WO2021036246A1 - 靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞及其制备方法和应用 - Google Patents

靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞及其制备方法和应用 Download PDF

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WO2021036246A1
WO2021036246A1 PCT/CN2020/081357 CN2020081357W WO2021036246A1 WO 2021036246 A1 WO2021036246 A1 WO 2021036246A1 CN 2020081357 W CN2020081357 W CN 2020081357W WO 2021036246 A1 WO2021036246 A1 WO 2021036246A1
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egfrviii
expression
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interferes
sirna
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曾滢
汪婷婷
杨忠华
张宏玲
唐超
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深圳宾德生物技术有限公司
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Definitions

  • the present invention claims the priority of the prior application of the application number 201910792807.6 with the title of "Chimeric antigen receptor T cell targeting EGFRvIII and interfering with IL-6 expression and its preparation method and application” filed on August 26, 2019, The content of the aforementioned earlier application is incorporated into this text by way of introduction.
  • the invention relates to the field of medical biology, in particular to a chimeric antigen receptor T cell that targets EGFRvIII and interferes with the expression of IL-6, and a preparation method and application thereof.
  • Chimeric Antigen Receptor T Cell (CAR-T) technology is a new type of cell therapy. It is to infuse T cells modified by chimeric antigen receptors into the human body to activate the own immune system and kill tumor cells. It is considered to be one of the most effective treatments for malignant tumors, which can make up for the shortcomings of traditional therapies such as antibody-drug conjugates.
  • GBM Glioblastoma
  • OS overall median survival
  • EGFRvIII is an epidermal growth factor receptor type III mutant, an oncogene with a high expression rate in glioma, and is closely related to various malignant phenotypes of glioma. About 30% of patients with glioblastoma express EGFRvIII EGFRvIII is not expressed by normal human cells, and its specific expression on the surface of tumor cells makes it an ideal target for tumor targeted therapy.
  • CAR-T cell therapy As a new tumor immunotherapy method, CAR-T cell therapy has achieved significant clinical effects in tumor treatment, but there are still many adverse reactions and complications.
  • the T cells In the initial stage of CAR-T cell transfusion, the T cells rapidly expand in a short period of time, and a large number of cytokines are secreted in the process of T cells killing tumors, which leads to cytokine release syndrome (CRS).
  • CRS cytokine release syndrome
  • the clinical manifestations mainly include fever and heartbeat. Overspeed, hypotension, and significantly increased levels of cytokines such as IL-6 in cells have all affected and limited the application of CAR-T cell therapy.
  • the present invention provides a chimeric antigen receptor T cell that targets EGFRvIII and interferes with the expression of IL-6, which has a chimeric antigen receptor targeting EGFRvIII, and can specifically target and express EGFRvIII T cells can activate T cells to exert cellular immunity, achieve efficient and specific killing of EGFRvIII positive tumor cells, and have long-lasting cell viability and lethality; at the same time, the chimeric antigen receptor T cells can interfere with IL -6 expression, to avoid the expression of a large number of cytokines during the action of chimeric antigen receptor T cells, change the tumor microenvironment, and prevent the chimeric antigen receptor T cells themselves from being immunosuppressed in the tumor microenvironment, making the chimeric antigen Recipient T cells can give full play to specific tumor cell killing effects, maintain long-lasting cell viability and killing power, and will not cause damage to normal cells.
  • the present invention provides a chimeric antigen receptor T cell that targets EGFRvIII and interferes with the expression of IL-6, including the chimeric antigen receptor CAR-EGFRvIII that targets EGFRvIII and siRNA that interferes with the expression of IL-6,
  • the CAR-EGFRvIII includes the amino acid sequence of the EGFRvIII-targeting single-chain antibody, the extracellular hinge region, the transmembrane region and the intracellular signal region, which are sequentially connected from the amino terminus to the carboxyl terminus.
  • the chain antibody includes the amino acid sequence shown in SEQ ID NO: 1, and the DNA sequence corresponding to the siRNA that interferes with IL-6 expression includes the DNA sequence shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 At least one of the nucleotide sequences.
  • the single-chain antibody encoding gene targeting EGFRvIII includes the nucleotide sequence shown in SEQ ID NO: 12.
  • the single-chain antibody coding gene targeting EGFRvIII should consider degenerate bases, that is, the coding gene of the amino acid sequence shown in SEQ ID NO: 1 includes the nucleotide sequence shown in SEQ ID NO: 12 , The scope of protection should also protect the nucleotide sequence that has a base degenerate nature with SEQ ID NO: 12, and the amino acid sequence corresponding to these nucleotide sequences is still SEQ ID NO: 1.
  • the "sequential connection from the amino terminus to the carboxy terminus" is specifically: the carboxy terminus of the amino acid sequence of the single-chain antibody targeting EGFRvIII is connected to the amino terminus of the amino acid sequence of the extracellular hinge region.
  • the carboxyl terminal of the amino acid sequence of the extracellular hinge region is connected to the amino terminal of the amino acid sequence of the transmembrane region, and the carboxyl terminal of the amino acid sequence of the transmembrane region is connected to the amino terminal of the amino acid sequence of the intracellular signal region. End connected.
  • the extracellular hinge region is used to promote the binding of the EGFRvIII-targeted single-chain antibody to EGFRvIII on the tumor.
  • the extracellular hinge region includes one or a combination of CD8 ⁇ hinge region, CD28 hinge region, CD4 hinge region, CD5 hinge region, CD134 hinge region, CD137 hinge region, and ICOS hinge region. Further, the extracellular hinge region is a CD8 ⁇ hinge region.
  • the transmembrane region is used to immobilize the chimeric antigen receptor CAR-EGFRvIII targeting EGFRvIII.
  • the transmembrane region includes one or a combination of a CD3 transmembrane region, a CD4 transmembrane region, a CD8 transmembrane region, and a CD28 transmembrane region. Further, the transmembrane region is a CD8 transmembrane region.
  • the intracellular signal region is used to provide signals for T cell activation, maintain the survival time of T cells and activate T cell proliferation signal pathways.
  • the intracellular signal area includes any of the 4-1BB signal area, the CD3 ⁇ signal area, the ICOS signal area, the CD27 signal area, the OX40 signal area, the CD28 signal area, the IL1R1 signal area, the CD70 signal area, and the TNFRSF19L signal area.
  • the 4-1BB signal area includes any of the 4-1BB signal area, the CD3 ⁇ signal area, the ICOS signal area, the CD27 signal area, the OX40 signal area, the CD28 signal area, the IL1R1 signal area, the CD70 signal area, and the TNFRSF19L signal area.
  • the intracellular signal area is a 4-1BB signal area and a CD3 ⁇ signal area.
  • the CD3 ⁇ signal region is the signal transduction domain (ie, the first signal) of T cells
  • the 4-1BB signal region is the costimulatory signal of T cells. Under their joint action, T cells are fully activated after recognizing antigens. .
  • amino acid sequence of the CAR-EGFRvIII includes the amino acid sequence shown in SEQ ID NO: 5.
  • the CAR-EGFRvIII encoding gene includes a nucleotide sequence as shown in SEQ ID NO: 13.
  • the CAR-EGFRvIII coding gene should consider degenerate bases, that is, the coding gene of the amino acid sequence shown in SEQ ID NO: 5 includes the nucleotide sequence shown in SEQ ID NO: 13, which protects The scope should also protect nucleotide sequences that have a degenerate base with SEQ ID NO: 13, and the amino acid sequence corresponding to these nucleotide sequences is still SEQ ID NO: 5.
  • the chimeric antigen receptor CAR-EGFRvIII targeting EGFRvIII allows T cells to specifically target tumor cells expressing EGFRvIII, and single-chain antibodies can specifically recognize the EGFRvIII protein on tumor cells and be specific to it. Sexual binding. After CAR-EGFRvIII and EGFRvIII are combined, the intracellular signal area is activated, which promotes the expansion of T cells in the patient's body, and efficiently and specifically kills tumor cells. EGFRvIII is widely expressed in malignant tumor cells, while the expression is very weak in ordinary cells. Therefore, the chimeric antigen receptor T cells targeting EGFRvIII provided by the present invention can specifically bind to tumor cells, and have a strong effect on malignant tumor cells expressing EGFRvIII. Strong affinity activity and internalization activity, have a killing effect on tumor cells, and will not cause damage to normal cells.
