Immune cells expressing immune checkpoint regulators and their uses

Abstract

The present invention relates to immune cells that express immune checkpoint regulators and overcome immune checkpoints, a method for producing the same, and a pharmaceutical composition containing the immune cells. More specifically, the present invention relates to immune cells that are genetically engineered to express an anti-HLA-G soluble scFv that specifically binds to HLA-G, which is an immune checkpoint, together with an antigen recognition receptor, a method for producing the same, and a pharmaceutical composition containing the immune cells. When the anti-HLA-G soluble scFv of the present invention is expressed alone or simultaneously with an antigen recognition receptor in T lymphocytes, natural killer cells, etc., immune cells inhibited by cancer cells expressing immune checkpoints can be activated, and cancer cells can be removed more efficiently. Thereby, activation of endogenous immune cells that have been inhibited by immune checkpoints can also be expected, and thus it can be usefully used as an immunotherapy method for various cancer diseases related to HLA-G.

Description

Technical Field

[0001] The present invention relates to immune cells that can express factors capable of controlling immune checkpoints, expression vectors for their production, compositions containing the same, and their uses.

Background Art

[0002] Methods for treating cancer have undergone a long process of development and change, and methods such as surgical operations, chemotherapy, and radiotherapy have continued to be used to date. However, such conventional cancer treatment methods are mostly effective only in the early stage when cancer has not metastasized. In the case where metastasis has already progressed, for example, even if a surgical operation is performed, there is a problem that the possibility of recurrence is high later. Therefore, research on methods using immune responses to treat cancer has continued.

[0003] In recent years, in the treatment of blood cancers, the therapeutic potential of T cells expressing chimeric antigen receptors (CAR-T) has been demonstrated, and CAR-T therapeutic agents targeting CD19 or B-cell maturation antigen (BCMA) have been commercially available. However, due to the characteristics of solid cancers different from blood cancers, especially the tumor microenvironment (TME) that suppresses the immune response, in addition to solid cancer CAR-T therapeutic agents, strategies that can efficiently overcome the TME and treat cancer are further required.

[0004] On the other hand, human leukocyte antigen G (HLA-G) is a non-classical HLA class I molecule and is known to be specifically expressed only in extravillous cytotrophoblasts at the basal part of the placenta. And through such expression, it is considered to play an important role in maintaining immune tolerance between the fetus and the mother.

[0005] Immune cell receptors for HLA-G include ILT2 (Ig-like transcript 2), ILT4, KIR2DL4 (Killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 4), etc. All of these receptors have an ITIM motif (immunoreceptor tyrosine-based inhibitory motif). When HLA-G binds to the receptor, a signal transduction pathway that suppresses immune cells is activated by this motif.

[0006] The expression of HLA-G is restricted in normal cells, but is overexpressed in multiple types of cancers (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)), which is associated with immune suppressive microenvironments, advanced cancer stages, inadequate treatment responses or prognosis.

[0007] In addition, due to its property of being overexpressed in various cancers, HLA-G may inhibit the efficacy of not only endogenous immune cells but also immune cell therapeutics targeting specific antigens. That is, HLA-G may play the role of an immune checkpoint that suppresses immunity, and thus may reduce the efficacy of endogenous and exogenous immune cells. Therefore, in order to effectively treat cancer using the immune response, a method capable of overcoming the immune checkpoint function of HLA-G is required.

Summary of the Invention

Problems to be Solved by the Invention

[0008] In such a situation, the present inventors made intensive research efforts to develop immune cells that can overcome the tumor microenvironment of HLA-G overexpressing cancers, particularly solid cancers. As a result, by producing immune cells that express and secrete a soluble scFv derived from an anti-HLA-G antibody that specifically binds to HLA-G, and clarifying that the produced immune cells exhibit higher anti-cancer activity than general immune cells against cell lines that express HLA-G, the present invention has been completed.

[0009] According to the present invention, the immune cells of the present invention secrete a soluble single-chain Fv (scFv) that can inhibit the interaction between HLA-G and its receptor, so that the tumor microenvironment is improved, immune cells such as tumor-infiltrating lymphocytes can be activated, and the activity of endogenous immune cells can also be further induced. Therefore, the immune cells of the present invention can more efficiently remove cancer cells and can be used for the treatment of various cancers, not only cancers related to high expression of HLA-G.

[0010] Therefore, an object of the present invention is to provide immune cells that express a soluble scFv that specifically binds to a target selected from the group consisting of HLA-G having immunosuppressive activity and its receptor, and have high anti-tumor activity.

[0011] Another object of the present invention is to provide immune cells that further express an antigen recognition receptor on the above-mentioned immune cells.

[0012] Another object of the present invention is to provide an expression vector for producing the above-mentioned immune cells.

[0013] Another object of the present invention is to provide a pharmaceutical composition for cancer treatment comprising the above-mentioned immune cells and a pharmaceutically acceptable carrier.

Means for Solving the Problems

[0014] To achieve the above object, one aspect of the present invention is an antigen recognition receptor; and An antibody or an antigen-binding fragment thereof that specifically binds to a target selected from the group consisting of human leukocyte antigen G (HLA-G) and its receptor; An immune cell that expresses and secretes

[0015] Another aspect of the present invention provides an immune cell that expresses and secretes an antibody or an antigen-binding fragment thereof that specifically binds to a target selected from the group consisting of human leukocyte antigen G (HLA-G) and its receptor.

[0016] In the present invention, the immune cell may be selected from the group consisting of natural killer cells, T cells, B cells, macrophages, dendritic cells, natural killer dendritic cells, mast cells, and progenitor cells thereof. More specifically, it may be an effector cell such as a T cell or a B cell.

[0017] In the present invention, the immune cell includes a population of autologous cells or a population of allogeneic cells. That is, the immune cell includes a population of autologous cells or a population of allogeneic cells that express and secrete an antibody or an antigen-binding fragment thereof that specifically binds to a target selected from the group consisting of human leukocyte antigen G (HLA-G) and its receptor.

[0018] As used herein, "autologous" refers to any substance derived from the same individual that is intended to be reintroduced into the individual. As used herein, "allogeneic" refers to any substance derived from a different animal of the same species as the individual into which the substance is introduced.

[0019] In the present invention, the difference between the two immune cells is the presence or absence of an antigen recognition receptor. In the present invention, the antigen recognition receptor is literally a receptor that recognizes and binds to a specific antigen present on the cell, and activates the immune cell when binding to the antigen.

[0020] In the present invention, the antigen recognition receptor may be exogenous or endogenous.

[0021] The exogenous antigen recognition receptor is not naturally expressed in the immune cells, but is a receptor that is artificially constructed into a receptor construct like a chimeric antigen receptor (CAR) and then transferred and expressed in immune cells.

[0022] The endogenous antigen recognition receptor refers to a receptor that is naturally expressed in the immune cells. For example, there are activation receptors such as the T cell receptor (TCR) in T cells or NKG2D that recognizes abnormal cells in NK cells.

[0023] The classification of the exogenous or endogenous antigen receptor is relative. The TCR in T cells corresponds to the endogenous antigen receptor, while the TCR expressed in other immune cells corresponds to the exogenous antigen receptor.

[0024] Generally, the antigen recognition receptor can be composed of an antigen-binding domain, a leader sequence (LS), a hinge, a transmembrane domain (TM), and an intracellular signaling domain (ICD), but its structure and composition are not limited to the above range.

[0025] The antigen-binding domain recognizes the antigen of the target cell and may be an antibody or an antibody fragment. The signal sequence refers to a sequence that enables the antigen recognition receptor to move to the cell membrane and be expressed and exposed on the cell surface. The transmembrane domain is a domain that connects the antigen-binding domain and the intracellular signaling domain across the cell membrane.

