CN110526975A - Target single-chain antibody, the Chimeric antigen receptor T cell and its preparation method and application of CEA - Google Patents

Target single-chain antibody, the Chimeric antigen receptor T cell and its preparation method and application of CEA Download PDF

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Publication number
CN110526975A
CN110526975A CN201810513874.5A CN201810513874A CN110526975A CN 110526975 A CN110526975 A CN 110526975A CN 201810513874 A CN201810513874 A CN 201810513874A CN 110526975 A CN110526975 A CN 110526975A
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cea
cell
targeting
gly
ser
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万晓春
唐超
李欣
刘绿艳
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Shenzhen Benta Biological Technology Co Ltd
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Shenzhen Benta Biological Technology Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57449—Specifically defined cancers of ovaries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
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    • C12N2510/00—Genetically modified cells
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Abstract

The present invention provides a kind of single-chain antibody for targeting CEA, the single-chain antibody of the targeting CEA includes the amino acid sequence as shown in SEQ ID NO:1.The present invention also provides a kind of Chimeric antigen receptor T cells of single-chain antibody including the targeting CEA, the Chimeric antigen receptor for wherein targeting CEA can in specific manner on target tumor CEA, T cell is activated to play immunization of cell, the malignant cell of the efficient and specific killing CEA positive, has lasting cell viability and lethality.The present invention also provides a kind of preparation method and application of Chimeric antigen receptor T cell for targeting CEA.

Description

Target the single-chain antibody of CEA, Chimeric antigen receptor T cell and preparation method thereof and Using
Technical field
The present invention relates to field of medical biotechnology, in particular to a kind of single-chain antibody, Chimeric antigen receptor T for targeting CEA is thin Born of the same parents and its preparation method and application.
Background technique
Carcinomebryonic antigen (carcinoembryonic antigen, CEA) is a kind of acid with human embryos antigenic characteristic Property glycoprotein, gains the name because being originally found in colon cancer and embryonic tissue.CEA is colorectal cancer (colon and rectum carcinoma), stomach The tumor markers of cancer, breast cancer, lung cancer, oophoroma, medullary carcinoma of thyroid gland etc., antidiastole, the state of an illness in malignant tumour Monitoring, therapeutic evaluation etc. have important clinical value.
CAR-T (Chimeric antigen receptor T cell) technology is a kind of novel cell therapy, it is will be by the T of CA R transformation Cell is fed back to human body, activates self immune system, kills to tumour cell, it is considered to be most effective at present pernicious swollen One of therapeutic modality of tumor, the drawbacks of traditional remedies can be made up.Currently, actually rare for the CAR-T cell of CEA design.
Summary of the invention
In view of this, the present invention provides a kind of single-chain antibodies for targeting CEA, and including the single-stranded of the targeting C EA The Chimeric antigen receptor T cell of antibody.The Chimeric antigen receptor of the targeting CEA can be swashed with the tumour cell of targeted expression CEA T cell living plays immunization of cell, efficiently and specifically kills CEA positive tumor cell, preferably maintenance chimeric antigen The vigor and lethality of recipient T cells.The present invention also provides a kind of preparation sides of Chimeric antigen receptor T cell for targeting CEA Method and related application.
In a first aspect, the present invention provides a kind of single-chain antibody for targeting CEA, the single-chain antibody of the targeting CEA includes The amino acid sequence as shown in SEQ ID NO:1.
Optionally, the encoding gene of the single-chain antibody of the targeting CEA includes the nucleotides sequence as shown in SEQ ID NO:2 Column.
Optionally, the single-chain antibody encoding gene of the targeting CEA should consider degeneracy base, i.e., such as SEQ ID NO:1 Shown in the encoding gene of amino acid sequence include the nucleotide sequence as shown in SEQ ID NO:2, protection scope should also protect Shield has the nucleotide sequence of base degeneracy matter with SEQ ID NO:2, and the corresponding amino acid sequence of these nucleotide sequences is still It is so SEQ ID NO:1.
The single-chain antibody for the targeting CEA that first aspect present invention provides, can be on specific recognition tumour cell CEA albumen, and specifically bound with it, there is stronger affine activity and internalization to the malignant cell of expression CEA Activity.
