CN107793483B - Chimeric antigen receptor and its gene and recombinant expression carrier, CARMSLN-NKT cell and its preparation method and application - Google Patents

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

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CN107793483B
CN107793483B CN201610807069.4A CN201610807069A CN107793483B CN 107793483 B CN107793483 B CN 107793483B CN 201610807069 A CN201610807069 A CN 201610807069A CN 107793483 B CN107793483 B CN 107793483B
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Abstract

The invention discloses a kind of Chimeric antigen receptor and its genes and recombinant expression carrier, CARMSLN-NKT cell and its preparation method and application, the Chimeric antigen receptor be MSLNScFv-CD8-CD137-CD3 ζ, including concatenated CD8a signal peptide, MSLNScFv, the CD8 hinge area of shortening and transmembrane region, the intracellular signal structural domain of CD137 and CD3 ζ intracellular signal structural domain.When co-culturing using the mesothelioma cell of CARMSLN-NKT cell and the MSLN positive of the invention, there is good specific killing activity to mesothelioma cell.

Description

Chimeric antigen receptor and its gene and recombinant expression carrier, CARMSLN-NKT cell and Preparation method and application
Technical field
The invention belongs to knubble biological arts, and in particular, to one of adoptive immunotherapy chimeric antigen by Body MSLNScFv-CD8-CD137-CD3 ζ and its gene and recombinant expression carrier, the NKT cell for being engineered MSLN targeting (CARMSLN-NKT cell) and its preparation method and application.
Background technique
Natural killer cells (NKT) is a kind of T lymphocyte subgroup of specific type, has T cell and NK cell Double property.NKT cell can express two kinds of receptors of NKR-P1 of TCR and the NK cell of T cell, in the case where TCR and NKR is mediated, NKT Cell can generate a large amount of IL-4 and INF γ, play killing functions of immunocytes to tumour cell.NKT cell passes through own face CD16 combined with the Fc of specific antibody section, play antibody dependent cell mediated cytotoxicity (ADCC, antibody-dependent cell-mediated cytotoxicity).But make in the cell-mediated killing of antibody-dependant With in the process, due to antibody can in conjunction with the corresponding antigens epitope specificity on target cell, NKT cell can kill it is any with it is anti- The target cell that body combines, therefore the antigen binding on antibody and target cell is specific, but killing of the NKT cell to target cell Effect is nonspecific.In addition, it is generally the case that the NKT cell of infusion is 2 weeks or so in patient's body half-life period, effectively Phase is of short duration, needs repeated multiple times infusion.Moreover, NKT cell itself lacks specific antibody, it is not enough to around tumour or tumor It is enriched in nest, constrains NKT cell to the targeted therapy of malignant tumour.Furthermore studies have shown that NKT cell is not to all Tumour have fragmentation effect, and weaker to the lethal effect of Partial tumors, specific killing activity is to be improved.
Mesothelin (MSLN) is the surface glycoprotein of a 40kDa, is expressed in kinds of tumor cells surface, such as pernicious mesothelium Tumor, Vipoma, oophoroma etc., wherein the expression rate of malignant pleural mesothelioma MSLN reaches 90% or more, and is in normal tissue The phenomenon that existing low expression.The study found that MSLN and progress, transfer and the lower survival rate of patient of tumour have very big correlation. Due to the above characteristic, MSLN antigen becomes the promising target treated based on antibody.Currently, numerous studies use MSLN monoclonal The tumour cell of the scheme target killing MSLN positive of antibody or MSLN vaccine, however, these schemes are generating certain effect On the basis of, it also suffers from certain drawbacks simultaneously, such as: the transience and MSLN monoclonal antibody of MSLN monoclonal antibody target effect In the deficiency etc. of tumor locus distribution.
Summary of the invention
The purpose of the invention is to overcome, the lethal effect of NKT cells against tumor in the prior art is weaker, specifically kills Wound activity is to be improved and existing clinical research in defect above-mentioned existing for MSLN monoclonal antibody and vaccine targeted therapy, A kind of Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3 ζ and its gene and recombinant expression carrier, engineering MSLN are provided NKT cell (CARMSLN-NKT cell) of targeting and its preparation method and application.
The present inventor has been surprisingly found that under study for action, using Chimeric antigen receptor MSLNScFv-CD8- of the invention When the NKT cell of CD137-CD3 ζ modification and the mesothelioma cell of the MSLN positive co-culture, have to mesothelioma cell good Specific killing activity.
