A novel anti-CD5 chimeric antigen receptor and immune cells expressing it

Abstract

The present invention relates to a CD5 antibody and a chimeric antigen receptor (CAR) comprising the same. The chimeric antigen receptor of the present invention can be usefully used in anti-cancer immunotherapy with reduced toxicity to normal T cells when expressed in immune cells.

Description

Detailed Description of the Invention

[0001] [Technical Field] The present invention relates to a novel antibody or an antigen-binding fragment thereof that specifically binds to CD5, a chimeric antigen receptor containing the antigen-binding fragment of the antibody, and an immune cell expressing the chimeric antigen receptor. [Background Art] Methods for treating cancer have undergone a continuous development and change process, and methods such as surgical operations, chemotherapy, and radiotherapy have continued to be used until now. However, such existing cancer treatment methods are mostly effective only in the early stage when cancer has not metastasized. In a state where metastasis has already progressed, for example, even if a surgical operation is performed, there is a problem that the possibility of recurrence in the future is high. As a result, recently, research on methods using the immune response for treating cancer has continued.

[0002] Among them, there has been an increasing interest in cell therapy methods that use immune cells, enhance them, or genetically modify them and then re-inject them into patients. For example, tumor infiltrating lymphocytes (TIL), chimeric antigen receptor (CAR), and T-cell receptor (TCR) technologies have been studied. In particular, in the case of CAR-T cells, which are T cells introduced with a chimeric antigen receptor (CAR), an artificial receptor designed to transmit antigen specificity, after a CAR-T cell targeting CD19 received FDA approval as an anti-cancer drug in 2017, its research has been actively promoted. However, there is a problem that it is almost effective only in vitro, and in vivo, it cannot achieve a significant therapeutic effect.

[0003] On the one hand, in the case of CAR-T cells, although effective against tumors, in some cases, there were also side effects of non-specifically attacking healthy tissues. The above-mentioned CD5 is a type of differentiation cluster expressed in T cells and B cells, mainly found in bone marrow and lymphoid tissues, and overexpressed in T cells rather than B cells, so it is mainly utilized as a marker for T cells. In particular, since CD5 is expressed not only in most T cell tumors but also in normal T cells, in the case of anti-CD5 CAR-T cells, there was a problem of auto-cross-reactivity in which not only T cell tumors but also normal T cells were attacked, or even the injected anti-CD5 CAR-T cells themselves were attacked (Reference 1). As a result, there was a problem that normal T cells and anti-CD5 CAR-T cells decreased, and ultimately, the anti-cancer effect was also reduced.

[0004] Therefore, there is a need for research and development of immune cells that can complement the above-mentioned problems of anti-CD5 CAR-T cells while maintaining the anti-cancer activity against cancer cells expressing CD5 as it is. [Prior Art Documents] [Patent Documents] [Patent Document 1] Korean Patent Publication No. 2018-0002604 [Patent Document 2] Korean Patent Registration No. 2,122,546 [Summary of the Invention] [Problems to be Solved by the Invention] An object of the present invention is to provide an antibody or an antigen-binding fragment thereof that can specifically bind to CD5.

[0005] Another object of the present invention is to provide a novel chimeric antigen receptor that amplifies cytotoxicity or cytolytic activity against cancer cells when expressed in immune cells.

[0006] Another object of the present invention is to provide a polynucleotide and an expression vector for expressing the chimeric antigen receptor.

[0007] Furthermore, an object of the present invention is to provide immune cells having an excellent therapeutic effect against cancer by expressing the chimeric antigen receptor on the surface.

[0008] In addition, an object of the present invention is to provide a pharmaceutical composition for treating cancer using the immune cells. [Means for Solving the Problems] To achieve the above object, one aspect of the present invention provides a heavy chain variable region comprising: i) a heavy chain CDR1 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 18, and SEQ ID NO: 26; ii) a heavy chain CDR2 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 19, and SEQ ID NO: 27; and iii) a heavy chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 20, and SEQ ID NO: 28; and iv) a light chain CDR1 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 22, and SEQ ID NO: 30; v) a light chain CDR2 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 23, and SEQ ID NO: 31; and vi) a light chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, and SEQ ID NO: 32; and an antibody or an antigen-binding fragment thereof that specifically binds to CD5.

[0009] The heavy chain variable region is any one selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 21, and SEQ ID NO: 29.

[0010] The light chain variable region is any one selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 25, and SEQ ID NO: 33.

[0011] Another aspect of the present invention is a chimeric antigen receptor (CAR) comprising an extracellular binding domain that specifically binds to CD5, a transmembrane domain, and an intracellular signaling domain, wherein the antigen-binding site that specifically binds to CD5 comprises: i) a heavy-chain CDR1 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 18, and SEQ ID NO: 26; ii) a heavy-chain CDR2 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 19, and SEQ ID NO: 27; and iii) a heavy-chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 20, and SEQ ID NO: 28; and a light-chain variable region comprising: iv) a light-chain CDR1 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 22, and SEQ ID NO: 30; v) a light-chain CDR2 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 23, and SEQ ID NO: 31; and vi) a light-chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, and SEQ ID NO: 32; and is a single-chain variable fragment (scFv) of an anti-CD5 antibody.

[0012] Another aspect of the present invention provides a polynucleotide comprising a nucleotide sequence encoding the chimeric antigen receptor, and an expression vector comprising the polynucleotide.

[0013] Another aspect of the present invention provides an immune cell that expresses the chimeric antigen receptor on its surface.

[0014] The immune cells are natural killer cells, T cells, natural killer T cells, cytokine-induced killer cells, macrophages or dendritic cells. Still another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, which contains the immune cells as an active ingredient.

[0015] The cancer is a blood cancer including leukemia or lymphoma, and the cancer is thymic carcinoma, non-Hodgkin lymphoma, diffuse large cell lymphoma, small lymphocytic lymphoma, T-cell neoplasma, peripheral T-cell lymphoma, mantle cell lymphoma, T-cell acute lymphoblastic lymphoma or chronic lymphoblastic lymphoma. [Advantages of the Invention] The antibody of the present invention, its antigen-binding fragment, and the chimeric antigen receptor using the same can specifically bind to CD5 expressed on cancer cells. As signal transduction occurs in immune cells expressing the chimeric antigen receptor, it significantly improves the cytotoxic or cytolytic activity of immune cells and shows the effect of promoting cytokine secretion. Further, it not only has the effect of increasing the degranulation of cancer cells co-cultured with the immune cells, but also shows reduced reactivity to normal cells expressing CD5 in the case of immune cells expressing the chimeric antigen receptor.

[0016] Therefore, the antibody of the present invention, the chimeric antigen receptor containing the same, and the immune cells expressing the same are used for the purpose of treating cancer.

