CN107793483A - Chimeric antigen receptor and its gene and recombinant expression carrier, CARMSLN NKT cells and its preparation method and application - Google Patents

Chimeric antigen receptor and its gene and recombinant expression carrier, CARMSLN NKT cells and its preparation method and application Download PDF

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CN107793483A
CN107793483A CN201610807069.4A CN201610807069A CN107793483A CN 107793483 A CN107793483 A CN 107793483A CN 201610807069 A CN201610807069 A CN 201610807069A CN 107793483 A CN107793483 A CN 107793483A
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    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
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Abstract

The invention discloses a kind of Chimeric antigen receptor and its gene and recombinant expression carrier, CARMSLN NKT cells and its preparation method and application, the Chimeric antigen receptor is MSLNScFv CD8 CD137 CD3 ζ, including the CD8a signal peptides of series connection, MSLNScFv, the intracellular signal domain of the CD8 hinge areas shortened and transmembrane region, CD137 intracellular signal domain and CD3 ζ.When being co-cultured using the CARMSLN NKT cells of the present invention with mesothelioma cell positive MSLN, there is good specific killing activity to mesothelioma cell.

Description

Chimeric antigen receptor and its gene and recombinant expression carrier, CARMSLN-NKT cells and Its preparation method and application
Technical field
The invention belongs to knubble biological arts, in particular it relates to a kind of chimeric antigen in adoptive immunotherapy by Body MSLNScFv-CD8-CD137-CD3 ζ and its gene and recombinant expression carrier, the NKT cells for being engineered MSLN targetings (CARMSLN-NKT cells) and its preparation method and application.
Background technology
NK (NKT) is a kind of t lymphocyte subset group of specific type, has T cell and NK cells Double property.NKT cells can express two kinds of acceptors of NKR-P1 of TCR and the NK cell of T cell, under TCR and NKR mediations, NKT Cell can produce substantial amounts of IL-4 and INF γ, and killing functions of immunocytes is played to tumour cell.NKT cells pass through its own face CD16 combined with the Fc sections of specific antibody, play antibody dependent cell mediated cytotoxicity (ADCC, antibody-dependent cell-mediated cytotoxicity).But make in the cell-mediated killing of antibody-dependant During, because antibody can be combined with the corresponding antigens epitope specificity on target cell, NKT cells can kill any with resisting The target cell that body combines, therefore antibody is specific, but killing of the NKT cells to target cell with the antigen binding on target cell Effect is nonspecific.In addition, it is generally the case that the NKT cells of infusion are 2 weeks or so in patient's body half-life period, effectively Phase is of short duration, it is necessary to repeated multiple times infusion.Moreover, NKT cell itselfs lack specific antibody, it is not enough to around tumour or knurl It is enriched with nest, constrains targeted therapy of the NKT cells to malignant tumour.Furthermore research shows, NKT cells are not to all Tumour have fragmentation effect, and weaker to the lethal effect of Partial tumors, specific killing activity has much room for improvement.
Mesothelin (MSLN) is 40kDa surface glycoprotein, is expressed in kinds of tumor cells surface, such as pernicious mesothelium Knurl, Vipoma, oophoroma etc., wherein MPM MSLN expression rate reaches more than 90%, and is in normal structure The phenomenon of existing low expression.Research finds that MSLN and progress, transfer and the relatively low survival rate of patient of tumour have very big correlation. Due to above characteristic, MSLN antigens turn into the promising target treated based on antibody.At present, numerous studies use MSLN monoclonals Tumour cell positive the scheme target killing MSLN of antibody or MSLN vaccines, however, these schemes are producing certain effect On the basis of, at the same there is also it is certain the defects of, such as：The transience and MSLN monoclonal antibodies of MSLN monoclonal antibody target effects In deficiency of tumor locus distribution etc..
The content of the invention
The invention aims to overcome, the lethal effect of NKT cells against tumor in the prior art is weaker, specifically kills Hinder activity have much room for improvement and existing clinical research in it is above-mentioned existing for MSLN monoclonal antibodies and vaccine targeted therapy the defects of, A kind of Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3 ζ and its gene and recombinant expression carrier, engineering MSLN are provided NKT cells (CARMSLN-NKT cells) of targeting and its preparation method and application.
The present inventor has been surprisingly found that under study for action, using the Chimeric antigen receptor MSLNScFv-CD8- of the present invention When the NKT cells of CD137-CD3 ζ modifications co-culture with mesothelioma cell positive MSLN, have to mesothelioma cell good Specific killing activity.
Therefore, to achieve these goals, it is described chimeric in a first aspect, the invention provides a kind of Chimeric antigen receptor Antigen receptor is MSLNScFv-CD8-CD137-CD3 ζ, including the CD8a signal peptides of series connection, MSLNScFv, the CD8 shortened hinge The intracellular signal domain of sequence (hinge areas) and transmembrane region, CD137 intracellular signal domain and CD3 ζ.
Second aspect, the invention provides the gene for encoding above-mentioned Chimeric antigen receptor.
The third aspect, the invention provides the recombinant expression carrier containing said gene.
Fourth aspect, the invention provides a kind of NKT cells of engineering MSLN targetings, the NKT cells are above-mentioned The NKT cells of Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3 ζ modifications.
