WO2021245580A1 - Chimeric antigen receptor comprising pa63 domain 4 variant as extracellular binding domain, and use thereof - Google Patents
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- WO2021245580A1 WO2021245580A1 PCT/IB2021/054846 IB2021054846W WO2021245580A1 WO 2021245580 A1 WO2021245580 A1 WO 2021245580A1 IB 2021054846 W IB2021054846 W IB 2021054846W WO 2021245580 A1 WO2021245580 A1 WO 2021245580A1
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Classifications
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- A—HUMAN NECESSITIES
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/463—Cellular immunotherapy characterised by recombinant expression
- A61K39/4631—Chimeric Antigen Receptors [CAR]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
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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
- A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
- A61K2239/54—Pancreas
Definitions
- the present invention relates to an ANTXR (Anthrax toxin receptor) known to be overexpressed in various cancer cells; Anthrax toxin receptor) relates to a chimeric antigen receptor that optimizes safety for normal cells while maintaining anticancer efficacy by controlling the binding affinity of the chimeric antigen receptor that recognizes and binds antigen, ANTXRKanthrax toxin receptor 1) or ANTXR2 ( anthrax toxin receptor 2) relates to a ligand-based chimeric antigen receptor comprising a mutant of PA63 domain 4 that specifically binds to a cancer antigen, immune cells comprising the same, and uses thereof. background
- Anthrax toxin receptor (ANTXR) antigens include ANTXR1 and ANTXR2, and anthrax toxin secreted by anthrax is known as a ligand for recognizing these antigens.
- Anthrax toxin is secreted by Gram-positive bacteria, Bacillus anthracis 6 ) ) and Edema factor [EF, 89 kDa] (Morton, N. , New Engl. J. Med. 345: 1621-1626 [2001]).
- PA protective antigen
- LF lethal factor
- ANTXR1 is an extracellular matrix protein, published in Brad St. It is known as TEM8 (Tumor endothelial marker 8) identified in colorectal cancer by Croix (Sciences 289:1197 [2000]). ANTXR1 is a type I transmembrane protein expressed in the endothelium of various tumors, and has been found to be a gene whose expression is increased during tumor angiogenesis (St. Croix et al., Sciences, 289).
- ANTXR1 acts as a receptor for extracellular ligand (extracellular ligand), it has become a target antigen for the treatment of angiogenesis (Car son-Walter, EB et al., Cancer Res 61:6649 [2001]) ).
- ANTXR2 Another name for ANTXR2 is CMG2 (capi 1 lary morphogenesis gene 2), and ANTXR2 is also known to be involved in angiogenesis. In addition, ANTXR2 is known to be involved in adhesion and migration of various types of cells, including epithelial cells and endothelial cells (Lin Ye et al., Int J Oncol 45: 1565-1573 [2014]).
- CMG2 capi 1 lary morphogenesis gene 2
- ANTXR2 is also known to be involved in angiogenesis.
- ANTXR2 is known to be involved in adhesion and migration of various types of cells, including epithelial cells and endothelial cells (Lin Ye et al., Int J Oncol 45: 1565-1573 [2014]).
- CAR-T immune cell therapy which has been booming in R&D by many global pharmaceutical companies, starting with FDA approval in 2017, is drawing attention from around the world as a genetically modified cell therapy.
- a chimeric antigen receptor (CAR) gene having specificity as a surface antigen of cancer cells and anticancer activity (effectiveness) of cells is transduced into immune T cells or NK cells, and these chimeric antigen receptors are expressed
- It is an anti-cancer immunotherapy (gene therapy) method in which T or NK cells are proliferated outside the body and administered to the patient for treatment.
- This treatment has excellent anti-cancer effects compared to antibody drugs with a single administration and shows long-lasting results, so there is a lot of anticipation for anti-cancer efficacy in clinical practice.
- the present inventors specifically recognize / bind to ANTXR, based on the CAR containing the PA63 domain 4, while maintaining the existing anti-cancer efficacy, differentiation / optimizing safety to minimize side effects on normal cells
- the affinity was controlled by mutation of the amino acid of PA63 domain 4 (D4), which is a ligand site binding to the antigen, and the cancer treatment effect was off while maintaining
- D4 the amino acid of PA63 domain 4
- An object of the present invention is to provide a chimeric antigen receptor (CAR) with optimized safety while maintaining efficacy as an immune cell therapy agent by developing CARs having various binding affinities.
- Another object of the present invention is a nucleic acid encoding the chimeric antigen receptor, 2021/245580 1» (: 1' year 2021/054846 To provide an expression vector containing a nucleic acid and a virus containing the expression vector.
- Another object of the present invention is a composition for preventing or treating cancer comprising the immune cells, a method for preventing or treating cancer comprising administering the immune cells, the use of the immune cells for preventing or treating cancer, and cancer prevention or to provide the use of the immune cells for the manufacture of a therapeutic agent.
- the present invention provides an extracellular binding domain containing a variant of PA63 ligand domain 4, a transmembrane domain, and an intracellular signaling domain comprising a It provides a chimeric antigen receptor (CAR).
- the present invention also provides a nucleic acid encoding the chimeric antigen receptor, an expression vector containing the nucleic acid, a virus containing the expression vector, and immune cells expressing the chimeric antigen receptor.
- the present invention also provides a composition for preventing or treating cancer comprising the immune cells, a method for preventing or treating cancer comprising administering the immune cells, the use of the immune cells for preventing or treating cancer, and preventing cancer or Provided is the use of said immune cells for the manufacture of a medicament for treatment.
- 1 is melanoma (526-mel), pancreatic cancer (PANC-1, Mia_PaCa2), breast cancer (SK-BR3, 2021/245580 1»(:1'year2021/054846
- FIG. 3 is a flow cytometry analysis of RFP fluorescent protein fused with each antigen using a flow cytometer in a chemochromatosis (526, el) cell line that simultaneously expresses ANTXRKTEM8) antigen protein or ANTXR2 (CMG2) antigen protein and RFP (Red Fluorescence Protein) fluorescent protein This is the result of confirming the expression.
- Figure 4 is a picture expressing the position of the mutation of PA63 domain 4 (D4) wil type CAR and D4 specific to ANTXRKTEM8) or ANTXR2 (CMG2) according to the present invention and a representative plasmid DNA vector map.
- the CAR of the present invention is an extracellular binding domain, a fragment containing D4 or D4 mutant essential for receptor binding of PA63 ligand, a transmembrane domain derived from CD8 a, a hinge region, a primary signaling domain of the CD3 chain, (3) 28 and CD137 costimulatory signaling domains.
- 5 is a graph comparing cytotoxicity to ANTXR TEM8) and ANTXR2 (CMG2) overexpressing cell lines of D4 wild and D4 mutant #1 - #6 CAR-T cells prepared using PBMC.
- Figure 6 shows the results of analyzing the anticancer activity against ANTXR TEM8) and ANTXR2 (CMG2) overexpressing cell lines of D4 wi ld and D4 mutant #1 - #6 CAR-T cells prepared using PBMC through IFN-gamma ELISA. This is the graph shown. 7 shows D4 wi ld and D4 prepared using PBMCs of 4 healthy donors. 2021/245580 1» (:1' year 2021/054846 mutant #4 and #6 This is a graph showing the results of analyzing the in vitro anticancer activity of CAR-T cells against ANTXR1 (TEM8) and ANTXR2 (CMG2).
- a chimeric antigen receptor comprising PA63 ligand domain 4 specifically binding to ANTXRKTEM8) antigen and ANTXR2 (CMG2) antigen expressed in cancer tissue as an extracellular binding domain was developed.
- anticancer activity efficiency
- cancer cell specificity is increased and attack power against normal cells is increased
- off-target toxicity was lowered, and it was confirmed that it can be maintained for a long time in the body by increasing the formation of memory T cells.
- the present invention provides an extracellular binding domain containing a variant of PA63 ligand domain 4 (extracellular binding domain), a transmembrane domain and It relates to a chimeric antigen receptor (CAR) comprising an intracellular signaling domain.
- extracellular binding domain containing a variant of PA63 ligand domain 4 (extracellular binding domain), a transmembrane domain and It relates to a chimeric antigen receptor (CAR) comprising an intracellular signaling domain.
- CAR chimeric antigen receptor
- CAR Chimeric antigen receptor
- the first-generation CAR an extracellular domain including an antigen recognition site specifically expressed in cancer cells, a transmembrane domain, and an intracellular signaling domain were used, and only (3) 3 ⁇ 4 was used as the signaling domain, but the therapeutic effect on cancer was insignificant, and there was a problem that the duration was short.
- This first-generation CAR is specifically described in US Patent No. 6,319,494, which is incorporated herein by reference.
- a costimulatory domain (CD28 or 2021/245580 1»(:1' year2021/054846
- a second-generation CAR combining (3) 137/4-1BB) and (3) 3 ⁇ 4 was prepared. Compared with the first-generation CAR, the number of CAR-containing immune cells remaining in the body significantly increased.
- the second-generation CAR used one co-stimulatory domain, whereas the third-generation CAR used two or more co-stimulatory domains.
- a costimulatory domain can be combined with 4-1BB, CD28 or 0X40, and the like.
- the second-generation CAR is specifically described in U.S. Patent Nos. 7,741,465, 7,446, 190 or 9,212,229, and the third-generation CAR is described in U.S. Patent No. 8,822,647 No., which is incorporated herein by reference.
- the CAR-based immune protein of the cytokine can be further expressed, and the 5th generation CAR is an interleukin for strengthening immune cells It further comprises a receptor chain, for example, IL-2Rp.
- 4th generation CAR is US Patent No. 10,316, 102,
- extracellular binding domain of the present invention means a portion of the CAR having the ability to specifically bind to a target antigen of interest (antigen binding domain; antiigen binding domain) .
- the extracellular binding domain is any protein that has the ability to specifically recognize and bind to a biological molecule (eg, a cell surface receptor or tumor protein, lipid, polysaccharide, or other cell surface target molecule, or a component thereof); Polypeptide, oligopeptide or 2021/245580 1» (: 1' year 2021/054846 peptide.
- a binding domain includes any naturally occurring, synthetic, semisynthetic or recombinantly produced binding partner for a biological molecule of interest.
- the term "specifically binds" refers to the binding of a molecule to another molecule with a greater binding affinity than background binding.
- the extracellular binding domain binds to or associates with the target molecule with an affinity of about 10 _ 5 M or more or Ka (ie, the equilibrium dissociation constant of a specific binding interaction having a unit of 1/M). binds specifically to Alternatively, affinity may be defined as the equilibrium dissociation constant (Kd) of a specific binding interaction with units of (eg, 10 5 M to 10, or less).
- the affinity of the extracellular binding domain and the CAR according to the present invention can be determined using conventional techniques, for example, competition ELISA (enzyme-linked immunosorbent assay) or labeled ligands, or using Biacore T100 (which is a piece of New Jersey). Combination using a surface-plasmon resonance device such as Cataway Biacore, Inc.) or optical biosensor technology such as EnSpire or the EPIC system available from Corning and Perkin Elmer, respectively. It can be readily determined by association or substitution analysis.
- the PA63 ligand domain 4 may be characterized in that it recognizes ARTXRKanthrax toxin receptor 1) or ARTXR2 (anthrax toxin receptor 2).
- the PA63 ligand domain 4 is the amino acid of SEQ ID NO: 1 2021/245580 1» (: 1' year 2021/054846 It can be characterized as represented by the sequence.
- the variant of the PA63 ligand domain 4 may be characterized in that the affinity of the PA63 ligand domain 4 to the target is adjusted.
- the affinity is a mutant regulated to recognize ANTXR expressed only in cancer cells, not in normal cells, but is not limited thereto.
- the variant of the PA63 ligand domain 4 is one in which some amino acids are mutated in the PA63 ligand domain 4 represented by the amino acid sequence of SEQ ID NO: 1, preferably selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 7 It may be characterized as comprising one or more amino acid sequences, but is not limited thereto.
- PA63 refers to a truncated 63kDa portion of protective anti-gen (PA, 83kDa), which is one of anthrax toxin proteins. Anthrax toxin is secreted by Bacillus anthracis 6 , a gram-positive bacterium.
- the pathogenicity of anthrax is determined by its toxin-producing ability and capsular formation.
- the proteins that affect the toxin production ability of anthrax are protective antigen (PA), edema factor (EF), and lethal factor (LF).
- PA protective antigen
- EF edema factor
- LF lethal factor
- PA is a receptor protein (Anthrax toxin) on the surface of infected cell lines such as macrophages.
- furin family protease bound to 2021/245580 1 ⁇ (:1 ⁇ 2021/054846 receptor)
- PA63 3 ⁇ 4 tamerized and is known to function as a toxin delivery channel that binds to LF or EF and transports them into cells.
- the complex enters the cell through the endocytosis mechanism and when the pH of the endosome is lowered, a structural change occurs in the PA63 3 ⁇ 4 tamer and a pore is formed in the endosomal membrane.
- Jiang J Atomic structure of anthrax protective antigen pore elucidates toxin translocation. Nature. 2015).
- the term “ANTXRl (anthrax toxin receptor 1)” refers to an extracellular matrix protein, and another name is TEM8 (Tumor endothelial marker 8). Its specific nucleic acid sequence is known from the NCBI (NCBI Reference Sequence: NM-032208.2).
- the term “anthrax toxin receptor 2 (ANTXR2)” refers to a protein involved in angiogenesis, and another name is CMG2 (capi1lary morphogenesis gene 2). A specific nucleic acid sequence thereof is known from NCBI (NCBI). Reference Sequence: NM_058172.5).
- the chimeric antigen receptor may be characterized in that it comprises a transmembrane domain.
- the term “transmembrane domain” refers to extracellular binding 2021/245580 1»(:1' year2021/054846 min and intracellular signaling domains fused and transferred to the plasma membrane of immune effector cells
- the transmembrane domain may be derived from a natural, synthetic, semi-synthetic or recombinant source.
- the transmembrane domain is ⁇ alpha, beta or zeta chain of the cell receptor, 0028, 003 epsilon, 0045, 004, 005, 008, 009, 0) 16, 0022, 0033, 0037, 0064, 0080, It may be characterized in that it is selected from the group consisting of 0086, 0)134, 0)137 and )154, but is not limited thereto. can be attached.
- the linker may be a short oligo_ or polypeptide linker of 2 to 10 amino acids in length, preferably a glycine 1 ⁇ 2) -serine) duplex ((101 ⁇ 1a), but is limited thereto no.
- the binding domain of a shomyo is generally followed by one or more "hinge domains( 1 ⁇ 6 (1011 11) ").
- the term "hinge domain” refers to an antigen-binding domain that plays an important role in positioning the antigen-binding domain away from the effector cell surface to enable proper cell/cell contact, antigen binding and activation. is a part It generally includes one or more hinge domains between the extracellular binding domain and the transmembrane domain.
- the hinge domain may be derived from natural, synthetic, semi-synthetic or recombinant sources.
- the hinge domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.
- a “changed hinge region” is ) an amino acid change of up to 30% (e.g. up to 25%, 20%, 15%,
- one or more cysteine residues in the naturally occurring immunoglobulin hinge region may be substituted with one or more other amino acid residues (eg, one or more serine residues).
- the altered immunoglobulin hinge region may alternatively or additionally have a proline residue of the wild-type immunoglobulin hinge region substituted with another amino acid residue (eg, a serine residue).
- the hinge domain is CD8a,
- any transmembrane domain including the hinge domain can be used as long as it can connect the extracellular domain and the intracellular domain between the cell membrane.
- it consists of a hinge domain and a transmembrane domain derived from (3) 8a, and may include the amino acid sequence represented by SEQ ID NO: 8, but is not limited thereto.
- the chimeric antigen receptor may be characterized in that it comprises an intracellular signaling domain (intracellular signaling domain).
- intracellular signaling domain is an effector cell function, for example, a CAR-bound target cell-to-cell Activation, including the release of toxic factors, cytokine production, proliferation and cytotoxic activity, or other cellular responses induced by antigen binding to the extracellular binding domain of the CAR into the interior of immune effector cells for the target antigen It refers to the portion of the CAR that is involved in the transmission of the message of effective CAR binding.
- the agonistic function refers to a specific function of a cell, for example, the agonistic function of a T cell may be cytolytic activity, may support an activity including secretion of cytokines, or may be active. Accordingly, the intracellular signaling domain refers to a portion of a protein that transmits an operative function signal and directs the cell to perform a specific function.
- T cell activation is mediated by two different classes of intracellular signaling domains.
- T cell activation is mediated by a primary signaling domain that initiates antigen-dependent primary activation through a T cell receptor and a costimulatory signaling domain that acts in an antigen-independent manner to provide a secondary signal.
- the intracellular signaling domain may be characterized as including a "primary signaling domain" and a "co-stimulatory signaling domain".
- primary signaling domain of the present invention, T 2021/245580 1» (: 1' year 2021/054846 refers to a signaling domain that regulates the primary activation of the cell receptor complex in a stimulating or inhibitory manner.
- a primary signaling domain that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITM.
- the ITAM containing the primary signaling domain is TCRC FcRy,
- the primary signaling domain is preferably SEQ ID NO:
- co-stimulatory signaling domain refers to an intracellular signaling domain of a co-stimulatory molecule.
- a costimulatory molecule is an antigen receptor or a cell surface molecule other than an Fc receptor that upon binding to an antigen provides a secondary signal required for efficient activation and function of T lymphocytes.
- the costimulatory signaling domain is 0X40, (3) 2, CD27, CD28, (3)S, ICAM-1, LFA-1 (CDlla/CD18), ICOS (CD278) , CD7, CD30, CD40, PD-1 , LIGHT, NKG2C, B7-H3 , (3) 83 and 4-1BB ((3) 137) may be selected from the group consisting of, but is not limited thereto.
