CN118055945A - Novel anti-EPHA 2 chimeric antigen receptor and immune cells expressing same - Google Patents

Abstract

The present invention relates to novel antibodies or antigen binding fragments thereof that specifically bind EphA2, chimeric antigen receptors comprising antigen binding variable fragments of the antibodies, and immune cells expressing the chimeric antigen receptors.

Description

Novel anti-EPHA 2 chimeric antigen receptor and immune cells expressing same

Technical Field

The present invention relates to novel antibodies or antigen binding fragments thereof that specifically bind EphA2, chimeric antigen receptors comprising antigen binding variable fragments of antibodies, and immune cells expressing chimeric antigen receptors.

Background

Methods for treating cancer have been steadily developed and varied, and methods such as surgery, chemotherapy, and radiotherapy have been used so far. However, these conventional methods for treating cancer are generally effective only at an early stage where the cancer has not metastasized, and if the cancer has metastasized, there is a high likelihood of recurrence in the future even if surgery is performed. In recent years, researchers have been exploring methods for treating cancer using immune responses.

In particular, there is increasing interest in using immune cells to boost them or genetically modify them and reinfusion them into cell therapies in patients such as Tumor Infiltrating Lymphocytes (TILs), chimeric Antigen Receptors (CARs) and T Cell Receptors (TCRs). In particular, chimeric antigen receptors are artificial receptors designed to specifically deliver antigen to T cells or natural killer cells (NK cells), consisting of an extracellular domain, a transmembrane domain, and an intracellular signaling domain, which allow the receptor to activate immune cells and provide specific immunity by binding to cancer cell specific antigens. And T cells expressing these chimeric antigen receptors have been 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), and natural killer cells expressing CARs have been named CAR-NK cells.

The intracellular signaling domain of the chimeric antigen receptor above is based primarily on the intracellular signaling domain of cd3ζ, the signaling subunit of the T cell receptor (first generation CAR). They have evolved to intracellular signaling domains comprising costimulatory molecules that promote immune cell growth and differentiation. For example, currently marketed CAR-T cell therapies use the intracellular signaling domains of CD28 and 4-1BB co-stimulatory molecules, respectively (second generation CARs), and subsequently CARs containing both CD28 and 4-1BB intracellular signaling domains (third generation CARs) are being tested (Stegen et al, nat. Rev. Drug discovery, 14 (7): 499-509, 2015).

On the other hand, ephA2 (ephrin) type a receptor 2), a protein expressed by the human EphA2 gene, is known to be overexpressed in various types of cancers including breast cancer, prostate cancer, lung cancer, and the like, and is known to promote growth and invasion of cancer cells. Furthermore, ephA2 expression has been reported to correlate with survival in cancer patients. Against this background, there is a need to investigate another aspect of strategies and methods for treating cancer by: antibodies targeting EphA2 above, particularly over-expressed in cancer cells, and CAR-T and CAR-NK treatments using the antibodies were developed.

Disclosure of Invention

Technical problem

It is an object of the present invention to provide antibodies or antigen binding fragments that can specifically bind EphA2 expressed primarily in cancer cells.

Furthermore, it is an object of the present invention to provide novel chimeric antigen receptors which, when expressed on immune cells, can enhance cytotoxicity or cytolytic activity against cancer cells.

Furthermore, it is an object of the present invention to provide polynucleotides and expression vectors for expressing chimeric antigen receptors.

The object of the present invention is also to provide immune cells having therapeutic effects against cancer by expressing chimeric antigen receptors on the surface of immune cells.

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

Technical proposal

To achieve the above objects, in one aspect of the present invention, there is provided an antibody or antigen-binding fragment that specifically binds to ephrin-a receptor 2 (EphA 2), 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; the light chain variable region comprises 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.

In another aspect of the invention, there is provided a Chimeric Antigen Receptor (CAR) comprising: an extracellular binding domain comprising an antigen binding site that specifically binds EphA2 (ephrin a type receptor 2); a transmembrane domain; and an intracellular signaling domain, wherein the antigen binding site that specifically binds EphA2 is a single chain variable fragment (scFv) 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.

In another aspect of the invention, polynucleotides comprising nucleotide sequences encoding chimeric antigen receptors and expression vectors comprising the polynucleotides are provided.

In another aspect, the invention provides an immune cell that expresses a chimeric antigen receptor on the surface of the immune cell.

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

Advantageous effects

The antibody, the antigen-binding fragment thereof, and the chimeric antigen receptor using the same of the present invention can specifically bind EphA2 expressed mainly in cancer cells, and thus have the effect of significantly enhancing the cytotoxicity or cytolytic activity of immune cells and promoting the secretion of cytokines as signal transduction occurs in immune cells expressing the chimeric antigen receptor. In addition, it has the effect of enhancing degranulation of cancer cells co-cultured with immune cells.

Thus, the antibodies of the invention, chimeric antigen receptors using the antibodies, and CAR-expressing immune cells may be useful for treating cancer.

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

Drawings

FIG. 1a is a schematic diagram showing the structure of a chimeric antigen receptor (EphA 2-CAR 1) of the present invention comprising an antigen-binding variable fragment (scFv) that specifically binds to EphA2 as an extracellular domain, and FIG. 1b is a vector map showing an expression vector for expressing the chimeric antigen receptor.

FIG. 2a is a schematic diagram showing the structure of a chimeric antigen receptor (EphA 2-CAR 2) of the present invention comprising an antigen-binding variable fragment (scFv) that specifically binds to EphA2 as an extracellular domain, and FIG. 2b is a vector map showing an expression vector for expressing the chimeric antigen receptor.

Fig. 3a shows the results of assaying the expression of two chimeric antigen receptors (EphA 2-CAR1 and EphA2-CAR 2) in natural killer cells (EphA 2#79-CAR1-NK cells and EphA2#85-CAR1-NK cells) into which a gene encoding the chimeric antigen receptor of the present invention has been introduced and expressed, and fig. 3b shows the results of assaying the expression of two chimeric antigen receptors (EphA 2-CAR1 and EphA2-CAR 2) in T cells (EphA 2#79-CAR2-T cells and EphA2#85-CAR2-T cells) into which a gene encoding the chimeric antigen receptor of the present invention has been introduced and expressed, respectively.

FIG. 4 shows the results of the expression of EphA2 in MDA-MB-231 cells (breast cancer cell line), A549 cells (lung cancer cell line) and K562 cells (chronic myelogenous leukemia cell line).

FIG. 5 shows the results of measuring and comparing cytotoxicity (cytolytic activity) of natural killer cells (EphA2#79-CAR 1-NK cells and EphA2#85-CAR1-NK cells) into which a gene encoding a chimeric antigen receptor of the present invention has been introduced and expressed, against an MDA-MB-231 cell expressing EphA2 and a K562 cell not expressing EphA 2.

