Chimeric antigen receptor targeting CD5 and immune cell expressing chimeric antigen receptor

Abstract

The present invention relates to an immune cell co-expressing IL-15 and a chimeric antigen receptor containing an OX40 ligand as an intracellular signal domain, and a composition for preventing or treating cancer comprising the same as an active ingredient. The immune cell shows synergistic tumor cell killing activity through co-expression of the chimeric antigen receptor and IL-15, and the survival rate and the in-vitro proliferation rate of the immune cell are remarkably improved, so that the immune cell can be used as an efficient anti-cancer cell therapy. Particularly, when the chimeric antigen receptor targeting CD5 is expressed, the immune cell can be used as an effective treatment composition for various CD5 positive tumors, including lymphocytic leukemia.

Description

Technical Field

[0001] The present invention relates to immune cells expressing a chimeric antigen receptor specifically recognizing CD5 and IL-15, and a method for treating CD5-positive tumors using such cells. Background Art

[0002] Natural killer (NK) cells are lymphoid cells that account for about 10% of blood cells and play a key role in immune responses. As a key player in innate immune defense, NK cells are suitable for adoptive cell immunotherapy, which can kill tumor cells or cells infected with exogenous pathogens. In the field of cell therapy using T cells (another type of immune cells), there has been research on technologies for introducing chimeric antigen receptors (CARs) into T cells to enhance the specific killing effect on cancer cells expressing specific tumor antigens. A CAR is an artificial receptor designed to confer antigen specificity to immune cells and consists of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, which are selected to provide specific immunity by activating immune cells such as T cells. The extracellular antigen-binding domain may contain a single-chain variable fragment (scFv) targeting an identified tumor antigen, thereby triggering the activation of immune cells specific for cancer cells expressing the tumor antigen.

[0003] Meanwhile, OX40 ligand (CD252), which belongs to the TNFR superfamily, is expressed within hours to days after activation of antigen-presenting cells (APCs), some natural killer cells, and B cells. OX40 (CD134) is the receptor for OX40 ligand and is expressed on T cells and also on CD28-activated T cells. The expression of OX40 further enhances the T cell response induced by CD28 activation, thereby increasing T cell proliferation, cytokine secretion, and survival.

[0004] CD5 is a type I glycoprotein and a member of the scavenger receptor family. CD5 is expressed by thymocytes, mature T cells, and mature B cells and is involved in regulating the activation and differentiation processes of lymphocytes. CD5 attenuates the activation signal of the BCR, so B-1 cells can only be activated by very strong stimuli (such as bacterial proteins) and not by normal tissue proteins. CD5 is highly expressed in a variety of hematological tumors (including acute T-lymphoblastic leukemia and peripheral T-cell lymphoma) and solid tumors (such as breast cancer and thymic carcinoma), and its expression is suppressed in normal cells. Therefore, CD5 has been actively studied as a therapeutic target for these tumors.

[0005] In this specification, many publications and patent documents are cited. The disclosures of the cited publications and patent documents are hereby incorporated by reference in their entirety to more clearly describe the state of the prior art to which the present invention pertains and the content of the present invention. Summary of the Invention

[0006] Technical Problem

[0007] The present inventors conducted in-depth research to develop an effective immunocyte therapy product for various CD5-positive tumors including lymphocytic leukemia. As a result, the present inventors found that when a fusion protein in which a chimeric antigen receptor containing OX40 ligand as an intracellular signaling domain is linked to IL-15 is expressed in immunocytes, the survival rate, in vitro expansion, and anti-tumor activity of the immunocytes can be significantly enhanced, so that a therapeutically effective amount of cells can be easily obtained, and even at a low dose, these cells can be used as an effective cell therapy product that exhibits excellent anti-cancer effects against CD5-positive tumors.

[0008] Therefore, an object of the present invention is to provide a fusion protein comprising: a chimeric antigen receptor comprising an intracellular signaling domain containing OX40 ligand and an extracellular antigen-binding domain that binds to CD5; and IL-15.

[0009] Another object of the present invention is to provide immunocytes expressing the fusion protein and a composition for preventing or treating CD5-positive tumors comprising the same as an active ingredient.

[0010] Other objects and advantages of the present invention will become more apparent from the following detailed description and the appended claims and drawings.

[0011] Technical Solution

[0012] In one aspect of the present invention, there is provided a fusion protein comprising:

[0013] (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

[0014] (b) interleukin (IL)-15.

[0015] The present inventors have conducted in-depth research to develop an effective immunocyte therapy product for various CD5-positive tumors including lymphocytic leukemia. As a result, the present inventors have found that when a fusion protein in which a chimeric antigen receptor containing OX40L as an intracellular signaling domain is linked to IL-15 is expressed in immunocytes, the survival rate, in vitro expansion, and anti-tumor activity of the immunocytes can be significantly enhanced, so that a therapeutically effective amount of cells can be easily obtained, and even at a low dose, these cells can be used as an effective cell therapy product that exhibits excellent anti-cancer effects against CD5-positive tumors.

[0016] 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 from different sources in the fusion protein can be directly linked to each other, or can be linked to each other through a linker sequence, a tag sequence, a self-cleaving sequence, or a combination thereof. In the fusion protein of the present invention, the amino acid sequence of the chimeric antigen receptor (CAR) and the amino acid sequence of the IL-15 protein can be directly linked to each other, or can be indirectly linked to each other through a linker, a tag, a self-cleaving sequence, or a combination thereof. The fusion protein can be produced by recombinant techniques known in the art or can be expressed in target cells.

[0017] As used herein, the term "extracellular antigen-binding domain" refers to a domain located in the extracellular portion of a chimeric antigen receptor expressed in a target cell (such as an immunocyte), which is capable of specifically recognizing a target antigen, thereby activating the apoptotic activity of the immunocyte in a target cell (such as a cancer cell)-specific manner. For example, the extracellular antigen-binding domain can be an antigen-binding fragment of an antibody, such as an Fc receptor or a single-chain variable fragment (ScFv). Therefore, the term "extracellular antigen-binding domain" in the present invention has the same meaning as the terms "extracellular domain", "antigen recognition fragment", or "antigen-binding fragment" when used.

[0018] According to a specific embodiment of the present invention, the extracellular antigen-binding domain is an antigen-binding fragment of an anti-CD5 antibody.

[0019] As used herein, the term "antibody" refers to an antibody against the CD5 protein, which is capable of specifically recognizing and binding to a specific epitope of the CD5 protein, and includes not only a complete full-length antibody, but also an antigen-binding fragment (antibody fragment) of the antibody molecule.

[0020] A full-length antibody has a structure composed of two full-length light chains and two full-length heavy chains, where each light chain is linked to a heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types and can be further divided into gamma 1 (γ 1 ), gamma 2 (γ 2 ), gamma 3 (γ 3 ), gamma 4 (γ 4 ), alpha 1 (α 1 ) and alpha 2 (α 2 ) subclasses. The light chain constant region has kappa (κ) and lambda (λ) types.

[0021] As used herein, the term "antigen-binding fragment of an antibody" refers to a fragment that retains the antigen-antibody binding function in the intact antibody molecule. Specific examples of antigen-binding fragments include Fab fragments, F(ab') fragments, F(ab')2 fragments, and Fv fragments.

[0022] Among antibody fragments, Fab has a structure that includes the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant domain (C H1 ) of the heavy chain and has one antigen-binding site. Fab′ differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain C H1 domain. F(ab′)2 antibodies are generated by forming a disulfide bond between the cysteine residues in the hinge region of Fab′. Fv refers to the smallest antibody fragment that contains only the variable regions of the heavy and light chains. The double-chain Fv is formed by non-covalently linking the variable region of the heavy chain and the variable region of the light chain, while the single-chain Fv (scFv) generally has the same structure as the dimer of the double-chain Fv, because the variable region of the heavy chain is covalently linked to the variable region of the light chain by a peptide linker or directly at the C-terminus. Such antibody fragments can be obtained by using proteases (for example, Fab can be obtained by limited cleavage of the intact antibody with papain, and F(ab′) 2 fragments can be obtained by pepsin cleavage), or can be produced by recombinant technology.

[0023] More specifically, the antibody antigen-binding fragment used in the present invention is the ScFv of an anti-CD5 antibody.

[0024] As used herein, the term "heavy chain" refers to a chain composed of a variable region V H and three constant regions C H1 , C H2 and C H3A full-length heavy chain, and fragments thereof, which comprise an amino acid sequence having a sufficient variable region sequence to confer antigen specificity. In this specification, the term "complementary determining region (CDR)" refers to the amino acid sequence of the hypervariable region of an immunoglobulin heavy or light chain. Each heavy chain (HCDR1, HCDR2, and HCDR3) and each light chain (LCDR1, LCDR2, and LCDR3) contain three CDRs, which provide the major contact residues by which the antibody binds to an antigen or epitope.

[0025] The scope of the antibodies or antibody fragments of the present invention includes variants having conservative amino acid substitutions in the CDR regions. In addition, the scope of the antibodies or antibody fragments of the present invention may include variants of the amino acid sequences listed in the attached sequence listing that are capable of specifically recognizing CD5. For example, the amino acid sequence of the antibody can be further modified to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid residues of the antibody. These amino acid variations are based on the relative similarity of the amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents shows 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 can be defined as biological function equivalents.

[0026] Amino acid substitutions in proteins that generally do not alter the molecular activity are well known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most common substitutions are those between the 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.