[0026] In the present invention, the signal sequence, hinge, transmembrane domain, and intracellular transmission domain used for the antigen recognition receptor may be those commonly used in the technical field to which the present invention belongs.

[0027] In the present invention, the antigen recognition receptor may bind to only one type of antigen or ligand, or may bind to two or more types of antigens or ligands. The antigen-binding domain can be selected from an antibody that recognizes the target antigen or a molecule that interacts with the antigen. This antigen includes, for example, viral antigens, bacterial (especially infectious bacteria) antigens, parasite antigens, cell surface markers on target cells related to specific pathological conditions (e.g., tumor antigens), and surface molecules of immune-related cells.

[0028] Specific antigens that can be recognized by the antigen recognition receptor may be selected from the group consisting of, but not limited to, CD19, CD20, CD22, CD7, CD10, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CD171, EGFR (epidermal growth factor receptor), PSMA (prostate specific membrane antigen), GD2, EGFR variant, ROR1 (Tyrosine-protein kinase transmembrane receptor 1), c-Met, HER2 (human epidermal growth factor receptor-2), CEA (Carcino embryonic antigen), mesothelin, GM2, MUC16, MUC1, CS1 (CD319), interleukin 13 receptor α2 (IL-13R α2), BCMA (B-cell maturation antigen), LewisY, IgG kappa chain, Folate receptor-alpha, PSCA (Prostate stem cell antigen), EpCAM (Epithelial cell adhesion molecule), NY-ES0-1 (New York esophageal squamous cell carcinoma 1), WT-1 (Wilms’ tumor gene1), MAGE-A1 (MAGE Family Member A1), TAG-72 (Tumor associated glycoprotein-72), NKG2D (KLRK1 (killer cell lectin like receptor K1)), Claudin 18.2, Claudin 3, Claudin 4, and Claudin 6.

[0029] The two immune cells are characterized by expressing and secreting an antibody or an antigen-binding fragment thereof that specifically binds to a target selected from the group consisting of human leukocyte antigen G (HLA-G) and its receptor.

[0030] HLA-G is overexpressed in various carcinomas. Among immune cells, receptors for HLA-G are known to include ILT2 (Ig-like transcript 2), ILT4, KIR2DL4 (Killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 4), etc. All of the above receptors have an ITIM motif (immunoreceptor tyrosine-based inhibitory motif). When HLA-G binds to the receptor, a signal transduction pathway that suppresses immune cells is activated by this motif.

[0031] The antibody or antigen-binding fragment thereof that specifically binds to the human leukocyte antigen G (HLA-G) or its receptor plays a role in blocking the binding of HLA-G and the receptor. As a result, to prevent the activation of the signal transduction pathway that suppresses immune cells, it enables immune cells to overcome immune checkpoints.

[0032] According to one embodiment of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to the human leukocyte antigen G (HLA-G) can include a sequence selected from the following: (a) A middle chain variable region containing a middle chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 21, a middle chain CDR2 having the amino acid sequence of SEQ ID NO: 22, and a middle chain CDR3 having the amino acid sequence of SEQ ID NO: 23; and A light chain variable region containing a light chain CDR1 having the amino acid sequence of SEQ ID NO: 24, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 25, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 26; (b) A middle-chain variable region comprising a middle-chain CDR1 having the amino acid sequence of SEQ ID NO: 27, a middle-chain CDR2 having the amino acid sequence of SEQ ID NO: 28, and a middle-chain CDR3 having the amino acid sequence of SEQ ID NO: 29; and A light-chain variable region comprising a light-chain CDR1 having the amino acid sequence of SEQ ID NO: 30, a light-chain CDR2 having the amino acid sequence of SEQ ID NO: 31, and a light-chain CDR3 having the amino acid sequence of SEQ ID NO: 32; or (c) A middle-chain variable region comprising a middle-chain CDR1 having the amino acid sequence of SEQ ID NO: 33, a middle-chain CDR2 having the amino acid sequence of SEQ ID NO: 34, and a middle-chain CDR3 having the amino acid sequence of SEQ ID NO: 35; and A light-chain variable region comprising a light-chain CDR1 having the amino acid sequence of SEQ ID NO: 36, a light-chain CDR2 having the amino acid sequence of SEQ ID NO: 37, and a light-chain CDR3 having the amino acid sequence of SEQ ID NO: 38.

[0033] More specifically, the antibody or antigen-binding fragment thereof that specifically binds to the human leukocyte antigen G (HLA-G) can comprise a sequence selected from the following: (a) A middle-chain variable region having the amino acid sequence of SEQ ID NO: 15 and a light-chain variable region having the amino acid sequence of SEQ ID NO: 16; (b) A middle-chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light-chain variable region having the amino acid sequence of SEQ ID NO: 18; or (c) A middle-chain variable region having the amino acid sequence of SEQ ID NO: 19 and a light-chain variable region having the amino acid sequence of SEQ ID NO: 20.

[0034] In the present invention, the antibody or antigen-binding fragment thereof that specifically binds to the human leukocyte antigen G (HLA-G) can comprise a sequence selected from the following: (a) A middle-chain variable region having the amino acid sequence of SEQ ID NO: 15 or a sequence having at least 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; and The light chain variable region having the amino acid sequence of SEQ ID NO: 16 or having at least 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; (b) The middle chain variable region having the amino acid sequence of SEQ ID NO: 17 or having at least 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; and The light chain variable region having the amino acid sequence of SEQ ID NO: 18 or having at least 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; or (c) The middle chain variable region having the amino acid sequence of SEQ ID NO: 19 or having at least 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; and The light chain variable region having the amino acid sequence of SEQ ID NO: 20 or having at least 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.

[0035] More specifically, the antibody or antigen-binding fragment thereof that specifically binds to the human leukocyte antigen G (HLA-G) may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3, and may also contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 6.

[0036] In addition, in the present invention, the antibody or antigen-binding fragment thereof that specifically binds to the human leukocyte antigen G (HLA-G) can contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 or an amino acid sequence having at least 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. It can also contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 6 or an amino acid sequence having at least 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.

[0037] In the present invention, the term "antibody" means a protein molecule that functions as a receptor that specifically recognizes an antigen, including immunoglobulin molecules that are reactive with a specifically defined antigen. The antibodies include monoclonal antibodies, polyclonal antibodies, mixtures of monoclonal and / or polyclonal antibodies, full-length antibodies, and antibody fragments. Full-length antibodies or intact antibodies have a structure substantially similar to the natural antibody structure or are used interchangeably to refer to antibodies having a heavy chain containing an Fc region as defined herein.

[0038] Also, the antibody can include bivalent or bispecific molecules (e.g., bispecific antibodies). Further, the antibody can be a human antibody, a humanized antibody, or a chimeric antibody depending on the origin of the sequence.

[0039] In the present invention, the term "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, and such monoclonal antibodies exhibit single binding and affinity for a specific epitope, unlike polyclonal antibodies that can bind to multiple epitopes. In the present invention, the term "full-length antibody" has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to the heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and as subclasses, has gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types. IgG includes IgG1, IgG2, IgG3, and IgG4 as subtypes.

[0040] In the present invention, the term "heavy chain" refers to both the full-length heavy chain and its fragments, which include the variable region domain VH containing an amino acid sequence of a sufficient variable region and three constant region domains CH1, CH2, and CH3 for conferring specificity to an antigen.

[0041] In the present invention, the term "light chain" refers to both the full-length light chain and its fragments, which include the variable region domain VL containing an amino acid sequence of a sufficient variable region and the constant region domain CL for conferring specificity to an antigen.

