CN105367661A - Chimeric antigen receptor, gene and recombinant expression vector of chimeric antigen receptor, engineering HER1 targeted NKT cells and application of engineering HER1 targeted NKT cells - Google Patents
Chimeric antigen receptor, gene and recombinant expression vector of chimeric antigen receptor, engineering HER1 targeted NKT cells and application of engineering HER1 targeted NKT cells Download PDFInfo
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Abstract
The invention discloses a chimeric antigen receptor, a gene and recombinant expression vector of the chimeric antigen receptor, engineering HER1 targeted NKT cells and application of the engineering HER1 targeted NKT cells. The chimeric antigen receptor is HER1ScFv-CD8-CD137-CD3 zeta and is formed by connecting HER1ScFv, a hinge region and a transmembrane region of CD8, an intracellular signal structure domain of CD137 and intracellular signal structure domain of CD3 zeta in series. When the NKT cells modified by the chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 zeta are used for treating an HER1 positive lung cancer in the progressive stage, the drug resistance caused when an HER1 tyrosine kinase inhibitor is adopted can be effectively avoided, and certain distinctive kill activity is achieved on lung cancer cells.
Description
Technical field
The invention belongs to knubble biological arts, particularly, a kind of Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ in adoptive immunotherapy and gene thereof and recombinant expression vector, the NKT cell (CARHER1-NKT cell) of through engineering approaches HER1 targeting and application thereof is related to.
Background technology
EGFR (epithelialgrowthfactorreceptor, EGF-R ELISA) i.e. HER1, it is the expression product of proto-oncogene c-erbB1, belong to human epidermal growth factor acceptor (HER) family, itself there is junket ammonia kinase enzyme active, can related gene in active cell core once combine with Urogastron (EGF), thus promote cell fission propagation.HER1 is high expression level in Several Kinds of Malignancy, and research finds that HER1 is expressed in most of patients with lung cancer, and expression level reaches 80%, the progress of its expression level and lung cancer disease and shift relevant.Although the treatment of lung cancer has obtained certain progress recently, still do not improve the survival rate of patient, therefore needed badly and inquire into new therapy to overcome this puzzlement.
At present, when treatment of advanced HER1 positive lung cancer patient, HER1 tyrosine kinase inhibitor (tyrosinekinaseinhibitors, TKIs) clinical investigation phase is in, but clinical effectiveness shows, only part patients with lung cancer adopts HER1 treatment with tyrosine kinase inhibitors effective, and patient finally can produce certain resistance, thus affect the curative effect of medicine.
Summary of the invention
The object of the invention is the defect in order to the resistance caused when overcoming in prior art and adopt HER1 treatment with tyrosine kinase inhibitors progressive stage HER1 positive lung cancer patient, a kind of Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ and gene thereof and recombinant expression vector are provided, the NKT cell (CARHER1-NKT cell) of through engineering approaches HER1 targeting and application thereof, the NKT cell that Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ modifies is when treatment of advanced HER1 positive lung cancer, effectively can avoid the resistance caused when adopting HER1 tyrosine kinase inhibitor, to lung carcinoma cell, there is certain special target killing activity.
The present inventor surprisingly finds under study for action, during the NKT cell therapy progressive stage HER1 positive lung cancer adopting Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ to modify, effectively can avoid the resistance caused when adopting HER1 tyrosine kinase inhibitor, to lung carcinoma cell, there is certain special target killing activity.
Therefore, to achieve these goals, first aspect, the invention provides a kind of Chimeric antigen receptor, described Chimeric antigen receptor is HER1ScFv-CD8-CD137-CD3 ζ, by the intracellular signal structural domain of the hinge area (hinge district) of HER1ScFv, CD8 and cross-film district, CD137 and the intracellular signal structural domain of CD3 ζ in series.
Second aspect, the invention provides the gene of above-mentioned Chimeric antigen receptor of encoding.
The third aspect, the invention provides the recombinant expression vector containing said gene.
Fourth aspect, the invention provides a kind of NKT cell of through engineering approaches HER1 targeting, and described NKT cell is the NKT cell that above-mentioned Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ modifies.
5th aspect, the invention provides the application of NKT cell in the preparation for the preparation for the treatment of tumour of above-mentioned through engineering approaches HER1 targeting.
When treatment of advanced HER1 positive lung cancer, the NKT cell that Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ of the present invention modifies, namely the NKT cell of through engineering approaches HER1 targeting can specific binding HER1 antigen, the obvious survival time of prolongation immunocyte in patient body, strengthen the ability of immunocyte targets identification lung carcinoma cell surface HER1 antigen, strengthen the specific killing of lung carcinoma cell active, and really effectively can avoid the resistance that causes when adopting HER1 tyrosine kinase inhibitor.The NKT cell of through engineering approaches HER1 targeting of the present invention is that treatment of advanced HER1 positive lung cancer provides a kind of selection newly, has good industrial application prospect.
