CN108341881A

CN108341881A - Chimeric antigen receptor and its expressing gene with safety switch, the NK cells of its modification and application

Info

Publication number: CN108341881A
Application number: CN201810096080.3A
Authority: CN
Inventors: 不公告发明人
Original assignee: Shenzhen Morecell Biomedical Technology Development Co Ltd
Current assignee: Shenzhen Morecell Biomedical Technology Development Co Ltd
Priority date: 2018-01-31
Filing date: 2018-01-31
Publication date: 2018-07-31
Anticipated expiration: 2038-01-31
Also published as: CN108341881B

Abstract

The present invention relates to a kind of Chimeric antigen receptor with safety switch and its expressing gene, the NK cells of its modification and applications.The Chimeric antigen receptor includes sequentially connected first signal peptide, c Met single-chain antibodies, CD8 structural domains, 41BB intracellular domain, DAP12 ITAM structural domains, T2A, second signal peptide and the EGFRt of transformation.The experimental results showed that the NK cells of above-mentioned Chimeric antigen receptor modification, Chimeric antigen receptor immune response is controllable, has a safety feature, and treatment side effect is few, and specificity is high.

Description

The NK cells of Chimeric antigen receptor and its expressing gene with safety switch, its modification And application

Technical field

The present invention relates to biotechnologies, more particularly to a kind of Chimeric antigen receptor with safety switch and its expression Gene, the NK cells of its modification and application.

Background technology

The human world matter epidermis transforming factor c-Met (Mesenchymal Epithelial Transition Factor, c- Met) be hepatocyte growth factor (Hepatocyte Growth Factor, HGF) receptor, it is super to belong to tyrosine kinase receptor Family member.C-Met activates HGF/c-Met signal paths, reduces intercellular adhesive force, be conducive to swell by being combined with HGF The transfer of oncocyte.Recent study finds that c-Met is in the higher liver cancer of China's incidence, lung cancer, breast cancer, gastric cancer, pancreas Overexpression, mutation or activity change are presented in the tumor tissues such as cancer, glioma.Therefore, c-Met has become antitumor One important target spot for the treatment of.

In recent years in adoptive cell therapy technology, Chimeric antigen receptor (chimeric antigen receptor, CAR) T cell (CAR-T) technology modified achieves significant breakthrough in the clinical application for the treatment of tumour, in clinical test Good targeting, lethal and persistence are shown, huge application potential and development prospect are illustrated.But it is traditional The T cell of Chimeric antigen receptor modification, cannot start or stop performance immunization as needed, and treatment side effect is more, special Property is poor.It finds that CAR-T technologies have in newest clinical trial and causes the high wind such as cytokine storm and systemic neurotoxicity Dangerous factor.

Natural killer cells (Natural Killer Cell, NK cell) is the core component of human body congenital immunity, It is that the third monoid lymphocyte arranged side by side with T, B cell is reached maturity from candidate stem cell in marrow.NK cells have There are many features：Inherent immunity cell, non-specific direct killing target cell, need not in advance by antigen sensibilization, need not resist Body participates in, is restricted without MHC, playing immunologic cytotoxicity effect early.Therefore, with Chimeric antigen receptor modification NK cells at For new antitumor research direction.

However, that there are security performances is poor, treatment side effect is more, specifically for the NK cells of traditional Chimeric antigen receptor modification The problems such as property is poor.

Invention content

Based on this, it is necessary to provide a kind of NK cells that can be modified, and make the security performance of the NK cells after modification It gets well, treatment side effect is few, specificity high Chimeric antigen receptor and its expressing gene with safety switch.

In addition, also providing the band that a kind of expression vector and a kind of security performance are preferable, treatment side effect is few, specificity is high The NK cells of the Chimeric antigen receptor modification of safety switch and its application.

A kind of Chimeric antigen receptor with safety switch, including sequentially connected first signal peptide, c-Met single-chain antibodies, CD8 structural domains, 41BB intracellular domain, DAP12-ITAM structural domains, T2A, second signal peptide and the EGFRt of transformation；Wherein, The amino acid sequence of the c-Met single-chain antibodies is as shown in SEQ ID No.1, and the amino acid sequence of the EGFRt of the transformation is such as Shown in SEQ ID No.2.

In one embodiment, the nucleotide sequence of the c-Met single-chain antibodies is encoded as shown in SEQ ID No.3.

In one embodiment, the nucleotide sequence of the EGFRt of the transformation is encoded as shown in SEQ ID No.4.

In one embodiment, the Chimeric antigen receptor is：

(a) protein that amino acid sequence forms shown in SEQ ID No.5；Or,

(b) by replacing, missing or adding one or several amino acid in the amino acid sequence shown in SEQ ID No.5 And there is the active protein derived from (a) of the Chimeric antigen receptor.

In one embodiment, the Chimeric antigen receptor is：

(a) the nucleotide sequence coded obtained protein shown in SEQ ID No.6；Or,

(b) in the nucleotide sequence shown in SEQ ID No.6 through replacing, missing or adding one or several bases and Can encode to obtain has the active protein derived from (a) of the Chimeric antigen receptor.

A kind of expressing gene, for expressing the Chimeric antigen receptor with safety switch, the Chimeric antigen receptor include according to First signal peptide of secondary connection, c-Met single-chain antibodies, CD8 structural domains, 41BB intracellular domain, DAP12-ITAM structural domains, The EGFRt of T2A, second signal peptide and transformation；Wherein, the amino acid sequence of the c-Met single-chain antibodies such as SEQ ID No.1 Shown, the amino acid sequence of the EGFRt of the transformation is as shown in SEQ ID No.2.

In one embodiment, the expressing gene is：

(a) nucleotide sequence as shown in SEQ ID No.6；Or,

(b) in the nucleotide sequence shown in SEQ ID No.6 through replacing, missing or adding one or several bases and Can encode to obtain has the active nucleotide sequence of the Chimeric antigen receptor.

