CN108203718A

CN108203718A - The CAR-T carriers and construction method of targeting EGFR vIII interference PD-1 reduction tumor immune escapes and application

Info

Publication number: CN108203718A
Application number: CN201611188014.6A
Authority: CN
Inventors: 吴小江; 王聪; 顾莉萍
Original assignee: SHANGHAI SHENGBO BIOMEDICAL TECHNOLOGY Co Ltd
Current assignee: SHANGHAI SHENGBO BIOMEDICAL TECHNOLOGY Co Ltd
Priority date: 2016-12-20
Filing date: 2016-12-20
Publication date: 2018-06-26

Abstract

The invention discloses a kind of targeting EGFR vIII interference PD 1 reduce tumor immune escape CAR carrier Ts, including：For people's EF1 α promoters of the eukaryotic transcription of Chimeric antigen receptor gene, as shown in SEQ ID NO.1；The sequence of PD 1shRNA, the PD 1shRNA in 3 ' UTR are as shown in SEQ ID NO.25；Gene delivery vector；And integrate identification, transmission, two generation CAR of startup or the Chimeric antigen receptor of three generations CAR for forming, which includes EGFRvIII single-chain antibody light chain VL and EGFRvIII single-chain antibody heavy chains VH.In addition, the construction method the invention also discloses the carrier and its application in preparing interference PD 1 and reducing the drug of tumor immune escape.CAR T technologies of the present invention for EGFRvIII, and pass through the expression for interfering PD 1 in T lymphocytes, inhibit the generation of immunosurveillance escape, killing of the enhancing T lymphocytes to tumour cell, so as to improve the anti-glioma effect of CAR T immunotherapies.

Description

Targeting EGFR vIII interference PD-1 reduces the CAR-T carriers and structure of tumor immune escape Construction method and application

Technical field

The invention belongs to field of medical biotechnology, and in particular to a kind of carrier more particularly to a kind of targeting EGFR vIII interference PD-1 reduces the CAR-T carriers of tumor immune escape.Moreover, it relates to construction method and the application of the carrier.

Background technology

With the development of tumor immunology theory and technology, effect of the immune cell therapy in oncotherapy is increasingly subject to Pay attention to.The study found that T lymphocytes are the natural enemies of tumour cell, play a major role in tumor immune response, to tumour cell There is extremely strong lethal effect.

Chimeric antigen receptor T cell immunotherapy (Chimeric Antigen Receptor T-Cell Immunotherapy, CAR-T immunotherapy) it is a kind of novel cell immunization therapy skill developed rapidly recent years Art [Eleanor J.Cheadle, et al.CAR T cells:driving the road from the laboratory to the clinic.Immunological Reviews 2014.Vol.257:91–106].It is identified based on immune system It is theoretical with activation, by technique for gene engineering, site-specific is identified into (single-chain antibody scFv), starts immunocompetence and attacks swollen The element of oncocyte is integrated into a gene, and in patient's autologous T lymphocytes of being transduceed, and defeated time patient's body, makes Patient regains specific recognition tumour cell, activates self T-cell, targetedly attacks and kill identified tumour The ability of cell.This is a revolutionary character and subversive oncotherapy technology, clinically has shown that its is powerful to B leaching The oncotherapy effect of bar property leukaemia, can achieve the effect that 80% healing B lymphatic leukemias.《Science》Magazine will be adoptive Target tumor immunization therapy is classified as the first place that ten big sciences in 2013 are broken through.

Most crucial theoretical foundation is the identification and activation of T lymphocytes in CAR-T immunotherapies, it is not mainly by Different specific antigen on same scFv tumor cells, then Hinge the and Transmembrane areas for passing through CD8 molecules Signal is reached to CD28 or CD137 the and TCR costimulations active region in T Lymphocyte Membranes, activates itself T lymph thin so as to reach Born of the same parents targetedly attack and kill the purpose of identified tumour cell.But it the different different target spots of researchers and is total to There are certain othernesses for result obtained by the research that stimulus signal combination is carried out, and different researchers is with different tumours in body The result obtained in interior and external research is not quite similar [Wilkie S, Picco G, Foster J, et al.Retargeting of human T cells to tumorassociated MΜC1:the evolution of a chimeric antigen receptor.J Immunol2008；180:4901–4909.].So between CAR-T immunotherapies Difference may come from signal transduction activation, the extracellular antigen bindings of scFv, the culture amplification for recombinating T lymphocytes incessantly, May also with body inner tumour cell to Chimeric antigen receptor T cell adaptive change (immunologic escape of such as tumour cell) or Complicated tumor microenvironment is related, so as to affect the final antitumous effect of CAR-T cells.

CAR-T immunotherapies achieve unprecedented success [Chimeric on treatment B cell leukemia and lymthoma antigen receptor-modified T cells in chronic lymphoid leukemia.N Engl J Med.2011；365(8):725-733.], however, but curative effect is not good enough for a large amount of patients with solid tumor for this therapeutic strategy [Prospects for gene-engineered T cell immunotherapy for solid cancers.Nat Med.2016；22(1):26-36.].The research of early stage is conceived to the T lymphocytes stablized and generate cancer target, and around tumour The microenvironment of inhibition but may make CAR-T cells become have no effectiveness.Therefore, the research direction of this following therapy is to produce Life can resist inhibition of the immune effector molecule to CAR-T cells and the induction to its apoptosis in tumor microenvironment.Recent monoclonal antibody The successful application of immune activation checkpoint (including CTLA-4 and PD-1) in treatment of solid tumors is targeted to pass through immunological approach Control cancer provides strong evidence.Programmed death 1 and its ligand (PD-1/PD-L1) are a pair of of negative immunes Costimulatory molecules are the important molecules participated in during tumor immune escape (Tumor escape).When T lymphocytic cell surfaces After the PD-1 and PD-L1 of tumor cell surface height expression is mutually distinguishable, inhibition signal is transferred in T lymphocytes by PD-1, Inhibit the function of T cell, inhibit the release of inflammatory factor, be the one of the major reasons for causing tumour that immunologic escape occurs.So The research to interact between PD-1 and PD-L1 is expected to provide important experimental basis for the targeted therapy of tumour.

EGFRvIII is the mutant three of epidermal growth factor, is most common EGFR mutant in human tumor.Due to Reading frame has lacked frame exon 2 to exon 7, causes to produce the transcript that a kind of exons 1 and extron 8 adjoin and dash forward Variant.This new extron arrangement mode causes the extracellular section of EGFR to form a kind of antigen table of new tumour-specific Position.About 30% glioblastoma patient is detected expression EGFRvIII, this can be used as CAR-T treatment entities to swell just The molecular target of knurl.(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 exists There is the effect of notable in the treatment of glioblastoma (glioblastoma), it is considered to be most promising glioblastoma One of therapeutic modality.