Other features and advantages of the present invention are described in detail in embodiment part subsequently.
Accompanying drawing explanation
Fig. 1 is the result that flow cytometry is analyzed the NKT cell phenotype of separation and Culture.
Fig. 2 is the electroresis appraisal figure of the restriction enzyme MluI/SalI double digestion fragment of Lentiviral pWPT-CD8-CD137-CD3 ζ of the present invention.
Fig. 3 is the electroresis appraisal figure of the restriction enzyme BamHI/SalI double digestion fragment of Lentiviral pWPT-HER1ScFv-CD8-CD137-CD3 ζ of the present invention.
Fig. 4 is the structural representation of Lentiviral pWPT-HER1ScFv-CD8-CD137-CD3 ζ of the present invention, and wherein, counterclockwise sequence is forward gene sheet degree, is cdna reverse fragment clockwise.
Fig. 5 is that Flow cytometry contains the viral concentration liquid of Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ to the efficiency of infection of NKT cell.
Fig. 6 is the result of NKT cell (CARHER1-NKT cell) phenotypic evaluation that Flow cytometry Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ modifies.
Fig. 7 is the cytotoxicity analysis figure of CARHER1-NKT cell of the present invention to human lung carcinoma cell lethal effect.
Fig. 8 be CARHER1-NKT cell of the present invention in progressive stage HER1 positive lung cancer patient treatment procedure, the change of patient's lesions position HER1 positive lung cancer cells number.
Fig. 9 be CARHER1-NKT cell of the present invention in progressive stage HER1 positive lung cancer patient treatment procedure, patient's lesions position striograph change.
Embodiment
Below the specific embodiment of the present invention is described in detail.Should be understood that, embodiment described herein, only for instruction and explanation of the present invention, is not limited to the present invention.
The invention provides a kind of Chimeric antigen receptor, described Chimeric antigen receptor is HER1ScFv-CD8-CD137-CD3 ζ, by the intracellular signal structural domain of the hinge area of HER1ScFv, CD8 and cross-film district, CD137 and the intracellular signal structural domain of CD3 ζ in series.Under preferable case, the aminoacid sequence of Chimeric antigen receptor is as shown in SEQIDNO.1.
The invention provides the gene of above-mentioned Chimeric antigen receptor of encoding.Under preferable case, the nucleotide sequence of the gene of above-mentioned Chimeric antigen receptor of encoding is as shown in SEQIDNO.2.
The invention provides the recombinant expression vector containing said gene.Under preferable case, recombinant expression vector is Lentiviral.For Lentiviral, there is no particular limitation, as long as can with assistant carrier cotransfection packing cell as 293T packing cell, obtain the NTK cell of viral concentration liquid and Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ modification, under preferable case, Lentiviral is pWPT-HER1ScFv-CD8-CD137-CD3 ζ.
For the preparation method of Lentiviral pWPT-HER1ScFv-CD8-CD137-CD3 ζ, there is no particular limitation, the various methods can be able to expected for those skilled in the art, under preferable case, the preparation method of Lentiviral pWPT-HER1ScFv-CD8-CD137-CD3 ζ comprises the following steps:
(1) increase respectively the hinge district of CD8 and cross-film district, the intracellular signal structural domain of CD137 and the intracellular signal structural domain of CD3 ζ from NKT cell cDNA, and be cloned in carrier pWPT-GFP, builds and obtain pWPT-CD8-CD137-CD3 ζ;
(2) nucleotide sequence of composite coding rat growth hormone signal peptide and HER1ScFv, and be cloned in pWPT-CD8-CD137-CD3 ζ, after sequence verification, obtain the pWPT-HER1ScFv-CD8-CD137-CD3 ζ that sequence is correct.
In step (1), for the method for the hinge district of the CD8 that increases respectively from NKT cell cDNA and cross-film district, the intracellular signal structural domain of CD137 and the intracellular signal structural domain of CD3 ζ, there is no particular limitation, the various methods can commonly used for this area can be such as RT-PCR method.Wherein, NKT cell by being separated the mononuclearcell in people's venous blood, then can carrying out cultivation and obtaining.