A kind of expression vector contains above-mentioned expressing gene in the expression vector.

A kind of NK cells of the Chimeric antigen receptor modification with safety switch, can express in the NK cells any of the above-described Chimeric antigen receptor described in has either imported expressing gene described in any one of the above embodiments or described in the NK cells Above-mentioned expression vector has been transfected in NK cells.

Chimeric antigen receptor described in any one of the above embodiments, expressing gene as described in any one of the above embodiments, the table described in above-mentioned 8 Up to carrier or NK cells the answering in preparing anti-tumor drug of the above-mentioned Chimeric antigen receptor modification with safety switch With.

Above-mentioned Chimeric antigen receptor includes sequentially connected first signal peptide, c-Met single-chain antibodies, CD8 structural domains, 41BB Intracellular domain, DAP12-ITAM structural domains, T2A, second signal peptide and the EGFRt of transformation.Utilize the anti-c-Met of screening The surface growth factor of transformation is added as the scFv sequences in Chimeric antigen receptor (CAR) carrier in monoclonal antibody sequences Receptor (EGFRt).Its structure of the EGFRt of transformation only includes extracellular domain III, extracellular domain IV and the transmembrane structure of EGFR Domain does not include intracellular tyrosine kinase domain.Extracellular domain III in the EGFRt of transformation is (shown in SEQ ID No.2 No. 1 amino acid~No. 172 amino acid) and (No. 173 amino acid~312nd shown in SEQ ID No.2 extracellular domain IV Number amino acid) be Cetuximab binding site.The experimental results showed that above-mentioned Chimeric antigen receptor can be steady in NK cells Fixed expression, to form the NK cells of Chimeric antigen receptor modification.And the Chimeric antigen receptor can target c-Met, and carry Safety switch, Chimeric antigen receptor structure and EGFRt constructions of switch are expressed in NK cell surfaces respectively with autothermic cracking peptide T 2A. Cytokine storm and system are generated after the NK cells (CAR-NK cells) of above-mentioned Chimeric antigen receptor modification are transfused to patient When the side effects such as nerve toxicity, then Cetuximab is commercialized in injectable, is acted on, can be removed by the ADCC of NK cells itself Internal genetic engineering transformation is to express the natural killer cells of CAR.Therefore Cetuximab can be used as chimeric antigen in NK cells " switch " of receptor behavior realizes that Chimeric antigen receptor immune response is controllable, has a safety feature that treatment side effect is few, specificity It is high.

Description of the drawings

Fig. 1 is the structural schematic diagram of the c-Met Chimeric antigen receptors with EGFRt switches in embodiment 1；

Fig. 2 is the plasmid table of the slow virus expression plasmid pLenti7.3-c-Met/CAR-EGFRt built in embodiment 1 Figure；

Fig. 3 is the electrophoresis knot of slow virus expression plasmid pLenti7.3-c-Met/CAR-EGFRt digestion products in embodiment 1 Fruit is schemed；

The expression quantity of detection c-Met after Fig. 4 behaviours HepG-2 cell and normal liver cell L-O2 cell crackings Western Blotting testing result figures；

Fig. 5 is the NK cells Cytotoxicity in vitro cell at different conditions of the Chimeric antigen receptor modification obtained in embodiment 1 Streaming scatterplot result comparison chart；

Fig. 6 is the system of the NK cells Cell viability at different conditions of the Chimeric antigen receptor modification obtained in embodiment 1 Count result comparison chart.

Specific implementation mode

In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, with reference to specific embodiment and Attached drawing is described in detail the specific implementation mode of the present invention.Elaborate in the following description many details in order to Fully understand the present invention.But the invention can be embodied in many other ways as described herein, art technology Personnel can do similar improvement without violating the connotation of the present invention, therefore the present invention is not by following public specific implementation Limitation.

The Chimeric antigen receptor (CAR) with safety switch of one embodiment, including sequentially connected first signal peptide, c- Met single-chain antibodies, CD8 structural domains, 41BB intracellular domain, DAP12-ITAM structural domains, T2A, second signal peptide and transformation EGFRt.Wherein, the amino acid sequence of c-Met single-chain antibodies is as shown in SEQ ID No.1, the amino acid sequence of the EGFRt of transformation Row are as shown in SEQ ID No.2.

Specifically, using the monoclonal antibody sequences of the anti-c-Met of screening, the sequence of wherein VH and VL are selected, for setting The scFv sequences in CAR carriers are counted, the amino acid sequence of the c-Met single-chain antibodies as shown in SEQ ID No.1 is obtained.Wherein, No. 1 amino acid~No. 97 amino acid is VH (heavy chain) sequence, No. 98 amino acid~No. 112 ammonia in SEQ ID No.1 Base acid is connection peptide, and No. 113 amino acid~No. 218 amino acid is VL (light chain) sequence.

Further, codon optimizes, and obtains the nucleotide sequence such as SEQ ID of coding c-Met single-chain antibodies Shown in No.3.

Specifically, the amino acid of the EGFRt of transformation only include EGFR extracellular domain III, extracellular domain IV and across Spanning domain does not include intracellular tyrosine kinase domain.Extracellular domain III (SEQ ID No.2 in the EGFRt of transformation Shown in No. 1 amino acid~No. 172 amino acid) and extracellular domain IV (No. 173 amino acid shown in SEQ ID No.2 ~the No. 312 amino acid) be Cetuximab binding site, be capable of specificity combination Cetuximab.

Further, codon optimizes, and obtains the nucleotide sequence such as SEQ ID No.4 of the EGFRt of coding transformation It is shown.

In present embodiment, Chimeric antigen receptor is：(a) egg that amino acid sequence forms shown in SEQ ID No.5 White matter；Or, (b) by replacing, missing or adding one or several amino acid in the amino acid sequence shown in SEQ ID No.5 And there is the active protein derived from (a) of Chimeric antigen receptor.

