CN109400712A

CN109400712A - The preparation method and applications of third generation LMP1 CAR-T cell

Info

Publication number: CN109400712A
Application number: CN201811175716.XA
Authority: CN
Inventors: 陈仁杰; 冯振卿; 朱进; 唐奇; 陈渊; 张舒; 张大为
Original assignee: 2nd Affiliated Hospital of Nanjing Medical University
Current assignee: 2nd Affiliated Hospital of Nanjing Medical University
Priority date: 2018-10-10
Filing date: 2018-10-10
Publication date: 2019-03-01

Abstract

The present invention utilizes technique for gene engineering, using the anti-LMP1 Fab of the full source of people with high-affinity as template, scFv sections of extracellular region LMP1 of the Chimeric antigen receptor of building targeting LMP1.Recombinate scFv sections of the extracellular region LMP1 and signal peptide CD8 α, transmembrane region CD8 of LMP1 CAR^TMAnd intracellular region CD28, CD137, CD3 ζ, the Chimeric antigen receptor of building third generation targeting LMP1.The Chimeric antigen receptor of prepared targeting LMP1 is reconstituted in linearisation slow virus carrier through overlap PCR, construct the slow virus recombinant expression carrier of anti-LMP1 CAR, and prepare LMP1 CAR virus liquid, it infects T cell and obtains LMP1 CAR-T cell, its selectively targeted lethal effect to LMP1 positive nasopharyngeal carcinoma's cell is verified in inside and outside, and new treatment thoughts and means are provided for treatment of nasopharyngeal carcinoma.

Description

The preparation method and applications of third generation LMP1 CAR-T cell

Technical field

The invention belongs to genetic engineering fields and immune targeted therapy field, are related to the system of third generation LMP1 CAR-T cell Preparation Method and its providing the application in a kind of novel treatment of nasopharyngeal carcinoma.

Background technique

Nasopharyngeal carcinoma is derived from the high malignancy tumour of nasopharyngeal epithelium, and tumour progression easily invades the important features such as basis cranii, And metastasic cervical lymph nodes and DISTANT METASTASES IN relatively early occurs.Nasopharyngeal carcinoma shows as the characteristics of race and Area distribution, is China ten One of big high-incidence malignant tumour, disease incidence 20/100,000 has caused serious health problems.

The primary treatment regimen of nasopharyngeal carcinoma is chemicotherapy at present, but not being true to type due to onset symptoms, and when patient assessment is past The main reason for toward course of disease middle and advanced stage is already belonged to, five year survival rate is not high, and the recurrence and transfer of lesion are its death.In recent years Come, Chimeric antigen receptor (chimeric antigen receptor, CAR) T based on T cell adoptive immunotherapy Cell therapy achieves significant achievement in Malignancy and part entity tumor in succession, is it in nasopharyngeal carcinoma Using providing possibility.

Nasopharyngeal carcinoma is by latent infection, environmental factor and the gene genetic of the host factor of Epstein-Ban viral (EBV) The multi-step reciprocation participated in jointly and the complex disease gradually formed.All nasopharyngeal carcinoma cells all contain EBV gene Group, and EBNA1, EBER1, EBER2 and LMP1 are expressed, LMP2A, LMP2B, this makes these virus proteins become ideal swollen Tumor markers, wherein LMP1 (latent membrane proteinl, LMP1) is in the conversion and canceration of nasopharyngeal epithelial cells Effect in journey is viral oncogene generally acknowledged at present by note.

LMP1 is prevalent in tissues of nasopharyngeal carcinoma, and is not expressed in normal nasopharyngeal epithelium, is had good special Property, become the good target of nasopharyngeal carcinoma CAR-T cell therapy.Currently, in the CAR-T cell therapy field of nasopharyngeal carcinoma, there has been no The report that the LMP1 CAR-T cell of three generations is successfully prepared.

Summary of the invention

The first purpose of the invention is to provide a kind of Chimeric antigen receptors (i.e. LMP1 CAR) for targeting LMP1.

A second object of the present invention is to provide the anti-LMP1 CAR's of the Chimeric antigen receptor containing aforementioned targeting LMP1 Slow virus recombinant expression carrier.

Third object of the present invention is to provide the preparation methods of the slow virus recombinant expression carrier of aforementioned anti-LMP1 CAR.

Fourth object of the present invention is to provide the slow virus recombinant expression carrier infection T cell of anti-LMP1 CAR above-mentioned The LMP1 CAR-T cell of acquisition.

Fifth object of the present invention is to provide the preparation methods of aforementioned LMP1 CAR-T cell.

Sixth object of the present invention is to provide Chimeric antigen receptor, the slow diseases of anti-LMP1 CAR of targeting LMP1 above-mentioned Malicious recombinant expression carrier, LMP1 CAR-T cell are preparing answering in nasopharyngeal carcinoma diagnosis reagent or nasopharyngeal carcinoma immunotherapy medicaments With.

Inventor is using the anti-LMP1 monoclonal antibody of the full source of people with high-affinity as template, according to the original of Infusion PCR Reason designs weight, light chain primer, respectively amplification weight, light chain variable region, and heavy, light with the connection of link peptide, that is, hinge area (Gly4Ser) 3 The LMP1scFv section of the Chimeric antigen receptor extracellular region of chain variable region building targeting LMP1.

In the end the 5' connection signal Peptide C D8 of the extracellular region LMP1scFv of the Chimeric antigen receptor for the targeting LMP1 that success constructs α sequence.Using the method for overlap PCR, in the CD8 that the LMP1scFv section of CD8 α label is synthesized with genetic engineering^TM, CD28, CD137 and CD3 ζ recombination constitutes the Chimeric antigen receptor of targeting LMP1.

In addition, the slow virus carrier of linearisation and the Chimeric antigen receptor of targeting LMP1 are recombinated, anti-LMP1 is obtained The slow virus expression plasmid of CAR.

Western blot verifies the slow virus expression plasmid of LMP1 CAR after the expression in X-293T cell, Jing Sanzhi Grain slow virus packaging system prepares LMP1 CAR slow virus liquid, infects T cell, prepares LMP1 CAR-T cell.

Detection LMP1 CAR-T cell is further tested to the specific target of LMP1 positive nasopharyngeal carcinoma's cell by inside and outside To killing ability.CCK8 method shows that the LMP1 CAR-T cell presses down the growth of human nasopharyngeal epithelioma 1 CNE1, CNE2 and HONE1 Production is used；After ELISA method detection shows that nasopharyngeal carcinoma cell and LMP1 CAR-T cell co-culture, the secretion of LMP1 CAR-T cell is thin The ability of intracellular cytokine (IL-2, IFN-γ), wherein IL-2 is used to maintain the activity of CAR-T cell, keeps its proliferative capacity, and rises Lethal effect is cell factor IFN-γ；Ex vivo animal experiment detection LMP1 CAR-T cell is in vivo respectively to the LMP1 positive With the tumor-inhibiting action of LMP1 Nasopharyngeal Carcinoma Patients with Negative transplantable tumor, it was demonstrated that LMP1 CAR-T cell is preparing nasopharyngeal carcinoma diagnosis reagent or nasopharynx Validity in cancer immunotherapy medicaments.

Specific technical solution of the present invention is as follows:

A kind of Chimeric antigen receptor targeting LMP1, the Chimeric antigen receptor of the targeting LMP1 is by from aminoterminal to carboxyl Hold sequentially connected signal peptide CD8 α, extracellular region LMP1scFv, transmembrane region CD8^TMIt is formed with intracellular region.

Further, the extracellular region LMP1scFv is by heavy chain variable region (i.e. V_H), link peptide (i.e. hinge area), light chain can Become area (i.e. V κ) and be sequentially connected composition,

The heavy chain variable amino acid sequence is as shown in SEQ ID NO.3:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRF TISRDNAKNSLYLRMNSLRAEDTAVYYCARAWGYSSYYFDYWGQGTLVTVSP,

Chain variable region amino acid sequence is as shown in SEQ ID NO.4:

ELQMTQSPSSLSASVGDRVAITCRASQSISSHLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIK,

Link peptide is concatenated three groups of pentapeptide repetitive units (Gly4Ser) 3.

