CN110157678A

CN110157678A - A kind of targeting T lymphocyte and its preparation method and application

Info

Publication number: CN110157678A
Application number: CN201810148376.5A
Authority: CN
Inventors: 张宏玲; 龙丽梅
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: 2019-08-23

Abstract

The present invention provides a kind of targeting T lymphocytes, including targeting the Chimeric antigen receptor CAR-CD33 of CD33 and/or targeting the Chimeric antigen receptor CAR-CD123 of CD123, it is wide to the targets identification of tumour cell and strong, there is target spot escape in avoidable tumour cell, efficiently and specifically killing tumor cell, it is lethal to expand its wide spectrum.The present invention also provides the preparation method and application of the targeting T lymphocyte.

Description

A kind of targeting T lymphocyte and its preparation method and application

Technical field

The present invention relates to field of medical biotechnology, in particular to a kind of targeting T lymphocyte and its preparation method and application.

Background technique

Leukaemia is a kind of malignant disease of hemopoietic system, can be divided into lymphocytic leukemia, marrow by sick cell series It is leukaemia, cell mixing leukaemia, higher in the death rate of each age group malignant tumour in China, especially acute myeloid is white Blood disease (AML) is most common, the highest quasi-leukemia of the death rate.CAR-T (Chimeric antigen receptor T cell) technology is a kind of Novel immune cell therapy, it is to activate self immune system for human body is fed back to by the T cell of CAR transformation, thin to tumour Born of the same parents kill, to achieve the purpose that remove malignant cell.

Although CAR-T technology achieves significant curative effect in the hematological system tumors such as leukaemia, myeloma, lymthoma, Identification, killing energy but in complicated tumor microenvironment (such as kinds of tumors antigen occurs simultaneously), to tumour cell Power substantially reduces, and limits its clinical application.Therefore, it is necessary to CAR-T technology stronger to tumour cell targeting is provided, with In treatment for malignant tumours such as leukaemia.

Summary of the invention

In consideration of it, the present invention provides a kind of for targeting T lymphocyte of marrow series leukemia and preparation method thereof and Using.The T lymphocyte can be targeted to two tumor targets of marrow series leukemia cell, knowledge high to the targeting of tumour cell Other property is relatively wide and strong, and target spot escape occurs in avoidable tumour cell, and the wide spectrum for expanding the T cell is lethal.

In a first aspect, the present invention provides a kind of targeting T lymphocyte, the Chimeric antigen receptor including targeting CD33 CAR-CD33 and/or target CD123 Chimeric antigen receptor CAR-CD123, wherein the CAR-CD33 include from aminoterminal to The sequentially connected targeting single-chain antibody of CD33 of c-terminus, extracellular hinge area, transmembrane region and intracellular signal area amino acid sequence, The CAR-CD123 include from aminoterminal to c-terminus the sequentially connected targeting single-chain antibody of CD123, extracellular hinge area, across The amino acid sequence in film area and intracellular signal area；Wherein, the amino acid sequence of the single-chain antibody of the targeting CD33 includes such as SEQ The amino acid sequence of amino acid sequence shown in ID NO:1, the single-chain antibody of the targeting CD123 includes such as SEQ ID NO:2 Shown in amino acid sequence.

Preferably, the targeting T lymphocyte can be double target spot inosculating antibodies with CAR-CD33 and CAR-CD123 Original receptor T cell (that is, double target spot Chimeric antigen receptor T cells of targeting CD33 and CD123), or with CAR-CD33's The mixing of Chimeric antigen receptor T cell and the Chimeric antigen receptor T cell with CAR-CD123, or for band CAR-CD33 and Double target spot Chimeric antigen receptor T cells of CAR-CD123, the Chimeric antigen receptor T cell with CAR-CD33 and with CAR-CD123 Chimeric antigen receptor T cell these three mixing.

At this point, the targeting T lymphocyte, can both identify that surface expression had the tumour cell of CD33 antigen protein, It can also identify that surface expression has the tumour cell of CD123 antigen protein, it is certainly anti-to having CD33 antigen protein and CD123 simultaneously The identity of the tumour cell of former albumen is also preferable, and tumour cell can effectively be avoided the generation of CD33 immunologic escape occur.

Wherein, it when the targeting T lymphocyte is double target spot CAR-T with CAR-CD33 and CAR-CD123, is fitted into The distributing position of antigen receptor CAR-CD33 and CAR-CD123 are not construed as limiting, can be and be alternately distributed (such as ABAB ..., AABABB ...) or it is sequentially distributed (such as AAAA ... BBB ...), but covalent linkage is had no between the two, they are corresponding at this time Also covalent linkage is had no between CD33 single-chain antibody and CD123 single-chain antibody, them can be made to keep preferable recognition capability in this way.

Above-mentioned " being sequentially connected with from aminoterminal to c-terminus " specifically: the single-chain antibody of the targeting CD33 or the targeting The c-terminus of the amino acid sequence of the single-chain antibody of CD123 is connected with the aminoterminal of the amino acid sequence of the extracellular hinge area, 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, it is described across The c-terminus of the amino acid sequence in film area is connected with the aminoterminal of the amino acid sequence in the intracellular signal area.

The above-mentioned CD33 referred to is myeloid cell differentiation antigen, and the Patients with Acute Myeloid Leukemia 90% or more has Expression is the good target spot of marrow series leukemia treatment.But there are also marrow series leukemia cells not to express CD33.And CD123 It is the related antigen of acute myeloid leukemia, height is expressed in leukemic stem cells, low or be not expressed in normal haematopoetic.Cause This, above-mentioned targeting T lymphocyte can target CD23 the and CD123 target spot on the tumour cells such as marrow series leukemia cell, Marrow series leukemia cell can be avoided the escape of CD23 feminine gender or the escape of CD123 feminine gender occur, effectively to expand its identification, killing model It encloses.

