CN116179495A - Transgenic immune cells and uses thereof - Google Patents

Transgenic immune cells and uses thereof Download PDF

Info

Publication number
CN116179495A
CN116179495A CN202211505642.8A CN202211505642A CN116179495A CN 116179495 A CN116179495 A CN 116179495A CN 202211505642 A CN202211505642 A CN 202211505642A CN 116179495 A CN116179495 A CN 116179495A
Authority
CN
China
Prior art keywords
cells
optionally
cell
nucleic acid
car
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202211505642.8A
Other languages
Chinese (zh)
Inventor
张彩
胡渊
陈敏华
王烃
伏永玲
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Enkai Cell Technology Co ltd
Original Assignee
Shanghai Enkai Cell Technology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Enkai Cell Technology Co ltd filed Critical Shanghai Enkai Cell Technology Co ltd
Priority to CN202211505642.8A priority Critical patent/CN116179495A/en
Publication of CN116179495A publication Critical patent/CN116179495A/en
Priority to PCT/CN2023/101463 priority patent/WO2024113777A1/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Veterinary Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Plant Pathology (AREA)
  • Epidemiology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The invention provides a transgenic immune cell and application thereof, wherein the immune cell expresses chimeric antigen receptor and an immune stimulating molecule, and the immune stimulating molecule comprises IL-15. The transgenic immune cells can express and secrete chimeric antigen receptors and immune stimulation molecules simultaneously, so that the immune cells can target corresponding antigens and be positioned on the surfaces of cells expressing the antigens, in addition, the immune stimulation molecules further promote the activation and proliferation of the immune cells, maintain the quantity and activity of the immune cells in a local tumor microenvironment, ensure that the immune cells keep strong tumor killing activity, and effectively avoid toxic and side effects caused by high-dose or repeated injections of the whole body.

