Novel chimeric antigen receptors and immune cells expressing them

Abstract

The present invention relates to a novel chimeric antigen receptor that contains an intracellular domain of a receptor that includes a death domain as an intracellular signaling domain, and an immune cell expressing the same. In the case of immune cells expressing the chimeric antigen receptor of the present invention, in an environment in which normal cells are present, the immune cells are apoptotically induced with little or no cytotoxicity, ensuring stability against normal cells, whereas in an environment in which target cells are present, the immune cells exhibit stronger cytotoxicity than existing techniques using a single chimeric antigen receptor.

Description

[Technical Field]

[0001] This invention relates to the intracellular domain of a receptor that includes a death domain. This paper relates to a novel chimeric antigen receptor containing an intracellular signaling domain and immune cells that express it. [Background technology]

[0002] Cancer treatment methods have undergone continuous development and evolution, and methods such as surgery, chemotherapy, and radiation therapy continue to be used today. However, most of these existing cancer treatments are only effective in the early stages when cancer has not yet metastasized. In cases where metastasis has already progressed, there is a high possibility of recurrence even after surgery. Therefore, research into methods that use immune responses to treat cancer has been ongoing recently.

[0003] Among these, there is growing interest in cell therapy methods that use immune cells to enhance them or genetically modify them and reinject them into patients. For example, tumor-infiltrating lymphocytes (TILs), chimeric antigen receptors (CARs), and T-cell receptors (TCRs) are being researched. In particular, T cells and Artificial receptors designed to transmit antigen specificity to natural killer cells (NK cells) In the case of chimeric antigen receptors, the receptor consists of an extracellular domain, a transmembrane domain, and an intracellular signaling domain, which can activate immune cells and provide specific immunity by binding to cancer cell-specific antigens. T cells expressing such chimeric antigen receptors are named CAR-T cells (Kershaw et al., Nat. Rev. Immunol., 5(12):928-940 (2005); Restifo et al., Nat. Rev. Immunol., 12(4):269-281 (2012)), while natural killer cells expressing such receptors are named CAR-NK cells.

[0004] The intracellular signaling domain of the aforementioned chimeric antigen receptor is primarily based on the intracellular signaling domain of CD3zeta, the signaling subunit of the T cell receptor (1st generation CAR). This has evolved to include the addition of intracellular signaling domains of co-stimulatory molecules that promote the growth and differentiation of immune cells. For example, currently available CAR-T cell therapies utilize the intracellular signaling domains of CD28 and 4-1BB co-stimulatory molecules, respectively (2nd generation CAR), and subsequently, CARs containing both CD28 and 4-1BB intracellular signaling domains simultaneously (3rd generation CAR) have been attempted (Stegen et al., Nat. Rev. Drug Discov., 14(7):499-509 (2015)).

[0005] However, while therapeutic agents like CAR-T are effective against cancer cells, they have sometimes caused side effects due to partially nonspecific attacks on healthy tissue. To overcome this, research continues to enhance the cancer cell antigen recognition ability and minimize the side effects of attacking normal cells. However, once CAR-T is injected, toxic T cells can persist their function even after the cancer cells are removed, causing cytotoxicity to normal cells as well, and side effects due to nonspecific attacks remain a problem. Furthermore, cancer recurrence is caused by the lack of specificity, diversity, and heterogeneity of antigens present in cancer cells, so in order to improve anticancer efficacy against this, "logic-gated CAR enhancement" technology, that is, Under the current circumstances, there is also a need for the development of a technology that improves the efficacy against cancer by designing CARs that multi-target antigens and secures safety against normal cells. Summary of the Invention Problem to be Solved by the Invention

[0006] An object of the present invention is to provide a novel chimeric antigen receptor that, when expressed in immune cells, does not exhibit cytotoxicity against normal cells and can amplify cytotoxic activity against cancer cells.

[0007] Another object of the present invention is to provide a polynucleotide and an expression vector for expressing the chimeric antigen receptor.

[0008] Another object of the present invention is to provide an immune cell that expresses the chimeric antigen receptor on its surface, which has an excellent therapeutic effect on cancer and is safe for normal cells, and thus can be usefully used as a cancer therapeutic agent.

[0009] Another object of the present invention is to provide a pharmaceutical composition for treating cancer using the immune cells. Means for Solving the Problem

[0010] To achieve the above object, one aspect of the present invention provides a chimeric antigen receptor (CAR) comprising: an extracellular domain comprising an antigen binding site (extracellular binding domain); a transmembrane domain; and an intracellular signaling domain, said intracellular signaling domain comprising an intracellular domain of a receptor that includes a death domain, .

[0011] In another aspect of the present invention, there are provided a polynucleotide comprising a nucleotide sequence encoding the chimeric antigen receptor, and an expression vector comprising the polynucleotide.

[0012] In another aspect of the present invention, there is provided an immune cell which expresses said chimeric antigen receptor as a first chimeric antigen receptor on the surface thereof, and further expresses on the surface a second chimeric antigen receptor, wherein the second chimeric antigen receptor comprises an extracellular domain containing an antigen binding site that specifically binds to an antigen expressed in cancer cells, a transmembrane domain, and an intracellular signaling domain.

[0013] In another aspect of the present invention, there is provided a pharmaceutical composition for treating cancer, comprising the immune cell. Effects of the Invention

[0014] The chimeric antigen receptor of the present invention is capable of transmitting signals caused by antigens that are expressed in both normal cells and cancer cells. When signals are transmitted by the chimeric antigen receptor alone, immune cells expressing the receptor can be rendered to exhibit no cytotoxicity or lower cytotoxicity. Furthermore, when the receptor binds to an antigen for a prolonged period, it can induce cell death of the immune cell, thereby exhibiting no cytotoxicity against normal cells rather than cancer cells, which has the advantage of ensuring stability.

[0015] On the other hand, when said chimeric antigen receptor binds to an antigen expressed in cancer cells, it acts in conjunction with another chimeric antigen receptor that can recognize and bind to a cancer cell-specific antigen to generate signal transduction. As a result, it can exhibit enhanced cytotoxicity compared to when only one cancer cell-specific chimeric antigen receptor acts, and exhibits a synergistic effect. Therefore, it has the effect of being extremely useful for use in the treatment of cancer.

[0016] However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned can be clearly understood by those skilled in the art from the following description. Brief Description of the Drawings

