WO2021172690A1 - Mesothelin-specific chimeric antigen receptor and uses thereof - Google Patents

Mesothelin-specific chimeric antigen receptor and uses thereof Download PDF

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WO2021172690A1
WO2021172690A1 PCT/KR2020/014394 KR2020014394W WO2021172690A1 WO 2021172690 A1 WO2021172690 A1 WO 2021172690A1 KR 2020014394 W KR2020014394 W KR 2020014394W WO 2021172690 A1 WO2021172690 A1 WO 2021172690A1
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antigen receptor
chimeric antigen
seq
mesothelin
cells
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PCT/KR2020/014394
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French (fr)
Korean (ko)
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안종성
이영은
김언교
권미선
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(주)녹십자셀
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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
    • A61K39/463—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
    • A61K39/461—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
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464466—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K39/464468—Mesothelin [MSLN]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/54—Pancreas

Definitions

  • the present invention relates to mesothelin-specific chimeric antigen receptors, immune cells expressing the same, and uses thereof.
  • T cells are cells that play an important role in mediating adaptive immunity. T cells are activated through stimulation of antigen-recognizing receptors (T cell receptors, TCRs), co-stimulatory molecules, and cytokines. In addition, the TCR of T cells causes an immune response through antigen-bound MHC molecules (major histocompatibility complex, MHC molecules), and cancer cells suppress the expression of MHC molecules as a mechanism to evade the immune response.
  • TCRs antigen-recognizing receptors
  • MHC molecules major histocompatibility complex
  • cancer cells suppress the expression of MHC molecules as a mechanism to evade the immune response.
  • Chimeric antigen receptors are being developed so that T cells can directly recognize antigens without MHC molecules.
  • the chimeric antigen receptor consists of an antigen-recognizing single chain variable fragment (scFv) part, a transmembrane domain, and a signaling domain that transmits a signal into a cell.
  • scFv antigen-recognizing single chain variable fragment
  • a transmembrane domain By artificially introducing a chimeric antigen receptor into T cells, T cells (CAR-T cells) can respond to cancer with specific antigens.
  • CAR-T cells T cells
  • Currently, multinational pharmaceutical companies such as Novartis, Gilead, Juno Therapeutics, and Celgene in the US are using CAR-T cells for blood cancer indications, but studies on solid cancer are still insufficient.
  • MSLN Mesothelin
  • ss1 CAR chimeric antigen receptor
  • HAMA response human anti-mouse antibody response
  • the present inventors produced T cells expressing a third-generation chimeric antigen receptor containing a human-derived scFv that specifically targets mesothelin, thereby exhibiting superior anticancer effect. It was confirmed that the present invention was completed.
  • An object of the present invention is to solve the problem of anticancer immune cell therapy using T cells expressing a chimeric antigen receptor containing a mesothelin binding domain, and a chimeric antigen receptor comprising a new mesothelin binding domain showing a high anticancer effect. And to provide an immune cell expressing the chimeric antigen receptor.
  • Another object of the present invention is to provide a composition for cancer treatment comprising the immune cells, a cancer treatment method using the same, the use of the immune cells for cancer treatment, and the use of the immune cells for the manufacture of a medicament for the treatment of cancer. have.
  • the present invention provides a chimeric antigen receptor comprising a binding domain that specifically binds to mesothelin (MSLN).
  • MSLN mesothelin
  • the present invention also provides a nucleic acid encoding a mesothelin-specific chimeric antigen receptor, an expression vector comprising the nucleic acid, and an immune cell expressing the chimeric antigen receptor.
  • the present invention also provides a cancer treatment composition comprising the immune cells, a cancer treatment method using the same, the use of the immune cells for cancer treatment, and the use of the immune cells for preparing a medicament for cancer treatment.
  • FIG. 1 shows the degree of binding of MS501 scFv according to the present invention
  • FIG. 1A shows the degree of binding to recombinant human mesothelin (rhMSLN) by enzyme-linked immunosorbent assay (ELISA).
  • FIG. 1B shows the degree of cell binding to MIA PaCa2-MSLN cells through flow cytometry.
  • Fig. 2 shows a schematic diagram of a CAR structure (501(28H)28BBz) of the present invention.
  • FIG. 3 shows the expression rate of 501(28H)28BBz transduced into human T cells through flow cytometry.
  • Figure 4 shows the killing ability according to the mesothelin-specific response in human T cells transduced with 501(28H)28BBz.
  • FIG. 5 is a diagram showing the efficacy of 501(28H)28BBz CAR-T cells in a pancreatic cancer orthotopic mouse model (off-target model) prepared using MIA PaCa-2 (Parental) cells, Vehicle, Mock CAR-T and After each injection of 501(28H)28BBz CAR-T of high dose (intraperitoneal injection and intravenous injection) and low dose (intraperitoneal injection), the tumor size of mice was monitored by Bioluminescence Images for 8 weeks.
  • FIG. 6 is a diagram showing the efficacy of 501(28H)28BBz CAR-T cells in a pancreatic cancer orthotopic mouse model (on-target model) prepared using Mesothelin-expressed MIA PaCa cells.
  • Vehicle, Mock CAR-T and High dose After intraperitoneal injection and intravenous injection
  • 501(28H)28BBz CAR-T of low dose injection
  • the tumor size of mice was monitored by Bioluminescence Images for 8 weeks.
  • FIG. 7 is a diagram showing the pathological efficacy of 501(28H)28BBz CAR-T cells in an orthotopic pancreatic cancer mouse model, Vehicle, Mock CAR-T and High dose (intraperitoneal injection and intravenous injection), Low dose (intraperitoneal injection) After injection of 501(28H)28BBz CAR-T, mice were sacrificed 8 or 12 weeks later, and the pancreas was H&E-stained.
  • the present invention provides a binding domain that specifically binds to mesothelin (MSLN), a signal peptide, a hinge, a transmembrane domain, and three or more intracellular domains. It relates to a chimeric antigen receptor (CAR) comprising:
  • the binding domain specifically binding to mesothelin is preferably an anti-mesothelin antibody or fragment thereof, but is not limited thereto.
  • fragment of an antibody refers to a fragment having an antigen-binding function, and is used to include scFv, Fab, F(ab') 2 and Fv fragment.
  • a “single chain Fv” or “scFv” antibody fragment comprises the VH and VL domains of an antibody, which domains are present within a single polypeptide chain.
  • the Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • Fv fragment is an antibody fragment that contains a complete antibody recognition and binding site. This region consists of a dimer in which one heavy chain variable domain and one light chain variable domain are tightly and substantially covalently associated, eg, scFv.
  • a “Fab” fragment contains the variable and constant domains of the light chain and the variable and first constant domains (CH1) of the heavy chain.
  • “F(ab′) 2 ” antibody fragments generally comprise a pair of Fab fragments covalently linked near their carboxy terminus by a hinge cysteine between them.
  • the anti-mesothelin antibody or fragment thereof contained in the chimeric antigen receptor is a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and the amino acid of SEQ ID NO: 23 a heavy chain variable region comprising a heavy chain CDR3 comprising the sequence, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26 It may be characterized as containing a light chain variable region comprising, but is not limited thereto.
  • the anti-mesothelin antibody or fragment thereof contained in the chimeric antigen receptor contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20.
  • a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19
  • a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20.
  • the anti-mesothelin antibody or fragment thereof contained in the chimeric antigen receptor is preferably in the form of an scFv (single-chain variable fragment), and may include the amino acid sequence shown in SEQ ID NO: 2, but is not limited thereto. .
  • the chimeric antigen receptor is additionally a signal peptide (SP), a hinge, a transmembrane domain (TM) and an intracellular binding domain that specifically binds to mesothelin. It may be characterized by including a domain.
  • the intracellular domain may be characterized as including three or more, but is not limited thereto.
  • the signal sequence is preferably a CD8 ⁇ signal sequence, but is not limited thereto, and the CD8 ⁇ signal sequence may include the amino acid sequence shown in SEQ ID NO: 4.
  • the signal sequence and the binding domain specifically binding to mesothelin constitute the extracellular domain of the chimeric antigen receptor.
  • the extracellular domain is a site through which a main signal is transmitted, exists outside the cell membrane, and is a domain for specifically recognizing mesothelin.
  • the transmembrane domain can be any type as long as it can connect the extracellular domain and the intracellular signaling domain between the cell membrane.
  • it consists of a CD28-derived transmembrane domain and/or a CD8 ⁇ -derived transmembrane domain, and may include all or part of the CD28-derived transmembrane domain and/or CD8 ⁇ -derived transmembrane domain.
  • the transmembrane domain may include the amino acid sequence represented by SEQ ID NO: 8, but is not limited thereto.
  • the extracellular domain and the transmembrane domain may be connected by a spacer domain.
  • the spacer domain may be a hinge domain.
  • the spacer domain that may be included in the chimeric antigen receptor may include a hinge domain derived from CD28 and/or a hinge domain derived from CD8 ⁇ , and all or part of the hinge domain derived from CD28 and/or a hinge domain derived from CD8 ⁇ may include. It may include all or part of CD28 and CD8 ⁇ .
  • the hinge domain may include the amino acid sequence represented by SEQ ID NO: 6, but is not limited thereto.
  • the intracellular signaling domain is a part located inside the cell membrane of immune cells, that is, in the cytoplasm, and when the binding domain bound to the extracellular domain binds to the target antigen, it activates the immune response of the immune cell. means the area to be made.
  • the chimeric antigen receptor according to the present invention may be characterized as comprising three or more intracellular signaling domains.
  • the intracellular and signaling domains may be serially connected to each other.
  • the intracellular signaling domain may include a sequence selected from the group consisting of intracellular signaling domain sequences of CD28, 4-1BB and CD3 ⁇ , or a combination thereof.
  • the intracellular signaling domain derived from CD28 and the intracellular signaling domain derived from 4-1BB may be linked to the intracellular signaling domain derived from CD3 ⁇ , and most preferably represented by SEQ ID NO: 10, 12 or 14 It may include, but is not limited to, an amino acid sequence.
  • MS501 scFv in an in-vivo experiment using various combinations of CAR structures including MS501 scFv, MS501 scFv was transformed into CD28 transmembrane domain, CD28-derived, 4-1BB-derived and CD3 ⁇ -derived cells.
  • the combination (501(28H)28BBz) linked to my signal transduction domain exhibited an unexpectedly excellent anticancer effect in reducing the size of the tumor compared to the control group.
  • the chimeric antigen receptor is 80% or more, preferably 90% or more, more preferably 95% or more, most preferably 99% or more of the amino acid sequence represented by SEQ ID NO: 16 or the amino acid sequence. It may include a variant thereof having sequence identity.
  • the present invention relates to a nucleic acid encoding the chimeric antigen receptor.
  • nucleic acid (polynucleotide) encoding the chimeric antigen receptor according to the present invention can be modified by codon optimization, which is due to the degeneracy of codons, and many nucleotide sequences encoding the polypeptide or variant fragment thereof. The existence of this will be well understood by those skilled in the art. Some of these polynucleotides (nucleic acids) possess minimal homology with the nucleotide sequence of any naturally occurring gene.
  • variable polynucleotides (nucleic acids) due to differences in codon utilization are preferred, for example, polynucleotides (nucleic acids) optimized for codon selection in humans, primates and/or mammals.
  • the nucleic acid sequence is the nucleotide sequence represented by 15, or 80% or more, preferably 90% or more, more preferably 95% or more, most preferably 99% of the nucleotide sequence and the nucleotide sequence. It may include a variant having more than one sequence identity.
  • the nucleic acid encoding the chimeric antigen receptor comprises:
  • a nucleotide sequence encoding a CD8 ⁇ signal sequence characterized in that represented by SEQ ID NO: 3;
  • a nucleotide sequence encoding a single-chain variable fragment (scFv) of an anti-mesothelin antibody characterized in that shown in SEQ ID NO: 1;
  • a nucleotide sequence encoding a CD28 hinge characterized in that shown in SEQ ID NO: 5;
  • a nucleotide sequence encoding the CD28 transmembrane domain characterized in that represented by SEQ ID NO: 7; SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, characterized in that the CD28, 4-1BB or CD3 ⁇ cell signal region encoding a nucleotide sequence;
  • the present invention relates to an expression vector comprising the nucleic acid and a virus comprising the expression vector.
  • vector refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule.
  • the transferred nucleic acid is generally linked to a vector nucleic acid molecule, eg, inserted into a vector nucleic acid molecule.
  • a vector may include sequences that direct autonomous replication in the cell, or it may include sequences sufficient to permit integration into host cell DNA.
  • the vector may be characterized in that it is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector and retroviral vector, but is not limited thereto.
  • virus means a genetically modified one to express the chimeric antigen receptor of the present invention for use in the treatment of cancer.
  • Genetically modified is the addition of foreign genetic material in the form of DNA or RNA into the entire genetic material in a cell.
  • the nucleic acid or the vector is transfected or transfected with a virus.
  • a number of different techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells for “transfection” or “transfection”, such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection or lipofection may be used.
  • the present invention relates to an immune cell expressing the chimeric antigen receptor on its surface.
  • the immune cells may be characterized as T cells, NK cells or NKT cells, but is not limited thereto, and preferably T cells.
  • Immune cells expressing the chimeric antigen receptor according to the present invention are CAR-T cells (Chimeric Antigen Receptor T Cells), CAR-NK cells (Chimeric Antigen Receptor Natural Killer Cells), or CAR-NKT cells (Chimeric Antigen Receptor Natural killer T Cells). ) may be characterized as
  • the T cells are cytotoxic T lymphocytes (CTL); It may be characterized in that it is selected from the group consisting of T cells isolated from tumor infiltrating lymphocytes (TIL) and peripheral blood mononuclear cells (PBMC).