[0042] In the present invention, the terms "fragment", "antibody fragment", "antigen-binding fragment", or "antigen-binding domain" are used interchangeably to refer to any fragment of an antibody that retains the antigen-binding function of the antibody. Exemplary antigen-binding fragments include, but are not limited to, Fab, Fab’, scFV, F(ab’)2, and Fv.

[0043] The Fab has a structure having the variable regions of the light chain and the middle chain and the constant region of the light chain and the first constant region (CH1 domain) of the middle chain, and has one antigen-binding site. Fab’ is different from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the middle chain CH1 domain. The F(ab’)2 antibody is generated while the cysteine residues in the hinge region of Fab’ form disulfide bonds. Fv is the smallest antibody fragment having only the variable region of the middle chain and the variable region of the light chain.

[0044] In the present invention, an antibody or an antigen-binding fragment thereof that specifically binds to human leukocyte antigen G (HLA-G) can include not only the sequence of the anti-HLA-G scFv described herein, but also its biological equivalents, as long as they can exhibit the ability to bind to HLA-G. For example, additional changes can be made to the amino acid sequence of the antibody in order to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody. Such amino acid mutations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on this point, it can be said that arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine are biologically functionally equivalent.

[0045] According to one embodiment of the present invention, the antibody or an antigen-binding fragment thereof that specifically binds to the human leukocyte antigen G (HLA-G) may be a single-chain Fv (single chain variable fragment (Fv), scFv) containing the selected middle chain variable and light chain variable sequences, and specifically, it may be a soluble single-chain Fv.

[0046] A single-chain Fv is a type of antibody fragment in which the variable regions of the antibody middle chain and light chain are linked by a short linker of 10 to 25 amino acids.

[0047] According to one embodiment of the present invention, the scFv that specifically binds to the human leukocyte antigen G (HLA-G) can be expressed and secreted by immune cells by a nucleic acid consisting of a nucleotide sequence encoding the scFv introduced from outside the cell.

[0048] Therefore, the immune cell can contain a nucleic acid consisting of a nucleotide sequence encoding an scFv that specifically binds to human leukocyte antigen G (HLA-G), and the nucleotide sequence can be selected from the following: A nucleotide sequence encoding a middle chain CDR1 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 21, 27, or 33; a nucleotide sequence encoding a middle chain CDR2 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 22, 28, or 34; and a nucleotide sequence encoding a middle chain CDR3 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 23, 29, or 35, and a nucleotide sequence encoding a middle chain variable region containing the nucleotide sequences; and A nucleotide sequence encoding a light chain CDR1 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 24, 30, or 36; a nucleotide sequence encoding a light chain CDR2 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 25, 31, or 37; and a nucleotide sequence encoding a light chain CDR3 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 26, 32, or 38, and a nucleotide sequence encoding a light chain variable region containing the nucleotide sequences; can be contained.

[0049] More specifically, the nucleotide sequence encoding an scFv that specifically binds to human leukocyte antigen G (HLA-G) can be selected from the following: (a) A nucleotide sequence encoding a middle chain CDR1 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 21, a nucleotide sequence encoding a middle chain CDR2 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 22, and a nucleotide sequence encoding a middle chain CDR3 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 23; and A nucleotide sequence encoding a light chain CDR1 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 24, a nucleotide sequence encoding a light chain CDR2 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 25, and a nucleotide sequence encoding a light chain CDR3 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 26; (b) A nucleotide sequence encoding a middle chain CDR1 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 27, a nucleotide sequence encoding a middle chain CDR2 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 28, and a nucleotide sequence encoding a middle chain CDR3 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 29; and A nucleotide sequence encoding a light chain CDR1 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 30, a nucleotide sequence encoding a light chain CDR2 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 31, and a nucleotide sequence encoding a light chain CDR3 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 32; or (c) A nucleotide sequence encoding a middle chain CDR1 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 33, a nucleotide sequence encoding a middle chain CDR2 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 34, and a nucleotide sequence encoding a middle chain CDR3 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 35; and A nucleotide sequence encoding a light chain CDR1 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 36, a nucleotide sequence encoding a light chain CDR2 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 37, and a nucleotide sequence encoding a light chain CDR3 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 38.

[0050] More specifically, the nucleotide sequence encoding the soluble scFv that specifically binds to the human leukocyte antigen G (HLA-G) can be selected from the following: (a) A nucleotide sequence encoding a middle chain variable region having the amino acid sequence of SEQ ID NO: 15 and a nucleotide sequence encoding a light chain variable region having the amino acid sequence of SEQ ID NO: 16; (b) A nucleotide sequence encoding a middle chain variable region having the amino acid sequence of SEQ ID NO: 17 and a nucleotide sequence encoding a light chain variable region having the amino acid sequence of SEQ ID NO: 18; or (c) A nucleotide sequence encoding a heavy-chain variable region having the amino acid sequence of SEQ ID NO: 19 and a nucleotide sequence encoding a light-chain variable region having the amino acid sequence of SEQ ID NO: 20.

[0051] In the present invention, the nucleotide sequence encoding the soluble scFv is a nucleotide sequence encoding a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 15, 17 or 19, or a mutant thereof having at least 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; and it can contain a nucleotide sequence encoding a light-chain variable region containing the amino acid sequence of SEQ ID NO: 16, 18 or 20, or a mutant thereof having at least 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.

[0052] In the present invention, the nucleotide sequence encoding the soluble scFv can be selected from the following sequences: (a) A nucleotide sequence encoding a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more sequence identity with SEQ ID NO: 15, and a nucleotide sequence encoding a light-chain variable region containing the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more sequence identity with SEQ ID NO: 16; (b) A nucleotide sequence encoding a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17, or 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 SEQ ID NO: 17, and the amino acid sequence of SEQ ID NO: 18, or 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 SEQ ID NO: 18; or (c) A nucleotide sequence encoding a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19, or 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 SEQ ID NO: 19, and the amino acid sequence of SEQ ID NO: 20, or 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 SEQ ID NO: 20; may be included.

[0053] On the other hand, the present inventors prepared an expression vector for expressing a soluble scFv specifically binding to human leukocyte antigen G (HLA-G) or the soluble scFv and an antigen recognition receptor in immune cells.

[0054] Therefore, another aspect of the present invention provides the following expression vectors for producing the immune cells: (a) An expression vector comprising a nucleotide sequence encoding an antigen recognition receptor; and a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof that specifically binds to a target selected from the group consisting of human leukocyte antigen G and its receptor; (b) An expression vector comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof that specifically binds to a target selected from the group consisting of human leukocyte antigen G and its receptor; and (c) The following two expression vectors of (c-1) and (c-2): (c-1) An expression vector containing a nucleotide sequence encoding an antigen recognition receptor; and An expression vector containing a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof that specifically binds to a target selected from the group consisting of human leukocyte antigen G and its receptor.

[0055] In the expression vectors of (a), (b) and (c) above, the nucleotide sequence encoding an antibody or an antigen-binding fragment thereof that specifically binds to human leukocyte antigen G is the same as the nucleotide sequence described for immune cells.

[0056] According to one embodiment of the present invention, in the expression vector of (a) above, the nucleotide sequence encoding an antigen recognition receptor and the nucleotide sequence encoding an antibody or an antigen-binding fragment thereof that specifically binds to a target selected from the group consisting of human leukocyte antigen G and its receptor may be linked via a nucleotide sequence encoding a self-cleaving peptide (Figs. 1a - 1c). The nucleotide sequence encoding the self-cleaving peptide is the sequence encoding the amino acid sequence of SEQ ID NO: 7.

[0057] According to one embodiment of the present invention, in the expression vectors of (a), (b) and (c) above, the nucleotide sequence encoding an antibody or an antigen-binding fragment thereof that specifically binds to human leukocyte antigen G may specifically be a nucleotide sequence encoding a signal sequence, a nucleotide sequence encoding an anti-HLA-G binding scFv, and a nucleotide sequence encoding a fragment crystallization region (Fc) of human immunoglobulin, which are sequentially linked (Figs. 1a - 1c).

