KR20230089464A - Transformed professional antigen presenting cells specifically binding to antigen containing chimeric antigen receptor(CAR) and uses thereof - Google Patents

Abstract

The present invention relates to a transformed antigen-specific professional antigen-expressing cell containing a chimeric antigen receptor (CAR), and specifically, the chimeric antigen receptor contains an antigen-binding domain that specifically binds to CD138. It relates to a pharmaceutical composition for the treatment of cell therapy or cancer comprising as an active ingredient.
Transformed cells according to the present invention provide an enhanced chimeric signaling domain that induces the activity of professional antigen-presenting cells when they specifically bind to an antigen. As an example of this, a dimer in which a pair of DR6 extracellular domains are linked to the human immunoglobulin G ₁ heavy chain constant region, respectively _, plays an important role in signal transduction of professional antigen-expressing cells. TLR-3, TLR-4 and 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) ) as a subunit of the immunoreceptor tyrosine-based activation motif (ITAM), and a novel synthetic intracellular signaling domain in which three ITAMs are connected by a linker. As a recombinant protein, , professional antigen-expressing cells transformed with a vector capable of overexpressing it have cytotoxicity specifically to carcinomas expressing CD138. Therefore, the professional antigen-expressing cells overexpressing the chimeric antigen receptor according to the present invention can be usefully used as an immune cell therapeutic agent for the treatment of CD138-expressing carcinoma.

Description

Transformed professional antigen presenting cells specifically binding to antigen containing chimeric antigen receptor (CAR) and uses thereof}

The present invention relates to a transformed antigen-specific professional antigen-expressing cell containing a chimeric antigen receptor (CAR), and specifically, the chimeric antigen receptor contains an antigen-binding domain that specifically binds to CD138. It relates to a pharmaceutical composition for the treatment of cell therapy or cancer comprising 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 the antigens into small pieces by phagocytosis, and the major histocompatibility complex (MHC) antigens expressed in macrophages and dendritic cells transfer antigenic determinants (epitopes) to T cells. (T cell) (Kambayashi and Laufer, 2014). In the case of naive B cells, antigens are recognized by the B cell receptor (membrane bound IgM), and then major histocompatibility (major histocompatibility), which is expressed in naive B cells by crushing the antigen into small pieces Complex, MHC) antigen delivers epitopes to T cells (Kambayashi and Laufer, 2014). Professional antigen-expressing cells also convert naïve T cells into effector T cells by delivery of antigenic determinants by major histocompatibility complex (MHC) antigens and transmission of co-stimulatory signals. effector T cells) (Kambayashi and Laufer, 2014). In addition, professional antigen-expressing cells secrete various cytokines and chemokines to induce inflammatory responses or activate other immune cells (Kambayashi and Laufer, 2014). Therefore, an effective immune response is initiated by activation of professional antigen-expressing cells, and an effective immune response is not induced without activation of professional antigen-presenting cells (Makala et al., 2004).

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

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

CD138, also called syndecan-1, is a type I transmembrane protein consisting of 288 amino acids and is known to be involved in the suppression of inflammatory responses by binding to ligands (Rops et al., 2007; Teng et al. , 2012). In other words, since it has been reported that when CD138 is deficient, lymphocyte activity is increased or antibody production is increased, resulting in a poor prognosis of autoimmune diseases, CD138 appears to play an immunosuppressive role in certain immune diseases, although the exact mechanism is not yet known. known (Xu et al., 2005; Zhang et al., 2013). CD138 is known to be expressed in various epithelial tissues and immune cells, and its expression is increased in some carcinomas, so it is used as a molecular marker or therapeutic target for carcinomas (Czarnowski, 2021). Carcinomas expressing CD138 reported so far include multiple myeloma (Dhodapkar et al., 1998), breast cancer (Malek-Hosseini et al., 2017), and pancreatic cancer (Conejo et al. ., 2000), lung cancer (Anttonen et al., 2001), hepatocellular carcinoma (Regφs et al., 2020), bladder cancer (Kim et al., 2014), ovarian cancer ( ovarian cancer) (Davies et al., 2004), prostate cancer (Szarvas et al., 2016), and colorectal cancer (Wei et al., 2015).

Death receptor 6 (DR6), also called tumor necrosis factor (TNF) receptor superfamily 21 (TNFRSF21), is a transmembrane protein composed of 392 amino acids. When a signal is received, it enters the cytoplasmic tail. It has a death domain that induces apoptosis (Pan et al., 1998). The currently known function of DR6 is known to be involved in the prognosis of several immune diseases by regulating T lymphocyte activity (Liu et al., 2001; Schmidt et al., 2005).

Currently, modified monocytes/macrophages expressing chimeric antigen receptors (Korean Patent Publication No. 10-2018-0028533) or CAR-T cells using chimeric antigen receptors (Korean Patent Publication No. 10-2017-0090506) Alternatively, although there are literatures on anticancer therapy using CAR-NK cells, immunocancer therapy using specialized antigen-expressing cells expressing chimeric antigen receptors including the extracellular domain of DR6 by enhancing the intracellular signaling domain has been described. does not exist.

Under this background, the present inventors devised a new treatment method combining the functions of professional antigen-expressing cells and chimeric antigen receptors. That is, after recognizing a receptor capable of recognizing a tumor cell surface protein as a ligand and a ligand, a novel chimeric signaling domain capable of inducing the activity of professional antigen-expressing cells by the chimeric antigen receptor was devised, It was demonstrated that professional antigen-expressing cells activated by these chimeric signaling domains can have cytotoxic ability against cancer. That is, using the fact that human CD138 and DR6 can bind, the extracellular domain of DR6, the transmembrane domain of toll-like receptor 2 (TLR-2), and the 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, expressing a recombinant protein combined with a novel synthetic intracellular signaling domain in which three ITAMs are linked by a linker A chimeric antigen receptor-expressing macrophage was constructed. At this time, the extracellular domain of DR6 expressed in macrophages recognizes CD138 and, when combined with specific cancer cells expressing CD138, can transmit activation signals into these macrophages, and eventually these signals activate macrophages to release CD138. It can have cytotoxicity against expressing cancer. In fact, it was confirmed through in vitro experiments that the macrophages have a cytotoxic ability specifically to cells expressing CD138.