5th aspect, the invention provides a kind of preparation method of the NKT cells of engineering MSLN targetings, methods described Including：Packaging carries PWPT-MSLNScFv-CD8-CD137-CD3 ζ slow virus, obtains viral concentration liquid；Utilize obtained disease Malicious concentrate infects NKT cells, makes NKT cells expression Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3 ζ.
6th aspect, the NKT cells for the engineering MSLN targetings being prepared the invention provides the above method.
7th aspect, the invention provides the NKT cells of above-mentioned engineering MSLN targetings to prepare for treating tumour Preparation in application.
When being co-cultured with mesothelioma cell positive MSLN, Chimeric antigen receptor MSLNScFv-CD8- of the invention The NKT cells of CD137-CD3 ζ modifications, that is, MSLN antigens can be specifically bound by being engineered the NKT cells of MSLN targetings, be increased The ability of strong immunocyte targets identification cancer cell surfaces MSLN antigens, strengthens the specific killing to MSLN positive mesothelioma cells Activity.The NKT cells of the engineering MSLN targetings of the present invention provide a kind of new selection for the positive tumours for the treatment of MSLN, With good industrial application prospect.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Brief description of the drawings
Fig. 1 is the result that flow cytometry is analyzed the NKT cell phenotypes being separately cultured.
Fig. 2 is the Lentiviral PWPT-CD8-CD137-CD3 ζ of present invention restriction enzyme MluI/SalI The electroresis appraisal figure of double digestion fragment.
Fig. 3 is the Lentiviral PWPT-MSLNScFv-CD8-CD137-CD3 ζ of present invention restriction enzyme The electroresis appraisal figure of BamHI/SalI double digestion fragments.
Fig. 4 is the Lentiviral PWPT-MSLNScFv-CD8-CD137-CD3 ζ of present invention structural representation, Wherein, sequence counterclockwise is positive gene piece degree, is clockwise cdna reverse fragment.
Fig. 5 is the viral concentration that Flow cytometry contains Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3 ζ Efficiency of infection of the liquid to NKT cells.
Fig. 6 is the NKT cells of Flow cytometry Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3 ζ modifications The result of (CARMSLN-NKT cells) phenotypic evaluation.
Fig. 7 is the cell toxicant of the lethal effect of the CARMSLN-NKT cells of the present invention mesothelioma cell positive to MSLN Property analysis chart.
Embodiment
The embodiment of the present invention is described in detail below.It is it should be appreciated that described herein specific Embodiment is merely to illustrate and explain the present invention, and is not intended to limit the invention.
The invention provides a kind of Chimeric antigen receptor, the Chimeric antigen receptor is MSLNScFv-CD8-CD137-CD3 ζ, including the CD8a signal peptides of series connection, MSLN single-chain antibodies MSLNScFv, the hinge area for shortening CD8 and transmembrane region, CD137 born of the same parents The intracellular signal domain of interior signal domain and CD3 ζ.
Under preferable case, Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3 ζ by CD8a signal peptides, MSLNScFv, The intracellular signal domain of CD8 hinge area and transmembrane region, CD137 intracellular signal domain and CD3 ζ is in series.Enter one Preferably, Chimeric antigen receptor has the amino acid sequence as shown in SEQ ID NO.1 to step, it is further preferred that inosculating antibody The amino acid sequence of original receptor is as shown in SEQ ID NO.1.
The invention provides the gene for encoding above-mentioned Chimeric antigen receptor.Under preferable case, the gene has such as SEQ Nucleotide sequence shown in ID NO.2, it is further preferred that encoding the nucleotide sequence of the gene of above-mentioned Chimeric antigen receptor such as Shown in SEQID NO.2.
The invention provides the recombinant expression carrier containing said gene.Under preferable case, recombinant expression carrier is slow disease Malicious expression vector.For Lentiviral, there is no particular limitation, as long as can be with assistant carrier cotransfection incasing cells Such as 293T incasing cells, the NKT for obtaining viral concentration liquid and Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3 ζ modifications is thin Born of the same parents, under preferable case, Lentiviral is PWPT-MSLNScFv-CD8-CD137-CD3 ζ.
Do not limited particularly for Lentiviral PWPT-MSLNScFv-CD8-CD137-CD3 ζ preparation method It is fixed, can be those skilled in the art it is conceivable that various methods, under preferable case, Lentiviral PWPT- MSLNScFv-CD8-CD137-CD3 ζ preparation method comprises the following steps：
(1) CD8 hinge areas and transmembrane region, CD137 intracellular signal domain is expanded respectively from NKT cell cDNAs With CD3 ζ intracellular signal domain, and it is cloned into carrier PWPT-GFP, structure obtains PWPT-CD8-CD137-CD3 ζ；
(2) composite coding CD8a signal peptides and MSLNScFv nucleotide sequence, and it is cloned into PWPT-CD8-CD137- In CD3 ζ, the correct PWPT-MSLNScFv-CD8-CD137-CD3 ζ of sequence are obtained after sequence verification.
In step (1), hinge areas and transmembrane region, CD137 intracellular for expanding CD8 respectively from NKT cell cDNAs There is no particular limitation for the method for signal domain and CD3 ζ intracellular signal domain, can be various sides commonly used in the art Method, such as can be RT-PCR methods.Wherein, then NKT cells can be carried out by separating the mononuclearcell in people's venous blood Culture obtains.
