Anti-endoglin antibody and uses thereof

Abstract

The present invention relates to: a novel antibody capable of binding to endoglin, or an antigen-binding fragment; and various uses thereof.

Description

Anti-endoglin antibodies and uses thereof

[0001] The present invention relates to antibodies or antigen-binding fragments capable of binding to endoglin and various uses thereof.

[0002] Endoglin, also known as CD105, is a transmembrane glycoprotein expressed on activated vascular endothelial cells. Endoglin is a co-receptor for the TGF-β receptor, forms heterodimers with type I and type II receptors for TGF-β signaling, and can modify the response to TGF-β. In endothelial cells, TGF-β activates the type I receptor pathway, the activin receptor-like kinases ALK5 and ALK1. Activation of ALK1 promotes Smad1 / 5 phosphorylation, stimulating cell proliferation and migration.

[0003] Accordingly, endoglin is known to be highly expressed in tumor vasculature and relatively low in a limited number of other cell types, including macrophages, fibroblasts, and syncytiotrophoblasts. Recent studies have also reported that endoglin is highly expressed in cancer fibroblasts from colon and pancreatic cancers (OncoTargets and Therapy 2021:14 5205-5220).

[0004] Angiogenesis is the physiological process by which new blood vessels develop from pre-existing ones. Angiogenesis has been proposed to play a role in both normal and pathological processes. Specifically, in pathological conditions, angiogenesis is promoted as a means of providing adequate blood and nutrient supply to cells within diseased tissues. Many of these pathological conditions involve abnormal cell proliferation and / or regulation. Therefore, inhibiting angiogenesis could be a potentially useful approach to treating diseases characterized by new blood vessel development. Diseases associated with angiogenesis include various forms of cancer, solid tumors, and metastases, as well as ocular and non-ocular diseases characterized by angiogenesis / neovascularization (e.g., macular degeneration, diabetic retinopathy), diabetic nephropathy, chronic inflammatory diseases (e.g., IBD), rheumatoid arthritis, and osteoarthritis.

[0005] Given the significant unmet clinical need for the treatment of these angiogenic diseases, particularly various cancers, the development of drugs targeting endoglin is urgently needed. While several monoclonal antibodies against endoglin have been developed in the past, most of these antibodies suffer from limitations such as immunogenicity, low affinity, and low specificity. Furthermore, no anti-endoglin antibody has been approved for human disease treatment to date. Therefore, the development of novel anti-endoglin antibodies with stronger binding affinity for endoglin, superior specificity, potent ADCC and CDC activities, and superior antitumor activity remains essential.

[0006] One object of the present invention is to provide an antibody or antigen-binding fragment that binds to Endoglin.

[0007] Another object of the present invention is to provide a nucleic acid molecule encoding the antibody or antigen-binding fragment, an expression vector comprising the same, and a host cell transfected with the vector.

[0008] Another object of the present invention is to provide a chimeric antigen receptor (CAR) comprising the antigen-binding fragment and an immune effector cell expressing the same.

[0009] Another object of the present invention is to provide a use for diagnosing or treating endoglin overexpression diseases as applicable uses thereof.

[0010] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0011] antibody or antigen-binding fragment

[0012] According to one embodiment of the present invention, it relates to an antibody or antigen-binding fragment that specifically binds to Endoglin.

[0013] As used herein, the term "Endoglin" refers to a type I membrane glycoprotein located on the cell surface and is part of the TGF beta receptor complex, also known as CD105. Endoglin plays a crucial role in angiogenesis, and is therefore known to be an important protein for cancer cell growth, survival, and metastasis to other locations in the body. In the present invention, the endoglin may be comprised of an amino acid sequence represented by SEQ ID NO: 207, but is not limited thereto.

[0014] The term "antibody" as used herein refers to a protein molecule that acts as a ligand that specifically recognizes an antigen, including an immunoglobulin molecule that immunologically has reactivity with a specific antigen, and includes polyclonal antibodies, monoclonal antibodies, whole antibodies, and antibody fragments. In addition, the term also includes, in the present invention, a chimeric antibody, a human antibody, a humanized antibody, a bivalent, a bispecific molecule, a minibody, a domain antibody, a bispecific or multispecific antibody, an immune cell engaging bispecific or multispecific antibody, an antibody mimetic, a unibody, a diabody, a triabody, or a tetrabody. The term further encompasses single-chain antibodies, scabs, derivatives of antibody constant regions, and artificial antibodies based on protein scaffolds, which possess binding function to the neonatal Fc receptor (FcRn). The whole antibody has a structure comprising two full-length light chains (LC) and two full-length heavy chains (HC), each light chain being linked to a heavy chain by a disulfide bond. The whole antibody comprises IgA, IgD, IgE, IgM, and IgG, and IgG comprises subtypes such as IgG1, IgG2, IgG3, and IgG4. In one embodiment, the antibody provided in the present invention may be an IgG antibody.

[0015] As used herein, the term "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, and such monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope. Typically, immunoglobulins have heavy and light chains, and each heavy and light chain comprises a constant region and a variable region (also known as a domain). The variable regions of the light and heavy chains comprise three variable regions and four structural regions, designated as complementarity-determining regions (CDRs).

