CN110423767A

CN110423767A - Express Chimeric antigen receptor CAR gene and the application of solubility PD-1

Info

Publication number: CN110423767A
Application number: CN201910631533.2A
Authority: CN
Inventors: 江文正; 王熙; 何聪; 刘明耀; 席在喜
Original assignee: Shanghai Bioray Biotechnology Co Ltd; East China Normal University
Current assignee: Shanghai Bioray Biotechnology Co Ltd; East China Normal University
Priority date: 2019-07-12
Filing date: 2019-07-12
Publication date: 2019-11-08

Abstract

This application discloses a kind of Chimeric antigen receptor CAR gene for expressing solubility PD-1 and applications.The CAR gene includes the antigen-binding domains gene being successively linked in sequence, transmembrane structure domain gene, intracellular domain gene and solubility PD-1 gene, the solubility PD-1 is the extracellular fragment region of PD-1, preferably, the solubility PD-1 includes amino acid sequence shown in SEQ ID No.1.The application can make CAR-T cell in tumor microenvironment endocrine sPD-1, through sPD-1 in conjunction with tumor cell surface PD-L1, Reverse transcriptase is in conjunction with CAR-T cell surface trans-membrane PD-1, to avoid CAR-T cell from being suppressed, the killing therapeutic effect for improving CAR-T cells against tumor cells, reaches to tumour especially treatment of solid tumors purpose.The application provides a kind of new way to improve CAR-T cell killing tumour cell effect.

Description

Express Chimeric antigen receptor CAR gene and the application of solubility PD-1

Technical field

The present invention relates to biomedicine fields, and in particular to a kind of Chimeric antigen receptor CAR base for expressing solubility PD-1 Cause and application.

Background technique

Tumour is a kind of disease for seriously threatening human life, and treatment method is constantly subjected to the height of medicine and biological educational circles Degree concern.In recent years, it deepens continuously with people to tumour immunity microenvironment understanding, a kind of new approaches that treating tumour are gradually The visual field of people is entered into, i.e. " immunotherapy of tumors ", 2013, U.S. FDA was called " breaking through sex therapy ", it is sufficient to see that its is important Property, and Chimeric antigen receptor T cell treatment (CAR-T) is one of the immunotherapy of tumors of current most prospect.

Chimeric antigen receptor CAR is generally by an antigen binding domain/extracellular region/transmembrane region and one after combining antigen It is capable of the intracellular signal district's groups of activating T cell at the more normal T cell of CAR-T cell has higher potential, because it is through one kind Non- MHC molecule dependence, specific, single signal pathway activation.Preclinical and clinical test some further investigations Display CAR-T therapy achieves encouraging therapeutic effect in treating to kinds cancer, is especially controlled with CD19-CAR-T Treat B cell malignant tumour.Currently, CAR-T cellular immunotherapy malignant hematologic disease has obtained significant clinical response.However, with The CAR-T cell therapy solid tumor immunosuppressive environment powerful by solid tumor mass structure is limited, the missing of ideal targets It is also another crucial deficiency for the treatment of solid tumor.Therefore more effective target spot is found to promote CAR-T cell therapy real Body tumor is a big significant challenge at this stage.

PD-1 is programmed death receptor 1, is a kind of important immunosuppression molecule, is CD28 superfamily member.PD-1 Belong to Ι type transmembrane protein, be made of extracellular fragment, transmembrane anchor area and intracellular signal transduction area three parts, main inducing expression in The T lymphocyte of activation, bone-marrow-derived lymphocyte surface.The ligand of PD-1 is B7-H1 (PD-L1) and B7-DC (PD-L2), and the two combines Inhibit T cell Proliferative Activated afterwards, down regulation is risen to t cell response, belongs to Inhibitory receptor.Tumor cell surface is often expressed PD-L1 inhibits T cell Proliferative Activated in conjunction with the PD-1 on T cell surface, realizes immunologic escape.Have currently on the market PD-1 and PD-L1 antibody blocks tumour cell to the inhibiting effect of T cell.In addition to there are Transmembrane binding prot eins PD- in human body 1, there is also the extracellular Ig V-Ig C spline structure domains that soluble PD-1 (sPD-1), sPD-1 remain Model Molecule, can combine In ligand PD-L1 and PD-L2.The initial research in relation to sPD-1 is mostly related to autoimmune disease, and such as rheumatoid arthritis is The autoimmune diseases such as system property lupus erythematosus, myasthenia gravis are proved to related with sPD-1, and sPD-1 is also considered as certainly In body immunological diseases, generated under the action of proinflammatory disease stimulating factor by immunocyte.Equally, sPD-1 has in conjunction with PD-L1 Ability, play inhibit the active function of T cell.

