WO2022203227A1 - Transformed antigen-specific professional antigen-presenting cell comprising chimeric antigen receptor (car) and use thereof - Google Patents

Abstract

The present invention relates to a transformed antigen-specific professional antigen-presenting cell comprising a chimeric antigen receptor (CAR), the CAR comprising an antigen binding domain specifically binding to a RANK ligand, and to a cell therapeutics or a pharmaceutical composition for treating cancer, comprising the cell as an active ingredient. The transformed cell according to the present invention provides an enhanced chimeric signaling domain which induces activity of the professional antigen-presenting cell when the professional antigen-presenting cell specifically binds to an antigen. For example, a recombinant protein comprises as subunits an extracellular domain of a RANK mutant (RANK.E125D+C127F) with improved RANK affinity to a RANK ligand, and TLR-3, TLR-4, and immunoreceptor tyrosine-based activation motifs (ITAMs) of IgE receptor gamma chain (Fcε receptor I gamma chain, FcεRIγ), IgG receptor 2A alpha chain (Fcγ receptor 2A alpha chain, FcγR2Aα), or IgE receptor beta chain (Fcε receptor I beta chain, FcεRIβ), which play an important role in signal transduction of professional antigen-presenting cells, wherein three ITAMs are connected via linkers, and the professional antigen-presenting cell transfected with a vector capable of overexpressing the recombinant protein exhibits cytotoxicity specific to cancers expressing the RANK ligand. Therefore, the professional antigen-presenting cell overexpressing the CAR of the present invention may be advantageously utilized as immune cell therapy for treatment of cancers expressing the RANK ligand.

Description

Transformed antigen-specific professional antigen-presenting cells containing chimeric antigen receptor (CAR) and uses thereof

The present invention is a transformed antigen-specific professional antigen-presenting cell comprising a chimeric antigen receptor (CAR), specifically, the chimeric antigen receptor comprises an antigen-binding domain that specifically binds to a RANK ligand It relates to a cell therapeutic agent or a pharmaceutical composition for the treatment of cancer comprising the cell as an active ingredient.

Professional antigen presenting cells include macrophages, dendritic cells, and naive B cells (Makala et al., 2004). When macrophages and dendritic cells recognize antigens, they break down antigens by phagocytosis, and major histocompatibility complex (MHC) antigens expressed in macrophages and dendritic cells transfer the antigenic determinant (epitope) to T cells. (T cell) (Kambayashi and Laufer, 2014). In the case of naive B cells, the antigen is recognized by the B cell receptor (membrane bound IgM), and then the antigen is crushed and expressed in the naïve B cells (major histocompatibility). complex, MHC) antigen delivers epitope to T cells (Kambayashi and Laufer, 2014). Professional antigen-presenting cells also induce naive T cells by transduction of antigenic determinants by major histocompatibility complex (MHC) antigens and co-stimulatory signal transduction into effector T cells ( effector T cells) (Kambayashi and Laufer, 2014). In addition, the specialized antigen-expressing cells secrete various cytokines and chemokines to induce an inflammatory response or activate other immune cells (Kambayashi and Laufer, 2014). Therefore, an effective immune response is initiated by activation of professional antigen-presenting cells, and an effective immune response cannot be induced without activation of professional antigen-presenting cells (Makala et al., 2004).

Recently, a method for treating tumors by inducing the activation of specialized antigen-expressing cells has been devised. That is, it is a method in which tumor-specific antigens are treated with cancer patient-derived specialized antigen-expressing cells, and then, professional antigen-expressing cells activated by tumor-specific antigens are put into the cancer patient's body (van Willigen et al., 2018). . Activated specialized antigen-expressing cells implanted in cancer patients induce the activation of cytotoxic T lymphocytes (CTL) and natural killer cells (NK cells) that directly target tumor cells, thereby causing tumor cells (Galluzzi et al., 2018; Lu et al., 2020).

However, the biggest challenge for such immunotherapy is that tumor-specific antigens have not been discovered so far. Therefore, recently, the single-chain variable fragment (scFv) portion of an antibody that recognizes a protein expressed on the cell surface of cancer has been replaced with CD3 zeta or the cytoplasmic signaling domain of another protein. ) grafted onto the chimeric antigen receptor (CAR) is expressed in cytotoxic T cells and natural killer cells to recognize proteins expressed on the surface of cancer cells and to communicate with cytotoxic T cells by signal transduction. A new anticancer treatment method that induces cancer-specific activity of natural killer cells has been introduced. That is, when the chimeric antigen receptor is grafted onto T cells or natural killer cells, the anticancer action of T cells or natural killer cells only by recognizing specific antigens of scFv regardless of signal transduction by specialized antigen-expressing cells that recognize cancer-specific antigens. In addition, since it is not limited to HLA type, it can be used as a more efficient treatment method that can be used universally by many people. Indeed, these chimeric antigen receptor-expressing T cells or natural killer cells have shown efficacy in several cancers (Holzinger et al., 2016; Pettitt et al., 2018; Wang et al., 2020).

