CN105920592B - Application of the CARHER1-NKT cell in the preparation that preparation is used for treatment of advanced HER1 positive lung cancer - Google Patents
Application of the CARHER1-NKT cell in the preparation that preparation is used for treatment of advanced HER1 positive lung cancer Download PDFInfo
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Abstract
The invention discloses application of the CARHER1-NKT cell in the preparation that preparation is used for treatment of advanced HER1 positive lung cancer, wherein, CARHER1-NKT cell is the NKT cell modified by Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ, and Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ includes the intracellular signal structural domain of concatenated rat growth hormone signal peptide, the hinge area of HER1ScFv, CD8 and transmembrane region, the intracellular signal structural domain of CD137 and CD3 ζ.When using CARHER1-NKT cell therapy progressive stage HER1 positive lung cancer of the invention, caused drug resistance when can effectively avoid using the existing drug for targeting HER1, there is certain specific killing activity to lung carcinoma cell, and have certain therapeutic effect to progressive stage HER1 positive lung cancer patient.
Description
Technical field
The invention belongs to knubble biological arts, and in particular, to CARHER1-NKT cell exists in adoptive immunotherapy
Preparation is for the application in the preparation for the treatment of of advanced HER1 positive lung cancer.
Background technique
EGFR (epithelial growth factor receptor, EGF-R ELISA) i.e. HER1, is former cancer
The expression product of gene c-erbB1 belongs to human epidermal growth factor acceptor (HER) family, itself has tyrosinase kinases
Activity, once correlation gene can be had in active cell core by combining with epidermal growth factor (EGF), so that cell division be promoted to increase
It grows.HER1 high expression, research in Several Kinds of Malignancy find that HER1 is expressed in most of patients with lung cancer, and expression reaches
80%, expression is related with the progress of lung cancer disease and transfer.Although the treatment of nearest lung cancer obtained it is certain into
Exhibition, but the survival rate of patient is not still improved, therefore needs to inquire into new therapy to overcome this puzzlement.
Currently, (such as HER1 tyrosine kinase inhibits the drug of anti-HER1 in treatment of advanced HER1 positive lung cancer patient
Agent (tyrosine kinase inhibitors, TKIs)) it has been in clinical investigation phase, still, clinical effectiveness shows only
Part patients with lung cancer is effective using HER1 treatment with tyrosine kinase inhibitors, and patient eventually generates certain drug resistance,
To influence the curative effect of drug.
Summary of the invention
The purpose of the invention is to overcome the Progress in Medication phase HER1 positive lung for using anti-HER1 in the prior art
The defect of caused drug resistance when cancer patient provides CARHER1-NKT cell in preparation for treatment of advanced HER1 positive lung
Application in the preparation of cancer can be effective when using CARHER1-NKT cell therapy progressive stage HER1 positive lung cancer of the invention
Caused drug resistance when avoiding using existing anti-HER1 drug has certain special target killing activity to lung carcinoma cell, and
Progressive stage HER1 sun to repeatedly treatment (such as radiotherapy, chemotherapy and other drugs symptomatic treatment) is had been subjected to but without obvious curative effects
Property patients with lung cancer has certain therapeutic effect.
Therefore, to achieve the goals above, the present invention provides CARHER1-NKT cells is used for treatment of advanced in preparation
Application in the preparation of HER1 positive lung cancer, the CARHER1-NKT cell are by Chimeric antigen receptor HER1ScFv-CD8-
The NKT cell of CD137-CD3 ζ modification, wherein the Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ includes series connection
Rat growth hormone signal peptide, HER1ScFv, CD8 hinge area (area hinge) and transmembrane region, CD137 intracellular signal knot
The intracellular signal structural domain in structure domain and CD3 ζ.
