CD5-targeting chimeric antigen receptor and immune cells expressing it

Abstract

The present invention relates to immune cells that co-express a chimeric antigen receptor (CAR) containing an OX40 ligand as an intracellular signaling domain and IL-15, and a composition for preventing or treating cancer containing the same as an active ingredient. The immune cells of the present invention not only exhibit synergistic tumor cell killing activity due to the co-expression of the CAR and IL-15, but also significantly improve survival rate and ex vivo proliferation rate, making them useful as an efficient anti-cancer cell therapy. In particular, when the immune cells of the present invention express a CAR targeting CD5, they can be used as an effective therapeutic composition for various CD5-positive tumors, including lymphocytic leukemia.

Description

[Technical Field]

[0001] The present invention relates to immune cells that express a chimeric antigen receptor that specifically recognizes CD5 and IL-15, and a method for treating CD5-positive tumors using the same. [Background technology]

[0002]

[0003] Natural killer (NK) cells are lymphoid cells that account for approximately 10% of blood cells and play a key role in the immune response. NK cells are an important component of innate immune defense and are suitable for adoptive cellular immunotherapy, which kills cancer cells and cells infected with exogenous pathogens. In the field of cell therapy using T cells, another type of immune cell, active research is being conducted on the technique of introducing chimeric antigen receptors (CARs) into T cells to enhance their specific killing effect against cancer cells expressing specific tumor antigens. CARs are artificial receptors designed to convey antigen specificity to immune cells. They contain an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain that are selected to activate immune cells such as T cells and provide specific immunity. The extracellular antigen-binding domain contains a single-chain variable fragment (scFv) that targets a confirmed tumor antigen, thereby inducing the activation of cancer cell-specific immune cells expressing the tumor antigen.

[0004] On the other hand, OX40 ligand (CD252), a member of the TNFR superfamily, is expressed on antigen-presenting cells (APCs), some natural killer cells, and B cells, and is known to be expressed within hours to days after activation of these cells. OX40 (CD134), the receptor for OX40 ligand, is expressed on T cells, especially on T cells activated by CD28. OX40 expression further enhances T cell responses induced by CD28 activation, increasing T cell proliferation, cytokine secretion, and survival.

[0005] CD5 is a type I glycoprotein and a member of the scavenger receptor family. It is expressed by thymocytes, mature T cells, and mature B cells and is known to be involved in the regulation of lymphocyte activation and differentiation processes. CD5 moderates the activation signal of the BCR, allowing B-1 cells to be activated only by very strong stimuli (e.g., bacterial proteins) and not by normal tissue proteins. CD5 is highly expressed in various hematological cancers, including acute T-lymphocytic leukemia and peripheral T-cell lymphoma, as well as solid tumors such as breast cancer and thymic carcinoma. Its expression is downregulated in normal cells, and it is being actively investigated as a therapeutic target for these tumors.

[0006] Numerous papers and patent documents are referenced throughout this specification and citations are provided, the disclosures of which are incorporated herein by reference in their entirety to more clearly describe the state of the art and the content of the present invention. Summary of the Invention [Problem to be solved by the invention]

[0007]

[0008] The present inventors have conducted extensive research to develop an effective immune cell therapy agent for various CD5-positive tumors, including lymphocytic leukemia. As a result, they discovered that when a fusion protein comprising a chimeric antigen receptor (CAR) containing OX40L as an intracellular signaling domain and IL-15 is expressed in immune cells, the survival rate, ex vivo proliferation rate, and antitumor activity of the immune cells are all significantly increased, making it easy to obtain a therapeutically effective amount of cells. Furthermore, the present invention was completed based on this discovery.

[0009] Therefore, an object of the present invention is to provide a fusion protein comprising a chimeric antigen receptor comprising an intracellular signaling domain comprising an OX40 ligand and an extracellular antigen-binding domain that binds to CD5; and IL-15.

[0010] Another object of the present invention is to provide immune cells expressing the fusion protein and compositions for preventing or treating CD5-positive tumors, which contain the same as an active ingredient.

[0011]

[0012] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, the claims and the drawings. [Means for solving the problem]

[0013]

[0014] According to one aspect of the invention, the invention provides a fusion protein comprising:

[0015] (a) a chimeric antigen receptor (CAR) comprising an intracellular signaling domain containing OX40 ligand (OX40L); and an extracellular antigen binding domain that binds to CD5; and

[0016] (b) IL (Interleukin)-15.

[0017] The present inventors have conducted extensive research to develop an effective immune cell therapy agent for various CD5-positive tumors, including lymphocytic leukemia. As a result, they discovered that when a fusion protein consisting of a chimeric antigen receptor (CAR) containing OX40L as an intracellular signaling domain and IL-15 is expressed in immune cells, the survival rate, ex vivo proliferation rate, and antitumor activity of the immune cells are all significantly improved, making it easy to obtain a therapeutically effective amount of cells. This finding also led to the discovery that the fusion protein can be used as an efficient cell therapy agent that exerts excellent anticancer effects against CD5-positive tumors even at low doses.

[0018] As used herein, the term "fusion protein" refers to a recombinant protein molecule in which an amino acid sequence derived from a specific protein or domain is fused with another amino acid sequence derived from a different protein or domain. The amino acid sequences of different origins may be directly linked within the fusion protein, or may be linked via a linker sequence, a tag sequence, a self-cleaving sequence, or a combination thereof. The fusion protein of the present invention may be formed by directly linking the amino acid sequence of a chimeric antigen receptor (CAR) and the amino acid sequence of an IL-15 protein, or may be indirectly linked via a linker, a tag, a self-cleaving sequence, or a combination thereof. The fusion protein may be prepared by recombinant techniques known in the art and expressed in target cells.

[0019] As used herein, the term "extracellular antigen-binding domain" refers to a domain that, when a chimeric antigen receptor is expressed in a target cell (e.g., an immune cell), is located in the extracellular portion and specifically recognizes an antigen of interest, thereby activating the apoptotic activity of the immune cell specifically against a target cell (e.g., a cancer cell), and may be, for example, an antigen-binding fragment of an antibody such as an Fc receptor or a single-chain variable fragment (ScFv). Thus, the term "extracellular antigen-binding domain" in the present invention is used interchangeably with "extracellular domain," "antigen-recognition fragment," or "antigen-binding fragment."

[0020] According to a specific embodiment of the invention, the extracellular antigen-binding domain is an antigen-binding fragment of an anti-CD5 antibody.

[0021] As used herein, the term "antibody" refers to an antibody against the CD5 protein that specifically recognizes and binds to a specific epitope thereof, and includes not only the complete, full-length antibody form but also an antigen-binding fragment (antibody fragment) of the antibody molecule.

[0022] A complete antibody has two full-length light chains and two full-length heavy chains, each connected to a heavy chain by a disulfide bond. The heavy chain constant regions are of the gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, with subclasses of gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The light chain constant regions are of the kappa (κ) and lambda (λ) types.

[0023] As used herein, the term "antigen-binding fragment of an antibody" refers to a fragment that retains the antigen-antibody binding function within the whole antibody molecule, and specifically includes Fab fragments, F(ab') fragments, F(ab')2 fragments, and Fv fragments.

[0024] Among antibody fragments, Fab contains the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region of the heavy chain (C H1 ) and has one antigen-binding site. Fab' has a heavy chain C H1 Fvs differ from Fabs in that they contain a hinge region containing one or more cysteine ​​residues at the C-terminus of the domain. F(ab')2 antibodies are formed by disulfide bonding between the cysteine ​​residues in the hinge region of Fab'. Fvs are the smallest antibody fragments, consisting only of the heavy and light chain variable regions. In two-chain Fvs, the heavy and light chain variable regions are linked non-covalently. In single-chain Fvs (ScFvs), the heavy and single chain variable regions are generally linked covalently via a peptide linker or directly at the C-terminus, forming a dimer-like structure like the two-chain Fv. Such antibody fragments can be obtained using protease enzymes (e.g., Fab fragments can be obtained by limited digestion of whole antibodies with papain, and F(ab')2 fragments can be obtained by digestion with pepsin), or they can be produced by genetic recombination techniques.

[0025] More specifically, the antigen-binding fragment of an antibody used in the present invention is an ScFv of an anti-CD5 antibody.

[0026] As used herein, the term "heavy chain" refers to a variable region domain V that contains an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. H and three constant region domains C H1 , C H2 and C H3The term "CDR" refers to a full-length heavy chain and all fragments thereof, including those containing the CDRs. As used herein, the term "CDR (complementarity determining region)" refers to the amino acid sequences of the hypervariable regions of immunoglobulin heavy and light chains. Each heavy chain (HCDR1, HCDR2, and HCDR3) and light chain (LCDR1, LCDR2, and LCDR3) contains three CDRs, which provide the main contact residues for antibody binding to an antigen or epitope.