On the other hand, RANK ligand (receptor activator of nuclear factor-kB ligand; RANK ligand; RANKL) is a type 2 membrane protein (membrane protein), and as a member of the tumor necrosis factor (TNF) family, TRANCE (TNF-related) activation-induced cytokine), OPGL (osteoprotegerin ligand), and ODF (osteoclast differentiation factor) are also known and are essential for osteoclastogenesis (Teitelbaum, 2007). That is, mice in which the RANK ligand gene has been removed lack osteoclasts and exhibit traits such as osteopetrosis and tooth eruption disorders. arthritis and osteomyelitis) (Pettit et al., 2001; Rinotas et al., 2014). In addition, RANK ligands are expressed in B lymphocyte tumors, particularly chronic lymphocytic leukemia (CLL) or multiple myeloma, and play a role in exacerbating these diseases (Croucher et al., 2001; Pearse). et al., 2001; Yaccoby et al., 2002; Sordillo et al., 2003; Secchiero et al., 2006). In particular, according to a recent study, expression of RANK ligand was observed in chronic lymphocytic leukemia in all 100% of chronic lymphocytic leukemia patients, and 80% of multiple myeloma patients express 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), non-small cell lung cancer ( Overexpression is observed in non-small cell lung cancer (Peng et al., 2013). In addition, receptor activator of nuclear factor kB (RANK) 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 binding of RANK ligand to RANK is an essential element for osteoclastogenesis (Teitelbaum, 2007; Ono et al., 2020). Recently, in order to increase the affinity of RANK for RANK ligand, a RANK mutant in which glutamic acid, the 125th amino acid of the extracellular domain of wild-tpe RANK, was substituted with aspartic acid, and cysteine, the 127th amino acid, was substituted with phenylalanine. RANK.E125D+C127F was constructed, and RANK.E125D+C127F has an affinity for RANK ligand approximately 4 times higher than that of wild-type RANK (Son et al., 2015).

Currently, immunotherapy using a chimeric antigen receptor-expressing modified mononuclear cell/macrophage (Korean Patent Publication No. 10-2018-0028533) or macrophage chimeric antigen receptor (Korea Patent Publication No. 10-2018-0054600) Ho) has been published, but there is no description of immunotherapy using specialized antigen-expressing cells that enhance the intracellular signaling domain and express a chimeric antigen receptor including the extracellular domain of RANK.

Against this background, the present inventors devised a new treatment method combining the function of a professional antigen-presenting cell and the function of a chimeric antigen receptor. That is, after recognizing a receptor and a ligand that can recognize a tumor cell surface protein as a ligand, a novel chimeric signaling domain that can induce the activity of a specialized antigen-presenting cell by the chimeric antigen receptor was designed, It was demonstrated that professional antigen-presenting cells activated by this chimeric signaling domain can have cytotoxicity against cancer. That is, the extracellular domain of RANK mutant (RANK.E125D+C127F), which has improved RANK affinity for RANK ligand, using the fact that human RANK ligand and RANK can bind, toll-like receptor-2 (toll-like) Transmembrane domain of receptor 2, TLR-2), intracellular domain of TLR-3, intracellular domain of TLR-4, IgE receptor gamma chain (Fcε receptor I gamma chain, FcεRIγ) or IgG receptor 2A alpha chain (Fcγ receptor) 2A alpha chain, FcγR2Aα) or IgE receptor beta chain (Fcε receptor I beta chain, FcεRIβ) immunoreceptor tyrosine-based activation motif (ITAM) as a subunit (subunit), three ITAM linkers A chimeric antigen receptor-expressing macrophage expressing a recombinant protein bound to a novel synthetic intracellular signal transduction domain linked by a linker was constructed. At this time, the extracellular domain of RANK expressed in macrophages recognizes RANK ligands and, when bound to specific cancer cells expressing RANK ligands, can deliver an activation signal to the inside of these macrophages, which in turn activates macrophages. It makes it possible to have cytotoxicity against cancers expressing RANK ligands. In fact, it was confirmed through an in vitro experiment that the macrophages have specific cytotoxicity to cells expressing the RANK ligand.

Accordingly, an object of the present invention is a transformed cell comprising a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain and an intracellular signaling domain that specifically binds to a RANK ligand. Including, wherein the cell is to provide a transformed cell, including a macrophage, dendritic cell or naive B cell (naive B cell) as an antigen-specific professional antigen-expressing cells having a targeted effector activity.

Another object of the present invention is to provide a pharmaceutical composition for the treatment of cancer that kills cancer cells expressing a cell therapeutic agent or RANK ligand comprising the transformed cell as an active ingredient.

In order to achieve the object of the present invention as described above, as a transformed cell comprising the present chimeric antigen receptor (CAR), the chimeric antigen receptor is an antigen binding domain that specifically binds to a RANK ligand, a transmembrane domain and an intracellular signaling domain, wherein the cell comprises a macrophage, a dendritic cell or a naive B cell as an antigen-specific professional antigen-presenting cell having a targeted effector activity. provides

In addition, the present invention provides a pharmaceutical composition for the treatment of cancer that kills cancer cells expressing cell therapeutics or HLA class II, including the transformed cells.

