CN112430579A - Chimeric antigen receptor T cell targeting Her2 and interfering IL-6 expression, and preparation method and application thereof - Google Patents

Chimeric antigen receptor T cell targeting Her2 and interfering IL-6 expression, and preparation method and application thereof Download PDF

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CN112430579A
CN112430579A CN201910792817.XA CN201910792817A CN112430579A CN 112430579 A CN112430579 A CN 112430579A CN 201910792817 A CN201910792817 A CN 201910792817A CN 112430579 A CN112430579 A CN 112430579A
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expression
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her2
interfering
antigen receptor
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曾滢
汪婷婷
杨忠华
张宏玲
唐超
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Shenzhen Bindebio Technology Co ltd
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Shenzhen Bindebio Technology Co ltd
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Abstract

The invention provides a chimeric antigen receptor T cell targeting Her2 and interfering with IL-6 expression, which comprises a chimeric antigen receptor CAR-Her2 targeting Her2 and siRNA interfering with IL-6 expression, wherein CAR-Her2 comprises amino acid sequences of a single-chain antibody targeting Her2, an extracellular hinge region, a transmembrane region and an intracellular signal region which are sequentially connected from an amino terminal to a carboxyl terminal, and the single-chain antibody targeting Her2 comprises the amino acid sequence shown as SEQ ID NO:1, and the DNA sequence corresponding to siRNA interfering IL-6 expression comprises the amino acid sequence shown in SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4; the tumor cell specifically targeted to express Her2 can interfere with the expression of IL-6, avoid the expression of a large number of cell factors and enable the T cell to have lasting cell activity and lethality.

Description

Chimeric antigen receptor T cell targeting Her2 and interfering IL-6 expression, 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 which targets Her2 and interferes with IL-6 expression, 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.
Her2 is epidermal growth factor receptor 2, is a tumor-associated antigen, is expressed on the surface of 80% of malignant glioma cells (GBMs), but is not expressed on the surface of normal neurons and glial cells after birth, and is proved to be an immunotherapy target with higher specificity through a plurality of experimental centers, so Her2 has remarkable curative effect on the treatment of malignant Glioma (GBM) in recent years and is considered to be one of the most potential treatment modes of GBM.
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 target Her2 and interfere with IL-6 expression and a preparation method thereof are needed.
Disclosure of Invention
In view of the above, the invention provides a chimeric antigen receptor T cell targeting Her2 and interfering with IL-6 expression, which has a chimeric antigen receptor targeting Her2, and further can specifically target tumor cells expressing Her2, activate T cells to exert a cellular immune effect, achieve efficient and specific killing on Her2 positive tumor cells, and have lasting cell viability and lethality; meanwhile, the chimeric antigen receptor T cell can interfere IL-6 expression, avoid the expression of a large number of cytokines in the action process of the chimeric antigen receptor T cell, change the tumor microenvironment, and simultaneously prevent the chimeric antigen receptor T cell from being in an immune suppression state in the tumor microenvironment, so that the chimeric antigen receptor T cell can fully exert the killing effect of specific tumor cells, maintain the lasting cell vitality and the killing power, and can not damage normal cells.
In a first aspect, the invention provides a Her 2-targeted chimeric antigen receptor T cell that interferes with IL-6 expression, comprising a Her 2-targeted chimeric antigen receptor CAR-Her2 and an siRNA that interferes with IL-6 expression, wherein the CAR-Her2 comprises the amino acid sequence of a Her 2-targeted single-chain antibody, an extracellular hinge region, a transmembrane region, and an intracellular signaling region, sequentially linked from amino-terminus to carboxy-terminus, and the Her 2-targeted single-chain antibody comprises the amino acid sequence as set forth in SEQ ID NO:1, and the DNA sequence corresponding to the siRNA for interfering the expression of IL-6 comprises the amino acid sequence shown as SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4.
