WO2019080538A1 - Chimeric antigen receptor, nk cell modified by same, coding dna, mrna, expression vector, preparation method and application - Google Patents

Chimeric antigen receptor, nk cell modified by same, coding dna, mrna, expression vector, preparation method and application

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WO2019080538A1
WO2019080538A1 PCT/CN2018/094005 CN2018094005W WO2019080538A1 WO 2019080538 A1 WO2019080538 A1 WO 2019080538A1 CN 2018094005 W CN2018094005 W CN 2018094005W WO 2019080538 A1 WO2019080538 A1 WO 2019080538A1
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chimeric antigen
antigen receptor
cells
nkg2d
amino acid
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PCT/CN2018/094005
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French (fr)
Chinese (zh)
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肖�琳
陈璨
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杭州优善生物科技有限公司
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Publication of WO2019080538A1 publication Critical patent/WO2019080538A1/en

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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
    • C12N5/0646—Natural killers cells [NK], NKT cells
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464429—Molecules with a "CD" designation not provided for elsewhere
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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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes
    • 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
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention belongs to the field of biotechnology, and in particular, to a chimeric antigen receptor, a modified NK cell thereof, a coding DNA, an mRNA, an expression vector, a preparation method and application.
  • Chimeric Antigen Receptor is an artificially engineered receptor, so any specific receptor can be grafted onto immune effector cells.
  • the extracellular specific portion of these receptors used to recognize an antigen is derived from the sequence of the antibody. Because different parts of this receptor have different origins, they are called chimeric receptors.
  • CAR-modified immune cells ie, immune cells expressing the chimeric antigen receptor (CAR) are now the most effective and promising tumor cell immunotherapy products.
  • NK Natural killer cells
  • T cells peripheral blood mononuclear cells
  • NK cells do not produce autocrine growth factors such as IL-2, and they do not increase in value when they encounter specific antigens, and have a short life span. They do not need to be equipped with suicide genes. Limit CAR toxicity.
  • the killing of tumors by NK cells does not depend on the specific antigen presented by MHC. Therefore, in the immunotherapy application of allogeneic NK cells, immune rejection is not produced, and it is a safer immunity than T cells. Treat candidate cells.
  • CAR can avoid the inhibitory signaling pathway in NK cells, while NK cells themselves can express activated receptors and can also utilize antibody-mediated cytotoxicity (ADCC). Therefore, CAR-expressing NK cells are treated in tumors. It is superior to T cells and has a broader application prospect.
  • CAR includes an extracellular portion, a transmembrane region, and an intracellular portion.
  • immune cells carrying CAR the selection of the extracellular and intracellular portions of CAR and its cooperation with immune cell types are extremely important, which is closely related to the specific killing ability of tumors. At present, in the immunotherapy of tumors, it is still necessary to develop a novel and effective CAR-immune cell drug.
  • the present invention provides a chimeric antigen receptor, a modified NK cell thereof, a coding DNA, an mRNA, an expression vector, a preparation method, and an application.
  • the present invention provides:
  • a chimeric antigen receptor comprising an operably linked, sequentially tandem antigen binding domain, a spacer, a transmembrane region and an intracellular domain, characterized in that said The antigen binding domain is derived from the ligand binding region of NKG2D, which is derived from the intracellular signaling region of DAP12.
  • An isolated DNA encoding a chimeric antigen receptor comprising operably linked, sequentially tandem antigen-binding domain coding elements, spacer coding elements, transmembrane region coding elements, and intracellular structures
  • a domain coding element characterized in that said antigen binding domain coding element is derived from a ligand binding region encoding DNA of NKG2D, said intracellular domain coding element being derived from the intracellular signal region encoding DNA of DAP12.
  • the acid sequence encodes the amino acid sequence of the spacer transmembrane region, the amino acid sequence of the spacer transmembrane region being identical to the amino acid sequence of position Y to position 210 of CD8 ⁇ , and 118 ⁇ Y ⁇ 128.
  • a recombinant expression vector comprising the DNA according to any one of (9) to (16), which is operably linked to a promoter.
  • a chimeric antigen receptor-modified NK cell the surface of which is modified by the chimeric antigen receptor according to any one of (1) to (8).
  • a pharmaceutical composition comprising the chimeric antigen receptor-modified NK cell according to (20) as an active ingredient, and a pharmaceutically acceptable adjuvant.
  • a method of treating a tumor and/or a cancer comprising administering a chimeric antigen receptor-modified NK cell according to (20) to a tumor and/or a cancer patient.
  • a tool vector comprising, in turn, an operably linked CMV promoter, a T7 promoter, a 5'UTR having a kozak sequence, a GM-CSF alpha chain signal peptide coding sequence, and an alpha having a PolyA signal The 3'UTR of the globulin.
  • the invention has the following advantages and positive effects:
  • the chimeric antigen receptor of the present invention enables the NK cells modified by it (also referred to as "engineered NKG2D ligand-targeted NK cells”) to have strong and specific targeting to tumors positive for expression of various NKG2D ligands.
  • the killing activity, the preclinical study of the present invention has fully demonstrated that the modified NK cells can significantly reduce or even eliminate the tumor burden in the animal and prolong the survival time of the animal.
  • the engineered NKG2D ligand-targeted NK cells of the invention provide a new choice for treating NKG2D ligand-positive tumor patients, and have good industrial application prospects.
  • FIG. 1 is a schematic diagram showing the construction of a chimeric antigen receptor NKG2D-CD8-DAP12 according to an embodiment of the present invention, wherein “CMV” indicates a CMV promoter sequence, “T7” indicates a T7 promoter sequence, and “5'UTR” indicates 5'UTR with Kozak sequence, “SP” indicates the GM-CSF alpha chain signal peptide coding sequence, “NKG2D” indicates the coding sequence of the ligand binding region of NKG2D, and “CD8” indicates the hinge region and transmembrane region coding sequence of CD8 ⁇ "DAP12” denotes the intracellular signal region coding sequence of DAP12, “alpha globulin 3'UTR” denotes the 3'UTR of the alpha globulin having a PolyA signal, and “pA 150 " denotes polyA (polyadenylation), wherein Contains 150 As.
  • CMV indicates a CMV promoter sequence
  • T7 indicates
  • Figure 2 is an electropherogram showing the identification fragment of the expression vector pFastbac1-CD8-DAP12 digested with restriction endonucleases SphI and SalI according to one embodiment of the present invention; wherein lane 1 is a DNA molecular weight marker and lane 2 is an identification fragment .
  • Figure 3 is an electropherogram showing the identification fragment of the expression vector pFastbac1-NKG2D-CD8-DAP12 digested with restriction endonucleases SphI and NheI according to an embodiment of the present invention; wherein lane 1 is a DNA molecular weight marker, and lane 2 is Identify the fragment.
  • Figure 4 is a schematic view showing the structure of a recombinant DNA vector pFastbac1-NKG2D-CD8-DAP12 of a chimeric antigen receptor according to an embodiment of the present invention; wherein the clockwise sequence is a forward gene fragment and the counterclockwise is a reverse gene fragment .
  • CMV denotes a CMV promoter sequence
  • T7 denotes a T7 promoter sequence
  • 5'UTR denotes a 5'UTR having a Kozak sequence
  • GM-CSF ⁇ denotes a GM-CSF alpha chain signal peptide coding sequence
  • NKG2D indicates the coding sequence of the ligand binding region of NKG2D
  • CD8 indicates the hinge region and transmembrane region coding sequence of CD8 ⁇
  • DAP12 indicates the intracellular signal region coding sequence of DAP12
  • alpha globulin 3'UTR Represents the 3'UTR of the alpha globulin with the PolyA signal.
  • Figure 5 shows the results of analysis of NK cell phenotype by flow cytometry.
  • Figure 5A shows the purity of NK cells when expanded from peripheral blood mononuclear cells for 17 days.
  • the abscissa indicates the CD3 expression intensity, and the ordinate indicates the CD56 expression intensity;
  • FIG. 5B shows the expression intensity of NK cells endogenous NKG2D and CD16 when expanded from peripheral blood mononuclear cells for 17 days, the abscissa indicates the intensity of NKG2D expression, and the ordinate indicates CD16. Expression intensity.
  • Figure 6 shows an electropherogram of a chimeric antigen receptor NKG2D-CD8-DAP12 linearized DNA template synthesized in vitro; Lane 1 is a DNA molecular weight marker and Lane 2 is a linearized DNA template.
  • Figure 7 shows an electropherogram of mRNA of the chimeric antigen receptor NKG2D-CD8-DAP12 synthesized in vitro; Lane 1 is a molecular weight marker and Lane 2 is an mRNA of NKG2D-CD8-DAP12.
  • Figure 8 shows the results of flow cytometry detection of chimeric antigen receptor NKG2D-CD8-DAP12 mRNA electroporation NK cells.
  • Figure 8A shows the intensity of endogenous expression of NKG2D by NK cells (where the left peak is the negative control curve and the right peak is the NK cell NKG2D expression intensity curve);
  • Figure 8B shows the intensity of NKG2D expression by NK cells after electroporation (where The left peak is the negative control curve, and the right peak is the NKG2D expression intensity curve after NK cell electrotransformation of NKG2D-CD8-DAP12 mRNA;
  • Figure 8C shows the comparison of the intensity of NKG2D expression after endogenous expression and electrotransfection of NK cells ( That is, 8A and 8B are combined together) (the left peak is a negative control curve, the middle peak is the NK cell NKG2D expression intensity curve, and the right peak is the NKG2D-CD8-DAP12 mRNA expression level after NK
  • the abscissa indicates the intensity of NKG2D expression, and the ordinate indicates the relative cell number.
  • “NKG2D fluorescence intensity” in the abscissa indicates the fluorescence reading displayed by the flow cytometer when detected with a fluorescent NKG2D antibody.
  • Figure 9 is a graph showing the results of Elispot analysis of IFN- ⁇ release after mixed culture of chimeric antigen receptor NKG2D-CD8-DAP12-modified NK (NKG2D-CAR-NK) cells and human tumor cells in an embodiment of the present invention.
  • mGFP-CAR-NK indicates an mGFP-CAR-modified NK cell group (control group)
  • “NKG2D-CAR-NK” indicates a chimeric antigen receptor NKG2D-CD8-DAP12 (i.e., NKG2D-CAR)-modified NK.
  • the abscissa indicates the tumor cell group
  • the ordinate indicates the relative amount of IFN- ⁇ (expressed as the number of spots per 2.5 ⁇ 10 4 NK cells).
  • FIGS. 10A-G show NKG2D-CAR-NK versus tumor cells HCT116(A), SKOV3(B), Fadu(C), Detroit(D), HepG2(E), MCF7(F), respectively, according to an embodiment of the present invention
  • Figure 11 shows the killing effect of adoptively NKG2D-CAR-NK cells on tumor cells; the dotted line shows the results of the chimeric antigen receptor NKG2D-CD8-DAP12 modified NK cell group (shown as "NKG2D-CAR-NK” (6 injections)"); the solid line shows the results of the NK cell reinfusion group (shown as "PBS control group”).
  • the abscissa indicates the number of days after inoculation of the tumor
  • the ordinate indicates the fluorescence intensity of the tumor cells in the animal recorded by the living imager
  • the radiance shown in the ordinate refers to the number of photons emitted from the surface of the animal per unit time, unit area, and unit radians.
  • Figure 12 AC shows chimeric antigen receptor NKG2D-CD8-DAP12 modified NK cells and chimeric antigen receptor NKG2D-CD8-CD3Z modified NK cells, respectively, on tumor cells SKOV3 (A), Detroit (B), HCT116 (C) Comparison of the test results of killing activity.
  • mGFP-CAR-NK indicates an mGFP-CAR-modified NK cell group
  • NKG2D-DAP12-CAR-NK indicates an NKG2D-DAP12-CAR-modified NK cell group
  • “NKG2D-CD3Z-CAR-NK” indicates NKG2D-CD3Z-CAR modified NK cell group.
  • the abscissa indicates the ratio of effector cells to tumor cells, and the ordinate indicates the proportion of tumor cells that are lysed after killing.
  • CAR includes an extracellular portion, a transmembrane region, and an intracellular portion.
  • the extracellular portion in turn includes an antigen binding domain for recognizing and binding an antigen, and a spacer for spacing the antigen binding domain and the transmembrane region;
  • the intracellular portion primarily includes an intracellular domain for signaling.
  • the inventors of the present invention have selected a specific combination of an antigen-binding domain and an intracellular domain for NK cells through theoretical research and experimental exploration, and successfully applied the thus developed CAR to NK cells to make the NK cells play.
  • a strong targeted tumoricidal activity thereby developing a novel and effective engineered NKG2D ligand-targeted NK cell that is available for selection in tumor immunotherapy.
  • the invention provides a chimeric antigen receptor comprising an operably linked, sequentially tandem antigen binding domain, a spacer, a transmembrane region and an intracellular domain, Characterized in that the antigen binding domain is from the ligand binding region of NKG2D, which is derived from the intracellular signaling region of DAP12.
  • NKG2D is an important receptor regulating NK cell killing activity.
  • NKG2D ligand is mainly expressed on the surface of tumor cells and stress cells, and is rarely expressed or even expressed on the surface of normal cells.
  • a large number of tumor cells such as colorectal cancer cells and ovarian cancer Cells, head and neck cancer cells, lymphatic cancer cells, glioma cells, and the like have a large number of ligands for NKG2D expression.
  • the amino acid sequence of the antigen-binding domain is preferably identical to the X-position 216 amino acid sequence of NKG2D, and 73 ⁇ X ⁇ 83, and X is an integer.
  • the amino acid sequence of NKG2D may be the amino acid sequence numbered NP_031386.2 in Genbank of NCBI (ie, National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov). That is, the amino acid sequence of the antigen-binding domain is preferably selected from amino acids 73-216 of NKG2D and comprises amino acids 83-216.
  • amino acid sequence of the antigen-binding domain is as shown in any one of the following amino acid sequence groups: amino acids 73-216 of NKG2D, amino acids 74-216, amino acids 75-216, 76- 216 amino acids, amino acids 77-216, amino acids 78-216, amino acids 79-216, amino acids 80-216, amino acids 81-216, amino acids 82-216, or 83- 216 amino acids. More preferably, the amino acid sequence of the antigen binding domain is set forth in SEQ ID NO: 3.
  • the intracellular domain acts as a signal to activate NK cells.
  • the intracellular domain of the CAR originally used for T cells has only one signaling molecule, usually the receptor-associated Fc ⁇ RI ⁇ of immunoglobulin E (a subunit of a receptor with high affinity for IgE) or T cell antigen receptor signaling.
  • the underlying conductive molecule DAP12 some intracellular domains comprise a T cell activation domain consisting of one or several T cell activation motifs.
  • the present inventors have found that by combining the antigen-binding domain derived from the ligand binding region of NKG2D described above with the intracellular signal region derived from DAP12, a CAR capable of exerting a strong targeted tumoricidal activity of NK cells can be obtained.
  • the amino acid sequence of the intracellular domain is selected from amino acids 62-113 of DAP12, and the amino acid sequence of the intracellular domain is more preferably as set forth in SEQ ID NO: 5.
  • the amino acid sequence of DAP12 has the Genbank number NP_003323.1.
  • the present invention further selects the spacer and transmembrane regions, thereby obtaining a CAR having a specific combination of antigen binding domain-spacer-transmembrane region-intracellular domain.
  • the spacer junction recognizes and binds to the antigen binding domain and transmembrane region of the antigen.
  • the structure of this region should be flexible so that the antigen binding domain can be adapted to different orientations to facilitate antigen recognition and binding.
  • the simplest form of spacer region is an immunoglobulin hinge region of IgGl (hinge), it may be a portion of an immunoglobulin C H2 C H3 region.
  • the transmembrane region is typically a hydrophobic alpha helix spanning the cell membrane.
  • the spacer is preferably derived from the hinge region of CD8[alpha], which is preferably from the transmembrane region of CD8[alpha].
  • CD8 is a transmembrane glycosylated membrane protein consisting of two subunits, alpha and beta, which interact with T cell surface receptors to bind T cells to specific antigens. CD8 specifically binds to MHCI and mediates cytotoxic T cells. Killing effect.
  • the spacer region and the transmembrane region constitute a spacer transmembrane region, and wherein the amino acid sequence of the spacer transmembrane region is identical to the amino acid sequence of the Yth to the 210th position of CD8 ⁇ , and 118 ⁇ Y ⁇ 128, Y is an integer.
  • the Genbank number of the amino acid sequence of CD8 ⁇ may be NP_001139345.1. That is, the amino acid sequence of the spacer transmembrane region is preferably selected from amino acids 118-210 of CD8 ⁇ and amino acids 128-210.
  • amino acid sequence of the spacer transmembrane region is as shown in any one of the following amino acid sequence groups: amino acids 118-210 of CD8 ⁇ , amino acids 119-210, amino acids 120-210, 121- 210 amino acids, amino acids 122-210, amino acids 123-210, amino acids 124-210, amino acids 125-210, amino acids 126-210, amino acids 127-210, or 128- 210 amino acids.
  • amino acid sequence of the spacer transmembrane region is set forth in SEQ ID NO:4.
  • the antigen-binding domain, the spacer, the transmembrane region and the intracellular domain are sequentially connected in series; between the antigen-binding domain and the spacer, between the spacer and the transmembrane region
  • the transmembrane region and the intracellular domain are operably linked, for example, may be connected by a joint or directly without a joint.
  • a linker is used between the antigen binding domain and the spacer (the linker is, for example, -Ala-Ser-), and the spacer and transmembrane region, the transmembrane region and the cell
  • the internal domains are directly connected without a joint.
  • the amino acid sequence of the chimeric antigen receptor is set forth in SEQ ID NO: 1.
  • the chimeric antigen receptor has an amino acid sequence obtained by replacing, deleting, and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1;
  • the chimeric antigen receptor has at least 90%, preferably at least 95%, more preferably at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 1.
  • the invention also provides an isolated DNA encoding a chimeric antigen receptor of the invention, the DNA comprising an operably linked, sequentially tandem antigen binding domain coding element, a spacer coding element, a transmembrane a region coding element and an intracellular domain coding element, wherein the antigen binding domain coding element is derived from a ligand binding region encoding DNA of NKG2D, and the intracellular domain coding element is derived from the intracellular signal region encoding DNA of DAP12 .
  • the nucleotide sequence of the antigen-binding domain coding element encodes an amino acid sequence of the antigen-binding domain, preferably, the amino acid sequence of the antigen-binding domain is identical to the X-position 216 amino acid sequence of NKG2D, And 73 ⁇ X ⁇ 83, and X is an integer.
  • amino acid sequence of the antigen-binding domain is as shown in any one of the following amino acid sequence groups: amino acids 73-216 of NKG2D, amino acids 74-216, amino acids 75-216, 76- 216 amino acids, amino acids 77-216, amino acids 78-216, amino acids 79-216, amino acids 80-216, amino acids 81-216, amino acids 82-216, or 83- 216 amino acids.
  • amino acids 73-216 of NKG2D amino acids 74-216, amino acids 75-216, 76- 216 amino acids, amino acids 77-216, amino acids 78-216, amino acids 79-216, amino acids 80-216, amino acids 81-216, amino acids 82-216, or 83- 216 amino acids.
  • amino acids 73-216 of NKG2D amino acids 74-216
  • amino acids 75-216 amino acids 77-216
  • amino acids 78-216 amino acids 79-216
  • amino acids 80-216 amino acids 81-216
  • the nucleotide sequence of the intracellular domain coding element encodes the amino acid sequence of the intracellular domain, preferably, the amino acid sequence of the intracellular domain is selected from positions 62-113 of the intracellular signal region of DAP12 Amino acid.
  • the nucleotide sequence of the intracellular domain coding element is set forth in SEQ ID NO: 8.
  • the spacer coding element is derived from the hinge region encoding DNA of CD8 ⁇
  • the transmembrane region coding element is derived from the transmembrane region encoding DNA of CD8 ⁇ .
  • the spacer coding element and the transmembrane region coding element comprise a spacer transmembrane region coding element, the nucleotide sequence of the spacer transmembrane region coding element encoding an amino acid sequence of the spacer transmembrane region, preferably, The amino acid sequence of the spacer transmembrane region is identical to the amino acid sequence of the Yth position to the 210th position of CD8 ⁇ , and 118 ⁇ Y ⁇ 128, and Y is an integer.
  • amino acid sequence of the spacer transmembrane region is as shown in any one of the following amino acid sequence groups: amino acids 118-210 of CD8 ⁇ , amino acids 119-210, amino acids 120-210, 121- 210 amino acids, amino acids 122-210, amino acids 123-210, amino acids 124-210, amino acids 125-210, amino acids 126-210, amino acids 127-210, or 128- 210 amino acids.
  • amino acids 118-210 of CD8 ⁇ amino acids 119-210
  • amino acids 120-210, 121- 210 amino acids, amino acids 122-210, amino acids 123-210, amino acids 124-210, amino acids 125-210, amino acids 126-210, amino acids 127-210, or 128- 210 amino acids amino acids 118-210 of CD8 ⁇
  • amino acids 119-210 amino acids 120-210, 121- 210 amino acids
  • amino acids 122-210 amino acids 123-210
  • amino acids 124-210 amino acids 125-210
  • the isolated nucleotide sequence encoding the chimeric antigen receptor of the invention is represented by SEQ ID NO: 2.
  • NKG2D, DAP12 and CD8 ⁇ of the present invention are preferably derived from humans, and their full-length amino acid sequences and nucleotide sequences are known, and can be found by public databases commonly used in the art.