[0058] Further, the nucleotide sequence encoding the anti-HLA-G binding scFv contains a nucleotide sequence encoding a linker sequence between the nucleotide sequences encoding the heavy chain and the light chain variable regions, and the linker sequence may be the sequence of SEQ ID NO: 13.

[0059] According to one embodiment of the present invention, the signal sequence serves to move and secrete the anti-HLA-G binding scFv expressed intracellularly to the outside of the cell. Conventional immune checkpoint inhibitory antibodies such as Pembrolizumab (trade name: Keytruda) administer the antibody itself into the body. However, in the present invention, since the anti-HLA-G binding scFv is secreted outside the cell even when immune cells are administered, all the effects of the immune cells and scFv can be expected. In addition, since the anti-HLA-G binding scFv is expressed and secreted only near cancer cells where immune cells are present, rather than throughout the body, systemic side effects can be reduced.

[0060] The nucleotide sequence encoding the signal sequence may be an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 12, or a nucleotide sequence encoding 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 selected from the group consisting of SEQ ID NOs: 8 to 12. In the examples of the present invention, the signal sequence of SEQ ID NO: 8 was used.

[0061] The Fc region maintains the stability and biological activity of the anti-HLA-G binding scFv, and the nucleotide sequence encoding the Fc region may be a sequence commonly used in the technical field to which the present invention belongs. In the examples of the present invention, the sequence of SEQ ID NO: 14 was used.

[0062] In the present invention, the anti-HLA-G binding scFv can also be referred to as anti-HLA-G scFv, antibody fragment scFv, etc.

[0063] In the present invention, an expression vector containing a nucleic acid encoding a soluble scFv that specifically binds to human leukocyte antigen G can also be referred to as a soluble scFv expression vector.

[0064] The vectors used in the present invention can use a variety of vectors known in the art, and according to the type of cells in which the antigen recognition receptor and / or soluble scFv are to be expressed, expression regulatory sequences such as promoters, terminators, enhancers, etc., sequences for membrane targeting or secretion, etc. can be appropriately selected and variously combined according to the purpose.

[0065] The method of introducing and expressing the vector of the present invention into cells is well-known in the relevant technical fields. The vector can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by methods known in the art. For example, the vector can be transferred 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 colloid dispersion systems, such as lipid-based systems including macromolecular complexes, nanocapsules, microspheres, beads, and oil-in-water emulsions, micelles, mixed micelles, and liposomes. Also, the biological means include the use of DNA or RNA vectors such as the aforementioned lentiviruses, retroviruses, etc.

[0066] The vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, virus vectors, etc. Suitable vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression regulatory elements such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers, and can be variously produced according to the purpose.

[0067] In one embodiment of the present invention, a lentiviral vector was used. In a specific embodiment of the present invention, the vector further comprises a promoter. The promoter may be, for example, but not limited to, the RSV promoter.

[0068] Another aspect of the present invention is an isolated nucleic acid comprising a nucleotide sequence encoding the anti-HLA-G binding soluble scFv.

[0069] As used herein, the term "nucleic acid" has the meaning of comprehensively including DNA and RNA molecules, and the nucleotide, which is the basic structural unit of nucleic acid molecules, includes 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).

[0070] The nucleotide sequence encoding the anti-HLA-G binding scFv of the present invention can be modified, and the modifications include addition, deletion, or non-conservative substitution or conservative substitution of nucleotides.

[0071] The nucleic acid (polynucleotide) comprising the nucleotide sequence encoding the soluble scFv according to the present invention can be modified by codon optimization, which is due to the degeneracy of codons. It will be well understood by those of ordinary skill that there are many nucleotide sequences encoding polypeptides or variant fragments thereof. Some of these nucleic acids have minimal homology with the nucleotide sequence of any naturally occurring gene.

[0072] In particular, nucleic acids that vary due to differences in codon usage, such as nucleic acids optimized for human, primate, and / or mammalian codon selection, are preferred.

[0073] In the present invention, the isolated nucleic acid can comprise the following sequences: (a) A nucleotide sequence encoding a middle-chain CDR1 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 21, a nucleotide sequence encoding a middle-chain CDR2 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 22, and a nucleotide sequence encoding a middle-chain CDR3 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 23; and A nucleotide sequence encoding a light-chain CDR1 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 24, a nucleotide sequence encoding a light-chain CDR2 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 25, and a nucleotide sequence encoding a light-chain CDR3 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 26; (b) A nucleotide sequence encoding a middle-chain CDR1 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 27, a nucleotide sequence encoding a middle-chain CDR2 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 28, and a nucleotide sequence encoding a middle-chain CDR3 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 29; and A nucleotide sequence encoding a light-chain CDR1 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 30, a nucleotide sequence encoding a light-chain CDR2 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 31, and a nucleotide sequence encoding a light-chain CDR3 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 32; or (c) A nucleotide sequence encoding a middle-chain CDR1 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 33, a nucleotide sequence encoding a middle-chain CDR2 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 34, and a nucleotide sequence encoding a middle-chain CDR3 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 35; and A nucleotide sequence encoding a light chain CDR1 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 36, a nucleotide sequence encoding a light chain CDR2 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 37, and a nucleotide sequence encoding a light chain CDR3 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 38.

[0074] More specifically, the isolated nucleic acid can comprise the following sequences: (a) A nucleotide sequence encoding a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and a nucleotide sequence encoding a light chain variable region having the amino acid sequence of SEQ ID NO: 16; (b) A nucleotide sequence encoding a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a nucleotide sequence encoding a light chain variable region having the amino acid sequence of SEQ ID NO: 18; or (c) A nucleotide sequence encoding a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and a nucleotide sequence encoding a light chain variable region having the amino acid sequence of SEQ ID NO: 20.

[0075] Also, according to one embodiment of the present invention, the isolated nucleic acid further comprises a nucleotide sequence encoding the linker sequence of SEQ ID NO: 13 between the nucleotide sequence encoding the heavy chain variable region and the nucleotide sequence encoding the light chain variable region.

[0076] On the other hand, the inventors have confirmed that the soluble scFv is well secreted extracellularly from anti-HLA-G soluble scFv-expressing immune cells (FIGS. 5 and 11), and that HER2 CAR-T cells expressing the soluble scFv do not cause a decrease in the function of immune cells by HLA-G-expressing cancer cell lines (FIG. 10).

[0077] That is, the immune cells of the present invention can co-express a soluble scFv that can inhibit the interaction between HLA-G and its receptor together with an antigen recognition receptor, and not only can exhibit an anti-cancer effect, but also when the soluble scFv is expressed alone, in the process of recognizing and removing the target (cancer cells) with an endogenous antigen recognition receptor, the soluble scFv can contribute to the enhancement of the anti-cancer efficacy. Such an anti-cancer efficacy enhancement effect can also act on endogenous immune cells such as tumor-infiltrating lymphocytes, so that the activation of endogenous immune cells can be further induced.

[0078] Therefore, the present invention also provides a pharmaceutical composition containing the aforementioned immune cells of the present invention.

[0079] According to one embodiment of the present invention, the pharmaceutical composition can be used for the prevention or treatment of cancer.

[0080] In the present invention, "cancer" and "tumor" are used interchangeably and typically refer to or mean a physiological state of a mammal characterized by unregulated cell growth / proliferation.

[0081] As used herein, the term "prevention" means any act of suppressing or delaying the progression of a disease by administration of the composition of the present invention, and "treatment" means suppressing, alleviating or removing the progression of a disease.

