CN109467604A

CN109467604A - Chimeric antigen receptor DAP12-T2A-CD8 α-CD19scFv-TREM1 and application thereof

Info

Publication number: CN109467604A
Application number: CN201811317120.9A
Authority: CN
Inventors: 王恩秀; 汪晨; 张海
Original assignee: Nanjing Katy Medical Technology Co Ltd
Current assignee: Nanjing Katy Medical Technology Co Ltd
Priority date: 2018-11-07
Filing date: 2018-11-07
Publication date: 2019-03-15

Abstract

The invention discloses Chimeric antigen receptor DAP12-T2A-CD8 α-CD19scFv-TREM1 of a kind of optimization and application thereof, and the Chimeric antigen receptor is in series by the second conducting structure intracellular domain DAP12, T2A, CD8 alpha signal peptide, anti human CD 19 monoclonal antibody FMC63 light chain and heavy chain variable region CD19scFv, the first conducting structure intracellular domain.For the Chimeric antigen receptor for modifying T lymphocyte, the T cell (CAR-T cell) after modification can be used for the treatment of the Hematological malignancies of the CD19 positive.In Hematological malignancies fragmentation test, hence it is evident that strengthen CAR-T cell to the killing ability of leukemia tumor cells, show good safety and anti-tumor activity in clinical application.

Description

Chimeric antigen receptor DAP12-T2A-CD8 α-CD19scFv-TREM1 and application thereof

Technical field

The present invention relates to immunotherapy of tumors technical fields.The present invention obtains encoding chimeric antigen using technique for gene engineering The genetic fragment is inserted into Lentiviral, packet by the fusion of receptor DAP12-T2A-CD8 α-CD19scFv-TREM1 Slow virus is dressed up, in the human T lymphocyte that transduces, T cell is made to express the Chimeric antigen receptor.It is such the invention further relates to encoding Nucleic acid, the carrier of transmembrane polypeptide, and the immunocyte of the CAR described in its surface expression for immunization therapy.Institute of the present invention The T cell for the Chimeric antigen receptor modification being related to can target and the tumour cell of the specific killing CD19 positive, is used for pernicious blood The treatment of liquid tumour opens the road to the effective adoptive immunotherapy strategy for treating cancer.

Background technique

With the development of tumour immunity theory and clinical technology, Chimeric antigen receptor T cell therapy (Chimeric Antigen receptor T-cell immunotherapy, CAR-T) become tumour immunity treatment most promising at present One of method [Schmitz M, et al.Chimeric antigen receptor-engineered T cells Forimmunotherapy of Cancer.J Biomed Biotechnol, 2010, doi:10.1155/2010/ 956304.].Chimeric antigen receptor (CAR) is the core component of CAR-T, and CAR can redirect it for selected immunocyte Specificity and reactivity, therefore the ability that the non-dependent mode of T cell HLA identifies tumour antigen is assigned, this to change by CAR The T cell made is compared different from (TCR) response of normal T-cell receptor, is not limited by MHC, has the ability of activation and proliferation, because This ability with efficient killing tumor cell.

Chimeric antigen receptor (CAR) expresses synthetic proteins in T cell, by antigen-recognizing fragments (such as antibody of antibody Single chain variable fragment) it is merged with intracellular signal structural domain.The study found that with the single-stranded variable of tumor specific monoclonal antibodies Area (scFv) replaces the α of TCR, β chain variable region, and scFv is directly connected with T cell signal transduction structural domain, forms inosculating antibody Original receptor (CAR) is expressed in T cell surface, and tumour specific antigen, directly the activation letter of generation T cell can be identified by scFv Number, promote T cell activation, proliferation, specific killing tumour cell.The process relies primarily on scFv pairs of CAR-T cell surface The specific recognition of tumour antigen, the specificity of immune response and lethal relatively strong.

Disease incidence of the B cell malignant tumour in Hematological malignancies rise year by year, but B cell Hematological malignancies are controlled Treatment means are still relatively limited to, at present based on chemotherapy, stem cell transplantation and biological therapy etc., though clinically common therapeutic scheme Can so have very high complete remission rate (CR), but such disease is easy to recur and successive treatment drug resistance, cause long-term prognosis compared with Difference.Based on this, the cellular immunotherapy of tumour becomes the hot spot studied now as a kind of new therapeutic strategy.CD19 is almost All B cell malignant cell surfaces are expressed in, and other parenchymas and candidate stem cell are hardly expressed, therefore are made It is high for the specificity of B cell tumour antigen.The Chimeric antigen receptor DAP12-T2A-CD8 α-constructed using anti-CD19scFv CD19scFv-TREM1 is expressed in T lymphocyte, can make the tumour cell of the T cell specific killing CD19 positive, foreign countries are There is two CD19CAR-T to obtain FDA approval listing, and the application in B cell tumour obtains significant curative effect.

The present invention provides a kind of Chimeric antigen receptor DAP12-T2A-CD8 α-CD19scFv-TREM1, includes intracellular second Conducting structure domain DAP12, T2A, CD8 alpha signal peptide, anti human CD 19 monoclonal antibody FMC63 light chain and heavy chain variable region CD19scFv, the first conducting structure intracellular domain TREM1.For the Chimeric antigen receptor for modifying T lymphocyte, the T after modification is thin Born of the same parents' (CAR-T cell) can be used for the treatment of the Hematological malignancies of the CD19 positive.In neoplastic hematologic disorder fragmentation test, hence it is evident that reinforce The killing abilities of CAR-T cells against tumor cells, shows good safety and anti-tumor activity in clinical application.

Summary of the invention

The technical problem to be solved by the invention is to provide the Chimeric antigen receptors of the targeting CD19 of optimization a kind of (DAP12-T2A-CD8 α-CD19scFv-TREM1), the T cell for expressing the Chimeric antigen receptor have it unique, grind first It is studied carefully in the tumour-specific killing ability of cellular level, further illustrates its application in clinical treatment.

It is an object of the present invention to a kind of Chimeric antigen receptor for optimizing CD19 targeting and preparation method thereof and use On the way.

The second object of the present invention is to provide the nucleic acid for encoding the Chimeric antigen receptor.

The third object of the present invention is to provide the cell containing the Chimeric antigen receptor and its application.

In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is that: a kind of Chimeric antigen receptor of optimization DAP12-T2A-CD8 α-CD19scFv-TREM1, the Chimeric antigen receptor by the second conducting structure intracellular domain DAP12, T2A, CD8 alpha signal peptide, anti human CD 19 monoclonal antibody FMC63 light chain and heavy chain variable region CD19scFv, the first conducting structure intracellular domain TREM1 is in series；The nucleic acid sequence of the Chimeric antigen receptor as shown in SEQ ID NO.16, the Chimeric antigen receptor Amino acid sequence is as shown in SEQ ID NO.20 in sequence table.

The antigen-binding domains (i.e. CD19scFv) of the targeting CD19 Chimeric antigen receptor are VL-VH or VH-VL, VL For antibody's light chain variable region；Wherein VH is antibody heavy chain variable region；"-" is link peptide or peptide bond.

