WO2018012895A1 - Immune cell surmounting immune checkpoint and pharmaceutical composition containing same immune cell - Google Patents

Immune cell surmounting immune checkpoint and pharmaceutical composition containing same immune cell Download PDF

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WO2018012895A1
WO2018012895A1 PCT/KR2017/007505 KR2017007505W WO2018012895A1 WO 2018012895 A1 WO2018012895 A1 WO 2018012895A1 KR 2017007505 W KR2017007505 W KR 2017007505W WO 2018012895 A1 WO2018012895 A1 WO 2018012895A1
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immune
car
cells
receptor
cell
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PCT/KR2017/007505
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French (fr)
Korean (ko)
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김찬혁
권용준
이영호
이지연
이형지
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주식회사 큐로셀
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    • 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
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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

Definitions

  • the present invention relates to immune cells that overcome immune barriers and pharmaceutical compositions comprising the immune cells, and more particularly, chimeric antigen receptor (CAR) or monoclonal T cell receptor (T cell receptor); And immune cells overcoming genetically engineered immune barriers to simultaneously express shothairpin ribonucleic acid (RNA) that inhibits the expression of immune gate receptors and pharmaceutical compositions comprising the immune cells.
  • CAR chimeric antigen receptor
  • T cell receptor monoclonal T cell receptor
  • RNA shothairpin ribonucleic acid
  • Anti-cancer therapy using immune cells that incubate T-cells or natural killer cells isolated from the patient or donor's body and administer them to the patient's body has recently attracted attention as a new cancer treatment. [1].
  • immune cells which have undergone proliferation after injecting new genetic information using viruses and the like during in vitro culture, have a higher anticancer effect than otherwise [2].
  • the genetic information injected into the T cells is mainly used a monoclonal T cell receptor (TCR) or chimeric antigen receptor (CAR) engineered to have a high affinity for the antigen (TARGET).
  • TCR monoclonal T cell receptor
  • CAR chimeric antigen receptor
  • the immune cells thus manipulated recognize and attack cancer cells, which are antigens, regardless of existing antigen specificity, thereby inducing cell death.
  • ISD consists of one or more costimulatory domains (CD28, CD137, or OX40: contribute to in vivo proliferation and long life of T cells) and TCR signaling domains (CD3 zeta: contribute to T cell activation).
  • the T cells engineered to express the car receptor thus made recognize and activate antigen-expressing cancer cells with high specificity, and then effectively induce the death of cancer cells, and at the same time proliferate exponentially in the body and live long. For example, it has been reported that cati cells (CART-19) made to target CD19, a specific antigen of B cells, multiply by 1,000 to 10,000 times when administered to patients with B-cell leukemia and have been living in the body for many years [4, 5].
  • CART-19 showed an amazing complete response of 90% in clinical trials in patients with terminal acute lymphocytic leukemia (B-ALL) who had not benefited from conventional chemotherapy. It is licensed to a global pharmaceutical company exceptionally at the clinical stage and is the first of its kind to be approved by the US Food and Drug Administration in 2017. In addition, most large pharmaceutical companies are engaged in this field, and investments and start-ups in biotech are very active, and it is expected that the treatment using cati cells may change the paradigm of the chemotherapy industry in the future. .
  • immune barrier receptors such as CTLA-4 or PD-1 exist on the surface of immune cells, such as T cells.
  • these receptors are safeguards for regulating over-activation of T cells to cause apoptosis or autoimmunity, but cancer cells, particularly solid cancers, are known to exploit this to avoid immune surveillance by T cells.
  • cancer cells particularly solid cancers, are known to exploit this to avoid immune surveillance by T cells.
  • cancer cells express the PDL-1 ligand on the surface, even if the T cells recognize and activate the cancer cells, they are soon exhausted due to inactivation signaling transmitted from the PD-1 receptor.
  • Monoclonal antibodies such as anti-CTLA4 and anti-PD-1, have been developed to prevent T cell activity from being inhibited by these immune checkpoints. Therapies that improve the overall immune function of the drug have been shown to be effective in several solid cancers.
  • cati cells are also therapeutic agents that depend on the cytotoxicity of activated T cells, the presence of an immunosuppressive environment around the cati cells acts as a significant inhibitor to the therapeutic effect. Indeed, compared to the surprising therapeutic effects seen in B-cell leukemia, CAR-Ts that are designed to target solid tumors are rarely clinically promising. It is presumed to be due to the formation of immunosuppressive tumor microenvironments, which inhibit the activity and proliferation of cati cells.
  • lymphoma lymphmphoma, 20-50% response rate
  • ALL acute lymphoblastic leukemia
  • immunosuppressive ligands such as programmed death ligand-1 (PDL-1) are expressed in tumor microenvironments formed by lymphomas, and thus, traits of T cells present in cancer tissues are depleted. [8]. In addition, it has been reported that T cells obtained from CLL patients have already exhibited much depleted traits and high levels of expression of immune gate receptors such as PD-1, CD160, and CD244 [9].
  • the technical problem to be achieved by the present invention is to improve the immune cell therapeutic effect by exerting the function of the chimeric antigen receptor while suppressing the expression of the immune gate receptor without using a separate antibody therapeutic agent for inhibiting the immune gate receptor expression.
  • Another aspect of the present invention is to provide an immune cell therapeutic agent including immune cells that overcome the immune barrier.
  • the present invention provides a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a monoclonal T cell receptor (TCR) in a nucleic acid sequence of an immune cell; And it provides a nucleic acid sequence characterized in that it comprises at the same time the base sequence encoding a short hairpin ribonucleic acid (shRNA) that inhibits the expression of the immune gateway receptor.
  • CAR chimeric antigen receptor
  • TCR monoclonal T cell receptor
  • the present invention is that the base sequence encoding the short hairpin ribonucleic acid (shRNA) is targeted to at least one immune gateway receptor selected from the group consisting of PD-1, CTLA-4, LAG-3 and TIM-3 It provides a nucleic acid sequence characterized by.
  • shRNA short hairpin ribonucleic acid
  • the present invention provides a nucleic acid sequence characterized in that the base sequence encoding the short hairpin ribonucleic acid (shRNA) targets PD-1 and TIM-3 at the same time.
  • shRNA short hairpin ribonucleic acid
  • the present invention is a base sequence encoding the short hairpin ribonucleic acid (shRNA) is PD-1 target nucleotide sequence of SEQ ID NO: 2 to 12, TIM-3 target nucleotide sequence of SEQ ID NO: 13 to 35, SEQ ID NO: 36 to 42 It provides a nucleic acid sequence comprising at least one nucleotide sequence selected from the group consisting of LAG-3 target nucleotide sequence of and the CTLA-4 target nucleotide sequence of SEQ ID NO: 43 to 51.
  • shRNA short hairpin ribonucleic acid
  • the present invention is the target of the chimeric antigen receptor (CAR) or monoclonal T cell receptor 5T4, alpha 5 ⁇ 1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, ⁇ -catenin / m, Bcr- abl, MN / CIX antigen, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27 / m, CD30, CD33, CD52, CD56, CD80, CDK4 / m, CEA, CT , Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2 / new, HLA-A * 0201-R170I, HPV-E7 , HSP70-2M, HST-2, hTERT (or h
  • the present invention also provides a nucleic acid sequence characterized in that the amino acid sequence of the CAR receptor is at least 90% identical to at least one amino acid sequence selected from the group consisting of SEQ ID NO: 52 to 54.
  • Another aspect of the invention includes a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a monoclonal T-cell receptor (TCR) and a nucleotide sequence encoding shorthairpin ribonucleic acid (RNA) that inhibits the expression of an immune gateway receptor.
  • CAR chimeric antigen receptor
  • TCR monoclonal T-cell receptor
  • RNA shorthairpin ribonucleic acid
  • the present invention is that the base sequence encoding the short hairpin ribonucleic acid (shRNA) is targeted to at least one immune gateway receptor selected from the group consisting of PD-1, CTLA-4, LAG-3 and TIM-3 A vector is provided.
  • shRNA short hairpin ribonucleic acid
  • the present invention provides a vector characterized in that the base sequence encoding the short hairpin ribonucleic acid (shRNA) targets PD-1 and TIM-3 at the same time.
  • shRNA short hairpin ribonucleic acid
  • the present invention is a base sequence encoding the short hairpin ribonucleic acid (shRNA) is PD-1 target nucleotide sequence of SEQ ID NO: 2 to 12, TIM-3 target nucleotide sequence of SEQ ID NO: 13 to 35, SEQ ID NO: 36 to 42 It provides a vector comprising at least one base sequence selected from the group consisting of LAG-3 target nucleotide sequence of and the CTLA-4 target nucleotide sequence of SEQ ID NO: 43 to 51.
  • shRNA short hairpin ribonucleic acid
  • the present invention is the target of the chimeric antigen receptor (CAR) or monoclonal T-cell receptor (TCR) is 5T4, alpha 5 ⁇ 1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, ⁇ -catenin / m , Bcr-abl, MN / CIX antigen, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27 / m, CD30, CD33, CD52, CD56, CD80, CDK4 / m, CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2 / new, HLA-A * 0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT
  • the present invention also provides a vector, wherein the amino acid sequence of the CAR receptor is at least 90% identical to at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 52 to 54.
  • the present invention provides a chimeric antigen receptor (CAR) or monoclonal T cell receptor (TCR); And an immune cell that overcomes the genetically engineered immune barrier to simultaneously express shorthairpin ribonucleic acid (RNA) that inhibits the expression of the immune barrier receptor.
  • CAR chimeric antigen receptor
  • TCR monoclonal T cell receptor
  • RNA shorthairpin ribonucleic acid
  • the present invention is that the short hairpin ribonucleic acid (shRNA) is a target of at least one immune gate receptor selected from the group consisting of PD-1, CTLA-4, LAG-3 and TIM-3 inhibited its expression It provides an immune cell that overcomes the characterized immune barrier.
  • shRNA short hairpin ribonucleic acid
  • the present invention provides immune cells overcoming the immune barrier, characterized in that the short hairpin ribonucleic acid (shRNA) targets the PD-1 and TIM-3 immune gate receptor at the same time, inhibiting its expression.
  • shRNA short hairpin ribonucleic acid
  • the present invention is the target of the chimeric antigen receptor (CAR) or monoclonal TCR is 5T4, alpha 5 ⁇ 1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, ⁇ -catenin / m, Bcr-abl, MN / CIX antigen, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27 / m, CD30, CD33, CD52, CD56, CD80, CDK4 / m, CEA, CT, Cyp -B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2 / new, HLA-A * 0201-R170I, HPV-E7, HSP70 -2M, HST-2, hTERT (or hT (or
  • the present invention provides an immune cell overcoming the immune barrier, characterized in that the amino acid sequence of the CAR receptor is at least 90% identical to at least one amino acid sequence selected from the group consisting of SEQ ID NO: 52 to 54.
  • the present invention provides an immune cell overcoming the immune barrier, characterized in that the immune cell is one or more selected from the group consisting of T cells, natural killer cells, cytotoxic T lymphocytes and regulatory T cells.
  • Another aspect of the invention provides a pharmaceutical composition comprising immune cells that overcome the immune barrier.
  • the present invention provides the pharmaceutical composition of the carrier, excipient, diluent, antioxidant, preservative, colorant, flavor and diluent, emulsifier, suspending agent, solvent, filler, bulking agent, buffer, delivery vehicle, isotonic agent, cosolvent It provides a pharmaceutical composition characterized in that it further comprises one or more pharmaceutically acceptable salts, excipients or vehicles selected from the group consisting of wetting agents, complexing agents, buffers, antibacterial agents and surfactants.
  • the present invention also provides a method for treating a subject, comprising administering an effective amount of the pharmaceutical composition to a subject in need thereof.
  • the present invention also provides a method for treating a subject in whom the condition is cancer.
  • the present invention is selected from the group consisting of breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and any combination thereof It provides a method for treating a subject.
  • the present invention provides a immune cell that overcomes the immune barrier capable of exerting the function of the chimeric antigen receptor while suppressing the expression of the immune barrier receptor without using a separate antibody therapeutic agent for inhibiting the immune barrier receptor expression by a simple method.
  • the treatment of cancer including the treatment of immune cells can maximize the effect.
  • FIG. 1 is a structural diagram of a two-in-one carvirus plasmid containing both a short hairpin RNA (shRNA) and an expression cassette of a CAR that inhibit the expression of the immune gate receptor of the present invention.
  • shRNA short hairpin RNA
  • Figure 2 confirms the maintenance and expression of CAR and reduced PD-1 expression in CAR T cells prepared using a two-in-one carvirus plasmid expressing shPD- 1 and CD19 target CAR at the same time inhibiting human PD-1 expression FACS DATA
  • FIG. 3 is a CAR T prepared using a two-in-one carvirus plasmid in which three genes ( ⁇ LNGFR: purification, CD19-CAR: CD19 antigen targeting, shPD-1 : PD-1 expression) are simultaneously expressed.
  • ⁇ LNGFR purification
  • CD19-CAR CD19 antigen targeting
  • shPD-1 PD-1 expression
  • FIG. 4 is a FACS DATA confirming the decrease of PD-1 expression in CAR-T cells after stimulation of ⁇ LNGFR-CART19 / shGFP and ⁇ LNGFR-CART19 / shPD - 1 T cells purified from Figure 3 with CD3 and CD28 antibody for 3 days
  • FIG. 5 shows FACS DATA confirming PD-L1 expression of PD-L1 overexpressing K562 cell line (K562-CD19-PD-L1).
  • FIG. 6 is an explanatory diagram of a CAR-T cell fabrication process in which CAR and PD-1 receptor mediated stimulation was repeatedly applied to ⁇ LNGFR-CART19 / shGFP and ⁇ LNGFR-CART19 / shPD - 1 T cells purified in FIG.
  • FIG. 8 shows that the decrease in cytotoxicity of CAR-T cells due to repetitive stimulation is significantly alleviated by inhibition of PD-1 expression.
  • Figure 9 shows the effect of PD-1 expression inhibition on the differentiation of CAR T cells prepared in Figure 6 FACS DATA confirmed by CD45RA and CCR7 surface antigen expression in CD8, CD4 subtype
  • FIG. 10 shows CSFE fluorescence by co-incubation with gamma-irradiated K562-CD19-PD-L1 cell line for 5 days after labeling CFSE to confirm the effect of PD-1 expression inhibition on the proliferation of CAR T cells prepared in FIG. 6.
  • FIG. 11 shows FACS DATA confirming a decrease in TIM-3 expression in CAR T cells expressing both shTIM - 3 and CD19 target CAR which inhibit human TIM-3 expression.
  • FIG. 12 is a schematic diagram of a two-in-one carvirus plasmid construct in which two shRNAs ( shPD- 1 and shTIM- 3 ), a CD19 CAR, and an LNGFR gene are simultaneously expressed.
  • Figure 13 is a process for preparing a pLV- ⁇ LNGFR_P2A_CD19-CAR_ shPD-1 _MCS two-in-one carvirus plasmid in which the multiple cloning site is inserted at the 3 ⁇ end of the mouse U6 promoter
  • Figure 14 is a 13-Plasmid hU6-shPD-1 and mU6-shTIM - 3 cassette -> ⁇ - (two-way), one pLV- ⁇ LNGFR_P2A_CD19-CAR_hU6-shPD- 1 insertion -> ⁇ - mU6-shTIM- 3 two Process for constructing -in-one carvirus plasmid
  • Figure 15 is a 13-Plasmid hU6 - shPD - 1 and mU6 - shTIM - 3 the cassette ⁇ -> pLV- ⁇ LNGFR_P2A_CD19-CAR_hU6 -shPD-1 inserted in (the two-way station) -> ⁇ - mU6-shTIM- 3 Construction of a two-in-one carvirus plasmid
  • FIG. 16 is a FACS DATA confirming purity after purifying CAR-T cells prepared with the two-in-one carvirus plasmids of FIGS. 14 and 15 using LNGFR antibodies.
  • FIG. 17 shows ⁇ LNGFR-CART19 / hU6-shPD-1-> ⁇ -shTIM-3-mU6 T cells and ⁇ LNGFR-CART19 / shPD-1-hU6 ⁇ --> U6-shTIM-3 T cells purified in FIG.
  • FACS DATA confirms simultaneous expression of PD-1 and TIM-3 in CAR-T cells after stimulation with CD3 and CD28 antibodies
  • an immune cell may refer to both human or animal harmful immune cells that have not been genetically engineered or treated, and unless otherwise specified, chimeric antigen receptor modified T cells, T cells, or T cells. It may mean a car-modified immune cell such as a TCR modified T-cell or a chimeric antigen receptor modified NK cell or a T cell receptor modified NK cell. have. Those skilled in the art to which the present invention pertains will be able to distinguish which immune cells mean without special mention. Examples of the immune cells include, but are not limited to, one or more selected from the group consisting of T cells, natural killer cells, cytotoxic T lymphocytes, and regulatory T cells.
  • FIG. 1 is a structural diagram of an embodiment of a two-in-one carvirus plasmid containing both a short hairpin RNA (shRNA) and an expression cassette of a CAR which inhibit the expression of the immune gate receptor of the present invention.
  • the present invention provides a nucleic acid sequence and a vector including a nucleotide sequence encoding a chimeric antigen receptor (CAR) and a nucleotide sequence encoding a short hairpin ribonucleic acid (RNA) that inhibits the expression of an immune gate receptor.
  • CAR chimeric antigen receptor
  • RNA short hairpin ribonucleic acid
  • the present invention also provides immune cells expressing shRNAs that inhibit expression of CAR and immune gate receptor.
  • the CAR or monoclonal T-cell receptor is not particularly limited in structure or structure, and is generally a single chain variable fragment (scFv) which is an extracellular domain. Grant) domain, spacer domain (controlling the distance between the scFv and the cell membrane), transmembrane domain (transmembrane domain) and intracellular signaling domain (intracellular signaling domain (ISD)).
  • the amino acid sequence of the CAR receptor comprises at least one amino acid sequence selected from the group consisting of 52 to 54 SEQ ID NOs corresponding to CD19, CD22 and / or LNGFR_P2A_CD19-CAR. Preferably at least 90% identical to the sequence, but is not limited thereto.
  • the extracellular domain of the CAR may be bound to only one antigen or ligand, or may be an extracellular domain that binds two or more antigens or ligands.
  • the extracellular domain can be selected from antibodies that recognize the targeting antigen or molecules interacting with the antigen.
  • antigens include, for example, viral antigens, bacterial (especially infectious bacterial) antigens, parasitic antigens, cell surface markers (e.g. tumor antigens) on target cells involved in a particular condition or surface molecules of immune related cells.
  • antigens in the present invention include, but are not limited to, retroviridae (eg, human immunodeficiency virus, such as HIV-1 and HIV-LP), Picornaviridae (eg, poliovirus, A). Hepatitis virus, enterovirus, human coxsackie virus, rhinovirus, echovirus), rubella virus, coronavirus, bullous stomatitis virus, rabies virus, ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory vesicle Virus, influenza virus, hepatitis B virus, parvovirus, Adenoviridae, herpesviridae, for example, herpes simplex virus (HSV), varicella zoster virus, cytomegalovirus (CMV), herpes virus], Poxviridae (eg smallpox virus, vaccinia) Oh, there may be a virus, poxvirus), an antigen derived from hepatitis C virus,
  • the species of Staphylococci As another type of antigen of the present invention, the species of Staphylococci, the species of Streptococcus, the species of Escherichia coli, the species of Pseudomonas and Salmonella It may be an antigen derived from the species of.
  • infectious bacteria such as Helicobacter pyloris, Legionella pneumophilia, Mycobacteria sps (eg, M. tuberculosis, M.avium, M.
  • M.kansaii M.gordonea, Staphylococcusaureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes, A. It may also be an antigen derived from group B streptococcus galactiae, Streptococcus pneumoniae, and Clostridium tetani.
  • 5T4 alpha 5 ⁇ 1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, ⁇ -catenin / m, Bcr-abl, MN / CIX antigen, CA125, CAMEL, CAP-1 as antigens of the present invention.
  • the antigen may be a tumor antigen of.
  • the antigen is preferably a human tumor antigen (cancer cell).
  • tumor antigens or cancer cells are not particularly limited, and may be ones that act as chimeric antigen receptor T-cells or immune cells that are natural killer cells to exhibit anticancer activity.
  • the cancer cells may be CD19-positive cells.
  • the cancer cell may be CD19-positive B lymphocytes.
  • the cancer cell may be Her2-positive cell.
  • Her2-positive cells can be Her2-positive breast cancer cells.
  • the target cell may be a BCMA-positive cell.
  • the cancer cell may be a BCMA-positive multiple myeloma cell.
  • the cancer cell may be a CS1-positive cell.
  • CS1-positive cells can be multiple myeloma cells.
  • the cancer cell may be an EGFRvIII-positive cell.
  • the cancer cell may be an EGFRvIII-positive glioblastoma cell.
  • the cancer cell may be a CD20-positive cell.
  • the cancer cell may be a CD22-positive cell.
  • the tumor antigen may be CD19-positive cells.
  • the cancer cell may be CD19-positive B lymphocytes.
  • the cancer cell may be Her2-positive cell.
  • Her2-positive cells can be Her2-positive breast cancer cells.
  • the target cell may be a BCMA-positive cell.
  • the cancer cell may be a BCMA-positive multiple myeloma cell.
  • the cancer cell may be a CS1-positive cell.
  • CS1-positive cells can be multiple myeloma cells.
  • the cancer cell may be an EGFRvIII-positive cell.
  • the cancer cell may be an EGFRvIII-positive glioblastoma cell.
  • the cancer cell may be a CD20-positive cell.
  • the cancer cell may be a CD22-positive cell.
  • the CAR can be designed to include a transmembrane domain that connects the antigen binding domain of the CAR to an intracellular domain.
  • the transmembrane domain naturally binds to one or more of the domains in the CAR.
  • the transmembrane domain is selected by amino acid substitutions to avoid such domains binding to the transmembrane domains of the same or different surface membrane proteins to minimize interaction with other members of the receptor complex, or It can be modified.
  • the intracellular domain or otherwise cytoplasmic domain of the CAR is responsible for activation of the cell in which the CAR is expressed. therefore
  • intracellular domain is meant to include any portion of the intracellular domain sufficient to carry the activation signal.
  • the intracellular domain of the CAR includes a domain responsible for signal activation and / or delivery.
  • the intracellular domain may be used to alter signal activation for protein-protein interactions, biochemical changes, or other responses to alterations in the metabolism, shape, or gene expression of the cells, or other three factors for activation of chimeric intracellular signaling molecules.
  • intracellular domains for use in the present invention include, but are not limited to, the cytoplasmic portion of the T cell receptor (TCR), and any co-stimulatory molecules that act in concert to initiate signal transduction following antigen receptor linkage. Any derivative or variant of these elements and any synthetic sequence having the same functional capacity.
  • the intracellular domain of the CAR is comprised of CD3, CD27, CD28, ICOS, 4-1BB, PD-1, T cell receptor (TCR), any derivative or variant thereof, any synthetic sequence thereof having the same functional capacity, And any portion of a co-stimulatory molecule, such as at least one signaling domain from any combination thereof.
  • the structure and expression method of the CAR and the method of manufacturing the same will be well known to those of ordinary skill in the art to which the present invention belongs will not be described further herein.
  • Immune cells that overcome the immune barrier of the present invention are designed to express a CAR or monoclonal TCR while simultaneously expressing shRNA that inhibits the expression of the immune barrier receptor.
  • the term 'inhibition' is less than 60%, more preferably 60%, 50%, 40%, 30%, 20%, 10% of the expression of the corresponding immune gate receptor compared to the 'inhibited' state Mean 5% or 1% or less.
  • the role of the shRNA is complementary to the gene mRNA of the immune gateway receptor induces the degradation of mRNA through the RNA interference mechanism in the cell, and ultimately inhibits the expression of the immune gateway receptor protein. do.
  • the shRNA targets immune gate receptors, such as PD-1, CTLA-4, LAG-3, or TIM-3, which are known immune gate receptors, to suppress the expression of immune gate receptors.
  • immune gate receptors such as PD-1, CTLA-4, LAG-3, or TIM-3, which are known immune gate receptors, to suppress the expression of immune gate receptors.
  • Cati cells engineered using have the ability to target cancer cells via CAR receptor or monoclonal TCRs, while at the same time exhibiting improved anti-cancer effects by making them less sensitive to immune gate signals derived from cancer cells.
  • By suppressing the expression of the immune gateway receptor without using an additional antibody therapeutic agent, it is possible to prevent the function of immune cells, including CAR-T cells, from deteriorating.
  • WO2016 / 069282 described above has a very complex process such as cell perforation during the introduction of CRISPR for endogenous gene inhibition after introduction of CAR in one step for CAR expression and endogenous gene expression inhibition, resulting in high productivity and yield. It is greatly reduced.
  • the present invention enables the simultaneous expression of the traits that inhibit CAR expression and the expression of the immune gateway receptor in one transduction process to immune cells using a single vector, so that the manufacturing process is simple and the productivity and yield are high.
  • the shRNA is preferably to inhibit the expression of at least one immune gateway receptor selected from the group consisting of PD-1, CTLA-4, LAG-3 and TIM-3, the PD-1, CTLA More preferably, it inhibits the expression of two or more immune gateway receptors selected from the group consisting of -4, LAG-3 and TIM-3.
  • the TIM-3 increases when only PD-1 is inhibited.
  • SEQ ID NO: 2 to 51 in the base sequence list attached to the present specification is a nucleotide sequence expressing shRNA to suppress the expression of the immune gateway receptor, PD-1 target nucleotide sequence of SEQ ID NO: 2 to 12, SEQ ID NO: 13 to 35 TIM-3 target nucleotide sequence of the DNA, LAG-3 target nucleotide sequence of SEQ ID NO: 36-42, and CTLA-4 target nucleotide sequence of SEQ ID NO: 43-51.
  • the base sequence is also summarized in Table 2 below.
  • the function, structure and sequence of the immune gate receptor, in particular the human immune gate receptor targeted by the shRNA are known facts (e.g., the base sequences of human PD-1, PDL-1 and TIM-3 are each NCBI Reference). Sequence: NM_005018.2, NCBI Reference Sequence: NM_014143.3 and NCBI Reference Sequence: NM_032782.4), and all of ordinary skill in the art to which the present invention pertains will be well known, and thus will not be described in further detail herein. I will not.
  • the present invention provides a pharmaceutical composition comprising immune cells that overcome the immune barrier.
  • the pharmaceutical composition may further comprise one or more pharmaceutically acceptable salts, excipients or vehicles.
  • Pharmaceutically acceptable salts, excipients, or vehicles for use in the pharmaceutical compositions of this invention include carriers, excipients, diluents, antioxidants, preservatives, colorants, flavors and diluents, emulsifiers, suspensions, solvents, fillers, bulks Agents, buffers, delivery vehicles, isotonic agents, cosolvents, wetting agents, complexing agents, buffers, antibacterial agents, and surfactants.
  • Neutral buffered saline or saline mixed with serum albumin is an exemplary suitable carrier.
  • compositions may comprise an antioxidant such as ascorbic acid; Low molecular weight polypeptides; Proteins such as serum albumin, gelatin or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, arginine or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; Chelating agents such as EDTA; Sugar alcohols such as mannitol or sorbitol; Salt-forming counterions such as sodium; And / or nonionic surfactants such as Tween, pluronics or polyethylene glycol (PEG).
  • an antioxidant such as ascorbic acid
  • Low molecular weight polypeptides such as serum albumin, gelatin or immunoglobulins
  • Hydrophilic polymers such as polyvinylpyrrolidone
  • Amino acids such as glycine, glutamine, asparagine, arg
  • suitable isotonic enhancers include alkali metal halides (preferably sodium chloride or potassium chloride), mannitol, sorbitol, and the like.
  • Suitable preservatives include benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid and the like. Hydrogen peroxide can also be used as a preservative.
  • Suitable cosolvents include glycerin, propylene glycol and PEG.
  • Suitable complexing agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxy-propyl-beta-cyclodextrin.
  • Suitable surfactants or wetting agents include sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapal and the like.
  • the buffer can be a traditional buffer, eg acetate, borate, citrate, phosphate, bicarbonate or tris-HCl.
  • Acetate buffer may be about pH 4 to 5.5 and Tris buffer may be about pH 7 to 8.5. Additional pharmaceutical agents may be referred to Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company, 1990.
  • the composition may be in liquid form or in lyophilized or lyophilized form and may include one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and / or bulking agents.
  • cryoprotectants which are non-reducing sugars such as sucrose, lactose or trehalose are included.
  • the amount of cryoprotectant is generally included so that upon reconstitution, the resulting formulation may be isotonic, although hypertonic or slightly hypertonic formulations may also be suitable.
  • the amount of lyoprotectant should be sufficient to prevent degradation and / or aggregation of unacceptable amounts of protein upon lyophilization.
  • Exemplary lyoprotectants for sugars are about 10 mM to about 400 mM.
