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WO2021036245A1 - 携带安全开关并靶向EGFRvⅢ的嵌合抗原受体T细胞及其制备方法和应用 - Google Patents

携带安全开关并靶向EGFRvⅢ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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WO2021036245A1
WO2021036245A1 PCT/CN2020/081345 CN2020081345W WO2021036245A1 WO 2021036245 A1 WO2021036245 A1 WO 2021036245A1 CN 2020081345 W CN2020081345 W CN 2020081345W WO 2021036245 A1 WO2021036245 A1 WO 2021036245A1
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egfrviii
safety switch
amino acid
gene
coding gene
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French (fr)
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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 201910792806.1 with the title of "Chimeric Antigen Receptor T Cell Carrying a Safety Switch and Targeting EGFRvIII and Its Preparation Method and Application” filed on August 26, 2019.
  • the content of the first 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 carrying a safety switch and targeting EGFRvIII, 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.
  • a chimeric antigen receptor T cell that can carry a safety switch and target EGFRvIII and a preparation method thereof to reduce the side effects of cytokine storm and make cell therapy more controllable and safe.
  • the present invention provides a chimeric antigen receptor T cell carrying a safety switch and targeting EGFRvIII, which has a chimeric antigen receptor targeting EGFRvIII, and can specifically target tumors expressing EGFRvIII Cells activate T cells to exert cellular immunity, and achieve efficient and specific killing of EGFRvIII positive tumor cells, with long-lasting cell viability and killing power; at the same time, the safety switch can be passed after the occurrence of cytokine release syndrome.
  • the dimerization induced by external drugs initiates the mitochondrial apoptosis pathway, which makes the chimeric antigen receptor T cells apoptotic without causing damage to normal cells, and improves the application safety of chimeric antigen receptor T cells.
  • the present invention provides a chimeric antigen receptor T cell carrying a safety switch and targeting EGFRvIII, including a chimeric antigen receptor CAR-EGFRvIII targeting EGFRvIII and a safety switch for inducing T cell apoptosis, wherein
  • 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 connected sequentially from the amino terminal to the carboxyl terminal, and the single-chain targeting EGFRvIII
  • the antibody includes the amino acid sequence shown in SEQ ID NO:1
  • the safety switch includes the amino acid sequence of the F36V mutant FK506 binding protein, the connecting peptide, and the CARD-removed caspase 9 sequentially connected from the amino terminal to the carboxy terminal.
  • the amino acid sequence of the F36V mutant FK506 binding protein includes the amino acid sequence shown in SEQ ID NO: 2, and the amino acid sequence of the C
  • the single-chain antibody encoding gene targeting EGFRvIII includes the nucleotide sequence shown in SEQ ID NO:6. Further, the single-chain antibody coding gene targeting EGFRvIII should include a nucleotide sequence with the nature of base degeneracy with SEQ ID NO:6.
  • the “CAR-EGFRvIII includes sequential connection from the amino terminus to the carboxyl terminus” is specifically: the carboxy terminus of the amino acid sequence of the single-chain antibody targeting EGFRvIII and the extracellular hinge region
  • the amino terminus of the amino acid sequence is connected, the carboxy terminus of the amino acid sequence of the extracellular hinge region is connected 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 connected to the intracellular signal
  • the amino terminus of the amino acid sequence of the region is 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: 4.
  • the CAR-EGFRvIII coding gene includes a nucleotide sequence as shown in SEQ ID NO:7. Furthermore, the CAR-EGFRvIII coding gene should include a nucleotide sequence that has a base degenerate nature with SEQ ID NO:7.
  • 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 coding gene of the F36V mutant FK506 binding protein includes the nucleotide sequence shown in SEQ ID NO: 8. Further, the gene encoding the F36V mutant FK506 binding protein should include a nucleotide sequence that has a base degenerate property with SEQ ID NO: 8.
  • the coding gene of caspase 9 (caspase 9 ⁇ CARD) from which CARD is removed includes a nucleotide sequence as shown in SEQ ID NO:9. Further, the coding gene of the caspase 9 from which CARD is removed should include a nucleotide sequence that has a base degenerate property with SEQ ID NO: 9.
  • the linker is used to connect different proteins or polypeptides, so that the connected proteins or polypeptides maintain their respective spatial conformations, so as to maintain the function or activity of the protein or polypeptide.