In one embodiment, the antibody's light chain variable region VL has amino acid sequence shown in SEQ ID NO.11 Column, or the polypeptide with its amino acid sequence with 85%-99% identity；Antibody heavy chain variable region VH has SEQ ID NO.12 Shown in amino acid sequence, or with its amino acid sequence have 85%-99% identity polypeptide.

In one embodiment, the light chain variable region (VL) of the single-chain antibody of the antigen-binding domains and heavy chain can Become between area (VH) and connected by link peptide (Linker), includes GS Linker such as (G₃S)₄Or (G₄S)₃, preferably (G₄S)₃Connection Peptide connects peptide ammino acid as shown in SEQ ID NO.13.

The Chimeric antigen receptor contains the single-chain antibody CD19scFv for people's CD19 antigen, and amino acid sequence is such as Shown in SEQ ID NO.2.

The intracellular signal structural domain preferably the first signal transduction structural domain include TREM1, TREM2, NKp44, NKp46, The sequence of NKp30 or NKG2D.It is further preferred that the first signal transduction structural domain includes the sequence of TREM1.

Shown in the preferred SEQ ID NO.9 of the amino acid sequence of first conducting structure domain TREM1, or with its amino acid sequence Arrange the polypeptide with 85%-99% identity.

The second conducting structure intracellular domain DAP12 is connected by T2A with extracellular signal peptide and antigen-binding domains.It is excellent Choosing, the DAP12 amino acid sequence is as shown in SEQ ID NO.2；The T2A amino acid sequence is as shown in SEQ ID NO.4.

Chimeric antigen receptor structure of the present invention is as follows:

Wherein antigen-binding domains can be by VH-Linker-VL or VL-Linker-VH or VHH1-Linker-VHH2 group At VHH1 and VHH2 can recognize same antigen, or not synantigen.

It is preferably carried out in scheme at of the invention one, further includes signal coding sequence in the carrier.Preferably, The signal peptide sequence is connected to the upstream of the antigen-binding domains nucleic acid sequence.The preferably described signal peptide is source of people CD8 alpha signal peptide or human GM-CSF signal peptide.

Preferably, the source of people CD8 alpha signal peptide nucleic acid sequence is as shown in SEQ ID NO.7, amino acid sequence such as SEQ ID Shown in NO.8, or with it with 85%-99% identity amino acid sequence；

Preferably, human GM-CSF signal peptide nucleic acid sequence SEQ ID NO.14 as shown, amino acid sequence such as SEQ ID NO.15, or with it with 85%-99% identity amino acid sequence；

In one embodiment, Chimeric antigen receptor of the present invention includes SEQ ID NO.16, SEQ ID NO.17, SEQ Nucleotide sequence shown in ID NO.18 or SEQ ID NO.19；Amino acid sequence such as SEQ ID NO.20, SEQ ID Shown in NO.21, SEQ ID NO.22 or SEQ ID NO.23, or with it with 85%-99% identity amino acid sequence

A kind of recombinant expression carrier, the preferred Lentiviral of the expression vector, comprising coding SEQ ID NO.16, The nucleotide sequence of SEQ ID NO.17, SEQ ID NO.18 or SEQ ID NO.19.

A kind of cell for expressing Chimeric antigen receptor, the preferred immunocyte of the cell；Further preferred T lymphocyte, NK Cell, NKT cell, macrophage, mescenchymal stem cell, candidate stem cell, multipotential stem cell or embryonic stem cell culture differentiation Immunocyte.

A method of the T cell of Chimeric antigen receptor DAP12-T2A-CD8 α-CD19scFv-TREM1 modification is prepared, it should Method includes separation and activates T cell to be finished, is then transduceed the T cell with foregoing expression vectors.

Exist containing the Chimeric antigen receptor DAP12-T2A-CD8 α-CD19scFv-TREM1, expression vector, the cell Purposes in the drug of preparation treatment tumour.

The tumour preferably includes B-lineage Acute Lymphocyte Leukemia, chronic lymphocytic leukemia, the white blood of anxious myelocyte Disease, Huppert's disease, Hodgkin lymphoma, diffusivity large B cell lymphoid tumor, follicular lymphoma, lymphoma mantle cell and Any combination thereof.

In a particular embodiment, it is related to the medicinal usage of preparation treatment blood system tumour.

Chimeric antigen receptor DAP12-T2A-CD8 α-CD19scFv-TREM1 of the present invention be related to prepare it is antitumor Application in drug and cellular immunotherapy.

Immune effector cell of the present invention is related to preparing the application in anti-tumor drug and cellular immunotherapy.

Beneficial effects of the present invention:

The present invention provides a kind of Chimeric antigen receptor, includes the second conducting structure intracellular domain, T2A, extracellular signal peptide, targeting CD19 antigen-binding domains, the first conducting structure intracellular domain, such as the second conducting structure intracellular domain DAP12, T2A, CD8 α letter Number peptide, CD19scFv, the first conducting structure intracellular domain TREM1 (DAP12-T2A-CD8 alpha signal peptide-CD19scFv-TREM1).It adopts With the CD19scFv gene order of targeting CD19 antigen, its VL and VH are combined rearrangement by link peptide, carried different Signal peptide combination, constructs anti human CD 19 CAR expression plasmid, using the packaging plasmid system of the plasmid and slow virus in 293T cell Middle packaging virus infects T cell, the CAR-T cell of preparation targeting CD19 antigen.By the external functional verification of CAR-T cell, Such as cytokine secretion and killing experiments, screening obtains the optimal Chimeric antigen receptor structure DAP12-T2A-CD8 α-of effect CD19scFv-TREM1.Secretion is lower when the Chimeric antigen receptor structure is stimulated conducive to CAR-T cell by extracellular tumour antigen Horizontal cell factor, can guarantee the safety of clinical application well, not only have good tumor-killing to act in vitro, And significant curative effect is also shown in clinical treatment Hematological malignancies patient, and cytokine release reaction is more warm With.Compared with prior art, which shows higher anti-tumor capacity, is had by its immunocyte modified The ability of higher targets identification tumour antigen, enhances the killing activity to tumour cell.

Detailed description of the invention

Fig. 1 is illustrated a variety of embedding involved in the present invention containing the CAR structure of targeting CD19 antigen-binding domains Close antigen receptor combination assumption diagram.

Fig. 2 is that CD19CAR-1, CD19CAR-2, CD19CAR-3, CD19CAR-4 cell infection slow virus passed through stream after 7 days Formula cell instrument detects the positive expression rate of T cell surface identification CAR structure.

Fig. 3 is cell proliferative conditions after CAR-T cell infection difference CAR slow virus, CAR-T and NTD cell expands in vitro Increase 10 days, wherein NTD is the T cell of untransfected CAR slow virus.

Fig. 4 is the secretion situation of CAR-T cell IFN-γ under CD19 antigenic stimulus, and CAR-T cell is trained altogether with target cell It supports for 24 hours.

Fig. 5 is CAR-T cell to CD19 antigen-positive cell strain lethal effect, and CAR-T cell is co-cultured with target cell and killed Hurt 8h.

Fig. 6 is human interleukin 6 emission levels figure, and after CD19CAR-T1 patient feeds back CD19CAR-1 cell, body temperature is last Start to increase within 3 days after primary feedback, interleukin 6 (IL-6) secretion level significantly improves 100 times to cytokines measurement as the result is shown.

