CN108395480A - Chimeric antigen receptor and its gene and recombinant expression carrier, CARHER2-NKT cells and its preparation method and application - Google Patents

Chimeric antigen receptor and its gene and recombinant expression carrier, CARHER2-NKT cells and its preparation method and application Download PDF

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CN108395480A
CN108395480A CN201710068756.3A CN201710068756A CN108395480A CN 108395480 A CN108395480 A CN 108395480A CN 201710068756 A CN201710068756 A CN 201710068756A CN 108395480 A CN108395480 A CN 108395480A
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nkt
nkt cells
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王晓慧
列浦昌
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Guangzhou 100 Neve Biotechnology Co Ltd
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Guangzhou 100 Neve Biotechnology Co Ltd
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Abstract

The invention belongs to knubble biological product technique fields, the invention discloses a kind of Chimeric antigen receptor and its gene and recombinant expression carrier, the NKT cells of engineering HER2 targetings and its applications, the Chimeric antigen receptor be HER2ScFv CD8 CD137 CD3 ζ, including concatenated CD8a signal peptides, HER2ScFv, shorten CD8 hinge area and transmembrane region, the intracellular signal structural domain of CD137 and CD3 ζ intracellular signal structural domain.The CARHER2 NKT cells of the present invention have certain therapeutic effect in the epithelial tumor of the treatment of advanced HER2 positives, to epithelial tumor.

Description

Chimeric antigen receptor and its gene and recombinant expression carrier, CARHER2-NKT cells and Preparation method and application
Technical field
The invention belongs to knubble biological product technique fields, and in particular, to a kind of inosculating antibody in adoptive immunotherapy Original receptor HER2ScFv-CD8-CD137-CD3 ζ and its gene and recombinant expression carrier, the NKT cells for being engineered HER2 targetings (CARHER2-NKT cells) and its preparation method and application.
Background technology
Natural killer cells (NKT) is a kind of t lymphocyte subset group of specific type, has both expressed T cell surface marker, The surface marker of NK cells is expressed again.The antigen recognizing of NKT cells is different from traditional T cell, cannot identify by classics The Antigenic Peptide that MHC-I, class Ⅱmolecule are offered, and it is merely able to the glycolipid antigen offered by non-classical MHC-I classes molecule CDId Activation, and cytokine profiles are generated rapidly, such as：IL-4, IFN-γ, IL-10, IL-13 etc. are prominent so as to identify and kill The tumour cell and virus infected cell of change, and normal autologous tissue's cytotoxic is acted on.NKT cells pass through own face CD16 and the Fc sections of specific antibody combine, play ADCC (antibody-dependent cell-mediated Cytotoxicity it) acts on.But during the cell-mediated lethal effect of antibody-dependant, due to antibody can on target cell Corresponding antigens epitope specificity combine, NKT cells can kill any target cell combined with antibody, although antibody is thin with target Antigen binding on born of the same parents is specific, but NKT cells are nonspecific to the lethal effect of target cell.Under normal conditions, The NKT cells of infusion are 2 weeks or so in patient's body half-life period, and the term of validity is of short duration, needs repeated multiple times infusion.In addition, NKT is thin Born of the same parents itself lack specific antibody, are not enough to be enriched with around tumour or in tumor nest, constrain NKT cells to malignant tumour Targeted therapy.Moreover, studies have shown that NKT cells are not to have fragmentation effect to all tumours, the killing to part entity tumor Effect is weaker, and specific killing activity is to be improved.
HER2 is one of human epidermal growth factor acceptor (HER) family member, and in normal cell, HER2 can Proliferation, migration, differentiation of cell etc. is controlled to maintain the normal bioactivity of cell.And after HER2 excessive activations, Neng Goutong It crosses the transcription for influencing gene and influences the stability of albumen, cause the change of gene expression, the relevant signal path of activationa and proliferation, Inhibit apoptosis pathway, to increase the generation of tumour, the formation of blood vessel and migration of tumour cell etc..Research hair In present many Several Epidermal Tumors and cancer, universal high the phenomenon that expressing, including cholangiocarcinoma, cancer of pancreas, breast cancer, ovum is presented in HER2 Nest cancer, lung cancer, liver cancer, the cancer of the esophagus, prostate cancer, gastric cancer or carcinoma of mouth etc..The patient of HER2 high expression, tumour invade profit Property, recurrence and prognosis mala degree are also very high.Based on the above phenomenon, the therapeutic strategy research for targeting HER2 has important meaning Justice.
Invention content
The purpose of the invention is to overcome in the prior art NKT cells it is weaker to the lethal effect of solid tumor, special Killing activity defect to be improved, provide a kind of Chimeric antigen receptor HER2ScFv-CD8-CD137-CD3 ζ and its gene and Recombinant expression carrier, the NKT cells (CARHER2-NKT cells) and its preparation method and application for being engineered HER2 targetings.
The present inventor has been surprisingly found that under study for action, Chimeric antigen receptor HER2ScFv-CD8- of the invention CD137-CD3 ζ have high efficiency of infection to NKT cells, and the CARHER2-NKT cells obtained after infecting have efficiently in vitro Kill tumor activity, when treating HER2 positive solid tumors, there is certain specific killing activity to tumour cell, especially controlling When treating the epithelial tumor patient of the progressive stage HER2 positive, there is certain therapeutic effect to epithelial tumor.
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 HER2ScFv-CD8-CD137-CD3 ζ, including concatenated CD8a signal peptides, HER2ScFv, the hinge for shortening CD8 The intracellular signal structural domain in area (areas hinge) and transmembrane region, the intracellular signal structural domain of CD137 and CD3 ζ.
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 of engineering HER2 targetings, and the NKT cells are above-mentioned The NKT cells of Chimeric antigen receptor HER2ScFv-CD8-CD137-CD3 ζ modifications.
5th aspect, the present invention provides a kind of preparation method of the NKT cells of engineering HER2 targetings, the methods Including：Packaging carries the slow virus of pWPT-HER2ScFv-CD8-CD137-CD3 ζ, obtains viral concentration liquid；Utilize obtained disease Malicious concentrate infects NKT cells, makes NKT cells expression Chimeric antigen receptor HER2ScFv-CD8-CD137-CD3 ζ.
6th aspect, the present invention provides the NKT cells for the engineering HER2 targetings that the above method is prepared.
7th aspect, the present invention provides the NKT cells of above-mentioned engineering HER2 targetings to prepare for treating tumour Preparation in application.
