CN103820393A - Engineered CD20 targeting NKT cell and its preparation method and application - Google Patents
Engineered CD20 targeting NKT cell and its preparation method and application Download PDFInfo
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Abstract
The invention relates to an engineered CD20 targeting NKT cell and its preparation method and application. The cell is an NKT cell modified by a chimeric antigen receptor CD20ScFv-CD8-CD137-CD3zeta. An amino acid sequence of the chimeric antigen receptor is as shown in SEQID NO.1 in a sequence table. The preparation method comprises the following steps: firstly constructing a chimeric antigen receptor pWPT-CD20ScFv-CD8-CD137-CD3zeta, carrying out infection of the NKT cell, inducing in vitro and carrying out amplification culture so as to obtain the engineered CD20 targeting NKT cell. During advanced CD20 positive malignant tumors, the engineered CD20 targeting NKT cell can be used to obviously prolong survival time of immune cells in a patient's body, enhance the immune cells' targeting recognition capability of tumor antigen and reinforce the killing activity of tumor cells.
Description
Technical field
The invention belongs to knubble biological goods field, relate to NKT cell (CAR20-NKT cell), its preparation and the application of a kind of through engineering approaches CD20 targeting in adoptive immunotherapy.
Background technology
Clinical in TCR
-, mIg
-, CD56
+, CD16
+, CD3
+lymphoidocyte is accredited as NKT cell.NKT cell had both been expressed T cell surface marker, expressed again the surface marker of NK cell, and NKT cell can be identified tumour cell and the virus infected cell with kill mutation, and to the acellular toxic action of normal autologous tissue cell.NKT cell is combined with the Fc of specific antibody section by the CD16 on self surface, performance ADCC (antibody-dependent cell-mediated cytotoxicity) effect.But, in antibody-mediated ADCC mechanism, the corresponding antigens epitope specificity of antibody capable on target cell is combined, can kill and wound any with the target cell of antibodies, it is specific that the antigen of antibody on target cell is combined, and NKT cell etc. is nonspecific to the lethal effect of target cell.Under normal circumstances, the NKT cell of infusion is generally about 2 weeks at patient's Half-life in vivo, and validity period is of short duration, needs repeated multiple times infusion.In addition, NKT cell itself lacks non-specific antibody, is not enough to, around tumour or enrichment in knurl nest, restrict the targeted therapy of NKT cell to progressive stage malignant tumour.
Chimeric antigen acceptor (chimeric antigen receptor, CAR) be to be connected in sequence by a series of signal structural domain in antigen recognition structural domain (part or single-chain antibody) and born of the same parents, specifically comprise the acceptor (as single-chain antibody ScFv) of antigen-specific, intervening sequence (spacer), costimulatory signal molecule in cross-film sequence (TM Domain) and born of the same parents.The lymphocyte that CAR modifies in vivo can pass through the special identification related neoplasms surface antigen of its single-chain antibody, then by costimulatory signal molecule in born of the same parents, the signal of identification is delivered in cell to activating cells lethal, target killing tumor cell.
CD20 monoclonal antibody therapy has been obtained gratifying effect in treatment B cell lymphoma, but a lot of patient finally can produce resistance.CD20 molecule is a kind of B cell differentiation antigen, is only expressed in pre B cell and mature B cell surface, is expressed in more than 95% B cell lymphoma, and does not express in hemopoietic stem cell, plasmocyte and other normal tissue cells.Importantly, CD20 molecule relatively exposes on film, easily approaches, be combined with monoclonal antibody rear without remarkable internalization and come off, can be because of antigenic modulation occurs with the combination of antibody, therefore become the desirable target spot for the treatment of B cell lymphoma yet.
Summary of the invention
The object of this invention is to provide NKT cell of a kind of through engineering approaches CD20 targeting and preparation method thereof, another object of the present invention is to provide the NKT cell of through engineering approaches CD20 targeting in the application for the preparation of in treatment tumour preparation.
For achieving the above object, the present invention is by the following technical solutions:
A kind of NKT cell of through engineering approaches CD20 targeting, this cell is the NKT cell that chimeric antigen acceptor CD20ScFv-CD8-CD137-CD3 ζ modifies, and this chimeric antigen acceptor is in series by hinge district and cross-film district, the intracellular signal structural domain of CD137 and the intracellular signal structural domain of CD3 ζ of CD20ScFv, CD8.
The NKT cell of through engineering approaches CD20 targeting as above, preferably, the nucleotide sequence of described chimeric antigen acceptor CD20ScFv-CD8-CD137-CD3 ζ is as shown in SEQ ID NO.2.
