Humanized antibodies targeting hepatic ligand protein A receptor 10

Abstract

The invention relates to a humanized antibody of a targeted human hepatic ligand protein A receptor 10 and application thereof, the specific antibody of the hepatic ligand protein A receptor 10 (EphA10) or a fragment with immunocompetence of the antibody is specifically combined with the EphA10, and the EphA10 is a very promising target spot for overcoming the drug resistance and resistance of the existing immune checkpoint inhibition factor targeted therapeutic agent. And when an ADC is prepared using the antibody, it is shown that it is capable of specifically killing cancer cells, and thus it is useful as an antibody therapeutic agent for treating cancer using a mechanism that causes cancer cell death by immune cell activation, an antibody therapeutic agent for treating EphA10-expressing cancer, or a cell therapeutic agent for treating EphA10-expressing cancer, and it is useful as an antibody therapeutic agent for treating EphA10-expressing cancer. And can also be used for cancer diagnosis.

Description

TECHNICAL FIELD

[0001] The present application relates to a humanized antibody targeting human ephrin A receptor 10 and uses thereof. BACKGROUND

[0002] Cancer is a state in which cells grow abnormally due to various changes in gene expression, resulting in uncontrolled cell proliferation and death, invade and destroy neighboring tissues, and metastasize to other sites, eventually leading to human death. The exact cause of cancer, i.e., the mechanism of transformation of normal cells into cancer cells, has not yet been identified, but it is currently known that cancer is caused by a complex interplay of exogenous factors such as environmental factors, chemicals, radiation, viruses, and endogenous factors such as genetic factors, immune factors. Cancer is roughly divided into blood cancer, which shows abnormality in the number of blood cells, and solid cancer, which has a certain hardness and shape of cell mass in the body. Cancer can occur almost anywhere in the body, including blood and tissues, such as lung cancer, stomach cancer, breast cancer, oral cancer, liver cancer, uterine cancer, esophageal cancer, skin cancer, etc. The main treatment methods for cancer include surgery, radiation therapy, and anticancer therapy using chemotherapy drugs that inhibit cell proliferation. Cancer treatment drugs are roughly divided into low molecular weight drugs and high molecular weight drugs, and high molecular weight drugs with higher specificity than low molecular weight drugs with insufficient specificity and greater side effects are attracting attention as therapeutic agents.

[0003] Protein therapeutic agents are rapidly replacing non-specific low-molecular compound therapeutic agents and are widely used in clinics because of their high specificity for disease targets and low side effects and toxicity. Among the protein therapeutic agents currently used in clinics, antibody therapeutic agents and Fc fusion protein therapeutic agents fused with the Fc region of antibodies are mainly used. Therapeutic antibodies are considered to be one of the most effective cancer treatment methods because they exhibit very high targeting specificity, have low biological toxicity and side effects, and have an excellent plasma half-life of about three weeks compared to existing low-molecular drugs. In fact, major pharmaceutical companies and research institutions around the world are accelerating the development of therapeutic antibodies that can specifically bind to cancer cells (including carcinogenic factors) and effectively eliminate cancer cells. Companies developing therapeutic antibody drugs include Roche, Amgen, Johnson & Johnson, Abbott, BMS, and other pharmaceutical companies, and in particular, Roche's representative products for anticancer treatment purposes, Herceptin, Avastin, Rituxan, etc. not only created huge profits, but also led the global antibody pharmaceutical market with sales of about $19.5 billion in 2012. Johnson & Johnson, which developed Remicade, also rapidly grew in the global antibody market due to sales growth, and Abbott and BMS, etc. are said to have various therapeutic antibodies in the final stages of development. Therefore, in the global pharmaceutical market dominated by low-molecular drugs, biopharmaceuticals, including therapeutic antibodies that have specificity for disease targets and have fewer side effects, are rapidly replacing low-molecular drugs.

[0004] Cancer cells express immune checkpoint proteins on their surface, and normal cells use these proteins to suppress the activation of immune cells, thereby evading the killing mechanism of immune cells. In recent years, research on immune checkpoint inhibitors as a cancer treatment method is actively ongoing (Immunity. 2018 Mar 20; 48(3): 434-452). Immune checkpoint inhibitors are drugs that activate T cells by blocking the activity of immune checkpoint proteins involved in T cell suppression to attack cancer cells, and representative antibodies that recognize CTLA 4, PD-1, PD-L1, etc. are used. However, the response rate of cancer patients to immune anticancer agents, including immune checkpoint inhibitors, immune cell therapies, therapeutic antibodies, and anticancer vaccines, still remains at 15 to 45%, and most solid tumors have problems of developing resistance to immune anticancer agents. Therefore, previous studies using a mouse model and human cancer cell beads that induce resistance to anti-PD-1 antibody therapeutic agents by repeatedly applying anti-PD-1 antibody therapeutic agents have found that the cell membrane protein EphA10 is a factor that causes resistance to therapeutic agents targeting immune checkpoint inhibitors. EphA10 is one of the Ephrin receptors, and the Ephrin receptor is the largest family of receptor tyrosine kinases (RTKs), unlike other Ephrin receptors, EphA10 is a very new receptor whose function has been little studied, and its expression is significantly increased in all breast cancers, including triple negative breast cancer (TNBC), which is a breast cancer that has no appropriate therapeutic agent and has a very poor prognosis, and it has been reported that the expression of EphA10 is also significantly increased in lung cancer, pancreatic cancer, prostate cancer, gastric cancer, and thyroid cancer compared to normal tissue. SUMMARY

[0005] TECHNICAL PROBLEM

[0006] The present application aims to provide an Eph receptor A10 (EphA10)-specific antibody or a fragment thereof having immunological activity.

[0007] Also, the present application aims to provide an Eph receptor A10-specific antibody drug conjugate.

[0008] Also, an object of the present application is to provide a bispecific or multispecific antibody.

[0009] Also, an object of the present application is to provide a chimeric antigen receptor (CAR).

[0010] Also, an object of the present application is to provide a chimeric antigen receptor-expressing cell.

[0011] Also, an object of the present application is to provide a T-cell engager.

[0012] Also, an object of the present application is to provide a pharmaceutical composition for preventing or treating cancer.

[0013] Also, an object of the present application is to provide a composition for diagnosing cancer.

[0014] Also, an object of the present application is to provide a use for preventing or treating cancer.

[0015] Also, an object of the present application is to provide a method for treating cancer.

[0016] Also, an object of the present application is to provide a use for diagnosing cancer.

[0017] Technical solutions

[0018] To solve the above problems, the present application provides a hepatoma A receptor 10 specific antibody or a fragment thereof having immunological activity.

[0019] Also, the present application provides a hepatoma A receptor 10 specific antibody drug conjugate comprising the antibody or a fragment thereof having immunological activity.

[0020] Also, the present application provides a bispecific or multispecific antibody comprising the antibody or a fragment thereof having immunological activity.

[0021] Also, the present application provides a chimeric antigen receptor comprising the antibody or a fragment thereof having immunological activity.

[0022] Also, the present application provides a chimeric antigen receptor-expressing cell.

[0023] Also, the present application provides a T-cell engager comprising the antibody or a fragment thereof having immunological activity.

[0024] Also, the present application provides a pharmaceutical composition for preventing or treating cancer.

[0025] Also, the present application provides a composition for diagnosing cancer.

[0026] Also, the present application provides a use of the antibody or the immunologically active fragment thereof, the antibody drug conjugate, the bispecific or multispecific antibody, the chimeric antigen receptor-expressing cell, or the T cell engager of the present application in preventing or treating cancer.

[0027] Also, the present application provides a method of treating cancer, comprising the step of administering a pharmaceutically effective amount of the antibody or the immunologically active fragment thereof, the antibody drug conjugate, the bispecific or multispecific antibody, the chimeric antigen receptor-expressing cell, or the T cell engager of the present application to an individual having cancer.

[0028] Meanwhile, the present application provides a use of the antibody or the immunologically active fragment thereof, the antibody drug conjugate, the bispecific or multispecific antibody, the chimeric antigen receptor-expressing cell, or the T cell engager of the present application in diagnosing cancer.

[0029] Effects of the Invention

[0030] The EphA10-specific antibody or the immunologically active fragment thereof of the present application specifically binds to EphA10, which is a very promising target for overcoming drug resistance and resistance of existing immune checkpoint inhibitor targeting therapeutic agents, and when an ADC is prepared using the antibody, it shows that it can specifically kill cancer cells, and thus, its effect lies in that it can be used as an antibody therapeutic agent for treating cancer using a mechanism in which cancer cell death is caused by activation of immune cells, an antibody therapeutic agent for treating cancer expressing EphA10, or a cell therapeutic agent for treating cancer expressing EphA10, and can also be used for diagnosis of cancer. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 A figure for analyzing the size and purity of 10 EphA10 antigen proteins expressed in animal cells and purified using SDS-PAGE gel.

[0032] Figure 2 Analysis results of the amino acid sequence of a mouse antibody targeting EphA10 and analysis results of the binding force of a chimeric #2 antibody prepared based on the results to EphA10.

[0033] Figure 3 A figure for showing the amino acid sequence of a humanized antibody targeting EphA10 and antibody information.

[0034] Figure 4Figures for confirming the expression amount of the humanized antibodies targeting EphA10 expressed in animal cells and analyzing the results after purification, and confirming the size and purity of the purified humanized antibodies using SDS-PAGE gel.

[0035] Figure 5 Figures for ELISA analysis results of the binding force of 5 humanized antibodies targeting EphA10 to EphA10.

[0036] Figure 6 Figures for confirming the binding force of 5 humanized antibodies targeting EphA10 expressed in human and mouse cells to EphA10.

[0037] Figure 7 Figures showing the analysis results of the quantitative binding constant of the humanized antibodies targeting EphA10 to EphA10.

[0038] Figure 8 Figures for the antibody-drug binding analysis results of the ADCs prepared using the EphA10-specific antibodies (1-1 and 1-2) selected due to their high binding force.

[0039] Figure 9 Figures for confirming the killing efficacy of cancer cells of the ADCs prepared using the EphA10-specific antibodies (1-1 and 1-2).

[0040] Figure 10 Figures for confirming the free cysteine residues present in the light chain by analyzing the sequence of the EphA10-specific humanized antibodies.

[0041] Figure 11 Figures for the ELISA analysis results of the binding force of the EphA10-specific humanized antibody (1-1) and the ADC antibody targeting EphA10 (1-1 (ADC)) prepared using the same to EphA10:

[0042] 1-1: EphA10-specific humanized antibody; and

[0043] 1-1 (ADC): ADC antibody targeting EphA10.

[0044] Figure 12 Figures for expressing the EphA10-specific humanized antibody in animal cells and confirming the size and purity thereof using SDS-PAGE gel after purification, the antibody having improved ADC efficacy due to the substitution of alanine (A) or serine (S) for the free cysteine residues present in the light chain of the EphA10-specific antibodies 1-1 and 1-2, respectively.

[0045] Figure 13Results of analysis of binding affinity of EphA10-specific humanized antibodies with improved ADC efficacy to EphA10.

[0046] Figure 14 Figure showing yeast library construction and screening strategy for increasing the binding affinity of humanized antibodies targeting EphA10.

[0047] Figure 15 Figure showing iterative screening process using flow cytometry to increase the binding affinity of humanized antibodies targeting EphA10.

[0048] Figure 16 Results of analysis of amino acid sequences of 10 antibodies (A.3, A.7, A.8, B.1, B.3, B.7, 2.1, 2.3, 1-N, and 2-W) discovered by flow cytometry and 6 (1-R, 1-W, 1-Y, 2-N, 2-R, and 2-Y) humanized antibodies further substituted with free cysteine.

[0049] Figure 17 Figure showing expression of 16 humanized antibodies with increased binding affinity to EphA10 in animal cells and confirmation of their size and purity using SDS-PAGE gel after purification.

[0050] Figure 18 Results of analysis of binding affinity of 16 humanized antibodies with increased binding affinity to EphA10 to EphA10.

[0051] Figure 19 Results of antibody-drug conjugation analysis of ADCs prepared using 2 (A7 and 2W) of the 16 humanized antibodies with increased binding affinity to EphA10.

[0052] Figure 20 Figure showing results of ELISA analysis of binding affinity of EphA10-specific antibody 1-1 and ADC (1-1_ADC) prepared using the same, humanized antibodies A7 and 2W with increased binding affinity to EphA10 and ADC antibodies (A7_ADC and 2W_ADC) targeting EphA10 prepared using the same to EphA10.

[0053] Figure 21 Figure showing killing efficacy of A7_ADC and 2W_ADC on cancer cells.

[0054] Figure 22 Figure showing results of quantitative binding constant analysis of humanized antibodies A7 and 2W with increased binding affinity to EphA10 to EphA10.

[0055] Figure 23A graph for confirming the size and purity of a fusion protein comprising four mouse EphA10 antigens expressed in animal cells and purified using SDS-PAGE gel and analyzing the activity thereof by ELISA.

[0056] Figure 24 A graph of the results of ELISA analysis of the binding ability of humanized antibodies A7 and 2W, which have increased binding ability to EphA10, to mouse EphA10. DETAILED DESCRIPTION

[0057] Hereinafter, the present application will be described in detail with reference to the accompanying drawings and examples of the present application. However, the following examples are only for exemplification of the present application, and if specific description of techniques or structures well known to those skilled in the art is considered to unnecessarily obscure the main idea of the present application, the detailed description thereof can be omitted, and the present application is not limited thereto. The present application can be variously modified and applied within the scope of the claims and their equivalent interpretations.

[0058] Further, the terminology used in the present specification is intended to describe only one preferred embodiment of the present application and can vary according to the intention of a user or an operator, or a custom in the field of the present application, etc. Therefore, the definition of the terminology should be made based on the content of the entire specification. In the entire specification, when a certain part "comprises" a certain structural element, unless otherwise explicitly stated, this does not mean that other structural elements are excluded, but can further include other structural elements.

