Novel Anti-GPC3 chimeric antigen receptor and immune cells expressing same

Abstract

The present invention relates to a novel antibody specifically binding to GPC3 or an antigen-binding fragment thereof, a chimeric antigen receptor comprising an antigen-binding variable fragment of the antibody, and immune cells expressing the chimeric antigen receptor.

Description

Novel anti-GPC3 chimeric antigen receptor and immune cells expressing the same

[0001] [Cross-reference with related applications]

[0002] This application claims the benefit of priority from Korean Patent Application No. 10-2023-0095124, filed July 21, 2023, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to a novel antibody or antigen-binding fragment thereof that specifically binds to GPC3, a chimeric antigen receptor comprising an antigen-binding variable fragment of the antibody, and an immune cell expressing the chimeric antigen receptor.

[0004] Cancer treatment methods have undergone constant development and change, with surgical procedures, chemotherapy, and radiation therapy still in use today. However, these existing cancer treatments are often only effective in the early stages, before metastasis. Furthermore, even with surgical intervention, the risk of recurrence is high in advanced metastases. Therefore, research into methods that utilize the immune response to treat cancer has been ongoing.

[0005] Among these, interest is growing in cell therapy methods that utilize immune cells to strengthen them or genetically modify them and then inject them back into the patient. For example, tumor infiltrating lymphocytes (TIL), chimeric antigen receptors (CAR), and T-cell receptor (TCR) technologies are being studied. In particular, chimeric antigen receptors, which are artificial receptors designed to convey antigen specificity to T cells or natural killer cells (NK cells), are composed of an extracellular domain that can activate the immune cells and provide specific immunity by binding to cancer cell-specific antigens, a transmembrane domain, and an intracellular signaling domain. And T cells expressing these chimeric antigen receptors were named CAR-T cells (Kershaw et al., Nat. Rev. Immunol., 5(12): 928-940 (2005); Restifo et al., Nat. Rev. Immunol., 12(4): 269-281 (2012)), and natural killer cells were named CAR-NK cells.

[0006] The intracellular signaling domain of the above chimeric antigen receptor is primarily based on the intracellular signaling domain of CD3zeta, a signaling subunit of the T cell receptor (first-generation CAR). However, some CARs have caused side effects due to nonspecific attacks on healthy tissues. To overcome this, research is currently underway on third-generation CARs. Two additional signaling domains that function as costimulatory signals and an artificial receptor are added to enhance cancer cell antigen recognition ability, thereby minimizing the side effects of attacking normal cells.

[0007] Meanwhile, GPC3 (Glypican-3) is a membrane-associated proteoglycan that is rarely or absent in normal liver tissue but is specifically overexpressed in hepatocellular carcinoma (HCC). It is a gene being studied as a diagnostic and therapeutic target for HCC. Accordingly, GPC3-based clinical trials, such as antibodies targeting GPC3 and CAR-NK therapeutics utilizing such antibodies, are underway, highlighting the need to explore alternative strategies and methods for treating cancer.

[0008] The present invention aims to provide an antibody or an antigen-binding fragment thereof that can specifically bind to GPC3, which is mainly expressed in cancer cells.

[0009] In addition, the present invention aims to provide a novel chimeric antigen receptor that, when expressed in immune cells, can amplify cytotoxicity or cytolytic activity against cancer cells.

[0010] In addition, the present invention aims to provide a polynucleotide and an expression vector for expressing the chimeric antigen receptor.

[0011] In addition, the present invention aims to provide immune cells with excellent therapeutic effects against cancer by expressing the chimeric antigen receptor on their surface.

[0012] In addition, the present invention aims to provide a pharmaceutical composition for treating cancer using the above immune cells.

[0013] In addition, the present invention aims to provide a method for preventing or treating cancer, which comprises a step of administering to a subject a pharmaceutical composition for preventing or treating cancer containing the immune cells as an active ingredient.

[0014] In order to achieve the above object, one aspect of the present invention is a heavy chain variable region comprising a heavy chain CDR1 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 13, and 19, a heavy chain CDR2 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, and 20, and a heavy chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, and 21; And a light chain variable region comprising a light chain CDR1 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 22, a light chain CDR2 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, 17, and 23, and a light chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 12, 18, and 24; and provides an antibody or an antigen-binding fragment thereof that specifically binds to GPC3.

[0015] Another aspect of the present invention provides a chimeric antigen receptor (CAR) comprising an extracellular binding domain comprising an antigen-binding fragment of the anti-GPC3 (Glypican 3) antibody; a transmembrane domain; and an intracellular signaling domain.

[0016] Another aspect of the present invention provides a polynucleotide comprising a base sequence encoding the chimeric antigen receptor and an expression vector comprising the polynucleotide.

[0017] Another aspect of the present invention provides an immune cell expressing the chimeric antigen receptor on its surface.

[0018] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the immune cell as an active ingredient.

[0019] Another aspect of the present invention provides a method for preventing or treating cancer, comprising administering to a subject a pharmaceutical composition for preventing or treating cancer.

[0020] The antibody of the present invention, its antigen-binding fragment, and chimeric antigen receptor using the same can specifically bind to GPC3, which is mainly expressed in cancer cells, and accordingly, when signal transduction occurs in immune cells expressing the chimeric antigen receptor, the cytolytic activity of the immune cells is significantly enhanced, and there is an effect of increasing degranulation of cancer cells co-cultured with the immune cells.

[0021] Therefore, it was confirmed that the antibody of the present invention, the chimeric antigen receptor using the antibody, and the immune cells expressing the antibody can specifically kill only cancer cells expressing GPC3, and thus can be usefully used for the treatment of cancer.

[0022]

[0023] However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0024] Figure 1 is a schematic diagram showing the structure of a chimeric antigen receptor (GPC3-CAR) of the present invention comprising an antigen-binding variable fragment (scFv) that specifically binds to GPC3 as an extracellular domain.

[0025] Figure 2 is a graph showing the results of confirming whether the chimeric antigen receptor (GPC3-CAR) of the present invention is expressed in four types of natural killer cells (GPC3-CAR-NK cells) into which each of the four genes of the chimeric antigen receptor of the present invention was introduced and expressed.

[0026] Figure 3 is a graph showing the results of confirming the expression of GPC3 in the HepG2 (GPC3+) cell line, a liver cancer cell line that expresses GPC3, and the HepG2 (GPC3-) and SK-Hep-1 cell lines, liver cancer cell lines that do not express GPC3.

[0027] FIG. 4 is a graph showing the results of comparing the cytolytic activity of four types of natural killer cells, each of which introduced and expressed four types of chimeric antigen receptor genes of the present invention, against liver cancer cell lines (puro, dEcto: NK92 cells not expressing CAR; #65: GPC3-CAR#65-NK cells; #5: GPC3-CAR#5-NK cells; #54: GPC3-CAR#54-NK cells; #55: GPC3-CAR#55-NK cells; HepG2+: HepG2 liver cancer cell line expressing GPC3; HepG2-: HepG2 liver cancer cell line not expressing GPC3; SK-hep-1: SK-hep-1 liver cancer cell line not expressing GPC3).

[0028] Figure 5 is a diagram showing the results of a flow cytometry analysis comparing the expression levels of CD107a, an indicator of degranulation, when four types of natural killer cells (GPC3-CAR-NK) each introducing and expressing the genes of four types of chimeric antigen receptors of the present invention and NK cells (puro, dEcto) that do not express CAR were cultured alone.

[0029] Figure 6 is a diagram showing the results of a flow cytometry analysis comparing the expression levels of CD107a, an indicator of degranulation, when four types of natural killer cells (GPC3-CAR-NK) each introducing and expressing the genes of four types of chimeric antigen receptors of the present invention and NK cells (puro, dEcto) that do not express CAR were co-cultured with SK-Hep-1 hepatoma cell line (SK-Hep-1) that does not express GPC3.

[0030] Figure 7 is a diagram showing the results of a flow cytometry analysis comparing the expression levels of CD107a, an indicator of degranulation, when four types of natural killer cells (GPC3-CAR-NK) each introducing and expressing the genes of four types of chimeric antigen receptors of the present invention and NK cells (puro, dEcto) that do not express CAR were co-cultured with a HepG2 liver cancer cell line (HepG2-) that does not express GPC3.

