CN105936649B - Chimeric antigen receptor and its gene and recombinant expression carrier, CAR133-NKT cell and its preparation method and application - Google Patents
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Abstract
The invention belongs to knubble biological arts, disclose a kind of Chimeric antigen receptor and its gene and recombinant expression carrier, CAR133-NKT cell and its preparation method and application, the Chimeric antigen receptor is CD133ScFv-CD8-CD137-CD3 ζ, the intracellular signal structural domain including concatenated rat growth hormone signal peptide, the hinge area of CD133ScFv, CD8 and transmembrane region, the intracellular signal structural domain of CD137 and CD3 ζ.When co-culturing using the epithelial tumor cell of CAR133-NKT cell and the CD133 positive of the invention, there is good specific killing activity to CD133 positive epithelial tumor cell.
Description
Technical field
The invention belongs to knubble biological arts, and in particular, to one of adoptive immunotherapy chimeric antigen by
Body CD133ScFv-CD8-CD137-CD3 ζ and its gene and recombinant expression carrier, the NKT cell for being engineered CD133 targeting
(CAR133-NKT cell) and its preparation method and application.
Background technique
Natural killer cells (NKT) is a kind of T lymphocyte subgroup of specific type, has T cell and NK cell
Double property.NKT cell can express two kinds of receptors of NKR-P1 of TCR and the NK cell of T cell, in the case where TCR and NKR is mediated, NKT
Cell can generate a large amount of IL-4 and INF γ, play killing functions of immunocytes to tumour cell.NKT cell passes through own face
CD16 combined with the Fc of specific antibody section, play antibody dependent cell mediated cytotoxicity (ADCC,
antibody-dependent cell-mediated cytotoxicity).But make in the cell-mediated killing of antibody-dependant
With in the process, due to antibody can in conjunction with the corresponding antigens epitope specificity on target cell, NKT cell can kill it is any with it is anti-
The target cell that body combines, therefore the antigen binding on antibody and target cell is specific, but killing of the NKT cell to target cell
Effect is nonspecific.In addition, it is generally the case that the NKT cell of infusion is 2 weeks or so in patient's body half-life period, effectively
Phase is of short duration, needs repeated multiple times infusion.Moreover, NKT cell itself lacks specific antibody, it is not enough to around tumour or tumor
It is enriched in nest, constrains NKT cell to the targeted therapy of malignant tumour.Furthermore studies have shown that NKT cell is not to all
Tumour have fragmentation effect, and weaker to the lethal effect of Partial tumors, specific killing activity is to be improved, NKT cell
Dispute is still had at present to the immunosurveillance of tumor stem cell.
Tumor stem cell is a kind of special tumour cell, and the ratio for accounting for tumour cell is lower, but has self-renewing,
And it is divided into the potential of other different tumour cells.Numerous studies point out, the recurrence and far-end transfer of tumor stem cell and tumour
It with very big correlation, and may be that tumour generates the cause resisted to chemicotherapy.CD133 is the sugared egg of cell surface
It is white, it can be used as the label of tumor stem cell in separation tumor tissues.In addition, high table is presented on kinds of tumor cells surface in CD133
It reaches, such as colorectal cancer, liver cancer, cholangiocarcinoma, cancer of pancreas, the cancer of the esophagus, gastric cancer, oophoroma, lung cancer, prostate cancer, bladder cancer, mammary gland
Cancer, carcinoma of endometrium, brain tumor, melanoma or glioma etc..Research is found in tumour initiator cell group containing a large amount of
The CD133 positive cell, meanwhile, the high expression of CD133 is related with the prognosis of oncotherapy difference, due to the above characteristic, CD133
Antigen becomes the promising target treated based on antibody, but at present still without a kind of pair of higher system of tomour specific killing activity
Agent.
Summary of the invention
The purpose of the invention is to overcome, the lethal effect of NKT cells against tumor in the prior art is weaker, specifically kills
Wound activity defect to be improved makes full use of the effect of the promising target of CD133 treatment tumour, provide a kind of chimeric antigen by
Body CD133ScFv-CD8-CD137-CD3 ζ and its gene and recombinant expression carrier, the NKT cell for being engineered CD133 targeting
(CAR133-NKT cell) and its preparation method and application.
The present inventor has been surprisingly found that under study for action, using Chimeric antigen receptor CD133ScFv-CD8- of the invention
When the NKT cell of CD137-CD3 ζ modification and the epithelial tumor cell of the CD133 positive co-culture, have to tumour cell good
Specific killing activity.
Therefore, to achieve the goals above, described chimeric in a first aspect, the present invention provides a kind of Chimeric antigen receptor
Antigen receptor is CD133ScFv-CD8-CD137-CD3 ζ, including concatenated rat growth hormone signal peptide, CD133ScFv, CD8
Hinge area (area hinge) and transmembrane region, the intracellular signal structural domain of CD137 and CD3 ζ intracellular signal structural domain.
Second aspect, the present invention provides the genes for encoding above-mentioned Chimeric antigen receptor.
The third aspect, the present invention provides the recombinant expression carriers containing said gene.
Fourth aspect, the present invention provides a kind of NKT cells for being engineered CD133 targeting, and the NKT cell is above-mentioned
The NKT cell of Chimeric antigen receptor CD133ScFv-CD8-CD137-CD3 ζ modification.
5th aspect, the present invention provides a kind of preparation method of NKT cell for being engineered CD133 targeting, the sides
Method includes: the slow virus that packaging carries pWPXL-CD133ScFv-CD8-CD137-CD3 ζ, obtains viral concentration liquid；Using obtaining
Viral concentration liquid inductance contaminate NKT cell, make NKT cell express Chimeric antigen receptor CD133ScFv-CD8-CD137-CD3 ζ.
6th aspect, the present invention provides the NKT cells for the engineering CD133 targeting that the above method is prepared.
7th aspect, the present invention provides the NKT cell of above-mentioned engineering CD133 targeting in preparation for treating tumour
Preparation in application.
Eighth aspect the present invention provides a kind of epithelial tumor for treating the CD133 positive and/or inhibits tumor recurrence and turns
The method of shifting, this method comprises: feeding back engineering CD133 targeting of the invention to the epithelial tumor patient's body of the CD133 positive
The NKT cell of property.
When with the co-cultivation of the epithelial tumor cell of the CD133 positive, Chimeric antigen receptor CD133ScFv- of the invention
The NKT cell of CD8-CD137-CD3 ζ modification, i.e. it is anti-that the NKT cell of engineering CD133 targeting can specifically bind CD133
Original enhances the ability of immunocyte targets identification cancer cell surfaces CD133 antigen, reinforces to the special of CD133 positive target cell
Killing activity.The NKT cell of engineering CD133 targeting of the invention is to treat the tumour of the CD133 positive and check tumour to turn
It moves and provides a kind of new selection with recurrence, there is good industrial application prospect.
Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.
Detailed description of the invention
Fig. 1 is result of the flow cytometry to the NKT cell phenotype analysis being separately cultured.
Fig. 2 is the restriction enzyme MluI/ of Lentiviral pWPXL-CD8-CD137-CD3 ζ of the invention
The electrophoretic identification of NdeI double digestion segment.