Particularly, the method obtaining pWPT-CD8-CD137-CD3 ζ can comprise: the total serum IgE extracting NKT cell, reverse transcription obtains NKT cell cDNA, with the NKT cell cDNA obtained for template, primer P1 (SEQIDNO.11) and P2 (SEQIDNO.12) is utilized to carry out hinge district and cross-film district (SEQIDNO.3) of pcr amplification acquisition CD8 gene; Primer P3 (SEQIDNO.13) and P4 (SEQIDNO.14) is utilized to carry out the intracellular signal structural domain (SEQIDNO.4) of pcr amplification acquisition CD137 gene; Primer P5 (SEQIDNO.15) and P6 (SEQIDNO.16) is utilized to carry out the intracellular signal structural domain (SEQIDNO.5) of pcr amplification acquisition CD3 ζ gene, the PCR primer of acquisition is carried out double digestion respectively, is then connected with the Lentiviral pWPT-GFP after MluI/SalI double digestion.
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 the conventional various methods in this area, such as, can be synthesized by full genome synthetic technology.
Particularly, the method obtaining the correct pWPT-HER1ScFv-CD8-CD137-CD3 ζ of sequence can comprise: by the nucleotide sequence (SEQIDNO.8) of full genome synthetic technology composite coding rat growth hormone signal peptide and HER1ScFv fusion gene, be cloned in carrier pGSI, obtain pGSI-HER1ScFv; Then pGSI-HER1ScFv is carried out EcoR V/MluI double digestion, with through restriction enzyme BamHI single endonuclease digestion, with KlenowFragment enzyme tack, connect with the recombinant plasmid pWPT-CD8-CD137-CD3 ζ that the step (1) after MluI single endonuclease digestion obtains again, through order-checking qualification, obtain the pWPT-HER1ScFv-CD8-CD137-CD3 ζ that sequence is correct.Wherein, the nucleotide sequence of rat growth hormone signal peptide is as shown in SEQIDNO.6, and HER1ScFv nucleotide sequence is as shown in SEQIDNO.7.
Present invention also offers a kind of NKT cell of through engineering approaches HER1 targeting, described NKT cell is the NKT cell (i.e. CARHER1-NKT cell) modified by above-mentioned Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ.
For the preparation method of the NKT cell of through engineering approaches HER1 targeting, there is no particular limitation, the any method can be able to expected for those skilled in the art, under preferable case, the method comprises: packaging carries the slow virus of pWPT-HER1ScFv-CD8-CD137-CD3 ζ encoding gene; Utilize the slow virus infection NKT cell obtained, make NKT cell expressing Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ.
For packing the method for carrying the slow virus of pWPT-HER1ScFv-CD8-CD137-CD3 ζ encoding gene, there is no particular limitation, the various methods can commonly used for those skilled in the art, under preferable case, by Lentiviral pWPT-HER1ScFv-CD8-CD137-CD3 ζ and helper plasmid (as psPAX2, pMD2.G) cotransfection 293T packing cell, viral supernatants is collected during transfection 48-72h, centrifugal, filter, in filtrate, add 5 × PEG6000-NaCl mix, supernatant is abandoned after centrifugal, the aseptic PBS of precipitation 0-4 DEG C of precooling dissolves, obtain viral concentration liquid.
Method for slow virus infection NKT cell is not particularly limited, and the various methods can commonly used for this area, under preferable case, the method comprises: get 1 × 10
7-5 × 10
7individual NKT cell, discards old nutrient solution, adds the fresh GT-T551 nutrient solution of 2-4mL, then adds 200-400 μ L viral concentration liquid, 2-4 μ L1 × 10
-6mg/mL protamine and final concentration are the IL-2 of 800-1200U/mL, be placed in 30-37 DEG C, saturated humidity is the CO of 3-6%
2after infecting 12-16h in incubator, abandon nutrient solution, cell gone to and does not wrap in the culturing bottle of quilt, add the GT-T551 substratum of 20-50mL, then add the IL-2 that final concentration is 800-1200U/mL, in 30-37 DEG C, saturated humidity is the CO of 3-6%
2cultivate 12-18h in incubator, obtain the NKT cell that Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ modifies.
Further preferably, the method of slow virus infection NKT cell also comprises: the final concentration of the NKT cell IL-2 of the slow virus infection above-mentioned cultivation obtained afterwards is that the GT-T551 nutrient solution of 800-1200U/mL carries out external evoked, proceed in cell culture bags by cell when the density of cell is 80-90%, the final concentration adding IL-2 every 1.5-2.5 days is that the fresh GT-T551 nutrient solution of 800-1200U/mL carries out amplification cultivation and is 1 × 10 by cell amplification to total amount
9-2 × 10
9individual cell.
