CN110144327A

CN110144327A - A kind of antitumor T cell of targeting and its preparation method and application

Info

Publication number: CN110144327A
Application number: CN201810148378.4A
Authority: CN
Inventors: 张宏玲; 钟春颖
Original assignee: Shenzhen Benta Biological Technology Co Ltd
Current assignee: Shenzhen Benta Biological Technology Co Ltd
Priority date: 2018-02-12
Filing date: 2018-02-12
Publication date: 2019-08-20

Abstract

The present invention provides a kind of antitumor T cells of targeting, Chimeric antigen receptor CAR-CD19 including targeting the Chimeric antigen receptor CAR-Mesothelin and targeting CD19 of Mesothelin, the T cell efficiently targets and kills pancreatic cancer cell, and remove the bone-marrow-derived lymphocyte near Pancreatic Adenocarcinoma, regulate and control surrounding immune microenvironment, reaches better therapeutic effect.The present invention also provides the preparation method and application of the antitumor T cell of the targeting.

Description

A kind of antitumor T cell of targeting and its preparation method and application

Technical field

The present invention relates to field of medical biotechnology, in particular to a kind of antitumor T cell of targeting and preparation method thereof and answer With.

Background technique

Cancer of pancreas is the malignant tumor of digestive tract that a kind of grade malignancy is high, diagnosing and treating is all highly difficult, and about 90% is Derived from the duct adenocarcinoma of glandular tube epithelium, morbidity and mortality obviously rise in recent years.(Chimeric antigen receptor T is thin by CAR-T Born of the same parents) technology is a kind of novel immune cell therapy, it is will to be fed back to human body by the T cell of CAR transformation, activates autoimmunity System kills tumour cell, to achieve the purpose that remove malignant cell, is considered most possibly thoroughly removing swollen The method of oncocyte.

But the application of current CAR-T technology is also limited to the hematological system tumors such as leukaemia, myeloma, lymthoma, all In such as cancer of pancreas entity tumor using less, and in complicated tumor microenvironment, the knowledge of CAR-T cells against tumor cells Not, killing ability substantially reduces, and limits its clinical application.

Summary of the invention

In consideration of it, the present invention provides a kind of antitumor T cell of the targeting for cancer of pancreas and preparation method thereof and answering With.The T cell can be efficiently targeted to pancreatic cancer cell, and removes the bone-marrow-derived lymphocyte near Pancreatic Adenocarcinoma, regulates and controls surrounding Immune microenvironment, reaches better therapeutic effect.

In a first aspect, the antitumor T cell of targeting is band the present invention provides a kind of targeting antitumor T cell Target the Chimeric antigen receptor CAR-Mesothelin of Mesothelin and the Chimeric antigen receptor CAR-CD19 with targeting CD19 Double target spot Chimeric antigen receptor T cells, or for the Chimeric antigen receptor T cell with the CAR-Mesothelin and with institute The mixing of the Chimeric antigen receptor T cell of CAR-CD19 is stated, or is the Chimeric antigen receptor T with the CAR-Mesothelin Cell and Chimeric antigen receptor T cell with the CAR-CD19 it is one or more with the CAR-Mesothelin and institute State the mixing of double target spot Chimeric antigen receptor T cells of CAR-CD19；Wherein, the CAR-Mesothelin includes from aminoterminal To the sequentially connected targeting single-chain antibody of Mesothelin of c-terminus, extracellular hinge area, transmembrane region and intracellular signal area ammonia Base acid sequence, the CAR-CD19 include the single-chain antibody of sequentially connected targeting CD19, extracellular hinge from aminoterminal to c-terminus The amino acid sequence of sequence, transmembrane region and intracellular signal area；Wherein, the amino of the single-chain antibody of the targeting Mesothe lin Acid sequence includes the amino acid sequence as shown in SEQ ID NO:1, the amino acid sequence packet of the single-chain antibody of the targeting CD19 Include the amino acid sequence as shown in SEQ ID NO:2.

Wherein, when the antitumor T cell of the targeting is double target spot CAR-T with CAR-Mesothelin and CAR-CD19 When, the distributing position of Chimeric antigen receptor CAR-Mesothelin and CAR-CD19 are not construed as limiting, can be and be alternately distributed (such as ABAB ..., AABABB ...) or be sequentially distributed (such as AAAA ... BB B ...), but have no covalent linkage between the two, at this time they Covalent linkage is also had no between corresponding Mesothelin single-chain antibody and CD19 single-chain antibody, them can be made to keep preferable in this way Recognition capability.

Above-mentioned " being sequentially connected with from aminoterminal to c-terminus " specifically: the single-chain antibody of the targeting Mesothelin or institute State the aminoterminal of the c-terminus of the amino acid sequence of the single-chain antibody of targeting CD19 and the amino acid sequence of the extracellular hinge area It is connected, the c-terminus of the amino acid sequence of the extracellular hinge area is connected with the aminoterminal of the amino acid sequence of the transmembrane region, The c-terminus of the amino acid sequence of the transmembrane region is connected with the aminoterminal of the amino acid sequence in the intracellular signal area.

The above-mentioned Mesothelin (mesothelin) referred to is a kind of cell surface glycoprotein that molecular weight is 40kDa, high table Up in kinds of tumors tissue, being the biological identification of Early pancreatic carcinoma.But the CAR-T cell of Mesothelin is targeted to pancreas The therapeutic effect of cancer patient is simultaneously bad, and the prognosis of patient can be excessively poor.CD19 is B cell system marker in hematological system, is passed through And the combination of the B-cell receptor in B cell participates in the processes such as B cell development, Cellular Signaling Transduction Mediated, and CD19 is that B system is pernicious swollen The more common target spot of tumor (such as lymphocytic leukemia).And the above-mentioned antitumor T cell of targeting of the present invention has CAR- Mesothelin and CAR-CD19, wherein CAR-Mesothelin can be targeted to expression Mesothelin's with preferably targeting Pancreatic cancer cell carries out the killing of pancreatic cancer cell；And the CAR-T cell with CAR-CD19 can remove Pancreatic Adenocarcinoma well Neighbouring bone-marrow-derived lymphocyte regulates and controls surrounding immune microenvironment, pancreatic tissue is avoided to be infiltrated by bone-marrow-derived lymphocyte, improves cancer of pancreas The prognosis situation of patient.Reach better therapeutic effect.

Optionally, the encoding gene of the single-chain antibody of the targeting Mesothelin includes as shown in SEQ ID NO:3 The encoding gene of nucleotide sequence, the single-chain antibody of the targeting CD19 includes the nucleotide sequence as shown in SEQ ID NO:4. The single-chain antibody of targeting Mesothelin of the present invention remains with the affine activity to Mesothelin antigen, can efficiently know Other surface expression has the tumour cell of Mesothelin antigen.Similarly, the single-chain antibody of the targeting CD19 also remains with pair The affine activity of CD19 antigen, can efficient identification surface expression have the B system malignant lymphocytic of CD19 antigen.

Optionally, the encoding gene of the amino acid sequence of the single-chain antibody of the targeting Mesothelin should consider degeneracy Base, the i.e. encoding gene of the amino acid sequence as shown in SEQ ID NO:1 include the nucleotides sequence as shown in SEQ ID NO:3 Column, protection scope should also protect the nucleotide sequence for having base degeneracy matter with SEQ ID NO:3, these nucleotide sequences Corresponding amino acid sequence remains as SEQ ID NO:1.The coding base of the amino acid sequence of the single-chain antibody of the targeting CD19 Because should equally consider degeneracy base.