[0017] [Figure 1]This figure shows a schematic diagram of the structure of the chimeric antigen receptor of the present invention that targets EGFR as an antigen, and the structure of a vector for inserting a polynucleotide that encodes the chimeric antigen receptor in order to express it. [Figure 2] This figure shows the results of confirming the expression levels of Myc and zsGreen in Dual-CAR NK92 cells 1, which were created by expressing the chimeric antigen receptor of the present invention in natural killer cells (α-Cot-CAR NK92(M2)) that express the anti-cotinine chimeric antigen receptor. In Figure 2, "M2-EGFR-P75NTR" is a natural killer cell that expresses the dual chimeric antigen receptor, and #1, #2, #3, and #5 are figures of natural killer cells expressing the dual chimeric antigen receptor of M2-EGFR-P75NTR separated into single cells. [Figure 3a] This figure shows the results of examining the expression levels of Myc and zsGreen in Dual-CAR NK92 cells 2, which were created by expressing the chimeric antigen receptor of the present invention in natural killer cells (α-EphA2-CAR NK92(79-14)) expressing the anti-EphA2 chimeric antigen receptor. [Figure 3b] This figure shows the results of examining the expression levels of Myc and zsGreen in Dual-CAR NK92 cells 2, which were created by expressing the chimeric antigen receptor of the present invention in natural killer cells (α-EphA2-CAR NK92(79-14)) expressing the anti-EphA2 chimeric antigen receptor. [Figure 4] The results show a comparison of cytotoxicity after co-culturing Dual-CAR NK92 cells 1 and α-Cot-CAR NK92(M2) cells with EGFR-expressing breast cancer cells AU565. Graph A compares the cytotoxicity of all cells, while graph B compares the cytotoxicity of single cells. [Figure 5a] This graph shows the results of co-culturing Dual-CAR NK92 cells 1 and α-Cot-CAR NK92(M2) cells with EGFR-expressing breast cancer cells AU565, and comparing the rate of cell death (apoptosis) over time. [Figure 5b] This graph shows the results of co-culturing Dual-CAR NK92 cells 1 and α-Cot-CAR NK92(M2) cells with EGFR-expressing breast cancer cells AU565, and comparing the rate of cell death (apoptosis) over time. [Figure 6] The graphs show the results of co-culturing EGFR and HER2-expressing breast cancer cells AU565 with HER2-Cot zygotes using Dual-CAR NK92 cells 1 and α-Cot-CAR NK92(M2) cells, and then comparing their cytotoxicity. Graph A compares the cytotoxicity of all cells, and graph B compares the cytotoxicity of single cells. [Figure 7] This figure compares the cytotoxicity observed after co-culturing Dual-CAR NK92 cells and α-Epha2-CAR NK92(79-14) cells with MDA-MB-231 breast cancer cells that fully express EGFR and EphA2. [Figure 8] This graph compares the incidence of cell death (apoptosis) after 17 hours of sclerosis, following co-culture of Dual-CAR NK92 cells and α-Epha2-CAR NK92(79-14) cells with MDA-MB-231 breast cancer cells that fully express EGFR and EphA2. [Figure 9a] This diagram shows a schematic representation of the structure of a chimeric antigen receptor (p75NTRDDD) in which the dead region of the intracellular domain of p75NTR has been removed, and the structure of a vector for inserting a polynucleotide that encodes the chimeric antigen receptor in order to express it. [Figure 9b] This diagram shows a schematic representation of the structure of a chimeric antigen receptor (p75NTRDDD) in which the dead region of the intracellular domain of p75NTR has been removed, and the structure of a vector for inserting a polynucleotide that encodes the chimeric antigen receptor in order to express it. [Figure 10]This figure shows the results of confirming the expression level of Myc in Dual-CAR NK92 cells, which were created by expressing the chimeric antigen receptor (p75NTRDDD) of the present invention, in which the dead region of the intracellular domain of p75NTR has been removed, in natural killer cells (α-Cot-CAR NK92(M2)) that express the anti-cotinine chimeric antigen receptor. [Figure 11] This figure compares the cytotoxicity observed after co-culturing EGFR and HER2-expressing breast cancer cells AU565 with HER2-Cot zygotes using Dual-CAR NK92 cells (p75NTRDDD), Dual-CAR NK92 cells 1, and α-Cot-CAR NK92(M2) cells, which were produced by expressing a chimeric antigen receptor (p75NTRDDD) in which the dead region of the intracellular domain of p75NTR was removed. [Figure 12a] This graph compares the rate of cell death (apoptosis) observed after co-culturing EGFR-expressing breast cancer cells AU565 using Dual-CAR NK92 cells (created by expressing a chimeric antigen receptor (p75NTRDDD) in which the cell death region of the intracellular domain of p75NTR has been removed), Dual-CAR NK92 cells 1, and α-Cot-CAR NK92(M2) cells. [Figure 12b] This graph compares the rate of cell death (apoptosis) observed after co-culturing EGFR-expressing breast cancer cells AU565 using Dual-CAR NK92 cells (created by expressing a chimeric antigen receptor (p75NTRDDD) in which the cell death region of the intracellular domain of p75NTR has been removed), Dual-CAR NK92 cells 1, and α-Cot-CAR NK92(M2) cells. [Figure 13] This diagram schematically illustrates the principle of action of Dual-CAR NK92 expressing the chimeric antigen receptor of the present invention. [Modes for carrying out the invention]

[0018] The present invention will be described in detail below.

[0019] 1. Novel chimeric antigen receptors (CARs), polynucleotides for expressing them, and expression vectors. One aspect of the present invention is to provide a novel chimeric antigen receptor that exhibits safety in normal cells but amplifies cytotoxicity in cancer cells in conjunction with other chimeric antigen receptors.

[0020] In this invention, the term "chimeric antigen receptor (CAR)" refers to a synthetic complex designed to recognize and bind to a target antigen and cells expressing the antigen, thereby inducing an immune response against them. The chimeric antigen receptor comprises an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain. The chimeric antigen receptor may include an intracellular signaling domain. Chimeric antigen receptors are expressed on the surface of immune cells and, via an antigen-binding site contained in the extracellular domain, recognize and bind to specific antigens, such as antigens specifically expressed on the surface of cancer cells. This can cause signal transduction within immune cells and alter the activity of immune cells, thus enabling the induction of an immune response targeting only specific antigens.

[0021] The chimeric antigen receptor of the present invention comprises an extracellular binding domain containing an antigen-binding site; a transmembrane domain; and an intracellular domain containing a death domain. It includes the intracellular signaling domain.

[0022] The receptor containing the aforementioned death domain is responsible for cell death (apopto). A receptor molecule that has an amino acid sequence involved in sis-inducing activity as its intracellular domain. It tastes like this. Examples of receptors containing the aforementioned death region include Fas, TNFRI, and p75. NTR These include DR3, DR4, DR5, DR6, EDAR, etc., but are not limited to these. In particular, of these, see p75 NTR The p75 neurotrophin receptor is a type of neurotrophin receptor involved in the differentiation of nerve cells, and is known as a type of low-affinity nerve growth factor receptor (LNGFR). NTR It consists of an extracellular domain, a transmembrane domain, and an intracellular domain, and in the present invention, the p75 NTR The intracellular domain is used. (See p75) NTR The intracellular domain may be a global-like domain, and specifically, it may be a protein containing the amino acid sequence of Sequence ID No. 1, but is not limited thereto. The aforementioned amino acid sequence may include variants having different sequences due to deletions, insertions, substitutions, or combinations thereof of amino acid residues, as long as they do not affect the structure, function, or activity of the protein containing it. Furthermore, the amino acid sequence may include amino acids that have undergone commonly known modifications in the art, such as phosphorylation, sulfation, acrylation, glycosylation, methylation, and farnesylation. p75 of the present invention NTRThe intracellular domain of includes not only the aforementioned amino acid sequence, but also sequences that are substantially identical to it or variants thereof. The meaning of substantially identical amino acid sequence is that it may include, but is not limited to, sequences that have 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more homology with the aforementioned amino acid sequence.

[0023] When the chimeric antigen receptor of the present invention is expressed on the surface of immune cells, the target antigen binds to the receptor, as described in p75. NTR When signal transduction occurs solely by the intracellular domain of the present invention, it may induce a reduction in the cytotoxicity of the immune cells and / or accelerate the cell death of the immune cells. The reduction in cytotoxicity may be such that the immune cells do not exhibit cytotoxicity, exhibit less cytotoxicity than that exhibited by immune cells in the absence of the antigen, or exhibit less cytotoxicity than when the immune cells are activated by the antigen. The acceleration of cell death (apoptosis) of the immune cells may be such that cell death is more induced than in immune cells in the absence of the antigen. The occurrence of signal transduction solely by the present invention may be such that signal transduction by other chimeric antigen receptors does not occur, and more specifically, signal transduction by chimeric antigen receptors that generate signals to promote the cytotoxicity of immune cells against cancer cells does not occur, and only signal transduction by the chimeric antigen receptor of the present invention occurs.

[0024] The extracellular domain comprises an antigen-binding site that specifically binds to a target antigen of interest, and is not limited to any particular type. The extracellular domain can be applied without limitation as long as it has a structure capable of specifically recognizing and binding to biological molecules, such as cell surface receptors, tumor proteins, lipids, polysaccharides, or other cell surface target molecules, and may for example be a protein, polypeptide, or a variant thereof having the aforementioned characteristics. Said "specific binding" may mean binding to another molecule with a greater binding affinity than background binding; for example, the extracellular domain has an affinity of about 10 for the target antigen -5 M or higher, or binds to the target antigen with Ka (the equilibrium dissociation constant of a specific binding interaction having units of 1 / M). Said affinity is such that the equilibrium dissociation constant (Kd) of the specific binding interaction having units of M is 10 -5 M to 10 -13 M, or may be equal to or lower than said range.

[0025] The antigen-binding site may be one that specifically binds to an antigen expressed in both cancer cells and normal cells. Specifically, the antigen expressed in both said cancer cells and normal cells may not be a cancer cell-specific antigen, that is, it may not be a cancer-specific marker or the like used for preventing, treating, ameliorating, or diagnosing cancer. Accordingly, the chimeric antigen receptor of the present invention can recognize and bind to both cancer cells and normal cells, thereby inducing signal transduction in immune cells in which said chimeric antigen receptor is expressed. The antigen-binding site may, for example, be one that specifically binds to EGFR, but is not limited thereto.

[0026] The antigen-binding site may be an antibody or an antigen-binding fragment thereof; more specifically, the antigen-binding site is a single chain variable fragment of an antibody (single chain variable f It may be a fragment (scFv). Specifically, the antigen-binding site may be a single-chain variable fragment of an antibody that specifically binds to EGFR, and more specifically, it may include the amino acid sequence of SEQ ID NO: 3.

[0027] The aforementioned antibody refers to an immunoglobulin molecule that is immunologically reactive by specifically binding to the epitope of an antigen. The aforementioned antibody may be a monoclonal antibody, a polyclonal antibody, or a full-length antibody (an antibody having a full-length chain structure). The antibody may include, at least, a functional fragment (antigen-binding fragment) having antigen-binding function and a recombination antibody, and specifically, the antibody of the present invention may be a monoclonal antibody or its antigen-binding fragment. The monoclonal antibody refers to an antibody molecule with a single molecular composition obtained from substantially the same antibody population, and such a monoclonal antibody exhibits single-binding specificity and affinity to a particular epitope. The full-length antibody has a structure having two full-length light chains and two full-length heavy chains, each light chain may be linked to a heavy chain by a disulfide bond. The antibody comprises a heavy chain (HC) and a light chain (LC) polypeptide, and the heavy chain and light chain may include a variable region and an invariant region.

