CN105950664B - A kind of replication defective recombinant slow virus CAR-T transgene carrier targeting CD123 and its construction method and application - Google Patents
A kind of replication defective recombinant slow virus CAR-T transgene carrier targeting CD123 and its construction method and application Download PDFInfo
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Abstract
The invention discloses a kind of CAR-T transgene carriers of replication defective recombinant slow virus for targeting CD123, comprising: the prokaryotic replions pUC Ori sequence for plasmid replication；The sequence of AmpR containing ampicillin resistance gene for purpose bacterial strain massive amplification；For enhancing the Viral Replicon SV40Ori sequence of the duplication in eukaryocyte；Slow virus for slow virus packaging packs cis element；ZsGreen1 green fluorescent protein for eukaryotic cell expression green fluorescence；IRES ribosome binding sequence for common transcriptional expression protein；People's EF1 α promoter of eukaryotic transcription for Chimeric antigen receptor gene；Integrate identification, transmitting, two generation CAR of starting or the Chimeric antigen receptor of three generations CAR for forming；For enhancing the eWPRE element of the expression efficiency of transgenosis.In addition, also disclosing construction method and the application of the carrier.The method can significantly improve the secretion of cell factor, the killing effect in vitro of CAR-T cell, clinical treatment acute myeloid leukemia (AML) effect is prominent.
Description
Technical field
The invention belongs to field of medical biotechnology, and in particular to a kind of carrier more particularly to a kind of duplication for targeting CD123 lack
Fall into the CAR-T transgene carrier of property recombinant slow virus.Moreover, it relates to construction method and the application of the carrier.
Background technique
The theoretical basis of immunotherapy of tumors is that immune system has identification tumor associated antigen, regulation body attack tumour
The ability of cell (cell dissolution of high degree of specificity).This bioprocess is sufficiently complex, at present still in research among.Previous generation
It records the nineties, multiple computer MSR Information systems have discovered that tumour antigen (t μm of or antigens), and T lymphocyte can be by main
Histocompatibility complex (major histocompatibility complex, MHC) dependence mode identifies these tumours
Antigen.
Immunotherapy of tumors is generally divided into two classes, nospecific immunity and specific immunity.Nonspecific immunotherapy for bronchus master
It to include interleukin 2 (interle μ kin-2, IL-2), interferon-' alpha ' (interferon α, IFN-α), tumor necrosis factor
Sub (t μm of or necrosis factor, TNF-α), the cell factors such as BCG vaccine and toxin, adoptive cellular immunotherapy etc..
Specific active immunotherapy is mainly tumor vaccine.
1.1 tumour Nonspecific immunotherapy for bronchus
Nonspecific immune response be it is inherent, its formation does not need antigenic stimulus, can be widely for more
Kind antigen, is the basis of immune response, but specificity is not strong, tends not to generate sufficient intensity to certain specific antigen substance
React nonspecific immunization therapy.In the cytokine profiles for entering clinical test, interleukin 2 and interferon are answered
With the most extensively [Rosenberg S A, Lotze M T, M μ μ l L M, et al.A 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 antibodies
Monoclonal antibody is used widely in therapeutic field of tumor over nearly more than 20 years.Antitumor monoclonal antibody medicine generally wraps
Include two classes: first is that antitumor monoclonal antibody, second is that antitumor monoclonal antibody conjugate or immune conjugate.Immune conjugate molecule is by list
Anti- to form with " bullet " drug two parts, " bullet " mainly includes radionuclide, drug and toxin, is distinguished after connecting with monoclonal antibody
Constitute radio-immunity conjugate, chemo-immunity conjugate and immunotoxin.In in November, 1997 and U.S. FDA in October, 1998 point
Two monoclonal antibodies for clinical cancer therapy-Rit μ ximab (rit μ xan) and Trast μ z μm ab is not passed through
(herceptin)[Dillman R O.Magic bμllets at last Finally—approval of 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 transduction effect and targeting
Deng three kinds of mechanism of action, without direct lethal effect.The problem of additionally there are in terms of pharmacology, mainly reaches tumour
Dose is insufficient.Since conjugate is foreign protein, can be absorbed by reticuloendothelial system, have it is a great deal of will accumulate in liver, spleen and
Marrow.Conjugate is macromolecular substances, by capillary endothelium layer and penetrates tumour cell external series gap and is restricted.
The adoptive immunotherapy of 1.3 tumours
The adoptive immunotherapy of tumour, which refers to, is transfused to patient for the self or alloimmune effector cell of Activation In Vitro, with
Kill the tumour cell of patient's body.A critical issue in tumour adoptive immunotherapy is to find suitable tumor-killing
Cell.Since the eighties in last century, including LAK, cell because induction killing cell (cytokine-ind μ ced
Killers, CIK), the cells such as TIL be successively applied to clinic, but it is lower since there is amplification speeds, cell origin is tired
Difficult, the not high problems of cytotoxicity, are restricted in clinical application.How the specific for tumour antigen of T cell is improved
With important clinical meaning.T cell the identification of tumour antigen is mainly passed through T cell receptor (T cell receptor,
TCR) human leukocyte antigen (h μm of an le μ kocyte antigen, the HLA)-peptide complexes on tumor cell surface, because
This, T cell depends on the TCR on T cell surface to the specificity that tumour antigen identifies.It is cloned using the means of molecular biology swollen
The TCR of tumor specific T-cells, and by viral vectors of the building containing TCR, TCR is transferred in normal T cell, keeps 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 to excite the specificity antineoplastic immunity of patient by importing tumour antigen to patient's body
Reaction.Due to vaccine therapy have many advantages, such as specificity, in vivo immunological effect hold time it is long, have become at present research heat
Point.In recent years polypeptide vaccine, nucleic acid vaccine, holoprotein vaccine, anti-unique antibody vaccine, recombinant viral vaccine, bacterial vaccine,
Tumor cell vaccine, Dendritic Cells (dendritic cells, DC) vaccine of gene modification etc. are widely studied and apply
[Robbins P F, Morgan R A, Feldman S A, et al.T μm or 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.].Tumour
The large-scale application of vaccine therapy is urgently to be resolved the problem of there are three aspects.It is each tumour, every firstly, tumor associated antigen
A hypotype, each neoplasm staging, these are different with respect to antigen presentation, so making an accurate selection of antigen, the patient crowd that makes an accurate selection of is to cause
It closes important.Second, how to reach tumour antigen efficient absorption and expression in Dendritic Cells? antigen is inhaled by Dendritic Cells
Receipts are with surface receptor for mediation.Dendritic Cells has more than ten of receptor, how according to the selection of specific antigen accordingly by
Body? third, for the regulation of differentiation of dendritic cells maturation.The differentiation and maturation of Dendritic Cells is an extremely complex mistake
Journey, it, which can both move towards activation T cell, can also move towards to inhibit T cell.[targeting DC cell therapeutic type tumor vaccine: bright spot with
It challenges and deposits.http://www.biodiscover.com/news/research/115794.html].
1.5 tumour CAR-T treatment
CAR-T, full name are Chimeric Antigen Receptor T-Cell Imm μ notherapy, chimeric antigen by
Body T cell immunotherapy, structure [Eleanor J.Cheadle, et al.CAR T cells:driving the as shown in Figure 1
road from the laboratory to the clinic.Immμnological Reviews 2014.Vol.257:91–
106]。
The t cell activation that first generation CAR is mediated is completed by the Tyrosine Activating Motifs on CD3z chain or FceRIg.
Letter needed for CD3z chain is capable of providing t cell activation, cracking target cell, adjusts IL-2 secretion and play anti-tumor activity in vivo
Number.But the anti-tumor activity of first generation CAR transformation T cell is restricted in vivo, and it is thin that T cell proliferation reduction eventually leads to T
The apoptosis of born of the same parents.
Second generation CAR increases a new costimulatory signal intracellular, it is demonstrated experimentally that this original to make to be originated from
" signal 1 " of TCR/CD3 complex expands, and many researchs all show the second generation CAR and first generation CAR that are equipped with " signal 2 "
It compares, antigentic specificity is constant, and T cell proliferation, cytokine secretion increase, and Anti-apoptotic proteins secretion increases, and cell is dead
Die delay.Common costimulatory molecules are CD28, but have research to be replaced CD28 with CD137 (4-1BB) later, except this it
Outside, a kind of thinking using NK cell receptor CD244 is also suggested.Although which is better and which is worse actually by different second generation CAR, no
Same researcher is not quite similar with result obtained in external research in vivo with different tumours.[depth full version: CAR-
The status and future biology paddy .2015-051-15 of T].
In order to further improve the design of CAR, it not only includes " letter that many study groups, which start to be conceived to, which develops third generation CAR,
Number 1 ", " signal 2 ", further comprise additional costimulatory signal.Different researchers are opened with different target spot and costimulatory signal
The having a certain difference property of comparison result of the obtained second generation CAR and third generation CAR of research of exhibition.Table is reported in some researchs
Recombination T cell up to third generation CAR is significantly increased in terms of anti-tumor activity, time to live and cytokine release；
Second generation CAR and the third generation CAR recombination T cell of the result of study display targeting M Μ C1 of Wilkie etc. is in antitumor cell poison
Property aspect and no significant difference, although expression third generation CAR T cell can secrete a greater amount of IFN-γ (Wilkie S,
Picco G,Foster J,et al.Retargeting of hμman T cells to tμmorassociated MΜC1:
the evolμtion of a chimeric antigen receptor.J Immμnol 2008；180:4901–4909.).
It is worth noting that, above-mentioned difference is only the conclusion obtained in experiment in vitro, not yet compare the second generation and in vivo at present
The report of three generations CAR.
Difference between this several generations CAR may be incessantly from signal transduction domain, extracellular antigen binding domain (scFv), again
The transfection method (slow virus VS retrovirus) of group T cell, feedback mode (the venous re-transfusion VS peritonaeum VS tumor for recombinating T cell
Body) etc. may influence the final antitumous effect of CAR-T cell.
It is entitled filed in the applicant on March 17th, 2016 that " one kind is based on replication defective recombinant slow virus
CAR-T transgene carrier and its construction method and application " application for a patent for invention, disclose the replication defective for CD19
The CAR-T transgene carrier and its construction method of recombinant slow virus and application.The present invention be directed to the CAR-T transgenosis 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 targeting CD123 a kind of
CAR-T transgene carrier.
The second technical problem to be solved by the present invention is to provide the replication defective recombinant slow virus of targeting CD123
The construction method of CAR-T transgene carrier.
The third technical problem to be solved by the present invention is to provide the replication defective recombinant slow virus of targeting CD123
The application of CAR-T transgene carrier.
