WO2022234976A1 - Transformed antigen-specific professional antigen-presenting cell containing chimeric antigen receptor (car) and use thereof - Google Patents

Transformed antigen-specific professional antigen-presenting cell containing chimeric antigen receptor (car) and use thereof Download PDF

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WO2022234976A1
WO2022234976A1 PCT/KR2022/005560 KR2022005560W WO2022234976A1 WO 2022234976 A1 WO2022234976 A1 WO 2022234976A1 KR 2022005560 W KR2022005560 W KR 2022005560W WO 2022234976 A1 WO2022234976 A1 WO 2022234976A1
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receptor
cell
cells
baff
antigen
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PCT/KR2022/005560
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French (fr)
Korean (ko)
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진화섭
전태훈
양준
이창희
이나영
박인병
강석진
박시원
이현정
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주식회사 이뮤노로지컬디자이닝랩
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Publication of WO2022234976A1 publication Critical patent/WO2022234976A1/en

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    • 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
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by targeting or presenting multiple antigens
    • A61K2239/28—Expressing multiple CARs, TCRs or antigens
    • 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
    • A61K39/4614—Monocytes; Macrophages
    • 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
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464416—Receptors for cytokines
    • A61K39/464417—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • 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

Definitions

  • the present invention relates to a transformed antigen-specific professional antigen-presenting cell comprising a chimeric antigen receptor (CAR).
  • the chimeric antigen receptor is a chimeric antigen receptor comprising an extracellular domain of BAFF, the BAFF receptor ( BAFF receptor), B-cell maturation antigen (BCMA) or transmembrane activator and CAML interactor (TACI) comprising an antigen-binding domain that specifically binds, a cell therapeutic agent comprising the cell as an active ingredient or cancer and systemic erythematosus It relates to a pharmaceutical composition for the treatment of lupus.
  • Professional antigen presenting cells include macrophages, dendritic cells, and naive B cells (Makala et al., 2004).
  • macrophages and dendritic cells recognize antigens, they break down antigens by phagocytosis, and major histocompatibility complex (MHC) antigens expressed in macrophages and dendritic cells transfer the antigenic determinant (epitope) to T cells. (T cell) (Kambayashi and Laufer, 2014).
  • MHC major histocompatibility complex
  • T cell T cell
  • naive B cells the antigen is recognized by the B cell receptor (membrane bound IgM), and then the antigen is crushed and expressed in the na ⁇ ve B cells (major histocompatibility).
  • MHC antigen delivers epitope to T cells (Kambayashi and Laufer, 2014).
  • Professional antigen-presenting cells also induce naive T cells by transduction of antigenic determinants by major histocompatibility complex (MHC) antigens and co-stimulatory signal transduction into effector T cells (effector T cells) (Kambayashi and Laufer, 2014).
  • MHC major histocompatibility complex
  • effector T cells effector T cells
  • the specialized antigen-expressing cells secrete various cytokines and chemokines to induce an inflammatory response or activate other immune cells (Kambayashi and Laufer, 2014). Therefore, an effective immune response is initiated by activation of professional antigen-presenting cells, and an effective immune response cannot be induced without activation of professional antigen-presenting cells (Makala et al., 2004).
  • a method for treating tumors by inducing the activation of specialized antigen-expressing cells has been devised. That is, it is a method in which tumor-specific antigens are treated with cancer patient-derived specialized antigen-expressing cells, and then, professional antigen-expressing cells activated by tumor-specific antigens are put into the cancer patient's body (van Willigen et al., 2018). .
  • Activated specialized antigen-expressing cells implanted in cancer patients induce the activation of cytotoxic T lymphocytes (CTL) and natural killer cells (NK cells) that directly target tumor cells, thereby causing tumor cells (Galluzzi et al., 2018; Lu et al., 2020).
  • CTL cytotoxic T lymphocytes
  • NK cells natural killer cells
  • scFv single-chain variable fragment
  • the chimeric antigen receptor when the chimeric antigen receptor is grafted onto T cells or natural killer cells, the anticancer action of T cells or natural killer cells only by recognizing specific antigens of scFv regardless of signal transduction by specialized antigen-expressing cells that recognize cancer-specific antigens.
  • it since it is not limited to HLA type, it can be used as a more efficient treatment method that can be used universally by many people. Indeed, these chimeric antigen receptor-expressing T cells or natural killer cells have shown efficacy in several cancers (Holzinger et al., 2016; Pettitt et al., 2018; Wang et al., 2020).
  • B-cell activating factor is a cytokine belonging to tumor necrosis factor (TNF) ligand, BLyS (B-lymphocyte stimulator), TALL-1 (TNF- and ApoL related leukocyte-expressed ligand 1), THANK ( It is called TNF homologue that activates apoptosis, nuclear factor- ⁇ B and c-Jun NH2-terminal kinase), TNFSF13B (TNFsuperfamily member 13B) or zTNF4 (Mackay and Browning, 2002).
  • TNF tumor necrosis factor
  • BLyS B-lymphocyte stimulator
  • TALL-1 TNF- and ApoL related leukocyte-expressed ligand 1
  • THANK It is called TNF homologue that activates apoptosis, nuclear factor- ⁇ B and c-Jun NH2-terminal kinase), TNFSF13B (TNFsuperfamily member 13B) or zTNF4 (Mackay and Browning, 2002).
  • BAFF is originally a type 2 transmembrane protein composed of 285 amino acids and is expressed on the cell surface as a homotrimer, and then the transmembrane portion is cut off and secreted out of the cell.
  • BAFF is secreted from macrophages, monocytes, and dendritic cells (Mackay and Browning, 2002).
  • Receptors that bind to BAFF include BAFF receptor, B-cell maturation antigen (BCMA), and Transmembrane activator and CAML interactor (TACI) (Mackay and Browning, 2002).
  • BCMA B-cell maturation antigen
  • TACI Transmembrane activator and CAML interactor
  • the BAFF receptor is expressed in most human B cells except plasma cells in the bone marrow, and BCMA is mainly expressed in antibody-secreting human cells (Mackay and Schneider, 2009).
  • TACI is also expressed in most peripheral B cells (Mackay and Schneider, 2009). Therefore, signal transduction by the interaction of BAFF and three BAFF-binding receptors, the BAFF receptor, BCMA, and TACI, is considered to be very important for B cell activity (Mackay and Schneider, 2009).
  • BCMA is a marker of multiple myeloma (Carpenter et al., 2013), breast cancer, T cell lymphoma, B cell Chronic lymphocytic leukemia, B -CLL), non-small cell lung cancer, etc. (Haiat et al., 2006; Dou et al., 2016; Kampa et al., 2020).
  • BAFF receptors are B-cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, B cell It is expressed in various lymphomas such as B-cell non-Hodgkin lymphoma, and also in leukemias such as acute lymphoblastic leukemia (ALL) and chronic lymphoblastic leukemia (CLL). to manifest
  • the BAFF receptor is overexpressed in B cells of autoimmune diseases such as systemic lupus erythematosus (SLE) (Haiat et al., 2006; Parameswaran et al., 2010; Takahata et al., 2010; Yang et al.
  • SLE systemic lupus erythematosus
  • TACI is found in multiple myeloma, breast cancer, thyroid carcinoma, non-small cell lung cancer, etc. (Moreaux et al., 2009; Dou et al., 2016; Kampa et al., 2020).
  • the present inventors devised a new treatment method that grafts the function of the professional antigen-presenting cell and the function of the chimeric antigen receptor. That is, after recognizing a receptor and a ligand that can recognize a tumor cell surface protein as a ligand, a novel chimeric signaling domain that can induce the activity of a specialized antigen-presenting cell by the chimeric antigen receptor was designed, It was demonstrated that professional antigen-presenting cells activated by this chimeric signaling domain can have cytotoxicity against cancer.
  • the extracellular domain of BAFF the transmembrane domain of toll-like receptor 2 (TLR-2), TLR- 3, intracellular domain of TLR-4, IgE receptor gamma chain (Fc ⁇ receptor I gamma chain, Fc ⁇ RI ⁇ ) or IgG receptor 2A alpha chain (Fc ⁇ receptor 2A alpha chain, Fc ⁇ R2A ⁇ ) or IgE receptor beta chain (Fc ⁇ receptor I beta chain, Fc ⁇ RI ⁇ ) with an immunoreceptor tyrosine-based activation motif (ITAM) as a subunit, a novel synthetic intracellular signal transduction domain linking three ITAMs with a linker and Chimeric antigen receptor-expressing macrophages expressing the bound recombinant protein were prepared.
  • ITAM immunoreceptor tyrosine-based activation motif
  • the extracellular domain of BAFF expressed in macrophages recognizes BAFF receptors, BCMA, and TACI, and when combined with specific cancer cells or systemic lupus erythematosus B cells expressing BAFF receptors, BCMA, TACI, these macrophages are activated Signals can be transmitted, which in turn activate macrophages to have cytotoxicity against specific cancer cells expressing BAFF receptors, BCMA, TACI, or systemic lupus erythematosus B cells. In fact, it was confirmed through an in vitro experiment that the macrophages have specific cytotoxicity to cells expressing the BAFF receptor, BCMA, and TACI.
  • an object of the present invention is a transformed cell comprising a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor is a BAFF receptor, an antigen binding domain that specifically binds BCMA or TACI, a transmembrane domain and an intracellular
  • CAR chimeric antigen receptor
  • the chimeric antigen receptor is a BAFF receptor
  • an antigen binding domain that specifically binds BCMA or TACI a transmembrane domain
  • a transmembrane domain and an intracellular
  • the cell comprises a macrophage, dendritic cell or naive B cell as an antigen-specific professional antigen-presenting cell having a targeted effector activity.
  • Another object of the present invention is to provide a pharmaceutical composition for the treatment of cancer and systemic lupus erythematosus expressing a cell therapeutic agent or BAFF receptor, BCMA or TACI comprising the transformed cell as an active ingredient.
  • the chimeric antigen receptor is an antigen binding domain that specifically binds to a BAFF receptor, BCMA or TACI.
  • a transmembrane domain and an intracellular signaling domain wherein the cell comprises a macrophage, dendritic cell or naive B cell as an antigen-specific professional antigen-presenting cell having a targeted effector activity.
  • the present invention provides a pharmaceutical composition for the treatment of cancer and systemic lupus erythematosus, comprising the transformed cell, a cell therapeutic agent or a BAFF receptor, BCMA or TACI.
  • the antigen-binding domain of the chimeric antigen receptor may be an extracellular domain of BAFF that specifically binds to the BAFF receptor, BCMA, or TACI.
  • the transmembrane domain may be TLR-2.
  • the intracellular signaling domain is TLR-3, TLR-4, IgE receptor gamma chain (Fc ⁇ receptor I gamma chain, Fc ⁇ RI ⁇ ), IgG receptor 2A alpha chain (Fc ⁇ receptor 2A alpha chain, Fc ⁇ R2A ⁇ ), an immunoreceptor tyrosine-based activation motif (ITAM) of the IgE receptor beta chain (Fc ⁇ receptor I beta chain, Fc ⁇ RI ⁇ ), or a combination thereof.
  • IgE receptor gamma chain Fc ⁇ receptor I gamma chain, Fc ⁇ RI ⁇
  • IgG receptor 2A alpha chain Fc ⁇ receptor 2A alpha chain, Fc ⁇ R2A ⁇
  • ITAM immunoreceptor tyrosine-based activation motif
  • BAFF receptor The "BAFF receptor”, “BCMA”, and “TACI” are observed in various malignant cells and autoimmune diseases, preferably in systemic lupus erythematosus (SLE).
  • SLE systemic lupus erythematosus
  • carcinomas expressing "BAFF receptor”, “BCMA”, and “TACI” have been classified into acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), and follicular lymphoma.
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphoblastic leukemia
  • follicular lymphoma follicular lymphoma
  • lymphoma mantle cell lymphoma
  • B-cell lymphoma diffuse large B-cell lymphoma (DLBCL)
  • B-cell non-Hodgkin lymphoma B-cell non-Hodgkin lymphoma
  • multiple myeloma T cell lymphoma
  • breast cancer thyroid carcinoma and non-small cell lung cancer
  • non-small cell lung cancer may be selected from the group consisting of , but not limited thereto.
  • the transformed cell according to the present invention provides an enhanced chimeric signaling domain that induces the activity of the professional antigen-presenting cell when the professional antigen-presenting cell specifically binds to an antigen.
  • the extracellular domain of BAFF and TLR-3, TLR-4, and IgE receptor gamma chain (Fc ⁇ receptor I gamma chain, Fc ⁇ RI ⁇ ) or IgG receptor 2A alpha chain which play an important role in signal transduction of professional antigen-presenting cells
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITAM immunoreceptor tyrosine-based activation motif
  • the specialized antigen-expressing cell overexpressing the chimeric antigen receptor according to the present invention can be usefully used as an immune cell therapeutic agent for the treatment of BAFF receptor, BCMA or TACI-expressing carcinoma and systemic lupus erythematosus.
  • FIG. 1 is a schematic diagram showing the form of one of the cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.
  • FIG. 2 is a schematic diagram showing the form of one of the cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.
  • FIG. 3 is a schematic diagram showing the form of one of the cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.
  • FIG. 4 is a schematic diagram showing the form of one of the cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.
  • FIG. 5 is a schematic diagram showing the form of one of the lentiviral vectors according to an embodiment of the present invention.
  • FIG. 6 is a schematic diagram showing the form of one of the lentiviral vectors according to an embodiment of the present invention.
  • FIG. 7 is a schematic diagram showing the form of one of the lentiviral vectors according to an embodiment of the present invention.
  • FIG. 8 is a schematic diagram illustrating a form of one of lentiviral vectors according to an embodiment of the present invention.
  • FIG. 9 is a schematic diagram showing the form of one of the chimeric antigen receptors expressed on the surface of a professional antigen-expressing cell according to an embodiment of the present invention.
  • FIG. 10 is a schematic diagram showing the form of one of the chimeric antigen receptors expressed on the surface of a professional antigen-expressing cell according to an embodiment of the present invention.
  • FIG. 11 is a schematic diagram showing the form of one of the chimeric antigen receptors expressed on the surface of a professional antigen-expressing cell according to an embodiment of the present invention.
  • FIG. 12 is a schematic diagram showing the form of one of the chimeric antigen receptors expressed on the surface of a professional antigen-expressing cell according to an embodiment of the present invention.
  • Figure 13a is a schematic diagram in which the BAFF receptor cDNA is inserted into a retroviral vector to express the BAFF receptor, a target of the chimeric antigen receptor provided in the present invention, in Chinese hamster ovary (CHO) cells.
  • FIG. 13B is a schematic diagram of BCMA cDNA inserted into a retroviral vector to express BCMA, a target of the chimeric antigen receptor provided in the present invention, in Chinese hamster ovary (CHO) cells.
  • FIG. 13c is a schematic diagram of TACI cDNA inserted into a retroviral vector to express TACI, a target of the chimeric antigen receptor provided in the present invention, in Chinese hamster ovary (CHO) cells.
  • FIG. 14A is a diagram showing the result of measuring the expression level of BAFF receptor in BAFF receptor positive cells (CHO cells) expressing BAFF receptor, a target of the chimeric antigen receptor provided in the present invention, using flow cytometry.
  • 14B is a diagram showing the results of measuring the expression level of BCMA in CHO cells (BAFF receptor positive cells) expressing BCMA, the target of the chimeric antigen receptor provided in the present invention, using flow cytometry.
  • 14c is a diagram showing the results of measuring the expression level of TACI in CHO cells (BAFF receptor positive cells) expressing TACI, a target of the chimeric antigen receptor provided in the present invention, using flow cytometry.
  • FIG. 15 is a graph showing the expression ratio of GFP in professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. It is a diagram showing the results of comparison.
  • BAFF CAR-pAPC professional antigen-presenting cells
  • FIG. 16a shows 3-4-3 of professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. It is a diagram showing whether the BAFF CAR (TLR-3+TLR-4+Fc ⁇ RI ⁇ ITAM trimer) expression specialized antigen-expressing cells recognize the BAFF receptor using flow cytometry.
  • BAFF CAR-pAPC professional antigen-presenting cells
  • FIG. 16b shows 3-4-3 of professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. It is a diagram showing whether BCMA is recognized by BAFF CAR (TLR-3+TLR-4+Fc ⁇ RI ⁇ ITAM trimer) expression specialized antigen-expressing cells using flow cytometry.
  • BAFF CAR-pAPC professional antigen-presenting cells
  • FIG. 16c shows 3-4-3 of professional antigen-presenting cells (BAFF CAR-pAPCs) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. It is a diagram showing whether BAFF CAR (TLR-3+TLR-4+Fc ⁇ RI ⁇ ITAM trimer) expression specialized antigen-expressing cells recognize TACI using flow cytometry.
  • BAFF CAR-pAPCs professional antigen-presenting cells
  • FIG. 17a shows 3-4-2A of professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. It is a diagram showing whether the BAFF CAR (TLR-3+TLR4+Fc ⁇ R2A ⁇ ITAM trimer) expression specialized antigen-expressing cells recognize the BAFF receptor using flow cytometry.
  • BAFF CAR-pAPC professional antigen-presenting cells
  • FIG. 17b shows 3-4-2A of professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. It is a diagram showing whether BCMA is recognized by BAFF CAR (TLR-3+TLR4+Fc ⁇ R2A ⁇ ITAM trimer) expression specialized antigen-expressing cells using flow cytometry.
  • BAFF CAR-pAPC professional antigen-presenting cells
  • FIG. 17c shows 3-4-2A of professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. It is a diagram showing whether BAFF CAR (TLR-3+TLR4+Fc ⁇ R2A ⁇ ITAM trimer) expression specialized antigen-expressing cells recognize TACI using flow cytometry.
  • BAFF CAR-pAPC professional antigen-presenting cells
  • FIG. 18A shows 3-4-1B of professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. It is a diagram showing whether BAFF CAR (TLR-3+TLR4+Fc ⁇ RI ⁇ ITAM trimer) expression specialized antigen-expressing cells recognize the BAFF receptor using flow cytometry.
  • BAFF CAR-pAPC professional antigen-presenting cells
  • FIG. 18B shows 3-4-1B of professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. It is a diagram showing whether BCMA is recognized by BAFF CAR (TLR-3+TLR4+Fc ⁇ RI ⁇ ITAM trimer) expression specialized antigen-expressing cells using flow cytometry.
  • BAFF CAR-pAPC professional antigen-presenting cells
  • FIG. 18c shows 3-4-1B of professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system.
  • BAFF CAR-pAPC professional antigen-presenting cells
  • FIG. 18c shows 3-4-1B of professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system.
  • BAFF CAR TLR-3+TLR4+Fc ⁇ RI ⁇ ITAM trimer
  • FIG. 19a shows 3-4-ABC among professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. It is a diagram showing whether BAFF CAR (TLR-3+TLR4+Fc ⁇ R2A ⁇ ITAM monomer+Fc ⁇ RI ⁇ ITAM monomer+Fc ⁇ RI ⁇ ITAM monomer) expression-specific antigen-expressing cells recognize the BAFF receptor using flow cytometry.
  • BAFF CAR-pAPC professional antigen-presenting cells
  • FIG. 19b shows 3-4-ABC among professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system.
  • a diagram showing whether BCMA is recognized by BAFF CAR (TLR-3+TLR4+Fc ⁇ R2A ⁇ ITAM monomer+Fc ⁇ RI ⁇ ITAM monomer+Fc ⁇ RI ⁇ ITAM monomer) expression-specific antigen-expressing cells using flow cytometry.
  • Figure 19c shows 3-4-ABC among professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system.
  • BAFF CAR-pAPC professional antigen-presenting cells
  • FIG. 19c A diagram showing whether BAFF CAR (TLR-3+TLR4+Fc ⁇ R2A ⁇ ITAM monomer+Fc ⁇ RI ⁇ ITAM monomer+Fc ⁇ RI ⁇ ITAM monomer) expression-specific antigen-expressing cells recognize TACI using flow cytometry.
  • FIG. 20 shows 3- of professional antigen-expressing cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors transduced with an empty vector (Mock pAPC) or a chimeric antigen receptor according to an embodiment of the present invention.
  • BAFF CAR-pAPC professional antigen-expressing cells
  • Specialized antigen-expressing cells were used as effector cells for BAFF receptor, BCMA or TACI-expressing Chinese hamster ovary (CHO) cells.
  • Figure 21 shows 3- of professional antigen-expressing cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors transduced with an empty vector (Mock pAPC) or a chimeric antigen receptor according to an embodiment of the present invention; 4-3 BAFF CAR (TLR-3+TLR-4+Fc ⁇ RI ⁇ ITAM trimer) expression Specialized antigen-expressing cells were used as effector cells for BAFF receptor, BCMA or TACI-expressing Chinese hamster ovary (CHO) cells. A diagram showing the results of measuring cytotoxicity after 48 hours.
  • BAFF CAR-pAPC professional antigen-expressing cells
  • CHO Chinese hamster ovary
  • BAFF CAR-pAPC professional antigen-expressing cells
  • 4-2A BAFF CAR (TLR-3+TLR4+Fc ⁇ R2A ⁇ ITAM trimer) expression cytotoxicity to Chinese hamster ovary (CHO) cells expressing BAFF receptor, BCMA or TACI using professional antigen-expressing cells as effector cells is a diagram showing the measurement result after 24 hours.
  • BAFF CAR-pAPC professional antigen-expressing cells
  • Mock pAPC empty vector
  • a chimeric antigen receptor according to an embodiment of the present invention
  • 4-2A BAFF CAR TLR-3+TLR4+Fc ⁇ R2A ⁇ ITAM trimer
  • CHO Chinese hamster ovary
  • Figure 24 shows 3- of professional antigen-expressing cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors transduced with an empty vector (Mock pAPC) or a chimeric antigen receptor according to an embodiment of the present invention; 4-1B BAFF CAR (TLR-3+TLR4+Fc ⁇ RI ⁇ ITAM trimer) expression Cytotoxicity to Chinese hamster ovary (CHO) cells expressing BAFF receptor, BCMA or TACI using professional antigen-expressing cells as effector cells is a diagram showing the measurement result after 24 hours.