  • the surfactant may be, for example, a nonionic surfactant and an ionic surfactant such as polysorbate (eg, polysorbate 20, polysorbate 80); Poloxamers (eg poloxamer 188); Poly (ethylene glycol) phenylether (eg triton); Sodium dodecyl sulfate (SDS); Sodium lauryl sulfate; Sodium octyl glycoside; Lauryl-, myristyl-, linoleyl- or stearyl-sulfobetaine; Lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; Linoleyl, myristyl- or cetyl-betaine; Lauroamidopropy
  • polysorbate eg, polysorbate 20, polysorbate 80
  • Poloxamers eg po
  • An exemplary amount of surfactant that may be present in the pre-lyophilized formulation is about 0.001 to 0.5%.
  • High molecular weight structural additives are, for example, acacia, albumin, alginic acid, calcium phosphate (bibasic), cellulose, carboxymethylcellulose, carboxymethylcellulose sodium, hydroxyethylcellulose, hydroxy Propylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, dextran, dextrin, dexrate, sucrose, tylos, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose, dihydrogen phosphate Sodium, sodium phosphate, sodium pyrosulfite, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressible sugar, magnesium aluminum silicate, maltodextrin, polyethylene oxide, polymethacrylate,
  • compositions may be in a form suitable for parenteral administration.
  • exemplary compositions include any route available to those of skill in the art, such as the intraarterial, subcutaneous, intravenous, intramuscular, intraperitoneal, cerebral (intraventricular), cerebrovascular, intramuscular, intraocular, intraarterial or intralesional routes. It is suitable for injection or infusion into the animal.
  • Parenteral formulations are typically sterile, pyrogen-free, isotonic aqueous solutions, optionally containing a pharmaceutically acceptable preservative.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include emulsions or suspensions comprising water, alcohol / aqueous solution, saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactic acid ringer or fixed oil.
  • Intravenous vehicles include fluid and nutrient supplements, electrolyte supplements such as those based on Ringer's dextrose, and the like. There may also be preservatives and other additives such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases and the like. In general, see Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980.
  • compositions described herein can be formulated for controlled or sustained delivery in a manner that provides increased stability or half-life at local concentrations of the product and / or at a particular local environment.
  • the composition provides controlled or sustained release of immune cells as well as active agents disclosed herein with particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, and the like, which may then be delivered as depot injections, such as biodegradable substrates, Formulations of injectable microspheres, microcapsule particles, microcapsules, biodegradable particle beads, liposomes and implantable delivery devices. Techniques for formulating such sustained- or controlled-delivery means are known and various polymers have been developed and used for the sustained release and delivery of drugs.
  • Such polymers are typically biodegradable and biocompatible.
  • Polymeric hydrogels including those formed by complexation of hydrogels with enantiomeric or polypeptide segments using temperature or pH sensitive properties, trapping bioactive protein agents (eg, antibodies comprising significantly longer CDR3). It may be desirable to provide drug depot effects because of the mild and aqueous conditions involved. See, for example, the description of controlled release porous polymeric microparticles for the delivery of pharmaceutical compositions in WO 93/15722. Suitable materials for this purpose include polylactide (see, eg, US Pat. No.
  • poly- (a-hydroxycarboxylic acids) such as poly-D-(-)-3-hydroxybutyric acid (EP 133,988A)
  • copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et
  • biodegradable polymers include poly (lactone), poly (acetal), poly (orthoesters) and poly (orthocarbonates).
  • Sustained release compositions may also include liposomes, which may be prepared by any of several methods known in the art (see, eg, Eppstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-92 (1985).
  • the carrier itself or its degradation product must be nontoxic in the target tissue and not further exacerbate the condition. This may be determined by routine screening in animal models of target disorders, or in normal animals if such models are not available.
  • Microencapsulation of recombinant proteins for sustained release can be successfully performed by human growth hormone (rhGH), interferon- (rhIFN-), interleukin-2 and MN rgp120.
  • Sustained release formulations of these proteins have been developed using poly-lactic-coglycolic acid (PLGA) polymers due to their biocompatibility and a wide range of biodegradable properties.
  • the degradation products of PLGA, lactic acid and glycolic acid can be quickly cleared in the human body.
  • the degradability of this polymer may depend on its molecular weight and composition.
  • Bioadhesive polymers are also contemplated for use in or with the compositions of the present disclosure.
  • Bioadhesives are synthetic and naturally occurring materials that can adhere to biological substrates for extended periods of time.
  • Carbopol and Polycarbophil are both synthetic crosslinked derivatives of poly (acrylic acid).
  • Bioadhesive delivery systems based on naturally occurring materials include, for example, hyaluronic acid, also known as hyaluronan.
  • Hyaluronic acid is a naturally occurring mucopolysaccharide consisting of residues of D-glucuronic acid and N-acetyl-D-glucosamine.
  • Hyaluronic acid is found in the extracellular tissue matrix of vertebrates, including in connective tissue as well as in the synovial fluid and in the vitreous and intraocular water of the eye. Esterified derivatives of hyaluronic acid have been used to generate microspheres for use in delivery that are biocompatible and biodegradable (see, eg, Cortivo et al., Biomaterials (1991) 12: 727-730; European Patent No. 517,565; WO96 / 29998; Illum et al., J. Controlled Rel. (1994) 29: 133-141).
  • Both biodegradable and non-biodegradable polymer matrices can be used to deliver the compositions of the invention, and such polymer matrices can include natural or synthetic polymers.
  • Biodegradable substrates are preferred.
  • the time period over which release occurs is based on the choice of polymer. Typically, release over time periods ranging from several hours to three to twelve months is most preferred.
  • Exemplary synthetic polymers that can be used to form biodegradable delivery systems include polymers of lactic acid and glycolic acid, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, poly Vinyl alcohol, polyvinyl ether, polyvinyl ester, poly-vinyl halide, polyvinylpyrrolidone, polyglycolide, polysiloxane, poly anhydride, polyurethane and copolymers thereof, poly (butyric acid), poly (valeric acid ), Alkyl cellulose, hydroxyalkyl cellulose, cellulose ethers, cellulose esters, polymers of nitro cellulose, acrylic acid and methacrylic acid esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose Cellulose acetate, cellulose propionate, cell Cellulose acetate but
  • Exemplary natural polymers include dextran and cellulose, collagen, chemicals, derivatives thereof (substitution, addition of chemical groups such as alkyl, alkylene, hydroxylation, oxidation and other modifications routinely made by those skilled in the art), Albumin and other hydrophilic proteins, zein and other prolamin and hydrophobic proteins, copolymers, and mixtures thereof. Generally, these materials are degraded by surface or bulk corrosion, either by enzymatic hydrolysis or exposure to water in vivo.
  • the polymer may optionally be in the form of a hydrogel that absorbs up to about 90% of its weight in water and optionally further crosslinks with polyvalent ions or other polymers (eg, WO 04/009664, WO 05/087201, Sawhney, et al., Macromolecules, 1993, 26, 581-587). Delivery also includes cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di- and tri-glycerides; Hydrogel release system; Silastic systems; Peptide based systems; Wax coatings; Compressed tablets using conventional binders and excipients; Non-polymeric systems that are lipids comprising sterols such as partially fused implants and the like. Liposomes containing the product can be produced, for example, by known methods such as prior art documents 13 to 24.
  • compositions disclosed herein may be formulated for inhalation, eg as a dry powder.
  • Inhalation solutions can also be formulated into liquefied propellants for aerosol delivery.
  • the solution may be nebulized.
  • Additional pharmaceutical compositions for pulmonary administration include, for example, those described in WO 94/20069 which initiates pulmonary delivery of chemically modified proteins.
  • the particle size must be suitable for delivery to the lungs.
  • the particle size may be between 1 ⁇ m and 5 ⁇ m; However, for example, if each particle is quite porous, larger particles can be used.
  • formulations disclosed herein can be administered orally.
  • Formulations administered in this manner may be formulated with or without a carrier customarily used in the preparation of solid dosage forms such as tablets and capsules.
  • the capsule can be designed to release the active portion of the formulation at a point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized.
  • Additional agents may be included to facilitate the absorption of the optional binder.
  • Diluents, flavors, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders may also be used.
  • Other formulations may involve an effective amount of the CAR-immune cells disclosed herein in admixture with non-toxic excipients suitable for the manufacture of tablets.
  • Suitable excipients include inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose or calcium phosphate; Or binders such as starch, gelatin or acacia; Or lubricants such as magnesium stearate, stearic acid or talc.
  • Preferred pharmaceutical formulations for the present invention can be determined in light of the general knowledge of the present disclosure and formulation techniques, depending on the intended route of administration, delivery format, and desired dosage. Despite the mode of administration, the effective dose can be calculated according to the patient weight, surface area or organ size. Further purification of the calculations to determine the appropriate dosage for treatment involving each formulation described herein is routinely made in the art and is within the scope of work routinely performed in the art. Appropriate dosages can be ascertained through the use of appropriate dose-response data.
  • Tables 1 to 5 are primers applied to the embodiments of the present invention, respectively, to target the respective immune gateway receptors, its expression is shRNA, the nucleic acid base encoding the CAR, for the simultaneous expression of the shRNA cassette and CAR and shRNA
  • shRNA the nucleic acid base encoding the CAR
  • Target gene shRNA SEQ ID # Sequence (5 ⁇ > 3 ⁇ ) Gfp One TCTCGGCATGGACGAGCTGTA hPD-1 2 TGGAACCCATTCCTGAAATTA 3 GGAACCCATTCCTGAAATTAT 4 GAACCCATTCCTGAAATTATT 5 ACCCATTCCTGAAATTATTTA 6 CCCATTCCTGAAATTATTTAA 7 CCTTCCCTGTGGTTCTATTAT 8 CTTCCCTGTGGTTCTATTATA 9 TTCCCTGTGGTTCTATTATAT 10 TCCCTGTGGTTCTATTATATT 11 CCCTGTGGTTCTATTATATTA 12 CCTGTGGTTCTATTATATTATATTAT hTIM-3 13 GATGAAAGGGATGTGAATTAT 14 GGGAGCCTCCCTGATATAAAT 15 GGAATTCGCTCAGAAGAAA 16 GGACCAAACTGAAGCTATATT 17 AGAACTTTGGTTTCCTTTAAT 18 ATGAAAGGGATGTGAATTATT 19 TCTTATCTTCGGCGCTTTAAT 20 CTTATCTTCGGCGCTT
  • shRNA cassetteSEQ ID Sequence (5 ⁇ -> 3 ⁇ ) shRNA cassette base sequence GTTAAC GATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGAGATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCT AACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAAAGCCTTGTTTGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN TTCAAGAAAAGCCTTGTTTGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN TTCAAGAAAAGCCTTGTTTGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN TTCAAGAAAAGCCTTGTTTGNNNNNNN
  • Example 1 Preparation of Engineered T Cells Simultaneously Expressing and CD19-CAR.
  • CD19-CAR gene was cloned to be regulated by the EF1-alpha promoter.
  • NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (19bp) is the sense shRNA sequence
  • NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (19bp) is an antisense shRNA sequence.
  • a TTCAAGAGA Linker was inserted between the sense and antisense shRNA seqs and placed in the U6 promoter 3 ′. And, the TTTTT required for termination of transcription by the mouse U6 promoter was placed in shRNA 3 ′.
  • pLV -CD19-CAR_ shGFP containing shRNA SEQ ID # 1 by ligation of the shRNA cassette product containing shGFP or shPD- 1 to Hpa1 restriction enzyme followed by HIP1 restriction enzyme and CIP-treated pLV-CD19-CAR plasmid: plasmid ID # containing 1 and shRNA SEQ ID # 12 pLV-CD19 -CAR_ shPD -1: to prepare a plasmid ID # 2.
  • Plasmids # 1, # 2 and packaging plasmids pMDL g / p, pRSVrev, and pMDG.1 were transfected into HEK293 T cells using lipofectamine, and cell cultures containing lentiviral were obtained after 48 hours.
  • Peripheral blood mononuclear cells (PBMC) were isolated from human blood using ficoll-paque solution, and T-cells were specifically activated using human CD3 and CD28 target antibodies. After 1-2 days of initial activation of T-cells, transduction was performed using the virus obtained earlier. The resulting CAR-T cells were cultured using AIM-V medium containing 5% human plasma and human IL2.
  • FIG. 2 confirms the maintenance and expression of CAR and reduced PD-1 expression in CAR T cells prepared using a two-in-one carvirus plasmid expressing shPD- 1 and CD19 target CAR at the same time inhibiting human PD-1 expression FACS DATA.
  • FIG 2 when the CD19-CAR T PD-1 + cells in cells expressing shGFP around 25%, shPD -1 expressing CD19-CAR T PD-1 + cells in cells which is 4% or less It was. Through this, CAR-T cell production technology was established that can simultaneously inhibit CAR expression and PD-1 expression in T cells.
  • Example 2 (( One) shPD -One Simultaneously expresses an expression cassette, (2) Low-affinity nerve growth factor receptor (LNGFR), and (3) CD19-CAR. Engineered T cell manufacturing)
  • ⁇ LNGFR engineered cell surface receptor, a cell surface receptor that does not exist in T cells, blocked signaling of the receptor without inducing CAR and TCR signal activation
  • pLV-CD19- CAR_shGFP and pLV-CD19- CAR_shPD-1 plasmids expressing cytoplasmic domain truncated LNGFR were prepared.
  • pMACS- ⁇ LNGFR plasmid (miltenyibiotec, Germany) was used as primers SEQ ID # 8, # 9 to obtain a PCR product.
  • Overlap PCR was performed using primers SEQ ID # 27, ⁇ LNGFR PCR product containing P2A, and primers SEQ ID # 8, # 10.
  • the overlap PCR product was ligation with BamH1, Swa1 restriction enzyme-treated plasmid ID # 1, # 2 after treatment with BamH1, Swa1 restriction enzyme and pLV- ⁇ LNGFR_P2A_CD19-CAR_ shGFP containing CAR SEQ ID # 3
  • the plasmid ID # 3 and pLV- ⁇ LNGFR_P2A_CD19-CAR_ shPD -1 plasmid # 4 was produced.
  • Lentiviruses were prepared using plasmid ID # 3 and # 4, followed by transduction into PBMC to prepare CAR-T cells.
  • MACSelect LNGFR System (miltenyibiotec, Germany) was used to obtain pure CAR-T cells, and the purity of CAR-T cells was confirmed by flow cytometry using LNGFR target antibody.
  • FIG. 3 CD19- CAR_shGFP or CD19- CAR_shPD - 1 T cells of about 90% LNGFR + could be obtained.
  • PD-1 expression of CAR-T cells isolated after stimulating T-cells for 3 days using human CD3 and CD28 target antibodies was confirmed.
  • Example 3 (Established human chronic myeloid leukemia cell line K562-CD19-PD-L1 expressing CD19, PD-L1 at the same time)
  • Human PD-L1 expressing lentiviral vectors were constructed to establish PD-L1 overexpression K562-CD19. After synthesizing the human PD-L1 sequence, PCR products were obtained using primers # 11 and # 12. The PCR product was ligation with Fse1, Pac1 restriction enzyme-treated lentiviral plasmid ID # 5 pSMOUW_IRES_Puro after Fse1, Pac1 restriction enzyme treatment to construct PD-L1 expressing lentiviral plasmid ID # 6.
  • Lentivirus plasmids # 5, # 6 and packaging plasmids pMDL g / p, pRSVrev, and pMDG.1 were transfected into HEK293 T cells with lipofectamine, and lentiviral was obtained from the cell culture after 48 hours.
  • the virus was transduced into the K562-CD19 cell line to establish the K562-CD19-PD-L1 cell line.
  • Flow cytometry was performed using CD19 and PD-L1 target antibodies of the K562-CD19-PD-L1 cell line.
  • PD-L1 was not expressed in the K562-CD19 cell line, while almost all cells expressed PD-L1 in the K562-CD19-PD-L1 cell line.
  • CD19 expression was confirmed that there is no difference between the K562-CD19-PD-L1 cell line and K562-CD19 cell line. Based on the above experiments, we established a K562 cell line expressing PD-L1 and CD19 simultaneously.
  • CAR-T cells were isolated by MACSelect LNGFR System (miltenyibiotec, Germany), and then cultured in AIM-V medium containing 5% human plasma and human IL2 for 4 days. By repeating this three times, CD19 CAR_sh GFP which received 1, 2 or 3 antigen-specific stimulation And CD19_ shPD - 1 to obtain the T cells (Fig. 8). In imposing antigen-specific stimulation it was confirmed that the PD-1 expression inhibition by shPD -1 being maintained.
  • CD19-CAR_ shPD - 1 T cells CD19-CAR_ shGFP T cells than it is seen that maintain a low level of the PD-1 expression of CD19-CAR_ shPD - 1 PD-1 expression in T cells It can be seen that the decrease is continuously made by shPD- 1 .
  • CellVue® Claret Far Red the CAR 2x10 5 T-cells were labeled with (sigma) were co-cultured five and K562-CD19-PD-L1 cell line 2x10. After 3 days, 7-AAD stained to determine the cytotoxic capacity of CAR-T cells.
  • Cytotoxicity% was calculated as 100 (100 x (1-(7-AAD-ClaretRed +% / ClaretRed +%)), and relative cytotoxic activity was determined by [different number of stimulated CAR-T cells % Cytotoxicity /% unstimulated CAR-T cytotoxicity] As shown in Figure 8, as the number of stimuli increased, the cytotoxicity of CAR-T cells decreased.
  • CD19-CAR_ shPD -1 T cells (stimulated before receiving CD19-CAR_ shPD - 1 T 17.7 % decrease cytotoxicity performance than the cells) CD19-shGFP CAR_ T cells (CD19-CAR_ shGFP before stimulation It was confirmed that the decrease in cytotoxicity was significantly lower than that of T cells (61.3% decrease).
  • Example 5 (Check the effect of decreased PD-1 expression on CD19-CAR T differentiation and proliferative capacity during repeated antigen stimulation of CAR-T cell exhaustion)
  • shTIM - 3 To construct a plasmid in which and CD19 target CAR is expressed simultaneously. Plasmid ID ## 4 was prepared using primers SEQ ID # 23, # 24, # 25, # 26, and # 1. shTIM-3 PCR products containing -mU6 cassettes were made. After the PCR product was treated with Hpa1 restriction enzyme, the TIM-3 target shRNA (shRNA SEQ ID # 13, # 14, # 15, # 16) and CD19-CAR were simultaneously ligation with Hpa1 restriction enzyme and CIP-treated plasmid ID # 4. Expressing pLV-CD19-CAR_ shGFP Plasmid IDs # 7, # 8, # 9, # 10 were prepared.
  • shPD- 1 , shTIM- 3 , and CD19-CAR are regulated by human U6 promoter (hU6), mouse U6 promoter (mU6), and EF1-alpha promoter, respectively.
  • hU6 human U6 promoter
  • mU6 mouse U6 promoter
  • EF1-alpha promoter EF1-alpha promoter
  • the multi-cloning seat insert (2) -1 switching shPD a mouse U6 promoter, the human U6 promoter (3) -3 shTIM insert (4) shTIM - cloning, such as the position of the switch 3 and shPD -1 was performed.
  • pLV- ⁇ LNGFR_P2A_CD19-CAR_ shPD -1 to insert a multiple cloning seat (MCS, Multiple cloning site) to shPD-1 -mU6 3 ⁇ portion of plasmid # 4, and primer SEQ ID # 13, by using a # 14 shPD -1 -mU6
  • the PCR product was then treated with BstZ171, Hpa1 restriction enzyme, and plasmid # 4 was treated with Hpa1 restriction enzyme, CIP and then blunt end ligation, followed by blunt end ligation, pLV containing shPD- 1 -mU6-MCS sequence (shRNA cassette SEQ ID # 2).
  • shRNA cassette SEQ ID # 2 shRNA cassette SEQ ID # 2
  • the - ⁇ LNGFR_P2A_CD19-CAR_ shPD -1 _MCS plasmid ID # 11 was prepared (Fig. 13).
  • the plasmid was then constructed so that expression of shPD- 1 was expressed by the human U6 promoter instead of the mouse U6 promoter.
  • LentiCRISPR V2 plasmid was used as primers SEQ ID # 15, # 16 to obtain a PCR product containing the human U6 promoter.
  • PCR product Hpa1 Spe1 Hpa1 restriction enzyme treatment, restriction enzyme Spe1 and ligation of the treated plasmid ID # 11 contains the shPD -1 -hU6 nucleotide sequence (shRNA cassette SEQ ID # 3) pLV- ⁇ LNGFR_P2A_CD19 -CAR_hU6- shPD - 1 _MCS prepare a plasmid ID # 12.
  • the shTIM- 3 -mU6 cassette was inserted into plasmid ID # 12 to construct a plasmid expressing both shTIM - 3 and shPD- 1 simultaneously.
  • PCR products containing the shTIM- 3 -mU6 cassette were obtained using plasmid ID # 9 and primers SEQ ID # 17, # 18.
  • the PCR product was ligation to the Bmt1, Spe1 restriction enzyme-treated plasmid ID # 12 after Bmt1, Spe1 restriction enzyme treatment and the bivalent ( shPD -1- hU6 shTIM- 3 -mU6) nucleotide sequence (shRNA cassette SEQ ID # 4) - ⁇ LNGFR_P2A_CD19-CAR_hU6- shPD- 1 mU6- shTIM-3 plasmid ID # 13 was prepared (FIG. 14). It was intended to construct a plasmid in which two shRNA cassettes were bidirectionally inverted ( shPD -1 -hU6 shTIM -3 -mU6).
  • PCR products were obtained using plasmid ID # 13 using primers SEQ ID # 19 and # 20. Spe1, Hpa1 restriction enzyme treatment and then inserted into the same restriction enzyme-treated plasmid ID # 11 was produced in the pLV- ⁇ LNGFR_P2A_CD19-CAR_hU6- shPD -1 ( reverse) _MCS plasmid ID # 14. Thereafter, PCR products were obtained using plasmid ID # 14 and primers SEQ ID # 21 and # 22.
  • ligation to the same restriction enzyme-treated plasmid ID # 14 finally contained a bidirectional reverse ( shPD -1 -hU6 shTIM -3 -mU6) nucleotide sequence (shRNA cassette SEQ ID # 5).
  • pLV- ⁇ LNGFR_P2A_CD19-CAR_hU6- shPD-1 mU6- shTIM-3 plasmid ID # 15 was prepared (FIG. 15).
  • Plasmids # 3, # 11, # 12, # 13, # 15 and the packaging plasmids pMDL g / p, pRSVrev, and pMDG.1 were transfected into HEK293 T cells with lipofectamine, and 48 hours later, the lentiviral was included.
  • Cell culture solution was obtained.
  • Peripheral blood mononuclear cells (PBMC) were isolated from human blood using ficoll-paque solution, and T-cells were specifically activated using human CD3 and CD28 target antibodies. After 1-2 days of initial activation of T-cells, transduction was performed using the virus obtained earlier.
  • the resulting CAR-T cells were cultured using AIM-V medium containing 5% human plasma and human IL2.
  • CAR-T cells Six days after transduction, MACSelect LNGFR System (miltenyibiotec, Germany) was used to obtain pure CAR-T cells, and the purity of CAR-T cells was confirmed by flow cytometry using LNGFR target antibody. As shown in FIG. 16, CAR-T cells of about 80% LNGFR + were obtained. The isolated CAR-T cells were stimulated with human CD3 and CD28 target antibodies for 3 days to induce PD-1 and TIM-3 expression, and then the PD-1 and TIM-3 expression of CAR-T cells was expressed in CAR, PD- 1, TIM-3 target antibody was used for flow cytometry. As can be seen in Figure 17, it was confirmed that the PD-1 expression decreased by the U6 promoter.
  • the human U6 promoter inhibited PD-1 expression similar to the existing mouse U6 promoter.
  • the effects of two simultaneously expressed shRNAs on the expression of PD-1 and TIM-3 were analyzed. Reduction of PD-1 and TIM-3 expression observed in CD19-CAR T cells expressing shPD - 1 and shTIM - 3 simultaneously decreased expression of CD19-CAR T cells expressing shPD- 1 or shTIM- 3 alone Similarity was confirmed to the degree.
  • shRNA cassette orientation on the expression of target genes was examined.
  • hU6- shPD - 1 mU6- shTIM- 3 and hU6- shPD - 1 mU6- shTIM- 3 expressing CD19-CAR T cells the degree of reduction in TIM-3 expression was similar, but the decrease in PD-1 was similar to hU6- shPD- . 1 mU6- shTIM-3 was found to be superior. As a result, it has secured a technology for producing immune cell therapeutics capable of inhibiting the expression of two immune gate genes and simultaneously expressing CAR.

Abstract

The present invention relates to an immune cell surmounting an immune checkpoint, a preparation method thereof, and a pharmaceutical composition comprising the immune cell and, more particularly, to an immune checkpoint-surmounting immune cell which is genetically modified to simultaneously express a chimeric antigen receptor (CAR) and a short hairpin ribonucleic acid (shRNA) suppressive of the expression of an immune checkpoint receptor, a method for preparing the same, and a pharmaceutical composition comprising the immune cell. Designed to genetically manipulate an immune cell to simultaneously express a chimeric antigen receptor or monoclonal TCR, and a shRNA suppressive of the expression of immune checkpoint receptors, the present invention can inhibit the expression of immune checkpoint receptors without the separate use of an antibody therapeutic agent suppressive of the expression of immune checkpoint receptors and thus can greatly improve the therapeutic effect of the manipulated immune cell using CAR or monoclonal TCR, such as an anticancer effect, without exerting side effects of antibody therapeutic agents, and reduce an economic load from patients.

Description

면역관문을 극복한 면역세포 및 상기 면역세포를 포함한 약제학적 조성물Immune Cells Overcoming Immune Gates and Pharmaceutical Compositions Comprising the Immune Cells
본 발명은 면역관문을 극복한 면역세포 및 상기 면역세포를 포함한 약제학적 조성물에 관한 것으로, 보다 상세하게는 키메라 항원 수용체(CAR) 또는 단일클론 티세포수용체(T cell receptor, TCR); 및 면역관문 수용체의 발현을 저해하는 숏헤어핀 리보핵산(RNA)이 동시에 발현되도록 유전적으로 조작된 면역관문을 극복한 면역세포 및 상기 면역세포를 포함한 약제학적 조성물에 관한 것이다.The present invention relates to immune cells that overcome immune barriers and pharmaceutical compositions comprising the immune cells, and more particularly, chimeric antigen receptor (CAR) or monoclonal T cell receptor (T cell receptor); And immune cells overcoming genetically engineered immune barriers to simultaneously express shothairpin ribonucleic acid (RNA) that inhibits the expression of immune gate receptors and pharmaceutical compositions comprising the immune cells.