[0059] Unless otherwise defined, all technical terms used in the present application have the same meanings as those generally understood by those skilled in the art. Although preferred methods and samples are described in the present specification, similar or equivalent methods and samples are also included in the scope of the present application. The contents of all publications cited as references in the present specification are incorporated by reference into the present application.

[0060] In the entire specification, the conventional one-letter and three-letter codes of natural amino acids, and the generally accepted three-letter codes of other amino acids such as Aib (alpha-aminoisobutyric acid) and Sar (N-methylglycine, N-methyl glycine) are used. In addition, the amino acids mentioned in abbreviations in the present application are described according to the IUPAC-IUB nomenclature as follows:

[0061] Alanine: A, Arginine: R, Asparagine: N, Aspartic acid: D, Cysteine: C, Glutamic acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine: M, Phenylalanine: F, Proline: P, Serine: S, Threonine: T, Tryptophan: W, Tyrosine: Y, and Valine: V.

[0062] In one aspect, the present application relates to an antibody specific to a Hepatocyte growth factor receptor A10 (Eph receptor A10, EphA10) or an immunologically active fragment thereof.

[0063] In one example, the EphA10 can be human EphA10 or mouse EphA10.

[0064] In one example, the antibody or an immunologically active fragment thereof of the present application can specifically bind to an Extra Cellular Domain (ECD) of EphA10 or a FNIII domain of the ECD.

[0065] In one example, the antibody or an immunologically active fragment thereof of the present application can comprise a VH domain comprising: a CDRH1 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 1 to 3; a CDRH2 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 4 to 6; and a CDRH3 comprising an amino acid sequence of SEQ ID NO: 7 or 8.

[0066] In one example, the antibody or an immunologically active fragment thereof of the present application can comprise a VL domain comprising: a CDRL1 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 9 to 14; a CDRL2 comprising an amino acid sequence of SEQ ID NO: 15 or 16; and a CDRL3 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 17 to 19.

[0067] In one example, the antibody or an immunologically active fragment thereof of the present application can comprise a VH domain comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 20 to 25.

[0068] In one example, the antibody or an immunologically active fragment thereof of the present application can comprise a VL domain comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 26 to 45.

[0069] In one example, the antibody 1-1 of the present application can comprise: a VH domain comprising an amino acid sequence of SEQ ID NO: 20; and a VL domain comprising an amino acid sequence of SEQ ID NO: 26.

[0070] In one example, the antibody 1-2 of the application can comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain comprising the amino acid sequence of SEQ ID NO: 27.

[0071] In one example, the antibody A.3 of the application can comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 21 ; and a VL domain comprising the amino acid sequence of SEQ ID NO: 28.

[0072] In one example, the antibody A.7 of the application can comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain comprising the amino acid sequence of SEQ ID NO: 29.

[0073] In one example, the antibody A.8 of the application can comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain comprising the amino acid sequence of SEQ ID NO: 30.

[0074] In one example, the antibody B.1 of the application can comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 22; and a VL domain comprising the amino acid sequence of SEQ ID NO: 31.

[0075] In one example, the antibody B.3 of the application can comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 23; and a VL domain comprising the amino acid sequence of SEQ ID NO: 32.

[0076] In one example, the antibody B.7 of the application can comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 24; and a VL domain comprising the amino acid sequence of SEQ ID NO: 33.

[0077] In one example, the antibody 2.1 of the application can comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 25; and a VL domain comprising the amino acid sequence of SEQ ID NO: 34.

[0078] In one example, the antibody 2.3 of the application can comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain comprising the amino acid sequence of SEQ ID NO: 35.

[0079] In one example, the antibody 1-A of the present application can comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain comprising the amino acid sequence of SEQ ID NO: 36.

[0080] In one example, the antibody 1-N of the present application can comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain comprising the amino acid sequence of SEQ ID NO: 37.

[0081] In one example, the antibody 1-R of the present application can comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain comprising the amino acid sequence of SEQ ID NO: 38.

[0082] In one example, the antibody 1-W of the present application can comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain comprising the amino acid sequence of SEQ ID NO: 39.

[0083] In one example, the antibody 1-Y of the present application can comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain comprising the amino acid sequence of SEQ ID NO: 40.

[0084] In one example, the antibody 2-A of the present application can comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain comprising the amino acid sequence of SEQ ID NO: 41.

[0085] In one example, the antibody 2-W of the present application can comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain comprising the amino acid sequence of SEQ ID NO: 42.

[0086] In one example, the antibody 2-N of the present application can comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain comprising the amino acid sequence of SEQ ID NO: 43.

[0087] In one example, the antibody 2-R of the present application can comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain comprising the amino acid sequence of SEQ ID NO: 44.

[0088] In one example, the antibody 2-Y of the present application can comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain comprising the amino acid sequence of SEQ ID NO: 45.

[0089] In one example, the antibody or an immunologically active fragment thereof of the present application can comprise a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof of human origin.

[0090] In one example, the immunologically active fragment can be any one selected from the group consisting of Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv (single chain fragment variable), Fv, single chain antibody, Fv dimer, complementarity determining region fragment, humanized antibody, chimeric antibody, and diabody, more preferably, a humanized antibody, Fab, or scFv.

[0091] In one example, the antibody of the present application can be a monoclonal antibody.

[0092] The antibody includes not only a whole antibody form but also a functional fragment of an antibody molecule. The whole antibody has a structure of two light chains and two heavy chains each having a full length, and each light chain is connected to a heavy chain by a disulfide bond. The functional fragment of the antibody molecule refers to a fragment having an antigen binding function, and examples of the antibody fragment include: (i) a Fab fragment consisting of a variable region of a light chain (VL) and a variable region of a heavy chain (VH), and a constant region of a light chain (CL) and a first constant region of a heavy chain (CH1); (ii) a Fd fragment consisting of VH and CH1 domains; (iii) a Fv fragment consisting of VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a VH domain (Ward ES et al., Nature 341:544-546 (1989)); (v) an isolated CDR region; (vi) a F(ab')2 fragment which is a bivalent fragment comprising two connected Fab fragments; (vii) a single chain Fv molecule (scFv) in which VH and VL domains are connected by a peptide linker to form an antigen binding site; (viii) a bispecific single chain Fv dimer (PCT / US92 / 09965); and (ix) a diabody which is a multivalent or multispecific fragment prepared by gene fusion (WO94 / 13804), etc.

[0093] The antibody or the immunologically active fragment thereof of the present application can be selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, a human-derived antibody, and immunologically active fragments thereof. The antibody can be produced by recombination or synthesis.

[0094] An animal-derived antibody produced by immunizing an animal with a desired antigen can cause an immune rejection reaction when administered to a human body for therapeutic purposes, and a chimeric antibody has been developed to suppress such an immune rejection reaction. The chimeric antibody is produced by replacing the constant region of an animal-derived antibody that causes an anti-isotype reaction with the constant region of a human-derived antibody using a genetic engineering method. Although the chimeric antibody has a significantly improved anti-isotype reaction compared to the animal-derived antibody, animal-derived amino acids still exist in the variable region, which has a side effect of a potential anti-idiotypic reaction. To improve this side effect, a humanized antibody has been developed. This is prepared by grafting the CDR (complementaritiy determining regions) site in the variable region of the chimeric antibody, which plays a key role in antigen binding, onto the framework of a human-derived antibody.

[0095] Among CDR grafting techniques for preparing a humanized antibody, the most important thing is to select an optimized human-derived antibody that can best accommodate the CDR site of an animal-derived antibody, and for this purpose, an antibody database, crystal structure analysis, and molecular modeling techniques, etc. are widely used. However, even if the CDR site of an animal-derived antibody is grafted onto the framework of an optimized human-derived antibody, there are many cases where the antigen binding ability cannot be maintained due to the presence of amino acids that affect antigen binding in the framework of the animal-derived antibody, and thus it can be said that the application of an additional antibody engineering technique to restore the antigen binding ability is essential.

[0096] The antibody or the immunologically active fragment thereof can be isolated from an organism (not present in an organism) or non-naturally occurring (non-naturally occurring), for example, can be produced by synthesis or recombination.

[0097] In the present invention, the term "antibody" refers to a substance produced by the immune system upon stimulation by an antigen, and the type of antibody is not particularly limited, and can be naturally obtained or non-naturally obtained (e.g., synthetic or recombinant). Antibodies are highly stable in vivo and in vitro, and have a long half-life, and thus are advantageous for large-scale expression and production. Also, antibodies have very high avidity because they essentially have a dimer structure. A complete antibody has a structure comprising two full-length light chains and two full-length heavy chains, and each light chain is connected to a heavy chain by a disulfide bond. The constant region of the antibody is divided into a heavy chain constant region and a light chain constant region. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and has gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2) subtypes. The constant region of the light chain has kappa (κ) and lambda (λ) types.

[0098] In the present invention, the term "heavy chain" is understood to mean a full-length heavy chain and fragments thereof, including: a variable region domain VH comprising an amino acid sequence having a variable region sequence sufficient to impart antigen specificity; three constant region domains CH1, CH2, and CH3; and a hinge. In addition, the term "light chain" is understood to mean a full-length light chain and fragments thereof, including: a variable region domain VL comprising an amino acid sequence having a variable region sequence sufficient to impart antigen specificity; and a constant region domain CL.

[0099] In the present invention, the term "variable region or variable domain" refers to a portion of an antibody molecule that exhibits a variety of sequence variations in performing the function of specifically binding to an antigen, and there are complementarity determining regions CDR1, CDR2, and CDR3 in the variable region. There are framework region (FR) portions that support the CDR loops between the CDRs. The "complementarity determining region" is a loop region involved in antigen recognition, and the specificity of the antibody for the antigen is determined by the sequence change in this region.

[0100] In the present invention, the term "scFv (single chain fragment variable)" refers to a single-chain antibody produced by expressing only the variable region of an antibody through genetic recombination, and refers to an antibody in a single-chain form in which the VH region and the VL region of an antibody are connected by a short peptide chain. Unless otherwise specified or otherwise understood from the context, the term "scFv" is intended to encompass scFv fragments including antigen-binding fragments. This is obvious to one skilled in the art.

[0101] In the present application, the term "complementarity determining region (CDR)" refers to the amino acid sequences of the hypervariable regions of the heavy and light chains of immunoglobulins. The heavy and light chains can each comprise three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3, respectively). The CDRs can provide important contact residues for the binding of an antibody to an antigen or antigenic determinant.

[0102] In the present application, the term "specifically binds" or "specifically recognizes" has the same meaning as commonly known by those skilled in the art, and refers to the specific interaction of an antigen and an antibody to cause an immune response.

[0103] In the present application, the term "antigen-binding fragment" refers to a fragment of an intact immunoglobulin structure, and refers to a polypeptide portion that comprises a portion capable of binding to an antigen. For example, it can be a scFv, (scFv)2, scFv-Fc, Fab, Fab', or F(ab')2, but is not limited thereto. In the antigen-binding fragment, Fab has a structure comprising a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (CH1) 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 CH1 domain of the heavy chain. F(ab')2 antibodies are produced by a disulfide bond between the cysteine residues of the hinge region of Fab'. Fv is the smallest antibody fragment comprising only a heavy chain variable region and a light chain variable region, and recombinant techniques for producing Fv fragments are widely known in the art. In two-chain Fv, the heavy chain variable region and the light chain variable region are connected by a non-covalent bond, and in single-chain Fv, the heavy chain variable region and the single chain variable region are generally covalently connected by a peptide linker or directly connected at the C-terminus, so that they can form a dimer-like structure like two-chain Fv. The linker can be a peptide linker consisting of 1 to 100 or 2 to 50 arbitrary amino acids, and suitable sequences are well known in the art. The antigen-binding fragment can be obtained using a proteolytic enzyme (for example, Fab fragments can be obtained by limited digestion of an intact antibody with papain, and F(ab')2 fragments can be obtained by digestion with pepsin), or can be prepared using genetic recombination techniques.

[0104] In the present application, the term "hinge region" refers to a region in the heavy chain of an antibody between the CH1 region and the CH2 region, which functions to provide flexibility to the antigen binding site within the antibody. For example, the hinge region can be derived from a human antibody, specifically, from IgA, IgE or IgG, for example, IgGl, IgG2, IgG3 or IgG4.

[0105] In the present application, the term "amino acid modification / variation" refers to substitution, insertion and / or deletion, preferably substitution, of an amino acid in a polypeptide sequence. The term "amino acid substitution" or "substitution" used in the present application refers to replacement of an amino acid at a specific position in a polypeptide sequence of an antibody with another amino acid.

[0106] The antibody of the present application or an immunologically active fragment thereof can be prepared by any method known in the art. The polynucleotide sequence encoding the antibody of the present application or an immunologically active fragment thereof, if necessary, can be cloned into a host cell to form an expression and identified nucleic acid. Various methods for this purpose are described in the literature (Molecular Cloning - A Laboratory Manual, 3rd Ed., Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001; Current Protocols in Molecular Biology, John Wiley & Sons).

[0107] The nucleic acid encoding the antibody of the present application or an immunologically active fragment thereof can be inserted into an expression vector for protein expression. The expression vector generally contains a protein operably linked (i.e., functionally linked) to a regulatory sequence, a selection marker, any fusion partner and / or additional elements. The antibody of the present application or an immunologically active fragment thereof can be produced by culturing a host cell transformed with the nucleic acid, preferably an expression vector containing a nucleic acid encoding the antibody of the present application or an immunologically active fragment thereof, under appropriate conditions, and inducing protein expression by a method that induces protein expression. Various suitable host cells can be used, including but not limited to mammalian cells, bacteria, insect cells and yeast. Methods for introducing foreign nucleic acids into host cells are well known in the art and will vary depending on the host cell used. Preferably, Escherichia coli, which is low in production cost and high in industrial value, is used as a host cell to produce the antibody of the present application or an immunologically active fragment thereof.

