CN112430576A

CN112430576A - Chimeric antigen receptor T cell carrying safety switch and targeting EGFRv III, and preparation method and application thereof

Info

Publication number: CN112430576A
Application number: CN201910792806.1A
Authority: CN
Inventors: 曾滢; 汪婷婷; 杨忠华; 张宏玲; 唐超
Original assignee: Shenzhen Bindebio Technology Co ltd
Current assignee: Shenzhen Bindebio Technology Co ltd
Priority date: 2019-08-26
Filing date: 2019-08-26
Publication date: 2021-03-02
Also published as: WO2021036245A1

Abstract

The present invention provides chimeric antigen receptor T cells carrying a safety switch and targeting EGFRvIII, including a chimeric antigen receptor CAR-EGFRvIII targeting EGFRvIII and a safety switch for inducing apoptosis of T cells. CAR-EGFRvIII includes targeting EGFRvIII The amino acid sequence of the single-chain antibody, extracellular hinge region, transmembrane region and intracellular signal region, the single-chain antibody targeting EGFRvIII includes the amino acid sequence shown in SEQ ID NO: 1, and the safety switch includes F36V mutant FK506 binding The amino acid sequence of protein, connecting peptide and CARD-removed caspase 9, the amino acid sequence of F36V mutant FK506 binding protein includes the amino acid sequence shown in SEQ ID NO: 2, and the amino acid sequence of CARD-removed caspase 9 includes as SEQ ID NO: 3 shows the amino acid sequence.

Description

Chimeric antigen receptor T cell carrying safety switch and targeting EGFRv III, and preparation method and application thereof

Technical Field

The invention relates to the field of medical biology, in particular to a chimeric antigen receptor T cell carrying a safety switch and targeting EGFRvIII, and a preparation method and application thereof.

Background

The chimeric antigen receptor T cell (CAR-T) technology is a novel cell therapy, which is characterized in that T cells modified by a chimeric antigen receptor are returned to a human body to activate an autoimmune system and kill tumor cells.

Glioblastoma (GBM) is the most malignant type of glioma, and is also the most common and aggressive one of primary brain tumors, with strong invasiveness and high recurrence rate. Although treatment of GBM has evolved into a complex treatment modality combining surgery, radiation therapy and chemotherapy, the prognosis remains poor, with an overall median survival (OS) of only 15 months and a 5-year survival rate of less than 10%. Development of drug resistance by invasive malignant cells is the source of recurrence of GBM, leading to treatment failure.

EGFRvIII is an epidermal growth factor receptor III type mutant, is an oncogene with high expression rate in glioma, is closely related to various malignant phenotypes of the glioma, about 30 percent of patients with glioblastoma express EGFRvIII, while normal cells of a human body do not express EGFRvIII, and the specific expression of the EGFRvIII on the surface of tumor cells enables the EGFRvIII to become an ideal target point for targeted therapy of the tumor.

CAR-T cell therapy is used as a new tumor immunotherapy method, has obvious clinical effect on tumor therapy, and still has a plurality of adverse reactions and complications. In the initial stage of CAR-T cell infusion, T cells rapidly expand in a short time, and secrete a large amount of cytokines in the process of killing tumors by the T cells, so that Cytokine Release Syndrome (CRS) is caused, the clinical manifestations mainly comprise fever, tachycardia, hypotension and obvious increase of cytokine levels such as IL-6 in cells, and the adverse reactions influence and limit the application of CAR-T cell therapy.

Therefore, a chimeric antigen receptor T cell which can carry a safety switch and target egfrviii and a preparation method thereof are needed to reduce the side effect of cytokine storm and make cell therapy more controllable and safe.

Disclosure of Invention

In view of the above, the invention provides a chimeric antigen receptor T cell carrying a safety switch and targeting EGFRv iii, which has a chimeric antigen receptor targeting EGFRv iii, and further can specifically target tumor cells expressing EGFRv iii, activate T cells to exert a cellular immune effect, and achieve efficient and specific killing of EGFRv iii positive tumor cells, and has lasting cell viability and lethality; meanwhile, the safety switch can start a mitochondrial apoptosis pathway through dimerization induced by external drugs after cytokine release syndrome occurs, so that the chimeric antigen receptor T cell is apoptotic, normal cells are not damaged, and the application safety of the chimeric antigen receptor T cell is improved.

In a first aspect, the present invention provides a chimeric antigen receptor T cell carrying a safety switch and targeting egfrviii, comprising a chimeric antigen receptor CAR-egfrviii targeting egfrviii and a safety switch inducing apoptosis of T cells, wherein the CAR-egfrviii comprises an amino acid sequence of a single-chain antibody targeting egfrviii, an extracellular hinge region, a transmembrane region and an intracellular signal region sequentially linked from amino terminus to carboxy terminus, and the single-chain antibody targeting egfrviii comprises the amino acid sequence as set forth in SEQ ID NO: 1, the safety switch comprising the amino acid sequence of the F36V mutant FK506 binding protein, the linker peptide, and the caspase 9 that removes the CARD, sequentially linked from the amino terminus to the carboxy terminus, the amino acid sequence of the F36V mutant FK506 binding protein comprising the amino acid sequence as set forth in SEQ ID NO: 2, and the amino acid sequence of caspase 9 with the CARD removed comprises the amino acid sequence shown as SEQ ID NO: 3.

Optionally, the coding gene of the EGFRvIII-targeting single-chain antibody comprises the sequence shown in SEQ ID NO: 6. Further, the gene encoding the EGFRvIII-targeting single-chain antibody comprises a nucleotide sequence with base degeneracy with SEQ ID NO. 6.

In the invention, the step of "said CAR-EGFRv iii comprises sequential connection from amino terminus to carboxy terminus" is specifically: the carboxyl end of the amino acid sequence of the single-chain antibody targeting the EGFRvIII is connected with the amino end of the amino acid sequence of the extracellular hinge region, the carboxyl end of the amino acid sequence of the extracellular hinge region is connected with the amino end of the amino acid sequence of the transmembrane region, and the carboxyl end of the amino acid sequence of the transmembrane region is connected with the amino end of the amino acid sequence of the intracellular signal region.