환자 혹은 공여자의 몸에서 분리한 티세포(T-cell) 혹은 자연살해세포(NK cell)를 체외에서 배양하여 다시 환자몸에 투여하는 과정을 거치는 면역세포를 이용한 항암 치료법이 최근 새로운 암치료법으로 주목을 받고 있다[1]. 특히 체외 배양과정 중에 바이러스 등을 이용하여 새로운 유전 정보를 주입한 후 증식하는 과정을 거친 면역 세포는 그렇지 않은 경우에 비해 높은 항암효과를 가진다는 것이 보고되었다[2]. 이 때 T세포에 주입되는 유전정보는 항원(TARGET)에 높은 친화력을 갖도록 조작된 단일 클론 T세포 수용체 (T cell receptor, TCR) 혹은 키메라 항원 수용체 (CAR, Chimeric Antigen Receptor)가 주로 사용된다. 이렇게 조작된 면역세포는 기존에 내재하는 항원 특이성에 구애받지 않고 항원인 암세포를 인식, 공격하여 세포 사멸을 유도하게 된다. 특히 T 세포를 CAR 수용체를 이용하여 유전적으로 조작하는 방법은 1989년 Eshhar및 그의 동료들이 최초로 제안하여 "T-body"라는 이름으로 불렀다[3]. 그 이후로 연구자들에 의해 CAR의 디자인은 여러 세대의 진화를 거치게 되는데, 현재 임상에서 주로 사용되는 CAR의 구조는 단일사슬단편항체(single chain variable fragment, scFv : 항원에 대한 특이성을 부여) domain, spacer domain(scFv와 세포막간의 거리를 조절), transmembrane domain, 그리고 intracellular signaling domain (ISD)으로 구성된다. ISD는 다시 한개 또는 여러개의 costimulatory domain (CD28, CD137, or OX40: T 세포의 in vivo 증식 및 긴 수명에 기여)과 TCR signaling domain (CD3 zeta: T세포의 활성화에 기여)으로 이루어진다. 이렇게 만들어진 카수용체를 발현하도록 조작된 T세포는 높은 특이성으로 스스로 항원발현 암세포를 인식하여 활성화된 다음 효율적으로 암세포의 사멸을 유도함과 동시에, 체내에서 기하급수적으로 증식하고 오래 살아있게 된다. 예를 들어, B세포의 특이 항원인 CD19을 타깃하도록 만들어진 카티세포(CART-19)가 B-세포 백혈병 환자에 투여되었을 때 1,000 ~ 10,000배 까지 증식되고 수년간 체내에서 살아있음이 보고 되었다 [4, 5]. 그 결과, CART-19은 기존의 화학치료 요법 등으로 효과를 보지 못한 말기 급성 림프구성 백혈병 (B-ALL) 환자들을 대상으로 한 임상시험에서 90%의 놀라운 완전 반응 (complete response) 을 보여 초기 연구자임상단계에서 이례적으로 글로벌 제약회사에 라이센스되었으며, 카티 세포치료제 최초로 2017년 미국 식약청의 승인을 앞두고 있다. 그 외에도 대부분의 대형 제약회사들이 이 분야에 뛰어들고 있고 바이오텍에 대한 투자와 창업 또한 매우 활발하게 이루어지고 있는 등, 카티세포를 이용한 치료법이 앞으로 항암 치료산업의 패러다임을 바꿀 수도 있을 것이라는 기대를 모으고 있다.Anti-cancer therapy using immune cells that incubate T-cells or natural killer cells isolated from the patient or donor's body and administer them to the patient's body has recently attracted attention as a new cancer treatment. [1]. In particular, it has been reported that immune cells, which have undergone proliferation after injecting new genetic information using viruses and the like during in vitro culture, have a higher anticancer effect than otherwise [2]. At this time, the genetic information injected into the T cells is mainly used a monoclonal T cell receptor (TCR) or chimeric antigen receptor (CAR) engineered to have a high affinity for the antigen (TARGET). The immune cells thus manipulated recognize and attack cancer cells, which are antigens, regardless of existing antigen specificity, thereby inducing cell death. In particular, a method of genetically manipulating T cells using a CAR receptor was first proposed in 1989 by Eshhar and his colleagues, called "T-body" [3]. Since then, the design of CAR has gone through several generations of evolution, and the structure of CAR, which is used mainly in the clinic, is a single chain variable fragment (scFv). It consists of a spacer domain (controlling the distance between the scFv and the cell membrane), a transmembrane domain, and an intracellular signaling domain (ISD). ISD consists of one or more costimulatory domains (CD28, CD137, or OX40: contribute to in vivo proliferation and long life of T cells) and TCR signaling domains (CD3 zeta: contribute to T cell activation). The T cells engineered to express the car receptor thus made recognize and activate antigen-expressing cancer cells with high specificity, and then effectively induce the death of cancer cells, and at the same time proliferate exponentially in the body and live long. For example, it has been reported that cati cells (CART-19) made to target CD19, a specific antigen of B cells, multiply by 1,000 to 10,000 times when administered to patients with B-cell leukemia and have been living in the body for many years [4, 5]. As a result, CART-19 showed an amazing complete response of 90% in clinical trials in patients with terminal acute lymphocytic leukemia (B-ALL) who had not benefited from conventional chemotherapy. It is licensed to a global pharmaceutical company exceptionally at the clinical stage and is the first of its kind to be approved by the US Food and Drug Administration in 2017. In addition, most large pharmaceutical companies are engaged in this field, and investments and start-ups in biotech are very active, and it is expected that the treatment using cati cells may change the paradigm of the chemotherapy industry in the future. .
그런데, 면역세포, 가령 T세포의 표면에는 CTLA-4 나 PD-1 같은 면역관문 수용체들이 존재한다. 본래 이 수용체들은 T 세포가 지나치게 활성화되어 세포사멸 되거나 자가 면역을 일으키는 것을 조절하기 위한 안전장치이나, 암세포들, 특히 고형암은 이 점을 악용하여 T 세포에 의한 면역감시를 피하는 것으로 알려져 있다. 예를 들어 암세포가 PDL-1 리간드를 표면에 발현하게 되면 T 세포가 암세포를 인식하여 활성화되더라도 곧 PD-1수용체로부터 전해지는 활성저해 시그널링으로 인해 소진(exhaustion)되게 되는 것이다. 이러한 면역관문(immune checkpoint)들에 의해 T 세포의 활성이 저해되는 것을 막기 위해 anti-CTLA4 나 anti-PD-1 등의 단일 클론 항체들이 개발되었고, 이들 항체를 이용하여 면역관문을 차단함으로써 T 세포의 전반적인 면역 기능을 향상시키는 치료가 여러 고형암에서 효능을 보이고 있다.However, immune barrier receptors such as CTLA-4 or PD-1 exist on the surface of immune cells, such as T cells. Originally, these receptors are safeguards for regulating over-activation of T cells to cause apoptosis or autoimmunity, but cancer cells, particularly solid cancers, are known to exploit this to avoid immune surveillance by T cells. For example, when cancer cells express the PDL-1 ligand on the surface, even if the T cells recognize and activate the cancer cells, they are soon exhausted due to inactivation signaling transmitted from the PD-1 receptor. Monoclonal antibodies, such as anti-CTLA4 and anti-PD-1, have been developed to prevent T cell activity from being inhibited by these immune checkpoints. Therapies that improve the overall immune function of the drug have been shown to be effective in several solid cancers.
카티세포도 결국 활성화된 T 세포의 세포 독성에 의존하는 치료제이기 때문에, 카티세포 주변에 면역억제 환경이 존재하게 되면 치료효과에 큰 저해 요인으로 작용한다. 실제로 B-세포 백혈병에서 보여졌던 놀라운 치료효과에 비해 아직 고형암 (solid tumor)을 타깃하도록 제작된 CAR-T 들은 임상에서 희망적인 치료효과를 보인 예가 드문데, 이는 혈액암과는 달리 고형암은 부분적으로 면역억제 환경 (immune-suppressive tumor microenvironments)을 형성하여 카티세포의 활성과 증식을 억제하기 때문인 것으로 추측되고 있다. 또한, B세포 혈액암 내에서도, CART-19을 이용한 치료가 90%에 달하는 반응을 보인 급성 백혈병 (acute lymphoblastic leukemia, ALL) 환자들과는 달리 림프종 (lymphoma, 20-50% 반응율)[6] 이나 만성 백혈병 (chronic lymphoblastic leukemia, CLL, ~20 %의 반응율)[7] 환자들의 경우에는 상대적으로 낮은 치료효과를 보인다는 것이 보고되었다. Because cati cells are also therapeutic agents that depend on the cytotoxicity of activated T cells, the presence of an immunosuppressive environment around the cati cells acts as a significant inhibitor to the therapeutic effect. Indeed, compared to the surprising therapeutic effects seen in B-cell leukemia, CAR-Ts that are designed to target solid tumors are rarely clinically promising. It is presumed to be due to the formation of immunosuppressive tumor microenvironments, which inhibit the activity and proliferation of cati cells. In addition, in B-cell hematologic cancer, lymphoma (lymphoma, 20-50% response rate) [6] or chronic leukemia, unlike patients with acute lymphoblastic leukemia (ALL), showed 90% of treatments with CART-19. (Chronic lymphoblastic leukemia, CLL, ~ 20% response rate) [7] Patients have been reported to have relatively low therapeutic effects.
그와 더불어 림프종에 의해 형성되는 tumor microenvironment에 programmed death ligand-1 (PDL-1)과 같은 면역억제 리간드가 발현되고, 그에 따라 암조직 내에 존재하는 T세포의 기능이 소진된 형질을 보인다는 것이 보고되었다[8]. 또한 CLL환자에게서 얻은 T 세포는 활성은 이미 많이 소진된 형질을 보이고 높은 정도의 PD-1, CD160, CD244와 같은 면역관문 수용체의 발현을 보임이 보고되었다[9]. In addition, immunosuppressive ligands such as programmed death ligand-1 (PDL-1) are expressed in tumor microenvironments formed by lymphomas, and thus, traits of T cells present in cancer tissues are depleted. [8]. In addition, it has been reported that T cells obtained from CLL patients have already exhibited much depleted traits and high levels of expression of immune gate receptors such as PD-1, CD160, and CD244 [9].
따라서 이렇게 낮아진 카티세포의 활성을 회복시키기 위해 anti-CTLA나 anti-PD-1 blocking 항체와 카티세포를 동시에 사용할 경우 항암 효과가 높아진다는 전임상 결과가 보고되었고[10, 11], 현재 이러한 컴비네이션을 이용한 임상시험이 진행 중이다. 그러나, 이러한 항체와 카티세포 co-therapy의 문제점은, 몸 전체에 퍼진 항체가 카티세포 뿐 아니라 다른 몸 안에 존재하는 모든 T 세포에 영향을 미치기 때문에 자가 면역 증상과 같은 부작용을 일으킬 수 있다는 점이다. 또한 고가의 항체치료제를 세포치료제와 병행하여 사용함으로 인해 생기는 치료비용 부담의 증가 또한 문제점으로 지적되고 있다. Therefore, preclinical results have been reported that anti-CTLA or anti-PD-1 blocking antibodies and cati cells can be used simultaneously to restore the activity of these lowered cati cells [10, 11]. Clinical trial is in progress. However, the problem with such antibody and catty cell co-therapy is that antibodies spread throughout the body can cause side effects such as autoimmune symptoms because it affects not only the cati cells but all T cells present in other bodies. In addition, the increase in the cost of treatment caused by the use of expensive antibody therapies in parallel with the cell therapy has also been pointed out as a problem.
따라서, 카티세포에 면역관문을 억제할 수 있도록 세포 내 유전자 발현을 조절하는 시도가 최근 있었다. 국제특허공개공보 WO2016/069282호에는 TCR α 쇄, TCR β 쇄, 베타-2 마이크로글로불린, 및 FAS로 이루어지는 그룹 중에서 선택된 내인성 유전자 발현을 하향조절할 수 있는 핵산을 갖고 표적 세포상의 표면 항원에 대한 친화성을 포함하는 변형된 T 세포 수용체(TCR)를 암호화하는 핵산 또는 키메릭 항원 수용체(CAR)를 암호화하는 전기천공된 핵산을 추가로 포함하는 변형된 T 세포의 생성을 위한 조성물 및 방법이 개시되어 있다[12]. 상기 문헌에 따르면 CRISPR/Cas9등의 유전자 가위를 이용하여 내인성 유전자의 발현을 knock-out할 수 있음을 보여주었다. 그러나, 전술한 문헌의 방법으로 제조된 카티세포는 제조방법이 다소 복잡할 뿐 아니라 제조수율이 낮고 생산 비용이 높은 문제가 있다. Thus, recent attempts have been made to modulate gene expression in cells to inhibit immune barriers in cati cells. International Patent Publication No. WO2016 / 069282 discloses affinity for surface antigens on target cells with nucleic acids capable of downregulating endogenous gene expression selected from the group consisting of TCR α chain, TCR β chain, beta-2 microglobulin, and FAS. Disclosed are compositions and methods for the production of modified T cells further comprising a nucleic acid encoding a modified T cell receptor (TCR) comprising an electroporated nucleic acid encoding a chimeric antigen receptor (CAR). [12]. According to the above literature, gene shears such as CRISPR / Cas9 can be used to knock-out expression of endogenous genes. However, the catty cells produced by the method described above have a problem that the manufacturing method is not only slightly complicated, but also has low production yield and high production cost.
<선행기술문헌><Preceding technical literature>
<특허문헌><Patent Documents>
12. WO2016/069282 A112.WO2016 / 069282 A1
15. DE 3,218,121 15.DE 3,218,121
16. 유럽특허 제52,322호 16. European Patent No. 52,322
17. 유럽 특허 제36,676호17. European Patent No. 36,676
18. 유럽 특허 제88,046호18. European Patent No. 88,046
19. 유럽 특허 제143,949호19. European Patent No. 143,949
20. 유럽 특허 제142,641호20. European Patent No. 142,641
21. 일본 특허 제83-118008호21. Japanese Patent No. 83-118008
22. 미국 특허 제4,485,045호22. US Patent No. 4,485,045
23. 미국특허 제4,544,545호23. US Patent 4,544,545
24. 유럽 특허 제102,324호24. European Patent No. 102,324
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11.Gargett, T., et al., GD2-specific CAR T Cells Undergo Potent Activation and Deletion Following Antigen Encounter but can be Protected From Activation-induced Cell Death by PD-1 Blockade. Mol Ther, 2016.11.Gargett, T., et al., GD2-specific CAR T Cells Undergo Potent Activation and Deletion Following Antigen Encounter but can be Protected From Activation-induced Cell Death by PD-1 Blockade. Mol Ther, 2016.
13.Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985).13.Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985).
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따라서, 본 발명이 이루고자 하는 기술적 과제는 면역관문 수용체 발현 억제를 위한 별도의 항체 치료제를 사용하지 않으면서도 면역관문 수용체의 발현을 억제하면서 키메라 항원 수용체의 기능을 발휘하도록 하여 면역세포 치료효과를 향상시킨 면역세포를 제공하는 것이다.Therefore, the technical problem to be achieved by the present invention is to improve the immune cell therapeutic effect by exerting the function of the chimeric antigen receptor while suppressing the expression of the immune gate receptor without using a separate antibody therapeutic agent for inhibiting the immune gate receptor expression. To provide immune cells.
본 발명의 또 다른 측면은 상기 면역관문을 극복한 면역세포를 포함한 면역세포 치료제를 제공하는 것이다.Another aspect of the present invention is to provide an immune cell therapeutic agent including immune cells that overcome the immune barrier.
상기 기술적 과제를 달성하기 위하여 본 발명은 면역세포의 핵산서열에 있어서, 키메라 항원 수용체(CAR) 또는 단일클론 티세포 수용체(TCR)을 코딩하는 염기서열; 및 면역관문 수용체의 발현을 저해하는 숏헤어핀 리보핵산(shRNA)을 코딩하는 염기서열을 동시에 포함한 것을 특징으로 하는 핵산서열을 제공한다.In order to achieve the above technical problem, the present invention provides a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a monoclonal T cell receptor (TCR) in a nucleic acid sequence of an immune cell; And it provides a nucleic acid sequence characterized in that it comprises at the same time the base sequence encoding a short hairpin ribonucleic acid (shRNA) that inhibits the expression of the immune gateway receptor.
또한, 본 발명은 상기 숏헤어핀 리보핵산(shRNA)을 코딩하는 염기서열은 PD-1, CTLA-4, LAG-3 및 TIM-3로 구성된 군으로부터 선택된 1종 이상의 면역관문 수용체를 타깃으로 한 것임을 특징으로 하는 핵산서열을 제공한다.In addition, the present invention is that the base sequence encoding the short hairpin ribonucleic acid (shRNA) is targeted to at least one immune gateway receptor selected from the group consisting of PD-1, CTLA-4, LAG-3 and TIM-3 It provides a nucleic acid sequence characterized by.
또한, 본 발명은 상기 숏헤어핀 리보핵산(shRNA)을 코딩하는 염기서열이 PD-1 및 TIM-3를 동시에 타깃으로 한 것을 특징으로 하는 핵산서열을 제공한다.In another aspect, the present invention provides a nucleic acid sequence characterized in that the base sequence encoding the short hairpin ribonucleic acid (shRNA) targets PD-1 and TIM-3 at the same time.
또한, 본 발명은 상기 숏헤어핀 리보핵산(shRNA)을 코딩하는 염기서열이 서열번호 2 내지 12의 PD-1 타깃 염기서열, 서열번호 13 내지 35의 TIM-3 타깃 염기서열, 서열번호 36 내지 42의 LAG-3 타깃 염기서열 및 서열번호 43 내지 51의 CTLA-4 타깃 염기서열로 구성된 군으로부터 선택된 1종 이상의 염기서열을 포함한 것을 특징으로 하는 핵산서열을 제공한다.In addition, the present invention is a base sequence encoding the short hairpin ribonucleic acid (shRNA) is PD-1 target nucleotide sequence of SEQ ID NO: 2 to 12, TIM-3 target nucleotide sequence of SEQ ID NO: 13 to 35, SEQ ID NO: 36 to 42 It provides a nucleic acid sequence comprising at least one nucleotide sequence selected from the group consisting of LAG-3 target nucleotide sequence of and the CTLA-4 target nucleotide sequence of SEQ ID NO: 43 to 51.
또한, 본 발명은 상기 키메라 항원 수용체(CAR) 또는 단일클론 T세포 수용체 의 타겟이 5T4, 알파 5β1-인테그린, 707-AP, AFP, ART-4, B7H4, BAGE, β-카테닌/m, Bcr-abl, MN/CIX 항원, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27/m, CD30, CD33, CD52, CD56, CD80, CDK4/m, CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2/new, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT(또는 hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/melan-A, MART-2/Ski, MC1R, 미오신/m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, PAP, 프로테이나제-3, p190 마이너 bcr-abl, Pml/RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1 또는 RU2, SAGE, SART-1 또는 SART-3, 서바이빈, TEL/AML1, TGFβ, TPI/m, TRP-1, TRP-2, TRP-2/INT2, VEGF, WT1, NY-Eso-1 및 NY-Eso-B로 구성된 군으로부터 선택된 1종 이상인 인간 종양항원인 것을 특징으로 하는 핵산서열을 제공한다.In addition, the present invention is the target of the chimeric antigen receptor (CAR) or monoclonal T cell receptor 5T4, alpha 5β1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, β-catenin / m, Bcr- abl, MN / CIX antigen, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27 / m, CD30, CD33, CD52, CD56, CD80, CDK4 / m, CEA, CT , Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2 / new, HLA-A * 0201-R170I, HPV-E7 , HSP70-2M, HST-2, hTERT (or hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR / FUT, MAGE, MART-1 / melan-A, MART-2 / Ski, MC1R, myosin / m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, PAP, proteinase-3, p190 minor bcr-abl, Pml / RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, Survivin, TEL / AML1, TGFβ, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, VEGF, Provided are nucleic acid sequences characterized in that at least one human tumor antigen selected from the group consisting of WT1, NY-Eso-1 and NY-Eso-B.
또한, 본 발명은 상기 CAR 수용체의 아미노산 서열이 서열번호 52 내지 54로 구성된 군으로부터 선택된 1종 이상의 아미노산 서열과 90% 이상 일치하는 것을 특징으로 하는 핵산서열을 제공한다.The present invention also provides a nucleic acid sequence characterized in that the amino acid sequence of the CAR receptor is at least 90% identical to at least one amino acid sequence selected from the group consisting of SEQ ID NO: 52 to 54.
본 발명의 또 다른 측면은 키메라 항원 수용체(CAR) 또는 단일클론 티세포 수용체(TCR)를 코딩하는 염기서열 및 면역관문 수용체의 발현을 저해하는 숏헤어핀 리보핵산(RNA)을 코딩하는 염기서열을 포함한 벡터를 제공한다.Another aspect of the invention includes a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a monoclonal T-cell receptor (TCR) and a nucleotide sequence encoding shorthairpin ribonucleic acid (RNA) that inhibits the expression of an immune gateway receptor. Provide a vector.
또한, 본 발명은 상기 숏헤어핀 리보핵산(shRNA)을 코딩하는 염기서열은 PD-1, CTLA-4, LAG-3 및 TIM-3로 구성된 군으로부터 선택된 1종 이상의 면역관문 수용체를 타깃으로 한 것임을 특징으로 하는 벡터를 제공한다.In addition, the present invention is that the base sequence encoding the short hairpin ribonucleic acid (shRNA) is targeted to at least one immune gateway receptor selected from the group consisting of PD-1, CTLA-4, LAG-3 and TIM-3 A vector is provided.
또한, 본 발명은 상기 숏헤어핀 리보핵산(shRNA)을 코딩하는 염기서열이 PD-1 및 TIM-3를 동시에 타깃으로 한 것을 특징으로 하는 벡터를 제공한다.In another aspect, the present invention provides a vector characterized in that the base sequence encoding the short hairpin ribonucleic acid (shRNA) targets PD-1 and TIM-3 at the same time.
또한, 본 발명은 상기 숏헤어핀 리보핵산(shRNA)을 코딩하는 염기서열은 서열번호 2 내지 12의 PD-1 타깃 염기서열, 서열번호 13 내지 35의 TIM-3 타깃 염기서열, 서열번호 36 내지 42의 LAG-3 타깃 염기서열 및 서열번호 43 내지 51의 CTLA-4 타깃 염기서열로 구성된 군으로부터 선택된 1종 이상의 염기서열을 포함한 것을 특징으로 하는 벡터를 제공한다.In addition, the present invention is a base sequence encoding the short hairpin ribonucleic acid (shRNA) is PD-1 target nucleotide sequence of SEQ ID NO: 2 to 12, TIM-3 target nucleotide sequence of SEQ ID NO: 13 to 35, SEQ ID NO: 36 to 42 It provides a vector comprising at least one base sequence selected from the group consisting of LAG-3 target nucleotide sequence of and the CTLA-4 target nucleotide sequence of SEQ ID NO: 43 to 51.
또한, 본 발명은 상기 키메라 항원 수용체(CAR) 또는 단일클론 티세포 수용체(TCR)의 타겟이 5T4, 알파 5β1-인테그린, 707-AP, AFP, ART-4, B7H4, BAGE, β-카테닌/m, Bcr-abl, MN/CIX 항원, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27/m, CD30, CD33, CD52, CD56, CD80, CDK4/m, CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2/new, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT(또는 hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/melan-A, MART-2/Ski, MC1R, 미오신/m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, PAP, 프로테이나제-3, p190 마이너 bcr-abl, Pml/RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1 또는 RU2, SAGE, SART-1 또는 SART-3, 서바이빈, TEL/AML1, TGFβ, TPI/m, TRP-1, TRP-2, TRP-2/INT2, VEGF, WT1, NY-Eso-1 및 NY-Eso-B로 구성된 군으로부터 선택된 1종 이상인 인간 종양항원인 것을 특징으로 하는 벡터를 제공한다.In addition, the present invention is the target of the chimeric antigen receptor (CAR) or monoclonal T-cell receptor (TCR) is 5T4, alpha 5β1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, β-catenin / m , Bcr-abl, MN / CIX antigen, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27 / m, CD30, CD33, CD52, CD56, CD80, CDK4 / m, CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2 / new, HLA-A * 0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT (or hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR / FUT, MAGE, MART-1 / melan-A, MART-2 / Ski, MC1R, Myosin / m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, PAP, Proteinase-3, p190 Minor bcr-abl, Pml / RARα, PRAME , PSA, PSM, PSMA, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, Survivin, TEL / AML1, TGFβ, TPI / m, TRP-1, TRP-2, TRP-2 / INT2 , VEGF, WT1, NY-Eso-1, and NY-Eso-B provides a vector characterized in that the human tumor antigens selected from the group consisting of.
또한, 본 발명은 상기 CAR 수용체의 아미노산 서열이 서열번호 52 내지 54로 구성된 군으로부터 선택된 1종 이상의 아미노산 서열과 90% 이상 일치하는 것을 특징으로 하는 벡터를 제공한다.The present invention also provides a vector, wherein the amino acid sequence of the CAR receptor is at least 90% identical to at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 52 to 54.
또한, 본 발명은 키메라 항원 수용체(CAR) 또는 단일클론 티세포 수용체(TCR); 및 면역관문 수용체의 발현을 저해하는 숏헤어핀 리보핵산(RNA)이 동시에 발현되도록 유전적으로 조작된 면역관문을 극복한 면역세포를 제공한다.In addition, the present invention provides a chimeric antigen receptor (CAR) or monoclonal T cell receptor (TCR); And an immune cell that overcomes the genetically engineered immune barrier to simultaneously express shorthairpin ribonucleic acid (RNA) that inhibits the expression of the immune barrier receptor.
또한, 본 발명은 상기 숏헤어핀 리보핵산(shRNA)은 PD-1, CTLA-4, LAG-3 및 TIM-3로 구성된 군으로부터 선택된 1종 이상의 면역관문 수용체를 타깃으로 하여 그 발현을 저해한 것을 특징으로 하는 면역관문을 극복한 면역세포를 제공한다.In addition, the present invention is that the short hairpin ribonucleic acid (shRNA) is a target of at least one immune gate receptor selected from the group consisting of PD-1, CTLA-4, LAG-3 and TIM-3 inhibited its expression It provides an immune cell that overcomes the characterized immune barrier.
또한, 본 발명은 상기 숏헤어핀 리보핵산(shRNA)이 PD-1 및 TIM-3 면역관문 수용체를 동시에 타깃으로 하여 그 발현을 저해한 것을 특징으로 하는 면역관문을 극복한 면역세포를 제공한다.In addition, the present invention provides immune cells overcoming the immune barrier, characterized in that the short hairpin ribonucleic acid (shRNA) targets the PD-1 and TIM-3 immune gate receptor at the same time, inhibiting its expression.
또한, 본 발명은 상기 키메라 항원 수용체(CAR) 또는 단일클론 TCR의 타겟이 5T4, 알파 5β1-인테그린, 707-AP, AFP, ART-4, B7H4, BAGE, β-카테닌/m, Bcr-abl, MN/CIX 항원, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27/m, CD30, CD33, CD52, CD56, CD80, CDK4/m, CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2/new, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT(또는 hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/melan-A, MART-2/Ski, MC1R, 미오신/m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, PAP, 프로테이나제-3, p190 마이너 bcr-abl, Pml/RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1 또는 RU2, SAGE, SART-1 또는 SART-3, 서바이빈, TEL/AML1, TGFβ, TPI/m, TRP-1, TRP-2, TRP-2/INT2, VEGF, WT1, NY-Eso-1 및 NY-Eso-B로 구성된 군으로부터 선택된 1종 이상인 인간 종양항원인 것을 특징으로 하는 면역관문을 극복한 면역세포를 제공한다.In addition, the present invention is the target of the chimeric antigen receptor (CAR) or monoclonal TCR is 5T4, alpha 5β1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, β-catenin / m, Bcr-abl, MN / CIX antigen, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27 / m, CD30, CD33, CD52, CD56, CD80, CDK4 / m, CEA, CT, Cyp -B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2 / new, HLA-A * 0201-R170I, HPV-E7, HSP70 -2M, HST-2, hTERT (or hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR / FUT, MAGE, MART-1 / melan-A, MART-2 / Ski , MC1R, myosin / m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, PAP, proteinase-3, p190 minor bcr-abl, Pml / RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, Survivin, TEL / AML1, TGFβ, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, VEGF, WT1, Immune cells that overcome the immune barrier, characterized in that the human tumor antigen is one or more selected from the group consisting of NY-Eso-1 and NY-Eso-B The ball.
또한, 본 발명은 상기 CAR 수용체의 아미노산 서열이 서열번호 52 내지 54로 구성된 군으로부터 선택된 1종 이상의 아미노산 서열과 90% 이상 일치하는 것을 특징으로 하는 면역관문을 극복한 면역세포를 제공한다.In addition, the present invention provides an immune cell overcoming the immune barrier, characterized in that the amino acid sequence of the CAR receptor is at least 90% identical to at least one amino acid sequence selected from the group consisting of SEQ ID NO: 52 to 54.
또한, 본 발명은 상기 면역세포가 T세포, 자연살해세포, 세포독성 T 림프구 및 조절용 T 세포로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는 면역관문을 극복한 면역세포를 제공한다.In addition, the present invention provides an immune cell overcoming the immune barrier, characterized in that the immune cell is one or more selected from the group consisting of T cells, natural killer cells, cytotoxic T lymphocytes and regulatory T cells.
본 발명의 또 다른 측면은 상기 면역관문을 극복한 면역세포를 포함한 약제학적 조성물을 제공한다. Another aspect of the invention provides a pharmaceutical composition comprising immune cells that overcome the immune barrier.
또한, 본 발명은 상기 약제학적 조성물이 담체, 부형제, 희석제, 항산화제, 보존제, 착색제, 향미제 및 희석제, 유화제, 현탁제, 용매, 충전제, 벌크화제, 완충제, 전달 비히클, 등장제, 공용매, 습윤제, 복합화제, 완충제, 항균제 및 계면활성제로 이루어진 군으로부터 선택된 1종 이상의 약제학적으로 허용가능한 염, 부형제 또는 비히클을 추가로 포함한 것을 특징으로 하는 약제학적 조성물을 제공한다.In addition, the present invention provides the pharmaceutical composition of the carrier, excipient, diluent, antioxidant, preservative, colorant, flavor and diluent, emulsifier, suspending agent, solvent, filler, bulking agent, buffer, delivery vehicle, isotonic agent, cosolvent It provides a pharmaceutical composition characterized in that it further comprises one or more pharmaceutically acceptable salts, excipients or vehicles selected from the group consisting of wetting agents, complexing agents, buffers, antibacterial agents and surfactants.
또한, 본 발명은 병증의 치료가 필요한 피험자에게 유효량의 상기 약제학적 조성물을 투여함을 포함하는, 피험자의 치료 방법을 제공한다.The present invention also provides a method for treating a subject, comprising administering an effective amount of the pharmaceutical composition to a subject in need thereof.
또한, 본 발명은 상기 병증이 암인 피험자의 치료방법을 제공한다.The present invention also provides a method for treating a subject in whom the condition is cancer.
또한, 본 발명은 상기 암이 유방암, 전립선암, 난소암, 자궁경부암, 피부암, 췌장암, 결장직장암, 신장암, 간암, 뇌암, 림프종, 백혈병, 폐암, 및 이들의 임의의 조합으로 이루어지는 그룹 중에서 선택되는 피험자의 치료방법을 제공한다.In addition, the present invention is selected from the group consisting of breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and any combination thereof It provides a method for treating a subject.
