CN109988242A - Joint Chimeric antigen receptor, expression vector, slow virus and T cell - Google Patents
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Abstract
The invention discloses a kind of joint Chimeric antigen receptor, expression vector, slow virus and T cells, joint Chimeric antigen receptor includes chimeric protein CAR19 and chimeric protein CAR22, the CAR19 are coupled by the segment of two kinds of albumen of single-chain antibody and T cell activation motif including at least specific recognition CD19；The CAR22 is coupled by the segment of two kinds of albumen of single-chain antibody and T cell activation motif including at least specific recognition CD22.The case where in addition, expression vector, slow virus and T cell are all based on CART technical principle, combining the sequential feedback of CAR19T and CAR22T, being recurred come CAR19T high before being effectively reduced, and prevent the severe CR of CAR19T.Significantly more efficient therapy approach is provided for tumor disease.
Description
Technical field
The invention belongs to fields of biomedicine, are related to two kinds of ligands in conjunction with CD19 and CD22, specifically with
The related joint Chimeric antigen receptor of CD19 and CD22, expression vector, slow virus and T cell.
Background technique
Immunotherapy of tumors
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 (tumor 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 (tumor necrosis factor, TNF-α), the cell factors such as BCG vaccine and toxin, adoptive cellular immunotherapy etc..
Specific active immunotherapy is mainly tumor vaccine.
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 157patients with advanced cancerμsing lymphokine-activated
Killer cells and interle μ kin-2or high-dose interle μ kin-2alone [J] .N Engl JMed,
1987,316 (15): 889-897].
The immunization therapy of anti-cancer monoclonal antibody
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 ofcancer [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 tumour
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 difficult,
The not high problems of cytotoxicity, are restricted in clinical application.The specific for tumour antigen for how improving T cell has
Important clinical meaning.T cell mainly passes through T cell receptor (T cell receptor, TCR) to the identification of tumour antigen
Human leukocyte antigen (h μm of an le μ kocyte antigen, the HLA)-peptide complexes on tumor cell surface, therefore, T
The specificity of cells against tumor antigen recognizing depends on the TCR on T cell surface.It is special using the means clonal tumours of molecular biology
The TCR of specific T cell, and by building the viral vectors containing TCR, TCR is transferred in normal T cell, make these T cells because
It carries tumour-specific and becomes 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.].
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.].
The large-scale application of tumor vaccine therapy is urgently to be resolved the problem of there are three aspects.Firstly, tumor associated antigen,
Each tumour, each hypotype, each neoplasm staging, these are different with respect to antigen presentation, so making an accurate selection of antigen, make an accurate selection of disease
Everybody group is of crucial importance.Second, how to reach tumour antigen efficient absorption and expression in Dendritic Cells? antigen is set
Prominent shape cell absorption is with surface receptor for mediation.Dendritic Cells has more than ten of receptor, how to be selected according to specific antigen
Select corresponding receptor? third, for the regulation of differentiation of dendritic cells maturation.The differentiation and maturation of Dendritic Cells be one very
Complicated process, it, which can both move towards activation T cell, can also move towards to inhibit T cell.[the therapeutic type tumour epidemic disease of targeting DC cell
Seedling: bright spot with challenge and deposit.http://www.biodiscover.com/news/research/115794.html].
Tumour CAR-T treatment
CAR-T, full name are ChimericAntigen Receptor T-Cell Immunotherapy, i.e. chimeric antigen
Recipient T cells immunotherapy.By the way that the antibody fragment scFv and CD3 intracellular signal domain ITAM of tumor associated antigen will be identified in body
Outer carry out genetic recombination generates recombinant plasmid, then is transfected into the T cell of patient in vitro, makes patient T cells' expression can be in conjunction with swollen
The receptor of tumor antigen.By purifying and the T cell namely CAR-T cell after extensive amplification after transfection.CAR-T technology utilizes
CAR is stablized expression in T cell by viral vectors, is expanded through overactivation, into Selective recognition and capable of killing tumour after in vivo
Cell.
Complete CAR structure includes: that (scFv, single-chain antibody identify the antibody piece of tumor associated antigen for antigen binding domain
Section)；Cross-film bonding pad (optional)；Intracellular signal area (T cell activation motif, CD3 intracellular signal domain ITAM).[Eleanor
J.Cheadle,et al.CAR T cells:driving the road from the laboratory to the
clinic.Immunological Reviews 2014.Vol.257:91–106]。
The t cell activation that first generation CAR is mediated is completed by the Tyrosine Activating Motifs on CD3 ζ chain or FceRIg.