Korean Patent Publication No. 10-2018-0028533 (2018. 03. 16) Korean Patent Publication Korean Patent Publication No. 10-2017-0090506 (2017. 08. 07)

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 that specifically binds to CD138, a transmembrane domain and an intracellular signaling domain. The cell is an antigen-specific professional antigen-expressing cell having a targeted effector activity, and is to provide a transformed cell including macrophages, dendritic cells or naive B cells.

Another object of the present invention is to provide a cell therapy or a pharmaceutical composition for treating cancer that kills cancer cells expressing CD138 containing the transformed cells as an active ingredient.

In order to achieve the object of the present invention as described above, as a transformed cell containing the chimeric antigen receptor (CAR), the chimeric antigen receptor is an antigen-binding domain that specifically binds to CD138, a transmembrane domain and An intracellular signaling domain, wherein the cells are antigen-specific specialized antigen-expressing cells having targeted effector activity, including macrophages, dendritic cells, or naive B cells, transformed cells to provide.

In addition, the present invention provides a cell therapy or a pharmaceutical composition for treating cancer that kills cancer cells expressing CD138, 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 death receptor 6 (DR6) that specifically binds to the CD138.

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), immunoreceptor tyrosine-based activation motif (ITAM) of IgE receptor beta chain (Fcε receptor I beta chain, FcεRIβ), or a combination thereof.

Carcinomas expressing the CD138 include multiple myeloma, breast cancer, pancreatic cancer, lung cancer, hepatocellular carcinoma, bladder cancer, and ovarian cancer. , prostate cancer and colorectal cancer.

Transformed cells according to the present invention provide an enhanced chimeric signaling domain that induces the activity of professional antigen-presenting cells when they specifically bind to an antigen. As an example of this, TLR-3, TLR-4 and IgE receptor gamma chain (Fcε receptor I gamma chain, FcεRIγ) or IgG receptor 2A alpha chain, which play an important role in signal transduction of the extracellular domain of DR6 and professional antigen-expressing cells (Fcγ receptor 2A alpha chain, FcγR2Aα) or immunoreceptor tyrosine-based activation motif (ITAM) of IgE receptor beta chain (Fcε receptor I beta chain, FcεRIβ) as a subunit, and 3 ITAMs as a linker As a recombinant protein comprising a newly synthesized intracellular signal transduction domain linked thereto, professional antigen-expressing cells transformed with a vector capable of overexpressing it have cytotoxicity specifically to carcinoma expressing CD138. Therefore, the professional antigen-expressing cells overexpressing the chimeric antigen receptor according to the present invention can be usefully used as an immune cell therapeutic agent for the treatment of CD138-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.
Figure 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.
Figure 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.
Figure 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 shape of one of lentiviral vectors according to an embodiment of the present invention.
6 is a schematic diagram showing the shape of one of lentiviral vectors according to an embodiment of the present invention.
7 is a schematic diagram showing the shape of one of lentiviral vectors according to an embodiment of the present invention.
8 is a schematic diagram showing the shape 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 professional antigen-expressing cells 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 professional antigen-expressing cells 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 professional antigen-expressing cells 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 professional antigen-expressing cells according to an embodiment of the present invention.
13 is a diagram showing the results of measuring the expression level of CD138 using flow cytometry in the AsPC-1 cell line, which is a cancer cell (CD138 positive cell) expressing CD138, which is a target of the chimeric antigen receptor provided in the present invention.
14 is a schematic diagram showing cDNA regions of each domain expressing a recombinant DR6 fusion protein prepared to measure whether DR6 recognizes CD138 according to an embodiment of the present invention.
15 is a schematic diagram of an expression vector (retroviral vector) expressing a recombinant DR6 fusion protein prepared to measure whether DR6 recognizes CD138 according to an embodiment of the present invention.
16 is a schematic diagram of a recombinant DR6 fusion protein prepared to measure whether DR6 recognizes CD138 according to an embodiment of the present invention.
17a is a diagram showing the result of expressing the prepared recombinant DR6 fusion protein in Chinese hamster ovary (CHO) cells and then purifying it by affinity chromatography using a protein A column.
Figure 17b is a diagram showing the result of Western blotting using an antibody (anti-human IgG heavy chain constant region antibody, anti-human IgG Fc region antibody) recognizing the human IgG heavy chain constant region of the prepared recombinant DR6 fusion protein.
17c is a diagram showing whether the prepared recombinant DR6 fusion protein recognizes the target cancer cells (AsPC-1 cell line) expressing CD138 using flow cytometry.
18 is a diagram showing the results of comparing the expression ratio of GFP in professional antigen-expressing cells transduced with four types of expression vectors according to an embodiment of the present invention using a lentivirus system.
Figure 19 shows the effector cells of professional antigen-expressing cells transduced with four types of chimeric antigen receptors or empty vectors according to an embodiment of the present invention, and cancer cells (AsPC-1 cell line) expressing CD138. It is a diagram showing the results of measuring the cytotoxicity for 24 hours after co-culture.
20 shows the effector cells of professional antigen-expressing cells transduced with four types of chimeric antigen receptors or empty vectors according to an embodiment of the present invention, and cancer cells (AsPC-1 cell line) expressing CD138. It is a diagram showing the results of measuring the cytotoxicity for 48 hours after co-culture.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining 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 provides a transformed cell comprising a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an antigen-binding domain that specifically binds to CD138, a transmembrane domain, and an intracellular signaling domain wherein the cells are antigen-specific specialized antigen-expressing cells having targeted effector activity, and include macrophages, dendritic cells, or naive B cells, to provide transformed cells.