  • the connecting peptide can be, but is not limited to, a polypeptide sequence composed mainly of glycine and serine.
  • glycine has the smallest molecular weight and is the amino acid with the shortest side chain, which can increase the flexibility of the side chain; serine is the affinity The strongest water-based amino acid can increase the hydrophilicity of the peptide chain.
  • the amino acid sequence of the connecting peptide includes the amino acid sequence shown in SEQ ID NO: 10.
  • the safety switch includes sequential connections from the amino terminal to the carboxy terminal
  • the carboxyl end of the amino acid sequence of the connecting peptide is connected to the amino end of the amino acid sequence of the caspase 9 from which CARD is removed.
  • the CAR-EGFRvIII and the safety switch are connected through an internal ribosome entry site or through a self-cleaving polypeptide.
  • the internal ribosome entry site mediates the binding of ribosome to RNA and initiates protein translation. After the protein translation before IRES, the ribosome does not break away from the mRNA and can bind to the IRES to enable translation to continue. , Which can translate the two proteins.
  • the coding gene of the internal ribosome entry site includes the nucleotide sequence shown in SEQ ID NO: 11.
  • the self-cleaving polypeptide is used in the construction of a polycistronic vector to express multiple proteins.
  • the chimeric antigen receptor T cell carrying a safety switch and targeting EGFRvIII further includes a dimerization chemical inducer, and the dimerization chemical inducer includes at least one of AP1903 and AP20187.
  • the safety switch (icaspase 9) includes F36V-FKBP and caspase 9 ⁇ CARD, where the F36V point mutation can increase the affinity between FKBP and the dimerization chemical inducer (CID), and the CARD (caspase recruitment domain, caspase Recruitment domain) is removed, its physiological function is replaced by FKBP, and gene expression can be improved.
  • CID can be introduced but not limited to by injection, so that icaspase 9 dimerizes, thereby activating downstream caspase 3 molecules, leading to CAR-T cell apoptosis, preventing the occurrence of adverse reactions, and improving Application safety of CAR-T cells.
  • the chimeric antigen receptor T cell carrying a safety switch and targeting EGFRvIII provided by the first aspect of the present invention can specifically target tumor cells expressing EGFRvIII, activate T cells to exert cellular immunity, and achieve the effect of EGFRvIII positive tumor cells Efficient and specific killing, with long-lasting cell viability and lethality; at the same time, the safety switch can induce dimerization through external drugs after the occurrence of cytokine release syndrome, start the mitochondrial apoptosis pathway, and make chimerism Antigen receptor T cells are apoptotic and will not cause damage to normal cells, which improves the application safety of chimeric antigen receptor T cells.
  • the present invention provides a method for preparing a chimeric antigen receptor T cell carrying a safety switch and targeting EGFRvIII, 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;
  • coding genes for safety switches that induce T cell apoptosis including F36V mutant FK506 binding protein coding genes, connecting peptide coding genes, and CARD-removed caspases that are sequentially connected from the 5'end to the 3'end.
  • the coding gene of the F36V mutant FK506 binding protein includes the nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 2, and the coding gene of the caspase 9 with CARD removed includes the coding gene of SEQ ID NO: 2.
  • the "encoding gene of the safety switch for inducing T cell apoptosis includes sequential connection from the 5'end to the 3'end” is specifically: the 3'end of the signal peptide encoding gene sequence and the The 5'end of the coding gene of the single-chain antibody targeting EGFRvIII is connected to the 5'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, the 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, 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. 'End connected.
  • the "encoding gene of the safety switch that induces T cell apoptosis includes sequential connection from the 5'end to the 3'end" is specifically: 3 of the encoding gene sequence of the F36V mutant FK506 binding protein The'end is connected to the 5'end of the coding gene of the connecting peptide, and the 3'end of the coding gene of the linking peptide is connected to the 5'end of the coding gene of the CARD-removed caspase 9.
  • 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: 12.
  • inserting the encoding gene of CAR-EGFRvIII and the encoding gene of the safety switch into a gene delivery vector includes:
  • the CAR-EGFRvIII coding gene and the safety switch coding gene are connected through an internal ribosome entry site or a self-cleaving polypeptide, and then inserted into the gene delivery vector.