Further, the nucleotide sequence of the heavy chain variable amino acid is encoded as shown in SEQ ID NO.1:

GAAGTGCAGCTGGTGGAGAGTGGGGGGGGACTGGTCCAGCCAGGAGGGTCACTGAGACTGAGCTGCGC CGCTTCCGGCTTCACCTTCAGCAGCTACTGGATGTCCTGGGTGAGGCAGGCTCCTGGAAAAGGCCTGGAGTGGGTC GCAAACATCAAACAGGACGGCTCTGAAAAGTACTATGTGGATAGTGTCAAAGGGCGGTTCACTATTTCCAGAGACA ACGCCAAGAATTCTCTGTATCTGAGGATGAATTCCCTGCGCGCCGAGGATACCGCCGTGTACTATTGCGCTCGCGC ATGGGGGTACTCTAGTTACTATTTTGACTATTGGGGGCAGGGAACTCTGGTGACCG TCAGCCCT,

The nucleotide sequence of the chain variable region amino acid is encoded as shown in SEQ ID NO.2:

GAACTGCAGATGACCCAGAGCCCATCAAGCCTGTCTGCCAGTGTGGGCGATCGAGTCGCAATCACATG TCGGGCCTCACAGAGCATTTCCTCTCACCTGAACTGGTACCAGCAGAAGCCCGGAAAAGCTCCTAAGCTGCTGATC TATGCAGCCAGTTCACTGCAGTCTGGCGTGCCAAGTCGATTCTCCGGCTCTGGGAGTGGAACAGACTTTACCCTGA CAATTAGCTCCCTGCAGCCCGAGGATTTCGCCACTTACTATTGTCAGCAGAGCTACTCAACCCCATACACCTTCGG GCAGGGAACTAAACTGGAAATCAAA,

The nucleotide sequence of the link peptide is encoded as shown in SEQ ID NO.7:

GGAGGAGGAGGATCAGGCGGCGGAGGCAGCGGAGGAGGAGGGTCC。

Preferably, the amino acid sequence of extracellular region LMP1scFv of the present invention encodes the born of the same parents as shown in SEQ ID NO.17 The nucleotide sequence of outskirt LMP1scFv amino acid is as shown in SEQ ID NO.16.

Further, the amino acid sequence of the signal peptide CD8 α is as shown in SEQ ID NO.6:

MALPVTALLLPLALLLHAARP,

The nucleotide sequence of encoded signal peptide CD8 α is as shown in SEQ ID NO.5:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGC CAGGCCG,

The nucleotide sequence for encoding the signal peptide CD8 α is connected to the end 5' of scFv heavy chain variable region.

Further, the transmembrane region CD8^TMAmino acid sequence as shown in SEQ ID NO.9:

FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQP YAPPRDFAAYRS,

Encode the transmembrane region CD8^TMThe nucleotide sequence of amino acid is as shown in SEQ ID NO.8:

TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTAT TATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGG CCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC。

Further, the intracellular region is sequentially connected by CD28, CD137, CD3 ζ and is formed；

The amino acid sequence of the CD28 is as shown in SEQ ID NO.11:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL YC,

The nucleotide sequence of the CD28 amino acid is encoded as shown in SEQ ID NO.10:

ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCG CCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATC TGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC；

The amino acid sequence of the CD137 is as shown in SEQ ID NO.13:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL,

The nucleotide sequence of the CD137 amino acid is encoded as shown in SEQ ID NO.12:

AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCA AGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG；

The amino acid sequence of the CD3 ζ is as shown in SEQ ID NO.15:

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR,

The nucleotide sequence of the CD3 ζ amino acid is encoded as shown in SEQ ID NO.14:

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGA GCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAG CCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGA TTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGA CACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC。

A second object of the present invention is to provide the slow virus recombinant expression carrier of anti-LMP1 CAR, the anti-LMP1 CAR Slow virus recombinant expression carrier contain it is aforementioned targeting LMP1 Chimeric antigen receptor.

Third object of the present invention is to provide the preparation method of the slow virus recombinant expression carrier of aforementioned anti-LMP1 CAR, The following steps are included:

S1: using the anti-LMP1 monoclonal antibody of full source of people as template, design heavy chain variable region specific primer to and light chain variable region Specific primer pair expands heavy chain variable region and light chain variable region respectively；Specific primer pair is connected using weight, light chain, is passed through Peptide sequence connection heavy chain variable region and light chain variable region are connected, extracellular region LMP1scFv segment is constructed；

S2: in the end the 5' connection signal Peptide C D8 α sequence for the extracellular region LMP1scFv that S1 is successfully constructed, CD8 α label is constructed Extracellular region LMP1scFv segment；

S3: the extracellular region LMP1scFv segment and transmembrane region CD8 that CD8 α is marked^TMWith intracellular region CD28, CD137, CD3 ζ It is connected, building obtains the nucleotide sequence of the Chimeric antigen receptor of targeting LMP1；

S4: with restriction endonuclease BamHI and XhoI double digestion slow virus carrier, the slow virus linearized is carried Body, by the method for In-Fusion PCR by the nucleotide sequence of the Chimeric antigen receptor of the S3 targeting LMP1 obtained and linearly The slow virus carrier of change recombinates, and obtains the slow virus recombinant expression carrier of anti-LMP1 CAR.

Further, heavy chain variable region specific primer pair described in S1 are as follows:

V_HUpstream primer: CACGCCGCCAGGCCGGAAGTGCAGCTGGTG (SEQ ID NO.18),

V_HDownstream primer: TGATCCTCCTCCTCCAGGGCTGACGGTCAC (SEQ ID NO.19)；

Light chain variable region specific primer pair are as follows:

V κ upstream primer: GGAGGAGGAGGGTCCGAACTGCAGATGACC (SEQ ID NO.20),

V κ downstream primer: CACCAGCACCCAAAATTTGATTTCCAGTTT (SEQ ID NO.21)；

The heavy, light chain connects specific primer centering, and weight, light chain connection primer 1 are using V shown in SEQ ID NO.18_H Upstream primer, weight, light chain connection primer 2 are using V κ downstream primer shown in SEQ ID NO.21.

Fourth object of the present invention is to provide LMP1 CAR-T cell, and the LMP1 CAR-T cell is above-mentioned anti- The slow virus recombinant expression carrier infection T cell of LMP1 CAR obtains.

Fifth object of the present invention is to provide the preparation method of aforementioned LMP1 CAR-T cell, the preparation method includes Following steps:

S1: the slow virus recombinant expression carrier of anti-LMP1 CAR above-mentioned and packaging plasmid psPAX2, pMD2.G are formed Three plasmid slow virus packaging systems, to X-293T cell, 37 DEG C of incubators are incubated for culture for transfection, replaced after 6-8h it is fresh non-fully Culture medium (1640+10%FBS) collects supernatant after cultivating 48h, replaces fresh non-fully culture medium (1640+10%FBS) and continues Culture collects supernatant to 72h；

S2: slow virus liquid is obtained after the supernatant that S1 is obtained is concentrated；

S3: the slow virus liquid inductance that S2 is obtained is contaminated into T lymphocyte to obtain LMP1 CAR-T cell.

The step of above-mentioned preparation method can realize by conventional method, a kind of preferred operations step provided herein:

The preparation method S1 the following steps are included:

1) X-293T cell is collected, 10cm culture dish is inoculated in and continues to cultivate, when cell confluency degree is up to 70%, virus packaging Preceding 30min replaces fresh non-fully culture medium (1640+10%FBS)；2) 1.5ml centrifuge tube A:470ul Opti-MEM+30ul PEI is incubated at room temperature 5min after resuspension；1.5ml centrifuge tube B:3.3ugpsPAX2+3.3ug pMD2.G+3.3ug LMP1 CAR, Opti-MEM constant volume is added to 250ul；3) reagent is added drop-wise in pipe B in pipe A, after mixing gently, is placed at room temperature for 15min；4) it mixes It closes object to be added drop-wise in X-293T cell, shakes gently culture dish, so that mixture is evenly distributed, 37 DEG C of incubators are incubated for culture； 5) fresh non-fully culture medium (1640+10%FBS) is replaced after 6-8h；6) for 24 hours after fluorescence microscopy microscopic observation transfection efficiency；7) Supernatant is collected after 48h, if cell is adherent good, is replaced fresh non-fully culture medium (1640+10%FBS) and is continued to cultivate to 72h, Collect supernatant；8) 4 DEG C of supernatant collected, 400g are centrifuged 5min, go to precipitate, and collect supernatant.