[0031] Figure 8 is a diagram showing the results of a flow cytometry analysis comparing the expression levels of CD107a, an indicator of degranulation, when four types of natural killer cells (GPC3-CAR-NK) each introducing and expressing the genes of four types of chimeric antigen receptors of the present invention and NK cells (puro, dEcto) that do not express CAR were co-cultured with HepG2 liver cancer cell lines (HepG2+) expressing GPC3.

[0032] Figure 9 is a graph showing the results of numerically quantifying the expression level (%) of CD107a according to the flow cytometry analysis results of Figures 5 to 8.

[0033] Figure 10 is a graph showing the results of analyzing the expression level of GPC3 in cell lines of liver cancer (HepG2), pancreatic cancer (MIA-PACA-2), prostate cancer (PC-3M), colon cancer (HCT116), ovarian cancer (A2780), and lung cancer (H460).

[0034] Figure 11 is a graph showing the results of comparing the cytolytic activity of natural killer cells introduced and expressed with the chimeric antigen receptor gene of the present invention against each of liver cancer (HepG2), pancreatic cancer (MIA-PACA-2), prostate cancer (PC-3M), colon cancer (HCT116), ovarian cancer (A2780), and lung cancer (H460) cell lines (△ECTO CAR: NK cell with the scFv of the CAR deleted; GPC3 CAR: NK cell expressing on its surface a chimeric antigen receptor that specifically binds to GPC3 (GPC3-CAR#54-NK cell)).

[0035] Hereinafter, the present invention will be described in detail.

[0036]

[0037] 1. Novel anti-GPC3 antibody or antigen-binding fragment thereof, and chimeric antigen receptor (CAR) comprising the same, and polynucleotide and expression vector for expressing the same

[0038] One aspect of the present invention provides an anti-GPC3 antibody and an antigen-binding fragment thereof capable of specifically binding to GPC3.

[0039] In the present invention, GPC3 (Glypican 3) is a membrane-associated proteoglycan protein that is rarely expressed or not expressed in normal liver tissue, but is specifically overexpressed in hepatocellular carcinoma.

[0040] The term "antibody" in the present invention refers to an immunoglobulin molecule that immunologically binds specifically to an epitope of an antigen and has reactivity. The antibody may include a monoclonal antibody, a polyclonal antibody, an antibody having a full-length chain structure (full-length antibody), a functional fragment having at least an antigen-binding function (antigen-binding fragment), and a recombinant antibody. Specifically, the antibody of the present invention may be a monoclonal antibody or an antigen-binding fragment thereof. The monoclonal antibody refers to an antibody molecule having a single molecular composition obtained from a substantially identical antibody population, and such a monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope. The full-length antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain may be linked to a heavy chain by a disulfide bond. The above antibody comprises a heavy chain (HC) and a light chain (LC) polypeptide, and the heavy chain and light chain may include a variable region and a constant region.

[0041] The constant region is a site that mediates binding of the antibody to various types of cells of the immune system (such as T cells), host tissues including components of the complement system, etc. The constant region has the same function regardless of the type of antigen as long as it is an antibody of the same type derived from the same species, and the amino acid sequence constituting the constant region is also the same or has a high degree of similarity between antibodies. The constant region can be divided into a heavy chain constant region (which may be abbreviated as CH) and a light chain constant region (which may be abbreviated as CL). The heavy chain constant region has a gamma (γ), mu (μ), alpha (α), delta (δ), and / or epsilon (ε) type, and has gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), and / or alpha2 (α2) as subclasses. The light chain constant region has a kappa (κ) and lambda (λ) type. IgG has subtypes, including IgG1, IgG2, IgG3, and IgG4.

[0042] The variable region is an antibody portion having specificity for an antigen, and can be divided into a heavy chain variable region (which may be abbreviated as VH) and a light chain variable region (which may be abbreviated as VL). The variable region may include three CDRs (complementary-determining regions) and four FRs (framework regions). The CDRs may be ring-shaped portions involved in antigen recognition, and specificity for an antigen may be determined according to the amino acid sequence of the CDRs. The CDRs may be referred to as CDR1, CDR2, and CDR3 according to their order, and depending on which polypeptide among the heavy and light chains they are CDRs, the heavy chain variable region may be referred to as CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region may be referred to as CDR-L1, CDR-L2, and CDR-L3. Likewise, FRs may be referred to as FR-H1, FR-H2, FR-H3, and FR-H4 for the heavy chain variable region, and as FR-L1, FR-L2, FR-L3, and FR-L4 for the light chain variable region. In addition, the CDRs and FRs may be arranged in the following order in each variable region.

[0043] The term "antigen-binding fragment" in the present invention refers to any fragment of the humanized antibody of the present invention that retains the antigen-binding function of the antibody. The antigen-binding fragment may be referred to interchangeably with terms such as "fragment", "antibody fragment", etc., and the antigen-binding fragment may be, but is not limited to, Fab, Fab', F(ab')2, Fv, etc.

[0044] The Fab has a structure with variable regions of the light and heavy chains, a constant region of the light chain, and the first constant region (CH1 domain) of the heavy chain, and has one antigen-binding site. The Fab' is different from the Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. The F(ab')2 is generated when the cysteine ​​residues of the hinge region of the Fab' form a disulfide bond. The Fv refers to the minimum antibody fragment having only a heavy chain variable region and a light chain variable region. A two-chain Fv has a heavy chain variable region and a light chain variable region linked by a non-covalent bond, and a single-chain Fv has a heavy chain variable region and a light chain variable region linked by a covalent bond, generally via a peptide linker, or linked directly at the C-terminus, so that it can form a dimer-like structure like a two-chain Fv. The above antigen-binding fragment can be produced using, but is not limited to, a protein hydrolytic enzyme (for example, Fab can be obtained by restriction digestion of the whole antibody with papain, and F(ab')2 fragment can be obtained by digestion with pepsin), or through genetic recombination technology.

[0045] The linker may be a peptide linker and may have a length of about 10 to 25 amino acids. For example, the linker may include a hydrophilic amino acid such as glycine (G) and / or serine (S). The linker may include, for example, (GS)n, (GGS)n, (GSGGS)n or (GnS)m (wherein n and m are each 1 to 10), for example, but not limited to, (GnS)m (wherein n and m are each 1 to 10).

[0046] As used herein, the term "epitope" refers to a specific site on an antigen to which an immunoglobulin, antibody, or antigen-binding fragment thereof can specifically recognize and bind. The epitope may be formed from consecutive amino acids or from discontinuous amino acids juxtaposed by the tertiary folding of the protein.

[0047] The above "specifically binds" may mean binding to another molecule with a binding affinity greater than background binding, for example, the extracellular domain may bind to a target antigen with an affinity or Ka (equilibrium dissociation constant of a specific binding interaction having units of 1 / M) of greater than or equal to about 10-5 M. The affinity may be such that the equilibrium dissociation constant (Kd) of a specific binding interaction having units of M is from 10-5 M to 10-13 M or less.

[0048] The antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region comprising a heavy chain CDR1 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 13, and 19, a heavy chain CDR2 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, and 20, and a heavy chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, and 21; A light chain variable region comprising a light chain CDR1 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 22, a light chain CDR2 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, 17, and 23, and a light chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 12, 18, and 24; and specifically binds to GPC3.

[0049] According to one specific example, the antibody or antigen-binding fragment thereof that specifically binds to GPC3 may include a heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 having an amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having an amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 6.

[0050] According to one specific example, the antibody or antigen-binding fragment thereof that specifically binds to GPC3 may include a heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 9; and a light chain variable region comprising a light chain CDR1 having an amino acid sequence of SEQ ID NO: 10, a light chain CDR2 having an amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 12.

[0051] According to one specific example, the antibody or antigen-binding fragment thereof that specifically binds to GPC3 may include a heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 13, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 14, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 15; and a light chain variable region comprising a light chain CDR1 having an amino acid sequence of SEQ ID NO: 16, a light chain CDR2 having an amino acid sequence of SEQ ID NO: 17, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 18.

[0052] According to one specific example, the antibody or antigen-binding fragment thereof that specifically binds to GPC3 may include a heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 19, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 20, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 21; and a light chain variable region comprising a light chain CDR1 having an amino acid sequence of SEQ ID NO: 22, a light chain CDR2 having an amino acid sequence of SEQ ID NO: 23, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 24.