Specific embodiment

The present invention provides a kind of Chimeric antigen receptor for targeting CD19, immune effector cell and its in clinical treatment blood Application in tumour with reference to specific embodiments the present invention is further explained.

Novel chimeric antigen receptor of the invention includes extracellular signal peptide structure, such as CD8 alpha signal peptide, 4-1BB signal Peptide, GM-CSF signal peptide or CD4 signal peptide, preferably CD8 alpha signal peptide and GM-CSF signal peptide.

The term as used herein " intracellular signal structural domain " refer to can transfer cell effector function signal and guide carefully The protein structure region of born of the same parents' execution specific function.Intracellular signal structural domain may include the first signal transduction structural domain, second Signal transduction structural domain and/or transmembrane domain.

" identity " (identity) of the term as used herein amino acid sequence can be used interchangeably with " similitude ", be referred to Be between amino acid sequence by sequence alignment program such as BLAST determine similarity degree.The side of amino acid alignment Method and software are well known to the skilled person.It can be by carrying out one or several (examples to known amino acid sequence Such as 1-15, such as 2,3,5,8,10 or 12) substitution of amino acid residue, deletion and/or addition and obtain modified ammonia Base acid sequence.For example, by conventional protein engineering means (such as conservative substitution etc.), to SEQ ID of the present invention CD19 antigen-binding domains shown in NO.2 are transformed, can obtain with SEQ ID NO.2 have at least 85% (such as 85%~99% or 90%~99% or 95%~99%) sequence identity, and there is essentially identical antigen binding structure The variant sequence thereof in domain.

The term as used herein " antigen-binding domains " be include the functional antibody moiety of tool, preferred antigens combine The variable region of complete antibody and/or.Antibody fragment include Fab, Fab ', F (ab ') 2, Fv fragments, single-chain antibody scFv, Single domain antibody VHH and multi-specificity antibody.

Below in conjunction with attached drawing, a preferred embodiment of the present invention will be described in detail.It is not specified in embodiment specific The experimental method of condition, usually according to normal condition, such as Molecular Cloning:A Laboratory guide (third edition, J. Pehanorm Brooker etc. write) Described in condition or according to the normal condition proposed by manufacturer.Test material as used in the following examples, such as without special theory It is bright, it is to be commercially available from routine biochemistry reagent shop.

Embodiment 1, Chimeric antigen receptor preparation

The present invention provides a kind of targeting CD19 antigen Chimeric antigen receptor of optimization.Chimeric antigen receptor of the invention is by born of the same parents Interior second conducting structure domain-T2A- extracellular signal peptide-targeting CD19 antigen-binding domains-the first conducting structure intracellular domain sequence It is composed in series.Therefore need to construct the viral vectors containing the combination of different stimulated signal respectively.In the present embodiment with TREM1 be system The structure in one the first conducting structure intracellular domain is respectively necessary for constructing following 4 Chimeric antigen receptors (Fig. 1):

1, the gene order containing targeting CD19 Chimeric antigen receptor

Design successively containing natural kill activated receptor (abbreviation DAP12), T2A, CD8 alpha signal peptide, GM-CSF signal peptide, VL-Linker-VH, VH-Linker-VL, human medullary cell triggering property receptor (abbreviation TREM1), structure are as shown in Figure 1.Its The nucleotide sequence of middle DAP12 is as shown in SEQ ID NO.1, and amino acid sequence is as shown in SEQ ID NO.2, the nucleotide of T2A Sequence is as shown in SEQ ID NO.3, and amino acid sequence is as shown in SEQ ID NO.4；The nucleotide sequence such as SEQ of CD8 alpha signal peptide Shown in ID NO.5, amino acid sequence is as shown in SEQ ID NO.6；The nucleotide sequence of GM-CSF signal peptide such as SEQ ID Shown in NO.14, amino acid sequence is as shown in SEQ ID NO.15；The amino acid sequence of the light chain variable region VL of CD19 single-chain antibody As shown in SEQ ID NO.11；The amino acid sequence of the heavy chain variable region VH of CD19 single-chain antibody is as shown in SEQ ID NO.12； The nucleotide sequence of TREM1 is as shown in SEQ ID NO.9, and amino acid sequence is as shown in SEQ ID NO.10.

2, the slow virus carrier of building expression Chimeric antigen receptor

PELNS-DAP12-T2A plasmid is saved by Nanjing Ka Ti medical science and technology Co., Ltd, or according to document (Enxiu Wang et al.Generation of Potent T-cell Immunotherapy for Cancer Using DAP12- Based,Multichain,Chimeric Immunoreceptors.2015,Cancer Immunology Research,3 (7): method disclosed in 815) is constructed, CD8 α-VL-VH-TREM1, CD8 α-VH-VL-TREM1, GM-CSF-VL-VH- TREM1, GM-CSF-VH-VL-TREM1 gene chemical synthesis are by biotechnology company, Sangon Biotech (Shanghai) Co., Ltd. It synthesizes and pUC19-CD19CAR-1, pUC19-CD19CAR-2, pUC19-CD19CAR-3, pUC19-CD19CAR-4 matter is provided The genetic fragment that plasmid pELNS-DAP12-T2A is synthesized with four respectively (is purchased from by grain by AvrII, SalI double digestion Takara company), endonuclease reaction by specification carries out, and reconnects after digestion, and the slow virus for obtaining expression Chimeric antigen receptor carries Body:

pELNS-DAP12-T2A-CD8α-VL-VH-TREM1(CD19CAR-1)

pELNS-DAP12-T2A-CD8α-VH-VL-TREM1(CD19CAR-2)

pELNS-DAP12-T2A-GM-CSF-VL-VH-TREM1(CD19CAR-3)

pELNS-DAP12-T2A-GM-CSF-VH-VL-TREM1(CD19CAR-4)。

5 μ L slow virus carriers are converted in Escherichia coli TOP10 competent cell (to be had purchased from the outstanding excellent biotechnology of Nanjing peace Limit company), picking monoclonal after 37 DEG C of culture 16h, the monoclonal of picking is tried after cultivating 12h under the conditions of 37 DEG C with plasmid extraction Agent box (being purchased from Takara company) extracting plasmid, specific method are shown in specification.

3, slow virus is packed

The present embodiment packs slow virus and uses calcium phosphate method, the specific steps are as follows:

(1) 293T cell passes on every other day

Each T150 cell bottle plantation 5 × 10⁶A cell.After 48 hours, cell number should reach million/bottle of 20-25.

(2) 293T cell spreads bottle

A) by taking 1 T150 Tissue Culture Flask as an example, cell is gently washed twice with 1 × PBS of about 15ml

B) 3ml0.25% pancreatin -2.21mM EDTA is added

C) cell detachment is waited until, the DMEM culture medium that 12ml 10% (wt) FBS (being purchased from Gibico) is added (is purchased from Corning) into the cell to have fallen off

D) it collects and cell is transferred to sterile centrifugation tube, 1000rpm is centrifuged 10 minutes

E) supernatant is sopped up, precipitating is resuspended in the DMEM culture solution of 10ml 10% (wt) FBS.

