WO2024078485A1 - 一种用于靶向肿瘤微环境的信号感受器和用于靶向治疗肿瘤的细胞 - Google Patents

一种用于靶向肿瘤微环境的信号感受器和用于靶向治疗肿瘤的细胞 Download PDF

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WO2024078485A1
WO2024078485A1 PCT/CN2023/123744 CN2023123744W WO2024078485A1 WO 2024078485 A1 WO2024078485 A1 WO 2024078485A1 CN 2023123744 W CN2023123744 W CN 2023123744W WO 2024078485 A1 WO2024078485 A1 WO 2024078485A1
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region
amino acid
differentiation antigen
leukocyte differentiation
signal
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French (fr)
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王永生
秦笛源
何霞
张本霞
李丹
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四川大学华西医院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001103—Receptors for growth factors
    • A61K39/001109—Vascular endothelial growth factor receptors [VEGFR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70517—CD8
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention belongs to the technical field of tumor immunotherapy, and specifically relates to a signal receptor for targeting tumor microenvironment and a cell for targeted tumor treatment.
  • lymphokine-activated killer cells LAK
  • CIK cytokine-activated killer cells
  • TIL tumor-infiltrating T cells
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • VEGFR is a highly specific transmembrane receptor that is highly expressed in the endothelium of tumor neovascularization and some tumor cell membranes.
  • the VEGFR family mainly includes three tyrosine kinase receptors: VEGFR-1, VEGFR-2 and VEGFR-3.
  • Its ligand, vascular epidermal growth factor (VEGF) is a signal transduction protein produced by cells to stimulate angiogenesis, mainly including 6 secretory glycoproteins: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor (PLGF).
  • VEGF interacts with receptors (Vascular endothelial growth factor receptor, VEGFR), causing signal cascade reactions and mediating physiological or pathological processes such as angiogenesis.
  • VEGF-VEGFR signaling pathway The presence of the VEGF-VEGFR signaling pathway in the tumor microenvironment will lead to a decrease in the effectiveness of adoptive immune cell therapy.
  • inhibitory molecules such as vascular epidermal growth factor (VEGF) and its receptors expressed by tumor and stromal cells produce an immunosuppressive microenvironment, which can damage the anti-tumor effect of the infused immune cells.
  • blood vessels in the tumor microenvironment highly express molecules such as VEGFR1 and VEGFR2, leading to abnormal vascular proliferation, obstacles for immune cells to enter the tumor, and hypoxia in the tumor microenvironment, all of which can inhibit the anti-tumor effect of immune cells.
  • the present invention provides a signal receptor for targeting the tumor microenvironment and a cell for targeted treatment of tumors, with the aim of realizing the function of immune cells targeting the VEGF-VEGFR signaling pathway and activating it after reaching the tumor microenvironment, thereby providing a new clinical drug option for the treatment of tumors.
  • a signal receptor for targeting tumor microenvironment comprising a signal peptide, an extracellular molecule recognition region, an extracellular hinge region, a transmembrane region and an intracellular co-stimulatory signal region;
  • the signal peptide is at least one of the protein domains having a sequence homology of more than 90% with the signal peptide of interleukin 2 (IL-2), the signal peptide of leukocyte differentiation antigen 8 (CD8), the signal peptide of granulocyte-macrophage colony-stimulating factor (GM-CSF) or the signal peptide of colony stimulating factor 2 receptor alpha subunit (CSF2RA);
  • IL-2 interleukin 2
  • CD8 the signal peptide of leukocyte differentiation antigen 8
  • CD8 the signal peptide of granulocyte-macrophage colony-stimulating factor
  • CSF2RA colony stimulating factor 2 receptor alpha subunit
  • the extracellular molecule recognition region is at least one of a monoclonal antibody single chain variable region, a nano single domain antibody recognition region, a receptor or a ligand that binds to VEGF or VEGFR;
  • the extracellular hinge region is at least one of the protein domains having a sequence homology of more than 90% with the extracellular hinge region of leukocyte differentiation antigen 8, the extracellular hinge region of leukocyte differentiation antigen 28, the extracellular hinge region of TRAIL, the extracellular hinge region of CD16, the extracellular hinge region of NKp30, the extracellular hinge region of NKG2C, the extracellular hinge region of NKG2D, the extracellular hinge region of 2B4 or the extracellular hinge region of DNAM-1;
  • the transmembrane region is at least one of the protein domains with a sequence homology of more than 90% with the leukocyte differentiation antigen 8 transmembrane region, the leukocyte differentiation antigen 28 transmembrane region, the TRAIL transmembrane region, the CD16 transmembrane region, the NKp30 transmembrane region, the NKG2C transmembrane region, the NKG2D transmembrane region, the 2B4 transmembrane region or the DNAM-1 transmembrane region;
  • the intracellular co-stimulatory signal region is co-stimulatory with leukocyte differentiation antigen 28, tumor necrosis factor receptor superfamily member 4 (TNFRSF4), tumor necrosis factor receptor superfamily member 9 (TNFRSF9), lymphocyte-specific protein tyrosine kinase (LCK), inducible T cell co-stimulatory molecule (ICOS), CD40 co-stimulatory signal region, CD27 co-stimulatory signal region, DAP10 co-stimulatory signal region, DAP12 co-stimulatory signal region, TRAIL co-stimulatory signal region, CD16 co-stimulatory signal region, NKp30 co-stimulatory signal region, NKG2C co-stimulatory signal region, NKG2D co-stimulatory signal region, 2B4 co-stimulatory signal region or DNAM-1 co-stimulatory signal region. At least one of the intracellular domains of the protein domain with a sequence homology of more than 90% to the stimulatory signal region.
  • the VEGF is at least one of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E or placental growth factor;
  • the VEGFR is at least one of VEGFR1, VEGFR2 or VEGFR3.
  • composition is selected in any of the following ways:
  • Composition method 1 is a composition of Composition method 1:
  • the signal peptide is a colony stimulating factor 2 receptor ⁇ subunit signal peptide, or a peptide having the same or similar function as the colony stimulating factor 2 receptor ⁇ subunit signal peptide obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the extracellular molecule recognition region is a single-chain variable region of a monoclonal antibody that recognizes VEGFR1, or a protein having the same or similar function as the single-chain variable region of a monoclonal antibody that recognizes VEGFR1 obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the extracellular hinge region is the extracellular hinge region of leukocyte differentiation antigen 28, or a protein having the same or similar function as the extracellular hinge region of leukocyte differentiation antigen 28 obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the transmembrane region is the transmembrane region of leukocyte differentiation antigen 28, or a protein having the same or similar function as the transmembrane region of leukocyte differentiation antigen 28 obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the intracellular co-stimulatory signal region is the intracellular domain of leukocyte differentiation antigen 28, or a protein having the same or similar function as the intracellular domain of leukocyte differentiation antigen 28 obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • Composition method 2
  • the signal peptide is a leukocyte differentiation antigen 8 signal peptide, or a peptide having the same or similar function as the leukocyte differentiation antigen 8 signal peptide obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the extracellular molecule recognition region is a nano single-domain antibody recognition region that recognizes VEGFR1, or a protein having the same or similar function as the nano single-domain antibody recognition region that recognizes VEGFR1 obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the extracellular hinge region is the transmembrane region of leukocyte differentiation antigen 8, or a protein having the same or similar function as the extracellular hinge region of leukocyte differentiation antigen 8 obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the transmembrane region is the transmembrane region of leukocyte differentiation antigen 8, or a protein having the same or similar function as the transmembrane region of leukocyte differentiation antigen 8 obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the intracellular co-stimulatory signal region is the intracellular co-stimulatory signal region of 4-1BB, or a protein having the same or similar function as the intracellular co-stimulatory signal region of 4-1BB obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the signal peptide is a leukocyte differentiation antigen 8 signal peptide, or a peptide having the same or similar function as the leukocyte differentiation antigen 8 signal peptide obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the extracellular molecule recognition region is a single-chain variable region of a monoclonal antibody or a single-domain antibody recognition region of a nanometer that recognizes VEGFR1, or a protein having the same or similar function as the single-chain variable region of a monoclonal antibody or a single-domain antibody recognition region of a nanometer that recognizes VEGFR1 obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the extracellular hinge region is the extracellular hinge region of leukocyte differentiation antigen 8, or a protein having the same or similar function as the extracellular hinge region of leukocyte differentiation antigen 8 obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the transmembrane region is the NKG2D transmembrane region, or a protein having the same or similar function as the NKG2D transmembrane region obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the intracellular co-stimulatory signal region is the intracellular co-stimulatory signal region of NKG2D, or a protein having the same or similar function as the intracellular co-stimulatory signal region of NKG2D obtained by substitution and/or deletion and/or addition of at least one amino acid.
  • Composition method four is a composition of Composition method four:
  • the signal peptide is a colony stimulating factor 2 receptor ⁇ subunit signal peptide, or a peptide having the same or similar function as the colony stimulating factor 2 receptor ⁇ subunit signal peptide obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the extracellular molecule recognition region is a single-chain variable region of a monoclonal antibody that recognizes VEGFR2, or a protein having the same or similar function as the single-chain variable region of a monoclonal antibody that recognizes VEGFR1 obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the extracellular hinge region is the extracellular hinge region of leukocyte differentiation antigen 28, or a protein having the same or similar function as the extracellular hinge region of leukocyte differentiation antigen 28 obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the transmembrane region is the transmembrane region of leukocyte differentiation antigen 28, or a protein having the same or similar function as the transmembrane region of leukocyte differentiation antigen 28 obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the intracellular costimulatory signal region is the intracellular domain of leukocyte differentiation antigen 28, or is replaced by and/or a protein having the same or similar function as the intracellular domain of leukocyte differentiation antigen 28 obtained by deleting and/or adding at least one amino acid;
  • Composition method five is a composition of Composition method five:
  • the signal peptide is a colony stimulating factor 2 receptor ⁇ subunit signal peptide, or a peptide having the same or similar function as the colony stimulating factor 2 receptor ⁇ subunit signal peptide obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the extracellular molecule recognition region is a single-chain variable region of a monoclonal antibody that recognizes VEGFR1, or a protein having the same or similar function as the single-chain variable region of a monoclonal antibody that recognizes VEGFR1 obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the extracellular hinge region is the extracellular hinge region of leukocyte differentiation antigen 8, or a protein having the same or similar function as the extracellular hinge region of leukocyte differentiation antigen 8 obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the transmembrane region is the transmembrane region of leukocyte differentiation antigen 8, or a protein having the same or similar function as the transmembrane region of leukocyte differentiation antigen 8 obtained by substitution and/or deletion and/or addition of at least one amino acid;
  • the intracellular co-stimulatory signal region is the intracellular domain of 4-1BB, or a protein having the same or similar function as the intracellular domain of 4-1BB obtained by substitution and/or deletion and/or addition of at least one amino acid.
  • the amino acid sequence of the colony stimulating factor 2 receptor ⁇ subunit signal peptide is as shown in SEQ ID NO.1.
  • the encoding nucleotide sequence of the colony stimulating factor 2 receptor ⁇ subunit signal peptide is shown as SEQ ID NO.2.
  • amino acid sequence of the single-chain variable region of the monoclonal antibody that recognizes VEGFR1 is shown as SEQ ID No.3.
  • the encoding nucleotide sequence that recognizes VEGFR1 is shown as SEQ ID No.4.
  • amino acid sequence of the extracellular hinge region of the leukocyte differentiation antigen 28 is shown in SEQ ID No.5.
  • the encoding nucleotide sequence of the extracellular hinge region of the leukocyte differentiation antigen 28 is shown as SEQ ID No.6.
  • amino acid sequence of the transmembrane region of the leukocyte differentiation antigen 28 is as shown in SEQ ID No.7.
  • nucleotide sequence encoding the transmembrane region of leukocyte differentiation antigen 28 is shown as SEQ ID No.8.
  • amino acid sequence of the intracellular domain of the leukocyte differentiation antigen 28 is shown in SEQ ID No.9.
  • the encoding nucleotide sequence of the intracellular domain of the leukocyte differentiation antigen 28 is shown in SEQ ID No.10.
  • the amino acid sequence of the leukocyte differentiation antigen 8 signal peptide is shown in SEQ ID NO.13.
  • the encoding nucleotide sequence of the leukocyte differentiation antigen 8 signal peptide is shown as SEQ ID NO.14.
  • amino acid sequence of the nano single-domain antibody recognition region of VEGFR1 is shown as SEQ ID NO.15 or SEQ ID NO.23.
