KR20230022810A - Novel Anti-EphA2 Chimeric Antigen Receptor and Immune Cell Expressing the Same - Google Patents

Abstract

The present invention relates to: a novel antibody or an antigen-binding fragment thereof that specifically binds to EphA2; a chimeric antigen receptor comprising an antigen-binding variable fragment of the antibody; and immune cells expressing the chimeric antigen receptor. The antibody of the present invention, the chimeric antigen receptor using the same, and the immune cells expressing the chimeric antigen receptor can be usefully used for cancer treatment.

Description

Novel Anti-EphA2 Chimeric Antigen Receptor and Immune Cell Expressing the Same}

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.

Methods for treating cancer have gone through a process of steady development and change, and methods such as surgery, chemotherapy, and radiation therapy are still being used, but these existing cancer treatment methods are only used in the early stage when the cancer has not metastasized. In most cases, it is effective, and in a state where metastasis has already progressed, there is a problem that, for example, even if surgical operation is performed, the possibility of recurrence is high. Accordingly, studies on methods of using an immune response to treat cancer have recently been conducted.

Among them, there is a growing interest in cell therapy methods that use immune cells to reinforce them or genetically modify them and inject them back into the patient. For example, tumor infiltrating lymphocytes (TIL), chimeric antigen receptors (Chimeric antigen receptor, CAR) and T-cell receptor (TCR) technologies are being studied. In particular, in the case of a chimeric antigen receptor, which is an artificial receptor designed to deliver antigen specificity to T cells or natural killer cells (NK cells), the receptor binds to a cancer cell-specific antigen to activate the immune cells and achieve specific immunity. It consists of an extracellular domain that can be provided, a transmembrane domain, and an intracellular signaling domain. And T cells expressing these chimeric antigen receptors were 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)), in the case of natural killer cells were named CAR-NK cells.

The intracellular signaling domain of the chimeric antigen receptor is mainly based on the intracellular signaling domain of CD3zeta, a signaling subunit of the T cell receptor (first-generation CAR). And it has evolved in the form of adding an intracellular signaling domain of a co-stimulatory molecule that promotes the growth and differentiation of immune cells. For example, currently marketed CAR-T cell therapeutics use the intracellular signaling domains of CD28 and 4-1BB co-stimulatory molecules, respectively (second-generation CARs), followed by CD28 and 4-1BB intracellular signaling domains. A CAR (3rd generation CAR) containing at the same time has been attempted (Stegen et al. , Nat. Rev. Drug Discov. , 14 (7): 499-509 (2015)).

On the other hand, EphA2 (ephrin type-A receptor 2) is a protein expressed from the human EPHA2 gene and is known to be overexpressed in various types of cancer, including breast cancer, prostate cancer, lung cancer, etc., and promotes the growth and penetration of cancer cells. It is known. In addition, it has been reported that the expression of EphA2 is related to the survival rate of cancer patients. Under this technical background, it is necessary to study another aspect of strategies and methods for treating cancer by developing antibodies targeting the EphA2, which is particularly overexpressed in cancer cells, or the CAR-T and CAR-NK therapeutics using the same. The need to do so emerges.

An object of the present invention is to provide an antibody or antigen-binding fragment thereof capable of specifically binding to EphA2, which is mainly expressed in cancer cells.

In addition, an object of the present invention is to provide a novel chimeric antigen receptor capable of amplifying cytotoxic or cytolytic activity against cancer cells when expressed in immune cells.

In addition, an object of the present invention is to provide a polynucleotide and an expression vector for expressing the chimeric antigen receptor.

In addition, an object of the present invention is to provide an immune cell with excellent therapeutic effect against cancer by expressing the chimeric antigen receptor on its surface.

In addition, an object of the present invention is to provide a pharmaceutical composition for treating cancer using the immune cells.

In order to achieve the above object, one aspect of the present invention is a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10, and SEQ ID NO: 3 or sequence A heavy chain variable region comprising a heavy chain CDR3 having the amino acid sequence of No. 11; and 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. region; and provides an antibody or antigen-binding fragment thereof that specifically binds to EphA2 (ephrin type-A receptor 2).

Another aspect of the present invention is an extracellular domain containing an antigen binding site that specifically binds to EphA2 (ephrin type-A receptor 2) (extracellular binding domain); transmembrane domain; And an intracellular signaling domain; as a Chimeric Antigen Receptor (CAR) comprising a; antigen binding site that binds specifically to EphA2, SEQ ID NO: 1 or SEQ ID NO: 9 A heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence, 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 SEQ ID NO: 4 or SEQ ID NO: 12 An anti-EphA2 antibody comprising a light chain variable region comprising light chain CDR1 having an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13 and light chain CDR3 having an amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 14. It provides a chimeric antigen receptor that is a single chain variable fragment (scFv) of.

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.

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 cancer treatment comprising the immune cells.

Antibodies, antigen-binding fragments thereof, and chimeric antigen receptors using the antibodies of the present invention can specifically bind to EphA2, which is mainly expressed in cancer cells, and thus signal transduction in immune cells expressing the chimeric antigen receptor. As this occurs, the cytotoxic or cytolytic activity of immune cells is significantly improved and the secretion of cytokines is promoted. In addition, this has an effect of increasing degranulation of cancer cells co-cultured with the immune cells.

Therefore, the antibody of the present invention or a chimeric antigen receptor using the same, and immune cells expressing the antibody can be usefully used for cancer treatment.

