KR20220131801A - 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 including a chimeric antigen receptor (CAR) and a use thereof. The transformed cell according to the present invention includes an enhanced chimeric signaling domain that induces the activity of the professional antigen-expressing cell. The professional antigen-expressing cell according to the present invention can be usefully used as immune cell treatment agent.

Description

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

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

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, a 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).

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 has been no description of immunotherapy using specialized antigen-expressing cells expressing a chimeric antigen receptor having binding specificity for HLA class II-expressing cancer cells by enhancing intracellular signaling domains.

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 such a chimeric signaling domain can have cytotoxicity against cancer. That is, the extracellular domain of lymphocyte activation gene-3 (LAG-3; CD223) having high affinity with the human major histocompatibility complex (MHC) antigen class II, HLA-DP, HLA-DQ, and HLA-DR. 1 to 4, transmembrane domain of toll-like 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, a chimeric antigen receptor-expressing macrophage expressing a recombinant protein bound to a novel synthetic intracellular signal transduction domain linking three ITAMs with a linker was constructed. At this time, the extracellular domain of LAG-3 expressed in macrophages recognizes HLA class II and, when combined with a specific cancer cell expressing HLA class II, can deliver an activation signal to the inside of these macrophages, and eventually these signals are By activating phagocytes, it is possible to have cytotoxicity against cancers expressing HLA class II. In fact, it was confirmed through an in vitro experiment that the macrophages have specific cytotoxicity to cells expressing HLA class II.

The HLA class II is mainly expressed in professional antigen-presenting cells and serves to deliver peptide antigens to CD4 ⁺ T cells (Trowsdale, 1993). However, HLA class II is expressed abnormally high in various carcinomas when normal cells are tumorigenic (Axelrod et al., 2019). To date, carcinomas expressing HLA class II have been classified as head and neck squamous cell carcinoma (Meissner et al., 2008), thymoma (Strobel et al., 2008) and esophageal squamous cell carcinoma ( Esophageal squamous cell carcinoma) (Hu et al., 2014), melanoma (Johnson et al., 2016; Rodig et al., 2018; Johnson et al., 2018), breast cancer (Park et al.) al., 2017; Loi et al., 2016; Bartek et al., 1987), colorectal cancer (Michel et al., 2010; Bustin et al., 2001), ovarian cancer (callahan et al.) al., 2008, Turner et al., 2017), prostate cancer (Younger et al., 2008), neuroblstoma (Yazawa et al., 2002), glioma (Soos et al.) al., 2001), non-small cell lung cancer (Yazawa et al., 1999), classic Hodgkin lymphoma (Roemer et al., 2018), T cell leukemia/lymphoma (T-cell leukemia/lymphoma) (Takeuchi et al., 2019), B cell lymphoma (Steidl et al., 2011), chronic myeloid leukemia (Petersdorf et al., 2001) , chronic lymphocytic leukemia (Hojjat-Farsangi et al., 2008), acute bone acute myeloid leukemia (van den Ancker et al., 2014), acute lymphoid leukemia (Hurwitz et al., 1988), and the like.

Korea Patent Publication No. 10-2018-0028533 (2018.03.16) Korean Patent Publication No. 10-2018-0054600 (May 24, 2018)

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

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 (No. 1 to No. 4) of LAG-3 that specifically binds to HLA class II.

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, CD3 zeta (zeta), IgE receptor gamma chain (Fcε receptor I gamma chain, FcεRIγ), IgG receptor 2A alpha chain ( Fcγ receptor 2A alpha chain, FcγR2Aα), IgE receptor beta chain (Fcε receptor I beta chain, FcεRIβ) immunoreceptor tyrosine-based activation motif (immunoreceptortyrosine-based activation motif, ITAM), or a combination thereof.

Cancer expressing the HLA class II is head and neck squamous cell carcinoma, thymoma, esophageal squamous cell carcinoma, melanoma, breast cancer ), colorectal cancer, ovarian cancer, prostate cancer, neuroblstoma, glioma, non-small cell lung cancer, classic Hodgkin Classic Hodgkin lymphoma, T-cell leukemia/lymphoma, B cell lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, acute and may be selected from the group consisting of acute myeloid leukemia and acute lymphoid leukemia.