FIGS. 6 and 7 show the results of measuring the expression levels of secreted cytokines (IFN-. Gamma., FIG. 6) and degranulation marker CD 107. Alpha. (FIG. 7) of NK cells (EphA2#79-CAR 1-NK cells and EphA2#85-CAR1-NK cells) into which genes encoding the chimeric antigen receptor of the present invention have been introduced and expressed, respectively, when co-cultured with either the MDA-MB-231 cells expressing EphA2 or the K562 cells not expressing EphA 2.

FIG. 8 shows the results of measuring cytotoxicity (cytolytic activity) against A549 cells expressing EphA2, T cells (EphA 2#79-CAR2-T cells and EphA2#85-CAR2-T cells) into which a gene encoding a chimeric antigen receptor of the present invention has been introduced and expressed.

FIG. 9a shows the results of confirming whether EphA2 is expressed in H460 cells (lung cancer cell line), respectively; FIG. 9b shows the results of confirming cytotoxicity (cytolytic activity) of natural killer cells (EphA2#79-CAR 1-NK cells) into which a gene encoding the chimeric antigen receptor of the present invention has been introduced and expressed against H460 cells; FIG. 9c is a schematic diagram showing an experimental procedure for confirming the activity of natural killer cells into which the gene encoding the chimeric antigen receptor of the present invention has been introduced and expressed in experimental animals injected with H460 cells; and FIGS. 9d and 9e show the results of confirming the anticancer activity of natural killer cells into which the gene encoding the chimeric antigen receptor of the present invention has been introduced and expressed by cytotoxicity (cytolytic activity) against H460 cells, as measured by size (FIG. 9 d) and weight (FIG. 9 e) of tumors.

FIGS. 10a and 10b show the results of anticancer activity of T cells (EphA2#79-CAR 2-T cells and EphA2#85-CAR2-T cells) into which a gene encoding a chimeric antigen receptor of the present invention has been introduced and expressed by cytotoxicity (cytolytic activity) against A549 cells, as measured by size (FIG. 10 a) and weight (FIG. 10 b) of tumors, respectively, in experimental animals injected with A549-luciferase cells. Furthermore, fig. 10c shows the results of confirming the presence of EphA2-CAR2-T cells in the blood of mice; and FIG. 10d shows the results of confirming the presence of EphA2-CAR2-T cells in tumors.

Detailed Description

Best mode

Hereinafter, the present invention will be described in detail.

1. Novel anti-EphA 2 antibodies, antigen-binding fragments thereof, and Chimeric Antigen Receptors (CARs) comprising the novel anti-EphA 2 antibodies and antigen-binding fragments thereof, as well as polynucleotides and expression vectors for expressing the CARs.

In one aspect of the invention, anti-EphA 2 antibodies and antigen-binding fragments thereof are provided that can specifically bind EphA 2.

As used herein, the term "antibody" refers to an immunoglobulin molecule that is immunoreactive by specifically binding an epitope of an antigen. Antibodies may include 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, and more specifically, antibodies of the invention may be monoclonal antibodies or antigen binding fragments thereof. Monoclonal antibodies refer to antibody molecules of a single molecular composition obtained from substantially the same population of antibodies, and such monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope. Full length antibodies are structures having two full length light chains and two full length heavy chains, wherein each light chain may be linked to a heavy chain by a disulfide bond. Antibodies comprise Heavy (HC) and Light (LC) polypeptides, wherein each heavy and light chain may comprise a variable region and a constant region.

Constant regions are sites that mediate binding of antibodies to various types of cells of the immune system (e.g., T cells) and host tissues including components of the complement system, and the like. If the antibodies are of the same type derived from the same species, the constant regions perform the same function regardless of the type of antigen, and their amino acid sequences are identical or highly similar in the various antibodies. The constant region can be divided into a heavy chain constant region (which can be abbreviated as C _H) and a light chain constant region (which can be abbreviated as C _L). The heavy chain constant regions may be of the gamma, mu, alpha, delta and/or epsilon type, wherein the subclasses are gamma 1, gamma 2, gamma 3, gamma 4, alpha 1 and/or alpha 2. The light chain constant regions are of the kappa and lambda type. IgG has subtypes, which include IgG1, igG2, igG3, and IgG4.

The variable region is an antibody site with antigen specificity, which can be divided into a heavy chain variable region (which can be abbreviated as V _H) and a light chain variable region (which can be abbreviated as V _L). The variable region may comprise three Complementarity Determining Regions (CDRs) and four Framework Regions (FR). CDRs may be loop regions involved in antigen recognition, and the amino acid sequence of the CDRs may determine their antigen specificity. CDRs may be referred to as CDR1, CDR2, CDR3 in that order, and depending on whether they are CDRs of a heavy chain polypeptide or CDRs of a light chain polypeptide, they may be referred to as CDR-H1, CDR-H2, CDR-H3 of a heavy chain variable region, and CDR-L1, CDR-L2, CDR-L3 of a light chain variable region. Similarly, FR can be referred to as FR-H1, FR-H2, FR-H3, FR-H4 of the heavy chain variable region and FR-L1, FR-L2, FR-L3, FR-L4 of the light chain variable region. In addition, CDRs and FR may be arranged in the following order in each variable region.

As used herein, the term "antigen-binding fragment" refers to any fragment of a humanized antibody of the invention that retains the antigen-binding function of the antibody. An antigen binding fragment may be interchangeably referred to with the terms "fragment," "antibody fragment," and the like, and an antigen binding fragment may be, but is not limited to, fab ', F (ab') ₂, fv, and the like.

Fab is a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (CH 1 domain) of a heavy chain, and has one antigen binding site. Fab' differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. F (ab ') ₂ is formed by disulfide bonding of the cysteine residues in the hinge region of the Fab'. Fv refers to the smallest antibody fragment having only the heavy and light chain variable regions. A double-chain Fv is an antibody fragment in which the heavy and light chain regions are joined by a non-covalent bond, and a single-chain Fv is an antibody fragment in which the heavy and light chain regions are joined, typically via a peptide linker or directly at the C-terminus, by a covalent bond, which can form a dimer-like structure like a double-chain Fv. Antigen binding fragments may be produced using, but are not limited to, proteolytic enzymes (e.g., proteolytic cleavage of full length antibodies with papain to produce Fab and proteolytic cleavage of full length antibodies with pepsin to produce F (ab') ₂ fragments) or by genetic recombination techniques.

The linker may be a peptide linker of about 10 to 25 amino acids in length. For example, the linker may comprise hydrophilic amino acids, such as glycine (G) and/or serine (S). The linker may comprise, for example, (GS) _n、(GGS)_n、(GSGGS)_n or (G _nS)_m (n, m are 1 to 10 respectively), such as, but not limited to (G _nS)_m (n, m are 1 to 10 respectively).