[0108] Accordingly, the scope of the present application includes a method of producing an EphA10 specific antibody or an immunologically active fragment thereof, comprising the steps of culturing a host cell into which a nucleic acid encoding the antibody or the immunologically active fragment thereof of the present application has been introduced under conditions suitable for protein expression; and purifying or isolating the antibody or the immunologically active fragment thereof expressed by the host cell.

[0109] The antibody can be isolated or purified using various methods known in the art. Standard purification methods include chromatographic techniques, electrophoresis, immunization, precipitation, dialysis, filtration, concentration, and chromatofocusing techniques. As is well known in the art, various natural proteins, such as bacterial proteins A, G, and L, can bind to antibodies and be used for purification. In general, purification using a specific fusion partner is also possible.

[0110] In one aspect, the present application relates to an antibody-drug conjugate (ADC) comprising the Ephrin A Receptor 10 specific antibody or the immunologically active fragment thereof of the present application and a drug.

[0111] In one example, the drug can be an immunogenic cell death inducer, a pro-apoptotic peptide, a microtubulin structure formation inhibitor, a mitosis inhibitor, a topoisomerase inhibitor, a DNA intercalator, a toxin, a radionuclide, or an anti-cancer agent.

[0112] In one example, the pro-apoptotic peptide can be selected from the group consisting of KLA, alpha-defensin-1, BMAP-28, Brevenin-2R, Buforin IIb, cecropin A-Magainin 2 (CA-MA-2), cecropin A, cecropin B, chrysophsin-1, D-K6L9, Gomesin, Lactoferricin B, LLL27, LTX-315, Magainin 2, Magainin II-bombesin conjugate (MG2B), Pardaxin, and combinations thereof.

[0113] In one example, the immunogenic cell death inducer can be selected from the group consisting of an anthracycline anticancer agent, a taxane anticancer agent, an anti-EGFR antibody, a BK channel agonist, Bortezomib, a cardiac glycoside, a cyclophosphamide anticancer agent, a GADD34 / PP1 inhibitor, an LV-tSMAC, a Measles virus, bleomycin, mitoxantrone, oxaliplatin, and combinations thereof.

[0114] In one example, the anti-cancer agent can be selected from the group consisting of 7- Ethyl-10-hydroxy-camptothecin (SN-38), daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, valrubicin, paclitaxel, docetaxel, mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, carboplatin, dactinomycin (actinomycin D), plicamycin, mitomycin C, methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, thioguanine, hydroxyurea, asparaginase, mitotane, aminoglutethimide, estramustine, leuprolide, goserelin, megestrol, medroxyprogesterone, tamoxifen, raloxifene, fulvestrant, anastrozole, letrozole, trastuzumab, bevacizumab, sunitinib, sorafenib, imatinib, gefitinib, erlotinib, crizotinib, lapatinib, vandetanib, afatinib, dasatinib, bosutinib, nilotinib, vemurafenib, dabrafenib, encorafenib, dabrafenib, encorafenib, regorafenib, cediranib, ramucirumab, nivolumab, pembrolizumab, atezolizumab, avelumab, ipilimumab, tremelimumab, blinatumomab, and any combination thereof.C) vincristine, vinblastine, teniposide, topotecan, iridotecan, uramustine, melphalan, bendamustine, dacarbazine, temozolomide, altretamine, duocarmycin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine, etoposide, mitoxantrone, izabepilone, vindesine, vinorelbine, estramustine, maytansine, mertansine (DM1), DM4, dolastatin, auristatin E, auristatin F, monomethyl auristatin E (MMAE), monomethyl auristatin F, and derivatives thereof.

[0115] In one example, the anti-cancer agent can be an immune anti-cancer agent, which can be an immune checkpoint inhibitor, an immune suppressor modulator, a cancer vaccine, an immunoadjuvant, an immune cell for treating cancer, an immune cell activity cofactor, an antibody for treating cancer, or a cytokine required for maintaining immune cell activity for treating cancer.

[0116] In one example, the immune suppressor modulator can be a drug that reduces the level of regulatory T cells (Tregs), which can activate the immune response of T effector cells by reducing regulatory T cells.

[0117] In one example, the immune checkpoint inhibitor can be an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA, or A2aR, and can be an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or a variant thereof.

[0118] In one example, the drug can be selected from the group consisting of 7-Ethyl-10- hydroxy-camptothecin (SN-38), daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, valrubicin, paclitaxel, docetaxel, mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, carboplatin, dactinomycin (actinomycin D), plicamycin, mitomycin C, vincristine, vinblastine, teniposide, topotecan, iridotecan, uramustine, melphalan, bendamustine, dacarbazine, temozolomide, altretamine, duocarmycin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate (triplatin), mitomycin, mitoguazone, procarbazine, estramustine, estramustine phosphate, estramustine sodium phosphate, estramustine diphosphate, estramustine sodium diphosphate, estramustine sodium phosphate, estramustine diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodium diphosphate, estramustine sodiumThe anti-cancer agent can be selected from the group consisting of alkylating agents, such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, uramustine, temozolomide, thiotepa, busulfan, carmustine, lomustine, streptozocin, dacarbazine, oxaliplatin, cisplatin, carboplatin, nedaplatin, triplatin, satraplatin, and derivatives thereof; antimetabolites, such as methotrexate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine, etoposide, mitoxantrone, izabepilone, vindesine, vinorelbine, estramustine, maytansine, mertansine (DM1), DM4, dolastatin, auristatin E, auristatin F, monomethyl auristatin E (MMAE), monomethyl auristatin F, and derivatives thereof.

[0119] In one example, the antibody drug conjugate can be formed by linking / binding the antibody or the immunologically active fragment thereof of the present application with a drug via a linker as a medium, and the linker can be any linker having a functional group capable of binding with an amine group, a carboxyl group, or a sulfhydryl group of a protein (e.g., a peptide or a ligand, an antibody, an antibody fragment), or a phosphate group, a hydroxyl group of a nucleic acid (e.g., an aptamer). The functional group of such a linker can be isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, fluorophenyl ester, hydroxymethyl phosphine, maleimide, haloacetyl, pyridyldisulfide, thiosulfonate, or vinylsulfone, etc. The linker can be a linker cleavable by a protease, a linker cleavable under acidic or basic conditions, a linker cleavable by high temperature or light, a linker cleavable under reducing or oxidizing conditions, or a linker not cleavable under such conditions. Examples of the cleavable linker can include a hydrazone linker cleavable under acidic conditions, a peptide linker cleavable by a protease, and a linker having a disulfide functional group cleavable under reducing conditions, etc., and examples of the non-cleavable linker can include a Maleimidomethyl cyclohexane-1-carboxylate (MCC) linker, a maleimidocaproyl (MC) linker, or a derivative thereof, such as a succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sMCC) linker, or a sulfo-sMCC.

[0120] In addition, the linker can be a self-immolative linker or a traceless linker that leaves no trace after cleavage. The self-immolative linker can include, for example, a linker disclosed in U.S. Patent No. 9,089,614 entitled "Hydrophilic self-immolative linkers and conjugates thereof," a linker disclosed in International Publication No. WO2015038426 entitled "SELF-IMMOLATIVE LINKERS CONTAINING MANDELIC ACID DERIVATIVES, DRUG-LIGAND CONJUGATES FOR TARGETED THERAPIES AND USES THEREOF," and the like. The traceless linker can include a phenylhydrazine linker, an aryltriazene linker, and a linker disclosed in the literature [Blaney, et al., "Traceless solid-phase organic synthesis," Chem Rev. 102: 2607-2024 (2002)], and the like.

[0121] The antibody or the immunologically active fragment thereof of the present application of the antibody drug conjugate of the present application can be bound by a biocompatible polymer as a medium or a carrier. The biocompatible polymer refers to a polymer having tissue compatibility and blood compatibility, which does not cause tissue necrosis or blood coagulation when it comes into contact with living tissue or blood. As the synthetic polymer of the biocompatible polymer, there are polyesters, polyhydroxyalkanoates (PHAs), poly(α-hydroxy acid), poly(β-hydroxy acid), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(3-hydroxypropionate) (PHP), poly(3-hydroxyhexanoate) (PHH), poly(4-hydroxy acid), poly(4-hydroxybutyrate), poly(4-hydroxyvalerate), poly(4-hydroxyhexanoate), poly(ester amide), polycaprolactone, polylactide, polyglycolide, poly(lactic-co-glycolic acid) (PLGA), polydioxanone, polyortho ester, polyanhydride, poly(glycolic acid-co-trimethylene carbonate), polyphosphoester, polyphosphoester urethane, poly(amino acid), polycyanoacrylate, poly(trimethylene carbonate), poly(imino carbonate), poly(tyrosine carbonate), polycarbonate, poly(tyrosine aryl ester), polyalkylene oxalate, polyphosphazene, PHA-PEG, ethylene-vinyl alcohol copolymer (EVOH), polyurethane, silica gel, polyester, polyolefin, polyisobutylene and ethylene-alpha olefin copolymer, styrene-isobutylene-styrene triblock copolymer, acrylic polymer and copolymer, halogen vinyl polymer and copolymer, polyvinyl chloride, polyvinyl ether, polyvinyl methyl ether, polyvinylidene halide, polyvinylidene fluoride, polyvinylidene chloride, polyfluoroolefin, polyperfluoroolefin, polyacrylonitrile, polyvinyl ketone, polyvinyl aromatic, polystyrene, polyvinyl ester, polyvinyl acetate, ethylene-methyl methacrylate copolymer, acrylonitrile-styrene copolymer, ABS resin, ethylene-vinyl acetate copolymer, polyamide, alkyd resin, polyformaldehyde, polyimide, polyether, polyacrylate, polymethacrylate, polyacrylic acid-maleic acid copolymer, or polyaminoamine, and the natural polymer is chitosan, dextran, cellulose, heparin, hyaluronic acid, alginate, inulin, starch, or glycogen.

[0122] In one example, the antibody drug conjugate can further comprise an ADC linker, which can be 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxy carbonyl (PAB), N-succinimidyl-4-(2-pyridyl dithio) valerate (SPP), N-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), valine-citrulline-p-aminobenzyloxy carbonyl (val-cit-PAB), or N-succinimidyl-(4-iodoacetyl) aminobenzoate (SIAB).

[0123] In one example, the antibody drug conjugate, the hepatoma A receptor 10 specific antibody or the immunologically active fragment thereof of the present application can form a complex with a drug through an ADC linker.

[0124] In one example, the hepatoma A receptor 10 specific antibody or the immunologically active fragment thereof of the present application can be further conjugated with RNA, DNA, antibody, effector, drug, prodrug, toxin, peptide, or delivery molecule (see Shoari et al., Pharmaceutics 13: 1391, pp. 1-32 (2021)).

[0125] In one aspect, the present application relates to a bispecific or multispecific antibody comprising the hepatoma A receptor 10 specific antibody or the immunologically active fragment thereof of the present application, and a moiety that binds to a target antigen other than hepatoma A receptor 10.

[0126] In one example, the moiety that binds to a target antigen can comprise an antibody or an immunologically active fragment thereof.

[0127] In one example, the target antigen can be one or more selected from the group consisting of 17-1A antigen, GD3 ganglioside R24, EGFRvIII, PSMA, PSCA, HLA-DR, EpCAM, MUC1 core protein, aberrantly glycosylated MUC1, fibronectin isoform containing ED-B domain, HER2 / neu, carcinoembryonic antigen (CEA), gastrin-releasing peptide (GRP) receptor antigen, mucin (mucine) antigen, epidermal growth factor receptor (EGF-R), HER3, HER4, MAGE antigen, SART antigen, MUC1 antigen, c-erb-2 antigen, TAG72, carbonic anhydrase IX, alpha-fetoprotein, A3, A33 antibody specific antigen, Ba 733, BrE3-antigen, CA125, CDl, CDla, CD3, CD5, CDl5, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD74, CD79a, CD80, CD138, colon-specific antigen-p (CSAp), CSAp, EGP-1, EGP-2, Ep-CAM, Fit-1, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and subunits thereof, hypoxia-inducible factor (HIF-I), Ia, IL-2, IL-6, IL-8, insulin-like growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, macrophage migration inhibitory factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, PAM-4 antibody specific antigen, placental growth factor, p53, prostatic acid phosphatase, PSA, RS5, SlOO, TAC, tenascin, TRAIL receptor, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigens, VEGF, ED-B fibronectin, angiogenesis marker, oncogene marker, or oncogene product.