Specifically, in amino acid sequence shown in SEQ ID No.5, No. 1 amino acid~No. 22 amino acid is first Signal peptide, No. 23 amino acid~No. 240 amino acid are c-Met single-chain antibodies, No. 245 amino acid~No. 315 amino Acid is CD8 structural domains (including hinge area and transmembrane domain), and No. 316 amino acid~No. 357 amino acid is 41BB intracellulars Structural domain (costimulatory signal intracellular domain), No. 358 amino acid~No. 405 amino acid are DAP12-ITAM structural domains (DAP12 immunoreceptor tyrosine activating motif ITAM structural domains), No. 410 amino acid~No. 430 amino acid are T2A (oneself Crack polypeptide), No. 431 amino acid~No. 452 amino acid is second signal peptide, No. 453 amino acid~No. 787 amino Acid is the EGFRt of transformation.In addition, there are two restriction enzyme site (No. 241 amino acid~No. 244 ammonia in Chimeric antigen receptor Base acid and No. 406 amino acid~No. 409 amino acid).

Further, Chimeric antigen receptor is：(a) the nucleotide sequence coded obtained egg shown in SEQ ID No.6 White matter；Or, (b) in the nucleotide sequence shown in SEQ ID No.6 through replacing, missing or adding one or several bases and It can encode to obtain with the active protein derived from (a) of Chimeric antigen receptor.

It is appreciated that there are many codons due to the same amino acid of coding, the coded sequence of polypeptide has polymorphism And the characteristics of variation.Therefore one or several by replacing, missing or adding in the amino acid sequence shown in SEQ ID No.5 Amino acid and there is the active protein of Chimeric antigen receptor recombinant antigen, or the nucleotide sequence shown in SEQ ID No.6 Middle process replaces, misses or adds one or several bases and can encode to obtain with the active derivative egg of recombinant antigen In vain, the albumen obtained does not have apparent function difference with Chimeric antigen receptor, is intended to be included within the scope of the present invention.

By continuous research and probe, successful design goes out the Chimeric antigen receptor with safety switch of above-mentioned functional structure (CAR).The Chimeric antigen receptor (CAR) with safety switch, including sequentially connected first signal peptide, c-Met single-chain antibodies, CD8 structural domains, 41BB intracellular domain, DAP12-ITAM structural domains, T2A, second signal peptide and the EGFRt of transformation.This is embedding Antigen receptor is closed experiment proves that c-Met can be targeted, and carries safety switch, Chimeric antigen receptor structure and EGFRt switches Structure is expressed in NK cell surfaces respectively with autothermic cracking peptide T 2A.As the NK cells (CAR-NK of above-mentioned Chimeric antigen receptor modification Cell) it is transfused to after patient when generating the side effects such as cytokine storm and systemic neurotoxicity, then injectable commercialization west Appropriate former times monoclonal antibody is acted on by the ADCC of NK cells itself, and it is thin to express the natural kill of CAR can to remove internal genetic engineering transformation Born of the same parents.

The above-mentioned Chimeric antigen receptor (CAR) with safety switch can be used in anti-tumor drug.

In addition, the application also provides the expressing gene of an embodiment, the expressing gene is for expressing with safety switch Chimeric antigen receptor.

Specifically, the feature of Chimeric antigen receptor refers to described above, and therefore not to repeat here.

Specifically, which is：(a) nucleotide sequence as shown in SEQ ID No.6；Or, (b) in SEQ ID By replacing, missing or adding one or several bases and can encode to obtain with chimeric in nucleotide sequence shown in No.6 The active nucleotide sequence of antigen receptor.

Above-mentioned expressing gene can express the Chimeric antigen receptor (CAR) with safety switch, and the Chimeric antigen receptor is through examination Verification can target c-Met, and carry safety switch, can stablize in NK cells and express, have a safety feature, treating secondary work With less, specificity it is good.

Above-mentioned expressing gene can be used in anti-tumor drug.

In addition, the application also provides the expression vector of an embodiment, above-mentioned expressing gene is contained in the expression vector.

In one embodiment, which is the slow virus containing above-mentioned expressing gene.

The expression vector can be used in expressing above-mentioned Chimeric antigen receptor, and expression efficiency is high, and expression quantity is stablized, and can use In anti-tumor drug.

In addition, the application also provides a kind of NK cells of Chimeric antigen receptor modification of the embodiment with safety switch.It should NK cells can express above-mentioned Chimeric antigen receptor and either import above-mentioned expressing gene or the NK in the NK cells Above-mentioned expression vector has been transfected in cell.

The experimental results showed that the NK cells against tumor of the above-mentioned Chimeric antigen receptor modification with safety switch has specificity Efficient killing effect effect.And it is generated after the NK cells (CAR-NK cells) of above-mentioned Chimeric antigen receptor modification are transfused to patient thin When the side effects such as intracellular cytokine storm and systemic neurotoxicity, then Cetuximab is commercialized in injectable, passes through NK cells itself ADCC effects, the transformation of internal genetic engineering can be removed to express the natural killer cells of CAR.Therefore Cetuximab can be used as " switch " of Chimeric antigen receptor behavior in NK cells is realized that Chimeric antigen receptor immune response is controllable, is had a safety feature, treat Few side effects, specificity is high, can be used in anti-tumor drug.

It is specific embodiment part below.

In following embodiment, unless otherwise instructed, test method without specific conditions, usually according to normal condition, For example, see Pehanorm Brooker, EF, not the written molecular cloning of Ritchie, T Mannies A Disi etc. (Jin Dongyan, Li Mengfeng etc. are translated) is real Test (the Beijing guide [M]：Science Press, 1992) method that condition or kit manufacturer described in are recommended is realized. Reagent used in embodiment is commercially available.Unit mM indicates that mmol/L, M indicate mol/L.