  • TIL tumor infiltrating lymphocytes
  • PBMC peripheral blood mononuclear cells
  • the immune cells of the present invention may be toxic to mesothelin-expressing tumor cells.
  • the immune cells (eg, T cells) of the present invention are toxic to pancreatic cancer cells, cervical cancer cells, mesothelioma cells or ovarian cancer cells.
  • the pancreatic cancer cells, cervical cancer cells, mesothelioma cells, or ovarian cancer cells may express mesothelin.
  • the present invention relates to a composition for treating cancer using immune cells (eg, T cells) expressing the chimeric antigen receptor.
  • immune cells eg, T cells
  • cancer and “tumor” are used interchangeably and refer to or mean a physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation.
  • cancer examples include, but are not limited to, carcinoma, lymphoma (eg, Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More preferred examples of cancer include squamous cell cancer, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer , hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, mesothelioma, leukemia and other lymphoproliferative disorders, and various types of including head and neck cancer.
  • lymphoma eg, Hodgkin's and non-Ho
  • the cancer is preferably mesothelin-positive cancer, and may be characterized in that it is selected from the group consisting of pancreatic cancer, ovarian cancer, lung cancer, gastric cancer, endometrial cancer and mesothelioma.
  • the therapeutic composition of the present invention is a composition for preventing or treating cancer, and the term, “prevention” of the present invention, refers to any action that inhibits or delays the progression of cancer by administration of the composition of the present invention, and “treatment” ” means inhibiting the development of cancer, alleviating or eliminating symptoms.
  • the number of immune cells expressing the chimeric antigen receptor according to the present invention is preferably 1 to 10 times the number of tumor cells (eg, pancreatic cancer, cervical cancer, mesothelioma or ovarian cancer) in the treatment target, but limited thereto it's not going to be
  • tumor cells eg, pancreatic cancer, cervical cancer, mesothelioma or ovarian cancer
  • the pharmaceutical composition comprising immune cells expressing the chimeric antigen receptor according to the present invention may additionally include a pharmaceutically acceptable excipient.
  • excipients include surfactants, preferably nonionic surfactants of the polysorbate series; buffers such as neutral buffered saline and human salt buffered saline; sugars or sugar alcohols such as glucose, mannose, sucrose or dextran and mannitol; amino acids, proteins, or polypeptides such as glycine and histidine; antioxidants; chelating agents such as EDTA or glutathione; penetrant; supplements; and preservatives, but are not limited thereto.
  • the pharmaceutical composition of the present invention is the number of the immune cells (eg, T cells) in one dose is the tumor cells in the subject to be treated, for example, pancreatic cancer cells, cervical cancer cells, mesothelioma cells Or it may be included in 1 to 10 times the number of ovarian cancer cells.
  • the immune cells eg, T cells
  • the present invention relates to a method for treating cancer comprising administering to a subject an immune cell expressing the chimeric antigen receptor.
  • the present invention also relates to the use of said immune cells for the treatment of cancer.
  • the present invention also relates to the use of said immune cells for the manufacture of a medicament for the treatment of cancer.
  • the subject may be a mammal having a tumor, specifically, a human, but is not limited thereto.
  • Immune cells expressing the chimeric antigen receptor according to the present invention or a composition comprising the same are administered orally, infusion, intravenous injection, intramuscular injection, subcutaneous injection, or intraperitoneal administration. It may be administered by intraperitoneal injectoon, intrarectal administration, topical administration, intranasal injection, etc., but is not limited thereto.
  • Example 1-1 Cell lines and cell line culture
  • the human pancreatic cancer cell line MIA PaCa2 was supplied from ATCC (American Type Culture Collection, Manassas, VA, USA) and used. MIA PaCa2 was maintained in DMEM (GIBCO, Grand Island, NY, USA) containing 10% fetal bovine serum (GIBCO, Grand Island, NY, USA).
  • the HEK293T cell line, a human embryonic kidney fibroblast, was supplied from ATCC and maintained in DMEM (GIBCO) containing 10% FBS (GIBCO, Grand Island, NY, USA).
  • Mesothelin-overexpressing human pancreatic cancer cell line MIA PaCa2-MSLN was supplied from Mok Cancer Research Institute (GC Green Cross, Korea) and used. MIA PaCa2-MSLN was maintained in DMEM (GIBCO) containing 10% FBS (GIBCO) and 200 ⁇ g/ml Hygromycin B (GIBCO).
  • Example 1-2 Human T Cell Isolation and Activation
  • PBMCs peripheral blood mononuclear cells
  • Ficoll-Hypaque GE healthcare, Chicago, IL, USA
  • the isolated PBMCs were immediately frozen. After thawing PBMCs at 37°C, positive selection was performed using QuadroMACSTM Separator (Miltenyi Biotec, Bergisch Gladbach, Germany), LS Columns (Miltenyi Biotec), CD4 MicroBeads (Miltenyi Biotec), and CD8 MicroBeads (Miltenyi Biotec). ) to isolated human T cells.
  • Human T cells are mixed with MACS GMP T cell TransAct (Miltenyi Biotec) and activated for 20 to 24 hours. Unless otherwise noted, human T cells were cultured in RPMI-1640 (Thermo Fisher Scientific) medium containing 10% FBS (Thermo Fisher Scientific) and 200 IU/ml IL-2 (Proleukin, Novartis, Basel, Switzerland). .
  • Example 1-3 Confirmation of binding of scFv to recombinant human mesothelin
  • MS501 scFv protein is coated on a 96-well immunoplate at 200ng/ml for 24 hours.
  • the recombinant human mesothelin detection antibody (Detection antibody) was diluted according to the certificate of analysis, dispensed into the wells, and treated for 1 hour.
  • Example 1-4 Confirmation of binding to mesothelin overexpressing cells
  • the degree of binding of MS501 scFv protein to MIA PaCa2-MSLN, a mesothelin-overexpressing cell line, is confirmed.
  • MS501 scFv protein was treated with 1 ⁇ g. Cells were washed twice using FACS buffer. Cells were stained using anti-6xHis tag antibody (Biolegend). A positive control sample was stained with an anti-mesothelin antibody (R&D system). Expression ratio and mean fluorescence intensity (MFI) of stained cells were measured using BD LSRFortessa and analyzed in FlowJo software.
  • MFI mean fluorescence intensity
  • the extracellular domain CD8 ⁇ signal sequence, scFv sequence (MS501 scFv sequence), CD28 hinge; the transmembrane domain of CD28;
  • Intracellular domains The intracellular signaling domains of CD28, 4-1BB, and CD3 ⁇ were artificially synthesized through SOE-PCR. They were digested with EcoR1 and BamH1 and then inserted into the EcoR1 and BamH1 sites of the EF1a-MCS vector, a third-generation self-inactivating lentiviral expression vector.
  • the chimeric antigen receptor (CAR) according to the present invention is summarized in Table 1 below.
  • the domains of the CAR according to the present embodiment are connected in tandem with each other and are also connected in-frame.
  • 501(28H)28BBz is the signal sequence domain of human CD8 ⁇ (890-952 nucleotides, GenBank NM 001768.6); MS501 IgG scFv domain (Application No.
  • Pseudotype-VSVG Lentivirus was prepared using 293T cells for gene transfer.
  • HEK293T cells were cultured in DMEM (GIBCO) medium containing 10% FBS (GIBCO).
  • HEK293T cells were co-transfected with the EF1 ⁇ -MSLN CAR construct vector and the HIV-based pPACKH1 lentivirus package kit (System Biosciences).
  • Lipofectamine 2000 (Invitrogen, Carlsbad, CA) was used for vector delivery.
  • the MSLN CAR construct is as follows; 501(28H)28BBz. 48 hours after transformation, the cell supernatant (Supernatant) containing the lentivirus was harvested.
  • the supernatant was removed from cell debris with a 0.45 ⁇ m filter unit (Millipore, Billerica, MA, USA).
  • the virus was concentrated 1,500-fold by ultracentrifugation at 10,600 rpm for 90 minutes. Concentrated virus was stored at -80°C.
  • Vectofusin-1 (Miltenyi Biotec) and lentivirus were added to 5 MOI and replaced with fresh media after 72 hours.
  • the transduced human T cells were replaced with fresh medium 3 times a week to maintain a concentration of 1x10 6 cells/ml. Unless otherwise noted, all cells were cultured in a 5% CO 2 incubator at 37°C.
  • Example 1-7 Receptor expression and T cell activity marker analysis including MSLN CAR
  • 501(28H)28BBz transduced human T cells and control vector transduced human T cells were washed twice using FACS buffer.
  • Cells were stained using anti-CD3 (BD Biosciences), anti-F(ab)2 (Jackson Immuno research).
  • Expression ratio and mean fluorescence intensity (MFI) of stained cells were measured using BD LSRFortessa and analyzed in FlowJo software.
  • MSLN CAR-expressing T cells on target cells that is, mesothelin-expressing cancer cells.
  • Target cells were engineered to produce luciferase using luciferase virus (BIOSETTIA).
  • Target cells were distributed at 1 x 10 4 cells/well in 96-well plates (Thermo Fisher Scientific).
  • effector cells 501(28H)28BBz transduced human T cells, control vector transduced human T cells and untreated human T cells
  • RPMI-1640 Thermo Fisher Scientific
  • Example 2 Evaluation of human T cells expressing mesothelin-specific CAR as cancer immunotherapeutic agents
  • Example 2-1 Evaluation of scFvs specifically targeting mesothelin
  • MS501 IgG was invented by existing patents (Mokam Research Institute, GC Green Cross, KOREA) and is known to be capable of mesothelin-specific binding.
  • the present inventors prepared MS501 IgG in the form of scFv and confirmed that it binds to recombinant human mesothelin even in the form of scFv by immunoprecipitation analysis (FIG. 1A).
  • the above scFv protein was bound to the mesothelin-overexpressing cell line, and it was confirmed through flow cytometry that it was more than 68% bound to the mesothelin-overexpressing cell line ( FIG. 1B ).
  • Example 2-2 Expression confirmation and expression maintenance evaluation of mesothelin-specific CAR
  • MS501 scFv is the signal sequence of CD8 ⁇ ; hinge, transmembrane domain, intracellular signaling domain of CD28; the intracellular signaling domain of 4-1BB; It was bound to the intracellular signaling domain of CD3 ⁇ (FIG. 2).
  • the 501(28H)28BBz gene was expressed in human T cells using a lentiviral vector with an EF1 ⁇ promoter. The amount of lentivirus used was 5 multiplicity of infection (MOI). Each CAR was detected using an anti-F(ab) 2 antibody. It was confirmed that CAR was transduced with an efficiency of 20% or more in human T cells through flow cytometry (FIG. 3).
  • Example 2-3 Evaluation of anticancer ability of human T cells expressing mesothelin-specific CAR
  • MIA PaCa2 cells or MIA PaCa2-MSLN cells were transfected with mesothelin receptors, respectively. After co-culture with the introduced human T cells, the target cell killing ability was evaluated. Human T cells expressing mesothelin receptor did not show killing ability against MIA PaCa2 cells, but killing ability was confirmed in MIA PaCa2-MSLN cells expressing mesothelin ( FIG. 4 ).
  • Example 3 Evaluation of in vivo anticancer efficacy of human T cells expressing mesothelin-specific CAR
  • MIA PaCa2-FLuc-GFP cells that rarely express mesothelin (off-target model) and MIA PaCa2 that overexpress mesothelin -MSLN-FLuc-GFP cell line (on-target model) was fabricated/constructed. Since these two cell lines stably express the firefly luciferase report gene, tumor growth was monitored in this example to evaluate anticancer efficacy.
  • MIA PaCa2-FLuc-GFP cell line and MIA PaCa2-MSLN-FLuc-GFP cell line were suspended in HBSS (Ca 2+ /Mg 2+ free Hank's balanced salt solution) at a concentration of 1.0x10 5 cells/ml, respectively. Then, it was prepared by injecting 50 ⁇ l into the pancreatic lobe of the mouse.
  • Mock CAR-T Human pancreatic cancer cell line MIA PaCa2-FLuc-GFP cell line and MIA PaCa2-MSLN-FLuc-GFP cell line were injected, and tumor formation was confirmed on the first day 11-12 days, and the second on day 25-26 Vehicle, Mock CAR-T, 501 (28H)28BBz CAR-T was administered by intraperitoneal (IP) and intravenous (IV) injections.
  • Mock CAR-T cells were prepared in DPBS at 1x10 7 cells/200 ⁇ l, and 501(28H)28BBz CAR-T cells were administered at high dose (1x10 7 cells/200 ⁇ l) and low dose (2x10 6 cells/200 ⁇ l) in DPBS, respectively.
  • Tumor size was monitored every week using an IVIS® Lumina LT Series III In Vivo Imaging System (PerkinElmer) for 8 weeks from CAR-T cell administration. This was expressed as a BLI signal to evaluate the mesothelin-specific anticancer efficacy of CAR-T cells ( FIGS. 5 and 6 ).
  • pancreatic tissue was isolated and H&E staining was performed. It was confirmed that the mouse BLI signal and the tumor size of the pancreas were consistent. From a pathological point of view, consistent with the BLI signal results, tumors were cured in the on-target high-dose group of the mesothelin-expressing pancreatic cancer mouse model, and the effect was also confirmed in the on-target low-dose group (FIG. 7).
  • 501(28H)28BBz CAR-T cells exhibited high therapeutic efficacy of complete remission by target specific trafficking to mesothelin-overexpressing pancreatic cancer, and showed efficacy in a dose-dependent manner.
  • the chimeric antigen receptor according to the present invention has excellent mesothelin-specific targeting efficiency as well as excellent expression persistence, and the T cells expressing the chimeric antigen receptor according to the present invention are cytotoxic to cancer cells. Because it is excellent, it can be usefully used for immune cell therapy for cancer treatment.