The transformed cell according to the present invention includes 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, extracellular domains 1 to 4 of LAG-3 that recognize HLA class II (HLA-DP, HLA-DQ, HLA-DR) and TLR- which plays an important role in signal transduction of professional antigen-presenting cells 3, Immunity of 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) It is a recombinant protein comprising a novel synthetic intracellular signal transduction domain in which three ITAMs are linked by a linker by using an immunoreceptortyrosine-based activation motif (ITAM) as a subunit. In the case of a professional antigen-expressing cell transformed with an effective vector, it has specific cytotoxicity to carcinoma expressing HLA class II (HLA-DP, HLA-DQ, HLA-DR). Therefore, the specialized antigen-expressing cells overexpressing the chimeric antigen receptor according to the present invention can be usefully used as immune cell therapeutics for the treatment of carcinomas expressing HLA class II (HLA-DP, HLA-DQ, HLA-DR). .

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 cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.
6 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.
7 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.
8 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.
9 is a schematic diagram illustrating a form of one of lentiviral vectors according to an embodiment of the present invention.
10 is a schematic diagram showing the form of one of the lentiviral vectors according to an embodiment of the present invention.
11 is a schematic diagram showing the shape of one of the lentiviral vectors according to an embodiment of the present invention.
12 is a schematic diagram showing the shape of one of the lentiviral vectors according to an embodiment of the present invention.
13 is a schematic diagram showing the shape of one of the lentiviral vectors according to an embodiment of the present invention.
14 is a schematic diagram showing the shape of one of the lentiviral vectors according to an embodiment of the present invention.
15 is a schematic diagram showing the shape of one of the lentiviral vectors according to an embodiment of the present invention.
16 is a schematic diagram showing the shape of one of the lentiviral vectors according to an embodiment of the present invention.
17 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.
18 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.
19 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.
20 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.
21 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.
22 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.
23 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.
24 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.
25 is a view of HLA class II in cancer cells expressing HLA class II (HLA class II positive cells) and cancer cells not expressing HLA class II (HLA class II negative cells), which are targets of the chimeric antigen receptor provided in the present invention. It is a diagram showing the result of measuring the expression level using flow cytometry.
26A 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, expressing it in Chinese hamster ovary (CHO) cells.
Figure 26b 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 26c 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 (HLA class II positive cells) expressing HLA class II, a target of the extracellular domain.
27 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.
28 is a cancer cell (HLA class II positive cell) expressing HLA class II using professional antigen-expressing cells transduced with a chimeric antigen receptor or empty vector according to an embodiment of the present invention as effector cells. It is a diagram showing the results of measuring the cytotoxicity of the cells 24 hours after the co-culture.
29 is a cancer cell (HLA class II positive cell) expressing HLA class II using professional antigen-expressing cells transduced with a chimeric antigen receptor or empty vector according to an embodiment of the present invention as effector cells. It is a diagram showing the results of measuring the cytotoxicity of the cells when 48 hours have elapsed after the co-culture.

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

In one aspect, the present invention provides a transformed cell comprising a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor has an antigen binding domain that specifically binds to HLA class II, a transmembrane domain and an intracellular signal. It provides a transformed cell comprising a delivery 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 HLA class II (HLA-DP, HLA-DQ, HLA-DR).

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 may comprise (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, wherein the substituted amino acid residues are encoded by the genetic code. may or may not be) or (ii) a polypeptide having substituent(s) at one or more amino acid residues, or (iii) another compound (a compound capable of extending the half-life of the polypeptide, such as polyethylene glycol); a polypeptide derived from the associated mature polypeptide, or (iv) an additional amino acid sequence (eg, a leader sequence, a secretion sequence, a sequence used to purify the polypeptide, a proteinogen sequence, or a fusion protein); It may be a polypeptide derived from said polypeptide to which it is bound. The fragments, derivatives and analogs as defined herein are well known to those skilled in the art.

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 include an extracellular domain (No. 1 to No. 4) of LAG-3 that specifically binds to HLA class II, but is not limited thereto.

Lymphocyte-activation gene 3 (LAG-3) protein is called lymphocyte activation gene-3, belongs to the immunoglobulin (Ig) superfamily, and has an extracellular domain consisting of four domains designated D1 to D4. It is an integral membrane protein.

