CN107365798A

CN107365798A - A kind of CD19 CAR T cells of carrying iCasp9 suicide genes and its application

Info

Publication number: CN107365798A
Application number: CN201710570484.7A
Authority: CN
Inventors: 谭毅; 张慧慧
Original assignee: Shandong Qilu Cell Therapy Engineering Technology Co Ltd
Current assignee: Shandong Qilu Cell Therapy Engineering Technology Co Ltd
Priority date: 2017-07-13
Filing date: 2017-07-13
Publication date: 2017-11-21
Anticipated expiration: 2037-07-13
Also published as: CN107365798B

Abstract

The invention discloses a kind of Lentiviral, include the gene and iCasp9 apoptogenes of encoding chimeric antigen acceptor.The invention also discloses a kind of CD19 CAR T cells of carrying iCasp9 suicide genes, to include the T lymphocytes of Lentiviral, or the gene of encoding chimeric antigen acceptor and the T lymphocytes of iCasp9 apoptogenes are integrated with chromosome.The invention also discloses application of the CD19 CAR T cells of carrying iCasp9 suicide genes in prevention and/or the medicine for the treatment of and/or adjuvant therapy of malignant tumor is prepared.Compared with existing CD19 CAR T cells, invention introduces suicide machinery, and a suicide gene iCasp9 is co-expressed while CAR is expressed, when serious side effect occurs, the generation of the side effects such as CRS is controlled by the apoptosis of inducing T cell, increases its security.

Description

A kind of CD19-CAR-T cells of carrying iCasp9 suicide genes and its application

Technical field

The present invention relates to a kind of Chimeric antigen receptor T for carrying iCasp9 (induciblecaspase9) suicide gene is thin Born of the same parents (Chimeric Antigen Receptor T cell, CAR-T), and its preparing prevention and/or treatment and/or aiding in controlling The application in the medicine of malignant tumour is treated, belongs to genetic engineering and cell biology.

Background technology

T cell (the anti-CD19chimeric antigen receptor modified for CD19 Chimeric antigen receptor Tcells, abbreviation CD19CAR-T cell) in the treatment of intractable B cell malignant tumour obtain immense success；And target The CAR-T technologies of other tumor targets also show good application prospect in the treatment of other solid tumors.With CD19CAR-T What the validity of therapy coexisted is its side effect and cytotoxicity.There may be thin for normal structure in clinical practice for it The mistake attack (effect of missing the target, on target/off-tumor effect) of born of the same parents, or because a large amount of release inflammatory cytokines draw The toxic side effects such as cytokine storm are played, turn into the principal element for restricting its application.

1989, Chimeric antigen receptor transformation T cell (CAR-T) this concept was proposed first, it is therefore an objective to establishes tumour The adoptive cellular therapy of specific recognition capability.First generation CAR design is simply to use monoclonal antibody specific source ScFv (single-chain antibody variable region fragment) section replaces TCR (T cell Receptor extracellular part), remains transmembrane structure and intracellular transmits the CD3 ζ regions of signal, some seminar's intracellular letters Number transmitting portions can also use FcR.But first generation CAR clinical test results are very disappointing, and effect is best once Clinical test, which also only has 2/11 patient, long-term alleviation.As what immunology was recognized deepens continuously, it has been found that CD3 Although ζ signals are capable of the activation of inducing T cell and of short duration propagation, the incapability (anergy) of inducing T cell is understood afterwards.1998 Year, the costimulation function of auxiliary can be provided for CD3 ζ signals by having two laboratories to report CD28 signal domains.Therefore, Two generation CAR design introduces the part of costimulatory molecules on the basis of the first generation, such as CD28 or CD137 (4-1BB) born of the same parents Interior signal domain.And third generation CAR is then the function signal domain for adding 2 costimulatory moleculeses, including CD27, CD28,4-1BB, ICOS or OX40 (15-18).The mainly second generation CAR design clinically tested now.

Be target spot due to choosing CD19 during design, the T cell of input can will normal B cells and tumour existing for itself in vivo Cell kills together, and after tumor disappearance, as long as CAR-T cells are still present, all without there is normal B cells to deposit in body Living, patient needs separated in time injecting immune globulin to maintain basic humoral immunity.On the other hand, it is thin in CAR-T Born of the same parents input initial stage, can secrete large amount of cell factor, patient in a large amount of amplifications and T cell killing neoplastic process due to T cell Cytokine release syndrome (Cytokine Release Syndrome, CRS) occurs, is embodied in heating, low blood Some significant rises of cytokine levels in pressure, anoxic and serum, these factors include interferon-γ (IFN-γ), divide shape Chemotactic factor (CF) (Fracktalkine), grain-macrophage growth factor (GM-CSF), interleukin 5 (IL-5), leucocyte Interleukin -6 (IL-6), people's FLT3L (Flt-3L) and interleukin 10 (IL-10).In July, 2016 Just, general headquarters be located at Seattle Juno Therapeutics issued be reported in the said firm progress clinical test JCAR015 In, successively there are three patients (being proved to be four afterwards) that death occurs after CAR-T cell therapies are received, FDA stopped immediately JCAR015 clinical test, it is because causing neurotoxin using chemotherapeutics fludarabine, but also have very that Juno, which is then explained, More people's conjectures are due to that strong cellular factor storm (CRS) causes patient death caused by the T cell of input.No matter reason such as What, it is most of all to trigger high fever because producing CRS so that producing violent secondary work in the patient for receiving CAR-T cell therapies With.How to control the extent of reaction of CAR-T cells becomes a problem.

Present invention contemplates that feeding back the state into internal T cell by control, ensureing CAR-T target killing function One " switch " is added to CAR-T simultaneously, CRS is controlled by the apoptosis of inducing T cell when serious side effect occurs Deng the generation of side effect, increase its security.

The content of the invention

For above-mentioned prior art, the invention provides one kind to carry iCasp9 (induciblecaspase9) suicide base The Chimeric antigen receptor T cell (Chimeric Antigen Receptor T cell, CAR-T) of cause, and its prevent preparing And/or the application in the medicine for the treatment of and/or adjuvant therapy of malignant tumor, present invention also offers carry iCasp9 suicide genes With the recombinant expression carrier of the gene of expression Chimeric antigen receptor.The present invention introduces suicide machinery in CAR-T therapeutic processes, CAR-T cells is being expressed outside CAR, co-express a Caspase9 genes (inducible by synthetic biology transformation Caspase9), after this gene expression, the mitochondria of icasp9 dimerization active cell can be induced by extraneous chemicals Apoptosis pathway makes CAR-T Apoptosis, improves the clinical safety of CAR-T cell therapies.Make present inventor surprised It is, after iCasp9 suicide genes are imported into CAR-T cells, the proliferation activity of CAR-T cell itselfs, the killing to tumour cell The secretion situation of cell factor is not affected during function and killing, and this has exceeded the pre- of those skilled in the art Material.

The present invention is achieved by the following technical solutions：

A kind of Chimeric antigen receptor, including signal peptide, single-chain antibody, extracellular hinge area, transmembrane region and the born of the same parents being sequentially connected in series Interior signaling zone, single-chain antibody include light chain variable district (VL), weight chain variable district (VH) and connect their hinge area；Wherein, The antigen of single-chain antibody identification be selected from CD19, CD20, CEA, GD2, CD22, CD23, CD30, CD33, CD44v7/8, It is more than one or both of CD70, VEGFR1, VEGFR2, IL-11R α, EGP-2, EGP-40, FBP, GD3.

Preferably, the antigen of single-chain antibody identification is CD19, the amino acid of the light chain variable district of the single-chain antibody Sequence such as SEQ ID NO:Shown in 1, the amino acid sequence such as SEQ ID NO of weight chain variable district:Shown in 2, the amino acid of hinge area Sequence such as SEQ ID NO:Shown in 3.