Optionally, the encoding gene of the single-chain antibody of the targeting CD33 includes the nucleotide as shown in SEQ ID NO:3 The encoding gene of sequence, the single-chain antibody of the targeting CD123 includes the nucleotide sequence as shown in SEQ ID NO:4.This hair It is bright it is described targeting CD33 single-chain antibody remain with the affine activity to CD33 antigen, can efficient identification surface expression have CD33 The tumour cell of antigen.Similarly, the single-chain antibody of the targeting CD123 also remains with the affine activity to CD123 antigen, energy Enough efficient identification surface expressions have the tumour cell of CD123 antigen.

Optionally, the encoding gene of the amino acid sequence of the single-chain antibody of the targeting CD33 should consider degeneracy base, I.e. the encoding gene of the amino acid sequence as shown in SEQ ID NO:1 includes the nucleotide sequence as shown in SEQ ID NO:3, is protected Shield range should also protect the nucleotide sequence for having base degeneracy matter with SEQ ID NO:3, these nucleotide sequences are corresponding Amino acid sequence remain as SEQ ID NO:1.The encoding gene of the amino acid sequence of the single-chain antibody of the targeting CD123 is same Sample should consider degeneracy base.

In the present invention, the extracellular hinge area in the CAR-CD33 is used to promote the single-chain antibody of the targeting CD33 and swells CD33 on oncocyte is combined；Similarly, the extracellular hinge area in the CAR-CD123 is used to promote the targeting CD123's Single-chain antibody is in conjunction with the CD123 on tumour cell.

Optionally, the extracellular hinge area include CD8 α hinge area, CD28 hinge area, CD4 hinge area, CD5 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-CD33 is used to fix the Chimeric antigen receptor CAR- of the targeting CD33 CD33；Similarly, the transmembrane region in the CAR-CD123 is used to fix the Chimeric antigen receptor CAR- of the targeting CD123 CD123。

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 letter Number area, CD27 signaling zone, OX40 signaling zone, CD28 signaling zone, IL1R1 signaling zone, CD70 signaling zone, TNFRSF19L signaling zone One of or a variety of combinations.

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 such as SEQ ID NO:13 Shown in amino acid sequence.

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 in the present invention, extracellular hinge area, transmembrane region and intracellular signal area in the CAR-CD33 Amino acid sequence, with extracellular hinge area corresponding in the CAR-CD123, the corresponding amino acid sequence of transmembrane region and intracellular signal area Column may be the same or different.

In an embodiment of the present invention, the amino acid sequence of the CAR-CD33 includes as shown in SEQ ID NO:5 Amino acid sequence.

Optionally, the encoding gene of the CAR-CD33 includes the nucleotide sequence as shown in SEQ ID NO:17.

Optionally, the encoding gene of the CAR-CD33 should consider degeneracy base, the i.e. ammonia as shown in SEQ ID NO:5 The encoding gene of base acid sequence includes the nucleotide sequence as shown in SEQ ID NO:17, and protection scope should also protect and SEQ ID NO:17 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:5。

In an embodiment of the present invention, the amino acid sequence of the CAR-CD123 includes as shown in SEQ ID NO:6 Amino acid sequence.

Optionally, the encoding gene of the CAR-CD123 includes the nucleotide sequence as shown in SEQ ID NO:18.

Optionally, the encoding gene of the CAR-CD123 should consider degeneracy base, i.e., as shown in SEQ ID NO:6 The encoding gene of amino acid sequence include the nucleotide sequence such as SEQ ID NO:18 shown in, 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。

The targeting T lymphocyte that first aspect present invention provides, the Chimeric antigen receptor CAR- including targeting CD33 The CD33 and/or Chimeric antigen receptor CAR-CD123 for targeting CD123, can identify two tumor targets, enhance to tumour cell Identification, avoid tumour cell from escaping.As CAR-CD33 and/or CAR-CD123 and antigen protein knot corresponding on tumour cell After conjunction, the intracellular signal area of the targeting T lymphocyte is activated, promote T cell patient's body amplification, and efficiently and Specifically killing tumor cell, especially expresses the tumour cell of CD33 and/or CD123, and the wide spectrum for expanding the T cell kills Wound property.In addition, CAR-CD33 and CAR-CD123 can also preferably destroy tumor microenvironment, mutual humidification makes target tumor Targeting T lymphocyte preferably function.Since the single-chain antibody of the CD33 and CD123 is anti-for Humanized single chain Body, this makes the targeting T cell be avoided the immune response for causing human organism, maintains ability, such as activity in body with lasting And lethality.

Targeting T lymphocyte of the present invention with efficient identification and can kill expression and have the tumour of CD33 and/or CD123 Cell, such as the tumour cell of marrow series leukemia, it is specific such as KG1, Kasumi-1 and THP-1 tumor cell line.

Second aspect, the present invention provides a kind of recombinant viral vectors, including targeting T lymph as described in relation to the first aspect The encoding gene of CAR-CD33 described in cell and/or CAR-CD123.

Optionally, the encoding gene of CAR-CD33 and the encoding gene of CAR-CD123 are contained when the recombinant viral vector When, on the recombinant viral vector between the encoding gene of the CAR-CD33 and the encoding gene of the CAR-CD123, also Including a special sequence, which is used to make the encoding gene of the CAR-CD33 encoding gene and the CAR-CD123 Available two independent PROTEIN C AR-CD33 and CAR-CD123 after transcription and translation.It is carried using such recombinant virus After body transfects CD3 positive t lymphocytes, resulting targeting T lymphocyte is double target spots with CAR-CD33 and CAR-CD123 Chimeric antigen receptor T cell.Still optionally further, the special sequence can be RBS sequence, IRES sequence, T2A sequence or its His Protease sequences etc..