[0017] However, the effects of the present invention are not limited to the effects described above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description. [Brief Description of Drawings] [FIG. 1] A schematic diagram showing the structure of the chimeric antigen receptor (CD5-CAR) of the present invention containing a single-chain variable fragment (ScFv) that specifically binds to CD5 as an extracellular domain. [FIG. 2] A drawing showing the results of confirming the presence or absence of expression of the chimeric antigen receptors (CD5#1-CAR, CD5#4-CAR, CD5#11-CAR, and CD5#14-CAR) of the present invention in natural killer cells (CD5#1-CAR-NK cells, CD5#4-CAR-NK cells, CD5#11-CAR-NK cells, and CD5#14-CAR-NK cells) into which four kinds of genes of the chimeric antigen receptor of the present invention have been introduced and expressed. [FIG. 3] A drawing showing the results of measuring and comparing the cytotoxicity (cytolytic activity) of natural killer cells (CD5#1-CAR-NK cells, CD5#4-CAR-NK cells, CD5#11-CAR-NK cells, and CD5#14-CAR-NK cells) into which four kinds of genes of the chimeric antigen receptor of the present invention have been introduced and expressed against MOLT4 cells expressing CD5 and U937 cells not expressing CD5. [FIG. 4] A drawing showing the results of measuring and comparing the cytotoxicity (cytolytic activity) of natural killer cells (CD5#11-CAR-NK cells and CD5#14-CAR-NK cells) into which the genes of the chimeric antigen receptors CD5#11-CAR and CD5#14-CAR of the present invention have been introduced and expressed against CCRF-CEM cells and Jurkat cells expressing CD5 and Daudi cells not expressing CD5. [Figures 5A and 5B to 5E] This is a drawing showing the results of measuring and comparing the amount of cytokine (IFN-γ) secreted from natural killer cells (CD5#1-CAR-NK cells, CD5#4-CAR-NK cells, CD5#11-CAR-NK cells, and CD5#14-CAR-NK cells) into which four kinds of chimeric antigen receptor genes of the present invention have been introduced and expressed, when co-cultured with MOLT4 cells expressing CD5 or U937 cells not expressing CD5 (Figure 5A), and the expression level of CD107α, which is a degranulation index (Figures 5B to 5E). [Figure 6] This is a drawing showing the results of measuring and comparing the cytotoxicity (cytolytic activity) of natural killer cells (CD5#11-CAR-NK cells and CD5#14-CAR-NK cells) into which the genes of chimeric antigen receptors CD5#11-CAR and CD5#14-CAR of the present invention have been introduced and expressed, against MNC (mononuclear cells) including normal cells expressing CD5. [Figure 7A] This is a drawing showing the results of confirming the expression level of CD5-CAR in single cell lines obtained by separating and proliferating natural killer cells (CD5#11-CAR-NK cells and CD5#14-CAR-NK cells) into which the genes of chimeric antigen receptors CD5#11-CAR and CD5#14-CAR of the present invention have been introduced and expressed, into single cells. [Figure 7B] This is a drawing showing the results of measuring and comparing the cytotoxicity (cytolytic activity) of each of the established single cell lines against MOLT4 cells and MNC. [Figure 7C] This is a drawing showing the process of selecting a leading cell line from among the single cell lines established as described above. [Figure 8] This is a schematic diagram showing the structure of an mRNA construct designed to temporarily express the chimeric antigen receptor (CD5-CAR) of the present invention. [Figure 9] This is a drawing showing the results of confirming the presence or absence of expression of the chimeric antigen receptors (CD5#11-CAR and CD5#14-CAR) of the present invention, for primary natural killer cells into which the genes of chimeric antigen receptors CD5#11-CAR and CD5#14-CAR of the present invention have been introduced and expressed. [FIG. 10] A drawing showing the results of measuring and comparing the cytotoxicity (cytolytic activity) of primary natural killer cells into which the genes of the chimeric antigen receptors CD5#11-CAR and CD5#14-CAR of the present invention were introduced and expressed against MOLT4 cells and CCRF-CEM cells expressing CD5. [FIG. 11A] A drawing showing the results of confirming the expression levels of ligands involved in the activation of natural killer cells between normal cells and cancer cell lines expressing CD5. [FIG. 11B] A drawing showing the results of measuring the expression level of B7-H6 for each leukocyte cell using MNCs. [FIG. 12] A drawing showing the results of confirming the change in the cytotoxicity of the CD5#11-CAR-NK cells of the present invention against the cancer cell line when the expression of B7-H6 was knocked down in MOLT4 cells and Jurkat cells, which are cancer cell lines expressing CD5. [FIG. 13] A drawing showing the results of measuring the expression levels of B7-H6 and CD5 over time after treating MOLT4, which is a cancer cell line expressing CD5, and MNCs with the CD5#11-CARNK cells of the present invention. [Mode for Carrying Out the Invention] Since the present invention can be subjected to various transformations and can have various embodiments, specific embodiments will be illustrated in the drawings and described in detail below. However, this is not intended to limit the present invention to specific embodiments, and it should be understood to include all transformations, equivalents, or alternatives included in the spirit and technical scope of the present invention. When it is determined that a specific description of related known technologies may obscure the gist of the present invention in explaining the present invention, the detailed description thereof will be omitted.

[0018] 1. Novel anti-CD5 antibodies, antigen-binding fragments thereof, chimeric antigen receptors (CARs) containing the same, and polynucleotides and expression vectors for expressing the same One aspect of the present invention provides an anti-CD5 antibody and an antigen-binding fragment thereof that can specifically bind to CD5.

[0019] In the present invention, the term "antibody" means an immunoglobulin molecule that immunologically binds specifically to an epitope of an antigen and has reactivity. The antibody can include any of monoclonal antibodies, polyclonal antibodies, antibodies having the structure of full-length chains (full-length antibodies), functional fragments having at least antigen-binding function (antigen-binding fragments), and recombinant antibodies. Specifically, the antibody of the present invention is a monoclonal antibody or its antigen-binding fragment. 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 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 is linked to the heavy chain by a disulfide bond. The antibody includes heavy chain (HC) and light chain (LC) polypeptides, and the heavy chain and light chain may include variable regions and constant regions.

[0020] The constant region is a site that mediates the ability of the antibody to bind to various cells of the immune system (such as T cells) and host tissues including components of the complement system. The constant region performs the same function regardless of the type of antigen as long as it is the same type of antibody derived from the same species, and the amino acid sequence constituting it is either identical or has a high degree of similarity for each antibody. The constant region is divided into a heavy chain constant region (abbreviated as CH) and a light chain constant region (abbreviated as CL). The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and / or epsilon (ε) types, and has subclasses such as gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and / or alpha 2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types. IgG is a subtype and includes IgG1, IgG2, IgG3, and IgG4.

[0021] The variable region is an antibody site having specificity for an antigen, and is divided into a variable region of a heavy chain (abbreviated as VH) and a variable region of a light chain (abbreviated as VL). The variable region contains three CDRs (complementary-determining regions, or complementarity-determining regions) and four FRs (framework regions). The CDR is a cyclic site involved in antigen recognition, and the specificity for the antigen is determined by the amino acid sequence of the CDR. The CDRs are designated CDR1, CDR2, and CDR3 in that order, and depending on whether they are CDRs of any polypeptide among the heavy and light chains, they are designated CDR-H1, CDR-H2, and CDR-H3 in the case of the heavy chain variable region, and CDR-L1, CDR-L2, and CDR-L3 in the case of the light chain variable region. Similarly, the FRs are designated FR-H1, FR-H2, FR-H3, and FR-H4 in the case of the heavy chain variable region, and FR-L1, FR-L2, FR-L3, and FR-L4 in the case of the light chain variable region. Further, the CDRs and FRs are arranged in the following order in their respective variable regions.

[0022] In the present invention, the term "antigen-binding fragment" means any fragment of the humanized antibody of the present invention that retains the antigen-binding function of the antibody. The antigen-binding fragment is interchangeably referred to with terms such as "fragment" and "antibody fragment", and the antigen-binding fragment is, for example, Fab, Fab’, F(ab’)2, Fv, etc., but is not limited thereto.

[0023] The Fab has a structure having the variable regions of the light and heavy chains and the 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" differs 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 while the cysteine residues in the hinge region of the Fab' form disulfide bonds. The Fv means the smallest antibody fragment having only the variable regions of the heavy and light chains. The two-chain Fv has the variable region of the heavy chain and the variable region of the light chain linked by a non-covalent bond, and the single-chain Fv generally has the variable region of the heavy chain and the variable region of the light chain linked by a covalent bond through a peptide linker or immediately linked at the C-terminus, and can form a dimer-like structure like the two-chain Fv. The antigen-binding fragment can be prepared by using a proteolytic enzyme (for example, if the whole antibody is digested with papain, Fab can be obtained, and if it is digested with pepsin, an F(ab')2 fragment can be obtained), or through genetic recombination technology, but is not limited thereto.

[0024] The linker is a peptide linker and has a length of about 10 to 25 amino acids. For example, the linker contains hydrophilic amino acids such as glycine (G) and / or serine (S). The linker can include, for example, (GS)n, (GGS)n, (GSGGS)n or (GnS)m (n, m are each 1 to 10), and for example, (GnS)m (n, m are each 1 to 10), but is not limited thereto.

[0025] In the present invention, the term "epitope" means a specific site on an antigen that can be specifically recognized and bound by an immunoglobulin, an antibody or an antigen-binding fragment thereof. The epitope can be formed from contiguous amino acids or from non-contiguous amino acids juxtaposed by the tertiary folding of a protein.

[0026] The term "specifically binds" can mean binding to other molecules with a binding affinity greater than the background binding. For example, the extracellular domain binds to a target antigen with an affinity of about 10 -5 M or greater or a Ka (equilibrium dissociation constant of a specific binding interaction with units of 1 / M) for the target antigen. The affinity is such that the equilibrium dissociation constant (Kd) of the specific binding interaction with units of M is 10 -5 ~10 -13 M or below that range.

[0027] The antibody or antigen-binding fragment thereof of the present invention comprises: i) a heavy-chain CDR1 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 18, and SEQ ID NO: 26; ii) a heavy-chain CDR2 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 19, and SEQ ID NO: 27; and iii) a heavy-chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 20, and SEQ ID NO: 28; and iv) a light-chain CDR1 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 22, and SEQ ID NO: 30; v) a light-chain CDR2 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 23, and SEQ ID NO: 31; and vi) a light-chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, and SEQ ID NO: 32; and specifically binds to CD5.

