CN107573419A - A kind of nucleic acid molecules for strengthening T cell antitumor activity - Google Patents

Abstract

The present invention relates to a novel chimeric antigen receptor (CAR) molecule containing the intracellular segment of DAP10 protein, its encoding nucleic acid, cells expressing it, and its use in preparing medicines for treating tumors. The CAR molecule of the present invention is realized by introducing the intracellular segment of DAP10 protein into the traditional second-generation CAR molecule, which activates the downstream signaling pathway of T cell DAP10 after the CAR molecule binds to the target antigen, so as to enhance the corresponding CART cell response The killing effect of solid tumors in vivo and in vitro enhances their proliferation and survival ability.

Description

A kind of nucleic acid molecules for strengthening T cell antitumor activity

Technical field

The present invention relates to the cellular immunotherapy technical field of tumour, in particular it relates to a kind of intracellular section of albumen containing DAP10 Chimeric antigen receptor, encode its nucleic acid and express its cell, and its prepare treatment tumour medicine in purposes.

Background technology

Chimeric antigen receptor (CAR, Chimeric Antigen Receptors) T cell is that surface expression identification is specific anti- Original simultaneously can transmit the T cell of the chimeric receptor of signal.CAR T cells by express Chimeric antigen receptor (CAR) molecule and Played a significant role in antitumor, CAR molecules generally include extracellular fragment, transmembrane region and intracellular section：Extracellular fragment is by heavy chain of antibody It is connected with light chain variable district by a peptide fragment and the single-stranded variable region (ScFv) formed；Intracellular section is various signal transductions point The intracellular section chimera of son, including CD3zeta, CD28, OX-40,4-1BB etc.；Transmembrane region then from other molecules (such as CD8, CD4, CD28 and CD3zeta) transmembrane region.The Gene Isolation of single chain variable fragment part identifies target antigen from such as generation The hybridoma of monoclonal antibody.The T cell of CAR molecules is expressed independently of the major histocompatibility antigen I types on tumour cell Expression and Direct Recognition tumor cell surface antigen, and activate T cell at the same time, and the T cell for therefore expressing CAR can be with Effective killing tumor cell.In short, CAR T cells are divided the special of tumor cell surface by Ag-Ab recognition mode Son is identified, and then enters line activating, propagation by the signal transduction of its intracellular and plays cell killing function.

The structure design of CAR molecules undergoes the research and development in more generations.It is thin that the structure of first generation CAR molecules includes identification tumour The single chain variable fragment (scFv) of cellular surface antigen, membrane spaning domain and activate T cell TCR compound CD3 ζ intracellular structure Domain.Because first generation CAR intracellular section only has CD3 ζ signal transfer regions domain, without costimulatory signal, thus first generation CAR T are thin There is very big defect in the function of born of the same parents, it all shows low-level in amplification in patient body, persistence and effector function etc.. In order to strengthen the purpose of the ability of first generation CAR activation T cells, second generation CAR is developed, second generation CAR adds in intracellular The intracellular molecular signal for having entered costimulatory molecules (such as CD28, CD134 (OX-40), CD137 (4-1BB)) source transmits domain.Face Bed experiment shows that second generation CAR T cells are showing preferably propagation, persistence and effector function in patient body.The second generation The clinical test of CAR T cells, it is largely treatments of the anti-CD19CAR T to B cell leukemia.Although CAR T cells are clinical Experiment obtains curative effect, but the space further improved also be present.Third generation CAR is treated to further improve CAR T cells The effect of method and develop.Third generation CAR intracellular section introduces the signal transfer region domain of two costimulatory moleculeses.Normal conditions Under, a costimulatory signal is CD28 intracellular regions, and another is then CD134, CD137 or ICOS etc. intracellular signal transmission region Domain.The various combination of costimulatory signal may influence the function and curative effect of CAR T cells, and research shows, and not all Three generations CAR is better than the second generation.

DAP10 is an independent cross-film adaptin expressed on hematopoietic cell, by making with activation receptor NKG2D With compound is formed, the associated ligands of overexpression can be identified in tumour generation, infection, autoimmune response reaction. DAP10 signals have important physiological role in autoimmune tolerance is maintained.According to the document reported, the NKG2D of people Acceptor is the unique combination acceptors of DAP10, and dimer is formed by the disulfide-bonded of extracellular fragment cysteine.DAP10 Intracellular region includes Tyr-Ile-Asn-Met motifs, is combined with PI3K regulation subunit p85 homologous section of 2 regions of Src.DAP10 After being combined with p85, the interaction with signaling molecule Grb2 and Vav1, downstream Rho families guanylic acid triphosphatase and C- are originated The activation and accumulation of the phosphatases of γ 2, with the activation of initial vaccination cell.And the combination of a NKG2D part can start 4 The phosphorylation of DAP10 molecules, in the case of low ligand concentration, expand signal transmission.

NKG2D-DAP10 receptor complexes are expressed in NK, gamma delta T, NKT and thymocyte, and can be stimulated with inherent immunity Up-regulated expression.In human body, NKG2D only interacts with DAP10 and produces immunological effect, and expresses two in mouse cell NKG2D isomers：NKG2D-1 and NKG2D-2, NKG2D-1 only act on DAP10, and NKG2D-2 then can be with DAP10, DAP12 Effect.The part of mouse source NKG2D-DAP10 acceptors is I classes MHC (MHC I).

Research is found, by knocking out NKG2D in C57BL/6 mouse, is improved mouse and is induced fiber meat in methyl cholanthrene Sensitiveness in knurl, this demonstrates NKG2D-DAP10 and plays an important roll (NKG2D in monitoring tumour occurs functionprotects the host from tumor initiation.J Exp Med 2005；202:583–588.).

NKG2D is also played in autoimmune reaction and acted on.Blocking NKG2D function will suppress in NOD Mice Bodies certainly The propagation of precursor reactant CD8+T cells, and improve the survival rate of spontaneous diabetes mellitus NOD mice.

Entitled " A Chimeric Receptor with NKG2D Specificity Enhances Natural One kind is elaborated in a Killer Cell Activation and Killing of Tumor Cells " scientific and technical literature Applied to the Novel chimeric antigen receptor on NK cells --- CAR NKG2D-DAP10-CD3 ε, it is mainly resisted by improving tumour Former specific recognition and activation capability are to strengthen outer anti-tumor function inside NK cells；And NKG2D can specific recognition swell Knurl specific antigen MIC and RAET1/ULBP, and downstream signaling pathway is activated by DAP10；DAP10 the chimeric antigen by Main function in body is positioned on cell membrane for help NKG2D acceptors.

At present, CART cells achieve the effect of fine in the preclinical and clinical test of neoplastic hematologic disorder, but for Its curative effect of entity tumor is simultaneously imprecise.According to tumor immunology and the general principle of tumor microenvironment, CART cells in vivo may be used The inhibitory action for overcoming entity tumor microenvironment can be difficult to, its effector function (such as cell killing, cytokine secretion etc.) is straight Connect suppression or because exhaustion effect is difficult to；In addition, the propagation survival ability of CART cells is also weakened by tumor microenvironment, Make quantity of the CART cells in tumour hard to carry on.Therefore, it is necessary to find approaches and methods to lift CART cells pair conscientiously The lethal effect of entity tumor in vivo, external, strengthen it and breed survival ability.

The content of the invention

It is an object of the invention to provide a kind of new Chimeric antigen receptor (CAR) molecule of the intracellular of albumen containing DAP10 section, And its nucleic acid is encoded, express its cell, and its purposes in the medicine for preparing treatment tumour.The present invention is in tradition Second generation CAR molecules in introduce the intracellular section of DAP10 albumen, activation T cell DAP10 after it is combined by CAR with target antigen Downstream signaling pathway, to reach the lethal effect for lifting corresponding CART cells to entity tumor in vivo, external, strengthen it Breed the purpose of survival ability.

The present invention is achieved through the following technical solutions above-mentioned purpose：

In a first aspect, the invention provides a kind of Chimeric antigen receptor, its include can combine antigen ectodomain, Membrane spaning domain and intracellular domain, wherein, the intracellular domain includes the intracellular section sequence of DAP10 albumen.

In above-mentioned Chimeric antigen receptor, the intracellular section sequence of the DAP10 albumen is: LCARPRRSPAQDGKVYINMPGRG (such as SEQ ID NO:Shown in 12), its corresponding nucleotides sequence is classified as： 5’- ctgtgcgcacgcccacgccgcagccccgcccaagaagatggcaaagtctacatcaacatgccaggcaggggc-3’ (such as SEQ ID NO:Shown in 13).

Preferably, the intracellular domain includes the intracellular section sequence of 1~9, preferably 1~3 DAP10 albumen；

Preferably, the intracellular domain includes two or more DAP10 eggs to be linked together by series system White intracellular section sequence.

In a particular embodiment, above-mentioned CAR molecules only can be made with the intracellular section sequence of one or more DAP10 albumen For its intracellular domain, one or more (such as 2 or 3 in addition to DAP10 albumen intracellular section sequences can also be included It is individual) other intracellular domain.For example, the intracellular domain may also include intracellular costimulatory signal transduction domain and/or CD3 ζ letters Number transduction domain；Preferably, the intracellular costimulatory signal transduction domain is selected from, but not limited to, the function signal biography of following protein Transduction domain：CD28、4-1BB、TLR1、TLR2、TLR3、TLR4、CD27、CD28、OX40、CD30、CD40、 PD-1、ICOS、 LFA-1、CD2、CD7、LIGHT、NKG2C、B7-H3、CDS、ICAM-1、GITR、BAFFR、 HVEM(LIGHTR)、SLAMF7、 NKp80(KLRF1)、CD160、CD19、CD4、CD8α、CD8β、IL2R β、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、 ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、 ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、 ITGAM、CD11b、ITGAX、 CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、TRANCE/ RANKL、 DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、 Ly9 (CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、 SLAM (SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、 SLP-76、PAG/ In Cbp, NKp44, NKp30, NKp46, NKG2D or CD83 any one or at least two combination, preferably TLR2,4- In 1BB or CD28 any one or at least two combination.

Preferably, the acceptor extracellular fragment that the antigen can be referred to reference to the ectodomain of antigen, Huo Zhesuo State the single chain variable fragment of the specific antibody of antigen.

Preferably, the antigen is tumour antigen and/or tumour correlation helper antigenic；The tumour antigen and/ Or tumour correlation helper antigenic is selected from, but not limited to,：The β 1- integrins of 5T4, α 5,707-AP, AFP, ART-4, B7H4, BAGE, beta-catenin/m, Bcr-abl, MN/C IX antibody, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22、CD25、CDC27/m、CD30、CD33、CD52、CD56、CD80、CDK4/m、CEA、 CT、Cyp-B、DAM、EGFR、 ErbB3、ELF2M、EMMPRIN、EpCam、ETV6-AML1、G250、 GAGE、GnT-V、Gp100、HAGE、HER-2/new、 HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT (or hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/melan-A, MART-2/Ski, MC1R, Mesothelin, flesh ball egg In vain/m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, PAP, proteinase-3, p190minor bcr-abl, Pml/RAR α, PRAME, PSA, PSM, PSMA, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, survivin protein, TEL/AML1, TGF β、TPI/m、TRP-1、TRP-2、TRP-2/INT2、VEGF、WT1、、BCMA、CD123、PD-1、PD-L1/2、 TIM3、LAG3、 A2ARA2AR、B7H3、B7H4、BTLA、IDO、KIR、CD160、2B4(CD244)、VISTA (C10orf54)、TSHR、CD171、 CS-1、CLL-1、GD3、Tn Ag、FLT3、CD38、CD44v6、B7-H3、 CD117、IL-13Ra2、IL-11Ra、PRSS21、 VEGFR2, Louis (Y) antigen, CD24, PDGFR- β, SSEA-4, NCAM, CAIX, EphA2, GM1, sLe, TGS5, HMWMAA, OAcGD2, folate receptor beta, CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, the compound of lymphocyte antigen 6, LY6K, OR51E2, TARP, ETS, SPA17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos related antigen 1, ERG, androgen receptor Body, cell periodic protein B 1, SART3, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, In BST2, EMR2, LY75, FCRL5, IGLL1, NY-Eso-1 or NY-Eso-B any one or at least two combination；

In addition, the antigen can also be the inflammatory cell surface molecular occurred in autoimmune disease or cause certainly The immune TCR of body.

It is further preferred that the antigen be in PCSA, Mesothelin antigen or GPC3 antigens any one or extremely Few two kinds combination；It is further preferred that the ectodomain be specific recognition antigen Mesothelin, PSCA or GPC3 acceptor extracellular fragment and/or antibody.

Preferably, the membrane spaning domain is membrane spaning domain corresponding to extracellular receptor seq or intracellular section costimulatory signal Membrane spaning domain corresponding to molecule, preferably CD28 transmembrane regions.

