CN105950664A - CD 123-targeting replication-defective recombinant lentivirus CAR-T transgenic vector as well as construction method and applications thereof - Google Patents

Abstract

The invention discloses a CD 123-targeting replication-defective recombinant lentivirus CAR-T transgenic vector. The CD 123-targeting replication-defective recombinant lentivirus CAR-T transgenic vector comprises a prokaryotic replicor pUC Ori sequence used for plasmid duplication; an amicillin resistance gene AmpR sequence used for amplification of a large number of target strains; a virus replicor SV40 Ori sequence used for enhancing the replication in eukaryocytes; a lentivirus packaging cis element used for lentivirus packaging; ZsGreen 1 green fluorescent protein used for green fluorescence expression of eukaryocytes; an IRES ribosome binding sequence used for co-transcription and co-expression of protein; a human EF1 alpha promoter used for eukaryotic transcription of genes of a chimeric antigen acceptor; the chimeric antigen acceptor used for forming second-generation CAR or third-generation CAR integrating recognition, delivery and promoting; an eWPRE element used for enhancing the expression efficiency of transgenes. In addition, the invention further discloses a construction method and applications of the vector. With the vector, the secretion of the cell factors and the in-vitro lethal effect of the CAR-T cells can be obviously improved, and the effect in treatingacute myelogenous leukemia (AML) clinically is outstanding.

Description

A kind of replication defective recombinant slow virus CAR-T transgene carrier of targeting CD123 and Its construction method and application

Technical field

The invention belongs to field of medical biotechnology, be specifically related to a kind of carrier, the duplication particularly relating to a kind of targeting CD123 lacks Fall into the CAR-T transgene carrier of property recombinant slow virus.Moreover, it relates to the construction method of this carrier and application.

Background technology

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

Immunotherapy of tumors is generally divided into two classes, nonspecific immunity and specific immunity.Nonspecific immunotherapy for bronchus master Interleukin II to be included (interle μ kin-2, IL-2), interferon-ALPHA (interferon α, IFN-α), tumor necrosis factor Son (t μm or necrosis factor, TNF-α), cytokine and the toxin such as bacillus calmette-guerin vaccine, adoptive cellular immunotherapy etc.. Specific active immunotherapy is mainly tumor vaccine.

1.1 tumor Nonspecific immunotherapy for bronchus

Nonspecific immune response is inherent, and its formation is not required to antigenic stimulus, can be widely for many Plant antigen, be the basis of immunne response, but specificity is strong, tend not to certain specific antigen material produce sufficient intensity React nonspecific immunization therapy.In the cytokine profiles entering clinical trial, interleukin II and interferon should With the most extensively [Rosenberg S A, Lotze M T, M μ μ l L M, et al.A progress report on the treatment of 157 patients with advanced cancerμsing lymphokine-activated killer cells and interleμkin-2or high-dose interleμkin-2alone[J].N Engl J Med, 1987,316 (15): 889-897.].

The immunization therapy of 1.2 anti-cancer monoclonal antibody

Over nearly more than 20 years, monoclonal antibody is used widely at therapeutic field of tumor.Antitumor monoclonal antibody medicine typically wraps Including two classes: one is antitumor monoclonal antibody, two is antitumor monoclonal antibody conjugate, or claims immune conjugate.Immune conjugate molecule is by list Anti-and " bullet " medicine two parts form, and " bullet " mainly includes radionuclide, medicine and toxin, after being connected with monoclonal antibody respectively Constitute radioimmunity conjugate, chemo-immunity conjugate and immunotoxin.Divide in November, 1997 and in October, 1998 U.S. FDA Two monoclonal antibody Rit μ ximab (rit μ xan) for clinical cancer therapy and Trast μ z μm ab are not passed through (herceptin)[Dillman R O.Magic bμllets at last Finally—approval of a Monoclonal antibody for the treatment of cancer [J] .Cancer Biother Radiopharm, 1997,12:223-225.].It is now recognized that the mechanism of action of monoclonal antibody has blocking effect, signal conduction and targeting Deng three kinds of mechanism of action, there is no direct lethal effect.Additionally there are the problem in terms of pharmacology, mainly arrive tumor Dose is not enough.Owing to conjugate is foreign protein, can be absorbed by reticuloendothelial system, have a great deal of will accumulate in liver, spleen and Bone marrow.Conjugate is macromolecular substances, by capillary endothelium layer and penetrate tumor cell external series gap and be all restricted.

The adoptive immunotherapy of 1.3 tumors

The adoptive immunotherapy of tumor refers to the autologous of Activation In Vitro or alloimmune effector lymphocyte's infusion to patient, with Kill tumor cell in the patient.A key issue in tumor adoptive immunotherapy is to find suitable tumor-killing Cell.Since the eighties in last century, including LAK, cell because of killing cell (the cytokine-ind μ ced of induction Killers, CIK), the cell such as TIL be successively applied to clinic, but amplification speed is relatively low, cell derived is stranded owing to also existing Difficult, the most high problems of cytotoxicity, are restricted in clinical practice.How to improve the specific for tumour antigen of T cell There is important clinical meaning.T cell to the identification of tumor antigen mainly by φt cell receptor (T cell receptor, TCR) human leukocyte antigen (h μm an le μ kocyte antigen, the HLA)-peptide complexes on tumor cell surface, because of This, the specificity of tumor antigen identification is depended on the TCR on T cell surface by T cell.Utilize the means clone of molecular biology swollen The TCR of tumor specific T-cells, and by building the viral vector containing TCR, TCR is proceeded in normal T cell, makes these T thin Born of the same parents become because carrying tumour-specific specific tumor killing cell [Johnson L A, Morgan R A, D μ dley M E, et al.Gene therapy with hμman and moμse T-cell receptors mediates cancer Regression and targets normal ti ss μ es expressing cognate antigen [J] .Blood, 2009,114 (3): 535-546.].

1.4 tumor vaccine therapy

Tumor vaccine therapy is the specificity antineoplastic immunity by exciting patient to importing tumor antigen in the patient Reaction.Hold time the advantage such as long owing to vaccine therapy has specificity, in vivo immunological effect, the most become research heat Point.In recent years polypeptide vaccine, nucleic acid vaccine, whole protein vaccine, anti-unique antibody vaccine, recombinant viral vaccine, bacterial vaccine, The tumour-cell vaccine of genetic modification, dendritic cell (dendritic cells, DC) vaccine etc. are widely studied and apply [Robbins P F, Morgan R A, Feldman S A, et al.T μm or regression in patients with metastatic synovial cell sarcoma and melanomaμsing genetically engineered Lymphocytes reactive with NY-ESO-1 [J] .J Clin Oncol, 2011,29 (7): 917-924.].Tumor The large-scale application of vaccine therapy also has the problems demand of three aspects to solve.First, tumor associated antigen, each tumor, every Individual hypotype, each neoplasm staging, these relative antigen presentations are different, so making an accurate selection of antigen, the patient crowd that makes an accurate selection of is to cause Close important.Second, how to reach tumor antigen and in dendritic cell, imitate absorption and express？Antigen is inhaled by dendritic cell Receipts are with surface receptor for mediation.Dendritic cell has ten several receptors, how to be subject to accordingly according to specific selection of antigen Body？3rd, for the regulation and control that differentiation of dendritic cells is ripe.The differentiation and maturation of dendritic cell is an extremely complex mistake Journey, it both can move towards to activate T cell and can also move towards to suppress T cell.[the therapeutic type tumor vaccine of targeting DC cell: bright spot with Challenge and deposit.http://www.biodiscover.com/news/research/115794.html].

1.5 tumor CAR-T treatments

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

The t cell activation of first generation CAR mediation is to be completed by the Tyrosine Activating Motifs on CD3z chain or FceRIg. CD3z chain can provide the letter needed for t cell activation, cracking target cell, regulation IL-2 secretion and internal performance anti-tumor activity Number.But the anti-tumor activity of first generation CAR transformation T cell is restricted in vivo, it is thin that T cell propagation minimizing ultimately results in T The apoptosis of born of the same parents.

Second filial generation CAR adds a new costimulatory signal in intracellular, it is demonstrated experimentally that this makes original making be derived from " signal 1 " of TCR/CD3 complex expands, and many researchs all show, is equipped with second filial generation CAR and first generation CAR of " signal 2 " Comparing, antigenic specificity is constant, and T cell propagation, cytokine secretion increase, and Anti-apoptotic proteins secretion increases, and cell is dead Die delay.Conventional costimulatory molecules is CD28, but has research to be replaced by CD28 CD137 (4-1BB) afterwards, except this it Outward, the thinking of a kind of NK of use cell receptor CD244 is also suggested.Although which is better and which is worse for different second filial generations CAR, no actually With researcher be not quite similar with the result that obtains in external research in vivo by different tumors.[degree of depth full version: CAR- The present situation of T and future. biological paddy .2015-051-15].

In order to improve the design of CAR further, many seminar start to be conceived to develop third generation CAR, not only include " letter Number 1 ", " signal 2 ", further comprises extra costimulatory signal.Different researchers open with different target spots and costimulatory signal There is certain diversity in second filial generation CAR and the comparative result of third generation CAR that the institute of exhibition obtains.Some research report tables The restructuring T cell reaching third generation CAR is significantly increased in terms of anti-tumor activity, time to live and release of cytokines； Second filial generation CAR of the result of study display targeting M Μ C1 of Wilkie etc. and third generation CAR restructuring T cell are at antitumor cell poison Property aspect no significant difference, although express the T cell of third generation CAR can secrete more substantial 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 should be noted that above-mentioned difference is only the conclusion obtained in experiment in vitro, compare the second filial generation and the most in vivo The report of three generations CAR.

Difference between this several generations CAR may come from signal transduction domain incessantly, the antigen binding domain (scFv) outside born of the same parents, weight The transfection method (slow virus VS retrovirus) of group T cell, feedback mode (the venous re-transfusion VS peritoneum VS tumor of restructuring T cell Body) etc. all may affect the final antitumous effect of CAR-T cell.

