Novel anti-EphA2 chimeric antigen receptor and immune cells expressing it

Abstract

The present invention relates to a novel antibody or antigen-binding fragment thereof that specifically binds to EphA2, a chimeric antigen receptor comprising an antigen-binding variable fragment of the antibody, and an immune cell expressing the chimeric antigen receptor, and the immune cell is useful for treating cancer.

Description

[Technical Field]

[0001] The present invention relates to novel antibodies or antigen-binding fragments thereof that specifically bind to EphA2, chimeric antigen receptors comprising the antigen-binding variable fragments of the antibodies, and immune cells that express the chimeric antigen receptors. [Background technology]

[0002] Cancer treatment methods have undergone continuous development and change, and methods such as surgery, chemotherapy, and radiation therapy are still in use today. However, these existing cancer treatment methods are often only effective in the early stages of cancer before it has metastasized, and in cases where metastasis has already progressed, there is a problem that even if surgery is performed, there is a high possibility of future recurrence. Therefore, research into methods that use immune responses to treat cancer has been ongoing recently.

[0003] Among these, there has been growing interest in cell therapy methods that use immune cells, strengthen them, or genetically modify them, and then inject them back into patients. For example, tumor infiltrating lymphocytes (TIL), chimeric antigen receptors (CAR), and T-cell receptor (TCR) technologies are being researched. In particular, chimeric antigen receptors (CARs), which are artificial receptors designed to convey antigen specificity to T cells and natural killer cells (NK cells), consist of an extracellular domain, a transmembrane domain, and an intracellular signaling domain that can activate immune cells and provide specific immunity by binding to cancer cell-specific antigens. T cells expressing such chimeric antigen receptors are called 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)), and in the case of natural killer cells, they are called CAR-NK cells.

[0004] The intracellular signaling domain of the chimeric antigen receptor (CAR) is primarily based on the intracellular signaling domain of CD3zeta, a signaling subunit of the T cell receptor (first-generation CAR). It has evolved to include the intracellular signaling domain of a costimulatory molecule that promotes immune cell growth and differentiation. For example, currently available CAR-T cell therapeutics use the intracellular signaling domains of CD28 and 4-1BB costimulatory molecules (second-generation CAR), respectively. Subsequently, attempts have been made to develop CARs that simultaneously contain both CD28 and 4-1BB intracellular signaling domains (third-generation CAR) (Stegen et al., Nat. Rev. Drug Discov., 14(7):499-509 (2015)).

[0005] Meanwhile, EphA2 (ephrin type-A receptor 2) is a protein expressed from the human EphA2 gene and is known to be overexpressed in various cancers, including breast cancer, prostate cancer, and lung cancer, and to promote the growth and penetration of cancer cells. Furthermore, EphA2 expression has been reported to be associated with the survival rate of cancer patients. Given this technical background, there is an emerging need to research additional strategies and methods for treating cancer by developing antibodies targeting EphA2, which is particularly overexpressed in cancer cells, and CAR-T and CAR-NK therapeutic agents utilizing the same. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide an antibody or antigen-binding fragment thereof that can specifically bind to EphA2, which is expressed primarily in cancer cells.

[0007] Another object of the present invention is to provide a novel chimeric antigen receptor that, when expressed in immune cells, amplifies cytotoxic or cytolytic activity against cancer cells.

[0008] Another object of the present invention is to provide a polynucleotide and an expression vector for expressing the chimeric antigen receptor.

[0009] Another object of the present invention is to provide immune cells that have excellent therapeutic effects against cancer by expressing the chimeric antigen receptor on the surface.

[0010] Another object of the present invention is to provide a pharmaceutical composition for treating cancer using the immune cells. [Means for solving the problem]

[0011] To achieve the above-mentioned objectives, one aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to EphA2, comprising: a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 11; and a light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 12, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 14.

[0012] Another aspect of the present invention provides a chimeric antigen receptor (CAR) comprising an extracellular binding domain comprising an antigen-binding site that specifically binds to EphA2; a transmembrane domain; and an intracellular signaling domain; wherein the antigen-binding site that specifically binds to EphA2 is a single-chain variable fragment (scFv) of an anti-EphA2 antibody comprising a heavy-chain variable region comprising a heavy-chain CDR1 having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9, a heavy-chain CDR2 having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10, and a heavy-chain CDR3 having the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 11, and a light-chain variable region comprising a light-chain CDR1 having the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 12, a light-chain CDR2 having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13, and a light-chain CDR3 having the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 14.

[0013] Another aspect of the present invention provides a polynucleotide comprising a nucleotide sequence encoding the chimeric antigen receptor, and an expression vector comprising the polynucleotide.

[0014] Another aspect of the present invention provides an immune cell expressing the chimeric antigen receptor on its surface. Another aspect of the present invention provides a pharmaceutical composition for treating cancer, comprising the immune cells. [Effects of the Invention]

[0015] The antibodies, antigen-binding fragments thereof, and chimeric antigen receptors utilizing the antibodies of the present invention can specifically bind to EphA2, which is primarily expressed in cancer cells, thereby significantly enhancing the cytotoxicity or cytolytic activity of immune cells and promoting cytokine secretion as signaling occurs in immune cells expressing the chimeric antigen receptor, thereby increasing the degranulation of cancer cells co-cultured with the immune cells.

[0016] Therefore, the antibody of the present invention, a chimeric antigen receptor utilizing the antibody, and immune cells expressing the antibody or chimeric antigen receptor will be useful for treating cancer.

[0017] However, the effects of the present invention are not limited to those described above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description. [Brief explanation of the drawings]

