CN105920592A - Application of CARHER1-NKT cells in preparation of preparations for treatment of HER1 positive lung cancer in progressive stage - Google Patents
Application of CARHER1-NKT cells in preparation of preparations for treatment of HER1 positive lung cancer in progressive stage Download PDFInfo
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Abstract
The invention discloses application of CARHER1-NKT cells in the preparation of preparations for the treatment of HER1 positive lung cancer in progressive stage. CARHER1-NKT cells are NKT cells modified by a chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 zeta; and the chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 zeta comprises a rat growth hormone signal peptide, HER1ScFv, a hinge area and a transmembrane domain of CD8, an intracellular signal structural domain of CD137 and an intracellular signal structural domain of CD3 zeta, which are in series connection. The CARHER1-NKT cells for the treatment of HER1 positive lung cancer in progressive stage can effectively avoid the drug resistance caused by the existing drugs targeting HER1, have certain specific killing activity on lung cancer cells, and certain therapeutic effect on patients with HER1 positive lung cancer in progressive stage.
Description
Technical field
The invention belongs to knubble biological arts, in particular it relates in adoptive immunotherapy
CARHER1-NKT cell is in preparation is used for the preparation for the treatment of of advanced HER1 positive lung cancer
Application.
Background technology
EGFR (epithelial growth factor receptor, EGF-R ELISA) i.e. HER1,
It is the expression product of proto-oncogene c-erbB1, belongs to human epidermal growth factor acceptor (HER) family,
Itself there is cheese ammonia kinase enzyme activity, once can active cell with epidermal growth factor (EGF) combination
There is correlation gene in core, thus promote that cell division is bred.HER1 is high expressed in Several Kinds of Malignancy,
Research find HER1 be expressed in most of patients with lung cancer, expression reaches 80%, its expression with
The progress of pulmonary carcinoma disease and shift relevant.Although the treatment of pulmonary carcinoma has obtained certain progress recently, but
It is the survival rate still not improving patient, therefore needs badly and inquire into new therapy to overcome this puzzlement.
At present, when treatment of advanced HER1 positive lung cancer patient, the medicine of anti-HER1 is (such as HER1
Tyrosine kinase inhibitor (tyrosine kinase inhibitors, TKIs)) already at clinical investigation phase,
But, clinical effectiveness shows, only part patients with lung cancer uses HER1 treatment with tyrosine kinase inhibitors to have
Imitate, and patient eventually produces certain drug resistance, thus affect the curative effect of medicine.
Summary of the invention
The invention aims to overcome the Progress in Medication phase using anti-HER1 in prior art
The defect of the drug resistance caused during HER1 positive lung cancer patient, it is provided that CARHER1-NKT cell exists
Preparation application in the preparation for the treatment of of advanced HER1 positive lung cancer, uses the present invention's
During CARHER1-NKT cell therapy progressive stage HER1 positive lung cancer, it is possible to be prevented effectively from employing existing
The drug resistance caused when having anti-HER1 medicine, has certain special target killing activity to lung carcinoma cell,
And to through repeatedly treatment (such as radiotherapy, chemotherapy and other drug symptomatic treatment etc.) but all without bright
The progressive stage HER1 positive lung cancer patient of aobvious curative effect has certain therapeutic effect.
Therefore, to achieve these goals, the invention provides CARHER1-NKT cell in preparation
Application in the preparation for the treatment of of advanced HER1 positive lung cancer, described CARHER1-NKT
Cell is the NKT cell modified by Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ,
Wherein, described Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ includes the rat of series connection
Growth hormone signal peptide, the hinge region (hinge district) of HER1ScFv, CD8 and cross-film district, CD137
Intracellular signal domain and the intracellular signal domain of CD3 ζ.
When treatment of advanced HER1 positive lung cancer, the CARHER1-NKT cell energy of the present invention
Enough specific binding HER1 antigen, hence it is evident that extend immunocyte in the time-to-live in the patient,
Strengthen the ability of immunocyte targets identification lung carcinoma cell surface HER1 antigen, strengthen thin to pulmonary carcinoma
The specific killing activity of born of the same parents, and the Drug therapy using existing anti-HER1 can actually be prevented effectively from
The drug resistance of Shi Yinqi, to through repeatedly treating (as radiotherapy, chemotherapy and other drug are controlled to the ill
Treat) but all progressive stage HER1 positive lung cancer patients without obvious curative effects have certain therapeutic effect.
The NKT cell (i.e. CARHER1-NKT cell) of the through engineering approaches HER1 targeting of the present invention is
Treatment of advanced HER1 positive lung cancer provides a kind of new selection, has good industrial application prospect.
Other features and advantages of the present invention will be described in detail in detailed description of the invention part subsequently.
Accompanying drawing explanation
Fig. 1 is the result that the NKT cell phenotype of separation and Culture is analyzed by flow cytometry.
Fig. 2 is the restricted interior of the Lentiviral pWPXL-CD8-CD137-CD3 ζ of the present invention
Cut the electroresis appraisal figure of enzyme MluI/NdeI double digestion fragment.
Fig. 3 is the Lentiviral pWPXL-HER1ScFv-CD8-CD137-CD3 ζ of the present invention
The electroresis appraisal figure of restricted enzyme MluI/NdeI double digestion fragment.
Fig. 4 is the Lentiviral pWPXL-HER1ScFv-CD8-CD137-CD3 ζ of the present invention
Structural representation, wherein, sequence counterclockwise is forward gene sheet degree, is clockwise cdna reverse fragment.