- the costimulatory signaling domain is preferably CD28 comprising the amino acid sequence shown in SEQ ID NO: 10 and ) 137 comprising the amino acid sequence shown in SEQ ID NO: 11, Therefore 2021/245580 1»(:1' year2021/054846 is not limited.
- the chimeric antigen receptor according to the present invention may be characterized in that it includes two or more intracellular signaling domains, and when it includes two or more intracellular signaling domains, the intracellular signaling domains may be connected in series with each other have. Alternatively, it may be linked through an oligopeptide linker or a polypeptide linker consisting of 2 to 10 amino acids, and examples of such a linker sequence include a glycine-serine continuous sequence.
- the CAR when designing the CAR according to the present invention, it may be characterized in that a nucleic acid encoding a signal peptide is inserted before the PA63 ligand domain 4 variant. Accordingly, the chimeric antigen receptor may be characterized in that it further comprises a signal peptide.
- the present invention relates to a nucleic acid encoding the chimeric antigen receptor.
- the nucleic acid (polynucleotide) encoding the chimeric antigen receptor according to the present invention may be modified by codon optimization, which is due to codon degeneracy, and many nucleotide sequences encoding polypeptides or variant fragments thereof Existence is well known to those of ordinary skill in the art.
- nucleic acid a polynucleotide (nucleic acid) that is variable due to the difference in codon utilization, for example, a polynucleotide (nucleic acid) optimized for codon selection in humans, primates and/or mammals is preferable.
- a nucleic acid encoding a mutant ( ⁇ 3 piece) of Sho63 domain 4 or 4) is introduced.
- the nucleic acid encoding the 11 and 1111 may preferably include a nucleotide sequence selected from the group consisting of SEQ ID NO: 14 to SEQ ID NO: 19, but is not limited thereto.
- the present invention relates to an expression vector comprising the nucleic acid and a virus comprising the expression vector.
- the term "vector” refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule.
- the transferred nucleic acid is generally linked to a vector nucleic acid molecule, for example, inserted into a vector nucleic acid molecule.
- a vector may include sequences that direct autonomous replication in a cell, or it may include sequences sufficient to permit integration into a host cell.
- the vector is It may be characterized in that it is selected from the group consisting of a plasmid, a lentiviral vector, an adenoviral vector and a retroviral vector, but is not limited thereto. 2021/245580 1» (: 1' year 2021/054846
- virus means genetically modified to express the chimeric antigen receptor of the present invention for use in the treatment of cancer.
- To be genetically modified is the addition of foreign genetic material in the form of DNA or RNA into the entire genetic material in a cell.
- the nucleic acid or the vector is transfected or transfected with a virus.
- the present invention relates to an immune cell expressing the chimeric antigen receptor on the surface.
- the immune cells are capable of inducing the desired cancer treatment effect by inducing immunity, for example, T cells, NK cells, NKT cells, cytokine-induced killer cells (Cytokine Induced Killer cell, CIK) , It may be selected from the group consisting of macrophages and dendritic cells, but is not limited thereto. Preferably, it may be characterized as a T cell. Therefore, the immune cells expressing the chimeric antigen receptor according to the present invention are CAR-T cells (Chimeric Antigen Receptor T Cells), CAR-NK cells (Chimeric Antigen Receptor Natural Killer Cells) or CAR-NKT cells (Chimeric Antigen Receptor cells).
- CAR-T cells Chimeric Antigen Receptor T Cells
- CAR-NK cells Chimeric Antigen Receptor Natural Killer Cells
- CAR-NKT cells Chimeric Antigen Receptor cells
- the T cells are cytotoxic T lymphocytes; CTL); It may be characterized in that it is selected from the group consisting of T cells isolated from tumor infiltrating lymphocytes (TIL) and peripheral blood mononuclear cells (PBMC).
- TIL tumor infiltrating lymphocytes
- PBMC peripheral blood mononuclear cells
- the present invention relates to a composition for preventing or treating cancer comprising immune cells (eg, T cells) expressing the chimeric antigen receptor.
- immune cells eg, T cells
- tumor infiltrating lymphocytes TIL
- PBMC peripheral blood mononuclear cells
- the term "prevention” refers to any action that inhibits or delays the progression of cancer by administering the composition of the present invention, and “treatment” refers to inhibition of cancer development, alleviation or elimination of symptoms.
- the variant of PA63 ligand domain 4 according to the present invention can specifically bind to cancer cells or cancer tissues expressing ANTXR (anthrax toxin receptor), and PA63 ligand domain according to the present invention It is apparent to those skilled in the art that CAR-containing cells comprising a mutant of 4 have a cytotoxic effect on cancer expressing ANTXR.
- the ANTXR includes ANTXR1 or ANTXR2.
- the cancer or carcinoma that can be treated with the composition of the present invention is not particularly limited. 2021/245580 1» (:1' year 2021/054846, including both solid cancer and blood cancer.
- the cancer may be a primary cancer or a metastatic cancer.
- solid cancer means a tumor that is composed of blood vessels or connective tissue and has a certain hardness and shape, but is not limited thereto, for example, pancreatic cancer, stomach cancer, colon cancer, lung cancer, breast cancer, germ cell cancer , liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, etc.
- the blood cancer include, but are not limited to, acute myeloid leukemia or multiple myeloma.
- composition for preventing or treating cancer of the present invention may further include a pharmaceutically acceptable carrier.
- pharmaceutically acceptable carrier refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound.
- a pharmaceutically acceptable carrier as sterile and biocompatible, saline, sterile water, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol and among these components
- One or more components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed.
- diluents may be additionally added to form an injection formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.
- the composition of the present invention may be in various oral or parenteral formulations.
- commonly used thickening agents, extenders, binders, wetting agents, disintegrants, 2021/245580 1» (: 1' year 2021/054846 Prepared using a diluent or excipient such as a surfactant.
- Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose or lactose. It is prepared by mixing (lactose), gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.
- Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included.
- Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories.
- Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
- injectable esters such as ethyl oleate.
- witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, etc. can be used as a base for the suppository.
- Such pharmaceutically acceptable carriers and agents are Remington's
- composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and rectal administration, etc. can be administered.
- parenteral administration intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and rectal administration, etc.
- the oral composition can be formulated to coat the active agent or to be protected from degradation in the stomach, and the composition of the present invention can be any device capable of moving the active substance to the target cell to be administered by 2021/245580 1» (:1' year 2021/054846 May.
- a suitable dosage of the composition for preventing or treating cancer of the present invention depends on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. It varies, and an ordinary skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention.
- the composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.
- the present invention relates to a method for preventing or treating cancer comprising administering the immune cells.
- the present invention relates to the use of the immune cells for the prevention or treatment of cancer.
- the present invention relates to the use of the immune cells for the manufacture of a medicament for the prevention or treatment of cancer. Since the above-described "immune cells" are used for the prevention or treatment methods, uses, and uses of the present invention, redundant descriptions thereof will be omitted.
- the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it is common knowledge in the art that the scope of the present invention is not to be construed as being limited by these examples. 2021/245580 1»(:1' year 20211/054846 It will be obvious to those who have it.
- Example 1 Analysis of target antigen protein expression in cancer cell lines Melanoma cell line 526-mel, pancreatic cancer cell line (PANC1, Mia_PaCa2), breast cancer cell line (SK-BR3, MDA-MB-231, MCF7, ANTXRKTEM8 of ZR-517), As a result of analyzing ANTXR2 (CMG2) protein expression using western blot, ANTXR1 and ANTXR2 expression were increased in pancreatic cancer and breast cancer cell lines, melanoma cell line 526-mel and breast cancer cell line SK-BR3 did not express ANTXR1 and ANTXR2 was confirmed (FIG. 1).
- Example 2 Preparation of target antigen protein overexpression cell line According to Example 1, TEM8-RFP, CMG2-RFP lentivirus was transducted into ANTXRKTEM8), ANTXR2 (CMG2) negative 526-mel cell line, and then western blot method was performed It was confirmed that the protein was normally expressed using the . After that, as a result of selection of positive cells using puromycin, a cell line with increased expression of 70% or more was prepared (FIG. 3) .
- Example 3 ANTXR specific CAR design
- the CAR specific for ANTXR1 or ANTXR2 was designed to include an extracellular binding domain, (3) a transmembrane domain derived from 8a, a hinge region, a primary signaling domain of the CD3 ⁇ 4 chain, (3) 28 and a CD137 costimulatory signaling domain.
- extracellular binding 2021/245580 1» (: 1' year 2021/054846 Domains essential for receptor binding of PA63 ligands D4 wild-type (wild) or D4 mutants (mutant) #1 - #6 are designed, respectively, the introduced CAR, Figure 4
- a vector was prepared by introducing it into the D4 site of the vector shown in .
- the signal peptide is a short amino acid sequence and plays an important role in transporting the synthesized protein to the seedlings.
- the signal peptide is removed, and the protein is transported to the Golgi complex through a carrier.
- GM-CSF was used as a signal peptide for transporting the synthesized protein to its destination.
- a nucleic acid encoding copGFP was introduced into the vector of FIG. 4 in order to check the expression of CAR, but it may be omitted in order to use the CAR as an actual therapeutic agent. in the present invention
- the amino acid sequences required for preparation are shown in Table 1.
- Example 4 ANTXR-specific CAR construction
- extracellular binding domain PA63 D4 wi ld and D4 mutant #1 - ⁇
- extracellular binding domain PA63 D4 wi ld and D4 mutant #1 - ⁇
- transmembrane domain CD8 transmembrane & hinge
- intracellular signaling domain CD3, CD134, CD28
- Gibson assembly was performed.
- the virus was prepared using the lentiviral vector for CAR introduction prepared above, and the packaging cell 293FT cell (4X10 6 ) has an efficiency of about 50% or more.
- 2021/245580 1» (:1' year 2021/054846 lentiviral vector was induced.
- the lentivirus was prepared and the suspended cellmedia was recovered, which was concentrated 10 times for 30 minutes at 1,000 X g using a Centicon, and the concentrated lentivirus was transduced into 293FT, NIH3T3 cells and CAR It was possible to confirm the lentivirus titer into which the gene was introduced.
- Each CAR-T cell was prepared by transduction of the prepared D4 wild or D4 mutant-containing CAR-introduced lentivirus into PBMCs isolated from healthy donors at a concentration of 3.5 M0I.
- Example 5 Confirmation of cytotoxicity against ANTXR1, 2TEM8, CMG2) overexpressing cell lines of CAR-T cells prepared using T cells isolated from PBMC D4 wild-containing CAR introduced T cells and D4 mutant # The purpose of this study was to confirm the anticancer cytotoxicity of 1 - #6 containing CAR-introduced T cells. Cytotoxicity was confirmed against the 526-mel melanoma cell lines, which were both negative for ANTXR1 and 2 (TEM8, CMG2), and the 526-mel/TEM8jFP and 526-me1/CMG2_RFP cell lines overexpressing ANTXR1 and 2 (TEM8, CMG2), respectively. .
- RFP Red Fluorescence Protein
- Example 6 Confirmation of anticancer activity against ANTXR1, 2TEM8, CMG2) overexpressing cell lines of CAR-T cells prepared using T cells isolated from PBMC It was intended to confirm the anticancer activity of the CAR-introduced T cells. Anticancer activity was confirmed against 526-mel/TEM8jFP and 526-me1/CMG2_RFP cell lines overexpressing ANTXR1 and 2 (TEM8, CMG2), respectively, in 526-mel melanoma cell lines negative for both ANTXR1 and 2 (TEM8, CMG2). .
- D4 mutant #1 - #6 CAR-T£1 IFN gamma secretion 2021/245580 1» (: 1' year, 2021/054846 decreased, which decreased as the binding force between the receptor and ligand for ANTXR1 and 2 (TEM8, CMG2) decreased, the anticancer activity of D4 mutant #1 #6 CAR-T decreased.
- Fig. 6 Example 7: In vitro anticancer activity and safety analysis of CAR-T cells prepared by selection Using PBMCs from 4 healthy donors, 2 types of D4 mutant CAR vectors selected from 6 types of D4 mutants CAR- T cells were prepared, and the difference in in vitro anticancer activity according to each donor PBMC was investigated.
- Example 6 In the same manner as in Example 6, NT (Non Transduced) T cell control and D4 (wild, mutant #4, #6) CAR-T cells were prepared and T cell activity was compared. As a result, it was possible to confirm the TEM8-specific activity of D4 mutants #4 and #6 in each of the CAR-T cells prepared from each of the four healthy donors, similar to the results of Example 6. In addition, it was reconfirmed that the CAR-T activity of D4 mutants maintained cytotoxicity compared to D4 wild, but the degree of IFNY secretion was decreased due to a decrease in affinity for antigen (Fig. 7 left) .
- Example 8 Analysis of T cell characteristics after co-culture of TEM8 overexpressing cancer cell line and two selected D4 mutant CAR-T 2021/245580 1»(:1' year2021/054846
- 526-me 1 /TEM8_RFP cells overexpressing ANTXR TEM8) in a 526-mel melanoma cell line negative for both ANTXR1 (TEM8) / ANTXR2 (CMG2) antigens were seeded in a 96-well plate with 4 lxlO cells each.
- the TEM8 antigen-overexpressing human pancreatic cancer cell line (Mia-PaCa2/TEM8) was implanted by subcutaneous injection at a dose of 5 x 10 6 cels/head. 21 days after transplantation (tumor size: about ⁇ 100mm 3 ) D4 2021/245580 1» (: 1' year 2021/054846 wi ld and D4 mutant #6 CAR-T cells finally selected from the above results 1 x 10 7 cel ls/head(total cel l: 2 x 10 7 cel ls/head) was administered to tumor-implanted mice by tail vein injection, and the tumor growth inhibitory effect was compared and investigated (FIG. 9A).
- the tumor growth was inhibited by about 61% (FIG. 9B).
- the D4 mutant-containing CAR-introduced T cells had excellent efficacy in an animal model using solid carcinoma expressing ANTXR1 (TEM8).
- TEM8 solid carcinoma expressing ANTXR1
- Industrial Applicability Most of the target antigens of solid cancer are cancer-associated antigens, not cancer-specific antigens, and the target antigen ANTXR of the present invention is also a cancer-associated antigen. It is important to secure safety while maintaining CAR-T anticancer efficacy.
- a chimeric antigen receptor comprising a mutant of PA63 ligand domain 4 as an extracellular binding domain, it shows an excellent anticancer effect against cancer expressing an ARTXR antigen, and controls the binding affinity to the ARTXR antigen to control normal cells Since safety can be optimized so that there is no effect on cancer, it is possible to treat cancer with very effective anticancer treatment and minimizing side effects of existing CAR-T immune cell therapies.
- a specific part of the content of the present invention has been described in detail, and for those of ordinary skill in the art, this specific description is only a preferred implementation.
Abstract
The present invention relates to a chimeric antigen receptor that optimizes safety for normal cells while maintaining anticancer effectiveness by controlling binding affinity of a chimeric antigen receptor recognizing an anthrax toxin receptor (ANTXR) antigen and binding thereto, and the present invention relates to: a ligand-based chimeric antigen receptor comprising a PA63 domain 4 variant specifically binding to an ANTXR1 or ANTXR2 cancer antigen; immune cells comprising same; and a use thereof. According to the present invention, the chimeric antigen receptor comprising a PA63 ligand domain 4 variant as an extracellular binding domain may be used to show an excellent anticancer effect for cancer expressing the ARTXR antigen and to optimize safety by controlling binding affinity for the ARTXR antigen such that normal cells are not affected, and, thereby, the chimeric antigen receptor is capable of cancer treatment that is very effective while minimizing side effects of an existing CAR-T immune cell treatment agent.
Description
2021/245580 1»（：1'해2021/054846쇼63 도메인 4 변이체를 세포외 결합 도메인으로 포함하는 키메라 항원 수용체 및 이의 용도 기술분야 본 발명은 다양한 암세포에서 과발현되는 것으로 알려진 ANTXR(Anthrax toxin receptor ; 탄저균 독소 수용체 ) 항원을 인식하여 결합하는 키메라 항원 수 용체의 결합 친화도를 조절하여 항암 유효성은 유지하면서 정상세포에 대한 안전 성을 최적화한 키메라 항원 수용체에 관한 것으로， ANTXRKanthrax toxin receptor 1) 또는 ANTXR2(anthrax toxin receptor 2) 암 항원에 특이적으로 결합 하는 PA63 도메인 4의 변이체를 포함하는 리간드 기반의 키메라 항원 수용체， 이 를 포함하는 면역세포 및 이의 용도에 관한 것이다. 배경기술 2021/245580 1 » (: 1' year 2021/054846 show 63 A chimeric antigen receptor comprising a domain 4 variant as an extracellular binding domain and uses thereof Technical field The present invention relates to an ANTXR (Anthrax toxin receptor) known to be overexpressed in various cancer cells; Anthrax toxin receptor) relates to a chimeric antigen receptor that optimizes safety for normal cells while maintaining anticancer efficacy by controlling the binding affinity of the chimeric antigen receptor that recognizes and binds antigen, ANTXRKanthrax toxin receptor 1) or ANTXR2 ( anthrax toxin receptor 2) relates to a ligand-based chimeric antigen receptor comprising a mutant of PA63 domain 4 that specifically binds to a cancer antigen, immune cells comprising the same, and uses thereof. background
ANTXR(anthrax toxin receptor) 항원은 ANTXR1과 ANTXR2를 포함하며 이 항원들을 인식하는 리간드로는 탄저균이 분비하는 탄저균 독소 (anthrax toxin)가 알려져 있다 . 탄저균 독소는 그람 양성균인 바실러스 안트라시스 (公 3C77A/S anthracis)6 의해 분비되며， 3가지 독소 단백질인 보호 항원 (Protect ive ant igen [PA, 83kDa] ) , 치사 인자 (Lethal factor [LF, 90kDa] ) ) , 부종 인자 (Edema factor [EF, 89kDa] )로 구성되어 있다 (Morton, N. , New Engl . J . Med. 345: 1621-1626 [2001] ) . 이 중 PA (보호 항원 )가 세포 표면의 ANTXR과 결합하면 퓨린 (furin) 계열의 단백질 가수분해 효소에 의해 아미노산 말단 20kDa(PA20) 단 백질이 잘려 나가고， 이꾸 631江 (?쇼63) 단백질은 헵타머 (116야311161 )를 형성하며
2021/245580 1»（：1'해2021/054846 Anthrax toxin receptor (ANTXR) antigens include ANTXR1 and ANTXR2, and anthrax toxin secreted by anthrax is known as a ligand for recognizing these antigens. Anthrax toxin is secreted by Gram-positive bacteria, Bacillus anthracis 6 ) ) and Edema factor [EF, 89 kDa] (Morton, N. , New Engl. J. Med. 345: 1621-1626 [2001]). Among these, when PA (protective antigen) binds to ANTXR on the cell surface, the amino acid terminal 20 kDa (PA20) protein is cut off by a furin-based proteolytic enzyme, and the Iku 631 River (? Show 63) protein is hep to form a tamer (116y311161) 2021/245580 1»（：1' year2021/054846
LF (치사 인자)가 결합하여 세포내로 이동하고 세포내 독성을 유도한다 (Collier, R.J. , Annu. Rev. Cell Dev. Biol. 19： 45-70 [2003]). LF (lethal factor) binds, migrates intracellularly and induces intracellular toxicity (Collier, R.J., Annu. Rev. Cell Dev. Biol. 19: 45-70 [2003]).