[0018] [Figure 1A] FIG. 1 is a schematic diagram showing the structure of the chimeric antigen receptor of the present invention (EphA2-CAR1) which comprises an antigen-binding variable fragment (scFv) that specifically binds to EphA2 as an extracellular domain. [Figure 1B] This is a diagram showing a map of the expression vector into which it was cloned. [Figure 2A] FIG. 1 is a schematic diagram showing the structure of the chimeric antigen receptor of the present invention (EphA2-CAR2) which comprises an antigen-binding variable fragment (scFv) that specifically binds to EphA2 as an extracellular domain. [Figure 2B] This is a diagram showing a map of the expression vector into which it was cloned. [Figure 3A]FIG. 1 shows the results of examining the expression of the chimeric antigen receptors of the present invention (EphA2-CAR1 and EphA2-CAR2) in natural killer cells (EphA2#79-CAR1-NK cells and EphA2#85-CAR1-NK cells) into which genes for two types of chimeric antigen receptors of the present invention were introduced and expressed. [Figure 3B] FIG. 1 shows the results of examining the expression of the chimeric antigen receptors of the present invention (EphA2-CAR1 and EphA2-CAR2) in T cells (EphA2#79-CAR2-T cells and EphA2#85-CAR2-T cells) into which genes for two types of chimeric antigen receptors of the present invention were introduced and expressed. [Figure 4] 1 shows the results of examining the presence or absence of EphA2 expression in breast cancer cell line MDA-MB-231 cells, lung cancer cell line A549 cells, and chronic myeloid leukemia cell line K562 cells. [Figure 5] FIG. 1 shows the results of measuring and comparing the cytotoxicity (cytolytic activity) of natural killer cells (EphA2#79-CAR1-NK cells and EphA2#85-CAR1-NK cells) into which genes for two types of chimeric antigen receptors of the present invention were introduced and expressed, against MDA-MB-231 cells that express EphA2 and K562 cells that do not express EphA2. [Figure 6-7] FIG. 1 shows the results of measuring and comparing the amount of cytokine (IFN-γ) secreted by natural killer cells and the expression level of CD107α, an indicator of degranulation, when natural killer cells (EphA2#79-CAR1-NK cells and EphA2#85-CAR1-NK cells) into which genes for two types of chimeric antigen receptors of the present invention have been introduced and which express the same were co-cultured with MDA-MB-231 cells that express EphA2 or K562 cells that do not express EphA2. [Figure 8] FIG. 1 shows the results of measuring the cytotoxicity (cytolytic activity) of T cells (EphA2#79-CAR2-T cells and EphA2#85-CAR2-T cells) into which genes for two types of chimeric antigen receptors of the present invention were introduced and expressed, against EphA2-expressing A549 cells. [Figure 9A] This result confirmed that EphA2 is expressed in H460 cells, a lung cancer cell line. [Figure 9B] This shows the results of confirming the cytotoxicity (cytolytic activity) of natural killer cells (EphA2#79-CAR1-NK cells) into which the gene for the chimeric antigen receptor of the present invention has been introduced and which express the gene for the chimeric antigen receptor of the present invention against H460 cells. [Figure 9C] 1 is a simplified diagram of an outline of an experiment in which H460 cells were injected into experimental animals to confirm the activity of natural killer cells into which the gene for the chimeric antigen receptor of the present invention was introduced and expressed. [Figure 9D-9E] 1 shows the results of confirming the anti-cancer activity of natural killer cells into which the gene for the chimeric antigen receptor of the present invention has been introduced and expressed, based on the cytotoxicity (cytolytic activity) of the cells against H460 cells, in terms of tumor size and weight. [Figures 10A-10B] The results show that the anti-cancer activity of T cells (EphA2#79-CAR2-T cells and EphA2#85-CAR2-T cells) introduced with and expressing genes for two types of chimeric antigen receptors of the present invention against A549 cells was confirmed by measuring the size and weight of the tumors in the experimental animals injected with A549-luciferase cells. [Figure 10C] These are the results of confirming the presence or absence of EphA2-CAR2-T cells in the mouse blood. [Figure 10D] These results confirm the presence or absence of EphA2-CAR2-T cells within the tumor. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention will be described in detail below.

[0020] 1. Novel anti-EphA2 antibodies, antigen-binding fragments thereof, chimeric antigen receptors (CARs) containing the same, and polynucleotides and expression vectors for expressing the same One aspect of the present invention provides anti-EphA2 antibodies and antigen-binding fragments thereof that are capable of specifically binding to EphA2.

[0021] In the present invention, the term "antibody" refers to an immunoglobulin molecule that immunologically binds specifically to and is reactive with an epitope of an antigen. The antibody may include monoclonal antibodies, polyclonal antibodies, antibodies with a full-length chain structure (full-length antibodies), functional fragments (antigen-binding fragments) that have at least the antigen-binding function, and recombinant antibodies. Specifically, the antibody of the present invention is a monoclonal antibody or an antigen-binding fragment thereof. A monoclonal antibody refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population. Such a monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope. A full-length antibody has a structure comprising two full-length light chains and two full-length heavy chains, each of which is connected to a heavy chain by a disulfide bond. The antibody comprises heavy chain (HC) and light chain (LC) polypeptides, and the heavy and light chains may comprise variable and constant regions.

[0022] The constant region mediates the antibody's binding to various cells of the immune system (e.g., T cells) and host tissues, including components of the complement system. The constant region performs the same function in antibodies of the same type derived from the same species, regardless of the type of antigen, and the amino acid sequence that constitutes it is identical or highly similar in each antibody. The constant region is divided into a heavy chain constant region (abbreviated as CH) and a light chain constant region (abbreviated as CL). The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and / or epsilon (ε) types, with gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and / or alpha 2 (α2) subclasses. The light chain constant region has kappa (κ) and lambda (λ) types. IgG has subtypes, including IgG1, IgG2, IgG3, and IgG4.

[0023] The variable region is an antibody site specific for an antigen and is divided into a heavy chain variable region (abbreviated as VH) and a light chain variable region (abbreviated as VL). The variable region contains three CDRs (complementary-determining regions or complementarity-determining regions) and four FRs (framework regions). The CDRs are cyclic regions involved in antigen recognition, and the specificity for the antigen is determined by the amino acid sequence of the CDR. The CDRs are referred to as CDR1, CDR2, and CDR3 depending on their order. Depending on which polypeptide the CDR belongs to, whether it is a heavy chain or a light chain, they are referred to as CDR-H1, CDR-H2, and CDR-H3 for the heavy chain variable region and CDR-L1, CDR-L2, and CDR-L3 for the light chain variable region. Similarly, FRs are designated as FR-H1, FR-H2, FR-H3, and FR-H4 for the heavy chain variable region, and as FR-L1, FR-L2, FR-L3, and FR-L4 for the light chain variable region. The CDRs and FRs are arranged in the following order in each variable region:

[0024] As used herein, the term "antigen-binding fragment" refers to any fragment of a humanized antibody of the present invention that retains the antigen-binding function of the antibody. The antigen-binding fragment is referred to interchangeably with terms such as "fragment" and "antibody fragment," and examples of the antigen-binding fragment include, but are not limited to, Fab, Fab', F(ab')2, and Fv.

[0025] The Fab has a structure comprising light-chain and heavy-chain variable regions, a light-chain constant region, and the first constant region of the heavy chain (CH1 domain), and has one antigen-binding site. The "Fab" differs from the Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy-chain CH1 domain. The F(ab')2 is formed when the cysteine residues in the hinge region of Fab' form disulfide bonds. The Fv refers to the smallest antibody fragment comprising only the heavy-chain variable region and the light-chain variable region. In a two-chain Fv, the heavy-chain variable region and the light-chain variable region are linked non-covalently, while in a single-chain Fv, the heavy-chain variable region and the light-chain variable region are generally linked covalently via a peptide linker or directly at the C-terminus, and can form a dimer-like structure like the two-chain Fv. The antigen-binding fragment can be produced by, but is not limited to, using protease hydrolysis (for example, limited cleavage of a whole antibody with papain to obtain Fab fragments, or cleavage with pepsin to obtain F(ab')2 fragments) or genetic recombination techniques.

[0026] The linker is a peptide linker having a length of about 10 to 25 amino acids. For example, the linker includes a hydrophilic 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.

[0027] In the present invention, the term "epitope" refers to a specific site on an antigen that can be specifically recognized and bound by an immunoglobulin, antibody, or antigen-binding fragment thereof. The epitope can be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein.

[0028] The term "specifically binds" can mean binding to another molecule with a binding affinity greater than background binding, e.g., the extracellular domain binds to a target antigen with a binding affinity of about 10 -5 It binds to a target antigen with an affinity or Ka (the equilibrium dissociation constant of a specific binding interaction, which has units of 1 / M) of at least M. The affinity is determined by the equilibrium dissociation constant (Kd) of a specific binding interaction, which has units of M, of at least 10 -5 ~10 -13 M or less than the above range.