Fig. 3 is the restriction enzyme of Lentiviral pWPXL-CD133ScFv-CD8-CD137-CD3 ζ of the invention
The electrophoretic identification of enzyme BamHI/NdeI double digestion segment.
Fig. 4 is the structural representation of Lentiviral pWPXL-CD133ScFv-CD8-CD137-CD3 ζ of the invention
Figure, wherein sequence counterclockwise is positive gene piece degree, is clockwise cdna reverse segment.
Fig. 5 is the viral concentration that Flow cytometry contains Chimeric antigen receptor CD133ScFv-CD8-CD137-CD3 ζ
Efficiency of infection of the liquid to NKT cell.
Fig. 6 is the NKT cell of Flow cytometry Chimeric antigen receptor CD133ScFv-CD8-CD137-CD3 ζ modification
The result of (CAR133-NKT cell) phenotypic evaluation.
Fig. 7 (A)-Fig. 7 (C) is the epithelial tumor cell of difference CD133 expression in the embodiment of the present invention 4
CD133 expression analysis chart.
Fig. 8 is CAR133-NKT cell in the embodiment of the present invention 4 to the epithelial tumor cell of different CD133 expressions
Lethal effect analysis chart (4 hours).
Fig. 9 is CAR133-NKT cell in the embodiment of the present invention 4 to the epithelial tumor cell of different CD133 expressions
Lethal effect analysis chart (8 hours).
Figure 10 is the CD133 expression analysis chart of SW620 and HT29 in the embodiment of the present invention 5.
After Figure 11 is injecting normal saline, NKT cell, Mock cell, CAR133-NKT cell in the embodiment of the present invention 5
Tumor tissues volume diagram.
Figure 12 is CAR133-NKT cell in the embodiment of the present invention 6 to the function effect analysis chart of hemopoietic system.
Figure 13 (A)-Figure 13 (D) is that the poison that CAR133-NKT cell treats liver cancer patient in the embodiment of the present invention 7 is secondary
The analysis chart of reaction, hemoglobin (Hgb), leucocyte (WBC), blood platelet when respectively illustrating number of days after different cells are fed back
(PLT) and net knits the variation tendency of Lactoferrin (Ret).
Figure 14 is CAR133-NKT cell copy number and liver cancer patient CD133 positive cell number in the embodiment of the present invention 7
Change curve.
Figure 15 is the analysis chart of the therapeutic effect of liver cancer patient in the embodiment of the present invention 7.
Figure 16 is CAR133-NKT cell in the embodiment of the present invention 8 to the therapeutic effect figure of Pancreas cancer patients.
Specific embodiment
Detailed description of the preferred embodiments below.It should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The present invention provides a kind of Chimeric antigen receptor, the Chimeric antigen receptor is CD133ScFv-CD8-CD137-
CD3 ζ, including concatenated rat growth hormone signal peptide, the hinge area of CD133 single-chain antibody CD133ScFv, CD8 and transmembrane region,
The intracellular signal structural domain of CD137 and the intracellular signal structural domain of CD3 ζ.
Under preferable case, Chimeric antigen receptor CD133ScFv-CD8-CD137-CD3 ζ by rat growth hormone signal peptide,
The intracellular signal structural domain of the hinge area and transmembrane region of CD133ScFv, CD8, the intracellular signal structural domain of CD137 and CD3 ζ is connected
It constitutes.It is further preferred that Chimeric antigen receptor has the amino acid sequence as shown in SEQ ID NO.1, still more preferably
Ground, the amino acid sequence of Chimeric antigen receptor is as shown in SEQ ID NO.1.
The present invention provides the genes for encoding above-mentioned Chimeric antigen receptor.Under preferable case, the gene has such as SEQ
Nucleotide sequence shown in ID NO.2, it is further preferred that encoding the nucleotide sequence of the gene of above-mentioned Chimeric antigen receptor such as
Shown in SEQID NO.2.
The present invention provides the recombinant expression carriers containing said gene.Under preferable case, recombinant expression carrier is slow disease
Malicious expression vector.For Lentiviral, there is no particular limitation, as long as can be with assistant carrier cotransfection incasing cells
Such as 293T incasing cells, the NKT of viral concentration liquid and Chimeric antigen receptor CD133ScFv-CD8-CD137-CD3 ζ modification is obtained
Cell, under preferable case, Lentiviral is pWPXL-CD133ScFv-CD8-CD137-CD3 ζ.
It is not special for the preparation method of Lentiviral pWPXL-CD133ScFv-CD8-CD137-CD3 ζ
Limit, can be those skilled in the art it is conceivable that various methods, under preferable case, Lentiviral pWPXL-
The preparation method of CD133ScFv-CD8-CD137-CD3 ζ the following steps are included:
(1) area hinge of CD8 and the intracellular signal structural domain of transmembrane region, CD137 are expanded respectively from NKT cell cDNA
It with the intracellular signal structural domain of CD3 ζ, and is cloned into carrier pWPXL-GFP, building obtains pWPXL-CD8-CD137-CD3 ζ；
(2) nucleotide sequence of composite coding rat growth hormone signal peptide and CD133ScFv, and it is cloned into pWPXL-
In CD8-CD137-CD3 ζ, the correct pWPXL-CD133ScFv-CD8-CD137-CD3 ζ of sequence is obtained after sequence verification.
In step (1), for expanded respectively from NKT cell cDNA CD8 the area hinge and transmembrane region, CD137 it is intracellular
There is no particular limitation for the method for the intracellular signal structural domain of signal domain and CD3 ζ, can be various sides commonly used in the art
Method, such as can be RT-PCR method.Wherein, then NKT cell can be carried out by the mononuclearcell in separation people's venous blood
Culture obtains.
Specifically, the method for obtaining pWPXL-CD8-CD137-CD3 ζ may include: to extract the total serum IgE of NKT cell, reverse
Record obtains NKT cell cDNA and utilizes primer P1 (SEQID NO.11) and P2 (SEQID using obtained NKT cell cDNA as template
NO.12 the area hinge and transmembrane region (SEQID NO.3) that PCR amplification obtains CD8 gene) are carried out；Utilize primer P3 (SEQID
NO.13) and P4 (SEQID NO.14) carries out the intracellular signal structural domain (SEQID NO.4) that PCR amplification obtains CD137 gene；
The intracellular signal structure that PCR amplification obtains CD3 ζ gene is carried out using primer P5 (SEQID NO.15) and P6 (SEQID NO.16)
Domain (SEQID NO.5), carries out double digestion for the PCR product of acquisition respectively, then with the slow virus after MluI/NdeI double digestion
Expression vector pWPXL-GFP connection.
In step (2), for the method for the nucleotide sequence of composite coding rat growth hormone signal peptide and CD133ScFv
There is no particular limitation, can be various methods commonly used in the art, such as can be synthesized by full genome synthetic technology.