ANTXR1은 세포외 기질 단백질로 2000년에 Brad St. Croix에 의해 대장암 에서 동정된 TEM8 (Tumor endothelial marker 8)로 알려져 있다 (Sciences 289:1197 [2000]). ANTXR1은 다양한 종양의 내피에서 발현되는 타입 I 세포막 단백질 (type I transmembrane protein)로， 종양의 혈관신생과정 (tumor angiogenesis) 동안 발현이 증가하는 유전자로 밝혀졌으며 (St. Croix et al . , Sciences, 289： 1197 [2000]), 종양의 혈관내피세포에서의 세포외 기질 단백질을 매개로 한 세포의 부착이나 이동 등에도 관여한다고 알려져 있다 (Bradely et al. , Nature 414:228-229 [2001]; Nanda et al . ,Curr Op in Oncol 16 : 44-49 [2004]). ANTXR1은 세포외 리간드 (extracel lular ligand)에 대한 수용체로 작용하기 때문 에， 혈관신생과정의 치료를 위한 표적 항원이 되고 있다 (Car son-Walter, E. B. et al. , Cancer Res 61:6649 [2001]). ANTXR2의 다른 명칭은 CMG2(capi 1 lary morphogenesis gene 2)이며， ANTXR2는 역시 혈관신생에 관여한다고 알려져 있다. 또한， ANTXR2는 상피세포와 내피세포를 포함한 다양한 종류의 세포 부착 (adhesion)과 이동 (moti lity)에도 관여한다고 알려져 있다 (Lin Ye et al . , Int J Oncol 45： 1565-1573 [2014]). 지난 수십 년간 암의 검출， 예방 및 치료 기술에 해난 진보적인 발전에도 불구하고 국내외 사망 원인 중 암에 의한사망이 1위를 차지하고 있으며 혈액암， 고형암을 포함한 난치성 암환자의 수는 지속적으로 증가하고 있는 추세이다. 기 존의 방사선 치료， 화학요법， 수술을 이용한 항암치료로서는 난치성 암의 치료와 암의 재발， 전이와 같은 심각한 부작용에 시달리고 있다. 최근 들어 항암치료용
2021/245580 1»（：1'해2021/054846 항체를 사용한 면역 치료법은 종양 부위 내로의 제한된 투과 및 종양 세포에 대 한 표적 항원의 발현 수준에 부분적으로 기인하여 제한된 성공을 제공하고 있다 . ANTXR1 is an extracellular matrix protein, published in Brad St. It is known as TEM8 (Tumor endothelial marker 8) identified in colorectal cancer by Croix (Sciences 289:1197 [2000]). ANTXR1 is a type I transmembrane protein expressed in the endothelium of various tumors, and has been found to be a gene whose expression is increased during tumor angiogenesis (St. Croix et al., Sciences, 289). : 1197 [2000]), is also known to be involved in cell adhesion and migration mediated by extracellular matrix proteins in tumor vascular endothelial cells (Bradely et al., Nature 414:228-229 [2001]; Nanda) et al., Curr Op in Oncol 16:44-49 [2004]). Since ANTXR1 acts as a receptor for extracellular ligand (extracelular ligand), it has become a target antigen for the treatment of angiogenesis (Car son-Walter, EB et al., Cancer Res 61:6649 [2001]) ). Another name for ANTXR2 is CMG2 (capi 1 lary morphogenesis gene 2), and ANTXR2 is also known to be involved in angiogenesis. In addition, ANTXR2 is known to be involved in adhesion and migration of various types of cells, including epithelial cells and endothelial cells (Lin Ye et al., Int J Oncol 45: 1565-1573 [2014]). Despite advances in cancer detection, prevention, and treatment technologies over the past few decades, cancer is the leading cause of death among domestic and foreign causes of death, and the number of patients with intractable cancer, including blood cancer and solid cancer, is continuously increasing. is the trend Conventional chemotherapy using radiation therapy, chemotherapy, and surgery suffers from serious side effects such as treatment of intractable cancer, recurrence of cancer, and metastasis. Recently, anticancer 2021/245580 1»（：1'year 20211/054846 Immunotherapy using antibodies has provided limited success due in part to limited penetration into the tumor site and expression levels of target antigens on tumor cells.
2017년 미 FDA승인을 시작으로 최근 많은 글로벌 제약회사에서 연구 개 발에 붐을 이루고 있는 CAR-T면역세포치료제의 경우 유전자변형 세포 치료 요법 으로 전세계의 주목을 받고 있다 . 이 치료법은 암세포의 표면항원으로의 특이성 과 세포의 항암 활성 (유효성 )을 갖는 키메라 항원 수용체(chimeric antigen receptor, CAR) 유전자를 면역 T세포 또는 NK세포에 형질 도입하고， 이들 키메 라 항원 수용체가 발현되고 있는 T혹은 NK세포를 체외에서 증식 배양하여 환자 에게 투여하여 치료하는 항암 면역 치료 (유전자치료)방법 이다 . 이 치료법은 한번 의 투여로 항체의 약에 비해 항암 효과가 탁월하고 효과도 보다 장기간 지속되는 결과들을 보여주고 있어 임상에 대한 항암 유효성에 많은 기대를 하고 있다 . 이 러한 기술적， 임상적 배경하에서， 본 발명자들은 ANTXR을 특이적으로 인식 /결합하는 리간드인 PA63 도메인 4를 포함하는 CAR에 관련된 개발내용을 2019년 국제특허출원 하였다 (PCT/KR2019八) 17215) . 하지만 기존의 췌장암을 표적으로 하는 세포치료제의 항원들의 경우 대부 분 종양 연관 항원 (Tumor-associated antigen)들로 암세포에서 과발현 되는 항원 이긴 하지만 모두 정상세포에도 발현이 되는 항원들로 정상세포를 공격하여 심각 한 부작용을 유도할 가능성 이 존재하고 있다 . 본 발명자들이 국제특허 출원한 ANTXR표적 항원도 종양 연관 항원으로 임상에 적용하기 위해 안전성을 최대화하 고 항암 유효성은 유지하는 더욱 진보된 CAR개발에 대한 필요성 이 요구된다 할 수 있다 . 특히 영국의 버밍험대학의 Steven P. Lee 그룹에서 항체기반의
세포가 마우스의 폐와 비장에서 염증반응을 유도하는
2021/245580 1»（：1'해2021/054846 부작용을 보고하였고 ‘on-target off-tumor’ 독성을 일으킬 수 있다는 가능성 을 제시하며 임상 시험 전에 이를 신중히 고려해야 한다고 보고하였다(PLoS One. 14, e0224015 [2019] ) . 이에 본 발명자들은 ANTXR을 특이적으로 인식 /결합하는 리간드인 PA63 도 메인 4를 포함하는 CAR를 기반으로 기존의 항암 유효성을 유지하면서， 정상세포 에 대한 부작용을 최소화할 수 있도록 안전성을 최적화한 차별화/진보된 키메라 항원 수용체를 개발하고자 하였다. 이에 ANTXR1과 ANTXR2 항원에 다양한 친화도 를 갖는 CAR개발하기 위해 항원에 결합하는 리간드 부위인 PA63 도메인 4(D4)의 아미노산을 변이 (mutation)시켜 친화도를 조절하였고， 암 치료 효과는 유지하면 서 off-target toxicity를 줄일 수 있는 CAR로서 본 발명을 완성하였다. 본 배경기술 부분에 기재된 상기 정보는 오직 본 발명의 배경에 대한 이 해를 향상시키기 위한 것이며， 이에 본 발명이 속하는 기술분야에서 통상의 지식 을 가지는 자에게 있어 이미 알려진 선행기술을 형성하는 정보를 포함하지 않을 수 있다. 발명의 요약 본 발명의 목적은 다양한 결합 친화력을 갖는 CAR를 개발하여 면역세포치 료제로서 유효성을 유지하면서 안전성을 최적화한 키메라 항원 수용체(chimeric antigen receptor , CAR)를 제공하는 데 있다. 본 발명의 다른 목적은 상기 키메라 항원 수용체를 코딩하는 핵산， 상기
2021/245580 1»（：1'해2021/054846 핵산을 포함하는 발현 벡터 및 상기 발현 벡터를 포함하는 바이러스를 제공하는 데 있다. 본 발명의 또 다른 목적은 상기 면역세포를 포함하는 암 예방 또는 치료 용 조성물， 상기 면역세포를 투여하는 단계를 포함하는 암 예방 또는 치료방법， 암 예방 또는 치료를 위한 상기 면역세포의 용도 및 암 예방 또는 치료용 약제 제조를 위한 상기 면역세포의 사용을 제공하는 데 있다. 상기 목적을 달성하기 위하여， 본 발명은 PA63 리간드 도메인 4의 변이체 를 함유하는 세포외 결합 도메인(extracellular binding domain) , 막횡단 도메인 (transmembrane domain) 및 세포내 신호전달 도메인(intracellular signaling domain)을 포함하는 키메라 항원 수용체(chimeric antigen receptor , CAR)를 제 공한다. 본 발명은 또한， 상기 키메라 항원 수용체를 코딩하는 핵산， 상기 핵산을 포함하는 발현 벡터， 상기 발현 벡터를 포함하는 바이러스 및 상기 키메라 항원 수용체를 발현하는 면역세포를 제공한다. 본 발명은 또한， 상기 면역세포를 포함하는 암 예방 또는 치료용 조성물， 상기 면역세포를 투여하는 단계를 포함하는 암 예방 또는 치료방법， 암 예방 또 는 치료를 위한 상기 면역세포의 용도 및 암 예방 또는 치료용 약제 제조를 위한 상기 면역세포의 사용을 제공한다. 도면의 간단한 설명 도 1은 흑색종 (526-mel) , 췌장암 (PANC-1, Mia_PaCa2) , 유방암 (SK-BR3,
2021/245580 1»（：1'해2021/054846 CAR-T immune cell therapy, which has been booming in R&D by many global pharmaceutical companies, starting with FDA approval in 2017, is drawing attention from around the world as a genetically modified cell therapy. In this treatment, a chimeric antigen receptor (CAR) gene having specificity as a surface antigen of cancer cells and anticancer activity (effectiveness) of cells is transduced into immune T cells or NK cells, and these chimeric antigen receptors are expressed It is an anti-cancer immunotherapy (gene therapy) method in which T or NK cells are proliferated outside the body and administered to the patient for treatment. This treatment has excellent anti-cancer effects compared to antibody drugs with a single administration and shows long-lasting results, so there is a lot of anticipation for anti-cancer efficacy in clinical practice. Under this technical and clinical background, the present inventors have filed an international patent application for development contents related to CAR including PA63 domain 4, which is a ligand that specifically recognizes / binds ANTXR (PCT/KR2019八) 17215). However, in the case of cell therapy antigens that target existing pancreatic cancer, most of them are tumor-associated antigens, which are overexpressed in cancer cells, but they all attack normal cells with antigens that are also expressed in normal cells. There is a possibility of inducing a side effect. In order to apply the ANTXR target antigen, which the present inventors applied for for an international patent, to clinical practice as a tumor-associated antigen, there is a need for more advanced CAR development that maximizes safety and maintains anticancer efficacy. In particular, in the Steven P. Lee group of the University of Birmingham in the UK, antibody-based Cells induce an inflammatory response in the lungs and spleen of mice. 2021/245580 1» (: 1' Year 2021/054846 reported side effects, suggested the possibility of causing 'on-target off-tumor' toxicity, and reported that it should be carefully considered before clinical trials (PLoS One. 14, e0224015) [2019]). Accordingly, the present inventors specifically recognize / bind to ANTXR, based on the CAR containing the PA63 domain 4, while maintaining the existing anti-cancer efficacy, differentiation / optimizing safety to minimize side effects on normal cells An attempt was made to develop an advanced chimeric antigen receptor. Accordingly, in order to develop CARs having various affinities for ANTXR1 and ANTXR2 antigens, the affinity was controlled by mutation of the amino acid of PA63 domain 4 (D4), which is a ligand site binding to the antigen, and the cancer treatment effect was off while maintaining The present invention has been completed as a CAR that can reduce -target toxicity. The information described in the background section is only for improving the understanding of the background of the present invention, and includes information forming the prior art known to those of ordinary skill in the art to which the present invention belongs may not SUMMARY OF THE INVENTION An object of the present invention is to provide a chimeric antigen receptor (CAR) with optimized safety while maintaining efficacy as an immune cell therapy agent by developing CARs having various binding affinities. Another object of the present invention is a nucleic acid encoding the chimeric antigen receptor, 2021/245580 1» (: 1' year 2021/054846 To provide an expression vector containing a nucleic acid and a virus containing the expression vector. Another object of the present invention is a composition for preventing or treating cancer comprising the immune cells, a method for preventing or treating cancer comprising administering the immune cells, the use of the immune cells for preventing or treating cancer, and cancer prevention or to provide the use of the immune cells for the manufacture of a therapeutic agent. In order to achieve the above object, the present invention provides an extracellular binding domain containing a variant of PA63 ligand domain 4, a transmembrane domain, and an intracellular signaling domain comprising a It provides a chimeric antigen receptor (CAR). The present invention also provides a nucleic acid encoding the chimeric antigen receptor, an expression vector containing the nucleic acid, a virus containing the expression vector, and immune cells expressing the chimeric antigen receptor. The present invention also provides a composition for preventing or treating cancer comprising the immune cells, a method for preventing or treating cancer comprising administering the immune cells, the use of the immune cells for preventing or treating cancer, and preventing cancer or Provided is the use of said immune cells for the manufacture of a medicament for treatment. 1 is melanoma (526-mel), pancreatic cancer (PANC-1, Mia_PaCa2), breast cancer (SK-BR3, 2021/245580 1»（：1'year2021/054846
MDA-MB-231 , MCF7, ZR571) 세포주에서 ANTXRKTEM8) 항원과 ANTXR2(CMG2) 항원들 의 단백질 발현을 확인한 결과이다. 도 2는 ANTXR TEM8)과 ANTXR2(CMG2) 항원이 모두 음성인 흑색종 (526-mel ) 세포주에서 ANTXRKTEM8) 항원과 ANTXR2(CMG2) 항원을 과발현 시켰을 때의 단백 질 발현을 확인한 결과이다. 도 3은 ANTXRKTEM8) 항원 단백질 혹은 ANTXR2(CMG2) 항원 단백질과 RFP(Red Fluorescence Protein) 형광 단백질을 동시에 발현시킨 폭색종 (526，el ) 세포주에서 유세포 분석기를 이용하여 각 항원과 fusion된 RFP 형광단백질의 발 현을 확인한 결과이다. 도 4는 본 발명에 따른 ANTXRKTEM8) 또는 ANTXR2(CMG2)에 특이적인 PA63 domain 4 (D4) wi ld type CAR와 D4의 mutat ion의 위치를 표현한 그림과 대표적인 플라스미드 DNA 벡터 맵이다. 본 발명의 CAR는 세포외 결합 도메인으로 PA63 리 간드의 수용체 결합에 필수적인 D4 또는 D4 mutant를 포함하는 단편， CD8 a로부 터 유래된 막횡단 도메인과 hinge region, CD3 사슬의 1차 신호전달 도메인， ⑶ 28 및 CD137 공자극 신호전달 도메인을 포함하도록 설계되었다. 도 5는 PBMC를 이용하여 제작한 D4 wi ld와 D4 mutant #1 - #6 CAR-T 세포 의 ANTXR TEM8)과 ANTXR2(CMG2) 과발현 세포주에 대한 세포독성을 비교 조사한 그래프이다. 도 6은 PBMC를 이용하여 제작한 D4 wi ld와 D4 mutant #1 - #6 CAR-T 세포 의 ANTXR TEM8)과 ANTXR2(CMG2) 과발현 세포주에 대한 항암활성을 IFN-gamma ELISA를 통해 분석한 결과를 나타낸 그래프이다. 도 7은 건강한 공여자 4명의 PBMC를 사용하여 제작한 D4 wi ld와 D4
2021/245580 1»（：1'해2021/054846 mutant #4 및 #6 CAR-T세포의 ANTXR1(TEM8)과 ANTXR2(CMG2)에 대한 in vitro항 암활성을 분석한 결과를 나타낸 그래프이다. 도 8은 ANTXRKTEM8) 과발현 암 세포주와 D4 mutant #4 및 #6 ◦쇼묘 의 공 배양 후 T세포 특성을 확인한 결과를 나타낸 그래프이다 . 도 9는 Xenograft마우스 모델을 이용한 D4 mutant #6 CAR-T의 항암 유효 성을 나타낸 그래프이다 . 발명의 상세한 설명 및 바람직한 구현예 다른 식으로 정의되지 않는 한， 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명 이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다 . 일반적으로 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다 . 본 발명의 일 실시 예에서는， 암 조직에서 발현하는 ANTXRKTEM8) 항원과 ANTXR2(CMG2) 항원에 특이적으로 결합하는 PA63 리간드 도메인 (domain) 4를 세포외 결합 도메인으로 포함하는 키메라 항원 수용체를 개발하였으며， 상기 PA63 리간드 도메인 4의 아미노산 잔기 일부를 변이시킨 변이체들을 포함하는 키메라 항원 수용체의 경우， affinity tuning을 통해 항암활성 (유효성 )은 유지되거나 약간 낮아지는 한편， 암 세포 특이성 이 높아지고 정상 세포에 대한 공격력 이 낮아져 off-target toxicity가 낮아짐을 확인하였으며， memory T 세포의 형성을 높여 체내에 오래 유지시킬 수 있음을 확인하였다 .
