CN105924529B - Chimeric antigen receptor and its gene and recombinant expression carrier, CAR138-NKT cell and its preparation method and application - Google Patents

Abstract

The invention discloses a kind of Chimeric antigen receptor and its genes and recombinant expression carrier, CAR138-NKT cell and its preparation method and application, the Chimeric antigen receptor is CD138ScFv-CD8-CD137-CD3 ζ, the intracellular signal structural domain including concatenated rat growth hormone signal peptide, the hinge area of CD138ScFv, CD8 and transmembrane region, the intracellular signal structural domain of CD137 and CD3 ζ.When co-culturing using the myeloma cell of CAR138-NKT cell and the CD138 positive of the invention, there is good specific killing activity to myeloma cell.

Description

Chimeric antigen receptor and its gene and recombinant expression carrier, CAR138-NKT cell and Preparation method and application

Technical field

The invention belongs to knubble biological arts, and in particular, to one of adoptive immunotherapy chimeric antigen by Body CD138ScFv-CD8-CD137-CD3 ζ and its gene and recombinant expression carrier, the NKT cell for being engineered CD138 targeting (CAR138-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.

Huppert's disease (Multiple myeloma, MM) is the B cell malignant tumour of thick liquid cell paraplasm, with bone A large amount of malignant plasma cell is gathered in marrow and secretes monoclonal immunoglobulin is characterized.Surface of myeloma cells expression is a series of Adhesion molecule such as CD138.CD138 is that one kind belongs to cross-film mucoprotein glycan family member, with the growth in bone marrow microenvironment The factor and matrix components combine, and the adherency of mediating multiple myeloma cell and go back to the nest.It is gradually deep with studying CD138 Enter, it has been found that CD138 has played key effect in the growth, migration of the injury repair and tumour of body.CD138 quilt at present It is considered the most special surface marker of thick liquid cell, expression of the studies have shown that CD138 molecule in B cell malignant tumour is very Complexity, the thick liquid cell expression in MM Bone Marrow of Patients and peripheral blood, and the not table of the hemopoietic forebody cell or lymphocyte in marrow It reaches.The raising of CD138 molecular level can be used as the efficiency index of myeloma prognosis mala, and when apoptosis is presented in MM cell When, film surface CD138 developed by molecule is lowered or is lost, and can reflect the load of MM patient's body tumour cell.

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 provides a kind of Chimeric antigen receptor CD138ScFv-CD8-CD137-CD3 ζ and its gene and again Group expression vector, the NKT cell (CAR138-NKT cell) for being engineered CD138 targeting and its preparation method and application.

The present inventor has been surprisingly found that under study for action, using Chimeric antigen receptor CD138ScFv-CD8- of the invention It is thin to Huppert's disease when the NKT cell of CD137-CD3 ζ modification and the multiple myeloma cells of the CD138 positive co-culture Born of the same parents have 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 CD138ScFv-CD8-CD137-CD3 ζ, including concatenated rat growth hormone signal peptide, CD138ScFv, 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 CD138 targeting, and the NKT cell is above-mentioned The NKT cell of Chimeric antigen receptor CD138ScFv-CD8-CD137-CD3 ζ modification.

5th aspect, the present invention provides a kind of preparation method of NKT cell for being engineered CD138 targeting, the sides Method includes:

Packaging carries the slow virus of pWPXL-CD138ScFv-CD8-CD137-CD3 ζ, obtains viral concentration liquid；It utilizes The viral concentration liquid inductance dye NKT cell arrived, makes NKT cell express Chimeric antigen receptor CD138ScFv-CD8-CD137-CD3 ζ.

6th aspect, the present invention provides the NKT cells for the engineering CD138 targeting that the above method is prepared.

7th aspect, the present invention provides the NKT cell of above-mentioned engineering CD138 targeting in preparation for treating tumour Preparation in application.

When with the co-cultivation of the multiple myeloma cells of the CD138 positive, Chimeric antigen receptor of the invention The NKT cell of CD138ScFv-CD8-CD137-CD3 ζ modification, i.e. the NKT cell of engineering CD138 targeting being capable of specificity In conjunction with CD138 antigen, enhance the ability of immunocyte targets identification surface of myeloma cells CD138 antigen, reinforces to CD138 sun Property myeloma cell specific killing activity.The NKT cell of engineering CD138 targeting of the invention is that treatment CD138 is positive Huppert's disease provide a kind of new selection, 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-CD138ScFv-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-CD138ScFv-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 CD138ScFv-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 CD138ScFv-CD8-CD137-CD3 ζ modification The result of (CAR138-NKT cell) phenotypic evaluation.

Fig. 7 is the lethal effect of CAR138-NKT cell of the invention to the multiple myeloma cells of the CD138 positive Cytotoxicity analysis figure.