Alternatively, the Her 2-targeted single-chain antibody encoding gene comprises the amino acid sequence shown in SEQ ID NO: 12. Furthermore, the gene encoding the Her 2-targeted single-chain antibody should take into consideration degenerate bases, that is, the gene encoding the amino acid sequence shown in SEQ ID NO. 1 includes the nucleotide sequence shown in SEQ ID NO. 12, and the protection scope should also protect the nucleotide sequence with base degeneracy with the SEQ ID NO. 12, and the corresponding amino acid sequence of the nucleotide sequence is still SEQ ID NO. 1.
In the present invention, the "connecting in sequence from amino terminus to carboxyl terminus" specifically includes: the carboxy terminus of the amino acid sequence of the Her 2-targeting single chain antibody is linked to the amino terminus of the amino acid sequence of the extracellular hinge region, the carboxy terminus of the amino acid sequence of the extracellular hinge region is linked to the amino terminus of the amino acid sequence of the transmembrane region, and the carboxy terminus of the amino acid sequence of the transmembrane region is linked to the amino terminus 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 Her2 targeted single chain antibody to Her2 on a tumor.
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 Her 2-targeted chimeric antigen receptor CAR-Her 2.
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-Her2 comprises the amino acid sequence set forth as SEQ ID NO: 5.
Further, the encoding gene of CAR-Her2 comprises the sequence shown in SEQ ID NO:13, or a nucleotide sequence as set forth in seq id no.
Alternatively, the CAR-Her2 encoding gene should take into account degenerate bases, i.e. the encoding gene of the amino acid sequence shown in SEQ ID NO. 5 comprises the nucleotide sequence shown in SEQ ID NO. 13, and the protection scope should also protect nucleotide sequences having base degeneracy with SEQ ID NO. 13, and the corresponding amino acid sequences of these nucleotide sequences are still SEQ ID NO. 5.
In the invention, the chimeric antigen receptor CAR-Her2 targeting Her2 enables T cells to specifically target Her 2-expressing tumor cells, the single-chain antibody can specifically recognize and specifically bind to Her2 protein on the tumor cells, and after the CAR-Her2 is bound to Her2, an intracellular signal region is activated, the expansion of the T cells in a patient body is promoted, and the tumor cells are killed efficiently and specifically. The Her2 is widely expressed in malignant cells and weakly expressed in common cells, so the chimeric antigen receptor T cell targeting the Her2 provided by the invention can be specifically combined with tumor cells, has stronger affinity activity and internalization activity on the Her 2-expressing malignant cells, generates a killing effect on the tumor cells, and does not damage normal cells.
Alternatively, when the DNA sequence corresponding to the siRNA interfering with the expression of IL-6 comprises the nucleotide sequence shown in SEQ ID NO: 2, the DNA sequence corresponding to the sense strand of the siRNA interfering the expression of IL-6 comprises the nucleotide sequence shown in SEQ ID NO: 6, and the DNA sequence corresponding to the antisense strand of the siRNA interfering the expression of IL-6 comprises the nucleotide sequence shown in SEQ ID NO: 7.
Alternatively, when the DNA sequence of the siRNA interfering with IL-6 expression comprises the nucleotide sequence shown in SEQ ID NO: 3, the DNA sequence corresponding to the sense strand of the siRNA interfering the expression of IL-6 comprises the nucleotide sequence shown as SEQ ID NO: 8, and the DNA sequence corresponding to the antisense strand of the siRNA interfering the expression of IL-6 comprises the nucleotide sequence shown as SEQ ID NO: 9, or a nucleotide sequence shown in the specification.
Alternatively, when the DNA sequence of the siRNA interfering with IL-6 expression comprises the nucleotide sequence shown in SEQ ID NO: 4, the DNA sequence corresponding to the sense strand of the siRNA interfering the expression of IL-6 comprises the nucleotide sequence shown as SEQ ID NO: 10, and the DNA sequence corresponding to the antisense strand of the siRNA interfering the expression of IL-6 comprises the nucleotide sequence shown in SEQ ID NO: 11.