Embodiment 1

1, the structure of the c-Met Chimeric antigen receptor slow virus carriers with EGFRt switches

1.1 c-Met scFv sequences

Using the monoclonal antibody sequences of the anti-c-Met of this laboratory screening, the sequence of wherein VH and VL are selected, for setting Count the scFv sequences in CAR carriers.

The 1.2 c-Met CAR sequent synthesis with EGFRt switches

According to the sequence of CAR each components, cut with scissors containing signal peptide SP, c-Met single-chain antibody (scFv), CD8 in full length sequence Sequence and transmembrane domain, 41BB intracellular domain and DAP12-ITAM structural domains, T2A, signal peptide SP, EGFRt, are collectively referred to as SP-c-Met scFv-CD8-41BB-DAP12-T2A-SP-EGFRt, structure chart are as shown in Figure 1.At entire frame 5 ' end and 3 ' Restriction enzyme site EcoR I and XhoI are added in end respectively, and commission Nanjing Genscript Biotechnology Co., Ltd. synthesizes whole table Up to frame, the sequence after synthesis is cloned in pUC57 carriers (as shown in SEQ ID No.6), and plasmid is named as pUC57-c-Met/ CAR-EGFRt。

The structure of 1.3 c-Met/CAR-EGFRt sequence Gateway entry clones

(1) pUC57-c-Met/CAR-EGFRt plasmids and Gateway entry vector pENTR 11vector use EcoR respectively I/Xho I double digestions, system are as shown in table 1 below.

Table 1：Digestion system

Plasmid or carrier	1ng
		EcoR I	1μL
Xho I	1μL
		10×Buffer	5μL
DdH is added₂O is to total volume	50μL

Digestion condition：2h is digested in 37 DEG C of water-baths.

(2) glue recycles large fragment after c-Met/CAR-EGFRt segments and 11 carrier digestions of pENTR respectively.

(3) c-Met/CAR-EGFRt segments are connected with 11 carrier segments of pENTR, and system is as shown in table 2 below.

Table 2：Linked system

10×T4 DNA ligase buffer	2.5μL
		C-Met/CAR-EGFRt segments	0.3pmol
11 carrier segments of pENTR	0.03pmol
		T4DNA ligase	1μL
DdH is added₂O is to total volume	25μL

Condition of contact：16 DEG C connect 12 hours.

(4) it takes connection product to convert into DH5 α competent bacterias, places and be incubated overnight in 37 DEG C of bacteriological incubators, picking Single bacterium colony expands culture, extracts the plasmid of positive colony, identified through digestion, and correct Gateway Entry clone plasmids are ordered Entitled pENTR-c-Met/CAR-EGFRt.

The structure of 1.4 pLenti7.3-c-Met/CAR-EGFRt slow virus expression plasmids

(1) LR reacts

LR reactions are entry clones pENTR-c-Met/CAR-EGFRt and purpose carrier pLenti7.3/V5-DEST The recombining reaction that vector occurs obtains slow virus expression plasmid pLenti7.3-c-Met/CAR-EGFRt, matter after recombination Grain spectrogram is as shown in Figure 2.

LR reaction systems are as shown in table 3 below.

Table 3：LR reaction systems

PENTR-c-Met/CAR-EGFRt (150ng/ reactions)	1 μ of μ L~7 L
		pLenti7.3/V5-DEST vector(150ng/μl)	1μL
TE Buffer, pH 8.0	Add to 8 μ L of total volume

It thaws on iceLR Clonase^TMII Plus Enzyme Mix draw 2 μ l and add to above-mentioned LR reactions In system, after soft mixing, 25 DEG C of placement 1h.1 μ L Proteinase Ks are added to above-mentioned reaction system, 37 DEG C of incubation 10min.

(2) in the conversion of LR reaction products to DH5 α competent bacterias, step is same as above.

(3) screening and amplification of positive colony

A) after dips 3 clones with pipette tips on a small quantity respectively, pipette tips is placed in 10 μ L sterile waters, are blown and beaten repeatedly.

B) draws 1 μ L bacterium solutions and is used for PCR, and reaction system and condition are as shown in table 4 below.

Table 4：The amplification reaction system of positive colony

Bacterium solution	1μL
		AttB1 primers (20 μM)	1μL
V5 reverse primers (20 μM)	1μL
		Bio-rad super mix	6.25μL
ddH₂O	15.75μL
		Total volume	25μL

Reaction condition is as shown in table 5 below.

Table 5：The amplification reaction condition of positive colony

C) PCR product of tri- clones of is reacted into row agarose gel electrophoresis, if being obtained at 2.6kb clearly single The positive colony identified is then chosen in the LB culture solutions containing ampicillin and is enlarged culture by band.

D) extracts plasmid, obtains slow virus expression plasmid pLenti7.3-c-Met/CAR-EGFRt, while preserving strain. Three clones on random picking LB tablets carry out PCR amplification with attB1 primers and V5 reverse primers, and product passes through agarose Gel electrophoresis identifies occur clearly band (Fig. 3) at 2.6kb, illustrate that c-Met/CAR-EGFRt genes are successfully plugged into In pLenti7.3/V5-DEST expression vectors.

It is prepared by the packaging of the c-Met Chimeric antigen receptor slow virus 2, with EGFRt switches

2.1 Day1：Take 5 × 10⁶A 293FT cells (Invitrogen, Cat.No.R700-07), supernatant is abandoned after centrifugation, With the complete medium of 37 DEG C of 10mL preheating (D-MEM+10%FBS+2mM L-Glutamine+0.1mM nonessential amino acids+ 1mM Sodium Pyruvates+1%P/S) it is resuspended, it is inoculated in 10cm culture dishes, 37 DEG C, 5%CO₂It is incubated overnight in incubator.