  • 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 a pWPXLD vector
  • the CAR-EGFRvIII encoding gene is inserted between the BamH I and EcoR I restriction sites in the pWPXLD vector
  • the safety switch encoding gene is inserted To between the Spe I and Nde I restriction sites in the pWPXLD vector.
  • 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
  • the 5'end of the coding gene of the safety switch can be added with a start codon (such as ATG), and the Spe I restriction site in the pWPXLD vector (actagt ) Connection, the 3'end can add a stop codon (such as TAA) to the Nde I restriction site
  • the gene fragments inserted into the gene delivery vector can be, but are not limited to, the start codon, the CAR-EGFRvIII coding gene and the stop codon, and the start codon and the safety switch coding gene. And the stop codon.
  • the CAR-EGFRvIII and the safety switch are connected through an internal ribosome entry site or through a self-cleaving polypeptide.
  • the gene fragment inserted into the gene delivery vector can be, but is not limited to, the start codon, the CAR-EGFRvIII coding gene and stop codon, the coding gene of the internal ribosome entry site, and the start codon.
  • the codon, the coding gene of the safety switch and the stop codon can be, but is not limited to, the start codon, the CAR-EGFRvIII coding gene and stop codon, the coding gene of the internal ribosome entry site, and the start codon.
  • 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 maintain 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 preparation method of the chimeric antigen receptor T cell carrying a safety switch and targeting EGFRvIII provided by the second aspect of the present invention is simple and can be applied on a large scale to obtain chimeric antigen receptor T cells with application safety.
  • the present invention provides a recombinant vector, including an inserted gene encoding a chimeric antigen receptor CAR-EGFRvIII targeting EGFRvIII and an encoding gene encoding a safety switch for inducing T cell apoptosis, 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 coding genes of the safety switch for inducing T cell apoptosis include the coding gene of F36V mutant FK506 binding protein,
  • the recombinant vector is obtained by inserting the coding gene of the chimeric antigen receptor CAR-EGFRvIII targeting EGFRvIII and the coding gene of the safety switch for inducing T cell apoptosis into the vector, and the insertion sequence of the two sequences Not limited.
  • the coding gene of the chimeric antigen receptor CAR-EGFRvIII targeting EGFRvIII is at the 5'end of the coding gene of the safety switch for inducing T cell apoptosis, or the chimeric antigen targeting EGFRvIII
  • the gene encoding the receptor CAR-EGFRvIII is at the 3'end of the gene encoding the safety switch for inducing T cell apoptosis.
  • 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 coding gene of the safety switch for inducing T cell apoptosis can be directly transfected into CD3 positive T lymphocytes, prepared chimeric antigen receptor T cells carrying a safety switch and targeting EGFRvIII.
  • the recombinant vector provided by the third aspect of the present invention is safe and efficient, can stably realize the introduction or replication of the encoding gene of CAR-EGFRvIII and the encoding gene of the safety switch into host cells, and can be used for the preparation of chimeric antigen receptor T cells.
  • 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 or 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 a chimeric antigen receptor T cell carrying a safety switch and targeting EGFRvIII prepared by the preparation method according to the first aspect or the preparation method according to the second 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 a chimeric antigen receptor T cell carrying a safety switch and targeting EGFRvIII prepared by the preparation method described in the first aspect or the preparation method described in the second aspect, as described in the third aspect
  • the application of the recombinant vector of, the host cell as described in the fourth aspect or the pharmaceutical composition as described in the fifth aspect in the preparation of drugs for the prevention and treatment of malignant tumors.
  • the application can specifically, but is not limited to, providing a kit, which includes the chimeric antigen receptor T cell carrying a safety switch and targeting EGFRvIII as described in the first aspect, and the chimeric antigen receptor T cell as described in the third aspect.
  • a kit which includes the chimeric antigen receptor T cell carrying a safety switch and targeting EGFRvIII as described in the first aspect, and the chimeric antigen receptor T cell as described in the third aspect.
  • One or more of the recombinant vector, the host cell according to the fourth aspect, and the pharmaceutical composition according to the fifth aspect are provided.
  • 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-icaspase 9 recombinant vector provided by the embodiment of the present invention.
