every genome conformity.

Titre (integration units per ml, IU ml^-1) calculation formula it is as follows:

IU ml^-1=(C × N × D × 1000)/V

Wherein: C=is averaged the viral copy number of every genome conformity

The number (about 1 × 10 of cell when N=infects⁵)

The extension rate of D=viral vectors

The volume number for the dilution virus that V=is added

(13) recombined lentivirus vector lvCAR-EGFRvIII-CLA, lvCAR-EGFRvIII-CLB, lvCAR- The titre results (as shown in Figure 8) of EGFRvIII-OLC, the result of ion-exchange chromatography are substantially better than supercentrifugation and height Fast centrifugal process；

Five, endotoxin measurement and compare；

(1), endotoxin working standard is 15EU/ branch；

(2), sensitivity of the limulus reagent λ=0.25EU/ml, 0.5ml/ pipe

(3), endotoxin standard dilutes: taking endotoxin standard one, is diluted to 4 λ and 2 λ in proportion with BET water respectively Dissolution, sealed membrane sealing, concussion dissolution 15min；One step of every dilution should all mix 30s on eddy mixer when dilution；

(4), it is loaded: taking reagents several, every addition BET water 0.5ml dissolution, packing to several endotoxin-frees tries Guan Zhong, every pipe 0.1ml.Wherein 2 are negative control pipe, and BET water 0.1ml is added；

2 are positive control pipe, and the endotoxin working standard solution 0.1ml of 2 λ concentration is added；

2 be Sample Positive control tube, be added 0.1ml containing 2 λ endotoxin standards sample solution (dilution 20 times to The endotoxin standard solution 1ml=2ml of sample 1ml+4 λ contains 40 times of samples of dilution of 2 λ endotoxin standards).

Be added 0.1ml sample in sample cell, dilution ratio be shown in Table 4,37 ± 1 DEG C of water-baths (or incubator) heat preservation 60 ± 1min；

(5), recombined lentivirus vector lvCAR-EGFRvIII-CLA, lvCAR-EGFRvIII-CLB, lvCAR- The endotoxin testing result (as shown in table 4) of EGFRvIII-OLC, the endotoxin content of supercentrifugation 20~40EU/ml it Between, the endotoxin content of supercentrifugal process between 20~40EU/ml, the endotoxin content of ion-exchange chromatography 1.25~ Supercentrifugation and supercentrifugal process are substantially better than between 2.5EU/ml；

The endotoxin testing result of the different way of purification of 4 recombined lentivirus vector of table

Six, mycoplasma measures and compares；

(1) first three day is being tested, cell sample is cultivated with antibiotic-free culture medium；

(2) (cell number is greater than 1*10 to collection 1ml cell suspending liquid⁵), it is placed in 1.5ml centrifuge tube；

(3) 13000 × g are centrifuged 1min, collect precipitating, discard culture medium；

(4) 500ul PBS pipette tips pressure-vaccum or vortex oscillation is added, precipitating is resuspended.13000 × g is centrifuged 5min；

(5) step 4 is repeated once；

(6) 50 μ l Cell Lysis Buffer are added after mixing well, to be incubated in 55 DEG C of water-baths with pipette tips pressure-vaccum 20min；

(7) sample is placed in 95 DEG C and heats 5min；

After (8) 13000 × g are centrifuged 5min, take 5 μ l supernatants as template, 25 μ lPCR reaction systems are as follows: 6.5 μ of ddH20 L, Myco Mix1 μ l, 2x Taq Plus Mix Master (Dye Plus) 12.5 μ l, 5 μ l of template；PCR cycle condition are as follows: 95 DEG C 30sec, (95 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 30sec) * 30cycle, 72 DEG C of 5min.

(9) detection of mycoplasma is as the result is shown (as shown in Fig. 9 and table 5), supercentrifugation, supercentrifugal process, ion exchange Chromatography

The recombined lentivirus vector of purifying is free of mycoplasma.