2.2Day2：The culture solution in culture dish is discarded, Opti-s of the 5mL containing 10%FBS is addedI culture solutions (Invitrogen,Cat.No.31985-062)。

2.3DNA-The preparation of 2000 compounds

(a) is by the Opti- of 1.5mL serum-freesI culture solutions are added in 5mL centrifuge tubes, and 9 μ g are added ViraPower^TMPackaging plasmid mixture and 3 μ g slow virus expression plasmid pLenti7.3-c-Met/CAR-EGFRt, it is soft mixed It closes.

(b) is by the Opti- of 1.5mL serum-freesI culture solutions are added in another 5mL centrifuge tube, and 36 μ L are added2000, it is incubated at room temperature 5min after soft mixing.

(c) solution that (a), (b) two step obtain is transferred in a centrifuge tube by, soft to be uniformly mixed.

(d) is incubated 20min at room temperature, obtains DNA-2000 compounds.

2.4 DNA- that will be obtained2000 complexes drop-wises are slowly added into culture dish, and gently Culture dish is shaken back and forth in ground.37 DEG C, 5%CO₂It is incubated overnight in incubator.

2.5 Day3：Culture dish is taken out, culture solution is discarded, 10mL DMEM complete mediums are added.37 DEG C, 5%CO₂Training It supports and is incubated 48h~72h in case.

2.6 Day5 or Day6：Culture solution in culture dish is transferred in 15mL centrifuge tubes, 2000g under the conditions of 4 DEG C Centrifuge 15min.

2.7 Aspirate supernatants are collected pLenti7.3-c-Met/CAR-EGFRt slow virus, are distributed into 1mL cryopreservation tubes, It is placed in -80 DEG C of long-term preservations.

3, the preparation of the c-Met specific C AR-NK cells with EGFRt switches

3.1 separating health volunteer's PBMC cells

(1) healthy volunteer peripheral blood 25mL, anticoagulant heparin are extracted, room temperature centrifuges (700g, 20min)；Draw upper layer blood Slurry, is placed in water-bath 56 DEG C, 30min；Then it after 4 DEG C of standing 15min, centrifuges (900g, 30min), takes 4 DEG C of guarantors of autologous plasma It deposits spare.

(2) above-mentioned 700g, 20min is taken to centrifuge rear lower cell component, D-PBS to 50mL, mixing is added to be added slowly to be equipped with In the 50mL centrifuge tubes of 20mL human lymphocyte separating liquids, room temperature centrifuges (800g, 15min).

(3) tunica albuginea confluent monolayer cells are drawn, are added in the 50mL centrifuge tubes equipped with 5mL RPMI 1640.

(4) RPMI 1640 washes twice (600g, 10min are centrifuged), and it is PBMC cells to collect cell.

The preparation of 3.2 CAR-NK cells

(1) with the monoclonal antibodies of CD16 containing 50ng/mL, 1000U/mL IL-2,100ng/mL IL-15 and 0.5% autologous plasma It is 1 × 10 that Alys505 culture solutions, which adjust PBMC cell densities,⁶/ mL is transferred in six orifice plates, and the holes 2mL/ are placed in saturated humidity, 37 DEG C, cultivate in 5.0%CO2 incubators.

(2) every 3 days adjustment cell densities are 1 × 10⁶/ mL adds IL-2 containing 1000U/mL and 0.5% autologous plasma Alys505 culture solutions.

(3) the 7th days, NK cells are divided into 2 groups, every group of cell presses 1 × 10⁵The density in/hole is transferred in 24 orifice plates, 100 holes μ L/, every group sets three multiple holes.

1st group：PLenti7.3-c-Met/CAR-EGFRt is transfected, c-Met/CAR-EGFRt groups are named as.According to MOI values =20 (ratios of MOI expressions herein viral number and cell quantity) are added sick slowly containing pLenti7.3-c-Met/CAR-EGFRt The stoste of malicious particle takes slow virus solution that Alys505 complete mediums is added to be configured to 100 μ L altogether, with pipette that slow virus is molten Liquid adds in cell, gently blows and beats mixing.

2nd group：Blank NK cell controls groups, are named as NC groups.

Every group of Polybrene for being separately added into final concentration of 6 μ g/mL, gently shakes after mixing, is placed in 37 back and forth DEG C, 5%CO₂It is incubated for 24 hours in incubator.

8th day, cell took out centrifugation, discards the supernatant containing slow virus, is resuspended, is added with 200 μ L Alys-505 culture solutions Enter in 24 orifice plates.Fluid infusion is carried out according to cell growth state.

It 10th day, harvests ripe cell and is used for subsequent analysis.

Test one：

The expression of c-Met in Western Blotting detection tumour cells

The human liver cancer cell HepG-2 and normal liver cell L-O2 for choosing exponential phase, with cell protein extracts reagent (RIPA) lytic cell extracts cell protein, carries out Western Blotting detections.The results are shown in Figure 4, liver cancer cells HepG-2 detects the expression of c-Met, and normal liver cell L-O2 can't detect the expression of c-Met.Illustrate have in liver cancer cells C-Met is expressed.

Test two：

C-Met specific C AR-NK cells in vitro antitumor activities detection with EGFRt switches

(1) the c-Met/CAR-EGFRt groups cell and NC groups cell prepared using embodiment 1 as effector cell, The HepG-2 cell and normal liver cell L-O2 of CFSE labels are as target cell, according to 20:1 effect target is thinner than melange effect Born of the same parents and target cell, gently mixing, is placed in 5%CO₂, it is incubated in 37 DEG C of incubators.

(2) after for 24 hours, 1 μ g/mL PI dye liquors, mixing is added and is examined using flow cytometer after room temperature is protected from light incubation 15min Survey CFSE⁺PI⁺The percentage of cell (dead HepG-2 cells and L-O2 cells).