A kind of preparation method of NKT cell described above, the method comprises the following steps: hinge district and the cross-film district of the CD8 that increases respectively from NKT cell cDNA by RT-PCR, and the intracellular signal structural domain of CD137 and CD3 ζ, enzyme is cut, glue is connected to carrier pWPT-GFP by T4DNA ligase enzyme after reclaiming, and builds and obtains pWPT-CD8-CD137-CD3 ζ; Again by the nucleotide sequence of full gene synthesis technology composite coding rat growth hormone signal peptide and CD20ScFv, enzyme is cut, glue reclaims rear clone to pWPT-CD8-CD137-CD3 ζ, sequence verification sequence exactness, obtains pWPT-CD20ScFv-CD8-CD137-CD3 ζ; Then be packaged into the slow virus of carrying pWPT-CD20ScFv-CD8-CD137-CD3 ζ encoding gene; Utilize this slow virus infection NKT cell, make this chimeric antigen acceptor of NKT cell expressing.
A preparation method for NKT cell described above, preferably, the method comprises the following steps:
1) preparation of NKT cell: the mononuclearcell in dividing vein blood, cultivate and obtain NKT cell;
2) structure of recombinant plasmid pWPT-CD8-CD137-CD3 ζ: after cultivating take extraction step 1, NKT cell cDNA is as template, utilize primer P1, P2 to carry out hinge district and the cross-film district of pcr amplification acquisition CD8 gene, nucleotide sequence is as shown in SEQ ID NO.3, and MluI and Bgl II restriction enzyme site and protection base are contained respectively in two ends, utilize primer P3, P4 to carry out pcr amplification and obtain gene C D137 intracellular signal structural domain, nucleotide sequence is as shown in SEQ ID NO.4, and Bgl II and EcoRI restriction enzyme site and protection base are contained respectively in two ends, utilize primer P5, P6 carries out the intracellular signal structural domain of pcr amplification gene C D3 ζ, nucleotide sequence is as shown in SEQ ID NO.5, EcoRI and SalI restriction enzyme site and protection base are contained respectively in two ends, to after the PCR product electrophoretic separation purifying of acquisition, carry out respectively double digestion, enzyme is connected by T4DNA ligase enzyme with the Lentiviral pWPT-GFP after MluI/SalI double digestion after cutting product purification, connect product and proceed to Trans1-T1Phage Resistant chemoreception state cell, after a large amount of cultivations of competent cell, extract plasmid, correct recombinant plasmid called after pWPT-CD8-CD137-CD3 ζ will be identified,
3) structure of chimeric antigen acceptor pWPT-CD20ScFv-CD8-CD137-CD3 ζ: the plasmid pGSI-CD20ScFv that contains rat growth hormone signal peptide and CD20ScFv is carried out to Bgl II/MluI double digestion, reclaim DNA fragmentation; Wherein, this rat growth hormone signal peptide nucleotide sequence is as SEQ ID NO.6, and 5 ' end contains Bgl II, kozak sequence, and this CD20ScFv nucleotide sequence is as SEQ ID NO.7, and its 3 ' end contains MluI restriction enzyme site; The recombinant plasmid pWPT-CD8-CD137-CD3 ζ that step 2 is reclaimed carries out BamHI/MluI double digestion, reclaims carrier segments; DNA fragmentation after enzyme is cut proceeds to Trans1-T1Phage Resistant chemoreception state cell after being connected with plasmid vector fragment, after a large amount of amplification cultivation of competent cell, extract plasmid, through identifying that correct recombinant plasmid is chimeric antigen acceptor pWPT-CD20ScFv-CD8-CD137-CD3 ζ;
4) chimeric antigen acceptor pWPT-CD20ScFv-CD8-CD137-CD3 ζ modifies NKT cell: the quality that slow virus expression plasmid pWPT-CD20ScFv-CD8-CD137-CD3 ζ and helper plasmid psPAX2, pMD2.G press 4:2:1 is than cotransfection 293T packing cell, when transfection 48h, 72h, collect viral supernatant, centrifugal and with 4.5 μ m filters filter, 5 × the PEG6000-NaCl that adds 1/4 volume in filtrate mixes, after centrifugal, abandon supernatant, precipitation is dissolved with the aseptic PBS of 4 ℃ of precoolings, obtains viral concentrated solution;
5) chimeric antigen acceptor pWPT-CD20ScFv-CD8-CD137-CD3 ζ modifies NKT cell: get 1 × 10
7-5 × 10
7nKT cell, discards old nutrient solution, adds the fresh GT-T551 nutrient solution of 2-4mL, the viral concentrated solution, 2-4 μ L1 × 10 that add 200-400 μ L step 4) to obtain
-6unit protamine, final concentration are 1000U/mL IL-2, are placed in 37 ℃, 5%CO
2in incubator, infect after 12-16 hour, abandon nutrient solution, cell is gone in not coated culturing bottle, add the GT-T551 substratum of 20-50mL, in 37 ℃, 5%CO
2incubator was proceeded after amplification cultivation 8-15 days, infected and obtained the NKT cell that chimeric antigen acceptor CD20ScFv-CD8-CD137-CD3 ζ modifies, and the maturation protein aminoacid sequence of this chimeric antigen acceptor is as shown in SEQ ID NO.1.