Fig. 5 is that Flow cytometry contains Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ
The viral concentration liquid efficiency of infection to NKT cell.
Fig. 6 is that Flow cytometry Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ repaiies
The result of NKT cell (CARHER1-NKT cell) phenotypic evaluation of decorations.
Fig. 7 is the CARHER1-NKT cell of the present invention cell toxicant to human lung carcinoma cell lethal effect
Property analysis chart.
Fig. 8 is the CARHER1-NKT cell of the present invention lung to the 11 example progressive stage HER1 positives
In the therapeutic process of cancer patient, during CARHER1-NKT cell sustainable existence in peripheral blood in patients
Half interval contour figure.
Fig. 9 is the CARHER1-NKT cell of the present invention pulmonary carcinoma to the two example progressive stage HER1 positives
In the therapeutic process of patient, the pneumonopathy stove of different time sections patient and pleura extractum variation diagram.
Detailed description of the invention
Hereinafter the detailed description of the invention of the present invention is described in detail.It should be appreciated that this place is retouched
The detailed description of the invention stated is merely to illustrate and explains the present invention, is not limited to the present invention.
The invention provides CARHER1-NKT cell in preparation for treatment of advanced HER1 sun
Application in the preparation of property pulmonary carcinoma, described CARHER1-NKT cell is by Chimeric antigen receptor
The NKT cell that HER1ScFv-CD8-CD137-CD3 ζ modifies, wherein, described chimeric antigen is subject to
Body HER1ScFv-CD8-CD137-CD3 ζ include series connection rat growth hormone signal peptide,
The hinge region of HER1ScFv, CD8 and cross-film district, the intracellular signal domain of CD137 and CD3 ζ
Intracellular signal domain.
In the application of the present invention, under preferable case, described Chimeric antigen receptor
HER1ScFv-CD8-CD137-CD3 ζ is by rat growth hormone signal peptide, HER1ScFv, CD8
The intracellular signal domain series connection of hinge region and cross-film district, the intracellular signal domain of CD137 and CD3 ζ
Constitute.It is further preferred that Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ have as
Aminoacid sequence shown in SEQ ID NO.1, it is further preferred that Chimeric antigen receptor
The aminoacid sequence of HER1ScFv-CD8-CD137-CD3 ζ is as shown in SEQ ID NO.1.
In the application of the present invention, under preferable case, encode described Chimeric antigen receptor
The gene of HER1ScFv-CD8-CD137-CD3 ζ has the nucleotides sequence as shown in SEQ ID NO.2
Row, it is further preferred that the base of encoding chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ
The nucleotide sequence of cause is as shown in SEQID NO.2.
In the application of the present invention, under preferable case, the preparation method of CARHER1-NKT cell includes:
Packaging carries the slow virus of pWPXL-HER1ScFv-CD8-CD137-CD3 ζ, obtains viral concentration liquid；
Utilize the viral concentration liquid inductance dye NKT cell obtained, make NKT cell express Chimeric antigen receptor
HER1ScFv-CD8-CD137-CD3 ζ, wherein, described pWPXL-HER1ScFv-CD8-CD137
-CD3 ζ is the slow of the gene containing encoding chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ
Virus expression carrier.
In the application of the present invention, Lentiviral pWPXL-HER1ScFv-CD8-CD137
CD3 ζ can be with assistant carrier cotransfection incasing cells such as 293T incasing cells, it is thus achieved that viral concentration liquid
And CARHER1-NKT cell.
Preparation side for Lentiviral pWPXL-HER1ScFv-CD8-CD137-CD3 ζ
There is no particular limitation for method, can be those skilled in the art it is conceivable that various methods, preferably
In the case of, the preparation side of Lentiviral pWPXL-HER1ScFv-CD8-CD137-CD3 ζ
Method comprises the following steps:
(1) from NKT cell cDNA, expand hinge district and cross-film district, the CD137 of CD8 respectively
Intracellular signal domain and the intracellular signal domain of CD3 ζ, and be cloned into carrier
In pWPXL-GFP, build and obtain pWPXL-CD8-CD137-CD3 ζ；
(2) composite coding rat growth hormone signal peptide and the nucleotide sequence of HER1ScFv, and
It is cloned in pWPXL-CD8-CD137-CD3 ζ, after sequence verification, obtains sequence correct
pWPXL-HER1ScFv-CD8-CD137-CD3ζ。
In step (1), for expanding the hinge district of CD8 from NKT cell cDNA respectively
Do not have with the method for the intracellular signal domain of cross-film district, the intracellular signal domain of CD137 and CD3 ζ
There is particularly restriction, can be various methods commonly used in the art, such as, can be RT-PCR method.
Wherein, then NKT cell can be cultivated by separating the mononuclearcell in people's venous blood
Obtain.
Specifically, the method obtaining pWPXL-CD8-CD137-CD3 ζ may include that extraction NKT
The total serum IgE of cell, reverse transcription obtains NKT cell cDNA, with the NKT cell cDNA obtained
For template, primer P1 (SEQID NO.11) and P2 (SEQID NO.12) is utilized to carry out PCR
Amplification obtains hinge district and cross-film district (the SEQID NO.3) of CD8 gene；Utilize primer P3
(SEQID NO.13) and P4 (SEQID NO.14) carry out PCR amplification and obtain CD137 gene
Intracellular signal domain (SEQID NO.4)；Utilize primer P5 (SEQID NO.15) and P6
(SEQID NO.16) carries out PCR amplification and obtains the intracellular signal domain of CD3 ζ gene
(SEQID NO.5), carries out double digestion respectively, then with MluI/NdeI by the PCR primer of acquisition
Lentiviral pWPXL-GFP after double digestion connects.