It is killed currently, there is no soluble PD-1 and Chimeric antigen receptor CAR co-expressing raising CAR-T cells against tumor cells The product of overstrain.

Summary of the invention

In view of the deficiencies in the prior art, the purpose of the present invention is to provide a kind of the chimeric of expression solubility PD-1 Antigen receptor CAR gene and application, the present invention can make CAR-T cell in tumor microenvironment endocrine sPD-1, by sPD-1 with Tumor cell surface PD-L1 is combined, and Reverse transcriptase is in conjunction with CAR-T cell surface trans-membrane PD-1, to avoid CAR-T cell It is suppressed, improves the killing therapeutic effect of CAR-T cells against tumor cells, reach to tumour especially treatment of solid tumors purpose.

To achieve the above objectives, the technical solution adopted by the present invention is that:

Present invention firstly provides a kind of Chimeric antigen receptor CAR gene for expressing solubility PD-1, the CAR gene packets Include antigen-binding domains (the i.e. one section of extracellular single chain antigen variable region fragment scFV) gene, transmembrane structure being successively linked in sequence Domain gene, intracellular domain gene and solubility PD-1 gene, the solubility PD-1 are the extracellular fragment region of PD-1,

Preferably, the solubility PD-1 includes amino acid sequence shown in SEQ ID No.1,

It is furthermore preferred that the sequence of the solubility PD-1 gene is as shown in 1264-1713 in SEQ ID No.2.

A kind of preferred embodiment is connected between the intracellular domain gene and the solubility PD-1 gene First signal peptide gene is expressed so that the extracellular fragment of PD-1 efficiently expresses and is secreted into extracellular rather than cross-film,

Preferably, first signal peptide be following albumen signal peptide: CD19, CD20, CD30, CD4, CD8a, CD28, CD137 or combinations thereof；

It is furthermore preferred that first signal peptide be CD8a signal peptide, amino acid sequence as shown in SEQ ID No.3, It is furthermore preferred that its gene order is as shown in 1201-1263 in SEQ ID No.2.

A kind of preferred embodiment is connected between the intracellular domain gene and first signal peptide gene Self cleavage protein gene, it is preferred that the self cleavage albumen is selected from T2A, P2A, E2A, F2A or combinations thereof, it is furthermore preferred that described Self cleavage albumen is T2A.

The antigen-binding domains can be specifically bound with some particular ligand of tumor cell surface, in the application In, some described particular ligand is specially NKG2D ligand, it is preferred that the antigen-binding domains are that NKG2D receptor is extracellular Section, it is furthermore preferred that the front end of the antigen-binding domains gene includes second signal peptide gene；

Preferably, the second signal peptide be following albumen signal peptide: CD19, CD20, CD30, CD4, CD8a, CD28, CD137 or combinations thereof；It is furthermore preferred that the second signal peptide is the signal peptide of CD8a, it is furthermore preferred that its amino acid sequence is such as Shown in SEQ ID No.3, it is furthermore preferred that its gene order is as shown in 1-63 in SEQ ID No.2；

NKG2D receptor has the cell surface of structural homology in a manner of a kind of " mixing " with a kind of and MHC I albuminoid Glycoprotein family combines.It is a kind of activated receptor mainly expressed on cytotoxin immunocyte, can induce and directly kill Extremely express the target cell of the ligand of its stress-induced.The ligand of NKG2D includes six members: MICA, MICB, ULBP1-6.DNA Damage, oncogene activation, hyper-proliferative etc. are the important symbols that cancer occurs, and the NKG2D ligand level of cell surface is significant It increases, and normal cell surface low expression these ligands.Therefore NKG2D receptor extracellular fragment is designed into CAR structure, when with match When body combines, T cell will be activated, generates a series of antitumor reaction.

