CN107141356A

CN107141356A - A kind of photoinduction dimer type Chimeric antigen receptor

Info

Publication number: CN107141356A
Application number: CN201710421652.6A
Authority: CN
Inventors: 应述欢; 高鹏; 李玉霞
Original assignee: Sheng Wu (beijing) Biotechnology Co Ltd
Current assignee: Sheng Wu (beijing) Biotechnology Co Ltd
Priority date: 2017-06-07
Filing date: 2017-06-07
Publication date: 2017-09-08
Anticipated expiration: 2037-06-07
Also published as: CN107141356B

Abstract

There is photoinduction dimer type Chimeric antigen receptor the invention discloses one kind, including two monomers, first monomer includes signal peptide, extracellular antigen binding domain, extracellular hinge area, transmembrane region, costimulatory signal conducting region and light-dependent control element and the functional domain of polymeric component 1 successively, and second comonomer includes signal peptide, extracellular affinity regions, transmembrane region, costimulatory signal conducting region, polymeric component 2 and T cell signal transduction area functional domain successively.Photoinduction dimer type of the present invention is embedding and antigen receptor is used for the immune response of mediate T cell, any monomer of the dimer is not when coupling, whether equal inactive, can be by being regulated and controled embedding and the polymerization and depolymerization of antigen receptor dimer by blue light illumination.

Description

A kind of photoinduction dimer type Chimeric antigen receptor

Technical field

The present invention relates to biomedical conversion field, more particularly to a kind of photoinduction dimer type Chimeric antigen receptor and its Encoding gene and application.

Background technology

Cancer rises year by year as public health problem, its morbidity and mortality, and cancer has become topmost dead Die one of reason.Adoptive immunotherapy is outside traditional operation treatment, radiotherapy and chemotherapy, to improve human body pair The critical treatment scheme of the immunological effect of tumour.Adoptive cellular immunotherapy（adoptive cellular Immunotherapy, ACI）Refer to the autologous or alloimmune cell infusion of Activation In Vitro to patient, to kill in patient's body Tumour cell, be one of most promising means of current treatment malignant tumour, in a variety of solid tumors and neoplastic hematologic disorder Good therapeutic effect is obtained in clinical treatment.Wherein, Chimeric antigen receptor（Chimeric antigen receptor, CAR）T cell Cure is to develop a kind of very fast new cellular immunotherapy technology in recent years, and effect is modified by technique for gene engineering T cell, overcomes tumor by local immunosupress microenvironment and host immune tolerance status, improves antitumor targeting ability of, killing Property and persistence.

CAR structures by extracellular antigen binding domain, transmembrane structure area and intracellular signal transduction district's groups into.Extracellular antigen binding domain The mainly single-stranded heavy chain of antigentic specificity monoclonal antibody（VL）And light chain（VH）Variable region composition, the two is connected with hinge area Connect to form single-chain antibody（Single chain fragment varible, scFv）.CAR by recognize tumour antigen scFv and Intracellular signal domain " ITAM（immunoreceptor tyrosine-based activation Motifs, ITAM）" genetic recombination is carried out in vitro, the T lymphocytes of patient are modified by Gene transfer techniques, patient T is drenched The thin cellular expression tumour antigen acceptor of bar, referred to as the T lymphocytes after purifying and extensive amplification is modified, Chimeric antigen receptor The T lymphocytes of modification（CAR-T）.

CAR technologies clinically achieve important achievement on the hematological system tumor even treatment of solid tumor, at present Develop into the third generation.First generation CAR passes through transmembrane domain and intracellular signal transduction area by single-chain antibody（ITAM）It is connected, ITAM is usually CD3 ζ or Fc ε RI γ；Second generation CAR intracellular signal transduction area introduces costimulatory molecules （Costimulatory molecule, CM）, predominantly CD28 molecules and 4-1BB molecules（CD137）；Third generation CAR is introduced Double costimulatory moleculeses（CM1 and CM2）, predominantly CD28 molecules are plus CD134 or CD137 etc..First generation CAR-T lymphocytes are ground Study carefully more, but most of experiments go back Shortcomings in terms of cell amplification, survival time in vitro, cytokine secretion, do not have Have and reach expected clinical effectiveness.Research shows that the complete activation of T lymphocytes depends on the work of dual signal and cell factor With.Wherein the first signal is specific signals, and the Antigenic Peptide-MHC compounds for recognizing antigen presenting cell surface by TCR are opened It is dynamic；Secondary signal is costimulatory signal, by the important costimulatory molecules such as CD28/B7, promotes IL-2 synthesis, and drench T Bar cell is fully activated and from apoptosis.Even if T lymphocytes and antigen contact, if without costimulatory signal, cell is difficult With function of bringing into normal play.Accordingly, the Chimeric antigen receptor only containing CD3 ζ sequences, if without costimulatory signal, it is also difficult With efficient activation CAR-T lymphocytes.Therefore, the dual signal theory according to T lymphocyte activations, the second generation and third generation CAR Such as CD28, CD137 costimulatory molecules is added in Chimeric antigen receptor, is lived with improving the cytotoxicity of T lymphocytes, propagation Property, maintain T lymphocyte responses, extension T lymphocyte time-to-live etc..Research confirms that the CAR-T lymphocytes of the second generation exist Tumor killing activity and internal time-to-live are superior to the first generation.At present, third generation CAR-T lymphocytes clinical practice is also compared It is few, therefore whether its security and validity are just necessarily better than second generation CAR-T lymphocytes, and what kind of costimulation point selected Sub-portfolio, in addition it is also necessary to further look at.

Although CAR-T targeting therapy on tumor cells are one of current most promising modality of cancer treatment, when too many Signal transduction reach after threshold value that the vivo immunization response triggered strongly may produce severes such as " cytokine storms ", very To initiation death.Therefore, activity " switch " control of increase CAR-T cells is the important interior of solution CAR-T toxicity problems Hold, be CAR-T significant in clinical popularization and application.

Chinese patent literature CN105142677A discloses a kind of heterodimer condition activation Chimeric antigen receptor, passes through two The activation of aggressiveness activator carries the Chimeric antigen receptor of dimer pair to realize the control to CAR-T cytoactives.For example, dimerization When body activator is rapamycin, dimer is to selection FKBP and FKBP, CnA, cyclophilin or FRG.But by exogenous Dimer activator is added to control Dimerized Chimeric antigen receptor to be influenceed by activation agent dose and drug absorption It is larger, and activator also likely to be present toxic side effect or adverse reaction in itself.Specific wavelength illumination is used as a kind of medical hand Section, with it is long-range, toxic side effect is small the features such as.The activity that CAR-T cells are controlled by photoinduction dimer is a kind of relative Safely and conveniently method, but this kind of research is relatively fewer at present, not yet has successful achievement in research to emerge.

The content of the invention

, should the invention provides the immune response that a kind of photoinduction dimer type is embedding and antigen receptor is for mediate T cell Any monomer of dimer is not when coupling, equal inactive, can be by whether embedding and anti-to regulate and control this by blue light illumination The polymerization and depolymerization of original receptor dimer.

Concrete technical scheme of the present invention is as follows：

A kind of photoinduction dimer type Chimeric antigen receptor, including two monomers, the first monomer includes signal peptide successively, extracellular anti- Former land, extracellular hinge area, transmembrane region, costimulatory signal conducting region and light-dependent control element and the functional structure of polymeric component 1 Domain, second comonomer includes signal peptide, extracellular affinity regions, transmembrane region, costimulatory signal conducting region, polymeric component 2 and T cell successively Signal transduction area functional domain, the light-dependent control element is ultraviolet and sensitive to blue light flavoprotein LOV2（aa 404-546 of Avena sativa Phototropin 1, NPH1-1, GenBank: AAC05083.1）, the polymeric component 1 is ssrA, LOV2 and ssrA keeps amino acid sequence such as SEQ ID No after rigidity fusion：Shown in 14, the polymeric component 2 is sspB, amino Acid sequence such as SEQ ID No：Shown in 22, each functional domain of first monomer and second comonomer is joined directly together or passed through Connect peptide connection.

Photoinduction dimer type Chimeric antigen receptor of the present invention, light-dependent control element and polymeric component 1（LOV2-ssrA） The light folding region of the blu-ray drives of formation is located at after the costimulatory signal conducting region of the first monomer, and adaptin sspB is single second Between body costimulatory signal conducting region and T cell signal transduction area.LOV2 C-terminal is the J α areas with αhelix, the rigidity Structure is the region that LOV2 is folded.SsrA and sspB is derived under bacterium, native state, and small peptide ssrA can be with sspB Adaptin is combined.

