Anti-HLA-G chimeric antigen receptor and its uses

Abstract

The present invention relates to a chimeric antigen receptor containing an antigen-binding site that specifically binds to HLA-G, an immune barrier, an immune cell into which the receptor has been introduced, and uses thereof. The immune cell can efficiently remove cancer cells expressing the immune barrier, and is therefore expected to activate endogenous immune cells that have been suppressed by the immune barrier, and therefore can be usefully used as an immunotherapy method for various cancer diseases associated with HLA-G.

Description

[Technical field]

[0001] The present invention relates to a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to HLA-G, an immune cell into which the receptor has been introduced, and uses thereof. [Background technology]

[0002] Methods for treating cancer have undergone steady development and change, and methods such as surgery, chemotherapy, and radiation therapy are still being used up to now. However, most of these existing cancer treatment methods are only effective in the early stages when cancer has not yet metastasized, and there is a problem that in cases where metastasis has already progressed, there is a high possibility of recurrence even after surgery. Therefore, research into methods that utilize immune responses to treat cancer has been ongoing recently.

[0003] Among these, there is growing interest in cell therapy methods that use immune cells to strengthen immune responses or genetically modify immune cells and then inject them back into patients. For the latter method, much research has been conducted on chimeric antigen receptor (CAR)-T cells. CAR-T cells are T cells that have been given a targeting function by injecting an artificially designed CAR molecule into them, and as the CAR molecule shows specificity for certain tumor antigens, it is able to eliminate only tumor cells.

[0004] Generally, a chimeric antigen receptor (CAR) comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The extracellular antigen-binding domain may comprise a single-chain variable fragment (scFv) that targets a recognized tumor antigen.

[0005] On the other hand, human major histocompatibility antigen, class I, G (HLA-G), also known as human leukocyte antigen-G (HLA-G), is an immune barrier factor that suppresses the activity and division of T cells, NK cells, cytotoxic T cells, etc. ILT2 (Ig-like transcript2), ILT4, KIR2DL4 (Killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 4), etc. are known as immune cell receptors for HLA-G, and all of these receptors have an ITIM (immunoreceptor tyrosine-based inhibitory motif). When HLA-G binds to the receptor, this motif activates a signaling system that suppresses immune cells.

[0006] It has been reported that HLA-G expression is limited in general cells, but is expressed in many types of cancer cells (Lin A, Yan WH.Human Leukocyte Antigen-G (HLA-G) Expression in Cancers; Roles in Immune Evasion, Metastasis and Target for Therapy. Mol. Med. 21(1), 782-791 (2015)). It has also been reported that the expression level of HLA-G in cancer cells of many cancer patients is correlated with the stage of cancer disease, and that patients who express HLA-G have a low survival rate (Ye, Sr., Yang, H., Li, K. et al.Human leukocyte antigen G expression: as a significant prognostic indicator for patients with colorectal cancer. Mod. Pathol. 20, 375-383 (2007)). Summary of the Invention [Problem to be solved by the invention]

[0007] The present inventors have made extensive research efforts to develop a chimeric antigen receptor and immune cells containing the same that can effectively treat diseases associated with HLA-G (human leukocyte antigen-G) expression. As a result, they have produced an anti-HLA-G chimeric antigen receptor that specifically binds to HLA-G, and T cells and NK cells expressing the chimeric antigen receptor. They have also confirmed that the T cells and NK cells exhibit high anticancer activity against cell lines expressing HLA-G, which has led to the completion of the present invention.

[0008] It is therefore an object of the present invention to provide an anti-HLA-G chimeric antigen receptor and an immune cell expressing said receptor.

[0009] Another object of the present invention is to provide a nucleic acid comprising a nucleotide sequence encoding an anti-HLA-G chimeric antigen receptor and a vector comprising said nucleic acid.

[0010] It is yet another object of the present invention to provide a pharmaceutical composition comprising said immune cells and a pharma- ceutically acceptable carrier. [Means for solving the problem]

[0011] To achieve the above object, one aspect of the present invention provides an anti-HLA-G chimeric antigen receptor, comprising an antigen-binding domain that specifically binds to HLA-G (human leukocyte antigen-G); a transmembrane domain; and an intracellular signaling domain.

[0012] In one embodiment of the invention, the antigen-binding domain that specifically binds to HLA-G may comprise a sequence selected from the following: (a) a heavy chain variable region comprising a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 31, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 32, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 33; and a light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 34, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 35, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 36; (b) a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 37, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 38, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39; and A light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 40, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 41, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 42; or (c) a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 43, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 44, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 45; and A light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 46, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 47, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 48.

[0013] In the present invention, the term "chimeric antigen receptor" refers to a receptor protein that combines a receptor site on an immune cell (e.g., T cell) with a recognition site for a specific antigen expressed on the surface of a cancer cell so that the immune cell can recognize the specific protein. This receptor has a chimeric nature because it combines an antigen binding site that can recognize an antigen expressed by a tumor with a signal transduction site that can activate T cells.

[0014] In one embodiment of the present invention, the chimeric antigen receptor comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The antigen-binding domain recognizes HLA-G of a target cell and may be an antibody or an antibody fragment. The antigen-binding fragment may be, but is not limited to, an scFv.

[0015] In one embodiment of the present invention, the antigen-binding site may comprise a heavy chain variable region sequence and a light chain variable region sequence, and the two sequences may be linked by a linker comprising the amino acid sequence of SEQ ID NO: 24. Thus, the antigen-binding site may be in the form of a single-chain fragment variable (scFv).

[0016] As used herein, the term "heavy chain" refers collectively to a full-length heavy chain and fragments thereof, including a variable region domain VH that contains sufficient variable region amino acid sequence to confer specificity for an antigen, and three constant region domains CH1, CH2, and CH3.

[0017] As used herein, the term "light chain" refers collectively to a full-length light chain and fragments thereof that includes a variable region domain VL and a constant region domain CL that contain sufficient variable region amino acid sequence to confer specificity for an antigen.

[0018] In one embodiment of the present invention, the antigen-binding domain may comprise the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, and more specifically, may consist of an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more sequence identity to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

[0019] The antigen-binding domain may also include (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:25 or an amino acid sequence having 80% or more identity to SEQ ID NO:25, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:26 or an amino acid sequence having 80% or more identity to SEQ ID NO:26; (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:27 or an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO:27, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:28 or an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO:28; or (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:29 or an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO:29, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:30 or an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO:30.

[0020] The chimeric antigen receptor may further include a signal sequence (leader sequence, LS). The signal sequence refers to a sequence that transports the chimeric antigen receptor to the cell membrane so that it can be expressed and exposed on the cell surface.

[0021] In one embodiment of the present invention, the signal sequence may be a signal sequence derived from CD8α, human immunoglobulin heavy chain or hGM-CSF receptor α-chain, in particular a signal sequence derived from CD8α.