[0028] The invariant region is a site that mediates the binding of the antibody to various types of cells in the immune system (such as T cells) and host tissues containing components of the complement system. The invariant region performs the same function regardless of the type of antigen for antibodies of the same species, and the amino acid sequences comprising it are the same or highly similar for each antibody. The invariant region is divided into a heavy chain invariant region (which can be abbreviated as CH) and a light chain invariant region (which can be abbreviated as CL). The heavy chain invariant region has gamma (γ), mu (μ), alpha (α), delta (δ), and / or epsilon (ε) types, and has subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and / or alpha 2 (α2). The light chain invariant region has kappa (κ) and lambda (λ) types. IgG is a subtype and includes IgG1, IgG2, IgG3, and IgG4.

[0029] The aforementioned variable region is an antibody site specific to the antigen, and is divided into a heavy chain variable region (which can be abbreviated as VH) and a light chain variable region (which can be abbreviated as VL). The aforementioned variable region contains three CDRs (complementary-determining regions). It includes the ons (or complementarity determination region) and four FR (framework regions). It may be included. The CDR may be a cyclic region involved in antigen recognition, and its specificity for the antigen may be determined according to the amino acid sequence of the CDR. The CDR may be referred to as CDR1, CDR2, CDR3, etc., depending on whether it is a heavy chain or light chain polypeptide CDR, and may be referred to as CDR-H1, CDR-H2, CDR-H3 if it is a variable region of the heavy chain, and as CDR-L1, CDR-L2, CDR-L3 if it is a variable region of the light chain. Similarly, the FR may be referred to as FR-H1, FR-H2, FR-H3, FR-H4 if it is a variable region of the heavy chain, and as FR-L1, FR-L2, FR-L3, FR-L4 if it is a variable region of the light chain.

[0030] The antigen-binding fragment refers to any fragment of an antibody that possesses the antigen-binding function of the antibody. The antigen-binding fragment may be referred to interchangeably with terms such as "fragment" or "antibody fragment," and may be, but is not limited to, Fab, Fab', F(ab')2, Fv, etc.

[0031] The Fab has a structure having variable regions of the light chain and heavy chain, an invariant region of the light chain and a first invariant region (CH1 domain) of the heavy chain, and has one antigen-binding site. The Fab' has a hinge region (hinge) containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. It differs from the aforementioned Fab in that it has a region. 2 Fab's Cysteine ​​residues in the hinge region are formed by disulfide bonds. Fv refers to the smallest antibody fragment having only the variable region of the heavy chain and the variable region of the light chain. The two-chain variable fragment is non-covalently bonded. The variable regions of the heavy chain and the variable regions of the light chain are linked, and the single chain variable fragment is Ain variable fragments (scFv) generally have a structure similar to a dimer, where the variable region of the heavy chain and the variable region of the light chain are covalently linked via a peptide linker or directly linked at the C-terminus. The antigen-binding fragment can be produced using a proteolytic enzyme (for example, restrictive cleavage of the entire antibody with papain yields Fab, and cleavage with pepsin yields the F(ab')2 fragment), or by gene rearrangement techniques, but is not limited to these methods.

[0032] The linker may be a peptide linker and may have a length of about 10 to 25 amino acids. For example, the linker may contain a new aqueous amino acid such as glycine (G) and / or serine (S). The linker may include, for example, (GS)n, (GGS)n, (GSGGS)n, or (GnS)m (n and m are each 1 to 10), for example, (GnS)m (n and m are each 1 to 10), but is not limited thereto.

[0033] The extracellular domain consists of a hinge domain and a spacer. Select at least one from a group consisting of domains (spacer domains) These may also be included. The antigen-binding site of the extracellular domain may be linked to the transmembrane domain via a hinge domain and / or a spacer domain.

[0034] The hinge domain is a portion that allows the antigen-binding site to be physically separated from the surface of the immune cell expressing the chimeric antigen receptor, enabling appropriate cell / cell contact, appropriate antigen / antigen-binding site binding, and appropriate activation of the chimeric antigen receptor, and can play an important role in positioning the extracellular domain. The chimeric antigen receptor may contain one or more hinge domains between the extracellular domain and the transmembrane domain. The hinge domain may be derived from natural, synthetic, semi-synthetic, or recombination sources. The hinge domain may contain the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. The modified hinge region refers to (a) a naturally occurring hinge region having up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), (b) a portion of a naturally occurring hinge region of at least 10 amino acids (e.g., at least 12, 13, 14, or 15 amino acids) in length having up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), or (c) a portion of a naturally occurring hinge region containing a core hinge region (which may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length). In certain embodiments, one or more cysteine ​​residues in the spontaneously occurring immunoglobulin hinge region may be one or more other amino acid residues (e.g., one or more The above serine residue may be substituted. The altered immunoglobulin hinge region may optionally or additionally have other amino acid residues, for example, a proline residue in the wild-type immunoglobulin hinge region substituted with cysteine. The hinge domain may be a hinge region derived from the extracellular domain of a type 1 membrane protein such as CD8, CD4, CD28, and CD7, but any can be used without limitation as long as it can link the antigen-binding site, the transmembrane domain, and the intracellular signaling domain between the cell membrane. Furthermore, this may be a wild-type hinge region from these molecules, or it may be altered.

[0035] The spacer domain may be referred to as a linking domain, and may, for example, include a hinge domain derived from CD28 and / or a hinge domain derived from CD8, and may include all or part of the hinge domain derived from CD28 and / or CD8.

[0036] The hinge domain and / or spacer domain may be at least one selected from the group consisting of a Myc epitope, a CD8 hinge domain, and Fc, and more specifically, may include a Myc epitope and a CD8 hinge domain. More specifically, the Myc epitope may include the amino acid sequence of SEQ ID NO: 5, and the CD8 hinge domain may include the amino acid sequence of SEQ ID NO: 7.

[0037] The aforementioned transmembrane domain is an extracellular domain This refers to a region that links and fuses the in and intracellular signaling domains to each other, thereby fixing the chimeric antigen receptor to the plasma membrane of immune cells. The transmembrane domain may be derived from natural, synthetic, semi-synthetic, or recombination sources. The transmembrane domain may be, but is not limited to, one selected from the group consisting of the alpha (α), beta (β), or zeta (ζ) chains of the T-cell receptor (TCR), CD28, CD3 epsilon (ε), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0038] The transmembrane domain may be attached to the extracellular domain via a linker. For example, the linker may be a short oligopeptide or polypeptide linker with a length of 2 to 10 amino acids, and may, for example, be a glycine (G)-serine (S) doublet, but is not limited thereto.

[0039] In a specific embodiment of the present invention, a chimeric antigen receptor was designed that includes a single-chain variable fragment (scFv) of an antibody that specifically binds to EGFR as an extracellular domain, CD28 as a transmembrane domain, and the single-chain variable fragment and CD28 linked by Myc and a hinge domain. Then, p75, one of the receptors that includes the death region in the transmembrane domain, was designed. NTR The intracellular domain of the present invention was linked with an intracellular signaling domain to produce the chimeric antigen receptor of the present invention.

[0040] As mentioned above, the chimeric antigen receptor of the present invention is p75 NTR When signal transduction occurs independently by the intracellular domain of a receptor containing a death region, as in the example above, it may be possible to induce a reduction in the cytotoxicity of immune cells and / or accelerate the cell death of said immune cells. Therefore, when normal cells are present, if the antigen-binding site of the chimeric antigen receptor recognizes and binds to an antigen expressed from normal cells, it may be possible to induce a reduction in the cytotoxicity of immune cells and / or accelerate the cell death of said immune cells through signal transduction. Since it does not have cytotoxicity to normal cells, it can be usefully used as a stable chimeric antigen receptor.

[0041] Another aspect of the present invention is the polynucleotide and for expressing the chimeric antigen receptor. We provide an expression vector.

[0042] The polynucleotide comprises a base sequence that encodes the chimeric antigen receptor.

[0043] In this invention, the term "polynucleotide" comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and the basic structural unit, the nucleoside, includes not only natural nucleosides but also analogues in which the sugar or base site has been altered.

[0044] Encrypting the chimeric antigen receptor means that the polynucleotide has encoded genetic information that allows for the synthesis of a protein having the amino acid sequence of the chimeric antigen receptor of the present invention through normal protein expression processes such as transcription and translation. In this case, the scope of the present invention may include not only proteins having the exact same amino acid sequence as the chimeric antigen receptor, but also, as mentioned above, proteins having substantially the same amino acid sequence as the protein, and polynucleotides that encode proteins having the same and / or similar activity as the protein.

