CN109535260A

CN109535260A - A kind of source of people Chimeric antigen receptor and its application targeting CD46

Info

Publication number: CN109535260A
Application number: CN201811396656.4A
Authority: CN
Inventors: 陈瑞; 吴炯; 孟庆涛
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2018-11-22
Filing date: 2018-11-22
Publication date: 2019-03-29
Anticipated expiration: 2038-11-22
Also published as: CN109535260B

Abstract

The invention discloses a kind of source of people Chimeric antigen receptors for targeting CD46 gene, the source of people Chimeric antigen receptor of the targeting CD46 gene contains single-chain antibody hCD46scFV and T cell stimulus signal the transduction structure for people's CD46 antigen, wherein, the single-chain antibody hCD46scFV and other structures are connected by link peptide.The invention also discloses the expression vector of the encoding gene of the source of people Chimeric antigen receptor of targeting CD46 gene and slow virus.The invention also discloses the applications of the Chimeric antigen receptor.ScFV in the Chimeric antigen receptor has the specificity of anti-human CD46, it can be specifically bound on target antigen, the cellulotoxic effect for being directed to colorectal cancer cell is played by the use of union and recombination adenovirus, the Chimeric antigen receptor can be used in modifying T lymphocyte, and the T cell after modification is used for the oncotherapy of the surface C D46 positive.

Description

A kind of source of people Chimeric antigen receptor and its application targeting CD46

Technical field

The invention belongs to field of immunology, and in particular to a kind of source of people Chimeric antigen receptor and its application for targeting CD46.

Background technique

The T cell that autoantigen-specificity can be generated except a kind of living body is designed, the T by cancer target specificity is thin The reaction of born of the same parents' mediated immunity is the research direction of the method for oncotherapy.Currently, the specific T-cells of certified cancer target point From and transfer in treatment melanoma, achieved good clinical effectiveness in neoplastic hematologic disorder.

Chimeric antigen receptor (CAR, chimeric antigen receptor) is by exogenous gene transfer technology T cell is set to obtain new specificity.CAR is by that can identify the relevant specific antigen of tumour as a kind of synthesis of receptor ScFV (is connected to form with VH region amino acid sequence by Linker) by the VL region amino acid sequence in antibody, is passed through it and is cut with scissors In conjunction with chain structure targeting moiety relevant to having the function of one or more signal transduction region of activating T cell, make ScFV is coupled by the signal area of activation and proliferation in trans-membrane region and T cell, and the specific antigen of tumour is identified by scFV, The signal of activating T cell is generated, then feeds back to patient's body, the T cell of activation and proliferation can limit sexual norm exhibition by non-MHC Existing stronger anti-tumor capacity.

Cytoplasmic compartment region or Fc receptor of the signal transduction structural region of first generation CAR technology from CD3zeta γ chain.However, lacking T cell costimulatory signal in the CAR receptor of the first generation, cause the activation of T cell that can only play its wink Between effect, there is the deficiencies of time-to-live is shorter, secretion of cell factor is few in vivo.Base of the CAR of the second generation in the first generation The intracellular structure region that costimulatory signal molecule is increased on plinth provides two kinds of signals for capableing of activating T cell, including CD28, CD134/OX40, CD137/4-1BB etc., improve the survival rate and proliferation rate of CAR transformation T cell, and enhance The secreting function of cell factor, so as to break through immunosupress present in the microenvironment of tumour.

CAR-T is most widely used in hematological system tumor at present, and clinical efficacy is definite (CD19, CD20), but It is that clinical research on solid tumor is relatively fewer, for the treatment of solid tumor, technology that CAR-T cell therapy technology encounters Bottleneck is that specific target spot is less, and T cell can not effectively infiltrate and inside tumor inhibitive ability of immunity.

CD46 is a kind of cyclin with transmembrane structure, is marker more special in tumour.It is a variety of There are higher expression, such as bladder cancer, oophoroma, mastadenoma, colorectal cancer etc. in entity tumor.The expression intensity of CD46 and swollen The grade malignancy of tumor, Epithelial and stromal are converted and are shifted related.

Summary of the invention

Goal of the invention: technical problem to be solved by the invention is to provide a kind of source of people inosculating antibodies for targeting CD46 gene Original receptor.There is anti-human CD46 specificity in the region scFV of the Chimeric antigen receptor, is capable of the combination of specificity on target antigen, To play its be directed to colorectal cancer cell cytotoxicity, the Chimeric antigen receptor simultaneously can to T lymphocyte into Row modification, T cell can be used for the treatment of cancer cell surfaces CD46 positive tumor after modification, especially have to colorectal cancer cell There is the lethal effect of specificity.The present invention reports that CD46 specific expressed increase in colorectal cancer, the expression of CD46 are strong for the first time Degree is related to the grade malignancy of tumour, and expresses in colorectal cancer cell surface height.Therefore, CD46 is that one kind can be used for tying directly The potential target antigen of the immunization therapy of the tumours such as intestinal cancer.And the antibody of source of mouse can generate host anti-graft after directly inputting human body Reaction seriously affects the killing ability that T cell after modifying is directed to the tumour cell of the CD46 positive.

There is provided the source of people chimeric antigens of the targeting CD46 gene described in one kind for the present invention also technical problems to be solved The expression vector of the encoding gene of receptor.

There is provided a kind of source of people Chimeric antigen receptors for targeting CD46 gene for the present invention also technical problems to be solved Preparation method.

Present invention solves the technical problem that being that weight is used in combination while targeting the source of people Chimeric antigen receptor of CD46 gene Group adenovirus, chemotactic factor (CF) (the regulated on of the recombined adhenovirus Expression modulation Activated normal T cells expression and secretion Activation normal T cell expressed and secreted, RANTES, NM_002985.2) and leucocyte Jie Element -15 (interleukin 15, IL-15, NM_000585.4), RANTES and IL-15 can promote the proliferation and migration of T cell, Enhance function of the car- modification T cell in entity tumor.

There is provided the source of people Chimeric antigen receptors of targeting CD46 gene to prepare for the present invention also technical problems to be solved Application in terms of the tumor reagent of the detection CD46 positive or the tumour medicine of the treatment CD46 positive.

Also there is provided a kind of detection kits for technical problems to be solved by the present invention, and it includes the targeting CD46 bases The source of people Chimeric antigen receptor of cause.