[0028] The heavy-chain variable region has the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 21, or SEQ ID NO: 29.

[0029] The light-chain variable region has the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 25, or SEQ ID NO: 33.

[0030] The light-chain variable region has the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 16.

[0031] The antibody or antigen-binding fragment thereof of the present invention can further contain, for example, the heavy-chain constant region and / or the 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 CD5, the heavy-chain constant region and / or the light-chain constant region of the antibody derived from a human can be used without limitation as to their types and amino acid sequences.

[0032] The above-described amino acid sequence can include variants having different sequences due to deletion, insertion, substitution of amino acid residues, or combinations thereof, as long as they do not affect the structure, function, activity, etc. of the polypeptide containing the sequence. Further, the above-described amino acid sequence includes amino acids that have undergone ordinary modifications known to those skilled in the art, and the amino acid modifications are, 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 above-described amino acid sequence, but also those having substantially the same amino acid sequence as this and variants thereof. The meaning of those having substantially the same amino acid sequence includes amino acid sequences 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 above-described amino acid sequence, but is not limited thereto.

[0033] In the present invention, the term "chimeric antigen receptor (CAR)" is a synthetic complex designed to induce an immune response against a target antigen and cells expressing said antigen when recognizing and binding thereto. The chimeric antigen receptor may include an extracellular 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 cancer cells, through an antigen-binding site contained in the extracellular domain, thereby causing signal transduction in immune cells and changing the activity of immune cells, so that an immune response can be induced targeting only a specific antigen.

[0034] The chimeric antigen receptor (CAR) of the present invention includes an extracellular domain containing an antigen-binding site that specifically binds to CD5 (ephrin type-A receptor 2); a transmembrane domain; and an intracellular signaling domain.

[0035] The antigen-binding site that specifically binds to CD5 is a single-chain variable fragment (scFv) of an anti-CD5 antibody, which includes a heavy-chain variable region including i) a heavy-chain CDR1 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 18, and SEQ ID NO: 26, ii) a heavy-chain CDR2 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 19, and SEQ ID NO: 27, and iii) a heavy-chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 20, and SEQ ID NO: 28, and a light-chain variable region including iv) a light-chain CDR1 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 22, and SEQ ID NO: 30, v) a light-chain CDR2 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 23, and SEQ ID NO: 31, and vi) a light-chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, and SEQ ID NO: 32.

[0036] When the chimeric antigen receptor of the present invention is expressed on the surface of an immune cell, if the target antigen CD5 binds to the receptor, signal transduction occurs within the immune cell, inducing an improvement in the cytotoxicity (or cytolytic activity) of the immune cell and / or promoting the secretion of cytokines by the immune cell. The improvement in the cytotoxicity (or cytolytic activity) or the promotion of cytokine secretion means that the cytotoxicity (or cytolytic activity) is shown at a higher level or cytokines are secreted at a higher level than the cytotoxicity (or cytolytic activity) or cytokine secretion exhibited by immune cells in the absence of antigen.

[0037] The extracellular domain may further include at least one selected from the group consisting of a hinge domain and a spacer domain. The antigen-binding site of the extracellular domain is linked to the transmembrane domain through the hinge domain and / or the spacer domain.

[0038] The hinge domain physically separates the antigen-binding site from the surface of the immune cell in which the chimeric antigen receptor is expressed so as to enable appropriate cell / cell contact, appropriate antigen / antigen-binding site binding, and appropriate activation of the chimeric antigen receptor, and can play an important role in the positioning of the extracellular domain. The chimeric antigen receptor may include one or more hinge domains between the extracellular domain and the transmembrane domain. The hinge domain is derived from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. The modified hinge region is (a) a naturally occurring hinge region having up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), (b) a portion of a naturally occurring hinge region having at least 10 amino acids (e.g., at least 12, 13, 14, or 15 amino acids) in length having up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), or (c) a core hinge region (which is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length) and refers to a portion of a naturally occurring hinge region. In certain embodiments, one or more cysteine residues in the naturally occurring immunoglobulin hinge region are replaced with one or more other amino acid residues (e.g., one or more serine residues). The modified immunoglobulin hinge region may alternatively or additionally further have other amino acid residues, for example, proline residues of the wild-type immunoglobulin hinge region replaced with cysteine. The hinge domain is a hinge region derived from the extracellular domain of type 1 membrane proteins such as CD8, CD4, CD28, and CD7, but any that can link the antigen-binding site, the transmembrane domain, and the intracellular signaling domain between the cell membranes can be used without limitation. Also, this can be the wild-type hinge region from these molecules or can be varied.

[0039] The spacer domain is referred to as a linker domain and includes, for example, a hinge domain derived from CD28 and / or a hinge domain derived from CD8, and includes all or part of the hinge domain derived from CD28 and / or the hinge domain derived from CD8.

[0040] The hinge domain and / or spacer domain is at least one selected from the group consisting of a Myc epitope, a CD8 hinge domain, and Fc, and specifically includes a Myc epitope and a CD8 hinge domain.

[0041] The transmembrane domain means a part of the region that connects and fuses the extracellular domain and the intracellular signaling domain to fix the chimeric antigen receptor to the plasma membrane of immune cells. The transmembrane domain is derived from natural, synthetic, semi-synthetic or recombinant sources. The transmembrane domain is any 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, but is not limited thereto.

[0042] The transmembrane domain is attached to the extracellular domain through a linker. For example, the linker is a short oligopeptide or polypeptide linker with a length of 2 to 10 amino acids, such as a glycine (G)-serine (S) doublet, but is not limited thereto.

[0043] The intracellular signaling domain corresponds to a portion that transmits the signal generated by the binding of the chimeric antigen receptor and the antigen into the immune cell in order to induce the functions of immune cells (for example, activation including the release of cytotoxic (or cytolytic activity) factors against target cells to which the chimeric antigen receptor and the antigen are bound, cytokine production, proliferation, and cytotoxic or cytolytic activity, or other cellular responses induced by the antigen binding). The intracellular signaling domain is a part of a protein that transmits an operative functional signal and instructs the cell to perform a special function.

[0044] The intracellular signaling domain uses the intracellular signaling domain that has already been used in the development process of chimeric antigen receptors. Specifically, it may only contain CD3ζ used in the first-generation CAR (chimeric antigen receptor). And, as used in the second-generation CAR, in order to improve the reactivity to immune cells, a form in which a co-stimulatory domain (CD28 or CD137 / 4-1BB) and CD3ζ are combined is used. Also, as used in the third-generation CAR, two or more co-stimulatory domains are used. At this time, in order to achieve the expansion and persistence of immune cells containing CAR in vivo, the co-stimulatory domain can be combined with 4-1BB, CD28, or OX40, etc. Furthermore, as utilized in the fourth-generation CAR, an additional gene encoding a cytokine such as IL-12 or IL-15 can be included to further express the CAR-based immune protein of the cytokine. And, as used in the fifth-generation CAR, for the enhancement of immune cells, it may further contain an interleukin receptor chain, for example, IL-2Rβ.

[0045] The intracellular signaling domain may include 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.

[0046] More specifically, the activation of the immune cells by the intracellular signaling domain is mediated by two different categories of intracellular signaling domains. For example, the activation of immune cells is mediated by a co-stimulatory signaling domain that acts in an antigen-independent manner in order to initiate primary signaling domains and secondary signals that stimulate antigen-dependent primary activation. Therefore, the intracellular signaling domain may include a primary signaling domain and a co-stimulatory signaling domain.

[0047] The primary signaling domain means a signaling domain that regulates the activation of immune cells in a stimulatory or inhibitory manner. The primary signaling domain that acts in a stimulatory manner can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. ITAMs containing a primary signaling domain include, but are not limited to, TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD66d, etc. More specifically, the primary signaling domain is CD3ζ (zeta), but is not limited thereto.

[0048] The co-stimulatory signaling domain means the intracellular signaling domain of a co-stimulatory molecule. The co-stimulatory signaling domain may include a co-stimulatory signaling domain 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, CD5, CD83, etc., but is not limited thereto. Specifically, the co-stimulatory molecule is DAP10, but is not limited thereto.

[0049] The chimeric antigen receptor can include two or more intracellular signaling domains. When two or more intracellular signaling domains are included, the intracellular signaling domains are connected in series with each other. Alternatively, they may be connected through a polypeptide linker consisting of 2 to 10 amino acids. The linker sequence is, for example, a glycine-serine continuous sequence. The linker can include, for example, (GS)n, (GGS)n, (GSGGS)n or (GnS)m (n, m are each 1 to 10), for example, (GnS)m (n, m are each 1 to 10), but is not limited thereto.