Description

Transgenic immune cells and uses thereof
Technical Field
The invention relates to the field of biotechnology, in particular to a transgenic immune cell and application thereof.
Background
In recent years, chimeric antigen receptor T (chimeric antigen receptorT, CAR-T) cells have achieved remarkable effects in the treatment of hematological malignancies. However, the CAR-T cells are prone to adverse reactions such as cytokine storm, neurotoxicity and GVHD in clinical application, and the therapeutic effect of the CAR-T cells on solid tumors is not ideal, so that the clinical application of the CAR-T cells still faces challenges.
Compared with the CAR-T cells, the CAR-NK cells have the advantage of good safety, and generally do not cause side effects such as cytokine storm, GVHD and the like; NK cells do not need antigen presentation and are not limited by MHC, so that the NK cells can play a role in directly killing tumor cells; the CAR-NK cells can identify and kill tumors by various identification mechanisms such as CAR dependence, NKR dependence and the like, and the anti-tumor spectrum is wide. Therefore, the CAR-NK cells have wide application prospect in anti-tumor treatment and become hot spots in the field of development of cellular immunotherapy. However, one of the difficulties faced in the development of CAR-NK cells is the short survival time of NK cells in vivo, affecting their in vivo effects.
IL-15 is a cytokine which can promote the survival, proliferation and function of T cells and NK cells, IL-15 shares IL-2/15Rβγc receptor with IL-2, IL-15 and IL-15Rβγc form dimer and then combine with IL-15Rβγc to activate downstream JAK1/JAK3 and STAT3/STAT5 signaling pathway, thereby promoting proliferation, activation and effector functions of NK cells. Thus, IL-15 has become a hot target for drug development to enhance the persistence and proliferative activity of lymphocytes in vivo.
However, the problems with the use of IL-15 in vivo are short half-life, limited in vivo efficacy, large doses, and frequent administration, resulting in various side effects including hypotension, thrombocytopenia, elevated AST and ALT, etc., which may lead to intolerance of such treatment in cancer patients. In clinical application and drug development, the activity of IL-15 for promoting lymphocyte proliferation and persistence and promoting immune response should be maintained as much as possible, and the side effects related to IL-15 should be reduced as much as possible.
Based on the above research and development status, there is a need to further study a safe and effective method for improving the persistence of CAR-NK cells in vivo.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the related art to some extent. Therefore, the invention provides the transgenic immune cell, the proliferation capacity and the in-vivo survival time of which are obviously improved compared with those of natural immune cells, and the transgenic immune cell has higher safety.
Thus, in a first aspect of the invention, the invention provides a transgenic immune cell. According to an embodiment of the invention, the immune cells express a chimeric antigen receptor and an immunostimulatory molecule, including IL-15. The transgenic immune cells according to embodiments of the present invention are capable of simultaneously expressing and secreting chimeric antigen receptors and immunostimulatory molecules. The chimeric antigen receptor can enable immune cells to target corresponding antigens and locate on the surfaces of cells expressing the antigens, and in addition, the immune stimulating molecules further promote the activation and proliferation of the immune cells, maintain the number and activity of the immune cells in a local tumor microenvironment, enable the immune cells to keep strong tumor killing activity, and can effectively avoid toxic and side effects caused by high-dose whole body or repeated multiple injection.
According to an embodiment of the present invention, the transgenic immune cell may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the chimeric antigen receptor comprises: an extracellular region capable of specifically binding to an antigen; a transmembrane region; and an intracellular region comprising an immune co-stimulatory molecule intracellular segment and a signal transduction domain; wherein the C end of the extracellular region is connected with the N end of the transmembrane region, and the C end of the transmembrane region is connected with the N end of the intracellular region. In the present application, the kind of antigen recognized by the chimeric antigen receptor is not particularly limited, and is suitable for specifically recognizing various antigens.
According to an embodiment of the invention, the antigen is a tumor-associated antigen. According to some embodiments of the invention, the kind of antigen is not particularly limited.
According to an embodiment of the invention, the extracellular region comprises a heavy chain variable region and a light chain variable region of an antibody,the antibody binds to the antigen. As will be appreciated by those skilled in the art, the extracellular region may include a binding region that recognizes the antigen, and the extracellular region may include a fully anti-antibody, a Fab 'antibody, a F (ab') 2 At least one of an antibody, fv antibody, single chain antibody, and nanobody. According to some preferred embodiments of the invention, the extracellular region comprises a single chain antibody.
According to an embodiment of the invention, the antigen comprises at least one selected from the group consisting of mesothelin, HER2, EGFR, GPC3, MUC1, CEA, CLDN 18.2, epCAM, GD2, PSCA, CD133, CD19, CD20, CD22, CD30, CD33, BCMA.
According to an embodiment of the invention, the antigen is mesothelin. According to some embodiments of the invention, when the antigen is mesothelin, the transgenic immune cells can effectively target mesothelin-positive tumors, retain higher proliferation activity and have higher anti-tumor capability.
According to an embodiment of the invention, the extracellular region comprises an anti-mesothelin single chain antibody.
According to an embodiment of the invention, the anti-mesothelin single chain antibody comprises a light chain variable region of an anti-mesothelin antibody, a connecting peptide 1 and a heavy chain variable region of an anti-mesothelin antibody.
According to an embodiment of the invention, the connecting peptide 1 has an amino acid sequence shown as (GGGGS) n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
According to an embodiment of the invention, the anti-mesothelin single chain antibody comprises the amino acid sequence shown in SEQ ID NO. 11. In some specific embodiments, when the anti-mesothelin single-chain antibody has the above amino acid sequence, the transgenic immune cell can effectively target mesothelin positive tumor, retain higher proliferation activity and have higher anti-tumor capability.
According to an embodiment of the invention, the extracellular region further comprises a hinge region segment, the N-terminus of the hinge region segment being linked to the C-terminus of the single chain antibody.
According to an embodiment of the invention, the hinge region segment comprises at least one of the hinge regions selected from the group consisting of CD8, CD28 and immunoglobulins.
According to an embodiment of the invention, the hinge segment comprises a hinge region of CD 8.
According to an embodiment of the invention, the hinge fragment comprises the amino acid sequence shown in SEQ ID NO. 12.
According to an embodiment of the invention, the transmembrane region comprises at least one selected from the group consisting of CD4, CD8 a, CD28 and CD3 ζ or a fragment thereof.
According to an embodiment of the invention, the transmembrane region comprises a CD8 transmembrane region or fragment thereof.
According to an embodiment of the invention, the transmembrane region has the amino acid sequence shown in SEQ ID NO. 13.
According to an embodiment of the invention, the immune co-stimulatory molecule comprises at least one selected from the group consisting of CD28, ICOS, 4-1BB, OX40 and CD 27.
According to an embodiment of the invention, the intracellular segment of the immune co-stimulatory molecule is an intracellular segment of 4-1BB or CD28 or a fragment thereof.
According to an embodiment of the invention, the intracellular segment of the immune co-stimulatory molecule comprises the amino acid sequence shown in SEQ ID NO. 14.
According to an embodiment of the invention, the C-terminal end of the intracellular segment of the immune co-stimulatory molecule is connected to the N-terminal end of the signaling domain.
According to an embodiment of the invention, the signal transduction domain comprises at least one selected from cd3ζ or fcsriy or a fragment thereof.
It will be appreciated by those skilled in the art that the choice of hinge region, transmembrane region, immune co-stimulatory molecule intracellular segment and signaling domain is not particularly limited and that hinge region, transmembrane region, immune co-stimulatory molecule intracellular segment and signaling domain may be used as are available in chimeric antigen receptors conventional in the art.
According to an embodiment of the invention, the signal transduction domain comprises cd3ζ or fragment thereof.
According to an embodiment of the invention, the signal transduction domain comprises the amino acid sequence shown in SEQ ID NO. 15.
According to an embodiment of the invention, the immune cells comprise at least one of T cells and NK cells. In the present application, the kind of the immunostimulatory molecule is not particularly limited, and an agent capable of promoting an immune function of at least one of T cells and NK cells may be used, and in some embodiments of the present application, the immunostimulatory molecule is IL-15.
According to an embodiment of the invention, the immune cells are preferably NK cells.
According to an embodiment of the present invention, the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC) -derived NK cells, and NK-92 cells.
According to an embodiment of the invention, the T cells comprise CD4 + T cells, CD8 + T cells and γδ T cells.
In a second aspect of the invention, the invention provides an isolated nucleic acid. According to an embodiment of the invention, the isolated nucleic acid comprises: 1) A first nucleic acid molecule encoding a chimeric antigen receptor; 2) A second nucleic acid molecule encoding an immunostimulatory molecule, the immunostimulatory molecule comprising IL-15.IL-15 is a pleiotropic cytokine with the function of activating T cells, B cells and NK cells and mediating proliferation and survival of these cells. In addition, IL-15 activates, maintains and amplifies CD8 + Memory T cells, but not regulatory T lymphocytes. The isolated nucleic acid according to the embodiments of the present invention can package higher titer viruses after being introduced into recipient cells and achieve specific infection of immune cells, such as NK cells, by the viruses. After the isolated nucleic acid is introduced into immune cells, the immune cells can simultaneously express and secrete chimeric antigen receptor and immune stimulating molecules, so that the immune cells can target corresponding antigens and be positioned on the surfaces of cells expressing the antigens, in addition, the immune stimulating molecules such as IL-15 further promote the activation and proliferation of the immune cells, maintain the quantity and activity of the immune cells in the local microenvironment of the tumor, ensure that the immune cells maintain strong tumor killing activity, and effectively avoid the whole tumorThe toxic and side effects caused by high dosage or repeated injection can be avoided.
According to an embodiment of the present invention, the above isolated nucleic acid may further include at least one of the following additional technical features:
according to an embodiment of the invention, the chimeric antigen receptor is as defined in the first aspect.
According to an embodiment of the invention, the first nucleic acid molecule and the second nucleic acid molecule are arranged to express the chimeric antigen receptor and the immunostimulatory molecule in immune cells, and the immunostimulatory molecule is in a non-fused form with the chimeric antigen receptor.
According to an embodiment of the invention, the isolated nucleic acid further comprises: an internal ribosome entry site sequence disposed between the first nucleic acid molecule and the second nucleic acid molecule, the internal ribosome entry site having the nucleotide sequence set forth in SEQ ID NO. 16.
According to an embodiment of the invention, the isolated nucleic acid further comprises a third nucleic acid molecule, which third nucleic acid molecule is arranged between the first nucleic acid molecule and the second nucleic acid molecule, which third nucleic acid molecule encodes a connecting peptide 2, which connecting peptide 2 is capable of being cleaved. The connecting peptide 2 can separate the first nucleic acid molecule from the second nucleic acid molecule, and reduces functional interference of the first nucleic acid molecule and the second nucleic acid molecule.