In preferred embodiment, the Chimeric antigen receptor includes as extracellular since the N-terminal side successively Domain, the single-stranded variable region of antibody for antigen or the acceptor extracellular fragment of antigen, the transmembrane region of CD28 molecules, CD3 ζ Chain, and one or more DAP10 intracellular section sequences being connected in series.

Second aspect, the invention provides a kind of nucleic acid for encoding Chimeric antigen receptor as described in relation to the first aspect or and its Nucleic acid with least 80% homogeneity, preferably at least 90% homogeneity.

In the present invention, the nucleic acid of the Chimeric antigen receptor with described in first aspect has at least 80% homogeneity, excellent The nucleic acid of choosing at least 90% homogeneity has phase for the Chimeric antigen receptor of coding with the Chimeric antigen receptor described in first aspect Same or similar functions, the change of its base will not have an impact to the 26S Proteasome Structure and Function of Chimeric antigen receptor.

The third aspect, the invention provides a kind of Chimeric antigen receptor expression cell, wherein introducing such as second aspect institute The nucleic acid stated；Preferably, the cell is immune effector cell, and more preferably T cell, B cell, NK cells, NKT is thin In born of the same parents, BMDC or macrophage any one or at least two combination.

Fourth aspect, the invention provides a kind of side of the Chimeric antigen receptor expression cell prepared as described in the third aspect Method, it include by as described in second aspect nucleic acid introduce cell the step of；

Preferably, the nucleic acid is introduced by cell by the transduction by lentivirus-mediated；

Preferably, the cell is immune effector cell, more preferably T cell, B cell, NK cells, NKT cells, In BMDC or macrophage any one or at least two combination.

5th aspect, the present invention provide a kind of recombinant vector, and it includes the nucleic acid described in claim 3；Preferably, institute Carrier is stated as any one in recombinant cloning vector, eukaryotic expression recombinant plasmid or recombined lentivirus vector or at least two Combination, preferably recombined lentivirus vector；

Preferably, the recombinant cloning vector be selected from, but not limited to, pUC18, pUC19, pMD19-T, pGM-T carrier, In pUC57, pMAX or pDC315 any one or at least two combination；

Preferably, the eukaryon expression plasmid be selected from, but not limited to, pCDNA3 serial carriers, pCDNA4 serial carriers, Any one in pCDNA5 serial carriers, pCDNA6 serial carriers, pRL serial carriers, pUC57 carriers, pMAX or pDC315 Or at least two combination；

Preferably, the recombined lentivirus vector is selected from, but not limited to, recombinant adenoviral vector, recombinant adeno-associated virus carries In body, recombinant retroviral vector, recombinant herpes simplex virus carrier or vaccinia virus recombinant carrier any one or extremely Few two kinds combination.

In the present invention, by the way that the nucleic acid construct of the Chimeric antigen receptor and the carrier are recombinated, so as to Carrier after the restructuring is transfected into cell by enough realizations obtains T cell, realizes the function of Chimeric antigen receptor.

6th aspect, the present invention provide a kind of recombinant virus, and it includes the recombinant vector and packaging as described in terms of the 5th The recombinant virus that helper plasmid cotransfection mammalian cell obtains.

7th aspect, the present invention provide a kind of Chimeric antigen receptor T cell, and it is by by the restructuring as described in terms of the 6th Virus transfection is expressed into T cell.

Eighth aspect, the present invention provide Chimeric antigen receptor as described in relation to the first aspect, the nucleic acid as described in second aspect, Recombinant vector as described in terms of the 5th or the recombinant virus as described in terms of the 6th is used to expand CAR T cells

9th aspect, the present invention provide a kind of pharmaceutical composition, and it includes Chimeric antigen receptor described in first aspect, such as Nucleic acid described in second aspect, the Chimeric antigen receptor expression cell as described in the third aspect or the restructuring as described in terms of the 5th Carrier, and optional pharmaceutically acceptable auxiliary material.

Tenth aspect, the invention provides a kind of Chimeric antigen receptor as described in relation to the first aspect, as described in second aspect Nucleic acid, the Chimeric antigen receptor expression cell as described in the third aspect or the recombinant vector as described in terms of the 5th is controlled in preparation Treat the purposes in the medicine of autoimmune disease or tumour.

Preferably, the tumour is solid tumor and/or blood tumor, the tumour can be selected from, but not limited to, lung cancer, liver cancer, Lymph cancer, colon cancer, colorectal cancer, breast cancer, oophoroma, cervical carcinoma, stomach cancer, cholangiocarcinoma, gallbladder cancer, the cancer of the esophagus, kidney, nerve Any one in colloid carcinoma, melanoma, cancer of pancreas, prostate cancer, leukaemia, Huppert's disease or malignant lymphoma etc. Or at least two combination.

In the specific embodiment of the purposes provided by the invention, the tumour is leukaemia, liver cancer or lung cancer.

Tenth aspect, the present invention provide a kind for the treatment of and suffer from autoimmune disease and/or the expression phase with tumour antigen The method of the subject of the disease of pass, it includes applying the subject medicine group included described in eighth aspect of effective dose The medicine of compound；

Preferably, the tumour is solid tumor and/or blood tumor.

In the specific embodiment of the purposes provided by the invention, the tumour is leukaemia, liver cancer or lung cancer.

It is worth noting that, the CAR molecules of the present invention are characterised by that it includes DAP10 intracellulars section as its intracellular structure Domain.The DAP10 intracellulars section includes its variant with identical function.Term " variant " refers to comprising one or several to multiple Any variant of the substitution of amino acid, missing or addition, condition is that the variant substantially retains phase possessed by original series Congenerous.

Beneficial effect

(1) present invention is used as independent common thorn by introducing DAP10 intracellular sections on traditional second generation CAR molecular basises Signal pathways, and acceptor is not only restricted to, other costimulatory signals can be further coupled, and can further increase by series system Strong tumour immunity effect；

(2) the CART cell anti-tumor effector functions of present invention integration DAP10 costimulatory molecules intracellular signal domains are bright Aobvious to improve, the cell is compared with the second generation CART for not integrating DAP10, to the cytotoxicity especially continuation of tumour target cell Cytotoxicity is remarkably reinforced, and secrete cytokines IL-2, IFN-y, Granzyme B, GM-CSF ability significantly improve, simultaneously Stronger tumor suppression ability and more preferable knurl internal breeding ability are shown in zoopery；

(3) the DAP10 costimulatory signal molecules in the present invention only have 72bp, and fragment is very small, will not be transfected because of exacerbation Carrier load, and cause to turn, under the transfection efficiency of liposome by various rotaring transfecting modes such as slow virus, retrovirus, electricity The influence of drop, and the CAR T cell tumour immunity effects for integrating DAP10 significantly improve, then DAP10 costimulatory signals molecule is carrying While high T cell tumour immunity effect, the potentiality more transformed are provided for CAR molecular vehicles.

Brief description of the drawings

Fig. 1 show a series of CAR molecules constructed in the embodiment of the present invention include CAR-Mes, CAR-Mes-41BB, CAR-Mes-DAP10、CAR-Mes-DAP10*3、CAR-Mes-DAP10*9、CAR-Mes-41BB-CD28、CAR-Mes-41BB- DAP10, CAR-PSCA, CAR-PSCA-DAP10, CAR-PSCA-DAP10*3, CAR-GPC3 (control group), CAR-GPC3- 41BB, CAR-GPC3-DAP10, CAR-GPC3-41BB-CD28, CAR-GPC3-41BB-DAP10 schematic arrangement.

Fig. 2 shows CAR-Mes T, CAR-Mes-DAP10 T, CAR-Mes-DAP10*3 T cells to A549GL cells Curve map Fig. 2 (A) and block diagram Fig. 2 (B) of Vitro Tumor lethal effect, and CAR-Mes, CAR-Mes-DAP10 slow virus The efficiency chart 2 (C) of carrier transfecting T cells.

Fig. 3 shows CAR-Mes T, CAR-Mes-DAP10 T, CAR-Mes-DAP10*9T cells to the bodies of A549GL cells Curve map Fig. 3 (A) and block diagram Fig. 3 (B) of outer Tumor cytotoxicity, and CAR-Mes T, CAR-Mes-DAP10 T cells 3 Related T-cell activated surface mark CD25 Fig. 3 (C), CD69 Fig. 3 (D), CD107a Fig. 3 after the secondary co-cultivation with tumour cell (E) expression compares.

Fig. 4 shows CAR-PSCA T, CAR-PSCA-DAP10 T cells to the Vitro Tumor lethal effects of A549GL cells Curve map Fig. 4 (A) and block diagram Fig. 4 (B).

Fig. 5 shows CAR-Mes T, CAR-Mes-41BB-CD28 T, CAR-Mes-DAP10 T cells respectively to A549GL Fig. 5 (A) and H460Mes+GL Fig. 5 (B) cell Vitro Tumor lethal effect.

Fig. 6 shows that CAR-GPC3 T, CAR-GPC3-41BB-CD28 T, CAR-GPC3-DAP10 T cells are right respectively HepG2GL Fig. 6 (A) and HC04-GL Fig. 6 (B) cell Vitro Tumor lethal effect.

Fig. 7 show ELISA detection GFP T, CAR-MesT, CAR-Mes-DAP10 T, CAR-Mes-DAP10*3 T or CAR-Mes-DAP10*9 T cells co-culture cell factor IL-2 Fig. 7 (A), IFN-γ Fig. 7 after 18h with A549-GL cells (B), GZMB Fig. 7 (C) and GM-CSF Fig. 7 (D) secretion situation.

Fig. 8 shows NC, GFP T, CAR-Mes T or CAR-Mes-41BB-CD28, CAR-Mes-DAP10 T cell pair Tumor weight Fig. 8 (A) and volume diagram 8 (B) that A549-GL cells are formed in immunodeficient mouse body influence.

Fig. 9 is shown with after processing lung cancer tumor heteroplastic transplantation model in CAR-Mes T or CAR-Mes-DAP10 T cells body The column diagram of the ratio of tumor-infiltrated T cell.

Figure 10 shows CAR-Mes-41BB T, CAR-Mes-41BB-DAP10 T cells to A549-GL Figure 10 (A), H460+ The curve map of the Vitro Tumor lethal effect of Mes-GL Figure 10 (B) cell, and CAR-Mes-CD28 T, CAR-Mes-41BB T, the curve map Figure 10 (C) of CAR-Mes-DAP10 T cells to external 3 times continuous lethal effects of A549-GL cells.

Figure 11 shows CAR-GPC3-41BB T, CAR-GPC3-DAP10 T cells to Hepg2-GL Figure 11 (A), HC04- The curve map of the Vitro Tumor lethal effect of GL Figure 11 (B) cell.

Figure 12 shows that CAR-GPC3 T, CAR-GPC3-DAP10 T cells kill effect to the Vitro Tumor of HC04-GL cells The curve map answered.

Embodiment

For ease of understanding the present invention, it is as follows that the present invention enumerates embodiment.Those skilled in the art are it will be clearly understood that the implementation Example is only to aid in understanding the present invention, is not construed as the concrete restriction to the present invention.

The structure of embodiment 1, CAR plasmids

1) by gene chemical synthesis, be respectively synthesized CAR-Mes (control group), CAR-Mes-DAP10, CAR-Mes-41BB, CAR-Mes-DAP10*3、CAR-Mes-DAP10*9、CAR-Mes-41BB-CD28、CAR-Mes-41BB-DAP10、 CAR- PSCA (control group), CAR-PSCA-DAP10, CAR-PSCA-DAP10*3, CAR-GPC3 (control group), CAR-GPC3-41BB, (each schematic arrangement is such as CAR-GPC3-DAP10, CAR-GPC3-41BB-CD28, CAR-GPC3-41BB-DAP10 molecule Shown in Fig. 1), the gene C end restriction enzyme sites of Pme1 containing restriction enzyme and its protection base and N-terminal of synthesis contain restriction enzyme Enzyme AsiSI restriction enzyme sites and its protection base；Wherein, " 41BB " refers to containing intracellular commonly used in the prior art believe in the molecule Number domain 4-1BB.

The gene order of synthesized each molecule refers to the SEQ ID NO in sequence table:2(CAR-Mes)、SEQ IDNO:3 (CAR-Mes-DAP10)、SEQ IDNO:4(CAR-Mes-41BB)、SEQ ID NO:5(CAR-Mes-DAP10*3)、 SEQ ID NO:6(CAR-Mes-DAP10*9)、SEQ ID NO：7(CAR-Mes-41BB-CD28)、SEQ ID NO:8 (CAR- Mes-41BB-DAP10)、SEQ ID NO:9(CAR-PSCA)、SEQ ID NO:10(CAR-PSCA-DAP10)、 SEQ ID NO:11(CAR-PSCA-DAP10*3)、SEQ ID NO:13 (CAR-GPC3 (control groups)), SEQ ID NO:14 (CAR- GPC3-41BB)、SEQ ID NO:15 (CAR-GPC3-DAP10) and SEQ ID NO:16 (CAR-GPC3-41BB-CD28)、 SEQ ID NO:17(CAR-GPC3-41BB-DAP10)。

2) the synthetic DNA fragment containing cohesive end is obtained by restriction enzyme Pme1 and AsiSI double digestion respectively (CAR-Mes、CAR-Mes-DAP10、CAR-Mes-41BB、CAR-Mes-DAP10*3、CAR-Mes-DAP10*9、 CAR-Mes- 41BB-CD28、CAR-Mes-41BB-DAP10、CAR-PSCA、CAR-PSCA-DAP10、CAR-PSCA- DAP10*3、CAR- GPC3 (control group), CAR-GPC3-41BB, CAR-GPC3-DAP10, CAR-GPC3-41BB-CD28, CAR-GPC3-41BB- DAP10) and the linearisation DNA of LEGO-2A-eGFP carriers (contains cohesive end, its sequence such as SEQ ID NO:Shown in 1).