[0050] The chimeric antigen receptor can further include an immune function promoting factor of immune cells. For example, the immune function promoting factor of the immune cells is an interleukin signal sequence. The interleukin signal sequence is characterized by inducing the expression of IL (interleukin)-12, IL-8, IL-2, etc., but is not limited thereto. Also, when the immune cell is a T cell, the immune function promoting factor is IL-7, CCL19, etc., but is not limited thereto.

[0051] On the other hand, the chimeric antigen receptor can further include a signal peptide for domain exposure. The signal peptide is any secreted or transmembrane protein, which can direct the chimeric antigen receptor to be transported to the cell membrane or cell surface and provide accurate site selection. Specific types are CD8 alpha or mouse light kappa signal peptide, but are not limited thereto.

[0052] In a specific embodiment of the present invention, a chimeric antigen receptor in a form in which scFvs of four types (#1, #4, #11, and #14) of anti-CD5 antibodies having the amino acid sequence as described above are included in the extracellular domain, CD28 linked with Myc and the hinge domain is included in the transmembrane domain and the intracellular signaling domain, and CD3-zeta and DAP10 are linked in the intracellular signaling domain was designed to produce the chimeric antigen receptor of the present invention (CD5-CAR).

[0053] As described above, when the chimeric antigen receptor of the present invention is expressed on the surface of immune cells and recognizes and binds to CD5, it can improve the cytotoxicity or cytolytic activity of immune cells or induce the promotion of cytokine secretion by immune cells. Therefore, when cancer cells expressing CD5 are present, when the antigen-binding site of the chimeric antigen receptor recognizes and binds to the antigen, signal transduction induces the improvement of the cytotoxicity or cytolytic activity of immune cells and / or the promotion of cytokine secretion, and it is usefully used as a chimeric antigen receptor having excellent cytotoxic or cytolytic efficacy for attacking cancer cells.

[0054] Another aspect of the present invention provides a polynucleotide and an expression vector for expressing the chimeric antigen receptor.

[0055] The polynucleotide contains a nucleotide sequence encoding the chimeric antigen receptor.

[0056] In the present invention, the term "polynucleotide" comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and the nucleotide which is the basic structural unit includes not only natural nucleotides but also analogues in which the sugar or base moiety is modified.

[0057] Encrypting the chimeric antigen receptor means that the genetic information for synthesizing a protein having the amino acid sequence of the chimeric antigen receptor of the present invention is encrypted through normal protein expression processes such as transcription and translation of the polynucleotide. At this time, not only a protein having exactly the same amino acid sequence as the chimeric antigen receptor, but also, as described above, a protein having substantially the same amino acid sequence as the protein, but having the same and / or similar activity as the protein, a polynucleotide encoding such a protein is included in the scope of the present invention.

[0058] Specifically, the polynucleotide of the present invention contains a base sequence encoding an antigen-binding fragment of an anti-CD5 antibody that specifically binds to CD5.

[0059] In addition, the polynucleotide of the present invention contains not only the base sequence encoding the antigen-binding fragment, but also a base sequence encoding another extracellular domain portion, the transmembrane domain and / or the intracellular signaling domain linked thereto.

[0060] The descriptions of the chimeric antigen receptor such as the antibody, antigen-binding fragment, extracellular domain, transmembrane domain, intracellular signaling domain, etc. are the same as those described above for these.

[0061] The polynucleotide of the present invention may contain a base sequence substantially the same as the listed base sequences. The substantially the same base sequence includes, for example, the case where the same amino acid is synthesized when transcribed and translated, and has 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 listed base sequences, but is not limited thereto.

[0062] The polynucleotide encoding the chimeric antigen receptor may contain an optimized nucleotide sequence depending on the type of organism into which it is to be introduced and expressed and the expression system such as transcription and translation of the organism. This is due to the degeneracy of codons, whereby there can exist various combinations of nucleotide sequences capable of encoding the protein to be expressed, and all of these are included within the scope of the present invention. The modification of the polynucleotide by codon optimization is determined by the type of organism in which the chimeric antigen receptor of the present invention is to be expressed and applied. For example, the polynucleotide of the present invention is a polynucleotide optimized and modified for codon selection in mammals and primates, and more specifically, is optimized and modified to be suitable for expression and action in humans.

[0063] The expression vector of the present invention contains the above polynucleotide.

[0064] Since the polynucleotide contains the nucleotide sequence encoding the chimeric antigen receptor of the present invention, the expression vector containing the same is used for expressing and producing the chimeric antigen receptor, and plays a role of transferring the polynucleotide to a specific cell or organism so that the chimeric antigen receptor is expressed, or is used for storing and preserving the polynucleotide.

[0065] The term "vector" means a DNA construct containing a DNA sequence operably linked to suitable regulatory sequences capable of expressing the DNA in a suitable host. The vector can be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome or, in some cases, integrate into the genome itself. Also, the "expression vector" is usually a recombinant carrier into which a fragment of heterologous DNA has been inserted, generally meaning a fragment of double-stranded DNA. Here, heterologous DNA means foreign DNA that is not naturally found in the host cell. Once inside the host cell, the expression vector can replicate independently of the host chromosomal DNA, and several copies of the vector and its inserted (heterologous) DNA can be produced.

[0066] The expression vector is constructed using a prokaryotic cell or a eukaryotic cell as the host.

[0067] For example, when the expression vector uses a prokaryotic cell as a host, it generally contains a strong promoter capable of promoting 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, T7 promoter, etc.), a ribosome binding position for the start of translation, and a transcription / translation termination sequence. When E. coli (e.g., HB101, BL21, DH5α, etc.) is used as the host cell, the promoter and operator regions of the E. coli tryptophan biosynthesis pathway (Yanofsky, C, J Bacteriol, (1984) 158: 1018 - 1024), and the leftward promoter of phage λ (pLλ promoter, Herskowitz, I and Hagen, D, Ann Rev Genet, (1980) 14: 399 - 445) are used as regulatory sites. When Bacillus bacteria are used as the 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 bacteria can be used as a regulatory site. The expression vector is prepared by manipulating plasmids frequently used by those skilled in the art (e.g., pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), phages (e.g., λgt4λB, λ-Charon, λΔz1, M13, etc.) or viruses (e.g., SV40, etc.).

[0068] When the expression vector uses eukaryotic cells as hosts, promoters derived from the genomes of mammalian cells (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 75K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) are used, and generally can have a polyadenylation sequence as a transcription termination sequence. The expression vector has a CMV promoter.

[0069] In addition, the expression vector is fused with other sequences to facilitate the purification of the antibody expressed thereby. Examples of the sequences to be fused include glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA). Also, since the protein expressed by the expression vector of the present invention is a chimeric antigen receptor, when considering its properties, the protein expressed without an additional sequence for purification can also be easily purified through a protein A column or the like.

[0070] The expression vector contains an antibiotic resistance gene commonly used by those skilled in the art as a selection marker, and can include, for example, resistance genes to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0071] In addition, the expression vector may further contain not only a promoter that promotes the expression of the gene to be transfected and a basal element necessary for transcription, but also an enhancer used for promoting and regulating the expression.

[0072] 2. Immune cells expressing a chimeric antigen receptor against CD5 on the surface Another aspect of the present invention provides immune cells characterized by exhibiting improved cytotoxicity or cytolytic activity against cancer cells expressing CD5.

[0073] The immune cells express the chimeric antigen receptor of the present invention described above on the surface. The chimeric antigen receptor is the same as that described above in "1. Novel anti-CD5 antibody, its antigen-binding fragment, and chimeric antigen receptor (CAR) containing the same and polynucleotide and expression vector for expressing the same". Specifically, the chimeric antigen receptor may contain an antigen-binding site capable of specifically binding to the CD5 antigen expressed on cancer cells.

[0074] The expression of the chimeric antigen receptor on the surface of immune cells means any immune cell engineered by the addition or modification of nucleic acid encoding the chimeric antigen receptor. Therefore, in order to express the chimeric antigen receptor on the surface of immune cells as described above, the polynucleotide encoding the chimeric antigen receptor or an expression vector containing the same can be transfected or transduced into the immune cells. The transfection can be carried out by various methods known in the art, such as calcium phosphate-DNA coprecipitation method, DEAE-dextran-mediated transfection method, polybrene-mediated transfection method, electroporation method, microinjection method, liposome fusion method, lipofectamine and protoplast fusion method. Also, the transfection means transferring a gene into a cell using a virus or virus vector particles by means of infection. Although transfection and transduction are used interchangeably in this specification, it is desirable that both are analyzed as transformation of foreign gene transfer into host cells in a broad sense, and cells into which a foreign gene has been introduced by transfection or transduction are referred to as transformants.