본 발명은 간단한 방법으로 면역관문 수용체 발현 억제를 위한 별도의 항체 치료제를 사용하지 않으면서도 면역관문 수용체의 발현을 억제하면서 키메라 항원 수용체의 기능을 발휘할 수 있는 면역관문을 극복한 면역세포를 제공하여 고형암을 비롯한 암치료에 있어 면역세포 치료의 효과를 극대화 할 수 있다.The present invention provides a immune cell that overcomes the immune barrier capable of exerting the function of the chimeric antigen receptor while suppressing the expression of the immune barrier receptor without using a separate antibody therapeutic agent for inhibiting the immune barrier receptor expression by a simple method. In the treatment of cancer, including the treatment of immune cells can maximize the effect.
도 1은 본 발명의 면역관문 수용체의 발현을 저해하는 short hairpin RNA(shRNA)와 CAR의 발현 카세트를 둘 다 포함하는 two-in-one 카바이러스 플라스미드의 구조도1 is a structural diagram of a two-in-one carvirus plasmid containing both a short hairpin RNA (shRNA) and an expression cassette of a CAR that inhibit the expression of the immune gate receptor of the present invention.
도 2는 인간 PD-1 발현을 억제하는 shPD -1과 CD19 표적 CAR가 동시에 발현되는 two-in-one 카바이러스 플라스미드를 이용해 제조한 CAR T 세포에서 CAR를 발현유지와 PD-1 발현 감소를 확인한 FACS DATAFigure 2 confirms the maintenance and expression of CAR and reduced PD-1 expression in CAR T cells prepared using a two-in-one carvirus plasmid expressing shPD- 1 and CD19 target CAR at the same time inhibiting human PD-1 expression FACS DATA
도 3은 3가지 유전자(ΔLNGFR : 정제용, CD19-CAR : CD19 항원 타겟팅용, shPD-1: PD-1 발현 억제용)가 동시에 발현되는 two-in-one 카바이러스 플라스미드를 이용해 제조한 CAR T세포를 LNGFR 특이적 항체로 정제한 후 순도를 확인한 FACS DATA3 is a CAR T prepared using a two-in-one carvirus plasmid in which three genes (ΔLNGFR: purification, CD19-CAR: CD19 antigen targeting, shPD-1 : PD-1 expression) are simultaneously expressed. FACS DATA Confirmed Purity after Purification of Cells with LNGFR Specific Antibodies
도 4는 도 3에서 정제한 ΔLNGFR-CART19/shGFP와 ΔLNGFR-CART19/shPD - 1 T 세포를 CD3과 CD28 항체로 3일간 자극을 준 후 CAR-T 세포 내 PD-1 발현 감소를 확인한 FACS DATA4 is a FACS DATA confirming the decrease of PD-1 expression in CAR-T cells after stimulation of ΔLNGFR-CART19 / shGFP and ΔLNGFR-CART19 / shPD - 1 T cells purified from Figure 3 with CD3 and CD28 antibody for 3 days
도 5는 PD-L1 과발현 K562 세포주 (K562-CD19-PD-L1)의 PD-L1 발현을 확인한 FACS DATA5 shows FACS DATA confirming PD-L1 expression of PD-L1 overexpressing K562 cell line (K562-CD19-PD-L1).
도 6은 도 3에서 정제한 ΔLNGFR-CART19/shGFP와 ΔLNGFR-CART19/shPD - 1 T 세포에 반복적으로 CAR와 PD-1 수용체 매개성 자극을 가한 CAR-T 세포 제작 과정에 대한 설명도FIG. 6 is an explanatory diagram of a CAR-T cell fabrication process in which CAR and PD-1 receptor mediated stimulation was repeatedly applied to ΔLNGFR-CART19 / shGFP and ΔLNGFR-CART19 / shPD - 1 T cells purified in FIG.
도 7은 shPD -1에 의한 PD-1 발현 억제가 반복적인 자극 시에도 지속적으로 유지되고 있음을 확인한 FACS DATA7 is a FACS DATA verifying that is sustained even in PD-1 expression inhibition of repetitive stimulation by shPD -1
도 8은 반복적인 자극에 따른 CAR-T 세포의 세포독성능 감소가 PD-1 발현 억제에 의해 현저히 완화됨을 확인한 DATA.FIG. 8 shows that the decrease in cytotoxicity of CAR-T cells due to repetitive stimulation is significantly alleviated by inhibition of PD-1 expression.
도 9는 도 6에서 제조된 CAR T 세포의 분화에 PD-1 발현 억제가 미치는 영향을 CD8, CD4 아형 내 CD45RA 및 CCR7 표면 항원 발현으로 확인한 FACS DATAFigure 9 shows the effect of PD-1 expression inhibition on the differentiation of CAR T cells prepared in Figure 6 FACS DATA confirmed by CD45RA and CCR7 surface antigen expression in CD8, CD4 subtype
도 10은 도 6에서 제조된 CAR T 세포의 증식에 PD-1 발현 억제가 미치는 영향을 확인하기 위해, CFSE를 라벨링한 후 감마 조사 K562-CD19-PD-L1 세포주와 5일간 동시 배양 하여 CSFE 형광 희석 정도로 세포 증식을 확인한 FACS DATAFIG. 10 shows CSFE fluorescence by co-incubation with gamma-irradiated K562-CD19-PD-L1 cell line for 5 days after labeling CFSE to confirm the effect of PD-1 expression inhibition on the proliferation of CAR T cells prepared in FIG. 6. FACS DATA Confirming Cell Proliferation to Dilution Level
도 11은 인간 TIM-3 발현을 억제하는 shTIM - 3와 CD19 표적 CAR가 동시에 발현하는 CAR T 세포 내 TIM-3 발현 감소를 확인한 FACS DATAFIG. 11 shows FACS DATA confirming a decrease in TIM-3 expression in CAR T cells expressing both shTIM - 3 and CD19 target CAR which inhibit human TIM-3 expression.
도 12는 두 가지 shRNA(shPD -1과 shTIM -3), CD19 CAR, 그리고 LNGFR 유전자가 동시에 발현되는 two-in-one 카바이러스 플라스미드 구성에 관한 모식도12 is a schematic diagram of a two-in-one carvirus plasmid construct in which two shRNAs ( shPD- 1 and shTIM- 3 ), a CD19 CAR, and an LNGFR gene are simultaneously expressed.
도 13은 마우스 U6 프로모터 3` 말단에 다중 클로닝 자리를 삽입한 pLV-ΔLNGFR_P2A_CD19-CAR_shPD-1_MCS two-in-one 카바이러스 플라스미드를 제작하는 과정 Figure 13 is a process for preparing a pLV-ΔLNGFR_P2A_CD19-CAR_ shPD-1 _MCS two-in-one carvirus plasmid in which the multiple cloning site is inserted at the 3` end of the mouse U6 promoter
도 14는 도 13의 플라스미드에 hU6 - shPD - 1와 mU6 - shTIM - 3 카세트를 -><-(양방향)으로 삽입한 pLV-ΔLNGFR_P2A_CD19-CAR_hU6-shPD-1-><-mU6-shTIM-3 two-in-one 카바이러스 플라스미드를 제작하는 과정Figure 14 is a 13-Plasmid hU6-shPD-1 and mU6-shTIM - 3 cassette -><- (two-way), one pLV-ΔLNGFR_P2A_CD19-CAR_hU6-shPD- 1 insertion -><- mU6-shTIM- 3 two Process for constructing -in-one carvirus plasmid
도 15는 도 13의 플라스미드에 hU6 - shPD - 1와 mU6 - shTIM - 3 카세트를 <-->(양방향의 역)으로 삽입한 pLV-ΔLNGFR_P2A_CD19-CAR_hU6-shPD-1-><-mU6-shTIM-3 two-in-one 카바이러스 플라스미드를 제작하는 과정Figure 15 is a 13-Plasmid hU6 - shPD - 1 and mU6 - shTIM - 3 the cassette <-> pLV-ΔLNGFR_P2A_CD19-CAR_hU6 -shPD-1 inserted in (the two-way station) -><- mU6-shTIM- 3 Construction of a two-in-one carvirus plasmid
도 16은 도 14과 도 15의 two-in-one 카바이러스 플라스미드로 제작한 CAR-T 세포를 LNGFR 항체를 이용하여 정제한 후 순도를 확인한 FACS DATAFIG. 16 is a FACS DATA confirming purity after purifying CAR-T cells prepared with the two-in-one carvirus plasmids of FIGS. 14 and 15 using LNGFR antibodies.
도 17은 도 16에서 정제한 ΔLNGFR-CART19/hU6-shPD-1-> <-shTIM-3-mU6 T 세포와 ΔLNGFR-CART19/shPD-1-hU6<- ->U6-shTIM-3 T 세포를 CD3와 CD28 항체로 3일간 자극을 준 후 CAR-T 세포 내 PD-1과 TIM-3의 발현이 동시에 감소함을 확인한 FACS DATA17 shows ΔLNGFR-CART19 / hU6-shPD-1-> <-shTIM-3-mU6 T cells and ΔLNGFR-CART19 / shPD-1-hU6 <--> U6-shTIM-3 T cells purified in FIG. FACS DATA confirms simultaneous expression of PD-1 and TIM-3 in CAR-T cells after stimulation with CD3 and CD28 antibodies
이하에서 본 명세서에 첨부된 도면을 참조하여 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.
본 명세서에서 면역세포는 유전공학적으로 처리되거나 처리되지 않은 인체 또는 동물 유해 면역세포 모두를 의미할 수 있으며, 특별한 언급이 없으면 경우에 따라 키메라 항원 수용체 변형 티세포(CAR modified T-cell), T 세포 수용체 변형 티세포 (TCR modified T-cell) 또는 키메라 항원 수용체 변형 자연살해세포(CAR modified NK cell), T 세포 수용체 변형 자연살해세포 (TCR modified NK cell) 등 카-변형된 면역세포를 의미할 수 있다. 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자라면 특별한 언급이 없더라도 어떤 면역세포를 의미하는지 구분이 가능할 것이다. 상기 면역세포의 예로는 T세포, 자연살해세포, 세포독성 T 림프구 및 조절용 T 세포로 이루어진 군으로부터 선택된 1종 이상을 들 수 있으나 이에 한정되는 것은 아니다. As used herein, an immune cell may refer to both human or animal harmful immune cells that have not been genetically engineered or treated, and unless otherwise specified, chimeric antigen receptor modified T cells, T cells, or T cells. It may mean a car-modified immune cell such as a TCR modified T-cell or a chimeric antigen receptor modified NK cell or a T cell receptor modified NK cell. have. Those skilled in the art to which the present invention pertains will be able to distinguish which immune cells mean without special mention. Examples of the immune cells include, but are not limited to, one or more selected from the group consisting of T cells, natural killer cells, cytotoxic T lymphocytes, and regulatory T cells.
도 1은 본 발명의 면역관문 수용체의 발현을 저해하는 short hairpin RNA(shRNA)와 CAR의 발현 카세트를 동시에 포함하는 two-in-one 카바이러스 플라스미드의 일실시예의 구조도이다. 도 1에서 보는 바와 같이, 본 발명은 키메라 항원 수용체(CAR)를 코딩하는 염기서열 및 면역관문 수용체의 발현을 저해하는 숏헤어핀 리보핵산(RNA)을 코딩하는 염기서열을 포함한 핵산서열과 벡터를 제공하며, 또한 상기 벡터를 이용하여 CAR와 면역관문 수용체의 발현을 저해하는 shRNA를 동시에 발현하는 면역세포를 제공한다. 1 is a structural diagram of an embodiment of a two-in-one carvirus plasmid containing both a short hairpin RNA (shRNA) and an expression cassette of a CAR which inhibit the expression of the immune gate receptor of the present invention. As shown in FIG. 1, the present invention provides a nucleic acid sequence and a vector including a nucleotide sequence encoding a chimeric antigen receptor (CAR) and a nucleotide sequence encoding a short hairpin ribonucleic acid (RNA) that inhibits the expression of an immune gate receptor. The present invention also provides immune cells expressing shRNAs that inhibit expression of CAR and immune gate receptor.
본 발명에 있어서 상기 CAR 또는 단일클론 티세포 수용체(TCR)는 특별히 그 구조나 구성이 제한되는 것은 아니며, 일반적으로 세포외 도메인인 단일사슬단편항체(single chain variable fragment, scFv : 항원에 대한 특이성을 부여) 도메인(domain), 스페이서 도메인(spacer domain:scFv와 세포막간의 거리를 조절), 막통과 도메인(transmembrane domain) 및 세포내 신호전달 도메인(intracellular signaling domain, ISD)으로 구성될 수 있다. 또한, 바람직하게는 상기 CAR 수용체의 아미노산 서열이 CD19, CD22 및/또는 LNGFR_P2A_CD19-CAR에 해당하는 서열번호인 52 내지 54로 구성된 군으로부터 선택된 1종 이상의 아미노산 서열을 포함하는 것이 바람직하고, 또한 상기 아미노산 서열과 90% 이상 일치하는 것이 바람직하나, 이에 한정되는 것은 아니다. In the present invention, the CAR or monoclonal T-cell receptor (TCR) is not particularly limited in structure or structure, and is generally a single chain variable fragment (scFv) which is an extracellular domain. Grant) domain, spacer domain (controlling the distance between the scFv and the cell membrane), transmembrane domain (transmembrane domain) and intracellular signaling domain (intracellular signaling domain (ISD)). In addition, preferably, the amino acid sequence of the CAR receptor comprises at least one amino acid sequence selected from the group consisting of 52 to 54 SEQ ID NOs corresponding to CD19, CD22 and / or LNGFR_P2A_CD19-CAR. Preferably at least 90% identical to the sequence, but is not limited thereto.
본 발명에 있어서 상기 CAR의 세포외 도메인은 1종의 항원 또는 리간드에만 결합하는 것일 수도 있고, 2종 이상의 항원 또는 리간드에 결합하는 세포외 도메인일 수도 있다. 세포외 도메인은 표적으로 하는 항원을 인식하는 항체 또는 상기 항원과 상호작용하는 분자로부터 선택할 수 있다. 이 항원은 예를 들면, 바이러스항원, 세균(특히 감염성 세균)항원, 기생충항원, 특정한 병상에 관계된 표적세포 상의 세포표면 마커(예를 들면 종양항원)이나 면역 관련 세포의 표면분자를 포함한다. 본 발명에 있어 상기 항원의 예를 들면, 레트로바이러스과(retroviridae, 예를 들면, HIV-1 및 HIV-LP와 같은, 인간 면역부전 바이러스), 피코루나바이러스과(Picornaviridae, 예를 들면, 폴리오바이러스, A형 간염바이러스, 엔테로바이러스, 인간 콕사키바이러스, 라이노바이러스, 에코바이러스), 풍진바이러스, 코로나바이러스, 수포성 구내염 바이러스, 광견병 바이러스, 에볼라 바이러스, 파라인플루엔자 바이러스, 멈프스바이러스, 홍역바이러스, 호흡기합포체 바이러스, 인플루엔자 바이러스, B형 간염 바이러스, 파보바이러스, 아데노위르스과(Adenoviridae), 포진바이러스과[Herpesviridae, 예를 들면, 1형 및 2형의 단순포진 바이러스(HSV), 수두대상포진 바이러스, 시토메갈로바이러스(CMV), 헤르페스바이러스], 폭스바이러스과(Poxviridae, 예를 들면, 천연두 바이러스, 백시니아바이러스, 폭스바이러스), C형 간염 바이러스에 유래하는 항원일 수 있다. 본 발명의 다른 형태의 항원으로서, 포도상구균속(Staphylococci)의 균종, 연쇄구균속(Streptococcus)의 균종, 대장균(Escherichia coli)의 균종, 슈우도모나드속(Pseudomonas)의 균종 및 살모넬라속(Salmonella)의 균종에 유래하는 항원일 수 있다. 특히, 감염성 세균, 예를 들면, 헬리코박터피로리(Helicobacter pyloris), 레지오넬라 뉴모필리아(Legionella pneumophilia), 마이코박테리움속(Mycobacteriasps)의 균종(예를 들면, M.tuberculosis, M.avium, M.intracellulare, M.kansaii, M.gordonea), 황색 포도상구균(Staphylococcusaureus), 임균(Neisseria gonorrhoeae), 수막염균(Neisseria meningitidis), 리스테리아균(Listeriamonocytogenes), 화농연쇄구균(Streptococcus pyogenes), A군 연쇄구균, B군 연쇄구균(Streptococcusagalactiae), 폐렴연쇄 구균(Streptococcus pneumoniae), 파상풍균(Clostridiumtetani)에 유래하는 항원일 수도 있다. 또한, 본 발명의 항원으로써 5T4, 알파 5β1-인테그린, 707-AP, AFP, ART-4, B7H4, BAGE, β-카테닌/m, Bcr-abl, MN/CIX 항원, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27/m, CD30, CD33, CD52, CD56, CD80, CDK4/m, CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2/new, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT(또는 hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/melan-A, MART-2/Ski, MC1R, 미오신/m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, PAP, 프로테이나제-3, p190 마이너 bcr-abl, Pml/RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1 또는 RU2, SAGE, SART-1 또는 SART-3, 서바이빈, TEL/AML1, TGFβ, TPI/m, TRP-1, TRP-2, TRP-2/INT2, VEGF, WT1, NY-Eso-1 또는 NY-Eso-B 등의 종양항원일 수도 있다. 바람직하게는 상기 항원은 인간 종양항원(암세포)인 것이 바람직하다. 본 명세서에서 종양항원 또는 암세포는 특별히 한정되지는 않으며 키메라 항원 수용체 티-세포 또는 자연살해 세포인 면역세포가 작용하여 항암활성을 나타내는 것이면 된다. 본 발명에 있어서 상기 암세포는 CD19-양성 세포일 수 있다. 암세포는 CD19-양성 B 림프구일 수 있다. 암세포는 Her2-양성 세포일 수 있다. Her2-양성 세포는 Her2-양성 유방암세포일 수 있다. 표적 세포는 BCMA-양성 세포일 수 있다. 암세포는 BCMA-양성 다발성 골수종 세포일 수 있다. 암세포는 CS1-양성 세포일 수 있다. CS1-양성 세포는 다발성 골수종 세포일 수 있다. 암세포는 EGFRvIII-양성 세포일 수 있다. 암세포는 EGFRvIII-양성 교모세포종 세포일 수 있다. 암세포는 CD20-양성 세포일 수 있다. 암세포는 CD22-양성 세포일 수 있다. 본 발명에 있어서 상기 종양항원는 CD19-양성 세포일 수 있다. 암세포는 CD19-양성 B 림프구일 수 있다. 암세포는 Her2-양성 세포일 수 있다. Her2-양성 세포는 Her2-양성 유방암세포일 수 있다. 표적 세포는 BCMA-양성 세포일 수 있다. 암세포는 BCMA-양성 다발성 골수종 세포일 수 있다. 암세포는 CS1-양성 세포일 수 있다. CS1-양성 세포는 다발성 골수종 세포일 수 있다. 암세포는 EGFRvIII-양성 세포일 수 있다. 암세포는 EGFRvIII-양성 교모세포종 세포일 수 있다. 암세포는 CD20-양성 세포일 수 있다. 암세포는 CD22-양성 세포일 수 있다. In the present invention, the extracellular domain of the CAR may be bound to only one antigen or ligand, or may be an extracellular domain that binds two or more antigens or ligands. The extracellular domain can be selected from antibodies that recognize the targeting antigen or molecules interacting with the antigen. These antigens include, for example, viral antigens, bacterial (especially infectious bacterial) antigens, parasitic antigens, cell surface markers (e.g. tumor antigens) on target cells involved in a particular condition or surface molecules of immune related cells. Examples of such antigens in the present invention include, but are not limited to, retroviridae (eg, human immunodeficiency virus, such as HIV-1 and HIV-LP), Picornaviridae (eg, poliovirus, A). Hepatitis virus, enterovirus, human coxsackie virus, rhinovirus, echovirus), rubella virus, coronavirus, bullous stomatitis virus, rabies virus, ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory vesicle Virus, influenza virus, hepatitis B virus, parvovirus, Adenoviridae, herpesviridae, for example, herpes simplex virus (HSV), varicella zoster virus, cytomegalovirus (CMV), herpes virus], Poxviridae (eg smallpox virus, vaccinia) Oh, there may be a virus, poxvirus), an antigen derived from hepatitis C virus. As another type of antigen of the present invention, the species of Staphylococci, the species of Streptococcus, the species of Escherichia coli, the species of Pseudomonas and Salmonella It may be an antigen derived from the species of. In particular, infectious bacteria such as Helicobacter pyloris, Legionella pneumophilia, Mycobacteria sps (eg, M. tuberculosis, M.avium, M. intracellulare, M.kansaii, M.gordonea, Staphylococcusaureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes, A. It may also be an antigen derived from group B streptococcus galactiae, Streptococcus pneumoniae, and Clostridium tetani. In addition, 5T4, alpha 5β1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, β-catenin / m, Bcr-abl, MN / CIX antigen, CA125, CAMEL, CAP-1 as antigens of the present invention. , CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27 / m, CD30, CD33, CD52, CD56, CD80, CDK4 / m, CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN , EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2 / new, HLA-A * 0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT (or hTRT) , iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR / FUT, MAGE, MART-1 / melan-A, MART-2 / Ski, MC1R, myosin / m, MUC1, MUM-1 , MUM-2, MUM-3, NA88-A, PAP, Proteinase-3, p190 Minor bcr-abl, Pml / RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1 or RU2, SAGE, SART- 1 or SART-3, Survivin, TEL / AML1, TGFβ, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, VEGF, WT1, NY-Eso-1 or NY-Eso-B, etc. It may be a tumor antigen of. Preferably, the antigen is preferably a human tumor antigen (cancer cell). In the present specification, tumor antigens or cancer cells are not particularly limited, and may be ones that act as chimeric antigen receptor T-cells or immune cells that are natural killer cells to exhibit anticancer activity. In the present invention, the cancer cells may be CD19-positive cells. The cancer cell may be CD19-positive B lymphocytes. The cancer cell may be Her2-positive cell. Her2-positive cells can be Her2-positive breast cancer cells. The target cell may be a BCMA-positive cell. The cancer cell may be a BCMA-positive multiple myeloma cell. The cancer cell may be a CS1-positive cell. CS1-positive cells can be multiple myeloma cells. The cancer cell may be an EGFRvIII-positive cell. The cancer cell may be an EGFRvIII-positive glioblastoma cell. The cancer cell may be a CD20-positive cell. The cancer cell may be a CD22-positive cell. In the present invention, the tumor antigen may be CD19-positive cells. The cancer cell may be CD19-positive B lymphocytes. The cancer cell may be Her2-positive cell. Her2-positive cells can be Her2-positive breast cancer cells. The target cell may be a BCMA-positive cell. The cancer cell may be a BCMA-positive multiple myeloma cell. The cancer cell may be a CS1-positive cell. CS1-positive cells can be multiple myeloma cells. The cancer cell may be an EGFRvIII-positive cell. The cancer cell may be an EGFRvIII-positive glioblastoma cell. The cancer cell may be a CD20-positive cell. The cancer cell may be a CD22-positive cell.
전술한 바와 같이, 상기 CAR는 상기 CAR의 항원 결합 도메인을 세포내 도메인에 연결하는 막관통 도메인을 포함하도록 설계될 수 있다. 상기 막관통 도메인은 상기 CAR 중의 도메인 중 하나 이상과 자연적으로 결합된다. 일부 예에서, 상기 막관통 도메인은, 상기와 같은 도메인이 동일하거나 상이한 표면 막 단백질의 막관통 도메인에 결합하여 수용체 복합체의 다른 구성원들과의 상호작용을 최소화하는 것을 피하기 위해서 아미노산 치환에 의해 선택되거나 변형될 수 있다. 상기 CAR의 세포내 도메인 또는 달리 세포질 도메인은 상기 CAR이 발현되는 세포의 활성화를 담당한다. 따라서As described above, the CAR can be designed to include a transmembrane domain that connects the antigen binding domain of the CAR to an intracellular domain. The transmembrane domain naturally binds to one or more of the domains in the CAR. In some instances, the transmembrane domain is selected by amino acid substitutions to avoid such domains binding to the transmembrane domains of the same or different surface membrane proteins to minimize interaction with other members of the receptor complex, or It can be modified. The intracellular domain or otherwise cytoplasmic domain of the CAR is responsible for activation of the cell in which the CAR is expressed. therefore
"세포내 도메인"이란 용어는 상기 활성화 신호를 전달하기에 충분한 세포내 도메인의 임의의 부분을 포함함을 의미한다. 상기 CAR의 세포내 도메인은 신호 활성화 및/또는 전달에 책임이 있는 도메인을 포함한다. 상기 세포내 도메인은 신호 활성화를 단백질-단백질 상호작용, 생화학적 변화, 또는 상기 세포의 대사, 모양, 유전자 발현의 변경에 대한 다른 반응, 또는 키메릭 세포내 신호전달 분자의 활성화에 대한 다른 세The term "intracellular domain" is meant to include any portion of the intracellular domain sufficient to carry the activation signal. The intracellular domain of the CAR includes a domain responsible for signal activation and / or delivery. The intracellular domain may be used to alter signal activation for protein-protein interactions, biochemical changes, or other responses to alterations in the metabolism, shape, or gene expression of the cells, or other three factors for activation of chimeric intracellular signaling molecules.
포 반응을 통해 전송할 수 있다. 본 발명에 사용하기 위한 세포내 도메인의 예는 비제한적으로 상기 T 세포 수용체(TCR)의 세포질 부분, 및 항원 수용체 연동에 따른 신호 전달을 개시하기 위해 협력하여 작용하는 임의의 보조-자극 분자뿐만 아니라 이들 요소의 임의의 유도체 또는 변체 및 동일한 기능 능력을 갖는 임의의 합성 서열을 포함한다. 상기 CAR의 세포내 도메인은 CD3, CD27, CD28, ICOS, 4-1BB, PD-1, T 세포 수용체(TCR), 이들의 임의의 유도체 또는 변체, 동일한 기능 능력을 갖는 이들의 임의의 합성 서열, 및 이들의 임의의 조합으로부터의 적어도 하나의 신호전달 도메인과 같은 보조-자극 분자의 임의의 부분을 포함할 수 있다. 상기 CAR의 구조와 발현방법 및 그 제조방법 등은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자라면 모두 잘 알 수 있을 것이므로 본 명세서에서 더 이상의 상세한 설명은 하지 않기로 한다.Can be sent via Four Reaction. Examples of intracellular domains for use in the present invention include, but are not limited to, the cytoplasmic portion of the T cell receptor (TCR), and any co-stimulatory molecules that act in concert to initiate signal transduction following antigen receptor linkage. Any derivative or variant of these elements and any synthetic sequence having the same functional capacity. The intracellular domain of the CAR is comprised of CD3, CD27, CD28, ICOS, 4-1BB, PD-1, T cell receptor (TCR), any derivative or variant thereof, any synthetic sequence thereof having the same functional capacity, And any portion of a co-stimulatory molecule, such as at least one signaling domain from any combination thereof. The structure and expression method of the CAR and the method of manufacturing the same will be well known to those of ordinary skill in the art to which the present invention belongs will not be described further herein.