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 CD138ScFv-CD8-CD137- CD3 ζ, including concatenated rat growth hormone signal peptide, the hinge area of CD138 single-chain antibody CD138ScFv, 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 CD138ScFv-CD8-CD137-CD3 ζ by rat growth hormone signal peptide, The intracellular signal structural domain of the hinge area and transmembrane region of CD138ScFv, 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 CD138ScFv-CD8-CD137-CD3 ζ modification is obtained Cell, under preferable case, Lentiviral is pWPXL-CD138ScFv-CD8-CD137-CD3 ζ.

It is not special for the preparation method of Lentiviral pWPXL-CD138ScFv-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 CD138ScFv-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 CD138ScFv, and it is cloned into pWPXL- In CD8-CD137-CD3 ζ, the correct pWPXL-CD138ScFv-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 CD138ScFv 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-CD138ScFv-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 CD138ScFv fusion (SEQID NO.8), is cloned into carrier pGSI, obtains pGSI-CD138ScFv；Then pGSI-CD138ScFv 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-CD138ScFv-CD8-CD137-CD3 ζ of sequence.Wherein, The nucleotide sequence of rat growth hormone signal peptide is as shown in SEQID NO.6, CD138ScFv nucleotide sequence such as SEQID Shown in NO.7.

The present invention also provides a kind of NKT cell for being engineered CD138 targeting, the NKT cell is by above-mentioned chimeric The NKT cell (i.e. CAR138-NKT cell) of antigen receptor CD138ScFv-CD8-CD137-CD3 ζ modification.

The present invention also provides a kind of preparation methods of NKT cell for being engineered CD138 targeting, this method comprises: packet Dress carries the slow virus of pWPXL-CD138ScFv-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 CD138ScFv-CD8-CD137-CD3 ζ.

The method for carrying the slow virus of pWPXL-CD138ScFv-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- CD138ScFv-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 carried out in incubator Culture.Those skilled in the art can be adaptively adjusted the time of culture, this is known to those skilled in the art, This is repeated 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 CD138ScFv-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 CD138 antigen carries out NKT cell infection, and efficiency of infection is up to 30%-60%, and obtain CAR138-NKT cell, CD3⁺CD56⁺The total CD3 of cell Zhan⁺The ratio of cell is 15% or more.

The Chimeric antigen receptor of the NKT cell expression of Chimeric antigen receptor CD138ScFv-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 CD138ScFv, 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 CD138 targeting that the above method is prepared.

The present invention also provides the NKT cells of engineering CD138 targeting in preparing the preparation for treating tumour Using.Under preferable case, tumour is the tumour of the CD138 positive, it is further preferred that the tumour is the multiple of the CD138 positive Property myeloma.

In application of the invention, for the composition of preparation of the tumour for treating the CD138 positive, there is no particular limitation, As long as being prepared containing CAR138-NKT cell of the present invention or by CAR138-NKT cell, specific group of preparation At known to those skilled in the art with preparation method, details are not described herein.

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.

RevertAid^TMFirst 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.

Lipofectamine^TM2000 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.

Myeloma cell line A266 is purchased from U.S. ATCC.

5-carboxyfluorescein succinimide ester is purchased from Shanghai Pu Zhen Biotechnology Co., Ltd.

Annexin V-RPE kit is purchased from U.S. company BD.

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-CD138ScFv-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 RNAisoReagent thin The total serum IgE of born of the same parents, -80 DEG C save backup.The total serum IgE of extraction Reverse Transcriptase kit RevertAid^TMFirst 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, nucleotide sequence as shown in SEQIDNO.3, both ends contain respectively MluI and II restriction enzyme site of Bgl and Protect base；PCR amplification is carried out with primer P3 and P4, obtains the CD137 intracellular signal structural domain of long 146bp, nucleotide sequence As shown in SEQID NO.4, Bgl II and EcoRI restriction enzyme site and protection base are contained in both ends respectively；It is carried out with primer P5 and P6 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 difference Contain EcoRI and NdeI restriction enzyme site and protection base.Each step pcr amplification reaction system is identical, to expand CD137 intracellular signal For 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-CD138ScFv-CD8-CD137-CD3 ζ