[0082] In the present invention, examples of the cancer (or tumor) include, but are not limited to, carcinomas, lymphomas (e.g., Hodgkin's and non-Hodgkin's lymphomas), blastomas, sarcomas, and leukemias. More preferred examples of cancer include pancreatic cancer, breast cancer, ovarian cancer, glioma, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, nasopharyngeal cancer, oral cancer, thyroid cancer, prostate cancer, kidney cancer, melanoma, gallbladder cancer, bile duct cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, bladder cancer, colon cancer, colorectal cancer, salivary gland cancer, vulvar cancer, mesothelioma, leukemia and other lymphoproliferative diseases, and various types of head and neck cancers.

[0083] More specifically, the pharmaceutical composition can be used for the treatment of cancers that are HLA-G expression positive, and the cancers can 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, colorectal cancer, nasopharyngeal cancer, oral cancer, thyroid cancer, prostate cancer, kidney cancer, gallbladder cancer, bile duct cancer, and melanoma.

[0084] The pharmaceutical composition of the present invention can further contain a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is one commonly used in formulation, and includes, but is 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, etc. The composition for cancer prevention or treatment of the present invention can further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in the literature [Remington’s Pharmaceutical Sciences (19th ed., 1995)].

[0085] The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intralung administration, rectal administration, etc. When administered orally, since proteins or peptides are digested, the oral composition can coat the active drug or be formulated to be protected from degradation in the stomach, and the composition of the present invention can be administered by any device capable of moving the active substance to the target cells.

[0086] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, mode of administration, age, weight, sex, medical condition, food, administration time, route of administration, excretion rate, and reaction sensitivity of the patient. Usually, a skilled physician can easily determine and prescribe a pharmaceutically effective amount effective for the desired treatment or prevention. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat, prevent, and diagnose a disease.

[0087] The pharmaceutical composition of the present invention may contain, within a single dose, the number of the immune cells (e.g., T cells) being 1 to 10 times the number of the tumor cells in the treatment target, such as pancreatic cancer cells, breast cancer cells, ovarian cancer cells, or glioma cells.

[0088] The pharmaceutical composition of the present invention is formulated using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily implemented by those having ordinary knowledge in the technical field to which the invention pertains, and can be manufactured in unit dosage form or enclosed in a multi-dose container. At this time, 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, acid agent, suppository, powder, granule, tablet, or capsule, and may further contain a dispersant or stabilizer.

[0089] The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents.

[0090] Another aspect of the present invention provides the use of the immune cells according to any one of claims 1 to 10 for the manufacture of a pharmaceutical composition for the prevention or treatment of cancer, and the prevention or treatment of cancer by the use of the immune cells according to any one of claims 1 to 10.

[0091] The present invention also provides a method for preventing or treating cancer, which includes the step of administering the aforementioned immune cells or pharmaceutical composition to a subject in need thereof.

[0092] The subject means all animals including monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs, including humans with cancer or those who may develop cancer.

[0093] The cancer may be, but is not limited to, a cancer in which HLA-G is overexpressed.

Advantages of the Invention

[0094] The present invention provides immune cells that overcome immune checkpoints and a pharmaceutical composition using the same. HLA-G acts as an immune checkpoint in various solid cancers and inhibits the normal function of tumor-infiltrating lymphocytes. However, the immune cells according to the present invention express and secrete a soluble scFv derived from an anti-HLA-G antibody, so that they can overcome the immunosuppressive reaction and more efficiently remove cancer cells, and can also further induce the activation of endogenous immune cells. Therefore, they can be used for the treatment of various cancers and the like related to the high expression of HLA-G.

Brief Description of the Drawings

[0095]

Figure 1a

Figure 1b

Figure 1c

Figure 2a

Figure 2b

Figure 2c

Figure 3a

Figure 3b

Figure 3c

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10a

Figure 10b

Figure 11

Mode for Carrying Out the Invention

[0096] Hereinafter, the present invention will be described in more detail through examples. It will be apparent to those having ordinary knowledge in the art that these examples are solely for illustrating the present invention and should not be construed as limiting the scope of the present invention by these examples.

[0097] Example 1. Cell Lines and Culture The human ovarian cancer cell line SK-OV-3 and the human acute leukemia cell line Jurkat were supplied and used from the Korea Cell Line Bank. The SK-OV-3-HLA-G cell line that stably overexpresses HLA-G and the Jurkat-ILT2 cell line that stably overexpresses ILT2 (Ig-like transcript 2), which is an HLA-G receptor, were prepared from the SK-OV-3 or Jurkat cell line, respectively, using a lentivral vector. The SK-OV-3-HLA-G and Jurkat-ILT2 cell lines were cultured in RPMI 1640 (Gibco, USA) medium containing 10% FBS (Gibco, USA), 1% antibiotic (Antibiotic-Antimycotic; Gibco, USA), and 0.5 μg / mL puromycin (Sigma-aldrich, USA).

[0098] Example 2. Construction of anti-HLA-G soluble scFv construct The antigen recognition receptor and the anti-HLA-G soluble scFv expression construct of the present invention were artificially synthesized. More specifically, the antigen recognition receptor and the soluble scFv were linked with a self-cleaving peptide, and the soluble scFv was artificially synthesized by SOE-PCR (Splicing by overlap extension PCR) in a state where a human immunoglobulin heavy chain signal sequence, an HLA-G antibody fragment scFv, and a human immunoglobulin Fc region were linked (Figs. 1a-1c).

[0099] After the PCR product was digested with MluI and XbaI, it was inserted into the MluI and XbaI sites of the pLV-EF1A-MCS vector, which is a third-generation self-inactivating lentiviral expression vector (Figs. 2a-2c).

[0100] For immune cells using endogenous antigen recognition receptors, constructs were also prepared that express only soluble scFv without antigen recognition receptors. The soluble scFv was artificially synthesized by SOE-PCR with the human immunoglobulin heavy chain signal sequence, HLA-G antibody fragment scFv, and human immunoglobulin Fc region linked together. After cleaving the PCR product with MluI and XbaI, it was inserted into the MluI and XbaI sites of the pLV-EF1A-MCS vector, a third-generation self-inactivating lentiviral expression vector (Figures 3a - 3c).

[0101] The information on the sequences of the anti-HLA-G soluble scFv according to the examples of the present invention and the domains used in its production was organized in Table 1. Each site constituting the soluble scFv is linked in tandem with each other and in frame. Specifically, it is linked with the signal sequence, antibody fragment scFv, Fc region, and the stop codon TAA.

[0102]

Table 1(1)

Table 1(2)

Table 1(3)

Table 1(4)

Table 1(5)

Table 1(6)

Table 1(7)

[0103] Example 3. Preparation of CAR-Jurkat cell line expressing anti-HLA-G soluble scFv 3-1. Production of Lentivirus Expressing Anti-HLA-G Soluble scFv Gene HER2 CAR was selected as an example of the antigen recognition receptor. Lentivirus for the transfer of these genes was prepared by the plasmid DNA transformation method so that HER2 CAR and anti-HLA-G soluble scFv could be co-expressed through a self-cleaving peptide. TransIT-293 Transfection Reagent (Mirus Bio LLC, USA) was used according to the manufacturer's protocol. One day before the experiment, Lenti-X TM 293T cell line (Clontech, USA) was seeded at 5×10 6 cells per 100 mm dish, and the next day, the pLV-EF1A-antigen recognition receptor-soluble scFv lentiviral vector, gag-pol expression vector, and VSV-G envelope expression vector were transfected. The antigen recognition receptor-soluble scFv lentiviral vectors were as follows: HER2 CAR, HER2 CAR-soluble scFv2, or HER CAR-soluble scFv3. After transfection, the cells were cultured for about 72 hours, and after the culture was completed, all the cell supernatants were collected. The supernatant was filtered through a 0.45 μm filter (Millipore, USA) to remove cell debris. Lenti-X TM Concentrator (Clontech, USA) was used to concentrate the lentivirus about 100-fold according to the manufacturer's protocol and stored frozen at -80 °C until use.