  • BAFF CAR-pAPC professional antigen-expressing cells
  • CHO Chinese hamster ovary
  • BAFF CAR-pAPC professional antigen-expressing cells
  • Mock pAPC empty vector
  • a chimeric antigen receptor according to an embodiment of the present invention
  • 4-1B BAFF CAR TLR-3+TLR4+Fc ⁇ RI ⁇ ITAM trimer
  • Cytotoxicity to Chinese hamster ovary (CHO) cells expressing BAFF receptor, BCMA or TACI using professional antigen-expressing cells as effector cells is a diagram showing the measurement result after 48 hours.
  • FIG. 26 shows 3- of professional antigen-expressing cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors transduced with an empty vector (Mock pAPC) or a chimeric antigen receptor according to an embodiment of the present invention
  • 4-ABC BAFF CAR TLR-3+TLR4+Fc ⁇ R2A ⁇ ITAM monomer+Fc ⁇ RI ⁇ ITAM monomer+Fc ⁇ RI ⁇ ITAM monomer
  • FIG. 26 shows 3- of professional antigen-expressing cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors transduced with an empty vector (Mock pAPC) or a chimeric antigen receptor according to an embodiment of the present invention
  • 4-ABC BAFF CAR TLR-3+TLR4+Fc ⁇ R2A ⁇ ITAM monomer+Fc ⁇ RI ⁇ ITAM monomer+Fc ⁇ RI ⁇ ITAM monomer
  • FIG. 27 shows 3- of professional antigen-expressing cells (BAFF CAR-pAPC) each expressing four types of chimeric antigen receptors transduced with an empty vector (Mock pAPC) or a chimeric antigen receptor according to an embodiment of the present invention; 4-ABC BAFF CAR (TLR-3+TLR4+Fc ⁇ R2A ⁇ ITAM monomer+Fc ⁇ RI ⁇ ITAM monomer+Fc ⁇ RI ⁇ ITAM monomer) expression Chinese hamster ovary expressing BAFF receptor, BCMA or TACI using professional antigen-expressing cells as effector cells It is a diagram showing the result of measuring cytotoxicity to (CHO) cells after 48 hours.
  • BAFF CAR-pAPC professional antigen-expressing cells
  • the present invention provides a transformed cell comprising a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an antigen binding domain that specifically binds to a BAFF receptor, BCMA or TACI, a transmembrane domain and A transformed cell comprising an intracellular signaling domain, wherein the cell comprises a macrophage, dendritic cell or naive B cell as an antigen-specific professional antigen-presenting cell having a targeted effector activity to provide.
  • CAR chimeric antigen receptor
  • the chimeric antigen receptor protein according to an embodiment of the present invention may include a functional equivalent.
  • “Functional equivalent” means at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably the amino acid sequence of the chimeric antigen receptor protein as a result of the addition, substitution, or deletion of amino acids. refers to a protein having a sequence homology of 95% or more and exhibiting substantially homogeneous physiological activity. 'Substantially homogenous physiological activity' means having an activity capable of specifically binding to a BAFF receptor, BCMA, or TACI.
  • the present invention also includes fragments, derivatives and analogues of chimeric antigen receptors.
  • fragments, derivatives and analogues of chimeric antigen receptors As used herein, the terms 'fragment', 'derivative' and 'analog' refer to a polypeptide that retains substantially the same biological function or activity as the chimeric antigen receptor protein of the present invention. Fragments, derivatives and analogs of the present invention may comprise (1) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, wherein the substituted amino acid residues are encoded by the genetic code.
  • polypeptide having substituent(s) at one or more amino acid residues may or may not be) or (2) a polypeptide having substituent(s) at one or more amino acid residues, or (3) another compound (a compound capable of extending the half-life of the polypeptide, such as polyethylene glycol); a polypeptide derived from the associated mature polypeptide, or (4) an additional amino acid sequence (eg, a leader sequence, a secretion sequence, a sequence used to purify the polypeptide, a proteinogen sequence, or a fusion protein); It may be a polypeptide derived from said polypeptide to which it is bound.
  • the fragments, derivatives and analogs as defined herein are well known to those skilled in the art.
  • BAFF receptor In the present invention, "BAFF receptor”, “BCMA”, and “TACI” are observed in various carcinomas (malignant cells) and autoimmune diseases, preferably systemic lupus erythematosus (SLE). Until now, carcinomas expressing "BAFF receptor”, “BCMA”, and “TACI” have been classified into acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), and follicular lymphoma.
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphoblastic leukemia
  • follicular lymphoma follicular lymphoma
  • lymphoma mantle cell lymphoma
  • B-cell lymphoma diffuse large B-cell lymphoma (DLBCL)
  • B-cell non-Hodgkin lymphoma B-cell non-Hodgkin lymphoma
  • multiple myeloma T cell lymphoma
  • breast cancer thyroid carcinoma and non-small cell lung cancer
  • non-small cell lung cancer may be selected from the group consisting of , but is not limited thereto.
  • chimeric signaling domain binds to a desired ligand, induces activation of professional antigen-presenting cells through ligand-receptor reaction, and is expressed in specialized antigen-expressing cells to attack cells expressing the ligand It may mean a fusion protein for In other words, when expressed in specialized antigen-expressing cells, it can be seen as a protein that binds to a ligand and induces activation of these cells. Therefore, the novel chimeric signaling domain provided by the present invention is a novel chimeric signaling domain in a general sense that can induce the activation of professional antigen-presenting cells using all ligand-receptor reactions including all antigen-antibody reactions. to provide.
  • the chimeric antigen receptor can be regarded as a protein that binds to an antigen and induces activation of these cells when expressed in professional antigen-expressing cells.
  • it may be a protein recognizing an antigen specific to a cell to cause an immune response
  • the cell to cause an immune response may refer to a cell existing in a specific tissue or constituting a tissue causing a lesion.
  • the antigen binding domain of the chimeric antigen receptor may be an extracellular domain of a B-cell activating factor (BAFF) that specifically binds to the BAFF receptor, BCMA, or TACI.
  • BAFF protein is a cytokine belonging to tumor necrosis factor (TNF) ligand, BLyS (B-lymphocyte stimulator), TALL-1 (TNF- and ApoL related leukocyte-expressed ligand 1), THANK (TNF homologue that activates apoptosis, nuclear factor- ⁇ B and c-Jun NH2-terminal kinase), TNFSF13B (TNFsuperfamily member 13B) or zTNF4.
  • TNF tumor necrosis factor
  • BLyS B-lymphocyte stimulator
  • TALL-1 TNF- and ApoL related leukocyte-expressed ligand 1
  • THANK TNF homologue that activates apoptosis, nuclear factor- ⁇ B and c-Jun NH2-terminal kinas
  • BAFF is originally a type 2 transmembrane protein composed of 285 amino acids and is expressed on the cell surface as a homotrimer, and then the transmembrane portion is cut off and secreted out of the cell. BAFF is secreted from macrophages, monocytes, and dendritic cells.
  • the extracellular domain of BAFF may consist of SEQ ID NO: 1 or an amino acid sequence having 95% or more homology thereto, but is not limited thereto.
  • the antigen binding domain may be in the form of a dimer in which a pair of extracellular domains of BAFF are linked to human immunoglobulin G 1 heavy chain constant region (IgG 1 heavy chain constant region) in order to amplify signal transduction, respectively. It is not limited.
  • the human immunoglobulin G 1 heavy chain constant region may consist of SEQ ID NO: 2 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto.
  • the intracellular signaling domain which is one component of the chimeric antigen receptor of the present invention, refers to a site that activates an immune response of an immune cell by binding to an antigen-binding domain.
  • the signaling domain may be TLR-3, TLR-4, tyrosine-based activation motif (ITAM), or a combination thereof.
  • ITAM tyrosine-based activation motif
  • the signaling domain may be in the form of connecting ITAM as a subunit to the structure of TLR-3-TLR-4 with a linker, wherein one or more ITAMs are linked may be, and preferably may be in the form of three connected, but is not limited thereto.
  • the ITAM is based on immunoreceptor tyrosine of IgE receptor gamma chain (Fc ⁇ receptor I gamma chain, Fc ⁇ RI ⁇ ), IgG receptor 2A alpha chain (Fc ⁇ receptor 2A alpha chain, Fc ⁇ R2A ⁇ ) or IgE receptor beta chain (Fc ⁇ receptor I beta chain, Fc ⁇ RI ⁇ ) It may be an activation motif, but is not limited thereto.
  • the TLR-3 is SEQ ID NO: 3 or more than 70%, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more sequence homology thereto, and the amino acid represented by SEQ ID NO: 3 may consist of an amino acid sequence exhibiting a function substantially equivalent to the sequence;
  • the TLR-4 is SEQ ID NO: 4 or more than 70%, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more sequence homology thereto, and the amino acid represented by SEQ ID NO: 4 may consist of an amino acid sequence exhibiting a function substantially equivalent to the sequence;
  • the ITAM is SEQ ID NO: 5; SEQ ID NO: 6; Alternatively, it may consist of an amino acid sequence exhibiting a function substantially equivalent to the amino acid sequence represented by SEQ ID NO: 7.
  • the linker may consist of an amino acid sequence having a function substantially equivalent to that of the amino acid sequence represented by SEQ ID NO: 8.
  • the transmembrane domain of the present invention is a site that connects the extracellular domain of BAFF and the costimulatory and essential signaling domains between the cell membrane, and the intracellular signaling domain is an immune response of immune cells by binding of the antigen-binding domain. site that activates
  • transmembrane domain of the chimeric antigen receptor of the present invention is CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and a transmembrane domain of a protein selected from the group consisting of TLR-2.
  • the transmembrane domain may be TLR-2, which may consist of SEQ ID NO: 9 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto.
  • the antigen-binding domain may include a signal peptide, and the signal peptide may be a TLR-4 signal peptide, and may consist of SEQ ID NO: 10 or an amino acid sequence showing 95% or more homology thereto, but is limited thereto not.
  • the vector used in the present invention a variety of vectors known in the art may be used, and depending on the type of host cell to produce the antigen receptor, such as a promoter, terminator, enhancer, etc. Expression control sequences, sequences for membrane targeting or secretion, etc. can be appropriately selected and variously combined according to the purpose.
  • the vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector. Suitable vectors include a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as promoter, operator, start codon, stop codon, polyadenylation signal and enhancer, and may be prepared in various ways depending on the purpose.
  • a lenti-virus vector can be used, and in the following examples of the present invention, pCDH-CMV-MCS-EF1-copGFP vector (lenti-virus vector) was used ( FIGS. 5 to 5 ). 8).
  • the cell can be transformed by introducing the chimeric antigen receptor that specifically binds to the BAFF receptor, BCMA or TACI of the present invention into the cell through the vector.
  • the cells may be cytotoxic T cells and NK cells, tumor infiltrating lymphocytes, B cells, NK cells, or NKT cells. It may be a naive B cell.
  • the cells may be obtained or prepared from bone marrow, peripheral blood, peripheral blood mononuclear cells or umbilical cord blood.
  • the cell is a human cell.
  • a chimeric antigen receptor that specifically binds to a BAFF receptor, BCMA or TACI can be transformed into a specialized antigen-expressing cell using the vector described above.
  • the transformed cells into which the chimeric antigen receptor of the present invention is introduced recognizes the BAFF receptor, BCMA or TACI as an antigen, and has a characteristic of strongly binding thereto.
  • chimeric antigen receptor professional antigen presenting cell (hereinafter abbreviated as 'CAR-pAPC cell') is a method such as transduction of normal professional antigen presenting cells It refers to a specialized antigen-presenting cell expressing a chimeric antigen receptor that specifically responds to cancer cells rather than the original specialized antigen-expressing cell surface receptor. Professional antigen-expressing cells with this receptor induce apoptosis of target cells and exhibit cytotoxicity.
  • the CAR-pAPC cell may be a cell in which the chimeric antigen receptor of the present invention is introduced into a professional antigen-expressing cell.
  • the cells have the advantage of anticancer-specific targeted therapy, which is the existing advantage of CAR-T therapeutics.
  • the specialized antigen-expressing cells equipped with the chimeric antigen receptor of the present invention recognize cancer cells expressing BAFF receptor, BCMA or TACI. can be effectively destroyed.
  • another aspect of the present invention is a cell therapy agent comprising the cell;
  • the cell may be a cytotoxic T cell (cytotoxic T cell) and NK cells, tumor infiltrating lymphocytes, B cells, NK cells, or NK-T cells, preferably, specialized antigen expression
  • the cells may be macrophages, dendritic cells, or naive B cells.
  • progenitor cells for example, hematopoietic stem cells, mesenchymal stromal cells, stem cells, pluripotent stem cells, and may be embryonic stem cells, which may be used in cell therapy such as anticancer therapy.
  • the cells may come from a donor, or they may be cells obtained from a patient. Cells can be used, for example, in regeneration, replacing the function of diseased cells. Cells can also be modified to express heterologous genes so that biological agents can be delivered to specific microenvironments, such as, for example, diseased bone marrow or metastatic deposits.
  • cell therapeutic refers to cells and tissues manufactured through isolation, culture, and special manipulation from an individual, and is a drug (US FDA regulations) used for the purpose of treatment, diagnosis, and prevention, restoring the function of cells or tissues. It refers to a drug used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro or changing the biological properties of cells in other ways.
  • prevention refers to any action that inhibits or delays the development of cancer and systemic lupus erythematosus and BAFF receptor, BCMA or TACI by administration of the composition.
  • treatment refers to any action in which symptoms caused by cancer and systemic lupus erythematosus, which express BAFF receptor, BCMA or TACI, are improved or beneficially changed by administration of the composition.
  • composition may include a pharmaceutically acceptable carrier.
  • the "pharmaceutically acceptable carrier” may mean a carrier or diluent that does not inhibit the biological activity and properties of the injected compound without irritating the organism.
  • the type of carrier usable in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable may be used.
  • Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or may be used in combination of two or more.
  • composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations.
  • formulation it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.
  • solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient to the compound, for example, starch, calcium carbonate, sucrose, lactose. , gelatin, etc. may be mixed and prepared.
  • excipients for example, starch, calcium carbonate, sucrose, lactose. , gelatin, etc.
  • lubricants such as magnesium stearate and talc may also be used.
  • Liquid formulations for oral use include suspensions, solutions, emulsions, and syrups.
  • various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories.
  • Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • Witepsol, Macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, etc. may be used as the base of the suppository.
  • composition may be administered in a pharmaceutically effective amount.
  • the "pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the subject's type and severity, age, sex, infected virus type, and drug. Activity, sensitivity to drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs and other factors well known in the medical field.
  • Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, may be administered intranasally, but is not limited thereto.
  • composition of the present invention may be administered daily or intermittently, and the number of administrations per day may be administered once or divided into two to three times.
  • the two active ingredients are single drugs, the number of administrations may be the same or different.
  • the composition of the present invention may be used alone or in combination with other drug treatments for the prevention or treatment of hematologic cancer. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.
  • the subject is a human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or Any animal, including guinea pigs. If the disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject, the type of subject is included without limitation.
  • Cells expressing BAFF receptor, BCMA, and TACI to be treated in the present invention include systemic lupus erythematosus and carcinoma, and types of cancer include acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia.
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphoblastic leukemia
  • follicular lymphoma mantle cell lymphoma
  • B-cell lymphoma diffuse large B-cell lymphoma
  • DLBCL diffuse large B-cell lympho
  • chimeric antigen receptor of the present invention four types of chimerics having sequences of domain 1 and transmembrane domains of TLR-2 among the extracellular domains of BAFF and encoding different intracellular signal transduction domains
  • the antigen receptor protein coding sequence was synthesized.
  • the chimeric antigen receptor is an antigen recognition site linking a pair of extracellular domains of BAFF to human immunoglobulin G 1 heavy chain constant region, respectively, a transmembrane domain of TLR-2, TLR-4 signal Peptides are commonly included.
  • Each of the signaling domains was constructed in four different configurations. Table 1 below describes the configuration of the four types of signaling domains.
  • FIGS. 1 to 4 Schematic diagrams of cDNAs of each domain expressing the chimeric antigen receptor constructed above are shown in FIGS. 1 to 4 .
  • the protein coding sequence of each domain part was confirmed in a gene database, and the accuracy of gene synthesis was confirmed through sequencing after a protein expression plasmid was prepared.
  • the gene was expressed in a lentivirus-derived expression vector. It was cloned into pCDH-CMV-MCS-EF1-copGFP (System Biosciences) using restriction enzymes Xba I and Not I cleavage sites.
  • each of the BAFF receptor, BCMA or TACI genes has a restriction enzyme Bgl II cleavage site sequence at the 5' end and a restriction enzyme Hind III cleavage site at the 3' end. The sequence was cloned into the pLNCX2 vector (see FIGS. 13A, 13B and 13C).
  • a retroviral system using 293GPG cells was used to introduce the pLNCX2 vector into which the BAFF receptor, BCMA, and TACI genes were inserted into Chinese hamster ovary (CHO) cells (ATCC).
  • 3 ⁇ 10 6 293GPG cells were dissolved in 10 ml of 10 ml of 10% calf serum-containing DMEM culture medium and inoculated in a 100 ⁇ cell culture dish and then cultured for 24 hours.
  • Each of the previously prepared recombinant vectors (BAFF-R.pLNCX2 vector, BCMA.pLNCX2 vector, TACI.pLNCX2 vector) (20 ⁇ g) was crystallized using calcium phosphate and HEPES-buffered solution, and then in the culture medium of the previously cultured 293GPG cells. was added Thereafter, the culture medium was changed over 72 hours at 24 hour intervals, and the culture supernatant of 293GPG cells containing retrovirus was collected and stored.
  • the collected retrovirus-containing supernatant was centrifuged at 21,000 rpm for 2 hours using an ultra-high speed centrifuge and then reconstituted in DMEM (welgene) containing 10% calf serum to be concentrated 100 times compared to before concentration.
  • DMEM fetal calf serum
  • To transduce the concentrated retrovirus into CHO cells CHO cells were inoculated into a 24 well cell culture dish at a density of 5 ⁇ 10 5 /ml. Then, a mixed solution in which 8 ⁇ g/ml of polybrene was added to the concentrated retrovirus was added to the culture medium of CHO cells in culture, and then cultured for 24 hours. After culturing, it was replaced with a new culture medium.
  • CHO cells For the selection of cells into which the gene was introduced, 24 hours after the culture medium was replaced, CHO cells treated with retrovirus using a DMEM culture medium containing 10% calf serum supplemented with neomycin antibiotic (600 ⁇ g/ml). was cultured. After 14 days of neomycin selection, CHO cells were transferred to fresh culture medium and grown for 1 week.
  • BAFF receptor, BCMA, and TACI in CHO cells were measured using flow cytometry, respectively, and recombinant vectors (BAFF-R.pLNCX2 vector, BCMA.pLNCX2 vector, TACI.pLNCX2 vector) were introduced into each CHO cell and BAFF receptor, BCMA, and TACI expression levels of CHO cells (Mock CHO cells) introduced with pLNCX2, an empty vector, were measured by anti-human BAFF receptor antibody (Biolegend), anti-human BCMA receptor antibody (Biolegend), and anti-human TACI receptor antibody (Biolegend). The results compared using a flow cytometer using
  • each of the CHO cell lines that strongly express the BAFF receptor, BCMA, and TACI were used as target cells, and the mock CHO cell line that does not express the BAFF receptor, BCMA, and TACI was used as the target cell. was used as a negative control of
  • Example 2 Lentivirus using 293FT cells (Thermo Fisher Scientific) to introduce the four recombinant chimeric antigen receptor expression vectors prepared in Example 2 into THP-1 cells (human macrophage cell line), a type of specialized antigen-expressing cells. system was used.
  • 293FT cells were inoculated to become 2.5 ⁇ 10 6 cells in a 100 ⁇ cell culture dish, and then cultured in DMEM medium containing 10% calf serum. After 24 hours of incubation, when the cells have grown to cover about 60-70% of the dish, 20 ⁇ g of each of the four vector DNAs of Example 2 is aliquoted, 10 ⁇ g of psPAX2 (Addgene) and 3 ⁇ g of pMD2.G ( Addgene) vector and calcium phosphate were crystallized using calcium phosphate and Hepes-buffered solution, and then introduced into 293FT cells.
  • psPAX2 Addgene
  • pMD2.G Addgene
  • the culture supernatant containing the virus (lentivirus) was collected at intervals of 24 hours after 48 hours of the 293FT cells introduced with the expression vector.
  • the collected supernatant was centrifuged at 21,000 rpm in an ultra-high speed centrifuge for 2 hours to concentrate the virus.
  • the concentrated virus was mixed with 4 ⁇ g/ml of polybrene and added to the culture medium of THP-1 cells, a macrophage cell line, for transduction.
  • the efficiency of transduction was increased by changing the culture medium of THP-1 cells to the culture medium in which the concentrated virus was added twice at 24 hour intervals. 24 hours after the last culture medium change, a portion of the transduced THP-1 cells was used to measure the transduction efficiency.
  • Transduction efficiency was measured using flow cytometry to measure the expression level of GFP inside the cell, and THP-1 cells (BAFF CAR) transduced with each of the four recombinant chimeric antigen receptor expression vectors were transfected with an empty vector (Mock). The results compared with the transduction ratio of the introduced THP-1 cells are shown in FIG. 15 .
  • Example 5 chimeric antigen receptors and BAFF receptor, BCMA or TACI Affinity measurement of expressing human cell lines
  • BAFF CAR-pAPC In order to confirm whether the prepared chimeric antigen receptor has affinity with a human cell line expressing the BAFF receptor, BCMA or TACI, the four types of BAFF CAR-pAPC [3-4-3 BAFF CAR ( TLR-3+TLR-4+Fc ⁇ RI ⁇ ITAM trimer), 3-4-2A BAFF CAR (TLR-3+TLR4+Fc ⁇ R2A ⁇ ITAM trimer), 3-4-1B BAFF CAR (TLR-3+TLR4+Fc ⁇ RI ⁇ ITAM trimer) , 3-4-ABC BAFF CAR (TLR-3+TLR4+Fc ⁇ R2A ⁇ ITAM monomer+Fc ⁇ RI ⁇ ITAM monomer+Fc ⁇ RI ⁇ ITAM monomer)].