Specifically, obtaining PWPT-CD8-CD137-CD3 ζ method can include：The total serum IgE of NKT cells is extracted, is reversed Record obtains NKT cell cDNAs, using obtained NKT cell cDNAs template, utilizes primer P1 (SEQID NO.11) and P2 (SEQID NO.12 hinge areas and transmembrane region (SEQID NO.3) that performing PCR amplification obtains CD8 genes) are entered；Utilize primer P3 (SEQID NO.13) and P4 (SEQID NO.14) enters the intracellular signal domain (SEQID NO.4) that performing PCR amplification obtains CD137 genes； Enter the intracellular signal structure of performing PCR amplification acquisition CD3 ζ genes using primer P5 (SEQID NO.15) and P6 (SEQID NO.16) Domain (SEQID NO.5), double digestion is carried out by the PCR primer of acquisition respectively, then with the slow virus after MluI/SalI double digestions Expression vector PWPT-GFP connections.
It is not special for the method for composite coding CD8a signal peptides and MSLNScFv nucleotide sequence in step (2) Restriction, can be various methods commonly used in the art, such as can be synthesized by full genome synthetic technology.
Specifically, obtaining the correct PWPT-MSLNScFv-CD8-CD137-CD3 ζ of sequence method can include：Pass through The nucleotide sequence of full genome synthetic technology composite coding CD8a signal peptides and MSLNScFv fusions (SEQID NO.8), gram In the grand pGSI to carrier, pGSI-MSLNScFv is obtained；Then by pGSI-MSLNScFv carry out BamHI/MluI double digestions, with The recombinant plasmid PWPT-CD8-CD137-CD3 ζ connections that step (1) after BamHI/MluI double digestions obtains, through sequencing identification, Obtain the correct PWPT-MSLNScFv-CD8-CD137-CD3 ζ of sequence.Wherein, the nucleotide sequence of CD8a signal peptides such as SEQID Shown in NO.6, MSLNScFv nucleotide sequences are as shown in SEQID NO.7.
Present invention also offers a kind of NKT cells of engineering MSLN targetings, the NKT cells are by above-mentioned inosculating antibody The NKT cells (i.e. CARMSLN-NKT cells) of original receptor MSLNScFv-CD8-CD137-CD3 ζ modifications.
Present invention also offers a kind of preparation method of the NKT cells of engineering MSLN targetings, this method includes：Packaging PWPT-MSLNScFv-CD8-CD137-CD3 ζ slow virus is carried, obtains viral concentration liquid；Utilize obtained viral concentration liquid NKT cells are infected, make NKT cells expression Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3 ζ.
The method of PWPT-MSLNScFv-CD8-CD137-CD3 ζ slow virus is carried for packaging, and there is no particular limitation, Can be the various methods commonly used of those skilled in the art, under preferable case, by Lentiviral PWPT-MSLNScFv- CD8-CD137-CD3 ζ and helper plasmid (such as psPAX2, pMD2.G) cotransfection 293T incasing cells, are collected when transfecting 48-72h Viral supernatants, centrifugation, filtering, 5 × PEG6000-NaCl of addition is mixed in filtrate, and supernatant, precipitation 0-4 are abandoned after centrifugation The sterile PBS dissolvings of DEG C precooling, obtain viral concentration liquid.
In the method for the present invention, in addition to it is prepared via a method which NKT cells：
(1) in the presence of CD3 monoclonal antibodies, proleulzin and interleukin-15, mononuclearcell is subjected to the first stage Culture；
(2) in the presence of proleulzin, the cell that the first stage is cultivated carries out second stage culture.
Under preferable case, the embodiment of the first stage culture includes：It is thin that mononuclearcell is incubated at the first NKT In born of the same parents' nutrient solution, the first NKT cell culture fluids contain NKT cell culture mediums, CD3 monoclonal antibodies, proleulzin and white Interleukin -15；It is further preferred that in the first NKT cell culture fluids, the concentration of the CD3 monoclonal antibodies is 30-70ng/mL, And/or the concentration of the proleulzin is 300-700U/mL, and/or the concentration of the interleukin-15 is 30-70ng/mL.
Under preferable case, the embodiment of the second stage culture includes：The cell that the first stage is cultivated is trained Support in the 2nd NKT cell culture fluids, NKT cell culture mediums and proleulzin are contained in the 2nd NKT cell culture fluids；Enter Preferably, in the 2nd NKT cell culture fluids, the concentration of the proleulzin is 300-700U/mL to one step.
For NKT cell culture mediums, there is no particular limitation, can be commonly used in the art various to be used to cultivate NKT cells Culture medium, such as can be GT-T551 culture mediums.
When preparing NKT cells, for first stage culture and the condition of second stage culture, there is no particular limitation, can Think various conditions commonly used in the art, such as can be in 30-37 DEG C, the CO that saturated humidity is 3-6%2Trained in incubator Support.Those skilled in the art can carry out accommodation to the time of culture, and this is known to those skilled in the art, herein Repeat no more.
In the NKT cells that the present invention is prepared, CD3+Cell average ratio>90%, CD3+CD8+Cell accounts for total CD3+Carefully The average ratio of born of the same parents>70%；CD3+CD56+Cell accounts for total CD3+The average ratio of cell>15%.