[0027] In view of the above-described variants having biological equivalent activity, it can be interpreted that the antigen recognition fragment of the antibody of the present invention and the nucleic acid molecule encoding the fragment also include sequences having substantial identity with the sequences listed in the sequence listing. As used herein, the term "substantial identity" refers to sequences that show at least 80% homology, in another specific embodiment at least 85% homology, in another specific embodiment at least 90% homology, in another specific embodiment at least 95% homology, and in another specific embodiment at least 99% homology, determined by aligning the sequences of the present invention with any other sequence as much as possible and analyzing the alignment using algorithms commonly used in the art. Alignment methods for sequence comparison are known in the art (Smith and Waterman, Adv. Appl. Math. (1981) 2:482; Huang et al., Comp. Appl. BioSci. (1992) 8:155-65; Pearson et al., Meth. Mol. Biol. (1994) 24:307-31).

[0028] According to a specific embodiment of the present invention, the extracellular antigen-binding domain comprises a heavy-chain variable region, the heavy-chain variable region comprising HCDR1 having the amino acid sequence shown in SEQ ID NO:26, HCDR2 having the amino acid sequence shown in SEQ ID NO:28, and HCDR3 having the amino acid sequence shown in SEQ ID NO:30.

[0029] More specifically, the heavy-chain variable region comprises the amino acid sequence shown in SEQ ID NO:32.

[0030] According to a specific embodiment of the present invention, the extracellular antigen-binding domain further comprises a light-chain variable region, the light-chain variable region comprising LCDR1 having the amino acid sequence shown in SEQ ID NO:34, LCDR2 having the amino acid sequence shown in SEQ ID NO:36, and LCDR3 having the amino acid sequence shown in SEQ ID NO:38.

[0031] More specifically, the light-chain variable region comprises the amino acid sequence shown in SEQ ID NO:40.

[0032] Most specifically, the extracellular antigen-binding domain used in the present invention comprises the amino acid sequence shown in SEQ ID NO:7.

[0033] According to a specific embodiment of the present invention, the intracellular signaling domain further comprises at least one domain selected from the group consisting of CD28, CD3-zeta, OX40, and 4-1BB. More specifically, the intracellular signaling domain further comprises CD28 and CD3-zeta.

[0034] Most specifically, the intracellular signaling domain contains CD28, OX40L, and CD3-zeta, which are arranged in sequence from the cell membrane towards the interior of the cell.

[0035] 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 IL-15. More specifically, the self-cleaving peptide comprises the amino acid sequence shown in SEQ ID NO:21.

[0036] According to the present invention, the amino acid sequence shown in SEQ ID NO:21 is the amino acid sequence of the T2A self-cleaving peptide. The T2A self-cleaving peptide is a 2A self-cleaving peptide that cleaves the full-length protein into short peptide fragments by inducing ribosomal skipping during intracellular protein translation. It comprises 18 amino acid residues (EGRGSLLTCGDVEENPGP) of SEQ ID NO:21. According to an embodiment of the present invention, a GSG (glycine-serine-glycine) sequence can be added at the N-terminus to enhance the self-cleaving activity of the amino acid sequence shown in SEQ ID NO:21. Through the T2A self-cleaving peptide, the chimeric antigen receptor and IL-15 of the present fusion protein can be expressed in a cleaved form.

[0037] In another aspect of the present invention, there is provided a nucleic acid molecule encoding the above-mentioned fusion protein of the present invention.

[0038] As used herein, the term "nucleic acid molecule" refers to both DNA (gDNA and cDNA) and RNA molecules. Nucleotides are the basic building blocks of nucleic acid molecules and include not only natural nucleotides but also analogs having modified sugar or base moieties (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)). The nucleic acid molecule of the present invention can be inserted into a gene delivery system and introduced into target cells, such as immune cells, so as to express the above-mentioned fusion protein of the present invention in the target cells.

[0039] As used herein, the term "expression" refers to allowing a target cell to express an exogenous gene or artificially introducing an endogenous gene using a gene delivery system to increase the natural expression level of the endogenous gene, so that the gene can replicate in the target cell as an extrachromosomal factor or through chromosomal integration. Thus, the term "expression" is synonymous with "transformation", "transfection", or "transduction".

[0040] As used herein, the term "gene delivery system" refers to any means of delivering a gene into a cell, and the term "gene delivery" has the same meaning as intracellular transduction of a gene. At the cellular or tissue level, gene delivery has the same meaning as the spread of a gene. Thus, the gene delivery system of the present invention may be referred to as a gene transduction system or a gene dissemination system.

[0041] To construct the gene delivery system of the present invention, the nucleotide sequence of the present invention is preferably operably linked to appropriate expression control sequences in a suitable expression construct. As used herein, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, or array of transcription regulatory factor binding sites) and a target nucleic acid sequence. By such linkage, the control sequence regulates the transcription and / or translation of the target nucleic acid sequence. A promoter linked to the target gene of the present invention is a promoter that can specifically act in animal cells (more specifically mammalian cells, most specifically immune cells) to regulate the transcription of the target gene, such as promoters from mammalian viruses and promoters from mammalian cell genomes. Examples of promoters include, but are not limited to, the mammalian cytomegalovirus (CMV) promoter, the adenovirus late promoter, the vaccinia virus 7.5K promoter, the SV40 promoter, the HSV tk promoter, the RSV promoter, the EF1α promoter, the metallothionein promoter, the β-actin promoter, the human IL-2 gene promoter, the human IFN gene promoter, the human IL-4 gene promoter, the human lymphotoxin gene promoter, and the human GM-CSF gene promoter.

[0042] The nucleotide sequence of the target gene can be applied to any gene delivery system commonly used for gene introduction. For example, the nucleotide sequence of the target gene can be applied to plasmids, adenoviruses (Lockett LJ et al., Clin. Cancer Res. 3:2075-2080 (1997)), adeno-associated viruses (AAV, Lashford LS et al., Gene Therapy Technology, Applications and Regulations, A. Meager (ed.), 1999), retroviruses (Gunzburg WH et al., Retroviral Vectors. Gene Therapy Technology, Applications and Regulations, A. Meager (ed.), 1999), lentiviruses (Wang G et al., J. Clin. Invest. 104(11):R55-62 (1999)), herpes simplex viruses (Chamber R et al., Proc. Natl. Acad. Sci USA 92:1411-1415 (1995)), vaccinia viruses (Puhlmann M et al., Human Gene Therapy 10:649-657 (1999)), liposomes (Methods in Molecular Biology, 199, S.C. Basu and M. Basu (eds.), Human Press 2002) or niosomes.

[0043] According to a specific embodiment of the present invention, the gene delivery system used in the present invention is a viral vector. More specifically, the virus is selected from the group consisting of: lentiviruses, adenoviruses, adeno-associated viruses (AAV), retroviruses, herpes simplex viruses and vaccinia viruses. Most specifically, the virus is a lentivirus.

[0044] The nucleotide sequence listings of the nucleic acid molecules encoding the full-length fusion protein or each domain of the fusion protein of the present invention are summarized in Table 2 below.

[0045] In another aspect of the present invention, there is provided an immune cell expressing the nucleic acid molecule of the present invention as described above.

[0046] In this specification, the term "immune cell" refers to any type of cell involved in initiating or promoting the immune response process. More specifically, it refers to immune effector cells. 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.

[0047] In another aspect of the present invention, there is provided an immune cell expressing:

[0048] (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

[0049] (b) Interleukin (IL)-15.

[0050] Since the immune cells used in the present invention have been described in detail above, to avoid over - repetition, they will not be elaborated here.

[0051] The immune cells of the present invention can express a chimeric antigen receptor and IL - 15 in the form of a single fusion protein, wherein the chimeric antigen receptor and IL - 15 are connected to each other, as described above for one aspect of the present invention. Additionally, the immune cells can also express the chimeric antigen receptor and IL - 15 as separate protein molecules that are not connected together. In the latter case, the chimeric antigen receptor and IL - 15 can be co - transfected into the immune cells by inserting the nucleic acid molecules encoding them into independent gene delivery systems, or they can be inserted together into one gene delivery system and then transfected into the immune cells.

[0052] According to a specific embodiment of the present invention, the immune cells used in the present invention are natural killer cells. More specifically, the natural killer cells are natural killer cells that are frozen and then thawed after being cultured for 23 to 35 days, more specifically 25 to 33 days, and most specifically 28 to 30 days.

[0053] In this specification, the term "culture" refers to inducing cells to grow and proliferate in an in vitro environment (such as a culture medium) while maintaining their biological activity.

[0054] The term "culture medium" used herein refers to a mixture that can effectively induce and promote cell growth and proliferation in vitro, which contains essential elements required for cell growth and proliferation, such as sugars, amino acids, minerals, and other nutrients. Cell culture media can be optimized according to the type, phenotype, and culture characteristics of specific cells. For example, they include basal media prepared to support cell growth, media prepared to promote the production of cell recombinant proteins, and concentrated media prepared by high - concentration concentrated nutrients.

[0055] As used herein, the terms "cryopreservation" or "low-temperature preservation" refer to the short-term or long-term maintenance of cell stability at low temperatures (especially ultra-low temperatures of -80°C to -200°C). Cells typically mutate at a rate of approximately 1 in every 10,000 cells during the culturing process. If cells are passaged for a long time, they will transform into a cell population different from the original cell population or may lose their inherent biological activity and functions. The risk of mycoplasma infection also increases with the increase in the passage time. Therefore, the method of cryopreserving cells is widely used to preserve the inherent characteristics of cells. The present inventors have found that when culturing immune cells, especially natural killer cells with low gene transduction efficiency using a lentiviral vector, a pre-cryopreservation process for an appropriate time can greatly improve the gene delivery rate and cell killing activity. Therefore, different from cryopreservation for long-term cell preservation, the cryopreservation step in the present invention can be carried out only for a short time.