When treating HER2 positive solid tumors, Chimeric antigen receptor HER2ScFv-CD8-CD137-CD3 ζ couple of the invention NKT cells have high efficiency of infection, and the CARHER2-NKT cells obtained after infecting, that is, are engineered the NKT of HER2 targetings Cell, can specific recognition and combine HER2 antigens, in vitro have efficiently kill tumor activity, hence it is evident that extension immunocyte exist The time-to-live of patient's body, the ability of enhancing immunocyte target recognition of tumor cell surface HER2 antigens are reinforced to tumour The specific killing activity of cell.Moreover, drug resistance caused by during being expected to the mab treatment for avoiding using HER2.This The NKT cells of the engineering HER2 targetings of invention provide a kind of new selection for treatment HER2 positive solid tumors, have good Good industrial application prospect.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Description of the drawings
Fig. 1 is result of the flow cytometry to the NKT cell phenotypes analysis being separately cultured.
Fig. 2 is the restriction enzyme MluI/SalI of the Lentiviral pWPT-CD8-CD137-CD3 ζ of the present invention The electroresis appraisal figure of double digestion segment.
Fig. 3 is the restriction enzyme of the Lentiviral pWPT-HER2ScFv-CD8-CD137-CD3 ζ of the present invention The electroresis appraisal figure of BamHI/SalI double digestion segments.
Fig. 4 is the structural schematic diagram of the Lentiviral pWPT-HER2ScFv-CD8-CD137-CD3 ζ of the present invention, Wherein, sequence counterclockwise is positive gene piece degree, is cdna reverse segment clockwise.
Fig. 5 is the viral concentration that Flow cytometry contains Chimeric antigen receptor HER2ScFv-CD8-CD137-CD3 ζ Efficiency of infection of the liquid to NKT cells.
Fig. 6 is the NKT cells of Flow cytometry Chimeric antigen receptor HER2ScFv-CD8-CD137-CD3 ζ modifications The result of (CARHER2-NKT cells) phenotypic evaluation.
Fig. 7 is cytotoxicity analysis figure of the CARHER2-NKT cells to human solid tumor's killing functions of immunocytes of the present invention.
Fig. 8 is the CARHER2-NKT cells of the present invention to progressive stage cholangiocarcinoma and advanced pancreatic carcinoma patient outcomes Analysis chart.
Fig. 9 is that the CARHER2-NKT cells of the present invention change the striograph that progressive stage cholangiocarcinoma Patients with Liver Metastasis is treated.
Specific implementation mode
The specific implementation mode of the present invention is described in detail 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 HER2ScFv-CD8-CD137-CD3 ζ, including concatenated CD8a signal peptides, HER2ScFv, shorten CD8 hinge area and transmembrane region, CD137 intracellular signal structural domain With the intracellular signal structural domain of CD3 ζ.
Under preferable case, Chimeric antigen receptor HER2ScFv-CD8-CD137-CD3 ζ by CD8a signal peptides, HER2ScFv, The intracellular signal structural domain of the hinge area and transmembrane region, the intracellular signal structural domain of CD137 and CD3 ζ that shorten CD8 is in series. It is further preferred that Chimeric antigen receptor has the amino acid sequence as shown in SEQ ID NO.1, it is further preferred that embedding The amino acid sequence of antigen receptor is closed 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 for obtaining viral concentration liquid and Chimeric antigen receptor HER2ScFv-CD8-CD137-CD3 ζ modifications is thin Born of the same parents, under preferable case, Lentiviral is pWPT-HER2ScFv-CD8-CD137-CD3 ζ.
The preparation method of Lentiviral pWPT-HER2ScFv-CD8-CD137-CD3 ζ is not limited particularly It is fixed, can be those skilled in the art it is conceivable that various methods, under preferable case, Lentiviral pWPT- The preparation method of HER2ScFv-CD8-CD137-CD3 ζ includes the following steps：
(1) the intracellular signal structural domain in the areas hinge and transmembrane region, CD137 of CD8 is expanded respectively from NKT cell cDNAs It with the intracellular signal structural domain of CD3 ζ, and is cloned into carrier pWPT-GFP, structure obtains pWPT-CD8-CD137-CD3 ζ；
(2) nucleotide sequence of composite coding CD8a signal peptides and HER2ScFv, and it is cloned into pWPT-CD8-CD137- In CD3 ζ, the correct pWPT-HER2ScFv-CD8-CD137-CD3 ζ of sequence are obtained after sequence verification.
In step (1), for expanding the areas hinge and transmembrane region, the intracellular of CD137 of CD8 respectively from NKT cell cDNAs 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 methods.Wherein, then NKT cells can be carried out by detaching the mononuclearcell in people's venous blood Culture obtains.
Specifically, the method for obtaining pWPT-CD8-CD137-CD3 ζ may include：The total serum IgE of NKT cells is extracted, is reversed Record obtains NKT cell cDNAs and utilizes primer P1 (SEQID NO.11) and P2 (SEQID using obtained NKT cell cDNAs template NO.12 the areas hinge and transmembrane region (SEQID NO.3) that PCR amplification obtains CD8 genes) 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 genes； The intracellular signal structure that PCR amplification obtains CD3 ζ genes is carried out using primer P5 (SEQID NO.15) and P6 (SEQID NO.16) Domain (SEQID NO.5), double digestion is carried out by the PCR product of acquisition respectively, then with the slow virus after MluI/SalI double digestions Expression vector pWPT-GFP connections.
It is not special for the method for the nucleotide sequence of composite coding CD8a signal peptides and HER2ScFv in step (2) Restriction, 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 pWPT-HER2ScFv-CD8-CD137-CD3 ζ of sequence may include：Pass through The nucleotide sequence (SEQID NO.8) of full genome synthetic technology composite coding CD8a signal peptides and HER2ScFv fusions, gram In the grand pGSI to carrier, pGSI-HER2ScFv is obtained；Then pGSI-HER2ScFv is subjected to BamHI/MluI double digestions, with The recombinant plasmid PWPT-CD8-CD137-CD3 ζ connections that step (1) after BamHI/MluI double digestions obtains, through identification is sequenced, Obtain the correct PWPT-HER2ScFv-CD8-CD137-CD3 ζ of sequence.Wherein, the nucleotide sequence of CD8a signal peptides such as SEQID Shown in NO.6, HER2ScFv nucleotide sequences are as shown in SEQID NO.7.