In the present invention the extracellular hinge region is used to facilitate the binding of the EGFRv iii-targeted single chain antibody to EGFRv iii on tumors.

Optionally, the extracellular hinge region comprises a combination of one or more of a CD8 a hinge region, a CD28 hinge region, a CD4 hinge region, a CD5 hinge region, a CD134 hinge region, a CD137 hinge region, an ICOS hinge region. Further, the extracellular hinge region is a CD8 a hinge region.

In the invention, the transmembrane region is used for fixing the EGFRv III targeted chimeric antigen receptor CAR-EGFRv III.

Optionally, the transmembrane region comprises one or more of a CD3 transmembrane region, a CD4 transmembrane region, a CD8 transmembrane region, and a CD28 transmembrane region. Further, the transmembrane region is the CD8 transmembrane region.

In the present invention, the intracellular signaling region is used to provide a signal for T cell activation, maintain the survival time of T cells, and activate a T cell proliferation signaling pathway.

Optionally, the intracellular signaling region comprises a combination of one or more of a 4-1BB signaling region, a CD3 zeta signaling region, an ICOS signaling region, a CD27 signaling region, an OX40 signaling region, a CD28 signaling region, an IL1R1 signaling region, a CD70 signaling region, and a TNFRSF19L signaling region.

Optionally, the intracellular signaling regions are the 4-1BB signaling region and the CD3 zeta signaling region. Among them, the CD3 zeta signal region is the signaling domain (i.e., the first signal) of T cells, and the 4-1BB signal region is the costimulatory signal of T cells, and under their combined action, T cells are completely activated after recognizing antigens.

Further, the amino acid sequence of CAR-EGFRv iii comprises the amino acid sequence set forth in SEQ ID NO: 4.

Further, the coding gene of the CAR-EGFRv III comprises a sequence shown in SEQ ID NO: 7. Furthermore, the gene encoding CAR-EGFRv III should include a nucleotide sequence having base degeneracy with SEQ ID NO. 7.

In the invention, the chimeric antigen receptor CAR-EGFRvIII targeting EGFRvIII enables T cells to specifically target tumor cells expressing EGFRvIII, the single-chain antibody can specifically recognize EGFRvIII protein on the tumor cells and is specifically combined with the EGFRvIII protein, and after the CAR-EGFRvIII is combined with the EGFRvIII, an intracellular signal region is activated, so that the T cells are promoted to be expanded in a patient body, and the tumor cells are efficiently and specifically killed. The EGFRv III is widely expressed in malignant tumor cells, and is weakly expressed in common cells, so that the chimeric antigen receptor T cell targeting the EGFRv III provided by the invention can be specifically combined with the tumor cells, has stronger affinity activity and internalization activity on the malignant tumor cells expressing the EGFRv III, generates killing effect on the tumor cells, and does not damage normal cells.

Alternatively, the gene encoding the F36V mutant FK 506-binding protein (F36V-FKBP) comprises the amino acid sequence set forth in SEQ ID NO: 8. Further, the gene encoding the F36V mutant FK 506-binding protein should include a nucleotide sequence having base degeneracy with SEQ ID NO. 8.

Optionally, the coding gene of the CARD-depleted caspase 9(caspase 9 Δ CARD) comprises a sequence as set forth in SEQ ID NO:9, or a nucleotide sequence shown in the specification. Further, the coding gene of the CARD-removed caspase 9 should include a nucleotide sequence having base degeneracy with SEQ ID NO. 9.

In the present invention, the linker peptide (linker) is used to link different proteins or polypeptides so that the linked proteins or polypeptides maintain their respective spatial conformations to maintain the functions or activities of the proteins or polypeptides. In the present invention, the linker peptide may be, but is not limited to, a polypeptide sequence mainly composed of glycine and serine, wherein glycine has the smallest molecular weight and is the amino acid with the shortest side chain, which can increase the flexibility of the side chain; serine is the most hydrophilic amino acid and increases the hydrophilicity of the peptide chain. Alternatively, the amino acid sequence of the linker peptide comprises the amino acid sequence set forth as SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof.

In the present invention, the step of "the safety switch comprises sequential connection from the amino terminal to the carboxyl terminal" specifically comprises: the carboxy terminus of the amino acid sequence of the F36V mutant FK506 binding protein is linked to the amino terminus of the amino acid sequence of the linker peptide, which is linked to the amino terminus of the amino acid sequence of the CARD-depleted caspase 9.

Optionally, the CAR-EGFRv iii and the safety switch are linked by an internal ribosomal entry site or by a self-cleaving polypeptide.

In the present invention, an Internal Ribosome Entry Site (IRES) mediates binding between a ribosome and RNA to initiate protein translation, and after translation of a protein before the IRES, the ribosome does not detach from mRNA and can bind to the IRES to continue translation, and thus both proteins can be translated. Alternatively, the coding gene of the internal ribosome entry site comprises the nucleotide sequence shown in SEQ ID NO: 11.

In the present invention, self-cleaving polypeptides are used in polycistronic vector construction for expression of multiple proteins.

Optionally, the chimeric antigen receptor T cell carrying a safety switch and targeting EGFRv iii further comprises a chemical inducer of dimerization comprising at least one of AP1903 and AP 20187.

In the present invention, the safety switch (icaspase 9) includes F36V-FKBP and caspase 9 Δ CARD, in which the F36V point mutation can increase the affinity between FKBP and dimerization Chemical Inducer (CID), remove CARD (caspase recovery domain), replace its physiological function with FKBP, and can increase the expression of gene. When cytokine release syndrome occurs, CID can be introduced in a mode of injection, so that icaspase 9 is dimerized, downstream caspase 3 molecules are activated, CAR-T cell apoptosis is caused, adverse reaction is prevented, and the application safety of CAR-T cells is improved.