Filed in 17 days March in 2016 of the applicant invention entitled " a kind of based on replication defective recombinant slow virus CAR-T transgene carrier and construction method and application " application for a patent for invention, disclose the replication defective for CD19 The CAR-T transgene carrier of recombinant slow virus and construction method thereof and application.The present invention be directed to the CAR-T transgenic of CD123 Carrier.

Summary of the invention

One of the technical problem to be solved in the present invention is to provide the replication defective recombinant slow virus of a kind of targeting CD123 CAR-T transgene carrier.

The two of the technical problem to be solved in the present invention are to provide the replication defective recombinant slow virus of this targeting CD123 The construction method of CAR-T transgene carrier.

The three of the technical problem to be solved in the present invention are to provide the replication defective recombinant slow virus of this targeting CD123 The application of CAR-T transgene carrier.

The present invention relates to the medical configuration product containing peptide, be specifically related to:

One, containing ampicillin resistance gene AmpR sequence, prokaryotic replions pUC Or i sequence, Viral Replicon SV40Or i sequence, RSV promoter, people's EF1 α promoter, slow virus 5 terminal LTR, slow virus 3 terminal Self-Inactivating LTR, Gag cis element, RRE cis element, env cis element, cPPT cis element, IRES core Sugar body binding sequence, ZsGreen1 green fluorescent protein, the recombinant lentiviral disease of eWPRE marmot hepatitis B virus posttranscriptional regulatory element Poisonous carrier skeleton, this recombined lentivirus vector skeleton can carry different therapeutic genes and be widely used in adoptive carefully Born of the same parents treat field.

Two, recombined lentivirus vector skeleton, CD8leader Chimerical receptor signal peptide, CD123 single-chain antibody light chain VL, list Chain antibody hinge LinkerA, single-chain antibody hinge Linker B, single-chain antibody hinge Linker C, CD123 single-chain antibody heavy chain VH, CD8Hinge Chimerical receptor hinge, CD8Transmembrane Chimerical receptor cross-film district, CD28 Chimerical receptor stimulate altogether because of Son, CD137 Chimerical receptor costimulating factor, TCR Chimerical receptor t cell activation territory build and form recombined lentivirus vector, the party The recombined lentivirus vector that method obtains can be implemented in human T lymphocyte's upper expression CD123 Chimeric antigen receptor, guides and activates The T lymphocyte lethal effect to CD123 positive cell, is used clinically for treating acute myeloid leukemia (AML).

CAR-T technology for CD123 of the present invention, is that a kind of immunity combining anti-cancer monoclonal antibody is controlled The targeted therapy new technique of the adoptive immunotherapy advantage for the treatment of and tumor.CD123 is the alpha chain of human interleukin-13 receptor, greatly All there is expression on most acute myeloid leukemia (AML) cells and a lot of hematopoietic cell surface, and CD123 is one and extraordinary exempts from Epidemic disease therapy target, even if because in CD123 expresses little cell, its expression can increase in time and gradually strengthen, and Acute myeloid leukemia is likely due to be in preleukelic hematopoietic stem cell through clone evolution, with CD123 For target spot, then can play effect (Saar Gill, the Carl H.June et al.Preclinical targeting of clear marrow of human acute myeloid leukemia and myeloablation using chimeric antigen receptor-modified T cells.Blood.2014；123 (15): 2343-2354.), recent years is white at acute myeloid Significant curative effect is had it is considered to be the most promising acute myeloid leukemia (AML) therapeutic modality in the treatment of disorders of blood (AML) One of.

Chimeric antigen receptor (CAR) is the core component (shown in Fig. 1) of CAR-T, gives T lymphocyte HLA non-dependent The ability of mode identification tumor antigen, this make through CAR transformation T cell can compared to nave T cell surface receptor TCR Identify widely target.The basic engineering of CAR includes tumor associated antigen (t μm or-associated Antigen, TAA) land (being typically derived from the scFV section of monoclonal antibody antigen calmodulin binding domain CaM), an outer hinge region of born of the same parents, One cross-film district and an intracellular signal transduction district.The design of scFV section is for specificity, effectiveness and the genetic modification of CAR It it is crucial determiner for the safety of T cell self.

In order to solve above-mentioned technical problem, the present invention is achieved through the following technical solutions:

In one aspect of the invention, it is provided that the CAR-T transgenic of the replication defective recombinant slow virus of a kind of targeting CD123 Carrier, including: for the prokaryotic replions pUC Ori sequence of plasmid replication, as shown in SEQ ID NO.2；For purpose bacterial strain A large amount of amplifications containing ampicillin resistance gene AmpR sequence, as shown in SEQ ID NO.1；In strengthening eukaryotic cell The Viral Replicon SV40Ori sequence replicated, as shown in SEQ ID NO.3；Cis for the slow virus packaging of slow virus packaging Element；For the ZsGreen1 green fluorescent protein of eukaryotic cell expression green fluorescence, as shown in SEQ ID NO.11；For altogether With the IRES ribosome binding sequence of transcriptional expression protein, as shown in SEQ ID NO.12；For Chimeric antigen receptor gene People's EF1 α promoter of eukaryotic transcription, as shown in SEQ ID NO.14；Increase for strengthening the eWPRE of the expression efficiency of transgenic Strong type marmot hepatitis B virus posttranscriptional regulatory element, as shown in SEQ ID NO.13；And be used for forming collection identify, transmission, Start the secondary CAR in one or the Chimeric antigen receptor of three generations CAR；Described integrate identification for composition, transmit, start The Chimeric antigen receptor of secondary CAR include: CD8leader Chimerical receptor signal peptide as shown in SEQ ID NO.15, such as SEQ CD123 single-chain antibody light chain VL shown in ID NO.20, the Optimal Linker C as shown in SEQ ID NO.19, such as SEQ CD123 single-chain antibody heavy chain VH shown in ID NO.16, the CD8Hinge Chimerical receptor hinge as shown in SEQ ID NO.21, as CD8Transmembrane Chimerical receptor cross-film district shown in SEQ ID NO.22, CD28, as shown in SEQ ID NO.23 CD137 Chimerical receptor costimulating factor, TCR Chimerical receptor t cell activation territory as shown in SEQ ID NO.24.Described for group Become to integrate identification, transmit, the Chimeric antigen receptor of three generations CAR that starts includes: as shown in SEQ ID NO.15 CD8leader Chimerical receptor signal peptide, CD123 single-chain antibody light chain VL as shown in SEQ ID NO.20, such as SEQ ID Optimal Linker C shown in NO.19, CD123 single-chain antibody heavy chain VH as shown in SEQ ID NO.16, such as SEQ ID CD8Hinge Chimerical receptor hinge shown in NO.21, CD8Transmembrane Chimerical receptor as shown in SEQ ID NO.22 Cross-film district, CD28, CD137 Chimerical receptor costimulating factor as shown in SEQ ID NO.23, as shown in SEQ ID NO.24 TCR Chimerical receptor t cell activation territory and the CD28 Chimerical receptor costimulating factor as shown in SEQ ID NO.25.The present invention The linker design of the scFV section used, can apply to second filial generation CAR design, can also be applied to the third generation equally CAR design.Third generation CAR design, compared with second filial generation design, adds CD28 Chimerical receptor costimulating factor (SEQ ID NO.25), in theory, higher signal amplification is had.

Further, described slow virus packaging cis element uses second filial generation slow virus carrier to include: such as SEQ ID NO.5 Shown 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 unit 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.Described slow virus Packaging cis element uses third generation slow virus carrier to include: slow virus 5 terminal 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, as Slow virus packaging cis element described in cPPT cis element shown in SEQ ID NO.10, and as shown in SEQ ID NO.4 RSV promoter.The present invention used CAR design can apply in third generation slow virus carrier structure, it is also possible to application In second filial generation slow virus carrier structure.The difference (as shown in Figure 2 B) structurally of the second filial generation and third generation slow virus carrier, Mainly third generation slow virus carrier replaces with RSV promoter the U3 region of second filial generation carrier 5 ' LTR, thus eliminating the need U3 Dependence to Tat albumen when transcribing, both can remove Tat sequence in the structural gene of slow virus, also improve slow virus base Because organizing transcriptional level and transcribing persistence.The second filial generation and third generation slow virus carrier are mainly the difference of subgenomic transcription mode, Therefore the present invention used CAR design can apply to this two generations slow virus carrier.Carrier framework of the present invention Being preferably third generation slow virus carrier (shown in Fig. 2 A), 3 ' SIN LTR eliminate U3 region, eliminate slow virus carrier oneself multiple The probability of system, substantially increases safety；Add cPPT and eWPRE element, improve the table of transduction efficiency and transgenic Reach efficiency；RSV promoter is used to ensure that the lasting efficient transcription of core RNA when slow virus carrier is packed；Use people's self EF1 α promoter, enables CAR gene long lasting for expression in human body.

Further, described eWPRE enhancement mode marmot hepatitis B virus posttranscriptional regulatory element has the enhancing of 6 nucleotide Sudden change, particularly as follows: g.396G > A, g.397C > T, g.398T > C, g.399G > A, g.400A > T, g.411A > T.

In another aspect of this invention, it is provided that the CAR-T of the replication defective recombinant slow virus of a kind of above-mentioned targeting CD123 The construction method of transgene carrier, comprises the following steps:

(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), for slow sick Slow virus packaging cis element, ZsGreen1 green fluorescent protein (as shown in SEQ ID NO.11), the IRES ribose of poison packaging Body binding sequence (as shown in SEQ ID NO.12), eWPRE enhancement mode marmot hepatitis B virus posttranscriptional regulatory element are (such as SEQ Shown in ID NO.13) it is stored on slow virus skeleton plasmid；

(2) by people's EF1 α promoter (as shown in SEQ ID NO.14), integrate identification for composition, transmit, start Secondary CAR or the Chimeric antigen receptor of three generations CAR be combined into secondary CAR or three generations's CAR design, through enzyme action, connection, Recombining reaction is cloned in slow virus skeleton plasmid, obtains the recombinant slow virus plasmid of secondary CAR or three generations CAR design；

(3) by the recombinant slow virus plasmid obtained 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, packs and successfully weighs in HEK293T/17 cell Group slow virus carrier can be discharged in cells and supernatant, collects the supernatant of the recombined lentivirus vector comprised；

(4) sucking filtration, absorption, the column purification mode of eluting is used to be purified the recombinant slow virus supernatant obtained, respectively Obtain recombined lentivirus vector.