Optionally, the recombinant viral vector has the encoding gene of the CAR-CD33, or has the CAR- The encoding gene of CD123.It is preferred that using the recombinant viral vector of CAR-CD33 encoding gene and with CAR-CD123 encoding gene Targeting T as described in the first aspect of the invention can be obtained in recombinant viral vector separately or simultaneously co-infection T lymphocyte Lymphocyte, and efficiency of infection is higher, gained targeting T lymphocyte can more fully hereinafter express CAR-CD33 and/or CAR- CD123。

Preferably, the encoding gene of the CAR-CD33 includes the nucleotide sequence as shown in SEQ ID NO:19.Such as SEQ Nucleotide sequence shown in ID NO:19 is compared with the nucleotide sequence as shown in SEQ ID NO:17, more companies described below Connect the encoding gene of peptide.The encoding gene of the signal peptide can be expressed with Chimeric antigen receptor CAR-CD33 described in guide To cell surface.

Preferably, the encoding gene of the CAR-CD123 includes the nucleotide sequence as shown in SEQ ID NO:20.Such as Nucleotide sequence shown in SEQ ID NO:20 is how described below compared with the nucleotide sequence as shown in SEQ ID NO:18 Link peptide encoding gene.The encoding gene of the signal peptide can be with Chimeric antigen receptor CAR- described in guide CD123 reaches 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-CD33 and/or CAR-CD123 therein can be inserted into host genome by genetic recombination, obtain Targeting T lymphocyte is stated, 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 targeting T lymphocyte, comprising:

(1) encoding gene of Chimeric antigen receptor CAR-CD33 and being fitted into for targeting CD123 of targeting CD33 are provided respectively The encoding gene of antigen receptor CAR-CD123；

The encoding gene of the CAR-CD33 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 CD33, the encoding gene of extracellular hinge area, the encoding gene of transmembrane region and intracellular signal area Encoding gene；The encoding gene of the CAR-CD123 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 CD123, the encoding gene of extracellular hinge area, the encoding gene of transmembrane region and intracellular signal area Encoding gene；

Wherein, the encoding gene of the single-chain antibody of the targeting CD33 includes the amino acid sequence as shown in SEQ ID NO:1 The encoding gene of the corresponding nucleotide sequence of column, the single-chain antibody of the targeting CD123 includes as shown in SEQ ID NO:2 Nucleotide sequence corresponding to amino acid sequence；

(2) encoding gene of the encoding gene of the CAR-CD33 and the CAR-CD123 is inserted respectively into pWPXLD In carrier, pWPXLD-CAR-CD33 recombinant plasmid and pWPXLD-CAR-CD123 recombinant plasmid are obtained；

(3) respectively to the pWPXLD-CAR-CD33 recombinant plasmid and the pWPXLD-CAR-CD123 recombinant plasmid into Row packaging, obtains the first recombinant slow virus with CAR-CD33 encoding gene and the second weight with CAR-CD123 encoding gene Group 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 targeting T lymphocyte.

It is above-mentioned " being sequentially connected with from 5 ' ends to 3 ' ends " specifically: the signal peptide by taking the encoding gene of CAR-CD33 as an example Coding gene sequence 3 ' end with it is described targeting CD33 single-chain antibody encoding gene 5 ' hold be connected, the targeting CD33 The 3 ' ends of encoding gene of single-chain antibody hold and be connected with the 5 ' of the extracellular hinge area encoding gene, the extracellular hinge area 3 ' ends of encoding gene are connected with 5 ' ends of the encoding gene of the transmembrane region, 3 ' ends of the encoding gene of the transmembrane region and institute 5 ' the ends for stating the encoding gene in intracellular signal area are connected.

In the present invention, the signal peptide is arrived for instructing the Chimeric antigen receptor CAR-CD33 or CAR-CD123 to express Cell surface, the signal peptide are cut in protein translation maturation by signal peptidase.And in the encoding gene of CAR-CD33 The amino acid sequence of signal peptide and signal peptide in the encoding gene of the CAR-CD123 may be the same or different, can also be with Be their signal peptide amino acid sequence it 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-CD33 includes as amino acid sequence shown in SEQ ID NO:7 is corresponding Nucleotide sequence.

Optionally, the encoding gene of the CAR-CD33 includes the nucleotide sequence as shown in SEQ ID NO:19.Certainly, The encoding gene of the CAR-CD33 may also comprise the nucleotide for having base degeneracy matter with sequence shown in SEQ ID NO:19 Sequence.

Optionally, the encoding gene of the CAR-CD123 includes as amino acid sequence shown in SEQ ID NO:8 is corresponding Nucleotide sequence.

Optionally, the encoding gene of the CAR-CD123 includes the nucleotide sequence as shown in SEQ ID NO:20.When So, the encoding gene of the CAR-CD123 may also comprise the core for having base degeneracy matter with sequence shown in SEQ ID NO:20 Nucleotide sequence.

By taking CAR-CD33 as an example, compared with SEQ ID NO:19 nucleotide sequence shown in the SEQ ID NO:17, Duo Liaolian Connect the encoding gene of peptide, but when Chimeric antigen receptor CAR-CD33 expression is to T cell surface, signal peptide is in protein translation maturation It is cut in the process by signal peptidase.Therefore, in amino acid sequence (the SEQ ID of the Chimeric antigen receptor CAR-CD33 translated into NO:5 in) and not with the amino acid sequence as shown in SEQ ID NO:21.The case where CAR-CD123, is similar therewith.