In treatment of advanced HER1 positive lung cancer, CARHER1-NKT cell of the invention can specifically bind HER1
Antigen, hence it is evident that extend immunocyte in the time-to-live of patient's body, enhance immunocyte targets identification lung carcinoma cell surface
The ability of HER1 antigen is reinforced the specific killing activity to lung carcinoma cell, and can actually effectively be avoided using existing anti-
Caused drug resistance when the drug therapy of HER1 repeatedly treats (such as radiotherapy, chemotherapy and other drugs symptomatic treatment to having been subjected to
Deng) but the progressive stage HER1 positive lung cancer patient without obvious curative effects has certain therapeutic effect.Engineering HER1 of the invention
The NKT cell (i.e. CARHER1-NKT cell) of targeting provides a kind of new selection for treatment of advanced HER1 positive lung cancer,
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/ of Lentiviral pWPXL-CD8-CD137-CD3 ζ of the invention
The electrophoretic identification of NdeI double digestion segment.
Fig. 3 is the restriction enzyme of Lentiviral pWPXL-HER1ScFv-CD8-CD137-CD3 ζ of the invention
The electrophoretic identification of enzyme MluI/NdeI double digestion segment.
Fig. 4 is the structural schematic diagram of Lentiviral pWPXL-HER1ScFv-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 HER1ScFv-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 HER1ScFv-CD8-CD137-CD3 ζ modification
The result of (CARHER1-NKT cell) phenotypic evaluation.
Fig. 7 is the cytotoxicity analysis figure of CARHER1-NKT cell of the invention to human lung carcinoma cell lethal effect.
Fig. 8 is the therapeutic process of CARHER1-NKT cell of the invention to the patients with lung cancer of 11 progressive stage HER1 positives
In, CARHER1-NKT cell continues that there are time plots in peripheral blood in patients.
Fig. 9 is the therapeutic process of CARHER1-NKT cell of the invention to the patients with lung cancer of two progressive stage HER1 positives
In, the tuberculosis stove and pleura extract variation diagram of different time sections patient.
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 CARHER1-NKT cells in the preparation that preparation is used for treatment of advanced HER1 positive lung cancer
Using, the CARHER1-NKT cell is the NKT cell modified by Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ,
Wherein, the Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ include concatenated rat growth hormone signal peptide,
The intracellular signal structural domain of the hinge area and transmembrane region of HER1ScFv, CD8, the intracellular signal structural domain of CD137 and CD3 ζ.
In application of the invention, under preferable case, the Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ is by big
The born of the same parents of rat growth hormone signal peptide, the hinge area of HER1ScFv, CD8 and transmembrane region, the intracellular signal structural domain of CD137 and CD3 ζ
Interior signal domain is in series.It is further preferred that Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ has such as
Amino acid sequence shown in SEQ ID NO.1, it is further preferred that Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3
The amino acid sequence of ζ is as shown in SEQ ID NO.1.
In application of the invention, under preferable case, the Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ is encoded
Gene have the nucleotide sequence as shown in SEQ ID NO.2, it is further preferred that encoding chimeric antigen receptor
The nucleotide sequence of the gene of HER1ScFv-CD8-CD137-CD3 ζ is as shown in SEQID NO.2.
In application of the invention, under preferable case, the preparation method of CARHER1-NKT cell includes: that packaging carries
The slow virus of pWPXL-HER1ScFv-CD8-CD137-CD3 ζ obtains viral concentration liquid；It is contaminated using obtained viral concentration liquid inductance
NKT cell makes NKT cell express Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ, wherein the pWPXL-
HER1ScFv-CD8-CD137-CD3 ζ is the gene containing encoding chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ
Lentiviral.
In application of the invention, Lentiviral pWPXL-HER1ScFv-CD8-CD137-CD3 ζ can be with auxiliary
Carrier cotransfection incasing cells such as 293T incasing cells obtains viral concentration liquid and CARHER1-NKT cell.
The preparation method of Lentiviral pWPXL-HER1ScFv-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 pWPXL-
The preparation method of HER1ScFv-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 pWPXL-GFP, building obtains pWPXL-CD8-CD137-CD3 ζ；
(2) nucleotide sequence of composite coding rat growth hormone signal peptide and HER1ScFv, and it is cloned into pWPXL-
In CD8-CD137-CD3 ζ, the correct pWPXL-HER1ScFv-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 pWPXL-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/NdeI double digestion
Expression vector pWPXL-GFP connection.