The antigen-binding domain is a dimer (dimer) in which a pair of extracellular domains (No. 1 to No. 4) of LAG-3 are linked to a human immunoglobulin G1 heavy chain constant region (IgG1 heavy chain constant region) to amplify signal transduction, respectively. ), but is not limited thereto.

The extracellular domain (No. 1 to No. 4) of the LAG-3 may consist of SEQ ID NO: 1 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto.

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.

In the present invention, “HLA class II” is an intrinsic membrane protein that is mainly expressed in professional antigen-expressing cells and delivers peptide antigens to CD4 ⁺ T cells (helper T cells), but HLA class II is used in various carcinomas when normal cells are tumorigenic. abnormally high expression. To date, carcinomas expressing HLA class II have been classified as head and neck squamous cell carcinoma, thymoma, esophageal squamous cell carcinoma, melanoma, and breast cancer. cancer, colorectal cancer, ovarian cancer, prostate cancer, neuroblstoma, glioma, non-small cell lung cancer, Classic Issue Classic Hodgkin lymphoma, T-cell leukemia/lymphoma, B-cell lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, acute myeloid leukemia or acute lymphoid leukemia. In addition, all HLA class II ((HLA-DP, HLA-DQ, HLA-DR) has a characteristic of strongly binding to extracellular domains 1 to 4 of LAG-3.

In addition, 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. In one embodiment of the present invention, the signaling domain may be TLR-3, TLR-4, CD3 zeta, an immunoreceptortyrosine-based activation motif (ITAM), or a combination thereof. In a preferred embodiment, the signaling domain comprises TLR-3-TLR-4, TLR-3-TLR 4-CD3 zeta or ITAM as a subunit in the composition of TLR-3-TLR-4. Thus, it may be in a form connected by a linker, and the ITAM may be in a form in which one or more are connected, preferably in a form in which three are 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 an amino acid represented by SEQ ID NO: 3, which has SEQ ID NO: 3 or more than 70%, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more sequence homology thereto. 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 CD3 zeta functions as a professional antigen-presenting cell activation domain, and has SEQ ID NO: 5 or more than 70%, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more of SEQ ID NO: 5 or more. It may consist of an amino acid sequence having substantially the same function as that of the amino acid sequence shown in SEQ ID NO: 5; The ITAM is SEQ ID NO: 6; SEQ ID NO: 7; Alternatively, it may consist of an amino acid sequence exhibiting a function substantially equivalent to the amino acid sequence represented by SEQ ID NO: 8.

The linker may consist of an amino acid sequence having a function substantially equivalent to that of the amino acid sequence shown in SEQ ID NO: 9.

The transmembrane domain of the present invention is a site that connects the extracellular domain of LAG-3 and the costimulatory and essential signaling domains between the cell membrane, and the intracellular signaling domain is the domain of immune cells by binding of the antigen-binding domain. It refers to a site that activates an immune response.

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: 10 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: 11 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 embodiment, 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. 9 to 16).

In addition, cells can be transformed by introducing a chimeric antigen receptor that specifically binds to HLA class II (HLA-DP, HLA-DQ, HLA-DR) of the present invention through the vector. The cells may be cytotoxic T cells and NK cells, tumor infiltrating lymphocytes, B cells, NK cells, or NK-T cells, preferably macrophages, dendritic cells, which 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, a chimeric antigen receptor that specifically binds to HLA class II (HLA-DP, HLA-DQ, HLA-DR) can be transformed into specialized antigen-expressing cells using the vector described above. can

As described above, the cells transformed by introducing the chimeric antigen receptor of the present invention recognize HLA class II (HLA-DP, HLA-DQ, HLA-DR) as antigens and have a characteristic of strongly binding to them.

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 are HLA class II (HLA-DP, HLA-DQ, HLA). -DR)-expressing cancer cells can be recognized and effectively destroyed.

Therefore, another aspect of the present invention is a cell therapy agent comprising the cell; A pharmaceutical composition for the treatment of cancer expressing HLA class II 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 suppressing or delaying the development of HLA class II-expressing cancer by administration of the composition.