F) cell count calculates 12 × 10 according to cell concentration⁶Volume required for a cell

G) the DMEM culture solution of cell and 10% (wt) FBS of 25ml are merged, is put into T150 cell bottle, jog makes It obtains cell and is evenly distributed to 37 DEG C of cell bottle bottom, overnight incubation in 5%CO2 incubator.

(3) cell transfecting

Cell is observed, cell density reaches about 80%-90%, can start to transfect at this time

A) 30-60 minutes before transfection, culture solution is softly sopped up.

B) it mixes Plasmid DNA and calcium chloride solution needs 28ug pRSV.rev (to be purchased from for one T150 bottles Invitrogen company), 28ug pGAG-Pol (is purchased from Invitrogen company), and 11ug pVSVG is (public purchased from Invitrogen Department), 23ug recombinant slow virus expression plasmid CD19CAR-1/CD19CAR-2/CD19CAR-3/CD19CAR-4 is added to 1.5ml In calcium chloride solution, mix.

C) 1.5ml BBS solution is added in 15ml sterile centrifugation tube, DNA- calcium chloride solution is mixed with 1ml pipette tips After be added drop-wise in BBS solution, rapidly mix 15-20 under, be incubated at room temperature 25-30 minutes.

D) with 5ml pipette that DNA- calcium chloride-BBS mixture (being purchased from the green skies Bioisystech Co., Ltd in Shanghai) is equal It is even to be added dropwise in T150 bottles.It is cultivated in 37 DEG C of cell incubators containing 5% carbon dioxide, 6h changes liquid.

E) liquid is changed after 6h.Culture plate is shaked gently for several times with some calcium phosphate precipitations that sufficiently suspend, and it is heavy to suck phosphoric acid calcium The culture solution in shallow lake is added the DMEM culture solution of 5% fresh (wt) FBS of 20ml, continues to cultivate.

(4) viral supernatants are collected for the first time

A) the 293T cells and supernatant that the previous day transfects is collected into centrifuge tube, 1000rpm is centrifuged 5 minutes, label, temporarily It is stored in 4 DEG C of refrigerators.

B) the DMEM culture medium of 20ml 5% (wt) FBS preheated in advance is added in cell bottle, 37 DEG C of cell incubators Continue overnight incubation.

(5) vial supernatant (48h/ the 4th day) is collected second.

(6) supernatant is filtered

The supernatant collected twice is concentrated in together, removes cell fragment with 0.45 μm of membrane filtration.

(7) viral concentration

4 DEG C, 12000-24000rpm centrifuged overnight

(8) virus storage

After centrifugation, whole supernatants are toppled over, the DMEM culture medium that fresh 5% (wt) FBS is added is resuspended, viral packing is carried out, It is spare to deposit in -80 DEG C of refrigerators rapidly

(9) slow virus titer determination

A) virus infection 293T cell

293T cell is spread into 24 orifice plates before infection, takes and has purified 200 μ L of concentrating virus and be added in 293T cell, for 24 hours Liquid is changed with the DMEM culture medium containing 10%FBS (wt) afterwards, is centrifuged 5min under the conditions of 1200r/min after infecting 72h to collect carefully Born of the same parents extract genome.

B) genome is extracted

Genome extraction agent box is to operate purchased from Takara company by kit specification

C) qPCR measures virus titer

Reaction system is as follows: 12.5 μ L of Probe qPCR Mix (is purchased from Takara), and 0.5 μ L of upstream primer is (raw by raw work The synthesis of object engineering (Shanghai) limited liability company), 0.5 μ L of downstream primer is (by Sangon Biotech (Shanghai) Co., Ltd. Synthesis), probe 1 μ L (being synthesized by Sangon Biotech (Shanghai) Co., Ltd.), 2 μ L of template, 8.5 μ L of aqua sterilisa, reaction System is 25 μ L, and reaction condition is arranged to specifications, after reaction, with analysis software data, according to standard curve Calculate virus titer.Calculated result shows that virus titer is 1 × 10⁷TU/ml。

Embodiment 2, virus infection T cell

1, the separation activation and virus infection of T cell

(1) separation of human peripheral blood mononuclear cell

Peripheral blood about 10ml, room temperature (18-25 DEG C) natural subsidence about 30min are acquired with the heparin tube containing anti-coagulants, is received Collect upper plasma, the upper plasma of collection is centrifuged 10min under the conditions of 5000r/min, 1:1 is added to lymphocyte by volume In separating liquid (being purchased from the ocean Tianjin Hao biological products science and technology limited Company), gradient centrifugation, 3000r/min, centrifugation 30min, after centrifugation, centrifuge tube is by upper lower leaf: first layer is plasma layer；The second layer is lymphocyte tunica albuginea layer；Third layer For transparent separation liquid layer；4th layer of red blood cell layer.Draw lymphocyte tunica albuginea layer, and washed 2 times with PBS, be centrifuged twice with 1500r/min is centrifuged 10min, and cell is resuspended in PBS, and it is complete that 5% autologous plasma+300IU/ml recombinant human il-2+KBM581 is added Culture medium culture human peripheral blood mononuclear cell.

(2) slow-virus infection T lymphocyte

With containing the 5% freshly prepd single core of autologous plasma+300IU/ml recombinant human il-2's+KBM581 complete medium culture Cell PBMC, IL-2 are purchased from R&D Systems, and KBM581 is purchased from Corning, and addition CD3/CD28Dynabeads is immune within the 0th day Magnetic bead (being purchased from invitrogen) activating T cell, progress slow-virus infection in first 3 days are added the corresponding slow virus of 0.25MOI and carry Culture medium is changed to containing 5% autologous plasma+300IU/ml after 48h as blank control by body, the T lymphocyte being uninfected by Recombinant human il-2's+KBM581 complete medium continues culture 7-9 days.

2, in T cell CAR positive rate detection

By the T cell of the virus infection of culture to the 7th day, 1200r/min is centrifuged 5min, and it is thin to collect to abandon supernatant to the greatest extent Born of the same parents are resuspended cell with the PBS solution containing volume fraction 1%FBS, and are 1 × 10 by cell adjustment density⁵Life is added in a/ml Object element marks Human CD19 (ACRO Biosystems), and adding Streptavidin-PE, (BD Biosciences is public Department), 4 DEG C of incubation 15min, PBS solution is washed 2 times, and flow cytometer is detected, pass through culture in 7 days as the result is shown, The positive rate of CAR-T cell CAR: CD19CAR-1 infection group and viral infection group positive rate 64.5%, CD19CAR-2 infection group and viral infection group are positive Rate 89.6%, CD19CAR-3 infection group and viral infection group the positive rate 57.4%, (figure of CD19CAR-4 infection group and viral infection group positive rate 51.5% 2)。

The influence of embodiment 3, virus infection CAR-T cell by cell proliferation

After the complete T cell of each group virus infection, by T cell 5% autologous plasma+300IU/ml recombined human containing volume fraction IL-2+KBM581 complete medium counts primary for every 1-2 days.Then T lymphocyte growing state is observed, as a result such as Fig. 3 institute Show.The result shows that cell is still capable of forming typical proliferating clones group, by cell after the virus of infection expression CAR It being counted, visible CD19CAR-1, CD19CAR-2, CD19CAR-3, CD19CAR-4 proliferation of drafting cell Proliferation curve is similar, It is slightly weaker than the T cell of uninfecting virus (NTD in Fig. 3) proliferative capacity.