2021/245580 1»（：1'해2021/054846 따라서， 본 발명은 일 관점에서， PA63 리간드 도메인 4의 변이체를 함유하는 세포외 결합 도메인 (extracel lular binding domain) , 막횡단 도메인 (transmembrane domain) 및 세포내 신호전달 도메인 (intracellular signaling domain)을 포함하는 키메라 항원 수용체 (chimeric antigen receptor , CAR)에 관한 것이다. This is the result of confirming the protein expression of ANTXRKTEM8) antigen and ANTXR2 (CMG2) antigen in MDA-MB-231 , MCF7, ZR571) cell lines. 2 is a result confirming the protein expression when the ANTXRKTEM8) antigen and the ANTXR2 (CMG2) antigen are overexpressed in a melanoma (526-mel) cell line in which both ANTXR TEM8) and ANTXR2 (CMG2) antigens are negative. 3 is a flow cytometry analysis of RFP fluorescent protein fused with each antigen using a flow cytometer in a chemochromatosis (526, el) cell line that simultaneously expresses ANTXRKTEM8) antigen protein or ANTXR2 (CMG2) antigen protein and RFP (Red Fluorescence Protein) fluorescent protein This is the result of confirming the expression. Figure 4 is a picture expressing the position of the mutation of PA63 domain 4 (D4) wil type CAR and D4 specific to ANTXRKTEM8) or ANTXR2 (CMG2) according to the present invention and a representative plasmid DNA vector map. The CAR of the present invention is an extracellular binding domain, a fragment containing D4 or D4 mutant essential for receptor binding of PA63 ligand, a transmembrane domain derived from CD8 a, a hinge region, a primary signaling domain of the CD3 chain, ⑶ 28 and CD137 costimulatory signaling domains. 5 is a graph comparing cytotoxicity to ANTXR TEM8) and ANTXR2 (CMG2) overexpressing cell lines of D4 wild and D4 mutant #1 - #6 CAR-T cells prepared using PBMC. Figure 6 shows the results of analyzing the anticancer activity against ANTXR TEM8) and ANTXR2 (CMG2) overexpressing cell lines of D4 wi ld and D4 mutant #1 - #6 CAR-T cells prepared using PBMC through IFN-gamma ELISA. This is the graph shown. 7 shows D4 wi ld and D4 prepared using PBMCs of 4 healthy donors. 2021/245580 1» (：1' year 2021/054846 mutant #4 and #6 This is a graph showing the results of analyzing the in vitro anticancer activity of CAR-T cells against ANTXR1 (TEM8) and ANTXR2 (CMG2). 8 is a graph showing the results of confirming the characteristics of T cells after co-culture of ANTXRKTEM8) overexpressing cancer cell lines and D4 mutants #4 and #6 ◦Shomyo. 9 is a graph showing the anticancer efficacy of D4 mutant #6 CAR-T using a Xenograft mouse model. DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art. In an embodiment of the present invention, a chimeric antigen receptor comprising PA63 ligand domain 4 specifically binding to ANTXRKTEM8) antigen and ANTXR2 (CMG2) antigen expressed in cancer tissue as an extracellular binding domain was developed. In the case of a chimeric antigen receptor comprising variants in which some of the amino acid residues of the PA63 ligand domain 4 are mutated, anticancer activity (efficiency) is maintained or slightly lowered through affinity tuning, while cancer cell specificity is increased and attack power against normal cells is increased It was confirmed that off-target toxicity was lowered, and it was confirmed that it can be maintained for a long time in the body by increasing the formation of memory T cells. 2021/245580 1 » (: 1' year 2021/054846 Therefore, in one aspect, the present invention provides an extracellular binding domain containing a variant of PA63 ligand domain 4 (extracellular binding domain), a transmembrane domain and It relates to a chimeric antigen receptor (CAR) comprising an intracellular signaling domain.
"키메라 항원 수용체 (Chimeric antigen receptor; CAR)"는 표적 항원 및 해당 항원을 발현하는 세포에 대하여 면역반응을 유도할 수 있도록 설계된 합성 construct이다. CAR는 세포외 도메인 (extracel lular domain) , 막횡단 도메인 (transmembrane domain) 및 세포내 신호전달 도메인 (intracellular signaling domain)을 포함한다. 암 세포의 표면에 특이적으로 발현되는 암 세포 표면 항원을 인식하는 수용체를 코딩하는 유전자를 면역세포에 도입하여， 암 세포를 사멸시킬 수 있다. 암 세포에서 특이적으로 발현하는 항원과 결합하는 수용체를 포함하는 면역세포를 통해， 암 세포만 표적하여 면역반응을 일으킬 수 있다. "Chimeric antigen receptor (CAR)" is a synthetic construct designed to induce an immune response against a target antigen and cells expressing the antigen. CAR includes an extracellular domain, a transmembrane domain and an intracellular signaling domain. By introducing a gene encoding a receptor recognizing a cancer cell surface antigen specifically expressed on the surface of cancer cells into immune cells, cancer cells can be killed. Through immune cells comprising a receptor that binds to an antigen specifically expressed in cancer cells, it is possible to induce an immune response by targeting only cancer cells.
1세대 CAR에서는 암 세포에서 특이적으로 발현하는 항원 인식 부위를 포함하는 세포외 도메인， 막횡단 도메인 및 세포내 신호전달 도메인을 포함하고， 신호전달 도메인으로서 ⑶ ¾만을 이용하였는데， 암에 대한 치료 효과가 미미하였고， 지속시간이 짧다는 문제가 있었다. 이러한 1세대 CAR는 미국등록특허 제 6 ,319 ,494호에 구체적으로 기재되어 있으며， 본 발명에 참조로서 도입된다. 면역세포에 대한 반응성 향상을 위하여 공자극 도메인 (CD28 또는
2021/245580 1»（：1'해2021/054846 In the first-generation CAR, an extracellular domain including an antigen recognition site specifically expressed in cancer cells, a transmembrane domain, and an intracellular signaling domain were used, and only ⑶ ¾ was used as the signaling domain, but the therapeutic effect on cancer was insignificant, and there was a problem that the duration was short. This first-generation CAR is specifically described in US Patent No. 6,319,494, which is incorporated herein by reference. To enhance responsiveness to immune cells, a costimulatory domain (CD28 or 2021/245580 1»（：1' year2021/054846
⑶ 137/4-1BB）과 ⑶ ¾를 결합한 2세대 CAR가 제조되었는데， 1세대 CAR와 비교하여 체내에 잔존하는 CAR 포함 면역세포의 수가 현저히 증가하였다. 2세대 CAR는 한 가지의 공자극 도메인을 이용한 것에 반해， 3세대 CAR에서는 두 가지 이상의 공자극 도메인을 이용하였다. 생체 내 CAR를 포함하는 면역세포의 확장 및 지속성 달성을 위해 공자극 도메인을 4-1BB, CD28 또는 0X40 등과 결합시킬 수 있다. 2세대 CAR는 미국등록특허 제 7 ,741 ,465호， 제 7 ,446 , 190호 또는 제 9 ,212 ,229호에 구체적으로 기재되어 있고， 3세대 CAR는 미국등록특허 제 8 ,822 ,647호에 구체적으로 기재되어 있으며， 본 발명에 참조로서 도입된다. A second-generation CAR combining ⑶ 137/4-1BB) and ⑶ ¾ was prepared. Compared with the first-generation CAR, the number of CAR-containing immune cells remaining in the body significantly increased. The second-generation CAR used one co-stimulatory domain, whereas the third-generation CAR used two or more co-stimulatory domains. In order to achieve expansion and persistence of immune cells including CAR in vivo, a costimulatory domain can be combined with 4-1BB, CD28 or 0X40, and the like. The second-generation CAR is specifically described in U.S. Patent Nos. 7,741,465, 7,446, 190 or 9,212,229, and the third-generation CAR is described in U.S. Patent No. 8,822,647 No., which is incorporated herein by reference.
4세대 CAR에서는 IL-12 또는 IL-15와 같은 사이토카인을 암호화하는 추가 유전자를 포함하여， 사이토카인의 CAR 기반 면역단백질이 추가로 발현될 수 있도록 하고， 5세대 CAR는 면역세포 강화를 위해 인터루킨 리셉터 체인， 예를 들어， IL-2Rp를 추가로 포함한다. 4세대 CAR는 미국등록특허 제 10 ,316, 102호，In the 4th generation CAR, including an additional gene encoding a cytokine such as IL-12 or IL-15, the CAR-based immune protein of the cytokine can be further expressed, and the 5th generation CAR is an interleukin for strengthening immune cells It further comprises a receptor chain, for example, IL-2Rp. 4th generation CAR is US Patent No. 10,316, 102,
5세대 CAR는 미국등록특허 제 10 ,336 ,810호에 구체적으로 기재되어 있으며， 본 발명에 참조로서 도입된다. 본 발명의 용어 "세포외 결합 도메인（extracel lular binding domain）"이 란， 목적하는 표적 항원에 특이적으로 결합하는 능력을 갖는 CAR의 부분을 의미하는 것이다（항원 결합 도메인 ; ant igen binding domain） . 세포외 결합 도메인은， 생물학적 분자（예 : 세포 표면 수용체 또는 종양 단백질， 지질， 다당류， 또는 기타 세포 표면 표적 분자， 또는 이의 성분）를 특이적으로 인식하고 이에 결합하는 능력을 보유하는 임의의 단백질， 폴리펩티드， 올리고펩티드 또는
2021/245580 1»（：1'해2021/054846 펩티드를 포함할 수 있다. 결합 도메인은 목적하는 생물학적 분자를 위한 임의의 천연 발생， 합성， 반합성 또는 재조합에 의해 생성된 결합 파트너를 포함한다. 본 발명의 용어 "특이적으로 결합하는"이란， 배경 결합보다 큰 결합 친화성으로 또 다른 분자에 대한 분자의 결합을 의미하는 것이다. 예를 들면， 세포외 결합 도메인은 약 10_5M 이상의 친화성 또는 Ka (즉， 1/M의 단위를 갖는 특정 결합 상호작용의 평형 해리 상수)로 표적 분자에 결합하거나 이와 연합하는 경우， 표적 분자에 대해 특이적으로 결합한다. 또는， 친화성은 의 단위를 갖는 특정 결합 상호작용의 평형 해리 상수 (Kd) (예 : 10 5M 내지 10, 또는 그 이하)로 정의될 수 있다. 본 발명에 따른 세포외 결합 도메인 및 CAR의 친화성은 통상의 기술， 예를 들면， 경쟁 ELISA (효소-결합된 면역흡착 검정 ) 또는 표지된 리간드를 사용하거나 바이아코어 (Biacore) T100 (이는 뉴저지주 피스카타웨이 바이아코어 인코포레이티드로부터 이용가능함) 등의 표면-플라스몬 공명 장치 또는 각각 코닝 (Corning) 및 퍼킨 엘머 (Perkin Elmer)로부터 이용가능한 EPIC 시스템 또는 EnSpire 등의 광학 바이오센서 기술을 사용하는 결합 회합 또는 치환 분석에 의해 용이하게 측정할 수 있다. 본 발명에 있어서， 상기 PA63 리간드 도메인 4는 ARTXRKanthrax toxin receptor 1) 또는 ARTXR2(anthrax toxin receptor 2)를 인지하는 것을 특징으로 할 수 있다. 본 발명에 있어서， 상기 PA63 리간드 도메인 4는 서열번호 1의 아미노산
2021/245580 1»（：1'해2021/054846 서열로 표시되는 것을 특징으로 할 수 있다. 본 발명에 있어서， 상기 PA63 리간드 도메인 4의 변이체는 PA63 리간드 도메인 4의 target에 대한 aff inity를 조절한 것을 특징으로 할 수 있다. 바람직하게는， 정상 세포가 아닌 암 세포에서만 발현된 ANTXR을 인식하도록 aff inity가 조절된 변이체인 것을 특징으로 할 수 있으나， 이에 제한되는 것은 아니다. 본 발명에 있어서， 상기 PA63 리간드 도메인 4의 변이체는 서열번호 1의 아미노산 서열로 표시되는 PA63 리간드 도메인 4에서 일부 아미노산이 변이된 것으로， 바람직하게는， 서열번호 2 내지 서열번호 7로 구성된 군에서 선택되는 하나 이상의 아미노산 서열을 포함하는 것을 특징으로 할 수 있으나， 이에 제한되는 것은 아니다. 본 발명의 용어 "PA63"이란， 탄저균 독소 (anthrax toxin) 단백질 중 하나인 protective ant i gen (PA, 83kDa)의 절단된 63kDa의 부분을 의미하는 것이다. 탄저균 독소는 그람 양성균인 Bacillus anthracis6 의해 분비된다. 일반적으로 탄저균의 병원성은 독소 생성 능력과 협막 형성에 의해 결정되는 것으로 알려져 있다. 이 중 탄저의 독소 생성 능력에 영향을 주는 단백질은 방어항원 (protective antigen, PA) , 부종요소 (edema factor , EF) 및 치사요소 ( lethal factor , LF)이다. 이들 각각은 독성을 나타내지 못하나， 방어항원과 부종요소가 결합하여 부종독소 (EdTx, edema toxin)를 형성하고， 방어항원과 치사요소가 결합하여 치사독소 (LeTx, ethal toxin)를 형성한다. The 5th generation CAR is specifically described in US Patent No. 10,336,810, and is incorporated herein by reference. The term "extracellular binding domain" of the present invention means a portion of the CAR having the ability to specifically bind to a target antigen of interest (antigen binding domain; antiigen binding domain) . The extracellular binding domain is any protein that has the ability to specifically recognize and bind to a biological molecule (eg, a cell surface receptor or tumor protein, lipid, polysaccharide, or other cell surface target molecule, or a component thereof); Polypeptide, oligopeptide or 2021/245580 1» (: 1' year 2021/054846 peptide. A binding domain includes any naturally occurring, synthetic, semisynthetic or recombinantly produced binding partner for a biological molecule of interest. As used herein, the term "specifically binds" refers to the binding of a molecule to another molecule with a greater binding affinity than background binding. For example, the extracellular binding domain binds to or associates with the target molecule with an affinity of about 10 _ 5 M or more or Ka (ie, the equilibrium dissociation constant of a specific binding interaction having a unit of 1/M). binds specifically to Alternatively, affinity may be defined as the equilibrium dissociation constant (Kd) of a specific binding interaction with units of (eg, 10 5 M to 10, or less). The affinity of the extracellular binding domain and the CAR according to the present invention can be determined using conventional techniques, for example, competition ELISA (enzyme-linked immunosorbent assay) or labeled ligands, or using Biacore T100 (which is a piece of New Jersey). Combination using a surface-plasmon resonance device such as Cataway Biacore, Inc.) or optical biosensor technology such as EnSpire or the EPIC system available from Corning and Perkin Elmer, respectively. It can be readily determined by association or substitution analysis. In the present invention, the PA63 ligand domain 4 may be characterized in that it recognizes ARTXRKanthrax toxin receptor 1) or ARTXR2 (anthrax toxin receptor 2). In the present invention, the PA63 ligand domain 4 is the amino acid of SEQ ID NO: 1 2021/245580 1» (: 1' year 2021/054846 It can be characterized as represented by the sequence. In the present invention, the variant of the PA63 ligand domain 4 may be characterized in that the affinity of the PA63 ligand domain 4 to the target is adjusted. Preferably, it may be characterized in that the affinity is a mutant regulated to recognize ANTXR expressed only in cancer cells, not in normal cells, but is not limited thereto. In the present invention, the variant of the PA63 ligand domain 4 is one in which some amino acids are mutated in the PA63 ligand domain 4 represented by the amino acid sequence of SEQ ID NO: 1, preferably selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 7 It may be characterized as comprising one or more amino acid sequences, but is not limited thereto. As used herein, the term "PA63" refers to a truncated 63kDa portion of protective anti-gen (PA, 83kDa), which is one of anthrax toxin proteins. Anthrax toxin is secreted by Bacillus anthracis 6 , a gram-positive bacterium. In general, it is known that the pathogenicity of anthrax is determined by its toxin-producing ability and capsular formation. Among these, the proteins that affect the toxin production ability of anthrax are protective antigen (PA), edema factor (EF), and lethal factor (LF). Each of these does not show toxicity, but the protective antigen and the edema factor combine to form an edema toxin (EdTx, edema toxin), and the protective antigen and the lethal factor combine to form a lethal toxin (LeTx, ethal toxin).