[0027] The scope of the antibodies or antibody fragments of the present invention includes variants with conservative amino acid substitutions in the CDR regions. Furthermore, the antibodies or antibody fragments of the present invention may include variants of the amino acid sequences set forth in the attached sequence listings, as long as they are capable of specifically recognizing phosphorylated PLCγ2. For example, additional changes can be made to the amino acid sequence of the antibody to further improve the antibody's binding affinity and / or other biological properties. Such modifications include, for example, deletion, insertion, and / or substitution of residues in the antibody's amino acid sequence. Such amino acid mutations are made based on the relative similarity of amino acid side chain substitutes, such as hydrophobicity, hydrophilicity, charge, and size. Analysis of the size, shape, and type of amino acid side chain substitutes reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

[0028] Amino acid exchanges in proteins that do not globally alter the activity of the molecule are known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0029] Considering the above-mentioned biologically equivalent mutations, the antigen-recognizing fragment of the antibody of the present invention or the nucleic acid molecule encoding the same is also understood to include sequences showing substantial identity to the sequences set forth in the sequence listing. The substantial identity means a sequence showing at least 80% homology, in one specific example, 85% homology, in another specific example, 90% homology, in yet another specific example, 95% homology, and in yet another specific example, 99% homology, when the above-mentioned sequences of the present invention are aligned with any other sequence for maximum correspondence and the aligned sequences are analyzed using algorithms commonly used in the art. Alignment methods for sequence comparison are well known in the art (Smith and Waterman, Adv. Appl. Math. (1981) 2:482; Huang et al. Comp. Appl. BioSci. (1992) 8:155-65 and Pearson et al. Meth. Mol. Biol. (1994) 24:307-31).

[0030]

[0031] According to a specific embodiment of the present invention, the extracellular antigen-binding domain comprises a heavy chain variable region comprising an HCDR1 having the amino acid sequence of sequence No. 26 of the Sequence Listing, an HCDR2 having the amino acid sequence of sequence No. 28 of the Sequence Listing, and an HCDR3 having the amino acid sequence of sequence No. 30 of the Sequence Listing.

[0032] More specifically, the heavy chain variable region comprises the amino acid sequence of sequence 32 in the sequence listing.

[0033] According to a specific embodiment of the present invention, the extracellular antigen-binding domain additionally comprises a light chain variable region comprising an LCDR1 having the amino acid sequence of Sequence No. 34, an LCDR2 having the amino acid sequence of Sequence No. 36, and an LCDR3 having the amino acid sequence of Sequence No. 38.

[0034] More specifically, the light chain variable region comprises the amino acid sequence of sequence no. 40 in the sequence listing.

[0035] Most specifically, the extracellular antigen-binding domain used in the present invention comprises the amino acid sequence of sequence no. 9 in the sequence listing.

[0036]

[0037] According to a specific embodiment of the present invention, the intracellular signaling domain additionally comprises one or more domains selected from the group consisting of CD28, CD3-zeta, OX40, and 4-1BB, more specifically, the intracellular signaling domain additionally comprises CD28 and CD3-zeta.

[0038] Most specifically, the intracellular signaling domain comprises CD28, OX40L and CD3-zeta in this order from the cell membrane to the cell.

[0039]

[0040] According to a specific embodiment of the present invention, the fusion protein further comprises a self-cleaving peptide located between the chimeric antigen receptor and the IL-15. More specifically, the self-cleaving peptide comprises the amino acid sequence of sequence 21 in the sequence listing.

[0041] According to the present invention, the amino acid sequence of Sequence No. 21 in Sequence Listing is the amino acid sequence of a T2A self-cleaving peptide. The T2A self-cleaving peptide is a type of T2A-self-cleaving peptide that cleaves full-length proteins into short peptide fragments by inducing ribosomal skipping during intracellular protein translation, and contains the 18 amino acids (EGRGSLLTCGDVEENPGP) of Sequence Listing No. 21. According to one embodiment of the present invention, a GSG (Gly-Ser-Gly) sequence can be added to the N-terminus to enhance the self-cleaving activity of the amino acid sequence of Sequence Listing No. 21. The chimeric antigen receptor and IL-15 of the present invention can be cleaved and expressed by the T2A self-cleaving peptide of the present invention.

[0042]

[0043] According to another aspect of the present invention, there is provided a nucleic acid molecule encoding the fusion protein of the present invention as described above.

[0044] As used herein, the term "nucleic acid molecule" encompasses DNA (gDNA and cDNA) and RNA molecules, and nucleotides, the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogs in which the sugar or base moiety is modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)). The nucleic acid molecules of the present invention may be inserted into a gene carrier and introduced into target cells, such as immune cells, to express the fusion proteins of the present invention described above.

[0045] As used herein, the term "express" refers to the artificial introduction of an exogenous gene into a target cell using a gene carrier, thereby enabling the gene to replicate in the target cell as an extrachromosomal element or by chromosomal integration, in order to express the exogenous gene or increase the natural expression level of an endogenous gene in the target cell. Therefore, the term "expression" has the same meaning as "transformation," "transfection," or "transduction."

[0046] As used herein, the term "gene delivery body" or "gene delivery system" refers to any means for transporting a gene into a cell, and gene delivery is synonymous with intracellular gene transduction. At the cellular or tissue level, gene delivery is synonymous with gene spread. Therefore, the gene delivery system of the present invention can be described as a gene penetration system and a gene spread system.

[0047] To prepare the gene delivery vector of the present invention, the nucleotide sequence of the present invention is preferably operably linked to a suitable expression control sequence within a suitable expression construct. As used herein, the term "operably linked" refers to the functional association between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the control sequence controls the transcription and / or translation of the other nucleic acid sequence. The promoter linked to the target gene of the present invention is specifically one that can operate in animal cells, more specifically mammalian cells, and most specifically immune cells to regulate the transcription of the target gene, and includes promoters derived from mammalian viruses and promoters derived from the genome of mammalian cells, such as, but not limited to, the CMV (mammalian cytomegalovirus) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1 alpha promoter, metallothionine promoter, beta-actin promoter, human IL-2 gene promoter, human IFN gene promoter, human IL-4 gene promoter, human lymphotoxin gene promoter, and human GM-CSF gene promoter.

[0048] The nucleotide sequence of the target gene can be applied to all gene transfer systems commonly used for gene transfer, such as plasmids, adenoviruses (Lockett LJ, et al., Clin. Cancer Res. 3:2075-2080 (1997)), adeno-associated viruses (AAV, Lashford LS., et al., Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), retroviruses (Gunzburg WH, et al., Retroviral vectors, Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), lentiviruses (Wang G., et al., J. Clin. Invest. 104(11):R55-62 (1999)), and herpes simplex viruses (Chamber R., et al., Proc. Natl. Act. Sci. USA 92:1411-1415 (1995)), vaccinia virus (Puhlmann M. et al., Human Gene Therapy 10:649-657 (1999)), liposomes (Methods in Molecular Biology, 199, SC Basu and M. Basu (Eds.), Human Press 2002) or niosomes.

[0049] According to a specific embodiment of the present invention, the gene delivery vehicle used in the present invention is a viral vector, more specifically, the virus is selected from the group consisting of lentivirus, adenovirus, adeno-associated virus (AAV), retrovirus, herpes simplex virus and vaccinia virus, most specifically, lentivirus.

[0050] The base sequences of nucleic acid molecules encoding the entire fusion protein of the present invention or each of the domains that make up the protein are listed in Table 2 below.

[0051] According to yet another aspect of the present invention, there is provided an immune cell expressing the above-described nucleic acid molecule of the present invention.

[0052] As used herein, the term "immune cell" refers to any cell involved in the initiation or promotion of an immune response, and more specifically, refers to an immune effector cell. Examples of immune cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, and mast cells. More specifically, the immune cell is a natural killer cell.

[0053] According to yet another aspect of the invention, the invention provides an immune cell expressing:

[0054] (a) a chimeric antigen receptor (CAR) comprising an intracellular signaling domain containing OX40 ligand (OX40L); and an extracellular antigen binding domain that binds to CD5; and

[0055] (b) IL (Interleukin)-15.

[0056] The immune cells used in the present invention have already been described above, so their description will be omitted to avoid excessive duplication.

[0057] As described in the above-mentioned embodiment of the present invention, the immune cells of the present invention may express the chimeric antigen receptor and IL-15 in the form of a single fusion protein in which they are linked to each other, or as in the present embodiment, the chimeric antigen receptor and IL-15 may be expressed as separate protein molecules in which they are not linked. In this case, the nucleic acid molecules encoding the chimeric antigen receptor and IL-15 may be inserted into separate gene carriers and co-transfected into the immune cells, or they may be inserted together into a single gene carrier and transfected.

[0058] According to a specific embodiment of the present invention, the immune cells used in the present invention are natural killer cells, more specifically, natural killer cells that have been cultured for 23 to 35 days, even more specifically, for 25 to 33 days, and most specifically, for 28 to 30 days, and then frozen and thawed.

[0059] As used herein, the term "culturing" means inducing cells to grow and proliferate in an in vitro environment, such as a culture medium, while maintaining biological activity.

[0060] As used herein, the term "culture medium" refers to a mixture containing essential elements for cell growth and proliferation, such as sugars, amino acids, minerals, and other nutrients, for efficiently inducing and promoting cell growth and proliferation in vitro. Cell culture media can be optimized for specific cell types and their phenotypes and culture characteristics, and include, for example, basal culture media formulated to support cell growth, culture media formulated to promote recombinant protein production from cells, and enriched media made by highly concentrated nutrients.

[0061] As used herein, the terms "freezing" or "cryopreservation" refer to the stable maintenance of cells at low temperatures, specifically, at ultralow temperatures between -80°C and -200°C, for short or long periods of time. Cells generally undergo mutation at a rate of 1 in 10,000 during culture. Prolonged cell passage can lead to degeneration into a different cell population from the original one, or to the loss of inherent biological activity and function. The risk of infection by mycoplasma and other pathogens also increases over time. Therefore, cell freezing is widely used to preserve the inherent characteristics of cells. The present inventors have found that immune cells, particularly natural killer cells, which have low gene transfer efficiency using lentiviral vectors, can be significantly improved in gene transfer efficiency and cell mortality rate by culturing them for an appropriate period of time and then freezing them. Therefore, the freezing step performed in the present invention may be performed for only a short period of time, unlike cryopreservation, which is performed for the purpose of long-term cell preservation.