The present invention also provides a kind of NKT cells of engineering HER2 targetings, and the NKT cells are by above-mentioned inosculating antibody The NKT cells (i.e. CARHER2-NKT cells) of original receptor HER2ScFv-CD8-CD137-CD3 ζ modifications.
For the preparation method of NKT cells that is engineered HER2 targetings, there is no particular limitation, can be this field skill Art personnel it is conceivable that any method, under preferable case, this method includes：Packaging carries PWPT-HER2ScFv-CD8- The slow virus of CD137-CD3 ζ obtains viral concentration liquid；NKT cells are contaminated using obtained viral concentration liquid inductance, make NKT cell tables Up to Chimeric antigen receptor HER2ScFv-CD8-CD137-CD3 ζ.
The method of the slow virus of pWPT-HER2ScFv-CD8-CD137-CD3 ζ encoding genes is carried for packaging without spy Other restriction, can be the common various methods of those skilled in the art, under preferable case, by Lentiviral pWPT- HER2ScFv-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 add 5 × PEG6000-NaCl progress mixings in filtrate, supernatant are abandoned after centrifugation, The sterile PBS dissolvings of 0-4 DEG C of precooling of precipitation, obtain viral concentration liquid.
Further include being prepared via a method which NKT cells in the method for the present invention：
(1) in the presence of CD3 monoclonal antibodies, 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 the first stage culture includes：It is thin that mononuclearcell is incubated at the first NKT In born of the same parents' culture solution, the first NKT cell culture fluids contain NKT cell culture mediums, CD3 monoclonal antibodies, proleulzin and white Interleukin -15；It is further preferred that in the first NKT cell culture fluids, a concentration of 30-70ng/mL of the CD3 monoclonal antibodies, And/or a concentration of 300-700U/mL of the proleulzin and/or a concentration of 30-70ng/mL of the interleukin-15.
Under preferable case, the embodiment of the second stage culture includes：The cell training that the first stage is cultivated It supports in the 2nd NKT cell culture fluids, NKT cell culture mediums and proleulzin is contained in the 2nd NKT cell culture fluids；Into One step preferably, in the 2nd NKT cell culture fluids, a concentration of 300-700U/mL of the proleulzin.
For NKT cell culture mediums, there is no particular limitation, can be commonly used in the art various for cultivating NKT cells Culture medium, such as can be GT-T551 culture mediums.
When preparing NKT cells, 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.The time of culture can be adaptively adjusted in those skilled in the art, this is known to those skilled in the art, herein It repeats no more.
In the NKT cells that the present invention is prepared, CD3+ cell average ratios>90%, CD3+CD8+ cell account for total CD3+ The average ratio of cell>70%；CD3+CD56+ cells account for the average ratio of total CD3+ cells>15%.
Method for infecting NKT cells is not particularly limited, and can be various methods commonly used in the art, preferable case Under, this method includes：
(1) in the presence of viral concentration liquid, nucleoprotamine and proleulzin, NKT cells are subjected to first stage infection training It supports；
(2) in the presence of CD3 monoclonal antibodies, 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 cells In culture solution, the 3rd NKT cell culture fluids contain NKT cell culture mediums, viral concentration liquid, nucleoprotamine and interleukin- 2；It is further preferred that in the 3rd NKT cell culture fluids, a concentration of 300-700U/mL of the proleulzin.
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 fluids.The concrete composition of first NKT cell culture fluids 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 cells 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.The time of culture can be adaptively adjusted in those skilled in the art, this is those skilled in the art Known, details are not described herein.
Specifically, the method for infection NKT cells includes：Take 1 × 107-5×107A NKT cells, discard old culture solution, The fresh GT-T551 culture solutions of 2-4mL are added, add 200-400 μ L viral concentrations liquid, L1 × 10 2-4 μ-6Mg/mL nucleoprotamine With the IL-2 of final concentration of 300-700U/mL, it is placed in 30-37 DEG C, infects 12- in the CO2 incubators that saturated humidity is 3-6% After 16h, culture solution is abandoned, cell is gone in not coated culture bottle, the GT-T551 culture mediums of 20-50mL are added, add end The IL-2 of a concentration of 300-700U/mL, the CD3 monoclonal antibodies of final concentration of 30-70ng/ml and final concentration of 30-70ng/mL Interleukin 15, cultivate 12-18h in 30-37 DEG C, the CO2 incubators that saturated humidity is 3-6%, obtain chimeric antigen The NKT cells of receptor CD133ScFv-CD8-CD137-CD3 ζ modifications.
It is further preferred that the method for infection NKT cells further includes：
(3) first in the presence of proleulzin, the cell progress of second stage infection culture is external evoked, wait for cell Density when being 80-90%, then in the presence of CD3 monoclonal antibodies, 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 fluids, the embodiment of the amplification cultivation includes：By cell culture in described first In NKT cell culture fluids.The concrete composition of first NKT cell culture fluids and the 2nd NKT cell culture fluids can be found in aforementioned corresponding Content, details are not described herein.
Specifically, the method for infection NKT cells further includes：By the slow-virus infection obtained after second stage infection culture NKT cells are carried out external evoked with the GT-T551 culture solutions of the final concentration of 300-700U/mL of IL-2, wait for that the density of cell is Cell is transferred in cell culture bags when 80-90%, every final concentration of 300-700U/mL, CD3 that 1.5-2.5 days are added IL-2 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 the slow virus pair of the present invention The Chimeric antigen receptor for targeting HER2 antigens carries out NKT cell infections, and efficiency of infection is up to 30%-60%, and the CAR obtained HER2-NKT cells, CD3+CD56+ cells account for the ratio of total CD3+ cells within the scope of 15%-40%.
The Chimeric antigen receptor of the NKT cells expression of Chimeric antigen receptor HER2ScFv-CD8-CD137-CD3 ζ modifications Maturation protein amino acid sequence is as shown in SEQID NO.1.Wherein, it will be understood by those skilled in the art that chimeric antigen by Body precursor protein is by CD8a signal peptides, HER2ScFv, the areas hinge for shortening 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 ripe Chimeric antigen receptor albumen, after secretion output and it is positioned on the cell membrane of NKT cells.The Chimeric antigen receptor The corresponding gene coded sequence of maturation protein amino acid sequence is as shown in SEQID NO.2.The Chimeric antigen receptor is with gene C D8 The areas hinge and the structure that is connected in series of the intracellular signal structural domain of transmembrane region and CD137 and CD3 ζ be signal transduction structural domain, Its 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 HER2 targetings that the above method is prepared.