Further, the DNA sequence corresponding to the siRNA interfering the expression of IL-6 comprises the nucleotide sequence shown in SEQ ID NO: 2.
In the present invention, small interfering RNA (siRNA) effectively silences or inhibits the expression of a target gene by selectively inactivating the corresponding mRNA of the target gene via double-stranded RNA (dsRNA). IL-6 is a proper target molecule of a cytokine storm, can quickly solve the toxic and side effects brought by CRS after blocking an IL-6 receptor and has no influence on the in-vivo proliferation of chimeric antigen receptor T cells.
The chimeric antigen receptor T cell which is targeted to Her2 and interferes with IL-6 expression provided by the invention has a chimeric antigen receptor targeted to Her2, can specifically target tumor cells expressing Her2, activates the T cell to play a cellular immune role, realizes efficient and specific killing on Her2 positive tumor cells, and has lasting cell activity and lethality; meanwhile, the chimeric antigen receptor T cell can interfere IL-6 expression, avoid the expression of a large number of cytokines in the action process of the chimeric antigen receptor T cell, change the tumor microenvironment, and simultaneously prevent the chimeric antigen receptor T cell from being in an immune suppression state in the tumor microenvironment, so that the chimeric antigen receptor T cell can fully exert the killing effect of specific tumor cells, maintain the lasting cell vitality and the killing power, and can not damage normal cells.
In a second aspect, the invention provides a method for making a chimeric antigen receptor T cell that targets Her2 and interferes with IL-6 expression, comprising:
(1) providing genes encoding a Her 2-targeted chimeric antigen receptor CAR-Her2, comprising a gene encoding a signal peptide, a gene encoding a Her 2-targeted single chain 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 'end to 3' end, wherein the gene encoding the Her 2-targeted single chain antibody comprises the amino acid sequence set forth in SEQ ID NO:1, and the nucleotide sequence corresponds to the amino acid sequence shown in the specification;
(2) providing a DNA sequence corresponding to siRNA interfering IL-6 expression, wherein the DNA sequence corresponding to siRNA interfering IL-6 expression comprises the nucleotide sequence shown in SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4;
(3) inserting the coding gene of the CAR-Her2 and a DNA sequence corresponding to the siRNA interfering the expression of the IL-6 into a gene delivery vector to obtain a recombinant gene delivery 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 which target Her2 and interfere IL-6 expression.
In the present invention, the "connecting in sequence from 5 'end to 3' end" specifically includes: the 3 'end of the coding gene sequence of the signal peptide is connected with the 5' end of the coding gene of the Her 2-targeting single-chain antibody, the 3 'end of the coding gene of the Her 2-targeting single-chain antibody 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 signal peptide is used to direct the expression of the chimeric antigen receptor CAR-Her2 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: as shown at 14.
Wherein, the specific selection of the extracellular hinge region, the transmembrane region and the intracellular signal region is as described in the first aspect of the present invention, and is not described herein again.
Optionally, the amino acid sequence corresponding to the coding gene of CAR-Her2 containing the signal peptide is shown in SEQ ID NO: shown at 15.
Optionally, the 5' end of the DNA sequence corresponding to the siRNA interfering the expression of IL-6 is connected with a human H1 promoter, a human U6 promoter or a mouse U6 promoter.