  • CAR-EGFRvIII coding gene that is, provide the nucleotide sequence shown in SEQ ID NO: 7, and add restriction site and start codon at the 5'end, and add restriction site at the 3'end Point and stop codon; insert it between the BamH I and EcoR I restriction sites of the pWPXLD vector. Then it was transformed into E. coli competent cells DH5 ⁇ , and the positive clones were identified by PCR and sequencing. After PCR product gel electrophoresis detection and sequencing to identify the size and sequence of the target fragment, the pWPXLD-CAR-EGFRvIII recombinant vector was successfully constructed, as shown in Figure 1.
  • the coding gene of the safety switch connected to the IRES that is, provide the nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 5, and add the enzyme cut site, shown in SEQ ID NO: 11 at its 5'end IRES nucleotide sequence, start codon and, and add restriction site and stop codon at the 3'end; insert it between the Spe I and Nde I restriction sites of pWPXLD vector. Then it was transformed into E. coli competent cells DH5 ⁇ , and the positive clones were identified by PCR and sequencing. After PCR product gel electrophoresis detection and sequencing to identify the size and sequence of the target fragment, the pWPXLD-CAR-EGFRvIII-icaspase 9 recombinant vector was successfully constructed, as shown in Figure 2.
  • the pWPXLD-CAR-EGFRvIII-icaspase 9 recombinant vector, the packaging plasmid psPAX2 and the envelope plasmid pMD2G were co-transfected into the 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; at 72h, the virus-containing supernatant was harvested for the second time, 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 the liquid, the virus preservation solution was added, gently resuspended repeated pipetting; titer was sufficiently dissolved, after centrifugation 10000 rpm for high-speed, centrifuge 5min, supernatant fluorescence, viruses are grouped according to 100 ⁇ l, 2 ⁇ 10 8 cells / mL Install and store in
  • 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 cells On the 3rd day of culture, perform cell count and change the medium, adjust the cell concentration to 1 ⁇ 10 6 cells/mL, inoculate and culture; on the 5th day of culture, observe the cell status. If the cell density increases, the diluted cell concentration is 1 ⁇ 10 6 cells/mL, check cell viability, and continue to culture. After expansion and culture to the 9th-11th day, the cells are collected, and the chimeric antigen receptor T cells carrying the safety switch and targeting EGFRvIII are obtained, and stored in the special cell cryopreservation solution for reinfusion.
  • a chimeric antigen receptor T cell carrying a safety switch and targeting EGFRvIII prepared by the method of the present invention and a chimeric antigen receptor T cell targeting EGFRvIII, a chimeric antigen carrying a safety switch and targeting EGFRvIII processed by AP20187 Comparing the in vitro tumor killing effects of receptor T cells, chimeric antigen receptor T cells targeting EGFRvIII treated by AP20187, and unprepared T lymphocytes (negative control group). Specifically: the effector cells are compared with those in vitro The ratio of the number of target cells is 1:10, 1:3, 1:1, 3:1, and 10:1. The co-cultivation is carried out at 37°C and 5% CO 2. In the first 15-18 hours after culture, Collect cells and perform flow cytometry to detect cell killing.