SsrA small peptide N-terminal sequences and the C-terminal sequence of J α in LOV2 have certain homology, and interconnection, which can be formed, to be had The peptide fragment of integral rigidity structure.

Under non-illuminated conditions, due to the curling of J α structures in LOV2, ssrA forms steric hindrance spatially, it is impossible to SspB is combined.

When blue light illumination of the structure by specific wavelength, LOV2 J α areas coiled structure is opened, and ssrA stretches, can Combined with sspB, form Light-inducible dimer.

Embedding and antigen receptor two monomers of restructuring of the present invention, can be gathered by the induction of specific wavelength blue light Close, so as to realize activity.When blue light illumination is closed, the dimer depolymerization, with frizzled receptor depolymerization, recombinate embedding and antigen by Body T cell specific activation path is eliminated.

Chimeric antigen receptor of the present invention, the antigen binding domain functional domain is selected from human antibody, humanization and resisted Body, antigen-binding fragment or its combination, can be complete antibody, Fab, Fab', (Fab')₂, Fv fragments or single-stranded variable Area fragment scFv.

It is preferred that, functional domain combination tumour antigen in antigen binding domain of the present invention.The tumour be prostate cancer, Clear-cell carcinoma, Hodgkin lymphoma, NHL, leukaemia（Chronic lymphocytic leukemia, acute lymphoblastic Cell leukemia, small lymphocyte leukaemia, acute myelocytic leukemia, Huppert's disease, gland cancer, colorectal cancer, Breast cancer, solid tumor, incidence cancer, glioblastoma, neuroblastoma, sarcoma, metastatic carcinoma, polymorphy colloid are female thin Born of the same parents' knurl etc..

It is preferred that, the tumour antigen is selected from CD19, CD20, CD22, CD33, CD138, ROR1, Her2/neu, mesothelium Element, CD33/IL3Ra, c-Met, PSMA, CAIX, CEA, PSCA, GD2, glycolipid F77, EGFRvIII, GD-2, FAP, FBP, NY- ESO-1TCR, MAGEA3TCR or its any combination.

It is preferred that, the signal peptide is selected from CD8 alpha signal peptides.

It is preferred that, the extracellular hinge area be Immunoglobulin IgG hinge area or the hinge area of CD8 protein moleculars or its Combination.

It is preferred that, the extracellular domain is selected from DAP10 extracellular domains.

The transmembrane structure area is selected from transmembrane structure area or its any combination of CD3, CD4, CD8 or CD28 protein molecular；

The costimulatory signal conducting region is selected from CD27, CD28, CD137 (4-1BB), CD134 (OX40), CD30, CD40, PD- The costimulatory signal conducting region of 1, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3 protein molecular and CD3 ζ are specifically bound Part or its any combination；The T cell signal transduction area functional domain is selected from CD28, CD137, Fc ε RI γ, ZAP70 Or the one or more in the T cell signal transduction area of CD3 ζ protein moleculars.T cell signal transduction area includes immunity receptor junket Propylhomoserin activation motifs.

Light-dependent control element and the fusion connection of polymeric component 1 in the monomer of Chimeric antigen receptor of the present invention, remaining Each functional domain（Signal peptide, antigen binding domain, extracellular hinge area, transmembrane structure area, costimulatory signal conducting region, T are thin Born of the same parents' signal transduction area and light-dependent control element, polymeric component 1 and polymeric component 2）It can be joined directly together, i.e., add when by clone gene Enter restriction enzyme site or fusion DNA vaccine technology these elements are connected after expressed, each work(can also be connected by connecting peptide Can domain.It is preferred that connection peptide refers to 1 ~ 50 flexible peptide rich in Gly and/or Ala and/or Ser amino acid residues.The present invention Preferred scheme, photoinduction dimer element is connected by connecting peptide with adjacent embedding and antigen receptor element, connects peptide ammonia Base acid sequence such as SEQ ID No：12、SEQ ID No：16、SEQ ID No：20、SEQ ID No：24 and SEQ ID No：28 institutes Show.

The preferred scheme of the present invention, the signal peptide is CD8 alpha signal peptides, and the extracellular affinity regions are DAP10 born of the same parents Foreign lands, the antigen binding domain is the scFv of anti-CD19 protein moleculars, extracellular hinge area is CD8 protein moleculars hinge area, The costimulation letter that transmembrane structure area is the transmembrane structure area of CD8 protein moleculars, costimulatory signal conducting region is 4-1BB protein moleculars Number conducting region, the signal transduction area that T cell signal transduction area is CD3 ζ protein moleculars, the CD8 alpha signals peptide amino acid sequence is such as SEQ ID No：Shown in 2, the amino acid sequence such as SEQ ID No of DAP10 extracellular domains：Shown in 18, anti-CD19 scFv amino acid sequences Row such as SEQ ID No：4 is shown, CD8 hinge region amino acids sequence such as SEQ ID No：6 is shown, CD8 transmembrane structures area amino acid Sequence such as SEQ ID No：8 is shown, 4-1BB costimulatory signals conduction region amino acid sequence such as SEQ ID No：10 is shown, CD3 ζ Signal transduction region amino acid sequence such as SEQ ID No：Shown in 26.

A specific embodiment of the present invention, the amino acid sequence such as SEQ of the monomer of Chimeric antigen receptor first ID No：Shown in 32, the amino acid sequence such as SEQ ID No of second comonomer：Shown in 34.

Another object of the present invention is to provide to encode the gene of above-mentioned photoinduction dimer type Chimeric antigen receptor.

The preferred scheme of the present invention, described Chimeric antigen receptor gene, the signal peptide is CD8 alpha signal peptides, core Nucleotide sequence such as SEQ ID No：Shown in 1, the extracellular affinity regions are DAP10 extracellular domains, nucleotide sequence such as SEQ ID No： Shown in 17, the antigen binding domain is anti-CD19 scFv, nucleotide sequence such as SEQ ID No：3 is shown, extracellular hinge area is CD8 hinge areas, nucleotide sequence such as SEQ ID No：5 is shown, transmembrane structure area is CD8 transmembrane structures area, and nucleotide sequence is such as SEQ ID No：7 is shown, costimulatory signal conducting region is 4-1BB costimulatory signal conducting regions, nucleotide sequence such as SEQ ID No：9 is shown, T cell signal transduction area is CD3 ζ signal transductions area, nucleotide sequence such as SEQ ID No：Shown in 25, light-operated member Part is LOV2, and the polymeric component 1 is ssrA, nucleotide sequence such as SEQ ID No after light-dependent control element and polymeric component 1 are merged： Shown in 13, the polymeric component 2 is sspB, nucleotide sequence such as SEQ ID No：Shown in 21.

The connection peptide is flexible peptide linker, connects the nucleotide sequence such as SEQ ID No of peptide：11、SEQ ID No： 15、SEQ ID No：19、SEQ ID No：23 and SEQ ID No：Shown in 27.

It is furthermore preferred that described Chimeric antigen receptor gene order such as SEQ ID No：Shown in 35.

The first monomer and/or second comonomer of Chimeric antigen receptor of the present invention can also include one or more signals Sequence, Epitope tag, affine domain or the polypeptide for producing detectable signal.

Another object of the present invention is to provide a kind of expression chimeric antigen of the present invention with photo-induction guiding element by Recombinant vector, expression cassette, slow virus carrier or the cell of body, contain above-mentioned Chimeric antigen receptor gene.

The preferred scheme of the present invention, expression vector is Lenti-EF1 α, and recombinant vector is Lenti-LID- CD19CAR, the Light-inducible restructuring of two structural order differences of design is embedding and antigen receptor expresses combination, nucleotide sequence difference Such as SEQ ID No：35 and SEQ ID No：Shown in 40, insertion expression vector Lenti-EF1 α BamH I and EcoR I digestions position Carrier obtained by between point, wherein each two monomer expression cassettes of combination are independently expressed.

The preferred scheme of the present invention, the cell is selected from autologous or transgenosis T cell, NK cells, cell toxicant Property T lymphocytes or regulatory T-cell, memory t cell, bispecific T cell, CIK cell.