Specifically, the method for obtaining the correct pWPXL-CD133ScFv-CD8-CD137-CD3 ζ of sequence may include: logical
Cross the nucleotide sequence of full genome synthetic technology composite coding rat growth hormone signal peptide and CD133ScFv fusion
(SEQID NO.8), is cloned into carrier pGSI, obtains pGSI-CD133ScFv；Then pGSI-CD133ScFv is carried out
BamHI/MluI double digestion, with the recombinant plasmid pWPXL-CD8- obtained with the step (1) after BamHI/MluI double digestion
CD137-CD3 ζ connection is identified through sequencing, obtains the correct pWPXL-CD133ScFv-CD8-CD137-CD3 ζ of sequence.Wherein,
The nucleotide sequence of rat growth hormone signal peptide is as shown in SEQID NO.6, CD133ScFv nucleotide sequence such as SEQID
Shown in NO.7.
The present invention also provides a kind of NKT cell for being engineered CD133 targeting, the NKT cell is by above-mentioned chimeric
The NKT cell (i.e. CAR133-NKT cell) of antigen receptor CD133ScFv-CD8-CD137-CD3 ζ modification.
The present invention also provides a kind of preparation methods of NKT cell for being engineered CD133 targeting, this method comprises: packet
Dress carries the slow virus of pWPXL-CD133ScFv-CD8-CD137-CD3 ζ, obtains viral concentration liquid；It is dense using obtained virus
Contracting liquid inductance contaminates NKT cell, and NKT cell is made to express Chimeric antigen receptor CD133ScFv-CD8-CD137-CD3 ζ.
The method for carrying the slow virus of pWPXL-CD133ScFv-CD8-CD137-CD3 ζ for packaging does not limit particularly
It is fixed, it can be the common various methods of those skilled in the art, under preferable case, by Lentiviral pWPXL-
CD133ScFv-CD8-CD137-CD3 ζ and helper plasmid (such as psPAX2, pMD2.G) cotransfection 293T incasing cells transfect 48-
Viral supernatants are collected when 72h, centrifugation, filtering are added 5 × PEG6000-NaCl in filtrate and are mixed, supernatant is abandoned after centrifugation,
The sterile PBS dissolution of 0-4 DEG C of pre-cooling of precipitating, obtains viral concentration liquid.
Further include being prepared via a method which NKT cell in method of the invention:
(1) in the presence of CD3 monoclonal antibody, proleulzin and interleukin-15, mononuclearcell is subjected to the first stage
Culture；
(2) in the presence of proleulzin, the cell that the first stage is cultivated carries out second stage culture.
Under preferable case, the embodiment of first stage culture includes: that mononuclearcell is incubated at the first NKT is thin
In born of the same parents' culture solution, the first NKT cell culture fluid contains NKT cell culture medium, CD3 monoclonal antibody, proleulzin and white
Interleukin -15；It is further preferred that the concentration of the CD3 monoclonal antibody is 30-70ng/mL in the first NKT cell culture fluid,
And/or the concentration of the proleulzin is 300-700U/mL and/or the concentration of the interleukin-15 is 30-70ng/mL.
Under preferable case, the embodiment of the second stage culture includes: the cell training for cultivating the first stage
It supports in the 2nd NKT cell culture fluid, NKT cell culture medium and proleulzin is contained in the 2nd NKT cell culture fluid；Into
Preferably, in the 2nd NKT cell culture fluid, the concentration of the proleulzin is 300-700U/mL to one step.
For NKT cell culture medium, there is no particular limitation, can be commonly used in the art various for cultivating NKT cell
Culture medium, such as can be GT-T551 culture medium.
When preparing NKT cell, for first stage culture and the condition of second stage culture, there is no particular limitation, can
Think various conditions commonly used in the art, such as can be in 30-37 DEG C, the CO that saturated humidity is 3-6%2It is trained in incubator
It supports.Those skilled in the art can be adaptively adjusted the time of culture, this is known to those skilled in the art, herein
It repeats no more.
In the NKT cell that the present invention is prepared, CD3+Cell average ratio > 90%, CD3+CD8+The total CD3 of cell Zhan+Carefully
Average ratio > 70% of born of the same parents；CD3+CD56+The total CD3 of cell Zhan+Average ratio > 15% of cell.
Method for infecting NKT cell is not particularly limited, and can be various methods commonly used in the art, preferable case
Under, this method comprises:
(1) in the presence of viral concentration liquid, nucleoprotamine and proleulzin, NKT cell is subjected to first stage infection training
It supports；
(2) in the presence of CD3 monoclonal antibody, proleulzin and interleukin-15, by first stage infection culture
Cell carries out second stage infection culture.
Preferably, the embodiment of the first stage infection culture includes: by NKT cell culture in the 3rd NKT cell
In culture solution, the 3rd NKT cell culture fluid contains NKT cell culture medium, viral concentration liquid, nucleoprotamine and interleukin-
2；It is further preferred that the concentration of the proleulzin is 300-700U/mL in the 3rd NKT cell culture fluid.
Preferably, the embodiment of the second stage infection culture includes: by the thin of first stage infection culture
Born of the same parents are incubated in the first NKT cell culture fluid.The concrete composition of first NKT cell culture fluid can be found in aforementioned corresponding interior
Hold, details are not described herein.
It is not special for the condition of first stage infection culture and second stage infection culture when infecting NKT cell
Restriction, can be various conditions commonly used in the art, such as can 30-37 DEG C, saturated humidity be 3-6% CO2Culture
It is cultivated in case.Those skilled in the art can be adaptively adjusted the time of culture, this is those skilled in the art
Known, details are not described herein.
Specifically, the method for infecting NKT cell includes: to take 1 × 107-5×107A NKT cell, discards old culture solution,
The fresh GT-T551 culture solution of 2-4mL is added, adds 200-400 μ L viral concentration liquid, 2-4 μ L 1 × 10-6Mg/mL milt egg
The IL-2 of white and final concentration of 300-700U/mL is placed in 30-37 DEG C, the CO that saturated humidity is 3-6%212- is infected in incubator
After 16h, culture solution is abandoned, cell is gone in not coated culture bottle, the GT-T551 culture medium of 20-50mL is added, adds end
Concentration is the IL-2 of 300-700U/mL, the CD3 monoclonal antibody of final concentration of 30-70ng/ml and final concentration of 30-70ng/mL
Interleukin 15, in 30-37 DEG C, saturated humidity be 3-6% CO212-18h is cultivated in incubator, obtains chimeric antigen
The NKT cell of receptor CD133ScFv-CD8-CD137-CD3 ζ modification.
It is further preferred that the method for infection NKT cell further include:
(3) first in the presence of proleulzin, the cell progress of second stage infection culture is external evoked, to cell
Density when being 80-90%, then in the presence of CD3 monoclonal antibody, proleulzin and interleukin-15, cell is expanded
Culture.
Under preferable case, the external evoked embodiment includes: by the cell training of second stage infection culture
It supports in the 2nd NKT cell culture fluid, the embodiment of the amplification cultivation includes: by cell culture in described first
In NKT cell culture fluid.The concrete composition of first NKT cell culture fluid and the 2nd NKT cell culture fluid can be found in aforementioned corresponding
Content, details are not described herein.