It is not particularly limited for the method for infecting NKT cells, can is various methods commonly used in the art, preferable case Under, this method includes：
(1) in the presence of viral concentration liquid, nucleoprotamine and proleulzin, NKT cells are subjected to first stage infection training Support；
(2) in the presence of CD3 monoclonal antibodies, proleulzin and interleukin-15, the first stage is infected into culture Cell carries out second stage infection culture.
Preferably, the embodiment of the first stage infection culture includes：By NKT cell culture in the 3rd NKT cells In nutrient solution, the 3rd NKT cell culture fluids contain NKT cell culture mediums, viral concentration liquid, nucleoprotamine and interleukin- 2；It is further preferred that in the 3rd NKT cell culture fluids, the concentration of the proleulzin is 300-700U/mL.
Preferably, the embodiment of the second stage infection culture includes：By the thin of first stage infection culture Born of the same parents are incubated in the first NKT cell culture fluids.The concrete composition of first NKT cell culture fluids can be found in foregoing corresponding interior Hold, will not be repeated here.
When infecting NKT cells, the condition that culture is infected for first stage infection culture and second stage is not special Restriction, can be various conditions commonly used in the art, for example, can 30-37 DEG C, saturated humidity be 3-6% CO2Culture Cultivated in case.Those skilled in the art can carry out accommodation to the time of culture, and this is those skilled in the art It is known, it will not be repeated here.
Specifically, infecting the method for NKT cells includes：Take 1 × 107-5×107Individual NKT cells, discard old nutrient solution, The fresh GT-T551 nutrient solutions of 2-4mL are added, add 200-400 μ L viral concentrations liquid, 2-4 μ L 1 × 10-6Mg/mL milt eggs White and final concentration of 300-700U/mL IL-2, it is placed in 30-37 DEG C, the CO that saturated humidity is 3-6%212- is infected in incubator After 16h, nutrient solution is abandoned, cell is gone in not coated blake bottle, 20-50mL GT-T551 culture mediums is added, adds end Concentration is 300-700U/mL IL-2, final concentration of 30-70ng/ml CD3 monoclonal antibodies and final concentration of 30-70ng/mL Interleukin 15, in 30-37 DEG C, saturated humidity be 3-6% CO212-18h is cultivated in incubator, obtains chimeric antigen The NKT cells of acceptor MSLNScFv-CD8-CD137-CD3 ζ modifications.
It is further preferred that the method for infection NKT cells also includes：
(3) it is first in the presence of proleulzin, the cell progress of second stage infection culture is external evoked, treat cell Density when being 80-90%, then in the presence of CD3 monoclonal antibodies, proleulzin and interleukin-15, cell is expanded Culture.
Under preferable case, the external evoked embodiment includes：The cell of second stage infection culture is trained Support in the 2nd NKT cell culture fluids, the embodiment of the amplification cultivation includes：By cell culture in described first In NKT cell culture fluids.The concrete composition of first NKT cell culture fluids and the 2nd NKT cell culture fluids can be found in foregoing corresponding Content, it will not be repeated here.
Specifically, infecting the method for NKT cells also includes：By the slow-virus infection obtained after second stage infection culture NKT cells are carried out external evoked with IL-2 final concentration of 300-700U/mL GT-T551 nutrient solutions, and the density for treating cell is Cell is transferred in cell culture bags during 80-90%, every final concentration of 300-700U/mL, CD3 that 1.5-2.5 days add IL-2 The final concentration of 30-70ng/ml of monoclonal antibody, the final concentration of 30-70ng/mL of IL-15 fresh GT-T551 Nutrient solution carries out amplification cultivation and cell is expanded into total amount for 1 × 109-2×109Individual cell.By the slow virus pair of the present invention The Chimeric antigen receptor for targetting MSLN antigens carries out NKT cell infections, and its efficiency of infection is up to 30%-60%, and obtain CARMSLN-NKT cells, its CD3+CD56+Cell accounts for total CD3+The ratio of cell is more than 15%.
The Chimeric antigen receptor of the NKT cells expression of Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3 ζ modifications Protein amino acid sequence is as shown in SEQID NO.1.Wherein, it will be understood by those skilled in the art that before Chimeric antigen receptor Body protein is by CD8a signal peptides, MSLNScFv, CD8 hinge areas and transmembrane region, CD137 intracellular signal domain and CD3 ζ Intracellular signal domain it is in series, after protein translation in the cell rough endoplasmic reticulum excision signal peptide after turn into maturation Chimeric antigen receptor albumen, after secretion output and it is positioned on the cell membrane of NKT cells.The histone amino of the Chimeric antigen receptor Gene coded sequence corresponding to acid sequence is as shown in SEQID NO.2.The Chimeric antigen receptor with gene C D8 hinge areas and across The structure that film area and CD137 and CD3 ζ intracellular signal domain are in series is signal transduction domain, its amino acid sequence As shown in SEQID NO.9, corresponding gene coded sequence is as shown in SEQID NO.10.
The NKT cells for the engineering MSLN targetings being prepared present invention also offers the above method.
Present invention also offers the NKT cells of engineering MSLN targetings to prepare for treating answering in the preparation of tumour With.Under preferable case, tumour is the positive celiotheliomas of MSLN, it is further preferred that the tumour is the positive pernicious chests of MSLN Intermembranous rind gall.
