KR102671410B1 - Chimeric antigen receptor specifically binding to RANK ligand and use thereof - Google Patents

Abstract

The present invention provides a chimeric antigen receptor that specifically binds to RANK ligand; A polynucleotide encoding the chimeric antigen receptor protein; A vector containing the above polynucleotide; T cells or natural killer cells transformed with the vector; and a cell therapy or pharmaceutical composition for treating cancer containing the cells as an active ingredient. The chimeric antigen receptor of the invention comprises the extracellular domain of RANK that recognizes RANK ligand. Therefore, cytotoxic T cells or natural killer cells transformed with a vector capable of overexpressing these cells have specific cytotoxicity against carcinomas expressing RANK ligands, so they can be useful as an immune cell therapy for cancer treatment. .

Description

Chimeric antigen receptor specifically binding to RANK ligand and use thereof {Chimeric antigen receptor specifically binding to RANK ligand and use thereof}

The present invention provides a chimeric antigen receptor that specifically binds to RANK ligand; A polynucleotide encoding the chimeric antigen receptor protein; A vector containing the above polynucleotide; T cells or natural killer cells transformed with the vector; and a cell therapy or pharmaceutical composition for treating cancer containing the cells as an active ingredient.

RANK ligand (receptor activator of nuclear factor-kB ligand; RANK ligand; RANKL) is a type 2 membrane protein and is a member of the tumor necrosis factor (TNF) family. TRANCE (TNF-related activation- It is also known as induced cytokine), OPGL (osteoprotegerin ligand), and ODF (osteoclast differentiation factor), and is an essential element in osteoclastogenesis (Teitelbaum, 2007). In other words, mice with the RANK ligand gene deleted are deficient in osteoclasts and exhibit traits such as osteopetrosis and tooth eruption disorders, and overproduction of RANK ligands is associated with various degenerative bone diseases (rheumatoid). arthritis and osteomyelitis (Pettit et al., 2001; Rinotas et al., 2014). Additionally, RANK ligand is expressed in B lymphocyte tumors, especially chronic lymphocytic leukemia (CLL) or multiple myeloma, and plays a role in worsening these diseases (Croucher et al., 2001; Pearse et al., 2001; Yaccoby et al., 2002; Secchiero et al., 2006). In particular, according to recent research results, the expression of RANK ligand was observed in 100% of chronic lymphocytic leukemia patients, and 80% of patients with multiple myeloma expressed RANK ligand in multiple myeloma (Schmiedel et al., 2013 ). In addition, bone metastasis of cancer (Roodman and Dougall, 2008), osteosarcoma (Branstetter et al., 2015), prostate cancer (Brown et al., 2001), and non-small cell lung cancer. Overexpression is observed in (non-small cell lung cancer) (Peng et al., 2013).

It has been reported that cytotoxic T lymphocytes (CTL) and natural killer cells (NK cells) that directly target cancer cells are important for effective cancer treatment. So far, research on anticancer treatment using cytotoxic T cells or natural killer cells has been conducted by delivering specific patient-derived cancer antigens to the patient's cytotoxic T cells to induce activation or induce antibody dependent cellular cytotoxicity. (Galluzzi et al., 2018; Lu et al., 2020). However, recently, a new anticancer treatment method using cytotoxic T cells and natural killer cells has been introduced. That is, a key grafted onto a domain grafted onto the cytoplasmic signaling domain of CD3 zeta or another protein using the single-chain variable fragment (scFv) portion of an antibody that recognizes the antigen. An attempt was made to deliver chimeric antigen receptor (CAR). When a chimeric antigen receptor is grafted onto a T cell or natural killer cell, the anticancer activity of the T cell or natural killer cell can be activated simply by recognizing a specific antigen by scFv, regardless of signal transduction by antigen presenting cells (APC). Also, since it is not limited by HLA type, it can be used as a more efficient treatment method that can be universally used by many people. In fact, these chimeric antigen receptor-expressing T cells or natural killer cells are showing efficacy in several cancers (Holzinger et al., 2016; Pettitt et al., 2018; Wang et al., 2020).

Meanwhile, RANK (receptor activator of nuclear factor kB) is a type 1 membrane protein, called TRANCE receptor or TNFRSF11A, and is a member of the tumor necrosis factor receptor superfamily (Ono et al., 2020). RANK has a high affinity for RANK ligand, and the signal transmitted by the combination of RANK ligand and RANK is an essential element in osteoclastogenesis (Teitelbaum, 2007; Ono et al., 2020). Recently, in order to increase the affinity of RANK for the RANK ligand, a RANK mutant was created in which the 125th amino acid, glutamic acid, in the extracellular domain of wild-tpe RANK was replaced with aspartic acid, and the 127th amino acid, cysteine, was replaced with phenylalanine. RANK.E125D+C127F was constructed, and RANK.E125D+C127F increases the affinity for the RANK ligand approximately 4-fold compared to wild-type RANK (Son et al., 2015).

Currently, a pharmaceutical composition for osteoporosis using a RANKL-RANK combination (Korean Patent Publication No. 10-2020-0054698) or a chemotherapy treatment for cancer using a RANKL inhibitor (Korean Patent Publication No. 10-2019-0030646) has been disclosed. However, there has been no description of an immunotherapy for cancer using a chimeric antigen receptor containing the extracellular domain of RANK.

Against this background, the present inventors took advantage of the fact that RANK ligand expression is confirmed or induced in various cancer cells and that RANK ligand and RANK can bind to create a RANK mutant (RANK) with improved affinity of RANK for RANK ligand. .E125D+C127F), cancer-specific chimeric antigen receptor-expressing T cells and natural killer cells expressing a RANK ligand were created using the extracellular domain, and the T cells or natural killer cells were used to treat cells expressing HLA class II. The present invention was completed by confirming through experiments that it had a specific cytotoxic ability.