  • the encoding nucleotide sequence of the nano single-domain antibody recognition region of VEGFR1 is shown as SEQ ID NO.16 or SEQ ID NO.24.
  • amino acid sequence of the leukocyte differentiation antigen 8 transmembrane region is as shown in SEQ ID NO.17.
  • the encoding nucleotide sequence of the leukocyte differentiation antigen 8 transmembrane region is as shown in SEQ ID NO.18.
  • amino acid sequence of the 4-1BB intracellular co-stimulatory signal region is as shown in SEQ ID NO.19.
  • the encoding nucleotide sequence of the 4-1BB intracellular co-stimulatory signal region is shown as SEQ ID NO.20.
  • amino acid sequence of the extracellular hinge region of the leukocyte differentiation antigen 8 is shown in SEQ ID NO.27.
  • the encoding nucleotide sequence of the extracellular hinge region of leukocyte differentiation antigen 8 is as shown in SEQ ID NO.28.
  • amino acid sequence of the NKG2D transmembrane region is shown in SEQ ID NO.29.
  • the encoding nucleotide sequence of the NKG2D transmembrane region is as shown in SEQ ID NO.30.
  • amino acid sequence of the NKG2D intracellular co-stimulatory signal region is shown in SEQ ID NO.31.
  • the encoding nucleotide sequence of the NKG2D intracellular co-stimulatory signal region is shown as SEQ ID NO.32.
  • amino acid sequence of the signal receptor is shown in SEQ ID NO.11, SEQ ID NO.21, SEQ ID NO.25, SEQ ID NO.33, SEQ ID NO.34 or SEQ ID NO.35.
  • the encoding nucleotide sequence of the signal receptor is shown as SEQ ID NO.12, SEQ ID NO.22, SEQ ID NO.26, SEQ ID NO.36, SEQ ID NO.37 or SEQ ID NO.38.
  • the present invention also provides an expression vector, which can express the above signal receptor.
  • it is a recombinant plasmid vector, a recombinant virus or a combination thereof.
  • the recombinant virus is a lentiviral vector or a retroviral vector.
  • the present invention also provides a cell for targeted tumor treatment, which is composed of the above expression vector and host cells.
  • the host cell is selected from ⁇ T cells, NKT cells or ⁇ T cells, tumor infiltrating lymphocytes, TCR-modified T cells, chimeric antibody receptor T cells or natural killer cells.
  • the present invention also provides a medicine for treating tumors, which is prepared by using the above cells for targeted tumor treatment as active ingredients and adding pharmaceutically acceptable excipients or auxiliary ingredients.
  • the tumor is lung cancer, liver cancer, lymphoma, colon cancer, rectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct cancer, esophageal cancer, kidney cancer, glioma, melanoma, pancreatic cancer, multiple myeloma or prostate cancer.
  • domain refers to a segment in a protein that is spatially distinct and has its own functions; changes (replacement, deletion and/or addition) of some amino acid residues in the domain will not significantly change the domain function. Therefore, it should be known that the antigen receptors involved in the present invention are within the scope of the present invention even if amino acid residues are changed, as long as the functions of the domains involved do not change significantly.
  • the present invention provides an artificially constructed signal receptor and a host immune cell containing the signal receptor.
  • the signal receptor can specifically recognize one or more immunosuppressive molecules such as VEGFR in the tumor microenvironment, convert the inhibitory signal in the microenvironment into a co-stimulatory signal inside the immune cell, participate in activating the immune cell, and enhance the anti-tumor activity of the immune cell.
  • the present invention has a good application prospect in the treatment of tumors.
  • FIG1 is a schematic structural diagram of a signal receptor of the present invention.
  • FIG2 is a schematic diagram of the gene structure of the viral vector plasmid constructed in Experimental Example 1;
  • Figure 3 shows the expression of EGFR CAR molecules and VEGFR1 signal receptors on the surface of Jurkat cell membranes in Experimental Example 1;
  • Figure 4 shows the expression of EGFR CAR molecules and VEGFR1 signal receptors on the human T lymphocyte membrane in Experimental Example 1;
  • FIG5 shows the expression of tumor cell target antigen EGFR and VEGFR1 in Experimental Example 2;
  • Figure 6 shows the secretion of T cell effector cytokines after co-culture of EGFR-VEGFR1CART and EGFR CAR-T with target cells H1975 in Experimental Example 2;
  • FIG. 7 is the experimental result of direct killing activity of CAR-T cells overexpressing signal receptors on target cells in Experimental Example 2;
  • FIG8 is a schematic diagram of the gene structure of the viral vector plasmid constructed in Experimental Example 3.
  • FIG. 9 shows the membrane expression of GPC3 CAR molecules and VEGFR1 signal receptors in Jurkat cell lines in Experimental Example 3;
  • FIG. 10 shows the expression of GPC3 CAR molecules and VEGFR1 signal receptors on the human T lymphocyte membrane in Experimental Example 3;
  • FIG11 shows the expression of tumor cell target antigen GPC3 and VEGFR1 in Experimental Example 4.
  • FIG12 shows the secretion of T cell effector cytokines after co-culture of different T cells with tumor target cells in Experimental Example 4;
  • FIG13 is the experimental result of direct killing activity of CAR-T cells overexpressing signal receptors on target cells in Experimental Example 4.
  • FIG14 is a schematic diagram of the gene structure of the viral vector plasmid constructed in Experimental Example 5;
  • FIG15 shows the expression of VEGFR1 signal receptors on the T lymphocyte membrane in Experimental Example 5.
  • FIG16 shows the expression of VEGFR1 in tumor cells in Experimental Example 6
  • FIG17 shows the formation of immune cell clones under different co-culture conditions of T cells and tumor target cells in Experimental Example 6;
  • FIG. 18 shows the secretion of T cell effector cytokines after co-culture of different T cells with tumor target cells in Experimental Example 6.
  • the signal receptor provided in this embodiment is shown in FIG1 and is composed of a signal peptide, an extracellular molecule recognition region, It consists of an extracellular hinge region, a transmembrane region and an intracellular co-stimulatory signal region.
  • the signal peptide is the colony stimulating factor 2 receptor ⁇ subunit signal peptide, and its amino acid sequence is shown in SEQ ID NO.1.
  • nucleotide coding sequence is shown in SEQ ID NO.2.
  • the extracellular molecule recognition region is a single-chain variable region of a monoclonal antibody that recognizes VEGFR1, and its amino acid sequence is shown in SEQ ID NO.3.
  • nucleotide coding sequence is shown in SEQ ID NO.4.
  • the extracellular hinge region is the leukocyte differentiation antigen 28 extracellular hinge region, and its amino acid sequence is shown in SEQ ID No.5.
  • nucleotide coding sequence is shown in SEQ ID NO.6.
  • the transmembrane region is the leukocyte differentiation antigen 28 transmembrane region, and its amino acid sequence is shown in SEQ ID NO.7.
  • nucleotide coding sequence is shown in SEQ ID NO.8.
  • the intracellular co-stimulatory signal region is the intracellular domain of leukocyte differentiation antigen 28, and its amino acid sequence is shown in SEQ ID NO.9.
  • nucleotide coding sequence is shown in SEQ ID NO.10.
  • sequence of the signal receptor in this embodiment is shown as SEQ ID NO.11.
  • nucleotide coding sequence is shown in SEQ ID NO.12.
  • the signal receptor provided in this embodiment is composed of a signal peptide, an extracellular molecule recognition region, an extracellular hinge region, a transmembrane region and an intracellular co-stimulatory signal region.
  • the signal peptide is the leukocyte differentiation antigen 8 signal peptide, and its amino acid sequence is shown in SEQ ID NO.13.
  • nucleotide coding sequence is shown in SEQ ID NO.14.
  • the extracellular molecule recognition region is a nano single-domain antibody recognition region that recognizes VEGFR1, and its amino acid sequence is shown in SEQ ID NO.15.
  • nucleotide coding sequence is shown in SEQ ID NO.16.
  • the extracellular hinge region is the leukocyte differentiation antigen 8 extracellular hinge region, and its amino acid sequence is shown in SEQ ID NO.27.
  • nucleotide coding sequence is shown in SEQ ID NO.28.
  • the transmembrane region is the leukocyte differentiation antigen 8 transmembrane region, and its amino acid sequence is as shown in SEQ ID NO.17 shown.
  • nucleotide coding sequence is shown in SEQ ID NO.18.
  • the intracellular co-stimulatory signal region is the 4-1BB intracellular co-stimulatory signal region, and its amino acid sequence is shown in SEQ ID NO.19.
  • nucleotide coding sequence is shown in SEQ ID NO.20.
  • sequence of the signal receptor of this embodiment is shown as SEQ ID NO.21.
  • nucleotide coding sequence is shown in SEQ ID NO.22.
  • the signal receptor provided in this embodiment is composed of a signal peptide, an extracellular molecule recognition region, an extracellular hinge region, a transmembrane region and an intracellular co-stimulatory signal region.
  • the signal peptide is the leukocyte differentiation antigen 8 signal peptide, and its amino acid sequence is shown in SEQ ID NO.13.
  • nucleotide coding sequence is shown in SEQ ID NO.14.
  • the extracellular molecule recognition region is a nano single-domain antibody recognition region that recognizes VEGFR1, and its amino acid sequence is shown in SEQ ID NO.23.
  • nucleotide coding sequence is shown in SEQ ID NO.24.
  • the extracellular hinge region is the leukocyte differentiation antigen 8 extracellular hinge region, and its amino acid sequence is shown in SEQ ID NO.27.
  • nucleotide coding sequence is shown in SEQ ID NO.28.
  • the transmembrane region is the leukocyte differentiation antigen 8 transmembrane region, and its amino acid sequence is shown in SEQ ID NO.17.
  • nucleotide coding sequence is shown in SEQ ID NO.18.
  • the intracellular co-stimulatory signal region is the 4-1BB intracellular co-stimulatory signal region, and its amino acid sequence is shown in SEQ ID NO.19.
  • nucleotide coding sequence is shown in SEQ ID NO.20.
  • sequence of the signal receptor in this embodiment is shown as SEQ ID NO.25.
  • nucleotide coding sequence is shown in SEQ ID NO.26.
  • the signal receptor provided in this embodiment is composed of a signal peptide, an extracellular molecule recognition region, an extracellular hinge region, a transmembrane region and an intracellular co-stimulatory signal region.
  • the signal peptide is the leukocyte differentiation antigen 8 signal peptide, and its amino acid sequence is shown in SEQ ID NO.13.
  • nucleotide coding sequence is shown in SEQ ID NO.14.
  • the extracellular molecule recognition region is a single-chain variable region of a monoclonal antibody that recognizes VEGFR1, and its amino acid sequence is shown in SEQ ID NO.3.
  • nucleotide coding sequence is shown in SEQ ID NO.4.
  • the extracellular molecule recognition region is a nano single-domain antibody recognition region that recognizes VEGFR1, and its amino acid sequence is shown in SEQ ID NO.15 or SEQ ID NO.23.
  • nucleotide coding sequence is shown as SEQ ID NO.16 or SEQ ID NO.24.
  • the extracellular hinge region is the leukocyte differentiation antigen 8 extracellular hinge region, and its amino acid sequence is shown in SEQ ID NO.27.
  • nucleotide coding sequence is shown in SEQ ID NO.28.
  • the transmembrane region is the NKG2D transmembrane region, and its amino acid sequence is shown in SEQ ID NO.29.
  • nucleotide coding sequence is shown in SEQ ID NO.30.
  • the intracellular co-stimulatory signal region is the NKG2D intracellular co-stimulatory signal region, and its amino acid sequence is shown in SEQ ID NO.31.
  • nucleotide coding sequence is shown in SEQ ID NO.32.
  • sequence of the signal receptor of this embodiment is shown as SEQ ID NO.33, SEQ ID NO.34 or SEQ ID NO.35.
  • nucleotide coding sequence is shown as SEQ ID NO.36, SEQ ID NO.37 or SEQ ID NO.38.
  • amino acid and nucleotide sequences are as follows:
  • Epidermal growth factor receptor is highly expressed in some epithelial tumor cells and can be used as an anti-tumor target. Based on the CART cells that recognize the tumor target antigen EGFR, this experimental example overexpresses the receptor for VEGFR1 signal (the signal receptor described in Example 1) and EGFR-VEGFR1 CART cells, converting the VEGFR1 molecular signal into a T cell activation signal to enhance the anti-tumor activity of CART cells and the secretion of anti-tumor cytokines.