[0029] The antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 11; and a light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 12, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 14; and specifically binds to EphA2.

[0030] The heavy chain variable region has the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:15.

[0031] The light chain variable region has the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:16.

[0032] The antibody or antigen-binding fragment thereof of the present invention may further comprise, for example, a heavy chain constant region and / or a light chain constant region of a human-derived antibody, and the heavy chain constant region and / or light chain constant region of the human-derived antibody may be used without limitation in terms of type or amino acid sequence, as long as they do not inhibit the specific binding property of the antibody or antigen-binding fragment thereof to EphA2.

[0033] The above-described amino acid sequences may include variants having different sequences due to deletion, insertion, substitution, or a combination thereof of amino acid residues, provided that such deletion, insertion, substitution, or a combination thereof does not affect the structure, function, or activity of the polypeptide containing the amino acid sequence. Furthermore, the above-described amino acid sequences include amino acids that have undergone common modifications known to those skilled in the art, such as phosphorylation, sulfation, acrylation, glycosylation, methylation, and farnesylation. The humanized antibodies or antigen-binding fragments thereof of the present invention include not only those containing the above-described amino acid sequences, but also those having substantially the same amino acid sequences as the above-described amino acid sequences and variants thereof. The term "substantially the same amino acid sequence" as used herein includes, but is not limited to, amino acid sequences that are 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 homologous to the above-described amino acid sequences.

[0034] In the present invention, the term "chimeric antigen receptor (CAR)" refers to a synthetic complex designed to induce an immune response when it recognizes and binds to a target antigen and a cell expressing the antigen. A chimeric antigen receptor may comprise an extracellular domain, a transmembrane domain, and an intracellular signaling domain. A chimeric antigen receptor is expressed on the surface of immune cells and recognizes and binds to a specific antigen, for example, an antigen specifically expressed on the surface of cancer cells, through the antigen-binding site contained in the extracellular domain, thereby inducing signal transduction within the immune cell and changing the activity of the immune cell, thereby enabling the induction of an immune response by targeting only a specific antigen.

[0035] The chimeric antigen receptor (CAR) of the present invention comprises an extracellular domain comprising an antigen-binding site that specifically binds to EphA2; a transmembrane domain; and an intracellular signaling domain.

[0036] The antigen-binding site that specifically binds to EphA2 is a single-chain variable fragment (scFv) of an anti-EphA2 antibody comprising a heavy chain variable region including a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 11, and a light chain variable region including a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 12, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 14.

[0037] When the chimeric antigen receptor of the present invention is expressed on the surface of immune cells, binding of the target antigen EphA2 to the receptor generates signal transduction within the immune cells, resulting in increased cytotoxicity (or cytolytic activity) and / or enhanced cytokine secretion from the immune cells. The increased cytotoxicity (or cytolytic activity) or enhanced cytokine secretion means that the immune cells exhibit a higher level of cytotoxicity (or cytolytic activity) or secrete a higher level of cytokine than the cytotoxicity (or cytolytic activity) or cytokine secretion exhibited by immune cells in the absence of antigen.

[0038] The extracellular domain may further comprise at least one selected from the group consisting of a hinge domain and a spacer domain, and the antigen-binding site of the extracellular domain is connected to the transmembrane domain via the hinge domain and / or the spacer domain.

[0039] The hinge domain physically separates the antigen-binding site from the surface of the immune cell on which the chimeric antigen receptor is expressed, allowing for proper cell-cell contact, proper antigen-antigen binding, and proper chimeric antigen receptor activation, and may 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 a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. The altered 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 at least 10 amino acids in length (e.g., at least 12, 13, 14, or 15 amino acids) 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 comprising the core hinge region (which is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, 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 a naturally occurring immunoglobulin hinge region are substituted with one or more other amino acid residues (e.g., one or more serine residues). The altered immunoglobulin hinge region may alternatively or additionally have other amino acid residues, such as proline residues, in the wild-type immunoglobulin hinge region substituted with cysteine. The hinge domain may be derived from the extracellular domain of a type 1 membrane protein such as CD8, CD4, CD28, or CD7, but any hinge domain capable of linking the antigen-binding site, transmembrane domain, and intracellular signaling domain to the cell membrane may be used without limitation. This may be a wild-type hinge region from these molecules or may be altered.

[0040] The spacer domain is called a linking domain and includes, for example, a CD28-derived hinge domain and / or a CD8-derived hinge domain, and includes the whole or a part of the CD28-derived hinge domain and / or the CD8-derived hinge domain.

[0041] The hinge domain and / or spacer domain is at least one selected from the group consisting of a Myc epitope, a CD8 hinge domain, and Fc, and specifically includes a Myc epitope and a CD8 hinge domain. More specifically, the Myc epitope includes the amino acid sequence of SEQ ID NO: 17, and the CD8 hinge domain includes the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19.

[0042] The transmembrane domain refers to a region that connects and fuses the extracellular domain and the intracellular signaling domain to each other and anchors the chimeric antigen receptor to the plasma membrane of immune cells. The transmembrane domain may be derived from a natural, synthetic, semi-synthetic, or recombinant source. The transmembrane domain may be any 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, but is not limited thereto.

[0043] The transmembrane domain is attached to the extracellular domain via a linker, such as a short oligopeptide or polypeptide linker of 2 to 10 amino acids in length, including, but not limited to, a glycine (G)-serine (S) doublet.

[0044] The intracellular signaling domain corresponds to a portion that plays a role in transmitting a signal generated by the binding of the chimeric antigen receptor with an antigen to the inside of the immune cell to induce immune cell functions (e.g., activation including the release of cytotoxic (or cytolytic) factors against target cells to which the chimeric antigen receptor and antigen have bound, cytokine production, proliferation, and cytotoxic or cytolytic activity, or other cellular responses induced by the antigen binding). The intracellular signaling domain is a part of a protein that transmits activating functional signals and instructs cells to perform specific functions.

[0045] The intracellular signaling domain may be the same as that used in the development of chimeric antigen receptors. Specifically, it may contain only CD3ζ, as used in first-generation CARs (chimeric antigen receptors). Furthermore, as used in second-generation CARs, a form in which a costimulatory domain (CD28 or CD137 / 4-1BB) is combined with CD3ζ is used to improve reactivity to immune cells. Furthermore, as used in third-generation CARs, two or more costimulatory domains may be used. In this case, the costimulatory domain may be combined with 4-1BB, CD28, or OX40 to achieve in vivo expansion and persistence of immune cells containing the CAR. Furthermore, as used in fourth-generation CARs, an additional gene encoding a cytokine such as IL-12 or IL-15 may be included to further express cytokine-based immune proteins on the CAR. As used in fifth-generation CARs, an interleukin receptor chain, e.g., IL-2Rβ, may be further included to enhance immune cells.

[0046] The intracellular signaling domain can comprise at least one selected from the group consisting of T cell receptor (TCR) zeta (ζ), FcR gamma (γ), FcR beta (β), CD3 gamma (γ), CD3 delta (δ), CD3 epsilon (ε), CD3 zeta (ζ), CD5, CD22, CD79a, CD79b, and CD66d.

[0047] More specifically, the activation of immune cells by the intracellular signaling domain is mediated by two different classes of intracellular signaling domains. For example, immune cell activation is 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 may include a primary signaling domain and a costimulatory signaling domain.