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-Her2 is inserted between BamHI and EcoRI cleavage sites in pWPXLD vector, and is located after EF1 α of pWPXLD vector, with EF1 α as promoter. When the coding gene of the CAR-Her2 is inserted into a pWPXLD vector, the 5 'end of the coding gene of the CAR-Her2 can be added with an initiation codon (such as ATG) and connected with a BamHI cleavage 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 cleavage site (gatattc) in the pWPXLD vector, so that the coding gene of the CAR-Her2 is positioned between the BamHI cleavage site and the EcoRI cleavage site, and the pWPXLD-CAR-Her2 recombinant plasmid is obtained. The DNA sequence corresponding to the siRNA for interfering the expression of the IL-6 is inserted between SmaI and NdeI enzyme cutting sites in a pWPXLD-CAR-Her2 recombinant plasmid, and the 5' end of the DNA sequence corresponding to the siRNA for interfering the expression of the IL-6 is connected with a human H1 promoter, a human U6 promoter or a mouse U6 promoter; and adding corresponding protective bases and SmaI enzyme cutting sites to the 5 'end and NdeI enzyme cutting sites to the 3' end of the DNA sequence corresponding to the siRNA interfering the expression of the IL-6, and adding the DNA sequence corresponding to the siRNA interfering the expression of the IL-6 into a pWPXLD-CAR-Her2 recombinant plasmid, wherein the siRNA sequence is positioned behind CAR-Her2 and takes H1 as a promoter. And then transferring the plasmid into DH5 alpha escherichia coli, carrying out vector amplification and enzyme digestion, and carrying out sequencing identification to obtain a pWPXLD-CAR-Her2-IL-6siRNA recombinant lentiviral plasmid vector.
In the present invention, the gene fragment inserted into the gene delivery vector can be, but not limited to, initiation codon, the gene encoding CAR-Her2 and stop codon, and a DNA sequence corresponding to a promoter and the siRNA interfering with IL-6 expression.
In the invention, the DNA sequence corresponding to the siRNA interfering with IL-6 expression can be inserted into a gene delivery vector, and then the coding gene of CAR-Her2 can be inserted into the gene delivery vector to obtain a recombinant gene delivery vector.
In the invention, a promoter can be additionally added in the coding gene of the CAR-Her2 and/or a DNA sequence corresponding to the siRNA interfering with IL-6 expression, so that the sequence of inserting the CAR-Her2 and the siRNA into a gene delivery vector is not limited, and the expression of the final protein CAR-Her2 and siRNA is not influenced.
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.
In a third aspect, the invention provides a recombinant vector, which comprises an inserted Her 2-targeting chimeric antigen receptor CAR-Her2 coding gene and a DNA sequence corresponding to siRNA interfering with IL-6 expression, wherein the Her 2-targeting chimeric antigen receptor CAR-Her2 coding gene comprises a coding gene of a signal peptide, a coding gene of a Her 2-targeting single-chain antibody, a coding gene of an extracellular hinge region, a coding gene of a transmembrane region, and a coding gene of an intracellular signal region which are sequentially connected from 5 'end to 3' end, and the coding gene of the Her 2-targeting single-chain antibody comprises 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 DNA sequence corresponding to the siRNA for interfering the expression of the IL-6 comprises the nucleotide sequence shown in SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4.
In the invention, the recombinant vector is obtained by inserting a gene coding for a chimeric antigen receptor CAR-Her2 targeting Her2 and a DNA sequence corresponding to siRNA interfering with IL-6 expression into the vector.
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 encoding gene of the chimeric antigen receptor CAR-Her2 targeting Her2 and the DNA sequence corresponding to the siRNA interfering with the expression of IL-6 can be directly transfected into CD3 positive T lymphocytes to prepare the chimeric antigen receptor T cells targeting Her2 and interfering with the expression of IL-6.
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 targeting Her2 and interfering with IL-6 expression, a recombinant vector as described in the third aspect, or a host cell as described in the fourth aspect, prepared according to the first aspect or the preparation method as described in the second aspect, for use in the preparation of a medicament for preventing and treating malignant tumors.
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 pWPXLD-CAR-Her2 recombinant vector provided by the embodiment of the present invention.