In one embodiment of the present invention, the antigen-binding domain of the chimeric antigen receptor (CAR) may be an extracellular domain of RANK that specifically binds to the RANK ligand. The extracellular domain of the RANK may be wild-type or mutant-type, and in the case of mutant-type, glutamic acid, which is the 125th amino acid, is preferably converted to aspartic acid. (aspartic acid) and may be a form in which the 127th amino acid, cysteine, is substituted with phenylalanine, but is not limited thereto.

In one embodiment of the present invention, the transmembrane domain may be TLR-2.

In one embodiment of the present invention, the intracellular signaling domain is TLR-3, TLR-4, IgE receptor gamma chain (Fcε receptor I gamma chain, FcεRIγ), IgG receptor 2A alpha chain (Fcγ receptor 2A alpha chain, FcγR2Aα), an immunoreceptor tyrosine-based activation motif (ITAM) of the IgE receptor beta chain (Fcε receptor I beta chain, FcεRIβ), or a combination thereof.

Carcinomas expressing the RANK ligand include chronic lymphocytic leukemia (CLL), multiple myeloma, bone metastasis of cancer, osteosarcoma, prostate cancer and non-small form. It may be selected from the group consisting of non-small cell lung cancer.

The transformed cell according to the present invention provides an enhanced chimeric signaling domain that induces the activity of the professional antigen-presenting cell when the professional antigen-presenting cell specifically binds to an antigen. As an example, the extracellular domain of RANK mutant (RANK.E125D+C127F) with improved RANK affinity for RANK ligand and TLR-3, TLR-4, and IgE that play an important role in signal transduction of specialized antigen-presenting cells Immunoreceptor tyrosine-based activation motif (ITAM) of receptor gamma chain (Fcε receptor I gamma chain, FcεRIγ) or IgG receptor 2A alpha chain (Fcγ receptor 2A alpha chain, FcγR2Aα) or IgE receptor beta chain (Fcε receptor I beta chain, FcεRIβ) ) as a subunit, and three ITAMs are linked with a linker as a recombinant protein comprising a new synthetic intracellular signal transduction domain, in the case of a specialized antigen-expressing cell transformed with a vector capable of overexpressing it It is cytotoxic specifically to carcinomas expressing RANK ligands. Therefore, the specialized antigen-expressing cell overexpressing the chimeric antigen receptor according to the present invention can be usefully used as a therapeutic agent for immune cells for the treatment of RANK ligand-expressing carcinoma.

1 is a schematic diagram showing the form of one of the cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.

2 is a schematic diagram showing the form of one of the cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.

3 is a schematic diagram showing the form of one of the cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.

4 is a schematic diagram showing the form of one of the cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.

5 is a schematic diagram showing the form of one of the lentiviral vectors according to an embodiment of the present invention.

6 is a schematic diagram showing the form of one of the lentiviral vectors according to an embodiment of the present invention.

7 is a schematic diagram showing the form of one of the lentiviral vectors according to an embodiment of the present invention.

8 is a schematic diagram illustrating a form of one of lentiviral vectors according to an embodiment of the present invention.

9 is a schematic diagram showing the form of one of the chimeric antigen receptors expressed on the surface of a professional antigen-expressing cell according to an embodiment of the present invention.

10 is a schematic diagram showing the form of one of the chimeric antigen receptors expressed on the surface of a professional antigen-expressing cell according to an embodiment of the present invention.

11 is a schematic diagram showing the form of one of the chimeric antigen receptors expressed on the surface of a professional antigen-expressing cell according to an embodiment of the present invention.

12 is a schematic diagram showing the form of one of the chimeric antigen receptors expressed on the surface of a professional antigen-expressing cell according to an embodiment of the present invention.

13 is flow cytometry analysis of the expression level of RANK ligand in cancer cells expressing RANK ligand (RANK ligand positive cells) and cancer cells not expressing RANK ligand (RANK ligand negative cells), which are targets of the chimeric antigen receptor provided in the present invention. It is a diagram showing the measurement result using .

14A is a diagram showing the results of purification by affinity chromatography using a protein A column after making the chimeric antigen receptor provided in the present invention water-soluble and expressing it in Chinese hamster ovary (CHO) cells.

Figure 14b is a water-soluble chimeric antigen receptor according to an embodiment of the present invention using an antibody recognizing the constant region of a human IgG heavy chain (anti-human IgG heavy chain constant region antibody, anti-human IgG Fc region antibody) Western blotting It is a diagram showing the results confirmed by .

Figure 14c is a diagram showing using flow cytometry whether the water-soluble chimeric antigen receptor according to an embodiment of the present invention recognizes cancer cells (RANK ligand positive cells) expressing RANK ligand, a target of the extracellular domain.