Korean Patent Publication No. 10-2020-0054698 (2020. 05. 20) Korean Patent Publication No. 10-2019-0030646 (2019. 03. 22)

Therefore, the purpose of the present invention is to provide a chimeric antigen receptor that specifically binds to RANK ligand.

Another object of the present invention is to provide a polynucleotide encoding the chimeric antigen receptor protein, a vector containing the polynucleotide, and T cells or natural killer cells transformed with the vector.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer that kills cancer cells expressing a cell therapy or RANK ligand, including the transformed T cells or natural killer cells.

In order to achieve the object of the present invention as described above, the present invention provides an antigen binding domain; transmembrane domain; and a chimeric antigen receptor (CAR) comprising an intracellular signaling domain, wherein the antigen binding domain comprises an extracellular domain of RANK that specifically binds to a RANK ligand.

Additionally, the present invention provides a polynucleotide encoding the chimeric antigen receptor protein, a vector containing the polynucleotide, and T cells or natural killer cells transformed with the vector.

In addition, the present invention provides a pharmaceutical composition for treating cancer that kills cancer cells expressing a cell therapy or RANK ligand, including the transformed T cells or natural killer cells.

The chimeric antigen receptor of the invention comprises the extracellular domain of RANK that recognizes RANK ligand. Therefore, cytotoxic T cells or natural killer cells transformed with a vector capable of overexpressing them have specific cytotoxicity against carcinomas expressing RANK ligands, and can be useful as an immune cell therapy for cancer treatment. .

Figure 1 is a schematic diagram showing the cDNA region of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.
Figure 2a is a schematic diagram of a lentiviral vector according to an embodiment of the present invention.
Figure 2b is a schematic diagram of a retroviral vector according to an embodiment of the present invention.
Figure 3 is a schematic diagram of a chimeric antigen receptor expressed on the surface of cytotoxic T cells or natural killer cells according to an embodiment of the present invention.
Figure 4 shows flow cytometric analysis of the expression level of RANK ligand in cancer cells (RANK ligand positive cells) expressing RANK ligand, which are the targets of the chimeric antigen receptor provided in the present invention, and cancer cells that do not express RANK ligand (RANK ligand negative cells). This diagram shows the results of measurement using .
Figure 5a is a diagram showing the results of making the chimeric antigen receptor provided by the present invention water-soluble, expressing it in Chinese hamster ovary (CHO) cells, and then purifying it by affinity chromatography using a protein A column.
Figure 5b shows Western blotting using an antibody that recognizes the constant region of the human IgG heavy chain (anti-human IgG heavy chain constant region antibody, anti-human IgG Fc region antibody) using the soluble chimeric antigen receptor according to an embodiment of the present invention. This diagram shows the results confirmed.
Figure 5c is a diagram showing, using flow cytometry, whether the soluble chimeric antigen receptor according to an embodiment of the present invention recognizes cancer cells (RANK ligand positive cells) expressing RANK ligand, which is an extracellular domain target.
Figure 6a is a diagram showing the results of comparing the expression ratio of GFP in cytotoxic T cells (RANK CAR-T cells) transduced with an expression vector according to an embodiment of the present invention using a lentivirus system.
Figure 6b is a diagram showing the results of comparing the expression ratio of RANK in natural killer cells (RANK CAR-NK cells) transduced with an expression vector according to an embodiment of the present invention using a retroviral system.
Figure 7a shows RANK ligand using a cytotoxic T cell (RANK CAR-T cell) or a cytotoxic T cell (Mock T cell) transduced with an empty vector as an effector cell according to an embodiment of the present invention. This diagram shows the results of measuring cytotoxicity against expressing cancer cells (RANK ligand positive cells).
Figure 7b shows a RANK ligand expressed using natural killer cells (RANK CAR-NK cells) or natural killer cells (Mock NK cells transduced with an empty vector) as effector cells according to an embodiment of the present invention. This diagram shows the results of measuring cytotoxicity against cancer cells (RANK ligand positive cells).

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

In one aspect, the present invention includes an antigen binding domain; transmembrane domain; and a chimeric antigen receptor (CAR) comprising an intracellular signaling domain, wherein the antigen binding domain comprises an extracellular domain of RANK that specifically binds to a RANK ligand.

In the present invention, “Chimeric antigen receptor (CAR)” naturally binds to the desired antigen without the mediation of antigen presenting cells (APC) or antibodies required for activation of T cells or natural killer cells, creating an antigen-antibody reaction. It may refer to a fusion protein for expression in T cells or natural killer cells in order to induce activation of T cells and attack cells expressing the corresponding antigen. In other words, it can be viewed as a protein that binds to antigen when expressed on T cells or natural killer cells and induces the activation of these cells. Through this, it may be a protein that recognizes an antigen specific to the cell to which an immune response is intended, and the cell to which an immune response is to be induced may refer to a cell present in a specific tissue or forming a tissue that has caused a lesion.

In the present invention, “RANK ligand” is an essential element in the production of osteoclasts, but RANK ligand is expressed at abnormally high levels in various carcinomas when normal cells become tumors. To date, carcinomas expressing RANK ligands include chronic lymphocytic leukemia (CLL), multiple myeloma, bone metastasis, osteosarcoma, prostate cancer, or Includes, but is not limited to, non-small cell lung cancer.