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

Figure 1a is a schematic diagram showing the structure of the chimeric antigen receptor (EphA2-CAR1) of the present invention comprising an antigen-binding variable fragment (scFv) that specifically binds to EphA2 as an extracellular domain, and Figure 1b is a cloned expression thereof. It represents a map of vectors.
Figure 2a is a schematic diagram showing the structure of the chimeric antigen receptor (EphA2-CAR2) of the present invention comprising an antigen-binding variable fragment (scFv) that specifically binds to EphA2 as an extracellular domain, and Figure 2b is a cloned expression thereof. It represents a map of vectors.
3a shows natural killer cells (EphA2#79-CAR1-NK cells and EphA2#85-CAR1-NK cells) into which two types of chimeric antigen receptor genes of the present invention were introduced and expressed, and FIG. 3b shows the present invention. The chimeric antigen receptors of the present invention (EphA2 -CAR1 and EphA2-CAR2) are shown as a result of confirming the expression.
4 shows the results of confirming the expression of EphA2 in MDA-MB-231 cells, a breast cancer cell line, A549 cells, a lung cancer cell line, and K562 cells, a chronic myelogenous leukemia cell line.
Figure 5 is MDA-MB expressing EphA2 in natural killer cells (EphA2#79-CAR1-NK cells and EphA2#85-CAR1-NK cells) into which two types of chimeric antigen receptor genes of the present invention were introduced and expressed. -231 cells and K562 cells that do not express EphA2 were measured for cytotoxicity (cytolytic activity), and the results were compared.
6 and 7 show MDA expressing EphA2 in natural killer cells (EphA2#79-CAR1-NK cells and EphA2#85-CAR1-NK cells) into which two types of genes of the chimeric antigen receptor of the present invention were introduced and expressed. -When co-cultured with MB-231 cells or K562 cells that do not express EphA2, the amount of cytokine (IFN-γ) secreted by natural killer cells and the expression level of CD107α, an indicator of degranulation, were measured and compared, respectively. that showed a result.
8 is a diagram of A549 cells expressing EphA2 in T cells (EphA2#79-CAR2-T cells and EphA2#85-CAR2-T cells) into which two types of chimeric antigen receptor genes of the present invention were introduced and expressed. It shows the result of measuring cytotoxicity (cytolytic activity).
9a is a result confirming that EphA2 is expressed in H460 cells, a lung cancer cell line, and FIG. 9b is a result of natural killer cells (EphA2#79-CAR1-NK cells) into which the chimeric antigen receptor gene of the present invention was introduced and expressed. This is the result of confirming cytotoxicity (cytolytic activity) for H460 cells. Figure 9c is a simplified schematic of an experiment confirming the activity of natural killer cells into which the gene of the chimeric antigen receptor of the present invention was introduced and expressed by injecting H460 cells into experimental animals, and Figures 9d and 9e show the results of the present invention. It is the result of confirming the anticancer activity by the cytotoxicity (cytolytic activity) of natural killer cells into H460 cells into which the gene of the chimeric antigen receptor was introduced and expressed, respectively, by the size and weight of the tumor.
10a and 10b show T cells (EphA2#79-CAR2-T cells and EphA2#85-CAR2-T cells) into which two types of chimeric antigen receptor genes of the present invention were introduced and expressed in experimental animals injected with A549-Luciferase cells. It is the result of confirming the anticancer activity by cytotoxicity (cytolytic activity) of A549 cells of T cells) by tumor size and weight, respectively. In addition, FIG. 10c is a result of confirming the presence or absence of the EphA2-CAR2-T cells in mouse blood, and FIG. 10d is a result of confirming the presence or absence of the EphA2-CAR2-T cells in a tumor.

Hereinafter, the present invention will be described in detail.

1. Novel anti-EphA2 antibodies, antigen-binding fragments thereof, and 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 capable of specifically binding to EphA2.

In the present invention, the term "antibody" refers to an immunoglobulin molecule having a reactivity by specifically binding to an epitope of an antigen immunologically. The antibody may include all of monoclonal antibodies, polyclonal antibodies, antibodies having a full-length chain structure (full-length antibodies), functional fragments having at least an antigen-binding function (antigen-binding fragments), and recombinant antibodies. Specifically, the antibody of the present invention may be a monoclonal antibody or an antigen-binding fragment thereof. The monoclonal antibody refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, and such monoclonal antibody exhibits a single binding specificity and affinity for a particular epitope. The full-length antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain may be connected to the heavy chain through a disulfide bond. The antibody includes a heavy chain (HC) and a light chain (LC) polypeptide, and the heavy chain and light chain may include a variable region and a constant region.

The constant region is a region that mediates binding of the antibody to various types of cells (T-cells, etc.) of the immune system, host tissues including components of the complement system, and the like. The constant region, if it is the same type of antibody derived from the same species, has the same function regardless of the type of antigen, and the amino acid sequence constituting it also has the same or high similarity for each antibody. The constant region may be divided into a heavy chain constant region (which may be abbreviated as CH) and a light chain constant region (which may be abbreviated as CL). The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and / or epsilon (ε) types, and subclasses include gamma 1 (γ1), gamma 2 (γ2), It has gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and/or alpha 2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types. IgG is a subtype and includes IgG1, IgG2, IgG3 and IgG4.

The variable region is an antibody region having specificity for an antigen, and may be divided into a heavy chain variable region (which may be abbreviated as VH) and a light chain variable region (which may be abbreviated as VL). The variable region may include three complementary-determining regions (CDRs) and four framework regions (FRs). The CDR may be a ring-shaped region involved in antigen recognition, and specificity for the antigen may be determined according to the amino acid sequence of the CDR. The CDRs may be referred to as CDR1, CDR2, and CDR3 according to their order, and depending on whether the heavy chain or light chain is a CDR of any polypeptide, in the case of a heavy chain variable region, CDR-H1, CDR-H2, CDR-H3, In the case of the light chain variable region, it may be referred to as CDR-L1, CDR-L2, or CDR-L3. Similarly, FR can be referred to as FR-H1, FR-H2, FR-H3, and FR-H4 for the heavy chain variable region, and FR-L1, FR-L2, FR-L3, and FR-L4 for the light chain variable region. there is. In addition, the CDRs and FRs may be arranged in the following order in each variable region.

As used herein, the term "antigen-binding fragment" refers to any fragment of the humanized antibody of the present invention that retains the antigen-binding function of the antibody. The antigen-binding fragment may be interchangeably referred to with terms such as "fragment" and "antibody fragment", and the antigen-binding fragment may be Fab, Fab', F(ab') ₂ , Fv, etc., but is not limited thereto. .

The Fab has a structure having light and heavy chain variable regions, a light chain constant region, and a first constant region (CH1 domain) of a heavy chain, and has one antigen binding site. The Fab' is different from the Fab in that it has a hinge region including one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. The F(ab') ₂ is generated when a cysteine residue in the hinge region of Fab' forms a disulfide bond. The Fv refers to a minimum antibody fragment having only a heavy chain variable region and a light chain variable region. Double-chain Fv (two-chain Fv) has a heavy chain variable region and light chain variable region connected by a non-covalent bond, and single-chain Fv (single-chain Fv) generally has a heavy chain variable region and a light chain variable region covalently bonded through a peptide linker. or directly linked at the C-terminus, it can form a dimer-like structure like double-chain Fv. The antigen-binding fragment may be prepared using a proteolytic enzyme (for example, Fab may be obtained by restriction digestion of whole antibody with papain, and F(ab')2 fragment may be obtained by digestion with pepsin), or It can be produced through genetic recombination technology, but is not limited thereto.

The linker may be a peptide linker and may have a length of about 10 to 25 amino acids. For example, the linker may include 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), and for example, (GnS)m( n and m may each be 1 to 10), but are not limited thereto.

As used herein, 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 may be formed from contiguous amino acids or from discontinuous amino acids juxtaposed by tertiary folding of a protein.