In the present invention, the extracellular hinge area in the CAR-Mesothelin is for promoting the targeting Mesothel in Single-chain antibody and tumour cell on Mesothelin antigen binding；Similarly, the extracellular hinge area in the CAR-CD19 For promoting the single-chain antibody of the targeting CD19 in conjunction with the CD19 on tumour cell.

Optionally, the extracellular hinge area include CD8 α hinge area, CD28 hinge area, CD4 hinge area, C D5 hinge area, One of CD134 hinge area, CD137 hinge area, ICOS hinge area or a variety of combinations.

Still optionally further, the extracellular hinge area is CD8 α hinge area.

Optionally, the amino acid sequence of the CD8 α hinge area includes the amino acid sequence as shown in SEQ ID NO:9.

Optionally, the encoding gene of the CD8 α hinge area includes the nucleotide sequence as shown in SEQ ID NO:10.

Optionally, the encoding gene of the CD8 α hinge area should consider degeneracy base, i.e., as shown in SEQ ID NO:9 The encoding gene of amino acid sequence include the nucleotide sequence such as SEQ ID NO:10 shown in, protection scope should also protect and SEQ ID NO:10 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:9。

In the present invention, the transmembrane region in the CAR-Mesothelin is used to fix the chimeric of the targeting Mesothelin Antigen receptor CAR-Mesothelin；Similarly, the transmembrane region in the CAR-CD19 is used to fix the embedding of the targeting CD19 Close antigen receptor CAR-CD19.

Optionally, the transmembrane region includes one of CD3 transmembrane region, CD4 transmembrane region, CD8 transmembrane region, CD28 transmembrane region Or a variety of combination.

Still optionally further, the transmembrane region is CD8 transmembrane region.

Optionally, the amino acid sequence of the CD8 transmembrane region includes the amino acid sequence as shown in SEQ ID NO:11.

Optionally, the encoding gene of the CD8 transmembrane region includes the nucleotide sequence as shown in SEQ ID NO:12.

Optionally, the encoding gene of the CD8 transmembrane region should consider degeneracy base, i.e., as shown in SEQ ID NO:11 The encoding gene of amino acid sequence include the nucleotide sequence such as SEQ ID NO:12 shown in, protection scope should also protect and SEQ ID NO:12 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:11。

In the present invention, the intracellular signal area for providing the signal of T cell activation, maintain T cell life span and Activate T cell proliferation signal access.

Optionally, the intracellular signal area includes 4-1BB signaling zone, CD3 ζ signaling zone, ICOS signaling zone, C D27 signal One of area, OX40 signaling zone, CD28 signaling zone, IL1R1 signaling zone, CD70 signaling zone, TNFRS F19L signaling zone are more The combination of kind.

In an embodiment of the present invention, the intracellular signal Qu Weicong aminoterminal to the sequentially connected 4-1BB of c-terminus Signaling zone and CD3 ζ signaling zone.Correspondingly, the encoding gene in the intracellular signal area includes sequentially connected from 5 ' ends to 3 ' ends The encoding gene of 4-1BB signaling zone and the encoding gene of CD3 ζ signaling zone.

In another embodiment of the present invention, the intracellular signal area can also be to be sequentially connected with from aminoterminal to c-terminus CD27 signaling zone and CD3 ζ signaling zone.

Optionally, the amino acid sequence of the 4-1BB signaling zone includes the amino acid sequence as shown in SEQ ID NO:13.

Optionally, the encoding gene of the 4-1BB signaling zone includes the nucleotide sequence as shown in SEQ ID NO:14.

Optionally, the encoding gene of the 4-1BB signaling zone should consider degeneracy base, i.e., as shown in SEQ ID NO:13 Amino acid sequence encoding gene include the nucleotide sequence such as SEQ ID NO:14 shown in, protection scope should also protect and SEQ ID NO:14 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:13。

Optionally, the amino acid sequence of the CD3 ζ signaling zone includes the amino acid sequence as shown in SEQ ID NO:15.

Optionally, the encoding gene of the CD3 ζ signaling zone includes the nucleotide sequence as shown in SEQ ID NO:16.

Optionally, the encoding gene of the CD3 ζ signaling zone should consider degeneracy base, i.e., as shown in SEQ ID NO:15 Amino acid sequence encoding gene include the nucleotide sequence such as SEQ ID NO:16 shown in, protection scope should also protect and SEQ ID NO:16 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:15。

It should be noted that extracellular hinge area, transmembrane region and letter intracellular in the present invention, in the CAR-Mesothelin The amino acid sequence in number area, with extracellular hinge area corresponding in the CAR-CD19, the corresponding ammonia of transmembrane region and intracellular signal area Base acid sequence may be the same or different.

In an embodiment of the present invention, the amino acid sequence of the CAR-Mesothelin includes such as SEQ ID NO:5 Shown in amino acid sequence.

Optionally, the encoding gene of the CAR-Mesothelin includes the nucleotides sequence as shown in SEQ ID NO:17 Column.

Optionally, the encoding gene of the CAR-Mesothelin should consider degeneracy base, i.e., such as SEQ ID NO:5 institute The encoding gene of the amino acid sequence shown includes the nucleotide sequence as shown in SEQ ID NO:17, and protection scope should also be protected There is the nucleotide sequence of base degeneracy matter with SEQ ID NO:17, the corresponding amino acid sequence of these nucleotide sequences is still For SEQ ID NO:5.

In an embodiment of the present invention, the amino acid sequence of the CAR-CD19 includes as shown in SEQ ID NO:6 Amino acid sequence.

Optionally, the encoding gene of the CAR-CD19 includes the nucleotide sequence as shown in SEQ ID NO:18.

Optionally, the encoding gene of the CAR-CD19 should consider degeneracy base, the i.e. ammonia as shown in SEQ ID NO:6 The encoding gene of base acid sequence includes the nucleotide sequence as shown in SEQ ID NO:18, and protection scope should also protect and SEQ ID NO:18 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:6。

First aspect present invention provide the antitumor T cell of targeting, including target Mesothelin chimeric antigen by The Chimeric antigen receptor CAR-CD19 of body CAR-Mesothelin and targeting CD19, the targeting antitumor T cell effect targeted to Pancreatic cancer cell, and the bone-marrow-derived lymphocyte near Pancreatic Adenocarcinoma is removed, regulate and control surrounding immune microenvironment, reaches better Therapeutic effect.After specifically in conjunction with CAR-Mesothelin and CAR-CD19 antigen protein corresponding on tumour cell, institute The intracellular signal area for stating the antitumor T cell of targeting is activated, and promotes T cell in the amplification of patient's body, and efficiently and special Property killing tumor cell, especially pancreatic cancer cell, therapeutic effect it is preferable.Additionally due to the Mesothelin and CD19 Single-chain antibody is Humanized single chain antibody, this makes the antitumor T cell be avoided the immune response for causing human organism, and it is lasting to have Body maintain ability, such as activity and lethality.

Second aspect, the present invention provides a kind of recombinant viral vectors, anti-swollen including targeting as described in relation to the first aspect The encoding gene of CAR-Mesothelin described in tumor T cell and/or CAR-CD19.