According to an embodiment of the invention, the connecting peptide 2 comprises a 2A peptide or a fragment thereof. It will be appreciated by those skilled in the art that the connecting peptide 2 is not particularly limited, and conventional peptides having a self-cleavage function may be used.
According to an embodiment of the invention, the connecting peptide 2 comprises at least one of P2A, T2A, E a and F2A or a fragment thereof.
According to an embodiment of the invention, the connecting peptide 2 comprises P2A or a fragment thereof.
According to an embodiment of the invention, the connecting peptide 2 comprises the amino acid sequence shown in SEQ ID NO. 17.
According to an embodiment of the invention, the isolated nucleic acid further comprises: a first promoter operably linked to the first nucleic acid molecule; and a second promoter operably linked to the second nucleic acid molecule.
According to an embodiment of the invention, the first promoter, the second promoter are each independently selected from the group consisting of U6, H1, CMV, EF-1, LTR or RSV promoters.
According to an embodiment of the invention, the isolated nucleic acid further comprises a fourth nucleic acid molecule encoding a signal peptide. According to an embodiment of the present invention, the signal peptide expressed by the gene encoding the signal peptide is located at the amino terminal of the chimeric antigen receptor, is a chimeric antigen receptor membrane localization terminal peptide, and is hydrolyzed and detached after helping the localization of the chimeric antigen receptor to the endoplasmic reticulum after maturation of the protein, so that the chimeric antigen receptor on the viral particle does not contain the signal peptide.
According to an embodiment of the invention, the fourth nucleic acid molecule is operably linked to the first nucleic acid molecule.
According to an embodiment of the invention, the signal peptide comprises at least one selected from CSF2R and CD8 a or a fragment thereof. It will be appreciated by those skilled in the art that the kind of the signal peptide is not particularly limited, and that conventional signal peptides in the art can be used.
According to an embodiment of the invention, the signal peptide comprises CSF2R or a fragment thereof.
According to an embodiment of the invention, the signal peptide comprises the amino acid sequence shown in SEQ ID NO. 10.
According to an embodiment of the invention, the first nucleic acid molecule has at least one of the nucleotide sequences shown in SEQ ID NO. 3, 4, 5, 6 and 7.
According to an embodiment of the invention, the second nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO. 9.
According to an embodiment of the invention, the third nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO. 8.
According to an embodiment of the invention, the fourth nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO. 2.
According to an embodiment of the invention, the isolated nucleic acid has the nucleotide sequence shown in SEQ ID NO. 1.
In a third aspect of the invention, the invention proposes a construct. According to an embodiment of the invention, the construct carries an isolated nucleic acid as described previously. In the case of the above-described isolated nucleic acid being linked to a vector, the isolated nucleic acid may be linked directly or indirectly to control elements on the vector, as long as these control elements are capable of controlling translation and expression of the isolated nucleic acid, etc., i.e., the isolated nucleic acid is operably linked to the control elements. Of course, these control elements may be directly from the carrier itself or may be exogenous, i.e. not from the carrier itself.
According to an embodiment of the present invention, the construct may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the vector of the construct is a non-pathogenic viral vector. According to some embodiments of the invention, the expression vector has a higher expression efficiency when it is a viral vector.
According to an embodiment of the invention, the viral vector comprises at least one selected from the group consisting of a retroviral vector, a lentiviral vector or an adenovirus-associated viral vector.
In a fourth aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell carries the isolated nucleic acid or construction vector described previously. Recombinant cells according to embodiments of the invention can be used to express and obtain in vitro under suitable conditions the proteins encoded by the aforementioned isolated nucleic acids, such as chimeric antigen receptors and immunostimulatory molecules, such as IL-15 and mesothelin, in large quantities.
According to an embodiment of the present invention, the recombinant cell may further include at least one of the following additional technical features:
according to an embodiment of the invention, the recombinant cells comprise eukaryotic cells, preferably mammalian cells.
It should be noted that the recombinant cells of the present invention are not particularly limited, and may be prokaryotic cells, eukaryotic cells, or phage. Illustratively, the prokaryotic cell may be escherichia coli, bacillus subtilis, streptomycete, proteus mirabilis, or the like; the eukaryotic cells comprise fungi such as pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces, trichoderma and the like, insect cells such as armyworm and the like, plant cells such as tobacco and the like, and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells and the like. In some embodiments, the recombinant cells of the invention are preferably mammalian cells, including T cells, B cells, NK cells, BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.
In a fifth aspect of the invention, the invention provides a CAR-NK or CAR-T cell. According to an embodiment of the invention, the CAR-NK cells carry the isolated nucleic acid or construct described previously. The CAR-NK cells according to embodiments of the present invention are capable of simultaneously expressing and secreting chimeric antigen receptors and immunostimulatory molecules. The inventor finds in experiments that the IL-15 modification strategy provided by the invention can enable NK cells or T cells to locally secrete IL-15 in tumors, obviously improve the in-vitro proliferation capacity of the CAR-NK cells or the T cells, enhance the in-vivo survival time of the NK cells or the T cells, and improve the in-vivo anti-tumor function of the NK cells or the T cells. And the IL-15 released slowly and continuously in local can avoid toxic and side effects caused by systemic application of recombinant IL-15 or repeated administration.
According to some embodiments of the invention, the NK cells comprise at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC) -derived NK cells, and NK-92 cells.
According to an embodiment of the invention, the T cells comprise CD4 + T cells, CD8 + T cells and γδ T cells.
In a sixth aspect of the invention, the invention provides a method of obtaining a virus. According to an embodiment of the invention, the construct described previously is introduced into a first recipient cell; the first recipient cell into which the construct is introduced is cultured so as to obtain the virus. Methods according to some preferred embodiments of the invention can achieve higher titers of virus.
According to an embodiment of the invention, the virus comprises a lentivirus.
According to an embodiment of the invention, the first recipient cell is 293T.
In a seventh aspect of the invention, the invention provides a virus. According to an embodiment of the invention, obtained by the method for obtaining viruses described above.
In an eighth aspect of the invention, the invention provides a virus. According to an embodiment of the invention, the virus comprises a nucleotide sequence having the sequence shown in SEQ ID NO. 1.
According to an embodiment of the invention, the virus comprises at least one of a retrovirus, a lentivirus and an adenovirus.
According to an embodiment of the invention, the virus comprises a lentivirus.
In an eighth aspect of the invention, the invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: an isolated transgenic immune cell, nucleic acid, construct, recombinant cell, CAR-NK or CAR-T cell or virus as described previously. As described above, the isolated nucleic acid, the expression vector or the cell or virus carrying the isolated nucleic acid or the expression vector can simultaneously express and secrete the chimeric antigen receptor and the immune stimulating molecule, so that the immune cells can target the corresponding antigen and be positioned on the surface of the cell expressing the antigen, in addition, the immune stimulating molecule such as IL-15 further promotes the activation and proliferation of the immune cells, maintains the quantity and the activity of the immune cells in the local microenvironment of the tumor, so that the immune cells maintain strong tumor killing activity, effectively avoids the toxic and side effects caused by high-dose or repeated multiple injections of the whole body, and also avoids the toxic and side effects caused by the application of the high-dose or repeated multiple injections of recombinant IL-15 to the whole body. Therefore, the pharmaceutical composition comprising the above-mentioned substances also has the above-mentioned functions, and will not be described here again.
According to an embodiment of the present invention, the above pharmaceutical composition may further include at least one of the following additional technical features:
according to an embodiment of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, except insofar as any conventional excipients are incompatible with the compounds of the invention, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a deleterious manner, the use of which is also contemplated by the present invention.
For example, the isolated nucleic acids, expression vectors, or cells carrying the isolated nucleic acids or expression vectors of the invention may be incorporated into a medicament suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms. Such as liquid, semi-solid, and solid dosage forms, and the like, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The drug is typically in the form of an injection solution or infusion solution. The isolated nucleic acid, expression vector, or cell carrying the isolated nucleic acid or expression vector may be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.
The effective amount of the isolated nucleic acid, expression vector or cell carrying the isolated nucleic acid or expression vector of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, depending on the urgent requirements of the treatment situation.
In a tenth aspect of the invention, the invention provides a kit. According to an embodiment of the invention, the kit comprises: isolated nucleic acids, constructs or viruses as described previously. The isolated nucleic acid, construct or virus can significantly promote the activation or proliferation of NK cells or T cells, and thus, the kit comprising the same also has the function of promoting the activation or proliferation of NK cells or T cells. The kit can be used for scientific research, such as reversing NK cells or T cells with low proliferation activity, so that the proliferation activity of the NK cells or the T cells is increased from low to obtain a biological sample meeting expectations.
In an eleventh aspect of the invention, the invention provides a method of introducing a virus into an activated immune cell. According to an embodiment of the invention, the activated immune cells are electrotransfected or transfected with the construct described above or infected with the virus described above.
According to an embodiment of the invention, the immune cells comprise at least one of T cells and NK cells.
According to an embodiment of the invention, the immune cells are preferably NK cells.
According to an embodiment of the present invention, the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC) -derived NK cells, and NK-92 cells.
According to an embodiment of the invention, the T cells comprise CD4 + T cells, CD8 + T cells and γδ T cells.
In a twelfth aspect of the invention, the invention provides a method of obtaining a chimeric antigen receptor and an immunostimulatory molecule. According to an embodiment of the invention, the method comprises: introducing the construct or virus described above into a second recipient cell; culturing the second receptor cells introduced into the construct or virus to obtain the chimeric antigen receptor and the immunostimulatory molecule. As previously described, the construct or virus is capable of simultaneously expressing the chimeric antigen receptor and the immunostimulatory molecule under suitable conditions, and thus the chimeric antigen receptor and immunostimulatory molecule can be obtained in large amounts according to the methods of embodiments of the present invention.