Second aspect, the present invention provides a kind of Chimeric antigen receptor T cells for targeting CEA, including the embedding of targeting C EA Close antigen receptor CAR-CEA, the CAR-CEA include from aminoterminal to c-terminus it is sequentially connected targeting CEA single-chain antibody, The amino acid sequence of extracellular hinge area, transmembrane region and intracellular signal area, wherein the single-chain antibody of the targeting CEA includes such as SEQ Amino acid sequence shown in ID NO:1.
Wherein, described " being sequentially connected with from aminoterminal to c-terminus " specifically: the amino of the single-chain antibody of the targeting CEA The c-terminus of acid sequence is connected with the aminoterminal of the amino acid sequence of the extracellular hinge area, the amino acid of the extracellular hinge area The c-terminus of sequence is connected with the aminoterminal of the amino acid sequence of the transmembrane region, the carboxyl of the amino acid sequence of the transmembrane region End is connected with the aminoterminal of the amino acid sequence in the intracellular signal area.
In the present invention, the extracellular hinge area is used to promote the C EA knot on the single-chain antibody and tumour of the targeting CEA It closes.Optionally, the extracellular hinge area includes CD8 α hinge area, CD28 hinge area, CD4 hinge area, CD5 hinge area, CD134 hinge One of sequence, 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:6.
Optionally, the encoding gene of the CD8 α hinge area includes the nucleotide sequence as shown in SEQ ID NO:7.
Optionally, the encoding gene of the CD8 α hinge area should consider degeneracy base, i.e., as shown in SEQ ID N O:6 Amino acid sequence encoding gene include the nucleotide sequence such as SEQ ID NO:7 shown in, protection scope should also protect and SEQ ID NO:7 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:6。
In the present invention, the transmembrane region is used to fix the Chimeric antigen receptor CAR-CEA of the targeting CEA.Optionally, institute Stating transmembrane region includes one of CD3 transmembrane region, CD4 transmembrane region, CD8 transmembrane region, CD28 transmembrane region or a variety of combinations.
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:8.
Optionally, the encoding gene of the CD8 transmembrane region includes the nucleotide sequence as shown in SEQ ID NO:9.
Optionally, the encoding gene of the CD8 transmembrane region should consider degeneracy base, i.e., as shown in SEQ ID NO:8 The encoding gene of amino acid sequence includes the nucleotide sequence as shown in SEQ ID NO:9, and protection scope should also protect and SEQ ID NO:9 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:8。
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.
Optionally, the intracellular signal Qu Weicong aminoterminal is believed to the sequentially connected 4-1BB signaling zone of c-terminus and CD3 ζ Number area.Correspondingly, the encoding gene in the intracellular signal area includes the volume from 5 ' ends to 3 ' the sequentially connected 4-1BB signaling zones in end The encoding gene of code gene and CD3 ζ signaling zone.
Wherein, CD3 ζ signaling zone is intracellular signal transduction structural domain (that is, first signaling zone), and 4-1BB signaling zone is to pierce altogether Swash structural domain, under their collective effect, T cell is activated completely after identifying antigen.Still optionally further, the cross-film The c-terminus of the amino acid sequence in area is connected with the aminoterminal of the amino acid sequence of the 4-1BB signaling zone, the 4-1BB signal The c-terminus of the amino acid sequence in area is connected with the aminoterminal of the amino acid sequence of the CD3 ζ signaling zone.
Optionally, the amino acid sequence of the 4-1BB signaling zone includes the amino acid sequence as shown in SEQ ID NO:10.
Optionally, the encoding gene of the 4-1BB signaling zone includes the nucleotide sequence as shown in SEQ ID NO:11.
Optionally, the encoding gene of the 4-1BB signaling zone should consider degeneracy base, i.e., such as SEQ ID N O:10 institute The encoding gene of the amino acid sequence shown includes the nucleotide sequence as shown in SEQ ID NO:11, and protection scope should also be protected There is the nucleotide sequence of base degeneracy matter with SEQ ID NO:11, the corresponding amino acid sequence of these nucleotide sequences is still For SEQ ID NO:10.
Optionally, the amino acid sequence of the CD3 ζ signaling zone includes the amino acid sequence as shown in SEQ ID NO:12.
Optionally, the encoding gene of the CD3 ζ signaling zone includes the nucleotide sequence as shown in SEQ ID NO:13.
Optionally, the encoding gene of the CD3 ζ signaling zone should consider degeneracy base, i.e., as shown in SEQ ID NO:12 Amino acid sequence encoding gene include the nucleotide sequence such as SEQ ID NO:13 shown in, protection scope should also protect and SEQ ID NO:13 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:12。
Optionally, the amino acid sequence of the CAR-CEA includes the amino acid sequence as shown in SEQ ID NO:3.