[0053] The antibody of the present invention or an antigen-binding fragment thereof may further include, for example, a heavy chain constant region and / or a light chain constant region of an antibody derived from a human, and, as long as the antibody or antigen-binding fragment thereof does not inhibit the property of specifically binding to GPC3, the heavy chain constant region and / or the light chain constant region of the antibody derived from a human may be used without limitation in type or amino acid sequence.

[0054] The amino acid sequences described above may include variants having different sequences by deletion, insertion, substitution, or a combination thereof of amino acid residues, as long as the structure, function, activity, etc. of the polypeptide including the same are not affected. In addition, the amino acid sequences may include amino acids that have undergone conventional modifications known in the art, and the amino acid modifications may be, for example, phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc. The humanized antibody or antigen-binding fragment thereof of the present invention includes not only those containing the amino acid sequences described above, but also those having substantially the same amino acid sequence as the above-described amino acid sequences or variants thereof. The meaning of having the substantially identical amino acid sequence may include, but is not limited to, an amino acid sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more homology with the amino acid sequence described above.

[0055] The term "chimeric antigen receptor (CAR)" used in the present invention refers to a synthetic complex designed to induce an immune response when it recognizes and binds to a target antigen and a cell expressing the antigen. The chimeric antigen receptor may include an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain. The chimeric antigen receptor is expressed on the surface of immune cells and recognizes and binds to a specific antigen, for example, an antigen specifically expressed on the surface of a cancer cell, through an antigen binding site included in the extracellular domain, thereby inducing signal transduction within the immune cell and changing the activity of the immune cell, thereby targeting only a specific antigen and inducing an immune response.

[0056] The chimeric antigen receptor (CAR) of the present invention comprises an extracellular binding domain including an antigen binding site of the antibody that specifically binds to Glypican 3 (GPC3); a transmembrane domain; and an intracellular signaling domain.

[0057] The antigen binding site that specifically binds to the above GPC3 comprises a heavy chain variable region including a heavy chain CDR1 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 13, and 19, a heavy chain CDR2 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, and 20, and a heavy chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, and 21; and a light chain variable region comprising a light chain CDR1 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 22, a light chain CDR2 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, 17, and 23, and a light chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 12, 18, and 24; and a single chain variable fragment (scFv) of an anti-GPC3 antibody.

[0058] According to one specific example, the antigen binding site that specifically binds to GPC3 may include a single chain variable fragment of an anti-GPC3 antibody, comprising a heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 having an amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having an amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 6.

[0059] According to one specific example, the antigen binding site that specifically binds to GPC3 may include a single chain variable fragment of an anti-GPC3 antibody, comprising a heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 9; and a light chain variable region comprising a light chain CDR1 having an amino acid sequence of SEQ ID NO: 10, a light chain CDR2 having an amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 12.

[0060] According to one specific example, the antigen binding site that specifically binds to GPC3 may include a single chain variable fragment of an anti-GPC3 antibody, comprising a heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 13, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 14, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 15; and a light chain variable region comprising a light chain CDR1 having an amino acid sequence of SEQ ID NO: 16, a light chain CDR2 having an amino acid sequence of SEQ ID NO: 17, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 18.

[0061] According to one specific example, the antigen binding site that specifically binds to GPC3 may include a single chain variable fragment of an anti-GPC3 antibody, comprising a heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 19, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 20, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 21; and a light chain variable region comprising a light chain CDR1 having an amino acid sequence of SEQ ID NO: 22, a light chain CDR2 having an amino acid sequence of SEQ ID NO: 23, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 24.

[0062] According to one specific example, the chimeric antigen receptor may further comprise a signal peptide located upstream of the antigen binding site (i.e., scFv region) that specifically binds to GPC3.

[0063] According to one specific example, the signal peptide may include an amino acid sequence represented by SEQ ID NO: 30, and the base sequence encoding the signal peptide may include a base sequence represented by SEQ ID NO: 29.

[0064] When the chimeric antigen receptor of the present invention is expressed on the surface of an immune cell, when the target antigen, GPC3, binds to the receptor, signal transmission occurs within the immune cell, which may induce an enhancement of cytotoxicity (or cytolytic activity) of the immune cell and / or promotion of cytokine secretion by the immune cell. The enhancement of cytotoxicity (or cytolytic activity) or promotion of cytokine secretion may exhibit cytolytic activity at a level higher than that exhibited by an immune cell in the absence of an antigen.

[0065] The extracellular domain may further comprise at least one selected from the group consisting of a hinge domain and a spacer domain. The antigen binding site of the extracellular domain may be linked to the transmembrane domain via the hinge domain and / or the spacer domain.

[0066] The hinge domain may play an important role in positioning the extracellular domain, which allows the antigen binding site to be physically separated from the surface of an immune cell on which the chimeric antigen receptor is expressed, to enable proper cell / cell contact, proper antigen / antigen binding site binding, and proper chimeric antigen receptor activation. The chimeric antigen receptor may comprise one or more hinge domains between the extracellular domain and the transmembrane domain. The hinge domain may be derived from a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. The altered hinge region is (a) a naturally occurring hinge region having at most 30% amino acid change (e.g., at most 25%, 20%, 15%, 10% or 5% amino acid substitutions or deletions), (b) a portion of a naturally occurring hinge region that is at least 10 amino acids long (e.g., at least 12, 13, 14 or 15 amino acids) having at most 30% amino acid change (e.g., at most 25%, 20%, 15%, 10% or 5% amino acid substitutions or deletions), or (c) a core hinge region (which is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or Refers to a portion of a naturally occurring hinge region comprising a cysteine ​​residue (which may be 15 amino acids in length). In certain embodiments, one or more cysteine ​​residues in the naturally occurring immunoglobulin hinge region may be substituted with one or more other amino acid residues (e.g., one or more serine residues). The altered immunoglobulin hinge region may alternatively or additionally have a proline residue of the wild-type immunoglobulin hinge region substituted with another amino acid residue, such as a cysteine.The hinge domain may be a hinge region derived from the extracellular domain of a type 1 membrane protein such as CD8, CD4, CD28, and CD7, but any hinge region capable of connecting the antigen binding site, the transmembrane domain, and the intracellular signaling domain across the cell membrane may be used without limitation. Furthermore, it may be a wild-type hinge region from these molecules or may be modified.

[0067] The spacer domain may be referred to as a linking domain, and may include, for example, a CD28-derived hinge domain and / or a CD8-derived hinge domain, and may include all or part of the CD28-derived hinge domain and / or the CD8-derived hinge domain.

[0068] The hinge domain and / or spacer domain may comprise at least one selected from the group consisting of Myc or an epitope thereof, a hinge domain of CD8 (i.e., a hinge region of CD8) and Fc, and may specifically comprise Myc or an epitope thereof and a hinge domain of CD8.

[0069] According to one specific example, the Myc may include an amino acid sequence represented by SEQ ID NO: 36, and the base sequence encoding the Myc may include a base sequence represented by SEQ ID NO: 35.

[0070] According to one specific example, the hinge domain of the CD8 may include an amino acid sequence represented by SEQ ID NO: 38, and the base sequence encoding the hinge domain of the CD8 may include a base sequence represented by SEQ ID NO: 37.

[0071] The above transmembrane domain refers to a portion of a region that connects and fuses the extracellular domain and the intracellular signaling domain and plays a role in anchoring the chimeric antigen receptor to the plasma membrane of an immune cell. The transmembrane domain may be derived from a natural, synthetic, semi-synthetic or recombinant source. The transmembrane domain may include at least one selected from the group consisting of the alpha (α), beta (β) or zeta (ζ) chain of the T-cell receptor (TCR), CD28, CD3 epsilon (ε), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a part or all of the amino acid sequence thereof, but is not limited thereto.

[0072] According to one specific example, the transmembrane domain may include an amino acid sequence represented by SEQ ID NO: 40 corresponding to a portion of the amino acid sequence of CD28, and the base sequence encoding the amino acid sequence of SEQ ID NO: 40 may include a base sequence represented by SEQ ID NO: 39.

[0073] The transmembrane domain may be attached to the extracellular domain via a linker. For example, the linker may be a short oligopeptide or polypeptide linker of 2 to 10 amino acids in length, such as, but not limited to, a glycine (G)-serine (S) doublet.