  • VEGFR1 signal the signal receptor described in Example 1
  • EGFR-VEGFR1 CART cells the receptor for VEGFR1 signal
  • T cell activation signal to enhance the anti-tumor activity of CART cells and the secretion of anti-tumor cytokines.
  • the gene fragment SEQ ID NO.12 was synthesized by whole gene synthesis method, and the synthesized gene fragment molecular clone was inserted into the pwpxld lentiviral core plasmid expressing EGFR CART to construct
  • the constructed lentiviral packaging core plasmid expressing the EGFR-VEGFR1 CART gene sequence, the lentiviral auxiliary plasmids PMD2.0G and PSPAX2 were transfected using lipo2000 transfection reagent (purchased from Invitrogen, catalog number: 11668019) were co-transfected with 293T virus packaging cells derived from human embryonic kidney. After the virus supernatant was collected and concentrated, it was infected with Jurkat cells (Jurkat cells are immortalized cell lines of lymphocytoma, which are transformed from lymphocytes. Their biological functions are similar to normal T lymphocytes to a certain extent.
  • Fluorescently labeled recombinant proteins purchased from Nearshore Protein Recombinant Human EGFR (C-6His), catalog number: CI61; Recombinant Human VEGFR1 (C-Fc) catalog number: CJ93) were used to detect the expression of EGFR CAR molecules and VEGFR1 signal transduction receptors on the surface of Jurkat cell membranes by flow cytometry. (Note: Since the transfection and infection steps will be used in subsequent experimental examples, and the process and reagents are the same as here, the subsequent similar experimental steps are described briefly.)
  • DMEM medium containing 10% FBS and without penicillin-streptomycin
  • the steps for lentivirus concentration are as follows:
  • the 293T culture supernatant was collected and filtered with a 0.45 ⁇ m filter, and the concentration reagent Lenti-X TM Concentrator (purchased from Takara, catalog number: 631232) was mixed with the virus supernatant in a volume ratio of 1:3. After inversion and mixing, it was incubated at 4°C overnight. After the mixture was centrifuged at 1500g for 45 minutes at 4°C, the supernatant was discarded, and the precipitate at the bottom of the centrifuge tube was resuspended with PBS and stored in a -80°C refrigerator.
  • the process of lentivirus infection of Jurkat is as follows:
  • the cells were centrifuged at 1500 rpm for 3 min, the supernatant was removed and replaced with 2 mL of 1640 complete medium, resuspended, and placed in a cell culture incubator for culture;
  • the experimental results based on the Jurkat cell line have shown that both EGFR CAR and VEGFR1 signal receptors can be highly expressed on the cell membrane.
  • the prepared EGFR CAR and EGFR-VEGFR1 CART lentiviruses were further used to infect T lymphocytes from human peripheral blood. After 48 hours of continuous culture, fluorescently labeled recombinant proteins (purchased from Nearshore Protein Recombinant Human EGFR (C-6His), Catalog No.: CI61; Recombinant Human VEGFR1 (C-Fc) Catalog No.: CJ93) were used to flow cytometry to detect the expression of EGFR CAR molecules and VEGFR1 signal transduction receptors on the T cell membrane.
  • the process of virus infection of T cells is as follows: (Note: Since the transfection and infection steps will be used in subsequent experimental examples, and the process and reagents are the same as here, the description of subsequent similar experimental steps is simplified.)
  • PBMCs peripheral blood mononuclear cells
  • the concentrated virus and the corresponding amount of T cells are mixed, they are added to a 12-well plate, the plate is sealed, and centrifuged at 1000g, 32°C, for 2 h. After the centrifugation is completed, the plate is removed and placed in a cell culture incubator for culture;
  • T cells were collected, washed once with PBS, and fluorescent antibodies or proteins were added. After incubation at 4°C for 30 min, the cells were resuspended in 1 ml PBS and centrifuged. The supernatant was removed and resuspended in 0.5 ml PBS for detection on a flow cytometer.
  • the present invention successfully constructed CAR-T cells that can simultaneously express EGFR CAR and VEGFR1 signal receptors in Jurkat cell lines and human T lymphocytes.
  • Experimental Example 2 The enhancing effect of expressing signal receptors on the anti-tumor activity of EGFR CAR-T cells.
  • EGFR CAR-T cells Most human lung cancer cell lines highly express EGFR antigens and can be used as target cells for EGFR CAR-T cells. In clinical practice, some patients also highly express VEGFR1 molecules, which can inhibit CAR-T immune responses. In this experimental case, EGFR CAR-T cells were modified with VEGFR1 signal receptors, which recognized VEGFR1 molecules and provided costimulatory signals for the activation of CAR-T cells, thereby enhancing the anti-tumor activity of CAR-T cells.
  • the detection process is as follows:
  • H1975 cells from a 100 mm culture dish, remove the culture medium containing 10% fetal bovine serum, Add 1ml PBS to rinse the cells once, remove the PBS and add 1ml 0.25% trypsin, incubate at 37°C for 1 minute, neutralize the digestion system with 5ml serum-containing culture medium, and centrifuge the cell suspension at 1500rpm for 3 minutes.
  • Count the cells take 1 ⁇ 10 6 cells into the flow tube, wash once with PBS, and use 1ul anti-human EGFR antibody (purchased from biolegend, catalog number: 352905) and 1ul anti-human VEGFR1 antibody (purchased from R&D Systems, catalog number: FAB321P) respectively, incubate at 4°C in the dark for 30 minutes. Wash the cells once with PBS and put them on the machine to detect the antigen expression of H1975 cells by flow cytometry.
  • the prepared EGFR-VEGFR1CART and EGFR CAR-T (prepared according to the method of lentiviral infection of T cells in Experimental Example 1, which has been described in detail above) were co-cultured with target cells H1975, and the secretion of T cell effector cytokines in the culture supernatant was detected.
  • T cells killing tumor cells a large number of inflammatory cytokines are released, such as interferon- ⁇ , tumor necrosis factor- ⁇ , interleukin-2, etc.
  • the release of these factors is positively correlated with the activity of T cells in killing tumors. Therefore, the cytokine content in the supernatant of co-culture of T cells and target cells is often detected to reflect the killing activity of T cells.
  • T cells that were not transfected with the virus were used as the control group T cells (UTD)
  • T cells that overexpressed EGFR CAR molecules were used as experimental group 1 (EGFR CAR-T)
  • T cells that overexpressed EGFR CAR molecules and VEGFR1 signal receptors were used as experimental group 2 (EGFR-VEGFR1CAR-T).
  • On-machine detection The wavelengths of 450 nm and 570 nm were selected as the absorbance values for detection on the ELISA instrument, and the data were obtained for analysis and statistics.
  • RTCA Real-Time Cell Analysis
  • This experimental example directly reflects the anti-tumor effect of T cells by monitoring the killing and lysis activity of different groups of T cells on target cells.
  • the RTCA instrument can monitor the changes in the impedance strength formed by the adherent cells and the metal microporous plate. The more adherent cells there are, the greater the impedance detected by the instrument, that is, the greater the cell index (cell index, CI) displayed by the instrument.
  • the cell index cell index
  • the prepared EGFR-VEGFR1CART and EGFR CAR-T were co-cultured with the target cells H1975, respectively. According to the effector-target ratio of 0.5:1, 100ul of the corresponding number of CAR-T or Control-T cells (CON-T) were added to each well and co-cultured with the target cells;
  • Flow cytometry was used to detect the antigen expression of H1975 cells.
  • the target cells H1975 cells
  • the cells highly express EGFR and VEGFR1 molecules and can be used as tumor target cells in this experimental example.
  • EGFR-VEGFR1CART and EGFR CAR-T were co-cultured with target cells H1975, and the secretion of T cell effector cytokines in the culture supernatant was detected
  • both EGFR-VEGFR1CART and EGFR CAR-T cells can effectively kill target cells.
  • EGFR CAR-T cells that overexpress VEGFR1 signal receptors have a greater change in cell index than conventional EGFR CAR-T cells, that is, they have a stronger ability to kill target tumor cells, indicating that VEGFR1 signal receptors can enhance the tumor killing activity of EGFR CAR-T cells.
  • GPC3 is mainly expressed in embryonic and fetal tissues and organs, and is significantly reduced in normal tissues and organs of adults.
  • GPC3 is highly expressed in liver cancer, kidney cancer, gastric cancer, colon cancer and other tissues, and can serve as an ideal target for immunotherapy.
  • the VEGFR1 signal transduction receptor is overexpressed on the CART cells targeting GPC3 to enhance the anti-tumor activity of such CART cells and the secretion of anti-tumor cytokines.
  • the experimental method is similar to that of Experimental Examples 1 and 2, and the specific implementation content is as follows:
  • the gene fragment SEQ ID NO.12 was synthesized by the whole gene synthesis method, and the synthesized gene fragment was cloned into the pwpxld lentiviral core plasmid expressing GPC3 CART to construct the GPC3-VEGFR1 CART plasmid.
  • APC fluorescently labeled human GPC-3 protein (Cat. No. C414) and PE-Cy7 labeled human VEGFR1 protein (Cat. No. CJ93) were purchased from Shanghai Jinan Protein Technology Co., Ltd., and the expression of GPC3 CAR and VEGFR1 receptors were detected by flow cytometry. The specific steps are as follows:
  • the plasmid was transfected into 293T cells, and the prepared GPC3-VEGFR1CART lentivirus was used to infect T lymphocytes from human peripheral blood. After continued culture for 48 hours, the expression of GPC3 CAR molecules and VEGFR1 signal transduction receptors on the T cell membrane was detected by flow cytometry. The flow staining and detection experimental procedures were the same as the second step above.
  • FIG8 A schematic diagram of the gene sequence structure of the viral vector plasmid is shown in FIG8 .
  • VEGFR1 signal receptor can be expressed in CAR-T cells with different target antigens and has wide applicability.
  • the detection process is as follows:
  • Hep3B-V1 cells Take Hep3B-V1 cells from a 100mm culture dish, digest with 0.25% trypsin, incubate at 37°C for 1 minute, and centrifuge the cell suspension at 1500rpm for 3 minutes. Take 1 ⁇ 10 6 cells into a flow tube, wash once with PBS, and use anti-human GPC3 antibody (primary antibody purchased from abcam, catalog number: ab95363; secondary antibody purchased from biolegend, catalog number: 406421) and anti-human VEGFR1 antibody (purchased from R&D Systems, catalog number: FAB321P) respectively. After adding antibodies, incubate at 4°C in the dark for 30 minutes. Wash the cells once with PBS and put them on the machine to detect the antigen expression of Hep3B cells by flow cytometry.
  • anti-human GPC3 antibody primary antibody purchased from abcam, catalog number: ab95363; secondary antibody purchased from biolegend, catalog number: 406421
  • anti-human VEGFR1 antibody purchased from R&D Systems
  • T cells that were not transfected with viruses were used as control T cells (Control-T)
  • T cells that overexpressed GPC3 CAR molecules were used as experimental group 1 (GPC3 CAR-T)
  • T cells that overexpressed GPC3 CAR molecules and VEGFR1 signal receptors were used as experimental group 2 (GPC3-VEGFR1 CAR-T).
  • tumor cells 5 ⁇ 10 3 tumor cells were plated in each well of a 96-well plate, and three groups of T cells, CON-T, GPC3 CAR-T, and GPC3-VEGFR1 CAR-T, were counted at the same time.
  • the ELISA detection process is as described in Experimental Example 2.
  • RTCA real-time cell analysis
  • T cells without virus transfection were used as control group T cells (CON-T)
  • T cells overexpressing GPC3 CAR molecules were used as experimental group 1 (GPC3 CAR-T)
  • T cells overexpressing GPC3 CAR molecules and VEGFR1 signal receptors were used as experimental group 2 (GPC3-VEGFR1 CAR-T).
  • the experimental operation process was the same as that of Experimental Example 2.
  • the target cell Hep3B cell line highly expresses GPC3 and VEGFR1 molecules and can be used as tumor target cells in this experimental example.
  • this experimental example uses nano single-domain antibodies as the molecular recognition region of the VEGFR1 signal receptor to construct a signal receptor.
  • two nano single-domain antibodies were selected as the molecular recognition regions of the signal receptors, respectively, as described in Example 2 and Example 3, respectively, combined with the signal peptide, transmembrane region and intracellular co-stimulatory signal region, and the gene fragments SEQ ID NO.22 and SEQ ID NO.26 were synthesized by the whole gene synthesis method, respectively, and the synthesized gene fragments were cloned into the lentiviral core plasmid pwpxld to construct the receptor plasmids targeting VEGFR1, named VE5 and VE6 respectively. Plasmids were transfected into 293T cells to prepare VE5 and VE6 lentiviral supernatants, and the process of infecting T cells after virus concentration was the same as in Experimental Example 1.