The maturation protein aminoacid sequence of the Chimeric antigen receptor of the NKT cell expressing that Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ modifies is as shown in SEQIDNO.1.Wherein, what those skilled in the art should understand that is, Chimeric antigen receptor precursor protein by the intracellular signal structural domain of the hinge district of signal peptide, HER1ScFv, CD8 and cross-film district, CD137 and the intracellular signal structural domain of CD3 ζ in series, in cell, become ripe Chimeric antigen receptor albumen after rough endoplasmic reticulum excision signal peptide after protein translation, secretion exports rear and is positioned on the cytolemma of NKT cell.Gene coded sequence corresponding to the maturation protein aminoacid sequence of this Chimeric antigen receptor is as shown in SEQIDNO.2.The structure that this Chimeric antigen receptor is in series with the intracellular signal structural domain of the hinge district of gene C D8 and cross-film district and CD137 and CD3 ζ is for intracellular signaling structural domain, its aminoacid sequence is as shown in SEQIDNO.9, and corresponding gene coded sequence is as shown in SEQIDNO.10.
Present invention also offers the NKT cell of the through engineering approaches HER1 targeting that aforesaid method prepares.
Present invention also offers the application of NKT cell in the preparation for the preparation for the treatment of tumour of through engineering approaches HER1 targeting.Under preferable case, tumour refers to progressive stage HER1 positive lung cancer, is especially the progressive stage HER1 positive lung cancer of recurrence refractory.
Embodiment
The present invention is further illustrated for following embodiment, but therefore do not limit the present invention.
Experimental technique in following examples, if no special instructions, is this area ordinary method.Experiment material used in following embodiment, if no special instructions, is the purchase from routine biochemistry reagent shop and obtains, 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) all purchased from TaKaRa company.
Recombinant human protein's interferon-γ, rhIL-2 are purchased from protech company.
Total RNA extraction reagent box RNAisoReagent, high-fidelity DNA polymerase (
hSDNAPolymerase), T4DNA ligase enzyme is purchased from TaKaRa company.
RevertAid
tMfirstStrandcDNASynthesisKit is purchased from Fermentas company.
Bgl II, EcoRI, MluI, BamHI, SalI, EcoR V are purchased from Fermentas company.
Modifying enzyme KlenowFragment is purchased from Fermentas company.
Sepharose DNA reclaims test kit, common DNA Product Purification Kit, the little extraction reagent kit of plasmid all purchased from Tian Gen biochemical technology company limited.
PWPT-GFP, psPAX2, pMD2.G are all purchased from Addgene company.
PGSI is purchased from Tian Yihuiyuan bio tech ltd, Beijing.
Trans1-T1PhageResistant Competent cell is purchased from Beijing Quanshijin Biotechnology Co., Ltd.
Lipofectamine
tM2000TransfectionReagent transfection reagent is purchased from Invitrogen company.
293T packing cell purchased from American ATCC.
In PEG6000-NaCl, PEG6000 final concentration is 25.5 quality %, NaCl final concentration is that 1.2M, PEG6000 and NaCl are all purchased from Suo Laibao bio tech ltd, Shanghai.
Foetal calf serum is purchased from German PAA company.
The human A549 cell lines purchased from American ATCC of high expression level HER1.
CellCountingKit-8 (CCK8) test kit is purchased from Beijing Wo Bisen Science and Technology Ltd..
All primers synthesize by Tian Yihuiyuan bio tech ltd, Beijing.
The preparation of embodiment 1NKT cell
(1) people's venous blood is got in the valve tube containing heparin.Adopt lymphocyte separation medium, be separated by density gradient centrifugation method and obtain mononuclearcell (PBMCs).
(2), after PBMCs washes three times, adopting the NKT cell culture medium GT-T551 of the foetal calf serum containing 0.6 volume % to adjust final concentration of cells is 2 × 10
6individual cell/mL; Cell is inoculated in advance through the 75cm of final concentration to be 5 μ g/mLCD3 monoclonal antibodies and final concentration the be retronectin bag quilt of 10 μ g/mL
2in Tissue Culture Flask.Then in substratum, add final concentration is recombinant human protein's interferon-γ of 1000U/mL and the rhIL-2 of 1000U/mL, 37 DEG C, saturated humidity is the CO of 5%
2cultivate in incubator.
(3) the 4th days, in culturing bottle, add the NKT cell culture medium GT-T551 that 100mL contains the foetal calf serum of 0.6 volume %, and add the rhIL-2 that final concentration is 1000U/mL.In 37 DEG C, saturated humidity is the CO of 5%
2cultivate 4 days in incubator, obtain NKT cell, flow cytometry is analyzed NKT cell phenotype.The results are shown in Figure 1, wherein CD3
+: 92.80%; CD3
+cD4
+: 28.25%; CD3
+cD8
+: 67.11%; CD3
+cD56
+: 6.51%; CD8
+cD56
+: 6.00%.