본 발명의 면역관문을 극복한 면역세포는 CAR 또는 단일클론 TCR을 발현함과 동시에 면역관문 수용체의 발현을 저해하는 shRNA를 발현하도록 설계되었다. 본 명세서에 있어서 '저해'라는 용어는 '저해되지' 않은 상태에 비해 해당 면역관문 수용체의 발현이 60%이하, 보다 바람직하게는 60%, 50%, 40%, 30%, 20%, 10%, 5% 또는 1% 이하, 수준으로 억제된 경우를 의미한다. 본 발명에 있어서 상기 shRNA의 역할은 면역관문 수용체의 유전자 mRNA에 상보적으로 결합함으로써 세포내의 RNA 간섭 (RNA interference) 작용기작을 통한 mRNA의 분해를 유도, 궁극적으로 해당 면역관문 수용체 단백질의 발현을 억제한다. 상기 shRNA는 면역관문 수용체들, 예를 들면 공지의 면역관문 수용체인 PD-1, CTLA-4, LAG-3 또는 TIM-3 등을 타깃하여 면역관문 수용체의 발현을 억제하도록 하였으며, 따라서 이러한 벡터를 이용해 엔지니어드된 카티세포는 CAR 수용체 또는 단일클론 TCR를 통하여 암세포를 타깃하는 능력을 가짐과 동시에, 암세포로부터 유도되는 면역관문 시그널에 덜 민감하게 반응함으로써 보다 향상된 항암효과를 보이게 된다. 이렇게 추가적인 항체 치료제를 사용하지 않고 면역관문 수용체의 발현을 억제함으로써 CAR-T 세포를 비롯한 면역세포의 기능이 저하되는 것을 방지할 수 있다. 또한, 본 발명에 따르면 면역관문 수용체의 차단 효과가 카티세포 등의 면역세포에만 국한되기 때문에 기존 관역관문 차단 항체치료제 사용시 수반되는 시스테믹한 부작용을 낮출 수 있고, 아울러 고가의 항체치료제를 요하지 않아 경제적인 이득이 크다 할 수 있다. 또한, 전술한 WO2016/069282호에는 CAR 발현 및 내인성 유전자 발형 억제를 위하여 1단계로 CAR 형질 도입후 내인성 유전자 억제를 위한 CRISPR의 도입시 세포 천공 과정을 거치는 등 매우 복잡한 과정을 거쳐야 해서 생산성 및 수율이 크게 저하된다. 반면, 본 발명은 CAR 발현 및 면역관문 수용체의 발현을 억제하는 형질을 하나의 벡터를 이용하여 면역세포에 한 번의 형질도입과정으로 동시발현이 가능하도록 하여 제조공정이 단순하고 생산성 및 수율이 높은 특징이 있다. 본 발명에 있어서, 상기 shRNA는 PD-1, CTLA-4, LAG-3 및 TIM-3로 구성된 군으로부터 선택된 1종 이상의 면역관문 수용체의 발현을 저해하도록 하는 것이 바람직하고, 상기 PD-1, CTLA-4, LAG-3 및 TIM-3로 구성된 군으로부터 선택된 2종 이상의 면역관문 수용체의 발현을 저해하는 것이 더욱 바람직하다. 특히, 최근 PD-1만을 저해했을 때는 TIM-3가 오히려 증가한다는 사실이 보고된 바 있다. 따라서, PD-1 면역관문 수용체의 저해를 목적으로 할 때, 적어도 PD-1 및 TIM-3 두 수용체를 동시에 저해하는 것이 바람직하다. 본 명세서에 첨부된 염기서열 목록 중 서열번호 2 내지 51은 상기 면역관문 수용체의 발현을 억제하도록 shRNA를 발현하는 염기서열로, 서열번호 2 내지 12의 PD-1 타깃 염기서열, 서열번호 13 내지 35의 TIM-3 타깃 염기서열, 서열번호 36 내지 42의 LAG-3 타깃 염기서열 및 서열번호 43 내지 51의 CTLA-4 타깃 염기서열이다. 상기 염기서열은 하기 표 2에도 정리되어 있다. 상기 shRNA가 타겟하는 면역관문 수용체, 특히 인간 면역관문 수용체의 기능, 구조 및 염기서열 등은 공지의 사실(예를 들면, 인간 PD-1, PDL-1 및 TIM-3의 염기서열은 각각 NCBI Reference Sequence: NM_005018.2, NCBI Reference Sequence: NM_014143.3 및 NCBI Reference Sequence: NM_032782.4)이고 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자라면 모두 잘 알 수 있을 것이므로 본 명세서에서 더 이상의 상세한 설명은 하지 않기로 한다.Immune cells that overcome the immune barrier of the present invention are designed to express a CAR or monoclonal TCR while simultaneously expressing shRNA that inhibits the expression of the immune barrier receptor. In the present specification, the term 'inhibition' is less than 60%, more preferably 60%, 50%, 40%, 30%, 20%, 10% of the expression of the corresponding immune gate receptor compared to the 'inhibited' state Mean 5% or 1% or less. In the present invention, the role of the shRNA is complementary to the gene mRNA of the immune gateway receptor induces the degradation of mRNA through the RNA interference mechanism in the cell, and ultimately inhibits the expression of the immune gateway receptor protein. do. The shRNA targets immune gate receptors, such as PD-1, CTLA-4, LAG-3, or TIM-3, which are known immune gate receptors, to suppress the expression of immune gate receptors. Cati cells engineered using have the ability to target cancer cells via CAR receptor or monoclonal TCRs, while at the same time exhibiting improved anti-cancer effects by making them less sensitive to immune gate signals derived from cancer cells. By suppressing the expression of the immune gateway receptor without using an additional antibody therapeutic agent, it is possible to prevent the function of immune cells, including CAR-T cells, from deteriorating. In addition, according to the present invention, since the blocking effect of the immune gateway receptor is limited to immune cells such as cati cells, it is possible to lower the cystemic side effects associated with the use of the existing gateway blocking antibody treatment, and also does not require expensive antibody treatment. It can be said that the benefit is large. In addition, WO2016 / 069282 described above has a very complex process such as cell perforation during the introduction of CRISPR for endogenous gene inhibition after introduction of CAR in one step for CAR expression and endogenous gene expression inhibition, resulting in high productivity and yield. It is greatly reduced. On the other hand, the present invention enables the simultaneous expression of the traits that inhibit CAR expression and the expression of the immune gateway receptor in one transduction process to immune cells using a single vector, so that the manufacturing process is simple and the productivity and yield are high. There is this. In the present invention, the shRNA is preferably to inhibit the expression of at least one immune gateway receptor selected from the group consisting of PD-1, CTLA-4, LAG-3 and TIM-3, the PD-1, CTLA More preferably, it inhibits the expression of two or more immune gateway receptors selected from the group consisting of -4, LAG-3 and TIM-3. In particular, it has recently been reported that the TIM-3 increases when only PD-1 is inhibited. Thus, for the purpose of inhibiting PD-1 immune gate receptors, it is desirable to simultaneously inhibit at least both PD-1 and TIM-3 receptors. SEQ ID NO: 2 to 51 in the base sequence list attached to the present specification is a nucleotide sequence expressing shRNA to suppress the expression of the immune gateway receptor, PD-1 target nucleotide sequence of SEQ ID NO: 2 to 12, SEQ ID NO: 13 to 35 TIM-3 target nucleotide sequence of the DNA, LAG-3 target nucleotide sequence of SEQ ID NO: 36-42, and CTLA-4 target nucleotide sequence of SEQ ID NO: 43-51. The base sequence is also summarized in Table 2 below. The function, structure and sequence of the immune gate receptor, in particular the human immune gate receptor targeted by the shRNA are known facts (e.g., the base sequences of human PD-1, PDL-1 and TIM-3 are each NCBI Reference). Sequence: NM_005018.2, NCBI Reference Sequence: NM_014143.3 and NCBI Reference Sequence: NM_032782.4), and all of ordinary skill in the art to which the present invention pertains will be well known, and thus will not be described in further detail herein. I will not.
본 발명은 상기 면역관문을 극복한 면역세포를 포함한 약제학적 조성물을 제공한다. 상기 약제학적 조성물은 하나 이상의 약제학적으로 허용가능한 염, 부형제 또는 비히클을 추가로 포함할 수 있다. 본 발명의 약제학적 조성물에서 사용하기 위한 약제학적으로 허용가능한 염, 부형제, 또는 비히클은 담체, 부형제, 희석제, 항산화제, 보존제, 착색제, 향미제 및 희석제, 유화제, 현탁제, 용매, 충전제, 벌크화제, 완충제, 전달 비히클, 등장제, 공용매, 습윤제, 복합화제, 완충제, 항균제 및 계면활성제를 포함한다. 중성 완충 식염수 또는 혈청 알부민과 혼합된 식염수는 예시적인 적절한 담체이다. 약제학적 조성물은 조성물은 항산화제, 예컨대 아스코브산; 저분자량 폴리펩타이드; 단백질, 예컨대 혈청 알부민, 젤라틴 또는 면역글로불린; 친수성 중합체, 예컨대 폴리비닐피롤리돈; 아미노산, 예컨대 글리신, 글루타민, 아스파라긴, 알기닌 또는 라이신; 단당류, 이당류, 및 글루코스, 만노스 또는 덱스트린을 비롯한 기타 탄수화물; 킬레이트제, 예컨대 EDTA; 당 알코올, 예컨대 만니톨 또는 솔비톨; 염-형성 반대이온, 예컨대 나트륨; 및/또는 비이온성 계면활성제, 예컨대 트윈(Tween), 플루로닉스(pluronics) 또는 폴리에틸렌글라이콜(PEG)을 포함할 수 있다. 또한, 예로서, 적합한 등장 증강제는 알칼리 금속 할로겐화물(바람직하게는 염화나트륨 또는 염화칼륨), 만니톨, 솔비톨 등을 포함한다. 적합한 보존제는 염화벤즈알코늄, 티메로살, 펜에틸 알코올, 메틸파라벤, 프로필파라벤, 클로르헥시딘, 솔브산 등을 포함한다. 과산화수소는 또한 보존제로서 사용될 수 있다. 적합한 공용매는 글리세린, 프로필렌 글리콜 및 PEG를 포함한다. 적합한 복합화제는 카페인, 폴리비닐피롤리돈, 베타-사이클로덱스트린 또는 하이드록시-프로필-베타-사이클로덱스트린을 포함한다. 적합한 계면활성제 또는 습윤제는 솔비탄 에스터, 폴리솔베이트, 예컨대 폴리솔베이트 80, 트로메타민, 레시틴, 콜레스테롤, 틸록사팔 등을 포함한다. 완충제는 전통적인 완충제, 예컹대 아세트산염, 붕산염, 시트르산염, 인산염, 중탄산염 또는 트리스-HCl일 수 있다. 아세트산염 완충제는 약 pH 4 내지 5.5일 수 있고, 트리스 완충제는 약 pH 7 내지 8.5일 수 있다. 추가적인 약제학적 작용제는 문헌[Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company, 1990]을 참고할 수 있다.The present invention provides a pharmaceutical composition comprising immune cells that overcome the immune barrier. The pharmaceutical composition may further comprise one or more pharmaceutically acceptable salts, excipients or vehicles. Pharmaceutically acceptable salts, excipients, or vehicles for use in the pharmaceutical compositions of this invention include carriers, excipients, diluents, antioxidants, preservatives, colorants, flavors and diluents, emulsifiers, suspensions, solvents, fillers, bulks Agents, buffers, delivery vehicles, isotonic agents, cosolvents, wetting agents, complexing agents, buffers, antibacterial agents, and surfactants. Neutral buffered saline or saline mixed with serum albumin is an exemplary suitable carrier. Pharmaceutical compositions may comprise an antioxidant such as ascorbic acid; Low molecular weight polypeptides; Proteins such as serum albumin, gelatin or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, arginine or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; Chelating agents such as EDTA; Sugar alcohols such as mannitol or sorbitol; Salt-forming counterions such as sodium; And / or nonionic surfactants such as Tween, pluronics or polyethylene glycol (PEG). Also by way of example, suitable isotonic enhancers include alkali metal halides (preferably sodium chloride or potassium chloride), mannitol, sorbitol, and the like. Suitable preservatives include benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid and the like. Hydrogen peroxide can also be used as a preservative. Suitable cosolvents include glycerin, propylene glycol and PEG. Suitable complexing agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxy-propyl-beta-cyclodextrin. Suitable surfactants or wetting agents include sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapal and the like. The buffer can be a traditional buffer, eg acetate, borate, citrate, phosphate, bicarbonate or tris-HCl. Acetate buffer may be about pH 4 to 5.5 and Tris buffer may be about pH 7 to 8.5. Additional pharmaceutical agents may be referred to Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company, 1990.
조성물은 액체 형태로 또는 동결건조 또는 냉동 건조된 형태일 수 있고, 하나 이상의 동결보호제, 부형제, 계면활성제, 고분자량 구조적 첨가제 및/또는 벌크화제를 포함할 수 있다. 일 실시형태에서, 수크로스, 락토스 또는 트레할로스와 같은 비환원당인 동결보호제가 포함된다. 동결보호제의 양은 일반적으로 재구성 시, 얻어진 제형이 고장성 또는 약간 고장성 제형이 또한 적합할 수 있다고 해도, 등장성일 수 있도록 포함된다. 추가로, 동결보호제의 양은 동결건조시 허용가능하지 않은 양의 단백질의 분해 및/또는 응집을 방지하기에 충분하여야 한다. 사전 동결건조된 제형에서 당(예를 들어, 수크로스, 락토스, 트레할로스)에 대한 예시적인 동결보호제는 약 10mM 내지 약 400mM이다. 다른 실시형태에서, 계면활성제는, 예를 들어, 비이온성 계면활성제 및 이온성 계면활성제, 예컨대 폴리솔베이트(예를 들어, 폴리솔베이트 20, 폴리솔베이트 80); 폴록사머(예를 들어, 폴록사머 188); 폴리(에틸렌 글리콜) 페닐에터(예를 들어, 트리톤); 도데실황산나트륨(SDS); 라우릴황산나트륨; 나트륨 옥틸 글리코사이드; 라우릴-, 미리스틸-, 리놀레일- 또는 스테아릴-설포베타인; 라우릴-, 미리스틸-, 리놀레일- 또는 스테아릴-사르코신; 리놀레일, 미리스틸- 또는 세틸-베타인; 라우로아미도프로필-, 코카미도프로필-, 리놀레아미도프로필-, 미리스타미도프로필-, 팔미도프로필-, 또는 아이소스테아르아미도프로필-베타인(예를 들어, 라우로아미도프로필); 미리스트아미도프로필-, 팔미도프로필-, 또는 아이소스테아르아미도프로필-다이메틸아민; 나트륨 메틸 코코일-, 또는 2나트륨 메틸 오페일-타우레이트; 및 모나쿠아트(MONAQUAT)(상표명)가 포함된다. 사전 동결건조된 제형에 존재할 수 있는 계면활성제의 예시적 양은 약 0.001 내지 0.5%이다. 고분자량 구조적 첨가제(예를 들어, 충전제, 결합제)는, 예를 들어, 아카시아, 알부민, 알긴산, 인산칼슘(2염기성), 셀룰로스, 카복시메틸셀룰로스, 카복시메틸셀룰로스 나트륨, 하이드록시에틸셀룰로스, 하이드록시프로필셀룰로스, 하이드록시프로필메틸셀룰로스, 미정질 셀룰로스, 덱스트란, 덱스트린, 덱스트레이트, 수크로스, 타일로스, 전호화 전분, 황산칼슘, 아밀로스, 글리신, 벤토나이트, 말토스, 솔비톨, 에틸셀룰로스, 인산수소이나트륨, 인산나트륨, 파이로아황산나트륨, 폴리비닐 알코올, 젤라틴, 글루코스, 구아검, 액체 글루코스, 압축성 당, 마그네슘 알루미늄 실리케이트, 말토덱스트린, 폴리에틸렌 옥사이드, 폴리메트아크릴레이트, 포비돈, 알긴산나트륨, 트래거캔스 미정질 셀룰로스, 전분 및 제인을 포함할 수 있다. 고분자량 구조적 첨가제의 예시적 농도는 0.1중량% 내지 10중량%이다. 다른 실시형태에서, 벌크화제(예를 들어, 만니톨, 글리신)이 포함될 수 있다.The composition may be in liquid form or in lyophilized or lyophilized form and may include one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and / or bulking agents. In one embodiment, cryoprotectants which are non-reducing sugars such as sucrose, lactose or trehalose are included. The amount of cryoprotectant is generally included so that upon reconstitution, the resulting formulation may be isotonic, although hypertonic or slightly hypertonic formulations may also be suitable. In addition, the amount of lyoprotectant should be sufficient to prevent degradation and / or aggregation of unacceptable amounts of protein upon lyophilization. Exemplary lyoprotectants for sugars (eg, sucrose, lactose, trehalose) in pre-lyophilized formulations are about 10 mM to about 400 mM. In other embodiments, the surfactant may be, for example, a nonionic surfactant and an ionic surfactant such as polysorbate (eg, polysorbate 20, polysorbate 80); Poloxamers (eg poloxamer 188); Poly (ethylene glycol) phenylether (eg triton); Sodium dodecyl sulfate (SDS); Sodium lauryl sulfate; Sodium octyl glycoside; Lauryl-, myristyl-, linoleyl- or stearyl-sulfobetaine; Lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; Linoleyl, myristyl- or cetyl-betaine; Lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristomidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (eg lauroamidopropyl); Myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; Sodium methyl cocoyl-, or disodium methyl opyl-taurate; And MONAQUAT ™. An exemplary amount of surfactant that may be present in the pre-lyophilized formulation is about 0.001 to 0.5%. High molecular weight structural additives (e.g. fillers, binders) are, for example, acacia, albumin, alginic acid, calcium phosphate (bibasic), cellulose, carboxymethylcellulose, carboxymethylcellulose sodium, hydroxyethylcellulose, hydroxy Propylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, dextran, dextrin, dexrate, sucrose, tylos, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose, dihydrogen phosphate Sodium, sodium phosphate, sodium pyrosulfite, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressible sugar, magnesium aluminum silicate, maltodextrin, polyethylene oxide, polymethacrylate, povidone, sodium alginate, tragacanth undecided Vaginal cellulose, starch and zein. Exemplary concentrations of high molecular weight structural additives are from 0.1% to 10% by weight. In other embodiments, bulking agents (eg, mannitol, glycine) may be included.
조성물은 비경구 투여에 적합한 형태일 수 있다. 예시적인 조성물은 당업자에게 이용가능한 임의의 경로, 예컨대 동맥내, 피하, 정맥내, 근육내, 복강내, 대뇌내(뇌실내), 뇌혈관내, 근육내, 안구내, 동맥내 또는 병변내 경로에 의해 동물 내로 주사 또는 주입에 적합하다. 비경구 제형은 전형적으로 선택적으로 약제학적으로 허용가능한 보존제를 함유하는 멸균, 무발열원, 등장성 수용액일 수 있다.The composition may be in a form suitable for parenteral administration. Exemplary compositions include any route available to those of skill in the art, such as the intraarterial, subcutaneous, intravenous, intramuscular, intraperitoneal, cerebral (intraventricular), cerebrovascular, intramuscular, intraocular, intraarterial or intralesional routes. It is suitable for injection or infusion into the animal. Parenteral formulations are typically sterile, pyrogen-free, isotonic aqueous solutions, optionally containing a pharmaceutically acceptable preservative.
비수성 용매의 예는 프로필렌 글리콜, 폴리에틸렌 글리콜, 식물성 오일, 예컨대 올리브 오일, 및 주사용 유기에스터, 예컨대 에틸 올레이트이다. 수성 담체는 물, 알코올/수용액, 식염수 및 완충 매질을 포함하는 에멀전 또는 현탁액을 포함한다. 비경구 비히클은 염화나트륨 용액, 링거 덱스트로스, 덱스트로스 및 염화나트륨, 젖산링거 또는 고정유를 포함한다. 정맥내 비히클은 유체 및 영양분 보충제, 전해질 보충제, 예컨대 링거 덱스트로스에 기반한 것 등을 포함한다. 또한, 예를 들어 항미생물제, 항산화제, 킬레이트제, 비활성 기체 등과 같은 보존제 및 다른 첨가제가 존재할 수 있다. 일반적으로, 문헌[Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980]을 참조한다. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include emulsions or suspensions comprising water, alcohol / aqueous solution, saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactic acid ringer or fixed oil. Intravenous vehicles include fluid and nutrient supplements, electrolyte supplements such as those based on Ringer's dextrose, and the like. There may also be preservatives and other additives such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases and the like. In general, see Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980.
본 발명의 명세서에 기재된 약제학적 조성물은 생성물의 국소 농도 및/또는 특정 국소환경에서 증가된 안정성 또는 반감기를 제공하는 방식으로 제어 또는 지속된 전달을 위해 제형화될 수 있다. 조성물은 폴리락트산, 폴리글리콜산 등과 같은 중합체 화합물의 미립자 제제와 함께 본 명세서에 개시된 면역세포 뿐만 아니라 활성제의 제어 또는 지속 방출을 제공하고, 이어서 데포 주사로서 전달될 수 있는 작용제, 예컨대 생분해성 기질, 주사용 마이크로스피어, 마이크로캡슐 입자, 마이크로캡슐, 생분해성 입자 비드, 리포좀 및 이식가능한 전달 장치의 제형을 포함할 수 있다. 이러한 지속- 또는 제어된-전달 수단을 제형화하기 위한 기법은 공지되어 있으며, 다양한 중합체가 약물의 지속적 방출 및 전달을 위해 개발되고 사용되었다. 이러한 중합체는 전형적으로는 생분해성 및 생체적합성이다. 온도 또는 pH 민감성 특성을 이용한 거울상체 중합체 또는 폴리펩타이드 세그먼트 및 하이드로겔의 복합체화에 의해 형성된 것을 포함하는 중합체 하이드로겔은 생활성 단백질 작용제(예를 들어, 상당히 긴 CDR3을 포함하는 항체)를 트래핑함 있어서 수반되는 약한 그리고 수성의 조건 때문에 약물 데포 효과를 제공하는데 바람직할 수 있다. 예를 들어, 제WO93/15722호에서 약제학적 조성물의 전달을 위한 제어 방출 다공성 중합체 마이크로입자의 설명을 참고한다. 이 목적을 위한 적합한 물질은 폴리락타이드(예를 들어, 미국 특허 제3,773,919호 참조), 폴리-(a-하이드록시카복실산)의 중합체, 예컨대 폴리-D-(-)-3-하이드록시뷰티르산 (EP 133,988A), L-글루탐산 및 감마 에틸-L-글루타메이트의 공중합체(Sidman et al., Biopolymers, 22: 547-556 (1983)), 폴리(2-하이드록시에틸-메트아크릴레이트)(Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981), 및 Langer, Chem. Tech., 12: 98-105 (1982)), 에틸렌 비닐 아세테이트, 또는 폴리-D(-)-3-하이드록시뷰티르산을 포함한다. 다른 생분해성 중합체는 폴리(락톤), 폴리(아세탈), 폴리(오쏘에스터) 및 폴리(오쏘카보네이트)를 포함한다. 지속 방출 조성물은 또한 당업계에 공지된 임의의 몇몇 방법에 의해 제조될 수 있는 리포좀을 포함할 수 있다(예를 들어, 문헌[Eppstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-92 (1985)] 참조). 담체 그 자체 또는 그의 분해 생성물은 표적 조직에서 비독성이어야 하며, 상태를 추가로 악화시키지 않아야 한다. 이는 표적 장애의 동물 모델에서 일상적 선별에 의해, 또는 이러한 모델이 이용가능하지 않다면 정상 동물에서 결정될 수 있다. 지속 방출을 위한 재조합 단백질의 마이크로캡슐화는 인간 성장 호르몬(rhGH), 인터페론-(rhIFN-), 인터류킨-2 및 MN rgp120에 의해 성공적으로 수행될 수 있다. 이들 단백질의 지속 방출 제형은 그의 생체적합성 및 넓은 범위의 생분해성 특성에 기인하여 폴리-락트-코글리콜산(PLGA) 중합체를 이용하여 개발되었다. PLGA, 락트산 및 글리콜산의 분해 생성물은 인간 신체 내에서 빠르게 클리어런스될 수 있다. 게다가, 이 중합체의 분해능력은 그의 분자량 및 조성에 따를 수 있다. The pharmaceutical compositions described herein can be formulated for controlled or sustained delivery in a manner that provides increased stability or half-life at local concentrations of the product and / or at a particular local environment. The composition provides controlled or sustained release of immune cells as well as active agents disclosed herein with particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, and the like, which may then be delivered as depot injections, such as biodegradable substrates, Formulations of injectable microspheres, microcapsule particles, microcapsules, biodegradable particle beads, liposomes and implantable delivery devices. Techniques for formulating such sustained- or controlled-delivery means are known and various polymers have been developed and used for the sustained release and delivery of drugs. Such polymers are typically biodegradable and biocompatible. Polymeric hydrogels, including those formed by complexation of hydrogels with enantiomeric or polypeptide segments using temperature or pH sensitive properties, trapping bioactive protein agents (eg, antibodies comprising significantly longer CDR3). It may be desirable to provide drug depot effects because of the mild and aqueous conditions involved. See, for example, the description of controlled release porous polymeric microparticles for the delivery of pharmaceutical compositions in WO 93/15722. Suitable materials for this purpose include polylactide (see, eg, US Pat. No. 3,773,919), polymers of poly- (a-hydroxycarboxylic acids), such as poly-D-(-)-3-hydroxybutyric acid (EP 133,988A), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers, 22: 547-556 (1983)), poly (2-hydroxyethyl-methacrylate) ( Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981), and Langer, Chem. Tech., 12: 98-105 (1982)), ethylene vinyl acetate, or poly-D ( -)-3-hydroxybutyric acid. Other biodegradable polymers include poly (lactone), poly (acetal), poly (orthoesters) and poly (orthocarbonates). Sustained release compositions may also include liposomes, which may be prepared by any of several methods known in the art (see, eg, Eppstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-92 (1985). The carrier itself or its degradation product must be nontoxic in the target tissue and not further exacerbate the condition. This may be determined by routine screening in animal models of target disorders, or in normal animals if such models are not available. Microencapsulation of recombinant proteins for sustained release can be successfully performed by human growth hormone (rhGH), interferon- (rhIFN-), interleukin-2 and MN rgp120. Sustained release formulations of these proteins have been developed using poly-lactic-coglycolic acid (PLGA) polymers due to their biocompatibility and a wide range of biodegradable properties. The degradation products of PLGA, lactic acid and glycolic acid can be quickly cleared in the human body. In addition, the degradability of this polymer may depend on its molecular weight and composition.
생접착성 중합체는 또한 본 개시내용에서 또는 본 개시내용의 조성물과 함께 사용을 위해 고려된다. 생접착제는 장기간의 시간 기간 동안 생물학적 기질에 접착될 수 있는 합성 및 천연 유래 물질이다. 예를 들어, 카보폴 및 폴리카보필은 둘 다 폴리(아크릴산)의 합성 가교 유도체이다. 천연 유래 물질에 기반한 생접착성 전달시스템은, 예를 들어 하이알루로난으로서도 알려진 하이알루론산을 포함한다. 하이알루론산은 D-글루쿠론산 및 N-아세틸-D-글루코사민의 잔기로 이루어진 천연 유래 뮤코다당류이다. 하이알루론산은 결합조직에서뿐만 아니라 활액 내 그리고 눈의 유리체 및 안방수 내를 포함하는, 척추동물의 세포밖 조직 기질에서 발견된다. 하이알루론산의 에스테르화된 유도체는 생체적합성 및 생분해성인 전달에서 사용을 위해 마이크로스피어를 생성하기 위해 사용되었다(예를 들어, 문헌[Cortivo et al., Biomaterials (1991) 12:727-730; 유럽 특허 제517,565호; 제WO96/29998호; Illum et al., J. Controlled Rel. (1994) 29:133-141] 참조). Bioadhesive polymers are also contemplated for use in or with the compositions of the present disclosure. Bioadhesives are synthetic and naturally occurring materials that can adhere to biological substrates for extended periods of time. For example, Carbopol and Polycarbophil are both synthetic crosslinked derivatives of poly (acrylic acid). Bioadhesive delivery systems based on naturally occurring materials include, for example, hyaluronic acid, also known as hyaluronan. Hyaluronic acid is a naturally occurring mucopolysaccharide consisting of residues of D-glucuronic acid and N-acetyl-D-glucosamine. Hyaluronic acid is found in the extracellular tissue matrix of vertebrates, including in connective tissue as well as in the synovial fluid and in the vitreous and intraocular water of the eye. Esterified derivatives of hyaluronic acid have been used to generate microspheres for use in delivery that are biocompatible and biodegradable (see, eg, Cortivo et al., Biomaterials (1991) 12: 727-730; European Patent No. 517,565; WO96 / 29998; Illum et al., J. Controlled Rel. (1994) 29: 133-141).