Method for concentration described in the S2 includes filtering and concentrating or centrifugal concentrating,

The filtering and concentrating the following steps are included:

1) supernatant collected is transferred in the super filter tube of 100kD after 0.45um membrane filtration and is concentrated into 1/3 volume；

2) concentrate collects filtered fluid, -80 DEG C freeze through 0.22um membrane filtration；

The centrifugal concentrating the following steps are included:

1) it takes 8.76g NaCl respectively and 50g PEG-8000 is dissolved in 200ml distilled water, high pressure sterilization, preparation 5 × PEG-8000NaCl solution；2) viral supernatants collected are mixed in the ratio of 1:25 and 5 × PEG-8000NaCl, every 20-30min Concussion is primary, and totally 5 times, 4 DEG C overnight；3) 6000rpm is centrifuged 20min, removes supernatant；4) precipitating is collected, is resuspended with culture medium, -80 It DEG C freezes.

The S3 the following steps are included:

1) 24 Kong Fei tissue culturing plates are added after the diluted RetroNectin of PBS (1:50), every hole 100ul, sealed membrane After closing, 4 DEG C of refrigerator overnights；2) it places, goes unless tissue culturing plate on ice after the 37 DEG C of water-bath dissolutions of the resulting slow virus liquid of S2 500ul virus liquid is added in middle liquid, every hole, and 37 DEG C of incubators are incubated for 30min；3) after removing virus liquid, fresh virus liquid is replaced Afterwards, 37 DEG C of incubators are incubated for 30min, remove liquid, and 2ml culture medium is added；4) T cell of culture, 1500rmp centrifugation are collected 5min after removing supernatant, after the non-fully culture medium (1640+10%FBS) (1:2500) containing IL-2 is resuspended, is added drop-wise to non-tissue In culture plate, 48h is cultivated；5) remove 1ml culture medium, after the CAR-T cell for collecting preparation, with it is fresh containing IL-2 non-fully After culture medium (1640+10%FBS) (1:10000) is resuspended, continue to expand culture.

Sixth object of the present invention is to provide the Chimeric antigen receptors of targeting LMP1 above-mentioned, anti-LMP1 CAR above-mentioned Slow virus recombinant expression carrier, LMP1 CAR-T cell above-mentioned preparing nasopharyngeal carcinoma diagnosis reagent or nasopharyngeal carcinoma immunization therapy Application in drug.

The present invention has the beneficial effect that:

The present invention utilizes technique for gene engineering, using the anti-LMP1 monoclonal antibody of the full source of people with high-affinity as template, building Target LMP1scFv sections of extracellular region of the Chimeric antigen receptor of LMP1.Recombinate LMP1scFv sections of the extracellular region and signal of LMP1 CAR Peptide C D8 α, transmembrane region CD8^TMAnd intracellular region CD28, CD137, CD3 ζ, the Chimeric antigen receptor of building third generation targeting LMP1. The Chimeric antigen receptor of prepared targeting LMP1 is reconstituted in linearisation slow virus carrier through Infusion PCR, constructs anti-LMP1 The slow virus recombinant expression carrier of CAR, and LMP1 CAR virus liquid is prepared, infection T cell obtains LMP1 CAR-T cell, in vivo Outer its selectively targeted lethal effect to LMP1 positive nasopharyngeal carcinoma's cell of verifying provides new treatment for treatment of nasopharyngeal carcinoma and thinks Road and means.

Detailed description of the invention

The Chimeric antigen receptor of LMP1 is targeted in the building of the Lentiviral of Fig. 1: embodiment 1LMP1 CAR The synthesis of (LMP1 CAR) is verified.

A: anti-LMP1Fab V_HChain variable region, V_KThe amplified production of chain variable region；Swimming lane M:DL2000DNA Marker；Swimming Road 1:LMP1V_KChain variable region；Swimming lane 2:LMP1V_HChain variable region.B: the verifying of the Chimeric antigen receptor of LMP1 is targeted；Swimming lane M: DL2000DNA Marker；Swimming lane 1: the Chimeric antigen receptor of LMP1 is targeted.C: the structure of the Chimeric antigen receptor of LMP1 is targeted Schematic diagram, including signal peptide CD28 α, extracellular region LMP1scFv, transmembrane region CD8^TM, intracellular region CD28, CD137, CD3 ζ.

Fig. 2: expression of the slow virus recombinant expression carrier of anti-LMP1 CAR in X-293T cell in embodiment 1.

Swimming lane 1: the X-293T cell of untransfected；The X- of the slow virus recombinant expression carrier transfection of swimming lane 2:CD19 CAR 293T cell；Swimming lane 3: the X-293T cell of the slow virus recombinant expression carrier transfection of anti-LMP1 CAR.

Fig. 3: the expression Flow cytometry map of nasopharyngeal carcinoma cell surface antigen LMP1 in embodiment 5.

Fig. 4: lethal effect of the LMP1 CAR-T cell to nasopharyngeal carcinoma in embodiment 7.

A:LMP1 CAR-T cell difference imitate target than when to the lethal effect of CNE1；B:LMP1 CAR-T cell difference imitates target Than when to the lethal effect of CNE2；C:LMP1 CAR-T cell difference imitate target than when to the lethal effect of HONE1, CD19 CAR-T Cell is used as with T cell and compares；When D:10:1 imitates target ratio, LMP1 CAR-T cell is to the lethal effect of nasopharyngeal carcinoma cell, with leaching Bar oncocyte Raji is as control.

Fig. 5: the secretion of IL-2, IFN-γ after LMP1 CAR-T cell and nasopharyngeal carcinoma cell co-culture in embodiment 8.

The nasopharyngeal carcinoma cell of the nasopharyngeal carcinoma cell CNE1 and CNE2, LMP1 feminine gender of A:LMP1 CAR-T cell and the LMP1 positive HONE1 is co-cultured in supernatant, the secretory volume of cell factor IL-2；The nasopharyngeal carcinoma cell of B:LMP1 CAR-T cell and the LMP1 positive CNE1 and the nasopharyngeal carcinoma cell HONE1 of CNE2, LMP1 feminine gender are co-cultured in supernatant, the secretory volume of cell factor FN- γ.

Fig. 6: the tumor-inhibiting action of unilateral nude mice Tumorigenesis detection LMP1 CAR-T cell in embodiment 9.

A: nude mice living imaging statistical chart；B: the quantitative analysis of living imaging fluorescence intensity；C: experiment terminates, and knurl is in kind Figure；D: tumor volume variation tendency；E: knurl weight.

Fig. 7: bilateral nude mice Tumorigenesis detects LMP1 CAR-T cell specific killing in vivo in embodiment 10,

A: nude mice living imaging statistical chart；B: the quantitative analysis of living imaging fluorescence intensity.

Specific embodiment

The building of the Lentiviral of the anti-LMP1 CAR of embodiment 1

The LMP1scFv section design of primers and synthesis of the Chimeric antigen receptor LMP1 CAR extracellular region of 1.1 targeting LMP1

The anti-LMP1 monoclonal antibody of full source of people constructed with 8.0 assay laboratory biological software DNAstar, design amplification are light Chain variable region (V κ) and heavy chain variable region (V_H) primer and optimize, primer holds up the new limited public affairs of industry biotechnology of section by Beijing Department completes final synthesis.

LMP1scFv weight, light chain variable region primer sequence are as follows:

Above-mentioned primer direction is 5 ' -3 '

The synthesis of LMP1scFv sections of CAR extracellular region of Chimeric antigen receptor the LMP1 weights, light chain variable region of 1.2 targeting LMP1 With amplification

1) reaction system (by taking 50ul system as an example):

2) reaction condition:

3) verifying and recycling purpose band

1% agarose gel is prepared, PCR product is added with constant pressure 120V electrophoresis 30min and observes purpose band under imager Size, be compared with theoretical value.If stripe size is consistent with theoretical value, glue recycling is carried out, recovery product part is for surveying Sequence.