[0056] The cryopreservation of cells can be carried out by treating the cells with a cryoprotectant, which can minimize the damage caused by the inevitable ice crystal formation and ion and osmotic pressure imbalance during the freezing and thawing processes. The cryopreservation medium can contain, for example, dextran, albumin, and / or DMSO to improve the safety and stability of the cells during cryopreservation.

[0057] As used herein, the term "thawing" refers to the process of raising the temperature of cryopreserved or low-temperature-preserved cells until the cells return to soft, living cells to resume their life activities. Thawing can usually be quickly completed by taking out the cryopreserved cells from a liquid nitrogen tank or the like and placing them in a 37°C constant temperature water bath.

[0058] Since the freezing and thawing processes of the present invention are aimed at improving the efficiency of gene transduction into cells and enhancing the activity of killing pathogenic cells, rather than long-term storage of cells, the thawing process can be initiated immediately after the freezing is completed without leaving a time interval between freezing and thawing, or if necessary, an appropriate time interval can be provided between the freezing process and the thawing process.

[0059] In another aspect of the present invention, there is provided a composition for preventing or treating CD5-positive tumors, which contains the above-mentioned immune cells of the present invention as an active ingredient.

[0060] In another aspect of the present invention, there is provided a method for preventing or treating CD5-positive tumors, which includes administering the above-mentioned immune cells of the present invention to a subject in need thereof.

[0061] As used herein, the term "treatment" refers to (a) inhibiting the progression of a disease, disorder or symptom; (b) alleviating a disease, disorder or symptom; or (c) eliminating 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, their function can induce the death of CD5-positive cancer cells, thereby inhibiting the progression of symptoms caused by various CD5-positive tumors (including lymphocytic leukemia), or eliminating or alleviating the symptoms. Therefore, the compositions of the present invention can be used alone as a cell therapy composition for the disease, or can be administered in combination with other pharmacological components and used as a therapeutic adjuvant for the disease. Therefore, the term "treatment" or "therapeutic agent" as used herein includes the meaning of "therapeutic adjuvant" or "therapeutic adjuvant agent".

[0062] As used herein, the term "administer" or "administration" refers to directly injecting a therapeutically effective amount of the composition of the present invention into a subject's body so as to form the same amount of the composition in the subject's body.

[0063] As used herein, the term "therapeutically effective amount" refers to the amount of the composition sufficient to provide a therapeutic or prophylactic effect to a subject to whom the composition of the present invention is administered. Therefore, the term "therapeutically effective amount" includes "prophylactically effective amount".

[0064] In the present specification, the term "subject" includes, but is not limited to, humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons or rhesus monkeys. Specifically, the subject of the present invention is a human.

[0065] As used herein, the term "CD5-positive tumor" refers to a solid cancer or blood cancer in which CD is highly expressed at a measurable level compared to normal cells or CD5-negative tumors. CD5 is a transmembrane receptor protein that is expressed in 85% of T-lineage acute lymphoblastic leukemia (T-ALL) and 75% of peripheral T-cell lymphoma, and is highly expressed in mantle cell lymphoma, B chronic lymphocytic leukemia (CLL) and hairy cell leukemia. Since the expression of CD5 in normal cells is inhibited, CD5 is used as an effective target for the treatment and diagnosis of tumors.

[0066] CD5-positive tumors that can be prevented or treated by the compositions of the present invention include blood cancers such as leukemia, lymphoma and multiple myeloma, and solid cancers such as breast cancer and thymic cancer, but are not limited thereto, and also include all malignant tumors that express CD5 at a measurable level on the surface of tumor cells and can thus be specifically recognized by the immune cells of the present invention.

[0067] More specifically, the CD5-positive tumor is T-lymphocytic leukemia, and more specifically, acute T-lymphocytic leukemia.

[0068] The immune cells of the present invention, particularly natural killer cells, can be included therein at a content of 10-95 wt% of the total weight of the composition for preventing or treating CD5-positive tumors. In addition, the composition of the present invention can be formulated to further contain one or more anti-cancer pharmacological components having the same or similar functions in addition to natural killer cells.

[0069] The dosage of the composition can be adjusted according to 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 in the composition, the type of formulation, the age, weight, general health status, gender, diet, administration time, administration route, secretion rate of the composition, treatment cycle, and drugs used simultaneously. However, in order to achieve the desired effect, the dosage of natural killer cells according to the present invention can be, for example, 0.01x10 7 cells / kg to 1.0x10 9 cells / kg, or 0.5x10 7 cells / kg to 1.0x10 8 cells / kg.

[0070] In addition, the composition of the present invention can be administered to a subject by various therapeutic cell injection methods known in the art. Those skilled in the art can appropriately select the administration route according to the administration method, the volume and viscosity of the body fluid, etc. For example, the composition of the present invention can be administered parenterally, particularly intravenously, subcutaneously or intraperitoneally, and most specifically intravenously.

[0071] Beneficial effects

[0072] The features and advantages of the present invention are summarized as follows:

[0073] (a) The present invention provides immune cells co-expressing a chimeric antigen receptor and IL-15, wherein the chimeric antigen receptor contains OX40 ligand as an intracellular signal domain, and provides a composition for preventing or treating cancer containing the immune cells as an active ingredient.

[0074] (b) The immune cells of the present invention not only exhibit synergistic tumor cell killing activity by co-expressing a chimeric antigen receptor and IL-15, but also significantly improve the survival rate and in vitro proliferation rate, and thus can be used as an effective anti-cancer cell therapy product.

[0075] (c) In particular, when expressing a chimeric antigen receptor targeting CD5, the immune cells of the present invention can be used as an effective therapeutic composition for various CD5-positive tumors including lymphocytic leukemia.

[0076] Brief description of the drawings

[0077] Figure 1Schematically shows the chimeric antigen receptor (CAR) constructs 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 signaling domains and containing only the IL-15 domain, and a fourth-generation CAR construct with an additional IL-15 domain connected to the third-generation CAR construct.

[0078] Figure 2 Shows the measurement results of the survival rate and amplification fold of third-generation CAR-NK cells and fourth-generation CAR-NK cells in the absence of the IL-2 cytokine.

[0079] Figure 3 Shows the measurement results of the tumor cell killing ability of third-generation CAR-NK cells and fourth-generation CAR-NK cells against HER2-positive tumor cell lines HCC1954 and SKOV.

[0080] Figure 4 Shows the change in the IFN-γ secretion amount of third-generation CAR-NK cells and fourth-generation CAR-NK cells that have contacted the HER2-positive tumor cell lines HCC1954 and SKOV.

[0081] Figure 5 Is a schematic diagram summarizing the NK cell culture process in the present invention.

[0082] Figure 6 Shows that through Figure 5 The natural killer cells (MCB) frozen on the 28th to 30th day after culture by the process shown in the upper figure and the natural killer cells (DP) frozen on the 42nd to 44th day after culture by the process shown in the lower figure Figure 5 Measurement results of the expression pattern of CD5 CAR.

[0083] Figure 7 Shows the results of analyzing the cell phenotypes of MCB and DP based on the expression pattern of each surface marker.

[0084] Figure 8 Shows the results of measuring the CD5 protein expression in each tumor cell line of K562, NALM6, CCRF-CEM, RPMI-8402_Luc, NALM6-NucLight, CCRF-CEM-NucLight, and RPMI-8402-NucLight by FACS analysis.

[0085] Figure 9 Shows the measurement results of the expression levels of CD107a, IFN-γ, and TNFα (n = 3) in CBNK or CD5 CAR-NK co-cultured with various tumor cell lines.

[0086] Figure 10 Shows a culture schematic diagram for evaluating the tumor cell killing ability of CD5 CAR-transduced cells.

[0087] Figure 11 Shows the evaluation results of the killing ability of NK cells against CD5-positive and CD-negative tumor cells.

[0088] Figure 12 Shows the measurement results of the average amount of IFN-γ secretion (pg / mL) in three donors.

[0089] Figure 13 Shows the measurement results of the average amount of IL-15 secreted (pg / mL) by NK cells co-cultured with each type of target cell.

[0090] Figure 14 shows the evaluation results of the long-term tumor cell killing ability after co-culturing CD5 CAR-NK cells with CD5-positive and CD5-negative tumor cells for 96 hours.

[0091] Figure 15 shows the measurement results of the survival rate (%) according to each drug substance until 140 days after tumor transplantation in each experimental group.

[0092] Figure 16 Shows the bioimaging results until 112 days after injecting the NK cells of the present invention after tumor transplantation.

[0093] Figure 17 shows the bioluminescence (photons / second) results measured over time until day 35 after tumor transplantation.

[0094] Figure 18 Shows the measurement results of the survival rate (%) until day 133 after injecting the NK cells of the present invention after tumor transplantation in experimental animals.

[0095] Figure 19 shows the bioimaging results until day 126 after injecting the NK cells of the present invention after tumor transplantation in experimental animals.

[0096] Embodiments of the invention Examples

[0097] Example 1: Preparation of CAR-NK cells expressing the third-generation chimeric antigen receptor (CAR) and fourth-generation CAR-NK cells co-expressing the third-generation chimeric antigen receptor and the IL-15 domain

[0098] The structure of the chimeric antigen receptor (CAR) used in the present invention is summarized in Table 1 below.