[0074] The intracellular signaling domain corresponds to a portion that transmits a signal generated upon binding of the chimeric antigen receptor to the interior of the immune cell to induce a function of the immune cell (e.g., activation including release of cytotoxic (or cytolytic) factors against a target cell to which the chimeric antigen receptor binds, cytokine production, proliferation and cytotoxicity or cytolytic activity, or other cellular responses induced by the antigen binding). The intracellular signaling domain may be a portion of a protein that transmits an effector function signal and instructs the cell to perform a specific function.

[0075] The intracellular signaling domain may be an intracellular signaling domain that has been previously used in the development of chimeric antigen receptors. Specifically, it may include only CD3zeta, which was used in the first-generation CAR (chimeric antigen receptor). In addition, as used in the second-generation CAR, a form combining a costimulatory domain (CD28 or CD137 / 4-1BB) and CD3zeta may be used to enhance responsiveness to immune cells. In addition, as used in the third-generation CAR, two or more costimulatory domains may be used, and in this case, the costimulatory domain may be combined with 4-1BB, CD28, or OX40, etc., to achieve expansion and persistence of immune cells containing the CAR in vivo. Furthermore, as used in fourth generation CARs, additional genes encoding cytokines such as IL-12 or IL-15 can be included to enable additional expression of cytokine-based immunoproteins, and as used in fifth generation CARs, interleukin receptor chains, for example, IL-2Rβ, can be additionally included to enhance immune cells.

[0076] The intracellular signaling domain may comprise at least one selected from the group consisting of T-cell receptor (TCR) zeta (ζ), FcR gamma (γ), FcR beta (β), CD3 gamma (γ), CD3 delta (δ), CD3 epsilon (ε), CD3 zeta (ζ), CD5, CD22, CD79a, CD79b and CD66d, or a part or all of an amino acid sequence thereof.

[0077] More specifically, the activation of the immune cell by the intracellular signaling domain may be mediated by two different classes of intracellular signaling domains. For example, immune cell activation may be mediated by a primary signaling domain that initiates antigen-dependent primary activation and a co-stimulatory signaling domain that acts in an antigen-independent manner to provide a secondary signal. Accordingly, the intracellular signaling domain may comprise a primary signaling domain and a co-stimulatory signaling domain.

[0078] The primary signaling domain refers to a signaling domain that regulates immune cell activation in a stimulatory or inhibitory manner. The primary signaling domain that acts in a stimulatory manner may include a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. The primary signaling domain may include, but is not limited to, at least one selected from the group consisting of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, and CD66d, or a part or all of an amino acid sequence thereof. More specifically, the primary signaling domain may include, but is not limited to, CD3ζ(zeta), or a part or all of an amino acid sequence thereof.

[0079] According to one specific example, the first signal transduction domain may include an amino acid sequence represented by SEQ ID NO: 44 corresponding to a part of the amino acid sequence of CD3ζ(zeta), and the base sequence encoding the amino acid sequence of SEQ ID NO: 44 may include a base sequence represented by SEQ ID NO: 43.

[0080] The co-stimulatory signaling domain refers to an intracellular signaling domain of a co-stimulatory molecule. The co-stimulatory signaling domain may include at least one selected from the group consisting of CD2, CD7, CD27, CD28, CD30, CD40, 4-1BB (CD137), OX40 (CD134), CDS, ICAM-1, ICOS (CD278), LFA-1 (CD11a / CD18), GITR, MyD88, DAP10, DAP12, PD-1, LIGHT, NKG2C, GPC3, and CD83, or a part or all of the amino acid sequence thereof, but is not limited thereto.

[0081] According to one specific example, the co-stimulatory signal transduction domain may include, but is not limited to, a portion of the amino acid sequence of CD28 and a portion of the amino acid sequence of DAP10.

[0082] According to one specific example, the co-stimulatory signal transduction domain may include an amino acid sequence represented by SEQ ID NO: 46 corresponding to a portion of the amino acid sequence of CD28, and the base sequence encoding the amino acid sequence of SEQ ID NO: 46 may include a base sequence represented by SEQ ID NO: 45.

[0083] According to one specific example, the co-stimulatory signal transduction domain may include an amino acid sequence represented by SEQ ID NO: 42 corresponding to a part of the amino acid sequence of DAP10, and the base sequence encoding the amino acid sequence of SEQ ID NO: 42 may include a base sequence represented by SEQ ID NO: 41.

[0084] The chimeric antigen receptor may comprise two or more intracellular signaling domains, and when comprising two or more intracellular signaling domains, the intracellular signaling domains may be connected in series with each other. Alternatively, they may be connected via a polypeptide linker consisting of 2 to 10 amino acids, and the linker sequence may be, for example, a glycine-serine continuous sequence. The linker may comprise, for example, (GS)n, (GGS)n, (GSGGS)n or (GnS)m (wherein n and m are each 1 to 10), and may be, for example, (GnS)m (wherein n and m are each 1 to 10), but is not limited thereto.

[0085] The above chimeric antigen receptor may additionally include an immune function promoting factor of an immune cell. For example, the immune function promoting factor of the immune cell may be an interleukin signal sequence. The interleukin signal sequence may be characterized by inducing the expression of IL (interleukin)-12, IL-8, IL-2, etc., but is not limited thereto. In addition, when the immune cell is a T cell, the immune function promoting factor may be IL-7, CCL19, etc., but is not limited thereto.

[0086] In a specific embodiment of the present invention, a chimeric antigen receptor was designed to include scFv of four types (#5, #54, #55 and #65) of anti-GPC3 antibodies having the amino acid sequences described above in the extracellular domain, CD28 linked to the extracellular domain with Myc and a hinge domain as a transmembrane domain and an intracellular signaling domain, and CD3-zeta and DAP10 were additionally linked as an intracellular signaling domain, thereby producing the chimeric antigen receptor of the present invention (GPC3-CAR).

[0087] As described above, the chimeric antigen receptor of the present invention can enhance the cytolytic activity of immune cells by recognizing and binding to GPC3 expressed on the surface of immune cells. Therefore, when cancer cells expressing GPC3 are present, if the antigen-binding site of the chimeric antigen receptor recognizes and binds to the antigen, the cytolytic activity of the immune cells can be enhanced through signal transduction, and thus, the chimeric antigen receptor can be usefully utilized as a chimeric antigen receptor with excellent cytolytic efficacy for attacking cancer cells.

[0088]

[0089] Another aspect of the present invention provides a polynucleotide and an expression vector for expressing the chimeric antigen receptor.

[0090] The polynucleotide comprises a base sequence encoding the chimeric antigen receptor.

[0091] In the present invention, the term "polynucleotide" comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and the basic structural unit, nucleotide, includes not only natural nucleotides but also analogues in which the sugar or base portion is modified.

[0092] Encoding the chimeric antigen receptor means that the polynucleotide encodes genetic information that allows a protein having the amino acid sequence of the chimeric antigen receptor of the present invention to be synthesized through conventional protein expression processes such as transcription and translation. In this case, not only a protein having an amino acid sequence completely identical to the chimeric antigen receptor, but also a polynucleotide encoding a protein having an amino acid sequence substantially identical to the protein as described above, or a protein having the same and / or similar activity as the protein, may be included within the scope of the present invention.

[0093] Specifically, the polynucleotide of the present invention may include a base sequence encoding an antigen-binding variable fragment of an anti-GPC3 antibody that specifically binds to GPC3, and more specifically, may include any one of the base sequences of SEQ ID NO: 31 to SEQ ID NO: 34.

[0094] In addition, the polynucleotide of the present invention may include not only a base sequence encoding the antigen-binding variable fragment, but also a base sequence encoding another extracellular domain portion linked thereto, the transmembrane domain, and / or the intracellular signaling domain.

[0095] The description of the chimeric antigen receptor, including the antibody, antigen-binding variable fragment, extracellular domain, transmembrane domain, and intracellular signaling domain, is the same as that described above.

[0096] The polynucleotide may include any one of the base sequences of SEQ ID NOs: 31 to 34. In a specific embodiment of the present invention, a chimeric antigen receptor was designed, which includes scFv of four types (#5, #54, #55, and #65) of anti-GPC3 antibodies in the extracellular domain, CD28 linked to the extracellular domain with Myc and a hinge domain as a transmembrane domain and an intracellular signaling domain, and CD3-zeta and DAP10 are additionally linked as an intracellular signaling domain, and a polynucleotide including any one of the base sequences of SEQ ID NOs: 31 to 34 was prepared to express the chimeric antigen receptor (GPC3-CAR).