[0045] Specifically, the polynucleotide of the present invention may include an intracellular domain of a receptor containing a death region, for example, p75 NTR It may contain a nucleotide sequence that encodes the intracellular domain of, and more specifically, it may contain the nucleotide sequence of Sequence ID No. 2.

[0046] Furthermore, the polynucleotide of the present invention may include an intracellular domain of a receptor containing a death region, for example, p75 NTR The sequence may include not only the nucleotide sequence that encodes the intracellular domain, but also the nucleotide sequence that encodes the transmembrane domain and / or the extracellular domain linked thereto.

[0047] See p75 NTR The explanation for the chimeric antigen receptor, including the intracellular signaling domain, transmembrane domain, and extracellular domain, which include the intracellular domain of the receptor containing the death region, is the same as that described above.

[0048] The polynucleotide may contain the base sequence of Sequence ID No. 13. In one specific embodiment of the present invention, the extracellular domain includes a single-chain variable fragment (scFv) of an antibody that specifically binds to EGFR, the transmembrane domain includes CD28, the single-chain variable fragment and CD28 are linked by Myc and a hinge domain, and the transmembrane domain contains p75, which is one of the receptors that includes the death region. NTRA chimeric antigen receptor was designed in which the intracellular domain of was linked by an intracellular signaling domain, and a polynucleotide containing the base sequence of Sequence ID No. 13 was produced to express it.

[0049] The polynucleotides of the present invention may include a nucleotide sequence substantially identical to the nucleotide sequence listed above. The substantially identical nucleotide sequence may include, for example, a nucleotide sequence that can synthesize the same amino acid when transcribed and translated, and may have, but is not limited to, a nucleotide sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more homology with the nucleotide sequence listed above.

[0050] The polynucleotides that encode the chimeric antigen receptor may include a base sequence optimized for the type of organism into which it is to be introduced and expressed, and for the expression system of said organism, such as transcription and translation. This is due to the degeneracy of codons, and there are a variety of nucleosyl combinations that can encode the resulting expressed protein. Codon sequences may exist, and all of these are within the scope of the present invention. The modification of the polynucleotide by optimizing the codons may be determined according to the type of organism to which the chimeric antigen receptor of the present invention is to be expressed and applied. For example, the polynucleotide of the present invention may be a polynucleotide modified to be optimized for codon selection in mammals and primates, and more specifically, it may be modified to be optimized for expression and action in humans.

[0051] The expression vector of the present invention comprises the aforementioned polynucleotide.

[0052] Since the polynucleotide contains a base sequence that encodes the chimeric antigen receptor of the present invention, the expression vector containing it may be used to produce the chimeric antigen receptor by expressing it, or it may be used to move the polynucleotide to a specific cell or organism so that the chimeric antigen receptor can be expressed, or to store and preserve the polynucleotide.

[0053] The expression vector can be constructed using prokaryotic or eukaryotic cells as a host.

[0054] For example, when the expression vector uses prokaryotic cells as its host, it typically includes a potent promoter capable of driving transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter), a ribosome binding site for translation disclosure, and a transcription / translation termination sequence. When E. coli (e.g., HB101, BL21, DH5α, etc.) is used as the host cell, E coli tryptophan Promoter and operator sites of the biosynthetic pathway (Yanofsky, C, J Bact) The eriol (1984) 158:1018-1024) and the left-directed promoter of phage λ (pLλ promoter, Herskowitz, I and Hagen, D, Ann Rev Genet, (1980) 14:399-445) may be used as regulatory sites. When Bacillus bacteria are used as host cells, the promoter of the Bacillus thuringiensis toxin protein gene (Appl Environ Microbiol (1998) 64:3932-3938; Mol Gen Genet (1996) 250:734-741) or any promoter expressible in Bacillus bacteria may be used as regulatory sites. The expression vector may be prepared by manipulating plasmids (e.g., pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), phages (e.g., λgt4·λB, λ-Charon, λΔz1, and M13, etc.), or viruses (e.g., SV40, etc.) that are sometimes used in the industry.

[0055] When the expression vector uses eukaryotic cells as its host, a promoter derived from the genome of a mammalian cell (e.g., metallothione promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., late adenovirus promoter, vaccinia virus 75K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rouss sacomavirus (RSV) promoter) may be used, and may generally have a polyadenylated sequence as the transcription termination sequence. The expression vector may have a CMV promoter.

[0056] Furthermore, the expression vector may be fused with other sequences to facilitate the purification of the antibodies expressed therefrom. Examples of fused sequences include glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6x His(hexahistidine;Quiag Examples include (en, USA). Furthermore, since the protein expressed by the expression vector of the present invention is a humanized antibody or its antigen-binding fragment, considering its characteristics, the expressed protein can be easily purified via a protein A column or the like without additional sequences for purification.

[0057] The expression vector may include antibiotic resistance genes commonly used in the industry as select labels, such as resistance genes to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, genethecin, neomycin, and tetracycline.

[0058] 2. Immune cells expressing dual-chimeric antigen receptors (Dual-CARs) Another aspect of the present invention is to provide immune cells that exhibit no cytotoxicity towards normal cells but more enhanced cytotoxicity towards cancer cells.

[0059] The aforementioned immune cells express the aforementioned chimeric antigen receptor of the present invention as a first chimeric antigen receptor on their surface, and also express a second chimeric antigen receptor on their surface, which includes an extracellular domain containing an antigen-binding site that specifically binds to antigens expressed on cancer cells, a transmembrane domain, and an intracellular signaling domain.

[0060] The first chimeric antigen receptor is the "1. Novel Chimeric Antigen Receptor (Chimeric The description of the chimeric antigen receptor is the same as that given in "Antigen receptor (CAR) and polynucleotides and expression vectors for expressing it." Specifically, the first chimeric antigen receptor may include an antigen-binding site that can specifically bind to antigens expressed in all normal and cancer cells, and the antigen to which the first chimeric antigen receptor binds may be different from the antigen to which the second chimeric antigen receptor binds.

[0061] The second chimeric antigen receptor can be described as follows:

[0062] Since the aforementioned second chimeric antigen receptor can typically target cancer cells and activate immune cells to induce an immune response, any second chimeric antigen receptor used as a cancer treatment agent can be used regardless of its type.

[0063] Specifically, the extracellular domain of the second chimeric antigen receptor includes an antigen-binding site that specifically binds to an antigen expressed on cancer cells. Specifically, the antigen may be a cancer cell-specific antigen, which may be a cancer-specific marker used to prevent, treat, improve, or diagnose cancer. That is, the cancer cell-specific antigen may be an antigen that is statistically expressed at a higher frequency on cancer cells than on normal cells, and therefore, the second chimeric antigen receptor can specifically recognize and bind to cancer cells more specifically than normal cells.

[0064] The antigen-binding site of the second chimeric antigen receptor can be applied without limitation as long as it can specifically bind to the cancer cell-specific antigen, for example, it may specifically bind to HER2, EphA2, ErbB3, IL-13Rα2, DLK1, B7H3, PD-L1, GPC3, CEACAM6, CD5, etc.

[0065] The extracellular domain of the second chimeric antigen receptor is described as follows, except for the description of the type of antigen to which the antigen-binding site specifically binds: "1. Novel Chimeric Antigen Receptor (Chimeric a CTIGEN receptor (CAR) and polynucleotides for expressing it The extracellular domain of the first chimeric antigen receptor is the same as described in "D and Expression Vectors," and for example, the antigen-binding site of the second chimeric antigen receptor may be a single-chain variable fragment of an antibody.

[0066] The description of the transmembrane domain of the second chimeric antigen receptor is the same as the description of the transmembrane domain of the first chimeric antigen receptor in "1. Novel Chimeric Antigen Receptors (CARs) and Polynucleotides and Expression Vectors for Expressing Them."

[0067] The intracellular signaling domain of the second chimeric antigen receptor is the portion that transmits signals generated by the binding of the chimeric antigen receptor to the immune cell in order to induce immune cell functions (for example, activation including the release of cytotoxic factors, cytokine production, proliferation and cytotoxic activity, or other cellular responses induced by the binding of the second chimeric antigen receptor to the antigen, etc.). The intracellular signaling domain may be part of a protein that transmits functional signals and instructs the cell to perform a specific function.

[0068] The intracellular signaling domain of the second chimeric antigen receptor may be an intracellular signaling domain that has been used in the development of existing chimeric antigen receptors. Specifically, it may contain only CD3ζ, which was used in first-generation CARs (chimeric antigen receptors). Furthermore, as used in second-generation CARs, a form in which CD3ζ is conjugated with a costimulatory domain (CD28 or CD137 / 4-1BB) may be used to improve responsiveness to immune cells. Also, as used in third-generation CARs, two or more costimulatory domains may be used, and in this case, the costimulatory domains may be conjugated with 4-1BB, CD28, or OX40, etc., to achieve expansion and persistence of immune cells, including in vivo CARs. In addition, as used in fourth-generation CARs, it may contain additional genes that encode cytokines such as IL-12 or IL-15, so that CAR-based immune proteins of cytokines are further expressed, and as used in fifth-generation CARs, it may further contain an interleukin receptor chain, such as IL-2Rβ, to enhance immune cells.