Agarose gel electrophoretogram is as shown in Figure 1A, the results show that the heavy chain variable region (VH) of anti-LMP1Fab, light chain The size of variable region (VK) about 360bp and 321bp respectively, size and theoretical value phase shown in SEQ ID NO.1, SEQ ID NO.2 Unanimously.

4) PCR glue recycling step: Nucleo Spin Gel and PCR Clean-up kit

1. weighing the weight of recycling glue, 200ul NT1/100mg, 50 DEG C of placement 5-10min are completely dissolved to glue.2. will The collecting pipe of 2ml is added in dissolved gum, and sample 700ul is added in every pipe, and 11000g is centrifuged 30s, removes liquid in collecting pipe.③ 700ul Buffer NT3,11000g are centrifuged 30s, remove liquid, repeatedly once.4. 11000g is centrifuged 1min, 70 DEG C of placement 2- 5min.5. 15-30ul deionized water, room temperature 1min, 11000g are centrifuged 1min.One drop surveys concentration and OD value.

LMP1scFv sections of CAR extracellular region of the Chimeric antigen receptor LMP1 synthesis of 1.3 targeting LMP1

It can with the heavy chain variable region of the above-mentioned synthesis of the connection of link peptide (Gly4Ser) 3, light chain using overlap PCR method Become area, building targets LMP1scFv sections of CAR extracellular region of Chimeric antigen receptor LMP1 of LMP1.

The sequence of link peptide (Gly4Ser) 3: GGAGGAGGAGGATCA

GGCGGCGGAGGCAGCGGAGGAGGAGGGTCC(SEQ ID NO.7)。

1) it is as follows to be related to primer sequence:

Weight, light chain connection primer 1 are aforementioned V_HUpstream primer: CACGCCGCCAGGCCGGAAGTGCAGCTGGTG (SEQ ID NO.18)

Weight, light chain connect primer 2, that is, aforementioned V κ downstream primer: CACCAGCACCCAAAATTTGATTTCCAGTTT (SEQ ID NO.21)

2) reaction system (for 50ul system):

3) reaction condition:

4) verifying and recycling purpose band:

1% agarose gel is prepared, PCR product is added with constant pressure 120V electrophoresis 30min and observes purpose band under imager Size, be compared with theoretical value.If stripe size is consistent with theoretical value, glue recycling is carried out, recovery product part is for surveying Sequencing result correct product as shown in SEQ ID NO.16 is extracellular region LMP1scFv by sequence, is used for subsequent experimental.

The extracellular region LMP1scFv segment of 1.4 building CD8 α labels

The end the 5' connection signal Peptide C D8 α sequence for the extracellular region LMP1scFv segment that 1.3 successes are constructed, building CD8 α mark The extracellular region LMP1scFv segment of note.

Construction method is as follows:

1) it is as follows to be related to primer sequence:

The LMP1scFv upstream primer of CD8 α label: GGACCATCCTCTAGGGATCCATGGCCTTACCAGTG (SEQ ID NO.22)

LMP1scFv downstream primer, that is, aforementioned V κ downstream primer of CD8 α label:

CACCAGCACCCAAAATTTGATTTCCAGTTT(SEQ ID NO.21)

2) reaction system (for 50ul system):

3) reaction condition:

4) verifying and recycling purpose band:

1% agarose gel is prepared, PCR product is added with constant pressure 120V electrophoresis 30min and observes purpose band under imager Size, be compared with theoretical value.If stripe size is consistent with theoretical value, glue recycling is carried out, recovery product part is for surveying Sequence will be sequenced the extracellular region LMP1scFv that correct product is CD8 α label, be used for subsequent experimental.

The Chimeric antigen receptor LMP1 CAR of 1.5 building targeting LMP1

The extracellular region LMP1scFv segment and transmembrane region CD8TM and intracellular region CD28 of the CD8 α label that 1.4 successes are constructed, CD137, CD3 ζ are connected, and building obtains the nucleotide sequence of the Chimeric antigen receptor LMP1 CAR of targeting LMP1；

Construction method is as follows:

1) it is as follows to be related to primer sequence:

LMP1 CAR upstream primer, that is, aforementioned CD8 α label LMP1scFv upstream primer:

GGACCATCCTCTAGGGATCCATGGCCTTACCAGTG(SEQ ID NO.22)

LMP1 CAR downstream primer: AATCCGGATCGATCTCGAGGTGCATGCTAACGC (SEQ ID NO.23) 2) it is anti- Answer system (for 50ul system):

3) reaction condition:

4) verifying and recycling purpose band:

1% agarose gel is prepared, PCR product is added with constant pressure 120V electrophoresis 30min and observes purpose band under imager Size, be compared with theoretical value.If stripe size is consistent with theoretical value, glue recycling is carried out, recovery product part is for surveying Sequence, it is the Chimeric antigen receptor (LMP1 CAR) for targeting LMP1 that correct product, which will be sequenced, is used for subsequent experimental.

The agarose gel electrophoretogram for targeting the Chimeric antigen receptor LMP1 CAR of LMP1 is as shown in Figure 1B, swimming lane M: DL2000DNA Marker；Swimming lane 1: the Chimeric antigen receptor LMP1 CAR of LMP1 is targeted.

The results show that the size of the Chimeric antigen receptor LMP1 CAR of targeting LMP1 is about 1455bp, size and theoretical value Consistent, successfully building targets the Chimeric antigen receptor LMP1 CAR of LMP1.

The Chimeric antigen receptor LMP1 CAR structural schematic diagram for targeting LMP1 is as shown in Figure 1 C, and LMP1 CAR includes successively connecting Signal peptide the CD8 α, extracellular region LMP1scFv, transmembrane region CD8 connect^TM, intracellular region CD28, CD137, CD3 ζ.

The building of the slow virus recombinant expression carrier of 1.6 anti-LMP1 CAR

The carrier that Lentiviral is linearized through restriction endonuclease BamHI and XhoI double digestion.Slowly Viral carrier is carrier can conventional slow virus carrier is carrier, this is sentenced for pLvx-IRES-ZsGreen.

1) reaction system (by taking 50ul system as an example):

2) reaction condition: 37 DEG C of water-baths are stayed overnight.

3) it recycles purpose band: preparing 1% agarose gel, PCR product is added, with constant pressure 120V electrophoresis 30min, imager The size of lower observation purpose band, is compared with theoretical value.If stripe size verifying is correct, recycling is saved.

The Chimeric antigen receptor for the targeting LMP1 that the slow virus carrier pLvx-IRES-ZsGreen of linearisation and 1.5 is obtained The nucleotide sequence of LMP1 CAR is recombinated by the method for In-Fusion PCR, chimeric antigen of the building containing targeting LMP1 by The Lentiviral of the anti-LMP1 CAR of body LMP1 CAR.

1) reaction system (for 10ul system):

2) reaction condition: 50 DEG C of water-bath 15min.

3) transformation experiment:

1ul plasmid 1. (10ul recombinant plasmid)+100ul competence bacteria --- it is operated in super-clean bench；2. ice compress 30min --- Heat shock 90s (42 DEG C of water-baths) --- ice compress 5min；3. 900ul liquid LB is added, 37 DEG C, 200r shakes 1h --- super-clean bench behaviour Make；4. 4000r is centrifuged 2min, 800ulLB is abandoned, is left LB and precipitating is resuspended, coated plate；5. in culture dish, 20ul Amp is first added, The solid-state LB (micro-wave oven is heated into liquid) to cool down is added, is trembled even；6. coated plate, after glass bar calcination is cooling, culture dish In it is applied；7. culture dish is put into 37 DEG C of insulating box 30-60min；8. being inverted overnight；9. second day picking monoclonal, inoculated liquid In LB culture medium, 37 DEG C, 200rmp shake culture stay overnight.