3) reclaimed by agargel electrophoresis, obtain respectively the synthesis with cohesive end DNA fragmentation (CAR-Mes, CAR-Mes-DAP10、CAR-Mes-41BB、CAR-Mes-DAP10*3、CAR-Mes-DAP10*9、 CAR-Mes-41BB- CD28, CAR-Mes-41BB-DAP10, CAR-PSCA, CAR-PSCA-DAP10, CAR-PSCA- DAP10*3, CAR-GPC3 are (right According to group), CAR-GPC3-41BB, CAR-GPC3-DAP10, CAR-GPC3-41BB-CD28, CAR-GPC3-41BB-DAP10) and The LEGO-2A-eGFP carrier DNA fragments of linearisation.

4) it is by T4 DNA ligases (Invitrogent companies), the LEGO-2A-eGFP of linearisation is viscous with band respectively The synthesis of property end DNA fragmentation (CAR-Mes, CAR-Mes-DAP10, CAR-Mes-41BB, CAR-Mes-DAP10*3, CAR-Mes-DAP10*9、CAR-Mes-41BB-CD28、CAR-Mes-41BB-DAP10、CAR-PSCA、 CAR-PSCA- DAP10, CAR-PSCA-DAP10*3, CAR-GPC3 (control group), CAR-GPC3-41BB, CAR-GPC3-DAP10, CAR- GPC3-41BB-CD28, CAR-GPC3-41BB-DAP10) connection, obtain CAR plasmid transfer vectors (LEGO-CAR-Mes-2A- EGFP、LEGO-CAR-Mes-41BB-2A-EGFP、 LEGO-CAR-Mes-DAP10-2A-EGFP、LEGO-CAR-Mes- DAP10*3-2A-EGFP、 LEGO-CAR-Mes-DAP10*9-2A-EGFP、LEGO-CAR-Mes-41BB-CD28-2A-EGFP、 LEGO-CAR-Mes-41BB-DAP10-2A-EGFP、LEGO-CAR-PSCA-2A-EGFP、 LEGO-CAR-PSCA-DAP10- 2A-EGFP、LEGO-CAR-PSCA-DAP10*3-2A-EGFP LEGO-CAR-GPC3-2A-EGFP、LEGO-CAR-GPC3- 41BB-2A-EGFP、 LEGO-CAR-GPC3-DAP10-2A-EGFP、LEGO-CAR-GPC3-41BB-CD28-2A-EGFP、 LEGO-CAR-GPC3-41BB-DAP10-2A-EGFP)。

Embodiment 2, the slow virus packaging of CAR plasmids

CAR plasmids and related control plasmid of the present invention prepared by embodiment 1 is subjected to slow virus packaging, specific steps It is as follows：

1) 293T cells are cultivated in 10cm culture dishes, culture medium is：DMEM high glucose mediums+10%FBS (tire ox bloods Clearly)+1% dual anti-(100 × Pen .- Strep mixed solution)；

2) when the 293T cell densities in 150mm culture dishes reach 80-90%, culture medium is changed：DMEM high glucose mediums + 1%FBS+1% is dual anti-；

3) after changing medium culture 2-6 hours, with PEI respectively by LEGO-CAR-EGFP plasmids (LEGO-CAR-Mes- 2A-EGFP、LEGO-CAR-Mes-41BB-2A-EGFP、 LEGO-CAR-Mes-DAP10-2A-EGFP、LEGO-CAR-Mes- DAP10*3-2A-EGFP、 LEGO-CAR-Mes-DAP10*9-2A-EGFP、LEGO-CAR-Mes-41BB-CD28-2A-EGFP、 LEGO-CAR-Mes-41BB-DAP10-2A-EGFP、LEGO-CAR-PSCA-2A-EGFP、 LEGO-CAR-PSCA-DAP10- 2A-EGFP、LEGO-CAR-PSCA-DAP10*3-2A-EGFP、 LEGO-CAR-GPC3-2A-EGFP、LEGO-CAR-GPC3- 41BB-2A-EGFP、 LEGO-CAR-GPC3-DAP10-2A-EGFP、LEGO-CAR-GPC3-41BB-CD28-2A-EGFP、 LEGO-CAR-GPC3-41BB-DAP10-2A-EGFP) or blank control plasmid LEGO-EGFP is packed with slow virus aid in respectively Plasmid psPAX2, pMD2.G co-transduction enters 293T cells, adds reagent and dosage is as follows：

4) 24,48 and 72 hours after conversion, culture medium supernatant is collected, and adds fresh culture (the high sugar of DMEM Culture medium+1%FBS+1% is dual anti-)；

5) culture medium supernatant is collected and finished, after supernatant 2500g is centrifuged 0.5 hour (optional step)；

6) centrifugation supernatant is taken, after being filtered with 0.45um filters, is centrifuged 1.5 hours using Ultracentrifuge 28000rpm (optional step)；

7) after ultracentrifugation, gently remove supernatant, add 200ul PBS, being placed in 4 degree of 12-16 hours dissolves, and produces CAR slow virus or blank control GFP slow virus (optional step)；

8) after virolysis, collect viral solution and be sub-packed in PCR pipe, freeze stand-by in -80 DEG C.

Embodiment 3, t cell activation and CAR slow-virus infections

1. T cell isolates and purifies：The mononuclearcell in blood is isolated by Ficoll density gradient methods, through red thin After the cracking of cellular lysate liquid removes red blood cell, then T cell gone out by MACS Pan-T magnetic bead sortings；

2. the T cell sorted out culture medium (AIM-V culture medium+5%FBS+ penicillin 100U/ml+ streptomysins 0.1mg/ml is diluted to cell concentration 2.5 × 10⁶Individual/ml is stand-by；

3. by the magnetic bead (rule of origin for being coated with CD2, CD3, CD28 antibody：Germany U.S. day Ni) stimulate T cell, that is, it is coated with Magnetic bead is with T cell with 1:2 ratios mix, and T cell final densities should be 5 × 10⁶Individual/ml/cm2.After mixing, be placed in 37 DEG C, 5% CO₂Incubator culture stimulates 48 hours.

4. slow-virus transfection T cell：Magnetic bead in the T cell of activation-magnetic bead mixed liquor is removed by magnetic fields, 300g centrifuges 5min, removes supernatant, is resuspended with fresh culture, is separately added into expression CAR and GFP (blank control) slow virus (disease Malicious addition is MOI=10) after, 8 μ g/ml of addition polybrene and 300IU/ml IL-2.37 DEG C are placed in, 5%CO₂Culture After case culture 24h, 300g centrifugation 5min, supernatant is removed, is resuspended with the fresh culture of the IL-2 containing 300IU/ml, produces overexpression The T cell of CAR plasmids.

5. CAR T cells expand：CAR T cell density is maintained 1 × 10⁶Individual/ml or so, carried out once per 2-3 days Half amount changes liquid.After two weeks, amplifiable 100 times of CAR T cell numbers.Cell positive GFP is the successful cell of transfection, and GFP is positive Ratio is detected by streaming, that is, obtains CAR T cells, respectively abbreviation CAR-Mes (also abbreviation M28z in accompanying drawing), CAR- Mes-41BB (also abbreviation MBBz in accompanying drawing), CAR-Mes-DAP10 (also abbreviation M28z10 in accompanying drawing), CAR-Mes-DAP10*3 (also abbreviation M28z10*3 in accompanying drawing), CAR-Mes-DAP10*9 (also abbreviation M28z10*9 in accompanying drawing), CAR-Mes-41BB- CD28 (also abbreviation MBB28z in accompanying drawing), CAR-Mes-41BB-DAP10 (also abbreviation MBBz10 in accompanying drawing), CAR-PSCA (accompanying drawings In also abbreviation P28z), CAR-PSCA-DAP10 (also abbreviation P28z10 in accompanying drawing), CAR-PSCA-DAP10*3 it is (also simple in accompanying drawing Claim P28z10*3), CAR-GPC3 (also abbreviation G28z in accompanying drawing), CAR-GPC3-41BB (also abbreviation GBBz in accompanying drawing), CAR- GPC3-DAP10 (also abbreviation G28z10 in accompanying drawing), CAR-GPC3-41BB-CD28 (also abbreviation GBB28z in accompanying drawing), CAR- GPC3-41BB-DAP10 (also abbreviation GBBz10 in accompanying drawing) or blank control T cell (GFP-T) ratio.

Efficiency (the figure of CAR-Mes, CAR-Mes-DAP10 slow virus carrier transfecting T cells is detected by Flow Cytometry 2C), as a result show：DAP10 integration has no effect on the transfection efficiency of CAR T cells.

Embodiment 4, CAR-Mes-DAP10 T cells identify the effect of killing tumour in vitro

External relatively GFP T (blank control), CAR-Mes T (negative control), CAR-Mes-DAP10, CAR-Mes- Identification killing ability of DAP10*3, CAR-Mes-DAP10*9 T cell to lung carcinoma cell, tumour cell select A549-GL bands The Lu-csf-1 of luciferase.

GFP T (blank control) prepared by embodiment 3, CAR-Mes T (negative control), CAR-Mes-DAP10T, CAR-Mes-DAP10*3 T, CAR-Mes-DAP10*9 T cells respectively with 1x10⁴Tumour cell A549-GL with 2:1、 1： 1、1：2、1：4、1：8 ratio (E：T ratios) mixing, it is added in 96 hole U-shaped boards, every group sets 3 multiple holes, 250g centrifugations 5min Afterwards, 37 DEG C, 5%CO are placed in₂Incubator co-cultures.

The quantitative killing-efficiency appraisal procedure of luciferase (Luciferase)：CAR T cells co-culture (real with tumour cell Control group is tested individually to cultivate for tumour cell) 18 hours afterwards, the luciferase in addition 100ul/ holes in 96 porocyte culture plates Substrate (1 ×), cell is resuspended and mixed, RLU (relative light unit) is determined by multi-function microplate reader immediately, surveyed Fix time and be set to 1 second.Killing ratio calculation formula：100% × (control wells reading-experimental port reading)/control wells reading (is not added with The blank group number-reading of cell can be ignored)；Shown in its result such as Fig. 2 (A)-(B), Fig. 3 (A)-(B).

By CAR-Mes-DAP10 and CAR-Mes T cells with E:T=1：2 are carried out 3 times with A549-GL tumour cells respectively Mixing co-cultures, and the mixing co-cultivation time is after 24 hours, 72 hours every time, is detected respectively by Flow Cytometry T cell activation surface marker CD25 (C), CD69 (D) and de- of T cells in CAR-Mes-DAP10 and CAR-Mes T cells Cell proportion positive grain surface marker CD107a (E), as a result such as Fig. 3 (C), 3 (D) and 3 (E).

As a result show, CAR-MES-DAP10 T, CAR-MES-DAP10*3, CAR-MES-DAP10*9 T cell are to tumour Cell A549-GL Cytotoxicity in vitro efficiency is all significantly higher than CAR-MES T cells, in E：T (i.e. Effector T cells with The ratio of Target target cells) in the case of very little, CAR-MES-DAP10, CAR-MES-DAP10*3, CAR-MES-DAP10* 9 T cells can also show very strong anti-tumor activity, be significantly higher than CAR-MES T cells, and CAR-MES-DAP10*3, The killing ability of CAR-MES-DAP10*9 T cells is then slightly above CAR-MES-DAP10 T cells respectively；CAR-Mes-DAP10 Cell its surface C D25, CD69 and CD107a marks after by target cell stimulation significantly raise, and this shows that DAP10 is total to Stimulation molecule can be obviously improved the activation level and cytotoxic effect of CAR T cells.

Embodiment 5, CAR-PSCA-DAP10 T cells identify the effect of killing tumour in vitro

External relatively GFP T (blank control), CAR-PSCA T (negative control), CAR-PSCA-DAP10 T cells are to lung The identification killing ability of cancer cell, tumour cell select Lu-csf-1s of the A549-GL with luciferase.Experimental method is such as Described in embodiment 4.