A kind of preferred embodiment, the transmembrane domain include the transmembrane region of following albumen: CD3 ε, CD4, CD8a, CD9, CD16, CD22, CD33, CD137, CTLA-4, PD-1, LAG-3 or combinations thereof, it is preferable that CD8a；

A kind of preferred embodiment, the intracellular domain include costimulatory signal molecule and endochylema signal transduction sequence Column；Preferably, the costimulatory signal molecule be selected from following albumen costimulatory signal molecule: OX40, CD28, CD30, CD40, CD70, CD134,4-1BB (CD137), PD1, Dap10, CDS, ICAM-1 or combinations thereof, it is further preferred that 4-1BB；Preferably, described Endochylema signal transduction sequence includes the endochylema signal transduction sequence derived from CD3 ζ.

A kind of preferred embodiment, the Chimeric antigen receptor CAR gene order such as SEQ of the expression solubility PD-1 Shown in ID No.2.

On the other hand, the present invention also provides a kind of recombinant vector, the recombinant vector contains the CAR of any description above Gene, the recombinant vector are preferably slow virus carrier,

In a preferred embodiment, the slow virus carrier that the present invention uses is pCDH-NKG2D-sPD-1- CAR, the carrier connect CAR shown in SEQ ID No.2 for I restriction enzyme site of Sal I and EcoR in Lentiviral pCDH Gene.

On the other hand, the present invention also provides a kind of recombinant cell or recombinant bacterium, the recombinant cell or the recombinant bacterium In the CAR gene containing any description above, or contain the recombinant vector,

The recombinant bacterium is engineering bacteria, the engineering bacteria concretely Escherichia coli,

Preferably, the recombinant cell is immunocyte, it is furthermore preferred that the recombinant cell is CAR-T cell.

On the other hand, the present invention also provides the CAR gene of any description above, the recombinant vector, the recombination are thin The application of born of the same parents or the recombinant bacterium in preparation oncotherapy product, the product concretely reagent, kit or drug.

In above-mentioned application, the oncotherapy product is that the tumour that selective killing tumor cytotoxicity effect improves is controlled Treat product.

In above-mentioned application, the tumour is solid tumor, it is preferable that osteosarcoma U2OS, osteosarcoma MG-3 or kidney cancer cell 786O, it is further preferred that osteosarcoma U2OS.

On the other hand, the present invention also provides a kind of methods for improving selective killing tumor cytotoxicity effect, including The step of applying any description above CAR gene, the recombinant vector or the recombinant cell to target cell.

In the above-mentioned methods, the target cell is the tumour cell for expressing PD-L1, it is preferred that the target cell derives from Osteosarcoma U2OS, osteosarcoma MG-3 or kidney cancer cell 786O, it is furthermore preferred that the target cell derives from osteosarcoma U2OS, it can High expression PD-L1 and NKG2D ligand simultaneously.

In the above-mentioned methods, the effect target ratio of the application is 1:0.5-5, it is preferable that 1:1-4, it is further preferred that 1:3.

It is described to rise to relative to being not connected with the solubility PD-1 gene in CAR gene in above-mentioned application and method Or the case where solubility PD-1, is not expressed or secreted to CAR-T cell.

Beneficial effects of the present invention are as follows:

CAR-T technology is the important way of current treatment tumour cell, however with CAR-T cell therapy solid tumor by reality Body tumor tissue structure, powerful immunosuppressive environment are limited, and the missing of ideal targets is also another for the treatment of solid tumor It is crucial insufficient.Therefore finding more effective target spot to promote CAR-T cell therapy solid tumor is that one at this stage big important chooses War.Soluble PD-1 is introduced CAR-T cell by the invention, is made it have the function of self secretion PD-1, is played drop The effect that low tumour cell inhibits CAR-T cell.Compared to PD-1 antibody, soluble PD-1 has stronger advantage and effect Fruit, first solubility PD-1 can avoid T cell activity inhibited in conjunction with the PD-L1 of tumor cell surface；Next is compared PD-1 antibody, soluble PD-1 is the effect of lower content can also keep persistent high efficiency, therefore brought by solubility PD-1 Side effect also can be lower than PD-1 antibody；Finally, due to which PD-1 antibody needs continuous injection, therefore the low dose of solubility PD-1 With higher cost performance.

The present invention adds PD-1 extracellular fragment sequence on NKG2D-CAR-T plasmid and then CAR-T cell is transformed, and has it The function of solubility PD-1 is secreted, and can achieve the purpose that reducing solid tumor cell inhibits CAR-T cell.It is solvable with not expressing The NKG2D-CAR-T cell of property PD-1 is compared, the fragmentation effect of improved NKG2D-sPD-1-CAR-T cells against tumor cells It is the 2 times or more of former NKG2D-CAR-T cell.