The present invention relates to the medical configuration product containing peptide, and in particular to:
One, the sequence of AmpR containing ampicillin resistance gene, prokaryotic replions pUC Or i sequence, Viral Replicon
SV40Or i sequence, RSV promoter, people EF1 α promoter, 5 terminal LTR of slow virus, 3 terminal of slow virus
Self-Inactivating LTR, Gag cis element, RRE cis element, env cis element, cPPT cis element, IRES core
The recombinant lentiviral disease of sugared body binding sequence, ZsGreen1 green fluorescent protein, eWPRE marmot hepatitis B posttranscriptional regulatory element
Poisonous carrier skeleton, this recombined lentivirus vector skeleton can carry different therapeutic genes and be widely used in adoptive thin
Born of the same parents' therapy 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 transmembrane region, the costimulation of CD28 Chimerical receptor because
Son, CD137 Chimerical receptor costimulating factor, TCR Chimerical receptor t cell activation domain construct to form recombined lentivirus vector, the party
The recombined lentivirus vector that method obtains may be implemented to express CD123 Chimeric antigen receptor on human T lymphocyte, guides and activates
T lymphocyte is used clinically for treatment acute myeloid leukemia (AML) to the lethal effect of CD123 positive cell.
CAR-T technology of the present invention for CD123 is that a kind of the immune of anti-cancer monoclonal antibody that combine is controlled
Treat the targeted therapy new technology with the adoptive immunotherapy advantage of tumour.CD123 is the alpha chain of human interleukin-13 receptor, big
Most acute myeloid leukemia (AML) cells and many hematopoietic cell surfaces has an expression, and CD123 is one and extraordinary exempts from
Epidemic disease therapy target, because even being expressed in seldom cell in CD123, its expression can increase at any time to be gradually increased, and
Acute myeloid leukemia is likely due to the candidate stem cell in preleukemia by clone evolution, with CD123
For target spot, then it can play the role of clear marrow (Saar Gill, Carl H.June et al.Preclinical targeting
of human acute myeloid leukemia and myeloablation using chimeric antigen
receptor-modified T cells.Blood.2014；123 (15): 2343-2354.), recent years is white in acute myeloid
There is significant curative effect in the treatment of blood disease (AML), it is considered to be most promising acute myeloid leukemia (AML) therapeutic modality
One of.
Chimeric antigen receptor (CAR) is the core component (shown in Fig. 1) of CAR-T, and it is non-dependent to assign T lymphocyte HLA
Mode identifies the ability of tumour antigen, this enables by the T cell of CAR transformation compared to nave T cell surface receptor TCR
Identify wider target.It include a tumor associated antigen (t μm of or-associated in the basic engineering of CAR
Antigen, TAA) combined area (the scFV section for being typically derived from monoclonal antibody antigen bond area), an extracellular hinge area,
One transmembrane region and an intracellular signal transduction area.Specificity, validity and genetic modification of scFV sections of the design for CAR
It is crucial determinant for the safety of T cell itself.
In order to solve the above-mentioned technical problem, the present invention is achieved through the following technical solutions:
In one aspect of the invention, a kind of CAR-T transgenosis of replication defective recombinant slow virus targeting CD123 is provided
Carrier, comprising: for the prokaryotic replions pUC Ori sequence of plasmid replication, as shown in SEQ ID NO.2；For purpose bacterial strain
The sequence of AmpR containing ampicillin resistance gene of massive amplification, as shown in SEQ ID NO.1；For enhancing in eukaryocyte
The Viral Replicon SV40Ori sequence of duplication, as shown in SEQ ID NO.3；Slow virus packaging for slow virus packaging is cis-
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
Eukaryotic transcription people's EF1 α promoter, as shown in SEQ ID NO.14；EWPRE for enhancing the expression efficiency of transgenosis increases
Strong type marmot hepatitis B posttranscriptional regulatory element, as shown in SEQ ID NO.13；And identify, transmit for forming collection,
Start in the Chimeric antigen receptor of the two generation CAR or three generations CAR of one；It is described to integrate identification, transmitting, starting for forming
The Chimeric antigen receptor of two generation CAR include: CD8leader Chimerical receptor signal peptide, such as SEQ as shown in SEQ ID NO.15
CD123 single-chain antibody light chain VL, Optimal Linker C, such as SEQ as shown in SEQ ID NO.19 shown in ID NO.20
CD123 single-chain antibody heavy chain VH shown in ID NO.16, the CD8Hinge Chimerical receptor hinge as shown in SEQ ID NO.21, such as
CD8Transmembrane Chimerical receptor transmembrane region, CD28 shown in SEQ ID NO.22, as shown in SEQ ID NO.23
CD137 Chimerical receptor costimulating factor, the TCR Chimerical receptor t cell activation domain as shown in SEQ ID NO.24.It is described to be used for group
Chimeric antigen receptor at the three generations CAR for integrating identification, transmitting, starting includes: as shown in SEQ ID NO.15
CD8leader Chimerical receptor signal peptide, CD123 single-chain antibody light chain VL, such as SEQ ID as shown in SEQ ID NO.20
Optimal Linker C shown in NO.19, CD123 single-chain antibody heavy chain VH, such as SEQ ID as shown in SEQ ID NO.16
CD8Hinge Chimerical receptor hinge, the CD8Transmembrane Chimerical receptor as shown in SEQ ID NO.22 shown in NO.21
Transmembrane region, CD28, the CD137 Chimerical receptor costimulating factor as shown in SEQ ID NO.23, as shown in SEQ ID NO.24
The Chimerical receptor t cell activation domain TCR and the CD28 Chimerical receptor costimulating factor as shown in SEQ ID NO.25.The present invention
Used scFV sections of linker design, can be applied to second generation CAR design scheme, equally also can be applied to the third generation
CAR design scheme.Third generation CAR design increases CD28 Chimerical receptor costimulating factor (SEQ ID compared with second generation design
NO.25), in theory, stronger signal amplification is had.
Further, the slow virus packaging cis element includes: such as SEQ ID NO.5 using second generation slow virus carrier
Shown in slow virus 5terminal LTR, the slow virus 3terminal Self- as shown in SEQ ID NO.6
Inactivating LTR, the Gag cis element as shown in SEQ ID NO.7, the cis- member of RRE as shown in SEQ ID NO.8
Part, the env cis element as shown in SEQ ID NO.9, the cPPT cis element as shown in SEQ ID NO.10.The slow virus
Packaging cis element using third generation slow virus carrier include: 5 terminal LTR of slow virus as shown in SEQ ID NO.5,
Slow virus 3terminal Self-Inactivating LTR as shown in SEQ ID NO.6, as shown in SEQ ID NO.7
Gag cis element, the RRE cis element as shown in SEQ ID NO.8, the env cis element as shown in SEQ ID NO.9, such as
Slow virus described in cPPT cis element shown in SEQ ID NO.10 packs cis element, and as shown in SEQ ID NO.4
RSV promoter.The used CAR design scheme of the present invention can be applied in third generation slow virus carrier structure, can also apply
In in second generation slow virus carrier structure.The difference (as shown in Figure 2 B) of the second generation and third generation slow virus carrier in structure,
Mainly the region U3 of 5 ' LTR of second generation carrier is replaced with RSV promoter by third generation slow virus carrier, and thus eliminating the need U3
To the dependence of Tat albumen when transcription, Tat sequence can be both removed in the structural gene of slow virus, also improves slow virus base
Because of group transcriptional level and transcription duration.The second generation and third generation slow virus carrier are mainly the difference of subgenomic transcription mode,
Therefore the used CAR design scheme of the present invention can be applied to this two generations slow virus carrier.Carrier framework of the present invention
Preferably third generation slow virus carrier (shown in Fig. 2A), 3 ' SIN LTR eliminate the region U3, eliminate slow virus carrier and self answer
A possibility that processed, substantially increases safety；CPPT and eWPRE element is increased, the table of transduction efficiency and transgenosis is improved
Up to efficiency；The lasting efficient transcription of core RNA when ensure that slow virus carrier packaging using RSV promoter；Using people's itself
EF1 α promoter enables CAR gene in human body long lasting for expression.
Further, the enhanced marmot hepatitis B posttranscriptional regulatory element of the eWPRE has the enhancing of 6 nucleotide
Mutation, specifically: g.396G > A, g.397C > T, g.398T > C, g.399G > A, g.400A > T, g.411A > T.
In another aspect of this invention, a kind of CAR-T of the replication defective recombinant slow virus of above-mentioned targeting CD123 is provided
The construction method of transgene carrier, comprising 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) are used for slow disease
The slow virus of poison packaging packs cis element, ZsGreen1 green fluorescent protein (as shown in SEQ ID NO.11), IRES ribose
Body binding sequence (as shown in SEQ ID NO.12), enhanced marmot hepatitis B posttranscriptional regulatory element (such as SEQ of eWPRE
Shown in ID NO.13) it is stored on slow virus skeleton plasmid；
(2) integrate identification, transmitting, starting by people EF1 α promoter (as shown in SEQ ID NO.14), for forming
Two generation CAR or the Chimeric antigen receptor of three generations CAR be combined into two generation CAR or three generations's CAR design scheme, by digestion, connection,
Recombining reaction is cloned into slow virus skeleton plasmid, obtains the recombinant slow virus plasmid of two generation CAR or three generations CAR design；
(3) by obtained recombinant slow virus plasmid and slow virus packaging plasmid pPac-GP, pPac-R and memebrane protein plasmid
PEnv-G transfects HEK293T/17 cell jointly, after carrying out gene transcript expression in HEK293T/17 cell, packs and successfully weighs
Group slow virus carrier can be discharged into cells and supernatant, collect the supernatant for the recombined lentivirus vector for including；
(4) obtained recombinant slow virus supernatant is purified using the column purification mode for filtering, adsorbing, eluting, respectively
Obtain recombined lentivirus vector.
In step (1), the slow virus packaging cis element includes: such as SEQ ID using second generation slow virus carrier
Slow virus 5terminal LTR shown in NO.5, the slow virus 3terminal Self- as shown in SEQ ID NO.6
Inactivating LTR, the Gag cis element as shown in SEQ ID NO.7, the cis- member of RRE as shown in SEQ ID NO.8
Part, the env cis element as shown in SEQ ID NO.9, the cPPT cis element as shown in SEQ ID NO.10；The slow virus
Packaging cis element using third generation slow virus carrier include: slow virus 5terminal LTR as shown in SEQ ID NO.5,
Slow virus 3terminal Self-Inactivating LTR as shown in SEQ ID NO.6, as shown in SEQ ID NO.7
Gag cis element, the RRE cis element as shown in SEQ ID NO.8, the env cis element as shown in SEQ ID NO.9, such as
Slow virus described in cPPT cis element shown in SEQ ID NO.10 packs cis element, and as shown in SEQ ID NO.4
RSV promoter.