5 detection of mycoplasma of table

Embodiment 3 recombined lentivirus vector lvCAR-EGFRvIII-CLA, lvCAR-EGFRvIII-CLB, lvCAR- The Function detection of EGFRvIII-OLC

One, the cellular level detection of expression of CAR gene:

(1) recombined lentivirus vector lvCAR-EGFRvIII-CLA, lvCAR-EGFRvIII-CLB, lvCAR- After EGFRvIII-OLC infection of PBMCs cell, the detection that cell carries out CAR mRNA transcriptional level using RT-PCR is collected, verifying The expression of CAR gene illustrates that the transcriptional level of CAR gene is expressed successfully if CAR mRNA transcriptional level increases；

(2) recombined lentivirus vector lvCAR-EGFRvIII-CLA, lvCAR-EGFRvIII-CLB, lvCAR- After EGFRvIII-OLC infection of PBMCs cell, the detection that cell carries out CAR protein expression level using western blot is collected, The expression of verifying CAR gene illustrates that the translation skill of CAR gene is expressed successfully if CAR protein expression level increases；

(3) respectively by lvCAR-EGFRvIII-CLA, lvCAR-EGFRvIII-CLB, lvCAR- of MOI=15 EGFRvIII-OLC and control virus MOCK infection cell, after 48h in extraction 6 orifice plates the total serum IgE of cell and total protein respectively into Row fluorescent quantitative PCR experiment and immunoblot experiment.Specific steps: being coated with four holes of 6 orifice plates, and each hole is added corresponding PBS and RN, 4 DEG C overnight.By MOI=15 coating virus after 12 hours, 37 DEG C of incubators place 5h；The 6 orifice plates of taking-up, discard disease Malicious supernatant is washed twice with PBS, by 1*10⁶/ hole is coated with PBMC (being separated from people's blood with lymphocyte separation medium), is added 500ul culture medium (contains 10% serum, 20U/ml IL-2, Polybrene 8ug/ml).Stand 20min, 20 DEG C of 1000g centrifugations 30min, 37 DEG C of culture 48h.

(4) Trizol method extracts the total serum IgE of PBMC cell in 6 orifice plates, reverse transcription amplification cDNA, with QPCR primer (sequence Fluorescent quantitative PCR experiment is carried out for SEQ ID NO.46---SEQ ID NO.49), reaction system is shown in Table 6, is with internal reference Actin Control group verifies the transcription situation of its mRNA.

6 20 μ l qPCR reaction system of table

(5) protein immunoblot (Western Blot) is total by what is extracted from PBMC by polyacrylamide gel electrophoresis Protein is separated by relative molecular mass.It, will be on protein delivery to pvdf membrane using wet turn (4 DEG C, 400mA, 120min).With envelope It closes the closing of liquid (the TBST solution containing 5% skim milk) room temperature pvdf membrane 1h, confining liquid 1:1000 and dilutes Biotinylated Then protein L is incubated at room temperature 4 DEG C overnight with the pvdf membrane closed.TBST washes film 3 times, each 10min.Confining liquid 1: The 500 corresponding SA-HRP of dilution, are incubated for pvdf membrane 2h, TBST washes film 3 times, each 10min at room temperature.Using Amersham company ECL+plusTM Western blotting system kit develops the color.X-ray development obtains the film of display band.

(6) RT-QPCR detection shows that the expression of the CAR after recombined lentivirus vector infection of PBMCs is than control virus MOCK and ghost are increased significantly (as shown in Figure 10 and table 7), illustrate that the transcriptional level of CAR gene is expressed successfully.

Table 7RT-QPCR detection

(7) protein immunoblot (Western Blot) the result shows that, CAR albumen is expressed in recombinant slow virus system (as shown in figure 11) illustrates that the translation skill of CAR gene is expressed successfully.