FIG. 2 is a plasmid map of pWPXLD-CAR-Her2-IL-6siRNA 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) Recombinant vector construction targeting Her2 and interfering with IL-6 expression
Providing a gene encoding CAR-Her2 comprising a signal peptide, i.e. providing a sequence as set forth in SEQ ID NO: 15, adding a restriction enzyme site and an initiation codon at the 5 'end of the nucleotide sequence corresponding to the amino acid sequence shown in the specification, and adding a restriction enzyme site and a termination codon at the 3' end of the nucleotide sequence; it was inserted between BamHI and EcoRI sites of pWPXLD vector and followed by EF1 α, using EF1 α as promoter. 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 which meets the target are identified through PCR product gel electrophoresis detection and sequencing, and the pWPXLD-CAR-Her2 recombinant plasmid is successfully constructed, and is shown as a pWPXLD-CAR-Her2 recombinant vector in figure 1.
Providing a DNA sequence corresponding to siRNA interfering with IL-6 expression, namely providing a DNA sequence shown as SEQ ID NO: 2, and adding a protective base, a Sma I enzyme cutting site and a human H1 promoter at the 5 'end of the nucleotide sequence, and adding a protective base and a Nde I enzyme cutting site at the 3' end of the nucleotide sequence, wherein the sequence is shown as SEQ ID NO: 16 is shown in the figure; it was inserted between the SmaI and Nde I cleavage sites of the pWPXLD-CAR-Her2 recombinant plasmid. 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 which meets the target are identified through PCR product gel electrophoresis detection and sequencing, and a pWPXLD-CAR-Her2-IL-6siRNA recombinant plasmid is successfully constructed, and is shown as a pWPXLD-CAR-Her2-IL-6siRNA recombinant vector in figure 2.
(2) Recombinant lentivirus construction
The pWPXLD-CAR-Her2-IL-6siRNA recombinant plasmid, 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 cells targeting Her2 and interfering with IL-6 expression
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 expanding and culturing to 9-11 days, collecting cells, obtaining chimeric antigen receptor T cells which target Her2 and interfere IL-6 expression, and storing in a cell freezing medium special for reinfusion.
Effect example 1
Evaluation of in vitro tumor cell killing of chimeric antigen receptor T cells targeting Her2 and interfering with IL-6 expression
Comparing the in vitro tumor killing effect of the chimeric antigen receptor T cell (abbreviated as CAR-T-Her2-siRNA) which is prepared by the method and targets Her2 and interferes with the expression of IL-6 with that of the chimeric antigen receptor T cell (abbreviated as CAR-T-Her2) which is targeted at Her2 and the T lymphocyte (negative control group) which is not prepared, the method specifically comprises the following steps: in vitro, effector cells were added to target cells at a ratio of 1:10, 1:3, 1:1, 3:1 and 10:1 at 37 deg.C with 5% CO2And then co-culturing, collecting cells at 15-18 hours after culturing, carrying out flow-type staining, and detecting the killing condition of the cells, and finding that the tumor killing effect of the CAR-T-Her2-siRNA cells prepared by the method is higher than that of CAR-T-Her2 cells and far higher than that of a negative control group, so that the CAR-T cells prepared by the method have strong tumor killing capability after the expression of IL-6 is inhibited.
Effect example 2
Evaluation of tumor cell killing in mice targeting Her2 and interfering with IL-6 expressing chimeric antigen receptor T cells
The chimeric antigen receptor T cell (CAR-T-Her2-siRNA) which is prepared by the method and targets Her2 and interferes with the expression of IL-6, the chimeric antigen receptor T cell (CAR-T-Her 2) which targets Her2, the T lymphocyte (negative control group) which is not prepared and physiological saline (blank control group) are administered in a mouse tumor modelIntravenous injection of 1X 10 to the tail of each mouse6And obtaining a survival curve of the mouse by using the cell (n-9), and finding that the CAR-T-Her2-siRNA cell prepared by the method can better protect the mouse from death caused by tumor, the average survival time is higher than that of CAR-T-Her2 cells, and the effect is better than that of a negative control group and a blank group.