The preparation method of through engineering approaches CD20 targeting as mentioned above, preferably, NKT cell after described step 5) transfection carries out external evoked with the GT-T551 nutrient solution that final concentration is 1000U IL-2, in the time that cell concentration accounts for culturing bottle 80-90%, cell is proceeded in cell culture bags, adding final concentration every 2 days is that the fresh GT-T551 nutrient solution of 1000U/mL IL-2 carries out amplification cultivation, treats cell amplification to 1 × 10
9behind left and right, adopt flow cytometer to identify the cell colony infecting.
A NKT cell for through engineering approaches CD20 targeting, preferably, this cell adopts method as above to prepare.
The NKT cell of through engineering approaches CD20 targeting as above is in the application for the preparation of in treatment tumour preparation.
Application as above, preferably, described tumour refers to the positive malignant tumour of progressive stage CD20.
Investigator of the present invention finds, in the positive malignant tumour immunotherapy of progressive stage CD20, the NKT cell of through engineering approaches CD20 targeting has more outstanding advantage than NKT cell, comprises special targeting, CD20 chimeric antigen acceptor rely on anti-tumor activity and infusion after long-lasting.These characteristics that the NKT cell of through engineering approaches CD20 targeting has, come from the functional structure of chimeric antigen acceptor, mainly comprise antibody recognition region and cell-stimulating region, antibody recognition region makes the NKT cell-targeting of through engineering approaches CD20 targeting in the positive malignant tumour of progressive stage CD20, in addition, in cell, active region can extend the survival time of cell in patient body.Visible, the NKT cell of through engineering approaches CD20 targeting provides a new selection for the positive malignant tumour for the treatment of of advanced CD20.
Beneficial effect of the present invention is: the NKT cell of through engineering approaches CD20 targeting of the present invention can specific binding CD20, in the positive treating malignant tumor of progressive stage CD20, adopt the NKT cell of through engineering approaches CD20 targeting significantly to extend the survival time of immunocyte in patient body, strengthen the ability of immunocyte target identification tumour antigen, strengthen the killing activity to tumour cell.In addition, the present invention, by optimal preparation technology, provides a kind of method of the efficient CD20 of preparation targeting NKT cell, and NKT cell prepared by the method has higher transfection efficiency, and is produced on a large scale, and has good industrial application prospect.
Accompanying drawing explanation
Fig. 1 is the NKT cell phenotype analytical results of flow cytometry to separation and Culture.
Fig. 2 is the restriction enzyme MluI/SalI double digestion fragment electrophoresis evaluation figure of Lentiviral pWPT-CD8-CD137-CD3 ζ of the present invention.
Fig. 3 is the restriction enzyme BamHI/SalI double digestion fragment electrophoresis evaluation figure of the Lentiviral pWPT-CD20ScFv-CD8-CD137-CD3 ζ of chimeric antigen acceptor of the present invention.
Fig. 4 is the structural representation of slow virus expression plasmid pWPT-CD20ScFv-CD8-CD137-CD3 ζ of the present invention, and wherein, sequence is forward gene sheet degree counterclockwise, is cdna reverse fragment clockwise.
Fig. 5 is that Flow cytometry is to CAR20-NKT cell phenotype qualification result.
Fig. 6 is the efficiency of infection of viral concentrated solution to NKT cell that Flow cytometry contains CD20ScFv-CD8-CD137-CD3 ζ.
Fig. 7 is that PT-PCR detects the expression efficiency of CD20ScFv-CD8-CD137-CD3 ζ in NKT cell.
Fig. 8 is the cytotoxicity analysis figure of the lethal effect of CAR20-NKT cell of the present invention to human tumor cells.
Fig. 9 is the B cell malignancies patient of CAR20-NKT cell of the present invention to the CD20 positive result for the treatment of.
Figure 10 be before CAR20-NKT cell therapy of the present invention (upper figure) with treatment after January CT comparison diagram.
Figure 11 is CAR20-NKT treatment Patients Before And After pathology lymphoglandula speedup figure of the present invention.
Embodiment
The invention provides a kind of NKT cell of through engineering approaches CD20 targeting, it is on NKT cell base, to build a kind of through engineering approaches cell forming, it is the NKT cell of being modified by chimeric antigen acceptor (chimeric antigen receptor, CAR) CD20ScFv-CD8-CD137-CD3 ζ.Chimeric antigen acceptor precursor albumen is in series by hinge district and cross-film district, the intracellular signal structural domain of CD137 and the intracellular signal structural domain of CD3 ζ of signal peptide, CD20ScFv, CD8, after protein translation, in cell, after rough surface endoplasmic reticulum excision signal peptide, become ripe chimeric antigen receptor protein, secretion is exported rear and is positioned on the cytolemma of NKT cell.The maturation protein aminoacid sequence of this chimeric antigen acceptor is as shown in SEQ ID NO.1, and its corresponding gene coded sequence is as shown in SEQ ID NO.2.44 amino acid whose one section, the extracellular region LOOP ring of this chimeric antigen receptor-specific ground identification CD20, its aminoacid sequence is as shown in SEQ ID NO.8.The structure that this chimeric antigen acceptor is in series take the hinge district of gene C D8 and the intracellular signal structural domain of cross-film district and CD137 and CD3 ζ is as signal conducting structure territory, its aminoacid sequence is as shown in SEQ ID NO.9, and its corresponding gene coded sequence is as shown in SEQ ID NO.10.