  • the DNA sequence corresponding to the siRNA that interferes with IL-6 expression includes the nucleotide sequence shown in SEQ ID NO: 2
  • the DNA sequence corresponding to the sense strand of the siRNA that interferes with IL-6 expression It includes the nucleotide sequence shown in SEQ ID NO: 6
  • the DNA sequence corresponding to the antisense strand of the siRNA that interferes with IL-6 expression includes the nucleotide sequence shown in SEQ ID NO: 7.
  • the DNA sequence of the siRNA that interferes with IL-6 expression includes the nucleotide sequence shown in SEQ ID NO: 3
  • the DNA sequence corresponding to the sense strand of the siRNA that interferes with IL-6 expression includes As shown in the nucleotide sequence of SEQ ID NO: 8
  • the DNA sequence corresponding to the antisense strand of the siRNA that interferes with IL-6 expression includes the nucleotide sequence as shown in SEQ ID NO: 9.
  • the DNA sequence of the siRNA that interferes with IL-6 expression includes the nucleotide sequence shown in SEQ ID NO: 4
  • the DNA sequence corresponding to the sense strand of the siRNA that interferes with IL-6 expression includes The nucleotide sequence shown in SEQ ID NO: 10
  • the DNA sequence corresponding to the antisense strand of the siRNA that interferes with IL-6 expression includes the nucleotide sequence shown in SEQ ID NO: 11.
  • DNA sequence corresponding to the siRNA that interferes with IL-6 expression includes the nucleotide sequence shown in SEQ ID NO: 2.
  • small interfering RNA selectively inactivates the corresponding mRNA of the target gene through double-stranded RNA (dsRNA), effectively silencing or inhibiting the expression of the target gene.
  • dsRNA double-stranded RNA
  • IL-6 is a suitable target molecule for cytokine storm. Blocking IL-6 receptor can quickly solve the toxic side effects caused by CRS and has no effect on the proliferation of chimeric antigen receptor T cells in vivo.
  • the chimeric antigen receptor T cells that target EGFRvIII and interfere with IL-6 expression provided in the first aspect of the present invention have chimeric antigen receptors that target EGFRvIII, and can specifically target tumor cells expressing EGFRvIII and activate T cells.
  • Cells play a cellular immune function, achieve high-efficiency and specific killing of EGFRvIII-positive tumor cells, and have long-lasting cell viability and lethality; at the same time, the chimeric antigen receptor T cells can interfere with IL-6 expression and avoid nesting.
  • the expression of a large number of cytokines during the action of the combined antigen receptor T cells can change the tumor microenvironment and prevent the chimeric antigen receptor T cells themselves from being immunosuppressed in the tumor microenvironment, so that the chimeric antigen receptor T cells can be fully Play specific tumor cell killing effect, maintain long-lasting cell viability and killing power, and will not cause damage to normal cells.
  • the present invention provides a method for preparing chimeric antigen receptor T cells that target EGFRvIII and interfere with IL-6 expression, including:
  • the coding gene of the chimeric antigen receptor CAR-EGFRvIII targeting EGFRvIII including the coding gene of the signal peptide connected sequentially from the 5'end to the 3'end, the coding gene of the single-chain antibody targeting EGFRvIII, The coding gene of the extracellular hinge region, the coding gene of the transmembrane region, and the coding gene of the intracellular signal region, wherein the coding gene of the single-chain antibody targeting EGFRvIII includes the coding gene shown in SEQ ID NO:1 The nucleotide sequence corresponding to the amino acid sequence;
  • the DNA sequence corresponding to the siRNA that interferes with the expression of IL-6 includes as shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 At least one of the nucleotide sequences of;
  • the "sequential connection from 5'end to 3'end” is specifically: the 3'end of the coding gene sequence of the signal peptide and 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 to the 5'end of the coding gene of the extracellular hinge region, and the 3'end of the coding gene of the extracellular hinge region is connected to The 5'end of the coding gene of the transmembrane region is connected, and the 3'end of the coding gene of the transmembrane region is connected to the 5'end of the coding gene of the intracellular signal region.
  • the signal peptide is used to instruct the chimeric antigen receptor CAR-EGFRvIII to be expressed on the cell surface, and the signal peptide is cleaved by signal peptidase during protein translation and maturation.
  • amino acid sequence corresponding to the gene encoding the signal peptide is shown in SEQ ID NO: 14.
  • amino acid sequence corresponding to the CAR-EGFRvIII encoding gene containing the signal peptide is shown in SEQ ID NO: 15.
  • the 5'end of the DNA sequence corresponding to the siRNA that interferes with IL-6 expression is connected to a human H1 promoter, a human U6 promoter, or a mouse U6 promoter.
  • the gene delivery vector includes at least one of a lentiviral vector, a retroviral vector and an adenoviral vector.
  • the gene delivery vector may be, but is not limited to, pWPXLD vector, pLEX-MCS vector, pSico vector and pCgpV vector.
  • the gene delivery vector is the pWPXLD vector
  • the CAR-EGFRvIII encoding gene is inserted between the BamH I and EcoR I restriction sites in the pWPXLD vector, and is located behind the EF1 ⁇ of the pWPXLD vector, Use EF1 ⁇ as the promoter.
  • the 5'end of the CAR-EGFRvIII coding gene can be added with a start codon (such as ATG), and the BamH I restriction site (ggatcc) in the pWPXLD vector Connected, a stop codon (such as TAA) can be added to the 3'end to connect to the EcoR I restriction site (gaattc) in the pWPXLD vector, so that the CAR-EGFRvIII coding gene is located at the BamH I and EcoR I restriction sites In between, the pWPXLD-CAR-EGFRvIII recombinant plasmid was obtained.
  • a start codon such as ATG
  • ggatcc BamH I restriction site
  • gaattc EcoR I restriction site
  • the DNA sequence corresponding to the siRNA that interferes with IL-6 expression is inserted between the Sma I and Nde I restriction sites in the pWPXLD-CAR-EGFRvIII recombinant plasmid, and the DNA sequence corresponding to the siRNA that interferes with IL-6 expression is 5
  • The'end is connected with human H1 promoter, human U6 promoter or mouse U6 promoter; the 5'end of the DNA sequence corresponding to the siRNA that interferes with IL-6 expression can be added with the corresponding protective base and Sma I restriction site, Protective bases and Nde I restriction sites can be added to the 3'end, and the DNA sequence corresponding to the siRNA that interferes with IL-6 expression can be added to the pWPXLD-CAR-EGFRvIII recombinant plasmid.
  • siRNA sequence is located after CAR-EGFRvIII, with H1 For the promoter. Then it was transferred to DH5 ⁇ Escherichia coli for vector amplification, restriction enzyme digestion, sequencing identification, and pWPXLD-CAR-EGFRvIII-IL-6 siRNA recombinant lentiviral plasmid vector was obtained.
  • the gene fragment inserted into the gene delivery vector may be, but not limited to, the start codon, the CAR-EGFRvIII coding gene and the stop codon, as well as the promoter and the siRNA that interferes with the expression of IL-6.
  • the corresponding DNA sequence may be, but not limited to, the start codon, the CAR-EGFRvIII coding gene and the stop codon, as well as the promoter and the siRNA that interferes with the expression of IL-6.
  • the DNA sequence corresponding to the siRNA that interferes with IL-6 expression can also be inserted into a gene delivery vector, and then the CAR-EGFRvIII coding gene can be inserted into the gene delivery vector to obtain a recombinant gene delivery vector.