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 targeting Her2 and interfering IL-6 expression, preparation method and application thereof
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Asp Ile Gln Leu Thr Gln Ser His Lys Phe Leu Ser Thr Ser Val Gly
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Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Tyr Asn Ala
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Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
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Tyr Ser Ala Ser Ser Arg Tyr Thr Gly Val Pro Ser Arg Phe Thr Gly
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Ser Gly Ser Gly Pro Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala
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Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln His Phe Arg Thr Pro Phe
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Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Gly Gly Gly Gly Ser Gly
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Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser
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Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys
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Ala Ser Gly Tyr Pro Phe Thr Asn Tyr Gly Met Asn Trp Val Lys Gln
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Ala Pro Gly Gln Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Ser Thr
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Gly Glu Ser Thr Phe Ala Asp Asp Phe Lys Gly Arg Phe Asp Phe Ser
180 185 190
Leu Glu Thr Ser Ala Asn Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys
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Ser Glu Asp Met Ala Thr Tyr Phe Cys Ala Arg Trp Glu Val Tyr His
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Gly Tyr Val Pro Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
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Asp Ile Gln Leu Thr Gln Ser His Lys Phe Leu Ser Thr Ser Val Gly
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Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Tyr Asn Ala
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Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
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Tyr Ser Ala Ser Ser Arg Tyr Thr Gly Val Pro Ser Arg Phe Thr Gly
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Ser Gly Ser Gly Pro Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala
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Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln His Phe Arg Thr Pro Phe
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Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Gly Gly Gly Gly Ser Gly
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Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser
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Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys
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Ala Ser Gly Tyr Pro Phe Thr Asn Tyr Gly Met Asn Trp Val Lys Gln
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Ala Pro Gly Gln Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Ser Thr
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Gly Glu Ser Thr Phe Ala Asp Asp Phe Lys Gly Arg Phe Asp Phe Ser
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Leu Glu Thr Ser Ala Asn Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys
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Ser Glu Asp Met Ala Thr Tyr Phe Cys Ala Arg Trp Glu Val Tyr His
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Gly Tyr Val Pro Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
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Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
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Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
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Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
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Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
290 295 300
Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
305 310 315 320
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
325 330 335
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
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Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln
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Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
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Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
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Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
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Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
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Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
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Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
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gacatccagc tgacccagtc tcacaaattc ctgtccactt cagtaggaga cagggtcagc 60
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ggacaatctc ctaaacttct gatttactcg gcatcctccc ggtacactgg agtcccttct 180
cgcttcactg gcagtggctc tgggccggat ttcactttca ccatcagcag tgtgcaggct 240
gaagacctgg cagtttattt ctgtcagcaa cattttcgta ctccattcac gttcggctcg 300