The NKT cell that utilizes this chimeric antigen acceptor to modify, can pass through the CD20 antigen on tumor cell surface, eliminates the tumour cell of CD20 antigen positive, carries out oncotherapy.
One of the present invention is preferred embodiment from people NKT cell cDNA, increase respectively by RT-PCR the hinge district of CD8 and the intracellular signal structural domain of cross-film district and CD137 and CD3 ζ, enzyme is cut, glue is connected to carrier pWPT-GFP by T4DNA ligase enzyme after reclaiming, structure obtains pWPT-CD8-CD137-CD3 ζ, again by the nucleotide sequence of full gene synthesis technology composite coding rat growth hormone signal peptide and CD20ScFv, enzyme is cut, glue reclaims rear clone to pWPT-CD8-CD137-CD3 ζ, sequence verification sequence exactness, obtain pWPT-CD20ScFv-CD8-CD137-CD3 ζ.Then be packaged into the slow virus of carrying pWPT-CD20ScFv-CD8-CD137-CD3 ζ encoding gene.Utilize this slow virus infection NKT cell, make this chimeric antigen acceptor of NKT cell expressing.Experimental results show that by cytotoxicity analysis the NKT cell that this chimeric antigen acceptor is modified has specific cytotoxicity to the tumour cell of the CD20 positive.Therefore the NKT cell of through engineering approaches CD20 targeting of the present invention can be applicable to the oncotherapy of the CD20 positive.
Below in conjunction with embodiment, the invention will be further described, following illustrated embodiment is for ease of understanding better the present invention, but be not used for limiting the present invention, those skilled in the art can make various modifications or change to the present invention, and these equivalent form of values fall within the application's claims limited range equally.
Experimental technique in following embodiment, if no special instructions, is ordinary method.Experiment material used in following embodiment, if no special instructions, is from routine biochemistry reagent shop and buys and obtain.
Reagent used in embodiments of the invention is as follows:
GT-T551 cell culture medium, TaKaRa company
Lymphocyte separation medium, TBD company
NK cell culture medium GT-T551, CD3 monoclonal antibody, retronectin, TAKORA company
Recombinant human protein's interferon-γ, rhIL-2, protech company
Total RNA extraction reagent box RNAiso Reagent, high-fidelity DNA polymerase (
hS DNA Polymerase), T4DNA ligase enzyme, TaKaRa company
RevertAid
tMfirst Strand cDNA Synthesis Kit, Fermentas company
Bgl II, EcoRI, MluI, BamHI, Fermentas company
Sepharose DNA reclaims test kit, common DNA product purification test kit, the little extraction reagent kit of plasmid, Tian Gen biochemical technology company limited
PWPT-GFP, Addgene company
Trans1-T1Phage Resistant chemoreception state cell, Beijing Quanshijin Biotechnology Co., Ltd
Lipofectamine
tM2000Transfection Reagent transfection reagent, Invitrogen company
Fluoresceincarboxylic acid succinimide ester, San Diego, CA, USA
Annexin V-RPE test kit, southern biotechnology, AL, USA
The preparation of embodiment 1 NKT cell
(1) get people's venous blood in containing the valve tube of heparin.Adopt lymphocyte separation medium, density gradient centrifugation method separates and obtains mononuclearcell (PBMCs).
(2) PBMCs washes after three times, and adopting the NKT cell culture medium GT-T551 that contains 0.6% conventional serum to adjust final concentration of cells is 2 × 10
6cell/mL; It is that 5 μ g/mLCD3 monoclonal antibodies and final concentration are that the retronectin(of 10 μ g/mL is purchased from TAKORA company that cell is inoculated in advance through final concentration) 75 coated cm cell culturing bottles.In substratum, adding final concentration is recombinant human protein's interferon-γ of 1000U/mL and the rhIL-2 of 1000U/mL, and 37 ℃, saturated humidity is 5%CO
2incubator is cultivated.
(3) the 4th days, in bottle, add the NKT cell culture medium that 100mL contains 0.6% conventional serum, adding final concentration is the rhIL-2 of 1000U/mL.In 37 ℃, 5%CO
2incubator is cultivated, and obtains NKT cell, and flow cytometry is analyzed NKT cell phenotype.The results are shown in Figure 1, wherein CD3:98.76%; CD3CD4:26.03%; CD3CD8:68.65%; CD3CD56:4.73%; CD8CD56:3.33%.