Therefore, to achieve the goals above, described chimeric in a first aspect, the present invention provides a kind of Chimeric antigen receptor Antigen receptor be MSLNScFv-CD8-CD137-CD3 ζ, including concatenated CD8a signal peptide, MSLNScFv, shortening CD8 hinge The intracellular signal structural domain in sequence (area hinge) and transmembrane region, the intracellular signal structural domain of CD137 and CD3 ζ.
Second aspect, the present invention provides the genes for encoding above-mentioned Chimeric antigen receptor.
The third aspect, the present invention provides the recombinant expression carriers containing said gene.
Fourth aspect, the present invention provides a kind of NKT cells for being engineered MSLN targeting, and the NKT cell is above-mentioned The NKT cell of Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3 ζ modification.
5th aspect, the present invention provides a kind of preparation method of NKT cell for being engineered MSLN targeting, the methods Include: the slow virus that packaging carries PWPT-MSLNScFv-CD8-CD137-CD3 ζ, obtains viral concentration liquid；Utilize obtained disease Malicious concentrate infects NKT cell, and NKT cell is made to express Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3 ζ.
6th aspect, the present invention provides the NKT cells for the engineering MSLN targeting that the above method is prepared.
7th aspect, the present invention provides the NKT cell of above-mentioned engineering MSLN targeting in preparation for treating tumour Preparation in application.
When with the co-cultivation of the mesothelioma cell of the MSLN positive, Chimeric antigen receptor MSLNScFv-CD8- of the invention The NKT cell of the NKT cell of CD137-CD3 ζ modification, i.e. engineering MSLN targeting can specifically bind MSLN antigen, increase The ability of strong immunocyte targets identification cancer cell surfaces MSLN antigen, reinforces the specific killing to MSLN positive mesothelioma cell Activity.The NKT cell of engineering MSLN targeting of the invention provides a kind of new selection to treat the tumour of the MSLN positive, With good industrial application prospect.
Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.
Detailed description of the invention
Fig. 1 is result of the flow cytometry to the NKT cell phenotype analysis being separately cultured.
Fig. 2 is the restriction enzyme MluI/SalI of Lentiviral PWPT-CD8-CD137-CD3 ζ of the invention The electrophoretic identification of double digestion segment.
Fig. 3 is the restriction enzyme of Lentiviral PWPT-MSLNScFv-CD8-CD137-CD3 ζ of the invention The electrophoretic identification of BamHI/SalI double digestion segment.
Fig. 4 is the structural schematic diagram of Lentiviral PWPT-MSLNScFv-CD8-CD137-CD3 ζ of the invention, Wherein, sequence counterclockwise is positive gene piece degree, is clockwise cdna reverse segment.
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 cell.
Fig. 6 is the NKT cell of Flow cytometry Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3 ζ modification The result of (CARMSLN-NKT cell) phenotypic evaluation.
Fig. 7 is the cell toxicant of CARMSLN-NKT cell of the invention to the lethal effect of the mesothelioma cell of the MSLN positive Property analysis chart.
Specific embodiment
Detailed description of the preferred embodiments below.It should be understood that described herein specific Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The present invention provides a kind of Chimeric antigen receptor, the Chimeric antigen receptor is MSLNScFv-CD8-CD137-CD3 ζ, including concatenated CD8a signal peptide, MSLN single-chain antibody MSLNScFv, shorten CD8 hinge area and transmembrane region, CD137 born of the same parents The intracellular signal structural domain of interior signal domain and CD3 ζ.
Under preferable case, Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3 ζ by CD8a signal peptide, MSLNScFv, The intracellular signal structural domain of the hinge area and transmembrane region of CD8, the intracellular signal structural domain of CD137 and CD3 ζ is in series.Into 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 present invention provides the genes 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 present invention provides the recombinant expression carriers 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 modification is thin Born of the same parents, under preferable case, Lentiviral is PWPT-MSLNScFv-CD8-CD137-CD3 ζ.
The preparation method of Lentiviral PWPT-MSLNScFv-CD8-CD137-CD3 ζ is not limited particularly It is fixed, can be those skilled in the art it is conceivable that various methods, under preferable case, Lentiviral PWPT- The preparation method of MSLNScFv-CD8-CD137-CD3 ζ the following steps are included:
(1) area hinge of CD8 and the intracellular signal structural domain of transmembrane region, CD137 are expanded respectively from NKT cell cDNA It with the intracellular signal structural domain of CD3 ζ, and is cloned into carrier PWPT-GFP, building obtains PWPT-CD8-CD137-CD3 ζ；
(2) nucleotide sequence of composite coding CD8a signal peptide and MSLNScFv, and it is cloned into PWPT-CD8-CD137- In CD3 ζ, the correct PWPT-MSLNScFv-CD8-CD137-CD3 ζ of sequence is obtained after sequence verification.