Optionally, the encoding gene of the CAR-CEA includes the nucleotide sequence as shown in SEQ ID NO:4.
Optionally, the encoding gene of the CAR-CEA should consider degeneracy base, the i.e. ammonia as shown in SEQ ID NO:3 The encoding gene of base acid sequence includes the nucleotide sequence as shown in SEQ ID NO:4, and protection scope should also protect and SEQ ID NO:4 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:3。
The Chimeric antigen receptor T cell (CAR-T cell) for the targeting CEA that second aspect of the present invention provides, including target To the Chimeric antigen receptor CAR-CEA of CEA, this receptor for the CAR-T cell, swell in specific manner by the pernicious of targeted expression CEA Oncocyte, after CAR-CEA and CEA protein binding, the intracellular signal area of the CAR-T cell is activated, and T cell is promoted to suffer from The intracorporal amplification of person, the malignant tumour of efficient and specific killing expression CEA.The CAR-T cell of the targeting CEA is being expressed The antidiastole of the malignant tumour of CEA, state of illness monitoring, therapeutic evaluation etc. have certain reference significance.
The third aspect, the present invention provides a kind of recombinant viral vector, the recombinant viral vector includes such as second aspect The encoding gene of the CAR-CEA of the Chimeric antigen receptor T cell of the targeting CEA.
Optionally, the encoding gene of the CAR-CEA includes the nucleotide sequence as shown in SEQ ID NO:4.
Optionally, the encoding gene of the CAR-CEA includes the nucleotide sequence as shown in SEQ ID NO:5.Such as SEQ Nucleotide sequence shown in ID NO:5 is compared with the nucleotide sequence as shown in SEQ ID NO:4, more coding bases of link peptide Cause.The encoding gene of the signal peptide can be expressed with Chimeric antigen receptor CAR-CEA described in guide to 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.
Further optionally, the slow virus carrier include pWPXLD carrier, pLEX-MCS carrier, pSico carrier and At least one of pCgpV carrier.Specifically, when the slow virus carrier is pWPXLD carrier, gained recombinant viral vector In, the encoding gene of CAR-CEA is located between I restriction enzyme site of I restriction enzyme site of BamH and EcoR of pWPXLD carrier.
The recombinant viral vector that third aspect present invention provides is a safe and reliable carrier tool, can be efficiently Shift the encoding gene of the CAR-CEA.The recombinant viral vector, which can be used for preparing, carries the CAR-CEA encoding gene Virus, and preparation targeting CEA Chimeric antigen receptor T cell, make the T cell continue, play consistently targeting, killing effect Power.
Fourth aspect, the present invention provides a kind of host cell, the host cell includes the weight as described in the third aspect Group viral vectors.
The host cell that fourth aspect present invention provides is used to provide and carries the recombinant virus as described in second aspect Body is assembled and is prepared the place for generating corresponding virus, and the something lost of the CAR-CEA is carried by virus prepared by host cell Communication breath, has strong infectivity.
Optionally, the host cell may include HEK293T cell, 293 cells, 293T cell, 293FT cell, SW480 cell, u87MG cell, HOS cell, COS1 cell or COS7 cell etc., but not limited to this.Still optionally further, described Host cell is HEK293T cell.
5th aspect, the present invention provides a kind of preparation methods of Chimeric antigen receptor T cell for targeting CEA, comprising:
(1) encoding gene of the Chimeric antigen receptor CAR-CEA of targeting CEA is provided, including is sequentially connected from 5 ' ends to 3 ' ends The encoding gene of the signal peptide connect, the encoding gene of single-chain antibody, the encoding gene of extracellular hinge area, transmembrane region for targeting CEA Encoding gene and intracellular signal area encoding gene, wherein it is described targeting CEA single-chain antibody encoding gene include such as Nucleotide sequence corresponding to amino acid sequence shown in SEQ ID NO:1；
(2) encoding gene of the CAR-CEA is inserted into pWPXLD carrier, obtains pWPXL D-CAR-CEA recombination Plasmid；
(3) it by the pWPXLD-CAR-CEA recombinant plasmid and envelope plasmid, packaging plasmid cotransfection host cell, obtains Recombinant slow virus；
(4) by the recombinant slow virus transfect CD3 positive t lymphocytes, through separation obtain targeting CEA chimeric antigen by Body T cell.