Therefore, how the generation of tumour cell immune escape inhibited using the method for low cost, enhances T lymphocytes pair The killing of tumour cell so as to improve the anti-glioma effect of CAR-T immunotherapies, becomes a technology difficulty of CAR-T treatments Topic.

At present, there is not yet in relation to that targeting EGFR vIII and PD-1 can be interfered to reduce the CAR-T carriers of tumor immune escape Report.

Invention content

One of the technical problem to be solved in the present invention, which is to provide a kind of targeting EGFR vIII interference PD-1, reduces tumour immunity The CAR-T carriers of escape.CAR-T technologies of the present invention for EGFRvIII, and pass through and interfere in T lymphocytes The expression of PD-1 inhibits the generation of immunosurveillance escape, killing of the enhancing T lymphocytes to tumour cell, so as to improve The anti-glioma effect of CAR-T immunotherapies.

The second technical problem to be solved by the present invention is to provide the construction method of the carrier.

The third technical problem to be solved by the present invention is to provide the application of the carrier.

In order to solve the above technical problems, the present invention adopts the following technical scheme that：

In one aspect of the invention, providing a kind of targeting EGFR vIII interference PD-1 reduces the CAR-T of tumor immune escape Carrier, including：

For people's EF1 α promoters of the eukaryotic transcription of Chimeric antigen receptor gene, as shown in SEQ ID NO.1；

The sequence of PD-1 shRNA, the PD-1 shRNA in 3 ' UTR are as shown in SEQ ID NO.25；

Gene delivery vector；

And integrate identification, transmission, two generation CAR of startup or the Chimeric antigen receptor of three generations CAR for forming, it should Chimeric antigen receptor includes EGFRvIII single-chain antibody light chain VL and EGFRvIII single-chain antibody heavy chains VH.

As currently preferred technical solution, the two generation CAR that identification, transmission, startup are integrated for composition Chimeric antigen receptor include：CD8 leader Chimerical receptors signal peptide, such as SEQ ID NO.3 as shown in SEQ ID NO.2 Shown EGFRvIII single-chain antibody light chains VL, single-chain antibody hinge Linker, such as SEQ ID as shown in SEQ ID NO.4 EGFRvIII single-chain antibody heavy chains VH shown in NO.5, the CD8 Hinge Chimerical receptors hinge as shown in SEQ ID NO.6, such as CD8 Transmembrane Chimerical receptors transmembrane region shown in SEQ ID NO.7, the CD137 as shown in SEQ ID NO.8 are fitted into Receptor costimulating factor, the TCR Chimerical receptor t cell activations domain as shown in SEQ ID NO.9；

As currently preferred technical solution, the three generations CAR that identification, transmission, startup are integrated for composition Chimeric antigen receptor include：CD8 leader Chimerical receptors signal peptide, such as SEQ ID NO.3 as shown in SEQ ID NO.2 Shown EGFRvIII single-chain antibody light chains VL, single-chain antibody hinge Linker, such as SEQ ID as shown in SEQ ID NO.4 EGFRvIII single-chain antibody heavy chains VH shown in NO.5, the CD8 Hinge Chimerical receptors hinge as shown in SEQ ID NO.6, such as CD8 Transmembrane Chimerical receptors transmembrane region shown in SEQ ID NO.7, the CD137 as shown in SEQ ID NO.8 are fitted into Receptor costimulating factor, the TCR Chimerical receptor t cell activation domains as shown in SEQ ID NO.9 and such as SEQ ID NO.24 institutes The CD28 Chimerical receptor costimulating factors shown.

The gene delivery vector includes slow virus carrier, retroviral vector, adenovirus vector, adeno-associated virus and carries Body etc., preferably third generation slow virus carrier, the third generation slow virus carrier include the sequences of AmpR containing ampicillin resistance gene, Prokaryotic replions pUC Ori sequences, Viral Replicon SV40 Ori sequences, RSV promoters, 5 terminal LTR of slow virus, 3 terminal Self-Inactivating LTR of slow virus, Gag cis elements, RRE cis elements, env cis elements, The enhanced marmot hepatitis B posttranscriptional regulatory element of cPPT cis elements, eWPRE.The enhanced marmot second of eWPRE Hepatovirus posttranscriptional regulatory element has the enhancing mutation of 6 nucleotide, specially：g.396G>A、g.397C>T、g.398T>C、 g.399G>A、g.400A>T、g.411A>T。

As currently preferred technical solution, the PD-1 shRNA in the 3 ' UTR increase mir-30 in 3 ' UTR Arm forms the PD-1 shRNA sequences as shown in SEQ ID NO.10.Increasing mir-30 arms helps to recruit related processing enzyme knot It closes on mir30 arms, promotes the processing shearings of entire shRNA in the cell, greatly increase the formation of ripe interference tiny RNA, carry The efficiency of high interference target gene.

In the second aspect of the present invention, providing a kind of above-mentioned targeting EGFR vIII interference PD-1 reduces tumor immune escape The construction method of CAR-T carriers, includes the following steps：

(1) gene delivery vector is provided；

(2) by as shown in SEQ ID NO.1 people EF1 α promoters, in 3 ' UTR plus PD-1 shRNA, know for forming collection Not, it transmits, startup is combined into two generation CAR or three generations CAR designs in the Chimeric antigen receptor of the two generation CAR or three generations CAR of one Scheme is cloned by digestion, connection, recombining reaction in gene delivery vector, obtains the base of two generation CAR or three generations CAR designs Because transmitting carrier；

(3) packaging gene transmits carrier；

(4) cmy vector obtains recombinant C AR-T carriers.

As currently preferred technical solution, gene delivery vector described in step (1) is third generation slow virus carrier, The third generation slow virus carrier includes the sequences of AmpR containing ampicillin resistance gene, prokaryotic replions pUC Ori sequences, virus Replicon SV40Ori sequences, RSV promoters, slow virus 5terminal LTR, 3 terminal Self- of slow virus Inactivating LTR, Gag cis elements, RRE cis elements, env cis elements, cPPT cis elements, eWPRE are enhanced Marmot hepatitis B posttranscriptional regulatory element；Step (3) is specially：The carrier that obtained two generation CAR or three generations CAR are designed HEK293T/17 cells are transfected jointly with slow virus packaging plasmid pPac-GP, pPac-R and memebrane protein plasmid pEnv-G respectively, After carrying out gene transcript expression in HEK293T/17 cells, packing successfully recombined lentivirus vector can be discharged into cell culture In clear, the supernatant of the recombined lentivirus vector included is collected.