In step (2), for composite coding rat growth hormone signal peptide and the core of HER1ScFv
There is no particular limitation for the method for nucleotide sequence, can be various methods commonly used in the art, such as
Can be synthesized by full genome synthetic technology.
Specifically, the side of the correct pWPXL-HER1ScFv-CD8-CD137-CD3 ζ of sequence is obtained
Method may include that by full genome synthetic technology composite coding rat growth hormone signal peptide and
The nucleotide sequence (SEQID NO.8) of HER1ScFv fusion gene, is cloned in carrier pGSI,
Obtain pGSI-HER1ScFv；Then pGSI-HER1ScFv is carried out MluI single endonuclease digestion, with warp
The recombiant plasmid pWPXL-CD8-CD137-CD3 ζ that restricted enzyme MluI single endonuclease digestion obtains is even
Connect, identify through order-checking, obtain the pWPXL-HER1ScFv-CD8-CD137-CD3 ζ that sequence is correct.
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.
Do not have for packing the method for the slow virus carrying pWPXL-HER1ScFv-CD8-CD137-CD3 ζ
There is particularly restriction, can be the various methods commonly used of those skilled in the art, under preferable case, will be slowly
Virus expression carrier pWPXL-HER1ScFv-CD8-CD137-CD3 ζ and helper plasmid (as
PsPAX2, pMD2.G) cotransfection 293T incasing cells, collect in virus during transfection 48-72h
Clearly, centrifugal, filtration, filtrate is added 5 × PEG6000-NaCl and mixes, abandon after being centrifuged
Clearly, the aseptic PBS of precipitation 0-4 DEG C pre-cooling dissolves, it is thus achieved that viral concentration liquid.
In the application of the present invention, the preparation method of CARHER1-NKT cell also includes by such as lower section
Method prepares NKT cell:
(1) in the presence of CD3 monoclonal antibody, interleukin-2 and interleukin-15, by single core
Cell carries out first stage cultivation；
(2) in the presence of interleukin-2, the cell cultivated the described first stage carries out second stage
Cultivate.
Under preferable case, the embodiment that the described first stage cultivates includes: trained by mononuclearcell
Supporting in a NKT cell culture fluid, a described NKT cell culture fluid contains NKT cell
Culture medium, CD3 monoclonal antibody, interleukin-2 and interleukin-15；It is further preferred that first
In NKT cell culture fluid, the concentration of described CD3 monoclonal antibody is 30-70ng/mL, and/or
The concentration of described interleukin-2 is 300-700U/mL, and/or the concentration of described interleukin-15 is
30-70ng/mL。
Under preferable case, the embodiment that described second stage is cultivated includes: by the described first stage
The cell cultivated is incubated in the 2nd NKT cell culture fluid, described 2nd NKT cell culture fluid
In containing NKT cell culture medium and interleukin-2；It is further preferred that the 2nd NKT cell is cultivated
In liquid, the concentration of described interleukin-2 is 300-700U/mL.
For NKT cell culture medium, there is no particular limitation, can be various use commonly used in the art
In the culture medium of cultivation NKT cell, such as, it can be GT-T551 culture medium.
When preparing NKT cell, the first stage is cultivated and the condition of second stage cultivation does not has
Particularly limit, can be various conditions commonly used in the art, such as can 30-37 DEG C, saturated
Humidity is the CO of 3-6%2Incubator is cultivated.Those skilled in the art can to cultivate time
Between carry out accommodation, this is known to those skilled in the art, does not repeats them here.
In the NKT cell that the present invention prepares, CD3+Cell average ratio > 90%, CD3+CD8+
Cell accounts for total CD3+The average ratio of cell > 70%；CD3+CD56+Cell accounts for total CD3+Cell
Average ratio > 15%.
In the application of the present invention, it is not particularly limited for infecting the method for NKT cell, Ke Yiwei
Various methods commonly used in the art, under preferable case, the method includes:
(1) in the presence of viral concentration liquid, protamine and interleukin-2, NKT cell is entered
The row first stage infects and cultivates；
(2) in the presence of CD3 monoclonal antibody, interleukin-2 and interleukin-15, by described
The second stage that carries out the cell that one stage infection is cultivated infects cultivates.
Preferably, the embodiment that the infection of described first stage is cultivated includes: cultivated by NKT cell
In the 3rd NKT cell culture fluid, described 3rd NKT cell culture fluid contains the training of NKT cell
Support base, viral concentration liquid, protamine and interleukin-2；It is further preferred that the 3rd NKT is thin
In born of the same parents' culture fluid, the concentration of described interleukin-2 is 300-700U/mL.
Preferably, the embodiment that the infection of described second stage is cultivated includes: by the described first stage
Infect the cell cultivated to be incubated in a described NKT cell culture fluid.Oneth NKT cell training
The particular make-up of nutrient solution can be found in aforementioned corresponding contents, does not repeats them here.
When infecting NKT cell, first stage infection cultivation and second stage infection are cultivated
There is no particular limitation for condition, can be various conditions commonly used in the art, such as can be
30-37 DEG C, saturated humidity be the CO of 3-6%2Incubator is cultivated.Those skilled in the art can
So that the time cultivated is carried out accommodation, this is known to those skilled in the art, at this not
Repeat again.