[0062] Cell freezing may be performed by treating cells with a cryoprotectant, which can minimize cell damage due to ice crystal formation and ionic and osmotic imbalances that inevitably accompany the freezing and thawing process. The freezing medium may contain, for example, dextran, albumin, and / or DMSO to improve safety and stability during cell freezing.

[0063] As used herein, the term "thawing" refers to the process of raising the temperature of frozen or cryopreserved cells until they become soft, viable cells, allowing them to resume their vital activities. Thawing can typically be rapidly performed by removing deep-frozen cells from a liquid nitrogen tank or the like and placing them in a 37°C thermostatic water bath.

[0064] The freezing and thawing process of the present invention is intended to improve the gene transfer efficiency of cells and enhance the killing activity of pathogenic cells without long-term storage of cells, so that the thawing process may be started immediately after the freezing is completed without any time interval, or an appropriate time lag may be left between the freezing and thawing processes, if necessary.

[0065]

[0066] In yet another aspect, the present invention provides a composition for preventing or treating CD5-positive tumors, which comprises the above-mentioned immune cells of the present invention as an active ingredient.

[0067] In yet another aspect, the present invention provides a method for preventing or treating CD5-positive tumors, comprising administering the above-described immune cells of the present invention to a subject.

[0068] As used herein, the term "treatment" refers to (a) the inhibition of the development of a disease, disorder, or symptom; (b) the alleviation of a disease, disorder, or symptom; or (c) the elimination of a disease, disorder, or symptom. When immune cells transfected with the target gene of the present invention (a nucleic acid molecule encoding a chimeric antigen receptor that specifically recognizes CD5) are administered to a subject, they induce the death of CD5-positive cancer cells, thereby inhibiting the development of, eliminating, or alleviating symptoms caused by various CD5-positive tumors, including lymphocytic leukemia. Therefore, the composition of the present invention may be used as a cell therapy composition for a disease by itself, or may be administered together with other pharmacological ingredients to be used as a therapeutic adjunct for the disease. Therefore, as used herein, the terms "treatment" or "therapeutic agent" encompass the meaning of "therapeutic adjunct" or "therapeutic adjunct."

[0069] As used herein, the term "administration" or "administering" refers to the direct injection of a therapeutically effective amount of a composition of the present invention into a subject, thereby allowing the same amount to be formed within the subject's body.

[0070] In the present invention, the term "therapeutically effective amount" means the content of the composition of the present invention contained in an amount sufficient to provide a therapeutic or prophylactic effect to an individual to whom the composition of the present invention is to be administered, and therefore includes a "prophylactically effective amount."

[0071] As used herein, the term "subject" includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus monkey. Specifically, the subject of the present invention is a human.

[0072] As used herein, the term "CD5-positive tumor" refers to a solid tumor or hematological cancer in which CD5 is measurably expressed at a higher level than normal cells or CD5-negative tumors. CD5 is a transmembrane receptor protein that is expressed in 85% of T-lineage acute lymphoblastic leukemias (T-ALL) and 75% of peripheral T-cell lymphomas, and is highly expressed in mantle cell lymphoma, B-CLL (chronic lymphocytic leukemia), and hairy cell leukemia. Its expression is suppressed in normal cells, making it an effective target for tumor therapy and diagnosis.

[0073] CD5-positive tumors that can be prevented or treated with the compositions of the present invention include, but are not limited to, blood cancers such as leukemia, lymphoma, and multiple myeloma, and solid cancers such as breast cancer and thymic carcinoma, and include all malignant tumors that can be specifically recognized by the immune cells of the present invention by expressing CD5 at a measurable level on the surface of tumor cells.

[0074] More particularly, said CD5-positive tumor is a T-lymphocytic leukemia, more particularly an acute T-lymphocytic leukemia.

[0075]

[0076] The immune cells of the present invention, specifically natural killer cells, can be contained in an amount of 10 to 95% by weight of the total weight of a composition for preventing or treating CD5-positive tumors containing the immune cells as an active ingredient. Furthermore, the composition of the present invention can be formulated to additionally contain one or more anti-cancer pharmacological ingredients that exhibit the same or similar functions in addition to the natural killer cells that are the pharmacological ingredient.

[0077] The dosage of the composition can be adjusted depending on various factors, including the type of CD5-positive tumor, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of dosage form, the patient's age, weight, general health condition, sex, and diet, the administration time, the administration route, the secretion rate of the composition, the treatment period, and concurrently used drugs. However, for a desirable effect, the dosage of natural killer cells according to the present invention is, for example, 0.01×10 7 cells / kg ~ 1.0 × 10 9 cells / kg, or 0.5 × 10 7 cells / kg ~ 1.0 × 10 8 It may be cells / kg.

[0078] Furthermore, the compositions of the present invention can be administered to an individual by various injection methods for cell therapy agents known in the art. The administration route can be appropriately selected by a skilled artisan, taking into consideration the administration method, volume, viscosity, etc. of the body fluid. For example, the compositions of the present invention can be administered parenterally, specifically, intravenously, subcutaneously, or intraperitoneally, and most specifically, intravenously. [Effects of the Invention]

[0079]

[0080] The features and advantages of the present invention can be summarized as follows:

[0081] (a) The present invention provides immune cells that co-express a chimeric antigen receptor containing an OX40 ligand as an intracellular signaling domain and IL-15, and a composition for preventing or treating cancer that contains the same as an active ingredient.

[0082] (b) The immune cells of the present invention not only exhibit synergistic tumor cell killing activity due to co-expression of the chimeric antigen receptor and IL-15, but also have significantly improved survival rates and in vitro proliferation rates, making them useful as an efficient anti-cancer cell therapy.

[0083] (c) In particular, when the immune cells of the present invention express a chimeric antigen receptor that targets CD5, they can be used as an effective therapeutic composition for various CD5-positive tumors, including lymphocytic leukemia. [Brief explanation of the drawings]

[0084]

[0085] [Figure 1] FIG. 1 shows the structure of the chimeric antigen receptor (CAR) used in the present invention, including a control containing only GFP, a third-generation CAR construct containing CD28, OX40L, and CD3ζ as signaling domains, a construct lacking the signaling domain and containing only the IL-15 domain, and a fourth-generation CAR construct in which the IL-15 domain has been additionally linked to the third-generation CAR construct.

[0086] [Figure 2] FIG. 1 shows the results of measuring the viability and fold expansion of third- and fourth-generation CAR-NK cells in the absence of IL-2 cytokine.

[0087] [Figure 3] Fig. 1 shows the results of measuring the tumor cell-killing ability of third-generation CAR-NK cells and fourth-generation CAR-NK cells against the HER2-positive tumor cell lines HCC1954 and SKOV.

[0088] [Figure 4]This figure shows the results of observing changes in the amount of IFN-γ secreted by third-generation CAR-NK cells and fourth-generation CAR-NK cells that were contacted with the HER2-positive tumor cell line HCC1954 and SKOV.

[0089] [Figure 5] FIG. 1 is a schematic diagram summarizing the NK cell culture process of the present invention.

[0090] [Figure 6] These are the results of measuring the expression pattern of CD5 CAR in natural killer cells (MCB) cultured for 28-30 days and then frozen according to the process shown in the upper diagram of Figure 5, and in natural killer cells (DP) cultured for 42-44 days and then frozen according to the process shown in the lower diagram of Figure 1.

[0091] [Figure 7] This is the result of analyzing the cell phenotype based on the expression patterns of each surface marker of MCB and DP.

[0092] [Figure 8] 1 shows the results of FACS analysis measuring CD5 protein expression in the tumor cell lines K562, NALM6, CCRF-CEM, RPMI-8402_Luc, NALM6-NucLight, CCRF-CEM-NucLight, and RPMI-8402-NucLight.

[0093] [Figure 9] The results of measuring the expression levels of CD107a, IFN-γ, and TNFα (n=3) in CBNK or CD5 CAR-NK co-cultured with various tumor cell lines are shown.

[0094] [Figure 10] FIG. 1 shows a schematic diagram of culture for confirming the tumor cell-killing ability of CD5 CAR-transduced cells.

[0095] [Figure 11]These results confirm the killing ability of NK cells against CD5-positive and CD5-negative tumor cells.

[0096] [Figure 12] The results are shown by measuring the average secretion level (pg / mL) of IFN-γ from three donors.

[0097] [Figure 13] The graph shows the results of measuring the average amount of IL-15 secreted (pg / mL) by NK cells co-cultured with each target cell.

[0098] [Figure 14A] The results show that CD5-positive and CD5-negative tumor cells were co-cultured with CD5 CAR-NK cells for 96 hours, and the long-term tumor cell killing ability was confirmed. [Figure 14B] Same as above [Figure 14C] Same as above [Figure 14D] Same as above

[0099] [Figure 15A] The survival rate (%) up to 140 days after tumor transplantation in each experimental group was measured for each administered substance, and the results are shown. [Figure 15B] Same as above [Figure 15C] Same as above

[0100] [Figure 16] The results of live animal imaging up to 112 days after tumor transplantation and injection of the NK cells of the present invention are shown.