Chimeric antigen receptor proteins according to one embodiment of the present invention may include functional equivalents. 'Functional equivalent' means at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably, the amino acid sequence of the chimeric antigen receptor protein as a result of addition, substitution, or deletion of amino acids. has a sequence homology of 95% or more, and refers to a protein that exhibits substantially the same physiological activity. 'Substantially homogeneous physiological activity' means having an activity capable of specifically binding to CD138.

The present invention also includes fragments, derivatives and analogs of chimeric antigen receptors. As used herein, the terms 'fragment', 'derivative' and 'analogue' refer to a polypeptide that retains substantially the same biological function or activity as the chimeric antigen receptor protein of the invention. Fragments, derivatives and analogs of the present invention include (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, the substituted amino acid residues being encoded by the genetic code. (ii) a polypeptide having substituent(s) on one or more amino acid residues, or (iii) another compound (a compound capable of extending the half-life of a polypeptide, such as polyethylene glycol) a polypeptide derived from the associated mature polypeptide, or (iv) an additional amino acid sequence (e.g., a leader sequence, a secreted sequence, a sequence used to purify the polypeptide, a proteinogen sequence, or a fusion protein) and It may be a polypeptide derived from the polypeptide to which it is linked. Such fragments, derivatives and analogues as defined herein are well known to those skilled in the art.

In the present invention, CD138 is found in various malignant cells. So far, carcinomas expressing CD138 include multiple myeloma, breast cancer, pancreatic cancer, lung cancer, hepatocellular carcinoma, bladder cancer, and ovarian cancer. ), prostate cancer or colorectal cancer, but is not limited thereto.

In the present invention, the "chimeric signaling domain" is expressed in professional antigen-expressing cells to induce activation of professional antigen-expressing cells through a ligand-receptor reaction by binding to a desired ligand and to attack cells expressing the corresponding ligand. It may mean a fusion protein for In other words, it can be seen as a protein that binds to a ligand when expressed in specialized antigen-expressing cells and induces the activation of these cells. Therefore, the novel chimeric signaling domain provided by the present invention is a novel chimeric signaling domain in a universal sense capable of inducing activation of professional antigen-expressing 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, when expressed in specialized antigen-expressing cells, binds to an antigen and induces activation of these cells. Through this, it may be a protein that recognizes an antigen specific to a cell to cause an immune response, and the cell to cause an immune response may refer to a cell present in a specific tissue or forming a tissue that causes a lesion.

The antigen-binding domain of the chimeric antigen receptor (CAR) may be an extracellular domain of death receptor 6 (DR6) that specifically binds to the CD138. The extracellular domain of DR6 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 DR6 are linked to a human immunoglobulin G ₁ heavy chain constant region, respectively, to amplify signal _transduction . It is not limited.

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

The intracellular signaling domain, which is one component of the chimeric antigen receptor of the present invention, means a site that activates the immune response of immune cells by binding to the 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 the ITAM as a subunit to the TLR-3-TLR-4 with a linker, and one or more ITAMs are linked. It may be, preferably three may be connected form, 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 an amino acid represented by SEQ ID NO: 3 having a sequence homology of at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% therewith. can consist of an amino acid sequence that exhibits a function substantially equivalent to that of the sequence; The TLR-4 is SEQ ID NO: 4 or an amino acid represented by SEQ ID NO: 4 having a sequence homology of at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% therewith. can consist of an amino acid sequence that exhibits a function substantially equivalent to that of the sequence; The ITAM is SEQ ID NO: 5; SEQ ID NO: 6; Alternatively, it may consist of an amino acid sequence that exhibits substantially equivalent functions to the amino acid sequence represented by SEQ ID NO: 7.

The linker may consist of an amino acid sequence that exhibits substantially the same function as 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 DR6 and the co-stimulatory and essential signaling domains between cell membranes, and the intracellular signaling domain is an immune response of immune cells by binding of the antigen-binding domain. indicates the site of activation.

The transmembrane domain, which is one component 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 not limited thereto no.

The vector used in the present invention may use various vectors known in the art, and depending on the type of host cell to produce the antigen receptor, a promoter, terminator, enhancer, etc. Expression control sequences, sequences for membrane targeting or secretion, etc. may 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 and viral vectors. Suitable vectors include expression control elements such as promoters, operators, initiation codons, stop codons, polyadenylation signals and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, 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, the pCDH-CMV-MCS-EF1-copGFP vector (a lenti-virus vector) was used (FIG. 5 to FIG. 8).

In addition, cells can be transformed by introducing the chimeric antigen receptor that specifically binds to CD138 of the present invention into cells through the vector. The cells may be cytotoxic T cells and NK cells, tumor-infiltrating lymphocytes, B cells, NK cells, or NKT cells, preferably macrophages, dendritic cells, and unsensitized cells that are specialized antigen-expressing 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, professional antigen-expressing cells can be transformed with a chimeric antigen receptor that specifically binds to CD138 using the vector described above.

As described above, cells transformed by introducing the chimeric antigen receptor of the present invention recognize CD138 as an antigen and bind strongly thereto.

In the present invention, "chimeric antigen receptor professional antigen presenting cell (chimeric antigen receptor professional antigen presenting cell, hereinafter simply abbreviated as 'CAR-pAPC cell')" means a method such as transduction of normal professional antigen presenting cells It refers to specialized antigen-expressing cells that express chimeric antigen receptors that specifically react to cancer cells, rather than the original specialized antigen-expressing cell surface receptors. Professional antigen-expressing cells having this receptor exhibit cytotoxicity by inducing apoptosis of target cells.