As a result show, CAR-PSCA-DAP10 T are significantly higher than to tumour cell A549-GL Cytotoxicity in vitro efficiency CAR-PSCA T cells, in E：In the case of T (i.e. Effector T cells and the ratio of Target target cells) very little, CAR- PSCA-DAP10 cells can also show very strong anti-tumor activity, be significantly higher than CAR-PSCA T cells (see Fig. 4 (A) and Fig. 4 (B)), show：Significantly increase effect of the DAP10- intracellular segment structure domains to CAR T cell killing abilities is not limited to list One tumor targets Mesothelin.

Embodiment 6, CAR-Mes-DAP10 T cells and CAR-Mes-41BB-CD28 identify killing tumor effect in vitro Compare

External relatively GFP T (blank control), CAR-Mes T (negative control), CAR-Mes-DAP10 T (can be referred to as PSCA-DAP10), identification killing ability of the CAR-Mes-41BB-CD28 T cells to lung carcinoma cell, tumour cell are selected The H460+Mes-GL human lung cancers of Lu-csf-1s of the A549-GL with luciferase and overexpression Mesothelin antigens are thin Born of the same parents system.Experimental method is as described in Example 4.

As a result show, in two kinds of cell line, CAR-Mes-DAP10 T and CAR-Mes-41BB-CD28 T cells are killed Hinder function and be all remarkably higher than CAR-Mes T cells, and CAR-Mes-DAP10 T and CAR-Mes-41BB-CD28 T cells are killed Hinder efficiency then substantially quite, but because DAP10 genetic fragments are small, do not influence transfection efficiency and CAR carrier plasticity, then DAP10 Costimulatory molecules has higher application value than 41BB costimulatory molecules in the application of CAR T cells.(Fig. 5 (A) and Fig. 5 (B))

Embodiment 7, CAR-GPC3-DAP10 cells to the Cytotoxicity in vitro effects of liver cancer cells and compare

External relatively GFP T (blank control), CAR-GPC3 T (negative control), CAR-GPC3-DAP10 T, CAR- Identification killing ability of the GPC3-41BB-CD28 T cells to liver cancer cells, tumour cell select the HepG2- with luciferase GL and HC04-GL human hepatocellular carcinoma cell line and HC04 people's liver cell system.Experimental method is as described in Example 4.

As a result show, in two kinds of cell line, CAR-GPC3-DAP10 T and CAR-GPC3-41BB-CD28 T cells Killing ability is all remarkably higher than CAR-GPC3 T cells (CAR-GPC3-CD28-CD3 ζ T cells of the prior art, abbreviation G28z), and the killing-efficiency of CAR-GPC3-DAP10 T and CAR-GPC3-41BB-CD28 T cells then substantially quite, further Confirm embodiment 6：DAP10 costimulatory moleculeses apply valency in the application of CAR T cells than 41BB costimulatory molecules with higher Value.(Fig. 6 (A) and Fig. 6 (B))

Embodiment 8, CAR-Mes-DAP10 T cells and the cytokine secretion after tumour cell co-cultivation

1) by 2 × 10⁵Individual tumour cell A549-GL is inoculated in 24 well culture plates per hole, overnight adhere-wall culture, then by the positive Cell number is up to 1 × 10⁵Individual GFP T (blank control), CAR-Mes T (negative control), CAR-Mes-DAP10 T, CAR- Mes-DAP10*3 T, CAR-Mes-DAP10*9 T structure cell (as prepared by embodiment 3) are with A549-GL cells with E：T= 1:2 mixing, are placed in 37 DEG C, 5%CO₂Incubator co-cultures；

2) co-culture 18 hours after, take GFPT, CAR-Mes T, CAR-Mes-DAP10 T, CAR-Mes-DAP10*3 T, The medium supernatant that CAR-Mes-DAP10*9 T cells co-culture with tumour cell respectively, interleukin-22 is detected by ELISA (IL-2), interferon gamma (IFN-γ), granzyme B (GZMB) and granulocyte-macrophage colony stimutaing factor (GM-CSF) water It is flat, as a result respectively as shown in Fig. 7 (A), 7 (B), 7 (C) and 7 (D)；

3) CAR-Mes-DAP10, CAR-Mes-DAP10*3, CAR-Mes-DAP10*9 T cell secretory immune effector Interleukin-22, interferon gamma, granzyme B, GM-CSF (CAR-Mes-DAP10) level are significantly higher than CAR-Mes T cells, this Illustrate that DAP10 intracellular domain can greatly improve CAR T cells interleukin-22, interferon gamma, granzyme B, GM-CSF secretions, can It is obviously promoted T cell propagation and tumor-killing effect.

Identification killing tumour in embodiment 9, CAR-Mes-DAP10 T cells body

1) by 5 × 10⁵It is subcutaneous that individual A549-GL cell transplantations enter NOD/SCID IL2rg-/- immunodeficient mouse groin, Build mice model of lung cancer；

2) it is respectively 3 × 10 in mice model of lung cancer medium sized vein injection GFP positive cell numbers after tumour transplatation 3 days⁶Individual GFP T, CAR-Mes T, CAR-Mes-41BB-CD28 T, CAR-Mes-DAP10 T cells, it is empty plus the NC for not injecting cell White control group, totally five experimental groups, four repetitions of every group of setting；

3) mouse was all put to death in the 36th day after tumour transplatation, peels off tumor mass, and analyze record tumor weight and body Product (respectively such as Fig. 8 (A) and Fig. 8 (B)), is made single cell suspension by the ground screen cloth of tumor mass, is detected by drain cell art Tumor-infiltrated T cell ratio (such as Fig. 9).

As a result show, CAR-Mes-DAP10 T cells and CAR-Mes-41BB-CD28 T cells are moved in lung cancer tumor xenogenesis Gross tumor volume can be reduced significantly by planting in model, prompt CAR-Mes-DAP10 T cells the killing-efficiency higher to tumour cell, and Inhibitory action of the tumor microenvironment to T cell can be overcome.By contrast, CAR-Mes T cells are in lung cancer tumor heterograft Then it is difficult to reduce tumour in model (relative to NC and GFPT control groups).To the analysis shows of tumor-infiltrated T cell, CAR-Mes There is very rare (total T cell ratio is less than 1%) in T cell, and CAR-Mes-DAP10 T cells have ratio and shown in tumour Write rise (total T cell ratio is more than 3%, Fig. 9)；These result explanations：DAP10 intracellular domain can lift T cell swollen Propagation survival ability in knurl microenvironment, the inhibitory action of tumor microenvironment is overcome, so as to strengthen the tumour in CAR T cell bodies Fragmentation effect.

Embodiment 10, contrast CAR-Mes-41BB-DAP10 T and CAR-Mes-41BB T cell identify killing tumour in vitro Effect

External relatively GFP T (blank control), CAR-Mes-41BB T (negative control), CAR-Mes-41BB-DAP10 T The identification killing ability of Cells on Lung Cancer cell, tumour cell select people lungs of the A549-GL and H460+Mes-GL with luciferase Gland cell system.As described in Example 4, as a result such as Figure 10 (A) and 10 (B) is shown for experimental method.

GFP T (blank control), CAR-Mes T, CAR-Mes-41BB T, CAR-Mes- are compared by external continuous killing The continuation Tumor cytotoxicity of DAP10 T cells, tumor cell line select human lung adenocarcinoma cells of the A549-GL with luciferase System.By by GFP T, CAR-Mes T, CAR-Mes-41BB T, CAR-Mes-DAP10 T cells respectively with E:T=1：2 three times After being co-cultured 24 hours with tumour cell, hundred of CAR T cell killing tumor cells after co-culturing every time is detected by ELIASA Divide ratio, such as Figure 10 (C) (luciferase detection method is as described in Example 4).

As a result show, CAR-Mes-41BB-DAP10 T and CAR-Mes-DAP10 T cell are to tumour cell A549-GL's Cytotoxicity in vitro efficiency is all significantly higher than CAR-Mes T, CAR-Mes-41BB T cells, and in external continuous killing experiments, CAR- Mes-DAP10 T lasting killing ability is stronger.Then show that the CAR T cells containing DAP10 can further enhance CAR T cells Tumour immunity lethal effect, continuation lethal effect especially for a long time, multiple, show the CAR T cells containing DAP10 in people There is higher application value in in-vivo tumour killing.

Embodiment 11, contrast body of CAR-GPC3-41BB-DAP10 T and the CAR-GPC3-41BB T cell to liver cancer cells Outer lethal effect

External relatively GFP T (blank control), CAR-GPC3-41BB T (negative control), CAR-GPC3-41BB-DAP10 Identification killing ability of the T cell to liver cancer cells, tumour cell select HepG2-G L and HC04-GL with luciferase mark Human hepatocellular carcinoma cell line.Experimental method is as described in Example 4.

As a result show, in two kinds of cell line, the killing ability of CAR-GPC3-41BB-DAP10 T cells is all remarkably higher than CAR-GPC3-41BB T cells (Figure 11 (A) and Figure 11 (B)), i.e., further prove that integrating DAP10 costimulatory moleculeses can effectively increase The tumor-killing effect of strong CAR T cells.

Identification killing tumour in embodiment 12, CAR-GPC3-DAP10 T cells body

1) by 5 × 10⁵Individual HepG2-GL cell transplantations enter NOD/SCID IL2rg-/- immunodeficient mouse groin skin Under, build liver cancer mouse model；

2) it is respectively 3 × 10 in liver cancer mouse model medium sized vein injection GFP positive cell numbers after tumour transplatation 3 days⁶Individual GFP T, CAR-GPC3 T, CAR-GPC3-DAP10 T cells, totally three experimental groups, four repetitions of every group of setting；

3) mouse was all put to death in the 36th day after tumour transplatation, peels off tumor mass, and analyze record gross tumor volume (as schemed 12)。

As a result show, CAR-GPC3 T, CAR-GPC3-DAP10 T cells can show in lung cancer tumor heteroplastic transplantation model Write and reduce gross tumor volume, and CAR-GPC3-DAP10 T cell effects are better than CAR-GPC3 T cells, prompt CAR-Mes- DAP10 T cells have higher killing-efficiency to tumour cells such as liver cancer.

Applicant states that the present invention illustrates product, purposes and its occupation mode of the present invention by above-described embodiment, but The invention is not limited in above-mentioned detailed use of commodity and occupation mode, that is, do not mean that the present invention have to rely on above-mentioned detailed use of commodity and Occupation mode could be implemented.Person of ordinary skill in the field produces it will be clearly understood that any improvement in the present invention to the present invention The equivalence replacement of each raw material of product and the addition of auxiliary element, the selection of concrete mode etc., all fall within protection scope of the present invention and Within the scope of open.

Sequence table

<110>Vivo biodistribution Pharmaceutical Technology Co., Ltd of Shenzhen

<120>A kind of nucleic acid molecules for strengthening T cell antitumor activity

<130> 2017

<141> 2017-09-30

<150> 2017100547591

<151> 2017-01-24

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8280

<212> DNA

<213>Artificial sequence ()