Embodiment 2: the structure of chimeric antigen acceptor (pWPT-CD20ScFv-CD8-CD137-CD3 ζ) Lentiviral
(1) preparation of NKT cell cDNA
The NKT cell that centrifugation embodiment 1 cultivates, with total RNA of total RNA extraction reagent box RNAiso Reagent extraction cell ,-80 ℃ save backup.The total RNA reverse transcription test kit RevertAid extracting
tMfirst Strand cDNA Synthesis Kit reverse transcription obtains NKT cell cDNA, and-20 ℃ save backup.
(2) preparation of slow virus plasmid pWPT-CD8-CD137-CD3 ζ
The primer is synthetic by Tian Yihuiyuan bio tech ltd, Beijing, primer sequence following (wherein, underscore is labeled as protection base, and square frame is restriction enzyme site):
Take NKT cell cDNA in step (1) as template, carry out pcr amplification with primer P1, P2, obtain hinge district and the cross-film district of the CD8 of long 287bp, nucleotide sequence is as shown in SEQ ID NO.3; MluI and Bgl II restriction enzyme site and protection base are contained respectively in two ends, carry out PCR reaction with primer P3, P4, the CD137 intracellular signal structural domain of the long 146bp of amplification, nucleotide sequence is as shown in SEQ ID NO.4, and Bgl II and EcoRI restriction enzyme site and protection base are contained respectively in two ends; Carry out PCR reaction with primer P5, P6, the intracellular signal structural domain of amplification CD3 ζ, nucleotide sequence is as shown in SEQ ID NO.5, and EcoRI and SalI restriction enzyme site and protection base are contained respectively in two ends.Each step pcr amplification reaction system is identical, take the CD137 intracellular signal structural domain that increases as example, carries out pcr amplification, the reference of PCR reaction conditions
the specification sheets of HS DNA Polymerase, (50 μ L) is as follows for reaction system:
Distilled water: 32.5 μ L
5 × reaction buffer:10 μ L
dNTP?Mixture（2.5mM?each）：4μL
P3（10mM）:1μL
P4（10mM）:1μL
NKT cell cDNA (200ng/ul): 1 μ L
HS?DNA?Polymerase：0.5μL
Above-mentioned PCR product is separated with 1% sepharose, reclaim test kit with sepharose DNA and carry out DNA fragmentation recovery.Obtain fragment and carry out respectively double digestion reaction, enzyme is cut the common DNA product purification of product test kit and is reclaimed for subsequent use.
Lentiviral pWPT-GFP MluI/SalI double digestion, enzyme is cut product and is separated through 1% sepharose, reclaim test kit with sepharose DNA and reclaim large carrier segments, then be connected by T4DNA ligase enzyme with CD8, the CD137, the CD3 ζ fragment that reclaim before, connect product and transform Trans1-T1Phage Resistant chemoreception state cell, picking mono-clonal after 37 ℃ of cultivation 16h, 37 ℃, after 250rpm cultivates 12h, the little extraction reagent kit of plasmid extracts plasmid.The plasmid extracting is through restriction enzyme MluI and the evaluation of SalI double digestion, and evaluation 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.To identify that correct plasmid send Beijing Tian Yihuiyuan Science and Technology Ltd. to check order to the fusion gene fragment of inserting.By recombinant plasmid called after pWPT-CD8-CD137-CD3 ζ correct sequencing result.
(3) preparation of slow virus plasmid pWPT-CD20ScFv-CD8-CD137-CD3 ζ
The nucleotide sequence of full gene composite coding rat growth hormone signal peptide and CD20ScFv, sequence is as shown in SEQ ID NO.6, synthesized by Tian Yihuiyuan Science and Technology Ltd., its 5 ' end contains Bgl II, kozak sequence, 3 ' end contains MluI restriction enzyme site, be cloned in plasmid pGSI called after pGSI-CD20ScFv.Plasmid is through Bgl II/MluI double digestion, and enzyme is cut product and separated through 1% sepharose, reclaims test kit recovery object fragment for subsequent use with sepharose DNA.