It is above-mentioned " from 5 ' end to 3 ' end be sequentially connected with " specifically: the coding gene sequence of the signal peptide 3 ' end with it is described Target CEA single-chain antibody encoding gene 5 ' end be connected, it is described targeting CEA single-chain antibody encoding gene 3 ' end with 5 ' ends of the encoding gene of the extracellular hinge area are connected, 3 ' ends of the encoding gene of the extracellular hinge area and the transmembrane region 5 ' ends of encoding gene be connected, the 3 ' ends and the 5 ' of the encoding gene in the intracellular signal area of the encoding gene of the transmembrane region End is connected.
The signal peptide is for instructing the Chimeric antigen receptor CAR-CEA expression to cell surface, institute in the present invention Signal peptide is stated to be cut in protein translation maturation by signal peptidase.
Optionally, the amino acid sequence of the signal peptide includes the amino acid sequence as shown in SEQ ID NO:14.
Optionally, the encoding gene of the signal peptide includes the nucleotide sequence as shown in SEQ ID NO:15.
Optionally, the encoding gene of the signal peptide should consider degeneracy base, the i.e. ammonia as shown in SEQ ID NO:14 The encoding gene of base acid sequence includes the nucleotide sequence as shown in SEQ ID NO:15, and protection scope should also protect and SEQ ID NO:15 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:14。
The extracellular hinge area, transmembrane region, the specific choice in intracellular signal area and corresponding coding gene sequence such as this hair Described in bright second aspect part, which is not described herein again.
Optionally, the encoding gene of the CAR-CEA includes corresponding to the amino acid sequence as shown in SEQ ID NO:16 Nucleotide sequence.
Still optionally further, the coding gene sequence of CAR-CEA nucleotide sequence as shown in SEQ ID NO:5. The nucleotide sequence as shown in SEQ ID NO:5 is compared with the nucleotide sequence as shown in SEQ ID NO:4, more connections The encoding gene of peptide, but when Chimeric antigen receptor CAR-CEA expression is to cell surface, the signal peptide is in protein translation It is cut in maturation by signal peptidase.Therefore, in the amino acid sequence of the Chimeric antigen receptor CAR-CEA translated into And not with the amino acid sequence as shown in SEQ ID N O:14.
The encoding gene of the CAR-CEA is inserted into pWPXLD carrier between I restriction enzyme site of BamH I and EcoR, and position After the EF1 α of pWPXLD carrier, using EF1 α as promoter.The encoding gene of the CAR-CEA is inserted into pWPXLD carrier When, initiation codon (such as ATG) can be added in 5 ' ends of the encoding gene of the CAR-CEA, with BamH1 digestion in pWPXLD carrier Site (ggatcc) is connected, and EcoR1 restriction enzyme site (g in terminator codon (such as TAA) and pWPXLD carrier can be added in 3 ' ends Aattc) it is connected, thus makes the encoding gene of the CAR-CEA between BamH1 and EcoR1 restriction enzyme site.
Optionally, the envelope plasmid is PMD2G, and the packaging plasmid is psPAX2, and the host cell is HEK293T Cell.
The envelope plasmid PMD2G encodes vesicular stomatitis virus glycoprotein capsid, the vesicular stomatitis virus sugar egg White capsid assists recombinant slow virus to adhere to cell membrane, and keeps 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 .
Optionally, the source of people peripheral blood mononuclear cells derives from autologous vein blood, autologous bone marrow, Cord blood and placenta Blood etc..
Still optionally further, the fresh peripheral acquired after cancer patient's operation one month, after chemicotherapy one month Blood or marrow.
Specifically, the acquisition process of the CD3 positive t lymphocytes is as follows: into peripheral blood mononuclear cells by certain CD3/CD28 immunomagnetic beads are added in ratio, 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.
6th aspect, the present invention provides the single-chain antibodies of targeting CEA as described in relation to the first aspect a kind of, such as second aspect Being fitted into for CEA is targeted made from the Chimeric antigen receptor T cell of the described targeting CEA or the preparation method as described in the 5th aspect Antigen receptor T cell, the recombinant viral vector as described in the third aspect or the host cell as described in fourth aspect are examined in preparation Application in disconnected and/treatment malignant tumour drug.Particularly, it can be used for diagnosing and/or treating the malignant tumour of expression CEA, Such as the cancers such as colorectal cancer (including colon and rectum carcinoma), gastric cancer, breast cancer, lung cancer, oophoroma.