[0022] The CD8α-derived signal sequence may comprise the amino acid sequence of SEQ ID NO:8, and specifically may consist of an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more sequence identity with the amino acid sequence of SEQ ID NO:8.

[0023] In the chimeric antigen receptor of the present invention, the antigen-binding domain that specifically binds to HLA-G may be linked to the transmembrane domain by a hinge. The hinge may be derived from the group consisting of CD8α hinge, IgG1 hinge, IgG4 hinge, IgD hinge, IgG1 CH3, the extracellular domain of CD28, or a combination thereof, and specifically may be derived from CD8α. The hinge derived from CD8α may comprise the amino acid sequence of SEQ ID NO: 10, and specifically may consist of an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 10.

[0024] In the chimeric antigen receptor of the present invention, the transmembrane domain (TM) is a domain that connects the antigen-binding domain and the intracellular signaling domain between the cell membrane. The transmembrane domain may be a transmembrane domain of a protein selected from the group consisting of CD8α, T cell receptor, CD28, CD3ε (epsilon), CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD99, CD134, CD137, and CD154 α, β, or ζ, and specifically, may be a transmembrane domain of CD8α.

[0025] The transmembrane domain of CD8α may comprise the amino acid sequence of SEQ ID NO:12, and specifically may consist of an amino acid sequence having a sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more with the amino acid sequence of SEQ ID NO:12.

[0026] In the chimeric antigen receptor of the present invention, the intracellular signaling domain (ICD) is a site that generates a signal that activates the immune response of immune cells when the antigen-binding site of the extracellular domain binds to HLA-G.

[0027] The intracellular signaling domains include CD3ζ, FcγR, ICOS (Inducible T-cell costimulator, CD278), 4-1BB (CD137, TNFRSF9), OX40 (CD134), CD27, CD28, IL-2Rβ, IL-15R-α, CD40, MyD88, DAP10 (DNAX-activating protein 10), DAP12, MHC class I molecule, TNF receptor (tumor necrosis factor receptor) protein, immunoglobulin-like protein, cytokine receptor, integrin, SLAM (Signaling lymphocytic activation molecule) protein, activating NK cell receptor, BTLA (B and T Lymphocyte Attenuator), Toll ligand receptor (Toll-like receptors, TLRs), CD2, CD7, CD30, CD99, CDS, ICAM-1 (Intercellular adhesion molecule-1), B7-H3 (CD276), GITR (Glucocorticoid-induced TNFR-related protein), BAFFR (BAFF (B-cell activating factor)-receptor), LIGHT, HVEM (LIGHTR), KIRDS2 (killer cell immunoglobulin like receptor, two Ig domains and short cytoplasmic tail 2), SLAMF7 (SLAM Family Member 7), NKp80 (KLRF1 (killer cell lectin-like receptor subfamily F, member 1)), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6 (integrin Subunit Alpha 6), VLA-6, CD49f, ITGAD (integrin subunit alpha D), CD11d, ITGAE (integrin subunit alpha E), CD103, ITGAL (integrinsubunit alpha L)、CD11a、LFA-1(lymphocyte function-associated antigen-1;CD11a / CD18)、ITGAM(integrin subunit alpha M)、CD11b、ITGAX(integrin subunit alpha X)、CD11c、ITGB1(integrin beta-1)、CD29、ITGB2(integrin beta-2)、CD18、ITGB7(integrin beta-7)、NKG2D(KLRK1(killer cell lectin like receptor K1)、NKG2C(KLRC2(Killer Cell Lectin Like Receptor C2))、TNFR2(Tumor necrosis factor receptor 2)、TRANCE(receptor activator of NF-kappa B ligand;RANKL(receptor activator of nuclear factors κB ligand))、DNAM1(DNAX accessory molecule-1、CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1(carcinoembryonic antigen-related cell adhesion molecule 1)、CRTAM(cytotoxic and regulatory T cell molecule)、Ly9(CD229)、CD160(BY55)、PSGL1(P-selectin glycoprotein Ligand 1、CD162)、CD100(SEMA4D(Semaphorin 4D))、CD69、SLAMF6(NTB-A(NK-T-B-antigen))、Ly108)、SLAMF1(CD150、IPO-3)、BLAME(SLAMF8)、LTBR(lymphotoxin-beta receptor)、LAT(Linker For Activation Of T Cells)、GADS(glutamate decarboxylase)、SLP-76(Src homology 2(SH2)domain-containing leukocyteThe domain may be derived from a protein selected from the group consisting of CD3ζ-derived intracellular signal transduction domain (PAG / Cbp), CD19a, and a CD83-specific ligand, and more specifically, may be an intracellular signal transduction domain derived from CD3ζ.

[0028] The CD3ζ-derived intracellular signaling domain may comprise the amino acid sequence of SEQ ID NO: 16, and more specifically, may consist of an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 16.

[0029] In addition, the intracellular signaling domain may further include a costimulatory domain to further activate immune cells and increase their survival potential. The costimulatory domain may be derived from a protein selected from the group consisting of OX40, CD2, CD27, CD28, CD99, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) and 4-1BB (CD137), and more specifically, may be derived from 4-1BB.

[0030] The 4-1BB-derived costimulatory domain may comprise the amino acid sequence of SEQ ID NO: 14, and more specifically, may consist of an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 14.

[0031] In one embodiment of the present invention, the chimeric antigen receptor may comprise the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22, and more specifically, may consist of an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22.

[0032] Yet another aspect of the invention is an isolated nucleic acid comprising a nucleotide sequence encoding the anti-HLA-G chimeric antigen receptor.

[0033] The term "nucleic acid" as used herein is intended to comprehensively include DNA and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogues in which the sugar or base moiety is modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews (1990) 90:543-584). The sequence of the nucleic acid molecule encoding the anti-HLA-G chimeric antigen receptor of the present invention may be modified, and such modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0034] In one embodiment of the present invention, the isolated nucleic acid comprising a nucleotide sequence encoding the anti-HLA-G chimeric antigen receptor comprises a nucleotide sequence encoding an antigen-binding site that specifically binds to HLA-G. The nucleotide sequence encoding the antigen-binding site may comprise the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, and may specifically comprise a nucleotide sequence having 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more sequence identity with the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

[0035] In one embodiment of the present invention, the isolated nucleic acid comprising a nucleotide sequence encoding the anti-HLA-G chimeric antigen receptor may comprise the nucleotide sequence of SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:21, and more specifically, may comprise a nucleotide sequence having 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more sequence identity with the nucleotide sequence of SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:21.

[0036] Another aspect of the present invention is a recombinant vector comprising the nucleic acid molecule.