[0101] [Figure 17A] The results of measuring the image signal value (photon / s) over time up to 35 days after tumor implantation are shown. [Figure 17B] Same as above

[0102] [Figure 18] This shows the results of measuring the survival rate (%) up to 133 days after tumor transplantation in experimental animals and injection of the NK cells of the present invention.

[0103] [Figure 19A] This shows the results of bioimaging up to 126 days after tumor transplantation into experimental animals and injection of the NK cells of the present invention. [Figure 19B] Same as above [Figure 19C] Same as above [Figure 19D] Same as above [Figure 19E] Same as above

[0104] MODE FOR CARRYING OUT THE INVENTION [Example]

[0105]

[0106] Example 1: Generation of CAR-NK cells expressing a third-generation chimeric antigen receptor (CAR) and fourth-generation CAR-NK cells simultaneously expressing a third-generation chimeric antigen receptor and an IL-15 domain

[0107] The structures of the chimeric antigen receptors (CARs) used in the present invention are summarized in Table 1 below.

[0108] [Table 1]

[0109] The HER2 CAR (third generation) was constructed to confirm the synergistic effect of co-expressing a CAR containing the OX40L of the present invention with IL-15. The HER2 CAR has a structure in which the signal sequence domain of CD8a (nucleotides 890-952, GenBank NM001768.6); the anti-HER2 scFv sequence (VH-(GGGGS)3-VL domain); the hinge domain derived from human CD8α (nucleotides 1292-1435, GenBank NM001768.6); the transmembrane and intracellular signaling domain derived from CD28 (nucleotides 679-882, GenBank NM006139.3); the intracellular signaling domain derived from CD252 (nucleotides 141-206, GenBank NM003326.4); and the intracellular signaling domain derived from CD3z (nucleotides 299-634, GenBank NM000734.3) are sequentially linked with the termination codon TGA. HER2 CAR(t)-IL-15 has a structure in which the signal sequence domain of CD8α (nucleotides 890-952, GenBank NM001768.6); anti-HER2 scFv sequence (VH-(GGGGS)3-VL domain); hinge domain from human CD8α (nucleotides 1292-1435, GenBank NM001768.6); transmembrane domain from CD28 (nucleotides 679-759, GenBank NM006139.3); T2A self-cleaving peptide sequence linked to glycine (G)-serine (S)-glycine (G); and human IL-15 sequence (nucleotides 375-860, GenBank NM000585.4) followed by the termination codon TGA are sequentially linked.

[0110] HER2 CAR-IL-15 (4th generation) contains the signal sequence domain of CD8α (890-952 nucleotides, GenBank NM001768.6); anti-HER2 scFv sequence (VH-(GGGGS)3-VL domain); hinge domain from human CD8α (1292-1435 nucleotides, GenBank NM001768.6); transmembrane and intracellular signaling domain from CD28 (679-882 ​​nucleotides, GenBank NM006139.3); intracellular signaling domain from CD252 (141-206 nucleotides, GenBank NM003326.4); and intracellular signaling domain from CD3z (299-634 nucleotides, GenBank NM000734.3); GSG-linked T2A self-cleaving peptide sequence; human IL-15 sequence (375-860 nucleotides, GenBank NM000585.4) and the termination codon TGA linked in sequence.

[0111] The CD5 CAR (4th generation) contains the signal sequence of CD8α (890-952 nucleotides, GenBank NM001768.6); the anti-CD5 scFv domain (VH-(GGGGS)3 linker-VL); the hinge domain from CD8α (1292-1435 nucleotides, GenBank NM001768.6); the transmembrane and intracellular signaling domain from CD28 (679-882 ​​nucleotides, GenBank NM006139.3); the intracellular signaling domain from CD252 (141-206 nucleotides, GenBank NM003326.4); the intracellular signaling domain from CD3z (299-634 nucleotides, GenBank NM000734.3); the T2A self-cleaving peptide sequence linked to GSG; and the human IL-15 sequence (375-860 nucleotides, GenBank NM000585.4) and the termination codon TGA linked in sequence.

[0112] The nucleotide sequence and amino acid sequence of each domain contained in the chimeric antigen receptor constructed in the present invention are summarized in Table 2 below.

[0113] [Table 2] JPEG2025535708000004.jpg131156

[0114] IL-15 is known to be a cytokine that activates NK cells and contributes to their survival and proliferation. Therefore, to determine whether co-expression of IL-15 in NK cells expressing a chimeric antigen receptor (CAR) improves their survival and in vivo persistence, we introduced a soluble form of IL-15 protein into a CAR containing an scFv fragment that specifically recognizes HER2 and a signaling domain, and evaluated its efficacy (Figure 1). NK cells expressing a bicistronic CAR were generated by linking the anti-HER2 scFv fragment and signaling domain to the soluble IL-15 domain using the T2A system, and we named these "fourth-generation CAR-NK cells" (Figure 1, "d"). As controls, we also generated third-generation CAR-NK cells expressing a chimeric antigen receptor (CARR) that expressed the anti-HER2 scFv fragment and signaling domain but did not contain the IL-15 domain (Figure 1, "b"). NK cells expressing the anti-HER2 scFv fragment but did not contain the intracellular signaling domain and expressed the IL-15 domain (Figure 1, "c"; designated CAR(t)-IL-15-NK cells). A gene expressing GFP was used as a control (Figure 1, "a").

[0115]

[0116] Example 2: Viability and growth results of 3rd and 4th generation CAN-NK cells

[0117] Cord blood-derived NK cells were cultured and transduced with each gene on day 7. From day 22 of culture, the cells were cultured without IL-2 in the culture medium, and cell viability and growth were monitored. Except for 4th generation CAR-NK cells and CAR(t)-NK cells, the viability of all cell groups decreased significantly from the point at which IL-2 was not added, and they did not grow any further, with the majority of cells dying by day 41 of culture (Figure 2). Only CAR-NK cells with a structure containing the IL-15 domain maintained viability and growth, confirming that secreted IL-15 contributes to the survival and proliferation of NK cells.

[0118]

[0119] Example 3: Comparison of the efficacy of third- and fourth-generation CAR-NK cells and synergistic effect of the IL-15 domain

[0120] 3-1) Evaluation of tumor cell killing ability

[0121] To confirm the efficacy of 4th-generation CAR-NK cells, we examined the cytotoxicity of NK cells by co-culture with HER2-expressing tumor cell lines. Two HER2-expressing tumor cell lines (HCC1954 and SKOV3) were used as targets, expressing RFP, which led to the disappearance of the fluorescent signal upon cell death. Starting from day 22 of culture, various NK cell groups were cultured without IL-2 and co-cultured with the tumor cell lines at an E:T ratio of 0.3:1 for six days, after which tumor cell proliferation was observed. In both HCC1954 and SKOV3 tumor cell lines, 4th-generation HER2 CAR-NK cells secreting IL-15 suppressed tumor cell proliferation and exhibited excellent cytotoxicity, whereas 3rd-generation HER2 CAR-NK cells and HER2 CAR(t)-NK cells exhibited relatively low cytotoxicity (Figure 3). It is thought that the third-generation HER2 CAR-NK could not effectively suppress tumor cell formation due to the absence of additional cytokines such as IL-2, and it could be assumed that the HER2 CAR(t)-NK cells exhibited a low cytotoxic effect due to the lack of a signaling domain.

[0122]

[0123] 3-2) Evaluation of IFN-γ secretion ability

[0124] When we measured the secretion level of IFN-γ, a functional cytokine secreted by NK cells upon contact with target tumor cells, we observed that third-generation HER2 CAR-NK cells and HER2 CAR(t)-NK cells secreted very little IFN-γ, whereas fourth-generation CAR-NK cells secreted very high levels of IFN-γ (Figure 4).This confirmed that, unlike third-generation CAR-NK cells, which have poor survival and proliferation, fourth-generation CAR-NK cells exhibit synergistic anti-tumor effects through co-expression of the chimeric antigen receptor of the present invention, which contains OX40L as a signaling domain, and IL-15.

[0125]

[0126] Example 4: NK cell culture and cell freezing at different time points

[0127] 4-1) Cell thawing

[0128] Frozen cells were quickly thawed in a preheated 37°C water bath. When thawed to at least 90%, the cells were transferred to a 50 mL tube on a biosafety work bench and 10x (v / v) ACD buffer was slowly added. After centrifugation at 1200 rpm and 4°C for 10 minutes, the supernatant was removed and the cells were suspended in CellGro medium and counted.

[0129]

[0130] 4-2) NK cell culture and transduction with lentiviral vectors

[0131] 1.0 x 10 on day 0 of culture 6 / mL NK cells and 2.5 × 10 6100 μg / mL of feeder cells and CellGro medium were mixed at a 1:1:1 ratio and cultured statically in a CO2 incubator at 37°C. The CellGro medium was supplemented with IL-2 (1,000 IU / mL), human plasma (2%), and OKT3 (10 μg / mL) in an amount equal to the total volume. On day 4 of culture, an equal amount of CellGro mixed medium containing IL-2 (1,000 IU / mL) and human plasma (1%) was added. On day 7 of culture, the total cells were harvested and counted without further dilution, resulting in a cell count of 0.66 × 10 6 On the 7th day of culture, the frozen cells were thawed and suspended in CellGro medium. The cell number was measured and the cell density was adjusted to 1.0 × 10 6 The cells were diluted to a concentration of 1.0 × 10 / mL and cultured. 6 To treat lentiviral vectors at an MOI of 20, 6.6 x 10 cells / mL 8 30 μL of lentiviral vector (TU / mL) was added to the mixture, and the composition of the mixture for transduction of lentiviral vector is shown in Table 3 below.