The structure of embodiment 2 Lentiviral pWPT-HER1ScFv-CD8-CD137-CD3 ζ
(1) preparation of NKT cell cDNA
Centrifugation embodiment 1 cultivates the NKT cell obtained, and extract the total serum IgE of cell with total RNA extraction reagent box RNAisoReagent ,-80 DEG C save backup.The total serum IgE Reverse Transcriptase kit RevertAid extracted
tMfirstStrandcDNASynthesisKit reverse transcription obtains NKT cell cDNA, and-20 DEG C save backup.
(2) preparation of slow virus plasmid pWPT-CD8-CD137-CD3 ζ
Design and synthesize following primer sequence (wherein, underscore is labeled as protection base, and square frame is restriction enzyme site):
P1(SEQIDNO.11)：
GATC CTGAGCAACTCCATCATGTACTTC
MluI
P2(SEQIDNO.12)：
GATC GCAGTAAAGGGTGATAACCAGTGA
BglII
P3(SEQIDNO.13)：
GATC AAACGGGGCAGAAAGAAACTCC
BglII
P4(SEQIDNO.14)：
GATC CAGTTCACATCCTCCTTCTTCTTCT
EcoRI
P5(SEQIDNO.15)：
GATC AGAGTGAAGTTCAGCAGGAGCG
EcoRI
P6(SEQIDNO.16)：
GATC TTAGCGAGGGGGCAGGGC
SalI
With NKT cell cDNA in step (1) for template, pcr amplification is carried out with primer P1 and P2, obtain hinge district and the cross-film district of the CD8 of long 287bp, nucleotide sequence is as shown in SEQIDNO.3, and two ends are respectively containing MluI and Bgl II restriction enzyme site and protection base; Carry out pcr amplification with primer P3 and P4, obtain the CD137 intracellular signal structural domain of long 146bp, nucleotide sequence is as shown in SEQIDNO.4, and two ends are contained Bgl II and EcoRI restriction enzyme site respectively and protected base; Carry out pcr amplification with primer P5 and P6, obtain the intracellular signal structural domain of the CD3 ζ of long 359bp, nucleotide sequence is as shown in SEQIDNO.5, and two ends are respectively containing EcoRI and SalI restriction enzyme site and protection base.Each step pcr amplification reaction system is identical, for the CD137 intracellular signal structural domain that increases, carries out pcr amplification, the reference of PCR reaction conditions
the specification sheets of HSDNAPolymerase, reaction system (50 μ L) is as follows:
Distilled water: 32.5 μ L
5 × reaction buffer:10 μ L
DNTP mixture (often kind of 2.5mM): 4 μ L
P3(10mM):1μL
P4(10mM):1μL
NKT cell cDNA (200ng/ul): 1 μ L
HSDNAPolymerase：0.5μL
By above-mentioned PCR primer with 1% sepharose be separated, with sepharose DNA reclaim test kit carry out DNA fragmentation recovery.Carry out double digestion reaction respectively after obtaining fragment, the common DNA Product Purification Kit of digestion products reclaims for subsequent use.
Lentiviral pWPT-GFP MluI/SalI double digestion, digestion products is separated through the sepharose of 1%, reclaim test kit with sepharose DNA and reclaim large carrier segments, then be connected by T4DNA ligase enzyme with CD8, CD137, CD3 ζ fragment reclaimed before, connect product conversion Trans1-T1PhageResistant Competent cell, picking mono-clonal after 37 DEG C of cultivation 16h, extracts plasmid with the little extraction reagent kit of plasmid after 250rpm cultivates 12h by 37 DEG C.The plasmid extracted is through restriction enzyme MluI and the qualification of SalI double digestion, and qualification electrophorogram is shown in Fig. 2, wherein, and 1 swimming lane: DNA molecular amount mark D2000; 2 swimming lanes: the endonuclease bamhi (835bp) of plasmid pWPT-GFP; 3 swimming lanes: the endonuclease bamhi (756bp) of plasmid pWPT-CD8-CD137-CD3 ζ.Tian Yihuiyuan bio tech ltd, Beijing is sent to check order to the fusion gene fragment inserted the correct plasmid of qualification.By recombinant plasmid called after pWPT-CD8-CD137-CD3 ζ correct for sequencing result.
(3) preparation of slow virus plasmid pWPT-HER1ScFv-CD8-CD137-CD3 ζ
The nucleotide sequence of full genome composite coding rat growth hormone signal peptide and HER1ScFv fusion gene, sequence is as shown in SEQIDNO.8, synthesized by Tian Yihuiyuan bio tech ltd, Beijing, its 5 ' end is containing EcoR V restriction enzyme site, kozak sequence, 3 ' end is containing MluI restriction enzyme site, by foregoing fusion gene clone in plasmid pGSI, called after pGSI-HER1ScFv.Plasmid is through EcoR V/MluI double digestion, and digestion products is separated through 1% sepharose, reclaims test kit recovery object fragment for subsequent use with sepharose DNA.