In step (1), the enhanced marmot hepatitis B posttranscriptional regulatory element of eWPRE has 6 nucleotide
Enhancing mutation, specifically: g.396G > A, g.397C > T, g.398T > C, g.399G > A, g.400A > T, g.411A > T.
In step (2), entire CAR gene expression is started by people's EF1 α promoter；CD8leader Chimerical receptor signal peptide
Positioned at the N-terminal of CAR coded sequence, for guiding CAR albumen to be positioned at cell membrane；CD123 single-chain antibody light chain VL, Optimal
Linker C, CD123 single-chain antibody heavy chain VH are combined into the region scfv, for identification CD123 antigen；CD8Hinge Chimerical receptor
Hinge is used to for scfv being anchored on the outside of cell membrane；CD8 Transmembrane Chimerical receptor transmembrane region will be for will entirely be fitted into
Receptor is fixed on cell membrane；CD137 Chimerical receptor costimulating factor is for stimulating T cell proliferation and cytokine secretion；TCR
Chimerical receptor t cell activation domain is used to activate the expression of downstream signaling pathway；When the region scfv and CD123 antigen binding, letter
It number is transferred into the cell by Chimerical receptor, to generate T cell proliferation, cytokine secretion increases, Anti-apoptotic proteins
Secretion increases, cell death delay, cracks a series of biological effects such as target cell.
In step (4), the slow virus carrier has the version with fluorescence labels zsGreen1 and without fluorescence labels
ZsGreen1 version, the version with fluorescence labels are used for experiment in vitro, are used for clinical trial without the version of fluorescence labels.
In step (4), it should be noted that several key points have, a: filter step to control supernatant volume (200ml~
2000ml) with vacuum degree (- 0.5MPA~-0.9MPA), prevent due to plug-hole bring carrier loss；B: adsorption step needs to control
The PH (6~8) of solution processed, prevents the variation of PH from carrier being caused to inactivate；C: elution step needs to control the ionic strength of eluent
(0.5M~1.0M) prevents the variation of ionic strength from causing elution not exclusively or carrier inactivation.
In another aspect of this invention, above-mentioned carrier is provided and is preparing the application in adoptive cellular therapeutic agent.It is preferred that
, the adoptive cellular therapeutic agent is acute myeloid leukemia (AML) therapeutic agent.It is verified through clinical test, the present invention
Carrier is applied to clinical treatment acute myeloid leukemia (AML), realizes the fully erased of tumour cell in patient's body, therefore
Carrier of the present invention is gathered around in CART therapy field to have broad application prospects.
Compared with prior art, the invention has the following beneficial effects:
The linker for the scFV section that the present invention uses is designed, and is the Linkers pool using Shi Ao company, by albumen
The analysis of matter Structure bioinformatics database (https: //www.predictprotein.org/), by protein two
The comparison of the protein properties such as level structure, Solvent accessibility, protein flexibility, disulfide bridge, binding site, preferably
Out.Detecting test and killing-efficiency test by cell in vitro cytokine secretion proves, can be significant compared with external design
Improve the secretion of cell factor, the killing effect in vitro of CAR-T cell.Also, also face than foreign countries in the effect of clinical treatment
The effect of bed experiment is good.
Slow virus carrier column purification system of the present invention is the slow virus large-scale production that the applicant develops
Technique.Slow virus carrier column purification mode of the present invention is different from the ultracentrifugation or ultracentrifugal generallyd use
Mode, common supercentrifugation or supercentrifugal process are to separate lentiviral particle using centrifugal sedimentation principle, unavoidably
Meeting remain impurity similar in many sedimentation coefficients, adverse effect is brought to subsequent experimental.Also, tubulature process is complicated, operation
Cumbersome, multiple conversions container brings more opportunities for contamination.And slow virus carrier column purification technique of the invention is semi-automation
Operation, all processes are completed hundred grades of Experimental Areas, are avoided manually-operated cumbersome and are made mistakes and pollute probability, are recycled
Clinical criteria is fully achieved in the indexs such as endotoxin, mycoplasma, titer determination in slow virus carrier.It is subsequent follow up develop it is complete from
Dynamic purifying 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 used by 725-33.) is compared, and bacteriophage f1 replication orgin is eliminated, using eukaryotic viral SV40
Replicon increases copy number of the target gene in eukaryocyte, enhances eukaryotic expression effect.
Third generation slow virus skeleton plasmid pLenti-3G basic of the present invention, using enhancedWPRE member
Part (i.e. the enhanced marmot hepatitis B posttranscriptional regulatory element of eWPRE), 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；Used by 365:725-33.)
WPRE element is compared, and has enhancing mutation (g.396G > A, g.397C > T, g.398T > C, g.399G > A, g.400A of 6 nucleotide
> T, g.411A > T), the poly-adenosine of primary transcript can be enhanced, increase the content of intracellular mRNA, enhance transgenosis
Expression efficiency.
The Lentival packaging system that the present invention uses is four plasmid packaging systems of non-auxiliary virus, passes through four kinds of plasmids
Common transfection generates recombined lentivirus vector into HEK293T/17 cell.Slow virus carrier after recombination is replication defect type
Exogenous sequences can be integrated into host gene by carrier, disposable, can not be replicated and be proliferated, safety improves a lot.
The slow virus carrier that the present invention uses has the version with fluorescence labels zsGreen1 and without fluorescence labels
ZsGreen1 version, the version with fluorescence labels are used for experiment in vitro, are used for clinical trial without the version of fluorescence labels, are applicable in
It is in extensive range.
Present invention preferably employs third generation slow virus carrier, 3 ' SIN LTR eliminate the region U3, eliminate slow virus carrier
A possibility that self-replacation, substantially increases safety；CPPT and eWPRE element is increased, transduction efficiency is improved and turns base
The expression efficiency of cause；The lasting efficient transcription of core RNA when ensure that slow virus carrier packaging using RSV promoter；Using people
The EF1 α promoter of itself, enables CAR gene in human body long lasting for expression.
It is provided as it can be seen that recombined lentivirus vector of the present invention will be treated to the CAR-T of acute myeloid leukemia (AML)
Reliable transgenosis guarantee.
Detailed description of the invention
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 schematic diagram of the present invention；Wherein Fig. 2A is that the third generation that the present invention uses is slow
Viral vectors structural schematic diagram, Fig. 2 B are the second generation and third generation slow virus carrier structure comparison schematic diagram；
Fig. 3 is the building flow chart that recombined lentivirus vector of the present invention is constructed in the embodiment of the present invention 1.Wherein,
Fig. 3 (A) is the structural schematic diagram of slow virus skeleton plasmid pLenti-3G basic；Fig. 3 (B) is the knot of pCAR22-CLA plasmid
Structure schematic diagram；Fig. 3 (C) is the structural schematic diagram 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 schematic diagram of slow virus packaging plasmid pPac-GP；Fig. 3 (F) is slow virus packaging plasmid pPac-R
Structural schematic diagram；Fig. 3 (G) is the structural schematic diagram of memebrane protein pEnv-G；
Fig. 4 is that the digestion prediction of recombinant slow virus plasmid pCAR123-CLA and digestion agarose are solidifying in the embodiment of the present invention 1
Gel electrophoresis figure；Wherein, Fig. 4 A is the digestion prediction schematic diagram of recombinant slow virus plasmid pCAR123-CLA, wherein lane1 is 1kb
DNA ladder Marker: band is from top to bottom successively are as follows: 10kb, 8Kb, 6kb, 5Kb, 4kb, 3.5Kb, 3Kb, 2.5kb,
2Kb, 1.5kb, 1Kb, 750bp, 500bp, 250bp, lane2 are the EcoR V+BamH I digestion predictions of pCAR123-CLA: item
Band is from top to bottom successively are as follows: 8950bp, 959bp；Fig. 4 B is the digestion Ago-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 is that the digestion prediction of recombinant slow virus plasmid pCAR123-CLB and digestion agarose are solidifying in the embodiment of the present invention 1
Gel electrophoresis figure；Wherein, Fig. 5 A is the digestion prediction schematic diagram of recombinant slow virus plasmid pCAR123-CLB, wherein lane1 is 1kb
DNA ladder Marker: band is from top to bottom successively are as follows: 10kb, 8Kb, 6kb, 5Kb, 4kb, 3.5Kb, 3Kb, 2.5kb,
2Kb, 1.5kb, 1Kb, 750bp, 500bp, 250bp, lane2 be pCAR123-CLB Pvu II digestion prediction: band on to
Under successively are as follows:: 6722bp, 2364bp, 823bp；Fig. 5 B is that the digestion agarose of recombinant slow virus plasmid pCAR123-CLB is solidifying
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 the digestion prediction and digestion 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 digestion prediction schematic diagram of recombinant slow virus plasmid pCAR123-OLC, wherein
Lane1 is 1kb DNA ladder Marker: band is from top to bottom successively are as follows: 10kb, 8Kb, 6kb, 5Kb, 4kb, 3.5Kb,
3Kb, 2.5kb, 2Kb, 1.5kb, 1Kb, 750bp, 500bp, 250bp, lane2 are the Kpn I digestion predictions of pCAR123-OLC:
Band is from top to bottom successively are as follows: 8483bp, 1426bp；Fig. 6 B is the digestion 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 2 intermediate ion exchange chromatography of embodiment of the present invention purifying recombined lentivirus vector；
Fig. 8 is the titre testing result schematic diagram of the different way of purification of recombined lentivirus vector in the embodiment of the present invention 2；
Fig. 9 is the detection of mycoplasma result signal of the different way of purification of recombined lentivirus vector in the embodiment of the present invention 2
Figure, lane1 DL2000marker, counterband tape is from top to bottom successively from top to bottom are as follows: 2kb, 1kb, 750bp, 500bp,
250bp,100bp；Lane2 is positive control；Lane3 is negative control；Lane4 is PBS；Lane5 is water；Lane6 is cracking
Liquid；Lane7 is the slow virus of ultracentrifugation purifying；Lane8 is the slow virus of high speed centrifugation purifying；Lane9 is ion exchange color
Compose the slow virus of purifying；Lane10 is ghost；
Figure 10 be the embodiment of the present invention 3 in mRNA relative expression quantity histogram, RT-QPCR the result shows that CAR in PBMC
Intracellular efficient transcription；
Figure 11 is that the WB of CAR expressing quantity in the embodiment of the present invention 3 detects figure, the results showed that CAR albumen is thin in PBMC
High efficient expression intracellular, in Figure 11 A, lane1 is PBMC ghost, and lane2 is control virus MOCK, lane3 lvCAR123-
CLA, lane4 lvCAR123-CLB, lane5 lvCAR123-OLC；Figure 11 B is beta-actin internal reference band；
Figure 12 is the killing-efficiency that LDH detects under the conditions of different effect target ratios in the embodiment of the present invention 3, and E is effector cell, T
For target cell；
Figure 13 is that qPCR detects Cytokine Expression Level schematic diagram under the conditions of different effect target ratios, E in the embodiment of the present invention 3
For effector cell, T is target cell；Wherein, Figure 13 A indicates the mRNA transcriptional level of IL-2；Figure 13 B indicates that the mRNA of IFN-γ turns
Record is horizontal；
Figure 14 is CAR123-T cell infusion acute myeloid leukemia (AML) cell xenograft in the embodiment of the present invention 4
After mouse model, intracorporal situation of change schematic diagram；After Figure 14 A indicates injection, experimental group and control group mice in-vivo tumour with
The living imaging result of time change；After Figure 14 B indicates injection, experimental group and control group mice vivo biodistribution luminous intensity are at any time
Between change curve；After Figure 14 C indicates injection, experimental group and control group mice life cycle change with time situation；
Figure 15 is the titre of the second generation of continuous 3 batches and third generation recombined lentivirus vector in the embodiment of the present invention 5
As a result comparison schematic diagram；
Figure 16 is the killing-efficiency schematic diagram that LDH detects under the conditions of different effect target ratios in the embodiment of the present invention 6, and E is effect
Cell, T are target cell.