The “specifically binding” may mean binding to another molecule with a binding affinity greater than background binding, for example, the extracellular domain has an affinity or Ka of about 10 ⁻⁵ M or higher for the target antigen. (equilibrium dissociation constant of a specific binding interaction having a unit of 1/M). The affinity may have an equilibrium dissociation constant (Kd) of a specific binding interaction having a unit of M of 10 ⁻⁵ M to 10 ⁻¹³ M or less than the above range.

The antibody or antigen-binding fragment thereof of the present invention is a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10, and an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 11 a heavy chain variable region comprising a heavy chain CDR3 having the sequence; and 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. domain; and specifically binds to EphA2 (ephrin type-A receptor 2).

The heavy chain variable region may have an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 15.

The light chain variable region may have an amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 16.

The antibody or antigen-binding fragment thereof of the present invention may further include, for example, a heavy chain constant region and/or a light chain constant region of an antibody derived from a human, and the antibody or antigen-binding fragment thereof specifically binds to EphA2. The heavy chain constant region and/or light chain constant region of the human-derived antibody may be used without limitation in type or amino acid sequence, as long as the properties are not impaired.

The above-described amino acid sequences may include variants having different sequences due to deletion, insertion, substitution, or combination thereof of amino acid residues within a range that does not affect the structure, function, activity, etc. of the polypeptide containing the same. . In addition, the amino acid sequences may include amino acids with conventional modifications known in the art, and the amino acid modifications include, for example, phosphorylation, sulfation, acrylation, glycosylation, methylation ( methylation), farnesylation, and the like. The humanized antibody or antigen-binding fragment thereof of the present invention includes those having amino acid sequences substantially identical thereto, as well as those comprising the above-described amino acid sequences, or variants thereof. The meaning of having the substantially identical amino acid sequence is 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 of the amino acid sequence described above. % or more, 99% or more, or may include an amino acid sequence having a homology of 99.5% or more, but is not limited thereto.

In the present invention, the term “Chimeric antigen receptor (CAR)” is a synthetic complex designed to induce an immune response to a target antigen and a cell expressing the antigen when recognized and bound thereto. A chimeric antigen receptor may include an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain. The chimeric antigen receptor is expressed on the surface of immune cells and recognizes and binds to a specific antigen, such as an antigen specifically expressed on the surface of cancer cells, through an antigen binding site included in an extracellular domain, thereby signaling within the immune cell. Transduction can cause changes in the activity of immune cells, and an immune response can be induced by targeting only a specific antigen.

The chimeric antigen receptor (CAR) of the present invention includes an extracellular binding domain including an antigen binding site that specifically binds to ephrin type-A receptor 2 (EphA2); transmembrane domain; and an intracellular signaling domain.

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

When the chimeric antigen receptor of the present invention is expressed on the surface of an immune cell, when EphA2, the target antigen, binds to the receptor, signal transduction occurs in the immune cell, and cytotoxicity (or cytolysis) of the immune cell occurs. activity) and/or stimulation of cytokine secretion of the immune cells may be induced. The enhancement of cytotoxicity (or cytolytic activity) or promotion of cytokine secretion is at a higher level than cytotoxicity (or cytolytic activity) or cytokine secretion exhibited by immune cells in the absence of antigen. activity) or secrete cytokines at high levels.

The extracellular domain may further include at least one selected from the group consisting of a hinge domain and a spacer domain. The antigen-binding site of the extracellular domain may be linked to the transmembrane domain through a hinge domain and/or a spacer domain.

The hinge domain is the antigen-binding site from the surface of the immune cell on which the chimeric antigen receptor is expressed, so as to enable proper cell/cell contact, binding of the appropriate antigen/antigen-binding site, and activation of the appropriate chimeric antigen receptor. As a part that allows to be physically separated, it can play an important role in determining the location of the extracellular domain. The chimeric antigen receptor may include one or more hinge domains between the extracellular domain and the transmembrane domain. Hinge domains may be derived from natural, synthetic, semi-synthetic or recombinant sources. A 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 is (a) a naturally occurring hinge region with up to 30% amino acid change (e.g., amino acid substitutions or deletions of up to 25%, 20%, 15%, 10% or 5%), (b) up to 30% Natural at least 10 amino acids (e.g., at least 12, 13, 14, or 15 amino acids) in length with % amino acid changes (e.g., amino acid substitutions or deletions of up to 25%, 20%, 15%, 10%, or 5%) part of the hinge region, or (c) 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 may be substituted with one or more other amino acid residues (eg, one or more serine residues). The altered immunoglobulin hinge region may alternatively or additionally have a proline residue of a wild-type immunoglobulin hinge region substituted with another amino acid residue, such as cysteine. The hinge domain may be a hinge region derived from the extracellular domain of type 1 membrane proteins such as CD8, CD4, CD28 and CD7, but if the antigen binding site, the transmembrane domain, and the intracellular signaling domain can be linked between the cell membrane Any of these may be used without limitation. Also, it can be the wild type hinge region from these molecules or it can be changed.

The spacer domain may be referred to as a linking domain, and may include, for example, a hinge domain derived from CD28 and/or a hinge domain derived from CD8, and the entire or It may contain some.

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 may specifically include a Myc epitope and a CD8 hinge domain. More specifically, the Myc epitope may include the amino acid sequence of SEQ ID NO: 17, and the CD8 hinge domain may include the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19.

The transmembrane domain refers to a partial region that connects and fuses an extracellular domain and an intracellular signaling domain with each other and serves to anchor a chimeric antigen receptor to the plasma membrane of an immune cell. The transmembrane domain may be from natural, synthetic, semisynthetic or recombinant sources. The transmembrane domain is 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 may be any one selected from the group consisting of CD154, but is not limited thereto.

The transmembrane domain may be attached to the extracellular domain through a linker. For example, the linker may be a short oligopeptide or polypeptide linker of 2 to 10 amino acids in length, such as, but not limited to, a glycine (G)-serine (S) doublet.

The intracellular signaling domain functions of immune cells (e.g., activation including release of cytotoxic (or cytolytic activity) factors for target cells to which chimeric antigen receptors and antigens are bound, cytokine production, Proliferation and cytotoxicity or cytolytic activity, or other cellular responses induced by the antigen binding), which serves to transmit signals generated according to the binding of the chimeric antigen receptor and the antigen to the inside of the immune cell corresponds to the part. The intracellular signaling domain may be a part of a protein that transmits an effector signal and directs the cell to perform a specific function.