15 is a diagram showing the results of comparing the expression ratio of GFP in professional antigen-expressing cells transduced with the expression vector according to an embodiment of the present invention using a lentiviral system.

16 is a cell for cancer cells expressing RANK ligand (RANK ligand positive cell) using, as an effector cell, a professional antigen-expressing cell transduced with a chimeric antigen receptor or an empty vector according to an embodiment of the present invention. A diagram showing the results of measuring toxicity when 24 hours have elapsed after co-culture.

17 is a cell for cancer cells expressing RANK ligand (RANK ligand positive cell) using, as an effector cell, a professional antigen-expressing cell transduced with a chimeric antigen receptor or an empty vector according to an embodiment of the present invention. A diagram showing the results of measuring toxicity when 48 hours have elapsed after co-culture.

In one aspect, the present invention provides a transformed cell comprising a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor has an antigen binding domain that specifically binds to a RANK ligand, a transmembrane domain and intracellular signaling. It provides a transformed cell comprising a domain, wherein the cell comprises a macrophage, a dendritic cell or a naive B cell as an antigen-specific professional antigen-presenting cell having a targeted effector activity.

The chimeric antigen receptor protein according to an embodiment of the present invention may include a functional equivalent. "Functional equivalent" means at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably the amino acid sequence of the chimeric antigen receptor protein as a result of the addition, substitution, or deletion of amino acids. refers to a protein having a sequence homology of 95% or more and exhibiting substantially homogeneous physiological activity. 'Substantially homogenous physiological activity' means having an activity capable of specifically binding to a RANK ligand.

The present 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 retains 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 include 1) 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) or 2) a polypeptide having substituent(s) at one or more amino acid residues, or 3) maturation associated with another compound (a compound capable of extending the half-life of the polypeptide, such as polyethylene glycol) a polypeptide derived from a polypeptide, or 4) the polypeptide associated with an additional amino acid sequence (eg, 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 fragments, derivatives and analogs as defined herein are well known to those skilled in the art.

In the present invention, the “RANK ligand” is an essential element for osteoclast generation, but the RANK ligand is abnormally high in expression in various carcinomas when normal cells are tumorigenic. To date, carcinomas expressing RANK ligands have been diagnosed with chronic lymphocytic leukemia (CLL), multiple myeloma, bone metastasis of cancer, osteosarcoma, prostate cancer or non-cancerous tumors. non-small cell lung cancer, but is not limited thereto.

In the present invention, "chimeric signaling domain" binds to a desired ligand, induces activation of professional antigen-presenting cells through ligand-receptor reaction, and is expressed in specialized antigen-expressing cells to attack cells expressing the ligand It may mean a fusion protein for In other words, when expressed in specialized antigen-expressing cells, it can be considered as a protein that binds to a ligand and induces activation of these cells. Therefore, the novel chimeric signaling domain provided by the present invention is a novel chimeric signaling domain in a general sense that can induce the activation of professional antigen-presenting cells using all ligand-receptor reactions including all antigen-antibody reactions. to provide.

That is, the chimeric antigen receptor can be regarded as a protein that binds to an antigen and induces activation of these cells when expressed in professional antigen-expressing cells. Through this, it may be a protein recognizing an antigen specific to a cell to cause an immune response, and the cell to cause an immune response may refer to a cell existing in a specific tissue or constituting a tissue causing a lesion.

The antigen binding domain of the chimeric antigen receptor (CAR) may be an extracellular domain of RANK that specifically binds to the RANK ligand. The extracellular domain of the RANK may be wild-type or mutant-type, and in the case of mutant-type, glutamic acid, which is the 125th amino acid, is preferably converted to aspartic acid. (aspartic acid) and may be a form in which the 127th amino acid, cysteine, is substituted with phenylalanine, but is not limited thereto.

The extracellular domain of the RANK may consist of SEQ ID NO: 1 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto.

The antigen-binding domain may be in the form of a dimer in which a pair of extracellular domains of RANK are linked to a human immunoglobulin G1 heavy chain constant region, respectively, in order to amplify signal transduction, but is limited thereto not.

The human immunoglobulin G1 heavy chain constant region (IgG1 heavy chain constant region) may consist of SEQ ID NO: 2 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto.

The intracellular signaling domain, which is a component of the chimeric antigen receptor of the present invention, refers to a site that activates an immune response of an immune cell by binding to an antigen-binding domain. The signaling domain may be TLR-3, TLR-4, tyrosine-based activation motif (ITAM), or a combination thereof. In a preferred embodiment, the signaling domain may be in the form of connecting ITAM as a subunit to the structure of TLR-3-TLR-4 with a linker, wherein one or more ITAMs are linked may be, and preferably may be in the form of three connected, but is not limited thereto.

The ITAM is based on immunoreceptor tyrosine of IgE receptor gamma chain (Fcε receptor I gamma chain, FcεRIγ), IgG receptor 2A alpha chain (Fcγ receptor 2A alpha chain, FcγR2Aα) or IgE receptor beta chain (Fcε receptor I beta chain, FcεRIβ) It may be an activation motif, but is not limited thereto.