  • Water-soluble recombinant human BAFF receptor IgG (Acrobiosystems), water-soluble recombinant BCMA IgG (R&D systems), and water-soluble recombinant TACI IgG (R&D systems) were each treated with BAFF CAR-pAPC and confirmed by flow cytometry. As a result, all of the water-soluble recombinant human BAFF receptor IgG, the water-soluble recombinant BCMA IgG, and the water-soluble recombinant TACI IgG did not bind to mock pAPC, but bound to BAFF CAR-pAPC.
  • 16A to 16C show the binding of 3-4-3 BAFF CAR (TLR-3+TLR-4+Fc ⁇ RI ⁇ ITAM trimer)-expressing specialized antigen-expressing cells to soluble recombinant human BAFF receptor IgG, soluble recombinant BCMA IgG, and soluble recombinant TACI IgG.
  • 17a to 17c show the binding of 3-4-2A BAFF CAR (TLR-3+TLR4+Fc ⁇ R2A ⁇ ITAM trimer) specialized antigen-expressing cells to soluble recombinant human BAFF receptor IgG, soluble recombinant BCMA IgG, and soluble recombinant TACI IgG.
  • FIGS. 18a to 18c are 3-4-1B BAFF CAR (TLR-3+TLR4+Fc ⁇ RI ⁇ ITAM trimer) expression specialized antigen-expressing cells and soluble recombinant human BAFF receptor IgG, soluble recombinant BCMA IgG, and soluble recombinant TACI IgG shows binding, and FIGS.
  • 19a to 19c show 3-4-ABC BAFF CAR (TLR-3+TLR4+Fc ⁇ R2A ⁇ ITAM monomer+Fc ⁇ RI ⁇ ITAM monomer+Fc ⁇ RI ⁇ ITAM monomer) expression specialized antigen-expressing cells and soluble recombinant human BAFF receptor IgG, water-soluble Binding to recombinant BCMA IgG and water-soluble recombinant TACI IgG is shown.
  • BAFF CAR-pAPC expressing a chimeric antigen receptor prepared based on the above results was used as an effector cell, a CHO cell line expressing BAFF receptor, BCMA or TACI was used as a target cell, and BAFF CAR-pAPC was used as a target cell.
  • BAFF receptor, BCMA or TACI-specific cytotoxicity was verified.
  • BAFF receptor, BCMA or TACI manifest CHO with cells co-culture BAFF receptors through, BCMA , TACI Specific cytotoxicity verification
  • 7-Aminoactinomycin D (7-AAD) After co-culture, treatment with 7-Aminoactinomycin D (7-AAD) 24 hours or 48 hours later and comparing the fluorescence expression level of 7-AAD by flow cytometry to measure the cytotoxicity of each chimeric antigen receptor-expressing THP-1 cell did.
  • 7-Aminoactinomycin D (7-AAD) binds to the DNA of the cell in which apoptosis is induced and becomes fluorescent (Zembruski et al., 2012).
  • each of the 4 types of chimeric antigen receptor-expressing THP-1 cells was dispensed at 5 ⁇ 10 5 into the wells of a 24-well cell culture dish, and each CHO cell expressing the target cell BAFF receptor, BCMA or TACI was dispensed with 1 ⁇ 10 5 each. Therefore, co-culture was performed with an effector: target ratio of 5:1. The volume per well of the co-culture was proceeded to be 1 ml, and centrifuged at 250 g for 4 minutes using a centrifuge to make the intercellular space close.
  • the cells in each well were centrifuged at 300 g for 3 minutes using a centrifuge to remove the supernatant, and then the cells were stained with 7-AAD (Biolegend). Thereafter, the cytotoxicity of the four chimeric antigen receptor-expressing THP-1 cells prepared by measuring the proportion of cells exhibiting fluorescence by 7-AAD using flow cytometry was quantified.
  • the prepared 3-4-3 BAFF CAR (TLR-3+TLR-4+Fc ⁇ RI ⁇ ITAM trimer) expressing THP-1 cells expressing BAFF receptor, BCMA or TACI
  • THP-1 cells not expressing the chimeric antigen receptor (Mock pAPC) showed less than 7.12% cytotoxicity
  • 3-4-3 BAFF CAR (TLR- 3+TLR-4+Fc ⁇ RI ⁇ ITAM trimer) expressing THP-1 cells showed cytotoxicity of 21.0% or more for each CHO cell expressing BAFF receptor, BCMA or TACI.
  • the prepared 3-4-2A BAFF CAR (TLR-3+TLR4+Fc ⁇ R2A ⁇ ITAM trimer) expressing THP-1 cells expressing BAFF receptor, BCMA or TACI
  • THP-1 cells not expressing the chimeric antigen receptor (Mock pAPC) showed less than 7.12% cytotoxicity
  • 3-4-2A BAFF CAR (TLR- 3+TLR4+Fc ⁇ R2A ⁇ ITAM trimer) THP-1 cells expressing BAFF receptor, BCMA or TACI were confirmed to show cytotoxicity of 23.7% or more for each CHO cell.
  • the prepared 3-4-1B BAFF CAR (TLR-3+TLR4+Fc ⁇ RI ⁇ ITAM trimer) expressing THP-1 cells expressed BAFF receptor, BCMA or TACI.
  • the THP-1 cells that do not express the chimeric antigen receptor (Mock pAPC) showed less than 7.12% cytotoxicity
  • the 3-4-1B BAFF CAR (TLR) -3+TLR4+Fc ⁇ RI ⁇ ITAM trimer) expressing THP-1 cells showed cytotoxicity of 25.7% or more for each CHO cell expressing BAFF receptor, BCMA or TACI.
  • the prepared 3-4-ABC BAFF CAR (TLR-3+TLR4+Fc ⁇ R2A ⁇ ITAM monomer+Fc ⁇ RI ⁇ ITAM monomer+Fc ⁇ RI ⁇ ITAM monomer) expressing THP-1 cells were At 24 hours after co-culture with individual CHO cells expressing BAFF receptor, BCMA or TACI, THP-1 cells not expressing chimeric antigen receptor (Mock pAPC) showed cytotoxicity of 7.12% or less, whereas 3 THP-1 cells expressing -4-ABC BAFF CAR (TLR-3+TLR4+Fc ⁇ R2A ⁇ ITAM monomer+Fc ⁇ RI ⁇ ITAM monomer+Fc ⁇ RI ⁇ ITAM monomer) showed cytotoxicity of more than 18.8% for each CHO cell expressing BAFF receptor, BCMA or TACI.
  • the prepared chimeric antigen recognition receptor-expressing THP-1 cells showed specific cytotoxicity to the BAFF receptor, BCMA or TACI protein.
  • the transformed THP-1 cells including the chimeric antigen receptor according to the present invention showed cytotoxicity specific to the BAFF receptor, BCMA or TACI protein, thereby producing the transformed antigen-specific professional antigen-expressing cells. It was confirmed that it is possible to treat related cancer cells using the

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Abstract

The present invention relates to a transformed antigen-specific professional antigen-expressing cell comprising a chimeric antigen receptor (CAR) and, more specifically, to a cell therapeutic agent or a pharmaceutical composition for the treatment of cancer and systemic lupus erythematosus, containing the cell as an active ingredient, wherein the CAR comprises an antigen-binding domain that specifically binds to a BAFF receptor, BCMA or TACI. The transformed cell according to the present invention provides an enhanced chimeric signaling domain that induces the activity of the professional antigen-presenting cell when the professional antigen-presenting cell specifically binds to an antigen. As an example therefor, provided is a recombinant protein containing a signal transduction domain in a novel synthetic cell, the recobminant protein being a protein wherein a dimer in which a pair of extracellular domains of BAFF are each linked to a human immunoglobulin G1 heavy chain constant region (lgG1 heavy chain constant region), and an immunoreceptor tyrosine-based activation motif (ITAM) of a TLR-3, a TLR-4, and an IgE receptor gamma chain (Fcε receptor I gamma chain, FcεRIγ), an IgG receptor 2A alpha chain (Fcγ receptor 2A alpha chain, FcγR2Aα) ) or an IgE receptor beta chain (Fcε receptor I beta chain, FcεRIβ), which serve as important roles in the signal transduction of the professional antigen-presenting cell, are subunits, and three ITAMs are linked with a linker, wherein the professional antigen-expressing cell transformed with a vector capable of overexpressing the protein has cytotoxicity specific to carcinomas and autoimmune diseases expressing BAFF receptor, BCMA or TACI, so as to be effectively used as an immunecyte therapeutic agent for the treatment of cancer and systemic lupus erythematosus.

Description

키메릭 항원 수용체(CAR)를 포함하는 형질전환된 항원 특이적 전문적 항원표출세포 및 이의 용도Transformed antigen-specific professional antigen-presenting cells containing chimeric antigen receptor (CAR) and uses thereof
본 발명은 키메릭 항원 수용체(CAR)를 포함하는 형질전환된 항원 특이적 전문적 항원표출세포로서, 구체적으로 상기 키메릭 항원 수용체는 BAFF의 세포외 도메인을 포함하는 키메릭 항원 수용체로서, BAFF 수용체(BAFF receptor), B-cell maturation antigen (BCMA) 또는 Transmembrane activator and CAML interactor (TACI)에 특이적으로 결합하는 항원 결합 도메인을 포함하는, 상기 세포를 유효성분으로 포함하는 세포 치료제 또는 암 및 전신 홍반성 루프스의 치료용 약학적 조성물에 관한 것이다.The present invention relates to a transformed antigen-specific professional antigen-presenting cell comprising a chimeric antigen receptor (CAR). Specifically, the chimeric antigen receptor is a chimeric antigen receptor comprising an extracellular domain of BAFF, the BAFF receptor ( BAFF receptor), B-cell maturation antigen (BCMA) or transmembrane activator and CAML interactor (TACI) comprising an antigen-binding domain that specifically binds, a cell therapeutic agent comprising the cell as an active ingredient or cancer and systemic erythematosus It relates to a pharmaceutical composition for the treatment of lupus.
전문적 항원표출세포(professional antigen presenting cell)에는 대식세포(macrophage), 수지상세포(dendritic cell), 미감작 B 세포(naive B cell)가 포함된다(Makala et al., 2004). 대식세포와 수지상세포는 항원을 인식하면 식작용(phagocytosis)에 의해 항원을 잘게 부수고, 대식세포와 수지상세포에서 발현하는 주조직적합성(major histocompatibility complex, MHC) 항원은 항원 결정부(epitope)를 T 세포(T cell)에 전달한다(Kambayashi and Laufer, 2014). 미감작 B 세포(naive B cell)의 경우는 B 세포 수용체(B cell receptor, membrane bound IgM)에 의해 항원을 인식하고, 그 후 항원을 잘게 부수어 미감작 B 세포에서 발현하는 주조직적합성(major histocompatibility complex, MHC) 항원은 항원 결정부(epitope)를 T 세포(T cell)에 전달한다(Kambayashi and Laufer, 2014). 전문적 항원표출세포는 또한 주조직적합성(major histocompatibility complex, MHC) 항원에 의한 항원 결정부 전달과 동시자극신호(co-stimulatory signal) 전달에 의해 미감작 T 세포(naive T cell)를 작동 T 세포(effector T cell)로 활성화시킨다(Kambayashi and Laufer, 2014). 전문적 항원표출세포는 그 외에도 다양한 사이토카인(cytokine)과 케모카인(chemokine)을 분비하여, 염증 반응을 유발하거나 다른 면역세포의 활성을 유발한다(Kambayashi and Laufer, 2014). 따라서, 효과적인 면역 반응은 전문적 항원표출세포의 활성화에 의해 시작되며, 전문적 항원표출세포의 활성화 없이는 효과적인 면역 반응이 유발되지 않는다(Makala et al., 2004). Professional antigen presenting cells include macrophages, dendritic cells, and naive B cells (Makala et al., 2004). When macrophages and dendritic cells recognize antigens, they break down antigens by phagocytosis, and major histocompatibility complex (MHC) antigens expressed in macrophages and dendritic cells transfer the antigenic determinant (epitope) to T cells. (T cell) (Kambayashi and Laufer, 2014). In the case of naive B cells, the antigen is recognized by the B cell receptor (membrane bound IgM), and then the antigen is crushed and expressed in the naïve B cells (major histocompatibility). complex, MHC) antigen delivers epitope to T cells (Kambayashi and Laufer, 2014). Professional antigen-presenting cells also induce naive T cells by transduction of antigenic determinants by major histocompatibility complex (MHC) antigens and co-stimulatory signal transduction into effector T cells ( effector T cells) (Kambayashi and Laufer, 2014). In addition, the specialized antigen-expressing cells secrete various cytokines and chemokines to induce an inflammatory response or activate other immune cells (Kambayashi and Laufer, 2014). Therefore, an effective immune response is initiated by activation of professional antigen-presenting cells, and an effective immune response cannot be induced without activation of professional antigen-presenting cells (Makala et al., 2004).
최근에는 전문적 항원표출세포의 활성을 유발하여 종양을 치료하는 방법이 고안되었다. 즉, 종양 특이적 항원을 암 환자 유래 전문적 항원표출세포에 처리하고, 다시 종양 특이적 항원에 의해 활성화된 전문적 항원표출세포를 암 환자의 몸에 넣어주는 방법이다(van Willigen et al., 2018). 암 환자에 이식된 활성화된 전문적 항원표출세포는 직접적으로 종양 세포를 표적하는 세포독성 T 세포(cytotoxic T lymphocytes, CTL)와 자연살생세포(natural killer cell, NK cell)의 활성을 유발하여, 종양 세포를 죽이게 된다(Galluzzi et al., 2018; Lu et al., 2020). Recently, a method for treating tumors by inducing the activation of specialized antigen-expressing cells has been devised. That is, it is a method in which tumor-specific antigens are treated with cancer patient-derived specialized antigen-expressing cells, and then, professional antigen-expressing cells activated by tumor-specific antigens are put into the cancer patient's body (van Willigen et al., 2018). . Activated specialized antigen-expressing cells implanted in cancer patients induce the activation of cytotoxic T lymphocytes (CTL) and natural killer cells (NK cells) that directly target tumor cells, thereby causing tumor cells (Galluzzi et al., 2018; Lu et al., 2020).
하지만, 이러한 면역 요법의 가장 큰 해결해야 할 과제는 종양 특이적인 항원이 지금까지 그렇게 많이 발견되지 않았다는 것이다. 따라서, 최근에는 암의 세포 표면에서 발현하는 단백질을 인식하는 항체의 단일사슬 Fv 단편(single-chain variable fragment, scFv) 부분을 CD3 제타(zeta) 또는 다른 단백질의 세포질내 신호전달 도메인(cytoplasmic signaling domain)에 접목시킨 키메릭 항원 수용체(chimeric antigen receptor, CAR)를 세포독성 T 세포와 자연살생세포에 발현시켜, 암의 세포 표면에서 발현하는 단백질을 인식하여 전달되는 신호 전달에 의해 세포독성 T 세포와 자연살생세포의 암 특이적 활성을 유발하는 새로운 항암 치료 방법이 도입되었다. 즉, 키메릭 항원 수용체를 T 세포 또는 자연살생세포에 접목시키면, 암특이적 항원을 인식하는 전문적 항원표출세포에 의한 신호 전달과 관계없이 scFv의 특정 항원 인지만으로 T 세포 또는 자연살생세포의 항암 작용을 활성화시킬 수 있으며, 또한 HLA type에 제한적이지 않아 많은 사람들이 보편적으로 사용할 수 있는 보다 효율적인 치료 방법으로 이용할 수 있다. 실제로, 이러한 키메릭 항원 수용체 발현 T 세포나 자연살생세포는 여러 암에서 효능을 보이고 있다(Holzinger et al., 2016; Pettitt et al., 2018; Wang et al., 2020).However, the biggest challenge for such immunotherapy is that tumor-specific antigens have not been discovered so far. Therefore, recently, a single-chain variable fragment (scFv) portion of an antibody that recognizes a protein expressed on the cell surface of cancer has been replaced with CD3 zeta or the cytoplasmic signaling domain of another protein. ) grafted onto the chimeric antigen receptor (CAR) is expressed in cytotoxic T cells and natural killer cells to recognize proteins expressed on the surface of cancer cells and to communicate with cytotoxic T cells by signal transduction. A new anticancer treatment method that induces cancer-specific activity of natural killer cells has been introduced. That is, when the chimeric antigen receptor is grafted onto T cells or natural killer cells, the anticancer action of T cells or natural killer cells only by recognizing specific antigens of scFv regardless of signal transduction by specialized antigen-expressing cells that recognize cancer-specific antigens. In addition, since it is not limited to HLA type, it can be used as a more efficient treatment method that can be used universally by many people. Indeed, these chimeric antigen receptor-expressing T cells or natural killer cells have shown efficacy in several cancers (Holzinger et al., 2016; Pettitt et al., 2018; Wang et al., 2020).
한편, B-cell activating factor (BAFF)는 tumor necrosis factor (TNF) 리간드에 속하는 사이토카인이며, BLyS (B-lymphocyte stimulator), TALL-1 (TNF- and ApoL related leukocyte-expressed ligand 1), THANK (TNF homologue that activates apoptosis, nuclear factor-κB and c-Jun NH2-terminal kinase), TNFSF13B (TNFsuperfamily member 13B) 또는 zTNF4로 불리운다(Mackay and Browning, 2002). BAFF는 원래 285개의 아미노산으로 구성된 type 2 막관통 단백질(transmembrane protein)로 동형3합체(homotrimer)로 세포 표면에 발현된 후, 막관통 부분이 잘려져 나가 세포외로 분비된다. BAFF는 대식세포(macrophage), 단핵구세포(monocyte), 수지상세포(dendritic cell)에서 분비된다(Mackay and Browning, 2002). BAFF와 결합하는 수용체는 BAFF 수용체(BAFF receptor), B-cell maturation antigen (BCMA), Transmembrane activator and CAML interactor (TACI) 등이 있다(Mackay and Browning, 2002). BAFF 수용체는 골수내 형질세포(plasma cell)를 제외한 대부분의 사람 B 세포에서 발현되며, BCMA는 주로 항체를 분비하는 사람 세포에서 발현된다(Mackay and Schneider, 2009). 또한 TACI는 대부분의 말초 B 세포에서 발현된다(Mackay and Schneider, 2009). 따라서, BAFF와 BAFF와 결합하는 3가지 수용체인 BAFF 수용체, BCMA, TACI의 상호 작용에 의한 신호 전달은 B 세포 활성에 매우 중요하다고 여겨진다(Mackay and Schneider, 2009). On the other hand, B-cell activating factor (BAFF) is a cytokine belonging to tumor necrosis factor (TNF) ligand, BLyS (B-lymphocyte stimulator), TALL-1 (TNF- and ApoL related leukocyte-expressed ligand 1), THANK ( It is called TNF homologue that activates apoptosis, nuclear factor-κB and c-Jun NH2-terminal kinase), TNFSF13B (TNFsuperfamily member 13B) or zTNF4 (Mackay and Browning, 2002). BAFF is originally a type 2 transmembrane protein composed of 285 amino acids and is expressed on the cell surface as a homotrimer, and then the transmembrane portion is cut off and secreted out of the cell. BAFF is secreted from macrophages, monocytes, and dendritic cells (Mackay and Browning, 2002). Receptors that bind to BAFF include BAFF receptor, B-cell maturation antigen (BCMA), and Transmembrane activator and CAML interactor (TACI) (Mackay and Browning, 2002). The BAFF receptor is expressed in most human B cells except plasma cells in the bone marrow, and BCMA is mainly expressed in antibody-secreting human cells (Mackay and Schneider, 2009). TACI is also expressed in most peripheral B cells (Mackay and Schneider, 2009). Therefore, signal transduction by the interaction of BAFF and three BAFF-binding receptors, the BAFF receptor, BCMA, and TACI, is considered to be very important for B cell activity (Mackay and Schneider, 2009).
BAFF와 결합하는 3가지 수용체인 BAFF 수용체, BCMA, TACI는 또한, 다양한 암종(malignant cells)에서 과발현이 관찰된다. 특히 BCMA는 다발성 골수종(multiple myeloma)의 표지 인자이며(Carpenter et al., 2013), 유방암(breast cancer), T 세포 림프종(T cell lymphoma), 만성 B 림프구성백혈병(B cell Chronic lymphocytic leukemia, B-CLL), 비소세포 폐암(non-small cell lung cancer) 등에서 발견된다(Haiat et al., 2006; Dou et al., 2016; Kampa et al., 2020). 또한, BAFF 수용체는 B 세포 림프종(B-cell lymphoma), 여포성 림프종(follicular lymphoma), 광범위큰 B 세포 림프종(diffuse large B-cell lymphoma, DLBCL), 외투세포림프종(mantle cell lymphoma), B 세포 비호지킨림프종(B-cell non-Hodgkin lymphoma) 등 다양한 림프종에서 발현하며, 또한, 급성림프아구성백혈병(acute lymphoblastic leukemia, ALL), 만성림프아구성백혈병(chronic lymphoblastic leukemia, CLL) 등과 같은 백혈병에서도 발현한다. 이 밖에도 BAFF 수용체는 전신 홍반성 루프스(systemic lupus erythematosus, SLE) 같은 자가면역질환의 B 세포에도 과발현 된다(Haiat et al., 2006; Parameswaran et al., 2010; Takahata et al., 2010; Yang et al., 2014). 한편, TACI는 다발성 골수종(multiple myeloma), 유방암(breast cancer), 갑상선암(thyroid carcinoma), 비소세포 폐암(non-small cell lung cancer) 등에서 발견된다(Moreaux et al., 2009; Dou et al., 2016; Kampa et al., 2020).Three BAFF-binding receptors, the BAFF receptor, BCMA, and TACI, are also overexpressed in various malignant cells. In particular, BCMA is a marker of multiple myeloma (Carpenter et al., 2013), breast cancer, T cell lymphoma, B cell Chronic lymphocytic leukemia, B -CLL), non-small cell lung cancer, etc. (Haiat et al., 2006; Dou et al., 2016; Kampa et al., 2020). In addition, BAFF receptors are B-cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, B cell It is expressed in various lymphomas such as B-cell non-Hodgkin lymphoma, and also in leukemias such as acute lymphoblastic leukemia (ALL) and chronic lymphoblastic leukemia (CLL). to manifest In addition, the BAFF receptor is overexpressed in B cells of autoimmune diseases such as systemic lupus erythematosus (SLE) (Haiat et al., 2006; Parameswaran et al., 2010; Takahata et al., 2010; Yang et al. al., 2014). On the other hand, TACI is found in multiple myeloma, breast cancer, thyroid carcinoma, non-small cell lung cancer, etc. (Moreaux et al., 2009; Dou et al., 2016; Kampa et al., 2020).