PWPT-CD8-CD137-CD3 ζ plasmid carries out enzyme through restriction enzyme BamHI/MluI and cuts.Enzyme is cut product and is separated through 1% sepharose, reclaims test kit recovery carrier segments for subsequent use with sepharose DNA.Then be connected by T4DNA ligase enzyme with the DNA fragmentation that contains rat growth hormone signal peptide and CD20ScFv reclaiming, concrete grammar is shown in specification sheets.To connect product and transform Trans1-T1Phage Resistant chemoreception state cell, picking mono-clonal after 37 ℃ of cultivation 16h, 37 ℃, 250rpm cultivates after 12h, with the little extraction reagent kit extraction of plasmid plasmid.Extract plasmid through restriction enzyme BamHI/MluI double digestion identify, qualification result as shown in Figure 3, wherein, 1 swimming lane: DNA molecular amount mark D15000; 2 swimming lanes: the endonuclease bamhi (756bp) of plasmid pWPT-CD8-CD137-CD3 ζ; 3 swimming lanes: the endonuclease bamhi (835bp) of plasmid pWPT-GFP; 4 swimming lanes: the endonuclease bamhi (1305bp) of plasmid pWPT-CD20ScFv-CD8-CD137-CD3 ζ.To identify that correct plasmid send Tian Yihuiyuan bio tech ltd, Beijing to check order to the fusion gene fragment of inserting.By recombinant plasmid called after pWPT-CD20ScFv-CD8-CD137-CD3 ζ correct sequencing result, also be called CAR-CD20, its structural representation as shown in Figure 4, comprising the hinge district of signal peptide, anti-CD20 single-chain antibody, CD8 and the intracellular signal structural domain of cross-film district and CD137 and CD3 ζ.
The preparation of the NKT cell that embodiment 3 chimeric antigen acceptor pWPT-CD20ScFv-CD8-CD137-CD3 ζ modify
(1) packing of slow virus and concentrated
Extract respectively slow virus expression plasmid pWPT-CD20ScFv-CD8-CD137-CD3 ζ and helper plasmid psPAX2, pMD2.G with the little extraction reagent kit of plasmid.Spectrophotometric determination plasmid concentration, three kinds of plasmids are with the mass ratio Lipofectamine of 4:2:1
tM2000Transfection Reagent transfection reagent cotransfection 293T packing cell.In the time of transfection 48h, 72h, collect viral supernatant in EP pipe respectively, 4 ℃, the centrifugal 10min of 2000g, shifts supernatant to new EP pipe, filters viral supernatant with 4.5 μ m filters; The viral supernatant and the 5 × PEG6000-NaCl that filter mix according to the volume ratio of 4:1,4 ℃ of standing 2h, and then 4 ℃, the centrifugal 20min of 10000g, abandons supernatant, and precipitation is dissolved with the aseptic PBS of 4 ℃ of precoolings, obtains viral concentrated solution, carries out packing, and-80 ℃ save backup.
(2) amplification cultivation of slow virus infection NKT cell and infected cell
Get embodiment 1 at 25cm
21 × 10 of the cultivation of culturing bottle
7nKT cell, discards old nutrient solution, adds the fresh GT-T551 nutrient solution of 2mL, 200 μ L virus liquids, 2 μ L1 × 10
-6unit protamine, final concentration is 1000U/mL IL-2, is placed in 37 ℃, 5%CO
2in incubator, infect after 12 hours, abandon nutrient solution, arrange containing the chimeric antigen acceptor virus concentrated solution of GFP green fluorescence mark simultaneously control group NKT cell is synchronously infected, for calculating this viral efficiency of infection.Metainfective cell is gone to not coated 75cm
2in culturing bottle, add the substratum of 20mL, in 37 ℃, 5%CO
2incubator is proceeded to cultivate.The efficiency of infection of this chimeric antigen acceptor virus concentrated solution to NKT cell, arrange containing the chimeric antigen acceptor virus concentrated solution of GFP green fluorescence mark control group NKT cell is synchronously infected, by Flow cytometry and calculate this viral efficiency of infection, as shown in Figure 6, efficiency of infection is 33.63% to result.
(3) external evoked amplification into is rich in the two positive CAR20-NKT cell masses of CD3CD56
By the application of metainfective NKT cell contain final concentration be 1000U/mL IL-2 GT-T551 nutrient solution to CART cell carry out external evoked, in the time that cell concentration accounts for culturing bottle 80-90%, cell is proceeded in cell culture bags, adding final concentration every 2 days is the GT-T551 nutrient solution amplification cultivation of 1000U/mL IL-2, treats cell amplification to 1 × 10
9behind left and right, adopt flow cytometer to identify the cell colony infecting, 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 5, CD3:96.9%; CD3CD4:20.93%; CD3CD8:70.52%; CD3CD56:6.24%; CD8CD56:5.09%.
(4) Flow cytometry chimeric antigen acceptor CD20ScFv-CD8-CD137-CD3 ζ is in the intracellular expression of NKT
Collect respectively 1 × 10
6infect pWPT-CD20ScFv-CD8-CD137-CD3 ζ NKT cell (being CAR20-NKT cell) and uninfecting virus cellular control unit (NKT), fix with 2% paraformaldehyde, carry out the expression that reverse transcription-pcr detects gene, detected result is as Fig. 7, and positive object of reference in figure (sun ginseng) refers to independent pWPT-CD20ScFv-CD8-CD137-CD3 ζ recombinant plasmid; NKT is the NKT cell that does not infect pWPT-CD20ScFv-CD8-CD137-CD3 ζ recombinant plasmid; CAR20-NKT is the NKT cell that infects pWPT-CD20ScFv-CD8-CD137-CD3 ζ recombinant plasmid.Result shows, chimeric antigen acceptor is at NKT cell inner expression, and the aminoacid sequence of this chimeric antigen acceptor is as shown in SEQ ID NO.1.