[0099] Table 1

[0100]

[0101] The HER2 CAR (third generation) constructed to evaluate the synergy of co-expression of the OX40L-containing CAR and IL-15 of the present invention has a structure in which the following elements are sequentially linked together: the signal sequence domain of CD8a (nucleotides 890-952, GenBank NM 001768.6); the anti-HER2 scFv sequence (VH-(GGGGS)3-VL domain); the hinge domain derived from human CD8α (nucleotides 1292-1435, GenBank NM 001768.6); the transmembrane and intracellular signaling domain derived from CD28 (nucleotides 679-882, GenBank NM 006139.3); the intracellular signaling domain derived from CD252 (nucleotides 141-206, GenBank NM 003326.4); the intracellular signaling domain derived from CD3ζ (nucleotides 299-634, GenBank NM000734.3); and the stop codon TGA. HER2 CAR(t)-IL-15 has a structure in which the following elements are sequentially linked together: the signal sequence domain of CD8α (nucleotides 890-952, GenBank NM 001768.6); 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 domain derived from CD28 (nucleotides 679-759, GenBank NM 006139.3); the T2A self-cleaving peptide sequence linked to glycine (G)-serine (S)-glycine (G); the human IL-15 sequence (nucleotides 375-860, GenBank NM000585.4); and the stop codon TGA.

[0102] HER2 CAR-IL-15 (Fourth Generation) has a structure in which the following elements are connected in sequence: the signal sequence domain of CD8α (nucleotides 890 - 952, GenBank NM 001768.6); the anti-HER2 scFv sequence (VH-(GGGGS)3-VL domain); the hinge domain derived from human CD8α (nucleotides 1292 - 1435, GenBank NM 001768.6); the transmembrane and intracellular signaling domain derived from CD28 (nucleotides 679 - 882, GenBank NM 006139.3); the intracellular signaling domain derived from CD252 (nucleotides 141 - 206, GenBank NM 003326.4); the intracellular signaling domain derived from CD3z (nucleotides 299 - 634, GenBank NM000734.3); the T2A self-cleaving peptide sequence linked with G-S-G; the human IL-15 sequence (nucleotides 375 - 860, GenBank NM000585.4); and the stop codon TGA.

[0103] CD5 CAR (Fourth Generation) has a structure in which the following elements are connected in sequence: the signal sequence of CD8α (nucleotides 890 - 952, GenBank NM 001768.6); the anti-CD5 scFv domain (VH-(GGGGS)3 linker - VL); the hinge domain derived from CD8α (nucleotides 1292 - 1435, GenBank NM 001768.6); the transmembrane and intracellular signaling domain derived from CD28 (nucleotides 679 - 882, GenBank NM 006139.3); the intracellular signaling domain derived from CD252 (nucleotides 141 - 206, GenBank NM 003326.4); the intracellular signaling domain derived from CD3z (nucleotides 299 - 634, GenBankNM000734.3); the T2A self-cleaving peptide sequence linked with G-S-G; the human IL-15 sequence (nucleotides 375 - 860, GenBankNM000585.4); and the stop codon TGA.

[0104] The nucleotide sequences and amino acid sequences of each domain contained in the chimeric antigen receptor of the present invention are summarized in Table 2 below.

[0105] [Table 2]

[0106]

[0107]

[0108]

[0109] IL-15 is a cytokine that can activate natural killer (NK) cells and promote their survival and proliferation. Therefore, to study whether co-expression of IL-15 in NK cells expressing chimeric antigen receptors can improve the survival rate and in vivo persistence of NK cells, the present inventors introduced a soluble form of IL-15 protein into a chimeric antigen receptor comprising a single-chain antibody fragment (scFv) specifically recognizing HER2 and a signaling domain, and evaluated its effect ( Figure 1 ). NK cells expressing the chimeric antigen receptor in a bicistronic form were constructed by connecting the anti-HER2 scFv fragment and the signaling domain to the soluble IL-15 domain through the T2A system, and were named "fourth-generation CAR-NK cells" ( Figure 1 "d" in Figure 1 ). As a control, third-generation CAR-NK cells expressing a chimeric antigen receptor comprising an anti-HER2 scFv fragment and a signaling domain but not expressing the IL-15 domain were also constructed ( Figure 1 "b" in Figure 1 ), and NK cells expressing the anti-HER2 scFv fragment but lacking the intracellular signaling domain and expressing the IL-15 domain (

[0110] Example 2: Evaluation of the survival rate and growth of third-generation and fourth-generation CAN-NK cells

[0111] Umbilical cord blood-derived NK cells were cultured, and each gene was introduced into the cells on the 7th day. Starting from the 22nd day of culture, the cells were cultured without adding IL-2 to the medium, and the survival rate and growth of the cells were observed. Except for the fourth-generation CAR-NK cells and CAR(t)-NK cells, other cell populations no longer grew, and starting from the time when IL-2 was not added, their survival rate decreased significantly, and most cells died on the 41st day of culture ( Figure 2 ). It was confirmed that only CAR-NK cells containing the IL-15 domain structure maintained their survival rate, indicating that secreted IL-15 contributes to the survival and proliferation of NK cells.

[0112] Example 3: Comparison of the efficacy of third-generation and fourth-generation CAR-NK cells and the synergistic effect induced by the IL-15 domain

[0113] 3-1) Evaluation of tumor cell killing ability

[0114] To evaluate the efficacy of the fourth-generation CAR-NK cells, NK cells were co-cultured with tumor cell lines expressing HER2 to measure the cytotoxicity of NK cells. Two tumor cell lines expressing HER2 (HCC1954 and SKOV3) were used as target cells. The cells expressed RFP, so that the fluorescence signal disappeared when the cells died. Starting from the 22nd day of culture, different types of NK cell populations were co-cultured with the tumor cell lines at a ratio of E:T = 0.3:1 without adding IL-2 for 6 days, and then the proliferation of tumor cells was detected. Among the two tumor cell lines (HCC1954 and SKOV3), the fourth-generation HER2 CAR-NK cells secreting IL-15 showed excellent cytotoxicity by inhibiting the proliferation of the tumor cell lines, while the third-generation HER2 CAR-NK cells and HER2 CAR(t)-NK cells showed relatively low cytotoxicity( Figure 3 ). The third-generation HER2 CAR-NK cells seemed unable to effectively inhibit the formation of tumor cells due to the lack of additional cytokines such as IL-2. It can be hypothesized that HER2 CAR(t)-NK cells showed a lower cytotoxic effect due to the lack of a signaling domain.

[0115] 3-2) Evaluation of IFN-γ secretion

[0116] By measuring the secretion results of the functional cytokine IFN-γ secreted by NK cells when NK cells contacted the target tumor cells, it was observed that the third-generation HER2 CAR-NK cells and HER2 CAR(t)-NK cells secreted very little IFN-γ or did not secrete IFN-γ, while the fourth-generation CAR-NK cells secreted a very high level of IFN-γ( Figure 4 ). Therefore, it can be confirmed that, contrary to the third-generation CAR-NK cells that could not survive and proliferate smoothly, the fourth-generation CAR-NK cells showed a synergistic anti-tumor effect due to the co-expression of IL-15 and the chimeric antigen receptor containing OX40L as a signaling domain.

[0117] Example 4: Culture of NK cells and cell cryopreservation at different culture time points

[0118] 4-1) Cell thawing

[0119] The cryopreserved cells were quickly thawed in a preheated 37 °C constant temperature water bath. When more than 90% of the cells were thawed, the cells were transferred to a 50 mL centrifuge tube in a biosafety cabinet, and 10 times the volume (v / v) of ACD buffer was slowly added. Then, centrifugation was performed at 1200 rpm and 4 °C for 10 minutes to completely remove the supernatant, and then the cells were resuspended in CellGro medium and counted.

[0120] 4-2) Culture of NK cells and transduction with lentiviral vector

[0121] On day 0 of culture, NK cells at 1.0 x 10 6 cells / mL, feeder cells at 2.5 x 10 6 cells / mL, and CellGro medium were mixed at a ratio of 1:1:1, and then statically cultured in a CO 2 incubator at 37°C. The Cellgro medium used was supplemented with IL-2 (1000 IU / mL), human plasma (2%), and OKT3 (10 μg / mL) corresponding to the total volume. On day 4 of culture, CellGro mixed medium containing IL-2 (1000 IU / mL) and human plasma (1%) equal to the total volume was added. On day 7 of culture, all cells were recovered and counted without additional dilution. When the cell count exceeded 0.66 x 10 6 cells / mL, the cells were cryopreserved. On day 7 of culture, the thawed original cells were resuspended in CellGro medium and counted, and then the cells were diluted to a concentration of 1.0 x 10 6 cells / mL for culture. On day 10 of culture, in order to treat cells at 1.0 x 10 6 cells / mL with a lentiviral vector at 20 MOI, the cells were treated with a mixture containing 6.6 x 10 8 TU / mL lentiviral vector (30 μL). The composition of the mixture for lentiviral vector transduction is shown in Table 3 below.