Two, cytokine secretion and fragmentation effect assessment.

(1) EGFRvIII+K562 cell and effector cell lvCAR-EGFRvIII-CLA-PBMC, lvCAR- are cultivated respectively EGFRvIII-CLB-PBMC,lvCAR-EGFRvIII-OLC-PBMC；

(2) target cell (EGFRvIII+K562) 4x10 is collected⁵Cells and effector cell's (CART cell) 2.8x10⁶Cells, 800g, 6min centrifugation, abandon supernatant；

(3) target cell and effector cell are resuspended respectively with 1ml 1xPBS solution, supernatant is abandoned in 800g, 6min centrifugation；

(4) it is primary to repeat step 3；

(5) effector cell is resuspended with 700ul culture medium (1640 culture medium+10%FBS), with (1640 cultures of 2ml culture medium Base+10%FBS) target cell is resuspended；

(6) setting effect target is than the experimental port for 1:1,5:1,10:1, and control group is arranged, every group of 3 multiple holes；

(7) centrifugation of 250xg, 5min plate；

It is cultivated 4 hours in (8) 37 DEG C of 5%CO2 incubators；

(9) centrifugation of 250xg, 5min plate；

(10) take the 50ul supernatant in each hole into new 96 orifice plate, and every hole is added 50ul substrate solution and (is protected from light behaviour Make)；

(11) it is protected from light incubation 25 minutes；

(12) 50ul terminate liquid is added in every hole；

(13) microplate reader detects 490nm absorbance；

(14) 3 multiple holes are averaged；The light absorption value of all experimental ports, Target cell wells and effector cell hole is subtracted into training Support the mean value of base background light absorption value；The light absorption value of target cell maximum value is subtracted to the mean value of volume correction control light absorption value.

(15) it brings the corrected value obtained in step 14 into following formula, it is thinner than generated to calculate each effect target Cellular toxicity percentage.As a result as shown in figure 12, the PBMC cell of recombined lentivirus vector lvCAR-EGFRvIII-OLC transduction exists Killing-efficiency is apparently higher than lvCAR-EGFRvIII-CLA and lvCAR-EGFRvIII-CLB transduction under the conditions of difference effect target ratio PBMC cell；

Killing-efficiency=(experimental port-effector cell hole-Target cell wells)/(target cell largest hole-Target cell wells) X100%

(16) sample for repeating to prepare a step 6 detects Cytokine Expression Level for qPCR, as a result such as Figure 13 institute Show, the PBMC cell of recombined lentivirus vector lvCAR-EGFRvIII-OLC transduction IL-2 and IFN- under the conditions of different effect target ratios The mRNA transcriptional level of γ is apparently higher than the PBMC cell of lvCAR-EGFRvIII-CLA and lvCAR-EGFRvIII-CLB transduction；

Three, (Laura A.Johnson, et al.Rational development and according to the literature characterization of humanized anti–EGFR variant III chimeric antigen receptor T cells for glioblastoma.Sci Transl Med.2015February18；7 (275): 275ra22.), CAR- EGFRvIII carrier pin has good effect to glioblastoma.

This research is #NCT02209376 in the number of registration of www.clinicaltrials.gov.Expression is used in this research The T lymphocyte of CAR-EGFRvIII gene is used to treat the patient of glioblastoma.Nuclear magnetic resonance image is shown, 11 after injection It, CAR-EGFRvIII-T lymphocyte in encephalic tumor-bearing mice body with do not transduce CAR-EGFRvIII experimental group and PBS blank group is compared, and gross tumor volume is obviously reduced (as shown in Figure 14 A)；Flow cytometry is the results show that 18 after injection It, in marrow, spleen, brain, the quantity of the CD4+CD8+T lymphocyte for CAR-EGFRvIII gene of having transduceed is than the CAR- that do not transduce EGFRvIII experimental group and PBS blank group will be higher by several times or even decades of times (as shown in Figure 14B)；Adoptive CAR- EGFRvIII-T cell therapy possesses good prospect in terms of the treatment for glioblastoma.