[0097] The polynucleotide of the present invention may include a base sequence substantially identical to the base sequences listed above. The substantially identical base sequence includes, for example, a case where the same amino acid can be synthesized when transcribed and translated, and may be a base sequence having a homology of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more with the base sequences listed above, but is not limited thereto.

[0098] The polynucleotide encoding the chimeric antigen receptor may include an optimized base sequence depending on the type of organism to be introduced and expressed and the expression system of the organism, such as transcription or translation. This is due to the degeneracy of codons, and accordingly, various combinations of nucleotide sequences capable of encoding the expressed protein may exist, all of which are included in the scope of the present invention. The modification of the polynucleotide according to the codon optimization may be determined depending on the type of organism to which the chimeric antigen receptor of the present invention is to be expressed and applied. For example, the polynucleotide of the present invention may be a polynucleotide modified to optimize codon selection for mammals and primates, and more specifically, may be optimized and modified to be suitable for expression and function in humans.

[0099] The expression vector of the present invention comprises the polynucleotide.

[0100] Since the polynucleotide includes a base sequence encoding the chimeric antigen receptor of the present invention, the expression vector including the same can be used to produce and express the chimeric antigen receptor, and can be used to transport the polynucleotide to a specific cell or organism so that the chimeric antigen receptor can be expressed, or to store and preserve the polynucleotide.

[0101] The above expression vector can be constructed using a prokaryotic cell or a eukaryotic cell as a host.

[0102] For example, when the expression vector uses a prokaryotic cell as a host, it usually includes a strong promoter capable of driving transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. When E. coli (e.g., HB101, BL21, DH5α, etc.) is used as a host cell, the promoter and operator regions of the E coli tryptophan biosynthetic pathway (Yanofsky, C, J Bacteriol, (1984) 158:1018-1024) and the left-hand promoter of phage λ (pLλ promoter, Herskowitz, I and Hagen, D, Ann Rev Genet, (1980) 14:399-445) can be used as regulatory regions. When Bacillus is used as a host cell, the promoter of the toxin protein gene of Bacillus thuringiensis (Appl Environ Microbiol (1998) 64:3932-3938; Mol Gen Genet (1996) 250:734-741) or any promoter that can be expressed in Bacillus can be used as a regulatory region. The above expression vector can be produced by manipulating plasmids (e.g., pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), phages (e.g., λgt4·λB, λ-Charon, λΔz1, and M13, etc.), or viruses (e.g., SV40, etc.) frequently used in the art.

[0103] When the above expression vector uses a eukaryotic cell as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 75K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, promoter of Epstein-Barr virus (EBV), and promoter of Rous sarcoma virus (RSV)) can be used, and can generally have a polyadenylation sequence as a transcription termination sequence. The above expression vector may have a CMV promoter.

[0104] In addition, the expression vector may be fused with other sequences to facilitate purification of the antibody expressed therefrom. Examples of the fused sequences include glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA). In addition, since the protein expressed by the expression vector of the present invention is a chimeric antigen receptor, considering its characteristics, the expressed protein can be easily purified through a protein A column or the like without additional sequences for purification.

[0105] The above expression vector includes an antibiotic resistance gene commonly used in the art as a selectable marker, and may include, for example, a resistance gene for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0106]

[0107] 2. Immune cells expressing a chimeric antigen receptor for GPC3 on their surface

[0108] Another aspect of the present invention provides an immune cell characterized by exhibiting enhanced cytotoxicity or cytolytic activity against cancer cells expressing GPC3.

[0109] The above immune cells express the chimeric antigen receptor of the present invention described above on their surface.

[0110] The above chimeric antigen receptor is the same as that described above in “1. Novel anti-GPC3 antibody, antigen-binding fragment thereof, and chimeric antigen receptor (CAR) comprising the same and polynucleotide and expression vector for expressing the same.” Specifically, the chimeric antigen receptor may include an antigen-binding site capable of specifically binding to a GPC3 antigen expressed on cancer cells.

[0111] The above immune cells can be used without limitation as long as they are cells that can induce immunity and induce the desired therapeutic effect, and for example, can be any one selected from the group consisting of natural killer cells (NK cells), T cells, natural killer T cells (NKT cells), cytokine-induced killer cells (CIK), macrophages, and dendritic cells, but are not limited thereto. Accordingly, the immune cells expressing the chimeric antigen receptor according to the present invention on the cell surface can be CAR-NK cells (Chimeric Antigen Receptor Natural Killer Cells), CAR-T cells (Chimeric Antigen Receptor T Cells), CAR-NKT cells (Chimeric Antigen Receptor Natural killer T Cells), CAR-macrophages (Chimeric Antigen Receptor Macrophages), etc.

[0112] The above T cells may be, but are not limited to, cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes (TILs), T cells isolated from peripheral blood mononuclear cells (PBMCs), etc.

[0113] The above CAR-NK cells refer to cells into which a chimeric antigen receptor has been introduced into natural killer cells, and when using existing T cell-based CAR-T treatment, the problems of persistent toxicity of cancer immunotherapy, the risk of autoimmune disease, the problem of graft-versus-host disease (GVHD) for xenogeneic cell transplantation, and the problem of non-target toxicity can be solved through a switch for initiating (on) / stopping (off) the reaction, and it has the advantage of being able to be used as a universal treatment because it can target various cancer cells.

[0114] The immune cells of the present invention express, on their cell surface, a chimeric antigen receptor comprising an antigen-binding variable fragment (scFv) of an antibody capable of specifically recognizing and binding to GPC3, which can be specifically expressed in cancer cells, as an extracellular domain. Therefore, in the presence of cancer cells expressing GPC3, signal transduction can occur via the chimeric antigen receptor, thereby further enhancing the cytolytic activity of the immune cells. Therefore, the immune cells of the present invention can have the activity of attacking and treating cancer cells.

[0115] In a specific embodiment of the present invention, four chimeric antigen receptors each containing an antigen-binding variable fragment of each of four GPC3-specific antibodies of the present invention as an extracellular domain were expressed on the surface of natural killer cells. When the four natural killer cells were co-cultured with HepG2 (GPC3+) cells, a liver cancer cell line expressing GPC3, the cytolytic activity was enhanced and the level of degranulation was also increased, confirming that the immune cells of the present invention have an excellent therapeutic effect on cancer cells expressing GPC3. In addition, the natural killer cells of the present invention, by expressing the chimeric antigen receptor that specifically binds to GPC3, showed an excellent killing ability against various cancer cells expressing GPC3, such as pancreatic cancer, prostate cancer, colon cancer, ovarian cancer, and lung cancer, not limited to liver cancer, confirming that they have an excellent therapeutic effect on various cancer cells expressing GPC3.

[0116]

[0117] 3. Use of the immune cells of the present invention for the treatment of cancer

[0118] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the immune cells as an active ingredient.

[0119] The description of the above-mentioned immune cells, chimeric antigen receptors expressed therefrom, etc. is the same as that described in '1. Novel anti-GPC3 antibodies, antigen-binding fragments thereof, and chimeric antigen receptors (CARs) containing the same, and polynucleotides and expression vectors for expressing the same' and '2. Immune cells expressing chimeric antigen receptors for GPC3 on their surfaces', so description is omitted to avoid repetition.

[0120] In the present invention, the term "cancer" is used with the same meaning as "tumor" and refers to or means a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation.

[0121] The cancer or cancer type that can be prevented or treated with the composition of the present invention is not particularly limited, and includes both solid cancers and blood cancers. For example, the cancer may be liver cancer, lung cancer, stomach cancer, ovarian cancer, cervical cancer, breast cancer, pancreatic cancer, colon cancer, colon cancer, esophageal cancer, skin cancer, thyroid cancer, kidney cancer, liver cancer, head and neck cancer, bladder cancer, prostate cancer, blood cancer, multiple myeloma, acute myeloid leukemia, malignant lymphoma, thymic cancer, osteosarcoma, fibrous tumor, and brain cancer, but is preferably liver cancer, and is not limited thereto. Any cancer cell that expresses GPC3 on its surface can be applied without limitation in the present invention.

[0122] In addition, GPC3 (Glypican-3) is a membrane-associated proteoglycan that is rarely expressed or not expressed in normal liver tissue, but is specifically overexpressed in hepatocellular carcinoma (HCC). It is a gene that is being studied as a diagnostic and therapeutic target for liver cancer, including hepatocellular carcinoma. Accordingly, the composition of the present invention, which includes an antibody targeting GPC3 or a CAR-NK cell using the same, can be used for the prevention or treatment of cancer that expresses GPC3 on its surface.