According to an embodiment of the invention, the introduction of the second recipient cell is by electrotransfection, transfection or infection. The "electrotransformation" or "transfection" is a method of introducing a viral vector into a recipient cell, and the "infection" refers to a process in which a virus actively binds to and fuses a cell membrane and enters the cell. Wherein "electrotransfection" refers to a method of introducing a viral packaging vector into a recipient cell by means of electrical stimulation, and "transfection" refers to a method of introducing a viral packaging vector into a recipient cell by means of a chemical mediator, such as a liposome.
According to an embodiment of the invention, the second recipient cell is at least one of a T cell and an NK cell.
According to an embodiment of the invention, the second recipient cell is an NK cell.
According to an embodiment of the present invention, the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC) -derived NK cells, and NK-92 cells.
According to an embodiment of the invention, the T cells comprise CD4 + T cells, CD8 + T cells and γδ T cells.
According to an embodiment of the invention, the virus comprises at least one selected from the group consisting of retrovirus, lentivirus and adenovirus.
According to an embodiment of the invention, the virus comprises a lentivirus.
In a thirteenth aspect of the invention, the invention proposes a method of obtaining CAR-NK or CAR-T cells of a chimeric antigen receptor and an immunostimulatory molecule. According to an embodiment of the invention, it comprises: introducing the construct or virus described above into an NK cell or T cell; NK cells or T cells introduced with the construct or virus are cultured to obtain the CAR-NK or CAR-T cells. According to some specific embodiments of the invention, a lentiviral expression vector targeting mesothelin and simultaneously expressing IL-15 is constructed, and virus particles are packaged by lentivirus to infect NK cells or T cells, so that high infection efficiency and CAR positive NK cells or T cells are obtained, and the CAR-NK or CAR-T cells not only can target and kill mesothelin positive malignant tumors, but also have higher proliferation capacity and killing activity than unmodified NK cells or T cells because the CAR-NK or CAR-T cells can locally and continuously secrete IL-15, and especially can maintain long-term survival of NK cells or T cells in vivo, so that the NK cells or T cells can maintain higher proliferation activity and killing activity and exert stronger capacity of continuously killing tumors. More importantly, the local secreted IL-15 plays an effective biological function in the tumor part, and can effectively avoid toxic and side effects caused by systemic application of high dose or repeated injection of recombinant IL-15.
According to embodiments of the invention, the introduction of NK cells or T cells is by electrotransfection, transfection or infection.
In a fourteenth aspect of the invention, the invention proposes the use of an isolated nucleic acid, construct, recombinant cell, CAR-NK or CAR-T cell or virus as described hereinbefore for the preparation of a pharmaceutical composition for the treatment or prevention of a tumor. As described above, the isolated nucleic acid, construct, or cell carrying the above can simultaneously express and secrete chimeric antigen receptor and immunostimulatory molecule under appropriate conditions, so that the cell can target the corresponding antigen and locate on the surface of the cell expressing the antigen, in addition, the immunostimulatory molecule, such as IL-15, further promotes the activation and proliferation of immune cells, maintains the number and activity of immune cells in the local microenvironment of tumor, so as to maintain strong tumor killing activity, effectively avoid the toxic and side effects caused by high-dose or repeated multiple injections of whole body, and avoid the toxic and side effects caused by high-dose or repeated multiple injections of recombinant IL-15 applied whole body.
According to an embodiment of the invention, the tumor comprises at least one of a mesothelin-positive tumor, a HER 2-positive tumor, an EGFR-positive tumor, a GPC 3-positive tumor, a MUC 1-positive tumor, a CEA-positive tumor, a CLDN 18.2-positive tumor, an EpCAM-positive tumor, a GD 2-positive tumor, a PSCA-positive tumor, a CD 133-positive tumor, a CD 19-positive tumor, a CD 20-positive tumor, a CD 22-positive tumor, a CD 30-positive tumor, a CD 33-positive tumor, and a BCMA-positive tumor.
According to an embodiment of the invention, the mesothelin-positive tumor comprises at least one of pancreatic cancer, ovarian cancer, mesothelioma, cholangiocarcinoma and lung cancer.
In a fifteenth aspect of the invention, the invention provides the use of an isolated nucleic acid, construct or virus as hereinbefore described in the preparation of a kit for promoting NK cell or T cell activation or proliferation. According to some embodiments of the invention, the isolated nucleic acid, construct or virus is capable of significantly promoting the activation or proliferation of NK cells or T cells, and therefore, a kit comprising the same also has the function of promoting the activation or proliferation of NK cells or T cells. The kit can be used for scientific research, such as reversing NK cells or T cells with low proliferation activity, so that the proliferation activity of the NK cells or the T cells is increased from low to obtain a biological sample meeting expectations.
Drawings
FIG. 1 is a schematic diagram of a CAR targeting MSLN and modified with IL-15 according to example 1 of the invention, wherein SP represents a nucleotide sequence encoding a signal peptide, alpha-MSLN-scFv represents a nucleotide sequence encoding an anti-MSLN single chain antibody, CD8 hinge +TM represents a nucleotide sequence encoding a CD8 hinge region and a transmembrane region, 4-1BB represents a nucleotide sequence encoding a 4-1BB costimulatory signal domain, CD3 ζ represents a nucleotide sequence encoding an intracellular region of CD3 ζ, P2A represents a nucleotide sequence encoding a self-cleaving region of P2A, and IL15 represents a nucleotide sequence encoding the full length of IL 15;
FIG. 2 is a graph showing the results of measuring secretion levels of NK-92, α -MSLN-CAR-NK-92 and α -MSLN-CAR-IL15-NK-92 cell IL-15 according to example 2 of the present invention;
FIG. 3 is a graph of detection results of NK-92, α -MSLN-CAR-NK-92 and α -MSLN-CAR-IL15-NK-92 cell STAT5 phosphorylation levels of IL-15 according to example 2 of the invention;
FIG. 4 is a graph of the results of in vitro killing ability assays of NK-92, α -MSLN-CAR-NK-92 and α -MSLN-CAR-IL15-NK-92 cells of IL-15 according to example 2 of the present invention;
FIG. 5 is a graph of the results of in vitro proliferation potency assays for NK-92, α -MSLN-CAR-NK-92, and α -MSLN-CAR-IL15-NK-92 cells according to example 2 of the present invention;
FIG. 6 is a flow chart of the operation of IL-15 expressing CAR-NK cell for the treatment of pancreatic cancer Aspc-1 cell tumor bearing mice according to example 3 of the present invention;
FIG. 7 is a graph of the viability assay of NK-92, α -MSLN-CAR-NK-92 and α -MSLN-CAR-IL15-NK-92 cells in pancreatic cancer Aspc-1 cell tumor bearing mice according to example 3 of the present invention; and
FIG. 8 is a graph showing the anti-tumor ability test results of NK-92, α -MSLN-CAR-NK-92 and α -MSLN-CAR-IL15-NK-92 cells against pancreatic cancer Aspc-1 cell tumor-bearing mice according to example 3 of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. Wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
In describing the present invention, the terms related thereto are explained and illustrated only for convenience of understanding the scheme and are not to be construed as limiting the protection scheme of the present invention.
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.
In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
"operably linked" herein refers to the linkage of a foreign gene to a vector such that control elements within the vector, such as transcription control sequences and translation control sequences, and the like, are capable of performing their intended functions of regulating transcription and translation of the foreign gene. The usual vectors may be, for example, viral vectors, plasmids, phages and the like. After the expression vector according to some embodiments of the present invention is introduced into a suitable recipient cell, the expression of the isolated nucleic acid described above can be effectively achieved under the mediation of a regulatory system, thereby achieving in vitro mass-production of the protein encoded by the isolated nucleic acid.
As used herein, the term "suitable conditions" refers to conditions suitable for expression of the proteins encoded by the isolated nucleic acids described herein. Those skilled in the art will readily appreciate that conditions suitable for expression of the protein encoded by the isolated nucleic acid include, but are not limited to, suitable transformation or transfection means, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the conditions for optimal expression of the protein encoded by the isolated nucleic acid according to the specific environment of the laboratory.
The application constructs a transgenic immune cell simultaneously expressing a chimeric antigen receptor and an immune stimulation molecule, wherein the chimeric antigen receptor can target a plurality of antigens, so that the immune cell can target the corresponding antigen and be positioned on the surface of the cell expressing the antigen, and the immune stimulation molecule can further promote the activation and proliferation of the immune cell, such as IL-15 used in the application, after experimental verification, the proliferation activity and tumor killing capacity of the immune cell simultaneously expressing the chimeric antigen receptor and the IL-15 are obviously improved, and the toxic and side effects caused by systemic high dose or repeated multiple injections are effectively avoided.
The amino acid or nucleic acid sequences referred to herein are shown below.
Figure BDA0003968075470000101
Figure BDA0003968075470000111
Figure BDA0003968075470000121
Figure BDA0003968075470000131
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro(SEQ IDNO:10)。
Asp Ile Gln Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ala Ser Val Gln ValSer Cys Arg Ala Ser Gly Tyr Ser Ile Asn Thr Tyr Tyr Met Gln Trp Val Arg Gln Ala Pro Gly Ala Gly Leu GluTrp Met Gly Val Ile Asn Pro Ser Gly Val Thr Ser Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Leu Thr Asn AspThr Ser Thr Asn Thr Val Tyr Met Gln Leu Asn Ser Leu Thr Ser Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg TrpAla Leu Trp Gly Asp Phe Gly MetAsp Val Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly SerGly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Ile GlyAsp Arg Val Thr Ile Thr Cys ArgAla Ser Glu Gly Ile Tyr His Trp LeuAla Trp Tyr Gln Gln Lys Pro Gly Lys AlaPro Lys Leu Leu Ile Tyr Lys Ala Ser Ser LeuAla Ser GlyAla Pro SerArg Phe Ser Gly Ser Gly Ser Gly ThrAspPhe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asn Tyr Pro LeuThr Phe Gly Gly Gly Thr Lys Leu Glu Ile LysArg(SEQ ID NO:11)。
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro GluAla Cys Arg Pro AlaAla Gly GlyAla Val His ThrArg Gly LeuAsp PheAla Cys Asp(SEQ ID NO:12)。
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys(SEQ ID NO:13)。
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe MetArg Pro Val Gln Thr Thr Gln Glu GluAsp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu(SEQ ID NO:14)。
Arg Val Lys Phe SerArg SerAlaAsp Ala Pro Ala Tyr Gln Gln Gly GlnAsn Gln Leu TyrAsn Glu Leu AsnLeu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro GlnArg Arg Lys Asn Pro Gln Glu Gly Leu TyrAsn Glu Leu Gln Lys Asp Lys MetAla Glu Ala Tyr Ser Glu Ile GlyMet Lys Gly Glu ArgArg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser ThrAla Thr Lys Asp Thr TyrAsp Ala Leu His Met GlnAla Leu Pro ProArg(SEQ ID NO:15)。
Figure BDA0003968075470000141
Ala ThrAsn Phe Ser Leu Leu Lys GlnAla GlyAsp Val Glu GluAsn Pro Gly Pro(SEQ ID NO:17)。
Embodiments of the present invention will be described in more detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
The "plasmid" and "vector" described in the following examples have the same meaning and are used interchangeably.
Example 1: preparation of CAR-NK cells
1.1 construction of CAR expression plasmids