Letter needed for CD3 ζ 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 (cross-film costimulatory signal) intracellular, it is demonstrated experimentally that this makes
Original " signal 1 " expansion made from TCR/CD3 complex is obtained, many researchs all show the second generation for being equipped with " signal 2 "
For CAR compared with first generation CAR, antigentic specificity is constant, and T cell proliferation, cytokine secretion increase, Anti-apoptotic proteins point
Secrete increase, cell death delay.Common costimulatory molecules are CD28, but have later research by CD28 with CD137 (4-1BB) into
Row replacement, in addition to this, a kind of thinking using NK cell receptor CD244 is also suggested.Although different second generation CAR study carefully
Unexpectedly which is better and which is worse, and different researchers is not quite similar with result obtained in external research in vivo with different tumours.It is [deep
Spend full version: the status and future biology paddy .2015-051-15 of CAR-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.
CAR-T has been developed for forth generation technology till now, including integrant expression immune factor, integrates costimulation
Factor ligand etc., it might even be possible to the mode of the more accurate regulation such as Small Molecule Switch is added, for the treatment of various tumours, and
Significant curative effect is achieved in clinical test.
The immunization therapy of CD19-CART
It is for treating hematologic malignancies that CART cell technology is clinically applied most successful at present, this may be with it
The factors such as tumor associated antigen specificity is relatively strong, tumor microenvironment immunosuppressive action is weaker are related.CD19 is specific expressed
In B cell, have an expression in all stages of B cell Development And Differentiation and most of B cell tumours, and in candidate stem cell and its
It is the hot spot in the treatment very promising target spot of B cell tumour, and CAR research at present without expression in his cell.Both at home and abroad just
There are 20 remainders in the clinical test using anti-CD19CAR-T cell therapy hematological system tumor of development, including chronic lymphatic is thin
Born of the same parents' leukaemia (CLL), non-Hodgkin lymphoma (NHL), B-lineage Acute Lymphocyte Leukemia (ALL) etc..
Curative effect is summarized:
With being constantly progressive for CART therapeutic scheme, reported in several large-scale clinical trials of the ALL of CART-19 treatment at present
Effective percentage have reached higher level.In October, 2014, the CART-19 that UP team reports 30 recurrent and refractory ALL are controlled
It treats as a result, thering are 30 children and adult to receive CART-19 adoptive therapy, wherein 27 (90%) obtain complete incidence graph
(CR), there are 8 (27%) that serious CRS adverse reaction has occurred, adverse reaction is effectively controlled after giving trastuzumab treatment
System, the generation of adverse reaction is positively correlated with heavier tumor load, can observe at least 24 months or more and continues in case
Alleviate.In December, 2015, Novartis disclose an II phase clinical study results of CART-19 treatment recurrence/intractable ALL, altogether
After having 59 children and Young Adults patient to receive CART-19 treatment, CR rate reaches 93%.This be it is current it has been reported that it is related
CART-19 treats the maximum clinical research of ALL, and follow up data shows, nothing when overall survival is 79%, 6 months at 12 months
Recurring survival rate is 76%, wherein 18 patients show to continue CR reaction after the treatment for 12 months.27% patient is due to tight
Weight CRS reacts and receives trastuzumab treatment, and adverse reaction is effectively controlled after treatment.According to statistics, CART-19 controls ALL
The average objective reactivity treated is 93% or so.
The clinical research quantity that CART-19 treats CLL is few compared with ALL, efficient also low compared with ALL.2013, MD
Anderson team recurs after reporting CART-19 treatment allotransplantation and 4 refractory CLL patients, 18 circumference of acquisition
Point alleviate (PR), 1 up to 15 months or more stable diseases (SD), 2 PD, this treated effect it is lower may be not carried out
It is related to imitate chemotherapy pretreatment.In January, 2015, NCI team report 4 patients of CART-19 treatment recurrence/intractable CLL,
After joint fludarabine/cyclophosphamide pretreating scheme, there is 3 acquisitions CR, 1 acquisition PR, wherein 1 CR patient show to
Few 23 months or more lasting remissions.In September, 2015, UP team report I phase of CART-19 treatment recurrence/intractable CLL and face
Bed receives 0.14 × 10^8~11 × 10^8 total amount CART- as a result, sharing after 14 patients combine different pretreatments scheme
19 cells, overall objective reactivity is 57% (n=8), wherein 4 CR, 4 PR；The amplification of internal CART is in clinical response
It is positively correlated, after receiving treatment 4 years, 4 CR patients are not recurred, and CART cell still shows anti-swollen in 2 CR patients
Tumor activity.According to statistics, the average objective reactivity that CART-19 treats CLL is 36% or so.
CART-19 is for B cell lymphoma also clinical response rate with higher.MD Anderson team is first within 2010
It is secondary to have treated 6 non-Hodgkin lymphoma (NHL) patients using CART-19,2 acquisitions when not receiving chemotherapy pretreatment
SD reaction, 4 acquisition PR.2012,2013 and 2015, the Rosenberg team of NCI reported total 19 CART-19 and controls
Treat the case of B cell lymphoma.Patient does not receive chemotherapy pretreatment in the article of report in 2013, and 2 diffuse large B cell lymph
1 PD, 1 SD in tumor (DLBCL) patient have 1 PR, 3 SD in 4 lymphoma mantle cells (MCL).2011 and 2015
Report in, all patients receive chemotherapy pretreatment before receiving CART treatment, and 2 Splenic marginal zone lymphomas obtain PR,
And 1 sustained response at least 23 months or more；3 evaluable FL are PR；There are 2 in 3 primary vertical big B lymthomas of diaphragm
CR, 1 SD；2 acquisitions CR, 2 acquisition PR in 4 DLBCL patients；1 low potential malignancy lymthoma obtains CR.According to statistics,
The average objective reactivity that CART-19 treats B cell lymphoma is 62% or so.