Optionally, the encoding gene of CAR-Mesothelin and the coding of CAR-CD19 are contained when the recombinant viral vector When gene, the coding base of the encoding gene of the CAR-Mesothelin and the CAR-CD19 on the recombinant viral vector It further include a special sequence, the special sequence is for making the CAR-Me sothelin encoding gene and the CAR- because between The encoding gene of CD19 available two independent PROTEIN C AR-Mesothelin and CAR-CD19 after transcription and translation.It is adopting After transfecting CD3 positive t lymphocytes with such recombinant viral vector, the resulting antitumor T cell of targeting is band CAR- Double target spot Chimeric antigen receptor T cells of Mesothelin and CAR-CD19.Still optionally further, the special sequence can be RBS sequence, IRES sequence, T2A sequence or other Protease sequences etc..

Optionally, the recombinant viral vector has the encoding gene of the CAR-Mesothelin, or with described The encoding gene of CAR-CD19.It is preferred that the recombinant viral vector using CAR-Mesothelin encoding gene is compiled with CAR-CD19 Recombinant viral vector separately or simultaneously the co-infection T lymphocyte of code gene, can be obtained as described in the first aspect of the invention The antitumor T cell of targeting, and efficiency of infection is higher, and the antitumor T cell of gained targeting can more fully hereinafter express CAR- Mesothelin and/or CAR-CD19.

Preferably, the encoding gene of the CAR-Mesothelin includes the nucleotides sequence as shown in SEQ ID NO:19 Column.The nucleotide sequence as shown in SEQ ID NO:19 is compared with the nucleotide sequence as shown in SEQ ID NO:17, under more The encoding gene of link peptide described in text.The encoding gene of the signal peptide can be with Chimeric antigen receptor described in guide CAR-Mesothelin is expressed to cell surface.

Preferably, the encoding gene of the CAR-CD19 includes the nucleotide sequence as shown in SEQ ID NO:20.Such as SEQ Nucleotide sequence shown in ID NO:20 is compared with the nucleotide sequence as shown in SEQ ID NO:18, more companies described below Connect the encoding gene of peptide.The encoding gene of the signal peptide can be reached with Chimeric antigen receptor CAR-CD19 described in guide Cell surface.

Optionally, the viral vectors in the recombinant viral vector includes slow virus carrier, adenovirus vector or reverse transcription Viral vectors.Still optionally further, the viral vectors is slow virus carrier.In the preparation method that fourth aspect present invention provides The step of (1)-(3) show the preparation process of the recombinant viral vector.

The recombinant viral vector that second aspect of the present invention provides, efficiency of infection and transcriptional efficiency with higher, The encoding gene segment of CAR-Mesothelin and/or CAR-CD19 therein can be inserted into host genome by genetic recombination, The antitumor T cell of above-mentioned targeting is obtained, continues it, play consistently targeting, killing effect.

The third aspect, the present invention provides a kind of host cell, the host cell includes the weight as described in second aspect Group slow virus carrier.

The host cell that third aspect present invention provides is used to assemble the recombinant viral vector as described in second aspect, Make it have infectivity.

Optionally, the host cell includes but is not limited to HEK293T cell, 293 cells, 293T cell, 293FT thin Born of the same parents, SW480 cell, u87MG cell, HOS cell, COS1 cell and COS7 cell.

Fourth aspect, the present invention provides a kind of preparation methods of the antitumor T cell of targeting, comprising:

(1) encoding gene and the targeting of the Chimeric antigen receptor CAR-Mesothelin of targeting Mesothelin are provided respectively The encoding gene of the Chimeric antigen receptor CAR-CD19 of CD19；

The encoding gene of the CAR-Mesothelin include from 5 ' end to 3 ' end be sequentially connected with signal peptide encoding gene, Target the encoding gene of single-chain antibody, the encoding gene of extracellular hinge area, the encoding gene of transmembrane region and the born of the same parents of Mesothelin The encoding gene of interior signaling zone；The encoding gene of the CAR-CD19 includes the coding that signal peptide is sequentially connected with from 5 ' ends to 3 ' ends Gene targets the encoding gene of single-chain antibody of CD19, the encoding gene of extracellular hinge area, the encoding gene of transmembrane region and intracellular The encoding gene of signaling zone；

Wherein, the encoding gene of the single-chain antibody of the targeting Mesothelin includes the ammonia as shown in SEQ ID NO:1 The encoding gene of nucleotide sequence corresponding to base acid sequence, the single-chain antibody of the targeting CD19 includes such as SEQ ID NO:2 Shown in nucleotide sequence corresponding to amino acid sequence；

(2) encoding gene of the encoding gene of the CAR-Mesothelin and the CAR-CD19 is inserted respectively into In pWPXLD carrier, pWPXLD-CAR-Mesothelin recombinant plasmid and pWPXLD-CAR-CD19 recombinant plasmid are obtained；

(3) matter is recombinated to the pWPXLD-CAR-Mesothelin recombinant plasmid and the pWPXLD-CAR-CD19 respectively Grain is packed, and obtains the first recombinant slow virus with CAR-Mesothelin encoding gene and with CAR-CD19 encoding gene The second recombinant slow virus；

(4) by first recombinant slow virus and second recombinant slow virus, separately or simultaneously co-transfection CD3 is positive Property T lymphocyte, through separation obtain the antitumor T cell of targeting.

It is above-mentioned " being sequentially connected with from 5 ' ends to 3 ' ends " specifically: described by taking the encoding gene of CAR-Mesothelin as an example 3 ' ends of the coding gene sequence of signal peptide are connected with 5 ' ends of the encoding gene of the single-chain antibody of the targeting Mesothelin, Phase is held with the 5 ' of the extracellular hinge area encoding gene in 3 ' ends of the encoding gene of the single-chain antibody of the targeting Mesothelin Even, 3 ' ends of the encoding gene of the extracellular hinge area are connected with 5 ' ends of the encoding gene of the transmembrane region, the transmembrane region 3 ' ends of encoding gene hold and be connected with the 5 ' of the encoding gene in the intracellular signal area.

In the present invention, the signal peptide is for instructing the Chimeric antigen receptor CAR-Mesothelin or CAR-CD19 table Reach cell surface, the signal peptide is cut in protein translation maturation by signal peptidase.And CAR-Mesothelin Signal peptide can be identical with the amino acid sequence of signal peptide in the encoding gene of the CAR-CD19 in encoding gene, can also not Together, the amino acid sequence for being also possible to their signal peptide is identical, and nucleotide sequence is different.

Optionally, the amino acid sequence of the signal peptide includes the amino acid sequence as shown in SEQ ID NO:21.

Optionally, the encoding gene of the signal peptide includes the nucleotide sequence as shown in SEQ ID NO:22.

Optionally, the encoding gene of the signal peptide should consider degeneracy base, the i.e. ammonia as shown in SEQ ID NO:21 The encoding gene of base acid sequence includes the nucleotide sequence as shown in SEQ ID NO:22, and protection scope should also protect and SEQ ID NO:22 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:21。

It, can for the extracellular hinge area, transmembrane region, the specific choice in intracellular signal area and corresponding coding gene sequence Referring to described in first aspect present invention, which is not described herein again.

Optionally, the encoding gene of the CAR-Mesothelin includes the amino acid sequence as shown in SEQ ID NO:7 Corresponding nucleotide sequence.

Optionally, the encoding gene of the CAR-Mesothelin includes the nucleotides sequence as shown in SEQ ID NO:19 Column.Certainly, the encoding gene of the CAR-Mesothelin, which may also comprise, has base letter with sequence shown in SEQ ID NO:19 And the nucleotide sequence of property.