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tctcaggtac aactgcagca gtctggacct gaactgaaga agcctggaga gacagtcaag 420
atctcctgca aggcctctgg gtatcctttc acaaactatg gaatgaactg ggtgaagcag 480
gctccaggac agggtttaaa gtggatgggc tggattaaca cttccactgg agagtcaaca 540
tttgctgatg acttcaaggg acggtttgac ttctctttgg aaacctctgc caacactgcc 600
tatttgcaga tcaacaacct caaaagtgaa gacatggcta catatttctg tgcaagatgg 660
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ggacaatctc ctaaacttct gatttactcg gcatcctccc ggtacactgg agtcccttct 180
cgcttcactg gcagtggctc tgggccggat ttcactttca ccatcagcag tgtgcaggct 240
gaagacctgg cagtttattt ctgtcagcaa cattttcgta ctccattcac gttcggctcg 300
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gctccaggac agggtttaaa gtggatgggc tggattaaca cttccactgg agagtcaaca 540
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tatttgcaga tcaacaacct caaaagtgaa gacatggcta catatttctg tgcaagatgg 660
gaggtttacc acggctacgt tccttactgg ggccaaggga ccacggtcac cgtttcctct 720
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 780
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 840
gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 900
ctgtcactgg ttatcaccct ttactgcaaa cggggcagaa agaaactcct gtatatattc 960
aaacaaccat ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga 1020
tttccagaag aagaagaagg aggatgtgaa ctgagagtga agttcagcag gagcgcagac 1080
gcccccgcgt acaagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 1140
gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 1200
agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 1260
gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 1320
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ccccctcgc 1389
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<213> Artificial Sequence
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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> 483
<212> PRT
<213> Artificial Sequence
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Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His
1 5 10 15
Ala Ala Arg Pro Asp Ile Gln Leu Thr Gln Ser His Lys Phe Leu Ser
20 25 30
Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp
35 40 45
Val Tyr Asn Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
50 55 60
Lys Leu Leu Ile Tyr Ser Ala Ser Ser Arg Tyr Thr Gly Val Pro Ser
65 70 75 80
Arg Phe Thr Gly Ser Gly Ser Gly Pro Asp Phe Thr Phe Thr Ile Ser
85 90 95
Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln His Phe
100 105 110
Arg Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln
130 135 140
Leu Gln Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys
145 150 155 160
Ile Ser Cys Lys Ala Ser Gly Tyr Pro Phe Thr Asn Tyr Gly Met Asn
165 170 175
Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met Gly Trp Ile
180 185 190
Asn Thr Ser Thr Gly Glu Ser Thr Phe Ala Asp Asp Phe Lys Gly Arg
195 200 205
Phe Asp Phe Ser Leu Glu Thr Ser Ala Asn Thr Ala Tyr Leu Gln Ile
210 215 220
Asn Asn Leu Lys Ser Glu Asp Met Ala Thr Tyr Phe Cys Ala Arg Trp
225 230 235 240
Glu Val Tyr His Gly Tyr Val Pro Tyr Trp Gly Gln Gly Thr Thr Val
245 250 255
Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala
260 265 270
Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg
275 280 285
Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys
290 295 300
Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu
305 310 315 320
Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu
325 330 335
Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln
340 345 350
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
355 360 365
Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
370 375 380
Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
385 390 395 400
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
405 410 415
Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
420 425 430
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
435 440 445
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
450 455 460
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
465 470 475 480
Pro Pro Arg
<210> 16
<211> 419
<212> DNA
<213> Artificial Sequence
<400> 16
tcccccgggt tatagggagc tgaagggaag ggggtcacag taggtggcat cgttcctttc 60
tgactgcccg ccccccgcat gccgtcccgc gatattgagc tccgaacctc tcgccctgcc 120
gccgccggtg ctccgtcgcc gccgcgccgc catggaattc gaacgctgac gtcatcaacc 180
cgctccaagg aatcgcgggc ccagtgtcac taggcgggaa cacccagcgc gcgtgcgccc 240
tggcaggaag atggctgtga gggacagggg agtggcgccc tgcaatattt gcatgtcgct 300
atgtgttctg ggaaatcacc ataaacgtga aatgtctttg gatttgggaa tcttataagt 360
tctgtatgag accactcttt cccacctcag attgttgttg ttaatgcata tggaattcc 419

Claims (10)

1. A Her 2-targeted chimeric antigen receptor T cell that interferes with IL-6 expression, comprising a Her 2-targeted chimeric antigen receptor CAR-Her2 and an siRNA that interferes with IL-6 expression, wherein the CAR-Her2 comprises the amino acid sequence of a Her 2-targeted single-chain antibody, an extracellular hinge region, a transmembrane region, and an intracellular signaling region, sequentially joined from amino-terminus to carboxy-terminus, and the Her 2-targeted single-chain antibody comprises the amino acid sequence as set forth in SEQ ID NO:1, and the DNA sequence corresponding to the siRNA for interfering the expression of IL-6 comprises the amino acid sequence shown as SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4.
2. The chimeric antigen receptor T-cell targeted Her2 and interfering with IL-6 expression of claim 1, wherein the amino acid sequence of CAR-Her2 comprises the amino acid sequence as set forth in SEQ ID NO: 5.