The present invention also provides NKT cells the answering in preparing the preparation for treating tumour of engineering HER2 targetings With.Under preferable case, tumour refers to HER2 positive solid tumors, refers in particular to recur refractory HER2 positive solid tumors；Further Preferably, tumour is cholangiocarcinoma, cancer of pancreas, breast cancer, oophoroma, lung cancer, liver cancer, the cancer of the esophagus, prostate cancer, gastric cancer or oral cavity Cancer.
In the application of the present invention, for the composition of preparation of the tumour for treating the HER2 positives, there is no particular limitation, As long as being prepared containing CAR HER2-NKT cells of the present invention or by CAR HER2-NKT cells, the tool of preparation Body forms and preparation method is well known to those skilled in the art, and details are not described herein.
It will be understood by those skilled in the art that epithelial tumor is cholangiocarcinoma, cancer of pancreas, breast cancer, oophoroma, lung Whens cancer, liver cancer, the cancer of the esophagus, prostate cancer, gastric cancer or carcinoma of mouth etc., CAR HER2-NKT cells of the invention can identify HER2 Positive tumour cell, and target killing activity is played, to have lethal effect to HER2 positive epitheliomas.
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 this field conventional method.Institute in following embodiments Experiment material, unless otherwise specified, be commercially available from routine biochemistry reagent shop, wherein：
NKT cell culture mediums GT-T551 is purchased from TaKaRa companies.
Lymphocyte separation medium is purchased from TBD companies.
Recombinant fiber connects albumen (retronectin) and is purchased from TaKaRa companies.
CD3, CD4, CD8, CD56 monoclonal antibody are purchased from BD companies.
Recombinant human protein's interferon-γ, rhIL-2, recombinant human interleukin 15 are purchased from protech companies.
Total RNA extraction reagent box RNAiso Reagent, high-fidelity DNA polymerase ( HS DNA Polymerase), T4DNA ligases are purchased from TaKaRa companies.
RevertAidTMFirst Strand cDNA Synthesis Kit are purchased from Fermentas companies.
Bgl II, EcoRI, MluI, BamHI, SalI are purchased from Fermentas companies.
Ago-Gel DNA QIAquick Gel Extraction Kits, common DNA product purification kit, the small extraction reagent kit of plasmid are purchased from day Root biochemical technology Co., Ltd.
PWPT-GFP, psPAX2, pMD2.G are purchased from Addgene companies.
PGSI is purchased from Beijing bio tech ltd Tian Yihuiyuan.
Trans1-T1Phage Resistant Competent cells are purchased from Beijing Quanshijin Biotechnology Co., Ltd.
LipofectamineTM2000Transfection Reagent transfection reagents are purchased from Invitrogen companies.
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 the Shanghai bio tech ltd Suo Laibao.
Fetal calf serum is purchased from PAA companies of Germany.
The human breast cancer cell SKBR3 of the HER2 positives, SGC-7901 cells, Proliferation of Human Ovarian Cell SKOV-3, no The HeLa cells of expression HER2 are purchased from U.S. ATCC.
Cell Counting Kit-8 (CCK8) kit is purchased from Beijing Wo Bisen Science and Technology Ltd.s.
All primers are synthesized by Beijing bio tech ltd Tian Yihuiyuan.
The preparation of embodiment 1NKT cells
(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 mediums GT-T551 of the Human autologous serum containing 0.6 volume % It is 2 × 10 to adjust final concentration of cells6A 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 antibodies 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 quantity 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 cells, flow cytometry analyzes NKT cell phenotypes 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 structure of 2 Lentiviral pWPT-HER2ScFv-CD8-CD137-CD3 ζ of embodiment
(1) preparation of NKT cell cDNAs
Centrifugation embodiment 1 cultivates obtained NKT cells, 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 RevertAidTMFirst Strand CDNA Synthesis Kit reverse transcriptions obtain NKT cell cDNAs, and -20 DEG C save backup.
(2) preparation of slow virus plasmid pWPT-CD8-CD137-CD3 ζ
Design and synthesize following primer sequence (wherein, underscore is labeled as protection base, and box is restriction enzyme site)：
Using NKT cell cDNAs in step (1) as template, PCR amplification is carried out with primer P1 and P2, obtains the CD8 of long 227bp The areas hinge and transmembrane region, nucleotide sequence as shown in SEQID NO.3, both ends respectively contain II restriction enzyme site of MluI and Bgl And protection base；PCR amplification is carried out with primer P3 and P4, obtains the CD137 intracellular signal structural domains of long 146bp, nucleotides sequence For row as shown in SEQID NO.4, Bgl II and EcoRI restriction enzyme sites 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 SalI restriction enzyme sites and protection base.Each step pcr amplification reaction system is identical, to expand CD137 intracellulars letter For number structural domain, PCR amplification, PCR reaction condition references are carried outThe explanation of HS DNA Polymerase Book, reaction system (50 μ L) are as follows：
Distilled water：32.5μL
5 × reaction buffer：10μL
DNTP mixtures (each 2.5mM)：4μL
P3(10mM):1μL
P4(10mM):1μL
NKT cell cDNAs (200ng/ul)：1μL
HS DNA Polymerase：0.5μL
Above-mentioned PCR product is detached with 1% Ago-Gel, is carried out with Ago-Gel DNA QIAquick Gel Extraction Kits DNA fragmentation recycles.Double digestion reaction is carried out respectively after obtaining segment, and digestion products are recycled with common DNA product purification kit It is spare.
Lentiviral pWPT-GFP MluI/SalI double digestions, digestion products are carried out through 1% Ago-Gel Separation, big carrier segments are recycled with Ago-Gel DNA QIAquick Gel Extraction Kits, then with the CD8, CD137, CD3 ζ that recycle before Segment is connected by T4DNA ligases, connection product conversion Trans1-T1Phage Resistant Competent cells, and 37 Picking monoclonal after DEG C culture 16h, 37 DEG C, 250rpm is cultivated and is extracted plasmid with the small extraction reagent kit of plasmid after 12h.The plasmid of extraction It is identified through restriction enzyme MluI and SalI double digestion, identification electrophoretogram is shown in Fig. 2, wherein M1：DNA molecular amount marks D4500；1 swimming lane：The non-endonuclease bamhi of plasmid pWPT-CD8-CD137-CD3 ζ；2 swimming lanes：Plasmid pWPT-CD8-CD137-CD3 ζ Endonuclease bamhi (672bp)；M2：DNA molecular amount marks D2000.It will identify that correct plasmid send Beijing day brightness far biological section The fusion segment of insertion is sequenced in skill Co., Ltd.The correct recombinant plasmid of sequencing result is named as pWPT- CD8-CD137-CD3 ζ, wherein the areas hinge of CD8 and the nucleotide sequence of transmembrane region as shown in SEQID NO.3, CD137's The nucleotide sequence of intracellular signal structural domain is as shown in SEQID NO.4, and the nucleotide sequence of the intracellular signal structural domain of CD3 ζ is such as Shown in SEQID NO.5.