4) plasmid extracts: according to Plasmid Miniprep Kit

1. 10ml bacterium solution is put into centrifuge tube, 12000rmp, 1min, supernatant is removed；2. 500ul is added in centrifuge tube to have added Enter the solution P1 of RNase A, concussion goes to precipitate；3. 500ul solution P2 is added in centrifuge tube, 6-8 lytic cell is spun upside down, Movement is mild；4. 700ul solution P3 is added in centrifuge tube, overturning is to there is flocculent deposit, after 12000rmp, 10min, in absorption Clearly；5. the supernatant drawn successively crosses adsorption column CP4,12000rmp is centrifuged 1min；6. 500ul PD solution is added in adsorption column, 12000rmp is centrifuged 1min；7. PW solution of the 600ul containing dehydrated alcohol is added in adsorption column, 12000rmp, 1min, 1 time repeatedly Afterwards, 12000rmp, 2min；8. 200ul deionized water is added in adsorption column, it is stored at room temperature 2min, 12000rmp is centrifuged 2min, receives Collect filtrate；9. One drop surveys concentration and OD value.

5) verifying and recycling purpose band:

1% agarose gel is prepared, PCR product is added, glue 30min is run with constant pressure 120V electrophoresis, observes purpose under imager The size of band, is compared with theoretical value.If stripe size is consistent with theoretical value, glue recycling is carried out, recovery product part is used In sequencing, correct product will be sequenced and be used for subsequent experimental.

2. the expression of the slow virus recombinant expression carrier of anti-LMP1 CAR is verified

This research is divided into 3 groups, and the respectively X-293T groups of cells of untransfected, CD19 CAR Lentiviral transfects The X-293T groups of cells of the recombinant expression carrier transfection of X-293T groups of cells, the slow disease of anti-LMP1 CAR.

2.1 LMP1 CAR Lentivirals, CD19 CAR Lentiviral transfect X-293T cell

The X-293T cell that 37 DEG C of water-bath recoveries freeze is inoculated in 6 orifice plates, cultivated after 10cm culture dish is cultivated Night is transfected.Steps are as follows:

1. drawing culture medium in 6 orifice plates, fresh non-fully culture medium is replaced；2. solution A: 1ug LMP1 CAR slow virus Expression vector+Opti-MEM culture medium, total volume 150ul；B solution: 2ul PEI (is purchased from the limited public affairs of Shanghai starting experiment reagent Department)+Opti-MEM culture medium, total volume 150ul；After B solution stands 5min, B solution is added dropwise to solution A, is mixed；3. mixing Solution is stored at room temperature 30min；4. mixed solution is added drop-wise in 6 orifice plates, 6 orifice plates are shaken by ten word directions, are mixed, 48h is cultivated；⑤ The transfection method of CD19 CAR Lentiviral is the same as LMP1 CAR Lentiviral；

2.2 X-293T cell proteins extract

By the X-293T cell of untransfected, the X-293T of the CD19 CAR Lentiviral transfection prepared in 2.1 is thin The X-293T cell of the Lentiviral of born of the same parents and anti-LMP1 CAR transfection proceeds as follows respectively:

1. removing culture medium, PBS cleaning removes remaining culture medium, and pancreatin digestion is added, and microscopically observation has cell de- It falls, pipettor is boasted and toadied all to fall off to cell, and culture medium is added and terminates digestion；2. cell is collected into centrifuge tube, 1200r, 5min；3. abandoning supernatant, PBS is added and is resuspended, is added in EP pipe, 1200r, 5min；4. removing supernatant, lysate RIPA weight is added Outstanding, ice bath 30min, every 10min concussion is primary, and 4 DEG C, 12000r, 30min；5. taking supernatant；6. RIPA according to illustrate protease press down Preparation, 10⁶The RIPA that cell/ml is added is less than 100ul.

2.3 Western blot

Western blot is for verifying expression of the LMP1 CAR Lentiviral in X-293T cell.

Experimental procedure is as follows:

1. preparing 10% protein adhesive, note: protein adhesive saves, and is wrapped up in paper bag, moistens, and is put into disposable bags, and refrigerator saves. 2. 95 DEG C are boiled sample 5min；3. being loaded, electrophoresis, electrophoresis liquid is 1 × Tris, and when electrophoresis first uses 90V, is adjusted to when being diffused into separation gel 120V；4. transferring film: 1 × transfer pours into porcelain dish, and clamping plate infiltrates wherein, takes out film, be placed on clamping plate, " black glue tunica albuginea ". 300A, 80min, when transferring film, transferring film instrument is placed in ice water；5. the film to take a turn for the better is closed with 5% milk confining liquid, shaking table closes 1- 2h；6. mouse anti human CD3 ζ antibody is diluted in the ratio of 1:1000 with 5% milk, 4 DEG C of overnight incubations；7. PBST rinses 3 It is secondary, each 10min；8. the goat anti-mouse antibody of HRP label is diluted in the ratio of 1:2000 with 5% milk, it is incubated at room temperature 60min；9. PBST is rinsed 3 times, each 10min；10. developing solution is added dropwise, it is imaged under imager.

As a result as shown in Fig. 2, swimming lane 1: the X-293T cell of untransfected；Swimming lane 2:CD19 CAR Lentiviral turns The X-293T cell of dye；Swimming lane 3: the X-293T cell of the Lentiviral transfection of anti-LMP1 CAR.

The results show that the slow virus recombinant expression carrier of anti-LMP1 CAR can express in X-293T cell, X-293T is thin Born of the same parents can be used for subsequent virus packaging.

The separation of 2.4 PBMC and the amplification of T cell

1) physiological saline, lymphocyte separation medium are incubated for 37 DEG C with incubator；2) 50ml centrifuge tube uses heparin wet in advance The peripheral blood of acquisition is transferred in centrifuge tube (total volume is no more than 45ml) by profit；At room temperature, 50ml centrifuge tube 2000rpm from Heart 10min removes upper plasma；4) physiological saline of haemocyte precipitating pre-temperature carries out 1:1 resuspension, turns upside down 3-4 times, fills Divide and mixes；5) by the mononuclearcell suspension after dilution, be added on isometric lymphocyte separation medium surface, 20 DEG C, 500g from The heart 23 minutes.The acceleration for starting and stopping is both configured to 1；6) blood plasma on upper layer is at the uniform velocity absorbed.7) speed draws tunica albuginea layer.Work as liquid When identity distance red blood cell layer 2-3cm, stop imbibition.Draw tunica albuginea layer into 50ml centrifuge tube, with physiological saline by total volume mend to 45ml, is centrifuged 5 minutes by 20 DEG C, 1200rpm.Start acceleration and be set as 9, acceleration at stall is set as 1；8) it after being centrifuged, absorbs Supernatant.Cell is resuspended in physiological saline, is centrifuged by 7 centrifugal conditions；9) after being centrifuged, supernatant will be absorbed.Cell is resuspended in complete medium Afterwards, overnight incubation；10) (1:1000) is coated with 24 Kong Fei tissue culturing plates, sealing after anti-human CD3 and CD28 antibody are diluted with PBS After film is sealed, 4 DEG C of refrigerator overnights；11) it goes unless non-tissue cultures are added in liquid, collection T cell in tissue culturing plate after resuspension In plate, 37 DEG C of incubators are stayed overnight；12) every hole sucks 1ml culture medium, and the culture medium that 1ml contains IL-2, IL-7, IL-15 is added (1:5000) continues to cultivate, and T cell aggregation is agglomerating, at " grape cluster " structure, replaces the culture medium containing IL-2 every other day, expands Culture prepares LMP1 CAR-T cell using slow-virus infection for subsequent.

2 LMP1 CAR virus of embodiment packaging

1) X-293T cell is collected, 10cm culture dish is inoculated in and continues to cultivate, when cell confluency degree is up to 70%, virus packaging Preceding 30min replaces fresh non-fully culture medium；2) 1.5ml centrifuge tube A:470ul Opti-MEM+30ul PEI, room temperature after resuspension It is incubated for 5min；It is fixed that Opti-MEM is added in 1.5ml centrifuge tube B:3.3ug psPAX2+3.3ug pMD2.G+3.3ug LMP1 CAR Hold 250ul；3) reagent is added drop-wise in pipe B in pipe A, after mixing gently, is placed at room temperature for 15min；4) mixture is added drop-wise to X- In 293T cell, culture dish is shaked gently, so that mixture is evenly distributed, 37 DEG C of incubators are incubated for culture；5) it is replaced after 6-8h Fresh non-fully culture medium (1640+10%FBS)；6) for 24 hours after fluorescence microscopy microscopic observation transfection efficiency；7) it is collected after 48h Clearly, it if cell is adherent good, replaces fresh non-fully culture medium (1640+10%FBS) and continues culture to 72h, collect supernatant；8) 4 DEG C of the supernatant of collection, 400g are centrifuged 5min, go to precipitate, and collect supernatant.