Optionally, the encoding gene of the CAR-CD19 includes as amino acid sequence shown in SEQ ID NO:8 is corresponding Nucleotide sequence.

Optionally, the encoding gene of the CAR-CD19 includes the nucleotide sequence as shown in SEQ ID NO:20.Certainly, The encoding gene of the CAR-CD19 may also comprise the nucleotide for having base degeneracy matter with sequence shown in SEQ ID NO:20 Sequence.

By taking CAR-Mesothelin as an example, compared with SEQ ID NO:19 nucleotide sequence shown in the SEQ ID NO:17, More encoding genes of link peptide, but when Chimeric antigen receptor CAR-Mesothelin expression is to T cell surface, signal peptide exists It is cut in protein translation maturation by signal peptidase.Therefore, the Chimeric antigen receptor CAR-Mesothelin's translated into In amino acid sequence (SEQ ID NO:5) and not with the amino acid sequence as shown in SEQ ID NO:21.The feelings of CAR-CD19 Condition is similar therewith.

By taking CAR-Mesothelin as an example, the coding gene sequence of the CAR-Mesothelin is inserted into pWPXLD carrier Between I restriction enzyme site of middle BamH I and EcoR, and it is located at after the extension factor 1 α (EF1 α) of pWPXLD carrier, is to open with EF1 α Mover.When the coding gene sequence of the CAR-Mesothelin is inserted into pWPXLD carrier, the CAR-Mesothelin's 5 ' ends of gene order can also be added initiation codon (such as ATG) and be connected with BamH1 restriction enzyme site in pWPXLD carrier, and 3 ' ends are also Terminator codon can be added to be connected with EcoR1 restriction enzyme site in pWPXLD carrier.The case where CAR-CD19, is same.

Optionally, described " respectively to the pWPXLD-CAR-Mesothelin recombinant plasmid and described in step (3) PWPXLD-CAR-CD19 recombinant plasmid is packed, and the first recombinant slow virus with CAR-Mesothelin encoding gene is obtained And the second recombinant slow virus with CAR-CD19 encoding gene ", comprising:

The pWPXLD-CAR-Mesothelin recombinant plasmid and envelope plasmid and packaging plasmid cotransfection host is thin Born of the same parents obtain first recombinant slow virus；The pWPXLD-CAR-CD19 recombinant plasmid is total to envelope plasmid and packaging plasmid Transfection host cell obtains second recombinant slow virus.

Using mode preparation and reorganization plasmid of the present invention and packaging virus, wherein the pWPXLD-CAR- On Mesothelin recombinant plasmid and pWPXLD-CAR-CD22 recombinant plasmid, the coding base of CAR-Mesotheli and CAR-CD22 Because have passed through codon optimization, and molecular weight is suitable for, and the packaging efficiency of recombinant slow virus is high, while as made from host cell The concentration of virus is higher.Correspondingly, using the first recombinant slow virus and the second recombinant slow virus come co-transfection CD3 positive T leaching When bar cell, the dosage of both recombinant slow virus is lower, can reduce experimental cost.

In an embodiment of the present invention, the envelope plasmid is PMD2G, and the packaging plasmid is psPAX2, the host Cell is HEK293T cell.

The envelope plasmid PMD2G encodes vesicular stomatitis virus glycoprotein capsid, the vesicular stomatitis virus sugar egg White capsid can assist recombinant slow virus to adhere to cell membrane, and keep the infectivity of recombinant slow virus.

Recombinant slow virus of the present invention can further contain the envelope protein from other viruses.For example, as this Kind protein, is preferably the virus enveloped protein for carrying out self-infection human cell.To this protein, there is no particular limitation, can example The amphophilic virus hand epithelium albumen etc. for enumerating retrovirus, can be used for example from mouse leukemia virus (MuMLV) 4070A plants of envelope protein.Alternatively, it is also possible to use the envelope protein from MuMLV 10Al.In addition, as herpetoviridae Albumen, it can be cited for example that, gB, gD, gH, gp85 albumen of herpes simplex virus, gp350, gp220 albumen of Epstein-Barr virus Deng.As the albumen of Hepadna Virus section, the S protein etc. of hepatitis B virus can be included.The envelope protein can also be morbilli It is formed after viral glycoprotein and other single chain antibody fusions.

The packaging of recombinant slow virus is generallyd use transient transfection or is packed using cell line.It may be used as wrapping when transient transfection Human cell's strain that dress cell uses, for example including 293 cells, 293T cell, 293FT cell, 293LTV cell, 293EBNA Cell and other clones separated from 293 cells；SW480 cell, u87MG cell, HOS cell, C8166 cell, MT-4 are thin Born of the same parents, Molt-4 cell, HeLa cell, HT1080 cell, TE671 cell etc..It can also be using the cell strain from monkey, example Such as, COS1 cell, COS7 cell, CV-1 cell, BMT10 cell etc..Moreover, the calcium phosphate and PEI transfection reagent that generally use, It is also well used there are also some transfection reagents such as Lipofectamine2000, FuGENE and S93fectin.

The packaging of recombinant slow virus also uses some slow virus package cell lines, such as most common Env glycoprotein of use, Stable cell lines caused by VSVG albumen or HIV-1gag-pol albumen.

For the sake of security, the slow virus carrier system of large-scale use is all the method using split gene group, i.e., will The assignment of genes gene mapping of different miscellaneous functions is played in different plasmids.There are four pUC pUCs (encoding gag-pol gene, Rev base at present Cause, VSVG gene, SIN metastatic gene be located at four different plasmids), three pUC pUCs (eliminate coding Rev gene Plasmid, gag-pol gene uses the codon of the preferences in people's cell in gag-pol plasmid) and two pUC pUCs are (slowly Auxiliary gene necessary to viral vectors is packed is located on the same plasmid, these auxiliary genes are single gene orders；Separately One is then transgenosis plasmid).Also the slow virus packaging system for having more than four pUC pUCs is using.

Optionally, in step (4), the CD3 positive t lymphocytes are to separate to obtain from source of people peripheral blood mononuclear cells ?.The source of people peripheral blood mononuclear cells is from autologous vein blood, autologous bone marrow, Cord blood and placental blood etc..Further Optionally, the fresh peripheral blood or marrow acquired after cancer patient's operation one month, after chemicotherapy one month.

Specifically, the acquisition process of the CD3 positive t lymphocytes is as follows: first by peripheral blood mononuclear cells by certain CD3/CD28 immunomagnetic beads are added in ratio, and after being incubated for a period of time, are put into magnet and are screened, it is coated to obtain immunomagnetic beads CD3 positive t lymphocytes after removing magnetic bead, obtain CD3 positive t lymphocytes.

Wherein, in step (4), first recombinant slow virus and second recombinant slow virus separately or simultaneously are joined Close transfection CD3 positive t lymphocytes, comprising:

After first transfecting CD3 positive t lymphocytes using first recombinant slow virus, then using the second recombinant lentiviral disease Poison is transfected；Or after first using second recombinant slow virus transfection CD3 positive t lymphocytes, then use first weight Group slow virus is transfected；Or CD3 sun is simultaneously transfected using first recombinant slow virus and second recombinant slow virus Property T lymphocyte.Here " co-transfection " refers to carries out for same group of cell.

Further, in step (4), the virus titer of used first recombinant slow virus and the first recombinant slow virus it Than being 1:(0.5-2).

The obtained antitumor T cell of targeting in step (4) of the present invention, as described in the first aspect of the invention.