[0132] [Table 3]

[0133] After preparing mixtures 1 and 2, they were slowly placed in a culture vessel. CBNK cells not transduced with a lentiviral vector were placed in a mixed medium (hereinafter referred to as CellGro mixed medium) prepared by mixing 1 mL of CellGro medium with human plasma (1%) and IL-2 (1,000 IU) and then cultured statically in a CO2 incubator at 37°C. On day 11 of culture, the same amount of CellGro mixed medium as that on day 10 of the 6-well standard culture was added, along with IL-21 at a concentration of 20 ng / mL. On day 13 of culture, the cells were harvested and CellGro mixed medium was added without further dilution to a concentration of 1 x 10 6On day 16 of culture, CD5-positive cells were isolated to obtain NK cells expressing CD5 CAR. To do this, all NK cells were collected and centrifuged at 1200 rpm at 4°C for 5 minutes. The supernatant was removed, and the cells were suspended in MACS buffer and counted to obtain a cell count of 1.0 x 10 7 MACS buffer was added to the cells so that the concentration was 1 μL / 10 s. Biotin-tagged recombinant human CD5 protein (His & AVI tag) (250 μg / mL) was then added at 1 μL / 10 s. 6 After adding 1.0 x 10 cells and mixing well, the mixture was incubated at 4°C for 30 minutes. MACS buffer was added in an amount 10 times the primary reaction volume, and the mixture was centrifuged at 340 g for 10 minutes at 4°C. The supernatant was then removed. 8 / mL in MACS buffer, and then anti-biotin beads (Miltenyi Biotec, 130-092-357) were added at 37.5 μL / 40 × 10 6 After adding 1.0 × 10 cells, the cells were incubated at 4°C for 15 minutes. A 10-fold volume of MACS buffer was added, and the mixture was centrifuged at 340 g for 10 minutes at 4°C. The supernatant was then removed. 8 After adding MACS buffer at a ratio of cells / 500 μL, the cells were thoroughly dissolved by pipetting several times. QuadroMACS TM An LS column (Miltenyi Biotec) was attached to a separator (Miltenyi Biotec) and 3 mL of MACS buffer was poured through the column to activate it. After thoroughly dissolving the cells reacted with the beads by pipetting, 500 μL was added to each column and the column was allowed to drain completely. To wash the column, 3 mL of MACS buffer was poured in, for a total of three washes. The column was then inserted into a QuadroMACS TM After separation with a separator, 5 mL of MACS buffer was added and the mixture was plunged down to collect CD5 CAR-positive cells. After centrifugation, the separated cells were collected at a concentration of 1 × 10 6CellGro mixed medium was added to the cells so that the cell volume reached 1 × 10 / mL, and restimulation using feeder cells was carried out in the same manner as on day 0 of culture, followed by static culture in a CO2 incubator at 37°C. On day 19 of culture, CellGro mixed medium was added in an amount equal to the culture volume on day 16 of culture. On day 21 of culture, the cells were harvested and counted without further dilution, and CellGro mixed medium was added to a cell volume of 1 × 10 6 / mL, and then, from day 28 to day 30 of culture, add CellGro mixed medium to 1 × 10 6 The NK cells were cultured to maintain a concentration of 1.0 × 10 / mL. After all NK cells were collected on days 28-30 of culture, the cells were frozen. Hereafter, the cells cultured on days 28-30 and then frozen are referred to as "MCB." The remaining cells were frozen at 1.0 × 10 6 CellGro medium was added to the cells so that the concentration was 1 × 10 / mL, and then restimulation using feeder cells was carried out as on day 16 of culture, followed by static culture in a CO2 incubator at 37°C. After that, CellGro mixed medium was added and the cells were cultured at 1 × 10 / mL for 14 days. 6 The cells were cultured to maintain a concentration of 100 / mL. After all NK cells were collected on days 42 to 44 of culture, the cells were frozen, and the frozen cells were designated "DP."

[0134] 4-3) Cell freezing

[0135] After centrifugation, cells were suspended in PBS and mixed with the freezing medium mixture at a ratio of 1:1, and 100 × 10 cells were frozen per freezing container. 6 The cells were dispensed at 1 / mL and frozen using an automated cell freezing machine (CRF).

[0136]

[0137] Example 5: Characteristics of CD5 CAR-transduced NK cells

[0138] 5-1) Confirmation of CD5 CAR expression

[0139] 1-5×10 5The cells were harvested and centrifuged, the supernatant removed, and then suspended in 2 mL of FACS buffer supplemented with 2% FBS. The supernatant was then removed by centrifugation, and 100 μL of FACS buffer and 1 μL of biotin-tagged recombinant human CD5 protein (His & AVI tag) (250 μg / mL) were added again and incubated at 4°C for 30 minutes in the dark. 2 mL of FACS buffer was then added, centrifuged, and the supernatant was removed. For secondary staining, 100 μL of cell buffer was added to the washed NK cells, and the antibody to be used for analysis was added and incubated at 4°C for 30 minutes in the dark.

[0140] [Table 4]

[0141] After thawing, the CD5 CAR expression rate in the MCB was 96.46 ± 2.00 (mean ± standard deviation), and that in the DP was 93.68 ± 2.23 (mean ± standard deviation). More than 90% of cells in both the MCB and DP expressed CD5 CAR (Figure 6). These values ​​were statistically significant using a two-tailed t-test (Table 5, *p<0.05, **p<0.01, ***<0.001, ns: not significant). These results confirmed that CD5 CAR expression was well maintained even with increasing culture period, and that CD5 CAR expression did not decrease rapidly even when frozen cells were thawed.

[0142] [Table 5]

[0143]

[0144] 5-2) Confirmation of the phenotype of CD5 CAR-transduced NK cells

[0145] 1.0-2.5 × 10 NK cells 6 The cells were suspended in FACS buffer at a concentration of 1 / mL and then dispensed in 100 μL aliquots into the plate containing the antibody mixture. The mixture was pipetted several times to thoroughly mix the cells and antibodies. The composition of the antibody mixture is shown in Table 6 below, and PE antibodies corresponding to each phenotype were added.

[0146] [Table 6]

[0147]

[0148] After incubation at 4°C for 30 minutes, 100 μL of FACS buffer was added to each well and mixed by pipetting. After centrifugation (2000 rpm, 3 minutes, 4°C), 200 μL of BD Cytofix solution was dispensed into each well. Flow cytometry was performed using an LSR Fortessa instrument and the results were analyzed. NK cells were gated in the following order: singlets, lymphocytes, live cells (7-AAD-), and NK cells (CD56+, CD3-). The expression of each marker was calculated based on the subtype antibody. To characterize the NK cells, the expression of 18 surface markers was analyzed using a flow cytometer and compared between the MCB and DP of CD5 CAR-NK and those of CBNK. The expression of each surface marker in CD5 CAR-NK was similar to that in CBNK, and there was no significant difference between MCB and DP (Figure 7, Table 7).

[0149] [Table 7]

[0150]

[0151] Example 6: In vitro efficacy evaluation of CD5 CAR-transduced cells

[0152] 6-1) Preparation and culture of tumor cell lines

[0153] Frozen K562, NALM6, CCRF-CEM, RPMI-8402_Luc, NALM6-NucLight, CCRF-CEM-NucLight, and RPMI-8402-NucLight cells were quickly thawed in a 37°C water bath, diluted with 10 mL of culture medium, centrifuged to remove the supernatant, and then 1 mL of medium was added to count the number of cells. 6 The cells were placed in a T75 flask and cultured in a CO2 incubator for 2-3 days. To generate NucLight-labeled tumor cell lines, the cells were transfected with Incucyte using 8 μg / mL polybrene (Santa Cruz, CA, USA). (登録商標) Cells were transduced with NuclightRed lentivirus (Sartorius, Göttingen, Germany) and treated with puromycin (Gibco, USA) at 0.5, 1, 1.5, or 2 μg / mL four days later. Seven days after treatment, the lowest concentration that killed 100% of tumor cells was selected to select only cells expressing red fluorescent protein, which were then cultured.

[0154]

[0155] 6-2) CD5 expression on tumor cells

[0156] Tumor cells were harvested and cultured at 1-5 × 10 5The cells were transferred to a FACS tube. After centrifugation, 100 μL of FACS buffer was added to 1 μL of anti-human CD5 PE, mixed, and incubated at 4°C for 30 minutes in the dark. 1 mL of FACS buffer was added, followed by centrifugation. The supernatant was discarded, and 200 μL of BD Cytofix solution was added and mixed before FACS analysis. The gray shading indicates the subtype control group, while the solid blue line indicates the results of anti-human CD5 antibody staining. The numbers indicate the expression rate (%) relative to the subtype control group. K562, NALM6, and NALM6-Nuclight showed little CD5 expression, whereas CCRF-CEM, CCRF-CEM-Nuclight, RPMI-8402_Luc, RPMI-8402-Nuclight, and Jurkat-Nuclight showed high CD5 expression (Figure 8).