3 LMP1 CAR viral concentration of embodiment

Method one:

1) supernatant that embodiment 2 is collected is transferred in the super filter tube of 100kD after 0.45um membrane filtration and is concentrated into 1/3 Volume；

2) concentrate collects filtered fluid, -80 DEG C freeze through 0.22um membrane filtration.

Method two:

1) it takes 8.76g NaCl respectively and 50g PEG-8000 is dissolved in 200ml distilled water, high pressure sterilization, preparation 5 × PEG-8000 NaCl solution；2) viral supernatants that embodiment 2 is collected are mixed in the ratio of 1:25 and 5 × PEG-8000NaCl, often 20-30min concussion is primary, and totally 5 times, 4 DEG C overnight；3) 6000rpm is centrifuged 20min, removes supernatant；4) precipitating is collected, with culture medium It is resuspended, -80 DEG C freeze.

The preparation of 4 LMP1 CAR-T cell of embodiment

1) 24 Kong Fei tissue culturing plates are added after the diluted RetroNectin of PBS (1:50), every hole 100ul, sealed membrane After closing, 4 DEG C of refrigerator overnights；2) it places, goes unless organizing training on ice after 37 DEG C of water-bath dissolutions of the virus liquid being concentrated in embodiment 3 Liquid in plate is supported, 500ul virus liquid is added in every hole, and 37 DEG C of incubators are incubated for 30min；3) after removing virus liquid, fresh disease is replaced After venom, 37 DEG C of incubators are incubated for 30min, remove liquid, and 2ml culture medium is added；4) collect culture T cell, 1500rmp from Heart 5min after removing supernatant, after the non-fully culture medium (1640+10%FBS) (1:2500) containing IL-2 is resuspended, is added drop-wise to non-group It knits in culture plate, cultivates 48h；5) 1ml culture medium is removed, after the CAR-T cell for collecting preparation, with fresh non-complete containing IL-2 After full culture medium (1640+10%FBS) (1:10000) is resuspended, continue to expand culture.

The expression of 5 Flow cytometry nasopharyngeal carcinoma cell surface antigen LMP1 of embodiment

This research is divided into nasopharyngeal carcinoma cell CNE1 group, nasopharyngeal carcinoma cell CNE2 group, nasopharyngeal carcinoma cell C666-1 group, nasopharyngeal carcinoma Cell HONE1 group and nasopharyngeal carcinoma cell SUNE1 group, each group take corresponding nasopharyngeal carcinoma cell to proceed as follows respectively:

1) recovery of nasopharyngeal carcinoma cell

Cryopreservation tube is placed in 37 DEG C of water-bath dissolutions, is added in the centrifuge tube containing 1ml culture medium, and 800rmp is centrifuged 5min, Supernatant is removed, is resuspended and is cultivated with culture medium.

2) passage of nasopharyngeal carcinoma cell

Culture medium is removed, PBS is added and removes remaining culture medium in order to avoid neutralizing pancreatin, 500ul pancreatin is added, incubator is incubated for It wait digest, is added culture medium (enzymolysis reaction), mixes, be transferred to centrifuge tube, 800rmp is centrifuged 5min, and culture medium is resuspended Afterwards, culture bottle is instilled to continue to cultivate.

3) collect logarithmic growth phase nasopharyngeal carcinoma cell, counted after being digested to single cell suspension, adjustment concentration be 1 × 10⁶Cells/ml is divided into experimental group and control group, 1ml cell suspension is added in every group of Ep pipe.

4) 800rmp is centrifuged 5min, and PBS is washed three times.

5) the anti-human LMP1 antibody in 1ul sheep source is added in the every pipe of experimental group, it is anti-that 1ul Isotype control is added in the every pipe of control group Body is incubated at room temperature 30min.

6) fluorescence antibody for the anti-sheep of source of mouse that 3ul FITC is marked, incubation at room temperature is added into experimental group and control group respectively 15min。

7) 800rmp is centrifuged 5min, and PBS washed once, and after being resuspended with 300ul PBS, streaming pipe is added, carries out streaming point Analysis.

The results show that nasopharyngeal carcinoma cell CNE1 is LMP1 high expression, nasopharyngeal carcinoma cell CNE2 and C666-1 is flow cytometer showed LMP1 low expression, and nasopharyngeal carcinoma cell HONE1 and SUNE1 do not express LMP1 substantially.

The nasopharyngeal carcinoma cell CNE1 of 6 Luciferase of embodiment label stablizes the screening of strain

1) the nasopharyngeal carcinoma cell CNE1 for collecting logarithmic growth phase, is inoculated in 96 orifice plates with the cell concentration in the hole 5000cells/ In, the puromycin of various concentration is added after cell is adherent, replaces the culture medium of purine-containing mycin every other day, daily observation cell Survival rate selects the minimum concentration of cell death after culture 5-6 days.2) the nasopharyngeal carcinoma cell CNE1 of logarithmic growth phase is collected, with The cell concentration in the hole 5000cells/ is inoculated in 96 orifice plates overnight, and a certain amount of Luciferase virus liquid is added (purchased from Beijing The luxuriant industry Biotechnology Co., Ltd of English), 37 DEG C of incubator cultures are for 24 hours.3) fresh complete medium is replaced, and is added in every hole The puromycin of minimum concentration.4) continue culture one week, the concentration of puromycin is kept in incubation.5) expand culture, it can Do not add puromycin.6) the CNE1 cell strain of Luciferase label freezes.

When cell confluency degree is up to 90% or more, after collecting cell, with frozen stock solution (30%FBS+60%DMEM+10%DMSO) Be resuspended, label Cell Name with freeze the Programmed cryopreservation box that the time is placed on room temperature, be put into -80 DEG C of refrigerators and freeze, the next day put Liquid nitrogen cryopreservation.

7 CCK8 method of embodiment detects LMP1 CAR-T cell to the lethal effect of nasopharyngeal carcinoma

1) target cell: human nasopharyngeal epithelioma 1 CNE1, CNE2 and HONE1, lymphoma cell strain Raji；Effector cell: this hair LMP1 CAR-T cell prepared by bright embodiment 4, the CD19 CAR-T cell of 4 same procedure of embodiment preparation, the T being separately cultured Cell.2) experiment the previous day, LMP1 CAR-T cell, CD19 CAR-T cell and T cell replace fresh culture (1640+ 10%FBS), 37 DEG C of incubator overnight incubations.3) CNE1, CNE2 and the HONE1 for collecting logarithmic growth phase, adjust cell concentration 5 ×10⁴Cells/ml is inoculated in 96 orifice plates, every hole 100ul, 37 DEG C of incubator overnight incubations.4) it is thin to collect LMP1 CAR-T Born of the same parents, CD19 CAR-T cell and T cell, count after resuspension, by the effect target ratio of 2:1,5:1,10:1,20:1, are separately added into 96 holes Plate, every group sets 4 multiple holes, 37 DEG C of incubator culture 6h.5) cell conditioned medium is drawn, PBS is cleaned 3 times, and it is adherent to wash Kong Zhongwei Cell, the mixed solution of 10+90 μ l RPMI of μ l CCK-8 solution, 1640 culture medium, 5%CO is added in every hole₂, 37 DEG C of cultures 4h is incubated in case.Microplate reader measures the absorbance in each hole at 450nm wavelength.

Killing rate=1- (OD_{Co-culture hole}-OD_{T cell hole})/OD_{Tumour cell hole}

Using CD19 CAR-T cell and T cell as compareing, when LMP1 CAR-T cell difference imitates target ratio to CNE1, The lethal effect of CNE2, HONE1, as a result as shown in Fig. 4 A, 4B, 4C.