Specific embodiment
Following embodiment is only illustrative of the invention and is not intended to limit the scope of the invention.It is not specified in embodiment specific
The experimental method of condition, usually according to normal condition, or according to the normal condition proposed by manufacturer.
Embodiment 1 constructs recombined lentivirus vector
One, material
1, slow virus skeleton plasmid pLenti-3G basic, slow virus packaging plasmid pPac-GP, pPac-R and film egg
White matter grain pEnv-G, HEK293T/17 cell, homologous recombination enzyme by generation take wing (Shanghai) biological medicine Science and Technology Ltd. provide；
2, primer: primer needed for designing amplification of DNA fragments and target site according to design of primers principle, the primer is by Shanghai
Biotech firm's synthesis, specifically:
EF1 α-F:5 '-ATTCAAAATTTTATCGATGCTCCGGTGCCCGTCAGT-3 ' (SEQ ID NO.26)
EF1 α-R:5 '-TCACGACACCTGAAATGGAAGA-3 ' (SEQ ID NO.27)
CD8leader-F:5 '-GGTGTCGTGAGGATCCGCCACCATGGCCTTACCAGTGACCGC-3 ' (SEQ ID
NO.28)
CD8leader-R:5 '-GTGTCATCTGGATGTCCGGCCTGGCGGCGTG-3 ' (SEQ ID NO.29)
VH-F:5 '-CACGCCGCCAGGCCGGAAGTGAAACTGGTGGAAAGC-3 ' (SEQ ID NO.30)
VH-R:5 '-GCTGCTCACGGTCACCAGG-3 ' (SEQ ID NO.31)
CLA-VL-F:5 '-GGTGACCGTGAGCAGCGGCGGTGGCTCGGGTGGTGGGTCGGGCGGCGGATCTGGGG
GAGGTTCTGATATTGTGATGACCCAGAGCC-3’(SEQ ID NO.32)
CLB-VL-F:5 '-GGTGACCGTGAGCAGC GGATCCACCTCCGGATCCGGAAAACCCGGATCCGGAGAA
GGATCCACCAAAGGAGATATTGTGATGACCCAGAGCC-3’(SEQ ID NO.33)
OLC-VL-F:5 '-GGTGACCGTGAGCAGCGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCG
GATCTGATATTGTGATGACCCAGAGCC-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 dna shown in ID NO.48, SEQ ID NO.49 is synthesized by Shanghai biotech firm, and with oligonucleotides dry powder or plasmid
Form saves；
4, toolenzyme BamH I, EcoR V, Pvu II, Kpn I, ApaL I, Sac I, Cla I, Sal I, T4DNA are purchased
From NEB company；
5, high fidelity enzyme PrimeSTAR, RN is purchased from Takara company；
6,0.22 μm of -0.8 μm of PES filter is purchased from millipore company；
7, plasmid extraction kit, Ago-Gel QIAquick Gel Extraction Kit are purchased from MN company；
8, competent cell TOP10 is purchased from Tiangen company；
9、NaCl、KCl、Na2HPO4.12H2O、KH2PO4、Trypsin、EDTA、CaCl2, NaOH, PEG6000 be purchased from
Hai Shenggong；
10, Opti-MEM, FBS, DMEM, 1640, Hepes, be purchased from invitrogen company；
11, Biotinylated protein L is purchased from GeneScript company；
12, the secondary antibody of horseradish peroxidase-labeled, DAB working solution are purchased from Beijing Zhong Shan Golden Bridge；
13, ECL+plusTM Western blotting system is purchased from Amersham company；
14, DNeasy kit is purchased from Shanghai JaRa company；
15, it is company that lymphocyte separation medium, which reaches section purchased from Shenzhen,；
16, phycoerythrin (PE)-conjugated streptavidin is purchased from BD Bioscience company；
17, SA-HRP is purchased from Shanghai Yi Sheng company；
18, mycoplasma test reagent box, endotoxin detection kit, CD123+K562 cell purchased from generation take wing (Shanghai) public affairs
Department；
19, LDH detection kit is purchased from promega company.
Two, the construction method of recombined lentivirus vector lvCAR123-CLA, lvCAR123-CLB, lvCAR123-OLC.
Referring to Fig. 3, the construction method of recombined lentivirus vector of the present invention is as follows:
1, by people EF1 α promoter, CD8leader Chimerical receptor signal peptide, CD123 single-chain antibody light chain VL, Common
Linker A, Common Linker B, Optimal Linker C, CD123 single-chain antibody heavy chain VH, CD8Hinge it is chimeric by
Body hinge, CD8Transmembrane Chimerical receptor transmembrane region, CD137 Chimerical receptor costimulating factor, TCR Chimerical receptor T are thin
Born of the same parents' activation domain segment is cloned into slow virus skeleton plasmid pLenti-3G basic, respectively obtains recombinant slow virus plasmid
PCAR123-CLA, pCAR123-CLB, pCAR123-OLC.
(1) slow virus skeleton plasmid pLenti-3G basic is carried out using Cla I and Sal I restriction enzyme double
Digestion, product pass through 1.5% agarose gel electrophoresis, confirm the segment V1 of 8303bp, and be tapped and recovered and be placed in Eppendorf
In pipe, corresponding segment (being shown in Table 1) is recycled with the Ago-Gel QIAquick Gel Extraction Kit of MN company, and measures the purity of product and dense
Degree；
1 Ago-Gel recycling step of table
(2) system in table 2, PCR are used using the SEQ ID NO.14 synthesized as template with primer EF1 α-F and EF1 α-R
Cycling condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 2min) * 35cycle, 72 DEG C of 10min.Product passes through
1.5% agarose gel electrophoresis, confirms the segment a of 1208bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company
Ago-Gel QIAquick Gel Extraction Kit recycle corresponding segment (being shown in Table 1), and measure the purity and concentration of product；
| Reagent | Volume (μ l) |
| H2O | 32.5 |
| 5×Bμffer(with Mg2+) | 10 |
| DNTP (each 2.5mM) | 4 |
| Primer1(+)(10μM) | 1 |
| Primer2 (-) (10 μM) | 1 |
| Template | 1 |
| PrimeSTAR | 0.5 |
2 50 μ l PCR reaction system of table
(3) with primer CD8leader-F and CD8leader-R using the SEQ ID NO.15 synthesized as template, using in table 2
System, PCR cycle condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C
5min.Product passes through 1.5% agarose gel electrophoresis, confirms the segment b of 101bp, and is tapped and recovered and is placed in Eppendorf pipe
It is interior, corresponding segment (being shown in Table 1) is recycled with the Ago-Gel QIAquick Gel Extraction Kit of MN company, and measure the purity of product and dense
Degree；
(4) system in table 2, PCR cycle are used using the SEQ ID NO.16 synthesized as template with primer VH-F and VH-R
Condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through
1.5% agarose gel electrophoresis, confirms the segment c of 336bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company
Ago-Gel QIAquick Gel Extraction Kit recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；
(5) system in table 2, PCR are used using the SEQ ID NO.20 synthesized as template with primer CLA-VL-F and VL-R
Cycling condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through
1.5% agarose gel electrophoresis, confirms the segment d of 424bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company
Ago-Gel QIAquick Gel Extraction Kit recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；
(6) system in table 2, PCR are used using the SEQ ID NO.20 synthesized as template with primer CLB-VL-F and VL-R
Cycling condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through
1.5% agarose gel electrophoresis, confirms the segment e of 430bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company
Ago-Gel QIAquick Gel Extraction Kit recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；
(7) system in table 2, PCR are used using the SEQ ID NO.20 synthesized as template with primer OLC-VL-F and VL-R
Cycling condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through
1.5% agarose gel electrophoresis, confirms the segment f of 421bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company
Ago-Gel QIAquick Gel Extraction Kit recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；
(8) with primer CD8Hinge-F and CD8Hinge-R using the SEQ ID NO.21 synthesized as template, using in table 2
System, PCR cycle condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.