[0122] [Table 3] Composition of each solution for lentiviral vector transduction

[0123]

[0124] Mixtures 1 and 2 were prepared separately and then slowly added to the culture container. A mixed medium obtained by mixing 1 mL of CellGro medium with human plasma (1%) and IL-2 (1000 IU) (hereinafter referred to as CellGro mixed medium) was added to CBNK without lentiviral vector transduction, and then statically cultured in a CO 2 incubator at 37°C. On day 11 of culture, according to the volume of the 6-well plate, CellGro mixed medium with the same volume as on day 10 was added, and IL-21 at a concentration of 20 ng / mL was added. On day 13 of culture, the cells were recovered and the cell concentration was adjusted to 1 x 10 6cells / mL, without further dilution. On day 16 of culture, CD5-positive cells were isolated to obtain NK cells expressing CD5 CAR. For this purpose, all NK cells were recovered and centrifuged at 1200 rpm for 5 minutes at 4 °C, and the supernatant was removed. Then the cells were resuspended in MACS buffer and counted. MACS buffer was added to adjust the cell concentration to 1.0x10 7 cells / mL. Then, biotinylated recombinant human CD5 protein (His and AVI tags) (250 μg / mL) was added to the cells at a dose of 1 μL / 10 6 cells, and after thorough mixing, the mixture was incubated at 4 °C for 30 minutes. 10 volumes of MACS buffer were added to the first incubation mixture, and then the mixture was centrifuged at 340 g for 10 minutes at 4 °C, and the supernatant was removed. The cells were resuspended in MACS buffer to 1.0x10 8 cells / mL, and then anti-biotin microbeads (Miltenyi Biotec, 130-092-357) were added at a dose of 37.5 μL / 40x10 6 cells, and the mixture was incubated at 4 °C for 15 minutes. 10 volumes of MACS buffer were added, and then the mixture was centrifuged at 340 g for 10 minutes at 4 °C, and the supernatant was removed. MACS buffer was added to adjust the cell concentration to 1.0x10 8 cells / 500 μL, and the cells were lysed thoroughly by pipetting several times. The LS column (Miltenyi Biotec) was installed on the QuadroMACS TM separator (Miltenyi Biotec), and the column was rinsed with 3 mL of MACS buffer to activate the column. The cells incubated with the microbeads were lysed thoroughly by pipetting, and 500 μL of the cell suspension was loaded onto the column until the cell suspension flowed out. To wash the column, the column was rinsed with 3 mL of MACS buffer, and a total of three washes were performed. The column was removed from the QuadroMACS TM separator, 5 mL of MACS buffer was added, and CD5 CAR-positive cells were recovered by pressing the plunger. After centrifugation of the isolated cells, CellGro mixed medium was added to adjust the cell concentration to 1x10 6 cells / mL. Then, the cells were restimulated with feeder cells in the same manner as on day 0 of culture, and then statically cultured in a CO 2 incubator at 37 °C. On day 19 of culture, the same volume of CellGro mixed medium as on day 16 of culture was added. On day 21 of culture, the cells were recovered and counted, without additional dilution, and the cell concentration was adjusted to 1x10 6cells / mL. Next, between days 28 and 30 of the culture, the cells were maintained at 1x10 6 cells / mL by adding CellGro mixed medium. All NK cells from days 28 to 30 of the culture were harvested and cryopreserved. The cells cryopreserved after days 28 to 30 of the culture are hereinafter referred to as "MCB". After adding CellGro medium to the remaining cells to reach a cell concentration of 1.0x10 6 cells / mL, they were restimulated with feeder cells in the same manner as on day 16 of the culture, and then statically cultured in a CO 2 incubator at 37°C. Next, the cells were maintained at 1x10 6 cells / mL by adding CellGro mixed medium and cultured for 14 days. All NK cells from days 42 to 44 of the culture were harvested and cryopreserved, and the cryopreserved cells were named "DP".

[0125] 4-3) Cell freezing

[0126] After centrifugation, the cells were resuspended in PBS and mixed with the cryopreservation medium mixture in a 1:1 ratio, and aliquoted into each cryovial at a concentration of 100x10 6 cells / mL, and then frozen using a programmable freezer (CRF).

[0127] Example 5: Characterization of CD5 CAR-Transduced NK Cells

[0128] 5-1) Identification of CD5 CAR expression

[0129] Collect 1 to 5x10 5 cells and centrifuge to remove the supernatant. Then resuspend the cells in FACS buffer supplemented with 2 mL of 2% FBS. Next, centrifuge the cell suspension and remove the supernatant. Add 100 μL of FACS buffer and 1 μL of biotinylated recombinant human CD5 protein (His and AVI tags) (250 μg / mL) to the cells, and incubate at 4°C for 30 minutes in the dark. Thereafter, add 2 mL of FACS buffer to the cells, centrifuge, and remove the supernatant. For the second staining, add 100 μL of cell buffer to the washed NK cells, then add the antibodies for analysis, and incubate at 4°C for 30 minutes in the dark.

[0130] [Table 4] Antibodies for Detecting CD5 CAR-NK Expression

[0131] Antibody Dosage Streptavidin-PE 0.5 μL Anti-human CD56 APC 5 μL Anti-human CD3-PE-Cy7 1 μL 7-AAD 5 μL FACS buffer Make up to 100 μL Total amount 100

[0132] Next, 2 mL of FACS buffer was added to the cells, and the supernatant was removed after centrifugation. Then, 200 μL of BD Cytofix solution was added to the cells and mixed well. Subsequently, the stained cells were analyzed using the LSRfortessa system. The raw data was analyzed using the Flowjo program, and gating was performed in the following order: "single cell → lymphocyte → live cell (7-AAD-) → NK cell (CD56+, CD3-) → CD5 CAR expression". After thawing, the CD5 CAR expression rate of MCB was 96.46 ± 2.00 (mean ± standard deviation), and that of DP was 93.68 ± 2.23 (mean ± standard deviation), indicating that the CD5 CAR expression rates of both MCB and DP cells were 90% or higher ( Figure 6 ). The statistical significance of these values was evaluated by two-sided t-test (Table 5, *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant). The above results confirmed that the expression of CD5 CAR could be well maintained even when the culture time was increased, and the expression of CD5 CAR did not decline rapidly after thawing of the frozen cells.

[0133] [Table 5] CAR expression of MCB and DP

[0134]

[0135] 5-2) Phenotype of CD5 CAR-transduced NK cells

[0136] The NK cells were resuspended in FACS buffer at a concentration of 1.0 to 2.5 x 10 6 cells / mL. 100 μL of the cell suspension was aliquoted into each well of the plate containing the antibody mixture. The cells and the antibody were mixed well by pipetting several times. The composition of the antibody mixture is shown in Table 6 below. The PE antibody corresponding to each phenotype was added.

[0137] [Table 6] Composition of the antibody mixture for phenotypic staining

[0138]

[0139]

[0140] After incubation at 4 °C for 30 minutes, 100 μL of FACS buffer was added to the cells and mixed by pipetting, followed by centrifugation (4 °C, 2000 rpm, 3 minutes). 200 μL of BD Cytofix solution was added to each well. Flow cytometry was performed using the LSRfortessa system and the results were analyzed. NK cells were gated in the following order: single cells → lymphocytes → live cells (7-AAD-) → NK cells (CD56+, CD3-). The expression of each marker was calculated based on the subtype antibodies. To characterize NK cells, the expression of 18 surface markers was analyzed by flow cytometry, and the MCB and DP of CD5CAR-NK were compared with those of CBNK. The results showed that there was no significant difference in the expression of each surface marker in CD5 CAR-NK compared with CBNK, and there was also no significant difference between MCB and DP ( Figure 7 and Table 7).

[0141] [Table 7] NK cell phenotype values (%) from three donors (2024P, 2044P, and 605463p)

[0142]

[0143] Example 6: In vitro efficacy evaluation of CD5 CAR-transduced cells

[0144] 6-1) Generation and culture of tumor cell lines

[0145] Frozen K562, NALM6, CCRF-CEM, RPMI-8402_Luc, NALM6-NucLight, CCRF-CEM-NucLight, or RPMI-8402-NucLight cells were rapidly thawed in a 37 °C constant temperature water bath and then diluted in 10 mL of medium. The cells were centrifuged, the supernatant was removed, and the cells were resuspended in 1 mL of medium and counted. 1 to 2x10 6 cells were placed in a T75 culture flask and cultured in a CO 2 incubator for 2 or 3 days. To generate NucLight-labeled tumor cell lines, the cell lines were transduced with NuclightRed lentivirus (Sartorius, Göttingen, Germany) using 8 μg / mL polybrene (Santa Cruz, CA, USA). Four days later, the cells were treated with 0.5, 1, 1.5, and 2 μg / mL puromycin (Gibco, USA). Seven days after puromycin treatment, the lowest concentration that killed 100% of the tumor cells was selected, and then the cells expressing red fluorescent protein were selected and cultured.

[0146] 6-2) Expression of CD5 in tumor cells

[0147] Transfer 1 to 5x10 5 recovered tumor cells into a FACS tube. After centrifugation, add 100 μL of FACS buffer containing 1 μL of anti-human CD5 PE to the cells and incubate for 30 minutes at 4 °C in the dark. Add 1 mL of FACS buffer to the cells, centrifuge and remove the supernatant. Add 200 μL of BD Cytofix solution and mix with the cells, then perform FACS analysis. In Figure 8 , the gray shade represents the isotype control, the blue solid line represents the result of anti-human CD5 antibody staining, and the numbers represent the expression rate (%) relative to the isotype control. The results confirmed that K562, NALM6, and NALM6-Nuclight hardly expressed CD5, while CCRF-CEM, CCRF-CEM-Nuclight, RPMI-8402-Luc, RPMI-8402-Nuclight, and Jurkat-Nuclight highly expressed CD5( Figure 8 ).