PWPT-CD8-CD137-CD3 ζ plasmid is through restriction enzyme BamHI single endonuclease digestion, use KlenowFragment enzyme tack again, then carry out MluI single endonuclease digestion, digestion products is separated through 1% sepharose, reclaims test kit recovery carrier segments for subsequent use with sepharose DNA.Then have the DNA fragmentation of rat growth hormone signal peptide and HER1ScFv to be connected by T4DNA ligase enzyme with recovery, concrete grammar is shown in specification sheets.Product conversion Trans1-T1PhageResistant Competent cell will be connected, picking mono-clonal after 37 DEG C of cultivation 16h, 37 DEG C, after 250rpm cultivates 12h, extract plasmid with the little extraction reagent kit of plasmid.Extract plasmid through restriction enzyme BamHI/SalI double digestion qualification, qualification result as shown in Figure 3, wherein, M1:DNA molecular weight marker D15000; 1 swimming lane: the endonuclease bamhi (920bp) of plasmid pWPT-HER1ScFv-CD8-CD137-CD3 ζ; 2 swimming lanes: the endonuclease bamhi (774bp) of plasmid pWPT-CD8-CD137-CD3 ζ; 3 swimming lanes: the endonuclease bamhi (853bp) of plasmid pWPT-GFP; M2:DNA molecular weight marker D2000.Tian Yihuiyuan bio tech ltd, Beijing is sent to check order to the fusion gene fragment inserted the correct plasmid of qualification.By recombinant plasmid called after pWPT-HER1ScFv-CD8-CD137-CD3 ζ correct for sequencing result, its structural representation as shown in Figure 4, comprising rat growth hormone signal peptide (nucleotide sequence is as shown in SEQIDNO.6), anti-HER1 single-chain antibody (nucleotide sequence is as shown in SEQIDNO.7), the intracellular signal structural domain of the hinge district of CD8 and the intracellular signal structural domain of cross-film district and CD137 and CD3 ζ, wherein, the structure that this Chimeric antigen receptor is in series with the intracellular signal structural domain of the hinge district of gene C D8 and cross-film district and CD137 and CD3 ζ is for intracellular signaling structural domain, its aminoacid sequence is as shown in SEQIDNO.9, corresponding gene coded sequence is as shown in SEQIDNO.10.
The preparation of the NKT cell that embodiment 3 Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ modifies
(1) packaging of slow virus and concentrated
Measure the concentration of slow virus expression plasmid pWPT-HER1ScFv-CD8-CD137-CD3 ζ and helper plasmid psPAX2, pMD2.G respectively with spectrophotometer, three kinds of plasmids are with the mass ratio Lipofectamine of 4:2:1
tM2000TransfectionReagent transfection reagent cotransfection 293T packing cell.Collect viral supernatants respectively when transfection 48h, 72h in 50mLEP pipe, 4 DEG C, the centrifugal 10min of 2000g, shift the supernatant that obtains for twice extremely in new EP pipe, with 4.5 μm of frit viral supernatants; The viral supernatants filtered and 5 × PEG6000-NaCl mix according to the volume ratio of 4:1,4 DEG C of standing 2h, then 4 DEG C, the centrifugal 20min of 10000g, abandon supernatant, the aseptic PBS of 4 DEG C of precoolings of precipitation 1mL dissolves, and obtains the viral concentration liquid of Chimeric antigen receptor, carry out packing by every pipe 100 μ L ,-80 DEG C save backup.
According to the method described above, utilize slow virus expression plasmid pWPT-GFP and helper plasmid psPAX2, pMD2.G cotransfection 293T packing cell, collect viral supernatants, concentrated, obtain the slow virus concentrated solution of expressing GFP green fluorescent protein.
(2) amplification cultivation of slow virus infection NKT cell and infected cell
Example 1 at 75cm
2cultivate in culturing bottle 1 × 10
7individual NKT cell, discards old nutrient solution, add 2mL fresh NKT cell culture medium GT-T551, viral concentration liquid that 200 μ L steps (1) obtain, 2 μ L1 × 10
-6mg/mL protamine, final concentration is the rhIL-2 of 1000U/mL, is placed in 37 DEG C, saturated humidity is the CO of 5%
2infect after 12 hours in incubator, abandon nutrient solution, the NKT cell obtained is called CARHER1-NKT cell.With the slow virus concentrated solution of expressing GFP green fluorescent protein, NKT cell is synchronously infected (the NKT cell obtained is called CART-GFP cell), for calculating the efficiency of infection of this virus simultaneously.Metainfective cell is gone to the 75cm without CD3 and retronectin bag quilt
2in culturing bottle, add the NKT cell culture medium GT-T551 of 20mL, then add the rhIL-2 that final concentration is 1000U/mL, in 37 DEG C, saturated humidity is the CO of 5%
218h is cultivated in incubator.With the efficiency of infection of this virus of Flow cytometry, result as shown in Figure 5, efficiency of infection 41.20%.