It is an object of the present invention to provide the Chimeric antigen receptor preparation method with photo-induction guiding element, it may include following Step：

1）The structure of coexpression vector

Photoinduction dimer element genes are cloned into two independences of plasmid Lenti-LID-CD19CAR with molecular cloning method Expression cassette in, build with photoinduction dimeric structure Chimeric antigen receptor coexpression vector；

2）The packaging of slow virus carrier

Packed using 293T cells and obtain the slow virus carrier with photoinduction CAR molecules.The slow virus carrier is will be above-mentioned Coexpression vector and slow virus pack necessary structural proteins expression plasmid corotation and efficiently assemble gained in the cell of slow virus Slow virus with expression recombination.The preferred scheme of the present invention, by above-mentioned coexpression vector and slow virus structure egg White expression vector pGP and pVSVG transfect HEK 293T cell culture and obtain slow virus carrier jointly；

3）Lentivirus mediated has the Chimeric antigen receptor transfectional cell of photo-induction guiding element.

The method that the present invention is provided comprises the following steps：Target gene slow virus package carrier is built, slow virus is packed, slowly Virus-mediated T cell transgenosis, recombinant C AR-T cells have reversible photocontrol special to the specific killing activity of target cell Property, i.e., there is killing ability only under illumination condition.

Advantage of the present invention：

The invention provides a kind of Chimeric antigen receptor with photo-induction guiding element, it is demonstrated experimentally that the recombinant protein passes through slow disease After poisonous carrier is expressed in T cell, the reversible control of the killing toxicity of T cell by LOV2.It is of the present invention that there is photo-induction The Chimeric antigen receptor of guiding element maintains CAR-T targeting and anti-personnel opened while adding control to this two kinds of characteristics Close, creative thinking is provided for the CAR-T securities for improving clinical anticancer.

Brief description of the drawings

Fig. 1 is photoinduction dimer type Chimeric antigen receptor structural representation of the present invention.

Fig. 2 is Jurkat-LID CD19-CAR cell killing activity analysis result.

Fig. 3 is the Jurkat-LID CD19-CAR different cell killing energy for imitating target ratios under the conditions of illumination and lucifuge respectively Power experimental result.

Embodiment

Used term, unless otherwise indicated, typically there are those of ordinary skill in the art generally to manage in the present invention The implication of solution.

In conjunction with specific embodiments and the present invention is described in further detail with reference to data in face.It should be understood that the embodiment is to be The present invention is illustrated, rather than limits the scope of the present invention in any way.

In the examples below, the various processes and method not being described in detail are conventional methods as known in the art.

With reference to specific embodiment, the present invention is further described.

Material, reagent used etc., unless otherwise specified, are commercially obtained in following examples.

Embodiment 1, photoinduction dimer type recombinate embedding and antigen receptor design

The present invention is from the CD19-CAR molecules studied, be most widely used at present as an example, building photoinduction dimerization respectively Build restructuring embedding and antigen receptor the first monomer and second comonomer.Design that two groups of photoinduction dimer types are embedding altogether and antigen by Body, is respectively designated as combination 1 and combination 2（Sequential structure is shown in Fig. 1）.

Combine 1 first monomer composed component as follows successively：CD8α signal peptide（Amino acid sequence such as SEQ ID No：Shown in 2, nucleotide sequence such as SEQ ID No：Shown in 1）, anti-CD19 scFv（Amino acid sequence such as SEQ ID No：4 institutes Show, nucleotide sequence such as SEQ ID No：Shown in 3）、CD8 hinge（Amino acid sequence such as SEQ ID No：Shown in 6, nucleotides Sequence such as SEQ ID No：Shown in 5）、CD8 transmembrane（Amino acid sequence such as SEQ ID No：Shown in 8, nucleotides sequence Row such as SEQ ID No：Shown in 7）、4-1BB intracellular chain（Amino acid sequence such as SEQ ID No：Shown in 10, Nucleotide sequence such as SEQ ID No：Shown in 9）, connection peptide（Amino acid sequence such as SEQ ID No：Shown in 12, nucleotide sequence is such as SEQ ID No：Shown in 11）LOV2-ssrA（Amino acid sequence such as SEQ ID No：Shown in 14, nucleotide sequence such as SEQ ID No：Shown in 13）.

Combine the amino acid sequence such as SEQ ID No of 1 first monomer：Shown in 32, nucleotide sequence such as SEQ ID No：31 It is shown.

Combine 1 second comonomer composed component as follows successively：CD8α signal peptide（Amino acid sequence such as SEQ ID No：Shown in 2, nucleotide sequence such as SEQ ID No：Shown in 1）, DAP10 extracellular domains（Amino acid sequence such as SEQ ID No：18 institutes Show, nucleotide sequence such as SEQ ID No：Shown in 17）、CD8α TM（Amino acid sequence such as SEQ ID No：Shown in 8, nucleotides sequence Row such as SEQ ID No：Shown in 7）、4-1BB intracellular chain（Amino acid sequence such as SEQ ID No：Shown in 10, Nucleotide sequence such as SEQ ID No：Shown in 9）, connection peptide（Amino acid sequence such as SEQ ID No：Shown in 20, nucleotide sequence is such as SEQ ID No：Shown in 19）、sspB（Amino acid sequence such as SEQ ID No：Shown in 22, nucleotide sequence such as SEQ ID No：21 It is shown）, connection peptide（Amino acid sequence such as SEQ ID No：Shown in 24, nucleotide sequence such as SEQ ID No：Shown in 23）CD3 zeta intracellular chain（Amino acid sequence such as SEQ ID No：Shown in 26, nucleotide sequence such as SEQ ID No： Shown in 25）.

Combine the amino acid sequence such as SEQ ID No of 1 second comonomer：Shown in 34, nucleotide sequence such as SEQ ID No：33 It is shown.

Combine between 1 first monomer and second comonomer by the connection of F2A connections peptide, F2A amino acid sequences such as SEQ ID No：Shown in 30, nucleotide sequence such as SEQ ID No：Shown in 29.

The nucleotide sequence such as SEQ ID No of combination 1：Shown in 35.

Combine 2 first monomer composed components as follows successively：CD8α signal peptide（Amino acid sequence such as SEQ ID No：Shown in 2, nucleotide sequence such as SEQ ID No：Shown in 1）, anti-CD19 scFv（Amino acid sequence such as SEQ ID No：4 institutes Show, nucleotide sequence such as SEQ ID No：Shown in 3）、CD8 hinge（Amino acid sequence such as SEQ ID No：Shown in 6, nucleotides Sequence such as SEQ ID No：Shown in 5）、CD8 transmembrane（Amino acid sequence such as SEQ ID No：Shown in 8, nucleotides sequence Row such as SEQ ID No：Shown in 7）、4-1BB intracellular chain（Amino acid sequence such as SEQ ID No：Shown in 10, Nucleotide sequence such as SEQ ID No：Shown in 9）, connection peptide（Amino acid sequence such as SEQ ID No：Shown in 12, nucleotide sequence is such as SEQ ID No：Shown in 11）、sspB（Amino acid sequence such as SEQ ID No：Shown in 22, nucleotide sequence such as SEQ ID No：21 It is shown）.

Combine the amino acid sequence such as SEQ ID No of 2 first monomers：Shown in 37, nucleotide sequence such as SEQ ID No：36 It is shown.

Combine 2 second comonomer composed components as follows successively：CD8α signal peptide（Amino acid sequence such as SEQ ID No：Shown in 2, nucleotide sequence such as SEQ ID No：Shown in 1）, DAP10 extracellular domains（Amino acid sequence such as SEQ ID No：18 institutes Show, nucleotide sequence such as SEQ ID No：Shown in 17）、CD8α TM（Amino acid sequence such as SEQ ID No：Shown in 8, nucleotides sequence Row such as SEQ ID No：Shown in 7）、4-1BB intracellular chain（Amino acid sequence such as SEQ ID No：Shown in 10, Nucleotide sequence such as SEQ ID No：Shown in 9）, connection peptide（Amino acid sequence such as SEQ ID No：Shown in 20, nucleotide sequence is such as SEQ ID No：Shown in 19）、LOV2-ssrA（Amino acid sequence such as SEQ ID No：Shown in 14, nucleotide sequence such as SEQ ID No：Shown in 13）, connection peptide（Amino acid sequence such as SEQ ID No：Shown in 24, nucleotide sequence such as SEQ ID No：Shown in 23） CD3 zeta intracellular chain（Amino acid sequence such as SEQ ID No：Shown in 26, nucleotide sequence such as SEQ ID No：Shown in 25）.

Combine the amino acid sequence such as SEQ ID No of 2 second comonomers：Shown in 39, nucleotide sequence such as SEQ ID No：38 It is shown.

Combine between 2 first monomers and second comonomer by the connection of F2A connections peptide.

The nucleotide sequence such as SEQ ID No of combination 2：Shown in 40.