As used herein, the term "epitope" refers to a specific site of an antigen to which an immunoglobulin, antibody or antigen binding fragment thereof can specifically recognize and bind. Epitopes can be formed by contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein.

"Specific binding" may mean binding to another molecule with a binding affinity greater than background binding, e.g., the extracellular domain may bind to the target antigen with an affinity of about 10 ^-5 M or higher or K _a (equilibrium dissociation constant for a specific binding interaction in 1/M). The affinity may be such that the equilibrium dissociation constant (K _d) for the specific binding interaction in M is 10 ^-5 M to 10 ^-13 M or less, or within the ranges above.

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; the light chain variable region comprises 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 ephrin-a receptor 2 (EphA 2).

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

The light chain variable region may have the 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 comprise, for example, a heavy chain constant region and/or a light chain constant region of an antibody derived from a human, and the heavy chain constant region and/or the light chain constant region of an antibody derived from a human may be used, regardless of type or amino acid sequence, as long as it does not interfere with the ability of the antibody or antigen-binding fragment to specifically bind EphA 2.

The foregoing amino acid sequences may include variants having different sequences by deletion, insertion, substitution, or combination thereof of amino acid residues unless the sequences affect the structure, function, activity, etc. of the polypeptides comprising them. Furthermore, the amino acid sequence may comprise amino acids that undergo conventional modifications known in the art, such as phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, and the like. Humanized antibodies or antigen binding fragments thereof according to the present invention include not only those comprising the amino acid sequences described above, but also those having substantially the same amino acid sequences or variants thereof. The meaning of having substantially identical amino acid sequences may include, but is not limited to, amino acid sequences having the following homology to the amino acid sequences described above: 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.

As used herein, the term "Chimeric Antigen Receptor (CAR)" refers to a synthetic protein designed to recognize a target antigen and cells expressing the antigen and, when bound, induce an immune response to the antigen. Chimeric antigen receptors can comprise an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain. Chimeric antigen receptors expressed on the surface of immune cells can recognize and bind specific antigens, such as antigens expressed on the surface of cancer cells, via antigen binding sites contained in the extracellular domain, and trigger signal transduction within immune cells to alter the activity of immune cells, thereby targeting only specific antigens to trigger an immune response.

The Chimeric Antigen Receptor (CAR) of the invention comprises an extracellular binding domain comprising an antigen binding site that specifically binds to ephrin a receptor 2 (EphA 2); a transmembrane domain; and intracellular signaling domains.

The antigen binding site that specifically binds EphA2 may be a single chain variable fragment (scFv) of an anti-EphA 2 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.

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 may cause signal transduction within immune cells, enhanced cytotoxicity (or cytolytic activity) of immune cells, and/or stimulation of cytokine secretion by immune cells. Enhancement of cytotoxicity (or cytolytic activity) and/or stimulation of cytokine secretion may result in immune cells exhibiting greater cytotoxicity (or cytolytic activity) and/or cytokine secretion than would be exhibited by immune cells in the absence of the antigen.

The extracellular domain may further comprise 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 via a hinge domain and/or a spacer domain.

The hinge domain may play an important role in locating the extracellular domain because it allows for physical separation of the antigen binding site from the surface of the immune cell on which the chimeric antigen receptor is expressed to enable proper cell/cell contact, proper antigen/antigen binding site binding, and proper chimeric antigen receptor activation. The chimeric antigen receptor can comprise one or more hinge domains between an extracellular domain and a transmembrane domain. The hinge domain may be derived from natural, synthetic, semisynthetic or recombinant sources. 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 may comprise (a) a naturally occurring hinge region having an amino acid change of up to 30% (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitution or deletion), (b) an amino acid change of up to 30% in length of at least 10 amino acids (e.g., at least 12, 13, 14, or 15 amino acids) (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitution or deletion), or (c) a portion of a naturally occurring hinge region comprising a core hinge region (which may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids). In certain embodiments, one or more cysteine residues in the naturally occurring immunoglobulin hinge region may be substituted with one or more other amino acid residues (e.g., one or more serine residues). Alternatively or additionally, the altered immunoglobulin hinge region may have another amino acid residue, for example a substitution of a proline residue in the wild-type immunoglobulin hinge region with cysteine. The hinge domain may be a hinge domain derived from an extracellular domain of a type 1 transmembrane protein such as CD8, CD4, CD28 and CD7, but any hinge domain capable of linking an antigen binding site, a transmembrane domain and an intracellular signaling domain across a cell membrane may be used without limitation. Furthermore, the hinge domain may be a wild-type hinge region from these molecules or may be modified.

The spacer domain may be referred to as a linking domain, may comprise a hinge domain derived from, for example, a CD 28-derived hinge domain and/or a CD 8-derived hinge domain, and may comprise all or a portion of a CD 28-derived and/or CD 8-derived hinge domain.

The hinge domain and/or spacer domain may be at least one selected from the group consisting of Myc epitopes, CD8 hinge domains and Fc, and more specifically may comprise Myc epitopes and CD8 hinge domains. More specifically, the Myc epitope may comprise the amino acid sequence of SEQ ID NO. 17 and the CD8 hinge domain may comprise the amino acid sequence of SEQ ID NO. 18 or SEQ ID NO. 19.

"Transmembrane domain" refers to a region of the plasma membrane that connects and fuses together an extracellular domain and an intracellular signaling domain and serves to anchor a chimeric antigen receptor to an immune cell. The transmembrane domain may be derived from natural, synthetic, semisynthetic or recombinant sources. The transmembrane domain may be derived from one selected from the group consisting of: the α, β or ζ chain of the T Cell Receptor (TCR), CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, are not limited thereto.

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

The intracellular signaling domain corresponds to the moiety used for: the signal generated when the chimeric antigen receptor binds to an antigen is transmitted into the cytoplasm of the immune cell in order to trigger immune cell function (e.g., activation including release of cytotoxic (or cytolytic) factors against the target cell in which the chimeric antigen receptor binds to the antigen, cytokine production, proliferation and cytotoxic or cytolytic activity, or other cellular responses triggered by antigen binding). Intracellular signaling may be part of a protein that transmits signals of operative functions and directs cells to perform specialized functions.

