CN109796535A

CN109796535A - The Chimeric antigen receptor and its purposes in preparation prevention or treatment malignant tumor medicine for targeting folacin receptor α

Info

Publication number: CN109796535A
Application number: CN201910094268.9A
Authority: CN
Inventors: 李伟强; 邵文陶; 冉启平; 周兴
Original assignee: Chongqing Fumei Stem Cell Biotechnology Development Co Ltd
Current assignee: Chongqing Fumei Stem Cell Biotechnology Development Co Ltd
Priority date: 2019-01-30
Filing date: 2019-01-30
Publication date: 2019-05-24
Anticipated expiration: 2039-01-30
Also published as: CN109796535B

Abstract

The invention belongs to field of biotechnology, and in particular to a kind of Chimeric antigen receptor for targeting folacin receptor α and its purposes in preparation prevention or treatment malignant tumor medicine.The composition of the Chimeric antigen receptor of targeting folacin receptor α of the invention successively includes: people's CD8 alpha signal peptide, the polypeptide in conjunction with folacin receptor α, 1Fc sections of immunoglobulin G, people CD8 α hinge area, people's CD28 cross-film section, intracellular section of people CD28, intracellular section of people CD137 and intracellular section of people CD3 ζ.Above-mentioned Chimeric antigen receptor is imported NK-92 cell by the present invention, with specific recognition and can kill the human tumor cell line and human ovarian cancer Transplanted tumor model of the folacin receptor alpha expression positive.The present invention provides a kind of safe, special, effective Chimeric antigen receptors, and modify NK-92 cell, the great application potential in the clinical treatment of folacin receptor α positive malignancies.

Description

It targets the Chimeric antigen receptor of folacin receptor α and its prevents or treat pernicious in preparation Purposes in tumour medicine

Technical field

The invention belongs to field of biotechnology, and in particular to it is a kind of target folacin receptor α Chimeric antigen receptor and its Purposes in preparation prevention or treatment malignant tumor medicine.

Background technique

Adoptive cell therapy (Adoptive with the development of tumor immunology theory and technology, in immunization therapy Cellular therapy, ACT) effect in oncotherapy is paid more and more attention.The especially inosculating antibody of rising in recent years Original receptor (chimeric antigen receptor, CAR) technical progress is swift and violent, and the immunocyte of CAR modification in vitro and faces The anti-tumor activity for being significantly better than traditional ACT is shown in bed test, has caused the new upsurge of oncotherapy Study on Transformation.

CAR generally comprises single-stranded variable region (the single-chain variable of targets identification tumour antigen Fragment, scFv) (from antibody) and different intracellular signal structural domain (deriving from lymphocyte-activating receptor).It is based on This special structure, express CAR immune effector cell can independent of MHC is restricted and antigen presentation and specific recognition And killing tumor cell.Having multinomial clinical test confirms, the T cell of CAR modification is a variety of to leukaemia, lymthoma etc. pernicious swollen Tumor has splendid curative effect.

Currently, three generations's CAR structure has been developed.First generation CAR is structure intracellular containing only CD3 ζ or Fc ε RI γ signal domain, The second generation, third generation CAR are then will such as CD28, CD134 and CD137 costimulatory molecules on the basis of first generation CAR structure Structural domain is integrated into CAR.It wherein, is second generation CAR comprising costimulatory molecules structural domain in CAR structure, in CAR structure It is third generation CAR comprising two costimulatory molecules structural domains.

When using CAR technology, it is necessary first to find suitable tumour antigen.And folacin receptor α (folate Receptor α, α FR) expression and distribution have the following characteristics that 1. α FR in 90% oophoroma in high expression status, this Outside, it is also expressed in the malignant tumour in the epithelial tissues such as carcinoma of endometrium, breast cancer, lung cancer and colon cancer source to some extent In；2. the expression of α FR is not influenced by chemotherapeutics in oophoroma, i.e., before and after chemotherapeutics use, α FR is High level expression；3. in the normal tissue, α FR is not expressed or restricted low expression level is in the top surface of polarized epithelial cell, And be usually herein in the circulatory system anti alpha FR antibody and folate conjugate be difficult to the position reached.Based on this, α FR is considered as The ideal tumour antigen of the α FR positive malignancies such as targeted therapy oophoroma, lung cancer and colon cancer.In early-stage study, research Person has constructed the monoclonal antibody of a variety of targeting α FR, and the CAR-T cell of targeting α FR, and utilizes these strategy treatment ovum Nest cancer, but unfortunately, clinical test does not show it with obvious curative effects.This may be transfected simultaneously with the CAR molecule of targeting α FR The inefficiency for being expressed in T cell is related.In addition, targeting α FR CAR-T cell can only individuation application, lead to preparation cost Valuableness also restricts its popularization and application.

In the recent period, have researcher's discovery, compared to T cell, natural killer cells (natural killer cell, NK) or Permitted to be better CAR load cells, and NK cell can be used for allosome, can be produced in batches, batch application, significantly reduces system Standby cost.But primary NK cells have defect identical with primary T cells, and e.g., the amplification ability of the NK cell of different patients has Height has low, and difference is obvious.In addition, the foreign gene transfection efficiency of primary NK cells is low and the time-to-live is limited, it is used as CAR The effect of load cells all need to be improved.

Summary of the invention

The technical problem to be solved in the present invention are as follows: the Chimeric antigen receptor of building high efficiency stable expression targeting folacin receptor α NK-92 cell strain, and provide the preparation method and purposes of Chimeric antigen receptor.

The present invention solution above-mentioned technical problem technical solution are as follows: provide it is a kind of target folacin receptor α chimeric antigen by Body, the composition of the Chimeric antigen receptor successively include: people's CD8 alpha signal peptide, the polypeptide in conjunction with folacin receptor α, people's immune globulin G1Fc sections white, people CD8 α hinge area, people's CD28 cross-film section, intracellular section of people CD28, intracellular section of people CD137 and intracellular section of people CD3 ζ.

Wherein, in the Chimeric antigen receptor of above-mentioned targeting folacin receptor α, the C-terminal of the polypeptide of the combination folacin receptor α It is connect with immunoglobulin G 1Fc sections of the N-terminal.

Wherein, in the Chimeric antigen receptor of above-mentioned targeting folacin receptor α, the amino acid sequence of the people CD8 alpha signal peptide is such as Shown in SEQ ID NO.13.Further, the coding nucleotide sequence of the people CD8 alpha signal peptide is as shown in SEQ ID NO.1.

Wherein, in the Chimeric antigen receptor of above-mentioned targeting folacin receptor α, the polypeptide of the combination folacin receptor α is anti-leaf The single-stranded variable region scFv of the antibody of acid acceptor α.The single-stranded variable region scFv of the anti-folacin receptor α include light chain variable region and Heavy chain variable region.

Wherein, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.15.Further, the light chain can Become the coding nucleotide sequence in area as shown in SEQ ID NO.2.

Wherein, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.16.Further, the heavy chain can Become the coding nucleotide sequence in area as shown in SEQ ID NO.3.