  • the present invention also provides an isolated mRNA which is transcribed from the DNA encoding the chimeric antigen receptor of the present invention.
  • the invention also provides a recombinant expression vector comprising a DNA encoding a chimeric antigen receptor according to the invention operably linked to a promoter.
  • the recombinant expression vector comprises, in sequence, a CMV promoter, a T7 promoter, a 5'UTR having a kozak sequence, and a GM-CSF alpha chain signal peptide encoding prior to the DNA encoding the chimeric antigen receptor according to the present invention.
  • the combination of the above-described functional elements of the recombinant expression vector of the present invention can promote transcription and translation of DNA and enhance the stability of mRNA.
  • the present invention also optimizes the structure of each of the above-described action elements to better perform their intended functions.
  • CMV promoter sequence TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTGTTTTGGCACCAAAACGGGACTTTCCAAAAAAAATGTCGTAACAACTCCCGCCACCCCACCATGGGGGGTTTT
  • the T7 promoter sequence is TAATACGACTCACTATAG (SEQ ID NO: 17).
  • the T7 promoter functions to initiate transcription of downstream DNA sequences.
  • the sequence of the 5' UTR having the kozak sequence is AAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA GCCA CCATG (SEQ ID NO: 18), wherein the sequence underlined is the kozak sequence.
  • the function of the 5'UTR with the kozak sequence is to enhance the translation efficiency of the mRNA.
  • the sequence of the GM-CSF ⁇ chain signal peptide coding sequence is ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCA (SEQ ID NO: 19), and the amino acid sequence thus obtained is MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 20).
  • the GM-CSF alpha chain signal peptide is a leader sequence that targets the CAR of the present invention to the secretory pathway, and its coding sequence is first translated into a protein together with the CAR in the cell, directing the synthesized protein into the endocrine pathway. The signal peptide has been removed before expression of the CAR on the cell surface.
  • the 3'UTR sequence of the alpha globulin is GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTG AATAAA GCCTGAGTAGGAAGT (SEQ ID NO: 21), wherein the sequence underlined is a polyA signal. Its role is to enhance the stability of mRNA.
  • the basic backbone of the recombinant expression vector is a commercially available pFastbac1 vector into which each of the above elements is inserted.
  • a DNA double-stranded template in which a positive strand carries a PolyA and an inverted strand carries a corresponding PolyT can be synthesized from the recombinant expression vector, for example, using a Tail-PCR technique, such that The instability of the DNA template is reduced, and mRNA can be synthesized in vitro.
  • the range of the number of A in the PolyA carried in the positive chain is 140-170, preferably 150-170, more preferably It is about 150 (for example, 150).
  • the present invention also provides a chimeric antigen receptor-modified NK cell whose surface is modified by the chimeric antigen receptor of the present invention.
  • modification means that an NK cell expresses a chimeric antigen receptor according to the present invention, that is, a transmembrane region of the chimeric antigen receptor is anchored to a cell membrane of the modified NK cell, The antigen binding domain is located on the cell surface and the intracellular domain is located in the cytoplasm.
  • the NK cells may be various types of NK cells known and can be obtained by conventional biological methods.
  • NK cells naturally killer cells
  • the phenotype is CD3 negative CD56.
  • Positive single cells mainly CD16-negative CD56bright (bright) and CD16-positive CD56 dim (dark) subtypes, have immunomodulatory and tumor killing in vivo efficacy. Since NK cell action is non-MHC-restricted, there is no need to match the histocompatibility complex of the individual patient in use, that is, NK cells can be used for cell therapy of allogeneic patients, and have wide clinical application value.
  • the invention also provides a method of preparing a chimeric antigen receptor-modified NK cell according to the invention, comprising the steps of:
  • the NK cells described in step 1) can be prepared from peripheral blood mononuclear cells.
  • the purity of the NK cells in the method of the invention may be > 70%, preferably > 80%.
  • NK cell purity refers to the proportion of NK cells in the total cell population.
  • the nucleic acid according to the step 2) is a DNA encoding the chimeric antigen receptor according to the present invention, or an mRNA obtained by transcription of the DNA.
  • the transfection described in step 3) can be carried out by cryoelectroporation techniques or lentiviral vectors.
  • Transfection using cryoelectroporation techniques can be carried out in a manner commonly used in the art, such as the literature "Nakazawa Y, Matsuda K, Kurata T, Sueki A, Tanaka M, Sakashita K, Imai C, Wilson MH, Koike K.
  • Anti- Proliferative effects of T cells expressing a ligand-based chimeric antigen receptor against CD116 on CD34(+) cells of juvenile myelomonocytic leukemia J Hematol Oncol. 2016 Mar.
  • Transfection using lentiviral vectors can be carried out in a manner commonly used in the art, such as the literature "James N. Kochenderfer, Steven A. Feldman, Yangbing Zhao, Hui Xu, Mary A.
  • the DNA corresponding to amino acids 83-216 of the human NKG2D protein, the DNA corresponding to amino acids 128-210 of human CD8 ⁇ , and the 62nd of human DAP12 are amplified by PCR from the PBMC cDNA library. DNA corresponding to -113 amino acids.
  • the three sequences amplified were ligated and ligated to the pFastbac1 vector by molecular cloning techniques to obtain a recombinant expression vector pFastbac1-NKG2D-CD8-DAP12.
  • the mRNA of the corresponding sequence of NKG2D-CD8-DAP12 was then synthesized.
  • the mRNA was electroporated into the NK cells expanded in vitro by high-efficiency cryoelectroporation to obtain engineered NKG2D ligand-targeted NK cells.
  • Each portion of the chimeric antigen receptor can also be amplified by genomic cDNA of mononuclear cells in venous blood.
  • the invention also provides the use of a chimeric antigen receptor-modified NK cell according to the invention for the preparation of a medicament for the treatment or prevention of a tumor and/or cancer.
  • the tumor and/or cancer is positive for NKG2D ligand, including colorectal cancer, ovarian cancer, head and neck cancer, myeloma, liver cancer, breast cancer, hematoma, cervical cancer, glioma, and the like.
  • the tumor and/or cancer is positive for NKG2D ligand comprising the tumor and/or cancer being untreated is NKG2D ligand positive and the tumor and/or cancer is treated It became positive for NKG2D ligand.
  • the treatment includes becoming NKG2D ligand positive after treatment with a drug, radiation or biological agent.
  • the invention also provides the use of a chimeric antigen receptor-modified NK cell according to the invention for the preparation of a medicament for detecting a tumor and/or cancer of a host.
  • the tumor and/or cancer is positive for NKG2D ligand, including colorectal cancer, ovarian cancer, head and neck cancer, myeloma, liver cancer, breast cancer, hematoma, cervical cancer, glioma, and the like.
  • the tumor and/or cancer is positive for NKG2D ligand comprising the tumor and/or cancer being untreated is NKG2D ligand positive and the tumor and/or cancer is treated It became positive for NKG2D ligand.
  • the treatment includes becoming NKG2D ligand positive after treatment with a drug, radiation or biological agent.
  • a sample of a tumor and/or a cancer cell taken out from a host may be contacted with a chimeric antigen receptor-modified NK cell of the present invention at a concentration according to the degree of reaction between the two. It can be judged whether the tumor and/or cancer is NKG2D ligand positive or NKG2D ligand negative.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the chimeric antigen receptor-modified NK cell according to the present invention as an active ingredient, and a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition preferably comprises the chimeric antigen receptor-modified NK cells in a total dose ranging from 1 x 10 6 to 1 x 10 11 per subject per subject.
  • each treatment is for 3 weeks for a total of 21 days, 1-2 times a week.
  • the patient may be treated for one or more courses depending on the actual situation and needs.
  • the pharmaceutical composition can be administered by a suitable route of administration including intravenous administration (for example, intravenous drip administration or intravenous administration) or topical administration (for example, topical administration or local injection). Administration).
  • intravenous administration for example, intravenous drip administration or intravenous administration
  • topical administration for example, topical administration or local injection. Administration.
  • the invention also provides a method of treating a tumor and/or cancer comprising administering to a tumor and/or cancer patient a chimeric antigen receptor modified NK cell according to the invention.
  • the tumor and/or cancer is positive for NKG2D ligand, including colorectal cancer, ovarian cancer, head and neck cancer, myeloma, liver cancer, breast cancer, hematoma, cervical cancer, glioma, and the like.
  • the tumor and/or cancer is positive for NKG2D ligand comprising the tumor and/or cancer being untreated is NKG2D ligand positive and the tumor and/or cancer is treated It became positive for NKG2D ligand.
  • the treatment includes becoming NKG2D ligand positive after treatment with a drug, radiation or biological agent.
  • the chimeric antigen receptor-modified NK cells are preferably administered at a dose ranging from 1 x 10 6 to 1 x 10 11 cells per patient per course of treatment.
  • each treatment is for 3 weeks for a total of 21 days, 1-2 times a week.
  • the patient may be treated for one or more courses depending on the actual situation and needs.
  • the chimeric antigen receptor-modified NK cells can be administered by a suitable administration route, such as intravenous administration (for example, intravenous drip administration or intravenous administration) or topical administration (for example, topical drip). Injection or local injection).
  • a suitable administration route such as intravenous administration (for example, intravenous drip administration or intravenous administration) or topical administration (for example, topical drip). Injection or local injection).
  • the invention also provides a tool vector comprising, in turn, an operably linked CMV promoter, a T7 promoter, a 5'UTR having a kozak sequence, a GM-CSF alpha chain signal peptide coding sequence, and a polyA signal The 3'UTR of alpha globulin.
  • tool vector refers to an empty vector for insertion of an exogenous DNA fragment in genetic engineering applications.
  • the foreign DNA fragment When the foreign DNA fragment is inserted, the foreign DNA fragment is inserted between the GM-CSF ⁇ chain signal peptide coding sequence of the tool vector and the 3'UTR of the ⁇ globulin having the polyA signal (GM-CSF ⁇ chain signal)
  • the peptide coding sequence may have a multiple cloning site between the 3' UTR of the alpha globulin having a polyA signal).
  • the nucleotide sequence of the CMV promoter is set forth in SEQ ID NO: 22, and the nucleotide sequence of the T7 promoter is set forth in SEQ ID NO: 17, the core of the 5'UTR having the kozak sequence.
  • the nucleotide sequence of SEQ ID NO: 18, the nucleotide sequence of the GM-CSF ⁇ chain signal peptide coding sequence is set forth in SEQ ID NO: 19, and the 3'UTR of the alpha globulin having a PolyA signal
  • the nucleotide sequence is shown in SEQ ID NO:21.
  • the tool vector of the present invention is capable of promoting transcription and translation of the inserted DNA and enhancing the stability of the mRNA.
  • the percent concentration (%) of each reagent refers to the volume percent concentration (% (v/v)) of the reagent.
  • the PBS used in the following examples was purchased from Lonza under the order number BW17-517Q.
  • PBMCs venous blood mononuclear cells
  • PBMCs Mononuclear cells
  • RNA extraction kit RNAiso Reagent purchased from Life Technologies
  • the extracted total RNA was reverse transcribed into cDNA of PBMCs using a reverse transcription kit RevertAicTFirst Strand cDNA Synthesis Kit (purchased from Life Technologies), and stored at -20 ° C until use.
  • PCR amplification was carried out with primers P1 (SEQ ID NO: 9) and P2 (SEQ ID NO: 10) to obtain an extracellular domain comprising a NKG2D protein of 402 bp in length.
  • a fragment of the corresponding DNA coding sequence (nucleotide sequence as shown in SEQ ID NO: 6 in the Sequence Listing) has a Sphl and NheI restriction site and a protective base at both ends, respectively.
  • PCR amplification using primers P5 (SEQ ID NO: 13) and P6 (SEQ ID NO: 14) gave an intracellular signal domain containing DAP12 of 156 bp in length (nucleotide sequence such as SEQ ID NO in the sequence listing: A fragment of 8).
  • the PCR amplification reaction system was the same in each step.
  • the PCR reaction conditions were as described in KAPA HiFi Hot Start Ready Mix (2X) (purchased from Kapa Biosystems), and each reaction system (50 ⁇ L) was as follows:
  • Double distilled water 21.5 ⁇ L
  • the above PCR product was separated on a 1% (w/v) agarose gel, and DNA fragment recovery was carried out using an agarose gel DNA recovery kit (purchased from Omega Bio Tek).
  • the fragment containing the extracellular domain DNA of the NKG2D protein of 402 bp in length was subjected to double digestion with Sphl and NheI, and the digested product was subjected to DNA fragment recovery using an agarose gel DNA recovery kit.
  • the commercial vector pFastbac1 (Life Technologies) was added to a CMV promoter, a T7 promoter, a 5'UTR with a Kozak sequence, and a coding sequence for a GM-CSF alpha chain signal peptide ( Figure 1 SP), and a 3'UTR of alpha globulin with a polyA signal to construct the pFastbac1 basic skeleton vector.
  • the pFastbac1 basic skeleton vector was subjected to double digestion with SphI and SalI, and the digested product was subjected to DNA fragment recovery using an agarose gel DNA recovery kit, and then passed through the CD8 and DAP12 fragments recovered by the previous PCR.
  • lane 1 1000 kb DNA molecular weight marker
  • lane 2 plasmid pFastbac1-CD8-DAP12 digestion fragment, vector backbone 5276 bp, CD8-DAP12 fragment 405 bp.
  • the correct plasmid was sent to AITbiotech for sequencing of the inserted fusion gene fragment, and the correct recombinant plasmid was named pFastbac1-CD8-DAP12.
  • the vector pFastbac1-CD8-DAP12 was digested with SphI and NheI, and the digested product was recovered by DNA fragmentation using an agarose gel DNA recovery kit, and then ligated to the extracellular domain fragment of the previously recovered NKG2D protein by T4 DNA.
  • Enzyme-linked, linked product-transformed One Chemically Competent TOP10 chemically competent cells were cultured at 37 ° C for 18 hours, and then picked up, and cultured at 37 ° C, 250 rpm for 6 hours, and then the plasmid was extracted with a plasmid mini-kit.
  • the extracted plasmid was identified by restriction endonuclease SphI and NheI digestion, and the electrophoresis pattern was identified as shown in Fig. 3; wherein, lane 1: 1000 kb DNA molecular weight marker; lane 2: restriction fragment of plasmid pFastbac1-NKG2D-CD8-DAP12, The vector backbone is 5682 bp and the NKG2D fragment is 402 bp.
  • the correct plasmid was sent to AITbiotech for sequencing of the inserted fusion gene fragment, and the recombinant plasmid with the correct sequencing result was named pFastbac1-NKG2D-CD8-DAP12, and the plasmid map is shown in Figure 4.
  • PBMCs cells with 20 ⁇ 10 6 cells and 2 mL NK cell activator I purchased from Shenzhen Daktronics, DKW35-CYT-NK001
  • NK culture medium was AIM
  • NK cell activator I was uniformly mixed in 400 ml of NK medium, inoculated back into a G-Rex 100 cell culture incubator, and culture was continued for 7 days under the same conditions.
  • NK cells cultured by this method was as high as 90%, as shown in Fig. 5.
  • Fig. 5A indicate that the proportion of CD3-negative and CD56-positive NK cells is 90.2%
  • Fig. 5B indicate that the ratio of NK cell surface receptor NKG2D and CD16 double positive is 96.4%.
  • Tail-PCR technology was used to synthesize large-dose DNA double-stranded templates with PolyA in the positive strand and PolyT in the reverse strand for in vitro RNA synthesis, which reduced the instability of the DNA template.
  • the chimeric antigen receptor NKG2D-CD8-DAP12 coding sequence was subjected to Tail-PCR amplification using the above pFastbac1-NKG2D-CD8-DAP12 vector as a DNA template to synthesize linearization of the chimeric antigen receptor NKG2D-CD8-DAP12 sequence.
  • DNA template, Tail-PCR reaction conditions refer to the instructions of KAPA HiFiHotStartReadyMix (2X), the reaction system (50 ⁇ L) is as follows:
  • pFastbac1-NKG2D-CD8-DAP12 vector DNA template 500ng/ ⁇ L: 0.5 ⁇ L
  • the above PCR product was isolated and identified on a 1% (w/v) agarose gel, as shown in FIG.
  • the correct product was identified for in vitro synthesis of the chimeric antigen receptor NKG2D-CD8-DAP12 mRNA.
  • In vitro mRNA synthesis kit with capped mRNA synthesis kit as mMESSAGEmMACHINE T7 ULTRA Transcription Kit (available from Invitrogen, USA) or mScript TM RNA system (available from the American Epicentre Corporation). According to the instructions of the kit, and using the reagents provided in the kit for synthesis.
  • the in vitro synthesized chimeric antigen receptor NKG2D-CD8-DAP12 mRNA product was isolated and identified on a 1% (w/v) agarose gel, as shown in FIG. The correct mRNA was identified and stored at -80 ° C for storage.
  • NK cells prepared in Preparation Example 2 (1 ⁇ 10 7 cells) and 4 ⁇ g of NKG2D-CD8-DAP12 mRNA were mixed in electroporation P3 (product name "P3 Primary Cell” X Kit L”, Lonza, item number V4XP-3012), placed in a 100 ⁇ l Nucleocuvette TM tube (P3Primary Cell) X Kit L, Lonza, item number V4XP-3012), and frozen in an ice bath for 5 minutes. Then using the 4D-Nucleofector TM electroporation instrument (available from Lonza, Inc., Switzerland), choose their own program NK cell electroporation electroporation.
  • P3 product name "P3 Primary Cell” X Kit L”, Lonza, item number V4XP-3012
  • Test Example 1 Detection of NKG2D-CAR NK cells in vitro killing ability of human tumor cells
  • an ELISPOT assay was performed to detect the secretion of IFN ⁇ , since the secretion of IFN- ⁇ was positively correlated with the anti-tumor activity of NKG2D-CAR NK cells in adoptive cellular immunotherapy.
  • the preparation method of mGFP-CAR NK cells is the same as that of NKG2D-CAR NK cells (NK cells transfected with NKG2D-CD8-DAP12 mRNA), except that the vector is constructed.
  • the outer domain used the mGFP sequence without antigen binding function (its Genbank accession number is YP_002302326.1) as a negative control.
  • NK cells transfected with NKG2D-CD8-DAP12 mRNA The preparation of NK cells transfected with NKG2D-CD8-DAP12 mRNA is shown in Preparation 3.
  • NK cells transfected with NKG2D-CD8-DAP12 mRNA or mGFP-CD8-DAP12 mRNA were associated with human ovarian cancer cells SKOV3, human colorectal cancer cells HCT116 and SW480, human head and neck cancer cells Detroit, human hepatoma cells HepG2 and human neutrophils
  • the stromal cell U87 was co-cultured on the ELISPOT assay plate, and the ratio of the above NK effector cells to the target cells was 5:1. Each set of experiments was repeated 3 times. After 24 hours of co-cultivation, development and use of the software immunospot for ELISPOT point counting.
  • NK cells transfected with NKG2D-CD8-DAP12 mRNA produced more and stronger IFN- ⁇ than NK cells transfected with mGFP-CD8-DAP12 mRNA (detected by ELISPOT assay kit, purchased from Mabtech The product number is 3420-4HST-1).
  • the ELISPOT spots produced by NKG2D-CD8-DAP12 mRNA-transfected NK cells were significantly higher than the control group, as shown in Figure 9.
  • NK cells transfected with NKG2D-CD8-DAP12 mRNA or mGFP-CD8-DAP12 mRNA were associated with human colorectal cancer cell HCT116, human ovarian cancer cell SKOV3, human head and neck cancer cells Fadu and Detroit, human hepatoma cell HepG2, human breast cancer
  • the cell MCF7 and the human myeloma cell KG1 were co-cultured in a U-shaped 96-well plate, and the ratio of the above-mentioned NK effector cells to the target cells ranged from 2.5:1 to 10:1. Each set of experiments was repeated 3 times.
  • NKG2D-CAR NK cells were examined using the DELFIA EuTDA cytotoxicity kit (available from PerkinElmer, USA), and the killing effect was calculated by the following formula:
  • % specific lysis ((experimental group release (reading) - blank group release (reading)) / (maximum release (reading) - blank group release (reading))) x 100
  • NKG2D-CAR modified NK cells have broad and strong tumoricidal activity.
  • the killing effect of NKG2D-CAR modified NK cells is At 10:1, it was significantly higher than the killing effect of the control group, as shown in Figure 10.
  • the above results fully demonstrate the universality and high efficiency of NKG2D-CAR NK on tumor killing.
  • Test Example 2 Detection and analysis of NKG2D-CAR NK cells in vivo killing ability of human tumor cells
  • NKG2D-CAR NK cells The in vivo antitumor effect of NKG2D-CAR NK cells was further tested using a mouse model implanted in human tumors.
  • the experimental mice were non-obese diabetic/severe combined immunodeficiency/IL-2R ⁇ cnull (NSG) mice (6-8 weeks, female), and each mouse was implanted with 1 ⁇ 10 7 ovarian cancer cells SKOV3-Luc. .
  • NSG non-obese diabetic/severe combined immunodeficiency/IL-2R ⁇ cnull mice (6-8 weeks, female), and each mouse was implanted with 1 ⁇ 10 7 ovarian cancer cells SKOV3-Luc. .
  • Seven days after tumor implantation tumor growth was observed on the IVIS Spectrum imaging platform using in vivo bioimaging (BLI), and tumor growth was photographed using an imager (available from PerkinElmer, USA).
  • mice with similar BLI intensity were randomly divided into 2 groups: phosphate buffered saline (PBS) group, and NKG2D-CAR NK.