Further, the amino acid sequence of the signal peptide such as SEQ ID NO:Shown in 4.

Further, the extracellular hinge area be selected from CD8 extracellular hinge area, CD28 extracellular hinge area, CD4 it is extracellular It is more than one or both of hinge area；It is preferred that CD8 extracellular hinge area, the amino acid sequence such as SEQ of CD8 extracellular hinge area ID NO:Shown in 5.

Further, transmembrane region of the transmembrane region selected from CD8, CD28 transmembrane region, one kind in CD4 transmembrane region or It is two or more；It is preferred that CD8 transmembrane regions, the amino acid sequence such as SEQ ID NO of CD8 transmembrane regions:Shown in 6.

Further, the intracellular signal area be selected from CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS, Intracellular signal area more than one or both of DAP10, CD3 ζ, Fc ε RI γ；It is preferred that 4-1BB intracellular signals area and CD3 ζ born of the same parents Interior signaling zone, or CD28 intracellular signals area and CD3 ζ intracellular signals area；4-1BB intracellular signals area and the ammonia in CD3 ζ intracellular signals area Base acid sequence is respectively such as SEQ ID NO:7 and SEQ ID NO:Shown in 8.

Further, the amino acid sequence of the Chimeric antigen receptor such as SEQ ID NO:It is signal peptide area, list shown in 9 The light chain area of chain antibody, hinge area, the heavy chain region of single-chain antibody, extracellular hinge area, CD8 transmembrane regions, 4-1BB intracellular signals area, CD3 ζ intracellular signals areas are sequentially connected in series.

A kind of gene for encoding above-mentioned Chimeric antigen receptor, its nucleotide sequence such as SEQ ID NO:Shown in 10.

A kind of iCasp9 (inducible caspase9) apoptogene, its nucleotide sequence such as SEQ ID NO:11 institutes Show.Its amino acid residue sequence such as SEQ ID NO expressed:Shown in 12.ICasp9 by F36V point mutation FKBPL (FKBP12v36) Serine-Glycine-Gly-Gly-serine (Ser-Gly-Gly-Gly-Ser) and removal are passed through The Caspase9 of CARD (Caspase recruitment domain) is connected.

A kind of Lentiviral, include the gene and iCasp9 apoptosis bases of above-mentioned encoding chimeric antigen acceptor Cause.

Further, the structure of the Lentiviral is pHR-iCasp9-2A-CD19CAR-4-1BB-CD3 ζ, Or：PHR-iCasp9-IRES-CD19CAR-4-1BB-CD3 ζ, wherein, 2A nucleotide sequence such as SEQ ID NO:Shown in 13, Its amino acid residue sequence such as SEQ ID NO expressed:14.IRES nucleotide sequence such as SEQ ID NO:Shown in 15.

Further, the nucleotide sequence of expressing gene is such as in the pHR-iCasp9-2A-CD19CAR-4-1BB-CD3 ζ SEQ ID NO:Shown in 16, its amino acid residue sequence such as SEQID NO expressed:Shown in 17.

Further, in the pHR-iCasp9-IRES-CD19CAR-4-1BB-CD3 ζ expressing gene nucleotide sequence Such as SEQ ID NO:Shown in 18, its amino acid residue sequence such as SEQID NO expressed:Shown in 19.

A kind of slow virus expresses kit, including above-mentioned Lentiviral.

The slow virus expresses kit, the preparation method of its recombinant slow virus, comprises the following steps：It will carry purposeful Lentiviral, pCMV carriers and the pMD.2G carriers of gene are transfected into 293FT cells after mixing, 6~8h after transfection Complete medium culture is replaced by, nutrient solution is collected after 48h, supernatant is retained after centrifugation and by the supernatant with 0.45 μm of filtering head Filtering, retain filtrate, the filtrate is the solution of recombinant slow virus.

A kind of CD19-CAR-T cells of carrying iCasp9 suicide genes, to include the T of above-mentioned Lentiviral Be integrated with lymphocyte, or chromosome above-mentioned encoding chimeric antigen acceptor gene and iCasp9 apoptogenes T lymphs it is thin Born of the same parents.It expresses above-mentioned iCasp9 apoptogenes and Chimeric antigen receptor simultaneously.

Above-mentioned Chimeric antigen receptor, the gene of encoding chimeric antigen acceptor, Lentiviral, carrying iCasp9 commit suiside Application of the CD19-CAR-T cells of gene in prevention and/or the medicine for the treatment of and/or adjuvant therapy of malignant tumor is prepared.Institute State malignant tumour and be selected from ALL, chronic lymphocytic leukemia.

A kind of iCasp9/CID suicide gene systems, the suicide gene system is by inducible caspase9 (iCasp9) the small-molecule chemical induced drug of gene and inactive forms.Wherein, small-molecule chemical induced drug is selected from AP1903, AP20187, preferably AP1903.

The beneficial effects of the invention are as follows：

1st, it is common while CAR is expressed invention introduces suicide machinery compared with existing CD19-CAR-T cells Express a suicide gene iCasp9 (inducible caspase9).One " switch " is added equivalent to CAR-T cells, When serious side effect occurs, the generation of the side effects such as CRS is controlled by the apoptosis of inducing T cell, increases its security.

2nd, compared with existing CD19-CAR-T cells, the present invention relates to a kind of iCasp9/CID suicide gene systems, institute Suicide gene system is stated by inducible caspase9 (iCasp9) genes and the small-molecule chemical revulsive of inactive Thing forms.Chemical induction medicine, such as AP1903, AP20187, for the small-molecule drug of inactive, it is injected intravenously this medicine The drug binding domains that thing can result in chimeric protein iCasp9 crosslink, so that dimerization occurs for iCasp9, from And the Caspase3 molecules in downstream are activated, cause Apoptosis.

3rd, compared with existing CD19-CAR-T cells, iCasp9-CD19CAR-T cells are in side effects such as control CRS Produce, while increasing its security, do not influence its characteristic in cell propagation, killing, cytokine secretion etc., exceed The expectation of those skilled in the art.

Some involved Essential Terms are described as follows in the present invention：

Term " Chimeric antigen receptor " is artificial reconstructed acceptor, can be by the specificity of tumor cell surface antigen point Sub (such as antibody) is anchored on immunocyte (such as T cell), is made immunocyte identification tumour antigen or viral antigen and is killed swollen Oncocyte or the cell of virus infection.

Term " single-chain antibody " (single-chain antibody variable region fragment, scFv) is Refer to by antibody light chain variable region (VL areas) amino acid sequence and weight chain variable district (V H areas) amino acid sequence through hinge connection and Into with the antibody fragment with reference to antigenic capacity.

Term " Linker " or hinge are the polypeptide fragments between the different albumen of connection or polypeptide, and the purpose is to make to be connected Albumen or polypeptide keep respective space conformation, to maintain the function of albumen or polypeptide or activity.

Term " CD19 " refers to human leukocytes differentiation antigen 19, it NCBI GeneBank ID number be 930, have 2 it is different Structure body (cDNA sequence/protein sequence), respectively NM_001178098.1/NP_001171569.1, NM_001770.5/NP_ 001761.3.When referring to CD19 amino acid sequence, it includes, the total length of CD19 albumen or the CD19 with CD19 functions Fragment；Also include the fusion protein of the total length or fragment.Also, it will be appreciated by those skilled in the art that in CD19 amino acid In sequence, can it is naturally-produced or be artificially introduced mutation or variation (including but not limited to replacing, lack and/or add), without shadow Ring its biological function.Also, when describing CD19 protein sequence fragments, in addition to it is corresponding in its natural or artificial variants Sequence fragment.