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Abstract

提供了携带安全开关并靶向EGFRvⅢ的嵌合抗原受体T细胞，其包括靶向EGFRvⅢ的嵌合抗原受体CAR-EGFRvⅢ以及诱导T细胞凋亡的安全开关，所述CAR-EGFRvⅢ包括靶向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，所述安全开关包括F36V突变的FK506结合蛋白、连接肽和去除CARD的caspase 9的氨基酸序列，所述F36V突变的FK506结合蛋白的氨基酸序列包括如SEQ ID NO：2所示的氨基酸序列，所述去除CARD的caspase 9的氨基酸序列包括如SEQ ID NO：3所示的氨基酸序列。

Description

携带安全开关并靶向EGFRvⅢ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“携带安全开关并靶向EGFRvⅢ的嵌合抗原受体T细胞及其制备方法和应用”的申请号201910792806.1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，特别涉及携带安全开关并靶向EGFRvⅢ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，亟需一种可以携带安全开关并靶向EGFRvⅢ的嵌合抗原受体T细胞及其制备方法，以降低细胞因子风暴的副作用，使得细胞疗法更加可控安全。
发明内容
有鉴于此，本发明提供了一种携带安全开关并靶向EGFRvⅢ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，安全开关可以在发生细胞因子释放综合症后，可以通过外界药物诱导的二聚化，启动线粒体凋亡通路，使嵌合抗原受体T细胞凋亡，且对正 常细胞不会造成损伤，提高嵌合抗原受体T细胞应用安全性。
第一方面，本发明提供了一种携带安全开关并靶向EGFRvⅢ的嵌合抗原受体T细胞，包括靶向EGFRvⅢ的嵌合抗原受体CAR-EGFRvⅢ以及诱导T细胞凋亡的安全开关，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，所述安全开关包括从氨基端到羧基端顺次连接的F36V突变的FK506结合蛋白、连接肽和去除CARD的caspase 9的氨基酸序列，所述F36V突变的FK506结合蛋白的氨基酸序列包括如SEQ ID NO：2所示的氨基酸序列，所述去除CARD的caspase 9的氨基酸序列包括如SEQ ID NO：3所示的氨基酸序列。
可选的，所述靶向EGFRvⅢ的单链抗体编码基因包括如SEQ ID NO：6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包括与SEQ ID NO:6具有碱基简并性质的核苷酸序列。
在本发明中，所述“所述CAR-EGFRvⅢ包括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：4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：7所示的核苷酸序列。更进一步的，所述CAR-EGFRvⅢ的编码基因应包括与SEQ ID NO:7具有碱基简并性质的核苷酸序列。
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，所述F36V突变的FK506结合蛋白(F36V-FKBP)的编码基因包括如SEQ ID NO：8所示的核苷酸序列。进一步的，所述F36V突变的FK506结合蛋白的编码基因应包括与SEQ ID NO:8具有碱基简并性质的核苷酸序列。
可选的，所述去除CARD的caspase 9(caspase 9ΔCARD)的编码基因包括如SEQ ID NO：9所示的核苷酸序列。进一步的，所述去除CARD的caspase 9的编码基因应包括与SEQ ID NO:9具有碱基简并性质的核苷酸序列。
在本发明中，所述连接肽(linker)用于连接不同蛋白或多肽，使所连接的蛋白或多肽保持各自的空间构象，以维持蛋白或多肽的功能或活性。在本发明中，所述连接肽可以但不限于为以甘氨酸和丝氨酸为主构成的多肽序列，其中，甘氨酸的分子量最小，是侧链最短的氨基酸，可以增加侧链的柔韧性；丝氨酸是亲水性最强的氨基酸，可以增加肽链的亲水性。可选的，所述连接肽的氨基酸序列包括如SEQ ID NO：10所示的氨基酸序列。
在本发明中，所述“所述安全开关包括从氨基端到羧基端顺次连接”具体为：所述F36V突变的FK506结合蛋白的氨基酸序列的羧基端与所述连接肽的氨基酸序列的氨基端相连，所述连接肽的氨基酸序列的羧基端与所述去除CARD的caspase 9的氨基酸序列的氨基端相连。
可选的，所述CAR-EGFRvⅢ和所述安全开关通过内部核糖体进入位点或通过自裂解多肽进行连接。
在本发明中，内部核糖体进入位点(IRES)介导核糖体与RNA结合，起始蛋白质翻译，在IRES前的蛋白质翻译后，核糖体不脱离mRNA，可与IRES结合，使翻译继续进行，进而可以翻译两种蛋白。可选的，所述内部核糖体进入位点的编码基因包括如SEQ ID NO：11所示的核苷酸序列。
在本发明中，自裂解多肽用于多顺反子载体构建中，用于表达多个蛋白。
可选的，所述携带安全开关并靶向EGFRvⅢ的嵌合抗原受体T细胞还包括二聚化化学诱导物，所述二聚化化学诱导物包括AP1903和AP20187中的至少一种。
在本发明中，安全开关(icaspase 9)包括F36V-FKBP和caspase 9ΔCARD，其中F36V点突变后可以提高FKBP与二聚化化学诱导物(CID)之间的亲和力，将CARD(caspase recruitment domain，caspase募集结构域)去除，其生理学功能被FKBP取代，且可以提高基因的表达。当发生细胞因子释放综合症时，可以但不限于通过注入的方式引入CID，使得icaspase 9发生二聚化，从而激活下游caspase 3分子，导致CAR-T细胞凋亡，阻止不良 反应的发生，提高CAR-T细胞的应用安全性。