Specifically, the method for NKT cell is infected further include: by the slow-virus infection obtained after second stage infection culture
NKT cell is carried out external evoked with the GT-T551 culture solution of the final concentration of 300-700U/mL of IL-2, and the density to cell is
Cell is transferred in cell culture bags when 80-90%, final concentration of 300-700U/mL, CD3 of IL-2 were added every 1.5-2.5 days
The fresh GT-T551 of the final concentration of 30-70ng/ml of monoclonal antibody, the final concentration of 30-70ng/mL of interleukin-15
Culture solution carries out amplification cultivation and cell is expanded to total amount to be 1 × 109-2×109A cell.By slow virus pair of the invention
The Chimeric antigen receptor for targeting CD133 antigen carries out NKT cell infection, and efficiency of infection is up to 30%-60%, and obtain
CAR133-NKT cell, CD3+CD56+The total CD3 of cell Zhan+The ratio of cell is within the scope of 15%-40%.
The Chimeric antigen receptor of the NKT cell expression of Chimeric antigen receptor CD133ScFv-CD8-CD137-CD3 ζ modification
Protein amino acid sequence is as shown in SEQID NO.1.Wherein, it will be understood by those skilled in the art that before Chimeric antigen receptor
Body protein is by rat growth hormone signal peptide, the area hinge of CD133ScFv, CD8 and transmembrane region, the intracellular signal structure of CD137
The intracellular signal structural domain of domain and CD3 ζ are in series, after protein translation after the signal peptide of rough endoplasmic reticulum excision in the cell
As mature Chimeric antigen receptor albumen, after secretion output and it is positioned on the cell membrane of NKT cell.The Chimeric antigen receptor
The corresponding gene coded sequence of protein amino acid sequence is as shown in SEQID NO.2.The Chimeric antigen receptor is with gene C D8's
The structure that the intracellular signal structural domain of the area hinge and transmembrane region and CD137 and CD3 ζ are connected in series is signal transduction structural domain,
Amino acid sequence is as shown in SEQID NO.9, and corresponding gene coded sequence is as shown in SEQID NO.10.
The present invention also provides the NKT cells for the engineering CD133 targeting that the above method is prepared.
The present invention also provides the NKT cells of engineering CD133 targeting in preparing the preparation for treating tumour
Using.Under preferable case, tumour is the epithelial tumor of the CD133 positive, it is further preferred that the epithelial tumor is Colon and rectum
Cancer, liver cancer, cholangiocarcinoma, cancer of pancreas, the cancer of the esophagus, gastric cancer, oophoroma, lung cancer, prostate cancer, bladder cancer, breast cancer, endometrium
Cancer, brain tumor, melanoma or glioma etc..
In application of the invention, for the composition of preparation of the tumour for treating the CD133 positive, there is no particular limitation,
As long as being prepared containing CAR133-NKT cell of the present invention or by CAR133-NKT cell, specific group of preparation
It is well known to those skilled in the art at preparation method, details are not described herein.
It will be understood by those skilled in the art that epithelial tumor is colorectal cancer, liver cancer, cholangiocarcinoma, cancer of pancreas, oesophagus
Cancer, gastric cancer, oophoroma, lung cancer, prostate cancer, bladder cancer, breast cancer, carcinoma of endometrium, brain tumor, melanoma or neuroglia
Whens matter tumor etc., CAR133-NKT cell of the invention can identify the cell of the CD133 positive, including tumour initiator cell, tumour
Stem cell, epithelial progenitor cells and tumour cell, and target killing activity is played, thus positive to aforementioned different types of CD133
Epithelioma has lethal effect, can not only remove in situ tumor and can inhibit the transfer of tumour cell, it is also possible to reduce swollen
The recurrence of tumor.
The present invention also provides the sides of a kind of epithelial tumor for treating the CD133 positive and/or inhibition tumor recurrence and transfer
Method, this method comprises: feeding back the NKT of engineering CD133 targeting of the invention to the epithelial tumor patient's body of the CD133 positive
Cell.Preferably, the epithelial tumor is colorectal cancer, liver cancer, cholangiocarcinoma, cancer of pancreas, the cancer of the esophagus, gastric cancer, oophoroma, lung
Cancer, prostate cancer, bladder cancer, breast cancer, carcinoma of endometrium, brain tumor, melanoma or glioma.
Embodiment
The present invention is further illustrated for embodiment below, but is not intended to limit the present invention.
Experimental method in following embodiment is unless otherwise specified conventional method in that art.Institute in following embodiments
Experimental material is unless otherwise specified to be commercially available from routine biochemistry reagent shop, in which:
NKT cell culture medium GT-T551 is purchased from TaKaRa company.
Lymphocyte separation medium is purchased from TBD company.
CD3 monoclonal antibody, recombinant fiber connection albumen (retronectin) are purchased from TaKaRa company.
Recombinant human protein's interferon-γ, rhIL-2, recombinant human interleukin 15 are purchased from protech company.
Total RNA extraction reagent box RNAiso Reagent, high-fidelity DNA polymerase (HS DNA
Polymerase), T4 DNA 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.
Ago-Gel DNA QIAquick Gel Extraction Kit, common DNA product purification kit, the small extraction reagent kit of plasmid are purchased from day
Root biochemical technology Co., Ltd.
PWPXL-GFP, psPAX2, pMD2.G are purchased from Addgene company.
PGSI is purchased from Beijing Tian Yihuiyuan Biotechnology Co., Ltd.
Trans1-T1 Phage Resistant Competent cell is purchased from the limited public affairs of Beijing Quan Shijin biotechnology
Department.
LipofectamineTM2000 Transfection Reagent transfection reagents are purchased from Invitrogen company.
293T incasing cells is purchased from U.S. ATCC.
In PEG6000-NaCl final concentration of 25.5 the mass %, NaCl of PEG6000 final concentration of 1.2M, PEG6000 and
NaCl is purchased from Shanghai Suo Laibao Biotechnology Co., Ltd.
Fetal calf serum is purchased from PAA company, Germany.
CD107a-PECy5 antibody is purchased from U.S. company BD.
The colorectal cancer cell system HT29 of the CD133 positive is purchased from U.S. ATCC company.
The human liver cancer cell Hep3B of the CD133 positive is purchased from U.S. ATCC company.
The human pancreatic cancer cell SW1990 of the CD133 positive is purchased from U.S. ATCC company.
The Human colorectal cancer cells DLD1 of the CD133 positive is purchased from U.S. ATCC company.
The Human colorectal cancer cells SW620 of the CD133 positive is purchased from U.S. ATCC company.
The Human colorectal cancer cells LOVO of CD133 feminine gender is purchased from U.S. ATCC company.
The human liver cancer cell HepG2 of CD133 feminine gender is purchased from U.S. ATCC company.
5-carboxyfluorescein succinimide ester is purchased from Shanghai Pu Zhen Biotechnology Co., Ltd.
Annexin V-RPE kit is purchased from U.S. company BD.
MethoCultTMH4434 classics cell culture medium is purchased from stem cell company.
All primers are synthesized by Beijing Tian Yihuiyuan Biotechnology Co., Ltd.
The preparation of 1 NKT cell of embodiment
(1) take people's venous blood in the vacuum tube containing heparin.Using lymphocyte separation medium, by density gradient centrifugation side
Method separation obtains mononuclearcell (PBMCs).