In the application of the present invention, for the composition of the preparation for treating the positive tumours of MSLN, there is no particular limitation, As long as being prepared containing CARMSLN-NKT cells of the present invention or by CARMSLN-NKT cells, preparation it is specific Composition and preparation method are known to those skilled in the art, will not be repeated here.
Embodiment
The present invention is further illustrated for following embodiment, but and is not so limited the present invention.
Experimental method in following examples, it is this area conventional method unless otherwise specified.Institute in following embodiments Experiment material, it is to be commercially available from routine biochemistry reagent shop unless otherwise specified, wherein：
NKT cell culture mediums GT-T551 is purchased from TaKaRa companies.
Lymphocyte separation medium is purchased from TBD companies.
CD3 monoclonal antibodies, recombinant fiber connection albumen (retronectin) are purchased from TaKaRa companies.
Recombinant human protein's interferon-γ, rhIL-2, recombinant human interleukin 15 are purchased from protech companies.
Total RNA extraction reagent box RNAiso Reagent, high-fidelity DNA polymerase ( HS DNA Polymerase), T4DNA ligases are purchased from TaKaRa companies.
RevertAidTMFirst Strand cDNA Synthesis Kit are purchased from Fermentas companies.
Bgl II, EcoRI, MluI, BamHI, SalI are purchased from Fermentas companies.
Ago-Gel DNA QIAquick Gel Extraction Kits, common DNA product purification kit, the small extraction reagent kit of plasmid are purchased from day Root biochemical technology Co., Ltd.
PWPT-GFP, psPAX2, pMD2.G are purchased from Addgene companies.
PGSI is purchased from Beijing Tian Yihuiyuan bio tech ltd.
Trans1-T1Phage Resistant Competents cell is purchased from Beijing Quanshijin Biotechnology Co., Ltd.
LipofectamineTM2000Transfection Reagent transfection reagents are purchased from Invitrogen companies.
293T incasing cells is purchased from U.S. ATCC.
In PEG6000-NaCl final concentration of 25.5 the mass %, NaCl of PEG6000 final concentration of 1.2M, PEG6000 and NaCl is purchased from Shanghai Suo Laibao bio tech ltd.
Hyclone is purchased from German PAA companies.
Mesothelioma cell lines NCI-H2452 positive MSLN is purchased from ATCC companies of the U.S..
CF succinimide ester is purchased from Shanghai Pu Zhen bio tech ltd.
Annexin V-RPE kits are purchased from U.S. company BD.
All primers synthesize by Beijing Tian Yihuiyuan bio tech ltd.
The preparation of the NKT cells of embodiment 1
(1) people's venous blood is taken in the vacuum tube containing heparin.Using lymphocyte separation medium, by density gradient centrifugation side Method separation obtains mononuclearcell (PBMCs).
(2) after PBMCs is washed three times, using the NKT cell culture mediums GT-T551 of the Human autologous serum containing 0.6 volume % It is 2 × 10 to adjust final concentration of cells6Individual cell/mL；Cell is inoculated in and pre- first passes through final concentration of 10 μ g/mL's The coated 75cm of retronectin2In Tissue Culture Flask.Then final concentration of 500U/mL recombined human is added in culture medium Recombination human interleukin -15 of interleukin-22,50ng/ml CD3 monoclonal antibodies and 50ng/mL, in 37 DEG C, saturated humidity 5% CO2Cultivated in incubator.
(3) cultivate the 4th day, cell is transferred in not coated blake bottle, added according to cell growth quantity within every 2 days NKT cell culture medium GT-T551, it is 1 × 10 to control cell concentration8Individual cell/mL, and add final concentration of 500U/ml weight Group human interleukin 2；Culture obtained NKT cells, flow cytometry is analyzed NKT cell phenotypes to the 12nd day.As a result figure is seen 1, wherein, CD3+：95.04%；CD3+CD8+：90.99%；CD3+CD56+：24.12%；CD8+CD56+：24.63%.
The Lentiviral PWPT-MSLNScFv-CD8-CD137-CD3 ζ of embodiment 2 structure
(1) preparation of NKT cell cDNAs
Centrifugation embodiment 1 cultivates obtained NKT cells, is extracted with total RNA extraction reagent box RNAiso Reagent The total serum IgE of cell, -80 DEG C save backup.The total serum IgE of extraction Reverse Transcriptase kit RevertAidTM First Strand CDNA Synthesis Kit reverse transcriptions obtain NKT cell cDNAs, and -20 DEG C save backup.
(2) slow virus plasmid PWPT-CD8-CD137-CD3 ζ preparation
Design and synthesize following primer sequence (wherein, underscore is labeled as protection base, and square frame is restriction enzyme site)：
Using NKT cell cDNAs in step (1) as template, enter performing PCR with primer P1 and P2 and expand, obtain long 227bp CD8 Hinge areas and shorten transmembrane region, for nucleotide sequence as shown in SEQID NO.3, MluI and the enzymes of Bgl II are contained in both ends respectively Enzyme site and protection base；Enter performing PCR with primer P3 and P4 to expand, obtain long 146bp CD137 intracellular signal domains, core For nucleotide sequence as shown in SEQID NO.4, Bgl II and EcoRI restriction enzyme sites and protection base are contained in both ends respectively；With primer P5 With P6 enter performing PCR amplification, obtain long 359bp CD3 ζ intracellular signal domain, nucleotide sequence as shown in SEQID NO.5, Contain EcoRI and SalI restriction enzyme sites and protection base respectively in both ends.Each step pcr amplification reaction system is identical, to expand CD137 Exemplified by intracellular signal domain, enter performing PCR amplification, PCR reaction condition references HS DNA Polymerase specification, reaction system (50 μ L) are as follows：
Distilled water：32.5μL
5 × reaction buffer：10μL
DNTP mixtures (every kind of 2.5mM)：4μL
P3(10mM):1μL
P4(10mM):1μL
NKT cell cDNAs (200ng/ul)：1μL
HS DNA Polymerase：0.5μL
Above-mentioned PCR primer is separated with 1% Ago-Gel, carried out with Ago-Gel DNA QIAquick Gel Extraction Kits DNA fragmentation reclaims.Double digestion reaction is carried out respectively after obtaining fragment, and digestion products are reclaimed with common DNA product purification kit It is standby.