Chimeric antigen receptor proteins according to the invention include functional equivalents. 'Functional equivalent' means at least 70%, preferably 80%, more preferably 90%, and even more preferably 90% or more of the amino acid sequence of the chimeric antigen receptor as a result of addition, substitution, or deletion of amino acids. It refers to a protein that has more than 95% sequence homology and exhibits substantially the same physiological activity. ‘Substantially homogeneous physiological activity’ means having the activity to specifically bind to RANK ligand.

The invention also includes fragments, derivatives and analogues of chimeric antigen receptors. As used herein, the terms ‘fragment’, ‘derivative’ and ‘analog’ refer to a polypeptide that possesses substantially the same biological function or activity as the chimeric antigen receptor protein of the present invention. Fragments, derivatives and analogs of the present invention are (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, wherein the substituted amino acid residues are encoded by the genetic code. may or may not be) or (ii) a polypeptide having substituent(s) at one or more amino acid residues, or (iii) another compound (a compound capable of extending the half-life of the polypeptide, e.g. polyethylene glycol) a polypeptide derived from the linked mature polypeptide, or (iv) an additional amino acid sequence (e.g., a leader sequence, a secretory sequence, a sequence used to purify the polypeptide, a proteinogen sequence, or a fusion protein) It may be a polypeptide derived from the bound polypeptide. The fragments, derivatives and analogs defined herein are well known to those skilled in the art.

The antigen binding domain may be in the form of a dimer in which a pair of extracellular domains of RANK are each linked to the human immunoglobulin G ₁ heavy chain constant region (IgG ₁ heavy chain constant region) to amplify signal transduction. It is not limited.

The extracellular domain of RANK is characterized by specifically binding to RANK ligand, and may be composed of SEQ ID NO: 1 or an amino acid sequence showing at least 95% homology thereto, but is not limited thereto.

The human immunoglobulin G ₁ heavy chain constant region (IgG ₁ heavy chain constant region) may be composed of SEQ ID NO: 2 or an amino acid sequence showing at least 95% homology thereto, but is not limited thereto.

The transmembrane domain of the present invention is a region that connects the extracellular domain of RANK and the co-stimulatory and essential signaling domains between the cell membranes, and the intracellular signaling domain is a region that responds to the immune response of immune cells by binding to the antigen-binding domain. refers to the area that activates.

The transmembrane domain, which is a component of the chimeric antigen receptor of the present invention, includes CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. It may include a transmembrane domain of a protein selected from the group consisting of. Preferably, the transmembrane domain may be the transmembrane domain of CD8, which may be composed of SEQ ID NO: 3 or an amino acid sequence showing at least 95% homology thereto, but is not limited thereto.

Additionally, the intracellular signaling domain, which is a component of the chimeric antigen receptor of the present invention, refers to a site that activates the immune response of immune cells by binding to the antigen-binding domain. In one embodiment of the present invention, the intracellular domain may be CD28, 4-1BB, CD3 zeta, or a combination thereof.

The chimeric antigen receptor of the present invention uses CD28, 4-1BB, and CD3 zeta as intracellular signaling domains, so it can exhibit a high activity and killing effect on cancer cells, especially cancer cells expressing RANK ligand.

The CD28 has sequence homology to SEQ ID NO: 4 or at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, and has an amino acid sequence represented by SEQ ID NO: 4 may consist of amino acid sequences that exhibit substantially equivalent functions; 4-1BB (CD137) has sequence homology to SEQ ID NO: 5 or at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, and is represented by SEQ ID NO: 5 It may be composed of an amino acid sequence that exhibits a function substantially equivalent to that of the amino acid sequence; CD3 zeta functions as an NK cell activation domain and has sequence homology to SEQ ID NO: 6 or at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%. As such, it may consist of an amino acid sequence that exhibits a function substantially equivalent to the amino acid sequence shown in SEQ ID NO: 6.

The antigen binding domain may include a signal peptide, and the signal peptide may be encoded by SEQ ID NO: 7 or an amino acid sequence showing at least 95% homology thereto, but is not limited thereto.

In addition, in another aspect according to the present invention, a polynucleotide encoding the chimeric antigen receptor protein is provided.

The polynucleotide encoding the antigen receptor of the present invention changes the amino acid sequence of the antigen receptor expressed from the coding region due to codon degeneracy or in consideration of the preferred codon in the organism in which the antigen receptor is to be expressed. Various modifications may be made to the coding region within the scope of not affecting the coding region, and various modifications or modifications may be made to parts other than the coding region within the scope of not affecting the expression of the gene, and such modified genes are also within the scope of the present invention. Those skilled in the art will be able to understand what is included. That is, as long as the polynucleotide of the present invention encodes a protein with equivalent activity, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included within the scope of the present invention.

In addition, as another aspect according to the present invention, a vector containing the polynucleotide and a cell transformed with the vector are provided.

The vector used in the present invention may be a variety of vectors known in the art, and may include a promoter, terminator, enhancer, etc. depending on the type of host cell intended to produce the antigen receptor. Expression control sequences, sequences for membrane targeting or secretion, etc. can be appropriately selected and combined in various ways depending on the purpose. Vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, etc. Suitable vectors include expression control elements such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and can be prepared in various ways depending on the purpose.

In the present invention, as a preferred embodiment, a lenti-virus vector or a retro-virus vector can be used, and in the following examples of the present invention, pCDH-CMV-MCS-EF1-copGFP vector (lenti-virus vector) pLNCX2 (vector for retro-virus) was used, but is not limited thereto.

Additionally, a chimeric antigen receptor that specifically binds to the RANK ligand of the present invention can be introduced into cells through the vector to transform cells. The cells may be T cells, tumor infiltrating lymphocytes, B cells, NK cells, or NK-T cells, preferably cytotoxic T cells and NK cells. The cells may be obtained or manufactured from bone marrow, peripheral blood, peripheral blood mononuclear cells, or umbilical cord blood, and the cells may be human cells, but are not limited thereto.