[0104] 3-2. Preparation and Culture of Anti-HLA-G Soluble scFv-Expressing CAR-Jurkat Cell Line The Jurkat-ILT2 cell line was placed in a 6-well plate at 1×10 6 cells per well together with lentivirus at an MOI (Multiplicity of infection) of about 5, and the culture medium was adjusted to 2 mL. Furthermore, polybrene was added to the medium at a concentration of 8 μg / mL. After static culture for 24 hours, the medium was replaced with fresh medium. The transduced Jurkat-ILT2 cell line was maintained by subculture at 2×10 5 cells per mL at intervals of 2 - 3 days.

[0105] 3-3. Confirmation of Secretion of Soluble scFv from HLA-G-Specific Soluble scFv-Expressing CAR-Jurkat Cell Line Using the previously generated HLA-G-specific soluble scFv-expressing CAR-Jurkat cell line, we confirmed whether HER CAR was expressed on the cell surface. Since HER2 CAR and the soluble scFv are linked by a self-cleaving peptide, if CAR is expressed normally, it can be predicted that the soluble scFv will also be expressed normally.

[0106] 1×10 5 After collecting 1×10⁶ cells, they were suspended in 100 μL of D-PBS to prepare a cell suspension. Recombinant Protein L (PE conjugated) (Sino Biological, China) and Human ILT2 APC-conjugated Antibody (R&D systems, USA) were added to the cell sample and reacted at 4°C for 30 minutes. After the reaction, the cells were washed twice with D-PBS, and the expression levels of HER2 CAR and ILT2 were confirmed by flow cytometry.

[0107] As a result, in the CAR-Jurkat cell line expressing the soluble scFv, almost all cells expressed HER2 CAR, and more than half of them also expressed ILT2 simultaneously (Figure 4).

[0108] Since the soluble scFv needs to be secreted extracellularly to function, we confirmed whether the soluble scFv was expressed normally and secreted extracellularly. After seeding 8×10⁵ cells in a 24-well plate, the medium was collected the next day, and Minute 5 cells were collected. TMThe protein in the medium was obtained using a High-Efficiency Protein Precipitation Kit (Invent Biotechnologies, USA). The protein obtained by Western blotting was detected using Goat Anti-Human IgG Antibody, Fc, HRP conjugate (Sigma-aldrich, USA).

[0109] As a result, it was confirmed that the soluble scFv was well secreted and stably present in the CAR-Jurkat cell line expressing the soluble scFv (Figure 5).

[0110] Example 4. Confirmation of the anti-cancer efficacy of the anti-HLA-G soluble scFv-expressing CAR-Jurkat cell line in vitro The SK-OV-3-HLA-G cell line was used to confirm the anti-cancer efficacy of the anti-HLA-G soluble scFv-expressing CAR-Jurkat cell line. The SK-OV-3-HLA-G cell line is a cell line positive for both HER2 and HLA-G. Immune cells can exhibit anti-cancer efficacy against the target cell line by HER2 CAR, and conversely, the efficacy of immune cells may be suppressed by HLA-G. Anti-HLA-G soluble scFv-expressing CAR-Jurkat or control group Jurkat cell lines were added to a 48-well plate at 2×10 5 cells / 150 uL per well, and then the SK-OV-3-HLA-G cell line was added at 2×10 5 cells / 150 uL and reacted for 48 hours.

[0111] The cell supernatant was collected, and the concentration of IL-2 in the collected supernatant was measured using a Human IL-2 Quantikine ELISA Kit (R&D systems, USA). As a result, it was confirmed that the secretion of IL-2 increased significantly when the HER2 CAR-Jurkat cell line expressing the anti-HLA-G soluble scFv was co-cultured with the target cell line compared to the Jurkat cell line expressing only HER2 CAR (Figure 6).

[0112] Simultaneously, after collecting the co-cultured cells, CD25, an activation marker of the Jurkat cell line, was detected by flow cytometry using CD25 Antibody, anti-human, PE-Vio®770, REAfinity TM (Miltenyi Biotec, Germany). The results showed that, compared with the Jurkat cell line expressing only HER2 CAR, the cell surface expression rate of CD25 increased significantly only when the HER2 CAR-Jurkat cell line expressing anti-HLA-G soluble scFv was co-cultured with the target cell line (Figure 7).

[0113] From the above results, it was found that when anti-HLA-G soluble scFv was expressed and secreted, a certain part of the reduction in the function of immune cells caused by HLA-G expressed by the target cell line was restored again.

[0114] Example 5. Preparation of CAR-T cells expressing anti-HLA-G soluble scFv 5-1. Production of lentivirus expressing ILT2 gene and lentivirus expressing anti-HLA-G soluble scFv gene Lentiviruses for the delivery of the ILT2 gene and the anti-HLA-G soluble scFv gene were prepared by the plasmid DNA transformation method. TransIT-293 Transfection Reagent (Mirus Bio LLC, USA) was used according to the manufacturer's protocol. One day before the experiment, Lenti-X TM 293T cell line (Clontech, USA) was seeded at 5×10 6Cells were seeded at a density of [number] cells per well, and the next day, they were transfected with the pLVX-puro-ILT2 lentiviral vector, the pLV-EF1A-antigen recognition receptor-soluble scFv lentiviral vector, the gag-pol expression vector, and the VSV-G envelope expression vector. The antigen recognition receptor-soluble scFv lentiviral vector was as follows: HER2 CAR, HER2 CAR-soluble scFv2, or HER CAR-soluble scFv3. After transfection, the cells were cultured for approximately 72 hours. After the culture was completed, all the cell supernatants were collected. The supernatants were filtered through a 0.45-μm filter (Millipore, USA) to remove cell debris. Lenti-X TM Concentrator (Clontech, USA) was used to concentrate the lentivirus approximately 100-fold according to the manufacturer's protocol and stored frozen at -80°C until use.

[0115] 5-2. Generation and culture of anti-HLA-G soluble scFv-expressing CAR-T cells Donors were recruited and leukocytes were obtained by leukapheresis. Peripheral blood mononuclear cells (PBMCs) were obtained from the leukocytes using SepMate TM -50 (STEMCELL technology, Canada) and Ficoll-Paque PLUS (GE healthcare, Sweden). Subsequently, human T cells were isolated by positive selection using the QuadroMACS TM Separator (Miltenyi Biotec, Germany), the LS column (Miltenyi Biotec, Germany), human CD4 MicroBeads (Miltenyi Biotec, Germany), and human CD8 MicroBeads (Miltenyi Biotec, Germany). The human T cells were cultured in TexMACS TM medium (Miltenyi Biotec, Germany) using T Cell TransAct TM, human (Miltenyi Biotec, Germany) was added and activated. Human IL-2 (R&D systems, USA) was added to the culture medium at 100 U / mL for the growth of T cells and cultured. After 24 hours of culture, the activated T cells were collected and used for lentiviral transduction.

[0116] To transfer both the ILT2 gene and the anti-HLA-G soluble scFv gene, gene transfer was performed at one-day intervals, and RetroNectin® (Takara, Japan) was used to increase the transfer efficiency. The lentivirus expressing the ILT2 gene with an MOI of about 10 was placed in a 24-well plate coated with RetroNectin, and the lentivirus was adsorbed according to the manufacturer's protocol. Then, 5×10 5 activated human T cells were placed in each well of the plate with the adsorbed lentivirus so that the culture medium volume was 1.25 mL. Then, centrifugation was performed at 1,000×g for 15 minutes to transfer the ILT2 gene-expressing lentivirus to the T cells. After 24 hours, in the same manner, the lentivirus expressing the anti-HLA-G soluble scFv gene was transferred to the T cells. Then, after the T cells were statically cultured for 48 hours, they were transferred to a new medium. The transduced human T cells were subcultured at 5×10 5 cells per mL at 2 - 3-day intervals and maintained so that the cell number did not exceed 2×10 6 cells per mL. Unless otherwise specified, human T cells were cultured with IL-2 (R&D systems, USA) added to the culture medium at 100 U / mL.