Preferably, the amino acid sequence of the single-stranded variable region scFv of the anti-folacin receptor α is as shown in SEQ ID NO.14. Further, the coding nucleotide sequence of the single-stranded variable region scFv of the anti-folacin receptor α is as shown in SEQ ID NO.4.

Wherein, immunoglobulin G 1Fc sections of the amino acid sequence is as shown in SEQ ID NO.17.Further, institute 1Fc sections of immunoglobulin G of coding nucleotide sequence is stated as shown in SEQ ID NO.5.

Wherein, the amino acid sequence of the people CD8 α hinge area is as shown in SEQ ID NO.18.Further, the people The coding nucleotide sequence of CD8 α hinge area is as shown in SEQ ID NO.6.

Wherein, the amino acid sequence of the people CD28 cross-film section is as shown in SEQ ID NO.19.Further, the people The coding nucleotide sequence of CD28 cross-film section is as shown in SEQ ID NO.7.

Wherein, the amino acid sequence of intracellular section of the people CD28 is as shown in SEQ ID NO.20.Further, the people The coding nucleotide sequence that intracellular section of CD28 is as shown in SEQ ID NO.8.

Wherein, the amino acid sequence of intracellular section of the people CD137 is as shown in SEQ ID NO.21.Further, the people The coding nucleotide sequence that intracellular section of CD137 is as shown in SEQ ID NO.9.

Wherein, intracellular section of people CD3 ζ of the amino acid sequence is as shown in SEQ ID NO.22.Further, the people The coding nucleotide sequence that intracellular section of CD3 ζ is as shown in SEQ ID NO.10.

Wherein, the amino acid sequence of the Chimeric antigen receptor of the targeting folacin receptor α is as shown in SEQ ID NO.12.

The present invention also provides a kind of polynucleotides, encode the Chimeric antigen receptor of above-mentioned targeting folacin receptor α.

The present invention also provides a kind of expression vectors for expressing above-mentioned polynucleotide.

The present invention also provides a kind of Chimeric antigen receptors containing above-mentioned targeting folacin receptor α, polynucleotide, expression The host cell of carrier.

Further, the host cell is that NK-92 cell, people's primary T cells, people's primary NK cells or cell factor lure One of killing cell led.

The present invention also provides a kind of method of Chimeric antigen receptor for preparing above-mentioned targeting folacin receptor α, including it is following Step: the expression vector of Chimeric antigen receptor polynucleotides encoding sequence of the building containing targeting folacin receptor α, then by institute Statement expression vector converts into host cell inducing expression, and separation obtains the targeting folacin receptor α's from expression product Chimeric antigen receptor.

The present invention also provides the Chimeric antigen receptor of above-mentioned targeting folacin receptor α a kind of, polynucleotide, expression to carry The purposes of body, host cell in the drug of preparation prevention or treatment malignant tumour.

Further, the malignant tumour is folacin receptor α positive malignancies.

Further, the malignant tumour includes in oophoroma, carcinoma of endometrium, breast cancer, lung cancer or colon cancer It is at least one.

Compared with prior art, the invention has the benefit that

The characteristics of present invention is according to oophoroma specificity overexpression tumour antigen-folacin receptor α, devises a kind of targeting leaf The Chimeric antigen receptor of acid acceptor α, and NK-92 cell is modified by the Chimeric antigen receptor of targeting folacin receptor α, it can be special Property identifies and kills the human tumor cell line and human ovarian cancer Transplanted tumor model of the folacin receptor alpha expression positive.The present invention provides A kind of safe, special, effective Chimeric antigen receptor, and NK-92 cell is modified, in the α FR positive malignancies such as oophoroma Great application potential in clinical treatment.

Detailed description of the invention

Fig. 1 show the structural schematic diagram of the Chimeric antigen receptor of targeting folacin receptor α.

Fig. 2 show the flow cytometry results figure of embodiment 3.As seen from the figure, target folacin receptor α chimeric antigen by Body modifies NK-92 cell (NK-92- α FR-CAR) high expression Chimeric antigen receptor.

Fig. 3 show the flow cytometry results figure of embodiment 3.As seen from the figure, in different human tumor cell lines, ovum The all high expression folacin receptor α of nest cancerous cell line SK-OV-3 and A2780.

Fig. 4 show the cell killing experimental result picture of embodiment 3.As seen from the figure, the chimeric antigen of folacin receptor α is targeted It is most strong to the lethal effect of SK-OV-3 cell and A2780 cell that receptor modifies NK-92 cell (NK-92- α FR-CAR).

Fig. 5 show the ELISA result figure of embodiment 3.As seen from the figure, the Chimeric antigen receptor for targeting folacin receptor α is repaired When adoring NK-92 cell (NK-92- α FR-CAR) and SK-OV-3 cell or the co-cultivation of A2780 cell, more IFN- can be secreted γ。

Fig. 6 show the schematic diagram of construction method of the human ovarian cancer Transplanted tumor model of embodiment 4.

Fig. 7 show the human ovarian cancer Transplanted tumor model of embodiment 4.The Chimeric antigen receptor of display targeting folacin receptor α Modification NK-92 cell (NK-92- α FR-CAR) can significantly inhibit tumour growth.

Fig. 8 show the human ovarian cancer Transplanted tumor model of embodiment 4.The Chimeric antigen receptor of display targeting folacin receptor α Modification NK-92 cell (NK-92- α FR-CAR) can significantly extend tumor-bearing mice life span.

Specific embodiment

The present invention provides a kind of Chimeric antigen receptor for targeting folacin receptor α, the composition of the Chimeric antigen receptor according to Secondary includes: people's CD8 alpha signal peptide, the polypeptide in conjunction with folacin receptor α, 1Fc sections of immunoglobulin G, people CD8 α hinge area, people CD28 cross-film section, intracellular section of people CD28, intracellular section of people CD137 and intracellular section of people CD3 ζ.

Although it is of the invention the study found that introduced in CAR CD28 structural domain can just be provided for immunocyte it is enough Activation signals, but the CD137 structural domains that are integrated into can make immunocyte survival more long and generate stronger antitumor reaction more again.This Outside, the immunocyte of CAR modification can be protected from the cell of CD3 ζ structural domain activation-inducing from the signal of CD137 structural domain Dead (activation-induced cell death, AICD).

Innovative proposition of the invention is pierced altogether using scFv, CD28 costimulatory molecules structural domain, the CD137 of targets identification α FR Swash the CAR in molecular structure domain and CD3 ζ signal domain building targeting α FR.Wherein, the scFv for targeting α FR can assign exempting from for CAR modification The ability of epidemic disease cell-specific identification α FR；CD28 costimulatory molecules structural domain, CD137 costimulatory molecules structural domain and CD3 ζ letter Number domain can assign the CAR stronger anti-tumor activity of immunocyte, proliferative capacity and the time-to-live of modification.