  • a promoter can be added to the CAR-EGFRvIII encoding gene and/or the DNA sequence corresponding to the siRNA that interferes with IL-6 expression, so as not to limit the sequence of their insertion into the gene delivery vector , Does not affect the expression of the final protein CAR-EGFRvIII and siRNA.
  • the recombinant gene delivery vector is packaged and transfected into a host cell to obtain a recombinant lentivirus, including:
  • the recombinant gene delivery vector, envelope plasmid and packaging plasmid are co-transfected into host cells to obtain recombinant lentivirus.
  • the envelope plasmid may be but not limited to PMD2G
  • the packaging plasmid may be but not limited to psPAX2
  • the host cell may be but not limited to HEK293T cells.
  • the envelope plasmid PMD2G encodes the vesicular stomatitis virus glycoprotein capsid, and the vesicular stomatitis virus glycoprotein capsid assists the recombinant lentivirus to adhere to the cell membrane and maintains the infectivity of the recombinant lentivirus.
  • the gene delivery vector when it includes a lentiviral vector, it may further contain envelope proteins from other viruses.
  • the protein is preferably a viral envelope protein that infects human cells.
  • This protein is not particularly limited, and examples include retroviral facultative virus hand skin membrane protein, and the like.
  • an envelope protein derived from mouse leukemia virus (MuMLV) 4070A strain can be used.
  • the envelope protein from MuMLV 10Al can also be used.
  • examples of the herpesvirus family proteins include, for example, the gB, gD, and gp85 proteins of herpes simplex virus, and the gp350 and gp220 proteins of the Epstein-Barr virus.
  • the protein of the family hepatoviridae the S protein of hepatitis B virus and the like can be mentioned.
  • the envelope protein can also be formed after the measles virus glycoprotein is fused with other single-chain antibodies.
  • the packaging of recombinant lentivirus usually adopts transient transfection or cell line packaging.
  • Human cell lines that can be used as packaging cells during transient transfection include 293 cells, 293T cells, etc. and other clones isolated from 293 cells; SW480 cells, TE671 cells, etc. Cell lines derived from monkeys, for example, COS1 cells, CV-1 cells, etc. can also be used.
  • the commonly used calcium phosphate and PEI transfection reagents, as well as some transfection reagents such as Lipofectamine2000, FuGENE and S93fectin, are also frequently used.
  • Recombinant lentivirus packaging also uses some lentiviral packaging cell lines, such as stable cell lines produced using the most common Env glycoprotein, VSVG protein or HIV-1 gag-pol protein.
  • large-scale lentiviral vector systems all adopt the method of segmenting the genome, which means that genes with different auxiliary functions are located on different plasmids.
  • genes with different auxiliary functions are located on different plasmids.
  • there are four-plasmid system encoding gag-pol gene, Rev gene, VSVG gene, and SIN transfer gene are located in four different plasmids
  • three-plasmid system the plasmid encoding Rev gene is removed, and the gag-pol plasmid is gag-pol.
  • the pol gene uses codons that are preferred in human cells) and a two-plasmid system (the auxiliary genes necessary for lentiviral vector packaging are located on the same plasmid, these auxiliary genes are a single gene sequence; the other is a transgenic plasmid) .
  • lentivirus packaging systems with more than four plasmid systems in use.
  • the CD3-positive T lymphocytes are obtained from human peripheral blood mononuclear cells.
  • the human peripheral blood mononuclear cells are derived from autologous venous blood, autologous bone marrow, umbilical cord blood, placental blood, and the like.
  • CD3 positive T lymphocytes are obtained.
  • CD3/CD28 immunomagnetic beads are added to the peripheral blood mononuclear cells in a certain proportion, after incubating for a period of time, they are placed in a magnet for screening, and immunomagnetic bead coatings are obtained. After removing the magnetic beads, CD3 positive T lymphocytes can be obtained.
  • the present invention provides a recombinant vector, including the inserted gene encoding the chimeric antigen receptor CAR-EGFRvIII targeting EGFRvIII and the DNA sequence corresponding to the siRNA that interferes with IL-6 expression, wherein the targeting
  • the coding genes of the chimeric antigen receptor CAR-EGFRvIII of EGFRvIII including the coding gene of the signal peptide connected sequentially from the 5'end to the 3'end, the coding gene of the single-chain antibody targeting EGFRvIII, and the coding of the extracellular hinge region
  • Genes, coding genes for transmembrane regions, coding genes for intracellular signal regions, the coding genes for the single-chain antibody targeting EGFRvIII include the nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO:1
  • the DNA sequence corresponding to the siRNA that interferes with IL-6 expression includes at least one of the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, and
  • the recombinant vector is obtained by inserting the coding gene of the chimeric antigen receptor CAR-EGFRvIII targeting EGFRvIII and the DNA sequence corresponding to the siRNA that interferes with IL-6 expression into the vector.
  • the vector may be, but is not limited to, the gene delivery vector described in the second aspect.
  • the vector is at least one of a viral vector and a non-viral vector.
  • the non-viral vectors include plasmid vectors and phage vectors.
  • the viral vector may be, but is not limited to, a lentiviral vector, a retroviral vector, and an adenoviral vector
  • the plasmid vector may be, but is not limited to, a eukaryotic plasmid vector, a prokaryotic plasmid vector, and a minicircle DNA.
  • the recombinant minicircle DNA inserted into the coding gene of the chimeric antigen receptor CAR-EGFRvIII targeting EGFRvIII and the DNA sequence corresponding to the siRNA that interferes with IL-6 expression can be directly transfected into CD3 positive T lymphocytes, prepared chimeric antigen receptor T cells that target EGFRvIII and interfere with IL-6 expression.
  • the present invention provides a host cell comprising the recombinant vector as described in the third aspect.
  • the host cell can be used to assemble the recombinant viral vector to make it infectious.
  • the host cells may include HEK293T cells, 293 cells, 293T cells, 293FT cells, SW480 cells, u87MG cells, HOS cells, COS7 cells, etc., but are not limited thereto.
  • the host cell is HEK293T cell.
  • the host cell is a corresponding eukaryotic host cell or a prokaryotic host cell.
  • the present invention provides a pharmaceutical composition comprising the chimeric antigen receptor that targets EGFRvIII and interferes with IL-6 expression prepared by the preparation method described in the first aspect or the preparation method described in the second aspect T cell, the recombinant vector according to the third aspect or the host cell according to the fourth aspect.
  • the pharmaceutical composition further includes a pharmaceutically acceptable carrier and/or adjuvant.
  • a pharmaceutically acceptable carrier is to transport the pharmaceutical composition to make it play its due role.
  • the carrier and/or adjuvant must be compatible with the components of the pharmaceutical composition, not affect the biological activity of the pharmaceutical composition, and it is relatively non-toxic, and does not cause toxic and side effects with the pharmaceutical composition it carries.
  • the carrier includes at least one of a solvent, a polymer, and a liposome.
  • the auxiliary material includes at least one of a diluent, an excipient and a stabilizer.
  • the pharmaceutical composition can be, but is not limited to, used to prepare drugs for the prevention and treatment of malignant tumors.
  • the malignant tumors can be, but are not limited to, glioma, breast cancer and the like.
  • the present invention provides chimeric antigen receptor T cells that target EGFRvIII and interfere with IL-6 expression prepared by the preparation method described in the first aspect or the preparation method described in the second aspect, such as the third aspect
  • the use of the recombinant vector, the host cell as described in the fourth aspect or the pharmaceutical composition as described in the fifth aspect in the preparation of a medicament for the prevention and treatment of malignant tumors.
  • the malignant tumor may be, but not limited to, glioma, breast cancer and the like.
  • Figure 1 is a plasmid map of the pWPXLD-CAR-EGFRvIII recombinant vector provided by the embodiment of the present invention.
  • Figure 2 is a plasmid map of the pWPXLD-CAR-EGFRvIII-IL-6 siRNA recombinant vector provided by the embodiment of the present invention.