The TLR-3 is SEQ ID NO: 3 or more than 70%, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more sequence homology thereto, and the amino acid represented by SEQ ID NO: 3 may consist of an amino acid sequence exhibiting a function substantially equivalent to the sequence; The TLR-4 is SEQ ID NO: 4 or more than 70%, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more sequence homology thereto, and the amino acid represented by SEQ ID NO: 4 may consist of an amino acid sequence exhibiting a function substantially equivalent to the sequence; The ITAM is SEQ ID NO: 5; SEQ ID NO: 6; Alternatively, it may consist of an amino acid sequence exhibiting a function substantially equivalent to the amino acid sequence represented by SEQ ID NO: 7.

The linker may consist of an amino acid sequence having a function substantially equivalent to that of the amino acid sequence represented by SEQ ID NO: 8.

The transmembrane domain of the present invention is a site that connects the extracellular domain of RANK and the costimulatory and essential signaling domains between the cell membrane, and the intracellular signaling domain is an immune response of immune cells by binding of the antigen-binding domain. site that activates

One component of the transmembrane domain of the chimeric antigen receptor of the present invention is CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and a transmembrane domain of a protein selected from the group consisting of TLR-2. Preferably, the transmembrane domain may be TLR-2, which may consist of SEQ ID NO: 9 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto.

The antigen-binding domain may include a signal peptide, and the signal peptide may be a TLR-4 signal peptide, and may consist of SEQ ID NO: 10 or an amino acid sequence showing 95% or more homology thereto, but is limited thereto not.

The vector used in the present invention may use a variety of vectors known in the art, and may include a promoter, a terminator, an enhancer, etc., depending on the type of host cell to produce the antigen receptor. Expression control sequences, sequences for membrane targeting or secretion, etc. can be appropriately selected and variously combined according to the purpose. The vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector. Suitable vectors include a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as promoter, operator, start codon, stop codon, polyadenylation signal and enhancer, and may be prepared in various ways depending on the purpose.

In the present invention, as a preferred example, a lenti-virus vector can be used, and in the following examples of the present invention, pCDH-CMV-MCS-EF1-copGFP vector (lenti-virus vector) was used ( FIGS. 5 to 5 ). 8).

In addition, a cell can be transformed by introducing a chimeric antigen receptor that specifically binds to the RANK ligand of the present invention into the cell through the vector. The cells may be cytotoxic T cells and NK cells, tumor infiltrating lymphocytes, B cells, NK cells, or NKT cells. It may be a naive B cell. In some embodiments, the cells may be obtained or prepared from bone marrow, peripheral blood, peripheral blood mononuclear cells or umbilical cord blood. In some embodiments, the cell is a human cell.

As an embodiment of the present invention, a chimeric antigen receptor that specifically binds to a RANK ligand can be transformed into a specialized antigen-expressing cell using the vector described above.

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

In the present invention, "chimeric antigen receptor professional antigen presenting cell (hereinafter abbreviated as 'CAR-pAPC cell')" is a method such as transduction of normal professional antigen presenting cells It refers to a specialized antigen-presenting cell expressing a chimeric antigen receptor that specifically responds to cancer cells rather than the original specialized antigen-expressing cell surface receptor. Professional antigen-expressing cells with this receptor induce apoptosis of target cells and exhibit cytotoxicity.

In the present invention, in particular, the CAR-pAPC cell may be a cell in which the chimeric antigen receptor of the present invention is introduced into a professional antigen-expressing cell. The cells have the advantage of anticancer-specific targeted therapy, which is the existing advantage of CAR-T therapeutics. In particular, the specialized antigen-expressing cells equipped with the chimeric antigen receptor of the present invention can recognize and effectively destroy cancer cells expressing RANK ligand. have.

Therefore, another aspect of the present invention is a cell therapy agent comprising the cell; A pharmaceutical composition for the treatment of cancer expressing a RANK ligand comprising it as an active ingredient; and administering the cells to a subject.

In the present invention, the cell, the cell may be a cytotoxic T cell (cytotoxic T cell) and NK cells, tumor infiltrating lymphocytes, B cells, NK cells, or NK-T cells, preferably, specialized antigen expression The cells may be macrophages, dendritic cells, or naive B cells. Or progenitor cells, for example, hematopoietic stem cells, mesenchymal stromal cells, stem cells, pluripotent stem cells, and may be embryonic stem cells, which may be used in cell therapy such as anticancer therapy. The cells may come from a donor, or they may be cells obtained from a patient. Cells can be used, for example, in regeneration, replacing the function of diseased cells. Cells can also be modified to express heterologous genes so that biological agents can be delivered to specific microenvironments, such as, for example, diseased bone marrow or metastatic deposits.

In the present invention, "cell therapeutic" refers to cells and tissues manufactured through isolation, culture, and special manipulation from an individual, and is a drug (US FDA regulations) used for the purpose of treatment, diagnosis, and prevention, restoring the function of cells or tissues. It refers to a drug used for the purpose 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 properties of cells in other ways.