Abstract

The present invention relates to a mesothelin-specific chimeric antigen receptor, an immune cell expressing same, and uses thereof. The chimeric antigen receptor according to the present invention has not only excellent mesothelin-specific targeting efficiency, but also excellent expression persistence. In addition, a T cell expressing the chimeric antigen receptor according to the present invention has excellent cytotoxicity for cancer cells, and thus can be useful in the treatment of immune cells for cancer treatment.

Description

메소텔린 특이적인 키메라 항원 수용체 및 이의 용도Mesothelin-specific chimeric antigen receptors and uses thereof
본 발명은 메소텔린 특이적인 키메라 항원 수용체 및 이를 발현하는 면역세포와 이의 용도에 관한 것이다.The present invention relates to mesothelin-specific chimeric antigen receptors, immune cells expressing the same, and uses thereof.
T 세포(T cells)는 적응성 면역반응(adaptive immunity)을 매개하는 중요한 역할을 하는 세포이다. T 세포는 항원을 인지할 수 있는 수용체(T cell receptor, TCR), 보조자극인자(co-stimulatory molecules), 그리고 사이토카인(cytokines) 자극을 통해서 활성화된다. 또한, T 세포의 TCR은 항원과 결합된 MHC 분자(major histocompatibility complex, MHC molecules)를 통해 면역반응을 일으키는데, 암 세포는 면역반응을 회피하기 위한 기전으로 MHC 분자의 발현을 억제한다. T 세포의 이러한 특성을 이용하여 면역세포 치료제(adoptive immune therapy, adoptive cellular therapy)가 개발되고 있다.T cells (T cells) are cells that play an important role in mediating adaptive immunity. T cells are activated through stimulation of antigen-recognizing receptors (T cell receptors, TCRs), co-stimulatory molecules, and cytokines. In addition, the TCR of T cells causes an immune response through antigen-bound MHC molecules (major histocompatibility complex, MHC molecules), and cancer cells suppress the expression of MHC molecules as a mechanism to evade the immune response. Using these characteristics of T cells, immune cell therapeutics (adoptive immune therapy, adoptive cellular therapy) are being developed.
T 세포가 MHC 분자 없이 직접 항원을 인지할 수 있도록 키메라 항원 수용체(chimeric antigen receptor, CAR)가 개발되고 있다. 키메라 항원 수용체는 항원을 인지하는 scFv(single chain variable fragment) 부분, 막관통 도메인(transmembrane domain), 세포 내로 신호를 전달하는 신호전달 도메인(signaling domain)으로 구성되어 있다. T 세포 내에 키메라 항원 수용체를 인위적으로 도입시켜 T 세포(CAR-T 세포)가 특정 항원을 가진 암에 대해 반응할 수 있도록 한다. 현재 미국의 Novartis, Gilead, Juno Therapeutics, Celgene 등과 같은 다국적 제약사들은 혈액암을 적응증으로 하여 CAR-T 세포를 이용하고 있지만, 고형암에 대한 연구는 아직 미진하다.Chimeric antigen receptors (CARs) are being developed so that T cells can directly recognize antigens without MHC molecules. The chimeric antigen receptor consists of an antigen-recognizing single chain variable fragment (scFv) part, a transmembrane domain, and a signaling domain that transmits a signal into a cell. By artificially introducing a chimeric antigen receptor into T cells, T cells (CAR-T cells) can respond to cancer with specific antigens. Currently, multinational pharmaceutical companies such as Novartis, Gilead, Juno Therapeutics, and Celgene in the US are using CAR-T cells for blood cancer indications, but studies on solid cancer are still insufficient.
메소텔린(Mesothelin, MSLN)은 40 kDa의 크기를 갖는 세포표면에 발현하는 당단백질이다. 일반 조직에서는 낮은 발현을 보이지만 중피암(Mesothelioma), 췌장암(Pancreatic cancer), 난소암(Ovarian cancer)을 비롯한 여러 종류의 고형암에서 과발현이 확인되었고, 항암표적으로 연구가 진행 중이다(Chang K 1996, Raffot H 2004). 이에 기반하여 Novartis와 협업하는 미국 펜실베니아 대학의 Carl H. June 박사그룹은 메소텔린을 표적하는 마우스 유래 항-메소텔린의 scFv을 가진 키메라 항원 수용체(ss1 CAR) 연구를 진행 중이다. ss1 CAR mRNA를 T 세포에 전달하여 일시적으로(transiently) CAR를 발현시켜 환자에게 투여했을 때, 환자로부터 항-마우스 항체 반응(human anti-mouse antibody response, HAMA response)이 일어났음이 확인되었다(Beatty GL 2014).Mesothelin (MSLN) is a glycoprotein expressed on the cell surface having a size of 40 kDa. Although it shows low expression in general tissues, overexpression has been confirmed in several types of solid cancer including mesothelioma, pancreatic cancer, and ovarian cancer, and research is ongoing as an anticancer target (Chang K 1996, Raffot) H 2004). Based on this, Dr. Carl H. June's group of the University of Pennsylvania, USA, in collaboration with Novartis, is conducting research on a chimeric antigen receptor (ss1 CAR) with a mouse-derived anti-mesothelin scFv that targets mesothelin. When ss1 CAR mRNA was delivered to T cells to transiently express CAR and administered to a patient, it was confirmed that a human anti-mouse antibody response (HAMA response) occurred from the patient (Beatty) GL 2014).
본 발명자들은 기존의 항-마우스 항체 반응 문제를 해소하기 위해, 메소텔린을 특이적으로 표적하는 인간 유래 scFv를 포함하는 3세대 키메라 항원 수용체를 발현하는 T 세포를 제작하여, 보다 우수한 항암 효과를 나타내는 것을 확인하고, 본 발명을 완성하였다.In order to solve the existing anti-mouse antibody response problem, the present inventors produced T cells expressing a third-generation chimeric antigen receptor containing a human-derived scFv that specifically targets mesothelin, thereby exhibiting superior anticancer effect. It was confirmed that the present invention was completed.
본 배경기술 부분에 기재된 상기 정보는 오직 본 발명의 배경에 대한 이해를 향상시키기 위한 것이며, 이에 본 발명이 속하는 기술분야에서 통상의 지식을 가지는 자에게 있어 이미 알려진 선행기술을 형성하는 정보를 포함하지 않을 수 있다.The information described in the background section is only for improving the understanding of the background of the present invention, and it does not include information forming prior art known to those of ordinary skill in the art to which the present invention pertains. may not be
발명의 요약Summary of the invention
본 발명의 목적은 기존의 메소텔린 결합 도메인을 포함하는 키메라 항원 수용체를 발현하는 T 세포를 이용한 항암 면역세포 치료의 문제점을 해결한, 높은 항암 효과를 나타내는 새로운 메소텔린 결합 도메인을 포함하는 키메라 항원 수용체 및 상기 키메라 항원 수용체를 발현하는 면역세포를 제공하는 데 있다.An object of the present invention is to solve the problem of anticancer immune cell therapy using T cells expressing a chimeric antigen receptor containing a mesothelin binding domain, and a chimeric antigen receptor comprising a new mesothelin binding domain showing a high anticancer effect. And to provide an immune cell expressing the chimeric antigen receptor.
본 발명의 또 다른 목적은 상기 면역세포를 포함하는 암 치료용 조성물, 이를 이용한 암 치료방법, 암 치료를 위한 상기 면역세포의 용도 및 암 치료용 약제 제조를 위한 상기 면역세포의 사용을 제공하는 데 있다.Another object of the present invention is to provide a composition for cancer treatment comprising the immune cells, a cancer treatment method using the same, the use of the immune cells for cancer treatment, and the use of the immune cells for the manufacture of a medicament for the treatment of cancer. have.
상기 목적을 달성하기 위하여, 본 발명은 메소텔린(Mesothelin, MSLN)에 특이적으로 결합하는 결합 도메인을 포함하는 키메라 항원 수용체를 제공한다.In order to achieve the above object, the present invention provides a chimeric antigen receptor comprising a binding domain that specifically binds to mesothelin (MSLN).
본 발명은 또한, 메소텔린 특이적인 키메라 항원 수용체를 코딩하는 핵산, 상기 핵산을 포함하는 발현 벡터 및 상기 키메라 항원 수용체를 발현하는 면역세포를 제공한다.The present invention also provides a nucleic acid encoding a mesothelin-specific chimeric antigen receptor, an expression vector comprising the nucleic acid, and an immune cell expressing the chimeric antigen receptor.
본 발명은 또한, 상기 면역세포를 포함하는 암 치료용 조성물, 이를 이용한 암 치료방법, 암 치료를 위한 상기 면역세포의 용도 및 암 치료용 약제 제조를 위한 상기 면역세포의 사용을 제공한다.The present invention also provides a cancer treatment composition comprising the immune cells, a cancer treatment method using the same, the use of the immune cells for cancer treatment, and the use of the immune cells for preparing a medicament for cancer treatment.
도 1은 본 발명에 따른 MS501 scFv의 결합 정도를 나타낸 것으로, 도 1A는 재조합 인간 메소텔린(recombinant human mesothelin, rhMSLN)에 대한 결합 정도를 효소 결합 면역 침강 분석법(Enzyme-linked immunosorbent assay, ELISA)으로 나타낸 것이며, 도 1B는 MIA PaCa2-MSLN 세포에 대한 세포 결합 정도를 유세포 분석을 통해 나타낸 것이다.1 shows the degree of binding of MS501 scFv according to the present invention, and FIG. 1A shows the degree of binding to recombinant human mesothelin (rhMSLN) by enzyme-linked immunosorbent assay (ELISA). and FIG. 1B shows the degree of cell binding to MIA PaCa2-MSLN cells through flow cytometry.
도 2는 본 발명의 CAR 구조(501(28H)28BBz)의 모식도를 나타낸다.Fig. 2 shows a schematic diagram of a CAR structure (501(28H)28BBz) of the present invention.
도 3은 인간 T 세포로 형질도입된 501(28H)28BBz의 발현율을 유세포 분석을 통해 나타낸 것이다.3 shows the expression rate of 501(28H)28BBz transduced into human T cells through flow cytometry.
도 4는 501(28H)28BBz로 형질도입된 인간 T 세포에서의 메소텔린 특이적인 반응에 따른 살해능을 나타낸다.Figure 4 shows the killing ability according to the mesothelin-specific response in human T cells transduced with 501(28H)28BBz.
도 5는 MIA PaCa-2(Parental) 세포를 이용해 제작한 췌장암 동소이식 마우스 모델(off-target 모델)에서 501(28H)28BBz CAR-T 세포의 효능을 나타낸 도면으로, Vehicle, Mock CAR-T 및 High dose(복강주사 및 정맥주사), Low dose(복강주사)의 501(28H)28BBz CAR-T를 각각 주입 후, 8주간 마우스의 종양 크기를 Bioluminescence Images로 모니터링 하였다.5 is a diagram showing the efficacy of 501(28H)28BBz CAR-T cells in a pancreatic cancer orthotopic mouse model (off-target model) prepared using MIA PaCa-2 (Parental) cells, Vehicle, Mock CAR-T and After each injection of 501(28H)28BBz CAR-T of high dose (intraperitoneal injection and intravenous injection) and low dose (intraperitoneal injection), the tumor size of mice was monitored by Bioluminescence Images for 8 weeks.
도 6은 Mesothelin expressed MIA PaCa 세포를 이용해 제작한 췌장암 동소이식 마우스 모델(on-target 모델)에서 501(28H)28BBz CAR-T 세포의 효능을 나타낸 도면으로, Vehicle, Mock CAR-T 및 High dose(복강주사 및 정맥주사), Low dose(복강주사)의 501(28H)28BBz CAR-T를 각각 주입 후, 8주간 마우스의 종양 크기를 Bioluminescence Images로 모니터링 하였다.6 is a diagram showing the efficacy of 501(28H)28BBz CAR-T cells in a pancreatic cancer orthotopic mouse model (on-target model) prepared using Mesothelin-expressed MIA PaCa cells. Vehicle, Mock CAR-T and High dose ( After intraperitoneal injection and intravenous injection) and 501(28H)28BBz CAR-T of low dose (intraperitoneal injection), respectively, the tumor size of mice was monitored by Bioluminescence Images for 8 weeks.
도 7은 췌장암 동소이식 마우스 모델에서 501(28H)28BBz CAR-T 세포의 병리학적 효능을 나타낸 도면으로, Vehicle, Mock CAR-T 및 High dose(복강주사 및 정맥주사), Low dose(복강주사)의 501(28H)28BBz CAR-T를 각각 주입 후, 8주 혹은 12주 뒤 마우스를 희생하여 췌장을 H&E 염색하였다.7 is a diagram showing the pathological efficacy of 501(28H)28BBz CAR-T cells in an orthotopic pancreatic cancer mouse model, Vehicle, Mock CAR-T and High dose (intraperitoneal injection and intravenous injection), Low dose (intraperitoneal injection) After injection of 501(28H)28BBz CAR-T, mice were sacrificed 8 or 12 weeks later, and the pancreas was H&E-stained.
발명의 상세한 설명 및 바람직한 구현예DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.
본 발명은 일 관점에서, 메소텔린(Mesothelin; MSLN)에 특이적으로 결합하는 결합 도메인, 신호서열(signal peptide), 힌지(hinge), 막관통 도메인(transmembrane domain) 및 3개 이상의 세포 내 도메인을 포함하는 키메라 항원 수용체(Chimeric antigen receptor; CAR)에 관한 것이다.In one aspect, the present invention provides a binding domain that specifically binds to mesothelin (MSLN), a signal peptide, a hinge, a transmembrane domain, and three or more intracellular domains. It relates to a chimeric antigen receptor (CAR) comprising:
본 발명에 있어서, 상기 메소텔린에 특이적으로 결합하는 결합 도메인은 항-메소텔린 항체 또는 그의 단편인 것이 바람직하지만, 이에 제한되는 것은 아니다.In the present invention, the binding domain specifically binding to mesothelin is preferably an anti-mesothelin antibody or fragment thereof, but is not limited thereto.