Term " iCasp9/CID suicide gene systems " is by inducible caspase9 (iCasp9) genes and without life The small-molecule chemical induced drug composition of thing activity.

Term " iCasp9 " (inducible caspase9) refers to by the FKBPL of F36V point mutation (FKBP12v36) by Serine-Glycine-Gly-Gly-serine and eliminating CARD (Caspase Recruitment domain) the connected fragments of Caspase9.FKBP12v36 is drug binding domains, F36V point mutation FKBP12 and small molecule AP1903 affinity can be improved.CARD is removed from Caspase9, because its physiologic function Substituted by FKBP12, and expression and the function of gene can be strengthened by removing it.Chemical induction medicine, as AP1903, AP20187, it is the small-molecule drug of inactive, is injected intravenously the medicine that this medicine can result in chimeric protein iCasp9 Thing binding structural domain crosslinks, so that dimerization occurs for iCasp9, so as to activate the Caspase3 molecules in downstream, causes thin Born of the same parents' apoptosis.

Term " 2A " refers to autothermic cracking polypeptide, is polypeptide of the segment length between 18 to 22 amino acid, sends out first Now in picornavirus, then it is found in insect viruses, rotavirus etc., it can be applicable to polycistronic vector structure In, and there is the advantages that structure is short and small, and upstream and downstream gene expression balance is good, available for multiple albumen are co-expressed, it is a kind of Build the effective tool of polycistronic vector.

Term " IRES " refers to internal ribosome entry site sequence (Internal ribosome entry site), one RNA sequence of the kind rich in secondary structure, this kind of RNA sequence can be folded into the structure similar to starting tRNA, so as to mediate ribose Body is combined with RNA, initiation protein translation.After the protein translation before IRES, ribosomes does not depart from mRNA, can be tied with IRES Close, continue translation, therefore a mRNA can translate two kinds of albumen.

Brief description of the drawings

Fig. 1：The ideograph of 2 kinds of iCasp9 suicide genes and CD19 specific Cs AR.

Fig. 2：The structure chart of pHR-iCasp9-2A-CD19CAR-4-1BB-CD3 ζ expression vectors.

Fig. 3：The structure chart of pHR-iCasp9-IRES-CD19CAR-4-1BB-CD3 ζ expression vectors.

Fig. 4：ICasp9-CD19CAR-K562 cell positives rate flow cytometer detection figure in K562, wherein, A：iCasp9- CD19CAR-K562—CD19CAR-FITC；B：K562—CD19CAR-FITC.

Fig. 5：AP1903 induces 2h iCasp9-CD19CAR-K562 Apoptosis flow cytometer detection figures, wherein, A：iCasp9- CD19CAR-K562-AP1903—CD19CAR-FITC；B：iCasp9-CD19CAR-K562+AP1903—CD19CAR-FITC； C：iCasp9-CD19CAR-K562+AP1903—Annexin V-APC；7-AAD.

Fig. 6：AP1903 induces 24h iCasp9-CD19CAR-K562 Apoptosis flow cytometer detection figures, wherein, A： iCasp9-CD19CAR-K562—CD19CAR-FITC；B：iCasp9-CD19CAR-K562+AP1903—CD19CAR-FITC.

Fig. 7：CD19CAR-T and iCasp9-CD19CAR-T cell positive rate flow cytometer detection figures, wherein, A：CD19CAR- T—CD19CAR-FITC；B：iCasp9-CD19CAR-T—CD19CAR-FITC.

Fig. 8：CD19CAR-T and iCasp9-CD19CAR-T cell proliferation times figures.

Fig. 9：AP1903 induces 2h iCasp9-CD19CAR-T Apoptosis flow cytometer detection figures, wherein A：CD19CAR-T— Annexin V-APC；7-AAD；B：iCasp9-CD19CAR-T—Annexin V-APC；7-AAD；C：CD19CAR-T+ AP1903—Annexin V-APC；7-AAD；D：iCasp9-CD19CAR-T+AP1903—Annexin V-APC；7-AAD.

Figure 10：AP1903 induces 24h iCasp9-CD19CAR-T Apoptosis flow cytometer detection figures, wherein A：CD19CAR- T；B：iCasp9-CD19CAR-T；C：CD19CAR-T+AP1903；D：iCasp9-CD19CAR-T+AP1903.

Figure 11：K562-CD19 monoclonal flow cytometer detection figures.

Figure 12：ICasp9-CD19CAR-T cell killing function flow cytometer detections figure (24h each group cell killings rate).

Figure 13：ELISA detection iCasp9-CD19CAR-T cells IL-2 secretion figures.

Figure 14：ELISA detection iCasp9-CD19CAR-T cells IFN-gama secretion figures.

Embodiment

With reference to embodiment, the present invention is further illustrated.

Involved instrument, reagent, material etc. in following embodiments, it is existing in the prior art unless otherwise noted Conventional instrument, reagent, material etc., can be obtained by regular commercial sources.Involved experimental method in following embodiments, inspection Survey method etc., it is existing normal experiment method, detection method etc. in the prior art unless otherwise noted.In embodiment not Particular technique or condition person are indicated, according to the technology described by document in the art or condition (such as with reference to J. Pehanorm cloth Shandong Gram wait and to write, what Huang Peitang etc. was translated《Molecular Cloning:A Laboratory guide》, the third edition, Science Press) or enter according to product description OK.

Embodiment 1：The CD19CAR design and the structure of expression vector of the suicide gene containing iCasp9

According to the construction strategies of iCasp9 with CD19CAR dual-expression vectors different, 2 kinds of design construction is committed suiside containing iCasp9 The CD19CAR of gene, it is respectively designated as pHR-iCasp9-2A-CD19CAR-4-1BB-CD3 ζ and pHR-iCasp9-IRES- CD19CAR-4-1BB-CD3 ζ, wherein, the amino acid residue sequence expressed by pHR-iCasp9-2A-CD19CAR-4-1BB-CD3 ζ Row such as SEQID NO:Shown in 17, the amino acid residue sequence expressed by pHR-iCasp9-IRES-CD19CAR-4-1BB-CD3 ζ Such as SEQID NO:Shown in 19, ideograph as shown in figure 1, structure chart as shown in Figure 2 and Figure 3.

Embodiment 2：The packaging of slow virus and concentration

Lentiviral (the pHR-iCasp9-2A-CD19CAR- that embodiment 1 is built of target gene will be carried 4-1BB-CD3 ζ, pHR-iCasp9-IRES-CD19CAR-4-1BB-CD3 ζ), pCMV carriers and pMD.2G carriers mixing after transfect Into 293FT cells, 6~8h is replaced by complete medium culture after transfection, and nutrient solution is collected after 48h, retains supernatant after centrifugation And filter the supernatant with 0.45 μm of filter, retain filtrate, the filtrate is the solution of recombinant slow virus.

Slow virus is concentrated according to Lenti-XTM Concentrator (takara, cat:631231) specification is carried out.

Embodiment 3：The checking of iCasp9/CID suicide gene systems

1. express the preparation of the K562 cells of slow virus carrier

Cell is resuspended to 1x106/mL in 1640 culture mediums.Add slow virus.37 DEG C, 5%CO₂Incubator culture 6-8h, from It is fresh K562 cells Multiplying culture liquid that the heart, which changes liquid,.Fresh K562 cells Multiplying culture liquid was added per 2-3 days, maintains cell density In 0.5x10⁶/ mL or so.After virus infection 48h, using the positive cell proportions of flow cytomery CAR (Fluorescein (FITC) AffiniPure Goat Anti-Mouse IgG, F (ab') 2 fragment specific, Jackson immunoresearch, cat：115-095-006).