The intracellular signaling domain may be the intracellular signaling domain previously used to develop chimeric antigen receptors. In particular, the intracellular signaling domain may comprise only the cytoplasmic domain of cd3ζ as utilized in first generation CARs. And as utilized in second generation CARs, a morphology in which the cytoplasmic domain of cd3ζ is combined with a costimulatory domain (CD 28 or CD137/4-1 BB) to enhance responsiveness to immune cells can be used. Furthermore, more than one co-stimulatory domain may be used, which has been used for third generation CARs, which may be fused with 4-1BB, CD28 or OX40 to achieve expansion and persistence of CAR-containing immune cells in vivo. Furthermore, as utilized in fourth generation CARs, additional genes encoding cytokines such as IL-12 or IL-15 may be used to additionally express CAR-based immune proteins (cytokines). In addition, the intracellular signaling domain may also contain an interleukin receptor chain, such as IL-2rβ, in order to enhance immune cells, as utilized in fifth generation CARs.

The intracellular signaling domain may be derived from at least one selected from the group consisting of: t Cell Receptor (TCR) ζ, fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, cd3ζ, CD5, CD22, CD79a, CD79b, and CD66d.

More specifically, activation of immune cells by intracellular signaling domains can be mediated by two different classes of intracellular signaling domains. For example, immune cell activation can be mediated by a primary signaling domain that initiates antigen-dependent primary activation and a costimulatory signaling domain that acts in an antigen-independent manner to provide secondary signaling. Thus, the intracellular signaling domain may comprise a primary signaling structure and a co-stimulatory signaling domain.

"Primary signaling domain" refers to a signaling domain that modulates immune cell activation in a stimulatory or inhibitory manner. The primary signaling domain acting in a stimulatory manner may comprise a signaling motif known as an immunoreceptor tyrosine activation motif or ITAM. The ITAM containing the primary signaling domain may be derived from, but is not limited to, at least one selected from the group consisting of: tcrζ, fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, cd3ζ, CD5, CD22, CD79a, CD79b, CD66d, and the like. More specifically, the primary signaling domain may be, but is not limited to, cd3ζ.

"Costimulatory signaling domain" refers to the intracellular signaling domain of a costimulatory molecule. Such co-stimulatory signaling domains may be derived from at least one ：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 selected from the group consisting of, but not limited to, and the like. Specifically, the costimulatory molecule can be, but is not limited to, DAP10.

The chimeric antigen receptor can comprise two or more intracellular signaling domains, and when two or more intracellular signaling domains are comprised, the intracellular signaling domains can be connected to each other in series. Alternatively, they may be linked via a polypeptide linker consisting of 2 to 10 amino acids, wherein 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 (G _nS)_m), where n and m are each 1 to 10, and may be, for example, (G _nS)_m (n and m are each 1 to 10), but is not limited thereto.

The chimeric antigen receptor may also comprise an immune function promoting factor of an immune cell, e.g., the immune function promoting factor may be an interleukin signal sequence. The interleukin signal sequence is characterized by inducing the expression of Interleukin (IL) -12, IL-8, IL-2, etc., but is not limited thereto. In addition, if the immune cell is a T cell, the immune function promoting factor may be a signal sequence of IL-7, CCL19, or the like, but is not limited thereto.

In a specific embodiment of the invention, the chimeric antigen receptor of the invention is prepared by designing a chimeric antigen receptor (EphA 2-CAR 1) comprising: scFv of two anti-EphA 2 antibodies (# 79 and # 85) having the same amino acid sequences as described previously as extracellular domains, CD28 linked to Myc and hinge domains as transmembrane domain and intracellular signaling domain, and CD3- ζ and DAP10 as intracellular signaling domain. In another specific embodiment of the invention, the chimeric antigen receptor of the invention is prepared by designing a chimeric antigen receptor (EphA 2-CAR 2) comprising: as extracellular domains there are scFv of two anti-EphA 2 antibodies (# 79 and # 85) with the same amino acid sequence as described previously, CD8 as hinge domain and transmembrane domain, CD3- ζ and 41-BB as intracellular signal transduction domain.

As described above, the chimeric antigen receptor of the present invention can be expressed on the surface of immune cells to recognize and bind EphA2, which can enhance the cytotoxicity or cytolytic activity of immune cells or induce immune cells to secrete cytokines. Thus, when the antigen binding site of a chimeric antigen receptor recognizes and binds an antigen in the presence of EphA 2-expressing cancer cells, signal transduction can be induced to enhance the cytotoxicity or cytolytic activity of immune cells and/or promote secretion of cytokines, thereby allowing the chimeric antigen receptor to be used as a chimeric antigen receptor with high cytotoxicity or cytolytic activity to attack cancer cells.

Another aspect of the invention provides polynucleotides and expression vectors for expressing chimeric antigen receptors.

The polynucleotide comprises a sequence encoding a chimeric antigen receptor.

As used herein, the term "polynucleotide" includes both DNA (gDNA and cDNA) and RNA molecules in which the basic building blocks, i.e., nucleotides, include not only naturally occurring nucleotides, but also sugar or base modified analogs thereof.

"Encoding a chimeric antigen receptor" means that the polynucleotide encodes genetic information such that a protein having the amino acid sequence of the chimeric antigen receptor of the present invention can be synthesized by conventional protein expression methods such as transcription and translation. The scope of the present invention includes polynucleotides encoding not only proteins having an amino acid sequence identical to a chimeric antigen receptor, but also proteins having substantially the same amino acid sequence as the proteins, or encoding proteins having the same and/or similar activity as the proteins, as described above.

In particular, polynucleotides of the invention may comprise sequences encoding antigen-binding variable fragments of anti-EphA 2 antibodies that specifically bind EphA2, and more particularly may comprise sequences of SEQ ID No. 20 or SEQ ID No. 21.

Furthermore, the polynucleotides of the invention may comprise not only nucleotide sequences encoding antigen-binding variable fragments, but also nucleotide sequences encoding other extracellular domain portions, transmembrane domains and/or intracellular signaling associated therewith, respectively.

The descriptions of antibodies, antigen-binding variable fragments, extracellular domains, transmembrane domains, intracellular signaling domains, and the like for chimeric antigen receptors are the same as those previously described.

The polynucleotide may comprise the sequence of SEQ ID NO. 22 or SEQ ID NO. 23 or SEQ ID NO. 24 or SEQ ID NO. 25. In a specific embodiment of the invention, a polynucleotide comprising SEQ ID NO. 22 or SEQ ID NO. 23 is prepared so as to design and express a chimeric antigen receptor (EphA 2-CAR 1) comprising: scFv of two anti-EphA 2 antibodies (# 79 and # 85) as extracellular domains, and CD28 linked to Myc and hinge domains as transmembrane and intracellular signaling domains, and CD3- ζ and DAP10 as intracellular signaling domains, respectively. In another specific embodiment of the invention, a polynucleotide comprising SEQ ID NO. 24 or SEQ ID NO. 25 is prepared so as to design and express a chimeric antigen receptor (EphA 2-CAR 2) comprising: scFv of two anti-EphA 2 antibodies (# 79 and # 85) as extracellular domains, CD8 as hinge and transmembrane domains, and CD3- ζ and DAP10 as intracellular signaling domains, respectively.