本发明第一方面提供的携带安全开关并靶向EGFRvⅢ的嵌合抗原受体T细胞，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，安全开关可以在发生细胞因子释放综合症后，可以通过外界药物诱导二聚化，启动线粒体凋亡通路，使嵌合抗原受体T细胞凋亡，且对正常细胞不会造成损伤，提高嵌合抗原受体T细胞应用安全性。
第二方面，本发明提供了一种携带安全开关并靶向EGFRvⅢ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)提供诱导T细胞凋亡的安全开关的编码基因，包括从5’端到3’端顺次连接的F36V突变的FK506结合蛋白的编码基因、连接肽的编码基因和去除CARD的caspase 9的编码基因，其中，所述F36V突变的FK506结合蛋白的编码基因包括如SEQ ID NO：2所示的氨基酸序列对应的核苷酸序列，所述去除CARD的caspase 9的编码基因包括如SEQ ID NO：3所示的氨基酸序列对应的核苷酸序列；
(3)将所述CAR-EGFRvⅢ的编码基因和所述安全开关的编码基因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，获得携带安全开关并靶向EGFRvⅢ的嵌合抗原受体T细胞。
在本发明中，所述“诱导T细胞凋亡的安全开关的编码基因包括从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“诱导T细胞凋亡的安全开关的编码基因，包括从5’端到3’端顺次连接”具体为：所述F36V突变的FK506结合蛋白的编码基因序列的3’端与所述连接肽的编码基因的5’端相连，所述连接肽的编码基因的3’端与所述去除CARD的caspase 9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：12所示。
其中，所述靶向EGFRvⅢ的单链抗体、胞外铰链区、跨膜区、胞内信号区、F36V突 变的FK506结合蛋白、连接肽、去除CARD的caspase 9的具体选择如本发明第一方面所述，这里不再赘述。
可选的，将所述CAR-EGFRvⅢ的编码基因和所述安全开关的编码基因插入到基因传递载体中，包括：
将所述CAR-EGFRvⅢ的编码基因和所述安全开关的编码基因通过内部核糖体进入位点或通过自裂解多肽进行连接后，再插入到所述基因传递载体中。
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；所述安全开关的编码基因插入到pWPXLD载体中Spe Ⅰ和Nde Ⅰ酶切位点之间。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；所述安全开关的编码基因插入到pWPXLD载体时，所述安全开关的编码基因的5’端可加入起始密码子(如ATG)，与pWPXLD载体中Spe Ⅰ酶切位点(actagt)相连，3’端可加入终止密码子(如TAA)与pWPXLD载体中Nde Ⅰ酶切位点(catatg)相连，这样就使所述CAR-EGFRvⅢ的编码基因位于Spe Ⅰ和Nde Ⅰ酶切位点之间，得到pWPXLD-CAR-EGFRvⅢ-icaspase 9重组基因传递载体。
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，以及起始密码子、所述安全开关的编码基因和终止密码子。
在本发明中，并不限制CAR-EGFRvⅢ的编码基因和安全开关的编码基因插入基因传递载体中的先后顺序。
可选的，所述CAR-EGFRvⅢ和所述安全开关通过内部核糖体进入位点或通过自裂解多肽进行连接。
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，内部核糖体进入位点的编码基因，以及起始密码子、所述安全开关的编码基因和终止密码子。
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。
本发明第二方面提供的携带安全开关并靶向EGFRvⅢ的嵌合抗原受体T细胞的制备方法简单，可以进行大规模应用，以得到具有应用安全性的嵌合抗原受体T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的编码基因和诱导T细胞凋亡的安全开关的编码基因，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；所述诱导T细胞凋亡的安全 开关的编码基因，包括从5’端到3’端顺次连接的F36V突变的FK506结合蛋白的编码基因、连接肽的编码基因和去除CARD的caspase 9的编码基因，其中，所述F36V突变的FK506结合蛋白的编码基因包括如SEQ ID NO：2所示的氨基酸序列对应的核苷酸序列，所述去除CARD的caspase 9的编码基因包括如SEQ ID NO：3所示的氨基酸序列对应的核苷酸序列。
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的编码基因和诱导T细胞凋亡的安全开关的编码基因插入载体中得到，且两个序列的插入顺序并不限定。可选的，所述靶向EGFRvⅢ的嵌合抗原受体CAR-EGFRvⅢ的编码基因在所述诱导T细胞凋亡的安全开关的编码基因的5’端，或所述靶向EGFRvⅢ的嵌合抗原受体CAR-EGFRvⅢ的编码基因在所述诱导T细胞凋亡的安全开关的编码基因的3’端。
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的编码基因和诱导T细胞凋亡的安全开关的编码基因的重组微环DNA直接转染CD3阳性T淋巴细胞，制得携带安全开关并靶向EGFRvⅢ的嵌合抗原受体T细胞。