Detailed description of the invention

Fig. 1 is CAR member in carrier pCDH-NKG2D-CAR (above) and carrier pCDH-NKG2D-sPD-1-CAR (following figure) The structural schematic diagram of part, wherein NKG2Dex is that front end contains signal peptide (signal peptide sequence is imitated in existing α CD19-CAR Fruit is significant) sequence expression NKG2D receptor extracellular fragment element, CD8a TM be expression PROTEIN C D8a transmembrane region element, 4- 1BB is the element for expressing the costimulatory signal molecule of albumen 4-1BB, and CD3 ζ is the member for expressing CD3 ζ endochylema signal transduction sequence Part, T2A are that (self cleavage albumen is being transferred to cell with the gene of T2A connection to the 2A element from any brown wing moth viral (TaV) After normally can translate and co-express), sPD-1 be express solubility PD-1 albumen element.

Fig. 2 is the electrophoresis result of colony PCR amplification sPD-1.Wherein, sample 1,3,5,6,8 is the positive, remaining is feminine gender.

Fig. 3 is the structural schematic diagram of carrier pCDH-NKG2D-sPD-1-CAR.

Fig. 4 is flow cytometer detection CAR-T cell positive rate result after the dyeing of APC-hNKG2D antibody.

Fig. 5 is the RT-PCR qualification result of NKG2D-sPD-1-CAR-T cell, wherein 1 is the T cell of uninfecting virus As a result, 2 be NKG2D-sPD-1-CAR-T cell results, 3 be molecular weight standard.

Fig. 6 is the ELISA qualification result of NKG2D-sPD-1-CAR-T cell.

Fig. 7 is tumor cell surface PD-L1 the selection result figure.

Fig. 8 is NKG2D-CAR-T and NKG2D-sPD-1-CAR-T cell killing tumour cell result figure.

Fig. 9 is NKG2D-CAR-T and NKG2D-sPD-1-CAR-T cell killing tumour cell result column diagram, wherein 1: Three column diagrams in 3 groups and 1:1 group in every group successively represent T cell, NKG2D-CAR-T cell and NKG2D- from left to right SPD-1CAR-T cell.

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

The acquisition of embodiment 1, the PD-1 gene extracellular fragment sequence of people

Pass through website https: //www.ncbi.nlm.nih.gov/pubmed/ finds PD-1 gene extracellular fragment sequence The area CDS, and in https: find the extracellular fragment region of PD-1 albumen in the //website www.uniprot.org/.It is soft with SnapGene Part designs and company's synthetic primer (including the signal peptide sequence of CD8a in primer), using the T cell cDNA of people as template, passes through The mode of PCR expands the extracellular fragment of PD-1 gene, and amplified production is sequenced to obtain containing 1201- in SEQ ID No.2 PD-1 shown in 1264-1713 is extracellular in the signal peptide gene sequence connection SEQ ID No.2 of CD8a shown in 1263 Section gene order, amino acid sequence is respectively as shown in SEQ ID No.3 and SEQ ID No.1.

The building of embodiment 2, pCDH-NKG2D-sPD-1-CAR carrier

The carrier PCDH-NKG2D-CAR having been had been built up using fast enzyme cutting BamHI and I double digestion of Sal is (in carrier CAR element shown in upper figure in connection figure 1 between the EcoR I and BamHI restriction enzyme site of pCDH), using ligase by the double digestion The pcr amplification product (sequence containing sPD-1) and T2A genetic fragment (SEQ ID of carrier PCDH-NKG2D-CAR and embodiment 1 afterwards Sequence shown in 1141-1200 of No.2) connection, recombinant vector is obtained, recombinant vector is converted into Escherichia coli, through bacterium colony Determine acquisition pCDH-NKG2D-sPD-1-CAR carrier (containing the member of CAR shown in the following figure in Fig. 1 after PCR and sequencing identification Part).

Above-mentioned recombinant vector pCDH-NKG2D-sPD-1-CAR is I enzyme of Sal I and EcoR in Lentiviral pCDH CAR component numbering gene shown in the following figure (i.e. CAR gene shown in SEQ ID No.2) in connection figure 1 between enzyme site,

In CAR gene shown in SEQ ID No.2,1-63 are CD8a signal peptide gene sequence, and 64-471 are NKG2D receptor extracellular region gene order, the costimulation of the 472-1140 transmembrane region genes, albumen 4-1B B for PROTEIN C D8a Signaling molecule gene and CD3 ζ endochylema signal transduction sequence gene, 1141-1200 are T2A gene order, 1201-1263 Position is CD8a signal peptide gene sequence, and 1264-1713 are solubility PD-1 (the extracellular fragment region of PD-1) gene order.