Specifically, using each group cell as effector cell, with CFSE labelling liver cancer cells HepG-2 and normal liver cell L- O2, as target cell, the killing using Flow cytometry NK cells to HepG-2 cell line and normal liver cell L-O2 Efficiency, experimental result is as shown in figure 5, c-Met/CAR-EGFRt groups cell is about to the killing rate of HepG-2 cell line 72.93%, NC group cell are about 24.87%, c-Met/CAR-EGFRt group cells to just to the killing rate of HepG-2 cell line It is about 1.7% to the killing rate of normal liver cell L-O2 that the killing rate of normal liver cell L-O2, which is about 1.2%, NC groups cell,.Explanation C-Met specific C AR-NK cells prepared by embodiment 1 have efficient lethal effect to liver cancer cells, and to normal liver cell Without lethal effect.

Test three：

EGFRt switchs efficiency assay

By c-Met/CAR-EGFRt groups cell and NC groups cell respectively according to 1 × 10⁵/ hole is inoculated into six orifice plates, respectively sets 2 A group, it is set as administration group and non-administration group, administration group gives the Cetuximab (Erbitux) of 1ug/ml；After cultivating 72h, platform is expected Indigo plant dyeing calculates live cell fraction.The results are shown in Figure 6, and the c-Met/CAR-EGFRt group Cell viabilities of administration are 23.8%, no The c-Met/CAR-EGFRt group Cell viabilities of administration are 96.4%.It is 95% or more that NC groups, which are administered and motility rate is not administered,.Explanation The c-Met specific C AR-NK cells of band EGFRt switches prepared by embodiment 1 can be by giving Cetuximab (Erbitux) eventually Only it plays a role, and realizes that Chimeric antigen receptor immune response is controllable, and treatment side effect is few, and specificity is high.

Test four：

Antitumor activity detection and switch safety in c-Met specific C AR-NK multicellular animal bodies with EGFRt switches Detection

The human liver cancer HepG-2 cells of logarithmic growth phase are made single cell suspension after pancreatin digestion, will contain 1 × 10⁷It is a The suspension 0.1mL of cancer cell is subcutaneously injected in nude mice scapular region.Experiment packet and treatment see the table below, and daily each group of observing is moved The variation of the diet of object, activity etc., the next day measure nude mice weight, observe changes of weight situation；Tumour was measured every 2 days most It the case where diameter (a) vertical greatly and maximum transverse diameter (b), observation tumour growth, is calculated according to formula：Knurl accumulates=1/12 π × a × b²。 (3) organize the 13rd day tumour inhibiting rate for calculating each group nude mice after treatment end, tumour inhibiting rate=(1- treatment group tumors average external volume/the moon Property control group tumor average volume) × 100%.

Experiment packet and treatment are as shown in table 6 below.

Table 6：Experiment packet and treatment

* FAC chemotherapy regimens：Fluorouracil 500mg/M2；Adriamycin 50mg/M2；Cyclophosphamide 500mg/M2, gives for first day Medicine, 21 days courses for the treatment of.

The experimental results showed that the c-Met specific C AR-NK cells with EGFRt switches carry out internal anti-tumor experiment hair Existing, each group nude mice survives, and using negative control group nude mice as reference, chemotherapy group nude mice weight loss, gross tumor volume reduces, but into Food and activity are reduced.

C-Met/CAR-EGFRt-NK treatment groups nude mice survival condition is better, and weight is without being decreased obviously, and tumour body Significantly smaller, the inhibitory rate to 76.0% of product.And switch safety test group nude mice survival condition is compared with c-Met/CAR-EGFRt- NK treatment groups are declined, and inhibitory rate is only 10.1%.Body weights and tumor control rate statistical result are shown in Table 7.

Table 7：The comparison of each group nude mice weight, gross tumor volume and tumour inhibiting rate

The above experimental result illustrates that the c-Met specific C AR-NK cells of the band EGFRt switches prepared can effectively inhibit The growth of in-vivo tumour, and switch with validity.Cetuximab can be used as " opening for Chimeric antigen receptor behavior in NK cells Close ", it realizes that Chimeric antigen receptor immune response is controllable, has a safety feature, treatment side effect is few, and specificity is high.

One or more of embodiments of the invention above described embodiment only expresses, description are more specific and detailed Carefully, but it cannot be construed as a limitation to the scope of the present invention.It should be pointed out that for the common skill of this field For art personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to this hair Bright protection domain.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.

Sequence table

<110>Shenzhen Mo Saier biomedicine developments in science and technology Co., Ltd

<120>Chimeric antigen receptor and its expressing gene with safety switch, the NK cells of its modification and application

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 218

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 1

Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp

1 5 10 15

Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Arg Asn Tyr Leu

20 25 30

Asn Trp Tyr Gln Gln Glu Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Ile Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Val Thr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