Meanwhile the Chimeric antigen receptor by targeting folacin receptor α of the present invention also creativeness has imported NK-92 cell.People NK cell line NK-92 cultural method is easy, can be cheap containing interleukin 2 (Interleukin-2, IL-2) Efficient amplification in complete medium.In addition, NK-92 cell is also easier to by virus or non-viral methods transfection expression external source base Cause.More importantly, early studies in man has been proven that in late malignant tumour, and NK-92 cell is as heterogenote The safety for the treatment of.Also, NK-92 cell has the characteristic of primary cell concurrently, and can stable massive amplification and passage in vitro, separately The outer efficiency for receiving foreign gene transfection is much higher than primary NK cells, and energy exogenous gene stablizes highly expressed cell strain, It is readily produced preparation and application.

The innovative CAR for proposing to express constructed targeting α FR using NK-92 cell as load cells of the invention, most The Chimeric antigen receptor modification NK-92 cell of the targeting folacin receptor α obtained eventually will specific efficient killing folacin receptor α sun Property ovarian cancer cell.In this way, which the operation for preparing the Chimeric antigen receptor modification NK cell of targeting folacin receptor α will be simpler Easily, efficiency will be obviously improved, cost will significantly reduce.More importantly, the chimeric antigen of constructed targeting folacin receptor α by Body modification NK-92 cell will be a kind of cell colony of height homogeneity, therefore be easier to carry out quality control management, and the targeting leaf The Chimeric antigen receptor modification NK-92 cell of acid acceptor α can stablize amplification, therefore pole is hopeful to develop into " instant to obtain " α FR positive malignancies immune cell therapy product.

Explanation will be further explained to a specific embodiment of the invention by embodiment below, but do not indicated this The protection scope of invention is limited in range described in embodiment.

Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of related fields.These technologies have perfect explanation in the prior art.

Embodiment 1 targets the building of the Chimeric antigen receptor expression vector of folacin receptor α

Chimeric antigen receptor (abbreviation α FR-CAR) expression vector of building targeting folacin receptor α.

As shown in Figure 1, α FR-CAR is immunized by sequentially connected people CD8 alpha signal peptide, the polypeptide in conjunction with folacin receptor α, people Fc sections of globulinG1, people CD8 α hinge area, people's CD28 cross-film section, intracellular section of people CD28, intracellular section of people CD137 and people CD3 ζ are intracellular Duan Zucheng.

Wherein, the coding nucleotide sequence of people CD8 alpha signal peptide is as shown in SEQ ID NO.1, specifically:

ATGGCCCTCCCAGTTACCGCCCTTCTCCTGCCCCTGGCCCTGCTGCTGCACGCCGCCCGCCCC。

Polypeptide in conjunction with folacin receptor α (α FR) is the single-stranded variable region (scFv) of the antibody of anti-folacin receptor α (α FR).Institute The single-stranded variable region (scFv) for stating anti-folacin receptor α (α FR) includes light chain variable region (V_L) and heavy chain variable region (V_H).It is described light Chain variable region (V_L) coding nucleotide sequence as shown in SEQ ID NO.2, specifically:

CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGAGCATCACCATCTCCTGCAC TGGAACCAGCAGTGATGTTGGGAGTTATAACCTGGTCTCCTGGTACCAGCAGCACCCAGGCAAAGCCCCCAAACTC ATGATTTATGAGGGCAGTAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACGCCGCCT CCCTGACAATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGAGTGT GGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTC。

Heavy chain variable region (the V_H) coding nucleotide sequence as shown in SEQ ID NO.3, specifically:

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTGCAGCCAGGGCGGTCCCTGAGACTCTCCTGCAC AACTTCTGGATTCACTTTTGGCGATTATGCTATGATCTGGGCCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTC TCATCCATTAGTAGTAGTAGTAGTTACATCTACTACGCAGACTCAGTGAAGGGCAGATTCACCATCTCCAGAGACA ACGCCAAGAACTCACTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCTGTGTATTACTGTGCCAGAGA ACGGTACGATTTTTGGAGTGGAATGGACGTCTGGGGCAAAGGGACCACCGTCACCGTGAGCAGT。

The coding nucleotide sequence such as SEQ ID NO.4 institute of the single-stranded variable region (scFv) of the anti-folacin receptor α (α FR) Show, specifically:

CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGAGCATCACCATCTCCTGCAC TGGAACCAGCAGTGATGTTGGGAGTTATAACCTGGTCTCCTGGTACCAGCAGCACCCAGGCAAAGCCCCCAAACTC ATGATTTATGAGGGCAGTAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACGCCGCCT CCCTGACAATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGAGTGT GGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTCGGAGGAGGCGGATCAGGCGGAGGAGGCTCTGGCGGAGGCGGA AGCCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTGCAGCCAGGGCGGTCCCTGAGACTCTCCTGCACAACTT CTGGATTCACTTTTGGCGATTATGCTATGATCTGGGCCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATC CATTAGTAGTAGTAGTAGTTACATCTACTACGCAGACTCAGTGAAGGGCAGATTCACCATCTCCAGAGACAACGCC AAGAACTCACTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCTGTGTATTACTGTGCCAGAGAACGGT ACGATTTTTGGAGTGGAATGGACGTCTGGGGCAAAGGGACCACCGTCACCGTGAGCAGT。

The coding nucleotide sequence that 1Fc sections of immunoglobulin G as shown in SEQ ID NO.5, specifically:

GAGCCCAAATCTAGCGACAAAACTCACACATGCCCACCTTGCCCAGCACCTGAGCTCCTGGGGGGACC TTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGGGGTCACATGCGTGGTG GTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGA CAAAGCCTCGGGAGGAGCAGTACAACAGCACCTACAGAGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCC AAAGGGCAGCCCAGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCC TGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCTGAGAACAA CTACAAGACCACCCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGC AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACCCAGAAGAGCC TCTCCCTGTCTCCTGGCAAA。

The coding nucleotide sequence of people's CD8 α hinge area as shown in SEQ ID NO.6, specifically:

GCCAAGCCCACCACTACACCCGCCCCACGCCCCCCCACCCCCGCCCCCACCATCGCCAGCCAGCCCCT GAGCCTGCGCCCCGAGGCCTGCCGCCCCGCCGCCGGCGGCGCCGTGCACACCCGCGGCCTGGACTTCGCCTGCGAC。

The coding nucleotide sequence of people's CD28 cross-film section as shown in SEQ ID NO.7, specifically:

TTCTGGGTGCTGGTGGTGGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACCGTGGCCTTCAT CATCTTCTGGGTG。

The coding nucleotide sequence that intracellular section of people CD28 as shown in SEQ ID NO.8, specifically:

CGCAGCAAGCGCAGCCGCCTGCTGCACAGCGACTACATGAACATGACCCCCCGCCGCCCCGGCCCCAC CCGCAAGCACTACCAGCCCTACGCCCCCCCCCGCGACTTCGCCGCCTACCGCAGC。

The coding nucleotide sequence that intracellular section of people CD137 as shown in SEQ ID NO.9, specifically:

AAGCGCGGCCGCAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTGCAGACCACCCA GGAGGAGGACGGCTGCAGCTGCCGCTTCCCCGAAGAAGAGGAGGGCGGCTGCGAGCTG。

The coding nucleotide sequence of intracellular section of people CD3 ζ as shown in SEQ ID NO.10, specifically:

CGCGTGAAGTTCAGCCGCAGCGCCGAGCCCCCCGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGA GCTGAACCTGGGCCGCCGCGAGGAGTACGACGTGCTGGACAAGCGCCGCGGCCGCGACCCCGAGATGGGCGGCAAG CCCCGCCGCAAGAACCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGA TCGGCATGAAGGGCGAGCGCCGCCGCGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGA CACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCCGCTGA。

Entrust the polynucleotide of α FR-CAR described in Shanghai and first Bioisystech Co., Ltd composite coding, insertion PLenti-EF1 α carrier (purchased from Shanghai and first Bioisystech Co., Ltd), after being sequenced correctly, uses TIANGEN company Plasmid purification kit extracts and plasmid purification, obtains the high-quality plasmid of recombinant expression carrier.