Embodiment 4, the cytokine secretion for detecting virus infection CD19CAR-T cell

(1) method that cytokines measurement uses Elisa is carried out using R&D company kit.

(2) dilution of standard items: preparing 1ml centrifuge tube 7, and first standard is added in each centrifuge tube in number consecutively number Then 500 μ L of product dilution takes 500 μ L of original content standard items to be added to 1 in the centrifuge tube for the number of finishing, mixes well, then It takes 500 μ L to be added in second centrifuge tube in the centrifuge tube, mixes well；Take 500 μ L that third is added in the centrifuge tube again In centrifuge tube, mix well；It takes 500 μ L to be added in the 4th centrifuge tube in the centrifuge tube again, mixes well；Again this from It takes 500 μ L to be added in the 5th centrifuge tube in heart pipe, mixes well；Take 500 μ L that the 6th centrifugation is added in the centrifuge tube again Guan Zhong is mixed well；It takes 500 μ L to be added in the 7th centrifuge tube in the centrifuge tube again, mixes well.

(3) it is marked with quasi- sample wells on enzyme mark coating plate, sequentially adds standard items 100 the μ L, each concentration 2-3 of various concentration A parallel hole.

(4) be loaded: blank well is respectively set, and (blank control wells are replaced with water, the antibody of enzyme marking reagent and biotin labeling Operation is as before), sample to be tested hole, 100 μ L of product is first loaded in sample to be tested hole on enzyme mark coating plate, sample is added on enzyme by sample-adding Target hole bottom, does not touch hole wall as far as possible, shakes gently mixing

(5) it is incubated for: being placed at room temperature for and be incubated for 2h

(6) it washs: discarding liquid, dry, every hole adds 200 μ L cleaning solutions, discards after static 30s, be so repeated 3 times, and claps It is dry

(7) add antibody: 100 μ L detection antibody being added on enzyme mark coating plate

(8) it is incubated for: biconditional operation (5)

(9) it washs: biconditional operation (6)

(10) label: 100 μ L horseradish peroxidase-labeled Streptavidins are added in every hole

(11) it is incubated for: being protected from light and be placed at room temperature for incubation 20min

(12) it washs: biconditional operation (6)

(13) develop the color: 100 μ L of developing solution is added in every hole, and gently concussion mixes, and is protected from light and is placed at room temperature for incubation 20min

(14) terminate: 50 μ L of terminate liquid is added in every hole, terminates reaction

(15) measure: with blank value school zero, 450nm wavelength sequentially measures the absorbance (OD value) in each hole, measures Ying Jia Enter and is carried out in 15min after terminate liquid.

Select CD19 antigenic expression have discrepant target cell and CD19CAR-1, CD19CAR-2, CD19CAR-3, The CAR-T cell of CD19CAR-4 co-cultures, and detection CAR-T is generated response effect secretion of gamma-IFN level by antigenic stimulus, CD19 antigen target cell selects 293T-CD19 (CD19 positive) and 293T (CD19 feminine gender), with this come show CAR-T by CD19 antigenic stimulus when institute specific secretion goes out IFN-γ, as a result reflects that 4 kinds of CAR-T cells have antigenic expression The target cell of difference produces different response effects.4 kinds of CAR-T cells are trained altogether with CD19 positive target cell 293T-CD19 Significant secretion of gamma-IFN (Fig. 4), shows that the CAR-T of CD19CAR-1, CD19CAR-2, CD19CAR-3, CD19CAR-4 are thin when supporting Born of the same parents all have response effect, but DAP12-T2A-CD8 α-VL-VH-TREM1 (CD19CAR- for the tumour cell of antigen positive 1) Chimeric antigen receptor effect structure is best.

Embodiment 5, virus infection targeting CD19CAR-T cells in vitro fragmentation effect assessment

(1) targeting CD19 antigen killing: culture target cell 293T-CD19 (CD19 is positive), 293T (CD19 is negative) and effect Answer the CAR-T of cell CD19CAR-1, CD19CAR-2, CD19CAR-3, CD19CAR-4；

(2) target cell and effector cell are collected, 1500rpm/min is centrifuged 5min, abandons supernatant

(3) target cell and effector cell is resuspended with 10%FBS+1640 complete medium

(4) real-time cell analysis system (RTCA) is utilized, in aerial addition 50 μ L, 1640 culture medium of E-Plate16

(5) baseline is detected using RTCA, determines that selected holes contact is normal

(6) setting effect target ratio is 0:1,1:1,5:1,10:1

(7) E-Plate16 is taken out, according to effect target ratio, uniformly mixed 100 μ L of target cell suspension is added in every hole, makes Every hole kind cell number is 10⁴cells/100μL。

(8) E-Plate16 is placed in incubator, with 37 DEG C, under the conditions of 5%CO2, is placed overnight

(9) second days, E-Plate16 is taken out, the 50 corresponding effector cells of μ L are added, calculated after effector cell 8h is added Killing rate.

(10)

Testing result is as shown in Figure 5.CAR-T pairs of CD19CAR-1, CD19CAR-2, CD19CAR-3, CD19CAR-4 CD19 antigen-positive cell lethal effect is significant, hence it is evident that is higher than NTD group, wherein Chimeric antigen receptor DAP12-T2A-CD8 α-VL- VH-TREM1 (CD19CAR-1) effect is best.Killing experiments in vitro is the result shows that Chimeric antigen receptor DAP12-T2A-CD8 α- CD19scFv-TREM1 targeting CD19 antigen has very strong anti-tumor activity.

Embodiment 6, CD19CAR-1 modification Autologous T cells to the B-lineage Acute Lymphocyte Leukemia patient's of the CD19 positive Clinical testing treatment effect

B-lineage Acute Lymphocyte Leukemia patient 100ml peripheral blood is acquired, isolates and purifies and obtains T cell (embodiment 2), add Add 5% autologous plasma, 300IU/ml recombinant human il-2 and CD3/CD28 magnetic bead Activation In Vitro after 1 day transduction of CD 19CAR-1 it is sick slowly Poisonous carrier.With 0.8~1.5 × 10⁶The culture density of/mL expands culture 9~12 days daily.

CD19CAR-1 is transduceed respectively into the T cell from 5 different B-lineage Acute Lymphocyte Leukemia patients, transduction Cell afterwards is named as CD19CAR-T1, CD19CAR-T2, CD19CAR-T3, CD19CAR-T4, CD19CAR-T5.

Below by taking the treatment of CD19CAR-T1 B-lineage Acute Lymphocyte Leukemia patient as an example.

CD19CAR-T1 patient:

(1) grain system: active proliferation, neutrophilic myelocyte ratio increase.Neutrophilic metamyelocyte ratio is normal.Mature rank Section ratio lowers.

(2) red system: active proliferation, early erythroblast ratio are normal.Rubricyte ratio lowers.Metarubricyte ratio Increase.The visible huge young sample of normoblast reddens cell, karyolobism red blood cell, ratio > 12%.Mature erythrocyte form is normal, Dyeing is good.