The chimeric antigen receptor T cell carrying the safety switch and targeting the EGFRvIII provided by the first aspect of the invention can specifically target tumor cells expressing the EGFRvIII, activate the T cell to play a role in cellular immunity, realize efficient and specific killing on EGFRvIII positive tumor cells, and have lasting cell activity and lethality; meanwhile, the safety switch can induce dimerization through external drugs after cytokine release syndrome occurs, a mitochondrial apoptosis pathway is started, the chimeric antigen receptor T cell is apoptotic, normal cells are not damaged, and the application safety of the chimeric antigen receptor T cell is improved.

In a second aspect, the present invention provides a method for preparing a chimeric antigen receptor T cell carrying a safety switch and targeting egfrviii, comprising:

(1) providing a gene encoding an EGFRvIII-targeted chimeric antigen receptor CAR-EGFRvIII, comprising a gene encoding a signal peptide, a gene encoding a single-chain antibody targeting EGFRvIII, a gene encoding an extracellular hinge region, a transmembrane region, and an intracellular signal region, which are connected in sequence from 5 'end to 3' end, wherein the gene encoding the EGFRvIII-targeted single-chain antibody comprises the sequence as shown in SEQ ID NO: 1, and the nucleotide sequence corresponds to the amino acid sequence shown in the specification;

(2) genes encoding a safety switch for inducing T cell apoptosis, comprising a gene encoding F36V mutant FK 506-binding protein, a gene encoding a linker peptide, and a gene encoding CARD-deleted caspase 9, which are sequentially linked from 5 'to 3', wherein the gene encoding F36V mutant FK 506-binding protein comprises the amino acid sequence as set forth in SEQ ID NO: 2, and the coding gene of caspase 9 without CARD comprises the nucleotide sequence shown as SEQ ID NO: 3, and a nucleotide sequence corresponding to the amino acid sequence shown in the figure;

(3) inserting the coding gene of the CAR-EGFRv III and the coding gene of the safety switch into a gene transfer vector to obtain a recombinant gene transfer vector;

(4) packaging the recombinant gene transfer vector and transfecting host cells to obtain recombinant lentiviruses;

(5) transfecting the recombinant lentivirus with CD3 positive T lymphocytes to obtain chimeric antigen receptor T cells carrying a safety switch and targeting EGFRv III.

In the present invention, the encoding genes of the "safety switch for inducing T cell apoptosis include a sequence linked from 5 'end to 3' end" specifically: the 3 'end of the coding gene sequence of the signal peptide is connected with the 5' end of the coding gene of the single-chain antibody targeting EGFRvIII, the 3 'end of the coding gene of the single-chain antibody targeting EGFRvIII is connected with the 5' end of the coding gene of the extracellular hinge region, the 3 'end of the coding gene of the extracellular hinge region is connected with the 5' end of the coding gene of the transmembrane region, and the 3 'end of the coding gene of the transmembrane region is connected with the 5' end of the coding gene of the intracellular signal region.

In the present invention, the encoding genes of the "safety switch for inducing T cell apoptosis, including the following genes, which are sequentially linked from 5 'end to 3' end" are specifically: the 3 'end of the coding gene sequence of the F36V mutant FK506 binding protein is connected with the 5' end of the coding gene of the connecting peptide, and the 3 'end of the coding gene of the connecting peptide is connected with the 5' end of the coding gene of the CARD-removed caspase 9.

In the present invention, the signal peptide is used to direct the expression of the chimeric antigen receptor CAR-EGFRv III to the cell surface, and the signal peptide is cleaved by a signal peptidase during the translational maturation of the protein.

Optionally, the amino acid sequence corresponding to the coding gene of the signal peptide is shown in SEQ ID NO: shown at 12.

Wherein, the specific selection of the EGFRvIII targeting single-chain antibody, the extracellular hinge region, the transmembrane region, the intracellular signal region, the F36V mutated FK506 binding protein, the connecting peptide and the CARD removing caspase 9 is as described in the first aspect of the invention, and is not repeated herein.

Optionally, inserting the CAR-EGFRv iii coding gene and the safety switch coding gene into a gene delivery vector, comprising:

and connecting the CAR-EGFRv III coding gene with the safety switch coding gene through an internal ribosome entry site or self-cleavage polypeptide, and then inserting the CAR-EGFRv III coding gene into the gene delivery vector.

Optionally, the gene delivery vector comprises at least one of a lentiviral vector, a retroviral vector, and an adenoviral vector.

Further, the gene delivery vector may be, but is not limited to, pWPXLD vector, pLEX-MCS vector, pSico vector, and pCgpV vector.

As an illustrative example, when the gene delivery vector is pWPXLD vector, the gene encoding CAR-EGFRvIII is inserted between BamHI and EcoRI cleavage sites in pWPXLD vector; the coding gene of the safety switch is inserted between SpeI and ndeI enzyme cutting sites in a pWPXLD vector. When the coding gene of the CAR-EGFRvIII is inserted into a pWPXLD vector, the 5 'end of the coding gene of the CAR-EGFRvIII can be added with an initiation codon (such as ATG) and connected with a BamHI enzyme cutting site (ggatcc) in the pWPXLD vector, and the 3' end can be added with a termination codon (such as TAA) and connected with an EcoRI enzyme cutting site (gattc) in the pWPXLD vector, so that the coding gene of the CAR-EGFRvIII is positioned between the BamHI enzyme cutting site and the EcoRI enzyme cutting site; when the coding gene of the safety switch is inserted into a pWPXLD vector, an initiation codon (such as ATG) can be added at the 5 'end of the coding gene of the safety switch and is connected with a SpeI enzyme cutting site (actagt) in the pWPXLD vector, a stop codon (such as TAA) can be added at the 3' end of the coding gene of the safety switch and is connected with a NdeI enzyme cutting site (catatg) in the pWPXLD vector, so that the coding gene of CAR-EGFRv III is positioned between the SpeI enzyme cutting site and the NdeI enzyme cutting site, and the pWPXLD-CAR-EGFRv III-icaspase 9 recombinant gene transfer vector is obtained.