[0128] In one example, the apoptosis-associated gene can be ABL1, AKT1, AKT2, BARD1, BAX, BCL11B, BCL2, BCL2A1, BCL2L1, BCL2L12, BCL3, BCL6, BIRC2, BIRC3, BIRC5, BRAF, CARD11, CAV1, CBL, CDC25A, CDKN1A, CFLAR, CNR2, CTNNB1, CUL4A, DAXX, DDIT3, E2F1, E2F3, E2F5, ESPL1, FOXO1, HDAC1, HSPA5, IGF1R, IGF2, JUN, JUNB, JUND, MALT1, MAP3K7, MCL1, MDM2, MDM4, MYB, MYC, NFKB2, NPM1, NTRK1, PAK1, PAX3, PML, PRKCA, PRKCE, PTK2B, RAF1, RHOA, TGFB1, TNFRSF1B, TP73, TRAF6, YWHAG, YWHAQ, or YWHAZ.The transcription factor gene can be AR, ARID3A, ASCL1, ATF1, ATF3, BCL11A, BCL11B, BCL3, BCL6, CDC5L, CDX2, CREB1, CUX1, DDIT3, DLX5, E2F1, E2F3, E2F5, ELF4, ELK1, ELK3, EN2, ERG, ETS1, ETS2, ETV1, ETV3, ETV4, ETV6, FEV, FEZF1, FLI1, FOS, FOSL1, FOXA1, FOXG1, FOXM1, FOXO1, FOXP1, FOXQ1, GATA1, GATA6, GFI1, GFI1B, GLI1, GLI2, GLI3, HES6, HHEX, HLF, HMGA1, HMGA2, HOXA1, HOXA9, HOXD13, HOXD9, ID1, ID2, IKZF1, IRF2, IRF4, JUN, JUNB, JUND, KAT6A, KDM2A, KDM5B, KLF2, KLF4, KLF5, KLF6, KLF8, KMT2A, LEF1, LHX1, LMX1B, MAF, MAFA, MAFB, MBD1, MECOM, MEF2C, MEIS1, MITF, MYB, MYC, MYCL, MYCN, NANOG, NCOA3, NFIB, NFKB2, NKX2-1, OTX2, PATZ1, PAX2, PAX3, PAX4, PAX8, PBX1, PBX2, PITX2, PLAG1, PLAGL2, PPARG, PPP1R13L, PRDM10, PRDM13, PRDM14, PRDM15, PRDM16, PRDM6, PRDM8, PRDM9, RARA, REL, RERE, RUNX1, RUNX3, SALL4, SATB1, SFPQ, SIX1, SNAI1, SOX2, SOX4, SPI1, SREBF1, STAT3, TAF1, TAL1, TAL2, TBX2, TBX3, TCF3, TFCP2, TFE3, THRA, TLX1, TP63, TP73, TWIST1, WT1, YBX1, YY1, ZBTB16, ZBTB7A, ZIC2, ZNF217, or ZNF268.The metastasis-associated gene can be AKT1, AKT2, AR, CBL, CDH1, CRK, CSF1, CTNNB1, CTTN, CXCR4, EGFR, FGFR1, FLT3, FYN, GLI1, ILK, ITGA3, JAK2, MET, PDGFRB, PLXNB1, PRKCI, PTCH1, PTPN11, RAC1, RHOA, RHOC, ROCK1, SMO, SNAI1, SRC, TCF3, or WT1; the angiogenesis-associated gene can be BRAF, CAV1, CTGF, EGFR, ERBB2, ETS1, FGF4, FGF6, FGFR1, FGFR3, FGFR4, ID1, NRAS, PDGFB, PDGFRA, PDGFRB, or SPARC; and the tyrosine kinase gene can be ABL1, ABL2, ALK, AXL, BLK, EGFR, EPHA2, ERBB2, ERBB3, ERBB4, FES, FGFR1, FGFR2, FGFR3, FGFR4, FGR, FLT3, FYN, ITK, JAK1, JAK2, KIT, LCK, MERTK, MET, MST1R, NTRK1, NTRK3, PDGFRA, PDGFRB, PTK2B, PTK7, RET, ROS1, SRC, SYK, TEC, or YES1.

[0129] In one example, the oncogene can be selected from the group consisting of SEPTIN9, ACOD1, ACTN4, ADAM28, ADAM9, ADGRF1, ADRBK2, AFF1, AFF3, AGAP2, AGFG1, AGRN, AHCYL1, AHI1, AIMP2, AKAP13, AKAP9, AKIRIN2, AKTIP, ALDH1A1, ALL1, ANIB1, ANP32C, ANP32D, AQP1, ARAF, ARHGEF1, ARHGEF2, ARHGEF5, ASPSCR1, AURKA, BAALC, BAIAP2L1, BANP, BCAR4, BCKDHB, BCL9, BCL9L, BCR, BMI1, BMP7, BOC, BRD4, BRF2, CABIN1, CAMK1D, CAPG, CBFB, CBLB, CBLL1, CBX7, CBX8, CCDC28A, CCDC6, CCNB1, CCNB2, CCND1, CCNE1, CCNL1, CD24, CDC25C, CDC6, CDH17, CDK1, CDK14, CDK4, CDK5R2, CDK6, CDK8, CDKN1B, CDKN3, CDON, CEACAM6, CENPW, CHD1L, CHIC1, CHL1, CKS1B, CMC4, CNTN2, COPS3, COPS5, CRKL, CRLF2, CROT, CRTC1, CRYAB, CSF1R, CSF3, CSF3R, CSNK2A1, CSNK2A2, CT45A1, CTBP2, CTNND2, CTSZ, CUL7, CXCL1, CXCL2, CXCL3, CYGB, CYP24A1, DCD, DCUN1D1DDB2, DDHD2, DDX6, DEK, DIS3, DNPH1, DPPA2, DPPA4, DSG3, DUSP12, DUSP26, ECHS1, ECT2, EEF1A1, EEF1A2, EEF1D, EIF3E, EIF3I, EIF4E, EIF5A2, ELAVL1, ELL, EML4, EMSY, ENTPD5, EPCAM, EPS8, ERAS, ERGIC1, ERVW-1, EVI2A, EVI5, EWSR1, EZH2, FAM189B, FAM72A, FAM83D, FASN, FDPS, FGF10, FGF3, FGF5, FGF8, FR1OP, FHL2, FIP1L1, FNDC3B, FRAT1, FUBP1, FUS, FZD2, GAB2, GAEC1, GALNT10, GALR2, GLO1, GMNN, GNA12, GNA13, GNAI2, GNAQ,GOLPH3, GOPC, GPAT4, GPM6A, GPM6B, GPR132, GREM1, GRM1, GSK3A, GSM1, H19, HAS1, HAX1, HDGFRP2, HMGN5, HNRNPA1, HOTAIR, HOTTIP, HOXA-AS2, HRAS, HSPA1A, HSPA4, HSPB1, HULC, IDH1, IFNG, IGF2BP1, IKBKE, IL7R, INPPL1, INTS1, INTS2, INTS3, INTS4, INTS5, INTS7, INTS8, IRS2, IST1, JUP, KDM4C, KIAA0101, KIAA1524, KIF14, KRAS, KSR2, LAMTOR5, LAPTM4B, LCN2, LDHB, LETMD1, LIN28A, LIN28B, LMO1, LMO2, LMO3, LMO4, LSM1, LUADT1, MACC1, MACROD1, MAGEA11, MALAT1, MAML2, MAP3K8, MAPRE1, MAS1, MCC, MCF2, MCF2L, MCTS1, MEFV, MFHAS1, MFNG, MIEN1, MINA, MKL2, MLANA, MLLT1, MLLT11, MLLT3, MLLT4, MMP12, MMS22L, MN1, MNAT1, MOS, MPL, MPST, MRAS, MRE11A, MSI1, MTCP1, MTDH, MTOR, MUC1, MUC4, MUM1, MYD88, NAAA, NANOGP8, NBPF12, NCOA4, NEAT1, NECTIN4, NEDD4, NEDD9, NET1, NINL, NME1, NOTCH1, NOTCH4, NOV, NSD1, NUAK2, NUP214, NUP98, NUTM1, OLR1, PA2G4, PADI2, PAK7, PARK7, PARM1, PBK, PCAT1, PCAT5, PDGFA, PDZK1IP1, PELP1, PFN1P3, PIGU, PIK3CA, PIK3R1, PIM1, PIM2, PIM3, PIR, PIWIL1, PLAC8, PLK1, PPM1D, PPP1R10, PPP1R14A, PPP2R1A, PRAME, PRDM12, PRMT5, PSIP1, PSMD10, PTCH2, PTMA, PTP4A1, PTP4A2, PTP4A3, PTTG1, PTTG1IP, PTTG2, PVT1, RAB11A, RAB18, RAB22A, RAB23, RAB8A, RALGDS,RAP1A, RASSF1, RBM14, RBM15, RBM3, RBMY1A1, RFC3, RGL4, RGR, RHO, RING1, RINT1, RIT1, RNF43, RPL23, RRAS, RRAS2, RSF1, RUNX1T1, S100A4, S100A7, S100A8, SAG, SART3, SBSN, SEA, SEC62, SERTAD1, SERTAD2, SERTAD3, SET, SETBP1, SETDB1, SGK1, SIRT1, SIRT6, SKI, SKIL, SKP2, SLC12A5, SLC3A2, SMR3B, SMURF1, SNCG, SNORA59A, SNORA80E, SPAG9, SPATA4, SPRY2, SQSTM1, SRSF1, SRSF2, SRSF3, SRSF6, SS18, SSX1, SSX2, SSX2B, STIL, STMN1, STRA6, STYK1, SUZ12, SWAP70, SYT1, TAC1, TACSTD2, TAF15, TALDO1, TAZ, TBC1D1, TBC1D15, TBC1D3, TBC1D3C, TBC1D7, TCL1A, TCL1B, TCL6, TCP1, TFG, TGM3, TINCR, TKTL1, TLE1, TMEM140, TMPOP2, TMPRSS2, TNS4, TPD52, TPR, TRE17, TREH, TRIB1, TRIB2, TRIM28, TRIM32, TRIM8, TRIO, TRIP6, TSPAN1, TSPY1, TXN, TYMS, TYRP1, UBE2C, UBE3C, UCA1, UCHL1, UHRF1, URI1, USP22, USP4, USP6, VAV1, VAV2, VAV3, VIM, WAPL, WHSC1, WHSC1L1, WISP1, WNT1, WNT10A, WNT10B, WNT2, WNT3, WNT5A, WWTR1, XCL1, XIAP, YAP1, YEATS4, YY1AP1, ZEB1-AS1, ZFAND4, ZFAS1, ZMYM2, ZNF703, and ZNHIT6.

[0130] In one example, the target antigen can be a cell surface antigen or a self-antigen.

[0131] In one example, the cell surface antigen can be one or more selected from the group consisting of CEA, ED-B fibronectin, CD20, CD22, CD19, EGFR, IGFlR, VEFGRl / Flt-1, VEGFR2 / KDR, VEGRF3 / Flt-4, HER2 / neu, CD30, CD33, CD3, CD16, CD64, CD89, CD2, adenovirus fiber knob, PfMSP-1, HN / NDV, EpCAM / 17-lA, hTR, IL-2R / Tac, CA19-9, MUC1, HLA class II, GD2, G250, TAG-72, PSMA, CEACAM6, HMWMAA, CD40, M13 coat protein, and GPIIb / IIIa.

[0132] In one aspect, the present application relates to an isolated nucleic acid molecule encoding an antibody of the present application or an immunologically active fragment thereof, or a bispecific or multispecific antibody of the present application, a vector comprising the nucleic acid molecule, a host cell transformed with the vector.

[0133] A nucleic acid molecule of the present application can be isolated or recombinant, including DNA and RNA in both single- and double-stranded form, and the corresponding complementary sequences. When an isolated nucleic acid is one that is isolated from a naturally occurring source, the nucleic acid is one that is separated from a surrounding gene sequences present in the individual genome from which the isolated nucleic acid is derived. For a nucleic acid synthesized by enzymatic or chemical means from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide, the nucleic acid produced by such methods is understood to be an isolated nucleic acid molecule. An isolated nucleic acid molecule is one which is in the form of a separate fragment or as a component of a larger nucleic acid construct. A nucleic acid is in operable connection when it is in a functional relationship with another nucleic acid sequence. For example, a DNA for a presequence or secretion leader sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein (i.e., a form of the polypeptide prior to secretion); a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked with a coding sequence if its position favors translation. Operably linked generally means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in the same reading frame. However, enhancers need not be contiguous. Linking can be accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0134] The isolated nucleic acid molecule encoding the antibody of the present application or an immunologically active fragment thereof, or the bispecific or multispecific antibody of the present application can be modified in various ways in the coding region, as long as the modification does not change the amino acid sequence of the antibody expressed from the coding region due to the degeneracy of codons or in consideration of the preferred codons for expression of the antibody in an organism, and various modifications or changes can be made to the portion other than the coding region within the range not affecting the gene expression, and it will be understood by those skilled in the art that such modified genes are also included within the scope of the present application. That is, the nucleic acid molecule of the present application can be mutated by substitution, deletion, insertion of one or more nucleic acid bases, or a combination thereof, as long as it encodes a protein having equivalent activity thereto, and these are also included within the scope of the present application. The sequence of such nucleic acid molecule can be single-stranded or double-stranded, and can be a DNA molecule or an RNA (mRNA) molecule.

[0135] The isolated nucleic acid molecule encoding the antibody of the present application or an immunologically active fragment thereof, or the bispecific or multispecific antibody of the present application can be inserted into an expression vector for protein expression. The expression vector generally comprises a protein operably linked (i.e., functionally linked) to a regulatory sequence, a selection marker, any fusion partner, and / or additional elements. The antibody of the present application or an immunologically active fragment thereof, or the bispecific or multispecific antibody of the present application can be produced by a method of inducing protein expression by culturing a host cell transformed with the nucleic acid, preferably an expression vector containing the isolated nucleic acid molecule encoding the antibody of the present application or an immunologically active fragment thereof, or the bispecific or multispecific antibody of the present application, under appropriate conditions. Various suitable host cells can be used, including but not limited to mammalian cells, bacteria, insect cells, and yeast. Methods of introducing foreign nucleic acids into host cells are well known in the art and will vary depending on the host cell used. Preferably, Escherichia coli, which is low in production cost and high in industrial value, is used as a host cell to produce the antibody of the present application or an immunologically active fragment thereof, or the bispecific or multispecific antibody of the present application.