The invention designs a CAR vector (anti-MSLN-CAR-IL 15) sequence targeting Mesothelin (MSLN) and expressing IL-15, comprising a Signal Peptide (SP), an extracellular domain (anti-MSLN single chain antibody, anti-MSLN scFv) targeting recognition MSLN, a CD8a Hinge (Hinge) region and transmembrane region (TM), a 4-1BB intracellular co-stimulatory signaling domain and intracellular signaling molecule CD3 ζ, and an IL-15 gene fragment linked by P2A. The structure of each genetic element in the CAR vector is shown in figure 1. Wherein:
the full-length sequence of the gene of the CAR vector formed by the elements is shown in SEQ ID NO:1 is shown in the specification;
the signal peptide is CSF2R, and the nucleotide sequence of the signal peptide is shown in SEQ ID NO:2 is shown in the figure;
the nucleotide sequence of the anti-MSLN scFv is shown in SEQ ID NO:3 is shown in the figure;
The nucleotide sequence of the CD8 hinge region is shown in SEQ ID NO:4 is shown in the figure;
the nucleotide sequence of the CD8 transmembrane region is shown in SEQ ID NO:5 is shown in the figure;
the nucleotide sequence of the 4-1BB co-stimulatory signal domain is shown in SEQ ID NO:6 is shown in the figure;
the nucleotide sequence of the CD3 zeta intracellular region is shown as SEQ ID NO: shown in figure 7;
the nucleotide sequence of the self-shearing region P2A is shown in SEQ ID NO: shown as 8;
the nucleotide sequence of the IL-15 is shown as SEQ ID NO: shown at 9.
Firstly, inserting an anti-MSLN-CAR fragment into a lentiviral vector pLent-EF1 alpha-P2A-CMV-GP to construct a pLent-anti-MSLN-CAR-P2A-CMV-GP vector. IL-15 gene fragment was amplified from human PBMC cell cDNA and inserted into pLent-anti-MSLN-CAR-P2A-CMV-GP vector through cleavage site Not I. The correct sequence is verified by PCR identification and sequencing, which shows that the pLent-anti-MSLN-CAR-P2A-IL15-CMV-GP vector is successfully constructed.
1.2 packaging of lentiviruses and concentration of viral fluids
Taking 293T cells in logarithmic growth phase 5X 10 6 Inoculating into 10cm culture dish, adding 10ml of LDMEM culture medium, and adding 5% CO at 37deg.C 2 Culturing overnight in an incubator. When the cell density reached 80%, 10mL of fresh DMEM medium was replaced for virus packaging and the cell culture dishes were kept in an incubator for further use. Preparing a slow virus packaging system, respectively adding 26 mug of slow virus packaging auxiliary plasmid psPAX and 6 mug of target gene vector plasmid into 250 mug serum-free DMEM culture medium to prepare plasmid mixed solution, and uniformly mixing. mu.L of PEIpro was added to 235. Mu.L of serum-free DMEM medium and mixed well. Will be
Figure BDA0003968075470000151
The mixed solution is added into the plasmid mixed solution at one time, evenly mixed, incubated for 15min at room temperature, and added into a 293T cell culture dish after the incubation is finished. After 24h, the liquid was changed and the dish was returned to 37℃with 5% CO 2 In the incubator, after culturing for 48 hours, the cell supernatant was collected, centrifuged at 400 Xg for 5 minutes, cell debris was removed, and the supernatant was filtered into a 50mL centrifuge tube with a 0.45 μm filter head.Adding 5 XPEG 8000 solution for concentrating virus liquid, reversing the centrifuge tube upside down, mixing, and standing in a refrigerator at 4deg.C overnight. Centrifuging at 4deg.C at 4000 Xg for 20min, discarding supernatant, adding appropriate amount of serum-free DMEM to resuspend virus precipitate, transferring into EP tube, and storing in refrigerator at-80deg.C.
1.3 lentiviral titer assay
Taking 293T cells in logarithmic growth phase, and adjusting concentration to 1×10 5 /mL. A24-well plate was used, and 1mL of cell suspension (1X 10) was added to each well 5 Well), 3 added viral volume gradients were set. Placing at 37deg.C and 5% CO 2 The incubator was cultured overnight. The concentrated virus solution is diluted 10 times: mu.L of the virus concentrate was pipetted into the EP tube using a 1mLEP tube, diluted with 540. Mu.L of LDMEM medium, and mixed well. Changing liquid of 293T cells with fresh DMEM culture medium, respectively sucking 5 μl, 50 μl and 500 μl diluted virus liquid, adding into corresponding well, marking, and placing the culture plate back to 37deg.C and 5% CO 2 In an incubator. After 24h, the well plates were blotted for virus and 1mL fresh DMEM medium was added. After 72h, cells were harvested by pancreatin digestion, 293T cells GFP expression was measured using a flow meter and viral titer was converted according to the following formula:
titer (TU/mL) = (c×n×d×1000)/V
Wherein: GFP positive rate for c=flow assay
N=number of cells at infection (about 1×10 5 )
D = dilution factor of viral vector
V = volume of diluted virus added.
1.4 lentivirus infection of human NK cells
NK-92 cells (purchased from ATCC) in the logarithmic phase were harvested by centrifugation at 100 Xg for 5min, and the cells were resuspended in an appropriate amount of alpha-MEM medium to adjust the cell density to 5X 10 5 And each mL. The respective 5X 10 holes are connected into 24 hole plates 5 NK-92 cells, 1mL of virus concentrate and protamine (purchased from Soxhoba, final concentration 8. Mu.g/mL) were mixed well, and placed at 37℃in 5% CO 2 Culturing in an incubator. After 24h, the cell state was observed, the solution was changed, and the infected cells were transferred into EP tubes, 100The cells were resuspended by centrifugation at Xg for 5min, a small amount of fresh alpha-MEM medium was added, the cells were transferred to a cell culture flask, and 10mL of fresh alpha-MEM medium and IL-2 (final concentration 200 IU/mL) were added for further culture for 48h. Cells were transferred into a inflow tube, 3mL of 1 XPBS solution was added, 100 XPS was centrifuged for 5min, the supernatant was discarded, the cell pellet was sprung, and washed once again with 1 XPBS solution. The expression rate of GFP was measured using a flow meter. And continuing to expand culture, and adjusting the state of NK-92 cells after infection to expand. The infected NK-92 cells were sorted by flow meter for GFP-positive CAR-NK-92 cells for later experiments.
Example 2: determination of IL-15 secretion level and cell proliferation ability of CAR-NK cells
This example uses the CAR-NK-92 (referred to as CAR-NK in short) cells obtained in example 1 to measure IL-15 secretion level and cell proliferation ability
2.1ELISA detection of secretion level of IL-15 by CAR-NK-92 cells
NK-92, alpha-MSLN-CAR-NK-92 (carrying MSLN-targeted CAR) and alpha-MSLN-CAR-IL 15-NK-92 (carrying MSLN-targeted CAR and expressing IL-15) cells were cultured separately, and supernatants were collected for 24 h. ELISA detects the IL-15 content in supernatants of different groups. The results of the experiment are shown in FIG. 2, in which IL-15 was hardly detected in NK-92 and alpha-MSLN-CAR-NK-92 cell supernatants. However, significant IL-15 was detected in the supernatant of CAR-IL15-NK-92 cells at a level of 74.10.+ -. 5.86pg/mL. Demonstrating the ability of the engineered modified alpha-MSLN-CAR-IL 15-NK cells of the invention to secrete IL-15.
2.2 detection of STAT5 phosphorylation level of CAR-NK cells
After IL-15 binds to the IL-15 receptor, downstream STAT5 phosphorylation (pSTAT 5) is activated, and pSTAT5 enters the nucleus to promote the expression of activation, proliferation and anti-apoptosis related genes. Thus, the inventors further observed whether the IL-15 secreted by the modified CAR-NK cells of the invention is biologically active, phosphorylating downstream STAT 5. The NK-92, alpha-MSLN-CAR-NK-92 and alpha-MSLN-CAR-IL 15-NK-92 cells were cultured in serum-free RPMI 1640 medium for 12 hours, respectively, to perform starvation treatment for reducing the phosphorylation level of autologous STAT 5. Cells were harvested and the level of pSTAT5 was detected by flow-through. The results are shown in FIG. 3, where the level of STAT5 phosphorylation was lower in the NK-92 cell group and the α -MSLN-CAR-NK-92 cell group, and significantly higher in the α -MSLN-CAR-IL15-NK-92 cell group than in the control group.
2.3 detection of in vitro killing Capacity of CAR-NK cells
The inventors incubated the above NK-92, alpha-MSLN-CAR-NK-92 and alpha-MSLN-CAR-IL 15-NK-92 cells with pancreatic cancer cell line Aspc-1 for 5 hours, respectively, and then examined the killing efficiency. As can be seen from fig. 4, the effective target ratio of α -MSLN-CAR-NK-92 and α -MSLN-CAR-IL15-NK-92 is 5: the killing efficiency of the Aspc-1 cells in the step 1 is 48.01 +/-2.00% and 48.60 +/-1.78%, which are obviously higher than that of NK-92 cells (35.59 +/-2.46%); however, the killing efficiency of the alpha-MSLN-CAR-IL 15-NK-92 cells was not significantly different from that of the alpha-MSLN-CAR-NK-92 group.
2.4 detection of in vitro proliferation Capacity of CAR-NK cells
The inventors further validated the pro-survival effect of autocrine IL-15 on NK-92 cells. Cell proliferation curves were drawn by plating the NK-92, alpha-MSLN-CAR-NK-92 and alpha-MSLN-CAR-IL-15-NK-92 cells of the same cell number into 96-well plates, counting cells every 3 days, culturing for 24 days, respectively. As can be seen from fig. 5, the number of α -MSLN-CAR-IL-15-NK-92 cells began to be different from the two groups of NK-92, α -MSLN-CAR-NK-92 cells by day 12 of culture. By day 21, there was a very significant difference starting. It was demonstrated that secreted IL-15 significantly promoted the survival and proliferation capacity of CAR-NK cells.
Example 3: IL-15-expressing CAR-NK cell in vivo anti-tumor ability and CAR-NK cell in vivo viability assay
This example establishes a pancreatic cancer Aspc-1 cell tumor-bearing mouse transplantation model to observe the therapeutic effect of CAR-NK-92 cells on pancreatic cancer. The specific experimental procedure is as follows:
BALB/c-nu nude mice of 6 weeks old were selected for underarm subcutaneous tumor-bearing at a dose of 2X 10 6 Cells/cells. After about 4 days, cell therapy was started after one week. Tumor volume size was measured prior to treatment and randomly divided into PBS group, NK-92 cell treatment group, alpha-MSLN-CAR-NK-92 cell treatment group and alpha-MSLN-CAR-IL 15-NK-92 cell treatment group according to tumor volume sizeAnd (5) treatment groups. Mice in the treatment group were given 1X 10 intravenous injection of effector cells from the tail 7 Untreated groups were injected with equal volumes of 1 XPBS, once every other week for 5 total treatments, and every 3 days for tail intravenous injection of IL-2 (5X 10) 4 IU/r), specific experimental setup and operational flow are described with reference to fig. 6. To investigate the pro-survival effect of IL-15 on CAR-NK-92 cells, peripheral blood was collected from mice on the first, third and seventh days after treatment, the PerCP/cyanine5.5anti-human CD56 antibody was labeled after erythrocyte lysis, and the proportion of CD56 cell population in peripheral blood lymphocytes, i.e., the proportion of NK-92 cells, was flow-detected. Tumor volume was measured every 3 days and tumor growth curves were drawn.
As a result, as shown in FIG. 7, the proportion of NK-92 cells, α -MSLN-CAR-NK-92 cells and α -MSLN-CAR-IL15-NK-92 cells in the mice at the time of NK cell treatment was 27.3%, 29.6% and 26.8%, respectively, on the peripheral blood lymphocytes, and the proportion of NK-92 cells in each group was similar in the body. On the third day, the in vivo NK-92 cells, alpha-MSLN-CAR-NK-92 cells and alpha-MSLN-CAR-IL 15-NK-92 cells in mice accounted for 8.32%, 9.83% and 11.5% of peripheral blood lymphocytes, respectively. It can be seen that the proportion of NK-92 cells was decreased in each group on the third day compared to the first day, the NK-92 group was decreased from 27.3% to 8.32%, the α -MSLN-CAR-NK-92 group was decreased from 29.6% to 9.83%, and the α -MSLN-CAR-IL15-NK-92 group was decreased from 28.7% to 11.762%. On day seven, the in vivo proportions of NK-92 cells, alpha-MSLN-CAR-NK-92 cells and alpha-MSLN-CAR-IL 15-NK-92 cells were 0.13%, 3.32%, and 9.27%, respectively. It can be seen that the proportion of IL-15 modified alpha-MSLN-CAR-IL 15-NK-92 cells in vivo is highest and the persistence in vivo is more prominent than in the NK-92 group and the alpha-MSLN-CAR-NK-92 group. Therefore, the expression of IL-15 can improve the viability of NK cells in vivo, and the locally secreted IL-15 has obvious effect of maintaining the survival of NK cells in vivo.
By measuring tumor size and plotting tumor growth curves, it can be seen from fig. 8 that compared with the control PBS group and NK-92 cell treatment group, the α -MSLN-CAR-NK92 cell and the α -MSLN-CAR-IL15-NK-92 cell treatment can significantly inhibit tumor growth, and the α -MSLN-CAR-IL15-NK-92 cell shows better antitumor effect than the α -MSLN-CAR-NK-92 cell. The results show that the mesothelin-targeted CAR-NK-92 cells can inhibit the growth of pancreatic cancer and have good treatment effect. The IL-15 gene modification can improve the persistence of the CAR-NK-92 cells in vivo, improve the viability of the CAR-NK-92 cells in vivo and improve the anti-tumor effect of the CAR-NK-92 cells.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present invention, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims (35)