In the present invention, the gene fragment inserted into the gene delivery vector may be, but not limited to, an initiation codon, the CAR-EGFRv iii coding gene and a stop codon, and an initiation codon, the safety switch coding gene and a stop codon.

In the present invention, the order of insertion of the CAR-EGFRv iii coding gene and the safety switch coding gene into the gene delivery vector is not limited.

In the present invention, the gene fragment inserted into the gene delivery vector may be, but not limited to, an initiation codon, the CAR-EGFRv iii coding gene and a stop codon, an internal ribosome entry site coding gene, and an initiation codon, the safety switch coding gene and a stop codon.

Optionally, packaging and transfecting the recombinant gene delivery vector into a host cell to obtain a recombinant lentivirus, comprising:

and co-transfecting the recombinant gene transfer vector, the envelope plasmid and the packaging plasmid to a host cell to obtain the recombinant lentivirus.

Further, the envelope plasmid may be, but is not limited to, PMD2G, the packaging plasmid may be, but is not limited to, psPAX2, and the host cell may be, but is not limited to, HEK 293T.

Wherein the enveloped plasmid PMD2G encodes a vesicular stomatitis virus glycoprotein capsid that aids in adhesion of the recombinant lentivirus to the cell membrane and maintains infectivity of the recombinant lentivirus.

In the present invention, when the gene delivery vector includes a lentiviral vector, it may further contain an envelope protein derived from another virus. For example, a viral envelope protein derived from a human cell infected with the protein is preferable. Such a protein is not particularly limited, and examples thereof include amphotropic virus hand membrane proteins of retroviruses, and envelope proteins derived from mouse leukemia virus (MuMLV)4070A strain can be used. In addition, envelope proteins derived from MuMLV 10Al may also be used. Examples of the proteins of the herpesviridae family include the gB, gD, and gp85 proteins of herpes simplex virus, and the gp350 and gp220 proteins of EB virus. Examples of the hepadnaviridae protein include hepatitis B virus S protein. The envelope protein may also be formed by fusion of measles virus glycoprotein with other single chain antibodies.

Packaging of recombinant lentiviruses is usually by transient transfection or by cell line packaging. Human cell lines that can be used as packaging cells upon transient transfection include, for example, 293 cells, 293T cells, and the like, and other clones isolated from 293 cells; SW480 cells, TE671 cells, and the like. Monkey-derived cell lines, for example, COS1 cells and CV-1 cells can also be used. Furthermore, commonly used calcium phosphate and PEI transfection reagents, as well as some transfection reagents such as Lipofectamine2000, FuGENE and S93fectin, are also commonly used.

Packaging of recombinant lentiviruses also employs some lentivirus packaging cell lines, such as stable cell lines produced using the most common Env glycoprotein, VSVG protein, or HIV-1gag-pol protein.

For safety reasons, the lentivirus vector systems used on a large scale all use a method of splitting the genome, i.e. locating genes with different helper functions on different plasmids. Currently, there are four-plasmid systems (where the coding gag-pol gene, Rev gene, VSVG gene, SIN transgene are located on four different plasmids), three-plasmid systems (where the plasmid coding for Rev gene is removed and the gag-pol gene in the gag-pol plasmid employs codons preferred in human cells), and two-plasmid systems (where the helper genes necessary for lentiviral vector packaging are located on the same plasmid, these helper genes being single gene sequences, and the other being a transgenic plasmid). There are also lentiviral packaging systems in use that exceed the four plasmid system.

Alternatively, the CD3 positive T lymphocytes are isolated from human peripheral blood mononuclear cells.

Optionally, the human-derived peripheral blood mononuclear cells are derived from autologous venous blood, autologous bone marrow, umbilical cord blood, placental blood, and the like.

Further, the blood is derived from fresh peripheral blood or bone marrow collected after one month of operation and one month of chemotherapy for cancer patients.

Specifically, the process for obtaining the CD3 positive T lymphocyte is as follows: adding CD3/CD28 immunomagnetic beads into peripheral blood mononuclear cells according to a certain proportion, incubating for a period of time, putting a magnet for screening to obtain CD3 positive T lymphocytes coated by the immunomagnetic beads, and removing the magnetic beads to obtain CD3 positive T lymphocytes.

The chimeric antigen receptor T cell carrying a safety switch and targeting EGFRvIII provided by the second aspect of the invention has a simple preparation method, and can be applied on a large scale to obtain the chimeric antigen receptor T cell with application safety.

In a third aspect, the present invention provides a recombinant vector, which includes an inserted gene encoding an egfrviii-targeting chimeric antigen receptor CAR-egfrviii and a gene encoding a safety switch for inducing T cell apoptosis, wherein the gene encoding the egfrviii-targeting chimeric antigen receptor CAR-egfrviii includes a gene encoding a signal peptide, a gene encoding a single-chain egfrviii-targeting antibody, a gene encoding an extracellular hinge region, a gene encoding a transmembrane region, and a gene encoding an intracellular signal region, which are sequentially linked from 5 'to 3', and the gene encoding the single-chain egfrviii-targeting antibody includes the amino acid sequence shown in SEQ ID NO: 1, and the nucleotide sequence corresponds to the amino acid sequence shown in the specification; the encoding gene of the safety switch for inducing T cell apoptosis comprises a gene encoding F36V mutant FK506 binding protein, a gene encoding connecting peptide and a gene encoding CARD-removed caspase 9 which are sequentially connected from 5 'end to 3', wherein the encoding gene of the F36V mutant FK506 binding protein comprises the nucleotide sequence shown in SEQ ID NO: 2, and the coding gene of caspase 9 without CARD comprises the nucleotide sequence shown as SEQ ID NO: 3, or a nucleotide sequence corresponding to the amino acid sequence shown in the figure.