[0148] 6-3) Expression of CD107a and cytokines on CD5 CAR-transduced cells

[0149] Resuspend NK cells and target tumor cell lines at a concentration of 2.5x10 6 cells / mL in RPMI-1640 + 10% FBS (hereinafter referred to as the assay medium). To detect whether CD5-specific cell activity occurs, use CCRF-CEM and RPMI-8402_Luc as CD5-positive tumor cell lines and NALM-6 as a CD5-negative tumor cell line. Add Golgistop (1.3 μ / mL) and Golgiplug (2 μ / mL) to the cells and mix to prevent the secretion of cytokines produced within NK cells to the outside of the cells. Dispense APC anti-human CD107a antibody (1 μL) into the (-) wells and target wells of a 96-well round bottom plate. To identify cytokines intrinsically expressed by NK cells, add 100 μL of assay medium and 100 μL of NK cells to the (-) wells of the plate to which the antibody has been dispensed, and add 100 μL of NK cells and 100 μL of target tumor cell line to the target wells. Wrap the 96-well plate with aluminum foil to protect from light, and incubate the cells at 37 °C in CO 2Co-culture in an incubator for 4 hours. After centrifugation (4°C, 2000 rpm, 3 minutes), the supernatant was removed, 200 μL of FACS buffer was added and mixed with the cells, and the supernatant was removed again after centrifugation. For cell surface staining, anti-human CD3 PerCP-Cy5.5 (1 μL), anti-human CD56-APC-e780 (1 μL), and 7-AAD (4 μL) were added to 100 μL of FACS buffer in each well and incubated at 4°C for 30 min. After adding 100 μL of FACS buffer, centrifugation was performed twice to remove the supernatant, 200 μL of fixation / permeabilization solution was added, and incubation was carried out at 4°C for 30 minutes. After centrifugation, the supernatant was removed, 200 μL of 1x Perm wash buffer was added, and centrifugation was performed again. 100 μL of 1x Perm wash buffer was added to each well, and then anti-human IFN-η FITC (1 μL) and anti-human TNF-α PE-Cy7 (1 μL) were added to each well and incubated at 4°C for 30 minutes. 100 μL of 1x Perm wash buffer was added, and the supernatant was removed after centrifugation. Then 200 μL of 1x Perm wash buffer was added, and centrifugation was performed again. The supernatant was removed, 200 μL of BD Cytofix was added, and measurement was performed using the LSR Fortessa FACS system. The results were analyzed using the FlowJo analysis program. The expression of CD107a and cytokines (IFN-γ η and TNF-α) against the K562 cell line for evaluating NK cell activity was higher in both CBNK and CD5 CAR-NK, but there was no significant difference. In addition, when NK cells were co-cultured with the positive cell lines CCRF-CEM and RPMI-8402_Luc, the expression of CD107a and cytokines (IFN-γ η and TNF-α) in the CD5 CAR-NK group was at least twice that of the CBNK group, while the expression level was lower in the negative cell line NALM-6, indicating that cytokines were expressed in a CD5-specific manner( Figure 9 ). The statistical significance of the measurement results of the expression levels of CD107a and two cytokines in three donors was evaluated using a two-sided t-test. The results are shown in Table 8 below in the form of mean ± standard deviation( Figure 9 , *p < 0.05, **p < 0.01, ns: no significance). The CD-specific expression of CD107a and cytokines was higher in CD5 CAR-NK than in CBNK, and there was no significant statistical difference between MCB and DP.

[0150] [Table 8] Mean expression levels of CD107α, IFN-η, and TNF-α (n = 3)

[0151]

[0152]

[0153] Evaluation of the Tumor Cell Killing Ability of CD5 CAR-Transduced Cells

[0154] Recover the NK cells (CBNK, CD5 CAR-NK) and centrifuge them at 1200 rpm for 5 minutes at room temperature. After removing the supernatant, resuspend the cells in 1 mL of assay medium and count them. Suspend the transduced cells in the assay medium according to each E (NK cell):T (target cell) ratio and add them to 5 mL FACS tubes. NK cells are used at 1x10 5 cells / 100 μL for the 10:1 ratio, 3x10 4 cells / 100 μL for the 3:1 ratio, 1x10 4 cells / 100 μL for the 1:1 ratio, 3x10 3 cells / 100 μL for the 0.3:1 ratio preparation.

[0155] The target cell line is fixed at 1x10 4 cells / well (1x10 5 cells / mL, 100 μL). Add 100 μL of the target cell line and 100 μL of NK cells to each well of a 96-well round-bottom culture plate according to the specified E:T ratio. To calibrate the Calcein-AM fluorescence value released by the Calcein-AM-stained target cells, the fluorescence value measured after mixing 100 μL of the target cells and 100 μL of the assay medium is used as the minimum release value. To measure the maximum fluorescence value of the target cells releasing Calcein-AM, mix 100 μL of the target cells and 100 μL of 2% Triton X-100 to completely lyse the cells, and the fluorescence value measured at this time is used as the maximum release value. To correct for the decrease in fluorescence value caused by Triton X-100, add 200 μL of the assay medium to each MM well and add 100 μL of the assay medium and 100 μL of 2% Triton X-100 to each MT well. Incubate the culture plate at 37 °C in the dark for 4 hours, and then centrifuge it at 2000 rpm for 3 minutes at 4 °C. Transfer 100 μL of the supernatant to a black 96-well flat-bottom culture plate and measure the fluorescence value using a fluorometer at a wavelength of 458 nm / 535 nm and a condition of 0.1 s. Calculate the cell killing ability according to the formula shown in Table 9 below.

[0156] [Table 9] Calculation of NK Cell Killing Ability

[0157]

[0158] Figure 11Shows the cytotoxicity of each of CBNK (MCB), CD5 CAR-NK (MCB), CBNK (DP), and CD5 CAR-NK (DP) against four target cells from 3 donors (2024P, 2044P, and 605463P). Table 10 below summarizes each cytotoxic activity value (%). As Figure 11 shown in Table 10, the tumor cytotoxicity 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 there was no significant difference in the tumor cytotoxicity between CD5 CAR-NK (MCB) and CD5 CAR-NK (DP) (two-tailed t-test, *p < 0.05, **p < 0.01, *** < 0.001, ns: not significant).

[0159] [Table 10] Cytotoxicity (%) against cells from three donors (2024P, 2044P, and 605463P)

[0160]

[0161]

[0162]

[0163] 6-5) Evaluation of the ability of CD5CAR-transduced cells to secrete IFN-γ

[0164] Dilute the target cells to a concentration of 1x10 5 cells / mL with the detection medium, and dilute the NK cells to a concentration of 3x10 5 cells / mL with the detection medium. Add 100 μL of the target cells and 100 μL of the NK cells to a 96-well round-bottom culture plate respectively, and co-culture them at a ratio of E:T = 3:1. After culturing the cells in an incubator at 37 °C and 5% CO 2 for 24 hours, collect the supernatant and store it at -20 °C.

[0165] Quantitative detection of IFN-γ was performed by ELISA. One day before the experiment, 200x capture antibody was diluted with 1x coating buffer A, and then 100 μL of the dilution was added to a 96-well plate and incubated at 2 to 8 °C for 16 to 18 hours. On the day of the experiment, the plate was washed four times with wash buffer, and 200 μL of 1x detection dilution A was added to each well to inhibit non-specific binding. After sealing the plate, it was placed on a plate shaker for 1 hour. Standards were prepared by serially diluting the undiluted interferon-γ standard with 1x detection dilution A (500, 250, 125, 62.5, 31.3, 15.6, 7.8, and 0 pg / mL). Subsequently, the supernatant stored at -20 °C was thawed and diluted 1 / 10 with 1x detection dilution A to prepare samples. 100 μL of the standard and 100 μL of the diluted sample were added to each well. After sealing the plate, it was incubated at room temperature on a plate shaker for 2 hours. The plate was washed four times with wash buffer, and 100 μL of 1x detection antibody was added to each well. After sealing the plate, it was incubated at room temperature on a plate shaker for 1 hour. The plate was washed four times with wash buffer, and 100 μL of 1x Avidin-HRP was added to each well. After sealing the plate, it was incubated at room temperature on a plate shaker for 30 minutes. After washing the plate five times with wash buffer, 100 μL of TMB substrate was added to each well and incubated 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 a wavelength of 450 nm using a spectrophotometer. The results showed that in 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, and 605463P), but no significant differences were found when the results of all donors were analyzed comprehensively. In the CD5-positive cell lines CCRF-CEM and RPMI-8402-Luciferase, CBNK (MCB, DP) from donor 2024P secreted 40 to 60 pg / mL of IFN-γ, while CD5 CAR-NK (MCB, DP) secreted 350 to 550 pg / mL of IFN-γ, indicating that the IFN-γ secretion of CD5 CAR-NK was approximately 8 to 9 times higher than that of CBNK. For donor 2044P, CBNK (MCB, DP) secreted 45 to 55 pg / mL of IFN-γ, while CD5 CAR-NK (MCB, DP) secreted 250 to 350 pg / mL, indicating that the IFN-γ secretion of CD5 CAR-NK was approximately 5 to 6 times higher than that of CBNK. For donor 605463P, CBNK (MCB, DP) secreted 70 to 120 pg / mL of IFN-γ, while CD5 CAR-NK (MCB, DP) secreted 600 to 1,300 pg / mL of IFN-γ, indicating that the IFN-γ secretion of CD5 CAR-NK was 8 to 10 times higher than that of CBNK.In summary, CD5 CAR-NKs from all three donors enhanced IFN-γ secretion in a CD5+ cell line-specific manner and showed statistically significant results ( Figure 12 , *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant). There was no statistically significant difference between CD5 CAR-NK (MCB) and CD5 CAR-NK (DP).