As used herein, the term “treatment” refers to any action in which the symptoms caused by cancer expressing HLA class II are improved or advantageously 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 is a human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig that has or can develop cancer expressing HLA class II. all animals, including 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, the types of HLA class II-expressing cancer to be treated include head and neck squamous cell carcinoma, thymoma, esophageal squamous cell carcinoma, melanoma, breast cancer, colorectal cancer, ovarian cancer, prostate cancer, neuroblstoma, glioma, non-small cell lung cancer -small cell lung cancer, classic Hodgkin lymphoma, T-cell leukemia/lymphoma, B cell lymphoma, chronic myeloid leukemia, chronic It may be, but is not limited to, chronic lymphocytic leukemia, acute myeloid leukemia, or acute lymphoid leukemia.

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

Example 1. Gene by gene synthesis method cloning

In order to prepare the chimeric antigen receptor of the present invention, it has sequences of extracellular domains 1 to 4 of LAG-3 and transmembrane domains of TLR-2, and has sequences encoding different intracellular signal transduction domains. A number of chimeric antigen receptor protein coding sequences were synthesized. The chimeric antigen receptor is an antigen recognition site that connects a pair of extracellular domains (No. 1 to No. 4) of LAG-3 to the human immunoglobulin G ₁ heavy chain constant region, respectively, TLR _- 2 It commonly contains a transmembrane domain, a TLR-4 signal peptide. The signal transduction domains were constructed with 8 different configurations, respectively. Table 1 below describes the composition of the eight types of signaling domains.

division Signaling domain configuration One TLR-3 / TLR4 2 CD3 zeta 3 TLR-3 / TLR4 / CD3 zeta 4 TLR-3 / TLR-4 / linker / FcεR1 γ chain ITAM motif 5
TLR-3 / TLR-4 / linker / FcεR1 γ chain ITAM motif / linker / FcεR1 γ chain ITAM motif / linker / FcεR1 γ chain ITAM motif 6 TLR-3 / TLR-4 / linker / FcγR2A α chain ITAM motif / linker / FcγR2A α chain ITAM motif / linker / FcγR2A α chain ITAM motif 7 TLR-3 / TLR-4 / linker/ FcεR1 β chain ITAM motif/ linker / FcεR1 β chain ITAM motif / linker / FcεR1 β chain ITAM motif 8 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 8 . 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 a recombinant protein obtained by fusion of extracellular domains 1 to 4 of LAG-3 of the 8 types of chimeric antigen receptors prepared in Example 1 and a macrophage signaling protein, the gene is lenti It was cloned into a virus-derived expression vector, pCDH-CMV-MCS-EF1-copGFP (System Biosciences). First, for cloning into the pCDH-CMV-MCS-EF1-copGFP vector, a primer that creates a cleavage site sequence of restriction enzyme Xba I at the 5' end was synthesized to create a restriction enzyme cleavage site by polymerase chain reaction, and also at the 3' end. Restriction enzyme cleavage site was created by polymerase chain reaction by synthesizing a primer that creates a cleavage site sequence of restriction enzyme Not I. After that, the 5' end of the gene having a restriction enzyme cleavage site was treated with Xba I, and the 3' end was treated with Not I due to the polymerase chain reaction. And the multicloning site of the expression vector was treated with Xba I and Not I to allow the gene to be inserted. After mixing the restriction enzyme-treated gene with the expression vector, it was ligated by treatment with a ligase.

As a result, a recombinant vector expressing 8 types of proteins in which extracellular domains 1 to 4 of LAG-3 having the structure of FIGS. 17 to 24 and a novel signal transduction protein were fused was completed (see FIGS. 9 to 16) .

Example 3. HLA screening of human cell lines expressing class II

In order to screen human cell lines expressing HLA class II on the cell surface, acute promyelocytic leukemia-derived cell line HL-60 (ATCC) and T cell lymphoma-derived cell line Jurkat (ATCC) Expression of HLA class II was confirmed in the cell line. To confirm expression, an antibody (biolegend) capable of binding to HLA class II to which a fluorescent protein is attached was treated in the cell line, and then flow cytometry (fluorescence-activated cell sorting) was used. As a result of the analysis, it was confirmed that HLA class II was expressed in the HL-60 cell line, and it was confirmed that the Jurkat cell line did not express HLA class II (see FIG. 25 ).

Based on the above results, the HL-60 cell line expressing HLA class II among the two cells was used as a target cell, and Jurkat cells not expressing HLA class II were used as a negative control.