[0069] More specifically, the activation of immune cells by the intracellular signaling domain can be mediated by two different classes of intracellular signaling domains. For example, immune cell activation can be mediated by a primary signaling domain that initiates antigen-dependent primary activation and a costimulatory signaling domain that acts in an antigen-independent manner to provide a secondary signal. Therefore, the intracellular signaling domain can be a primary signaling domain. It may include an ng domain and a co-stimulatory signaling domain.

[0070] The primary signaling domain refers to a signaling domain that regulates immune cell activation through stimulation or suppression. Primary signaling domains that act through stimulation may include immune receptor tyrosine-based activation motives or signaling motives known in ITAMs. ITAMs containing primary signaling domains may include, but are not limited to, TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD66d, etc. More specifically, the primary signaling domain may be, but is not limited to, CD3ζ(zeta).

[0071] The aforementioned co-stimulatory signaling domain refers to the intracellular signaling domain of the co-stimulatory molecule. The co-stimulatory signaling domain may include, but is not limited to, a co-stimulatory signaling domain selected from the group consisting of ligands that specifically bind to CD2, CD7, CD27, CD28, CD30, CD40, 4-1BB (CD137), OX40 (CD134), CDS, ICAM-1, ICOS (CD278), LFA-1 (CD11a / CD18), GITR, MyD88, DAP10, DAP12, PD-1, LIGHT, NKG2C, B7-H3, CD83, etc. Specifically, the co-stimulatory molecule may be, but is not limited to, DAP10.

[0072] According to one embodiment of the present invention, the second chimeric antigen receptor can be made to exhibit a cancer cell killing effect with high activity of natural killer cells by using DAP10 and CD3ζ as intracellular signaling domains.

[0073] The second chimeric antigen receptor may include two or more intracellular signaling domains, and if it includes two or more intracellular signaling domains, the intracellular signaling domains may be linked in series with each other. Alternatively, they may be linked via a polypeptide linker consisting of 2 to 10 amino acids, the linker sequence of which may be, for example, a glycine-serine sequence. The linker may include, for example, (GS)n, (GGS)n, (GSGGS)n, or (GnS)m (n and m are each 1 to 10), for example, (GnS)m (n and m are each 1 to 10), but is not limited thereto.

[0074] The second chimeric antigen receptor may further contain an immune function-promoting factor for immune cells, for example, the immune function-promoting factor for immune cells may be an interleukin signal sequence. The interleukin signal sequence may be characterized by inducing the expression of IL-12, IL-8, IL-2, etc., but is not limited thereto. Furthermore, if the immune cell is a T cell, the immune function-promoting factor may be IL-7, CCL19, etc., but is not limited thereto.

[0075] The aforementioned immune cells can be any cells capable of inducing immunity and triggering the desired therapeutic effect, such as natural killer cells (NK cells), T cells, and natural killer cells. NKT cells (KRT cells), cytokine-induced killer cells (Cytokin e-Induced Killer cell (CIK), macrophages and Dendrous Cells The immune cells expressing the first chimeric antigen receptor and the second chimeric antigen receptor on the cell surface according to the present invention include CAR-NK cells (Chimeric Antigen Receptor Natural Killer Cells), CAR-T cells (Chimeric Antigen Receptor T Cells), and CAR-NKT cells (Chimeric Antigen Receptor T Cells). ic Antigen Receptor Natural killer T Cell), CAR-Macrophage (Chimeric Antigen Receptor Macrop) It can be something like "hage" (or something similar).

[0076] The aforementioned T cells may be, but are not limited to, cytotoxic T lymphocytes (CTLs), tumor-infiltrating lymphocytes (TILs), or T cells isolated from peripheral blood mononuclear cells (PBMCs). It is not something that can be done.

[0077] The aforementioned CAR-NK cells refer to cells into which a chimeric antigen receptor has been introduced into natural killer cells, and cancer immunotherapy using existing T cell-based CAR-T therapies has the same properties. This method not only solves problems such as persistent toxicity, the risk of autoimmune diseases, graft-versus-host disease (GVHD) issues in xenotransplantation, and non-targeted toxicity issues through an on / off switch, but it also has the advantage of being able to target a variety of cancer cells and be used as a general-purpose therapeutic agent.

[0078] The immune cells of this invention are p75 NTR Death domains, etc. A first chimeric antigen receptor, which includes an antigen-binding site that specifically binds to antigens expressed on both normal and cancer cells, and a second chimeric antigen receptor, which includes an antigen-binding site that specifically binds to cancer cell-specific antigens, can be expressed on the surface of immune cells. In this case, if only normal cells are present, only the first chimeric antigen receptor of the two chimeric antigen receptors can recognize and bind to antigens on the surface of normal cells, thereby reducing the cytotoxicity of the immune cells or promoting the cell death of immune cells as described above, and thus potentially not inducing an immune response against normal cells. On the other hand, if cancer cells are present, all antigens that can be recognized by the first chimeric antigen receptor and all antigens that can be recognized by the second chimeric antigen receptor can be present on the surface of cancer cells, so signal transduction occurs at all two chimeric antigen receptors. In this case, when signal transduction occurs at all two chimeric antigen receptors, the cytotoxicity of the immune cells can be further enhanced compared to when the second chimeric antigen receptor activates immune cells via signal transduction alone. Therefore, the immune cells of the present invention exhibit stability against normal cells and can show increased cytotoxicity against cancer cells compared to existing chimeric antigen receptors, thus possessing improved efficacy in attacking and treating cancer cells.

[0079] In a specific embodiment of the present invention, natural killer cells expressing both the first chimeric antigen receptor, which can specifically bind to EGFR (expressed in all normal and cancer cells), and the second chimeric antigen receptor, which can specifically bind to HER2 and EphA2 (expressed in all cancer cells), were used to confirm cytotoxicity against breast cancer cells expressing EGFR and HER2, or breast cancer cells expressing both EGFR and EphA2. The results showed that natural killer cells expressing both the first and second chimeric antigen receptors exhibited higher cytotoxicity compared to natural killer cells expressing only the second chimeric antigen receptor. Therefore, it can be confirmed that the immune cells of the present invention are extremely useful for treating cancer.

[0080] On the other hand, the immune cells of the present invention may express a plurality of additional chimeric antigen receptors, such as third, fourth, and fifth chimeric antigen receptors, on their surface, in addition to the first and second chimeric antigen receptors mentioned above. The plurality of additional chimeric antigen receptors may specifically bind to antigens of a completely different type from the first and second chimeric antigen receptors, and each of the plurality of chimeric antigen receptors may also specifically bind to antigens of a different type from each other. When such plurality of additional chimeric antigen receptors are expressed on the surface of the immune cells, the immune cells of the present invention can target a plurality of antigens simultaneously. In short, the immune cells of the present invention can target p75 NTR The intracellular domain of the receptor, which includes the death domain, is further subdivided as shown above. By simultaneously expressing the second, third, and fourth chimeric antigen receptors, etc., along with the first chimeric antigen receptor, which is included as an intracellular signaling domain, the cytotoxicity of immune cells mediated by the second, third, and fourth chimeric antigen receptors, etc., against target cells that have antigens specific to the second, third, and fourth chimeric antigen receptors, etc., can be further enhanced. This further expands the range of target cells in which the immune cells of the present invention can exhibit cytotoxicity.

[0081] 3. Therapeutic use of immune cells in the present invention against cancer Another aspect of the present invention provides a pharmaceutical composition for cancer treatment comprising the immune cells.

[0082] The explanations regarding the aforementioned immune cells and the chimeric antigen receptors they express are the same as those described in "1. Novel Chimeric Antigen Receptors (CARs) and Polynucleotides and Expression Vectors for Expressing Them" and "2. Immune Cells Expressing Dual-CARs," so to avoid repetition, they will be omitted here.

[0083] In this invention, the terms "cancer" and "tumor" are used interchangeably and refer to or mean a physiological condition in mammals typically characterized by unregulated cell growth / proliferation.

[0084] The cancer or type of cancer that can be treated with the composition of the present invention is not particularly limited and includes all solid tumors and hematological cancers. For example, the cancers include lung cancer, gastric cancer, ovarian cancer, cervical cancer, breast cancer, pancreatic cancer, and organs cancer, large intestines The cancer may be at least one selected from the group consisting of cancer, colon cancer, esophageal cancer, skin cancer, thyroid cancer, kidney cancer, liver cancer, head and neck cancer, bladder cancer, prostate cancer, hematological cancer, multiple myeloma, acute osteomyeloleukemia, malignant lymphoma, thymic carcinoma, osteosarcoma, fibrous tumor, and brain cancer, but is not limited thereto. The present invention can be applied without limitation to any cancer cell containing an antigen recognizable by the second chimeric antigen receptor.