The application specifically: provide a kind of kit, the kit includes targeting CEA described in first aspect Single-chain antibody, the Chimeric antigen receptor T cell for targeting CEA as described in second aspect, the recombinant virus as described in the third aspect One of carrier, host cell as described in fourth aspect 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-CEA 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.
Embodiment one
A kind of preparation method for the Chimeric antigen receptor T cell targeting CEA, comprising the following steps:
(1) gene order of the Chimeric antigen receptor CAR-CEA of preparation targeting CEA
Prepare respectively signal peptide, target the single-chain antibody of CEA, CD8 α hinge area, CD8 transmembrane region, 4-1B B signal area and The encoding gene of CD3 ζ signaling zone, for the encoding gene of the signal peptide as shown in SEQ ID NO:15, the targeting CEA's is single-stranded The encoding gene of antibody is as shown in SEQ ID NO:2, and the encoding gene of the CD8 α hinge area is as shown in SEQ ID NO:7, institute The encoding gene of CD8 transmembrane region is stated as shown in SEQ I D NO:9, the encoding gene of the 4-1BB signaling zone such as SEQ ID NO: Shown in 11, the encoding gene of the CD3 ζ signaling zone is as shown in SEQ ID NO:13.
By the method for PCR by above-mentioned signal peptide, target single-chain antibody, the CD8 α hinge area, CD8 transmembrane region, 4- of CEA 1BB signaling zone is successively connected together from 5 ' ends to 3 ' ends with the encoding gene of CD3 ζ signaling zone, obtains the inosculating antibody of targeting CEA The encoding gene of original receptor CAR-CEA, the encoding gene of the CAR-CEA is as shown in SEQ ID NO:5.
(2) pWPXLd-CAR-CEA recombinant plasmid is constructed
The encoding gene of CAR-CEA is inserted between BamH1 the and EcoR1 restriction enzyme site of pWPXLD carrier, and After pWPXLD carrier EF1 α, using EF1 α as promoter.When the encoding gene of the CAR-CEA is inserted into pWPXLD carrier, institute Initiation codon (such as ATG) and BamH1 restriction enzyme site phase in pWPXLD carrier can be added in the 5 ' ends for stating the encoding gene of CAR-CEA Even, 3 ' ends can also be added terminator codon (such as TAA) and be connected with EcoR1 restriction enzyme site in pWPXLD carrier.Then it is transferred to big Enterobacteria competent cell DH5 α carries out positive colony PCR identification and sequencing identification.By PCR product detected through gel electrophoresis and Sequencing identification meets target fragment size and sequence, successfully constructs pWPXLd-CAR-CEA recombinant plasmid, is as shown in Figure 1 PWPXLd-CAR-CEA recombinant plasmid.
(3) recombinant slow virus constructs
PWPXLd-CAR-CEA recombinant plasmid, packaging plasmid psPAX2, envelope plasmid pMD2G three cotransfection are entered into culture Good HEK293T cell.48h harvest saves in -80 DEG C of ultra low temperature freezers containing the supernatant of virus through 0.45 μm of membrane filtration； It is super to merge addition together with the viral supernatants of 48h harvest for supernatant of the 72h aftercrop containing virus, 0.45 μm of membrane filtration In fast centrifuge tube, be put into Beckman ultracentrifuge one by one, setting parameter of noncentricity be 25000rpm, centrifugation time 2h, Centrifuging temperature is 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 and saves Liquid, gently piping and druming is resuspended repeatedly；Through after completely dissolution, high speed centrifugation 10000rpm takes supernatant fluorescence spectrometry after being centrifuged 5min Titre, virus is according to 100 μ l, and 2 × 108A/mL packing, is stored in -80 DEG C of ultra low temperature freezers, obtains recombinant slow virus.
(4) preparation of the Chimeric antigen receptor T cell of CEA is targeted
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, are added and CD3 positive cell number phase The recombinant slow virus for the virus titer answered is cultivated.
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 by the 9-11 days, obtained the Chimeric antigen receptor T cell of targeting CEA, and protect In the presence of in the dedicated cells frozen storing liquid of feedback.