There are problems:
CD19 is the CART therapy significant effect of target spot, studies extensively, it has also become most clinical research institutions conduct genes
Modify the multimodal therapy of T cell Therapy study.However, CART cellular immunotherapy is there are still many problems, such as Problems Concerning Their Recurrence and
Safety issue.With reasons such as decline, CAR loss or the target antigen mutation of CAR-T cell in blood, many patients are swollen
It is recurred after a period of time after tumor is removed.Have in safety issue, especially CRS problem and CAR-T clinical application
The major issue faced.Treatment for B cell tumour, CD19 is a promising target, but CART-19 is unable to satisfy
The patient of CD19 target spot loss patients with recurrent and plasma cell tumor.
Summary of the invention
In order to solve the above problem in the prior art, the present invention combines CD19 and CD22, is based on Chimeric antigen receptor T
Cellular immunity technology, provides following technical scheme:
A kind of joint Chimeric antigen receptor, including chimeric protein CAR19 and chimeric protein CAR22, the CAR19 are by extremely
The segment of two kinds of albumen of single-chain antibody and T cell activation motif including specific recognition CD19 is coupled less；The CAR22
It is to be coupled by the segment of two kinds of albumen of single-chain antibody and T cell activation motif including at least specific recognition CD22.
CD22 has high expression on B-ALL cell, and joint Chimeric antigen receptor of the invention targets CD19 and CD22 simultaneously
It can effectively prevent the recurrence of target spot loss type.
Preferably, in above-mentioned joint Chimeric antigen receptor, the T cell activation motif is CD3 ζ.
Preferably, in above-mentioned joint Chimeric antigen receptor, the structure of the CAR19 are as follows: specific recognition CD19's is single-stranded
Antibody, the first cross-film costimulatory signal and T cell activation motif are successively coupled；
The first cross-film costimulatory signal is CD28, one or several combinations in 4-1BB, OX40.
Preferably, in above-mentioned joint Chimeric antigen receptor, which is characterized in that the structure of the CAR22 are as follows: specific recognition
The single-chain antibody of CD22, the second cross-film costimulatory signal and T cell activation motif are successively coupled；
The second cross-film costimulatory signal is CD28, one or several combinations in 4-1BB, OX40.
The present invention also provides the encoding gene of the joint Chimeric antigen receptor of any description above, the volume including CAR19
The encoding gene of code gene and CAR22.
The present invention also provides a kind of Combined expression carriers comprising expresses the first vector and table of the CAR19 gene
Up to the Second support of the CAR22 gene, or
The third carrier of CAR19 gene and CAR22 gene described in claim 1 is expressed simultaneously.
Preferably, in above-mentioned Combined expression carrier, the carrier that sets out is recombined lentivirus vector pLVX-EF1 α-IRES-
Puro。
The present invention also provides a kind of union and recombination slow virus, simultaneously or separately and slowly by above-mentioned Combined expression carrier
The package body combination obtained after virus auxiliary packaging plasmid cotransfection host cell.
Preferably, in above-mentioned recombinant slow virus, the slow virus auxiliary packaging plasmid is gag plasmid and vsvg plasmid, institute
Stating host cell is 293T cell.
The present invention also provides the T cells that transfection has above-mentioned union and recombination slow virus.
The present invention also provides a kind of oncotherapy preparations, contain above-described T cell.The tumour includes but not
It is limited to B cell acute leukemia and/or B cell lymphoma.
Compared with prior art, the invention has the following advantages:
Joint Chimeric antigen receptor provided by the invention, Combined expression carrier, joint slow virus, joint T cell, and it is swollen
Tumor treats preparation, is all based on CART technical principle, combines the sequential feedback of CAR19T and CAR22T, before being effectively reduced
The case where CAR19T high recurs, and prevent the severe CRS of CAR19T.Its superiority outstanding is more embodied in the following areas:
1, the complete remission rate of recurrent intractable B cell acute leukemia and B cell lymphoma is increased.
2, the long-term survival rate of recurrent intractable B cell acute leukemia and B cell lymphoma is increased.