Specifically, the method infecting NKT cell includes: take 1 × 107-5×107Individual NKT cell,
Discard old culture fluid, add 2-4mL fresh GT-T551 culture fluid, add 200-400 μ L
Viral concentration liquid, 2-4 μ L 1 × 10-6Mg/mL protamine and final concentration of 300-700U/mL's
IL-2, be placed in 30-37 DEG C, saturated humidity be the CO of 3-6%2After incubator infects 12-16h,
Abandon culture fluid, cell is gone in the most coated culture bottle, add the GT-T551 training of 20-50mL
Support base, add the IL-2 of final concentration of 300-700U/mL, final concentration of 30-70ng/ml
CD3 monoclonal antibody and the interleukin 15 of final concentration of 30-70ng/mL, in 30-37 DEG C, full
It is the CO of 3-6% with humidity2Incubator is cultivated 12-18h, it is thus achieved that Chimeric antigen receptor
The NKT cell that HER1ScFv-CD8-CD137-CD3 ζ modifies.
It is further preferred that the method infecting NKT cell also includes:
(3) first in the presence of interleukin-2, described second stage is infected the cell cultivated and carries out body
Outer induction, when the density of cell is 80-90%, then at CD3 monoclonal antibody, interleukin-2
In the presence of interleukin-15, cell is carried out amplification cultivation.
Under preferable case, described external evoked embodiment includes: described second stage is infected training
The cell supported is incubated in described 2nd NKT cell culture fluid, the embodiment of described amplification cultivation
Including: cell is incubated in a described NKT cell culture fluid.Oneth NKT cell is cultivated
The particular make-up of liquid and the 2nd NKT cell culture fluid can be found in aforementioned corresponding contents, the most superfluous at this
State.
Specifically, the method infecting NKT cell also includes: second stage is infected and obtains after cultivating
The GT-T551 of final concentration of 300-700U/mL of NKT cell IL-2 of slow virus infection
Culture fluid carries out external evoked, when the density of cell is 80-90%, cell is proceeded to cell culture bags
In, final concentration of 300-700U/mL, CD3 monoclonal antibody of IL-2 was added every 1.5-2.5 days
Final concentration of 30-70ng/ml, final concentration of 30-70ng/mL fresh of Interleukin-15
GT-T551 culture fluid carries out amplification cultivation and is 1 × 10 by cell amplification to total amount9-2×109Individual carefully
Born of the same parents.It is thin that slow virus through the present invention carries out NKT to the Chimeric antigen receptor of targeting HER1 antigen
Born of the same parents infect, and its efficiency of infection is up to 30%-60%, and the CARHER1-NKT cell obtained, its
CD3+CD56+Cell accounts for total CD3+The ratio of cell is within the scope of 15%-40%.
The NKT cell that Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ modifies is expressed
The protein amino acid sequence of Chimeric antigen receptor is as shown in SEQID NO.1.Wherein, art technology
Personnel it should be understood that Chimeric antigen receptor precursor protein by rat growth hormone signal peptide,
The hinge district of HER1ScFv, CD8 and cross-film district, the intracellular signal domain of CD137 and CD3 ζ
Intracellular signal domain in series, excise at intracellular rough endoplasmic reticulum after protein translation
Become ripe Chimeric antigen receptor albumen after signal peptide, secrete after exporting and be positioned NKT cell
On cell membrane.Gene coded sequence corresponding to the protein amino acid sequence of this Chimeric antigen receptor is such as
Shown in SEQID NO.2.This Chimeric antigen receptor with the hinge district of gene C D8 and cross-film district and
The structure that the intracellular signal domain of CD137 and CD3 ζ is in series is signal conducting structure territory, its
Aminoacid sequence as shown in SEQID NO.9, corresponding gene coded sequence such as SEQID NO.10
Shown in.
In the application of the present invention, it will be understood by those skilled in the art that progressive stage HER1 is positive
Pulmonary carcinoma refers to that Locally Advanced cannot be carried out excision, transitivity or the pulmonary carcinoma in recurrent stage, symptom table
Now for cough, expectoration, uncomfortable in chest, tachypnea.The progressive stage HER1 positive lung cancer of the present invention is especially for recurrence
The progressive stage HER1 positive lung cancer of refractory.
In the application of the present invention, for the composition of the preparation for treatment of advanced HER1 positive lung cancer
There is no particular limitation, if containing CARHER1-NKT cell of the present invention or by
CARHER1-NKT cell is prepared from, and the particular make-up of preparation and preparation method are ability
Well known to field technique personnel, do not repeat them here.
Embodiment
The present invention is further illustrated for below example, but and is not so limited the present invention.
Experimental technique in following example, if no special instructions, is this area conventional method.Following
Experiment material used in embodiment, if no special instructions, is and buys from routine biochemistry reagent shop
Arrive, wherein:
NKT cell culture medium GT-T551 is purchased from TaKaRa company.
Lymphocyte separation medium is purchased from TBD company.
CD3 monoclonal antibody, recombinant fiber connect albumen (retronectin) and 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.
Agarose gel DNA reclaims test kit, common DNA product purification kit, plasmid is little carries
Test kit is purchased from Tian Gen biochemical technology company limited.
PWPXL-GFP, psPAX2, pMD2.G are purchased from Addgene company.
PGSI is purchased from Tian Yihuiyuan bio tech ltd, Beijing.
Trans1-T1 Phage Resistant Competent cell has purchased from Beijing full formula gold biotechnology
Limit company.
LipofectamineTM2000 Transfection Reagent transfection reagents are public purchased from Invitrogen
Department.
293T incasing cells is purchased from U.S. ATCC.
Final concentration of 25.5 mass % of PEG6000 in PEG6000-NaCl, NaCl is final concentration of
1.2M, PEG6000 and NaCl are purchased from Suo Laibao bio tech ltd, Shanghai.