In step (1), for expanded respectively from NKT cell cDNA CD8 the area hinge and transmembrane region, CD137 it is intracellular There is no particular limitation for the method for the intracellular signal structural domain of signal domain and CD3 ζ, can be various sides commonly used in the art Method, such as can be RT-PCR method.Wherein, then NKT cell can be carried out by the mononuclearcell in separation people's venous blood Culture obtains.
Specifically, the method for obtaining PWPT-CD8-CD137-CD3 ζ may include: to extract the total serum IgE of NKT cell, reverse Record obtains NKT cell cDNA and utilizes primer P1 (SEQID NO.11) and P2 (SEQID using obtained NKT cell cDNA as template NO.12 the area hinge and transmembrane region (SEQID NO.3) that PCR amplification obtains CD8 gene) are carried out；Utilize primer P3 (SEQID NO.13) and P4 (SEQID NO.14) carries out the intracellular signal structural domain (SEQID NO.4) that PCR amplification obtains CD137 gene； The intracellular signal structure that PCR amplification obtains CD3 ζ gene is carried out using primer P5 (SEQID NO.15) and P6 (SEQID NO.16) Domain (SEQID NO.5), carries out double digestion for the PCR product of acquisition respectively, then with the slow virus after MluI/SalI double digestion Expression vector PWPT-GFP connection.
It is not special for the method for the nucleotide sequence of composite coding CD8a signal peptide and MSLNScFv in step (2) Restriction, can be various methods commonly used in the art, such as can be synthesized by full genome synthetic technology.
Specifically, the method for obtaining the correct PWPT-MSLNScFv-CD8-CD137-CD3 ζ of sequence may include: to pass through The nucleotide sequence (SEQID NO.8) of full genome synthetic technology composite coding CD8a signal peptide and MSLNScFv fusion, gram It is grand into carrier pGSI, obtain pGSI-MSLNScFv；Then by pGSI-MSLNScFv carry out BamHI/MluI double digestion, with The recombinant plasmid PWPT-CD8-CD137-CD3 ζ connection that step (1) after BamHI/MluI double digestion obtains, through identification is sequenced, Obtain the correct PWPT-MSLNScFv-CD8-CD137-CD3 ζ of sequence.Wherein, the nucleotide sequence of CD8a signal peptide such as SEQID Shown in NO.6, MSLNScFv nucleotide sequence is as shown in SEQID NO.7.
The present invention also provides a kind of NKT cells for being engineered MSLN targeting, and the NKT cell is by above-mentioned inosculating antibody The NKT cell (i.e. CARMSLN-NKT cell) of original receptor MSLNScFv-CD8-CD137-CD3 ζ modification.
The present invention also provides a kind of preparation methods of NKT cell for being engineered MSLN targeting, this method comprises: packaging The slow virus for carrying PWPT-MSLNScFv-CD8-CD137-CD3 ζ, obtains viral concentration liquid；Utilize obtained viral concentration liquid NKT cell is infected, NKT cell is made to express Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3 ζ.
Carrying the method for slow virus of PWPT-MSLNScFv-CD8-CD137-CD3 ζ for packaging, there is no particular limitation, It can be the common various methods 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 are added 5 × PEG6000-NaCl in filtrate and are mixed, and supernatant is abandoned after centrifugation, and precipitating uses 0-4 DEG C pre-cooling sterile PBS dissolution, obtain viral concentration liquid.
Further include being prepared via a method which NKT cell in method of the invention:
(1) in the presence of CD3 monoclonal antibody, 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 first stage culture includes: that mononuclearcell is incubated at the first NKT is thin In born of the same parents' culture solution, the first NKT cell culture fluid contains NKT cell culture medium, CD3 monoclonal antibody, proleulzin and white Interleukin -15；It is further preferred that the concentration of the CD3 monoclonal antibody is 30-70ng/mL in the first NKT cell culture fluid, 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 training for cultivating the first stage It supports in the 2nd NKT cell culture fluid, NKT cell culture medium and proleulzin is contained in the 2nd NKT cell culture fluid；Into Preferably, in the 2nd NKT cell culture fluid, the concentration of the proleulzin is 300-700U/mL to one step.
For NKT cell culture medium, there is no particular limitation, can be commonly used in the art various for cultivating NKT cell Culture medium, such as can be GT-T551 culture medium.
When preparing NKT cell, 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%2It is trained in incubator It supports.Those skilled in the art can be adaptively adjusted the time of culture, this is known to those skilled in the art, herein It repeats no more.