4 third generation slow virus skeleton carrier 3rdLV-CAR-EGFRvIII of embodiment and second generation slow virus skeleton carrier 2ndLV-CAR-EGFRvIII titre compares

3rdLV-CAR-EGFRvIII and 2ndLV-CAR-EGFRvIII is constructed respectively；Plasmid construction, virus packaging, virus Ultracentrifugation concentration, titration procedure reference implementation example 1；The titre results of continuous 3 batches are as shown in figure 15, and the third generation is slow The titre of viral backbone carrier 3rdLV-CAR-EGFRvIII is better than second generation slow virus skeleton carrier 2ndLV-CAR- EGFRvIII；Therefore, this patent selects the carrying skeleton for using third generation slow virus skeleton carrier as CAR gene.

5 second generation CAR slow virus carrier 3rdLV-2ndCAR-EGFRvIII of embodiment and third generation CAR slow virus carrier 3rdLV-3rd CAR-EGFRvIII killing-efficiency compares

3rdLV-2ndCAR-EGFRvIII and 3rdLV-3rdCAR-EGFRvIII is constructed respectively；Plasmid construction, virus packet Dress, viral ultracentrifugation concentration, titration procedure reference implementation example 1；Killing experiments reference implementation example 3；Second generation CAR is sick slowly Poisonous carrier

3rdLV-2ndCAR-EGFRvIII and third generation CAR slow virus carrier 3rdLV-3rd CAR-EGFRvIII is killed Efficiencies are more as shown in figure 16, and 3rdLV-3rd CAR-EGFRvIII is directed to the killing-efficiency of target cell in different effect target ratios Under the conditions of the ratio 3rdLV-2ndCAR-EGFRvIII that does not show it is more efficient；Therefore, this patent selection is set using second generation CAR The slow virus carrier of meter is as clinical trial carrier.

The preferred embodiment of the present invention has been described in detail above, but the invention be not limited to it is described Embodiment, those skilled in the art can also make various equivalent on the premise of not violating the inventive spirit of the present invention Variation or replacement, these equivalent variation or replacement are all included in the scope defined by the claims of the present application.

Claims (11)

1. a kind of anti-EGFRvIII Chimeric antigen receptor, including be sequentially connected in series CD8 leader Chimerical receptor signal peptide, EGFRvIII single-chain antibody light chain VL, Optimal Linker C, EGFRvIII single-chain antibody heavy chain VH, CD8 Hinge are chimeric Receptor hinge, CD8 Transmembrane Chimerical receptor transmembrane region, CD137 Chimerical receptor costimulating factor and TCR are chimeric Recipient T cells activation domain；The amino acid sequence of the anti-EGFRvIII Chimeric antigen receptor is as shown in SEQ ID NO.50.

2. a kind of anti-EGFRvIII Chimeric antigen receptor, including be sequentially connected in series CD8 leader Chimerical receptor signal peptide, EGFRvIII single-chain antibody light chain VL, Optimal Linker C, EGFRvIII single-chain antibody heavy chain VH, CD8 Hinge are chimeric Receptor hinge, CD8 Transmembrane Chimerical receptor transmembrane region, CD28 Chimerical receptor costimulating factor, CD137 Chimerical receptor Costimulating factor and TCR Chimerical receptor t cell activation domain；The amino acid sequence of the anti-EGFRvIII Chimeric antigen receptor As shown in SEQ ID NO.51.

3. encoding the gene of anti-EGFRvIII Chimeric antigen receptor of any of claims 1 or 2.

4. gene according to claim 3, it is characterised in that: the nucleotide sequence of the gene such as SEQ ID NO.52 or Shown in SEQ ID NO.53.