[0085] In this invention, the term "treatment" means the suppression of cancer development, the alleviation or elimination of symptoms.

[0086] The pharmaceutical composition may contain, but is not limited to, immune cells in an amount that is 1 to 10 times, 2 to 10 times, or 5 to 8 times the number of tumor cells in the individual being treated.

[0087] The aforementioned composition may be in forms other than pharmaceutical compositions, such as quasi-pharmaceutical compositions or health food compositions.

[0088] The cancer therapeutic composition of the present invention may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not have a toxicity greater than that which is appropriate for the target of application (prescription), and the "carrier" is defined as a compound that facilitates the addition of the compound to cells or tissues.

[0089] The pharmaceutical composition of the present invention may be administered alone or in mixture with a convenient carrier, and such dosage form may be a single-dose or repeated-dose form. The pharmaceutical composition may be a solid or liquid preparation. Solid preparations include, but are not limited to, powders, granules, tablets, capsules, suppositories, etc. Solid preparations may contain, but are not limited to, carriers, flavoring agents, binders, preservatives, disintegrants, activators, fillers, etc. Liquid preparations include, but are not limited to, water, solutions such as propylene glycol solution, suspensions, oils, etc., and may be manufactured by adding appropriate coloring agents, flavoring agents, stabilizers, viscosifiers, etc. For example, a powder can be manufactured by simply mixing a tri-hydroxy derivative of a polyunsaturated fatty acid, which is the active ingredient of the present invention, with a suitable pharmaceutically acceptable carrier such as lactose, starch, or uncrystalline cellulose. The granules can be produced by mixing the tri-hydroxy derivative of the polyunsaturated fatty acid of the present invention, a pharmaceutically acceptable suitable carrier, and a pharmaceutically acceptable suitable binder such as polyvinylpyrrolidone or hydroxypropylcellulose, and then using a wet granulation method with a solvent such as water, ethanol, or isopropanol, or a dry granulation method using compressive force. Tablets can also be produced by mixing the granules with a pharmaceutically acceptable suitable activator such as magnesium stearic acid, and then compressing them using a tablet press.

[0090] The aforementioned pharmaceutical composition may be prepared as an oral preparation or an injectable preparation (for example) depending on the disease to be treated and the individual's condition. It may be administered by intramuscular injection, intraperitoneal injection, intravenous injection, infusion, subcutaneous injection, implant, inhalation, nasal administration, vaginal administration, rectal administration, sublingual administration, torndomal, topical administration, etc., but is not limited thereto. Depending on the route of administration, it may be formulated into a suitable administration unit dosage form containing a pharmaceutically acceptable carrier, excipient, and vehicle that is normally used and non-toxic.

[0091] The pharmaceutical composition may be administered daily at a dose of approximately 0.0001 mg / kg to approximately 10 g / kg, or at a daily dose of approximately 0.001 mg / kg to approximately 1 g / kg. However, the dosage may vary depending on the degree of purification of the mixture, the patient's condition (age, sex, weight, etc.), and the severity of the condition being treated. If necessary, for convenience, the total daily dose may be divided into multiple doses per day.

[0092] The present invention will be described in detail below with reference to examples.

[0093] However, the following embodiments are merely illustrative examples of the present invention, and the content of the present invention is not limited by these embodiments.

[0094] [Example 1] Chimeric receptors containing the intracellular domain of the receptor including the death domain Design of protoreceptors Chimeric antigen receptors (CARs) include an extracellular domain (ectodomain), a transmembrane domain, and an intracellular signaling domain (endodomain). p75 is one of the receptors that includes a death region as an intracellular signaling domain. NTR To include the intracellular domain We designed the chimeric antigen receptor of the present invention.

[0095] The extracellular domain includes an antigen-binding site, and the chimeric antigen receptor of the present invention can be manufactured regardless of the type of antigen or antigen-binding site. In this embodiment, a single-chain variable fragment of an antibody that can specifically bind to EGFR is used. ment;scFv) was used. As a transmembrane domain, CD28 was used to link the scFv for EGFR with the CD28 via Myc and a hinge domain, and p75 was used in the transmembrane domain. NTRThe intracellular domains were configured to be linked, and a gene construct containing the nucleotide sequence of Sequence ID No. 13, which encodes this, was inserted into the pLVX-EF1a-IRES-zsGREEN vector (Figure 1).

[0096] [Example 2] Production of immune cells that express dual chimeric antigen receptors (dual CARs) on their surface.

[0097] [2-1] Production of immune cells expressing both α-Cot-CAR and p75NTR intracellular domains Immune cells capable of expressing the chimeric antigen receptor of the present invention, designed via Example 1, were produced. In this process, the vector designed in Example 1 was introduced into α-Cot-CAR NK92 cells (M2), which are natural killer cells (NK cells) expressing the anti-cotinine chimeric antigen receptor, thereby producing Dual-CAR NK92 cells 1, which are natural killer cells capable of expressing two types of chimeric antigen receptors.

[0098] The aforementioned α-Cot-CAR NK92 cells are described in Korean Registered Patent No. 2,122,546. Natural killer cells that can be manufactured and / or acquired by the described method, which have a chimeric antigen receptor on their surface containing an scFv, which can specifically bind to cotinine, as an extracellular domain. It exhibits the characteristic of automating the rapid generation of the α-Cot-CAR NK92 cells. The cells were separated into single cells via a parenchyma cell separator (FacsAria fusion) (M2), A vector encoding the chimeric antigen receptor of the present invention, designed via Example 1 using a lentivirus, is introduced into the α-Cot-CAR NK92 cells. As a result, in the case of natural killer cells into which the vector has been successfully introduced, not only the anti-cotinine chimeric antigen receptor that is already expressed, but also the p75 of the present invention. NTR A chimeric antigen receptor containing the intracellular domain can be expressed on the cell surface.

[0099] After introducing a vector encoding the chimeric antigen receptor of the present invention into a natural killer cell line, the expression of the chimeric antigen receptor was confirmed. Specifically, cell lines were stained using a Myc antibody that exhibits fluorescence to detect the tag protein Myc contained in the chimeric antigen receptor, and its expression on the cell surface was analyzed by flow cytometry. This was confirmed. Furthermore, the expression of EGFR-p75NTR was confirmed by analyzing the fluorescence of zsGreen expressed from the inserted vector via parenchyma cell analysis (flow cytometry). By measuring the two types of chimeric antigen receptors, we were able to confirm that they were successfully expressed (Figure 2).

[0100] [2-2] Production of immune cells expressing both α-EphA2-CAR and p75NTR intracellular domains Furthermore, other immune cells capable of expressing the chimeric antigen receptor of the present invention, designed via Example 1, were produced.

[0101] First, the anti-EphA2 single-chain variable fragment (single-chain variable f We designed a chimeric antigen receptor (CAR) to be introduced into natural killer cells, with an extracellular domain containing scFv as the antigen-binding site. Specifically, we linked the extracellular domain containing scFv as the antigen-binding site with CD28 as a transmembrane domain via Myc and a hinge domain, and added CD3-zeta and the co-stimulatory molecule CD28 DAP10 to the transmembrane domain via an intracellular signaling domain, thereby creating a chimeric antigen receptor classified as a third-generation CAR. The chimeric antigen receptor of the present invention was designed to have an intracellular signaling domain of the antigen receptor (Figure 3a). Then, the base sequence of the gene construct that encodes this was inserted into a lentiviral vector, and a viral packaging vector (viral packa ging vectors (pMDLG / RRE, pRSV / REV, VSVG) HEK293T cells were transformed. Then, a lentivirus expressing EphA2-CAR1 was obtained, which was concentrated using an ultrafast centrifuge, and then the multiplicity of infection was determined by spinoculation (360g, 90min, RT). Natural killer cells are infected so that the MOI (Moment of Infection) becomes 30. As described above, the infected natural killer cells were subjected to 5 hours of incubation at 37°C and 5% CO2 conditions. After intermittent culture, the culture medium was changed to fresh medium, and after 3 days, the cells were treated with 3ug / ml puromycin to select for confirmed infected natural killer cells, and the culture was continued. Natural killer cells in the control group that were not infected were also treated with puromycin, and the culture was continued in the puromycin-treated medium until all natural killer cells in the control group were killed by puromycin. Once all natural killer cells in the control group had been killed, infected natural killer cells were selected and the experiment was conducted. The α-EphA2-CAR NK92 cells selected as described above underwent automated high-speed parenchyma cell separation. After separating into single cells via a container (79-14), the vector designed in Example 1 is introduced using the same method as in Example [2-1], thereby introducing the existing anti-EphA2 chimeric antigen receptor and the p75 of the present invention. NTR Dual-CAR NK92 cells are natural killer cells capable of expressing all aspects of a chimeric antigen receptor, including its intracellular domain, on their cell surface. They manufactured it.