  • the CAR-EGFRvIII encoding gene including the signal peptide that is, provide the nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 15, and add the restriction site and start codon at its 5'end, and Add a restriction site and a stop codon at the 3'end; insert it between the BamH I and EcoR I restriction sites of the pWPXLD vector, and use EF1 ⁇ as the promoter after the pWPXLD vector EF1 ⁇ . Then it was transformed into E. coli competent cells DH5 ⁇ , and the positive clones were identified by PCR and sequencing.
  • the pWPXLD-CAR-EGFRvIII recombinant plasmid was successfully constructed, as shown in Figure 1 for the pWPXLD-CAR-EGFRvIII recombinant vector.
  • DNA sequence corresponding to the siRNA that interferes with IL-6 expression that is, provide the nucleotide sequence shown in SEQ ID NO: 2, and add a protective base, Sma I restriction site and human H1 promoter to its 5'end
  • the sequence is shown in SEQ ID NO: 16; insert it into the Sma I and Nde I restriction sites of the pWPXLD-CAR-EGFRvIII recombinant plasmid. between. Then it was transformed into E. coli competent cells DH5 ⁇ , and the positive clones were identified by PCR and sequencing.
  • the pWPXLD-CAR-EGFRvIII-IL-6 siRNA recombinant plasmid was successfully constructed, as shown in Figure 2 is the pWPXLD-CAR-EGFRvIII-IL-6 siRNA recombinant vector .
  • the pWPXLD-CAR-EGFRvIII-IL-6 siRNA recombinant plasmid, packaging plasmid psPAX2 and envelope plasmid pMD2G were co-transfected into cultured HEK293T cells.
  • the virus-containing supernatant was harvested at 48h, filtered through a 0.45 ⁇ m filter and stored in an ultra-low temperature refrigerator at -80°C; the virus-containing supernatant was harvested for the second time at 72h, filtered with a 0.45 ⁇ m filter, and combined with the virus supernatant harvested at 48h Put them into the ultracentrifuge tube together, put them into the Beckman ultracentrifuge one by one, set the centrifugal parameters to 25000rpm, the centrifugation time to 2h, and the centrifugal temperature to be controlled at 4°C; after centrifugation, discard the supernatant and try to remove the residue on the tube wall Add the virus preservation solution to the upper liquid, gently pipetting to resuspend; after fully dissolving, centrifuge at high speed at 10000 rpm, centrifuge for 5 min, take the supernatant to determine the titer of the virus according to 100 ⁇ l, 2 ⁇ 10 8 /mL Install and store in -80°C
  • PBMC peripheral blood mononuclear cells
  • PBMC comes from autologous venous blood, autologous bone marrow, umbilical cord blood and placental blood. It is best derived from fresh peripheral blood or bone marrow collected from cancer patients one month after surgery and one month after radiotherapy and chemotherapy.
  • the patient's blood is drawn and sent to the blood separation chamber; the peripheral blood mononuclear cells are collected, Ficoll centrifugal separation, and the middle layer cells are collected; after washing with PBS, PBMCs are obtained.
  • PBMC blood pressure
  • serum-free basal medium to prepare a cell suspension
  • CD3/CD28 immunomagnetic beads according to the ratio of magnetic beads to cells of 3:1, and incubate for 1-2h at room temperature; incubate with a magnet pair.
  • the cells of the magnetic beads are screened; after washing with PBS and removing the immunomagnetic beads, CD3 positive T lymphocytes are obtained.
  • the CD3 positive T lymphocytes obtained by the immunomagnetic bead separation method are taken, and the recombinant lentivirus with the virus titer corresponding to the number of CD3 positive cells is added for culture.
  • the chimeric antigen receptor T cells (CAR-T-EGFRvIII-siRNA) that target EGFRvIII and interfere with IL-6 expression prepared by the method of the present invention and the chimeric antigen receptor T cells that target EGFRvIII (abbreviated as CAR-T-EGFRvIII) and unprepared T lymphocytes (negative control group) in vitro tumor killing effects are compared.