As currently preferred technical solution, step (4) is specially：The purifying is using the column for filtering, adsorbing, eluting Way of purification.

As currently preferred technical solution, in step (2), entire CAR gene expressions are started by people EF1 α promoters； CD8 leader Chimerical receptors signal peptide is located at the N-terminal of CAR coded sequences, for CAR albumen to be guided to be positioned at cell membrane； EGFRvIII single-chain antibody light chains VL, single-chain antibody hinge Linker, EGFRvIII single-chain antibody heavy chain VH are combined into scfv areas Domain, for identifying EGFRvIII antigens；CD8 Hinge Chimerical receptors hinge is used to scfv being anchored on the outside of cell membrane；CD8 Transmembrane Chimerical receptors transmembrane region is used to entire Chimerical receptor being fixed on cell membrane；CD137 Chimerical receptors pierce altogether The factor is swashed for stimulating T cell proliferation and cytokine secretion；TCR Chimerical receptor t cell activations domain is used to activate downstream signal The expression of access；When EGFRvIII scFv regions and EGFRvIII antigen bindings, signal is transferred to cell by Chimerical receptor Interior, so as to generate T cell proliferation, cytokine secretion increases, Anti-apoptotic proteins secretion increases, cell death delay, splits Solve a series of biological effects of target cell；PD-1 shRNA in 3 ' UTR inhibit PD-1 in T lymphocytes to express, and reduce tumour Immunologic escape enhances the killing to tumour cell.

As currently preferred technical solution, in step (4), the suction filtration step will control supernatant volume in 200ml ~2000ml, control vacuum degree prevent the carrier loss brought due to plug-hole in -0.5MPA~-0.9MPA；The adsorption step Controlling the pH value of solution, preventing the variation of PH causes carrier to inactivate 6~8；The elution step to control eluent from Sub- intensity prevents the variation of ionic strength from causing elution not exclusively or carrier inactivation in 0.5M~1.0M.

In the third aspect of the present invention, above-mentioned carrier is provided in preparing interference PD-1 and reducing the drug of tumor immune escape Application.

Compared with prior art, the present invention has the advantages that：

Chimeric antigen receptor (CAR) is the core component (shown in Fig. 1) of CAR-T, and it is non-dependent to assign T lymphocytes HLA Mode identifies the ability of tumour antigen, this enables by the T cell that CAR is transformed compared to nave T cell surface receptor TCR Identify wider target.The CAR designs of the present invention include an EGFRvIII combined area and (are typically derived from monoclonal antibody The scFV sections of antigen binding regions), an extracellular hinge area, a transmembrane region, in an intracellular signal transduction area and 3 ' UTR PD-1 shRNA areas.ScFV sections of design is for the specificity of CAR, validity and the genetic modification T cell safety of itself Being crucial determinant.And the PD-1 shRNA areas in 3 ' UTR are that PD-1 in T lymphocytes is inhibited to express, and resist tumour Cellular immunity escape plays a crucial role.

Currently preferred gene delivery vector is that (third generation slow virus carrier exists third generation slow virus carrier " a kind of CAR-T transgene carriers and its construction method based on replication defective recombinant slow virus filed in 17 days March in 2016 And application " disclosed in patent of invention, number of patent application：201610008360.5), 3 ' SIN LTR eliminate U3 regions, eliminate The possibility of slow virus carrier self-replacation, substantially increases safety；CPPT and WPRE elements are increased, improve transduction effect The expression efficiency of rate and transgenosis；Core RNA continues efficiently to turn when ensure that slow virus carrier packaging using RSV promoters Record；Using the EF1 α promoters of people itself, enable CAR genes in human body long lasting for expression.

The present invention recombined lentivirus vector can realize on human T lymphocyte express EGFRvIII chimeric antigens by Body, guides and activated T lymphocytes are to the lethal effects of EGFRvIII positive cells, while lower the PD-1 in T lymphocytes Expression, killing of the enhancing T lymphocytes to EGFRvIII positive cells reduce the immunologic escape of tumour cell, clinically The effect of improving CAR-T- treatment glioblastomas.

Experiments verify that the PD-1 shRNA (lvCAR-EGFRvIII-PD1-shRNA) in the 3 ' UTR that the present invention uses PD-1 in T lymphocytes can effectively be inhibited to express, tumor immune escape is reduced, enhance the killing to tumour cell.With it is simple LvCAR-EGFRvIII and control lvCAR-EGFRvIII-NC-shRNA compare, can significantly improve cell factor secretion, The killing effect in vitro and clinical therapeutic efficacy of CAR-T cells, have reached unexpected technique effect.

Description of the drawings

The schematic diagram of Fig. 1 CAR of the present invention, wherein Figure 1A are the basic block diagrams of CAR, and Figure 1B is the generation of CAR Improve schematic diagram；

Slow virus carrier structure diagram in Fig. 2 embodiment of the present invention 1；

Fig. 3 is the structure flow chart that recombined lentivirus vector of the present invention is built in the embodiment of the present invention 1.Wherein, Fig. 3 A are the structure diagrams of slow virus skeleton plasmid pLenti-3G basic；Fig. 3 B are pLenti-3G basic plasmid inscribes Digestion removes ZsGreen1 green fluorescent protein schematic diagrames；Fig. 3 C are CAR-EGFRvIII, CAR-EGFRvIII- of gene chemical synthesis NC-shRNA, CAR-EGFRvIII-PD1-shRNA fragmentary views；Fig. 3 D are pCAR-EGFRvIII plasmid construct schematic diagrames； Fig. 3 E are pCAR-EGFRvIII-NC-shRNA plasmid construct schematic diagrames；Fig. 3 F are pCAR-EGFRvIII-PD1-shRNA plasmids Structure diagram；Fig. 3 G are slow virus packaging plasmid pPac-GP plasmid construct schematic diagrames；Fig. 3 H are slow virus packaging plasmids PPac-R plasmid construct schematic diagrames；Fig. 3 I are memebrane protein pEnv-G plasmid construct schematic diagrames；

Fig. 4 is that recombinant slow virus detection of mycoplasma is as a result, lane1 is DL2000marker in the embodiment of the present invention 3, from upper It is followed successively by from top to bottom to lower counterband tape：2kb、1kb、750bp、500bp、250bp、100bp；Lane2 is positive control； Lane3 is negative control；Lane4 is lvCAR-EGFRvIII；Lane5 is lvCAR-EGFRvIII-NC-shRNA；Lane6 is lvCAR-EGFRvIII-PD1-shRNA；Lane7 is PBS；Lane8 is H₂O；Lane9 is Lysis buffer；