The expression of embodiment 2, photoinduction dimer type Chimeric antigen receptor

First, the structure of coexpression vector

Lenti-EF1 α plasmid vectors are purchased from Ai Kang get biomedical technologies（Suzhou）Co., Ltd（http:// www.icartab.com.cn）.

Respectively according to SEQ ID NO：35 and SEQ ID NO：The sequence of 40 codings, commission Beijing AudioCodes biotechnology is limited Company carries out full genome synthesis, is inserted into Lenti-EF1 α plasmid EcoRI and BamHI restriction enzyme sites.It is transformed into E.coli （TOP10, purchased from Quan Shi King Companies）, it is correct through sequencing, extracted using the plasmid purification kit of Qiagen companies and purify matter Grain, the recombinant expression carrier for obtaining two combinations removes endotoxin plasmid, i.e. Lenti-LID-CD19CAR-1 and Lenti-LID- CD19CAR-2。

2nd, slow virus carrier Lentivirus packaging

5 × 10 are inoculated with the pre-coated 15-cm culture dishes of gelatin⁶HEK 293T cells, in 37 DEG C, CO2 incubators overnight Culture.When degree of converging reaches 70%, prepare plasmid transfection.2 hours before transfection, fresh serum-free media is changed, is turned Dye.

100 μM of PEI of transfection reagent are taken out from refrigerator（Polyethylenimine）, in 60 °C of water-bath heating 15min To being completely dissolved.Plasmid Lenti-LID-CD19CAR-1, Lenti-LID-CD19CAR-2, pGP, pVSVG are taken out from refrigerator, Room-temperature dissolution.

Prepare PEI/DNA compounds：2ml PBS are taken, respectively with 8 μ g Lenti-LID-CD19CAR-1 and Lenti-LID- CD19CAR-2,4 μ g pGP, 2 μ g pVSVG prepare DNA mixed liquors, after fully piping and druming is mixed, and add 18 μ l, 100 μM of PEI, stand I.e. pressure-vaccum is mixed, and is stored at room temperature 10min.The PEI/DNA complexes drop-wises obtained after standing are added and have changed the thin of culture medium In born of the same parents' culture, culture dish is gently rocked, is fully mixed.In 37 DEG C, CO₂Cultivated in incubator after 6-8h, transfection examination will be contained The culture medium of agent changes fresh complete medium into.

48h collects the cells and supernatant containing virion after transfection, and adds new complete medium.72h is again Secondary collection cells and supernatant, twice gleanings pass through 0.45 μm filtering membrane filtration.Respectively by lentiviral particle Lenti-LID- CD19CAR-1 and Lenti-LID-CD19CAR-2 is concentrated using Amicon Ultra Centrifugal Filters 100KDa Virion, then packing is stored in -80 °C of ultra low temperature freezers.

3rd, the cell transfecting of lentivirus mediated

Polybrene to final concentration of 8 μ g/ml is added in the RPMI 1640 of the serum-free of appropriate volume, with the culture medium weight The outstanding Jurkat cell being collected by centrifugation, and with 5 × 10⁵The density in/hole is seeded in 6 orifice plates.According to virus titer and cell number Mesh, adds the slow virus of MOI=10（Lenti-LID-CD19CAR-1 and Lenti-LID-CD19CAR-2）Concentrate, gently shakes Culture plate is moved to mix.It is placed on 37 DEG C, 5%CO2 incubator cultures change the RPMI 1640 containing 10% FBS into after 8h and cultivated Base continues to cultivate, and LID CD19-CAR-1 Jurkat T cells and LID CD19-CAR-2 Jurkat T cells are obtained respectively. Amplification cultivation is carried out to it.

The cell killing activity analysis of embodiment 3, LID CD19-CAR Jurkat T cells

（1）IL-2 secretory volumes are detected

The degree that the assessment of levels immunocyte for secreting IL-2 using cell is activated.By K562 and K562-CD19（Pass through transfection The stabilization of acquisition has the K562 cells of CD19 phenotypes）According to 1 × 10⁵The concentration in/hole is plated in 96 orifice plates, according to every hole 100 μ l target cells add.By LID CD19-CAR Jurkat T cells（LID CD19-CAR-1 Jurkat T cells and LID CD19-CAR-2 Jurkat T cells）According to target cell：Effector cell=1:1、1:3、1:10 ratio, it is common with target cell respectively Culture.Light group and non-light group are set simultaneously.Light group is when co-culturing, using blue LED lamp（1.2mW/cm² at 450nm）It is irradiated 6h；Non- light group then wraps up Tissue Culture Plate with masking foil.In 37 DEG C, 5%CO₂Incubator co-cultures 18- After 20h, suspension culture is collected respectively and collects supernatant, user IL-2 ELISA kit by centrifugation（BD 555190）Inspection Survey IL-2 contents, the degree being activated by the assessment of levels Jurkat cell of IL-2 secretory volumes.The specific steps that IL-2 is determined Referring to human IL-2's ELISA kit operation instructions.

Concrete outcome as shown in Fig. 2 as a result illustrate expression Light-inducible dimer Chimeric antigen receptor T cell with CD19⁺During the target cell of phenotype co-cultures, compared with classical CD19-CAR-T, secrete cytokines are white after it is activated The comparison of the secretory volume of interleukin -2.As a result show LID CD19-CAR-1 Jurkat T cells can under blue light illumination with Classic CAR-T cells have similar proleulzin secretory volume, without under blue light illumination without then with typical CAR-T cell effects Difference is huge.Other LID CD19-CAR-2 Jurkat T cells then illumination whether can not all produce stronger interleukin- 2 secretions.Two groups of Light-inducible dimer CAR-T interleukins are secreted effects and shown, only the photoinduction dimerization of specific structure Body could form tool functional under specific wavelength blue light illumination and recombinate embedding and antigen receptor, and then further activate T cell.

（2）Cytotoxicity is detected

LID CD19-CAR Jurkat are evaluated to CD19 using cell release LDH amounts⁺The killing activity of B cell.With K562 with K562-CD19 is target cell, according to 1 × 10⁵The cell concentration in/hole is seeded in 96 orifice plates.By the LID CD19-CAR of structure Jurkat T cells（LID CD19-CAR-1 Jurkat T cells and LID CD19-CAR-2 Jurkat T cells）According to 10: 1、3:1、1:1 effect target ratio and target cell co-incubation.And light group and non-light group are set.Light group is adopted when co-culturing Use 450nm blue-ray LEDs（1.2mW/cm²）Carry out parallel radiation 6h；Non- light group then carries out lucifuge using masking foil.Co-culture thin Born of the same parents are in 37 DEG C, 5%CO₂LDH burst sizes are detected after incubator culture 4h, specific steps are detected referring to lactic dehydrogenase cytotoxicity Kit（Promega G1780）Operation instructions, compared with control group, calculate percentage burst size.

As a result concrete outcome as shown in figure 3, illustrate the T cell and CD19 for the inosculating antibody antigen receptor for being overexpressed anti-CD19 During the target cell of phenotype co-cultures, the cytolysis that target cell is showed by the killing of effector cell, to table Show the killing activity of T cell.As a result show, compared to classical CD19 CAR-T cells, LID CD19-CAR-1 Jurkat T are thin Born of the same parents have shown cell killing activity similar under illumination condition, and significant difference and Jurkat T under non-illumination condition are thin Born of the same parents.Meanwhile, whether LID CD19-CAR-2 Jurkat T cells illumination all fails to produce obvious cell killing activity Lifting.The cell killing vigor that LID CD19-CAR-1 Jurkat T cells are shown under the conditions of blue light illumination and lucifuge Difference shows that the Light-inducible restructuring of the present invention is embedding and antigen receptor activation pathway can be controlled effectively by blue light illumination. What LID CD19-CAR-1 Jurkat T cells and LID CD19-CAR-2 Jurkat T cells were shown under blue light illumination The dimer formation of the Discrepancy Description of cell killing activity needs specific structural order.

Although at large describing experimental implementation scheme in the specific embodiment of the present invention, same domain technical staff can be fast Speed understands.According to available data, details is modified and replaced, these change within protection scope of the present invention.