(3) lymphocyte: ratio increased significantly.Visible lymphoblast, accounts for 32.5%.

(4) megacaryocyte: full sheet is shown in megacaryocyte 7；Classify 7 megacaryocytes；Initial cell is 0；Inmature macronucleus Cell is 0；Graininess megacaryocyte is 6；Producing platelet type megakaryocyte is 0；Bare nucleus megacaryocyte is 1.Blood is small Plate is visible

(5) Blood piece: leukocyte count is normal, and rare in granulocyte classification, percentage of lymphocyte obviously increases, monocyte ratio Example is reduced, and blood platelet is visible.

(6) impression: acute lymphoblastic leukemia relapse

Recommend through hospital, by Ethic review, patient signs informed consent form, then carries out CD19CAR-1 cell therapy Clinical experimental study.

The Autologous T cells therapeutic process of CD19CAR-1 modification are as follows: acquisition peripheral blood in patients, external sorting obtain T cell, T Cells in vitro activation and amplification prepare CD19CAR-T1 cell preparation, and the quality of the pharmaceutical preparations controls and lets pass, and two time points is divided to be divided to two It is secondary by venous re-transfusion in patient body, amount to infusion about 2.5 × 10⁸CAR-T cell.

After patient CD19CAR-T1 feeds back CD19CAR-1 cell, there is mild exothermic reaction, body temperature is returned in last time Start to increase within 2 days after defeated, interleukin 6 (IL-6) secretion level is compared with 80 times of baseline period raising as the result is shown for cytokines measurement, such as Shown in Fig. 6, patient, which persistently has a fever 3 days, is less than 38.5 DEG C, and subsequent body temperature is gradually reduced and reaches normal body temperature.Foreign countries' report CD19CAR-T treatment B-lineage Acute Lymphocyte Leukemia patient side reaction it is serious when in 3 days occur continue high fever.This clinic Test result shows the T cell of Novel chimeric antigen receptor modification according to the present invention in therapeutic bone-marrow-derived lymphocyte leukaemia In have the reaction of milder, it is less side effects to patient.The peripheral blood and marrow of patient are carried out behind 14 days after feedback Detection.As shown in 1 result of table, percentage shared by the CD19 positive (CD19+) cell is reduced to by treating preceding 24% in peripheral blood 0%, percentage shared by positive (CD19+) cell of CD19 is reduced to < 1% by treating preceding 30.5% in marrow.In conjunction with hematology Testing result shows: the patient is after CD19CAR-1 cell therapy provided by the invention, and CD19 is positive in peripheral blood and marrow Leukaemia cell be completely removed, reach clinically complete remission.

The variation of the 1 pretherapy and post-treatment bone-marrow-derived lymphocyte of B-lineage Acute Lymphocyte Leukemia patient of table

Detection project	Peripheral blood (before treatment)	Peripheral blood (after treatment)	Marrow (before treatment)	Marrow (after treatment)
					CD19+ cell	24%	0%	30.5%	< 1%

Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be Various changes are made to it in form and in details, without departing from claims limited range of the present invention.

Sequence table

<110>Nanjing Ka Ti medical science and technology Co., Ltd

<120>Chimeric antigen receptor DAP12-T2A-CD8 α-CD19scfv-TREM1 and application thereof

<130> 2018

<160> 23

<170> SIPOSequenceListing 1.0

<210> 1

<211> 726

<212> DNA

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<400> 1

gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300

gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360

ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420

tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480

cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540

tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600

ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660

tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720

tcctca 726

<210> 2

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<212> PRT

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

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys

130 135 140

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

145 150 155 160

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

165 170 175

Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

195 200 205

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

210 215 220

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

225 230 235 240

Ser Ser

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atggggggac ttgaaccctg cagcaggttc ctgctcctgc ctctcctgct ggctgtaagt 60

ggtctccgtc ctgtccaggt ccaggcccag agcgattgca gttgctctac ggtgagcccg 120

ggcgtgctgg cagggatcgt gatgggagac ctggtgctga cagtgctcat tgccctggcc 180

gtgtacttcc tgggccggct ggtccctcgg gggcgagggg ctgcggaggc agcgacccgg 240

aaacagcgta tcactgagac cgagtcgcct tatcaggagc tccagggtca gaggtcggat 300

gtctacagcg acctcaacac acagaggccg tattacaaa 339

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Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu

1 5 10 15

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

20 25 30

Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met

35 40 45

Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu

50 55 60

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

65 70 75 80

Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly

85 90 95

Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr

100 105 110

Lys

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ggagagggca gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccct 57

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

1 5 10 15

Pro Gly Pro

<210> 7

<211> 63

<212> DNA

<213>artificial sequence (artificial sequence)

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atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 8

<211> 21

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 8

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

1 5 10 15

His Ala Ala Arg Pro

20

<210> 9

<211> 153

<212> DNA

<213>artificial sequence (artificial sequence)

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actcctgact ctgaaatcaa ccttacaaat gtgacagata tcatcagggt tccggtgttc 60

aacattgtca ttctcctggc tggtggattc ctgagtaaga gcctggtctt ctctgtcctg 120

tttgctgtca cgctgaggtc atttgtaccc tag 153

<210> 10

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<213>artificial sequence (artificial sequence)

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Thr Pro Asp Ser Glu Ile Asn Leu Thr Asn Val Thr Asp Ile Ile Arg

1 5 10 15

Val Pro Val Phe Asn Ile Val Ile Leu Leu Ala Gly Gly Phe Leu Ser

20 25 30

Lys Ser Leu Val Phe Ser Val Leu Phe Ala Val Thr Leu Arg Ser Phe

35 40 45

Val Pro

50

<210> 11

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<212> PRT

<213>artificial sequence (artificial sequence)

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

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

100 105

<210> 12

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<212> PRT

<213>artificial sequence (artificial sequence)

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

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

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

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 13

<211> 15

<212> PRT

<213>artificial sequence (artificial sequence)

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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 14

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<212> DNA

<213>artificial sequence (artificial sequence)

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atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atccca 66

<210> 15

<211> 22

<212> PRT

<213>artificial sequence (artificial sequence)