Product passes through 1.5% agarose gel electrophoresis, confirms the segment g of 147bp, and be tapped and recovered and be placed in Eppendorf pipe, uses
The Ago-Gel QIAquick Gel Extraction Kit of MN company recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；
(9) it is with the SEQ ID NO.22 synthesized with primer CD8Transmembrane-F and CD8Transmembrane-R
Template uses the system in table 2, PCR cycle condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) *
35cycle, 72 DEG C of 5min.Product passes through 1.5% agarose gel electrophoresis, confirms the segment h of 100bp, and be tapped and recovered and set
In in Eppendorf pipe, corresponding segment (being shown in Table 1) is recycled with the Ago-Gel QIAquick Gel Extraction Kit of MN company, and measure production
The purity and concentration of object；
(10) with primer CD137-F and CD137-R using the SEQ ID NO.23 synthesized as template, using the system in table 2,
PCR cycle condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product
By 1.5% agarose gel electrophoresis, the segment i of 142bp is confirmed, and be tapped and recovered and be placed in Eppendorf pipe, with MN public affairs
The Ago-Gel QIAquick Gel Extraction Kit of department recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；
(11) system in table 2, PCR are used using the SEQ ID NO.24 synthesized as template with primer TCR-F and TCR-R
Cycling condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through
1.5% agarose gel electrophoresis, confirms the segment j of 355bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company
Ago-Gel QIAquick Gel Extraction Kit recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；
(12) Eppendorf is added in addition to primer using the system in table 3 using each 1 μ l of DNA fragmentation b, c, d as template
In pipe, PCR cycle condition are as follows: primer is added in 98 DEG C of 3min, (98 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 30sec) * 6cycle
CD8leader-F/VL-R, (98 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 40sec) * 24cycle, 72 DEG C of 5min.Product passes through
1.5% agarose gel electrophoresis, confirms the segment k of 814bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company
Ago-Gel QIAquick Gel Extraction Kit recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；
| Reagent | Volume (μ l) |
| H2O | 33.5-1* template number |
| 5×Bμffer(with Mg2+) | 10 |
| DNTP (each 2.5mM) | 4 |
| Primer1(+)(10μM) | 1 |
| Primer2 (-) (10 μM) | 1 |
| Template | 1* template number |
| PrimeSTAR | 0.5 |
3 50 μ l over-lap PCR reaction system of table
(13) Eppendorf is added in addition to primer using the system in table 3 using each 1 μ l of DNA fragmentation b, c, e as template
In pipe, PCR cycle condition are as follows: primer is added in 98 DEG C of 3min, (98 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 30sec) * 6cycle
CD8leader-F/VL-R, (98 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 40sec) * 24cycle, 72 DEG C of 5min.Product passes through
1.5% agarose gel electrophoresis, confirms the segment l of 820bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company
Ago-Gel QIAquick Gel Extraction Kit recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；
(14) Eppendorf is added in addition to primer using the system in table 3 using each 1 μ l of DNA fragmentation b, c, f as template
In pipe, PCR cycle condition are as follows: primer is added in 98 DEG C of 3min, (98 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 30sec) * 6cycle
CD8leader-F/VL-R, (98 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 40sec) * 24cycle, 72 DEG C of 5min.Product passes through
1.5% agarose gel electrophoresis, confirms the segment m of 811bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company
Ago-Gel QIAquick Gel Extraction Kit recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；
(15) it using each 1 μ l of DNA fragmentation g, h, i, j as template, using the system in table 3, is added in addition to primer
In Eppendorf pipe, PCR cycle condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 30sec) * 6cycle,
Primer CD8Hinge-F/TCR-R, (98 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 30sec) * 24cycle, 72 DEG C of 5min is added.It produces
Object passes through 1.5% agarose gel electrophoresis, confirms the segment n of 704bp, and be tapped and recovered and be placed in Eppendorf pipe, uses MN
The Ago-Gel QIAquick Gel Extraction Kit of company recycles corresponding segment (being shown in Table 1), and measures the purity and concentration of product；
(16) Eppendorf pipe is added with the ratio of 5 μ l total volumes and molar ratio 1:1:1:1 in DNA fragmentation V1, a, k, n
It is interior, 15 μ l of homologous recombination enzyme reaction solution is added, is incubated for 30 minutes after mixing at 42 DEG C, is transferred to and places 2-3 minutes on ice, it will be anti-
It answers liquid to be added in 50 μ l TOP10, gently rotates to mix content, placed 30 minutes in ice, pipe is put into pre-heating to 42
DEG C thermostat water bath in heat shock 90 seconds, quickly pipe is transferred in ice bath, makes cell cooling 2-3 minutes, every pipe adds 900 μ l
Then pipe is transferred on 37 DEG C of shaking tables by LB culture solution, incubating 1 hour makes bacteria resuscitation, and the transformed bacteria solution of 100 μ l is taken to be coated on
On Amp LB agar plate, it is inverted plate, 37 DEG C of cultures in constant incubator, 16 hours.Picked clones carry out bacterium colony PCR mirror
It is fixed, it identifies that correctly clone is recombinant slow virus plasmid pCAR123-CLA, digestion identification is carried out (see figure to correct clone
4)；
(17) Eppendorf pipe is added with the ratio of 5 μ l total volumes and molar ratio 1:1:1:1 in DNA fragmentation V1, a, l, n
It is interior, 15 μ l of homologous recombination enzyme reaction solution is added, is incubated for 30 minutes after mixing at 42 DEG C, is transferred to and places 2-3 minutes on ice, it will be anti-
It answers liquid to be added in 50 μ l TOP10, gently rotates to mix content, placed 30 minutes in ice, pipe is put into pre-heating to 42
DEG C thermostat water bath in heat shock 90 seconds, quickly pipe is transferred in ice bath, makes cell cooling 2-3 minutes, every pipe adds 900 μ l
Then pipe is transferred on 37 DEG C of shaking tables by LB culture solution, incubating 1 hour makes bacteria resuscitation, and the transformed bacteria solution of 100 μ l is taken to be coated on
On Amp LB agar plate, it is inverted plate, 37 DEG C of cultures in constant incubator, 16 hours.Picked clones carry out bacterium colony PCR mirror
It is fixed, it identifies that correctly clone is recombinant slow virus plasmid pCAR123-CLB, digestion identification is carried out (see figure to correct clone
5)；
(18) Eppendorf pipe is added with the ratio of 5 μ l total volumes and molar ratio 1:1:1:1 in DNA fragmentation V1, a, m, n
It is interior, 15 μ l of homologous recombination enzyme reaction solution is added, is incubated for 30 minutes after mixing at 42 DEG C, is transferred to and places 2-3 minutes on ice, it will be anti-
It answers liquid to be added in 50 μ l TOP10, gently rotates to mix content, placed 30 minutes in ice, pipe is put into pre-heating to 42
DEG C thermostat water bath in heat shock 90 seconds, quickly pipe is transferred in ice bath, makes cell cooling 2-3 minutes, every pipe adds 900 μ l
Then pipe is transferred on 37 DEG C of shaking tables by LB culture solution, incubating 1 hour makes bacteria resuscitation, and the transformed bacteria solution of 100 μ l is taken to be coated on
On Amp LB agar plate, it is inverted plate, 37 DEG C of cultures in constant incubator, 16 hours.Picked clones carry out bacterium colony PCR mirror
It is fixed, it identifies that correctly clone is recombinant slow virus plasmid pCAR123-OLC, digestion identification is carried out (see figure to correct clone
6)。
2, the packaging of recombined lentivirus vector lvCAR123-CLA, lvCAR123-CLB, lvCAR123-OLC.
(1) complete medium: taking out preheated fresh culture, and 10%FBS+5ml Pen-Srep is added, runs up and down
Mix；
(2) NaCl 8g, KCl 0.2, Na 1XPBS solution: are weighed2HPO4.12H2O 3.58g, KH2PO4 0.24g is placed in
In 1000ml beaker, the dissolution of 900ml Milli-Q grade ultrapure water is added, after the completion of dissolution, uses 1000ml graduated cylinder constant volume
To 1000ml, 121 DEG C of high-temperature heat sterilization 20min；
(3) 0.25%Trypsin solution: weighing Trypsin 2.5g, and EDTA 0.19729g is placed in 1000ml beaker,
900ml 1XPBS dissolution is added, after the completion of dissolution, is settled to 1000ml using 1000ml graduated cylinder, 0.22 μM of filtration sterilization, for a long time
Using can be reserved for -20 DEG C of refrigerators；(4) 0.5M CaCl2 solution: 36.75g CaCl is weighed2With 400ml Milli-Q grade
Ultrapure water dissolution；Total volume is settled to 500ml with Milli-Q grade ultrapure water, is mixed；0.22 μm of filtration sterilization, packing
It is saved in 50ml centrifuge tube, every pipe 45ml or so, 4 DEG C of preservations.
(5) 4.09g NaCl, 0.269g Na 2XHBS solution: are weighed2HPO4,5.96g Hepes, with 400ml Milli-
The dissolution of Q grade ultrapure water；After calibrating pH instrument, the pH of HBS solution is transferred to 7.05 with 2M NaOH solution.Adjust every bottle of HBS
PH consumption 2M NaOH be 3ml or so；
(6) the HEK293T/17 cell frozen is taken out from liquid nitrogen container, is quickly transferred in 37 DEG C of water-baths, after 1~2min
It is transferred in super-clean bench, the liquid in cryopreservation tube is fully transferred to 10cm by sterile working2In culture dish, supply containing 10%FBS
DMEM to 8mL/10cm2Dish, rear micro- sem observation cell, the degree of cell confluency are greater than 80% and are passed on for 24 hours；
(7) selection cell state is good, free of contamination HEK293T/17 cell, and every 2-6 culture dish is one group, by cell
After pancreatin digestion, 4-12ml complete medium is drawn with electric pipettor, 2ml is added into each postdigestive culture dish, is avoided
Culture dish dries out；All cells are blown and beaten into single cell suspension using 1ml pipettor, are transferred in medium bottle；
(8) remaining cell in above-mentioned 2-6 culture dish is transferred in medium bottle, and rinsed again with culture medium once
Culture dish；
(9) culture medium bottle cap is covered tightly, turns upside down 10 times or so and mixes well cell suspension, cell is passed to 8-24
10cm2In culture dish, the cell density of every ware should about 4 × 106A/10ml complete medium or so.If cell density and pre-
The difference of phase is larger, then needs to count cell, then according to 4 × 106The amount of a/ware is inoculated with；
(10) every 6 culture dishes arrange piles up for one, pays attention to keeping the cooperation between ware up and down.By culture dish or so, front and back
It shakes for several times, spreads out cell sufficiently, be then placed in 5%CO2Incubator.Remaining cell does same processing；
(11) checking that institute's passage cell, cell confluency degree should be 70-80%, profile is full, and it is adherent good, it is trained in cell
It supports and is uniformly distributed in ware；
(12) liquid is changed for cell, culture medium is replaced with into fresh complete medium, every ware 9ml, and by the CO of incubator2It is dense
Degree setting value is increased to 8%；(13) match DNA/CaCl according to N+0.52Solution.Every ware HEK293T/17 cell transfecting plasmid amount is pressed
It is used 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).