[0075] The immune cells can be used without limitation as long as they are cells capable of inducing immunity to induce a desired therapeutic effect, and can be obtained from peripheral blood, umbilical cord blood, bone marrow, tumor-infiltrating lymphocytes, lymph node tissue or thymus tissue, and can be obtained by differentiating from placental cells, embryonic stem cells, induced pluripotent stem cells or hematopoietic stem cells. Also, the immune cells can be obtained not only from humans, monkeys, chimpanzees, dogs, cats, mice, rats and their genetically engineered species, but also from established cell lines.

[0076] Methods for obtaining immune cells can use any means known to those of ordinary skill in the art and can be obtained autologously, allogeneically, or xenogeneically. "Autologous" means any cell derived from the same individual that will later be reintroduced into that individual, "allogeneic" means any cell derived from another animal of the same species as the individual into which the cell is introduced, and "xenogeneic" means a cell derived from an animal of another species.

[0077] In particular, the immune cells are any one selected from natural killer cells (NK cells), T cells, natural killer T cells (NKT cells), cytokine-induced killer cells (CIK), macrophages, dendritic cells, etc., but are not limited thereto. Therefore, the immune cells expressing the chimeric antigen receptor according to the present invention are CAR-NK cells (Chimeric Antigen Receptor Natural Killer Cell), CAR-T cells (Chimeric Antigen Receptor T Cell), CAR-NKT cells (Chimeric Antigen Receptor Natural killer T Cell), CAR-macrophages (Chimeric Antigen ReceptorMacrophage), etc.

[0078] The immune cells provided by the present invention can regulate the start (on) / stop (off) of the reaction of normal chimeric antigen receptors to target cells, and thus include a very beneficial safety switch in subsequent cell therapies and situations where the activity of therapeutic cells needs to be increased or decreased. For example, when immune cells expressing a chimeric antigen receptor are provided to a patient, in certain situations, there are side effects, such as off-target toxicity. Or, for example, the therapeutic cells can act to reduce the number of tumor cells or the tumor size and are no longer needed. In such situations, the activation of the therapeutic cells can be regulated so that they are no longer activated. In particular, the CAR-NK cells in which a chimeric antigen receptor is introduced into natural killer cells not only solve problems such as the continuous toxicity problems, the risk of autoimmune diseases, the problem of graft-versus-host disease (GVHD) for allogeneic cell transplantation, and off-target toxicity problems in cancer immunotherapy when using existing CAR-T therapeutics based on T cells through the start (on) / stop (off) switch, but also have the advantage of being able to target various cancer cells and being utilized as a general therapeutic agent.

[0079] The immune cells of the present invention express, on the cell surface, a chimeric antigen receptor that includes, as an extracellular domain, an antigen-binding variable fragment (scFv) of an antibody that can specifically recognize and bind to CD5 specifically expressed in cancer cells. Therefore, when cancer cells expressing the CD5 are present, signal transduction can occur through the chimeric antigen receptor, through which the cytotoxicity or cytolytic activity of the immune cells is further improved, and the secretion amount of cytokines is increased. Therefore, the immune cells of the present invention can have the activity of attacking and treating cancer cells.

[0080] In a specific embodiment of the present invention, a chimeric antigen receptor containing four antigen-binding variable fragments of the CD5-specific antibody of the present invention as an extracellular domain was expressed on the surface of natural killer cells. When each of the four natural killer cells was co-cultured with cancer cell lines expressing CD5, such as MOLT4 cells, CCRF-CEM, and Jurkat cells, not only was the cytotoxicity (or cytolytic activity) improved, and the secretion amount of cytokines and the degree of degranulation were significantly increased, but it was also confirmed that they showed decreased cytotoxicity against MNCs containing normal cells expressing CD5. Thus, it was confirmed that the immune cells of the present invention have a selectively excellent therapeutic effect on cancer cells expressing CD5.

[0081] 3. Preventive or therapeutic use of the immune cells of the present invention against cancer Still another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the immune cells as an active ingredient.

[0082] The descriptions of the immune cells, the chimeric antigen receptors expressed thereby, etc. are the same as those described in the items of "1. Novel anti-CD5 antibody, its antigen-binding fragment, and chimeric antigen receptor (CAR) containing the same and polynucleotide and expression vector for expressing the same" and "2. Immune cells expressing a chimeric antigen receptor against CD5 on the surface". In order to avoid repeated description, the description is omitted.

[0083] In the present invention, the terms "cancer" and "tumor" are used interchangeably and refer to or denote a physiological state of a mammal characterized by typically unregulated cell growth / proliferation.

[0084] The cancer or carcinoma that can be treated with the pharmaceutical composition of the present invention is not particularly limited and includes both solid cancers and blood cancers. In particular, it is a blood cancer such as the leukemia or lymphoma, specifically, selected from thymic carcinoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, small lymphocytic lymphoma, T-cell tumor, peripheral T-cell lymphoma, mantle cell lymphoma, T-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, and the like.

[0085] In addition, in the pharmaceutical composition of the present invention, the number of the immune cells is included in an amount of 1 to 10 times, 2 to 10 times, or 5 to 8 times, compared to the number of tumor cells of an individual to be treated, but is not limited thereto.

[0086] On the other hand, in the treatment of cancer, it is common to perform chemotherapy and radiotherapy in parallel. The anticancer agents used in chemotherapy induce cell death of cells with active proliferation, and radiotherapy for enhancing the therapeutic effect of the anticancer agents increases such cell death. At this time, the cells in which cell death is induced by the anticancer agents and radiation are not limited to cancer cells, and may also affect the immune cell therapy agents administered to an individual for immunotherapy.

[0087] Chemotherapy includes, but is not limited to, CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone), EPOCH (etoposide, vincristine, doxorubicin, cyclophosphamide, prednisone), or any other various drug therapies.

[0088] In the present invention, a "cell therapy agent" is a pharmaceutical product (regulated by the US FDA) that is used for the purposes of treatment, diagnosis, and prevention with cells and tissues produced through separation, culture, and special operations from an individual, and through a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro or changing the biological characteristics of cells by other methods to restore the functions of the cells or tissues, it means a pharmaceutical product used for the purposes of treatment, diagnosis, and prevention.

[0089] On the other hand, the pharmaceutical composition of the present invention may further contain a pharmaceutically or pharmacologically acceptable carrier. The meaning of "pharmaceutically acceptable" is that it does not have more toxicity than the applicable (formulated) subject can tolerate without suppressing the activity of the active ingredient, and the "carrier" is defined as a compound that facilitates the addition of a compound into cells or tissues.

[0090] The pharmaceutical composition of the present invention is administered alone or in admixture with a convenient carrier or the like, and such dosage forms are single-dose or repeated-dose forms. The pharmaceutical composition is a solid preparation or a liquid preparation. Solid preparations include, but are not limited to, powders, granules, tablets, capsules, suppositories, etc. Solid preparations 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 and propylene glycol solutions, suspensions, emulsions, etc., and can be manufactured by adding appropriate coloring agents, flavoring agents, stabilizers, thickening agents, etc. For example, a powder can be manufactured by simply mixing a tri-hydroxy 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, microcrystalline cellulose, etc. Granules can be manufactured by mixing the tri-hydroxy derivative of the polyunsaturated fatty acid of the present invention, a pharmaceutically acceptable appropriate carrier, and a pharmaceutically acceptable appropriate binder such as polyvinylpyrrolidone and hydroxypropylcellulose, and then using a wet granulation method using a solvent such as water, ethanol, isopropanol, etc. or a dry granulation method using compressive force. Further, tablets can be manufactured by mixing the granules with a pharmaceutically acceptable appropriate lubricant such as magnesium stearate and then tableting using a tableting machine.

[0091] The pharmaceutical composition of the present invention is administered by, but not limited to, oral agents, injections (e.g., intramuscular injection, intraperitoneal injection, intravenous injection, infusion, subcutaneous injection, implant), inhalants, nasal administrations, vaginal agents, rectal administrations, sublingual agents, transdermal agents, topical agents, etc., depending on the disease to be treated and the condition of the individual. It is formulated into an appropriate dosage unit form containing a pharmaceutically acceptable carrier, additive, vehicle that is commonly used depending on the administration route and is non-toxic.

[0092] In particular, the pharmaceutical composition of the present invention is used through an injection ampoule. The injection ampoule can be mixed and prepared with an injection solution immediately before use, and as the injection solution, physiological saline, glucose, mannitol, Ringer's solution, etc. can be used. The pharmaceutical composition or preparation of the present invention thus produced is administered in the form of a mixture with cells used for transplantation and other uses using a conventional administration method by those skilled in the art. The actual dosage of the active ingredient must be determined in light of various relevant factors such as the disease to be treated, the severity of the disease, the administration route, the weight, age, and gender of the patient.