Technical solution: to solve the above-mentioned problems, the technical solution of the present invention is to provide the targeting CD46 genes Source of people Chimeric antigen receptor contains single-chain antibody hCD46scFV and T cell stimulus signal the transduction structure for people's CD46 antigen, The T cell stimulus signal transduction structure behaviour CD8 hinge area and the transmembrane structure CD8, CD137 and people's CD3zeta intracellular region knot The structural domain that structure is composed in series, the single-chain antibody hCD46scFV are connect with CD8 hinge area by link peptide.Targeting CD46 Its structure of the source of people Chimeric antigen receptor of gene is as shown in Figure 1.

Wherein, in above-mentioned single-chain antibody hCD46scFV, the amino acid sequence of light chain such as SEQ ID NO.1 or SEQ ID Shown in NO.2, as shown in SEQ ID NO.3 or SEQ ID NO.4, the heavy chain and light chain pass through the amino acid sequence of heavy chain GGGGSGGGGSGGGGS connection.

Wherein, the source of people Chimeric antigen receptor of above-mentioned targeting CD46 gene is by the single-chain antibody for people CD46 antigen HCD46scFV, CD8 hinge area and transmembrane structure, CD137 and people's CD3zeta intracellular region structures in series form hCD46scFV- CD8Hinge-CD8membrane-CD137-CD3zeta。

Wherein, the nucleotide sequence of the encoding gene of the source of people Chimeric antigen receptor of above-mentioned targeting CD46 gene such as SEQ ID Shown in NO.5.

Wherein, the nucleotide sequence of above-mentioned link peptide is as shown in SEQ ID NO.6.

Wherein, signal peptide CD8a is contained at the 5 ' ends of above-mentioned single-chain antibody hCD46scFV, and the amino acid sequence of the signal peptide is Shown in MALPVTALLLPLALLLHAARP.

A kind of expression vector of the encoding gene of the source of people Chimeric antigen receptor containing the targeting CD46 gene.Especially It is the table containing Chimeric antigen receptor hCD46scFV-CD8 Hinge-CD8 membrane-CD137-CD3zeta encoding gene Up to carrier.

A kind of expression vector of the encoding gene of the source of people Chimeric antigen receptor containing the described targeting CD46 gene it is slow Virus.

The content of present invention further includes the expression of the encoding gene of the source of people Chimeric antigen receptor of the targeting CD46 gene The packing method of the slow virus of carrier, comprising the following steps:

1) for the acquisition of the single-chain antibody hCD46scFV of people CD46 antigen；

2) building of the anti-human slow virus plasmid of Chimeric antigen receptor hCD46-CAR；

3) packaging of slow virus, concentration and titer determination.

Wherein, the tumor reagent of the source of people Chimeric antigen receptor preparation detection CD46 positive of above-mentioned targeting CD46 gene or Treat the application in terms of the tumour medicine of the CD46 positive.It is thin that Chimeric antigen receptor of the present invention can be used in modification T lymph Born of the same parents, the T cell after modification are used for the oncotherapy of the surface C D46 positive.

Wherein, the source of people Chimeric antigen receptor of above-mentioned targeting CD46 gene is in preparation detection carcinoma of the rectum reagent or treatment knot Application in terms of rectum cancer drug.

A kind of detection kit, it includes the source of people Chimeric antigen receptors of the targeting CD46 gene.

The single-chain antibody of source of mouse can generate host anti-graft response (HAMA), seriously affect modification after being transfused human body Lethal effect of the T cell afterwards to CD46 positive tumor cell, scFV gene of the present invention for anti-mouse CD46 monoclonal antibody The frame of CDR region amino acid and source of people is combined using frame displacement and the scheme of random mutation, is constructed source of people by sequence The antibody sequence of change, and linker is added by overlap extension pcr and forms anti-human CD46 specificity scFV, the list of humanization Framework region in antibody is replaced with the amino acid sequence of source of people by chain antibody hCD46LC2scFV, can utmostly volume be avoided HAMA。

Wherein, the scFV light-chain amino acid sequence of the source of mouse CD46 monoclonal antibody is referring to the SEQ ID in sequence table NO.1 and NO.2, the amino acid sequence of heavy chain such as SEQ ID NO.3 and NO.4.

The present invention constructs Chimeric antigen receptor i.e. hCD46-CAR using anti-human CD46 single-chain antibody, and uses signal peptide CD8a guidance Chimeric antigen receptor cross-film expression connects the EGFR of truncated-type and Chimeric antigen receptor reading frame, Ke Yitong The expression for crossing detection EGFR reflects the expression efficiency of Chimeric antigen receptor indirectly.The T cell for expressing the Chimeric antigen receptor has Uniqueness can be used for the oncotherapy of the tumor surface CD46 positive with tumour-specific killing ability in clinic.

The invention also includes obtain encoding chimeric antigen receptor hCD46scFV-CD8 using genetic engineering editing technique The genetic fragment is inserted into Lentiviral, packet by the fusion of Hinge-CD8 membrane-CD137-CD3zeta Slow virus is dressed up, human T-cell is infected, T cell is made to express the Chimeric antigen receptor.

The present invention also pass through flow cytometry, LDH cytotoxicity analysis experiment, ELISA detection T cell secretion cell because Son, it was demonstrated that the T cell modified through Chimeric antigen receptor of the invention has specific cytotoxicity, Neng Goute to colorectal cancer cell The opposite sex identifies and kills the tumour cell of the surface C D46 positive, and therefore, Chimeric antigen receptor of the present invention can be applied to tie In carcinoma of the rectum immunization therapy.

The utility model has the advantages that the present invention compared with the existing technology, has the advantage that the source of people of targeting CD46 of the invention is chimeric The source of mouse frame area for having stronger immunogenicity is replaced with the amino acid sequence of source of people, avoided as far as possible by antigen receptor Host anti-graft response.Antibody after humanization has the specificity of anti-human CD46, can specifically binding on target antigen, and The cytotoxicity for colorectal cancer cell is played, can be used in the modification of T lymphocyte, T through the humanized antibody Cell can be used for the treatment of cancer cell surfaces CD46 positive tumor after modification, especially have specificity to colorectal cancer cell Lethal effect.