[0157]

[0158] 6-3) Confirmation of CD107a and cytokine expression in CD5 CAR-transduced cells

[0159] NK cells and target tumor cell lines were each cultured in RPMI-1640 + 10% FBS (hereinafter referred to as assay medium) at 2.5 × 10 6The cells were suspended at 100 μL / mL. To confirm CD5-specific cell activity, CCRF-CEM and RPMI-8402_Luc were used as CD5-positive tumor cell lines, and NALM-6 was used as a CD5-negative tumor cell line. Golgistop (1.3 μL / mL) and Golgiplug (2 μL / mL) were added and mixed to prevent the secretion of cytokines produced within NK cells. APC anti-human CD107a antibody (1 μL) was dispensed into the (-) well and target well of a 96-well round-bottom plate. To confirm cytokine expression in NK cells themselves, 100 μL of assay medium and 100 μL of NK cells were placed in the (-) well of the antibody-dispensed plate, and 100 μL each of NK cells and the target tumor cell line were placed in the target well. The 96-well plate was wrapped in foil to block light and then co-cultured for 4 hours in a CO2 incubator at 37°C. After centrifugation (2000 rpm, 3 minutes, 4°C), the supernatant was removed, and 200 μL of FACS buffer was added. After mixing and centrifugation, the supernatant was removed. For cell surface staining, 1 μL of anti-human CD3 PerCP-Cy5.5, 1 μL of anti-human CD56-APC-e780, and 4 μL of 7-AAD were added to 100 μL of FACS buffer per well and incubated at 4°C for 30 minutes. After adding 100 μL of FACS buffer and centrifuging twice, the supernatant was removed. 200 μL of fixation / permeabilization solution was added and incubated at 4°C for 30 minutes. After centrifugation, the supernatant was removed, and 200 μL of 1x Perm wash buffer was added, followed by centrifugation. 100 μL of 1x Perm wash buffer was added per well, followed by anti-human IFN-η FITC (1 μL) and anti-human TNF-α PE-Cy7 (1 μL) and incubation at 4°C for 30 minutes. 100 μL of 1x Perm wash buffer was added, followed by centrifugation. The supernatant was removed, and 200 μL of 1x Perm wash buffer was added and centrifuged. The supernatant was removed, and 200 μL of BD cytofix was added. The cells were analyzed using an LSRFortessa FACS instrument and the results were analyzed using the FlowJo analysis program.As a result, CD107a expression and cytokine (IFN-γη, TNF-α) expression in the K562 cell line, which is used to evaluate NK cell activity, were similarly high in CBNK and CD5 CAR-NK. When cocultured with the positive cell lines CCRF-CEM and RPMI-8402_Luc, the CD5 CAR-NK group showed more than two-fold higher expression than the CBNK, whereas the negative cell line NALM-6 showed similar low levels of expression, confirming CD5-specific cytokine expression (Figure 9). Statistical significance was confirmed for the CD107a expression levels and two cytokine measurements for three donors using a two-tailed t-test, and the results are shown in Table 8 as mean ± standard deviation (Figure 9, *p<0.05, **p<0.01, ns: not significant). Compared to CBNK, CD5 CAR-NK showed higher CD107a expression and increased cytokine expression specifically in CD5, but there was no statistical significance between MCB and DP.

[0160] [Table 8]

[0161]

[0162] 6-4) Evaluation of tumor cell killing ability of CD5 CAR-transduced cells

[0163] NK cells (CBNK, CD5 CAR-NK) were collected and centrifuged at room temperature for 5 minutes at 1200 rpm. The supernatant was removed, and the cells were suspended in 1 mL of assay medium and counted. The transduced cells were added to a 5 mL FACS tube in assay medium at a ratio of E (NK cells):T (target cells). NK cells were added at a ratio of 10:1, with 1 x 10 cells. 5 cells / 100 μL, 3:1 ratio is 3 × 10 4 cells / 100 μL, 1:1 ratio is 1 × 10 4 cells / 100 μL, 0.3:1 ratio is 3 × 10 3 The solution was prepared so as to have a concentration of cells / 100 μL.

[0164] 1 x 10 cells of the target cell line in a 96-well round-bottom plate 4 cells / well (1 × 10 5 The cells were fixed at 100μL / mL (100μL of cells / mL), and NK cells were added at an E:T ratio of 100μL each. To correct for the fluorescence intensity of Calcein-AM released from Calcein-AM-stained target cells, 100μL of target cells and 100μL of assay medium were added, and the value measured at this point was defined as the minimum release value. To measure the maximum Calcein-AM fluorescence intensity from the target cells, 100μL of target cells were added to 100μL of 2% Triton X-100 to completely lyse the cells, and the fluorescence intensity measured at this point was defined as the maximum release value. To correct for the decrease in fluorescence intensity due to Triton X-100, 200μL of assay medium was added to the MM wells, and 100μL of assay medium and 100μL of 2% Triton X-100 were added to the MT wells. After 4 hours of incubation in a 37°C incubator in the dark, the wells were centrifuged at 2000 rpm at 4°C for 3 minutes. 100 μL of the supernatant was transferred to a 96-well flat-bottomed plate for testing, and the fluorescence value was measured using a fluorometer under conditions of 458 nm / 535 nm and 0.1 s. The cell killing activity was calculated using the formula in Table 9.

[0165] [Table 9]

[0166] The cell-killing ability of CBNK (MCB), CD5 CAR-NK (MCB), CBNK (DP), and CD5 CAR-NK (DP) against four types of target cells in three donors (2024P, 2044P, 605463P) is shown in Figure 7, and the cell-killing ability values ​​(%) are summarized in Table 10 below. As shown in Figure 11 and Table 10, the tumor-killing ability of CD5 CAR-NK cells against the CD5-positive cell lines CCRF-CEM and RPMI-8402-Luciferase cells was higher than that of CBNK, and the difference between CD5 CAR-NK (MCB) and CD5 CAR-NK (DP) was not significant (two-tailed t-test *p<0.05, **p<0.01, ***<0.001, ns: not significant).

[0167] [Table 10]

[0168]

[0169] 6-5) Confirmation of IFN-γ secretion ability of CD5 CAR-transduced cells

[0170] Dilute target cells in assay medium to 1 x 10 5 NK cells were prepared at 3 x 10 / mL and diluted in assay medium. 5 After preparing the mixture at a concentration of 1 / mL, 100 μL of each was dispensed into a 96-well round-bottom plate and co-cultured at an E:T ratio of 3:1. After culturing for 24 hours in a 37°C, 5% CO2 incubator, the supernatant was collected and stored at -20°C.

[0171] ELISA was performed to quantify IFN-γ. One day before the experiment, 200x capture antibody was diluted in 1x coating buffer A, and 100 μL was added to a 96-well plate and incubated at 2-8°C for 16-18 hours. On the day of the experiment, the plate was washed four times with wash buffer, and 200 μL of 1x assay diluent A was added to each well to suppress nonspecific binding. The plate was sealed and left on a plate shaker for 1 hour. Standards were prepared by serially diluting the interferon gamma standard stock solution (500, 250, 125, 62.5, 31.3, 15.6, 7.8, and 0 pg / mL) into 1x assay diluent A. Subsequently, the supernatants stored at -20°C were dissolved and diluted 1 / 10 in 1x assay diluent A to prepare samples. 100 μL of the standard and diluted sample were dispensed into each well, the plate was sealed, and the reaction was carried out on a plate shaker at room temperature for 2 hours. The plate was washed four times with wash buffer. 100 μL of 1x detection antibody was added to each well, the plate was sealed, and the reaction was carried out on a plate shaker at room temperature for 1 hour. The plate was washed four times with wash buffer. 100 μL of 1x Avidin-HRP was added to each well, the plate was sealed, and the reaction was carried out on a plate shaker at room temperature for 30 minutes. The plate was washed five times with wash buffer, and 100 μL of TMB Substrate was added to each well and the reaction was carried out at room temperature for 20 minutes in the dark. 100 μL of stop solution was added to each well to terminate the reaction, and the absorbance was measured at 450 nm using a spectrophotometer. As a result, for the CD5-negative cell lines K562 and NALM6, there were differences in IFN-γ secretion between CBNK (MCB, DP) and CD5 CAR-NK (MCB, DP) from three donors (2024P, 2044P, 605463P), but when the results from all donors were analyzed together, there were no significant differences.For the CD5-positive cell lines CCRF-CEM and RPMI-8402-Luciferase, in donor 2024P, CBNK (MCB, DP) secreted 40-60 pg / mL of IFN-γ, while CD5 CAR-NK (MCB, DP) secreted 350-550 pg / mL of IFN-γ, indicating that the IFN-γ secretion levels of CD5 CAR-NK were approximately 8-9 times higher than those of CBNK.In donor 2044P, CBNK (MCB, DP) secreted 45-55 pg / mL of IFN-γ, while CD5 CAR-NK (MCB, DP) secreted 250-350 pg / mL, demonstrating approximately 5-6 times more IFN-γ than CBNK. In donor 605463P, CBNK (MCB, DP) secreted 70-120 pg / mL of IFN-γ, while CD5 CAR-NK (MCB, DP) secreted 600-1300 pg / mL of IFN-γ, demonstrating 8-10 times more IFN-γ than CBNK. Overall, all three donors demonstrated improved IFN-γ secretion ability in CD5+ cell lines specifically with CD5 CAR-NK, with statistically significant results (Figure 12, *p<0.05, **p<0.01, ***<0.001, ****<0.0001, ns: not significant). There was no statistical significance between CD5 CAR-NK (MCB) and CD5 CAR-NK (DP).