By taking CAR-CD33 as an example, the coding gene sequence of the CAR-CD33 is inserted into I He of BamH in pWPXLD carrier Between I restriction enzyme site of EcoR, and it is located at after the extension factor 1 α (EF1 α) of pWPXLD carrier, using EF1 α as promoter.It is described When the coding gene sequence of CAR-CD33 is inserted into pWPXLD carrier, 5 ' ends of the gene order of the CAR-CD33 can be also added Initiation codon (such as ATG) is connected with BamH1 restriction enzyme site in pWPXLD carrier, 3 ' end can also be added terminator codon with EcoR1 restriction enzyme site is connected in pWPXLD carrier.The case where CAR-CD123, is same.

Optionally, described " respectively to the pWPXLD-CAR-CD33 recombinant plasmid and the pWPXLD- in step (3) CAR-CD123 recombinant plasmid is packed, and obtains the first recombinant slow virus with CAR-CD33 encoding gene and with CAR- Second recombinant slow virus of CD123 encoding gene ", comprising:

By the pWPXLD-CAR-CD33 recombinant plasmid and envelope plasmid and packaging plasmid cotransfection host cell, obtain First recombinant slow virus；By the pWPXLD-CAR-CD123 recombinant plasmid and envelope plasmid and packaging plasmid cotransfection place Chief cell obtains second recombinant slow virus.

Using method preparation and reorganization plasmid of the present invention and packaging virus, wherein the pWPXLD-CAR-CD33 weight On group plasmid and pWPXLD-CAR-CD22 recombinant plasmid, it is excellent that the encoding gene of CAR-CD33 and CAR-CD22 have passed through codon Change, and molecular weight is suitable for, the packaging efficiency of recombinant slow virus is high, while viral concentration as made from host cell is higher. Correspondingly, when using the first recombinant slow virus and the second recombinant slow virus come co-transfection CD3 positive t lymphocytes, both The dosage of 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).

In the present invention, the targeting T lymphocyte being prepared includes with the CAR-CD33 and the CAR- Double target spot Chimeric antigen receptor T cells of CD123, the Chimeric antigen receptor T cell with the CAR-CD33 and with the CAR- At least one of Chimeric antigen receptor T cell of CD123.Optionally, the targeting T lymphocyte is with the CAR- Double target spot Chimeric antigen receptor T cells of the CD33 and CAR-CD123, or for the chimeric antigen with the CAR-CD33 by The mixing of body T cell and the Chimeric antigen receptor T cell with the CAR-CD123, or the inosculating antibody with the CAR-CD33 One or both of original receptor T cell and Chimeric antigen receptor T cell with the CAR-CD123 are chimeric with double target spots The mixing of antigen receptor T cell.

Preferably, the targeting T lymphocyte is chimeric for double target spots with the CAR-CD33 and CAR-CD123 Antigen receptor T cell, or it is chimeric for the Chimeric antigen receptor T cell with the CAR-CD33 and with the CAR-CD123 The mixing of antigen receptor T cell, or be double target spot Chimeric antigen receptor T with the CAR-CD33 and CAR-CD123 Cell, the Chimeric antigen receptor T cell with the CAR-CD33 and the Chimeric antigen receptor T cell with the CAR-CD123 Mixing.

At this point, the surface tool of targeting T lymphocyte there are two it is independent, not covalently bound Chimeric antigen receptor ( Be there are two independent single-chain antibody), do not influence they to the identification of respective target, combine, can simultaneously, efficiently identify it is swollen CD33 and CD123 target on oncocyte.The targeting T lymphocyte is to one or two kinds of in expression CD33 and CD123 Tumour cell can identify and kill, and target spot escape occurs in avoidable tumour cell, improve the range of its targets identification and strong Degree, and killing broad spectrum activity, also have stronger tumor-killing ability under complicated tumor microenvironment.

In another embodiment of the present invention, when required targeting T lymphocyte is the inosculating antibody with the CAR-CD33 When the mixing of original receptor T cell and Chimeric antigen receptor T cell with the CAR-CD123, it can also make in the following ways : using above-mentioned first recombinant slow virus transfect CD3 positive t lymphocytes, obtain the chimeric antigen with the CAR-CD33 by Body T cell；CD3 positive t lymphocytes are transfected using above-mentioned second recombinant slow virus, are obtained chimeric with the CAR-CD123 Antigen receptor T cell；Then both Chimeric antigen receptor T cells are mixed.

In the preparation method for the targeting T lymphocyte that fourth aspect present invention provides, base is encoded using band CAR-CD33 First recombinant slow virus of cause and the second recombinant slow virus with CAR-CD123 encoding gene either separately or simultaneously co-transfection CD3 positive t lymphocytes may make Chimeric antigen receptor CAR-CD33 and/or CAR- in targeting T lymphocyte obtained The expression efficiency of CD123 is higher, with the identification of preferable tumour, killing ability.

5th aspect, the present invention provides a kind of targeting T lymphocytes as described in the first aspect of the invention, such as this hair Recombinant viral vector described in bright second aspect, host cell as described in the third aspect of the present invention or such as fourth aspect present invention Application of the targeting T lymphocyte made from the preparation method in the drug that preparation diagnoses and/or treats malignant tumour.

Particularly, suitable for the malignant tumour for expressing CD33 and/or CD123.Such as anxious/chronic myelogenous leukemia etc. Diagnosing and treating.

The application can be with specifically: provides a kind of kit, the kit includes target as described in relation to the first aspect Tropism T lymphocyte or the targeting T lymphocyte transfected using the recombinant viral vector as described in second aspect are adopted The targeting T lymphocyte obtained by the preparation method as described in fourth aspect, recombination as described in respect of the second aspect of the invention One of viral vectors, host cell as described in the fourth aspect of the present invention are 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-CD33 recombinant plasmid provided in an embodiment of the present invention.