생분해성과 비-생분해성 중합체 기질은 둘 다 본 발명의 조성물을 전달하기 위해 사용될 수 있고, 이러한 중합체 기질은 천연 또는 합성 중합체를 포함할 수 있다. 생분해성 기질이 바람직하다. 방출이 일어나는 시간 기간은 중합체의 선택에 기반한다. 전형적으로, 몇 시간 내지 3 내지 12개월의 범위에 있는 기간에 따른 방출이 가장 바람직하다. 생분해성 전달 시스템을 형성하기 위해 사용될 수 있는 예시적인 합성 중합체는 락트산 및 글리콜산의 중합체, 폴리아마이드, 폴리카보네이트, 폴리알킬렌, 폴리알킬렌 글리콜, 폴리알킬렌 옥사이드, 폴리알킬렌 테레프탈레이트, 폴리비닐 알코올, 폴리비닐 에터, 폴리비닐 에스터, 폴리-비닐 할로겐화물, 폴리비닐피롤리돈, 폴리글리콜라이드, 폴리실록산, 폴리무수물, 폴리우레탄 및 이들의 공중합체, 폴리(부트산), 폴리(발레르산), 알킬 셀룰로스, 하이드록시알킬 셀룰로스, 셀룰로스 에터, 셀룰로스 에스터, 나이트로 셀룰로스, 아크릴산 및 메트아크릴산 에스터의 중합체, 메틸 셀룰로스, 에틸 셀룰로스, 하이드록시프로필 셀룰로스, 하이드록시프로필 메틸 셀룰로스, 하이드록시뷰틸 메틸 셀룰로스, 셀룰로스 아세트산염, 셀룰로스 프로피온산염, 셀룰로스아세테이트 뷰티레이트, 셀룰로스 아세테이트 프탈레이트, 카복실에틸 셀룰로스, 셀룰로스 트라이아세테이트, 셀룰로스 설페이트 나트륨염, 폴리(메틸 메트아크리레이트), 폴리(에틸 메트아크릴레이트), 폴리(뷰틸메트아크릴레이트), 폴리(아이소뷰틸 메트아크릴레이트), 폴리(헥실메트아크릴레이트), 폴리(아이소데실 메트아크릴레이트), 폴리(라우릴 메트아크릴레이트), 폴리(페닐 메트아크릴레이트), 폴리(메틸 아크릴레이트), 폴리(아이소프로필 아크릴레이트), 폴리(아이소뷰틸 아크릴레이트), 폴리(옥타데실 아크릴레이트), 폴리에틸렌, 폴리프로필렌, 폴리(에틸렌 글리콜), 폴리(에틸렌 옥사이드), 폴리(에틸렌 테레프탈레이트), 폴리(비닐 알코올), 폴리비닐 아세테이트, 염화폴리 비닐, 폴리스타이렌 및 폴리비닐피롤리돈의 중합체를 포함한다. 예시적인 천연 중합체는 덱스트란 및 셀룰로스, 콜라겐, 화학물질, 이의 유도체(화학적 기의 치환, 첨가, 예를 들어, 알킬, 알킬렌, 하이드록실화, 산화 및 당업자에 의해 일상적으로 이루어지는 다른 변형), 알부민 및 다른 친수성 단백질, 제인 및 다른 프롤라민 및 소수성 단백질, 공중합체 및 이들의 혼합물을 포함한다. 일반적으로, 이들 물질은 효소적 가수분해 또는 생체내에서 물에 대한 노출에 의해, 표면 또는 벌크 부식에 의해 분해된다. 중합체는 선택적으로 수 중에서 그의 중량의 약 90%까지를 흡수하고, 추가로 선택적으로 다가 이온 또는 다른 중합체와 가교되는 하이드로겔의 형태일 수 있다(예를 들어, 제WO 04/009664호, 제WO 05/087201호, Sawhney, et al., Macromolecules, 1993, 26, 581-587 참조). 전달은 또한 콜레스테롤, 콜레스테롤 에스터 및 지방산 또는 중성 지방, 예컨대 모노-다이-및 트라이-글리세라이드; 하이드로겔 방출 시스템; 실라스틱 시스템; 펩타이드 기반 시스템; 왁스 코팅; 통상적인 결합제 및 부형제를 이용하는 압축 정제; 부분적으로 융합된 이식물 등과 같은 스테롤을 포함하는 지질인 비-중합체 시스템을 포함한다. 생성물을 함유하는 리포좀은, 예를 들어 선행기술문헌 13 내지 24와 같은 공지된 방법에 의해 제조될 수 있다. Both biodegradable and non-biodegradable polymer matrices can be used to deliver the compositions of the invention, and such polymer matrices can include natural or synthetic polymers. Biodegradable substrates are preferred. The time period over which release occurs is based on the choice of polymer. Typically, release over time periods ranging from several hours to three to twelve months is most preferred. Exemplary synthetic polymers that can be used to form biodegradable delivery systems include polymers of lactic acid and glycolic acid, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, poly Vinyl alcohol, polyvinyl ether, polyvinyl ester, poly-vinyl halide, polyvinylpyrrolidone, polyglycolide, polysiloxane, poly anhydride, polyurethane and copolymers thereof, poly (butyric acid), poly (valeric acid ), Alkyl cellulose, hydroxyalkyl cellulose, cellulose ethers, cellulose esters, polymers of nitro cellulose, acrylic acid and methacrylic acid esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose Cellulose acetate, cellulose propionate, cell Cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly ( Isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly ( Isopropyl acrylate), poly (isobutyl acrylate), poly (octadecyl acrylate), polyethylene, polypropylene, poly (ethylene glycol), poly (ethylene oxide), poly (ethylene terephthalate), poly (vinyl alcohol ), Including polymers of polyvinyl acetate, polyvinyl chloride, polystyrene and polyvinylpyrrolidone All. Exemplary natural polymers include dextran and cellulose, collagen, chemicals, derivatives thereof (substitution, addition of chemical groups such as alkyl, alkylene, hydroxylation, oxidation and other modifications routinely made by those skilled in the art), Albumin and other hydrophilic proteins, zein and other prolamin and hydrophobic proteins, copolymers, and mixtures thereof. Generally, these materials are degraded by surface or bulk corrosion, either by enzymatic hydrolysis or exposure to water in vivo. The polymer may optionally be in the form of a hydrogel that absorbs up to about 90% of its weight in water and optionally further crosslinks with polyvalent ions or other polymers (eg, WO 04/009664, WO 05/087201, Sawhney, et al., Macromolecules, 1993, 26, 581-587). Delivery also includes cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di- and tri-glycerides; Hydrogel release system; Silastic systems; Peptide based systems; Wax coatings; Compressed tablets using conventional binders and excipients; Non-polymeric systems that are lipids comprising sterols such as partially fused implants and the like. Liposomes containing the product can be produced, for example, by known methods such as prior art documents 13 to 24.
본 명세서에 개시된 약제학적 조성물은 흡입, 예를 들어 건조 분말로서 제형화될 수 있다. 흡입 용액은 또한 에어로졸 전달을 위한 액화 추진제로 제형화될 수 있다. 또 다른 제형에서, 용액은 네뷸라이징될 수 있다. 폐 투여를 위한 추가적인 약제학적 조성물은, 예를 들어, 화학적으로 변형된 단백질의 폐 전달을 개시하는 제WO 94/20069호에 기재된 것을 포함한다. 폐 전달을 위해, 입자 크기는 폐에 대한 전달에 적합하여야 한다. 예를 들어, 입자 크기는 1㎛ 내지 5㎛일 수 있지만; 그러나, 예를 들어, 각각의 입자가 상당히 다공성이라면, 더 큰 입자가 사용될 수 있다.The pharmaceutical compositions disclosed herein may be formulated for inhalation, eg as a dry powder. Inhalation solutions can also be formulated into liquefied propellants for aerosol delivery. In another formulation, the solution may be nebulized. Additional pharmaceutical compositions for pulmonary administration include, for example, those described in WO 94/20069 which initiates pulmonary delivery of chemically modified proteins. For lung delivery, the particle size must be suitable for delivery to the lungs. For example, the particle size may be between 1 μm and 5 μm; However, for example, if each particle is quite porous, larger particles can be used.
본 명세서에 개시된 특정 제형은 경구로 투여될 수 있다. 본 방식으로 투여되는 제형은 정제 및 캡슐과 같은 고체 제형의 조제에서 관습적으로 사용되는 담체와 함께 또는 담체 없이 제형화될 수 있다. 예를 들어, 캡슐은 생체이용가능성이 최대화되고, 사전-전신성 분해가 최소화될 때 위장관 내 지점에서 제형의 활성 부분을 방출하도록 설계될 수 있다. 추가적인 작용제는 선택적 결합제의 흡수를 용이하게 하기 위해 포함될 수 있다. 희석제, 향미제, 저융점 왁스, 식물성 오일, 윤활제, 현탁제, 정제 붕해제 및 결합제가 또한 사용될 수 있다. 다른 제제는 정제의 제조에 적합한 비 독성 부형제와의 혼합물로 본 명세서에 개시된 CAR-면역세포의 효과적인 양을 수반할 수 있다. 멸균수 또는 다른 적절한 비히클 중에서 정제를 용해시킴으로써, 용액은 단위 용량 형태로 제조될 수 있다. 적합한 부형제는 비활성 희석제, 예컨대 탄산칼슘, 탄산나트륨 또는 중탄산나트륨, 락토스 또는 인산칼슘; 또는 결합제, 예컨대 전분, 젤라틴 또는 아카시아; 또는 윤활제, 예컨대 스테아르산마그네슘, 스테아르산 또는 탈크를 포함하지만, 이들로 제한되지 않는다. Certain formulations disclosed herein can be administered orally. Formulations administered in this manner may be formulated with or without a carrier customarily used in the preparation of solid dosage forms such as tablets and capsules. For example, the capsule can be designed to release the active portion of the formulation at a point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to facilitate the absorption of the optional binder. Diluents, flavors, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders may also be used. Other formulations may involve an effective amount of the CAR-immune cells disclosed herein in admixture with non-toxic excipients suitable for the manufacture of tablets. By dissolving the tablets in sterile water or other suitable vehicle, the solution may be prepared in unit dose form. Suitable excipients include inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose or calcium phosphate; Or binders such as starch, gelatin or acacia; Or lubricants such as magnesium stearate, stearic acid or talc.
본 발명에 있어서 바람직한 약제학적 제형은 의도된 투여 경로, 전달 형식 및 목적으로 하는 투약량에 따라서 본 개시내용 및 제형화 기법의 일반적인 지식에 비추어 결정될 수 있다. 투여방식에도 불구하고, 효과적인 용량은 환자 체중, 표면적 또는 기관 크기에 따라서 계산될 수 있다. 본 명세서에 기재된 각각의 제형을 수반하는 치료를 위한 적절한 투약량을 결정하기 위한 계산의 추가적인 정제는 당업계에서 일상적으로 이루어지며, 당업계에서 일상적으로 수행되는 작업의 영역 내에 있다. 적절한 투약량은 적절한 용량-반응 데이터의 사용을 통해 확인될 수 있다.Preferred pharmaceutical formulations for the present invention can be determined in light of the general knowledge of the present disclosure and formulation techniques, depending on the intended route of administration, delivery format, and desired dosage. Despite the mode of administration, the effective dose can be calculated according to the patient weight, surface area or organ size. Further purification of the calculations to determine the appropriate dosage for treatment involving each formulation described herein is routinely made in the art and is within the scope of work routinely performed in the art. Appropriate dosages can be ascertained through the use of appropriate dose-response data.
이하에서 본 발명의 바람직한 태양인 실시예를 통해 본 발명을 더욱 상세히 설명한다. 다만, 하기 실시예는 본 발명의 예를 들기 위한 것일 뿐, 본 발명의 범위가 하기 실시예로 제한되는 것은 아니며 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자라면 실험 조건 등을 변형 또는 변경하더라도 그러한 변형 등이 본 발명의 범위에 속함을 알 수 있을 것이다. Hereinafter, the present invention will be described in more detail with reference to examples which are preferred embodiments of the present invention. However, the following examples are only intended to give examples of the present invention, and the scope of the present invention is not limited to the following examples, and those skilled in the art to which the present invention pertains may modify or change the experimental conditions. Even if such modifications and such will be within the scope of the present invention.
하기 표1 내지 5는 각각 본 발명의 실시예에 적용된 프라이머, 각 면역관문 수용체를 타겟으로 하여 그 발현을 억제하는 shRNA, CAR을 코딩하는 핵산염기, 상기 shRNA 카세트 및 CAR 및 shRNA의 동시발현을 위한 조합 플라스미드의 경우를 정리한 것이다. Tables 1 to 5 are primers applied to the embodiments of the present invention, respectively, to target the respective immune gateway receptors, its expression is shRNA, the nucleic acid base encoding the CAR, for the simultaneous expression of the shRNA cassette and CAR and shRNA The case of the combination plasmid is summarized.
프라이머SEQPrimer ID # ID # 염기서열(5` > 3`)Sequence (5`> 3`)
1One GTACCGTTAACGATCCGACGCCGCCATCTCT GTACCGTTAACGATCCGACGCCGCCATCTCT
22 TACTGGTTAACCAAAAAGGAACCCATTCCTGAAATTATTCTCTTGAAATAATTTCAGGAATGGGTTCCAAACAAGGCTTTTCTCCAAGG TACTGGTTAACCAAAAAGGAACCCATTCCTGAAATTATTCTCTTGAAATAATTTCAGGAATGGGTTCCAAACAAGGCTTTTCTCCAAGG
33 TACTGGTTAACCAAAAACCTTCCCTGTGGTTCTATTATTCTCTTGAAATAATAGAACCACAGGGAAGGCAAACAAGGCTTTTCTCCAAGG TACTGGTTAACCAAAAACCTTCCCTGTGGTTCTATTATTCTCTTGAAATAATAGAACCACAGGGAAGGCAAACAAGGCTTTTCTCCAAGG
44 TACTGGTTAACCAAAAACCTGTGGTTCTATTATATTATTCTCTTGAAATAATATAATAGAACCACAGGCAAACAAGGCTTTTCTCCAAGG TACTGGTTAACCAAAAACCTGTGGTTCTATTATATTATTCTCTTGAAATAATATAATAGAACCACAGGCAAACAAGGCTTTTCTCCAAGG
55 TACTGGTTAACCAAAAATCTCGGCATGGACGAGCTGTATCTCTTGAAATAATATAATAGAACCACAGGCAAACAAGGCTTTTCTCCAAGGTACTGGTTAACCAAAAATCTCGGCATGGACGAGCTGTATCTCTTGAAATAATATAATAGAACCACAGGCAAACAAGGCTTTTCTCCAAGG
66 CTTGGTTCATTCTCAAGCCTCCTTGGTTCATTCTCAAGCCTC
77 GTTGATTGTCGACTTAGCGAGGGGTTGATTGTCGACTTAGCGAGGG
88 GGGGATCCCCCCATCAGTCCGCAAAG GGGGATCCCCCCATCAGTCCGCAAAG
99 AAGTTAGTAGCTCCGCTTCCCCACCTCTTGAAGGCTATGTAGG AAGTTAGTAGCTCCGCTTCCCCACCTCTTGAAGGCTATGTAGG
1010 CCAATTTAAATATTTACTAATGTCCTGACTTGCCCTGCAACTGCCAATTTAAATATTTACTAATGTCCTGACTTGCCCTGCAACTG
1111 TCGGCCGGCCATGAGGATATTTGCTCGGCCGGCCATGAGGATATTTGC
1212 TCTTAATTAATTACGTCTCCTCCAAATGTGTATCACTTTGTCTTAATTAATTACGTCTCCTCCAAATGTGTATCACTTTG
1313 GGTATACTCTAGACATATGGCTAGCACTAGTCAAAAACCTGTGGTTCGGTATACTCTAGACATATGGCTAGCACTAGTCAAAAACCTGTGGTTC
1414 TTGTACCGTTAACGATCCGACGCCGC TTGTACCGTTAACGATCCGACGCCGC
1515 TGACTAGTCAAAAACCTGTGGTTCTATTATATTATTCTCTTGAAATAATATAATAGAACCACAGGCGGTGTTTCGTCCTTTCCACAAGATATATAAAGCCAA TGACTAGTCAAAAACCTGTGGTTCTATTATATTATTCTCTTGAAATAATATAATAGAACCACAGGCGGTGTTTCGTCCTTTCCACAAGATATATAAAGCCAA
1616 GTACCGTTAACAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCT GTACCGTTAACAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCT
1717 AGGACTAGTCAAAAAGGAATTCGCTCAGAAGAAATCTCTAGGACTAGTCAAAAAGGAATTCGCTCAGAAGAAATCTCT
1818 CTAGCTAGCGATCCGACGCCGCCATCT CTAGCTAGCGATCCGACGCCGCCATCT
1919 ATGTTAACCAAAAACCTGTGGTTCTATTATATTATTCTCTTG ATGTTAACCAAAAACCTGTGGTTCTATTATATTATTCTCTTG
2020 TCACTAGTAAGGTCGGGCAGGAAGAGGGCCTATTTCACTAGTAAGGTCGGGCAGGAAGAGGGCCTATT
2121 TAGGCCCTCACTAGTGATCCGACGCCGCCTAGGCCCTCACTAGTGATCCGACGCCGCC
2222 CTAGCTAGCCAAAAAGGAATTCGCTCAGAAGAAATCTCCTAGCTAGCCAAAAAGGAATTCGCTCAGAAGAAATCTC
2323 CGGTTAACCAAAAAGATGAAAGGGATGTGAATTATTCTCTTGAAATAATTCACATCCCTTTCATCAAACAAGGCTTTTCTCCAAGGGATATTTACGGTTAACCAAAAAGATGAAAGGGATGTGAATTATTCTCTTGAAATAATTCACATCCCTTTCATCAAACAAGGCTTTTCTCCAAGGGATATTTA
2424 CGGTTAACCAAAAAGGGAGCCTCCCTGATATAAATTCTCTTGAAATTTATATCAGGGAGGCTCCCAAACAAGGCTTTTCTCCAAGGGATATTTACGGTTAACCAAAAAGGGAGCCTCCCTGATATAAATTCTCTTGAAATTTATATCAGGGAGGCTCCCAAACAAGGCTTTTCTCCAAGGGATATTTA
2525 CGGTTAACCAAAAAGGAATTCGCTCAGAAGAAATCTCTTGAATTTCTTCTGAGCGAATTCCAAACAAGGCTTTTCTCCAAGGGATATTTACGGTTAACCAAAAAGGAATTCGCTCAGAAGAAATCTCTTGAATTTCTTCTGAGCGAATTCCAAACAAGGCTTTTCTCCAAGGGATATTTA
2626 CGGTTAACCAAAAAGGACCAAACTGAAGCTATATTTCTCTTGAAAATATAGCTTCAGTTTGGTCCAAACAAGGCTTTTCTCCAAGGGATATTTACGGTTAACCAAAAAGGACCAAACTGAAGCTATATTTCTCTTGAAAATATAGCTTCAGTTTGGTCCAAACAAGGCTTTTCTCCAAGGGATATTTA
2727 AAGAGGTGGGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGCGACGTGGAGGAGAACCCTGGACCTGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGAAGAGGTGGGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGCGACGTGGAGGAGAACCCTGGACCTGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCAGTCTGCATAGGT
타깃유전자Target gene shRNA SEQ ID #shRNA SEQ ID # 염기서열 (5`> 3`)Sequence (5`> 3`)
GFPGfp 1One TCTCGGCATGGACGAGCTGTATCTCGGCATGGACGAGCTGTA
hPD-1hPD-1 22 TGGAACCCATTCCTGAAATTA TGGAACCCATTCCTGAAATTA
33 GGAACCCATTCCTGAAATTAT GGAACCCATTCCTGAAATTAT
44 GAACCCATTCCTGAAATTATT GAACCCATTCCTGAAATTATT
55 ACCCATTCCTGAAATTATTTAACCCATTCCTGAAATTATTTA
66 CCCATTCCTGAAATTATTTAACCCATTCCTGAAATTATTTAA
77 CCTTCCCTGTGGTTCTATTATCCTTCCCTGTGGTTCTATTAT
88 CTTCCCTGTGGTTCTATTATA CTTCCCTGTGGTTCTATTATA
99 TTCCCTGTGGTTCTATTATAT TTCCCTGTGGTTCTATTATAT
1010 TCCCTGTGGTTCTATTATATTTCCCTGTGGTTCTATTATATT
1111 CCCTGTGGTTCTATTATATTACCCTGTGGTTCTATTATATTA
1212 CCTGTGGTTCTATTATATTATCCTGTGGTTCTATTATATTAT
hTIM-3hTIM-3 1313 GATGAAAGGGATGTGAATTATGATGAAAGGGATGTGAATTAT
1414 GGGAGCCTCCCTGATATAAAT GGGAGCCTCCCTGATATAAAT
1515 GGAATTCGCTCAGAAGAAA GGAATTCGCTCAGAAGAAA
1616 GGACCAAACTGAAGCTATATT GGACCAAACTGAAGCTATATT
1717 AGAACTTTGGTTTCCTTTAATAGAACTTTGGTTTCCTTTAAT
1818 ATGAAAGGGATGTGAATTATT ATGAAAGGGATGTGAATTATT
1919 TCTTATCTTCGGCGCTTTAAT TCTTATCTTCGGCGCTTTAAT
2020 CTTATCTTCGGCGCTTTAATTCTTATCTTCGGCGCTTTAATT
2121 TTATCTTCGGCGCTTTAATTTTTATCTTCGGCGCTTTAATTT
2222 GAGGAGCCCAATGAGTATTATGAGGAGCCCAATGAGTATTAT
2323 AGGAGCCCAATGAGTATTATTAGGAGCCCAATGAGTATTATT
2424 ATAGATCCAACCACCTTATTTATAGATCCAACCACCTTATTT
2525 ATGTCATTGCCTCTGTATTTAATGTCATTGCCTCTGTATTTA
2626 TGTCATTGCCTCTGTATTTAATGTCATTGCCTCTGTATTTAA
2727 ACCACCATGCCCAGCTAATTTACCACCATGCCCAGCTAATTT
2828 TGTTGAGATTTAGGCTTATTTTGTTGAGATTTAGGCTTATTT
2929 GACCAAACTGAAGCTATATTT GACCAAACTGAAGCTATATTT
3030 AGGCCTTCAGCAATCTATATTAGGCCTTCAGCAATCTATATT
3131 GGCCTTCAGCAATCTATATTAGGCCTTCAGCAATCTATATTA
3232 GAGTGGTCCCTAAACTTAAATGAGTGGTCCCTAAACTTAAAT
3333 AGTGGTCCCTAAACTTAAATTAGTGGTCCCTAAACTTAAATT
3434 GTGGTCCCTAAACTTAAATTTGTGGTCCCTAAACTTAAATTT
3535 CTAACACAAATATCCACATCTAACACAAATATCCACAT
hLAG-3hLAG-3 3636 TCAGCAGCCCAGTCCAAATAATCAGCAGCCCAGTCCAAATAA
3737 CAGCAGCCCAGTCCAAATAAACAGCAGCCCAGTCCAAATAAA
3838 TCAACGTCTCCATCATGTATATCAACGTCTCCATCATGTATA
3939 CAACGTCTCCATCATGTATAACAACGTCTCCATCATGTATAA
4040 CTGGAGACAATGGCGACTTTACTGGAGACAATGGCGACTTTA
4141 CTCAGCAGCCCAGTCCAAATACTCAGCAGCCCAGTCCAAATA
4242 AGCAGCCCAGTCCAAATAAACAGCAGCCCAGTCCAAATAAAC
hCTLA-4hCTLA-4 4343 GGGATCAAAGCTATCTATATAGGGATCAAAGCTATCTATATA
4444 GGATCAAAGCTATCTATATAAGGATCAAAGCTATCTATATAA
4545 GGCAACGGAACCCAGATTTATGGCAACGGAACCCAGATTTAT
4646 TGAAGAAGAGAGTCCATATTTTGAAGAAGAGAGTCCATATTT
4747 TTGGATGCGGAACCCAAATTATTGGATGCGGAACCCAAATTA
4848 AGCATCACTTGGGATTAATATAGCATCACTTGGGATTAATAT
4949 TGATGTGGGTCAAGGAATTAATGATGTGGGTCAAGGAATTAA
5050 AGCGAGGGAGAAGACTATATTAGCGAGGGAGAAGACTATATT
5151 TTTACGTATGAGACGTTTATATTTACGTATGAGACGTTTATA
CAR CAR SEQSEQ ID # ID # 서열order
#52 # 52 (CD22-CAR)(CD22-CAR) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEITTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEITTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
#53# 53 (CD19-CAR)(CD19-CAR) MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRMALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
#54# 54 (( LNGFRLNGFR __ P2AP2A _CD19-CAR)_CD19-CAR) MGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKRWGSGATNFSLLKQAGDVEENPGPALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRMGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKRWGSGATNFSLLKQAGDVEENPGPALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
shRNA 카세트SEQ ID shRNA cassetteSEQ ID 서열(5`-> 3`)Sequence (5`-> 3`)
shRNA 카세트염기서열shRNA cassette base sequence GTTAAC GATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGAGATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCT AACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAAAGCCTTGTTTGNNNNNNNNNNNNNNNNNNNNNTTCAAGAGA NNNNNNNNNNNNNNNNNNNNN TTTTTGGTTAAC GTTAAC GATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGAGATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCT AACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAAAGCCTTGTTTGNNNNNNNNNNNNNNNNNNNNN TTCAAGAGA NNNNNNNNNNNNNNNNNNNNN TTTTT GGTTAAC
#55 # 55 PD-1 shRNA-hU6 염기서열PD-1 shRNA-hU6 Sequence GTTAACAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCCTGTGGTTCTATTATATTATTTCAAGAGAATAATATAATAGAACCACAGGTTTTTGACTAGTGTTAACAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCCTGTGGTTCTATTATATTATTTCAAGAGAATAATATAATAGAACCACAGGTTTTTGACTAGT
#56# 56 PD-1 shRNA-hU6 -> <-PD-1 shRNA-hU6-> <- TIM-3 shRNA-mU6 염기서열TIM-3 shRNA-mU6 Sequence GTTAACAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCCTGTGGTTCTATTATATTATTTCAAGAGAATAATATAATAGAACCACAGGTTTTTGACTAGTCAAAAAGGAATTCGCTCAGAAGAAATCTCTTGAATTTCTTCTGAGCGAATTCCAAACAAGGCTTTTCTCCAAGGGATATTTATAGTCTCAAAACACACAATTACTTTACAGTTAGGGTGAGTTTCCTTTTGTGCTGTTTTTTAAAATAATAATTTAGTATTTGTATCTCTTATAGAAATCCAAGCCTATCATGTAAAATGTAGCTAGTATTAAAAAGAACAGATTATCTGTCTTTTATCGCACATTAAGCCTCTATAGTTACTAGGAAATATTATATGCAAATTAACCGGGGCAGGGGAGTAGCCGAGCTTCTCCCACAAGTCTGTGCGAGGGGGCCGGCGCGGGCCTAGAGATGGCGGCGTCGGATCGCTAGCGTTAACAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCCTGTGGTTCTATTATATTATTTCAAGAGAATAATATAATAGAACCACAGGTTTTTGACTAGTCAAAAAGGAATTCGCTCAGAAGAAATCTCTTGAATTTCTTCTGAGCGAATTCCAAACAAGGCTTTTCTCCAAGGGATATTTATAGTCTCAAAACACACAATTACTTTACAGTTAGGGTGAGTTTCCTTTTGTGCTGTTTTTTAAAATAATAATTTAGTATTTGTATCTCTTATAGAAATCCAAGCCTATCATGTAAAATGTAGCTAGTATTAAAAAGAACAGATTATCTGTCTTTTATCGCACATTAAGCCTCTATAGTTACTAGGAAATATTATATGCAAATTAACCGGGGCAGGGGAGTAGCCGAGCTTCTCCCACAAGTCTGTGCGAGGGGGCCGGCGCGGGCCTAGAGATGGCGGCGTCGGATCGCTAGC
#57# 57 PD-1 shRNA-hU6 <- ->PD-1 shRNA-hU6 <--> TIM-3 shRNA-mU6 염기서열TIM-3 shRNA-mU6 Sequence GTTAACCAAAAACCTGTGGTTCTATTATATTATTCTCTTGAAATAATATAATAGAACCACAGGCGGTGTTTCGTCCTTTCCACAAGATATATAAAGCCAAGAAATCGAAATACTTTCAAGTTACGGTAAGCATATGATAGTCCATTTTAAAACATAATTTTAAAACTGCAAACTACCCAAGAAATTATTACTTTCTACGTCACGTATTTTGTACTAATATCTTTGTGTTTACAGTCAAATTAATTCTAATTATCTCTCTAACAGCCTTGTATCGTATATGCAAATATGAAGGAATCATGGGAAATAGGCCCTCTTCCTGCCCGACCTTACTAGTGATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGAGATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAAAGCCTTGTTTGGAATTCGCTCAGAAGAAATTCAAGAGATTTCTTCTGAGCGAATTCCTTTTTGGCTAGCGTTAACCAAAAACCTGTGGTTCTATTATATTATTCTCTTGAAATAATATAATAGAACCACAGGCGGTGTTTCGTCCTTTCCACAAGATATATAAAGCCAAGAAATCGAAATACTTTCAAGTTACGGTAAGCATATGATAGTCCATTTTAAAACATAATTTTAAAACTGCAAACTACCCAAGAAATTATTACTTTCTACGTCACGTATTTTGTACTAATATCTTTGTGTTTACAGTCAAATTAATTCTAATTATCTCTCTAACAGCCTTGTATCGTATATGCAAATATGAAGGAATCATGGGAAATAGGCCCTCTTCCTGCCCGACCTTACTAGTGATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGAGATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAAAGCCTTGTTTGGAATTCGCTCAGAAGAAATTCAAGAGATTTCTTCTGAGCGAATTCCTTTTTGGCTAGC
플라스미드 ID #Plasmid ID # 플라스미드Plasmid
#1#One pLV-CD19-CAR_pLV-CD19-CAR_ shGFPshGFP
#2#2 pLV-CD19-CAR_pLV-CD19-CAR_ shPD-1shPD-1
#3# 3 pLV-ΔLNGFR_P2A_CD19-CAR_pLV-ΔLNGFR_P2A_CD19-CAR_ shGFPshGFP
#4#4 pLV-ΔLNGFR_P2A_CD19-CAR_pLV-ΔLNGFR_P2A_CD19-CAR_ shPD-1shPD-1
#5# 5 pSMOUW_IRES_PuropSMOUW_IRES_Puro
#6# 6 pSMOUW_hPD-L1_IRES_PuropSMOUW_hPD-L1_IRES_Puro
#7# 7 pLV-CD19-CAR_pLV-CD19-CAR_ shTIM-3 #13shTIM-3 # 13
#8#8 pLV-CD19-CAR_pLV-CD19-CAR_ shTIM-3 #14shTIM-3 # 14
#9# 9 pLV-CD19-CAR_pLV-CD19-CAR_ shTIM-3 #15shTIM-3 # 15
#10# 10 pLV-CD19-CAR_pLV-CD19-CAR_ shTIM-3 #16shTIM-3 # 16
#11# 11 pLV-ΔLNGFR_P2A_CD19-CAR_pLV-ΔLNGFR_P2A_CD19-CAR_ shPD-1shPD-1 _MCS_MCS
#12# 12 pLV-ΔLNGFR_P2A_CD19-CAR_hU6-pLV-ΔLNGFR_P2A_CD19-CAR_hU6- shPD-1shPD-1 _MCS_MCS
#13# 13 pLV-ΔLNGFR_P2A_CD19-CAR_hU6-pLV-ΔLNGFR_P2A_CD19-CAR_hU6- shPD-1shPD-1 -> <-mU6--> <-mU6- shTIM-3shTIM-3
#14# 14 pLV-ΔLNGFR_P2A_CD19-CAR_hU6-pLV-ΔLNGFR_P2A_CD19-CAR_hU6- shPD-1(reverse)shPD-1 (reverse) _MCS _MCS
#15# 15 pLV-ΔLNGFR_P2A_CD19-CAR_hU6-pLV-ΔLNGFR_P2A_CD19-CAR_hU6- shPD-1shPD-1 <- ->mU6-<--> mU6- shTIM-3shTIM-3
실시예 1. Example 1. shPD-1shPD-1 와 CD19-CAR를 동시에 발현하는 엔지니어드 T 세포의 제조.Preparation of Engineered T Cells Simultaneously Expressing and CD19-CAR.