1. A transgenic immune cell, wherein the immune cell expresses a chimeric antigen receptor and an immunostimulatory molecule, the immunostimulatory molecule comprising IL-15.
2. The transgenic immune cell of claim 1, wherein the chimeric antigen receptor comprises:
an extracellular region capable of specifically binding to an antigen;
a transmembrane region; and
an intracellular region comprising an immune co-stimulatory molecule intracellular segment and a signal transduction domain;
wherein the C end of the extracellular region is connected with the N end of the transmembrane region, and the C end of the transmembrane region is connected with the N end of the intracellular region.
3. The transgenic immune cell of claim 2, wherein the antigen is a tumor-associated antigen.
4. The transgenic immune cell of claim 2, wherein the extracellular region comprises a heavy chain variable region and a light chain variable region of an antibody that binds the antigen.
5. The transgenic immune cell of claim 2, wherein the antibody is a single chain antibody.
6. The transgenic immune cell of claim 2, wherein the antigen comprises at least one selected from the group consisting of mesothelin, HER2, EGFR, GPC3, MUC1, CEA, CLDN 18.2, epCAM, GD2, PSCA, CD133, CD19, CD20, CD22, CD30, CD33, BCMA;
preferably, the antigen is mesothelin;
optionally, the extracellular region comprises an anti-mesothelin single chain antibody;
optionally, the anti-mesothelin single chain antibody comprises a light chain variable region of an anti-mesothelin antibody, a connecting peptide 1, and a heavy chain variable region of an anti-mesothelin antibody;
optionally, the connecting peptide 1 has an amino acid sequence shown as (GGGGS) n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
optionally, the anti-mesothelin single chain antibody comprises the amino acid sequence shown in SEQ ID NO. 11.
7. The transgenic immune cell of claim 6, wherein the extracellular region further comprises a hinge region segment, the N-terminus of the hinge region segment being linked to the C-terminus of the single chain antibody;
optionally, the hinge region segment comprises at least one of a hinge region selected from CD8, CD28 and an immunoglobulin;
Optionally, the hinge segment comprises a hinge region of CD 8;
optionally, the hinge fragment comprises the amino acid sequence shown in SEQ ID NO. 12.
8. The transgenic immune cell of claim 2, wherein the transmembrane region comprises at least one member selected from the group consisting of CD4, CD8 a, CD28 and cd3ζ or fragment thereof;
optionally, the transmembrane region comprises a CD8 transmembrane region or fragment thereof;
optionally, the transmembrane region has the amino acid sequence shown in SEQ ID NO. 13.
9. The transgenic immune cell of claim 2, wherein the immune co-stimulatory molecule comprises at least one member selected from the group consisting of CD28, ICOS, 4-1BB, OX40 and CD 27;
optionally, the intracellular segment of the immune co-stimulatory molecule is an intracellular segment of 4-1BB or CD28 or fragment thereof;
optionally, the intracellular segment of the immune co-stimulatory molecule comprises the amino acid sequence of SEQ ID NO. 14.
10. The transgenic immune cell of claim 2, wherein the C-terminus of the intracellular segment of the immune co-stimulatory molecule is linked to the N-terminus of the signaling domain;
optionally, the signal transduction domain comprises at least one selected from cd3ζ or fcsriy or a fragment thereof;
Optionally, the signal transduction domain comprises cd3ζ or fragment thereof;
optionally, the signal transduction domain comprises the amino acid sequence shown in SEQ ID NO. 15.
11. The transgenic immune cell of claim 1, wherein the immune cell comprises at least one of a T cell and an NK cell, preferably an NK cell;
optionally, the NK cells comprise at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC) -derived NK cells, and NK-92 cells;
optionally, the T cells comprise CD4 + T cells, CD8 + T cells and γδ T cells.
12. An isolated nucleic acid, wherein the isolated nucleic acid comprises:
a first nucleic acid molecule encoding a chimeric antigen receptor; and
a second nucleic acid molecule encoding an immunostimulatory molecule, the immunostimulatory molecule comprising IL-15.
13. The isolated nucleic acid of claim 12, wherein the chimeric antigen receptor is as defined in any one of claims 2 to 10.
14. The isolated nucleic acid of claim 12, wherein the first nucleic acid molecule and the second nucleic acid molecule are disposed to express the chimeric antigen receptor and the immunostimulatory molecule in an immune cell, and the immunostimulatory molecule is in a non-fused form with the chimeric antigen receptor.
15. The isolated nucleic acid of claim 12, further comprising:
an internal ribosome entry site sequence disposed between the first nucleic acid molecule and the second nucleic acid molecule, the internal ribosome entry site having the nucleotide sequence set forth in SEQ ID NO. 16.
16. The isolated nucleic acid of claim 12, further comprising:
a third nucleic acid molecule disposed between the first nucleic acid molecule and the second nucleic acid molecule, and the third nucleic acid molecule encodes a connecting peptide 2, the connecting peptide 2 being capable of being cleaved in the immune cell;
optionally, the linker peptide 2 comprises a 2A peptide or fragment thereof;
optionally, the connecting peptide 2 comprises at least one of P2A, T2A, E2A and F2A or a fragment thereof;
optionally, the connecting peptide 2 comprises P2A or a fragment thereof;
optionally, the connecting peptide 2 comprises an amino acid sequence shown in SEQ ID NO. 17.
17. The isolated nucleic acid of claim 12, further comprising:
a first promoter operably linked to the first nucleic acid molecule; and
A second promoter operably linked to the second nucleic acid molecule.
18. The isolated nucleic acid of claim 12, wherein the first promoter and the second promoter are each independently selected from the group consisting of U6, H1, CMV, EF-1, LTR, and RSV promoters.
19. The isolated nucleic acid of claim 12, further comprising:
a fourth nucleic acid molecule encoding a signal peptide;
optionally, the fourth nucleic acid molecule is operably linked to the first nucleic acid molecule;
optionally, the signal peptide comprises at least one selected from CSF2R and CD8 a or a fragment thereof;
optionally, the signal peptide comprises CSF2R or a fragment thereof;
optionally, the signal peptide comprises the amino acid sequence shown in SEQ ID NO. 10.
20. The isolated nucleic acid of any one of claims 12 to 19, wherein the first nucleic acid molecule has at least one of the nucleotide sequences set forth in SEQ id nos 3, 4, 5, 6 and 7;
optionally, the second nucleic acid molecule has the nucleotide sequence shown as SEQ ID NO. 9;
optionally, the third nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO. 8;
Optionally, the fourth nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO. 2.
21. A construct carrying the isolated nucleic acid of any one of claims 12 to 20.
22. The construct of claim 21, wherein the vector of the construct is a non-pathogenic viral vector;
optionally, the viral vector comprises at least one selected from the group consisting of a retroviral vector, a lentiviral vector, and an adenovirus-associated viral vector.
23. A recombinant cell carrying the isolated nucleic acid of any one of claims 12 to 20 or the construct of claim 21 or 22;
optionally, the recombinant cell comprises a eukaryotic cell, preferably a mammalian cell.
24. A CAR-NK or CAR-T cell carrying the isolated nucleic acid of any one of claims 12 to 20 or the construct of claim 21 or 22;
optionally, the NK cells comprise at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC) -derived NK cells, and NK-92 cells;
optionally, the T cells comprise CD4 + T cells, CD8 + T cells and γδ T cells.
25. A method of obtaining a virus, wherein the construct of claim 21 or 22 is introduced into a first recipient cell; culturing the first recipient cell into which the construct is introduced to obtain the virus.
26. The method of claim 25, wherein the virus comprises a lentivirus;
optionally, the first recipient cell is 293T.
27. A virus obtainable by the method of claim 25 or 26.
28. A virus comprising a nucleotide sequence represented by SEQ ID NO. 1.
29. A pharmaceutical composition comprising an effective amount of the transgenic immune cell of any one of claims 1-11, the isolated nucleic acid of any one of claims 12-20, the construct of claim 21 or 22, the recombinant cell of claim 23, the CAR-NK or CAR-T cell of claim 24, or the virus of any one of claims 27-28.
30. A kit, comprising: the isolated nucleic acid of any one of claims 12 to 20, the construct of claim 21 or 22, or the virus of any one of claims 27 to 28.
31. A method of introducing a virus into an activated immune cell, wherein the activated immune cell is electrotransfected or transfected with the construct of claim 21 or 22 or infected with the virus of any one of claims 27 to 28;
optionally, the immune cells comprise at least one of T cells and NK cells, preferably NK cells;
optionally, the NK cells comprise at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC) -derived NK cells, and NK-92 cells;
optionally, the T cells comprise CD4 + T cells, CD8 + T cells and γδ T cells.
32. A method of obtaining a chimeric antigen receptor and an immunostimulatory molecule, comprising:
introducing the construct of claim 21 or 22 or the virus of any one of claims 27 to 28 into a second recipient cell;
culturing a second receptor cell introduced into an expression vector or virus to obtain the chimeric antigen receptor and the immunostimulatory molecule;
optionally, the introducing of the second recipient cell is by electrotransfection, transfection or infection;
optionally, the second recipient cell is at least one of a T cell and an NK cell;
Optionally, the second recipient cell is an NK cell;
optionally, the NK cells comprise at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC) -derived NK cells, and NK-92 cells;
optionally, the T cells comprise CD4 + T cells, CD8 + T cells and γδ T cells;
optionally, the virus comprises a lentivirus.
33. A method of obtaining a CAR-NK or CAR-T cell expressing a chimeric antigen receptor and an immunostimulatory molecule, comprising:
introducing the construct of claim 21 or 22 or the virus of any one of claims 27 to 28 into NK cells or T cells;
culturing NK cells or T cells introduced into the construct or virus so as to obtain the CAR-NK or CAR-T cells;
optionally, the introduction of NK cells or T cells by electrotransfection, transfection or infection;
optionally, the NK cells comprise at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC) -derived NK cells, and NK-92 cells;
optionally, the T cells comprise CD4 + T cells, CD8 + T cells and γδ T cells.
34. Use of the transgenic immune cell of any one of claims 1 to 11, the isolated nucleic acid of any one of claims 12 to 20, the construct of claim 21 or 22, the recombinant cell of claim 23, the CAR-NK or CAR-T cell of claim 24, or the virus of any one of claims 27 to 28 in the preparation of a pharmaceutical composition for the treatment or prevention of a tumor;
Optionally, the tumor comprises at least one of a mesothelin-positive tumor, a HER 2-positive tumor, an EGFR-positive tumor, a GPC 3-positive tumor, a MUC 1-positive tumor, a CEA-positive tumor, a CLDN 18.2-positive tumor, an EpCAM-positive tumor, a GD 2-positive tumor, a PSCA-positive tumor, a CD 133-positive tumor, a CD 19-positive tumor, a CD 20-positive tumor, a CD 22-positive tumor, a CD 30-positive tumor, a CD 33-positive tumor, and a BCMA-positive tumor;
optionally, the mesothelin-positive tumor comprises at least one of pancreatic cancer, ovarian cancer, mesothelioma, cholangiocarcinoma, and lung cancer.
35. Use of the isolated nucleic acid of any one of claims 12 to 20, the construct of claim 21 or 22 or the virus of any one of claims 27 to 28 in the preparation of a kit for promoting NK cell or T cell activation or proliferation;
optionally, the NK cells comprise at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC) -derived NK cells, and NK-92 cells;
optionally, the T cells comprise CD4 + T cells, CD8 + T cells and γδ T cells.
CN202211505642.8A 2022-11-28 2022-11-28 Transgenic immune cells and uses thereof Pending CN116179495A (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202211505642.8A CN116179495A (en) 2022-11-28 2022-11-28 Transgenic immune cells and uses thereof
PCT/CN2023/101463 WO2024113777A1 (en) 2022-11-28 2023-06-20 Transgenic immune cell and use thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202211505642.8A CN116179495A (en) 2022-11-28 2022-11-28 Transgenic immune cells and uses thereof