Preferably, the antitumor T cell of the targeting is double with the CAR-Mesothelin and CAR-CD19 Target spot Chimeric antigen receptor T cell, or for the Chimeric antigen receptor T cell with the CAR-Mesothelin and with described The mixing of the Chimeric antigen receptor T cell of CAR-CD19, or for the CAR-Mesothelin's and CAR-CD19 Double target spot Chimeric antigen receptor T cells, the Chimeric antigen receptor T cell with the CAR-Mesothelin and with the CAR- The mixing of the Chimeric antigen receptor T cell of CD19.

At this point, there are two independent, not covalently bound Chimeric antigen receptors for the surface tool of the antitumor T cell of targeting (that is to say that there are two independent single-chain antibodies) does not influence their identification, combinations to respective target, can simultaneously, efficiently know Mesothelin and CD19 target on other tumour cell.The antitumor T cell of targeting efficiently can be targeted and be killed Pancreatic cancer cell, and the bone-marrow-derived lymphocyte near Pancreatic Adenocarcinoma can be removed, regulate and control the immune microenvironment in Pancreatic Adenocarcinoma, reaches To better therapeutic effect.

In another embodiment of the present invention, when the antitumor T cell of required targeting is with the CAR-Mesot helin Chimeric antigen receptor T cell and Chimeric antigen receptor T cell with the CAR-CD19 mixing when, can also use following Mode is made: transfecting CD3 positive t lymphocytes using above-mentioned first recombinant slow virus, obtains with the CAR-Mesothelin Chimeric antigen receptor T cell；CD3 positive t lymphocytes are transfected using above-mentioned second recombinant slow virus, are obtained with the CAR- The Chimeric antigen receptor T cell of CD19；Then both Chimeric antigen receptor T cells are mixed.

In the preparation method for the antitumor T cell of targeting that fourth aspect present invention provides, using band CAR- First recombinant slow virus of Mesothelin encoding gene and with CAR-CD19 encoding gene the second recombinant slow virus difference Or simultaneously co-transfection CD3 positive t lymphocytes, it may make Chimeric antigen receptor in the antitumor T cell of targeting obtained The expression efficiency of CAR-Mesothelin and CAR-CD19 is higher, with the identification of preferable tumour, killing ability, gained targeting Property antitumor T cell also there is stronger tumor-killing ability under complicated tumor microenvironment.

5th aspect, the present invention provides a kind of antitumor T cell of targeting as described in the first aspect of the invention, such as originally Recombinant viral vector described in invention second aspect, host cell as described in the third aspect of the present invention or such as present invention four directions The antitumor T cell of targeting made from preparation method described in face is in the drug that preparation diagnoses and/or treats malignant tumour Using.Particularly, suitable for expressing the diagnosing and treating of the malignant tumour (such as cancer of pancreas etc.) of Mesothelin.

The application can be with specifically: provides a kind of kit, the kit includes target as described in relation to the first aspect The antitumor T cell of tropism or the antitumor T cell of targeting transfected using the recombinant viral vector as described in second aspect Or using the antitumor T cell of targeting obtained by the preparation method as described in fourth aspect, as described in respect of the second aspect of the invention Recombinant viral vector, one of host cell as described in the fourth aspect of the present invention or a variety of.

Advantages of the present invention will be illustrated partially in the following description, and a part is apparent according to specification , or can implementation through the embodiment of the present invention and know.

Detailed description of the invention

Fig. 1 is the plasmid map of pWPXLd-CAR-Mesothelin recombinant plasmid provided in an embodiment of the present invention.

Fig. 2 is the plasmid map of pWPXLd-CAR-CD19 recombinant plasmid provided in an embodiment of the present invention.

Specific embodiment

The following is a preferred embodiment of the present invention, it is noted that for those skilled in the art For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as Protection scope of the present invention.

A kind of preparation method of the one antitumor T cell of targeting of embodiment, comprising the following steps:

(1) gene order of the Chimeric antigen receptor CAR-Mesothelin of preparation targeting Mesothelin

Signal peptide, the single-chain antibody for targeting Mesothelin, CD8 α hinge area, CD8 transmembrane region, 4-1BB letter are prepared respectively The encoding gene in number area and CD3 ζ signaling zone, in the CAR-Mesothelin, the encoding gene of signal peptide such as SEQ ID NO: Shown in 22, the encoding gene of the single-chain antibody of Mesothelin is targeted as shown in SEQ ID NO:3, the coding base of CD8 α hinge area Because as shown in SEQ ID NO:10, the encoding gene of the CD8 transmembrane region is as shown in SEQ ID NO:12, the 4-1BB signal The encoding gene in area is as shown in SEQ ID NO:14, and the encoding gene of the CD3 ζ signaling zone is as shown in SEQ ID NO:16.

By the method for PCR by above-mentioned signal peptide, target single-chain antibody, the CD8 α hinge area, CD8 cross-film of Mesothelin Area, 4-1BB signaling zone and the encoding gene of CD3 ζ signaling zone are successively connected together from 5 ' ends to 3 ' ends, obtain CAR- The encoding gene of Mesothelin, the encoding gene of the CAR-Mesothelin is as shown in SEQ ID NO:19.

(2) gene order of the Chimeric antigen receptor CAR-CD19 of preparation targeting CD19

Prepare respectively signal peptide, target the single-chain antibody of CD19, CD8 α hinge area, CD8 transmembrane region, 4-1BB signaling zone and The encoding gene of CD3 ζ signaling zone, the encoding gene of signal peptide used in the CAR-CD19 is as shown in SEQ ID NO:22, target To CD19 single-chain antibody encoding gene as shown in SEQ ID NO:4, the encoding gene of CD8 α hinge area such as SEQ ID NO: Shown in 10, the encoding gene of CD8 transmembrane region is as shown in SEQ ID NO:12, the encoding gene such as SEQ of the 4-1BB signaling zone Shown in ID NO:14, the encoding gene of the CD3 ζ signaling zone is as shown in SEQ ID NO:16.

By the method for PCR by above-mentioned signal peptide, target single-chain antibody, the CD8 α hinge area, CD8 transmembrane region, 4- of CD19 1BB signaling zone is successively connected together from 5 ' ends to 3 ' ends with the encoding gene of CD3 ζ signaling zone, obtains the coding of CAR-CD19 Gene, the encoding gene of the CAR-CD19 is as shown in SEQ ID NO:20.

(3) pWPXLd-CAR-Mesothelin recombinant plasmid and pWPXLd-CAR-CD19 recombinant plasmid are constructed

The encoding gene of CAR-Mesothelin is inserted between I restriction enzyme site of BamH I and EcoR of pWPXLD carrier, And after pWPXLD carrier EF1 α, using EF1 α as promoter.The encoding gene of the CAR-Mesothelin is inserted into When pWPXLD carrier, the encoding gene of the CAR-Mesothelin 5 ' end can be added initiation codon (such as ATG) with I restriction enzyme site of BamH is connected in pWPXLD carrier, and EcoR I in terminator codon (such as TAA) and pWPXLD carrier can be added in 3 ' ends Restriction enzyme site is connected.Then it is transferred to competent escherichia coli cell DH5 α, carries out positive colony PCR identification and sequencing identification.Through It crosses PCR product detected through gel electrophoresis and sequencing identification meets target fragment size and sequence, successfully construct as shown in Figure 1 PWPXLd-CAR-Mesothelin recombinant plasmid.