The cytotoxicity analysis of embodiment 4 CAR20-NKT cells to human tumor cells lethal effect
Get respectively in embodiment 3 the NKT cell of cultivating in the CAR20-NKT cell of preparation and embodiment 1 and be inoculated in 96 orifice plates, dye with Fluoresceincarboxylic acid succinimide ester (CFSE), with Molt-4, K562, K562-CD20+ and Reji cell be with 1:1,5:1,10:1,20:1 ratio carries out common cultivation, after the common cultivation of 24 hours, by annexin V-RPE test kit dyeing for cell.Flow cytometry detects apoptosis, and the amount of necrocytosis is calculated according to formula below: mortality ratio=(contrast-sample)/contrast × 100%), contrast the tumour cell for not adding CAR20-NKT; Sample, for adding the tumour cell of the CAR20-NKT that effect target is 20:1 than (killer cell: target cell), is shown in Fig. 8.The NKT cell that chimeric antigen acceptor CD20ScFv-CD8-CD137-CD3 ζ modifies has specific killing activity to the tumour cell of high expression level CD20.
The result for the treatment of of the malignant tumor patient of embodiment 5 CAR20-NKT cells to the CD20 positive
Get 5 × 10
8the NKT lymphocyte that individual CD20ScFv-CD8-CD137-CD3 ζ modifies, within continuous three days, vein feeds back in the tumour patient body of the CD20 positive, treats and analyzes after one month.
As shown in Figure 9, the NKT cell that immunohistochemical methods detects through engineering approaches CD20 targeting feeds back to the situation of going back to the nest in patient body, result shows, lymph node biopsy display target is to a large amount of CD3 after immunocyte treatment, CD20 positive cell appears at tumor tissues, the NKT cell that CD20 targeting the is described lesions position performance lethal effect of can going back to the nest.
As shown in figure 10, the result for the treatment of of the NKT cell that CT shows through engineering approaches CD20 targeting to CD20 malignant tumor patient, result shows (upper figure) and treatment CT(figure below in rear January before the treatment of target immunocyte) contrasting: enlarged lymph node dwindles (specifically seeing shown in arrow).
As shown in figure 11, after the NKT cell therapy of through engineering approaches CD20 targeting, patient's pathology lymphoglandula speedup obviously slows down before, and after treatment 3 weeks, enlarged lymph node start to disappear voluntarily (the longest through as representative take superficial lymph knot on the clavicle of enlargement in figure).
Claims (8)
1. the NKT cell of a through engineering approaches CD20 targeting, it is characterized in that, this cell is the NKT cell that chimeric antigen acceptor CD20ScFv-CD8-CD137-CD3 ζ modifies, and this chimeric antigen acceptor is in series by hinge district and cross-film district, the intracellular signal structural domain of CD137 and the intracellular signal structural domain of CD3 ζ of CD20ScFv, CD8.
2. the NKT cell of through engineering approaches CD20 targeting as claimed in claim 1, is characterized in that, the nucleotide sequence of described chimeric antigen acceptor CD20ScFv-CD8-CD137-CD3 ζ is as shown in SEQ ID NO.2.
3. the preparation method of NKT cell as claimed in claim 1, it is characterized in that, the method comprises the following steps: hinge district and the cross-film district of the CD8 that increases respectively from NKT cell cDNA by RT-PCR, and the intracellular signal structural domain of CD137 and CD3 ζ, enzyme is cut, glue is connected to carrier pWPT-GFP by T4DNA ligase enzyme after reclaiming, and builds and obtains pWPT-CD8-CD137-CD3 ζ; Again by the nucleotide sequence of full gene synthesis technology composite coding rat growth hormone signal peptide and CD20ScFv, enzyme is cut, glue reclaims rear clone to pWPT-CD8-CD137-CD3 ζ, sequence verification sequence exactness, obtains pWPT-CD20ScFv-CD8-CD137-CD3 ζ; Then be packaged into the slow virus of carrying pWPT-CD20ScFv-CD8-CD137-CD3 ζ encoding gene; Utilize this slow virus infection NKT cell, make this chimeric antigen acceptor of NKT cell expressing.