Fig. 5 be the embodiment of the present invention 4 in mRNA relative expression quantities block diagram, QPCR the result shows that CAR in PBMC cells Interior high efficient expression；

Fig. 6 is PD-1 interference effect QPCR block diagrams in the embodiment of the present invention 4, the results showed that is done in the intracellular PD-1 of PBMC It disturbs with obvious effects；

Fig. 7 is that the killing-efficiency schematic diagram under the conditions of the different effect target ratios of LDH detections, E are thin for effect in the embodiment of the present invention 4 Born of the same parents, T are target cell the result shows that the killing-efficiency of lvCAR-EGFRvIII-PD1-shRNA groups is apparently higher than lvCAR- EGFRvIII groups and lvCAR-EGFRvIII-NC-shRNA groups；

Fig. 8 is the different Cytokine Expression Level schematic diagrames under the conditions of imitating target ratio of QPCR detections, E in the embodiment of the present invention 4 For effector cell, T is target cell；Wherein, Fig. 8 A represent the mRNA transcriptional levels of IL-2；Fig. 8 B represent the mRNA transcriptions of IFN-γ It is horizontal；The result shows that compared with lvCAR-EGFRvIII groups and lvCAR-EGFRvIII-NC-shRNA groups, lvCAR- EGFRvIII-PD-1 shRNA groups can significantly raise the expression of IL-2 and IFN-γ inflammatory factor, contribute to CAR-T effector cell Killing to tumour cell.

Specific embodiment

The invention is expanded on further with reference to specific embodiment.It should be understood that particular implementation described here It represents by way of example, is not intended as limitation of the present invention.Without departing from the scope of the invention, it is of the invention Main feature can be used for various embodiments.Embodiment 1 builds CAR recombined lentivirus vectors

First, material

1st, slow virus skeleton plasmid pLenti-3G basic (slow virus carrier structure is shown in Fig. 2), slow virus packaging plasmid PPac-GP, pPac-R and memebrane protein plasmid pEnv-G, HEK293T/17 cell, homologous recombination enzyme are taken wing (Shanghai) biology by generation Pharmaceutical Technology Co., Ltd provides；

2nd, people EF1 α promoters (SEQ ID NO.1), CD8 leader Chimerical receptors signal peptide (SEQ ID NO.2), EGFRvIII single-chain antibody light chains VL (SEQ ID NO.3), single-chain antibody hinge Linker (SEQ ID NO.4), EGFRvIII Heavy chain VH (SEQ ID NO.5), CD8 Hinge Chimerical receptors hinge (SEQ ID NO.6), CD8 Transmembrane are fitted into Receptor transmembrane area (SEQ ID NO.7), CD137 Chimerical receptors costimulating factor (SEQ ID NO.8), TCR Chimerical receptor T cells Add mir- in activation domain (SEQ ID NO.9), 3 ' UTR plus in mir-30 arms and PD-1shRNA (SEQ ID NO.10), 3 ' UTR 30 arms and NC shRNA (SEQ ID NO.11)；

3rd, primer：Primer according to needed for design of primers principle designs amplification of DNA fragments, the primer is by Shanghai biotech firm Synthesis, specially：

WPRE-QPCR-F：5’-CCTTTCCGGGACTTTCGCTTT-3’(SEQ ID NO.12)

WPRE-QPCR-R：5’-GCAGAATCCAGGTGGCAACA-3’(SEQ ID NO.13)

Actin-QPCR-F：5’-CATGTACGTTGCTATCCAGGC-3’(SEQ ID NO.14)

Actin-QPCR-R：5’-CTCCTTAATGTCACGCACGAT-3’(SEQ ID NO.15)

CAR-QPCR-F：5’-GACTTGTGGGGTCCTTCTCCT-3’(SEQ ID NO.16)

CAR-QPCR-R：5’-GCAGCTACAGCCATCTTCCTC-3’(SEQ ID NO.17)

PD-1-QPCR-F：5’-CCCTGGTGGTTGGTGTCGTG-3’(SEQ ID NO.18)

PD-1-QPCR-R：5’-GCCTGGCTCCTATTGTCCCTC-3’(SEQ ID NO.19)

IL2-QPCR-F：5’-GGACTTAATCAGCAATATCAAC-3’(SEQ ID NO.20)

IL2-QPCR-R：5’-AAGGTAATCCATCTGTTCAG-3’(SEQ ID NO.21)

IFN-γ-QPCR-F：5’-TTCTCTTGGCTGTTACTG-3’(SEQ ID NO.22)

IFN-γ-QPCR-R：5’-TTCTGTCACTCTCCTCTT-3’(SEQ ID NO.23)

4、SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、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, shown in SEQ ID NO.23 DNA sequence dna is synthesized by Shanghai biotech firm, and is preserved with oligonucleotides dry powder or plasmid form；

5th, toolenzyme Cla I, Sal I are purchased from NEB companies；

6th, high-fidelity enzyme PrimeSTAR, RN are purchased from Takara companies；

7th, 0.22 μm of -0.8 μm of PES filter is purchased from millipore companies；

8th, plasmid extraction kit, Ago-Gel QIAquick Gel Extraction Kit are purchased from MN companies；

9th, competent cell TOP10 is purchased from tiangen companies；

10、NaCl、KCl、Na₂HPO₄.12H₂O、KH₂PO₄、Trypsin、EDTA、CaCl₂, NaOH be purchased from Shanghai life work；

11st, Opti-MEM, FBS, DMEM, RPMI-1640, Hepes, purchased from invitrogen companies；

12nd, DNeasy kits are purchased from Shanghai JaRa company；

13rd, it is company that lymphocyte separation medium reaches section purchased from Shenzhen；

14th, mycoplasma test reagent box, endotoxin detection kit, EGFRvIII⁺K562 cells are taken wing in (Shanghai) purchased from generation Company；

15th, LDH detection kits are purchased from promega companies.

2nd, recombined lentivirus vector pCAR-EGFRvIII, pCAR-EGFRvIII-NC-shRNA, pCAR-EGFRvIII- The construction method of PD1-shRNA.