  • VEGFR1 signal receptor on the membrane of T lymphocytes.
  • the viral core plasmid carries a FLAG protein tag, which can be expressed simultaneously with the signal receptor.
  • APC fluorescently labeled anti-FLAG antibody is used to stain T cells, and flow cytometry is used to detect the expression of VEGFR1 receptors. The specific steps are as follows:
  • VEGFR1 signal receptors on T lymphocyte membranes are shown in FIG15 . It can be seen that the expression positive rates of VEGFR1 signal receptor molecules VE5 and VE6 expressed on the surface of T cell membranes are 8.9% and 5.73% respectively.
  • VEGFR1 signal receptor composed of nano single-domain antibodies can also be expressed on the surface of T cells and has wide application.
  • T cells When T cells recognize tumor cells and become activated, they will proliferate to form T cell clones.
  • the size of the clones can be used to preliminarily determine the strength of T cell activation. The larger the clones, the stronger the activation signal and the stronger the anti-tumor immune effect activity.
  • the experimental process is similar to the EGFR CAR-T co-culture process in Experimental Example 2:
  • T cells that are not transfected with viruses are used as control group T cells (CON-T)
  • T cells that overexpress VE5 signal receptor molecules are used as experimental group 1 (VE5-T)
  • T cells that overexpress VE6 signal receptor molecules are used as experimental group 2 (VE5-T).
  • Group 2 (VE6-T) T cells that overexpress VE6 signal receptor molecules
  • tumor cells 5 ⁇ 10 3 tumor cells were plated in each well of a 96-well plate, and three groups of T cells, CON-T, VE5-T, and VE6-T, were counted at the same time.
  • the plates were plated at a ratio of T cell number to tumor cell number of 2:1 and placed in a 37°C incubator for co-culture. After 24 hours, the formation of cell clones was photographed under a light microscope.
  • the detection results are shown in Figure 16, and the target cell SKOV3-V1 cell highly expresses VEGFR1 molecules and can be used as the tumor target cell in this experimental example.
  • the signal receptors of the present invention that overexpress VEGFR1 in CAR-T cells targeting different target antigens can enhance the anti-tumor activity of CAR-T cells.
  • the signal receptors based on nano single-domain antibodies can also enhance the anti-tumor activity of CAR-T cells.
  • the receptor and the cell containing the signal receptor can be used to prepare anti-tumor drugs and have a good application prospect in the treatment of tumors.

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Abstract

本发明属于肿瘤免疫治疗技术领域，具体涉及一种用于靶向肿瘤微环境的信号感受器和用于靶向治疗肿瘤的细胞。该信号感受器包括信号肽、胞外分子识别区、胞外铰链区、跨膜区和胞内共刺激信号区；该信号感受器可以特异性识别肿瘤微环境中的VEGFR等分子，将微环境中的抑制信号转换为免疫细胞内部的共刺激信号，参与活化免疫细胞，增强免疫细胞抗肿瘤活性。本发明还提供了表达该信号感受器的细胞。本发明的细胞可用于制备抗肿瘤药物，在肿瘤的治疗中具有很好的应用前景。

Description

一种用于靶向肿瘤微环境的信号感受器和用于靶向治疗肿瘤的细胞 技术领域
本发明属于肿瘤免疫治疗技术领域，具体涉及一种用于靶向肿瘤微环境的信号感受器和用于靶向治疗肿瘤的细胞。
背景技术
目前多种免疫细胞疗法已被运用于抗肿瘤治疗并取得一定疗效。从上世纪八十年代年运用淋巴因子激活的非特异性杀伤细胞(Lymphokine-activated killer cells，LAK)治疗黑色素瘤，到随后利用特定细胞因子激活的杀伤细胞(Cytokine-induced killer cells，CIK)治疗B细胞淋巴瘤，随后，美国国立卫生研究院的Rosenberg教授团队应用特异性更高的肿瘤浸润T细胞(Tumor-infiltrating lymphocytes，TIL)进行过继抗肿瘤细胞治疗。近年来，基于基因工程技术改造的T细胞受体(T cell receptor，TCR)或嵌合抗原受体(Chimericantigen receptor,CAR)T细胞免疫疗法成为肿瘤过继免疫治疗的热点，此外，NK细胞作为固有免疫系统的重要组成，能够比T细胞更快速、直接地发挥抗肿瘤作用，已有团队运用基因工程技术对NK细胞进行改造并用于抗肿瘤过继疗法。
VEGFR是高度特异性的跨膜受体，在肿瘤新生血管内皮及部分肿瘤细胞膜高表达。VEGFR家族主要包括三个酪氨酸激酶受体：VEGFR-1、VEGFR-2和VEGFR-3。其配体，血管表皮生长因子(VEGF)，是一种由细胞产生的刺激血管生成的信号转导蛋白，主要包括6种分泌型糖蛋白：VEGF-A、VEGF-B、VEGF-C、VEGF-D、VEGF-E和胎盘生长因子(PLGF)。VEGF与受体(Vascular endothelial growth factor receptor，VEGFR)相互作用，引起信号级联反应，介导血管生成等生理或病理过程。
肿瘤微环境中，VEGF-VEGFR信号通路的存在的会导致免疫细胞过继疗法的效果下降。一方面，肿瘤及基质细胞表达的血管表皮生长因子(Vascular endothelial growth factor，VEGF)及其受体等抑制性分子，产生免疫抑制微环境，可损伤回输的免疫细胞的抗肿瘤作用。另一方面，肿瘤微环境中的血管高表达VEGFR1、VEGFR2等分子，导致异常血管增生，免疫细胞进入肿瘤障碍以及肿瘤微环境乏氧，均可抑制免疫细胞抗肿瘤效果。
然而，当前尚无针对VEGF-VEGFR信号通路分子以优化改造免疫细胞的解决方案。本领域亟需发展新的免疫细胞或修饰免疫细胞的方法，使得免疫细胞克服上述VEGF-VEGFR信号通路的活性抑制现象，提升免疫细胞抗肿瘤活性，从而实现更好的抗肿瘤效果。
发明内容
针对现有技术的问题，本发明提供一种用于靶向肿瘤微环境的信号感受器和用于靶向治疗肿瘤的细胞，目的在于实现免疫细胞靶向VEGF-VEGFR信号通路并且在达到肿瘤微环境后激活的功能，为肿瘤的治疗提供新的临床用药选择。
一种用于靶向肿瘤微环境的信号感受器，它包括信号肽、胞外分子识别区、胞外铰链区、跨膜区和胞内共刺激信号区；
所述信号肽为与白介素2(Interleukin 2，IL-2)信号肽、白细胞分化抗原8(CD8)信号肽、粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor，GM-CSF)信号肽或集落刺激因子2受体α亚基(Colony Stimulating Factor 2 Receptor Subunit Alpha,CSF2RA)信号肽中序列同源性90％以上的蛋白结构域中的至少一种；
所述胞外分子识别区是与VEGF或VEGFR结合的单克隆抗体单链可变区、纳米单域抗体识别区、受体或配体中的至少一种；
所述胞外铰链区为与白细胞分化抗原8胞外铰链区、白细胞分化抗原28胞外铰链区、TRAIL胞外铰链区、CD16胞外铰链区、NKp30胞外铰链区、NKG2C胞外铰链区、NKG2D胞外铰链区、2B4胞外铰链区或DNAM-1胞外铰链区序列同源性90％以上的蛋白结构域中的至少一种；
所述跨膜区为与白细胞分化抗原8跨膜区、白细胞分化抗原28跨膜区、TRAIL跨膜区、CD16跨膜区、NKp30跨膜区、NKG2C跨膜区、NKG2D跨膜区、2B4跨膜区或DNAM-1跨膜区序列同源性90％以上的蛋白结构域中的至少一种；
所述胞内共刺激信号区为与白细胞分化抗原28、肿瘤坏死因子受体超家族成员4(TNFRSF4)、肿瘤坏死因子受体超家族成员9(TNFRSF9)、淋巴细胞特异性蛋白酪氨酸激酶(LCK)、诱导性T细胞共刺激分子(ICOS)、CD40共刺激信号区、CD27共刺激信号区、DAP10共刺激信号区、DAP12共刺激信号区、TRAIL共刺激信号区、CD16共刺激信号区、NKp30共刺激信号区、NKG2C共刺激信号区、NKG2D共刺激信号区、2B4共刺激信号区或DNAM-1共刺 激信号区序列同源性90％以上的蛋白结构域的胞内结构域中的至少一种。
优选的，所述VEGF为VEGF-A、VEGF-B、VEGF-C、VEGF-D、VEGF-E或胎盘生长因子中的至少一种；
所述VEGFR为VEGFR1、VEGFR2或VEGFR3中的至少一种。
优选的，它的组成按照如下任意一种方式选择：
组成方式一：
所述信号肽为集落刺激因子2受体α亚基信号肽，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与集落刺激因子2受体α亚基信号肽功能相同或相似的肽；
所述胞外分子识别区为识别VEGFR1的单克隆抗体单链可变区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与识别VEGFR1的单克隆抗体单链可变区功能相同或相似的蛋白；
所述胞外铰链区为白细胞分化抗原28胞外铰链区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原28胞外铰链区功能相同或相似的蛋白；
所述跨膜区为白细胞分化抗原28跨膜区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原28跨膜区功能相同或相似的蛋白；
所述胞内共刺激信号区为白细胞分化抗原28的胞内结构域，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原28的胞内结构域功能相同或相似的蛋白；
组成方式二：
所述信号肽为白细胞分化抗原8信号肽，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原8信号肽功能相同或相似的肽；