As a result as shown in figure 4, the ratio of the CD19CAR-K562 cells of the suicide gene containing iCasp9 is 34%, show to contain The CD19CAR-K562 cells of iCasp9 suicide genes have successfully obtained, and are named as iCasp9-CD19CAR-K562 cells.As Effector cell in the present embodiment.

2.AP1903 induce the detection of iCasp9-CD19CAR-K562 Apoptosis

Take 800ul iCasp9-CD19CAR-K562 cells to be laid in 24 orifice plates, add AP1903 20nM, 37 DEG C, 5% CO2 incubator cultures, respectively at the positive cell proportion (Fluorescein of 2h, 24h application flow cytomery CAR (FITC) AffiniPure Goat Anti-Mouse IgG, F (ab') 2 fragment specific, jackson Immunoresearch, cat：115-095-006) and Apoptosis situation (Annexin-V, Biolegend, cat：640920； 7-AAD, Biolegend, 420404).With iCasp9-CD19CAR-K562 cells, AP1903 groups are not added with；K562 cells, add AP1903 20nM groups；K562 cells, AP1903 groups are added without as control group, are examined respectively at 2h, 24h application flow cytometer Survey positive cell proportion (Fluorescein (FITC) AffiniPure Goat the Anti-Mouse IgG, F (ab') 2 of CAR Fragment specific, jackson immunoresearch, cat：115-095-006) and Apoptosis situation (Annexin-V, Biolegend, cat：640920；7-AAD, Biolegend, 420404).

As Fig. 5 results are shown, iCasp9-CD19CAR-K562 cells add AP1903 groups and cell death occur after 2h, with Living cells draws door, and iCasp9-CD19CAR-K562 positive cell ratios drop to 25.8%, with iCasp9-CD19CAR-K562 Positive cell draws door, and apoptosis occurs in the cell for having 33.3%.And iCasp9-CD19CAR-K562 cells, it is not added with AP1903 groups； K562 cells, add AP1903 20nM groups；K562 cells, it is added without AP1903 groups and does not occur phenomena of apoptosis.Fig. 6 shows Showing, after 24h, iCasp9-CD19CAR-K562 cells add AP1903 groups and cell mortality occur, and door is drawn with living cells, ICasp9-CD19CAR-K562 positive cell ratios drop to 3.24%.Show that positive cell apoptosis occurs by successfully induction. And iCasp9-CD19CAR-K562 cells, it is not added with AP1903 groups；K562 cells, add AP1903 20nM groups；K562 cells, no Add AP1903 groups and do not occur phenomena of apoptosis.As a result show, AP1903 administrations can successfully induce iCasp9-CD19CAR- K562 Apoptosis, without causing K562 Apoptosis.

Embodiment 4：Express the preparation of the T cell of slow virus carrier

The PBMC of fresh separated is adjusted cell density to 1-2x10 with lonza culture mediums⁶/mL.Anti-CD3 is added to resist Body (Ebioscience), anti-CD28 antibody (Novoprotein) to final concentration 50ng/mL.After culture 2 days, cell is collected, Dead cell is removed by the method for Ficoll density gradient centrifugations.Cell is resuspended to 1x106/mL.Add slow virus (MOI 5- 10), DEAE-Dextran, final concentration 10ug/mL (or polybrene, final concentration 4ug/mL), IL-2 to final concentration 1000U/ mL.37 DEG C, 5%CO²Incubator culture 6-8h, it is fresh T cells Multiplying culture liquid that liquid is changed in centrifugation.It is thin that fresh T was added per 2-3 days Born of the same parents' Multiplying culture liquid, cell density is maintained in 0.5x10⁶/ mL or so, expand 10-12 days.

After virus infection 48h, using the positive cell proportion (Fluorescein (FITC) of flow cytomery CAR AffiniPure Goat Anti-Mouse IgG, F (ab') 2 fragment specific, jackson Immunoresearch, cat：115-095-006).

As shown in fig. 7, the ratio of the CD19CAR-T cells of the suicide gene containing iCasp9 shows containing iCasp9 certainly for 82.9% The CD19CAR-T cells for killing gene have successfully obtained.ICasp9-CD19CAR-T is named as, as the effect in embodiment 5,6 Cell.

CD19CAR-T groups of cells and iCasp9-CD19CAR-T groups of cells cultivated in cell cultivation process 0d, 7d, 9d, 11d, 14d count T cell quantity respectively, draw T cell growth curve.As shown in figure 8, CD19CAR-T groups of cells and iCasp9- CD19CAR-T groups of cells cell breeds 126 times and 133 times respectively, is not significantly different between two groups.Express iCasp9 genes The propagation of cell is not influenceed afterwards.

Embodiment 5：AP1903 induction iCasp9-CD19CAR-T apoptosis detections

Take 800ul iCasp9-CD19CAR-T cells to be laid in 24 orifice plates, add AP1903 20nM, 37 DEG C, 5%CO₂ Incubator culture, respectively at the positive cell proportion (Fluorescein (FITC) of 2h, 24h application flow cytomery CAR AffiniPure Goat Anti-Mouse IgG, F (ab') 2 fragment specific, jackson Immunoresearch, cat：115-095-006) and Apoptosis situation (Annexin-V, Biolegend, cat：640920； 7-AAD, Biolegend, 420404).With iCasp9-CD19CAR-T cells, AP1903 groups are not added with；CD19CAR-T cells, add Enter AP1903 20nM groups；CD19CAR-T cells, AP1903 groups are added without as control group, it is thin respectively at 2h, 24h application streaming Cell proportion (Fluorescein (FITC) AffiniPure Goat Anti-Mouse IgG, F positive born of the same parents' instrument detection CAR (ab') 2 fragment specific, jackson immunoresearch, cat：115-095-006) and Apoptosis feelings Condition (Annexin-V, Biolegend, cat：640920；7-AAD, Biolegend, 420404).

As a result as shown in figure 9, drawing door after 2h with iCasp9-CD19CAR-T positive cells, the cell for having 29.3% withers Die.And iCasp9-CD19CAR-T cells, it is not added with AP1903 groups；CD19CAR-T, add AP1903；CD19CAR-T, it is added without AP1903 groups do not occur obvious phenomena of apoptosis.As shown in Figure 10, after 24h, iCasp9-CD19CAR-T cells, it is not added with The live cell fraction of AP1903 groups is that 37.6%, iCasp9-CD19CAR-T cells addition AP1903 group live cell fractions drop to 24.2%.And CD19CAR-T, AP1903 groups and CD19CAR-T are added, is added without AP1903 groups live cell fraction without obvious poor It is different.Show that iCasp9-CD19CAR-T cells apoptosis occur by successfully induction.As a result show, AP1903 administrations can be induced successfully ICasp9-CD19CAR-T Apoptosis, without causing CD19CAR-T apoptosis.

Embodiment 6：ICasp9-CD19CAR-T killing ability detection

1. the structure of the K562 cells (target cell) of stable expression CD19 genes

1) the DNA encoding sequence of CD19 extracellular regions and transmembrane region is synthesized, inserts pHR carriers, builds the carrier name of acquisition For pHR-CD19.

CD19 extracellular regions and the nucleotide coding sequence of transmembrane region such as SEQID NO:Shown in 20.

2) slow virus packaging and concentration

It is transfected into after the pHR-CD19 carriers, pCMV carriers and pMD.2G carriers are mixed in 293FT cells, after transfection 6~8h is replaced by complete medium culture, and nutrient solution is collected after 48h, supernatant is retained after centrifugation and by the supernatant with 0.45 μm Filter filters, and retains filtrate, the filtrate is the solution of recombinant slow virus.