PA는 대식세포와 같은 감염 세포주 표면의 수용체 단백질 (Anthrax toxin
2021/245580 1^(:1^ 2021/054846 receptor)에 결합한 꾸 단백질 분해효소(furin family protease)에 의해PA is a receptor protein (Anthrax toxin) on the surface of infected cell lines such as macrophages. By furin family protease bound to 2021/245580 1^ (:1^ 2021/054846 receptor)
PA20(20kDa)과 PA63(63kDa)으로 잘리는데,이 중 PA63이 ¾타머화되면서 LF혹은 EF와 결합하여 이를 세포 내로 운반시키는 독소 단백질 운반체(Toxin delivery channel)의 역할을 수행하는 것으로 알려져 있다.이들 복합체는 엔도시토시스 기전으로 세포 내로 들어가고 엔도좀의 pH가 낮아지면 PA63 ¾타머에 구조적 변화가 생기며 엔도좀 멤브레인에 기공을 형성하고,결과적으로 LF혹은 EF는 세포질로 유리되어 독성 효과를 나타내게 된다(Jiang J. Atomic structure of anthrax protective antigen pore elucidates toxin translocation. Nature. 2015). 본 발명의 용어 "ANTXRl(anthrax toxin receptor 1)"이란,세포외 기질 단백질을 의미하는 것으로,다른 명칭은 TEM8(Tumor endothelialmarker 8)이다. 그의 구체적인 핵산서열은 NCBI에 공지되어 있다(NCBI Reference Sequence: NM-032208.2). 본 발명의 용어 "ANTXR2(anthrax toxin receptor 2)"란,혈관신생에 관여하는 단백질을 의미하는 것으로,다른 명칭은 CMG2(capi1lary morphogenesis gene 2)이다.그의 구체적인 핵산서열은 NCBI에 공지되어 있다(NCBI Reference Sequence:NM_058172.5). 본 발명에 있어서, 상기 키메라 항원 수용체는 막횡단 도메인(transmembrane domain)을 포함하는 것을 특징으로 할 수 있다. 본 발명의 용어 "막횡단 도메인(transmembrane domain)"이란,세포외 결합
2021/245580 1»（：1'해2021/054846 분 및 세포내 신호전달 도메인을 융합시키고 면역 작동 세포의 원형질 막에It is cleaved into PA20 (20 kDa) and PA63 (63 kDa), of which PA63 is ¾ tamerized and is known to function as a toxin delivery channel that binds to LF or EF and transports them into cells. The complex enters the cell through the endocytosis mechanism and when the pH of the endosome is lowered, a structural change occurs in the PA63 ¾ tamer and a pore is formed in the endosomal membrane. Jiang J. Atomic structure of anthrax protective antigen pore elucidates toxin translocation. Nature. 2015). As used herein, the term "ANTXRl (anthrax toxin receptor 1)" refers to an extracellular matrix protein, and another name is TEM8 (Tumor endothelial marker 8). Its specific nucleic acid sequence is known from the NCBI (NCBI Reference Sequence: NM-032208.2). As used herein, the term “anthrax toxin receptor 2 (ANTXR2)” refers to a protein involved in angiogenesis, and another name is CMG2 (capi1lary morphogenesis gene 2). A specific nucleic acid sequence thereof is known from NCBI (NCBI). Reference Sequence: NM_058172.5). In the present invention, the chimeric antigen receptor may be characterized in that it comprises a transmembrane domain. As used herein, the term "transmembrane domain" refers to extracellular binding 2021/245580 1»（：1' year2021/054846 min and intracellular signaling domains fused and transferred to the plasma membrane of immune effector cells
◦쇼묘를 고정시키는
일부이다. 막횡단 도메인은 천연， 합성， 반합성 또는 재조합 공급원으로부터 유래할 수 있다. 본 발명에 있어서， 상기 막횡단 도메인은 ^세포 수용체의 알파， 베타 또는 제타 사슬， 0028, 003 엡실론， 0045, 004, 005, 008, 009, 0）16, 0022, 0033 , 0037, 0064, 0080, 0086, 0）134, 0）137 및 ）154로 구성된 군에서 선택되는 것을 특징으로 할 수 있으나， 이에 제한되는 것은 아니다.
부착될 수 있다. 예를 들어， 링커는 2 내지 10개 아미노산 길이의 짧은 올리고_ 또는 폴리펩티드 링커일 수 있으며， 바람직하게는 글라이신 ½） -세린比） 이중체（（101止1아）일 수 있으나， 이에 제한되는 것은 아니다. ◦ Fixing the shomyo is a part The transmembrane domain may be derived from a natural, synthetic, semi-synthetic or recombinant source. In the present invention, the transmembrane domain is ^ alpha, beta or zeta chain of the cell receptor, 0028, 003 epsilon, 0045, 004, 005, 008, 009, 0) 16, 0022, 0033, 0037, 0064, 0080, It may be characterized in that it is selected from the group consisting of 0086, 0)134, 0)137 and )154, but is not limited thereto. can be attached. For example, the linker may be a short oligo_ or polypeptide linker of 2 to 10 amino acids in length, preferably a glycine ½) -serine) duplex ((101止1a), but is limited thereto no.
◦쇼묘의 결합 도메인은 일반적으로 하나 이상의 "힌지 도메인（ 1语6 （1011 11） "이 후속한다. 본 발명의 용어 "힌지 도메인"이란， 적절한 세포/세포 접촉， 항원 결합 및 활성화를 가능하게 하도록 작동 세포 표면으로부터 떨어져 항원 결합 도메인의 위치결정에 중요한 역할을 담당하는
일부이다.
일반적으로 세포외 결합 도메인과 막횡단 도메인 사이에 하나 이상의 힌지 도메인을 포함한다. 힌지 도메인은 천연， 합성， 반-합성 또는 재조합 공급원으로부터 유래될 수 있다. 힌지 도메인은 천연 발생 면역글로불린 힌지 영 역 또는 변화된 면역글로불린 힌지 영 역의 아미노산 서열을 포함할 수 있다.◦The binding domain of a shomyo is generally followed by one or more "hinge domains（ 1语6 (1011 11) "). As used herein, the term "hinge domain" refers to an antigen-binding domain that plays an important role in positioning the antigen-binding domain away from the effector cell surface to enable proper cell/cell contact, antigen binding and activation. is a part It generally includes one or more hinge domains between the extracellular binding domain and the transmembrane domain. The hinge domain may be derived from natural, synthetic, semi-synthetic or recombinant sources. The hinge domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.
"변화된 힌지 영 역"은 ） 최대 30%의 아미노산 변화（예 : 최대 25%, 20%, 15%,A "changed hinge region" is ) an amino acid change of up to 30% (e.g. up to 25%, 20%, 15%,
10% 또는 5%의 아미노산 치환 또는 결실）을 갖는 천연 발생 힌지 영 역， （ 최대
2021/245580 1»（：1'해2021/054846 naturally occurring hinge region with 10% or 5% amino acid substitutions or deletions), (maximum 2021/245580 1»（：1' year2021/054846
30% 아미노산 변화（예 : 최대 25%, 20%, 15%, 10% 또는 5%의 아미노산 치환 또는 결실）을 갖는 적어도 10개 아미노산（예 : 적어도 12, 13 , 14 또는 15개 아미노산） 길이의 천연 발생 힌지 영 역의 일부， 또는 （c） 코어 힌지 영 역（이는 4, 5, 6, 7, 8， 9， 10, 11， 12， 13， 14 또는 15， 또는 적어도 4， 5， 6， 7, 8， 9， 10, 11， 12 , 13 , 14 또는 15개 아미노산 길이일 수 있다）을 포함하는 천연 발생 힌지 영 역의 일부를 지칭한다. 특정 실시형태에서， 천연 발생 면역글로불린 힌지 영 역에서 하나 이상의 시스테인 잔기는 하나 이상의 다른 아미노산 잔기（예 : 하나 이상의 세린 잔기）로 치환될 수 있다. 변화된 면역글로불린 힌지 영 역은 대안적으로 또는 추가적으로 또 다른 아미노산 잔기（예 : 세린 잔기）로 치환된 야생형 면역글로불린 힌지 영 역의 프롤린 잔기를 가질 수 있다. 힌지 도메인은 CD8a,of at least 10 amino acids (eg at least 12, 13 , 14 or 15 amino acids) with a 30% amino acid change (eg at most 25%, 20%, 15%, 10% or 5% amino acid substitutions or deletions) part of a naturally occurring hinge region, or (c) a core hinge region (which is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in length). In certain embodiments, one or more cysteine residues in the naturally occurring immunoglobulin hinge region may be substituted with one or more other amino acid residues (eg, one or more serine residues). The altered immunoglobulin hinge region may alternatively or additionally have a proline residue of the wild-type immunoglobulin hinge region substituted with another amino acid residue (eg, a serine residue). The hinge domain is CD8a,
⑶ 4, CD28 및 CD7 등의 유형 1 막단백질의 세포외 영 역으로부터 유래하는 힌지 영 역을 포함하고， 이는 이들 분자로부터의 야생형 힌지 영 역일 수 있거나 변화될 수 있다. 본 발명에 있어서， 상기 힌지 도메인을 포함하는 막횡단 도메인은 상기 세포외 도메인과 세포내 도메인을 세포막 사이로 연결 가능하다면 어떠한 것이든 사용 가능하다. 바람직하게는 ⑶ 8a로부터 유래된 힌지 도메인 및 막횡단 도메인으로 이루어지며， 서열번호 8로 표시되는 아미노산 서열을 포함할 수 있지만， 이에 제한되는 것은 아니다. 본 발명에 있어서， 상기 키메라 항원 수용체는 세포내 신호전달 도메인（intracel lular signal ing domain）을 포함하는 것을 특징으로 할 수 있다.
2021/245580 1»（：1'해2021/054846 본 발명의 용어 "세포내 신호전달 도메인 ( intracel lular signal ing domain)"이란， 작동 세포 기능， 예를 들면， CAR-결합된 표적 세포에 대한 세포독성 인자의 방출을 포함하는 활성화， 사이토카인 생성， 증식 및 세포독성 활성， 또는 CAR의 세포외 결합 도메인에 대한 항원 결합으로 유발된 기타 세포 반응을 유발하기 위해 면역 작동 세포의 내부로 표적 항원에 대한 효과적 CAR 결합의 메시지의 전달에 관여하는 CAR의 부분을 의미하는 것이다. 작동 기능은 세포의 특수한 기능을 지칭하며， 예를 들면， T 세포의 작동 기능은 세포용해 활성일 수 있거나， 사이토카인의 분비를 포함하는 활성을 지원할 수 있거나 활성일 수 있다. 따라서， 세포내 신호전달 도메인은 작동 기능 신호를 전달하고 세포가 특수한 기능을 수행하도록 지시하는 단백질의 일부를 의미한다. ⑶ 4, including a hinge region derived from the extracellular region of type 1 membrane proteins such as CD28 and CD7, which may be a wild-type hinge region from these molecules or may be changed. In the present invention, any transmembrane domain including the hinge domain can be used as long as it can connect the extracellular domain and the intracellular domain between the cell membrane. Preferably, it consists of a hinge domain and a transmembrane domain derived from ⑶ 8a, and may include the amino acid sequence represented by SEQ ID NO: 8, but is not limited thereto. In the present invention, the chimeric antigen receptor may be characterized in that it comprises an intracellular signaling domain (intracellular signaling domain). 2021/245580 1» (: 1' year 2021/054846 The term "intracellular signaling domain" of the present invention is an effector cell function, for example, a CAR-bound target cell-to-cell Activation, including the release of toxic factors, cytokine production, proliferation and cytotoxic activity, or other cellular responses induced by antigen binding to the extracellular binding domain of the CAR into the interior of immune effector cells for the target antigen It refers to the portion of the CAR that is involved in the transmission of the message of effective CAR binding. The agonistic function refers to a specific function of a cell, for example, the agonistic function of a T cell may be cytolytic activity, may support an activity including secretion of cytokines, or may be active. Accordingly, the intracellular signaling domain refers to a portion of a protein that transmits an operative function signal and directs the cell to perform a specific function.
T 세포 수용체 단독으로 생성된 신호는 T 세포를 충분하게 활성화할 수 없다고 알려졌기에 공자극 신호가 추가로 요구되는 것으로 공지되어 있다. 따라서， T 세포 활성화는 세포내 신호전달 도메인의 2개 상이한 부류에 의해 매개된다. 예를 들면， T 세포 수용체를 통한 항원-의존성 일차 활성화를 개시하는 1차 신호전달 도메인 및 2차 신호를 제공하기 위해 항원-독립적 방식으로 작용하는 공자극 신호전달 도메인에 의해 T 세포 활성화가 매개된다. 따라서， 상기 세포내 신호전달 도메인은 "1차 신호전달 도메인 (primary signal ing domain)" 및 "공자극 신호전달 도메인 (co-st imulatory signal ing domain)"을 포함하는 것을 특징으로 할 수 있다. 본 발명의 용어 "1차 신호전달 도메인 (primary signal ing domain)"이란， T
2021/245580 1»（：1'해2021/054846 세포 수용체 복합체의 1차 활성화를 자극 또는 억제 방식으로 조절하는 신호전달 도메인을 의미하는 것이다. 자극 방식으로 작용하는 1차 신호전달 도메인은 면역수용체 티로신-기반 활성화 모티브 또는 ITM으로 공지되어 있는 신호전달 모티브를 함유할 수 있다. 1차 신호전달 도메인을 함유하는 ITAM은 TCRC FcRy,It is known that a costimulatory signal is additionally required since it is known that a signal generated by T cell receptor alone cannot sufficiently activate T cells. Thus, T cell activation is mediated by two different classes of intracellular signaling domains. For example, T cell activation is mediated by a primary signaling domain that initiates antigen-dependent primary activation through a T cell receptor and a costimulatory signaling domain that acts in an antigen-independent manner to provide a secondary signal. . Accordingly, the intracellular signaling domain may be characterized as including a "primary signaling domain" and a "co-stimulatory signaling domain". The term "primary signaling domain" of the present invention, T 2021/245580 1» (: 1' year 2021/054846 refers to a signaling domain that regulates the primary activation of the cell receptor complex in a stimulating or inhibitory manner. A primary signaling domain that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITM. The ITAM containing the primary signaling domain is TCRC FcRy,
FcRp, CD3y, CD36, CD3e, CD¾, ⑶ 5, CD22, ⑶ 79a, CD79b및 CD66d로 구성된 군에서 선택될 수 있으나， 이에 제한되는 것은 아니다. 본 발명에 있어서， 상기 1차 신호전달 도메인은 바람직하게는， 서열번호It may be selected from the group consisting of FcRp, CD3y, CD36, CD3e, CD¾, ⑶ 5, CD22, ⑶ 79a, CD79b and CD66d, but is not limited thereto. In the present invention, the primary signaling domain is preferably SEQ ID NO:
9로 표시되는 아미노산 서열을 포함하는 ⑶ ¾(zeta)인 것을 특징으로 할 수 있으나， 이에 제한되는 것은 아니다. 본 발명의 용어 "공자극 신호전달 도메인 (co-stimulatory signaling domain)"이란， 공자극 분자의 세포내 신호전달 도메인을 의미한다. 공자극 분자는 항원 수용체 또는 항원에 결합시 T림프구의 효율적 활성화 및 기능에 요구되는 2차 신호를 제공하는 Fc수용체 이외의 세포 표면 분자이다. 본 발명에 있어서， 상기 공자극 신호전달 도메인은 0X40， ⑶ 2， CD27, CD28, ⑶S, ICAM-1 , LFA-l(CDlla/CD18) , IC0S(CD278) , CD7, CD30, CD40, PD-1 , LIGHT, NKG2C, B7-H3 , ⑶ 83 및 4-1BB (⑶ 137)로 구성된 군에서 선택되는 것을 특징으로 할 수 있으나， 이에 제한되는 것은 아니다. 본 발명에 있어서， 상기 공자극 신호전달 도메인은 바람직하게는， 서열번호 10으로 표시되는 아미노산 서열을 포함하는 CD28 및 서열번호 11로 표시되는 아미노산 서열을 포함하는 )137인 것을 특징으로 할 수 있으나， 이에
2021/245580 1»（：1'해2021/054846 제한되는 것은 아니다. 본 발명에 따른 키메라 항원 수용체는 2개 이상의 세포내 신호전달 도메인을 포함하는 것을 특징으로 할 수 있으며， 2개 이상의 세포내 신호전달 도메인을 포함하는 경우에는 세포내 신호전달 도메인들이 서로 직렬로 연결될 수 있다. 또는 2~10 아미노산으로 이루어지는 올리고 펩티드 링커 또는 폴리펩티드 링커를 통해 연결되어 있을 수도 있고， 이러한 링커 서열의 예로서 글리신-세린 연속 서열을 들 수 있다. 본 발명에 따른 CAR 설계시 PA63 리간드 도메인 4 변이체 앞에 신호 펩티드 (signal pept ide)를 코딩하는 핵산을 삽입하는 것을 특징으로 할 수 있다. 따라서， 상기 키메라 항원 수용체는 신호 펩티드를 더 포함하는 것을 특징으로 할 수 있다. 상기 signal pept ide로는 GM-CSF, CD8 alpha signal pept ide 등이 사용되나， 대부분의 CAR 설계에 GM-CSF를 사용하고 있으며， 본 발명의 실시 예에서는 GM-CSF를 signal pept ide로 사용하였다. 본 발명은 다른 관점에서， 상기 키메라 항원 수용체를 코딩하는 핵산에 관한 것이다. 본 발명에 따른 키메라 항원 수용체를 코딩하는 핵산 (폴리뉴클레오타이드)은 코돈 최적화에 의해 변형될 수 있으며， 이는 코돈의 축퇴성 (degeneracy)에서 기인한 것으로， 폴리펩티드 또는 이의 변이체 단편을 코딩하는 많은 뉴클레오타이드 서열이 존재한다는 것은 통상의 기술자가 잘
2021/245580 1»（：1'해2021/054846 이해할 수 있을 것이다. 이들 폴리뉴클레오타이드（핵산）의 일부는 임의의 자연 발생형 유전자의 뉴클레오타이드 서열과 최소 상동성을 보유한다 . 특히 코돈 활용법의 차이로 인해 가변적인 폴리뉴클레오타이드（핵산）， 예를 들어 인간， 영장류 및/또는 포유동물의 코돈 선택에 최적화된 폴리뉴클레오타이드（핵산）가 바람직하다. 본 발명의 실시예에서는 쇼63 도메인 4여4）의 변이체（■^3삵）를 코딩하는 핵산이 도입된
구축하였으며 ， 상기 11와£1111；를 코딩하는 핵산은 바람직하게는 서열번호 14 내지 서열번호 19로 구성된 군에서 선택되는 뉴클레오타이드 서열을 포함하는 것을 특징으로 할 수 있으나， 이에 제한되는 것은 아니다. 본 발명은 또 다른 관점에서， 상기 핵산을 포함하는 발현 벡터 및 상기 발현 벡터를 포함하는 바이러스에 관한 것이다. 본 발명의 용어 "벡터"란， 다른 핵산 분자를 전이시키거나 수송할 수 있는 핵산 분자를 의미하는 것이다. 전이된 핵산은 일반적으로 벡터 핵산 분자에 연결되는데， 예를 들면， 벡터 핵산 분자 내에 삽입된다. 벡터는 세포에서의 자율적 복제를 지시하는 서열을 포함할 수 있거나， 숙주 세포 내로의 통합을 가능하게 하는데 충분한 서열을 포함할 수 있다. 상기 벡터는
플라스미드， 렌티바이러스 벡터， 아데노바이러스 벡터 및 레트로바이러스 벡터로 구성된 군에서 선택되는 것을 특징으로 할 수 있으나， 이에 제한되는 것은 아니다.