본 발명에서, 항체의 “단편”은, 항원 결합 기능을 보유하고 있는 단편을 의미하며, scFv, Fab, F(ab’)2 및 Fv 단편 등을 포함하는 의미로 사용된다.In the present invention, the term "fragment" of an antibody refers to a fragment having an antigen-binding function, and is used to include scFv, Fab, F(ab') 2 and Fv fragment.
“단일쇄 Fv” 또는 “scFv” 항체 단편은 항체의 VH 및 VL 도메인을 포함하는데, 이들 도메인은 단일 폴리펩티드 쇄 내에 존재한다. Fv 폴리펩티드는 scFv가 항원 결합을 위해 목적하는 구조를 형성할 수 있도록 하는 VH 도메인과 VL 도메인 사이에 폴리펩티드 링커를 추가로 포함할 수 있다.A “single chain Fv” or “scFv” antibody fragment comprises the VH and VL domains of an antibody, which domains are present within a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
“Fv” 단편은 완전한 항체 인식 및 결합 부위를 함유하는 항체 단편이다. 이러한 영역은 1개의 중쇄 가변 도메인과 1개의 경쇄 가변 도메인이, 예를 들어 scFv로 단단하게 사실상 공유적으로 연합된 이량체로 이루어진다.An “Fv” fragment is an antibody fragment that contains a complete antibody recognition and binding site. This region consists of a dimer in which one heavy chain variable domain and one light chain variable domain are tightly and substantially covalently associated, eg, scFv.
“Fab” 단편은 경쇄의 가변 및 불변 도메인과, 중쇄의 가변 및 제1 불변 도메인(CH1)을 함유한다. “F(ab’)2” 항체 단편은 일반적으로 그들 사이에 힌지 시스테인에 의해 그들의 카복시 말단 근처에 공유적으로 연결되는 한 쌍의 Fab 단편을 포함한다.A “Fab” fragment contains the variable and constant domains of the light chain and the variable and first constant domains (CH1) of the heavy chain. “F(ab′) 2 ” antibody fragments generally comprise a pair of Fab fragments covalently linked near their carboxy terminus by a hinge cysteine between them.
본 발명에 있어서, 상기 키메라 항원 수용체에 포함된 항-메소텔린 항체 또는 그의 단편은 서열번호 21의 아미노산 서열을 포함하는 중쇄 CDR1, 서열번호 22의 아미노산 서열을 포함하는 중쇄 CDR2, 서열번호 23의 아미노산 서열을 포함하는 중쇄 CDR3를 포함하는 중쇄 가변영역, 및 서열번호 24의 아미노산 서열을 포함하는 경쇄 CDR1, 서열번호 25의 아미노산 서열을 포함하는 경쇄 CDR2 및 서열번호 26의 아미노산 서열을 포함하는 경쇄 CDR3를 포함하는 경쇄 가변영역을 함유하는 것을 특징으로 할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the anti-mesothelin antibody or fragment thereof contained in the chimeric antigen receptor is a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and the amino acid of SEQ ID NO: 23 a heavy chain variable region comprising a heavy chain CDR3 comprising the sequence, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26 It may be characterized as containing a light chain variable region comprising, but is not limited thereto.
본 발명에 있어서, 상기 키메라 항원 수용체에 포함된 항-메소텔린 항체 또는 그의 단편은 서열번호 19의 아미노산 서열을 포함하는 중쇄 가변영역 및 서열번호 20의 아미노산 서열을 포함하는 경쇄 가변영역을 함유하는 것을 특징으로 할 수 있다.In the present invention, the anti-mesothelin antibody or fragment thereof contained in the chimeric antigen receptor contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20. can be characterized.
상기 키메라 항원 수용체에 포함된 항-메소텔린 항체 또는 그의 단편은 바람직하게는 scFv(단일-사슬 가변 단편)의 형태로서, 서열번호 2로 표시되는 아미노산 서열을 포함할 수 있지만, 이에 제한되는 것은 아니다.The anti-mesothelin antibody or fragment thereof contained in the chimeric antigen receptor is preferably in the form of an scFv (single-chain variable fragment), and may include the amino acid sequence shown in SEQ ID NO: 2, but is not limited thereto. .
본 발명에 있어서, 상기 키메릭 항원 수용체는 메소텔린에 특이적으로 결합하는 결합 도메인과 함께 추가적으로 신호서열(signal peptide; SP), 힌지(hinge), 막관통 도메인(transmembrane domain; TM) 및 세포 내 도메인을 포함하는 것을 특징으로 할 수 있다. 상기 세포 내 도메인은 3개 이상을 포함하는 것을 특징으로 할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the chimeric antigen receptor is additionally a signal peptide (SP), a hinge, a transmembrane domain (TM) and an intracellular binding domain that specifically binds to mesothelin. It may be characterized by including a domain. The intracellular domain may be characterized as including three or more, but is not limited thereto.
본 발명에 있어서, 상기 신호서열은 CD8α 신호서열인 것이 바람직하지만, 이에 제한되는 것은 아니며, 상기 CD8α 신호서열은 서열번호 4로 표시되는 아미노산 서열을 포함하는 것을 특징으로 할 수 있다.In the present invention, the signal sequence is preferably a CD8α signal sequence, but is not limited thereto, and the CD8α signal sequence may include the amino acid sequence shown in SEQ ID NO: 4.
본 발명에 있어서, 상기 신호서열과 메소텔린에 특이적으로 결합하는 결합 도메인은 키메라 항원 수용체의 세포 외 도메인을 구성한다. 세포 외 도메인은 주신호가 전달되는 부위로 세포막 외부에 존재하며 메소텔린을 특이적으로 인식하기 위한 도메인이다.In the present invention, the signal sequence and the binding domain specifically binding to mesothelin constitute the extracellular domain of the chimeric antigen receptor. The extracellular domain is a site through which a main signal is transmitted, exists outside the cell membrane, and is a domain for specifically recognizing mesothelin.
본 발명에 있어서, 상기 막관통 도메인은 상기 세포 외 도메인과 상기 세포 내 신호전달 도메인을 세포막 사이로 연결 가능하다면 어떠한 것이든 사용 가능하다. 바람직하게는 CD28 유래의 막관통 도메인 및/또는 CD8α 유래의 막관통 도메인으로 이루어지며, 상기 CD28 유래의 막관통 도메인 및/또는 CD8α 유래의 막관통 도메인의 전체 또는 일부를 포함하는 것일 수 있다.In the present invention, the transmembrane domain can be any type as long as it can connect the extracellular domain and the intracellular signaling domain between the cell membrane. Preferably, it consists of a CD28-derived transmembrane domain and/or a CD8α-derived transmembrane domain, and may include all or part of the CD28-derived transmembrane domain and/or CD8α-derived transmembrane domain.
가장 바람직하게는 상기 막관통 도메인은 서열번호 8로 표시되는 아미노산 서열을 포함할 수 있지만, 이에 제한되는 것은 아니다.Most preferably, the transmembrane domain may include the amino acid sequence represented by SEQ ID NO: 8, but is not limited thereto.
본 발명의 일 실시예에 따르면, 상기 세포 외 도메인과 상기 막관통 도메인은 스페이서 도메인으로 연결될 수 있다.According to an embodiment of the present invention, the extracellular domain and the transmembrane domain may be connected by a spacer domain.
바람직하게는 상기 스페이서 도메인은 힌지 도메인일 수 있다. 상기 키메릭 항원 수용체에 포함될 수 있는 스페이서 도메인은 CD28 유래의 힌지 도메인 및/또는 CD8α 유래의 힌지 도메인을 포함할 수 있으며, 상기 CD28 유래의 힌지 도메인 및/또는 CD8α 유래의 힌지 도메인의 전체 또는 일부를 포함하는 것일 수 있다. CD28 및 CD8α로서 전체 또는 일부를 포함하는 것일 수 있다.Preferably, the spacer domain may be a hinge domain. The spacer domain that may be included in the chimeric antigen receptor may include a hinge domain derived from CD28 and/or a hinge domain derived from CD8α, and all or part of the hinge domain derived from CD28 and/or a hinge domain derived from CD8α may include. It may include all or part of CD28 and CD8α.
가장 바람직하게는 상기 힌지 도메인은 서열번호 6으로 표시되는 아미노산 서열을 포함할 수 있으나, 이에 제한되는 것은 아니다.Most preferably, the hinge domain may include the amino acid sequence represented by SEQ ID NO: 6, but is not limited thereto.
본 발명에 있어서, 상기 세포 내 신호전달 도메인은 면역세포의 세포막 안쪽, 즉 세포질에 위치하게 되는 부분으로서, 세포 외 도메인에 결합된 결합 도메인이 표적 항원에 결합하였을 때, 면역세포의 면역반응을 활성화시키는 부위를 의미한다.In the present invention, the intracellular signaling domain is a part located inside the cell membrane of immune cells, that is, in the cytoplasm, and when the binding domain bound to the extracellular domain binds to the target antigen, it activates the immune response of the immune cell. means the area to be made.
본 발명에 따른 키메라 항원 수용체는 3개 이상의 세포 내 신호전달 도메인을 포함하는 것을 특징으로 할 수 있다. 3개 이상의 세포 내 신호전달 도메인을 포함하는 경우에는 세포 내, 신호전달 도메인들이 서로 직렬로 연결될 수 있다.The chimeric antigen receptor according to the present invention may be characterized as comprising three or more intracellular signaling domains. In the case of including three or more intracellular signaling domains, the intracellular and signaling domains may be serially connected to each other.
본 발명에 있어서, 상기 세포 내 신호전달 도메인은 CD28, 4-1BB 및 CD3ζ의 세포 내 신호영역 서열, 또는 이들의 조합으로 구성된 군에서 선택되는 서열을 포함하는 것을 특징으로 할 수 있다. 바람직하게는 CD28 유래의 세포 내 신호전달 도메인 및 4-1BB 유래의 세포 내 신호전달 도메인이 CD3ζ 유래의 세포 내 신호전달 도메인과 연결된 것일 수 있으며, 가장 바람직하게는 서열번호 10, 12 또는 14로 표시되는 아미노산 서열을 포함할 수 있지만, 이에 제한되는 것은 아니다.In the present invention, the intracellular signaling domain may include a sequence selected from the group consisting of intracellular signaling domain sequences of CD28, 4-1BB and CD3ζ, or a combination thereof. Preferably, the intracellular signaling domain derived from CD28 and the intracellular signaling domain derived from 4-1BB may be linked to the intracellular signaling domain derived from CD3ζ, and most preferably represented by SEQ ID NO: 10, 12 or 14 It may include, but is not limited to, an amino acid sequence.
구체적인 일 실시예에 따르면, MS501 scFv를 포함하는 다양한 조합의 CAR 구조를 이용한 인-비보(in-vivo) 실험에서, MS501 scFv를 CD28 막관통 도메인, CD28 유래, 4-1BB 유래 및 CD3ζ 유래의 세포 내 신호 전달 도메인과 연결한 조합(501(28H)28BBz)이 대조군에 비해 종양의 크기를 감소시키는 예기치 못한 우수한 항암 효과를 나타냈다.According to a specific embodiment, in an in-vivo experiment using various combinations of CAR structures including MS501 scFv, MS501 scFv was transformed into CD28 transmembrane domain, CD28-derived, 4-1BB-derived and CD3ζ-derived cells. The combination (501(28H)28BBz) linked to my signal transduction domain exhibited an unexpectedly excellent anticancer effect in reducing the size of the tumor compared to the control group.
본 발명에 있어서, 상기 키메라 항원 수용체는 서열번호 16으로 표시되는 아미노산 서열, 또는 상기 아미노산 서열과 80% 이상, 바람직하게는 90% 이상, 더욱 바람직하게는 95% 이상, 가장 바람직하게는 99% 이상의 서열 동일성을 갖는 이의 변이체를 포함하는 것일 수 있다.In the present invention, the chimeric antigen receptor is 80% or more, preferably 90% or more, more preferably 95% or more, most preferably 99% or more of the amino acid sequence represented by SEQ ID NO: 16 or the amino acid sequence. It may include a variant thereof having sequence identity.
본 발명은 다른 관점에서, 상기 키메라 항원 수용체를 코딩하는 핵산에 관한 것이다.In another aspect, the present invention relates to a nucleic acid encoding the chimeric antigen receptor.
본 발명에 따른 키메라 항원 수용체를 코딩하는 핵산(폴리뉴클레오타이드)은 코돈 최적화에 의해 변형될 수 있으며, 이는 코돈의 축퇴성(degeneracy)에서 기인한 것으로, 폴리펩타이드 또는 이의 변이체 단편을 코딩하는 많은 뉴클레오타이드 서열이 존재한다는 것은 통상의 기술자가 잘 이해할 수 있을 것이다. 이들 폴리뉴클레오타이드(핵산)의 일부는 임의의 자연 발생형 유전자의 뉴클레오타이드 서열과 최소 상동성을 보유한다.The nucleic acid (polynucleotide) encoding the chimeric antigen receptor according to the present invention can be modified by codon optimization, which is due to the degeneracy of codons, and many nucleotide sequences encoding the polypeptide or variant fragment thereof. The existence of this will be well understood by those skilled in the art. Some of these polynucleotides (nucleic acids) possess minimal homology with the nucleotide sequence of any naturally occurring gene.
특히 코돈 활용법의 차이로 인해 가변적인 폴리뉴클레오타이드(핵산), 예를 들어 인간, 영장류 및/또는 포유동물의 코돈 선택에 최적화된 폴리뉴클레오타이드(핵산)가 바람직하다.In particular, variable polynucleotides (nucleic acids) due to differences in codon utilization are preferred, for example, polynucleotides (nucleic acids) optimized for codon selection in humans, primates and/or mammals.