所述胞外分子识别区为识别VEGFR1的纳米单域抗体识别区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与识别VEGFR1的纳米单域抗体识别区功能相同或相似的蛋白；
所述胞外铰链区为白细胞分化抗原8跨膜区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原8胞外铰链区功能相同或相似的蛋白；
所述跨膜区为白细胞分化抗原8跨膜区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原8跨膜区功能相同或相似的蛋白；
所述胞内共刺激信号区为4-1BB胞内共刺激信号区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与4-1BB胞内共刺激信号区功能相同或相似的蛋白；
组成方式三：
所述信号肽为白细胞分化抗原8信号肽，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原8信号肽功能相同或相似的肽；
所述胞外分子识别区为识别VEGFR1的单克隆抗体单链可变区或纳米单域抗体识别区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与识别VEGFR1的单克隆抗体单链可变区或纳米单域抗体识别区功能相同或相似的蛋白；
胞外铰链区为白细胞分化抗原8胞外铰链区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原8胞外铰链区功能相同或相似的蛋白；
所述跨膜区为NKG2D跨膜区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与NKG2D跨膜区功能相同或相似的蛋白；
所述胞内共刺激信号区为NKG2D胞内共刺激信号区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与NKG2D胞内共刺激信号区功能相同或相似的蛋白。
组成方式四：
所述信号肽为集落刺激因子2受体α亚基信号肽，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与集落刺激因子2受体α亚基信号肽功能相同或相似的肽；
所述胞外分子识别区为识别VEGFR2的单克隆抗体单链可变区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与识别VEGFR1的单克隆抗体单链可变区功能相同或相似的蛋白；
所述胞外铰链区为白细胞分化抗原28胞外铰链区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原28胞外铰链区功能相同或相似的蛋白；
所述跨膜区为白细胞分化抗原28跨膜区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原28跨膜区功能相同或相似的蛋白；
所述胞内共刺激信号区为白细胞分化抗原28的胞内结构域，或经过取代 和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原28的胞内结构域功能相同或相似的蛋白；
组成方式五：
所述信号肽为集落刺激因子2受体α亚基信号肽，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与集落刺激因子2受体α亚基信号肽功能相同或相似的肽；
所述胞外分子识别区为识别VEGFR1的单克隆抗体单链可变区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与识别VEGFR1的单克隆抗体单链可变区功能相同或相似的蛋白；
所述胞外铰链区为白细胞分化抗原8胞外铰链区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原8胞外铰链区功能相同或相似的蛋白；
所述跨膜区为白细胞分化抗原8跨膜区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原8跨膜区功能相同或相似的蛋白；
所述胞内共刺激信号区为4-1BB的胞内结构域，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与4-1BB的胞内结构域功能相同或相似的蛋白。
优选的，所述集落刺激因子2受体α亚基信号肽的氨基酸序列如SEQ ID NO.1所示。
优选的，所述集落刺激因子2受体α亚基信号肽的编码核苷酸序列如SEQ ID NO.2所示。
优选的，所述识别VEGFR1的单克隆抗体单链可变区的氨基酸序列如SEQ ID No.3所示。
优选的，所述识别VEGFR1的编码核苷酸序列如SEQ IDNo.4所示。
优选的，所述白细胞分化抗原28胞外铰链区的氨基酸序列如SEQ ID No.5所示。
优选的，所述白细胞分化抗原28胞外铰链区的编码核苷酸序列如SEQ ID No.6所示。
优选的，所述白细胞分化抗原28跨膜区的氨基酸序列如SEQ ID No.7所示。
优选的，所述白细胞分化抗原28跨膜区的编码核苷酸序列如SEQ ID No.8所示。
优选的，所述白细胞分化抗原28的胞内结构域的氨基酸序列如SEQ ID No.9所示。
优选的，所述白细胞分化抗原28的胞内结构域的编码核苷酸序列如SEQ ID No.10所示。
优选的，所述白细胞分化抗原8信号肽的氨基酸序列如SEQ ID NO.13所示。
优选的，所述白细胞分化抗原8信号肽的编码核苷酸序列如SEQ ID NO.14所示。
优选的，所述VEGFR1的纳米单域抗体识别区的氨基酸序列如SEQ ID NO.15或SEQ ID NO.23所示。
优选的，所述VEGFR1的纳米单域抗体识别区的编码核苷酸序列如SEQ ID NO.16或SEQ ID NO.24所示。
优选的，所述白细胞分化抗原8跨膜区的氨基酸序列如SEQ ID NO.17所示。
优选的，所述白细胞分化抗原8跨膜区的编码核苷酸序列如SEQ ID NO.18所示。
优选的，所述4-1BB胞内共刺激信号区的氨基酸序列如SEQ ID NO.19所示。
优选的，所述4-1BB胞内共刺激信号区的编码核苷酸序列如SEQ ID NO.20所示。
优选的，所述白细胞分化抗原8胞外铰链区的氨基酸序列如SEQ ID NO.27所示。
优选的，所述白细胞分化抗原8胞外铰链区的编码核苷酸序列如SEQ ID NO.28所示。
优选的，所述NKG2D跨膜区的氨基酸序列如SEQ ID NO.29所示。
优选的，所述NKG2D跨膜区的编码核苷酸序列如SEQ ID NO.30所示。
优选的，所述NKG2D胞内共刺激信号区的氨基酸序列如SEQ ID NO.31所示。
优选的，所述NKG2D胞内共刺激信号区的编码核苷酸序列如SEQ ID NO.32所示。
优选的，所述信号感受器的氨基酸序列如SEQ ID NO.11、SEQ ID NO.21、SEQ ID NO.25、SEQ ID NO.33、SEQ ID NO.34或SEQ ID NO.35所示。
优选的，所述信号感受器的编码核苷酸序列如SEQ ID NO.12、SEQ ID NO.22、SEQ ID NO.26、SEQ ID NO.36、SEQ ID NO.37或SEQ ID NO.38所示。
本发明还提供一种表达载体，它能够表达上述信号感受器。
优选的，它是重组质粒载体、重组病毒或它们的组合。
优选的，所述重组病毒为慢病毒载体或逆转录病毒载体。
本发明还提供一种用于靶向治疗肿瘤的细胞，它由上述表达载体和宿主细胞构成。
优选的，所述宿主细胞选自αβT细胞、NKT细胞或γδT细胞、肿瘤浸润淋巴细胞、TCR修饰的T细胞、嵌合抗体受体T细胞或自然杀伤细胞。
本发明还提供一种用于治疗肿瘤的药物，它是以上述用于靶向治疗肿瘤的细胞作为活性成分，加上药学上可接受的辅料或辅助性成分制成的。
优选的，所述肿瘤为肺癌、肝癌、淋巴瘤、结肠癌、直肠癌、乳腺癌、卵巢癌、宫颈癌、胃癌、胆管癌、食管癌、肾癌、神经胶质瘤、黑色素瘤、胰腺癌、多发性骨髓瘤或前列腺癌。
本发明中，术语“结构域”是指蛋白质中空间上可明显区分，又具有各自功能的区段；结构域中部分氨基酸残基的改变(替换、缺失和/或增加)并不会明显引起结构域功能的变化。因此应当知晓：本发明涉及的抗原受体，即使发生氨基酸残基改变，只要涉及的结构域功能无明显变化，均在本发明的范围内。
本发明提供一种人工构建的信号感受器及含有该信号感受器的宿主免疫细胞。该信号感受器可以特异性识别肿瘤微环境中的VEGFR等免疫抑制性分子的一种或多种，将微环境中的抑制信号转换为免疫细胞内部的共刺激信号，参与活化免疫细胞，增强免疫细胞抗肿瘤活性。本发明在肿瘤的治疗中具有很好的应用前景。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为本发明的信号感受器的结构示意图；
图2为实验例1中构建的病毒载体质粒的基因结构示意图；
图3为实验例1中EGFR CAR分子及VEGFR1信号感受器在Jurkat细胞膜表面表达情况；
图4为实验例1中EGFR CAR分子及VEGFR1信号感受器在人T淋巴细胞膜表达情况；
图5为实验例2中肿瘤细胞靶抗原EGFR的表达及VEGFR1的表达情况；
图6为实验例2中EGFR-VEGFR1CART及EGFR CAR-T分别与靶细胞H1975共培养后T细胞效应细胞因子分泌情况；
图7为实验例2中过表达信号感受器的CAR-T细胞对靶细胞的直接杀伤活性实验结果；
图8为实验例3中构建的病毒载体质粒的基因结构示意图；
图9为实验例3中GPC3 CAR分子及VEGFR1信号感受器在Jurkat细胞系的膜表达情况；
图10为实验例3中GPC3 CAR分子及VEGFR1信号感受器在人T淋巴细胞膜表达情况；
图11为实验例4中肿瘤细胞靶抗原GPC3的表达及VEGFR1的表达情况；
图12为实验例4中不同T细胞与肿瘤靶细胞共培养后T细胞效应细胞因子分泌情况；
图13为实验例4中过表达信号感受器的CAR-T细胞对靶细胞的直接杀伤活性实验结果；
图14为实验例5中构建的病毒载体质粒的基因结构示意图；
图15为实验例5中VEGFR1信号感受器在T淋巴细胞膜表达情况；
图16为实验例6中肿瘤细胞VEGFR1的表达情况；
图17为实验例6中不同T细胞与肿瘤靶细胞共培养条件下免疫细胞克隆团形成情况；
图18为实验例6中不同T细胞与肿瘤靶细胞共培养后T细胞效应细胞因子分泌情况。
具体实施方式
实施例1
本实施例提供的信号感受器如图1所示，由信号肽、胞外分子识别区、 胞外铰链区、跨膜区和胞内共刺激信号区组成。
所述信号肽为集落刺激因子2受体α亚基信号肽，其氨基酸序列如SEQ ID NO.1所示。
其核苷酸编码序列如SEQ ID NO.2所示。
所述胞外分子识别区为识别VEGFR1的单克隆抗体单链可变区，其氨基酸序列如SEQ ID NO.3所示。
其核苷酸编码序列如SEQ ID NO.4所示。
所述胞外铰链区为白细胞分化抗原28胞外铰链区，其氨基酸序列如SEQ ID No.5所示。
其核苷酸编码序列如SEQ ID NO.6所示。
所述跨膜区为白细胞分化抗原28跨膜区，其氨基酸序列如SEQ ID NO.7所示。
其核苷酸编码序列如SEQ ID NO.8所示。
所述胞内共刺激信号区为白细胞分化抗原28的胞内结构域，其氨基酸序列如SEQ ID NO.9所示。
其核苷酸编码序列如SEQ ID NO.10所示。
本实施例的信号感受器的序列如SEQ ID NO.11所示。
其核苷酸编码序列如SEQ ID NO.12所示。
实施例2
本实施例提供的信号感受器由信号肽、胞外分子识别区、胞外铰链区、跨膜区和胞内共刺激信号区组成。
所述信号肽为白细胞分化抗原8信号肽，其氨基酸序列如SEQ ID NO.13所示。
其核苷酸编码序列如SEQ ID NO.14所示。
所述胞外分子识别区为识别VEGFR1的纳米单域抗体识别区，其氨基酸序列如SEQ ID NO.15所示。
其核苷酸编码序列如SEQ ID NO.16所示。
所述胞外铰链区为白细胞分化抗原8胞外铰链区，其氨基酸序列如SEQ ID NO.27所示。
其核苷酸编码序列如SEQ ID NO.28所示。
所述跨膜区为白细胞分化抗原8跨膜区，其氨基酸序列如SEQ ID NO.17 所示。
其核苷酸编码序列如SEQ ID NO.18所示。
所述胞内共刺激信号区为4-1BB胞内共刺激信号区，其氨基酸序列如SEQ ID NO.19所示。
其核苷酸编码序列如SEQ ID NO.20所示。
本实施例的信号感受器的序列如SEQ ID NO.21所示。
其核苷酸编码序列如SEQ ID NO.22所示。
实施例3
本实施例提供的信号感受器由信号肽、胞外分子识别区、胞外铰链区、跨膜区和胞内共刺激信号区组成。
所述信号肽为白细胞分化抗原8信号肽，其氨基酸序列如SEQ ID NO.13所示。
其核苷酸编码序列如SEQ ID NO.14所示。
所述胞外分子识别区为识别VEGFR1的纳米单域抗体识别区，其氨基酸序列如SEQ ID NO.23所示。
其核苷酸编码序列如SEQ ID NO.24所示。