In the present invention, the recombinant vector is obtained by inserting a gene encoding a chimeric antigen receptor CAR-egfrviii targeting egfrviii and a gene encoding a safety switch inducing T cell apoptosis into a vector, and the insertion order of the two sequences is not limited. Optionally, the coding gene of the chimeric antigen receptor targeting egfrviii CAR-egfrviii is at the 5 'end of the coding gene of the safety switch for inducing T cell apoptosis, or the coding gene of the chimeric antigen receptor targeting egfrviii CAR-egfrviii is at the 3' end of the coding gene of the safety switch for inducing T cell apoptosis.

In the present invention, the vector may be, but is not limited to, the gene delivery vector of the second aspect. Optionally, the vector is at least one of a viral vector and a non-viral vector. Further, the non-viral vector includes a plasmid vector and a phage vector. In particular, the viral vector may be, but is not limited to, a lentiviral vector, a retroviral vector, and an adenoviral vector, and the plasmid vector may be, but is not limited to, a eukaryotic plasmid vector, a prokaryotic plasmid vector, and a micro-loop DNA. When the vector is micro-ring DNA, the recombinant micro-ring DNA inserted with the coding gene of the chimeric antigen receptor CAR-EGFRvIII targeting EGFRvIII and the coding gene of the safety switch inducing T cell apoptosis can be directly transfected into CD3 positive T lymphocytes to prepare the chimeric antigen receptor T cells carrying the safety switch and targeting EGFRvIII.

The recombinant vector provided by the third aspect of the invention is safe and efficient, can stably realize the introduction or replication of the coding gene of CAR-EGFRvIII and the coding gene of a safety switch, and can be used for preparing chimeric antigen receptor T cells.

In a fourth aspect, the present invention provides a host cell comprising a recombinant vector as described in the third aspect.

Alternatively, when the recombinant vector is a recombinant viral vector, the host cell can be used to assemble the recombinant viral vector so that it is infectious. Further, the host cell may include, but is not limited to, HEK293T cell, 293T cell, 293FT cell, SW480 cell, u87MG cell, HOS cell, or COS7 cell, and the like. Further, the host cell is a HEK293T cell.

Optionally, when the recombinant plasmid is a recombinant eukaryotic plasmid vector, a recombinant prokaryotic plasmid vector, or a recombinant minicircle DNA, the host cell is a corresponding eukaryotic host cell or prokaryotic host cell.

In a fifth aspect, the present invention provides a chimeric antigen receptor T cell carrying a safety switch and targeting egfrviii, a recombinant vector according to the third aspect, or a host cell according to the fourth aspect, prepared by the preparation method according to the first aspect or the second aspect, for use in the preparation of a medicament for the prevention and treatment of malignant tumors.

The use may be particularly, but not exclusively, to provide a kit comprising one or more of the chimeric antigen receptor T cells carrying a safety switch and targeting egfrviii according to the first aspect, the recombinant vector according to the third aspect, the host cell according to the fourth aspect.

In the present invention, the malignant tumor may be, but is not limited to, brain glioma, breast cancer, etc.

Advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of embodiments of the invention.

Drawings

FIG. 1 is a plasmid map of a pWPXLD-CAR-EGFRvIII recombinant vector provided by the embodiment of the invention.

FIG. 2 is a plasmid map of pWPXLD-CAR-EGFRv III-icaspase 9 recombinant vector provided by the embodiment of the invention.

Detailed Description

While the following is a description of the preferred embodiments of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

Preparation examples

(1) Construction of pWPXLD-CAR-EGFRv III-icaspase 9 recombinant vector

Providing a gene encoding CAR-EGFRv iii, i.e. providing a sequence as set forth in SEQ ID NO:7, adding a restriction enzyme cutting site and an initiation codon at the 5 'end of the nucleotide sequence, and adding a restriction enzyme cutting site and a termination codon at the 3' end of the nucleotide sequence; it was inserted between the BamHI and EcoRI sites of pWPXLD vector. Then transferred into escherichia coli competent cell DH5 alpha, and positive clone PCR identification and sequencing identification are carried out. The size and the sequence of the fragment according with the purpose are identified through PCR product gel electrophoresis detection and sequencing, and the pWPXLD-CAR-EGFRv III recombinant vector is successfully constructed, as shown in figure 1.

Providing a gene encoding a safety switch linked to an IRES, i.e. providing a sequence as set forth in SEQ ID NO: 5, and adding a restriction enzyme cutting site and a nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 11, the nucleotide sequence of IRES, initiation codon and, at the 3' end, addition of a cleavage site and a stop codon; it was inserted between the Spe I and Nde I cleavage sites of pWPXLD vector. Then transferred into escherichia coli competent cell DH5 alpha, and positive clone PCR identification and sequencing identification are carried out. And (3) successfully constructing a pWPXLD-CAR-EGFRv III-icaspase 9 recombinant vector according with the size and the sequence of the target fragment through PCR product gel electrophoresis detection and sequencing identification.

(2) Recombinant lentivirus construction

The pWPXLD-CAR-EGFRv III-icaspase 9 recombinant vector, the packaging plasmid psPAX2 and the envelope plasmid pMD2G are co-transfected into the cultured HEK293T cell. Collecting virus-containing supernatant in 48h, filtering with 0.45 μm filter membrane, and storing in an ultra-low temperature refrigerator at-80 deg.C; harvesting virus-containing supernatants for the second 72h, filtering with 0.45 μm filter membrane, mixing with the virus supernatants harvested for the 48h, adding into an ultracentrifuge tube, placing into a Beckman ultracentrifuge one by one, setting the centrifugation parameters to be 25000rpm, the centrifugation time to be 2h, and controlling the centrifugation temperature to be 4 ℃; after the centrifugation is finished, removing the supernatant, removing the liquid remained on the tube wall as much as possible, adding a virus preservation solution, and lightly and repeatedly blowing and resuspending; after fully dissolving, centrifuging at high speed 10000rpm for 5min, taking supernatant to measure titer by a fluorescence method, and measuring virus according to 100 mul, 2 multiplied by 10⁸Subpackaging each/mL, and storing in an ultra-low temperature refrigerator at-80 ℃ to obtain the recombinant lentivirus.