Example 4. chimeric antigen receptors and HLA Affinity measurement of human cell lines expressing class II

In order to confirm that the constructed chimeric antigen receptor has affinity with a human cell line expressing HLA class II, each of the extracellular domains (domains 1 to 4) of LAG-3 is human immunoglobulin G ₁ heavy chain constant. By linking with the region (IgG ₁ heavy chain constant region), two extracellular domains of LAG-3 (domains 1 to 4) per chimeric antigen receptor make the chimeric antigen receptor water-soluble After expression in Chinese hamster ovary (CHO) cells, the protein was purified by affinity chromatography (GE healthcare) using a protein A column (see FIG. 26a ).

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. 26b).

Thereafter, the purified water-soluble chimeric antigen receptor was treated with a cell line expressing HLA class II (HL-60) and a cell line not expressing HLA class II (Jurkat) to prevent the water-soluble chimeric antigen receptor from binding to the cell. The results confirmed through flow cytometry analysis are shown in FIG. 26C .

As shown in FIG. 26c , it was confirmed that HL-60 cells expressing MHC class II bound to the water-soluble chimeric antigen receptor, but Jurkat cells not expressing MHC class II 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 a high affinity with the human cell line expressing HLA class II, which was the MHC class II of the immune cell therapy prepared with HL-60 cells and Jurkat cells. This means that it can be used for specific cytotoxicity verification of proteins.

Example 5. chimeric Antigen receptor expression specialized antigen-expressing cell production

A lentivirus system using 293FT cells (Thermo Fisher Scientific) was used to introduce the 8 recombinant vectors prepared in Example 2 into THP-1 cells (human macrophage cell line), a type of specialized antigen-expressing cells.

First, 293FT cells were inoculated to become 2.5Х10 ⁶ cells in a 100ð 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 eight 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. The transduction efficiency was measured by using flow cytometry to measure the GFP expression level inside the cells, and compared the transduction ratio of THP-1 cells transduced with an empty vector (Mock) for 8 types of transduced THP-1 cells. is shown in FIG. 27 .

Example 6. MHC Cells expressing class II ( MHC class II ⁺ cell) co-culture through MHC class II ⁺ Cell-specific cytotoxicity verification

In order to check whether the produced 8 types of chimeric antigen receptor-expressing THP-1 cells selectively recognize MHC class II-expressing cells (MHC class II ⁺ cells) and show toxicity, the target cells selected in Example 3, HL- 60 cells were co-cultured with 8 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 8 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 MHC class II ⁺ cell (HL-60), a target cell, was dispensed by 1Х10 ⁵ to effector: Co-culture was performed with a target 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 the prepared chimeric antigen receptor-expressing THP-1 cells was quantified by measuring the proportion of cells showing fluorescence by 7-AAD using flow cytometry.

As a result, as shown in FIG. 28 , when the prepared chimeric antigen receptor-expressing THP-1 cells were co-cultured with HL-60 cells expressing MHC class II (MHC class II ⁺ cell), when 24 hours had elapsed, the chimeric THP-1 cells that do not express antigen receptors (Mock/THP-1) showed about 5.21% cytotoxicity, whereas 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 cell (TLR-3+TLR4+FcγR2A ITAM monomer+FcεRIβ ITAM monomer+FcγR2Aα ITAM monomer) showed very high cytotoxicity at 20.5%, 15.9%, 16.7%, and 20.6%, respectively. In addition, it was confirmed that THP-1 cells transduced with different types of intracellular signaling domains had higher cytotoxicity compared to Mock.

In addition, as shown in FIG. 29 , when the cytotoxicity of HL-60 cells co-cultured after 48 hours of co-culture was confirmed, Mock showed about 8.78% cytotoxicity, whereas 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) and 3-4-ABC THP-1 cells (TLR-3+TLR4+FcγR2A ITAM monomer+FcεRIβ ITAM monomer+FcγR2Aα ITAM monomer) were 64.0%, 62.7%, 52.1%, and 49.8, respectively. % was confirmed to show a very high cytotoxicity. In addition, it was confirmed that THP-1 cells transduced with different types of intracellular signaling domains had higher cytotoxicity compared to Mock.

Through the above results, the transformed THP-1 cells containing the chimeric antigen receptor according to the present invention showed cytotoxicity specific to the MHC class II protein, and thus, using the transformed antigen-specific specialized antigen-expressing cells It was confirmed that cancer cell treatment 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.