4. a preparation method for NKT cell as claimed in claim 3, is characterized in that, the method comprises the following steps:
1) preparation of NKT cell: the mononuclearcell in dividing vein blood, cultivate and obtain NKT cell;
2) structure of recombinant plasmid pWPT-CD8-CD137-CD3 ζ: after cultivating take extraction step 1, NKT cell cDNA is as template, utilize primer P1, P2 to carry out hinge district and the cross-film district of pcr amplification acquisition CD8 gene, nucleotide sequence is as shown in SEQ ID NO.3, and MluI and Bgl II restriction enzyme site and protection base are contained respectively in two ends, utilize primer P3, P4 to carry out pcr amplification and obtain gene C D137 intracellular signal structural domain, nucleotide sequence is as shown in SEQ ID NO.4, and Bgl II and EcoRI restriction enzyme site and protection base are contained respectively in two ends, utilize primer P5, P6 carries out the intracellular signal structural domain of pcr amplification gene C D3 ζ, nucleotide sequence is as shown in SEQ ID NO.5, EcoRI and SalI restriction enzyme site and protection base are contained respectively in two ends, to after the PCR product electrophoretic separation purifying of acquisition, carry out respectively double digestion, enzyme is connected by T4DNA ligase enzyme with the Lentiviral pWPT-GFP after MluI/SalI double digestion after cutting product purification, connect product and proceed to Trans1-T1Phage Resistant chemoreception state cell, after a large amount of cultivations of competent cell, extract plasmid, correct recombinant plasmid called after pWPT-CD8-CD137-CD3 ζ will be identified,
3) structure of chimeric antigen acceptor pWPT-CD20ScFv-CD8-CD137-CD3 ζ: the plasmid pGSI-CD20ScFv that contains rat growth hormone signal peptide and CD20ScFv is carried out to Bgl II/MluI double digestion, reclaim DNA fragmentation; Wherein, this rat growth hormone signal peptide nucleotide sequence is as SEQ ID NO.6, and 5 ' end contains Bgl II, kozak sequence, and this CD20ScFv nucleotide sequence is as SEQ ID NO.7, and its 3 ' end contains MluI restriction enzyme site; The recombinant plasmid pWPT-CD8-CD137-CD3 ζ that step 2 is reclaimed carries out BamHI/MluI double digestion, reclaims carrier segments; DNA fragmentation after enzyme is cut proceeds to Trans1-T1Phage Resistant chemoreception state cell after being connected with plasmid vector fragment, after a large amount of amplification cultivation of competent cell, extract plasmid, through identifying that correct recombinant plasmid is chimeric antigen acceptor pWPT-CD20ScFv-CD8-CD137-CD3 ζ;
4) chimeric antigen acceptor pWPT-CD20ScFv-CD8-CD137-CD3 ζ modifies NKT cell: the quality that slow virus expression plasmid pWPT-CD20ScFv-CD8-CD137-CD3 ζ and helper plasmid psPAX2, pMD2.G press 4:2:1 is than cotransfection 293T packing cell, when transfection 48h, 72h, collect viral supernatant, centrifugal and with 4.5 μ m filters filter, 5 × the PEG6000-NaCl that adds 1/4 volume in filtrate mixes, after centrifugal, abandon supernatant, precipitation is dissolved with the aseptic PBS of 4 ℃ of precoolings, obtains viral concentrated solution;
5) chimeric antigen acceptor pWPT-CD20ScFv-CD8-CD137-CD3 ζ modifies NKT cell: get 1 × 10
7-5 × 10
7nKT cell, discards old nutrient solution, adds the fresh GT-T551 nutrient solution of 2-4mL, the viral concentrated solution, 2-4 μ L1 × 10 that add 200-400 μ L step 4) to obtain
-6unit protamine, final concentration are 1000U/mL IL-2, are placed in 37 ℃, 5%CO
2in incubator, infect after 12-16 hour, abandon nutrient solution, cell is gone in not coated culturing bottle, add the GT-T551 substratum of 20-50mL, in 37 ℃, 5%CO
2incubator was proceeded after amplification cultivation 8-15 days, infected and obtained the NKT cell that chimeric antigen acceptor CD20ScFv-CD8-CD137-CD3 ζ modifies, and the maturation protein aminoacid sequence of this chimeric antigen acceptor is as shown in SEQ ID NO.1.
5. preparation method as claimed in claim 4, it is characterized in that, NKT cell after described step 5) transfection carries out external evoked with the GT-T551 nutrient solution that final concentration is 1000U IL-2, in the time that cell concentration accounts for culturing bottle 80-90%, cell is proceeded in cell culture bags, adding final concentration every 2 days is that the fresh GT-T551 nutrient solution of 1000U/mL IL-2 carries out amplification cultivation, treats cell amplification to 1 × 10
9behind left and right, adopt flow cytometer to identify the cell colony infecting.
6. a NKT cell for through engineering approaches CD20 targeting, is characterized in that, this cell is to adopt the method described in any one in claim 3-5 to prepare.
7. the NKT cell of the through engineering approaches CD20 targeting described in claim 1 or 6 is in the application for the preparation of in treatment tumour preparation.
8. application as claimed in claim 7, is characterized in that, described tumour refers to the positive malignant tumour of progressive stage CD20.
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| CN106279434A (en) | 2017-01-04 |
| CN103820393B (en) | 2016-09-07 |
| CN106279434B (en) | 2020-07-03 |
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