  • the ratio of effector cells to target cells in vitro is 1:10, 1:3,
  • the ratio of 1:1, 3:1 and 10:1 was co-cultured at 37°C and 5% CO 2.
  • the cells were collected and subjected to flow cytometry to detect cell killing.
  • CAR-T-EGFRvIII-siRNA cells prepared by the method of the present invention is higher than that of CAR-T-EGFRvIII cells, and is much higher than that of the negative control group. Therefore, after IL-6 expression is inhibited, the method of the present invention
  • the prepared CAR-T cells have strong tumor killing ability.
  • CAR-T-EGFRvIII-siRNA chimeric antigen receptor T cells that target EGFRvIII and interfere with IL-6 expression prepared by the method of the present invention and the chimeric antigen receptor T cells that target EGFRvIII (abbreviated as CAR-T) -EGFRvIII), unprepared T lymphocytes (negative control group) and normal saline (blank control group).

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Abstract

公开了靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞，包括靶向EGFRvⅢ的嵌合抗原受体CAR-EGFRvⅢ以及干扰IL-6表达的siRNA，CAR-EGFRvⅢ包括从氨基端到羧基端顺次连接的靶向EGFRvⅢ的单链抗体、胞外铰链区、跨膜区和胞内信号区的氨基酸序列，靶向EGFRvⅢ的单链抗体包括如SEQ ID NO：1所示的氨基酸序列，干扰IL-6表达的siRNA对应的DNA序列包括如SEQ ID NO：2、SEQ ID NO：3和SEQ ID NO：4所示的核苷酸序列中的至少一种。

Description

靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞及其制备方法和应用
本发明要求2019年8月26日递交的发明名称为“靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞及其制备方法和应用”的申请号201910792807.6的在先申请优先权，上述在先申请的内容以引入的方式并入本文本中。
技术领域
本发明涉及医学生物领域，特别涉及靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞及其制备方法和应用。
背景技术
嵌合抗原受体T细胞(CAR-T)技术是一种新型细胞疗法，它是将经过嵌合抗原受体改造的T细胞回输至人体，激活自身免疫系统，对肿瘤细胞进行杀伤，被认为是目前最有效的恶性肿瘤的治疗方式之一，可弥补诸如抗体药物偶联物等传统疗法的弊端。
胶质母细胞瘤(GBM)是胶质瘤中恶性程度最高的病理类型，也是原发性脑肿瘤中最常见、最具侵袭性的一种肿瘤，侵袭性强，极易复发。虽然GBM的治疗已发展为手术、放疗和化疗相结合的综合治疗模式，其预后仍很差，整体中位生存期(OS)仅为15个月，5年生存率不到10％。侵袭性恶性肿瘤细胞产生耐药性是GBM复发的根源，从而导致治疗失败。
EGFRvⅢ是表皮生长因子受体Ⅲ型突变体，是胶质瘤中表达率很高的癌基因，与胶质瘤的各种恶性表型密切相关，大约30％的胶质母细胞瘤患者表达EGFRvIII，而人体正常细胞不会表达EGFRvIII，其在肿瘤细胞表面的特异性表达使其成为肿瘤靶向治疗的理想靶点。
CAR-T细胞治疗作为一种新的肿瘤免疫治疗方法，在肿瘤治疗上取得显著的临床效果，但仍然存在多种不良反应及并发症。病人在CAR-T细胞输入初期，T细胞短时间内迅速扩增，在T细胞杀伤肿瘤的过程中分泌大量的细胞因子，从而导致细胞因子释放综合症(CRS)，临床表现主要有发热、心动过速、低血压及细胞中IL-6等细胞因子水平显著升高，这些不良反应均影响和限制了CAR-T细胞疗法的应用。
因此，亟需一种可以靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞及其制备方法。
发明内容
有鉴于此，本发明提供了一种靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞，其具有靶向EGFRvⅢ的嵌合抗原受体，进而可以专一性地靶向表达EGFRvⅢ的肿瘤细胞，激活T细胞发挥细胞免疫作用，对EGFRvⅢ阳性肿瘤细胞实现高效且特异性的杀伤，具有持久的细胞活力和杀伤力；与此同时，所述嵌合抗原受体T细胞可以干扰IL-6表达，避免 嵌合抗原受体T细胞作用过程中大量细胞因子的表达，改变肿瘤微环境，同时防止嵌合抗原受体T细胞自身在肿瘤微环境中处于免疫抑制状态，使得嵌合抗原受体T细胞可以充分发挥特异性肿瘤细胞杀伤作用，保持持久的细胞活力和杀伤力，并且对正常细胞不会造成损伤。
第一方面，本发明提供了一种靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞，包括靶向EGFRvⅢ的嵌合抗原受体CAR-EGFRvⅢ以及干扰IL-6表达的siRNA，其中，所述CAR-EGFRvⅢ包括从氨基端到羧基端顺次连接的靶向EGFRvⅢ的单链抗体、胞外铰链区、跨膜区和胞内信号区的氨基酸序列，所述靶向EGFRvⅢ的单链抗体包括如SEQ ID NO：1所示的氨基酸序列，所述干扰IL-6表达的siRNA对应的DNA序列包括如SEQ ID NO：2、SEQ ID NO：3和SEQ ID NO：4所示的核苷酸序列中的至少一种。
可选的，所述靶向EGFRvⅢ的单链抗体编码基因包括如SEQ ID NO：12所示的核苷酸序列。进一步的，所述靶向EGFRvⅢ的单链抗体编码基因应该考虑简并碱基，即如SEQ ID NO:1所示的氨基酸序列的编码基因包括如SEQ ID NO:12所示的核苷酸序列，保护范围还应该保护与SEQ ID NO:12具有碱基简并性质的核苷酸序列，这些核苷酸序列对应的氨基酸序列仍然为SEQ ID NO:1。
在本发明中，所述“从氨基端到羧基端顺次连接”具体为：所述靶向EGFRvⅢ的单链抗体的氨基酸序列的羧基端与所述胞外铰链区的氨基酸序列的氨基端相连，所述胞外铰链区的氨基酸序列的羧基端与所述跨膜区的氨基酸序列的氨基端相连，所述跨膜区的氨基酸序列的羧基端与所述胞内信号区的氨基酸序列的氨基端相连。
在本发明中所述胞外铰链区用于促进所述靶向EGFRvⅢ的单链抗体与肿瘤上的EGFRvⅢ结合。
可选的，所述胞外铰链区包括CD8α铰链区、CD28铰链区、CD4铰链区、CD5铰链区、CD134铰链区、CD137铰链区、ICOS铰链区中的一种或多种的组合。进一步的，所述胞外铰链区为CD8α铰链区。
在本发明中所述跨膜区用于固定所述靶向EGFRvⅢ的嵌合抗原受体CAR-EGFRvⅢ。
可选的，所述跨膜区包括CD3跨膜区、CD4跨膜区、CD8跨膜区、CD28跨膜区中的一种或多种的组合。进一步的，所述跨膜区为CD8跨膜区。
在本发明中所述胞内信号区用于提供T细胞活化的信号，维持T细胞的生存时间和激活T细胞增殖信号通路。
可选的，所述胞内信号区包括4-1BB信号区、CD3ζ信号区、ICOS信号区、CD27信号区、OX40信号区、CD28信号区、IL1R1信号区、CD70信号区、TNFRSF19L信号区中的一种或多种的组合。
可选的，所述胞内信号区为4-1BB信号区和CD3ζ信号区。其中，CD3ζ信号区为T细胞的信号传导结构域(即，第一信号)，4-1BB信号区为T细胞的共刺激信号，在它们的共同作用下，T细胞在识别抗原后被完全活化。