Through sequencing it is found that the full-length gene order of α FR-CAR is correct, it is consistent with expection.

Specifically, the coding nucleotide sequence of α FR-CAR is as shown in SEQ ID NO.11, specifically:

ATGGCCCTCCCAGTTACCGCCCTTCTCCTGCCCCTGGCCCTGCTGCTGCACGCCGCCCGCCCCCAGTC TGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGAGCATCACCATCTCCTGCACTGGAACCAGCAGT GATGTTGGGAGTTATAACCTGGTCTCCTGGTACCAGCAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGG GCAGTAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACGCCGCCTCCCTGACAATCTC TGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGAGTGTGGTGTTCGGCGGA GGGACCAAGCTGACCGTCCTCGGAGGAGGCGGATCAGGCGGAGGAGGCTCTGGCGGAGGCGGAAGCCAGGTGCAGC TGGTGGAGTCTGGGGGAGGCCTGGTGCAGCCAGGGCGGTCCCTGAGACTCTCCTGCACAACTTCTGGATTCACTTT TGGCGATTATGCTATGATCTGGGCCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGT AGTAGTTACATCTACTACGCAGACTCAGTGAAGGGCAGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGT ATCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCTGTGTATTACTGTGCCAGAGAACGGTACGATTTTTGGAG TGGAATGGACGTCTGGGGCAAAGGGACCACCGTCACCGTGAGCAGTGAGCCCAAATCTAGCGACAAAACTCACACA TGCCCACCTTGCCCAGCACCTGAGCTCCTGGGGGGACCTTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCC TCATGATCTCCCGGACCCCTGGGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAA CTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCTCGGGAGGAGCAGTACAACAGCACCTACAGA GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAG CCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCAGAGAACCACAGGTGTACACCCTGCC CCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGAT GCATGAGGCTCTGCACAACCACTACACCCAGAAGAGCCTCTCCCTGTCTCCTGGCAAAGCCAAGCCCACCACTACA CCCGCCCCACGCCCCCCCACCCCCGCCCCCACCATCGCCAGCCAGCCCCTGAGCCTGCGCCCCGAGGCCTGCCGCC CCGCCGCCGGCGGCGCCGTGCACACCCGCGGCCTGGACTTCGCCTGCGACTTCTGGGTGCTGGTGGTGGTGGGCGG CGTGCTGGCCTGCTACAGCCTGCTGGTGACCGTGGCCTTCATCATCTTCTGGGTGCGCAGCAAGCGCAGCCGCCTG CTGCACAGCGACTACATGAACATGACCCCCCGCCGCCCCGGCCCCACCCGCAAGCACTACCAGCCCTACGCCCCCC CCCGCGACTTCGCCGCCTACCGCAGCAAGCGCGGCCGCAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCG CCCCGTGCAGACCACCCAGGAGGAGGACGGCTGCAGCTGCCGCTTCCCCGAAGAAGAGGAGGGCGGCTGCGAGCTG CGCGTGAAGTTCAGCCGCAGCGCCGAGCCCCCCGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACC TGGGCCGCCGCGAGGAGTACGACGTGCTGGACAAGCGCCGCGGCCGCGACCCCGAGATGGGCGGCAAGCCCCGCCG CAAGAACCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATG AAGGGCGAGCGCCGCCGCGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACG ACGCCCTGCACATGCAGGCCCTGCCCCCCCGCTGA。

Using following methods, to the high-quality plasmid transfection of recombinant expression carrier into host cell inducing expression, specifically Step are as follows:

After the 293T cell pancreatin of culture is digested and washed, with DMEM complete medium (containing 10% fetal calf serum) weight Cell suspension is made in outstanding cell, counts cell and calculates concentration of cell suspension.With 1 × 10⁵/cm²Density by 293T cell inoculation In in 10cm culture dish.According to PolyJet^TMDescribed in DNA Transfection Reagent (SignaGen company) specification, The high-quality plasmid of recombinant expression carrier described in 5 μ g is added in 0.25ml plasma-free DMEM medium, gently concussion mixes. 15 μ l PolyJet are added in 0.25ml plasma-free DMEM medium^TMTransfection reagent, gently concussion mixes.Then it will contain transfection The 0.25ml plasma-free DMEM medium of reagent is added in the 0.25ml plasma-free DMEM medium containing plasmid, mixes gently rear chamber Temperature is stood, and compound to be transfected is formed.After twenty minutes, transfection composite is added dropwise into inoculated 293T cell, and Culture medium total volume is mended to 10ml, is put into incubator, 5%CO₂, 37 DEG C of cultures.It moves back within 18 hours except containing transfection composite Culture medium supernatant rejoins fresh DMEM complete medium.After 72 hours, is isolated and purified from expression product and obtain α FR- CAR。

It is isolated and purified from expression product and obtains α FR-CAR.To the α FR-CAR of acquisition through N/C terminal sequence analysis, as a result Show that expressed α FR-CAR amino acid sequence is errorless, it is consistent with theoretical N/C terminal amino acid sequence.

Therefore, it can be seen that, the amino acid sequence of α FR-CAR as shown in SEQ ID NO.12, specifically:

MALPVTALLLPLALLLHAARPQSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPK LMIYEGSKRPSGVSNRFSGSKSGNAASLTISGLQAEDEADYYCQSYDSSLSVVFGGGTKLTVLGGGGSGGGGSGGG GSQVQLVESGGGLVQPGRSLRLSCTTSGFTFGDYAMIWARQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDN AKNSLYLQMNSLRAEDTAVYYCARERYDFWSGMDVWGKGTTVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAKPTTTPAPRPPTPAPTIASQPLSLR PEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHY QPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAEPPAYQQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR。

The amino acid sequence of people's CD8 alpha signal peptide as shown in SEQ ID NO.13, specifically:

MALPVTALLLPLALLLHAARP。

The amino acid sequence of the single-stranded variable region (scFv) of anti-folacin receptor α (α FR) is as shown in SEQ ID NO.14, specifically Are as follows:

QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEGSKRPSGVSNRFSGSK SGNAASLTISGLQAEDEADYYCQSYDSSLSVVFGGGTKLTVLGGGGSGGGGSGGGGSQVQLVESGGGLVQPGRSLR LSCTTSGFTFGDYAMIWARQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYY CARERYDFWSGMDVWGKGTTVTVSS。

Light chain variable region (the V of the single-stranded variable region (scFv) of anti-folacin receptor α (α FR)_L) amino acid sequence such as SEQ Shown in ID NO.15, specifically:

QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEGSKRPSGVSNRFSGSK SGNAASLTISGLQAEDEADYYCQSYDSSLSVVFGGGTKLTVL。

Heavy chain variable region (the V of the single-stranded variable region (scFv) of anti-folacin receptor α (α FR)_H) amino acid sequence such as SEQ Shown in ID NO.16, specifically:

QVQLVESGGGLVQPGRSLRLSCTTSGFTFGDYAMIWARQAPGKGLEWVSSISSSSSYIYYADSVKGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCARERYDFWSGMDVWGKGTTVTVSS。

The amino acid sequence that 1Fc sections of immunoglobulin G as shown in SEQ ID NO.17, specifically:

EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK。

The amino acid sequence of people's CD8 α hinge area as shown in SEQ ID NO.18, specifically:

AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD。

The amino acid sequence of people's CD28 cross-film section as shown in SEQ ID NO.19, specifically:

FWVLVVVGGVLACYSLLVTVAFIIFWV。

The amino acid sequence that intracellular section of people CD28 as shown in SEQ ID NO.20, specifically:

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS。

The amino acid sequence that intracellular section of people CD137 as shown in SEQ ID NO.21, specifically:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL。

The amino acid sequence of intracellular section of people CD3 ζ as shown in SEQ ID NO.22, specifically:

RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

Embodiment 2 targets the slow virus preparation of the Chimeric antigen receptor expression of folacin receptor α

1, equipment and material are as shown in table 1 below.

Equipment and material needed for table 1 expresses slow virus carrier

Reagent and material	Company	Article No.
			DMEM culture medium	Gibco	11960044
Fetal calf serum (FBS)	Gibco	16140
			1X D-PBS	Beyotime	C0221D
PolyJet^TM DNATransfection Reagent	SignaGen	SL100688
			Disposable aspiration needle filter 0.45um	Millipore	SLHV033RB
Ultra-clear SW28 centrifuge tube	Beckman
			L90K supercentrifuge, band SW28 rotor	Beckman
293T cell strain	FuHengBioLogy	FH0823

2, concrete operation step:

2.1. the packaging of slow virus:

After the 293T cell pancreatin of culture is digested and washed, it is resuspended with DMEM complete medium (containing 10%FBS) thin Cell suspension is made in born of the same parents, counts cell and calculates concentration of cell suspension.With 1 × 10⁵/cm²Density by 293T cell inoculation in In 10cm culture dish.According to PolyJet^TMDescribed in DNATransfection Reagent specification, in 1.5ml serum-free DMEM Plasmid DNA, the 3 μ g pLP1 plasmids, 3 μ g pLP2 plasmids, 3 μ g pLP/VSVG plasmids of 3 μ g building are added in culture medium, gently Concussion mixes.36 μ l PolyJet are added in 1.5ml plasma-free DMEM medium^TMTransfection reagent, gently concussion mixes.Then 1.5ml plasma-free DMEM medium containing transfection reagent is added in the 1.5ml plasma-free DMEM medium containing plasmid, is gently mixed It is stored at room temperature after even, compound to be transfected is formed.After twenty minutes, it is compound that transfection is added dropwise into inoculated 293T cell Object, and culture medium total volume is mended to 10ml, it is put into incubator, 5%CO₂, 37 DEG C of cultures.It moves back within 18 hours except compound containing transfecting The culture medium supernatant of object rejoins fresh DMEM complete medium.After 72 hours, on the culture medium of collection 293T cell Clearly, spare after being filtered with the filter that aperture is 0.45 μm.

2.2. the concentration of slow virus:

2.2.1. 6 Ultra-clear SW28 centrifuge tubes is taken to put and beat in superclean bench after 70% ethanol disinfection It opens ultraviolet lamp and continues disinfection 30 minutes.

2.2.2. the pretreated vial supernatant of about 32ml is added in each Ultra-clear SW28 centrifuge tube.

2.2.3. the pipette of a 10ml is taken, the sucrose solution of 12ml 20% is drawn.Pipette is inserted into always from The bottom of heart pipe, slowly gets 4ml for sucrose solution.Similarly, by the sucrose solution of remaining 8ml be added separately to another two from In heart pipe.A clean pipette separately is taken, remaining 3 pipes are equally handled.

2.2.4. the weight that each pipe is adjusted with PBS differs the weight between corresponding centrifuge tube and is no more than 0.1g.

2.2.5. all 6 centrifuge tubes are put into Beckman SW28 ultracentrifugation rotary head in order.

2.2.6.4 DEG C, 25000rpm (82700g) is centrifuged 2 hours.

2.2.7. carefully pipe is taken out from rotary head.Supernatant is outwelled, centrifuge tube is tipped upside down on paper handkerchief and is placed 10 minutes Remaining supernatant is set to drain off.Sop up remaining drop.There should be visible precipitating in tube bottom.

2.2.8. 100ml being added in every pipe, the PBS of calcic and magnesium does not wash lower precipitating.

2.2.9. SW28 ultracentrifugation pipe is inserted into 50ml cone bottom centrifuge tube, is closed the lid.

2.2.10. it dissolves 2 hours at 4 DEG C, was gently shaken every 20 minutes.

2.2.11.4 DEG C, 500g is centrifuged 1 minute, and solution is made to concentrate on tube bottom.

2.2.12. softly being blown and beaten with 200 μ l pipettors is resuspended precipitating.It avoids generating foam.By the liquid in all pipes It focuses in a SW28 centrifuge tube.

2.2.13. the viral suspension after concentrating is distributed into 50 every part of μ l, is stored in production tube.With being stored up after broken dry ice quick-frozen In the presence of -80 DEG C.

By aforesaid operations, the Chimeric antigen receptor expression slow virus of targeting folacin receptor α is prepared.

Embodiment 3 targets the building of the Chimeric antigen receptor modification NK-92 cell of folacin receptor α

It is thin that the Chimeric antigen receptor for the targeting folacin receptor α being prepared using embodiment 2 expresses slow-virus infection NK-92 Born of the same parents, concrete operations are as follows:

1. by NK-92 cell inoculation in 24 orifice plates, every hole 5 × 10⁶A cell.

2. the interleukin 2 that 200U/ml is added in every hole maintains cell Proliferation.

3.5%CO₂, 37 DEG C are cultivated 24 hours.

4. the Chimeric antigen receptor of the targeting folacin receptor α of 50 μ l embodiments 2 preparation is added into NK-92 cell culture well Slow virus concentrate and the polybrene of 5mg/ml are expressed, is mixed.

5.5%CO₂, 37 DEG C of overnight incubations.

6. removing old culture medium supernatant, the fresh NK-92 cell that the interleukin 2 containing 200U/ml is added is cultivated completely Base.