In step (1), described slow virus packaging cis element uses second filial generation slow virus carrier to include: such as SEQ ID Slow virus 5terminal LTR shown in NO.5, 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 unit 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；Described slow virus Packaging cis element uses third generation slow virus carrier to 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, as Slow virus packaging cis element described in cPPT cis element shown in SEQ ID NO.10, and as shown in SEQ ID NO.4 RSV promoter.

In step (1), described eWPRE enhancement mode marmot hepatitis B virus posttranscriptional regulatory element has 6 nucleotide Strengthen sudden change, particularly as follows: g.396G > A, g.397C > T, g.398T > C, g.399G > A, g.400A > T, g.411A > T.

In step (2), people's EF1 α promoter start whole CAR gene expression；CD8leader Chimerical receptor signal peptide It is positioned at the N end of CAR coded sequence, is used for guiding CAR protein localization in cell membrane；CD123 single-chain antibody light chain VL, Optimal Linker C, CD123 single-chain antibody heavy chain VH is combined into scfv region, is used for identifying CD123 antigen；CD8Hinge Chimerical receptor Hinge is for being anchored to scfv outside cell membrane；CD8 Transmembrane Chimerical receptor cross-film district is for by whole chimeric Receptor is fixed on cell membrane；CD137 Chimerical receptor costimulating factor is used for stimulating T cell propagation and cytokine secretion；TCR Chimerical receptor t cell activation territory is for activating the expression of downstream signaling pathway；When scfv region is combined with CD123 antigen, letter Number be transferred to intracellular by Chimerical receptor, thus produce T cell propagation, cytokine secretion increases, Anti-apoptotic proteins A series of biological effects such as secretion increases, cell death postpones, cracking target cell.

In step (4), described slow virus carrier has the version of band fluorescence labels zsGreen1 and without fluorescence labels ZsGreen1 version, the version of band fluorescence labels is used for experiment in vitro, and the version without fluorescence labels is used for clinical experiment.

In step (4), the several key points that should be noted that have, a: sucking filtration step supernatant to be controlled volume (200ml～ 2000ml) with vacuum (-0.5MPA～-0.9MPA), prevent the carrier loss brought due to plug-hole；B: adsorption step needs control The PH (6～8) of solution processed, prevents the change of PH from causing carrier to inactivate；C: elution step needs to control the ionic strength of eluent (0.5M～1.0M), prevents the change of ionic strength from causing eluting not exclusively or carrier inactivation.

In another aspect of this invention, it is provided that the application in preparing adoptive cellular therapeutic agent of the above-mentioned carrier.Preferably , described adoptive cellular therapeutic agent is acute myeloid leukemia (AML) medicine.Verify through clinical trial, the present invention Carrier is applied to clinical treatment acute myeloid leukemia (AML), is achieving the fully erased of tumor cell in the patient, therefore Carrier of the present invention is gathered around in CART treatment field and is had broad application prospects.

Compared with prior art, there is advantages that

The linker design of the scFV section that the present invention uses, is the Linkers pool using Shi Ao company, through albumen The analysis of matter Structure bioinformatics data base (https: //www.predictprotein.org/), by protein two The comparison of the protein properties such as level structure, Solvent accessibility, protein pliability, disulfide bond bridge, binding site, preferably Go out.Proved, compared with external design by the detection test of cell in vitro cytokine secretion and killing-efficiency test, it is possible to notable Improve the secretion of cytokine, the killing effect in vitro of CAR-T cell.Further, in the effect of clinical treatment, also ratio abroad faces It is effective that bed is tested.

Slow virus carrier column purification system of the present invention, is the slow virus large-scale production developed of the applicant Technique.Slow virus carrier column purification mode of the present invention is different from the ultracentrifugation of generally employing or ultracentrifugal Mode, conventional supercentrifugation or supercentrifugal process, be to utilize centrifugal sedimentation principle separation lentiviral particle, unavoidably Meeting remain the close impurity of a lot of sedimentation coefficients, subsequent experimental is brought adverse effect.Further, tubulature process is complicated, operation Loaded down with trivial details, multiple conversions container brings more opportunities for contamination.And the slow virus carrier column purification technique of the present invention is semi-automatic Operation, all processes completes hundred grades of Experimental Areas, it is to avoid manually-operated loaded down with trivial details and error and pollution probability, is reclaimed Slow virus carrier is fully achieved clinical criteria in the indexs such as endotoxin, mycoplasma, titer determination.Follow-up exploitation of following up is complete certainly Dynamic purification instrument.

Third generation slow virus skeleton plasmid pLenti-3G basic of the present invention, with University of Pennsylvania Carl H.June et al. (Porter DL, Levine BL, Kalos M, Bagg A, June CH.Chimeric antigen receptormodified T cells in chronic lymphoid leukemia.N Engl J Med 2011；365: Third generation slow virus carrier 725-33.) used is compared, and eliminates phage f1 origin of replication, uses eukaryotic viral SV40 Replicon, adds genes of interest copy number in eukaryotic cell, enhances eukaryotic expression effect.

Third generation slow virus skeleton plasmid pLenti-3G basic of the present invention, uses enhancedWPRE unit Part (i.e. eWPRE enhancement mode marmot hepatitis B virus posttranscriptional regulatory element), with University of Pennsylvania Carl H.June et al. (Porter DL,Levine BL,Kalos M,Bagg A,June CH.Chimeric antigen receptormodified T cells in chronic lymphoid leukemia.N Engl J Med 2011；365:725-33.) used WPRE element is compared, have the enhancing of 6 nucleotide suddenly change (g.396G > A, g.397C > T, g.398T > C, g.399G > A, g.400A > T, g.411A > T), it is possible to strengthen the poly-adenosine of primary transcript, increase the content of intracellular mRNA, strengthen transgenic Expression efficiency.

The Lentival packaging system that the present invention uses is four plasmid packaging systems of non-auxiliary virus, by four kinds of plasmids Jointly transfect to HEK293T/17 cell, produce recombined lentivirus vector.Slow virus carrier after restructuring is replication defect type Carrier, can be integrated into host gene, single use, it is impossible to replicating and breed, safety improves a lot by exogenous sequences.

The slow virus carrier that the present invention uses has the version of band fluorescence labels zsGreen1 and without fluorescence labels ZsGreen1 version, the version of band fluorescence labels is used for experiment in vitro, and the version without fluorescence labels is used for clinical experiment, is suitable for In extensive range.

Present invention preferably employs third generation slow virus carrier, 3 ' SIN LTR eliminate U3 region, eliminate slow virus carrier The probability of self replication, substantially increases safety；Add cPPT and eWPRE element, improve transduction efficiency and turn base The expression efficiency of cause；RSV promoter is used to ensure that the lasting efficient transcription of core RNA when slow virus carrier is packed；Use people The EF1 α promoter of self, enables CAR gene long lasting for expression in human body.

Visible, recombined lentivirus vector of the present invention will be treated to the CAR-T of acute myeloid leukemia (AML) and be provided Transgenic guarantee reliably.

Accompanying drawing explanation

Fig. 1 is the schematic diagram of CAR of the present invention, and wherein Figure 1A is the basic block diagram of CAR, and Figure 1B is the generation of CAR Secondary improvement schematic diagram；

Fig. 2 is slow virus carrier structural representation of the present invention；Wherein Fig. 2 A is that the third generation that the present invention uses is slow Viral vector structural representation, Fig. 2 B is the second filial generation and third generation slow virus carrier structure comparison schematic diagram；

Fig. 3 is the structure flow chart building recombined lentivirus vector of the present invention in the embodiment of the present invention 1.Wherein, Fig. 3 (A) is the structural representation of slow virus skeleton plasmid pLenti-3G basic；Fig. 3 (B) is the knot of pCAR22-CLA plasmid Structure schematic diagram；Fig. 3 (C) is the structural representation of pCAR123-CLB plasmid；Fig. 3 (D) is that the structure of pCAR123-OLC plasmid is shown It is intended to；Fig. 3 (E) is the structural representation of slow virus packaging plasmid pPac-GP；Fig. 3 (F) is slow virus packaging plasmid pPac-R Structural representation；Fig. 3 (G) is the structural representation of memebrane protein pEnv-G；

Fig. 4 be recombinant slow virus plasmid pCAR123-CLA in the embodiment of the present invention 1 enzyme action prediction and enzyme action agarose coagulate Gel electrophoresis figure；Wherein, Fig. 4 A is the enzyme action prediction schematic diagram of recombinant slow virus plasmid pCAR123-CLA, and wherein, lane1 is 1kb DNA ladder Marker: band is followed successively by from top to bottom: 10kb, 8Kb, 6kb, 5Kb, 4kb, 3.5Kb, 3Kb, 2.5kb, 2Kb, 1.5kb, 1Kb, 750bp, 500bp, 250bp, lane2 is the EcoR V+BamH I enzyme action prediction of pCAR123-CLA: bar Band is followed successively by from top to bottom: 8950bp, 959bp；Fig. 4 B is the enzyme action agarose gel of recombinant slow virus plasmid pCAR123-CLA Electrophoretogram, wherein, lane1 is the electrophoresis result of 1kb DNA ladder Marker, and lane2 is the EcoR V of pCAR123-CLA + BamH I restriction enzyme digestion and electrophoresis result；