In the present invention, in particular, CAR-pAPC cells may be cells into which the chimeric antigen receptor of the present invention has been introduced into professional antigen-expressing cells. The cells have the advantage of anticancer-specific targeted therapy, which is an existing advantage of CAR-T therapeutics. In particular, professional antigen-expressing cells equipped with the chimeric antigen receptor of the present invention can recognize and effectively destroy cancer cells expressing CD138. .

Therefore, another aspect of the present invention is a cell therapy agent comprising the cells; A pharmaceutical composition for the treatment of cancer expressing CD138 comprising the same as an active ingredient; and administering the cells to a subject.

In the present invention, the cells may be cytotoxic T cells and NK cells, tumor infiltrating lymphocytes, B cells, NK cells, or NK-T cells, preferably, specialized antigen expression It may be a macrophage cell, a dendritic cell, or a naive B cell. or progenitor cells, for example, hematopoietic stem cells, mesenchymal stromal cells, stem cells, pluripotent stem cells, and embryonic stem cells, which can be used for cell therapy such as anticancer treatment. The cells may come from a donor or may be cells obtained from a patient. Cells can be used for regeneration, eg 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 therapy" refers to cells and tissues prepared by isolation, culture, and special manipulation from a subject, and is a drug (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention, and restores the function of cells or tissues. It refers to medicines used for the purpose of treatment, diagnosis, and prevention through a series of actions, such as proliferating and selecting living autologous, allogeneic, or heterogeneous cells in vitro or changing the biological characteristics of cells in other ways.

The term "prevention" of the present invention refers to any activity that inhibits or delays the development of cancer expressing CD138 by administration of the composition.

As used herein, the term "treatment" refers to all activities that improve or beneficially change symptoms caused by cancer expressing CD138 by administration of the composition.

The composition may include a pharmaceutically acceptable carrier.

The "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not inhibit the biological activity and properties of the compound to be injected without irritating living organisms. The type of the 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, 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 containing a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. 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, and the like, and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose, and lactose in the compound. , can be prepared by mixing gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solutions for internal use, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may 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 dependent on the type and severity of the subject, age, sex, infected virus type, and drug activity, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including concomitantly used drugs and other factors well known in the medical arts.

The administration means introducing the composition of the present invention to a 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, intranasal administration may be administered, 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 2 to 3 times. The number of administrations when the two active ingredients are each a single agent may be the same or may be different. In addition, 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. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors, and can be easily determined by those skilled in the art.

The subject refers to all humans, including humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs that have or may develop cancer expressing CD138. means animals. The type of subject is included without limitation as long as the disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject.

Carcinoma expressing CD138 to be treated in the present invention is multiple myeloma, breast cancer, pancreatic cancer, lung cancer, hepatocellular carcinoma, bladder cancer , ovarian cancer, prostate cancer, and colorectal cancer, but is not limited thereto.

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

Example 1. Gene synthesis by gene synthesis method cloning

In order to prepare the chimeric antigen receptor of the present invention, four types of antigen receptors having the sequences of the extracellular domain of DR6 (Death receptor 6) and the transmembrane domain of TLR-2 and encoding sequences for different intracellular signal transduction domains were prepared. A chimeric antigen receptor protein coding sequence was synthesized. The chimeric antigen receptor is an antigen recognition site in which a pair of extracellular domains of DR6 are linked to the human immunoglobulin G ₁ heavy chain constant region, respectively, a transmembrane domain of TLR-2, and a _TLR -4 signal peptide. Including in common. Signal transduction domains were constructed in four different configurations. Table 1 below shows the configuration of the four types of signaling domains.

division Signaling domain organization 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

1 to 4 show cDNA schematics of each domain expressing the constructed chimeric antigen receptor. 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 constructing a protein expression plasmid.

Example 2. Preparation of protein expression plasmids

In order to express the recombinant protein (Figs. 5 to 8) fused with the extracellular domain of DR6 of the four chimeric antigen receptors prepared in Example 1 and the macrophage signaling protein in macrophages, the corresponding gene was Lenti It was cloned into pCDH-CMV-MCS-EF1-copGFP (System Biosciences), a virus-derived expression vector, using restriction enzymes Xba I and Not I cleavage sites.

As a result, a recombinant vector expressing four fusion proteins of the extracellular domain of DR6 having the structure of FIGS. 9 to 12 and a novel signal transduction protein was completed (see FIGS. 5 to 8).

Example 3. Screening human cell lines expressing CD138

In order to screen a human cell line expressing CD138 on the cell surface, CD138 expression was confirmed in the human pancreatic adenocarcinoma (AsPC-1) (ATCC) cell line, which is a cell line derived from pancreatic cancer. To confirm expression, after treating the cell line with an antibody (Biolegend) capable of binding to CD138 to which a fluorescent protein is attached, flow cytometry analysis (fluorescence-activated cell sorting) was used. As a result of the analysis, it was confirmed that CD138 was expressed in the AsPC-1 cell line (see FIG. 13). Based on the above results, the AsPC-1 cell line expressing CD138 was used as a target cell.

Example 4. Soluble recombinant DR6 fusion protein gene by gene synthesis method cloning

To confirm that DR6 binds to CD138, a soluble recombinant DR6 fusion protein was prepared. First, a protein coding sequence in which each of the extracellular domains of DR6 is linked to the human immunoglobulin G1 heavy chain constant region was identified in a gene database. In addition, gene synthesis was performed by connecting the remaining sequences except for the stop codon portion of the base sequence constituting the domains into one. The accuracy of gene synthesis was confirmed through sequencing after constructing a protein expression plasmid. A schematic diagram showing the cDNA region of each domain expressing the recombinant protein is shown in FIG. 14 .