The bacterium colony PCR is after picking positive monoclonal bacterium colony with 1201-1713 in PCR amplification SEQ ID No.2 Shown sequence, to examine whether the plasmid for being transformed into Escherichia coli is successfully connected with sPD-1-CAR2, agarose electrophoresis runs glue It obtains stripe size and meets expected (Fig. 2), about 500bp.Plasmid is extracted to the monoclonal colonies for having band and carries out sequencing identification, most Confirmation obtains purpose recombinant vector pCDH-NKG2D-sPD-1-CAR (Fig. 3) eventually.G2D-CAR2-T2A-SIPD1 in Fig. 3 is CAR element shown in the following figure in Fig. 1.

Embodiment 3, virus packaging

1, plasmid transfection

By three plasmid transfection systems by recombinant vector (the carrier pCDH-NKG2D-sPD-1-CAR or carrier of embodiment 2 PCDH-NKG2D-CAR) carrier feeding 293T is intracellular as a purpose, according to helper plasmid psPAX2: helper plasmid pMD2.G: The addition of purpose carrier=5:3:3 mass ratio enters Opti-MEM culture medium, and 50ul PEI solution is added, and blows and beats 20 times and blows repeatedly It beats after mixing, is stored at room temperature 20min, obtain DNA/PEI mixture.It takes and 1mlDNA/PEI mixture is slowly instilled to the previous day paving It in good 293T culture dish, mixes gently, 37 DEG C of incubators are incubated for, and are replaced fresh culture after 6-8h, are put into 37 DEG C of incubators Continue to be incubated for.

2, collection virus and concentration

After plasmid transfection 48h, after collecting supernatant, the addition fresh Opti-MEM culture medium of 10ml continues culture to 72h, again Supernatant is collected, after being mixed with the 48h supernatant collected, is placed in 4 DEG C of refrigerators stand-by；4 DEG C, 4000g is centrifuged 10min, removes cell Fragment；The supernatant being obtained by filtration with 0.45 μm of filter；Filtered viral supernatants are transferred in ultracentrifugation pipe, 25000 leave Heart 2h is diluted with the PBS of 1/100 supernatant volume, is transferred in closed centrifuge tube and is stayed overnight after blowing and beating repeatedly for 4 DEG C, obtains disease Venom；Virus liquid is dispensed to suitable volume, is placed in -80 DEG C and saves, and 200 μ l virus liquids is taken to carry out titer determination.

3, virus titer measures

293T cell is digested, is counted after centrifugation, is made cell suspension with serum-containing media, adjustment cell density is 2 × 10⁵0.5ml cell suspension is added into every hole of 24 well culture plates by/ml；With full culture medium, dilution step 2 is obtained in the following proportions The virus liquid arrived: 1:3,1:9,1:27, the virus liquid dilution after being diluted respectively by 100 μ l virus liquid stostes and by different proportion Liquid is added in 24 orifice plates of inoculating cell；Infection supernatant is discarded after 16h, adds the fresh full culture medium of 0.5ml；It is used after 48h Flow cytometer detection is infected the destination gene expression of cell after the dyeing of NKG2D antibody.Calculate titre, titre=2 × 10⁵× infection effect Rate × extension rate.

As a result: it collects and is concentrated through step 2, virus liquid (the slow disease that transfection carrier pCDH-NKG2D-sPD-1-CAR is obtained Malicious NKG2D-sPD-1-CAR) titre be T=1.89 × 10⁹TU/mL, the virus that transfection carrier PCDH-NKG2D-CAR is obtained The titre of liquid (slow virus NKG2D-CAR) is T=1.87 × 10⁹TU/mL。

Embodiment 4, the preparation of CAR-T cell and detection

1, prepared by CAR-T cell

Two kinds of slow virus NKG2D-CAR and NKG2D-sPD-1-CAR prepared by embodiment 3 are respectively according to MOI (effect target Than) ratio of=5:1 is added to the culture medium containing source of people T cell (cell density is 1 × 10 when infection⁶A/mL) in, 16h Liquid is changed in centrifugation afterwards, and fresh complete medium is added and continues to cultivate, obtains two kinds of CAR-T cells, is respectively designated as NKG2D- CAR-T and NKG2D-sPD-1-CAR-T.