The encoding gene of CAR-CD19 is inserted between I restriction enzyme site of BamH I and EcoR of pWPXLD carrier, and After pWPXLD carrier EF1 α, using EF1 α as promoter.Wherein carried when the encoding gene of the CAR-CD19 is inserted into pWPXLD When body, I enzyme of BamH in initiation codon (such as ATG) and pWPXLD carrier can be added in 5 ' ends of the encoding gene of the CAR-CD19 Enzyme site is connected, and 3 ' ends can be added terminator codon (such as TAA) and be connected with I restriction enzyme site of EcoR in pWPXLD carrier.Then turn Enter competent escherichia coli cell DH5 α, carries out positive colony PCR identification and sequencing identification.It is examined by PCR product gel electrophoresis It surveys and sequencing identification meets target fragment size and sequence, successfully construct pWPXLd-CAR-CD19 recombination matter as shown in Figure 2 Grain.

(4) recombinant slow virus constructs

PWPXLd-CAR-CD19 recombinant plasmid, packaging plasmid psPAX2, envelope plasmid pMD2G three cotransfection are entered into training The HEK293T cell supported.48h harvest is protected in -80 DEG C of ultra low temperature freezers containing the supernatant of virus through 0.45 μm of membrane filtration It deposits；Supernatant of the 72h aftercrop containing virus, 0.45 μm of membrane filtration merge with the viral supernatants of 48h harvest and are added together It in ultracentrifugation pipe, is put into Beckman ultracentrifuge one by one, setting parameter of noncentricity is 25000rpm, and centrifugation time is 2h, centrifuging temperature are controlled at 4 DEG C；It after centrifugation, discards supernatant, removal as far as possible remains in the liquid on tube wall, and virus is added Liquid is saved, gently piping and druming is resuspended repeatedly；Through after completely dissolution, high speed centrifugation 10000rpm takes supernatant fluorescence method after being centrifuged 5min Titre is measured, by virus according to 100 μ L, 2 × 10⁸TU/mL packing, is stored in -80 DEG C of ultra low temperature freezers, obtains band CAR- The first recombinant slow virus of Mesothelin.

PWPXLd-CAR-CD19 recombinant plasmid, packaging plasmid psPAX2, envelope plasmid pMD2G three cotransfection are entered into training The HEK293T cell supported.48h harvest is protected in -80 DEG C of ultra low temperature freezers containing the supernatant of virus through 0.45 μm of membrane filtration It deposits；Supernatant of the 72h aftercrop containing virus, 0.45 μm of membrane filtration merge with the viral supernatants of 48h harvest and are added together It in ultracentrifugation pipe, is put into Beckman ultracentrifuge one by one, setting parameter of noncentricity is 25000rpm, and centrifugation time is 2h, centrifuging temperature are controlled at 4 DEG C；It after centrifugation, discards supernatant, removal as far as possible remains in the liquid on tube wall, and virus is added Liquid is saved, gently piping and druming is resuspended repeatedly；Through after completely dissolution, high speed centrifugation 10000rpm takes supernatant fluorescence method after being centrifuged 5min Titre is measured, by virus according to 100 μ L, 2 × 10⁸TU/mL packing, is stored in -80 DEG C of ultra low temperature freezers, obtains band CAR-CD19 The second recombinant slow virus.

(5) preparation of the antitumor T cell of targeting

A) separation of PBMC (peripheral blood mononuclear cells)

PBMC is from autologous vein blood, autologous bone marrow, Cord blood and placental blood etc..Preferably derive from cancer patient's hand The fresh peripheral blood or marrow acquired after art one month, after chemicotherapy one month.

Extract patient blood, sample presentation to blood separating chamber；Peripheral blood mononuclear cells is acquired, is taken after Ficoll centrifuge separation Intermediate layer cell；After PBS is washed, PBMC is obtained.

B) immunomagnetic beads antigenspecific T lymphocyte

Above-mentioned PBMC is taken, the basal medium for being free of serum is added, is made into cell suspension；Ratio in magnetic bead and cell is 3:1, is added CD3/CD28 immunomagnetic beads, and room temperature incubates 1-2h；The cell for being incubated for magnetic bead is screened using magnet；PBS is washed It washs, after removing immunomagnetic beads, obtains CD3 positive t lymphocytes.

C) virus transfection method prepares antigenspecific T lymphocyte

The above-mentioned CD3 positive t lymphocytes obtained by magnetic activated cell seperation are taken, while being added and CD3 positive cell The first recombinant slow virus with CAR-Mesothelin and the disease of the second recombinant lentiviral with CAR-CD19 of the corresponding virus titer of number Poison carries out co-incubation, wherein the ratio between the first recombinant slow virus and the dosage (titre) of the second recombinant slow virus are 1:1.

The 3rd day of culture carries out cell count and changes liquid, and adjustment cell concentration is 1 × 10⁶A/mL is inoculated with, culture；Training Cell state is observed in feeding the 5th day, if cell density increases, diluting cells concentration is 1 × 10⁶A/mL detects cell Activity continues to cultivate.Amplification cultivation collected cell, and obtained the antitumor T cell of targeting, and save it in by the 9-11 days It feeds back in dedicated cells frozen storing liquid.

Effect example

Effect example one: the tumor cell in vitro of the assessment antitumor T cell of targeting of the present invention kills situation

By the antitumor T cell of targeting made from the embodiment of the present invention one (experimental group) and without the T lymphocyte of preparation (negative control group), the T cell (independent group of Mesothelin CAR-T) with individual CAR-Mesothelin and have The T cell (independent group of CD19CAR-T) of individual CAR-CD19 is compared, in vitro that above-mentioned four groups of effector cells and target is thin Born of the same parents' (Panc1 cell) in quantity than the ratio for 1:10,1:3,1:1,3:1 and 10:1, in 37 DEG C, 5%CO₂Under carry out total training It supports, after incubation 15-18 hours, collects cell, carry out streaming dyeing, detect cell killing situation.As a result, it has been found that passing through The tumor-killing power of the antitumor T cell of targeting provided by the invention is significantly larger than other control groups, this explanation is through present invention side The antitumor T cell of the targeting tumor-killing ability with super strength of method preparation.

Effect example two, assess the antitumor T cell of targeting of the present invention to mouse interior tumor cell killing situation

The antitumor T cell of the targeting of preparation made from the embodiment of the present invention one (experimental group) and the T without preparation are drenched Bar cell (negative control group), the T cell (independent group of Mesothelin CAR-T) with individual CAR-Mesothelin with And the T cell (independent group of CD19CAR-T) with individual CAR-CD19 gives every mouse tail in mouse pancreas cancer model Intravenous injection 1 × 10⁶A cell (n=9), obtains the survivorship curve of mouse.As a result, it has been found that targeting provided by the invention is anti-swollen Tumor T cell remains to keep the survival rate of mouse more stable and still higher in injection Mice Body after a period of time, and considerably beyond yin Property control group and independent group of two above.This shows that the antitumor T cell of targeting provided can be protected mice against preferably It is dead due to tumour.

The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.