In step (2), for the method for the nucleotide sequence of composite coding rat growth hormone signal peptide and HER1ScFv
There is no particular limitation, 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 pWPXL-HER1ScFv-CD8-CD137-CD3 ζ of sequence may include: to pass through
Nucleotide sequence (the SEQID of full genome synthetic technology composite coding rat growth hormone signal peptide and HER1ScFv fusion
NO.8), it is cloned into carrier pGSI, obtains pGSI-HER1ScFv；Then pGSI-HER1ScFv is subjected to MluI single endonuclease digestion, with
The recombinant plasmid pWPXL-CD8-CD137-CD3 ζ connection obtained through restriction enzyme MluI single endonuclease digestion is identified through sequencing, is obtained
To the correct pWPXL-HER1ScFv-CD8-CD137-CD3 ζ of sequence.Wherein, the nucleotide sequence of rat growth hormone signal peptide
As shown in SEQID NO.6, HER1ScFv nucleotide sequence is as shown in SEQID NO.7.
The method for carrying the slow virus of pWPXL-HER1ScFv-CD8-CD137-CD3 ζ for packaging does not limit particularly
It is fixed, it can be the common various methods of those skilled in the art, under preferable case, by Lentiviral pWPXL-
HER1ScFv-CD8-CD137-CD3 ζ and helper plasmid (such as psPAX2, pMD2.G) cotransfection 293T incasing cells transfect 48-
Viral supernatants are collected when 72h, centrifugation, filtering are added 5 × PEG6000-NaCl in filtrate and are mixed, supernatant is abandoned after centrifugation,
The sterile PBS dissolution of 0-4 DEG C of pre-cooling of precipitating, obtains viral concentration liquid.
In application of the invention, the preparation method of CARHER1-NKT cell further includes being prepared via a method which that NKT is thin
Born of the same parents:
(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.
In application of the invention, the method for infecting NKT cell is not particularly limited, and can be commonly used in the art each
Kind of method, under preferable case, 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 HER1ScFv-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 HER1 antigen carries out NKT cell infection, and efficiency of infection is up to 30%-60%, and obtain
CARHER1-NKT cell, CD3+CD56+The total CD3 of cell Zhan+The ratio of cell is within the scope of 15%-40%.
The Chimeric antigen receptor of the NKT cell expression of Chimeric antigen receptor HER1ScFv-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 rat growth hormone signal peptide, the area hinge of HER1ScFv, CD8 and transmembrane region, the intracellular signal structure of CD137
The intracellular signal structural domain of domain and CD3 ζ are in series, after protein translation after the signal peptide of rough endoplasmic reticulum excision in the cell
As mature Chimeric antigen receptor albumen, after secretion output and it is positioned on the cell membrane of NKT cell.The Chimeric antigen receptor
The corresponding gene coded sequence of protein amino acid sequence is as shown in SEQID NO.2.The Chimeric antigen receptor is with gene C D8's
The structure that the intracellular signal structural domain of the area hinge and transmembrane region and CD137 and CD3 ζ are connected in series is signal transduction structural domain,
Amino acid sequence is as shown in SEQID NO.9, and corresponding gene coded sequence is as shown in SEQID NO.10.
In application of the invention, it will be understood by those skilled in the art that progressive stage HER1 positive lung cancer refers to part
Advanced stage can not carry out operation excision, metastatic or the lung cancer for recurring the sexual stage, and Symptoms are cough, expectoration, uncomfortable in chest, shortness of breath.
Progressive stage HER1 positive lung cancer of the invention in particular recurs refractory progressive stage HER1 positive lung cancer.
In application of the invention, the composition of the preparation for treatment of advanced HER1 positive lung cancer is not limited particularly
It is fixed, as long as being prepared containing CARHER1-NKT cell of the present invention or by CARHER1-NKT cell, preparation
Concrete 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, NdeI, EcoR V are purchased from Fermentas company.
Modification enzyme Klenow Fragment is 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.
PWPXL-GFP, psPAX2, pMD2.G are purchased from Addgene company.
PGSI is purchased from Beijing Tian Yihuiyuan Biotechnology Co., Ltd.
Trans1-T1 Phage Resistant Competent cell is purchased from the limited public affairs of Beijing Quan Shijin biotechnology
Department.