In the NKT cell that the present invention is prepared, CD3+Cell average ratio > 90%, CD3+CD8+The total CD3 of cell Zhan+Carefully Average ratio > 70% of born of the same parents；CD3+CD56+The total CD3 of cell Zhan+Average ratio > 15% of cell.
Method for infecting NKT cell is not particularly limited, and can be various methods commonly used in the art, preferable case Under, this method comprises:
(1) in the presence of viral concentration liquid, nucleoprotamine and proleulzin, NKT cell is subjected to first stage infection training It supports；
(2) in the presence of CD3 monoclonal antibody, proleulzin and interleukin-15, by first stage infection 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 cell In culture solution, the 3rd NKT cell culture fluid contains NKT cell culture medium, viral concentration liquid, nucleoprotamine and interleukin- 2；It is further preferred that the concentration of the proleulzin is 300-700U/mL in the 3rd NKT cell culture fluid.
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 fluid.The concrete composition of first NKT cell culture fluid can be found in aforementioned corresponding interior Hold, details are not described herein.
It is not special for the condition of first stage infection culture and second stage infection culture when infecting NKT cell Restriction, can be various conditions commonly used in the art, such as can 30-37 DEG C, saturated humidity be 3-6% CO2Culture It is cultivated in case.Those skilled in the art can be adaptively adjusted the time of culture, this is those skilled in the art Known, details are not described herein.
Specifically, the method for infecting NKT cell includes: to take 1 × 107-5×107A NKT cell, discards old culture solution, The fresh GT-T551 culture solution of 2-4mL is added, adds 200-400 μ L viral concentration liquid, 2-4 μ L 1 × 10-6Mg/mL milt egg The IL-2 of white and final concentration of 300-700U/mL is placed in 30-37 DEG C, the CO that saturated humidity is 3-6%212- is infected in incubator After 16h, culture solution is abandoned, cell is gone in not coated culture bottle, the GT-T551 culture medium of 20-50mL is added, adds end Concentration is the IL-2 of 300-700U/mL, the CD3 monoclonal antibody of final concentration of 30-70ng/ml 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 cell of receptor MSLNScFv-CD8-CD137-CD3 ζ modification.
It is further preferred that the method for infection NKT cell further include:
(3) first in the presence of proleulzin, the cell progress of second stage infection culture is external evoked, to cell Density when being 80-90%, then in the presence of CD3 monoclonal antibody, proleulzin and interleukin-15, cell is expanded Culture.
Under preferable case, the external evoked embodiment includes: by the cell training of second stage infection culture It supports in the 2nd NKT cell culture fluid, the embodiment of the amplification cultivation includes: by cell culture in described first In NKT cell culture fluid.The concrete composition of first NKT cell culture fluid and the 2nd NKT cell culture fluid can be found in aforementioned corresponding Content, details are not described herein.
Specifically, the method for NKT cell is infected further include: by the slow-virus infection obtained after second stage infection culture NKT cell is carried out external evoked with the GT-T551 culture solution of the final concentration of 300-700U/mL of IL-2, and the density to cell is Cell is transferred in cell culture bags when 80-90%, final concentration of 300-700U/mL, CD3 of IL-2 were added every 1.5-2.5 days The fresh GT-T551 of the final concentration of 30-70ng/ml of monoclonal antibody, the final concentration of 30-70ng/mL of interleukin-15 Culture solution carries out amplification cultivation and cell is expanded to total amount to be 1 × 109-2×109A cell.By slow virus pair of the invention The Chimeric antigen receptor for targeting MSLN antigen carries out NKT cell infection, and efficiency of infection is up to 30%-60%, and obtain CARMSLN-NKT cell, CD3+CD56+The total CD3 of cell Zhan+The ratio of cell is 15% or more.
The Chimeric antigen receptor of the NKT cell expression of Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3 ζ modification 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 peptide, the area hinge of MSLNScFv, CD8 and transmembrane region, the intracellular signal structural domain and CD3 ζ of CD137 Intracellular signal structural domain it is in series, after protein translation in the cell rough endoplasmic reticulum excision signal peptide after become maturation Chimeric antigen receptor albumen after secretion output and is positioned on the cell membrane of NKT cell.The histone amino of the Chimeric antigen receptor The corresponding gene coded sequence of acid sequence is as shown in SEQID NO.2.The Chimeric antigen receptor with the area hinge of gene C D8 and across The structure that the intracellular signal structural domain of film area and CD137 and CD3 ζ are connected in series is signal transduction structural domain, amino acid sequence As shown in SEQID NO.9, corresponding gene coded sequence is as shown in SEQID NO.10.