100 105 110

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

115 120 125

Phe Ser Lys Tyr Asp Met Leu Trp Val Arg Gln Ala Pro Gly Lys Gly

130 135 140

Leu Glu Trp Val Ser Tyr Ile Tyr Pro Ser Gly Gly Leu Thr Glu Tyr

145 150 155 160

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

165 170 175

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

180 185 190

Val Tyr Tyr Cys Ala Arg Arg Ala Pro Arg Ser Leu Ser Phe Asp Ile

195 200 205

Trp Gly Gln Gly Thr Met Val Thr Val Ser

210 215

<210> 2

<211> 335

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 2

Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu

1 5 10 15

Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile

20 25 30

Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe

35 40 45

Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr

50 55 60

Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn

65 70 75 80

Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg

85 90 95

Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile

100 105 110

Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val

115 120 125

Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp

130 135 140

Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn

145 150 155 160

Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu

165 170 175

Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser

180 185 190

Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu

195 200 205

Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln

210 215 220

Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly

225 230 235 240

Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro

245 250 255

His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr

260 265 270

Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His

275 280 285

Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro

290 295 300

Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala

305 310 315 320

Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met

325 330 335

<210> 3

<211> 654

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 3

atccagatga cccagtctcc atcctccctg tctgcatctg taggagacag agtcaccatc 60

acttgtcggg caagtcagag cattaggaac tatttaaatt ggtatcagca ggaaccaggg 120

aaagccccta agctcctgat ctatgctgca tccagtttgc aaagtggggt cccatcaagg 180

ttcagtggca gtggatctgg gacagacttc attctcacca tcagcagtct gcagcctgaa 240

gattttgcaa cttactactg tcaacagagt tacgttaccc cgctcacttt cggtggtggt 300

ggttctggcg gcggcggctc cggtggtggt ggatctcagc ctggtggttc tttacgtctt 360

tcttgcgctg cttccggatt cactttctct aagtacgata tgctttgggt tcgccaagct 420

cctggtaaag gtttggagtg ggtttcttat atctatcctt ctggtggcct tactgagtat 480

gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac 540

ttgcagatga acagcttaag ggctgaggac acggccgtgt attactgtgc gagacgggct 600

ccccggtccc tttcttttga tatctggggc caagggacaa tggtcaccgt ctcc 654

<210> 4

<211> 1005

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

cgcaaagtgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60

acgaatatta aacacttcaa aaactgcacc tccatcagtg gcgatctcca catcctgccg 120

gtggcattta ggggtgactc cttcacacat actcctcctc tggatccaca ggaactggat 180

attctgaaaa ccgtaaagga aatcacaggg tttttgctga ttcaggcttg gcctgaaaac 240

aggacggacc tccatgcctt tgagaaccta gaaatcatac gcggcaggac caagcaacat 300

ggtcagtttt ctcttgcagt cgtcagcctg aacataacat ccttgggatt acgctccctc 360

aaggagataa gtgatggaga tgtgataatt tcaggaaaca aaaatttgtg ctatgcaaat 420

acaataaact ggaaaaaact gtttgggacc tccggtcaga aaaccaaaat tataagcaac 480

agaggtgaaa acagctgcaa ggccacaggc caggtctgcc atgccttgtg ctcccccgag 540

ggctgctggg gcccggagcc cagggactgc gtctcttgcc ggaatgtcag ccgaggcagg 600

gaatgcgtgg acaagtgcaa ccttctggag ggtgagccaa gggagtttgt ggagaactct 660

gagtgcatac agtgccaccc agagtgcctg cctcaggcca tgaacatcac ctgcacagga 720

cggggaccag acaactgtat ccagtgtgcc cactacattg acggccccca ctgcgtcaag 780

acctgcccgg caggagtcat gggagaaaac aacaccctgg tctggaagta cgcagacgcc 840

ggccatgtgt gccacctgtg ccatccaaac tgcacctacg gatgcactgg gccaggtctt 900

gaaggctgtc caacgaatgg gcctaagatc ccgtccatcg ccactgggat ggtgggggcc 960

ctcctcttgc tgctggtggt ggccctgggg atcggcctct tcatg 1005

<210> 5

<211> 787

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 5

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

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

35 40 45

Ser Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Glu Pro Gly Lys Ala

50 55 60

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

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ile Leu Thr Ile

85 90 95

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

100 105 110

Tyr Val Thr Pro Leu Thr Phe Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

130 135 140

Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr Asp Met Leu Trp Val Arg

145 150 155 160

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr Ile Tyr Pro Ser

165 170 175

Gly Gly Leu Thr Glu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

180 185 190

Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

195 200 205

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Ala Pro Arg

210 215 220

Ser Leu Ser Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

225 230 235 240

Pro Lys Leu Gly Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro

245 250 255

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

260 265 270

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

275 280 285

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

290 295 300

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Lys Arg Gly Arg Lys

305 310 315 320

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

325 330 335

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

340 345 350

Gly Gly Cys Glu Leu Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu

355 360 365

Ala Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln

370 375 380

Glu Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln

385 390 395 400

Arg Pro Tyr Tyr Lys Ser Pro Gly Gly Gly Ser Gly Glu Gly Arg Gly

405 410 415

Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Leu

420 425 430

Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe

435 440 445

Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe

450 455 460

Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn

465 470 475 480

Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg

485 490 495

Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp

500 505 510

Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala

515 520 525

Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile

530 535 540

Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val

545 550 555 560

Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser

565 570 575

Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn

580 585 590

Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys

595 600 605

Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val

610 615 620

Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg

625 630 635 640

Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp

645 650 655

Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser

660 665 670

Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile

675 680 685

Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr

690 695 700

Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly

705 710 715 720

Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys

725 730 735

His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu

740 745 750

Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly

755 760 765

Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly

770 775 780

Leu Phe Met

785

<210> 6

<211> 2361

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 6

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccaatcc agatgaccca gtctccatcc tccctgtctg catctgtagg agacagagtc 120