[0136] The vector of the present application includes, but is not limited to, a plasmid vector, a cosmid vector, a phage vector, and a viral vector, etc. A suitable vector contains, in addition to expression regulatory elements such as a promoter, an operator, a start codon, a stop codon, a polyadenylation signal, an enhancer, etc., a signal sequence or a leader sequence for membrane targeting or secretion, which can be prepared in various ways according to the purpose. The promoter of the vector can be a constitutive promoter or an inducible promoter. The signal sequence includes, but is not limited to, a PhoA signal sequence, an OmpA signal sequence, etc. when the host is Escherichia sp. bacteria, an alpha-amylase signal sequence, a subtilisin signal sequence, etc. when the host is Bacillus sp. bacteria, an MF alpha signal sequence, a SUC2 signal sequence, etc. when the host is yeast, and an insulin signal sequence, an alpha-interferon signal sequence, an antibody molecule signal sequence, etc. when the host is an animal cell. In addition, the vector can include a selection marker for selecting a host cell containing the vector, and if it is a replicable expression vector, a replication origin.

[0137] In the present application, the term "vector" refers to a delivery body capable of inserting a nucleic acid sequence and introducing it into a cell capable of replicating the nucleic acid sequence. The nucleic acid sequence can be exogenous or heterologous. Examples of the vector can include, but are not limited to, a plasmid, a cosmid, and a virus (e.g., a phage). A person skilled in the art can construct a vector using standard recombination techniques (Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994, etc.).

[0138] In one example, in making the vector, expression regulatory sequences such as a promoter, a terminator, an enhancer, etc., a sequence for membrane targeting or secretion, etc. can be appropriately selected and combined in various ways according to the type of the host cell used to produce the antibody, as desired.

[0139] In the present invention, the term "expression vector" refers to a vector containing a nucleic acid sequence encoding at least part of a transcribed gene product. In some cases, the RNA molecule is subsequently translated into a protein, polypeptide, or peptide. Expression vectors can contain various regulatory sequences. In addition to regulatory sequences that control transcription and translation, vectors and expression vectors can also contain nucleic acid sequences that serve additional functions.

[0140] In the present invention, the term "host cell" includes eukaryotic and prokaryotic organisms, and refers to any transformable organism that is capable of replicating the vector or expressing a gene encoded by the vector. The host cell can be transfected or transformed with the vector, which refers to a process by which exogenous nucleic acid molecules are transferred or introduced into a host cell.

[0141] In one example, the host cell can be a bacterial or animal cell, and the animal cell bead can be a CHO cell, a HEK cell, or an NSO cell, and the bacteria can be E. coli.

[0142] In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating a disease or disorder, comprising the antibody or the immunologically active fragment thereof of the present invention, an antibody drug conjugate, or a bispecific or multispecific antibody as an effective ingredient, the disease or disorder being selected from the group consisting of an autoimmune disease, a neurodegenerative disease, Alzheimer's disease, a metabolic disease, a cardiovascular disease, atherosclerosis, organ transplant rejection, a disease or symptom caused by a fungus, a virus, a bacterium, or a parasite.

[0143] In one aspect, the present invention relates to a chimeric antigen receptor (CAR) comprising the antibody or the immunologically active fragment thereof of the present invention as an antigen binding domain.

[0144] In one aspect, the present invention relates to a recombinant vector comprising a gene encoding the chimeric antigen receptor.

[0145] In one aspect, the present invention relates to a chimeric antigen receptor-expressing cell transformed with the recombinant vector.

[0146] In one example, the chimeric antigen receptor-expressing cell can be a chimeric antigen receptor-expressing macrophage (CAR-macrophage), a chimeric antigen receptor-expressing T (CAR-T) cell, a chimeric antigen receptor-expressing Gamma-delta T (CAR-Gamma-delta T) cell, or a natural killer (CAR-NK) cell.

[0147] In the present application, the term "chimeric antigen receptor (CAR)" refers to a non-naturally occurring receptor that can impart specificity for a specific antigen to an immune effector cell. In general, the CAR refers to a receptor for transferring the specificity of a monoclonal antibody to a T cell. The CAR is generally composed of an ectodomain, a transmembrane domain, and an endodomain.

[0148] The ectodomain includes an antigen recognition region, and the transmembrane domain of the CAR is connected to the ectodomain, which can be derived from a natural or synthetic source. If derived from a naturally occurring source, it can be derived from a membrane-bound protein or a membrane-permeable protein, and can be a portion derived from a membrane-permeable region of various proteins, such as the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or CD8, etc. The sequence of such a transmembrane domain can be obtained from the literature on the membrane-permeable region portion of the publicly known membrane-permeable protein, but is not limited thereto.

[0149] In addition, if the transmembrane domain is synthetic, it can mainly include hydrophobic amino acid residues, such as leucine and valine, for example, a triplet of phenylalanine, tryptophan, and valine can be present in the synthetic transmembrane domain, but is not limited thereto. The sequence information of such a transmembrane domain can be obtained from the literature on the synthetic transmembrane domain known in the art, but is not limited thereto.

[0150] In the CAR of the present application, the intracellular domain is a part of the CAR domain present in the cell and is connected to the transmembrane domain. The intracellular domain of the present application can comprise an intracellular signaling domain characterized by inducing T cell activation, preferably inducing T cell proliferation, when an antigen binds to the antigen binding site of the CAR. The type of the intracellular signaling domain is not particularly limited as long as it is a part that transmits a signal capable of causing T cell activation when an antibody binds to an antigen binding site present outside the cell, and various types of intracellular signaling domains can be used, examples of which can be based on an immunoreceptor tyrosine-based activation motif or ITAM, and the ITAM includes but is not limited to ITAM derived from CD3ξ (zeta), FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, CD66d, or FcεRIγ.

[0151] The CAR comprises a single-chain fragment variable region (scFv) of a tumor-associated antigen (TAA)-specific antibody coupled to the cytoplasmic domain of a T cell signaling molecule via a hinge region and a transmembrane region. Most conventional lymphocyte activation moieties consist of a T cell costimulatory domain (e.g., CD28, CD137, OX40, ICOS, and CD27) in series with a T cell trigger domain (e.g., CD3ζ). CAR-mediated adoptive immunotherapy allows CAR-transplanted cells to directly recognize TAA on target tumor cells in a non-HLA-restricted manner.

[0152] In the present application, the term "chimeric antigen receptor-expressing T (CAR-T) cell" refers to a T cell expressing a CAR. The chimeric antigen receptor-expressing T (CAR-T) cell has the following advantages: i) recognizes a cancer antigen in a human leukocyte antigen (HLA)-independent manner, thus being able to treat a cancer that escapes the action of an anticancer agent by reducing the expression of cell surface HLA; ii) is independent of the type of HLA, thus being able to be used for treatment regardless of the HLA type of the patient; iii) is able to produce a large number of cancer-specific T cells in a short time, thus being able to exert an excellent anticancer effect.

[0153] The T cell includes a CD4+ T cell (helper T cell, TH cell), a CD8+ T cell (cytotoxic T cell, CTL), a memory T cell, a regulatory T cell (Treg cell), a natural killer T cell, etc., and the T cell into which the CAR is introduced in the present application is preferably a CD8+ T cell, but is not limited thereto.

[0154] In one example, the present application relates to a T-cell engager comprising an antibody or an immunologically active fragment thereof.

[0155] In one example, the T-cell engager can be, for example, a bispecific T-cell engager (BiTE). The BiTE is a class of artificial bispecific monoclonal antibodies that are fusion proteins composed of amino acid sequences from four different genes or single-chain variable fragment (scFv) of two different antibodies on a single peptide chain of about 55 kilodaltons. One scFv binds to T cells through the CD3 receptor and the other scFv binds to tumor cells through a tumor-specific molecule. Like other bispecific antibodies, unlike traditional monoclonal antibodies, BiTE establishes a connection between T cells and tumor cells. This enables T cells to exert cytotoxic activity on tumor cells by producing proteins such as perforin and granzyme, independent of the presence of MHC I or costimulatory molecules. These proteins enter tumor cells and initiate cell death.

[0156] In one aspect, the present application relates to a pharmaceutical composition for preventing or treating cancer, comprising the antibody or an immunologically active fragment thereof, an antibody drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor, a chimeric antigen receptor-expressing cell, or a T-cell engager of the present application as an active ingredient.

[0157] In one example, the cancer can be any one selected from the group consisting of brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, gastric cancer, colon cancer, breast cancer, triple negative breast cancer (TNBC), lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, large intestine cancer, small intestine cancer, rectal cancer, fallopian tube cancer, perianal cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brain stem glioma, and pituitary adenoma, and can be a cancer resistant to an immune checkpoint inhibitor.

[0158] The pharmaceutical composition of the present application can be used as a monotherapy or in combination with other conventional biological therapy, chemotherapy or radiotherapy. Such combination therapy can be more effective in treating cancer. When the present application is used for the prevention and treatment of cancer, the chemotherapeutic agents that can be used in combination with the composition include cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate, etc. The radiotherapy that can be used in combination with the composition of the present application is X-ray irradiation and gamma-ray irradiation, etc.

[0159] In one example, the composition of the present application can further comprise an immunogenic cell death inducer, which can be one or more selected from the group consisting of an anthracycline anticancer agent, a taxane anticancer agent, an anti-EGFR antibody, a BK channel agonist, Bortezomib, a cardiac glycoside, a cyclophosphamide anticancer agent, a GADD34 / PP1 inhibitor, LV-tSMAC, a Measles virus, bleomycin, mitoxantrone or oxaliplatin. The anthracycline anticancer agent can be daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin or valrubicin. The taxane anticancer agent can be paclitaxel or docetaxel.

[0160] The pharmaceutical composition for preventing or treating cancer of the present application can be administered in combination with a chemotherapeutic drug (anti-cancer agent) or the like to enhance the cancer treatment effect of the existing anti-cancer agent through its cancer cell-killing effect. The administration in combination can be simultaneous administration or sequential administration with the anti-cancer agent. Examples of the anti-cancer agent include, but are not limited to, DNA alkylating agents such as mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, and carboplatin; anti-cancer antibiotics such as dactinomycin (actinomycin D), plicamycin, and mitomycin C; and plant alkaloids such as vincristine, vinblastine, etoposide, teniposide, topotecan, and iridotecan, etc.

[0161] In the present application, the term "prevention" refers to any action of inhibiting or delaying the occurrence, spread, and recurrence of cancer by administering the pharmaceutical composition of the present application.

[0162] The term "treatment" used in the present application refers to any action of killing cancer cells or improving or beneficially changing the symptoms of cancer by administering the composition of the present application. The skilled person in the art to which the present application pertains can determine the exact criteria for the effectiveness of the composition of the present application on a disease and judge the degree of improvement, enhancement, and treatment by referring to the materials provided by the Korean Medical Association or the like.

[0163] The term "therapeutically effective amount" in the present invention used in combination with the active ingredient means an amount of the pharmaceutically acceptable salt of the composition effective in preventing or treating the target disease, and the therapeutically effective amount of the composition of the present invention can vary depending on various factors such as administration method, target site, and patient condition. Therefore, the appropriate dose for humans should be determined while taking into account safety and effectiveness. The dose for humans can also be estimated from the effective amount determined through animal experiments. These considerations in determining the effective dose are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

[0164] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" used in the present invention means a dose sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment without causing side effects, and the effective dose level can be determined depending on various factors including the patient's health condition, the type and severity of cancer, the drug activity and sensitivity to the drug, the administration method, the administration time, the administration route and the excretion rate, the treatment period, the combination or simultaneous use of drugs, and other factors known in the medical field. The composition of the present invention can be administered as a separate therapeutic agent, or can be administered in combination with other therapeutic agents, and can be administered sequentially or simultaneously with existing therapeutic agents, or can be administered once or multiple times. Considering all the above factors, it is important to achieve the maximum therapeutic effect with the minimum dose without side effects, which can be easily determined by those skilled in the art.

[0165] The pharmaceutical composition of the present invention can further include a pharmaceutically acceptable additive, and in this case, the pharmaceutically acceptable additive can include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, maltose, acacia, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, glucose, sorbitol, and talc, etc. The pharmaceutically acceptable additive of the present invention is preferably contained in the composition in an amount of 0.1 to 90 parts by weight, but is not limited thereto.

[0166] The composition of the present application can also contain a carrier, diluent, excipient commonly used in biologics, or a combination of two or more thereof. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for the delivery of the composition in vivo, and examples thereof include compounds described in the Merck Index, 13th ed., Merck & Co. Inc., physiological saline, sterile water, Ringer's solution, buffered saline, glucose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components, and other commonly used additives such as antioxidants, buffers, and bacteriostats can be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants can be added to formulate the composition into a primary dosage form such as an aqueous solution, a suspension, an emulsion, etc., a pill, a capsule, a granule, or a tablet. Further, the composition can be formulated according to each disease or ingredient, preferably using a suitable method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0167] The composition of the present application can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or locally in the form of an injection) or orally according to the intended method, and the dosage range depends on the body weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of the disease of the patient. The daily dose of the composition of the present application is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, and more preferably administered once or more times a day.

[0168] Liquid formulations of the composition of the present application for oral administration include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. can be added in addition to simple diluents such as water, liquid paraffin, etc. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, etc.

[0169] In one aspect, the present application relates to an anticancer adjuvant comprising the antibody or an immunologically active fragment thereof, an antibody drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor, a chimeric antigen receptor-expressing cell, or a T cell engager of the present application as an active ingredient.

[0170] In one example, the anticancer adjuvant of the present application can be administered alone or can be administered in combination with an immune anticancer agent simultaneously or sequentially.

[0171] In one example, the anticancer adjuvant of the present application can improve the resistance to an immune anticancer agent.

[0172] In one example, the immune anti-cancer agent can be an immune checkpoint inhibitor, an immune suppressor modulator, a cancer vaccine, an immunoadjuvant, an immune cell for treating cancer, an immune cell activity cofactor, an antibody for treating cancer, or a cytokine required for maintaining immune cell activity for treating cancer.