SEQUENCE LISTING

<110>Victory is military（Beijing）Bio tech ltd

<120>A kind of photoinduction dimer type Chimeric antigen receptor

<130>

<160> 40

<170> PatentIn version 3.5

<210> 1

<211> 63

<212> DNA

<213>Artificial sequence

<400> 1

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

ccc

<210> 2

<211> 21

<212> PRT

<213>Artificial sequence

<400> 2

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

His Ala Ala Arg Pro

<210> 3

<211> 726

<212> DNA

<213>Artificial sequence

<400> 3

gaaattgtga tgacccagtc acccgccact cttagccttt cacccggtga gcgcgcaacc

ctgtcttgca gagcctccca agacatctca aaatacctta attggtatca acagaagccc

ggacaggctc ctcgccttct gatctaccac accagccggc tccattctgg aatccctgcc

aggttcagcg gtagcggatc tgggaccgac tacaccctca ctatcagctc actgcagcca

gaggacttcg ctgtctattt ctgtcagcaa gggaacaccc tgccctacac ctttggacag

ggcaccaagc tcgagattaa aggtggaggt ggcagcggag gaggtgggtc cggcggtgga

ggaagccagg tccaactcca agaaagcgga ccgggtcttg tgaagccatc agaaactctt

tcactgactt gtactgtgag cggagtgtct ctccccgatt acggggtgtc ttggatcaga

cagccaccgg ggaagggtct ggaatggatt ggagtgattt ggggctctga gactacttac

tactcttcat ccctcaagtc acgcgtcacc atctcaaagg acaactctaa gaatcaggtg

tcactgaaac tgtcatctgt gaccgcagcc gacaccgccg tgtactattg cgctaagcat

tactattatg gcgggagcta cgcaatggat tactggggac agggtactct ggtcaccgtg

tccagc

<210> 4

<211> 242

<212> PRT

<213>Artificial sequence

<400> 4

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

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

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

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

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

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

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

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

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

Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

Ser Ser

<210> 5

<211> 135

<212> DNA

<213>Artificial sequence

<400> 5

accactaccc cagcaccgag gccacccacc ccggctccta ccatcgcctc ccagcctctg

tccctgcgtc cggaggcatg tagacccgca gctggtgggg ccgtgcatac ccggggtctt

gacttcgcct gcgat

<210> 6

<211> 45

<212> PRT

<213>Artificial sequence

<400> 6

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

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

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

<210> 7

<211> 72

<212> DNA

<213>Artificial sequence

<400> 7

atctacattt gggcccctct ggctggtact tgcggggtcc tgctgctttc actcgtgatc

actctttact gt

<210> 8

<211> 24

<212> PRT

<213>Artificial sequence

<400> 8

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

Ser Leu Val Ile Thr Leu Tyr Cys

<210> 9

<211> 126

<212> DNA

<213>Artificial sequence

<400> 9

aagcgcggtc ggaagaagct gctgtacatc tttaagcaac ccttcatgag gcctgtgcag

actactcaag aggaggacgg ctgttcatgc cggttcccag aggaggagga aggcggctgc

gaactg

<210> 10

<211> 42

<212> PRT

<213>Artificial sequence

<400> 10

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

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

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

<210> 11

<211> 21

<212> DNA

<213>Artificial sequence

<400> 11

ggtagtggta gtggggagtt t

<210> 12

<211> 7

<212> PRT

<213>Artificial sequence

<400> 12

Gly Ser Gly Ser Gly Glu Phe

<210> 13

<211> 438

<212> DNA

<213>Artificial sequence

<400> 13

cttgcaacca ccttggaaag aatcgagaag aacttcgtca tcaccgaccc aaggctgccc

gacaacccta tcatcttcgc cagcgacagt ttcctccagc tgaccgaata ctccagggag

gaaatcctgg gaagaaactg ccggttcctc caaggccctg agactgacag ggctactgtg

aggaagatcc gggatgcaat tgacaaccag acagaggtga cagtgcagct gattaactac

acaaagtctg gcaagaagtt ttggaatgtg ttccacctgc agccgatgcg cgattataag

ggcgacgtcc agtacttcat tggcgtgcag ctggatggca ccgaacgtct tcatggcgcc

gctgagcgtg aggcggtctg cctgatcaaa aagacagcct ttcagattgc tgaggcagcg

aacgacgaaa attacttt

<210> 14

<211> 146

<212> PRT

<213>Artificial sequence

<400> 14

Leu Ala Thr Thr Leu Glu Arg Ile Glu Lys Asn Phe Val Ile Thr Asp

Pro Arg Leu Pro Asp Asn Pro Ile Ile Phe Ala Ser Asp Ser Phe Leu

Gln Leu Thr Glu Tyr Ser Arg Glu Glu Ile Leu Gly Arg Asn Cys Arg

Phe Leu Gln Gly Pro Glu Thr Asp Arg Ala Thr Val Arg Lys Ile Arg

Asp Ala Ile Asp Asn Gln Thr Glu Val Thr Val Gln Leu Ile Asn Tyr

Thr Lys Ser Gly Lys Lys Phe Trp Asn Val Phe His Leu Gln Pro Met

Arg Asp Tyr Lys Gly Asp Val Gln Tyr Phe Ile Gly Val Gln Leu Asp

Gly Thr Glu Arg Leu His Gly Ala Ala Glu Arg Glu Ala Val Cys Leu

Ile Lys Lys Thr Ala Phe Gln Ile Ala Glu Ala Ala Asn Asp Glu Asn

Tyr Phe

<210> 15

<211> 18

<212> DNA

<213>Artificial sequence

<400> 15

ggaagcggga gtgggagc

<210> 16

<211> 6

<212> PRT

<213>Artificial sequence

<400> 16

Gly Ser Gly Ser Gly Ser

<210> 17

<211> 144

<212> DNA

<213>Artificial sequence

<400> 17

atgatccatc tgggtcacat cctcttcctg cttttgctcc cagtggctgc agctcagacg

actccaggag agagatcatc actccctgcc ttttaccctg gcacttcagg ctcttgttcc

ggatgtgggt ccctctctct gccg

<210> 18

<211> 48

<212> PRT

<213>Artificial sequence

<400> 18

Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala

Ala Ala Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr

Pro Gly Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro

<210> 19

<211> 21

<212> DNA

<213>Artificial sequence

<400> 19

ggtagtggta gtggggagtt t

<210> 20

<211> 7

<212> PRT

<213>Artificial sequence

<400> 20

Gly Ser Gly Ser Gly Glu Phe

<210> 21

<211> 333

<212> DNA

<213> Haemophilus influenzae

<400> 21

agctccccga aacgccctaa gctgctgcgt gaatattacg attggctggt tgataacagc

tttaccccat atctggtggt ggatgccaca tacctgggcg tgaacgtgcc cgtggagtat

gtgaaagacg gtcagatcgt gctgaatctg tctgcaagtg cgaccggcaa cctgcaactg

acaaatgatt ttatccagtt caacgcccgc tttaagggcg tgtctcgtga actgtatatc

ccgatgggtg ccgctctggc catttacgct cgcgagaacg gcgatggtgt gatgttcgaa

ccagaagaaa tctatgacga gctgaatatt ggt

<210> 22

<211> 111

<212> PRT

<213> Haemophilus influenzae

<400> 22

Ser Ser Pro Lys Arg Pro Lys Leu Leu Arg Glu Tyr Tyr Asp Trp Leu

Val Asp Asn Ser Phe Thr Pro Tyr Leu Val Val Asp Ala Thr Tyr Leu

Gly Val Asn Val Pro Val Glu Tyr Val Lys Asp Gly Gln Ile Val Leu

Asn Leu Ser Ala Ser Ala Thr Gly Asn Leu Gln Leu Thr Asn Asp Phe