(3) Preparation of chimeric antigen receptor T cell carrying safety switch and targeting EGFRv III

a) Isolation of PBMC (peripheral blood mononuclear cells)

PBMC is derived from autologous venous blood, autologous bone marrow, umbilical cord blood, placental blood, etc. Preferably fresh peripheral blood or bone marrow taken from cancer patients after one month of surgery and one month of chemotherapy.

Drawing blood from a patient and sending the blood to a blood separation chamber; collecting peripheral blood mononuclear cells, and taking intermediate layer cells after Ficoll centrifugal separation; PBMC were obtained after PBS wash.

b) Separation of antigen specific T lymphocyte by immunomagnetic bead method

Taking the PBMC, adding a serum-free basal culture medium to prepare a cell suspension; adding CD3/CD28 immunomagnetic beads according to the ratio of the magnetic beads to the cells being 3:1, and incubating for 1-2h at room temperature; screening the cells incubated with the magnetic beads by using a magnet; after washing with PBS and removal of immunomagnetic beads, CD 3-positive T lymphocytes were obtained.

c) Preparation of antigen-specific T lymphocytes by virus transfection method

And (3) adding the recombinant lentivirus with the virus titer corresponding to the number of the CD3 positive cells into the CD3 positive T lymphocytes obtained by the immunomagnetic bead separation method for culture.

On the 3 rd day of the culture, cell counting and medium exchange were performed to adjust the cell concentration to 1X 10⁶Inoculating and culturing the seeds per mL; on the 5 th day of culture, the state of cells was observed, and if the cell density increased, the cell concentration was diluted to 1X 10⁶And (4) detecting the activity of the cells per mL, and continuing to culture. And performing amplification culture on the cells until 9-11 days, collecting the cells to obtain chimeric antigen receptor T cells which carry a safety switch and target EGFRv III, and storing the chimeric antigen receptor T cells in a cell freezing medium special for reinfusion.

Effects of the embodiment

Evaluation of in vitro tumor cell killing of chimeric antigen receptor T cells carrying a safety switch and targeting EGFRv III

Comparing the in vitro tumor killing effects of the chimeric antigen receptor T cell carrying the safety switch and targeting the EGFRvIII prepared by the method, the chimeric antigen receptor T cell carrying the safety switch and targeting the EGFRvIII and treated by the AP20187, the chimeric antigen receptor T cell targeting the EGFRvIII and the T lymphocyte (negative control group) not prepared, specifically: in vitro number ratio of effector cells to target cellsAt a ratio of 1:10, 1:3, 1:1, 3:1 and 10:1, 5% CO at 37 deg.C₂Then, co-culture was performed, and at 15 to 18 hours after the culture, cells were collected, flow-stained, and cell killing was examined.

The results show that the killing rates of the chimeric antigen receptor T cell carrying the safety switch and targeting the EGFRvIII, the chimeric antigen receptor T cell targeting the EGFRvIII and the chimeric antigen receptor T cell targeting the EGFRvIII treated by the AP20187 are all 100 percent and are far higher than that of a negative control group; the killing rate of the chimeric antigen receptor T cells which are treated by the AP20187 and carry the safety switch and target the EGFRv III is reduced to 10%, which indicates that the chimeric antigen receptor T cells which carry the safety switch and target the EGFRv III are not influenced by the safety switch and can be normally used, and when adverse reactions occur, such as cytokine release syndrome, a dimerization chemical inducer is injected to induce apoptosis, so that the killing power of the chimeric antigen receptor T cells is remarkably reduced and effectively stopped.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Shenzhen Binje Biotechnology Limited

<120> chimeric antigen receptor T cell carrying safety switch and targeting EGFRvIII, and preparation method and application thereof

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 246

<212> PRT

<213> Artificial Sequence

<400> 1

Gly Ile Gly Leu Val Gly Ser Gly Ala Gly Val Leu Leu Pro Gly Gly

1 5 10 15

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

20 25 30

Thr Ile His Thr Val Ala Gly Met Pro Gly Leu Gly Leu Gly Thr Met

35 40 45

Gly Ala Ile Ala Pro Gly Ala Ala Gly Thr Leu Thr Gly Pro Ile Pro

50 55 60

Gly Gly His Val Thr Ile Ser Ala Ala Thr Ser Ile Ala Thr Val Thr

65 70 75 80

Leu Gly Thr Ser Ser Leu Leu Ala Ser Ala Thr Ala Met Thr Thr Cys

85 90 95

Ala Pro Ala Gly Gly Val Thr Thr Gly Gly Gly Thr Thr Val Thr Val

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Gly Ser Leu Leu Ala Ser Ala Gly Leu Thr Thr Leu Ala Thr Leu Gly

165 170 175

Gly Leu Pro Gly Gly Pro Pro Leu Ala Leu Ile Ser Leu Val Ser Leu

180 185 190

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

195 200 205

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

210 215 220

Thr Thr Cys Thr Gly Gly Thr His Pro Pro Gly Thr Pro Gly Gly Gly

225 230 235 240

Thr Leu Val Gly Ile Leu

245

<210> 2

<211> 107

<212> PRT

<213> Artificial Sequence

<400> 2

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu

100 105

<210> 3

<211> 277

<212> PRT

<213> Artificial Sequence

<400> 3

Gly Ala Leu Glu Ser Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu

1 5 10 15

Ser Met Glu Pro Cys Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe

20 25 30

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

35 40 45

Glu Lys Leu Arg Arg Arg Phe Ser Ser Leu His Phe Met Val Glu Val

50 55 60

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

65 70 75 80

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

85 90 95

Ser His Gly Cys Gln Ala Ser His Leu Gln Phe Pro Gly Ala Val Tyr

100 105 110

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

115 120 125

Asn Gly Thr Ser Cys Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe

130 135 140

Ile Gln Ala Cys Gly Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala

145 150 155 160

Ser Thr Ser Pro Glu Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro Asp

165 170 175

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

180 185 190

Ile Ser Ser Leu Pro Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser Thr

195 200 205

Phe Pro Gly Phe Val Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Tyr

210 215 220

Val Glu Thr Leu Asp Asp Ile Phe Glu Gln Trp Ala His Ser Glu Asp

225 230 235 240

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

245 250 255

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

260 265 270

Phe Phe Lys Thr Ser

275

<210> 4

<211> 469

<212> PRT

<213> Artificial Sequence

<400> 4

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

1 5 10 15

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

20 25 30

Tyr Ile His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Glu Asn Asp Glu Thr Lys Tyr Gly Pro Ile Phe