As an embodiment of the present invention, a chimeric antigen receptor that specifically binds to RANK ligand can be transformed into cytotoxic T cells or natural killer cells (NK cells) using the vector described above. there is.

As described above, cells transformed by introducing the chimeric antigen receptor of the present invention have the characteristic of recognizing RANK ligand as an antigen and strongly binding to it.

In the present invention, "chimeric antigen receptor T cells (chimeric antigen receptor T cells, hereinafter simply abbreviated as 'CAR-T cells')" or "chimeric antigen receptor NK cells (chimeric antigen receptor NK cells, hereinafter). (abbreviated as 'CAR-NK cell')" refers to a chimeric antigen receptor that reacts specifically to cancer cells rather than the original T cell receptor or NK cell receptor through methods such as transduction of normal T cells or natural killer cells. refers to T cells or NK cells that express . T cells or NK cells having the above receptors exhibit cytotoxicity by inducing apoptosis of target cells.

In the present invention, in particular, CAR-T cells or CAR-NK cells may be cells into which the chimeric antigen receptor of the present invention has been introduced into cytotoxic T cells or NK cells. The cells have the advantage of anti-cancer specific target treatment, which is an existing advantage of CAR-T treatment. In particular, cytotoxic T cells or NK cells equipped with the chimeric antigen receptor of the present invention recognize cancer cells expressing RANK ligand and effectively It can be destroyed.

Therefore, as another aspect of the present invention, a cell therapeutic agent comprising the above cells; A pharmaceutical composition for preventing or treating cancer expressing RANK ligand, comprising the same as an active ingredient; and administering the cells to a subject.

In the present invention, "cell therapy" is a medicine (US FDA regulations) used for treatment, diagnosis, and prevention purposes with cells and tissues manufactured through isolation, culture, and special manipulation from an individual, and restores the function of cells or tissues. It refers to a medicine used for the purposes of treatment, diagnosis, and prevention through a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro or changing the biological characteristics of cells by other methods.

As used herein, the term “treatment” refers to any action in which symptoms caused by cancer expressing RANK ligand are improved or beneficially changed by administration of the composition.

The composition may include a pharmaceutically acceptable carrier.

The term “pharmaceutically acceptable carrier” may mean a carrier or diluent that does not irritate living organisms and does not inhibit the biological activity and properties of the injected compound. The type of carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable can be used. Non-limiting examples of the carrier include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, etc. These may be used individually or in combination of two or more types.

The composition containing a pharmaceutically acceptable carrier may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Specifically, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include the compound with at least one excipient, such as starch, calcium carbonate, sucrose, and lactose. It can be prepared by mixing , gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, Withepsol, Macrogol, Tween 61, cacao, laurin, glycerogelatin, etc. can be used.

The composition can be administered in a pharmaceutically effective amount.

The "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the individual, age, gender, type of infected virus, and the drug's It can be determined based on factors including activity, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the field of medicine.

The administration means introducing the composition of the present invention into the patient by any appropriate method, and the composition may be administered through any general route as long as it can reach the target tissue. It may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, locally, or intranasally, but is not limited thereto.

The composition of the present invention may be administered daily or intermittently, and the number of administrations per day may be once or divided into two to three doses. When both active ingredients are single drugs, the number of administrations may be the same or may be different. Additionally, the composition of the present invention can be used alone or in combination with other drug treatments for the prevention or treatment of blood cancer. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The above-mentioned individuals include humans who have or may develop cancer expressing RANK ligands, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs. This means all animals. If the disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject, the type of subject is included without limitation.

Types of cancer expressing RANK ligand that are targeted for treatment in the present invention include chronic lymphocytic leukemia (CLL), multiple myeloma, bone metastasis of cancer, and osteosarcoma. , prostate cancer, non-small cell lung cancer, etc., but are not limited thereto.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrating the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example 1. By gene synthesis method chimeric antigen receptor gene cloning

To increase the affinity of RANK for the RANK ligand, RANK.E125D+, in which the 125th amino acid, glutamic acid, in the extracellular domain of wild-tpe RANK was replaced with aspartic acid, and the 127th amino acid, cysteine, was replaced with phenylalanine. In order to prepare the chimeric antigen receptor of the present invention using C127F, an antigen recognition site in which each extracellular domain of RANK.E125D+C127F is linked to the human immunoglobulin G ₁ heavy chain constant region (IgG ₁ heavy chain constant region). The protein coding sequences of the transmembrane domain of CD8α, CD28, 4-1BB, and intracellular domain of CD3 zeta were confirmed in a genetic database.

Afterwards, gene synthesis was performed by connecting the remaining sequences excluding the stop codon portion of the base sequences forming the domains. The accuracy of gene synthesis was confirmed through sequencing after producing a protein expression plasmid. A schematic diagram showing the cDNA region of each domain expressing the chimeric antigen receptor is shown in Figure 1.

Example 2. chimeric Preparation of antigen receptor protein expression plasmid

In order to recognize the RANK ligand and express a recombinant protein fused with the human immunoglobulin G ₁ heavy chain constant region and signaling protein domain in cytotoxic T cells and natural killer cells, the gene was transferred to a lentivirus-derived expression vector, pCDH-. It was cloned into CMV-MCS-EF1-copGFP (System Biosciences) or pLNCX2 (Addgene), a retrovirus-derived expression vector.