所述胞外铰链区为白细胞分化抗原8胞外铰链区，其氨基酸序列如SEQ ID NO.27所示。
其核苷酸编码序列如SEQ ID NO.28所示。
所述跨膜区为白细胞分化抗原8跨膜区，其氨基酸序列如SEQ ID NO.17所示。
其核苷酸编码序列如SEQ ID NO.18所示。
所述胞内共刺激信号区为4-1BB胞内共刺激信号区，其氨基酸序列如SEQ ID NO.19所示。
其核苷酸编码序列如SEQ ID NO.20所示。
本实施例的信号感受器的序列如SEQ ID NO.25所示。
其核苷酸编码序列如SEQ ID NO.26所示。
实施例4
本实施例提供的信号感受器由信号肽、胞外分子识别区、胞外铰链区、跨膜区和胞内共刺激信号区组成。
所述信号肽为白细胞分化抗原8信号肽，其氨基酸序列如SEQ ID NO.13所示。
其核苷酸编码序列如SEQ ID NO.14所示。
所述胞外分子识别区为识别VEGFR1的单克隆抗体单链可变区，其氨基酸序列如SEQ ID NO.3所示。
其核苷酸编码序列如SEQ ID NO.4所示。
所述胞外分子识别区为识别VEGFR1的纳米单域抗体识别区，其氨基酸序列如SEQ ID NO.15或SEQ ID NO.23所示。
其核苷酸编码序列如SEQ ID NO.16或SEQ ID NO.24所示。
胞外区铰链区为白细胞分化抗原8胞外铰链区，其氨基酸序列如SEQ ID NO.27所示。
其核苷酸编码序列如SEQ ID NO.28所示。
所述跨膜区为NKG2D跨膜区，其氨基酸序列如SEQ ID NO.29所示。
其核苷酸编码序列如SEQ ID NO.30所示。
所述胞内共刺激信号区为NKG2D胞内共刺激信号区，其氨基酸序列如SEQ ID NO.31所示。
其核苷酸编码序列如SEQ ID NO.32所示。
本实施例的信号感受器的序列如SEQ ID NO.33、SEQ ID NO.34或SEQ ID NO.35所示。
其核苷酸编码序列如SEQ ID NO.36、SEQ ID NO.37或SEQ ID NO.38所示。
上述实施例中，氨基酸和核苷酸序列具体如下：











下面通过实验对本发明的技术方案做进一步的说明。
实验例1表达信号转换器的靶向EGFR的CAR-T细胞的制备
表皮生长因子受体(EGFR)在部分上皮来源的肿瘤细胞高表达，可以作为抗肿瘤靶点。本实验例在识别肿瘤靶抗原EGFR的CART细胞基础上，过表达针对VEGFR1信号的感受器(实施例1所述的信号感受器)，及EGFR-VEGFR1 CART细胞，将VEGFR1分子信号，转换为T细胞激活信号，以达到增强CART细胞抗肿瘤活性及抗肿瘤细胞因子分泌增强的作用。具体实施内容如下：
一、实验方法
1.构建病毒载体质粒。
通过全基因合成方法，合成基因片段SEQ ID NO.12，将合成的基因片段分子克隆插入表达EGFR CART的pwpxld慢病毒核心质粒中，构建
EGFR-VEGFR1 CART质粒。
2.检测EGFR CAR分子及VEGFR1信号感受器在T细胞系的膜表达。
将构建好的表达EGFR-VEGFR1 CART基因序列的慢病毒包装核心质粒，与慢病毒辅助质粒PMD2.0G及PSPAX2使用lipo2000转染试剂(购自 invitrogen，货号：11668019)共同转染人胚肾来源的293T病毒包装细胞，收取病毒上清浓缩后，感染Jurkat细胞(Jurkat细胞为淋巴细胞瘤的永生化的细胞系，其由淋巴细胞转化而来，生物学功能与正常T淋巴细胞具有一定程度相似性，较原代的T淋巴细胞更容易培养，因此常用于初步测试基因工程过表达分子在淋巴细胞表达情况)。分别用荧光标记的重组蛋白(购于近岸蛋白Recombinant Human EGFR(C-6His)，货号：CI61；Recombinant Human VEGFR1(C-Fc)货号:CJ93)，流式细胞数术检测Jurkat细胞膜表面EGFR CAR分子及VEGFR1信号转换感受器的表达。(注意：因后续实验例中会使用转染及感染步骤，且与此处流程及试剂相同，后续类似实验步骤描述从简。)
具体地，质粒转染293T细胞的流程如下：
(1)选择处于对数生长期的293T细胞进行传代，24h时细胞密度为70％-80％，准备转染，所有试剂平衡至室温；
(2)转染前2h将细胞从培养箱中取出，吸掉培养基，加入10mL 37℃已预热的含10％FBS无青链霉素抗性的DMEM培养基；
(3)转染体系：向500ul DMEM培养基中加入pWPXLd质粒6μg，psPAX2质粒3μg，pMD2.0G质粒1.5μg，移液器吹打混匀；另取500ul DMEM培养基加入30ullipo2000吹打混匀；将质粒混合体系加入lipo2000混合体系后混匀，室温静置15min；
(4)将转染混合物轻轻均匀地滴入培养皿，摇匀后放入细胞培养箱中继续培养；
(8)转染后6-8h，倒掉培养基上清，加入10mL 37℃预热的DMEM培养基(含10％FBS，无青链霉素)。
慢病毒浓缩步骤如下：
转染48小时后收293T培养上清用0.45um滤器过滤，按照浓缩试剂Lenti-XTM Concentrator(购于Takara，货号：631232)与病毒上清体积比1:3混合，颠倒混匀后置于4℃过夜孵育，将混合物在4℃环境下以1500g转速离心45min后，弃上清，将离心管底部沉淀用PBS重悬后于-80℃冰箱保存。
慢病毒感染Jurkat流程如下：
(1)1×106个Jurkat细胞，置于含有1ml1640培养基的12孔板中，加入10ul病毒浓缩液，同时设置对照组(未经感染的Jurkat)；
(2)将孔板封口，离心1000g，2h，32℃；
(3)离心完后，取出12孔板，放入细胞培养箱培养；
(4)病毒感染24h后，吸取细胞离心，1500rpm，3min，吸去上清换液，加入2mL 1640完全培养基重悬，放入细胞培养箱培养；
(5)病毒感染48h后，吸取5×105个慢病毒感染后和未经感染的Jurkat细胞于流式管中，离心300g，5min；
(6)弃上清，每管加入2mL预冷后的PBS缓冲液，离心300g，5min；重复此操作；
(7)弃上清，每管用100ul PBS重悬，然后加入荧光抗体，4℃避光孵育30min；
(8)PBS清洗2次，离心300g，5min；
(9)弃上清，每管用150ul PBS重悬，流式细胞仪上机检测。
3.检测EGFR CAR分子及VEGFR1信号感受器在人T淋巴细胞膜表达。
在基于jurkat细胞系的实验结果中已经明确，EGFR CAR及VEGFR1信号感受器均能在细胞膜高表达。进一步将制备好的EGFR CAR及EGFR-VEGFR1 CART慢病毒，分别感染人外周血来源的T淋巴细胞，继续培养48小时后，分别用荧光标记的重组蛋白(购于近岸蛋白Recombinant Human EGFR(C-6His)，货号：CI61；Recombinant Human VEGFR1(C-Fc)货号:CJ93)，流式检测T细胞膜上EGFR CAR分子及VEGFR1信号转换感受器的表达。
具体地，病毒感染T细胞流程如下：(注意：因后续实验例中会使用转染及感染步骤，且与此处流程及试剂相同，后续类似实验步骤描述从简。)
(1)复苏外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)，加入5mL预热的无血清无抗生素T细胞培养基，吹匀后计数，离心300g，5min；
(2)按T细胞数与磁珠数1:1的比例吸取CD3/CD28磁珠(Novoprotein CD3/CD28beads，货号：GMP-B038)于1mL EP管中，再加入适量无血清无抗生素T细胞培养基与磁珠充分混匀，离心300g，5min，弃上清洗涤磁珠；
(3)PBMC离心后，弃上清，分别向细胞沉淀和磁珠中加入适量T细胞完全培养基，调整T细胞培养密度为0.5-1×106/mL，然后将T细胞与磁珠混匀后放入12孔板中，放入细胞培养箱中培养；
(4)T细胞刺激48h后，取出保存于-80℃的浓缩慢病毒，置于冰上，自然缓慢解冻：
(5)将浓缩病毒与相应量的T细胞混匀后，加入12孔板，将孔板封口，离心1000g，32℃，2h，离心结束后，取出孔板，放于细胞培养箱培养；
(6)感染24h后，观察T细胞状态，完全换液，置于细胞培养箱继续培养。
(7)感染48h后，取T细胞，PBS洗涤一次后，加入荧光抗体或蛋白，4℃孵育30min后，1mlPBS重悬离心后，去上清，加入0.5ml PBS重悬，流式上机检测。
二、实验结果
构建好的EGFR-VEGFR1CART质粒基因结构示意图如图2所示。
流式细胞数术检测Jurkat细胞膜表面EGFR CAR分子及VEGFR1信号转换感受器的表达，结果如图3所示，可以看到，Jurkat细胞膜表面表达的EGFR CAR分子阳性率为83.17％，VEGFR1信号感受器分子表达阳性率为92.68％，即CAR分子与信号感受器分子均能在Jurkat细胞膜表达。
流式检测T细胞膜上EGFR CAR分子及VEGFR1信号转换感受器的表达，结果如图4所示，可以看到，T细胞膜表面表达的EGFR CAR分子阳性率为23.41％，VEGFR1信号感受器分子表达阳性率为29.63％。
通过上述实验可以看到，本发明在jurkat细胞系和人T淋巴细胞中成功构建了能够同时表达EGFR CAR及VEGFR1信号感受器的CAR-T细胞。
实验例2表达信号感受器对于EGFR CAR-T细胞抗肿瘤活性的增强作用。
人肺癌细胞系多数高表达EGFR抗原，可以作为EGFR CAR-T细胞的靶细胞，在临床实践过程中，部分患者会同时高表达VEGFR1分子，VEGFR1分子可抑制CAR-T免疫反应。本实验例中，运用VEGFR1的信号感受器修饰EGFR CAR-T细胞，通过信号感受器识别VEGFR1分子，为CAR-T细胞的激活提供共刺激信号，以增强CAR-T细胞抗肿瘤活性。
一、实验方法
1、检测肿瘤细胞靶抗原EGFR的表达及VEGFR1的表达
将人肺癌细胞H1975细胞系用荧光抗体染色标记后，流式仪检测靶抗原EGFR分子及VEGFR1分子的表达。
检测过程如下：
取100mm培养皿中的H1975细胞，吸去含有10％胎牛血清的培养基后， 加入1ml PBS润洗细胞一次，吸去PBS后加入1ml 0.25％胰酶，37℃孵育1分钟后，使用5ml含血清的培养基中和消化体系，将细胞悬液1500rpm离心3分钟。计数细胞，取1×106细胞入流式管，用PBS洗一次后，分别使用1ul抗人EGFR抗体(购自biolegend，货号：352905)及1ul抗人VEGFR1抗体(购自R&D Systems，货号：FAB321P)，4℃避光孵育30分钟。使用PBS洗细胞一次后上机，流式检测H1975细胞的抗原表达情况。
2、将制备好的EGFR-VEGFR1CART及EGFR CAR-T(按照实验例中1慢病毒感染T细胞的方法制备，前已详述)分别与靶细胞H1975共培养，检测培养上清中T细胞效应细胞因子分泌情况
T细胞杀伤肿瘤细胞过程中，会释放大量炎性细胞因子，比如γ干扰素，肿瘤坏死因子α，白细胞介素2等，这些因子的释放与T细胞杀伤肿瘤的活性成正相关，所以常检测T细胞与靶细胞共培养的上清中细胞因子含量，以反映T细胞杀伤活性。
具体地，本实验实施流程如下：
将100mm培养皿中的H1975细胞系，去培养基上清，PBS缓冲液润洗一次后，加入1ml 0.25％胰酶，37℃孵育1分钟后，使用5ml含血清的培养基中和消化体系，将细胞悬液1500rpm离心3分钟。计数细胞，为共培养做准备。
取制备好的CAR-T细胞计数为后续共培养铺板做准备。本实验例中，使用未经病毒转染的T细胞作为对照组T细胞(UTD),过表达EGFR CAR分子的T细胞作为实验组1(EGFR CAR-T)，过表达EGFR CAR分子及VEGFR1信号感受器的T细胞作为实验组2(EGFR-VEGFR1CAR-T)。
取胰酶消化后的H1975计数，将96孔板中每孔铺板5×103肿瘤细胞，同时计数CON-T、EGFR CAR-T、EGFR-VEGFR1CAR-T三组T细胞，分别按照T细胞数：肿瘤细胞数比值＝1:8、1:4、1:2、1:1、2:1、4:1铺板放入37℃培养箱共培养，24h后取培养上清做ELISA。ELISA检测流程如下：
(1)取ELISA板子，每孔加入100ul含包被抗体的包被液，4℃冰箱，静置过夜；
(2)加入清洗液300ul/孔、中速震荡1min，共清洗4次，后续清洗，按该条件进行；
(3)每孔加入200ul 1×ELISA/ELISPOT溶液，室温封闭1h；
(4)上述清洗条件清洗4次；
(5)加入待测样品(稀释后)和标准品，100ul/孔，每个样品设置3个复孔，放于4℃冰箱，孵育过夜；
(6)上述清洗条件清洗4次；
(7)加入检测抗体100ul/孔，室温孵育1h；
(8)清洗4次，加入Avidin-HRP二抗100μL/孔，室温孵育30min；
(9)清洗6次，每孔加入100ul的TMB显色液，室温孵育20min；
(10)待各孔显色后，每孔加入50μL终止液；
(11)上机检测：酶标仪上选取450nm、570nm的波长作为检测吸光值，获得数据进行分析统计。
3、运用安捷伦的实时细胞毒性监测系统(Real-Time Cell Analysis,RTCA)检测过表达信号感受器的CAR-T细胞对靶细胞的直接杀伤活性。