[0173] In one example, the immune suppressor modulator can be a drug that reduces the level of regulatory T cells (Treg).

[0174] In one example, the immune checkpoint inhibitor can be an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA, or A2aR.

[0175] In one aspect, the present application relates to a composition for diagnosing cancer, comprising the antibody or the immunologically active fragment thereof of the present application, an antibody drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor, a chimeric antigen receptor-expressing cell, or a T cell engager as an effective ingredient.

[0176] In one example, the marker can be a chromogenic enzyme, a radioisotope, a chromopore, a luminescent substance, a fluorescent substance, a probe, or a tag, and the fluorescent substance can be a fluorescent substance of the cyanine (Cy) series, the Rhodamine series, the Alexa series, the BODIPY series, or the ROX series, Nile Red, BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene), cyanine, fluorescein, rhodamine, coumarine, or Alexa.

[0177] In one aspect, the present application relates to a method of producing a ephrin A receptor 10 specific antibody or an immunologically active fragment thereof, comprising: step a) culturing a host cell transformed with a vector comprising an isolated nucleic acid molecule encoding the antibody or the immunologically active fragment thereof of the present application; and step b) recovering the polypeptide expressed by the host cell.

[0178] In one aspect, the present application relates to a method of producing a bispecific or multispecific antibody, comprising: step a) culturing a host cell transformed with a vector comprising an isolated nucleic acid molecule encoding the bispecific or multispecific antibody of the present application; and step b) recovering the polypeptide expressed by the host cell.

[0179] In one example, the purification of the antibody can comprise filtration, HPLC, anion exchange or cation exchange, high performance liquid chromatography (HPLC), affinity chromatography, or a combination thereof, preferably affinity chromatography using Protein A.

[0180] In one aspect, the present application relates to use of the antibody or an immunologically active fragment thereof, an antibody drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor-expressing cell, or a T cell engager of the present application in preventing or treating cancer.

[0181] In one aspect, the present application relates to a method of treating cancer, comprising the step of administering to an individual having cancer a pharmaceutically effective amount of the antibody or an immunologically active fragment thereof, an antibody drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor-expressing cell, or a T cell engager of the present application.

[0182] In one aspect, the present application relates to use of the antibody or an immunologically active fragment thereof, an antibody drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor-expressing cell, or a T cell engager of the present application in diagnosing cancer.

[0183] Embodiments of the application

[0184] The present application is explained in more detail by the following examples. However, the following examples are intended to specifically illustrate the present application and not to limit the present application.

[0185] Example 1. Expression and purification of EphA10 antigen protein

[0186] To prepare an antigen for preparing an antibody targeting Eph receptor A10 (EphA10) of a hepatocytin A, a vector for expressing an extra cellular domain (ECD) or a FNIII domain of the extra cellular domain of EphA10, which is a cell membrane protein, resulting in resistance to a therapeutic agent targeting an immune checkpoint inhibitor, was prepared, expressed in an animal cell, and purified. To this end, a vector for expressing the ECD or the FNIII domain of EphA10 in an animal cell was prepared, and in order to perform more efficient antibody screening through avidity, a vector for expressing a dimeric form of an EphA10 protein fused with glutathione-S-transferase (GST) or human Fc, respectively, in an animal cell, and a vector for expressing a tetrameric form of an EphA10 protein fused with streptavidin in an animal cell were also prepared. Specifically, Expi293F cells were subcultured at a density of 2 x 10 6 cells / ml, and 1 day later, the above-prepared vectors were transfected using polyethylenimine (PEI, Polyscience, 23966). After 7 days of shaking culture at 37°C, 125 rpm, and 8% CO2 in a CO2 incubator, the supernatant was collected by centrifugation, 25 x PBS was added at a volume of 1 / 25 of the supernatant, and then the solution was filtered using a 0.2 μm bottle mouth filter (Merck Millipore). In the filtered solution, 1 ml of GSH resin, Protein A resin, or Ni-NTA resin was added, respectively, and stirred at 4°C for 16 hours, and then each resin was recovered by column. After washing with 5 ml of 1 x PBS, elution was performed using 4 ml of 50 mM Tris-HCl (pH 8.0) containing 10 mM reduced glutathione, 200 mM glycine (pH 2.7), or 250 mM imidazole, respectively. After replacing the buffer with 1 x PBS using a centrifugal filter unit 3K (Merck Millipore), the size and purity of 10 kinds of purified antigen proteins were analyzed by SDS-PAGE (FIG. 1). Figure 1 ).

[0187] Example 2. Acquisition and humanization of EphA10-binding mouse antibodies

[0188] Using the 10 antigens prepared in Example 1, a mouse antibody m#2 specific to EphA10 was obtained through mouse immunization and hybridoma technology (Kohler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975; 256: 495-497), on the basis of which a Chimeric #2 antibody introducing a human constant region was obtained through animal cell expression and antibody purification. In order to minimize the possibility of immunogenicity caused by humanization of the sequence of the framework region of the prepared EphA10-specific mouse antibody, germline sequence analysis was performed on the EphA10-specific mouse antibody, thereby obtaining 4 EphA10-specific humanized antibodies (1-1, 1-2, 2-1, and 2-2) in which the framework of the two human heavy chains (IGHV2-5*09, IGHV4-38-2*02) and light chains (IGKV2-30*02, IGKV2-29*02) having the highest similarity to the EphA10-specific mouse antibody were introduced, respectively. In addition, a humanized antibody (TRA #2) in which the framework of trastuzumab, which is a humanized antibody having excellent physical properties among therapeutic antibodies widely used in the clinic at present, was introduced was prepared. Figure 3 ).

[0189] Example 3. Expression and purification of EphA10-binding humanized antibodies

[0190] In order to express the 5 EphA10-specific humanized antibodies obtained in the above Example 2 in the form of IgG and analyze whether the binding force to EphA10 is maintained, 3 heavy chain and light chain expression vectors were prepared, respectively. Expi293F cells were transfected with the prepared expression vectors at 2 x 106cells / mL, and the cells were cultured for 6 days in a 37℃, 8% CO2incubator. After the culture, the culture supernatant was collected, and the antibodies were purified through protein A affinity chromatography. 6The prepared heavy chain expression vector and light chain expression vector were added to polyethylenimine (PEI, Polyscience, 23966) and transfected after 1 day of subculture at a density of 1 x 106cells / ml, and then cultured in a CO2incubator at 37°C, 125 rpm, and 8% CO2for 7 days. After the culture, the supernatant was collected by centrifugation, 25x PBS was added at a volume of 1 / 25 of the supernatant, and then filtered using a 0.2 μm bottle mouth filter (Merck Millipore). 500 μl of Protein A resin was added to the filtered solution, stirred at 4°C for 16 hours, and then recovered by column. After washing with 5 ml of 1x PBS, eluted with 3 ml of 100 mM glycine (pH 2.7), and neutralized with 1M Tris-HCl (pH 8.0). After replacing the buffer with 1x PBS using a centrifugal filter unit 3K (Merck Millipore), the size and purity of the five purified antibodies were analyzed by SDS-PAGE Figure 4 ).

[0191] Example 4. Screening of EphA10-specific humanized antibodies

[0192] 4-1. Analysis of binding ability to EphA10

[0193] The binding affinity of the five purified EphA10 antibodies to humanized antibodies from Example 3 above to EphA10 was analyzed by ELISA. Specifically, 4 μg / ml of EphA10 fused with human Fc was diluted with 0.05M Na2CO3 (pH 9.6) solution, and 50 μl was taken from each well and fixed onto a 96-well COSTA microplate. The plate was fixed at 4°C for 16 hours, and then blocked with 100 μl of 4% skim milk (GenomicBase) at room temperature for 2 hours. After washing four times with 180 μl of 1×PBS (PBST) containing 0.05% Tween 20, 50 μl of each of the five antibodies (IMGT 1-1, IMGT 1-2, IMGT 2-1, IMGT 2-2, and TRA #2) serially diluted in PBS containing 1% skim milk was injected into each well and reacted at room temperature for 1 hour. After washing, 50 μl of goat anti-human kappa chain HRP conjugate (Millipore) was added, and the mixture was incubated at room temperature for 1 hour, followed by washing. 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added for color development, and 50 μl of 2M H2SO4 was added to each well to stop the reaction. Analysis was then performed using an Epoch Microplate Spectrophotometer (BioTek). The results confirmed that the binding affinity of the humanized antibody to EphA10 was not significantly reduced compared to the mouse antibody before humanization (ch#2), indicating successful humanization. Figure 5 ).

[0194] 4-2. Analysis of binding ability to cell-expressing EphA10 and antibody screening

[0195] The binding affinity of the five purified EphA10-binding humanized antibodies from Example 3 above to cell-expressed EphA10 was analyzed, and antibodies with strong binding affinity were screened. Specifically, the MDA-MB-231 human EphA10 overexpressing cell line and the mouse EphA10 overexpressing cell line were suspended in DPBS (Corning), and 100 μl was aliquoted into each e-tube to achieve a cell count of 9 × 10⁶ cells / tube. 4 The five purified EphA10 antibodies from Example 3 above were combined with humanized antibodies as primary antibodies (1... st), and IgG as a control group, each at 500 ng, were incubated at 4°C for 30 minutes. Alexa Fluor 488 anti-human IgG (Invitrogen) was used as a secondary antibody (2 nd ), at a ratio of 1:100, and incubated at 4°C for 30 minutes in the dark. After washing twice with DPBS, CytoFLEX flow cytometer (Beckman Coulter) was used for analysis. The results showed that the mouse antibody before humanization (Chimeric #2) and the five humanized antibodies could specifically bind to human and mouse EphA10, and the binding force of all humanized antibodies to human EphA10 was higher than that to mouse EphA10. In addition, among the five humanized antibodies, 1-1 and 1-2 had the strongest binding force to EphA10, and therefore were selected as the final candidate antibodies (Figure 2). Figure 6

[0196] Example 5. Analysis of the quantitative binding constant of EphA10-specific humanized antibodies to EphA10

[0197] Octet Red 96 (Satorius), a Bio-Layer Interferometry (BLI) device, was used to analyze the quantitative binding constant of the two screened EphA10-specific humanized antibodies 1-1 and 1-2. ® The BLI system R8 (Satorius) analyzed the quantitative binding constant of two screened EphA10-specific humanized antibodies 1-1 and 1-2. Specifically, for immobilization, the two EphA10-specific humanized antibodies (1-1 and 1-2) were diluted to 5 μg / ml in 200 μl of 1xPBS, and then incubated on Octet ® Protein A bio-sensor (Satorius) (Satorius) for 300 seconds. After baseline stabilization for 150 seconds with 1xPBS, EphA10-His monomeric antigen protein was serially diluted from 1000 nM to 200 μl in 1xPBS and associated with the EphA10-specific humanized antibody-coated biosensor for 300 seconds. Then, dissociation was performed by incubating 200 μl of 1xPBS for 300 seconds, and the quantitative binding constant was analyzed using Octet BLI Analysis 12.2 software (Satorius).​Figure 7 ).

[0198] Example 6. Preparation of ADC antibody targeting EphA10

[0199] To prepare an antibody drug conjugate (ADC) using the EphA10-specific humanized antibodies 1-1 and 1-2 screened in Example 4 above, an ADC was prepared using a method commonly used to prepare ADCs, i.e., cysteine conjugation after partial reduction. Specifically, the buffer of the EphA10-specific humanized antibodies 1-1 and 1-2 stored in 1x PBS was exchanged with a reduction buffer (5mM DTPA, pH 8.0 PBS) using a PD MidiTrap G-25 (Cytiva, 28918008), and concentrated to 4mg / ml using a centrifugal filter units 10K (Centrifugal filter units 10K, Merck Millipore). The concentrated antibody at 2mg / ml and Tris(2-carboxyethyl)phosphine (TCEP, sigma) were mixed with the reduction buffer to 68.7μM, and a reduction reaction was performed at 37℃ for 2 hours. Then, TCEP was removed to prevent further reduction and conjugation, and the exchange and concentration were performed with 1x PBS buffer (pH 7.4) using a PD MidiTrap G-25 column and a centrifugal filter units 10K in the same manner as the above buffer exchange. The reduced antibody at a concentration of 2mg / ml was mixed with 63.2μM of MC-Val-Cit-PAB-MMAE (HY-15575, medchemexpress) (linker-drug reacted with cysteine) in 1x PBS, and reacted at 4℃ for 1 hour. To prevent further reaction, cysteine was dissolved in 1x PBS, and treated at a concentration of 126.4μM (twice the concentration of the linker-drug after treatment), and quenched at room temperature for 10 minutes. After the reaction was completed, the solution was subjected to size exclusion chromatography (SEC) purification, and the reaction solution was injected into a Superdex200 increase 10 / 300 (28990944, Cytiva) column of an FPLC (Biorad) system, and subjected to SEC chromatography separation at a speed of 0.4ml / min using 1x PBS, and then analyzed by SDS-PAGE (FIG. 6). Figure 8). The eluted ADC was concentrated by centrifugal filter units 10K and stored at -20°C until analyzed. The purified ADC was analyzed by hydrophobic interaction chromatography, and the result showed that the peaks of the ADC were separated by the difference in hydrophobicity according to DAR Figure 8 ).