[0166] [Table 11] IFN-γ secretion levels (pg / mL) of NK cells from three donors

[0167]

[0168] 6-6) Evaluation of the ability of CD5-CAR-transduced cells to secrete IL-15

[0169] Target cells and NK cells at concentrations of 2 x 10 6 cells / mL and 6 x 10 6 cells / mL were prepared by dilution in the assay medium. 100 μL of target cells and 100 μL of NK cells were added to a 96-well round-bottom culture plate at an E:T ratio of 1:3 and co-cultured in a 37 °C, 5% CO 2 2 incubator for 24 hours. Subsequently, the supernatant was collected and stored at -20 °C.

[0170] ELISA was performed using an IL-15 ELISA kit. The microplate strips were inserted into the plate holder, and 100 μL of assay diluent RD1-19 was added to each well. 50 μL of IL-15 standard and 50 μL of the stored supernatant were added to each well of the plate, and then the plate was 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 of the plate, and the plate was 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 of the plate and incubated at room temperature for 30 minutes in the dark. 50 μL of stop solution was added to terminate the reaction, and the absorbance was measured at a wavelength of 450 nm. As Figure 13As shown in Table 12 below, under the condition of culturing NK cells alone without target cells (only effector cells), CBNK (MCB, DP) from three donors secreted 4 to 6 pg / mL of IL-15, while CD5 CAR-NK (MCB, DP) secreted 5 to 17 pg / mL of IL-15. In the CD5-negative cell lines K562 and NALM6, the levels of IL-15 secreted by CBNK (MCB, DP) and CD5 CAR-NK (MCB, DP) from three donors were similar to those in the group of NK cells cultured alone (only effector cells). In the CD5-positive cell lines CCRF-CEM and RPMI-8402-Luciferase, the levels of IL-15 secreted by CBNK (MCB, DP) were similar to those in the CD5-negative cell lines, while the IL-15 secretion of CD5 CAR-NK (MCB, DP) increased to 25 to 60 pg / mL in donor 2024P, 8 to 13 pg / mL in donor 2044P, and 20 to 50 pg / mL in donor 605463P, indicating that both MCB and DP of CD5 CAR-NK showed statistically significant results compared with CBNK. There was no statistically significant difference between CD5 CAR-NK (MCB) and CD5 CAR-NK (DP) Figure 13 , *p<0.05, **p<0.01, ns: no significance).

[0171] [Table 12] IL-15 secretion of MCB and DP (pg / mL)

[0172]

[0173] 6-7) Evaluation of the long-term tumor cell killing activity of NK cells transduced with the CD5CAR gene

[0174] To measure the long-term killing ability of NK cells transduced with the CD5 CAR gene against tumor cell lines, tumor cell lines expressing red fluorescent protein were co-cultured with CD5 CAR cells from three donors for 96 hours. In the experiment, the CD5-negative B cell-derived cancer cell line NALM6, and the CD5-positive acute T lymphocyte leukemia cell lines CCRF-CEM and RPMI-8402 were used. The tumor cells were suspended in the detection medium (RPMI1640 (Gibco, 11875093) containing 10% FBS) at a concentration of 1x10 5 cells / mL. To make the tumor cell lines spherical, 100 μL of tumor cells were added to each well of a 96-well ultra-low attachment (ULA) plate (Corning), and then centrifuged at 125 g and 4 °C for 10 minutes. Diluted with the detection medium, the NK cells to be co-cultured with NALM6 or RPMI-8402 were prepared as 1x10 5cells / mL (E:T ratio = 1:1). Dilute using the assay medium and prepare the NK cells to be co-cultured with CCRF-CEM at a concentration of 0.3x10 5 cells / mL (E:T ratio = 0.3:1). Then, add 100 μL of NK cells to each well containing tumor cells. In the control group with only tumor cells cultured, add 100 μL of assay medium (instead of NK cells) and 100 μL of tumor cells to each well. Place the plate in an Incucyte S3 instrument (Sartorius) and analyze the red fluorescence intensity of the tumor cell spheroids after 96 hours of co-culture using the Oncocyte spheroid analysis program (v2019B) (total red cumulative intensity of all brightfield objects, RCU x μm 2 / imaging). The graph in Figure 14 shows the values obtained by normalizing the red fluorescence intensity of the co-culture wells at each time point relative to the red fluorescence intensity of the wells containing only tumor cells. As shown in Figure 14, the tumor killing ability of NK cells isolated and generated from three donors against CD5-negative NALM6 cells is similar to that of CBNK cells. For the CD5-positive tumor cell line CCRF-CEM, CD5 CAR-NK cells (MCB, DP) more continuously inhibit the growth of tumor cells than CBNK cells (MCB, DP). The CD5 CAR-NK cells (MCB) and CD5 CAR-NK cells (DP) from donor 1 have similar abilities in inhibiting tumor cell proliferation. On the other hand, for the cells from donor 2 and donor 3, when co-cultured with CCRF-CEM, the tumor cell killing ability of CD5 CAR-NK cells (MCB) is higher than that of CD5 CAR-NK cells (DP). For the CD5-positive tumor cell line RPMI-8402, in the cells from two donors (donor 1 and donor 3), the tumor cell killing ability of CD5 CAR-NK cells (MCB) compared to CBNK cells (MCB, DP) is higher than that of CD5 CAR-NK cells (DP), but the CD5 CAR-NK cells (DP) from donor 2 did not show tumor killing activity (Figure 14). Significance was evaluated by two-sided t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant). In summary, the long-term tumor cell killing ability of CD5 CAR NK cells against CD5-positive tumor cells was confirmed, and the killing ability of CD5 CAR-NK (MCB) cells is higher than that of CD5 CAR-NK (DP) cells (Table 13).

[0175] [Table 13] Long-term tumor cell killing ability of CD5 CAR NK cells

[0176]

[0177]

[0178] Example 7: In Vivo Efficacy Evaluation of CD5 CAR-Transduced Cells

[0179] 7-1) Culture and freezing of tumor cells

[0180] The RPMI-8402-Luc cell line was generated by inserting the CML-Luc lentiviral vector into the human T-cell acute lymphoblastic leukemia cancer cell line RPMI-8402. As the culture medium for the cancer cell line, IMDM medium (ATCC) containing 10% (v / v) fetal bovine serum (FBS) (Gibco) and 2.0 μg / mL puromycin (Gibco) was used. The cancer cells were adjusted to a concentration of 1x10 5 cells / mL and cultured in a culture container of an appropriate volume. The cancer cell line was cryopreserved using the cancer cell line culture medium containing 20% DMSO (Avantor) and stored in a Mr. Frosty (cryo-container) (Thermo Fisher Scientific) in an ultra-low temperature freezer for one week, and then transferred to an ultra-low temperature liquid nitrogen tank for storage.

[0181] 7-2) Tumor cell xenograft and administration of NK cells

[0182] Six-week-old female specific pathogen-free (SPF) NOD.Cg-Prkdc scid IL2γg tm1Sug / JicKoat mice (hereinafter referred to as NOG mice) (provided by Saeron Bio) (produced by Coretech Co., Ltd.) were used for the experiment. The cancer cells for tumor transplantation were prepared by thawing the cryopreserved cell line and passaging every 3 to 4 days. On the day of tumor transplantation into the mice, all cancer cells were collected, centrifuged, and resuspended in PBS. The administration experiments of tumor cells and NK cells were divided into two parts: ① comparison according to the number of administrations and dose (MCB), ② comparison according to the culture conditions (MCB, DP).

[0183] ① For the comparison according to the number of administrations and dose, a cell suspension of the tumor cell line with a concentration of 5x10 6 cells / mL was prepared. Each mouse in each experimental group was injected with 0.2 mL of the prepared cell suspension via the tail vein, so that the cancer cell transplantation amount was 1x10 6cells / mouse. The mice were divided into three test groups: (A) a group administered CBNK(MCB) and CD5 CAR-NK(MCB) cells once 3 days after transplantation; (B) a group administered CBNK(MCB) and CD5 CAR-NK(MCB) cells three times at an interval of twice a week starting from 3 days after transplantation; (C) a group administered CBNK(MCB) and CD5 CAR-NK(MCB) cells three times at an interval of once a week starting from 3 days after transplantation (Table 14). After thawing the cryopreserved NK cells, the cells were resuspended in cryopreservation medium under specified conditions, and then 0.2 mL of various cell suspensions were injected into the tail vein of the mice once or three times in total. For a total of three administrations, the NK cells were administered twice a week on days 3, 7, and 10 after transplantation, or once a week on days 3, 10, and 17. For the experimental group with a total of three weekly administrations, the dose of CD5 CAR-NK(MCB) was divided into 2x10 6 , 5x10 6 , and 1x10 7 cells / mouse. As a control group, a group injected with only PBS and a group injected with the cancer cell line at a dose of 1x10 6 cells / mouse (tumor-only group) were added. Each group included 3 or 5 animals.

[0184] ② According to the comparison of culture time (MCB and DP), a cell suspension of the cancer cell line with a concentration of 1.5x10 7 cells / mL was prepared. Each mouse in each experimental group was injected with 0.2 mL of the prepared cell suspension through the tail vein, so that the amount of cancer cell transplantation was 3x10 6 cells / mouse. Starting from 3 days after transplantation, CBNK(MCB) and CD5 CAR-NK(MCB, DP) cells were administered once at a dose of 1x10 7 cells / mouse (Table 15). As a control group, a group injected with only PBS and a tumor-only group injected with the cancer cell line at a dose of 3x10 6 cells / mouse were added. Each group included 3 or 5 animals.