7.5%CO₂, 37 DEG C are cultivated 3 days.

8. adding into the NK-92 cell culture medium for the Chimeric antigen receptor expression slow virus for having infected targeting folacin receptor α Enter 5 μ g/ml puromycins, 5%CO₂, 37 DEG C are cultivated 14 days.Wherein, when changing liquid or passage every time, 5 μ are added into culture medium G/ml puromycin.

9. collecting cell and being detected using flow cytometry to analyze the Chimeric antigen receptor of targeting folacin receptor α and exist Expression in NK-92 cell.

Be screened out from it the NK-92 cell for being successfully made genetic modification, that is, target the chimeric antigen of folacin receptor α by Body modifies NK-92 cell (NK-92- α FR-CAR).

Next, the folacin receptor alpha expression for identifying different human tumor cell lines is horizontal, and by its with targeting folic acid by Chimeric antigen receptor modification NK-92 cell (NK-92- α FR-CAR) of body α co-cultures, the inosculating antibody of observation targeting folacin receptor α Original receptor modifies the effector function of NK-92 cell (NK-92- α FR-CAR): specific operating procedure are as follows:

1. by abortion syndrome SK-OV-3 and A2780, human colon cancer cell line HCT 116, the people's epidermal carcinoma of culture After cell line A-431 is digested and is washed with pancreatin respectively, with phosphate buffered saline solution (phosphate buffer saline, PBS cell) is resuspended, cell suspension is made.

2. counting cell and calculating every kind of concentration of cell suspension, 1 × 10 is extracted from every kind of cell suspension respectively⁶It is a thin Born of the same parents are respectively resuspended in 0.1ml PBS.

3. being separately added into 1 μ l PE- into the 0.1ml cell suspension of the resulting every kind of tumor cell line of step 2 Conjugated mouse anti-human FOLR1 antibody (R&D SYSTEMS company) is incubated at room temperature 30 minutes.

4. washed once with PBS, cell then is resuspended with 0.1ml PBS.

5. the selection detection PE fluorescence channel in the operating software of flow cytometer, then loading respectively, is obtained different The folacin receptor alpha expression of human tumor cell line is horizontal.

Fig. 3 is flow cytometry results, display, in different human tumor cell lines, ovarian cancer cell line SK-OV-3 and The all high expression folacin receptor α of A2780.

Fig. 4 is cell killing experimental result, and the Chimeric antigen receptor of display, targeting folacin receptor α modifies NK-92 cell (NK-92- α FR-CAR) is most strong to the lethal effect of SK-OV-3 cell and A2780 cell.

Fig. 5 is ELISA as a result, display, the Chimeric antigen receptor of targeting folacin receptor α modify NK-92 cell (NK-92- α When FR-CAR) co-culturing with SK-OV-3 cell or A2780 cell, more IFN-γ can be secreted.

The Chimeric antigen receptor of 4 experiment in vivo of embodiment verifying targeting folacin receptor α modifies NK-92 cell (NK-92- α FR-CAR antitumor action)

NK-92 cell (NK-92- α is modified using the Chimeric antigen receptor of targeting folacin receptor α constructed in embodiment 3 FR-CAR), human ovarian cancer transplantable tumor is established in severe immunodeficient mouse B-NDG using abortion syndrome SK-OV-3 Model, experiment in vivo inquire into Chimeric antigen receptor modification NK-92 cell (NK-92- α FR-CAR) of targeting folacin receptor α to ovum The lethal effect of nest cancer cell.

Fig. 6 show 20 B-NDG mouse peritoneal inoculations 1 × 10⁶A SK-OV-3 cell, after inoculation 14 days, tumor-bearing mice Grouping receives different treatments, wherein and phosphate buffered saline solution (PBS) organizes tumor-bearing mice abdominal cavity and is transfused phosphate buffered saline solution, The group tumor-bearing mice abdominal cavity NK-92 infusion 1 × 10⁶A NK-92 cell, the group tumor-bearing mice abdominal cavity NK-92-EV infusion 1 × 10⁶A NK- 92-EV cell, the group tumor-bearing mice abdominal cavity α FR-CAR NK-92- infusion 1 × 10⁶The Chimeric antigen receptor of a targeting folacin receptor α It modifies NK-92 cell (NK-92- α FR-CAR), above-mentioned four kinds of therapeutic modalities are to carry out within every 3 days once, and totally 3 times.

Fig. 7 is human ovarian cancer Transplanted tumor model, and the Chimeric antigen receptor of display, targeting folacin receptor α modifies NK-92 cell (NK-92- α FR-CAR) can significantly inhibit tumour growth.

Fig. 8 is human ovarian cancer Transplanted tumor model, and the Chimeric antigen receptor of display, targeting folacin receptor α modifies NK-92 cell (NK-92- α FR-CAR) can significantly extend tumor-bearing mice life span.

From experimental result of the invention: being expressed using the Chimeric antigen receptor for the targeting folacin receptor α being prepared After slow-virus infection NK-92 cell, the Chimeric antigen receptor of targeting folacin receptor α can be expressed in NK-92 cell, this says It is bright, NK-92 cell (NK-92- α FR- is modified using the Chimeric antigen receptor that this method can successfully construct targeting folacin receptor α CAR).In addition, experiment in vitro shows to target Chimeric antigen receptor modification NK-92 cell (the NK-92- α FR- of folacin receptor α When CAR) co-culturing from different human tumor cell lines, the ovarian cancer cell line SK- of high expression folacin receptor α can be significantly killed OV-3 and A2780 simultaneously secretes more cell factor IFN-γ；Human ovarian cancer Transplanted tumor model shows, infusion targeting folic acid by After Chimeric antigen receptor modification NK-92 cell (NK-92- α FR-CAR) of body α, the intracorporal tumour growth of mouse is obviously inhibited, And receives PBS or compare the mouse of NK-92 cell infusion then without this phenomenon, moreover, receiving the chimeric antigen of targeting folacin receptor α The mouse survival time that receptor modifies NK-92 cell (NK-92- α FR-CAR) infusion is more long, these results explanation, the target of building It can specific efficient killing folacin receptor to the Chimeric antigen receptor of folacin receptor α modification NK-92 cell (NK-92- α FR-CAR) α positive tumor cell.