[0037] The vector used in the present invention may be a variety of vectors known in the art. Depending on the type of host cell in which the antigen receptor is to be produced, expression regulatory sequences such as promoters, terminators, enhancers, and sequences for membrane targeting or secretion may be appropriately selected and combined in various ways according to the purpose.

[0038] Methods for injecting and expressing the vectors of the present invention into cells are well known in the art. The vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by methods known in the art. For example, the vectors can be delivered into host cells by physical, chemical or biological means. The physical means include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. The chemical means include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspears, beads and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The biological means also include the use of DNA or RNA vectors, such as lentiviruses and retroviruses, as described above.

[0039] The vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, a virus vector, etc. Suitable vectors include a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as a promoter, an operator, an initiation codon, a stop codon, a polyadenylation signal, and an enhancer, and can be prepared in a variety of ways depending on the purpose.

[0040] In one embodiment of the present invention, a lentiviral vector is used, which further comprises a promoter, for example, but not limited to, the EF-1 promoter.

[0041] The present invention also provides an immune cell expressing the above-mentioned anti-HLA-G chimeric antigen receptor on the cell surface.

[0042] In the present invention, the immune cells may be selected from the group consisting of natural killer cells, T cells, natural killer T cells, B cells, macrophages, dendritic cells, natural killer dendritic cells, mast cells, and precursor cells thereof.

[0043] The anti-HLA-G chimeric antigen receptor can be introduced into immune cells via the above-mentioned vector, and the method of introduction can be selected from standard techniques suitable for the host cell as known in the art.

[0044] In one embodiment of the present invention, T cells and NK cells expressing chimeric antigen receptors were produced by infecting T cells and NK cells with a recombinant vector containing a polynucleotide capable of encoding an anti-HLA-G chimeric antigen receptor. Specifically, the coding sequence of the anti-HLA-G chimeric antigen receptor contained in the recombinant vector is transcribed into mRNA, and the polynucleotide of the anti-HLA-G chimeric antigen receptor is translated from the mRNA and expressed on the surface of the T cells and NK cells. If necessary, the T cells and NK cells infected with the recombinant vector can be cultured.

[0045] Immune cells expressing anti-HLA-G chimeric antigen receptors can recognize HLA-G as an antigen and specifically bind to HLA-G. After binding, they can induce activation of immune cells through the intracellular signaling domain, thereby inducing tumor-specific death.

[0046] Thus, another aspect of the present invention provides a pharmaceutical composition for use in the prevention or treatment of cancer comprising immune cells and a pharma- ceutically acceptable carrier.

[0047] In the present invention, the pharmaceutical composition can be used for the treatment of cancers that overexpress HLA-G, but is not limited thereto, which may be selected from the group consisting of pancreatic cancer, breast cancer, ovarian cancer, glioma, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, liver cancer, lung cancer, colon cancer, nasopharyngeal cancer, oral cancer, thyroid cancer, prostate cancer, kidney cancer, gallbladder cancer, bile duct cancer, blood cancer, and melanoma.

[0048] The blood cancer may be acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), multiple myeloma (MM), or lymphoma.

[0049] As used herein, the term "prevention" refers to any action of suppressing or slowing the progression of a disease by administering a composition of the present invention, and "treatment" refers to suppressing, reducing or eliminating the development of a disease.

[0050] The pharmaceutical composition of the present invention may further include a pharma- ceutically acceptable carrier, which is a carrier that is commonly used in formulation, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition for preventing or treating cancer of the present invention may further include, in addition to the above ingredients, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharma- ceutically acceptable carriers and formulations are described in detail in the literature [Remington's Pharmaceutical Sciences (19th ed., 1995)].

[0051] In addition, the pharmaceutical composition according to the present invention may be administered by, but is not limited to, oral administration, infusion, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intrarectal administration, topical administration, intranasal injection, etc. During oral administration, proteins or peptides are digested, so oral compositions may be formulated to coat or protect the active agent from degradation in the stomach, and the compositions of the present invention may be administered by any device that allows the active agent to be delivered to target cells.

[0052] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity of the patient, and generally, a skilled doctor can easily determine and prescribe an effective dosage for the desired treatment or prevention. The composition preferably contains 1 to 10 times the number of immune cells expressing the chimeric antigen receptor of the present invention as compared with the number of tumor cells (e.g., pancreatic cancer, breast cancer, ovarian cancer or glioma) in the subject to be treated, but is not limited thereto.

[0053] According to one embodiment of the present invention, the number of the immune cells (e.g., T cells or natural killer cells) in a single dose of the pharmaceutical composition of the present invention may be 1 to 10 times the number of the tumor cells, e.g., pancreatic cancer cells, breast cancer cells, ovarian cancer cells, or glioma cells, in a subject to be treated.

[0054] As used herein, the term "pharmaceutical effective amount" refers to an amount sufficient to treat, prevent, and diagnose a disease.

[0055] The pharmaceutical composition of the present invention may be prepared in a unit dose form or in a multi-capacity container by formulating it using pharma- ceutically acceptable carriers and / or excipients in a manner that can be easily carried out by a person having ordinary skill in the art to which the invention pertains. In this case, the dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may further include a dispersant or stabilizer.

[0056] The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, either sequentially or simultaneously with conventional therapeutic agents.

[0057] Another aspect of the present invention is a method for diagnosing, preventing or treating a disease associated with HLA-G overexpression, such as cancer, comprising administering a pharma- ceutical effective amount of the anti-HLA-G chimeric antigen receptor of the present invention, or an immune cell expressing the chimeric antigen receptor, or the pharmaceutical composition to a subject, such as a human or a non-human mammal.The present invention also provides a method for diagnosing a disease associated with HLA-G overexpression, such as cancer, comprising administering the anti-HLA-G chimeric antigen receptor of the present invention or an immune cell expressing the chimeric antigen receptor to an individual in need thereof, such as a human or a non-human mammal.