2, FCM analysis

Two kinds of CAR-T cells are cultivated two days later respectively, collect cell, extract RNA, and the table of sPD-1 is detected by RT-PCR It reaches.With flow cytometer detection CAR-T cell positive rate after the dyeing of APC-hNKG2D antibody.As a result: as shown in figure 4, two kinds of CAR-T cells Positive rate be respectively 80.3% and 86.3%, T cell (negative control UT-T) result of uninfecting virus is 1.22%.

3, sPD-1 secretion identification

The T cell of NKG2D-sPD-1-CAR-T cell and uninfecting virus that counting step 1 obtains, according to 1 × 10⁶A/ ML density spreads 96 orifice plates.After culture for 24 hours, Cell extraction RNA is taken, using reverse transcription reagent box by RNA reverse transcription at cDNA, with CDNA is that template carries out PCR amplification, compares the mRNA expression contents of three kinds of intracellular sPD-1 genes.As a result as shown in figure 5, The mRNA of NKG2D-sPD-1-CAR-T cell height expression sPD-1.

By the T cell of two kinds of CAR-T cells of NKG2D-CAR-T and NKG2D-sPD-1-CAR-T and uninfecting virus according to Effect target ratio 1:3 is seeded in cell line of human osteosarcoma U2OS, at the same setting do not add tumour cell only contain NKG2D-sPD- The negative control of 1-CAR-T takes supernatant culture solution, is examined with ELISA kit after co-culturing for 24 hours in 37 DEG C of incubators The expression of sPD-1.As a result: as shown in Figure 6 a, compared to the T cell of NKG2D-CAR-T and uninfecting virus, NKG2D- SPD-1-CAR-T cell height expresses sPD-1, and sPD-1 is expressed as 94.19 μ g/mL in NKG2D-sPD-1-CA R-T group；Such as Shown in Fig. 6 b, addition cell line of human osteosarcoma U2OS can significantly improve the secretion of sPD-1.

The killing ability research of embodiment 5, the selection of CAR-T target cell and two kinds of CAR-T cells

By consulting literatures, NKG2D ligand and the tumour cell of PD-L1 are expressed using antibody screening height, wherein PD- The expression quantity measuring method of L1 is to carry out the dyeing of PD-L1 antibody after room temperature dyes 15min to tumour cell to pass through fluidic cell Art Positive rate, as a result as shown in fig. 7, finally determining that the strain of CAR-T target cell is that human osteosarcoma is thin according to the expression quantity of PD-L1 Born of the same parents' strain U2OS.

Target cell cell line of human osteosarcoma U2OS is marked with fluorescein based dye CFSE, by NKG2D-CAR-T and NKG2D-sPD- The T cell of two kinds of CAR-T cells of 1-CAR-T and uninfecting virus is seeded to target cell U2OS according to effect target ratio 1:3,1:1 respectively In, every group sets two repetitions, every hole fluid infusion to 200ul；Culture plate after mixing with cells is put into 37 DEG C of incubators and is cultivated 24h；After for 24 hours, all cells in every hole are collected, cell is transferred in streaming pipe, uses apoptosis antibody A nnexin V at normal temperature 15min is dyed, is changed by the Apoptosis ratio of flow cytomery target cell.

As a result: as shown in Figure 8 and Figure 9, with the raising of effect target ratio, NKG2D-sPD-1-CAR-T compares uninfecting virus T cell and NKG2D-CAR-T have preferable fragmentation effect to target cell U2OS.

When imitating target ratio is 1:3, the Apoptosis ratio of the target cell of NKG2D-CAR-T treatment is 22.3%, NKG2D- The Apoptosis ratio of the target cell of sPD-1-CAR-T treatment is 52.5%.

To sum up, the application pass through RT-PCR it can be found that in NKG2D-sPD-1-CAR-T cell PD-1 mRNA compared to The expression of T cell height, and directly detect that sPD-1 expression contents increase by ELISA, it can think to have constructed purpose matter Grain.It is co-cultured using the osteosarcoma cell of the CAR-T cell constructed and high expression NKG2D ligand and PD-L1 ligand, killing It is observed that NKG2D-sPD-1-CAR-T has better fragmentation effect with increasing for target ratio of effect, and it is pre- to meet experiment afterwards for 24 hours Phase；Fragmentation effect compared to NKG2D-CAR-T, NKG2D-sPD-1-CAR-T are significantly improved.It is also worth noting that In Under the stimulation of tumour cell, NKG2D-sPD-CAR-T cell secretion sPD-1 obviously increases, this illustrates that the cell need to be swollen Further stimulation plays effect in tumor microenvironment, and compared to systemic injection PD-1 antibody, toxic side effect is lower, possesses more Safe and efficient advantage.