The polynucleotides of the invention may comprise sequences substantially identical to those listed above. Sequences that are substantially identical to the sequences include, for example, sequences that, when transcribed and translated, can result in the synthesis of a protein having the same amino acid sequence as the CAR, and can have a nucleotide sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% homology to the sequences listed above, but are not limited thereto.

Polynucleotides encoding chimeric antigen receptors may comprise optimized sequences depending on the type of organism into which the polynucleotide is to be introduced and expressed and the transcription, translation or other expression system of the organism. This is due to the degeneracy of the codons, whereby various combinations of nucleotide sequences may exist which may encode the protein to be expressed, all of which are included within the scope of the present invention. The modification of the polynucleotide according to codon optimisation may be determined by the type of organism in which the chimeric antigen receptor of the invention is to be expressed and applied, for example, the polynucleotide of the invention may be a polynucleotide modified by optimisation of codon usage in mammals, primates, and more particularly by optimisation of expression and function in humans.

The expression vectors of the invention comprise polynucleotides.

Since the polynucleotide comprises a sequence encoding a chimeric antigen receptor of the invention, expression vectors comprising polynucleotides that can be used to produce chimeric antigen receptors can be used to transfer the polynucleotide to a particular cell or organism for expression of the chimeric antigen receptor, or can be used to store the polynucleotide.

Expression vectors may be constructed in prokaryotic or eukaryotic cells as hosts.

For example, if an expression vector is constructed in a prokaryotic cell, a strong promoter capable of driving transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLlambda promoter, pRlambda promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter), a ribosome binding site for initiating translation, and a transcription/translation termination sequence. When E.coli (e.g., HB101, BL21, DH 5. Alpha. Etc.) is used as the host cell, promoters and operator sites (Yanofsky, C., J.Bacteriol.,158;1018-1024,1984) of the E.coli tryptophan biosynthesis pathway and the left-hand helical promoters of phage lambda (pLlambda promoter, herskowitz, I. And Hagen, D., ann. Rev. Genet.,14:399-445,1980) can be used as regulatory sites. If Bacillus (Bacillus) is used as host cell, the promoter of the toxin protein gene of Bacillus thuringiensis (Bacillus churrigensis) (appl. Environ. Microbiol.,64:3932-3938,1998; mol. Gen. Genet.,250:734-741,1996) or any promoter expressible in Bacillus may be used as regulatory site. Expression vectors can be constructed from plasmids commonly 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 pUC 19), phages (e.g., λgt4λB, λ -Charon, λΔz1, and M13), or viruses (e.g., SV 40).

If the expression vector involves eukaryotic cells as hosts, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter, beta-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian 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 promoter of HIV, moloney virus promoter, epstein-Barr virus (EBV) promoter, and Rous Sarcoma Virus (RSV) promoter) may be utilized, and typically have polyadenylation sequences as transcription termination sequences. The expression vector may be an expression vector having a CMV promoter.

Furthermore, the expression vector may be fused to additional sequences to facilitate purification of the antibodies expressed thereby. Additional sequences to be fused may include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6 XHis (hexahistidine; quiagen, USA). Furthermore, in view of the fact that the protein expressed by the expression vector of the present invention is a chimeric antigen receptor, the expressed protein can be easily purified by a protein a column or the like in consideration of this property, without requiring an additional sequence for purification.

The expression vector may contain antibiotic resistance genes conventionally used in the art as selectable markers, such as ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline resistance genes.

2. Immune cells expressing chimeric antigen receptor for EphA2 on the surface of immune cells

In another aspect of the invention, immune cells are provided, characterized in that they exhibit enhanced cytotoxicity or cytolytic activity against EphA 2-expressing cancer cells.

The immune cells express on their surface the chimeric antigen receptor of the invention as described above.

The chimeric antigen receptor is the same as previously described in "1. Novel anti-EphA 2 antibodies, antigen-binding fragments thereof, and Chimeric Antigen Receptors (CARs) comprising the novel anti-EphA 2 antibodies and antigen-binding fragments thereof, as well as polynucleotides and expression vectors for expressing the CARs. In particular, the chimeric antigen receptor may comprise an antigen binding site capable of specifically binding to EphA2 antigen expressed on cancer cells.

The immune cell may be any cell capable of inducing immunity to elicit a desired therapeutic effect, such as, 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. Thus, an immune cell expressing a chimeric antigen receptor on the cell surface of an immune cell according to the present invention may be a CAR-NK cell (chimeric antigen receptor-natural killer cell), a CAR-T cell (chimeric antigen receptor-T cell), a CAR-NKT cell (chimeric antigen receptor-natural killer T cell), a CAR-macrophage (chimeric antigen receptor-macrophage), or the like.

T cells may be, but are not limited to, cytotoxic T Lymphocytes (CTLs), tumor Infiltrating Lymphocytes (TILs), T cells isolated from Peripheral Blood Mononuclear Cells (PBMCs), and the like.

CAR-NK cells, which are cells in which a chimeric antigen receptor is introduced into natural killer cells, have the following advantages: can solve the problems of persistent toxicity of cancer immunotherapy using conventional T cell-based CAR-T therapy, risk of autoimmune diseases, graft Versus Host Disease (GVHD) and off-target toxicity, and can target various cancer cells through on/off response, and can be used as a general therapeutic agent.

The immune cells of the invention express on their cell surface chimeric antigen receptors comprising: an antigen binding variable fragment (scFv) that is an antibody capable of specifically recognizing and binding EphA2 as an extracellular domain, said EphA2 can be specifically expressed in cancer cells. Thus, in the presence of EphA 2-expressing cancer cells, signal transduction may occur through chimeric antigen receptors that further enhance the cytotoxic or cytolytic activity of immune cells and increase cytokine secretion. Thus, the immune cells of the invention may have activity in attacking and treating cancer cells.

In a specific embodiment of the invention, chimeric antigen receptors are expressed on the surface of natural killer cells and T cells comprising as extracellular domains antigen-binding variable fragments of two EphA 2-specific antibodies of the invention. The two natural killer cells were each co-cultured with the breast cancer cell line MDA-MB-231 expressing EphA2, and the two T cells were each co-cultured with the lung cancer cell line lung A549 cells expressing Eph A2. The enhanced cytotoxicity (or cytolytic activity), significantly increased amount of cytokine secretion, and increased extent of degranulation, confirm that the immune cells of the present invention have excellent therapeutic effects on EphA 2-expressing cancer cells. Furthermore, the therapeutic effect on cancer cells as described above was clearly demonstrated in an animal model in which cancer cells were implanted.

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

Another aspect of the invention provides a pharmaceutical composition comprising immune cells for use in the treatment of cancer.