[0058] In the present invention, the term "individual" refers to any animal, including monkeys, cows, horses, sheep, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits, or guinea pigs, including humans, that have or may develop an HLA-G overexpression associated disease. Effect of the Invention

[0059] Since the chimeric antigen receptor according to the present invention has excellent specificity and affinity for HLA-G, immune cells expressing the anti-HLA-G chimeric antigen receptor have excellent cytotoxicity against cancer cells and can be used in the treatment of various cancers associated with high expression of HLA-G. [Brief description of the drawings]

[0060] [Figure 1a] FIG. 1 shows the structures of the HLA-G CAR (antibody fragment 1 CAR), HLA-G CAR (antibody fragment 2 CAR) and HLA-G CAR (antibody fragment 3 CAR) constructs of the present invention. [Figure 1b] FIG. 1 shows the structures of the HLA-G CAR (antibody fragment 1 CAR), HLA-G CAR (antibody fragment 2 CAR) and HLA-G CAR (antibody fragment 3 CAR) constructs of the present invention. [Figure 1c] FIG. 1 shows the structures of the HLA-G CAR (antibody fragment 1 CAR), HLA-G CAR (antibody fragment 2 CAR) and HLA-G CAR (antibody fragment 3 CAR) constructs of the present invention. [Figure 2a] FIG. 1 shows the structures of the pLV-EF1A-HLA-G CAR (antibody fragment 1 CAR), pLV-EF1A-HLA-G CAR (antibody fragment 2 CAR) and pLV-EF1A-HLA-G CAR (antibody fragment 3 CAR) plasmids of the present invention. [Figure 2b] FIG. 1 shows the structures of the pLV-EF1A-HLA-G CAR (antibody fragment 1 CAR), pLV-EF1A-HLA-G CAR (antibody fragment 2 CAR) and pLV-EF1A-HLA-G CAR (antibody fragment 3 CAR) plasmids of the present invention. [Figure 2c] FIG. 1 shows the structures of the pLV-EF1A-HLA-G CAR (antibody fragment 1 CAR), pLV-EF1A-HLA-G CAR (antibody fragment 2 CAR) and pLV-EF1A-HLA-G CAR (antibody fragment 3 CAR) plasmids of the present invention. [Diagram 3] FIG. 1 shows the structure of the pLV-EF1A-B2M-IRES-HLA-G-CMV-EGFP-T2A-PuroR plasmid of the present invention. [Figure 4] FIG. 1 is a graph confirming HLA-G expression rates in the SK-OV-3-HLA-G and MDA-MB-231-HLA-G overexpressing cell lines of the present invention. [Figure 5a]This figure shows the expression rate of CAR on the surface of anti-HLA-G CAR-expressing T cells of the present invention. [Figure 5b] This figure shows the expression rate of CAR on the surface of anti-HLA-G CAR-expressing T cells of the present invention. [Figure 5c] This figure shows the expression rate of CAR on the surface of anti-HLA-G CAR-expressing T cells of the present invention. [Figure 6a] FIG. 1 shows the anti-cancer efficacy of anti-HLA-G CAR-expressing T cells of the present invention against SK-OV-3-HLA-G, MDA-MB-231-HLA-G, SK-OV-3 and MDA-MB-231 cell lines. [Figure 6b] FIG. 1 shows the anti-cancer efficacy of anti-HLA-G CAR-expressing T cells of the present invention against SK-OV-3-HLA-G, MDA-MB-231-HLA-G, SK-OV-3 and MDA-MB-231 cell lines. [Figure 6c] FIG. 1 shows the anti-cancer efficacy of anti-HLA-G CAR-expressing T cells of the present invention against SK-OV-3-HLA-G, MDA-MB-231-HLA-G, SK-OV-3 and MDA-MB-231 cell lines. [Figure 7] FIG. 1 shows the activity of the anti-HLA-G CAR-expressing T cells of the present invention against SK-OV-3-HLA-G, MDA-MB-231-HLA-G, SK-OV-3 and MDA-MB-231 cell lines, as confirmed by the secretion of interferon gamma. [Figure 8] This figure shows the expression rate of CAR on the surface of anti-HLA-G CAR-expressing T cells cultured with the addition of IL-2 and IL-7 / IL-15 cytokines in the present invention. [Figure 9] FIG. 1 shows the anti-cancer efficacy of anti-HLA-G CAR-expressing T cells cultured with the addition of IL-2 and IL-7 / IL-15 cytokines against SK-OV-3-HLA-G and SK-OV-3 cell lines in the present invention. [Figure 10]This figure shows the activity of anti-HLA-G CAR-expressing T cells cultured with the addition of IL-2 and IL-7 / IL-15 cytokines against SK-OV-3-HLA-G and SK-OV-3 cell lines, as confirmed by the secretion of interferon gamma. [Figure 11] This figure shows the expression rates of CD3 and CD56 in NK cells expressing the anti-HLA-G CAR of the present invention. [Figure 12] This figure shows the expression rate of CAR on the surface of anti-HLA-G CAR-expressing NK cells of the present invention. [Figure 13] FIG. 1 shows the anti-cancer efficacy of anti-HLA-G CAR-expressing NK cells of the present invention against SK-OV-3-HLA-G and SK-OV-3 cell lines. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] The present invention will be described in more detail with reference to the following examples. However, it will be obvious to those skilled in the art that these examples are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention.

[0062] Example 1: Cell lines and culture The human ovarian cancer cell line SK-OV-3 and the human triple-negative breast cancer cell line MDA-MB-231 were supplied by the Korea Cell Line Bank (Korea). SK-OV-3 and MDA-MB-231 were cultured in RPMI 1640 (Gibco, USA) medium containing 10% FBS (Gibco, USA) and 1% Antibiotic-Antimycotic (Gibco, USA).

[0063] <Example 2: Construction and culture of HLA-G overexpressing cell line> 2-1. Preparation of lentivirus expressing B2M-HLA-G-EGFP-PuroR gene The lentivirus for the transfection of the B2M-HLA-G-EGFP-PuroR gene was produced using the plasmid DNA transformation method. TransIT-293 Transfection Reagent (Mirus Bio LLC, USA) was used, and the procedure was carried out according to the manufacturer's protocol. The day before the experiment, a 100 mm dish was transfected with Lenti-X. TM 293T cell line (Clontech, USA) was cultured at 5 × 10 6 The next day, the cells were transfected with the pLV-EF1A-B2M-IRES-HLA-G-CMV-EGFP-T2A-PuroR lentiviral vector (Figure 3), gag-pol expression vector, and VSV-G envelope expression vector. After culturing the cells for about 72 hours, the entire supernatant was harvested. The supernatant was filtered through a 0.45 μm filter (Millipore, USA) to remove cell debris, and the filtrate containing the B2M-HLA-G-EGFP-PuroR lentivirus was frozen and stored at -80°C until use.

[0064] 2-2. Construction and culture of B2M-HLA-G-EGFP-PuroR gene expressing SK-OV-3 and MDA-MB-231 cell lines The day before the experiment, 1x10 SK-OV-3 and MDA-MB-231 cell lines were each placed in a 100 mm dish. 6The prepared B2M-HLA-G-EGFP-PuroR lentivirus was mixed with 7 mL of fresh medium at 3 mL per dish and placed in the dish. Polybrene was added to the medium at a concentration of 8 ug / mL. After 24 hours, the medium was replaced with fresh medium containing 2 μg / mL of puromycin (Sigma-Aldrich, USA). When the selection by puromycin progressed to a certain extent, the cells were subcultured at a concentration of 0.5 cells / well in a 96-well plate to separate HLA-G overexpressing cells into single cells. After the final selection was completed, puromycin was added to the medium at a concentration of 0.5 ug / mL. Several wells with high EGFP fluorescent signals were selected under a fluorescent microscope, and when the corresponding wells were filled with cells, the cells were collected and cultured in 48-well, 24-well, 12-well, 6-well, T25, and T75 flasks in order. Finally, the cells were cultured in a T175 flask to prepare a cell stock.