Sequence table

<110>Chongqing good fortune U.S. stem cell biological development in science and technology Co., Ltd

<120>Chimeric antigen receptor and its use in preparation prevention or treatment malignant tumor medicine for targeting folacin receptor α On the way

<130> A190058K

<141> 2019-01-30

<160> 22

<170> SIPOSequenceListing 1.0

<210> 1

<211> 63

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

atggccctcc cagttaccgc ccttctcctg cccctggccc tgctgctgca cgccgcccgc 60

ccc 63

<210> 2

<211> 330

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagag catcaccatc 60

tcctgcactg gaaccagcag tgatgttggg agttataacc tggtctcctg gtaccagcag 120

cacccaggca aagcccccaa actcatgatt tatgagggca gtaagcggcc ctcaggggtt 180

tctaatcgct tctctggctc caagtctggc aacgccgcct ccctgacaat ctctgggctc 240

caggctgagg acgaggctga ttattactgc cagtcctatg acagcagcct gagtgtggtg 300

ttcggcggag ggaccaagct gaccgtcctc 330

<210> 3

<211> 360

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

caggtgcagc tggtggagtc tgggggaggc ctggtgcagc cagggcggtc cctgagactc 60

tcctgcacaa cttctggatt cacttttggc gattatgcta tgatctgggc ccgccaggct 120

ccagggaagg ggctggagtg ggtctcatcc attagtagta gtagtagtta catctactac 180

gcagactcag tgaagggcag attcaccatc tccagagaca acgccaagaa ctcactgtat 240

ctgcagatga acagcctgag agccgaggac accgctgtgt attactgtgc cagagaacgg 300

tacgattttt ggagtggaat ggacgtctgg ggcaaaggga ccaccgtcac cgtgagcagt 360

<210> 4

<211> 735

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagag catcaccatc 60

tcctgcactg gaaccagcag tgatgttggg agttataacc tggtctcctg gtaccagcag 120

cacccaggca aagcccccaa actcatgatt tatgagggca gtaagcggcc ctcaggggtt 180

tctaatcgct tctctggctc caagtctggc aacgccgcct ccctgacaat ctctgggctc 240

caggctgagg acgaggctga ttattactgc cagtcctatg acagcagcct gagtgtggtg 300

ttcggcggag ggaccaagct gaccgtcctc ggaggaggcg gatcaggcgg aggaggctct 360

ggcggaggcg gaagccaggt gcagctggtg gagtctgggg gaggcctggt gcagccaggg 420

cggtccctga gactctcctg cacaacttct ggattcactt ttggcgatta tgctatgatc 480

tgggcccgcc aggctccagg gaaggggctg gagtgggtct catccattag tagtagtagt 540

agttacatct actacgcaga ctcagtgaag ggcagattca ccatctccag agacaacgcc 600

aagaactcac tgtatctgca gatgaacagc ctgagagccg aggacaccgc tgtgtattac 660

tgtgccagag aacggtacga tttttggagt ggaatggacg tctggggcaa agggaccacc 720

gtcaccgtga gcagt 735

<210> 5

<211> 696

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

gagcccaaat ctagcgacaa aactcacaca tgcccacctt gcccagcacc tgagctcctg 60

gggggacctt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120

acccctgggg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagcctcg ggaggagcag 240