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

1 5 10 15

Ala Phe Leu Leu Ile Pro

20

<210> 16

<211> 1395

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 16

atggggggac ttgaaccctg cagcaggttc ctgctcctgc ctctcctgct ggctgtaagt 60

ggtctccgtc ctgtccaggt ccaggcccag agcgattgca gttgctctac ggtgagcccg 120

ggcgtgctgg cagggatcgt gatgggagac ctggtgctga cagtgctcat tgccctggcc 180

gtgtacttcc tgggccggct ggtccctcgg gggcgagggg ctgcggaggc agcgacccgg 240

aaacagcgta tcactgagac cgagtcgcct tatcaggagc tccagggtca gaggtcggat 300

gtctacagcg acctcaacac acagaggccg tattacaaag tcgagggcgg cggagagggc 360

agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggccctag gatggcctta 420

ccagtgaccg ccttgctcct gccgctggcc ttgctgctcc acgccgccag gccgggatcc 480

gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 540

atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 600

gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 660

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 720

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 780

gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 840

ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 900

tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 960

cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 1020

tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 1080

ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 1140

tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 1200

tcctcagcta gcggtggcgg aggttctgga ggtgggggtt ccactcctga ctctgaaatc 1260

aaccttacaa atgtgacaga tatcatcagg gttccggtgt tcaacattgt cattctcctg 1320

gctggtggat tcctgagtaa gagcctggtc ttctctgtcc tgtttgctgt cacgctgagg 1380

tcatttgtac cctag 1395

<210> 17

<211> 1395

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 17

atggggggac ttgaaccctg cagcaggttc ctgctcctgc ctctcctgct ggctgtaagt 60

ggtctccgtc ctgtccaggt ccaggcccag agcgattgca gttgctctac ggtgagcccg 120

ggcgtgctgg cagggatcgt gatgggagac ctggtgctga cagtgctcat tgccctggcc 180

gtgtacttcc tgggccggct ggtccctcgg gggcgagggg ctgcggaggc agcgacccgg 240

aaacagcgta tcactgagac cgagtcgcct tatcaggagc tccagggtca gaggtcggat 300

gtctacagcg acctcaacac acagaggccg tattacaaag tcgagggcgg cggagagggc 360

agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggccctag gatggcctta 420

ccagtgaccg ccttgctcct gccgctggcc ttgctgctcc acgccgccag gccgggatcc 480

gaggtgaaac tgcaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccgtc 540

acatgcactg tctcaggggt ctcattaccc gactatggtg taagctggat tcgccagcct 600

ccacgaaagg gtctggagtg gctgggagta atatggggta gtgaaaccac atactataat 660

tcagctctca aatccagact gaccatcatc aaggacaact ccaagagcca agttttctta 720

aaaatgaaca gtctgcaaac tgatgacaca gccatttact actgtgccaa acattattac 780

tacggtggta gctatgctat ggactactgg ggccaaggaa cctcagtcac cgtctcctca 840

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgacat ccagatgaca 900

cagactacat cctccctgtc tgcctctctg ggagacagag tcaccatcag ttgcagggca 960

agtcaggaca ttagtaaata tttaaattgg tatcagcaga aaccagatgg aactgttaaa 1020

ctcctgatct accatacatc aagattacac tcaggagtcc catcaaggtt cagtggcagt 1080

gggtctggaa cagattattc tctcaccatt agcaacctgg agcaagaaga tattgccact 1140

tacttttgcc aacagggtaa tacgcttccg tacacgttcg gaggggggac caagctggag 1200

atcacagcta gcggtggcgg aggttctgga ggtgggggtt ccactcctga ctctgaaatc 1260

aaccttacaa atgtgacaga tatcatcagg gttccggtgt tcaacattgt cattctcctg 1320

gctggtggat tcctgagtaa gagcctggtc ttctctgtcc tgtttgctgt cacgctgagg 1380

tcatttgtac cctag 1395

<210> 18

<211> 1398

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 18

atggggggac ttgaaccctg cagcaggttc ctgctcctgc ctctcctgct ggctgtaagt 60

ggtctccgtc ctgtccaggt ccaggcccag agcgattgca gttgctctac ggtgagcccg 120

ggcgtgctgg cagggatcgt gatgggagac ctggtgctga cagtgctcat tgccctggcc 180

gtgtacttcc tgggccggct ggtccctcgg gggcgagggg ctgcggaggc agcgacccgg 240

aaacagcgta tcactgagac cgagtcgcct tatcaggagc tccagggtca gaggtcggat 300

gtctacagcg acctcaacac acagaggccg tattacaaag tcgagggcgg cggagagggc 360

agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggccctag gatgcttctc 420

ctggtgacaa gccttctgct ctgtgagtta ccacacccag cattcctcct gatcccagga 480

tccgacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 540

accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 600

ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 660

tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 720

caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 780

ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 840

ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc 900

ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 960

cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 1020

tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 1080

gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 1140

cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc 1200

gtctcctcag ctagcggtgg cggaggttct ggaggtgggg gttccactcc tgactctgaa 1260

atcaacctta caaatgtgac agatatcatc agggttccgg tgttcaacat tgtcattctc 1320

ctggctggtg gattcctgag taagagcctg gtcttctctg tcctgtttgc tgtcacgctg 1380

aggtcatttg taccctag 1398

<210> 19

<211> 1398

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 19

atggggggac ttgaaccctg cagcaggttc ctgctcctgc ctctcctgct ggctgtaagt 60

ggtctccgtc ctgtccaggt ccaggcccag agcgattgca gttgctctac ggtgagcccg 120

ggcgtgctgg cagggatcgt gatgggagac ctggtgctga cagtgctcat tgccctggcc 180

gtgtacttcc tgggccggct ggtccctcgg gggcgagggg ctgcggaggc agcgacccgg 240

aaacagcgta tcactgagac cgagtcgcct tatcaggagc tccagggtca gaggtcggat 300

gtctacagcg acctcaacac acagaggccg tattacaaag tcgagggcgg cggagagggc 360

agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggccctag gatgcttctc 420

ctggtgacaa gccttctgct ctgtgagtta ccacacccag cattcctcct gatcccagga 480

tccgaggtga aactgcagga gtcaggacct ggcctggtgg cgccctcaca gagcctgtcc 540

gtcacatgca ctgtctcagg ggtctcatta cccgactatg gtgtaagctg gattcgccag 600

cctccacgaa agggtctgga gtggctggga gtaatatggg gtagtgaaac cacatactat 660

aattcagctc tcaaatccag actgaccatc atcaaggaca actccaagag ccaagttttc 720

ttaaaaatga acagtctgca aactgatgac acagccattt actactgtgc caaacattat 780

tactacggtg gtagctatgc tatggactac tggggccaag gaacctcagt caccgtctcc 840

tcaggtggcg gtggctcggg cggtggtggg tcgggtggcg gcggatctga catccagatg 900

acacagacta catcctccct gtctgcctct ctgggagaca gagtcaccat cagttgcagg 960

gcaagtcagg acattagtaa atatttaaat tggtatcagc agaaaccaga tggaactgtt 1020

aaactcctga tctaccatac atcaagatta cactcaggag tcccatcaag gttcagtggc 1080

agtgggtctg gaacagatta ttctctcacc attagcaacc tggagcaaga agatattgcc 1140

acttactttt gccaacaggg taatacgctt ccgtacacgt tcggaggggg gaccaagctg 1200

gagatcacag ctagcggtgg cggaggttct ggaggtgggg gttccactcc tgactctgaa 1260

atcaacctta caaatgtgac agatatcatc agggttccgg tgttcaacat tgtcattctc 1320

ctggctggtg gattcctgag taagagcctg gtcttctctg tcctgtttgc tgtcacgctg 1380

aggtcatttg taccctag 1398

<210> 20

<211> 464

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 20

Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu

1 5 10 15

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

20 25 30

Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met

35 40 45

Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu

50 55 60

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

65 70 75 80

Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly

85 90 95

Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr

100 105 110

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

115 120 125

Asp Val Glu Glu Asn Pro Gly Pro Arg Met Ala Leu Pro Val Thr Ala

130 135 140

Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Gly Ser

145 150 155 160

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

165 170 175

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

180 185 190

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

195 200 205

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

210 215 220

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys

290 295 300

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

305 310 315 320

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

325 330 335

Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile

340 345 350

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

355 360 365

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

370 375 380

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

385 390 395 400

Ser Ser Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Pro

405 410 415

Asp Ser Glu Ile Asn Leu Thr Asn Val Thr Asp Ile Ile Arg Val Pro

420 425 430

Val Phe Asn Ile Val Ile Leu Leu Ala Gly Gly Phe Leu Ser Lys Ser

435 440 445

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

450 455 460

<210> 21

<211> 464

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 21

Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu

1 5 10 15

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

20 25 30

Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met

35 40 45

Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu

50 55 60

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

65 70 75 80

Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly

85 90 95

Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr

100 105 110

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

115 120 125

Asp Val Glu Glu Asn Pro Gly Pro Arg Met Ala Leu Pro Val Thr Ala

130 135 140

Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Gly Ser

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala

245 250 255

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

260 265 270

Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

275 280 285

Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Thr Thr Ser

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln

370 375 380

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

385 390 395 400

Ile Thr Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Pro

405 410 415

Asp Ser Glu Ile Asn Leu Thr Asn Val Thr Asp Ile Ile Arg Val Pro

420 425 430

Val Phe Asn Ile Val Ile Leu Leu Ala Gly Gly Phe Leu Ser Lys Ser

435 440 445

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

450 455 460

<210> 22

<211> 465

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 22

Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu

1 5 10 15

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

20 25 30

Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met

35 40 45

Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu

50 55 60

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

65 70 75 80

Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly

85 90 95

Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr

100 105 110

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

115 120 125

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

130 135 140

Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro Gly

145 150 155 160

Ser Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu

165 170 175

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

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu

195 200 205

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

210 215 220

Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu

225 230 235 240

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

245 250 255

Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile

340 345 350

Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu

355 360 365

Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr

370 375 380

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

385 390 395 400

Val Ser Ser Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr

405 410 415

Pro Asp Ser Glu Ile Asn Leu Thr Asn Val Thr Asp Ile Ile Arg Val

420 425 430

Pro Val Phe Asn Ile Val Ile Leu Leu Ala Gly Gly Phe Leu Ser Lys

435 440 445

Ser Leu Val Phe Ser Val Leu Phe Ala Val Thr Leu Arg Ser Phe Val

450 455 460

Pro

465

<210> 23

<211> 465

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 23

Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu

1 5 10 15

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

20 25 30

Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met

35 40 45

Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu

50 55 60

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

65 70 75 80

Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly

85 90 95

Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr

100 105 110

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

115 120 125

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

130 135 140

Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro Gly

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu

210 215 220

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

225 230 235 240

Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys

245 250 255

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

260 265 270

Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

275 280 285

Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Thr Thr

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser

355 360 365

Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys

370 375 380

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

385 390 395 400

Glu Ile Thr Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr

405 410 415

Pro Asp Ser Glu Ile Asn Leu Thr Asn Val Thr Asp Ile Ile Arg Val

420 425 430

Pro Val Phe Asn Ile Val Ile Leu Leu Ala Gly Gly Phe Leu Ser Lys

435 440 445

Ser Leu Val Phe Ser Val Leu Phe Ala Val Thr Leu Arg Ser Phe Val

450 455 460

Pro

465

Claims

1. a kind of Chimeric antigen receptor DAP12-T2A-CD8 α-CD19scFv-TREM1, which is characterized in that the chimeric antigen by Body is by the second conducting structure intracellular domain DAP12, T2A, CD8 alpha signal peptide, anti human CD 19 monoclonal antibody FMC63 light chain and heavy chain Variable region CD19scFv, the first conducting structure intracellular domain TREM1 are in series.

2. Chimeric antigen receptor DAP12-T2A-CD8 α-CD19scFv-TREM1 according to claim 1, feature exist In the Chimeric antigen receptor contains the single-chain antibody CD19scFv for people's CD19 antigen, nucleic acid sequence such as SEQ ID Shown in NO.1, amino acid sequence as shown in SEQ ID NO.2, or with its amino acid sequence with 85%-99% identity；Institute It states and is connected between the light chain variable region (VL) of single-chain antibody and heavy chain variable region (VH) by link peptide (Linker), include GS Linker such as (G₃S)₄Or (G₄S)₃, preferably (G₄S)₃Link peptide.

3. Chimeric antigen receptor DAP12-T2A-CD8 α-CD19scFv-TREM1 according to claim 1, feature exist In the second conducting structure domain DAP12 is connected by T2A with extracellular signal peptide and antigen-binding domains；Preferably, described DAP12 nucleotide sequence as shown in SEQ ID NO.3, amino acid sequence as shown in SEQ ID NO.4, or with its amino acid sequence Polypeptide with 85%-99% identity；The T2A nucleotide sequence is as shown in SEQ ID NO.5, amino acid sequence such as SEQ Shown in ID NO.6, or the polypeptide with its amino acid sequence with 85%-99% identity.

4. Chimeric antigen receptor DAP12-T2A-CD8 α-CD19scFv-TREM1 according to claim 1, feature exist In extracellular signal peptide is selected from nucleotide CD8 alpha signal peptide as shown in SEQ ID NO.7, amino acid sequence such as SEQ ID NO.8 It is shown, or the polypeptide with its amino acid sequence with 85%-99% identity；First conducting structure intracellular domain is selected from nucleotide such as TREM1 shown in SEQ ID NO.9, TREM1 amino acid sequence have as shown in SEQ ID NO.10, or with its amino acid sequence The polypeptide of 85%-99% identity.

5. a kind of coding such as the nucleic acid of targeting CD19 Chimeric antigen receptor of any of claims 1-4.Its feature exists In the nucleic acid sequence of the Chimeric antigen receptor is as shown in SEQ ID NO.16；The amino acid sequence of the Chimeric antigen receptor As shown in SEQ ID NO.20 in sequence table, or with its amino acid sequence with 85%-99% identity.

6. a kind of recombinant expression carrier, it is characterised in that include the nucleic acid molecules described in claim 5.

7. a kind of method for the T cell for preparing Chimeric antigen receptor modification described in claim 1, it is characterised in that this method packet Separation and activation T cell to be finished are included, is then transduceed the T cell with recombinant expression carrier as claimed in claim 6.

8. a kind of cell of the Chimeric antigen receptor of recombinant expression carrier as claimed in claim 6；It is characterized in that described Cell is selected from immunocyte；It is dry thin that the cell is selected from T lymphocyte, NK cell, NKT cell, macrophage, mesenchyma The immunocyte that born of the same parents, candidate stem cell, multipotential stem cell or embryonic stem cell culture are broken up.

9. a kind of pharmaceutical composition, it is characterised in that include cell and pharmacy described in any one of a effective amount of claim 8 Upper acceptable carrier.

10. targeting CD19 Chimeric antigen receptor of any of claims 1-4, described in any one of claim 5 Nucleic acid, recombinant expression carrier as claimed in claim 6 and host cell according to any one of claims 8 are preparing mammal tumor Application in therapeutic agent；It is preferred that preparing the application in hematologic malignancies therapeutic agent.