<110> IMMUNOLOGICAL DESIGNINGLAB CO., LTD <120> LAG-3.CAR-MAC <130> KP21-0025-ILDL <160> 22 <170> KoPatentIn 3.0 <210> 1 <211> 428 <212> PRT <213 > Artificial Sequence <220> <223> Extracellular domain of LAG-3 (#1 to #4) <400> 1 Leu Gln Pro Gly Ala Glu Val Pro Val Val Trp Ala Gln Glu Gly Ala 1 5 10 15 Pro Ala Gln Leu Pro Cys Ser Pro Thr Ile Pro Leu Gln Asp Leu Ser 20 25 30 Leu Leu Arg Arg Ala Gly Val Thr Trp Gln His Gln Pro Asp Ser Gly 35 40 45 Pro Pro Ala Ala Ala Pro Gly His Pro Leu Ala Pro Gly Pro His Pro 50 55 60 Ala Ala Pro Ser Ser Ser Trp Gly Pro Arg Pro Arg Arg Tyr Thr Val Leu 65 70 75 80 Ser Val Gly Pro Gly Gly Leu Arg Ser Gly Arg Leu Pro Leu Gln Pro 85 90 95 Arg Val Gln Leu Asp Glu Arg Gly Arg Gln Arg Gly Asp Phe Ser Leu 100 105 110 Trp Leu Arg Pro Ala Arg Arg Ala Asp Ala Gly Glu Tyr Arg Ala Ala 115 120 125 Val His Leu Arg Asp Arg Ala Leu Ser Cys Arg Leu Arg Leu Arg Leu 130 135 140 Gly Gln Ala Ser Met Thr Ala Ser Pro Pro Gly Ser Leu Arg Ala Ser 145 150 155 160 Asp Trp Val Ile Leu Asn Cys Ser Phe Ser Arg Pro Asp Arg Pro Ala 165 170 175 Ser Val His Trp Phe Arg Asn Arg Gly Gln Gly Arg Val Pro Val Arg 180 185 190 Glu Ser Pro His His His Leu Ala Glu Ser Phe Leu Phe Leu Pro Gln 195 200 205 Val Ser Pro Met Asp Ser Gly Pro Trp Gly Cys Ile Leu Thr Tyr Arg 210 215 220 Asp Gly Phe Asn Val Ser Ile Met Tyr Asn Leu Thr Val Leu Gly Leu 225 230 235 240 Glu Pro Pro Thr Pro Leu Thr Val Tyr Ala Gly Ala Gly Ser Arg Val 245 250 255 Gly Leu Pro Cys Arg Leu Pro Ala Gly Val Gly Thr Arg Ser Phe Leu 260 265 270 Thr Ala Lys Trp Thr Pro Pro Gly Gly Gly Pro Asp Leu Leu Val Thr 275 280 285 Gly Asp Asn Gly Asp Phe Thr Leu Arg Leu Glu Asp Val Ser Gln Ala 290 295 300 Gln Ala Gly Thr Tyr Thr Cys His Ile His Leu Gln Glu Gln Gln Leu 305 310 315 320 Asn Ala Thr Val Thr Leu Ala Ile Ile Thr Val Thr Pro Lys Ser Phe 325 330 335 Gly Ser Pro Gly Ser Leu Gly Lys Leu Leu Cys Glu Val Thr Pro Val 340 345 350 Ser Gly Gln Glu Arg Phe Val Trp Ser Ser Leu Asp Thr Pro Ser Gln 355 360 365 Arg Ser Phe Ser Gly Pro Trp Leu Glu Ala Gln Glu Ala Gln Leu Leu 370 375 380 Ser Gln Pro Trp Gln Cys Gln Leu Tyr Gln Gly Glu Arg Leu Leu Gly 385 390 395 400 Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser Pro Gly Ala Gln Arg Ser 405 410 415 Gly Arg Ala Pro Gly Ala Leu Pro Ala Gly His Leu 420 425 <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 Gl n 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 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 > 112 <212> PRT <213> Artificial Sequence <220> <223> Human CD3z Cytoplasmic domain <400> 5 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 6 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> ITAM motif <400> 6 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> 7 <211> 23 <212 > PRT <213> Artificial Sequence <220> <223> ITAM motif <400> 7 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> 8 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> ITAM motif <400> 8 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> 9 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Flexible linker <400> 9 Gly Gly Gly Gly Ser 1 5 <210> 10 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> hTLR-2 transmembrane domain <400> 10 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> 11 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> hTLR-4 Signal peptide <400 > 11 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> 12 <211> 1284 <212> DNA <213> Artificial Sequence <220> <223> Extracellular domain of LAG-3 (#1 to #4) <400> 12 ctccagccag gggctgaggt cccggtggtg tgggccca gg agggggctcc tgcccagctc 60 ccctgcagcc ccacaatccc cctccaggat ctcagccttc tgcgaagagc aggggtcact 120 tggcagcatc agccagacag tggcccgccc gctgccgccc