현재, BCMA 또는 BAFF 수용체를 종양항원으로 인식하는 CAR-T 세포(미국공개특허 제2020-0283534호)나 수용성 TACI 이뮤노글로불린 조성물과 사용 방법(미국등록특허 제08524232호)가 공개되어 있으나, BAFF 수용체, BCMA, TACI가 발현되는 암세포에 대한 결합 특이성을 갖는 BAFF의 세포외 도메인(extracellular domain)을 항원 결합 도메인에 포함하여 면역 항암 치료제로서 이용하는 발명에 대해서는 기재된 바 없다.Currently, CAR-T cells that recognize BCMA or BAFF receptors as tumor antigens (US Patent Publication No. 2020-0283534) or water-soluble TACI immunoglobulin compositions and methods of use (US Patent No. 08524232) have been disclosed, but BAFF The invention using the extracellular domain of BAFF, which has binding specificity for cancer cells expressing receptor, BCMA, and TACI, as an antigen-binding domain, has not been described.
이러한 배경 하에, 본 발명자들은 전문적 항원표출세포의 기능과 키메릭 항원 수용체의 기능을 접목시킨 새로운 치료 방법을 고안하였다. 즉, 종양 세포 표면 단백질을 리간드(ligand)로 인식할 수 있는 수용체와 리간드를 인식한 후, 키메릭 항원 수용체에 의해 전문적 항원표출세포의 활성을 유발할 수 있는 신규 키메릭 신호전달 도메인을 고안하였고, 이러한 키메릭 신호전달 도메인에 의해 활성화된 전문적 항원표출세포가 암에 대한 세포독성 능력을 가질수 있다는 것을 증명하였다. 즉, 사람의 BAFF 수용체, BCMA, TACI와 BAFF의 결합이 가능하다는 점을 이용하여 BAFF의 세포외 도메인, 톨유사수용체-2(toll-like receptor 2, TLR-2)의 막관통 도메인, TLR-3의 세포 내 도메인, TLR-4의 세포 내 도메인, IgE 수용체 gamma chain (Fcε receptor I gamma chain, FcεRIγ) 또는 IgG 수용체 2A alpha chain (Fcγ receptor 2A alpha chain, FcγR2Aα) 또는 IgE 수용체 beta chain (Fcε receptor I beta chain, FcεRIβ)의 면역수용체 타이로신 기반 활성화 모티프(immunoreceptor tyrosine-based activation motif, ITAM)를 소단위(subunit)로 하여, 3개의 ITAM을 링커(linker)로 연결시킨 신규 합성 세포 내 신호 전달 도메인과 결합한 재조합 단백질을 발현하는 키메릭 항원 수용체 발현 대식세포를 제작하였다. 이때 대식세포에서 발현하는 BAFF의 세포 외 도메인은 BAFF 수용체, BCMA, TACI를 인지하여 BAFF 수용체, BCMA, TACI를 발현하는 특정 암세포 또는 전신 홍반성 루프스의 B 세포와 결합 시, 이들 대식세포 내부로 활성화 신호를 전달해 줄 수 있고, 결국 이러한 신호는 대식세포를 활성화시켜 BAFF 수용체, BCMA, TACI를 발현하는 특정 암세포 또는 전신 홍반성 루프스의 B 세포에 대한 세포독성을 가질 수 있게 한다. 실제로, 상기 대식세포가 BAFF 수용체, BCMA, TACI를 발현하는 세포에 특이적으로 세포독성 능력이 있음을 체외 실험을 통해 확인하였다. Under this background, the present inventors devised a new treatment method that grafts the function of the professional antigen-presenting cell and the function of the chimeric antigen receptor. That is, after recognizing a receptor and a ligand that can recognize a tumor cell surface protein as a ligand, a novel chimeric signaling domain that can induce the activity of a specialized antigen-presenting cell by the chimeric antigen receptor was designed, It was demonstrated that professional antigen-presenting cells activated by this chimeric signaling domain can have cytotoxicity against cancer. That is, the extracellular domain of BAFF, the transmembrane domain of toll-like receptor 2 (TLR-2), TLR- 3, intracellular domain of TLR-4, IgE receptor gamma chain (Fcε receptor I gamma chain, FcεRIγ) or IgG receptor 2A alpha chain (Fcγ receptor 2A alpha chain, FcγR2Aα) or IgE receptor beta chain (Fcε receptor I beta chain, FcεRIβ) with an immunoreceptor tyrosine-based activation motif (ITAM) as a subunit, a novel synthetic intracellular signal transduction domain linking three ITAMs with a linker and Chimeric antigen receptor-expressing macrophages expressing the bound recombinant protein were prepared. At this time, the extracellular domain of BAFF expressed in macrophages recognizes BAFF receptors, BCMA, and TACI, and when combined with specific cancer cells or systemic lupus erythematosus B cells expressing BAFF receptors, BCMA, TACI, these macrophages are activated Signals can be transmitted, which in turn activate macrophages to have cytotoxicity against specific cancer cells expressing BAFF receptors, BCMA, TACI, or systemic lupus erythematosus B cells. In fact, it was confirmed through an in vitro experiment that the macrophages have specific cytotoxicity to cells expressing the BAFF receptor, BCMA, and TACI.
따라서 본 발명의 목적은 키메릭 항원 수용체(CAR)를 포함하는 형질전환된 세포로서, 상기 키메릭 항원 수용체는 BAFF 수용체, BCMA 또는 TACI에 특이적으로 결합하는 항원 결합 도메인, 막관통 도메인 및 세포 내 신호전달 도메인을 포함하며, 상기 세포는 표적화된 효과기 활성을 갖는 항원 특이적 전문적 항원표출세포로서 대식세포, 수지상세포 또는 미감작 B 세포(naive B cell)를 포함하는, 형질전환된 세포를 제공하는 것이다.Accordingly, an object of the present invention is a transformed cell comprising a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor is a BAFF receptor, an antigen binding domain that specifically binds BCMA or TACI, a transmembrane domain and an intracellular To provide a transformed cell comprising a signaling domain, wherein the cell comprises a macrophage, dendritic cell or naive B cell as an antigen-specific professional antigen-presenting cell having a targeted effector activity will be.
본 발명의 다른 목적은 상기 형질전환된 세포를 유효성분으로 포함하는 세포 치료제 또는 BAFF 수용체, BCMA 또는 TACI를 발현하는 암 및 전신 홍반성 루프스의 치료용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for the treatment of cancer and systemic lupus erythematosus expressing a cell therapeutic agent or BAFF receptor, BCMA or TACI comprising the transformed cell as an active ingredient.
상기와 같은 본 발명의 목적을 달성하기 위해서, 본 키메릭 항원 수용체(CAR)를 포함하는 형질전환된 세포로서, 상기 키메릭 항원 수용체는 BAFF 수용체, BCMA 또는 TACI에 특이적으로 결합하는 항원 결합 도메인, 막관통 도메인 및 세포 내 신호전달 도메인을 포함하며, 상기 세포는 표적화된 효과기 활성을 갖는 항원 특이적 전문적 항원표출세포로서 대식세포, 수지상세포 또는 미감작 B 세포(naive B cell)를 포함하는, 형질전환된 세포를 제공한다.In order to achieve the object of the present invention as described above, as a transformed cell comprising the present chimeric antigen receptor (CAR), the chimeric antigen receptor is an antigen binding domain that specifically binds to a BAFF receptor, BCMA or TACI. , a transmembrane domain and an intracellular signaling domain, wherein the cell comprises a macrophage, dendritic cell or naive B cell as an antigen-specific professional antigen-presenting cell having a targeted effector activity, Transformed cells are provided.
또한, 본 발명은 상기 형질전환된 세포를 포함하는, 세포 치료제 또는 BAFF 수용체, BCMA 또는 TACI를 발현하는 암 및 전신 홍반성 루프스의 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for the treatment of cancer and systemic lupus erythematosus, comprising the transformed cell, a cell therapeutic agent or a BAFF receptor, BCMA or TACI.
본 발명의 일실시 예에 있어서, 상기 키메릭 항원 수용체(CAR)의 항원 결합 도메인은 상기 BAFF 수용체, BCMA 또는 TACI에 특이적으로 결합하는 BAFF의 세포외 도메인일 수 있다.In one embodiment of the present invention, the antigen-binding domain of the chimeric antigen receptor (CAR) may be an extracellular domain of BAFF that specifically binds to the BAFF receptor, BCMA, or TACI.
본 발명의 일실시 예에 있어서, 상기 막관통 도메인은 TLR-2일 수 있다.In one embodiment of the present invention, the transmembrane domain may be TLR-2.
본 발명의 일실시 예에 있어서, 상기 세포 내 신호전달 도메인은 TLR-3, TLR-4, IgE 수용체 gamma chain (Fcε receptor I gamma chain, FcεRIγ), IgG 수용체 2A alpha chain (Fcγ receptor 2A alpha chain, FcγR2Aα), IgE 수용체 beta chain (Fcε receptor I beta chain, FcεRIβ)의 면역수용체 타이로신 기반 활성화 모티프(ITAM) 또는 이들의 조합일 수 있다.In one embodiment of the present invention, the intracellular signaling domain is TLR-3, TLR-4, IgE receptor gamma chain (Fcε receptor I gamma chain, FcεRIγ), IgG receptor 2A alpha chain (Fcγ receptor 2A alpha chain, FcγR2Aα), an immunoreceptor tyrosine-based activation motif (ITAM) of the IgE receptor beta chain (Fcε receptor I beta chain, FcεRIβ), or a combination thereof.
상기 "BAFF 수용체", "BCMA", "TACI"는 다양한 암종(malignant cells)과 자가면역질환, 바람직하게는 전신 홍반성 루프스(systemic lupus erythematosus, SLE)에서 관찰된다. 지금까지 "BAFF 수용체", "BCMA", "TACI"를 발현하는 암종은 급성림프아구성백혈병(acute lymphoblastic leukemia, ALL), 만성림프아구성백혈병(chronic lymphoblastic leukemia, CLL), 여포성 림프종(follicular lymphoma), 외투세포림프종(mantle cell lymphoma), B 세포 림프종(B-cell lymphoma), 광범위큰 B 세포 림프종(diffuse large B-cell lymphoma, DLBCL), B 세포 비호지킨림프종(B-cell non-Hodgkin lymphoma), 다발성 골수종(multiple myeloma), T 세포 림프종(T cell lymphoma), 유방암(breast cancer), 갑상선암(thyroid carcinoma) 및 비소세포 폐암(non-small cell lung cancer)으로 이루어지는 군으로부터 선택될 수 있으나, 이에 제한되지 않는다. The "BAFF receptor", "BCMA", and "TACI" are observed in various malignant cells and autoimmune diseases, preferably in systemic lupus erythematosus (SLE). Until now, carcinomas expressing "BAFF receptor", "BCMA", and "TACI" have been classified into acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), and follicular lymphoma. lymphoma), mantle cell lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), B-cell non-Hodgkin lymphoma), multiple myeloma, T cell lymphoma, breast cancer, thyroid carcinoma and non-small cell lung cancer may be selected from the group consisting of , but not limited thereto.
본 발명에 따른 형질전환된 세포는 전문적 항원표출세포가 항원에 특이적으로 결합할 때, 전문적 항원표출세포의 활성을 유발하는 강화된 키메릭 신호전달 도메인을 제공한다. 이에 대한 예시로, BAFF의 세포외 도메인과 전문적 항원표출세포의 신호 전달에 중요한 작용을 하는 TLR-3, TLR-4 및 IgE 수용체 gamma chain (Fcε receptor I gamma chain, FcεRIγ) 또는 IgG 수용체 2A alpha chain (Fcγ receptor 2A alpha chain, FcγR2Aα) 또는 IgE 수용체 beta chain (Fcε receptor I beta chain, FcεRIβ)의 면역수용체 타이로신 기반 활성화 모티프(ITAM)를 소단위(subunit)로 하여, 3개의 ITAM을 링커(linker)로 연결시킨 신규 합성 세포 내 신호 전달 도메인을 포함하는 재조합 단백질로서, 이를 과발현시킬 수 있는 벡터로 형질전환된 전문적 항원표출세포의 경우 BAFF 수용체, BCMA 또는 TACI를 발현하는 암종에 특이적으로 세포독성을 갖는다. 따라서, 본 발명에 의한 키메릭 항원 수용체를 과발현하는 전문적 항원표출세포는 BAFF 수용체, BCMA 또는 TACI를 발현하는 암종 및 전신홍반성 루프스 치료를 위한 면역세포 치료제로서 유용하게 사용될 수 있다.The transformed cell according to the present invention provides an enhanced chimeric signaling domain that induces the activity of the professional antigen-presenting cell when the professional antigen-presenting cell specifically binds to an antigen. As an example, the extracellular domain of BAFF and TLR-3, TLR-4, and IgE receptor gamma chain (Fcε receptor I gamma chain, FcεRIγ) or IgG receptor 2A alpha chain, which play an important role in signal transduction of professional antigen-presenting cells Using the immunoreceptor tyrosine-based activation motif (ITAM) of (Fcγ receptor 2A alpha chain, FcγR2Aα) or IgE receptor beta chain (Fcε receptor I beta chain, FcεRIβ) as a subunit, three ITAMs as a linker Recombinant protein containing a new synthetic intracellular signal transduction domain that is linked. In the case of a professional antigen-presenting cell transformed with a vector capable of overexpressing it, it is specifically cytotoxic to carcinoma expressing BAFF receptor, BCMA or TACI. . Therefore, the specialized antigen-expressing cell overexpressing the chimeric antigen receptor according to the present invention can be usefully used as an immune cell therapeutic agent for the treatment of BAFF receptor, BCMA or TACI-expressing carcinoma and systemic lupus erythematosus.
도 1은 본 발명의 일 실시예에 따른 키메릭 항원 수용체를 발현시키는 각 도메인의 cDNA 구역 중 하나의 형태를 나타낸 모식도이다. 1 is a schematic diagram showing the form of one of the cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.
도 2는 본 발명의 일 실시예에 따른 키메릭 항원 수용체를 발현시키는 각 도메인의 cDNA 구역 중 하나의 형태를 나타낸 모식도이다.2 is a schematic diagram showing the form of one of the cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.
도 3은 본 발명의 일 실시예에 따른 키메릭 항원 수용체를 발현시키는 각 도메인의 cDNA 구역 중 하나의 형태를 나타낸 모식도이다.3 is a schematic diagram showing the form of one of the cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.
도 4는 본 발명의 일 실시예에 따른 키메릭 항원 수용체를 발현시키는 각 도메인의 cDNA 구역 중 하나의 형태를 나타낸 모식도이다.4 is a schematic diagram showing the form of one of the cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.
도 5는 본 발명의 일 실시예에 따른 렌티바이럴 벡터 중 하나의 형태를 나타낸 모식도이다.5 is a schematic diagram showing the form of one of the lentiviral vectors according to an embodiment of the present invention.
도 6은 본 발명의 일 실시예에 따른 렌티바이럴 벡터 중 하나의 형태를 나타낸 모식도이다.6 is a schematic diagram showing the form of one of the lentiviral vectors according to an embodiment of the present invention.
도 7은 본 발명의 일 실시예에 따른 렌티바이럴 벡터 중 하나의 형태를 나타낸 모식도이다.7 is a schematic diagram showing the form of one of the lentiviral vectors according to an embodiment of the present invention.
도 8은 본 발명의 일 실시예에 따른 렌티바이럴 벡터 중 하나의 형태를 나타낸 모식도이다.8 is a schematic diagram illustrating a form of one of lentiviral vectors according to an embodiment of the present invention.
도 9는 본 발명의 일 실시예에 따른 전문적 항원표출세포 표면에 발현하는 키메릭 항원 수용체 중 하나의 형태를 나타낸 모식도이다.9 is a schematic diagram showing the form of one of the chimeric antigen receptors expressed on the surface of a professional antigen-expressing cell according to an embodiment of the present invention.
도 10은 본 발명의 일 실시예에 따른 전문적 항원표출세포 표면에 발현하는 키메릭 항원 수용체 중 하나의 형태를 나타낸 모식도이다.10 is a schematic diagram showing the form of one of the chimeric antigen receptors expressed on the surface of a professional antigen-expressing cell according to an embodiment of the present invention.
도 11은 본 발명의 일 실시예에 따른 전문적 항원표출세포 표면에 발현하는 키메릭 항원 수용체 중 하나의 형태를 나타낸 모식도이다.11 is a schematic diagram showing the form of one of the chimeric antigen receptors expressed on the surface of a professional antigen-expressing cell according to an embodiment of the present invention.
도 12는 본 발명의 일 실시예에 따른 전문적 항원표출세포 표면에 발현하는 키메릭 항원 수용체 중 하나의 형태를 나타낸 모식도이다. 12 is a schematic diagram showing the form of one of the chimeric antigen receptors expressed on the surface of a professional antigen-expressing cell according to an embodiment of the present invention.
도 13a는 본 발명에서 제공하는 키메릭 항원 수용체의 표적인 BAFF 수용체를 Chinese hamster ovary (CHO) 세포에 발현하기 위해 BAFF 수용체 cDNA를 레트로바이럴 벡터에 삽입한 모식도이다.Figure 13a is a schematic diagram in which the BAFF receptor cDNA is inserted into a retroviral vector to express the BAFF receptor, a target of the chimeric antigen receptor provided in the present invention, in Chinese hamster ovary (CHO) cells.
도 13b는 본 발명에서 제공하는 키메릭 항원 수용체의 표적인 BCMA를 Chinese hamster ovary (CHO) 세포에 발현하기 위해 BCMA cDNA를 레트로바이럴 벡터에 삽입한 모식도이다.13B is a schematic diagram of BCMA cDNA inserted into a retroviral vector to express BCMA, a target of the chimeric antigen receptor provided in the present invention, in Chinese hamster ovary (CHO) cells.
도 13c는 본 발명에서 제공하는 키메릭 항원 수용체의 표적인 TACI를 Chinese hamster ovary (CHO) 세포에 발현하기 위해 TACI cDNA를 레트로바이럴 벡터에 삽입한 모식도이다.13c is a schematic diagram of TACI cDNA inserted into a retroviral vector to express TACI, a target of the chimeric antigen receptor provided in the present invention, in Chinese hamster ovary (CHO) cells.
도 14a는 본 발명에서 제공하는 키메릭 항원 수용체의 타겟인 BAFF 수용체를 발현하는 CHO 세포(BAFF receptor positive cell)에서 BAFF 수용체의 발현량을 유세포 분석을 이용해 측정한 결과를 나타낸 도이다.14A is a diagram showing the result of measuring the expression level of BAFF receptor in BAFF receptor positive cells (CHO cells) expressing BAFF receptor, a target of the chimeric antigen receptor provided in the present invention, using flow cytometry.
도 14b는 본 발명에서 제공하는 키메릭 항원 수용체의 타겟인 BCMA를 발현하는 CHO 세포(BAFF receptor positive cell)에서 BCMA의 발현량을 유세포 분석을 이용해 측정한 결과를 나타낸 도이다.14B is a diagram showing the results of measuring the expression level of BCMA in CHO cells (BAFF receptor positive cells) expressing BCMA, the target of the chimeric antigen receptor provided in the present invention, using flow cytometry.
도 14c는 본 발명에서 제공하는 키메릭 항원 수용체의 타겟인 TACI를 발현하는 CHO 세포(BAFF receptor positive cell)에서 TACI의 발현량을 유세포 분석을 이용해 측정한 결과를 나타낸 도이다.14c is a diagram showing the results of measuring the expression level of TACI in CHO cells (BAFF receptor positive cells) expressing TACI, a target of the chimeric antigen receptor provided in the present invention, using flow cytometry.
도 15는 본 발명의 일 실시예에 따른 발현 벡터를 렌티바이러스 시스템을 이용하여 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC)에서의 GFP의 발현 비율을 비교한 결과를 나타낸 도이다.15 is a graph showing the expression ratio of GFP in professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. It is a diagram showing the results of comparison.
도 16a는 본 발명의 일 실시예에 따른 발현 벡터를 렌티바이러스 시스템을 이용하여 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC) 중 3-4-3 BAFF CAR (TLR-3+TLR-4+FcεRIγ ITAM trimer) 발현 전문적 항원표출세포가 BAFF 수용체를 인식하는지 여부를 유세포 분석을 이용해 나타낸 도이다.FIG. 16a shows 3-4-3 of professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. It is a diagram showing whether the BAFF CAR (TLR-3+TLR-4+FcεRIγ ITAM trimer) expression specialized antigen-expressing cells recognize the BAFF receptor using flow cytometry.
도 16b는 본 발명의 일 실시예에 따른 발현 벡터를 렌티바이러스 시스템을 이용하여 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC) 중 3-4-3 BAFF CAR (TLR-3+TLR-4+FcεRIγ ITAM trimer) 발현 전문적 항원표출세포가 BCMA를 인식하는지 여부를 유세포 분석을 이용해 나타낸 도이다.FIG. 16b shows 3-4-3 of professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. It is a diagram showing whether BCMA is recognized by BAFF CAR (TLR-3+TLR-4+FcεRIγ ITAM trimer) expression specialized antigen-expressing cells using flow cytometry.