[0200] Example 7. Analysis of efficacy and sequence analysis of ADC antibody targeting EphA10

[0201] 7-1. Confirmation of cancer cell killing effect of ADC

[0202] To confirm the in vitro efficacy of the ADC, cell survival analysis was performed by ATP measurement to confirm the killing effect on cancer cells. Specifically, MDA-MB-231 naive cells (control group), mock cell line (negative control group), and hEphA10 overexpressing cell line were each added at 2 x 10 4 cells / well to a 96-well white plate (SPL), and the next day, the ADC prepared in Example 6 above was diluted in RPMI (Capricorn) to various concentrations and treated at 100 μl / well, and then incubated at 37°C for 48 hours. The ADC-treated well plate was incubated at room temperature for 5 minutes outside the incubator, 50 μl of CellTiter-Glo solution was added per well, and shaken at 500 rpm for 2 minutes. After incubation at room temperature on the bench for 10 minutes in the dark, ATP was measured using a Synergy HTX multimode reader (Biotek).

[0203] As a result, no significant cell killing effect was observed in the ADC Figure 9 ).

[0204] 7-2. Sequence analysis

[0205] To analyze the reason why the ADC antibody prepared using the EphA10-specific humanized antibody in Example 7-1 above failed to exhibit in vitro ADC efficacy, the sequence of the EphA10-specific humanized antibody was re-analyzed. The sequence analysis result confirmed that there was a free cysteine amino acid (Cys) not involved in disulfide bonding in the CDR region of the light chain of the EphA10-specific humanized antibody Figure 10). Since the EphA10-targeting ADC antibody was generated by linking a low-molecular payload through a linker, it is presumed that in this process, the payload is coupled to the free cysteine in the CDR region, thereby blocking the site of binding to the antigenic epitope of the EphA10-targeting humanized antibody, thus losing the antigen-binding ability. To verify this, the binding ability of the EphA10-specific humanized antibody (1-1) and the EphA10-targeting ADC antibody (1-1 (ADC)) after preparation of the ADC to EphA10 was compared by ELISA analysis, and as a result, it was confirmed that due to the presence of the free cysteine in the CDR of the antibody light chain, the binding ability of the ADC antibody to EphA10 was greatly reduced as expected, and this also significantly reduced the in vitro ADC efficacy Figure 11 ).

[0206] Example 8. Preparation of EphA10-specific humanized antibodies with improved ADC efficacy

[0207] According to the results confirmed in the above-described Example 7, the free cysteine present in the light chain of the EphA10-specific humanized antibodies 1-1 and 1-2 screened in Example 4 was replaced with serine (1-1-S and 1-2-S) similar in properties to cysteine or alanine (1-1-A and 1-2-A) in an attempt to minimize the impact on the overall structure of the antibody, respectively, and each was cloned into a light chain animal cell expression vector and expressed in animal cells according to the above-described Example 3, and each antibody was purified, and then the size and purity of the purified antibodies (1-1-A, 1-1-S, 1-2-A, and 1-2-S) were analyzed by SDS-PAGE (FIG. 2). Figure 12 ).

[0208] Example 9. Analysis of the binding ability of EphA10-specific humanized antibodies with improved ADC efficacy to EphA10

[0209] In the above-described Example 8, the binding ability of the antibodies in which the free cysteine present in the light chain of the two EphA10-specific humanized antibodies 1-1 and 1-2 was replaced with serine and alanine (1-1-A, 1-1-S, 1-2-A, and 1-2-S), respectively, to EphA10 was analyzed by ELISA.

[0210] The results showed that when the free cysteine of the EphA10-specific humanized antibodies 1-1 and 1-2 was replaced with serine, the binding ability to EphA10 was almost completely lost, whereas when it was replaced with alanine, the binding ability to EphA10 was restored (FIG. 3). Figure 13 ).

[0211] Example 10. Construction of a yeast display library for enhancing the binding affinity to EphA10

[0212] In order to mine antibodies with further enhanced binding affinity to EphA10 from the EphA10-specific humanized antibodies 1-1 and 1-2 mined in the above-described Example, the variable regions (VH and VL) of 1-1 and 1-2 antibodies were amplified by error-prone PCR, inducing random mutations at a rate of 0.2%, and a library of antibodies was constructed. The constructed DNA library was transformed into 400 μl of Saccharomyces cerevisiae competent cells by electroporation using a MicroPulser Electroporator (Bio-Rad), and then cultured in 500 ml of SD medium [Difco Yeast nitrogen base (BD Biosciences) 6.7 g / l, Bacto casamino acid (BD Biosciences) 5.0 g / l, Na2HPO4 (Junsei) 5.4 g / l, NaH2PO4-H2O (Samchun) 8.56 g / l, Glucose (Duksan) 20 g / l] to selectively allow only the transformed yeast strains to survive, and libraries of 7.5 x 10 7 and 1.0 x 10 8 Figure 14

[0213] Example 11. Screening of humanized antibodies with enhanced binding affinity to EphA10

[0214] First, in order to perform screening using a flow cytometer, the antigen protein EphA10-Fc fused with human Fc was labeled with an Alexa 488 fluorescent molecule. In addition, 5.5 x 10 8 cells in the yeast library prepared in the above-described Example 10 were cultured in 100 ml of SD medium lacking tryptophan at 30°C for 16 hours, and then 7 x 10 8 ​​One cell was cultured in 100 ml of SG medium [Difco Yeast nitrogen base (BDBiosciences) 6.7 g / l, Bacto casamino acid (BD) 5.0 g / l, Na2HPO4(Junsei) 5.4 g / l, NaH2PO4·H2O (SAMCHUN) 8.56 g / l, Galactose (Sigma-Aldrich) 20 g / l] at 20°C for 48 hours. From the cultured cells, 1 x 10 8 After centrifugation of one cell at 4°C at 14000 x g for 30 seconds, the supernatant was discarded, washed with 1 ml of PBSB (1 x PBS, 0.1% bovine serum albumin (Gibco) (pH 7.4), and then 200 nM of Alexa488-conjugated EphA10-Fc and anti-FLAG-iFlour (GenScript) diluted at a ratio of 1:1000 in 1 ml of PBSB (pH 7.4) were added, and incubated for 1 hour. After incubation, the cells were washed with 1 ml of PBSB (pH 7.4), resuspended in 1 ml of PBSB (pH 7.4), and cells showing a high fluorescence signal by inducing binding with Alexa48-conjugated EphA10-Fc were recovered by a flow cytometer (Bio-Rad S3 sorter, Bio-Rad). The recovered sub-library was subjected to a total of five rounds of screening using a reduced concentration of Alexa488-conjugated EphA10-Fc in the same manner, thereby enriching cells (A.3, A.7, A.8, B.1, B.3, B.7, 2.1, 2.3, 1-N, and 2-W) displaying Fab variants having superior binding ability to EphA10. Figure 15

[0215] Example 12. Confirmation of the gene sequence of a humanized antibody having enhanced binding ability to EphA10

[0216] The gene sequence of the Fab variant clone having superior binding signal to EphA10 in the above-described Example 11 was confirmed by Sanger sequencing.

[0217] The analysis result confirmed that antibodies having a specific antibody sequence were successfully enriched, and 10 kinds of antibodies (A.3, A.7, A.8, B.1, B.3, B.7, 2.1, 2.3, 1-N, and 2-W) were obtained through the screening for enhancing the binding ability to EphA10. Figure 16 ​). In addition, it was confirmed that most of the clones of the discovered antibodies had a free cysteine in the light chain CDR of the 1-1 antibody and the 1-2 antibody substituted with one of asparagine (N), arginine (R), tryptophan (W), or tyrosine (Y), and thus, six variants (1-R, 1-W, 1-Y, 2-N, 2-R, and 2-Y) were prepared by mutating the free cysteine in the light chain CDR of the 1-1 antibody and the 1-2 antibody, respectively, to be substituted with a single amino acid, such as asparagine, arginine, tryptophan, or tyrosine. Figure 16

[0218] Example 13. Expression and purification of humanized antibodies with enhanced binding affinity to EphA10

[0219] To confirm whether the 16 Fab antibody variants screened in Example 12 above also have binding affinity to EphA10 in the IgG form, heavy chain and light chain expression vectors for each of the Fab antibody variants were constructed, and after expression of each of the antibodies in animal cells and purification according to the method of Example 3, the sizes and purities of the 16 purified antibodies were analyzed by SDS-PAGE (FIG. 6). Figure 17

[0220] Example 14. Analysis of EphA10 binding affinity of humanized antibodies with enhanced binding affinity to EphA10

[0221] To confirm whether the 16 antibodies purified in Example 13 above have enhanced binding affinity to EphA10, an ELISA analysis was performed, and as a result, it was confirmed that the humanized antibodies selected by additional engineering in Example 10 have enhanced binding affinity to EphA10 (FIG. 7). Figure 18

[0222] Example 15. Preparation of ADCs of humanized antibodies with enhanced binding affinity to EphA10

[0223] Two EphA10-specific humanized antibodies (A.7 and 2-W) were selected by CDR region cysteine substitution and further engineering, and ADCs were prepared using the cysteine coupling method of Example 6, and analyzed by SDS-PAGE (FIG. 8). Figure 19 In addition, the purified ADCs were analyzed by hydrophobic interaction chromatography (FIG. 9). Figure 19 ​​​). The peaks of the prepared ADCs were separated by the difference in hydrophobicity according to the DAR, and the distribution and ratio of the Drug to Antibody ratio (DAR) of the ADCs were confirmed by HIC analysis.

[0224] Example 16. Analysis of the binding ability of ADC to EphA10 antigen based on the enhanced binding ability to EphA10

[0225] The binding ability of the following antibodies to EphA10 was analyzed by ELISA to determine whether it was maintained at the same level as before the ADC: an EphA10-targeting ADC (1-1_ADC) antibody prepared using the humanized antibody 1-1 screened in Example 4, an EphA10-targeting ADC (2W_ADC) antibody prepared using an antibody in which a free cysteine present in the light chain CDR of the humanized antibody 1-2 screened in Example 4 was substituted with tryptophan (2-W), and an EphA10-targeting ADC (A7_ADC) antibody prepared using the antibody A7 (A.7) screened by the yeast display library.

[0226] The results confirmed that, unlike the case where the binding ability to EphA10 was significantly reduced when ADCs were prepared using the humanized antibodies 1-1 and 1-2 screened in Example 4, the binding ability to EphA10 was maintained well when ADCs were prepared using the screened antibodies by applying additional engineering (amino acid substitution, yeast display library) (). Figure 20 ).

[0227] Example 17. Confirmation of the cancer cell-killing effect of ADC based on the enhanced binding ability to EphA10

[0228] To verify the effects of two EphA10-targeting ADCs (A7-MMAE and 2W_MMAE) prepared using the A7 and 2W antibodies, the cell viability of cancer cells was analyzed by measuring ATP as described in Example 7-1 above.

[0229] The results showed that, although the control antibodies (A7 and W2) had no significant effect on cell survival, all of the EphA10-targeting ADCs using them exhibited a clear cancer cell-killing effect, and the cancer cell-killing effect of the ADCs increased in proportion to the expression of EphA10 (). Figure 21 ).

[0230] Example 18. Analysis of the quantitative binding constant of humanized antibody with enhanced binding ability to EphA10 to EphA10

[0231] To analyze the quantitative binding constants of two selected EphA10 specific humanized antibodies (A.7 and 2-W) to EphA10, experiments were performed using Octet® as a Bio-Layer Interferometry (BLI) instrument ® BLI system R8 (Satorius). Specifically, for immobilization, two EphA10 specific humanized antibodies (A.7 and 2-W) were diluted to 5 pg / ml in 200 mΐ of 1 x PBS and then incubated for 300 seconds on Octet® Protein A bio-sensors (Satorius). After baseline stabilization for 150 seconds with 1 x PBS, EphA10-His monomeric antigen protein was bound to the EphA10 specific humanized antibody (A.7 and 2-W) coated bio-sensors for 300 seconds from 1000 nM serially diluted to 200 mΐ in 1 x PBS. Then, dissociation was performed by incubating 200 mΐ of 1 x PBS for 300 seconds and the quantitative binding constants were analyzed using Octet BLI Analysis 12.2 software (Satorius). ® BLI system R8 (Satorius). Specifically, for immobilization, two EphA10 specific humanized antibodies (A.7 and 2-W) were diluted to 5 pg / ml in 200 mΐ of 1 x PBS and then incubated for 300 seconds on Octet® Protein A bio-sensors (Satorius). After baseline stabilization for 150 seconds with 1 x PBS, EphA10-His monomeric antigen protein was bound to the EphA10 specific humanized antibody (A.7 and 2-W) coated bio-sensors for 300 seconds from 1000 nM serially diluted to 200 mΐ in 1 x PBS. Then, dissociation was performed by incubating 200 mΐ of 1 x PBS for 300 seconds and the quantitative binding constants were analyzed using Octet BLI Analysis 12.2 software (Satorius). ® BLI system R8 (Satorius). Specifically, for immobilization, two EphA10 specific humanized antibodies (A.7 and 2-W) were diluted to 5 pg / ml in 200 mΐ of 1 x PBS and then incubated for 300 seconds on Octet® Protein A bio-sensors (Satorius). After baseline stabilization for 150 seconds with 1 x PBS, EphA10-His monomeric antigen protein was bound to the EphA10 specific humanized antibody (A.7 and 2-W) coated bio-sensors for 300 seconds from 1000 nM serially diluted to 200 mΐ in 1 x PBS. Then, dissociation was performed by incubating 200 mΐ of 1 x PBS for 300 seconds and the quantitative binding constants were analyzed using Octet BLI Analysis 12.2 software (Satorius).

[0232] The results confirmed that the binding constants of both antibodies to human EphA10 were higher than that of #2 antibody (A.7: 1.2 x 1010M-1, 2-W: 1.1 x 1010M-1, #2: 1.0 x 109M-1). Figure 22 ).