LipofectamineTM2000 Transfection Reagent transfection reagents are 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 typeⅡ pneumocyte of height expression HER1 is purchased from U.S. ATCC company.
Cell Counting Kit-8 (CCK8) kit is purchased from Beijing Wo Bisen Science and Technology Ltd..
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 pWPXL-HER1ScFv-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 pWPXL-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 287bp
The area hinge and transmembrane region, for nucleotide sequence as shown in SEQID NO.3, II restriction enzyme site of MluI and Bgl is contained at both ends respectively
With protection base；PCR amplification is carried out with primer P3 and P4, obtains the CD137 intracellular signal structural domain of long 146bp, nucleotides sequence
For column 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 and P6 into
Row PCR amplification obtains the intracellular signal structural domain of the CD3 ζ of long 359bp, and nucleotide sequence is as shown in SEQID NO.5, both ends point
It Han You not EcoRI and NdeI restriction enzyme site and protection base.Each step pcr amplification reaction system is identical, to expand CD137 letter intracellular
For number structural domain, PCR amplification, PCR reaction condition reference are carried outThe explanation of HS DNA Polymerase
Book, reaction system (50 μ L) are 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 pWPXL-GFP MluI/NdeI double digestion, digestion products through 1% Ago-Gel into
Row separation, recycle big carrier segments with Ago-Gel DNA QIAquick Gel Extraction Kit, then with the CD8, CD137 recycled before,
CD3 ζ segment is connected by T4DNA ligase, and it is thin that connection product converts Trans1-T1 Phage Resistant Competent
Born of the same parents, picking monoclonal after 37 DEG C of culture 16h, 37 DEG C, 250rpm is cultivated and is extracted plasmid with the small extraction reagent kit of plasmid after 12h.It extracts
Plasmid through restriction enzyme MluI and NdeI double digestion identify, identification electrophoretogram see Fig. 2, wherein M1:DNA molecular weight mark
Remember D15000；1 swimming lane: the non-endonuclease bamhi of plasmid pWPXL-CD8-CD137-CD3 ζ；2 swimming lanes: plasmid pWPXL-CD8-
The endonuclease bamhi (751bp) of CD137-CD3 ζ；M2:DNA molecular weight marker D2000.It will identify that correct plasmid send Beijing day one
The fusion segment of insertion is sequenced in Hui Yuan Biotechnology Co., Ltd.By the correct recombinant plasmid name of sequencing result
For pWPXL-CD8-CD137-CD3 ζ, wherein the area hinge of CD8 and the nucleotide sequence of transmembrane region as shown in SEQID NO.3,
The nucleotide sequence of the intracellular signal structural domain of CD137 is as shown in SEQID NO.4, the nucleosides of the intracellular signal structural domain of CD3 ζ
Acid sequence is as shown in SEQID NO.5.
(3) preparation of slow virus plasmid pWPXL-HER1ScFv-CD8-CD137-CD3 ζ
The nucleotide sequence of full genome composite coding rat growth hormone signal peptide and HER1ScFv fusion, sequence is such as
Shown in SEQID NO.8, by Beijing, Tian Yihuiyuan Biotechnology Co., Ltd is synthesized, 5 ' end containing MluI restriction enzyme site,
Kozak sequence, 3 ' ends are named as pGSI- by foregoing fusion gene cloning in plasmid pGSI containing MluI restriction enzyme site
HER1ScFv.Plasmid is separated through MluI single endonuclease digestion, digestion products through 1% Ago-Gel, is recycled with Ago-Gel DNA
It is spare that kit recycles target fragment.