The present invention also provides the NKT cells for the engineering MSLN targeting that the above method is prepared.
The present invention also provides the NKT cells of engineering MSLN targeting to prepare answering in the preparation for treating tumour With.Under preferable case, tumour is the celiothelioma of the MSLN positive, it is further preferred that the tumour is the pernicious chest of the MSLN positive Film celiothelioma.
In application of the invention, for the composition of preparation of the tumour for treating the MSLN positive, there is no particular limitation, As long as being prepared containing CARMSLN-NKT cell of the present invention or by CARMSLN-NKT cell, preparation it is specific Composition and preparation method are known to those skilled in the art, and details are not described herein.
Embodiment
The present invention is further illustrated for embodiment below, but is not intended to limit the present invention.
Experimental method in following embodiment is unless otherwise specified conventional method in that art.Institute in following embodiments Experimental material is unless otherwise specified to be commercially available from routine biochemistry reagent shop, in which:
NKT cell culture medium GT-T551 is purchased from TaKaRa company.
Lymphocyte separation medium is purchased from TBD company.
CD3 monoclonal antibody, recombinant fiber connection albumen (retronectin) are purchased from TaKaRa company.
Recombinant human protein's interferon-γ, rhIL-2, recombinant human interleukin 15 are purchased from protech company.
Total RNA extraction reagent box RNAiso Reagent, high-fidelity DNA polymerase (HS DNA Polymerase), T4DNA ligase is purchased from TaKaRa company.
RevertAidTMFirst Strand cDNA Synthesis Kit is purchased from Fermentas company.
Bgl II, EcoRI, MluI, BamHI, SalI are purchased from Fermentas company.
Ago-Gel DNA QIAquick Gel Extraction Kit, 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 company.
PGSI is purchased from Beijing Tian Yihuiyuan Biotechnology Co., Ltd.
Trans1-T1Phage Resistant Competent cell is purchased from Beijing Quanshijin Biotechnology Co., Ltd.
LipofectamineTM2000Transfection Reagent transfection reagent is purchased from Invitrogen company.
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 Biotechnology Co., Ltd.
Fetal calf serum is purchased from PAA company, Germany.
The mesothelioma cell lines NCI-H2452 of the MSLN positive is purchased from U.S. ATCC company.
5-carboxyfluorescein succinimide ester is purchased from Shanghai Pu Zhen Biotechnology Co., Ltd.
Annexin V-RPE kit is purchased from U.S. company BD.
All primers are synthesized by Beijing Tian Yihuiyuan Biotechnology Co., Ltd.
The preparation of 1 NKT cell of embodiment
(1) take people's venous blood 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 medium GT-T551 of the Human autologous serum containing 0.6 volume % Adjusting final concentration of cells is 2 × 106A cell/mL；By cell inoculation in first passing through final concentration of 10 μ g/mL's in advance The coated 75cm of retronectin2In Tissue Culture Flask.Then the recombined human of final concentration of 500U/mL is added in culture medium The recombination human interleukin -15 of interleukin-22,50ng/ml CD3 monoclonal antibody and 50ng/mL, in 37 DEG C, saturated humidity 5% CO2It is cultivated in incubator.
(3) it cultivates the 4th day, cell is transferred in not coated culture bottle, be added according to cell growth population within every 2 days NKT cell culture medium GT-T551, control cell concentration are 1 × 108A cell/mL, and the weight of final concentration of 500U/ml is added Group human interleukin 2；Culture obtained NKT cell, flow cytometry analyzes NKT cell phenotype to the 12nd day.As a result see figure 1, wherein CD3+: 95.04%；CD3+CD8+: 90.99%；CD3+CD56+: 24.12%；CD8+CD56+: 24.63%.
The building of 2 Lentiviral PWPT-MSLNScFv-CD8-CD137-CD3 ζ of embodiment
(1) preparation of NKT cell cDNA
Centrifugation embodiment 1 cultivates obtained NKT cell, 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 transcription obtains NKT cell cDNA, and -20 DEG C save backup.