(2) after PBMCs is washed three times, using the NKT cell culture medium GT-T551 of the Human autologous serum containing 0.6 volume %
Adjusting final concentration of cells is 2 × 106A cell/mL；By cell inoculation in first passing through final concentration of 10 μ g/mL's in advance
The coated 75cm of retronectin2In Tissue Culture Flask.Then the recombined human of final concentration of 500U/mL is added in culture medium
The recombination human interleukin -15 of interleukin-22,50ng/ml CD3 monoclonal antibody and 50ng/mL, in 37 DEG C, saturated humidity 5%
CO2It is cultivated in incubator.
(3) it cultivates the 4th day, cell is transferred in not coated culture bottle, be added according to cell growth population within every 2 days
NKT cell culture medium GT-T551, control cell concentration are 1 × 108A cell/mL, and the weight of final concentration of 500U/ml is added
Group human interleukin 2；Culture obtained NKT cell, flow cytometry analyzes NKT cell phenotype to the 12nd day.As a result see figure
1, wherein CD3+: 95.04%；CD3+CD8+: 90.99%；CD3+CD56+: 24.12%；CD8+CD56+: 24.63%.
The building of 2 Lentiviral pWPXL-CD133ScFv-CD8-CD137-CD3 ζ of embodiment
(1) preparation of NKT cell cDNA
Centrifugation embodiment 1 cultivates obtained NKT cell, is extracted with total RNA extraction reagent box RNAiso Reagent
The total serum IgE of cell, -80 DEG C save backup.The total serum IgE of extraction Reverse Transcriptase kit RevertAidTM First Strand
CDNA Synthesis Kit reverse transcription obtains NKT cell cDNA, and -20 DEG C save backup.
(2) preparation of slow virus plasmid pWPXL-CD8-CD137-CD3 ζ
Design and synthesize following primer sequence (wherein, for underscore labeled as protection base, box is restriction enzyme site):
Using NKT cell cDNA in step (1) as template, PCR amplification is carried out with primer P1 and P2, obtains the CD8 of long 287bp
The area hinge and transmembrane region, for nucleotide sequence as shown in SEQID NO.3, II restriction enzyme site of MluI and Bgl is contained at both ends respectively
With protection base；PCR amplification is carried out with primer P3 and P4, obtains the CD137 intracellular signal structural domain of long 146bp, nucleotides sequence
For column as shown in SEQID NO.4, Bgl II and EcoRI restriction enzyme site and protection base are contained in both ends respectively；With primer P5 and P6 into
Row PCR amplification obtains the intracellular signal structural domain of the CD3 ζ of long 359bp, and nucleotide sequence is as shown in SEQID NO.5, both ends point
It Han You not EcoRI and NdeI restriction enzyme site and protection base.Each step pcr amplification reaction system is identical, to expand CD137 letter intracellular
For number structural domain, PCR amplification, PCR reaction condition reference are carried outThe explanation of HS DNA Polymerase
Book, reaction system (50 μ L) are as follows:
Distilled water: 32.5 μ L
5 × reaction buffer:10 μ L
DNTP mixture (every kind of 2.5mM): 4 μ L
P3(10mM):1μL
P4(10mM):1μL
NKT cell cDNA (200ng/ul): 1 μ L
HS DNA Polymerase:0.5 μ L
Above-mentioned PCR product is separated with 1% Ago-Gel, is carried out with Ago-Gel DNA QIAquick Gel Extraction Kit
DNA fragmentation recycling.Double enzyme digestion reaction is carried out respectively after obtaining segment, and digestion products are recycled with common DNA product purification kit
It is spare.
Lentiviral pWPXL-GFP MluI/NdeI double digestion, digestion products through 1% Ago-Gel into
Row separation, recycle big carrier segments with Ago-Gel DNA QIAquick Gel Extraction Kit, then with the CD8, CD137 recycled before,
CD3 ζ segment is connected by T4 DNA ligase, and connection product converts Trans1-T1 Phage Resistant Competent
Cell, picking monoclonal after 37 DEG C of culture 16h, 37 DEG C, 250rpm is cultivated and is extracted plasmid with the small extraction reagent kit of plasmid after 12h.It mentions
The plasmid taken identifies that identification electrophoretogram is shown in Fig. 2, wherein M1:DNA molecular weight through restriction enzyme MluI and NdeI double digestion
Mark D15000；1 swimming lane: the non-endonuclease bamhi of plasmid pWPXL-CD8-CD137-CD3 ζ；2 swimming lanes: plasmid pWPXL-CD8-
The endonuclease bamhi (751bp) of CD137-CD3 ζ；M2:DNA molecular weight marker D2000.It will identify that correct plasmid send Beijing day one
The fusion segment of insertion is sequenced in Hui Yuan Biotechnology Co., Ltd.By the correct recombinant plasmid name of sequencing result
For pWPXL-CD8-CD137-CD3 ζ, wherein the area hinge of CD8 and the nucleotide sequence of transmembrane region as shown in SEQID NO.3,
The nucleotide sequence of the intracellular signal structural domain of CD137 is as shown in SEQID NO.4, the nucleosides of the intracellular signal structural domain of CD3 ζ
Acid sequence is as shown in SEQID NO.5.
(3) preparation of slow virus plasmid pWPXL-CD133ScFv-CD8-CD137-CD3 ζ
The nucleotide sequence of full genome composite coding rat growth hormone signal peptide and CD133ScFv fusion, sequence
As shown in SEQID NO.8, by Beijing, Tian Yihuiyuan Biotechnology Co., Ltd is synthesized, and BamHI, kozak sequence are contained in 5 ' ends
Column, 3 ' ends are named as pGSI-CD133ScFv by foregoing fusion gene cloning in plasmid pGSI containing MluI restriction enzyme site.
Plasmid is separated through BamHI/MluI double digestion, digestion products through 1% Ago-Gel, is recycled and is tried with Ago-Gel DNA
It is spare that agent box recycles target fragment.
PWPXL-CD8-CD137-CD3 ζ plasmid is through restriction enzyme BamHI/MluI digestion, and digestion products are through 1% fine jade
Sepharose is separated, spare with Ago-Gel DNA QIAquick Gel Extraction Kit recycling carrier segments.Then with recycling containing big
The DNA fragmentation of rat growth hormone signal peptide and CD133ScFv are attached by T4 DNA ligase, and specific method is shown in explanation
Book.Connection product is converted into Trans1-T1 Phage Resistant Competent cell, picking list after 37 DEG C of culture 16h
Clone, after 250rpm cultivates 12h, extracts plasmid with the small extraction reagent kit of plasmid by 37 DEG C.The plasmid of extraction is through restriction enzyme
The identification of BamHI/NdeI double digestion, qualification result are as shown in Figure 3, wherein M1:DNA molecular weight marker D15000；1 swimming
Road: the non-endonuclease bamhi of plasmid pWPXL-CD133ScFv-CD8-CD137-CD3 ζ (11281p)；2 swimming lanes: plasmid pWPXL-
The endonuclease bamhi (650bp, 956bp) of CD133ScFv-CD8-CD137-CD3 ζ；M2:DNA molecular weight marker D2000.It will identification
Correct plasmid send Beijing Tian Yihuiyuan Biotechnology Co., Ltd that the fusion segment of insertion is sequenced.Sequencing is tied
The correct recombinant plasmid of fruit is named as pWPXL-CD133ScFv-CD8-CD137-CD3 ζ, structural schematic diagram as shown in figure 4, its
In include rat growth hormone signal peptide (nucleotide sequence is as shown in SEQID NO.6), anti-CD133 single-chain antibody (nucleotides sequence
Arrange as shown in SEQID NO.7), the letter intracellular of the intracellular signal structural domain and CD3 ζ of the area hinge of CD8 and transmembrane region and CD137
Number structural domain, wherein the Chimeric antigen receptor is with the area hinge of gene C D8 and transmembrane region and the intracellular signal of CD137 and CD3 ζ
The structure that structural domain is connected in series is signal transduction structural domain, and amino acid sequence is as shown in SEQID NO.9, corresponding gene
Coded sequence is as shown in SEQID NO.10.