As the intracellular signaling domain, an intracellular signaling domain previously used in the development process of a chimeric antigen receptor may be used. Specifically, it may include only CD3ζ used in the first generation CAR (chimeric antigen receptor). And, as used in the second-generation CAR, a form in which a co-stimulatory domain (CD28 or CD137/4-1BB) and CD3ζ are combined can be used to improve responsiveness to immune cells. In addition, two or more co-stimulatory domains may be used as in the third-generation CAR, and at this time, to achieve expansion and persistence of immune cells including the CAR in vivo, the co-stimulatory domain is 4-1BB, CD28 or OX40, etc. can be combined. Furthermore, as used in the 4th generation CAR, additional genes encoding cytokines such as IL-12 or IL-15 can be included to allow the expression of additional CAR-based immunoproteins of cytokines, and the 5th generation CAR As used in, an interleukin receptor chain, for example, IL-2Rβ may be further included to enhance immune cells.

The intracellular signaling domain is T-cell receptor (TCR) zeta (ζ), FcR gamma (γ), FcR beta (β), CD3 gamma (γ), CD3 delta (δ), CD3 epsilon (ε), CD3 It may include at least one selected from the group consisting of zeta (ζ), CD5, CD22, CD79a, CD79b, and CD66d.

More specifically, activation of the immune cell by the intracellular signaling domain may 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 an antigen-dependent primary activation and a costimulatory signaling domain that acts in an antigen-independent manner to provide a secondary signal. Accordingly, the intracellular signaling domain may include a primary signaling domain and a co-stimulatory signaling domain.

The primary signaling domain refers to a signaling domain that regulates immune cell activation in a stimulating or inhibiting manner. Primary signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. The ITAM containing the primary signaling domain may be TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD66d, etc., but is not limited thereto. More specifically, the primary signaling domain may be CD3ζ(zeta), but is not limited thereto.

The co-stimulatory signaling domain refers to an intracellular signaling domain of a co-stimulatory molecule. The costimulatory signaling domain is 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, may include a co-stimulatory signaling domain selected from the group consisting of CD83, etc., but is not limited thereto . Specifically, the co-stimulatory molecule may be DAP10, but is not limited thereto.

The chimeric antigen receptor may include two or more intracellular signaling domains, and in the case of including two or more intracellular signaling domains, the intracellular signaling domains may be serially connected to each other. Alternatively, they may be connected through a polypeptide linker consisting of 2 to 10 amino acids, and the linker sequence may be, for example, a glycine-serine continuous sequence. The linker may include, for example, (GS)n, (GGS)n, (GSGGS)n or (GnS)m (n and m are each 1 to 10), and for example, (GnS)m( n and m may each be 1 to 10), but are not limited thereto.

The chimeric antigen receptor may further include an immune function stimulating factor of immune cells, and for example, the immune function stimulating factor of immune cells may be an interleukin signal sequence. The interleukin signal sequence may induce expression of IL-12, IL-8, IL-2, etc., but is not limited thereto. In addition, when the immune cells are T cells, the immune function promoting factor may be IL-7, CCL19, etc., but is not limited thereto.

In a specific embodiment of the present invention, scFvs of two types of anti-EphA2 antibodies (#79 and #85) having the same amino acid sequence as described above are included in the extracellular domain, and CD28 linked to Myc and hinge domains are included. A chimeric antigen receptor of the present invention was prepared by designing a chimeric antigen receptor comprising a transmembrane domain and an intracellular signaling domain, and CD3-zeta and DAP10 linked to an intracellular signaling domain (EphA2-CAR1 ). In addition, in another specific embodiment of the present invention, scFvs of two anti-EphA2 antibodies having the same amino acid sequence as described above (#79 and #85) are included in the extracellular domain, and CD8 is included in the hinge domain and membrane The chimeric antigen receptor of the present invention was prepared by designing a chimeric antigen receptor in which CD3-zeta and 41-BB were linked as an intracellular signaling domain and including the transit domain (EphA2-CAR2).

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 cytotoxic or cytolytic activity of immune cells or induce cytokine secretion of immune cells. can Therefore, when the antigen-binding site of the chimeric antigen receptor recognizes and binds to an antigen when cancer cells expressing EphA2 exist, cytotoxicity or cytolytic activity of immune cells is enhanced and/or cytokine secretion is promoted by signal transduction. can be induced, so that it can be usefully used as a chimeric antigen receptor with excellent cytotoxicity or cytolytic activity to attack cancer cells.

Another aspect of the present invention provides a polynucleotide and an expression vector for expressing the chimeric antigen receptor.

The polynucleotide includes a nucleotide sequence encoding the chimeric antigen receptor.

In the present invention, the term "polynucleotide" comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units, include not only natural nucleotides, but also analogs in which sugar or base sites are modified. do.

Encoding the chimeric antigen receptor means that the polynucleotide undergoes a normal protein expression process such as transcription and translation to encode genetic information that can synthesize a protein having the amino acid sequence of the chimeric antigen receptor of the present invention. means there is At this time, as described above, as well as a protein having an amino acid sequence completely identical to that of the chimeric antigen receptor, a protein having substantially the same amino acid sequence as the protein, or a protein encoding a protein having the same and/or similar activity as the protein Even polynucleotides may be included in the scope of the present invention.

Specifically, the polynucleotide of the present invention may include a nucleotide sequence encoding an antigen-binding variable fragment of an anti-EphA2 antibody that specifically binds to EphA2, and more specifically, the base of SEQ ID NO: 20 or SEQ ID NO: 21 It may contain sequences.

In addition, the polynucleotide of the present invention includes not only a nucleotide sequence encoding the antigen-binding variable fragment, but also a nucleotide sequence encoding another extracellular domain portion linked thereto, the transmembrane domain, and/or the intracellular signaling domain. can

Descriptions of the chimeric antigen receptors, such as the antibodies, antigen-binding variable fragments, extracellular domains, transmembrane domains, and intracellular signaling domains, are the same as those described above.

The polynucleotide may include a nucleotide sequence of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25. In a specific embodiment of the present invention, scFvs of two types of anti-EphA2 antibodies (#79 and #85) are included in the extracellular domain, and CD28 linked to Myc and hinge domains are transmembrane domains and intracellular signal transduction A polynucleotide containing the nucleotide sequence of SEQ ID NO: 22 or SEQ ID NO: 23 is prepared to design and express a chimeric antigen receptor in which CD3-zeta and DAP10 are linked to an intracellular signaling domain and to express the receptor. (EphA2-CAR1). In another specific embodiment of the present invention, scFvs of two types of anti-EphA2 antibodies (#79 and #85) are included in the extracellular domain, and the hinge domain, transmembrane domain, and intracellular signaling domain of CD8 are included. In addition, a polynucleotide containing the nucleotide sequence of SEQ ID NO: 24 or SEQ ID NO: 25 is prepared to design and express a chimeric antigen receptor in which CD3-zeta and 41-BB are linked to an intracellular signaling domain. (EphA2-CAR2).