(3) preparation of slow virus plasmid pWPT-HER2ScFv-CD8-CD137-CD3 ζ
The nucleotide sequence of full genome composite coding CD8a signal peptides and HER2ScFv fusions, sequence such as SEQID It shown in NO.8, is synthesized by Beijing bio tech ltd Tian Yihuiyuan, BamHI, kozak sequence are contained in 5 ' ends, and 3 ' ends contain There are MluI restriction enzyme sites, by foregoing fusion gene cloning in plasmid pGSI, is named as pGSI-HER2ScFv.Plasmid passes through BamHI/MluI double digestions, digestion products are detached through 1% Ago-Gel, are returned with Ago-Gel DNA QIAquick Gel Extraction Kits It is spare to receive target fragment.
PWPT-CD8-CD137-CD3 ζ plasmids are through restriction enzyme BamHI/MluI digestions, and digestion products are through 1% agar Sugared gel is detached, spare with Ago-Gel DNA QIAquick Gel Extraction Kits recycling carrier segments.Then contain CD8a with recycling The DNA fragmentation of signal peptide and HER2ScFv are attached by T4DNA ligases, and specific method is shown in specification.By connection product Convert Trans1-T1Phage Resistant Competent cells, 37 DEG C culture 16h after picking monoclonal, 37 DEG C, After 250rpm cultivates 12h, plasmid is extracted with the small extraction reagent kit of plasmid.The plasmid of extraction is bis- through restriction enzyme BamHI/SalI Digestion identifies that qualification result is as shown in Figure 3, wherein 1 swimming lane：The digestion of plasmid pWPT-HER2ScFv-CD8-CD137-CD3 ζ Segment (1485bp)；2 swimming lanes：The non-endonuclease bamhis of plasmid pWPT-HER2ScFv-CD8-CD137-CD3 ζ (10114bp)；M2：DNA Molecular weight marker D2000.The fusion base that will identify that correct plasmid send Beijing bio tech ltd Tian Yihuiyuan to insertion Because segment is sequenced.The correct recombinant plasmid of sequencing result is named as pWPT-HER2ScFv-CD8-CD137-CD3 ζ, Structural schematic diagram is as shown in figure 4, single-stranded including CD8a signal peptides (nucleotide sequence is as shown in SEQID NO.6), anti-HER2 The intracellular signal structural domain in the areas hinge and transmembrane region and CD137 of antibody (nucleotide sequence is as shown in SEQID NO.7), CD8 With the intracellular signal structural domain of CD3 ζ, wherein the Chimeric antigen receptor with the areas hinge of gene C D8 and transmembrane region and CD137 and The structure that the intracellular signal structural domain of CD3 ζ is connected in series is signal transduction structural domain, amino acid sequence such as SEQID NO.9 institutes Show, corresponding gene coded sequence is as shown in SEQID NO.10.
The preparation of the NKT cells of 3 Chimeric antigen receptor HER2ScFv-CD8-CD137-CD3 ζ modifications of embodiment
(1) packaging of slow virus and concentration
Measure slow virus expression plasmid pWPT-HER2ScFv-CD8-CD137-CD3 ζ and auxiliary respectively with spectrophotometer The concentration of plasmid psPAX2, pMD2.G, three kinds of plasmids are with 4:2:1 mass ratio is used LipofectamineTM2000Transfection Reagent transfection reagent cotransfection 293T incasing cells.It is transfecting respectively Viral supernatants are collected when 48h, 72h in 50mL EP pipes, 4 DEG C, 2000g centrifuges 10min, shifts the supernatant that obtains twice to new In EP pipes, with 4.5 μm of filter filter virus supernatants；The viral supernatants and 5 × PEG6000-NaCl of filtering are according to 4:1 volume ratio Mixing, 4 DEG C of standing 2h, then 4 DEG C, 10000g centrifuges 20min, abandons supernatant, and precipitation is molten with the sterile PBS of 4 DEG C of precoolings of 1mL Solution is dispensed, -80 DEG C save backup to get the viral concentration liquid of Chimeric antigen receptor by often 100 μ L of pipe.
According to the method described above, slow virus expression plasmid pWPT-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 proteins.
(2) amplification cultivation of slow-virus infection NKT cells and infected cell
Example 1 in 75cm21 × 10 cultivated in culture bottle7A NKT cells discard old culture solution, and 2mL is added Viral concentration liquid that fresh NKT cell culture mediums GT-T551,200 μ L steps (1) obtain, 2 L1 × 10 μ-6Mg/mL nucleoprotamine, The rhIL-2 of final concentration of 500U/mL is placed in 37 DEG C, the CO that saturated humidity is 5%2It is infected 12 hours in incubator Afterwards, culture solution is abandoned.Infection is synchronized to NKT cells with the slow virus concentrate of expression GFP green fluorescent proteins simultaneously (to obtain NKT cells be known as CART-GFP cells), the efficiency of infection for calculating the virus.Metainfective cell is gone to without CD3 With the coated 75cm of retronectin2In culture bottle, the NKT cell culture medium GT-T551 of 20mL are added, add final concentration For the weight of the CD3 monoclonal antibodies and final concentration of 50ng/mL of the rhIL-2 of 500U/mL, final concentration of 50ng/ml Group human interleukin 15, in 37 DEG C, the CO that saturated humidity is 5%218h is cultivated in incubator, obtained NKT cells are known as CARHER2-NKT cells.CAR33-NKT cells are prepared as Mock cells according to method disclosed in CN 105384823A.With The efficiency of infection of the Flow cytometry virus, the results are shown in Figure 5, and the efficiency of infection of CARHER2-NKT cells is 55.32%.