50 55 60

Gln Gly His Val Thr Ile Ser Ala Asp Thr Ser Ile Asn Thr Val Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Tyr Tyr Cys Trp Gln Gly Thr His Phe Pro Gly Thr Phe Gly Gly Gly

225 230 235 240

Thr Lys Val Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Ala Leu Pro Pro Arg

465

<210> 5

<211> 389

<212> PRT

<213> Artificial Sequence

<400> 5

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Ala Leu Glu Ser Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu

115 120 125

Ser Met Glu Pro Cys Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe

130 135 140

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

145 150 155 160

Glu Lys Leu Arg Arg Arg Phe Ser Ser Leu His Phe Met Val Glu Val

165 170 175

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

180 185 190

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

195 200 205

Ser His Gly Cys Gln Ala Ser His Leu Gln Phe Pro Gly Ala Val Tyr

210 215 220

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

225 230 235 240

Asn Gly Thr Ser Cys Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe

245 250 255

Ile Gln Ala Cys Gly Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala

260 265 270

Ser Thr Ser Pro Glu Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro Asp

275 280 285

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

290 295 300

Ile Ser Ser Leu Pro Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser Thr

305 310 315 320

Phe Pro Gly Phe Val Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Tyr

325 330 335

Val Glu Thr Leu Asp Asp Ile Phe Glu Gln Trp Ala His Ser Glu Asp

340 345 350

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

355 360 365

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

370 375 380

Phe Phe Lys Thr Ser

385

<210> 6

<211> 738

<212> DNA

<213> Artificial Sequence

<400> 6

gagattcagc tcgtgcaatc gggagcggaa gtcaagaagc caggagagtc cttgcggatc 60

tcatgcaagg gtagcggctt taacatcgag gattactaca tccactgggt gaggcagatg 120

ccggggaagg gactcgaatg gatgggacgg atcgacccag aaaacgacga aactaagtac 180

ggtccgatct tccaaggcca tgtgactatt agcgccgata cttcaatcaa taccgtgtat 240

ctgcaatggt cctcattgaa agcctcagat accgcgatgt actactgtgc tttcagagga 300

ggggtctact ggggacaggg aactaccgtg actgtctcgt ccggcggagg cgggtcagga 360

ggtggcggca gcggaggagg agggtccggc ggaggtgggt ccgacgtcgt gatgacccag 420

agccctgaca gcctggcagt gagcctgggc gaaagagcta ccattaactg caaatcgtcg 480

cagagcctgc tggactcgga cggaaaaacg tacctcaatt ggctgcagca aaagcctggc 540

cagccaccga agcgccttat ctcactggtg tcgaagctgg attcgggagt gcccgatcgc 600

ttctccggct cgggatcggg tactgacttc accctcacta tctcctcgct tcaagcagag 660

gacgtggccg tctactactg ctggcaggga acccactttc cgggaacctt cggcggaggg 720

acgaaagtgg agatcaag 738

<210> 7

<211> 1407

<212> DNA

<213> Artificial Sequence

<400> 7

gagattcagc tcgtgcaatc gggagcggaa gtcaagaagc caggagagtc cttgcggatc 60

tcatgcaagg gtagcggctt taacatcgag gattactaca tccactgggt gaggcagatg 120

ccggggaagg gactcgaatg gatgggacgg atcgacccag aaaacgacga aactaagtac 180

ggtccgatct tccaaggcca tgtgactatt agcgccgata cttcaatcaa taccgtgtat 240

ctgcaatggt cctcattgaa agcctcagat accgcgatgt actactgtgc tttcagagga 300

ggggtctact ggggacaggg aactaccgtg actgtctcgt ccggcggagg cgggtcagga 360

ggtggcggca gcggaggagg agggtccggc ggaggtgggt ccgacgtcgt gatgacccag 420

agccctgaca gcctggcagt gagcctgggc gaaagagcta ccattaactg caaatcgtcg 480

cagagcctgc tggactcgga cggaaaaacg tacctcaatt ggctgcagca aaagcctggc 540

cagccaccga agcgccttat ctcactggtg tcgaagctgg attcgggagt gcccgatcgc 600

ttctccggct cgggatcggg tactgacttc accctcacta tctcctcgct tcaagcagag 660

gacgtggccg tctactactg ctggcaggga acccactttc cgggaacctt cggcggaggg 720

acgaaagtgg agatcaagac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 780

atcgcgtcgc agcccctgtc cctgcgccca gaggcgtgcc ggccagcggc ggggggcgca 840

gtgcacacga gggggctgga cttcgcctgt gatatctaca tctgggcgcc cttggccggg 900

acttgtgggg tccttctcct gtcactggtt atcacccttt actgcaaacg gggcagaaag 960

aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 1020

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gagagtgaag 1080

ttcagcagga gcgcagacgc ccccgcgtac aagcagggcc agaaccagct ctataacgag 1140

ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1200

gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1260

aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1320

aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1380

cttcacatgc aggccctgcc ccctcgc 1407

<210> 8

<211> 321

<212> DNA

<213> Artificial Sequence

<400> 8

ggagtgcagg tggaaaccat ctccccagga gacgggcgca ccttccccaa gcgcggccag 60

acctgcgtgg tgcactacac cgggatgctt gaagatggaa agaaatttga ttcctcccgg 120

gacagaaaca agccctttaa gtttatgcta ggcaagcagg aggtgatccg aggctgggaa 180

gaaggggttg cccagatgag tgtgggtcag agagccaaac tgactatatc tccagattat 240

gcctatggtg ccactgggca cccaggcatc atcccaccac atgccactct cgtcttcgat 300

gtggagcttc taaaactgga a 321

<210> 9

<211> 831

<212> DNA

<213> Artificial Sequence

<400> 9

ggtgctcttg agagtttgag gggaaatgca gatttggctt acatcctgag catggagccc 60

tgtggccact gcctcattat caacaatgtg aacttctgcc gtgagtccgg gctccgcacc 120

cgcactggct ccaacatcga ctgtgagaag ttgcggcgtc gcttctcctc gctgcatttc 180

atggtggagg tgaagggcga cctgactgcc aagaaaatgg tgctggcttt gctggagctg 240