The polynucleotide of the present invention may include a nucleotide sequence substantially identical to the nucleotide sequence listed above. The substantially identical nucleotide sequence includes, for example, cases in which the same amino acid can be synthesized when transcribed and translated, and is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more than the listed nucleotide sequences. It may be a nucleotide sequence having a homology of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, but is not limited thereto.

The polynucleotide encoding the chimeric antigen receptor may include an optimized nucleotide sequence according to the type of organism to be introduced and expressed, and the expression system of the organism such as transcription and translation. This is due to the degeneracy of codons, and therefore, there may be various combinations of nucleotide sequences capable of encoding proteins to be expressed, and all of them are included in the scope of the present invention. Modification of the polynucleotide according to the codon optimization 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 is optimized for codon selection of mammals and primates. It may be a modified polynucleotide, and more specifically, it may be a polynucleotide optimized and modified to be suitable for expression and function in humans.

The expression vector of the present invention contains the above polynucleotide.

Since the polynucleotide contains the nucleotide sequence encoding the chimeric antigen receptor of the present invention, the expression vector containing the nucleotide sequence can be used to express and prepare the chimeric antigen receptor, and the chimeric antigen receptor It can serve to move the polynucleotide to a specific cell or organism so that it can be expressed, or can be used to store and store the polynucleotide.

The expression vector may be constructed using a prokaryotic or eukaryotic cell as a host.

For example, when the expression vector uses a prokaryotic cell as a host, a strong promoter capable of promoting 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, etc.), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence. When E. coli (eg, HB101, BL21, DH5α, etc.) is used as the host cell, the promoter and operator regions of the E coli tryptophan biosynthetic pathway (Yanofsky, C, J Bacteriol, (1984) 158: 1018-1024) , and the leftward promoter of phage λ (pLλ promoter, Herskowitz, I and Hagen, D, Ann Rev Genet, (1980) 14:399-445) can be used as a control region. When Bacillus bacteria are used as host cells, 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 expression in Bacillus bacteria Any possible promoter can be used as a regulatory region. The expression vector is a plasmid often used in the art (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 (eg, λgt4·λB, λ-Charon, λΔz1, and M13, etc.) or viruses (eg, SV40, etc.).

When the expression vector is a eukaryotic cell as a host, a promoter derived from the genome of a mammalian cell (eg, metallotionine promoter, β-actin promoter, human hemoglobin promoter and human muscle creatine promoter) or mammalian Promoters derived from animal viruses (e.g., adenovirus late promoter, vaccinia virus 75K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR of HIV A promoter, a promoter of Moloney virus, a promoter of Epstein Barr virus (EBV) and a promoter of Rouss sarcoma virus (RSV)) may be used, and may generally have a polyadenylation sequence as a transcription termination sequence. The expression vector may have a CMV promoter.

In addition, the expression vector may be fused with other sequences to facilitate purification of the antibody expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA). In addition, since the protein expressed by the expression vector of the present invention is a chimeric antigen receptor, considering its characteristics, the expressed protein can be easily purified through a protein A column or the like without an additional sequence for purification.

The expression vector contains an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline. resistance genes may be included.

2. Immune cells expressing a chimeric antigen receptor for EphA2 on their surface

Another aspect of the present invention provides immune cells characterized by exhibiting enhanced cytotoxic or cytolytic activity against cancer cells expressing EphA2.

The immune cell expresses the above-described chimeric antigen receptor of the present invention on its surface.

The chimeric antigen receptor is described in “1. Novel anti-EphA2 antibodies, antigen-binding fragments thereof, and chimeric antigen receptors (CARs) containing the same, and polynucleotides and expression vectors for expressing the same.” Specifically, the chimeric antigen receptor may include an antigen binding site capable of specifically binding to the EphA2 antigen expressed in cancer cells.

Any cell that can induce the desired therapeutic effect by inducing immunity can be used as the immune cell without limitation. For example, natural killer cells (NK cells), T cells, natural killer T cells (NKT cells), cytotoxic cells It may be any one selected from the group consisting of cytokine induced killer cells (CIK), macrophages and dendritic cells, but is not limited thereto. Therefore, immune cells expressing the chimeric antigen receptor according to the present invention on the cell surface include CAR-NK cells (Chimeric Antigen Receptor Natural Killer Cell), CAR-T cells (Chimeric Antigen Receptor T Cell), and CAR-NKT cells (Chimeric Antigen Receptor Cell). Receptor Natural killer T Cell), CAR-macrophage (Chimeric Antigen Receptor Macrophage), and the like.

The T cells may be cytotoxic T lymphocytes (CTL), tumor infiltrating lymphocytes (TIL), T cells isolated from peripheral blood mononuclear cells (PBMC), etc., but are limited thereto. It is not.

The CAR-NK cell refers to a cell into which a chimeric antigen receptor is introduced into a natural killer cell, and a problem due to the persistent toxicity of cancer immunotherapy when using a conventional T cell-based CAR-T therapeutic agent , the risk of autoimmune disease, the problem of graft-versus-host disease (GVHD) for xenogeneic cell transplantation, the problem of off-target toxicity, etc. can be solved through the on/off switch, as well as various cancers. It has the advantage of being able to be used as a general-purpose therapeutic agent by allowing it to target cells.

The immune cell of the present invention comprises a chimeric antigen receptor comprising an antigen-binding variable fragment (scFv) of an antibody capable of specifically recognizing and binding to EphA2, which can be specifically expressed in cancer cells, as an extracellular domain, expressed on the cell surface. Therefore, when the EphA2-expressing cancer cells exist, signal transduction may occur by the chimeric antigen receptor, which further enhances the cytotoxic or cytolytic activity of immune cells and increases the amount of cytokine secretion. . Therefore, the immune cells of the present invention may have an activity to attack and treat cancer cells.

In a specific embodiment of the present invention, a chimeric antigen receptor comprising the antigen-binding variable fragments of two types of EphA2-specific antibodies of the present invention as extracellular domains was expressed on the surface of natural killer cells and on T cells. When each of the two natural killer cells were co-cultured with EphA2-expressing breast cancer cell line MDA-MB-231 cells, and each of the two T cells were co-cultured with EphA2-expressing lung cancer cell line A549 cells. When cultured, both cytotoxicity (or cytolytic activity) was improved, the amount of cytokine secretion was remarkably increased, and the degree of degranulation was also increased. It was confirmed that there was an excellent treatment effect. In addition, the therapeutic effect on cancer cells as described above was clearly confirmed in an animal model transplanted with cancer cells.

3. Therapeutic use of the immune cells of the present invention for cancer

Another aspect of the present invention provides a pharmaceutical composition for treating cancer comprising the immune cells.