Publications (1)

Publication Number Publication Date
CN116179495A true CN116179495A (en) 2023-05-30

Family

ID=86444936

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202211505642.8A Pending CN116179495A (en) 2022-11-28 2022-11-28 Transgenic immune cells and uses thereof

Country Status (2)

Country Link
CN (1) CN116179495A (en)
WO (1) WO2024113777A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116410336A (en) * 2023-06-02 2023-07-11 云南赛元生物技术有限公司 Construction and application of chimeric antigen receptor capable of being efficiently expressed and CAR-NK cell secreting function stimulating factor IL-15
WO2024113777A1 (en) * 2022-11-28 2024-06-06 上海恩凯细胞技术有限公司 Transgenic immune cell and use thereof

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106132423A (en) * 2014-02-14 2016-11-16 贝里坤制药股份有限公司 Method with induction type chimeric polyeptides activating T cell
CN107475276A (en) * 2017-09-05 2017-12-15 深圳大学 The T cell and its application that Chimeric antigen receptor and its expressing gene, the Chimeric antigen receptor of tetracycline regulation are modified
CN109762844A (en) * 2019-01-31 2019-05-17 北京呈诺医学科技有限公司 A kind of CAR-NK cell preparation method of target mesothelin
CN110684790A (en) * 2019-10-31 2020-01-14 山东兴瑞生物科技有限公司 Encoding gene of anti-B7-H3 chimeric antigen receptor, preparation method, plasmid with encoding gene, immune cell and application of immune cell
CN110904048A (en) * 2019-12-04 2020-03-24 合肥中科干细胞再生医学有限公司 CAR-CD123T2 chimeric antigen receptor T cell and application thereof
US20200223918A1 (en) * 2017-06-21 2020-07-16 Icell Gene Therapeutics Llc CHIMERIC ANTIGEN RECEPTORS (CARs), COMPOSITIONS AND METHODS THEREOF
CN112080525A (en) * 2019-06-14 2020-12-15 南京艾德免疫治疗研究院有限公司 Preparation of improved chimeric antigen receptor T cells
CN113122504A (en) * 2019-12-30 2021-07-16 博雅辑因（北京）生物科技有限公司 Method for purifying UCART cells and application
CN113501884A (en) * 2021-06-30 2021-10-15 徐州医科大学 Fully human chimeric antigen receptor targeting B7H3, iNKT cell and application thereof
CN113913379A (en) * 2020-07-07 2022-01-11 深圳市菲鹏生物治疗股份有限公司 T lymphocyte and application thereof