(3) external evoked amplification CARHER2-NKT cell masses
By the NKT cell culture mediums of the final concentration of 500U/mL of the NKT cell rhIL-2s 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%, and recombination was added every 2 days The final concentration of 50ng/ml of final concentration of 500U/mL, CD3 monoclonal antibody of human interleukin 2, the end of recombinant human interleukin 15 The fresh NKT cell culture mediums GT-T551 of a concentration of 50ng/mL carries out amplification cultivation, wait for cell amplification to total amount be 1.5 × 109After a cell or so, the cell colony of infection is identified using flow cytometer, cell phenotype commonly reaches CD3 sun Property cell proportion ＞ 90%；CD3CD8 positive cell ratios>70%；CD3CD56 double positive cells ratios>15%, as a result see figure 6, CD3+:95.43%；CD3+CD8+:82.44%；CD3+CD56+:20.65%；CD8+CD56+:19.97%.
Cytotoxicity analysis of the embodiment 4CARHER2-NKT cells to human tumor cells lethal effect
The human breast cancer cell SKBR3, SGC-7901 cells, Proliferation of Human Ovarian Cell of the HER2 positives are taken respectively SKOV-3 and do not express HER2 HeLa cell inoculations after 96 orifice plates, 37 DEG C of incubator overnight incubations, prepared in Example 3 CARHER2-NKT cells, the NKT cells cultivated in CAR33-NKT cells and embodiment 1, to imitate target ratio (killing cell：Target Cell) 1:1,5:1,10:1,20:1 with human breast cancer cell SKBR3, SGC-7901 cells, the people of the above-mentioned HER2 positives Ovarian cancer cell SKOV-3 and the HeLa cells for not expressing HER2 are co-cultured, and after the co-cultivation of 4h, each hole is added The CCK8 of 10 μ L is dyed.It is respectively the CARHER2-NKT that is prepared in embodiment 3 thin that killing cell controls group is arranged simultaneously The NKT cells cultivated in born of the same parents, CAR33-NKT cells and embodiment 1, and same amount of CCK8 is added and is dyed；And setting Target cell control group is human breast cancer cell SKBR3, SGC-7901 cells, the people that immunocyte killing processing is not added Ovarian cancer cell SKOV-3 and HeLa cell, and same amount of CCK8 is added and is dyed.Microplate reader to Apoptosis situation into Row detection, the amount of Apoptosis are calculated according to the following equation：Apoptosis rate={ [(experimental group-killing cell controls group-target is thin by 1- Born of the same parents' control group)/experimental group] × 100%, in the formula, killing cell controls group be only killing cell and plus target cell is surveyed The light absorption value obtained, target cell control group are the light absorption value for there was only target cell and killing cell not being added to measure；Experimental group is that phase is added Corresponding effect target ratio (killing cell：Target cell) immunocyte killing processing after the light absorption value that measures, see Fig. 7.The result shows that The NKT cells of Chimeric antigen receptor HER2ScFv-CD8-CD137-CD3 ζ modifications have the tumour cell of the HER2 positives special Killing activity, and the specific killing activity of CARHER2-NKT cells is substantially better than NKT cells and Mock cells (CAR33-NKT is thin Born of the same parents).
The CAR HER2-NKT cells of 5 present invention of embodiment are to the epithelial tumor of the HER2 positives (with cholangiocarcinoma and cancer of pancreas To represent) patient outcomes
CAR HER2-NKT cells are taken, after 100ml normal saline dilutions, continuous three days quiet in the form of dosage escalation Arteries and veins feeds back to the progressive stage cholangiocarcinoma of the HER2 positives and Pancreas cancer patients (are carried out using the CAR HER2-NKT cells of the present invention Before targeting immunization therapy, repeatedly treatment (such as radiotherapy, chemotherapy and other drugs symptomatic treatment) is had been subjected to, but treated without apparent Effect) in vivo, it is 0.8 × 10 that continuous three days cells, which feed back total amount,7/ kg assesses the treatment situation of patient after feedback.
Table 1 is CAR HER2-NKT cell therapy progressive stage cholangiocarcinomas and the toxicity analysis of advanced pancreatic carcinoma patient Figure respectively illustrates the adverse reaction in pretreatment chemotherapy stage, prolonging after the adverse reaction in cell infusion stage and cell feedback Slow adverse reaction.As shown in table 1, the toxicity of hematological system caused by patient's infusion is mainly slight anaemia and blood platelet Horizontal reduction, toxicity is relatively low, and patient is tolerable；Other toxicities such as feed back relevant high fever, fatigue, shiver with cold, weary Power etc., patient tolerability is good, is alleviated by giving antipyretic-antalgic preparation.
Table 1
Fig. 8 is the therapeutic response figure of 11 cholangiocarcinomas and Pancreas cancer patients, wherein a patient treats disease after the period 1 Disease occur complete incidence graph part alleviate；5 patients treat stable disease after the period 1；After remaining 5 patients treatment disease into Exhibition.Illustrate that CAR133-NKT cells have cholangiocarcinoma and Pancreas cancer patients certain therapeutic effect.
Fig. 9 is therapeutic effect figure of the CAR HER2-NKT cells to progressive stage cholangiocarcinoma Patients with Liver Metastasis, as shown in Figure 9, Compared with baseline before treatment, cholangiocarcinoma patients hepatic metastases lesion is obviously reduced after treating four weeks.Illustrate CAR HER2-NKT cells There is certain therapeutic effect to cholangiocarcinoma patients.
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 can carry out a variety of simple variants to technical scheme of the present invention within the scope of the technical concept of the present invention, 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 of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can The combination of energy no longer separately illustrates.
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.