Hyclone is purchased from PAA company of Germany.
The typeⅡ pneumocyte of high expressed HER1 is purchased from ATCC company of the U.S..
Cell Counting Kit-8 (CCK8) test kit is purchased from Beijing Wo Bisen Science and Technology Ltd..
All primers are synthesized by Tian Yihuiyuan bio tech ltd, Beijing.
The preparation of embodiment 1 NKT cell
(1) people's venous blood is taken in the vacuum tube containing heparin.Use lymphocyte separation medium, by close
Degree gradient centrifugation procedure separates and obtains mononuclearcell (PBMCs).
(2), after PBMCs washes three times, the NKT cell of the Human autologous serum containing 0.6 volume % is used
It is 2 × 10 that culture medium GT-T551 adjusts final concentration of cells6Individual cell/mL；Cell is inoculated in warp in advance
Cross the coated 75cm of retronectin of final concentration of 10 μ g/mL2In Tissue Culture Flask.Then in training
Support and add the rhIL-2 of final concentration of 500U/mL, 50ng/ml CD3 monoclonal anti in base
Body and recombination human interleukin-15 of 50ng/mL, 37 DEG C, saturated humidity be the CO of 5%2Incubator
Middle cultivation.
(3) cultivating the 4th day, be transferred to by cell in the most coated culture bottle, every 2 days according to cell
Growth population adds NKT cell culture medium GT-T551, and controlling cell concentration is 1 × 108Individual cell/mL,
And add the rhIL-2 of final concentration of 500U/ml；Cultivate to the 12nd day, obtain NKT thin
Born of the same parents, NKT cell phenotype is analyzed by flow cytometry.Result is shown in Fig. 1, wherein CD3+: 95.04%；
CD3+CD8+: 90.99%；CD3+CD56+: 24.12%；CD8+CD56+: 24.63%.
The structure of embodiment 2 Lentiviral pWPXL-HER1ScFv-CD8-CD137-CD3 ζ
Build
(1) preparation of NKT cell cDNA
Centrifugation embodiment 1 cultivates the NKT cell obtained, with total RNA extraction reagent box RNAiso
Reagent extracts the total serum IgE of cell, and-80 DEG C save backup.The total serum IgE reverse transcription reagents extracted
Box RevertAidTMFirst Strand cDNA Synthesis Kit reverse transcription obtains NKT cell cDNA,
-20 DEG C save backup.
(2) preparation of slow virus plasmid pWPXL-CD8-CD137-CD3 ζ
(wherein, underscore is labeled as protecting base, and square frame is enzyme action to design and synthesize following primer sequence
Site):
As template, PCR is carried out with primer P1 and P2 with NKT cell cDNA in step (1)
Amplification, obtains hinge district and the cross-film district of the CD8 of long 287bp, nucleotide sequence such as SEQID
Shown in NO.3, two ends contain MluI and Bgl II restriction enzyme site and protection base respectively；Use primer P3
Carry out PCR amplification with P4, obtain the CD137 intracellular signal domain of long 146bp, nucleotide
Sequence is as shown in SEQID NO.4, and two ends contain Bgl II and EcoRI restriction enzyme site and protection respectively
Base；Carry out PCR amplification with primer P5 and P6, obtain the intracellular letter of the CD3 ζ of long 359bp
Number domain, nucleotide sequence is as shown in SEQID NO.5, EcoRI and NdeI is contained at two ends respectively
Restriction enzyme site and protection base.Each step pcr amplification reaction system is identical, to expand CD137 intracellular
As a example by signal structure territory, carry out PCR amplification, PCR reaction condition referenceHS
The description of DNA Polymerase, 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 primer is separated with the agarose gel of 1%, returns with agarose gel DNA
Receive test kit and carry out DNA fragmentation recovery.Carrying out double digestion reaction after obtaining fragment respectively, enzyme action produces
Thing reclaims standby with common DNA product purification kit.
Lentiviral pWPXL-GFP MluI/NdeI double digestion, digestion products is through 1%
Agarose gel separate, reclaim test kit with agarose gel DNA and reclaim big carrier-pellet
Section, then with CD8, CD137, CD3 ζ fragment reclaimed before by T4DNA ligase even
Connect, connect product and convert Trans1-T1 Phage Resistant Competent cell, 37 DEG C of cultivations
Picking monoclonal after 16h, 37 DEG C, 250rpm extracts matter with the little extraction reagent kit of plasmid after cultivating 12h
Grain.The plasmid extracted is identified through restricted enzyme MluI and NdeI double digestion, identifies that electrophoretogram is shown in
Fig. 2, wherein, M1:DNA molecular weight marker D15000；1 swimming lane: plasmid
The non-endonuclease bamhi of pWPXL-CD8-CD137-CD3 ζ；2 swimming lanes: plasmid
The endonuclease bamhi (751bp) of pWPXL-CD8-CD137-CD3 ζ；M2:DNA molecular weight mark
Note D2000.To identify that correct plasmid send Tian Yihuiyuan bio tech ltd, Beijing to insertion
Fusion gene fragment check order.By named for recombiant plasmid correct for sequencing result
PWPXL-CD8-CD137-CD3 ζ, wherein, the hinge district of CD8 and the nucleotides sequence in cross-film district
Arrange as shown in SEQID NO.3, the nucleotide sequence such as SEQID of the intracellular signal domain of CD137
Shown in NO.4, the nucleotide sequence of the intracellular signal domain of CD3 ζ is as shown in SEQID NO.5.