The descriptions for immune cells, chimeric antigen receptors expressed thereon, and the like are the same as those described for them in "1. Novel anti-EphA 2 antibodies, antigen-binding fragments thereof, and Chimeric Antigen Receptors (CARs) comprising the novel anti-EphA 2 antibodies and antigen-binding fragments thereof, as well as polynucleotides and expression vectors for expressing CARs" and "2. Immune cells expressing chimeric antigen receptors for EphA2 on the surface of immune cells", and thus omitted to avoid repetition.

As used herein, the term "cancer" is used interchangeably with "tumor" and refers to or means a physiological condition of a mammal typically characterized by uncontrolled cell growth/proliferation.

Cancers or tumors that may be treated with the compositions of the present invention include both solid and hematological cancers, but are not particularly limited thereto. For example, the cancer may be at least one selected from the group consisting of: lung cancer, stomach cancer, ovarian cancer, cervical cancer, breast cancer, pancreatic 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 myelogenous leukemia, malignant lymphoma, thymus cancer, osteosarcoma, fibromatous tumor, and brain cancer, but are not limited thereto, and any cancer cell containing an antigen that can be recognized by a chimeric antigen receptor can be used in the present invention without limitation.

As used herein, the term "treating" means inhibiting the development of cancer, alleviating or eliminating its symptoms.

The pharmaceutical composition may comprise 1 to 10 times, 2 to 10 times or 5 to 10 times the number of immune cells as tumor cells in the subject to be treated, but is not limited thereto.

The composition may be in the form of a pharmaceutical composition, a prodrug composition, a nutritional composition, and the like.

The compositions of the present invention for treating cancer may also comprise a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant that it does not inhibit the activity of the active ingredient and does not have toxicity beyond acceptable toxicity to the subject to which it is applied (at the outset), and that a "carrier" is defined as a compound that facilitates the incorporation of the compound into cells or tissues.

The pharmaceutical compositions of the present invention may be administered alone or in combination with any convenient carrier, and such dosage forms may be single or repeated administration formulations. The pharmaceutical composition of the present invention may be a solid dosage formulation or a liquid dosage formulation. Solid dosage formulations include, but are not limited to, powders, granules, tablets, capsules, suppositories, and the like. Solid dosage formulations may include, but are not limited to, carriers, flavoring agents, binders, preservatives, disintegrants, gloss agents, and fillers. Liquid dosage formulations include, but are not limited to, water, solvents such as propylene glycol solutions, suspensions, emulsions, and the like, and may be prepared by adding suitable colorants, flavors, stabilizers, tackifiers, and the like. For example, powders may be prepared by simply mixing the active ingredient of the invention with a suitable pharmaceutically acceptable carrier such as lactose, starch, microcrystalline cellulose and the like. The particles may be prepared by: the active ingredient of the present invention is mixed with a pharmaceutically acceptable suitable carrier and a pharmaceutically acceptable suitable binder such as polyvinylpyrrolidone, hydroxypropylcellulose, and then subjected to wet granulation using a solvent such as water, ethanol, isopropanol, etc., or dry granulation using a compressive force. Tablets may also be prepared by mixing the granules with a suitable pharmaceutically acceptable lubricant such as magnesium stearate and tabletting using a tabletting machine.

The pharmaceutical composition may be administered as an oral dosage form, an injectable dosage form (e.g., intramuscular, intraperitoneal, intravenous, infusion, subcutaneous, implant), an inhaled dosage form, a nasal dosage form, a vaginal dosage form, a rectal dosage form, a sublingual dosage form, a transdermal dosage form, a topical dosage form, or in other ways, depending on the condition to be treated and the condition of the subject to be treated, but is not limited thereto. Depending on the route of administration, the pharmaceutical compositions may be formulated in any suitable dosage unit formulation comprising the conventionally used, non-toxic, pharmaceutically acceptable carriers, excipients and vehicles.

The pharmaceutical composition may be administered at a daily dose of about 0.0001mg/kg to about 10g/kg, and may be administered at a daily dose of about 0.001mg/kg to about 1 g/kg. However, the dosage may vary depending on the degree of purification of the mixture, the condition of the patient (age, sex, weight, etc.), the severity of the condition under treatment, and the like. If necessary, the total daily administration may be divided into several administrations throughout the day for convenience.

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

However, the following examples are specific illustrations of the present invention, and the present invention is not limited by the following examples.

Example 1

1-1: Preparation of Single-chain variable fragments of antibodies that specifically bind to EphA2

In an anti-EphA 2 antibody sequence that can specifically bind to an ephrin a type receptor 2 (EphA 2) protein expressed in cancer cells, two single chain variable fragments (scFv) were prepared using sequences that play an important role in specific binding (# 79, # 85).

For a #79scFv, the heavy chain variable region comprises 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 the light chain variable region comprises 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. And for a #85scFv, the heavy chain variable region comprises 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, the light chain variable region comprises 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. 3 and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 14.

1-2: Design of chimeric antigen receptor to be introduced into natural killer cells

The inventors used two scFv as designed above as the extracellular domain containing the antigen binding site to design a Chimeric Antigen Receptor (CAR) to be introduced into natural killer cells. Specifically, the present inventors designed chimeric antigen receptors (epha2#79-CAR 1 and epha2#85-CAR1, respectively, and collectively referred to as "EphA2-CAR 1") to have an intracellular signaling domain of a chimeric antigen receptor classified as a so-called third generation CAR according to embodiments of the present invention by using an extracellular domain comprising two scFv as antigen binding sites, CD28 as a transmembrane domain linked to Myc and a hinge domain, and CD28DAP10 as an intracellular signaling domain additionally linked to the transmembrane domain. And then the polynucleotides having the nucleotide sequences encoding their genetic constructs are subsequently inserted into a lentiviral vector (FIG. 1).

1-3: Design of chimeric antigen receptor to be introduced into T cells

Furthermore, the present inventors used two scFv designed as described above as the extracellular domain containing the antigen binding site to design a chimeric antigen receptor to be introduced into T cells. Specifically, the present inventors designed chimeric antigen receptors (epha2#79-CAR 2 and epha2#85-CAR2, respectively, and collectively referred to as "EphA2-CAR 2") to have intracellular signaling domains of chimeric antigen receptors classified as so-called second generation CARs according to embodiments of the present invention by using an extracellular domain comprising two scFv as antigen binding sites, CD8 as a transmembrane domain and a hinge domain, and CD3- ζ as an intracellular signaling domain and 4-1BB as a costimulatory molecule additionally linked to the transmembrane domain. And then the polynucleotides having the nucleotide sequences encoding their genetic constructs are subsequently inserted into a lentiviral vector (FIG. 2).