본 발명의 일 실시예에 따르면, 상기 핵산 서열은 15로 표시되는 뉴클레오타이드 서열, 또는 상기 뉴클레오타이드 서열과 80% 이상, 바람직하게는 90% 이상, 더욱 바람직하게는 95% 이상, 가장 바람직하게는 99% 이상의 서열 동일성을 갖는 변이체를 포함하는 것일 수 있다.According to an embodiment of the present invention, the nucleic acid sequence is the nucleotide sequence represented by 15, or 80% or more, preferably 90% or more, more preferably 95% or more, most preferably 99% of the nucleotide sequence and the nucleotide sequence. It may include a variant having more than one sequence identity.
본 발명에 있어서, 상기 키메라 항원 수용체를 코딩하는 핵산은,In the present invention, the nucleic acid encoding the chimeric antigen receptor comprises:
서열번호 3으로 표시되는 것을 특징으로 하는 CD8α 신호서열을 코딩하는 뉴클레오타이드 서열; 서열번호 1로 표시되는 것을 특징으로 하는 항-메소텔린 항체의 단일-사슬 가변 단편(scFv)을 코딩하는 뉴클레오타이드 서열; 서열번호 5로 표시되는 것을 특징으로 하는 CD28 힌지를 코딩하는 뉴클레오타이드 서열; 서열번호 7로 표시되는 것을 특징으로 하는 CD28 막관통 도메인을 코딩하는 뉴클레오타이드 서열; 서열번호 9, 서열번호 11 또는 서열번호 13으로 표시되는 것을 특징으로 하는 CD28, 4-1BB 또는 CD3ζ 세포 내 신호영역을 코딩하는 뉴클레오타이드 서열;을 포함하는 것을 특징할 수 있으나, 이에 제한되는 것은 아니다.A nucleotide sequence encoding a CD8α signal sequence, characterized in that represented by SEQ ID NO: 3; A nucleotide sequence encoding a single-chain variable fragment (scFv) of an anti-mesothelin antibody, characterized in that shown in SEQ ID NO: 1; A nucleotide sequence encoding a CD28 hinge, characterized in that shown in SEQ ID NO: 5; A nucleotide sequence encoding the CD28 transmembrane domain, characterized in that represented by SEQ ID NO: 7; SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, characterized in that the CD28, 4-1BB or CD3ζ cell signal region encoding a nucleotide sequence;
본 발명은 또 다른 관점에서, 상기 핵산을 포함하는 발현 벡터 및 상기 발현 벡터를 포함하는 바이러스에 관한 것이다.In another aspect, the present invention relates to an expression vector comprising the nucleic acid and a virus comprising the expression vector.
본 발명의 용어 “벡터”란, 다른 핵산 분자를 전이시키거나 수송할 수 있는 핵산 분자를 의미하는 것이다. 전이된 핵산은 일반적으로 벡터 핵산 분자에 연결되는데, 예를 들면, 벡터 핵산 분자 내에 삽입된다. 벡터는 세포에서의 자율적 복제를 지시하는 서열을 포함할 수 있거나, 숙주 세포 DNA 내로의 통합을 가능하게 하는데 충분한 서열을 포함할 수 있다. 상기 벡터는 DNA, RNA, 플라스미드, 렌티바이러스 벡터, 아데노바이러스 벡터 및 레트로바이러스 벡터로 구성된 군에서 선택되는 것을 특징으로 할 수 있으나, 이에 제한되는 것은 아니다.As used herein, the term “vector” refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to a vector nucleic acid molecule, eg, inserted into a vector nucleic acid molecule. A vector may include sequences that direct autonomous replication in the cell, or it may include sequences sufficient to permit integration into host cell DNA. The vector may be characterized in that it is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector and retroviral vector, but is not limited thereto.
본 발명에 있어서, “바이러스”는 암의 치료 등에 사용하기 위해, 본 발명의 키메라 항원 수용체를 발현하도록 유전적으로 변형된 것을 의미한다. 유전적으로 변형되었다는 것은 세포에서 전체 유전자 재료로 DNA 또는 RNA의 형태로 외부 유전자 재료를 부가하는 것이다.In the present invention, "virus" means a genetically modified one to express the chimeric antigen receptor of the present invention for use in the treatment of cancer. Genetically modified is the addition of foreign genetic material in the form of DNA or RNA into the entire genetic material in a cell.
본 발명에 있어서, 상기 핵산 또는 상기 벡터는 바이러스에 형질주입 또는 트랜스펙션(transfection)된다. “형질주입” 또는 “트랜스펙션”시키기 위해 원핵 또는 진핵 숙주세포 내로 외인성 핵산(DNA 또는 RNA)을 도입하는 데에 통상 사용되는 여러 종류의 다양한 기술, 예를 들어 전기 영동법, 인산칼슘 침전법, DEAE-덱스트란 트랜스펙션 또는 리포펙션(lipofection) 등을 사용할 수 있다.In the present invention, the nucleic acid or the vector is transfected or transfected with a virus. A number of different techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells for “transfection” or “transfection”, such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection or lipofection may be used.
본 발명은 또 다른 관점에서, 상기 키메라 항원 수용체를 표면에 발현하는 면역세포에 관한 것이다.In another aspect, the present invention relates to an immune cell expressing the chimeric antigen receptor on its surface.
본 발명에 있어서, 상기 면역세포는 T 세포, NK 세포 또는 NKT 세포인 것을 특징으로 할 수 있으나, 이에 제한되는 것은 아니며, 바람직하게는 T 세포인 것을 특징으로 할 수 있다.In the present invention, the immune cells may be characterized as T cells, NK cells or NKT cells, but is not limited thereto, and preferably T cells.
본 발명에 따른 키메라 항원 수용체를 발현하는 면역세포는 CAR-T 세포(Chimeric Antigen Receptor T Cell), CAR-NK 세포(Chimeric Antigen Receptor Natural Killer Cell) 또는 CAR-NKT 세포(Chimeric Antigen Receptor Natural killer T Cell)인 것을 특징으로 할 수 있다.Immune cells expressing the chimeric antigen receptor according to the present invention are CAR-T cells (Chimeric Antigen Receptor T Cells), CAR-NK cells (Chimeric Antigen Receptor Natural Killer Cells), or CAR-NKT cells (Chimeric Antigen Receptor Natural killer T Cells). ) may be characterized as
본 발명에 있어서, 상기 T 세포는 세포독성 T 림프구(Cytotoxic T lymphocyte; CTL); 종양 침윤 림프구(Tumor infiltrating lymphocyte; TIL) 및 말초혈액 단핵세포(Peripheral blood mononuclear cell; PBMC)에서 분리한 T 세포로 구성된 군에서 선택되는 것을 특징으로 할 수 있다.In the present invention, the T cells are cytotoxic T lymphocytes (CTL); It may be characterized in that it is selected from the group consisting of T cells isolated from tumor infiltrating lymphocytes (TIL) and peripheral blood mononuclear cells (PBMC).
본 발명의 면역세포는 메소텔린을 발현하는 종양세포에 대해 독성을 나타내는 것일 수 있다. 일 실시예에 따르면, 본 발명의 면역세포(예컨대, T 세포)가 췌장암(Pancreatic cancer) 세포, 자궁경부암(Cervical cancer) 세포, 중피종(Mesothelioma) 세포 또는 난소암(Ovarian cancer) 세포에 대해 독성을 나타낼 수 있다. 예컨대, 상기 췌장암 세포, 자궁경부암 세포, 중피종 세포 또는 난소암 세포는 메소텔린을 발현하는 것일 수 있다.The immune cells of the present invention may be toxic to mesothelin-expressing tumor cells. According to one embodiment, the immune cells (eg, T cells) of the present invention are toxic to pancreatic cancer cells, cervical cancer cells, mesothelioma cells or ovarian cancer cells. can indicate For example, the pancreatic cancer cells, cervical cancer cells, mesothelioma cells, or ovarian cancer cells may express mesothelin.
본 발명은 또 다른 관점에서, 상기 키메라 항원 수용체를 발현하는 면역세포(예컨대, T 세포)를 암 치료용 조성물에 관한 것이다.In another aspect, the present invention relates to a composition for treating cancer using immune cells (eg, T cells) expressing the chimeric antigen receptor.
본 발명에 있어서, “암”과 “종양”은 동일한 의미로 사용되며, 전형적으로 조절되지 않은 세포 성장/증식을 특징으로 하는 포유동물의 생리학적 상태를 지칭하거나 의미한다.In the present invention, “cancer” and “tumor” are used interchangeably and refer to or mean a physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation.
암(또는 종양)의 예는 암종, 림프종(예를 들어, 호지킨 및 비-호지킨 림프종), 모세포종, 육종 및 백혈병을 포함하나, 이에 한정되지는 않는다. 보다 바람직한 암의 예는 편평세포암, 소세포 폐암, 비소세포 폐암, 폐의 선암종, 폐의 편평세포 암종, 복막암, 간세포성암, 위장암, 췌장암, 신경교종, 자궁경부암, 난소암, 간암, 방광암, 간세포암, 유방암, 결장암, 결장직장암, 자궁내막 또는 자궁 암종, 타액선 암종, 신장암, 간암, 전립선암, 외음부암, 갑상선암, 간 암종, 중피종, 백혈병 및 다른 림프구증식성 장애, 및 다양한 유형의 두경부암을 포함한다. 본 발명에서 상기 암은 바람직하게는 메소텔린-양성 암이고, 췌장암, 난소암, 폐암, 위암, 자궁내막암 및 중피종으로 구성된 군에서 선택되는 것을 특징으로 할 수 있다.Examples of cancer (or tumors) include, but are not limited to, carcinoma, lymphoma (eg, Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More preferred examples of cancer include squamous cell cancer, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer , hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, mesothelioma, leukemia and other lymphoproliferative disorders, and various types of including head and neck cancer. In the present invention, the cancer is preferably mesothelin-positive cancer, and may be characterized in that it is selected from the group consisting of pancreatic cancer, ovarian cancer, lung cancer, gastric cancer, endometrial cancer and mesothelioma.
본 발명의 치료용 조성물은 암의 예방 또는 치료를 위한 조성물로서, 본 발명의 용어, “예방”은 본 발명의 조성물의 투여로 암을 억제시키거나 진행을 지연시키는 모든 행위를 의미하며, “치료”는 암의 발전의 억제, 증상의 경감 또는 제거를 의미한다.The therapeutic composition of the present invention is a composition for preventing or treating cancer, and the term, “prevention” of the present invention, refers to any action that inhibits or delays the progression of cancer by administration of the composition of the present invention, and “treatment” ” means inhibiting the development of cancer, alleviating or eliminating symptoms.
상기 조성물에는 본 발명에 따른 키메라 항원 수용체를 발현하는 면역세포의 수가 치료 대상 내의 종양 세포(예컨대, 췌장암, 자궁경부암, 중피종 또는 난소암)수의 1배 내지 10배로 포함되는 것이 바람직하지만, 이에 한정되는 것은 아니다. In the composition, the number of immune cells expressing the chimeric antigen receptor according to the present invention is preferably 1 to 10 times the number of tumor cells (eg, pancreatic cancer, cervical cancer, mesothelioma or ovarian cancer) in the treatment target, but limited thereto it's not going to be
본 발명에 따른 키메라 항원 수용체를 발현하는 면역세포를 포함하는 약학 조성물에는 약제학적으로 허용되는 부형제가 추가적으로 포함될 수 있다. 그러한 부형제의 예로는, 계면활성제, 바람직하게는 폴리소르베이트 계열의 비이온성 계면활성제; 중성 완충 염수, 인간염 완충 염수 등의 완충제; 글루코스, 만노스, 수크로스 또는 덱스트란, 만니톨 등의 당 또는 당알콜류; 글리신, 히스티딘 등의 아미노산이나 단백질 또는 폴리펩티드; 항산화제; EDTA 또는 글루타티온 등의 킬레이트제 예컨대; 침투제; 보조제; 및 보존제가 포함될 수 있지만, 이에 한정되는 것은 아니다.The pharmaceutical composition comprising immune cells expressing the chimeric antigen receptor according to the present invention may additionally include a pharmaceutically acceptable excipient. Examples of such excipients include surfactants, preferably nonionic surfactants of the polysorbate series; buffers such as neutral buffered saline and human salt buffered saline; sugars or sugar alcohols such as glucose, mannose, sucrose or dextran and mannitol; amino acids, proteins, or polypeptides such as glycine and histidine; antioxidants; chelating agents such as EDTA or glutathione; penetrant; supplements; and preservatives, but are not limited thereto.
본 발명의 일 실시예에 따르면, 본 발명의 약학 조성물은 1회 투여량 내에 상기 면역세포(예컨대, T 세포)의 수가 치료 대상 내의 상기 종양세포, 예를 들어 췌장암 세포, 자궁경부암 세포, 중피종 세포 또는 난소암 세포 수의 1배 내지 10배로 포함된 것일 수 있다.According to one embodiment of the present invention, the pharmaceutical composition of the present invention is the number of the immune cells (eg, T cells) in one dose is the tumor cells in the subject to be treated, for example, pancreatic cancer cells, cervical cancer cells, mesothelioma cells Or it may be included in 1 to 10 times the number of ovarian cancer cells.
본 발명은 또 다른 관점에서, 상기 키메라 항원 수용체를 발현하는 면역세포를 대상체에 투여하는 단계를 포함하는 암 치료방법에 관한 것이다.In another aspect, the present invention relates to a method for treating cancer comprising administering to a subject an immune cell expressing the chimeric antigen receptor.