3) preparation of CD19 K562 cells is expressed

K562 cells are resuspended to 1x10 in 1640 (Gibco) culture mediums⁶/ mL, adds slow virus (MOI 5-10), 37 DEG C, 5% CO₂Incubator culture 6-8h, it is fresh K562 cells Multiplying culture liquid that liquid is changed in centrifugation.Fresh K562 cells were added per 2-3 days to increase Nutrient solution is grown, maintains cell density in 0.5x10⁶/ mL or so.After 5 generations, monoclonal screening is carried out using limiting dilution assay.Treat list After clone cell length to certain amount, (anti-CD19PE, biolegend cat is screened by flow cytometer： 302254) cell clone that, expression quantity is high and purity is high, as stable expression CD19 K562 cells, is named as K562-CD19 (as shown in figure 11), as the target cell in the present embodiment.

2.iCasp9-CD19CAR-T killing ability Validation in vitro

20nM AP1903 are separately added into iCasp9-CD19CAR-T cells, CD19CAR-T cells, 37 DEG C, 5%CO₂Training Case culture 24h is supported, is named as iCasp9-CD19CAR-T+AP1903 groups and CD19CAR-T+AP1903 groups.By iCasp9- CD19CAR-T groups cell, iCasp9-CD19CAR-T+AP1903 groups cell and K562-CD19, K562 cell are respectively with 3:1:1； 1：1：1 mixing, 37 degree, 5%CO2 incubator cultures, apply after 24h flow cytomery killing-efficiency (anti-CD3FITC, biolegend cat：300306；Anti-CD19PE, biolegend cat：302254).Negative control is used as by the use of T cell group Group, by the use of CD19CAR-T groups of cells and CD19CAR-T+AP1903 groups of cells as positive controls, as iCasp9- The parallel control of CD19CAR-T groups of cells, iCasp9-CD19CAR-T+AP1903 groups of cells.

As shown in figure 12, using the T cell group without specific killing as negative control group, the specificity of the group in theory Kill as 0.E:T=3:When 1, CD19CAR-T groups of cells, iCasp9-CD19CAR-T groups of cells, CD19CAR-T+AP1903 are thin It is that 100%, iCasp9-CD19CAR-T+AP1903 groups of cells killing rates are about 42% that born of the same parents, which organize killing rate,.E:T=1:When 1, CD19CAR-T groups of cells, iCasp9-CD19CAR-T groups of cells, CD19CAR-T+AP1903 groups of cells killing rates are left 60% The right side, without obvious group difference, and iCasp9-CD19CAR-T+AP1903 groups of cells killing rates are about 26%.Statistical result table Bright, iCasp9-CD19CAR-T groups of cells and iCasp9-CD19CAR-T+AP1903 groups of cells killing rates have significant difference.Cause This, iCasp9-CD19CAR-T cells have the killing-efficiency equal with CD19CAR-T cells, i.e., with CD19CAR-T groups of cells phases Than the CD19CAR-T cells for expressing iCasp9 do not interfere with the killing ability of CAR-T cells.Can be effectively whole after AP1903 administrations The only killing of iCasp9-CD19CAR-T cells, had an impact without the killing to CD19CAR-T cells.

Embodiment 7：ELISA detects iCasp9-CD19CAR-T cell cytokine secretions

20nM AP1903 are separately added into iCasp9-CD19CAR-T cells, CD19CAR-T cells, 37 DEG C, 5%CO₂Training Case culture 24h is supported, is named as iCasp9-CD19CAR-T+AP1903 groups and CD19CAR-T+AP1903 groups.By iCasp9- CD19CAR-T cells, iCasp9-CD19CAR-T+AP1903 groups cell and K562-CD19, K562 cell are with 3:1:1 mixing, 37 DEG C, 5%CO₂Incubator culture, ELISA detection cytokine secretions (human IL-2ELISA-Kit, Biolegend after 24h cat：431804；Human IFN-gama ELISA-Kit, Biolegend cat：430104).With T cell group and K562+ K562-CD19 groups of cells is used as sun as negative control group by the use of CD19CAR-T groups of cells and CD19CAR-T+AP1903 groups of cells Property control group, the parallel control as iCasp9-CD19CAR-T groups of cells, iCasp9-CD19CAR-T+AP1903 groups of cells. As shown in figure 13, CD19CAR-T groups of cells, iCasp9-CD19CAR-T groups of cells and CD19CAR-T+AP1903 groups of cells IL-2 Burst size be respectively 1605pg/ml, 1635pg/ml and 1629pg/ml, there was no significant difference.iCasp9-CD19CAR-T+ AP1903 groups of cells IL-2 burst size is 214pg/ml, and CD19CAR-T groups of cells, iCasp9-CD19CAR-T groups of cells and CD19CAR-T+AP1903 groups of cells has significant difference.Compared with CD19CAR-T groups of cells, express iCasp9's CD19CAR-T cells do not interfere with IL-2 release.ICasp9-CD19CAR-T cells can be effectively terminated after AP1903 administration energy IL-2 release, had an impact without the release to CD19CAR-T cells IL-2.As shown in figure 14, CD19CAR-T cells The burst size of group, iCasp9-CD19CAR-T groups of cells and CD19CAR-T+AP1903 groups of cells IFN-gama is respectively 12370pg/ml, 15510pg/ml and 11170pg/ml, there was no significant difference.ICasp9-CD19CAR-T+AP1903 groups of cells IFN-gama burst size is 503.2pg/ml, and CD19CAR-T groups of cells, iCasp9-CD19CAR-T groups of cells and CD19CAR-T+AP1903 groups of cells has significant difference.Compared with CD19CAR-T groups of cells, express iCasp9's CD19CAR-T cells do not interfere with IFN-gama release.ICasp9-CD19CAR-T can be effectively terminated after AP1903 administration energy Cell IFN-gama release, had an impact without the release to CD19CAR-T cells IFN-gama.

Although the embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.But It is evident that other embodiment and the deformation of the present invention can be designed by those skilled in the art, without departing from the present invention's True spirit and scope.Claim is intended to be interpreted as including all such embodiments and equivalent variations.

Sequence table

<110>Shandong Qilu Cell Therapy Engineering Technology Co., Ltd.

<120>A kind of CD19-CAR-T cells of carrying iCasp9 suicide genes and its application

<141> 2017-07-13

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 115

<212> PRT

<213> Artificial Sequence

<400> 1

Lys Leu Ile Ser Glu Glu Asp Leu Asp Ile Gln Met Thr Gln Thr Thr

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Glu Ile Thr

115

<210> 2

<211> 120

<212> PRT

<213> Artificial Sequence

<400> 2

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

1 5 10 15

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> 3

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 3

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

1 5 10 15

<210> 4

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 4

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 Glu Gln

20

<210> 5

<211> 46

<212> PRT

<213> Artificial Sequence

<400> 5

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

1 5 10 15

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

20 25 30

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

35 40 45

<210> 6

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 6

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

1 5 10 15

Leu Val Ile Thr Leu Tyr Cys

20

<210> 7

<211> 43

<212> PRT

<213> Artificial Sequence

<400> 7

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

1 5 10 15

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

20 25 30

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

35 40

<210> 8

<211> 114

<212> PRT

<213> Artificial Sequence

<400> 8

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Arg Gly

<210> 9

<211> 499

<212> PRT

<213> Artificial Sequence

<400> 9

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 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asp