The description of the immune cells, chimeric antigen receptors expressed therefrom, and the like, 'a novel anti-EphA2 antibody, an antigen-binding fragment thereof, and a chimeric antigen receptor (CAR) containing the same and for expressing the same' Polynucleotides and Expression Vectors' and '2. Immune cells expressing the chimeric antigen receptor for EphA2 on their surface' are the same as those described for them, so description is omitted to avoid repetitive explanation.

In the present invention, the term “cancer” is used synonymously with “tumor” and refers to or refers to a mammalian physiological condition typically characterized by unregulated cell growth/proliferation.

Cancer or carcinoma that can be treated with the composition of the present invention is not particularly limited, and includes both solid cancer and hematological cancer. For example, the cancer is lung cancer, stomach cancer, ovarian cancer, cervical cancer, breast cancer, pancreatic cancer, colon cancer, colon cancer, esophageal cancer, skin cancer, thyroid cancer, kidney cancer, liver cancer, head and neck cancer, bladder cancer, prostate cancer, blood cancer, and multiple myeloma. , acute myelogenous leukemia, malignant lymphoma, thymic cancer, osteosarcoma, fibrotic tumors, and at least one selected from the group consisting of brain cancer, but is not limited thereto, and an antigen that can be recognized by the second chimeric antigen receptor Any cancer cell containing a cancer cell can be applied without limitation in the present invention.

In the present invention, the term "treatment" means suppression of the development of cancer, relief or elimination of symptoms.

The pharmaceutical composition may include 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 of the subject to be treated, but is not limited thereto.

In addition to pharmaceutical compositions, the composition may be in the form of quasi-drug compositions, health food compositions, and the like.

The composition for treating cancer of the present invention may further include a pharmaceutically acceptable carrier. The meaning of 'pharmaceutically acceptable' is that it does not inhibit the activity of the active ingredient and does not have toxicity more than is adaptable to the subject of application (prescription), and the 'carrier' facilitates the addition of the compound into cells or tissues. defined as a compound that

The pharmaceutical composition of the present invention may be administered alone or in combination with any convenient carrier, and such dosage form may be a single or repeated dosage form. The pharmaceutical composition may be a solid preparation or a liquid preparation. Solid preparations include, but are not limited to, powders, granules, tablets, capsules, and suppositories. Solid preparations may include carriers, flavoring agents, binders, preservatives, disintegrants, lubricants, fillers, and the like, but are not limited thereto. Liquid preparations include solutions, suspensions, and emulsions such as water and propylene glycol solutions, but are not limited thereto, and may be prepared by adding appropriate colorants, flavoring agents, stabilizers, viscosifiers, and the like. For example, a powder may be prepared by simply mixing a tri-hydroxy derivative of polyunsaturated fatty acid, which is an active ingredient of the present invention, with a suitable pharmaceutically acceptable carrier such as lactose, starch, or microcrystalline cellulose. Granules are obtained by mixing the tri-hydroxy derivative of the polyunsaturated fatty acid of the present invention, a suitable pharmaceutically acceptable carrier, and a suitable pharmaceutically acceptable binder such as polyvinylpyrrolidone or hydroxypropylcellulose, followed by mixing water, It may be prepared using a wet granulation method using a solvent such as ethanol or isopropanol or a dry granulation method using compression force. In addition, tablets may be prepared by mixing the granules with a suitable pharmaceutically acceptable lubricant such as magnesium stearate and then tableting them using a tableting machine.

The pharmaceutical composition is an oral agent, an injection (eg, intramuscular injection, intraperitoneal injection, intravenous injection, infusion, subcutaneous injection, implant), inhalant, nasal agent, It may be administered as a vaginal agent, a rectal agent, a sublingual agent, a transdermal agent, a topical agent, and the like, but is not limited thereto. Depending on the route of administration, it may be formulated into an appropriate dosage unit formulation containing a pharmaceutically acceptable carrier, excipients, and vehicles that are commonly used and non-toxic.

The pharmaceutical composition may be administered at about 0.0001 mg/kg to about 10 g/kg daily, and at 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, and the like. If necessary, for convenience, the total daily dose may be administered in several divided doses throughout the day.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[Example 1]

[1-1] Construction of a single-chain variable fragment of an antibody that specifically binds to EphA2

Among the anti-EphA2 antibody sequences that can specifically bind to the EphA2 (ephrin type-A receptor 2) protein expressed in cancer cells, a single chain variable fragment (single chain variable fragment) using a sequence that plays an important role in specific binding fragment; scFv) two types (#79, #85) were prepared.

In the case of #79 scFv, 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; It was designed to include a light chain variable region including light chain CDR1 having the amino acid sequence, light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 6. And in the case of #85 scFv, a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 9, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 10, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 11, and SEQ ID NO: 12 It was designed to include a light chain variable region including light chain CDR1 having the amino acid sequence of SEQ ID NO: 13, light chain CDR2 having the amino acid sequence of SEQ ID NO: 13, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 14.

[1-2] Design of chimeric antigen receptors for introduction into natural killer cells

A chimeric antigen receptor (CAR) to be introduced into natural killer cells was designed by using the two types of scFvs designed as above as extracellular domains containing antigen binding sites. Specifically, an extracellular domain containing the two scFvs as antigen-binding sites and CD28 as a transmembrane domain were linked to Myc and a hinge domain, and the transmembrane domain was linked with CD3-zeta as an intracellular signaling domain. By adding and linking CD28 DAP10 as a co-stimulatory molecule, the chimeric antigen receptor of the present invention was designed to have an intracellular signaling domain of a chimeric antigen receptor classified as a so-called third-generation CAR ('EphA2#79-CAR1, respectively). ' and 'EphA2#85-CAR1', collectively referred to as 'EphA2-CAR1'). Then, the nucleotide sequence of the gene construct encoding it was inserted into the lentiviral vector (FIG. 1).

[1-3] Design of chimeric antigen receptors for introduction into T cells

Additionally, a chimeric antigen receptor to be introduced into T cells was designed with the extracellular domain containing the two types of scFvs designed as described above as antigen binding sites. Specifically, an extracellular domain containing the two scFvs as an antigen-binding site and CD8 as a transmembrane domain were linked to a hinge domain, and the transmembrane domain was coupled to CD3-zeta as an intracellular signaling domain and co-stimulation By adding and linking 4-1BB as a molecule, the chimeric antigen receptor of the present invention was designed to have an intracellular signaling domain of a chimeric antigen receptor classified as a so-called second-generation CAR ('EphA2#79-CAR2', respectively). and 'EphA2#85-CAR2', collectively referred to as 'EphA2-CAR2'). Then, the nucleotide sequence of the gene construct encoding it was inserted into the lentiviral vector (FIG. 2).

[Example 2]

Preparation of immune cells expressing a chimeric antigen receptor for EphA2 on the surface

Immune cells capable of expressing on the surface the chimeric antigen receptors of the present invention designed in Example 1 were prepared.