(3) preparation of slow virus plasmid pWPXL-HER1ScFv-CD8-CD137-CD3 ζ
Full genome composite coding rat growth hormone signal peptide and the nucleoside of HER1ScFv fusion gene
Acid sequence, sequence, as shown in SEQID NO.8, is closed by Tian Yihuiyuan bio tech ltd, Beijing
Becoming, its 5 ' end contains MluI restriction enzyme site containing MluI restriction enzyme site, kozak sequence, 3 ' ends,
Foregoing fusion gene is cloned in plasmid pGSI, named pGSI-HER1ScFv.Plasmid warp
MluI single endonuclease digestion, digestion products separates through 1% agarose gel, uses agarose gel DNA
Reclaim test kit recovery purpose fragment standby.
PWPXL-CD8-CD137-CD3 ζ plasmid is through restricted enzyme MluI single endonuclease digestion, enzyme action
Product separates through 1% agarose gel, reclaims test kit with agarose gel DNA and reclaims
Carrier segments is standby.Then there is the DNA of rat growth hormone signal peptide and HER1ScFv with recovery
Fragment is attached by T4DNA ligase, and concrete grammar is shown in description.Connection product is turned
Change Trans1-T1 Phage Resistant Competent cell, cultivate picking Dan Ke after 16h for 37 DEG C
Grand, 37 DEG C, after 250rpm cultivates 12h, extract plasmid with the little extraction reagent kit of plasmid.The matter extracted
Grain is identified through restricted enzyme MluI/NdeI double digestion, qualification result as it is shown on figure 3, wherein,
M1:DNA molecular weight marker D2000；1 swimming lane: plasmid
The endonuclease bamhi (825bp, 751bp) of pWPXL-HER1ScFv-CD8-CD137-CD3 ζ；2 swimming
Road: the non-endonuclease bamhi of plasmid pWPXL-HER1ScFv-CD8-CD137-CD3 ζ；M2:DNA
Molecular weight marker D15000.To identify that correct plasmid send sky, Beijing one brightness limited public affairs of remote biotechnology
Take charge of the fusion gene fragment to inserting to check order.By named for recombiant plasmid correct for sequencing result
PWPXL-HER1ScFv-CD8-CD137-CD3 ζ, its structural representation as shown in Figure 4, wherein
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), the hinge district of CD8 and cross-film district
And the intracellular signal domain of CD137 and the intracellular signal domain of CD3 ζ, wherein, this is fitted together to
Antigen receptor is with the hinge district of gene C D8 and cross-film district and the intracellular signal of CD137 and CD3 ζ
The structure that domain is in series is signal conducting structure territory, its aminoacid sequence such as SEQID NO.9
Shown in, corresponding gene coded sequence is as shown in SEQID NO.10.
The NKT that embodiment 3 Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ modifies
The preparation of cell
(1) packaging of slow virus and concentration
Slow virus expression plasmid is measured respectively with spectrophotometer
PWPXL-HER1ScFv-CD8-CD137-CD3 ζ and helper plasmid psPAX2, pMD2.G's is dense
Degree, three kinds of plasmids are with the mass ratio Lipofectamine of 4:2:1TM 2000 Transfection Reagent
Transfection reagent cotransfection 293T incasing cells.Viral supernatants is collected respectively when transfecting 48h, 72h
In 50mL EP pipe, 4 DEG C, 2000g is centrifuged 10min, shifts the supernatant obtained for twice to newly
In EP pipe, with 4.5 μm filter filter virus supernatants；Filter viral supernatants with
5 × PEG6000-NaCl mixes according to the volume ratio of 4:1,4 DEG C of standing 2h, then 4 DEG C, and 10000
G is centrifuged 20min, abandons supernatant, and the aseptic PBS of 4 DEG C of pre-coolings of precipitation 1mL dissolves, and to obtain final product
The viral concentration liquid of Chimeric antigen receptor, carries out subpackage by often pipe 100 μ L, and-80 DEG C save backup.
According to the method described above, slow virus expression plasmid pWPXL-GFP and helper plasmid are utilized
PsPAX2, pMD2.G cotransfection 293T incasing cells, collects viral supernatants, concentrates, it is thus achieved that table
Reach the slow virus concentrated solution of GFP green fluorescent protein.
(2) slow virus infection NKT cell and the amplification cultivation of infected cell
Example 1 at 75cm21 × 10 cultivated in culture bottle7Individual NKT cell, discards old
Culture fluid, add 2mL fresh NKT cell culture medium GT-T551,200 μ L step (1)
The viral concentration liquid that obtains, 2 μ L 1 × 10-6Mg/mL protamine, final concentration of 500U/mL's
RhIL-2, be placed in 37 DEG C, saturated humidity be the CO of 5%2Incubator infects 12 hours
After, abandon culture fluid.Simultaneously thin to NKT with the slow virus concentrated solution expressing GFP green fluorescent protein
Born of the same parents carry out synchronizing to infect (the NKT cell obtained is referred to as CART-GFP cell), are used for calculating this
The efficiency of infection of virus.Metainfective cell is gone to without CD3 and retronectin coated 75
cm2In culture bottle, add the NKT cell culture medium GT-T551 of 20mL, add final concentration
For the rhIL-2 of 500U/mL, the CD3 monoclonal antibody of final concentration of 50ng/ml and
The recombinant human interleukin 15 of final concentration of 50ng/mL, in 37 DEG C, saturated humidity be the CO of 5%2
Cultivating 18h in incubator, the NKT cell obtained is referred to as CARHER1-NKT cell.With identical
Method prepare CARHER1-T cell (the preparation method reference literature of T cell: Yajing Zhang,
et al.Autologous CIK Cell Immunotherapy in Patients with Renal Cell
Carcinoma after Radical Nephrectomy.Clinical Study, 2.4 part CIK cell in 2013
Preparation method).With the efficiency of infection of this virus of Flow cytometry, result as it is shown in figure 5,
The efficiency of infection of CARHER1-NKT cell is 46.52%.