Detailed description of the invention

Fig. 1 is the structural schematic diagram of anti-human hCD46-CAR of the invention；

Fig. 2 is the restriction enzyme digestion and electrophoresis figure of the hCD46 antibody expression vector of the embodiment of the present invention 1；Swimming lane 1:pCAR-46-puro,

Swimming lane 2:pCAR-46-puro Digested with EcoRI/XbaI, swimming lane 3:DNA Marker；

Fig. 3 is the target egg of 1 flow cytomery hCD46LC1scFV antibody and SW620 cell surface of the embodiment of the present invention The white horizontal result figure of combination；

Fig. 4 is 1 flow cytomery hCD46LC2scFV antibody of the embodiment of the present invention and SW620 cell surface target protein In conjunction with horizontal result figure；

Fig. 5 is 3 fluorescent quantitative PCR result WPRE gene canonical plotting of the embodiment of the present invention；

Fig. 6 is 3 fluorescent quantitative PCR result ALB gene canonical plotting of the embodiment of the present invention；

Fig. 7 is that the CD46 CAR-T cell of the embodiment of the present invention 5 compares experimental result picture to SW620 cell killing effect target；

Fig. 8 is that the ELISA of the embodiment of the present invention 6 detects the level of CAR-T cell secretion of cytokines interleukin-22；

Fig. 9 is the reality that CAR-CD46 union and recombination adenovirus verifies tumor cell killing potential in lotus knurl model of the present invention Test flow chart；

Figure 10 is the detection figure that CAR-CD46 union and recombination adenovirus of the present invention is imaged in NCG mouse fluorescent vital；

Figure 11 is that CART cell of the present invention handles the mouse tumor volume diagram after NCG mouse；

Figure 12 is that CART cell of the present invention handles the mouse survival curve graph after NCG mouse；

Figure 13 is that CART cell of the present invention handles the mouse tumor histogenic immunity group result figure after NCG mouse.

Specific embodiment

The present invention will be further explained with reference to the accompanying drawing.

Acquisition of the embodiment 1 for the single-chain antibody hCD46scFV of people CD46 antigen

Humanization design is carried out by antibody of the analysis of biological information means to source of mouse, to obtain humanized antibody The sequence of hCD46scFV coding nucleotide, by purifying single-chain antibody hCD46scFV, by being verified, specifically such as Under:

1, humanized antibody sequence is designed

It is logical according to the gene order (referring to the NM_010778.4 of the genbank of NCBI) of existing Anti-CD46 source of mouse antibody Frame displacement and random mutation method are crossed, CDR region domain amino acid and source of people framework are combined, source of people is designed Change antibody hCD46scFV sequence, the amino acid sequence of light chain as shown in SEQ ID NO.1 or SEQ ID NO.2, heavy chain Amino acid sequence is as shown in SEQ ID NO.3 or SEQ ID NO.4.

2, construct humanized antibody expression vector pFUSE-hIgG1-FC1-hCD46scFV and

pFUSE-hIgG1-FC2-hCD46scFV

The nucleotide sequence information of the humanized antibody of above-mentioned design is subjected to gene chemical synthesis, is cloned respectively after synthesis To expression vector pFUSE-hIgG1-FC1 (purchase of Invivogen company) and pFUSE-hIgG1-FC2 (Invivogen company purchase Buy) in, recombinant vector send to Jin Sirui Biotechnology Co., Ltd and is sequenced after digestion identification confirmation is errorless.Carrier By digestion verification, target gene Insert Fragment size is correct as the result is shown, uses the big odd test agent of plasmid of endotoxin-free later Box, preparation transfection grade plasmid pFUSE-hIgG1-FC1-hCD46scFV and pFUSE-hIgG1-FC2-hCD46scFV.

3, the expression and purification of Humanized single chain antibody hCD46scFV

Specific step is as follows:

1) the recovery 293T cell from liquid nitrogen, using FreeStyle293 culture medium (purchase of Thermo Fisher company), Contain in 5%CO2 incubator at 37 DEG C and is cultivated.Cell was continuously passed to 5 generations.

2) the previous day for carrying out cell transfecting, fresh culture medium is replaced to cell, and cell density is adjusted to 5 × 10⁵A/mL, then be incubated overnight；

3) the plasmid pFUSE-hIgG1-FC1-hCD46scFV that 100 μM of steps 2 are prepared is taken out in -80 DEG C of refrigerators (Vector) and pFUSE-hIgG1-FC2-hCD46scFV (Vector) and polyethyleneimine (PEI), after thaw at RT according to PEI/Vector=3:1 prepares transfection composite, and plasmid dosage is adjusted to 2.5 μ g/mL, then PEI is added separately in plasmid, uses Pipettor blows and beats rapidly mixing, is stored at room temperature 15min, and transfection composite is softly added dropwise in 293TBE cell, continues to train It supports 8 hours.

4) the fresh culture FreeStyle293 culture medium of 1 times of volume is added into culture dish respectively, after shaking up overnight Culture.

5) cell is taken out by incubator, the tryptone Tryptone that concentration is 0.5% is added, is cultivated 5 days after shaking up.

6) cell being taken out by incubator, supernatant is transferred to new 50mL centrifuge tube after being centrifuged 15 minutes by 4 DEG C of 2000 × g, And the PBS buffering dilution of same volume pH7.4 is added.

7) Protein A pillar (Jin Sirui biotechnology company, article No. L00464) is taken out from 4 DEG C of refrigerators, sets flow velocity For 1mL/min, pillar is washed using 5 times of volume PBS, the supernatant in previous step is crossed into pillar later；After all crossing column, pH is used =3.0 glycine buffer gets off the antibody elution combined on pillar, and using pH=9.0 Tris buffer it is rapid in With.

8) antibody purification is used to the semi-permeable membrane dialysed overnight of 15kDa respectively, the dialysis medium used is PBS buffer solution.

9) liquid in bag filter is transferred in the centrifugal filtration column that molecular cut off is 15KDa, 3000 × g is centrifuged 10 points Clock is concentrated, and finally obtains humanized antibody hCD46LC1scFV (containing light chain 1) and hCD46LC2scFV respectively (containing light Chain 2).

4, humanized antibody hCD46LC1scFV and hCD46LC2scFV are subjected to SDS-PAGE electrophoresis

1) preparation of gel

The preparation of separation gel: 20mL10% polyacrylamide solution is prepared, will be matched using elongated head dropper after mixing The solution set adds in the gap between length glass plate, until about 8cm high, takes distilled water with 1mL syringe, along long glass wooden partition It is slowly injected into, until about 3~4mm high carries out water seal.After waiting 30min, occur different bright of refractive index between gel and water seal layer Border display lines remove water seal layer distilled water, absorb extra distilled water with filter paper item.