[2-1] Preparation of natural killer cells expressing EphA2-CAR1

HEK293T cells were transfected with the lentivirus vector prepared in Example 1-2 together with a viral packaging vector (pMDLG/RRE, pRSV/REV, VSVG), and a lentivirus expressing EphA2-CAR1 was obtained therefrom. After obtaining, it was concentrated using an ultra-high-speed centrifugal separator, and then infected with natural killer cells by a spinoculation method (360 g, 90 min, RT) such that the multiplicity of infection (MOI) was 30. After culturing the infected natural killer cells as described above for 5 hours at 37°C and 5% CO ₂ , the medium was replaced with a fresh medium, and after 3 days, puromycin at a concentration of 3ug/ml was used to select properly infected natural killer cells. ) to continue the culture. Puromycin was also treated with uninfected natural killer cells as a control group, and culture was continued using the puromycin-treated medium until all natural killer cells in the control group were killed by puromycin. The experiment was performed by selecting infected natural killer cells at the time point when all natural killer cells of the control group died.

Natural killer cells expressing EphA2#79-CAR1 and EphA2#85-CAR1 prepared and selected as described above (referred to as 'EphA2#79-CAR1-NK cells' and 'EphA2#85-CAR1-NK cells', respectively, These collectively referred to as 'EphA2-CAR1-NK cells') were treated with an anti-myc antibody (CST; 9B11) that specifically binds to myc of EphA2-CAR1 (4°C, 30 minutes in the dark), and flow cytometry Analysis confirmed the expression of Myc. At this time, natural killer cells that do not express EphA2-CAR1 were used as a control group.

As a result, as shown in FIG. 3a , it was confirmed that EphA2-CAR1 was properly expressed in both types of EphA2-CAR1-NK cells compared to the control group.

[2-2] Preparation of T cells expressing EphA2-CAR2

Peripheral blood mononuclear cells (PBMC) were differentiated into T cells, and then the lentiviral vector prepared in Example 1-3 was obtained using the same virus obtained in Example 2-1 and Spinoculation method (5MOI, 300g, 32°C, 90min), and infected T cells expressing EphA2#79-CAR2 and EphA2#85-CAR2 ('EphA2#79-CAR2-T cells' and 'EphA2#85-CAR2, respectively). -T cells', collectively referred to as 'EphA2-CAR2-T cells') were produced. Then, the expression of EphA2-CAR2 was confirmed by flow cytometry using anti-EphA2 antibodies (His-tag recombinant Protein EphA2 (NKMAX) and Anti-6X His tag ^® antibody (FITC) (abcam)). used native T cells that do not express EphA2-CAR2.

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

[Example 3]

Confirmation of cytotoxicity (or cytolytic activity) of EphA2-CAR1-NK cells and EphA2-CAR2-T cells against cancer cells expressing EphA2

As prepared in Example 2, the natural killer cells and T cells expressing the chimeric antigen receptor of the present invention on their surface contain scFv that specifically binds to EphA2 as an antigen binding site, and the EphA2 Cytotoxicity to cancer cells expressing was confirmed.

[3-1] Selection of cancer cells expressing EphA2

First, breast cancer cell line MDA-MB-231 cells (Korea Cell Line Bank) and lung cancer cell line A549 cells (Korea Cell Line Bank) were used as EphA2-expressing cancer cells, and chronic myeloid leukemia cell line as cancer cells that do not express EphA2. After selecting K562 cells (Korea Cell Line Bank) and reacting with anti-EphA2 antibody (Human EphA2/Mouse IgG2A Alexa Fluor ^® 488-conjugated antibody (R&D systems)) in an amount of 1ul/100ul (4°C, cow 30 min), the expression level of EphA2 in the three cell lines was confirmed by flow cytometry.

As a result, as shown in FIG. 4 , it was confirmed that MDA-MB-231 cells and A549 cells expressed EphA2, and K562 cells did not express EphA2.

[3-2] Confirmation of the activity of EphA2-CAR1-NK cells against EphA2-expressing cancer cells

Regarding the MDA-MB-231 cells and K562 cells for which expression of EphA2 was confirmed as described above, the cytotoxicity of the two types of EphA2-CAR1-NK cells prepared in Example 2-1 was evaluated by Calcein AM assay. Confirmed. Specifically, MDA-MB-231 cells and K562 cells were reacted with calcein at a concentration of 5ug/ml (37°C, 5% CO2, 1 hour in the dark), and each cancer cell stained with calcein The original natural killer cells and each of the two types of EphA2-CAR1-NK cells were treated and reacted at a ratio of 5:1, 1:1, and 0.5:1 (natural killer cells: cancer cells), respectively (37 ° C. , 4 hours under 5% CO ₂ condition), taking 100ul of the supernatant to confirm the amount of calcein present in the supernatant.

As a result, as shown in FIG. 5 below, for MDA-MB-231 cells expressing EphA2, the two types of EphA2#79-CAR1-NK cells and EphA2#85-CAR1-NK cells were both the control natural killer cells. In addition to exhibiting much higher cytotoxicity compared to cells, this cytotoxicity was found to be concentration dependent on treated EphA2-CAR1-NK cells. In contrast, it was confirmed that both the control group and the two types of EphA2-CAR1-NK cells showed little cytotoxicity to K562 cells that do not express EphA2.

Furthermore, the secretion of cytokines and granules was confirmed with respect to the two types of EphA2-CAR1-NK cells. Specifically, MDA-MB-231 cells and K562 cells were reacted by treating the original natural killer cells or each of the two types of EphA2-CAR1-NK cells prepared in Example 2-1 1: 1 (37 ℃, 5% CO ₂ conditions for 16 hours), the supernatant was collected and INF-γ (Interferon-γ) present in the supernatant was confirmed through ELISA. At this time, the amount of cytokine secreted from the original natural killer cells and EphA2-CAR1-NK cells alone was used as a control.

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

In addition, MDA-MB-231 cells, K562 cells, 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). After reaction (4 hours at 37°C, 5% CO ₂ conditions), anti-CD56 antibody was treated and stained to select natural killer cells, and through flow cytometry, original natural killer cells and the above two types The degree of CD107a expression in EphA2-CAR1-NK cells was analyzed.

As a result, as shown in FIG. 7, as in the case of INF-γ, it was confirmed that the expression of CD107a was significantly enhanced only in EphA2-CAR1-NK cells treated with MDA-MB-231 cells expressing EphA2. .