A new 5ml centrifuge tube is taken, 0.5M CaCl2:0.25ml, 20 μ g:pPac-GP of recombinant slow virus plasmid, 15 μ g is added:
10 μ g:pEnv-G of pPac-R, 7.5 μ g, supplement ultrapure water close the lid to 0.5ml, mix well；
(14) a 5ml centrifuge tube is separately taken, 0.5ml DNA/CaCl2 solution is added.Turbula shaker is opened, a hand is taken
The firmly upper end of 5ml centrifuge tube makes tube bottom contact oscillating end, and liquid is made to scatter on tube wall flowing, and another hand moves 1mL by one
Liquid rifle is drawn 2 × HBS of 0.5mL solution, is slowly added dropwise into centrifuge tube, coutroi velocity, being dripped off with half a minute is advisable.2×HBS
It after addition, after persistent oscillation 5 seconds, stops oscillation, can be directly added into the cell for needing to transfect；
(15) take a ware cell, the 1mL calcium in centrifuge tube turned into drop and is added, make as far as possible calcium turn reagent be distributed to it is whole
In a culture dish；
(16) it after calcium turns liquid addition, is marked in ware lid, culture dish is released to another 5%CO2In incubator.
Ensure that culture dish is horizontal positioned, every pile culture dish does not exceed 6.In 5%CO2(6-8h) is placed in incubator；
(17) by the CO of first incubator2Concentration set point adjusts back to 5%；
After (18) 24 hours, cell state is checked.Cell confluency degree should be 80-85% or so, in good condition.It will culture
Base siphons away, replacement 10ml fresh DMEM complete medium；
After (19) 48 hours, transfection efficiency is observed.Most cells are still adherent.It can be seen that thin more than 95%
Born of the same parents can have green fluorescence.By the same virus packaging supernatant collection to together, and continue addition 10mL into culture dish
Fresh culture；
After (20) 72 hours, the same vial supernatant is collected into the virus together, collected twice again to be placed on
Together, culture dish is abandoned；Recombined lentivirus vector lvCAR123-CLA, lvCAR123- are contained in the supernatant collected at this time
CLB、lvCAR123-OLC。
The concentration and detection of 2 recombined lentivirus vector of embodiment
One, supercentrifugation purifies recombined lentivirus vector；
(1) supernatant of collection is dispensed into 50ml centrifuge tube, 500g room temperature is centrifuged 10min, removes cell and big
Fragment；
(2) with 0.22 μm of -0.8 μm of filter filtering supernatant；
(3) 6 Hitachi 40PA ultracentrifugation pipes are taken, 70% ethanol disinfection is sprayed on surface, is placed on super-clean bench with ultraviolet
Light irradiation is sterilized 30 minutes.High-temperature heat sterilization can also be passed through；
(4) the cell conditioned medium sample packing 32ml handled step 2 well is into centrifuge tube；
(5) metal cover is covered, by centrifuge tube together with metal cover trim, makes deviation of weight in 0.02g with 1XPBS adjustment
In range；
(6) centrifuge tube of trim is symmetrically placed in ultracentrifugation rotor P50AT2.Centrifugal rotational speed 100,000g is set,
4 DEG C are centrifuged 2 hours；
(7) after being centrifuged, carefully centrifuge tube is taken out from rotor, it can be seen that have a small group heavy at centrifuge tube bottom
It forms sediment, is made marks on outer tube wall with Marker, outwell supernatant.Centrifuge tube is tipped upside down on the paper handkerchief completed in advance, remnants are made
Liquid pours off.The drop hung on wall can be siphoned away with liquid-transfering gun；
(8) Opti-MEM of 200 μ l is added into each centrifuge tube, dissolves precipitating with the piping and druming of 200 μ l pipettors, as far as possible
Reduce the generation of foam；
(9) ultracentrifugation pipe is inserted into 50ml centrifuge tube, closes the lid, is put into 4 DEG C of refrigerator overnights；
(10) 500g, room temperature are centrifuged 1min, virus liquid are made to focus on tube bottom；
(11) all identical viral concentration liquid are brought together, are filtered with 0.22 μm -0.8 μm of PES filter；It will be sick
Poison is divided into 25 to 50 μ l mono- pipe, freezes -80 DEG C of refrigerators, carries out long-term preservation；
Two, supercentrifugal process purifies recombined lentivirus vector；
(1) the supernatant 204ml of collection is filtered using 0.22 μm -0.8 μm of PES filter；
(2) 6000 solution of PEG of 51ml 50% is added；
(3) 21.7ml 4M NaCl solution is added；
(4) be added 23.3ml PBS solution, at this moment overall solution volume be 300ml.PEG 6000 final concentration 8.5%,
NaCl final concentration 0.3M；
(5) solution is dispensed into 250ml wide-mouth bottle, every part of 150ml；
(6) 4 DEG C are placed 1.5 hours, and every 20-30 minutes mixes once；
(7) 4 DEG C, 7000g centrifugation 10min；
(8) it can see that tube bottom has white precipitate after being centrifuged；
(9) liquid is carefully discarded supernatant, it is heavy acutely to rock resuspension for every bottle of addition 1.2ml 50mM Tris-HCl (pH 7.4)
It forms sediment；
(10) it is vortexed concussion 20-30 seconds and precipitating is further resuspended；
(11) virus is divided into 25 to 50 μ l mono- to manage, freezes -80 DEG C of refrigerators, carry out long-term preservation；
Three, ion exchange chromatography recombined lentivirus vector (as shown in Figure 7)；
(1) supernatant of collection is used into Thermo vacuum pump, is filtered through 0.22 μm -0.8 μm of PES filter, remove impurity elimination
Matter；
(2) 1.5M NaCl 250mM Tris-HCl (pH 6-8) is added into supernatant in the ratio of 1:1~1:10；
(3) 2 ion exchange columns are placed in series, with 4ml 1M NaOH, 4ml 1M NaCl, 5ml 0.15M NaCl
25mM Tris-HCl (pH 6-8) solution successively crosses column；
(4) solution obtained in step 2 is given to ion exchange column loading with the speed of 1-10ml/min by peristaltic pump；
(5) it after whole supernatants cross column, is cleaned using 10ml 0.15M NaCl 25mM Tris-HCl (pH 6-8) solution
One time；
(6) it is eluted according to applied sample amount using 1-5ml 1.5M NaCl 25mM Tris-HCl (pH 6-8), collection is washed
De- liquid；
(7) eluent is divided into 25 to 50 μ l mono- to manage, freezes -80 DEG C of refrigerators, carry out long-term preservation.
Four, titer determination and compare；
(1) 24 orifice plates is taken to be inoculated with 293T cell.Every hole cell is 5 × 104A, added culture volume is 500ul, different
Difference, cell confluency when carrying out virus infection are 40%-60% to the vitro growth rates of type；
(2) prepare 3 sterile EP tubes, the fresh complete medium (DMEM in high glucose+10% of 90ul is added in each pipe
FBS) after inoculating cell 24 hours, the cell in two holes is taken to be counted with blood counting chamber, the actual number of cell when determining infection,
It is denoted as N；
(3) it takes virus stock solution used 10ul to be determined to be added in first pipe, after mixing gently, 10ul is taken to be added to second
In a pipe, a to the last pipe is then successively operated；410ul complete medium (DMEM in high glucose+10% is added in every pipe
), FBS final volume 500ul；
(4) 20 hours after infection starts, culture supernatant is removed, 500 μ l complete medium (DMEM in high glucose+10% are changed to
FBS), 5%CO2Continue culture 48 hours；
After (5) 72 hours, luciferase expression situation is observed, under normal circumstances, fluorecyte number increases and phase with extension rate
It should reduce, and take pictures；
(6) 0.25% pancreas enzyme -EDTA solution digestion cell of 0.2ml is used, is placed 1 minute at 37 DEG C.It is purged with culture medium whole
A cell face, is collected by centrifugation cell.Illustrate extracting genomic DNA according to DNeasy kit.200 are added in each sample cell
μ l eluent is washed lower DNA and is quantified；
(7) preparing target DNA detection qPCRmix general pipeline I, (QPCR primer sequence is SEQ ID NO.44---SEQ ID
NO.45):
N=number of reactions. is for example: overall reaction number is 40, by 2 × TaqMan of 1ml Universal
PCR Master Mix, 4 μ l forward primer, 4 μ l reverse primer, 4 μ l probe and 788 μ l H2O is mixed
With.It is placed on ice after concussion；
(8) preparing internal reference DNA detection qPCRmix pipe II, (QPCR primer sequence is SEQ ID NO.46---SEQ ID
NO.47):
2×TaqMan Master Mix 25μl×n
10×RNaseP primer/probe mix 2.5μl×n
H2O 17.5μl×n
N=number of reactions. is for example: overall reaction number is 40, by 2 × TaqMan of 1ml Universal
PCR Master Mix, 100 μ l 10 × RNaseP primer/probe mix and 700 μ l H2O is mixed.Ice is placed on after concussion
On；
(9) PCR system is completed in 96 hole PCR plates of pre-cooling to establish.45 μ l are respectively taken to be added to each row of A-D from general pipeline I
Hole in, from respectively taking 45 μ l to be added in the hole of each row of E-G in general pipeline II.
(10) 5 μ l plasmid standards and sample to be tested genomic DNA are taken to be added in A-D row respectively, each sample repeats 1
It is secondary.Separately stay 1 hole that the water of 5 μ l is added as no template control (no-template control).
(11) 5 μ l genome standard items and sample to be tested genomic DNA are taken to be added in E-G row respectively, each sample weight
It is 1 time multiple.Separately stay 1 hole that the water of 5 μ l is added as no template control (no-template control).
(12) used quantitative PCR apparatus is 7000 quantitative system of ABI PRISM.Cycling condition setting are as follows: 50 DEG C 2 minutes,
95 DEG C 10 minutes, followed by 95 DEG C 15 seconds, 60 DEG C of 40 of 1 minute circulations.
Data analysis: the slow virus carrier copy number integrated in the DNA sample measured is demarcated with genome number, is obtained
The viral copy number of every genome conformity.
Titre (integration units per ml, IU ml-1) calculation formula it is as follows:
IU ml-1=(C × N × D × 1000)/V
Wherein: C=is averaged the viral copy number of every genome conformity
The number (about 1 × 10 of cell when N=infects5)
The extension rate of D=viral vectors
The volume number for the dilution virus that V=is added
(13) titre results of recombined lentivirus vector lvCAR123-CLA, lvCAR123-CLB, lvCAR123-OLC are such as
Shown in Fig. 8, the result of ion-exchange chromatography is substantially better than supercentrifugation and supercentrifugal process.