The preparation of glue is concentrated: preparing 10mL3% polyacrylamide solution, will be concentrated using elongated head dropper after mixing Glue is added to above the separation gel solidified, until being gently inserted into sample slot template dense at glass plate upper limb about 0.5cm In contracting glue, solidified completely after about 50min.The sample slot template for carefully taking out top, by the Tris- glycine buffer of pH=8.3 Liquid pours into storage tank up and down, prepares sample-adding.

2) processing of sample

The 100 μ L of antibody-solutions being concentrated in step 3 is taken respectively, and after 10 μ 100 × sample-loading buffers of L of addition, at 100 DEG C It is taken out after placing 5min in boiling water bath, carries out electrophoresis after cooling.

3) it is loaded

The Tris-HCl buffer (SDS for including 0.1%) that pH=8.3 is poured into slot electrode, is added in loading hole About 50 μ L samples, electric current is set as 10mA when electrophoresis initially starts；30mA is changed to after sample enters separation gel；To dye front Power supply is closed when reaching apart from silica gel frame bottom edge about 1.5cm stops electrophoresis.

4) it develops the color

Gel is removed, dyeing liquor poststaining is added and stays overnight, next day distilled water rinses for several times, is decolourized later with destainer to egg Informal voucher band is clear, learns that antibody purification band is clear, no foreign protein pollution.

5, humanized antibody hCD46LC1scFV and hCD46LC2scFV are detected using FACS

Steps are as follows:

1) recovery CHO-K1 cell (the ATCC cell bank) from liquid nitrogen, is placed in 37 DEG C of incubators containing 5%CO2 and cultivates； Cell is adjusted to logarithmic growth phase after 2 generations of continuous biography.

2) pFUSE-hIgG1-FC1-hCD46scFV and pFUSE-hIgG1-FC2-hCD46scFV matter is transfected using PEI Grain is replaced culture completely, is incubated overnight after 6 hours；

3) the culture medium F12K culture medium of serum-free, continuous culture 72 hours are replaced；

4) supernatant is taken to be incubated for altogether with SW620 cell strain, 4 DEG C 1 hour；

5) it after PBS is washed 3 times, is added PE-Anti-human IgG1 secondary antibody (Biolegend company, HP6017), is incubated for 30min；

6) it is washed cell 5 minutes using PBS, totally 3 times, later plus 500 μ L PBS are resuspended to obtain humanized antibody HCD46scFv (hCD46LC1scFV (containing light chain 1) and hCD46LC2scFV (containing light chain 2)).

7) by flow cytometry analysis, to humanized antibody hCD46scFv (hCD46LC1scFV (containing light chain 1) and HCD46LC2scFV (containing light chain 2)) it is compared, analysis result is as shown in Figure 3, Figure 4.

As seen from Figure 3, total with the antibody hCD46LC1scFV and target cell SW620 of Light chain1 (light chain 1) construction It is unobvious in conjunction with the target protein of cell surface after incubation；As seen from Figure 4, with Light chain2 (light chain 2) construction Human single chain variable fragments antibody hCD46LC2scFV can significant combination cell surface target protein.

Embodiment 2 constructs humanized antibody CD46scFV-CD137-CD3zeta slow virus plasmid

According to the humanized antibody hCD46scFV sequence information in embodiment 1, CD46scFv-CD137-CD3Zeta is constructed Lentiviral, and the preparation of carrier is carried out, steps are as follows:

The antibody sequence in conjunction with target proteins is selected by the result of the fluidic cell fluorescence sorting technology FACS of embodiment 1 (light chain2) is used as template, constructs anti-human hCD46 Chimeric antigen receptor (hCD46-CAR, abbreviation CAR, similarly hereinafter), T is thin Born of the same parents' stimulus signal transduction structure is with people CD8 hinge area and transmembrane structure, CD137 and people's CD3zeta intracellular region structures in series group At structural domain, the hCD46-CAR structure of acquisition as shown in Figure 1, its nucleotide sequence as shown in SEQ ID NO.5.

It is expressed using the cross-film of signal peptide (its nucleotide sequence is as shown in SEQ ID NO.6) guidance CAR, it will be truncated EGFR is connected with Chimeric antigen receptor reading frame, embedding so as to be reflected indirectly by the expression intensity of detection EGFR Close the expression efficiency of antigen receptor.The CAR sequence of synthesis is inserted into Lentiviral Lenti-EF1 α-puro (love health Obtain biomedical technology (Suzhou) Co., Ltd), CAR Lentiviral Lenti-EF1 α-CAR is obtained, is sent to Jin Weizhi Biotechnology Co., Ltd is sequenced, by sequencing result and the hCD46scFV-CD8 Hinge-CD8 being fitted to Membrane-CD137-CD3zeta sequence is compared, it was demonstrated that sequence is errorless.

The slow virus packaging plasmid Lenti-EF1 α-CAR plasmid of endotoxin-free is prepared, wherein the expression of CAR is opened by EF1 α Mover driving, and be attached by 2A peptide fragment and skin factor growth receptors, it include signal peptide in CAR sequence, humanization is anti- Body scFv sequence, the intracellular region of CD137 and the intracellular region of CD3zeta.It is compared with reference sequences, the Lenti- of building PCAR-46-puro (hCD46scFV-CD8 Hinge-CD8 membrane- in EF1 α-CAR plasmid Lentiviral CD137-CD3zeta), the CAR sequence of insertion is accurate.

Sequencing use primer sequence are as follows: 5'-GTGGAGACTGAAGTTAGGCCAGC-3' and

5'-CCAAGAGCAGCAGGCTCTGC-3'。

The CAR Lentiviral pCAR-46-puro of preparation is double using restriction enzyme EcoRI, XbaI progress Digestion identification, it was demonstrated that aim sequence insertion is correct in the recombinant vector.Qualification result is as shown in Figure 2.

Embodiment 3 packs slow virus, concentration and titer determination

Produce hCD46scFV-CD8 Hinge-CD8 membrane-CD137-CD3zeta slow virus (Lenti-CD46- CAR), it is ready for preparation and reorganization CAR-T cell.

1. preparing slow virus Lenti-CD46-CAR

5*10 is inoculated in 15cm Tissue Culture Dish⁶Complete medium (the DMEM high containing 10%FBS is added in a cell/ware Sugar culture-medium), 37 DEG C are placed in containing 5%CO₂Incubator be incubated overnight.