[0065] 2-3. Confirmation of HLA-G expression in B2M-HLA-G-EGFP-PuroR gene expressing SK-OV-3 and MDA-MB-231 cell lines The expression of HLA-G was confirmed in the HLA-G overexpressing cell line prepared in 2-2 above as follows.

[0066] 1 × 10 SK-OV-3 and MDA-MB-231 cell lines expressing B2M-HLA-G-EGFP-PuroR gene 5A cell sample was prepared by suspending the cells in 100uL D-PBS (Gibco, USA). 1ug of anti-HLA-G (TANKIO17) antibody was added to the cell sample and reacted at 4℃ for 30 minutes. After the reaction was completed, the cells were washed twice with D-PBS and then suspended again. 0.25ug of R-Phycoerythrin AffiniPure F(ab')2Fragment Goat Anti-Human IgG(H+L) (Jackson ImmunoResearch, USA) was added to the cell sample and reacted at 4℃ for 30 minutes. After the reaction, the cells were washed twice with D-PBS and the expression of HLA-G was confirmed by cell-based analysis. As a result, it was confirmed that HLA-G was expressed at approximately 99.8% on the surface of SK-OV-3 cells transduced with B2M-HLA-G-EGFP-PuroR lentivirus, and at approximately 98.9% on the surface of MDA-MB-231 cells (Figure 4).

[0067] Example 3: Construction of chimeric antigen receptor The chimeric antigen receptor (CAR) of the present invention is artificially synthesized.

[0068] More specifically, the coding sequences of the extracellular and intracellular domains were artificially synthesized through SOE-PCR (Splicing by overlap extension PCR) to produce CAR (Figure 1a-c). The PCR product was cleaved with MluI and XbaI and then inserted into the MluI and XbaI sites of the pLV-EF1A-MCS vector, a third generation self-inactivating lentivirus expression vector (Figure 2a-c).

[0069] -Extracellular domain: signal sequence (leader sequence, LS) derived from CD8α, HLA-G binding domain (antibody fragment 1, antibody fragment 2 or antibody fragment 3; scFv), hinge derived from CD8α and transmembrane (TM) domain derived from CD8α -Intracellular domain: Intracellular signaling domain of 4-1BB and CD3ζ A chimeric antigen receptor according to an embodiment of the present invention is summarized in Table 1 below. The domains of the chimeric antigen receptor are linked in tandem and in frame with each other. In detail, a signal sequence derived from CD8α, an HLA-G binding domain (scFv form), a hinge derived from CD8α, a transmembrane domain derived from CD8α, an intracellular signaling domain derived from 4-1BB, an intracellular signaling domain derived from CD3ζ, and a TAA which is a stop codon are linked.

[0070] [Table 1]

[0071] The sequence information of the chimeric antigen receptor according to the embodiment of the present invention and the domains used in its construction are summarized in Tables 2 and 3.

[0072] [Table 2]

[0073] [Table 3(1)] [Table 3(2)] [Table 3(3)] [Table 3(4)]

[0074] The sequences of the heavy chain, light chain and CDRs of the antibody fragment variable regions used in the production of the chimeric antigen receptor according to the embodiments of the present invention are summarized in Tables 4 and 5.

[0075] [Table 4]

[0076] [Table 5]

[0077] Example 4: Preparation of anti-HLA-G CAR-T cells 4-1. Production of lentivirus expressing anti-HLA-G CAR gene Lentivirus for the transfer of the anti-HLA-G CAR gene was produced by the plasmid DNA transfection method. TransIT-293 Transfection Reagent (Mirus Bio LLC, USA) was used, and the procedure was carried out according to the manufacturer's protocol. The day before the experiment, Lenti-X was placed in a 100 mm dish. TM 293T cell line (Clontech, USA) was cultured at 5 × 10 6 The next day, the cells were transfected with the pLV-EF1A-HLA-G CAR lentiviral vector, the gag-pol expression vector, and the VSV-G envelope expression vector. The HLA-G CAR lentiviral vectors were as follows: antibody fragment 1, antibody fragment 2, or antibody fragment 3. The transfected cells were cultured for about 72 hours, and after the culture was completed, the entire supernatant of the cells was harvested. The supernatant was filtered through a 0.45 μm filter (Millipore, USA) to remove cell debris. Lenti-X TM The lentivirus was concentrated approximately 100-fold using a Concentrator (Clontech, USA) according to the manufacturer's protocol, and stored frozen at -80°C until use.

[0078] 4-2. Generation and culture of anti-HLA-G CAR-expressing T cells Donors were recruited and leukocytes were obtained through leukapheresis. TM Peripheral blood mononuclear cells (PBMCs) were obtained using Ficoll-Paque PLUS (GE healthcare, Sweden) and Ficoll-50 (STEMCELL technology, Canada). TMHuman T cells were isolated by a positive selection method using a separator (Miltenyi Biotec, Germany) and an LS column (Miltenyi Biotec, Germany), human CD4 MicroBeads (Miltenyi Biotec, Germany), and human CD8 MicroBeads (Miltenyi Biotec, Germany). TM The medium (Miltenyi Biotec, Germany) was used as the culture medium for T Cell TransAct TM The cells were activated by adding human IL-2 (Miltenyi Biotec, Germany) to the culture medium for T cell growth. After 48 hours of culture, the activated T cells were harvested and used for lentiviral transduction.

[0079] Human T cells activated with lentivirus at an MOI (multiplicity of infection) of about 3 were cultured in a 12-well plate at 1 × 10 per well. 6 The culture medium was adjusted to 2.5 mL by adding 5 × 10 cells per mL. The lentivirus was transduced into the T cells by centrifugation at 1,000 × g for 15 min. The T cells were cultured for 48 h and then replaced with fresh medium. The transduced human T cells were cultured at 5 × 10 cells per mL at 2–3 day intervals. 5 The cells were subcultured at 2 × 10 per mL. 6 Unless otherwise specified, human T cells were cultured in a culture medium supplemented with IL-2 (R&D systems, USA) at 100 U / mL.

[0080] 4-3. Confirmation of CAR expression in anti-HLA-G CAR-expressing T cells We confirmed whether anti-HLA-G CAR was expressed on the cell surface in anti-HLA-G CAR-expressing T cells produced from leukocytes obtained from three donors.