[0102] The above example uses Dual-CAR NK92 cells 2 produced as described above. Using the same method as in [2-1], we confirmed that the expression of the chimeric antigen receptors was successfully observed, and we were able to confirm that both types of chimeric antigen receptors were expressed (Figure 3b).

[0103] [Example 3] Functional confirmation of the p75NTR signal in natural killer cells when only antigens expressed in both cancer cells and normal cells are present. The natural killer cells produced in the above example [2-1] were p75 NTR This cell is characterized by expressing on its surface a chimeric antigen receptor (first CAR) containing the intracellular domain as a signaling domain, and a chimeric antigen receptor (second CAR) containing an antigen-binding site that can specifically bind to cotinine. In this case, the first CAR contains a single-chain variable fragment (scFv) of an antibody that can recognize and bind to EGFR, which is expressed in all normal and cancer cells. Therefore, if normal cells or cancer cells expressing EGFR on their surface are present, signal transduction can occur within natural killer cells as a result.

[0104] Therefore, p75 NTR To confirm the activity of natural killer cells when only the chimeric antigen receptor (second CAR) of the present invention, which contains the intracellular domain, is activated alone, AU565, a breast cancer cell expressing EGFR on its surface, was used as the target cell. Under conditions where cotinine zygotes were not added to prevent signal transduction by the second CAR, the AU565 cells were stained with calcein and allowed to proceed. They were then co-cultured for 4 hours with natural killer cells (Dual-CAR NK92) or α-Cot-CAR NK92 cells (M2) from Example [2-1] in a ratio of 1:3 (target cells:natural killer cells). The cytotoxicity of the natural killer cells was confirmed by the amount of calcein secreted into the supernatant after 4 hours of co-culture. As a result, α-Cot-CAR NK92 cells (M2) In this case, it cannot bind to EGFR, and since there is no cotinin zygote, signal transduction does not occur, and therefore, cytotoxic activity of natural killer cells has not been shown, p75 NTR Similarly, natural killer cells (Dual-CAR NK92) in Example [2-1], which expressed a chimeric antigen receptor including the intracellular domain, did not exhibit cytotoxic activity (Figure). 4).

[0105] Furthermore, after culturing the natural killer cells with AU565 cells, the degree of apoptosis of the natural killer cells was confirmed at 6 hours and 24 hours, respectively. The degree of apoptosis was determined by a ratio of AU565 cells to the natural killer cells (Dual-CAR NK92) or M2 cells of Example [2-1], in a ratio of 1:0.2 (target cells: natural killer cells). The procedure was performed after co-culturing the cells (natural killer cells) in proportion to each cell type for 6 or 24 hours. After co-culturing each cell type, the cells were stained with annexin V and 7AAD, and the degree of cell death was confirmed by parenchymal cell analysis. In this procedure, the M2 cells were stained with an antibody expressing fluorescence of CD56-FITC, and the natural killer cells of Example [2-1] were distinguished from the target cells AU565 using zsGreen, which is expressed automatically, and the degree of cell death was confirmed by compartmentalizing only the natural killer cells. As a result, when the natural killer cells (Dual-CAR NK92) of Example [2-1] were exposed to EGFR-expressing AU565 cells for a long period of time... When it comes to producing it, compared to α-Cot-CAR NK92 cells (M2), natural keratin —Increased cell death was confirmed (Figure 5).

[0106] Considering the results mentioned above, p75 NTR When a chimeric antigen receptor containing the intracellular domain is expressed on the surface of natural killer cells, and signal transduction is activated solely by the chimeric antigen receptor, no cytotoxic activity of natural killer cells is observed. Rather, prolonged exposure to the antigen induces cell death in natural killer cells, confirming that natural killer cells are unable to perform their original functions. (See p75) NTR The intracellular domain The chimeric antigen receptor of the present invention is designed to recognize and bind to antigens expressed in both cancer cells and normal cells. Therefore, if only normal cells are present, the natural killer cells will not exhibit toxicity to normal cells, and cell death will be induced. Thus, it was confirmed that natural killer cells expressing the chimeric antigen receptor of the present invention on their surface do not attack normal cells.

[0107] [Example 4] Confirmation of cytotoxicity of natural killer cells expressing dual CARs when cancer cell-specific antigens are present. In the above example 3, p75 NTR In addition to confirming that the cytotoxicity of natural killer cells decreases and cell death increases when signal transduction by a chimeric antigen receptor containing an intracellular domain occurs alone, we also investigated how the activity of natural killer cells changes when cancer cell-specific antigens are also present and all signal transduction by dual CAR occurs in natural killer cells.

[0108] Specifically, AU565 cells, which are breast cancer cells expressing both EGFR and HER2, were used as target cells, along with α-Cot-CAR NK92 cells (M2) and Example [2-1]. Natural killer cells (Dual-CAR NK92) were co-cultured separately. , the α-Cot-CAR NK92 cells (M2) and the natural cells of Example [2-1] Recognition by chimeric antigen receptors expressed on the surface of Rah cells (Dual-CAR NK92) To enable recognition and binding, a HER2-cotinine (HER2-cot) zygote was used in conjunction with the HER2 expressed in AU565 cells (see Korean Patent No. 2,122,546).

[0109] The cytotoxicity of natural killer cells was confirmed using the same method as in Example 3, with natural killer cells co-cultured with AU565 cells.

[0110] As a result, in the presence of HER2-cotinine zygotes, the cytotoxicity of α-Cot-CAR NK92 cells (M2) expressing only the anti-cotinine chimeric antigen receptor was compared to that of HER2-cotinine zygotes. Cytotoxicity of natural killer cells (Dual-CAR NK92) in Example [2-1] A significant increase was confirmed, and even when examined individually, all Dual-CAR NK92 cells showed higher cytotoxicity compared to the aforementioned α-Cot-CAR NK92 cells (M2) (Figure 6).

[0111] In natural killer cells expressing the anti-cotinine chimeric antigen receptor, the recognition of HER2 on the surface of cancer cells via the HER2-cotinine zygote leads to signal transduction that makes them cytotoxic and increases their ability to attack cancer cells. However, the experimental results mentioned above show that p75 NTR It has been found that when a chimeric antigen receptor containing the intracellular domain is expressed in a dual manner, and signal transduction occurs via EGFR on the surface of cancer cells, the cytotoxicity of natural killer cells increases.

[0112] [Example 5] Further verification of the cytotoxicity of natural killer cells expressing dual CARs. p75 confirmed in Example 3 above NTR The fact that cell death of natural killer cells increases when signal transduction occurs solely by the chimeric antigen receptor containing the intracellular domain, and that the cytotoxicity of natural killer cells improves when both cancer-specific antigens are present and signal transduction occurs in both types of CARs, as confirmed in Example 4, was observed in natural killer cells expressing a CAR with an extracellular domain of a different type (α-EphA2-CAR NK92 cells) rather than natural killer cells expressing α-Cot-CAR. We verified it again using [the specified method].

[0113] Specifically, using MDA-MB-231 cells, which are breast cancer cells expressing both EGFR and EphA2, as target cells, i) NK92 cells, ii) EGFR-p75NTR NK92 cells prepared by introducing the vector encoding the chimeric antigen receptor of the present invention designed in Example 1 into NK92 cells in the same manner as in Example [2-1], and iii) the aforementioned α-EphA2-CAR NK92 cells (79-14) prepared in the example [2-2], iv ) Dual-CAR NK92 cells 2 (79-14-7) prepared in the above example [2-2] 5) were co-cultured, and the cytotoxicity of each natural killer cell was confirmed using the same method as in Example 3. As a result, i) NK92 cells and ii) EGFR-p75NTR NK9 When comparing the cellular activity of two cells, p75 NTR When signaling occurred solely by the chimeric antigen receptor containing the intracellular domain, cytotoxicity did not increase, but iv) Dual-CAR NK92 cells 2 (79-14-75) showed increased anti-EphA2 chimeric antigen receptor iii) Compared to α-Epha2-CAR NK92 cells (79-14) in which only the body expresses it. All were confirmed to exhibit significantly improved cytotoxicity (Figure 7).

[0114] Furthermore, similar to Example 3, after culturing the native cells with MDA-MB-231 cells, cell death was confirmed after 17 hours. As a result, i) in the case of NK92 cells, cell death did not increase after co-culturing with target cells compared to before co-culturing, but ii) in the case of EGFR-p75NTR NK92 cells, cell death increased after co-culturing with target cells. This was confirmed (Figure 8).