[0117] 5-3. Confirmation of Secretion of Soluble scFv from Anti-HLA-G Soluble scFv-Expressing CAR-T Cells It was confirmed whether HER CAR was expressed on the cell surface in the previously prepared anti-HLA-G soluble scFv-expressing CAR-T cells. Since HER2 CAR and soluble scFv are linked by a self-cleaving peptide, it can be predicted that if CAR is normally expressed, soluble scFv will also be normally expressed.

[0118] 1×10 5 After collecting 1×10 5 cells, they were suspended in 100 uL of D-PBS to prepare a cell suspension. Recombinant Protein L (PE conjugated) (Sino Biological, China) and Human ILT2 APC-conjugated Antibody (R&D systems, USA) were added to the cell sample and reacted at 4°C for 30 minutes. After the reaction, the cells were washed twice with D-PBS, and the expression levels of HER2 CAR and ILT2 were confirmed by flow cytometry.

[0119] As a result, in the cells transfected with HER2 CAR, the CAR expression rate was at the level of 35 - 45%, and in the cells transfected with ILT2, the ILT2 expression rate was at the level of 20 - 30% (Figure 8).

[0120] Since soluble scFv needs to be secreted extracellularly to function, it was confirmed whether soluble scFv was normally expressed and secreted extracellularly. After seeding 8×10 5 cells in a 24-well plate, all the culture medium was collected the next day, and proteins in the culture medium were obtained using the Minute TM High-Efficiency Protein Precipitation Kit (Invent Biotechnologies, USA). The proteins obtained by Western blotting were detected using Goat Anti-Human IgG Antibody, Fc, HRP conjugate (Sigma-aldrich, USA).

[0121] As a result, it was confirmed that soluble scFv was well secreted from the CAR-T cells expressing soluble scFv and stably existed (Figure 9).

[0122] Example 6. Confirmation of the anti-cancer efficacy of anti-HLA-G soluble scFv-expressing CAR-T cells in vitro The SK-OV-3-HLA-G cell line was used to confirm the anti-cancer efficacy of anti-HLA-G soluble scFv-expressing CAR-T cells. The SK-OV-3-HLA-G cell line is a cell line positive for both HER2 and HLA-G. Immune cells can exhibit anti-cancer efficacy against the target cell line by HER2 CAR. Conversely, the efficacy of immune cells may be suppressed by HLA-G in the target cell line. The SK-OV-3-HLA-G cell line (target cell = T) was added to a 96-well plate at 1×10 4 cells / 150 uL per well. After 24 hours, anti-HLA-G soluble scFv-expressing CAR-T or control group T cells (effector cells = E) were added at 5×10 4 cells / 70 uL or 3×10 4 cells / 70 uL (E:T ratio = 5:1 or 3:1) and reacted for 48 hours.

[0123] The cell supernatant was collected, and the concentration of IL-2 was measured from the collected supernatant using the Human IL-2 Quantikine ELISA Kit (R&D systems, USA). As a result, in both cases of E:T ratios 5 and 3, compared with CAR-T cells expressing only HER2 CAR, the secretion of IL-2 by HER2 CAR-T-ILT2 cells that further express ILT2 decreased, and it was confirmed that such a decrease was significantly restored again in HER2 CAR-T cells expressing anti-HLA-G soluble scFv (Figure 10; Figure 10a - E:T = 5:1, Figure 10b - E:T = 3:1).

[0124] From the above results, it was found that when anti-HLA-G soluble scFv is expressed and secreted, the decrease in the function of immune cells due to HLA-G expressed by the target cell line is restored to a certain extent again.

[0125] Example 7. Preparation of a Jurkat cell line expressing anti-HLA-G soluble scFv 7-1. Production of a lentivirus expressing the anti-HLA-G soluble scFv gene An anti-HLA-G soluble scFv single-expression construct prepared for immune cells that use endogenous antigen recognition receptors without exogenous antigen recognition receptors such as CAR was used, and lentiviruses for the transfer of these genes were prepared by the plasmid DNA transformation method. It was performed according to the manufacturer's protocol using TransIT-293 Transfection Reagent (Mirus Bio LLC, USA). One day before the experiment, Lenti-X TM 293T cell line (Clontech, USA) was seeded at 5×10 6 cells per 100 mm dish, and the next day, it was transformed with the pLV-EF1A-soluble scFv lentiviral vector, the gag-pol expression vector, and the VSV-G envelope expression vector. The soluble scFv lentiviral vectors are as follows: soluble scFv2 or soluble scFv3. After transformation, the cells were cultured for about 72 hours, and after the culture was completed, all the cell supernatants were collected. The supernatant was filtered through a 0.45 μm filter (Millipore, USA) to remove cell debris. Lenti-X TM Concentrator (Clontech, USA) was used to concentrate the lentivirus about 100-fold according to the manufacturer's protocol and stored frozen at -80 °C until use.

[0126] 7-2. Preparation and culture of anti-HLA-G soluble scFv-expressing Jurkat cell line The Jurkat-ILT2 cell line was placed in a 6-well plate at 1×10 6 cells per well together with lentivirus at a multiplicity of infection (MOI) of about 5, and the culture medium was adjusted to 2 mL. Furthermore, polybrene was added to the medium at a concentration of 8 μg / mL. After static culture for 24 hours, the medium was replaced with fresh medium. The transduced Jurkat-ILT2 cell line was maintained by subculturing at 2×10 5 cells per mL at intervals of 2 to 3 days.

[0127] 7-3. Confirmation of secretion of soluble scFv from anti-HLA-G soluble scFv-expressing Jurkat cell line Since the soluble scFv needs to be secreted extracellularly to function, we confirmed whether the soluble scFv was normally expressed and secreted extracellularly. After seeding 8×10 5 cells in a 24-well plate, the next day all the culture medium was collected, and the proteins in the medium were obtained using the Minute TM High-Efficiency Protein Precipitation Kit (Invent Biotechnologies, USA). The proteins obtained by Western blotting were detected with Goat Anti-Human IgG Antibody, Fc, HRP conjugate (Sigma-aldrich, USA).

[0128] As a result, it was confirmed that the soluble scFv was well secreted and stably present in the Jurkat cell line expressing the soluble scFv (Figure 11).

Claims

1. An antigen - recognizing receptor; and An antibody or an antigen - binding fragment thereof that specifically binds to a target selected from the group consisting of human leukocyte antigen G (HLA - G) and its receptor; An immune cell that expresses and secretes.

2. An immune cell that expresses and secretes an antibody or an antigen - binding fragment thereof that specifically binds to a target selected from the group consisting of human leukocyte antigen G (HLA - G) and its receptor.