Sequence table

<110>Shenzhen Bin De Bioisystech Co., Ltd

<120>antitumor T cell of a kind of targeting and its preparation method and application

<160> 22

<170> SIPOSequenceListing 1.0

<210> 1

<211> 265

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 1

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

1 5 10 15

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

20 25 30

Gly Pro Glu Leu Glu Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys

35 40 45

Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln

50 55 60

Ser His Gly Lys Ser Leu Glu Trp Ile Gly Leu Ile Thr Pro Tyr Asn

65 70 75 80

Gly Ala Ser Ser Tyr Asn Gln Lys Phe Arg Gly Lys Ala Thr Leu Thr

85 90 95

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

100 105 110

Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Gly Gly Tyr Asp Gly

115 120 125

Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser Asp

145 150 155 160

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

165 170 175

Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His

180 185 190

Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp

195 200 205

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

210 215 220

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

225 230 235 240

Asp Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Lys His Pro Leu Thr Phe

245 250 255

Gly Ala Gly Thr Lys Leu Glu Ile Lys

260 265

<210> 2

<211> 242

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 2

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

<210> 3

<211> 795

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

atggttctgc tggtgacatc tctcctgctc tgtgaactgc ctcatcccgc ttttctgctc 60

attcccgaca ttcagcaggt ccagctccag cagtctggcc ctgaactcga aaaacctggc 120

gctagcgtga aaatttcctg taaagcctcc ggctactctt ttactggcta cacaatgaat 180

tgggtgaaac agtctcacgg caaatccctc gaatggatcg gactcatcac accctacaat 240

ggcgcctctt cctacaacca gaaattccgg ggcaaggcaa cactcactgt ggacaaatca 300

tcctctaccg cctacatgga tctgctctcc ctcacatctg aggactccgc tgtctacttt 360

tgtgcccgag gaggatacga cggacgagga ttcgattact ggggacaggg aacaactgtg 420

accgtgtcta gtggcggcgg agggagtgga ggcggaggat cttctggcgg gggatccgat 480

attgaactca cacagtctcc cgctatcatg tctgcttctc ccggcgagaa agtgactatg 540

acttgctctg cttcctcttc tgtgtcctac atgcactggt accagcagaa atctggcaca 600

tcccctaaac ggtggatcta cgatactagc aaactggcat ccggcgtgcc tgggcgattc 660

tctggctctg gctctggcaa ctcttactct ctcacaatct catctgtcga ggctgaggac 720

gatgccacat actactgtca gcagtggtct aaacacccac tcacattcgg cgctggcact 780

aaactggaaa taaaa 795

<210> 4

<211> 726

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

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> 5

<211> 488

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 5

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

1 5 10 15

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

20 25 30

Gly Pro Glu Leu Glu Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys

35 40 45

Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln

50 55 60

Ser His Gly Lys Ser Leu Glu Trp Ile Gly Leu Ile Thr Pro Tyr Asn

65 70 75 80

Gly Ala Ser Ser Tyr Asn Gln Lys Phe Arg Gly Lys Ala Thr Leu Thr

85 90 95

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

100 105 110

Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Gly Gly Tyr Asp Gly

115 120 125

Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser Asp

145 150 155 160

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

165 170 175

Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His

180 185 190

Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp

195 200 205

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

210 215 220

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

225 230 235 240

Asp Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Lys His Pro Leu Thr Phe

245 250 255

Gly Ala Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

His Met Gln Ala Leu Pro Pro Arg

485

<210> 6

<211> 465

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 6

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Arg

465

<210> 7

<211> 508

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 7

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

1 5 10 15

Ala Ala Arg Pro Met Val Leu Leu Val Thr Ser Leu Leu Leu Cys Glu

20 25 30

Leu Pro His Pro Ala Phe Leu Leu Ile Pro Asp Ile Gln Gln Val Gln

35 40 45

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

50 55 60

Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn

65 70 75 80

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

85 90 95

Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe Arg Gly Lys

100 105 110

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

115 120 125

Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Gly

130 135 140

Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val

145 150 155 160

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

165 170 175

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

180 185 190

Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val

195 200 205

Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg

210 215 220

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

225 230 235 240

Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Val

245 250 255

Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Lys His

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505

<210> 8

<211> 485

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 8

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Ala Leu Pro Pro Arg

485

<210> 9

<211> 45

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 9

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

35 40 45

<210> 10

<211> 135

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 11

<211> 24

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 11

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

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 12

<211> 72

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 13

<211> 42

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 13

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

1 5 10 15

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

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 14

<211> 126

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 15

<211> 112

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 15

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 16

<211> 336

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 17

<211> 1464

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 17

atggttctgc tggtgacatc tctcctgctc tgtgaactgc ctcatcccgc ttttctgctc 60

attcccgaca ttcagcaggt ccagctccag cagtctggcc ctgaactcga aaaacctggc 120

gctagcgtga aaatttcctg taaagcctcc ggctactctt ttactggcta cacaatgaat 180

tgggtgaaac agtctcacgg caaatccctc gaatggatcg gactcatcac accctacaat 240

ggcgcctctt cctacaacca gaaattccgg ggcaaggcaa cactcactgt ggacaaatca 300

tcctctaccg cctacatgga tctgctctcc ctcacatctg aggactccgc tgtctacttt 360

tgtgcccgag gaggatacga cggacgagga ttcgattact ggggacaggg aacaactgtg 420

accgtgtcta gtggcggcgg agggagtgga ggcggaggat cttctggcgg gggatccgat 480

attgaactca cacagtctcc cgctatcatg tctgcttctc ccggcgagaa agtgactatg 540

acttgctctg cttcctcttc tgtgtcctac atgcactggt accagcagaa atctggcaca 600

tcccctaaac ggtggatcta cgatactagc aaactggcat ccggcgtgcc tgggcgattc 660

tctggctctg gctctggcaa ctcttactct ctcacaatct catctgtcga ggctgaggac 720

gatgccacat actactgtca gcagtggtct aaacacccac tcacattcgg cgctggcact 780

aaactggaaa taaaaaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 840

gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg 900

cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact 960

tgtggggtcc ttctcctgtc actggttatc accctttact gcaaacgggg cagaaagaaa 1020

ctcctgtata tattcaaaca accatttatg agaccagtac aaactactca agaggaagat 1080

ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgag agtgaagttc 1140

agcaggagcg cagacgcccc cgcgtacaag cagggccaga accagctcta taacgagctc 1200

aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1260

atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 1320

gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1380

gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 1440

cacatgcagg ccctgccccc tcgc 1464

<210> 18

<211> 1395

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 18

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

tcctcaacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 780

cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 840

gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 900

cttctcctgt cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat 960

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1020

tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1080

gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 1140

cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1200

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1260

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1320

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1380

gccctgcccc ctcgc 1395

<210> 19

<211> 1524

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 19

gccttaccag tgaccgcctt gctcctgccg ctggccttgc tgctccacgc cgccaggccg 60

atggttctgc tggtgacatc tctcctgctc tgtgaactgc ctcatcccgc ttttctgctc 120

attcccgaca ttcagcaggt ccagctccag cagtctggcc ctgaactcga aaaacctggc 180