Example 5. Preparation of soluble recombinant DR6 fusion protein expression plasmid

In order to express the recombinant DR6 fusion protein obtained by fusing the human CD138 recognition domain of Example 4 with the human immunoglobulin G ₁ heavy chain constant region in Chinese hamster ovary (CHO) cells, the corresponding gene was used in pLNCX2, a retrovirus-derived expression vector. (Addgene).

First, in order to clone into the pLNCX2 vector, a primer that creates a restriction enzyme Xho I cleavage site sequence is synthesized at the 5' end, a restriction enzyme cleavage site is created by polymerase chain reaction (PCR), and restriction is also made at the 3' end. A restriction enzyme cleavage site was prepared by synthesizing a primer that creates the cleavage site sequence of the enzyme Not I and polymerase chain reaction. After that, the 5' end of the gene having a restriction enzyme cleavage site due to polymerase chain reaction was treated with Xho I, and the 3' end was treated with Not I. In addition, the multicloning site of the expression vector was treated with xho I and Not I so that the gene could be inserted. After mixing the restriction enzyme-treated gene and the expression vector, they were ligated by treatment with a ligase.

As a result, a recombinant vector DR6-IgG.pLNCX2 expressing a protein obtained by fusing the extracellular domain of DR6 recognizing human CD138 with the human immunoglobulin G ₁ heavy chain constant region (see FIG. 16) was completed (see FIG. 15).

Example 6. Affinity measurement of soluble recombinant DR6 fusion protein and human cell line expressing CD138

In order to confirm the affinity of the soluble recombinant DR6 fusion protein prepared in Example 5 with a human cell line expressing CD138, after expressing the soluble recombinant DR6 fusion protein in CHO cells, affinity chromatography was performed using a protein A column ( affinity chromatography) (GE healthcare) (see Fig. 17a).

In addition, the purified water-soluble chimeric antigen receptor was confirmed by Western blotting using an antibody (anti-human IgG heavy chain constant region antibody, anti-human IgG Fc region antibody, abcam) recognizing the human IgG heavy chain constant region (Fig. see 17b).

Thereafter, the purified soluble recombinant DR6 fusion protein was treated with a cell line (AsPC-1 cell line) expressing CD138, and it was confirmed through flow cytometry whether the soluble recombinant DR6 fusion protein could bind to the cell. The results are shown in FIG. 17C. .

As shown in FIG. 17c , AsPC-1 cells expressing CD138 did not bind to control Ig (isotype control), but were confirmed to bind to soluble DR6 fusion protein (DR6-Ig).

As confirmed from the above results, it was confirmed that the prepared soluble DR6 fusion protein had high affinity with the human cell line expressing CD138, which recognized AsPC-1 cells expressing CD138, and recognized the prepared CD138 as a human immunocompetent. This means that it can be used to verify CD138 protein-specific cytotoxicity in professional antigen-expressing cells (DR6.CAR-pAPC cells) expressing a recombinant protein in which a globulin G ₁ heavy chain constant region and a signaling protein domain are fused.

Example 7. chimeric Production of specialized antigen-expressing cells expressing 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 professional antigen-expressing cells system was used.

First, 293FT cells were inoculated into 2.5Х10 ⁶ cells in a 100π cell culture dish, and cultured in DMEM medium containing 10% calf serum. After 24 hours of culture, when the cells grow to cover about 60-70% of the dish, 20 μg of each of the 4 vector DNAs of Example 2 were dispensed, and 10 μg of psPAX2 (Addgene) and 3 μg of pMD2.G ( After crystallization of Calcium phosphate with Addgene) vector using Calcium phosphate and Hepes-buffered solution, it was introduced into 293FT cells. Thereafter, the culture supernatant containing the virus (lentivirus) was collected at 24 hour intervals after 48 hours of 293FT cells into which the expression vector was introduced. The collected supernatant was centrifuged for 2 hours at 21,000 rpm in an ultra-high-speed centrifuge to concentrate the virus. Transduction was performed by mixing the concentrated virus with 4 μg/ml of polybrene and adding it to the culture medium of THP-1 cells, a macrophage cell line. The efficiency of transduction was increased by changing the culture medium of THP-1 cells to a culture medium into which the virus was concentrated twice at 24-hour intervals. 24 hours after the last change of the culture medium, some of the transduced THP-1 cells were used to measure the transduction efficiency. The transduction efficiency was measured by flow cytometry to measure the amount of GFP expression inside the cell. THP-1 cells transduced with each of the four recombinant chimeric antigen receptor expression vectors and THP-1 cells transduced with an empty vector (Mock) were analyzed for transduction efficiency. The result of comparison with the transduction rate of 1 cell is shown in FIG. 18 .

Example 8. Cells expressing CD138 (CD138 positive cells) co-culture Verification of CD138-specific cytotoxicity through

AsPC-1, the target cell selected in Example 3, to confirm whether the four types of chimeric antigen receptor-expressing THP-1 cells prepared above show toxicity by selectively recognizing CD138-expressing cells (CD138 positive cell, CD138 ⁺ cell). One cell was co-cultured with 4 types of chimeric antigen receptor-expressing THP-1 cells or THP-1 cells (Mock) into which an empty vector was introduced. After co-culture, 7-Aminoactinomycin D (7-AAD) was treated 24 or 48 hours later, and the fluorescence expression level of 7-AAD was compared by flow cytometry to measure the cytotoxicity of each chimeric antigen receptor-expressing THP-1 cell. did 7-Aminoactinomycin D (7-AAD) binds to the DNA of apoptosis-induced cells and becomes fluorescent (Zembruski et al., 2012).