Fig. 5 be recombinant slow virus plasmid pCAR123-CLB in the embodiment of the present invention 1 enzyme action prediction and enzyme action agarose coagulate Gel electrophoresis figure；Wherein, Fig. 5 A is the enzyme action prediction schematic diagram of recombinant slow virus plasmid pCAR123-CLB, and wherein, lane1 is 1kb DNA ladder Marker: band is followed successively by from top to bottom: 10kb, 8Kb, 6kb, 5Kb, 4kb, 3.5Kb, 3Kb, 2.5kb, 2Kb, 1.5kb, 1Kb, 750bp, 500bp, 250bp, lane2 be pCAR123-CLB Pvu II enzyme action prediction: band to Under be followed successively by:: 6722bp, 2364bp, 823bp；Fig. 5 B is that the enzyme action agarose of recombinant slow virus plasmid pCAR123-CLB coagulates Gel electrophoresis figure, wherein, lane1 is the electrophoresis result of 1kb DNA ladder Marker, and lane2 is the Pvu of pCAR123-CLB II restriction enzyme digestion and electrophoresis result；Fig. 6 is enzyme action prediction and the enzyme action of recombinant slow virus plasmid pCAR123-OLC in the embodiment of the present invention 1 Agarose gel electrophoresis figure；Wherein, Fig. 6 A is the enzyme action prediction schematic diagram of recombinant slow virus plasmid pCAR123-OLC, wherein, Lane1 is 1kb DNA ladder Marker: band is followed successively by from top to bottom: 10kb, 8Kb, 6kb, 5Kb, 4kb, 3.5Kb, 3Kb, 2.5kb, 2Kb, 1.5kb, 1Kb, 750bp, 500bp, 250bp, lane2 is the Kpn I enzyme action prediction of pCAR123-OLC: Band is followed successively by from top to bottom: 8483bp, 1426bp；Fig. 6 B is the enzyme action agarose of recombinant slow virus plasmid pCAR123-OLC Gel electrophoresis figure, wherein, lane1 is the electrophoresis result of 1kb DNA ladder Marker, and lane2 is pCAR123-OLC Kpn I restriction enzyme digestion and electrophoresis result；

Fig. 7 is the flow chart of the embodiment of the present invention 2 intermediate ion exchange chromatography purification of Recombinant slow virus carrier；

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, lane1 is DL2000marker, and bar counterband tape is followed successively by from top to bottom from top to bottom: 2kb, 1kb, 750bp, 500bp, 250bp、100bp；Lane2 is positive control；Lane3 is negative control；Lane4 is PBS；Lane5 is water；Lane6 is cracking Liquid；Lane7 is the slow virus of ultracentrifugation purification；Lane8 is the slow virus of high speed centrifugation purification；Lane9 is that ion exchanges color The slow virus of spectrum purification；Lane10 is ghost；

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

Figure 11 is the WB detection figure of CAR expressing quantity in the embodiment of the present invention 3, and result shows that CAR albumen is thin at PBMC Intracellular height efficient expression, in Figure 11 A, lane1 is PBMC ghost, and lane2 is lvCAR123-for comparison virus MOCK, lane3 CLA, lane4 be lvCAR123-CLB, lane5 be lvCAR123-OLC；Figure 11 B is beta-actin internal reference band；

Figure 12 is the killing-efficiency in the embodiment of the present invention 3 under the conditions of LDH detection difference effect target ratio, and E is effector lymphocyte, T For target cell；

Figure 13 is that in the embodiment of the present invention 3, qPCR detection difference imitates Cytokine Expression Level schematic diagram under the conditions of target ratio, E For effector lymphocyte, T is target cell；Wherein, Figure 13 A represents the mRNA transcriptional level of IL-2；Figure 13 B represents that the mRNA of IFN-γ turns Record level；

Figure 14 is CAR123-T cell infusion acute myeloid leukemia (AML) cell xenograft in the embodiment of the present invention 4 After mouse model, internal situation of change schematic diagram；After Figure 14 A represents injection, experimental group and control group mice in-vivo tumour with The living imaging result of time change；After Figure 14 B represents injection, experimental group and control group mice vivo biodistribution luminous intensity are at any time Between change curve；After Figure 14 C represents injection, experimental group and the control group mice life cycle of situation over time；

Figure 15 is the second filial generation and the titre of third generation recombined lentivirus vector of continuous 3 batches in the embodiment of the present invention 5 Results contrast schematic diagram；

Figure 16 is the killing-efficiency schematic diagram in the embodiment of the present invention 6 under the conditions of LDH detection difference effect target ratio, and E is effect Cell, T is target cell.

Detailed description of the invention

Following example are merely to illustrate the present invention rather than limit the scope of the present invention.In embodiment unreceipted specifically The experimental technique of condition, generally according to normal condition, or according to the condition proposed by manufacturer.

Embodiment 1 builds 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: according to the primer needed for design of primers principle design amplification of DNA fragments and target site, this primer is by Shanghai Biotech firm synthesizes, particularly as follows:

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)

VH-F:5 '-CACGCCGCCAGGCCGGAAGTGAAACTGGTGGAAAGC-3 ' (SEQ ID NO.30)

VH-R:5 '-GCTGCTCACGGTCACCAGG-3 ' (SEQ ID NO.31)

CLA-VL-F:5 '-GGTGACCGTGAGCAGCGGCGGTGGCTCGGGTGGTGGGTCGGGCGGCGGATCTGGGG G AGGTTCTGATATTGTGATGACCCAGAGCC-3’(SEQ ID NO.32)

CLB-VL-F:5 '-GGTGACCGTGAGCAGC GGATCCACCTCCGGATCCGGAAAACCCGGATCCGGAGAAG GATCCACCAAAGGAG ATATTGTGATGACCCAGAGCC-3’(SEQ ID NO.33)

OLC-VL-F:5 '-GGTGACCGTGAGCAGCGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCG G ATCTGATATTGTGATGACCCAGAGCC-3’(SEQ ID NO.34)

VL-R:5 '-GCGTTTAATTTCCACTTTGGT-3 ' (SEQ ID NO.35)

CD8Hinge-F:5 '-GTGGAAATTAAACGCACCACGACGCCAGCGCC-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 shown in ID NO.48, SEQ ID NO.49 is synthesized by Shanghai biotech firm, and with oligonucleotide dry powder or plasmid Form preserves；

4, toolenzyme BamH I, EcoR V, Pvu II, Kpn I, ApaL I, Sac I, Cla I, Sal I, T4DNA all purchase From NEB company；

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

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

7, plasmid extraction test kit, agarose gel reclaim test kit and 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, purchased from invitrogen company；

11, Biotinylated protein L is purchased from GeneScript company；

12, the two of horseradish peroxidase-labeled resist, DAB working solution is purchased from Beijing Zhong Shan Golden Bridge；

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

14, DNeasy test kit is purchased from Shanghai JaRa company；

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

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, CD123⁺K562 cell purchased from generation take wing (Shanghai) public Department；

19, LDH detection kit is purchased from promega company.

Two, the construction method of recombined lentivirus vector lvCAR123-CLA, lvCAR123-CLB, lvCAR123-OLC.

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

1, by people's EF1 α promoter, CD8leader Chimerical receptor signal peptide, CD123 single-chain antibody light chain VL, Common Linker A, Common Linker B, Optimal Linker C, CD123 single-chain antibody heavy chain VH, CD8Hinge are fitted together to and are subject to Body hinge, CD8Transmembrane Chimerical receptor cross-film district, CD137 Chimerical receptor costimulating factor, TCR Chimerical receptor T are thin Born of the same parents' activation domain fragment is cloned into slow virus skeleton plasmid pLenti-3G basic, respectively obtains recombinant slow virus plasmid PCAR123-CLA, pCAR123-CLB, pCAR123-OLC.

(1) Cla I and Sal I restricted enzyme is used to carry out slow virus skeleton plasmid pLenti-3G basic double Enzyme action, product, through the agarose gel electrophoresis of 1.5%, confirms fragment V1 of 8303bp, and recovery of tapping rubber is placed in Eppendorf In pipe, reclaim test kit with the agarose gel of MN company and reclaim corresponding fragment (being shown in Table 1), and measure the purity of product and dense Degree；

Table 1 agarose gel recycling step

(2) use primer EF1 α-F and EF1 α-R with the SEQ ID NO.14 of synthesis as template, use the system in table 2, PCR Cycling condition is: 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 The agarose gel electrophoresis of 1.5%, confirms fragment a of 1208bp, and recovery of tapping rubber is placed in Eppendorf pipe, uses MN company Agarose gel reclaim test kit and reclaim corresponding fragment (being shown in Table 1), and measure purity and the concentration of product；

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

Table 2 50 μ l PCR reaction system

(3) use primer CD8leader-F and CD8leader-R with the SEQ ID NO.15 of synthesis as template, use in table 2 System, PCR cycle condition is: 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, through the agarose gel electrophoresis of 1.5%, confirms fragment b of 101bp, and recovery of tapping rubber is placed in Eppendorf pipe In, reclaim test kit with the agarose gel of MN company and reclaim corresponding fragment (being shown in Table 1), and measure the purity of product and dense Degree；

(4) use primer VH-F and VH-R with the SEQ ID NO.16 of synthesis as template, use the system in table 2, PCR cycle Condition is: 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 The agarose gel electrophoresis of 1.5%, confirms fragment c of 336bp, and recovery of tapping rubber is placed in Eppendorf pipe, with MN company Agarose gel reclaims test kit and reclaims corresponding fragment (being shown in Table 1), and measures purity and the concentration of product；

(5) use primer CLA-VL-F and VL-R with the SEQ ID NO.20 of synthesis as template, use the system in table 2, PCR Cycling condition is: 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 The agarose gel electrophoresis of 1.5%, confirms fragment d of 424bp, and recovery of tapping rubber is placed in Eppendorf pipe, with MN company Agarose gel reclaims test kit and reclaims corresponding fragment (being shown in Table 1), and measures purity and the concentration of product；