The nucleotide sequence of full genome composite coding rat growth hormone signal peptide and CD138ScFv 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-CD138ScFv 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 CD138ScFv 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 lane: plasmid The non-endonuclease bamhi (11227bp) of pWPXL-CD138ScFv-CD8-CD137-CD3 ζ；2 swimming lanes: plasmid pWPXL- The endonuclease bamhi (1552bp) of CD138ScFv-CD8-CD137-CD3 ζ；；M2:DNA molecular weight marker D2000.It will identify correct Plasmid send Beijing Tian Yihuiyuan Biotechnology Co., Ltd that the fusion segment of insertion is sequenced.Just by sequencing result True recombinant plasmid is named as pWPXL-CD138ScFv-CD8-CD137-CD3 ζ, and structural schematic diagram is as shown in figure 4, wherein wrap Include that rat growth hormone signal peptide (nucleotide sequence is as shown in SEQID NO.6), (nucleotide sequence is such as anti-CD138 single-chain antibody Shown in SEQID NO.7), the intracellular signal knot of the intracellular signal structural domain and CD3 ζ of the area hinge of CD8 and transmembrane region and CD137 Structure domain, wherein the Chimeric antigen receptor is with the area hinge of gene C D8 and transmembrane region and the intracellular signal structure of CD137 and CD3 ζ The structure that domain is connected in series is signal transduction structural domain, and amino acid sequence is as shown in SEQID NO.9, corresponding gene coding Sequence is as shown in SEQID NO.10.

The preparation of the NKT cell of 3 Chimeric antigen receptor CD138ScFv-CD8-CD137-CD3 ζ of embodiment modification

(1) packaging and concentration of slow virus

Measure slow virus expression plasmid pWPXL-CD138ScFv-CD8-CD137-CD3 ζ and auxiliary respectively with spectrophotometer The concentration of plasmid psPAX2, pMD2.G are helped, three kinds of plasmids are used with the mass ratio of 4:2:1 Lipofectamine^TM2000Transfection Reagent transfection reagent cotransfection 293T incasing cells.It is transfecting respectively Viral supernatants are collected when 48h, 72h in 50mL EP pipe, 4 DEG C, 2000g is centrifuged 10min, shifts the supernatant that obtains twice to new In EP pipe, with 4.5 μm of filter filter virus supernatants；The viral supernatants and 5 × PEG6000-NaCl of filtering according to 4:1 volume ratio It mixes, 4 DEG C of standing 2h, then 4 DEG C, 10000g is centrifuged 20min, abandons supernatant, and precipitating is molten with the sterile PBS of 4 DEG C of pre-coolings of 1mL Solution is dispensed, -80 DEG C save backup to get the viral concentration liquid of Chimeric antigen receptor by every 100 μ L of pipe.

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 CAR138-NKT cell.CAR138-T cell (the preparation method reference literature of T cell: Yajing is prepared with identical method Zhang,et al.Autologous CIK Cell Immunotherapy in Patients with Renal Cell Carcinoma after Radical Nephrectomy.Clinical Study, the preparation of 2.4 part CIK cells in 2013 Method).With the efficiency of infection of the Flow cytometry virus, as a result as shown in figure 5, the efficiency of infection of CAR138-NKT cell It is 46.75%.

(3) external evoked amplification CAR138-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⁺: 93.94%；CD3⁺CD4⁺: 6.44%；CD3⁺CD8⁺: 86.65%；CD3⁺CD56⁺: 61.49%；CD8⁺CD56⁺: 55.62%.

Cytotoxicity analysis of the 4 CAR138-NKT cell of embodiment to myeloma cell's lethal effect

The NKT cultivated in CAR138-NKT cell, CAR138-T cell and the embodiment 1 prepared in Example 3 respectively Cell inoculation is dyed in 96 orifice plates with 5-carboxyfluorescein succinimide ester (CFSE), with myeloma cell line A266 To imitate target ratio (killing cell: target cell) 5:1, the ratio of 10:1,20:1,40:1 were co-cultured, by total training in 24 hours After supporting, cell annexin V-RPE kit is dyed, while control group is set and is not added at immunocyte killing respectively The myeloma cell line A266 of reason, and cell annexin V-RPE kit is dyed.Flow cytometry is to Apoptosis It is detected, the amount of Apoptosis is calculated according to the following equation: apoptosis rate=(control-sample)/control × 100%, control For the cell survival number for not adding immunocyte killing processing；Sample is that (killing cell: target is thin for the corresponding effect target ratio of addition Born of the same parents) immunocyte killing processing cell survival number, see Fig. 7.As shown in Figure 7, Chimeric antigen receptor CD138ScFv- The NKT cell of CD8-CD137-CD3 ζ modification has specific killing activity, and the spy of CAR138-NKT cell to myeloma cell Different killing activity is substantially better than CAR138-T cell and NKT cell.

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 CD138 targeting, which is characterized in that the described method includes:

Packaging carries the slow virus of pWPXL-CD138ScFv-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 CD138ScFv-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 CD138 targeting that method as claimed in claim 1 or 2 is prepared.

4. the NKT cell of engineering CD138 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 CD138 positive.

6. application according to claim 5, wherein the tumour is the Huppert's disease of the CD138 positive.