Lentiviral PWPT-GFP MluI/SalI double digestions, digestion products are carried out through 1% Ago-Gel Separation, big carrier segments are reclaimed with Ago-Gel DNA QIAquick Gel Extraction Kits, then CD8, CD137, CD3 ζ with reclaiming before Fragment is connected by T4DNA ligases, connection product conversion Trans1-T1Phage Resistant Competent cells, and 37 Picking monoclonal after DEG C culture 16h, 37 DEG C, with plasmid small extraction reagent kit extraction plasmid after 250rpm cultures 12h.The plasmid of extraction To be identified through restriction enzyme MluI and SalI double digestion, identification electrophoretogram is shown in Fig. 2, wherein, M1：DNA molecular amount marks D15000；1 swimming lane：Plasmid PWPT-CD8-CD137-CD3 ζ non-endonuclease bamhi；2 swimming lanes：Plasmid PWPT-CD8-CD137-CD3 ζ endonuclease bamhi (672bp)；M2：DNA molecular amount marks D2000.It will identify that correct plasmid send the brightness of Beijing day one far biological section The fusion fragment of insertion is sequenced for skill Co., Ltd.The correct recombinant plasmid of sequencing result is named as PWPT- CD8-CD137-CD3 ζ, wherein, CD8 hinge areas and the nucleotide sequence of transmembrane region as shown in SEQID NO.3, CD137's The nucleotide sequence of intracellular signal domain is as shown in SEQID NO.4, and the nucleotide sequence of CD3 ζ intracellular signal domain is such as Shown in SEQID NO.5.
(3) slow virus plasmid PWPT-MSLNScFv-CD8-CD137-CD3 ζ preparation
The nucleotide sequence of full genome composite coding CD8a signal peptides and MSLNScFv fusions, sequence such as SEQID Shown in NO.8, synthesized by Beijing Tian Yihuiyuan bio tech ltd, BamHI, kozak sequence are contained in its 5 ' end, and 3 ' ends contain There are MluI restriction enzyme sites, by foregoing fusion gene cloning in plasmid pGSI, be named as pGSI-MSLNScFv.Plasmid passes through BamHI/MluI double digestions, digestion products are separated through 1% Ago-Gel, are returned with Ago-Gel DNA QIAquick Gel Extraction Kits It is standby to receive purpose fragment.
PWPT-CD8-CD137-CD3 ζ plasmids are through restriction enzyme BamHI/MluI digestions, and digestion products are through 1% agar Sugared gel is separated, and it is standby to reclaim carrier segments with Ago-Gel DNA QIAquick Gel Extraction Kits.Then CD8a is contained with recovery Signal peptide and MSLN ScFv DNA fragmentation are attached by T4DNA ligases, and specific method is shown in specification.By connection product Convert Trans1-T1Phage Resistant Competent cells, 37 DEG C culture 16h after picking monoclonal, 37 DEG C, After 250rpm cultures 12h, plasmid is extracted with the small extraction reagent kit of plasmid.The plasmid of extraction is double through restriction enzyme BamHI/SalI Digestion identifies, qualification result as shown in figure 3, wherein, wherein, 1 swimming lane：Plasmid PWPT-MSLNScFv-CD8-CD137-CD3 ζ are not Endonuclease bamhi (10117bp)；2 swimming lanes：Plasmid PWPT-MSLNScFv-CD8-CD137-CD3 ζ endonuclease bamhi (1488bp)； M2：DNA molecular amount marks D2000.It will identify that correct plasmid send Beijing Tian Yihuiyuan bio tech ltd to insertion Fusion fragment is sequenced.The correct recombinant plasmid of sequencing result is named as PWPT-MSLNScFv-CD8-CD137- CD3 ζ, its structural representation is as shown in figure 4, including CD8a signal peptides (nucleotide sequence is as shown in SEQID NO.6), resist MSLN single-chain antibodies (nucleotide sequence is as shown in SEQID NO.7), CD8 hinge areas and transmembrane region and CD137 intracellular letter The intracellular signal domain of number domain and CD3 ζ, wherein, the Chimeric antigen receptor is with gene C D8 hinge areas and transmembrane region And the structure that CD137 and CD3 ζ intracellular signal domain is in series is signal transduction domain, its amino acid sequence is such as Shown in SEQID NO.9, corresponding gene coded sequence is as shown in SEQID NO.10.