Fig. 2 is the plasmid map of pWPXLd-CAR-CD123 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 targeting T lymphocyte of embodiment one, comprising the following steps:

(1) gene order of the Chimeric antigen receptor CAR-CD33 of preparation targeting CD33

Prepare respectively signal peptide, target the single-chain antibody of CD33, CD8 α hinge area, CD8 transmembrane region, 4-1BB signaling zone and The encoding gene of CD3 ζ signaling zone, in the CAR-CD33, the encoding gene of signal peptide is as shown in SEQ ID NO:22, targeting The encoding gene of the single-chain antibody of CD33 is as shown in SEQ ID NO:3, the encoding gene of CD8 α hinge area such as SEQ ID NO:10 Shown, the encoding gene of the 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 CD33 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-CD33 Gene, the encoding gene of the CAR-CD33 is as shown in SEQ ID NO:19.

(2) gene order of the Chimeric antigen receptor CAR-CD123 of preparation targeting CD123

Prepare respectively signal peptide, target the single-chain antibody of CD123, 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-CD123 is as shown in SEQ ID NO:22, target To CD123 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 CD123 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-CD123 Gene, the encoding gene of the CAR-CD123 is as shown in SEQ ID NO:20.

(3) pWPXLd-CAR-CD33 recombinant plasmid and pWPXLd-CAR-CD123 recombinant plasmid are constructed

The encoding gene of CAR-CD33 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.When the encoding gene of the CAR-CD33 is inserted into pWPXLD carrier, institute I restriction enzyme site of BamH in initiation codon (such as ATG) and pWPXLD carrier can be added in the 5 ' ends for stating the encoding gene of CAR-CD33 It is connected, 3 ' ends can be added terminator codon (such as TAA) and be connected with I restriction enzyme site of EcoR in pWPXLD carrier.Then it is transferred to large intestine Bacillus competent cell DH5 α carries out positive colony PCR identification and sequencing identification.By PCR product detected through gel electrophoresis and survey Sequence identification meets target fragment size and sequence, successfully constructs pWPXLd-CAR-CD33 recombinant plasmid as shown in Figure 1.

The encoding gene of CAR-CD123 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-CD123 is inserted into pWPXLD When body, BamH I in initiation codon (such as ATG) and pWPXLD carrier can be added in 5 ' ends of the encoding gene of the CAR-CD123 Restriction 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 It is transferred to competent escherichia coli cell DH5 α, carries out positive colony PCR identification and sequencing identification.By PCR product gel electrophoresis Detection and sequencing identification meet target fragment size and sequence, successfully construct pWPXLd-CAR-CD123 recombination as shown in Figure 2 Plasmid.

(4) recombinant slow virus constructs

PWPXLd-CAR-CD123 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-CD33 The first recombinant slow virus.

PWPXLd-CAR-CD123 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 second recombinant slow virus of CD123.

(5) preparation of targeting T lymphocyte

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-CD33 and the second recombinant slow virus with CAR-CD123 of the corresponding virus titer of number carry 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 targeting T lymphocyte, i.e. CAR-CD33 Dan Yang by the 9-11 days Property T lymphocyte and the mono- positive t lymphocytes of CAR-CD123, the bis- positive t lymphocytes cells of CAR-CD33/CAR-CD123, And it saves it in and feeds back in dedicated cells frozen storing liquid.

Effect example

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

By targeting T lymphocyte (experimental group) made from the embodiment of the present invention one and the T lymphocyte (yin without preparation Property control group), the T cell (independent group of CD33CAR-T) with individual CAR-CD33 and with individual CAR-CD123's T cell (independent group of CD123CAR-T) is compared, and by above-mentioned four groups of effector cells and target cell, (THP-1 cell is (anxious in vitro Property monocytic leukemia cell)) in quantity than the ratio for 1:10,1:3,1:1,3:1 and 10:1, in 37 DEG C, 5%CO₂Under It is co-cultured, after incubation 15-18 hours, collects cell, carry out streaming dyeing, detect cell killing situation.As a result It was found that the tumor-killing power of targeting T lymphocyte provided by the invention is significantly larger than other control groups, this explanation is through the present invention The targeting T lymphocyte tumor-killing ability with super strength of method preparation.

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

By targeting T lymphocyte (experimental group) made from the embodiment of the present invention one and the T lymphocyte (yin without preparation Property control group), the T cell (independent group of CD33CAR-T) with individual CAR-CD33 and with individual CAR-CD123's T cell (independent group of CD123CAR-T) gives every mouse tail vein injection 1 × 10 in mouse medullary system Leukemia Model⁶It is a thin Born of the same parents (n=9), obtain the survivorship curve of mouse.As a result, it has been found that targeting T lymphocyte prepared by the present invention is in injection Mice Body After interior a period of time, remain to keep the survival rate of mouse more stable and still higher, considerably beyond negative control group and two above list Only group.This shows dead caused by the targeting T lymphocyte provided can be protected mice against preferably because of marrow series leukemia.