3, it reduces in CART therapeutic process since cytokine storm causes the incidence of serious side reaction.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of recombined lentivirus vector pCAR19；
Fig. 2 is CAR19 partial structure diagram in Fig. 1；
Fig. 3 is that comparison diagram is sequenced in the part pCAR19 in Fig. 1 comprising CAR19 sequence, and lower end black lines indicate mark in figure
Quasi- sequence, grey lines indicate sequencing sequence comparison result；
Fig. 4 is the structural schematic diagram of recombined lentivirus vector pCAR22；
Fig. 5 is CAR22 partial structure diagram in Fig. 4；
Fig. 6 is the pCAR22 partial sequence sequencing comparison diagram in 4 comprising CAR222, and lower end black lines indicate standard in figure
Sequence, grey lines indicate sequencing sequence comparison result；
Fig. 7 is the Concentration Testing result of plasmid pCAR19；
Fig. 8 is the Concentration Testing result of plasmid pCAR22；
Fig. 9 is the digestion testing result of plasmid pCAR19 and plasmid pCAR22；
Figure 10 be CAR19T cell and CAR22T transduction after transduction efficiency testing result, above 3 width figures be CAR22T, below
For CAR19T；
Figure 11 is that CAR19T cell and CAR22T cell kill tumor testing result；
Figure 12 is the experimental result for the influence that CAR19T and CAR22T grow tumour in mouse, wherein right side color column from
Top to bottm is followed successively by red, yellow, and green, blueness, indigo plant, in corresponding Mice Body the color of color development area ring layer from inside to outside be also it is red, yellow,
Green, green, blue, wherein CD22 and CD19 are only displayed in blue.
Figure 13 is CD19-scFv sequence fragment structure chart；
Figure 14 is CD22-scFv sequence fragment structure chart.
Specific embodiment
The present invention is further explained in the light of specific embodiments, so that those skilled in the art can be better
Understand the present invention and can be practiced, but illustrated embodiment is not as a limitation of the invention.
Biology and reagent material source:
PLVX-EF1 α-IRES-Puro: it is purchased from Clontech.
DH5alpha competent cell: it is purchased from Takara.
EndoFree plasmid mega kit: Qiagen, including QIAfilter Cartridge, Buffers are purchased from
P1、Buffers P2、Buffers P3、Buffer FW、Buffer ER、Buffers QBT、Buffer QC、Buffer QN、
Endotoxin-free water、Buffer TE。
Gag plasmid and vsvg plasmid: it is purchased from Addgene:
293T cell: it is purchased from Takara.
The building of embodiment 1, recombined lentivirus vector pCAR19, pCAR22
By sequence 1 (CAR19 is labeled as CAR19, SEQ ID NO.1 referring to Figure 13) and (CAR22, referring to figure of sequence 2
14, it is labeled as CAR22, SEQ ID NO.2), gene chemical synthesis, the sequence gram of synthesis are carried out by Nanjing Jin Sirui biotechnology company
It is grand in carrier T.
1, CAR19 sequence fragment length 1596bp, both ends separately design EcoRI and MluI restriction enzyme site, are cloned into slow disease
The multiple cloning sites of malicious skeleton plasmid pLVX-EF1 α-IRES-Puro complete the building of carrier.Complete map is constructed referring to Fig. 1.
Wherein CAR19 part-structure is shown in Fig. 2, and CD19 is the three generations CAR using CD28 and 4-1BB as costimulatory signal.
CAR19 sequence comparison diagram is as shown in Figure 3: lower end black lines indicate standard sequence, grey lines table in figure
Show sequencing sequence comparison result, sequences match is completely the same as the result is shown.
2, CAR22 sequence fragment length 1443bp, both ends separately design EcoRI and MluI enzyme site, are cloned into slow virus
The multiple cloning sites of skeleton plasmid pLVX-EF1 α-IRES-Puro complete the building of carrier.It is as shown in Figure 4 to construct complete map.
Wherein CAR22 part-structure is shown in Fig. 5, and CD22 is the two generation CAR using CD28 as costimulatory signal.
CAR22 sequence comparison diagram is as shown in Figure 6: lower end black lines indicate standard sequence, grey lines table in figure
Show sequencing sequence comparison result, sequences match is completely the same as the result is shown.
Slow virus skeleton plasmid pLVX-EF1 α-IRES-Puro and composition sequence are limited using EcoR I-HF and Mlu I
Property restriction endonuclease carry out double digestion, product passes through 1.5% agarose gel electrophoresis, and be tapped and recovered and be placed in Eppendorf pipe,
Corresponding segment is recycled with the Ago-Gel QIAquick Gel Extraction Kit of QIAGEN company, and measures the purity and concentration of product.
Eppendorf pipe is added with 1:1 molar ratio in segment, T4 DNA ligase (NEB) and T4 DNA ligase is added
Buffer, 22 DEG C are reacted 2 hours；Connection liquid is taken out 8 μ L to be added 42 after 100 μ L DH5alpha competent cell ice bath 30min
500 37 DEG C of μ L soc culture mediums are added in DEG C heat shock 90s after the completion, 220rpm is cultivated 2 hours；Eppendorf is managed after 2 hours
4000g is centrifuged 1min and removes 400 μ L surplus liquids.Remaining liq is coated on 37 DEG C of LB plate to cultivate 12 hours；On plate
Picking single colonie is inoculated into 5mL LB liquid medium 37 DEG C, 220rpm culture 12 hours.