As a result, it has been found that when effect target ratio is 20:1,10:1, nasopharyngeal carcinoma cell CNE1 of the LMP1 CAR-T cell to the LMP1 positive There is statistical difference compared with the control group with the lethal effect of CNE2, and the killing to LMP1 Nasopharyngeal Carcinoma Patients with Negative cell HONE1 Act on no difference of science of statistics compared with the control group.Confirm LMP1 CAR-T cell in vitro to the spy of LMP1 positive nasopharyngeal carcinoma's cell Anisotropic targeting killing effect.

2) in the same way, measure each effector cell in the effect target ratio of 10:1 to human nasopharyngeal epithelioma 1 CNE1, CNE2, The killing rate of HONE1 and lymphoma cell strain Raji.

Using CD19 CAR-T cell, T cell as control, when 10:1 imitates target ratio, LMP1 CAR-T cell is thin to nasopharyngeal carcinoma The lethal effect of born of the same parents CNE1, CNE2, HONE1, lymphoma cell Raji, as a result as shown in Figure 4 D.

As the result is shown: when effect target ratio is 10:1, nasopharyngeal carcinoma cell CNE1, the CNE2 of LMP1 CAR-T cell to the LMP1 positive There is statistical difference compared with the control group with the lethal effect of lymphoma cell strain Raji, and to LMP1 Nasopharyngeal Carcinoma Patients with Negative cell The lethal effect of HONE1 no difference of science of statistics compared with the control group.Confirm LMP1 CAR-T cell in vitro to LMP1 positive nose The selectively targeted lethal effect of pharynx cancer cell.

Point of IL-2 after 8 ELISA method detection LMP1 CAR-T cell of embodiment and nasopharyngeal carcinoma cell co-culture, IFN-γ It secretes

1) CNE1, CNE2 and the HONE1 for collecting logarithmic growth phase, adjust cell concentration 5 × 10⁴Cells/ml is inoculated in In 96 orifice plates, every hole 100ul, 37 DEG C of incubator overnight incubations；2) LMP1 CAR-T cell, CD19 CAR-T cell and T are collected Cell counts after resuspension, imitates target ratio by 10:1, is added separately to 96 orifice plates, 37 DEG C of incubator cultures are for 24 hours；3) 96 orifice plates are horizontal Centrifugation, 1500rmp are centrifuged 5min, draw supernatant, -20 DEG C freeze；4) anti-IL-2/IFN- gamma antibodies are added after diluted 96 orifice plates, the hole 100ul/ close 4 DEG C overnight；5) remove supernatant, PBST washs 3 times, at least the hole 250ul//time, every time no less than 1min；6) 1 × ELISA/ELISPOT of 200ul solution is added in every hole, is placed at room temperature for 1h；7) supernatant is removed, PBST is washed 1 time；8) The collected co-cultivation supernatant of 100ul is added in every hole, 2 secondary orifices are set, and 4 DEG C overnight.Standard items are added according to gradient；9) it goes Supernatant, PBST are washed 5 times, no less than 1min every time；10) every hole is added 100ul and detects antibody, is placed at room temperature for 1h；11) it goes Clearly, PBST is washed 5 times；12) Avidin of 100ul HRP label is added in every hole, is placed at room temperature for 30min；13) supernatant, PBST are removed Washing 7 times；14) 1 × TMB of 100ul developing solution is added in every hole, is placed at room temperature for 15min；15) 50ul terminate liquid, enzyme is added in every hole Mark the absorbance of instrument measurement 450mn.

As a result as shown in Figure 5: on the nasopharyngeal carcinoma cell CNE1 and CNE2 co-cultivation of LMP1 CAR-T cell and the LMP1 positive In clear, cell factor IL-2, IFN-γ secretory volume there is statistical difference compared with the control group, and the nasopharynx with LMP1 feminine gender Cancer cell HONE1 is co-cultured in supernatant, the secretory volume no difference of science of statistics compared with the control group of cell factor IL-2, IFN-γ.

The tumor-inhibiting action of the unilateral in vivo test LMP1 CAR-T cell of embodiment 9

1) experimental subjects selection lacks the BALB/c nude mice of T cell, is divided into LMP1 CAR-T cell processing group, CD19 CAR-T cell processing group, T cell processing group and physiological saline processing group, every group 6；2) before tumor formation, nude mice abdominal cavity injection 2.5mg/kg cyclophosphamide, for three days on end, the 4th day progress roentgen radiation x；3) the Luciferase label of logarithmic growth phase is collected Human nasopharyngeal epithelioma 1 CNE1, adjustment concentration are 10⁷/ ml, it is 100ul that total volume is injected in oxter；4) the 0th, 3,6 day after tumor formation, respectively It is 10 that experimental group, which distinguishes tumor week injection cell concentration,⁷LMP1 CAR-T cell, CD19 CAR-T cell, T cell and same volume Physiological saline；5) it is observed and measured daily after tumor formation, tumorous size is obtained by vernier caliper measurement, tumor volume (mm³)=1/2 × Major diameter × minor axis².(Fig. 6 D) 6) the 0th, 3,6,9 day after tumor formation, nude mice abdominal cavity injected fluorescein potassium (3mg/ is only), carry out living body at As (Fig. 6 A, B)；7) the 9th day, all processing groups are put to death, dissection obtains each group knurl, observes tumorous size (Fig. 6 C), knurl weight Amount (Fig. 6 E) simultaneously calculates tumour inhibiting rate, tumour inhibiting rate=(1- treatment group average knurl weight/physiological saline group average knurl weight) × 100%

As the result is shown: LMP1 CAR-T cell processing group knurl fluorescence intensity, volume, weight have compared with each control group Statistical difference, it was demonstrated that LMP1 CAR-T cell in vivo makees LMP1 positive nasopharyngeal carcinoma cell transplantation tumor with growth inhibition With.

10 bilateral in vivo test LMP1 CAR-T cell of embodiment specific killing in vivo

1) experimental subjects selection lacks the BALB/c nude mice of T cell, is divided into LMP1 CAR-T cell processing group, CD19 CAR-T cell processing group, T cell processing group and physiological saline processing group, every group 3；2) before tumor formation, nude mice abdominal cavity injection 2.5mg/kg cyclophosphamide, for three days on end, the 4th day progress roentgen radiation x；3) the Luciferase label of logarithmic growth phase is collected Human nasopharyngeal epithelioma 1 CNE1 and nasopharyngeal carcinoma cell HONE1, adjusting separately concentration is 10⁷/ ml, oxter injection total volume are 100ul, wherein 100%CEN1 cell is injected in left side oxter, 50%CNE1+50%HONE1 is injected in right side oxter；4) after tumor formation 0,3,6 days, each experimental group difference intratumor injection cell concentration was 10⁷LMP1 CAR-T cell, CD19 CAR-T cell, T cell With same volume physiological saline；5) the 0th, 9 day after tumor formation, nude mice abdominal cavity injected fluorescein potassium (3mg/ is only) carries out living body fluorescent Imaging, and shoot light field figure (Fig. 7 A)；6) the 9th day, all processing groups are put to death, each group knurl are obtained by dissection, knurl is with 4% Paraformaldehyde is fixed, and 4 DEG C of placements are used for subsequent immunohistochemistry.

Bilateral nude mice Tumorigenesis detects LMP1 CAR-T cell specific killing in vivo, the results show that LMP1 CAR- Though the fluorescence intensity of T cell group mixed tumour side knurl weakens, tumor volume, which has no, to be obviously reduced, it was demonstrated that LMP1 CAR-T cell Inhibit the growth of LMP1 positive nasopharyngeal carcinoma cell transplantation tumor in vivo, and LMP1 Nasopharyngeal Carcinoma Patients with Negative cell transplantation tumor is had no obviously Effect.