(6) use primer CLB-VL-F and VL-R with the SEQ ID NO.20 of synthesis as template, use the system in table 2, PCR Cycling condition is: 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 The agarose gel electrophoresis of 1.5%, confirms fragment e of 430bp, and recovery of tapping rubber is placed in Eppendorf pipe, with MN company Agarose gel reclaims test kit and reclaims corresponding fragment (being shown in Table 1), and measures purity and the concentration of product；

(7) use primer OLC-VL-F and VL-R with the SEQ ID NO.20 of synthesis as template, use the system in table 2, PCR Cycling condition is: 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 The agarose gel electrophoresis of 1.5%, confirms fragment f of 421bp, and recovery of tapping rubber is placed in Eppendorf pipe, with MN company Agarose gel reclaims test kit and reclaims corresponding fragment (being shown in Table 1), and measures purity and the concentration of product；

(8) use primer CD8Hinge-F and CD8Hinge-R with the SEQ ID NO.21 of synthesis as template, use in table 2 System, PCR cycle condition is: 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, through the agarose gel electrophoresis of 1.5%, confirms fragment g of 147bp, and recovery of tapping rubber is placed in Eppendorf pipe, uses The agarose gel of MN company reclaims test kit and reclaims corresponding fragment (being shown in Table 1), and measures purity and the concentration of product；

(9) with primer CD8Transmembrane-F and CD8Transmembrane-R with the SEQ ID NO.22 of synthesis it is Template, uses the system in table 2, and PCR cycle condition is: 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, through the agarose gel electrophoresis of 1.5%, confirms fragment h of 100bp, and recovery of tapping rubber is put In Eppendorf manages, reclaim test kit with the agarose gel of MN company and reclaim corresponding fragment (being shown in Table 1), and measure product The purity of thing and concentration；

(10) use primer CD137-F and CD137-R with the SEQ ID NO.23 of synthesis as template, use the system in table 2, PCR cycle condition is: 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 Through the agarose gel electrophoresis of 1.5%, confirm fragment i of 142bp, and recovery of tapping rubber is placed in Eppendorf pipe, public with MN The agarose gel of department reclaims test kit and reclaims corresponding fragment (being shown in Table 1), and measures purity and the concentration of product；

(11) use primer TCR-F and TCR-R with the SEQ ID NO.24 of synthesis as template, use the system in table 2, PCR Cycling condition is: 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 The agarose gel electrophoresis of 1.5%, confirms fragment j of 355bp, and recovery of tapping rubber is placed in Eppendorf pipe, with MN company Agarose gel reclaims test kit and reclaims corresponding fragment (being shown in Table 1), and measures purity and the concentration of product；

(12) using each to DNA fragmentation b, c, d 1 μ l as template, use the system in table 3, in addition to primer, add Eppendorf In pipe, PCR cycle condition is: 98 DEG C of 3min, (98 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 30sec) * 6cycle, adds primer 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 The agarose gel electrophoresis of 1.5%, confirms fragment k of 814bp, and recovery of tapping rubber is placed in Eppendorf pipe, with MN company Agarose gel reclaims test kit and reclaims corresponding fragment (being shown in Table 1), and measures purity and the concentration of product；

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

Table 3 50 μ l over-lap PCR reaction system

(13) using each to DNA fragmentation b, c, e 1 μ l as template, use the system in table 3, in addition to primer, add Eppendorf In pipe, PCR cycle condition is: 98 DEG C of 3min, (98 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 30sec) * 6cycle, adds primer 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 The agarose gel electrophoresis of 1.5%, confirms fragment l of 820bp, and recovery of tapping rubber is placed in Eppendorf pipe, with MN company Agarose gel reclaims test kit and reclaims corresponding fragment (being shown in Table 1), and measures purity and the concentration of product；

(14) using each to DNA fragmentation b, c, f 1 μ l as template, use the system in table 3, in addition to primer, add Eppendorf In pipe, PCR cycle condition is: 98 DEG C of 3min, (98 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 30sec) * 6cycle, adds primer 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 The agarose gel electrophoresis of 1.5%, confirms fragment m of 811bp, and recovery of tapping rubber is placed in Eppendorf pipe, with MN company Agarose gel reclaims test kit and reclaims corresponding fragment (being shown in Table 1), and measures purity and the concentration of product；

(15) using each to DNA fragmentation g, h, i, j 1 μ l as template, use the system in table 3, add in addition to primer In Eppendorf pipe, PCR cycle condition is: 98 DEG C of 3min, (98 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 30sec) * 6cycle, Add 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.Produce Thing, through the agarose gel electrophoresis of 1.5%, confirms fragment n of 704bp, and recovery of tapping rubber is placed in Eppendorf pipe, uses MN The agarose gel of company reclaims test kit and reclaims corresponding fragment (being shown in Table 1), and measures purity and the concentration of product；

(16) DNA fragmentation V1, a, k, n are added Eppendorf pipe with the ratio of 5 μ l cumulative volumes and mol ratio 1:1:1:1 In, add homologous recombination enzyme reaction solution 15 μ l, hatch 30 minutes at 42 DEG C after mixing, be transferred to place on ice 2-3 minute, will be anti- Answer liquid to add in 50 μ l TOP10, rotate gently to mix content, place 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 cool down 2-3 minute, often pipe adds 900 μ l LB culture fluid, then transfers to pipe on 37 DEG C of shaking tables, and incubation makes bacteria resuscitation in 1 hour, and the conversion bacterium solution taking 100 μ l is coated On Amp LB agar plate, inversion plate, 37 DEG C of cultivations in constant incubator, 16 hours.Picked clones carries out bacterium colony PCR mirror Fixed, identify that correct clone is recombinant slow virus plasmid pCAR123-CLA, correct clone is carried out enzyme action qualification (see figure 4)；

(17) DNA fragmentation V1, a, l, n are added Eppendorf pipe with the ratio of 5 μ l cumulative volumes and mol ratio 1:1:1:1 In, add homologous recombination enzyme reaction solution 15 μ l, hatch 30 minutes at 42 DEG C after mixing, be transferred to place on ice 2-3 minute, will be anti- Answer liquid to add in 50 μ l TOP10, rotate gently to mix content, place 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 cool down 2-3 minute, often pipe adds 900 μ l LB culture fluid, then transfers to pipe on 37 DEG C of shaking tables, and incubation makes bacteria resuscitation in 1 hour, and the conversion bacterium solution taking 100 μ l is coated On Amp LB agar plate, inversion plate, 37 DEG C of cultivations in constant incubator, 16 hours.Picked clones carries out bacterium colony PCR mirror Fixed, identify that correct clone is recombinant slow virus plasmid pCAR123-CLB, correct clone is carried out enzyme action qualification (see figure 5)；

(18) DNA fragmentation V1, a, m, n are added Eppendorf pipe with the ratio of 5 μ l cumulative volumes and mol ratio 1:1:1:1 In, add homologous recombination enzyme reaction solution 15 μ l, hatch 30 minutes at 42 DEG C after mixing, be transferred to place on ice 2-3 minute, will be anti- Answer liquid to add in 50 μ l TOP10, rotate gently to mix content, place 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 cool down 2-3 minute, often pipe adds 900 μ l LB culture fluid, then transfers to pipe on 37 DEG C of shaking tables, and incubation makes bacteria resuscitation in 1 hour, and the conversion bacterium solution taking 100 μ l is coated On Amp LB agar plate, inversion plate, 37 DEG C of cultivations in constant incubator, 16 hours.Picked clones carries out bacterium colony PCR mirror Fixed, identify that correct clone is recombinant slow virus plasmid pCAR123-OLC, correct clone is carried out enzyme action qualification (see figure 6)。

2, the packaging of recombined lentivirus vector lvCAR123-CLA, lvCAR123-CLB, lvCAR123-OLC.

(1) complete medium: take out preheated fresh culture, adds 10%FBS+5ml Pen-Srep, runs up and down Mix；

(2) 1XPBS solution: weigh NaCl 8g, KCl 0.2, Na₂HPO₄.12H₂O 3.58g, KH₂PO4 0.24g is placed in In 1000ml beaker, add 900ml Milli-Q grade ultra-pure water and dissolve, after having dissolved, use 1000ml graduated cylinder constant volume To 1000ml, 121 DEG C of high-temperature heat sterilization 20min；

(3) 0.25%Trypsin solution: weigh Trypsin 2.5g, EDTA 0.19729g and be placed in 1000ml beaker, Add 900ml 1XPBS to dissolve, after having dissolved, use 1000ml graduated cylinder to be settled to 1000ml, 0.22 μM of filtration sterilization, for a long time Use can preserve to-20 DEG C of refrigerators；(4) 0.5M CaCl2 solution: weigh 36.75g CaCl₂With 400ml Milli-Q grade Ultra-pure water dissolves；With Milli-Q grade ultra-pure water, cumulative volume is settled to 500ml, mixing；0.22 μm filtration sterilization, subpackage It is saved in 50ml centrifuge tube, often pipe about 45ml, 4 DEG C of preservations.