Plasmid is extracted with the small extraction reagent kit of QIAGEN, obtains pCAR19 and pCAR22 plasmid；Send Nanjing Jin Sirui biology section
After skill company generation sequence verification is errorless, the DH5alpha bacterial strain conservation of the plasmid containing pCAR19 or pCAR22 is carried out.
The plasmid preparation of embodiment 2, pCAR19, pCAR22
The DH5alpha strain of pCAR19 or pCAR22 plasmid is seeded to LB of the 250mL containing 100 μ g/mL ammonia benzyl mycins to train
In nutrient solution, 37 DEG C, 220rpm overnight incubation.Culture solution is centrifuged 20min in 6000g at 4 DEG C, abandons supernatant.
Take out the Buffers P1 in EndoFree plasmid mega kit (Qiagen), the large intestine obtained to centrifugation
The Buffers P1 for adding 120mL to be pre-chilled in advance in bacillus precipitating, covers centrifugation bottle cap, and acutely vibrating centrifugal bottle keeps Escherichia coli heavy
Shallow lake is completely dispersed in Buffers P1.
Into centrifugal bottle plus 120mL Buffers P2, cover bottle cap and be placed on roller bearing blending instrument, slowly raise speed to
50rpm is placed at room temperature for 5min after thoroughly mixing.
Add 120mL Buffers P3 into centrifugal bottle, cover bottle cap and be placed on roller bearing blending instrument, slowly speed-raising to rolling
Axis blending instrument maximum (top) speed 70rpm is thoroughly mixed until white not sticky fluffy mixed liquor.It is centrifuged at 4 DEG C in 9000g
15min。
50mL Buffer FW is poured into QIAfilter Cartridge, centrifugation gained supernatant is poured into QIAfilter
In Cartridge, lightly stir and evenly mix.Mixed liquor is filtered in marked good corresponding vial.
20mL Buffer ER is added into each vial, mixing 6 times of turning upside down, in -20 DEG C of incubation 30min.
The mega column marked is put on corresponding shelf, it is flat that 35mL Buffers QBT is added into each mega column
Weighing apparatus, gravity are allowed to flow to end.
Liquid batch in vial is all poured into the mega column of correspondence markings, after liquid is flow to end in column, to every
A mega column is added portionwise 200mL Buffer QC and is cleaned.After liquid is flow to end in column, by the waste liquid in waste collection disk
It pours into 50mL cleaning centrifuge tube.
40mL Buffer QN is added into each mega column again, collects efflux using 50mL cleaning centrifuge tube, up and down
Reverse 6 mixings, dispense in 20mL to the marked 50mL centrifuge tube of another cleaning.
14mL isopropanol (room temperature) is added to each 50mL centrifuge tube, turns upside down 6 times and mixes.4 DEG C in 15000g from
Heart 50min.
Supernatant is exhausted in superclean bench, 3.5mL Endotoxin-free water rinsing is added in every pipe, should not be the bottom of by
Portion's precipitating is broken up.30min is centrifuged in 15000g at 4 DEG C.Buffer TE in EndoFree plasmid mega kit is put
Enter preheating in baking oven.
In superclean bench exhaust centrifugation after supernatant, in superclean bench drying (volatilize remaining dehydrated alcohol,
Time is in 10min or so).
Buffer TE is taken out in baking oven, and 1mL Buffer TE is added to every pipe in superclean bench, is blown and beaten with rifle
It is put into 65 DEG C of baking ovens after 10 times, during which taps tube wall incessantly and precipitating is promoted to be completely dissolved.1min is centrifuged in 4000g at 4 DEG C
Liquid on tube wall is thrown to blow and beat after tube bottom and is mixed.
Liquid is fully transferred in EP pipe of the endotoxin-free without heat source nuclease free correspondence markings in superclean bench.
2 μ L are sucked out, surveys plasmid concentration with micro-spectrophotometer, and mark on corresponding EP pipe, obtains pCAR19 or pCAR22 matter
Grain.
Plasmid inspection:
1. plasmid concentration inspection
Sample is received, takes 1 μ L to carry out concentration mensuration, using ultramicron ultraviolet specrophotometer (Nanodrop), into core
Parameter is arranged in sour measurement module, and loading is detected after blank correction, as a result sees Fig. 7, Fig. 8 and following table:
2. Plasmid DNA (digestion) checks
Principle: agarose gel electrophoresis is the standard method for separating, identifying and purifying DNA fragmentation.Agarose be from
A kind of polysaccharide extracted in agar has hydrophily, but neutral, is a kind of good electrophoresis support.DNA is in alkaline condition
Under (buffer of pH8.0) it is negatively charged, it is mobile to anode by gel media in the electric field, different DNA molecular segments due to
Molecule and configuration difference, swimming rate liquid in the electric field are different.It is formed between the embeddable DNA molecular base-pair of ethidium bromide (EB)
Fluorescent complex can separate different zone after ultraviolet light irradiates, and reach separation, identification molecular weight, screen the mesh of recon
's.