도 16c는 본 발명의 일 실시예에 따른 발현 벡터를 렌티바이러스 시스템을 이용하여 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC) 중 3-4-3 BAFF CAR (TLR-3+TLR-4+FcεRIγ ITAM trimer) 발현 전문적 항원표출세포가 TACI를 인식하는지 여부를 유세포 분석을 이용해 나타낸 도이다.FIG. 16c shows 3-4-3 of professional antigen-presenting cells (BAFF CAR-pAPCs) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. It is a diagram showing whether BAFF CAR (TLR-3+TLR-4+FcεRIγ ITAM trimer) expression specialized antigen-expressing cells recognize TACI using flow cytometry.
도 17a는 본 발명의 일 실시예에 따른 발현 벡터를 렌티바이러스 시스템을 이용하여 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC) 중 3-4-2A BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM trimer) 발현 전문적 항원표출세포가 BAFF 수용체를 인식하는지 여부를 유세포 분석을 이용해 나타낸 도이다.FIG. 17a shows 3-4-2A of professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. It is a diagram showing whether the BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM trimer) expression specialized antigen-expressing cells recognize the BAFF receptor using flow cytometry.
도 17b는 본 발명의 일 실시예에 따른 발현 벡터를 렌티바이러스 시스템을 이용하여 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC) 중 3-4-2A BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM trimer) 발현 전문적 항원표출세포가 BCMA를 인식하는지 여부를 유세포 분석을 이용해 나타낸 도이다.FIG. 17b shows 3-4-2A of professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. It is a diagram showing whether BCMA is recognized by BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM trimer) expression specialized antigen-expressing cells using flow cytometry.
도 17c는 본 발명의 일 실시예에 따른 발현 벡터를 렌티바이러스 시스템을 이용하여 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC) 중 3-4-2A BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM trimer) 발현 전문적 항원표출세포가 TACI를 인식하는지 여부를 유세포 분석을 이용해 나타낸 도이다.FIG. 17c shows 3-4-2A of professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. It is a diagram showing whether BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM trimer) expression specialized antigen-expressing cells recognize TACI using flow cytometry.
도 18a는 본 발명의 일 실시예에 따른 발현 벡터를 렌티바이러스 시스템을 이용하여 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC) 중 3-4-1B BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer) 발현 전문적 항원표출세포가 BAFF 수용체를 인식하는지 여부를 유세포 분석을 이용해 나타낸 도이다.18A shows 3-4-1B of professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. It is a diagram showing whether BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer) expression specialized antigen-expressing cells recognize the BAFF receptor using flow cytometry.
도 18b는 본 발명의 일 실시예에 따른 발현 벡터를 렌티바이러스 시스템을 이용하여 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC) 중 3-4-1B BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer) 발현 전문적 항원표출세포가 BCMA를 인식하는지 여부를 유세포 분석을 이용해 나타낸 도이다.18B shows 3-4-1B of professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. It is a diagram showing whether BCMA is recognized by BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer) expression specialized antigen-expressing cells using flow cytometry.
도 18c는 본 발명의 일 실시예에 따른 발현 벡터를 렌티바이러스 시스템을 이용하여 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC) 중 3-4-1B BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer) 발현 전문적 항원표출세포가 TACI를 인식하는지 여부를 유세포 분석을 이용해 나타낸 도이다.FIG. 18c shows 3-4-1B of professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. A diagram showing whether BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer) expression specialized antigen-expressing cells recognize TACI using flow cytometry.
도 19a는 본 발명의 일 실시예에 따른 발현 벡터를 렌티바이러스 시스템을 이용하여 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC) 중 3-4-ABC BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer) 발현 전문적 항원표출세포가 BAFF 수용체를 인식하는지 여부를 유세포 분석을 이용해 나타낸 도이다.FIG. 19a shows 3-4-ABC among professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. It is a diagram showing whether BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer) expression-specific antigen-expressing cells recognize the BAFF receptor using flow cytometry.
도 19b는 본 발명의 일 실시예에 따른 발현 벡터를 렌티바이러스 시스템을 이용하여 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC) 중 3-4-ABC BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer) 발현 전문적 항원표출세포가 BCMA를 인식하는지 여부를 유세포 분석을 이용해 나타낸 도이다.FIG. 19b shows 3-4-ABC among professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. A diagram showing whether BCMA is recognized by BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer) expression-specific antigen-expressing cells using flow cytometry.
도 19c는 본 발명의 일 실시예에 따른 발현 벡터를 렌티바이러스 시스템을 이용하여 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC) 중 3-4-ABC BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer) 발현 전문적 항원표출세포가 TACI를 인식하는지 여부를 유세포 분석을 이용해 나타낸 도이다.Figure 19c shows 3-4-ABC among professional antigen-presenting cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors, each of which was transduced with an expression vector according to an embodiment of the present invention using a lentiviral system. A diagram showing whether BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer) expression-specific antigen-expressing cells recognize TACI using flow cytometry.
도 20은 본 발명의 일 실시예에 따른 공벡터(Mock pAPC) 또는 키메릭 항원 수용체를 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC) 중 3-4-3 BAFF CAR (TLR-3+TLR-4+FcεRIγ ITAM trimer) 발현 전문적 항원표출세포를 작동세포(effector cell)로 하여 BAFF 수용체, BCMA 또는 TACI를 발현하는 Chinese hamster ovary (CHO) 세포에 대한 세포독성을 24시간 후 측정한 결과를 나타낸 도이다.FIG. 20 shows 3- of professional antigen-expressing cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors transduced with an empty vector (Mock pAPC) or a chimeric antigen receptor according to an embodiment of the present invention. 4-3 BAFF CAR (TLR-3+TLR-4+FcεRIγ ITAM trimer) expression Specialized antigen-expressing cells were used as effector cells for BAFF receptor, BCMA or TACI-expressing Chinese hamster ovary (CHO) cells. A diagram showing the results of measuring cytotoxicity after 24 hours.
도 21은 본 발명의 일 실시예에 따른 공벡터(Mock pAPC) 또는 키메릭 항원 수용체를 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC) 중 3-4-3 BAFF CAR (TLR-3+TLR-4+FcεRIγ ITAM trimer) 발현 전문적 항원표출세포를 작동세포(effector cell)로 하여 BAFF 수용체, BCMA 또는 TACI를 발현하는 Chinese hamster ovary (CHO) 세포에 대한 세포독성을 48시간 후 측정한 결과를 나타낸 도이다.Figure 21 shows 3- of professional antigen-expressing cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors transduced with an empty vector (Mock pAPC) or a chimeric antigen receptor according to an embodiment of the present invention; 4-3 BAFF CAR (TLR-3+TLR-4+FcεRIγ ITAM trimer) expression Specialized antigen-expressing cells were used as effector cells for BAFF receptor, BCMA or TACI-expressing Chinese hamster ovary (CHO) cells. A diagram showing the results of measuring cytotoxicity after 48 hours.
도 22는 본 발명의 일 실시예에 따른 공벡터(Mock pAPC) 또는 키메릭 항원 수용체를 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC) 중 3-4-2A BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM trimer) 발현 전문적 항원표출세포를 작동세포(effector cell)로 하여 BAFF 수용체, BCMA 또는 TACI를 발현하는 Chinese hamster ovary (CHO) 세포에 대한 세포독성을 24시간 후 측정한 결과를 나타낸 도이다.22 shows 3- of professional antigen-expressing cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors transduced with an empty vector (Mock pAPC) or a chimeric antigen receptor according to an embodiment of the present invention. 4-2A BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM trimer) expression cytotoxicity to Chinese hamster ovary (CHO) cells expressing BAFF receptor, BCMA or TACI using professional antigen-expressing cells as effector cells is a diagram showing the measurement result after 24 hours.
도 23은 본 발명의 일 실시예에 따른 공벡터(Mock pAPC) 또는 키메릭 항원 수용체를 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC) 중 3-4-2A BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM trimer) 발현 전문적 항원표출세포를 작동세포(effector cell)로 하여 BAFF 수용체, BCMA 또는 TACI를 발현하는 Chinese hamster ovary (CHO) 세포에 대한 세포독성을 48시간 후 측정한 결과를 나타낸 도이다.23 shows 3- of professional antigen-expressing cells (BAFF CAR-pAPC) each expressing four types of chimeric antigen receptors transduced with an empty vector (Mock pAPC) or a chimeric antigen receptor according to an embodiment of the present invention; 4-2A BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM trimer) expression cytotoxicity to Chinese hamster ovary (CHO) cells expressing BAFF receptor, BCMA or TACI using professional antigen-expressing cells as effector cells is a diagram showing the measurement result after 48 hours.
도 24는 본 발명의 일 실시예에 따른 공벡터(Mock pAPC) 또는 키메릭 항원 수용체를 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC) 중 3-4-1B BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer) 발현 전문적 항원표출세포를 작동세포(effector cell)로 하여 BAFF 수용체, BCMA 또는 TACI를 발현하는 Chinese hamster ovary (CHO) 세포에 대한 세포독성을 24시간 후 측정한 결과를 나타낸 도이다.Figure 24 shows 3- of professional antigen-expressing cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors transduced with an empty vector (Mock pAPC) or a chimeric antigen receptor according to an embodiment of the present invention; 4-1B BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer) expression Cytotoxicity to Chinese hamster ovary (CHO) cells expressing BAFF receptor, BCMA or TACI using professional antigen-expressing cells as effector cells is a diagram showing the measurement result after 24 hours.
도 25는 본 발명의 일 실시예에 따른 공벡터(Mock pAPC) 또는 키메릭 항원 수용체를 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC) 중 3-4-1B BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer) 발현 전문적 항원표출세포를 작동세포(effector cell)로 하여 BAFF 수용체, BCMA 또는 TACI를 발현하는 Chinese hamster ovary (CHO) 세포에 대한 세포독성을 48시간 후 측정한 결과를 나타낸 도이다.25 shows 3- of professional antigen-expressing cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors transduced with an empty vector (Mock pAPC) or a chimeric antigen receptor according to an embodiment of the present invention; 4-1B BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer) expression Cytotoxicity to Chinese hamster ovary (CHO) cells expressing BAFF receptor, BCMA or TACI using professional antigen-expressing cells as effector cells is a diagram showing the measurement result after 48 hours.
도 26은 본 발명의 일 실시예에 따른 공벡터(Mock pAPC) 또는 키메릭 항원 수용체를 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC) 중 3-4-ABC BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer) 발현 전문적 항원표출세포를 작동세포(effector cell)로 하여 BAFF 수용체, BCMA 또는 TACI를 발현하는 Chinese hamster ovary (CHO) 세포에 대한 세포독성을 24시간 후 측정한 결과를 나타낸 도이다.26 shows 3- of professional antigen-expressing cells (BAFF CAR-pAPC) expressing four types of chimeric antigen receptors transduced with an empty vector (Mock pAPC) or a chimeric antigen receptor according to an embodiment of the present invention; 4-ABC BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer) expression Chinese hamster ovary expressing BAFF receptor, BCMA or TACI using professional antigen-expressing cells as effector cells (CHO) is a diagram showing the result of measuring the cytotoxicity to the cell after 24 hours.
도 27은 본 발명의 일 실시예에 따른 공벡터(Mock pAPC) 또는 키메릭 항원 수용체를 형질도입시킨 4종의 키메릭 항원 수용체를 각각 발현시킨 전문적 항원표출세포(BAFF CAR-pAPC) 중 3-4-ABC BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer) 발현 전문적 항원표출세포를 작동세포(effector cell)로 하여 BAFF 수용체, BCMA 또는 TACI를 발현하는 Chinese hamster ovary (CHO) 세포에 대한 세포독성을 48시간 후 측정한 결과를 나타낸 도이다.27 shows 3- of professional antigen-expressing cells (BAFF CAR-pAPC) each expressing four types of chimeric antigen receptors transduced with an empty vector (Mock pAPC) or a chimeric antigen receptor according to an embodiment of the present invention; 4-ABC BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer) expression Chinese hamster ovary expressing BAFF receptor, BCMA or TACI using professional antigen-expressing cells as effector cells It is a diagram showing the result of measuring cytotoxicity to (CHO) cells after 48 hours.
본 발명은 하나의 양태로서, 키메릭 항원 수용체(CAR)를 포함하는 형질전환된 세포로서, 상기 키메릭 항원 수용체는 BAFF 수용체, BCMA 또는 TACI에 특이적으로 결합하는 항원 결합 도메인, 막관통 도메인 및 세포 내 신호전달 도메인을 포함하며, 상기 세포는 표적화된 효과기 활성을 갖는 항원 특이적 전문적 항원표출세포로서 대식세포, 수지상세포 또는 미감작 B 세포(naive B cell)를 포함하는, 형질전환된 세포를 제공한다.In one aspect, the present invention provides a transformed cell comprising a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an antigen binding domain that specifically binds to a BAFF receptor, BCMA or TACI, a transmembrane domain and A transformed cell comprising an intracellular signaling domain, wherein the cell comprises a macrophage, dendritic cell or naive B cell as an antigen-specific professional antigen-presenting cell having a targeted effector activity to provide.
본 발명의 일 실시예에 따른 키메릭 항원 수용체 단백질은 기능적 동등물을 포함할 수 있다. '기능적 동등물'이란 아미노산의 부가, 치환, 또는 결실의 결과, 상기 키메릭 항원 수용체 단백질의 아미노산 서열과 적어도 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 더더욱 바람직하게는 95% 이상의 서열 상동성을 갖는 것으로, 실질적으로 동질의 생리활성을 나타내는 단백질을 말한다. '실질적으로 동질의 생리활성'이란 BAFF 수용체, BCMA 또는 TACI에 특이적으로 결합할 수 있는 활성을 가진 것을 의미한다.The chimeric antigen receptor protein according to an embodiment of the present invention may include a functional equivalent. "Functional equivalent" means at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably the amino acid sequence of the chimeric antigen receptor protein as a result of the addition, substitution, or deletion of amino acids. refers to a protein having a sequence homology of 95% or more and exhibiting substantially homogeneous physiological activity. 'Substantially homogenous physiological activity' means having an activity capable of specifically binding to a BAFF receptor, BCMA, or TACI.
본 발명은 또한 키메릭 항원 수용체의 단편, 유도체 및 유사체(analogues)를 포함한다. 본원에 사용된, 용어 '단편', '유도체' 및 '유사체'는 본 발명의 키메릭 항원 수용체 단백질과 실질적으로 같은 생물학적 기능 또는 활성을 보유하는 폴리펩티드를 말한다. 본 발명의 단편, 유도체 및 유사체는 (1) 하나 이상의 보존적(conservative) 또는 비보존적 아미노산 잔기(바람직하게는 보존적 아미노산 잔기)가 치환된 폴리펩티드(상기 치환된 아미노산 잔기는 유전 암호에 의해 암호화될 수도, 되지 않을 수도 있다) 또는 (2) 하나 이상의 아미노산 잔기에서 치환기(들)를 가지는 폴리펩티드, 또는 (3) 또 다른 화합물(폴리펩티드의 반감기를 연장할 수 있는 화합물, 예를 들면 폴리에틸렌 글리콜)과 결합된 성숙 폴리펩티드로부터 유래된 폴리펩티드, 또는 (4) 부가적인 아미노산 서열(예를 들면, 선도 서열, 분비 서열, 상기 폴리펩티드를 정제하는데 사용된 서열, 프로테이노젠(proteinogen) 서열 또는 융합 단백질)과 결합된 상기 폴리펩티드로부터 유래된 폴리펩티드일 수 있다. 본 발명에 정의된 상기 단편, 유도체 및 유사체는 당업자에 잘 알려져 있다.The present invention also includes fragments, derivatives and analogues of chimeric antigen receptors. As used herein, the terms 'fragment', 'derivative' and 'analog' refer to a polypeptide that retains substantially the same biological function or activity as the chimeric antigen receptor protein of the present invention. Fragments, derivatives and analogs of the present invention may comprise (1) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, wherein the substituted amino acid residues are encoded by the genetic code. may or may not be) or (2) a polypeptide having substituent(s) at one or more amino acid residues, or (3) another compound (a compound capable of extending the half-life of the polypeptide, such as polyethylene glycol); a polypeptide derived from the associated mature polypeptide, or (4) an additional amino acid sequence (eg, a leader sequence, a secretion sequence, a sequence used to purify the polypeptide, a proteinogen sequence, or a fusion protein); It may be a polypeptide derived from said polypeptide to which it is bound. The fragments, derivatives and analogs as defined herein are well known to those skilled in the art.
본 발명에서 "BAFF 수용체", "BCMA", "TACI"는 다양한 암종(malignant cells)과 자가면역질환, 바람직하게는 전신 홍반성 루프스(systemic lupus erythematosus, SLE)에서 관찰된다. 지금까지 "BAFF 수용체", "BCMA", "TACI"를 발현하는 암종은 급성림프아구성백혈병(acute lymphoblastic leukemia, ALL), 만성림프아구성백혈병(chronic lymphoblastic leukemia, CLL), 여포성 림프종(follicular lymphoma), 외투세포림프종(mantle cell lymphoma), B 세포 림프종(B-cell lymphoma), 광범위큰 B 세포 림프종(diffuse large B-cell lymphoma, DLBCL), B 세포 비호지킨림프종(B-cell non-Hodgkin lymphoma), 다발성 골수종(multiple myeloma), T 세포 림프종(T cell lymphoma), 유방암(breast cancer), 갑상선암(thyroid carcinoma) 및 비소세포 폐암(non-small cell lung cancer)으로 이루어지는 군으로부터 선택될 수 있으나, 이에 제한되는 것은 아니다.In the present invention, "BAFF receptor", "BCMA", and "TACI" are observed in various carcinomas (malignant cells) and autoimmune diseases, preferably systemic lupus erythematosus (SLE). Until now, carcinomas expressing "BAFF receptor", "BCMA", and "TACI" have been classified into acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), and follicular lymphoma. lymphoma), mantle cell lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), B-cell non-Hodgkin lymphoma), multiple myeloma, T cell lymphoma, breast cancer, thyroid carcinoma and non-small cell lung cancer may be selected from the group consisting of , but is not limited thereto.
본 발명에서 "키메릭 신호전달 도메인"은 원하는 리간드에 결합하여 리간드-수용체 반응을 통해 전문적 항원표출세포의 활성화를 유도하고 해당 리간드를 발현하는 세포를 공격할 수 있도록 하기 위해 전문적 항원표출세포에 발현시키기 위한 융합 단백질을 의미할 수 있다. 곧, 전문적 항원표출세포에 발현 시 리간드에 결합하여 이들 세포들의 활성화를 유도하는 단백질이라고 볼 수 있다. 따라서, 본 발명에서 제공하는 신규 키메릭 신호전달 도메인은 모든 항원-항체 반응을 포함한 모든 리간드-수용체 반응을 이용한 전문적 항원표출세포의 활성화를 유도할 수 있는 범용적 의미의 신규 키메릭 신호전달 도메인을 제공한다.In the present invention, "chimeric signaling domain" binds to a desired ligand, induces activation of professional antigen-presenting cells through ligand-receptor reaction, and is expressed in specialized antigen-expressing cells to attack cells expressing the ligand It may mean a fusion protein for In other words, when expressed in specialized antigen-expressing cells, it can be seen as a protein that binds to a ligand and induces activation of these cells. Therefore, the novel chimeric signaling domain provided by the present invention is a novel chimeric signaling domain in a general sense that can induce the activation of professional antigen-presenting cells using all ligand-receptor reactions including all antigen-antibody reactions. to provide.
즉, 상기 키메릭 항원 수용체는 전문적 항원표출세포에 발현 시 항원에 결합하여 이들 세포들의 활성화를 유도하는 단백질이라고 볼 수 있다. 이를 통해 면역 반응을 일으키고자 하는 세포에 특이적인 항원을 인식하는 단백질일 수 있으며, 상기 면역 반응을 일으키고자 하는 세포는 특정 조직에 존재하거나 병변을 일으킨 조직을 이루는 세포를 의미할 수 있다.That is, the chimeric antigen receptor can be regarded as a protein that binds to an antigen and induces activation of these cells when expressed in professional antigen-expressing cells. Through this, it may be a protein recognizing an antigen specific to a cell to cause an immune response, and the cell to cause an immune response may refer to a cell existing in a specific tissue or constituting a tissue causing a lesion.
상기 키메릭 항원 수용체(CAR)의 항원 결합 도메인은 상기 BAFF 수용체, BCMA, TACI에 특이적으로 결합하는 BAFF(B-cell activating factor)의 세포외 도메인일 수 있다. 상기 BAFF 단백질은 tumor necrosis factor (TNF) 리간드에 속하는 사이토카인이며, BLyS (B-lymphocyte stimulator), TALL-1 (TNF- and ApoL related leukocyte-expressed ligand 1), THANK (TNF homologue that activates apoptosis, nuclear factor-κB and c-Jun NH2-terminal kinase), TNFSF13B (TNFsuperfamily member 13B) 또는 zTNF4로 불리운다. BAFF는 원래 285개의 아미노산으로 구성된 type 2 막관통 단백질(transmembrane protein)로 동형3합체(homotrimer)로 세포 표면에 발현된 후, 막관통 부분이 잘려져 나가 세포외로 분비된다. BAFF는 대식세포(macrophage), 단핵구세포(monocyte), 수지상세포(dendritic cell)에서 분비된다. The antigen binding domain of the chimeric antigen receptor (CAR) may be an extracellular domain of a B-cell activating factor (BAFF) that specifically binds to the BAFF receptor, BCMA, or TACI. The BAFF protein is a cytokine belonging to tumor necrosis factor (TNF) ligand, BLyS (B-lymphocyte stimulator), TALL-1 (TNF- and ApoL related leukocyte-expressed ligand 1), THANK (TNF homologue that activates apoptosis, nuclear factor-κB and c-Jun NH2-terminal kinase), TNFSF13B (TNFsuperfamily member 13B) or zTNF4. BAFF is originally a type 2 transmembrane protein composed of 285 amino acids and is expressed on the cell surface as a homotrimer, and then the transmembrane portion is cut off and secreted out of the cell. BAFF is secreted from macrophages, monocytes, and dendritic cells.