[0233] Example 19. Analysis of binding force of humanized antibody with enhanced binding force to EphA10 to mouse EphA10

[0234] 19-1. Expression and purification of mouse EphA10

[0235] Since human EphA10 has 95% similarity to mouse EphA10, if an antibody binds to mouse EphA10, it can be a good indicator for selecting a mouse model before in vivo experiments of antibodies targeting EphA10. Therefore, an animal cell expression vector capable of expressing the extracellular domain (ECD) of mouse EphA10 and various fusion proteins was prepared, transfected into Expi293F cells, and after 7 days of culture, the supernatant was collected by centrifugation. After adding 1 / 25 of the volume of 25x PBS to the supernatant, it was filtered using a 0.2 pm bottle top filter (Merck Millipore), and after adding 1 ml of GSH resin, Protein A resin, or Ni-NTA resin to the filtered supernatant, it was stirred at 4°C for 16 hours. Then, each resin was recovered by column. After washing with 5 ml of 1x PBS, elution was performed with 4 ml of 50 mM Tris-HCl (pH 8.0) containing 10 mM reduced glutathione, 200 mM glycine (pH 2.7), or 250 mM imidazole, respectively. After replacing the buffer with 1x PBS using a centrifugal filter unit 3K (Merck Millipore), the size and purity of the four purified antigen proteins were analyzed by SDS-PAGE, and as a result, only mEphA10-Fc (mouse EphA10 fused with human Fc) was purified, and its activity as an antigen was confirmed by ELISA analysis using an EphA10-specific mouse antibody (m#2) before humanization (Fig. 23).

[0236] 19-2. Analysis of binding ability of humanized antibody with enhanced binding ability to EphA10 to mouse EphA10

[0237] To confirm whether the two selected EphA10 specific humanized antibodies (A.7 and 2-W) also bind to the mouse EphA10 expressed and purified in Example 19-1 above, an ELISA was performed. Specifically, 4 pg / ml of mEphA10-Fc was diluted with 0.05 M Na2CO3(pH 9.6) solution, 50 mI / well was immobilized on a flat bottom polystyrene high bind 96-well microplate (costar) at 4°C for 16 hours, and then blocked with 100 mI of PBS containing 4% skim milk (Genomic Base) at room temperature for 2 hours. After washing with 180 mI of PBST four times, EPHA10 binding antibodies (A.7 and 2-W) diluted serially in 1% skim milk were injected 50 mI / well, respectively, and reacted at room temperature for 1 hour. After washing, 50 mI of goat anti-human kappa chain HRP conjugate (Millipore) was added, incubated at room temperature for 1 hour, and washed. After color development by adding 50 mI of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific), the reaction was stopped by adding 50 mI of 2M H2SO4 / well, and then analyzed using an Epoch microplate spectrophotometer (BioTek). As a result, it was confirmed that both the A7 antibody (A.7) and the 2W antibody (2-W) also bind to mouse EphA10 Figure 24 ).

Claims

1. A liver ligand A receptor 10 specific antibody or an immunologically active fragment thereof.

2. The liver glycoprotein A receptor 10 specific antibody or its immunologically active fragment according to claim 1, characterized in that, It contains the VH structural field. The VH domain includes: CDRH1 comprises any one of the groups consisting of amino acid sequences selected from SEQ ID NO: 1 to 3; CDRH2, comprising any one of the groups consisting of amino acid sequences selected from SEQ ID NO: 4 to 6; and CDRH3 contains the amino acid sequence of SEQ ID NO: 7 or 8.

3. The liver glycoprotein A receptor 10 specific antibody or its immunologically active fragment according to claim 1, characterized in that, Includes the VL structure domain. The VL structure domain includes: CDRL1 comprises any one of the groups consisting of the amino acid sequences selected from SEQ ID NO: 9 to 14; CDRL2, containing the amino acid sequence of SEQ ID NO: 15 or 16; and CDRL3 comprises any one of the groups consisting of amino acid sequences selected from SEQ ID NO: 17 to 19.

4. The liver glycoprotein A receptor 10 specific antibody or its immunologically active fragment according to claim 1, characterized in that, It includes a VH domain, which comprises any one of the amino acid sequences selected from SEQ ID NO: 20 to 25.

5. The liver glycoprotein A receptor 10 specific antibody or its immunologically active fragment according to claim 1, characterized in that, It includes a VL domain, which comprises any one of the amino acid sequences selected from SEQ ID NO: 26 to 45.

6. The liver glycoprotein A receptor 10 specific antibody or its immunologically active fragment according to claim 1, characterized in that, The immunologically active fragment is selected from any one of the following groups: Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv, Fv, single-chain antibody, Fv dimer, complementarity-determining region fragment, humanized antibody, chimeric antibody, and bivalent antibody.

7. The liver glycoprotein A receptor 10 specific antibody or its immunologically active fragment according to claim 1, characterized in that, It is a humanized antibody.

8. An antibody-drug conjugate, characterized in that, It comprises the antibody or an immunologically active fragment thereof as described in claim 1, and a drug.

9. The antibody-drug conjugate according to claim 8, characterized in that, The drugs are immunogenic cell death inducers, apoptotic peptides, microtubule formation inhibitors, mitosis inhibitors, topoisomerase inhibitors, DNA intercalators, toxins, radionuclides, or anticancer agents.

10. The antibody-drug conjugate according to claim 9, characterized in that, The drugs selected are 7-ethyl-10-hydroxycamptothecin, daunorubicin, doxorubicin, epirubicin, idarubicin, pisacaproicin, sababicin, penoxorubicin, paclitaxel, docetaxel, dichloromethyldiethylamine, chlorambucil, phenylalanine, nitrogen mustard, cyclophosphamide, ifosfamide, carmustine, lomustine, streptozotocin, busulfan, thiotepa, cisplatin, carboplatin, actinomycin D, procainamide, mitomycin C, vincristine, vinblastine, teniposide, topotecan, irinotecan, uramustine, melphalan, bendamustine, dacarbazine, temozolomide, and hexachlorocyclohexane. The group consisting of melamine, docamycin, nedaplatin, oxaliplatin, ceterplatin, triplatinum tetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cytarabine, fluorouridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine, etoposide, mitoxantrone, ixaprone, vinorelbine, estradiol, maytansine, maytansine DM1, DM4, dolalastatin, oliquistatin E, oliquistatin F, monomethyl oliquistatin E, monomethyl oliquistatin F and their derivatives.

11. The antibody-drug conjugate according to claim 8, characterized in that, It also includes an ADC connector.

12. The antibody-drug conjugate according to claim 11, characterized in that, The ADC linker is 6-maleimide hexanoyl, maleimide propionyl, valine-citrulline, alanine-phenylalanine, p-aminobenzyloxycarbonyl, N-succinimide-4-(2-pyridyl dithio)valerate, N-succinimide-4-(N-maleimide methyl)cyclohexane-1-carboxylate, valine-citrulline-p-aminobenzyloxycarbonyl, or N-succinimide-(4-iodoacetyl)aminobenzoate.

13. The antibody-drug conjugate according to claim 11, characterized in that, Antibodies or their immunologically active fragments form complexes with drugs via ADC linkers.

14. A bispecific or multispecific antibody, characterized in that, It comprises the antibody as described in claim 1 or an immunologically active fragment thereof, and a portion that binds to a target antigen other than liver ligand A receptor 10.

15. The bispecific or multispecific antibody according to claim 14, characterized in that, The portion that binds to the target antigen contains an antibody or an immunologically active fragment thereof.

16. The bispecific or multispecific antibody according to claim 14, characterized in that, Target antigens are selected from 17-1A antigen, GD3 ganglioside R24, EGFRvIII, PSMA, PSCA, HLA-DR, EpCAM, MUC1 core protein, aberrantly glycosylated MUC1, fibronectin isoforms containing the ED-B domain, HER2 / neu, carcinoembryonic antigen, gastrin-releasing peptide receptor antigen, mucin antigen, epidermal growth factor receptor, HER3, HER4, MAGE antigen, SART antigen, MUC1 antigen, c-erb-2 antigen, TAG 72, carbonic anhydrase IX, alpha-fetoprotein, A3, A33 antibody-specific antigen, and Ba. 733, BrE3-antigen, CA125, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD74, CD79a, CD80, CD138, colon-specific antigen-p, CSAp, EGP-1, EGP-2, Ep-CAM, FIT-1, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin and its subunits, hypoxia-inducible factor, Ia, IL-2, IL-6, IL-8, insulin-like substances One or more of the following groups: growth factor-1, KC4 antigen, KS-1 antigen, KSl-4, Le-Y, macrophage migration inhibitory factor, MAGE, MUC1, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, PAM-4 antibody-specific antigen, placental growth factor, p53, prostatic acid phosphatase, PSA, RS5, S100, TAC, tendon glycoprotein, TRAIL receptor, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, VEGF, ED-B fibronectin, angiogenesis markers, oncogene markers, or oncogene products.

17. The bispecific or multispecific antibody according to claim 14, characterized in that, The target antigen is a cell surface antigen or an autoantigen.

18. An isolated nucleic acid molecule, characterized in that, Encoding the antibody according to claim 1 or its immunologically active fragment, or the bispecific or multispecific antibody according to claim 14.

19. A pharmaceutical composition for the prevention or treatment of a disease or ailment, characterized in that, The active ingredient comprises the antibody or its immunologically active fragment as claimed in claim 1, the antibody-drug conjugate as claimed in claim 8, or the bispecific or multispecific antibody as claimed in claim 14, wherein the disease or symptom is selected from the group consisting of autoimmune diseases, neurodegenerative diseases, Alzheimer's disease, metabolic diseases, cardiovascular diseases, atherosclerosis, organ transplant rejection, and diseases or symptoms caused by fungi, viruses, bacteria or parasites.

20. A chimeric antigen receptor, characterized in that, It contains the antibody or an immunologically active fragment thereof as an antigen-binding domain according to claim 1.

21. A recombinant vector, characterized in that, It includes a gene encoding the chimeric antigen receptor as described in claim 20.

22. A chimeric antigen receptor-expressing cell, characterized in that, Transformation using the recombinant vector according to claim 21.

23. The chimeric antigen receptor expression cell according to claim 22, characterized in that, Chimeric antigen receptor expressing cells are either chimeric antigen receptor expressing T cells or natural killer cells.

24. A T-cell connector, characterized in that, It contains the antibody or an immunologically active fragment thereof as described in claim 1.

25. A pharmaceutical composition for the prevention or treatment of cancer, characterized in that, The active ingredient comprises the antibody or an immunologically active fragment thereof according to claim 1, the antibody-drug conjugate according to claim 8, the bispecific or multispecific antibody according to claim 14, the chimeric antigen receptor according to claim 20, the chimeric antigen receptor expressing cell according to claim 22, or the T cell connector according to claim 24.

26. The pharmaceutical composition for the prevention or treatment of cancer according to claim 25, characterized in that, The cancer is selected from any one of the following groups: brain tumors, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, triple-negative breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, colorectal cancer, small bowel cancer, rectal cancer, fallopian tube cancer, perianal cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumors, primary central nervous system lymphoma, spinal cord tumors, brainstem gliomas, and pituitary adenomas.

27. The pharmaceutical composition for the prevention or treatment of cancer according to claim 25, characterized in that, The cancer is one that is resistant to immune checkpoint inhibitors.

28. An anticancer adjuvant, characterized in that, The active ingredient comprises the antibody or an immunologically active fragment thereof according to claim 1, the antibody-drug conjugate according to claim 8, the bispecific or multispecific antibody according to claim 14, the chimeric antigen receptor according to claim 20, the chimeric antigen receptor expressing cell according to claim 22, or the T cell connector according to claim 24.

29. The anticancer adjuvant according to claim 28, characterized in that, The anticancer adjuvant may be administered alone or in combination with an immunotherapeutic agent, either simultaneously or sequentially.

30. The anticancer adjuvant according to claim 28, characterized in that, Improve resistance to immunotherapeutic agents.

31. The anticancer adjuvant according to claim 28, characterized in that, Immunotherapy agents include immune checkpoint inhibitors, immunosuppressive factor modulators, cancer vaccines, immune adjuvants, immune cells for cancer treatment, immune cell activity cofactors, antibodies for cancer treatment, or cytokines required to maintain the activity of immune cells for cancer treatment.

32. The anticancer adjuvant according to claim 31, characterized in that, Immune checkpoint inhibitors are inhibitors of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA, or A2aR.

33. A composition for diagnosing cancer, characterized in that, The active ingredient comprises the antibody or an immunologically active fragment thereof according to claim 1, the antibody-drug conjugate according to claim 8, the bispecific or multispecific antibody according to claim 14, the chimeric antigen receptor according to claim 20, the chimeric antigen receptor expressing cell according to claim 22, or the T cell connector according to claim 24.

34. The composition for diagnosing cancer according to claim 33, characterized in that, It also includes signs.

35. The composition for diagnosing cancer according to claim 34, characterized in that, The markers are chromogenic enzymes, radioactive isotopes, chromophores, luminescent substances, fluorescent substances, probes, or tags.

36. Use of an antibody according to claim 1 or an immunologically active fragment thereof, an antibody-drug conjugate according to claim 8, a bispecific or multispecific antibody according to claim 14, a chimeric antigen receptor expressing cell according to claim 22, or a T-cell connector according to claim 24 in the prevention or treatment of cancer.

37. A method for treating cancer, characterized in that, The procedure includes administering to an individual with cancer a pharmaceutically effective amount of the antibody or an immunologically active fragment thereof according to claim 1, the antibody-drug conjugate according to claim 8, the bispecific or multispecific antibody according to claim 14, the chimeric antigen receptor expressing cell according to claim 22, or the T-cell connector according to claim 24.

38. Use of an antibody or an immunologically active fragment thereof according to claim 1, an antibody-drug conjugate according to claim 8, a bispecific or multispecific antibody according to claim 14, a chimeric antigen receptor according to claim 20, a chimeric antigen receptor expressing cell according to claim 22, or a T-cell connector according to claim 24 in the diagnosis of cancer.

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