(5) 2XHBS solution: weigh 4.09g NaCl, 0.269g Na₂HPO4,5.96g Hepes, uses 400ml Milli- Q grade ultra-pure water dissolves；After calibration pH instrument, by 2M NaOH solution, the pH of HBS solution is transferred to 7.05.Adjust every bottle of HBS PH consume 2M NaOH be about 3ml；

(6) from liquid nitrogen container, take out frozen HEK293T/17 cell, be quickly transferred in 37 DEG C of water-baths, after 1～2min Transferring 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/10cm²Microscope observation of cell after dish, 24h, the degree of cell confluency passes on more than 80%；

(7) selecting cell state HEK293T/17 cell good, free of contamination, every 2-6 culture dish is one group, by cell After trypsinization, draw 4-12ml complete medium with electric pipettor, in each postdigestive culture dish, add 2ml, it is to avoid Culture dish becomes dry；Use 1ml pipettor that all cells is blown and beaten into single cell suspension, transfer in medium bottle；

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

(9) cover tightly culture medium bottle cap, turn upside down about 10 times and fully mix cell suspension, cell is passed to 8-24 10cm²In culture dish, the cell density of every ware should about 4 × 10⁶About individual/10ml complete medium.If cell density is with pre- The difference of phase is relatively big, then need to count cell, then according to 4 × 10⁶The amount inoculation of individual/ware；

(10) every 6 culture dishs arrange is a pile, notes keeping the cooperation between upper and lower ware.By about culture dish, front and back Rock for several times, make cell fully spread out, be then placed in 5%CO₂Incubator.Remaining cell does same process；

(11) checking institute passage cell, cell confluency degree should be 70-80%, and profile is full, adherent well, train at cell Support in ware and be uniformly distributed；

(12) it is that cell changes liquid, culture medium is replaced with fresh complete medium, every ware 9ml, and by the CO of incubator₂Dense Degree setting value brings up to 8%；(13) DNA/CaCl is joined according to N+0.5₂Solution.Every ware HEK293T/17 cell transfecting plasmid amount is pressed Use according to following ratio: recombinant slow virus plasmid (20 μ g), pPac-GP (15 μ g), pPac-R (10 μ g), pEnv-G (7.5 μ g). Take a new 5ml centrifuge tube, addition 0.5M CaCl2:0.25ml, recombinant slow virus plasmid 20 μ g:pPac-GP 15 μ g: PPac-R 10 μ g:pEnv-G 7.5 μ g, supplements ultra-pure water and closes the lid to 0.5ml, fully mix；

(14) separately take a 5ml centrifuge tube, add 0.5ml DNA/CaCl2 solution.Opening turbula shaker, one hand-held Living the upper end of 5ml centrifuge tube, make to contact at the bottom of pipe oscillating end, make liquid scatter on tube wall flowing, the hand-held 1mL of another moves Liquid rifle, draws 0.5mL 2 × HBS solution, is slowly added dropwise entrance centrifuge tube, coutroi velocity, drips off with half a minute and be advisable.2×HBS After addition, continue vibration 5 seconds, stop oscillation, can be directly added in the cell needing transfection；

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

(16), after calcium turns liquid addition, ware lid carries out labelling, culture dish is released to another 5%CO₂In incubator. Guaranteeing culture dish horizontal positioned, often pile culture dish does not exceeds 6.At 5%CO₂Incubator places (6 8h)；

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

After (18) 24 hours, check cell state.Cell confluency degree should be about 80 85%, in good condition.To cultivate Base siphons away, and changes the fresh DMEM complete medium of 10ml；

After (19) 48 hours, observe transfection efficiency.Most cells remain adherent.It can be seen that it is thin more than 95% Born of the same parents can be with green fluorescence.By same virus packaging supernatant collection to together, and in culture dish, continue interpolation 10mL Fresh culture；

After (20) 72 hours, again collecting together by same vial supernatant, the virus of twice collection can be placed on Together, culture dish is abandoned；Recombined lentivirus vector lvCAR123-CLA, lvCAR123-is contained in the supernatant now collected CLB、lvCAR123-OLC。

The concentration of embodiment 2 recombined lentivirus vector and detection

One, supercentrifugation purification of Recombinant slow virus carrier；

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

(2) by 0.22 μm-0.8 μm filter filtering supernatant；

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

(4) the cell conditioned medium sample subpackage 32ml the 2nd step handled well is in centrifuge tube；

(5) cover crown cap, by centrifuge tube together with crown cap trim, adjust with 1XPBS and make deviation of weight at 0.02g In the range of；

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

(7) after centrifugal end, carefully centrifuge tube is taken out from rotor, it can be seen that sink there being a little group at the bottom of centrifuge tube Form sediment, make marks on outer tube wall with Marker pen, outwell supernatant.Centrifuge tube is tipped upside down on the napkin completed in advance, makes remnants Liquid pours off.With liquid-transfering gun, the drop hung on wall can be siphoned away；

(8) in each centrifuge tube, add the Opti-MEM of 200 μ l, make resolution of precipitate, as far as possible with 200 μ l pipettor piping and druming Reduce the generation of foam；

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

(10) 500g, room temperature is centrifuged 1min, makes virus liquid focus at the bottom of pipe；

(11) all identical viral concentration liquid are brought together, filter with the PES filter of 0.22 μm-0.8 μm；By disease Poison is divided into 25 to 50 μ l mono-pipes, frozen to-80 DEG C refrigerators, preserves for a long time；

Two, supercentrifugal process purification of Recombinant slow virus carrier；

(1) the PES filter that the supernatant 204ml of collection uses 0.22 μm-0.8 μm filters；

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

(3) 21.7ml 4M NaCl solution is added；

(4) add 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 subpackage is entered in 250ml wide mouthed bottle, every part of 150ml；

Placing 1.5 hours for (6) 4 DEG C, mixing in every 20-30 minute is once；

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

(8) can see at the bottom of pipe, there is white precipitate after being centrifuged；

(9) careful abandoning supernatant, every bottle adds 1.2ml 50mM Tris-HCl (pH 7.4), and it is resuspended heavy acutely to rock Form sediment；

(10) vortex shakes 20 30 seconds further resuspended precipitations；

(11) virus is divided into 25 to 50 μ l mono-pipes, frozen to-80 DEG C refrigerators, preserves for a long time；

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

(1) supernatant collected is used Thermo vacuum pump, through the PES filter sucking filtration of 0.22 μm-0.8 μm, remove miscellaneous Matter；

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

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

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

(5), after all supernatant crosses post, 10ml 0.15M NaCl 25mM Tris-HCl (pH 6-8) solution is used to clean One time；

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

(7) eluent is divided into 25 to 50 μ l mono-pipes, frozen to-80 DEG C refrigerators, preserves for a long time.

Four, titer determination and comparing；

(1) 24 orifice plate inoculation 293T cells are taken.Every porocyte is 5 × 10⁴Individual, added culture volume is 500ul, different The vitro growth rates of kind difference, carrying out cell confluency when virus infects is 40%-60%；

(2) prepare 3 aseptic EP pipes, each pipe adds the fresh complete medium (DMEM in high glucose+10% of 90ul FBS) inoculating cell is after 24 hours, and the cell blood counting chamber taking two holes counts, the actual number of cell when determining infection, It is designated as N；

(3) take virus stock solution used 10ul to be determined and join in first pipe, gently after mixing, take 10ul and join second In individual pipe, operate a to the last pipe the most successively；410ul complete medium (DMEM in high glucose+10% is added in often pipe FBS), final volume is 500ul；

(4) infection starts latter 20 hours, removes culture supernatant, is replaced by 500 μ l complete medium (DMEM in high glucoses+10% FBS), 5%CO₂Continue to cultivate 48 hours；

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

(6) with 0.2ml 0.25% pancreas enzyme-EDTA solution digestion cell, place 1 minute at 37 DEG C.Whole with culture medium purging Individual cell face, centrifugal collecting cell.Genomic DNA is extracted according to the explanation of DNeasy test kit.Each sample cell adds 200 μ l eluent washes lower DNA the most quantitatively；

(7) (QPCR primer sequence is SEQ ID NO.44---SEQ ID to prepare target DNA detection house steward qPCRmix I NO.45):

N=number of reactions. is such as: overall reaction number is 40, by 1ml 2 × TaqMan Universal PCR Master Mix, 4 μ l forward primer, 4 μ l reverse primer, 4 μ l probe and 788 μ l H₂O mixes With.It is placed on ice after concussion；

(8) (QPCR primer sequence is SEQ ID NO.46---SEQ ID to prepare internal reference DNA detection qPCRmix pipe II 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 such as: overall reaction number is 40, by 1ml 2 × TaqMan Universal PCR Master Mix, 100 μ l 10 × RNaseP primer/probe mix and 700 μ l H₂O mixes.Ice it is placed on after concussion On；

(9) in 96 hole PCR plate of pre-cooling, complete PCR system set up.From house steward I, respectively take 45 μ l join each row of A-D Hole in, respectively take from house steward II in the hole that 45 μ l join each row of E-G.

(10) taking 5 μ l plasmid standards respectively and testing sample genomic DNA joins in A-D row, each sample repeats 1 Secondary.1 hole is separately stayed to add the water of 5 μ l as no template control (no-template control).

(11) take 5 μ l genome standard substance respectively and testing sample genomic DNA joins in E-G row, each sample weight Multiple 1 time.1 hole is separately stayed to add the water of 5 μ l as no template control (no-template control).

(12) used quantitative PCR apparatus is ABI PRISM 7000 quantitative system.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 circulations of 1 minute.

Data analysis: the slow virus carrier copy number genome number integrated in the DNA sample recorded is demarcated, and obtains The viral copy number of every genome conformity.

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

IU ml^-1=(C × N × D × 1000)/V

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

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

The extension rate of D=viral vector

The volume number of the virus dilution that V=adds

(13) titre results of recombined lentivirus vector lvCAR123-CLA, lvCAR123-CLB, lvCAR123-OLC is such as Shown in Fig. 8, the result of ion exchange chromatography is substantially better than supercentrifugation and supercentrifugal process.

Five, endotoxin measurement and comparing；

(1), endotoxin working standard is that 15EU/ props up；

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

(3), endotoxin standard dilution: take endotoxin standard one, be diluted to 4 λ and 2 λ in proportion with BET water respectively Dissolving, sealed membrane seal, concussion dissolve 15min；Often dilute a step during dilution and all should mix 30s on eddy mixer；

(4), sample-adding: if taking the tachypleus amebocyte lysate Heavenly Stems and Earthly Branches, every adds BET water 0.5ml and dissolves, and the subpackage as Heavenly Stems and Earthly Branches try without endotoxin Guan Zhong, often pipe 0.1ml.Wherein 2 is negative control pipe, adds BET water 0.1ml；

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

2 is Sample Positive control tube, adds the 0.1ml sample solution containing 2 λ endotoxin standards and (dilutes 20 times treat The endotoxin standard solution 1ml=2ml of test sample product 1ml+4 λ 40 times of samples of dilution containing 2 λ endotoxin standards).