实验原理：本实验例通过监测不同组T细胞对靶细胞的杀伤裂解活性，直接反应T细胞抗肿瘤效应。RTCA仪器可以监测贴壁细胞与金属微孔板的形成的阻抗强度大小变化，贴壁细胞越多，仪器检测到的阻抗越大，即仪器显示的细胞指数(cellindex，CI)越大。当T细胞杀伤贴壁的肿瘤细胞时，随着肿瘤细胞死亡裂解，RTCA仪器监测到的细胞阻抗会逐渐变小，即细胞指数逐渐变小。
具体地，实验流程如下：
(1)取胰酶消化后的H1975计数；
(2)在RTCA培养板各孔加入50ul完全T细胞培养基，将板子放回检测仪，调节各孔初始CI值为0±0.02；
(3)取出RTCA培养板，向每孔加入靶细胞悬液(1x10^4/孔，50μL/孔)，静置10min后，将培养板放回检测仪，检测仪置于细胞培养箱中；
(4)当靶细胞的CI值达到1.5-2左右时，暂停监测，将培养板取出；
(5)将制备好的EGFR-VEGFR1CART及EGFR CAR-T分别与靶细胞H1975共培养，按照0.5:1效靶比，各孔加入100ul对应数量的CAR-T或Control-T细胞(CON-T)与靶细胞共培养；
(6)将培养板及监测仪放回细胞培养箱，继续监测，观察并记录各组CI值变化，分析数据。
二、实验结果
1、检测肿瘤细胞靶抗原EGFR的表达及VEGFR1的表达
流式检测H1975细胞的抗原表达情况，如图5所示，靶细胞H1975细胞 系高表达EGFR分子及VEGFR1分子。可以作为本实验例的肿瘤靶细胞。
2、EGFR-VEGFR1CART及EGFR CAR-T分别与靶细胞H1975共培养，检测培养上清中T细胞效应细胞因子分泌情况
检测结果如图6，与靶细胞共培养后，EGFR-VEGFR1CART及EGFR CAR-T两种CAR-T细胞的炎性细胞因子分泌量均高于未表达CAR的UTD细胞组。其中，过表达VEGFR1信号感受器的EGFR CAR-T细胞相较于常规EGFR CAR-T细胞能分泌更多细胞因子，说明，VEGFR1信号感受器能够增强EGFR CAR-T细胞的抗肿瘤效应。
3、过表达信号感受器的CAR-T细胞对靶细胞的直接杀伤活性
实验结果如图7所示，与靶细胞共培养后，EGFR-VEGFR1CART及EGFR CAR-T两种CAR-T细胞均能有效杀伤靶细胞。其中，过表达VEGFR1信号感受器的EGFR CAR-T细胞相较于常规EGFR CAR-T细胞，细胞指数变化值更大，即对于靶肿瘤细胞的杀伤能力更强，说明，VEGFR1信号感受器能够增强EGFR CAR-T细胞的杀伤肿瘤活性。
以上实验结果表明，对于高表达VEGFR1分子的肿瘤细胞，表达VEGFR1信号感受器的EGFR CAR-T细胞相比于常规EGFR CAR-T细胞具有更好的抗肿瘤活性。
实验例3表达信号转换器的靶向Glypican-3(GPC3)的CAR-T细胞的制备
GPC3在正常情况下主要表达于胚胎及胎儿组织器官，在成年人正常组织器官中显著降低，但是GPC3高表达于肝癌、肾癌、胃癌、结肠癌等组织，可以作为免疫治疗理想靶点。
本实验例将VEGFR1信号转换感受器过表达于靶向GPC3的CART细胞上，以达到增强该类CART细胞抗肿瘤活性及抗肿瘤细胞因子分泌增强的作用。实验方法与实验例1和实验例2类似，具体实施内容如下：
一、实验方法
1.构建病毒载体质粒。
通过全基因合成方法，合成基因片段SEQ ID NO.12，将合成的基因片段克隆入表达GPC3 CART的pwpxld慢病毒核心质粒中，构建GPC3-VEGFR1 CART质粒。
2.检测GPC3 CAR分子及VEGFR1信号感受器在T淋巴瘤Jurkat细胞系 的膜表达
在上海近岸蛋白质科技有限公司购买APC荧光标记的人GPC-3蛋白(货号：C414)和PE-Cy7标记的人VEGFR1蛋白(货号：CJ93)，通过流式细胞术分别检测GPC3 CAR及VEGFR1感受器的表达。具体操作步骤如下：
(1)病毒感染72小时后，吸取5×105个慢病毒感染后和未经感染的Jurkat细胞于流式管中，300g，离心5min；
(2)弃上清，每管加入2mL预冷后的PBS缓冲液，300g，离心5min；重复此操作；
(3)弃上清，每管用100ulPBS重悬，然后加入APC-GPC-3蛋白及PE-Cy7-VEGFR1蛋白，4℃避光孵育30min；
(4)PBS清洗2次，300g，离心5min；
(5)弃上清，每管用150ul PBS重悬，流式细胞仪上机检测。
3.检测GPC3 CAR分子及VEGFR1信号感受器在人T淋巴细胞膜表达。
将质粒转染293T细胞，制备好的GPC3-VEGFR1CART慢病毒，感染人外周血来源的T淋巴细胞，继续培养48小时后，流式检测T细胞膜上GPC3 CAR分子及VEGFR1信号转换感受器的表达，流式染色及检测实验流程如上第二步。
二、实验结果
病毒载体质粒的基因序列结构示意图如图8所示。
Jurkat细胞膜表面表达结果如图9所示，可以看到，Jurkat细胞膜表面表达的GPC3 CAR分子阳性率为68.90％，VEGFR1信号感受器分子表达阳性率为67.56％。
人T淋巴细胞膜表达结果如图10所示，可以看到，T细胞膜表面表达的GPC3 CAR分子阳性率为13.86％，VEGFR1信号感受器分子表达阳性率为12.03％
以上实验结果说明，VEGFR1信号感受器可表达于不同靶抗原的CAR-T细胞，具有广泛适用性。
实验例4表达信号感受器对于GPC3 CAR-T细胞抗肿瘤活性的增强作用
1、检测肿瘤细胞靶抗原GPC3的表达及VEGFR1的表达。
将构建好的人源肝肿瘤细胞Hep3B-V1细胞系用荧光抗体染色标记后， 流式仪检测靶抗原GPC3分子及VEGFR1分子的表达。
检测过程如下：
取100mm培养皿中的Hep3B-V1细胞，0.25％胰酶消化，37℃孵育1分钟后，将细胞悬液1500rpm离心3分钟。取1×106细胞入流式管，用PBS洗一次后，分别使用抗人GPC3抗体(一抗购自abcam，货号：ab95363；二抗购自biolegend，货号：406421)及抗人VEGFR1抗体(购自R&D Systems，货号：FAB321P)，加入抗体后4℃避光分别孵育30分钟。使用PBS洗细胞一次后上机，流式检测Hep3B细胞的抗原表达情况。
2、检测不同T细胞与肿瘤靶细胞共培养上清中T细胞效应细胞因子分泌情况
实验流程与实验例2中EGFR CAR-T与靶细胞共培养流程类似：
将100mm培养皿中的Hep3B细胞系，去培养基上清，胰酶消化后计数，取制备好的CAR-T细胞，细胞计数为后续共培养铺板做准备。本实验例中，使用未经病毒转染的T细胞作为对照组T细胞(Control-T),过表达GPC3 CAR分子的T细胞作为实验组1(GPC3 CAR-T)，过表达GPC3 CAR分子及VEGFR1信号感受器的T细胞作为实验组2(GPC3-VEGFR1 CAR-T)。
将96孔板中每孔铺板5×103肿瘤细胞，同时计数CON-T、GPC3 CAR-T、GPC3-VEGFR1 CAR-T三组T细胞，分别按照T细胞数：肿瘤细胞数比值＝1:4、1:2、1:1、2:1铺板放入37℃培养箱共培养，24h后取培养上清做ELISA。ELISA检测流程如实验例2中所述。
3、运用实时细胞毒性监测系统(Real-Time Cell Analysis,RTCA)检测过表达信号感受器的CAR-T细胞对靶细胞的直接杀伤活性。
本实验中使用未经病毒转染的T细胞作为对照组T细胞(CON-T),过表达GPC3 CAR分子的T细胞作为实验组1(GPC3 CAR-T)，过表达GPC3 CAR分子及VEGFR1信号感受器的T细胞作为实验组2(GPC3-VEGFR1 CAR-T)，实验操作流程同实验例2。
二、实验结果
1、肿瘤细胞靶抗原GPC3的表达及VEGFR1的表达
流式检测Hep3B细胞的抗原表达情况，如图11所示。靶细胞Hep3B细胞系高表达GPC3分子及VEGFR1分子。可以作为本实验例的肿瘤靶细胞。
2、不同T细胞与肿瘤靶细胞共培养上清中T细胞效应细胞因子分泌情况
检测结果如图12，与靶细胞共培养后，两种CAR-T细胞的炎性细胞因子分泌量均高于不表达CAR的CON-T细胞组。其中，过表达VEGFR1信号感受器的GPC3 CAR-T细胞相较于常规GPC3 CAR-T细胞能分泌更多细胞因子，说明VEGFR1信号感受器能够增强GPC3 CAR-T细胞的抗肿瘤效应。
3、过表达信号感受器的CAR-T细胞对靶细胞的直接杀伤活性
实验结果如图13所示，与靶细胞共培养后，两种CAR-T细胞均能有效杀伤靶细胞。其中，过表达VEGFR1信号感受器的GPC3 CAR-T细胞相较于常规GPC3 CAR-T细胞，细胞指数变化值更大，即对于靶肿瘤细胞的杀伤能力更强，说明，VEGFR1信号感受器能够增强GPC3 CAR-T细胞的杀伤肿瘤活性。
以上实验结果表明，对于高表达VEGFR1分子的肿瘤细胞，表达VEGFR1信号感受器的GPC3 CAR-T细胞相比于常规GPC3 CAR-T细胞具有更好的抗肿瘤活性。
实验例5制备胞外分子识别区为纳米单域抗体的信号感受器。
如实施例2和实施例3所述，本实验例运用纳米单域抗体作为VEGFR1信号感受器的分子识别区，构建信号感受器。
具体实施内容如下：
一、实验方法
1.构建病毒载体质粒，构建表达信号感受器的T细胞。
本实验例中，优选了两条纳米单域抗体分别作为信号感受器的分子识别区，分别如实施例2和实施例3中所述，与信号肽、跨膜区和胞内共刺激信号区组合，通过全基因合成方法，分别合成基因片段SEQ ID NO.22及SEQ ID NO.26，将合成的基因片段分别克隆入慢病毒核心质粒pwpxld中，构建靶向VEGFR1的感受器质粒，分别命名为VE5和VE6。质粒转染293T细胞制备VE5和VE6慢病毒上清，病毒浓缩后感染T细胞的流程与实验例1相同。
2.检测VEGFR1信号感受器在T淋巴细胞膜表达。
为便于检测VEGFR1纳米单域抗体的信号感受器表达，病毒核心质粒中带有FLAG蛋白标签，该标签可以与信号感受器同时表达，使用APC荧光标记的anti-FLAG抗体染色T细胞，通过流式细胞术分别检测VEGFR1感受器的表达。具体操作步骤如下：
(1)病毒感染72小时后，吸取5×105个慢病毒感染后和未经感染的 T细胞于流式管中，300g，离心5min；
(2)弃上清，每管加入2mL预冷后的PBS缓冲液，300g，离心5min；重复此操作；
(3)弃上清，每管用100ulPBS重悬，然后加入anti-FLAG抗体，4℃避光孵育30min；
(4)PBS清洗2次，300g，离心5min；
(5)弃上清，每管用150ul PBS重悬，流式细胞仪上机检测。
二、实验结果
构建好的病毒载体质粒结构示意图如图14所示。
VEGFR1信号感受器在T淋巴细胞膜表达结果如图15所示，可以看到T细胞膜表面表达的VEGFR1信号感受器分子VE5及VE6的表达阳性率分别为8.9％和5.73％。
以上实验结果说明，纳米单域抗体构成的VEGFR1信号感受器同样可表达于T细胞表面，具有应用广泛性。
实验例6纳米单域抗体的VEGFR1信号感受器对于免疫细胞抗肿瘤活性的增强作用
一、实验方法
1、检测肿瘤细胞VEGFR1的表达。
取1×106构建好的过表达VEGFR1的人源肿瘤细胞SKOV3-V1，加入1ul抗人VEGFR1抗体(购自R&D Systems，货号：FAB321P)，4℃避光孵育30分钟。使用PBS洗细胞一次后上机，荧光抗体染色标记后，流式仪检测靶抗原VEGFR1分子的表达。
2、观察不同T细胞与肿瘤靶细胞共培养条件下免疫细胞克隆团形成。
当T细胞识别肿瘤细胞激活后，会增殖形成T细胞克隆团，可以通过观察克隆团的大小，初步判断T细胞激活的强弱程度，克隆团越大，激活信号越强，抗肿瘤免疫效应活性越强。实验流程与实验例2中EGFR CAR-T共培养流程类似：
将培养皿中的肿瘤细胞系，去培养基上清，胰酶消化后计数，取制备好的T细胞，细胞计数为后续共培养铺板做准备。本实验例中，使用未经病毒转染的T细胞作为对照组T细胞(CON-T),过表达VE5信号感受器分子的T细胞作为实验组1(VE5-T)，过表达VE6信号感受器的T细胞作为实验 组2(VE6-T)。
将96孔板中每孔铺板5×103肿瘤细胞，同时计数CON-T、VE5-T、VE6-T三组T细胞，分别按照T细胞数：肿瘤细胞数比值＝2:1铺板放入37℃培养箱共培养，24h后显微镜光镜下拍摄细胞克隆团形成。
3、检测不同T细胞与肿瘤靶细胞共培养上清中T细胞效应细胞因子分泌情况。
将培养皿中的肿瘤细胞系，去培养基上清，胰酶消化后计数，取制备好的T细胞，细胞计数为后续共培养铺板做准备。将96孔板中每孔铺板5×103肿瘤细胞，同时计数CON-T、VE5-T、VE6-T三组T细胞，分别按照T细胞数：肿瘤细胞数比值＝1:1、2:1铺板放入37℃培养箱共培养，24h后取上清ELISA检测IFN-γ分泌。
二、实验结果
1、肿瘤细胞VEGFR1的表达
检测结果如图16所示，靶细胞SKOV3-V1细胞高表达VEGFR1分子。可以作为本实验例的肿瘤靶细胞。
2、不同T细胞与肿瘤靶细胞共培养条件下免疫细胞克隆团形成
结果如图17所示，与靶细胞共培养后，VE5及VE6T细胞的克隆团形成均高于未表达信号感受器的CON-T细胞组。说明，基于纳米单域抗体的VEGFR1信号感受器能够增强T细胞的抗肿瘤效应。
3、不同T细胞与肿瘤靶细胞共培养上清中T细胞效应细胞因子分泌情况