PEI (polyethyleneimine is bought in PolyScience company, article No. 23966) and slow virus are taken out from refrigerator Packaging plasmid (Lenti-EF1 α-CAR, Lenti-Mix, Ai Kang get biology, article No. LVPACKAGING50), is used after thaw at RT Liquid-transfering gun is blown and beaten mix completely up and down.PBS or HBSS buffer is taken out, is warmed to room temperature before use.Take 2mLPBS to 6 orifice plates One hole is separately added into 10 μ g Lenti-EF1 α-CAR, 11 μ g Lenti-Mix, liquid-transfering gun is blown and beaten up and down mix well after plus Enter 26 μ L, 100 μM of PEI, piping and druming is uniformly mixed so as to obtain DNA/PEI mixture up and down again with pipettor immediately, stands 10 points at room temperature Clock.

The DNA/PEI mixture of above-mentioned preparation is slowly added dropwise in 15cm culture dish, culture dish is shaked gently and fills Divide and mixes.Culture dish is placed in 37 DEG C containing 5%CO₂It is cultivated 6~8 hours in incubator, later by the culture containing transfection reagent Base discards, and is changed to fresh complete medium, culture dish is placed back in incubator and is cultivated.

The culture medium supernatant containing virus in ware is collected in continuous culture after 48 hours, carried out using 0.45 μm of filter membrane Filter, filtrate go in sterile centrifugation tube, and 4 DEG C of 50000 × g are centrifuged 2 hours.After centrifugation, carefully will in Biohazard Safety Equipment Liquid in centrifuge tube sucks, and 500 μ L PBS buffer solution are added by precipitating resuspension and obtain slow virus Lenti-CD46-CAR, will be slow Viral Lenti-CD46-CAR is placed in -80 DEG C of preservations.

2. slow virus titer determination

1) by cell culture to logarithmic growth phase after recovery HT1080 cell (ATCC cell bank) in liquid nitrogen.

2) according to 50000, every hole cell inoculation HT1080 cell in 24 orifice plates, supplementing culture medium to final volume is 500 24 orifice plates are placed in 37 DEG C containing 5%CO by μ L₂It is incubated overnight in incubator.

3) slow virus is pressed 10^-2、10^-1, 1, the amount of 10 μ L added in above-mentioned 24 orifice plate respectively, while it is 6 μ that concentration, which is added, The polybrene (polybrene, Sigma company, article No. H9268) of g/mL.

4) 24 orifice plates are reentered into 37 DEG C containing 5%CO₂In incubator, continuous culture 96 hours.

5) after cultivating, the cell in every hole is washed with PBS buffer solution, then uses Genomic DNA Purification Kit (Lifetech, CAT#K0512) extracts genomic DNA, measures mentioned base using NanoDrop2000 Because of the concentration of group DNA.

6) pUC-ALB and pUC-WPRE plasmid (Ai Kang get biology, CAT#LVTR12) is taken out from refrigerator, dilutes 10 respectively Times, standard curve sample needed for preparing quantitative fluorescent PCR.

7) PCR reaction mixture is prepared according to PCR reaction system in table 1, the sequence and fluorophor of the primer are such as Shown in table 2.5 μ L genome DNA samples or standard curve sample is added in every hole in 96 hole PCR plates, then 15 μ are added into every hole The PCR reaction mixture of the above-mentioned preparation of L, using sealer be sealed it is rear it is of short duration centrifugation about 1 minute, according to the PCR program in table 3 into Row PCR reaction.PCR after reaction, using the matched software of fluorescence quantitative PCR instrument exports the Ct value of all samples to electronics In table, the slow virus titre obtained is calculated using following slow virus titre calculation formula.The first step is by the Ct of WPRE gene Value and the log value of copy number are drawn standard curve (as shown in Figure 5) respectively as transverse and longitudinal coordinate；Then by the Ct of ALB gene and The log value of copy number is drawn standard curve (as shown in Figure 6) respectively as transverse and longitudinal coordinate.

Table 1

Table 2

Table 3

Using the slow-virus infection HT1080 cell after concentration, qPCR experiment is carried out after extracting its genomic DNA, acquisition QPCR standard curve is as shown in Figure 5 and Figure 6, and the Ct value of sample Lenti-CD46-CAR is as shown in table 4,

Table 4

As shown in Table 4, in HT1080 cellular genome it is amplifiable go out stable integration in HT1080 cellular genome Lentivirus sequences.

The copy number of Copy WPRE:WPRE gene；

The copy number of Copy ALB:ALB gene；

The cell number of Cell No:HT1080 cell；

Volume of virus: the volume of virus liquid；

It can be calculated by table 4 and above-mentioned formula, slow virus titre is 1.87 × 10⁸TU/mL。

Embodiment 4 obtains CAR-T cell

The peripheral blood lymphocytes of isolated healthy donors is further separated out T cell therein and preparation and reorganization CAR-T cell.

1, the separation of primary T cells

1) T lymphocyte that turns upside down separating liquid for several times, by Lymphoprep reagent (LymphoPrep, Axis-shield Company, article No. 07801) it mixes well.

2) in Biohazard Safety Equipment, add in 50mL centrifuge tube (or 15mL centrifuge tube, depending on separating the volume of blood sample) Enter 15mL Lymphoprep reagent, it is spare.

3) blood sample is diluted using the PBS buffer solution in equal volume containing 2% fetal calf serum FBS.

4) upper layer that separation agent is added slowly to along tube wall with liquid-transfering gun that the blood sample after dilution is careful, avoids in the process Blood sample is mixed with separation agent.

5) set most slow for centrifuge speed decrease speed, 800 × g of room temperature is centrifuged 20 minutes.

6) it after being centrifuged, collects lurid serum in upper layer and is stored in -80 DEG C into another sterile centrifugation tube.

7) will be drawn in a new centrifuge tube in the mononuclear cell layer at serum and separation agent interface gently, uses It is primary that culture medium washs cell.

8) cell density is adjusted to 1 × 10⁸/ mL (total volume is no more than 2.5mL), is resuspended in the round tube of 5mL.

9) 100 μ l/mL antibody cocktail (StemCell company) are added and mix sufficiently, are incubated at room temperature 15 minutes.

10) it is blown and beaten at least 5 times, is mixed well, and take magnetic bead spare with liquid-transfering gun.

11) magnetic bead for drawing 50 μ l/mL is mixed well into above-mentioned sample, is incubated at room temperature 10 minutes.

12) addition complete medium (Lonza company, X-VIVO15) to liquid in pipe total volume is 2.5mL, by what is uncapped Pipe is inserted into magnetic pole, is stored at room temperature 5 minutes.