  • PBS phosphate buffered saline
  • NKG2D-CAR NK cell group 5 mice per group.
  • NKG2D-CAR NK cell group NKG2D-CAR-modified NK cells prepared by the method shown in Preparation Example 3 were intraperitoneally injected with 1 ⁇ 10 7 cells per mouse, and each mouse in the PBS group was intraperitoneally injected with a dose of 100 ⁇ L. PBS.
  • the cell injection protocol was: two to five injections per week for a total of three weeks (ie, six injections per group).
  • mice were closely observed and the development of the tumors was recorded by BLI. All light signals and images were analyzed by Xenogen in vivo imaging software v2.5. As shown in Figure 11, on the 28th day after tumor implantation, tumor growth showed that the tumor growth of mice in the PBS group was rapid, the light signal intensity increased about 10 times that of the 7th day, and all 5 of the NKG2D-CAR NK cell group. Tumor growth in mice was not only inhibited, but the original tumors were eliminated and the light signal intensity decreased to about 10% on day 7. Thus, NKG2D-CAR-modified NK cells can effectively kill tumors in vivo.
  • Test Example 3 Comparison of different chimeric antigen receptors
  • the NK cells modified with the chimeric antigen receptor NKG2D-CD8-DAP12 and the NK cells modified with the chimeric antigen receptor NKG2D-CD8-CD3Z were tested on tumor cells to compare the killing viability.
  • NK cells transfected with NKG2D-CD8-DAP12 mRNA, NKG2D-CD8-CD3z mRNA, or mGFP-CD8-DAP12 mRNA were co-cultured with human colorectal cancer cell line HCT116, human ovarian cancer cell line SKOV3, and human head and neck cancer cell line Detroit, respectively.
  • HCT116 human colorectal cancer cell line
  • SKOV3 human ovarian cancer cell line SKOV3
  • human head and neck cancer cell line Detroit respectively.
  • the ratio of the above NK cells to the target cells ranges from 2.5:1 to 10:1.
  • Each set of experiments was repeated 3 times.
  • the ability of NKG2D-CAR NK cells to lyse tumor cells was examined using the DELFIA EuTDA Cytotoxicity Kit (available from PerkinElmer, USA). The killing effect was calculated using the following formula:
  • % specific lysis ((experimental group release (reading) - blank group release (reading)) / (maximum release (reading) - blank group release (reading))) x 100
  • NKG2D-CD8-DAP12 mRNA-modified NK cells had significantly stronger tumoricidal activity than NKG2D-CD8- in the tumoricidal experiments of human colorectal cancer cell HCT116, human ovarian cancer cell SKOV3, and human head and neck cancer cell Detroit.
  • CD3z mRNA modified NK cells It is indicated that the chimeric antigen receptor NKG2D-CD8-DAP12 having the specific constituent unit combination created by the present invention has remarkable curative effect and good commercialization prospect.
  • NKG2D-CD8-CD3z modified NK cells were prepared in the same manner as NKG2D-CD8-DAP12 modified NK cells except that the intracellular signal domain was constructed using CD3z (the full-length amino acid sequence of Genbank number: NP_932170). .1) The intracellular signal sequence (shown as SEQ ID NO: 23).

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Abstract

Provided are a chimeric antigen receptor, an NK cell modified by same, a coding DNA, an mRNA, an expression vector, a preparation method and application. The chimeric antigen receptor comprises an antigen binding structural domain, a silent region, a transmembrane domain and an intracellular structural domain which are operatably connected and sequentially connected to each other in series. The antigen binding structural domain is from an NKG2D ligand binding domain, and the intracellular structural domain is from a DAP12 intracellular signal area.

Description

嵌合抗原受体、其修饰的NK细胞、编码DNA、mRNA、表达载体、制备方法和应用Chimeric antigen receptor, modified NK cell, coding DNA, mRNA, expression vector, preparation method and application thereof 技术领域Technical field
本发明属于生物技术领域，具体而言，涉及嵌合抗原受体、其修饰的NK细胞、编码DNA、mRNA、表达载体、制备方法和应用。The present invention belongs to the field of biotechnology, and in particular, to a chimeric antigen receptor, a modified NK cell thereof, a coding DNA, an mRNA, an expression vector, a preparation method and application.
背景技术Background technique
近几年随着对免疫机制的了解，导致了继手术治疗、化疗、放射性治疗之后又一治疗癌症的新技术的产生，即免疫治疗。癌症的免疫治疗被《Science》杂志评为2013年世界年度科技突破。嵌合抗原受体(Chimeric Antigen Receptor，CAR)是经人工改造的受体，因此可将任意的特异性受体嫁接到免疫效应细胞上。通常，这些受体的用于识别抗原的胞外特异性部分来自抗体的序列。由于这种受体的不同部分有不同的来源，因此被称为嵌合受体。CAR修饰的免疫细胞即表达嵌合性抗原受体(CAR)的免疫细胞，是现在最有效，最有希望的肿瘤细胞免疫治疗产品。In recent years, with the understanding of the immune mechanism, it has led to the emergence of another new technology for treating cancer after surgery, chemotherapy and radiotherapy, namely immunotherapy. The immunotherapy of cancer was named the 2013 World Technology Breakthrough by Science magazine. Chimeric Antigen Receptor (CAR) is an artificially engineered receptor, so any specific receptor can be grafted onto immune effector cells. Typically, the extracellular specific portion of these receptors used to recognize an antigen is derived from the sequence of the antibody. Because different parts of this receptor have different origins, they are called chimeric receptors. CAR-modified immune cells, ie, immune cells expressing the chimeric antigen receptor (CAR), are now the most effective and promising tumor cell immunotherapy products.
自然杀伤细胞(NK)是机体重要的免疫细胞，在外周血单个核细胞组成中占10％左右，是人体抗肿瘤、抗病毒感染的第一道防线。NK细胞与T细胞不同，不会产生自分泌生长因子如IL-2，而且相比T细胞，在遇到特异性抗原时不会增值过快，寿命较短，不需要装备自杀基因，也能限制CAR毒性。除此以外，NK细胞对肿瘤的杀伤不依赖于MHC呈递的特异性抗原，因此，在异体NK细胞的免疫治疗应用上，不会产生免疫排斥作用，是一种比T细胞更为安全的免疫治疗候选细胞。理论上，CAR可以避开NK细胞内抑制性信号通路，同时NK细胞自身可表达激活型受体，还能利用抗体介导的细胞毒作用(ADCC)，因此，CAR表达的NK细胞在肿瘤治疗上更优于T细胞，有着更为广阔的应用前景。Natural killer cells (NK) are important immune cells in the body, accounting for about 10% of the composition of peripheral blood mononuclear cells, which is the first line of defense against anti-tumor and anti-viral infections. Unlike T cells, NK cells do not produce autocrine growth factors such as IL-2, and they do not increase in value when they encounter specific antigens, and have a short life span. They do not need to be equipped with suicide genes. Limit CAR toxicity. In addition, the killing of tumors by NK cells does not depend on the specific antigen presented by MHC. Therefore, in the immunotherapy application of allogeneic NK cells, immune rejection is not produced, and it is a safer immunity than T cells. Treat candidate cells. In theory, CAR can avoid the inhibitory signaling pathway in NK cells, while NK cells themselves can express activated receptors and can also utilize antibody-mediated cytotoxicity (ADCC). Therefore, CAR-expressing NK cells are treated in tumors. It is superior to T cells and has a broader application prospect.
CAR包括胞外部分、跨膜区和胞内部分。对于携带CAR的免疫细胞而言，CAR的胞外部分和胞内部分的选择、及其与免疫细胞种类的配合极为重要，这与对肿瘤的特异性杀伤能力密切相关。目前在肿瘤的免疫治疗中，仍然需要开发出可供选择的新型且效果良好的CAR-免疫细胞药物。CAR includes an extracellular portion, a transmembrane region, and an intracellular portion. For immune cells carrying CAR, the selection of the extracellular and intracellular portions of CAR and its cooperation with immune cell types are extremely important, which is closely related to the specific killing ability of tumors. At present, in the immunotherapy of tumors, it is still necessary to develop a novel and effective CAR-immune cell drug.
发明内容Summary of the invention
为解决上述现有技术中所存在的问题，本发明提供了嵌合抗原受体、其修饰的NK细胞、编码DNA、mRNA、表达载体、制备方法和应用。In order to solve the problems in the prior art described above, the present invention provides a chimeric antigen receptor, a modified NK cell thereof, a coding DNA, an mRNA, an expression vector, a preparation method, and an application.
具体而言，本发明提供了：In particular, the present invention provides:
(1)一种嵌合抗原受体，该嵌合抗原受体包括可操作地连接的、依次串联的抗原结合结构域、间隔区、跨膜区和胞内结构域，其特征在于，所述抗原结合结构域来自NKG2D的配体结合区，所述胞内结构域来自DAP12的胞内信号区。(1) A chimeric antigen receptor comprising an operably linked, sequentially tandem antigen binding domain, a spacer, a transmembrane region and an intracellular domain, characterized in that said The antigen binding domain is derived from the ligand binding region of NKG2D, which is derived from the intracellular signaling region of DAP12.
(2)根据(1)所述的嵌合抗原受体，其中所述抗原结合结构域的氨基酸序列与NKG2D的第X位-第216位氨基酸序列一致，且73≤X≤83。(2) The chimeric antigen receptor according to (1), wherein the amino acid sequence of the antigen-binding domain is identical to the X-position 216 amino acid sequence of NKG2D, and 73 ≤ X ≤ 83.
(3)根据(1)或(2)所述的嵌合抗原受体，其中所述抗原结合结构域的氨基酸序列如SEQ ID NO：3所示。(3) The chimeric antigen receptor according to (1) or (2), wherein the amino acid sequence of the antigen-binding domain is as shown in SEQ ID NO: 3.
(4)根据(1)所述的嵌合抗原受体，其中所述胞内结构域的氨基酸序列选自DAP12的第62-113位氨基酸；优选地，所述胞内结构域的氨基酸序列如SEQ ID NO：5所示。(4) The chimeric antigen receptor according to (1), wherein the amino acid sequence of the intracellular domain is selected from amino acids 62 to 113 of DAP12; preferably, the amino acid sequence of the intracellular domain is as SEQ ID NO: 5 is shown.
(5)根据(1)所述的嵌合抗原受体，其中所述间隔区来自CD8α的铰链区，所述跨膜区来自CD8α的跨膜区。(5) The chimeric antigen receptor according to (1), wherein the spacer is derived from a hinge region of CD8α, and the transmembrane region is derived from a transmembrane region of CD8α.
(6)根据(1)或(5)所述的嵌合抗原受体，其中所述间隔区和跨膜区构成间隔跨膜区，并且其中所述间隔跨膜区的氨基酸序列与CD8α的第Y位-第210位氨基酸序列一致，且118≤Y≤128。(6) The chimeric antigen receptor according to (1) or (5), wherein the spacer and the transmembrane region constitute a spacer transmembrane region, and wherein the amino acid sequence of the spacer transmembrane region is the same as the CD8α The Y-position-210th amino acid sequence is identical, and 118≤Y≤128.
(7)根据(6)所述的嵌合抗原受体，其中所述间隔跨膜区的氨基酸序列如SEQ ID NO：4所示。(7) The chimeric antigen receptor according to (6), wherein the amino acid sequence of the spacer transmembrane region is as shown in SEQ ID NO: 4.
(8)根据(1)所述的嵌合抗原受体，其中所述嵌合抗原受体的氨基酸序列如SEQ ID NO：1所示。(8) The chimeric antigen receptor according to (1), wherein the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO: 1.
(9)一种分离的、编码嵌合抗原受体的DNA，该DNA包括可操作地连接的、依次串联的抗原结合结构域编码元件、间隔区编码元件、跨膜区编码元件和胞内结构域编码元件，其特征在于，所述抗原结合结构域编码元件来自NKG2D的配体结合区编码DNA，所述胞内结构域编码元件来自DAP12的胞内信号区编码DNA。(9) An isolated DNA encoding a chimeric antigen receptor comprising operably linked, sequentially tandem antigen-binding domain coding elements, spacer coding elements, transmembrane region coding elements, and intracellular structures A domain coding element characterized in that said antigen binding domain coding element is derived from a ligand binding region encoding DNA of NKG2D, said intracellular domain coding element being derived from the intracellular signal region encoding DNA of DAP12.
(10)根据(9)所述的DNA，其中所述抗原结合结构域编码元件的核苷酸序列编码所述抗原结合结构域的氨基酸序列，所述抗原结合结构域的氨基酸序列与NKG2D的第X位-第216位氨基酸序列一致，且73≤X≤83。(10) The DNA according to (9), wherein the nucleotide sequence of the antigen-binding domain coding element encodes an amino acid sequence of the antigen-binding domain, the amino acid sequence of the antigen-binding domain and the NKG2D The X-position - amino acid sequence at position 216 is identical, and 73 < X < 83.
(11)根据(9)或(10)所述的DNA，其中所述抗原结合结构域编码元件的核苷酸序列如SEQ ID NO：6所示。(11) The DNA according to (9) or (10), wherein the nucleotide sequence of the antigen-binding domain coding element is as shown in SEQ ID NO: 6.
(12)根据(9)所述的DNA，其中所述胞内结构域编码元件编码所述胞内结构域的氨基酸序列，所述胞内结构域的氨基酸序列选自DAP12的第62-113位氨基酸；优选地，所述胞内结构域编码元件的核苷酸序列如SEQ ID NO：8所示。(12) The DNA according to (9), wherein the intracellular domain coding element encodes an amino acid sequence of the intracellular domain, and the amino acid sequence of the intracellular domain is selected from positions 62-113 of DAP12. Amino acid; preferably, the nucleotide sequence of the intracellular domain coding element is set forth in SEQ ID NO: 8.
(13)根据(9)所述的DNA，其中所述间隔区编码元件来自CD8α的铰链区编码DNA，所述跨膜区编码元件来自CD8α的跨膜区编码DNA。(13) The DNA according to (9), wherein the spacer encoding element is derived from a hinge region encoding DNA of CD8α, and the transmembrane region coding element is derived from a transmembrane region encoding DNA of CD8α.
(14)根据(9)或(13)所述的DNA，其中所述间隔区编码元件和所述跨膜区编码元件构成间隔跨膜区编码元件，所述间隔跨膜区编码元件的核苷酸序列编码所述间隔跨膜区的氨基酸序列，所述间隔跨膜区的氨基酸序列与CD8α的第Y位-第210位氨基酸序列一致，且118≤Y≤128。(14) The DNA according to (9) or (13), wherein the spacer coding element and the transmembrane region coding element constitute a spacer transmembrane region coding element, the spacer transmembrane region coding element nucleoside The acid sequence encodes the amino acid sequence of the spacer transmembrane region, the amino acid sequence of the spacer transmembrane region being identical to the amino acid sequence of position Y to position 210 of CD8α, and 118 ≤ Y ≤ 128.
(15)根据(14)所述的DNA，其中所述间隔跨膜区编码元件的核苷酸序列如SEQ ID NO：7所示。(15) The DNA according to (14), wherein the nucleotide sequence of the spacer transmembrane region coding element is as shown in SEQ ID NO: 7.
(16)根据(9)所述的DNA，其核苷酸序列如SEQ ID NO：2所示。(16) The DNA according to (9), which has a nucleotide sequence as shown in SEQ ID NO: 2.
(17)一种分离的、由根据(9)-(16)中任一项所述的DNA 转录的mRNA。(17) An isolated mRNA transcribed by the DNA according to any one of (9) to (16).
(18)一种重组表达载体，其含有与启动子有效连接的根据(9)-(16)中任一项所述的DNA。(18) A recombinant expression vector comprising the DNA according to any one of (9) to (16), which is operably linked to a promoter.
(19)根据(18)所述的重组表达载体，其中所述重组表达载体在根据(9)-(16)中任一项所述的DNA之前依次包含CMV启动子、T7启动子、具有kozak序列的5’UTR和GM-CSF α链信号肽编码序列；并且在根据(9)-(16)中任一项所述的DNA之后包含具有PolyA信号的α球蛋白的3’UTR。(19) The recombinant expression vector according to (18), wherein the recombinant expression vector comprises a CMV promoter, a T7 promoter, and a kozak in sequence before the DNA according to any one of (9) to (16). The 5'UTR and GM-CSF alpha chain signal peptide coding sequences of the sequence; and the 3'UTR of the alpha globulin having a PolyA signal is included after the DNA according to any one of (9)-(16).
(20)一种嵌合抗原受体修饰的NK细胞，该NK细胞的表面被(1)-(8)中任一项所述的嵌合抗原受体修饰。(20) A chimeric antigen receptor-modified NK cell, the surface of which is modified by the chimeric antigen receptor according to any one of (1) to (8).
(21)一种制备根据(20)所述的嵌合抗原受体修饰的NK细胞的方法，包括以下步骤：(21) A method of producing a chimeric antigen receptor-modified NK cell according to (20), comprising the steps of:
1)提供NK细胞；1) providing NK cells;
2)提供编码根据(1)-(8)中任一项所述的嵌合抗原受体的核酸；2) A nucleic acid encoding the chimeric antigen receptor according to any one of (1) to (8);
3)将所述核酸转染入所述NK细胞中。3) Transfecting the nucleic acid into the NK cells.
(22)根据(21)所述的方法，其中步骤1)所述的NK细胞由外周血单个核细胞制备。(22) The method according to (21), wherein the NK cells of the step 1) are prepared from peripheral blood mononuclear cells.
(23)根据(21)所述的方法，其中所述转染通过冷冻电穿孔技术或慢病毒载体进行。(23) The method according to (21), wherein the transfection is carried out by a freeze electroporation technique or a lentiviral vector.
(24)根据(21)所述的方法，其中步骤2)所述的核酸为根据(9)-(16)中任一项所述的DNA、或根据(17)所述的mRNA。(24) The method according to (21), wherein the nucleic acid according to any one of (9) to (16), or the mRNA according to (17).
(25)根据(20)所述的嵌合抗原受体修饰的NK细胞在制备用于治疗或预防肿瘤和/或癌症的药物中的用途。(25) Use of the chimeric antigen receptor-modified NK cell according to (20) for the preparation of a medicament for treating or preventing a tumor and/or cancer.
(26)根据(25)所述的用途，其中所述肿瘤和/或癌症是NKG2D配体阳性的，包括所述肿瘤和/或癌症未经处理是NKG2D配体阳性的以及所述肿瘤和/或癌症经处理而成为NKG2D配体阳性的。(26) The use according to (25), wherein the tumor and/or cancer is NKG2D ligand positive, including the tumor and/or cancer being untreated is NKG2D ligand positive and the tumor and/or Or the cancer is treated to become positive for NKG2D ligand.
(27)根据(20)所述的嵌合抗原受体修饰的NK细胞在制备用于检测宿主的肿瘤和/或癌症的药物中的用途。(27) Use of the chimeric antigen receptor-modified NK cell according to (20) for the preparation of a medicament for detecting a tumor and/or cancer of a host.
(28)一种药物组合物，其中该药物组合物包括作为活性成分 的根据(20)所述的嵌合抗原受体修饰的NK细胞，及可药用辅料。(28) A pharmaceutical composition comprising the chimeric antigen receptor-modified NK cell according to (20) as an active ingredient, and a pharmaceutically acceptable adjuvant.
(29)根据(28)所述的药物组合物，其中所述药物组合物包含每人每个疗程总剂量范围为1×10 6-1×10 11个的所述嵌合抗原受体修饰的NK细胞。 (29) The pharmaceutical composition according to (28), wherein the pharmaceutical composition comprises the chimeric antigen receptor modified in a total dose ranging from 1×10 6 to 1×10 11 per subject per subject. NK cells.
(30)根据(28)所述的药物组合物，其中所述药物组合物的给药方式包括静脉给药或局部给药。(30) The pharmaceutical composition according to (28), wherein the pharmaceutical composition is administered by intravenous or topical administration.
(31)一种治疗肿瘤和/或癌症的方法，包括对肿瘤和/或癌症患者施用根据(20)所述的嵌合抗原受体修饰的NK细胞。(31) A method of treating a tumor and/or a cancer comprising administering a chimeric antigen receptor-modified NK cell according to (20) to a tumor and/or a cancer patient.
(32)根据(31)所述的方法，其中所述嵌合抗原受体修饰的NK细胞的施用剂量为每人每个疗程总剂量范围为1×10 6-1×10 11个细胞。 (32) The method according to (31), wherein the chimeric antigen receptor-modified NK cells are administered at a dose ranging from 1 x 10 6 to 1 x 10 11 cells per patient per course of treatment.
(33)根据(31)所述的方法，其中所述嵌合抗原受体修饰的NK细胞的给药方式包括静脉给药或局部给药。(33) The method according to (31), wherein the chimeric antigen receptor-modified NK cells are administered by intravenous or topical administration.
(34)根据(31)所述的方法，其中所述肿瘤和/或癌症是NKG2D配体阳性的，包括所述肿瘤和/或癌症未经处理是NKG2D配体阳性的以及所述肿瘤和/或癌症经处理而成为NKG2D配体阳性的。(34) The method of (31), wherein the tumor and/or cancer is NKG2D ligand positive, including the tumor and/or cancer being untreated is NKG2D ligand positive and the tumor and/ Or the cancer is treated to become positive for NKG2D ligand.