<400> 1

gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60

atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120

gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 180

tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 240

attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 300

atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 360

acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 420

tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 480

tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 540

attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 600

tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 660

ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 720

accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 780

gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 840

ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 900

aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 960

tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1020

gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1080

ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1140

ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1200

ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1260

aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1320

tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1380

caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1440

aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1500

aaagtaagac caccgcacag caagcggccg gccgcgctga tcttcagacc tggaggagga 1560

gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca 1620

ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg 1680

ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg 1740

tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac 1800

aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc 1860

aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg 1920

gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt 1980

tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga 2040

gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa 2100

gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt 2160

aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta 2220

ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca 2280

ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata 2340

gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatcggca 2400

ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg 2460

ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca 2520

aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga 2580

cagcagagat ccagtttggt tagtaccggg cccgctctag tcgaggtcga cggtatcgat 2640

aagctcgctt cacgagattc cagcaggtcg agggacctaa taacttcgta tagcatacat 2700

tatacgaagt tatattaagg gttccaagct taagcggccg ctgaaagacc ccacctgtag 2760

gtttggcaag ctagcaggat tgtttatctt aggaggcatg cttactgtta aaagacagga 2820

tatgtttgaa gtggcttctg agaaaaatgg ttaagaaaat tatgacttaa aaatgtgaga 2880

gattttcaag tatattaatt tttttaactg tccaagtatt tgaaattctt atcatttgat 2940

taacacccat gagtgatatg tgtctggaat tgaggccaaa gcaagctcag ctaagaaata 3000

ctagcacagt gctgtcggcc ccgatgcggg actgcgtttt gaccatcata aatcaagttt 3060

atttttttaa ttaattgagc gaagctggaa gcagatgatg aattagagtc aagatggctg 3120

catgggggtc tccggcaccc acagcaggtg gcaggaagca ggtcaccgcg agagtctatt 3180

ttaggaagca aaaaaacaca attggtaaat ttatcacttc tggttgtgaa gaggtggttt 3240

tgcccaggcc cagatctgaa agtgctctac tgagcaaaac aacacctgga caatttgcgt 3300

ttctaaaata aggcgaggct gaccgaaact gaaaaggctt tttttaacta tctgaatttc 3360

atttccaatc ttagcttatc aactgctagt ttgtgcaaac agcatatcaa cttctaaact 3420

gcattcattt ttaaagtaag atgtttaaga aattaaacag tcttagggag agtttatgac 3480

tgtattcaaa aagtttttta aattagcttg ttatcccttc atgtgataac taatctcaaa 3540

tactttttcg atacctcaga gcattatttt cataatgact gtgttcacaa tctttttagg 3600

ttaactcgtt ctctctttgt gattaaggag aaacactttg atattctgat agagtggcct 3660

tcattttagt atttttcaag accacttttc aactactcac tttaggataa gttttaggta 3720

aaatgtgcat cattatcctg aattatttca gttaagcatg ttagttggtg gcataagaga 3780

aaactcaatc agatagctag ctgcagtaac gccattttgc aaggcatgga aaaataccaa 3840

accaagaata gagaagttca gatcaagggc gggtacatga aaatagctaa cgttgggcca 3900

aacaggatat ctgcggtgag cagtttcggc cccggcccgg ggccaagaac agatggtcac 3960

cgcagtttcg gccccggccc gaggccaaga acagatggtc cccagatatg gcccaaccct 4020

cagcagtttc ttaagaccca tcagatgttt ccaggctccc ccaaggacct gaaatgaccc 4080

tgcgccttat ttgaattaac caatcagcct gcttctcgct tctgttcgcg cgcttctgct 4140

tcccgagctc tataaaagag ctcacaaccc ctcactcggc gcgccagtcc tccgattgac 4200

tgagtcgccc ggatccgcga gctttgttta aacgcgatcg cgaaaattgt cgctcctgtc 4260

aaacaaactc ttaactttga tttactcaaa ctggctgggg atgtagaaag caatccaggt 4320

ccagctagca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc 4380

gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat 4440

gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc 4500

tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac 4560

cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc 4620

accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc 4680

gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc 4740

ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag 4800

cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg 4860

cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc 4920

gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat 4980

cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg 5040

tacaagtaac tcgagcggcg gaattcgtcg agggacctaa taacttcgta tagcatacat 5100

tatacgaagt tatacatgtt taagggttcc ggttccacta ggtacaattc gatatcaagc 5160

ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact 5220

atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg 5280

cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg 5340

aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa 5400

cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc 5460

ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg 5520

ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt 5580

ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt 5640

cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc 5700

cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc 5760

gataccgtcg acctcgatcg agacctagaa aaacatggag caatcacaag tagcaataca 5820

gcagctacca atgctgattg tgcctggcta gaagcacaag aggaggagga ggtgggtttt 5880

ccagtcacac ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc 5940

cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat 6000

atccttgatc tgtggatcta ccacacacaa ggctacttcc ctgattggca gaactacaca 6060

ccagggccag ggatcagata tccactgacc tttggatggt gctacaagct agtaccagtt 6120

gagcaagaga aggtagaaga agccaatgaa ggagagaaca cccgcttgtt acaccctgtg 6180

agcctgcatg ggatggatga cccggagaga gaagtattag agtggaggtt tgacagccgc 6240

ctagcatttc atcacatggc ccgagagctg catccggact gtactgggtc tctctggtta 6300

gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa 6360

taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac 6420

tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagcat gtgagcaaaa 6480

ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 6540

cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 6600

ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 6660

accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 6720

catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 6780

gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 6840

tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 6900

agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 6960

actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 7020

gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 7080

aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 7140

gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 7200

aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 7260

atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 7320

gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 7380

atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 7440

ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 7500

cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 7560

agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 7620

cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 7680

tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 7740

agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 7800

gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 7860

gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 7920

ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 7980

tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 8040

tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 8100

gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 8160

caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 8220

atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 8280

<210> 2

<211> 1550

<212> DNA

<213>Artificial sequence ()

<400> 2

gtttaaacat gcttctcctg gtgacaagcc ttctgctctg tgagttacca cacccagcat 60

tcctcctgat cccaagccag gtacagctgc agcagtcagg tccaggactc gtgacgccct 120

cgcagaccct ctcactcacc tgtgccatct ccggggacag tgtctctagc aacagtgcta 180

cttggaactg gatcaggcag tccccatcga gaggccttga gtggctggga aggacatact 240

acaggtccaa gtggtataac gactatgcag tatctgtgaa aagtcgaatg agcatcaacc 300

cagacacatc caagaaccag ttctccctgc agctgaactc tgtgactccc gaggacacgg 360

ctgtgtatta ctgtgcaaga ggaatgatga cttactatta cggtatggac gtctggggcc 420

aagggaccac ggtcaccgtc tcctcaggaa ttctaggatc cggtggcggt ggcagcggcg 480

gtggtggttc cggaggcggc ggttctcagc ctgtgctgac tcagtcgtct tccctctctg 540

catctcctgg agcatcagcc agtctcacct gcaccttgcg cagtggcatc aatgttggtc 600

cctacaggat atactggtac cagcagaagc cagggagtcc tccccagtat ctcctgaact 660

acaaatcaga ctcagataag cagcagggct ctggagtccc cagccgcttc tctggatcca 720

aagatgcttc ggccaatgca ggggttttac tcatctctgg gctccggtct gaggatgagg 780

ctgactatta ctgtatgatt tggcacagca gcgctgctgt gttcggagga ggcacccaac 840

tgaccgtcct ctccggaatt ctagaacaac agggtgcggc cgcaattgaa gttatgtatc 900

ctcctcctta cctagacaat gagaagagca atggaaccat tatccatgtg aaagggaaac 960

acctttgtcc aagtccccta tttcccggac cttctaagcc cttttgggtg ctggtggtgg 1020

ttgggggagt cctggcttgc tatagcttgc tagtaacagt ggcctttatt attttctggg 1080

tgaggagtaa gaggagcagg ctcctgcaca gtgactacat gaacatgact ccccgccgcc 1140

ccgggcccac ccgcaagcat taccagccct atgccccacc acgcgacttc gcagcctatc 1200

gctccagagt gaagttcagc aggagcgcag acgcccccgc gtaccagcag ggccagaacc 1260

agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg gacaagagac 1320

gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt 1380

acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg atgaaaggcg 1440

agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca gccaccaagg 1500

acacctacga cgcccttcac atgcaggccc tgccccctcg cgcgatcgcg 1550

<210> 3

<211> 1550

<212> DNA

<213>Artificial sequence ()

<400> 3

gtttaaacat gcttctcctg gtgacaagcc ttctgctctg tgagttacca cacccagcat 60

tcctcctgat cccaagccag gtacagctgc agcagtcagg tccaggactc gtgacgccct 120

cgcagaccct ctcactcacc tgtgccatct ccggggacag tgtctctagc aacagtgcta 180

cttggaactg gatcaggcag tccccatcga gaggccttga gtggctggga aggacatact 240

acaggtccaa gtggtataac gactatgcag tatctgtgaa aagtcgaatg agcatcaacc 300

cagacacatc caagaaccag ttctccctgc agctgaactc tgtgactccc gaggacacgg 360

ctgtgtatta ctgtgcaaga ggaatgatga cttactatta cggtatggac gtctggggcc 420

aagggaccac ggtcaccgtc tcctcaggaa ttctaggatc cggtggcggt ggcagcggcg 480

gtggtggttc cggaggcggc ggttctcagc ctgtgctgac tcagtcgtct tccctctctg 540

catctcctgg agcatcagcc agtctcacct gcaccttgcg cagtggcatc aatgttggtc 600

cctacaggat atactggtac cagcagaagc cagggagtcc tccccagtat ctcctgaact 660

acaaatcaga ctcagataag cagcagggct ctggagtccc cagccgcttc tctggatcca 720

aagatgcttc ggccaatgca ggggttttac tcatctctgg gctccggtct gaggatgagg 780

ctgactatta ctgtatgatt tggcacagca gcgctgctgt gttcggagga ggcacccaac 840

tgaccgtcct ctccggaatt ctagaacaac agggtgcggc cgcaattgaa gttatgtatc 900

ctcctcctta cctagacaat gagaagagca atggaaccat tatccatgtg aaagggaaac 960

acctttgtcc aagtccccta tttcccggac cttctaagcc cttttgggtg ctggtggtgg 1020

ttgggggagt cctggcttgc tatagcttgc tagtaacagt ggcctttatt attttctggg 1080

tgaggagtaa gaggagcagg ctcctgcaca gtgactacat gaacatgact ccccgccgcc 1140

ccgggcccac ccgcaagcat taccagccct atgccccacc acgcgacttc gcagcctatc 1200

gctccagagt gaagttcagc aggagcgcag acgcccccgc gtaccagcag ggccagaacc 1260

agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg gacaagagac 1320

gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt 1380

acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg atgaaaggcg 1440

agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca gccaccaagg 1500

acacctacga cgcccttcac atgcaggccc tgccccctcg cgcgatcgcg 1550

<210> 4

<211> 1659

<212> DNA

<213>Artificial sequence ()

<400> 4

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccaagcc aggtacagct gcagcagtca ggtccaggac tcgtgacgcc ctcgcagacc 120

ctctcactca cctgtgccat ctccggggac agtgtctcta gcaacagtgc tacttggaac 180

tggatcaggc agtccccatc gagaggcctt gagtggctgg gaaggacata ctacaggtcc 240

aagtggtata acgactatgc agtatctgtg aaaagtcgaa tgagcatcaa cccagacaca 300

tccaagaacc agttctccct gcagctgaac tctgtgactc ccgaggacac ggctgtgtat 360

tactgtgcaa gaggaatgat gacttactat tacggtatgg acgtctgggg ccaagggacc 420

acggtcaccg tctcctcagg aattctagga tccggtggcg gtggcagcgg cggtggtggt 480

tccggaggcg gcggttctca gcctgtgctg actcagtcgt cttccctctc tgcatctcct 540

ggagcatcag ccagtctcac ctgcaccttg cgcagtggca tcaatgttgg tccctacagg 600

atatactggt accagcagaa gccagggagt cctccccagt atctcctgaa ctacaaatca 660

gactcagata agcagcaggg ctctggagtc cccagccgct tctctggatc caaagatgct 720

tcggccaatg caggggtttt actcatctct gggctccggt ctgaggatga ggctgactat 780

tactgtatga tttggcacag cagcgctgct gtgttcggag gaggcaccca actgaccgtc 840

ctctccggaa ttctagaaca acagggtgcg gccgcaattg aagttatgta tcctcctcct 900

tacctagaca atgagaagag caatggaacc attatccatg tgaaagggaa acacctttgt 960

ccaagtcccc tatttcccgg accttctaag cccttttggg tgctggtggt ggttggggga 1020

gtcctggctt gctatagctt gctagtaaca gtggccttta ttattttctg ggtgaggagt 1080

aagaggagca ggctcctgca cagtgactac atgaacatga ctccccgccg ccccgggccc 1140

acccgcaagc attaccagcc ctatgcccca ccacgcgact tcgcagccta tcgctccaga 1200

gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 1260

aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1320

gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1380

ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1440

aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1500

gacgcccttc acatgcaggc cctgccccct cgcaaacggg gcagaaagaa actcctgtat 1560

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1620

tgccgatttc cagaagaaga agaaggagga tgtgaactg 1659

<210> 5

<211> 1778

<212> DNA

<213>Artificial sequence ()

<400> 5

gtttaaacat gcttctcctg gtgacaagcc ttctgctctg tgagttacca cacccagcat 60

tcctcctgat cccaagccag gtacagctgc agcagtcagg tccaggactc gtgacgccct 120

cgcagaccct ctcactcacc tgtgccatct ccggggacag tgtctctagc aacagtgcta 180

cttggaactg gatcaggcag tccccatcga gaggccttga gtggctggga aggacatact 240

acaggtccaa gtggtataac gactatgcag tatctgtgaa aagtcgaatg agcatcaacc 300

cagacacatc caagaaccag ttctccctgc agctgaactc tgtgactccc gaggacacgg 360

ctgtgtatta ctgtgcaaga ggaatgatga cttactatta cggtatggac gtctggggcc 420

aagggaccac ggtcaccgtc tcctcaggaa ttctaggatc cggtggcggt ggcagcggcg 480

gtggtggttc cggaggcggc ggttctcagc ctgtgctgac tcagtcgtct tccctctctg 540

catctcctgg agcatcagcc agtctcacct gcaccttgcg cagtggcatc aatgttggtc 600

cctacaggat atactggtac cagcagaagc cagggagtcc tccccagtat ctcctgaact 660

acaaatcaga ctcagataag cagcagggct ctggagtccc cagccgcttc tctggatcca 720

aagatgcttc ggccaatgca ggggttttac tcatctctgg gctccggtct gaggatgagg 780

ctgactatta ctgtatgatt tggcacagca gcgctgctgt gttcggagga ggcacccaac 840

tgaccgtcct ctccggaatt ctagaacaac agggtgcggc cgcaattgaa gttatgtatc 900

ctcctcctta cctagacaat gagaagagca atggaaccat tatccatgtg aaagggaaac 960

acctttgtcc aagtccccta tttcccggac cttctaagcc cttttgggtg ctggtggtgg 1020

ttgggggagt cctggcttgc tatagcttgc tagtaacagt ggcctttatt attttctggg 1080

tgaggagtaa gaggagcagg ctcctgcaca gtgactacat gaacatgact ccccgccgcc 1140

ccgggcccac ccgcaagcat taccagccct atgccccacc acgcgacttc gcagcctatc 1200

gctccagagt gaagttcagc aggagcgcag acgcccccgc gtaccagcag ggccagaacc 1260

agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg gacaagagac 1320

gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt 1380

acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg atgaaaggcg 1440

agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca gccaccaagg 1500

acacctacga cgcccttcac atgcaggccc tgccccctcg cttcgaactg tgcgcacgcc 1560

cacgccgcag ccccgcccaa gaagatggca aagtctacat caacatgcca ggcaggggcc 1620

tgtgcgcacg cccacgccgc agccccgccc aagaagatgg caaagtctac atcaacatgc 1680

caggcagggg cctgtgcgca cgcccacgcc gcagccccgc ccaagaagat ggcaaagtct 1740

acatcaacat gccaggcagg ggcacgcgtg cgatcgcg 1778

<210> 6

<211> 2204

<212> DNA

<213>Artificial sequence ()