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 Xianjin Technology Academe
<120>single-chain antibody, the Chimeric antigen receptor T cell and its preparation method and application of CEA are targeted
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 250
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Gly Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
1 5 10 15
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Thr Ser Ser Ser
20 25 30
Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
35 40 45
Leu Leu Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg
50 55 60
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser
65 70 75 80
Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys His Gln Trp Ser Ser
85 90 95
Tyr Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly
100 105 110
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro
130 135 140
Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Ile Ser
145 150 155 160
Ser Gly Tyr Ser Trp His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu
165 170 175
Glu Trp Ile Gly Tyr Ile Gln Tyr Ser Gly Ile Thr Asn Tyr Asn Pro
180 185 190
Ser Leu Lys Ser Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln
195 200 205
Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
210 215 220
Tyr Cys Ala Arg Glu Asp Tyr Asp Tyr His Trp Tyr Phe Asp Val Trp
225 230 235 240
Gly Gln Gly Ser Leu Val Thr Val Thr Val
245 250
<210> 2
<211> 750
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggtgtccact ccgacatcca gatgacccag agcccaagca gcctgagcgc cagcgtgggt 60
gacagagtga ccatcacctg tagtaccagc tcgagtgtaa gttacatgca ctggtaccag 120
cagaagccag gtaaggctcc aaagctgctg atctacagca catccaacct ggcttctggt 180
gtgccaagca gattcagcgg tagcggtagc ggtaccgact tcaccttcac catcagcagc 240
ctccagccag aggacatcgc cacctactac tgccatcagt ggagtagtta tcccacgttc 300
ggccaaggga ccaaggtgga aatcaaacgt ggaggcggag gatctggcgg cggaggaagt 360
ggcggagggg gatctggggg aggcggaagc caggtccaac tgcaggagag cggtccaggt 420
cttgtgagac ctagccagac cctgagcctg acctgcaccg tgtctggctt caccatcagc 480
agtggttata gctggcactg ggtgagacag ccacctggac gaggtcttga gtggattgga 540
tacatacagt acagtggtat cactaactac aacccctctc tcaaaagtag agtgacaatg 600
ctggtagaca ccagcaagaa ccagttcagc ctgagactca gcagcgtgac agccgccgac 660
accgcggtct attattgtgc aagagaagac tatgattacc actggtactt cgatgtctgg 720
ggtcaaggca gcctcgtcac agtcacagtc 750
<210> 3
<211> 473
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Gly Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
1 5 10 15
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Thr Ser Ser Ser
20 25 30
Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
35 40 45
Leu Leu Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg
50 55 60
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser
65 70 75 80
Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys His Gln Trp Ser Ser
85 90 95
Tyr Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly
100 105 110
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro
130 135 140
Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Ile Ser
145 150 155 160
Ser Gly Tyr Ser Trp His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu
165 170 175
Glu Trp Ile Gly Tyr Ile Gln Tyr Ser Gly Ile Thr Asn Tyr Asn Pro
180 185 190
Ser Leu Lys Ser Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln
195 200 205
Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
210 215 220
Tyr Cys Ala Arg Glu Asp Tyr Asp Tyr His Trp Tyr Phe Asp Val Trp
225 230 235 240
Gly Gln Gly Ser Leu Val Thr Val Thr Val Thr Thr Thr Pro Ala Pro
245 250 255
Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu
260 265 270
Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg
275 280 285
Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly
290 295 300
Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys
305 310 315 320
Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg
325 330 335
Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro
340 345 350
Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser
355 360 365
Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu
370 375 380
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
385 390 395 400
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
405 410 415
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
420 425 430
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
435 440 445
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
450 455 460
Leu His Met Gln Ala Leu Pro Pro Arg
465 470
<210> 4
<211> 1419
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ggtgtccact ccgacatcca gatgacccag agcccaagca gcctgagcgc cagcgtgggt 60
gacagagtga ccatcacctg tagtaccagc tcgagtgtaa gttacatgca ctggtaccag 120
cagaagccag gtaaggctcc aaagctgctg atctacagca catccaacct ggcttctggt 180
gtgccaagca gattcagcgg tagcggtagc ggtaccgact tcaccttcac catcagcagc 240
ctccagccag aggacatcgc cacctactac tgccatcagt ggagtagtta tcccacgttc 300
ggccaaggga ccaaggtgga aatcaaacgt ggaggcggag gatctggcgg cggaggaagt 360
ggcggagggg gatctggggg aggcggaagc caggtccaac tgcaggagag cggtccaggt 420
cttgtgagac ctagccagac cctgagcctg acctgcaccg tgtctggctt caccatcagc 480
agtggttata gctggcactg ggtgagacag ccacctggac gaggtcttga gtggattgga 540
tacatacagt acagtggtat cactaactac aacccctctc tcaaaagtag agtgacaatg 600
ctggtagaca ccagcaagaa ccagttcagc ctgagactca gcagcgtgac agccgccgac 660
accgcggtct attattgtgc aagagaagac tatgattacc actggtactt cgatgtctgg 720
ggtcaaggca gcctcgtcac agtcacagtc accacgacgc cagcgccgcg accaccaaca 780
ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg 840
gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgatatcta catctgggcg 900
cccttggccg ggacttgtgg ggtccttctc ctgtcactgg ttatcaccct ttactgcaaa 960