PWPXL-CD8-CD137-CD3 ζ plasmid is through restriction enzyme MluI single endonuclease digestion, and digestion products are through 1% agarose
Gel is separated, spare with Ago-Gel DNA QIAquick Gel Extraction Kit recycling carrier segments.Then Growth in Rats swashs with recycling
The DNA fragmentation of plain signal peptide and HER1ScFv are attached by T4DNA ligase, and specific method is shown in specification.Connection is produced
Object converts Trans1-T1 Phage Resistant Competent cell, picking monoclonal after 37 DEG C of culture 16h, and 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 MluI/NdeI
Digestion identification, qualification result are as shown in Figure 3, wherein M1:DNA molecular weight marker D2000；1 swimming lane: plasmid pWPXL-
The endonuclease bamhi (825bp, 751bp) of HER1ScFv-CD8-CD137-CD3 ζ；2 swimming lanes: plasmid pWPXL-HER1ScFv-CD8-
The non-endonuclease bamhi of CD137-CD3 ζ；M2:DNA molecular weight marker D15000.It will identify that correct plasmid send Beijing day brightness remote
The fusion segment of insertion is sequenced in Biotechnology Co., Ltd.The correct recombinant plasmid of sequencing result is named as
PWPXL-HER1ScFv-CD8-CD137-CD3 ζ, structural schematic diagram is as shown in figure 4, including rat growth hormone signal
Peptide (nucleotide sequence is as shown in SEQID NO.6), anti-HER1 single-chain antibody (nucleotide sequence is as shown in SEQID NO.7), CD8
The area hinge and transmembrane region and CD137 intracellular signal structural domain and CD3 ζ intracellular signal structural domain, wherein the inosculating antibody
Original receptor is with the structure that the intracellular signal structural domain of the area hinge of gene C D8 and transmembrane region and CD137 and CD3 ζ is connected in series
Signal transduction structural domain, amino acid sequence is as shown in SEQID NO.9, corresponding gene coded sequence such as SEQID NO.10 institute
Show.
The preparation of the NKT cell of 3 Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ of embodiment modification
(1) packaging and concentration of slow virus
Measure slow virus expression plasmid pWPXL-HER1ScFv-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 2000
Transfection Reagent transfection reagent cotransfection 293T incasing cells.It is collected in virus when transfecting 48h, 72h respectively
Clearly in 50mL EP pipe, 4 DEG C, 2000g is centrifuged 10min, the supernatant obtained twice is shifted into new EP pipe, with 4.5 μm of filter mistakes
Filter viral supernatants；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, and then 4
DEG C, 10000g is centrifuged 20min, abandons supernatant, and the precipitating sterile PBS of 4 DEG C of pre-coolings of 1mL dissolves to get Chimeric antigen receptor
Viral concentration liquid 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 pWPXL-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
CARHER1-NKT cell.CARHER1-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 CARHER1-NKT cell is imitated
Rate is 46.52%.
(3) external evoked amplification CARHER1-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+: 90.83%；CD3+CD4+: 14.48%；CD3+CD8+: 80.90%；CD3+CD56+: 34.48%；CD8+CD56+:
34.25%.
Cytotoxicity analysis of the 4 CARHER1-NKT cell of embodiment to human lung carcinoma cell lethal effect
It takes the human lung cancer cell A549 of high expression HER1 to be inoculated in 96 orifice plates, after 37 DEG C of incubator overnight incubations, takes respectively
The NKT cell cultivated in CARHER1-NKT cell, CARHER1-T cell and the embodiment 1 prepared in embodiment 3, to imitate target ratio
(killing cell: target cell) 5:1,10:1,20:1,40:1 is co-cultured with A549 cell, after the co-cultivation of 4h, each
The CCK8 that 10 μ L are added in hole is dyed.It is respectively the CARHER1- prepared in embodiment 3 that killing cell controls group is arranged simultaneously
The NKT cell cultivated in NKT cell, CARHER1-T cell and embodiment 1, and same amount of CCK8 is added and is dyed；And
It is the A549 cell that immunocyte killing processing is not added that target cell control group, which is arranged, and same amount of CCK8 is added and is contaminated
Color.Microplate reader detects Apoptosis situation, and the amount of Apoptosis is calculated according to the following equation: apoptosis rate={ 1-
[(experimental group-killing cell controls group-target cell control group)/experimental group] } × 100%, in the formula, kill cell controls group
The light absorption value for target cell not being added to measure there was only killing cell, target cell control group are there was only target cell and do not add killing cell
The light absorption value measured；Experimental group is after the immunocyte killing processing of corresponding effect target ratio (killing cell: target cell) is added
The light absorption value measured, is shown in Fig. 7.The NKT cell of Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ modification expresses height
The lung carcinoma cell of HER1 has specific killing activity, and the specific killing activity of CARHER1-NKT cell is substantially better than
CARHER1-T cell and NKT cell.