<400> 6

gtttaaacat gcttctcctg gtgacaagcc ttctgctctg tgagttacca cacccagcat 60

tcctcctgat cccaagccag gtacagctgc agcagtcagg tccaggactc gtgacgccct 120

cgcagaccct ctcactcacc tgtgccatct ccggggacag tgtctctagc aacagtgcta 180

cttggaactg gatcaggcag tccccatcga gaggccttga gtggctggga aggacatact 240

acaggtccaa gtggtataac gactatgcag tatctgtgaa aagtcgaatg agcatcaacc 300

cagacacatc caagaaccag ttctccctgc agctgaactc tgtgactccc gaggacacgg 360

ctgtgtatta ctgtgcaaga ggaatgatga cttactatta cggtatggac gtctggggcc 420

aagggaccac ggtcaccgtc tcctcaggaa ttctaggatc cggtggcggt ggcagcggcg 480

gtggtggttc cggaggcggc ggttctcagc ctgtgctgac tcagtcgtct tccctctctg 540

catctcctgg agcatcagcc agtctcacct gcaccttgcg cagtggcatc aatgttggtc 600

cctacaggat atactggtac cagcagaagc cagggagtcc tccccagtat ctcctgaact 660

acaaatcaga ctcagataag cagcagggct ctggagtccc cagccgcttc tctggatcca 720

aagatgcttc ggccaatgca ggggttttac tcatctctgg gctccggtct gaggatgagg 780

ctgactatta ctgtatgatt tggcacagca gcgctgctgt gttcggagga ggcacccaac 840

tgaccgtcct ctccggaatt ctagaacaac agggtgcggc cgcaattgaa gttatgtatc 900

ctcctcctta cctagacaat gagaagagca atggaaccat tatccatgtg aaagggaaac 960

acctttgtcc aagtccccta tttcccggac cttctaagcc cttttgggtg ctggtggtgg 1020

ttgggggagt cctggcttgc tatagcttgc tagtaacagt ggcctttatt attttctggg 1080

tgaggagtaa gaggagcagg ctcctgcaca gtgactacat gaacatgact ccccgccgcc 1140

ccgggcccac ccgcaagcat taccagccct atgccccacc acgcgacttc gcagcctatc 1200

gctccagagt gaagttcagc aggagcgcag acgcccccgc gtaccagcag ggccagaacc 1260

agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg gacaagagac 1320

gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt 1380

acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg atgaaaggcg 1440

agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca gccaccaagg 1500

acacctacga cgcccttcac atgcaggccc tgccccctcg cttcgaactg tgcgcacgcc 1560

cacgccgcag ccccgcccaa gaagatggca aagtctacat caacatgcca ggcaggggcc 1620

tgtgcgcacg cccacgccgc agccccgccc aagaagatgg caaagtctac atcaacatgc 1680

caggcagggg cctgtgcgca cgcccacgcc gcagccccgc ccaagaagat ggcaaagtct 1740

acatcaacat gccaggcagg ggcctgtgcg cacgcccacg ccgcagcccc gcccaagaag 1800

atggcaaagt ctacatcaac atgccaggca ggggcctgtg cgcacgccca cgccgcagcc 1860

ccgcccaaga agatggcaaa gtctacatca acatgccagg caggggcctg tgcgcacgcc 1920

cacgccgcag ccccgcccaa gaagatggca aagtctacat caacatgcca ggcaggggcc 1980

tgtgcgcacg cccacgccgc agccccgccc aagaagatgg caaagtctac atcaacatgc 2040

caggcagggg cctgtgcgca cgcccacgcc gcagccccgc ccaagaagat ggcaaagtct 2100

acatcaacat gccaggcagg ggcctgtgcg cacgcccacg ccgcagcccc gcccaagaag 2160

atggcaaagt ctacatcaac atgccaggca ggggcgcgat cgcg 2204

<210> 7

<211> 1659

<212> DNA

<213>Artificial sequence ()

<400> 7

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccaagcc aggtacagct gcagcagtca ggtccaggac tcgtgacgcc ctcgcagacc 120

ctctcactca cctgtgccat ctccggggac agtgtctcta gcaacagtgc tacttggaac 180

tggatcaggc agtccccatc gagaggcctt gagtggctgg gaaggacata ctacaggtcc 240

aagtggtata acgactatgc agtatctgtg aaaagtcgaa tgagcatcaa cccagacaca 300

tccaagaacc agttctccct gcagctgaac tctgtgactc ccgaggacac ggctgtgtat 360

tactgtgcaa gaggaatgat gacttactat tacggtatgg acgtctgggg ccaagggacc 420

acggtcaccg tctcctcagg aattctagga tccggtggcg gtggcagcgg cggtggtggt 480

tccggaggcg gcggttctca gcctgtgctg actcagtcgt cttccctctc tgcatctcct 540

ggagcatcag ccagtctcac ctgcaccttg cgcagtggca tcaatgttgg tccctacagg 600

atatactggt accagcagaa gccagggagt cctccccagt atctcctgaa ctacaaatca 660

gactcagata agcagcaggg ctctggagtc cccagccgct tctctggatc caaagatgct 720

tcggccaatg caggggtttt actcatctct gggctccggt ctgaggatga ggctgactat 780

tactgtatga tttggcacag cagcgctgct gtgttcggag gaggcaccca actgaccgtc 840

ctctccggaa ttctagaaca acagggtgcg gccgcaattg aagttatgta tcctcctcct 900

tacctagaca atgagaagag caatggaacc attatccatg tgaaagggaa acacctttgt 960

ccaagtcccc tatttcccgg accttctaag cccttttggg tgctggtggt ggttggggga 1020

gtcctggctt gctatagctt gctagtaaca gtggccttta ttattttctg ggtgaggagt 1080

aagaggagca ggctcctgca cagtgactac atgaacatga ctccccgccg ccccgggccc 1140

acccgcaagc attaccagcc ctatgcccca ccacgcgact tcgcagccta tcgctccaga 1200

gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 1260

aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1320

gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1380

ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1440

aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1500

gacgcccttc acatgcaggc cctgccccct cgcaaacggg gcagaaagaa actcctgtat 1560

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1620

tgccgatttc cagaagaaga agaaggagga tgtgaactg 1659

<210> 8

<211> 1737

<212> DNA

<213>Artificial sequence ()

<400> 8

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccaagcc aggtacagct gcagcagtca ggtccaggac tcgtgacgcc ctcgcagacc 120

ctctcactca cctgtgccat ctccggggac agtgtctcta gcaacagtgc tacttggaac 180

tggatcaggc agtccccatc gagaggcctt gagtggctgg gaaggacata ctacaggtcc 240

aagtggtata acgactatgc agtatctgtg aaaagtcgaa tgagcatcaa cccagacaca 300

tccaagaacc agttctccct gcagctgaac tctgtgactc ccgaggacac ggctgtgtat 360

tactgtgcaa gaggaatgat gacttactat tacggtatgg acgtctgggg ccaagggacc 420

acggtcaccg tctcctcagg aattctagga tccggtggcg gtggcagcgg cggtggtggt 480

tccggaggcg gcggttctca gcctgtgctg actcagtcgt cttccctctc tgcatctcct 540

ggagcatcag ccagtctcac ctgcaccttg cgcagtggca tcaatgttgg tccctacagg 600

atatactggt accagcagaa gccagggagt cctccccagt atctcctgaa ctacaaatca 660

gactcagata agcagcaggg ctctggagtc cccagccgct tctctggatc caaagatgct 720

tcggccaatg caggggtttt actcatctct gggctccggt ctgaggatga ggctgactat 780

tactgtatga tttggcacag cagcgctgct gtgttcggag gaggcaccca actgaccgtc 840

ctctccggaa ttctagaaca acagggtgcg gccgcaattg aagttatgta tcctcctcct 900

tacctagaca atgagaagag caatggaacc attatccatg tgaaagggaa acacctttgt 960

ccaagtcccc tatttcccgg accttctaag cccttttggg tgctggtggt ggttggggga 1020

gtcctggctt gctatagctt gctagtaaca gtggccttta ttattttctg ggtgaggagt 1080

aagaggagca ggctcctgca cagtgactac atgaacatga ctccccgccg ccccgggccc 1140

acccgcaagc attaccagcc ctatgcccca ccacgcgact tcgcagccta tcgctccaga 1200

gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 1260

aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1320

gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1380

ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1440

aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1500

gacgcccttc acatgcaggc cctgccccct cgcaaacggg gcagaaagaa actcctgtat 1560

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1620

tgccgatttc cagaagaaga agaaggagga tgtgaactgt tcgaactgtg cgcacgccca 1680

cgccgcagcc ccgcccaaga agatggcaaa gtctacatca acatgccagg caggggc 1737

<210> 9

<211> 1796

<212> DNA

<213>Artificial sequence ()

<400> 9

cgcgatcgca cgcgtttcga agcgaggggg cagggcctgc atgtgaaggg cgtcgtaggt 60

gtccttggtg gctgtactga gaccctggta aaggccatcg tgccccttgc ccctccggcg 120

ctcgcctttc atcccaatct cactgtaggc ctccgccatc ttatctttct gcagttcatt 180

gtacaggcct tcctgagggt tcttccttct cggctttccc cccatctcag ggtcccggcc 240

acgtctcttg tccaaaacat cgtactcctc tcttcgtcct agattgagct cgttatagag 300

ctggttctgg ccctgctggt acgcgggggc gtctgcgctc ctgctgaact tcactctgga 360

gcgataggct gcgaagtcgc gtggtggggc atagggctgg taatgcttgc gggtgggccc 420

ggggcggcgg ggagtcatgt tcatgtagtc actgtgcagg agcctgctcc tcttactcct 480

cacccagaaa ataataaagg ccactgttac tagcaagcta tagcaagcca ggactccccc 540

aaccaccacc agcacccaaa agggcttaga aggtccggga aataggggac ttggacaaag 600

gtgtttccct ttcacatgga taatggttcc attgctcttc tcattgtcta ggtaaggagg 660

aggatacata acttcaattg cggccgcttt acccggagac agggagaggc tcttctgcgt 720

gtagtggttg tgcagagcct catgcatcac ggagcatgag aagacgttcc cctgctgcca 780

cctgctcttg tccacggtga gcttgctgta gaggaagaag gagccgtcgg agtccagcac 840

gggaggcgtg gtcttgtagt tgttctccgg ctgcccattg ctctcccact ccacggcgat 900

gtcgctggga tagaagcctt tgaccaggca ggtcaggctg acctggttct tggtcagctc 960

atcccgggat gggggcaggg tgtacacctg tggttctcgg ggctgcccgc cggatccacc 1020

gccagagcta cctccgccgc acggtgggca tgtgtgagtt ttgtcacaag atttgggctc 1080

gctcgagacg gtgaccaggg ttccttgacc ccagaacccc cccgttttac aataatagac 1140

ggcagtgtcc tcagcacgca ggctgttcat ctgcaggtag gctgtgtttt tggatgtgtc 1200

tgcgcttata gtggcacggc cctggaactt cgggacaaat tcagtgtcac cattctcagg 1260

atcaatccat gcaacccatt ccaggccctt accaggggcc tgacgcaccc agtgtatata 1320

gtagtcttta atgttgaagc cagaagctgc gcaggacaaa cggagtgagc cccctggctg 1380

cacaaggcca cccccagact ccaccagctg aacctctgag ctgccgccac ctccggagcc 1440

gcctccggag ccaccgccgc tagtactgcc ttttatctcc accttggtcc cctgtccgaa 1500

cgtgaatggg ctactactcc actgctgaca gtaataggtg gcgaaatctt ccggctgcag 1560

actgctaatg gtgagggtga agtctgtccc agacccggag ccactgaacc tagaagggac 1620

gccagaagcc agtttggatg tgtcatagat gagtcttttg ggagcttttc ctggtttctg 1680

ctggtaccag tgaatgaatc ttacacttga actggcactg caggtgatgg tgaccctatc 1740

ccccacagag gcggacaaag agcttggaga ttgggtcagc tgaatgtcgt ttaaac 1796

<210> 10

<211> 1862

<212> DNA

<213>Artificial sequence ()