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu

355 360 365

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

370 375 380

Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser

465 470 475 480

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

485 490 495

Pro Arg Gly

<210> 10

<211> 1500

<212> DNA

<213> Artificial Sequence

<400> 10

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggagcaga agctgatcag cgaggaggac ctggacatcc agatgacaca gactacatcc 120

tccctgtctg cctctctggg agacagagtc accatcagtt gcagggcaag tcaggacatt 180

agtaaatatt taaattggta tcagcagaaa ccagatggaa ctgttaaact cctgatctac 240

catacatcaa gattacactc aggagtccca tcaaggttca gtggcagtgg gtctggaaca 300

gattattctc tcaccattag caacctggag caagaagata ttgccactta cttttgccaa 360

cagggtaata cgcttccgta cacgttcgga ggggggacca agctggagat cacaggtggc 420

ggtggctcgg gcggtggtgg gtcgggtggc ggcggatctg aggtgaaact gcaggagtca 480

ggacctggcc tggtggcgcc ctcacagagc ctgtccgtca catgcactgt ctcaggggtc 540

tcattacccg actatggtgt aagctggatt cgccagcctc cacgaaaggg tctggagtgg 600

ctgggagtaa tatggggtag tgaaaccaca tactataatt cagctctcaa atccagactg 660

accatcatca aggacaactc caagagccaa gttttcttaa aaatgaacag tctgcaaact 720

gatgacacag ccatttacta ctgtgccaaa cattattact acggtggtag ctatgctatg 780

gactactggg gccaaggaac ctcagtcacc gtctcctcaa ccacgacgcc agcgccgcga 840

ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc 900

cggccagcgg cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatatctac 960

atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 1020

tactgctccc taaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg 1080

agaccagtac aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa 1140

gaaggaggat gtgaactgag agtgaagttc agcaggagcg cagacgcccc cgcgtacaag 1200

cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 1260

ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 1320

caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 1380

gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 1440

acagccacca aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgcggctaa 1500

<210> 11

<211> 1197

<212> DNA

<213> Artificial Sequence

<400> 11

atgggcgtgc aggtggaaac aatcagccct ggcgacggca ggacattccc caagaggggc 60

cagacctgtg tggtgcacta taccggcatg ctggaggacg gcaagaaggt ggacagcagc 120

agggacagga acaagccctt caagttcatg ctgggcaaac aggaagtcat cagaggctgg 180

gaggaaggcg tcgcccagat gagcgtgggc cagagagcca agctgacaat cagccccgat 240

tacgcctacg gcgccacagg ccatcctggc atcattcccc cccacgccac cctggtgttc 300

gatgtggagc tgctgaagct ggagtccgga ggcggttctg gaggctttgg cgatgtgggc 360

gccctggagt ccctcagagg aaacgctgac ctggcctaca tcctgagcat ggagccctgc 420

ggccactgcc tgatcatcaa caacgtgaac ttctgcaggg agtccggcct gaggaccaga 480

acaggctcca acatcgactg cgaaaagctg aggaggaggt tctcctccct gcactttatg 540

gtggaggtga agggcgatct gaccgccaag aagatggtgc tcgccctgct cgaactggcc 600

agacaggacc acggcgccct ggactgttgc gtggtcgtga tcctgagcca cggctgtcag 660

gcttcccacc tgcagttccc tggcgccgtg tacggcaccg acggatgtcc tgtgagcgtg 720

gagaagatcg tgaatatctt caacggcacc tcctgtccca gcctgggcgg aaagcctaag 780

ctgttcttca tccaggcctg cggaggcgag cagaaggacc acggctttga ggtggcttcc 840

acctcccccg aagacgagtc ccccggaagc aaccccgaac ctgacgccac ccccttccag 900

gagggactga ggacattcga ccagctggcc gccattagct ccctgcccac accttccgac 960

atcttcgtga gctacagcac ctttcccgga ttcgtgagct ggagagaccc caagtccgga 1020

agctggtacg tggagaccct ggacgacatc tttgagcagt gggcccatag cgaggacctg 1080

cagagcctgc tgctgagagt ggccaacgcc gtcagcgtga agggcatcta caagcagatg 1140

cctggctgct tcaacttcct gaggaagaag ctcttcttca agaccagcgc cagcaga 1197

<210> 12

<211> 399

<212> PRT

<213> Artificial Sequence

<400> 12

Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe

1 5 10 15

Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu

20 25 30

Asp Gly Lys Lys Val Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys

35 40 45

Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val

50 55 60

Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp

65 70 75 80

Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala

85 90 95

Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Ser Gly Gly Gly

100 105 110

Ser Gly Gly Phe Gly Asp Val Gly Ala Leu Glu Ser Leu Arg Gly Asn

115 120 125

Ala Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys Gly His Cys Leu

130 135 140

Ile Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly Leu Arg Thr Arg

145 150 155 160

Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg Arg Phe Ser Ser

165 170 175

Leu His Phe Met Val Glu Val Lys Gly Asp Leu Thr Ala Lys Lys Met

180 185 190

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

195 200 205

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

210 215 220

Gln Phe Pro Gly Ala Val Tyr Gly Thr Asp Gly Cys Pro Val Ser Val

225 230 235 240

Glu Lys Ile Val Asn Ile Phe Asn Gly Thr Ser Cys Pro Ser Leu Gly

245 250 255

Gly Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Gly Gly Glu Gln Lys

260 265 270

Asp His Gly Phe Glu Val Ala Ser Thr Ser Pro Glu Asp Glu Ser Pro

275 280 285

Gly Ser Asn Pro Glu Pro Asp Ala Thr Pro Phe Gln Glu Gly Leu Arg

290 295 300

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

305 310 315 320

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

325 330 335

Pro Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu Asp Asp Ile Phe Glu

340 345 350

Gln Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu Leu Arg Val Ala

355 360 365

Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met Pro Gly Cys Phe

370 375 380

Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser Ala Ser Arg

385 390 395

<210> 13

<211> 57

<212> DNA

<213> Artificial Sequence

<400> 13

gccgagggaa ggggttctct cctgacctgc ggcgatgtgg aggagaaccc cggaccc 57

<210> 14

<211> 19

<212> PRT

<213> Artificial Sequence

<400> 14

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

1 5 10 15

Pro Gly Pro

<210> 15

<211> 585

<212> DNA

<213> Artificial Sequence

<400> 15

gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt 60

gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc 120

ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag 180

gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac 240

aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc 300

tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc 360

acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca 420

aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt 480

gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg 540

gggacgtggt tttcctttga aaaacacgat gataatatgg ccaca 585

<210> 16

<211> 2760

<212> DNA

<213> Artificial Sequence

<400> 16

atgggcgtgc aggtggaaac aatcagccct ggcgacggca ggacattccc caagaggggc 60

cagacctgtg tggtgcacta taccggcatg ctggaggacg gcaagaaggt ggacagcagc 120

agggacagga acaagccctt caagttcatg ctgggcaaac aggaagtcat cagaggctgg 180

gaggaaggcg tcgcccagat gagcgtgggc cagagagcca agctgacaat cagccccgat 240

tacgcctacg gcgccacagg ccatcctggc atcattcccc cccacgccac cctggtgttc 300

gatgtggagc tgctgaagct ggagtccgga ggcggttctg gaggctttgg cgatgtgggc 360

gccctggagt ccctcagagg aaacgctgac ctggcctaca tcctgagcat ggagccctgc 420

ggccactgcc tgatcatcaa caacgtgaac ttctgcaggg agtccggcct gaggaccaga 480

acaggctcca acatcgactg cgaaaagctg aggaggaggt tctcctccct gcactttatg 540

gtggaggtga agggcgatct gaccgccaag aagatggtgc tcgccctgct cgaactggcc 600

agacaggacc acggcgccct ggactgttgc gtggtcgtga tcctgagcca cggctgtcag 660

gcttcccacc tgcagttccc tggcgccgtg tacggcaccg acggatgtcc tgtgagcgtg 720

gagaagatcg tgaatatctt caacggcacc tcctgtccca gcctgggcgg aaagcctaag 780