cggggcagaa agaaactcct gtatatattc aaacaaccat ttatgagacc agtacaaact 1020
actcaagagg aagatggctg tagctgccga tttccagaag aagaagaagg aggatgtgaa 1080
ctgagagtga agttcagcag gagcgcagac gcccccgcgt acaagcaggg ccagaaccag 1140
ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 1200
ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1260
aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1320
cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1380
acctacgacg cccttcacat gcaggccctg ccccctcgc 1419
<210> 5
<211> 1479
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60
ggtgtccact ccgacatcca gatgacccag agcccaagca gcctgagcgc cagcgtgggt 120
gacagagtga ccatcacctg tagtaccagc tcgagtgtaa gttacatgca ctggtaccag 180
cagaagccag gtaaggctcc aaagctgctg atctacagca catccaacct ggcttctggt 240
gtgccaagca gattcagcgg tagcggtagc ggtaccgact tcaccttcac catcagcagc 300
ctccagccag aggacatcgc cacctactac tgccatcagt ggagtagtta tcccacgttc 360
ggccaaggga ccaaggtgga aatcaaacgt ggaggcggag gatctggcgg cggaggaagt 420
ggcggagggg gatctggggg aggcggaagc caggtccaac tgcaggagag cggtccaggt 480
cttgtgagac ctagccagac cctgagcctg acctgcaccg tgtctggctt caccatcagc 540
agtggttata gctggcactg ggtgagacag ccacctggac gaggtcttga gtggattgga 600
tacatacagt acagtggtat cactaactac aacccctctc tcaaaagtag agtgacaatg 660
ctggtagaca ccagcaagaa ccagttcagc ctgagactca gcagcgtgac agccgccgac 720
accgcggtct attattgtgc aagagaagac tatgattacc actggtactt cgatgtctgg 780
ggtcaaggca gcctcgtcac agtcacagtc accacgacgc cagcgccgcg accaccaaca 840
ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg 900
gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgatatcta catctgggcg 960
cccttggccg ggacttgtgg ggtccttctc ctgtcactgg ttatcaccct ttactgcaaa 1020
cggggcagaa agaaactcct gtatatattc aaacaaccat ttatgagacc agtacaaact 1080
actcaagagg aagatggctg tagctgccga tttccagaag aagaagaagg aggatgtgaa 1140
ctgagagtga agttcagcag gagcgcagac gcccccgcgt acaagcaggg ccagaaccag 1200
ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 1260
ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1320
aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1380
cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1440
acctacgacg cccttcacat gcaggccctg ccccctcgc 1479
<210> 6
<211> 45
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 6
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> 7
<211> 135
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 8
<211> 24
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 8
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> 9
<211> 72
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 10
<211> 42
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 10
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> 11
<211> 126
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 12
<211> 112
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 12
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> 13
<211> 336
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
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> 14
<211> 20
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 14
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> 15
<211> 60
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60
<210> 16
<211> 493
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 16
Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His
1 5 10 15
Ala Ala Arg Pro Gly Val His Ser Asp Ile Gln Met Thr Gln Ser Pro
20 25 30
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser
35 40 45
Thr Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly
50 55 60
Lys Ala Pro Lys Leu Leu Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly
65 70 75 80
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe
85 90 95
Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys His
100 105 110
Gln Trp Ser Ser Tyr Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
115 120 125
Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
145 150 155 160
Leu Val Arg Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
165 170 175
Phe Thr Ile Ser Ser Gly Tyr Ser Trp His Trp Val Arg Gln Pro Pro
180 185 190
Gly Arg Gly Leu Glu Trp Ile Gly Tyr Ile Gln Tyr Ser Gly Ile Thr
195 200 205
Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Met Leu Val Asp Thr
210 215 220
Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp
225 230 235 240
Thr Ala Val Tyr Tyr Cys Ala Arg Glu Asp Tyr Asp Tyr His Trp Tyr
245 250 255
Phe Asp Val Trp Gly Gln Gly Ser Leu Val Thr Val Thr Val Thr Thr
260 265 270
Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
275 280 285
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
290 295 300
Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala
305 310 315 320
Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr
325 330 335
Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
340 345 350
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
355 360 365
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
370 375 380
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln
385 390 395 400
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
405 410 415
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
420 425 430
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
435 440 445
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
450 455 460
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
465 470 475 480
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490

Claims (10)

1. a kind of single-chain antibody for targeting CEA, which is characterized in that the single-chain antibody of the targeting CEA includes such as SEQ ID NO:1 Shown in amino acid sequence.