Ile Gln Phe Asn Ala Arg Phe Lys Gly Val Ser Arg Glu Leu Tyr Ile

Pro Met Gly Ala Ala Leu Ala Ile Tyr Ala Arg Glu Asn Gly Asp Gly

Val Met Phe Glu Pro Glu Glu Ile Tyr Asp Glu Leu Asn Ile Gly

<210> 23

<211> 30

<212> DNA

<213>Artificial sequence

<400> 23

ggaagcgggt ccggtagcgg atcttcccta

<210> 24

<211> 10

<212> PRT

<213>Artificial sequence

<400> 24

Gly Ser Gly Ser Gly Ser Gly Ser Ser Leu

<210> 25

<211> 336

<212> DNA

<213>Artificial sequence

<400> 25

cgcgtgaaat tcagccgcag cgcagatgct ccagcctacc agcaggggca gaaccagctc

tacaacgaac tcaatcttgg tcggagagag gagtacgacg tgctggacaa gcggagagga

cgggacccag aaatgggcgg gaagccgcgc agaaagaatc cccaagaggg cctgtacaac

gagctccaaa aggataagat ggcagaagcc tatagcgaga ttggtatgaa aggggaacgc

agaagaggca aaggccacga cggactgtac cagggactca gcaccgccac caaggacacc

tatgacgctc ttcacatgca ggccctgccg cctcgg

<210> 26

<211> 112

<212> PRT

<213>Artificial sequence

<400> 26

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

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

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

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

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

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

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

<210> 27

<211> 30

<212> DNA

<213>Artificial sequence

<400> 27

tcgcgaggaa gcgggtccgg tagcggatct

<210> 28

<211> 10

<212> PRT

<213>Artificial sequence

<400> 28

Ser Arg Gly Ser Gly Ser Gly Ser Gly Ser

<210> 29

<211> 66

<212> DNA

<213>Artificial sequence

<400> 29

gtgaaacaga ctttgaattt tgaccttctc aagttggcgg gagacgtgga gtccaaccca

gggccc

<210> 30

<211> 22

<212> PRT

<213>Artificial sequence

<400> 30

Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val

Glu Ser Asn Pro Gly Pro

<210> 31

<211> 1581

<212> DNA

<213>Artificial sequence

<400> 31

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

ggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact

tactactctt catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc

gtgtccagca ccactacccc agcaccgagg ccacccaccc cggctcctac catcgcctcc

cagcctctgt ccctgcgtcc ggaggcatgt agacccgcag ctggtggggc cgtgcatacc

cggggtcttg acttcgcctg cgatatctac atttgggccc ctctggctgg tacttgcggg

gtcctgctgc tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg

tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga ggacggctgt

tcatgccggt tcccagagga ggaggaaggc ggctgcgaac tgggtagtgg tagtggggag

tttcttgcaa ccaccttgga aagaatcgag aagaacttcg tcatcaccga cccaaggctg

cccgacaacc ctatcatctt cgccagcgac agtttcctcc agctgaccga atactccagg

gaggaaatcc tgggaagaaa ctgccggttc ctccaaggcc ctgagactga cagggctact

gtgaggaaga tccgggatgc aattgacaac cagacagagg tgacagtgca gctgattaac

tacacaaagt ctggcaagaa gttttggaat gtgttccacc tgcagccgat gcgcgattat

aagggcgacg tccagtactt cattggcgtg cagctggatg gcaccgaacg tcttcatggc

gccgctgagc gtgaggcggt ctgcctgatc aaaaagacag cctttcagat tgctgaggca

gcgaacgacg aaaattactt t

<210> 32

<211> 527

<212> PRT

<213>Artificial sequence

<400> 32

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

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

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

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

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

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

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

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

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

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

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

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

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

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

Glu Gly Gly Cys Glu Leu Gly Ser Gly Ser Gly Glu Phe Leu Ala Thr

Thr Leu Glu Arg Ile Glu Lys Asn Phe Val Ile Thr Asp Pro Arg Leu

Pro Asp Asn Pro Ile Ile Phe Ala Ser Asp Ser Phe Leu Gln Leu Thr

Glu Tyr Ser Arg Glu Glu Ile Leu Gly Arg Asn Cys Arg Phe Leu Gln

Gly Pro Glu Thr Asp Arg Ala Thr Val Arg Lys Ile Arg Asp Ala Ile

Asp Asn Gln Thr Glu Val Thr Val Gln Leu Ile Asn Tyr Thr Lys Ser

Gly Lys Lys Phe Trp Asn Val Phe His Leu Gln Pro Met Arg Asp Tyr

Lys Gly Asp Val Gln Tyr Phe Ile Gly Val Gln Leu Asp Gly Thr Glu

Arg Leu His Gly Ala Ala Glu Arg Glu Ala Val Cys Leu Ile Lys Lys

Thr Ala Phe Gln Ile Ala Glu Ala Ala Asn Asp Glu Asn Tyr Phe

<210> 33

<211> 1128

<212> DNA

<213>Artificial sequence

<400> 33

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

cccatgatcc atctgggtca catcctcttc ctgcttttgc tcccagtggc tgcagctcag

acgactccag gagagagatc atcactccct gccttttacc ctggcacttc aggctcttgt

tccggatgtg ggtccctctc tctgccgatc tacatttggg cccctctggc tggtacttgc

ggggtcctgc tgctttcact cgtgatcact ctttactgtg gtagtggtag tggggagttt

agctccccga aacgccctaa gctgctgcgt gaatattacg attggctggt tgataacagc

tttaccccat atctggtggt ggatgccaca tacctgggcg tgaacgtgcc cgtggagtat

gtgaaagacg gtcagatcgt gctgaatctg tctgcaagtg cgaccggcaa cctgcaactg

acaaatgatt ttatccagtt caacgcccgc tttaagggcg tgtctcgtga actgtatatc

ccgatgggtg ccgctctggc catttacgct cgcgagaacg gcgatggtgt gatgttcgaa

ccagaagaaa tctatgacga gctgaatatt ggtggaagcg ggtccggtag cggatcttcc

ctaaagcgcg gtcggaagaa gctgctgtac atctttaagc aacccttcat gaggcctgtg

cagactactc aagaggagga cggctgttca tgccggttcc cagaggagga ggaaggcggc

tgcgaactgc gcgtgaaatt cagccgcagc gcagatgctc cagcctacca gcaggggcag

aaccagctct acaacgaact caatcttggt cggagagagg agtacgacgt gctggacaag

cggagaggac gggacccaga aatgggcggg aagccgcgca gaaagaatcc ccaagagggc

ctgtacaacg agctccaaaa ggataagatg gcagaagcct atagcgagat tggtatgaaa

ggggaacgca gaagaggcaa aggccacgac ggactgtacc agggactcag caccgccacc

aaggacacct atgacgctct tcacatgcag gccctgccgc ctcggtag

<210> 34

<211> 375

<212> PRT

<213>Artificial sequence

<400> 34

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

His Ala Ala Arg Pro Met Ile His Leu Gly His Ile Leu Phe Leu Leu

Leu Leu Pro Val Ala Ala Ala Gln Thr Thr Pro Gly Glu Arg Ser Ser

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

Ser Leu Ser Leu Pro Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Gly Ser Gly