(3) external evoked amplification CARHER1-NKT cell mass
The NKT cell culture medium GT-T551 being 1000U/mL by the final concentration of the NKT cell rhIL-2 after above-mentioned cultivation carries out external evoked, when the density of cell is 85%, cell is proceeded in cell culture bags, the final concentration adding rhIL-2 every 2 days is that the fresh NKT cell culture medium GT-T551 of 1000U/mL carries out amplification cultivation, treats that cell amplification is 1.5 × 10 to total amount
9after about individual cell, adopt flow cytometer to identify the cell colony infected, cell phenotype generally reaches CD3 positive cell ratio > 90%; CD3CD8 positive cell ratio >70%; The two positive cell ratio >5% of CD3CD56, the results are shown in Figure 6, CD3
+: 96.46%; CD3
+cD4
+: 16.71%; CD3
+cD8
+: 75.68%; CD3
+cD56
+: 5.44%; CD8
+cD56
+: 3.65%.
Embodiment 4CARHER1-NKT cell is to the cytotoxicity analysis of human lung carcinoma cell lethal effect
The human A549 cell lines getting high expression level HER1 is inoculated in 96 orifice plates, after 37 DEG C of incubator overnight incubation, the NKT cell cultivated in CARHER1-NKT cell, CART-GFP cell and the embodiment 1 prepared in Example 3 respectively, to imitate target ratio (killer cell: target cell) 2.5:1,5:1,10:1,20:1 and A549 carry out Dual culture, after the Dual culture of 4h, the CCK8 that each hole adds 10 μ L dyes.Killer cell control group is set simultaneously and is respectively the NKT cell cultivated in CARHER1-NKT cell, CART-GFP cell and the embodiment 1 prepared in embodiment 3, and the CCK8 adding identical amount dyes; And to arrange target cell control group be do not add the human A549 cell lines that immunocyte kills and wounds process, and the CCK8 adding identical amount dyes.Microplate reader detects apoptosis situation, apoptotic amount is according to formulae discovery below: apoptosis rate={ 1-[(experimental group-killer cell control group-target cell control group)/experimental group] } × 100%, in this formula, killer cell control group is the light absorption value only having killer cell not add target cell to record, and target cell control group is the light absorption value only having target cell not add killer cell to record; Experimental group is the light absorption value recorded after the immunocyte adding corresponding effect target ratio (killer cell: target cell) kills and wounds process, sees Fig. 7.The lung carcinoma cell of NKT cell to high expression level HER1 that Chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 ζ modifies has specific killing activity, and the specific killing activity of CARHER1-NKT cell is obviously better than NKT cell.
Embodiment 5CARHER1-NKT cell is to the result for the treatment of of progressive stage HER1 positive lung cancer patient
Get 5 × 10
8the NKT cell (i.e. CARHER1-NKT cell) that individual HER1ScFv-CD8-CD137-CD3 ζ modifies, after 100ml normal saline dilution, within continuous three days, venous re-transfusion is to progressive stage HER1 positive lung cancer patient (the 4 phase patients with lung cancer HER1 tyrosine kinase inhibitor being produced to resistance, before utilizing CARHER1-NKT cell of the present invention to carry out target immunotherapy, through repeatedly treating (as operative treatment, radiotherapy, chemotherapy and HER1 treatment with tyrosine kinase inhibitors etc.), but all without obvious curative effects) in body, after feedback, the treatment situation of patient is assessed.
Fig. 8 be immunohistochemical methods detect CARHER1-NKT cell feed back to resistance produces to HER1 tyrosine kinase inhibitor progressive stage HER1 positive lung cancer patient body in after, the change of patient same lesions position HER1 positive lung cancer cells number.Patient's same lesions position puncture sample result is shown, compared with before treatment, through target immune cell therapy after three months, in patient body, the lung carcinoma cell of the HER1 positive obviously reduces, and illustrates that CARHER1-NKT cell can play killing activity to the target cell produced HER1 tyrosine kinase inhibitor in the progressive stage HER1 positive lung cancer patient body of resistance.