2021/245580 1»（：1'해2021/054846 본 발명에 있어서， "바이러스"는 암의 치료 등에 사용하기 위해， 본 발명의 키메라 항원 수용체를 발현하도록 유전적으로 변형된 것을 의미한다. 유전적으로 변형되었다는 것은 세포에서 전체 유전자 재료로 DNA 또는 RNA의 형태로 외부 유전자 재료를 부가하는 것이다. 본 발명에 있어서， 상기 핵산 또는 상기 벡터는 바이러스에 형질주입 또는 트랜스펙션 (transfection)된다. "형질주입" 또는 "트랜스펙션 "시키기 위해 원핵 또는 진핵 숙주세포 내로외인성 핵산 (DNA또는 RNA)을 도입하는 데에 통상 사용되는 여러 종류의 다양한 기술， 예를 들어 전기 영동법， 인산칼슘 침전법， DEAE-텍스트란 트랜스펙션 또는 리포펙션 (lipofection) 등을사용할수 있다. 본 발명은 또 다른 관점에서， 상기 키메라 항원 수용체를 표면에 발현하는 면역세포에 관한 것이다. 본 발명에 있어서， 상기 면역세포는 면역을 유도하여 목적하는 암 치료 효과를 유발할 수 있는 것으로， 예를 들어， T 세포， NK 세포， NKT 세포， 사이토카인 유도 살해세포 (Cytokine Induced Killer cell, CIK)， 마크로파지 및 수지상세포로 구성된 군에서 선택될 수 있으나， 이에 제한되는 것은 아니다. 바람직하게는 T세포인 것을 특징으로 할수 있다. 따라서， 본 발명에 따른 키메라 항원 수용체를 발현하는 면역세포는 CAR- T 세포 (Chimeric Antigen Receptor T Cell), CAR-NK 세포 (Chimeric Antigen Receptor Natural Killer Cell) 또는 CAR-NKT 세포 (Chimeric Antigen ReceptorIt may be characterized as ⑶ ¾ (zeta) including the amino acid sequence represented by 9, but is not limited thereto. As used herein, the term "co-stimulatory signaling domain" refers to an intracellular signaling domain of a co-stimulatory molecule. A costimulatory molecule is an antigen receptor or a cell surface molecule other than an Fc receptor that upon binding to an antigen provides a secondary signal required for efficient activation and function of T lymphocytes. In the present invention, the costimulatory signaling domain is 0X40, ⑶ 2, CD27, CD28, ⑶S, ICAM-1, LFA-1 (CDlla/CD18), ICOS (CD278) , CD7, CD30, CD40, PD-1 , LIGHT, NKG2C, B7-H3 , ⑶ 83 and 4-1BB (⑶ 137) may be selected from the group consisting of, but is not limited thereto. In the present invention, the costimulatory signaling domain is preferably CD28 comprising the amino acid sequence shown in SEQ ID NO: 10 and ) 137 comprising the amino acid sequence shown in SEQ ID NO: 11, Therefore 2021/245580 1»（：1' year2021/054846 is not limited. The chimeric antigen receptor according to the present invention may be characterized in that it includes two or more intracellular signaling domains, and when it includes two or more intracellular signaling domains, the intracellular signaling domains may be connected in series with each other have. Alternatively, it may be linked through an oligopeptide linker or a polypeptide linker consisting of 2 to 10 amino acids, and examples of such a linker sequence include a glycine-serine continuous sequence. When designing the CAR according to the present invention, it may be characterized in that a nucleic acid encoding a signal peptide is inserted before the PA63 ligand domain 4 variant. Accordingly, the chimeric antigen receptor may be characterized in that it further comprises a signal peptide. GM-CSF, CD8 alpha signal pept ide, etc. are used as the signal pept ide, but GM-CSF is used for most CAR designs, and GM-CSF was used as the signal pept ide in the embodiment of the present invention. In another aspect, the present invention relates to a nucleic acid encoding the chimeric antigen receptor. The nucleic acid (polynucleotide) encoding the chimeric antigen receptor according to the present invention may be modified by codon optimization, which is due to codon degeneracy, and many nucleotide sequences encoding polypeptides or variant fragments thereof Existence is well known to those of ordinary skill in the art. 2021/245580 1»（：1'year2021/054846 You will understand. Some of these polynucleotides (nucleic acids) have minimal homology with the nucleotide sequence of any naturally occurring gene. In particular, a polynucleotide (nucleic acid) that is variable due to the difference in codon utilization, for example, a polynucleotide (nucleic acid) optimized for codon selection in humans, primates and/or mammals is preferable. In an embodiment of the present invention, a nucleic acid encoding a mutant (■^3 piece) of Sho63 domain 4 or 4) is introduced. The nucleic acid encoding the 11 and 1111; may preferably include a nucleotide sequence selected from the group consisting of SEQ ID NO: 14 to SEQ ID NO: 19, but is not limited thereto. In another aspect, the present invention relates to an expression vector comprising the nucleic acid and a virus comprising the expression vector. As used herein, the term "vector" refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to a vector nucleic acid molecule, for example, inserted into a vector nucleic acid molecule. A vector may include sequences that direct autonomous replication in a cell, or it may include sequences sufficient to permit integration into a host cell. the vector is It may be characterized in that it is selected from the group consisting of a plasmid, a lentiviral vector, an adenoviral vector and a retroviral vector, but is not limited thereto. 2021/245580 1» (: 1' year 2021/054846 In the present invention, "virus" means genetically modified to express the chimeric antigen receptor of the present invention for use in the treatment of cancer. To be genetically modified is the addition of foreign genetic material in the form of DNA or RNA into the entire genetic material in a cell. In the present invention, the nucleic acid or the vector is transfected or transfected with a virus. A variety of techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells for "transfection" or "transfection", for example, electrophoresis, calcium phosphate precipitation, DEAE-text can be used for transfection or lipofection. In another aspect, the present invention relates to an immune cell expressing the chimeric antigen receptor on the surface. In the present invention, the immune cells are capable of inducing the desired cancer treatment effect by inducing immunity, for example, T cells, NK cells, NKT cells, cytokine-induced killer cells (Cytokine Induced Killer cell, CIK) , It may be selected from the group consisting of macrophages and dendritic cells, but is not limited thereto. Preferably, it may be characterized as a T cell. Therefore, the immune cells expressing the chimeric antigen receptor according to the present invention are CAR-T cells (Chimeric Antigen Receptor T Cells), CAR-NK cells (Chimeric Antigen Receptor Natural Killer Cells) or CAR-NKT cells (Chimeric Antigen Receptor cells).
¾1；11데 11 I 0611) 등일 수 있다.
2021/245580 1»（：1'해2021/054846 본 발명에 있어서， 상기 T 세포는 세포독성 T 림프구 (Cytotoxic T lymphocyte； CTL) ; 종양 침윤 림프구 (Tumor inf i ltrat ing lymphocyte； TIL) 및 말초혈액 단핵세포 (Per ipheral blood mononuclear cel l ; PBMC)에서 분리한 T 세포로 구성된 군에서 선택되는 것을 특징으로 할 수 있다. 본 발명은 또 다른 관점에서， 상기 키메라 항원 수용체를 발현하는 면역세포 (예컨대， T 세포)를 포함하는 암 예방 또는 치료용 조성물에 관한 것이다. 본 발명에 있어서， "암"과 "종양"은 동일한 의미로 사용되며， 전형적으로 조절되지 않은 세포 성장/증식을 특징으로 하는 포유동물의 생리학적 상태를 지칭하거나 의미한다. 본 발명의 용어， "예방"은 본 발명의 조성물의 투여로 암을 억제시키거나 진행을 지연시키는 모든 행위를 의미하며， "치료"는 암의 발전의 억제， 증상의 경감 또는 제거를 의미한다. 상기 PA63의 세포독성 작용 기전에 따라 본 발명에 따른 PA63 리간드 도메인 4의 변이체는 ANTXR(anthrax toxin receptor)을 발현하는 암 세포 또는 암 조직에 특이적으로 결합할 수 있으며， 본 발명에 따른 PA63 리간드 도메인 4의 변이체를 포함하는 CAR 함유 세포는 ANTXR을 발현하는 암에 대하여 세포독성 효과가 있음은 통상의 기술자에게 자명하다. 상기 ANTXR은 ANTXR1 또는 ANTXR2를 포함한다. 본 발명의 조성물로 치료할 수 있는 암 또는 암종은 특별히 제한되지
2021/245580 1»（：1'해2021/054846 않으며， 고형암 및 혈액암을 모두 포함한다 . 상기 암은 원발성 암 또는 전이성 암일 수 있다. 본 발명에 있어서， 고형암이란， 혈관이나 결합성 조직으로 이루어져 일정한 경도와 형태를 지니는 종양을 의미하는 것으로， 이에 제한되지 않으나， 예를 들어， 췌장암， 위암， 대장암， 폐암， 유방암， 배세포암， 간암， 피부암， 방광암， 전립선암， 자궁암， 자궁경암， 난소암 등이 있다. 상기 혈액암에는 급성골수성백혈병 또는 다발성골수종 등을 예로 들 수 있으나， 이에 제한되는 것은 아니다. 본 발명의 암 예방 또는 치료용 조성물은 약제학적으로 허용되는 담체를 추가로 포함할 수 있다. 본 발명에서 용어， "약학적으로 허용 가능한 담체"란 생물체를 자극하지 않고 투여 화합물의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 말한다. 액상 용액으로 제제화되는 조성물에 있어서 약학적으로 허용가능한 담체로는， 멸균 및 생체에 적합한 것으로서， 식염수， 멸균수， 완충식염수， 알부민 주사용액， 텍스트로즈 용액， 말토텍스트린 용액， 글리세롤 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며， 필요에 따라 항산화제， 완충액， 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제， 분산제， 계면활성제， 결합제 및 윤활제를 부가적으로 첨가하여 수용액， 현탁액， 유탁액 등과 같은 주사용 제형， 환약， 캡슐， 과립 또는 정제로 제제화할 수 있다. 본 발명의 상기 조성물은 경구 또는 비경구의 여러 가지 제형일 수 있다. 제제화할 경우에는 보통 사용하는 중진제， 증량제， 결합제， 습윤제， 붕해제，
2021/245580 1»（：1'해2021/054846 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다 . 경구투여를 위한 고형제제에는 정제， 환제， 산제， 과립제， 캡슐제 등이 포함되며， 이 러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면， 전분， 탄산칼슘， 수크로오스 (sucrose) 또는 락토오스 (lactose)， 젤라틴 등을 섞어 조제된다 . 또한 단순한 부형제 이외에 스테아린산 마그네슘， 탈크 등과 같은 윤활제들도 사용된다 . 경구투여를 위한 액상제제로는 현탁제， 내용액제， 유제， 시 럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물， 리퀴드 파라핀 이외에 여 러 가지 부형제， 예를 들면 습윤제， 감미제， 방향제， 보존제 등이 포함될 수 있다 . 비경구투여를 위한 제제에는 멸균된 수용액， 비수성용제， 현탁제， 유제， 동결건조제제， 좌제가 포함된다 . 비수성용제 ， 현탁용제로는 프로필렌글리콜(propylene glycol ) , 폴리에틸렌 글리콜， 올리브 오일과 같은 식물성 기름， 에틸올레이트와 같은 주사 가능한 에스테로 등이 사용될 수 있다 . 좌제의 기제로는 위 텝솔 (witepsol )， 마크로골， 트원 (tween) 61， 카카오지， 라우린지， 글리세로젤라틴 등이 사용될 수 있다 . 이 러한 약학적으로 허용가능한 담체 및 제제는 Remington's¾1; 11 de 11 I 0611) and the like. 2021/245580 1» (: 1' year 2021/054846 In the present invention, the T cells are cytotoxic T lymphocytes; CTL); It may be characterized in that it is selected from the group consisting of T cells isolated from tumor infiltrating lymphocytes (TIL) and peripheral blood mononuclear cells (PBMC). In another aspect, the present invention relates to a composition for preventing or treating cancer comprising immune cells (eg, T cells) expressing the chimeric antigen receptor. In the present invention, "cancer" and "tumor" are used interchangeably, and typically refer to or mean a physiological condition of mammals characterized by unregulated cell growth/proliferation. As used herein, the term "prevention" refers to any action that inhibits or delays the progression of cancer by administering the composition of the present invention, and "treatment" refers to inhibition of cancer development, alleviation or elimination of symptoms. According to the cytotoxic action mechanism of PA63, the variant of PA63 ligand domain 4 according to the present invention can specifically bind to cancer cells or cancer tissues expressing ANTXR (anthrax toxin receptor), and PA63 ligand domain according to the present invention It is apparent to those skilled in the art that CAR-containing cells comprising a mutant of 4 have a cytotoxic effect on cancer expressing ANTXR. The ANTXR includes ANTXR1 or ANTXR2. The cancer or carcinoma that can be treated with the composition of the present invention is not particularly limited. 2021/245580 1» (：1' year 2021/054846, including both solid cancer and blood cancer. The cancer may be a primary cancer or a metastatic cancer. In the present invention, solid cancer means a tumor that is composed of blood vessels or connective tissue and has a certain hardness and shape, but is not limited thereto, for example, pancreatic cancer, stomach cancer, colon cancer, lung cancer, breast cancer, germ cell cancer , liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, etc. Examples of the blood cancer include, but are not limited to, acute myeloid leukemia or multiple myeloma. The composition for preventing or treating cancer of the present invention may further include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound. In the composition formulated as a liquid solution, as a pharmaceutically acceptable carrier, as sterile and biocompatible, saline, sterile water, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol and among these components One or more components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injection formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets. The composition of the present invention may be in various oral or parenteral formulations. In the case of formulation, commonly used thickening agents, extenders, binders, wetting agents, disintegrants, 2021/245580 1» (: 1' year 2021/054846 Prepared using a diluent or excipient such as a surfactant. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose or lactose. It is prepared by mixing (lactose), gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, etc. can be used. Such pharmaceutically acceptable carriers and agents are Remington's
Pharmaceutical Sciences(19th ed. , 1995)에 상세히 기재되어 있다 . 본 발명의 조성물은 경구 또는 비경구로 투여할 수 있고， 비경구 투여 인 경우에는 정맥내 주입， 피하주입， 근육 주입， 복강 주입， 내피 투여， 국소 투여， 비내 투여， 폐내 투여 및 직장 내 투여 등으로 투여할 수 있다 . 경구 투여시， 단백질 또는 펩타이드는 소화가 되기 때문에 경구용 조성물은 활성 약제를 코팅하거나 위에서의 분해로부터 보호되도록 제형화 될 수 있으며， 본 발명의 조성물은 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될