Super spirial plasmid content in stoste can be tentatively judged by digestion qualification result, and supercoil content is higher, plasmid
Purity it is better, it is better to viral packaging efficiency below.
Method: taking 200ng sample respectively, with EcoRI, tri- digestion of KpnI, MluI, EcoRI single endonuclease digestion, using 0.7% fine jade
Sepharose electrophoresis detection.Be labeled as sample number into spectrum above glue hole, M1:10000kb DNA Marker (10000,8000,
6000、5000、4000、3500、3000、2000、1500、1200、1000、900、800、700、600、500、400、300、200、
100), (5000,3000,2000,1000,750,500,250,100) M2:Dl5000 DNA Marker.Sample Ago-Gel
Electrophoretogram (sample loading about 100ng), is as a result shown in Fig. 9.
3. target gene is sequenced
20 μ L (500ng) Plasmid DNA are taken, send sequencing outside, according to primordial seed sequence, check plasmid production products obtained therefrom
Target gene is whether there is or not changing, and under stable technique, work seed is in carrying out fermented and cultured amplification process, and target gene is not
It can change, can be used for the production of next link and correctly express albumen.
The preparation of embodiment 3, recombinant slow virus LVCAR19, LVCAR22
130.0~140.0 × 10 are accessed at multi-layer cellular culture bottle (Hyperflask) (Corning)6The 293T of number
Cell (Takara), total 560mL DMEM complete medium (50mL fetal calf serum, 5mL Antibiotic-Antimycotic
(100 ×)), contain 5%CO at 37 DEG C2It is cultivated 24 hours in incubator.320 μ g plasmid (pCAR19 or pCAR22:gag will be mixed with
Plasmid: vsvg plasmid=6:3:2) DMEM complete medium be added 960 μ g PEI pipes in, whirlpool concussion, equilibrium at room temperature
10min.The mixed liquor of 35mL PEI and plasmid and 525mL DMEM complete medium are mixed, above-mentioned multi-layer cellular culture is changed to
In bottle.Multi-layer cellular culture bottle is placed in 37 DEG C containing 5%CO2After incubator culture 3 days, cell culture supernatant is collected.
After supernatant 4000rpm (or 3000g) is centrifuged 30min, to centrifugation after cryonase enzyme is added in supernatant
(Takara) 4 DEG C are placed in.After 6 hours, slow virus supernatant is filtered using 0.22 μm of filter membrane, 4 DEG C in 30000g
It is centrifuged 2.5h.Supernatant is removed, 1mLT cell culture medium is added, precipitating is resuspended.After resuspension, 20 μ L is stayed to do titre Activity determination, it is remaining
The packing of slow virus concentrate, is placed in -80 DEG C labeled as LVCAR19 or LVCAR22 and saves backup.
The detection of slow virus activity titers:
Principle: there is label on Protein-L, and protein-L can be with the Kappa of single-chain antibody light chain in CAR
Area's specific binding, by flow cytomery to fluorescence signal indirect reaction expression feelings of the CAR in 293T cell
Condition.
Method: 5.0 × 10 are accessed in 6 orifice plates5A/hole 293T cell, the every hole of slow virus concentrate are separately added into 0.1 μ
L, 0.5 μ L, 1 μ L, and set 1 negative control.37 DEG C are placed in containing 5%CO2Culture in incubator.After three days, with Versene solution
(Gibco) collect 293T cell send flow cyctometry detect CAR positive 293T cell proportion, and convert obtain LVCAR19 or
LVCAR22 slow virus concentrate activity titers.
Current slow virus stoste activity titers are in 1~10E+05 range, and concentrate activity titers are in 1~10E+08 range
It is interior, it is as follows to test and analyze result:
CD22:
CD19:
The preparation of embodiment 4, CAR19T and CAR22T
Health donors peripheral blood 100mL is acquired, mononuclearcell is separated using Ficoll lymphocyte separation medium.It counts
Afterwards, CD3 positive cell is sorted using appropriate CD3 MicroBeads, human (U.S. day Ni), and with 1.0~2.0 × 106A/mL
Density is in T cell complete culture solution (OpTmizerTM CTSTMT-Cell Expansion Basal Medium, OpTmizerTM
CTS T-Cell Expansion Supplement (Invitrogen), the IL-2 (double aigret medicine companies) of 500 units) in culture, together
When press 25 μ L/106It is thin that Dynabeads Human T-Activator CD3/CD28 (Invitrogen) activation T is added in a cell
Born of the same parents.
It is that 3 addition LVCAR19 or LVCAR22 viruses are transduceed by MOI, mixing is placed on CO after 24 hours2Incubator
It is incubated for, suitable T cell complete medium is added after 4 hours and is cultivated.