Example 2

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

Immune cells capable of expressing the chimeric antigen receptor of the invention designed in example 1 were prepared.

2-1: Preparation of EphA2-CAR 1-expressing Natural killer cells

The lentiviral vectors and viral packaging vectors (pMDLG/RRE, pRSV/REV, VSVG) prepared in examples 1-2 above were transfected into HEK293T cells and EphA2-CAR1 expressing lentiviruses were obtained therefrom, concentrated using ultracentrifugation, and subsequently infected into natural killer cells by the rotary inoculation (spinoculation) method (360 g,90min, RT) at a multiplicity of infection (MOI) of 30. Infected natural killer cells were incubated at 37 ℃ for 5 hours at 5% CO ₂ and then replaced with fresh medium and cultivation continued by treating natural killer cells with puromycin at a concentration of 3ug/ml for selection of appropriately infected natural killer cells after 3 days. As a control, uninfected natural killer cells were also treated with puromycin and culture was continued using puromycin-treated medium until all natural killer cells in the control were killed by puromycin. At the time point when all natural killer cells in the control group were killed, infected natural killer cells were selected and subjected to experiments.

Natural killer cells expressing epha2#79-CAR1 and epha2#85-CAR1 (referred to as 'epha2#79-CAR1-NK cells' and 'epha2#85-CAR1-NK cells', respectively, and collectively referred to as 'EphA2-CAR1-NK cells') prepared and screened as described above were treated (30 min at 4 ℃ in vivo) with an anti-Myc antibody (CST; 9B 11) that specifically binds to Myc of EphA2-CAR1, and expression of Myc was confirmed by flow cytometry. As a control, intact natural killer cells that do not express EphA2-CAR1 were used.

As a result, ephA2-CAR1 was found to be properly expressed in both EphA2-CAR1-NK cells as compared to the control, as shown in FIG. 3 a.

2-2: Preparation of EphA2-CAR 2-expressing T cells

Peripheral Blood Mononuclear Cells (PBMCs) were differentiated into T cells, and then the lentiviral vector prepared in example 1-3 above was transfected into the differentiated T cells using the same virus acquisition and rotary inoculation method (5 moi,300g,32 ℃,90 min) as in example 2-1 above to infect the differentiated T cells to produce T cells expressing epha2#79-CAR2 and epha2#85-CAR2 (referred to as 'epha2#79-CAR2-T cells' and 'epha2#85-CAR2-T cells', respectively, and collectively referred to as 'EphA2-CAR2-T cells'). Subsequent use of anti-EphA 2 antibodies (His-tag recombinant protein EphA2 (NKMAX) and anti-6X)FITC conjugated antibody (abcam)) expression of EphA2-CAR2 was confirmed by flow cytometry using natural T cells that did not express EphA2-CAR2 as controls.

As a result, ephA2-CAR2 was found to be properly expressed in both EphA2-CAR2-T cells as compared to the control, as shown in FIG. 3 b.

Example 3

Cytotoxicity (or cytolytic activity) of EphA2-CAR1-NK cells and EphA2-CAR2-T cells against EphA 2-expressing cancer cells was determined.

Since natural killer cells and T cells expressing the chimeric antigen receptor of the present invention on the surface of natural killer cells and T cells, such as those prepared in example 2, contain scFv specific for EphA2 as an antigen binding site, the present inventors studied their cytotoxicity against EphA 2-expressing cancer cells.

3-1 Selection of EphA 2-expressing cancer cells

First, the present inventors selected breast cancer cell line MDA-MB-231 cells (Korea Cell Line Bank) and lung cancer cell line A549 cells (Korea Cell Line Bank) as EphA 2-expressing cancer cells, and chronic myelogenous leukemia cell line K562 cells (Korea Cell Line Bank) as non-EphA 2-expressing cancer cells, in an amount of 1. Mu.l/100. Mu.l of anti-EphA 2 antibody (human EphA 2/mouse IgG2A Alexa)488 Conjugated antibody (R & Dsystems)) and cells were incubated (4 ℃ for 30 minutes in the dark) and EphA2 expression levels in the three cell lines were determined by flow cytometry. /(I)

As a result, MDA-MB-231 cells and A549 cells were found to express EphA2, and K562 cells were found not to express EphA2, as shown in FIG. 4.

3-2: Confirmation of Activity of EphA2-CAR1-NK cells against EphA 2-expressing cancer cells

The calcein AM assay was used to confirm cytotoxicity of two types of EphA2-CAR1-NK cells prepared in example 2-1 against MDA-MB-231 cells and K562 cells whose expression of EphA2 was confirmed as described above. Specifically, MDA-MB-231 cells and K562 cells were treated and incubated with calcein at a concentration of 5 μg/ml (37 ℃,5% CO ₂, 1 hour in the dark), and then with primary natural killer cells and each of the two EphA2-CAR1-NK cells above at 5: 1. 1: 1. 0.5:1 (natural killer cells: cancer cells) and incubating (37 ℃ C., 5% CO ₂ for 4 hours) each cancer cell stained with calcein, and then taking 100 μl of the supernatant to determine the amount of calcein present in the supernatant.

As a result, both epha2#79-CAR1-NK cells and epha2#85-CAR1-NK cells were found to exhibit significantly higher cytotoxicity against EphA 2-expressing MDA-MB-231 cells as shown in fig. 5, as compared to control natural killer cells, and this cytotoxicity was dependent on the concentration of EphA2-CAR1-NK cells treated. In contrast, control NK cells and both EphA2-CAR1-NK cells showed slight cytotoxicity against K562 cells that did not express EphA 2.

In addition, cytokine and granule secretion was confirmed for both types of EphA2-CAR1-NK cells. Specifically, MDA-MB-231 cells and K562 cells were subjected to 1's with either original natural killer cells or each of the two types of EphA2-CAR1-NK cells prepared in example 2-1 above: 1 treatment and incubation (37 ℃,5% CO ₂ for 16 hours) and then the supernatant was collected and the presence of INF-gamma (interferon-gamma) in the supernatant was confirmed by ELISA. The amounts of cytokines secreted by control natural killer cells and EphA2-CAR1-NK cells alone were used as controls.

As shown in fig. 6, secretion of INF- γ was found to be significantly enhanced only in EphA2-CAR1-NK cells treated with EphA 2-expressing MDA-MB-231 cells.

In addition, each of MDA-MB-231 cells and K562 cells, and control natural killer cells or two types of EphA2-CAR1-NK cells prepared in example 2-1, was expressed as 1 in RPMI (10% FBS): 1 in RPMI (10% FBS) and subsequent incubation (4 hours at 37 ℃,5% CO ₂), treatment and staining with anti-CD 56 antibody to select natural killer cells, and analysis of CD107a expression levels by flow cytometry of control natural killer cells and both types of EphA2-CAR1-NK cells.