Ser Gly Glu Phe Ser Ser Pro Lys Arg Pro Lys Leu Leu Arg Glu Tyr

Tyr Asp Trp Leu Val Asp Asn Ser Phe Thr Pro Tyr Leu Val Val Asp

Ala Thr Tyr Leu Gly Val Asn Val Pro Val Glu Tyr Val Lys Asp Gly

Gln Ile Val Leu Asn Leu Ser Ala Ser Ala Thr Gly Asn Leu Gln Leu

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

Glu Leu Tyr Ile Pro Met Gly Ala Ala Leu Ala Ile Tyr Ala Arg Glu

Asn Gly Asp Gly Val Met Phe Glu Pro Glu Glu Ile Tyr Asp Glu Leu

Asn Ile Gly Gly Ser Gly Ser Gly Ser Gly Ser Ser Leu Lys Arg Gly

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

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

Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp

Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

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

Met Gln Ala Leu Pro Pro Arg

<210> 35

<211> 2775

<212> DNA

<213>Artificial sequence

<400> 35

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

ggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact

tactactctt catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc

gtgtccagca ccactacccc agcaccgagg ccacccaccc cggctcctac catcgcctcc

cagcctctgt ccctgcgtcc ggaggcatgt agacccgcag ctggtggggc cgtgcatacc

cggggtcttg acttcgcctg cgatatctac atttgggccc ctctggctgg tacttgcggg

gtcctgctgc tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg

tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga ggacggctgt

tcatgccggt tcccagagga ggaggaaggc ggctgcgaac tgggtagtgg tagtggggag

tttcttgcaa ccaccttgga aagaatcgag aagaacttcg tcatcaccga cccaaggctg

cccgacaacc ctatcatctt cgccagcgac agtttcctcc agctgaccga atactccagg

gaggaaatcc tgggaagaaa ctgccggttc ctccaaggcc ctgagactga cagggctact

gtgaggaaga tccgggatgc aattgacaac cagacagagg tgacagtgca gctgattaac

tacacaaagt ctggcaagaa gttttggaat gtgttccacc tgcagccgat gcgcgattat

aagggcgacg tccagtactt cattggcgtg cagctggatg gcaccgaacg tcttcatggc

gccgctgagc gtgaggcggt ctgcctgatc aaaaagacag cctttcagat tgctgaggca

gcgaacgacg aaaattactt tgtgaaacag actttgaatt ttgaccttct caagttggcg

ggagacgtgg agtccaaccc agggcccatg gccctccctg tcaccgccct gctgcttccg

ctggctcttc tgctccacgc cgctcggccc atgatccatc tgggtcacat cctcttcctg

cttttgctcc cagtggctgc agctcagacg actccaggag agagatcatc actccctgcc

ttttaccctg gcacttcagg ctcttgttcc ggatgtgggt ccctctctct gccgatctac

atttgggccc ctctggctgg tacttgcggg gtcctgctgc tttcactcgt gatcactctt

tactgtggta gtggtagtgg ggagtttagc tccccgaaac gccctaagct gctgcgtgaa

tattacgatt ggctggttga taacagcttt accccatatc tggtggtgga tgccacatac

ctgggcgtga acgtgcccgt ggagtatgtg aaagacggtc agatcgtgct gaatctgtct

gcaagtgcga ccggcaacct gcaactgaca aatgatttta tccagttcaa cgcccgcttt

aagggcgtgt ctcgtgaact gtatatcccg atgggtgccg ctctggccat ttacgctcgc

gagaacggcg atggtgtgat gttcgaacca gaagaaatct atgacgagct gaatattggt

ggaagcgggt ccggtagcgg atcttcccta aagcgcggtc ggaagaagct gctgtacatc

tttaagcaac ccttcatgag gcctgtgcag actactcaag aggaggacgg ctgttcatgc

cggttcccag aggaggagga aggcggctgc gaactgcgcg tgaaattcag ccgcagcgca

gatgctccag cctaccagca ggggcagaac cagctctaca acgaactcaa tcttggtcgg

agagaggagt acgacgtgct ggacaagcgg agaggacggg acccagaaat gggcgggaag

ccgcgcagaa agaatcccca agagggcctg tacaacgagc tccaaaagga taagatggca

gaagcctata gcgagattgg tatgaaaggg gaacgcagaa gaggcaaagg ccacgacgga

ctgtaccagg gactcagcac cgccaccaag gacacctatg acgctcttca catgcaggcc

ctgccgcctc ggtag

<210> 36

<211> 1476

<212> DNA

<213>Artificial sequence

<400> 36

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

ggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact

tactactctt catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc

gtgtccagca ccactacccc agcaccgagg ccacccaccc cggctcctac catcgcctcc

cagcctctgt ccctgcgtcc ggaggcatgt agacccgcag ctggtggggc cgtgcatacc

cggggtcttg acttcgcctg cgatatctac atttgggccc ctctggctgg tacttgcggg

gtcctgctgc tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg

tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga ggacggctgt

tcatgccggt tcccagagga ggaggaaggc ggctgcgaac tgggtagtgg tagtggggag

tttagctccc cgaaacgccc taagctgctg cgtgaatatt acgattggct ggttgataac

agctttaccc catatctggt ggtggatgcc acatacctgg gcgtgaacgt gcccgtggag

tatgtgaaag acggtcagat cgtgctgaat ctgtctgcaa gtgcgaccgg caacctgcaa

ctgacaaatg attttatcca gttcaacgcc cgctttaagg gcgtgtctcg tgaactgtat

atcccgatgg gtgccgctct ggccatttac gctcgcgaga acggcgatgg tgtgatgttc

gaaccagaag aaatctatga cgagctgaat attggt

<210> 37

<211> 492

<212> PRT

<213>Artificial sequence

<400> 37

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

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

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

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

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

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

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

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

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

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

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

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

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

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

Glu Gly Gly Cys Glu Leu Gly Ser Gly Ser Gly Glu Phe Ser Ser Pro

Lys Arg Pro Lys Leu Leu Arg Glu Tyr Tyr Asp Trp Leu Val Asp Asn

Ser Phe Thr Pro Tyr Leu Val Val Asp Ala Thr Tyr Leu Gly Val Asn

Val Pro Val Glu Tyr Val Lys Asp Gly Gln Ile Val Leu Asn Leu Ser

Ala Ser Ala Thr Gly Asn Leu Gln Leu Thr Asn Asp Phe Ile Gln Phe

Asn Ala Arg Phe Lys Gly Val Ser Arg Glu Leu Tyr Ile Pro Met Gly

Ala Ala Leu Ala Ile Tyr Ala Arg Glu Asn Gly Asp Gly Val Met Phe

Glu Pro Glu Glu Ile Tyr Asp Glu Leu Asn Ile Gly

<210> 38

<211> 1233

<212> DNA

<213>Artificial sequence

<400> 38

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

cccatgatcc atctgggtca catcctcttc ctgcttttgc tcccagtggc tgcagctcag

acgactccag gagagagatc atcactccct gccttttacc ctggcacttc aggctcttgt

tccggatgtg ggtccctctc tctgccgatc tacatttggg cccctctggc tggtacttgc

ggggtcctgc tgctttcact cgtgatcact ctttactgtg gtagtggtag tggggagttt

cttgcaacca ccttggaaag aatcgagaag aacttcgtca tcaccgaccc aaggctgccc

gacaacccta tcatcttcgc cagcgacagt ttcctccagc tgaccgaata ctccagggag

gaaatcctgg gaagaaactg ccggttcctc caaggccctg agactgacag ggctactgtg

aggaagatcc gggatgcaat tgacaaccag acagaggtga cagtgcagct gattaactac

acaaagtctg gcaagaagtt ttggaatgtg ttccacctgc agccgatgcg cgattataag

ggcgacgtcc agtacttcat tggcgtgcag ctggatggca ccgaacgtct tcatggcgcc

gctgagcgtg aggcggtctg cctgatcaaa aagacagcct ttcagattgc tgaggcagcg

aacgacgaaa attactttgg aagcgggtcc ggtagcggat cttccctaaa gcgcggtcgg

aagaagctgc tgtacatctt taagcaaccc ttcatgaggc ctgtgcagac tactcaagag

gaggacggct gttcatgccg gttcccagag gaggaggaag gcggctgcga actgcgcgtg

aaattcagcc gcagcgcaga tgctccagcc taccagcagg ggcagaacca gctctacaac

gaactcaatc ttggtcggag agaggagtac gacgtgctgg acaagcggag aggacgggac

ccagaaatgg gcgggaagcc gcgcagaaag aatccccaag agggcctgta caacgagctc

caaaaggata agatggcaga agcctatagc gagattggta tgaaagggga acgcagaaga

ggcaaaggcc acgacggact gtaccaggga ctcagcaccg ccaccaagga cacctatgac

gctcttcaca tgcaggccct gccgcctcgg tag

<210> 39

<211> 410

<212> PRT

<213>Artificial sequence

<400> 39

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

His Ala Ala Arg Pro Met Ile His Leu Gly His Ile Leu Phe Leu Leu

Leu Leu Pro Val Ala Ala Ala Gln Thr Thr Pro Gly Glu Arg Ser Ser

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

Ser Leu Ser Leu Pro Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Gly Ser Gly