gctagcgtga aaatttcctg taaagcctcc ggctactctt ttactggcta cacaatgaat 240

tgggtgaaac agtctcacgg caaatccctc gaatggatcg gactcatcac accctacaat 300

ggcgcctctt cctacaacca gaaattccgg ggcaaggcaa cactcactgt ggacaaatca 360

tcctctaccg cctacatgga tctgctctcc ctcacatctg aggactccgc tgtctacttt 420

tgtgcccgag gaggatacga cggacgagga ttcgattact ggggacaggg aacaactgtg 480

accgtgtcta gtggcggcgg agggagtgga ggcggaggat cttctggcgg gggatccgat 540

attgaactca cacagtctcc cgctatcatg tctgcttctc ccggcgagaa agtgactatg 600

acttgctctg cttcctcttc tgtgtcctac atgcactggt accagcagaa atctggcaca 660

tcccctaaac ggtggatcta cgatactagc aaactggcat ccggcgtgcc tgggcgattc 720

tctggctctg gctctggcaa ctcttactct ctcacaatct catctgtcga ggctgaggac 780

gatgccacat actactgtca gcagtggtct aaacacccac tcacattcgg cgctggcact 840

aaactggaaa taaaaaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 900

gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg 960

cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact 1020

tgtggggtcc ttctcctgtc actggttatc accctttact gcaaacgggg cagaaagaaa 1080

ctcctgtata tattcaaaca accatttatg agaccagtac aaactactca agaggaagat 1140

ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgag agtgaagttc 1200

agcaggagcg cagacgcccc cgcgtacaag cagggccaga accagctcta taacgagctc 1260

aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1320

atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 1380

gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1440

gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 1500

cacatgcagg ccctgccccc tcgc 1524

<210> 20

<211> 1455

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 20

gccttaccag tgaccgcctt gctcctgccg ctggccttgc tgctccacgc cgccaggccg 60

gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 120

atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 180

gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 240

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 300

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 360

gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 420

ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 480

tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 540

cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 600

tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 660

ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 720

tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 780

tcctcaacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 840

cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 900

gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 960

cttctcctgt cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat 1020

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1080

tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1140

gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 1200

cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1260

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1320

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1380

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1440

gccctgcccc ctcgc 1455

<210> 21

<211> 20

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 21

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

1 5 10 15

Ala Ala Arg Pro

20

<210> 22

<211> 60

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 22

gccttaccag tgaccgcctt gctcctgccg ctggccttgc tgctccacgc cgccaggccg 60

Claims

1. a kind of antitumor T cell of targeting, which is characterized in that the antitumor T cell of targeting is band targeting Pair of the Chimeric antigen receptor CAR-Mesothelin of Mesothelin and the Chimeric antigen receptor CAR-CD19 with targeting CD19 Target spot Chimeric antigen receptor T cell, or for the Chimeric antigen receptor T cell with the CAR-Mesothelin and with described The mixing of the Chimeric antigen receptor T cell of CAR-CD19, or it is thin for the Chimeric antigen receptor T with the CAR-Mesothelin Born of the same parents and Chimeric antigen receptor T cell with the CAR-CD19 it is one or more with double target spot Chimeric antigen receptor T The mixing of cell；Wherein, the CAR-Mesothelin includes the sequentially connected targeting from aminoterminal to c-terminus The single-chain antibody of Mesothelin, extracellular hinge area, transmembrane region and intracellular signal area amino acid sequence, the CAR-CD19 packet Include single-chain antibody, extracellular hinge area, transmembrane region and the intracellular signal area of the sequentially connected targeting CD19 from aminoterminal to c-terminus Amino acid sequence；

Wherein, the amino acid sequence of the single-chain antibody of the targeting Mesothelin includes the amino as shown in SEQ ID NO:1 The amino acid sequence of acid sequence, the single-chain antibody of the targeting CD19 includes the amino acid sequence as shown in SEQ ID NO:2.

2. the antitumor T cell of targeting as described in claim 1, which is characterized in that the targeting Mesot helin's is single-stranded The encoding gene of antibody includes the nucleotide sequence as shown in SEQ ID NO:3, the coding of the single-chain antibody of the targeting CD19 Gene includes the nucleotide sequence as shown in SEQ ID NO:4.

3. the antitumor T cell of targeting as described in claim 1, which is characterized in that the amino of the CAR-Mesot helin Acid sequence includes the amino acid sequence as shown in SEQ ID NO:5, and the amino acid sequence of the CAR-CD19 includes such as SEQ ID Amino acid sequence shown in NO:6.

4. a kind of recombinant viral vector, which is characterized in that including the antitumor T of targeting as described in any one of claims 1-3 The encoding gene of CAR-Mesothelin described in cell and/or CAR-CD19.

5. a kind of host cell, which is characterized in that the host cell includes recombinant viral vector as claimed in claim 4.

6. a kind of preparation method of the antitumor T cell of targeting characterized by comprising

(1) encoding gene and targeting CD19 of the Chimeric antigen receptor CAR-Mesothelin of targeting Mesothelin are provided respectively Chimeric antigen receptor CAR-CD19 encoding gene；

The encoding gene of the CAR-Mesothelin includes encoding gene, the targeting that signal peptide is sequentially connected with from 5 ' ends to 3 ' ends The encoding gene of the single-chain antibody of Mesothelin, the encoding gene of extracellular hinge area, transmembrane region encoding gene and letter intracellular The encoding gene in number area；The encoding gene of the CAR-CD19 includes the coding base that signal peptide is sequentially connected with from 5 ' ends to 3 ' ends Because, the encoding gene and letter intracellular of the targeting encoding gene of single-chain antibody of CD19, the encoding gene of extracellular hinge area, transmembrane region The encoding gene in number area；

Wherein, the encoding gene of the single-chain antibody of the targeting Mesothelin includes the amino acid as shown in SEQ ID NO:1 The encoding gene of nucleotide sequence corresponding to sequence, the single-chain antibody of the targeting CD19 includes as shown in SEQ ID NO:2 Amino acid sequence corresponding to nucleotide sequence；

(2) encoding gene of the encoding gene of the CAR-Mesothelin and the CAR-CD19 is inserted respectively into pWPXLD In carrier, pWPXLD-CAR-Mesothelin recombinant plasmid and pWPXLD-CAR-CD19 recombinant plasmid are obtained；

(3) respectively to the pWPXLD-CAR-Mesothelin recombinant plasmid and the pWPXLD-CAR-CD19 recombinant plasmid into Row packaging, obtains the first recombinant slow virus with CAR-Mesothelin encoding gene and the with CAR-CD19 encoding gene Two recombinant slow virus；

(4) by first recombinant slow virus and second recombinant slow virus, separately or simultaneously co-transfection CD3 positive T drenches Bar cell obtains the antitumor T cell of targeting through separation.

7. the preparation method of the antitumor T cell of targeting as claimed in claim 6, which is characterized in that the CAR- The encoding gene of Mesothelin includes the nucleotide sequence as shown in SEQ ID NO:7, the encoding gene of the CA R-CD19 Including the nucleotide sequence as shown in SEQ ID NO:8.

8. the preparation method of the antitumor T cell of targeting as claimed in claim 6, which is characterized in that described in step (4) The ratio between first recombinant slow virus and the virus titer of the first recombinant slow virus are 1:(0.5-2).

9. the preparation method of the antitumor T cell of targeting as claimed in claim 6, which is characterized in that the targeting is anti-swollen Tumor T cell is double target spot Chimeric antigen receptor T cells with the CAR-Mesothelin and CAR-CD19, or is band The Chimeric antigen receptor T cell of the CAR-Mesothelin and Chimeric antigen receptor T cell with the CAR-CD19 it is mixed It closes, or for described in double target spot Chimeric antigen receptor T cells with the CAR-Mesothelin and CAR-CD19, band The mixing of the Chimeric antigen receptor T cell of CAR-Mesothelin and the Chimeric antigen receptor T cell with the CAR-CD19.

10. recombinant viral vector as claimed in claim 4, host cell as claimed in claim 5, such as claim 1-3 The antitumor T of targeting made from the antitumor T cell of described in any item targetings or preparation method as described in claim 6-9 Application of the cell in the drug that preparation diagnoses and/or treats malignant tumour.