상기 BAFF의 세포외 도메인은 서열번호 1 또는 이와 95% 이상의 상동성을 나타내는 아미노산 서열로 이루어진 것일 수 있으나, 이에 제한되는 것은 아니다.The extracellular domain of BAFF may consist of SEQ ID NO: 1 or an amino acid sequence having 95% or more homology thereto, but is not limited thereto.
상기 항원 결합 도메인은 신호전달을 증폭하기 위해 BAFF의 세포외 도메인 한 쌍을 인간 이뮤노글로불린 G1 중쇄 불변 영역(IgG1 heavy chain constant region)에 각각 연결시킨 이합체(dimer) 형태일 수 있으나, 이에 제한되는 것은 아니다.The antigen binding domain may be in the form of a dimer in which a pair of extracellular domains of BAFF are linked to human immunoglobulin G 1 heavy chain constant region (IgG 1 heavy chain constant region) in order to amplify signal transduction, respectively. It is not limited.
상기 인간 이뮤노글로불린 G1 중쇄 불변 영역(IgG1 heavy chain constant region)은 서열번호 2 또는 이와 95% 이상의 상동성을 나타내는 아미노산 서열로 이루어진 것일 수 있으나, 이에 제한되는 것은 아니다.The human immunoglobulin G 1 heavy chain constant region (IgG 1 heavy chain constant region) may consist of SEQ ID NO: 2 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto.
본 발명의 키메릭 항원 수용체의 일 구성요소인 세포내 신호전달 도메인은 항원 결합 도메인에 결합에 의해 면역 세포의 면역반응을 활성화시키는 부위를 의미한다. 상기 신호전달 도메인은 TLR-3, TLR-4, 타이로신 기반 활성화 모티프(ITAM) 또는 이들의 조합일 수 있다. 바람직한 일 구현예에서, 상기 신호전달 도메인은 TLR-3-TLR-4의 구성에 ITAM을 소단위(subunit)로 하여 링커(linker)로 연결시킨 형태일 수 있으며, 상기 ITAM은 한 개 이상이 연결된 형태일 수 있으며, 바람직하게는 3개가 연결된 형태일 수 있으나, 이에 제한되는 것은 아니다.The intracellular signaling domain, which is one component of the chimeric antigen receptor of the present invention, refers to a site that activates an immune response of an immune cell by binding to an antigen-binding domain. The signaling domain may be TLR-3, TLR-4, tyrosine-based activation motif (ITAM), or a combination thereof. In a preferred embodiment, the signaling domain may be in the form of connecting ITAM as a subunit to the structure of TLR-3-TLR-4 with a linker, wherein one or more ITAMs are linked may be, and preferably may be in the form of three connected, but is not limited thereto.
상기 ITAM은 IgE 수용체 gamma chain (Fcε receptor I gamma chain, FcεRIγ), IgG 수용체 2A alpha chain (Fcγ receptor 2A alpha chain, FcγR2Aα) 또는 IgE 수용체 beta chain (Fcε receptor I beta chain, FcεRIβ)의 면역수용체 타이로신 기반 활성화 모티프일 수 있으나, 이에 제한되는 것은 아니다.The ITAM is based on immunoreceptor tyrosine of IgE receptor gamma chain (Fcε receptor I gamma chain, FcεRIγ), IgG receptor 2A alpha chain (Fcγ receptor 2A alpha chain, FcγR2Aα) or IgE receptor beta chain (Fcε receptor I beta chain, FcεRIβ) It may be an activation motif, but is not limited thereto.
상기 TLR-3은 서열번호 3 또는 이와 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 더더욱 바람직하게는 95% 이상의 서열 상동성을 갖는 것으로, 서열번호 3으로 표시되는 아미노산 서열과 실질적으로 동등한 기능을 나타내는 아미노산 서열로 이루어질 수 있고; 상기 TLR-4는 서열번호 4 또는 이와 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 더더욱 바람직하게는 95% 이상의 서열 상동성을 갖는 것으로, 서열번호 4로 표시되는 아미노산 서열과 실질적으로 동등한 기능을 나타내는 아미노산 서열로 이루어질 수 있고; 상기 ITAM은 서열번호 5; 서열번호 6; 또는 서열번호 7로 표시되는 아미노산 서열과 실질적으로 동등한 기능을 나타내는 아미노산 서열로 이루어질 수 있다.The TLR-3 is SEQ ID NO: 3 or more than 70%, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more sequence homology thereto, and the amino acid represented by SEQ ID NO: 3 may consist of an amino acid sequence exhibiting a function substantially equivalent to the sequence; The TLR-4 is SEQ ID NO: 4 or more than 70%, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more sequence homology thereto, and the amino acid represented by SEQ ID NO: 4 may consist of an amino acid sequence exhibiting a function substantially equivalent to the sequence; The ITAM is SEQ ID NO: 5; SEQ ID NO: 6; Alternatively, it may consist of an amino acid sequence exhibiting a function substantially equivalent to the amino acid sequence represented by SEQ ID NO: 7.
상기 링커는 서열번호 8로 표시되는 아미노산 서열과 실질적으로 동등한 기능을 나타내는 아미노산 서열로 이루어질 수 있다.The linker may consist of an amino acid sequence having a function substantially equivalent to that of the amino acid sequence represented by SEQ ID NO: 8.
본 발명의 막관통 도메인 (Transmembrane domain)은 BAFF의 세포외 도메인과 보조자극, 필수 신호전달 도메인을 세포막 사이로 연결하는 부위이며, 세포내 신호 전달 도메인은 항원 결합 도메인의 결합에 의해 면역 세포의 면역반응을 활성화시키는 부위를 의미한다.The transmembrane domain of the present invention is a site that connects the extracellular domain of BAFF and the costimulatory and essential signaling domains between the cell membrane, and the intracellular signaling domain is an immune response of immune cells by binding of the antigen-binding domain. site that activates
본 발명의 키메릭 항원 수용체의 일 구성요소인 막관통 도메인은 CD28, CD3 엡실론, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 및 TLR-2로 이루어진 군으로부터 선택되는 단백질의 막관통 도메인을 포함하는 것일 수 있다. 바람직하게는, 상기 막통과 도메인은 TLR-2일 수 있고, 이는 서열번호 9 또는 이와 95% 이상의 상동성을 나타내는 아미노산 서열로 이루어진 것일 수 있으나, 이에 제한되는 것은 아니다.One component of the transmembrane domain of the chimeric antigen receptor of the present invention is CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and a transmembrane domain of a protein selected from the group consisting of TLR-2. Preferably, the transmembrane domain may be TLR-2, which may consist of SEQ ID NO: 9 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto.
상기 항원 결합 도메인은 신호 펩타이드를 포함할 수 있으며, 상기 신호 펩타이드는 TLR-4 신호 펩타이드일 수 있으며, 서열번호 10 또는 이와 95% 이상의 상동성을 나타내는 아미노산 서열로 이루어진 것일 수 있으나, 이에 제한되는 것은 아니다.The antigen-binding domain may include a signal peptide, and the signal peptide may be a TLR-4 signal peptide, and may consist of SEQ ID NO: 10 or an amino acid sequence showing 95% or more homology thereto, but is limited thereto not.
본 발명에서 사용되는 벡터는 당 분야에 공지된 벡터를 다양하게 사용할 수 있고, 상기 항원 수용체를 생산하고자 하는 숙주세포의 종류에 따라 프로모터 (promoter), 종결자(terminator), 인핸서(enhancer) 등과 같은 발현조절 서열, 막 표적화 또는 분비를 위한 서열 등을 적절히 선택하고 목적에 따라 다양하게 조합할 수 있다. 본 발명의 벡터는 플라스미드 벡터, 코즈미드 벡터, 박테리오 파아지 벡터 및 바이러스 벡터 등을 포함하나 이에 제한되지 않는다. 적합한 벡터는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널 및 인핸서 같은 발현 조절 엘리먼트 외에도 막 표적화 또는 분비를 위한 시그널 서열 또는 리더 서열을 포함하며 목적에 따라 다양하게 제조될 수 있다.As the vector used in the present invention, a variety of vectors known in the art may be used, and depending on the type of host cell to produce the antigen receptor, such as a promoter, terminator, enhancer, etc. Expression control sequences, sequences for membrane targeting or secretion, etc. can be appropriately selected and variously combined according to the purpose. The vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector. Suitable vectors include a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as promoter, operator, start codon, stop codon, polyadenylation signal and enhancer, and may be prepared in various ways depending on the purpose.
본 발명에서는 바람직한 일 예로서, 렌티-바이러스용 벡터를 사용할 수 있으며, 본 발명의 하기 실시예에서는 pCDH-CMV-MCS-EF1-copGFP 벡터(렌티-바이러스용 벡터)를 사용하였다(도 5 내지 도 8 참조).In the present invention, as a preferred example, a lenti-virus vector can be used, and in the following examples of the present invention, pCDH-CMV-MCS-EF1-copGFP vector (lenti-virus vector) was used ( FIGS. 5 to 5 ). 8).
또한, 본 발명의 BAFF 수용체, BCMA 또는 TACI에 특이적으로 결합하는 키메릭 항원 수용체를 상기 벡터를 통해 세포에 도입하여 세포를 형질전환 시킬 수 있다. 상기 세포는 세포독성 T 세포(cytotoxic T cell)와 NK 세포, 종양 침윤 림프구, B 세포, NK 세포, 또는 NKT 세포일 수 있으며, 바람직하게는, 전문적 항원표출세포인 대식세포, 수지상세포, 미감작 B 세포(naive B cell) 일 수 있다. 일부 실시 양태에서, 상기 세포는 골수, 말초혈액, 말초혈액단핵세포 또는 제대혈로부터 얻거나 제조될 수 있다. 일부 실시양태에서, 세포는 인간 세포이다.In addition, the cell can be transformed by introducing the chimeric antigen receptor that specifically binds to the BAFF receptor, BCMA or TACI of the present invention into the cell through the vector. The cells may be cytotoxic T cells and NK cells, tumor infiltrating lymphocytes, B cells, NK cells, or NKT cells. It may be a naive B cell. In some embodiments, the cells may be obtained or prepared from bone marrow, peripheral blood, peripheral blood mononuclear cells or umbilical cord blood. In some embodiments, the cell is a human cell.
본 발명의 일 구현예로서, 상기 기술한 벡터를 이용하여 BAFF 수용체, BCMA 또는 TACI에 특이적으로 결합하는 키메릭 항원 수용체를 전문적 항원표출세포에 형질전환 시킬 수 있다.As an embodiment of the present invention, a chimeric antigen receptor that specifically binds to a BAFF receptor, BCMA or TACI can be transformed into a specialized antigen-expressing cell using the vector described above.
상기와 같이 본 발명의 키메릭 항원 수용체가 도입되어 형질전환된 세포는 BAFF 수용체, BCMA 또는 TACI를 항원으로 인식하고 이와 강하게 결합하는 특징을 가진다.As described above, the transformed cells into which the chimeric antigen receptor of the present invention is introduced recognizes the BAFF receptor, BCMA or TACI as an antigen, and has a characteristic of strongly binding thereto.
본 발명에서, "키메릭 항원 수용체 발현 전문적 항원표출세포 (chimeric antigen receptor professional antigen presenting cell, 이하 간략하게 'CAR-pAPC 세포’라 약칭함)" 란 정상의 전문적 항원표출세포를 형질도입 등의 방법으로 본래의 전문적 항원표출세포 표면 수용체가 아닌 암세포에 특이적으로 반응하는 키메라 항원 수용체를 발현하는 전문적 항원표출세포를 의미한다. 이 수용체를 갖는 전문적 항원표출세포는 타겟 세포의 세포자살을 유도하여 세포독성을 나타낸다.In the present invention, "chimeric antigen receptor professional antigen presenting cell (hereinafter abbreviated as 'CAR-pAPC cell')" is a method such as transduction of normal professional antigen presenting cells It refers to a specialized antigen-presenting cell expressing a chimeric antigen receptor that specifically responds to cancer cells rather than the original specialized antigen-expressing cell surface receptor. Professional antigen-expressing cells with this receptor induce apoptosis of target cells and exhibit cytotoxicity.
본 발명에 있어, 특히 CAR-pAPC 세포는 전문적 항원표출세포에 본 발명의 키메릭 항원 수용체가 도입된 세포일 수 있다. 상기 세포는 CAR-T 치료제의 기존 장점인 항암 특이적 표적 치료의 장점을 가지며, 특히, 본 발명의 키메라 항원 수용체를 장착시킨 전문적 항원표출세포는 BAFF 수용체, BCMA 또는 TACI를 발현하는 암세포를 인지하여 효과적으로 파괴할 수 있다.In the present invention, in particular, the CAR-pAPC cell may be a cell in which the chimeric antigen receptor of the present invention is introduced into a professional antigen-expressing cell. The cells have the advantage of anticancer-specific targeted therapy, which is the existing advantage of CAR-T therapeutics. In particular, the specialized antigen-expressing cells equipped with the chimeric antigen receptor of the present invention recognize cancer cells expressing BAFF receptor, BCMA or TACI. can be effectively destroyed.
따라서 본 발명의 다른 하나의 양태는 상기 세포를 포함하는 세포치료제; 이를 유효성분으로 포함하는 BAFF 수용체, BCMA 또는 TACI를 발현하는 암 및 전신 홍반성 루프스의 치료용 약학적 조성물; 및 상기 세포를 개체에 투여하는 단계를 포함하는 암 및 전신 홍반성 루프스를 치료하는 방법이다. Therefore, another aspect of the present invention is a cell therapy agent comprising the cell; A pharmaceutical composition for the treatment of cancer and systemic lupus erythematosus expressing BAFF receptor, BCMA, or TACI comprising it as an active ingredient; and administering the cells to a subject. It is a method of treating cancer and systemic lupus erythematosus.
본 발명에 있어 상기 세포는, 상기 세포는 세포독성 T 세포(cytotoxic T cell)와 NK 세포, 종양 침윤 림프구, B 세포, NK 세포, 또는 NK-T 세포일 수 있으며, 바람직하게는, 전문적 항원표출세포인 대식세포, 수지상세포, 미감작 B 세포(naive B cell) 일 수 있다. 또는 전구 세포(progenitor cell), 예를 들어, 조혈 줄기세포, 중간엽 간질 세포, 줄기세포, 전분화능 줄기세포, 및 배아 줄기세포일 수 있으며, 이들은 항암치료 등의 세포 요법에 사용될 수 있다. 세포는 공여자로부터 올 수 있거나, 환자로부터 얻어진 세포가 될 수 있다. 세포는 예를 들어, 병에 걸린 세포의 기능을 대체하는 재생에 사용될 수 있다. 세포는 또한 이종 유전자를 발현하도록 변형되어 생물학 제제가, 예를 들어, 병에 걸린 골수 또는 전이성 침착물과 같은 특정 미세 환경으로 전달될 수 있다.In the present invention, the cell, the cell may be a cytotoxic T cell (cytotoxic T cell) and NK cells, tumor infiltrating lymphocytes, B cells, NK cells, or NK-T cells, preferably, specialized antigen expression The cells may be macrophages, dendritic cells, or naive B cells. Or progenitor cells, for example, hematopoietic stem cells, mesenchymal stromal cells, stem cells, pluripotent stem cells, and may be embryonic stem cells, which may be used in cell therapy such as anticancer therapy. The cells may come from a donor, or they may be cells obtained from a patient. Cells can be used, for example, in regeneration, replacing the function of diseased cells. Cells can also be modified to express heterologous genes so that biological agents can be delivered to specific microenvironments, such as, for example, diseased bone marrow or metastatic deposits.
본 발명에서, "세포 치료제"는 개체로부터 분리, 배양 및 특수한 조작을 통해 제조된 세포 및 조직으로 치료, 진단 및 예방의 목적으로 사용되는 의약품(미국 FDA 규정)으로서, 세포 혹은 조직의 기능을 복원시키기 위하여 살아있는 자가, 동종, 또는 이종 세포를 체외에서 증식 선별하거나 다른 방법으로 세포의 생물학적 특성을 변화시키는 등의 일련의 행위를 통하여 치료, 진단 및 예방의 목적으로 사용되는 의약품을 의미한다.In the present invention, "cell therapeutic" refers to cells and tissues manufactured through isolation, culture, and special manipulation from an individual, and is a drug (US FDA regulations) used for the purpose of treatment, diagnosis, and prevention, restoring the function of cells or tissues. It refers to a drug used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro or changing the biological properties of cells in other ways.
본 발명의 용어 "예방"은 상기 조성물의 투여에 의해 BAFF 수용체, BCMA 또는 TACI를 발현하는 암 및 전신 홍반성 루프스를 억제시키거나 발생을 지연시키는 모든 행위를 의미한다.As used herein, the term “prevention” refers to any action that inhibits or delays the development of cancer and systemic lupus erythematosus and BAFF receptor, BCMA or TACI by administration of the composition.
본 발명의 용어, "치료"는 상기 조성물의 투여에 의해 BAFF 수용체, BCMA 또는 TACI를 발현하는 암 및 전신 홍반성 루프스에 의한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term “treatment” refers to any action in which symptoms caused by cancer and systemic lupus erythematosus, which express BAFF receptor, BCMA or TACI, are improved or beneficially changed by administration of the composition.
상기 조성물은 약학적으로 허용 가능한 담체를 포함할 수 있다.The composition may include a pharmaceutically acceptable carrier.
상기 "약학적으로 허용 가능한 담체"란 생물체를 자극하지 않으면서, 주입되는 화합물의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 의미할 수 있다. 본 발명에 사용 가능한 상기 담체의 종류는 특별히 제한되지 아니하며 당해 기술 분야에서 통상적으로 사용되고 약학적으로 허용되는 담체라면 어느 것이든 사용할 수 있다. 상기 담체의 비제한적인 예로는, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사 용액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 등을 들 수 있다. 이들은 단독으로 사용되거나 2 종 이상을 혼합하여 사용될 수 있다.The "pharmaceutically acceptable carrier" may mean a carrier or diluent that does not inhibit the biological activity and properties of the injected compound without irritating the organism. The type of carrier usable in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable may be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or may be used in combination of two or more.
약학적으로 허용 가능한 담체를 포함하는 상기 조성물은 경구 또는 비경구의 여러 가지 제형일 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다.The composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.
상세하게는, 경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 화합물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트, 수크로오스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 흔히 사용되는 단순 희석제인 물, 액체 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제 및 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 오일, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로골, 트윈 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.Specifically, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient to the compound, for example, starch, calcium carbonate, sucrose, lactose. , gelatin, etc. may be mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, etc. may be used.
상기 조성물은 약학적으로 유효한 양으로 투여할 수 있다.The composition may be administered in a pharmaceutically effective amount.
상기 "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 감염된 바이러스 종류, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다.The "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the subject's type and severity, age, sex, infected virus type, and drug. Activity, sensitivity to drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs and other factors well known in the medical field.
상기 투여는 어떠한 적절한 방법으로 환자에게 본 발명의 조성물을 도입하는 것을 의미하며, 상기 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 복강 내 투여, 정맥내 투여, 근육 내 투여, 피하 투여, 피내 투여, 경구 투여, 국소 투여, 비 내 투여될 수 있으나, 이에 제한되지는 않는다.The administration means introducing the composition of the present invention to the patient by any suitable method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, may be administered intranasally, but is not limited thereto.
본 발명의 조성물을 매일 투여 또는 간헐적으로 투여해도 좋고, 1일당 투여 횟수는 1회 또는 2~3회로 나누어 투여하는 것이 가능하다. 두 유효성분이 각각 단제인 경우의 투여횟수는 같은 횟수여도 좋고, 다른 횟수로 해도 된다. 또한, 본 발명의 조성물은 혈액암의 예방 또는 치료를 위하여 단독으로, 또는 다른 약물 치료와 병용하여 사용할 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다.The composition of the present invention may be administered daily or intermittently, and the number of administrations per day may be administered once or divided into two to three times. When the two active ingredients are single drugs, the number of administrations may be the same or different. In addition, the composition of the present invention may be used alone or in combination with other drug treatments for the prevention or treatment of hematologic cancer. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.
상기 개체란, BAFF 수용체, BCMA 또는 TACI를 발현하는 암이 발병하였거나 발병할 수 있는 인간과, 원숭이, 소, 말, 양, 돼지, 닭, 칠면조, 메추라기, 고양이, 개, 마우스, 쥐, 토끼 또는 기니아 피그를 포함한 모든 동물을 의미한다. 본 발명의 약학적 조성물을 개체에게 투여함으로써 상기 질환을 효과적으로 예방 또는 치료할 수 있다면 개체의 종류는 제한 없이 포함된다.The subject is a human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or Any animal, including guinea pigs. If the disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject, the type of subject is included without limitation.