2021/245580 1»（：1'해2021/054846 수 있다 . 본 발명의 암 예방 또는 치료용 조성물의 적합한 투여량은 제제화 방법， 투여 방식， 환자의 연령， 체중， 성， 병적 상태， 음식， 투여 시간， 투여 경로， 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며， 보통으로 숙련된 의사는 소망하는 치료 또는 예방에 효과적 인 투여량을 용이하게 결정 및 처방할 수 있다 . 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다 . 본 발명은 또 다른 관점에서， 상기 면역세포를 투여하는 단계를 포함하는 암 예방 또는 치료방법에 관한 것이다 . 본 발명은 또 다른 관점에서， 암 예방 또는 치료를 위한 상기 면역세포의 용도에 관한 것이다 . 본 발명은 또 다른 관점에서， 암 예방 또는 치료용 약제 제조를 위한 상기 면역세포의 사용에 관한 것이다 . 본 발명의 예방 또는 치료방법， 용도 및 사용은 상술한 "면역세포"를 이용하기 때문에， 중복된 내용은 그 기재를 생략한다 . 이하， 실시 예를 통하여 본 발명을 더욱 상세히 설명하고자 한다 . 이들 실시 예는 오로지 본 발명을 예시하기 위한 것으로서， 본 발명의 범위가 이들 실시 예에 의해 제한되는 것으로 해석되지 않는 것은 당업 계에서 통상의 지식을
2021/245580 1»（：1'해2021/054846 가진 자에 있어서 자명할 것이다. 실시예 1： 암 세포주에서 타겟 항원 단백질 발현 분석 흑색종 세포주 526-mel , 췌장암 세포주 (PANC1 , Mia_PaCa2) , 유방암 세포주 (SK-BR3 , MDA-MB-231 , MCF7, ZR-517)의 ANTXRKTEM8) , ANTXR2(CMG2) 단백질 발현을 western Blot을 이용하여 분석한 결과， 췌장암 및 유방암 세포주에서는 ANTXR1 , ANTXR2 발현이 증가되어 있었으며， 흑색종 세포주 526 - mel과 유방암 세포주 SK-BR3는 ANTXR1 , ANTXR2가 발현되지 않는 것을 확인하였다 (도 1) . 실시예 2： 타겟 항원 단백질 과발현 세포주 제작 실시 예 1에 따라， ANTXRKTEM8) , ANTXR2(CMG2) 음성인 526-mel 세포주에 TEM8-RFP , CMG2-RFP 렌티바이러스를 transduct ion 하였으며， 그 후 western blot 방법을 이용하여 정상적으로 단백질이 발현됨을 확인하였다 (도 2) . 그 후 puromycin을 이용하여 posit ive cel l을 select ion한 결과 70% 이상의 발현이 증가된 세포주를 제작하였다 (도 3) . 실시예 3： ANTXR 특이적 CAR 설계 It is described in detail in Pharmaceutical Sciences (19th ed., 1995). The composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and rectal administration, etc. can be administered. When administered orally, since the protein or peptide is digested, the oral composition can be formulated to coat the active agent or to be protected from degradation in the stomach, and the composition of the present invention can be any device capable of moving the active substance to the target cell to be administered by 2021/245580 1» (：1' year 2021/054846 May. A suitable dosage of the composition for preventing or treating cancer of the present invention depends on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. It varies, and an ordinary skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. In another aspect, the present invention relates to a method for preventing or treating cancer comprising administering the immune cells. In another aspect, the present invention relates to the use of the immune cells for the prevention or treatment of cancer. In another aspect, the present invention relates to the use of the immune cells for the manufacture of a medicament for the prevention or treatment of cancer. Since the above-described "immune cells" are used for the prevention or treatment methods, uses, and uses of the present invention, redundant descriptions thereof will be omitted. Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it is common knowledge in the art that the scope of the present invention is not to be construed as being limited by these examples. 2021/245580 1»（：1' year 20211/054846 It will be obvious to those who have it. Example 1: Analysis of target antigen protein expression in cancer cell lines Melanoma cell line 526-mel, pancreatic cancer cell line (PANC1, Mia_PaCa2), breast cancer cell line (SK-BR3, MDA-MB-231, MCF7, ANTXRKTEM8 of ZR-517), As a result of analyzing ANTXR2 (CMG2) protein expression using western blot, ANTXR1 and ANTXR2 expression were increased in pancreatic cancer and breast cancer cell lines, melanoma cell line 526-mel and breast cancer cell line SK-BR3 did not express ANTXR1 and ANTXR2 was confirmed (FIG. 1). Example 2: Preparation of target antigen protein overexpression cell line According to Example 1, TEM8-RFP, CMG2-RFP lentivirus was transducted into ANTXRKTEM8), ANTXR2 (CMG2) negative 526-mel cell line, and then western blot method was performed It was confirmed that the protein was normally expressed using the . After that, as a result of selection of positive cells using puromycin, a cell line with increased expression of 70% or more was prepared (FIG. 3) . Example 3: ANTXR specific CAR design
ANTXR1 또는 ANTXR2에 특이적인 CAR는 세포외 결합 도메인， ⑶ 8a로부터 유래된 막횡단 도메인과 hinge region, CD¾ 사슬의 1차 신호전달 도메인， ⑶ 28 및 CD137 공자극 신호전달 도메인을 포함하도록 설계되었다. 세포외 결합
2021/245580 1»（：1'해2021/054846 도메인으로는 PA63 리간드의 수용체 결합에 필수적인 D4 야생형 (wild) 또는 D4 변이체 (mutant) #1 - #6이 각각 도입된 CAR를 설계하고， 도 4에 나타낸 벡터의 D4 자리에 도입하여 벡터를 제작하였다. 또한， 추가로 D4 wild 또는 D4 mutant #1 #6 앞에 신호 펩타이드 (signal peptide)를 코딩하는 핵산을 포함하도록 설계하였다. 상기 신호 펩타이드는 짧은 아미노산 서열로， 합성된 단백질을 묘묘로 운반하는 중요한 역할을 담당한다. 단백질이 묘묘에 도착하면 signal peptide는 제거되며， 단백질은 운반체를 통해 골지 (Golgi complex)로 운반된다. 이후 세포내 소기관이 아닌 세포막에 발현되어야 하는 단백질의 경우에는 목적지로 운반이 되어야 한다. 본 실시예에서는 합성된 단백질을 목적지로 운반을 하기 위한 신호 펩타이드로 GM-CSF를 사용하였다. 도 4의 벡터에는 CAR의 발현 여부를 확인하기 위하여 copGFP를 코딩하는 핵산을 도입하였으나， CAR를 실제 치료제로서 사용하기 위해서는 생략할 수 있다. 본 발명에
제조에 필요한 아미노산 서열을 표 1에 나타내었다.The CAR specific for ANTXR1 or ANTXR2 was designed to include an extracellular binding domain, ⑶ a transmembrane domain derived from 8a, a hinge region, a primary signaling domain of the CD¾ chain, ⑶ 28 and a CD137 costimulatory signaling domain. extracellular binding 2021/245580 1» (: 1' year 2021/054846 Domains essential for receptor binding of PA63 ligands D4 wild-type (wild) or D4 mutants (mutant) #1 - #6 are designed, respectively, the introduced CAR, Figure 4 A vector was prepared by introducing it into the D4 site of the vector shown in . In addition, it was designed to include a nucleic acid encoding a signal peptide in front of D4 wild or D4 mutant #1 #6. The signal peptide is a short amino acid sequence and plays an important role in transporting the synthesized protein to the seedlings. When the protein arrives at the seedling, the signal peptide is removed, and the protein is transported to the Golgi complex through a carrier. Afterwards, in the case of a protein that needs to be expressed on the cell membrane rather than the intracellular organelle, it must be transported to the destination. In this example, GM-CSF was used as a signal peptide for transporting the synthesized protein to its destination. A nucleic acid encoding copGFP was introduced into the vector of FIG. 4 in order to check the expression of CAR, but it may be omitted in order to use the CAR as an actual therapeutic agent. in the present invention The amino acid sequences required for preparation are shown in Table 1.
【표 1】
2021/245580 1»(：1'해2021/054846
상기 표 1의 04
#1 - 將에서 밑줄 및 굵은 글씨로 표시된 아미노산 잔기는 ?쇼63 04 1(1에서 변이된 아미노산 잔기이다. 【Table 1】 2021/245580 1»(：1' year2021/054846 04 of Table 1 above #1 - Amino acid residues underlined and bold in 將 are amino acid residues mutated from ?show63 04 1(1).
PA63 리간드 D4 wi ld 및 D4 mutant #1 - 將를 코딩하는 핵산은 하기 표 2에 나타내었다. Nucleic acids encoding PA63 ligand D4 wi ld and D4 mutant #1 - 將 are shown in Table 2 below.
【표 2】
2021/245580 1^(:1^ 2021/054846
2021/245580 1»(：1'해2021/054846
2021/245580 1»（：1'해2021/054846
상기 표 2의
#1 - 將에서 밑줄 및 굵은 글씨로 표시된 뉴클레오타이드는 ?쇼63 04 1(1에서 변이된 뉴클레오타이드이다. 실시예 4： ANTXR 특이적 CAR 제작 실시 예 3에서 설계한 CAR의 신호 펩타이드， 세포외 결합도메인 (PA63 D4 wi ld 및 D4 mutant #1 - 將)은 PCR을 이용하여 증폭하였고， 막횡단 도메인 (CD8 transmembrane & hinge)과 세포내 신호전달 도메인 (CD3 ， CD134, CD28)은 합성하였으며， Gibson assembly를 이용하여 렌티바이러스 vector에 삽입하였다. 위에서 제작된 CAR 도입용 렌티바이러스 vector를 이용하여 바이러스를 제작하였으며， packaging 세포인 293FT 세포 (4X106)에 약 50% 이상의 효율로
2021/245580 1»（：1'해2021/054846 렌티바이러스 vector를 유도하였다. 렌티바이러스 vector 유도 2일차에 렌티바이러스가 제작되어 부유되어 있는 cellmedia를 회수 후， 이를 Centicon을 이용하여 1,000 X g에서 30분 동안 10배 농축하였으며， 농축된 렌티바이러스를 293FT, NIH3T3세포에 transduction하여 CAR유전자가 도입된 렌티바이러스 Titer를 확인할 수 있었다. 제작된 D4 wild또는 D4 mutant함유 CAR가 도입된 렌티바이러스를 3.5 M0I의 농도로 건강한 공여자로부터 분리한 PBMC에 각각 transduction시켜 각각의 CAR-T세포를 제작하였다. 실시예 5： PBMC에서 분리한 T세포를 이용하여 제작한 CAR-T세포의 ANTXR1, 2TEM8, CMG2) 과발현 세포주에 대한 세포독성 확인 실시예 4에서 제작된 D4 wild함유 CAR도입 T세포와 D4 mutant #1 - #6 함유 CAR도입 T세포의 항암 세포독성을 확인하고자 하였다. ANTXR1, 2(TEM8, CMG2) 모두 음성인 526-mel흑색종 세포주에 ANTXR1, 2(TEM8, CMG2)를 각각 과발현시킨 세포주 526-mel/TEM8jFP와 526-me1/CMG2_RFP 세포주에 대한 세포독성을 확인하였다. 흑색종 세포주인 526-mel에 형광을 나타낼 수 있도록 RFP 유전자를 도입하여 제작한 세포주(Red Fluorescence Protein(RFP)을 발현하는 526-mel 세포주; 526-me1/TEM8_RFP, 526-mel/CMG2JFP)를 96 well plate에 5xl03개 세포 씩 분주한 뒤， 건강한 공여자의 PBMC (혈액내 면역세포; peripheral blood mononuclear celIs)로 제작된 NT(non transduced) T세포 또는 CAR(D4 wild및 D4 mutant #1 #6 CAR-T)를 각각 Effector(CAR positive cell)：Target(Tumor
2021/245580 1»（：1'해2021/054846 cell) ratio = 0.5:1， 1:1， 2:1， 4:1개의 세포로 48시간 공배양하였다.96 well plate에 남아있는 과발현 세포주 (526，el/TEM8jFP, 526，el/CMG2jFP)를 수거하여 Flow cytometry를 이용하여 RFP positive cell counting하였다. 그 결과， D4 wild에 비해 D4 mutant #1 - #6 CAR-T£1 killing이 감소하였으며， 이는 ANTXR1, 2(TEM8, CMG2)에 대한 수용체， 리간드 간의 결합력이 감소함에 따라 D4 mutant #1 - #6 ◦쇼묘 의 세포독성이 감소한 것을 시사한다 (도 5). 실시예 6： PBMC에서 분리한 T세포를 이용하여 제작한 CAR-T세포의 ANTXR1, 2TEM8, CMG2) 과발현 세포주에 대한 항암활성 확인 제작된 D4 wild함유 CAR도입 T세포와 D4 mutant #1 - #6함유 CAR 도입 T 세포의 항암활성을 확인하고자 하였다. ANTXR1, 2(TEM8, CMG2)모두 음성인 526-mel흑색종 세포주에 ANTXR1, 2(TEM8, CMG2)를 각각 과발현시킨 세포주 526-mel/TEM8jFP와 526-me1/CMG2_RFP 세포주에 대한 항암활성을 확인하였다. 【Table 2】 2021/245580 1^(:1^ 2021/054846 2021/245580 1»(：1' year2021/054846 2021/245580 1»（：1' year2021/054846 of Table 2 above #1 - Nucleotides underlined and bold in 將 are nucleotides mutated in ?show 63 04 1 ( 1. Example 4: ANTXR-specific CAR construction The signal peptide of the CAR designed in Example 3, extracellular binding domain (PA63 D4 wi ld and D4 mutant #1 - 將) was amplified using PCR, and the transmembrane domain (CD8 transmembrane & hinge) and intracellular signaling domain (CD3, CD134, CD28) were synthesized, and Gibson assembly was performed. Using the lentiviral vector, the virus was prepared using the lentiviral vector for CAR introduction prepared above, and the packaging cell 293FT cell (4X10 6 ) has an efficiency of about 50% or more. 2021/245580 1» (：1' year 2021/054846 lentiviral vector was induced. On the 2nd day of lentivirus vector induction, the lentivirus was prepared and the suspended cellmedia was recovered, which was concentrated 10 times for 30 minutes at 1,000 X g using a Centicon, and the concentrated lentivirus was transduced into 293FT, NIH3T3 cells and CAR It was possible to confirm the lentivirus titer into which the gene was introduced. Each CAR-T cell was prepared by transduction of the prepared D4 wild or D4 mutant-containing CAR-introduced lentivirus into PBMCs isolated from healthy donors at a concentration of 3.5 M0I. Example 5: Confirmation of cytotoxicity against ANTXR1, 2TEM8, CMG2) overexpressing cell lines of CAR-T cells prepared using T cells isolated from PBMC D4 wild-containing CAR introduced T cells and D4 mutant # The purpose of this study was to confirm the anticancer cytotoxicity of 1 - #6 containing CAR-introduced T cells. Cytotoxicity was confirmed against the 526-mel melanoma cell lines, which were both negative for ANTXR1 and 2 (TEM8, CMG2), and the 526-mel/TEM8jFP and 526-me1/CMG2_RFP cell lines overexpressing ANTXR1 and 2 (TEM8, CMG2), respectively. . A cell line (526-mel cell line expressing Red Fluorescence Protein (RFP); 526-me1/TEM8_RFP, 526-mel/CMG2JFP) prepared by introducing the RFP gene to show fluorescence in the melanoma cell line 526-mel was 96 After dispensing 3 cells of 5xl0 in a well plate, NT (non transduced) T cells or CAR (D4 wild and D4 mutant #1 #6 CARs) prepared from PBMCs (immune cells in blood; peripheral blood mononuclear celIs) from healthy donors -T) to each Effector (CAR positive cell): Target (Tumor) 2021/245580 1» (: 1' year 2021/054846 cells) ratio = 0.5:1, 1:1, 2:1, 4:1 cells were co-cultured for 48 hours. Overexpression cell lines remaining in 96 well plate ( 526, el/TEM8jFP, 526, el/CMG2jFP) were collected and RFP positive cells were counted using flow cytometry. As a result, compared to D4 wild, D4 mutant #1 - #6 CAR-T£1 killing was reduced, which decreased as the binding force between the receptor and ligand for ANTXR1, 2 (TEM8, CMG2) decreased, D4 mutant #1 - # 6 ◦ This suggests that the cytotoxicity of shomyo is reduced (FIG. 5). Example 6: Confirmation of anticancer activity against ANTXR1, 2TEM8, CMG2) overexpressing cell lines of CAR-T cells prepared using T cells isolated from PBMC It was intended to confirm the anticancer activity of the CAR-introduced T cells. Anticancer activity was confirmed against 526-mel/TEM8jFP and 526-me1/CMG2_RFP cell lines overexpressing ANTXR1 and 2 (TEM8, CMG2), respectively, in 526-mel melanoma cell lines negative for both ANTXR1 and 2 (TEM8, CMG2). .