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>a kind of targeting T lymphocyte and its preparation method and application

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<210> 1

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<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 1

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

1 5 10 15

Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Phe Phe Ser

20 25 30

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

35 40 45

Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

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

65 70 75 80

Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln

85 90 95

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

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser

115 120 125

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

130 135 140

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

145 150 155 160

Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Val

165 170 175

Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe

180 185 190

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

195 200 205

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

210 215 220

Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val Trp Gly Gln Gly Thr

225 230 235 240

Thr Val Thr Val Ser Ser

245

<210> 2

<211> 244

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 2

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Asn Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

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

65 70 75 80

Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

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

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser Gln Ile

115 120 125

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

130 135 140

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

145 150 155 160

Asn Trp Val Lys Gln Ala Pro Gly Lys Ser Phe Lys Trp Met Gly Trp

165 170 175

Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr Ser Ala Asp Phe Lys Gly

180 185 190

Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu His

195 200 205

Ile Asn Asp Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg

210 215 220

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

225 230 235 240

Thr Val Ser Ser

<210> 3

<211> 738

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

gagatcgtgc tgacacagag ccctggaagc ctggccgtgt ctcctggcga gcgcgtgaca 60

atgagctgca agagcagcca gagcgtgttc ttcagcagct cccagaagaa ctacctggcc 120

tggtatcagc agatccccgg ccagagcccc agactgctga tctactgggc cagcaccaga 180

gaaagcggcg tgcccgatag attcaccggc agcggctctg gcaccgactt caccctgaca 240

atcagcagcg tgcagcccga ggacctggcc atctactact gccaccagta cctgagcagc 300

cggacctttg gccagggcac caagctggaa atcaagagag gcggcggagg ctctggcgga 360

ggcggatcta gtggcggagg atctcaggtg cagctgcagc agcctggcgc cgaggtcgtg 420

aaacctggcg cctctgtgaa gatgtcctgc aaggccagcg gctacacctt caccagctac 480

tacatccact ggatcaagca gacccctgga cagggcctgg aatgggtggg agtgatctac 540

cccggcaacg acgacatcag ctacaaccag aagttccagg gcaaggccac cctgaccgcc 600

gacaagtcta gcaccaccgc ctacatgcag ctgtccagcc tgaccagcga ggacagcgcc 660

gtgtactact gcgccagaga agtgcggctg cggtacttcg atgtgtgggg ccagggaacc 720

accgtgaccg tgtcatct 738

<210> 4

<211> 732

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

gacattgtac tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60

atatcctgca gagccagtga aagtgttgat aattatggca atacttttat gcactggtac 120

cagcagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaatct 180

gggttccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccattaat 240

cctgtggagg ctgatgatgt tgcaacctat tactgtcagc aaagtaatga ggttcctccc 300

acgttcggtg ctgggaccaa gctggagctg aaaggaggcg gaggatctgg cggcggagga 360

agttctggcg gagggtctca gatccagttg gtgcagtctg gacctgagct gaagaagcct 420

ggagagacag tcaagatctc ctgcaaggct tctgggtata ttttcacaaa ctatggaatg 480

aactgggtga agcaggctcc aggaaagagt tttaagtgga tgggctggat aaacacctac 540

actggagagt caacatatag tgctgacttc aagggacggt ttgccttctc tttggaaacc 600

tctgccagca ctgcctattt gcatatcaac gacctcaaaa atgaggacac ggctacatat 660

ttctgtgcaa gatcgggggg ttacgacccc atggactact ggggtcaagg aacctcagtc 720

accgtctcct ca 732

<210> 5

<211> 469

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 5

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

1 5 10 15

Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Phe Phe Ser

20 25 30

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

35 40 45

Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

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

65 70 75 80

Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln

85 90 95

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

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser

115 120 125

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

130 135 140

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

145 150 155 160

Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Val

165 170 175

Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe

180 185 190

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

195 200 205

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

210 215 220

Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val Trp Gly Gln Gly Thr

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Ala Leu Pro Pro Arg

465

<210> 6

<211> 467

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 6

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Asn Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

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

65 70 75 80

Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

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

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser Gln Ile

115 120 125

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

130 135 140

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

145 150 155 160

Asn Trp Val Lys Gln Ala Pro Gly Lys Ser Phe Lys Trp Met Gly Trp

165 170 175

Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr Ser Ala Asp Phe Lys Gly

180 185 190

Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu His

195 200 205

Ile Asn Asp Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Pro Pro Arg

465

<210> 7

<211> 489

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

20 25 30

Val Ser Pro Gly Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser

35 40 45

Val Phe Phe Ser Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln

50 55 60

Ile Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg

65 70 75 80

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

85 90 95

Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile Tyr

100 105 110

Tyr Cys His Gln Tyr Leu Ser Ser Arg Thr Phe Gly Gln Gly Thr Lys

115 120 125

Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser

130 135 140

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

145 150 155 160

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

165 170 175

Phe Thr Ser Tyr Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln Gly

180 185 190

Leu Glu Trp Val Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser Tyr

195 200 205

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

210 215 220

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

225 230 235 240

Val Tyr Tyr Cys Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val Trp

245 250 255

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Leu His Met Gln Ala Leu Pro Pro Arg

485

<210> 8

<211> 487

<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 Val Leu Thr Gln Ser Pro Ala Ser Leu Ala

20 25 30

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

35 40 45

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

50 55 60

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser

65 70 75 80

Gly Phe Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr

85 90 95

Leu Thr Ile Asn Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys

100 105 110

Gln Gln Ser Asn Glu Val Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu

115 120 125

Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Gly

130 135 140

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

145 150 155 160

Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr

165 170 175

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

180 185 190

Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr Ser Ala

195 200 205

Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr

210 215 220

Ala Tyr Leu His Ile Asn Asp Leu Lys Asn Glu Asp Thr Ala Thr Tyr

225 230 235 240

Phe Cys Ala Arg Ser Gly Gly Tyr Asp Pro Met Asp Tyr Trp Gly Gln

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Met Gln 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> 1407