CAR19T cell after transduction or CAR22T cell change fresh T cells are cultivated completely after lentiviruses transduction 24 hours
Liquid, and adjusting viable cell density is 1.0 × 106/ mL continues culture amplification 10~20 days, is observed and counted daily, and root
Fluid infusion is carried out according to the cell quantity counted and expands culture, remains that cell culture density is 1.0 × 106/mL。
The preparation of CAR-T cell preparation:
Cell dosage collects CAR19T cell or CAR22T cell on the estimation, is resuspended in containing 2% human serum albumin
In 100mL physiological saline, it is transferred to cell and feeds back in bag, CAR19T cell or CAR22T cell preparation finished product are made after heat-sealing.
The detection of CAR19T, CAR22T transduction efficiency
Take 1.0 × 106T cell after a transduction is incubated at room temperature 30 minutes, physiology salt with 1ug/ml FITC-Protein-L
After water cleaning twice, by flow cytomery FITC fluorescence signal, FITC positive cell ratio is measured, it is thin to reflect CART
Ratio of the born of the same parents in total cell.Testing result is as shown in Figure 10 and following table.Illustrate to be successfully prepared CAR19T and CAR22T cell.
| Number | Transduction type | Transduction efficiency |
| 2017112007 | CAR19T | 51.0% |
| 2017112008 | CAR22T | 38.6% |
The external Function detection of embodiment 5, CAR19T and CAR22T
CAR19T and CAR22T is carried out using calcein-AM to kill tumor Function detection in vitro.
Appropriate K562 target cell is taken, 1 × 106Calcein-AM is added in the cell suspension (PBS, 5% fetal calf serum) of/mL
To 25 μM of final concentration, 30min is incubated in incubator.Room temperature is resuspended after washing twice to 1.5 × 105/mL.Add by different effect target ratios
Enter CAR19T or CAR22T cell, 200g is centrifuged 30 seconds, and 37 DEG C are incubated for 2~3 hours.Supernatant is taken after the completion of being incubated for, measurement is wherein
The fluorescence intensity of calcein, and according to spontaneous release control and maximum release control, calculate target cell lysis percentage.
1. killing tumor experimental data
It needs progress cell line to kill the functional detection such as tumor before feedback different batches virus, is detected using calcein
Method.As a result referring to Figure 11 and following table:
Positive target cell
Negative targets
Embodiment 6, CART19T and the CAR22T sequential feedback in mouse-borne tumor model
In order to detect the anti-tumor effect of CAR-T cell in vivo, we select immune deficiency (NOD-SCID) mouse with
Raji-luc cell establishes tumor model, CAR-T cell is injected after modeling successfully, and use IVIS small animal living body imaging system
(Xenogen, Hopkinton, USA) test experience result.Experimental procedure is as follows:
1. modeling, to the NOD-SCID mouse subcutaneous injection 5 × 10 of 5 week old6A Raji-luc cell/only.
D-luciferin (Molecular Imaging is injected intraperitoneally with the dosage of 150mg/kg in animal imaging after 2.7 days
Products, Bend, USA), yellow Jackets anesthetized mice is injected intraperitoneally in 75mg/kg, waits and uses IVIS toy after ten minutes
Living imaging system (Xenogen, Hopkinton, USA) collects optical signal.
3. the same day injects CAR-T cell after modeling successfully, it is grouped as follows:
(1) CD19 group injects CAR19T cell 5 × 106A/only；
(2) CD22 group injects CAR22T cell 5 × 106A/only；
(3) CD19+CD22 group first injects CAR19T cell 5 × 106A/only, CAR22T cell 5 × 10 is injected after 3 days6
A/only.
4. injection is imaged after CAR-T cell 21 days, experimental result is analyzed, referring to Figure 12, wherein NT: general T cell infusion
Group；CD22:CAR22T injection group；GFP:GFP slow-virus transfection T cell injection group；CD19+CD22:CAR19T and CAR22T note
Penetrate group；CD19:CAR19T injection group；.
The results show that compared with general T cell infusion group and GFP-T injection group, CAR19T and CAR22T injection group tumour
Load significantly reduces, CAR19T and CAR22T group cases of complete remission.Illustrate that double sequential feedback curative effects of target spot CART are better than
Single target spot feeds back and does not feed back group.
The clinical application of embodiment 7, CAR19T and CAR22T
59 are drenched with recurrence/refractory/high-risk acute B using the sequential anti-CD19 CAR-T and anti-CD22 CAR-T of infusion
Bar cell tumour is treated, including 42 B-ALL patients and 17 aggressive B cell lymphoma patients.
Patient in group's clinical settings:
About B-ALL leukaemic: the median age is 28 years old, and male to female ratio is about 3:2.Nearly half Patients on Recurrence is more than
2 times or suffer from refractory primary disease.Before participating in this test, there are 7 patients to receive transplanting, 6 receive
CAR19 treatment, but be all reoccurred.Early stage B cell out of control from marrow is differed from 0 to 92%.It is sent out in 6 Cerebrospinal Fluid in Patients
Existing early stage B cell out of control.High-risk genetic distortion, including BCR/ABL transposition, T315I mutation occur for Most patients, and 17p is lacked
It loses or TP53 is mutated.Also detect that MLL is reset, mainly MLL/AF4 and hypodiploid.