First, each of the four types of chimeric antigen receptor-expressing THP-1 cells was dispensed at 5Х10 ⁵ into wells of a 24-well cell culture dish, and target cells, CD138 ⁺ cells (AsPC-1 cells) were dispensed at 1Х10 ⁵ each, effector: target Coculture was performed at a ratio of 5:1. The volume per well of the co-culture was 1 ml, and centrifugation was performed at 250 g for 4 minutes using a centrifuge to close the cell spacing. Then, after 24 hours or 48 hours of co-culture, the cells in each well were centrifuged for 3 minutes at 300 g using a centrifuge to remove the supernatant, and then the cells were stained with 7-AAD (Biolegend). Thereafter, the degree of cytotoxicity of the four types of chimeric antigen receptor-expressing THP-1 cells prepared was quantified by measuring the ratio of cells showing fluorescence by 7-AAD using flow cytometry.

As a result, as shown in FIG. 19, after 24 hours of co-cultivation of THP-1 cells expressing the chimeric antigen receptor with CD138 ⁺ cells (ASPC-1 cells), THP that does not express the chimeric antigen receptor is shown in FIG. -1 cells (Mock) showed cytotoxicity of about 11.0%, whereas 3-4-3 THP-1 cells (TLR-3+TLR-4+FcεRIγ ITAM), which are THP-1 cells expressing four types of chimeric antigen receptors trimer), 3-4-2A THP-1 cell (TLR-3+TLR4+FcγR2Aα ITAM trimer), 3-4-1B THP-1 cell (TLR-3+TLR4+FcεRIβ ITAM trimer), 3-4-ABC In THP-1 cells (TLR-3 + TLR4 + FcγR2A ITAM monomer + FcεRIβ ITAM monomer + FcγR2Aα ITAM monomer), it was confirmed that they showed very high cytotoxicity of 19.6%, 17.5%, 20.1%, and 22.7%, respectively.

In addition, as shown in FIG. 20, when confirming the cytotoxicity of CD138 ⁺ cells (AsPC-1 cells) co-cultured after 48 hours of co-culture, Mock showed cytotoxicity of about 18.5%, whereas , 4 types of chimeric 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 40.8%, 38.9%, 44.3%, and 43.6%, respectively. Through this, the chimeric antigen recognition receptor-expressing THP-1 cells produced were CD138 It was confirmed that the protein showed specific cytotoxicity.

Through the above results, the transformed THP-1 cells containing the chimeric antigen receptor according to the present invention were CD138 By showing protein-specific cytotoxicity, it was confirmed that cancer cells can be treated using the transformed antigen-specific specialized antigen-expressing cells.

So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can 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 limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