본 발명은 또한, 암 치료를 위한 상기 면역세포의 용도에 관한 것이다.The present invention also relates to the use of said immune cells for the treatment of cancer.
본 발명은 또한, 암 치료용 약제 제조를 위한 상기 면역세포의 사용에 관한 것이다.The present invention also relates to the use of said immune cells for the manufacture of a medicament for the treatment of cancer.
상기 대상체는 종양을 가진 포유류일 수 있으며, 구체적으로는 인간일 수 있으나, 이에 한정되는 것은 아니다.The subject may be a mammal having a tumor, specifically, a human, but is not limited thereto.
본 발명에 따른 키메라 항원 수용체를 발현하는 면역세포 또는 이를 포함하는 조성물은 경구투여, 주입(infusion), 정맥내 투여(intravenous injection), 근육내 투여(intramuscular injection), 피하 투여(subcutaneous injection), 복강내 투여(intraperitoneal injectoon), 직장내 투여(Intrarectal administration), 국소 투여(topical administration), 비내 투여(intranasal injection) 등으로 투여될 수 있지만, 이에 한정되는 것은 아니다.Immune cells expressing the chimeric antigen receptor according to the present invention or a composition comprising the same are administered orally, infusion, intravenous injection, intramuscular injection, subcutaneous injection, or intraperitoneal administration. It may be administered by intraperitoneal injectoon, intrarectal administration, topical administration, intranasal injection, etc., but is not limited thereto.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예 1: 방법 및 시약Example 1: Methods and Reagents
실시예 1-1: 세포주 및 세포주 배양Example 1-1: Cell lines and cell line culture
인간 췌장암 세포주 MIA PaCa2는 ATCC(American Type Culture Collection, Manassas, VA, USA)로부터 공급받아 사용하였다. MIA PaCa2는 10% FBS(fetal bovine serum; GIBCO, Grand Island, NY, USA)를 포함하는 DMEM(GIBCO, Grand Island, NY, USA)에서 유지되었다. 인간 배아 신장 섬유아세포(Human embryonic kidney fibroblast)인 HEK293T 세포주는 ATCC로부터 공급받아 사용하였고 10% FBS(GIBCO, Grand Island, NY, USA)를 포함하는 DMEM(GIBCO)에서 유지되었다. 메소텔린 과발현 인간 췌장암 세포주 MIA PaCa2-MSLN은 목암연구소(GC녹십자, Korea)로 부터 공급받아 사용하였다. MIA PaCa2-MSLN은 10% FBS(GIBCO)와 200 μg/ml Hygromycin B(GIBCO)를 포함하는 DMEM(GIBCO)에서 유지되었다.The human pancreatic cancer cell line MIA PaCa2 was supplied from ATCC (American Type Culture Collection, Manassas, VA, USA) and used. MIA PaCa2 was maintained in DMEM (GIBCO, Grand Island, NY, USA) containing 10% fetal bovine serum (GIBCO, Grand Island, NY, USA). The HEK293T cell line, a human embryonic kidney fibroblast, was supplied from ATCC and maintained in DMEM (GIBCO) containing 10% FBS (GIBCO, Grand Island, NY, USA). Mesothelin-overexpressing human pancreatic cancer cell line MIA PaCa2-MSLN was supplied from Mok Cancer Research Institute (GC Green Cross, Korea) and used. MIA PaCa2-MSLN was maintained in DMEM (GIBCO) containing 10% FBS (GIBCO) and 200 μg/ml Hygromycin B (GIBCO).
실시예 1-2: 인간 T 세포 분리 및 활성화Example 1-2: Human T Cell Isolation and Activation
기관생명윤리위원회(Institutional review board, IRB, Korea)의 승인을 받아 공여자를 모집하여 말초혈 단핵세포(Peripheral blood mononuclear cell, PBMC)를 피콜-하이파크(Ficoll-Hypaque; GE healthcare, Chicago, IL, USA)로 분리하였다. 분리된 PBMC는 바로 동결하였다. PBMC를 37℃에서 해동한 후, QuadroMACS™ Separator(Miltenyi Biotec, Bergisch Gladbach, Germany)와 LS Columns(Miltenyi Biotec), CD4 MicroBeads(Miltenyi Biotec), CD8 MicroBeads(Miltenyi Biotec)를 이용하여 양성선택(Positive selection) 방법으로 인간 T 세포를 분리하였다. 인간 T 세포는 MACS GMP T cell TransAct(Miltenyi Biotec)와 섞어 20~24시간 활성화시킨다. 특별한 언급이 없는 한, 인간 T 세포는 10% FBS(Thermo Fisher Scientific)과 200 IU/ml IL-2(Proleukin, Novartis, Basel, Switzerland)이 포함된 RPMI-1640(Thermo Fisher Scientific) 배지에서 배양되었다.With the approval of the institutional review board (IRB, Korea), donors were recruited and peripheral blood mononuclear cells (PBMCs) were transferred to Ficoll-Hypaque (GE healthcare, Chicago, IL, USA) was isolated. The isolated PBMCs were immediately frozen. After thawing PBMCs at 37°C, positive selection was performed using QuadroMACS™ Separator (Miltenyi Biotec, Bergisch Gladbach, Germany), LS Columns (Miltenyi Biotec), CD4 MicroBeads (Miltenyi Biotec), and CD8 MicroBeads (Miltenyi Biotec). ) to isolated human T cells. Human T cells are mixed with MACS GMP T cell TransAct (Miltenyi Biotec) and activated for 20 to 24 hours. Unless otherwise noted, human T cells were cultured in RPMI-1640 (Thermo Fisher Scientific) medium containing 10% FBS (Thermo Fisher Scientific) and 200 IU/ml IL-2 (Proleukin, Novartis, Basel, Switzerland). .
실시예 1-3: 재조합 인간 메소텔린에 대한 scFv의 결합 확인Example 1-3: Confirmation of binding of scFv to recombinant human mesothelin
다음의 과정을 거쳐 재조합 인간 메소텔린에 본 발명에 따른 scFv가 결합하는 것을 확인하였다.It was confirmed that the scFv according to the present invention binds to recombinant human mesothelin through the following procedure.
(i) MS501 scFv 단백질을 96-well immuno plate에 200ng/ml로 24시간 코팅한다.(i) MS501 scFv protein is coated on a 96-well immunoplate at 200ng/ml for 24 hours.
(ii) 세척용액으로 well을 세척한 후, 1% BSA(Bovine serum albumin, Sigma) 용액으로 1시간 블로킹(Blocking)하였다.(ii) After washing the wells with a washing solution, blocking was performed for 1 hour with 1% BSA (Bovine serum albumin, Sigma) solution.
(iii) 세척용액으로 well을 세척한 후, 100ng/ml의 재조합 인간 메소텔린 단백질을 2시간 처리하였다.(iii) After washing the wells with a washing solution, 100 ng/ml of recombinant human mesothelin protein was treated for 2 hours.
(iv) 세척용액으로 well을 세척한 후, 재조합 인간 메소텔린 탐지 항체(Detection antibody)를 분석증명서에 따라 희석하여 well에 분주한 후 1시간 처리하였다.(iv) After washing the wells with the washing solution, the recombinant human mesothelin detection antibody (Detection antibody) was diluted according to the certificate of analysis, dispensed into the wells, and treated for 1 hour.
(v) 세척용액으로 well을 세척한 후, 스트렙타비딘-HRP(Streptavidin-HRP)이 30분간 처리되었다.(v) After washing the wells with a washing solution, streptavidin-HRP was treated for 30 minutes.
(vi) 세척용액으로 well을 세척한 후, 기질 용액(Substrate solution)이 15분간 처리되었다.(vi) After washing the wells with the washing solution, the substrate solution was treated for 15 minutes.
(vii) 정지 용액(Stop solution)을 처리하고 450nm 흡광도에서 측정되었다.(vii) Stop solution was treated and the absorbance was measured at 450 nm.
실시예 1-4: 메소텔린 과발현 세포와의 결합 확인Example 1-4: Confirmation of binding to mesothelin overexpressing cells
MS501 scFv 단백질의 메소텔린 과발현 세포주인 MIA PaCa2-MSLN에 대한 결합 정도를 확인한다. MIA PaCa2-MSLN 세포주를 1 x 105 cells/100 μl로 FACS 완충용액에 부유하여 준비한다. MS501 scFv 단백질을 1 μg 처리하였다. 세포들은 FACS 완충액을 이용하여 두 차례 세척되었다. 세포들은 항-6xHis tag 항체(Biolegend)를 사용하여 염색하였다. 양성대조군(positive control) 시료는 항-메소텔린 항체(R&D system)로 염색하였다. 염색된 세포들의 발현비율 및 평균 형광강도(Mean fluorescence intensity, MFI)는 BD LSRFortessa를 사용하여 측정되었고, 플로우조(FlowJo) 소프트웨어에서 분석되었다.The degree of binding of MS501 scFv protein to MIA PaCa2-MSLN, a mesothelin-overexpressing cell line, is confirmed. Prepare the MIA PaCa2-MSLN cell line by floating in FACS buffer at 1 x 10 5 cells/100 μl. MS501 scFv protein was treated with 1 μg. Cells were washed twice using FACS buffer. Cells were stained using anti-6xHis tag antibody (Biolegend). A positive control sample was stained with an anti-mesothelin antibody (R&D system). Expression ratio and mean fluorescence intensity (MFI) of stained cells were measured using BD LSRFortessa and analyzed in FlowJo software.
실시예 1-5: 키메라 항원 수용체 구축Example 1-5: Chimeric Antigen Receptor Construction
본 발명의 키메라 항원 수용체를 제작하기 위하여 SOE-PCR(Splicing by overlapping extension by polymerase chain reaction)을 통해 인공적으로 합성되었다. 좀 더 상세하게는, 세포 외 도메인: CD8α의 신호서열, scFv 서열 (MS501 scFv서열), CD28의 힌지; CD28의 막관통 도메인; 세포 내 도메인: CD28, 4-1BB, CD3ζ의 세포 내 신호전달 도메인은 SOE-PCR을 통해 인공적으로 합성되었다. 이들은 EcoR1 및 BamH1으로 절단된 후 3세대 자가-불활성화 렌티바이러스 발현 벡터인 EF1a-MCS 벡터의 EcoR1 및 BamH1 사이트에 삽입되었다.In order to produce the chimeric antigen receptor of the present invention, it was artificially synthesized through SOE-PCR (Splicing by overlapping extension by polymerase chain reaction). More specifically, the extracellular domain: CD8α signal sequence, scFv sequence (MS501 scFv sequence), CD28 hinge; the transmembrane domain of CD28; Intracellular domains: The intracellular signaling domains of CD28, 4-1BB, and CD3ζ were artificially synthesized through SOE-PCR. They were digested with EcoR1 and BamH1 and then inserted into the EcoR1 and BamH1 sites of the EF1a-MCS vector, a third-generation self-inactivating lentiviral expression vector.
본 발명에 따른 키메라 항원 수용체(chimeric antigen receptor, CAR)를 하기 표 1에 정리하였다. 본 실시예에 따른 CAR의 도메인들은 서로 직렬로(in tandem) 연결된 것이며, 또한 인프레임(in frame)으로 연결된 것이다. 501(28H)28BBz는 인간 CD8α의 신호서열 도메인(890-952 뉴클레오타이드, GenBank NM 001768.6); MS501 IgG scFv 도메인(출원번호: 10-2015-0135755); 인간 CD28 유래의 힌지; 인간 CD28 유래의 막관통 도메인; 인간 CD28 유래의 세포 내 신호 전달 도메인(439-759 뉴클레오타이드, GenBank J02988.1); 인간 4-1BB 유래의 세포 내 신호전달 도메인(901-1026 뉴클레오타이드, GenBank NM001561.5); 및 CD3ζ 유래의 세포 내 신호전달 도메인(299-634 뉴클레오타이드, GenBank NM000734.3)과 stop codon TGA가 연결된 것이다.The chimeric antigen receptor (CAR) according to the present invention is summarized in Table 1 below. The domains of the CAR according to the present embodiment are connected in tandem with each other and are also connected in-frame. 501(28H)28BBz is the signal sequence domain of human CD8α (890-952 nucleotides, GenBank NM 001768.6); MS501 IgG scFv domain (Application No. 10-2015-0135755); hinge from human CD28; transmembrane domain from human CD28; intracellular signaling domain derived from human CD28 (439-759 nucleotides, GenBank J02988.1); an intracellular signaling domain derived from human 4-1BB (901-1026 nucleotides, GenBank NM001561.5); and a CD3ζ-derived intracellular signaling domain (nucleotides 299-634, GenBank NM000734.3) linked to a stop codon TGA.
Figure PCTKR2020014394-appb-T000001
Figure PCTKR2020014394-appb-T000001
본 실시예에 따른 CAR 및 그의 제조에 사용된 도메인들의 서열 정보를 하기 표 2에 정리하였다.The sequence information of the domains used for the CAR and its preparation according to this Example is summarized in Table 2 below.
Figure PCTKR2020014394-appb-T000002
Figure PCTKR2020014394-appb-T000002
Figure PCTKR2020014394-appb-I000001
Figure PCTKR2020014394-appb-I000001
Figure PCTKR2020014394-appb-I000002
Figure PCTKR2020014394-appb-I000002
Figure PCTKR2020014394-appb-I000003
Figure PCTKR2020014394-appb-I000003
Figure PCTKR2020014394-appb-I000004
Figure PCTKR2020014394-appb-I000004
본 실시예에 따른 CAR의 제조에 사용된 scFv 가변영역의 중쇄, 경쇄, 및 CDRs의 서열을 표 3 및 표4에 정리하였다.The sequences of the heavy chain, light chain, and CDRs of the scFv variable region used in the preparation of the CAR according to this Example are summarized in Tables 3 and 4.