First, in order to clone into the pCDH-CMV-MCS-EF1-copGFP vector, a primer that creates a restriction enzyme Primers that create the NotI cleavage site sequence were synthesized, and a restriction enzyme cleavage site was created through polymerase chain reaction. Afterwards, the 5' end of the gene with the restriction enzyme cut site was treated with XbaI and the 3' end with NotI through polymerase chain reaction. Then, the multiple cloning site of the expression vector was treated with XbaI and NotI to enable gene insertion. The restriction enzyme-treated gene and the expression vector were mixed and then linked by treatment with a linking enzyme (ligase).

Next, in order to clone into the pLNCX2 vector, a primer was synthesized to create a restriction enzyme Bgl II cleavage site sequence at the 5' end, a restriction enzyme cleavage site was created through polymerase chain reaction, and a restriction enzyme NotI cleavage site sequence was also created at the 3' end. The primers were synthesized and a restriction enzyme cut site was created through polymerase chain reaction. Afterwards, the 5' end of the gene with the restriction enzyme cut site was treated with BglII and the 3' end with NotI through polymerase chain reaction. Then, the multiple cloning site of the expression vector was treated with BglII and NotI to enable gene insertion. The restriction enzyme-treated gene and the expression vector were mixed and then linked by treatment with a linking enzyme (ligase).

As a result, a recombinant vector RANK.E125D+C127F.CAR-T.pCDH-CMV-MCS-EF1- recognizes the RANK ligand and expresses a protein fused with the human immunoglobulin G ₁ heavy chain constant region and signaling protein domain. copGFP and RANK.E125D+C127F.CAR-NK.pLNCX2 were completed (see Figures 2a and 2b).

Example 3. Screening of human cell lines expressing RANK ligand

To screen human cell lines expressing RANK ligand on the cell surface, HL-60 (ATCC), a cell line derived from acute promyelocytic leukemia, and U937 (ATCC), a cell line derived from histiocytic lymphoma, were used. Expression of RANK ligand was confirmed. To confirm expression, cell lines were treated with an antibody (biolegend) capable of binding to the RANK ligand attached to a fluorescent protein, and then flow cytometry (fluorescence-activated cell sorting) was used.

As a result of the analysis, it was confirmed that the HL-60 cell line expressed the RANK ligand, and the U937 cell line was confirmed to not express the RANK ligand (see Figure 4).

Based on the above results, among the two cells, the HL-60 cell line, which expresses RANK ligand, was used as the target cell, and the U937 cell line, which does not express RANK ligand, was used as a negative control.

Example 4. chimeric Affinity measurements of human cell lines expressing antigen receptors and RANK ligands

In order to confirm whether the constructed chimeric antigen receptor has affinity with human cell lines expressing RANK ligand , each extracellular domain of RANK.E125D+C127F was cloned into the human immunoglobulin G ₁ heavy chain constant region (IgG ₁ heavy chain constant region). ), the chimeric antigen receptor was made water-soluble so that two extracellular domains of RANK.E125D+C127F per chimeric antigen receptor recognize the antigen, and expressed in Chinese hamster ovary (CHO) cells, then protein A It was purified by affinity chromatography (GE healthcare) using a column (see Figure 5a).

In addition, the purified soluble chimeric antigen receptor was confirmed by Western blotting using an antibody that recognizes the constant region of the human IgG heavy chain (anti-human IgG heavy chain constant region antibody, anti-human IgG Fc region antibody, abcam) (Figure 5b) reference).

Afterwards, the purified soluble chimeric antigen receptor was treated with a cell line expressing RANK ligand (HL-60 cell line) and a cell line not expressing RANK ligand (U937 cell line) to determine whether the soluble chimeric antigen receptor could bind to the corresponding cells. The results confirmed through flow cytometry are shown in Figure 5c.

As shown in Figure 5c, it was confirmed that HL-60 cells expressing RANK ligand bound to the soluble chimeric antigen receptor, but U937 cells not expressing RANK ligand did not bind to the soluble chimeric antigen receptor.

As confirmed in the above results, it was confirmed that the produced chimeric antigen receptor has high affinity with human cell lines expressing RANK ligand, and the specificity of the RANK ligand protein of the immune cell therapy produced in HL-60 cells and U937 cells was confirmed. This means that it can be used to verify cytotoxicity.

Example 5. Cytotoxic T cell isolation and activation

To produce T cells expressing a chimeric antigen receptor according to an embodiment of the present invention, cytotoxic T cells were first isolated from human peripheral blood mononuclear cells. After purchasing human peripheral blood mononuclear cells (Medilab Korea), magnetic-activated cell sorting was used to isolate cytotoxic T cells. Peripheral blood mononuclear cells were combined with an antibody (CD8 ⁺ T cell biotin-conjugated antibody cocktail, Miltenyi Biotec) that can bind to immune cells other than cytotoxic T cells, and then these antibodies were again combined with magnetic microbeads (anti -biotin microbeads) (Miltenyi Biotec). The microbeads, antibodies attached to them, and cells were passed through a magnetic separation column (Miltenyi Biotec) to obtain cytotoxic T cells that were not labeled with antibodies. To confirm the purity of the isolated cytotoxic T cells, flow cytometry was performed using CD8 and CD3 epsilon, cell surface factors of cytotoxic T cells, and the results showed a purity of over 95%.

For activation of isolated cytotoxic T cells, 1 × ¹⁰⁶ /ml human CD3 and CD28 antibody-coated magnetic beads (Thermo Fisher Scientific) and 100 U/μl recombinant human IL-2 were added to 10% calf. Cells were reconstituted at a density of 1.5×10 ⁶ cells/ml in serum-containing RPMI (Welgene), inoculated into a 24-well cell culture dish, and cultured for 24 hours.