(35)一种工具载体，该工具载体依次包含可操作地连接的CMV启动子、T7启动子、具有kozak序列的5’UTR、GM-CSF α链信号肽编码序列、和具有PolyA信号的α球蛋白的3’UTR。(35) A tool vector comprising, in turn, an operably linked CMV promoter, a T7 promoter, a 5'UTR having a kozak sequence, a GM-CSF alpha chain signal peptide coding sequence, and an alpha having a PolyA signal The 3'UTR of the globulin.
(36)根据(35)所述的工具载体，其中所述CMV启动子的核苷酸序列如SEQ ID NO：22所示，T7启动子的核苷酸序列如SEQ ID NO：17所示，所述具有kozak序列的5’UTR的核苷酸序列如SEQ ID NO：18所示，所述GM-CSF α链信号肽编码序列的核苷酸序列如SEQ ID NO：19所示，所述具有PolyA信号的α球蛋白的3’UTR的核苷酸序列如SEQ ID NO：21所示。(36) The tool vector according to (35), wherein the nucleotide sequence of the CMV promoter is as shown in SEQ ID NO: 22, and the nucleotide sequence of the T7 promoter is as shown in SEQ ID NO: The nucleotide sequence of the 5'UTR having a kozak sequence is set forth in SEQ ID NO: 18, and the nucleotide sequence of the GM-CSF α chain signal peptide coding sequence is set forth in SEQ ID NO: 19, The nucleotide sequence of the 3'UTR of the alpha globulin having the PolyA signal is shown in SEQ ID NO:21.
本发明与现有技术相比具有以下优点和积极效果：Compared with the prior art, the invention has the following advantages and positive effects:
本发明的嵌合抗原受体能够使被其修饰的NK细胞(也称为“工程化NKG2D配体靶向性NK细胞”)对多种NKG2D配体表达阳性的肿瘤具有强烈且特异的靶向杀伤活性，本发明的临床前研究已充分 证明该修饰的NK细胞可以明显减小甚至消除动物体内的肿瘤负荷，延长动物的存活时间。The chimeric antigen receptor of the present invention enables the NK cells modified by it (also referred to as "engineered NKG2D ligand-targeted NK cells") to have strong and specific targeting to tumors positive for expression of various NKG2D ligands. The killing activity, the preclinical study of the present invention has fully demonstrated that the modified NK cells can significantly reduce or even eliminate the tumor burden in the animal and prolong the survival time of the animal.
用本发明的工程化NKG2D配体靶向性NK细胞治疗肿瘤时，可有效避免采用CAR-T治疗时所引起的细胞因子风暴和免疫排斥反应。When treating tumors with the engineered NKG2D ligand-targeted NK cells of the present invention, cytokine storms and immune rejection caused by CAR-T treatment can be effectively avoided.
本发明的工程化NKG2D配体靶向性的NK细胞为治疗NKG2D配体阳性肿瘤患者提供了一种新的选择，具有良好的产业应用前景。The engineered NKG2D ligand-targeted NK cells of the invention provide a new choice for treating NKG2D ligand-positive tumor patients, and have good industrial application prospects.
附图说明DRAWINGS
图1为示出本发明一个实施方案的嵌合抗原受体NKG2D-CD8-DAP12的构建示意图，其中“CMV”表示CMV启动子序列，“T7”表示T7启动子序列，“5’UTR”表示具有Kozak序列的5’UTR，“SP”表示GM-CSF α链信号肽编码序列，“NKG2D”表示NKG2D的配体结合区的编码序列，“CD8”表示CD8α的铰链区和跨膜区编码序列，“DAP12”表示DAP12的胞内信号区编码序列，“α球蛋白3’UTR”表示具有PolyA信号的α球蛋白的3’UTR，“pA 150”表示polyA(多聚腺苷酸)，其中包含150个A。 BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic diagram showing the construction of a chimeric antigen receptor NKG2D-CD8-DAP12 according to an embodiment of the present invention, wherein "CMV" indicates a CMV promoter sequence, "T7" indicates a T7 promoter sequence, and "5'UTR" indicates 5'UTR with Kozak sequence, "SP" indicates the GM-CSF alpha chain signal peptide coding sequence, "NKG2D" indicates the coding sequence of the ligand binding region of NKG2D, and "CD8" indicates the hinge region and transmembrane region coding sequence of CD8α "DAP12" denotes the intracellular signal region coding sequence of DAP12, "alpha globulin 3'UTR" denotes the 3'UTR of the alpha globulin having a PolyA signal, and "pA 150 " denotes polyA (polyadenylation), wherein Contains 150 As.
图2为示出本发明一个实施方案的表达载体pFastbac1-CD8-DAP12经限制性内切酶SphI和SalI双酶切的鉴定片段的电泳图；其中泳道1为DNA分子量标记，泳道2为鉴定片段。Figure 2 is an electropherogram showing the identification fragment of the expression vector pFastbac1-CD8-DAP12 digested with restriction endonucleases SphI and SalI according to one embodiment of the present invention; wherein lane 1 is a DNA molecular weight marker and lane 2 is an identification fragment .
图3为示出本发明一个实施方案的表达载体pFastbac1-NKG2D-CD8-DAP12经限制性内切酶SphI和NheI双酶切的鉴定片段的电泳图；其中泳道1为DNA分子量标记，泳道2为鉴定片段。Figure 3 is an electropherogram showing the identification fragment of the expression vector pFastbac1-NKG2D-CD8-DAP12 digested with restriction endonucleases SphI and NheI according to an embodiment of the present invention; wherein lane 1 is a DNA molecular weight marker, and lane 2 is Identify the fragment.
图4为示出本发明一个实施方案的嵌合型抗原受体的重组DNA载体pFastbac1-NKG2D-CD8-DAP12的结构示意图；其中，顺时针序列为正向基因片段，逆时针为反向基因片段。其中“CMV”表示CMV启动子序列，“T7”表示T7启动子序列，“5’UTR”表示具有Kozak序列的5’UTR，“GM-CSF α”表示GM-CSF α链信号肽编码序列，“NKG2D”表示NKG2D的配体结合区的编码序列，“CD8”表示CD8α的铰链区和跨膜区编码序列，“DAP12”表示DAP12的胞内 信号区编码序列，“α球蛋白3’UTR”表示具有PolyA信号的α球蛋白的3’UTR。Figure 4 is a schematic view showing the structure of a recombinant DNA vector pFastbac1-NKG2D-CD8-DAP12 of a chimeric antigen receptor according to an embodiment of the present invention; wherein the clockwise sequence is a forward gene fragment and the counterclockwise is a reverse gene fragment . Wherein "CMV" denotes a CMV promoter sequence, "T7" denotes a T7 promoter sequence, "5'UTR" denotes a 5'UTR having a Kozak sequence, and "GM-CSF α" denotes a GM-CSF alpha chain signal peptide coding sequence, "NKG2D" indicates the coding sequence of the ligand binding region of NKG2D, "CD8" indicates the hinge region and transmembrane region coding sequence of CD8α, and "DAP12" indicates the intracellular signal region coding sequence of DAP12, "alpha globulin 3'UTR" Represents the 3'UTR of the alpha globulin with the PolyA signal.
图5示出流式细胞术对NK细胞表型的分析结果。图5A示出从外周血单个核细胞扩增17天时NK细胞的纯度。横坐标表示CD3表达强度，纵坐标表示CD56表达强度；图5B示出从外周血单个核细胞扩增17天时NK细胞内源NKG2D和CD16的表达强度，横坐标表示NKG2D表达强度，纵坐标表示CD16表达强度。Figure 5 shows the results of analysis of NK cell phenotype by flow cytometry. Figure 5A shows the purity of NK cells when expanded from peripheral blood mononuclear cells for 17 days. The abscissa indicates the CD3 expression intensity, and the ordinate indicates the CD56 expression intensity; FIG. 5B shows the expression intensity of NK cells endogenous NKG2D and CD16 when expanded from peripheral blood mononuclear cells for 17 days, the abscissa indicates the intensity of NKG2D expression, and the ordinate indicates CD16. Expression intensity.
图6示出体外合成的嵌合抗原受体NKG2D-CD8-DAP12线性化DNA模板的电泳图；其中泳道1为DNA分子量标记，泳道2为线性化DNA模板。Figure 6 shows an electropherogram of a chimeric antigen receptor NKG2D-CD8-DAP12 linearized DNA template synthesized in vitro; Lane 1 is a DNA molecular weight marker and Lane 2 is a linearized DNA template.
图7示出体外合成的嵌合抗原受体NKG2D-CD8-DAP12的mRNA的电泳图；其中泳道1为分子量标记，泳道2为NKG2D-CD8-DAP12的mRNA。Figure 7 shows an electropherogram of mRNA of the chimeric antigen receptor NKG2D-CD8-DAP12 synthesized in vitro; Lane 1 is a molecular weight marker and Lane 2 is an mRNA of NKG2D-CD8-DAP12.
图8示出流式细胞术对嵌合抗原受体NKG2D-CD8-DAP12 mRNA电转NK细胞效率的检测结果。图8A示出NK细胞内源表达NKG2D的强度(其中左侧峰为阴性对照曲线，右侧峰为NK细胞NKG2D表达强度曲线)；图8B示出电转染后NK细胞表达NKG2D的强度(其中左侧峰为阴性对照曲线，右侧峰为NK细胞电转NKG2D-CD8-DAP12 mRNA之后NKG2D的表达强度曲线)；图8C示出NK细胞内源表达和电转染后表达NKG2D的强度的比较(即，将图8A和8B合并在一起)(其中左侧峰为阴性对照曲线，中间峰为NK细胞NKG2D表达强度曲线，右侧峰为NK细胞电转NKG2D-CD8-DAP12 mRNA之后NKG2D的表达强度曲线)。图中横坐标表示NKG2D表达强度，纵坐标表示相对细胞数。横坐标中“NKG2D荧光强度”表示用带荧光的NKG2D抗体检测时，流式细胞仪所显示的荧光读数。Figure 8 shows the results of flow cytometry detection of chimeric antigen receptor NKG2D-CD8-DAP12 mRNA electroporation NK cells. Figure 8A shows the intensity of endogenous expression of NKG2D by NK cells (where the left peak is the negative control curve and the right peak is the NK cell NKG2D expression intensity curve); Figure 8B shows the intensity of NKG2D expression by NK cells after electroporation (where The left peak is the negative control curve, and the right peak is the NKG2D expression intensity curve after NK cell electrotransformation of NKG2D-CD8-DAP12 mRNA; Figure 8C shows the comparison of the intensity of NKG2D expression after endogenous expression and electrotransfection of NK cells ( That is, 8A and 8B are combined together) (the left peak is a negative control curve, the middle peak is the NK cell NKG2D expression intensity curve, and the right peak is the NKG2D-CD8-DAP12 mRNA expression level after NK cell electroporation. ). In the figure, the abscissa indicates the intensity of NKG2D expression, and the ordinate indicates the relative cell number. "NKG2D fluorescence intensity" in the abscissa indicates the fluorescence reading displayed by the flow cytometer when detected with a fluorescent NKG2D antibody.
图9示出本发明实施例的嵌合抗原受体NKG2D-CD8-DAP12修饰的NK(NKG2D-CAR-NK)细胞与人肿瘤细胞混合培养后，释放IFN-γ的Elispot分析结果。图中“mGFP-CAR-NK”表示mGFP-CAR修饰的NK细胞组(对照组)，“NKG2D-CAR-NK”表示嵌合抗原 受体NKG2D-CD8-DAP12(即NKG2D-CAR)修饰的NK细胞组。图中横坐标表示肿瘤细胞组别，纵坐标表示相对IFN-γ数量(以每2.5×10 4个NK细胞中的斑点数表示)。 Figure 9 is a graph showing the results of Elispot analysis of IFN-γ release after mixed culture of chimeric antigen receptor NKG2D-CD8-DAP12-modified NK (NKG2D-CAR-NK) cells and human tumor cells in an embodiment of the present invention. In the figure, "mGFP-CAR-NK" indicates an mGFP-CAR-modified NK cell group (control group), and "NKG2D-CAR-NK" indicates a chimeric antigen receptor NKG2D-CD8-DAP12 (i.e., NKG2D-CAR)-modified NK. Cell group. In the figure, the abscissa indicates the tumor cell group, and the ordinate indicates the relative amount of IFN-γ (expressed as the number of spots per 2.5 × 10 4 NK cells).
图10A-G分别示出本发明实施例的NKG2D-CAR-NK对肿瘤细胞HCT116(A)、SKOV3(B)、Fadu(C)、Detroit(D)、HepG2(E)、MCF7(F)、KG1(G)的杀伤活力检测结果；其中纵坐标表示肿瘤细胞被杀伤后裂解的比例；横坐标表示效应细胞和肿瘤细胞的比例；实线即“NKG2D-CAR-NK”示出嵌合抗原受体NKG2D-CAR修饰的NK细胞组的结果；虚线即“mGFP-CAR-NK”示出嵌合抗原受体mGFP-CAR修饰的NK细胞组(对照组)的结果。10A-G show NKG2D-CAR-NK versus tumor cells HCT116(A), SKOV3(B), Fadu(C), Detroit(D), HepG2(E), MCF7(F), respectively, according to an embodiment of the present invention, The killing activity test result of KG1(G); the ordinate indicates the proportion of tumor cells ruptured after killing; the abscissa indicates the ratio of effector cells to tumor cells; the solid line is “NKG2D-CAR-NK” showing chimeric antigen Results of the NKG2D-CAR-modified NK cell group; the dotted line, "mGFP-CAR-NK", shows the results of the chimeric antigen receptor mGFP-CAR-modified NK cell group (control group).
图11示出过继回输NKG2D-CAR-NK细胞对肿瘤细胞的杀伤作用；虚线示出嵌合抗原受体NKG2D-CD8-DAP12修饰NK细胞组的结果(图中示为“NKG2D-CAR-NK(6次注射)”)；实线示出未进行NK细胞回输组的结果(图中示为“PBS对照组”)。图中横坐标表示接种肿瘤后的天数，纵坐标表示活体成像仪记录的动物体内肿瘤细胞荧光强度，其中纵坐标中所示辐射率(单位为“p/秒/cm 2/sr”，即，photons/sec/cm 2/steradian)指单位时间、单位面积、单位弧度从动物体表发出的光子数。 Figure 11 shows the killing effect of adoptively NKG2D-CAR-NK cells on tumor cells; the dotted line shows the results of the chimeric antigen receptor NKG2D-CD8-DAP12 modified NK cell group (shown as "NKG2D-CAR-NK" (6 injections)"); the solid line shows the results of the NK cell reinfusion group (shown as "PBS control group"). In the figure, the abscissa indicates the number of days after inoculation of the tumor, and the ordinate indicates the fluorescence intensity of the tumor cells in the animal recorded by the living imager, wherein the radiance shown in the ordinate (in units of "p/sec/cm 2 /sr", ie, Photons/sec/cm 2 /steradian) refers to the number of photons emitted from the surface of the animal per unit time, unit area, and unit radians.
图12 A-C分别示出嵌合抗原受体NKG2D-CD8-DAP12修饰的NK细胞和嵌合抗原受体NKG2D-CD8-CD3Z修饰的NK细胞分别对肿瘤细胞SKOV3(A)、Detroit(B)、HCT116(C)的杀伤活力的检测结果比较。图中“mGFP-CAR-NK”表示mGFP-CAR修饰的NK细胞组，“NKG2D-DAP12-CAR-NK”表示NKG2D-DAP12-CAR修饰的NK细胞组，“NKG2D-CD3Z-CAR-NK”表示NKG2D-CD3Z-CAR修饰的NK细胞组。图中横坐标表示效应细胞和肿瘤细胞的比例，纵坐标表示肿瘤细胞被杀伤后裂解的比例。Figure 12 AC shows chimeric antigen receptor NKG2D-CD8-DAP12 modified NK cells and chimeric antigen receptor NKG2D-CD8-CD3Z modified NK cells, respectively, on tumor cells SKOV3 (A), Detroit (B), HCT116 (C) Comparison of the test results of killing activity. In the figure, "mGFP-CAR-NK" indicates an mGFP-CAR-modified NK cell group, "NKG2D-DAP12-CAR-NK" indicates an NKG2D-DAP12-CAR-modified NK cell group, and "NKG2D-CD3Z-CAR-NK" indicates NKG2D-CD3Z-CAR modified NK cell group. In the figure, the abscissa indicates the ratio of effector cells to tumor cells, and the ordinate indicates the proportion of tumor cells that are lysed after killing.
具体实施方式Detailed ways
以下通过具体实施方式的描述并参照附图对本发明作进一步说明，但这并非是对本发明的限制，本领域技术人员根据本发明的基本 思想，可以做出各种修改或改进，但是只要不脱离本发明的基本思想，均在本发明的范围之内。The invention is further described by the following description of the embodiments and with reference to the accompanying drawings, but this is not a limitation of the invention, and those skilled in the art can make various modifications or improvements according to the basic idea of the invention, but The basic idea of the invention is within the scope of the invention.
在本发明中，词语“肿瘤”、“癌症”、“肿瘤细胞”、“癌细胞”涵盖本领域通常认为的含义。In the present invention, the words "tumor", "cancer", "tumor cell", "cancer cell" encompass the meanings generally recognized in the art.
CAR包括胞外部分、跨膜区和胞内部分。胞外部分又包括用于识别和结合抗原的抗原结合结构域、以及用于间隔抗原结合结构域和跨膜区的间隔区；胞内部分主要包括用于信号传递的胞内结构域。对于携带CAR的免疫细胞而言，CAR的功能性部分的选择、及其与免疫细胞种类的配合极为重要，这与对肿瘤的特异性杀伤能力密切相关。本发明的发明人通过理论研究和实验摸索，针对NK细胞选择了特定的抗原结合结构域和胞内结构域的组合，并将由此开发的CAR成功地应用于了NK细胞，使该NK细胞发挥了强烈的靶向杀瘤活性，由此开发出在肿瘤免疫治疗中可供选择的新型且效果良好的工程化NKG2D配体靶向性的NK细胞。CAR includes an extracellular portion, a transmembrane region, and an intracellular portion. The extracellular portion in turn includes an antigen binding domain for recognizing and binding an antigen, and a spacer for spacing the antigen binding domain and the transmembrane region; the intracellular portion primarily includes an intracellular domain for signaling. For immune cells carrying CAR, the selection of the functional part of CAR and its cooperation with immune cell types are extremely important, which is closely related to the specific killing ability of tumors. The inventors of the present invention have selected a specific combination of an antigen-binding domain and an intracellular domain for NK cells through theoretical research and experimental exploration, and successfully applied the thus developed CAR to NK cells to make the NK cells play. A strong targeted tumoricidal activity, thereby developing a novel and effective engineered NKG2D ligand-targeted NK cell that is available for selection in tumor immunotherapy.
本发明所述术语“抗原结合结构域”、“间隔区”、“跨膜区”、“胞内结构域”的定义可参考“《免疫学导论》，于善谦，高等教育出版社，2008”；以及“《Immunobiology》，第七版，Kenneth Murphy，Paul Travers，Mark Walport等”。The definitions of the terms "antigen-binding domain", "spacer", "transmembrane region" and "intracellular domain" of the present invention can be referred to "Introduction to Immunology", Yu Shanqian, Higher Education Press, 2008 "; and "Immunobiology, seventh edition, Kenneth Murphy, Paul Travers, Mark Walport, etc.".
具体而言，本发明提供了一种嵌合抗原受体，该嵌合抗原受体包括可操作地连接的、依次串联的抗原结合结构域、间隔区、跨膜区和胞内结构域，其特征在于，所述抗原结合结构域来自NKG2D的配体结合区，所述胞内结构域来自DAP12的胞内信号区。In particular, the invention provides a chimeric antigen receptor comprising an operably linked, sequentially tandem antigen binding domain, a spacer, a transmembrane region and an intracellular domain, Characterized in that the antigen binding domain is from the ligand binding region of NKG2D, which is derived from the intracellular signaling region of DAP12.
NKG2D是调节NK细胞杀伤活性的重要受体，NKG2D的配体主要表达于肿瘤细胞和应激细胞表面，很少表达甚至不表达于正常细胞表面，大量的肿瘤细胞如结直肠癌细胞、卵巢癌细胞、头颈癌细胞、淋巴癌细胞、神经胶质瘤细胞等都有大量NKG2D的配体的表达。NKG2D is an important receptor regulating NK cell killing activity. NKG2D ligand is mainly expressed on the surface of tumor cells and stress cells, and is rarely expressed or even expressed on the surface of normal cells. A large number of tumor cells such as colorectal cancer cells and ovarian cancer Cells, head and neck cancer cells, lymphatic cancer cells, glioma cells, and the like have a large number of ligands for NKG2D expression.