进一步的，所述CAR-EGFRvⅢ的氨基酸序列包括如SEQ ID NO：5所示的氨基酸序列。
进一步的，所述CAR-EGFRvⅢ的编码基因包括如SEQ ID NO：13所示的核苷酸序列。
可选的，所述CAR-EGFRvⅢ的编码基因应该考虑简并碱基，即如SEQ ID NO:5所示的氨基酸序列的编码基因包括如SEQ ID NO:13所示的核苷酸序列，保护范围还应该保护与SEQ ID NO:13具有碱基简并性质的核苷酸序列，这些核苷酸序列对应的氨基酸序列仍然为SEQ ID NO:5。
在本发明中，靶向EGFRvⅢ的嵌合抗原受体CAR-EGFRvⅢ使得T细胞专一性地靶向表达EGFRvⅢ的肿瘤细胞，单链抗体能特异性识别肿瘤细胞上的EGFRvⅢ蛋白，并与其发生特异性结合，在CAR-EGFRvⅢ与EGFRvⅢ结合后，胞内信号区被激活，促进T细胞在患者体内的扩增，并高效且特异性的杀伤肿瘤细胞。EGFRvⅢ在恶性肿瘤细胞中广泛表达，而在普通细胞中表达很微弱，因此本发明提供的靶向EGFRvⅢ的嵌合抗原受体T细胞可以特异性的结合肿瘤细胞，对表达EGFRvⅢ的恶性肿瘤细胞具有较强的亲和活性及内化活性，对肿瘤细胞产生杀伤效果，对正常细胞不会造成损伤。
可选的，当所述干扰IL-6表达的siRNA对应的DNA序列包括如SEQ ID NO：2所示的核苷酸序列时，所述干扰IL-6表达的siRNA的正义链对应的DNA序列包括如SEQ ID NO：6所示的核苷酸序列，所述干扰IL-6表达的siRNA的反义链对应的DNA序列包括如SEQ ID NO：7所示的核苷酸序列。
可选的，当所述干扰IL-6表达的siRNA的DNA序列包括如SEQ ID NO：3所示的核苷酸序列时，所述干扰IL-6表达的siRNA的正义链对应的DNA序列包括如SEQ ID NO：8所示的核苷酸序列，所述干扰IL-6表达的siRNA的反义链对应的DNA序列包括如SEQ ID NO：9所示的核苷酸序列。
可选的，当所述干扰IL-6表达的siRNA的DNA序列包括如SEQ ID NO：4所示的核苷酸序列时，所述干扰IL-6表达的siRNA的正义链对应的DNA序列包括如SEQ ID NO：10所示的核苷酸序列，所述干扰IL-6表达的siRNA的反义链对应的DNA序列包括如SEQ ID NO：11所示的核苷酸序列。
进一步的，所述干扰IL-6表达的siRNA对应的DNA序列包括如SEQ ID NO：2所示的核苷酸序列。
在本发明中，小干扰RNA(siRNA)通过双链RNA(dsRNA)使得靶基因相应的mRNA选择性失活，有效沉默或抑制靶基因表达。IL-6是一种细胞因子风暴的合适靶分子，阻断IL-6受体后能迅速解决CRS带来的毒副作用并且对嵌合抗原受体T细胞体内增殖没有影响。
本发明第一方面提供的靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞具有靶向EGFRvⅢ的嵌合抗原受体，可以专一性地靶向表达EGFRvⅢ的肿瘤细胞，激活T细胞发挥细胞免疫作用，对EGFRvⅢ阳性肿瘤细胞实现高效且特异性的杀伤，具有持久的细胞活力和杀伤力；与此同时，所述嵌合抗原受体T细胞可以干扰IL-6表达，避免嵌合抗原受体T细胞作用过程中大量细胞因子的表达，改变肿瘤微环境，同时防止嵌合抗原受体T细胞自身在肿瘤微环境中处于免疫抑制状态，使得嵌合抗原受体T细胞可以充分发挥特异性肿瘤细胞杀伤作用，保持持久的细胞活力和杀伤力，并且对正常细胞不会造成损伤。
第二方面，本发明提供了一种靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞的制备方法，包括：
(1)提供靶向EGFRvⅢ的嵌合抗原受体CAR-EGFRvⅢ的编码基因，包括从5’端到3’端顺次连接的信号肽的编码基因、靶向EGFRvⅢ的单链抗体的编码基因、胞外铰链区的编码基因、跨膜区的编码基因、胞内信号区的编码基因的编码基因，其中，所述靶向EGFRvⅢ的单链抗体的编码基因包括如SEQ ID NO：1所示的氨基酸序列对应的核苷酸序列；
(2)提供干扰IL-6表达的siRNA对应的DNA序列，所述干扰IL-6表达的siRNA对应的DNA序列包括如SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4所示的核苷酸序列中的至少一种；
(3)将所述CAR-EGFRvⅢ的编码基因和所述干扰IL-6表达的siRNA对应的DNA序列插入到基因传递载体中，得到重组基因传递载体；
(4)将所述重组基因传递载体进行包装并转染宿主细胞，得到重组慢病毒；
(5)将所述重组慢病毒转染CD3阳性T淋巴细胞，获得靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞。
在本发明中，所述“从5’端到3’端顺次连接”具体为：所述信号肽的编码基因序列的3’端与所述靶向EGFRvⅢ的单链抗体的编码基因的5’端相连，所述靶向EGFRvⅢ的单链抗体的编码基因的3’端与所述胞外铰链区的编码基因的5’端相连，所述胞外铰链区的编码基因的3’端与所述跨膜区的编码基因的5’端相连，所述跨膜区的编码基因的3’端与所述胞内信号区的编码基因的5’端相连。
在本发明中所述信号肽用于指导所述嵌合抗原受体CAR-EGFRvⅢ表达到细胞表面，所述信号肽在蛋白翻译成熟过程中被信号肽酶切割。
可选的，所述信号肽的编码基因对应的氨基酸序列如SEQ ID NO：14所示。
其中，所述胞外铰链区、跨膜区、胞内信号区的具体选择如本发明第一方面所述，这里不再赘述。
可选的，含有所述信号肽的所述CAR-EGFRvⅢ的编码基因对应的氨基酸序列如SEQ ID NO：15所示。
可选的，所述干扰IL-6表达的siRNA对应的DNA序列的5’端连接有人H1启动子、人U6启动子或鼠U6启动子。
可选的，所述基因传递载体包括慢病毒载体、逆转录病毒载体和腺病毒载体中的至少一种。
进一步的，所述基因传递载体可以但不限于为pWPXLD载体、pLEX-MCS载体、pSico载体和pCgpV载体。
作为示例性的例子，当所述基因传递载体为pWPXLD载体时，所述CAR-EGFRvⅢ的编码基因插入到pWPXLD载体中BamH Ⅰ和EcoR Ⅰ酶切位点之间，且位于pWPXLD载体的EF1α之后，以EF1α为启动子。所述CAR-EGFRvⅢ的编码基因插入到pWPXLD载体时，所述CAR-EGFRvⅢ的编码基因的5’端可加入起始密码子(如ATG)，与pWPXLD载体中 BamH Ⅰ酶切位点(ggatcc)相连，3’端可加入终止密码子(如TAA)与pWPXLD载体中EcoR Ⅰ酶切位点(gaattc)相连，这样就使所述CAR-EGFRvⅢ的编码基因位于BamH Ⅰ和EcoR Ⅰ酶切位点之间，得到pWPXLD-CAR-EGFRvⅢ重组质粒。所述干扰IL-6表达的siRNA对应的DNA序列插入到pWPXLD-CAR-EGFRvⅢ重组质粒中Sma Ⅰ和Nde Ⅰ酶切位点之间，所述干扰IL-6表达的siRNA对应的DNA序列的5’端连接有人H1启动子、人U6启动子或鼠U6启动子；所述干扰IL-6表达的siRNA对应的DNA序列的5’端可加入相应的保护碱基、Sma Ⅰ酶切位点，3’端可加入保护碱基、Nde Ⅰ酶切位点，将所述干扰IL-6表达的siRNA对应的DNA序列至pWPXLD-CAR-EGFRvⅢ重组质粒中，siRNA序列位于CAR-EGFRvⅢ之后，以H1为启动子。随后转入DH5α大肠杆菌中，进行载体扩增以及酶切，测序鉴定，获得pWPXLD-CAR-EGFRvⅢ-IL-6 siRNA重组慢病毒质粒载体。
在本发明中，插入所述基因传递载体的基因片段可以但不限于为起始密码子、所述CAR-EGFRvⅢ的编码基因和终止密码子，以及启动子和所述干扰IL-6表达的siRNA对应的DNA序列。
在本发明中，也可以先将所述干扰IL-6表达的siRNA对应的DNA序列插入基因传递载体中，再将所述CAR-EGFRvⅢ的编码基因插入基因传递载体中，得到重组基因传递载体。
在本发明中，可以通过在所述CAR-EGFRvⅢ的编码基因和/或所述干扰IL-6表达的siRNA对应的DNA序列额外添加启动子，从而不限制两者插入基因传递载体中的先后顺序，也不影响最终蛋白CAR-EGFRvⅢ和siRNA的表达。
可选地，将所述重组基因传递载体进行包装并转染宿主细胞，得到重组慢病毒，包括：
将所述重组基因传递载体与包膜质粒、包装质粒共转染宿主细胞，得到重组慢病毒。
更进一步的，所述包膜质粒可以但不限于为PMD2G，所述包装质粒可以但不限于为psPAX2，所述宿主细胞可以但不限于为HEK293T细胞。
其中，所述包膜质粒PMD2G编码水疱性口炎病毒糖蛋白衣壳，所述水疱性口炎病毒糖蛋白衣壳协助重组慢病毒向细胞膜粘附，并保持重组慢病毒的感染性。