본 발명에 있어 치료 대상이 되는 BAFF 수용체, BCMA, TACI를 발현하는 세포로는 전신 홍반성 루프스 및 암종이 있으며, 암의 종류로는 급성림프아구성백혈병(acute lymphoblastic leukemia, ALL), 만성림프아구성백혈병(chronic lymphoblastic leukemia, CLL), 여포성 림프종(follicular lymphoma), 외투세포림프종(mantle cell lymphoma), B 세포 림프종(B-cell lymphoma), 광범위큰 B 세포 림프종(diffuse large B-cell lymphoma, DLBCL), B 세포 비호지킨림프종(B-cell non-Hodgkin lymphoma), 다발성 골수종(multiple myeloma), T 세포 림프종(T cell lymphoma), 유방암(breast cancer), 갑상선암(thyroid carcinoma) 및 비소세포 폐암(non-small cell lung cancer)으로 이루어진 군으로부터 선택될 수 있으나, 이에 제한되는 것은 아니다.Cells expressing BAFF receptor, BCMA, and TACI to be treated in the present invention include systemic lupus erythematosus and carcinoma, and types of cancer include acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia. Chronic lymphoblastic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma, DLBCL), B-cell non-Hodgkin lymphoma, multiple myeloma, T cell lymphoma, breast cancer, thyroid carcinoma and non-small cell lung cancer ( It may be selected from the group consisting of non-small cell lung cancer), but is not limited thereto.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
실시예 1. 유전자 합성 방법에 의한 유전자의 클로닝Example 1. Cloning of a gene by a gene synthesis method
본 발명의 키메릭 항원 수용체를 제조하기 위해서 BAFF의 세포외 도메인 중 도메인 1과 TLR-2의 막관통 도메인의 서열을 가지며, 서로 다른 세포 내 신호 전달 도메인을 암호화하는 서열을 가진 4종류의 키메릭 항원 수용체 단백질 암호화 서열을 합성하였다. 키메릭 항원 수용체는 BAFF의 세포외 도메인 한 쌍을 각각 인간 이뮤노글로불린 G1 중쇄 불변 영역(IgG1 heavy chain constant region)에 연결시킨 항원 인식 부위, TLR-2의 막관통 도메인, TLR-4 신호 펩타이드를 공통적으로 포함하며. 신호전달 도메인을 각각 다른 4가지 구성으로 하여 제작하였다. 하기 표 1에 상기 4종류의 신호전달 도메인의 구성을 기재하였다.In order to prepare the chimeric antigen receptor of the present invention, four types of chimerics having sequences of domain 1 and transmembrane domains of TLR-2 among the extracellular domains of BAFF and encoding different intracellular signal transduction domains The antigen receptor protein coding sequence was synthesized. The chimeric antigen receptor is an antigen recognition site linking a pair of extracellular domains of BAFF to human immunoglobulin G 1 heavy chain constant region, respectively, a transmembrane domain of TLR-2, TLR-4 signal Peptides are commonly included. Each of the signaling domains was constructed in four different configurations. Table 1 below describes the configuration of the four types of signaling domains.
구분division 신호전달 도메인 구성Signaling domain configuration
1One TLR-3 / TLR-4 / linker / FcεR1 γ chain ITAM motif / linker / FcεR1 γ chain ITAM motif / linker / FcεR1 γ chain ITAM motifTLR-3 / TLR-4 / linker / FcεR1 γ chain ITAM motif / linker / FcεR1 γ chain ITAM motif / linker / FcεR1 γ chain ITAM motif
22 TLR-3 / TLR-4 / linker / FcγR2A α chain ITAM motif / linker / FcγR2A α chain ITAM motif / linker / FcγR2A α chain ITAM motifTLR-3 / TLR-4 / linker / FcγR2A α chain ITAM motif / linker / FcγR2A α chain ITAM motif / linker / FcγR2A α chain ITAM motif
33 TLR-3 / TLR-4 / linker/ FcεR1 β chain ITAM motif/ linker / FcεR1 β chain ITAM motif / linker / FcεR1 β chain ITAM motifTLR-3 / TLR-4 / linker/ FcεR1 β chain ITAM motif/ linker / FcεR1 β chain ITAM motif / linker / FcεR1 β chain ITAM motif
44 TLR-3 / TLR-4 / linker / FcγR2A α chain ITAM motif / linker / FcεR1 β chain ITAM motif / linker / FcεR1 γ chain ITAM motifTLR-3 / TLR-4 / linker / FcγR2A α chain ITAM motif / linker / FcεR1 β chain ITAM motif / linker / FcεR1 γ chain ITAM motif
상기 제작한 키메릭 항원 수용체를 발현시키는 각 도메인의 cDNA 모식도를 도 1 내지 도 4에 나타내었다. 각 도메인 부분의 단백질 암호화 서열은 유전자 데이터베이스(database)에서 확인하였으며, 유전자 합성의 정확도는 단백질 발현 플라스미드를 제작한 후 시퀀싱을 통해 확인하였다.Schematic diagrams of cDNAs of each domain expressing the chimeric antigen receptor constructed above are shown in FIGS. 1 to 4 . The protein coding sequence of each domain part was confirmed in a gene database, and the accuracy of gene synthesis was confirmed through sequencing after a protein expression plasmid was prepared.
실시예 2. 단백질 발현 플라스미드 제조Example 2. Preparation of protein expression plasmids
상기 실시예 1에서 제작한 4종의 키메릭 항원 수용체의 BAFF의 세포외 도메인 중 도메인 1과 대식 세포 신호전달 단백질을 융합시킨 재조합 단백질을 대식 세포에서 발현시키기 위해, 해당 유전자를 렌티바이러스 유래 발현벡터인 pCDH-CMV-MCS-EF1-copGFP(System Biosciences)에 제한효소 XbaI과 NotI의 절단 부위를 이용하여 클로닝하였다. In order to express in macrophages the recombinant protein obtained by fusion of domain 1 and macrophage signaling protein among the extracellular domains of BAFF of the four chimeric antigen receptors prepared in Example 1, the gene was expressed in a lentivirus-derived expression vector. It was cloned into pCDH-CMV-MCS-EF1-copGFP (System Biosciences) using restriction enzymes Xba I and Not I cleavage sites.
그 결과, 도 9 내지 도 12의 구조로 된 BAFF의 세포외 도메인과 신규 신호 전달 단백질이 융합된 단백질 4종을 발현하는 재조합 벡터를 완성하였다(도 5 내지 도 8 참조).As a result, a recombinant vector expressing four proteins in which the extracellular domain of BAFF having the structure of FIGS. 9 to 12 and a novel signal transduction protein was fused was completed (see FIGS. 5 to 8 ).
실시예Example 3. 3. BAFFBAFF 수용체, receptor, BCMABCMA 또는 or TACI를TACI 발현하는 세포주 제작 Creation of an expressing cell line
BAFF 수용체, BCMA 또는 TACI를 세포 표면에 발현하는 세포주를 제작하기 위해서, BAFF 수용체, BCMA 또는 TACI 유전자 각각을 5′말단에 제한효소 Bgl II 절단 부위 서열과 3′말단에 제한효소 Hind III의 절단 부위 서열을 이용하여 pLNCX2 벡터에 클로닝 하였다(도 13a, 13b 및 13c 참조). In order to construct a cell line expressing BAFF receptor, BCMA or TACI on the cell surface, each of the BAFF receptor, BCMA or TACI genes has a restriction enzyme Bgl II cleavage site sequence at the 5' end and a restriction enzyme Hind III cleavage site at the 3' end. The sequence was cloned into the pLNCX2 vector (see FIGS. 13A, 13B and 13C).
그 후, BAFF 수용체, BCMA, TACI 유전자가 삽입된 pLNCX2 벡터를 Chinese hamster ovary (CHO) 세포(ATCC)에 도입하기 위하여 293GPG 세포를 사용한 레트로바이러스 시스템을 이용하였다. 먼저, 293GPG 세포 3Х106개를 10ml의 10% 송아지 혈청 함유 DMEM 배양액 10ml에 풀어 100π 세포 배양 접시에 접종 후 24시간 동안 배양하였다. 앞서 제작한 재조합 벡터(BAFF-R.pLNCX2 벡터, BCMA.pLNCX2 벡터, TACI.pLNCX2 벡터) (20 μg) 각각을 calcium phosphate 및 HEPES-buffered solution을 이용하여 결정화시킨 후 앞서 배양한 293GPG 세포의 배양액에 첨가해주었다. 이후 24시간 간격으로 72시간에 걸쳐 배양액을 교체하며 레트로바이러스를 함유한 293GPG 세포의 배양 상층액을 채취 및 보관하였다. 상기 채취한 레트로바이러스 함유 상층액은 초고속 원심분리기를 이용하여 21,000 rpm으로 2시간 동안 원심분리 후 농축 이전 대비 100배로 농축될 수 있도록 10% 송아지 혈청 함유 DMEM (welgene)에 재구성하였다. 농축한 레트로바이러스를 CHO 세포에 형질도입하기 위해 CHO 세포를 24 well 세포 배양 접시에 5Х105/ml의 밀도로 접종하였다. 그 다음 농축된 레트로바이러스에 polybrene을 8 μg/ml만큼 첨가한 혼합액을 배양 중인 CHO 세포의 배양액에 추가한 후 24시간 동안 배양하였다. 배양 후 새 배양액으로 교체하였으며 유전자가 도입된 세포의 선별을 위해 배양액 교체 후 24시간 후부터는 neomycin 항생제(600 μg/ml)가 첨가된 10% 송아지 혈청 함유 DMEM 배양액을 이용하여 레트로바이러스를 처리해준 CHO 세포를 배양하였다. 14일 간의 neomycin 선별과정을 마친 CHO 세포는 다시 신선한 배양액에 옮겨 1주일간 증식시켰다. Thereafter, a retroviral system using 293GPG cells was used to introduce the pLNCX2 vector into which the BAFF receptor, BCMA, and TACI genes were inserted into Chinese hamster ovary (CHO) cells (ATCC). First, 3Х10 6 293GPG cells were dissolved in 10 ml of 10 ml of 10% calf serum-containing DMEM culture medium and inoculated in a 100π cell culture dish and then cultured for 24 hours. Each of the previously prepared recombinant vectors (BAFF-R.pLNCX2 vector, BCMA.pLNCX2 vector, TACI.pLNCX2 vector) (20 μg) was crystallized using calcium phosphate and HEPES-buffered solution, and then in the culture medium of the previously cultured 293GPG cells. was added Thereafter, the culture medium was changed over 72 hours at 24 hour intervals, and the culture supernatant of 293GPG cells containing retrovirus was collected and stored. The collected retrovirus-containing supernatant was centrifuged at 21,000 rpm for 2 hours using an ultra-high speed centrifuge and then reconstituted in DMEM (welgene) containing 10% calf serum to be concentrated 100 times compared to before concentration. To transduce the concentrated retrovirus into CHO cells, CHO cells were inoculated into a 24 well cell culture dish at a density of 5Х10 5 /ml. Then, a mixed solution in which 8 μg/ml of polybrene was added to the concentrated retrovirus was added to the culture medium of CHO cells in culture, and then cultured for 24 hours. After culturing, it was replaced with a new culture medium. For the selection of cells into which the gene was introduced, 24 hours after the culture medium was replaced, CHO cells treated with retrovirus using a DMEM culture medium containing 10% calf serum supplemented with neomycin antibiotic (600 μg/ml). was cultured. After 14 days of neomycin selection, CHO cells were transferred to fresh culture medium and grown for 1 week.
증식 후, 유세포 분석을 이용해 CHO 세포의 BAFF 수용체, BCMA, TACI 발현량을 각각 측정하였으며 재조합 벡터(BAFF-R.pLNCX2 벡터, BCMA.pLNCX2 벡터, TACI.pLNCX2 벡터)를 도입한 각각의 CHO 세포와 공벡터인 pLNCX2를 도입한 CHO 세포(Mock CHO cell)의 BAFF 수용체, BCMA, TACI 발현량을 항 인간 BAFF 수용체 항체(Biolegend), 항 인간 BCMA 수용체 항체(Biolegend), 항 인간 TACI 수용체 항체(Biolegend)를 이용한 유세포분석기를 이용하여 비교한 각각의 결과를 도 14a 내지 14c에 나타내었다. After proliferation, expression levels of BAFF receptor, BCMA, and TACI in CHO cells were measured using flow cytometry, respectively, and recombinant vectors (BAFF-R.pLNCX2 vector, BCMA.pLNCX2 vector, TACI.pLNCX2 vector) were introduced into each CHO cell and BAFF receptor, BCMA, and TACI expression levels of CHO cells (Mock CHO cells) introduced with pLNCX2, an empty vector, were measured by anti-human BAFF receptor antibody (Biolegend), anti-human BCMA receptor antibody (Biolegend), and anti-human TACI receptor antibody (Biolegend). The results compared using a flow cytometer using
상기 결과를 근거로 BAFF 수용체, BCMA, TACI를 강하게 발현하는 CHO 세포주 각각을 표적세포(target cell)로 이용하였으며, BAFF 수용체, BCMA, TACI를 발현하지 않는 Mock CHO cell 세포주를 표적세포(target cell)의 음성 대조군으로 사용하였다.Based on the above results, each of the CHO cell lines that strongly express the BAFF receptor, BCMA, and TACI were used as target cells, and the mock CHO cell line that does not express the BAFF receptor, BCMA, and TACI was used as the target cell. was used as a negative control of
실시예 4. 키메릭 항원 수용체 발현 전문적 항원표출세포 제작Example 4. Production of specialized antigen-expressing cells expressing chimeric antigen receptors
상기 실시예 2를 통해 제작한 재조합 키메릭 항원 수용체 발현 벡터 4종을 전문적 항원표출세포의 일종인 THP-1 세포(사람 대식 세포주)에 도입하기 위하여 293FT 세포(Thermo Fisher Scientific社)를 사용한 렌티 바이러스 시스템을 이용하였다. Lentivirus using 293FT cells (Thermo Fisher Scientific) to introduce the four recombinant chimeric antigen receptor expression vectors prepared in Example 2 into THP-1 cells (human macrophage cell line), a type of specialized antigen-expressing cells. system was used.
먼저, 293FT 세포를 100π 세포 배양 접시에 2.5Х106 세포가 되도록 접종한 후, 10% 송아지 혈청을 함유한 DMEM 배지에서 배양하였다. 배양 24시간 후, 세포가 접시의 60~70% 정도를 덮을 정도로 자라면, 상기 실시예 2의 4종의 벡터 DNA 각각을 20μg으로 분주하여, 10μg의 psPAX2(Addgene)와 3μg의 pMD2.G(Addgene) 벡터와 함께 Calcium phosphate를 Calcium phosphate 및 Hepes-buffered solution을 이용하여 결정화시킨 후 293FT 세포에 도입하였다. 그 후, 상기 발현 벡터가 도입된 293FT 세포를 48시간 후 24시간 간격으로 바이러스(렌티바이러스)를 포함하는 배양 상층액을 모았다. 모은 상층액을 초고속 원심분리기를 21,000rpm으로 2시간 동안 원심분리하여 바이러스를 농축시켰다. 농축된 바이러스를 polybrene 4μg/ml과 혼합하여 대식 세포주인 THP-1 세포의 배양액에 추가하여 형질도입을 진행하였다. 24시간 간격으로 2회 농축된 바이러스를 넣어준 배양액으로 THP-1 세포의 배양액을 변경하여 형질도입의 효율을 증가시켰다. 마지막 배양액 교체 후, 24시간이 지난 다음, 형질도입한 THP-1 세포의 일부를 형질도입 효율 측정에 사용하였다. 형질도입 효율은 세포 내부의 GFP 발현량을 유세포 분석을 이용해 측정했으며, 4종의 재조합 키메릭 항원 수용체 발현 벡터가 각각 형질도입된 THP-1 세포 (BAFF CAR)를, empty vetor (Mock)를 형질도입한 THP-1 세포의 형질도입 비율과 비교한 결과를 도 15에 나타내었다.First, 293FT cells were inoculated to become 2.5Х10 6 cells in a 100π cell culture dish, and then cultured in DMEM medium containing 10% calf serum. After 24 hours of incubation, when the cells have grown to cover about 60-70% of the dish, 20 µg of each of the four vector DNAs of Example 2 is aliquoted, 10 µg of psPAX2 (Addgene) and 3 µg of pMD2.G ( Addgene) vector and calcium phosphate were crystallized using calcium phosphate and Hepes-buffered solution, and then introduced into 293FT cells. Then, the culture supernatant containing the virus (lentivirus) was collected at intervals of 24 hours after 48 hours of the 293FT cells introduced with the expression vector. The collected supernatant was centrifuged at 21,000 rpm in an ultra-high speed centrifuge for 2 hours to concentrate the virus. The concentrated virus was mixed with 4 μg/ml of polybrene and added to the culture medium of THP-1 cells, a macrophage cell line, for transduction. The efficiency of transduction was increased by changing the culture medium of THP-1 cells to the culture medium in which the concentrated virus was added twice at 24 hour intervals. 24 hours after the last culture medium change, a portion of the transduced THP-1 cells was used to measure the transduction efficiency. Transduction efficiency was measured using flow cytometry to measure the expression level of GFP inside the cell, and THP-1 cells (BAFF CAR) transduced with each of the four recombinant chimeric antigen receptor expression vectors were transfected with an empty vector (Mock). The results compared with the transduction ratio of the introduced THP-1 cells are shown in FIG. 15 .
실시예Example 5. 5. 키메릭chimeric 항원 수용체와 antigen receptors and BAFFBAFF 수용체, receptor, BCMABCMA 또는 or TACI를TACI 발현하는 사람 세포주의 친화도 측정 Affinity measurement of expressing human cell lines
상기 제작한 키메릭 항원 수용체가 BAFF 수용체, BCMA 또는 TACI를 발현하는 사람 세포주와 친화도가 있는지 확인하기 위하여, 실시예 4에서 제작한 4종의 BAFF CAR-pAPC [3-4-3 BAFF CAR (TLR-3+TLR-4+FcεRIγ ITAM trimer), 3-4-2A BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM trimer), 3-4-1B BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer), 3-4-ABC BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer)] 발현 전문적 항원표출세포 각각이 BAFF 수용체, BCMA, TACI와 결합하는지 여부를 측정하기 위해, 수용성 재조합 human BAFF 수용체 IgG (Acrobiosystems), 수용성 재조합 BCMA IgG (R&D systems), 수용성 재조합 TACI IgG (R&D systems) 각각을 BAFF CAR-pAPC에 처리하여 유세포 분석을 통하여 확인하였다. 그 결과, 수용성 재조합 human BAFF 수용체 IgG, 수용성 재조합 BCMA IgG, 수용성 재조합 TACI IgG 모두는 Mock pAPC와 결합하지 않지만, BAFF CAR-pAPC와 결합하였다. 도 16a 내지 16c는 3-4-3 BAFF CAR (TLR-3+TLR-4+FcεRIγ ITAM trimer) 발현 전문적 항원표출세포와 수용성 재조합 human BAFF 수용체 IgG, 수용성 재조합 BCMA IgG, 수용성 재조합 TACI IgG와의 결합을 나타낸 것이고, 도 17a 내지 17c는 3-4-2A BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM trimer) 발현 전문적 항원표출세포와 수용성 재조합 human BAFF 수용체 IgG, 수용성 재조합 BCMA IgG, 수용성 재조합 TACI IgG와의 결합을 나타낸 것이고, 도 18a 내지 18c는 3-4-1B BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer) 발현 전문적 항원표출세포와 수용성 재조합 human BAFF 수용체 IgG, 수용성 재조합 BCMA IgG, 수용성 재조합 TACI IgG와의 결합을 나타낸 것이고, 도 19a 내지 19c는 3-4-ABC BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer) 발현 전문적 항원표출세포와 수용성 재조합 human BAFF 수용체 IgG, 수용성 재조합 BCMA IgG, 수용성 재조합 TACI IgG와의 결합을 나타낸 것이다. In order to confirm whether the prepared chimeric antigen receptor has affinity with a human cell line expressing the BAFF receptor, BCMA or TACI, the four types of BAFF CAR-pAPC [3-4-3 BAFF CAR ( TLR-3+TLR-4+FcεRIγ ITAM trimer), 3-4-2A BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM trimer), 3-4-1B BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer) , 3-4-ABC BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer)]. Water-soluble recombinant human BAFF receptor IgG (Acrobiosystems), water-soluble recombinant BCMA IgG (R&D systems), and water-soluble recombinant TACI IgG (R&D systems) were each treated with BAFF CAR-pAPC and confirmed by flow cytometry. As a result, all of the water-soluble recombinant human BAFF receptor IgG, the water-soluble recombinant BCMA IgG, and the water-soluble recombinant TACI IgG did not bind to mock pAPC, but bound to BAFF CAR-pAPC. 16A to 16C show the binding of 3-4-3 BAFF CAR (TLR-3+TLR-4+FcεRIγ ITAM trimer)-expressing specialized antigen-expressing cells to soluble recombinant human BAFF receptor IgG, soluble recombinant BCMA IgG, and soluble recombinant TACI IgG. 17a to 17c show the binding of 3-4-2A BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM trimer) specialized antigen-expressing cells to soluble recombinant human BAFF receptor IgG, soluble recombinant BCMA IgG, and soluble recombinant TACI IgG. 18a to 18c are 3-4-1B BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer) expression specialized antigen-expressing cells and soluble recombinant human BAFF receptor IgG, soluble recombinant BCMA IgG, and soluble recombinant TACI IgG shows binding, and FIGS. 19a to 19c show 3-4-ABC BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer) expression specialized antigen-expressing cells and soluble recombinant human BAFF receptor IgG, water-soluble Binding to recombinant BCMA IgG and water-soluble recombinant TACI IgG is shown.
상기 결과를 근거로 제작한 키메릭 항원 수용체를 발현하는 BAFF CAR-pAPC를 작동세포(effector cell)로 BAFF 수용체, BCMA 또는 TACI를 발현하는 CHO 세포주를 표적세포(target cell)로, BAFF CAR-pAPC의 BAFF 수용체, BCMA 또는 TACI 특이적 세포독성을 검증하였다.Based on the above results, BAFF CAR-pAPC expressing a chimeric antigen receptor prepared based on the above results was used as an effector cell, a CHO cell line expressing BAFF receptor, BCMA or TACI was used as a target cell, and BAFF CAR-pAPC was used as a target cell. BAFF receptor, BCMA or TACI-specific cytotoxicity was verified.