[0123] In the present invention, the term “prevention” means any act of inhibiting or delaying the onset of cancer by administering the composition.

[0124] The term "treatment" in the present invention refers to any form of treatment that provides an effect to a subject suffering from cancer or at risk of developing cancer, including improving the condition of the subject (e.g., one or more symptoms), delaying the progression of cancer, delaying or slowing the onset or progression of symptoms, inhibiting the development of cancer, or reducing cancer. Therefore, the terms "treatment" and "prevention" are not intended to mean a cure or complete elimination of symptoms.

[0125] The pharmaceutical composition may include, but is not limited to, a number of immune cells that is 1 to 10 times, 2 to 10 times, or 5 to 8 times the number of tumor cells of the subject to be treated.

[0126] The above composition may be in the form of a pharmaceutical composition, a quasi-drug composition, a health food composition, etc.

[0127] The pharmaceutical composition for preventing or treating cancer of the present invention may additionally include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means that the composition does not inhibit the activity of the active ingredient and does not exhibit toxicity exceeding the adaptability of the subject of application (prescription). The "carrier" is defined as a compound that facilitates the addition of the compound into cells or tissues.

[0128] The pharmaceutical composition of the present invention may be administered alone or mixed with any convenient carrier, and such dosage form may be a single-dose or repeated-dose dosage form. The pharmaceutical composition may be a solid preparation or a liquid preparation. Solid preparations include, but are not limited to, powders, granules, tablets, capsules, suppositories, etc. Solid preparations may include, but are not limited to, carriers, flavoring agents, binders, preservatives, disintegrants, lubricants, fillers, etc. Liquid preparations include, but are not limited to, solutions such as water or propylene glycol solutions, suspensions, emulsions, etc., and may be prepared by adding appropriate colorants, flavoring agents, stabilizers, viscosifiers, etc. For example, a powder may be prepared by simply mixing a trihydroxy derivative of a polyunsaturated fatty acid, which is an active ingredient of the present invention, with a pharmaceutically acceptable appropriate carrier such as lactose, starch, or microcrystalline cellulose. Granules can be prepared by mixing the tri-hydroxy derivative of the polyunsaturated fatty acid of the present invention, a suitable pharmaceutically acceptable carrier, and a suitable pharmaceutically acceptable binder such as polyvinylpyrrolidone or hydroxypropylcellulose, and then using a wet granulation method using a solvent such as water, ethanol, or isopropanol, or a dry granulation method using compression force. In addition, tablets can be prepared by mixing the granules with a suitable pharmaceutically acceptable lubricant such as magnesium stearate, and then compressing them using a tableting machine.

[0129] The pharmaceutical composition may be administered orally, by injection (e.g., intramuscular injection, intraperitoneal injection, intravenous injection, infusion, subcutaneous injection, implant), inhalant, nasal administration, vaginal administration, rectal administration, sublingual administration, transdermal administration, topical administration, etc., depending on the disease to be treated and the condition of the subject, but is not limited thereto. Depending on the route of administration, the composition may be formulated into an appropriate dosage unit formulation containing a pharmaceutically acceptable carrier, additive, or vehicle that is commonly used and non-toxic.

[0130] The pharmaceutical composition may be administered daily at a dose of about 0.0001 mg / kg to about 10 g / kg, with a daily dosage of about 0.001 mg / kg to about 1 g / kg. However, the dosage may vary depending on the degree of purification of the mixture, the patient's condition (age, sex, weight, etc.), the severity of the condition being treated, etc. If necessary, the total daily dosage may be divided and administered several times throughout the day for convenience.

[0131]

[0132] 4. How to prevent or treat cancer

[0133] Another aspect of the present invention provides a method for preventing or treating cancer, comprising administering to a subject a pharmaceutical composition for preventing or treating cancer comprising the immune cell as an active ingredient.

[0134] Descriptions of the above-described immune cells, chimeric antigen receptors expressed therefrom, pharmaceutical compositions for preventing or treating cancer, etc. are the same as those described in '1. Novel anti-GPC3 antibodies, antigen-binding fragments thereof, and chimeric antigen receptors (CARs) containing the same and polynucleotides and expression vectors for expressing the same', '2. Immune cells expressing chimeric antigen receptors for GPC3 on their surfaces', and '3. Use of the immune cells of the present invention for treating cancer', so descriptions are omitted to avoid repetition.

[0135] The above-mentioned entity may include, without limitation, mammals including cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, cats, rats, livestock, humans, etc., farmed fish, mammals other than humans, etc. that are prone to or at risk of developing the above-mentioned cancer.

[0136] The cancer may be liver cancer, lung cancer, stomach cancer, ovarian cancer, cervical cancer, breast cancer, pancreatic cancer, colon cancer, colon cancer, esophageal cancer, skin cancer, thyroid cancer, kidney cancer, liver cancer, head and neck cancer, bladder cancer, prostate cancer, blood cancer, multiple myeloma, acute myeloid leukemia, malignant lymphoma, thymic cancer, osteosarcoma, fibrous tumor, brain cancer, etc., but is preferably liver cancer, but is not limited thereto. In addition, the cancer may be a cancer caused by cancer cells expressing GPC3 on the surface.

[0137] The term "administration" refers to introducing a given substance into a subject in an appropriate manner, and the composition may be administered via any conventional route as long as it can reach the target tissue. Examples of such routes include, but are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, and rectal administration. However, since components such as proteins are digested when administered orally, it is preferable that oral compositions be formulated to coat the active agent or protect it from degradation in the stomach. Furthermore, the composition may be administered via any device capable of transporting the active agent to target cells. Furthermore, the composition may be administered to a subject via intravenous injection, but is not limited thereto.

[0138] The composition may be administered in a pharmaceutically effective amount, wherein the term “pharmaceutically effective amount” may mean an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention.

[0139] The dosage of the above composition may be determined according to the purpose of use, the degree of toxicity of the disease, the patient's age, weight, health, sex, medical history, the type and / or activity of the substance used as the active ingredient, the patient's sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the treatment period, the components of other drugs combined or other drugs used simultaneously, and other factors well known in the medical field. The above composition may be administered alone or in combination with a component known to exhibit a preventive or therapeutic effect against the known cancer. It is important to take all of the above factors into consideration and administer an amount that can achieve the maximum effect with the minimum amount without side effects.

[0140] For example, the composition may be administered daily at a dose of about 0.0001 mg / kg to about 10 g / kg, with a daily dosage of about 0.001 mg / kg to about 1 g / kg. However, the dosage may vary depending on the degree of purification of the mixture, the patient's condition (age, sex, weight, etc.), the severity of the condition being treated, etc. If necessary, the total daily dosage may be divided and administered several times throughout the day for convenience.

[0141] The above protein and composition can be administered as individual therapeutic agents or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. They can be administered singly or in multiple doses. Taking all of the above factors into account, it is important to administer the amount that achieves maximum effect with the minimum amount possible without causing side effects, a decision that can be readily made by those skilled in the art.

[0142]

[0143] Hereinafter, the present invention will be described in detail by examples.

[0144] However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0145]

[0146] [Example 1]

[0147] [1-1] Production of a single-chain variable fragment of an antibody that specifically binds to GPC3

[0148] Among the anti-GPC3 antibody sequences capable of specifically binding to the GPC3 protein expressed in cancer cells, four types of single chain variable fragments (scFv) (#5, #54, #55, #65) were prepared using sequences that play an important role in specific binding. Each scFv was designed to include heavy and light chain variable regions having CDRs of the amino acid sequences shown in Table 1 below.