[0093] Also, the desirable dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the degree of the disease, the form of the drug, the administration route, and the period, but can be appropriately selected by those skilled in the art. For example, the pharmaceutical composition is administered at about 0.0001 mg / kg to about 10 g / kg per day and at a daily dosage volume of about 0.001 mg / kg to about 1 g / kg. However, the dosage varies depending on the degree of purification of the mixture, the patient's condition (age, gender, weight, etc.), the severity of the condition being treated, etc. If necessary, for convenience, the total daily dosage is divided into several administrations during the day.

[0094] On the other hand, the composition can be in the form of a quasi-drug composition, a composition for health foods, etc. in addition to the pharmaceutical composition.

[0095] Furthermore, another aspect of the present invention provides a method for preventing or treating cancer including the step of administering the immune cells to an individual in need of treatment.

[0096] The immune cells are in the form of the aforementioned pharmaceutical composition, and those skilled in the art can determine the appropriate administration route and volume of the pharmaceutical composition.

[0097] Hereinafter, the present invention will be described in more detail through examples.

[0098] However, the following examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited to the following examples.

[0099] [Example 1] Production of immune cells expressing a chimeric antigen receptor against CD5 on the surface [1-1] Preparation of single-chain variable fragments of antibodies specifically binding to CD5 Among the anti-CD5 antibody sequences that can specifically bind to CD5, four single-chain variable fragments (scFv) (#1, #4, #11, #14) were produced using the sequences that play an important role in specific binding

[0100] As described in Table 1 below, for #1 scFv, it was designed to include a heavy chain variable region containing a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 3, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 4, and a light chain variable region containing a light chain CDR1 having the amino acid sequence of SEQ ID NO: 6, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 8. For #4 scFv, it was designed to include a heavy chain variable region containing a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 10, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 11, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 12, and a light chain variable region containing a light chain CDR1 having the amino acid sequence of SEQ ID NO: 14, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 15, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 16. For #11 scFv, it was designed to include a heavy chain variable region containing a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 18, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 19, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 20, and a light chain variable region containing a light chain CDR1 having the amino acid sequence of SEQ ID NO: 22, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 23, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 24. And for #14 scFv, it was designed to include a heavy chain variable region containing a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 26, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 27, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 28, and a light chain variable region containing 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.

[0101]

Table 1

[0102] [1-2] Design of Chimeric Antigen Receptor Four types of scFv designed as described above were used as the extracellular domain containing the antigen-binding site, and a chimeric antigen receptor (CAR) was designed to be introduced into natural killer cells using the CAR region of the sequence as described in Table 2 below. Specifically, the extracellular domain containing the four types of scFv as the antigen-binding site was linked with Myc and the hinge domain using CD28 as the transmembrane domain, and the intracellular signaling domain CD3-zeta and the co-stimulatory molecule CD28 DAP10 were added and linked to the transmembrane domain, so that the chimeric antigen receptor of the present invention has the intracellular signaling domain of the so-called third-generation CAR (each being designated as "CD5#1-CAR", "CD5#4-CAR", "CD5#11-CAR", and "CD5#14-CAR", and collectively referred to as "CD5-CAR"). Then, the nucleotide sequence of the gene construct encoding it was inserted into a lentiviral vector (Clontech, 632155) (Figure 1).

[0103]

Table 2

[0104] [1-3] Production of natural killer cells expressing CD5-CAR The lentiviral vector prepared in Example 1-2 was transformed into HEK293T cells together with a viral packaging vector (PMDLg / RRE, RSV / REV, VSVG), and then a lentivirus expressing CD5-CAR was obtained. The lentivirus was concentrated using an ultra-high-speed centrifuge. After infecting HEK293T cells with the concentrated lentivirus, the amount of the Myc epitope of CD5-CAR was confirmed by flow cytometry to calculate the infection unit. The number of natural killer cells and the amount of lentivirus were calculated so that the multiplicity of infection (MOI) was 30, and the lentivirus expressing CD5-CAR was infected into natural killer cells (NK92 cells) by the spinoculation method (360 g, 90 min, RT). The natural killer cells infected as described above were cultured at 37 °C and 5% CO2 for 5 hours, then replaced with fresh culture medium. Three days later, for the selection of well-infected natural killer cells, puromycin at a concentration of 3 μg / ml was treated to continue the culture.

[0105] As a control group, puromycin was also treated on uninfected natural killer cells, and the culture was continued using the medium treated with puromycin until all natural killer cells in the control group were killed by puromycin. When all natural killer cells in the control group were killed, the infected natural killer cells were selected, and the selected natural killer cells were cultured again in a medium without puromycin for proliferation or expansion. For the proliferation or expansion of the selected natural killer cells, a medium containing 12.5% fetal bovine serum, 12.5% horse serum, 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, and 200 U / ml recombinant IL-2 in Alpha-MEM was used.

[0106] Natural killer cells expressing the CD5#1-CAR, CD5#4-CAR, CD5#11-CAR, and CD5#14-CAR prepared and selected as described above (collectively referred to as "CD5-CAR-NK cells", hereinafter referred to as "CD5#1-CAR-NK cells", "CD5#4-CAR-NK cells", "CD5#11-CAR-NK cells", and "CD5#14-CAR-NK cells", respectively) were treated with an anti-myc antibody (CST; 9B11) that specifically binds to the myc of CD5-CAR (4°C, 30 minutes in female cows), and the expression of CD5-CAR was confirmed by confirming the expression of Myc through flow cytometry. At this time, as a control group, original natural killer cells that do not express CD5-CAR were used.

[0107] As a result, as shown in Figure 2, it was confirmed that CD5-CAR was sufficiently expressed in all four types of CD5-CAR-NK cells compared to the control group.

[0108] [Example 2] Confirmation of the activity of immune cells expressing CD5-CAR against cancer cells expressing CD5 [2-1] Confirmation of the activity of CD5-CAR-NK cells against cancer cells expressing CD5 Regarding the cancer cell line MOLT4, which is known to express CD5, and the cancer cell line U937, which is known not to express CD5, the cytotoxicity (or cell killing ability, cell lysis activity) of the four types of CD5-CAR-NK cells produced in Examples 1-3 was confirmed by the Calcein AM assay. Specifically, after treating MOLT4 cells and U937 cells with Calcein-AM (Life Technologies; C1430) at a concentration of 5 μg / ml and reacting them (37°C, 5% CO2, female bovine for 1 hour), the four types of each CD5-CAR-NK cell were treated with the Calcein-stained MOLT4 cells and U937 cells at ratios of 2:1, 1:1, and 0.5:1 (natural killer cells: cancer cells) respectively and reacted with 200 μl of RPMI1640 (10% FBS) (37°C, 5% CO2 for 2 hours). Then, 100 μl of the supernatant was taken to confirm the amount of Calcein present in the supernatant, and the cytotoxicity under each condition was calculated by the following method.

[0109] Cytotoxicity (%) = (Calcein release value by condition - spontaneous value) / (maximum value - spontaneous value) x 100 As a control group, an empty lentiviral vector that does not contain the gene construct encoding the CD5-CAR of the present invention, or a lentiviral vector containing a gene construct in which the extracellular domain has been removed from the CD5-CAR of the present invention, was introduced into natural killer cells by the method as in Examples [1-3] and selected with puromycin. The natural killer cells obtained through this process were used, and these were referred to as "PURO NK cells" and "dEcto-NK cells" respectively.

[0110] As a result, as shown in Fig. 3, for MOLT4 cells expressing CD5, all of the four types of CD5-CAR-NK cells not only showed significantly higher cytotoxicity compared to the control group (PURO NK cells or dEcto-NK cells), but also such cytotoxicity was confirmed to be concentration-dependent in the treated CD5-CAR-NK cells. In contrast, for U937 cells not expressing CD5, it was confirmed that neither the control group nor the four types of CD5-CAR-NK cells could show significant cytotoxicity.

[0111] In addition, among the four types of CD5-CAR-NK cells as described above, two types (CD5#11-CAR-NK cells, CD5#14-CAR-NK cells) that were confirmed to have the best cytotoxicity were selected, and CCRF-CEM and Jurkat, which are other cancer cell lines known to express CD5, and Daudi, which is another cancer cell line known not to express CD5, were used as targets. Each of the two types of CD5-CAR-NK cells was treated at a ratio of 2:1 (natural killer cells: cancer cells), and the cytotoxicity of the CD5-CAR-NK cells was confirmed again by the same method as above.

