WO2021036245A1 - Lymphocyte t exprimant un récepteur chimérique à l'antigène portant un commutateur de sécurité et ciblant egfrviii, son procédé de préparation et son utilisation - Google Patents

Lymphocyte t exprimant un récepteur chimérique à l'antigène portant un commutateur de sécurité et ciblant egfrviii, son procédé de préparation et son utilisation 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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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

L'invention concerne un lymphocyte T exprimant un récepteur chimérique à l'antigène portant un commutateur de sécurité et ciblant EGFRvIII, comprenant un récepteur d'antigène chimèrique ciblant EGFRvIII CAR-EGFRvIII, et un commutateur de sécurité induisant l'apoptose de lymphocytes T. Le CAR-EGFRvIII comprend des séquences d'acides aminés d'un anticorps à chaîne unique ciblant EGFRvIII, une région de charnière extracellulaire, une région transmembranaire et une région de signal intracellulaire, l'anticorps à chaîne unique ciblant EGFRvIII comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO: 1. Le commutateur de sécurité comprend des séquences d'acides aminés d'une protéine de liaison FK506 mutante F36V, un peptide de liaison et une caspase 9 avec CARD retiré, la séquence d'acides aminés de la protéine de liaison FK506 mutante F36V comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO: 2, et la séquence d'acides aminés de la caspase 9 avec CARD retiré comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO: 3.
PCT/CN2020/081345 2019-08-26 2020-03-26 Lymphocyte t exprimant un récepteur chimérique à l'antigène portant un commutateur de sécurité et ciblant egfrviii, son procédé de préparation et son utilisation WO2021036245A1 (fr)

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