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

By the people EF1 α promoters (SEQ ID NO.1) of synthesis, CD8 leader Chimerical receptors signal peptides (SEQ ID NO.2), EGFRvIII single-chain antibodies light chain VL (SEQ ID NO.3), single-chain antibody hinge Linker (SEQ ID NO.4), EGFRvIII single-chain antibody heavy chains VH (SEQ ID NO.5), CD8 Hinge Chimerical receptors hinge (SEQ ID NO.6), CD8 Transmembrane Chimerical receptors transmembrane region (SEQ ID NO.7), CD137 Chimerical receptors costimulating factor (SEQ ID NO.8), add mir-30 arms and NC shRNA (SEQ in TCR Chimerical receptors t cell activation domain segment (SEQ ID NO.9), 3 ' UTR ID NO.11), in 3 ' UTR plus mir-30 arms and PD-1 shRNA (SEQ ID NO.10) are cloned into slow virus skeleton plasmid PLenti-3G basic, respectively obtain recombinant slow virus plasmid pCAR-EGFRvIII, pCAR-EGFRvIII-NC-shRNA, pCAR-EGFRvIII-PD1-shRNA。

SEQ ID NO.10 sequences are seen below, wherein, underscore part represents 5'mir30, and italicized item represents 3'mir30, Bolded section represents PD-1 shRNA.

SEQ ID NO.11 sequences are seen below, wherein, underscore part represents 5'mir30, and italicized item represents 3'mir30, Bolded section represents NC shRNA.

(1) slow virus skeleton plasmid pLenti-3G basic are carried out using Cla I and Sal I restriction enzymes double Digestion (Fig. 3 B), product pass through 1.5% agarose gel electrophoresis, and are tapped and recovered large fragment V1 (5833bp), are placed in In Eppendorf pipes, corresponding segment (being shown in Table 1) is recycled, and measure product with the Ago-Gel QIAquick Gel Extraction Kit of MN companies Purity and concentration；

1st, 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.
		2nd, with reference to DNA	11000g is centrifuged 30 seconds, discards filtrate.
3rd, film is washed	700 μ l NT3,11000g centrifugation 30 seconds is added in, discards filtrate.
		4th, film is washed	It is primary to repeat third step
5th, it dries	11000g is centrifuged 1 minute, and the collecting pipe renewed is placed at room temperature for 1 minute.
		6th, eluted dna	15-30 μ l NE are added in, are placed at room temperature for 1 minute, 11000g is centrifuged 1 minute, collects filtrate.

1 Ago-Gel recycling step of table

(2) by DNA fragmentation V1 respectively with CAR-EGFRvIII, CAR-EGFRvIII-NC-shRNA, CAR-EGFRvIII- PD1-shRNA segments are with 5 μ l total volumes and molar ratio 1:4 ratio is added in Eppendorf pipes, adds in homologous recombination enzyme reaction 15 μ l of liquid are incubated 30 minutes at 42 DEG C after mixing, are transferred to and are placed 2-3 minute on ice, by 50 μ l TOP10 of reaction solution addition, It gently rotates with mixing content, is placed 30 minutes in ice, pipe is put into heat shock in pre-heating to 42 DEG C of thermostat water bath 90 seconds, quickly pipe is transferred in ice bath, cell is made to cool down 2-3 minutes, often pipe plus 900 μ l LB culture solutions, then shift pipe Onto 37 DEG C of shaking tables, incubating 1 hour makes bacteria resuscitation, and the transformed bacteria solution of 100 μ l is taken to be coated on Amp LB agar plates, is inverted Plate, 37 DEG C of cultures in constant incubator, 16 hours.

In picking monoclonal to 1ml LB (Amp+) fluid nutrient medium, 37 DEG C of shaking tables 200 turn over night, and inspection in second day is surveyed Sequence identifies correctly clone；

(3) by corresponding recombinant slow virus plasmid glycerol stock 20ul inoculation with 200ml LB (Amp+) fluid nutrient medium in, 37 DEG C of shaking tables 200 turn over night, second day harvest bacterium solution, and it is pure to detect plasmid for row plasmid extraction (MN companies plasmid extraction kit) Degree and concentration, -20 DEG C spare.

Embodiment 2 recombinant slow virus lvCAR-EGFRvIII, lvCAR-EGFRvIII-NC-shRNA, lvCAR- The packaging of EGFRvIII-PD1-shRNA.

(1) complete medium：Preheated fresh culture is taken out, 10%FBS+5ml Pen-Srep is added in, runs up and down Mixing；

(2) 1XPBS solution：Weigh NaCl 8g, KCl 0.2, Na₂HPO₄.12H₂O 3.58g, KH₂PO4 0.24g are placed in In 1000ml beakers, the ultrapure water dissolutions of 900ml Milli-Q grade are added in, after the completion of dissolving, use 1000ml graduated cylinder constant volumes To 1000ml, 121 DEG C of high-temperature heat sterilization 20min；

(3) 0.25%Trypsin solution:Trypsin 2.5g, EDTA 0.19729g is weighed to be placed in 1000ml beakers, 900ml 1XPBS dissolving is added in, after the completion of dissolving, 1000ml is settled to using 1000ml graduated cylinders, 0.22 μM of filtration sterilization, for a long time Using can preserve to -20 DEG C of refrigerators；

(4) 0.5M CaCl2 solution：Weigh 36.75g CaCl₂With the ultrapure water dissolutions of 400ml Milli-Q grade；With Total volume is settled to 500ml, mixing by Milli-Q grade ultra-pure waters；0.22 μm of filtration sterilization, packing are saved in 50ml centrifugations Guan Zhong, often pipe 45ml or so, 4 DEG C of preservations.

(5) 2XHBS solution：Weigh 4.09g NaCl, 0.269g Na₂HPO4,5.96g Hepes, with 400ml Milli- The ultrapure water dissolutions of Q grade；After calibrating pH instrument, the pH of HBS solution is transferred to 7.05 with 2M NaOH solutions.Adjust every bottle of HBS's PH consumption 2M NaOH are 3ml or so；

(6) the HEK293T/17 cells frozen are taken out from liquid nitrogen container, are 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 passed on more than 80% for 24 hours；

(7) selection cell state is good, free of contamination HEK293T/17 cells, is one group per 2-6 culture dish, by cell After pancreatin digestion, 4-12ml complete mediums are drawn with electric pipettor, 2ml is added into each postdigestive culture dish, is avoided Culture dish is dried；All cells are blown and beaten into single cell suspension using 1ml pipettors, 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, turn upside down 10 times or so abundant mixing cell suspensions, and cell is passed to 8-24 10cm²In culture dish, the cell density per ware should about 4 × 10⁶A/10ml complete mediums or so.If cell density and pre- The difference of phase is larger, then needs to count cell, then according to 4 × 10⁶The amount inoculation of a/ware；

(10) every 6 culture dishes are arranged piles up for one, pays attention to keeping the cooperation between ware up and down.It is front and rear by culture dish or so It shakes for several times, cell is made fully to spread out, is then placed in 5%CO₂Incubator.Remaining cell does similary processing；