[0048] The primary signaling domain refers to a signaling domain that regulates immune cell activation in a stimulatory or inhibitory manner. A primary signaling domain that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM). ITAMs containing a primary signaling domain include, but are not limited to, TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, and CD66d. More specifically, the primary signaling domain is CD3ζ (zeta), but is not limited to these.

[0049] The costimulatory signaling domain refers to the intracellular signaling domain of a costimulatory molecule. The costimulatory signaling domain may include, but is not limited to, a costimulatory signaling domain 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, EphA2, CD83, etc. Specifically, the costimulatory molecule is, but is not limited to, DAP10.

[0050] The chimeric antigen receptor may contain two or more intracellular signaling domains. When the chimeric antigen receptor contains two or more intracellular signaling domains, the intracellular signaling domains are linked in tandem. Alternatively, the intracellular signaling domains may be linked via a polypeptide linker consisting of 2 to 10 amino acids, such as a glycine-serine contiguous sequence. The linker may include, for example, (GS)n, (GGS)n, (GSGGS)n, or (GnS)m (n and m are each 1 to 10), such as, but not limited to, (GnS)m (n and m are each 1 to 10).

[0051] The chimeric antigen receptor may further comprise an immune cell immune function-enhancing factor, for example, an interleukin signal sequence. The interleukin signal sequence is characterized by inducing the expression of IL (interleukin)-12, IL-8, IL-2, etc., but is not limited to these. Furthermore, when the immune cell is a T cell, the immune function-enhancing factor may be, but is not limited to, IL-7, CCL19, etc.

[0052] In one specific example of the present invention, a chimeric antigen receptor of the present invention was designed to contain, in its extracellular domain, scFvs of two anti-EphA2 antibodies (#79 and #85) having the amino acid sequences described above, CD28 linked to this antibody via a hinge domain as a transmembrane domain and an intracellular signaling domain, and CD3-zeta and DAP10 linked to this antibody via an intracellular signaling domain (EphA2-CAR1). In another specific example of the present invention, a chimeric antigen receptor of the present invention was designed to contain, in its extracellular domain, scFvs of two anti-EphA2 antibodies (#79 and #85) having the amino acid sequences described above, CD8 linked to this antibody via a hinge domain and a transmembrane domain, and CD3-zeta and 41-BB linked to this antibody via an intracellular signaling domain (EphA2-CAR2).

[0053] As described above, when the chimeric antigen receptor of the present invention is expressed on the surface of immune cells and recognizes and binds to EphA2, it can enhance the cytotoxicity or cytolytic activity of immune cells or induce the promotion of cytokine secretion in immune cells. Therefore, when the antigen-binding site of the chimeric antigen receptor recognizes and binds to an antigen in the presence of EphA2-expressing cancer cells, signal transduction is induced, thereby enhancing the cytotoxicity or cytolytic activity of immune cells and / or promoting cytokine secretion, making the chimeric antigen receptor useful as a chimeric antigen receptor with excellent cytotoxic or cytolytic efficacy for attacking cancer cells.

[0054] Another aspect of the present invention provides polynucleotides and expression vectors for expressing the chimeric antigen receptor. The polynucleotide comprises a nucleotide sequence encoding the chimeric antigen receptor.

[0055] In the present invention, the term "polynucleotide" comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and the basic structural unit, nucleotide, includes not only natural nucleotides but also analogues in which the sugar or base moiety is modified.

[0056] "Encoding the chimeric antigen receptor" means that the polynucleotide encodes genetic information for synthesizing a protein having the amino acid sequence of the chimeric antigen receptor of the present invention through normal protein expression processes such as transcription, translation, etc. In this regard, the scope of the present invention includes not only a protein having the exact same amino acid sequence as the chimeric antigen receptor, but also a polynucleotide encoding a protein having substantially the same amino acid sequence as the chimeric antigen receptor, as described above, but having the same and / or similar activity as the protein.

[0057] Specifically, the polynucleotide of the present invention comprises a base sequence encoding an antigen-binding variable fragment of an anti-EphA2 antibody that specifically binds to EphA2, and more specifically comprises the base sequence of SEQ ID NO: 20 or SEQ ID NO: 21.

[0058] Furthermore, the polynucleotide of the present invention includes not only a base sequence encoding the antigen-binding variable fragment but also base sequences encoding other extracellular domain portions linked thereto, the transmembrane domain, and / or the intracellular signaling domain.

[0059] The description of the chimeric antigen receptor, including the antibody, antigen-binding variable fragment, extracellular domain, transmembrane domain, and intracellular signaling domain, is the same as that previously described.

[0060] The polynucleotide comprises the nucleotide sequence of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25. In one specific example of the present invention, a chimeric antigen receptor was designed that contains the scFvs of two types of anti-EphA2 antibodies (#79 and #85) in the extracellular domain, CD28 linked to Myc and a hinge domain as the transmembrane domain and intracellular signaling domain, and CD3-zeta and DAP10 linked via the intracellular signaling domain, and a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 22 or SEQ ID NO: 23 was prepared for expression of the chimeric antigen receptor (EphA2-CAR1). In another specific embodiment of the present invention, a chimeric antigen receptor was designed that contained the scFvs of two anti-EphA2 antibodies (#79 and #85) in the extracellular domain, together with the hinge domain, transmembrane domain, and intracellular signaling domain of CD8, and was linked to CD3-zeta and 41-BB via the intracellular signaling domain. To express this chimeric antigen receptor, a polynucleotide containing the base sequence of SEQ ID NO: 24 or SEQ ID NO: 25 was prepared (EphA2-CAR2).

[0061] The polynucleotide of the present invention may comprise a nucleotide sequence that is substantially the same as the listed nucleotide sequence, including, for example, a nucleotide sequence that has 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 to the listed nucleotide sequence, including when the same amino acids are synthesized upon transcription and translation, but is not limited thereto.

[0062] The polynucleotide encoding the chimeric antigen receptor may contain a nucleotide sequence optimized depending on the type of organism into which it is to be introduced and expressed, and the expression system of the organism, such as transcription and translation. This is due to codon degeneracy, which allows for a variety of combinations of nucleotide sequences capable of encoding the expressed protein, all of which are within the scope of the present invention. The modification of the polynucleotide through codon optimization is determined depending on the type of organism into which the chimeric antigen receptor of the present invention is to be expressed and applied. For example, the polynucleotide of the present invention is a polynucleotide that has been modified and optimized for codon selection in mammals and primates, and more specifically, is a polynucleotide that has been optimized and modified to be suitable for expression and function in humans.

[0063] The expression vector of the present invention comprises the polynucleotide.

[0064] Since the polynucleotide contains a nucleotide sequence encoding the chimeric antigen receptor of the present invention, the expression vector containing the polynucleotide is used to express and prepare the chimeric antigen receptor, and serves to transfer the polynucleotide into a specific cell or organism so that the chimeric antigen receptor is expressed, or to store and preserve the polynucleotide.

[0065] The expression vectors are constructed for use in prokaryotic or eukaryotic hosts.