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 17

gagatcgtgc tgacacagag ccctggaagc ctggccgtgt ctcctggcga gcgcgtgaca 60

atgagctgca agagcagcca gagcgtgttc ttcagcagct cccagaagaa ctacctggcc 120

tggtatcagc agatccccgg ccagagcccc agactgctga tctactgggc cagcaccaga 180

gaaagcggcg tgcccgatag attcaccggc agcggctctg gcaccgactt caccctgaca 240

atcagcagcg tgcagcccga ggacctggcc atctactact gccaccagta cctgagcagc 300

cggacctttg gccagggcac caagctggaa atcaagagag gcggcggagg ctctggcgga 360

ggcggatcta gtggcggagg atctcaggtg cagctgcagc agcctggcgc cgaggtcgtg 420

aaacctggcg cctctgtgaa gatgtcctgc aaggccagcg gctacacctt caccagctac 480

tacatccact ggatcaagca gacccctgga cagggcctgg aatgggtggg agtgatctac 540

cccggcaacg acgacatcag ctacaaccag aagttccagg gcaaggccac cctgaccgcc 600

gacaagtcta gcaccaccgc ctacatgcag ctgtccagcc tgaccagcga ggacagcgcc 660

gtgtactact gcgccagaga agtgcggctg cggtacttcg atgtgtgggg ccagggaacc 720

accgtgaccg tgtcatctac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 780

atcgcgtcgc agcccctgtc cctgcgccca gaggcgtgcc ggccagcggc ggggggcgca 840

gtgcacacga gggggctgga cttcgcctgt gatatctaca tctgggcgcc cttggccggg 900

acttgtgggg tccttctcct gtcactggtt atcacccttt actgcaaacg gggcagaaag 960

aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 1020

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gagagtgaag 1080

ttcagcagga gcgcagacgc ccccgcgtac aagcagggcc agaaccagct ctataacgag 1140

ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1200

gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1260

aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1320

aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1380

cttcacatgc aggccctgcc ccctcgc 1407

<210> 18

<211> 1401

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 18

gacattgtac tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60

atatcctgca gagccagtga aagtgttgat aattatggca atacttttat gcactggtac 120

cagcagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaatct 180

gggttccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccattaat 240

cctgtggagg ctgatgatgt tgcaacctat tactgtcagc aaagtaatga ggttcctccc 300

acgttcggtg ctgggaccaa gctggagctg aaaggaggcg gaggatctgg cggcggagga 360

agttctggcg gagggtctca gatccagttg gtgcagtctg gacctgagct gaagaagcct 420

ggagagacag tcaagatctc ctgcaaggct tctgggtata ttttcacaaa ctatggaatg 480

aactgggtga agcaggctcc aggaaagagt tttaagtgga tgggctggat aaacacctac 540

actggagagt caacatatag tgctgacttc aagggacggt ttgccttctc tttggaaacc 600

tctgccagca ctgcctattt gcatatcaac gacctcaaaa atgaggacac ggctacatat 660

ttctgtgcaa gatcgggggg ttacgacccc atggactact ggggtcaagg aacctcagtc 720

accgtctcct caaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 780

tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 840

acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 900

ggggtccttc tcctgtcact ggttatcacc ctttactgca aacggggcag aaagaaactc 960

ctgtatatat tcaaacaacc atttatgaga ccagtacaaa ctactcaaga ggaagatggc 1020

tgtagctgcc gatttccaga agaagaagaa ggaggatgtg aactgagagt gaagttcagc 1080

aggagcgcag acgcccccgc gtacaagcag ggccagaacc agctctataa cgagctcaat 1140

ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 1200

gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1260

aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1320

cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1380

atgcaggccc tgccccctcg c 1401

<210> 19

<211> 1467

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 19

gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60

gagatcgtgc tgacacagag ccctggaagc ctggccgtgt ctcctggcga gcgcgtgaca 120

atgagctgca agagcagcca gagcgtgttc ttcagcagct cccagaagaa ctacctggcc 180

tggtatcagc agatccccgg ccagagcccc agactgctga tctactgggc cagcaccaga 240

gaaagcggcg tgcccgatag attcaccggc agcggctctg gcaccgactt caccctgaca 300

atcagcagcg tgcagcccga ggacctggcc atctactact gccaccagta cctgagcagc 360

cggacctttg gccagggcac caagctggaa atcaagagag gcggcggagg ctctggcgga 420

ggcggatcta gtggcggagg atctcaggtg cagctgcagc agcctggcgc cgaggtcgtg 480

aaacctggcg cctctgtgaa gatgtcctgc aaggccagcg gctacacctt caccagctac 540

tacatccact ggatcaagca gacccctgga cagggcctgg aatgggtggg agtgatctac 600

cccggcaacg acgacatcag ctacaaccag aagttccagg gcaaggccac cctgaccgcc 660

gacaagtcta gcaccaccgc ctacatgcag ctgtccagcc tgaccagcga ggacagcgcc 720

gtgtactact gcgccagaga agtgcggctg cggtacttcg atgtgtgggg ccagggaacc 780

accgtgaccg tgtcatctac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 840

atcgcgtcgc agcccctgtc cctgcgccca gaggcgtgcc ggccagcggc ggggggcgca 900

gtgcacacga gggggctgga cttcgcctgt gatatctaca tctgggcgcc cttggccggg 960

acttgtgggg tccttctcct gtcactggtt atcacccttt actgcaaacg gggcagaaag 1020

aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 1080

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gagagtgaag 1140

ttcagcagga gcgcagacgc ccccgcgtac aagcagggcc agaaccagct ctataacgag 1200

ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1260

gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1320

aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380

aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1440

cttcacatgc aggccctgcc ccctcgc 1467

<210> 20

<211> 1461

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 20

gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60

gacattgtac tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 120

atatcctgca gagccagtga aagtgttgat aattatggca atacttttat gcactggtac 180

cagcagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaatct 240

gggttccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccattaat 300

cctgtggagg ctgatgatgt tgcaacctat tactgtcagc aaagtaatga ggttcctccc 360

acgttcggtg ctgggaccaa gctggagctg aaaggaggcg gaggatctgg cggcggagga 420

agttctggcg gagggtctca gatccagttg gtgcagtctg gacctgagct gaagaagcct 480

ggagagacag tcaagatctc ctgcaaggct tctgggtata ttttcacaaa ctatggaatg 540