Example 6. chimeric Production of antigen receptor-expressing cytotoxic T cells

To introduce the recombinant vector constructed in Example 2 into cytotoxic T cells, a lentivirus system using 293FT cells (Thermo Fisher Scientific) was used.

First, 293FT cells were inoculated into a 100π cell culture dish at 2.5×10 ⁶ cells and then cultured in DMEM medium containing 10% calf serum. After 24 hours of incubation, when the cells have grown to cover about 60-70% of the dish, 20 μg of RANK.E125D+C127F.CAR-T.pCDH-CMV-MCS-EF1-copGFP vector DNA was added to 10 μg of psPAX2 (Addgene). and 3 μg of pMD2.G (Addgene) vector were crystallized using calcium phosphate and Hepes-buffered solution and then introduced into 293FT cells. Afterwards, 48 hours after the 293FT cells into which the expression vector was introduced, the culture supernatant containing the lentivirus was collected at 24-hour intervals. The collected supernatant was centrifuged in an ultra-high-speed centrifuge at 21,000 rpm for 2 hours to concentrate the virus. The concentrated virus was mixed with 4μg/ml of polybrene and added to the culture medium of activated cytotoxic T cells, and then centrifuged at 1,800g for 75 minutes using a centrifuge to proceed with transduction. After centrifugation, the cytotoxic T cells were cultured for an additional 4 hours and then replaced with RPMI medium containing 10% calf serum. After 48 hours, a portion of the transduced cytotoxic T cells was used to measure transduction efficiency.

Transduction efficiency was measured using flow cytometry to determine the amount of GFP expression inside the cell. Cytotoxic T cells were transduced with a virus produced using the transduced cytotoxic T cells (RANK.E125D+C127F CAR-T cell) as an empty vector. The results compared to the transduction rate of (Mock T cell) are shown in Figure 6a.

Example 7. chimeric Antigen receptor expression natural killer cells produce

To introduce the recombinant vector prepared in Example 2 into NK cells, a retroviral system using 293GPG cells was used.

First, 3 × 10 ⁶ 293GPG cells were dissolved in 10 ml of DMEM culture medium containing 10% calf serum, inoculated into a 100π cell culture dish, and cultured for 24 hours. 20 μg of the previously prepared recombinant vector (RANK.E125D+C127F.CAR-NK.pLNCX2 vector) was crystallized using calcium phosphate and Hepes-buffered solution and then added to the culture medium of the previously cultured 293GPG cells. Afterwards, the culture medium was replaced every 24 hours for 72 hours, and the culture supernatant of 293GPG cells containing the retrovirus was collected and stored. The collected retrovirus-containing supernatant was centrifuged at 21,000 rpm for 2 hours using an ultra-high-speed centrifuge, and then reconstituted in Myelocult H5100 culture medium (STEMCELL) containing 10% calf serum so that it could be concentrated 100 times compared to before concentration. To transduce the concentrated retrovirus into NK92MI cells (American type culture collection, ATCC), NK92MI cells were inoculated into a 24-well cell culture dish at a density of 5×10 ⁵ /ml. Next, a mixture containing 8ug/ml of polybrene added to the concentrated retrovirus was added to the culture medium of NK92MI cells and cultured for 24 hours. After 24 hours, it was replaced with a new culture medium, and NK92MI cells treated with retrovirus were cultured using Myelocult H5100 culture medium supplemented with neomycin antibiotic (600 μg/ml) from 24 hours after replacing the culture medium to select cells into which the gene had been introduced. NK92MI cells that completed the 14-day neomycin selection process were transferred back to fresh culture medium and grown for 1 week.

After proliferation, the level of RANK expression in NK92MI cells was measured using flow cytometry. NK92MI cells (RANK.E125D+C127F CAR-NK cell) into which LAG-3.CAR-NK.pLNCX2 was introduced and NK92MI cells into which pLNCX2, an empty vector, were introduced. The results of comparing the RANK expression level of (Mock CAR-NK cell) using a flow cytometer using an anti-human RANK antibody (Novus biologicals) are shown in Figure 6b.

Example 8. Verification of cell-specific cytotoxicity of CAR-T cells expressing RANK ligand

The chimeric antigen receptor- expressing cytotoxic T cells produced above express RANK ligand. In order to confirm whether cells specifically recognize cells and exhibit toxicity, HL-60 cells and RANK, which are target cells selected in Example 3, were used to determine whether cells expressing RANK ligand ( RANK ligand positive cells) were selectively toxic. U937 cells, a negative control cell that does not express a ligand ( RANK ligand negative cell), were transformed into a chimeric antigen receptor-expressing cytotoxic T cell (RANK.E125D+C127F CAR-T cell) or an empty vector-introduced cytotoxic T cell (Mock). T cell) and co-cultured for 6 hours. A non-radioactive cytotoxicity assay was used to measure the degree of cytotoxicity of cytotoxic T cells to target cells based on the amount of lactate dehydrogenase present in the supernatant after co-culture.

First, add cytotoxic T cells (1x10 ⁵ cells) and target cells (1x10 ⁴ cells) to each well of a 96-well cell culture dish, adjust the effector:target ratio to 10:1, and centrifuge after inoculating the volume to 100 μl per well. Centrifuge for 4 minutes at 250g using a separator to close the gap between cells. After cultivating for 6 hours, 50 μl of the supernatant from each well was removed and transferred to a transparent 96-well plate for absorbance measurement, followed by treatment with the analysis solution and 1M hydrochloric acid solution to proceed and stop the enzyme reaction. After stopping the enzyme reaction, the absorbance in the 490 nm wavelength range was measured and converted to a value using a fluorescence/luminescence/absorbance meter (multi-detection plate reader) to quantify the degree of cytotoxicity of cytotoxic T cells in each well.