The preparation of the NKT cells of the Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3 ζ of embodiment 3 modifications
(1) packaging of slow virus and concentration
Determine slow virus expression plasmid PWPT-MSLNScFv-CD8-CD137-CD3 ζ and auxiliary respectively with spectrophotometer Plasmid psPAX2, pMD2.G concentration, three kinds of plasmids are with 4:2:1 mass ratio LipofectamineTM 2000Transfection Reagent transfection reagent cotransfection 293T incasing cells.Disease is collected when transfecting 48h, 72h respectively Malicious supernatant is in 50mL EP pipes, 4 DEG C, 2000g centrifugation 10min, shifts the supernatant obtained twice into new EP pipes, with 4.5 μm of filters Device filter virus supernatant；The viral supernatants and 5 × PEG6000-NaCl of filtering are according to 4:1 volume ratio mixes, 4 DEG C of standing 2h, Then 4 DEG C, 10000g centrifugation 20min, abandon supernatant, the sterile PBS dissolvings of precipitation 1mL 4 DEG C of precoolings, produce chimeric antigen by The viral concentration liquid of body, dispensed by the often μ L of pipe 100, -80 DEG C save backup.
According to the method described above, slow virus expression plasmid PWPT-GFP and helper plasmid psPAX2, pMD2.G cotransfection are utilized 293T incasing cells, viral supernatants are collected, concentration, obtain the slow virus concentrate for expressing GFP green fluorescent proteins.
(2) amplification cultivation of slow-virus infection NKT cells and infected cell
Example 1 in 75cm21 × 10 cultivated in blake bottle7Individual NKT cells, old nutrient solution is discarded, add 2mL Viral concentration liquid, the 2 μ L 1 × 10 that fresh NKT cell culture mediums GT-T551,200 μ L steps (1) obtain-6Mg/mL milt eggs In vain, final concentration of 500U/mL rhIL-2,37 DEG C are placed in, the CO that saturated humidity is 5%2Infection 12 is small in incubator Shi Hou, abandon nutrient solution.NKT cells are synchronized with the slow virus concentrate of expression GFP green fluorescent proteins simultaneously and infect ( To NKT cells be referred to as CART-GFP cells), for calculating the viral efficiency of infection.By metainfective cell go to without The coated 75cm of CD3 and retronectin2In blake bottle, 20mL NKT cell culture medium GT-T551 are added, are added dense eventually Spend the rhIL-2 for 500U/mL, final concentration of 50ng/ml CD3 monoclonal antibodies and final concentration of 50ng/mL Recombinant human interleukin 15, in 37 DEG C, the CO that saturated humidity is 5%218h is cultivated in incubator, obtained NKT cells are referred to as CARMSLN-NKT cells.CARMSLN-T cells (the preparation method reference literature of T cell is prepared with identical method：Yajing Zhang,et al.Autologous CIK Cell Immunotherapy in Patients with Renal Cell Carcinoma after Radical Nephrectomy.Clinical Study, the preparation of 2.4 part CIK cells in 2013 Method).Viral efficiency of infection with Flow cytometry, as a result as shown in figure 5, the infection effect of CARMSLN-NKT cells Rate is 48.45%.
(3) external evoked amplification CARMSLN-NKT cell masses
By the final concentration of 500U/mL of the NKT cell rhIL-2s after above-mentioned culture NKT cell culture mediums GT-T551 progress is external evoked, and cell is transferred in cell culture bags when the density of cell is 85%, and restructuring was added every 2 days The final concentration of 50ng/ml of final concentration of 500U/mL, CD3 monoclonal antibody of human interleukin 2, the end of recombinant human interleukin 15 Concentration be 50ng/mL fresh NKT cell culture mediums GT-T551 carry out amplification cultivation, treat cell amplification to total amount be 1.5 × 109After individual cell or so, the cell colony of infection is identified using flow cytometer, cell phenotype commonly reaches CD3 sun Property cell proportion ＞ 90%；CD3CD8 double positive cells ratios>70%；CD3CD56 double positive cells ratios>15%, as a result see Fig. 6, CD3+：98.23%；CD3+CD4+：15.11%；CD3+CD8+：88.02%；CD3+CD56+：23.98%；CD8+CD56+： 26.55%.
Cytotoxicity analysis of the CARMSLN-NKT cells of embodiment 4 to mesothelioma cell lethal effect
Cultivated in the CARMSLN-NKT cells, CARMSLN-T cells and the embodiment 1 that are prepared respectively in Example 3 NKT cells are inoculated in 96 orifice plates, are dyed with CF succinimide ester (CFSE), the positive mesothelium with MSLN Oncocyte system NCI-H2452 (kills cell to imitate target ratio：Target cell) 5:1,10:1,20:Isosorbide-5-Nitrae 0:1 ratio is co-cultured, After the co-cultivation of 24 hours, cell is dyed with annexin V-RPE kits, while set control group not add respectively Enter the mesothelioma cell lines NCI-H2452 of immunocyte killing processing, and cell is dyed with annexin V-RPE kits. Flow cytometry detects to Apoptosis, and the amount of Apoptosis is calculated according to the following equation：Apoptosis rate=(control-sample Product)/control × 100%, compare not add the cell survival number of immunocyte killing processing；Sample is the corresponding effect of addition Target ratio (killing cell：Target cell) immunocyte killing processing cell survival number, see Fig. 7.As shown in Figure 7, inosculating antibody The mesothelioma cell that the NKT cells of original receptor MSLNScFv-CD8-CD137-CD3 ζ modifications are positive to MSLN has specific killing Activity, and the specific killing activity of CARMSLN-NKT cells is substantially better than CARMSLN-T cells and NKT cells.