(2) preparation of slow virus plasmid PWPT-CD8-CD137-CD3 ζ
Design and synthesize following primer sequence (wherein, for underscore labeled as protection base, box is restriction enzyme site):
Using NKT cell cDNA in step (1) as template, PCR amplification is carried out with primer P1 and P2, obtains the CD8 of long 227bp The area hinge and shortening transmembrane region, for nucleotide sequence as shown in SEQID NO.3, II enzyme of MluI and Bgl is contained at both ends respectively Enzyme site and protection base；PCR amplification is carried out with primer P3 and P4, obtains the CD137 intracellular signal structural domain of long 146bp, core For nucleotide sequence as shown in SEQID NO.4, Bgl II and EcoRI restriction enzyme site and protection base are contained in both ends respectively；With primer P5 With P6 carry out PCR amplification, obtain the intracellular signal structural domain of the CD3 ζ of long 359bp, nucleotide sequence as shown in SEQID NO.5, Contain EcoRI and SalI restriction enzyme site and protection base respectively in both ends.Each step pcr amplification reaction system is identical, to expand CD137 For intracellular signal structural domain, PCR amplification, PCR reaction condition reference are carried outHS DNA Polymerase Specification, reaction system (50 μ L) is as follows:
Distilled water: 32.5 μ L
5 × reaction buffer:10 μ L
DNTP mixture (every kind of 2.5mM): 4 μ L
P3(10mM):1μL
P4(10mM):1μL
NKT cell cDNA (200ng/ul): 1 μ L
HS DNA Polymerase:0.5 μ L
Above-mentioned PCR product is separated with 1% Ago-Gel, is carried out with Ago-Gel DNA QIAquick Gel Extraction Kit DNA fragmentation recycling.Double enzyme digestion reaction is carried out respectively after obtaining segment, and digestion products are recycled with common DNA product purification kit It is spare.
Lentiviral PWPT-GFP MluI/SalI double digestion, digestion products are carried out through 1% Ago-Gel Separation, recycles big carrier segments with Ago-Gel DNA QIAquick Gel Extraction Kit, then with the CD8, the CD137, CD3 ζ that recycle before Segment is connected by T4DNA ligase, connection product conversion Trans1-T1Phage Resistant Competent cell, and 37 Picking monoclonal after DEG C culture 16h, 37 DEG C, 250rpm is cultivated and is extracted plasmid with the small extraction reagent kit of plasmid after 12h.The plasmid of extraction It is identified through restriction enzyme MluI and SalI double digestion, identification electrophoretogram is shown in Fig. 2, wherein M1:DNA molecular weight marker D15000；1 swimming lane: the non-endonuclease bamhi of plasmid PWPT-CD8-CD137-CD3 ζ；2 swimming lanes: plasmid PWPT-CD8-CD137-CD3 The endonuclease bamhi (672bp) of ζ；M2:DNA molecular weight marker D2000.It will identify that correct plasmid send Beijing day brightness far biological section The fusion segment of insertion is sequenced in skill Co., Ltd.The correct recombinant plasmid of sequencing result is named as PWPT- CD8-CD137-CD3 ζ, wherein the area hinge of CD8 and the nucleotide sequence of transmembrane region as shown in SEQID NO.3, CD137's The nucleotide sequence of intracellular signal structural domain is as shown in SEQID NO.4, and the nucleotide sequence of the intracellular signal structural domain of CD3 ζ is such as Shown in SEQID NO.5.
(3) preparation of slow virus plasmid PWPT-MSLNScFv-CD8-CD137-CD3 ζ
The nucleotide sequence of full genome composite coding CD8a signal peptide and MSLNScFv fusion, sequence such as SEQID Shown in NO.8, by Beijing, Tian Yihuiyuan Biotechnology Co., Ltd is synthesized, and BamHI, kozak sequence are contained in 5 ' ends, and 3 ' ends contain There is MluI restriction enzyme site, by foregoing fusion gene cloning in plasmid pGSI, is named as pGSI-MSLNScFv.Plasmid warp BamHI/MluI double digestion, digestion products are separated through 1% Ago-Gel, are returned with Ago-Gel DNA QIAquick Gel Extraction Kit It is spare to receive target fragment.