Family Cites Families (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010111282A1 (en) * 2009-03-24 2010-09-30 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Anti-mesothelin antibodies
WO2013063419A2 (en) * 2011-10-28 2013-05-02 The Trustees Of The University Of Pennsylvania A fully human, anti-mesothelin specific chimeric immune receptor for redirected mesothelin-expressing cell targeting
WO2014117121A1 (en) * 2013-01-28 2014-07-31 St. Jude Children's Research Hospital, Inc. A chimeric receptor with nkg2d specificity for use in cell therapy against cancer and infectious disease
US9944690B2 (en) * 2013-03-14 2018-04-17 Bellicum Pharmaceuticals, Inc. Methods for controlling T cell proliferation
RS61223B1 (en) * 2013-12-20 2021-01-29 Hutchinson Fred Cancer Res Tagged chimeric effector molecules and receptors thereof
NZ725079A (en) * 2014-04-10 2018-03-23 Seattle Children’S Hospital Dba Seattle Children’S Res Institute Transgene genetic tags and methods of use
GB2547179A (en) * 2015-10-26 2017-08-16 Quethera Ltd Genetic construct
US11246896B2 (en) * 2015-10-28 2022-02-15 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Tumor-specific adenovirus vectors and therapeutic uses
US11384156B2 (en) * 2016-07-25 2022-07-12 The Nemours Foundation Adoptive T-cell therapy using EMPD-specific chimeric antigen receptors for treating IgE-mediated allergic diseases
CN107793481A (en) * 2016-08-31 2018-03-13 南京传奇生物科技有限公司 A kind of Chimerical receptor part for targetting people CD123 and its application
EP3545082A4 (en) * 2016-11-22 2020-07-01 National University of Singapore Blockade of cd7 expression and chimeric antigen receptors for immunotherapy of t-cell malignancies
WO2019157691A1 (en) * 2018-02-14 2019-08-22 宜明细胞生物科技有限公司 Recombinant chimeric antigen receptor gene and use thereof
CN111304248B (en) * 2018-12-25 2021-08-24 百奥赛图江苏基因生物技术有限公司 Construction method and application of humanized cytokine IL15 gene modified non-human animal
EP3962938A4 (en) * 2019-05-01 2023-07-05 PACT Pharma, Inc. Compositions and methods for the treatment of cancer using a cd8 engineered t cell therapy
EP4271795A1 (en) * 2020-12-31 2023-11-08 CRISPR Therapeutics AG Universal donor cells
WO2022192439A1 (en) * 2021-03-11 2022-09-15 Kite Pharma, Inc. Improving immune cell function
CN114592010A (en) * 2022-03-03 2022-06-07 南方医科大学南方医院 NK-CAR-MbIL-15 cell and preparation method and application thereof
CN114921496B (en) * 2022-04-29 2023-03-10 上海驯鹿生物技术有限公司 Construction method and application of humanized immune system animal model with NK (natural killer) cell and ADCC (advanced cellular ADCC) capabilities
CN116179495A (en) * 2022-11-28 2023-05-30 上海恩凯细胞技术有限公司 Transgenic immune cells and uses thereof

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106132423A (en) * 2014-02-14 2016-11-16 贝里坤制药股份有限公司 Method with induction type chimeric polyeptides activating T cell
US20200223918A1 (en) * 2017-06-21 2020-07-16 Icell Gene Therapeutics Llc CHIMERIC ANTIGEN RECEPTORS (CARs), COMPOSITIONS AND METHODS THEREOF
CN107475276A (en) * 2017-09-05 2017-12-15 深圳大学 The T cell and its application that Chimeric antigen receptor and its expressing gene, the Chimeric antigen receptor of tetracycline regulation are modified
CN109762844A (en) * 2019-01-31 2019-05-17 北京呈诺医学科技有限公司 A kind of CAR-NK cell preparation method of target mesothelin
CN112080525A (en) * 2019-06-14 2020-12-15 南京艾德免疫治疗研究院有限公司 Preparation of improved chimeric antigen receptor T cells
CN110684790A (en) * 2019-10-31 2020-01-14 山东兴瑞生物科技有限公司 Encoding gene of anti-B7-H3 chimeric antigen receptor, preparation method, plasmid with encoding gene, immune cell and application of immune cell
CN110904048A (en) * 2019-12-04 2020-03-24 合肥中科干细胞再生医学有限公司 CAR-CD123T2 chimeric antigen receptor T cell and application thereof
CN113122504A (en) * 2019-12-30 2021-07-16 博雅辑因（北京）生物科技有限公司 Method for purifying UCART cells and application
CN113913379A (en) * 2020-07-07 2022-01-11 深圳市菲鹏生物治疗股份有限公司 T lymphocyte and application thereof
CN113501884A (en) * 2021-06-30 2021-10-15 徐州医科大学 Fully human chimeric antigen receptor targeting B7H3, iNKT cell and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ENLI LIU 等: "Cord blood NK cells engineered to express IL-15 and a CD19-targeted CAR show long-term persistence and potent anti-tumor activity", LEUKEMIA, vol. 32, no. 2, 20 July 2017 (2017-07-20) *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024113777A1 (en) * 2022-11-28 2024-06-06 上海恩凯细胞技术有限公司 Transgenic immune cell and use thereof
CN116410336A (en) * 2023-06-02 2023-07-11 云南赛元生物技术有限公司 Construction and application of chimeric antigen receptor capable of being efficiently expressed and CAR-NK cell secreting function stimulating factor IL-15
CN116410336B (en) * 2023-06-02 2023-09-22 云南赛元生物技术有限公司 Chimeric antigen receptor encoding nucleotide, CAR-NK cell, construction method and application thereof

Also Published As

Publication number Publication date
WO2024113777A1 (en) 2024-06-06

Similar Documents

Publication Publication Date Title
CN110818802B (en) Chimeric T cell receptor STAR and application thereof
CN109306016B (en) NKG2D-CAR-T cells co-expressing cytokine IL-7 and uses thereof
CN108004259B (en) Chimeric antigen receptor targeting B cell maturation antigen and uses thereof
CN109880804B (en) Preparation method and application of B7H 3-targeted CAR-T cells
WO2018145649A1 (en) Construction of chimeric antigen receptor targeting cd20 antigen and activity identification of engineered t cells thereof
WO2024113649A1 (en) Method for improving viability and anti-tumor activity of nk cells and use thereof
KR20240046645A (en) Chimeric antigen receptor for solid cancer and t cells expressing chimeric antigen receptor
CN113913379A (en) T lymphocyte and application thereof
CN108441505B (en) Chimeric antigen receptor targeting ROR1 and application thereof
CN114478806B (en) Chimeric receptor for improving killing activity of immune cells and application thereof
CN116179495A (en) Transgenic immune cells and uses thereof
CN110923255A (en) Chimeric antigen receptor targeting BCMA and CD19 and uses thereof
CN116064620A (en) Preparation and application of CAR-NK cells for enhancing infiltration capacity to tumor parts
CN108707619B (en) Chimeric antigen receptor targeting ROR1 and application thereof
WO2017071173A1 (en) Tumor therapeutic agent modified by il-12/cd62l fusion protein and preparation method and use thereof
CN112522208A (en) Transgenic tumor infiltrating lymphocyte and application thereof
CN114591444B (en) Humanized chimeric antigen receptor based on CD7 and application thereof
CN111632135A (en) Application of chimeric antigen receptor T cell targeting NKG2D in treatment of prostate cancer and medicine for treating prostate cancer
CN116041542A (en) NK cell preparation method for reversing tumor microenvironment inhibitory signals and application
CN114149510B (en) Condition-controlled splicing chimeric antigen receptor molecule and application thereof
CN114249807B (en) Hypoxia triggered artificial transcription factor, transcription control system and application thereof
CN108165568B (en) Method for culturing CD19CAR-iNKT cells and application
CN115975056A (en) Construction of TSHR-targeted CAR-T cells Using native protein TSH as antigen binding site
CN111996170B (en) Genetically engineered NK cells, methods of preparation and uses thereof
CN116254230A (en) Method for preparing and amplifying universal humanized anti-CD 19CAR-NK cells and uses thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 40092408

Country of ref document: HK