本发明第三方面提供的重组载体安全高效，可以稳定地实现将CAR-EGFRvIII的编码基因和安全开关的编码基因导入宿主细胞中或复制，并可以用于嵌合抗原受体T细胞的制备。
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携带安全开关并靶向EGFRvⅢ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携带安全开关并靶向EGFRvⅢ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。
所述应用具体可以但不限于为提供了一种试剂盒，所述试剂盒包括第一方面所述的携带安全开关并靶向EGFRvⅢ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、如第五方面所述的药物组合物中的一种或多种。
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-icaspase 9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)pWPXLD-CAR-EGFRvⅢ-icaspase 9重组载体构建
提供CAR-EGFRvⅢ的编码基因，即提供如SEQ ID NO：7所示的核苷酸序列，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。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重组载体，如图1所示。
提供连接IRES的安全开关的编码基因，即提供如SEQ ID NO：5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、SEQ ID NO：11所示的IRES的核苷酸序列、起始密码子以及，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Spe 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-icaspase 9重组载体，如图2所示。
(2)重组慢病毒构建
将pWPXLD-CAR-EGFRvⅢ-icaspase 9重组载体、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)携带安全开关并靶向EGFRvⅢ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，得到携带安全开关并靶向EGFRvⅢ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
评估携带安全开关并靶向EGFRvⅢ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携带安全开关并靶向EGFRvⅢ的嵌合抗原受体T细胞与靶向EGFRvⅢ嵌合抗原受体T细胞、由AP20187处理的携带安全开关并靶向EGFRvⅢ的嵌合抗原受体T细胞、由AP20187处理的靶向EGFRvⅢ的嵌合抗原受体T细胞、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。
结果发现，携带安全开关并靶向EGFRvⅢ的嵌合抗原受体T细胞、靶向EGFRvⅢ嵌合抗原受体T细胞、由AP20187处理的靶向EGFRvⅢ的嵌合抗原受体T细胞的杀伤率均为100％，且远远高于阴性对照组；经过AP20187处理的携带安全开关并靶向EGFRvⅢ的嵌合抗原受体T细胞的杀伤率降低至10％，表明携带安全开关并靶向EGFRvⅢ的嵌合抗原受体T细胞不受安全开关的影响，可以正常使用，而当发生不良反应，如细胞因子释放综合症时，通过 注入二聚化化学诱导物可以诱导细胞凋亡，显著降低并有效终止嵌合抗原受体T细胞的杀伤力。
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. 一种携带安全开关并靶向EGFRvⅢ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以及诱导T细胞凋亡的安全开关，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，所述安全开关包括从氨基端到羧基端顺次连接的F36V突变的FK506结合蛋白、连接肽和去除CARD的caspase 9的氨基酸序列，所述F36V突变的FK506结合蛋白的氨基酸序列包括如SEQ ID NO：2所示的氨基酸序列，所述去除CARD的caspase 9的氨基酸序列包括如SEQ ID NO：3所示的氨基酸序列。
  2. 如权利要求1所述的携带安全开关并靶向EGFRvⅢ的嵌合抗原受体T细胞，其特征在于，所述CAR-EGFRvⅢ和所述安全开关通过内部核糖体进入位点或通过自裂解多肽进行连接。
  3. 如权利要求1所述的携带安全开关并靶向EGFRvⅢ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：4所示的氨基酸序列。
  4. 如权利要求1所述的携带安全开关并靶向EGFRvⅢ的嵌合抗原受体T细胞，其特征在于，所述安全开关的氨基酸序列包括如SEQ ID NO：5所示的氨基酸序列。
  5. 如权利要求1所述的携带安全开关并靶向EGFRvⅢ的嵌合抗原受体T细胞，其特征在于，还包括二聚化化学诱导物，所述二聚化化学诱导物包括AP1903和AP20187中的至少一种。
  6. 一种携带安全开关并靶向EGFRvⅢ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)提供诱导T细胞凋亡的安全开关的编码基因，包括从5’端到3’端顺次连接的F36V突变的FK506结合蛋白的编码基因、连接肽的编码基因和去除CARD的caspase 9的编码基因，其中，所述F36V突变的FK506结合蛋白的编码基因包括如SEQ ID NO：2所示的氨基酸序列对应的核苷酸序列，所述去除CARD的caspase 9的编码基因包括如SEQ ID NO：3所示的氨基酸序列对应的核苷酸序列；