[0172] [Table 11]

[0173]

[0174] 6-6) Confirmation of IL-15 secretion ability of CD5 CAR-transduced cells

[0175] Dilute target cells and NK cells in assay medium. Target cells were diluted to 2 x 10 6 / mL, and NK cells were 6 × 10 6 100 μL of each was dispensed into a 96-well round-bottom plate and co-cultured at an E:T ratio of 1:3 in a 37°C, 5% CO2 incubator for 24 hours, after which the supernatant was obtained and stored at -20°C.

[0176] ELISA analysis was performed using an IL-15 ELISA kit. The microplate strips were sandwiched between plate frames, and 100 μL of assay diluent RD1-19 was dispensed into each well. 50 μL of the IL-15 standard and the stored supernatant were dispensed into the plate and incubated on a shaker at room temperature for 3 hours. The plate was washed four times with wash buffer. 200 μL of IL-15 conjugate was added to each well and incubated on a shaker at room temperature for 45 minutes. After washing the plate four times with wash buffer, 200 μL of "Substrate solution" was added to each well and incubated in the dark at room temperature for 30 minutes. 50 μL of "Stop solution" was added to each well to terminate the reaction, and the absorbance was measured at a wavelength of 450 nm. As a result, as shown in Figure 13 and Table 12 below, when NK cells were cultured alone without target cells (effector only), CBNK (MCB, DP) and CD5 CAR-NK (MCB, DP) secreted IL-15 at levels of 4-6 pg / mL and 5-17 pg / mL, respectively, from three donors. The CD5-negative cell lines, K562 and NALM6, secreted IL-15 at levels similar to those of the group in which NK cells alone (effector only) were cultured with CBNK (MCB, DP) and CD5 CAR-NK (MCB, DP) from three donors. In response to the CD5-positive cell lines CCRF-CEM and RPMI-8402-Luciferase, CBNK (MCB, DP) secreted IL-15 at levels similar to those of CD5-negative cell lines. For CD5 CAR-NK (MCB, DP), donor 2024P secreted IL-15 at levels of 25-60 pg / mL, donor 2044P at levels of 8-13 pg / mL, and donor 605463P at levels of 20-50 pg / mL. Compared to CBNK, CD5 CAR-NK secreted IL-15 at levels of both MCB and DP, demonstrating statistical significance. There was no statistical significance between CD5 CAR-NK (MCB) and CD5 CAR-NK (DP) (Figure 13, *p<0.05, **p<0.01, ns: not significant).

[0177] [Table 12]

[0178]

[0179] 6-7) Analysis of the long-term tumor cell line killing ability of CD5 CAR gene-transduced NK cells

[0180] To evaluate the long-term tumor cell line killing ability of CD5 CAR gene-transduced NK cells, red fluorescent protein-expressing tumor cell lines were co-cultured with CD5 CAR cells from three donors for 96 hours to confirm tumor cell line killing ability. NALM6, a CD5-negative B cell-derived cancer cell line, and CCRF-CEM and RPMI-8402, CD5-positive acute T lymphocytic leukemia cell lines, were used in the experiment. Tumor cells were cultured at 1 x 10 in assay medium (RPMI1640 containing 10% FBS (Gibco, 11875093)). 5 The tumor cell lines were suspended at a concentration of 1 × 10 cells / mL. To form spheroids, 100 μL of the cells were placed in each well of a 96-well ULA (ultra-low attachment) plate (Corning) and centrifuged at 125 g, 4°C, for 10 minutes. NK cells co-cultured with NALM6 and RPMI-8402 were at a concentration of 1 × 10 5 The NK cells were prepared at a concentration of 0.3 × 10 cells / mL (E:T ratio = 1:1) and co-cultured with CCRF-CEM. 5 The NK cells were suspended in assay medium at a concentration of 100 cells / mL (E:T ratio = 0.3:1) and then added in 100 μL to each well containing tumor cells. For the tumor cell-only control group, 100 μL of assay medium was added to 100 μL of tumor cells instead of NK cells. The plates were placed in an Incucyte S3 instrument (Sartorius) and co-cultured for 96 hours. The red fluorescence intensity (All Brightfield Object Total Red Integrated Intensity, RCU × μm) within the tumor cell spheroids was measured. 2The images were analyzed using Oncocyte spheroid analysis software (v2019B). The graphs show the red fluorescence intensity of the co-cultured wells normalized to that of the wells containing tumor cells alone at each time point. As shown in Figure 10, the tumor-killing ability of NK cells isolated from three donors against CD5-negative NALM6 cells was similar compared to that of CBNK cells. When used with CD5-positive tumor cells, CCRF-CEM, CD5 CAR-NK cells (MCB, DP) persistently suppressed tumor cell growth compared to CBNK cells (MCB, DP). The CD5 CAR-NK cells from MCB and DP of donor 1 had similar tumor cell proliferation-inhibitory abilities. In contrast, in donors 2 and 3, CD5 CAR-NK cells (MCB) had higher tumor cell-killing ability than CD5 CAR-NK cells (DP) when incubated blank with CCRF-CEM. In two donors (donors 1 and 3), the tumor cell-killing ability of CD5 CAR-NK cells (MCB) was higher than that of CD5 CAR-NK cells (DP) when used with CD5-positive tumor cells, RPMI-8402, compared with that of CBNK cells (MCB, DP). However, the CD5 CAR-NK cells (DP) from donor 2 did not exhibit tumor-killing ability (Figure 14). Significance was confirmed by a two-tailed t-test (*p<0.05, **p<0.01, ***<0.001, ns: not significant). In conclusion, the long-term tumor cell-killing ability of CD5 CAR NK cells against CD5-positive tumor cells was confirmed, and CD5 CAR-NK (MCB) cells exhibited higher tumor cell-killing ability than CD5 CAR-NK (DP) cells (Table 13).

[0181] [Table 13]

[0182]

[0183] Example 7: In vivo efficacy evaluation of CD5 CAR-transduced cells

[0184] 7-1) Cultivation and freezing of tumor cells

[0185] The RPMI-8402-Luc cell line, which was generated by inserting the CML-Luc lentiviral vector into RPMI-8402, a human T-cell acute lymphoblastic leukemia cancer cell line, was cultured in IMDM medium (ATCC) containing 10% (v / v) fetal bovine serum (FBS) (Gibco) and 2.0 μg / mL puromycin (Gibco). The cancer cell line was cultured at a cell density of 1 × 10 5 The cancer cell lines were frozen in a cancer cell line culture medium containing 20% ​​DMSO (Avantor) using a Mr. Frosty (freezing container) (Thermo Fisher) in an ultra-low temperature freezer for up to one week, and then transferred to an ultra-low temperature liquid nitrogen tank for storage.

[0186]

[0187] 7-2) Tumor cell line xenografts and NK cell administration

[0188] For experimental animals, specific pathogen-free (SPF) NOD.Cg-Prkdc scid IL2γg tm1Sug Six-week-old female JicKoat (NOG) mice (supplied by Seronbio (produced by KOATECH Co., Ltd.)) were used. The cancer cells used for tumor implantation in mice were prepared by thawing frozen cell lines and subculturing them every 3–4 days. On the day of tumor implantation in mice, all cancer cells were collected, centrifuged, and resuspended in PBS. Experiments were conducted under two different conditions for tumor cell and NK cell administration: (1) a comparison of the number of injections and the dosage (MCB) and (2) a comparison of the culture conditions (MCB and DP).

[0189] (1) For comparisons based on the number of doses and the dose, tumor cell lines were 5 × 10 6 A cell suspension was prepared at a concentration of 1 × 10 cells / mL. 0.2 mL of the prepared cell suspension was injected into the tail vein of each mouse in each experimental group. 6Cancer cells were transplanted at 1000x1000 cells / head. Starting three days after transplantation, CBNK (MCB) and CD5 CAR-NK (MCB) cells were administered in three test groups: (A) a single administration; (B) three administrations twice weekly; and (C) three administrations once weekly (Table 14). After thawing the frozen NK cells, the cell suspension was resuspended in freezing medium to adjust the concentration to suit the experimental conditions, and then 0.2 mL of each cell was injected into the tail vein of each mouse either once or three times in total. For the three-time administration, NK cells were administered twice weekly on days 3, 7, and 10, and once weekly on days 3, 10, and 17. For the test group administered three times weekly, 2x10 cells were used. 6 , 5×10 6 , 1×10 7 The CD5 CAR-NK (MCB) was administered at different doses based on cells / head. The experimental group received only PBS as a control, and the cancer cell line 1×10 6 A tumor-only experimental group was added in which only cells / heads were administered, and each experimental group contained 3 or 5 individuals.

[0190] (2) For comparison by culture period (MCB, DP), tumor cell lines were cultured at 1.5 × 10 7 A cell suspension was prepared at a concentration of 3 × 10 cells / mL. 0.2 mL of the prepared cell suspension was injected into the tail vein of each mouse in each experimental group. 6 Cancer cells were transplanted at 1 × 10 cells / head. From 3 days after transplantation, CBNK (MCB) and CD5 CAR-NK (MCB, DP) cells were administered in a single dose. 7 The control group was administered PBS alone, and the other group was administered 3 × 10 cancer cell lines per head (Table 15). 6 A tumor-only experimental group was added in which only cells / heads were administered, and each experimental group contained 3 or 5 individuals.