2세대 lentiviral CAR 플라스미드에 기반하여 CD19-CAR 유전자가 EF1-alpha 프로모터에 의해 조절되도록 클로닝하였다. CD19 표적 단일가닥 가변부위 (FMC63 scFv)_CD8 hinge-세포막 도메인_4-1BB 신호 도메인-CD3z 신호 도메인으로 구성된 CAR SEQ ID #2와 렌티바이러스 플라스미드 pLV-GFP를 BamH1, Sal1 제한효소 처리 한 후 ligation하여 pLV-CD19-CAR 렌티바이러스 플라스미드 ID #1를 제작하였다. 이를 기반으로 하여 CD19-CAR와 shRNA의 발현이 각각 EF1-alpha 프로모터와 마우스 U6 프로모터에 의해 조절되는 도 1의 플라스미드를 제작하고자 하였다. 마우스 U6 프로모터 기반 shRNA 발현 플라스미드를 프라이머 SEQ ID #1, #4, #5로 PCR 증폭하여 PD-1 또는 GFP 타깃 shRNA를 포함하는 shRNA 카세트 PCR 산물을 얻었다. shRNA 카세트 염기서열 (표 4의 shRNA 카세트 염기서열 #1) 은 표4에 정리하였으며, 아래와 같이 구성되어 있다. NNNNNNNNNNNNNNNNNNNNN(21bp) 또는 NNNNNNNNNNNNNNNNNNNNN(19bp) 은 센스 shRNA 염기서열이며, NNNNNNNNNNNNNNNNNNNNN(21bp) 또는 NNNNNNNNNNNN NNNNNNN(19bp)는 안티센스 shRNA 염기서열이다. 센스와 안티센스 shRNA seq 중간에 TTCAAGAGA Linker를 삽입하여 U6 프로모터 3`에 위치시켰다. 그리고, 마우스 U6 프로모터에 의한 전사종결 시 필요한 TTTTT를 shRNA 3`에 위치시켰다. shGFP 또는 shPD -1 포함하는 shRNA 카세트 산물을 Hpa1 제한효소 처리 후 Hpa1 제한효소 와 CIP 처리한 pLV-CD19-CAR 플라스미드를 ligation하여 shRNA SEQ ID #1을 포함하는 pLV-CD19-CAR_shGFP: 플라스미드 ID #1와 shRNA SEQ ID #12를 포함하는 pLV-CD19-CAR_shPD -1 : 플라스미드 ID #2를 제작하였다. 플라스미드 #1, #2와 패키징 플라스미드 pMDL g/p, pRSVrev, 그리고 pMDG.1를 HEK293 T세포에 lipofectamine을 이용하여 transfection 하였고, 48시간 경과 후 렌티바이러스가 포함된 세포배양액을 얻었다. 인간 혈액으로부터 ficoll-paque 용액을 이용하여 말초혈액단색세포 (peripheral blood mononuclear cell, PBMC)를 분리하였고, 인간 CD3, CD28 타깃 항체를 이용하여 T-세포를 특이적으로 활성화하였다. T-세포의 초기 활성화 1-2일 경과 후 앞서 얻어진 바이러스를 이용해 transduction 하였다. 이후 만들어진 CAR-T세포는 5% 인간 혈장 그리고 인간 IL2를 포함하는 AIM-V 배양액을 이용하여 배양하였다. Transduction 후 7일 째, 인간 CD3, CD28 타깃 항체로 3일간 자극을 준 후, CAR, PD-1 타깃 항체를 활용하여 유세포분석을 하였다. 도 2는 인간 PD-1 발현을 억제하는 shPD -1과 CD19 표적 CAR가 동시에 발현되는 two-in-one 카바이러스 플라스미드를 이용해 제조한 CAR T 세포에서 CAR를 발현유지와 PD-1 발현 감소를 확인한 FACS DATA이다. 도 2에서 보여지는 바와 같이, shGFP를 발현하는 CD19-CAR T 세포 중 PD-1+ 세포가 25% 내외 일 때, shPD -1 발현하는 CD19-CAR T 세포 중 PD-1+ 세포는 4% 내외 였다. 이를 통해, T 세포에서 CAR 발현과 PD-1 발현 억제를 동시에 할 수 있는 CAR-T세포 제조 기술을 확립하였다. Based on the second generation lentiviral CAR plasmid, the CD19-CAR gene was cloned to be regulated by the EF1-alpha promoter. CD19 target single-stranded variable region (FMC63 scFv) _CD8 hinge-cell membrane domain_4-1BB signal domain-CD3z signal domain CAR SEQ ID # 2 and lentiviral plasmid pLV-GFP treated with BamH1, Sal1 restriction enzyme and ligation pLV-CD19-CAR lentiviral plasmid ID # 1 was constructed. Based on this, the plasmid of FIG. 1 in which the expression of CD19-CAR and shRNA is regulated by the EF1-alpha promoter and the mouse U6 promoter, respectively, was prepared. Mouse U6 promoter based shRNA expression plasmids were PCR amplified with primers SEQ ID # 1, # 4, # 5 to obtain shRNA cassette PCR products containing PD-1 or GFP target shRNAs. The shRNA cassette sequences (shRNA cassette sequences # 1 in Table 4) are summarized in Table 4 and are configured as follows. NNNNNNNNNNNNNNNNNNNNN (21bp) or NNNNNNNNNNNNNNNNNNNNN (19bp) is the sense shRNA sequence, NNNNNNNNNNNNNNNNNNNNN (21bp) or NNNNNNNNNNNN NNNNNNN ( 19bp ) is an antisense shRNA sequence. A TTCAAGAGA Linker was inserted between the sense and antisense shRNA seqs and placed in the U6 promoter 3 ′. And, the TTTTT required for termination of transcription by the mouse U6 promoter was placed in shRNA 3 ′. pLV -CD19-CAR_ shGFP containing shRNA SEQ ID # 1 by ligation of the shRNA cassette product containing shGFP or shPD- 1 to Hpa1 restriction enzyme followed by HIP1 restriction enzyme and CIP-treated pLV-CD19-CAR plasmid: plasmid ID # containing 1 and shRNA SEQ ID # 12 pLV-CD19 -CAR_ shPD -1: to prepare a plasmid ID # 2. Plasmids # 1, # 2 and packaging plasmids pMDL g / p, pRSVrev, and pMDG.1 were transfected into HEK293 T cells using lipofectamine, and cell cultures containing lentiviral were obtained after 48 hours. Peripheral blood mononuclear cells (PBMC) were isolated from human blood using ficoll-paque solution, and T-cells were specifically activated using human CD3 and CD28 target antibodies. After 1-2 days of initial activation of T-cells, transduction was performed using the virus obtained earlier. The resulting CAR-T cells were cultured using AIM-V medium containing 5% human plasma and human IL2. Seven days after transduction, after stimulation with human CD3 and CD28 target antibodies for 3 days, flow cytometry was performed using CAR and PD-1 target antibodies. Figure 2 confirms the maintenance and expression of CAR and reduced PD-1 expression in CAR T cells prepared using a two-in-one carvirus plasmid expressing shPD- 1 and CD19 target CAR at the same time inhibiting human PD-1 expression FACS DATA. As also shown in Figure 2, when the CD19-CAR T PD-1 + cells in cells expressing shGFP around 25%, shPD -1 expressing CD19-CAR T PD-1 + cells in cells which is 4% or less It was. Through this, CAR-T cell production technology was established that can simultaneously inhibit CAR expression and PD-1 expression in T cells.
실시예Example 2. (( 2. (( 1)One) shPDshPD -1-One 발현 카세트, （２）LNGFR (Low-affinity nerve growth factor receptor), 그리고 　(3) CD19-CAR를 동시에 발현하는  Simultaneously expresses an expression cassette, (2) Low-affinity nerve growth factor receptor (LNGFR), and (3) CD19-CAR. 엔지니어드Engineered T 세포 제조) T cell manufacturing)
PBMC에 Transduction한 후 순수한 CAR-T 세포만 분리하고자, T 세포에 존재하지 않는 세포 표면 수용체 중, CAR 및 TCR 신호 활성화를 유도하지 않으면서 해당 수용체의 신호전달이 차단된 엔지니어드 세포 표면 수용체인 ΔLNGFR (cytoplasmic domain truncated LNGFR)이 발현하는 pLV-CD19-CAR_shGFP, pLV-CD19-CAR_shPD-1 플라스미드를 제작하고자 하였다. pMACS-ΔLNGFR 플라스미드 (miltenyibiotec, Germany)를 프라이머 SEQ ID #8, #9를 활용해 PCR 산물을 얻었다. P2A를 포함하는 프라이머 SEQ ID #27, ΔLNGFR PCR product, 그리고 프라이머 SEQ ID #8, #10을 이용해 Overlap PCR을 하였다. Overlap PCR 산물을 BamH1, Swa1 제한 효소 처리 후 BamH1, Swa1 제한 효소 처리된 플라스미드 ID #1, #2와 ligation 해서 CAR SEQ ID #3이 포함된 pLV-ΔLNGFR_P2A_CD19-CAR_shGFP 플라스미드 ID #3과 pLV-ΔLNGFR_P2A_CD19-CAR_shPD -1 플라스미드 #4를 제작하였다. 플라스미드 ID #3, #4를 이용해 렌티바이러스를 만든 후 PBMC에 Transduction해 CAR-T 세포를 제조하였다. Transduction 후 6일 째, 순수한 CAR-T 세포를 획득하고자 MACSelect LNGFR System (miltenyibiotec, Germany)을 사용하였고, LNGFR 타깃 항체를 이용해 유세포분석을 통해 CAR-T 세포의 순도를 확인하였다. 도 3에서 보여지는 바와 같이, 90% 내외의 LNGFR+인 CD19-CAR_shGFP 또는 CD19-CAR_shPD - 1 T 세포를 얻을 수 있었다. 또한, 인간 CD3, CD28 타깃 항체를 이용하여 T-세포를 3일간 자극을 준 후 분리한 CAR-T 세포의 PD-1 발현을 확인해 보았다. 도 4에서 보여지는 바와 같이, ΔLNGFR-P2A-CD19-CAR_shGFP T 세포 중 PD-1+ T 세포는 25~27% 내외였지만, ΔLNGFR-P2A-CD19-CAR_shPD - 1 T 세포 중 PD-1+ T 세포는 2% 내외임을 알 수 있었다.To isolate only pure CAR-T cells after transduction to PBMC, ΔLNGFR (engineered cell surface receptor, a cell surface receptor that does not exist in T cells, blocked signaling of the receptor without inducing CAR and TCR signal activation) pLV-CD19- CAR_shGFP and pLV-CD19- CAR_shPD-1 plasmids expressing cytoplasmic domain truncated LNGFR) were prepared. pMACS-ΔLNGFR plasmid (miltenyibiotec, Germany) was used as primers SEQ ID # 8, # 9 to obtain a PCR product. Overlap PCR was performed using primers SEQ ID # 27, ΔLNGFR PCR product containing P2A, and primers SEQ ID # 8, # 10. The overlap PCR product was ligation with BamH1, Swa1 restriction enzyme-treated plasmid ID # 1, # 2 after treatment with BamH1, Swa1 restriction enzyme and pLV-ΔLNGFR_P2A_CD19-CAR_ shGFP containing CAR SEQ ID # 3 The plasmid ID # 3 and pLV-ΔLNGFR_P2A_CD19-CAR_ shPD -1 plasmid # 4 was produced. Lentiviruses were prepared using plasmid ID # 3 and # 4, followed by transduction into PBMC to prepare CAR-T cells. Six days after transduction, MACSelect LNGFR System (miltenyibiotec, Germany) was used to obtain pure CAR-T cells, and the purity of CAR-T cells was confirmed by flow cytometry using LNGFR target antibody. As shown in FIG. 3, CD19- CAR_shGFP or CD19- CAR_shPD - 1 T cells of about 90% LNGFR + could be obtained. In addition, PD-1 expression of CAR-T cells isolated after stimulating T-cells for 3 days using human CD3 and CD28 target antibodies was confirmed. As shown in Figure 4, ΔLNGFR-P2A-CD19- CAR_ shGFP T cells of PD-1 + T cells, but around 25 ~ 27%, ΔLNGFR-P2A -CD19-CAR_ shPD - 1 T cells, PD-1 of the + T cells were found to be around 2%.
실시예Example 3. (CD19, PD-L1을 동시에 발현하는 인간 만성 골수성 백혈병 세포주 K562-CD19-PD-L1 확립) 3. (Established human chronic myeloid leukemia cell line K562-CD19-PD-L1 expressing CD19, PD-L1 at the same time)
인간 PD-L1 발현 렌티바이러스 벡터를 구축하여 PD-L1 과발현 K562-CD19을 확립하고자 하였다. 인간 PD-L1 염기서열을 합성한 후 프라이머 #11, #12를 활용해 PCR 산물을 얻었다. PCR 산물을 Fse1, Pac1 제한효소 처리 후 Fse1, Pac1 제한효소 처리된 렌티바이러스 플라스미드 ID #5 pSMOUW_IRES_Puro와 ligation 해서 PD-L1 발현 렌티바이러스 플라스미드 ID #6을 구축하였다. 렌티바이러스 플라스미드 #5, #6과 패키징 플라스미드 pMDL g/p, pRSVrev, 그리고 pMDG.1를 HEK293 T세포에 lipofectamine을 이용하여 transfection 하였고, 48시간 경과 후 세포배양액으로부터 렌티바이러스를 얻었다. 해당 바이러스를 K562-CD19 세포주에 Transduction 하여 K562-CD19-PD-L1 세포주를 확립하였다. K562-CD19-PD-L1 세포주의 CD19, PD-L1 타깃 항체를 이용해 유세포분석을 하였다. 도 5에서 보여지는 바와 같이, K562-CD19 세포주에서 PD-L1은 발현하지 않는 반면, K562-CD19-PD-L1 세포주에서는 거의 모든세포가 PD-L1을 발현하였다. 또한, CD19 발현은 K562-CD19-PD-L1 세포주와 K562-CD19 세포주 간 차이가 없음을 확인하였다. 위 실험을 바탕으로, PD-L1과 CD19이 동시에 발현하는 K562 세포주를 확립하게 되었다.Human PD-L1 expressing lentiviral vectors were constructed to establish PD-L1 overexpression K562-CD19. After synthesizing the human PD-L1 sequence, PCR products were obtained using primers # 11 and # 12. The PCR product was ligation with Fse1, Pac1 restriction enzyme-treated lentiviral plasmid ID # 5 pSMOUW_IRES_Puro after Fse1, Pac1 restriction enzyme treatment to construct PD-L1 expressing lentiviral plasmid ID # 6. Lentivirus plasmids # 5, # 6 and packaging plasmids pMDL g / p, pRSVrev, and pMDG.1 were transfected into HEK293 T cells with lipofectamine, and lentiviral was obtained from the cell culture after 48 hours. The virus was transduced into the K562-CD19 cell line to establish the K562-CD19-PD-L1 cell line. Flow cytometry was performed using CD19 and PD-L1 target antibodies of the K562-CD19-PD-L1 cell line. As shown in FIG. 5, PD-L1 was not expressed in the K562-CD19 cell line, while almost all cells expressed PD-L1 in the K562-CD19-PD-L1 cell line. In addition, CD19 expression was confirmed that there is no difference between the K562-CD19-PD-L1 cell line and K562-CD19 cell line. Based on the above experiments, we established a K562 cell line expressing PD-L1 and CD19 simultaneously.
실시예Example 4. (반복적인 항원 자극에 의한 CAR-T 세포 탈진(Exhaustion)시 PD-1 발현 감소가 CD19-CAR T 세포독성능에 미치는 영향 확인) 4. (Identify the effect of decreased PD-1 expression on CD19-CAR T cytotoxic ability upon repeated antigen stimulation of CAR-T cell exhaustion)
ΔLNGFR을 활용해 분리한 ΔLNGFR-P2A-CD19-CAR_shGFP T 세포와 ΔLNGFR-P2A-CD19-CAR_shPD-1 T세포에 지속적으로 항원 특이적 자극을 가해 세포 탈진을 유도한 후 PD-1 발현 감소가 CD19-CAR T 세포 기능에 미치는 영향을 확인해보고자 하였다. CAR-T 세포 2x106개를 감마 조사한 K562-CD19-PD-L1 세포주 2x106개와 공동 배양 하였다. 3일 후 MACSelect LNGFR System (miltenyibiotec, Germany)로 CAR-T 세포만 분리 한 후 4일간 5% 인간 혈장 그리고 인간 IL2를 포함하는 AIM-V 배양액에 넣어 배양하였다. 이를 3회 반복함으로서 항원 특이적 자극을 1, 2, 3회 받은 CD19 CAR_shGFP 와 CD19_shPD - 1 T 세포를 획득하였다 (도 8). 항원 특이적 자극을 가함에도 shPD -1에 의한 PD-1 발현 억제가 유지되고 있는지 확인하였다. 도 9에 보여지는 바와 같이, CD19-CAR_shPD - 1 T 세포는 CD19-CAR_shGFP T 세포 대비 낮은 수준의 PD-1 발현을 유지하는 것으로 보아 CD19-CAR_shPD - 1 T 세포에서 PD-1의 발현 감소가 shPD -1에 의해 지속적으로 이루어지고 있음을 알 수 있었다. 그 다음으로, 항원 특이적 자극을 받은 횟수에 따라 PD-1 발현이 CAR-T 세포독성능에 미치는 영향을 확인해 보았다. CellVue® Claret Far Red (sigma) 로 라벨링한 2x105 CAR-T 세포를 K562-CD19-PD-L1 세포주 2x105 개와 공동 배양하였다. 3일 후 7-AAD를 염색하여 CAR-T 세포의 세포독성능을 확인해 보았다. 세포독성능 %는 100 (100 x ( 1 - (7-AAD-ClaretRed+% / ClaretRed+ %))로 계산하였으며, 상대적 세포독성능 (Relative cytotoxic activity)은 [각기 다른 횟수로 자극을 받은 CAR-T 세포 세포독성능 % / 자극을 받지 않은 CAR-T　세포독성능 %]으로 계산하였다. 도 8에 보여지는 바와 같이, 자극을 받은 횟수가 증가할수록 CAR-T 세포의 세포독성능이 감소하였다. 이러한 상황에서, PD-1의 발현이 억제된 CD19-CAR_shPD -1 T세포 (자극 받기전 CD19-CAR_shPD - 1 T 세포의 세포독성능 대비 17.7% 감소)는 CD19-CAR_shGFP T 세포 (자극 받기 전 CD19-CAR_shGFP T 세포의 세포독성능 대비 61.3% 감소)보다 세포독성능의 감소폭이 현저히 낮음을 확인하였다.After continuously applying the antigen-specific stimulation induces cell exhaustion in the year of ΔLNGFR-P2A-CD19-CAR_ shGFP T cells with ΔLNGFR-P2A-CD19-CAR_ shPD -1 T separated utilizing cell ΔLNGFR PD-1 expression decreases the The purpose of this study was to determine the effect on CD19-CAR T cell function. 2x10 6 CAR-T cells were co-cultured with 2x10 6 K562-CD19-PD-L1 cell lines gamma irradiated. After 3 days, only CAR-T cells were isolated by MACSelect LNGFR System (miltenyibiotec, Germany), and then cultured in AIM-V medium containing 5% human plasma and human IL2 for 4 days. By repeating this three times, CD19 CAR_sh GFP which received 1, 2 or 3 antigen-specific stimulation And CD19_ shPD - 1 to obtain the T cells (Fig. 8). In imposing antigen-specific stimulation it was confirmed that the PD-1 expression inhibition by shPD -1 being maintained. As shown in Figure 9, CD19-CAR_ shPD - 1 T cells, CD19-CAR_ shGFP T cells than it is seen that maintain a low level of the PD-1 expression of CD19-CAR_ shPD - 1 PD-1 expression in T cells It can be seen that the decrease is continuously made by shPD- 1 . Next, we examined the effect of PD-1 expression on CAR-T cytotoxic ability according to the number of antigen-specific stimulation. CellVue® Claret Far Red the CAR 2x10 5 T-cells were labeled with (sigma) were co-cultured five and K562-CD19-PD-L1 cell line 2x10. After 3 days, 7-AAD stained to determine the cytotoxic capacity of CAR-T cells. Cytotoxicity% was calculated as 100 (100 x (1-(7-AAD-ClaretRed +% / ClaretRed +%)), and relative cytotoxic activity was determined by [different number of stimulated CAR-T cells % Cytotoxicity /% unstimulated CAR-T cytotoxicity] As shown in Figure 8, as the number of stimuli increased, the cytotoxicity of CAR-T cells decreased. in, the expression of PD-1 is suppressed CD19-CAR_ shPD -1 T cells (stimulated before receiving CD19-CAR_ shPD - 1 T 17.7 % decrease cytotoxicity performance than the cells) CD19-shGFP CAR_ T cells (CD19-CAR_ shGFP before stimulation It was confirmed that the decrease in cytotoxicity was significantly lower than that of T cells (61.3% decrease).
실시예Example 5. (반복적인 항원 자극에 의한 CAR-T 세포 탈진(Exhaustion)시 PD-1 발현 감소가 CD19-CAR T 분화 및 증식능에 미치는 영향 확인) 5. (Check the effect of decreased PD-1 expression on CD19-CAR T differentiation and proliferative capacity during repeated antigen stimulation of CAR-T cell exhaustion)
CD45RA, CCR7, CD8, CD4 타깃 항체를 이용하여 반복적인 자극을 받은 CD8+ 또는 CD4+ CAR-T 세포의 분화 정도를 유세포 분석기로 분석하였다. 도 9에 보여지는 바와 같이, 반복적인 자극을 가함에도 불구하고 CD19 -CAR_shPD - 1 T 세포는 CD19-CAR_shGFP T 세포 보다 더 많은 Effector T 세포 (TEFF ; CCR7-CD45RA+ T 세포) 아형으로 구성되어 있었다. 2회 반복 자극을 준 CAR-T 세포의 증식능 변화를 관찰하고자, CellTrace™ CFSE (Thermofisher)를 라벨링한 CAR-T 세포를 감마 조사 K562-CD19-PD-L1 세포주와 동시 배양 하였다. 5일 후 유세포 분석기로 CAR-T 세포의 증식정도를 확인해 본 결과, CD19-CAR_shPD - 1 T 세포가 CD19-CAR_shGFP T 세포보다 더 잘 증식하였다 (도 10). Differentiation of repetitively stimulated CD8 + or CD4 + CAR-T cells using CD45RA, CCR7, CD8, and CD4 target antibodies was analyzed by flow cytometry. Composed of subtypes; - as shown in Figure 9, repeated despite imposing stimulation and CD19 -CAR_ shPD 1 T cells, CD19-CAR_ shGFP more Effector T cells than T-cells (CCR7-CD45RA + T cells, T EFF) It was. In order to observe the proliferative change of CAR-T cells with two repeated stimulation, CAR-T cells labeled with CellTrace ™ CFSE (Thermofisher) were co-cultured with gamma irradiation K562-CD19-PD-L1 cell line. Five days later, flow cytometry confirmed the extent of CAR-T cell proliferation. CD19- CAR_shPD - 1 T cells showed CD19- CAR_shGFP Proliferated better than T cells (FIG. 10).
실시예 6. (Example 6. shTIM-3shTIM-3 및 CD19-CAR를 발현하는 엔지니어드 T 세포의 제조) And preparation of engineered T cells expressing CD19-CAR)
shTIM - 3와 CD19 표적 CAR가 동시에 발현되는 플라스미드를 구축하고자 하였다. 플라스미드 ID　#４를 프라이머 SEQ ID #23, #24, #25, #26, #1을 활용해 shTIM-3-mU6 카세트가 포함된 PCR 산물을 만들었다. PCR 산물을 Hpa1 제한효소 처리 한 후 Hpa1 제한효소, CIP 처리된 플라스미드　ID #4와 ligation 해서 TIM-3 표적 shRNA (shRNA SEQ ID #13, #14, #15, #16)와 CD19-CAR가 동시에 발현하는 pLV-CD19-CAR_shGFP 플라스미드 ID #7, #8, #9, #10을 제작하였다. PBMC에 Transduction 후 7일 째, 인간 CD3, CD28 타깃 항체로 3일간 자극을 준 후, CAR, TIM-3 타깃 항체로 이용해 유세포분석을 하였다. 도 11에서 보여지는 바와 같이, shRNA SEQ ID #14가 CD19-CAR-T 세포의 TIM-3 발현을 가장 잘 억제하였다. shTIM - 3To construct a plasmid in which and CD19 target CAR is expressed simultaneously. Plasmid ID ## 4 was prepared using primers SEQ ID # 23, # 24, # 25, # 26, and # 1.shTIM-3PCR products containing -mU6 cassettes were made. After the PCR product was treated with Hpa1 restriction enzyme, the TIM-3 target shRNA (shRNA SEQ ID # 13, # 14, # 15, # 16) and CD19-CAR were simultaneously ligation with Hpa1 restriction enzyme and CIP-treated plasmid ID # 4. Expressing pLV-CD19-CAR_shGFPPlasmid IDs # 7, # 8, # 9, # 10 were prepared. Seven days after transduction into PBMC, human CD3 and CD28 target antibodies were stimulated for 3 days, followed by flow cytometry using CAR and TIM-3 target antibodies. As shown in FIG. 11, shRNA SEQ ID # 14 best inhibited TIM-3 expression of CD19-CAR-T cells.