ctgttcttca tccaggcctg cggaggcgag cagaaggacc acggctttga ggtggcttcc 840

acctcccccg aagacgagtc ccccggaagc aaccccgaac ctgacgccac ccccttccag 900

gagggactga ggacattcga ccagctggcc gccattagct ccctgcccac accttccgac 960

atcttcgtga gctacagcac ctttcccgga ttcgtgagct ggagagaccc caagtccgga 1020

agctggtacg tggagaccct ggacgacatc tttgagcagt gggcccatag cgaggacctg 1080

cagagcctgc tgctgagagt ggccaacgcc gtcagcgtga agggcatcta caagcagatg 1140

cctggctgct tcaacttcct gaggaagaag ctcttcttca agaccagcgc cagcagagcc 1200

gagggaaggg gttctctcct gacctgcggc gatgtggagg agaaccccgg acccggatcc 1260

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 1320

ccggagcaga agctgatcag cgaggaggac ctggacatcc agatgacaca gactacatcc 1380

tccctgtctg cctctctggg agacagagtc accatcagtt gcagggcaag tcaggacatt 1440

agtaaatatt taaattggta tcagcagaaa ccagatggaa ctgttaaact cctgatctac 1500

catacatcaa gattacactc aggagtccca tcaaggttca gtggcagtgg gtctggaaca 1560

gattattctc tcaccattag caacctggag caagaagata ttgccactta cttttgccaa 1620

cagggtaata cgcttccgta cacgttcgga ggggggacca agctggagat cacaggtggc 1680

ggtggctcgg gcggtggtgg gtcgggtggc ggcggatctg aggtgaaact gcaggagtca 1740

ggacctggcc tggtggcgcc ctcacagagc ctgtccgtca catgcactgt ctcaggggtc 1800

tcattacccg actatggtgt aagctggatt cgccagcctc cacgaaaggg tctggagtgg 1860

ctgggagtaa tatggggtag tgaaaccaca tactataatt cagctctcaa atccagactg 1920

accatcatca aggacaactc caagagccaa gttttcttaa aaatgaacag tctgcaaact 1980

gatgacacag ccatttacta ctgtgccaaa cattattact acggtggtag ctatgctatg 2040

gactactggg gccaaggaac ctcagtcacc gtctcctcaa ccacgacgcc agcgccgcga 2100

ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc 2160

cggccagcgg cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatatctac 2220

atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 2280

tactgctccc taaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg 2340

agaccagtac aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa 2400

gaaggaggat gtgaactgag agtgaagttc agcaggagcg cagacgcccc cgcgtacaag 2460

cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 2520

ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 2580

caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 2640

gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 2700

acagccacca aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgcggctaa 2760

<210> 17

<211> 919

<212> PRT

<213> Artificial Sequence

<400> 17

Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe

1 5 10 15

Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu

20 25 30

Asp Gly Lys Lys Val Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys

35 40 45

Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val

50 55 60

Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp

65 70 75 80

Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala

85 90 95

Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Ser Gly Gly Gly

100 105 110

Ser Gly Gly Phe Gly Asp Val Gly Ala Leu Glu Ser Leu Arg Gly Asn

115 120 125

Ala Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys Gly His Cys Leu

130 135 140

Ile Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly Leu Arg Thr Arg

145 150 155 160

Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg Arg Phe Ser Ser

165 170 175

Leu His Phe Met Val Glu Val Lys Gly Asp Leu Thr Ala Lys Lys Met

180 185 190

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

195 200 205

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

210 215 220

Gln Phe Pro Gly Ala Val Tyr Gly Thr Asp Gly Cys Pro Val Ser Val

225 230 235 240

Glu Lys Ile Val Asn Ile Phe Asn Gly Thr Ser Cys Pro Ser Leu Gly

245 250 255

Gly Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Gly Gly Glu Gln Lys

260 265 270

Asp His Gly Phe Glu Val Ala Ser Thr Ser Pro Glu Asp Glu Ser Pro

275 280 285

Gly Ser Asn Pro Glu Pro Asp Ala Thr Pro Phe Gln Glu Gly Leu Arg

290 295 300

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

305 310 315 320

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

325 330 335

Pro Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu Asp Asp Ile Phe Glu

340 345 350

Gln Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu Leu Arg Val Ala

355 360 365

Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met Pro Gly Cys Phe

370 375 380

Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser Ala Ser Arg Ala

385 390 395 400

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

405 410 415

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

420 425 430

Ala Leu Leu Leu His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

Gln Ala Leu Pro Pro Arg Gly

915

<210> 18

<211> 3282

<212> DNA

<213> Artificial Sequence

<400> 18

atgggcgtgc aggtggaaac aatcagccct ggcgacggca ggacattccc caagaggggc 60

cagacctgtg tggtgcacta taccggcatg ctggaggacg gcaagaaggt ggacagcagc 120

agggacagga acaagccctt caagttcatg ctgggcaaac aggaagtcat cagaggctgg 180

gaggaaggcg tcgcccagat gagcgtgggc cagagagcca agctgacaat cagccccgat 240

tacgcctacg gcgccacagg ccatcctggc atcattcccc cccacgccac cctggtgttc 300

gatgtggagc tgctgaagct ggagtccgga ggcggttctg gaggctttgg cgatgtgggc 360

gccctggagt ccctcagagg aaacgctgac ctggcctaca tcctgagcat ggagccctgc 420

ggccactgcc tgatcatcaa caacgtgaac ttctgcaggg agtccggcct gaggaccaga 480

acaggctcca acatcgactg cgaaaagctg aggaggaggt tctcctccct gcactttatg 540

gtggaggtga agggcgatct gaccgccaag aagatggtgc tcgccctgct cgaactggcc 600

agacaggacc acggcgccct ggactgttgc gtggtcgtga tcctgagcca cggctgtcag 660

gcttcccacc tgcagttccc tggcgccgtg tacggcaccg acggatgtcc tgtgagcgtg 720

gagaagatcg tgaatatctt caacggcacc tcctgtccca gcctgggcgg aaagcctaag 780

ctgttcttca tccaggcctg cggaggcgag cagaaggacc acggctttga ggtggcttcc 840

acctcccccg aagacgagtc ccccggaagc aaccccgaac ctgacgccac ccccttccag 900

gagggactga ggacattcga ccagctggcc gccattagct ccctgcccac accttccgac 960

atcttcgtga gctacagcac ctttcccgga ttcgtgagct ggagagaccc caagtccgga 1020

agctggtacg tggagaccct ggacgacatc tttgagcagt gggcccatag cgaggacctg 1080

cagagcctgc tgctgagagt ggccaacgcc gtcagcgtga agggcatcta caagcagatg 1140

cctggctgct tcaacttcct gaggaagaag ctcttcttca agaccagcta agcccctctc 1200

cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg 1260

tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg 1320

gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag 1380

gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt 1440

ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc 1500

aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga 1560

gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga 1620

aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct 1680

ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg 1740

ttttcctttg aaaaacacga tgataatatg gccacaggat ccatggcctt accagtgacc 1800

gccttgctcc tgccgctggc cttgctgctc cacgccgcca ggccggagca gaagctgatc 1860

agcgaggagg acctggacat ccagatgaca cagactacat cctccctgtc tgcctctctg 1920

ggagacagag tcaccatcag ttgcagggca agtcaggaca ttagtaaata tttaaattgg 1980

tatcagcaga aaccagatgg aactgttaaa ctcctgatct accatacatc aagattacac 2040

tcaggagtcc catcaaggtt cagtggcagt gggtctggaa cagattattc tctcaccatt 2100

agcaacctgg agcaagaaga tattgccact tacttttgcc aacagggtaa tacgcttccg 2160

tacacgttcg gaggggggac caagctggag atcacaggtg gcggtggctc gggcggtggt 2220

gggtcgggtg gcggcggatc tgaggtgaaa ctgcaggagt caggacctgg cctggtggcg 2280

ccctcacaga gcctgtccgt cacatgcact gtctcagggg tctcattacc cgactatggt 2340

gtaagctgga ttcgccagcc tccacgaaag ggtctggagt ggctgggagt aatatggggt 2400

agtgaaacca catactataa ttcagctctc aaatccagac tgaccatcat caaggacaac 2460

tccaagagcc aagttttctt aaaaatgaac agtctgcaaa ctgatgacac agccatttac 2520