As used herein, the term “prevention” refers to any action of inhibiting or delaying the development of a cancer expressing RANK ligand by administration of the composition.

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

The composition may include a pharmaceutically acceptable carrier.

The "pharmaceutically acceptable carrier" may mean a carrier or diluent that does not inhibit the biological activity and properties of the injected compound without irritating the organism. The type of carrier usable in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable may be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or in combination of two or more.

The composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.

Specifically, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient to the compound, for example, starch, calcium carbonate, sucrose, lactose. , gelatin, etc. may be mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, etc. may be used.

The composition may 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 dependent on the subject's type and severity, age, sex, infected virus type, and drug. Activity, sensitivity to drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs and other factors well known in the medical field.

The administration means introducing the composition of the present invention to the patient by any suitable method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, may be administered 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 administered once or divided into two to three times. When the two active ingredients are single drugs, the number of administrations may be the same or different. In addition, the composition of the present invention may be used alone or in combination with other drug treatments for the prevention or treatment of hematologic cancer. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.

The subject includes humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs that have or can develop cancer expressing RANK ligands. 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.

In the present invention, as the type of cancer expressing the RANK ligand to be treated, carcinoma is chronic lymphocytic leukemia (CLL), multiple myeloma, bone metastasis of cancer, osteosarcoma ( osteosarcoma), prostate cancer, or non-small cell lung cancer, but is not limited thereto.

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. .

Example 1. Cloning of a gene by a gene synthesis method

In order to prepare the chimeric antigen receptor of the present invention, four types of chimeric antigen receptor proteins have sequences of an extracellular domain of RANK and a transmembrane domain of TLR-2, and have sequences encoding different intracellular signal transduction domains. The coding sequence was synthesized. The chimeric antigen receptor is an antigen recognition site linking _a pair of extracellular domains of RANK to a human immunoglobulin G ₁ heavy chain constant region, respectively, a transmembrane domain of TLR-2, a TLR-4 signal peptide. includes in common. Each of the signaling domains was constructed in four different configurations. Table 1 below describes the configuration of the four types of signaling domains.

division	Signaling domain configuration
One	TLR-3 / TLR-4 / linker / FcεR1 γ chain ITAM motif / linker / FcεR1 γ chain ITAM motif / linker / FcεR1 γ chain ITAM motif
2	TLR-3 / TLR-4 / linker / FcγR2A α chain ITAM motif / linker / FcγR2A α chain ITAM motif / linker / FcγR2A α chain ITAM motif
3	TLR-3 / TLR-4 / linker/ FcεR1 β chain ITAM motif/ linker / FcεR1 β chain ITAM motif / linker / FcεR1 β chain ITAM motif
4	TLR-3 / TLR-4 / linker / FcγR2A α chain ITAM motif / linker / FcεR1 β chain ITAM motif / linker / FcεR1 γ chain ITAM motif

Schematic diagrams of cDNAs of each domain expressing the chimeric antigen receptor constructed above are shown in FIGS. 1 to 4 . The protein coding sequence of each domain part was confirmed in a gene database, and the accuracy of gene synthesis was confirmed through sequencing after a protein expression plasmid was prepared.

Example 2. Preparation of protein expression plasmids

In order to express in macrophages the recombinant protein obtained by fusion of the RANK extracellular domain of the four chimeric antigen receptors prepared in Example 1 and the macrophage signaling protein, the gene was transferred to the lentivirus-derived expression vector pCDH- CMV-MCS-EF1-copGFP (System Biosciences) was cloned using restriction enzymes Xba I and Not I cleavage site.

As a result, a recombinant vector expressing four proteins in which the extracellular domain of RANK having the structure of FIGS. 9 to 12 and a novel signal transduction protein were fused was completed (see FIGS. 5 to 8 ).

Example 3. Screening of human cell lines expressing RANK ligand

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

As a result of the analysis, it was confirmed that the RANK ligand was expressed in the HL-60 cell line, and the RANK ligand was not expressed in the U937 cell line (see FIG. 13 ).

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

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

In order to confirm that the constructed chimeric antigen receptor has affinity with a human cell line expressing the RANK ligand, each of the extracellular domains of RANK.E125D+C127F is human immunoglobulin G ₁ heavy chain constant region (IgG ₁ heavy chain constant region), two extracellular domains of RANK.E125D+C127F per chimeric antigen receptor made the antigen-recognizing chimeric antigen receptor water-soluble and expressed in Chinese hamster ovary (CHO) cells. It was purified by affinity chromatography (GE healthcare) using column A (see FIG. 14a ).

In addition, the purified water-soluble chimeric antigen receptor was confirmed by Western blotting using an antibody recognizing the constant region of human IgG heavy chain (anti-human IgG heavy chain constant region antibody, anti-human IgG Fc region antibody, abcam) (Fig. 14b). Reference).