本发明进一步发现，所述抗原结合结构域的氨基酸序列优选与NKG2D的第X位-第216位氨基酸序列一致，且73≤X≤83，X为整数。其中，NKG2D的氨基酸序列可为NCBI(即，美国国立生物技术信息中心，网址：https：//www.ncbi.nlm.nih.gov)的Genbank中编号为 NP_031386.2的氨基酸序列。也就是说，所述抗原结合结构域的氨基酸序列优选选自NKG2D的第73-216位氨基酸、并且包含第83-216位氨基酸。例如，所述抗原结合结构域的氨基酸序列如以下氨基酸序列组中任一氨基酸序列所示：NKG2D的第73-216位氨基酸、第74-216位氨基酸、第75-216位氨基酸、第76-216位氨基酸、第77-216位氨基酸、第78-216位氨基酸、第79-216位氨基酸、第80-216位氨基酸、第81-216位氨基酸、第82-216位氨基酸、或第83-216位氨基酸。更优选地，所述抗原结合结构域的氨基酸序列如SEQ ID NO：3所示。The present invention further found that the amino acid sequence of the antigen-binding domain is preferably identical to the X-position 216 amino acid sequence of NKG2D, and 73 ≤ X ≤ 83, and X is an integer. The amino acid sequence of NKG2D may be the amino acid sequence numbered NP_031386.2 in Genbank of NCBI (ie, National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov). That is, the amino acid sequence of the antigen-binding domain is preferably selected from amino acids 73-216 of NKG2D and comprises amino acids 83-216. For example, the amino acid sequence of the antigen-binding domain is as shown in any one of the following amino acid sequence groups: amino acids 73-216 of NKG2D, amino acids 74-216, amino acids 75-216, 76- 216 amino acids, amino acids 77-216, amino acids 78-216, amino acids 79-216, amino acids 80-216, amino acids 81-216, amino acids 82-216, or 83- 216 amino acids. More preferably, the amino acid sequence of the antigen binding domain is set forth in SEQ ID NO: 3.
胞内结构域起到信号传递从而活化NK细胞的作用。最初用于T细胞的CAR的胞内结构域只有一个信号分子，通常为免疫球蛋白E的受体相关FcεRIγ(与IgE有高亲和力的受体的一个亚基)或T细胞抗原受体信号的基础传导分子DAP12；有的胞内结构域包含由一种或几种T细胞活化基序组成的T细胞活化结构域。本发明发现，将上述来自NKG2D的配体结合区的抗原结合结构域与来自DAP12的胞内信号区进行组合，可得到能够使NK细胞发挥强烈的靶向杀瘤活性的CAR。优选地，所述胞内结构域的氨基酸序列选自DAP12的第62-113位氨基酸，所述胞内结构域的氨基酸序列更优选如SEQ ID NO：5所示。其中，DAP12的氨基酸序列的Genbank编号为NP_003323.1。The intracellular domain acts as a signal to activate NK cells. The intracellular domain of the CAR originally used for T cells has only one signaling molecule, usually the receptor-associated FcεRIγ of immunoglobulin E (a subunit of a receptor with high affinity for IgE) or T cell antigen receptor signaling. The underlying conductive molecule DAP12; some intracellular domains comprise a T cell activation domain consisting of one or several T cell activation motifs. The present inventors have found that by combining the antigen-binding domain derived from the ligand binding region of NKG2D described above with the intracellular signal region derived from DAP12, a CAR capable of exerting a strong targeted tumoricidal activity of NK cells can be obtained. Preferably, the amino acid sequence of the intracellular domain is selected from amino acids 62-113 of DAP12, and the amino acid sequence of the intracellular domain is more preferably as set forth in SEQ ID NO: 5. Among them, the amino acid sequence of DAP12 has the Genbank number NP_003323.1.
本发明更进一步选择了间隔区和跨膜区，从而获得了具有抗原结合结构域-间隔区-跨膜区-胞内结构域的特定组合的CAR。间隔区连接识别和结合抗原的抗原结合结构域和跨膜区，这一区域的结构应该是易弯曲的，这样可以使抗原结合结构域适应不同的方向，以促进抗原的识别和结合。形式最简单的间隔区是免疫球蛋白IgGl的铰链区(hinge)，也可以是免疫球蛋白C H2C H3区的一部分。跨膜区一般是跨越细胞膜的疏水性α螺旋。通过研究和实验摸索，本发明发现，所述间隔区优选来自CD8α的铰链区，所述跨膜区优选来自CD8α的跨膜区。CD8为跨膜的糖基化膜蛋白，由α和β两个亚基组成，与T细胞表面受体共同作用使T细胞与特定抗原结合，CD8特异性结合MHCI，介导细胞毒性T细胞的杀伤作用。 The present invention further selects the spacer and transmembrane regions, thereby obtaining a CAR having a specific combination of antigen binding domain-spacer-transmembrane region-intracellular domain. The spacer junction recognizes and binds to the antigen binding domain and transmembrane region of the antigen. The structure of this region should be flexible so that the antigen binding domain can be adapted to different orientations to facilitate antigen recognition and binding. The simplest form of spacer region is an immunoglobulin hinge region of IgGl (hinge), it may be a portion of an immunoglobulin C H2 C H3 region. The transmembrane region is typically a hydrophobic alpha helix spanning the cell membrane. Through research and experimental exploration, the present inventors have found that the spacer is preferably derived from the hinge region of CD8[alpha], which is preferably from the transmembrane region of CD8[alpha]. CD8 is a transmembrane glycosylated membrane protein consisting of two subunits, alpha and beta, which interact with T cell surface receptors to bind T cells to specific antigens. CD8 specifically binds to MHCI and mediates cytotoxic T cells. Killing effect.
更优选地，所述间隔区和跨膜区构成间隔跨膜区，并且其中所 述间隔跨膜区的氨基酸序列与CD8α的第Y位-第210位氨基酸序列一致，且118≤Y≤128，Y为整数。其中，CD8α的氨基酸序列的Genbank编号可为NP_001139345.1。也就是说，所述间隔跨膜区的氨基酸序列优选选自CD8α的第118-210位氨基酸、并且包含第128-210位氨基酸。例如，所述间隔跨膜区的氨基酸序列如以下氨基酸序列组中任一氨基酸序列所示：CD8α的第118-210位氨基酸、第119-210位氨基酸、第120-210位氨基酸、第121-210位氨基酸、第122-210位氨基酸、第123-210位氨基酸、第124-210位氨基酸、第125-210位氨基酸、第126-210位氨基酸、第127-210位氨基酸、或第128-210位氨基酸。More preferably, the spacer region and the transmembrane region constitute a spacer transmembrane region, and wherein the amino acid sequence of the spacer transmembrane region is identical to the amino acid sequence of the Yth to the 210th position of CD8α, and 118≤Y≤128, Y is an integer. Wherein, the Genbank number of the amino acid sequence of CD8α may be NP_001139345.1. That is, the amino acid sequence of the spacer transmembrane region is preferably selected from amino acids 118-210 of CD8α and amino acids 128-210. For example, the amino acid sequence of the spacer transmembrane region is as shown in any one of the following amino acid sequence groups: amino acids 118-210 of CD8α, amino acids 119-210, amino acids 120-210, 121- 210 amino acids, amino acids 122-210, amino acids 123-210, amino acids 124-210, amino acids 125-210, amino acids 126-210, amino acids 127-210, or 128- 210 amino acids.
更优选地，所述间隔跨膜区的氨基酸序列如SEQ ID NO：4所示。More preferably, the amino acid sequence of the spacer transmembrane region is set forth in SEQ ID NO:4.
在本发明的嵌合抗原受体中，抗原结合结构域、间隔区、跨膜区和胞内结构域是依次串联的；抗原结合结构域和间隔区之间、间隔区和跨膜区之间、跨膜区和胞内结构域之间是可操作地连接的，例如可以采用接头连接，也可以不采用接头而直接连接。在本发明一个实施方案中，抗原结合结构域和间隔区之间采用接头连接(所述接头(例如)为-Ala-Ser-)，而间隔区和跨膜区之间、跨膜区和胞内结构域之间未采用接头而直接连接。In the chimeric antigen receptor of the present invention, the antigen-binding domain, the spacer, the transmembrane region and the intracellular domain are sequentially connected in series; between the antigen-binding domain and the spacer, between the spacer and the transmembrane region The transmembrane region and the intracellular domain are operably linked, for example, may be connected by a joint or directly without a joint. In one embodiment of the invention, a linker is used between the antigen binding domain and the spacer (the linker is, for example, -Ala-Ser-), and the spacer and transmembrane region, the transmembrane region and the cell The internal domains are directly connected without a joint.
在本发明一个优选的实施方案中，所述嵌合抗原受体的氨基酸序列如SEQ ID NO：1所示。在本发明另一个优选的实施方案中，所述嵌合抗原受体具有在SEQ ID NO：1所示氨基酸序列中替换、删除、和/或添加一个或多个氨基酸而得到的氨基酸序列；例如，所述嵌合抗原受体具有与SEQ ID NO：1所示氨基酸序列至少90％、优选至少95％、更优选至少99％的一致性。In a preferred embodiment of the invention, the amino acid sequence of the chimeric antigen receptor is set forth in SEQ ID NO: 1. In another preferred embodiment of the present invention, the chimeric antigen receptor has an amino acid sequence obtained by replacing, deleting, and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1; The chimeric antigen receptor has at least 90%, preferably at least 95%, more preferably at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 1.
本发明还提供了一种分离的、编码本发明所述的嵌合抗原受体的DNA，该DNA包括可操作地连接的、依次串联的抗原结合结构域编码元件、间隔区编码元件、跨膜区编码元件和胞内结构域编码元件，其特征在于，所述抗原结合结构域编码元件来自NKG2D的配体结合区编码DNA，所述胞内结构域编码元件来自DAP12的胞内信号区编码DNA。The invention also provides an isolated DNA encoding a chimeric antigen receptor of the invention, the DNA comprising an operably linked, sequentially tandem antigen binding domain coding element, a spacer coding element, a transmembrane a region coding element and an intracellular domain coding element, wherein the antigen binding domain coding element is derived from a ligand binding region encoding DNA of NKG2D, and the intracellular domain coding element is derived from the intracellular signal region encoding DNA of DAP12 .
所述抗原结合结构域编码元件的核苷酸序列编码所述抗原结合结构域的氨基酸序列，优选地，所述抗原结合结构域的氨基酸序列与NKG2D的第X位-第216位氨基酸序列一致，且73≤X≤83，X为整数。例如，所述抗原结合结构域的氨基酸序列如以下氨基酸序列组中任一氨基酸序列所示：NKG2D的第73-216位氨基酸、第74-216位氨基酸、第75-216位氨基酸、第76-216位氨基酸、第77-216位氨基酸、第78-216位氨基酸、第79-216位氨基酸、第80-216位氨基酸、第81-216位氨基酸、第82-216位氨基酸、或第83-216位氨基酸。优选地，所述抗原结合结构域编码元件的核苷酸序列如SEQ ID NO：6所示。The nucleotide sequence of the antigen-binding domain coding element encodes an amino acid sequence of the antigen-binding domain, preferably, the amino acid sequence of the antigen-binding domain is identical to the X-position 216 amino acid sequence of NKG2D, And 73 ≤ X ≤ 83, and X is an integer. For example, the amino acid sequence of the antigen-binding domain is as shown in any one of the following amino acid sequence groups: amino acids 73-216 of NKG2D, amino acids 74-216, amino acids 75-216, 76- 216 amino acids, amino acids 77-216, amino acids 78-216, amino acids 79-216, amino acids 80-216, amino acids 81-216, amino acids 82-216, or 83- 216 amino acids. Preferably, the nucleotide sequence of the antigen binding domain coding element is set forth in SEQ ID NO: 6.
所述胞内结构域编码元件的核苷酸序列编码所述胞内结构域的氨基酸序列，优选地，所述胞内结构域的氨基酸序列选自DAP12的胞内信号区的第62-113位氨基酸。优选地，所述胞内结构域编码元件的核苷酸序列如SEQ ID NO：8所示。The nucleotide sequence of the intracellular domain coding element encodes the amino acid sequence of the intracellular domain, preferably, the amino acid sequence of the intracellular domain is selected from positions 62-113 of the intracellular signal region of DAP12 Amino acid. Preferably, the nucleotide sequence of the intracellular domain coding element is set forth in SEQ ID NO: 8.
优选地，所述间隔区编码元件来自CD8α的铰链区编码DNA，所述跨膜区编码元件来自CD8α的跨膜区编码DNA。Preferably, the spacer coding element is derived from the hinge region encoding DNA of CD8α, and the transmembrane region coding element is derived from the transmembrane region encoding DNA of CD8α.
所述间隔区编码元件和所述跨膜区编码元件构成间隔跨膜区编码元件，所述间隔跨膜区编码元件的核苷酸序列编码所述间隔跨膜区的氨基酸序列，优选地，所述间隔跨膜区的氨基酸序列与CD8α的第Y位-第210位氨基酸序列一致，且118≤Y≤128，Y为整数。例如，所述间隔跨膜区的氨基酸序列如以下氨基酸序列组中任一氨基酸序列所示：CD8α的第118-210位氨基酸、第119-210位氨基酸、第120-210位氨基酸、第121-210位氨基酸、第122-210位氨基酸、第123-210位氨基酸、第124-210位氨基酸、第125-210位氨基酸、第126-210位氨基酸、第127-210位氨基酸、或第128-210位氨基酸。优选地，所述间隔区编码元件的核苷酸序列包含SEQ ID NO：7所示的序列。The spacer coding element and the transmembrane region coding element comprise a spacer transmembrane region coding element, the nucleotide sequence of the spacer transmembrane region coding element encoding an amino acid sequence of the spacer transmembrane region, preferably, The amino acid sequence of the spacer transmembrane region is identical to the amino acid sequence of the Yth position to the 210th position of CD8α, and 118≤Y≤128, and Y is an integer. For example, the amino acid sequence of the spacer transmembrane region is as shown in any one of the following amino acid sequence groups: amino acids 118-210 of CD8α, amino acids 119-210, amino acids 120-210, 121- 210 amino acids, amino acids 122-210, amino acids 123-210, amino acids 124-210, amino acids 125-210, amino acids 126-210, amino acids 127-210, or 128- 210 amino acids. Preferably, the nucleotide sequence of the spacer coding element comprises the sequence set forth in SEQ ID NO:7.
在本发明一个优选的实施方案中，所述分离的、编码本发明所述的嵌合抗原受体的DNA的核苷酸序列如SEQ ID NO：2所示。In a preferred embodiment of the invention, the isolated nucleotide sequence encoding the chimeric antigen receptor of the invention is represented by SEQ ID NO: 2.
本发明所述的NKG2D、DAP12和CD8α均优选来源于人，其全长氨基酸序列和核苷酸序列均为已知的，可以由本领域常用的公开数 据库查询到。The NKG2D, DAP12 and CD8α of the present invention are preferably derived from humans, and their full-length amino acid sequences and nucleotide sequences are known, and can be found by public databases commonly used in the art.
本发明还提供了一种分离的、由本发明所述的编码嵌合抗原受体的DNA转录得到的mRNA。The present invention also provides an isolated mRNA which is transcribed from the DNA encoding the chimeric antigen receptor of the present invention.
本发明还提供了一种重组表达载体，其含有与启动子有效连接的根据本发明所述的编码嵌合抗原受体的DNA。The invention also provides a recombinant expression vector comprising a DNA encoding a chimeric antigen receptor according to the invention operably linked to a promoter.
优选地，所述重组表达载体在根据本发明所述的编码嵌合抗原受体的DNA之前依次包含CMV启动子、T7启动子、具有kozak序列的5’UTR和GM-CSF α链信号肽编码序列；并且在根据本发明所述的编码嵌合抗原受体的DNA之后包含具有polyA信号的α球蛋白的3’UTR。Preferably, the recombinant expression vector comprises, in sequence, a CMV promoter, a T7 promoter, a 5'UTR having a kozak sequence, and a GM-CSF alpha chain signal peptide encoding prior to the DNA encoding the chimeric antigen receptor according to the present invention. a sequence; and comprising a 3'UTR of an alpha globulin having a polyA signal following the DNA encoding the chimeric antigen receptor according to the invention.
本发明的重组表达载体的上述作用元件的组合能够促进DNA的转录和翻译，并增强mRNA的稳定性。本发明还对上述各作用元件的结构作了如下优化，从而更好地发挥其应有的功能。The combination of the above-described functional elements of the recombinant expression vector of the present invention can promote transcription and translation of DNA and enhance the stability of mRNA. The present invention also optimizes the structure of each of the above-described action elements to better perform their intended functions.
优选地，在本发明中，CMV启动子序列为TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATC(SEQ ID NO：22)。CMV启动子功能为使下游的DNA序列开始转录。Preferably, in the present invention, CMV promoter sequence TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATC (SEQ ID NO: 22). The CMV promoter functions to initiate transcription of downstream DNA sequences.
优选地，在本发明中，T7启动子序列为 TAATACGACTCACTATAG(SEQ ID NO：17)。T7启动子功能为使下游的DNA序列开始转录。Preferably, in the present invention, the T7 promoter sequence is TAATACGACTCACTATAG (SEQ ID NO: 17). The T7 promoter functions to initiate transcription of downstream DNA sequences.
优选地，在本发明中，具有kozak序列的5’UTR的序列为AAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA GCCA CCATG(SEQ ID NO：18)，其中下划线所示序列为kozak序列。具有kozak序列的5’UTR的功能为增强mRNA的翻译效率。 Preferably, in the present invention, the sequence of the 5' UTR having the kozak sequence is AAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA GCCA CCATG (SEQ ID NO: 18), wherein the sequence underlined is the kozak sequence. The function of the 5'UTR with the kozak sequence is to enhance the translation efficiency of the mRNA.
优选地，在本发明中，GM-CSF α链信号肽编码序列的序列为ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCA(SEQ ID NO：19)，由此得出的氨基酸序列为MLLLVTSLLLCELPHPAFLLIP(SEQ ID NO：20)。GM-CSF α链信号肽是将本发明的CAR靶向至分泌途径的前导序列，其编码序列首先与CAR一起在细胞内被翻译成蛋白质，引导合成的蛋白进入胞内分泌途径。在细胞表面表达CAR前信号肽已被去除。Preferably, in the present invention, the sequence of the GM-CSF α chain signal peptide coding sequence is ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCA (SEQ ID NO: 19), and the amino acid sequence thus obtained is MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 20). The GM-CSF alpha chain signal peptide is a leader sequence that targets the CAR of the present invention to the secretory pathway, and its coding sequence is first translated into a protein together with the CAR in the cell, directing the synthesized protein into the endocrine pathway. The signal peptide has been removed before expression of the CAR on the cell surface.
优选地，在本发明中，α球蛋白的3’UTR序列为GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTG AATAAAGCCTGAGTAGGAAGT(SEQ ID NO：21)，其中下划线所示序列为polyA信号。其作用是增强mRNA的稳定性。 Preferably, in the present invention, the 3'UTR sequence of the alpha globulin is GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTG AATAAA GCCTGAGTAGGAAGT (SEQ ID NO: 21), wherein the sequence underlined is a polyA signal. Its role is to enhance the stability of mRNA.
在一个具体实施方案中，所述重组表达载体的基本骨架为市售可得的pFastbac1载体，然后在其中插入上述各元件。In a specific embodiment, the basic backbone of the recombinant expression vector is a commercially available pFastbac1 vector into which each of the above elements is inserted.
由于本发明优化了3’UTR和5’UTR结构，因此可以例如应用Tail-PCR技术由所述重组表达载体合成其中正链带有PolyA、反链带有对应的PolyT的DNA双链模板，这样降低了DNA模板的不稳定性，进而可在体外合成mRNA。所述的正链中所带有的PolyA中的A的个数范围(或者其反链中对应的PolyT中的T的个数范围)为140-170个，优选为150-170个，更优选为150个左右(例如150个)。Since the present invention optimizes the 3'UTR and 5'UTR structures, a DNA double-stranded template in which a positive strand carries a PolyA and an inverted strand carries a corresponding PolyT can be synthesized from the recombinant expression vector, for example, using a Tail-PCR technique, such that The instability of the DNA template is reduced, and mRNA can be synthesized in vitro. The range of the number of A in the PolyA carried in the positive chain (or the range of the number of T in the corresponding PolyT in the reverse chain) is 140-170, preferably 150-170, more preferably It is about 150 (for example, 150).
本发明还提供了一种嵌合抗原受体修饰的NK细胞，该NK细胞的表面被本发明所述的嵌合抗原受体修饰。The present invention also provides a chimeric antigen receptor-modified NK cell whose surface is modified by the chimeric antigen receptor of the present invention.
本文所用的术语“修饰”是指，NK细胞表达有本发明所述的嵌合抗原受体，即，所述嵌合抗原受体的跨膜区锚定在所修饰的NK细 胞的细胞膜上，抗原结合结构域位于细胞表面，胞内结构域位于细胞质内。The term "modification" as used herein means that an NK cell expresses a chimeric antigen receptor according to the present invention, that is, a transmembrane region of the chimeric antigen receptor is anchored to a cell membrane of the modified NK cell, The antigen binding domain is located on the cell surface and the intracellular domain is located in the cytoplasm.
所述NK细胞可以为已知的各种类型的NK细胞，并且可以通过常规的生物学方法得到。NK细胞(自然杀伤细胞)是人体内的一种非特异性的先天免疫细胞，来源于骨髓，在体内几乎所有器官中都有存在，是非特异性免疫系统的重要组成部分，表型为CD3阴性CD56阳性的单一细胞，主要有CD16阴性CD56bright(亮)和CD16阳性CD56 dim(暗)两种亚型，分别有免疫调节和肿瘤杀伤的体内功效。由于NK细胞作用是非MHC-限制性，使用上没有必要与病人个体的组织相容性复合物相匹配，也就是NK细胞可用于异体病人细胞治疗，具有广泛的临床应用价值。The NK cells may be various types of NK cells known and can be obtained by conventional biological methods. NK cells (natural killer cells) are non-specific innate immune cells in the human body. They are derived from bone marrow and are present in almost all organs of the body. They are an important part of the non-specific immune system. The phenotype is CD3 negative CD56. Positive single cells, mainly CD16-negative CD56bright (bright) and CD16-positive CD56 dim (dark) subtypes, have immunomodulatory and tumor killing in vivo efficacy. Since NK cell action is non-MHC-restricted, there is no need to match the histocompatibility complex of the individual patient in use, that is, NK cells can be used for cell therapy of allogeneic patients, and have wide clinical application value.