(3) external evoked amplification CARHER1-NKT cell mass
By the final concentration of 500U/mL's of the NKT cell rhIL-2 after above-mentioned cultivation
NKT cell culture medium GT-T551 carries out external evoked, by cell when the density of cell is 85%
Proceed in cell culture bags, every 2 days add rhIL-2 final concentration of 500U/mL,
The final concentration of 50ng/ml of CD3 monoclonal antibody, final concentration of the 50 of recombinant human interleukin 15
The fresh NKT cell culture medium GT-T551 of ng/mL carries out amplification cultivation, treats that cell amplification arrives always
Amount is 1.5 × 109After about individual cell, use flow cytometer that the cell colony infected is identified,
Cell phenotype commonly reaches CD3 positive cell ratio ＞ 90%；The double positive cell ratio of CD3CD8
Example > 70%；The double positive cell ratio of CD3CD56 > 15%, result is shown in Fig. 6, CD3+: 90.83%；
CD3+CD4+: 14.48%；CD3+CD8+: 80.90%；CD3+CD56+: 34.48%；CD8+CD56+:
34.25%.
The embodiment 4 CARHER1-NKT cell cytotoxicity to human lung carcinoma cell lethal effect
Analyze
The human lung cancer cell A549 taking high expressed HER1 is inoculated in 96 orifice plates, 37 DEG C of incubator trainings
After supporting overnight, the CARHER1-NKT cell of preparation, CARHER1-T in Example 3 respectively
The NKT cell cultivated in cell and embodiment 1, to imitate target ratio (killing cell: target cell) 5:1,
10:1,20:1,40:1 and A549 cell co-cultures, after the co-culturing of 4h, each
Hole adds the CCK8 of 10 μ L and dyes.Killing cell controls group is set simultaneously and is respectively embodiment
In 3, CARHER1-NKT cell, CARHER1-T cell and the embodiment 1 of preparation are cultivated
NKT cell, and add same amount of CCK8 and dye；And target cell matched group is set is
Do not add the A549 cell that immunocyte killing processes, and add same amount of CCK8 and dye.
Apoptosis situation is detected by microplate reader, and apoptotic amount is calculated according to the following equation:
Apoptosis rate={ 1-[(experimental group-killing cell controls group-target cell matched group)/experimental group] } × 100%,
In this formula, killing cell controls group is only to kill cell and do not add the light absorption value that target cell records,
Target cell matched group be only target cell and do not add and kill the light absorption value that records of cell；Experimental group is for adding
Record after entering the immunocyte killing process of corresponding effect target ratio (killing cell: target cell)
Light absorption value, is shown in Fig. 7.The NKT that Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ modifies
Cell has specific killing activity, and CARHER1-NKT to the lung carcinoma cell of high expressed HER1
The specific killing activity of cell is substantially better than CARHER1-T cell and NKT cell.
Controlling of the patients with lung cancer that embodiment 5 CARHER1-NKT cell is positive to progressive stage HER1
Therapeutic effect
Take 5 × 108NKT cell (the i.e. CAR that individual HER1ScFv-CD8-CD137-CD3 ζ modifies
HER1-NKT cell), after 100ml normal saline dilution, within continuous three days, venous re-transfusion is to entering
The duration of an exhibition, the patients with lung cancer of the HER1 positive (was carried out at the CARHER1-NKT cell utilizing the present invention
Before targeting immunization therapy, through repeatedly treatment (such as radiotherapy, chemotherapy and other drug symptomatic treatment
Deng), but all without obvious curative effects) internal, after feedback, the treatment situation to patient is estimated.
Fig. 8 is the CARHER1-NKT cell of the present invention pulmonary carcinoma to the 11 example progressive stage HER1 positives
In the therapeutic process of patient, the CARHER1-NKT cell sustainable existence time in peripheral blood in patients
Curve chart.Fig. 8 result shows, after CARHER1-NKT cell therapy, and 11 example progressive stages
CARHER1-NKT the most permanent the depositing of cell copy number in the peripheral blood from patients with lung cancer that HER1 is positive
, illustrate that the CARHER1-NKT cell of the present invention can be in pulmonary carcinoma positive for progressive stage HER1
Continue substantial amounts of propagation in the patient and play killing activity.
The patients with lung cancer that the CARHER1-NKT cell of the present invention is positive to 11 example progressive stage HER1
Therapeutic process in, the clinical toxicity of patient is shown in Table 1.As shown in table 1, through the present invention's
After CARHER1-NKT cell therapy, the toxicity that great majority reaction is 1-2 level that patient presents,
Illustrate that patient can be good at tolerating the treatment of CARHER1-NKT cell, the i.e. present invention
CARHER1-NKT cell, for the treatment of patients with lung cancer positive for progressive stage HER1, is safe and feasible
's.