[0112] As a result, as shown in Fig. 4, for CCRF-CEM cells and Jurkat cells expressing CD5, it was confirmed that both of the two types of CD5-CAR-NK cells showed significantly higher cytotoxicity compared to the control group (PURO NK cells or dEcto-NK cells). In contrast, for Daudi cells not expressing CD5, it was confirmed that neither the control group nor the two types of CD5-CAR-NK cells could show significant cytotoxicity.

[0113] Furthermore, cytokine and granule secretion of the four types of CD5-CAR-NK cells were confirmed. Specifically, Puro NK cells, dEcto-NK cells or each of the four types of CD5-CAR-NK cells prepared in Examples 1-3 were treated with MOLT4 cells and U937 cells at a ratio of 1:1 and reacted with RPMI1640 (10% FBS). After that (at 37°C, 5% CO2 for 12 hours), the supernatant was collected and IFN-γ, a cytokine present in the supernatant, was confirmed through ELISA. At this time, the amount of cytokine secreted from Puro NK cells, dEcto-NK cells and CD5-CAR-NK cells alone was used as a control group.

[0114] As a result, as shown in Fig. 5A, it was confirmed that the secretion amount of INF-γ was significantly improved only in the CD5-CAR-NK cells treated with MOLT4 cells expressing CD5.

[0115] Also, Puro NK cells, dEcto-NK cells or each of the four types of CD5-CAR-NK cells prepared in Examples 1-3 were treated with MOLT4 cells and U937 cells at a ratio of 1:1 and reacted with RPMI1640 (10% FBS). After that (at 37°C, 5% CO2 for 4 hours), an anti-CD56 antibody was treated and stained so that natural killer cells could be sorted, and the degree of CD107a expression in Puro NK cells, dEcto-NK cells and the four types of CD5-CAR-NK cells was analyzed using flow cytometry.

[0116] As a result, as shown in Figs. 5B to 5E, similar to the case of INF-γ, it was confirmed that the expression of CD107a was significantly improved only in the CD5-CAR-NK cells treated with MOLT4 cells expressing CD5.

[0117] [2-2]Confirmation of the activity of CD5-CAR-NK cells against normal T cells expressing CD5 To confirm the activity of the CD5-CAR-NK cells of the present invention against normal T cells expressing CD5, which are not cancer cells expressing CD5, first, NMC (mononuclear cells) were isolated from umbilical cord blood using the Ficoll-Paque (registered trademark) Gradient method. Specifically, the umbilical cord blood was carefully added onto ficoll so that they did not mix, and after centrifugation at 2000 rpm for 30 minutes at room temperature, a buffy coat (Enriched MNC fraction) containing white blood cells and platelets was separated. The fraction separated as described above was treated with 1X ACK (Ammonium-Chloride-Potassium) lysis buffer solution at 37°C for 10 minutes to break down the remaining red blood cells and isolate only pure MNCs.

[0118] Using the MNCs separated as described above, the CD5#11-CAR-NK cells and CD5#14-CAR-NK cells produced in Examples 1-3 were each treated at a ratio of 1:1 (natural killer cells: MNC), and the cytotoxicity of the CD5-CAR-NK cells was confirmed in the same manner as in Example [2-1].

[0119] As a result, as shown in FIG. 6, it was confirmed that both the CD5#11-CAR-NK cells and the CD5#14-CAR-NK cells showed reduced cytotoxicity against MNCs compared to MOLT4, which is a cancer cell expressing CD5.

[0120] [2-3] Confirmation of the activity of CD5-CAR-NK cells according to the expression level of CD5-CAR Using a high-speed flow cell sorter (BD FACS Aria Fusion), each of the CD5#11-CAR-NK cells and CD5#14-CAR-NK cells produced in Examples 1-3 was separated into single cells in a 96-well plate. Then, among them, cells growing at similar speeds were repeatedly selected and grown in a 48-well plate, 24-well plate, 6-well plate, and 25T flask in that order.

[0121] Subsequently, the expression levels of CD5-CAR were confirmed for each of the single cells of the CD5#11-CAR-NK cells and CD5#14-CAR-NK cells separated as described above, and based on this, cell lines with different expression levels of CD5-CAR were constructed (Figure 7A).

[0122] Subsequently, using the MOLT4 cancer cell line expressing CD5 and the MNCs separated from Example [2-2] above, the single cell lines of the CD5#11-CAR-NK cells and CD5#14-CAR-NK cells constructed as described above were each treated at a ratio of 1:1 (natural killer cells: cancer cells or MNCs), and the cytotoxicity of the individual single cell lines of the CD5-CAR-NK cells was confirmed in the same manner as in Example [2-1].

[0123] As a result, as shown in Figure 7B, even though the individual single cell lines of the CD5-CAR-NK cells showed improved cytotoxicity against MOLT4, which is a cancer cell expressing CD5, it was confirmed that the cytotoxicity itself was not proportional to the expression level of CD5-CAR. Also, against MNCs containing normal T cells, all the individual single cell lines of the CD5#11-CAR-NK cells showed decreased cytotoxicity, but the individual single cell lines of the CD5#14-CAR-NK cells were confirmed to have relatively little decrease in cytotoxicity.

[0124] From the results as described above, among the two types of CD5-CAR-NK cells, for the individual single cell lines of the CD5#11-CAR-NK cells showing low cytotoxicity by normal T cells, while showing high cytotoxicity against MOLT4, which is a cancer cell expressing CD5, single cell lines showing low cytotoxicity against MNCs containing normal T cells were selected. As a result, as shown in Figure 7C, the L4 single cell line was selected as the leading cell line.

[0125] [2-4]Confirmation of the Activity of CD5-CAR-NK Cells Based on Primary Natural Killer Cells Furthermore, when the CD5-CAR-NK cells of the present invention were prepared using primary natural killer cells other than NK92 cells, it was confirmed whether they could exhibit excellent activities as confirmed in Examples [2-1] to [2-3].

[0126] For this purpose, first, as shown in FIG. 8, mRNA constructs of CD5#11-CAR and CD5#14-CAR having a structure that combines the UTRs of mRNA to improve protein expression and mRNA safety were designed and prepared, and the nucleotide sequences of the mRNA constructs as described above were inserted into a lentiviral vector (Clontech, 632155). Then, using an mRNA synthesis Kit (EZ TM High Yield In Vitro Transcription kit), mRNA of CD5-CAR having the structure shown in FIG. 8 was synthesized using the gene construct as a template.

[0127] The synthesized CD5-CAR mRNA was introduced into primary natural killer cells by electroporation. After 7 hours, an anti-myc antibody (CST; 9B11) that specifically binds to Myc of CD5-CAR was treated (4°C, 30 minutes with female bovine), and the expression of CD5-CAR was confirmed by confirming the expression of Myc through flow cytometry. As a result, as shown in FIG. 9, it was confirmed that CD5-CAR was sufficiently expressed in primary natural killer cells.

[0128] Also, two types of CD5-CAR-NK cells produced based on primary natural killer cells as described above were treated at ratios of 0.5:1 and 1:1 (primary natural killer cells: cancer cells) with respect to the cancer cell lines MOLT4 and CCRF-CEM, which are known to express CD5, and MNC isolated from Example [2-2], and the cytotoxicity of CD5-CAR-NK cells based on primary natural killer cells was confirmed by the same method as in Example [2-1].

[0129] As a result, as shown in FIG. 10, against MOLT4 cells and CCRF-CEM cells, which are cancer cells expressing CD5, both of the two types of primary natural killer cell-based CD5-CAR-NK cells not only exhibited significantly higher cytotoxicity compared to the control groups (PURO NK cells or dEcto-NK cells), but it was also confirmed that such cytotoxicity was concentration-dependent in the treated CD5-CAR-NK cells. In contrast, it was confirmed that MNCs containing normal T cells expressing CD5 exhibited reduced cytotoxicity.

[0130] [Example 3] Confirmation of ligands that regulate the activity of the CD5-CAR-NK cells of the present invention [3-1] Ligand screening for the CD5-CAR-NK cells of the present invention Although both normal T cells and T cell-based cancer cells express CD5, in order to investigate the reason why the CD5-CAR-NK cells of the present invention exhibit particularly high cytotoxicity against cancer cells expressing CD5, as confirmed in Example 2 above, the expression of ligands involved in the activation of natural killer cells was confirmed in MNCs separated from Example [2-2], which are normal cells, and in cancer cells expressing CD5, namely MOLT4, CCRF-CEM, and Jurkat. Antibodies specifically binding to each ligand were treated with each of the above cells, and the expression level of the ligand was analyzed by flow cytometry.