SEQUENCE LISTING
<110>Hundred Ni Fu Bioisystech Co., Ltd of Guangzhou
<120>It Chimeric antigen receptor and its gene and recombinant expression carrier, CARHER2-NKT cells and preparation method thereof and answers With
<130> I41973RMJ
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 492
<212> PRT
<213>Chimeric antigen receptor HER2ScFv-CD8-CD137-CD3 ζ
<400> 1
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
20 25 30
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
35 40 45
Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
50 55 60
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile
85 90 95
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
100 105 110
Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
130 135 140
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
145 150 155 160
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr
165 170 175
Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala
180 185 190
Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys
195 200 205
Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu
210 215 220
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser
225 230 235 240
Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly
245 250 255
Thr Leu Val Thr Val Ser Ser Thr Arg Thr Thr Thr Pro Ala Pro Arg
260 265 270
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
275 280 285
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
290 295 300
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
305 310 315 320
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Ser
325 330 335
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
340 345 350
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
355 360 365
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Glu Phe Arg Val Lys Phe
370 375 380
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu
385 390 395 400
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
405 410 415
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
420 425 430
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
435 440 445
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
450 455 460
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
465 470 475 480
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 2
<211> 1479
<212> DNA
<213>Chimeric antigen receptor HER2ScFv-CD8-CD137-CD3 ζ
<400> 2
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggacatcc agatgaccca gagcccaagc tctctgtctg cctctgtggg cgacagagtg 120
accatcacct gcagagccag ccaggacgtg aacacagccg tggcctggta tcagcagaag 180
ccaggcaagg ccccaaagct gctgatctac agcgccagct tcctgtacag cggcgtgcca 240
agcagattca gcggcagcag aagcggcaca gacttcaccc tgaccatcag cagcctgcag 300
ccagaggact tcgccaccta ctactgccag cagcactaca ccaccccacc aaccttcgga 360
cagggcacca aggtggagat caagggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420
ggcggatctg aagtgcaatt ggtggaaagc ggcggcggcc tggtgcaacc gggcggcagc 480
ctgcgtctga gctgcgcggc ctccggattt aacataaagg acacatacat ccactgggtg 540
cgccaagcac ctgggaaggg tctcgagtgg gtggctcgga tttacccaac aaatggctac 600
accaggtatg cggatagcgt gaaaggccgt tttaccattt cagctgatac ttcgaagaac 660
accgcctatc tgcaaatgaa cagcctgcgt gcggaagata cggccgtgta ttattgctcg 720
cgttggggag gagacgggtt ctatgctatg gattactggg gccaaggcac cctggtgacg 780
gttagctcaa cgcgtaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 840
gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg 900
cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact 960
tgtggggtcc ttctcctgtc actggttatc accctttact gcagatctaa acggggcaga 1020
aagaaactcc tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag 1080
gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actggaattc 1140
agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 1200
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 1260
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 1320
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 1380
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 1440
tacgacgccc ttcacatgca ggccctgccc cctcgctaa 1479
<210> 3
<211> 227
<212> DNA
<213>The areas hinge of CD8 and transmembrane region
<400> 3
gatcacgcgt accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc 60
gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac 120
gagggggctg gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg 180
ggtccttctc ctgtcactgg ttatcaccct ttactgcaga tctgatc 227
<210> 4
<211> 146
<212> DNA
<213>The intracellular signal structural domain of CD137
<400> 4
gatcagatct aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag 60
accagtacaa actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga 120
aggaggatgt gaactggaat tcgatc 146
<210> 5
<211> 359
<212> DNA
<213>The intracellular signal structural domain of CD3 ζ
<400> 5
gatcgaattc agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca 60
gaaccagctc tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa 120
gagacgtggc cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg 180
cctgtacaat gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa 240
aggcgagcgc cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac 300
caaggacacc tacgacgccc ttcacatgca ggccctgccc cctcgctaag tcgacgatc 359
<210> 6
<211> 63
<212> DNA
<213>CD8a signal peptides
<400> 6
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccg 63
<210> 7
<211> 726
<212> DNA
<213> HER2ScFv
<400> 7
gacatccaga tgacccagag cccaagctct ctgtctgcct ctgtgggcga cagagtgacc 60
atcacctgca gagccagcca ggacgtgaac acagccgtgg cctggtatca gcagaagcca 120
ggcaaggccc caaagctgct gatctacagc gccagcttcc tgtacagcgg cgtgccaagc 180
agattcagcg gcagcagaag cggcacagac ttcaccctga ccatcagcag cctgcagcca 240
gaggacttcg ccacctacta ctgccagcag cactacacca ccccaccaac cttcggacag 300
ggcaccaagg tggagatcaa gggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360
ggatctgaag tgcaattggt ggaaagcggc ggcggcctgg tgcaaccggg cggcagcctg 420
cgtctgagct gcgcggcctc cggatttaac ataaaggaca catacatcca ctgggtgcgc 480
caagcacctg ggaagggtct cgagtgggtg gctcggattt acccaacaaa tggctacacc 540
aggtatgcgg atagcgtgaa aggccgtttt accatttcag ctgatacttc gaagaacacc 600
gcctatctgc aaatgaacag cctgcgtgcg gaagatacgg ccgtgtatta ttgctcgcgt 660
tggggaggag acgggttcta tgctatggat tactggggcc aaggcaccct ggtgacggtt 720
agctca 726
<210> 8
<211> 789
<212> DNA
<213>CD8a signal peptides and HER2ScFv fusions
<400> 8
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggacatcc agatgaccca gagcccaagc tctctgtctg cctctgtggg cgacagagtg 120
accatcacct gcagagccag ccaggacgtg aacacagccg tggcctggta tcagcagaag 180
ccaggcaagg ccccaaagct gctgatctac agcgccagct tcctgtacag cggcgtgcca 240
agcagattca gcggcagcag aagcggcaca gacttcaccc tgaccatcag cagcctgcag 300
ccagaggact tcgccaccta ctactgccag cagcactaca ccaccccacc aaccttcgga 360
cagggcacca aggtggagat caagggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420
ggcggatctg aagtgcaatt ggtggaaagc ggcggcggcc tggtgcaacc gggcggcagc 480
ctgcgtctga gctgcgcggc ctccggattt aacataaagg acacatacat ccactgggtg 540
cgccaagcac ctgggaaggg tctcgagtgg gtggctcgga tttacccaac aaatggctac 600
accaggtatg cggatagcgt gaaaggccgt tttaccattt cagctgatac ttcgaagaac 660
accgcctatc tgcaaatgaa cagcctgcgt gcggaagata cggccgtgta ttattgctcg 720
cgttggggag gagacgggtt ctatgctatg gattactggg gccaaggcac cctggtgacg 780
gttagctca 789
<210> 9
<211> 229
<212> PRT
<213>Signal transduction structural domain
<400> 9
Thr Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
1 5 10 15
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
20 25 30
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile
35 40 45
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser
50 55 60