[0066] For example, when the expression vector is used in a prokaryotic cell as a host, it generally contains a strong 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, T7 promoter, etc.), a ribosome binding site for translation initiation, and a transcription / translation termination sequence. When E. coli (e.g., HB101, BL21, DH5α, etc.) is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol. (1984) 158:1018-1024) and the left-facing promoter of phage lambda (pLλ promoter; Herskowitz, I. and Hagen, D., Ann. Rev. Genet. (1980) 14:399-445) are used as regulatory sites. When Bacillus bacteria are used as the host cell, the promoter of the toxin protein gene of Bacillus thuringiensis (Appl Environ Microbiol (1998) 64: 3932-3938; Mol Gen Genet (1996) 250: 734-741) or any promoter that can be expressed in Bacillus bacteria can be used as the regulatory site. The expression vector can be prepared by manipulating a plasmid (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.), a phage (e.g., λgt4·λB, λ-Charon, λΔz1, and M13, etc.), or a virus (e.g., SV40, etc.) that is frequently used by those skilled in the art.

[0067] When the expression vector is used in a eukaryotic cell as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late 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 Rous sarcoma virus (RSV) promoter) is used, and generally has a polyadenylation sequence as a transcription termination sequence. The expression vector has a CMV promoter.

[0068] The expression vector may also be fused with other sequences to facilitate purification of the antibody expressed thereby. Examples of fused sequences include glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA). Furthermore, since the protein expressed by the expression vector of the present invention is a chimeric antigen receptor, given its characteristics, the expressed protein can be easily purified through a protein A column or the like without any additional sequences for purification.

[0069] The expression vector may contain an antibiotic resistance gene commonly used by those skilled in the art as a selection marker, for example, resistance genes to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0070] 2. Immune cells expressing chimeric antigen receptors for EphA2 on their surface Another aspect of the invention provides immune cells characterized as exhibiting enhanced cytotoxic or cytolytic activity against cancer cells that express EphA2.

[0071] The immune cells express the chimeric antigen receptor of the present invention described above on their surfaces. The chimeric antigen receptor is the same as that previously described in "1. Novel anti-EphA2 antibodies, antigen-binding fragments thereof, chimeric antigen receptors (CARs) containing the same, and polynucleotides and expression vectors for expressing the same." Specifically, the chimeric antigen receptor may contain an antigen-binding site capable of specifically binding to EphA2 antigen expressed in cancer cells.

[0072] The immune cells may be any cells capable of inducing immunity and producing the desired therapeutic effect, including, but not limited to, any one selected from the group consisting of natural killer cells (NK cells), T cells, natural killer T cells (NKT cells), cytokine-induced killer cells (CIK), macrophages, and dendritic cells. Therefore, immune cells expressing a 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), CAR-NKT cells (chimeric antigen receptor natural killer T cells), and CAR-macrophages (chimeric antigen receptor macrophages).

[0073] The T cells include, but are not limited to, cytotoxic T lymphocytes (CTLs), tumor-infiltrating lymphocytes (TILs), and T cells isolated from peripheral blood mononuclear cells (PBMCs).

[0074] CAR-NK cells refer to cells in which a chimeric antigen receptor has been introduced into natural killer cells. They not only solve the problems of persistent toxicity, the risk of autoimmune disease, graft-versus-host disease (GVHD) in xenogeneic cell transplants, and non-target toxicity that exist in cancer immunotherapy using existing T cell-based CAR-T therapeutic agents, but also have the advantage of being able to target a variety of cancer cells and be used as a versatile therapeutic agent by switching on / off the response.

[0075] The immune cells of the present invention express a chimeric antigen receptor (CAR) on their cell surface, which comprises an antigen-binding variable fragment (scFv) of an antibody as an extracellular domain, capable of specifically recognizing and binding to EphA2, which is specifically expressed in cancer cells. Therefore, in the presence of cancer cells expressing EphA2, signal transduction can occur via the CAR, thereby further enhancing the cytotoxicity or cytolytic activity of the immune cells and increasing cytokine secretion. Therefore, the immune cells of the present invention can have the activity of attacking and treating cancer cells.

[0076] In a specific example of the present invention, chimeric antigen receptors containing antigen-binding variable fragments of two EphA2-specific antibodies of the present invention as extracellular domains were expressed on the surface of natural killer cells and on T cells. When each of the two natural killer cells was co-cultured with MDA-MB-231 cells, a breast cancer cell line that expresses EphA2, and when each of the two T cells was co-cultured with A549 cells, a lung cancer cell line that expresses EphA2, both showed improved cytotoxicity (or cytolytic activity), significantly increased cytokine secretion, and increased degranulation, demonstrating that the immune cells of the present invention have a significant therapeutic effect against EphA2-expressing cancer cells. Furthermore, the therapeutic effect against cancer cells was confirmed in an animal model transplanted with cancer cells.

[0077] 3. Therapeutic use of immune cells of the present invention against cancer Yet another aspect of the present invention provides a pharmaceutical composition for treating cancer, comprising the immune cells. The explanations regarding the immune cells and the chimeric antigen receptors expressed thereby are the same as those explained in "1. Novel anti-EphA2 antibodies, antigen-binding fragments thereof, chimeric antigen receptors (CARs) containing them, and polynucleotides and expression vectors for expressing them" and "2. Immune cells expressing chimeric antigen receptors against EphA2 on their surface," and therefore will be omitted to avoid repetition.

[0078] As used herein, the term "cancer", used interchangeably with "tumor", refers to or means the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Cancers or carcinomas that can be treated with the composition of the present invention are not particularly limited and include both solid cancers and blood cancers, for example, at least one selected from the group consisting of lung cancer, gastric 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, blood cancer, multiple myeloma, acute myeloid leukemia, malignant lymphoma, thymic cancer, osteosarcoma, fibroid tumor, and brain cancer, but are not limited thereto, and any cancer cells containing an antigen that can be recognized by the second chimeric antigen receptor can be used without limitation in the present invention.

[0079] In the present invention, the term "treatment" means inhibiting the development of cancer, reducing or eliminating symptoms. The pharmaceutical composition contains 1 to 10 times, 2 to 10 times, or 5 to 8 times the number of immune cells compared to the number of tumor cells in the individual to be treated, but is not limited to these.

[0080] The composition may be in the form of a quasi-drug composition, a health food composition, or the like, in addition to a pharmaceutical composition.

[0081] The cancer therapeutic composition of the present invention may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means that the compound does not inhibit the activity of the active ingredient and does not have toxicity greater than that applicable to the subject to which it is applied (prescribed), and the "carrier" is defined as a compound that facilitates the addition of the compound into cells or tissues.

[0082] The pharmaceutical composition of the present invention may be administered alone or in admixture with a suitable carrier, etc., and may be administered in single-dose or multiple-dose forms. The pharmaceutical composition may be a solid or liquid formulation. Solid formulations include, but are not limited to, powders, granules, tablets, capsules, suppositories, etc. Solid formulations may contain, but are not limited to, carriers, flavoring agents, binders, preservatives, disintegrants, lubricants, fillers, etc. Liquid formulations include, but are not limited to, solutions such as water and propylene glycol solutions, suspensions, and emulsions, which may be prepared by adding appropriate colorants, flavoring agents, stabilizers, thickeners, etc. For example, powders may be prepared by simply mixing the active ingredient, a tri-hydroxy derivative of a polyunsaturated fatty acid, with a suitable pharmaceutically acceptable carrier, such as lactose, starch, or microcrystalline cellulose. Granules can be prepared by mixing the tri-hydroxy derivative of polyunsaturated fatty acid of the present invention, a suitable pharmaceutically acceptable carrier, and a suitable pharmaceutically acceptable binder such as polyvinylpyrrolidone, hydroxypropyl cellulose, etc., followed by a wet granulation method using a solvent such as water, ethanol, isopropanol, etc., or a dry granulation method using compression force. Tablets can be prepared by mixing the granules with a suitable pharmaceutically acceptable lubricant such as magnesium stearate, followed by tableting using a tablet press.