3. The chimeric antigen receptor T cell targeted to Her2 and interfering with IL-6 expression of claim 1, wherein when the DNA sequence corresponding to the siRNA interfering with IL-6 expression comprises the sequence set forth in SEQ ID NO: 2, the DNA sequence corresponding to the sense strand of the siRNA interfering the expression of IL-6 comprises the nucleotide sequence shown in SEQ ID NO: 6, and the DNA sequence corresponding to the antisense strand of the siRNA interfering the expression of IL-6 comprises the nucleotide sequence shown in SEQ ID NO: 7.
4. The chimeric antigen receptor T cell targeted to Her2 and interfering with IL-6 expression of claim 1, wherein when the DNA sequence of the siRNA interfering with IL-6 expression comprises the sequence set forth in SEQ ID NO: 3, the DNA sequence corresponding to the sense strand of the siRNA interfering the expression of IL-6 comprises the nucleotide sequence shown as SEQ ID NO: 8, and the DNA sequence corresponding to the antisense strand of the siRNA interfering the expression of IL-6 comprises the nucleotide sequence shown as SEQ ID NO: 9, or a nucleotide sequence shown in the specification.
5. The chimeric antigen receptor T cell targeted to Her2 and interfering with IL-6 expression of claim 1, wherein when the DNA sequence of the siRNA interfering with IL-6 expression comprises the sequence set forth in SEQ ID NO: 4, the DNA sequence corresponding to the sense strand of the siRNA interfering the expression of IL-6 comprises the nucleotide sequence shown as SEQ ID NO: 10, and the DNA sequence corresponding to the antisense strand of the siRNA interfering the expression of IL-6 comprises the nucleotide sequence shown in SEQ ID NO: 11.
6. A method of making a chimeric antigen receptor T cell that targets Her2 and interferes with IL-6 expression, comprising:
(1) providing genes encoding a Her 2-targeted chimeric antigen receptor CAR-Her2, comprising a gene encoding a signal peptide, a gene encoding a Her 2-targeted single chain 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 'end to 3' end, wherein the gene encoding the Her 2-targeted single chain antibody comprises the amino acid sequence set forth in SEQ ID NO:1, and the nucleotide sequence corresponds to the amino acid sequence shown in the specification;
(2) providing a DNA sequence corresponding to siRNA interfering IL-6 expression, wherein the DNA sequence corresponding to siRNA interfering IL-6 expression comprises the nucleotide sequence shown in SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4;
(3) inserting the coding gene of the CAR-Her2 and a DNA sequence corresponding to the siRNA interfering the expression of the IL-6 into a gene delivery vector to obtain a recombinant gene delivery 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 which target Her2 and interfere IL-6 expression.
7. The method of claim 6, wherein the siRNA interfering with the expression of IL-6 has a DNA sequence corresponding to the DNA sequence linked at its 5' end to a human H1 promoter, a human U6 promoter or a murine U6 promoter.
8. A recombinant vector, which comprises an inserted Her 2-targeting chimeric antigen receptor CAR-Her2 coding gene and a DNA sequence corresponding to siRNA interfering with IL-6 expression, wherein the Her 2-targeting chimeric antigen receptor CAR-Her2 coding gene comprises a coding gene of a signal peptide, a coding gene of a Her 2-targeting single-chain antibody, a coding gene of an extracellular hinge region, a coding gene of a transmembrane region, a coding gene of an intracellular signal region, which are sequentially connected from 5 'end to 3' end, and the coding gene of the Her 2-targeting single-chain antibody comprises 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 DNA sequence corresponding to the siRNA for interfering the expression of the IL-6 comprises the nucleotide sequence shown in SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4.
9. A host cell comprising the recombinant vector of claim 8.
10. Use of a chimeric antigen receptor T-cell targeting Her2 and interfering with IL-6 expression, produced according to the process of any one of claims 1-5 or 6-7, 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.
CN201910792817.XA 2019-08-26 2019-08-26 Chimeric antigen receptor T cell targeting Her2 and interfering IL-6 expression, and preparation method and application thereof Withdrawn CN112430579A (en)

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