[0115] The results described above are identical to those obtained with Dual-CAR NK92 cells 1 of α-Cot-CAR NK92 cells. These results suggest that p75 may also be effective against other antigens besides cotinine. NTR It can be seen that the Dual-CAR using the chimeric antigen receptor of the present invention, which includes the intracellular domain as a signaling domain, exhibits the same efficacy.

[0116] [Example 6] Subdivision of the P75NTR intracellular domain by the death domain Confirmation of cell death effect P75 NTR The intracellular domain of this cell includes a death domain. Furthermore, this domain is known to be involved in cell death. In the present invention, p75 is also used through Examples 3 and 5. NTR We confirmed that cell death increases with sole signaling, and that this phenomenon is actually present in p75 NTR We attempted to determine whether or not the effect was due to the aforementioned cell death region located in the intracellular domain.

[0117] Specifically, in order to confirm the function of the death region, the chimeric antigen receptor designed in Example 1 was used to test p75 NTR A chimeric antigen receptor (p75) in which the dead region of the intracellular domain has been removed. NTR A DDD was designed, and a gene construct to encrypt it was inserted into a pBlueScript SK(-) vector (Figure 9). Separately, the above-mentioned Example 1 The gene construct that encodes the chimeric antigen receptor designed was also inserted into the pBlueScript SK(-) vector, and in vitro transcription was performed from each of these two vectors. via mR of EGFR-p75NTR CAR and EGFR-p75NTR DDD CAR NA was produced. The mRNA produced as described above was introduced into α-Cot-CAR NK92 cells. After transformation to (M2), the expression of the introduced chimeric antigen receptor was confirmed 17 hours later by detecting the tag protein Myc using the same method as in Example 2. As a result, p75 NTR It was confirmed that both types of chimeric antigen receptors, from which the dead region had been removed, were successfully expressed (Figure 10).

[0118] As described above, Dual-CAR NK92 cells expressing each chimeric antigen receptor are used In the same manner as in Example 4, the target cells, AU565 cells, and their respective Dual-C AR NK92 cells 1:1, 1:0.5, 1:0.25 (Target cells: Natural killer cells) Cytotoxicity was confirmed by co-culturing the cells in a specific ratio. As a result, regardless of whether the dead regions were removed, Dual-CAR NK92 cells showed higher cytotoxicity than α-Cot-CAR NK92 cells, and because HER2-cotinine (HER2-cot) zygotes were not included, p75 NTR It was confirmed that when only the chimeric antigen receptor containing the intracellular domain was activated, cytotoxicity did not increase regardless of whether the dead region was removed (Figure 11). This is consistent with the results obtained in Examples 3 to 5.

[0119] On the other hand, since the aforementioned death region is known to affect cell death, after co-culturing the Dual-CAR NK92 cells with the target cell AU565, 24 hours had elapsed. At that point, the degree of cell death (apoptosis) of the Dual-CAR NK92 cells was confirmed. Confirmed. The degree of cell death was confirmed using the same method as in Example 3, p75 NTR Co-culture Dual-CAR NK92 cells and target cells in a 0.5:1 ratio, without introducing HER2-cotinine (HER2-cot) zygotes, so that only intracellular signals are transmitted. This was confirmed. As a result, each Dual-CAR NK9 that was not co-cultured with AU565 While the degree of apoptosis in the two cells was similar in all cases, when co-cultured with AU565, p75 NTR Dual-CAR NK92 cells expressing a chimeric antigen receptor with an intracellular domain of p75 NTR α-Cot without an intracellular domain - Increased cell death was observed compared to CAR NK92 cells, and the dead cell regions were removed. In the case of Dual-CAR NK92 cells expressing chimeric antigen receptors, the degree of cell death is A decrease was confirmed (Figure 12). Based on the results described above, p75 NTR When intracellular signaling is activated solely by this, cell death is induced, and this cell death induction phenomenon is described in p75. NTRIt appears that this is due to a death region located in the intracellular domain.

[0120] Therefore, p75 NTR When using the chimeric antigen receptor of the present invention, which includes an intracellular signaling domain containing an intracellular domain of the receptor that includes a death region such as the one described above, it was confirmed that it exhibits little to no cytotoxicity to normal cells and induces cell death of natural killer cells, thus ensuring stability to normal cells while showing stronger cytotoxicity to cancer cells compared to existing technologies utilizing single chimeric antigen receptors. Therefore, it was confirmed that the chimeric antigen receptor and natural killer cells can be usefully used as cancer treatment agents with excellent efficacy.

[0121] Although the present invention has been described in detail only with respect to the embodiments described above, it will be obvious to those skilled in the art that various modifications and alterations are possible within the scope of the technical concept of the present invention, and such modifications and alterations naturally fall within the scope of the attached claims.

Claims

1. An extracellular domain containing an antigen-binding site that specifically binds to antigens expressed in both cancer cells and normal cells, a transmembrane domain, and p75 ＮＴＲ intracellular The first chimeric antigen receptor, including the main (intracellular domain) The body (Chimeric antigen receptor; CAR) and, A second chimeric antigen receptor comprising an extracellular domain containing an antigen-binding site that specifically binds to an antigen expressed differently in cancer cells than in normal cells, a transmembrane domain, and an intracellular signaling domain including a primary signaling domain and a costimulatory signaling domain, Immune cells that express on their surface, The intracellular domain of the aforementioned p75 NTR consists of the amino acid sequence of Sequence ID No.

1. The aforementioned immune cells include natural killer cells (NK cells), T cells, and natural killer cells. - Selected from the group consisting of T cells (NKT cells) and macrophages One of them is the immune cell.

2. The primary signal transfer domain is one selected from the group consisting of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, and CD66d. The immune cell according to claim 1, wherein the co-stimulatory signaling domain is one selected from the group consisting of CD2, CD7, CD27, CD28, CD30, CD40, 4-1BB (CD137), OX40 (CD134), CDS, ICAM-1, ICOS (CD278), LFA-1 (CD11a / CD18), GITR, MyD88, DAP10, DAP12, PD-1, LIGHT, NKG2C, B7-H3, and CD83.

3. The primary signal transmission domain is CD3ζ, The immune cell according to claim 2, wherein the aforementioned co-stimulatory signaling domain is DAP10.

4. The immune cell according to claim 1, wherein the antigen-binding site of the first chimeric antigen receptor specifically binds to EGFR.

5. The antigen-binding site of the first chimeric antigen receptor is a single-chain variable fragment of the antibody (single c The immune cell according to claim 1, wherein the immune cell is a hain variable fragment (scFv).

6. The immune cell according to claim 1, wherein the extracellular domain of the first chimeric antigen receptor or the second chimeric antigen receptor further comprises at least one selected from the group consisting of a hinge domain and a spacer domain.

7. The immune cell according to claim 6, wherein the hinge domain or spacer domain is at least one selected from the group consisting of Myc epitope, CD8 hinge domain, and Fc.

8. The immune cell according to claim 1, wherein the transmembrane domain of the first chimeric antigen receptor or the second chimeric antigen receptor is one selected from the group consisting of the alpha (α), beta (β), or zeta (ζ) chain of the T-cell receptor (TCR), CD28, CD3 epsilon (ε), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

9. The immune cells are natural killer cells (NK cells) as described in claim 1. immune cells.

10. The immune cell according to claim 1, wherein the immune cell further expresses at least one chimeric antigen receptor on its surface, the chimeric antigen receptor comprising an extracellular domain including an antigen-binding site that specifically binds to an antigen different from the first chimeric antigen receptor and the second chimeric antigen receptor, a transmembrane domain, and an intracellular signaling domain.

11. The immune cell according to claim 10, wherein the different antigen is an antigen expressed only in cancer cells.

12. The immune cell according to claim 1, wherein the antigen-binding site of the second chimeric antigen receptor specifically binds to one selected from the group consisting of HER2, EphA2, ErbB3, IL-13Rα2, DLK1, B7H3, PD-L1, GPC3, CEACAM6, and CD5.

13. When only the first chimeric antigen receptor is activated, the immune cells exhibit reduced cytotoxicity or undergo cell death (apoptosis). The immune cells according to claim 1, wherein when both the first chimeric antigen receptor and the second chimeric antigen receptor are activated, they exhibit even greater cytotoxicity than when only the second chimeric antigen receptor is activated alone.

14. A pharmaceutical composition for cancer treatment comprising immune cells according to any one of claims 1 to 13.

15. The cancer is at least one selected from the group consisting of lung cancer, stomach cancer, ovarian cancer, cervical cancer, breast cancer, pancreatic cancer, colorectal cancer, colon cancer, esophageal cancer, skin cancer, thyroid cancer, kidney cancer, liver cancer, head and neck cancer, bladder cancer, prostate cancer, hematological cancer, multiple myeloma, acute osteomyeloleukemia, malignant lymphoma, thymic carcinoma, osteosarcoma, fibrous tumor, and brain cancer, according to claim 14.

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