Adding 0.1ml sample in sample cell, dilution ratio is shown in Table 4, and 37 ± 1 DEG C of water-baths (or incubator) are incubated 60 ± 1min；

Table 4 endotoxin dilution ratio and corresponding endotoxin content

(5), the endotoxin detection of recombined lentivirus vector lvCAR123-CLA, lvCAR123-CLB, lvCAR123-OLC Result (as shown in table 5), the endotoxin content of supercentrifugation is between 20～40EU/ml, and the endotoxin of supercentrifugal process contains Amount is between 20～40EU/ml, and the endotoxin content of ion exchange chromatography is substantially better than super between 1.25～2.5EU/ml Speed centrifuging and supercentrifugal process.

The endotoxin testing result of the different way of purification of table 5 recombined lentivirus vector

Six, mycoplasma measures and compares；

(1) on pretreatment three days, cell sample antibiotic-free culture medium was cultivated；

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

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

(4) 500ul PBS rifle head pressure-vaccum or vortex oscillation, resuspended precipitation are added.13000 × g is centrifuged 5min；

(5) step 4 is repeated once；

(6) add 50 μ l Cell Lysis Buffer, after rifle head pressure-vaccum, fully mixing, hatch in 55 DEG C of water-baths 20min；

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

After (8) 13000 × g are centrifuged 5min, taking 5 μ l supernatants as template, 25 μ lPCR reaction systems are: ddH20 6.5 μ L, Myco Mix 1 μ l, 2x Taq Plus Mix Master (Dye Plus) 12.5 μ l, template 5 μ l；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 shows (as shown in Figure 9), supercentrifugation, supercentrifugal process, ion exchange chromatography The recombined lentivirus vector of purification is all without mycoplasma.

Visible, the present invention uses ion exchange chromatography recombined lentivirus vector, with traditional supercentrifugal process and Supercentrifugation purification of Recombinant slow virus carrier is compared, and titer determination result and endotoxin measurement result are significantly superior.

The function inspection of embodiment 3 recombined lentivirus vector lvCAR123-CLA, lvCAR123-CLB, lvCAR123-OLC Survey.

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

(1) recombined lentivirus vector lvCAR123-CLA, lvCAR123-CLB, lvCAR123-OLC infection of PBMCs cell After, collect cell and use RT-PCR to carry out the detection of CAR mRNA transcriptional level, verify the expression of CAR gene, if CAR MRNA transcriptional level increases, then the transcriptional level of explanation CAR gene is expressed successfully；

(2) recombined lentivirus vector lvCAR123-CLA, lvCAR123-CLB, lvCAR123-OLC infection of PBMCs cell After, collect cell and use western blot to carry out the detection of CAR protein expression level, verify the expression of CAR gene, if CAR protein expression level increases, then the translation skill of explanation CAR gene is expressed successfully；

(3) respectively by lvCAR123-CLA, lvCAR123-CLB, lvCAR123-OLC and the comparison virus of MOI=15 MOCK infection cell, the total serum IgE and the total protein that extract cell in 6 orifice plates after 48h carry out fluorescent quantitative PCR experiment and immunity respectively Blot experiment.Concrete steps: be coated four holes of 6 orifice plates, each hole adds corresponding PBS and RN, and 4 DEG C overnight.After 12 hours Being coated virus by MOI=15,37 DEG C of incubators place 5h；6 orifice plates taken out, discard viral supernatants, wash twice with PBS, by 1* 10⁶/ hole, is coated PBMC (separating from human blood with lymphocyte separation medium), adds 500ul culture medium (containing 10% serum, 20U/ ml IL-2、Polybrene 8ug/ml).Stand 20 DEG C of centrifugal 30min of 20min, 1000g, cultivate 48h for 37 DEG C.

(4) total serum IgE of PBMC cell during Trizol method extracts 6 orifice plates, reverse transcription amplification cDNA, by QPCR primer (sequence For SEQ ID NO.46---SEQ ID NO.49) carry out fluorescent quantitative PCR experiment, reaction system is shown in Table 6, with internal reference Actin is Matched group, that verifies its mRNA transcribes situation.

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

Table 6 20 μ l qPCR reaction system

(5) protein immunoblot (Western Blot) is total by extract from PBMC by polyacrylamide gel electrophoresis Protein is pressed relative molecular mass and is separated.Use wet turn (4 DEG C, 400mA, 120min), by protein delivery to pvdf membrane.By envelope Close liquid (the TBST solution containing 5% skim milk) room temperature closing pvdf membrane 1h, confining liquid 1:1000 and dilute Biotinylated Protein L, then with the pvdf membrane incubated at room closed 4 DEG C overnight.TBST washes film 3 times, each 10min.Confining liquid 1: 500 dilute corresponding SA-HRP, and incubated at room temperature pvdf membrane 2h, TBST wash film 3 times, each 10min.Use Amersham company ECL+plusTM Western blotting system test kit develops the color.X-ray development obtains the film of display band.

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

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

Two, cytokine secretion and fragmentation effect assessment.

(1) CD123+K562 cell and effector lymphocyte lvCAR123-CLA-PBMC, lvCAR123-CLB-are cultivated respectively PBMC、lvCAR123-OLC-PBMC；(2) target cell (CD123+K562) 4x10 is collected⁵(CART is thin for cells and effector lymphocyte Born of the same parents) 2.8x10⁶Cells, 800g, 6min are centrifugal, abandon supernatant；

(3) with 1ml 1xPBS solution resuspended target cell and effector lymphocyte respectively, 800g, 6min are centrifugal, abandon supernatant；

(4) step 3 is repeated once；

(5) with 700ul culture medium (1640 culture medium+10%FBS) resuspended effector lymphocyte, by 2ml culture medium, (1640 cultivate Base+10%FBS) resuspended target cell；

(6) effect target is set than the experimental port for 1:1,5:1,10:1, and matched group is set, often the multiple holes of group 3；

(7) 250xg, 5min flat board is centrifuged；

(8) 37 DEG C of 5%CO2 incubators are cultivated 4 hours；

(9) 250xg, 5min flat board is centrifuged；

(10) take the 50ul supernatant in each hole in new 96 orifice plates, and every hole adds 50ul substrate solution, and (lucifuge is grasped Make)；

(11) lucifuge hatches 25 minutes；

(12) every hole adds 50ul stop buffer；

(13) microplate reader detection 490nm absorbance；

(14) are averaged in 3 multiple holes；The light absorption value in all experimental ports, Target cell wells and effector lymphocyte hole is deducted training Support the average of base background light absorption value；The light absorption value of target cell maximum is deducted the average of volume correction comparison light absorption value.

(15) by step 14 obtain corrected value bring formula below into, calculate each effect target than produced carefully Cellular toxicity percentage ratio.Result as shown in figure 12, imitate in difference by the PBMC cell of recombined lentivirus vector lvCAR123-OLC transduction The PBMC cell that under the conditions of target ratio, killing-efficiency is transduceed apparently higher than lvCAR123-CLA and lvCAR123-CLB；

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

(16) sample repeating to prepare a step 6 detects Cytokine Expression Level for qPCR, result such as Figure 13 institute Showing, the PBMC cell of recombined lentivirus vector lvCAR123-OLC transduction is IL-2 and IFN-γ under the conditions of difference effect target ratio The PBMC cell that mRNA transcriptional level is transduceed apparently higher than lvCAR123-CLA and lvCAR123-CLB.

Embodiment 4, the clinic of recombined lentivirus vector lvCAR123-OLC (removing the version of fluorescence labels zsGreen1) Application

Root (Saar Gill, Carl H.June et al.Preclinical targeting of according to the literature human acute myeloid leukemia and myeloablation using chimeric antigen receptor-modified T cells.Blood.2014；123 (15): 2343-2354.), CAR123 carrier pin is to acute marrow It is that leukemia (AML) has good effect.

This research is #NCT02623582 at the number of registration of www.clinicaltrials.gov.This research uses and expresses The T lymphocyte of CAR123 gene has good tumor to kill for people's AML cell line MOLM14 expressing CD123 in Mice Body Hinder effect.Inject latter 6 days, 14 days, living imaging result after 21 days show, CART123 experimental group is right in Mice Body The tumor that MOLM14 cell line is formed has obviously tumor-killing effect compared with CART19 experimental group and PBS control group (as shown in Figure 14 A)；The mice of CART123 experimental group, compared with CART19 experimental group and PBS control group mice, over time Increase, bioluminescence change in signal strength amplitude is the least, illustrates that tumor cell is substantially suppressed (as shown in Figure 14B)； Kaplan-Meier survival curve result shows, CART123 experimental mice and CART19 experimental group and PBS control group mice phase The life cycle (as shown in Figure 14 C) more longer than having；Adoptive CAR123-T cell therapy is for acute myeloid leukemia (AML) treatment aspect has good prospect.

Embodiment 5 third generation slow virus skeleton carrier 3rdLV-CAR123 and second filial generation slow virus skeleton carrier 2ndLV- CAR123 titre compares.

Build 3rdLV-CAR123 and 2ndLV-CAR123 respectively；Plasmid construction, virus packaging, virus ultracentrifugation are dense Contracting, titration procedure reference example 1；The titre results of continuous 3 batches as shown in figure 15, third generation slow virus skeleton The titre of carrier 3rdLV-CAR123 is better than second filial generation slow virus skeleton carrier 2ndLV-CAR123；Therefore, the present invention is preferred Use third generation slow virus skeleton carrier as the lift-launch skeleton of CAR gene.

Embodiment 6 second filial generation CAR slow virus carrier 3rdLV-2ndCAR123 and third generation CAR slow virus carrier 3rdLV- 3rd CAR123 killing-efficiency compares.