Leu Val Ile Thr Leu Tyr Cys Arg Ser Lys Arg Gly Arg Lys Lys Leu
65 70 75 80
Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln
85 90 95
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
100 105 110
Cys Glu Leu Glu Phe Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro
115 120 125
Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
130 135 140
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
145 150 155 160
Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
165 170 175
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
180 185 190
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
195 200 205
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
210 215 220
Ala Leu Pro Pro Arg
225
<210> 10
<211> 690
<212> DNA
<213>Signal transduction structural domain
<400> 10
acgcgtacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 60
cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 120
gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 180
cttctcctgt cactggttat caccctttac tgcagatcta aacggggcag aaagaaactc 240
ctgtatatat tcaaacaacc atttatgaga ccagtacaaa ctactcaaga ggaagatggc 300
tgtagctgcc gatttccaga agaagaagaa ggaggatgtg aactggaatt cagagtgaag 360
ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 420
ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 480
gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 540
aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 600
aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 660
cttcacatgc aggccctgcc ccctcgctaa 690
<210> 11
<211> 30
<212> DNA
<213>Artificial sequence
<400> 11
gatcacgcgt accacgacgc cagcgccgcg 30
<210> 12
<211> 34
<212> DNA
<213>Artificial sequence
<400> 12
gatcagatct gcagtaaagg gtgataacca gtga 34
<210> 13
<211> 32
<212> DNA
<213>Artificial sequence
<400> 13
gatcagatct aaacggggca gaaagaaact cc 32
<210> 14
<211> 35
<212> DNA
<213>Artificial sequence
<400> 14
gatcgaattc cagttcacat cctccttctt cttct 35
<210> 15
<211> 32
<212> DNA
<213>Artificial sequence
<400> 15
gatcgaattc agagtgaagt tcagcaggag cg 32
<210> 16
<211> 30
<212> DNA
<213>Artificial sequence
<400> 16
gatcgtcgac ttagcgaggg ggcagggcct 30

Claims (10)

1. a kind of Chimeric antigen receptor, which is characterized in that the Chimeric antigen receptor is HER2ScFv-CD8-CD137-CD3 ζ, Including concatenated CD8a signal peptides, HER2ScFv, shorten CD8 hinge area and transmembrane region, CD137 intracellular signal structural domain and The intracellular signal structural domain of CD3 ζ；Preferably, the Chimeric antigen receptor has the amino acid sequence as shown in SEQ ID NO.1 Row, it is further preferred that the amino acid sequence of the Chimeric antigen receptor is as shown in SEQ ID NO.1.
2. the gene of coding Chimeric antigen receptor described in claim 1, it is preferable that the gene has such as SEQ ID NO.2 Shown in nucleotide sequence, it is further preferred that the nucleotide sequence of the gene is as shown in SEQ ID NO.2.
3. the recombinant expression carrier containing the gene described in claim 2, it is preferable that the recombinant expression carrier is slow virus table Up to carrier, it is further preferred that the Lentiviral is pWPT-HER2ScFv-CD8-CD137-CD3 ζ.
4. a kind of NKT cells of engineering HER2 targetings, which is characterized in that the NKT cells are by described in claim 1 The NKT cells of Chimeric antigen receptor modification.
5. a kind of preparation method of the NKT cells of engineering HER2 targetings, which is characterized in that the method includes：
Packaging carries the slow virus of pWPT-HER2ScFv-CD8-CD137-CD3 ζ, obtains viral concentration liquid；Utilize obtained disease Malicious concentrate infects NKT cells, makes NKT cells expression Chimeric antigen receptor HER2ScFv-CD8-CD137-CD3 ζ.
6. according to the method described in claim 5, wherein, the method further includes being prepared via a method which NKT cells：
(1) in the presence of CD3 monoclonal antibodies, proleulzin and interleukin-15, mononuclearcell is subjected to first stage training It supports；Preferably, the embodiment of the first stage culture includes：Mononuclearcell is incubated at the first NKT cell culture fluids In, the first NKT cell culture fluids contain NKT cell culture mediums, CD3 monoclonal antibodies, proleulzin and interleukin-15； It is further preferred that in the first NKT cell culture fluids, a concentration of 30-70ng/mL of the CD3 monoclonal antibodies and/or institute State a concentration of 300-700U/mL of the proleulzin and/or a concentration of 30-70ng/mL of the interleukin-15；
(2) in the presence of proleulzin, the cell that the first stage is cultivated carries out second stage culture；Preferably, described The embodiment of second stage culture includes：The cell culture that the first stage is cultivated in the 2nd NKT cell culture fluids, Contain NKT cell culture mediums and proleulzin in the 2nd NKT cell culture fluids；It is further preferred that the 2nd NKT cells are trained In nutrient solution, a concentration of 300-700U/mL of the proleulzin.
7. according to the method described in claim 6, wherein, the method for the infection NKT cells includes：
(1) in the presence of viral concentration liquid, nucleoprotamine and proleulzin, NKT cells are subjected to first stage infection culture；It is excellent Selection of land, the embodiment that the first stage infection is cultivated include：By NKT cell culture in the 3rd NKT cell culture fluids, institute It states the 3rd NKT cell culture fluids and contains NKT cell culture mediums, viral concentration liquid, nucleoprotamine and proleulzin；Further preferably Ground, in the 3rd NKT cell culture fluids, a concentration of 300-700U/mL of the proleulzin；
(2) in the presence of CD3 monoclonal antibodies, proleulzin and interleukin-15, by the cell of first stage infection culture Carry out second stage infection culture；Preferably, the embodiment of the second stage infection culture includes：By the first stage The cell culture of culture is infected in the first NKT cell culture fluids.
8. according to the method described in claim 7, wherein, the method for the infection NKT cells further includes：
(3) first in the presence of proleulzin, the cell progress of second stage infection culture is external evoked, wait for the close of cell When degree is 80-90%, then in the presence of CD3 monoclonal antibodies, proleulzin and interleukin-15, cell is subjected to amplification cultivation； Preferably, the external evoked embodiment includes：By the cell culture of second stage infection culture in described second In NKT cell culture fluids, the embodiment of the amplification cultivation includes：By cell culture in the first NKT cell culture fluids In.
9. the NKT cells for the engineering HER2 targetings that any one of claim 5-8 the methods are prepared.
10. the NKT cells of the engineering HER2 targetings described in claim 4 or 9 are in preparing the preparation for treating tumour Application, it is preferable that the tumour is the epithelial tumor of the progressive stage HER2 positive, it is further preferred that the epithelial tumor is Cholangiocarcinoma, cancer of pancreas, breast cancer, oophoroma, lung cancer, liver cancer, the cancer of the esophagus, prostate cancer, gastric cancer or carcinoma of mouth.
CN201710068756.3A 2017-02-08 2017-02-08 Chimeric antigen receptor and its gene and recombinant expression carrier, CARHER2-NKT cells and its preparation method and application Pending CN108395480A (en)

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