Sequence table

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<210> 12

<211> 69

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 12

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Leu Tyr Cys

65

<210> 12

<211> 126

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 13

<211> 42

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 13

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 14

<211> 336

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 15

<211> 112

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 15

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 16

<211> 726

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

gaagtgcagc tggtggagag tgggggggga ctggtccagc caggagggtc actgagactg 60

agctgcgccg cttccggctt caccttcagc agctactgga tgtcctgggt gaggcaggct 120

cctggaaaag gcctggagtg ggtcgcaaac atcaaacagg acggctctga aaagtactat 180

gtggatagtg tcaaagggcg gttcactatt tccagagaca acgccaagaa ttctctgtat 240

ctgaggatga attccctgcg cgccgaggat accgccgtgt actattgcgc tcgcgcatgg 300

gggtactcta gttactattt tgactattgg gggcagggaa ctctggtgac cgtcagccct 360

ggaggaggag gatcaggcgg cggaggcagc ggaggaggag ggtccgaact gcagatgacc 420

cagagcccat caagcctgtc tgccagtgtg ggcgatcgag tcgcaatcac atgtcgggcc 480

tcacagagca tttcctctca cctgaactgg taccagcaga agcccggaaa agctcctaag 540

ctgctgatct atgcagccag ttcactgcag tctggcgtgc caagtcgatt ctccggctct 600

gggagtggaa cagactttac cctgacaatt agctccctgc agcccgagga tttcgccact 660

tactattgtc agcagagcta ctcaacccca tacaccttcg ggcagggaac taaactggaa 720

atcaaa 726

<210> 17

<211> 242

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Arg Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Trp Gly Tyr Ser Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Pro Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Glu Leu Gln Met Thr Gln Ser Pro Ser

130 135 140

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Ala Ile Thr Cys Arg Ala

145 150 155 160

Ser Gln Ser Ile Ser Ser His Leu Asn Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly

180 185 190

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln

210 215 220

Gln Ser Tyr Ser Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu

225 230 235 240

Ile Lys

<210> 18

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 18

cacgccgcca ggccggaagt gcagctggtg 30

<210> 19

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 19

tgatcctcct cctccagggc tgacggtcac 30

<210> 20

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 20

ggaggaggag ggtccgaact gcagatgacc 30

<210> 21

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 21

caccagcacc caaaatttga tttccagttt 30

<210> 22

<211> 35

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 22

ggaccatcct ctagggatcc atggccttac cagtg 35

<210> 23

<211> 33

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 23

aatccggatc gatctcgagg tgcatgctaa cgc 33

Claims

1. a kind of Chimeric antigen receptor for targeting LMP1, which is characterized in that the Chimeric antigen receptor of the targeting LMP1 is by from ammonia Cardinal extremity is to sequentially connected signal peptide CD8 α of c-terminus, extracellular region LMP1 scFv, transmembrane region CD8^TMIt is formed with intracellular region.

2. the Chimeric antigen receptor of targeting LMP1 according to claim 1, which is characterized in that the extracellular region LMP1 ScFv is sequentially connected by heavy chain variable region, link peptide, light chain variable region and is formed, the heavy chain variable amino acid sequence such as SEQ Shown in ID NO.3, for chain variable region amino acid sequence as shown in SEQ ID NO.4, link peptide is the repetition of concatenated three groups of pentapeptides Unit (Gly4Ser) 3.

3. the Chimeric antigen receptor of targeting LMP1 according to claim 2, which is characterized in that encode the heavy chain variable region The nucleotide sequence of amino acid encodes the nucleotide sequence such as SEQ of the chain variable region amino acid as shown in SEQ ID NO.1 Shown in ID NO.2, the nucleotide sequence of the link peptide is encoded as shown in SEQ ID NO.7.

4. the Chimeric antigen receptor of targeting LMP1 according to claim 1, which is characterized in that the ammonia of the signal peptide CD8 α Base acid sequence is as shown in SEQ ID NO.6, and the nucleotide sequence of encoded signal peptide CD8 α is as shown in SEQ ID NO.5, the volume The nucleotide sequence of code signal Peptide C D8 α is connected to the end 5' of LMP1 scFv heavy chain variable region.

5. the Chimeric antigen receptor of targeting LMP1 according to claim 1, which is characterized in that the transmembrane region CD8^TMAmmonia Base acid sequence encodes the transmembrane region CD8 as shown in SEQ ID NO.9^TMThe nucleotide sequence of amino acid such as SEQ ID NO.8 institute Show.

6. it is according to claim 1 targeting LMP1 Chimeric antigen receptor, which is characterized in that the intracellular region by CD28, CD137, CD3 ζ are sequentially connected composition；

The amino acid sequence of the CD28 encodes the nucleotide sequence of the CD28 amino acid such as shown in SEQ ID NO.11 Shown in SEQ ID NO.10；

The amino acid sequence of the CD137 encodes the nucleotide sequence of the CD137 amino acid such as shown in SEQ ID NO.13 Shown in SEQ ID NO.12；

The amino acid sequence of the CD3 ζ encodes the nucleotide sequence of the CD3 ζ amino acid such as shown in SEQ ID NO.15 Shown in SEQ ID NO.14.

7. the slow virus recombinant expression carrier of anti-LMP1 CAR, which is characterized in that the slow virus of the anti-LMP1 CAR recombinates table Contain the Chimeric antigen receptor of any one of claim 1 to 6 targeting LMP1 up to carrier.

8. the preparation method of the slow virus recombinant expression carrier of anti-LMP1 CAR described in claim 7, which is characterized in that including with Lower step:

S1: using the anti-LMP1 monoclonal antibody of full source of people as template, design heavy chain variable region specific primer to and light chain variable region it is special Property primer pair, expand heavy chain variable region and light chain variable region respectively；Specific primer pair is connected using weight, light chain, passes through connection Peptide sequence connects heavy chain variable region and light chain variable region, constructs extracellular region LMP1 scFv segment；

S2: in the end the 5' connection signal Peptide C D8 α sequence for the anti-extracellular region LMP1 scFv segment that S1 is successfully constructed, CD8 α mark is constructed The extracellular region LMP1 scFv segment of note；

S3: the extracellular region LMP1 scFv segment and transmembrane region CD8 that CD8 α is marked^TMWith intracellular region CD28, CD137, CD3 ζ phase Even, building obtains the nucleotide sequence of the Chimeric antigen receptor of targeting LMP1；

S4: with restriction endonuclease BamHI and XhoI double digestion slow virus carrier, the slow virus carrier linearized, By the method for In-Fusion PCR by the nucleotide sequence of the Chimeric antigen receptor of the S3 targeting LMP1 obtained and linearisation Slow virus carrier recombination, obtains the slow virus recombinant expression carrier of anti-LMP1 CAR.

9. preparation method according to claim 8, which is characterized in that heavy chain variable region specific primer pair described in S1 are as follows:

V_HUpstream primer: CACGCCGCCAGGCCGGAAGTGCAGCTGGTG (SEQ ID NO.18),

V_HDownstream primer TGATCCTCCTCCTCCAGGGCTGACGGTCAC (SEQ ID NO.19)；

Light chain variable region specific primer pair are as follows:

V κ upstream primer: GGAGGAGGAGGGTCCGAACTGCAGATGACC (SEQ ID NO.20),

V κ downstream primer: CACCAGCACCCAAAATTTGATTTCCAGTTT (SEQ ID NO.21).

10.LMP1 CAR-T cell, which is characterized in that the LMP1 CAR-T cell is anti-LMP1 as claimed in claim 7 The slow virus recombinant expression carrier infection T cell of CAR obtains.

11. the preparation method of LMP1 CAR-T cell described in claim 10, which is characterized in that the method includes following steps It is rapid:

S1: by the slow virus recombinant expression carrier and packaging plasmid psPAX2, pMD2.G of anti-LMP1 CAR as claimed in claim 7 Three plasmid slow virus packaging systems, transfection to X-293T cell are formed, 37 DEG C of incubators are incubated for culture, replace after 6-8h fresh non- Complete medium (1640+10%FBS) collects supernatant after cultivating 48h, replaces fresh non-fully culture medium (1640+10%FBS) Continue culture to 72h, collects supernatant；

12. the Chimeric antigen receptor of targeting LMP1 described in any one of claims 1 to 6, anti-LMP1 as claimed in claim 7 The slow virus recombinant expression carrier of CAR, LMP1 CAR-T cell described in any one of claim 10 prepare nasopharyngeal carcinoma diagnosis reagent or Application in nasopharyngeal carcinoma immunotherapy medicaments.