[3-3] Confirmation of activity of EphA2-CAR2-T cells against EphA2-expressing cancer cells

GFP was expressed in A549 cells in which EphA2 expression was confirmed as described above, and the EphA2-CAR2-T cells prepared in Example 2-2 were respectively 2:1, 1:1, and 0.5:1. Treatment was performed at a ratio of 0.25: 1 (T cells: cancer cells), and data were collected at 4-hour intervals while culturing for 48 hours in an IncuCyte instrument, and the cytotoxicity of EphA2-CAR2-T cells was analyzed using the IncuCyte ZOOM program. did

As a result, as shown in FIG. 8 , it was confirmed that the two types of EphA2-CAR2-T cells were cytotoxic to A549 cells expressing EphA2.

[Example 4]

in vivo ( in vivo ) Confirmation of cytotoxicity (or cytolytic activity) of EphA2-CAR1-NK cells and EphA2-CAR2-T cells in

The cytotoxicity of cancer cells expressing EphA2 of natural killer cells and T cells expressing the chimeric antigen receptor of the present invention confirmed in Example 3 on the surface was confirmed again in an animal model.

[4-1] Confirmation of the activity of EphA2-CAR1-NK cells against EphA2-expressing cancer cells

First, it was confirmed that EphA2 was expressed in lung cancer cell line H460 cells (Korea Cell Line Bank) in the same manner as in Example [3-1] (Fig. 9a), and the same method as in Example [3-2] As a result, it was confirmed that the EphA2#79-CAR1-NK cells showed much higher cytotoxicity to the H460 cells than the control natural killer cells (dECTO) expressing CAR from which the extracellular domain was removed (FIG. 9b).

Next, as shown in FIG. 9c, 3x10 ⁶ of the H460 cells were subcutaneously injected into the flank of about 6-week-old female Balb/c nude mice (Saeron Bio), and after 10 days, the size of the tumor was about 50 mm ³ 2x10 ⁶ of the EphA2-CAR1-NK cells or control natural killer cells were intravenously injected 5 times every 3 or 4 days. Then, the size and weight of the tumors were measured over 24 days.

As a result, as shown in FIGS. 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. .

[4-2] Confirmation of activity of EphA2-CAR2-T cells against EphA2-expressing cancer cells

Next, 1x10 ⁶ of A549-Luciferase cells (PerkinElmer) into which luciferase was introduced into A549 cells, a lung cancer cell line expressing EphA2, were subcutaneously injected into the right flank of 6-week-old male NOG mice (Coretech Co., Ltd.). Inject, and when the size of the tumor is about 200 mm ³ , the EphA2-CAR2-T cells or

Control T cells (dECTO) expressing CAR ^from which the extracellular domain was removed were injected intravenously. Then, tumor size, mouse weight and IVIS were measured twice a week over 45 days.

As a result, as shown in FIGS. 10A and 10B , it was confirmed that the EphA2-CAR2-T cells effectively suppressed tumors, and as shown in FIG. 10C , the EphA2-CAR2-T cells were found in mouse blood after intravenous injection. The presence of cells and control T cells was also confirmed. In addition, as shown in FIG. 10d , it was confirmed that the EphA2-CAR2-T cells were present in a larger amount in the tumor than the control T cells.

[organize]

In view of the above experimental results, the two anti-EphA2 antibodies (#79 and #85) of the present invention not only exhibit specific binding ability to EphA2, but also inhibit EphA2 in cancer cells expressing EphA2. When recognizing and binding, the chimeric antigen receptor containing the antigen-binding site of the antibody triggers signal transduction in immune cells (natural killer cells or T cells) expressed on the surface, and thus the immune cells attack cancer cells. It was clearly confirmed not only in vitro but also in vivo that there is an effect of inducing various immune responses that can be produced.

In the above, the present invention has been described in detail only for the described embodiments, but it is obvious to those skilled in the art that various changes and modifications are possible within the scope of the technical idea of the present invention, and it is natural that these changes and modifications fall within the scope of the appended claims.

Claims (17)

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 including;
An antibody or antigen-binding fragment thereof that specifically binds to ephrin type-A receptor 2 (EphA2). The method of claim 1,
The heavy chain variable region will have an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 15, antibody or antigen-binding fragment thereof. The method of claim 1,
The light chain variable region will have an amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 16, antibody or antigen-binding fragment thereof. an extracellular binding domain containing an antigen binding site that specifically binds to ephrin type-A receptor 2 (EphA2);
transmembrane domain; and
Including; intracellular signaling domain (intracellular signaling domain)
As a chimeric antigen receptor (CAR),
The antigen-binding site that specifically binds to EphA2 is 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 SEQ ID NO: 3 or SEQ ID NO: 11 A heavy chain variable region comprising a heavy chain CDR3 having an amino acid sequence of and a light chain CDR1 having an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 12, a light chain CDR2 having an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13, and SEQ ID NO: 6 or SEQ ID NO: A chimeric antigen receptor that is a single chain variable fragment (scFv) of an anti-EphA2 antibody comprising a light chain variable region including a light chain CDR3 having an amino acid sequence of 14. The method of claim 4,
The antigen-binding site that specifically binds to EphA2,
A chimeric antigen receptor that 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. The method of claim 4,
The antigen-binding site that specifically binds to EphA2,
A chimeric antigen receptor that 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. The method of claim 1,
The extracellular domain further comprises at least one selected from the group consisting of a hinge domain and a spacer domain. The method of claim 7,
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, the chimeric antigen receptor. The method of claim 4,
The transmembrane domain is 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, Any one selected from the group consisting of CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, chimeric antigen receptor. The method of claim 4,
The intracellular signaling domain,
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, A chimeric antigen receptor comprising at least one selected from the group consisting of CD22, CD79a, CD79b and CD66d. The method of claim 4,
The intracellular signaling domain,
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, At least one selected from the group consisting of CD22, CD79a, CD79b and CD66d as a primary signaling domain;
CD2, CD7, CD27, CD28, CD30, CD40, 4-1BB (CD137), OX40 (CD134), CDS, ICAM-1, ICOS (CD278), LFA-1 (CD11a/CD18), GITR, MyD88, DAP10, A chimeric antigen receptor comprising at least one selected from the group consisting of DAP12, PD-1, LIGHT, NKG2C, EphA2 and CD83 as a co-stimulatory signaling domain. A polynucleotide comprising a nucleotide sequence encoding the chimeric antigen receptor of any one of claims 4 to 11. An expression vector comprising the polynucleotide of claim 12 . An immune cell expressing the chimeric antigen receptor according to any one of claims 4 to 11 on its surface. The method of claim 14,
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. Which is, immune cells. A pharmaceutical composition for treating cancer, comprising the immune cells of claim 14. The method of claim 16
The cancers include lung cancer, stomach cancer, ovarian cancer, cervical cancer, breast cancer, pancreatic cancer, colon 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, at least one selected from the group consisting of fibrotic tumors and brain cancer, the pharmaceutical composition for the treatment of cancer.

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