PWPT-CD8-CD137-CD3 ζ plasmid is through restriction enzyme BamHI/MluI digestion, and digestion products are through 1% agar Sugared gel is separated, spare with Ago-Gel DNA QIAquick Gel Extraction Kit recycling carrier segments.Then contain CD8a with recycling The DNA fragmentation of signal peptide and MSLN ScFv are attached by T4DNA ligase, and specific method is shown in specification.By connection product Trans1-T1Phage Resistant Competent cell is converted, picking monoclonal after 37 DEG C of culture 16h, 37 DEG C, After 250rpm cultivates 12h, plasmid is extracted with the small extraction reagent kit of plasmid.The plasmid of extraction is bis- through restriction enzyme BamHI/SalI Digestion identification, qualification result are as shown in Figure 3, wherein 1 swimming lane: plasmid PWPT-MSLNScFv-CD8-CD137-CD3 ζ is not Endonuclease bamhi (10117bp)；2 swimming lanes: the endonuclease bamhi (1488bp) of plasmid PWPT-MSLNScFv-CD8-CD137-CD3 ζ； M2:DNA molecular weight marker D2000.It will identify that correct plasmid send Beijing Tian Yihuiyuan Biotechnology Co., Ltd to insertion Fusion segment is sequenced.The correct recombinant plasmid of sequencing result is named as PWPT-MSLNScFv-CD8-CD137- CD3 ζ, structural schematic diagram is as shown in figure 4, including CD8a signal peptide (nucleotide sequence is as shown in SEQID NO.6), resist MSLN single-chain antibody (nucleotide sequence is as shown in SEQID NO.7), the area hinge of CD8 and transmembrane region and CD137 letter intracellular The intracellular signal structural domain of number structural domain and CD3 ζ, wherein the Chimeric antigen receptor is with the area hinge of gene C D8 and transmembrane region And the structure that the intracellular signal structural domain of CD137 and CD3 ζ is connected in series is signal transduction structural domain, 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 cell of 3 Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3 ζ of embodiment modification
(1) packaging and concentration of slow virus
Measure slow virus expression plasmid PWPT-MSLNScFv-CD8-CD137-CD3 ζ and auxiliary respectively with spectrophotometer The concentration of plasmid psPAX2, pMD2.G, three kinds of plasmids are with the mass ratio Lipofectamine of 4:2:1TM 2000Transfection Reagent transfection reagent cotransfection 293T incasing cells.Disease is collected when transfecting 48h, 72h respectively Malicious supernatant is in 50mL EP pipe, and 4 DEG C, 2000g is centrifuged 10min, shifts the supernatant obtained twice into new EP pipe, is filtered with 4.5 μm Device filter virus supernatant；The viral supernatants and 5 × PEG6000-NaCl of filtering are mixed according to the volume ratio of 4:1,4 DEG C of standing 2h, Then 4 DEG C, 10000g is centrifuged 20min, abandons supernatant, the sterile PBS dissolutions of 4 DEG C of 1mL pre-coolings of precipitating to get chimeric antigen by The viral concentration liquid of body is dispensed by every 100 μ L of pipe, and -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, collects viral supernatants, and concentration obtains the slow virus concentrate of expression GFP green fluorescent protein.
(2) amplification cultivation of slow-virus infection NKT cell and infected cell
Example 1 in 75cm21 × 10 cultivated in culture bottle7A NKT cell discards old culture solution, and 2mL is added Viral concentration liquid, the 2 μ L 1 × 10 that fresh NKT cell culture medium GT-T551,200 μ L steps (1) obtain-6Mg/mL milt egg White, the rhIL-2 of final concentration of 500U/mL is placed in 37 DEG C, the CO that saturated humidity is 5%2Infection 12 is small in incubator Shi Hou abandons culture solution.NKT cell is synchronized with the slow virus concentrate of expression GFP green fluorescent protein simultaneously and infects ( To NKT cell be known as CART-GFP cell), for calculating the efficiency of infection of the virus.By metainfective cell go to without The coated 75cm of CD3 and retronectin2In culture bottle, the NKT cell culture medium GT-T551 of 20mL is added, adds dense eventually Degree is the rhIL-2 of 500U/mL, the CD3 monoclonal antibody of final concentration of 50ng/ml 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 cell is known as CARMSLN-NKT cell.CARMSLN-T cell (the preparation method reference literature of T cell: Yajing is prepared with identical method 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).With the efficiency of infection of the Flow cytometry virus, as a result as shown in figure 5, the infection of CARMSLN-NKT cell is imitated Rate is 48.45%.