[0185] [Table 14] NK cell administration conditions (① According to the comparison of administration times and doses)

[0186]

[0187] [Table 15] NK cell administration conditions (② According to the comparison of culture time (MCB, DP))

[0188]

[0189] 7-3) Evaluation of the efficacy of CD5CAR-NK cells in mice according to the number and dose of administrations

[0190] For all animals, general symptoms were measured twice a day during the test (once on weekends and holidays), and body weight changes were measured three times a week. Starting from the 60th day after cancer cell transplantation, body weight changes were measured once a week. After confirming whether the animals died, the median survival time of all groups was calculated using GraphPad Prism to evaluate the survival rate. D-luciferin (GOLDBIO) at a concentration of 15 mg / ml was intraperitoneally injected into both sides of the abdomen of the mice (50 μL), and then small animal bioimaging (Perkin Elmer, IVIS Spectrum series) was performed for 7 minutes under 2% isoflurane inhalation anesthesia. Imaging was performed once a week starting from the day of cancer cell transplantation, for a total of 13 times (on days 0, 7, 14, 21, 28, 35, 42, 49, 56, 70, 84, 102, and 112 after transplantation). The results showed that in the CBNK (MCB) administration group, regardless of the number of administrations, all animals showed hind limb paralysis symptoms around the 40th day and all died within 2 weeks after the symptoms appeared. The median survival time was 48 to 50 days because the death time was earlier than that of the tumor-only group (Table 16). In the CD5 CAR-NK (MCB) administration group, hind limb paralysis symptoms were not observed in the single administration group, while only some animals showed hind limb paralysis symptoms in the group administered CD5 CAR-NK (MCB) three times at an interval of twice a week and the group administered CD5 CAR-NK (MCB) three times at an interval of once a week. According to the results of dose comparison, more animals showed hind limb paralysis symptoms at low doses. At the final observation point (day 137), the median survival time of the CD5 CAR-NK (MCB) administration group was: 129 days for the single administration group, >137 days for the group administered three times at an interval of twice a week, >137 days for the group administered three times at an interval of once a week (experimental group administered at a dose of 5x10 6 cells and 1x10 7 cells / mouse), and 100 days for the group administered three times at an interval of once a week (experimental group administered at a dose of 2x10 6 cells / mouse), which was at least twice as long as the median survival period (50 days) of the tumor-only group and the CBNK (MCB) administration group (Table 16). From the results of the group administered three times at an interval of once a week, it was confirmed that the survival period showed a tendency to increase with the increase in dose and the number of administrations (Figure 15) (*p<0.05, **p<0.01, ***p<0.001, ns: not significant).

[0191] Regarding weight changes, only the tumor group and the CBNK(MCB)-administered group started to lose weight around day 37. All animals in these groups died on day 56 and day 53 respectively, showing a trend of weight loss as the disease progressed and then died. In the CD5 CAR-NK(MCB)-administered group, some animals died without significant weight loss.

[0192] Bioluminescence imaging results showed that bioluminescence was detected starting from day 21, while no bioluminescence was detected in all CD5 CAR-NK(MCB)-administered groups, indicating that CD5 CAR-NK(MCB) showed significant inhibitory ability against tumor formation ( Figure 16 ). It was confirmed that even when all animals in the CBNK(MCB)-administered group died on day 56, tumor formation in the CD5 CAR-NK(MCB)-administered group was inhibited, except for some animals in the experimental group administered at a dose of 2x10 6 cells / animal. The measurement results of bioluminescence (photons / second) showed that bioluminescence in the CBNK(MCB)-administered group increased starting from day 21, while bioluminescence in all single-dose and triple-dose CD5 CAR-NK(MCB) groups remained at a level similar to that on the day of tumor transplantation until day 35 ( Figure 16 ). It was confirmed that even at the final imaging time point of day 112, the surviving animals in the CD5 CAR-NK(MCB)-administered group still maintained the inhibitory ability against tumor formation. Statistical significance was evaluated by one-way analysis of variance (ANOVA) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant).

[0193] [Table 16] Median survival time

[0194]

[0195] [Table 17] Bioluminescence intensity (photons / second) at different administration frequencies after tumor transplantation

[0196]

[0197] [Table 18] Bioluminescence intensity (photons / second) at different administration doses after tumor transplantation

[0198]

[0199]

[0200] 7-4) The efficacy of CD5 CAR-NK cells in mice depends on the culture period

[0201] According to the same procedure as in Example 7-3, the general symptoms and body weight changes of all animals were measured twice a week during the experiment, the median survival time was calculated, and the survival rates (MCB, DP) were compared according to the culture period. Imaging was performed once a week from the day of cancer cell transplantation 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). The results showed that in the CBNK (MCB) administration group, regardless of the number of administrations, all animals developed hind limb paralysis symptoms at around 40 days. All animals in this group died within 2 weeks after the appearance of symptoms, and the median survival time of this group was 49 days, the same as that of the tumor-only group (Table 19). In the CD5CAR-NK (MCB) administration group, hind limb paralysis first appeared on day 69, and one mouse died for the first time on day 72. In the CD5 CAR-NK (DP) administration group, one mouse died for the first time on day 83, and no hind limb paralysis symptoms appeared before death. Different from the CBNK (MCB) administration group, only some mice in the CD5 CAR-NK (MCB) administration group and the CD5 CAR-NK (DP) administration group developed hind limb paralysis symptoms before death. The median survival time of the CD5CAR-NK (DP) administration group was 90 days, while the median survival time of the CD5 CAR-NK (MCB) administration group could not be calculated because more than half of the mice did not die before day 133( Figure 18 ). Therefore, the median survival time of the CD5 CAR-NK (MCB) administration group was longer than that of the CD5CAR-NK (DP) administration group, but this difference was not statistically significant. However, the survival rates of both the CD5 CAR-NK (MCB) administration group and the CD5CAR-NK (DP) administration group were statistically significantly better than those of the CBNK (MCB) administration group. Statistical significance was evaluated by the log-rank (mantel-cox) test (*p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant).

[0202] For the tumor-only group and the CBNK(MCB)-administered group, bioluminescence was detected near the hind limbs of the mice starting from day 14, while no bioluminescence was detected in the CD5 CAR-NK(MCB)-administered group, indicating that CD5 CAR-NK(MCB) has a significant inhibitory ability against tumor formation (Figure 19). In the CD5 CAR-NK(DP)-administered group, the bioluminescence signal was first detected on day 35 after tumor cell transplantation. In the CD5 CAR-NK(MCB)-administered group, the bioluminescence signal was first observed on day 42, one week later. It can be seen that even on day 49, when all the mice in the CBNK(MCB)-administered group had died, bioluminescence signals were detected only in some mice in the CD5 CAR-NK(MCB) and CD5 CAR-NK(DP)-administered groups, indicating that tumor formation in the CD5 CAR-NK(MCB, DP)-administered group was inhibited compared to the CBNK(MCB)-administered group. It was confirmed that the ability to inhibit tumor formation could be maintained even on day 126 (the final imaging time point) in the surviving mice. Statistical significance was evaluated by one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant).

[0203] [Table 19] Median survival time

[0204]

[0205] Although the present invention has been described in detail with reference to specific features, it will be apparent to those skilled in the art that this description is only a preferred embodiment of the present invention and does not limit the scope of the present invention. Therefore, the substantial scope of the present invention will be 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 containing OX40 ligand (OX40L) and an extracellular antigen-binding domain that binds CD5; and (b) Interleukin (IL)-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 according to claim 2, wherein the antigen-binding fragment is a Fab fragment, F(ab') fragment, F(ab')2 fragment or Fv fragment.

4. The fusion protein according to claim 2, wherein the extracellular antigen-binding domain comprises a heavy chain variable region, the heavy chain variable region comprising HCDR1 having the amino acid sequence shown in SEQ ID NO:26, HCDR2 having the amino acid sequence shown in SEQ ID NO:28, and HCDR3 having the amino acid sequence shown in SEQ ID NO:

30.

5. The fusion protein according to claim 4, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

32.

6. The fusion protein according to claim 2, wherein the extracellular antigen-binding domain further comprises a light chain variable region, the light chain variable region comprising LCDR1 having the amino acid sequence shown in SEQ ID NO:34, LCDR2 having the amino acid sequence shown in SEQ ID NO:36, and LCDR3 having the amino acid sequence shown in SEQ ID NO:

38.

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

40.

8. The fusion protein according to claim 2, wherein the extracellular antigen-binding domain comprises the amino acid sequence shown in SEQ IDNO:

7.

9. The fusion protein according to claim 1, wherein the intracellular signaling domain further comprises at least one domain selected from the group consisting of CD28, CD3-zeta, OX40, and 4-1BB.

10. The fusion protein according to claim 9, wherein the intracellular signaling domain further comprises CD28 and CD3-zeta.

11. The fusion protein according to claim 10, wherein the intracellular signaling domain comprises CD28, OX40L, and CD3-zeta, which are arranged in sequence from the cell membrane towards the cell interior.

12. The fusion protein according to claim 1, wherein the fusion protein further comprises a self-cleaving peptide located between the chimeric antigen receptor and IL-15.

13. The fusion protein according to claim 12, wherein the self-cleaving peptide comprises the amino acid sequence shown in SEQ ID NO:

21.

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

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

16. An immune cell that expresses: (a) a chimeric antigen receptor (CAR) that includes an intracellular signaling domain containing OX40 ligand (OX40L), and an extracellular antigen-binding domain that binds to CD5; and (b) interleukin (IL)-15.

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

18. The immune cell according to claim 17, wherein, the natural killer cell is a natural killer cell that has been cryopreserved and then thawed after 23 to 35 days of culture.

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

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

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