Therapeutic effect of the 5 CARHER1-NKT cell of embodiment to the patients with lung cancer of the progressive stage HER1 positive
Take 5 × 108The NKT cell (i.e. CAR HER1-NKT cell) of a HER1ScFv-CD8-CD137-CD3 ζ modification, warp
After crossing 100ml normal saline dilution, the patients with lung cancer of continuous three days venous re-transfusions to the progressive stage HER1 positive (is utilizing the present invention
CARHER1-NKT cell carry out targeting immunization therapy before, have been subjected to repeatedly treatment (such as radiotherapy, chemotherapy and other drugs are suited the medicine to the illness
Treatment etc.), but without obvious curative effects) in vivo, the treatment situation of patient is assessed after feedback.
Fig. 8 is the therapeutic process of CARHER1-NKT cell of the invention to the patients with lung cancer of 11 progressive stage HER1 positives
In, CARHER1-NKT cell continues that there are time plots in peripheral blood in patients.Fig. 8 is the results show that by CARHER1-
After NKT cell therapy, CARHER1-NKT cell copy number is persistently grown in the peripheral blood from patients with lung cancer of 11 progressive stage HER1 positives
Long presence illustrates that CARHER1-NKT cell of the invention can continue greatly in the patients with lung cancer body of the progressive stage HER1 positive
The proliferation of amount plays killing activity.
CARHER1-NKT cell of the invention is suffered in the therapeutic process of the patients with lung cancer of 11 progressive stage HER1 positives
The clinical toxicity of person is shown in Table 1.As shown in table 1, after CARHER1-NKT cell therapy of the invention, what patient was presented
The toxicity that most of reactions are 1-2 grades illustrates that patient can be good at the treatment for being resistant to CARHER1-NKT cell, i.e., originally
Treatment of the CARHER1-NKT cell of invention for the patients with lung cancer of the progressive stage HER1 positive, is safe and feasible.
Table 1
Fig. 9 is the therapeutic process of CARHER1-NKT cell of the invention to the patients with lung cancer of two progressive stage HER1 positives
In, the tuberculosis stove and pleura extract variation diagram of different time sections patient.As shown in figure 9, the lung of two progressive stage HER1 positives
Cancer patient is after two courses for the treatment of of CARHER1-NKT cell therapy, and pleura extract significantly reduces, while tumor focus size
Also it is reduced significantly, clinical diagnosis PR.Illustrate CARHER1-NKT cell of the invention to the patients with lung cancer of the progressive stage HER1 positive
With good treatment curative effect.
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 (4)
- Application of the 1.CARHER1-NKT cell in the preparation that preparation is used for treatment of advanced HER1 positive lung cancer, feature exist In, the CARHER1-NKT cell is the NKT cell modified by Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ, In, the Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ include concatenated rat growth hormone signal peptide, The intracellular signal structural domain of the hinge area and transmembrane region of HER1ScFv, CD8, the intracellular signal structural domain of CD137 and CD3 ζ；The amino acid sequence of the Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ is as shown in SEQ ID NO.1；The preparation method of the CARHER1-NKT cell includes: that packaging carries pWPXL-HER1ScFv-CD8-CD137-CD3 ζ Slow virus obtains viral concentration liquid；Contaminate NKT cell using obtained viral concentration liquid inductance, make NKT cell expression chimeric antigen by Body HER1ScFv-CD8-CD137-CD3 ζ, wherein the pWPXL-HER1ScFv-CD8-CD137-CD3 ζ is embedding containing encoding Close the Lentiviral of the gene of antigen receptor HER1ScFv-CD8-CD137-CD3 ζ；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 μ L 1 × 10-6The 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. application according to claim 1, wherein encode the Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 The gene of ζ has the nucleotide sequence as shown in SEQ ID NO.2.
- 3. application according to claim 2, wherein the nucleotide sequence of the gene is as shown in SEQ ID NO.2.
- 4. application according to claim 1, wherein the preparation method of the CARHER1-NKT cell further includes by such as Lower section method prepares 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.
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