13) pipe continues to stay in magnetic pole after being incubated for, and is gently inverted, pours out the primary T cells in pipe.

14) primary T cells are resuspended in containing 10%FBS, 300U/mL IL-2 (Peprotech, article No. 96-200-02- 100), 5ng/mL IL-15 (Peprotech, article No. 96-200-15-10) and 10ng/mL IL-7 (Peprotec, article No. 96- 200-07-10) in the culture medium (Lonza, 04-418Q) of X-vivo 15.

2, primary T cells and slow-virus infection are activated

Primary T cells are in unactivated state, are difficult to be infected with slow virus；It is easier to after activation by slow virus sense Dye.

1) primary T cells density is adjusted to 1 × 10⁶/ mL, antibody complex is added, and (Anti-CD3, Anti-CD28's is mixed Close object) and cell factor (300U/mLIL-2,10ng/mL IL-7,5ng/mL IL-15,500ng/mL Anti-CD3,2 μ G/mL Anti-CD28), it is continuous later to cultivate 48 hours.

2) virus quantity needed for calculating by MOI=20, formula are as follows:

Required virus quantity (mL)=(MOI* cell quantity)/virus titer

3) melt in 37 DEG C of water-baths rapidly after taking out slow virus prepared by embodiment 3 from -80 DEG C of refrigerators.In six orifice plates It is middle that the above-mentioned resulting virus quantity of calculating is added, it is mixed well after adding the polybrene of 6 μ g/mL, using sealed membrane by six orifice plates The sealing of four sides, 800 × g are centrifuged 1 hour.

4) after being centrifuged, sealed membrane is torn, six orifice plates are placed in 37 DEG C containing 5%CO₂Incubator in continue culture 24 Hour.

5) 250 × g is centrifuged 10 minutes, removes the culture medium supernatant containing virus, cell is resuspended with fresh culture, by its turn It moves in six new orifice plates and continues culture 3 to 6 days, obtain CAR-T cell.

Cracking of the 5 CAR-T cell of embodiment to target cell

By the co-cultivation of CAR-T cell and target cell SW620 cell, CAR-T cell is assessed to the killing energy of target cell Power.

1) the target cell SW620 cell tumour cell of CD46 (expression) is adjusted to logarithmic growth phase, and before the experiment Cell needs continuous passage 2 times；

2) digestion of adherent target cell is resuspended in complete medium with pancreatin (Thermo company, 25300054), adjustment is thin Born of the same parents' density is to 5 × 10⁵A/mL is inoculated with target cell by the amount in 100 holes μ L/ in 96 new orifice plates.The 96 unused holes of orifice plate surrounding 100 μ L sterile waters are added, prevent moisture in experimental port from evaporating.96 orifice plates are placed in containing 5%CO₂It is trained overnight in 37 DEG C of incubators It supports.

3) the CAR-T cell prepared in embodiment 4 is collected by centrifugation, is resuspended using 1640 culture medium of serum-free；From incubator 96 orifice plates of middle taking-up, culture medium in hole are sucked out, respectively according to E/T ratio after target cell is gently washed one time with sterile PBS The CAR-T cell being resuspended is added in (ratio of effector cell and target cell) 1:1,5:1,10:1, and final volume is mended to 100 μ L/ Hole；The same amount of target cell of inoculation in the hole Maxi lysis and Mini lysis (maximum cracking hole and minimum cracking hole), but CAR-T cell is not added.Orifice plate is placed in 37 DEG C of incubators containing 5%CO2 and is cultivated 6 hours,

4) 96 orifice plates are taken out after cultivating, LDH detection kit (Promega, G1780) is added to the hole Maxi lysis In lysate, 96 orifice plate 5 minutes will be centrifuged by 1200 × g of room temperature after wherein target cell cracks completely, after taking out plate gently From 50 μ L are shifted in every hole into another 96 new orifice plate, LDH detection reagent is added, OD value is detected by microplate reader.

5) target cell lysis rate calculation formula:

Each parameter meaning is as follows in above-mentioned formula:

Cracking percentage of the Lysis%:CAR-T cell to target cell；

ODeach well: the OD value of every sample well；

ODmini lysis: the OD value of blank control wells；

ODmaxi lysis: the OD value of maximum cracking control wells；

6) above-mentioned target cell lysis data are mapped using GraphPad 6.0.

As a result as shown in fig. 7, as shown in Figure 7, when target effect is than being 10:1, there are one to target cell for the T cell of untransfected Determine the killing of degree non-specificity, cleavage rate 30%, and CAR-T cell of the invention is capable of the identification of specificity and to kill target thin Born of the same parents, for cleavage rate up to 90% or more, cleavage rate increases about 300%, and therefore, CAR-T cell of the invention can be to target cell Significant lethal effect is generated, and as effect target is than improving, lethal effect is become apparent.

The detection of 6 CAR-T cell secretion of cytokines interleukin-22 level of embodiment

Steps are as follows:

1) the target cell SW620 cell tumour cell of CD46 (expression) is adjusted to logarithmic growth phase, and before the experiment Cell needs continuous passage 2 times.

2) digestion of adherent target cell is resuspended in complete medium, adjustment cell density to 5 × 10 with pancreatin⁵A/mL, Target cell is inoculated with by the amount in 100 holes μ L/ in 96 new orifice plates.100 μ L sterile waters are added in the unused hole of 96 orifice plate surroundings, prevent reality Middle moisture of verifying evaporates.96 orifice plates are placed in 5%CO₂, it is incubated overnight in 37 DEG C of incubators.

3) the CAR-T cell prepared in embodiment 4 is collected by centrifugation, is resuspended using 1640 culture medium of serum-free；From incubator 96 orifice plates of middle taking-up, culture medium in hole are sucked out, according to the E/T of embodiment 5 after target cell is gently washed one time with sterile PBS The CAR-T cell being resuspended is added in ratio, and final volume is mended to 100 holes μ L/；Orifice plate is placed in 37 DEG C containing 5%CO₂In incubator Culture 6 hours, while control T cell group is set.

4) 96 orifice plates are taken out after cultivating, 1200 × g of room temperature is centrifuged 96 orifice plate 5 minutes, gently takes out plate, from every hole 50 μ L culture medium supernatants of middle transfer are passed through using the expression of ELISA kit (IL2ELISA kit, R&D, D2050) detection IL-2 Microplate reader detects OD value, as a result as shown in Figure 8.