[0081] 1×10 5After harvesting, the cells were suspended in 100uL D-PBS to prepare a cell suspension. PE fluorescence was conjugated to the B2M-HLA-G antigen used in the production of anti-HLA-G antibody using a PE conjugation kit (Abcam, USA). The B2M-HLA-G antigen-PE conjugate was added to the cell suspension and reacted at 4℃ for 30 minutes. After the reaction, the cells were washed twice with D-PBS, and the expression level of anti-HLA-G CAR was confirmed by cell-based analysis.

[0082] As a result, in donor 1, it was confirmed that anti-HLA-G CAR was expressed at approximately 34.9% of the T cells that had been delivered with the anti-HLA-G CAR (antibody fragment 1 CAR) gene, at approximately 45.8% of the T cells that had been delivered with the anti-HLA-G CAR (antibody fragment 2 CAR) gene, and at approximately 43.3% of the T cells that had been delivered with the anti-HLA-G CAR (antibody fragment 3 CAR) gene (Figure 5a).

[0083] As a result, in donor 2, it was confirmed that anti-HLA-G CAR was expressed in approximately 37.6% of T cells that had been delivered with the anti-HLA-G CAR (antibody fragment 1 CAR) gene, approximately 57.3% of T cells that had been delivered with the anti-HLA-G CAR (antibody fragment 2 CAR) gene, and approximately 38.1% of T cells that had been delivered with the anti-HLA-G CAR (antibody fragment 3 CAR) gene (Figure 5b).

[0084] As a result, in donor 5, it was confirmed that anti-HLA-G CAR was expressed at approximately 30.4% on the surface of T cells that had been delivered with the anti-HLA-G CAR (antibody fragment 1 CAR) gene, at approximately 46.2% on the surface of T cells that had been delivered with the anti-HLA-G CAR (antibody fragment 2 CAR) gene, and at approximately 37.4% on the surface of T cells that had been delivered with the anti-HLA-G CAR (antibody fragment 3 CAR) gene (Figure 5c).

[0085] <Example 5: Confirmation of anti-cancer efficacy of anti-HLA-G CAR-expressing T cells in vitro> The anti-cancer efficacy of anti-HLA-G CAR-expressing T cells (effector cells, E) against target cells (T) was confirmed using the following method.

[0086] (i) All target cells were engineered to overexpress EGFP.

[0087] (ii) Plate target cells at 1 × 10 per well in a 48-well plate. 5 Cells were added at 150 uL.

[0088] (iii) 1×10 anti-HLA-G CAR-expressing T cells and control T cells per well 6 cells / 150uL, 5×10 5 cells / 150uL, 1×10 5 The solution was added at 150 uL per cell (E:T ratio = 10, 5, 1) and reacted with the target cells for 24 hours.

[0089] (iv) When the target cells died, the EGFP fluorescent signal disappeared, so the number of cells expressing EGFP was counted 24 hours later using a cell-based assay.

[0090] [Formula 1] Cytotoxicity(%)=100-{(EGFP after co-culture of target and effector cells) + Cell number / EGFP after monoculture of target cells + number of cells)×100} The experimental results showed that the anti-cancer efficacy of T cells expressing three types of anti-HLA-G CAR (antibody fragment 1 CAR), anti-HLA-G CAR (antibody fragment 2 CAR), and anti-HLA-G CAR (antibody fragment 3 CAR) made from white blood cells of donor 1 against the HLA-G overexpressing SK-OV-3-HLA-G cell line was much higher than that of the control T cells not expressing anti-HLA-G CAR. However, there was no difference in the killing ability of the three types of anti-HLA-G CAR expressing T cells and the control T cells against the SK-OV-3 cell line that does not express HLA-G (Figure 6a).

[0091] The anti-cancer efficacy of the three anti-HLA-G CAR (antibody fragment 1 CAR), anti-HLA-G CAR (antibody fragment 2 CAR, and anti-HLA-G CAR (antibody fragment 3 CAR)-expressing T cells prepared from the leukocytes of donor 2 against the HLA-G overexpressing MDA-MB-231-HLA-G cell line was significantly higher than that of the control T cells that did not express the anti-HLA-G CAR. On the other hand, there was no difference in the killing ability of the three anti-HLA-G CAR-expressing T cells and the control T cells against the MDA-MB-231 cell line that does not express HLA-G (Figure 6b).

[0092] The anti-cancer efficacy of the three anti-HLA-G CAR (antibody fragment 1 CAR), anti-HLA-G CAR (antibody fragment 2 CAR), and anti-HLA-G CAR (antibody fragment 3 CAR)-expressing T cells prepared from the leukocytes of donor 5 against the HLA-G overexpressing SK-OV-3-HLA-G and MDA-MB-231-HLA-G cell lines was also significantly higher than that of the control T cells not expressing the anti-HLA-G CAR. However, there was no difference in the killing ability of the three anti-HLA-G CAR-expressing T cells and the control T cells against the SK-OV-3 and MDA-MB-231 cell lines that do not express HLA-G (Figure 6c).

[0093] These results confirmed that anti-HLA-G CAR-expressing T cells have a high killing ability specifically against cells that highly express HLA-G.

[0094] The cell supernatant was harvested from the experimental group in which anti-HLA-G CAR-expressing T cells produced by donor 5 and control T cells were treated with target cells at an E:T ratio of 1:1. The interferon gamma concentration in the harvested supernatant was measured using a Human IFN-gamma Quantikine ELISA Kit (R&D systems, USA).

[0095] As a result, it was confirmed that the secretion of interferon gamma was significantly increased only when the three types of anti-HLA-G CAR (antibody fragment 1 CAR), anti-HLA-G CAR (antibody fragment 2 CAR, and anti-HLA-G CAR (antibody fragment 3 CAR)-expressing T cells were co-cultured with the SK-OV-3-HLA-G or MDA-MB-231-HLA-G target cell lines that overexpress HLA-G (Figure 7).

[0096] Example 6: Comparison of anti-cancer efficacy of anti-HLA-G CAR-expressing T cells according to in vitro T cell culture conditions Anti-HLA-G CAR (antibody fragment 2 CAR) expressing T cells were produced in the manner described in Example 4-2. At this time, human IL-2 (R&D systems, USA) was added at 100 U / mL, or IL-7 (Miltenyi Biotec, Germany) and IL-15 (Miltenyi Biotec, Germany) were added at 12.5 ng / mL, respectively, to the culture medium for T cell growth.

[0097] We confirmed whether anti-HLA-G CAR was expressed on the cell surface of anti-HLA-G CAR-expressing T cells. We found that anti-HLA-G CAR was expressed in approximately 48.3% of anti-HLA-G CAR-expressing T cells cultured with IL-2 in the culture medium, and that anti-HLA-G CAR was expressed in approximately 47.9% of anti-HLA-G CAR-expressing T cells cultured with IL-7 and IL-15 in the culture medium (Figure 8).