About aggressive B cell lymphoma patient: Lymphoma the median age is 38 years old, and the ratio between men and women is about 1:1.Institute
Having subtypes is high malignancy B cell lymphoma, wherein 1 is dual lymthoma.Nearly 65% Patients on Recurrence is more than two
It is secondary or have refractory primary disease.Three patients once received transfer operation, but recurred.Nearly half patient carries Myc transposition,
17p missing or TP53 mutation.
CAR-T cell therapy validity:
For leukaemic, before feeding back CART cell, 40 patients receive fludarabine and cyclophosphamide is located in advance
Reason.The mean dose of CAR19T and CAR22T is respectively 2.44 ± 1.11 × 106/ kg and 2.19 ± 1.22 × 106/kg。
A patient of 38/42 (90.5%) obtains complete incidence graph (CR or CRi), than the CD19CART drug of Novartis's listing
The complete remission rate 83% of Kymriah slightly improves.A patient of 33/42 (78.6%) obtains molecules and alleviates, thin by streaming
Born of the same parents' instrument detects MRD feminine gender.
6 months overall survival rates and be respectively 80.0% and 60.8% without event survival rate.
At 12 months, overall survival rate and without event survival rate respectively close to 60% and 40%.
During follow-up, although be up to the present not detected in recurrent cases after mixing CAR-T cell therapy CD19 and
The antigen of CD22 is escaped, but 23 patients are finally still recurred.
For aggressive lymphomas patient, the patient more than half reaches CR.W-response rate is more than 80%, is higher than Kate
The 72% of the CD19 CART drug Yescarta of listing.At 6 months, OS and EFS respectively may be about 90% and 60%.It is higher than
The 79% of Yescarta and 55%.
CAR-T cell therapy safety:
For leukaemic, most of (88%) patients have CRS, but only 20% has serious CRS (>=3 grades).Compare promise
The serious CRS reactivity of the CD19 CART drug Kymriah75% (47/63) of China's listing is substantially reduced.
All aggressive lymphomas patient experience CRS, only 10% has serious CRS but reversible, 13% summary than Yescarta
There is reduction, but only 17.6% occurs neurotoxicity, hence it is evident that lower than the 87% of Yescarta.
Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection model of the invention
It encloses without being limited thereto.Those skilled in the art's made equivalent substitute or transformation on the basis of the present invention, in the present invention
Protection scope within.Protection scope of the present invention is subject to claims.
Claims (10)
1. a kind of joint Chimeric antigen receptor, which is characterized in that including Chimeric antigen receptor CAR19 and Chimeric antigen receptor
CAR22,
The CAR19 is by the piece of two kinds of albumen of single-chain antibody and T cell activation motif including at least specific recognition CD19
Section is coupled；The CAR22 is two hatching egg of single-chain antibody and T cell activation motif by including at least specific recognition CD22
White segment is coupled.
2. joint Chimeric antigen receptor according to claim 1, which is characterized in that the T cell activation motif is CD3 ζ.
3. joint Chimeric antigen receptor according to claim 1 or 2, which is characterized in that the structure of the CAR19 are as follows: special
Single-chain antibody, the first cross-film costimulatory signal and the T cell activation motif of opposite sex identification CD19 is successively coupled；
The first cross-film costimulatory signal is CD28, one or several combinations in 4-1BB, OX40.
4. joint Chimeric antigen receptor according to claim 1 or 2, which is characterized in that the structure of the CAR22 are as follows: special
Single-chain antibody, the second cross-film costimulatory signal and the T cell activation motif of opposite sex identification CD22 is successively coupled；
The second cross-film costimulatory signal is CD28, one or several combinations in 4-1BB, OX40.
5. the encoding gene of any joint Chimeric antigen receptor of Claims 1 to 4, which is characterized in that including CAR19's
The encoding gene of encoding gene and CAR22.
6. Combined expression carrier, which is characterized in that first vector and expression including CAR19 gene described in expression claim 1
The Second support of CAR22 gene described in claim 1, or
The third carrier of CAR19 gene and CAR22 gene described in claim 1 is expressed simultaneously.
7. Combined expression carrier according to claim 6, which is characterized in that the carrier that sets out is recombined lentivirus vector
pLVX-EF1α-IRES-Puro。
8. union and recombination slow virus, which is characterized in that the Combined expression carrier as described in claim 6 or 7 simultaneously or separately with
The package body combination obtained after slow virus auxiliary packaging plasmid cotransfection host cell.
9. transfection has the right to require the T cell of the 8 union and recombination slow virus.
10. a kind of oncotherapy preparation, which is characterized in that contain T cell as claimed in claim 9.
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Application publication date: 20190709 |