5. the recombinant expression carrier containing gene described in claim 3 or 4, which is characterized in that the expression vector is slow disease Malicious expression vector, the Lentiviral include: the prokaryotic replions pUC Ori sequence for plasmid replication；For The sequence of AmpR containing ampicillin resistance gene of purpose bacterial strain massive amplification；For enhancing the virus of the duplication in eukaryocyte Replicon SV40Ori sequence；Slow virus for slow virus packaging packs cis element；For eukaryotic cell expression green fluorescence ZsGreen1 green fluorescent protein；IRES ribosome binding sequence for common transcriptional expression protein；For inosculating antibody People's EF1 α promoter of the eukaryotic transcription of original receptor gene；For form integrate identification, transmitting, starting two generation CAR or The encoding gene of the Chimeric antigen receptor as described in claim 3 or 4 of three generations CAR；For enhancing the expression efficiency of transgenosis The enhanced marmot hepatitis B posttranscriptional regulatory element of eWPRE, after the enhanced marmot hepatitis B transcription of the eWPRE Controlling element have 6 nucleotide enhancing be mutated, specifically: g.396G > A, g.397C > T, g.398T > C, g.399G > A, g.400A>T、g.411A>T。

6. recombinant expression carrier according to claim 5, which is characterized in that slow virus packaging cis element is using the Two generation slow virus carriers include: slow virus 5terminal LTR, slow virus 3terminal Self-Inactivating LTR, Gag cis element, RRE cis element, env cis element and cPPT cis element.

7. recombinant expression carrier according to claim 5, which is characterized in that slow virus packaging cis element is using the Three generations's slow virus carrier include: slow virus 5terminal LTR, slow virus 3terminal Self-Inactivating LTR, Gag cis element, RRE cis element, env cis element, cPPT cis element and RSV promoter.

8. a kind of construction method of the recombinant expression carrier as described in any one of claim 5-7, comprising the following steps:

(1) ampicillin resistance gene AmpR sequence, prokaryotic replions pUC Ori sequence, Viral Replicon SV40 will be contained Ori sequence, slow virus packaging cis element, ZsGreen1 green fluorescent protein, IRES ribosomes knot for slow virus packaging Close sequence, the enhanced marmot hepatitis B posttranscriptional regulatory element of eWPRE is stored on slow virus skeleton plasmid；

(2) by people EF1 α promoter, be used for form integrate identification, transmitting, two generation CAR of starting or three generations CAR be fitted into Antigen receptor is combined into two generation CAR or three generations's CAR design scheme, is cloned into slow virus skeleton by digestion, connection, recombining reaction In plasmid, the recombinant slow virus plasmid of two generation CAR or three generations CAR design is obtained；

(3) by obtained recombinant slow virus plasmid and slow virus packaging plasmid pPac-GP, pPac-R and memebrane protein plasmid PEnv-G transfects HEK293T/17 cell jointly, after carrying out gene transcript expression in HEK293T/17 cell, packs and successfully weighs Group slow virus carrier can be discharged into cells and supernatant, collect the supernatant for the recombined lentivirus vector for including；

(4) obtained recombinant slow virus supernatant is purified using the ion-exchange method for filtering, adsorbing, eluting, respectively To recombined lentivirus vector；

Wherein, step (1), slow virus skeleton plasmid described in step (2) are third generation slow virus skeleton plasmid pLenti-3G basic。

9. construction method according to claim 8, which is characterized in that in step (4), the suction filtration step controls supernatant body Product in 200ml~2000ml, vacuum degree control in -0.5MPA~-0.9MPA,；The pH value of the adsorption step control solution is 6 ~8；The ionic strength of the elution step control eluent is in 0.5M~1.0M.

10. a kind of CAR-T cell, the CAR-T cell is fitted by the described in any item anti-EGFRvIII of claim 1-4 The T lymphocyte of antigen receptor modification.

11. application of the CAR-T cell according to claim 10 in preparation glioblastoma therapeutic agent.