Five, endotoxin measurement and compare；
(1), endotoxin working standard is 15EU/ branch；
(2), sensitivity of the limulus reagent λ=0.25EU/ml, 0.5ml/ pipe
(3), endotoxin standard dilutes: taking endotoxin standard one, is diluted to 4 λ and 2 λ in proportion with BET water respectively
Dissolution, sealed membrane sealing, concussion dissolution 15min；One step of every dilution should all mix 30s on eddy mixer when dilution；
(4), it is loaded: taking reagents several, every addition BET water 0.5ml dissolution, packing to several endotoxin-frees tries
Guan Zhong, every pipe 0.1ml.Wherein 2 are negative control pipe, and BET water 0.1ml is added；
2 are positive control pipe, and the endotoxin working standard solution 0.1ml of 2 λ concentration is added；
2 be Sample Positive control tube, be added 0.1ml containing 2 λ endotoxin standards sample solution (dilution 20 times to
The endotoxin standard solution 1ml=2ml of sample 1ml+4 λ contains 40 times of samples of dilution of 2 λ endotoxin standards).
Be added 0.1ml sample in sample cell, dilution ratio be shown in Table 4,37 ± 1 DEG C of water-baths (or incubator) heat preservation 60 ±
1min；
4 endotoxin dilution ratio of table and corresponding endotoxin content
(5), the endotoxin detection of recombined lentivirus vector lvCAR123-CLA, lvCAR123-CLB, lvCAR123-OLC
As a result (as shown in table 5), between 20~40EU/ml, the endotoxin of supercentrifugal process contains the endotoxin content of supercentrifugation
Between 20~40EU/ml, the endotoxin content of ion-exchange chromatography is substantially better than super amount between 1.25~2.5EU/ml
Fast centrifugal process and supercentrifugal process.
The endotoxin testing result of the different way of purification of 5 recombined lentivirus vector of table
Six, mycoplasma measures and compares；
(1) first three day is being tested, cell sample is cultivated with antibiotic-free culture medium；
(2) (cell number is greater than 1*10 to collection 1ml cell suspending liquid5), it is placed in 1.5ml centrifuge tube；
(3) 13000 × g are centrifuged 1min, collect precipitating, discard culture medium；
(4) 500ul PBS pipette tips pressure-vaccum or vortex oscillation is added, precipitating is resuspended.13000 × g is centrifuged 5min；
(5) step 4 is repeated once；
(6) 50 μ l Cell Lysis Buffer are added after mixing well, to be incubated in 55 DEG C of water-baths with pipette tips pressure-vaccum
20min；
(7) sample is placed in 95 DEG C and heats 5min；
After (8) 13000 × g are centrifuged 5min, take 5 μ l supernatants as template, 25 μ lPCR reaction systems are as follows: 6.5 μ of ddH20
L, 1 μ l of Myco Mix, 2x Taq Plus Mix Master (Dye Plus) 12.5 μ l, 5 μ l of template；PCR cycle condition are as follows:
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 as the result is shown (as shown in Figure 9), supercentrifugation, supercentrifugal process, ion-exchange chromatography
The recombined lentivirus vector of purifying is free of mycoplasma.
As it can be seen that the present invention uses ion exchange chromatography recombined lentivirus vector, with traditional supercentrifugal process and
Supercentrifugation purifying recombined lentivirus vector is compared, and titer determination result and endotoxin measurement result are significantly superior.
The function of embodiment 3 recombined lentivirus vector lvCAR123-CLA, lvCAR123-CLB, lvCAR123-OLC are examined
It surveys.
One, the cellular level detection of expression of CAR gene:
(1) recombined lentivirus vector lvCAR123-CLA, lvCAR123-CLB, lvCAR123-OLC infection of PBMCs cell
Afterwards, the detection that cell carries out CAR mRNA transcriptional level using RT-PCR is collected, the expression of CAR gene is verified, if CAR
MRNA transcriptional level increases, then illustrates that the transcriptional level of CAR gene is expressed successfully；
(2) recombined lentivirus vector lvCAR123-CLA, lvCAR123-CLB, lvCAR123-OLC infection of PBMCs cell
Afterwards, the detection that cell carries out CAR protein expression level using western blot is collected, the expression of CAR gene is verified, if
CAR protein expression level increases, then illustrates that the translation skill of CAR gene is expressed successfully；
(3) respectively by lvCAR123-CLA, lvCAR123-CLB, lvCAR123-OLC of MOI=15 and control virus
MOCK infection cell extracts the total serum IgE of cell and total protein in 6 orifice plates after 48h and carries out fluorescent quantitative PCR experiment and immune respectively
Blot experiment.Specific steps: being coated with four holes of 6 orifice plates, and corresponding PBS and RN is added in each hole, and 4 DEG C overnight.After 12 hours
It is coated with virus by MOI=15,37 DEG C of incubators place 5h；The 6 orifice plates of taking-up, discard viral supernatants, are washed twice with PBS, by 1*
106/ hole is coated with PBMC (being separated from people's blood with lymphocyte separation medium), and 500ul culture medium is added and (contains 10% serum, 20U/
ml IL-2,Polybrene 8ug/ml).Stand 20min, 1000g 20 DEG C of centrifugations 30min, 37 DEG C of culture 48h.
(4) Trizol method extracts the total serum IgE of PBMC cell in 6 orifice plates, reverse transcription amplification cDNA, with QPCR primer (sequence
Fluorescent quantitative PCR experiment is carried out for SEQ ID NO.46---SEQ ID NO.49), reaction system is shown in Table 6, is with internal reference Actin
Control group verifies the transcription situation of its mRNA.
| Reagent | Volume (μ l) |
| SYBR premix ex taq: | 10μl |
| ROX Reverse Dye(50x) | 0.4μl |
| Upstream primer (2.5 μM): | 0.5μl |
| Downstream primer (2.5 μM): | 0.5μl |
| cDNA | 1.0μl |
| ddH2O | 7.6μl |
6 20 μ l qPCR reaction system of table
(5) protein immunoblot (Western Blot) is total by what is extracted from PBMC by polyacrylamide gel electrophoresis
Protein is separated by relative molecular mass.It, will be on protein delivery to pvdf membrane using wet turn (4 DEG C, 400mA, 120min).With envelope
It closes the closing of liquid (the TBST solution containing 5% skim milk) room temperature pvdf membrane 1h, confining liquid 1:1000 and dilutes Biotinylated
Then protein L is incubated at room temperature 4 DEG C overnight with the pvdf membrane closed.TBST washes film 3 times, each 10min.Confining liquid 1:
The 500 corresponding SA-HRP of dilution, are incubated for pvdf membrane 2h, TBST washes film 3 times, each 10min at room temperature.Using Amersham company
ECL+plusTM Western blotting system kit develops the color.X-ray development obtains the film of display band.
(6) RT-QPCR detection shows that the expression of the CAR after recombined lentivirus vector infection of PBMCs is than control virus
MOCK and ghost are increased significantly (as shown in Figure 10), illustrate that the transcriptional level of CAR gene is expressed successfully.
(7) protein immunoblot (Western Blot) the result shows that, CAR albumen is expressed in recombinant slow virus system
(as shown in figure 11) illustrates that the translation skill of CAR gene is expressed successfully.
Two, cytokine secretion and fragmentation effect assessment.
(1) CD123+K562 cell and effector cell lvCAR123-CLA-PBMC, lvCAR123-CLB- are cultivated respectively
PBMC,lvCAR123-OLC-PBMC；(2) target cell (CD123+K562) 4x10 is collected5(CART is thin by cells and effector cell
Born of the same parents) 2.8x106Cells, 800g, 6min centrifugation, abandon supernatant；
(3) target cell and effector cell are resuspended respectively with 1ml 1xPBS solution, supernatant is abandoned in 800g, 6min centrifugation；
(4) it is primary to repeat step 3；
(5) effector cell is resuspended with 700ul culture medium (1640 culture medium+10%FBS), with (1640 cultures of 2ml culture medium
Base+10%FBS) target cell is resuspended；
(6) setting effect target is than the experimental port for 1:1,5:1,10:1, and control group is arranged, every group of 3 multiple holes；
(7) centrifugation of 250xg, 5min plate；
It is cultivated 4 hours in (8) 37 DEG C of 5%CO2 incubators；
(9) centrifugation of 250xg, 5min plate；
(10) take the 50ul supernatant in each hole into new 96 orifice plate, and every hole is added 50ul substrate solution and (is protected from light behaviour
Make)；
(11) it is protected from light incubation 25 minutes；
(12) 50ul terminate liquid is added in every hole；
(13) microplate reader detects 490nm absorbance；
(14) 3 multiple holes are averaged；The light absorption value of all experimental ports, Target cell wells and effector cell hole is subtracted into training
Support the mean value of base background light absorption value；The light absorption value of target cell maximum value is subtracted to the mean value of volume correction control light absorption value.
(15) it brings the corrected value obtained in step 14 into following formula, it is thinner than generated to calculate each effect target
Cellular toxicity percentage.As a result as shown in figure 12, the PBMC cell of recombined lentivirus vector lvCAR123-OLC transduction is in different effects
Killing-efficiency is apparently higher than the PBMC cell of lvCAR123-CLA and lvCAR123-CLB transduction under the conditions of target ratio；
Killing-efficiency=(experimental port-effector cell hole-Target cell wells)/(target cell largest hole-Target cell wells) X100%
(16) sample for repeating to prepare a step 6 detects Cytokine Expression Level for qPCR, as a result such as Figure 13 institute
Show, the PBMC cell of recombined lentivirus vector lvCAR123-OLC transduction IL-2 and IFN-γ under the conditions of different effect target ratios
MRNA transcriptional level is apparently higher than the PBMC cell of lvCAR123-CLA and lvCAR123-CLB transduction.
The clinic of embodiment 4, recombined lentivirus vector lvCAR123-OLC (version of removal fluorescence labels zsGreen1)
Using
(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 leukaemia (AML) has good effect.
This research is #NCT02623582 in the number of registration of www.clinicaltrials.gov.Expression is used in this research
The T lymphocyte of CAR123 gene has good tumour to kill in Mice Body for people's AML cell line MOLM14 of expression CD123
Hurt effect.Living imaging after injection after 6 days, 14 days, 21 days is the results show that CART123 experimental group is right in Mice Body
The tumour 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 mouse of CART123 experimental group, compared with CART19 experimental group and PBS control group mouse, with the time
Increase, bioluminescence change in signal strength amplitude very little illustrates that tumour cell is inhibited (as shown in Figure 14B) by obvious；
Kaplan-Meier survivorship curve is the results show that CART123 experimental mice and CART19 experimental group and PBS control group mouse phase
Than there is longer life cycle (as shown in Figure 14 C)；Adoptive CAR123-T cell therapy is being directed to acute myeloid leukemia
(AML) possess good prospect in terms for the treatment of.