[0083] The pharmaceutical composition may be administered by, but is not limited to, oral administration, injection (e.g., intramuscular injection, intraperitoneal injection, intravenous injection, infusion, subcutaneous injection, implant), inhalation, nasal administration, vaginal administration, rectal administration, sublingual administration, transdermal powder, topical administration, etc. depending on the disease to be treated and the condition of the individual. It is formulated into an appropriate dosage unit form containing non-toxic pharmaceutically acceptable carriers, additives, and vehicles commonly used depending on the administration route.

[0084] The pharmaceutical composition is administered daily at a dose of about 0.0001 mg / kg to about 10 g / kg, i.e., a daily dosage of about 0.001 mg / kg to about 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.), the severity of the condition being treated, etc. If necessary and for convenience, the total daily dosage may be administered several times throughout the day. [Example]

[0085] The present invention will be described in detail below with reference to examples.

[0086] However, the following examples are provided to specifically illustrate the present invention, and the content of the present invention is not limited to the following examples.

[0087] [Example 1] [1-1] Generation of single-chain variable fragments of antibodies that specifically bind to EphA2 Two single-chain variable fragments (scFv) (#79, #85) were prepared using sequences that play an important role in specific binding among anti-EphA2 antibody sequences that can specifically bind to EphA2 protein expressed in cancer cells.

[0088] scFv #79 was designed to comprise a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6. scFv #85 was designed to comprise a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 10, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 13, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 14.

[0089] [1-2] Design of chimeric antigen receptors for transfection into natural killer cells We designed chimeric antigen receptors (CARs) to be introduced into natural killer cells as extracellular domains containing the two scFvs designed above as antigen-binding sites. Specifically, the extracellular domains containing the two scFvs as antigen-binding sites were linked to CD28 as a transmembrane domain via Myc and a hinge domain, and the transmembrane domain was further linked to CD3-zeta as an intracellular signaling domain and CD28 DAP10 as a costimulatory molecule. These chimeric antigen receptors of the present invention were designed to have the intracellular signaling domain of a chimeric antigen receptor classified as a so-called third-generation CAR (designated "EphA2#79-CAR1" and "EphA2#85-CAR1," respectively, and collectively referred to as "EphA2-CAR1"). The nucleotide sequences of the gene constructs encoding them were then inserted into a lentiviral vector (Figure 1).

[0090] [1-3] Design of chimeric antigen receptors for transfection into T cells Additionally, we designed chimeric antigen receptors (CARs) to be introduced into T cells as extracellular domains containing the two scFvs designed above as antigen-binding sites. Specifically, the extracellular domains containing the two scFvs as antigen-binding sites were linked to CD8 as a transmembrane domain via a hinge domain, and the intracellular signaling domains CD3-zeta and 4-1BB as a costimulatory molecule were added to the transmembrane domain to design the CARs of the present invention to have the intracellular signaling domains of so-called second-generation CAR-classified chimeric antigen receptors (designated "EphA2#79-CAR2" and "EphA2#85-CAR2," respectively, and collectively referred to as "EphA2-CAR2"). The nucleotide sequences of the gene constructs encoding them were then inserted into lentiviral vectors (Figure 2).

[0091] [Example 2] Generation of immune cells expressing chimeric antigen receptors for EphA2 on their surface Immune cells capable of expressing the chimeric antigen receptor of the present invention on the surface, as designed in Example 1, were prepared.

[0092] [2-1] Production of EphA2-CAR1-expressing natural killer cells The lentiviral vectors prepared in Examples 1-2 were cotransfected into HEK293T cells with viral packaging vectors (pMDLG / RRE, pRSV / REV, VSVG). EphA2-CAR1-expressing lentivirus was then harvested and concentrated using an ultracentrifuge. Natural killer cells were then infected using the spinoculation method (360 g, 90 min, RT) at a multiplicity of infection (MOI) of 30. The infected natural killer cells were cultured at 37°C and 5% CO for 5 hours, then replaced with fresh medium. After 3 days, the cells were treated with 3 μg / ml of puromycin to select fully infected natural killer cells, and the culture was continued. As a control, uninfected natural killer cells were also treated with puromycin, and the control natural killer cells were cultured in the puromycin-treated medium until they were completely killed by puromycin. When all natural killer cells in the control group had died, the infected natural killer cells were selected and the experiment was carried out.

[0093] The natural killer cells expressing EphA2#79-CAR1 and EphA2#85-CAR1 (EphA2#79-CAR1-NK cells and EphA2#85-CAR1-NK cells, respectively, collectively referred to as EphA2-CAR1-NK cells) were treated with an anti-myc antibody (CST; 9B11) that specifically binds to the myc domain of EphA2-CAR1 (4°C, 30 minutes in heifers), and Myc expression was confirmed by flow cytometry. Natural killer cells that did not express EphA2-CAR1 were used as a control.

[0094] As a result, as shown in Figure 3A, it was confirmed that EphA2-CAR1 was sufficiently expressed in both types of EphA2-CAR1-NK cells compared to the control group.

[0095] [2-2] Production of EphA2-CAR2-expressing T cells After peripheral blood mononuclear cells (PBMCs) were differentiated into T cells, the lentiviral vectors prepared in Example 1-3 were used to infect the differentiated T cells using the same virus harvesting and spinoculation method as in Example 2-1 (5 MOI, 300 g, 32°C, 90 min). EphA2#79-CAR2- and EphA2#85-CAR2-expressing T cells (EphA2#79-CAR2-T cells and EphA2#85-CAR2-T cells, respectively, are collectively referred to as EphA2-CAR2-T cells) were then generated. Subsequently, anti-EphA2 antibodies (His-tag recombinant protein EphA2 (NKMAX) and Anti-6X His tag) were used. (登録商標) The expression of EphA2-CAR2 was confirmed using a flow cytometer with a FITC antibody (Abcam). As a control, naive T cells that did not express EphA2-CAR2 were used.

[0096] As a result, as shown in Figure 3B, it was confirmed that EphA2-CAR2 was sufficiently expressed in both types of EphA2-CAR2-T cells compared to the control group.

[0097] [Example 3] Confirmation of the cytotoxicity (or cytolytic activity) of EphA2-CAR1-NK cells and EphA2-CAR2-T cells against EphA2-expressing cancer cells As prepared in Example 2, natural killer cells and T cells expressing the chimeric antigen receptor of the present invention on their surface contain an scFv that specifically binds to EphA2 as an antigen-binding site, and therefore, their cytotoxicity against cancer cells expressing EphA2 was confirmed.

[0098] [3-1] Selection of EphA2-expressing cancer cells First, we selected two cancer cells that express EphA2: breast cancer cell line MDA-MB-231 (Korean Cell Line Bank) and lung cancer cell line A549 (Korean Cell Line Bank), and cancer cells that do not express EphA2: chronic myeloid leukemia cell line K562 (Korean Cell Line Bank), and then used anti-EphA2 antibodies (Human EphA2 / Mouse IgG2A Alexa Fluor 1000). (登録商標) After incubation with 1 ul / 100 ul of 488-conjugated antibody (R&D systems) at 4°C for 30 minutes in heifers, the expression levels of EphA2 in the three cell lines were confirmed using a flow cytometer.