As shown in Figure 9, after CT analyzes the progressive stage HER1 positive lung cancer patient of CARHER1-NKT cell therapy to HER1 tyrosine kinase inhibitor generation resistance, the striograph change of patient's lesions position.Striograph result shows, compared with before treatment, after target immune cell therapy, patient many places focus bounces back to some extent; And compared with latter one month for the treatment of, treating focus after three months reduces more obvious, the hydrothorax of patient also significantly reduces simultaneously, illustrates that CARHER1-NKT cell therapy can make to alleviate to some extent the disease of the progressive stage HER1 positive lung cancer patient of HER1 tyrosine kinase inhibitor generation resistance.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each concrete technical characteristic described in above-mentioned embodiment, in reconcilable situation, can be combined by any suitable mode, in order to avoid unnecessary repetition, the present invention illustrates no longer separately to various possible array mode.
In addition, also can carry out arbitrary combination between various different embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (10)
1. a Chimeric antigen receptor, it is characterized in that, described Chimeric antigen receptor is HER1ScFv-CD8-CD137-CD3 ζ, by the intracellular signal structural domain of the hinge area of HER1ScFv, CD8 and cross-film district, CD137 and the intracellular signal structural domain of CD3 ζ in series.
2. Chimeric antigen receptor according to claim 1, wherein, the aminoacid sequence of described Chimeric antigen receptor is as shown in SEQIDNO.1.
3. the gene of the Chimeric antigen receptor of coding described in claim 1 or 2.
4. gene according to claim 3, wherein, the nucleotide sequence of described gene is as shown in SEQIDNO.2.
5. the recombinant expression vector containing the gene described in claim 3 or 4.
6. recombinant expression vector according to claim 5, wherein, described recombinant expression vector is Lentiviral.
7. recombinant expression vector according to claim 6, wherein, described Lentiviral is pWPT-HER1ScFv-CD8-CD137-CD3 ζ.
8. a NKT cell for through engineering approaches HER1 targeting, is characterized in that, described NKT cell is the NKT cell modified by the Chimeric antigen receptor described in claim 1 or 2.
9. the application of NKT cell in the preparation for the preparation for the treatment of tumour of through engineering approaches HER1 targeting according to claim 8.
10. application according to claim 9, wherein, described tumour refers to progressive stage HER1 positive lung cancer.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410426060.XA CN105367661B (en) | 2014-08-26 | 2014-08-26 | Chimeric antigen receptor and its gene and recombinant expression carrier, the NKT cell of engineering HER1 targeting and its application |
| PCT/CN2015/088172 WO2016029855A1 (en) | 2014-08-26 | 2015-08-26 | Her1-targeted chimeric antigen receptor and nkt cell as well as preparation method therefor and application thereof |
Applications Claiming Priority (1)
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| CN105924526A (en) * | 2015-09-11 | 2016-09-07 | 中国人民解放军总医院 | Chimeric antigen receptor, gene and recombinant expression vector thereof, CARHER1-NKT cell and preparation method and application thereof |
| WO2022068870A1 (en) * | 2020-09-30 | 2022-04-07 | 中国科学院动物研究所 | Egfr-targeting chimeric antigen receptor |
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| CN113651893B (en) * | 2021-08-12 | 2023-08-01 | 上海生物制品研究所有限责任公司 | HER2 and MESO combined double-target CAR-T vector, construction method thereof and application thereof in cancers |
| CN115558641B (en) * | 2022-11-14 | 2023-05-12 | 四川新生命干细胞科技股份有限公司 | High-purity effector immune cell population, culture method, reagent composition and application thereof |
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| CN103483453A (en) * | 2012-06-12 | 2014-01-01 | 上海吴孟超医学科技基金会 | Chimeric antigen receptor combining EGFR (epidermal growth factor receptor) family proteins and composition and uses thereof |
| CN103820393A (en) * | 2014-02-24 | 2014-05-28 | 中国人民解放军总医院 | Engineered CD20 targeting NKT cell and its preparation method and application |
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| CN103483453A (en) * | 2012-06-12 | 2014-01-01 | 上海吴孟超医学科技基金会 | Chimeric antigen receptor combining EGFR (epidermal growth factor receptor) family proteins and composition and uses thereof |
| CN103820393A (en) * | 2014-02-24 | 2014-05-28 | 中国人民解放军总医院 | Engineered CD20 targeting NKT cell and its preparation method and application |
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| CN105924526A (en) * | 2015-09-11 | 2016-09-07 | 中国人民解放军总医院 | Chimeric antigen receptor, gene and recombinant expression vector thereof, CARHER1-NKT cell and preparation method and application thereof |
| CN105924526B (en) * | 2015-09-11 | 2019-08-06 | 中国人民解放军总医院 | Chimeric antigen receptor and its gene and recombinant expression carrier, CARHER1-NKT cell and its preparation method and application |
| WO2022068870A1 (en) * | 2020-09-30 | 2022-04-07 | 中国科学院动物研究所 | Egfr-targeting chimeric antigen receptor |
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