Thereafter, the purified water-soluble chimeric antigen receptor is 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 water-soluble chimeric antigen receptor can bind to the cell. The results confirmed through flow cytometry are shown in FIG. 14C .

As shown in FIG. 14c , it was confirmed that the HL-60 cells expressing the RANK ligand bound to the water-soluble chimeric antigen receptor, but the U937 cells not expressing the RANK ligand did not bind to the water-soluble chimeric antigen receptor.

As confirmed from the above results, it was confirmed that the prepared chimeric antigen receptor had high affinity with human cell lines expressing RANK ligand, and chimeric antigen receptor-expressing specialized antigen-expressing cells prepared from HL-60 and U937 cells. This means that it can be used for specific cytotoxicity verification of RANK ligand proteins.

Example 5. Production of specialized antigen-expressing cells expressing chimeric antigen receptors

Lentivirus using 293FT cells (Thermo Fisher Scientific) to introduce the four recombinant chimeric antigen receptor expression vectors prepared in Example 2 into THP-1 cells (human macrophage cell line), a type of specialized antigen-expressing cells. system was used.

First, 293FT cells were inoculated to become 2.5Х10 ⁶ cells in a 100π cell culture dish, 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 each of the four vector DNAs of Example 2 is aliquoted, 10 µg of psPAX2 (Addgene) and 3 µg of pMD2.G ( Addgene) vector and calcium phosphate were crystallized using calcium phosphate and Hepes-buffered solution, and then introduced into 293FT cells. Then, the culture supernatant containing the virus (lentivirus) was collected at intervals of 24 hours after 48 hours of the 293FT cells introduced with the expression vector. The collected supernatant was centrifuged at 21,000 rpm in an ultra-high speed centrifuge for 2 hours to concentrate the virus. The concentrated virus was mixed with 4 μg/ml of polybrene and added to the culture medium of THP-1 cells, a macrophage cell line, for transduction. The efficiency of transduction was increased by changing the culture medium of THP-1 cells to the culture medium in which the concentrated virus was added twice at 24 hour intervals. 24 hours after the last culture medium change, a portion of the transduced THP-1 cells was used to measure the transduction efficiency. Transduction efficiency was measured using flow cytometry for the expression level of GFP inside the cells, and THP-1 cells transduced with each of the four recombinant chimeric antigen receptor expression vectors, and THP- cells transduced with an empty vector (Mock) The results compared with the transduction rate of 1 cell are shown in FIG. 15 .

Example 6. Cells Expressing RANK Ligand (RANK ligand RANK ligand positive cell-specific cytotoxicity verification through co-culture with positive cells)

The target cells selected in Example 3 were selected in Example 3 to confirm that the four prepared chimeric antigen receptor-expressing THP-1 cells selectively recognize RANK ligand-expressing cells (RANK ligand positive cells, RANKL ⁺ cells) and show toxicity. HL-60 cells were co-cultured with 4 types of chimeric antigen receptor-expressing THP-1 cells or THP-1 cells (Mock) introduced with an empty vector. After co-culture, treatment with 7-Aminoactinomycin D (7-AAD) was performed 24 or 48 hours later, and the cytotoxicity of each chimeric antigen receptor-expressing THP-1 cell was measured by comparing the fluorescence expression level of 7-AAD by flow cytometry. did. 7-Aminoactinomycin D (7-AAD) binds to the DNA of the cell in which apoptosis is induced and becomes fluorescent (Zembruski et al., 2012).

First, each of the 4 types of chimeric antigen receptor-expressing THP-1 cells was dispensed at 5Х10 ⁵ into the wells of a 24-well cell culture dish, and RANKL ⁺ cells (HL-60), the target cells, were dispensed by 1Х10 ⁵ each to effector: target ratio. Co-culture was carried out at a ratio of 5:1. The volume per well of the co-culture was proceeded to be 1 ml, and centrifuged at 250 g for 4 minutes using a centrifuge to make the intercellular space close. After co-culture for 24 hours or 48 hours, the cells in each well were centrifuged at 300 g for 3 minutes using a centrifuge to remove the supernatant, and then the cells were stained with 7-AAD (Biolegend). Thereafter, the cytotoxicity of four chimeric antigen receptor-expressing THP-1 cells prepared by measuring the proportion of cells exhibiting fluorescence by 7-AAD using flow cytometry was quantified.

As a result, as shown in FIG. 16 , when the prepared chimeric antigen receptor-expressing THP-1 cells were co-cultured with RANKL ⁺ cells (HL-60), 24 hours passed, THP- that did not express the chimeric antigen receptor 1 cell (Mock) showed cytotoxicity of about 14.7%, whereas 3-4-3 THP-1 cells (TLR-3+TLR-4+FcεRIγ ITAM trimer), which are THP-1 cells expressing 4 types of chimeric antigen receptors ), 3-4-2A THP-1 cells (TLR-3+TLR4+FcγR2Aα ITAM trimer), 3-4-1B THP-1 cells (TLR-3+TLR4+FcεRIβ ITAM trimer), 3-4-ABC THP -1 cells (TLR-3+TLR4+FcγR2A ITAM monomer+FcεRIβ ITAM monomer+FcγR2Aα ITAM monomer) showed very high cytotoxicity of 28.3%, 26.7%, 22.9%, and 21.6%, respectively.