Figure PCTKR2020014394-appb-T000003
Figure PCTKR2020014394-appb-T000003
Figure PCTKR2020014394-appb-T000004
Figure PCTKR2020014394-appb-T000004
실시예 1-6: 인간 T 세포 형질도입(transduction) 및 배양Examples 1-6: Human T cell transduction and culture
유전자 전달을 위해 293T 세포를 사용하여 위형-VSVG 렌티바이러스(pseudotype-VSVG Lentivirus)를 제조하였다. HEK293T 세포는 10% FBS(GIBCO)가 포함된 DMEM(GIBCO) 배지에서 배양하였다. HEK293T 세포는 EF1α-MSLN CAR construct 벡터 및 HIV 기반 pPACKH1 렌티바이러스 패키지 키트(System Biosciences)와 공-형질감염(co-transfected)되었다. Vector 전달을 위해 리포펙타민 2000(Invitrogen, Carlsbad, CA)이 사용되었다. MSLN CAR construct는 다음과 같다; 501(28H)28BBz. 형질전환 48시간 후, 렌티바이러스를 포함하는 세포 상층액(Supernatant)을 수확하였다. 상층액은 0.45㎛ 필터 유닛(Millipore, Billerica, MA, USA)으로 세포 조각(cell debris)를 제거하였다. 10,600 rpm에서 90분간 ultracentrifugation 하여 바이러스를 1,500배 농축하였다. 농축된 바이러스는 -80℃에서 보관되었다.Pseudotype-VSVG Lentivirus was prepared using 293T cells for gene transfer. HEK293T cells were cultured in DMEM (GIBCO) medium containing 10% FBS (GIBCO). HEK293T cells were co-transfected with the EF1α-MSLN CAR construct vector and the HIV-based pPACKH1 lentivirus package kit (System Biosciences). Lipofectamine 2000 (Invitrogen, Carlsbad, CA) was used for vector delivery. The MSLN CAR construct is as follows; 501(28H)28BBz. 48 hours after transformation, the cell supernatant (Supernatant) containing the lentivirus was harvested. The supernatant was removed from cell debris with a 0.45 μm filter unit (Millipore, Billerica, MA, USA). The virus was concentrated 1,500-fold by ultracentrifugation at 10,600 rpm for 90 minutes. Concentrated virus was stored at -80°C.
1x106 cells/ml 농도로 20~24시간 활성화시킨 인간 T 세포에 Vectofusin-1(Miltenyi Biotec)과 함께 렌티바이러스를 5 MOI 되도록 첨가하고 72시간 후에 새로운 배지(Fresh media)로 교체하였다. 형질도입된 인간 T 세포가 1x106 cells/ml 농도로 유지되도록 주 3회 새로운 배지로 교체하였다. 특별한 언급이 없는 한, 모든 세포는 37℃의 5% CO2 배양기에서 배양하였다.To human T cells activated for 20 to 24 hours at a concentration of 1x10 6 cells/ml, Vectofusin-1 (Miltenyi Biotec) and lentivirus were added to 5 MOI and replaced with fresh media after 72 hours. The transduced human T cells were replaced with fresh medium 3 times a week to maintain a concentration of 1x10 6 cells/ml. Unless otherwise noted, all cells were cultured in a 5% CO 2 incubator at 37°C.
실시예 1-7: MSLN CAR를 포함하는 수용체 발현 및 T 세포 활성마커 분석Example 1-7: Receptor expression and T cell activity marker analysis including MSLN CAR
501(28H)28BBz 형질도입된 인간 T 세포 및 대조군 벡터 형질도입된 인간 T 세포는 FACS 완충액을 사용하여 두 차례 세척하였다. 세포들은 항-CD3(BD Biosciences), 항-F(ab)2(Jackson Immuno research)를 사용하여 염색하였다. 염색된 세포들의 발현비율 및 평균 형광강도(Mean fluorescence intensity, MFI)는 BD LSRFortessa 를 사용하여 측정되었고, 플로우조(FlowJo) 소프트웨어에서 분석되었다.501(28H)28BBz transduced human T cells and control vector transduced human T cells were washed twice using FACS buffer. Cells were stained using anti-CD3 (BD Biosciences), anti-F(ab)2 (Jackson Immuno research). Expression ratio and mean fluorescence intensity (MFI) of stained cells were measured using BD LSRFortessa and analyzed in FlowJo software.
실시예 1-8: 루시퍼라제 세포독성 에세이Examples 1-8: Luciferase Cytotoxicity Assay
다음 과정을 거쳐서, MSLN CAR 발현 T 세포의 표적세포, 즉 메소텔린을 발현하는 암 세포에 대한 활성능을 확인하였다.Through the following procedure, the activity of MSLN CAR-expressing T cells on target cells, that is, mesothelin-expressing cancer cells, was confirmed.
(i) 표적 세포들은 루시퍼라제 바이러스(BIOSETTIA)를 사용하여 루시퍼라제를 만들어 내도록 제작되었다.(i) Target cells were engineered to produce luciferase using luciferase virus (BIOSETTIA).
(ii) 표적 세포들은 96-well plate(Thermo Fisher Scientific)에 1 x 104 cells/well로 분배하였다. (ii) Target cells were distributed at 1 x 10 4 cells/well in 96-well plates (Thermo Fisher Scientific).
(iii) 효과세포들(501(28H)28BBz 형질도입 된 인간 T 세포, 대조군 벡터 형질도입된 인간 T 세포 및 아무 처리하지 않은 인간 T 세포)은 수확(harvest)되고, RPMI-1640(Thermo Fisher Scientific) 배지로 세척된 후 다양한 E/T(effector-to-target) 비율 조건으로 첨가되어 함께 배양되었다.(iii) effector cells (501(28H)28BBz transduced human T cells, control vector transduced human T cells and untreated human T cells) were harvested, and RPMI-1640 (Thermo Fisher Scientific ) was washed with the medium, then added under various E/T (effector-to-target) ratio conditions and incubated together.
(iv) 24 시간 후에, 플레이트들에 세포 용해 용액(Lysis buffer)을 처리하고 4℃에서 반응시켰다.(iv) 24 hours later, the plates were treated with a cell lysis solution (Lysis buffer) and reacted at 4°C.
(v) 20분 뒤 기질(Luciferin)을 넣어 SPARK® 마이크로 플레이트 리더(TECAN, Mannedorf, Switzerland)로 Luminescence 값을 측정하였다.(v) After 20 minutes, the substrate (Luciferin) was added and the luminescence value was measured with a SPARK® microplate reader (TECAN, Mannedorf, Switzerland).
실시예 2: 메소텔린 특이적인 CAR를 발현하는 인간 T 세포의 암 면역치료제로서의 평가Example 2: Evaluation of human T cells expressing mesothelin-specific CAR as cancer immunotherapeutic agents
실시예 2-1: 메소텔린을 특이적으로 표적하는 scFv 평가Example 2-1: Evaluation of scFvs specifically targeting mesothelin
MS501 IgG는 기존의 특허(목암연구소, GC녹십자, KOREA)에서 발명되어 메소텔린 특이적인 결합을 할 수 있다고 알려져 있다. 본 발명자들은 MS501 IgG를 scFv 형태로 제작하고 scFv 형태에서도 재조합 인간 메소텔린에 결합함을 면역 침강 분석으로 확인하였다(도 1A). 메소텔린을 과발현하는 세포주에 위 scFv 단백질을 결합하고, 유세포 분석을 통해 메소텔린 과발현 세포주에 68% 이상 결합함을 확인하였다(도 1B).MS501 IgG was invented by existing patents (Mokam Research Institute, GC Green Cross, KOREA) and is known to be capable of mesothelin-specific binding. The present inventors prepared MS501 IgG in the form of scFv and confirmed that it binds to recombinant human mesothelin even in the form of scFv by immunoprecipitation analysis (FIG. 1A). The above scFv protein was bound to the mesothelin-overexpressing cell line, and it was confirmed through flow cytometry that it was more than 68% bound to the mesothelin-overexpressing cell line ( FIG. 1B ).
실시예 2-2: 메소텔린 특이적인 CAR의 발현 확인과 발현 유지능 평가Example 2-2: Expression confirmation and expression maintenance evaluation of mesothelin-specific CAR
MS501 scFv는 CD8α의 신호서열; CD28의 힌지, 막관통 도메인, 세포 내 신호전달 도메인; 4-1BB의 세포 내 신호전달 도메인; CD3ζ의 세포 내 신호전달 도메인과 결합시켰다(도 2). 501(28H)28BBz 유전자는 EF1α 프로모터를 가지는 렌티바이러스 벡터를 이용하여 인간 T 세포에서 발현되었다. 사용된 렌티바이러스 양은 5 감염 배수(Multiplicity of infection, MOI)가 사용되었다. 각각의 CAR는 항-F(ab)2 항체를 사용하여 검출되었다. 유세포 분석을 통해 인간 T 세포에서 CAR가 20% 이상의 효율로 형질도입된 것이 확인되었다(도 3).MS501 scFv is the signal sequence of CD8α; hinge, transmembrane domain, intracellular signaling domain of CD28; the intracellular signaling domain of 4-1BB; It was bound to the intracellular signaling domain of CD3ζ (FIG. 2). The 501(28H)28BBz gene was expressed in human T cells using a lentiviral vector with an EF1α promoter. The amount of lentivirus used was 5 multiplicity of infection (MOI). Each CAR was detected using an anti-F(ab) 2 antibody. It was confirmed that CAR was transduced with an efficiency of 20% or more in human T cells through flow cytometry (FIG. 3).
실시예 2-3: 메소텔린 특이적인 CAR를 발현하는 인간 T 세포의 항암 능력 평가Example 2-3: Evaluation of anticancer ability of human T cells expressing mesothelin-specific CAR
본 발명에 따른 메소텔린 키메라 항원 수용체(501(28H)28BBz)를 발현하는 인간 T 세포의 메소텔린 특이적인 항암능력을 평가하기 위하여, MIA PaCa2 세포 또는 MIA PaCa2-MSLN 세포를 각각 메소텔린 수용체가 형질도입된 인간 T 세포와 공배양 후 표적세포 살해능을 평가하였다. 메소텔린 수용체를 발현하는 인간 T 세포가 MIA PaCa2 세포에 대해 살해능을 보이지 않았지만, 메소텔린을 발현하는 MIA PaCa2-MSLN 세포에서는 살해능이 확인되었다(도 4).In order to evaluate the mesothelin-specific anticancer ability of human T cells expressing the mesothelin chimeric antigen receptor (501(28H)28BBz) according to the present invention, MIA PaCa2 cells or MIA PaCa2-MSLN cells were transfected with mesothelin receptors, respectively. After co-culture with the introduced human T cells, the target cell killing ability was evaluated. Human T cells expressing mesothelin receptor did not show killing ability against MIA PaCa2 cells, but killing ability was confirmed in MIA PaCa2-MSLN cells expressing mesothelin ( FIG. 4 ).
실시예 3: 메소텔린 특이적인 CAR를 발현하는 인간 T 세포의 in vivo 항암효능 평가 Example 3: Evaluation of in vivo anticancer efficacy of human T cells expressing mesothelin-specific CAR
본 발명에 따른 메소텔린 키메라 항원 수용체(501(28H)28BBz)를 발현하는 인간 T 세포의 메소텔린 특이적인 항암효능을 in vivo 상으로 평가하기 위하여, 인간 췌장암 세포인 MIA PaCa2 세포주를 사용하여 동소이식 췌장암 마우스 모델(Orthotopic Pancreatic Cancer Mouse Model)을 설립하였다. 사용한 면역결핍 마우스는 6주령의 암컷 NOG(NOD.Cg-Prkdcscid II2rgtm1Sug/Jic) mice로, T 세포, B 세포, NK 세포가 결손된 모델이다.In order to evaluate the mesothelin-specific anticancer efficacy of human T cells expressing the mesothelin chimeric antigen receptor (501(28H)28BBz) according to the present invention in vivo , orthotopic transplantation using the human pancreatic cancer cell MIA PaCa2 cell line An Orthotopic Pancreatic Cancer Mouse Model was established. The immunodeficient mice used were 6-week-old female NOG (NOD.Cg-Prkdcscid II2rgtm1Sug/Jic) mice, a model lacking T cells, B cells, and NK cells.
501(28H)28BBz CAR-T가 메소텔린을 타겟하여 항암효능이 있는지 평가하기 위하여, 메소텔린을 거의 발현하지 않는 MIA PaCa2-FLuc-GFP 세포(off-target 모델) 및 메소텔린을 과발현하는 MIA PaCa2-MSLN-FLuc-GFP 세포주(on-target 모델)를 제작/구축하였다. 이 두 종류의 세포주는 firefly luciferase 리포트 유전자를 안정적으로 발현하므로, 본 실시예에서 종양의 성장을 모니터링하며 항암효능을 평가하였다. 동소이식 췌장암 마우스모델은 MIA PaCa2-FLuc-GFP 세포주 및 MIA PaCa2-MSLN-FLuc-GFP 세포주를 각각 HBSS(Ca2+/Mg2+ free Hank’s balanced salt solution)에 1.0x105cells/ml 농도로 부유시킨 뒤, 마우스의 췌장엽에 50μl씩 주사하여 제작하였다. 인간 췌장암 세포주 MIA PaCa2-FLuc-GFP 세포주 및 MIA PaCa2-MSLN-FLuc-GFP 세포주를 주입하고 종양형성이 확인된 11~12일째 1차, 25~26일째에 2차로 Vehicle, Mock CAR-T, 501(28H)28BBz CAR-T를 복강주사(IP)와 정맥주사(IV)로 투여하였다. Mock CAR-T 세포는 1x107cells/200μl, 501(28H)28BBz CAR-T 세포는 각각 High dose(1x107cells/200μl), Low dose(2x106cells/200μl)로 DPBS에 준비하여 투여하였다.To evaluate whether 501(28H)28BBz CAR-T has anticancer efficacy by targeting mesothelin, MIA PaCa2-FLuc-GFP cells that rarely express mesothelin (off-target model) and MIA PaCa2 that overexpress mesothelin -MSLN-FLuc-GFP cell line (on-target model) was fabricated/constructed. Since these two cell lines stably express the firefly luciferase report gene, tumor growth was monitored in this example to evaluate anticancer efficacy. In the orthotopic pancreatic cancer mouse model, MIA PaCa2-FLuc-GFP cell line and MIA PaCa2-MSLN-FLuc-GFP cell line were suspended in HBSS (Ca 2+ /Mg 2+ free Hank's balanced salt solution) at a concentration of 1.0x10 5 cells/ml, respectively. Then, it was prepared by injecting 50 μl into the pancreatic lobe of the mouse. Human pancreatic cancer cell line MIA PaCa2-FLuc-GFP cell line and MIA PaCa2-MSLN-FLuc-GFP cell line were injected, and tumor formation was confirmed on the first day 11-12 days, and the second on day 25-26 Vehicle, Mock CAR-T, 501 (28H)28BBz CAR-T was administered by intraperitoneal (IP) and intravenous (IV) injections. Mock CAR-T cells were prepared in DPBS at 1x10 7 cells/200μl, and 501(28H)28BBz CAR-T cells were administered at high dose (1x10 7 cells/200μl) and low dose (2x10 6 cells/200μl) in DPBS, respectively.