gcgcagcagg accacggtgc tctggactgc tgcgtggtgg tcattctctc tcacggctgt 300

caggccagcc acctgcagtt cccaggggct gtctacggca cagatggatg ccctgtgtcg 360

gtcgagaaga ttgtgaacat cttcaatggg accagctgcc ccagcctggg agggaagccc 420

aagctctttt tcatccaggc ctgtggtggg gagcagaaag accatgggtt tgaggtggcc 480

tccacttccc ctgaagacga gtcccctggc agtaaccccg agccagatgc caccccgttc 540

caggaaggtt tgaggacctt cgaccagctg gacgccatat ctagtttgcc cacacccagt 600

gacatctttg tgtcctactc tactttccca ggttttgttt cctggaggga ccccaagagt 660

ggctcctggt acgttgagac cctggacgac atctttgagc agtgggctca ctctgaagac 720

ctgcagtccc tcctgcttag ggtcgctaat gctgtttcgg tgaaagggat ttataaacag 780

atgcctggtt gctttaattt cctccggaaa aaacttttct ttaaaacatc a 831

<210> 10

<211> 5

<212> PRT

<213> Artificial Sequence

<400> 10

Ser Gly Gly Gly Ser

1 5

<210> 11

<211> 247

<212> DNA

<213> Artificial Sequence

<400> 11

ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag 60

cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg 120

gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac 180

aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa 240

gcgtatt 247

<210> 12

<211> 60

<212> DNA

<213> Artificial Sequence

<400> 12

gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60

Claims

1. A chimeric antigen receptor T-cell carrying a safety switch and targeting egfrviii, comprising a chimeric antigen receptor CAR-egfrviii targeting egfrviii and a safety switch inducing apoptosis of T-cells, wherein said CAR-egfrviii comprises, sequentially joined from amino terminus to carboxy terminus, an amino acid sequence of a single-chain antibody targeting egfrviii, an extracellular hinge region, a transmembrane region and an intracellular signal region, said single-chain antibody targeting egfrviii comprising the amino acid sequence as set forth in SEQ ID NO: 1, the safety switch comprising the amino acid sequence of the F36V mutant FK506 binding protein, the linker peptide, and the caspase 9 that removes the CARD, sequentially linked from the amino terminus to the carboxy terminus, the amino acid sequence of the F36V mutant FK506 binding protein comprising the amino acid sequence as set forth in SEQ ID NO: 2, and the amino acid sequence of caspase 9 with the CARD removed comprises the amino acid sequence shown as SEQ ID NO: 3.

2. The chimeric antigen receptor T cell carrying a safety switch and targeting EGFRv iii according to claim 1, wherein said CAR-EGFRv iii and said safety switch are linked by an internal ribosomal entry site or by a self-cleaving polypeptide.

3. The chimeric antigen receptor T cell carrying a safety switch and targeting egfrviii according to claim 1, wherein the amino acid sequence of CAR-egfrviii comprises the amino acid sequence as set forth in SEQ ID NO: 4.

4. The chimeric antigen receptor T cell carrying a safety switch and targeting EGFRv iii according to claim 1, wherein the amino acid sequence of the safety switch comprises the amino acid sequence as set forth in SEQ ID NO: 5.

5. The chimeric antigen receptor T cell carrying a safety switch and targeting EGFRv iii of claim 1, further comprising a dimerization chemical inducer comprising at least one of AP1903 and AP 20187.

6. A method for preparing a chimeric antigen receptor T cell carrying a safety switch and targeting EGFRv III, comprising:

7. The method of claim 6, wherein inserting the CAR-EGFRv iii coding gene and the safety switch coding gene into a gene delivery vector comprises:

8. A recombinant vector, which comprises an inserted EGFRvIII-targeting chimeric antigen receptor CAR-EGFRvIII coding gene and a coding gene of a safety switch for inducing T cell apoptosis, wherein the EGFRvIII-targeting chimeric antigen receptor CAR-EGFRvIII coding gene comprises a coding gene of a signal peptide, a coding gene of an EGFRvIII-targeting single-chain antibody, a coding gene of an extracellular hinge region, a transmembrane region and an intracellular signal region which are sequentially connected from 5 'end to 3' end, and the coding gene of the EGFRvIII-targeting single-chain antibody comprises the following genes: 1, and the nucleotide sequence corresponds to the amino acid sequence shown in the specification; the encoding gene of the safety switch for inducing T cell apoptosis comprises a gene encoding F36V mutant FK506 binding protein, a gene encoding connecting peptide and a gene encoding CARD-removed caspase 9 which are sequentially connected from 5 'end to 3', wherein the encoding gene of the F36V mutant FK506 binding protein comprises the nucleotide sequence shown in SEQ ID NO: 2, and the coding gene of caspase 9 without CARD comprises the nucleotide sequence shown as SEQ ID NO: 3, or a nucleotide sequence corresponding to the amino acid sequence shown in the figure.

9. A host cell comprising the recombinant vector of claim 8.

10. Use of a chimeric antigen receptor T cell carrying a safety switch and targeting egfrviii, produced by the process of manufacture of any one of claims 1 to 5 or 6 to 7, a recombinant vector according to claim 8 or a host cell according to claim 9 for the preparation of a medicament for the prevention and treatment of malignancies.