Build 3rdLV-2ndCAR123 and 3rdLV-3rdCAR123 respectively；Plasmid construction, virus packaging, virus hypervelocity from The heart concentrates, titration procedure reference example 1；Killing experiments reference example 3；Second filial generation CAR slow virus carrier 3rdLV- 2ndCAR123 and third generation CAR slow virus carrier 3rdLV-3rdCAR123 killing-efficiency results contrast as shown in figure 16, 3rdLV-3rdCAR123 is for killing-efficiency ratio 3rdLV-of not showing under the conditions of difference effect target ratio of target cell 2ndCAR123 is more efficient；Therefore, present invention preferably uses the slow virus carrier of second filial generation CAR design as clinical experiment carrier.

Claims (12)

1. the CAR-T transgene carrier of the replication defective recombinant slow virus of a targeting CD123, it is characterised in that including: use In the prokaryotic replions pUC Ori sequence of plasmid replication, as shown in SEQ ID NO.2；For containing that purpose bacterial strain expands in a large number Ampicillin resistance gene AmpR sequence, as shown in SEQ ID NO.1；The virus of the duplication in strengthening eukaryotic cell is multiple System SV40Ori sequence, as shown in SEQ ID NO.3；Slow virus packaging cis element for slow virus packaging；For eucaryon Cell expresses the ZsGreen1 green fluorescent protein of green fluorescence, as shown in SEQ ID NO.11；For common transcriptional expression egg The IRES ribosome binding sequence of white matter, as shown in SEQ ID NO.12；Eukaryotic transcription for Chimeric antigen receptor gene People's EF1 α promoter, as shown in SEQ ID NO.14；For strengthening the eWPRE enhancement mode marmot second of the expression efficiency of transgenic Hepatovirus posttranscriptional regulatory element, as shown in SEQ ID NO.13；And integrate identification for composition, transmit, start Secondary CAR or the Chimeric antigen receptor of three generations CAR；Described for composition integrate identification, transmit, the secondary CAR that starts Chimeric antigen receptor includes: CD8leader Chimerical receptor signal peptide as shown in SEQ ID NO.15, such as SEQ ID NO.20 institute The CD123 single-chain antibody light chain VL shown, the Optimal Linker C as shown in SEQ ID NO.19, such as SEQ ID NO.16 institute The CD123 single-chain antibody heavy chain VH shown, the CD8Hinge Chimerical receptor hinge as shown in SEQ ID NO.21, such as SEQ ID CD8Transmembrane Chimerical receptor cross-film district shown in NO.22, CD28, CD137 as shown in SEQ ID NO.23 are fitted together to Receptor costimulating factor, TCR Chimerical receptor t cell activation territory as shown in SEQ ID NO.24；Described for form collection identify, Transmission, start the Chimeric antigen receptor of three generations CAR in one and include: the CD8leader as shown in SEQ ID NO.15 is fitted together to Receptor signal peptide, CD123 single-chain antibody light chain VL as shown in SEQ ID NO.20, as shown in SEQ ID NO.19 Optimal Linker C, CD123 single-chain antibody heavy chain VH as shown in SEQ ID NO.16, as shown in SEQ ID NO.21 CD8Hinge Chimerical receptor hinge, CD8 Transmembrane Chimerical receptor cross-film district as shown in SEQ ID NO.22, CD28, the CD137 Chimerical receptor costimulating factor as shown in SEQ ID NO.23, the TCR as shown in SEQ ID NO.24 are chimeric Recipient T cells activation domain and the CD28 Chimerical receptor costimulating factor as shown in SEQ ID NO.25.

2. carrier as claimed in claim 1, it is characterised in that described slow virus packaging cis element uses second filial generation slow virus Carrier includes: the slow virus 5terminal LTR as shown in SEQ ID NO.5, the slow virus as shown in SEQ ID NO.6 3terminal Self-Inactivating LTR, Gag cis element as shown in SEQ ID NO.7, such as SEQ ID NO.8 Shown RRE cis element, the env cis element as shown in SEQ ID NO.9, cPPT as shown in SEQ ID NO.10 are cis Element.

3. carrier as claimed in claim 1, it is characterised in that described slow virus packaging cis element uses third generation slow virus Carrier includes: the slow virus 5terminal LTR as shown in SEQ ID NO.5, the slow virus as shown in SEQ ID NO.6 3terminal Self-Inactivating LTR, Gag cis element as shown in SEQ ID NO.7, such as SEQ ID NO.8 Shown RRE cis element, the env cis element as shown in SEQ ID NO.9, cPPT as shown in SEQ ID NO.10 are cis Slow virus packaging cis element described in element, and the RSV promoter as shown in SEQ ID NO.4.

4. the carrier as described in any one of claim 1-3, it is characterised in that described eWPRE enhancement mode marmot hepatitis B virus Posttranscriptional regulatory element has the enhancing of 6 nucleotide to suddenly change, particularly as follows: g.396G > A, g.397C > T, g.398T > C, g.399G > A、g.400A>T、g.411A>T。

5. the CAR-T transgene carrier of the replication defective recombinant slow virus of a targeting CD123 as claimed in claim 1 Construction method, it is characterised in that comprise the following steps:

(1) ampicillin resistance gene AmpR sequence (as shown in SEQ ID NO.1), prokaryotic replions pUC Ori sequence will be contained Arrange (as shown in SEQ ID NO.2), Viral Replicon SV40Ori sequence (as shown in SEQ ID NO.3), pack for slow virus Slow virus packaging cis element, ZsGreen1 green fluorescent protein (as shown in SEQ ID NO.11), IRES ribosome combine Sequence (as shown in SEQ ID NO.12), eWPRE enhancement mode marmot hepatitis B virus posttranscriptional regulatory element are (such as SEQ ID Shown in NO.13) it is stored on slow virus skeleton plasmid；

(2) by people's EF1 α promoter (as shown in SEQ ID NO.14), for composition integrate identification, transmit, start two Chimeric antigen receptor for CAR or three generations CAR is combined into secondary CAR or three generations's CAR design, through enzyme action, connects, recombinates Reaction is cloned in slow virus skeleton plasmid, obtains the recombinant slow virus plasmid of secondary CAR or three generations CAR design；

(3) by the recombinant slow virus plasmid obtained 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, packs and successfully weighs in HEK293T/17 cell Group slow virus carrier can be discharged in cells and supernatant, collects the supernatant of the recombined lentivirus vector comprised；

(4) sucking filtration, absorption, the ion-exchange method of eluting is used to be purified the recombinant slow virus supernatant obtained, respectively To recombined lentivirus vector.

6. method as claimed in claim 5, it is characterised in that in step (1), described slow virus packaging cis element uses the Secondary slow virus carrier includes: slow virus 5terminal LTR as shown in SEQ ID NO.5, as shown in SEQ ID NO.6 Slow virus 3terminal Self-Inactivating LTR, Gag cis element as shown in SEQ ID NO.7, such as SEQ ID RRE cis element shown in NO.8, the env cis element as shown in SEQ ID NO.9, the cPPT as shown in SEQ ID NO.10 Cis element；Described slow virus packaging cis element uses third generation slow virus carrier to include: slow as shown in SEQ ID NO.5 Virus 5 terminal LTR, slow virus 3terminal Self-Inactivating LTR as shown in SEQ ID NO.6, as Gag cis element shown in SEQ ID NO.7, RRE cis element as shown in SEQ ID NO.8, as shown in SEQ ID NO.9 Env cis element, slow virus packaging cis element as described in cPPT cis element shown in SEQ ID NO.10, Yi Jiru RSV promoter shown in SEQ ID NO.4.

7. method as claimed in claim 5, it is characterised in that in step (1), described eWPRE enhancement mode marmot B-type hepatitis Poison posttranscriptional regulatory element has the enhancing of 6 nucleotide to suddenly change, particularly as follows: g.396G > A, g.397C > T, g.398T > C, g.399G>A、g.400A>T、g.411A>T。

8. method as claimed in claim 7, it is characterised in that in step (2), people's EF1 α promoter start whole CAR base Because expressing；CD8leader Chimerical receptor signal peptide is positioned at the N end of CAR coded sequence, is used for guiding CAR protein localization in cell Film；CD123 single-chain antibody light chain VL, Optimal Linker C, CD123 single-chain antibody heavy chain VH is combined into scfv region, uses In identifying CD123 antigen；CD8Hinge Chimerical receptor hinge is for being anchored to scfv outside cell membrane； CD8Transmembrane Chimerical receptor cross-film district is for being fixed on cell membrane by whole Chimerical receptor；CD137 Chimerical receptor Costimulating factor is used for stimulating T cell propagation and cytokine secretion；TCR Chimerical receptor t cell activation territory is used for activating downstream The expression of signal path；When scfv region is combined with CD123 antigen, signal is transferred to intracellular by Chimerical receptor, thus Generation includes that T cell propagation, cytokine secretion increase, Anti-apoptotic proteins secretion increases, cell death postpones, cracking target A series of biological effects of cell.

9. method as claimed in claim 5, it is characterised in that in step (4), described slow virus carrier has band fluorescence labels The version of zsGreen1 and without fluorescence labels zsGreen1 version, the version of band fluorescence labels is used for experiment in vitro, without glimmering The version of optical label is used for clinical experiment.

10. method as claimed in claim 5, it is characterised in that in step (4), described sucking filtration step supernatant to be controlled volume At 200ml～2000ml, control vacuum, at-0.5MPA～-0.9MPA, prevents the carrier loss brought due to plug-hole；Described The pH value of adsorption step solution to be controlled, 6～8, prevents the change of PH from causing carrier to inactivate；Described elution step to control to wash The ionic strength of de-liquid, at 0.5M～1.0M, prevents the change of ionic strength from causing eluting not exclusively or carrier inactivation.

The application in preparing adoptive cellular therapeutic agent of 11. carriers as described in any one of claim 1-4.

12. apply as claimed in claim 11, it is characterised in that described adoptive cellular therapeutic agent is the white blood of acute myeloid Sick (AML) medicine.