The preferred embodiment of the present invention described in detail above, still, the present invention are not limited in above-mentioned embodiment Detail, in the range of the technology design of the present invention, a variety of simple variants can be carried out to technical scheme, this A little simple variants belong to protection scope of the present invention.
It is further to note that each particular technique feature described in above-mentioned embodiment, in not lance In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally The thought of invention, it should equally be considered as content disclosed in this invention.

Claims (10)

  1. A kind of 1. Chimeric antigen receptor, it is characterised in that the Chimeric antigen receptor is MSLNScFv-CD8-CD137-CD3 ζ, Hinge area and transmembrane region, CD137 the intracellular signal domain and CD3 ζ of CD8a signal peptides, MSLNScFv, CD8 including series connection Intracellular signal domain；Preferably, the Chimeric antigen receptor has the amino acid sequence as shown in SEQ ID NO.1, enters Preferably, the amino acid sequence of the Chimeric antigen receptor is as shown in SEQ ID NO.1 for one step.
  2. 2. encode the gene of the Chimeric antigen receptor described in claim 1, it is preferable that the gene has such as SEQ ID NO.2 Shown nucleotide sequence, it is further preferred that the nucleotide sequence of the gene is as shown in SEQ ID NO.2.
  3. 3. the recombinant expression carrier containing the gene described in claim 2, it is preferable that the recombinant expression carrier is slow virus table Up to carrier, it is further preferred that the Lentiviral is PWPT-MSLNScFv-CD8-CD137-CD3 ζ.
  4. 4. a kind of NKT cells of engineering MSLN targetings, it is characterised in that the NKT cells are as described in claim 1 The NKT cells of Chimeric antigen receptor modification.
  5. 5. a kind of preparation method of the NKT cells of engineering MSLN targetings, it is characterised in that methods described includes：
    Packaging carries PWPT-MSLNScFv-CD8-CD137-CD3 ζ slow virus, obtains viral concentration liquid；Utilize obtained disease Malicious concentrate infects NKT cells, makes NKT cells expression Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3 ζ.
  6. 6. according to the method for claim 5, wherein, methods described also includes being prepared via a method which NKT cells：
    (1) in the presence of CD3 monoclonal antibodies, proleulzin and interleukin-15, mononuclearcell is subjected to first stage training Support；Preferably, the embodiment of the first stage culture includes：Mononuclearcell is incubated at the first NKT cell culture fluids In, the first NKT cell culture fluids contain NKT cell culture mediums, CD3 monoclonal antibodies, proleulzin and interleukin-15； It is further preferred that in the first NKT cell culture fluids, the concentration of the CD3 monoclonal antibodies is 30-70ng/mL, and/or institute The concentration for stating proleulzin is 300-700U/mL, and/or the concentration of the interleukin-15 is 30-70ng/mL；
    (2) in the presence of proleulzin, the cell that the first stage is cultivated carries out second stage culture；Preferably, it is described The embodiment of second stage culture includes：By the cell culture that the first stage cultivates in the 2nd NKT cell culture fluids, Contain NKT cell culture mediums and proleulzin in the 2nd NKT cell culture fluids；It is further preferred that the 2nd NKT cells are trained In nutrient solution, the concentration of the proleulzin is 300-700U/mL.
  7. 7. according to the method for claim 6, wherein, the method for the infection NKT cells includes：
    (1) in the presence of viral concentration liquid, nucleoprotamine and proleulzin, NKT cells are subjected to first stage infection culture；It is excellent Selection of land, the embodiment of the first stage infection culture include：By NKT cell culture in the 3rd NKT cell culture fluids, institute State the 3rd NKT cell culture fluids and contain NKT cell culture mediums, viral concentration liquid, nucleoprotamine and proleulzin；Further preferably Ground, in the 3rd NKT cell culture fluids, the concentration of the proleulzin is 300-700U/mL；
    (2) in the presence of CD3 monoclonal antibodies, proleulzin and interleukin-15, infected to the cell of culture the first stage Carry out second stage infection culture；Preferably, the embodiment of the second stage infection culture includes：By the first stage The cell culture of culture is infected in the first NKT cell culture fluids.
  8. 8. according to the method for claim 7, wherein, the method for the infection NKT cells also includes：
    (3) it is first in the presence of proleulzin, the cell progress of second stage infection culture is external evoked, treat the close of cell Spend for 80-90% when, then in the presence of CD3 monoclonal antibodies, proleulzin and interleukin-15, cell is subjected to amplification cultivation； Preferably, the external evoked embodiment includes：By the cell culture of second stage infection culture in described second In NKT cell culture fluids, the embodiment of the amplification cultivation includes：By cell culture in the first NKT cell culture fluids In.
  9. 9. the NKT cells for the engineering MSLN targetings that any one methods described is prepared in claim 5-8.
  10. 10. the NKT cells of the engineering MSLN targetings described in claim 4 or 9 are being prepared for treating in the preparation of tumour Application, it is preferable that the tumour is the positive celiotheliomas of MSLN, it is further preferred that the tumour is the positive evils of MSLN Property mesothelioma of pleura.
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