As seen from Figure 8, CAR-T cell generates a large amount of IL-2 after co-culturing with target cell, and with target cell amount It improves, IL-2 secretion level significantly increases, and dosage effect is presented with the increased ratio of target cell, and it is thin to target to compare T cell Although born of the same parents have certain non-specific killing, its IL-2 secretion level is substantially less than CAR-T cell, and without obvious dosage Effect relation.Therefore, the ability of recombinant C AR-T cell of the invention secretion IL-2 significantly improves.

7 RANTES-IL15 of embodiment is overexpressed recombined adhenovirus

RANTES-IL15 is overexpressed recombinant adenovirus toxic action: stimulation T cell is proliferated, and promotes T cell transfer.

1, RANTES-IL15 shuttle vector constructs

According to RANTES (NM_002985.2) and IL15 (NM_000585.4) reference sequences information, the area overall length ORF is synthesized, And be subcloned into adenovirus shuttle vector pShuttle (Ai Kang get biology, VP1225), obtain pShuttle- RANTESIRES-IL15, and prepare the plasmid of transfection rank.

2. prepared by recombined adhenovirus skeleton carrier

Based on the shuttle vector of adenovirus of above-mentioned preparation, in conjunction with adenoviral backbone plasmid pADBackbone, lead to Vitro recombination is crossed, recombinant adenovirus plasmid is constructed.The big pumping of plasmid is carried out to the expression vector of above-mentioned preparation, preparation transfection rank Plasmid.

3. prepared by recombined adhenovirus

It uses PacI to carry out linearization for enzyme restriction respectively the recombinant adenoviral vector of above-mentioned preparation, uses liposome after purification Transfection is into 293A cell (Thermo, R70507), after CPE phenomenon (cytopathic effect inhibition assay) occurs in cell, collects cell, and Prepare adenovirus seed.Based on this seed virus, extensive adenovirus preparation is carried out.

4. the purifying of recombined adhenovirus

The recombinant adenoviral vector of above-mentioned preparation is purified using pillar respectively, carries out buffer exchange using PBS, And (10 are measured to the virus titer finally obtained¹⁰Recombined adhenovirus, PBS are resuspended).

Killing ability of the CAR-CD46 union and recombination adenovirus to tumour cell in 8 lotus knurl model of embodiment

Animal species: NOD-prkdcem26Cd52Il2rgem26Cd22/Nju (NCG) mouse

Experiment flow such as Fig. 9: CAR-CD46 group: the mouse bilateral oxter NCG inoculates colorectal cancer cell SW620 (Luciferase ATCC cell bank) 5 × 10⁵A/side/only, the 10th day, the side intratumor injection 30ul//recombined adhenovirus RANTES-IL15, stimulation T cell are proliferated and T cell are promoted to shift, and tail vein injection infection CAR-CD46 virus is within the 13rd day The T cell 5 × 10 of Lenti-CD46-CAR virus⁶A/only, the side 30ul//recombined adhenovirus is equally injected again within the 18th day RANTES-IL15 carries out the CAR-T cell infusion of second of isodose on the 21st day, from the 7th day, 12 days, 17 days, carries out within 23 days The detection (Figure 10) of toy fluorescent vital imaging, while concurrent control group is set: it is straight that the mouse bilateral oxter NCG inoculates knot Colon-cancer cell SW620 (Luciferase ATCC cell bank) 5 × 10⁵A/side/only, the 10th day, the side intratumor injection 30ul//only Recombined adhenovirus RANTES-IL15, stimulation T cell are proliferated and T cell are promoted to shift, the 13rd day tail vein injection uninfecting virus Control T cell 5 × 10⁶A/only, the side 30ul//recombined adhenovirus RANTES-IL15 is equally injected again within the 18th day, The control T cell injection for carrying out the uninfecting virus of second of isodose on the 21st day, from the 7th day, 12 days, 17 days, 23 days Carry out the detection (Figure 10) of toy fluorescent vital imaging.The results show that the fluorescence intensity of CAR-CD46 group mouse is significantly lower than Control group, display CAR-CD46 have more significant killing inhibiting effect to the tumour cell in lotus knurl model.It is swollen by detecting The tumor speed of growth finds the tumor volume growth of CAR-CD46 group mouse slowly (Figure 11) compared with control group, and CAR-CD46 group mouse Life cycle be obviously prolonged (Figure 12).Immunohistochemistry (anti-CD31 is done to the tumor tissues of different groups of NCG mouse Antibody, ab28364, Abcam；Anti-CD3, ab5690, Abcam；Anti-caspase3, ab13847, Abcam； Anti-ki67 antibody, ab15580, Abcam) (CD31 is higher, and tumor blood vessels generate more；CD3 prompts T cell Infiltration；Caspase3 is higher, and apoptosis of tumor cells is more；Ki67 is higher, and tumor cell proliferation is faster), as the result is shown (referring to figure 13), compared with the control group, occurs T cell infiltration in the tumor tissues of CAR-CD46 group mouse, apoptosis increases, and is proliferated less, blood Pipe, which generates, to be reduced.Figure 13 further illustrates that CAR-CD46 has more significant killing to inhibit to make the tumour cell in lotus knurl model With.

The present invention provides one by screening and the closely related marker for having treatment potentiality of colorectal cancer occurrence and development It plants and recombinates Chimeric antigen receptor gene C D46, after the virus for transfecting recombinant C AR gene and the carrier containing recombinant C AR gene T cell have apparent fragmentation effect to the tumour cell of high expression CD46.In invention, experimenter is by adjusting test side Recombined adhenovirus is first injected into mouse tumor tissue and (promotes T cell proliferation and transfer), then further injects CART by case Cell, the therapeutic effect of the promotion CART cell of potentiality.NCG mouse is IL2rg knock out mice, lacks mature T, B It is that internationally recognized immune deficiency degree is high at present, is suitble to the tool mouse of human archeocyte or tissue transplantation with NK cell.

There is exogenous T in the mouse tumor tissue of CART group Showed by immune group result in the present invention, CAR-CD46 group The infiltration of cell, and control group further illustrates the T cell energy that the CAR-CD46 group of recombined adhenovirus is used in combination without this phenomenon Effectively infiltration tumor tissues, to realize fragmentation effect, the target spot specificity that can effectively improve in treatment of solid tumors is poor, and T is thin Born of the same parents can not effectively infiltrate and the shortcomings that inside tumor inhibitive ability of immunity, can not effectively apply in solid tumor with other current schemes It is contrasted.