[0099] As a result, as shown in FIG. 4, it was confirmed that MDA-MB-231 cells and A549 cells express EphA2, whereas K562 cells do not express EphA2.

[0100] [3-2] Confirmation of activity of EphA2-CAR1-NK cells against EphA2-expressing cancer cells The cytotoxicity of the two types of EphA2-CAR1-NK cells prepared in Example 2-1 was confirmed using calcein AM assay against MDA-MB-231 cells and K562 cells, whose EphA2 expression was confirmed as described above. Specifically, MDA-MB-231 cells and K562 cells were treated with calcein at a concentration of 5 μg / ml and incubated (37°C, 5% CO2, heifer, 1 hour). Calcein-stained cancer cells were then treated with natural natural killer cells and the two types of EphA2-CAR1-NK cells at a ratio of 5:1, 1:1, or 0.5:1 (natural killer cells:cancer cells), respectively, and incubated (37°C, 5% CO2, 4 hours). The amount of calcein present in the supernatant was then determined by sampling 100 μl of the supernatant.

[0101] As a result, as shown in Figure 5 below, both the EphA2#79-CAR1-NK cells and EphA2#85-CAR1-NK cells exhibited significantly higher cytotoxicity against EphA2-expressing MDA-MB-231 cells than the control natural killer cells, and this cytotoxicity was confirmed to be concentration-dependent for the treated EphA2-CAR1-NK cells.In contrast, both the control and the two EphA2-CAR1-NK cells exhibited almost no cytotoxicity against K562 cells, which do not express EphA2.

[0102] Furthermore, cytokine and granule secretion from the two types of EphA2-CAR1-NK cells was confirmed. Specifically, MDA-MB-231 cells and K562 cells were treated with native natural killer cells or each of the two types of EphA2-CAR1-NK cells prepared in Example 2-1 in a 1:1 ratio, and then reacted (37°C, 5% CO2 for 16 hours). The supernatant was collected and the presence of INF-γ (Interferon-γ) in the supernatant was confirmed by ELISA. The amount of cytokine secreted from native natural killer cells and EphA2-CAR1-NK cells alone was used as a control.

[0103] As a result, as shown in Figure 6, it was confirmed that the amount of INF-γ secretion was significantly improved only in EphA2-CAR1-NK cells treated with EphA2-expressing MDA-MB-231 cells.

[0104] In addition, MDA-MB-231 cells, K562 cells, and original natural killer cells or each of the two types of EphA2-CAR1-NK cells prepared in Example 2-1 were mixed 1:1 in RPMI (10% FBS) and reacted (37°C, 5% CO2 for 4 hours). After that, the cells were treated with anti-CD56 antibody and stained to select natural killer cells, and the level of CD107a expression in the original natural killer cells and the two types of EphA2-CAR1-NK cells was analyzed using flow cytometry.

[0105] As a result, as shown in Figure 7, similar to the case of INF-γ, it was confirmed that CD107a expression was significantly increased only in EphA2-CAR1-NK cells treated with EphA2-expressing MDA-MB-231 cells.

[0106] [3-3] Confirmation of activity of EphA2-CAR2-T cells against EphA2-expressing cancer cells GFP was expressed in A549 cells in which the expression of EphA2 had been confirmed as described above, and the EphA2-CAR2-T cells prepared in Example 2-2 were treated with GFP at ratios of 2:1, 1:1, 0.5:1, and 0.25:1 (T cells:cancer cells). The cells were cultured for 48 hours using an IncuCyte device, and data were collected at 4-hour intervals. The cytotoxicity of the EphA2-CAR2-T cells was analyzed using the IncuCyte ZOOM program.

[0107] As a result, as shown in Figure 8, it was confirmed that the two types of EphA2-CAR2-T cells exhibited cytotoxicity against EphA2-expressing A549 cells.

[0108] [Example 4] Confirmation of the cytotoxicity (or cytolytic activity) of EphA2-CAR1-NK cells and EphA2-CAR2-T cells in vivo The cytotoxicity of natural killer cells and T cells expressing the chimeric antigen receptor of the present invention on their surface, as confirmed in Example 3, against EphA2-expressing cancer cells was confirmed again in an animal model.

[0109] [4-1] Confirmation of activity of EphA2-CAR1-NK cells against EphA2-expressing cancer cells First, using the same method as in Example [3-1], we confirmed that EphA2 is expressed in H460 cells (Korea Cell Line Bank), a lung cancer cell line (Figure 9A). Using the same method as in Example [3-2], we confirmed that EphA2#79-CAR1-NK cells exhibited significantly higher cytotoxicity against H460 cells than control natural killer cells expressing a CAR with its extracellular domain deleted (dECTO) (Figure 9B).

[0110] Next, as shown in Figure 9C, 3 x 10 6The cells were injected subcutaneously into the flank of approximately 6-week-old female Balb / c nude mice (Selon Bio). After 10 days, the tumor size reached 50 mm. 3 When the cells reached the target concentration, 2 x 10 EphA2-CAR1-NK cells or control natural killer cells were added. 6 The mice were intravenously injected with the drug at 3- or 4-day intervals for five times, and tumor size and weight were measured over a 24-day period.

[0111] As a result, as shown in Figures 9D and 9E, it was confirmed that the tumor size and weight were significantly reduced in mice administered with the EphA2-CAR1-NK cells of the present invention compared to mice administered with control natural killer cells.

[0112] [4-2] Confirmation of activity of EphA2-CAR2-T cells against EphA2-expressing cancer cells Next, 1x10 A549-Luciferase cells (PerkinElmer), in which luciferase was introduced into A549 cells, a lung cancer cell line that expresses EphA2, were cultured. 6 The 1000mg IgG1 was subcutaneously injected into the right flank of 6-week-old male NOG mice (Coretec Co., Ltd.) until the tumor size reached 200mm. 3 When the target cells reached a certain level, 5x10 EphA2-CAR2 T cells or control T cells expressing ectodomain-deleted CAR (dECTO) were cultured. 6 The tumor size, mouse weight, and IVIS were then measured twice a week for 45 days.

[0113] As a result, as shown in Figures 10A and 10B, the EphA2-CAR2-T cells were confirmed to effectively suppress tumors, and as shown in Figure 10C, the presence of the EphA2-CAR2-T cells and control T cells was confirmed in the mouse blood after intravenous injection. Furthermore, as shown in Figure 10D, the EphA2-CAR2-T cells were confirmed to be present in greater amounts in tumors than control T cells.

[0114] [Organize] Taking the results of the above experiments into consideration, it was confirmed not only in vitro but also in vivo that the two anti-EphA2 antibodies of the present invention (#79, #85) not only exhibit specific binding affinity to EphA2, but also, when they recognize and bind to EphA2 on cancer cells expressing the EphA2, induce signal transduction in immune cells (natural killer cells or T cells) that express chimeric antigen receptors containing the antigen-binding site of the antibody on their surface, thereby inducing various immune responses that enable immune cells to attack cancer cells.