<110> IMMUNOLOGICAL DESIGNINGLAB CO., LTD <120> DR6.CAR-MAC <130> KP21-0051-ILDL <160> 20 <170> KoPatentIn 3.0 <210> 1 <211> 308 <212> PRT <213> artificial sequence <220> <223> DR6 Extracellular domain <400> 1 Gln Pro Glu Gln Lys Ala Ser Asn Leu Ile Gly Thr Tyr Arg His Val 1 5 10 15 Asp Arg Ala Thr Gly Gln Val Leu Thr Cys Asp Lys Cys Pro Ala Gly 20 25 30 Thr Tyr Val Ser Glu His Cys Thr Asn Thr Ser Leu Arg Val Cys Ser 35 40 45 Ser Cys Pro Val Gly Thr Phe Thr Arg His Glu Asn Gly Ile Glu Lys 50 55 60 Cys His Asp Cys Ser Gln Pro Cys Pro Trp Pro Met Ile Glu Lys Leu 65 70 75 80 Pro Cys Ala Ala Leu Thr Asp Arg Glu Cys Thr Cys Pro Pro Gly Met 85 90 95 Phe Gln Ser Asn Ala Thr Cys Ala Pro His Thr Val Cys Pro Val Gly 100 105 110 Trp Gly Val Arg Lys Lys Gly Thr Glu Thr Glu Asp Val Arg Cys Lys 115 120 125 Gln Cys Ala Arg Gly Thr Phe Ser Asp Val Pro Ser Ser Val Met Lys 130 135 140 Cys Lys Ala Tyr Thr Asp Cys Leu Ser Gln Asn Leu Val Val Ile Lys 145 150 155 160 Pro Gly Thr Lys Glu Thr Asp Asn Val Cys Gly Thr Leu Pro Ser Phe 165 170 175 Ser Ser Ser Thr Ser Pro Ser Pro Gly Thr Ala Ile Phe Pro Arg Pro 180 185 190 Glu His Met Glu Thr His Glu Val Pro Ser Ser Thr Tyr Val Pro Lys 195 200 205 Gly Met Asn Ser Thr Glu Ser Asn Ser Ser Ala Ser Val Arg Pro Lys 210 215 220 Val Leu Ser Ser Ile Gln Glu Gly Thr Val Pro Asp Asn Thr Ser Ser 225 230 235 240 Ala Arg Gly Lys Glu Asp Val Asn Lys Thr Leu Pro Asn Leu Gln Val 245 250 255 Val Asn His Gln Gln Gly Pro His Arg His Ile Leu Lys Leu Leu 260 265 270 Pro Ser Met Glu Ala Thr Gly Gly Glu Lys Ser Ser Thr Pro Ile Lys 275 280 285 Gly Pro Lys Arg Gly His Pro Arg Gln Asn Leu His Lys His Phe Asp 290 295 300 Ile Asn Glu His 305 <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> 143 <212> PRT <213> artificial sequence <220> <223> hTLR-3 TIR domain <400> 3 Phe Glu Tyr Ala Ala Tyr Ile Ile His Ala Tyr Lys Asp Lys Asp Trp 1 5 10 15 Val Trp Glu His Phe Ser Ser Met Glu Lys Glu Asp Gln Ser Leu Lys 20 25 30 Phe Cys Leu Glu Glu Arg Asp Phe Glu Ala Gly Val Phe Glu Leu Glu 35 40 45 Ala Ile Val Asn Ser Ile Lys Arg Ser Arg Lys Ile Ile Phe Val Ile 50 55 60 Thr His His Leu Leu Lys Asp Pro Leu Cys Lys Arg Phe Lys Val His 65 70 75 80 His Ala Val Gln Gln Ala Ile Glu Gln Asn Leu Asp Ser Ile Ile Leu 85 90 95 Val Phe Leu Glu Glu Ile Pro Asp Tyr Lys Leu Asn His Ala Leu Cys 100 105 110 Leu Arg Arg Gly Met Phe Lys Ser His Cys Ile Leu Asn Trp Pro Val 115 120 125 Gln Lys Glu Arg Ile Gly Ala Phe Arg His Lys Leu Gln Val Ala 130 135 140 <210> 4 <211> 147 <212> PRT <213> artificial sequence <220> <223> hTLR-4 TIR domain <400> 4 Asn Ile Tyr Asp Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp 1 5 10 15 Val Arg Asn Glu Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe 20 25 30 Gln Leu Cys Leu His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala 35 40 45 Ala Asn Ile Ile His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val 50 55 60 Val Val Ser Gln His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr 65 70 75 80 Glu Ile Ala Gln Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile 85 90 95 Phe Ile Val Leu Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val 100 105 110 Glu Leu Tyr Arg Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp 115 120 125 Ser Val Leu Gly Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu 130 135 140 Leu Asp Gly 145 <210> 5 <211> 29 <212> PRT <213> artificial sequence <220> <223> ITAM of IgE receptor gamma chain <400> 5 Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly Leu Ser 1 5 10 15 Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu 20 25 <210> 6 <211> 23 <212> PRT <213> artificial sequence <220> <223> ITAM of IgG receptor 2A alpha chain <400> 6 Asp Gly Gly Tyr Met Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp 1 5 10 15 Lys Asn Ile Tyr Leu Thr Leu 20 <210> 7 <211> 29 <212> PRT <213> artificial sequence <220> <223> ITAM of IgE receptor beta chain <400> 7 Lys Val Pro Glu Asp Arg Val Tyr Glu Glu Leu Asn Ile Tyr Ser Ala 1 5 10 15 Thr Tyr Ser Glu Leu Glu Asp Pro Gly Glu Met Ser Pro 20 25 <210> 8 <211> 5 <212> PRT <213> artificial sequence <220> <223> flexible linker <400> 8 Gly Gly Gly Gly Ser 1 5 <210> 9 <211> 21 <212> PRT <213> artificial sequence <220> <223> hTLR-2 transmembrane domain <400> 9 Ala Leu Val Ser Gly Met Cys Cys Ala Leu Phe Leu Leu Ile Leu Leu 1 5 10 15 Thr Gly Val Leu Cys 20 <210> 10 <211> 23 <212> PRT <213> artificial sequence <220> <223> hTLR-4 Signal peptide <400> 10 Met Met Ser Ala Ser Arg Leu Ala Gly Thr Leu Ile Pro Ala Met Ala 1 5 10 15 Phe Leu Ser Cys Val Arg Pro 20 <210> 11 <211> 924 <212> DNA <213> artificial sequence <220> <223> DR6 Extracellular domain <400> 11 cagccagaac agaaggcctc gaatctcatt ggcacatacc gccatgttga ccgtgccacc 60 ggccaggtgc taacctgtga caagtgtcca gcaggaacct atgtctctga gcattgtacc 120 aacacaagcc tgcgcgtctg cagcagttgc cctgtgggga cctttaccag gcatgagaat 180 ggcatagaga aatgccatga ctgtagtcag ccatgcccat ggccaatgat tgagaaatta 240 ccttgtgctg ccttgactga ccgagaatgc acttgcccac ctggcatgtt ccagtctaac 300 gctacctgtg ccccccatac ggtgtgtcct gtgggttggg gtgtgcggaa gaaagggaca 360 gagactgagg atgtgcggtg taagcagtgt gctcggggta ccttctcaga tgtgccttct 420 agtgtgatga aatgcaaagc atacacagac tgtctgagtc agaacctggt ggtgatcaag 480 ccggggacca aggagacaga caacgtctgt ggcacactcc cgtccttctc cagctccacc 540 tcaccttccc ctggcacagc catctttcca cgccctgagc acatggaaac ccatgaagtc 600 ccttcctcca cttatgttcc caaaggcatg aactcaacag aatccaactc ttctgcctct 660 gttagaccaa aggtactgag tagcatccag gaagggacag tccctgacaa cacaagctca 720 gcaaggggga aggaagacgt gaacaagacc ctcccaaacc ttcaggtagt caaccaccag 780 caaggccccc accacagaca catcctgaag ctgctgccgt ccatggaggc cactgggggc 840 gagaagtcca gcacgcccat caagggcccc aagaggggac atcctagaca gaacctacac 900 aagcattttg acatcaatga gcat 924 <210> 12 <211> 702 <212> DNA <213> artificial sequence <220> <223> IgG1 heavy chain constant region <400> 12 ggatccgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 60 ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 120 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 180 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcggggag 240 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 300 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 360 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 420 tcacgagatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 480 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 540 acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 600 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 660 aaccactaca cgcagaagag cctctccctg tctccgggta aa 702 <210> 13 <211> 429 <212> DNA <213> artificial sequence <220> <223> hTLR-3 TIR domain <400> 13 tttgaatatg cagcatatat aattcatgcc tataaagata aggattgggt ctgggaacat 60 ttctcttcaa tggaaaagga agaccaatct ctcaaatttt gtctggaaga aagggacttt 120 gaggcgggtg tttttgaact agaagcaatt gttaacagca tcaaaagaag cagaaaaatt 180 atttttgtta taacacacca tctattaaaa gacccatat gcaaaagatt caaggtacat 240 catgcagttc aacaagctat tgaacaaaat ctggattcca ttatattggt tttccttgag 300 gagattccag attataaact gaaccatgca ctctgtttgc gaagaggaat gtttaaatct 360 cactgcatct tgaactggcc agttcagaaa gaacggatag gtgcctttcg tcataaattg 420 caagtagca 429 <210> 14 <211> 441 <212> DNA <213> artificial sequence <220> <223> hTLR-4 TIR domain <400> 14 aacatctatg atgcctttgt tatctactca agccaggatg aggactgggt aaggaatgag 60 ctagtaaaga atttagaaga aggggtgcct ccatttcagc tctgccttca ctacagagac 120 tttatcccg gtgtggccat tgctgccaac atcatccatg aaggtttcca taaaagccga 180 aaggtgattg ttgtggtgtc ccagcacttc atccagagcc gctggtgtat ctttgaatat 240 gagattgctc agacctggca gtttctgagc agtcgtgctg gtatcatctt cattgtcctg 300 cagaaggtgg agaagaccct gctcaggcag caggtggagc tgtaccgcct tctcagcagg 360 aacacttacc tggagtggga ggacagtgtc ctggggcggc acatcttctg gagacgactc 420 agaaaagccc tgctggatgg t 441 <210> 15 <211> 87 <212> DNA <213> artificial sequence <220> <223> ITAM of IgE receptor gamma chain <400> 15 gctataacca gctatgagaa atcagatggt gtttacacgg gcctgagcac caggaaccag 60 gagacttacg agactctgaa gcatgag 87 <210> 16 <211> 69 <212> DNA <213> artificial sequence <220> <223> ITAM of IgG receptor 2A alpha chain <400> 16 gacggcggct acatgactct gaaccccagg gcacctactg acgatgataa aaacatctac 60 ctgactctt 69 <210> 17 <211> 87 <212> DNA <213> artificial sequence <220> <223> ITAM of IgE receptor beta chain <400> 17 aaggttccag aggatcgtgt ttatgaagaa ttaaacatat attcagctac ttacagtgag 60 ttggaagacc caggggaaat gtctcct 87 <210> 18 <211> 15 <212> DNA <213> artificial sequence <220> <223> flexible linker <400> 18 ggtggcggtg gctcg 15 <210> 19 <211> 63 <212> DNA <213> artificial sequence <220> <223> hTLR-2 transmembrane domain <400> 19 gcactggtgt ctggcatgtg ctgtgctctg ttcctgctga tcctgctcac cggtgtcctg 60 tgc 63 <210> 20 <211> 69 <212> DNA <213> artificial sequence <220> <223> hTLR-4 Signal peptide <400> 20 atgatgtctg cctcgcgcct ggctgggact ctgatcccag ccatggcctt cctctcctgc 60 gtgagacca 69