<400> 10

gtttaaacga cattcagctg acccaatctc caagctcttt gtccgcctct gtgggggata 60

gggtcaccat cacctgcagt gccagttcaa gtgtaagatt cattcactgg taccagcaga 120

aaccaggaaa agctcccaaa agactcatct atgacacatc caaactggct tctggcgtcc 180

cttctaggtt cagtggctcc gggtctggga cagacttcac cctcaccatt agcagtctgc 240

agccggaaga tttcgccacc tattactgtc agcagtggag tagtagccca ttcacgttcg 300

gacaggggac caaggtggag ataaaaggca gtactagcgg cggtggctcc ggaggcggct 360

ccggaggtgg cggcagctca gaggttcagc tggtggagtc tgggggtggc cttgtgcagc 420

cagggggctc actccgtttg tcctgcgcag cttctggctt caacattaaa gactactata 480

tacactgggt gcgtcaggcc cctggtaagg gcctggaatg ggttgcatgg attgatcctg 540

agaatggtga cactgaattt gtcccgaagt tccagggccg tgccactata agcgcagaca 600

catccaaaaa cacagcctac ctgcagatga acagcctgcg tgctgaggac actgccgtct 660

attattgtaa aacggggggg ttctggggtc aaggaaccct ggtcaccgtc tcgagcgagc 720

ccaaatcttg tgacaaaact cacacatgcc caccgtgcgg cggaggtagc tctggcggtg 780

gatccggcgg gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggatgagc 840

tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg 900

ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc 960

tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc 1020

agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac cactacacgc 1080

agaagagcct ctccctgtct ccgggtaaag cggccgcaat tgaagttatg tatcctcctc 1140

cttacctaga caatgagaag agcaatggaa ccattatcca tgtgaaaggg aaacaccttt 1200

gtccaagtcc cctatttccc ggaccttcta agcccttttg ggtgctggtg gtggttgggg 1260

gagtcctggc ttgctatagc ttgctagtaa cagtggcctt tattattttc tgggtgagga 1320

gtaagaggag caggctcctg cacagtgact acatgaacat gactccccgc cgccccgggc 1380

ccacccgcaa gcattaccag ccctatgccc caccacgcga cttcgcagcc tatcgctcca 1440

gagtgaagtt cagcaggagc gcagacgccc ccgcgtacca gcagggccag aaccagctct 1500

ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag agacgtggcc 1560

gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg 1620

aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc 1680

ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc aaggacacct 1740

acgacgccct tcacatgcag gccctgcccc ctcgcctgtg cgcacgccca cgccgcagcc 1800

ccgcccaaga agatggcaaa gtctacatca acatgccagg caggggcacg cgtgcgatcg 1860

cg 1862

<210> 11

<211> 2012

<212> DNA

<213>Artificial sequence ()

<400> 11

gtttaaacga cattcagctg acccaatctc caagctcttt gtccgcctct gtgggggata 60

gggtcaccat cacctgcagt gccagttcaa gtgtaagatt cattcactgg taccagcaga 120

aaccaggaaa agctcccaaa agactcatct atgacacatc caaactggct tctggcgtcc 180

cttctaggtt cagtggctcc gggtctggga cagacttcac cctcaccatt agcagtctgc 240

agccggaaga tttcgccacc tattactgtc agcagtggag tagtagccca ttcacgttcg 300

gacaggggac caaggtggag ataaaaggca gtactagcgg cggtggctcc ggaggcggct 360

ccggaggtgg cggcagctca gaggttcagc tggtggagtc tgggggtggc cttgtgcagc 420

cagggggctc actccgtttg tcctgcgcag cttctggctt caacattaaa gactactata 480

tacactgggt gcgtcaggcc cctggtaagg gcctggaatg ggttgcatgg attgatcctg 540

agaatggtga cactgaattt gtcccgaagt tccagggccg tgccactata agcgcagaca 600

catccaaaaa cacagcctac ctgcagatga acagcctgcg tgctgaggac actgccgtct 660

attattgtaa aacggggggg ttctggggtc aaggaaccct ggtcaccgtc tcgagcgagc 720

ccaaatcttg tgacaaaact cacacatgcc caccgtgcgg cggaggtagc tctggcggtg 780

gatccggcgg gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggatgagc 840

tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg 900

ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc 960

tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc 1020

agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac cactacacgc 1080

agaagagcct ctccctgtct ccgggtaaag cggccgcaat tgaagttatg tatcctcctc 1140

cttacctaga caatgagaag agcaatggaa ccattatcca tgtgaaaggg aaacaccttt 1200

gtccaagtcc cctatttccc ggaccttcta agcccttttg ggtgctggtg gtggttgggg 1260

gagtcctggc ttgctatagc ttgctagtaa cagtggcctt tattattttc tgggtgagga 1320

gtaagaggag caggctcctg cacagtgact acatgaacat gactccccgc cgccccgggc 1380

ccacccgcaa gcattaccag ccctatgccc caccacgcga cttcgcagcc tatcgctcca 1440

gagtgaagtt cagcaggagc gcagacgccc ccgcgtacca gcagggccag aaccagctct 1500

ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag agacgtggcc 1560

gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg 1620

aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc 1680

ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc aaggacacct 1740

acgacgccct tcacatgcag gccctgcccc ctcgcttcga actgtgcgca cgcccacgcc 1800

gcagccccgc ccaagaagat ggcaaagtct acatcaacat gccaggcagg ggcctgtgcg 1860

cacgcccacg ccgcagcccc gcccaagaag atggcaaagt ctacatcaac atgccaggca 1920

ggggcctgtg cgcacgccca cgccgcagcc ccgcccaaga agatggcaaa gtctacatca 1980

acatgccagg caggggcacg cgtgcgatcg cg 2012

<210> 12

<211> 23

<212> PRT

<213>Artificial sequence ()

<400> 12

Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln Asp Gly Lys Val Tyr

1 5 10 15

Ile Asn Met Pro Gly Arg Gly

20

<210> 13

<211> 72

<212> DNA

<213>Artificial sequence ()

<400> 13

ctgtgcgcac gcccacgccg cagccccgcc caagaagatg gcaaagtcta catcaacatg 60

ccaggcaggg gc 72

<210> 14

<211> 1449

<212> DNA

<213>Artificial sequence ()

<400> 14

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccagatg ttgtgatgac ccaaactcca ctctccctgc ctgtcagtct tggagatcaa 120

gcctccatct cttgcagatc tagtcagagc cttgtacaca gtaatggaaa cacctattta 180

cattggtacc tgcagaagcc aggccagtct ccaaagctcc tgatctacaa agtttccaac 240

cgattttctg gggtcccaga caggttcagt ggcagtggat cagggacaga tttcacactc 300

aagatcagca gagtggaggc tgaggatctg ggagtttatt tctgctctca aaatacacat 360

gttcctccta cgttcggatc ggggaccaag ctggaaataa aaggtggagg cggttcaggc 420

ggaggtggca gcggcggtgg cgggtcgcag gttcaactgc agcagtctgg ggctgagctg 480

gtgaggcctg gggcttcagt gaagctgtcc tgcaaggctt cgggctacac atttactgac 540

tatgaaatgc actgggtgaa gcagacacct gtgcatggcc taaaatggat tggagctctt 600

gatcctaaaa ctggtgatac tgcctacagt cagaagttca agggcaaggc cacactgact 660

gcagacaaat cctccagcac agcctacatg gagctccgca gcctgacatc tgaggactct 720

gccgtctatt actgtacaag attctactcc tatacttact ggggccaagg gactctggtc 780

actgtctctg caattgaagt tatgtatcct cctccttacc tagacaatga gaagagcaat 840

ggaaccatta tccatgtgaa agggaaacac ctttgtccaa gtcccctatt tcccggacct 900

tctaagccct tttgggtgct ggtggtggtt gggggagtcc tggcttgcta tagcttgcta 960

gtaacagtgg cctttattat tttctgggtg aggagtaaga ggagcaggct cctgcacagt 1020

gactacatga acatgactcc ccgccgcccc gggcccaccc gcaagcatta ccagccctat 1080

gccccaccac gcgacttcgc agcctatcgc tccagagtga agttcagcag gagcgcagac 1140

gcccccgcgt accagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 1200

gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 1260

agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 1320

gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 1380

taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 1440

ccccctcgc 1449

<210> 15

<211> 1575

<212> DNA

<213>Artificial sequence ()

<400> 15

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccagatg ttgtgatgac ccaaactcca ctctccctgc ctgtcagtct tggagatcaa 120

gcctccatct cttgcagatc tagtcagagc cttgtacaca gtaatggaaa cacctattta 180

cattggtacc tgcagaagcc aggccagtct ccaaagctcc tgatctacaa agtttccaac 240

cgattttctg gggtcccaga caggttcagt ggcagtggat cagggacaga tttcacactc 300

aagatcagca gagtggaggc tgaggatctg ggagtttatt tctgctctca aaatacacat 360

gttcctccta cgttcggatc ggggaccaag ctggaaataa aaggtggagg cggttcaggc 420

ggaggtggca gcggcggtgg cgggtcgcag gttcaactgc agcagtctgg ggctgagctg 480

gtgaggcctg gggcttcagt gaagctgtcc tgcaaggctt cgggctacac atttactgac 540

tatgaaatgc actgggtgaa gcagacacct gtgcatggcc taaaatggat tggagctctt 600

gatcctaaaa ctggtgatac tgcctacagt cagaagttca agggcaaggc cacactgact 660

gcagacaaat cctccagcac agcctacatg gagctccgca gcctgacatc tgaggactct 720

gccgtctatt actgtacaag attctactcc tatacttact ggggccaagg gactctggtc 780

actgtctctg caattgaagt tatgtatcct cctccttacc tagacaatga gaagagcaat 840

ggaaccatta tccatgtgaa agggaaacac ctttgtccaa gtcccctatt tcccggacct 900

tctaagccct tttgggtgct ggtggtggtt gggggagtcc tggcttgcta tagcttgcta 960

gtaacagtgg cctttattat tttctgggtg aggagtaaga ggagcaggct cctgcacagt 1020

gactacatga acatgactcc ccgccgcccc gggcccaccc gcaagcatta ccagccctat 1080

gccccaccac gcgacttcgc agcctatcgc tccagagtga agttcagcag gagcgcagac 1140

gcccccgcgt accagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 1200

gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 1260

agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 1320

gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 1380

taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 1440

ccccctcgca aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga 1500

ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc gatttccaga agaagaagaa 1560

ggaggatgtg aactg 1575

<210> 16

<211> 1528

<212> DNA

<213>Artificial sequence ()

<400> 16

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccagatg ttgtgatgac ccaaactcca ctctccctgc ctgtcagtct tggagatcaa 120

gcctccatct cttgcagatc tagtcagagc cttgtacaca gtaatggaaa cacctattta 180

cattggtacc tgcagaagcc aggccagtct ccaaagctcc tgatctacaa agtttccaac 240

cgattttctg gggtcccaga caggttcagt ggcagtggat cagggacaga tttcacactc 300

aagatcagca gagtggaggc tgaggatctg ggagtttatt tctgctctca aaatacacat 360

gttcctccta cgttcggatc ggggaccaag ctggaaataa aaggtggagg cggttcaggc 420

ggaggtggca gcggcggtgg cgggtcgcag gttcaactgc agcagtctgg ggctgagctg 480

gtgaggcctg gggcttcagt gaagctgtcc tgcaaggctt cgggctacac atttactgac 540

tatgaaatgc actgggtgaa gcagacacct gtgcatggcc taaaatggat tggagctctt 600

gatcctaaaa ctggtgatac tgcctacagt cagaagttca agggcaaggc cacactgact 660

gcagacaaat cctccagcac agcctacatg gagctccgca gcctgacatc tgaggactct 720

gccgtctatt actgtacaag attctactcc tatacttact ggggccaagg gactctggtc 780

actgtctctg caattgaagt tatgtatcct cctccttacc tagacaatga gaagagcaat 840

ggaaccatta tccatgtgaa agggaaacac ctttgtccaa gtcccctatt tcccggacct 900

tctaagccct tttgggtgct ggtggtggtt gggggagtcc tggcttgcta tagcttgcta 960

gtaacagtgg cctttattat tttctgggtg aggagtaaga ggagcaggct cctgcacagt 1020

gactacatga acatgactcc ccgccgcccc gggcccaccc gcaagcatta ccagccctat 1080

gccccaccac gcgacttcgc agcctatcgc tccagagtga agttcagcag gagcgcagac 1140

gcccccgcgt accagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 1200

gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 1260

agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 1320

gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 1380

taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 1440

ccccctcgct tcgaactgtg cgcacgccca cgccgcagcc ccgcccaaga agatggcaaa 1500

gtctacatca acatgccagg caggggcc 1528

<210> 17

<211> 1575

<212> DNA

<213>Artificial sequence ()