3. The immune cell according to claim 1 or claim 2, wherein the antibody or antigen - binding fragment that specifically binds to the human leukocyte antigen G comprises a sequence selected from the following: (a) A middle - chain variable region comprising a middle - chain complementarity - determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 21, a middle - chain CDR2 having the amino acid sequence of SEQ ID NO: 22, and a middle - chain CDR3 having the amino acid sequence of SEQ ID NO: 23; and A light - chain variable region comprising a light - chain CDR1 having the amino acid sequence of SEQ ID NO: 24, a light - chain CDR2 having the amino acid sequence of SEQ ID NO: 25, and a light - chain CDR3 having the amino acid sequence of SEQ ID NO: 26; (b) A middle - chain variable region comprising a middle - chain CDR1 having the amino acid sequence of SEQ ID NO: 27, a middle - chain CDR2 having the amino acid sequence of SEQ ID NO: 28, and a middle - chain CDR3 having the amino acid sequence of SEQ ID NO: 29; and A light - chain variable region comprising a light - chain CDR1 having the amino acid sequence of SEQ ID NO: 30, a light - chain CDR2 having the amino acid sequence of SEQ ID NO: 31, and a light - chain CDR3 having the amino acid sequence of SEQ ID NO: 32; or (c) A middle - chain variable region comprising a middle - chain CDR1 having the amino acid sequence of SEQ ID NO: 33, a middle - chain CDR2 having the amino acid sequence of SEQ ID NO: 34, and a middle - chain CDR3 having the amino acid sequence of SEQ ID NO: 35; and A light - chain variable region comprising a light - chain CDR1 having the amino acid sequence of SEQ ID NO: 36, a light - chain CDR2 having the amino acid sequence of SEQ ID NO: 37, and a light - chain CDR3 having the amino acid sequence of SEQ ID NO:

38.

4. The immune cell according to claim 1 or claim 2, wherein the antibody or its antigen - binding fragment that specifically binds to the human leukocyte antigen G is a single - chain Fv (scFv).

5. The immune cell according to claim 4, wherein the single - chain Fv is secreted from the immune cell.

6. The single-chain Fv improves the immune response of the immune cells and tumor-infiltrating lymphocytes, and the immune cell according to claim 4.

7. The immune cell according to claim 1 or claim 2, wherein the immune cell is selected from the group consisting of natural killer cells, T lymphocytes, B lymphocytes, macrophages, dendritic cells, natural killer dendritic cells, mast cells, and precursor cells thereof.

8. The immune cell according to claim 1, wherein the antigen recognition receptor is an exogenous or endogenous antigen recognition receptor.

9. The immune cell according to claim 1, wherein the antigen recognition receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

10. The antigen recognition receptor recognizes an antigen selected from the group consisting of CD19, CD20, CD22, CD7, CD10, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CD171, EGFR (epidermal growth factor receptor), PSMA (prostate specific membrane antigen), GD2, EGFR variant, ROR1 (Tyrosine-protein kinase transmembrane receptor 1), c-Met, HER2 (human epidermal growth factor receptor-2), CEA (Carcinoembryonic antigen), mesothelin, GM2, MUC16, MUC1, CS1 (CD319), interleukin 13 receptor α2 (IL-13Rα2), BCMA (B-cell maturation antigen), LewisY, IgG kappa chain, folate receptor α (Folate receptor-alpha), PSCA (Prostate stem cell antigen), EpCAM (Epithelial cell adhesion molecule), NY-ESO-1 (New York esophageal squamous cell carcinoma 1), WT-1 (Wilms’ tumor gene1), MAGE-A1 (MAGE Family Member A1), TAG-72 (Tumor associated glycoprotein-72), NKG2D (KLRK1 (killer cell lectin like receptor K1)), claudin 18.2, claudin 3, claudin 4, and claudin 6. The immune cell according to claim 1.

11. The following expression vector (a) to (c) for producing the immune cell according to claim 1 or claim 2: An expression vector containing a nucleotide sequence encoding an antigen recognition receptor and a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof that specifically binds to a target selected from the group consisting of human leukocyte antigen G and its receptor; An expression vector containing a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof that specifically binds to a target selected from the group consisting of human leukocyte antigen G and its receptor; and (c) The following two expression vectors of (c-1) and (c-2): (c-1) An expression vector containing a nucleotide sequence encoding an antigen recognition receptor; and (c-2) An expression vector containing a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof that specifically binds to a target selected from the group consisting of human leukocyte antigen G and its receptor.

12. The expression vector according to claim 11, wherein in the expression vector of (a), the nucleotide sequence encoding an antigen recognition receptor and the nucleotide sequence encoding an antibody or an antigen-binding fragment thereof that specifically binds to a target selected from the group consisting of human leukocyte antigen G and its receptor are linked via a self-cleaving peptide-encoding nucleotide sequence.

13. The expression vector according to claim 11 or claim 12, wherein the nucleotide sequence encoding an antibody or an antigen-binding fragment thereof that specifically binds to human leukocyte antigen G has a nucleotide sequence encoding a signal sequence, a nucleotide sequence encoding an anti-HLA-G binding scFv, and a nucleotide sequence encoding an Fc region linked thereto.

14. The nucleotide sequence encoding an antibody or an antigen-binding fragment thereof that specifically binds to human leukocyte antigen G is (a) A nucleotide sequence encoding a middle chain variable region containing a nucleotide sequence encoding a middle chain CDR1 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 21, 27, or 33; a nucleotide sequence encoding a middle chain CDR2 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 22, 28, or 34; and a nucleotide sequence encoding a middle chain CDR3 containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 23, 29, or 35; and The light chain variable region encoding nucleotide sequence comprising: a nucleotide sequence encoding a light chain CDR1 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 24, 30 or 36; a nucleotide sequence encoding a light chain CDR2 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 25, 31 or 37; and a nucleotide sequence encoding a light chain CDR3 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 26, 32 or 38; The expression vector according to claim 11.

15. The nucleotide sequence encoding an antibody or an antigen-binding fragment thereof that specifically binds to the human leukocyte antigen G is (a)a nucleotide sequence encoding a middle chain CDR1 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 21, a nucleotide sequence encoding a middle chain CDR2 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 22, and a nucleotide sequence encoding a middle chain CDR3 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 23; and a nucleotide sequence encoding a light chain CDR1 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 24, a nucleotide sequence encoding a light chain CDR2 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 25, and a nucleotide sequence encoding a light chain CDR3 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 26; (b)a nucleotide sequence encoding a middle chain CDR1 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 27, a nucleotide sequence encoding a middle chain CDR2 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 28, and a nucleotide sequence encoding a middle chain CDR3 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 29; and a nucleotide sequence encoding a light chain CDR1 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 30, a nucleotide sequence encoding a light chain CDR2 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 31, and a nucleotide sequence encoding a light chain CDR3 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 32; or (c) A nucleotide sequence encoding a middle-chain CDR1 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 33, a nucleotide sequence encoding a middle-chain CDR2 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 34, and a nucleotide sequence encoding a middle-chain CDR3 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 35; and A nucleotide sequence encoding a light-chain CDR1 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 36, a nucleotide sequence encoding a light-chain CDR2 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 37, and a nucleotide sequence encoding a light-chain CDR3 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 38; The expression vector according to claim 14, comprising: **Claim 16** A pharmaceutical composition comprising the immune cells according to any one of claims 1 to 10. **Claim 17** The pharmaceutical composition according to claim 16, wherein the pharmaceutical composition is for the prevention or treatment of cancer. **Claim 18** 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, colorectal cancer, nasopharyngeal cancer, oral cancer, thyroid cancer, prostate cancer, kidney cancer, gallbladder cancer, bile duct cancer, melanoma. The pharmaceutical composition according to claim 17. **Claim 19** The pharmaceutical composition according to any one of claims 16 to 18, further comprising a pharmaceutically acceptable carrier. **Claim 20** Use of the immune cells according to any one of claims 1 to 10 for the manufacture of a pharmaceutical composition for the prevention or treatment of cancer. **Claim 21** Use of the immune cells according to any one of claims 1 to 10 for the prevention or treatment of cancer. **Claim 22** A method for treating cancer, comprising administering the immune cells according to any one of claims 1 to 10 or the pharmaceutical composition according to any one of claims 16 to 18 to a subject in need thereof.

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