[0098] Anti-HLA-G CAR expressing T cells were co-cultured with target cells to confirm the anti-cancer efficacy. The anti-HLA-G CAR expressing T cells (IL-2) and anti-HLA-G CAR expressing T cells (IL-7 / IL-15) had similar and excellent anti-cancer efficacy against the SK-OV-3-HLA-G cell line, which overexpresses HLA-G. However, the killing ability against the SK-OV-3 cell line, which does not express HLA-G, was very low, so there was no difference in killing ability between the two types of anti-HLA-G CAR expressing T cells (Figure 9).

[0099] In the experimental group where anti-HLA-G CAR-expressing T cells were treated with target cells at an E:T ratio of 1:1, the cell supernatant was harvested and the concentration of interferon gamma was measured using a Human IFN-gamma Quantikine ELISA Kit (R&D systems, USA). As a result, it was confirmed that interferon gamma secretion was significantly increased only when anti-HLA-G CAR-expressing T cells (IL-2) or anti-HLA-G CAR-expressing T cells (IL-7 / IL-15) were co-cultured with the SK-OV-3-HLA-G target cell line that overexpresses HLA-G, and there was almost no difference between the two types of anti-HLA-G CAR-expressing T cells (Figure 10).

[0100] These results confirmed that anti-HLA-G CAR-expressing T cells showed similar levels of CAR expression regardless of the type of cytokine (IL-2 or IL-7 / IL-15) added to the culture medium for T cell growth, and that both types of anti-HLA-G CAR-expressing T cells showed good anti-cancer efficacy in vitro.

[0101] <Example 7: Preparation of anti-HLA-G CAR-NK cells and confirmation of anti-cancer efficacy> 7-1. Generation and culture of anti-HLA-G CAR-expressing NK cells Donors were recruited and leukocytes were obtained through leukapheresis. TMPeripheral blood mononuclear cells (PBMCs) were obtained using Ficoll-Paque PLUS (GE healthcare, Sweden) and Ficoll-Paque 100% Ficoll-50 (STEMCELL technology, Canada). Human natural killer (NK) cells were then isolated by negative selection using an NK cell isolation kit (Miltenyi Biotec, Germany). Human NK cells were cultured in NK MACS® Medium (Miltenyi Biotec, Germany). For efficient growth of NK cells, GemCell TM The cells were cultured with 5% Human Serum AB (GeminiBio, USA), and human IL-2 (R&D systems, USA) and IL-15 (Miltenyi Biotec, Germany) at concentrations of 500 U / mL and 140 U / mL, respectively.

[0102] RetroNectin® (Takara, Japan) and BX-795 (Sigma-Aldrich, USA) were used to efficiently deliver lentivirus to NK cells. Lentivirus at MOI of 10 was placed in a RetroNectin-coated 24-well plate and adsorbed according to the manufacturer's protocol. 5 × 10 NK cells on day 10 of culture were cultured. 5 2.5uM BX-795 was added to each well and treated for 30 minutes. Then, the BX-795-treated NK cells were placed on the plate with the lentivirus adsorbed and centrifuged at 1,000xg for 15 minutes to deliver the lentivirus to the NK cells. The NK cells were then cultured for 24 hours and then replaced with fresh culture. The transduced human NK cells were appropriately subcultured at 3-4 day intervals.

[0103] 7-2. Confirmation of CAR expression in anti-HLA-G CAR-expressing NK cells First, we confirmed whether anti-HLA-G CAR-expressing NK cells were cultured correctly.5 After harvesting, cells were suspended in 100uL D-PBS to prepare a cell suspension. VioBlue-conjugated CD3 antibody (Miltenyi Biotec, Germany) and FITC-conjugated CD56 antibody (Miltenyi Biotec, Germany) were added to the cell suspension and reacted at 4℃ for 30 minutes. After the reaction, the cells were washed twice with D-PBS, and the expression levels of CD3 and CD56 were confirmed by cell-based analysis.

[0104] As a result, CD3 - CD56 + The percentage of NK cells with CD3+ expression was 99.1% in the control group NK cells, 99.1% in the NK cells transfected with the anti-HLA-G CAR (antibody fragment 1 CAR) gene, 98.9% in the NK cells transfected with the anti-HLA-G CAR (antibody fragment 2 CAR) gene, and 99.1% in the NK cells transfected with the anti-HLA-G CAR (antibody fragment 3 CAR) gene. Almost all cells were CD3+ - CD56 + It was confirmed that they were NK cells (Figure 11).

[0105] We then confirmed whether anti-HLA-G CAR was expressed on the surface of each cell. 5 After harvesting, the cells were suspended in 100uL D-PBS to prepare a cell suspension. Using a PE conjugation kit (Abcam, USA), PE fluorescence was conjugated to the B2M-HLA-G antigen used to produce the anti-HLA-G antibody. The B2M-HLA-G antigen PE conjugate was added to the cell suspension and reacted at 4℃ for 30 minutes. After the reaction was completed, the cells were washed twice with D-PBS, and the expression of anti-HLA-G CAR was confirmed by cell-based analysis.

[0106] As a result, it was confirmed that anti-HLA-G CAR was expressed in approximately 18.8% of NK cells that had been transfected with the anti-HLA-G CAR (antibody fragment 1 CAR) gene, approximately 25.5% of NK cells that had been transfected with the anti-HLA-G CAR (antibody fragment 2 CAR) gene, and approximately 22.4% of NK cells that had been transfected with the anti-HLA-G CAR (antibody fragment 3 CAR) gene (Figure 12).

[0107] 7-3. Confirmation of the anti-cancer efficacy of anti-HLA-G CAR-expressing NK cells in vitro The anti-cancer efficacy of anti-HLA-G CAR-expressing NK cells was confirmed using the same method as in Example 5.

[0108] As a result, the anti-cancer efficacy of NK cells expressing three types of anti-HLA-G CARs (antibody fragment 1 CAR), anti-HLA-G CAR (antibody fragment 2 CAR), and anti-HLA-G CAR (antibody fragment 3 CAR) against the SK-OV-3-HLA-G cell line, which overexpresses HLA-G, was significantly higher than that of control NK cells that did not express anti-HLA-G CAR. However, in the SK-OV-3 cell line, there was no difference in the killing ability of anti-HLA-G CAR-expressing NK cells and control NK cells (Figure 13).

Claims

1. an antigen-binding domain that specifically binds to HLA-G (Human leukocyte antigen-G); A transmembrane domain; and an intracellular signaling domain; The antigen-binding domain that specifically binds to HLA-G comprises a sequence selected from the following: (a) a heavy chain variable region comprising a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:31, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:32, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:33; and a light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 34, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 35, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 36; (b) a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 37, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 38, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39; and A light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 40, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 41, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 42; or (c) a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 43, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 44, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 45; and A light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO:46, a light chain CDR2 having the amino acid sequence of SEQ ID NO:47, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

48.