Table 1
Fig. 9 is the CARHER1-NKT cell of the present invention pulmonary carcinoma to the two example progressive stage HER1 positives
In the therapeutic process of patient, the pneumonopathy stove of different time sections patient and pleura extractum variation diagram.Such as Fig. 9
Shown in, patients with lung cancer positive for two example progressive stage HER1 is through CARHER1-NKT cell therapy
After two courses for the treatment of, pleura extractum significantly reduces, and tumor focus size is also reduced significantly simultaneously, and clinic is examined
Break as PR.Illustrate that pulmonary carcinoma positive for progressive stage HER1 is suffered from by the CARHER1-NKT cell of the present invention
Person has and well treats curative effect.
The preferred embodiment of the present invention described in detail above, but, the present invention is not limited to above-mentioned reality
Execute the detail in mode, in the technology concept of the present invention, can be to the technical side of the present invention
Case carries out multiple simple variant, and these simple variant belong to protection scope of the present invention.
It is further to note that each the concrete technology described in above-mentioned detailed description of the invention is special
Levy, in the case of reconcilable, can be combined by any suitable means, in order to avoid need not
The repetition wanted, various possible compound modes are illustrated by the present invention the most separately.
Additionally, combination in any can also be carried out between the various different embodiment of the present invention, as long as its
Without prejudice to the thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (7)
1.CARHER1-NKT cell is used for the system for the treatment of of advanced HER1 positive lung cancer in preparation
Application in agent, it is characterised in that described CARHER1-NKT cell is by Chimeric antigen receptor
The NKT cell that HER1ScFv-CD8-CD137-CD3 ζ modifies, wherein, described chimeric antigen is subject to
Body HER1ScFv-CD8-CD137-CD3 ζ include series connection rat growth hormone signal peptide,
The hinge region of HER1ScFv, CD8 and cross-film district, the intracellular signal domain of CD137 and CD3 ζ
Intracellular signal domain.
Application the most according to claim 1, wherein, described Chimeric antigen receptor
HER1ScFv-CD8-CD137-CD3 ζ has the aminoacid sequence as shown in SEQ ID NO.1,
Preferably, the aminoacid sequence of described Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ
As shown in SEQ ID NO.1.
Application the most according to claim 1, wherein, encodes described Chimeric antigen receptor
The gene of HER1ScFv-CD8-CD137-CD3 ζ has the nucleotides sequence as shown in SEQ ID NO.2
Row, it is preferable that the nucleotide sequence of described gene is as shown in SEQ ID NO.2.
4. according to the application described in any one in claim 1-3, wherein, described
The preparation method of CARHER1-NKT cell includes: packaging carries pWPXL-HER1ScFv-CD8
The slow virus of-CD137-CD3 ζ, obtains viral concentration liquid；Utilize the viral concentration liquid inductance dye NKT obtained
Cell, makes NKT cell express Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ, its
In, described pWPXL-HER1ScFv-CD8-CD137-CD3 ζ is containing encoding chimeric antigen receptor
The Lentiviral of the gene of HER1ScFv-CD8-CD137-CD3 ζ.
Application the most according to claim 4, wherein, described CARHER1-NKT cell
Preparation method also includes being prepared via a method which NKT cell:
(1) in the presence of CD3 monoclonal antibody, interleukin-2 and interleukin-15, by single core
Cell carries out first stage cultivation；Preferably, the embodiment that the described first stage cultivates includes:
Being incubated at by mononuclearcell in a NKT cell culture fluid, a described NKT cell is cultivated
Liquid contains NKT cell culture medium, CD3 monoclonal antibody, interleukin-2 and interleukin-15；Enter
Preferably, in a NKT cell culture fluid, the concentration of described CD3 monoclonal antibody is one step
30-70ng/mL, and/or the concentration of described interleukin-2 is 300-700U/mL, and/or described white Jie
The concentration of element-15 is 30-70ng/mL；
(2) in the presence of interleukin-2, the cell cultivated the described first stage carries out second stage
Cultivate；Preferably, the embodiment that described second stage is cultivated includes: trained the described first stage
The cell supported is incubated in the 2nd NKT cell culture fluid, in described 2nd NKT cell culture fluid
Containing NKT cell culture medium and interleukin-2；It is further preferred that the 2nd NKT cell culture fluid
In, the concentration of described interleukin-2 is 300-700U/mL.
Application the most according to claim 5, wherein, the method for described infection NKT cell
Including:
(1) in the presence of viral concentration liquid, protamine and interleukin-2, NKT cell is entered
The row first stage infects and cultivates；Preferably, the embodiment that the infection of described first stage is cultivated includes:
NKT cell is incubated in the 3rd NKT cell culture fluid, described 3rd NKT cell culture fluid
Containing NKT cell culture medium, viral concentration liquid, protamine and interleukin-2；Further preferably
Ground, in the 3rd NKT cell culture fluid, the concentration of described interleukin-2 is 300-700U/mL；
(2) in the presence of CD3 monoclonal antibody, interleukin-2 and interleukin-15, by described
The second stage that carries out the cell that one stage infection is cultivated infects cultivates；Preferably, described second stage
Infect the embodiment cultivated to include: the described first stage infects the cell cultivated and is incubated at described
In oneth NKT cell culture fluid.
Application the most according to claim 6, wherein, the method for described infection NKT cell
Also include:
(3) first in the presence of interleukin-2, described second stage is infected the cell cultivated and carries out body
Outer induction, when the density of cell is 80-90%, then at CD3 monoclonal antibody, interleukin-2
In the presence of interleukin-15, cell is carried out amplification cultivation；Preferably, described external evoked reality
The mode of executing includes: described second stage is infected the cell cultivated and is incubated at described 2nd NKT cell
In culture fluid, the embodiment of described amplification cultivation includes: cell is incubated at a described NKT
In cell culture fluid.
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