CAR-T 세포 투여로부터 8주 동안 IVIS® Lumina LT Series III In Vivo Imaging System(PerkinElmer)을 이용하여 1주일 마다 종양의 크기를 모니터링하였다. 이를 BLI Signal로 나타내어 CAR-T 세포의 메소텔린 특이적 항암효능을 평가하였다(도 5 및 도 6).Tumor size was monitored every week using an IVIS® Lumina LT Series III In Vivo Imaging System (PerkinElmer) for 8 weeks from CAR-T cell administration. This was expressed as a BLI signal to evaluate the mesothelin-specific anticancer efficacy of CAR-T cells ( FIGS. 5 and 6 ).
메소텔린을 거의 발현하지 않는 췌장암 마우스 모델(off-target model)에서, 501(28H)28BBz High dose 그룹은 5주까지 전혀 항암효능을 보이지 않았고, 8주에는 22%의 항암효능(Partial Remission, PR)을 보였으며(도 5C), low dose 그룹에서는 전혀 항암효능을 나타내지 않았다(도 5E).In a pancreatic cancer mouse model (off-target model) that hardly expresses mesothelin, the 501(28H)28BBz high dose group showed no anticancer effect until 5 weeks, and at 8 weeks, 22% of the anticancer efficacy (Partial Remission, PR) ) was shown (FIG. 5C), and the low-dose group did not show any anticancer effect (FIG. 5E).
메소텔린을 발현하는 췌장암 마우스 모델에서, 501(28H)28BBz CAR-T 세포를 High dose로 넣어준 그룹은 CAR-T 세포 주입 후 1주일만에 암세포의 BLI signal이 나타나지 않았다(도 6C). CAR-T 투여후 8주까지 완전관해(Complete Remission, CR)를 유지하였다(도 6C). 투여 경로에 무관하게 IP 및 IV 모두 완전관해로 나타났다(도 6C 및 도 6D). 한편, 501(28H)28BBz CAR-T 세포를 Low dose로 넣어준 그룹에서는 22%의 항암효능을 보였다(도 6E).In the mesothelin-expressing pancreatic cancer mouse model, the group to which 501(28H)28BBz CAR-T cells were administered at a high dose did not show the BLI signal of cancer cells within one week after CAR-T cell injection ( FIG. 6C ). Complete remission (CR) was maintained until 8 weeks after CAR-T administration ( FIG. 6C ). Regardless of the route of administration, both IP and IV showed complete remission ( FIGS. 6C and 6D ). On the other hand, the group to which the 501(28H)28BBz CAR-T cells were given at a low dose showed an anticancer efficacy of 22% (FIG. 6E).
CAR-T 세포 투여 후 8주 및 12주 후, 췌장조직을 분리하여 H&E 염색을 진행하였다. 마우스의 BLI signal과 췌장의 종양크기가 일치하는 것을 확인하였다. 병리학적으로 보았을 때에도 BLI signal 결과와 일치하게, 메소텔린을 발현하는 췌장암 마우스 모델의 on-target High dose 그룹에서 종양이 치유되었으며, on-target Low dose 그룹에서도 효과를 확인하였다(도 7).After 8 and 12 weeks after CAR-T cell administration, pancreatic tissue was isolated and H&E staining was performed. It was confirmed that the mouse BLI signal and the tumor size of the pancreas were consistent. From a pathological point of view, consistent with the BLI signal results, tumors were cured in the on-target high-dose group of the mesothelin-expressing pancreatic cancer mouse model, and the effect was also confirmed in the on-target low-dose group (FIG. 7).
결과적으로, 501(28H)28BBz CAR-T 세포는 메소텔린 과발현 췌장암에 표적 특이적으로 이동(target specific trafficking)하여 완전관해의 높은 치료효능을 나타내며, dose dependent하게 효능을 나타낸다.As a result, 501(28H)28BBz CAR-T cells exhibited high therapeutic efficacy of complete remission by target specific trafficking to mesothelin-overexpressing pancreatic cancer, and showed efficacy in a dose-dependent manner.
본 발명에 따른 키메라 항원 수용체는 메소텔린 특이적인 표적 효율이 우수할 뿐 아니라, 발현지속율이 우수한 장점을 가지고 있으며, 본 발명에 따른 키메라 항원 수용체를 발현하는 T 세포는 암 세포에 대한 세포독성이 우수하여, 암 치료를 위한 면역세포 치료에 유용하게 사용될 수 있다.The chimeric antigen receptor according to the present invention has excellent mesothelin-specific targeting efficiency as well as excellent expression persistence, and the T cells expressing the chimeric antigen receptor according to the present invention are cytotoxic to cancer cells. Because it is excellent, it can be usefully used for immune cell therapy for cancer treatment.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. will be. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.
전자파일 첨부하였음.An electronic file is attached.

Claims (20)

  1. 메소텔린(Mesothelin; MSLN)에 특이적으로 결합하는 결합 도메인, 신호서열(signal peptide), 힌지(hinge), 막관통 도메인(transmembrane domain) 및 3개 이상의 세포 내 도메인을 포함하는 키메라 항원 수용체(Chimeric antigen receptor; CAR).A chimeric antigen receptor (Chimeric) comprising a binding domain that specifically binds to Mesothelin (MSLN), a signal peptide, a hinge, a transmembrane domain, and three or more intracellular domains antigen receptor (CAR).
  2. 제1항에 있어서, 상기 세포 내 도메인은 CD28, 4-1BB 및 CD3ζ의 세포 내 신호영역 서열, 또는 이들의 조합으로 구성된 군에서 선택되는 서열을 포함하는 것을 특징으로 하는 키메라 항원 수용체.The chimeric antigen receptor according to claim 1, wherein the intracellular domain comprises a sequence selected from the group consisting of intracellular signaling domain sequences of CD28, 4-1BB and CD3ζ, or a combination thereof.
  3. 제2항에 있어서, 상기 CD28, 4-1BB 및 CD3ζ의 세포 내 신호영역 서열은 각각 서열번호 10, 서열번호 12 및 서열번호 14로 표시되는 아미노산 서열을 포함하는 것을 특징으로 하는 키메라 항원 수용체.The chimeric antigen receptor according to claim 2, wherein the intracellular signal region sequences of CD28, 4-1BB and CD3ζ include amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14, respectively.
  4. 제1항에 있어서, 상기 메소텔린에 특이적으로 결합하는 결합 도메인은 다음의 중쇄 CDR을 포함하는 중쇄 가변영역 및 경쇄 CDR을 포함하는 경쇄 가변영역을 함유하는 항-메소텔린 항체, 또는 이의 단편인 것을 특징으로 하는 키메라 항원 수용체:The method of claim 1, wherein the binding domain specifically binding to mesothelin is an anti-mesothelin antibody, or a fragment thereof, comprising a heavy chain variable region comprising the following heavy chain CDRs and a light chain variable region comprising a light chain CDR. A chimeric antigen receptor characterized in that:
    서열번호 21의 아미노산 서열을 포함하는 중쇄 CDR1, 서열번호 22의 아미노산 서열을 포함하는 중쇄 CDR2, 서열번호 23의 아미노산 서열을 포함하는 중쇄 CDR3, 및 서열번호 24의 아미노산 서열을 포함하는 경쇄 CDR1, 서열번호 25의 아미노산 서열을 포함하는 경쇄 CDR2 및 서열번호 26의 아미노산 서열을 포함하는 경쇄 CDR3.A heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 22, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 23, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 24, sequence A light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 25 and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26.
  5. 제1항에 있어서, 상기 메소텔린에 특이적으로 결합하는 결합 도메인은 서열번호 19의 아미노산 서열을 포함하는 중쇄 가변영역 및 서열번호 20의 아미노산 서열을 포함하는 경쇄 가변영역을 함유하는 항-메소텔린 항체, 또는 이의 단편인 것을 특징으로 하는 키메라 항원 수용체.The anti-mesothelin according to claim 1, wherein the binding domain specifically binding to mesothelin contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20. A chimeric antigen receptor, characterized in that it is an antibody, or a fragment thereof.
  6. 제1항에 있어서, 상기 메소텔린에 특이적으로 결합하는 결합 도메인은 항-메소텔린 항체의 단일-사슬 가변 단편(scFv)인 것을 특징으로 하는 키메라 항원 수용체.The chimeric antigen receptor according to claim 1, wherein the binding domain specifically binding to mesothelin is a single-chain variable fragment (scFv) of an anti-mesothelin antibody.
  7. 제6항에 있어서, 상기 항-메소텔린 항체의 단일-사슬 가변 단편(scFv)은 서열번호 2로 표시되는 아미노산 서열을 포함하는 것을 특징으로 하는 키메라 항원 수용체.The chimeric antigen receptor according to claim 6, wherein the single-chain variable fragment (scFv) of the anti-mesothelin antibody comprises the amino acid sequence represented by SEQ ID NO: 2.
  8. 제1항에 있어서, 상기 신호서열은 CD8α의 신호서열을 포함하는 것을 특징으로 하는 키메라 항원 수용체.The chimeric antigen receptor according to claim 1, wherein the signal sequence comprises a CD8α signal sequence.
  9. 제8항에 있어서, 상기 CD8α의 신호서열은 서열번호 4로 표시되는 아미노산 서열을 포함하는 것을 특징으로 하는 키메라 항원 수용체.The chimeric antigen receptor according to claim 8, wherein the CD8α signal sequence comprises the amino acid sequence shown in SEQ ID NO: 4.
  10. 제1항에 있어서, 상기 힌지는 CD28의 힌지를 포함하는 것을 특징으로 하는 키메라 항원 수용체.The chimeric antigen receptor of claim 1 , wherein the hinge comprises a hinge of CD28.
  11. 제10항에 있어서, 상기 CD28의 힌지는 서열번호 6으로 표시되는 아미노산 서열을 포함하는 것을 특징으로 하는 키메라 항원 수용체.The chimeric antigen receptor according to claim 10, wherein the hinge of CD28 comprises the amino acid sequence shown in SEQ ID NO: 6.
  12. 제1항에 있어서, 상기 막관통 도메인은 CD28의 막관통 도메인을 포함하는 것을 특징으로 하는 키메라 항원 수용체.The chimeric antigen receptor of claim 1 , wherein the transmembrane domain comprises the transmembrane domain of CD28.
  13. 제12항에 있어서, 상기 CD28의 막관통 도메인은 서열번호 8로 표시되는 아미노산 서열을 포함하는 것을 특징으로 하는 키메라 항원 수용체.The chimeric antigen receptor according to claim 12, wherein the transmembrane domain of CD28 comprises the amino acid sequence shown in SEQ ID NO: 8.
  14. 제1항에 있어서, 서열번호 16으로 표시되는 아미노산 서열을 포함하는 것을 특징으로 하는 키메라 항원 수용체.The chimeric antigen receptor according to claim 1, comprising the amino acid sequence represented by SEQ ID NO: 16.
  15. 제1항 내지 제14항 중 어느 한 항의 키메라 항원 수용체를 코딩하는 핵산.A nucleic acid encoding the chimeric antigen receptor of any one of claims 1 to 14 .
  16. 제15항의 핵산을 포함하는 발현 벡터.An expression vector comprising the nucleic acid of claim 15 .
  17. 제16항의 발현 벡터를 포함하는 바이러스.A virus comprising the expression vector of claim 16 .
  18. 제1항 내지 제14항 중 어느 한 항의 키메라 항원 수용체를 표면에 발현하는 면역세포.An immune cell expressing the chimeric antigen receptor of any one of claims 1 to 14 on its surface.
  19. 제18항에 있어서, 상기 면역세포는 T 세포, NK 세포, 또는 NKT 세포인 것을 특징으로 하는 면역세포.The immune cell according to claim 18, wherein the immune cell is a T cell, an NK cell, or an NKT cell.
  20. 제18항의 면역세포를 포함하는 암 치료용 조성물.A composition for treating cancer comprising the immune cell of claim 18 .
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XU DANDAN, JIN GUOLIANG, CHAI DAFEI, ZHOU XIAOWAN, GU WEIYU, CHONG YANYUN, SONG JINGYUAN, ZHENG JUNNIAN: "The development of CAR design for tumor CAR-T cell therapy", ONCOTARGET, vol. 9, no. 17, 2 March 2018 (2018-03-02), pages 13991 - 14004, XP055844944, DOI: 10.18632/oncotarget.24179 *

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