2. the single-chain antibody of targeting CEA as described in claim 1, which is characterized in that the volume of the single-chain antibody of the targeting CEA Code gene includes the nucleotide sequence as shown in SEQ ID NO:2.
3. a kind of Chimeric antigen receptor T cell for targeting CEA, which is characterized in that the Chimeric antigen receptor CAR- including targeting CEA CEA, the CAR-CEA include from aminoterminal to c-terminus the sequentially connected targeting single-chain antibody of CEA, extracellular hinge area, across The amino acid sequence in film area and intracellular signal area, wherein the single-chain antibody of the targeting CEA includes as shown in SEQ ID NO:1 Amino acid sequence.
4. the Chimeric antigen receptor T cell of targeting CEA as claimed in claim 3, which is characterized in that the extracellular hinge area packet CD8 α hinge area is included, the transmembrane region includes CD8 transmembrane region, and the intracellular signal area includes sequentially connecting from aminoterminal to c-terminus The 4-1BB signaling zone and CD3 ζ signaling zone connect.
5. the Chimeric antigen receptor T cell of targeting CEA as claimed in claim 4, which is characterized in that the ammonia of the CAR-CEA Base acid sequence includes the amino acid sequence as shown in SEQ ID NO:3.
6. the Chimeric antigen receptor T cell of targeting CEA as described in claim 3 or 4, which is characterized in that the CAR-CEA's Encoding gene includes the nucleotide sequence as shown in SEQ ID NO:4.
7. a kind of recombinant viral vector, which is characterized in that the recombinant viral vector includes as described in claim any one of 3-6 Targeting CEA Chimeric antigen receptor T cell CAR-CEA encoding gene.
8. a kind of host cell, which is characterized in that the host cell includes recombinant viral vector as claimed in claim 7.
9. a kind of preparation method for the Chimeric antigen receptor T cell for targeting CEA characterized by comprising
(1) encoding gene of the Chimeric antigen receptor CAR-CEA of targeting CEA is provided, including sequentially connected from 5 ' ends to 3 ' ends The encoding gene of signal peptide, the encoding gene of single-chain antibody for targeting CEA, the encoding gene of extracellular hinge area, transmembrane region volume The encoding gene of code gene and intracellular signal area, wherein the encoding gene of the single-chain antibody of the targeting CEA includes such as SEQ ID Nucleotide sequence corresponding to amino acid sequence shown in NO:1；
(2) encoding gene of the CAR-CEA is inserted into pWPXLD carrier, obtains pWPXLD-CAR-CEA recombinant plasmid；
(3) it by the pWPXLD-CAR-CEA recombinant plasmid and envelope plasmid, packaging plasmid cotransfection host cell, is recombinated Slow virus；
(4) recombinant slow virus is transfected into CD3 positive t lymphocytes, the Chimeric antigen receptor T of targeting CEA is obtained through separation Cell.
10. a kind of target the single-chain antibody of CEA, as described in claim any one of 3-6 as claim 1-2 is described in any item Or preparation method as claimed in claim 9 targeting CEA obtained Chimeric antigen receptor T cell or such as claim 7 institute The recombinant viral vector or host cell as claimed in claim 8 stated diagnose and/or treat the drug of malignant tumour in preparation In application.
CN201810513874.5A 2018-05-25 2018-05-25 Target single-chain antibody, the Chimeric antigen receptor T cell and its preparation method and application of CEA Withdrawn CN110526975A (en)

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