本发明还提供了一种制备根据本发明所述的嵌合抗原受体修饰的NK细胞的方法，包括以下步骤：The invention also provides a method of preparing a chimeric antigen receptor-modified NK cell according to the invention, comprising the steps of:
1)提供NK细胞；1) providing NK cells;
2)提供编码根据本发明所述的嵌合抗原受体的核酸；2) providing a nucleic acid encoding a chimeric antigen receptor according to the invention;
3)将所述核酸转染入所述NK细胞中。3) Transfecting the nucleic acid into the NK cells.
步骤1)所述的NK细胞可以由外周血单个核细胞制备。本发明方法中NK细胞的纯度可为≥70％，优选为≥80％。NK细胞纯度指NK细胞在总细胞群体中所占比例。步骤2)所述的核酸为根据本发明所述的编码所述嵌合抗原受体的DNA、或由该DNA转录得到的mRNA。步骤3)所述的转染可通过冷冻电穿孔技术或慢病毒载体进行。采用冷冻电穿孔技术进行的转染可采用本领域常用的方式进行，如文献“Nakazawa Y，Matsuda K，Kurata T，Sueki A，Tanaka M，Sakashita K，Imai C，Wilson MH，Koike K.Anti-proliferative effects of T cells expressing a ligand-based chimeric antigen receptor against CD116 on CD34(+) cells of juvenile myelomonocytic leukemia.J Hematol Oncol.2016 Mar.”所述。采用慢病毒载体进行的转染可采用本领域常用的方式进行，如文献“James N.Kochenderfer，Steven A.Feldman，Yangbing Zhao，Hui Xu，Mary A.Black，Richard A.Morgan，Wyndham H.Wilson，Ψ and Steven A.Rosenberg.Construction and  Pre-clinical Evaluation of an Anti-CD19 Chimeric Antigen Receptor.2009 J Immunother.2009 Sep；32(7)：689-702.”和文献“Cooper LJ，Topp MS，Serrano LM，Gonzalez S，Chang WC，Naranjo A，Wright C，Popplewell L，Raubitschek A，Forman SJ，Jensen MC.T-cell clones can be rendered specific for CD19：toward the selective augmentation of the graft-versus-B-lineage leukemia effect.Blood.2003 Feb 15；101(4)：1637-44.”所述。The NK cells described in step 1) can be prepared from peripheral blood mononuclear cells. The purity of the NK cells in the method of the invention may be > 70%, preferably > 80%. NK cell purity refers to the proportion of NK cells in the total cell population. The nucleic acid according to the step 2) is a DNA encoding the chimeric antigen receptor according to the present invention, or an mRNA obtained by transcription of the DNA. The transfection described in step 3) can be carried out by cryoelectroporation techniques or lentiviral vectors. Transfection using cryoelectroporation techniques can be carried out in a manner commonly used in the art, such as the literature "Nakazawa Y, Matsuda K, Kurata T, Sueki A, Tanaka M, Sakashita K, Imai C, Wilson MH, Koike K. Anti- Proliferative effects of T cells expressing a ligand-based chimeric antigen receptor against CD116 on CD34(+) cells of juvenile myelomonocytic leukemia. J Hematol Oncol. 2016 Mar. Transfection using lentiviral vectors can be carried out in a manner commonly used in the art, such as the literature "James N. Kochenderfer, Steven A. Feldman, Yangbing Zhao, Hui Xu, Mary A. Black, Richard A. Morgan, Wyndham H. Wilson. , Ψ and Steven A. Rosenberg. Construction and Pre-clinical Evaluation of an Anti-CD19 Chimeric Antigen Receptor. 2009 J Immunother. 2009 Sep; 32(7): 689-702." and the literature "Cooper LJ, Topp MS, Serrano LM, Gonzalez S, Chang WC, Naranjo A, Wright C, Popplewell L, Raubitschek A, Forman SJ, Jensen MC. T-cell clones can be rendered specific for CD19: toward the selective augmentation of the graft-versus-B-lineage Leukemia effect. Blood.2003 Feb 15;101(4):1637-44."
在本发明一个具体实施方案中，通过PCR由PBMC cDNA文库扩增得到人NKG2D蛋白的第83-216位氨基酸对应的DNA、人CD8α的第128-210位氨基酸对应的DNA、人DAP12的第62-113位氨基酸对应的DNA。将扩增得到的三种序列连接，然后通过分子克隆技术连接到pFastbac1载体上，得到pFastbac1-NKG2D-CD8-DAP12重组表达载体。然后合成NKG2D-CD8-DAP12对应序列的mRNA。再利用高效冷冻电穿孔技术将mRNA电转进入体外扩增培养的NK细胞，获得工程化NKG2D配体靶向性的NK细胞。In a specific embodiment of the present invention, the DNA corresponding to amino acids 83-216 of the human NKG2D protein, the DNA corresponding to amino acids 128-210 of human CD8α, and the 62nd of human DAP12 are amplified by PCR from the PBMC cDNA library. DNA corresponding to -113 amino acids. The three sequences amplified were ligated and ligated to the pFastbac1 vector by molecular cloning techniques to obtain a recombinant expression vector pFastbac1-NKG2D-CD8-DAP12. The mRNA of the corresponding sequence of NKG2D-CD8-DAP12 was then synthesized. The mRNA was electroporated into the NK cells expanded in vitro by high-efficiency cryoelectroporation to obtain engineered NKG2D ligand-targeted NK cells.
所述嵌合抗原受体的各部分也可由静脉血中单个核细胞的基因组cDNA扩增得到。Each portion of the chimeric antigen receptor can also be amplified by genomic cDNA of mononuclear cells in venous blood.
本发明还提供了根据本发明所述的嵌合抗原受体修饰的NK细胞在制备用于治疗或预防肿瘤和/或癌症的药物中的用途。The invention also provides the use of a chimeric antigen receptor-modified NK cell according to the invention for the preparation of a medicament for the treatment or prevention of a tumor and/or cancer.
所述肿瘤和/或癌症是NKG2D配体阳性的，包括结直肠癌、卵巢癌、头颈癌、骨髓瘤、肝癌、乳腺癌、血液瘤、宫颈癌、神经胶质瘤等。在本发明的一个实施方案中，所述肿瘤和/或癌症是NKG2D配体阳性的情况包括所述肿瘤和/或癌症未经处理是NKG2D配体阳性的以及所述肿瘤和/或癌症经处理而成为NKG2D配体阳性的。所述处理包括经药物、放射或生物制剂处理后成为NKG2D配体阳性的。The tumor and/or cancer is positive for NKG2D ligand, including colorectal cancer, ovarian cancer, head and neck cancer, myeloma, liver cancer, breast cancer, hematoma, cervical cancer, glioma, and the like. In one embodiment of the invention, the tumor and/or cancer is positive for NKG2D ligand comprising the tumor and/or cancer being untreated is NKG2D ligand positive and the tumor and/or cancer is treated It became positive for NKG2D ligand. The treatment includes becoming NKG2D ligand positive after treatment with a drug, radiation or biological agent.
本发明还提供了根据本发明所述的嵌合抗原受体修饰的NK细胞在制备用于检测宿主的肿瘤和/或癌症的药物中的用途。The invention also provides the use of a chimeric antigen receptor-modified NK cell according to the invention for the preparation of a medicament for detecting a tumor and/or cancer of a host.
所述肿瘤和/或癌症是NKG2D配体阳性的，包括结直肠癌、卵巢癌、头颈癌、骨髓瘤、肝癌、乳腺癌、血液瘤、宫颈癌、神经胶质瘤等。在本发明的一个实施方案中，所述肿瘤和/或癌症是NKG2D 配体阳性的情况包括所述肿瘤和/或癌症未经处理是NKG2D配体阳性的以及所述肿瘤和/或癌症经处理而成为NKG2D配体阳性的。所述处理包括经药物、放射或生物制剂处理后成为NKG2D配体阳性的。The tumor and/or cancer is positive for NKG2D ligand, including colorectal cancer, ovarian cancer, head and neck cancer, myeloma, liver cancer, breast cancer, hematoma, cervical cancer, glioma, and the like. In one embodiment of the invention, the tumor and/or cancer is positive for NKG2D ligand comprising the tumor and/or cancer being untreated is NKG2D ligand positive and the tumor and/or cancer is treated It became positive for NKG2D ligand. The treatment includes becoming NKG2D ligand positive after treatment with a drug, radiation or biological agent.
在本发明的一个实施方案中，可将从宿主取出的肿瘤和/或癌症细胞的样本与本发明所述的嵌合抗原受体修饰的NK细胞以一定浓度进行接触，根据二者的反应程度可以判断所述肿瘤和/或癌症是NKG2D配体阳性的还是NKG2D配体阴性的。In one embodiment of the present invention, a sample of a tumor and/or a cancer cell taken out from a host may be contacted with a chimeric antigen receptor-modified NK cell of the present invention at a concentration according to the degree of reaction between the two. It can be judged whether the tumor and/or cancer is NKG2D ligand positive or NKG2D ligand negative.
本发明还提供了一种药物组合物，其中该药物组合物包括作为活性成分的根据本发明所述的嵌合抗原受体修饰的NK细胞，及可药用辅料。The present invention also provides a pharmaceutical composition comprising the chimeric antigen receptor-modified NK cell according to the present invention as an active ingredient, and a pharmaceutically acceptable adjuvant.
所述药物组合物优选包含每人每个疗程总剂量范围为1×10 6-1×10 11个的所述嵌合抗原受体修饰的NK细胞。优选的是，每个疗程3周共21天，每周施用1-2次。可以根据实际情况和需要对患者进行一个或多个疗程的治疗。 The pharmaceutical composition preferably comprises the chimeric antigen receptor-modified NK cells in a total dose ranging from 1 x 10 6 to 1 x 10 11 per subject per subject. Preferably, each treatment is for 3 weeks for a total of 21 days, 1-2 times a week. The patient may be treated for one or more courses depending on the actual situation and needs.
所述药物组合物可通过合适的给药途径给药，其给药方式包括静脉给药(例如静脉滴注给药或静脉注射给药)或局部给药(例如局部滴注给药或局部注射给药)。The pharmaceutical composition can be administered by a suitable route of administration including intravenous administration (for example, intravenous drip administration or intravenous administration) or topical administration (for example, topical administration or local injection). Administration).
本发明还提供了一种治疗肿瘤和/或癌症的方法，包括对肿瘤和/或癌症患者施用根据本发明所述的嵌合抗原受体修饰的NK细胞。The invention also provides a method of treating a tumor and/or cancer comprising administering to a tumor and/or cancer patient a chimeric antigen receptor modified NK cell according to the invention.
所述肿瘤和/或癌症是NKG2D配体阳性的，包括结直肠癌、卵巢癌、头颈癌、骨髓瘤、肝癌、乳腺癌、血液瘤、宫颈癌、神经胶质瘤等。在本发明的一个实施方案中，所述肿瘤和/或癌症是NKG2D配体阳性的情况包括所述肿瘤和/或癌症未经处理是NKG2D配体阳性的以及所述肿瘤和/或癌症经处理而成为NKG2D配体阳性的。所述处理包括经药物、放射或生物制剂处理后成为NKG2D配体阳性的。The tumor and/or cancer is positive for NKG2D ligand, including colorectal cancer, ovarian cancer, head and neck cancer, myeloma, liver cancer, breast cancer, hematoma, cervical cancer, glioma, and the like. In one embodiment of the invention, the tumor and/or cancer is positive for NKG2D ligand comprising the tumor and/or cancer being untreated is NKG2D ligand positive and the tumor and/or cancer is treated It became positive for NKG2D ligand. The treatment includes becoming NKG2D ligand positive after treatment with a drug, radiation or biological agent.
所述嵌合抗原受体修饰的NK细胞的施用剂量优选为每人每个疗程总剂量范围为1×10 6-1×10 11个细胞。优选的是，每个疗程3周共21天，每周施用1-2次。可以根据实际情况和需要对患者进行一个或多个疗程的治疗。 The chimeric antigen receptor-modified NK cells are preferably administered at a dose ranging from 1 x 10 6 to 1 x 10 11 cells per patient per course of treatment. Preferably, each treatment is for 3 weeks for a total of 21 days, 1-2 times a week. The patient may be treated for one or more courses depending on the actual situation and needs.
所述嵌合抗原受体修饰的NK细胞可通过合适的给药途径给药，其给药方式包括静脉给药(例如静脉滴注给药或静脉注射给药)或局部给药(例如局部滴注给药或局部注射给药)。The chimeric antigen receptor-modified NK cells can be administered by a suitable administration route, such as intravenous administration (for example, intravenous drip administration or intravenous administration) or topical administration (for example, topical drip). Injection or local injection).
本发明还提供了一种工具载体，该工具载体依次包含可操作地连接的CMV启动子、T7启动子、具有kozak序列的5’UTR、GM-CSF α链信号肽编码序列、和具有polyA信号的α球蛋白的3’UTR。The invention also provides a tool vector comprising, in turn, an operably linked CMV promoter, a T7 promoter, a 5'UTR having a kozak sequence, a GM-CSF alpha chain signal peptide coding sequence, and a polyA signal The 3'UTR of alpha globulin.
术语“工具载体”是指基因工程应用中，用于插入外源DNA片段的空载体。The term "tool vector" refers to an empty vector for insertion of an exogenous DNA fragment in genetic engineering applications.
在插入外源DNA片段时，将外源DNA片段插入到所述工具载体的GM-CSF α链信号肽编码序列与具有polyA信号的α球蛋白的3’UTR之间(GM-CSF α链信号肽编码序列与具有polyA信号的α球蛋白的3’UTR之间可以具有多克隆位点)。When the foreign DNA fragment is inserted, the foreign DNA fragment is inserted between the GM-CSF α chain signal peptide coding sequence of the tool vector and the 3'UTR of the α globulin having the polyA signal (GM-CSF α chain signal) The peptide coding sequence may have a multiple cloning site between the 3' UTR of the alpha globulin having a polyA signal).
优选地，所述CMV启动子的核苷酸序列如SEQ ID NO：22所示，T7启动子的核苷酸序列如SEQ ID NO：17所示，所述具有kozak序列的5’UTR的核苷酸序列如SEQ ID NO：18所示，所述GM-CSF α链信号肽编码序列的核苷酸序列如SEQ ID NO：19所示，所述具有PolyA信号的α球蛋白的3’UTR的核苷酸序列如SEQ ID NO：21所示。Preferably, the nucleotide sequence of the CMV promoter is set forth in SEQ ID NO: 22, and the nucleotide sequence of the T7 promoter is set forth in SEQ ID NO: 17, the core of the 5'UTR having the kozak sequence. The nucleotide sequence of SEQ ID NO: 18, the nucleotide sequence of the GM-CSF α chain signal peptide coding sequence is set forth in SEQ ID NO: 19, and the 3'UTR of the alpha globulin having a PolyA signal The nucleotide sequence is shown in SEQ ID NO:21.
本发明的工具载体能够促进插入DNA的转录和翻译，并增强mRNA的稳定性。The tool vector of the present invention is capable of promoting transcription and translation of the inserted DNA and enhancing the stability of the mRNA.
以下通过例子的方式进一步解释或说明本发明的内容，但这些例子不应被理解为对本发明的保护范围的限制。The contents of the present invention are further explained or illustrated by way of examples, but the examples are not to be construed as limiting the scope of the invention.
例子example
以下除非特别说明，否则以下例子中所用实验方法均使用生物工程领域的常规实验流程、操作、材料和条件进行。Unless otherwise stated, the experimental methods used in the following examples were carried out using conventional experimental procedures, procedures, materials and conditions in the field of bioengineering.
以下除非特别说明，否则各试剂的百分浓度(％)均指该试剂的体积百分浓度(％(v/v))。Unless otherwise indicated, the percent concentration (%) of each reagent refers to the volume percent concentration (% (v/v)) of the reagent.
以下例子中所用人类卵巢癌细胞SKOV3、人类结直肠癌细胞HCT116和SW480、人类头颈癌细胞Fadu和Detroit、人类肝癌细胞 HepG2、人类神经胶质瘤细胞U87、人类乳腺癌细胞MCF7、和人类骨髓瘤细胞KG1购自ATCC。Human ovarian cancer cell line SKOV3, human colorectal cancer cells HCT116 and SW480, human head and neck cancer cells Fadu and Detroit, human liver cancer cell HepG2, human glioma cell line U87, human breast cancer cell MCF7, and human myeloma used in the following examples Cell KG1 was purchased from ATCC.
以下例子中所用PBS购自Lonza，货号为BW17-517Q。The PBS used in the following examples was purchased from Lonza under the order number BW17-517Q.
制备例1：嵌合抗原受体质粒构建Preparation Example 1: Construction of chimeric antigen receptor plasmid
1)静脉血单个核细胞(PBMCs)cDNA的制备1) Preparation of venous blood mononuclear cells (PBMCs) cDNA
取人静脉血于含肝素的真空管中。采用淋巴细胞分离液，密度梯度离心方法分离获得单个核细胞(PBMCs)。Take human venous blood into a vacuum tube containing heparin. Mononuclear cells (PBMCs) were isolated by lymphocyte separation and density gradient centrifugation.
离心沉淀上述PBMCs，用总RNA提取试剂盒RNAiso Reagent(购自Life Technologies)提取细胞的总RNA，-80℃保存备用。提取的总RNA用逆转录试剂盒RevertAicTFirst Strand cDNASynthesis Kit(购自Life Technologies)逆转录得PBMCs的cDNA，-20℃保存备用。The above PBMCs were pelleted by centrifugation, and total RNA of the cells was extracted with a total RNA extraction kit RNAiso Reagent (purchased from Life Technologies), and stored at -80 ° C until use. The extracted total RNA was reverse transcribed into cDNA of PBMCs using a reverse transcription kit RevertAicTFirst Strand cDNA Synthesis Kit (purchased from Life Technologies), and stored at -20 ° C until use.
2)嵌合抗原受体的重组DNA载体pFastbac1-NKG2D-CD8-DAP12的构建2) Construction of recombinant DNA vector pFastbac1-NKG2D-CD8-DAP12 of chimeric antigen receptor
以步骤1)中单个核细胞cDNA为模板，用引物P1(SEQ ID NO：9)、P2(SEQ ID NO：10)进行PCR扩增，获得其中包含长度为402bp的NKG2D蛋白的胞外结构域对应的DNA编码序列(核苷酸序列如序列表中SEQ ID NO：6所示)的片段，其两端分别具有Sphl和NheI酶切位点和保护碱基。利用引物P3(SEQ ID NO：11)、P4(SEQ ID NO：12)进行PCR扩增，获得其中包含长度为249bp的CD8α基因的铰链(hinge)区和跨膜区(核苷酸序列如序列表中SEQ ID NO：7所示)的片段。利用引物P5(SEQ ID NO：13)、P6(SEQ ID NO：14)进行PCR扩增得到其中包含长度为156bp的DAP12的胞内信号结构域(核苷酸序列如序列表中SEQ ID NO：8所示)的片段。各步PCR扩增反应体系相同，PCR反应条件参照KAPA HiFiHotStart ReadyMix(2X)(购自Kapa Biosystems)的说明书，各反应体系(50μL)如下：Using the single nuclear cell cDNA in step 1) as a template, PCR amplification was carried out with primers P1 (SEQ ID NO: 9) and P2 (SEQ ID NO: 10) to obtain an extracellular domain comprising a NKG2D protein of 402 bp in length. A fragment of the corresponding DNA coding sequence (nucleotide sequence as shown in SEQ ID NO: 6 in the Sequence Listing) has a Sphl and NheI restriction site and a protective base at both ends, respectively. PCR amplification using primers P3 (SEQ ID NO: 11) and P4 (SEQ ID NO: 12) to obtain a hinge region and a transmembrane region in which a CD8α gene of 249 bp in length was obtained (nucleotide sequence A fragment of SEQ ID NO: 7 in the list). PCR amplification using primers P5 (SEQ ID NO: 13) and P6 (SEQ ID NO: 14) gave an intracellular signal domain containing DAP12 of 156 bp in length (nucleotide sequence such as SEQ ID NO in the sequence listing: A fragment of 8). The PCR amplification reaction system was the same in each step. The PCR reaction conditions were as described in KAPA HiFi Hot Start Ready Mix (2X) (purchased from Kapa Biosystems), and each reaction system (50 μL) was as follows:
双蒸水：21.5μLDouble distilled water: 21.5μL
2×KAPA HiFiHotStart Uracil+ReadyMix：25μL2×KAPA HiFiHotStart Uracil+ReadyMix: 25μL
上游引物(10μM)：1.5μLUpstream primer (10μM): 1.5μL
下游引物(10μM)：1.5μLDownstream primer (10μM): 1.5μL
单个核细胞cDNA模板(120ng/ul)：0.5μLMononuclear cell cDNA template (120ng/ul): 0.5μL
将上述PCR产物用1％(w/v)的琼脂糖凝胶进行分离，用琼脂糖凝胶DNA回收试剂盒(购自Omega Bio Tek)进行DNA片段回收。将回收片段中包含长度为402bp的NKG2D蛋白的胞外结构域DNA的片段进行Sphl和NheI双酶切反应，酶切产物用琼脂糖凝胶DNA回收试剂盒进行DNA片段回收备用。The above PCR product was separated on a 1% (w/v) agarose gel, and DNA fragment recovery was carried out using an agarose gel DNA recovery kit (purchased from Omega Bio Tek). The fragment containing the extracellular domain DNA of the NKG2D protein of 402 bp in length was subjected to double digestion with Sphl and NheI, and the digested product was subjected to DNA fragment recovery using an agarose gel DNA recovery kit.