In addition, as shown in FIG. 17 , when the cytotoxicity of HL-60 cells co-cultured 48 hours after co-culture was checked, Mock showed about 17.8% cytotoxicity, while 4 types of height Merrick antigen receptor expressing THP-1 cells, 3-4-3 THP-1 cells (TLR-3+TLR-4+FcεRIγ ITAM trimer), 3-4-2A THP-1 cells (TLR-3+TLR4+FcγR2Aα ITAM) trimer), 3-4-1B THP-1 cells (TLR-3+TLR4+FcεRIβ ITAM trimer), 3-4-ABC THP-1 cells (TLR-3+TLR4+FcγR2A ITAM monomer+FcεRIβ ITAM monomer+FcγR2Aα ITAM) monomer) showed very high cytotoxicity of 55.3%, 50.3%, 46.5%, and 53.8%, respectively. Through this, it was confirmed that the prepared chimeric antigen recognition receptor-expressing THP-1 cells showed specific cytotoxicity to the RANK ligand protein.

Through the above results, the transformed THP-1 cells containing the chimeric antigen receptor according to the present invention showed cytotoxicity specific to the RANK ligand protein. It was confirmed that cell therapy was possible.

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill 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 are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (17)

1. A transformed cell comprising a chimeric antigen receptor (CAR), comprising:

   The chimeric antigen receptor includes an antigen binding domain, a transmembrane domain and an intracellular signaling domain that specifically binds to a RANK ligand, and the cell is an antigen-specific professional antigen-presenting cell having a targeted effector activity, and is a macrophage. , Transformed cells, including dendritic cells or naive B cells.
2. According to claim 1,

   The antigen-binding domain is an extracellular domain of RANK that specifically binds to a RANK ligand, the transformed cell.
3. 3. The method of claim 2,

   The extracellular domain of the RANK is a mutant-type in which the 125th amino acid, glutamic acid, is substituted with aspartic acid, and the 127th amino acid, cysteine, is substituted with phenylalanine. which is a transformed cell.
4. 3. The method of claim 2,

   The extracellular domain of the RANK will include the amino acid sequence shown in SEQ ID NO: 1, the transformed cell.
5. 3. The method of claim 2,

   The antigen-binding domain is a pair of extracellular domains of RANK in the form of a dimer, respectively, human immunoglobulin G ₁ heavy chain constant region (IgG ₁ heavy chain constant region) Transformed cells, characterized in that connected to.
6. 6. The method of claim 5,

   Human immunoglobulin G ₁ heavy chain constant region (IgG ₁ heavy chain constant region) is a transformed cell comprising the amino acid sequence represented by SEQ ID NO: 2.
7. According to claim 1,

   The intracellular signaling domain is linked to the structure of TLR-3-TLR-4 with an immunoreceptor tyrosine-based activation motif (ITAM) as a subunit and linked by a linker. transformed cells.
8. 8. The method of claim 7,

   The ITAM is based on immunoreceptor tyrosine of IgE receptor gamma chain (Fcε receptor I gamma chain, FcεRIγ), IgG receptor 2A alpha chain (Fcγ receptor 2A alpha chain, FcγR2Aα) or IgE receptor beta chain (Fcε receptor I beta chain, FcεRIβ) An activation motif, the transformed cell.
9. 8. The method of claim 7,

   The ITAM is a transformed cell that connects three with a linker.
10. 8. The method of claim 7,

    The TLR-3 is SEQ ID NO: 3; And TLR-4 is a transformed cell comprising the amino acid sequence shown in SEQ ID NO: 4.
11. 8. The method of claim 7,

    The ITAM is SEQ ID NO: 5; SEQ ID NO: 6; Or comprising the amino acid sequence represented by SEQ ID NO: 7, the transformed cell.
12. 8. The method of claim 7,

    Wherein the linker comprises the amino acid sequence represented by SEQ ID NO: 8, the transformed cell.
13. The method of claim 1,

    The transmembrane domain is TLR-2, and the transformed cell comprising the amino acid sequence shown in SEQ ID NO: 9.
14. The method of claim 1,

    The chimeric antigen receptor comprises a signal peptide, the transformed cell comprising the amino acid sequence shown in SEQ ID NO: 10.
15. A pharmaceutical composition for the treatment of cancer expressing a RANK ligand comprising the cell of any one of claims 1 to 14 as an active ingredient.
16. 16. The method of claim 15,

    The cancer is chronic lymphocytic leukemia (CLL), multiple myeloma, bone metastasis of cancer, osteosarcoma, prostate cancer and non-small cell lung cancer (non-small cell lung cancer). A composition selected from the group consisting of small cell lung cancer).
17. A cell therapeutic agent comprising the cell of any one of claims 1 to 14 as an active ingredient.

Images