Claims (16)

A transformed cell comprising a chimeric antigen receptor (CAR),
The chimeric antigen receptor includes an antigen-binding domain that specifically binds to CD138, a transmembrane domain and an intracellular signaling domain, and the cells are antigen-specific professional antigen-expressing cells having targeted effector activity, macrophages, Transformed cells, including dendritic cells or naive B cells. According to claim 1,
The antigen-binding domain is an extracellular domain of DR6 (Death receptor 6) that specifically binds to CD138, transformed cells. According to claim 2,
Wherein the extracellular domain of DR6 comprises the amino acid sequence represented by SEQ ID NO: 1, the transformed cell. According to claim 2,
The antigen-binding domain is a transformed cell _, characterized in that a pair of extracellular domains of DR6 are linked to a human immunoglobulin G ₁ heavy chain constant region, respectively, in the form of a dimer. According to claim 4,
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. According to claim 1,
The intracellular signaling domain is linked to the structure of TLR-3-TLR-4 with a linker using an immunoreceptortyrosine-based activation motif (ITAM) as a subunit, transformed cells. According to claim 6,
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). A transformed cell that is an activation motif. According to claim 6,
Transformed cells, wherein three ITAMs are linked by a linker. According to claim 6,
The TLR-3 is SEQ ID NO: 3; and TLR-4 comprises the amino acid sequence represented by SEQ ID NO: 4. According to claim 6,
The ITAM is SEQ ID NO: 5; SEQ ID NO: 6; Or, a transformed cell comprising the amino acid sequence represented by SEQ ID NO: 7. According to claim 6,
Wherein the linker comprises the amino acid sequence represented by SEQ ID NO: 8, the transformed cell. According to claim 1,
The transmembrane domain is TLR-2 and comprises the amino acid sequence represented by SEQ ID NO: 9, the transformed cell. According to claim 1,
The chimeric antigen receptor comprises a signal peptide and comprises an amino acid sequence represented by SEQ ID NO: 10, the transformed cell. A pharmaceutical composition for the treatment of cancer expressing CD138, comprising the cell of any one of claims 1 to 13 as an active ingredient. According to claim 14,
The cancer includes multiple myeloma, breast cancer, pancreatic cancer, lung cancer, hepatocellular carcinoma, bladder cancer, ovarian cancer, prostate cancer ( A composition characterized in that it is selected from the group consisting of prostate cancer) and colon cancer (colorectal cancer). A cell therapy product comprising the cell of any one of claims 1 to 13 as an active ingredient.

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