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

Val Ile Thr Asp Pro Arg Leu Pro Asp Asn Pro Ile Ile Phe Ala Ser

Asp Ser Phe Leu Gln Leu Thr Glu Tyr Ser Arg Glu Glu Ile Leu Gly

Arg Asn Cys Arg Phe Leu Gln Gly Pro Glu Thr Asp Arg Ala Thr Val

Arg Lys Ile Arg Asp Ala Ile Asp Asn Gln Thr Glu Val Thr Val Gln

Leu Ile Asn Tyr Thr Lys Ser Gly Lys Lys Phe Trp Asn Val Phe His

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

Val Gln Leu Asp Gly Thr Glu Arg Leu His Gly Ala Ala Glu Arg Glu

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

Asn Asp Glu Asn Tyr Phe Gly Ser Gly Ser Gly Ser Gly Ser Ser Leu

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

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

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

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

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

Ala Leu His Met Gln Ala Leu Pro Pro Arg

<210> 40

<211> 2775

<212> DNA

<213>Artificial sequence

<400> 40

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

ggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact

tactactctt catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc

gtgtccagca ccactacccc agcaccgagg ccacccaccc cggctcctac catcgcctcc

cagcctctgt ccctgcgtcc ggaggcatgt agacccgcag ctggtggggc cgtgcatacc

cggggtcttg acttcgcctg cgatatctac atttgggccc ctctggctgg tacttgcggg

gtcctgctgc tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg

tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga ggacggctgt

tcatgccggt tcccagagga ggaggaaggc ggctgcgaac tgggtagtgg tagtggggag

tttagctccc cgaaacgccc taagctgctg cgtgaatatt acgattggct ggttgataac

agctttaccc catatctggt ggtggatgcc acatacctgg gcgtgaacgt gcccgtggag

tatgtgaaag acggtcagat cgtgctgaat ctgtctgcaa gtgcgaccgg caacctgcaa

ctgacaaatg attttatcca gttcaacgcc cgctttaagg gcgtgtctcg tgaactgtat

atcccgatgg gtgccgctct ggccatttac gctcgcgaga acggcgatgg tgtgatgttc

gaaccagaag aaatctatga cgagctgaat attggtgtga aacagacttt gaattttgac

cttctcaagt tggcgggaga cgtggagtcc aacccagggc ccatggccct ccctgtcacc

gccctgctgc ttccgctggc tcttctgctc cacgccgctc ggcccatgat ccatctgggt

cacatcctct tcctgctttt gctcccagtg gctgcagctc agacgactcc aggagagaga

tcatcactcc ctgcctttta ccctggcact tcaggctctt gttccggatg tgggtccctc

tctctgccga tctacatttg ggcccctctg gctggtactt gcggggtcct gctgctttca

ctcgtgatca ctctttactg tggtagtggt agtggggagt ttcttgcaac caccttggaa

agaatcgaga agaacttcgt catcaccgac ccaaggctgc ccgacaaccc tatcatcttc

gccagcgaca gtttcctcca gctgaccgaa tactccaggg aggaaatcct gggaagaaac

tgccggttcc tccaaggccc tgagactgac agggctactg tgaggaagat ccgggatgca

attgacaacc agacagaggt gacagtgcag ctgattaact acacaaagtc tggcaagaag

ttttggaatg tgttccacct gcagccgatg cgcgattata agggcgacgt ccagtacttc

attggcgtgc agctggatgg caccgaacgt cttcatggcg ccgctgagcg tgaggcggtc

tgcctgatca aaaagacagc ctttcagatt gctgaggcag cgaacgacga aaattacttt

ggaagcgggt ccggtagcgg atcttcccta aagcgcggtc ggaagaagct gctgtacatc

tttaagcaac ccttcatgag gcctgtgcag actactcaag aggaggacgg ctgttcatgc

cggttcccag aggaggagga aggcggctgc gaactgcgcg tgaaattcag ccgcagcgca

gatgctccag cctaccagca ggggcagaac cagctctaca acgaactcaa tcttggtcgg

agagaggagt acgacgtgct ggacaagcgg agaggacggg acccagaaat gggcgggaag

ccgcgcagaa agaatcccca agagggcctg tacaacgagc tccaaaagga taagatggca

gaagcctata gcgagattgg tatgaaaggg gaacgcagaa gaggcaaagg ccacgacgga

ctgtaccagg gactcagcac cgccaccaag gacacctatg acgctcttca catgcaggcc

ctgccgcctc ggtag

Claims

1. one kind has photoinduction dimer type Chimeric antigen receptor, it is characterised in that including two monomers, the first monomer is successively Including signal peptide, extracellular antigen binding domain, extracellular hinge area, transmembrane region, costimulatory signal conducting region and light-dependent control element and The functional domain of polymeric component 1, second comonomer includes signal peptide, extracellular affinity regions, transmembrane region, costimulatory signal conduction successively Area, polymeric component 2 and T cell signal transduction area functional domain, the light-dependent control element are LOV2, and the polymeric component 1 is SsrA, the polymeric component 2 is sspB, amino acid sequence such as SEQ ID No：Shown in 22, light-dependent control element in first monomer With the fusion connection of polymeric component 1, each functional domain of remaining first monomer and second comonomer is joined directly together or by connection Peptide is connected, amino acid sequence such as SEQ ID No after light-dependent control element and the fusion of polymeric component 1：Shown in 14.

2. Chimeric antigen receptor as claimed in claim 1, it is characterised in that the signal peptide is selected from CD8 α；

The antigen binding domain is Fab, Fab', (Fab')₂, Fv or scFv, the extracellular antigen binding domain functional domain combines Tumour antigen, the tumour antigen be selected from CD19, CD20, CD22, CD23, ROR1, CD30, CD56, kappa light chains, CD44, Pi Su, Penn/UCSF, NKG2D part, CD33/IL3Ra, c-Met, PAMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO- 1TCR, MAGEA3TCR or its any combination；

The extracellular affinity regions are DAP10 extracellular domains；

The extracellular hinge area is Immunoglobulin IgG hinge area or the hinge area of CD8 protein moleculars or its combination；

The costimulatory signal conducting region is selected from CD27, CD28,4-1BB, CD134, CD30, CD40, PD-1, LFA-1, CD2, The costimulatory signal conducting region of CD7, LIGHT, NKG2C, B7-H3 protein molecular, with CD3 ζ specifically bind part or its Meaning combination；

The T cell signal transduction area functional domain is selected from CD28, CD137, Fc ε RI γ, ZAP70 or CD3 ζ protein moleculars T cell signal transduction area or its any combination.

3. Chimeric antigen receptor as claimed in claim 1, it is characterised in that the connection peptide be 1 ~ 50 rich in Gly and/or The flexible peptide of Ala and/or Ser amino acid residues.

4. Chimeric antigen receptor as claimed in claim 1, it is characterised in that the signal peptide is CD8 α, the extracellular affinity regions For DAP10 extracellular domains, the antigen binding domain is the scFv of anti-CD19 protein moleculars, and the extracellular hinge area is CD8 albumen The hinge area of molecule, the transmembrane structure area is the transmembrane structure area of CD8 protein moleculars, and the costimulatory signal conducting region is 4- The costimulatory signal conducting region of 1BB protein moleculars, the T cell signal transduction area is the signal transduction area of CD3 ζ protein moleculars.

5. Chimeric antigen receptor as claimed in claim 4, it is characterised in that the CD8 alpha signals peptide amino acid sequence such as SEQ ID No：Shown in 2, the amino acid sequence such as SEQ ID No of DAP10 extracellular domains：Shown in 18, anti-CD19 scFv amino acid sequences are such as SEQ ID No：4 is shown, CD8 hinge region amino acids sequence such as SEQ ID No：6 is shown, CD8 transmembrane structures region amino acid sequence Such as SEQ ID No：8 is shown, 4-1BB costimulatory signals conduction region amino acid sequence such as SEQ ID No：10 is shown, CD3 ζ signals Conduct region amino acid sequence such as SEQ ID No：Shown in 26, the amino acid sequence such as SEQ ID No of the connection peptide：12、SEQ ID No：16、SEQ ID No：20、SEQ ID No：24 and SEQ ID No：Shown in 28.

6. Chimeric antigen receptor as claimed in claim 1, it is characterised in that the amino of the monomer of Chimeric antigen receptor first Acid sequence such as SEQ ID No：Shown in 32, the amino acid sequence such as SEQ ID No of second comonomer：Shown in 34.

7. one kind has photoinduction dimer type Chimeric antigen receptor gene, it is characterised in that coding such as claim 1-6 is any Chimeric antigen receptor described in.

8. Chimeric antigen receptor gene as claimed in claim 7, it is characterised in that the signal peptide is CD8 alpha signal peptides, nucleosides Acid sequence such as SEQ ID No：Shown in 1, the extracellular affinity regions are DAP10 extracellular domains, nucleotide sequence such as SEQ ID No：17 Shown, the antigen binding domain is anti-CD19 scFv, nucleotide sequence such as SEQ ID No：3 is shown, extracellular hinge area is CD8 hinge areas, nucleotide sequence such as SEQ ID No：5 is shown, transmembrane structure area is CD8 transmembrane structures area, and nucleotide sequence is such as SEQ ID No：7 is shown, costimulatory signal conducting region is 4-1BB costimulatory signal conducting regions, nucleotide sequence such as SEQ ID No：9 is shown, T cell signal transduction area is CD3 ζ signal transductions area, nucleotide sequence such as SEQ ID No：Shown in 25, peptide is connected Nucleotide sequence such as SEQ ID No：11、SEQ ID No：15、SEQ ID No：19、SEQ ID No：23 and SEQ ID No：27 Shown, light-dependent control element is LOV2, and the polymeric component 1 is ssrA, and nucleotide sequence is such as after light-dependent control element and polymeric component 1 are merged SEQ ID No：Shown in 13, the polymeric component 2 is sspB, nucleotide sequence such as SEQ ID No：Shown in 21.

9. Chimeric antigen receptor gene as claimed in claim 7, it is characterised in that described Chimeric antigen receptor gene order Such as SEQ ID No：Shown in 35.

10. recombinant vector, expression cassette, slow virus carrier or the cell of Chimeric antigen receptor of the expression with photo-induction guiding element, its It is characterised by the gene containing the Chimeric antigen receptor as described in claim any one of 7-9.