本发明中，当所述基因传递载体包括慢病毒载体时，可以进一步含有来自其它病毒的被膜蛋白。例如，作为这种蛋白质，最好是来自感染人类细胞的病毒被膜蛋白。对这种蛋白质没有特别的限定，可例举出逆转录病毒的兼嗜性病毒手皮膜蛋白等，例如可以使用来自小鼠白血病病毒(MuMLV)4070A株的被膜蛋白。另外，也可以使用来自MuMLV 10Al的被膜蛋白。另外，作为疱疹病毒科的蛋白，可以举出例如，单纯性疱疹病毒的gB、gD、gp85蛋白，EB病毒的gp350、gp220蛋白等。作为嗜肝病毒科的蛋白，可以例举出B型肝炎病毒的S蛋白等。所述被膜蛋白还可为麻疹病毒糖蛋白与其他单链抗体融合后形成。
重组慢病毒的包装通常采用瞬时转染或采用细胞系包装。瞬时转染时可以用作包装细胞使用的人类细胞株，例如包括293细胞、293T细胞等及其他的从293细胞分离的克隆；SW480细胞、TE671细胞等。也可以采用来源于猴子的细胞株，例如，COS1细胞、CV-1细胞等。而且，通常采用的磷酸钙和PEI转染试剂，还有一些转染试剂如Lipofectamine2000、FuGENE和S93fectin也被经常使用。
重组慢病毒的包装也采用一些慢病毒包装细胞系，如使用最普遍的Env糖蛋白、VSVG蛋白或HIV-1gag-pol蛋白所产生的稳定细胞系。
为了安全起见，大规模使用的慢病毒载体系统都是采用分割基因组的方法，即将起不同辅助功能的基因定位于不同的质粒。目前有四质粒系统(编码gag-pol基因、Rev基因、VSVG基因、SIN转移基因分别位于四个不同的质粒)、三质粒系统(去掉了编码Rev基因的质粒，在gag-pol质粒中gag-pol基因采用了在人细胞中偏爱性的密码子)和二质粒系统(慢病毒载体包装所必需的辅助基因位于同一个质粒上，这些辅助基因是单一的基因序列；另一个则是转基因质粒)。也有超过四质粒系统的慢病毒包装系统在使用。
可选的，所述CD3阳性T淋巴细胞是从人源外周血单个核细胞中分离获得。
可选的，所述人源外周血单个核细胞来源于自体静脉血、自体骨髓、脐带血和胎盘血等。
进一步的，来源于癌症患者手术一个月后、放化疗一个月后采集的新鲜外周血或骨髓。
具体的，所述CD3阳性T淋巴细胞的获得过程如下：向外周血单个核细胞中按一定比例加入CD3/CD28免疫磁珠，孵育一段时间后，放入磁铁进行筛选，得到免疫磁珠包被的CD3阳性T淋巴细胞，去除磁珠后，获得CD3阳性T淋巴细胞。
第三方面，本发明提供了一种重组载体，包括插入的靶向EGFRvⅢ的嵌合抗原受体CAR-EGFRvⅢ的编码基因和干扰IL-6表达的siRNA对应的DNA序列，其中，所述靶向EGFRvⅢ的嵌合抗原受体CAR-EGFRvⅢ的编码基因，包括从5’端到3’端顺次连接的信号肽的编码基因、靶向EGFRvⅢ的单链抗体的编码基因、胞外铰链区的编码基因、跨膜区的编码基因、胞内信号区的编码基因的编码基因，所述靶向EGFRvⅢ的单链抗体的编码基因包括如SEQ ID NO：1所示的氨基酸序列对应的核苷酸序列；所述干扰IL-6表达的siRNA对应的DNA序列包括如SEQ ID NO：2、SEQ ID NO：3和SEQ ID NO：4所示的核苷酸序列中的至少一种。
在本发明中，所述重组载体通过将靶向EGFRvⅢ的嵌合抗原受体CAR-EGFRvⅢ的编码基因和干扰IL-6表达的siRNA对应的DNA序列插入载体中得到。
在本发明中，所述载体可以但不限于为第二方面所述的基因传递载体。可选的，所述载体为病毒载体和非病毒载体中的至少一种。进一步的，所述非病毒载体包括质粒载体和噬菌体载体。具体的，所述病毒载体可以但不限于为慢病毒载体、逆转录病毒载体和腺病毒载体，所述质粒载体可以但不限于为真核质粒载体、原核质粒载体和微环DNA。当所述载体为微环DNA时，可以将插入靶向EGFRvⅢ的嵌合抗原受体CAR-EGFRvⅢ的编码基因和干扰IL-6表达的siRNA对应的DNA序列的重组微环DNA直接转染CD3阳性T淋巴细胞，制得靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞。
第四方面，本发明提供了一种宿主细胞，包括如第三方面所述的重组载体。
可选的，当所述重组载体为重组病毒载体时，所述宿主细胞可以用于组装所述重组病毒载体，使其具有感染性。进一步的，所述宿主细胞可以包括HEK293T细胞、293细胞、293T细胞、293FT细胞、SW480细胞、u87MG细胞、HOS细胞或COS7细胞等，但不限 于此。更进一步的，所述宿主细胞为HEK293T细胞。
可选的，当所述重组质粒为重组真核质粒载体、重组原核质粒载体和重组微环DNA时，所述宿主细胞为相应的真核宿主细胞或原核宿主细胞。
第五方面，本发明提供了一种药物组合物，包括如第一方面所述的或如第二方面所述的制备方法制得的靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞、如第三方面所述的重组载体或如第四方面所述的宿主细胞。
可选的，所述药物组合物还包括药学上可接受的载体和/或辅料。在本申请中，“药学上可接受的载体”的作用是运输所述药物组合物，使其发挥应有的作用。载体和/或辅料必须和药物组合物成分相兼容，不影响药物组合物的生物学活性，而且本身是相对无毒的，且不与其携带的药物组合物发生引起毒副作用的反应。进一步的，所述载体包括溶剂、聚合物和脂质体中的至少一种。进一步的，所述辅料包括稀释剂、赋形剂和稳定剂中的至少一种。
在本发明中，所述药物组合物可以但不限于用于制备预防和治疗恶性肿瘤的药物，所述恶性肿瘤可以但不限于为脑胶质瘤、乳腺癌等。
第六方面，本发明提供了如第一方面所述的或如第二方面所述的制备方法制得的靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞、如第三方面所述的重组载体、如第四方面所述的宿主细胞或如第五方面所述的药物组合物在制备预防和治疗恶性肿瘤的药物中的应用。
在本发明中，所述恶性肿瘤可以但不限于为脑胶质瘤、乳腺癌等。
本发明的优点将会在下面的说明书中部分阐明，一部分根据说明书是显而易见的，或者可以通过本发明实施例的实施而获知。
附图说明
图1为本发明实施例提供的pWPXLD-CAR-EGFRvⅢ重组载体的质粒图谱。
图2为本发明实施例提供的pWPXLD-CAR-EGFRvⅢ-IL-6 siRNA重组载体的质粒图谱。
具体实施方式
以下所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明的保护范围。
制备实施例
(1)靶向EGFRvⅢ并干扰IL-6表达的重组载体构建
提供包括信号肽的CAR-EGFRvⅢ的编码基因，即提供如SEQ ID NO：15所示的氨基酸序列对应的核苷酸序列，并在其5’端加入酶切位点和起始密码子，以及在3’端加入酶切位点和终止密码子；将其插入到pWPXLD载体的BamH Ⅰ和EcoR Ⅰ酶切位点之间，并在pWPXLD载体EF1α之后，以EF1α为启动子。然后转入大肠杆菌感受态细胞DH5α，进行阳性克隆PCR鉴定和测序鉴定。经过PCR产物凝胶电泳检测和测序鉴定符合目的片段大小和序列，成功 构建pWPXLD-CAR-EGFRvⅢ重组质粒，如图1所示为pWPXLD-CAR-EGFRvⅢ重组载体。
提供干扰IL-6表达的siRNA对应的DNA序列，即提供如SEQ ID NO：2所示的核苷酸序列，并在其5’端加入保护碱基、Sma Ⅰ酶切位点和人H1启动子，以及在3’端加入保护碱基和Nde Ⅰ酶切位点，序列如SEQ ID NO：16所示；将其插入到pWPXLD-CAR-EGFRvⅢ重组质粒的Sma Ⅰ和Nde Ⅰ酶切位点之间。然后转入大肠杆菌感受态细胞DH5α，进行阳性克隆PCR鉴定和测序鉴定。经过PCR产物凝胶电泳检测和测序鉴定符合目的片段大小和序列，成功构建pWPXLD-CAR-EGFRvⅢ-IL-6 siRNA重组质粒，如图2所示为pWPXLD-CAR-EGFRvⅢ-IL-6 siRNA重组载体。
(2)重组慢病毒构建
将pWPXLD-CAR-EGFRvⅢ-IL-6 siRNA重组质粒、包装质粒psPAX2、包膜质粒pMD2G三者共转染入培养好的HEK293T细胞。第48h收获含病毒的上清，经0.45μm滤膜过滤，-80℃超低温冰箱中保存；第72h二次收获含病毒的上清，0.45μm滤膜过滤，与第48h收获的病毒上清合并一起加入超速离心管中，逐一放入至Beckman超速离心机内，设置离心参数为25000rpm，离心时间为2h，离心温度控制在4℃；离心结束后，弃去上清，尽量去除残留在管壁上的液体，加入病毒保存液，轻轻反复吹打重悬；经充分溶解后，高速离心10000rpm，离心5min后，取上清荧光法测定滴度，病毒按照100μl，2×10 8个/mL分装，保存于-80℃超低温冰箱，得到重组慢病毒。
(3)靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞的制备
a)PBMC(外周血单个核细胞)的分离
PBMC来源于自体静脉血、自体骨髓、脐带血和胎盘血等。最好是来源于癌症患者手术一个月后、放化疗一个月后采集的新鲜外周血或骨髓。
抽取病人血液，送样至血液分离室；采集外周血单个核细胞，Ficoll离心分离后取中间层细胞；经PBS洗涤后，得到PBMC。