ccggccatcc cctggccccc 180 ggccctcacc cggcggcgcc ctcctcctgg gggcccaggc cccgccgcta cacggtgctg 240 agcgtgggtc ccggaggcct gcgcagcggg aggctgcccc tgcagccccg cgtccagctg 300 gatgagcgcg gccggcagcg cggggacttc tcgctatggc tgcgcccagc ccggcgcgcg 360 gacgccggcg agtaccgcgc cgcggtgcac ctcagggacc gcgccctctc ctgccgcctc 420 cgtctgcgcc tgggccaggc ctcgatgact gccagccccc caggatctct cagagcctcc 480 gactgggtca ttttgaactg ctccttcagc cgccctgacc gcccagcctc tgtgcattgg 540 ttccggaacc ggggccaggg ccgagtccct gtccgggagt ccccccatca ccacttagcg 600 gaaagcttcc tcttcctgcc ccaagtcagc cccatggact ctgggccctg gggctgcatc 660 ctcacctaca gagatggctt caacgtctcc atcatgtata acctcactgt tctgggtctg 720 gagcccccaa ctcccttgac agtgtacgct ggagcaggtt ccagggtggg gctgccctgc 780 cgcctgcctg ctggtgtggg gacccggtct ttcctcactg ccaagtggac tcctcctggg 840 ggaggccctg acctcctggt gactggagac aatggcgact ttacccttcg actagag gat 900 gtgagccagg cccaggctgg gacctacacc tgccatatcc atctgcagga acagcagctc 960 aatgccactg tcacattggc aatcatcaca gtgactccca aatcctttgg gtcacctgga 1020 tccctgggga agctgctttg tgaggtgact ccagtatctg gacaagaacg ctttgtgtgg 1080 agctctctgg acaccccatc ccagaggagt ttctcaggac cttggctgga ggcacaggag 1140 gcccagctcc tttcccagcc ttggcaatgc cagctgtacc agggggagag gcttcttgga 1200 gcagcagtgt acttcacaga gctgtctagc ccaggtgccc aacgctctgg gagagcccca 1260 ggtgccctcc cagcaggcca cctc 1284 <210 > 13 <211> 702 <212> DNA <213> Artificial Sequence <220> <223> IgG1 heavy chain constant region <400> 13 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 gccgcgggag 240 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 300 ctgaatggca aggagtccacaa aggagtccag gtgcaacca tct 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> 14 <211> 429 < 212> DNA <213> Artificial Sequence <220> <223> hTLR-3 TIR domain <400> 14 tttgaatatg cagcatatat aattcatgcc tataaagata aggattgggt ctgggaacat 60 ttctcttcaa tggaaaagga agaccaatct ctcaaatttt gtctggaaga aagggacttt 120 gaggcgggtg tttttgaact agaagcaatt gttaacagca tcaaaagaag cagaaaaatt 180 atttttgtta taacacacca tctattaaaa gacccattat gcaaaagatt caaggtacat 240 catgcagttc aacaagctat tgaacaaaat ctggattcca ttatattggt tttccttgag 300 gagattccag attataaact gaaccatgca ctctgtttgc gaagaggaat gttaaatct 360 cactgcatct tgaactggcc agttg gttag catag 4 <211> 441 <212> DNA <213> Artificial Sequence <220> <223> hTLR-4 TIR domain <400> 15 aacatctatg atgcctttgt tatctactca agccaggatg aggactgggt aaggaatgag 60 ctagtaaaga atttagaaga aggggtgcct ccatttcagc tctgccttca ctacagagac 120 tttattcccg 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> 16 <211> 339 <212> DNA <213> Artificial Sequence <220 > <223> CD3 Human CD3z Cytoplasmic domain <400> 16 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240 cggaggggc a aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgctaa 339 <210> 17 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> ggttactactagactag gtga cagtagctataactagact gtga cagtagtataacgactac gtga c gcatgag 87 <210> 18 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> ITAM motif <400> 18 gacggcggct acatgactct gaaccccagg gcacctactg acgatgataa aaacatctac 60 ctgactctt 69 <210> 19 <211> 87 <212 > DNA <213> Artificial Sequence <220> <223> ITAM motif <400> 19 aaggttccag aggatcgtgt ttatgaagaa ttaaacatat attcagctac ttacagtgag 60 ttggaagacc caggggaaat gtctcct 87 <210> 20 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Flexible linker <400> 20 ggtggcggtg gctcg 15 <210> 21 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> hTLR-2 transmembrane domain <400> 21 gcactggtgt ctggcatgtg ctgtgctctg ttcctgctga tcctgtcctcac cgg 60 tgc 63 <210> 22 <211> 69 <212> DNA <213> Artificial Sequence <220 > <223> hTLR-4 Signal peptide <400> 22 atgatgtctg cctcgcgcct ggctgggact ctgatcccag ccatggcctt cctctcctgc 60gtgagacca 69