按照图1的顺序，将商业化载体pFastbac1(Life Technologies)加入一个CMV启动子，T7启动子，一个拥有Kozak序列的5’UTR，一个GM-CSF α链信号肽的编码序列(图1中的SP)，和一个具有polyA信号的阿尔法球蛋白的3’UTR来构建pFastbac1基本骨架载体。将pFastbac1基本骨架载体进行SphI和SalI双酶切反应，酶切产物用琼脂糖凝胶DNA回收试剂盒进行DNA片段回收，然后与之前PCR回收的CD8、DAP12片段通过
Figure PCTCN2018094005-appb-000001
Seamless Cloning and Assembly Kit(购自Life Technologies)进行连接，连接产物转化One 
Figure PCTCN2018094005-appb-000002
Chemically Competent TOP10化学感受态细胞(购自Life Technologies)，37℃培养18小时后挑取单克隆，37℃，250rpm培养6小时后用质粒小提试剂盒(购自Omega Bio Tek)提取质粒。提取的质粒经限制性内切酶SphI和SalI双酶切鉴定，鉴定电泳图见图2；其中，泳道1：1000kb DNA分子量标记；泳道2：质粒pFastbac1-CD8-DAP12的酶切片段，载体骨架5276bp，CD8-DAP12片段405bp。将鉴定正确的质粒送AITbiotech公司对插入的融合基因片段进行测序，将测序结果正确的重组质粒命名为pFastbac1-CD8-DAP12。 According to the sequence of Figure 1, the commercial vector pFastbac1 (Life Technologies) was added to a CMV promoter, a T7 promoter, a 5'UTR with a Kozak sequence, and a coding sequence for a GM-CSF alpha chain signal peptide (Figure 1 SP), and a 3'UTR of alpha globulin with a polyA signal to construct the pFastbac1 basic skeleton vector. The pFastbac1 basic skeleton vector was subjected to double digestion with SphI and SalI, and the digested product was subjected to DNA fragment recovery using an agarose gel DNA recovery kit, and then passed through the CD8 and DAP12 fragments recovered by the previous PCR.
Figure PCTCN2018094005-appb-000001
Seamless Cloning and Assembly Kit (purchased from Life Technologies) for connection and connection of product conversion to One
Figure PCTCN2018094005-appb-000002
Chemically Competent TOP10 chemically competent cells (purchased from Life Technologies), picked up for 18 hours at 37 ° C, picked up the monoclonal, and cultured at 37 ° C, 250 rpm for 6 hours, and then extracted the plasmid with a plasmid miniprep kit (purchased from Omega Bio Tek). The extracted plasmid was identified by restriction endonuclease SphI and SalI digestion, and the electrophoresis pattern was identified as shown in Fig. 2; wherein, lane 1: 1000 kb DNA molecular weight marker; lane 2: plasmid pFastbac1-CD8-DAP12 digestion fragment, vector backbone 5276 bp, CD8-DAP12 fragment 405 bp. The correct plasmid was sent to AITbiotech for sequencing of the inserted fusion gene fragment, and the correct recombinant plasmid was named pFastbac1-CD8-DAP12.
将载体pFastbac1-CD8-DAP12进行SphI和NheI双酶切反应，酶切产物用琼脂糖凝胶DNA回收试剂盒进行DNA片段回收，然后与之前回收的NKG2D蛋白的胞外结构域片段通过T4 DNA连接酶连接，连接产物转化One
Figure PCTCN2018094005-appb-000003
Chemically Competent TOP10化学感受态细胞，37℃培养18小时后挑取单克隆，37℃，250rpm培养6小时后用质粒小提试剂盒提取质粒。提取的质粒经限制性内切酶SphI和 NheI双酶切鉴定，鉴定电泳图见图3；其中，泳道1：1000kb DNA分子量标记；泳道2：质粒pFastbac1-NKG2D-CD8-DAP12的酶切片段，载体骨架5682bp，NKG2D片段402bp。将鉴定正确的质粒送AITbiotech公司对插入的融合基因片段进行测序，将测序结果正确的重组质粒命名为pFastbac1-NKG2D-CD8-DAP12，其质粒图谱示意图见图4。 The vector pFastbac1-CD8-DAP12 was digested with SphI and NheI, and the digested product was recovered by DNA fragmentation using an agarose gel DNA recovery kit, and then ligated to the extracellular domain fragment of the previously recovered NKG2D protein by T4 DNA. Enzyme-linked, linked product-transformed One
Figure PCTCN2018094005-appb-000003
Chemically Competent TOP10 chemically competent cells were cultured at 37 ° C for 18 hours, and then picked up, and cultured at 37 ° C, 250 rpm for 6 hours, and then the plasmid was extracted with a plasmid mini-kit. The extracted plasmid was identified by restriction endonuclease SphI and NheI digestion, and the electrophoresis pattern was identified as shown in Fig. 3; wherein, lane 1: 1000 kb DNA molecular weight marker; lane 2: restriction fragment of plasmid pFastbac1-NKG2D-CD8-DAP12, The vector backbone is 5682 bp and the NKG2D fragment is 402 bp. The correct plasmid was sent to AITbiotech for sequencing of the inserted fusion gene fragment, and the recombinant plasmid with the correct sequencing result was named pFastbac1-NKG2D-CD8-DAP12, and the plasmid map is shown in Figure 4.
制备例2：NK细胞的制备Preparation Example 2: Preparation of NK cells
将细胞数为20×10 6个的PBMCs细胞和2mL NK细胞活化剂I(购自深圳达科为，DKW35-CYT-NK001)均匀混合于400ml NK培养液(NK培养液为AIM
Figure PCTCN2018094005-appb-000004
培养基(购自Life Technologies)+1％人类血清(购自Valley Biomedical，货号HP1022HI)中，接种于一个G-Rex100细胞培养器(购自Wilson Wolf)，加入IL2(终浓度：50IU/ml)，放置于37℃细胞培养箱中，每隔一天补充全液体积的IL2(终浓度：50IU/ml)，培养10天，收集细胞计数，计数完毕取出收集细胞中的20×10 6个细胞与另外的2mL NK细胞活化剂I均匀混合于400ml NK培养液中，接种回G-Rex100细胞培养器，相同条件继续培养7天。上述第10天时的剩余细胞冻存备用，第17天时的细胞收集计数，取出2×10 6个细胞用于流式细胞仪(购自BD，型号C6 Samplar)进行细胞表型分析。结果显示，用该方法培养出来的NK细胞平均纯度高达90％，见图5。图5A所示结果说明CD3阴性且CD56阳性的NK细胞比例为90.2％，图5B所示结果说明NK细胞表面受体NKG2D和CD16双阳性的比例为96.4％。 PBMCs cells with 20×10 6 cells and 2 mL NK cell activator I (purchased from Shenzhen Daktronics, DKW35-CYT-NK001) were uniformly mixed in 400 ml NK medium (NK culture medium was AIM).
Figure PCTCN2018094005-appb-000004
Medium (purchased from Life Technologies) + 1% human serum (purchased from Valley Biomedical, item number HP1022HI), inoculated into a G-Rex100 cell culture device (purchased from Wilson Wolf), added with IL2 (final concentration: 50 IU/ml) Place in a 37 ° C cell culture incubator, replenish the whole liquid volume of IL2 every other day (final concentration: 50 IU / ml), culture for 10 days, collect the cell count, and count the 20 × 10 6 cells in the collected cells. An additional 2 mL of NK cell activator I was uniformly mixed in 400 ml of NK medium, inoculated back into a G-Rex 100 cell culture incubator, and culture was continued for 7 days under the same conditions. The remaining cells on the 10th day were stored frozen, and the cells were counted on the 17th day, and 2 × 10 6 cells were taken out for flow cytometry (purchased from BD, model C6 Samplar) for cell phenotypic analysis. The results showed that the average purity of NK cells cultured by this method was as high as 90%, as shown in Fig. 5. The results shown in Fig. 5A indicate that the proportion of CD3-negative and CD56-positive NK cells is 90.2%, and the results shown in Fig. 5B indicate that the ratio of NK cell surface receptor NKG2D and CD16 double positive is 96.4%.
制备例3：制备嵌合抗原受体修饰的NK细胞Preparation Example 3: Preparation of chimeric antigen receptor-modified NK cells
1)嵌合抗原受体NKG2D-CD8-DAP12 mRNA的体外合成1) In vitro synthesis of chimeric antigen receptor NKG2D-CD8-DAP12 mRNA
优化了3’UTR和5’UTR结构，其序列如上文所述。应用Tail-PCR技术大剂量合成其中正链带有PolyA、反链带有对应的PolyT的DNA双链模板用来进行试管内RNA合成，减少了DNA模板的不稳定性。嵌合抗原受体NKG2D-CD8-DAP12编码序列以上述 pFastbac1-NKG2D-CD8-DAP12载体作为DNA模板进行Tail-PCR扩增，用以合成编码嵌合抗原受体NKG2D-CD8-DAP12序列的线性化DNA模板，Tail-PCR反应条件参照KAPA HiFiHotStartReadyMix(2X)的说明书，反应体系(50μL)如下：The 3' UTR and 5' UTR structures were optimized and the sequences are as described above. Tail-PCR technology was used to synthesize large-dose DNA double-stranded templates with PolyA in the positive strand and PolyT in the reverse strand for in vitro RNA synthesis, which reduced the instability of the DNA template. The chimeric antigen receptor NKG2D-CD8-DAP12 coding sequence was subjected to Tail-PCR amplification using the above pFastbac1-NKG2D-CD8-DAP12 vector as a DNA template to synthesize linearization of the chimeric antigen receptor NKG2D-CD8-DAP12 sequence. DNA template, Tail-PCR reaction conditions refer to the instructions of KAPA HiFiHotStartReadyMix (2X), the reaction system (50 μL) is as follows:
双蒸水(nuclease free)：25μLDouble distilled water (nuclease free): 25μL
2X KAPA HiFiHotStart Uracil+ReadyMix：25μL2X KAPA HiFiHotStart Uracil+ReadyMix: 25μL
P7(SEQ ID NO：15)(100μM)：0.15μLP7 (SEQ ID NO: 15) (100 μM): 0.15 μL
P8(SEQ ID NO：16)(100μM)：0.15μLP8 (SEQ ID NO: 16) (100 μM): 0.15 μL
pFastbac1-NKG2D-CD8-DAP12载体DNA模板(500ng/μL)：0.5μLpFastbac1-NKG2D-CD8-DAP12 vector DNA template (500ng/μL): 0.5μL
将上述PCR产物用1％(w/v)的琼脂糖凝胶进行分离鉴定，见图6。鉴定正确的产物用于嵌合抗原受体NKG2D-CD8-DAP12 mRNA的体外合成。用mRNA体外合成试剂盒合成戴帽的mRNA，试剂盒为mMESSAGEmMACHINE T7 ULTRA转录试剂盒(可得自美国Invitrogen公司)或者mScript TM RNA system(可得自美国Epicentre公司)。按照试剂盒的说明，并用试剂盒提供的试剂进行合成即可。 The above PCR product was isolated and identified on a 1% (w/v) agarose gel, as shown in FIG. The correct product was identified for in vitro synthesis of the chimeric antigen receptor NKG2D-CD8-DAP12 mRNA. In vitro mRNA synthesis kit with capped mRNA synthesis kit as mMESSAGEmMACHINE T7 ULTRA Transcription Kit (available from Invitrogen, USA) or mScript TM RNA system (available from the American Epicentre Corporation). According to the instructions of the kit, and using the reagents provided in the kit for synthesis.
将体外合成的嵌合抗原受体NKG2D-CD8-DAP12 mRNA产物用1％(w/v)的琼脂糖凝胶进行分离鉴定，见图7。鉴定正确的mRNA存放于-80℃保存备用。The in vitro synthesized chimeric antigen receptor NKG2D-CD8-DAP12 mRNA product was isolated and identified on a 1% (w/v) agarose gel, as shown in FIG. The correct mRNA was identified and stored at -80 ° C for storage.
2)对NK细胞进行嵌合抗原受体修饰2) Chimeric antigen receptor modification of NK cells
将制备例2制备的NK细胞(1×10 7个)与4μg NKG2D-CD8-DAP12 mRNA混合于电转液P3(产品名称“P3Primary Cell
Figure PCTCN2018094005-appb-000005
X Kit L”，Lonza，货号V4XP-3012)，置于100μl Nucleocuvette TM管(P3Primary Cell
Figure PCTCN2018094005-appb-000006
X Kit L，Lonza，货号V4XP-3012)，并进行冰浴冷冻5分钟。然后使用4D-Nucleofector TM电转仪(得自瑞士Lonza公司)，选择其自带的NK细胞电转程序进行电转。电转后，将细胞取出置于制备例2所述NK培养液中，加入IL2 50IU/mL和0.5μg/mL的DNase，置于37℃，5％CO 2孵箱中恢复过夜。24小时后，收集细胞，使用流式细胞仪对电转细胞进行鉴定，见图8。图8所示结果说明转染NKG2D-CAR  mRNA后，NKG2D的表达强度明显增加。符合要求的细胞(NKG2D-CAR NK细胞)可用于对相关肿瘤的治疗。 NK cells prepared in Preparation Example 2 (1 × 10 7 cells) and 4 μg of NKG2D-CD8-DAP12 mRNA were mixed in electroporation P3 (product name "P3 Primary Cell"
Figure PCTCN2018094005-appb-000005
X Kit L”, Lonza, item number V4XP-3012), placed in a 100μl Nucleocuvette TM tube (P3Primary Cell)
Figure PCTCN2018094005-appb-000006
X Kit L, Lonza, item number V4XP-3012), and frozen in an ice bath for 5 minutes. Then using the 4D-Nucleofector TM electroporation instrument (available from Lonza, Inc., Switzerland), choose their own program NK cell electroporation electroporation. After electroporation, the cells were removed and placed in the NK medium described in Preparation Example 2, and IL 2 50 IU/mL and 0.5 μg/mL DNase were added, and placed at 37 ° C in a 5% CO 2 incubator to recover overnight. After 24 hours, cells were harvested and electroporated cells were identified using flow cytometry, see Figure 8. The results shown in Figure 8 indicate that the expression intensity of NKG2D is significantly increased after transfection of NKG2D-CAR mRNA. Eligible cells (NKG2D-CAR NK cells) can be used to treat related tumors.
试验例1：NKG2D-CAR NK细胞对人肿瘤细胞体外杀伤能力的检测分析Test Example 1: Detection of NKG2D-CAR NK cells in vitro killing ability of human tumor cells
1)对NKG2D-CAR NK细胞杀伤性细胞因子IFN-γ的释放能力的检测1) Detection of the release ability of NKG2D-CAR NK cell killing cytokine IFN-γ
为了评价肿瘤细胞杀伤效率，进行了ELISPOT实验来检测IFNγ的分泌，因为IFN-γ的分泌与过继细胞免疫治疗时NKG2D-CAR NK细胞的抗肿瘤活性成正相关。In order to evaluate the tumor cell killing efficiency, an ELISPOT assay was performed to detect the secretion of IFNγ, since the secretion of IFN-γ was positively correlated with the anti-tumor activity of NKG2D-CAR NK cells in adoptive cellular immunotherapy.
mGFP-CAR NK细胞(转染mGFP-CD8-DAP12 mRNA的NK细胞)的制备方法与NKG2D-CAR NK细胞(转染NKG2D-CD8-DAP12 mRNA的NK细胞)的制备方法相同，只是载体构建时胞外结构域使用无抗原结合功能的mGFP序列(其Genbank编号为YP_002302326.1)，以此作为阴性对照。The preparation method of mGFP-CAR NK cells (NK cells transfected with mGFP-CD8-DAP12 mRNA) is the same as that of NKG2D-CAR NK cells (NK cells transfected with NKG2D-CD8-DAP12 mRNA), except that the vector is constructed. The outer domain used the mGFP sequence without antigen binding function (its Genbank accession number is YP_002302326.1) as a negative control.
转染NKG2D-CD8-DAP12 mRNA的NK细胞的制备如制备例3所示。The preparation of NK cells transfected with NKG2D-CD8-DAP12 mRNA is shown in Preparation 3.
将转染NKG2D-CD8-DAP12 mRNA或者mGFP-CD8-DAP12 mRNA的NK细胞分别与人类卵巢癌细胞SKOV3、人类结直肠癌细胞HCT116和SW480、人类头颈癌细胞Detroit、人类肝癌细胞HepG2和人类神经胶质瘤细胞U87共培养于ELISPOT检测板，上述NK效应细胞与靶标细胞的数量比例为5∶1。每组实验重复3次。经过24小时的共培养后，显影并使用软件immunospot进行ELISPOT点计数。结果显示，NKG2D-CD8-DAP12 mRNA转染的NK细胞与mGFP-CD8-DAP12 mRNA转染的NK细胞相比，产生了更多更强的IFN-γ(通过ELISPOT检测试剂盒检测，购自Mabtech，货号为3420-4HST-1)，统计后发现NKG2D-CD8-DAP12 mRNA转染的NK细胞产生的ELISPOT点显著高于对照组，见图9。NK cells transfected with NKG2D-CD8-DAP12 mRNA or mGFP-CD8-DAP12 mRNA were associated with human ovarian cancer cells SKOV3, human colorectal cancer cells HCT116 and SW480, human head and neck cancer cells Detroit, human hepatoma cells HepG2 and human neutrophils The stromal cell U87 was co-cultured on the ELISPOT assay plate, and the ratio of the above NK effector cells to the target cells was 5:1. Each set of experiments was repeated 3 times. After 24 hours of co-cultivation, development and use of the software immunospot for ELISPOT point counting. The results showed that NK cells transfected with NKG2D-CD8-DAP12 mRNA produced more and stronger IFN-γ than NK cells transfected with mGFP-CD8-DAP12 mRNA (detected by ELISPOT assay kit, purchased from Mabtech The product number is 3420-4HST-1). After counting, the ELISPOT spots produced by NKG2D-CD8-DAP12 mRNA-transfected NK cells were significantly higher than the control group, as shown in Figure 9.
2)对NKG2D-CAR NK细胞溶解肿瘤细胞能力的检测2) Detection of the ability of NKG2D-CAR NK cells to lyse tumor cells
将转染NKG2D-CD8-DAP12 mRNA或者mGFP-CD8-DAP12  mRNA的NK细胞分别与人类结直肠癌细胞HCT116、人类卵巢癌细胞SKOV3、人类头颈癌细胞Fadu和Detroit、人类肝癌细胞HepG2、人类乳腺癌细胞MCF7、和人类骨髓瘤细胞KG1共培养于U型96孔板，上述NK效应细胞与靶标细胞的个数比例范围为由2.5∶1到10∶1。每组实验重复3次。经过2小时的共培养后，用DELFIA EuTDA细胞毒性试剂盒(得自美国的PerkinElmer公司)检测NKG2D-CAR NK细胞溶解肿瘤细胞的能力，杀伤效果用如下公式计算：NK cells transfected with NKG2D-CD8-DAP12 mRNA or mGFP-CD8-DAP12 mRNA were associated with human colorectal cancer cell HCT116, human ovarian cancer cell SKOV3, human head and neck cancer cells Fadu and Detroit, human hepatoma cell HepG2, human breast cancer The cell MCF7 and the human myeloma cell KG1 were co-cultured in a U-shaped 96-well plate, and the ratio of the above-mentioned NK effector cells to the target cells ranged from 2.5:1 to 10:1. Each set of experiments was repeated 3 times. After 2 hours of co-cultivation, the ability of NKG2D-CAR NK cells to lyse tumor cells was examined using the DELFIA EuTDA cytotoxicity kit (available from PerkinElmer, USA), and the killing effect was calculated by the following formula:
％特异性裂解＝((实验组释放(读数)-空白组释放(读数))/(最大释放(读数)-空白组释放(读数)))×100% specific lysis = ((experimental group release (reading) - blank group release (reading)) / (maximum release (reading) - blank group release (reading))) x 100
结果显示，NKG2D-CAR修饰的NK细胞具有广泛并且强烈的杀瘤活性。如，当结直肠癌细胞HCT116、卵巢癌细胞SKOV3、头颈癌细胞Fadu和Detroit、肝癌细胞HepG2、乳腺癌细胞MCF7、以及骨髓瘤细胞KG1作为靶标时，NKG2D-CAR修饰的NK细胞的杀伤效果在10∶1时均已显著高于对照组的杀伤效果，见图10。以上结果充分说明了NKG2D-CAR NK对肿瘤杀伤的普遍性和高效性。The results show that NKG2D-CAR modified NK cells have broad and strong tumoricidal activity. For example, when colorectal cancer cell HCT116, ovarian cancer cell SKOV3, head and neck cancer cells Fadu and Detroit, liver cancer cell HepG2, breast cancer cell MCF7, and myeloma cell KG1 are targeted, the killing effect of NKG2D-CAR modified NK cells is At 10:1, it was significantly higher than the killing effect of the control group, as shown in Figure 10. The above results fully demonstrate the universality and high efficiency of NKG2D-CAR NK on tumor killing.