    (3)将所述CAR-EGFRvⅢ的编码基因和所述安全开关的编码基因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，获得携带安全开关并靶向EGFRv Ⅲ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，将所述CAR-EGFRvⅢ的编码基因和所述安全开关的编码基因插入到基因传递载体中，包括：
    将所述CAR-EGFRvⅢ的编码基因和所述安全开关的编码基因通过内部核糖体进入位点或通过自裂解多肽进行连接后，再插入到所述基因传递载体中。
  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的编码基因和诱导T细胞凋亡的安全开关的编码基因，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；所述诱导T细胞凋亡的安全开关的编码基因，包括从5’端到3’端顺次连接的F36V突变的FK506结合蛋白的编码基因、连接肽的编码基因和去除CARD的caspase 9的编码基因，其中，所述F36V突变的FK506结合蛋白的编码基因包括如SEQ ID NO：2所示的氨基酸序列对应的核苷酸序列，所述去除CARD的caspase 9的编码基因包括如SEQ ID NO：3所示的氨基酸序列对应的核苷酸序列。
  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携带安全开关并靶向EGFRvⅢ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携带安全开关并靶向EGFRvⅢ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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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106535925A (zh) * 2014-05-23 2017-03-22 佛罗里达大学研究基金会有限公司 基于car的免疫治疗
CN106755023A (zh) * 2015-10-15 2017-05-31 中国人民解放军军事医学科学院附属医院 带安全开关的嵌合抗原受体免疫细胞及其制备方法与应用
WO2018208849A1 (en) * 2017-05-09 2018-11-15 Bellicum Pharmaceuticals, Inc. Methods to augment or alter signal transduction
CN109265561A (zh) * 2018-09-25 2019-01-25 山东兴瑞生物科技有限公司 抗EGFRvⅢ安全型嵌合抗原受体、其制备方法、利用其修饰的NK细胞及应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109957546A (zh) * 2017-12-25 2019-07-02 深圳宾德生物技术有限公司 一种靶向cd317的嵌合抗原受体t细胞及其制备方法和应用
CN110144326A (zh) * 2018-02-12 2019-08-20 深圳宾德生物技术有限公司 一种靶向性抗肿瘤t细胞及其制备方法和应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106535925A (zh) * 2014-05-23 2017-03-22 佛罗里达大学研究基金会有限公司 基于car的免疫治疗
CN106755023A (zh) * 2015-10-15 2017-05-31 中国人民解放军军事医学科学院附属医院 带安全开关的嵌合抗原受体免疫细胞及其制备方法与应用
WO2018208849A1 (en) * 2017-05-09 2018-11-15 Bellicum Pharmaceuticals, Inc. Methods to augment or alter signal transduction
CN109265561A (zh) * 2018-09-25 2019-01-25 山东兴瑞生物科技有限公司 抗EGFRvⅢ安全型嵌合抗原受体、其制备方法、利用其修饰的NK细胞及应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHENG YAN, GAO NING, FU YU-LONG, ZHANG BING-YONG, LI XIU-LING, GUPTA PUJA, WONG ALBERT J., LI TIAN-FANG, HAN SHUANG-YIN: "Generation of regulable EGFRvIII targeted chimeric antigen receptor T cells for adoptive cell therapy of glioblastoma", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ELSEVIER, AMSTERDAM NL, vol. 507, no. 1-4, 1 December 2018 (2018-12-01), Amsterdam NL, pages 59 - 66, XP055785770, ISSN: 0006-291X, DOI: 10.1016/j.bbrc.2018.10.151 *

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