[0131] As a result, as shown in FIG. 11A, among various ligands, B7-H6 was significantly highly expressed in all three types of cancer cells as described above, and it was confirmed that HLA class1, an inhibitory ligand, was highly expressed in normal cells.

[0132] In addition, antibodies that specifically bind to CD3, which is a marker for T cells, CD34, which is a marker for hematopoietic stem cells (HSCs), CD33, which is a marker for myeloid cells, and CD56, which is a marker for natural killer cells, were treated with MNCs isolated from the above Example [2-2], and the expression level of B7-H6 was measured for each type of white blood cell by flow cytometry. As a result, as shown in FIG. 11B, it was confirmed that B7-H6 was not expressed in most normal white blood cells.

[0133] [3-2] Confirmation of the effect of B7-H6 expression on the activity of CD5-CAR-NK cells It was confirmed how the expression of B7-H6, a natural killer cell activation ligand confirmed in the above Example [3-1], affects the activity of the CD5-CAR-NK cells of the present invention.

[0134] Therefore, siRNA having a sequence complementary to the B7-H6 gene was introduced into MOLT4 cells and Jurkat cells, which are cancer cell lines expressing CD5, to temporarily knockdown the expression of B7-H6. Thereafter, CD5#11-CAR-NK cells were treated with the cancer cells in which the expression of B7-H6 was decreased as described above at a ratio of 1:1 (natural killer cells: cancer cells), and the cytotoxicity of CD5#11-CAR-NK cells was confirmed by the same method as in Example [2-1].

[0135] As a result, as shown in FIG. 12, it was confirmed that the cytotoxicity of CD5#11-CAR-NK cells decreased in cancer cells in which the expression of B7-H6 was decreased compared to cancer cells in which the expression of B7-H6 was not decreased.

[0136] Furthermore, CD5#11-CARNK cells were treated with MOLT4, which is a cancer cell line expressing CD5, and MNCs isolated from the above Example [2-2] at a ratio of 1:1 (natural killer cells: cancer cells or MNCs), and the expression levels of B7-H6 and CD5 in living MOLT4 cells and MNCs were measured every 2 hours by flow cytometry.

[0137] As a result, as shown in FIG. 13, it was confirmed that the expression of B7-H6 and the expression of CD5 decreased over time in living MOLT4. From the above results, it can be seen that cells with high B7-H6 expression were preferentially killed by CD5#11-CAR-NK cells.

[0138] Consideration of [Example 2] and [Example 3] The CD5-CAR-NK cells of the present invention not only showed improved cytotoxicity in a concentration-dependent manner against cancer cells expressing CD5, but also increased the secretion of IFN-γ and degranulation. Thus, it can be seen that the CD5-CAR-NK cells of the present invention specifically react against cancer cells expressing CD5.

[0139] CD5 is expressed in most T cell malignancies but is also expressed in normal T cells. All CD5-CARs developed to date have been reported to show side effects on normal T cells. However, in the case of the CD5-CAR-NK cells of the present invention, it was confirmed that they show reduced cytotoxicity against MNCs isolated from umbilical cord blood. In particular, it was confirmed that CD5#11-CAR-NK cells show even lower cytotoxicity compared to CD5#14-CAR-NK cells. The difference between CD5#11-CAR-NK cells and CD5#14-CAR-NK cells as described above is considered to be due to the difference in the structure of the single-chain variable fragment, and includes a heavy-chain variable region containing a heavy-chain CDR1 having the amino acid sequence of SEQ ID NO: 18, a heavy-chain CDR2 having the amino acid sequence of SEQ ID NO: 19, and a heavy-chain CDR3 having the amino acid sequence of SEQ ID NO: 20, and a light-chain variable region containing a light-chain CDR1 having the amino acid sequence of SEQ ID NO: 22, a light-chain CDR2 having the amino acid sequence of SEQ ID NO: 23, and a light-chain CDR3 having the amino acid sequence of SEQ ID NO: 24. The #11 ScFv is judged to have a more advantageous effect on safety. Furthermore, a similar pattern was shown in the evaluation of the cytotoxicity of individual cell lines established by separating and proliferating CD5-CAR-NK cells into single cells. In particular, in the case of CD5#11-CAR-NK cells, the cytotoxicity against cancer cells expressing CD5 is about 10 times higher compared to the control group, while the cytotoxicity against normal cells expressing CD5 increases by only about 2 times compared to the control group. From the above results, it is judged that the CD5#11-CAR-NK cells of the present invention are much safer than the previously reported CD5-CARs. In addition, by showing that not only CD5-CAR permanently expressed in NK92 cells but also primary natural killer cell-based CD5-CAR-NK cells in which the mRNA of CD5-CAR is introduced into primary natural killer cells to temporarily express CD5-CAR have cytotoxicity against cancer cells expressing CD5, it was confirmed that there is a possibility of reducing side effects that also decrease to normal cells expressing CD5.

[0140] On the one hand, it was confirmed that, unlike normal cells, cancer cells expressing CD5 highly express B7-H6 among various ligands involved in the activation of natural killer cells. Based on this difference in B7-H6 expression, it was predicted that the CD5-CAR-NK cells of the present invention would react differently to cancer cells expressing CD5 and normal cells. Furthermore, when the expression level of B7-H6 was regulated in cancer cells expressing CD5, it was confirmed that the cytotoxicity of the CD5-CAR-NK cells of the present invention changed depending on the expression level of B7-H6. Thus, B7-H6 acts as a major factor in the activity of the CD5-CAR-NK cells of the present invention and is relatively highly expressed in cancer cells expressing CD5, indicating that the CD5-CAR-NK cells of the present invention react to cancer cells earlier than to normal cells.

[0141] That is, due to the difference in the activity of CD5-CAR caused by the difference in the CDR sequence of the antibody against CD5, the cytotoxicity against cancer cells expressing CD5 and normal cells is completely differentiated. By utilizing single cell lines with different cytotoxicities depending on the expression level of CD5-CAR, it is determined that a new therapeutic agent with high anti-cancer effect against cancer cells and low reactivity against normal cells can be developed for T cell leukemia.

[0142] As described in detail above for specific parts of the content of the present invention, it is clear to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the claims and their equivalents.

Brief Description of the Drawings

[0143]

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Claims

1. i) a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 18, ii) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 19, and iii) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 20, and iv) a light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 22, v) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 23, and vi) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 24, and an antibody or an antigen-binding fragment thereof that specifically binds to CD5.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:

21.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region has the amino acid sequence of SEQ ID NO:

25.

4. A chimeric antigen receptor (CAR) comprising an extracellular domain comprising an antigen-binding site that specifically binds to CD5, a transmembrane domain, and an intracellular signaling domain, wherein the antigen-binding site that specifically binds to CD5 is a single-chain variable fragment (scFv) of an anti-CD5 antibody comprising i) a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 18, ii) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 19, and iii) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 20, and iv) a light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 22, v) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 23, and vi) a light chain CDR3 having the amino acid sequence of SEQ ID NO:

24.

5. The antigen-binding site that specifically binds to CD5 is a single-chain variable fragment of an anti-CD5 antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21, the chimeric antigen receptor according to claim 4.

6. The antigen-binding site that specifically binds to CD5 is a single-chain variable fragment of an anti-CD5 antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 25, the chimeric antigen receptor according to claim 4.

7. A polynucleotide comprising a nucleotide sequence encoding the chimeric antigen receptor according to any one of claims 4 to 6.

8. An expression vector comprising the polynucleotide according to claim 7.

9. An immune cell that expresses on its surface the chimeric antigen receptor according to any one of claims 4 to 6.

10. The immune cell is at least one selected from the group consisting of natural killer cells (NK cells), T cells, natural killer T cells (NKT cells), macrophages, and dendritic cells, and is the immune cell according to claim 9. **Claim 11** A pharmaceutical composition for preventing or treating cancer, comprising the immune cell according to claim 9 as an active ingredient. **Claim 12** The cancer is a hematological cancer including leukemia or lymphoma, and is the pharmaceutical composition for preventing or treating cancer according to claim 11. **Claim 13** The cancer is at least one selected from the group consisting of thymic cancer, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, small lymphocytic lymphoma, T-cell tumor, peripheral T-cell lymphoma, mantle cell lymphoma, T-cell acute lymphoblastic leukemia, and chronic lymphocytic leukemia, and is the pharmaceutical composition for preventing or treating cancer according to claim 12. **Claim 14** The pharmaceutical composition for preventing or treating cancer according to claim 11, further comprising an anticancer chemotherapeutic agent.

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