The preparation of the NKT cell of 3 Chimeric antigen receptor CD133ScFv-CD8-CD137-CD3 ζ of embodiment modification
(1) packaging and concentration of slow virus
Measure slow virus expression plasmid pWPXL-CD133ScFv-CD8-CD137-CD3 ζ and auxiliary respectively with spectrophotometer
The concentration of plasmid psPAX2, pMD2.G are helped, three kinds of plasmids are with the mass ratio Lipofectamine of 4:2:1TM 2000
Transfection Reagent transfection reagent cotransfection 293T incasing cells.It is collected in virus when transfecting 48h, 72h respectively
Clearly in 50mL EP pipe, 4 DEG C, 2000g is centrifuged 10min, the supernatant obtained twice is shifted into new EP pipe, with 4.5 μm of filter mistakes
Filter viral supernatants；The viral supernatants and 5 × PEG6000-NaCl of filtering are mixed according to the volume ratio of 4:1,4 DEG C of standing 2h, and then 4
DEG C, 10000g is centrifuged 20min, abandons supernatant, and the precipitating sterile PBS of 4 DEG C of pre-coolings of 1mL dissolves to get Chimeric antigen receptor
Viral concentration liquid is dispensed by every 100 μ L of pipe, and -80 DEG C save backup.
According to the method described above, slow virus expression plasmid pWPXL-GFP and helper plasmid psPAX2, pMD2.G cotransfection are utilized
293T incasing cells, collects viral supernatants, and concentration obtains the slow virus concentrate of expression GFP green fluorescent protein.
(2) amplification cultivation of slow-virus infection NKT cell and infected cell
Example 1 in 75cm21 × 10 cultivated in culture bottle7A NKT cell discards old culture solution, and 2mL is added
Viral concentration liquid, the 2 μ L 1 × 10 that fresh NKT cell culture medium GT-T551,200 μ L steps (1) obtain-6Mg/mL milt egg
White, the rhIL-2 of final concentration of 500U/mL is placed in 37 DEG C, the CO that saturated humidity is 5%2Infection 12 is small in incubator
Shi Hou abandons culture solution.NKT cell is synchronized with the slow virus concentrate of expression GFP green fluorescent protein simultaneously and infects (
To NKT cell be known as CART-GFP cell), for calculating the efficiency of infection of the virus.By metainfective cell go to without
The coated 75cm of CD3 and retronectin2In culture bottle, the NKT cell culture medium GT-T551 of 20mL is added, adds dense eventually
Degree is the rhIL-2 of 500U/mL, the CD3 monoclonal antibody of final concentration of 50ng/ml and final concentration of 50ng/mL
Recombinant human interleukin 15, in 37 DEG C, the CO that saturated humidity is 5%218h is cultivated in incubator, obtained NKT cell is known as
CAR133-NKT cell.According to the preparation CAR33-NKT cell of method disclosed in 105384823 A of CN as Mock cell.With
The efficiency of infection of the Flow cytometry virus, as a result as shown in figure 5, the efficiency of infection of CAR133-NKT cell is
36.32%.
(3) external evoked amplification CAR133-NKT cell mass
By the NKT cell culture medium of the final concentration of 500U/mL of the NKT cell rhIL-2 after above-mentioned culture
GT-T551 progress is external evoked, and cell is transferred in cell culture bags when the density of cell is 85%, is recombinated every addition in 2 days
The end of the final concentration of 50ng/ml of final concentration of 500U/mL, CD3 monoclonal antibody of human interleukin 2, recombinant human interleukin 15
Concentration be 50ng/mL fresh NKT cell culture medium GT-T551 carry out amplification cultivation, to cell amplification to total amount be 1.5 ×
109It after a cell or so, is identified using cell colony of the flow cytometer to infection, cell phenotype commonly reaches CD3 sun
Property cell proportion > 90%；CD3CD8 double positive cells ratio > 70%；CD3CD56 double positive cells ratio > 15%, is as a result shown in
Fig. 6, CD3+: 94.09%；CD3+CD4+: 8.79%；CD3+CD8+: 79.95%；CD3+CD56+: 27.68%；CD8+CD56+:
24.41%.
4 CAR133-NKT cell of embodiment analyzes the epithelial tumor cell lethal effect of different CD133 expressions
The NKT cultivated in CAR133-NKT cell, CAR33-NKT cell and the embodiment 1 prepared in Example 3 respectively
Cell inoculation in 96 orifice plates, from the epithelial tumor cells of different CD133 expressions (cancerous cell line of high level expression CD133:
Hep3B,SW1990；The cancerous cell line of medium level expression CD133: HT29, DLD1；And do not express the cancerous cell line of CD133
LOVO, HepG2) it is co-cultured with the ratio for imitating target ratio (killing cell: target cell) 20:1, after co-cultivation in 4 hours,
Coban is added, final concentration of 2 μm of ol/L are incubated for 2 hours, then with CD107a antibody incubation 15 minutes of PE label, use
After PBS buffer solution is washed 3 times, the expression of flow cytometer showed CD107a.
The NKT cultivated in CAR133-NKT cell, CAR33-NKT cell and the embodiment 1 prepared in Example 3 respectively
Cell inoculation is dyed in 96 orifice plates with 5-carboxyfluorescein succinimide ester (CFSE), from different CD133 expressions
The epithelial tumor cell (cancerous cell line of high level expression CD133: Hep3B, SW1990；The cancer that medium level expresses CD133 is thin
Born of the same parents system: HT29, DLD1；And do not express cancerous cell line LOVO, HepG2 of CD133) to imitate target ratio (killing cell: target cell) 20:
1 ratio is co-cultured, and after co-cultivation in 8 hours, cell annexin V-RPE kit is dyed, is set simultaneously
The epithelial tumor cell that the variant CD133 expression of immunocyte killing processing is not added respectively in control group is set, and will
Cell is dyed with annexin V-RPE kit.Flow cytometry detects Apoptosis, and the amount of Apoptosis is under
The formula in face calculates: apoptosis rate=(control-sample)/control × 100%, compares the cell not add immunocyte killing processing
Viable count；Sample is that the cell for the immunocyte killing processing that corresponding effect target ratio (killing cell: target cell) is added is deposited
Number living.