tacaacagca cctacagagt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360

atctccaaag ccaaagggca gcccagagaa ccacaggtgt acaccctgcc cccatcccgg 420

gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 480

gacatcgccg tggagtggga gagcaatggg cagcctgaga acaactacaa gaccacccct 540

cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660

tacacccaga agagcctctc cctgtctcct ggcaaa 696

<210> 6

<211> 144

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

gccaagccca ccactacacc cgccccacgc ccccccaccc ccgcccccac catcgccagc 60

cagcccctga gcctgcgccc cgaggcctgc cgccccgccg ccggcggcgc cgtgcacacc 120

cgcggcctgg acttcgcctg cgac 144

<210> 7

<211> 81

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

ttctgggtgc tggtggtggt gggcggcgtg ctggcctgct acagcctgct ggtgaccgtg 60

gccttcatca tcttctgggt g 81

<210> 8

<211> 123

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

cgcagcaagc gcagccgcct gctgcacagc gactacatga acatgacccc ccgccgcccc 60

ggccccaccc gcaagcacta ccagccctac gccccccccc gcgacttcgc cgcctaccgc 120

agc 123

<210> 9

<211> 126

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

aagcgcggcc gcaagaagct gctgtacatc ttcaagcagc ccttcatgcg ccccgtgcag 60

accacccagg aggaggacgg ctgcagctgc cgcttccccg aagaagagga gggcggctgc 120

gagctg 126

<210> 10

<211> 339

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

cgcgtgaagt tcagccgcag cgccgagccc cccgcctacc agcagggcca gaaccagctg 60

tacaacgagc tgaacctggg ccgccgcgag gagtacgacg tgctggacaa gcgccgcggc 120

cgcgaccccg agatgggcgg caagccccgc cgcaagaacc cccaggaggg cctgtacaac 180

gagctgcaga aggacaagat ggccgaggcc tacagcgaga tcggcatgaa gggcgagcgc 240

cgccgcggca agggccacga cggcctgtac cagggcctga gcaccgccac caaggacacc 300

tacgacgccc tgcacatgca ggccctgccc ccccgctga 339

<210> 11

<211> 2307

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

atggccctcc cagttaccgc ccttctcctg cccctggccc tgctgctgca cgccgcccgc 60

ccccagtctg ccctgactca gcctgcctcc gtgtctgggt ctcctggaca gagcatcacc 120

atctcctgca ctggaaccag cagtgatgtt gggagttata acctggtctc ctggtaccag 180

cagcacccag gcaaagcccc caaactcatg atttatgagg gcagtaagcg gccctcaggg 240

gtttctaatc gcttctctgg ctccaagtct ggcaacgccg cctccctgac aatctctggg 300

ctccaggctg aggacgaggc tgattattac tgccagtcct atgacagcag cctgagtgtg 360

gtgttcggcg gagggaccaa gctgaccgtc ctcggaggag gcggatcagg cggaggaggc 420

tctggcggag gcggaagcca ggtgcagctg gtggagtctg ggggaggcct ggtgcagcca 480

gggcggtccc tgagactctc ctgcacaact tctggattca cttttggcga ttatgctatg 540

atctgggccc gccaggctcc agggaagggg ctggagtggg tctcatccat tagtagtagt 600

agtagttaca tctactacgc agactcagtg aagggcagat tcaccatctc cagagacaac 660

gccaagaact cactgtatct gcagatgaac agcctgagag ccgaggacac cgctgtgtat 720

tactgtgcca gagaacggta cgatttttgg agtggaatgg acgtctgggg caaagggacc 780

accgtcaccg tgagcagtga gcccaaatct agcgacaaaa ctcacacatg cccaccttgc 840

ccagcacctg agctcctggg gggaccttca gtcttcctct tccccccaaa acccaaggac 900

accctcatga tctcccggac ccctggggtc acatgcgtgg tggtggacgt gagccacgaa 960

gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 1020

aagcctcggg aggagcagta caacagcacc tacagagtgg tcagcgtcct caccgtcctg 1080

caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1140

gcccccatcg agaaaaccat ctccaaagcc aaagggcagc ccagagaacc acaggtgtac 1200

accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 1260

aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gcctgagaac 1320

aactacaaga ccacccctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 1380

ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1440

gaggctctgc acaaccacta cacccagaag agcctctccc tgtctcctgg caaagccaag 1500

cccaccacta cacccgcccc acgccccccc acccccgccc ccaccatcgc cagccagccc 1560

ctgagcctgc gccccgaggc ctgccgcccc gccgccggcg gcgccgtgca cacccgcggc 1620

ctggacttcg cctgcgactt ctgggtgctg gtggtggtgg gcggcgtgct ggcctgctac 1680

agcctgctgg tgaccgtggc cttcatcatc ttctgggtgc gcagcaagcg cagccgcctg 1740

ctgcacagcg actacatgaa catgaccccc cgccgccccg gccccacccg caagcactac 1800

cagccctacg cccccccccg cgacttcgcc gcctaccgca gcaagcgcgg ccgcaagaag 1860

ctgctgtaca tcttcaagca gcccttcatg cgccccgtgc agaccaccca ggaggaggac 1920

ggctgcagct gccgcttccc cgaagaagag gagggcggct gcgagctgcg cgtgaagttc 1980

agccgcagcg ccgagccccc cgcctaccag cagggccaga accagctgta caacgagctg 2040

aacctgggcc gccgcgagga gtacgacgtg ctggacaagc gccgcggccg cgaccccgag 2100

atgggcggca agccccgccg caagaacccc caggagggcc tgtacaacga gctgcagaag 2160

gacaagatgg ccgaggccta cagcgagatc ggcatgaagg gcgagcgccg ccgcggcaag 2220

ggccacgacg gcctgtacca gggcctgagc accgccacca aggacaccta cgacgccctg 2280

cacatgcagg ccctgccccc ccgctga 2307

<210> 12

<211> 768

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 12

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

1 5 10 15

His Ala Ala Arg Pro Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser

20 25 30

Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser

35 40 45

Asp Val Gly Ser Tyr Asn Leu Val Ser Trp Tyr Gln Gln His Pro Gly

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Gly Arg Ser Leu Arg Leu Ser Cys Thr Thr Ser Gly Phe Thr Phe Gly

165 170 175

Asp Tyr Ala Met Ile Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu Glu

180 185 190

Trp Val Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp

195 200 205

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

210 215 220

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

225 230 235 240

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

245 250 255

Gly Lys Gly Thr Thr Val Thr Val Ser Ser Glu Pro Lys Ser Ser Asp

260 265 270

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

275 280 285

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

290 295 300

Ser Arg Thr Pro Gly Val Thr Cys Val Val Val Asp Val Ser His Glu

305 310 315 320

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

325 330 335

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

340 345 350

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

355 360 365

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

370 375 380

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

385 390 395 400

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

405 410 415

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

420 425 430

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

435 440 445

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

450 455 460

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

465 470 475 480

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

485 490 495

Gly Lys Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro

500 505 510

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

515 520 525

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

530 535 540

Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr

545 550 555 560

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

565 570 575

Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg

580 585 590

Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp

595 600 605

Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

<210> 13

<211> 21

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 13

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

1 5 10 15

His Ala Ala Arg Pro

20

<210> 14

<211> 245

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 14

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

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr

20 25 30

Asn Leu Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

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

50 55 60

Ser Gly Ser Lys Ser Gly Asn Ala Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser

85 90 95

Leu Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly

100 105 110

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

115 120 125

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

130 135 140

Leu Ser Cys Thr Thr Ser Gly Phe Thr Phe Gly Asp Tyr Ala Met Ile

145 150 155 160

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

165 170 175

Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys Gly Arg

180 185 190

Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met

195 200 205

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

210 215 220

Arg Tyr Asp Phe Trp Ser Gly Met Asp Val Trp Gly Lys Gly Thr Thr

225 230 235 240

Val Thr Val Ser Ser

245

<210> 15

<211> 110

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 15

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

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr

20 25 30

Asn Leu Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

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

50 55 60

Ser Gly Ser Lys Ser Gly Asn Ala Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser

85 90 95

Leu Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 16

<211> 120

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 16

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

1 5 10 15

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

20 25 30

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

35 40 45

Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Tyr Asp Phe Trp Ser Gly Met Asp Val Trp Gly Lys

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 17

<211> 232

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 17

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

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

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Gly Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

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

115 120 125

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

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

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

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 18

<211> 48

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 18

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

1 5 10 15

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

20 25 30

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

35 40 45

<210> 19

<211> 27

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 19

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 20

<211> 41

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 20

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 21

<211> 42

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 21

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> 22

<211> 112

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 22

Arg Val Lys Phe Ser Arg Ser Ala Glu Pro Pro Ala Tyr Gln 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

Claims

1. targeting the Chimeric antigen receptor of folacin receptor α, it is characterised in that: the composition of the Chimeric antigen receptor successively includes: People's CD8 alpha signal peptide, the polypeptide in conjunction with folacin receptor α, 1Fc sections of immunoglobulin G, people CD8 α hinge area, people's CD28 cross-film Section, intracellular section of people CD28, intracellular section of people CD137 and intracellular section of people CD3 ζ.

2. the Chimeric antigen receptor of targeting folacin receptor α according to claim 1, it is characterised in that: the combination folic acid The C-terminal of the polypeptide of receptor alpha is connect with immunoglobulin G 1Fc sections of the N-terminal.

3. the Chimeric antigen receptor of targeting folacin receptor α according to claim 1 or 2, it is characterised in that: the combination leaf The polypeptide of acid acceptor α is the single-stranded variable region scFv of the antibody of anti-folacin receptor α.

4. it is according to claim 3 targeting folacin receptor α Chimeric antigen receptor, it is characterised in that: the anti-folic acid by The single-stranded variable region scFv of body α includes light chain variable region and heavy chain variable region.

5. the Chimeric antigen receptor of targeting folacin receptor α according to claim 1-4, it is characterised in that: described The amino acid sequence of the Chimeric antigen receptor of folacin receptor α is targeted as shown in SEQ ID NO.12.

6. encoding the polynucleotide of the described in any item Chimeric antigen receptors of claim 1-5.

7. polynucleotide according to claim 6, which is characterized in that its nucleotide sequence such as SEQ ID NO.11 institute Show.

8. expressing the expression vector of the described in any item Chimeric antigen receptors of claim 1-5.

9. being wanted containing nucleotide described in the described in any item Chimeric antigen receptors of claim 1-5, claim 6 or 7, right The host cell of expression vector described in asking 8.

10. host cell according to claim 9, it is characterised in that: the host cell is NK-92 cell, the primary T of people One of cell, people's primary NK cells or cytokine induced kill cell.

11. the method for preparing the described in any item Chimeric antigen receptors of claim 1-5, which comprises the following steps: The expression vector of Chimeric antigen receptor polynucleotides encoding sequence of the building containing targeting folacin receptor α, then will be stated Expression vector converts into host cell inducing expression, and separation obtains the chimeric of the targeting folacin receptor α from expression product Antigen receptor.

12. nucleotide, claim 8 described in the described in any item Chimeric antigen receptors of claim 1-5, claim 6 or 7 Host cell described in the expression vector, claim 9 or 10 is in the drug of preparation prevention or treatment malignant tumour Purposes.

13. purposes according to claim 12, it is characterised in that: the malignant tumour is folacin receptor α positive malignant Tumour.

14. purposes according to claim 12, it is characterised in that: the malignant tumour includes oophoroma, endometrium At least one of cancer, breast cancer, lung cancer or colon cancer.