[0149] Antibody region sequence sequence number 1st antibody (#5) heavy chain variable region CDR1-HNYGMH1 CDR2-HAIKSSGSSIYYADSVKG2 CDR3-HRSYREYGEVFDI3 light chain variable region CDR1-LRASQSIASDLN4 CDR2-LAASRLQS5 CDR3-LQQSYSFPFT6 2nd antibody (#54) heavy chain variable region CDR1-HSYAMN7 CDR2-HRISSSGGSKYYADSVKG8 CDR3-HRRLGAEGGYFDI9 light chain variable region CDR1-LRASQSISNYLN10 CDR2-LDASSLQS11 CDR3-LQQSYSFPWT12 3rd antibody (#55) heavy chain variable region CDR1-HSYAMH13 CDR2-HAISQSGGSKYYADSVKG14 CDR3-HGLGGGLGAFDY15 light chain Variable region CDR1-LRASQSISNYLN16CDR2-LAASRLQS17CDR3-LQQSYSFPWT18Fourth antibody (#65) Heavy chain variable region CDR1-HSYAMH19CDR2-HRISSSGGNTYYADSVKG20CDR3-HLRYGEAYSYFDI21Light chain variable region CDR1-LRASQDISNDLN22CDR2-LATSTLQS23CDR3-LQQSYTFPFT24

[0150]

[0151] Specifically, using the heavy chain and light chain variable regions of the anti-GPC3 antibodies of the present invention designed as described above, scFv of anti-GPC3 antibodies having an amino acid sequence as shown in Table 2 below (hereinafter referred to as 'anti-GPC3 scFv') was designed.

[0152] 항체서열서열번호제1 항체 (#5)의 scFvMAEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSAIKSSGSSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRSYREYGEVFDIWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSIASDLNWYQQKPGKAPKLLIYAASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSFPFTFGQGTKVEIKAST25제2 항체(#54)의 scFvMAEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSRISSSGGSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRLGAEGGYFDIWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSFPWTFGQGTKVEIKAST26제3 항체(#55)의 scFvMAEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSAISQSGGSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGLGGGLGAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYAASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSFPWTFGQGTKVEIKAST27제4 항체(#65)의scFvMAEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSRISSSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLRYGEAYSYFDIWGQGTLVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISNDLNWYQQKPGKAPKLLIYATSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTFPFTFGQGTKVEIKAST28

[0153]

[0154] [1-2] Design of chimeric antigen receptors to be introduced into natural killer cells

[0155] As a chimeric antigen receptor (CAR) to be introduced into natural killer cells, a CAR was designed that includes an ectodomain including the scFv designed as described above as an antigen-binding site. Specifically, the ectodomain including any one of the four types of scFv as an antigen-binding site was linked to a transmembrane domain (TM) with Myc and a hinge domain, and at this time, a CD28-derived sequence was used as the transmembrane domain, and some sequences of the CD28-derived sequence were designed to belong to the transmembrane domain region and the remaining sequences to belong to the intracellular signaling domain region. In addition, by additionally linking a DAP10-derived sequence and a CD3zeta-derived sequence involved in killing ability as the intracellular signaling domain, the chimeric antigen receptor of the present invention was designed to have an intracellular signaling domain of a chimeric antigen receptor classified as a so-called third-generation CAR (collectively referred to as 'GPC3-CAR'). Then, the base sequence of the gene construct encoding this was inserted into a lentiviral vector (Fig. 1).

[0156]

[0157] [Example 2]

[0158] Production of immune cells expressing a chimeric antigen receptor for GPC3 on their surface

[0159] The lentiviral vector prepared in the above Example 1-2 was transfected into HEK293T cells together with a viral packaging vector (pMDLG / RRE, pRSV / REV, VSVG), and a lentivirus expressing GPC3-CAR was obtained therefrom. This was concentrated using an ultracentrifuge and then infected into natural killer cells using the spinoculation method (360 g, 90 min, RT) at a multiplicity of infection (MOI) of 30. The natural killer cells infected as described above were cultured for 5 hours under 5% CO2 conditions at 37°C, replaced with fresh medium, and 3 days later, the culture was continued by treating with puromycin at a concentration of 3 ug / ml to select properly infected natural killer cells. Uninfected natural killer cells in the control group were also treated with puromycin, and culture was continued using puromycin-treated medium until all natural killer cells in the control group were killed by puromycin. At the point when all natural killer cells in the control group were killed, infected natural killer cells were selected and the experiment was performed.

[0160] Natural killer cells expressing GPC3-CAR#5, GPC3-CAR#54, GPC3-CAR#55, and GPC3-CAR#65 produced and selected as described above (referred to as GPC3-CAR#5-NK cells, GPC3-CAR#54-NK cells, GPC3-CAR#55-NK cells, and GPC3-CAR#65-NK cells, respectively, and collectively referred to as 'GPC3-CAR-NK cells') were treated with an anti-myc antibody (CST; 9B11) that specifically binds to myc of GPC3-CAR (4℃ in the dark for 30 minutes), and the expression of Myc was confirmed through flow cytometry. At this time, original natural killer cells that do not express GPC3-CAR were used as a control.

[0161] As a result, as shown in Fig. 2, it was confirmed that GPC3-CAR was properly expressed in all four types of GPC3-CAR-NK cells compared to the control group.

[0162]

[0163] [Example 3]

[0164] In vitro killing rate of GPC3-CAR-NK cells against GPC3-expressing cancer cells

[0165] As prepared in Example 2 above, natural killer cells expressing the chimeric antigen receptor of the present invention on their surface contain scFv that specifically binds to GPC3 as an antigen binding site, and thus the cell death rate against cancer cells expressing GPC3 was confirmed.

[0166] [3-1] Selection of cancer cells expressing GPC3

[0167] First, we wanted to confirm the cancer cell killing ability of GPC3-CAR-NK against HepG2 (GPC3+), a liver cancer cell line that expresses GPC3, and HepG2 (GPC3-) and SK-Hep-1, liver cancer cell lines that do not express GPC3. To this end, first, 1 ul / 100 ul of anti-GPC3 antibody (Recombinant Anti-Glypican3 / GPC3 Antibody (FITC), Rabbit Monoclonal; Cat: 100393-R024-F (SinoBiological)) was treated and reacted (30 min in a dark place at 4℃), and then the expression level of GPC3 in the above cell lines was confirmed through flow cytometry.

[0168] As a result, as shown in Fig. 3, it was confirmed that HepG2 (GPC3+) cells express GPC3, while HepG2 (GPC3-) and SK-Hep-1 cells do not express GPC3.

[0169] [3-2] Confirmation of specific cell lysis in GPC3-expressing cancer cells

[0170] As above, the cell lytic activity of the GPC3-CAR-NK cells produced in the present invention was confirmed for HepG2 (GPC3+) cells, a liver cancer cell line in which GPC3 expression was confirmed, and HepG2 (GPC3-) and SK-hep-1, liver cancer cell lines in which GPC3 expression was confirmed not to occur.

[0171] Specifically, the 'specific cell lysis' rate of NK cells for each cancer cell line was measured as an indicator of the cell death rate. For the liver cancer cell lines of HepG2 (GPC3+), HepG2 (GPC3-), and SK-hep-1, NK92 cells (hereinafter referred to as 'PURO-92' and 'dEcto-92') that do not express CAR were treated as a control group, and GPC3-CAR-NK expressing the CAR of the present invention was treated as an experimental group. After the cancer cell lines were stained with calcein (5 ug / ml) for 1 hour in a dark place under conditions of 37°C and 5% CO2, the E:T ratio (Effector to Target Ratio) of the NK cells and the cancer cells was set to 1:1, and the NK cells of the experimental group or the control group were treated to each cancer cell, and then cultured under conditions of 37°C and 5% CO2 for 4 hours. After culture, 100 μl of the supernatant was taken and the amount of calcein present in the supernatant was confirmed.

[0172] As a result, as shown in Fig. 4, all four types of GPC3-CAR-NK were confirmed to have a superior killing rate against HepG2 (GPC3+) compared to the control groups PURO-92 and dEcto-92. Meanwhile, there was no significant difference in the killing rate against HepG2 (GPC3-) and SK-hep-1 cell lines when treated with GPC3-CAR-NK and the control groups PURO-92 and dEcto-92. Therefore, it was confirmed that GPC3-CAR-NK specifically kills cancer cells expressing GPC3.

[0173] [3-3] Checking the degree of degranulation

[0174] Furthermore, in order to confirm that the death of the cancer cell line was caused by the GPC3-CAR-NK of the present invention, the cancer cell line and GPC3-CAR-NK were co-cultured to confirm the degree of degranulation.