结果如图18所示，与靶细胞共培养后，两种过表达信号感受器的T细胞的IFN-γ分泌量均高于未表达信号感受器的CON-T细胞组，说明VEGFR1信号感受器能够增强EGFR CAR-T细胞的抗肿瘤效应。其中，VE5T细胞相较于VE6T细胞能分泌更多细胞因子。
以上实验结果表明，对于高表达VEGFR1分子的肿瘤细胞，表达纳米单域抗体构成的VEGFR1信号感受器的免疫细胞相比于常规免疫细胞具有更好的抗肿瘤活性。
综上，本发明在针对不同靶抗原的CAR-T细胞过表达识别VEGFR1的信号感受器，均能增强CAR-T细胞的抗肿瘤活性，此外，基于纳米单域抗体的信号感受器同样能增强CAR-T细胞抗肿瘤活性。因此，本发明提供的信号 感受器及包含该信号感受器的细胞，可用于制备抗肿瘤药物，在肿瘤的治疗中具有很好的应用前景。

Claims (10)

  1. 一种用于靶向肿瘤微环境的信号感受器，其特征在于，它包括信号肽、胞外分子识别区、胞外铰链区、跨膜区和胞内共刺激信号区；
    所述信号肽为与白介素2信号肽、白细胞分化抗原8信号肽、粒细胞-巨噬细胞集落刺激因子信号肽或集落刺激因子2受体α亚基信号肽中序列同源性90％以上的蛋白结构域中的至少一种；
    所述胞外分子识别区是与VEGF或VEGFR结合的单克隆抗体单链可变区、纳米单域抗体识别区、受体或配体中的至少一种；
    所述胞外铰链区是与白细胞分化抗原8胞外铰链区、白细胞分化抗原28胞外铰链区、TRAIL胞外铰链区、白细胞分化抗原16胞外铰链区、NKp30胞外铰链区、NKG2C胞外铰链区、NKG2D胞外铰链区、2B4胞外铰链区或DNAM-1胞外铰链区序列同源性90％以上的蛋白结构域中的至少一种；
    所述跨膜区为与白细胞分化抗原8跨膜区、白细胞分化抗原28跨膜区、TRAIL跨膜区、白细胞分化抗原16跨膜区、NKp30跨膜区、NKG2C跨膜区、NKG2D跨膜区、2B4跨膜区或DNAM-1跨膜区序列同源性90％以上的蛋白结构域中的至少一种；
    所述胞内共刺激信号区为与白细胞分化抗原28、肿瘤坏死因子受体超家族成员4、肿瘤坏死因子受体超家族成员9、淋巴细胞特异性蛋白酪氨酸激酶、诱导性T细胞共刺激分子、白细胞分化抗原40共刺激信号区、白细胞分化抗原27共刺激信号区、DAP10共刺激信号区、DAP12共刺激信号区、TRAIL共刺激信号区、白细胞分化抗原16共刺激信号区、NKp30共刺激信号区、NKG2C共刺激信号区、NKG2D共刺激信号区、2B4共刺激信号区或DNAM-1共刺激信号区序列同源性90％以上的蛋白结构域的胞内结构域中的至少一种。
  2. 按照权利要求1所述的信号感受器，其特征在于：所述VEGF为VEGF-A、VEGF-B、VEGF-C、VEGF-D、VEGF-E或胎盘生长因子中的至少一种；
    所述VEGFR为VEGFR1、VEGFR2或VEGFR3中的至少一种。
  3. 按照权利要求1所述的信号感受器，其特征在于：
    它的组成按照如下任意一种方式选择：
    组成方式一：
    所述信号肽为集落刺激因子2受体α亚基信号肽，或经过取代和/或缺失 和/或添加至少一个氨基酸所得的与集落刺激因子2受体α亚基信号肽功能相同或相似的肽；
    所述胞外分子识别区为识别VEGFR1的单克隆抗体单链可变区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与识别VEGFR1的单克隆抗体单链可变区功能相同或相似的蛋白；
    所述胞外铰链区为白细胞分化抗原28胞外铰链区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原28胞外铰链区功能相同或相似的蛋白；
    所述跨膜区为白细胞分化抗原28跨膜区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原28跨膜区功能相同或相似的蛋白；
    所述胞内共刺激信号区为白细胞分化抗原28的胞内结构域，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原28的胞内结构域功能相同或相似的蛋白；
    组成方式二：
    所述信号肽为白细胞分化抗原8信号肽，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原8信号肽功能相同或相似的肽；
    所述胞外分子识别区为识别VEGFR1的纳米单域抗体识别区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与识别VEGFR1的纳米单域抗体识别区功能相同或相似的蛋白；
    所述胞外铰链区为白细胞分化抗原8铰链区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原8胞外铰链区功能相同或相似的蛋白；
    所述跨膜区为白细胞分化抗原8跨膜区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原8跨膜区功能相同或相似的蛋白；
    所述胞内共刺激信号区为4-1BB胞内共刺激信号区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与4-1BB胞内共刺激信号区功能相同或相似的蛋白；
    组成方式三：
    所述信号肽为白细胞分化抗原8信号肽，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原8信号肽功能相同或相似的肽；
    所述胞外分子识别区为识别VEGFR1的单克隆抗体单链可变区或纳米 单域抗体识别区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与识别VEGFR1的单克隆抗体单链可变区或纳米单域抗体识别区功能相同或相似的蛋白；
    胞外铰链区为白细胞分化抗原8胞外铰链区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原8胞外铰链区功能相同或相似的蛋白；
    所述跨膜区为NKG2D跨膜区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与NKG2D跨膜区功能相同或相似的蛋白；
    所述胞内共刺激信号区为NKG2D胞内共刺激信号区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与NKG2D胞内共刺激信号区功能相同或相似的蛋白。
    组成方式四：
    所述信号肽为集落刺激因子2受体α亚基信号肽，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与集落刺激因子2受体α亚基信号肽功能相同或相似的肽；
    所述胞外分子识别区为识别VEGFR2的单克隆抗体单链可变区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与识别VEGFR2的单克隆抗体单链可变区功能相同或相似的蛋白；
    所述胞外铰链区为白细胞分化抗原28胞外铰链区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原28胞外铰链区功能相同或相似的蛋白；
    所述跨膜区为白细胞分化抗原28跨膜区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原28跨膜区功能相同或相似的蛋白；
    所述胞内共刺激信号区为白细胞分化抗原28的胞内结构域，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原28的胞内结构域功能相同或相似的蛋白；
    组成方式五：
    所述信号肽为集落刺激因子2受体α亚基信号肽，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与集落刺激因子2受体α亚基信号肽功能相同或相似的肽；
    所述胞外分子识别区为识别VEGFR1的单克隆抗体单链可变区，或经过 取代和/或缺失和/或添加至少一个氨基酸所得的与识别VEGFR1的单克隆抗体单链可变区功能相同或相似的蛋白；
    所述胞外铰链区为白细胞分化抗原8胞外铰链区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原8胞外铰链区功能相同或相似的蛋白；
    所述跨膜区为白细胞分化抗原8跨膜区，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与白细胞分化抗原8跨膜区功能相同或相似的蛋白；
    所述胞内共刺激信号区为4-1BB的胞内结构域，或经过取代和/或缺失和/或添加至少一个氨基酸所得的与4-1BB的胞内结构域功能相同或相似的蛋白。
  4. 按照权利要求1-3任一项所述的信号感受器，其特征在于：所述集落刺激因子2受体α亚基信号肽的氨基酸序列如SEQ ID NO.1所示；
    和/或，所述识别VEGFR1的单克隆抗体单链可变区的氨基酸序列如SEQ ID No.3所示；
    和/或，所述白细胞分化抗原28胞外铰链区的氨基酸序列如SEQ ID No.5所示；
    和/或，所述白细胞分化抗原28跨膜区的氨基酸序列如SEQ ID No.7所示；
    和/或，所述白细胞分化抗原28的胞内结构域的氨基酸序列如SEQ ID No.9所示；
    和/或，所述白细胞分化抗原8信号肽的氨基酸序列如SEQ ID NO.13所示；
    和/或，所述VEGFR1的纳米单域抗体识别区的氨基酸序列如SEQ ID NO.15或SEQ ID NO.23所示；
    和/或，所述白细胞分化抗原8跨膜区的氨基酸序列如SEQ ID NO.17所示；
    和/或，所述4-1BB胞内共刺激信号区的氨基酸序列如SEQ ID NO.19所示；
    和/或，所述白细胞分化抗原8胞外铰链区的氨基酸序列如SEQ ID NO.27所示；
    和/或，所述NKG2D跨膜区的氨基酸序列如SEQ ID NO.29所示；
    和/或，所述NKG2D胞内共刺激信号区的氨基酸序列如SEQ ID NO.31 所示；
    和/或，所述信号感受器的氨基酸序列如SEQ ID NO.11、SEQ ID NO.21、SEQ ID NO.25、SEQ ID NO.33、SEQ ID NO.34或SEQ ID NO.35所示。
  5. 一种表达载体，其特征在于：它能够表达权利要求1-4任一项所述的信号感受器。
  6. 按照权利要求5所述的表达载体，其特征在于：它是重组质粒载体、重组病毒或它们的组合。
  7. 按照权利要求6所述的表达载体，其特征在于：所述重组病毒为慢病毒载体或逆转录病毒载体。
  8. 一种用于靶向治疗肿瘤的细胞，其特征在于：它由权利要求5-7任一项所述的表达载体和宿主细胞构成。
  9. 按照权利要求8所述的用于靶向治疗肿瘤的细胞，其特征在于：所述宿主细胞选自αβT细胞、NKT细胞或γδT细胞、肿瘤浸润淋巴细胞、TCR修饰的T细胞、嵌合抗体受体T细胞或自然杀伤细胞。
  10. 一种用于治疗肿瘤的药物，其特征在于：它是以权利要求8或9任一项所述的用于靶向治疗肿瘤的细胞作为活性成分，加上药学上可接受的辅料或辅助性成分制成的。
PCT/CN2023/123744 2022-10-11 2023-10-10 一种用于靶向肿瘤微环境的信号感受器和用于靶向治疗肿瘤的细胞 WO2024078485A1 (zh)

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CN107988165A (zh) * 2013-02-06 2018-05-04 人类起源公司 具有改进特异性的修饰的t淋巴细胞
CN109485732A (zh) * 2018-12-20 2019-03-19 四川大学华西医院 基因工程修饰的双靶点嵌合抗原受体及其用途
CN110004167A (zh) * 2019-04-16 2019-07-12 中国医学科学院血液病医院（血液学研究所） 靶向vegfr-2和/或vegfr-3嵌合抗原受体及其应用
CN110520533A (zh) * 2017-01-05 2019-11-29 赫利克斯生物药品公司 治疗癌症的vegfr-2 car免疫细胞
CN114874335A (zh) * 2022-05-06 2022-08-09 北京大学深圳研究生院 三靶点可调控car免疫细胞的组合物及其应用

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CN107988165A (zh) * 2013-02-06 2018-05-04 人类起源公司 具有改进特异性的修饰的t淋巴细胞
CN110520533A (zh) * 2017-01-05 2019-11-29 赫利克斯生物药品公司 治疗癌症的vegfr-2 car免疫细胞
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