The content being not described in detail in this specification belongs to the prior art well known to professional and technical personnel in the field.More than Described is only embodiments herein, is not intended to limit this application.To those skilled in the art, the application can To there is various modifications and variations.All any modification, equivalent replacement, improvement and so within the spirit and principles of the present application, It should be included within the scope of the claims of this application.

SEQUENCE LISTING

<110>East China Normal University Shanghai Bang Yao Biotechnology Co., Ltd

<120>Chimeric antigen receptor CAR gene and the application of solubility PD-1 are expressed

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<170> PatentIn version 3.5

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aggtcagccg gccagttcca aaccctggtg gtttaa 1716

<210> 3

<211> 21

<212> PRT

<213>artificial sequence

<400> 3

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

1 5 10 15

His Ala Ala Arg Pro

20

Claims

1. a kind of Chimeric antigen receptor CAR gene for expressing solubility PD-1, which is characterized in that the CAR gene includes successively Antigen-binding domains gene, transmembrane structure domain gene, intracellular domain gene and the solubility PD-1 gene of sequential connection, institute The extracellular fragment region that soluble PD-1 is PD-1 is stated,

2. CAR gene according to claim 1, it is characterised in that: in the intracellular domain gene and the solubility The first signal peptide gene is connected between PD-1 gene,

It is furthermore preferred that first signal peptide is the signal peptide of CD8a, amino acid sequence is more excellent as shown in SEQ ID No.3 Choosing, gene order is as shown in 1201-1263 in SEQ ID No.2.

3. CAR gene according to claim 2, it is characterised in that: in the intracellular domain gene and first letter Be connected with self cleavage protein gene between number peptide gene, it is preferred that the self cleavage albumen be selected from T2A, P2A, E2A, F2A or its Combination, it is furthermore preferred that the self cleavage albumen is T2A.

4. CAR gene according to claim 1 to 3, it is characterised in that: the antigen-binding domains can be with NKG2D ligand specificity combines, it is preferred that the antigen-binding domains are NKG2D receptor extracellular fragment, it is furthermore preferred that described The front end of antigen-binding domains gene includes second signal peptide gene；

The transmembrane domain includes the transmembrane region of following albumen: CD3 ε, CD4, CD8a, CD9, CD16, CD22, CD33, CD137, CTLA-4, PD-1, LAG-3 or combinations thereof, it is preferable that CD8a；

The intracellular domain includes costimulatory signal molecule and endochylema signal transduction sequence；Preferably, the costimulatory signal Molecule be selected from following albumen costimulatory signal molecule: OX40, CD28, CD30, CD40, CD70, CD134,4-1BB, PD1, Dap10, CDS, ICAM-1 or combinations thereof, it is further preferred that 4-1BB；Preferably, the endochylema signal transduction sequence includes being derived from CD3 ζ Endochylema signal transduction sequence.

5. CAR gene according to any one of claims 1-4, it is characterised in that: the expression solubility PD-1's is chimeric Antigen receptor CAR gene order is as shown in SEQ ID No.2.

6. a kind of recombinant vector, it is characterised in that: the recombinant vector contains CAR gene as claimed in any one of claims 1 to 5, The recombinant vector is preferably slow virus carrier.

7. a kind of recombinant cell or recombinant bacterium, it is characterised in that: contain claim in the recombinant cell or the recombinant bacterium Any CAR gene in 1-5, or contain recombinant vector described in claim 6,

8. described in CAR gene as claimed in any one of claims 1 to 5, recombinant vector as claimed in claim 6 or claim 7 Recombinant cell or recombinant bacterium preparation oncotherapy product in application.

9. application according to claim 8, it is characterised in that: the oncotherapy product is selective killing tumour cell The oncotherapy product that fragmentation effect improves.

10. application according to claim 8 or claim 9, it is characterised in that: the tumour is solid tumor, it is preferable that osteosarcoma U2OS, osteosarcoma MG-3, kidney cancer cell 786O, it is further preferred that osteosarcoma U2OS.