[0115] Although the present invention has been described in detail above only with reference to the embodiments, it will be apparent to those skilled in the art that various modifications and variations are possible within the scope of the technical concept of the present invention, and it is to be understood that such modifications and variations are also within the scope of the appended claims. For example, the present disclosure provides the following embodiments. [Section 1] a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 11; a light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 12, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 14; An antibody or antigen-binding fragment thereof that specifically binds to EphA2. [Section 2] Item 1, the antibody or antigen-binding fragment thereof according to Item 1, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 15. [Section 3] Item 1, the antibody or antigen-binding fragment thereof according to Item 1, wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 16. [Section 4] an extracellular domain comprising an antigen-binding site that specifically binds to EphA2; a transmembrane domain; an intracellular signaling domain; and a chimeric antigen receptor (CAR) comprising: The antigen-binding site that specifically binds to EphA2 is a single-chain variable fragment (scFv) of an anti-EphA2 antibody comprising a heavy chain variable region including a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 11, and a light chain variable region including a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 12, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 14, said chimeric antigen receptor. [Section 5] The antigen-binding site that specifically binds to EphA2 is The chimeric antigen receptor of Item 4, which is a single-chain variable fragment of an anti-EphA2 antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 15. [Section 6] The antigen-binding site that specifically binds to EphA2 is The chimeric antigen receptor of Item 4, which is a single-chain variable fragment of an anti-EphA2 antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:16. [Section 7] Item 1. The chimeric antigen receptor according to Item 1, wherein the extracellular domain further comprises at least one selected from the group consisting of a hinge domain and a spacer domain. [Section 8] Item 8. The chimeric antigen receptor according to Item 7, wherein the hinge domain or spacer domain is at least one selected from the group consisting of a Myc epitope, a CD8 hinge domain, and Fc. [Section 9] Item 5. The chimeric antigen receptor according to Item 4, wherein the transmembrane domain is any one selected from the group consisting of the alpha (α), beta (β), or zeta (ζ) chain of a T cell receptor (TCR), CD28, CD3 epsilon (ε), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. [Section 10] The intracellular signaling domain comprises: The chimeric antigen receptor of Item 4, comprising at least one selected from the group consisting of T cell receptor (TCR) zeta (ζ), FcR gamma (γ), FcR beta (β), CD3 gamma (γ), CD3 delta (δ), CD3 epsilon (ε), CD3 zeta (ζ), CD5, CD22, CD79a, CD79b, and CD66d. [Section 11] The intracellular signaling domain comprises: comprising as a first signaling domain at least one selected from the group consisting of T cell receptor (TCR) zeta (ζ), FcR gamma (γ), FcR beta (β), CD3 gamma (γ), CD3 delta (δ), CD3 epsilon (ε), CD3 zeta (ζ), CD5, CD22, CD79a, CD79b, and CD66d; The chimeric antigen receptor according to Item 4, comprising at least one costimulatory signaling domain 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, EphA2, and CD83. [Section 12] A polynucleotide comprising a nucleotide sequence encoding the chimeric antigen receptor according to any one of Items 4 to 11. [Section 13] Item 13. An expression vector comprising the polynucleotide according to Item 12. [Section 14] Item 12. An immune cell that expresses the chimeric antigen receptor according to any one of Items 4 to 11 on its surface. [Section 15] Item 15. The immune cell according to Item 14, wherein the immune cell is any one selected from the group consisting of natural killer cells (NK cells), T cells, natural killer T cells (NKT cells), cytokine-induced killer cells (CIK), macrophages, and dendritic cells. [Section 16] A pharmaceutical composition for cancer treatment, comprising the immune cells of item 14. [Section 17] Item 17. The pharmaceutical composition for cancer treatment according to Item 16, wherein the cancer is at least one selected from the group consisting of lung cancer, gastric 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, blood cancer, multiple myeloma, acute myeloid leukemia, malignant lymphoma, thymic cancer, osteosarcoma, fibroid tumor, and brain cancer.

Claims

1. A heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3; a light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6; An antibody or antigen-binding fragment thereof that specifically binds to EphA2.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:

7.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the light chain variable region has the amino acid sequence of SEQ ID NO:

8.

4. an extracellular domain comprising an antigen-binding site that specifically binds to EphA2; a transmembrane domain; an intracellular signaling domain; and a chimeric antigen receptor (CAR) comprising: The antigen-binding site that specifically binds to EphA2 is a single-chain variable fragment (scFv) of an anti-EphA2 antibody comprising a heavy chain variable region including a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region including a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6, wherein the chimeric antigen receptor is a single-chain variable fragment (scFv) of an anti-EphA2 antibody comprising:

5. The antigen-binding site that specifically binds to EphA2 is The chimeric antigen receptor of claim 4, which is a single-chain variable fragment of an anti-EphA2 antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:

7.

6. The antigen-binding site that specifically binds to EphA2 is The chimeric antigen receptor of claim 4, which is a single-chain variable fragment of an anti-EphA2 antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO:

8.

7. The chimeric antigen receptor of claim 4, wherein the extracellular domain further comprises at least one selected from the group consisting of a hinge domain and a spacer domain.

8. The chimeric antigen receptor of claim 7, wherein the hinge domain or spacer domain is at least one selected from the group consisting of a Myc epitope, a CD8 hinge domain, and Fc.

9. The chimeric antigen receptor of claim 4, wherein the transmembrane domain is any one selected from the group consisting of the alpha (α), beta (β), or zeta (ζ) chain of a T cell receptor (TCR), CD28, CD3 epsilon (ε), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

10. The intracellular signaling domain The chimeric antigen receptor of claim 4, comprising at least one selected from the group consisting of T cell receptor (TCR) zeta (ζ), FcR gamma (γ), FcR beta (β), CD3 gamma (γ), CD3 delta (δ), CD3 epsilon (ε), CD3 zeta (ζ), CD5, CD22, CD79a, CD79b, and CD66d.

11. The intracellular signaling domain comprises: comprising at least one selected from the group consisting of T cell receptor (TCR) zeta (ζ), FcR gamma (γ), FcR beta (β), CD3 gamma (γ), CD3 delta (δ), CD3 epsilon (ε), CD3 zeta (ζ), CD5, CD22, CD79a, CD79b, and CD66d as a first signaling domain; The chimeric antigen receptor of claim 4, which comprises at least 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, EphA2, and CD83 as a costimulatory signaling domain.

12. A polynucleotide comprising a base sequence encoding the chimeric antigen receptor of any one of claims 4 to 11.

13. An expression vector comprising the polynucleotide of claim 12.

14. An immune cell expressing the chimeric antigen receptor according to any one of claims 4 to 11 on its surface.

15. The immune cell according to claim 14, wherein the immune cell is any one selected from the group consisting of natural killer cells (NK cells), T cells, natural killer T cells (NKT cells), cytokine-induced killer cells (CIK), macrophages, and dendritic cells.

16. A pharmaceutical composition for cancer treatment comprising the immune cells of claim 14.

17. 17. The pharmaceutical composition for cancer therapy according to claim 16, wherein the cancer is at least one selected from the group consisting of lung cancer, gastric 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, blood cancer, multiple myeloma, acute myeloid leukemia, malignant lymphoma, thymic cancer, osteosarcoma, fibroid tumor, and brain cancer.

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