As shown in fig. 7, the expression of CD107a was found to be significantly enhanced only in EphA2-CAR1-NK cells treated with EphA 2-expressing MDA-MB-231 cells, as was the case with INF- γ.

3-3: Confirmation of EphA2-CAR2-T cell Activity against EphA 2-expressing cancer cells

GFP was expressed in a549 cells confirmed to express EphA2 as described above, and 2: 1. 1: 1. 0.5:1 and 0.25: ratio of 1 (T cells: cancer cells) EphA2-CAR2-T cells prepared in example 2-2 were treated and incubated on an incuCyte apparatus for 48 hours, and data were collected every 4 hours and analyzed for EphA2-CAR2-T cell cytotoxicity using the incuCyte ZOOM program.

The results confirm that both types of EphA2-CAR2-T cells exhibit cytotoxicity against EphA 2-expressing a549 cells, as shown in fig. 8.

Example 4

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

Cytotoxicity of natural killer cells and T cells expressing the chimeric antigen receptor of the present invention on the surfaces of natural killer cells and T cells identified in example 3 against EphA 2-expressing cancer cells was confirmed again in an animal model.

4-1: Confirmation of Activity of EphA2-CAR1-NK cells against EphA 2-expressing cancer cells

First, the present inventors confirmed that EphA2 was expressed in lung cancer cell line H460 cells (Korea Cell Line Bank) using the same method as in example 3-1 (fig. 9 a), and confirmed that EphA2#79-CAR1-NK cells exhibited much higher cytotoxicity against H460 cells compared to control natural killer cells expressing CARs with the extracellular domain removed (dECTO) using the same method as in example 3-2 (fig. 9 b).

Subsequently, as shown in fig. 9c, 3x10 ⁶ of the above H460 cells were subcutaneously injected into the flank region of a female Balb/c nude mouse (SaronBio) about 6 weeks old, and 2x10 ⁶ of the above EphA2-CAR1-NK cells or control natural killer cells were intravenously injected 5 times at 3-day or 4-day intervals after 10 days when the tumor size was about 50mm ³. The tumor size and weight were then measured over 24 days.

The results are shown in fig. 9D and 9E, where tumor size and weight were significantly reduced in mice treated with EphA2-CAR1-NK cells of the invention compared to mice treated with control natural killer cells.

4-2: Confirmation of EphA2-CAR2-T cell Activity against EphA 2-expressing cancer cells

Subsequently, 1x10 ⁶ EphA 2-expressing lung cancer cell line a 549-luciferase cells transfected with luciferase (PerkinElmer) were subcutaneously injected into the right flank of 6 week old male NOG mice (Coatech), and 5x10 ⁶ above EphA2-CAR2-T cells or control T cells expressing CARs with removed extracellular domains (dECTO) were intravenously injected when the tumor size was about 200mm ³.

Tumor size, mouse body weight and IVIS were then measured twice weekly over 45 days.

The results confirm that EphA2-CAR2-T cells effectively inhibited tumors as shown in fig. 10a and 10b, and also confirm the presence of EphA2-CAR2-T cells and control T cells in the blood of mice after intravenous injection as shown in fig. 10 c. Furthermore, as shown in fig. 10d, ephA2-CAR2-T cells were confirmed to be present in the tumor in higher amounts than control T cells.

Summary

In summary, the above experimental results show that the two anti-EphA 2 antibodies (# 79, # 85) of the present invention not only exhibit specific binding to EphA2, but also recognize and bind EphA2 on cancer cells expressing EphA2, and that, upon binding, the chimeric antigen receptor comprising the antigen binding site of the antibody triggers signal transduction in immune cells (natural killer cells or T cells) whose surfaces are expressed, and thus it has been clearly demonstrated in vitro as well as in vivo that the chimeric antigen receptor comprising the antigen binding site of the antibody triggers various immune responses in immune cells capable of attacking cancer cells.

While the invention has been described in detail with respect to only the embodiments described above, it will be apparent to those skilled in the art that various changes and modifications are possible within the scope of the invention, and it will be apparent that such changes and modifications fall within the scope of the appended claims.

Claims (17)

1. An antibody or antigen-binding fragment thereof that specifically binds to ephrin-a receptor 2 (EphA 2), 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.

2. The antibody or antigen-binding fragment of claim 1, wherein the heavy chain variable region has the amino acid sequence of SEQ ID No. 7 or SEQ ID No. 15.

3. The antibody or antigen-binding fragment of claim 1, wherein the light chain variable region has the amino acid sequence of SEQ ID No. 8 or SEQ ID No. 16.

4. A chimeric antigen receptor comprising:

An extracellular binding domain comprising an antigen binding site that specifically binds to ephrin-a receptor 2 (EphA 2);

A transmembrane domain; and

An intracellular signaling domain;

Wherein the antigen binding site that specifically binds EphA2 is a single chain variable fragment (scFv) 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; the light chain variable region comprises 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.

5. The chimeric antigen receptor of claim 4, wherein the antigen binding site that specifically binds EphA2 is a single chain variable fragment of an anti-EphA 2 antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID No. 7 or SEQ ID No. 15.

6. The chimeric antigen receptor of claim 4, wherein the antigen binding site that specifically binds EphA2 is a single chain variable fragment of an anti-EphA 2 antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID No. 8 or SEQ ID No. 16.

7. The chimeric antigen receptor according to claim 1, wherein the extracellular domain further comprises at least one selected from a hinge domain and a spacer domain.

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

9. The chimeric antigen receptor according to claim 4, wherein the transmembrane domain is derived from at least one transmembrane domain selected from the group consisting of: the α, β or ζ chain of the T Cell Receptor (TCR), CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

10. The chimeric antigen receptor according to claim 4, wherein the intracellular signaling domain is derived from at least one selected from the group consisting of: t Cell Receptor (TCR) ζ, fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, cd3ζ, CD5, CD22, CD79a, CD79b, and CD66d.

11. The chimeric antigen receptor according to claim 4, wherein the intracellular signaling domain comprises at least one primary signaling domain derived from one selected from the group consisting of: t Cell Receptor (TCR) ζ, fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, cd3ζ, CD5, CD22, CD79a, CD79b, and CD66d; and

At least one costimulatory 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、DAP12、PD-1、LIGHT、NKG2C、EphA2 and CD83 derived from one selected from the group consisting of.

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

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

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

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

16. A pharmaceutical composition for treating cancer comprising the immune cell of claim 14.

17. The pharmaceutical composition of claim 16, wherein the cancer is at least one selected from the group consisting of: lung cancer, stomach cancer, ovarian cancer, cervical cancer, breast cancer, pancreatic 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, thymus cancer, osteosarcoma, fibromatous tumor, and brain cancer.