(3) external evoked amplification CARMSLN-NKT cell mass
By the NKT cell culture medium of the final concentration of 500U/mL of the NKT cell rhIL-2 after above-mentioned culture GT-T551 progress is external evoked, and cell is transferred in cell culture bags when the density of cell is 85%, is recombinated every addition in 2 days The end of the final concentration of 50ng/ml of final concentration of 500U/mL, CD3 monoclonal antibody of human interleukin 2, recombinant human interleukin 15 Concentration be 50ng/mL fresh NKT cell culture medium GT-T551 carry out amplification cultivation, to cell amplification to total amount be 1.5 × 109It after a cell or so, is identified using cell colony of the flow cytometer to infection, cell phenotype commonly reaches CD3 sun Property cell proportion > 90%；CD3CD8 double positive cells ratio > 70%；CD3CD56 double positive cells ratio > 15%, is as a result shown in 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 4 CARMSLN-NKT cell of embodiment to mesothelioma cell lethal effect
It is cultivated in CARMSLN-NKT cell, CARMSLN-T cell and the embodiment 1 prepared in Example 3 respectively NKT cell inoculation is dyed in 96 orifice plates with 5-carboxyfluorescein succinimide ester (CFSE), the mesothelium with the MSLN positive To imitate target ratio (killing cell: target cell) 5:1, the ratio of 10:1,20:1,40:1 are co-cultured oncocyte system NCI-H2452, After co-cultivation in 24 hours, cell annexin V-RPE kit is dyed, while control group is set and is not added respectively Enter the mesothelioma cell lines NCI-H2452 of immunocyte killing processing, and cell annexin V-RPE kit is dyed. Flow cytometry detects Apoptosis, and the amount of Apoptosis is calculated according to the following equation: apoptosis rate=(control-sample Product)/control × 100%, compare the cell survival number not add immunocyte killing processing；Sample is that corresponding effect is added The cell survival number of the immunocyte killing processing of target ratio (killing cell: target cell), is shown in Fig. 7.As shown in Figure 7, inosculating antibody The NKT cell of original receptor MSLNScFv-CD8-CD137-CD3 ζ modification has specific killing to the mesothelioma cell of the MSLN positive Activity, and the specific killing activity of CARMSLN-NKT cell is substantially better than CARMSLN-T cell and NKT cell.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can No further explanation will be given for the combination of energy.
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 also be regarded as the disclosure of the present invention.

Claims (6)

1. a kind of preparation method for the NKT cell for being engineered MSLN targeting, which is characterized in that the described method includes:
Packaging carries the slow virus of PWPT-MSLNScFv-CD8-CD137-CD3 ζ, obtains viral concentration liquid；Utilize obtained disease Malicious concentrate infects NKT cell, and NKT cell is made to express Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3 ζ；
The amino acid sequence of the Chimeric antigen receptor is as shown in SEQ ID NO.1；
It is described infection NKT cell method include:
It takes in 75cm21 × 10 cultivated in culture bottle7A NKT cell discards old culture solution, and the fresh NKT cell training of 2mL is added Support base GT-T551,200 μ L viral concentration liquid, 2 L1 × 10 μ-6The recombined human of mg/mL nucleoprotamine, final concentration of 500U/mL is white Interleukin 2 is placed in 37 DEG C, the CO that saturated humidity is 5%2After infecting 12 hours in incubator, culture solution is abandoned；By metainfective cell It goes to and connects the coated 75cm of albumen with recombinant fiber without CD32In culture bottle, the NKT cell culture medium GT- of 20mL is added T551 adds the rhIL-2 of final concentration of 500U/mL, the CD3 monoclonal antibody of final concentration of 50ng/ml and end Concentration is the recombinant human interleukin 15 of 50ng/mL, in 37 DEG C, the CO that saturated humidity is 5%218h is cultivated in incubator；
By the NKT cell culture medium GT- of the final concentration of 500U/mL of the NKT cell rhIL-2 after above-mentioned culture T551 progress is external evoked, and cell is transferred in cell culture bags when the density of cell is 85%, white every 2 days addition recombined humans The final concentration of the final concentration of 50ng/ml of final concentration of 500U/mL, CD3 monoclonal antibody of interleukin 2, recombinant human interleukin 15 Amplification cultivation is carried out for the fresh NKT cell culture medium GT-T551 of 50ng/mL.
2. according to the method described in claim 1, wherein, the method also includes being prepared via a method which NKT cell:
(1) in the presence of CD3 monoclonal antibody, proleulzin and interleukin-15, mononuclearcell is subjected to first stage training It supports；The embodiment of the first stage culture includes: that mononuclearcell is incubated in the first NKT cell culture fluid, described First NKT cell culture fluid contains NKT cell culture medium, CD3 monoclonal antibody, proleulzin and interleukin-15；First NKT In cell culture fluid, the concentration of the CD3 monoclonal antibody is 30-70ng/mL and/or the concentration of the proleulzin is The concentration of 300-700U/mL and/or 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；The second stage The embodiment of culture includes: the cell culture of cultivating the first stage in the 2nd NKT cell culture fluid, and described second Contain NKT cell culture medium and proleulzin in NKT cell culture fluid；In 2nd NKT cell culture fluid, the proleulzin Concentration is 300-700U/mL.
3. the NKT cell for the engineering MSLN targeting that method as claimed in claim 1 or 2 is prepared.
4. the NKT cell of engineering MSLN targeting as claimed in claim 3 is preparing answering in the preparation for treating tumour With.
5. application according to claim 4, wherein the tumour is the celiothelioma of the MSLN positive.
6. application according to claim 5, wherein the tumour is the malignant pleural mesothelioma of the MSLN positive.
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