As a result, as shown in Figure 7a, the produced chimeric antigen receptor-expressing cytotoxic T cells (RANK.E125D+C127F CAR-T cells) were mixed with U937 cells that do not express RANK ligand ( RANK ligand negative cells) or RANK ligand. When co-cultured with HL-60 cells, which are RANK ligand positive cells, cytotoxicity was about 4.3% against cells that do not express RANK ligand, whereas it was more than 48% against target cell lines expressing MHC class II. It was confirmed that it showed very high cytotoxicity (p<0.01).

On the other hand, in the case of cytotoxic T cells introduced with the control empty vector, it was measured to have no cytotoxicity regardless of the presence or absence of RANK ligand expression.

The above results show that cytotoxic T cells expressing a chimeric antigen recognition receptor containing the RANK extracellular domain show specific cytotoxicity to the RANK ligand protein, making it possible to treat related cancer cells using the CAR-T T cells. was able to confirm.

Example 9.CAR- NK Verification of cell-specific cytotoxicity of RANK ligand expression in cells

In order to confirm whether the chimeric antigen receptor-expressing NK cells produced above specifically recognize RANK ligand-expressing cells and exhibit toxicity, HL-60 cells ( RANK ligand positive cells) and U937 cells, which are target cells selected in Example 3, were used. ( RANK ligand negative cells) were co-cultured with NK cells expressing chimeric antigen receptors (RANK.E125D+C127F CAR-T cells) or NK cells introduced with an empty vector (Mock NK cells).

A non-radioactive cytotoxicity assay was used to measure the degree of cytotoxicity of NK cells against target cells based on the amount of lactate dehydrogenase present in the supernatant after co-culture.

First, add natural killer cells (1x10 ⁵ cells) and target cells (1x10 ⁴ cells) to each well of a 96-well cell culture dish, adjust the effector:target ratio to 10:1, inoculate the volume to 100 μl per well, and then centrifuge. Centrifuge for 4 minutes at 250g to close the gap between cells. After 6 hours of incubation, 50 μl of the supernatant from each well is removed and transferred to a transparent 96-well plate for absorbance measurement, followed by treatment with the analysis solution and 1M hydrochloric acid solution to proceed and stop the enzyme reaction. After stopping the enzyme reaction, the absorbance in the 490 nm wavelength range was measured and converted to a value using a fluorescence/luminescence/absorbance meter (multi-detection plate reader) to quantify the degree of cytotoxicity of NK cells in each well.

As a result, as shown in Figure 7b, the produced chimeric antigen receptor-expressing NK cells (LAG-3 CAR-NK cells) were replaced with U937 cells ( RANK ligand negative cells) that do not express RANK ligand or HL- expressing RANK ligand . When 60 cells ( RANK ligand positive cells) were co-cultured, almost no cytotoxicity was observed for U937 cells, while very high cytotoxicity of more than 47% was observed for HL-60 cells (p<0.01).

On the other hand, NK cells introduced with the empty vector, which is the control group, showed no cytotoxicity against cells that do not express MHC class II and were measured to have no cytotoxicity regardless of whether RANK ligand was expressed or not.

Through the above results, it was confirmed that NK cells expressing a chimeric antigen recognition receptor containing the extracellular domain of RANK showed specific cytotoxicity to the RANK ligand protein, making it possible to treat related cancer cells using the CAR-NK cells. I was able to.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

