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WO2021172690A1 - Récepteur antigénique chimérique spécifique de la mésothéline et ses utilisations - Google Patents

Récepteur antigénique chimérique spécifique de la mésothéline et ses utilisations 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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Korean (ko)
Inventor
안종성
이영은
김언교
권미선
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GC Cell Corp
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Green Cross Cell Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4254—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K40/4255—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 A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 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 A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 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 A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 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.

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Abstract

La présente invention concerne un récepteur antigénique chimérique spécifique de la mésothéline, une cellule immunitaire l'exprimant, et leurs utilisations. Le récepteur antigénique chimérique selon la présente invention possède non seulement une excellente efficacité de ciblage spécifique de la mésothéline, mais également une excellente persistance d'expression. De plus, un lymphocyte T exprimant le récepteur antigénique chimérique selon la présente invention a une excellente cytotoxicité pour des cellules cancéreuses, et peut donc être utile dans le traitement de cellules immunitaires pour le traitement du cancer.
PCT/KR2020/014394 2020-02-25 2020-10-21 Récepteur antigénique chimérique spécifique de la mésothéline et ses utilisations Ceased WO2021172690A1 (fr)

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