<400> 17

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccagatg ttgtgatgac ccaaactcca ctctccctgc ctgtcagtct tggagatcaa 120

gcctccatct cttgcagatc tagtcagagc cttgtacaca gtaatggaaa cacctattta 180

cattggtacc tgcagaagcc aggccagtct ccaaagctcc tgatctacaa agtttccaac 240

cgattttctg gggtcccaga caggttcagt ggcagtggat cagggacaga tttcacactc 300

aagatcagca gagtggaggc tgaggatctg ggagtttatt tctgctctca aaatacacat 360

gttcctccta cgttcggatc ggggaccaag ctggaaataa aaggtggagg cggttcaggc 420

ggaggtggca gcggcggtgg cgggtcgcag gttcaactgc agcagtctgg ggctgagctg 480

gtgaggcctg gggcttcagt gaagctgtcc tgcaaggctt cgggctacac atttactgac 540

tatgaaatgc actgggtgaa gcagacacct gtgcatggcc taaaatggat tggagctctt 600

gatcctaaaa ctggtgatac tgcctacagt cagaagttca agggcaaggc cacactgact 660

gcagacaaat cctccagcac agcctacatg gagctccgca gcctgacatc tgaggactct 720

gccgtctatt actgtacaag attctactcc tatacttact ggggccaagg gactctggtc 780

actgtctctg caattgaagt tatgtatcct cctccttacc tagacaatga gaagagcaat 840

ggaaccatta tccatgtgaa agggaaacac ctttgtccaa gtcccctatt tcccggacct 900

tctaagccct tttgggtgct ggtggtggtt gggggagtcc tggcttgcta tagcttgcta 960

gtaacagtgg cctttattat tttctgggtg aaacggggca gaaagaaact cctgtatata 1020

ttcaaacaac catttatgag accagtacaa actactcaag aggaagatgg ctgtagctgc 1080

cgatttccag aagaagaaga aggaggatgt gaactgagga gtaagaggag caggctcctg 1140

cacagtgact acatgaacat gactccccgc cgccccgggc ccacccgcaa gcattaccag 1200

ccctatgccc caccacgcga cttcgcagcc tatcgctcca gagtgaagtt cagcaggagc 1260

gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1320

cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1380

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1440

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1500

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1560

gccctgcccc ctcgc 1575

<210> 18

<211> 1653

<212> DNA

<213>Artificial sequence ()

<400> 18

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccagatg ttgtgatgac ccaaactcca ctctccctgc ctgtcagtct tggagatcaa 120

gcctccatct cttgcagatc tagtcagagc cttgtacaca gtaatggaaa cacctattta 180

cattggtacc tgcagaagcc aggccagtct ccaaagctcc tgatctacaa agtttccaac 240

cgattttctg gggtcccaga caggttcagt ggcagtggat cagggacaga tttcacactc 300

aagatcagca gagtggaggc tgaggatctg ggagtttatt tctgctctca aaatacacat 360

gttcctccta cgttcggatc ggggaccaag ctggaaataa aaggtggagg cggttcaggc 420

ggaggtggca gcggcggtgg cgggtcgcag gttcaactgc agcagtctgg ggctgagctg 480

gtgaggcctg gggcttcagt gaagctgtcc tgcaaggctt cgggctacac atttactgac 540

tatgaaatgc actgggtgaa gcagacacct gtgcatggcc taaaatggat tggagctctt 600

gatcctaaaa ctggtgatac tgcctacagt cagaagttca agggcaaggc cacactgact 660

gcagacaaat cctccagcac agcctacatg gagctccgca gcctgacatc tgaggactct 720

gccgtctatt actgtacaag attctactcc tatacttact ggggccaagg gactctggtc 780

actgtctctg caattgaagt tatgtatcct cctccttacc tagacaatga gaagagcaat 840

ggaaccatta tccatgtgaa agggaaacac ctttgtccaa gtcccctatt tcccggacct 900

tctaagccct tttgggtgct ggtggtggtt gggggagtcc tggcttgcta tagcttgcta 960

gtaacagtgg cctttattat tttctgggtg aggagtaaga ggagcaggct cctgcacagt 1020

gactacatga acatgactcc ccgccgcccc gggcccaccc gcaagcatta ccagccctat 1080

gccccaccac gcgacttcgc agcctatcgc tccagagtga agttcagcag gagcgcagac 1140

gcccccgcgt accagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 1200

gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 1260

agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 1320

gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 1380

taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 1440

ccccctcgca aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga 1500

ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc gatttccaga agaagaagaa 1560

ggaggatgtg aactgttcga actgtgcgca cgcccacgcc gcagccccgc ccaagaagat 1620

ggcaaagtct acatcaacat gccaggcagg ggc 1653

Claims (10)

1. a kind of Chimeric antigen receptor, it includes ectodomain, membrane spaning domain and the intracellular domain that can combine antigen, Wherein, the intracellular domain includes the intracellular section sequence of DAP10 albumen.

2. Chimeric antigen receptor according to claim 1, it is characterised in that the intracellular section sequence of the DAP10 albumen is such as SEQ ID NO:Shown in 12；

Preferably, the intracellular domain includes the intracellular section sequence of 1~9, preferably 1~3 DAP10 albumen；

Preferably, the intracellular domain includes two or more DAP10 albumen to be linked together by series system Intracellular section sequence；

Preferably, the intracellular domain also includes intracellular costimulatory signal transduction domain and/or CD3 ζ signal transductions domain；

Preferably, the intracellular costimulatory signal transduction domain is selected from the function signal conducting structure domain of following protein：CD28、 4-1BB、TLR1、TLR2、TLR3、TLR4、CD27、CD28、OX40、CD30、CD40、PD-1、ICOS、LFA-1、CD2、CD7、 LIGHT、NKG2C、B7-H3、CDS、ICAM-1、GITR、BAFFR、HVEM、SLAMF7、NKp80、CD160、CD19、CD4、CD8 α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、 ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、 ITGB2、CD18、LFA-1、ITGB7、TNFR2、TRANCE/RANKL、DNAM1、SLAMF4、CD84、CD96、CEACAM1、 CRTAM、Ly9、CD160、PSGL1、CD100、CD69、SLAMF6、SLAM、BLAME、SELPLG、LTBR、LAT、GADS、SLP- 76th, any one in PAG/Cbp, NKp44, NKp30, NKp46, NKG2D or CD83 or at least two combination, it is preferably In TLR2,4-1BB or CD28 any one or at least two combination；

Preferably, the acceptor extracellular fragment that the antigen can be referred to reference to the ectodomain of antigen, or the antigen Specific antibody single chain variable fragment；

Preferably, the antigen is tumour antigen and/or tumour correlation helper antigenic；

Preferably, the tumour antigen and/or tumour correlation helper antigenic be selected from the β 1- integrins of 5T4, α 5,707-AP, AFP, ART-4, BAGE, beta-catenin/m, Bcr-abl, MN/C IX antibody, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19、CD20、CD22、CD25、CDC27/m、CD30、CD33、CD52、CD56、CD80、CDK4/m、CEA、CT、Cyp-B、DAM、 EGFR、ErbB3、ELF2M、EMMPRIN、EpCam、ETV6-AML1、G250、GAGE、GnT-V、Gp100、HAGE、HER-2/ new、HLA-A*0201-R170I、HPV-E7、HSP70-2M、HST-2、hTERT、hTRT、iCE、IGF-1R、IL-2R、IL-5、 KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/melan-A, MART-2/Ski, MC1R, Mesothelin, flesh ball egg In vain/m, PSCA, GPC3, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, PAP, proteinase-3, p190minor bcr-abl, Pml/RAR α, PRAME, PSA, PSM, PSMA, RAGE, RU1, RU2, SAGE, SART-1, SART-3, survivin protein, TEL/ AML1、TGFβ、TPI/m、TRP-1、TRP-2、TRP-2/INT2、VEGF、WT1、BCMA、CD123、PD-1、PD-L1/2、TIM3、 LAG3、A2ARA2AR、B7H3、B7H4、BTLA、IDO、KIR、CD160、2B4、VISTA、TSHR、CD171、CS-1、CLL-1、 GD3, Tn Ag, FLT3, CD38, CD44v6, B7-H3, CD117, IL-13Ra2, IL-11Ra, PRSS21, VEGFR2, Louis Antigen, CD24, PDGFR- β, SSEA-4, NCAM, CAIX, EphA2, GM1, sLe, TGS5, HMWMAA, OAcGD2, folacin receptor β、CD248、TEM7R、CLDN6、GPRC5D、CXORF61、CD97、CD179a、ALK、PLAC1、GloboH、NY-BR-1、UPK2、 HAVCR1, ADRB3, PANX3, GPR20, the compound of lymphocyte antigen 6, LY6K, OR51E2, TARP, ETS, SPA17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos related antigen 1, ERG, androgen receptor, cell periodic protein B 1, SART3、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、 In FCRL5, IGLL1, NY-Eso-1 or NY-Eso-B any one or at least two combination；

It is further preferred that the antigen be in PCSA, Mesothelin or GPC3 antigen any one or at least two Combination；It is further preferred that the ectodomain is specific recognition antigen Mesothelin, PSCA or GPC3 acceptor Extracellular fragment and/or antibody；

Preferably, the membrane spaning domain is membrane spaning domain corresponding to extracellular receptor seq or intracellular section costimulatory signal molecule pair The membrane spaning domain answered, preferably CD28 transmembrane regions.

3. the nucleic acid of coding Chimeric antigen receptor as claimed in claim 1 or 2 has at least 80% homogeneity with it, preferably At least nucleic acid of 90% homogeneity.

A kind of 4. Chimeric antigen receptor expression cell, wherein introducing nucleic acid as claimed in claim 3；Preferably, it is described thin Born of the same parents are immune effector cell, more preferably T cell, B cell, NK cells, NKT cells, BMDC or macrophage In any one or at least two combination.

5. preparing the method for Chimeric antigen receptor expression cell as claimed in claim 4, it includes will be as claimed in claim 7 Nucleic acid introduce cell the step of；Preferably, the cell is immune effector cell, more preferably T cell, B cell, NK In cell, NKT cells, BMDC or macrophage any one or at least two combination.

6. a kind of recombinant vector, it includes the nucleic acid described in claim 3；Preferably, the carrier be recombinant cloning vector, In eukaryotic expression recombinant plasmid or recombined lentivirus vector any one or at least two combination, preferably recombinant slow virus Carrier；

Preferably, the recombinant cloning vector include pUC18, pUC19, pMD19-T, pGM-T carrier, pUC57, pMAX or In pDC315 any one or at least two combination；

Preferably, the eukaryon expression plasmid include pCDNA3 serial carriers, pCDNA4 serial carriers, pCDNA5 serial carriers, In pCDNA6 serial carriers, pRL serial carriers, pUC57 carriers, pMAX or pDC315 any one or at least two group Close；

Preferably, the recombined lentivirus vector includes recombinant adenoviral vector, recombined glandulae correlation viral vectors, recombinant retroviral In viral vector, recombinant herpes simplex virus carrier or vaccinia virus recombinant carrier any one or at least two combination.

7. a kind of recombinant virus, it includes recombinant vector as claimed in claim 6 and packaging helper plasmid cotransfection lactation is thin The recombinant virus that born of the same parents obtain；

Preferably, a kind of Chimeric antigen receptor T cell, it by recombinant virus as claimed in claim 7 by being transfected into T cell Middle expression；

Preferably, the Chimeric antigen receptor as described in right 1 or 2, nucleic acid as claimed in claim 3, as claimed in claim 6 Recombinant vector or recombinant virus as claimed in claim 7 be used for expand CAR T cells.

8. a kind of pharmaceutical composition, it includes the Chimeric antigen receptor described in claim 1 or 2, core as claimed in claim 3 Chimeric antigen receptor expression cell sour, as claimed in claim 4 or expression vector as claimed in claim 6, and optionally Pharmaceutically acceptable auxiliary material.

9. Chimeric antigen receptor as claimed in claim 1 or 2, nucleic acid as claimed in claim 3, as claimed in claim 4 Chimeric antigen receptor expression cell or expression vector as claimed in claim 6 prepare treatment and/or prevention autoimmunity Purposes in the medicine of property disease or tumour；

Preferably, the tumour is solid tumor and/or blood tumor, preferably leukaemia, liver cancer or lung cancer.

A kind of 10. side for treating the subject with autoimmune disease and/or the disease related to the expression of tumour antigen Method, it includes the medicine for including the pharmaceutical composition described in claim 8 that effective dose is applied to the subject；

Preferably, the tumour is solid tumor and/or blood tumor, preferably leukaemia, liver cancer or lung cancer.