accatcactt gtcgggcaag tcagagcatt aggaactatt taaattggta tcagcaggaa 180

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 240

tcaaggttca gtggcagtgg atctgggaca gacttcattc tcaccatcag cagtctgcag 300

cctgaagatt ttgcaactta ctactgtcaa cagagttacg ttaccccgct cactttcggt 360

ggtggtggtt ctggcggcgg cggctccggt ggtggtggat ctcagcctgg tggttcttta 420

cgtctttctt gcgctgcttc cggattcact ttctctaagt acgatatgct ttgggttcgc 480

caagctcctg gtaaaggttt ggagtgggtt tcttatatct atccttctgg tggccttact 540

gagtatgctg actccgttaa aggtcgcttc actatctcta gagacaactc taagaatact 600

ctctacttgc agatgaacag cttaagggct gaggacacgg ccgtgtatta ctgtgcgaga 660

cgggctcccc ggtccctttc ttttgatatc tggggccaag ggacaatggt caccgtctcc 720

cccaagcttg gggcgaagcc caccacgacg ccagcgccgc gaccaccaac accggcgccc 780

accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 840

gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccttggcc 900

gggacttgtg gggtccttct cctgtcactg gttatcaccc tttacaaacg gggcagaaag 960

aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 1020

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gcggctggtc 1080

cctcgggggc gaggggctgc ggaggcagcg acccggaaac agcgtatcac tgagaccgag 1140

tcgccttatc aggagctcca gggtcagagg tcggatgtct acagcgacct caacacacag 1200

aggccgtatt acaaatcccc cgggggagga agcggagagg gcagaggaag tctgctaaca 1260

tgcggtgacg tcgaggagaa tcctggacct atgcttctcc tggtgacaag ccttctgctc 1320

tgtgagttac cacacccagc attcctcctg atcccacgca aagtgtgtaa cggaataggt 1380

attggtgaat ttaaagactc actctccata aatgctacga atattaaaca cttcaaaaac 1440

tgcacctcca tcagtggcga tctccacatc ctgccggtgg catttagggg tgactccttc 1500

acacatactc ctcctctgga tccacaggaa ctggatattc tgaaaaccgt aaaggaaatc 1560

acagggtttt tgctgattca ggcttggcct gaaaacagga cggacctcca tgcctttgag 1620

aacctagaaa tcatacgcgg caggaccaag caacatggtc agttttctct tgcagtcgtc 1680

agcctgaaca taacatcctt gggattacgc tccctcaagg agataagtga tggagatgtg 1740

ataatttcag gaaacaaaaa tttgtgctat gcaaatacaa taaactggaa aaaactgttt 1800

gggacctccg gtcagaaaac caaaattata agcaacagag gtgaaaacag ctgcaaggcc 1860

acaggccagg tctgccatgc cttgtgctcc cccgagggct gctggggccc ggagcccagg 1920

gactgcgtct cttgccggaa tgtcagccga ggcagggaat gcgtggacaa gtgcaacctt 1980

ctggagggtg agccaaggga gtttgtggag aactctgagt gcatacagtg ccacccagag 2040

tgcctgcctc aggccatgaa catcacctgc acaggacggg gaccagacaa ctgtatccag 2100

tgtgcccact acattgacgg cccccactgc gtcaagacct gcccggcagg agtcatggga 2160

gaaaacaaca ccctggtctg gaagtacgca gacgccggcc atgtgtgcca cctgtgccat 2220

ccaaactgca cctacggatg cactgggcca ggtcttgaag gctgtccaac gaatgggcct 2280

aagatcccgt ccatcgccac tgggatggtg ggggccctcc tcttgctgct ggtggtggcc 2340

ctggggatcg gcctcttcat g 2361

Claims

1. a kind of Chimeric antigen receptor with safety switch, which is characterized in that including sequentially connected first signal peptide, c-Met Single-chain antibody, CD8 structural domains, 41BB intracellular domain, DAP12-ITAM structural domains, T2A, second signal peptide and transformation EGFRt；Wherein, the amino acid sequence of the c-Met single-chain antibodies is as shown in SEQ ID No.1, the ammonia of the EGFRt of the transformation Base acid sequence is as shown in SEQ ID No.2.

2. the Chimeric antigen receptor according to claim 1 with safety switch, which is characterized in that it is mono- to encode the c-Met The nucleotide sequence of chain antibody is as shown in SEQ ID No.3.

3. the Chimeric antigen receptor according to claim 1 with safety switch, which is characterized in that encode the transformation The nucleotide sequence of EGFRt is as shown in SEQ ID No.4.

4. the Chimeric antigen receptor according to claim 1 with safety switch, which is characterized in that the Chimeric antigen receptor For：

(a) protein that amino acid sequence forms shown in SEQ ID No.5；Or,

(b) by replacing, missing or adding one or several amino acid and tool in the amino acid sequence shown in SEQ ID No.5 There is the active protein derived from (a) of the Chimeric antigen receptor.

5. the Chimeric antigen receptor according to claim 1 or 4 with safety switch, which is characterized in that the chimeric antigen Receptor is：

(a) the nucleotide sequence coded obtained protein shown in SEQ ID No.6；Or,

(b) by replacing, missing or adding one or several bases and can in the nucleotide sequence shown in SEQ ID No.6 Coding obtains having the active protein derived from (a) of the Chimeric antigen receptor.

6. a kind of expressing gene, for expressing the Chimeric antigen receptor with safety switch, which is characterized in that the chimeric antigen by Body includes sequentially connected first signal peptide, c-Met single-chain antibodies, CD8 structural domains, 41BB intracellular domain, DAP12-ITAM Structural domain, T2A, second signal peptide and the EGFRt of transformation；Wherein, the amino acid sequence such as SEQ of the c-Met single-chain antibodies Shown in ID No.1, the amino acid sequence of the EGFRt of the transformation is as shown in SEQ ID No.2.

7. expressing gene according to claim 6, which is characterized in that the expressing gene is：

(a) nucleotide sequence as shown in SEQ ID No.6；Or,

(b) by replacing, missing or adding one or several bases and can in the nucleotide sequence shown in SEQ ID No.6 Coding obtains having the active nucleotide sequence of the Chimeric antigen receptor.

8. a kind of expression vector, which is characterized in that contain expressing gene as claimed in claims 6 or 7 in the expression vector.

9. a kind of NK cells of the Chimeric antigen receptor modification with safety switch, which is characterized in that being capable of table in the NK cells Up to having imported such as claim 6~7 in such as Claims 1 to 5 any one of them Chimeric antigen receptor or the NK cells Expression vector as claimed in claim 8 has been transfected in any one of them expressing gene or the NK cells.

10. as Claims 1 to 5 any one of them Chimeric antigen receptor, such as claim 6~7 any one of them are expressed Gene, expression vector as claimed in claim 8 or the Chimeric antigen receptor with safety switch is repaiied as claimed in claim 9 Application of the NK cells of decorations in preparing anti-tumor drug.