b)免疫磁珠法分离抗原特异性T淋巴细胞
取上述PBMC，加入不含血清的基础培养基，配成细胞悬液；按磁珠与细胞的比例为3:1，加入CD3/CD28免疫磁珠，室温孵1-2h；采用磁铁对孵育好磁珠的细胞进行筛选；PBS洗涤，去除免疫磁珠后，得到CD3阳性T淋巴细胞。
c)病毒转染法制备抗原特异性T淋巴细胞
取上述经过免疫磁珠分离法得到的CD3阳性T淋巴细胞，加入与CD3阳性细胞数相应的病毒滴度的所述重组慢病毒进行培养。
培养的第3天，进行细胞计数和换液，调整细胞浓度为1×10 6个/mL，接种，培养；培养的第5天，观察细胞状态，如果细胞密度增大，则稀释细胞浓度为1×10 6个/mL，检测细胞活性，继续培养。扩增培养到第9-11天，收集细胞，得到靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞，并保存在回输专用的细胞冻存液中。
效果实施例1
评估靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞的体外肿瘤细胞杀伤情况
将经过本发明方法制得的靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞(简写为CAR-T-EGFRvⅢ-siRNA)与靶向EGFRvⅢ嵌合抗原受体T细胞(简写为CAR-T-EGFRvⅢ)、未经制备的T淋巴细胞(阴性对照组)的体外肿瘤杀伤效果进行比较，具体的：在体外将效应细胞与靶细胞数量比为为1:10、1:3、1:1、3:1和10:1比例，在37℃，5％CO 2下进行共培养，在培养后的第15-18小时，收集细胞，进行流式染色，检测细胞杀伤情况，发现本发明所述的方法制备的CAR-T-EGFRvⅢ-siRNA细胞肿瘤杀伤效果高于CAR-T-EGFRvⅢ细胞，且远远高于阴性对照组，因此在IL-6表达抑制后，经本发明方法制备的CAR-T细胞具有强的肿瘤杀伤能力。
效果实施例2
评估靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞的小鼠体内肿瘤细胞杀伤情况
将经过本发明方法制备的靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞(CAR-T-EGFRvⅢ-siRNA)与靶向EGFRvⅢ嵌合抗原受体T细胞(简写为CAR-T-EGFRvⅢ)、未经制备的T淋巴细胞(阴性对照组)以及生理盐水(空白对照组)，在小鼠肿瘤模型中，给每只小鼠尾静脉注射1×10 6个细胞(n＝9)，得到小鼠的生存曲线，发现经过本方法制备的CAR-T-EGFRvⅢ-siRNA细胞能够更好的保护小鼠免于因肿瘤导致的死亡，平均生存期高于CAR-T-EGFRvⅢ细胞，效果优于阴性对照组和空白组。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (13)

  1. 一种靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞，其特征在于，包括靶向EGFRvⅢ的嵌合抗原受体CAR-EGFRvⅢ以及干扰IL-6表达的siRNA，其中，所述CAR-EGFRvⅢ包括从氨基端到羧基端顺次连接的靶向EGFRvⅢ的单链抗体、胞外铰链区、跨膜区和胞内信号区的氨基酸序列，所述靶向EGFRvⅢ的单链抗体包括如SEQ ID NO：1所示的氨基酸序列，所述干扰IL-6表达的siRNA对应的DNA序列包括如SEQ ID NO：2、SEQ ID NO：3和SEQ ID NO：4所示的核苷酸序列中的至少一种。
  2. 如权利要求1所述的靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞，其特征在于，所述CAR-EGFRvⅢ的氨基酸序列包括如SEQ ID NO：5所示的氨基酸序列。
  3. 如权利要求1所述的靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞，其特征在于，当所述干扰IL-6表达的siRNA对应的DNA序列包括如SEQ ID NO：2所示的核苷酸序列时，所述干扰IL-6表达的siRNA的正义链对应的DNA序列包括如SEQ ID NO：6所示的核苷酸序列，所述干扰IL-6表达的siRNA的反义链对应的DNA序列包括如SEQ ID NO：7所示的核苷酸序列。
  4. 如权利要求1所述的靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞，其特征在于，当所述干扰IL-6表达的siRNA的DNA序列包括如SEQ ID NO：3所示的核苷酸序列时，所述干扰IL-6表达的siRNA的正义链对应的DNA序列包括如SEQ ID NO：8所示的核苷酸序列，所述干扰IL-6表达的siRNA的反义链对应的DNA序列包括如SEQ ID NO：9所示的核苷酸序列。
  5. 如权利要求1所述的靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞，其特征在于，当所述干扰IL-6表达的siRNA的DNA序列包括如SEQ ID NO：4所示的核苷酸序列时，所述干扰IL-6表达的siRNA的正义链对应的DNA序列包括如SEQ ID NO：10所示的核苷酸序列，所述干扰IL-6表达的siRNA的反义链对应的DNA序列包括如SEQ ID NO：11所示的核苷酸序列。
  6. 一种靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞的制备方法，其特征在于，包括：
    (1)提供靶向EGFRvⅢ的嵌合抗原受体CAR-EGFRvⅢ的编码基因，包括从5’端到3’端顺次连接的信号肽的编码基因、靶向EGFRvⅢ的单链抗体的编码基因、胞外铰链区的编码基因、跨膜区的编码基因、胞内信号区的编码基因的编码基因，其中，所述靶向EGFRvⅢ的单链抗体的编码基因包括如SEQ ID NO：1所示的氨基酸序列对应的核苷酸序列；
    (2)提供干扰IL-6表达的siRNA对应的DNA序列，所述干扰IL-6表达的siRNA对应的DNA序列包括如SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4所示的核苷酸序列中的至少一种；
    (3)将所述CAR-EGFRvⅢ的编码基因和所述干扰IL-6表达的siRNA对应的DNA序 列插入到基因传递载体中，得到重组基因传递载体；
    (4)将所述重组基因传递载体进行包装并转染宿主细胞，得到重组慢病毒；
    (5)将所述重组慢病毒转染CD3阳性T淋巴细胞，获得靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞。
  7. 如权利要求6所述的制备方法，其特征在于，所述干扰IL-6表达的siRNA对应的DNA序列的5’端连接有人H1启动子、人U6启动子或鼠U6启动子。
  8. 一种重组载体，其特征在于，包括插入的靶向EGFRvⅢ的嵌合抗原受体CAR-EGFRvⅢ的编码基因和干扰IL-6表达的siRNA对应的DNA序列，其中，所述靶向EGFRvⅢ的嵌合抗原受体CAR-EGFRvⅢ的编码基因，包括从5’端到3’端顺次连接的信号肽的编码基因、靶向EGFRvⅢ的单链抗体的编码基因、胞外铰链区的编码基因、跨膜区的编码基因、胞内信号区的编码基因的编码基因，所述靶向EGFRvⅢ的单链抗体的编码基因包括如SEQ ID NO：1所示的氨基酸序列对应的核苷酸序列；所述干扰IL-6表达的siRNA对应的DNA序列包括如SEQ ID NO：2、SEQ ID NO：3和SEQ ID NO：4所示的核苷酸序列中的至少一种。
  9. 一种宿主细胞，其特征在于，包括如权利要求8所述的重组载体。
  10. 一种药物组合物，其特征在于，包括如权利要求1-5任一项所述的或权利要求6-7任一项所述的制备方法制得的靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞、如权利要求8所述的重组载体或如权利要求9所述的宿主细胞。
  11. 如权利要求10所述的药物组合物，其特征在于，所述药物组合物还包括药学上可接受的载体和/或辅料。
  12. 如权利要求11所述的药物组合物，其特征在于，所述载体包括溶剂、聚合物和脂质体中的至少一种，所述辅料包括稀释剂、赋形剂和稳定剂中的至少一种。
  13. 如权利要求1-5任一项所述的或权利要求6-7任一项所述的制备方法制得的靶向EGFRvⅢ并干扰IL-6表达的嵌合抗原受体T细胞、如权利要求8所述的重组载体、如权利要求9所述的宿主细胞或如权利要求10-12任一项所述的药物组合物在制备预防和治疗恶性肿瘤的药物中的应用。
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