The above is only a preferred embodiment of the present invention, it should be pointed out that: for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Sequence table

<110>Southeast China University

<120>a kind of source of people Chimeric antigen receptor and its application for targeting CD46

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Gln Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Gly His Ser

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Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

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

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Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Ser Gln Ser

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Thr His Val Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

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Lys

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Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

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

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accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag 180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct 240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag 300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga 360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt 420

ggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact 480

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc 540

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact 600

tactactctt catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag 660

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag 720

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc 780

gtgtccagca ccactacccc agcaccgagg ccacccaccc cggctcctac catcgcctcc 840

cagcctctgt ccctgcgtcc ggaggcatgt agacccgcag ctggtggggc cgtgcatacc 900

cggggtcttg acttcgcctg cgatatctac atttgggccc ctctggctgg tacttgcggg 960

gtcctgctgc tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg 1020

tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga ggacggctgt 1080

tcatgccggt tcccagagga ggaggaaggc ggctgcgaac tgcgcgtgaa attcagccgc 1140

agcgcagatg ctccagccta caagcagggg cagaaccagc tctacaacga actcaatctt 1200

ggtcggagag aggagtacga cgtgctggac aagcggagag gacgggaccc agaaatgggc 1260

gggaagccgc gcagaaagaa tccccaagag ggcctgtaca acgagctcca aaaggataag 1320

atggcagaag cctatagcga gattggtatg aaaggggaac gcagaagagg caaaggccac 1380

gacggactgt accagggact cagcaccgcc accaaggaca cctatgacgc tcttcacatg 1440

caggccctgc cgcctcggga gggcagaggc agcctgctga catgtggcga cgtggaagag 1500

aaccctggcc ccatgtggct gcagagcctg ctgctcttgg gcactgtggc ctgcagcatc 1560

tctcgcaaag tgtgtaacgg aataggtatt ggtgaattta aagactcact ctccataaat 1620

gctacgaata ttaaacactt caaaaactgc acctccatca gtggcgatct ccacatcctg 1680

ccggtggcat ttaggggtga ctccttcaca catactcctc ctctggatcc acaggaactg 1740

gatattctga aaaccgtaaa ggaaatcaca gggtttttgc tgattcaggc ttggcctgaa 1800

aacaggacgg acctccatgc ctttgagaac ctagaaatca tacgcggcag gaccaagcaa 1860

catggtcagt tttctcttgc agtcgtcagc ctgaacataa catccttggg attacgctcc 1920

ctcaaggaga taagtgatgg agatgtgata atttcaggaa acaaaaattt gtgctatgca 1980

aatacaataa actggaaaaa actgtttggg acctccggtc agaaaaccaa aattataagc 2040

aacagaggtg aaaacagctg caaggccaca ggccaggtct gccatgcctt gtgctccccc 2100

gagggctgct ggggcccgga gcccagggac tgcgtctctt gccggaatgt cagccgaggc 2160

agggaatgcg tggacaagtg caaccttctg gagggtgagc caagggagtt tgtggagaac 2220

tctgagtgca tacagtgcca cccagagtgc ctgcctcagg ccatgaacat cacctgcaca 2280

ggacggggac cagacaactg tatccagtgt gcccactaca ttgacggccc ccactgcgtc 2340

aagacctgcc cggcaggagt catgggagaa aacaacaccc tggtctggaa gtacgcagac 2400

gccggccatg tgtgccacct gtgccatcca aactgcacct acggatgcac tgggccaggt 2460

cttgaaggct gtccaacgaa tgggcctaag atcccgtcca tcgccactgg gatggtgggg 2520

gccctcctct tgctgctggt ggtggccctg gggatcggcc tcttcatg 2568

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<213>artificial sequence (Artificial Sequence)

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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

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His Ala Ala Arg Pro

20

Claims

1. a kind of source of people Chimeric antigen receptor for targeting CD46 gene, which is characterized in that the source of people of the targeting CD46 gene is embedding Conjunction antigen receptor contains single-chain antibody hCD46scFV and T cell stimulus signal the transduction structure for people's CD46 antigen, the T Cytositimulation signal transduction structure behaviour CD8 hinge area and the transmembrane structure CD8, CD137 and people's CD3zeta intracellular region structures in series The structural domain of composition, the single-chain antibody hCD46scFV are connect with CD8 hinge area by link peptide.

2. the source of people Chimeric antigen receptor of targeting CD46 gene according to claim 1, which is characterized in that described single-stranded anti- In body hCD46scFV, the amino acid sequence of light chain is as shown in SEQ ID NO.1 or SEQ ID NO.2, the amino acid of heavy chain As shown in SEQ ID NO.3 or SEQ ID NO.4, the heavy chain is connected with light chain by GGGGSGGGGSGGGGS sequence.

3. the source of people Chimeric antigen receptor of targeting CD46 gene according to claim 1, which is characterized in that the targeting The nucleotide sequence of the encoding gene of the source of people Chimeric antigen receptor of CD46 gene is as shown in SEQ ID NO.5.

4. the source of people Chimeric antigen receptor of targeting CD46 gene according to claim 1, which is characterized in that the link peptide Nucleotide arrange as shown in SEQ ID NO.6.

5. the source of people Chimeric antigen receptor of targeting CD46 gene according to claim 1, which is characterized in that described single-stranded anti- Signal peptide CD8a is contained at the 5 ' ends of body hCD46scFV, and the amino acid sequence of the signal peptide is MALPVTALLLPLALLLHAARP institute Show.

6. a kind of expression vector of the encoding gene containing the source of people Chimeric antigen receptor for having the right to target CD46 gene described in 5.

7. a kind of expression of the encoding gene of the source of people Chimeric antigen receptor containing targeting CD46 gene as claimed in claim 6 carries The slow virus of body.

8. the slow disease of the expression vector of the encoding gene of the source of people Chimeric antigen receptor of targeting CD46 gene as claimed in claim 7 The packing method of poison, which comprises the following steps:

2) building of Chimeric antigen receptor hCD46-CAR slow virus plasmid；

3) packaging of slow virus, concentration and titer determination.

9. targeting tumour of the source of people Chimeric antigen receptor in the preparation detection CD46 positive of CD46 gene described in claim 1 ~ 5 Application in terms of reagent or the tumour medicine of the treatment CD46 positive.

10. targeting the source of people Chimeric antigen receptor of CD46 gene described in claim 1 ~ 5, expression as claimed in claim 6 carries Body, application of the slow virus as claimed in claim 7 in preparation detection carcinoma of the rectum reagent or in terms for the treatment of colorectal cancer drug.