실시예Example 6. 6. BAFFBAFF 수용체, receptor, BCMABCMA 또는 or TACI를TACI 발현하는 manifest CHOCHO 세포와의 with cells 공배양을co-culture 통한 BAFF 수용체, BAFF receptors through, BCMABCMA , , TACITACI 특이적 세포독성 검증 Specific cytotoxicity verification
상기 제작한 키메릭 항원 수용체 발현 THP-1 세포 4종[3-4-3 BAFF CAR (TLR-3+TLR-4+FcεRIγ ITAM trimer), 3-4-2A BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM trimer), 3-4-1B BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer), 3-4-ABC BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer)]이 BAFF 수용체, BCMA 또는 TACI를 발현하는 CHO 세포를 선택적으로 인지하여 독성을 나타내는지 확인하기 위해 실시예 3에서 선정한 타겟 세포인 BAFF 수용체, BCMA 또는 TACI를 발현하는 각각의 CHO 세포를, 키메릭 항원 수용체 발현 THP-1 세포 4종 혹은 공벡터를 도입한 THP-1 세포(Mock)와 공배양하였다. 공배양 후, 24시간 혹은 48시간 뒤에 7-Aminoactinomycin D (7-AAD)를 처리하여 7-AAD의 형광 발현양을 유세포 분석으로 비교하여 각 키메릭 항원 수용체 발현 THP-1 세포의 세포독성을 측정하였다. 7-Aminoactinomycin D (7-AAD)는 세포자살(apoptosis)이 유도된 세포의 DNA와 결합하여 형광을 띠게 된다(Zembruski et al., 2012).4 types of chimeric antigen receptor-expressing THP-1 cells prepared above [3-4-3 BAFF CAR (TLR-3+TLR-4+FcεRIγ ITAM trimer), 3-4-2A BAFF CAR (TLR-3+TLR4+) FcγR2Aα ITAM trimer), 3-4-1B BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer), 3-4-ABC BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer) ] to selectively recognize CHO cells expressing BAFF receptor, BCMA or TACI and show toxicity It was co-cultured with 4 types of antigen receptor-expressing THP-1 cells or THP-1 cells (Mock) introduced with an empty vector. After co-culture, treatment with 7-Aminoactinomycin D (7-AAD) 24 hours or 48 hours later and comparing the fluorescence expression level of 7-AAD by flow cytometry to measure the cytotoxicity of each chimeric antigen receptor-expressing THP-1 cell did. 7-Aminoactinomycin D (7-AAD) binds to the DNA of the cell in which apoptosis is induced and becomes fluorescent (Zembruski et al., 2012).
먼저, 24 well 세포 배양 접시의 well에 키메릭 항원 수용체 발현 THP-1 세포 4종 각각을 5Х105로 분주하고, 표적세포인 BAFF 수용체, BCMA 또는 TACI를 발현하는 각각의 CHO 세포를 1Х105씩 분주하여 effector: target ratio가 5:1로 공배양 진행하였다. 공배양의 well당 부피는 1ml가 되도록 진행하였고, 원심분리기를 이용해 250g에서 4분 동안 원심분리하여 세포 간 간격을 가깝게 만들었다. 그 후, 24시간 또는 48시간 공배양 진행 후 각 well의 세포를 원심분리기를 이용하여 300g, 3분 원심분리하여 상층액을 제거한 뒤, 7-AAD (Biolegend)로 세포를 염색하였다. 그 후 유세포 분석을 이용하여 7-AAD에 의해 형광을 나타내는 세포의 비율을 측정하여 제작한 키메릭 항원 수용체 발현 THP-1 세포 4종의 세포독성을 정도를 정량화하였다.First, each of the 4 types of chimeric antigen receptor-expressing THP-1 cells was dispensed at 5Х10 5 into the wells of a 24-well cell culture dish, and each CHO cell expressing the target cell BAFF receptor, BCMA or TACI was dispensed with 1Х10 5 each. Therefore, co-culture was performed with an effector: target ratio of 5:1. The volume per well of the co-culture was proceeded to be 1 ml, and centrifuged at 250 g for 4 minutes using a centrifuge to make the intercellular space close. Then, after co-culture for 24 hours or 48 hours, the cells in each well were centrifuged at 300 g for 3 minutes using a centrifuge to remove the supernatant, and then the cells were stained with 7-AAD (Biolegend). Thereafter, the cytotoxicity of the four chimeric antigen receptor-expressing THP-1 cells prepared by measuring the proportion of cells exhibiting fluorescence by 7-AAD using flow cytometry was quantified.
그 결과 도 20 및 도 21에서 나타낸 바와 같이, 상기 제작한 3-4-3 BAFF CAR (TLR-3+TLR-4+FcεRIγ ITAM trimer) 발현 THP-1 세포를 BAFF 수용체, BCMA 또는 TACI를 발현하는 각각의 CHO 세포와 공배양 후 24시간이 지났을 때, 키메릭 항원 수용체를 발현하지 않는 THP-1 세포(Mock pAPC)는 7.12% 이하의 세포독성을 보인 반면 3-4-3 BAFF CAR (TLR-3+TLR-4+FcεRIγ ITAM trimer) 발현 THP-1 세포는 BAFF 수용체, BCMA 또는 TACI를 발현하는 각각의 CHO 세포 모두 21.0% 이상의 세포독성을 보임을 확인하였다. 또한, 공배양 48시간 후 공배양한 BAFF 수용체, BCMA 또는 TACI를 발현하는 각각의 CHO 세포의 세포독성을 확인해 보았을 때, 키메릭 항원 수용체를 발현하지 않는 THP-1 세포(Mock pAPC)의 경우 17.2% 이하의 세포독성을 보인 반면, 3-4-3 BAFF CAR (TLR-3+TLR-4+FcεRIγ ITAM trimer) 발현 THP-1 세포는 BAFF 수용체, BCMA 또는 TACI를 발현하는 각각의 CHO 세포 모두 45.4% 이상의 세포독성을 보였다. As a result, as shown in FIGS. 20 and 21, the prepared 3-4-3 BAFF CAR (TLR-3+TLR-4+FcεRIγ ITAM trimer) expressing THP-1 cells expressing BAFF receptor, BCMA or TACI At 24 hours after co-culture with each CHO cell, THP-1 cells not expressing the chimeric antigen receptor (Mock pAPC) showed less than 7.12% cytotoxicity, whereas 3-4-3 BAFF CAR (TLR- 3+TLR-4+FcεRIγ ITAM trimer) expressing THP-1 cells showed cytotoxicity of 21.0% or more for each CHO cell expressing BAFF receptor, BCMA or TACI. In addition, when the cytotoxicity of each CHO cell expressing the co-cultured BAFF receptor, BCMA or TACI was checked after 48 hours of co-culture, 17.2 for THP-1 cells (Mock pAPC) that do not express the chimeric antigen receptor. % or less of cytotoxicity, whereas THP-1 cells expressing 3-4-3 BAFF CAR (TLR-3+TLR-4+FcεRIγ ITAM trimer) were all 45.4 cells expressing BAFF receptor, BCMA or TACI. % or higher cytotoxicity.
다른 결과로, 도 22 및 도 23에서 나타낸 바와 같이, 상기 제작한 3-4-2A BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM trimer) 발현 THP-1 세포를 BAFF 수용체, BCMA 또는 TACI를 발현하는 각각의 CHO 세포와 공배양 후 24시간이 지났을 때, 키메릭 항원 수용체를 발현하지 않는 THP-1 세포(Mock pAPC)는 7.12% 이하의 세포독성을 보인 반면 3-4-2A BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM trimer) 발현 THP-1 세포는 BAFF 수용체, BCMA 또는 TACI를 발현하는 각각의 CHO 세포 모두 23.7% 이상의 세포독성을 보임을 확인하였다. 또한, 공배양 48시간 후 공배양한 BAFF 수용체, BCMA 또는 TACI를 발현하는 각각의 CHO 세포의 세포독성을 확인해 보았을 때, 키메릭 항원 수용체를 발현하지 않는 THP-1 세포(Mock pAPC)의 경우 17.2% 이하의 세포독성을 보인 반면, 3-4-2A BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM trimer) 발현 THP-1 세포는 BAFF 수용체, BCMA 또는 TACI를 발현하는 각각의 CHO 세포 모두 43.0% 이상의 세포독성을 보였다.As another result, as shown in Figures 22 and 23, the prepared 3-4-2A BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM trimer) expressing THP-1 cells expressing BAFF receptor, BCMA or TACI At 24 hours after co-culture with each CHO cell, THP-1 cells not expressing the chimeric antigen receptor (Mock pAPC) showed less than 7.12% cytotoxicity, whereas 3-4-2A BAFF CAR (TLR- 3+TLR4+FcγR2Aα ITAM trimer) THP-1 cells expressing BAFF receptor, BCMA or TACI were confirmed to show cytotoxicity of 23.7% or more for each CHO cell. In addition, when the cytotoxicity of each CHO cell expressing the co-cultured BAFF receptor, BCMA or TACI was checked after 48 hours of co-culture, 17.2 for THP-1 cells (Mock pAPC) that do not express the chimeric antigen receptor. % or less of cytotoxicity, whereas THP-1 cells expressing 3-4-2A BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM trimer) showed more than 43.0% of each CHO cell expressing BAFF receptor, BCMA or TACI. showed cytotoxicity.
또 다른 결과로, 도 24 및 도 25에서 나타낸 바와 같이, 상기 제작한 3-4-1B BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer) 발현 THP-1 세포를 BAFF 수용체, BCMA 또는 TACI를 발현하는 각각의 CHO 세포와 공배양 후 24시간이 지났을 때, 키메릭 항원 수용체를 발현하지 않는 THP-1 세포(Mock pAPC)는 7.12% 이하의 세포독성을 보인 반면 3-4-1B BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer) 발현 THP-1 세포는 BAFF 수용체, BCMA 또는 TACI를 발현하는 각각의 CHO 세포 모두 25.7% 이상의 세포독성을 보임을 확인하였다. 또한, 공배양 48시간 후 공배양한 BAFF 수용체, BCMA 또는 TACI를 발현하는 각각의 CHO 세포의 세포독성을 확인해 보았을 때, 키메릭 항원 수용체를 발현하지 않는 THP-1 세포(Mock pAPC)의 경우 17.2% 이하의 세포독성을 보인 반면, 3-4-1B BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer) 발현 THP-1 세포는 BAFF 수용체, BCMA 또는 TACI를 발현하는 각각의 CHO 세포 모두 45.1% 이상의 세포독성을 보였다.As another result, as shown in FIGS. 24 and 25 , the prepared 3-4-1B BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer) expressing THP-1 cells expressed BAFF receptor, BCMA or TACI. At 24 hours after co-culture with each CHO cell, the THP-1 cells that do not express the chimeric antigen receptor (Mock pAPC) showed less than 7.12% cytotoxicity, whereas the 3-4-1B BAFF CAR (TLR) -3+TLR4+FcεRIβ ITAM trimer) expressing THP-1 cells showed cytotoxicity of 25.7% or more for each CHO cell expressing BAFF receptor, BCMA or TACI. In addition, when the cytotoxicity of each CHO cell expressing the co-cultured BAFF receptor, BCMA or TACI was checked after 48 hours of co-culture, 17.2 for THP-1 cells (Mock pAPC) that do not express the chimeric antigen receptor. % or less of cytotoxicity, whereas THP-1 cells expressing 3-4-1B BAFF CAR (TLR-3+TLR4+FcεRIβ ITAM trimer) showed more than 45.1% of each CHO cell expressing BAFF receptor, BCMA or TACI. showed cytotoxicity.
또 다른 결과로, 도 26 및 도 27에 나타낸 바와 같이, 상기 제작한 3-4-ABC BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer) 발현 THP-1 세포를 BAFF 수용체, BCMA 또는 TACI를 발현하는 각각의 CHO 세포와 공배양 후 24시간이 지났을 때, 키메릭 항원 수용체를 발현하지 않는 THP-1 세포(Mock pAPC)는 7.12% 이하의 세포독성을 보인 반면 3-4-ABC BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer) 발현 THP-1 세포는 BAFF 수용체, BCMA 또는 TACI를 발현하는 각각의 CHO 세포 모두 18.8% 이상의 세포독성을 보임을 확인하였다. 또한, 공배양 48시간 후 공배양한 BAFF 수용체, BCMA 또는 TACI를 발현하는 각각의 CHO 세포의 세포독성을 확인해 보았을 때, 키메릭 항원 수용체를 발현하지 않는 THP-1 세포(Mock pAPC)의 경우 17.2% 이하의 세포독성을 보인 반면, 3-4-ABC BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer) 발현 THP-1 세포는 BAFF 수용체, BCMA 또는 TACI를 발현하는 각각의 CHO 세포 모두 50.4% 이상의 세포독성을 보였다.As another result, as shown in FIGS. 26 and 27, the prepared 3-4-ABC BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer) expressing THP-1 cells were At 24 hours after co-culture with individual CHO cells expressing BAFF receptor, BCMA or TACI, THP-1 cells not expressing chimeric antigen receptor (Mock pAPC) showed cytotoxicity of 7.12% or less, whereas 3 THP-1 cells expressing -4-ABC BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer) showed cytotoxicity of more than 18.8% for each CHO cell expressing BAFF receptor, BCMA or TACI. was confirmed to be visible. In addition, when the cytotoxicity of each CHO cell expressing the co-cultured BAFF receptor, BCMA or TACI was checked after 48 hours of co-culture, 17.2 for THP-1 cells (Mock pAPC) that do not express the chimeric antigen receptor. % or less, whereas THP-1 cells expressing 3-4-ABC BAFF CAR (TLR-3+TLR4+FcγR2Aα ITAM monomer+FcεRIβ ITAM monomer+FcεRIγ ITAM monomer) All CHO cells showed cytotoxicity of 50.4% or more.
이를 통해 제작한 키메릭 항원 인지 수용체 발현 THP-1 세포가 BAFF 수용체, BCMA 또는 TACI 단백질에 특이적인 세포독성을 보임을 확인할 수 있었다.Through this, it was confirmed that the prepared chimeric antigen recognition receptor-expressing THP-1 cells showed specific cytotoxicity to the BAFF receptor, BCMA or TACI protein.
상기 결과를 통해 본 발명에 따른 키메릭 항원 수용체를 포함한 형질전환된 THP-1 세포가 BAFF 수용체, BCMA 또는 TACI 단백질에 특이적인 세포독성을 보임으로써, 상기 형질전환된 항원 특이적 전문적 항원표출세포를 이용하여 관련된 암 세포 치료가 가능함을 확인할 수 있었다.Through the above results, the transformed THP-1 cells including the chimeric antigen receptor according to the present invention showed cytotoxicity specific to the BAFF receptor, BCMA or TACI protein, thereby producing the transformed antigen-specific professional antigen-expressing cells. It was confirmed that it is possible to treat related cancer cells using the
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (16)

  1. 키메릭 항원 수용체(CAR)를 포함하는 형질전환된 세포로서,A transformed cell comprising a chimeric antigen receptor (CAR), comprising:
    상기 키메릭 항원 수용체는 BAFF 수용체, BCMA 또는 TACI에 특이적으로 결합하는 항원 결합 도메인, 막관통 도메인 및 세포 내 신호전달 도메인을 포함하며, 상기 세포는 표적화된 효과기 활성을 갖는 항원 특이적 전문적 항원표출세포로서 대식세포, 수지상세포 또는 미감작 B 세포(naive B cell)를 포함하는, 형질전환된 세포.The chimeric antigen receptor includes an antigen binding domain that specifically binds to a BAFF receptor, BCMA or TACI, a transmembrane domain and an intracellular signaling domain, wherein the cell is an antigen-specific professional antigen-expressing agent having a targeted effector activity. A transformed cell comprising macrophages, dendritic cells or naive B cells as cells.
  2. 제1항에 있어서,According to claim 1,
    상기 항원 결합 도메인은 BAFF 수용체, BCMA 또는 TACI에 특이적으로 결합하는 BAFF(B-cell activating factor)의 세포외 도메인인 것인, 형질전환된 세포.The antigen-binding domain is an extracellular domain of BAFF (B-cell activating factor) that specifically binds to a BAFF receptor, BCMA or TACI, the transformed cell.
  3. 제2항에 있어서,3. The method of claim 2,
    상기 BAFF의 세포외 도메인은 서열번호 1로 표시되는 아미노산 서열을 포함하는 것인, 형질전환된 세포.The extracellular domain of the BAFF will include the amino acid sequence represented by SEQ ID NO: 1, the transformed cell.
  4. 제2항에 있어서,3. The method of claim 2,
    상기 항원 결합 도메인은 BAFF의 세포외 도메인 한 쌍이 이합체(dimer)의 형태로 각각 인간 이뮤노글로불린 G1 중쇄 불변 영역(IgG1 heavy chain constant region)에 연결된 것을 특징으로 하는 형질전환된 세포.The antigen-binding domain is a pair of extracellular domains of BAFF in the form of dimers, respectively, human immunoglobulin G 1 heavy chain constant region (IgG 1 heavy chain constant region) Transformed cells, characterized in that connected to.
  5. 제4항에 있어서,5. The method of claim 4,
    인간 이뮤노글로불린 G1 중쇄 불변 영역(IgG1 heavy chain constant region)은 서열번호 2로 표시되는 아미노산 서열을 포함하는 것인, 형질전환된 세포.Human immunoglobulin G 1 heavy chain constant region (IgG 1 heavy chain constant region) is a transformed cell comprising the amino acid sequence represented by SEQ ID NO: 2.
  6. 제1항에 있어서,According to claim 1,
    상기 세포 내 신호전달 도메인은 TLR-3-TLR-4의 구성에, 면역수용체 타이로신 기반 활성화 모티프(immunoreceptortyrosine-based activation motif, ITAM)를 소단위(subunit)로 하여 링커(linker)로 연결시킨 것인, 형질전환된 세포.The intracellular signaling domain is linked to the structure of TLR-3-TLR-4 with an immunoreceptor tyrosine-based activation motif (ITAM) as a subunit and linked by a linker. transformed cells.
  7. 제6항에 있어서,7. The method of claim 6,
    상기 ITAM은 IgE 수용체 gamma chain (Fcε receptor I gamma chain, FcεRIγ), IgG 수용체 2A alpha chain (Fcγ receptor 2A alpha chain, FcγR2Aα) 또는 IgE 수용체 beta chain (Fcε receptor I beta chain, FcεRIβ)의 면역수용체 타이로신 기반 활성화 모티프인 것인, 형질전환된 세포.The ITAM is based on immunoreceptor tyrosine of IgE receptor gamma chain (Fcε receptor I gamma chain, FcεRIγ), IgG receptor 2A alpha chain (Fcγ receptor 2A alpha chain, FcγR2Aα) or IgE receptor beta chain (Fcε receptor I beta chain, FcεRIβ) An activation motif, the transformed cell.
  8. 제6항에 있어서,7. The method of claim 6,
    상기 ITAM은 링커로 3개를 연결시킨 것인, 형질전환된 세포.The ITAM is a transformed cell that connects three with a linker.
  9. 제6항에 있어서, 7. The method of claim 6,
    상기 TLR-3는 서열번호 3; 및 TLR-4는 서열번호 4로 표시되는 아미노산 서열을 포함하는 것인, 형질전환된 세포.The TLR-3 is SEQ ID NO: 3; And TLR-4 is a transformed cell comprising the amino acid sequence shown in SEQ ID NO: 4.
  10. 제6항에 있어서,7. The method of claim 6,
    상기 ITAM은 서열번호 5; 서열번호 6; 또는 서열번호 7로 표시되는 아미노산 서열을 포함하는 것인, 형질전환된 세포.The ITAM is SEQ ID NO: 5; SEQ ID NO: 6; Or comprising the amino acid sequence represented by SEQ ID NO: 7, the transformed cell.
  11. 제6항에 있어서, 7. The method of claim 6,
    상기 링커는 서열번호 8로 표시되는 아미노산 서열을 포함하는 것인, 형질전환된 세포.Wherein the linker comprises the amino acid sequence represented by SEQ ID NO: 8, the transformed cell.
  12. 제1항에 있어서,According to claim 1,
    상기 막관통 도메인은 TLR-2이며, 서열번호 9로 표시되는 아미노산 서열을 포함하는 것인, 형질전환된 세포.The transmembrane domain is TLR-2, and the transformed cell comprising the amino acid sequence shown in SEQ ID NO: 9.
  13. 제1항에 있어서,According to claim 1,
    상기 키메릭 항원 수용체는 신호 펩타이드를 포함하며, 서열번호 10으로 표시되는 아미노산 서열을 포함하는 것인, 형질전환된 세포.The chimeric antigen receptor comprises a signal peptide and will comprise the amino acid sequence shown in SEQ ID NO: 10, the transformed cell.
  14. 제1항 내지 제13항 중 어느 한 항의 세포를 유효성분으로 포함하는 BAFF 수용체, BCMA 또는 TACI를 발현하는 암 또는 전신 홍반성 루프스(systemic lupus erythematosus, SLE)의 치료용 약학적 조성물.A pharmaceutical composition for treatment of cancer or systemic lupus erythematosus (SLE) expressing a BAFF receptor, BCMA or TACI comprising the cell of any one of claims 1 to 13 as an active ingredient.
  15. 제14항에 있어서, 15. The method of claim 14,
    상기 BAFF 수용체, BCMA 또는 TACI를 발현하는 암은 급성림프아구성백혈병(acute lymphoblastic leukemia, ALL), 만성림프아구성백혈병(chronic lymphoblastic leukemia, CLL), 여포성 림프종(follicular lymphoma), 외투세포림프종(mantle cell lymphoma), B 세포 림프종(B-cell lymphoma), 광범위큰 B 세포 림프종(diffuse large B-cell lymphoma, DLBCL), B 세포 비호지킨림프종(B-cell non-Hodgkin lymphoma), 다발성 골수종(multiple myeloma), T 세포 림프종(T cell lymphoma), 유방암(breast cancer), 갑상선암(thyroid carcinoma) 및 비소세포 폐암(non-small cell lung cancer)으로 이루어진 군으로부터 선택되는 것을 특징으로 하는 조성물. Cancer expressing the BAFF receptor, BCMA or TACI is acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), follicular lymphoma, mantle cell lymphoma ( mantle cell lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), B-cell non-Hodgkin lymphoma, multiple myeloma myeloma), T cell lymphoma (T cell lymphoma), breast cancer (breast cancer), thyroid cancer (thyroid carcinoma) and non-small cell lung cancer (non-small cell lung cancer), characterized in that the composition selected from the group consisting of.
  16. 제1항 내지 제13항 중 어느 한 항의 세포를 유효성분으로 포함하는 세포 치료제.A cell therapeutic agent comprising the cell of any one of claims 1 to 13 as an active ingredient.
PCT/KR2022/005560 2021-05-04 2022-04-18 Transformed antigen-specific professional antigen-presenting cell containing chimeric antigen receptor (car) and use thereof WO2022234976A1 (en)

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