[0175] The liver cancer cell lines HepG2 (GPC3+), HepG2 (GPC3-), and SK-hep-1 were each treated with anti-CD107a antibody at an E:T ratio of 1:1 together with GPC3-CAR-NK (#5, 54, 55, 65) and Puro-92 and dEcto-92 that do not express GPC3-CAR, and then cultured at 37°C for 4 hours. After 4 hours, natural killer cells were selected and stained with anti-CD56 antibody, and flow cytometry was performed to analyze the degree of CD107a expression in the original NK cells and the GPC3-CAR-NK cells produced in the present invention. NK cells and GPC3-CAR-NK cells were distinguished by cells stained with anti-CD56 antibody, and the degree of expression was analyzed using the degree of anti-CD107a antibody staining in the distinguished cells.

[0176] As a result, as shown in FIGS. 5 to 9, only when the HepG2 (GPC3+) liver cancer cell line expressing GPC3 was cultured with GPC3-CAR-NK, a significant increase in the degranulation expression of CD107a was observed compared to the control group (Puro-92 and dEcto-92).

[0177] [3-4] Confirmation of GPC3 expression patterns in various cancer types

[0178] Cell lines of liver cancer (HepG2), pancreatic cancer (MIA-PACA-2), prostate cancer (PC-3M), colon cancer (HCT116), ovarian cancer (A2780), and lung cancer (H460) were selected, and the expression of GPC3 was confirmed.

[0179] Specifically, each of the above cell lines was cultured at approximately 1x10 5After collecting at a concentration of cell / 100ul, anti-GPC3 antibody (Human Glypican 3 Allophycocyanin; Cat#: FAB119A (R&D system)) was treated at an amount of approximately 1ul / 100ul to the cells, and the cells were reacted in the dark at 4°C for 30 minutes. Then, the expression level of GPC3 in each cell line was confirmed through flow cytometry analysis.

[0180] As a result, as shown in Fig. 10, it was confirmed that GPC3 was expressed in all cell lines of liver cancer, pancreatic cancer, prostate cancer, colon cancer, ovarian cancer, and lung cancer, ranging from as high as 80% to as low as 8%.

[0181] [3-5] Confirmation of the apoptotic effect on various cancers expressing GPC3

[0182] As above, the cancer cell killing effect of the GPC3-CAR-NK cells produced in the present invention was confirmed for various cancer cell lines in which GPC3 expression was confirmed.

[0183] Specifically, for each cancer cell line of liver cancer (HepG2), pancreatic cancer (MIA-PACA-2), prostate cancer (PC-3M), colon cancer (HCT116), ovarian cancer (A2780), and lung cancer (H460), the NK cells (dECTO CAR) in which the scFv of the CAR was deleted as a control group or the GPC3-CAR#54-NK cells of the present invention as an experimental group were treated to measure the 'specific cell lysis' rate for the cancer cell lines. After staining each cancer cell line with calcein (5 ug / ml) for 1 hour in a dark place at 37°C and 5% CO2, the E:T ratio (Effector to Target Ratio) of the NK cells and the cancer cells was set to 5:1, and the NK cells of the experimental group or the control group were treated to the cancer cells, and then cultured for 4 hours at 37°C and 5% CO2. After culture, 100 μl of the supernatant was taken and the amount of calcein present in the supernatant was confirmed.

[0184] As a result, as shown in Fig. 11, it was confirmed that GPC3-CAR-NK showed superior killing ability compared to the control group for all cancer cell lines expressing GPC3, including liver cancer, pancreatic cancer, prostate cancer, colon cancer, ovarian cancer, and lung cancer. In particular, GPC3-CAR-NK showed significantly superior killing ability against liver cancer and prostate cancer cell lines expressing GPC3, and showed the best killing ability against liver cancer cell lines expressing GPC3. Therefore, it was found that GPC3-CAR-NK showed superior killing ability against cancer cell lines expressing GPC3 by expressing a CAR that specifically binds to GPC3, and it was found that it could effectively kill GPC3-expressing cancer cells of various cancers, not limited to liver cancer.

[0185] [3-6] Summary

[0186] In summary of the above experimental results, it was confirmed that the four anti-GPC3 antibodies of the present invention not only exhibit specific binding affinity for GPC3, but also, when the chimeric antigen receptor containing the antigen binding site of the antibody recognizes and binds to GPC3 of cancer cells expressing GPC3, signal transduction of immune cells expressed on the surface of the chimeric antigen receptor is induced, thereby inducing various immune responses that enable the immune cells to attack cancer cells.

[0187]

[0188] Although the present invention has been described in detail only with respect to the described embodiments and experimental examples, it will be apparent to those skilled in the art that various modifications and variations are possible within the technical scope of the present invention, and it is natural that such modifications and variations fall within the scope of the appended claims.

Claims

1. A heavy chain variable region comprising a heavy chain CDR1 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 13, and 19, a heavy chain CDR2 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, and 20, and a heavy chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, and 21; and A light chain variable region comprising a light chain CDR1 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 22, a light chain CDR2 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, 17, and 23, and a light chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 12, 18, and 24; An antibody or antigen-binding fragment thereof that specifically binds to GPC3 (Glypican-3).

2. In claim 1, An antibody or an antigen-binding fragment thereof, wherein the antigen-binding fragment of the antibody is a single chain variable fragment (scFv).

3. An extracellular binding domain comprising an antigen-binding fragment of the antibody of claim 1 or claim 2; transmembrane domain; and comprising an intracellular signaling domain; Chimeric antigen receptor (CAR).

4. In claim 3, A chimeric antigen receptor, wherein the extracellular domain further comprises at least one selected from the group consisting of a hinge domain and a spacer domain.

5. In claim 4, A chimeric antigen receptor, wherein the hinge domain or spacer domain comprises at least one selected from the group consisting of Myc or an epitope thereof, a hinge domain of CD8, and Fc.

6. In claim 3, A chimeric antigen receptor, wherein the transmembrane domain comprises at least one selected from the group consisting of the alpha (α), beta (β) or zeta (ζ) chain of a T-cell receptor (TCR), CD28, CD3 epsilon (ε), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or all or part of an amino acid sequence thereof.

7. In claim 3, The intracellular signaling domain is, A chimeric antigen receptor comprising at least one selected from the group consisting of T-cell receptor (TCR) zeta (ζ), FcR gamma (γ), FcR beta (β), CD3 gamma (γ), CD3 delta (δ), CD3 epsilon (ε), CD3 zeta (ζ), CD5, CD22, CD79a, CD79b, and CD66d, or all or part of an amino acid sequence thereof.

8. In claim 3, The intracellular signaling domain comprises a primary signaling domain and a co-stimulatory signaling domain, The first signal transduction domain comprises at least one selected from the group consisting of T-cell receptor (TCR) zeta (ζ), FcR gamma (γ), FcR beta (β), CD3 gamma (γ), CD3 delta (δ), CD3 epsilon (ε), CD3 zeta (ζ), CD5, CD22, CD79a, CD79b and CD66d, or all or part of an amino acid sequence thereof, A chimeric antigen receptor, wherein the co-stimulatory signal transduction domain comprises at least one selected from the group consisting of CD2, CD7, CD27, CD28, CD30, CD40, 4-1BB (CD137), OX40 (CD134), CDS, ICAM-1, ICOS (CD278), LFA-1 (CD11a / CD18), GITR, MyD88, DAP10, DAP12, PD-1, LIGHT, NKG2C, GPC3, and CD83, or all or part of an amino acid sequence thereof.

9. A polynucleotide comprising a base sequence encoding a chimeric antigen receptor according to any one of claims 3 to 8.

10. An expression vector comprising the polynucleotide of claim 9.

11. An immune cell expressing on its surface a chimeric antigen receptor according to any one of claims 3 to 8.

12. In claim 11, The above immune cell is an immune cell selected from the group consisting of a natural killer cell (NK cell), a natural killer T cell (NKT cell), a cytokine-induced killer cell (CIK), a macrophage, and a dendritic cell.

13. A pharmaceutical composition for preventing or treating cancer, comprising the immune cell of claim 11 as an active ingredient.

14. A pharmaceutical composition for preventing or treating cancer according to claim 13, wherein the cancer is at least one selected from the group consisting of liver cancer, lung cancer, stomach cancer, ovarian cancer, cervical cancer, breast cancer, pancreatic cancer, colon cancer, colon cancer, esophageal cancer, skin cancer, thyroid cancer, kidney cancer, liver cancer, head and neck cancer, bladder cancer, prostate cancer, blood cancer, multiple myeloma, acute myeloid leukemia, malignant lymphoma, thymic cancer, osteosarcoma, fibrous tumor, and brain cancer.

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