aactgggtga agcaggctcc aggaaagagt tttaagtgga tgggctggat aaacacctac 600

actggagagt caacatatag tgctgacttc aagggacggt ttgccttctc tttggaaacc 660

tctgccagca ctgcctattt gcatatcaac gacctcaaaa atgaggacac ggctacatat 720

ttctgtgcaa gatcgggggg ttacgacccc atggactact ggggtcaagg aacctcagtc 780

accgtctcct caaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840

tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900

acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 960

ggggtccttc tcctgtcact ggttatcacc ctttactgca aacggggcag aaagaaactc 1020

ctgtatatat tcaaacaacc atttatgaga ccagtacaaa ctactcaaga ggaagatggc 1080

tgtagctgcc gatttccaga agaagaagaa ggaggatgtg aactgagagt gaagttcagc 1140

aggagcgcag acgcccccgc gtacaagcag ggccagaacc agctctataa cgagctcaat 1200

ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 1260

gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1320

aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1380

cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1440

atgcaggccc tgccccctcg c 1461

<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

gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60

Claims

1. a kind of targeting T lymphocyte, which is characterized in that including target CD33 Chimeric antigen receptor CAR-CD33 and/or Target the Chimeric antigen receptor CAR-CD123 of CD123, wherein the CAR-CD33 includes sequentially connecting from aminoterminal to c-terminus The targeting single-chain antibody of CD33 that connects, extracellular hinge area, transmembrane region and intracellular signal area amino acid sequence, the CAR- CD123 includes single-chain antibody, extracellular hinge area, transmembrane region and the born of the same parents of the sequentially connected targeting CD123 from aminoterminal to c-terminus The amino acid sequence of interior signaling zone；

Wherein, the amino acid sequence of the single-chain antibody of the targeting CD33 includes the amino acid sequence as shown in SEQ ID NO:1, The amino acid sequence of the single-chain antibody of the targeting CD123 includes the amino acid sequence as shown in SEQ ID NO:2.

2. targeting T lymphocyte as described in claim 1, which is characterized in that the volume of the single-chain antibody of the targeting CD33 Code gene includes the nucleotide sequence as shown in SEQ ID NO:3, and the encoding gene of the single-chain antibody of the targeting CD123 includes The nucleotide sequence as shown in SEQ ID NO:4.

3. targeting T lymphocyte as described in claim 1, which is characterized in that the amino acid sequence packet of the CAR-CD33 The amino acid sequence as shown in SEQ ID NO:5 is included, the amino acid sequence of the CAR-CD123 includes such as SEQ ID NO:6 institute The amino acid sequence shown.

4. a kind of recombinant viral vector, which is characterized in that thin including targeting T lymph as described in any one of claims 1-3 The encoding gene of CAR-CD33 described in born of the same parents and/or CAR-CD123.

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 targeting T lymphocyte characterized by comprising

(1) encoding gene of the Chimeric antigen receptor CAR-CD33 of targeting CD33 and the chimeric antigen of targeting CD123 are provided respectively The encoding gene of receptor CAR-CD123；

The encoding gene of the CAR-CD33 includes encoding gene, the targeting CD33 that signal peptide is sequentially connected with from 5 ' ends to 3 ' ends The encoding gene of single-chain antibody, the encoding gene of extracellular hinge area, the encoding gene of transmembrane region and the coding base in intracellular signal area Cause；The encoding gene of the CAR-CD123 includes encoding gene, the targeting CD123 that signal peptide is sequentially connected with from 5 ' ends to 3 ' ends The encoding gene of single-chain antibody, the encoding gene of extracellular hinge area, the encoding gene of transmembrane region and intracellular signal area coding Gene；

Wherein, the encoding gene of the single-chain antibody of the targeting CD33 includes the amino acid sequence institute as shown in SEQ ID NO:1 The encoding gene of corresponding nucleotide sequence, the single-chain antibody of the targeting CD123 includes the amino as shown in SEQ ID NO:2 Nucleotide sequence corresponding to acid sequence；

(2) encoding gene of the encoding gene of the CAR-CD33 and the CAR-CD123 is inserted respectively into pWPXLD carrier In, obtain pWPXLD-CAR-CD33 recombinant plasmid and pWPXLD-CAR-CD123 recombinant plasmid；

(3) the pWPXLD-CAR-CD33 recombinant plasmid and the pWPXLD-CAR-CD123 recombinant plasmid are wrapped respectively Dress, obtains the first recombinant slow virus with CAR-CD33 encoding gene and the second recombinant lentiviral with CAR-CD123 encoding gene Virus；

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

7. the preparation method of targeting T lymphocyte as claimed in claim 6, which is characterized in that the volume of the CAR-CD33 Code gene includes the nucleotide sequence as shown in SEQ ID NO:7, and the encoding gene of the CAR-CD123 includes such as SEQ ID Nucleotide sequence shown in NO:8.

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

9. the preparation method of targeting T lymphocyte as claimed in claim 6, which is characterized in that the targeting T lymph is thin Born of the same parents are double target spot Chimeric antigen receptor T cells with the CAR-CD33 and CAR-CD123, or for the CAR- The mixing of the Chimeric antigen receptor T cell of CD33 and the Chimeric antigen receptor T cell with the CAR-CD123, or be band institute State double target spot Chimeric antigen receptor T cells of the CAR-CD33 and CAR-CD123, the chimeric antigen with the CAR-CD33 by The mixing of body T cell and the Chimeric antigen receptor T cell with the CAR-CD123.

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