(11) institute's passage cell is checked, cell confluency degree should be 70-80%, and profile is full, adherent good, 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, per 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.Per ware HEK293T/17 cell transfecting plasmid amounts 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 5ml centrifuge tubes add in 0.5M CaCl2：0.25ml, 20 μ g of recombinant slow virus plasmid：pPac-GP 15μg：pPac-R 10μg： 7.5 μ g of pEnv-G, supplement ultra-pure water to 0.5ml close the lid, abundant mixing；

(14) it is another to take a 5ml centrifuge tube, add in 0.5ml DNA/CaCl2 solution.Turbula shaker is opened, a hand is taken The firmly upper end of 5ml centrifuge tubes 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 0.5mL 2 × HBS 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 is turned drop adds, 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, covers and marks in ware, culture dish is released to another 5%CO₂In incubator. Ensure that culture dish is horizontal positioned, often pile up culture dish and do 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, and replaces the fresh DMEM complete mediums of 10ml；

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

After (20) 72 hours, 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, lvCAR- are contained in the supernatant collected at this time EGFRvIII-NC-shRNA、lvCAR-EGFRvIII-PD1-shRNA。

The concentration and detection of 3 recombined lentivirus vector of embodiment

1st, ion exchange chromatography recombinant slow virus

(1) supernatant of collection is filtered, except impurity elimination using Thermo vacuum pumps through 0.22 μm -0.8 μm of PES filters Matter；

(2) by 1:1~1:10 ratio adds in 1.5M NaCl 250mM Tris-HCl (pH 6-8) into supernatant；

(3) 2 ion exchange columns are placed in series, with 4ml 1M NaOH, 4ml 1M NaCl, 5ml 0.15M NaCl 25mM Tris-HCl (pH6-8) solution crosses column successively；

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

(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, preserved for a long time.

2nd, recombinant slow virus titer determination；

(1) 24 orifice plates is taken to be inoculated with 293T cells.It is 5 × 10 per hole cell⁴A, 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 pipes, the fresh complete medium (DMEM in high glucose+10% of 90ul is added in each pipe FBS) inoculating cell takes the cell in two holes to be counted with blood counting chamber after 24 hours, determines the actual number of cell during infection, It is denoted as N；

(3) virus stock solution used 10ul to be determined is taken to be added in first pipe, gently after mixing, 10ul is taken to be added to second In a pipe, a to the last pipe is then operated successively；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, is changed to 500 μ l complete medium (DMEM in high glucose+10% 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) it with 0.25% pancreas enzyme -EDTA solution digestion cells of 0.2ml, 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 kits.200 are added in each sample cell μ l eluents are washed lower DNA and are quantified；

(7) prepare target DNA detection qPCRmix manifold I (QPCR primers are SEQ ID NO.12-SEQ ID NO.13)：

N=number of reactions. are 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 are mixed With.It is placed on ice after concussion；

(8) preparing internal reference DNA detection qPCRmix pipes II, (QPCR primer sequences are SEQ ID NO.14-SEQ ID NO.15)：

2×TaqMan Master Mix 25μl×n

10×RNaseP primer/probe mix 2.5μl×n

H₂O 17.5μl×n

N=number of reactions. are for example：Overall reaction number is 40, by 2 × TaqMan of 1ml Universal PCR Master Mix, 100 μ l 10 × 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 precooling to establish.45 μ l is respectively taken to be added to each rows of A-D from manifold I Hole in, 45 μ l is respectively taken to be added in the hole of each rows of E-G from manifold II.

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

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

(12) it is 7500 quantitative systems of ABI PRISM to use quantitative PCR apparatus.Cycling condition is set as：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 cycles.

Data analysis：The slow virus carrier copy number integrated in the DNA sample measured is demarcated with genome number, is obtained Viral copy number per 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：The average viral copy numbers per genome conformity of C=

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

The extension rate of D=viral vectors

The volume number of dilution virus that V=is added in

3rd, recombinant slow virus endotoxin measurement；

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

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

(3), endotoxin standard dilutes：Endotoxin standard one is taken, is diluted to 4 λ and 2 λ in proportion with BET water respectively Dissolving, sealed membrane sealing, concussion dissolving 15min；Often diluted during dilution a step should all on eddy mixer mixing 30s；

(4), it is loaded：Take reagents several, every adds in BET water 0.5ml dissolvings, dispenses to several endotoxin-frees and tries Guan Zhong, often pipe 0.1ml.

Wherein 2 are negative control pipe, add in BET water 0.1ml；

2 are positive control pipe, add in the endotoxin working standard solution 0.1ml of 2 λ concentration；

2 are Sample Positive control tube, and adding in sample solutions of the 0.1ml containing 2 λ endotoxin standards, (20 times of dilution is treated The endotoxin standard solution 1ml=2ml of sample 1ml+4 λ contains 40 times of samples of dilution of 2 λ endotoxin standards).

Add in 0.1ml samples in sample cell, dilution ratio be shown in Table 2,37 ± 1 DEG C of water-baths (or incubator) heat preservation 60 ± 1min；

2 endotoxin dilution ratio of table and corresponding endotoxin content

(5), recombined lentivirus vector pCAR-EGFRvIII, pCAR-EGFRvIII-NC-shRNA, pCAR-EGFRvIII- The endotoxin testing result (as shown in table 3) of PD1-shRNA, endotoxin content meet the requirements between 0~2.5EU/ml；

Extension rate	Stoste	5	10	20	40	80	160
								Corresponding EU/ml	0.25	1.25	2.5	5	10	20	40
pCAR-EGFRvIII	+	+	-	-	-	-	-
								pCAR-EGFRvIII-NC-shRNA	+	+	-	-	-	-	-
pCAR-EGFRvIII-PD1-shRNA	+	+	-	-	-	-	-

The endotoxin testing result of 3 recombined lentivirus vector of table

4th, recombinant slow virus mycoplasma measures；

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

(2) (cell number is more than 1*10 to collection 1ml cell suspending liquids⁵), it is placed in 1.5ml centrifuge tubes；

(3) 13000 × g centrifuge 1min, collect precipitation, discard culture medium；

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

(5) step 4 is repeated once；

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

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

After (8) 13000 × g centrifugations 5min, taking 5 μ l supernatants, 25 μ lPCR reaction systems are as template：ddH20 6.5μ L, 1 μ l of Myco Mix, 2x Taq Plus Mix Master (Dye Plus) 12.5 μ l, 5 μ l of template；PCR cycle condition is： 95 DEG C of 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 result is shown (as shown in Figure 4), and the recombinant slow virus of purifying is free of mycoplasma.

Embodiment 4 recombinant slow virus 1vCAR-EGFRvIII, 1vCAR-EGFRvIII-NC-shRNA, 1vCAR- The Function detection of EGFRvIII-PD1-shRNA.