tactgtgcca aacattatta ctacggtggt agctatgcta tggactactg gggccaagga 2580

acctcagtca ccgtctcctc aaccacgacg ccagcgccgc gaccaccaac accggcgccc 2640

accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 2700

gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccttggcc 2760

gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgctc cctaaaacgg 2820

ggcagaaaga aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact 2880

caagaggaag atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 2940

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 3000

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 3060

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 3120

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 3180

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 3240

tacgacgccc ttcacatgca ggccctgccc cctcgcggct aa 3282

<210> 19

<211> 897

<212> PRT

<213> Artificial Sequence

<400> 19

Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe

1 5 10 15

Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu

20 25 30

Asp Gly Lys Lys Val Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys

35 40 45

Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val

50 55 60

Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp

65 70 75 80

Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala

85 90 95

Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Ser Gly Gly Gly

100 105 110

Ser Gly Gly Phe Gly Asp Val Gly Ala Leu Glu Ser Leu Arg Gly Asn

115 120 125

Ala Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys Gly His Cys Leu

130 135 140

Ile Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly Leu Arg Thr Arg

145 150 155 160

Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg Arg Phe Ser Ser

165 170 175

Leu His Phe Met Val Glu Val Lys Gly Asp Leu Thr Ala Lys Lys Met

180 185 190

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

195 200 205

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

210 215 220

Gln Phe Pro Gly Ala Val Tyr Gly Thr Asp Gly Cys Pro Val Ser Val

225 230 235 240

Glu Lys Ile Val Asn Ile Phe Asn Gly Thr Ser Cys Pro Ser Leu Gly

245 250 255

Gly Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Gly Gly Glu Gln Lys

260 265 270

Asp His Gly Phe Glu Val Ala Ser Thr Ser Pro Glu Asp Glu Ser Pro

275 280 285

Gly Ser Asn Pro Glu Pro Asp Ala Thr Pro Phe Gln Glu Gly Leu Arg

290 295 300

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

305 310 315 320

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

325 330 335

Pro Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu Asp Asp Ile Phe Glu

340 345 350

Gln Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu Leu Arg Val Ala

355 360 365

Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met Pro Gly Cys Phe

370 375 380

Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser Gly Ser Met Ala

385 390 395 400

Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala

405 410 415

Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asp Ile Gln

420 425 430

Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val

435 440 445

Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp

450 455 460

Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr His Thr

465 470 475 480

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

485 490 495

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

500 505 510

Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp

705 710 715 720

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

725 730 735

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

740 745 750

Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

Gly

<210> 20

<211> 960

<212> DNA

<213> Artificial Sequence

<400> 20

atgccacctc ctcgcctcct cttcttcctc ctcttcctca cccccatgga agtcaggccc 60

gaggaacctc tagtggtgaa ggtggaagag ggagataacg ctgtgctgca gtgcctcaag 120

gggacctcag atggccccac tcagcagctg acctggtctc gggagtcccc gcttaaaccc 180

ttcttaaaac tcagcctggg gctgccaggc ctgggaatcc acatgaggcc cctggccatc 240

tggcttttca tcttcaacgt ctctcaacag atggggggct tctacctgtg ccagccgggg 300

cccccctctg agaaggcctg gcagcctggc tggacagtca atgtggaggg cagcggggag 360

ctgttccggt ggaatgtttc ggacctaggt ggcctgggct gtggcctgaa gaacaggtcc 420

tcagagggcc ccagctcccc ttccgggaag ctcatgagcc ccaagctgta tgtgtgggcc 480

aaagaccgcc ctgagatctg ggagggagag cctccgtgtc tcccaccgag ggacagcctg 540

aaccagagcc tcagccagga cctcaccatg gcccctggct ccacactctg gctgtcctgt 600

ggggtacccc ctgactctgt gtccaggggc cccctctcct ggacccatgt gcaccccaag 660

gggcctaagt cattgctgag cctagagctg aaggacgatc gcccggccag agatatgtgg 720

gtaatggaga cgggtctgtt gttgccccgg gccacagctc aagacgctgg aaagtattat 780

tgtcaccgtg gcaacctgac catgtcattc cacctggaga tcactgctcg gccagtacta 840

tggcactggc tgctgaggac tggtggctgg aaggtctcag ctgtgacttt ggcttatctg 900

atcttctgcc tgtgttccct tgtgggcatt cttcatcttc aaagagccct ggtcctgagg 960

Claims

A kind of 1. Lentiviral, it is characterised in that：Include the gene and iCasp9 apoptosis of encoding chimeric antigen acceptor Gene, the gene of the encoding chimeric antigen acceptor, its nucleotide sequence such as SEQ ID NO:Shown in 10；The iCasp9 apoptosis Gene, its nucleotide sequence such as SEQ ID NO:Shown in 11.
2. Lentiviral according to claim 1, it is characterised in that：The structure of the Lentiviral is PHR-iCasp9-2A-CD19CAR-4-1BB-CD3 ζ, the nucleotide sequence such as SEQ ID NO of its expressing gene:Shown in 16；Or： PHR-iCasp9-IRES-CD19CAR-4-1BB-CD3 ζ, the nucleotide sequence such as SEQ ID NO of its expressing gene:Shown in 18.
3. a kind of slow virus expresses kit, including the Lentiviral described in claim 1 or 2.
A kind of 4. recombinant slow virus, it is characterised in that：It is prepared by the following method to obtain：By the slow disease described in claim 1 or 2 It is transfected into after malicious expression vector, pCMV carriers and the mixing of pMD.2G carriers in 293FT cells, 6~8h is replaced by completely after transfection Medium culture, nutrient solution is collected after 48h, supernatant is retained after centrifugation and filters the supernatant with 0.45 μm of filtering head, reservation Filtrate, the filtrate are the solution of recombinant slow virus.
A kind of 5. CD19-CAR-T cells of carrying iCasp9 suicide genes, it is characterised in that：To include claim 1 or 2 Lentiviral T lymphocytes, or the gene of encoding chimeric antigen acceptor is integrated with chromosome and iCasp9 withers Die the T lymphocytes of gene, the gene of the encoding chimeric antigen acceptor, its nucleotide sequence such as SEQ ID NO:Shown in 10； The iCasp9 apoptogenes, its nucleotide sequence such as SEQ ID NO:Shown in 11.
6. the Lentiviral described in claim 1 or 2 is pernicious swollen in preparation prevention and/or treatment and/or auxiliary treatment Application in the medicine of knurl.
7. application according to claim 6, it is characterised in that：The malignant tumour is selected from ALL, Chronic lymphocytic leukemia.
8. the CD19-CAR-T cells of the carrying iCasp9 suicide genes described in claim 5 prepare prevention and/or treatment and/ Or the application in the medicine of adjuvant therapy of malignant tumor.
9. application according to claim 8, it is characterised in that：The malignant tumour is selected from ALL, Chronic lymphocytic leukemia.
A kind of 10. iCasp9/CID suicide gene systems, it is characterised in that：By the small molecule of iCasp9 genes and inactive Chemical induction medicine forms；Wherein, the nucleotide sequence of iCasp9 genes such as SEQ ID NO:Shown in 11；Small-molecule chemical induces Medicine is selected from AP1903, AP20187.