Wherein, the CD133 expression analysis chart of the epithelial tumor cell of different CD133 expressions is shown in Fig. 7 (A)-Fig. 7
(C), CAR133-NKT cell is shown in the lethal effect analysis chart (4 hours) of the epithelial tumor cell of different CD133 expressions
Fig. 8, CAR133-NKT cell are shown in the lethal effect analysis chart (8 hours) of the epithelial tumor cell of different CD133 expressions
Fig. 9.By Fig. 7-9 it is found that the NKT cell of Chimeric antigen receptor CD133ScFv-CD8-CD137-CD3 ζ modification is to high level expression
All have higher specific killing activity with the CD133 cancer cell of medium level expression, and to do not express the cell line of CD133 without
Killing activity, and the specific killing activity of CAR133-NKT cell is substantially better than NKT cell and Mock cell (CAR33-NKT is thin
Born of the same parents).
5 zoopery horizontal analysis CAR133-NKT cell of embodiment is to colorectal cancer mouse model therapeutic effect
It is respectively adopted and cultivates to the Human colorectal cancer cells SW620 and HT29 of the CD133 positive expression of logarithmic growth phase (number
Amount is 5 × 106A, wherein the expression rate of CD133 is respectively 99.43% and 66.67%, wherein SW620's and HT29
CD133 expression analysis chart is shown in Figure 10), it is diluted in 100 μ l physiological saline, is injected into nude mice by subcutaneous.Through 15 days, subcutaneous shape
At colorectal cancer mouse model.
Physiological saline, NKT cell, Mock cell, CAR133- are given respectively by abdominal cavity in colorectal cancer mouse model
(cell quantity is 2 × 10 to NKT cell7) treated, injection is primary daily, continuous injection 2 days, in 40 days execution mouse,
Tumor tissues are removed, tumor tissues volume size, the result is shown in Figure 11 are measured.As shown in Figure 11, CAR133-NKT cell is injected
The knurl of colorectal cancer mouse model is obviously reduced, and illustrates that CAR133-NKT cell of the invention in zoopery level can
Play the function of target killing tumor cell.
Function effect of the CAR133-NKT cell of the invention of embodiment 6 to hemopoietic system
Cord blood is taken to separate primitive progenitors using Ficoll monokaryon separating liquid, it is thin with CAR133-NKT cell, NKT respectively
Born of the same parents, physiological saline and Mock cell (CAR33-NKT cell) are mixed with the ratio of 1:20, are inoculated in MethoCultTMH4434 warp
Allusion quotation cell culture medium, in 37 DEG C, 5%CO2Incubator culture 2 weeks, then clone is counted respectively.
CAR133-NKT cell is shown in Figure 12 to the function effect analysis chart of hemopoietic system, and the figure shows grams of hematopoietic cell
Grand number, wherein BFU-E is erythroid colony forming unit (burst forming unit:early erythroid
Progenitor cell), CFU-GM be Granulocyte macrophage-colony forming unit (colony forming unit:
Granulocyte and macrophage), CFU-GEMM is granulocyte, red blood cell, monocyte and megakaryocyte colony shape
At unit (colony forming unit:granulocyte, erythrocyte, monocyte and
Megakaryocyte), CFU-E is early stage erythroid progenitor cells colony forming unit (colony forming unit:early
erythroid progenitor cell).As shown in figure 12, CAR133-NKT cell of the invention is to hemopoietic system without obvious
Adverse effect.Candidate stem cell also expresses CD133, therefore, eliminates the influence to hemopoietic system, ensure that CAR133-NKT is thin
The safety of born of the same parents' treatment.
Epithelial tumor (using liver cancer as representative) patient of the CAR133-NKT cell of the invention of embodiment 7 to the CD133 positive
Therapeutic effect
CAR133-NKT cell is taken, after 100ml normal saline dilution, continuous three days veins in the form of dosage escalation
Feed back to the liver cancer patient of the CD133 positive (before carrying out targeting immunization therapy using CAR133-NKT cell of the invention,
Cross repeatedly treatment (such as radiotherapy, chemotherapy and other drugs symptomatic treatment), but without obvious curative effects) in vivo, wherein patient 1, suffer from
Person 2, patient 3 have passed through safety dose infusion, and continuous three days infusion dosage total amounts are respectively as follows: 0.03 × 107/kg、0.06
×107/ kg and 0.03 × 107/kg；The lowest dose level that patient 1, patient 2, patient 3, patient 4 play therapeutic effect is defeated
Note, continuous three days infusion dosage total amounts are respectively as follows: 0.39 × 107/kg、0.25×107/kg、0.4×107/ kg and 0.33 ×
107/kg；Patient 5, patient 6, patient 7, patient 8 play the effective dose infusion of clinical efficacy, continuous three days infusions
Dosage total amount is respectively as follows: 0.5 × 107/kg、0.5×107/kg、0.5×107/ kg and 0.79 × 107/kg；To patient after feedback
Treatment situation assessed.
The dosage for treating patient's infusion CAR133-NKT cell is divided into three gradients: 1) safety dose infusion, and cell returns
Throughput rate is 0.01-0.06 (× 107/ kg), 2) play therapeutic effect lowest dose level infusion, cell feedback amount be 0.1-0.5 (×
107/ kg), 3) play clinical efficacy effective dose infusion, cell feedback amount be 0.5-1.0 (× 107/kg)。
Figure 13 (A)-Figure 13 (D) is the toxicity analysis chart of CAR133-NKT cell therapy liver cancer patient, is shown respectively
Hemoglobin (Hgb), leucocyte (WBC), blood platelet (PLT) and net knit Lactoferrin (Ret) when number of days after different cells are fed back
Variation tendency.As shown in Figure 13 (A)-Figure 13 (D), the toxicity of hematological system is mainly that slight hemoglobin and blood are small
The reduction of plate level, toxicity is lower, and patient is tolerable；Other toxicities such as feed back it is relevant shiver, high fever, headache,
Dizzy, out of strength, Nausea and vomiting etc., patient tolerability is good, is alleviated by giving antipyretic-antalgic preparation.Poison is secondary after feedback
Reaction such as fash, ascites, bilirubin increase, and can alleviate after giving symptomatic treatment.Other toxicity datas are as shown in table 1.
Table 1
| Feed back related heating paresthesia 8/8 | Grade 1 |
| Headache 6/8 | < grade 1 |
| Dizziness 7/8 | < grade 1 |
| Low blood pressure 4/8 | < grade 1 |
| Acute ascites 1/8 | Grade 3 |
Figure 14 is the change curve of CAR133-NKT cell copy number and liver cancer patient CD133 positive cell number, wherein
Negative correlation is presented in the percentage of CAR133-NKT cell copy number and CD133 positive cell, shows that CAR133-NKT can be killed
The target cell of the CD133 positive.
Figure 15 is the therapeutic response figure of 8 liver cancer patients, wherein a patient treats progression of disease after the period 1, the
Stable disease after two cycles；Remaining seven patients treat stable disease after the period 1.Illustrate CAR133-NKT cell to liver cancer
Patient has certain therapeutic effect.