실시예Example 7.(7. ( shPDshPD -1-One , , shTIMshTIM -3-3 , CD19-CAR가 동시에 발현하는 , Simultaneously expressed by CD19-CAR 렌티바이러스Lentivirus 플라스미드 제작) Plasmid production)
shPD -1, shTIM -3, 그리고 CD19-CAR의 발현이 각각 인간 U6 프로모터 (hU6), 마우스 U6 프로모터 (mU6), 그리고 EF1-alpha 프로모터에 의해 조절되는 렌티바이러스를 구축하고자 하였다. 두 가지 shRNA가 동시에 발현되는 플라스미드는 도 12에 보여주는 바와 같이 각 shRNA 카세트가 (1) 양방향성(shPD-1-hU6 shTIM -3-mU6)와 (2) 양방향성의 역(shPD -1-hU6 shTIM -3-mU6) 염기서열로 구성되도록 제작하였다. 이를 위해, (1) 다중클로닝자리 삽입 (2) shPD -1의 마우스 U6 프로모터를 인간 U6 프로모터로 전환 (3) shTIM -3 삽입 (4) shTIM - 3와 shPD -1의 위치 전환과 같은 클로닝을 수행하였다. pLV-ΔLNGFR_P2A_CD19-CAR_shPD -1 플라스미드 #4의 shPD-1-mU6 3` 부분에 다중클로닝자리 (MCS, Multiple cloning site)를 삽입하고자, 프라이머 SEQ ID #13, #14를 이용해 shPD -1-mU6 3`의 Hpa1 제한효소 인식부위가 BstZ171-Xba1-Nde1-Bmt1-Spe1 다중클로닝자리로 변형된 PCR 산물 (416 bp)을 만들었다. 이후 PCR 산물은 BstZ171, Hpa1 제한효소 처리하고 플라스미드 #4는 Hpa1 제한효소, CIP를 처리한 후 blunt end ligation을 통해 shPD -1-mU6-MCS 염기서열 (shRNA 카세트 SEQ ID #2)을 포함하는 pLV-ΔLNGFR_P2A_CD19-CAR_shPD -1_MCS 플라스미드 ID #11을 제작하였다 (도 13). 그 후, shPD -1의 발현이 마우스 U6 프로모터 대신에 인간 U6 프로모터에 의해서 발현되도록 플라스미드를 구축하였다. LentiCRISPR V2 플라스미드를 프라이머 SEQ ID #15, #16을 사용해 인간 U6 프로모터를 포함하는 PCR 산물을 얻었다. PCR 산물을 Hpa1, Spe1 제한효소 처리 후 Hpa1, Spe1 제한효소 처리된 플라스미드 ID #11에 ligation 하여 shPD -1-hU6 염기서열 (shRNA 카세트 SEQ ID #3)가 포함된 pLV-ΔLNGFR_P2A_CD19-CAR_hU6-shPD -1_MCS 플라스미드 ID #12을 제작하였다. shTIM -3-mU6 카세트를 플라스미드 ID #12에 삽입하여 shTIM - 3와 shPD -1이 동시에 발현하는 플라스미드를 구축하고자 하였다. shTIM -3-mU6 카세트를 포함하는 PCR 산물은 플라스미드 ID #9와 프라이머 SEQ ID #17, #18을 이용해 얻었다. PCR 산물을 Bmt1, Spe1 제한효소 처리 후 Bmt1, Spe1 제한효소 처리된 플라스미드 ID #12에 ligation 하여 양방향(shPD -1- hU6 shTIM -3-mU6) 염기서열 (shRNA 카세트 SEQ ID #4)로 구성된 pLV-ΔLNGFR_P2A_CD19-CAR_hU6-shPD -1mU6-shTIM-3 플라스미드 ID #13을 제작하였다 (도 14). 두가지 shRNA 카세트가 양방향의 역(shPD -1-hU6 shTIM -3-mU6)으로 구성된 플라스미드를 구축하고자 하였다. 이를 위해 플라스미드 ID #13을 프라이머 SEQ ID #19, #20 사용하여 PCR 산물을 얻었다. Spe1, Hpa1 제한 효소 처리한 후 같은 제한 효소 처리한 플라스미드 ID #11에 삽입하여 pLV-ΔLNGFR_P2A_CD19-CAR_hU6-shPD -1 (reverse)_MCS 플라스미드 ID #14를 제작하였다. 그 후 플라스미드 ID #14와 프라이머 SEQ ID #21, #22을 이용해 PCR 산물을 얻었다. Bmt1, Spe1 제한 효소 처리한 후 같은 제한효소 처리한 플라스미드 ID #14에 ligation 해서 최종적으로 양방향의 역(shPD -1-hU6 shTIM -3-mU6) 염기서열 (shRNA 카세트 SEQ ID #5) 이 포함된 pLV-ΔLNGFR_P2A_CD19-CAR_hU6-shPD-1mU6-shTIM-3 플라스미드 ID #15를 제작하였다 (도 15). We have constructed lentiviruses in which the expression of shPD- 1 , shTIM- 3 , and CD19-CAR are regulated by human U6 promoter (hU6), mouse U6 promoter (mU6), and EF1-alpha promoter, respectively. Two kinds of the shRNA that each shRNA cassette 1, as shown in Figure 12. This plasmid is expressed at the same time, bi-directional (shPD-1 -hU6 shTIM -3 -mU6 ) and (2) of the bi-directional station (shPD -1 -hU6 shTIM - 3 -mU6) was constructed to consist of the base sequence. To this end, (1) the multi-cloning seat insert (2) -1 switching shPD a mouse U6 promoter, the human U6 promoter (3) -3 shTIM insert (4) shTIM - cloning, such as the position of the switch 3 and shPD -1 Was performed. pLV-ΔLNGFR_P2A_CD19-CAR_ shPD -1 to insert a multiple cloning seat (MCS, Multiple cloning site) to shPD-1 -mU6 3` portion of plasmid # 4, and primer SEQ ID # 13, by using a # 14 shPD -1 -mU6 A PCR product (416 bp) in which 3 ′ Hpa1 restriction enzyme recognition sites were modified with BstZ171-Xba1-Nde1-Bmt1-Spe1 multicloning site was generated. The PCR product was then treated with BstZ171, Hpa1 restriction enzyme, and plasmid # 4 was treated with Hpa1 restriction enzyme, CIP and then blunt end ligation, followed by blunt end ligation, pLV containing shPD- 1 -mU6-MCS sequence (shRNA cassette SEQ ID # 2). the -ΔLNGFR_P2A_CD19-CAR_ shPD -1 _MCS plasmid ID # 11 was prepared (Fig. 13). The plasmid was then constructed so that expression of shPD- 1 was expressed by the human U6 promoter instead of the mouse U6 promoter. LentiCRISPR V2 plasmid was used as primers SEQ ID # 15, # 16 to obtain a PCR product containing the human U6 promoter. After the PCR product Hpa1, Spe1 Hpa1 restriction enzyme treatment, restriction enzyme Spe1 and ligation of the treated plasmid ID # 11 contains the shPD -1 -hU6 nucleotide sequence (shRNA cassette SEQ ID # 3) pLV-ΔLNGFR_P2A_CD19 -CAR_hU6- shPD - 1 _MCS prepare a plasmid ID # 12. The shTIM- 3 -mU6 cassette was inserted into plasmid ID # 12 to construct a plasmid expressing both shTIM - 3 and shPD- 1 simultaneously. PCR products containing the shTIM- 3 -mU6 cassette were obtained using plasmid ID # 9 and primers SEQ ID # 17, # 18. The PCR product was ligation to the Bmt1, Spe1 restriction enzyme-treated plasmid ID # 12 after Bmt1, Spe1 restriction enzyme treatment and the bivalent ( shPD -1- hU6 shTIM- 3 -mU6) nucleotide sequence (shRNA cassette SEQ ID # 4) -ΔLNGFR_P2A_CD19-CAR_hU6- shPD- 1 mU6- shTIM-3 plasmid ID # 13 was prepared (FIG. 14). It was intended to construct a plasmid in which two shRNA cassettes were bidirectionally inverted ( shPD -1 -hU6 shTIM -3 -mU6). For this purpose, PCR products were obtained using plasmid ID # 13 using primers SEQ ID # 19 and # 20. Spe1, Hpa1 restriction enzyme treatment and then inserted into the same restriction enzyme-treated plasmid ID # 11 was produced in the pLV-ΔLNGFR_P2A_CD19-CAR_hU6- shPD -1 ( reverse) _MCS plasmid ID # 14. Thereafter, PCR products were obtained using plasmid ID # 14 and primers SEQ ID # 21 and # 22. After treatment with Bmt1 and Spe1 restriction enzymes, ligation to the same restriction enzyme-treated plasmid ID # 14 finally contained a bidirectional reverse ( shPD -1 -hU6 shTIM -3 -mU6) nucleotide sequence (shRNA cassette SEQ ID # 5). pLV-ΔLNGFR_P2A_CD19-CAR_hU6- shPD-1 mU6- shTIM-3 plasmid ID # 15 was prepared (FIG. 15).
실시예 8.(Example 8. shPD-1, shTIM-3shPD-1, shTIM-3 , CD19-CAR가 동시에 발현된 CAR-T 세포 제조.), Production of CAR-T cells expressing CD19-CAR simultaneously.)
플라스미드 #3, #11, #12, #13, #15와 패키징 플라스미드 pMDL g/p, pRSVrev, 그리고 pMDG.1를 HEK293 T세포에 lipofectamine을 이용하여 transfection 하였고, 48시간 경과 후 렌티바이러스가 포함된 세포배양액을 얻었다. 인간 혈액으로부터 ficoll-paque 용액을 이용하여 말초혈액단색세포 (peripheral blood mononuclear cell, PBMC)를 분리하였고, 인간 CD3, CD28 타깃 항체를 이용하여 T-세포를 특이적으로 활성화하였다. T-세포의 초기 활성화 1-2일 경과 후 앞서 얻어진 바이러스를 이용해 transduction 하였다. 이후 만들어진 CAR-T세포는 5% 인간 혈장 그리고 인간 IL2를 포함하는 AIM-V 배양액을 이용하여 배양하였다. Transduction 후 6일 째, 순수한 CAR-T 세포를 획득하고자 MACSelect LNGFR System (miltenyibiotec, Germany)을 사용하였고, LNGFR 타깃 항체를 이용해 유세포분석을 통해 CAR-T 세포의 순도를 확인하였다. 도 16에서 보여지는 바와 같이, 80% 내외의 LNGFR+인 CAR-T 세포를 얻을 수 있었다. 분리한 CAR-T 세포에 인간 CD3, CD28 타깃 항체로 3일간 자극을 주어 PD-1, TIM-3 발현을 유도한 후, CAR-T 세포의 PD-1, TIM-3 발현을 CAR, PD-1, TIM-3 타깃 항체를 활용하여 유세포분석을 하였다. 도 17에서 볼 수 있는 바와 같이, U6 프로모터에 따른 PD-1 발현 감소를 확인해 보았다. 인간 U6 프로모터는 기존 마우스 U6 프로모터와 유사하게 PD-1 발현을 억제했다. 두번째로, 동시에 발현시킨 두 개의 shRNA가 PD-1과 TIM-3의 발현 감소에 미치는 영향에 대해서 분석하였다. shPD - 1와 shTIM - 3를 동시에 발현하는 CD19-CAR T 세포에서 관찰되는 PD-1, TIM-3 발현 감소 정도가 shPD -1 또는 shTIM -3가 단독으로 발현하는 CD19-CAR T 세포의 발현 감소 정도와 유사함을 확인하였다. 마지막으로, shRNA 카세트의 방향 구성이 타겟 유전자의 발현감소에 미치는 영향에 대해서 확인해 보았다. hU6-shPD - 1mU6-shTIM -3 와 hU6-shPD - 1mU6-shTIM -3 발현 CD19-CAR T 세포에서 TIM-3의 발현 감소 정도는 유사하였으나, PD-1의 발현 감소는 hU6-shPD -1mU6-shTIM-3가 더 뛰어난 것으로 확인되었다. 결과적으로, 두 가지 면역 관문 유전자의 발현을 억제함과 동시에 CAR를 발현할 수 있는 면역세포 치료제 제조 기술을 확보하게 되었다. Plasmids # 3, # 11, # 12, # 13, # 15 and the packaging plasmids pMDL g / p, pRSVrev, and pMDG.1 were transfected into HEK293 T cells with lipofectamine, and 48 hours later, the lentiviral was included. Cell culture solution was obtained. Peripheral blood mononuclear cells (PBMC) were isolated from human blood using ficoll-paque solution, and T-cells were specifically activated using human CD3 and CD28 target antibodies. After 1-2 days of initial activation of T-cells, transduction was performed using the virus obtained earlier. The resulting CAR-T cells were cultured using AIM-V medium containing 5% human plasma and human IL2. Six days after transduction, MACSelect LNGFR System (miltenyibiotec, Germany) was used to obtain pure CAR-T cells, and the purity of CAR-T cells was confirmed by flow cytometry using LNGFR target antibody. As shown in FIG. 16, CAR-T cells of about 80% LNGFR + were obtained. The isolated CAR-T cells were stimulated with human CD3 and CD28 target antibodies for 3 days to induce PD-1 and TIM-3 expression, and then the PD-1 and TIM-3 expression of CAR-T cells was expressed in CAR, PD- 1, TIM-3 target antibody was used for flow cytometry. As can be seen in Figure 17, it was confirmed that the PD-1 expression decreased by the U6 promoter. The human U6 promoter inhibited PD-1 expression similar to the existing mouse U6 promoter. Second, the effects of two simultaneously expressed shRNAs on the expression of PD-1 and TIM-3 were analyzed. Reduction of PD-1 and TIM-3 expression observed in CD19-CAR T cells expressing shPD - 1 and shTIM - 3 simultaneously decreased expression of CD19-CAR T cells expressing shPD- 1 or shTIM- 3 alone Similarity was confirmed to the degree. Finally, the effect of shRNA cassette orientation on the expression of target genes was examined. In hU6- shPD - 1 mU6- shTIM- 3 and hU6- shPD - 1 mU6- shTIM- 3 expressing CD19-CAR T cells, the degree of reduction in TIM-3 expression was similar, but the decrease in PD-1 was similar to hU6- shPD- . 1 mU6- shTIM-3 was found to be superior. As a result, it has secured a technology for producing immune cell therapeutics capable of inhibiting the expression of two immune gate genes and simultaneously expressing CAR.
앞에서 설명된 본 발명의 일실시예는 본 발명의 기술적 사상을 한정하는 것으로 해석되어서는 안 된다. 본 발명의 보호범위는 청구범위에 기재된 사항에 의하여만 제한되고, 본 발명의 기술분야에서 통상의 지식을 가진 자는 본 발명의 기술적 사상을 다양한 형태로 개량 변경하는 것이 가능하다. 따라서 이러한 개량 및 변경은 통상의 지식을 가진 자에게 자명한 것인 한 본 발명의 보호범위에 속하게 될 것이다.One embodiment of the present invention described above should not be construed as limiting the technical spirit of the present invention. The protection scope of the present invention is limited only by the matters described in the claims, and those skilled in the art can change and change the technical idea of the present invention in various forms. Therefore, such improvements and modifications will fall within the protection scope of the present invention, as will be apparent to those skilled in the art.

Claims (23)

  1. 면역세포의 핵산서열에 있어서, In nucleic acid sequences of immune cells,
    키메라 항원 수용체(CAR) 또는 단일클론 티세포 수용체(TCR)을 코딩하는 염기서열; 및 면역관문 수용체의 발현을 저해하는 숏헤어핀 리보핵산(shRNA)을 코딩하는 염기서열을 동시에 포함한 것을 특징으로 하는 핵산서열.A nucleotide sequence encoding a chimeric antigen receptor (CAR) or monoclonal T cell receptor (TCR); And a nucleotide sequence encoding short hairpin ribonucleic acid (shRNA) that inhibits expression of an immune gateway receptor.
  2. 제1항에 있어서,The method of claim 1,
    상기 숏헤어핀 리보핵산(shRNA)을 코딩하는 염기서열은 PD-1, CTLA-4, LAG-3 및 TIM-3로 구성된 군으로부터 선택된 1종 이상의 면역관문 수용체를 타깃으로 한 것임을 특징으로 하는 핵산서열. The base sequence encoding the short hairpin ribonucleic acid (shRNA) is a nucleic acid sequence characterized in that the target is at least one immune gateway receptor selected from the group consisting of PD-1, CTLA-4, LAG-3 and TIM-3 .
  3. 제2항에 있어서,The method of claim 2,
    상기 숏헤어핀 리보핵산(shRNA)을 코딩하는 염기서열이 PD-1 및 TIM-3를 동시에 타깃으로 한 것을 특징으로 하는 핵산서열.The nucleic acid sequence characterized in that the base sequence encoding the short hairpin ribonucleic acid (shRNA) targets PD-1 and TIM-3 simultaneously.
  4. 제2항에 있어서,The method of claim 2,
    상기 숏헤어핀 리보핵산(shRNA)을 코딩하는 염기서열은 서열번호 2 내지 12의 PD-1 타깃 염기서열, 서열번호 13 내지 35의 TIM-3 타깃 염기서열, 서열번호 36 내지 42의 LAG-3 타깃 염기서열 및 서열번호 43 내지 51의 CTLA-4 타깃 염기서열로 구성된 군으로부터 선택된 1종 이상의 염기서열을 포함한 것을 특징으로 하는 핵산서열. The base sequence encoding the short hairpin ribonucleic acid (shRNA) is a PD-1 target sequence of SEQ ID NO: 2 to 12, a TIM-3 target sequence of SEQ ID NO: 13 to 35, and a LAG-3 target of SEQ ID NO: 36 to 42 A nucleic acid sequence comprising at least one nucleotide sequence selected from the group consisting of a nucleotide sequence and a CTLA-4 target nucleotide sequence of SEQ ID NOs: 43 to 51.
  5. 제1항에 있어서,The method of claim 1,
    상기 키메라 항원 수용체(CAR) 또는 단일클론 티세포 수용체(TCR)의 타겟은 5T4, 알파 5β1-인테그린, 707-AP, AFP, ART-4, B7H4, BAGE, β-카테닌/m, Bcr-abl, MN/CIX 항원, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27/m, CD30, CD33, CD52, CD56, CD80, CDK4/m, CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2/new, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT(또는 hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/melan-A, MART-2/Ski, MC1R, 미오신/m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, PAP, 프로테이나제-3, p190 마이너 bcr-abl, Pml/RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1 또는 RU2, SAGE, SART-1 또는 SART-3, 서바이빈, TEL/AML1, TGFβ, TPI/m, TRP-1, TRP-2, TRP-2/INT2, VEGF, WT1, NY-Eso-1 및 NY-Eso-B로 구성된 군으로부터 선택된 1종 이상인 인간 종양항원인 것을 특징으로 하는 핵산서열.The target of the chimeric antigen receptor (CAR) or monoclonal T cell receptor (TCR) is 5T4, alpha 5β1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, β-catenin / m, Bcr-abl, MN / CIX antigen, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27 / m, CD30, CD33, CD52, CD56, CD80, CDK4 / m, CEA, CT, Cyp -B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2 / new, HLA-A * 0201-R170I, HPV-E7, HSP70 -2M, HST-2, hTERT (or hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR / FUT, MAGE, MART-1 / melan-A, MART-2 / Ski , MC1R, myosin / m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, PAP, proteinase-3, p190 minor bcr-abl, Pml / RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, Survivin, TEL / AML1, TGFβ, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, VEGF, WT1, Nucleic acid sequence, characterized in that one or more human tumor antigens selected from the group consisting of NY-Eso-1 and NY-Eso-B.
  6. 제1항에 있어서,The method of claim 1,
    상기 CAR의 아미노산 서열은 서열번호 52 내지 54로 구성된 군으로부터 선택된 하나 이상의 서열과 90% 이상 일치하는 것을 특징으로 하는 핵산서열.Wherein the amino acid sequence of the CAR is at least 90% identical to at least one sequence selected from the group consisting of SEQ ID NOs: 52-54.
  7. 키메라 항원 수용체(CAR) 또는 단일클론 티세포 수용체(TCR)을 코딩하는 염기서열; 및 면역관문 수용체의 발현을 저해하는 숏헤어핀 리보핵산(shRNA)을 코딩하는 염기서열을 포함한 벡터.A nucleotide sequence encoding a chimeric antigen receptor (CAR) or monoclonal T cell receptor (TCR); And a vector comprising a nucleotide sequence encoding shorthairpin ribonucleic acid (shRNA) that inhibits expression of an immune gateway receptor.
  8. 제7항에 있어서,The method of claim 7, wherein
    상기 숏헤어핀 리보핵산(shRNA)을 코딩하는 염기서열은 PD-1, CTLA-4, LAG-3 및 TIM-3로 구성된 군으로부터 선택된 1종 이상의 면역관문 수용체를 타깃으로 한 것임을 특징으로 하는 벡터. The nucleotide sequence encoding the short hairpin ribonucleic acid (shRNA) is a vector, characterized in that targeting at least one immune gateway receptor selected from the group consisting of PD-1, CTLA-4, LAG-3 and TIM-3.
  9. 제7항에 있어서,The method of claim 7, wherein
    상기 숏헤어핀 리보핵산(shRNA)을 코딩하는 염기서열이 PD-1 및 TIM-3를 동시에 타깃으로 한 것을 특징으로 하는 벡터.The vector encoding the short hairpin ribonucleic acid (shRNA) is characterized in that the PD-1 and TIM-3 targets at the same time.
  10. 제7항에 있어서,The method of claim 7, wherein
    상기 숏헤어핀 리보핵산(shRNA)을 코딩하는 염기서열은 서열번호 2 내지 12의 PD-1 타깃 염기서열, 서열번호 13 내지 35의 TIM-3 타깃 염기서열, 서열번호 36 내지 42의 LAG-3 타깃 염기서열 및 서열번호 43 내지 51의 CTLA-4 타깃 염기서열로 구성된 군으로부터 선택된 1종 이상의 염기서열을 포함한 것을 특징으로 하는 벡터. The base sequence encoding the short hairpin ribonucleic acid (shRNA) is a PD-1 target sequence of SEQ ID NO: 2 to 12, a TIM-3 target sequence of SEQ ID NO: 13 to 35, and a LAG-3 target of SEQ ID NO: 36 to 42 A vector comprising a base sequence and at least one base sequence selected from the group consisting of CTLA-4 target base sequences of SEQ ID NOs: 43 to 51.
  11. 제7항에 있어서,The method of claim 7, wherein
    상기 키메라 항원 수용체(CAR) 또는 단일클론 티세포 수용체(TCR)의 타겟은 5T4, 알파 5β1-인테그린, 707-AP, AFP, ART-4, B7H4, BAGE, β-카테닌/m, Bcr-abl, MN/CIX 항원, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27/m, CD30, CD33, CD52, CD56, CD80, CDK4/m, CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2/new, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT(또는 hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/melan-A, MART-2/Ski, MC1R, 미오신/m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, PAP, 프로테이나제-3, p190 마이너 bcr-abl, Pml/RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1 또는 RU2, SAGE, SART-1 또는 SART-3, 서바이빈, TEL/AML1, TGFβ, TPI/m, TRP-1, TRP-2, TRP-2/INT2, VEGF, WT1, NY-Eso-1 및 NY-Eso-B로 구성된 군으로부터 선택된 1종 이상인 인간 종양항원인 것을 특징으로 하는 벡터.The target of the chimeric antigen receptor (CAR) or monoclonal T cell receptor (TCR) is 5T4, alpha 5β1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, β-catenin / m, Bcr-abl, MN / CIX antigen, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27 / m, CD30, CD33, CD52, CD56, CD80, CDK4 / m, CEA, CT, Cyp -B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2 / new, HLA-A * 0201-R170I, HPV-E7, HSP70 -2M, HST-2, hTERT (or hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR / FUT, MAGE, MART-1 / melan-A, MART-2 / Ski , MC1R, myosin / m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, PAP, proteinase-3, p190 minor bcr-abl, Pml / RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, Survivin, TEL / AML1, TGFβ, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, VEGF, WT1, A vector characterized in that it is at least one human tumor antigen selected from the group consisting of NY-Eso-1 and NY-Eso-B.
  12. 제7항에 있어서,The method of claim 7, wherein
    상기 CAR의 아미노산 서열이 서열번호 52 내지 54로 구성된 군으로부터 선택된 1종 이상의 아미노산 서열과 90% 이상 일치하는 것을 특징으로 하는 벡터.The amino acid sequence of the CAR is at least 90% identical to at least one amino acid sequence selected from the group consisting of SEQ ID NO: 52 to 54.
  13. 키메라 항원 수용체(CAR) 또는 단일클론 티세포 수용체(TCR); 및 면역관문 수용체의 발현을 저해하는 숏헤어핀 리보핵산(RNA)이 동시에 발현되도록 유전적으로 조작된 면역관문을 극복한 면역세포.Chimeric antigen receptor (CAR) or monoclonal T cell receptor (TCR); And immune cells overcoming the genetically engineered immune barrier to simultaneously express shorthairpin ribonucleic acid (RNA) that inhibits the expression of the immune barrier receptor.
  14. 제13항에 있어서,The method of claim 13,
    상기 숏헤어핀 리보핵산(shRNA)은 PD-1, CTLA-4, LAG-3 및 TIM-3로 구성된 군으로부터 선택된 1종 이상의 면역관문 수용체를 타깃으로 하여 그 발현을 저해한 것을 특징으로 하는 면역관문을 극복한 면역세포. The short hairpin ribonucleic acid (shRNA) is an immune gateway characterized in that its expression is inhibited by targeting at least one immune gateway receptor selected from the group consisting of PD-1, CTLA-4, LAG-3 and TIM-3. Overcome immune cells.
  15. 제14항에 있어서,The method of claim 14,
    상기 숏헤어핀 리보핵산(shRNA)은 PD-1 및 TIM-3 면역관문 수용체를 동시에 타깃으로 하여 그 발현을 저해한 것을 특징으로 하는 면역관문을 극복한 면역세포.The short hairpin ribonucleic acid (shRNA) is an immune cell that overcomes the immune barrier, characterized in that the target to the PD-1 and TIM-3 immune gate receptor at the same time inhibited its expression.
  16. 제13항에 있어서,The method of claim 13,
    상기 CAR의 아미노산 서열이 서열번호 52 내지 54로 구성된 군으로부터 선택된 1종 이상과 90% 이상 일치하는 것을 특징으로 하는 면역관문을 극복한 면역세포.Immune cell overcoming the immune barrier, characterized in that the amino acid sequence of the CAR is at least 90% identical to at least one selected from the group consisting of SEQ ID NO: 52 to 54.
  17. 제13항에 있어서,The method of claim 13,
    상기 키메라 항원 수용체(CAR) 또는 단일클론 티세포 수용체(TCR)의 타겟은 5T4, 알파 5β1-인테그린, 707-AP, AFP, ART-4, B7H4, BAGE, β-카테닌/m, Bcr-abl, MN/CIX 항원, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27/m, CD30, CD33, CD52, CD56, CD80, CDK4/m, CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2/new, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT(또는 hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/melan-A, MART-2/Ski, MC1R, 미오신/m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, PAP, 프로테이나제-3, p190 마이너 bcr-abl, Pml/RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1 또는 RU2, SAGE, SART-1 또는 SART-3, 서바이빈, TEL/AML1, TGFβ, TPI/m, TRP-1, TRP-2, TRP-2/INT2, VEGF, WT1, NY-Eso-1 및 NY-Eso-B로 구성된 군으로부터 선택된 1종 이상인 인간 종양항원인 것을 특징으로 하는 면역관문을 극복한 면역세포.The target of the chimeric antigen receptor (CAR) or monoclonal T cell receptor (TCR) is 5T4, alpha 5β1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, β-catenin / m, Bcr-abl, MN / CIX antigen, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27 / m, CD30, CD33, CD52, CD56, CD80, CDK4 / m, CEA, CT, Cyp -B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2 / new, HLA-A * 0201-R170I, HPV-E7, HSP70 -2M, HST-2, hTERT (or hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR / FUT, MAGE, MART-1 / melan-A, MART-2 / Ski , MC1R, myosin / m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, PAP, proteinase-3, p190 minor bcr-abl, Pml / RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, Survivin, TEL / AML1, TGFβ, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, VEGF, WT1, Immune cells that overcome the immune barrier, characterized in that the human tumor antigen is one or more selected from the group consisting of NY-Eso-1 and NY-Eso-B.
  18. 제13항에 있어서,The method of claim 13,
    상기 면역세포는 T세포, 자연살해세포, 세포독성 T 림프구 및 조절용 T 세포로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는 면역관문을 극복한 면역세포.The immune cells are immune cells overcoming the immune barrier, characterized in that one or more selected from the group consisting of T cells, natural killer cells, cytotoxic T lymphocytes and regulatory T cells.
  19. 제13항 내지 제18항 중 어느 한 항의 면역관문을 극복한 면역세포를 포함한 약제학적 조성물.A pharmaceutical composition comprising immune cells overcoming the immune barrier of claim 13.
  20. 제19항에 있어서,The method of claim 19,
    상기 약제학적 조성물은 담체, 부형제, 희석제, 항산화제, 보존제, 착색제, 향미제 및 희석제, 유화제, 현탁제, 용매, 충전제, 벌크화제, 완충제, 전달 비히클, 등장제, 공용매, 습윤제, 복합화제, 완충제, 항균제 및 계면활성제로 이루어진 군으로부터 선택된 1종 이상의 약제학적으로 허용가능한 염, 부형제 또는 비히클을 추가로 포함한 것을 특징으로 하는 약제학적 조성물.The pharmaceutical compositions include carriers, excipients, diluents, antioxidants, preservatives, colorants, flavoring and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, isotonic agents, cosolvents, wetting agents, complexing agents And at least one pharmaceutically acceptable salt, excipient or vehicle selected from the group consisting of buffers, antimicrobials and surfactants.
  21. 병증의 치료가 필요한 피험자에게 유효량의 제19항 또는 제20항의 약제학적 조성물을 투여함을 포함하는, 피험자의 치료 방법.A method of treating a subject, comprising administering an effective amount of the pharmaceutical composition of claim 19 to a subject in need thereof.
  22. 제21항에 있어서,The method of claim 21,
    상기 병증은 암인 피험자의 치료방법.The condition is a method of treating a subject is cancer.
  23. 제22항에 있어서,The method of claim 22,
    상기 암이 유방암, 전립선암, 난소암, 자궁경부암, 피부암, 췌장암, 결장직장암, 신장암, 간암, 뇌암, 림프종, 백혈병, 폐암, 및 이들의 임의의 조합으로 이루어지는 그룹 중에서 선택되는 피험자의 치료방법.The cancer is selected from the group consisting of breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and any combination thereof. .
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