ANTXR1, 2(TEM8,CMG2)를 과발현하는 526-mel세포주; 526-me1/TEM8_RFP, 526-mel/CMG2jFP)를 96 well plate에 lxlO5개 세포 씩 분주한 뒤， 건강한 공여자의 PBMC로 제작된 NT(non transduced) T세포 또는 CAR(D4 wild및 D4 mutant #1 #6 CAR-T)를 각각 Effector(CAR positive cell)：Target(Tumor cell) ratio = 1:1개의 세포로 24시간 공배양하였다.공배양 꾸 96 well plate에서 공배양 배지를 수거하고 ELISA를 이용하여 IFN gamma분비를 분석하였다. 그 결과， D4 wild에 비해 D4 mutant #1 - #6 CAR-T£1 IFN gamma분비가
2021/245580 1»（：1'해2021/054846 감소하였으며， 이는 ANTXR1 , 2(TEM8, CMG2)에 대한 수용체， 리간드 간의 결합력이 감소함에 따라 D4 mutant #1 #6 CAR-T의 항암활성이 감소한 것을 시사한다 (도 6) . 실시예 7： 선별 제작한 CAR-T세포의 in vitro항암활성 및 CMG2에 대한 안전성 분석 건강한 공여자 4명의 PBMC를 사용하여 6종의 D4 mutant중 선별된 2종의 D4 mutant CAR벡터를 이용하여 CAR-T세포를 제조하고， 각 공여자 PBMC에 따른 in vitro항암활성 차이를 조사하고자 하였다. 실시예 6과 동일한 방법으로 NT(Non Transduced) T 세포 대조군과 D4(wild, mutant #4, #6) CAR-T세포를 제작하여 T세포의 활성을 비교하였다. 그 결과， 각 4명의 건강한 공여자로부터 제작된 각각의 CAR-T세포에서도 실시예 6의 결과와 유사하게 D4 mutant #4와 #6의 TEM8 특이적인 활성을 확인할 수 있었다. 또한， D4 mutant들의 CAR-T활성이 D4 wild과 비교하여 세포독성은 유지되나， 항원에 대한 affinity감소로 인해 IFNY분비 정도는 감소되었음을 재확인하였다 (도 7 왼쪽) . CMG2 표적 세포에 대한 항암활성은 wild type에 비해 mutant #4와 #6에서는 NT수준으로 매우 낮게 관찰되어 안전성도 향상되었음을 확인하였다 (도 7 오른쪽) . 실시예 8： TEM8 과발현 암 세포주와 선별된 2종의 D4 mutant CAR-T의 공배양 후 T세포 특성 분석
2021/245580 1»（：1'해2021/054846 526-mel cell line overexpressing ANTXR1, 2 (TEM8, CMG2); After dispensing 526-me1/TEM8_RFP, 526-mel/CMG2jFP) in a 96-well plate by 5 cells each, NT (non transduced) T cells or CAR (D4 wild and D4 mutant #1) prepared from PBMCs from healthy donors #6 CAR-T) was co-cultured with each Effector (CAR positive cell):Target (Tumor cell) ratio = 1:1 cells for 24 hours. Thus, IFN gamma secretion was analyzed. As a result, compared to D4 wild, D4 mutant #1 - #6 CAR-T£1 IFN gamma secretion 2021/245580 1» (: 1' year, 2021/054846 decreased, which decreased as the binding force between the receptor and ligand for ANTXR1 and 2 (TEM8, CMG2) decreased, the anticancer activity of D4 mutant #1 #6 CAR-T decreased. suggest that (Fig. 6). Example 7: In vitro anticancer activity and safety analysis of CAR-T cells prepared by selection Using PBMCs from 4 healthy donors, 2 types of D4 mutant CAR vectors selected from 6 types of D4 mutants CAR- T cells were prepared, and the difference in in vitro anticancer activity according to each donor PBMC was investigated. In the same manner as in Example 6, NT (Non Transduced) T cell control and D4 (wild, mutant #4, #6) CAR-T cells were prepared and T cell activity was compared. As a result, it was possible to confirm the TEM8-specific activity of D4 mutants #4 and #6 in each of the CAR-T cells prepared from each of the four healthy donors, similar to the results of Example 6. In addition, it was reconfirmed that the CAR-T activity of D4 mutants maintained cytotoxicity compared to D4 wild, but the degree of IFNY secretion was decreased due to a decrease in affinity for antigen (Fig. 7 left) . Anticancer activity against CMG2 target cells was observed to be very low at NT level in mutants #4 and #6 compared to wild type, confirming that safety was also improved (FIG. 7 right) . Example 8: Analysis of T cell characteristics after co-culture of TEM8 overexpressing cancer cell line and two selected D4 mutant CAR-T 2021/245580 1»（：1' year2021/054846
TEM8 항원 발현 암 세포주와 2종의 D4 mutant (D4 mutant #4 및 #6) CAR-T 세포의 공배양 후 T 세포의 기능성 차이를 비교 분석하기 위해， T 세포 exhaust ion biomarker인 PD-1과 LAG-3 단백질의 발현 정도를 조사하였다. To compare and analyze the functional difference between T cells after co-culture of TEM8 antigen-expressing cancer cell lines and two types of D4 mutant (D4 mutant #4 and #6) CAR-T cells, PD-1 and LAG, which are T cell exhaustion biomarkers -3 The expression level of the protein was investigated.
ANTXR1 ( TEM8 ) / ANTXR2 ( CMG2 ) 항원이 모두 음성인 526-mel 흑색종 세포주에 ANTXR TEM8)을 과발현시킨 526-me 1 /TEM8_RFP 세포를 96 wel l plate에 lxlO4개 세포씩 분주하였다. 그 후， NT(non transduced) T 세포 또는 CAR-T 세포 (D4 wi ld 및 D4 mutant #4， #6)를 각각 Effector (CAR posit ive cel l )： Target (Tumor cel l ) rat io = 1 : 1 비율로 시간대별 (24， 48， 72시간)로 공배양 후 CD3 T 세포에서 PD- 1과 LAG-3의 발현을 FACS 분석하였다. 그 결과， 卵 -1의 경우 24시간， 48시간， 72시간 공배양 모든 경우에서 wi ld CAR-T 세포 대비 D4 mutant #4， #6 CAR-T에서 발현이 감소되는 경향성을 보였다 (도 8 왼쪽) . LAG-3의 경우 24시간 공배양의 경우에서만 wi ld CAR-T 세포 대비 D4 mutant들에서에서 발현이 감소됨을 확인하였다 (도 8 오른쪽) . 이는 ANTXRKTEM8) 항원에 대한 D4 리간드 간의 결합력을 조절하여 T 세포 과활성으로 인한 부작용을 낮추고 체내에서 WT 대비 T 세포의 지속성을 유지하여 체내에서 더 효율적이고 지속적인 항암효과를 유도할 수 있음을 시사한다. 실시예 9： 이종이식 (Xenograft) 마우스 모델을 이용한 항암 유효성 평가 면역부전 유전자변형 마우스 N0G(N0D/Shi-scid, IL_2Ry nul l； male)에526-me 1 /TEM8_RFP cells overexpressing ANTXR TEM8) in a 526-mel melanoma cell line negative for both ANTXR1 (TEM8) / ANTXR2 (CMG2) antigens were seeded in a 96-well plate with 4 lxlO cells each. Then, NT (non transduced) T cells or CAR-T cells (D4 wi ld and D4 mutant #4, #6) were respectively Effector (CAR positive cel l ): Target (Tumor cel l ) rat io = 1: After co-culture at a ratio of 1 for each time period (24, 48, 72 hours), the expression of PD-1 and LAG-3 in CD3 T cells was analyzed by FACS. As a result, in the case of 卵-1, the expression of D4 mutant #4, #6 CAR-T was decreased in all cases of co-culture for 24 hours, 48 hours, and 72 hours (Fig. 8 left side) ). In the case of LAG-3, it was confirmed that the expression was reduced in D4 mutants compared to wi ld CAR-T cells only in the case of co-culture for 24 hours (FIG. 8 right). This suggests that ANTXRKTEM8) can induce more efficient and sustained anticancer effects in the body by controlling the binding force between the D4 ligands to the antigen, lowering the side effects caused by T cell overactivity and maintaining the persistence of T cells compared to WT in the body. Example 9: Anticancer efficacy evaluation using a xenograft mouse model Immunodeficiency transgenic mouse N0G (N0D/Shi-scid, IL_2Ry nul l; male)
TEM8 항원 과발현 인간 췌장암 세포주 (Mia-PaCa2/TEM8)를 5 x 106 cel ls/head 용량으로 피하 주사하여 이식하였다. 이식 후 21일 (종양크기 : 약 〜 100mm3)째 D4
2021/245580 1»（：1'해2021/054846 wi ld와 위의 결과들로부터 최종 선별된 D4 mutant #6 CAR-T 세포 1 x 107 cel ls/head（total cel l： 2 x 107 cel ls/head）를 종양 이식 마우스에 꼬리 정맥 주사 방법으로 투여 후 종양 성장 억제 효과를 비교 조사하였다（도 9A）. The TEM8 antigen-overexpressing human pancreatic cancer cell line (Mia-PaCa2/TEM8) was implanted by subcutaneous injection at a dose of 5 x 10 6 cels/head. 21 days after transplantation (tumor size: about ~ 100mm 3 ) D4 2021/245580 1» (: 1' year 2021/054846 wi ld and D4 mutant #6 CAR-T cells finally selected from the above results 1 x 10 7 cel ls/head（total cel l: 2 x 10 7 cel ls/head) was administered to tumor-implanted mice by tail vein injection, and the tumor growth inhibitory effect was compared and investigated (FIG. 9A).
CAR-T 투여 3주 경과 후， D4 wi ld CAR-T 투여군의 종양 크기（평균값:After 3 weeks of CAR-T administration, the tumor size of the D4 wi ld CAR-T administration group (average value:
505.63 ± 290.21mm3） 대비 D4 mutant #6 CAR-T 투여군의 종양 크기（평균값:505.63 ± 290.21mm 3 ) compared to the tumor size of the D4 mutant #6 CAR-T group (mean value:
310.49±68.13·3）를 비교한 결과， 약 61%의 종양성장을 억제하였다（도 9B）. 이를 통해 D4 mutant 함유 CAR 도입 T세포가 ANTXR1（TEM8）을 발현하는 고형암종을 이용한 동물 모델에서도 효능이 우수함을 확인할 수 있었다. 산업상 이용가능성 대부분 고형암의 표적항원은 암 특이적 항원이 아닌 암 연관항원이며， 본 발명의 표적항원 ANTXR도 암 연관항원인
CAR-T 항암 유효성은 유지하면서 안전성을 확보하는 것이 중요하다. 본 발명에 따르면， PA63 리간드 도메인 4의 변이체를 세포외 결합 도메인으로 포함하는 키메라 항원 수용체를 이용하여 ARTXR 항원을 발현하는 암에 대해 탁월한 항암 효과를 보이면서， ARTXR 항원에 대한 결합 친화도 조절로 정상세포에는 영향이 없도록 안전성을 최적화할 수 있으므로， 항암치료에 매우 유효하면서 기존 CAR-T 면역세포치료제가 갖는 부작용들을 최소화한 항암 치료가 가능하다. 이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바， 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시
2021/245580 1^(:1^ 2021/054846 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.
310.49±68.13· 3 ) compared, the tumor growth was inhibited by about 61% (FIG. 9B). Through this, it was confirmed that the D4 mutant-containing CAR-introduced T cells had excellent efficacy in an animal model using solid carcinoma expressing ANTXR1 (TEM8). Industrial Applicability Most of the target antigens of solid cancer are cancer-associated antigens, not cancer-specific antigens, and the target antigen ANTXR of the present invention is also a cancer-associated antigen. It is important to secure safety while maintaining CAR-T anticancer efficacy. According to the present invention, by using a chimeric antigen receptor comprising a mutant of PA63 ligand domain 4 as an extracellular binding domain, it shows an excellent anticancer effect against cancer expressing an ARTXR antigen, and controls the binding affinity to the ARTXR antigen to control normal cells Since safety can be optimized so that there is no effect on cancer, it is possible to treat cancer with very effective anticancer treatment and minimizing side effects of existing CAR-T immune cell therapies. Above, a specific part of the content of the present invention has been described in detail, and for those of ordinary skill in the art, this specific description is only a preferred implementation. It will be apparent that 2021/245580 1^(:1^ 2021/054846 are only aspects, and not thereby limiting the scope of the present invention. Accordingly, the substantial scope of the present invention is defined by the appended claims and their equivalents. would be defined.
Claims
【청구항 1] [Claim 1]
PA63 리간드 도메인 4 의 변이체를 함유하는 세포외 결합 도메인 (extracel lular binding domain) , 막횡단 도메인 (transmembrane domain) 및 세포내 신호전달 도메인 (intracel lular signal ing domain)을 포함하는 키메라 항원 수용체 (chimeric antigen receptor , CAR) . A chimeric antigen receptor comprising an extracellular binding domain, a transmembrane domain and an intracellular signaling domain containing a variant of PA63 ligand domain 4 , CAR).
【청구항 2] 제 1 항에 있어서， 상기 PA63 리간드 도메인 4 는 ARTXRKanthrax toxin receptor 1) 또는 ARTXR2(anthrax toxin receptor 2)를 인지하는 것을 특징으로 하는 키메라 항원 수용체 . [Claim 2] The chimeric antigen receptor according to claim 1, wherein the PA63 ligand domain 4 recognizes ARTXRKanthrax toxin receptor 1) or ARTXR2 (anthrax toxin receptor 2).
【청구항 3] 제 1 항에 있어서， 상기 쇼63 리간드 도메인 4 의 변이체는 서열번호 2 내지 서열번호 7 로 구성된 군에서 선택되는 하나 이상의 아미노산 서열을 포함하는 것을 특징으로 하는 키메라 항원 수용체 . [Claim 3] The chimeric antigen receptor according to claim 1, wherein the variant of Show63 ligand domain 4 comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 7.
【청구항 4]
2021/245580 1»（：1'해2021/054846 제 1 항에 있어서， 상기 막횡단 도메인은 세포 수용체의 알파， 베타 또는 제타 사슬， 0028, 003 엡실론， 0045, ⑶ 4, ⑶ 5, ⑶ 8, ⑶ 9, 0)16, 0022, 0033, 0037, ⑶ 64, 0080, 0086, ⑶ 134, 0)137 및 0)154 로 구성된 군에서 선택되는 것을 특징으로 하는 키메라 항원 수용체. [Claim 4] 2021/245580 1» (: 1' year 2021/054846 according to claim 1, wherein the transmembrane domain is an alpha, beta or zeta chain of a cell receptor, 0028, 003 epsilon, 0045, ⑶ 4, ⑶ 5, ⑶ 8, A chimeric antigen receptor, characterized in that it is selected from the group consisting of ⑶ 9, 0)16, 0022, 0033, 0037, ⑶ 64, 0080, 0086, ⑶ 134, 0)137 and 0)154.
【청구항 5] 제 1항에 있어서， 상기 세포내 신호전달 도메인은 1차 신호전달 도메인 (primary signaling domain) 및 공자극 신호전달 도메인 (co-stimulatory signaling domain)을 포함하는 것을 특징으로 하는 키메라 항원 수용체. [Claim 5] The chimeric antigen receptor according to claim 1, wherein the intracellular signaling domain comprises a primary signaling domain and a co-stimulatory signaling domain. .
【청구항 6] 제 5 항에 있어서， 상기 1 차 신호전달 도메인은 TCIU， FcRy , FcRp , CD3y , ⑶ 35 , CD3e , CD3 ^ , ⑶ 5, CD22, ⑶ 79a, CD79b 및 CD66d 로 구성된 군에서 선택되는 것을 특징으로 하는 키메라 항원 수용체. [Claim 6] The method of claim 5, wherein the primary signaling domain is selected from the group consisting of TCIU, FcRy, FcRp, CD3y, ⑶ 35, CD3e, CD3 ^, ⑶ 5, CD22, ⑶ 79a, CD79b and CD66d. Chimeric antigen receptor, characterized in that.
【청구항 7] 제 5 항에 있어서， 상기 공자극 신호전달 도메인은 0X40, ⑶ 2, 0027, 0028, ⑶ 比能-1, -1(0)113/0)18)， 1008(00278), 0X)7, 0030, 0040, 1)-1， 1그抑 ■ ，
2021/245580 1»（：1'해2021/054846 [Claim 7] The method of claim 5, wherein the costimulatory signaling domain is 0X40, ⑶ 2, 0027, 0028, ⑶ 比能-1, -1(0)113/0)18), 1008 (00278), 0X )7, 0030, 0040, 1)-1, 1 next ■ ， 2021/245580 1»（：1' year2021/054846
87-113， ⑶ 83 및 4-내표（⑶ 137）로 구성된 군에서 선택되는 것을 특징으로 하는 키메라 항원 수용체. Chimeric antigen receptor, characterized in that it is selected from the group consisting of 87-113, ⑶ 83 and 4-inner table (⑶ 137).
【청구항 8] 제 1항에 있어서， 상기 키메라 항원 수용체는 신호 펩티드（ 요 peptide）를 더 포함하는 것을 특징으로 하는 키메라 항원 수용체. [Claim 8] The chimeric antigen receptor according to claim 1, wherein the chimeric antigen receptor further comprises a signal peptide.
【청구항 9] 제 1항의 키메라 항원 수용체를 코딩하는 핵산. [Claim 9] A nucleic acid encoding the chimeric antigen receptor of claim 1.
【청구항 10】 제 9항의 핵산을 포함하는 발현 벡터 . [Claim 10] An expression vector comprising the nucleic acid of claim 9.
【청구항 11】 제 10항의 발현 벡터를 포함하는 바이러스. [Claim 11] A virus comprising the expression vector of claim 10.
【청구항 12】 제 1항 내지 제 8항 중 어느 한 항의 키메라 항원 수용체를 발현하는 면역세포.
2021/245580 1»（：1'해2021/054846 [Claim 12] An immune cell expressing the chimeric antigen receptor according to any one of claims 1 to 8. 2021/245580 1»（：1' year2021/054846
【청구항 13】 제 12항에 있어서， 상기 면역세포는 T세포， NK세포， NKT세포， 사이토카인 유도 살해세포(Cytokine Induced Killer cell, CIK)， 마크로파지 및 수지상세포로 구성된 군에서 선택되는 것을 특징으로 하는 면역세포. [Claim 13] The method according to claim 12, wherein the immune cells are selected from the group consisting of T cells, NK cells, NKT cells, cytokine-induced killer cells (CIK), macrophages and dendritic cells. immune cells that do.
【청구항 14】 제 13항의 면역세포를 포함하는 암 예방 또는 치료용 조성물. [Claim 14] A composition for preventing or treating cancer comprising the immune cell of claim 13.
【청구항 15】 제 14항에 있어서， 상기 암은 ANTXR(anthrax toxin receptor)을 발현하는 암인 것을 특징으로 하는 암 예방 또는 치료용 조성물. [Claim 15] The composition for preventing or treating cancer according to claim 14, wherein the cancer is a cancer expressing anthrax toxin receptor (ANTXR).
【청구항 16】 제 15항에 있어서， 상기 암은 췌장암， 위암， 대장암， 폐암， 유방암， 배세포암， 간암， 피부암， 방광암， 전립선암， 자궁암， 자궁경암 및 난소암으로 구성된 군에서 선택되는 것을 특징으로 하는 암 예방 또는 치료용 조성물.
[Claim 16] The method of claim 15, wherein the cancer is selected from the group consisting of pancreatic cancer, stomach cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer and ovarian cancer A composition for preventing or treating cancer, characterized in that.
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| KR20080052566A (en) * | 2005-08-02 | 2008-06-11 | 플래닛 바이오테크놀로지, 인코포레이티드 | Improved chimeric toxin receptor proteins and chimeric toxin receptor proteins for treatment and prevention of anthrax |
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