试验例2：NKG2D-CAR NK细胞对人肿瘤细胞体内杀伤能力的检测分析Test Example 2: Detection and analysis of NKG2D-CAR NK cells in vivo killing ability of human tumor cells
进一步利用植入人体肿瘤的小鼠模型测试了NKG2D-CAR NK细胞的体内杀瘤效果。实验用小鼠为非肥胖型糖尿病/重症联合免疫缺陷/IL-2Rγcnull(NSG)小鼠(6-8周，雌性)，每只小鼠被植入1×10 7个卵巢癌细胞SKOV3-Luc。肿瘤植入7天后，利用活体生物成像技术(BLI)，在IVIS Spectrum成像平台观测到了肿瘤的生长，并用成像仪(得自美国PerkinElmer公司)拍照记录肿瘤的生长情况。拥有相似BLI强度(BLI强度指的是活体成像仪记录的小鼠体内肿瘤细胞荧光强度)的小鼠被随机分为2组，分别为：磷酸盐缓冲液(PBS)组，和NKG2D-CAR NK细胞组，每组5只小鼠。NKG2D-CAR NK细胞组以每只小鼠每次1×10 7细胞数腹腔注射按制备例3所示方法制备的NKG2D-CAR修饰的NK细胞，PBS组每只小鼠每次腹腔注射100μL量的PBS。细胞注射方案为：每周二、五注射，总共注射三 周(即，每组共6次注射)。密切地观察小鼠的行为和生存情况，并将肿瘤的发展状况由BLI记录。所有的光信号和图片都由Xenogen活体成像软件v2.5记录分析。如图11所示，肿瘤植入后第28天，肿瘤生长状况显示PBS组小鼠肿瘤生长迅速，光信号强度增长约为第7天的10倍，而NKG2D-CAR NK细胞组的全部5只小鼠肿瘤生长不但被抑制，而且原有肿瘤消除明显，光信号强度下降到第7天的约10％。由此可见，NKG2D-CAR修饰的NK细胞能够有效杀死体内肿瘤。 The in vivo antitumor effect of NKG2D-CAR NK cells was further tested using a mouse model implanted in human tumors. The experimental mice were non-obese diabetic/severe combined immunodeficiency/IL-2Rγcnull (NSG) mice (6-8 weeks, female), and each mouse was implanted with 1×10 7 ovarian cancer cells SKOV3-Luc. . Seven days after tumor implantation, tumor growth was observed on the IVIS Spectrum imaging platform using in vivo bioimaging (BLI), and tumor growth was photographed using an imager (available from PerkinElmer, USA). Mice with similar BLI intensity (BLI intensity refers to the fluorescence intensity of tumor cells in mice recorded by a live imager) were randomly divided into 2 groups: phosphate buffered saline (PBS) group, and NKG2D-CAR NK. Cell group, 5 mice per group. In the NKG2D-CAR NK cell group, NKG2D-CAR-modified NK cells prepared by the method shown in Preparation Example 3 were intraperitoneally injected with 1 × 10 7 cells per mouse, and each mouse in the PBS group was intraperitoneally injected with a dose of 100 μL. PBS. The cell injection protocol was: two to five injections per week for a total of three weeks (ie, six injections per group). The behavior and survival of the mice were closely observed and the development of the tumors was recorded by BLI. All light signals and images were analyzed by Xenogen in vivo imaging software v2.5. As shown in Figure 11, on the 28th day after tumor implantation, tumor growth showed that the tumor growth of mice in the PBS group was rapid, the light signal intensity increased about 10 times that of the 7th day, and all 5 of the NKG2D-CAR NK cell group. Tumor growth in mice was not only inhibited, but the original tumors were eliminated and the light signal intensity decreased to about 10% on day 7. Thus, NKG2D-CAR-modified NK cells can effectively kill tumors in vivo.
试验例3：不同嵌合抗原受体的比较Test Example 3: Comparison of different chimeric antigen receptors
用嵌合抗原受体NKG2D-CD8-DAP12修饰的NK细胞和嵌合抗原受体NKG2D-CD8-CD3Z修饰的NK细胞分别对肿瘤细胞进行实验，比较杀伤活力。The NK cells modified with the chimeric antigen receptor NKG2D-CD8-DAP12 and the NK cells modified with the chimeric antigen receptor NKG2D-CD8-CD3Z were tested on tumor cells to compare the killing viability.
将转染NKG2D-CD8-DAP12 mRNA、NKG2D-CD8-CD3z mRNA、或者mGFP-CD8-DAP12 mRNA的NK细胞分别与人类结直肠癌细胞HCT116、人类卵巢癌细胞SKOV3、和人类头颈癌细胞Detroit共培养于U型96孔板，上述NK细胞与靶标细胞的个数比例范围为由2.5∶1到10∶1。每组实验重复3次。经过2小时的共培养后，用DELFIA EuTDA细胞毒性试剂盒(得自美国的PerkinElmer公司)检测NKG2D-CAR NK细胞溶解肿瘤细胞的能力，杀伤效果的计算应用如下公式：NK cells transfected with NKG2D-CD8-DAP12 mRNA, NKG2D-CD8-CD3z mRNA, or mGFP-CD8-DAP12 mRNA were co-cultured with human colorectal cancer cell line HCT116, human ovarian cancer cell line SKOV3, and human head and neck cancer cell line Detroit, respectively. In the U-shaped 96-well plate, the ratio of the above NK cells to the target cells ranges from 2.5:1 to 10:1. Each set of experiments was repeated 3 times. After 2 hours of co-cultivation, the ability of NKG2D-CAR NK cells to lyse tumor cells was examined using the DELFIA EuTDA Cytotoxicity Kit (available from PerkinElmer, USA). The killing effect was calculated using the following formula:
％特异性裂解＝((实验组释放(读数)-空白组释放(读数))/(最大释放(读数)-空白组释放(读数)))×100% specific lysis = ((experimental group release (reading) - blank group release (reading)) / (maximum release (reading) - blank group release (reading))) x 100
结果显示，在人类结直肠癌细胞HCT116、人类卵巢癌细胞SKOV3、和人类头颈癌细胞Detroit的杀瘤实验中，NKG2D-CD8-DAP12 mRNA修饰的NK细胞的杀瘤活性明显强于NKG2D-CD8-CD3z mRNA修饰的NK细胞。说明本发明所创建的具有所述特定构成单元组合的嵌合抗原受体NKG2D-CD8-DAP12具有显著的疗效和良好的商业化前景。The results showed that NKG2D-CD8-DAP12 mRNA-modified NK cells had significantly stronger tumoricidal activity than NKG2D-CD8- in the tumoricidal experiments of human colorectal cancer cell HCT116, human ovarian cancer cell SKOV3, and human head and neck cancer cell Detroit. CD3z mRNA modified NK cells. It is indicated that the chimeric antigen receptor NKG2D-CD8-DAP12 having the specific constituent unit combination created by the present invention has remarkable curative effect and good commercialization prospect.
上述NKG2D-CD8-CD3z修饰的NK细胞的制备方法与 NKG2D-CD8-DAP12修饰的NK细胞的制备方法相同，只是载体构建时胞内信号结构域使用CD3z(全长氨基酸序列的Genbank编号为：NP_932170.1)的胞内信号序列(如SEQ ID NO：23所示)。The above NKG2D-CD8-CD3z modified NK cells were prepared in the same manner as NKG2D-CD8-DAP12 modified NK cells except that the intracellular signal domain was constructed using CD3z (the full-length amino acid sequence of Genbank number: NP_932170). .1) The intracellular signal sequence (shown as SEQ ID NO: 23).

Claims (36)

  1. 一种嵌合抗原受体，该嵌合抗原受体包括可操作地连接的、依次串联的抗原结合结构域、间隔区、跨膜区和胞内结构域，其特征在于，所述抗原结合结构域来自NKG2D的配体结合区，所述胞内结构域来自DAP12的胞内信号区。A chimeric antigen receptor comprising an operably linked, sequentially tandem antigen binding domain, a spacer, a transmembrane domain and an intracellular domain, characterized in that said antigen binding structure The domain is from the ligand binding region of NKG2D, which is derived from the intracellular signaling region of DAP12.
  2. 根据权利要求1所述的嵌合抗原受体，其中所述抗原结合结构域的氨基酸序列与NKG2D的第X位-第216位氨基酸序列一致，且73≤X≤83。The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the antigen-binding domain is identical to the X-position 216 amino acid sequence of NKG2D, and 73 ≤ X ≤ 83.
  3. 根据权利要求1或2所述的嵌合抗原受体，其中所述抗原结合结构域的氨基酸序列如SEQ ID NO：3所示。The chimeric antigen receptor according to claim 1 or 2, wherein the amino acid sequence of the antigen-binding domain is as shown in SEQ ID NO: 3.
  4. 根据权利要求1所述的嵌合抗原受体，其中所述胞内结构域的氨基酸序列选自DAP12的第62-113位氨基酸；优选地，所述胞内结构域的氨基酸序列如SEQ ID NO：5所示。The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of said intracellular domain is selected from amino acids 62 to 113 of DAP12; preferably, the amino acid sequence of said intracellular domain is SEQ ID NO :5 is shown.
  5. 根据权利要求1所述的嵌合抗原受体，其中所述间隔区来自CD8α的铰链区，所述跨膜区来自CD8α的跨膜区。The chimeric antigen receptor according to claim 1, wherein the spacer is derived from a hinge region of CD8α, and the transmembrane region is derived from a transmembrane region of CD8α.
  6. 根据权利要求1或5所述的嵌合抗原受体，其中所述间隔区和跨膜区构成间隔跨膜区，并且其中所述间隔跨膜区的氨基酸序列与CD8α的第Y位-第210位氨基酸序列一致，且118≤Y≤128。The chimeric antigen receptor according to claim 1 or 5, wherein the spacer and transmembrane regions constitute a spacer transmembrane region, and wherein the amino acid sequence of the spacer transmembrane region and the Y-position of CD8α-210 The amino acid sequence is identical and 118 ≤ Y ≤ 128.
  7. 根据权利要求6所述的嵌合抗原受体，其中所述间隔跨膜区的氨基酸序列如SEQ ID NO：4所示。The chimeric antigen receptor according to claim 6, wherein the amino acid sequence of the spacer transmembrane region is as shown in SEQ ID NO: 4.
  8. 根据权利要求1所述的嵌合抗原受体，其中所述嵌合抗原受体的氨基酸序列如SEQ ID NO：1所示。The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO: 1.
  9. 一种分离的、编码嵌合抗原受体的DNA，该DNA包括可操作地连接的、依次串联的抗原结合结构域编码元件、间隔区编码元件、跨膜区编码元件和胞内结构域编码元件，其特征在于，所述抗原结合结构域编码元件来自NKG2D的配体结合区编码DNA，所述胞内结构域编码元件来自DAP12的胞内信号区编码DNA。An isolated DNA encoding a chimeric antigen receptor comprising an operably linked, sequentially tandem antigen binding domain coding element, a spacer coding element, a transmembrane region coding element, and an intracellular domain coding element And wherein the antigen binding domain coding element is derived from a ligand binding region encoding DNA of NKG2D, and the intracellular domain coding element is derived from the intracellular signal region encoding DNA of DAP12.
  10. 根据权利要求9所述的DNA，其中所述抗原结合结构域编码元件的核苷酸序列编码所述抗原结合结构域的氨基酸序列，所述抗原结合结构域的氨基酸序列与NKG2D的第X位-第216位氨基酸序列一致，且73≤X≤83。The DNA according to claim 9, wherein the nucleotide sequence of the antigen-binding domain coding element encodes an amino acid sequence of the antigen-binding domain, the amino acid sequence of the antigen-binding domain and the X-th position of NKG2D- The amino acid sequence at position 216 is identical, and 73 ≤ X ≤ 83.
  11. 根据权利要求9或10所述的DNA，其中所述抗原结合结构域编码元件的核苷酸序列如SEQ ID NO：6所示。The DNA according to claim 9 or 10, wherein the nucleotide sequence of the antigen-binding domain coding element is as shown in SEQ ID NO: 6.
  12. 根据权利要求9所述的DNA，其中所述胞内结构域编码元件编码所述胞内结构域的氨基酸序列，所述胞内结构域的氨基酸序列选自DAP12的第62-113位氨基酸；优选地，所述胞内结构域编码元件的核苷酸序列如SEQ ID NO：8所示。The DNA according to claim 9, wherein said intracellular domain coding element encodes an amino acid sequence of said intracellular domain, said amino acid sequence of said intracellular domain being selected from amino acids 62 to 113 of DAP12; The nucleotide sequence of the intracellular domain coding element is set forth in SEQ ID NO: 8.
  13. 根据权利要求9所述的DNA，其中所述间隔区编码元件来自CD8α的铰链区编码DNA，所述跨膜区编码元件来自CD8α的跨膜区编码DNA。The DNA according to claim 9, wherein said spacer coding element is derived from the hinge region encoding DNA of CD8α, and said transmembrane region coding element is derived from the transmembrane region encoding DNA of CD8α.
  14. 根据权利要求9或13所述的DNA，其中所述间隔区编码元件和所述跨膜区编码元件构成间隔跨膜区编码元件，所述间隔跨膜区编码元件的核苷酸序列编码所述间隔跨膜区的氨基酸序列，所述间隔跨膜区的氨基酸序列与CD8α的第Y位-第210位氨基酸序列一致，且118≤Y≤128。The DNA according to claim 9 or 13, wherein said spacer coding element and said transmembrane region coding element constitute a spacer transmembrane region coding element, said nucleotide sequence of said spacer transmembrane region coding element encoding said The amino acid sequence of the transmembrane region is separated from the amino acid sequence of the Y-position to the 210th amino acid sequence of CD8α, and 118≤Y≤128.
  15. 根据权利要求14所述的DNA，其中所述间隔跨膜区编码元件的核苷酸序列如SEQ ID NO：7所示。The DNA according to claim 14, wherein the nucleotide sequence of the spacer transmembrane region coding element is set forth in SEQ ID NO: 7.
  16. 根据权利要求9所述的DNA，其核苷酸序列如SEQ ID NO：2所示。The DNA according to claim 9, which has the nucleotide sequence shown as SEQ ID NO: 2.
  17. 一种分离的、由根据权利要求9-16中任一项所述的DNA转录的mRNA。An isolated mRNA transcribed from the DNA of any one of claims 9-16.
  18. 一种重组表达载体，其含有与启动子有效连接的根据权利要求9-16中任一项所述的DNA。A recombinant expression vector comprising the DNA of any one of claims 9-16 operably linked to a promoter.
  19. 根据权利要求18所述的重组表达载体，其中所述重组表达载体在根据权利要求9-16中任一项所述的DNA之前依次包含CMV启动子、T7启动子、具有kozak序列的5’UTR和GM-CSFα链信号肽编码序列；并且在根据权利要求9-16中任一项所述的DNA之后包含具有PolyA信号的α球蛋白的3’UTR。The recombinant expression vector according to claim 18, wherein the recombinant expression vector comprises a CMV promoter, a T7 promoter, and a 5'UTR having a kozak sequence in sequence before the DNA according to any one of claims 9-16. And a GM-CSF alpha chain signal peptide coding sequence; and comprising the 3'UTR of the alpha globulin having a PolyA signal following the DNA according to any one of claims 9-16.
  20. 一种嵌合抗原受体修饰的NK细胞，该NK细胞的表面被权利要求1-8中任一项所述的嵌合抗原受体修饰。A chimeric antigen receptor-modified NK cell whose surface is modified with the chimeric antigen receptor of any one of claims 1-8.
  21. 一种制备根据权利要求20所述的嵌合抗原受体修饰的NK细胞的方法，包括以下步骤：A method of preparing a chimeric antigen receptor-modified NK cell according to claim 20, comprising the steps of:
    1)提供NK细胞；1) providing NK cells;
    2)提供编码根据权利要求1-8中任一项所述的嵌合抗原受体的核酸；2) providing a nucleic acid encoding the chimeric antigen receptor according to any one of claims 1-8;
    3)将所述核酸转染入所述NK细胞中。3) Transfecting the nucleic acid into the NK cells.
  22. 根据权利要求21所述的方法，其中步骤1)所述的NK细胞由外周血单个核细胞制备。The method according to claim 21, wherein the NK cells of step 1) are prepared from peripheral blood mononuclear cells.
  23. 根据权利要求21所述的方法，其中所述转染通过冷冻电穿孔技术或慢病毒载体进行。The method of claim 21, wherein the transfection is performed by a freeze electroporation technique or a lentiviral vector.
  24. 根据权利要求21所述的方法，其中步骤2)所述的核酸为根据权利要求9-16中任一项所述的DNA、或根据权利要求17所述的mRNA。The method according to claim 21, wherein the nucleic acid according to any one of claims 9 to 16, or the mRNA according to claim 17, is the nucleic acid according to any one of claims 9 to 16.
  25. 根据权利要求20所述的嵌合抗原受体修饰的NK细胞在制备用于治疗或预防肿瘤和/或癌症的药物中的用途。Use of the chimeric antigen receptor-modified NK cell according to claim 20 for the preparation of a medicament for the treatment or prevention of a tumor and/or cancer.
  26. 根据权利要求25所述的用途，其中所述肿瘤和/或癌症是NKG2D配体阳性的，包括所述肿瘤和/或癌症未经处理是NKG2D配体阳性的以及所述肿瘤和/或癌症经处理而成为NKG2D配体阳性的。The use according to claim 25, wherein the tumor and/or cancer is NKG2D ligand positive, including the tumor and/or cancer being untreated is NKG2D ligand positive and the tumor and/or cancer Treated to become NKG2D ligand positive.
  27. 根据权利要求20所述的嵌合抗原受体修饰的NK细胞在制备用于检测宿主的肿瘤和/或癌症的药物中的用途。Use of the chimeric antigen receptor-modified NK cell according to claim 20 for the preparation of a medicament for detecting a tumor and/or cancer of a host.
  28. 一种药物组合物，其中该药物组合物包括作为活性成分的根据权利要求20所述的嵌合抗原受体修饰的NK细胞，及可药用辅料。A pharmaceutical composition comprising the chimeric antigen receptor-modified NK cell according to claim 20 as an active ingredient, and a pharmaceutically acceptable adjuvant.
  29. 根据权利要求28所述的药物组合物，其中所述药物组合物包含每人每个疗程总剂量范围为1×10 6-1×10 11个的所述嵌合抗原受体修饰的NK细胞。 The pharmaceutical composition according to claim 28, wherein said pharmaceutical composition comprises said chimeric antigen receptor-modified NK cells in a total dose ranging from 1 x 10 6 to 1 x 10 11 per subject per subject.
  30. 根据权利要求28所述的药物组合物，其中所述药物组合物的给药方式包括静脉给药或局部给药。The pharmaceutical composition according to claim 28, wherein the pharmaceutical composition is administered by intravenous or topical administration.
  31. 一种治疗肿瘤和/或癌症的方法，包括对肿瘤和/或癌症患者施用根据权利要求20所述的嵌合抗原受体修饰的NK细胞。A method of treating a tumor and/or cancer comprising administering a chimeric antigen receptor modified NK cell according to claim 20 to a tumor and/or cancer patient.
  32. 根据权利要求31所述的方法，其中所述嵌合抗原受体修饰的NK细胞的施用剂量为每人每个疗程总剂量范围为1×10 6-1×10 11个细胞。 The method according to claim 31, wherein said chimeric antigen receptor-modified NK cells are administered at a dose ranging from 1 x 10 6 to 1 x 10 11 cells per patient per course of treatment.
  33. 根据权利要求31所述的方法，其中所述嵌合抗原受体修饰的NK细胞的给药方式包括静脉给药或局部给药。The method according to claim 31, wherein the administration of the chimeric antigen receptor-modified NK cells comprises intravenous administration or topical administration.
  34. 根据权利要求31所述的方法，其中所述肿瘤和/或癌症是NKG2D配体阳性的，包括所述肿瘤和/或癌症未经处理是NKG2D配体阳性的以及所述肿瘤和/或癌症经处理而成为NKG2D配体阳性的。The method according to claim 31, wherein said tumor and/or cancer is NKG2D ligand positive, including said tumor and/or cancer being untreated is NKG2D ligand positive and said tumor and/or cancer Treated to become NKG2D ligand positive.
  35. 一种工具载体，该工具载体依次包含可操作地连接的CMV启动子、T7启动子、具有kozak序列的5’UTR、GM-CSFα链信号肽编码序列、和具有PolyA信号的α球蛋白的3’UTR。A tool vector comprising, in turn, an operably linked CMV promoter, a T7 promoter, a 5'UTR having a kozak sequence, a GM-CSF alpha chain signal peptide coding sequence, and an alpha globulin having a PolyA signal. 'UTR.
  36. 根据权利要求35所述的工具载体，其中所述CMV启动子的核苷酸序列如SEQ ID NO：22所示，T7启动子的核苷酸序列如SEQ ID NO：17所示，所述具有kozak序列的5’UTR的核苷酸序列如SEQ ID NO：18所示，所述GM-CSFα链信号肽编码序列的核苷酸序列如SEQ ID NO：19所示，所述具有PolyA信号的α球蛋白的3’UTR的核苷酸序列如SEQ ID NO：21所示。The tool vector according to claim 35, wherein the nucleotide sequence of the CMV promoter is as shown in SEQ ID NO: 22, and the nucleotide sequence of the T7 promoter is as shown in SEQ ID NO: 17, The nucleotide sequence of the 5'UTR of the kozak sequence is set forth in SEQ ID NO: 18, and the nucleotide sequence of the GM-CSF alpha chain signal peptide coding sequence is set forth in SEQ ID NO: 19, which has a PolyA signal. The nucleotide sequence of the 3'UTR of the α-globulin is shown in SEQ ID NO:21.
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WO2022182976A1 (en) * 2021-02-25 2022-09-01 Riboz, Llc Engineered expression constructs to increase protein expression from synthetic ribonucleic acid (rna)

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