The CAR133-NKT cell of the invention of embodiment 8 suffers from the epithelial tumor of the CD133 positive (using cancer of pancreas as representative)
Person's therapeutic effect
CAR133-NKT cell is taken, after 100ml normal saline dilution, continuous three days veins in a manner of dosage escalation
Feed back to the Pancreas cancer patients of the CD133 positive (before carrying out targeting immunization therapy using CAR133-NKT cell of the invention,
By repeatedly treating (such as radiotherapy, chemotherapy and other drugs symptomatic treatment), but without obvious curative effects) in vivo, it is continuous three days thin
It is 0.8 × 10 that born of the same parents, which feed back total amount,7/ kg assesses the treatment situation of patient after feedback.
Figure 16 is therapeutic effect figure of the CAR133-NKT cell to Pancreas cancer patients, as shown in Figure 16, Pancreas cancer patients warp
It crosses 4 weeks treatment posterior tuberosity stoves to reduce, tumor regression is more obvious after treatment in 10 weeks.Illustrate CAR133-NKT cell to cancer of pancreas
Patient has certain therapeutic effect.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (6)
1. a kind of preparation method for the NKT cell for being engineered CD133 targeting, which is characterized in that the described method includes:
Packaging carries the slow virus of pWPXL-CD133ScFv-CD8-CD137-CD3 ζ, obtains viral concentration liquid；Utilize what is obtained
Viral concentration liquid inductance contaminates NKT cell, and NKT cell is made to express Chimeric antigen receptor CD133ScFv-CD8-CD137-CD3 ζ；
The amino acid sequence of the Chimeric antigen receptor is as shown in SEQ ID NO.1；
It is described infection NKT cell method include:
It takes in 75cm21 × 10 cultivated in culture bottle7A NKT cell discards old culture solution, and the fresh NKT cell training of 2mL is added
Support base GT-T551,200 μ L viral concentration liquid, 2 μ L 1 × 10-6The recombined human of mg/mL nucleoprotamine, final concentration of 500U/mL is white
Interleukin 2 is placed in 37 DEG C, the CO that saturated humidity is 5%2After infecting 12 hours in incubator, culture solution is abandoned；By metainfective cell
It goes to and connects the coated 75cm of albumen with recombinant fiber without CD32In culture bottle, the NKT cell culture medium GT- of 20mL is added
T551 adds the rhIL-2 of final concentration of 500U/mL, the CD3 monoclonal antibody of final concentration of 50ng/ml and end
Concentration is the recombinant human interleukin 15 of 50ng/mL, in 37 DEG C, the CO that saturated humidity is 5%218h is cultivated in incubator；
By the NKT cell culture medium GT- of the final concentration of 500U/mL of the NKT cell rhIL-2 after above-mentioned culture
T551 progress is external evoked, and cell is transferred in cell culture bags when the density of cell is 85%, white every 2 days addition recombined humans
The final concentration of the final concentration of 50ng/ml of final concentration of 500U/mL, CD3 monoclonal antibody of interleukin 2, recombinant human interleukin 15
Amplification cultivation is carried out for the fresh NKT cell culture medium GT-T551 of 50ng/mL.
2. according to the method described in claim 1, wherein, the method also includes being prepared via a method which NKT cell:
(1) in the presence of CD3 monoclonal antibody, proleulzin and interleukin-15, mononuclearcell is subjected to first stage training
It supports；The embodiment of the first stage culture includes: that mononuclearcell is incubated in the first NKT cell culture fluid, described
First NKT cell culture fluid contains NKT cell culture medium, CD3 monoclonal antibody, proleulzin and interleukin-15；First NKT
In cell culture fluid, the concentration of the CD3 monoclonal antibody is 30-70ng/mL and/or the concentration of the proleulzin is
The concentration of 300-700U/mL and/or the interleukin-15 is 30-70ng/mL；
(2) in the presence of proleulzin, the cell that the first stage is cultivated carries out second stage culture；The second stage
The embodiment of culture includes: the cell culture of cultivating the first stage in the 2nd NKT cell culture fluid, and described second
Contain NKT cell culture medium and proleulzin in NKT cell culture fluid；In 2nd NKT cell culture fluid, the proleulzin
Concentration is 300-700U/mL.
3. the NKT cell for the engineering CD133 targeting that method as claimed in claim 1 or 2 is prepared.
4. the NKT cell of engineering CD133 targeting as claimed in claim 3 is preparing answering in the preparation for treating tumour
With.
5. application according to claim 4, wherein the tumour is the tumour of the CD133 positive.
6. application according to claim 5, wherein the tumour is the colorectal cancer of the CD133 positive.
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| CN107793482A (en) * | 2016-09-06 | 2018-03-13 | 广州百尼夫生物科技有限公司 | Chimeric antigen receptor and its gene and recombinant expression carrier, CAR133 NKT cells and its preparation method and application |
| IL311608A (en) | 2017-03-13 | 2024-05-01 | Poseida Therapeutics Inc | Compositions and methods for selective elimination and replacement of hematopoietic stem cells |
| CN110526970A (en) * | 2018-05-25 | 2019-12-03 | 深圳宾德生物技术有限公司 | Target single-chain antibody, the Chimeric antigen receptor T cell and its preparation method and application of CD133 |
| CN110526987A (en) * | 2018-05-25 | 2019-12-03 | 深圳宾德生物技术有限公司 | Target Chimeric antigen receptor, the Chimeric antigen receptor T cell and its preparation method and application of CD133 |
| CN113214408B (en) * | 2021-04-30 | 2022-12-27 | 清华大学深圳国际研究生院 | Chimeric antigen receptor macrophage and preparation method and application thereof |
| CN116814553A (en) * | 2023-03-30 | 2023-09-29 | 广州百吉生物制药有限公司 | Killing and viability-enhanced CAR-T cells, preparation and use thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103068850A (en) * | 2010-05-26 | 2013-04-24 | 明尼苏达大学评议会 | Single -chain variable fragment anti-cd133 antibodies 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 |
| CN105384823A (en) * | 2014-08-26 | 2016-03-09 | 中国人民解放军总医院 | Chimeric antigen receptor and gene and recombinant expression vector thereof, engineered CD33 targeting NKT cell and application thereof |
| CN105384822A (en) * | 2014-08-26 | 2016-03-09 | 中国人民解放军总医院 | Chimeric antigen receptor and gene and recombinant expression vector thereof, engineered CD138 targeting NKT cell and application thereof |
-
2016
- 2016-04-20 CN CN201610248750.XA patent/CN105936649B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103068850A (en) * | 2010-05-26 | 2013-04-24 | 明尼苏达大学评议会 | Single -chain variable fragment anti-cd133 antibodies 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 |
| CN105384823A (en) * | 2014-08-26 | 2016-03-09 | 中国人民解放军总医院 | Chimeric antigen receptor and gene and recombinant expression vector thereof, engineered CD33 targeting NKT cell and application thereof |
| CN105384822A (en) * | 2014-08-26 | 2016-03-09 | 中国人民解放军总医院 | Chimeric antigen receptor and gene and recombinant expression vector thereof, engineered CD138 targeting NKT cell and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Patient-derived glioblastoma stem cells are killed by CD133-specific CAR T cells but induce the T cell aging marker CD57;Zhu 等;《Oncotarget》;20141115;第6卷(第1期);第171页摘要、左栏第1段，第172页第2段，第173页Figure 1 |
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