1st, the cellular level detection of expression of CAR genes：

(1) recombinant slow virus 1vCAR-EGFRvIII, lvCAR-EGFRvIII-NC-shRNA, 1vCAR-EGFRvIII- After PD1-shRNA infection of PBMCs cells, the detection that cell carries out CAR mRNA transcriptional levels using QPCR is collected, verifies CAR bases The expression of cause if CAR mRNA transcriptional levels increase, illustrates that the transcriptional level of CAR genes is expressed successfully；

(2) respectively by lvCAR-EGFRvIII, lvCAR-EGFRvIII-NC-shRNA, lvCAR- of MOI=15 EGFRvIII-PD1-shRNA and comparison virus MOCK infection cells, the total serum IgE that cell in 6 orifice plates is extracted after 48h carry out fluorescence Quantitative PCR experiment.Specific steps：Four holes of 6 orifice plates are coated with, each hole adds in corresponding PBS and RN, and 4 DEG C overnight.12 hours Afterwards by MOI=15 coating viruses, 37 DEG C of incubators place 5h；6 orifice plates taken out, discard viral supernatants, are washed twice, pressed with PBS 1*10⁶/ hole, coating PBMC (being detached from people's blood with lymphocyte separation medium), add in 500ul culture mediums (containing 10% serum, 20U/ml IL-2、Polybrene 8ug/ml).Stand 20min, 1000g20 DEG C of centrifugation 30min, 37 DEG C of culture 48h.

(3) Trizol methods extract the total serum IgE of PBMC cells in 6 orifice plates, reverse transcription amplification cDNA, with QPCR primer (sequences For SEQ ID NO.16-SEQ ID NO.17, SEQ ID NO.18-SEQ ID NO.19) fluorescent quantitative PCR experiment is carried out, instead System is answered to be shown in Table 4, using internal reference Actin as control group, verifies the transcription situation of its mRNA.

Reagent	Volume (μ l)
		SYBR premix ex taq:	10μl
ROX Reverse Dye(50x)	0.4μl
		Sense primer (2.5 μM)：	0.5μl
Downstream primer (2.5 μM)：	0.5μl
		cDNA	1.0μl
ddH₂O	7.6μl

4 20 μ l qPCR reaction systems of table

(4) QPCR detections show that the expression of the CAR-EGFRvIII after recombinant slow virus infection of PBMCs is than control disease Malicious MOCK and ghost are increased significantly (as shown in Figure 5), illustrate CAR gene expressions success；

(5) QPCR, which is detected, shows, the PD- after recombinant slow virus infection of PBMCs in lvCAR-EGFRvIII-PD1-shRNA groups 1 expression than comparison virus MOCK and ghost, lvCAR-EGFRvIII, lvCAR-EGFRvIII-NC-shRNA virus group have it is bright Aobvious decline (as shown in Figure 6) illustrates that PD1-shRNA can significantly interfere with PBMC cells in lvCAR-EGFRvIII-PD1-shRNA The expression of middle PD-1, this contributes to the immunologic escape for reducing tumour cell；

2nd, fragmentation effect assessment and cytokine secretion detection.

(1) EGFRvIII+K562 cells and effector cell lvCAR-EGFRvIII, lvCAR-EGFRvIII- are cultivated respectively NC-shRNA、lvCAR-EGFRvIII-PD1-shRNA；

(2) target cell (EGFRvIII+K562) 4x10 is collected⁵Cells and effector cell's (CART cells) 2.8x10⁶Cells, 800g, 6min are centrifuged, and 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 mediums (1640 culture medium+10%FBS), with (1640 cultures of 2ml culture mediums Base+10%FBS) target cell is resuspended；

(6) setting effect target ratio is 1:1、5:1、10:1 experimental port, and control group is set, every group of 3 multiple holes；

(7) 250xg, 5min tablet centrifuge；

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

(9) 250xg, 5min tablet centrifuge；

(10) it takes in the 50ul supernatants to new 96 orifice plate in each hole, and adds in 50ul substrate solutions per hole and (be protected from light behaviour Make)；

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

(12) 50ul terminate liquids are added in per hole；

(13) microplate reader detection 490nm absorbances；

(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 formula below, it is thinner than generated to calculate each effect target Cellular toxicity percentage.The results are shown in Figure 7, and the PBMC cells of recombinant slow virus lvCAR-EGFRvIII-PD1-shRNA infection exist Killing-efficiency is apparently higher than lvCAR-EGFRvIII and lvCAR-EGFRvIII-NC-shRNA infection under the conditions of difference effect target ratio PBMC cells；Illustrate that PD1-shRNA effectively enhances lethal effect of the CAR-T effector cell to tumour 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 Fig. 8 institutes Show, recombinant slow virus lvCAR-EGFRvIII-PD1-shRNA infection PBMC cells it is different effect target ratios under the conditions of IL-2 and The mRNA transcriptional levels of IFN-γ are apparently higher than the PBMC of lvCAR-EGFRvIII and lvCAR-EGFRvIII-NC-shRNA infection Cell.Illustrate that PD1-shRNA can significantly raise the expression of IL-2 and IFN-γ inflammatory factor, effectively enhance the table of inflammatory factor It reaches, so as to significantly improve lethal effect of the CAR-T effector cell to tumour cell.

From more than contrast and experiment Fig. 7 and Fig. 8 as it can be seen that PD-1 shRNA (lvCAR- in the 3 ' UTR that use of the present invention EGFRvIII-PD1-shRNA PD-1 in T lymphocytes) can effectively be inhibited to express, tumor immune escape is reduced, enhance to tumour The killing of cell.It, can be notable compared with simple lvCAR-EGFRvIII and control lvCAR-EGFRvIII-NC-shRNA Improve the secretion of cell factor, the killing effect in vitro and clinical therapeutic efficacy of CAR-T cells.

Sequence table

<110>Shanghai Shengbo Biomedical Technology Co., Ltd.

<120>The CAR-T carriers and construction method of targeting EGFR vIII interference PD-1 reduction tumor immune escapes and application

<130>HJ16-12449

<160> 25

<170> PatentIn version 3.5

<210> 1

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<213>Artificial sequence

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gcagctacag ccatcttcct c 21

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gcctggctcc tattgtccct c 21

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<213>Artificial sequence

<220>

<221> misc_feature

<223>Primer

<400> 23

ttctgtcact ctcctctt 18

<210> 24

<211> 123

<212> DNA

<213>Artificial sequence

<400> 24

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 25

<211> 57

<212> DNA

<213>Artificial sequence

<400> 25

accggaggcg cagatcaaag agagctcgag ctctctttga tctgcgcctt tttttgc 57

Claims

1. a kind of targeting EGFR vIII interference PD-1 reduces the CAR-T carriers of tumor immune escape, which is characterized in that including：

The sequence of PD-1shRNA in 3 ' UTR, the PD-1shRNA are as shown in SEQ ID NO.25；

Gene delivery vector；

And integrating identification, transmission, two generation CAR of startup or the Chimeric antigen receptor of three generations CAR for forming, this is chimeric Antigen receptor includes EGFRvIII single-chain antibody light chain VL and EGFRvIII single-chain antibody heavy chains VH.