<110> IMMUNOLOGICAL DESIGNINGLAB CO., LTD <120> Chimeric antigen receptor specifically binding to RANK ligand and use that <130> KP21-0023-ILDL <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 184 <212> PRT <213> Artificial Sequence <220> <223> Extracellular domain of RANK mutant <400> 1 Ile Ala Pro Pro Cys Thr Ser Glu Lys His Tyr Glu His Leu Gly Arg 1 5 10 15 Cys Cys Asn Lys Cys Glu Pro Gly Lys Tyr Met Ser Ser Lys Cys Thr 20 25 30 Thr Thr Ser Asp Ser Val Cys Leu Pro Cys Gly Pro Asp Glu Tyr Leu 35 40 45 Asp Ser Trp Asn Glu Glu Asp Lys Cys Leu Leu His Lys Val Cys Asp 50 55 60 Thr Gly Lys Ala Leu Val Ala Val Val Ala Gly Asn Ser Thr Thr Pro 65 70 75 80 Arg Arg Cys Ala Cys Thr Ala Gly Tyr His Trp Ser Gln Asp Cys Asp 85 90 95 Cys Phe Arg Arg Asn Thr Glu Cys Ala Pro Gly Leu Gly Ala Gln His 100 105 110 Pro Leu Gln Leu Asn Lys Asp Thr Val Cys Lys Pro Cys Leu Ala Gly 115 120 125 Tyr Phe Ser Asp Ala Phe Ser Ser Thr Asp Lys Cys Arg Pro Trp Thr 130 135 140 Asn Cys Thr Phe Leu Gly Lys Arg Val Glu His His Gly Thr Glu Lys 145 150 155 160 Ser Asp Ala Val Cys Ser Ser Ser Leu Pro Ala Arg Lys Pro Pro Asn 165 170 175 Glu Pro His Val Tyr Leu Pro Thr 180 <210> 2 <211> 234 <212> PRT <213> Artificial Sequence <220> <223> IgG1 heavy chain constant region <400> 2 Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 1 5 10 15 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 20 25 30 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 35 40 45 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 50 55 60 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 65 70 75 80 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 85 90 95 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 100 105 110 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 115 120 125 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 130 135 140 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 145 150 155 160 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 165 170 175 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 180 185 190 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 195 200 205 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 210 215 220 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 3 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Human CD8-alpha transmembrane domain <400> 3 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr 20 <210> 4 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Human CD28 Cytoplasmic domain <400> 4 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 5 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Human CD137 Cytoplasmic domain <400> 5 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 6 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Human CD3z Cytoplasmic domain <400> 6 Arg Ala Lys Phe Ser Arg Ser Ala Glu Thr Ala Ala Asn Leu Gln Asp 1 5 10 15 Pro Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Glu Lys Lys Arg Ala Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Gln Gln Arg Arg Arg Asn Pro Gln Glu Gly Val Tyr Asn Ala Leu Gln 50 55 60 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Thr Lys Gly Glu 65 70 75 80 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 85 90 95 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Thr Leu Ala Pro 100 105 110 Arg <210> 7 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Human LAG-3 Signal peptide <400> 7 Met Trp Glu Ala Gln Phe Leu Gly Leu Leu Phe Leu Gln Pro Leu Trp 1 5 10 15 Val Ala Pro Val Lys Pro 20 <210> 8 <211> 552 <212> DNA <213> Artificial Sequence <220> <223> Extracellular domain of RANK mutant <400> 8 atcgctcctc catgtaccag tgagaagcat tatgagcatc tgggacggtg ctgtaacaaa 60 tgtgaaccag gaaagtacat gtcttctaaa tgcactacta cctctgacag tgtatgtctg 120 ccctgtggcc cggatgaata cttggatagc tggaatgaag aagataaatg cttgctgcat 180 aaagtttgtg atacaggcaa ggccctggtg gccgtggtcg ccggcaacag cacgaccccc 240 cggcgctgcg cgtgcacggc tgggtaccac tggagccagg actgcgattg cttccgccgc 300 aacaccgagt gcgcgccggg cctgggcgcc cagcacccgt tgcagctcaa caaggacaca 360 gtgtgcaaac cttgccttgc aggctacttc tctgatgcct tttcctccac ggacaaatgc 420 agaccctgga ccaactgtac cttccttgga aagagagtag aacatcatgg gacagagaaa 480 tccgatgcgg tttgcagttc ttctctgcca gctagaaaac caccaaatga accccatgtt 540 tacttgcccg gt 552 <210> 9 <211> 234 <212> DNA <213> Artificial Sequence <220> <223> IgG1 heavy chain constant region <400> 9 GSEPKSCDKT HTCPPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV 60 KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE 120 KTISKAKGQP REPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT 180 TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK 234 <210> 10 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Human CD8-alpha transmembrane domain <400> 10 atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60 acc 63 <210> 11 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> Human CD28 Cytoplasmic domain <400> 11 aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60 gggccccacc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120 tcc 123 <210> 12 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> Human CD137 Cytoplasmic domain <400> 12 aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60 actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120 gaactg 126 <210> 13 <211> 342 <212> DNA <213> Artificial Sequence <220> <223> Human CD3z Cytoplasmic domain <400> 13 agagcaaaat tcagcaggag tgcagagact gctgccaacc tgcaggaccc caaccagctc 60 tacaatgagc tcaatctagg gcgaagagag gaatatgacg tcttggagaa gaagcgggct 120 cgggatccag agatgggagg caaacagcag aggaggagga acccccagga aggcgtatac 180 aatgcactgc agaaagacaa gatggcagaa gcctacagtg agatcggcac aaaaggcgag 240 aggcggagag gcaaggggca cgatggcctt taccagggtc tcagcactgc caccaaggac 300 acctatgatg ccctgcatat gcagaccctg gcccctcgct aa 342 <210> 14 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> Human LAG-3 Signal peptide <400> 14 atgtgggagg ctcagttcct gggcttgctg tttctgcagc cgctttgggt ggctccagtg 60 aagcct 66

Claims (15)

antigen binding domain;
CD8 transmembrane domain; and
A chimeric antigen receptor (CAR) comprising an intracellular signaling domain consisting of CD28, 4-1BB and CD3-zeta or a combination thereof,
The antigen-binding domain includes an extracellular domain of RANK containing the amino acid sequence represented by SEQ ID NO: 1 that specifically binds to the RANK ligand,
A pair of extracellular domains of RANK are connected to a human immunoglobulin G1 heavy chain constant region (IgG1 heavy chain constant region) each containing the amino acid sequence shown in SEQ ID NO: 2 in the form of a dimer,
The CD8 transmembrane domain includes the amino acid sequence represented by SEQ ID NO: 3,
The CD28 is SEQ ID NO: 4; 4-1BB is SEQ ID NO: 5; and CD3-zeta is a chimeric antigen receptor comprising the amino acid sequence represented by SEQ ID NO: 6. delete delete delete delete delete delete According to paragraph 1,
A chimeric antigen receptor, wherein the antigen-binding domain includes a signal peptide and the amino acid sequence represented by SEQ ID NO: 7. A polynucleotide encoding the chimeric antigen receptor protein of any one of claims 1 or 8. A vector containing the polynucleotide of claim 9. Cells transformed with the vector of claim 10. According to clause 11,
A cell, characterized in that the cell is a cytotoxic T cell or NK cell. A pharmaceutical composition for the treatment of cancer expressing RANK ligand containing the cells of claim 12 as an active ingredient, wherein the cancer includes chronic lymphocytic leukemia (CLL), multiple myeloma, and bone metastasis of cancer. A pharmaceutical composition for the treatment of cancer, which is selected from the group consisting of bone metastasis, osteosarcoma, prostate cancer, and non-small cell lung cancer. delete A cell therapeutic agent comprising the cells of claim 11.