5 third generation slow virus skeleton carrier 3rdLV-CAR123 of embodiment and second generation slow virus skeleton carrier 2ndLV-
CAR123 titre compares.
3rdLV-CAR123 and 2ndLV-CAR123 is constructed respectively；Plasmid construction, virus packaging, viral ultracentrifugation are dense
Contracting, titration procedure reference implementation example 1；The titre results of continuous 3 batches are as shown in figure 15, third generation slow virus skeleton
The titre of carrier 3rdLV-CAR123 is better than second generation slow virus skeleton carrier 2ndLV-CAR123；Therefore, the present invention is preferred
Use third generation slow virus skeleton carrier as the carrying skeleton of CAR gene.
6 second generation CAR slow virus carrier 3rdLV-2ndCAR123 of embodiment and third generation CAR slow virus carrier 3rdLV-
3rd CAR123 killing-efficiency compares.
3rdLV-2ndCAR123 and 3rdLV-3rdCAR123 is constructed respectively；Plasmid construction, virus packaging, virus exceed the speed limit from
Heart concentration, titration procedure reference implementation example 1；Killing experiments reference implementation example 3；Second generation CAR slow virus carrier 3rdLV-
2ndCAR123 is as shown in figure 16 compared with third generation CAR slow virus carrier 3rdLV-3rdCAR123 killing-efficiency result,
The ratio 3rdLV- that 3rdLV-3rdCAR123 is not showed under the conditions of different effect target ratios for the killing-efficiency of target cell
2ndCAR123 is more efficient；Therefore, present invention preferably uses the slow virus carriers of second generation CAR design as clinical trial carrier.
Claims (10)
1. a kind of CAR-T transgene carrier for the replication defective recombinant slow virus for targeting CD123 characterized by comprising such as
It is used for the prokaryotic replions pUC Ori sequence of plasmid replication shown in SEQ ID NO.2, is used for as shown in SEQ ID NO.1
The sequence of AmpR containing ampicillin resistance gene of purpose bacterial strain massive amplification is true for enhancing as shown in SEQ ID NO.3
The Viral Replicon SV40 Ori sequence of duplication in nucleus, the slow virus for slow virus packaging pack cis element, such as
The IRES ribosome binding sequence of common transcriptional expression protein is used for shown in SEQ ID NO.12, such as SEQ ID NO.14 institute
People's EF1 α promoter of the eukaryotic transcription for Chimeric antigen receptor gene shown, for enhancing as shown in SEQ ID NO.13
The enhanced marmot hepatitis B posttranscriptional regulatory element of the eWPRE of the expression efficiency of transgenosis, and collect for composition and identify,
Transmitting starts in the Chimeric antigen receptor of the two generation CAR or three generations CAR of one；
It is described for form the Chimeric antigen receptor for the two generation CAR for integrating identification, transmitting, starting to include: such as SEQ ID
CD8 leader Chimerical receptor signal peptide shown in NO.15, the CD123 single-chain antibody light chain VL as shown in SEQ ID NO.20,
Optimal Linker C as shown in SEQ ID NO.19, the CD123 single-chain antibody heavy chain VH as shown in SEQ ID NO.16,
CD8 Hinge Chimerical receptor hinge, the CD8 as shown in SEQ ID NO.22 as shown in SEQ ID NO.21
Transmembrane Chimerical receptor transmembrane region, CD28, the CD137 Chimerical receptor costimulating factor as shown in SEQ ID NO.23,
With the TCR Chimerical receptor t cell activation domain as shown in SEQ ID NO.24；
It is described for form the Chimeric antigen receptor for the three generations CAR for integrating identification, transmitting, starting to include: such as SEQ ID
CD8 leader Chimerical receptor signal peptide shown in NO.15, the CD123 single-chain antibody light chain VL as shown in SEQ ID NO.20,
Optimal Linker C as shown in SEQ ID NO.19, the CD123 single-chain antibody heavy chain VH as shown in SEQ ID NO.16,
CD8 Hinge Chimerical receptor hinge, the CD8 as shown in SEQ ID NO.22 as shown in SEQ ID NO.21
Transmembrane Chimerical receptor transmembrane region, CD28, the CD137 Chimerical receptor costimulating factor as shown in SEQ ID NO.23,
The Chimerical receptor t cell activation domain TCR and the CD28 as shown in SEQ ID NO.25 as shown in SEQ ID NO.24 it is chimeric by
Body costimulating factor.
2. carrier as described in claim 1, which is characterized in that the slow virus packaging cis element includes: such as SEQ ID
5 terminal LTR of slow virus, the 3 terminal Self- of slow virus as shown in SEQ ID NO.6 shown in NO.5
Inactivating LTR, the Gag cis element as shown in SEQ ID NO.7, the cis- member of RRE as shown in SEQ ID NO.8
Part, the env cis element as shown in SEQ ID NO.9 and the cPPT cis element as shown in SEQ ID NO.10.
3. carrier as described in claim 1, which is characterized in that the slow virus packaging cis element includes: such as SEQ ID
5 terminal LTR of slow virus, the 3 terminal Self- of slow virus as shown in SEQ ID NO.6 shown in NO.5
Inactivating LTR, the Gag cis element as shown in SEQ ID NO.7, the cis- member of RRE as shown in SEQ ID NO.8
Part, the env cis element as shown in SEQ ID NO.9, the cPPT cis element as shown in SEQ ID NO.10, and such as SEQ
RSV promoter shown in ID NO.4.
4. carrier as described in claim 1, which is characterized in that further include: eukaryocyte is used for as shown in SEQ ID NO.11
Express the ZsGreen1 green fluorescent protein of green fluorescence.
5. a kind of CAR-T transgene carrier of the replication defective recombinant slow virus of targeting CD123 as described in claim 1
Construction method, which comprises the following steps:
(1) the ampicillin resistance gene AmpR sequence as shown in SEQ ID NO.1 will be contained, as shown in SEQ ID NO.2
Prokaryotic replions pUC Ori sequence, is used for slow virus packet at the Viral Replicon SV40 Ori sequence as shown in SEQ ID NO.3
The slow virus of dress packs cis element, the IRES ribosome binding sequence as shown in SEQ ID NO.12, such as SEQ ID NO.13
Shown in the enhanced marmot hepatitis B posttranscriptional regulatory element of eWPRE be stored on slow virus skeleton plasmid；
(2) the people EF1 α promoter as shown in SEQ ID NO.14, and integrate identification, transmitting, starting for forming
The Chimeric antigen receptor of two generation CAR or three generations CAR is combined into two generation CAR or three generations CAR, by digestion, connection, recombining reaction gram
It is grand into slow virus skeleton plasmid, obtain two generation CAR or three generations CAR design recombinant slow virus plasmid；
(3) by obtained recombinant slow virus plasmid and slow virus packaging plasmid pPac-GP, pPac-R and memebrane protein plasmid
PEnv-G transfects HEK293T/17 cell jointly, after carrying out gene transcript expression in HEK293T/17 cell, packs and successfully weighs
Group slow virus carrier can be discharged into cells and supernatant, collect the supernatant for the recombined lentivirus vector for including；
(4) obtained recombinant slow virus supernatant is purified using the ion-exchange method for filtering, adsorbing, eluting, respectively
To recombined lentivirus vector.
6. method as claimed in claim 5, which is characterized in that in step (1), the slow virus packaging cis element includes:
5 terminal LTR of slow virus, 3 terminal of slow virus as shown in SEQ ID NO.6 as shown in SEQ ID NO.5
Self-Inactivating LTR, the Gag cis element as shown in SEQ ID NO.7, as RRE shown in SEQ ID NO.8 it is suitable
Formula element, the env cis element as shown in SEQ ID NO.9 and the cPPT cis element as shown in SEQ ID NO.10；Or
The slow virus packaging cis element includes: slow virus 5 terminal LTR, such as SEQ ID as shown in SEQ ID NO.5
3 terminal Self-Inactivating LTR of slow virus, the cis- member of Gag as shown in SEQ ID NO.7 shown in NO.6
Part, the RRE cis element as shown in SEQ ID NO.8, the env cis element as shown in SEQ ID NO.9, such as SEQ ID
CPPT cis element shown in NO.10, and the RSV promoter as shown in SEQ ID NO.4.
7. method as claimed in claim 5, which is characterized in that in step (2), start entire CAR base by people's EF1 α promoter
Because of expression；CD8 leader Chimerical receptor signal peptide is located at the N-terminal of CAR coded sequence, for guiding CAR albumen to be positioned at cell
Film；CD123 single-chain antibody light chain VL, Optimal Linker C, CD123 single-chain antibody heavy chain VH are combined into the region scfv, use
In identification CD123 antigen；CD8 Hinge Chimerical receptor hinge is used to for scfv being anchored on the outside of cell membrane；CD8
Transmembrane Chimerical receptor transmembrane region is used to entire Chimerical receptor being fixed on cell membrane；CD137 Chimerical receptor pierces altogether
The factor is swashed for stimulating T cell proliferation and cytokine secretion；The Chimerical receptor t cell activation domain TCR is for activating downstream signal
The expression of access；When the region scfv and CD123 antigen binding, signal is transferred into the cell by Chimerical receptor, to generate
Increase including T cell proliferation, cytokine secretion, Anti-apoptotic proteins secretion increases, cell death delay, cracking target cell
A series of biological effects.
8. method as claimed in claim 5, which is characterized in that in step (4), the slow virus carrier has band fluorescence labels
The version of zsGreen1 and without fluorescence labels zsGreen1 version, the version with fluorescence labels is used for experiment in vitro, without glimmering
The version of optical label is used for clinical trial；The version with fluorescence labels zsGreen1 is used as shown in SEQ ID NO.11
ZsGreen1 green fluorescent protein.
9. method as claimed in claim 5, which is characterized in that in step (4), the suction filtration step will control supernatant volume and exist
200ml ~ 2000ml controls vacuum degree in -0.5MPA ~ -0.9MPA, prevents due to plug-hole bring carrier loss；The absorption
Step will control the pH value of solution 6 ~ 8, prevent the variation of PH from carrier being caused to inactivate；The elution step will control eluent
Ionic strength prevents the variation of ionic strength from causing elution not exclusively or carrier inactivation in 0.5M ~ 1.0M.
10. carrier according to any one of claims 1-4 is preparing the application in acute myeloid leukemia therapeutic agent.
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| CN107177632B (en) * | 2017-05-27 | 2020-02-07 | 上海优卡迪生物医药科技有限公司 | OCTS technology-based myeloid leukemia CAR-T treatment vector and construction method and application thereof |
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