[0191] [Table 14]

[0192] [Table 15]

[0193]

[0194] 7-3) Efficacy evaluation of CD5 CAR-NK cells in mice depending on the number and dose of administration

[0195] All animals were monitored twice daily (once on weekends and holidays) for general symptoms and three times weekly for body weight changes during the study period. Starting on day 60 after cancer cell implantation, weight changes were monitored once weekly. Mortality was also observed, and survival rates were assessed by calculating the median survival time for all groups using GraphPad Prism. Mice were intraperitoneally administered 50 μL of 15 mg / mL D-luciferin (GOLDBIO) into each abdominal cavity. After seven minutes of inhalation anesthesia with 2% isoflurane, small animal bioimaging (Perkin Elmer, IVIS Spectrum Series) was performed. Images were taken weekly for a total of 13 times (days 0, 7, 14, 21, 28, 35, 42, 49, 56, 70, 84, 102, and 112) from the day of implantation. As a result, in the CBNK (MCB) administration group, hind limb paralysis symptoms were confirmed in all animals around day 40, regardless of the number of doses administered, and all animals died within two weeks of symptom onset, earlier than the tumor-only group, with a median survival time of 48-50 days (Table 16). In the CD5 CAR-NK (MCB) administration group, hind limb paralysis symptoms were not observed in the single-dose group, and hind limb paralysis symptoms were observed only in some animals in the group administered a total of three times twice weekly and the group administered a total of three times weekly. A comparison by dose showed that more animals showed hind limb paralysis symptoms when administered at a low dose. On day 137, the final observation time, the median survival time in the CD5 CAR-NK (MCB) administration group was 129 days for the single-dose group, >137 days for the group administered a total of three times twice weekly, and 5 x 10 days for the group administered a total of three times weekly. 6 , 1×10 7 cells / head in both experimental groups >137 days, 2 × 10 6At 100 days, the cells / head experimental group showed a survival time extension effect of more than two times compared to the tumor-only and CBNK (MCB) groups, whose median survival time was around 50 days (Table 16).Across the group administered once a week for a total of three times, a trend toward increased survival time was observed with higher doses and frequency of administration (Figure 15) (*p<0.05, **p<0.01, ***<0.001, ns: not significant).

[0196] In terms of weight change, the tumor-only group and the CBNK (MCB)-administered group began to lose weight around day 37, and all animals died on days 56 and 53, respectively, indicating a tendency for weight loss and death to occur as the disease progresses. In the CD5 CAR-NK (MCB)-administered group, some animals died without any obvious weight loss being observed.

[0197] As a result of imaging measurements, in the single and triple-administered CBNK(MCB) groups, imaging signals were measured from day 21, whereas no imaging signals were measured in any of the CD5 CAR-NK(MCB) administered groups, confirming a significant tumor formation suppression ability (Figure 16). Even on day 56, when all of the CBNK(MCB) administered groups had died, the CD5 CAR-NK(MCB) administered group still had 2 x 10 6 It was confirmed that tumor formation was suppressed, with the exception of a few individuals in the cells / head experimental group. Measurement of the image signal (photon / s) showed an increase in the CBNK (MCB)-administered group from day 21, while the CD5 CAR-NK (MCB)-administered group maintained a similar level to that on the day of tumor administration until day 35 in both the single- and triple-administered groups (Figure 16). It was confirmed that tumor formation suppression was maintained in surviving individuals in the CD5 CAR-NK (MCB)-administered group even on day 112, the final imaging observation point, and statistical significance was confirmed by one-way analysis of variance (ANOVA) (*p<0.05, **p<0.01, ***<0.001, ****<0.0001, ns: not significant).

[0198] [Table 16]

[0199] Table 17

[0200] Table 18

[0201]

[0202] 7-4) Efficacy evaluation of CD5 CAR-NK cells depending on the culture period in mice

[0203] General symptoms and body weight changes were measured twice weekly for all animals during the study period using the same procedures as in Example 7-3. Median survival time was calculated and survival rates (MCB, DP) were compared based on the culture period. Images were taken weekly for a total of 17 times (days 0, 7, 14, 27, 35, 42, 49, 56, 65, 72, 78, 86, 94, 100, 107, 118, and 126) from the day of cancer cell implantation. As a result, hind limb paralysis symptoms were observed in all animals in the CBNK (MCB)-treated group, regardless of the number of implantations, around day 40. All animals died within two weeks of symptom onset, and the median survival time was 49 days, the same as that of the tumor-only group (Table 19). In the CD5 CAR-NK (MCB)-treated group, hind limb paralysis first appeared on day 69, and one mouse died on day 72. In the CD5 CAR-NK(DP) group, one mouse died on day 83, but no hind limb paralysis symptoms were observed immediately prior to death. Unlike CBNK(MCB), hind limb paralysis was observed only in a portion of CD5 CAR-NK(MCB) and CD5 CAR-NK(DP) mice before death. The median survival time for CD5 CAR-NK(DP) was measured at 90 days, but for CD5 CAR-NK(MCB), more than half of the mice had not died until day 133, making it impossible to calculate the median survival time (Figure 18). Therefore, although the median survival time for CD5 CAR-NK(MCB) was longer than that for CD5 CAR-NK(DP), this was not statistically significant. However, both CD5 CAR-NK (MCB) and CD5 CAR-NK (DP) showed a statistically significant improvement in survival rate compared to CBNK (MCB), and statistical significance was confirmed by log-rank (Mantel-Cox) test (*p<0.05, **p<0.01, ***<0.001, ns: not significant).

[0204] In imaging measurements, video signals were detected near the hind limbs of mice in the tumor-only and CBNK(MCB)-injected groups from day 14, whereas no video signals were detected in any of the CD5 CAR-NK(MCB)-injected groups, confirming a significant tumor formation suppression ability (Figure 19). In the CD5 CAR-NK(DP)-injected group, video signals began to be observed on day 35 after tumor cell injection, and in the case of CD5 CAR-NK(MCB), video signals began to be observed one week later, on day 42. Even on day 49, when all CBNK(MCB) mice had died, video signals were observed in only some mice in the CD5 CAR-NK(MCB) and CD5 CAR-NK(DP)-injected groups, indicating that tumor formation was suppressed in the CD5 CAR-NK(MCB, DP)-injected groups compared to CBNK(MCB). It was confirmed that tumor formation suppression ability was maintained in surviving individuals on day 126, the final observation point, and statistical significance was confirmed by one-way analysis of variance (*p<0.05, **p<0.01, ***<0.001, ****<0.0001, ns: not significant).

[0205] [Table 19]

[0206] Although certain parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention, and therefore the true scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A fusion protein comprising: (a) a chimeric antigen receptor (CAR) comprising an intracellular signaling domain that includes an OX40 ligand (OX40L); and an extracellular antigen binding domain that binds to CD5; and (b) IL (Interleukin)-15.

2. The fusion protein according to claim 1, wherein the extracellular antigen-binding domain is an antigen-binding fragment of an anti-CD5 antibody.

3. The fusion protein of claim 2, wherein the antigen-binding fragment is a Fab fragment, a F(ab') fragment, a F(ab')2 fragment, or a Fv fragment.

4. The fusion protein of claim 2, wherein the extracellular antigen-binding domain comprises a heavy chain variable region including HCDR1 having the amino acid sequence of sequence 26 of the sequence listing, HCDR2 having the amino acid sequence of sequence 28 of the sequence listing, and HCDR3 having the amino acid sequence of sequence 30 of the sequence listing.

5. The fusion protein of claim 4, wherein the heavy chain variable region comprises the amino acid sequence of sequence 32 of the sequence listing.

6. The fusion protein of claim 2, wherein the extracellular antigen-binding domain additionally comprises a light chain variable region including LCDR1 having the amino acid sequence of sequence No. 34 of the sequence listing, LCDR2 having the amino acid sequence of sequence No. 36 of the sequence listing, and LCDR3 having the amino acid sequence of sequence No. 38 of the sequence listing.

7. The fusion protein of claim 6, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

40.

8. The fusion protein of claim 2, wherein the extracellular antigen-binding domain comprises the amino acid sequence of sequence number 7 in the sequence listing.

9. The fusion protein of claim 1, wherein the intracellular signaling domain additionally comprises one or more domains selected from the group consisting of CD28, CD3-zeta, OX40, and 4-1BB.

10. The fusion protein of claim 9, wherein the intracellular signaling domain additionally comprises CD28 and CD3-zeta.

11. The fusion protein according to claim 10, wherein the intracellular signaling domain comprises CD28, OX40L, and CD3-zeta in that order from the cell membrane to the cell.

12. The fusion protein of claim 1, further comprising a self-cleaving peptide located between the chimeric antigen receptor and the IL-15.

13. The fusion protein of claim 12, wherein the self-cleaving peptide comprises the amino acid sequence of sequence 21 in the sequence listing.

14. A nucleic acid molecule encoding the fusion protein of any one of claims 1 to 13.

15. An immune cell expressing the nucleic acid molecule of claim 14.

16. Immune cells that express: (a) a chimeric antigen receptor (CAR) comprising an intracellular signaling domain that includes an OX40 ligand (OX40L); and an extracellular antigen binding domain that binds to CD5; and (b) IL (Interleukin)-15.

17. The immune cell of claim 16, wherein the immune cell is a natural killer cell.

18. The immune cells according to claim 17, characterized in that the natural killer cells are natural killer cells that have been cultured for 23 to 35 days and then frozen and thawed.

19. A composition for preventing or treating CD5-positive tumors, comprising the immune cells according to claim 15 or 16 as an active ingredient.

20. 20. The composition of claim 19, wherein the CD5-positive tumor is lymphocytic leukemia.

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