2. carrier as described in claim 1, which is characterized in that

It is described for form integrate identification, transmission, startup the Chimeric antigen receptor of two generation CAR include：Such as SEQ ID CD8 leader Chimerical receptors signal peptide shown in NO.2, the EGFRvIII single-chain antibody light chains VL as shown in SEQ ID NO.3, Single-chain antibody hinge Linker as shown in SEQ ID NO.4, the EGFRvIII single-chain antibody heavy chains as shown in SEQ ID NO.5 VH, the CD8 Hinge Chimerical receptors hinge as shown in SEQ ID NO.6, as shown in SEQ ID NO.7 CD8Transmembrane Chimerical receptors transmembrane region, the CD137 Chimerical receptors costimulating factor as shown in SEQ ID NO.8, such as TCR Chimerical receptor t cell activations domain shown in SEQ ID NO.9；

It is described for form integrate identification, transmission, startup the Chimeric antigen receptor of three generations CAR include：Such as SEQ ID CD8 leader Chimerical receptors signal peptide shown in NO.2, the EGFRvIII single-chain antibody light chains VL as shown in SEQ ID NO.3, Single-chain antibody hinge Linker as shown in SEQ ID NO.4, the EGFRvIII single-chain antibody heavy chains as shown in SEQ ID NO.5 VH, CD8 Hinge Chimerical receptors hinge, the CD8 as shown in SEQ ID NO.7 as shown in SEQ ID NO.6 Transmembrane Chimerical receptors transmembrane region, CD137 Chimerical receptors costimulating factor, such as SEQ as shown in SEQ ID NO.8 TCR Chimerical receptor t cell activation domains shown in ID NO.9 and the CD28 Chimerical receptors as shown in SEQ ID NO.24 pierce altogether Swash the factor.

3. carrier as described in claim 1, which is characterized in that the gene delivery vector includes slow virus carrier, reverse transcription Viral vectors, adenovirus vector, gland relevant viral vector.

4. carrier as claimed in claim 3, which is characterized in that the gene delivery vector is third generation slow virus carrier, should It is multiple that third generation slow virus carrier includes the sequences of AmpR containing ampicillin resistance gene, prokaryotic replions pUC Ori sequences, virus System SV40 Ori sequences, RSV promoters, slow virus 5terminal LTR, slow virus 3terminal Self- Inactivating LTR, Gag cis elements, RRE cis elements, env cis elements, cPPT cis elements, eWPRE are enhanced Marmot hepatitis B posttranscriptional regulatory element.

5. carrier as described in claim 1, which is characterized in that the PD-1shRNA in the 3 ' UTR increases in 3 ' UTR Mir-30 arms form the PD-1shRNA sequences as shown in SEQ ID NO.10.

6. a kind of CAR- that tumor immune escape is reduced such as claim 1-5 any one of them targeting EGFRs vIII interference PD-1 The construction method of carrier T, which is characterized in that include the following steps：

(1) gene delivery vector is provided；

(2) by as shown in SEQ ID NO.1 people EF1 α promoters, in 3 ' UTR plus PD-1shRNA, for forming collection identification, passing It passs, start and be combined into two generation CAR or three generations's CAR designing schemes in the two generation CAR or the Chimeric antigen receptor of three generations CAR of one, It is cloned into gene delivery vector by digestion, connection, recombining reaction, obtains the gene delivery of two generation CAR or three generations CAR designs Carrier；

(3) packaging gene transmits carrier；

(4) cmy vector obtains recombinant C AR-T carriers.

7. method as claimed in claim 6, which is characterized in that gene delivery vector described in step (1) is sick slowly for the third generation Poisonous carrier, the third generation slow virus carrier include the sequences of AmpR containing ampicillin resistance gene, prokaryotic replions pUC Ori sequences Row, Viral Replicon SV40 Ori sequences, RSV promoters, slow virus 5terminal LTR, slow virus 3terminal Self- Inactivating LTR, Gag cis elements, RRE cis elements, env cis elements, cPPT cis elements, eWPRE are enhanced Marmot hepatitis B posttranscriptional regulatory element；Step (3) is specially：The carrier that obtained two generation CAR or three generations CAR are designed HEK293T/17 cells are transfected jointly with slow virus packaging plasmid pPac-GP, pPac-R and memebrane protein plasmid pEnv-G respectively, After carrying out gene transcript expression in HEK293T/17 cells, packing successfully recombined lentivirus vector can be discharged into cell culture In clear, the supernatant of the recombined lentivirus vector included is collected.

8. method as claimed in claim 6, which is characterized in that step (4) is specially：The purifying, which uses, to be filtered, adsorbs, washing De- column purification mode.

9. method as claimed in claim 6, which is characterized in that in step (2), entire CAR bases are started by people EF1 α promoters Because of expression；CD8 leader Chimerical receptors signal peptide is located at the N-terminal of CAR coded sequences, for CAR albumen to be guided to be positioned at cell Film；EGFRvIII single-chain antibody light chains VL, single-chain antibody hinge Linker, EGFRvIII single-chain antibody heavy chain VH are combined into scfv Region, for identifying EGFRvIII antigens；CD8 Hinge Chimerical receptors hinge is used to scfv being anchored on the outside of cell membrane；CD8 Transmembrane Chimerical receptors transmembrane region is used to entire Chimerical receptor being fixed on cell membrane；CD137 Chimerical receptors pierce altogether The factor is swashed for stimulating T cell proliferation and cytokine secretion；TCR Chimerical receptor t cell activations domain is used to activate downstream signal The expression of access；When EGFRvIII scFv regions and EGFRvIII antigen bindings, signal is transferred to cell by Chimerical receptor Interior, so as to generate T cell proliferation, cytokine secretion increases, Anti-apoptotic proteins secretion increases, cell death delay, splits Solve a series of biological effects of target cell；PD-1shRNA in 3 ' UTR inhibits PD-1 in T lymphocytes to express, and reduces tumour and exempts from Epidemic disease is escaped, and enhances the killing to tumour cell.

10. if claim 1-4 any one of them carrier is in preparing interference PD-1 and reducing the drug of tumor immune escape Using.