2. The anti-HLA-G chimeric antigen receptor according to claim 1, wherein the antigen-binding domain that specifically binds to HLA-G is an antibody or an antigen-binding fragment thereof.

3. The anti-HLA-G chimeric antigen receptor according to claim 2, wherein the antigen-binding fragment is an scFv.

4. The anti-HLA-G chimeric antigen receptor according to claim 1, further comprising a signal sequence.

5. Anti-HLA-G chimeric antigen receptor according to claim 4, characterized in that the signal sequence is from CD8α, human immunoglobulin heavy chain or hGM-CSF receptor α-chain.

6. The anti-HLA-G chimeric antigen receptor according to claim 5, wherein the signal sequence derived from CD8α comprises the amino acid sequence of SEQ ID NO:

8.

7. The anti-HLA-G chimeric antigen receptor according to claim 1, wherein the antigen-binding domain that specifically binds to HLA-G is connected to the transmembrane domain by a hinge.

8. The anti-HLA-G chimeric antigen receptor according to claim 7, wherein the hinge is selected from the group consisting of a CD8α hinge, an IgG1 hinge, an IgG4 hinge, an IgD hinge, an IgG1 CH3, an extracellular domain of CD28, or a combination thereof.

9. The anti-HLA-G chimeric antigen receptor according to claim 8, wherein the hinge of CD8α comprises the amino acid sequence of SEQ ID NO:

10.

10. The anti-HLA-G chimeric antigen receptor according to claim 1, wherein the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of CD8α, T cell receptor, CD28, CD3ε (epsilon), CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD99, CD134, CD137, and CD154 α, β, or ζ.

11. The anti-HLA-G chimeric antigen receptor according to claim 10, wherein the transmembrane domain of CD8α comprises the amino acid sequence of SEQ ID NO:

12.

12. The intracellular signaling domains include CD3ζ, FcγR, ICOS (Inducible T-cell costimulator, CD278), 4-1BB (CD137, TNFRSF9), OX40 (CD134), CD27, CD28, IL-2Rβ, IL-15R-α, CD40, MyD88, DAP10 (DNAX-activating protein 10), DAP12, MHC class I molecule, TNF receptor (tumor necrosis factor receptor) protein, immunoglobulin-like protein, cytokine receptor, integrin, SLAM (Signaling lymphocytic activation protein, activating NK cell receptor, BTLA (B and T Lymphocyte Attenuator), Toll-like receptors (TLRs), CD2, CD7, CD30, CD99, CDS, ICAM-1 (Intercellular adhesion molecule-1), B7-H3 (CD276), GITR (Glucocorticoid-induced TNFR-related protein), BAFFR (BAFF (B-cell activating factor)-receptor), LIGHT, HVEM (LIGHTR), KIRDS2 (killer cell immunoglobulin like receptor, two Ig domains and short cytoplasmic tail 2), SLAMF7 (SLAM Family Member 7), NKp80 (KLRF1 (killer cell lectin-like receptor subfamily F, member 1)), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6 (integrin Subunit Alpha 6), VLA-6, CD49f, ITGAD (integrin subunit alpha D), CD11d, ITGAE (integrin subunit alpha E), CD103, ITGAL (integrinsubunit alpha L)、CD11a、LFA-1(lymphocyte function-associated antigen-1;CD11a / CD18)、ITGAM(integrin subunit alpha M)、CD11b、ITGAX(integrin subunit alpha X)、CD11c、ITGB1(integrin beta-1)、CD29、ITGB2(integrin beta-2)、CD18、ITGB7(integrin beta-7)、NKG2D(KLRK1(killer cell lectin like receptor K1)、NKG2C(KLRC2(Killer Cell Lectin Like Receptor C2))、TNFR2(Tumor necrosis factor receptor 2)、TRANCE(receptor activator of NF-kappa B ligand;RANKL(receptor activator of nuclear factors κB ligand))、DNAM1(DNAX accessory molecule-1、CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1(carcinoembryonic antigen-related cell adhesion molecule 1)、CRTAM(cytotoxic and regulatory T cell molecule)、Ly9(CD229)、CD160(BY55)、PSGL1(P-selectin glycoprotein Ligand 1、CD162)、CD100(SEMA4D(Semaphorin 4D))、CD69、SLAMF6(NTB-A(NK-T-B-antigen))、Ly108)、SLAMF1(CD150、IPO-3)、BLAME(SLAMF8)、LTBR(lymphotoxin-beta receptor)、LAT(Linker For Activation Of T Cells)、GADS(glutamate decarboxylase)、SLP-76(Src homology 2(SH2)domain-containing leukocyteThe anti-HLA-G chimeric antigen receptor according to claim 1, which is derived from a protein selected from the group consisting of phosphoprotein of 76-kDa, PAG / Cbp, CD19a and a CD83 specific ligand.

13. The anti-HLA-G chimeric antigen receptor according to claim 12, wherein the CD3ζ-derived intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

16.

14. The anti-HLA-G chimeric antigen receptor according to claim 1, wherein the intracellular signaling domain further comprises a signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CD99, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) and 4-1BB (CD137) as a costimulatory domain.

15. The anti-HLA-G chimeric antigen receptor according to claim 14, wherein the 4-1BB-derived costimulatory domain comprises the amino acid sequence of SEQ ID NO:

14.

16. An isolated nucleic acid comprising a nucleotide sequence encoding an anti-HLA-G chimeric antigen receptor according to any one of claims 1 to 15.

17. A vector comprising the isolated nucleic acid of claim 16.

18. An immune cell, characterized in that it expresses the anti-HLA-G chimeric antigen receptor according to any one of claims 1 to 15 on its surface.

19. The immune cell according to claim 18, characterized in that the immune cell is selected from the group consisting of natural killer cells, T cells, natural killer T cells, B cells, macrophages, dendritic cells, natural killer dendritic cells, mast cells and precursor cells thereof.

20. 20. A pharmaceutical composition for use in the prevention or treatment of cancer comprising the immune cells of claim 18 or 19 and a pharma- ceutically acceptable carrier.

21. The pharmaceutical composition of claim 20, wherein the cancer is selected from the group consisting of pancreatic cancer, breast cancer, ovarian cancer, glioma, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, liver cancer, lung cancer, colon cancer, nasopharyngeal cancer, oral cancer, thyroid cancer, prostate cancer, kidney cancer, gallbladder cancer, bile duct cancer, blood cancer and melanoma.

22. 20. A method for preventing or treating cancer, comprising administering an immune cell according to claim 18 or claim 19, or a pharmaceutical composition according to claim 20, to a subject in need thereof.

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