Claims (17)

A transformed cell comprising a chimeric antigen receptor (CAR), comprising:
The chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain and an intracellular signaling domain that specifically binds to HLA class II, and the cell is an antigen-specific professional antigen-presenting cell having a targeted effector activity. Transformed cells, including phagocytes, dendritic cells or naive B cells. According to claim 1,
The antigen-binding domain of the chimeric antigen receptor (CAR) is a transformed cell comprising the extracellular domain of LAG-3 (No. 1 to No. 4) that specifically binds to HLA class II. 3. The method of claim 2,
The extracellular domain of the LAG-3 (No. 1 to No. 4) will include the amino acid sequence shown in SEQ ID NO: 1, the transformed cell. According to claim 1,
The antigen binding domain is characterized in that a pair of extracellular domains (No. 1 to No. 4) of LAG-3 are connected to each human immunoglobulin G ₁ heavy chain constant region in the form of a dimer (IgG ₁ heavy chain constant region). A chimeric antigen receptor. 5. The method of 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 for activating antigen-specific professional antigen-presenting cells, and is TLR-3-TLR-4, CD3 zeta (zeta) or TLR 3-TLR 4-CD3 zeta (zeta). converted cells. 7. The method of claim 6,
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. 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. 8. The method of claim 7,
The ITAM is a transformed cell that connects three with a linker. 7. The method of claim 6,
The TLR-3 is SEQ ID NO: 3; TLR-4 is SEQ ID NO: 4; And CD3 zeta (zeta) will include the amino acid sequence shown in SEQ ID NO: 5, the transformed cell. 8. The method of claim 7,
The ITAM is SEQ ID NO: 6; SEQ ID NO: 7; Or comprising the amino acid sequence represented by SEQ ID NO: 8, the transformed cell. 8. The method of claim 7,
Wherein the linker comprises the amino acid sequence represented by SEQ ID NO: 9, the transformed cell. According to claim 1,
The transmembrane domain is TLR-2, which comprises the amino acid sequence shown in SEQ ID NO: 10, the transformed cell. 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: 11. A pharmaceutical composition for the treatment of cancer expressing HLA class II comprising the cell of any one of claims 1 to 14 as an active ingredient. 16. The method of claim 15,
Cancer expressing the HLA class II is head and neck squamous cell carcinoma, thymoma, esophageal squamous cell carcinoma, melanoma, breast cancer ), colorectal cancer, ovarian cancer, prostate cancer, neuroblstoma, glioma, non-small cell lung cancer, classic Hodgkin Lymphoma (classic Hodgkin lymphoma), T-cell leukemia/lymphoma, B-cell lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, acute A composition selected from the group consisting of acute myeloid leukemia and acute lymphoid leukemia. A cell therapeutic agent comprising the cell of any one of claims 1 to 14 as an active ingredient.

Images