[0016] As used herein, the term "complementarity determining region" or "CDR region" or "CDR" refers to a highly variable region of sequence within an antibody variable region that forms structurally defined loops ("hypervariable loops") and / or contains antigen-contacting residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigenic epitopes. The complementarity determining regions are interspersed between relatively conserved regions called constant regions (FRs). Each heavy chain variable region (VH) and light chain variable region (VL) consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The CDRs of the heavy chain variable region may be referred to as HCDR1, HCDR2, HCDR3, the CDRs of the light chain variable region may be referred to as LCDR1, LCDR2, LCDR3, the FRs of the heavy chain variable region may be referred to as HFR1, HFR2, HFR3, HFR4, and the FRs of the light chain variable region may be referred to as LFR1, LFR2, LFR3, LFR4. The numbers are sequentially numbered from the N-terminus. The boundaries of the exact amino acid sequence of each CDR in a given light chain variable region and heavy chain variable region amino acid sequence can be determined, for example, by Chothia (Chothia et al. (1989) Nature 342: 877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability, AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (imgt.cines.fr / on the World Wide Web), and North CDR definition based on affinity propagation clustering using multiple crystal structures, or a combination thereof.

[0017] The antibody of the present invention may comprise a constant region, wherein the constant region may be, but is not limited to, a constant region derived from IgG, IgA, IgD, IgE, IgM, or a combination thereof or a hybrid thereof. Here, the term "combination" means that a polypeptide encoding a single-chain immunoglobulin constant region of the same origin forms a bond with a single-chain polypeptide of a different origin when forming a dimer or multimer. For example, a dimer or multimer may be formed from two or more constant regions selected from the group consisting of constant regions of IgG, IgA, IgD, IgE, and IgM. Additionally, the term "hybrid" or "hybrid" means that within a single-chain immunoglobulin heavy chain constant region, there are sequences corresponding to immunoglobulin heavy chain constant regions of two or more different origins, for example, a hybrid of domains consisting of one to four domains selected from the group consisting of CH1, CH2, CH3 and CH4 of IgG, IgA, IgD, IgE and IgM is possible.

[0018] The term "full-length antibody" as used herein refers to a structure having two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond, and includes IgA, IgD, IgE, IgM, and IgG. The IgG subtypes include IgG1, IgG2, IgG3, and IgG4.

[0019] As used herein, the terms "fragment," "binding fragment of polypeptide," and "antibody fragment" are used interchangeably to refer to any fragment of an antibody of the present invention that retains the antigen-binding activity of the antibody. Exemplary antibody fragments include, but are not limited to, single chain antibodies, Fd, Fab, Fab', F(ab')2, dsFv, or scFv. The Fd refers to the heavy chain portion included in a Fab fragment. As used herein, the term "Fab" typically refers to a fragment comprising a heavy chain variable domain and a light chain variable domain, and further comprising a light chain constant domain and a heavy chain first constant domain (CH1). The term "Fab" typically refers to a fragment that differs from a Fab by the addition of a few residues (including one or more cysteines from the antibody hinge region) to the carboxy terminus of the heavy chain CH1 domain. The term "F(ab')2" typically refers to a dimeric antibody fragment of Fab' comprising two Fab fragments linked by a disulfide bridge at the hinge region. The term "Fv" typically refers to the smallest antibody fragment containing a complete antigen recognition and binding site. In certain cases, the fragment may be configured as a dimer in which the heavy chain variable region and the light chain variable region are closely non-covalently associated. The term "dsFv" typically refers to an Fv fragment in which the disulfide bond is stabilized, wherein the linkage between a single light chain variable region and a single heavy chain variable region is a disulfide bond. The term "dAb fragment" typically refers to an antibody fragment consisting of a VH domain. In the present invention, the term "scFv" typically refers to a monovalent molecule formed by covalent pairing of the heavy chain variable domain and the light chain variable domain of an antibody via a flexible peptide linker. These scFv molecules can have the general structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.

[0020] The term "chimeric antibody" as used herein refers to an antibody in which the variable region of a mouse antibody and the constant region of a human antibody are recombined, and which has a significantly improved immune response compared to a mouse antibody.

[0021] Additionally, the term "humanized antibody" as used herein refers to an antibody in which the protein sequence of an antibody derived from a non-human species has been modified to resemble a naturally produced antibody variant in humans. For example, the humanized antibody can be produced by recombining a mouse-derived CDR with a human antibody-derived FR to produce a humanized variable region, which can then be recombined with a desired human antibody constant region.

[0022] The terms "anti-endoglin antibody," "anti-endoglin," "endoglin antibody," or "antibody that binds to endoglin," as used herein, refer to an antibody that recognizes endoglin or binds to endoglin with sufficient affinity such that the antibody can be used as a therapeutic agent targeting endoglin. In one embodiment of the invention, the endoglin antibody binds to endoglin with high affinity in vitro or in vivo. Here, the binding can be determined by, but is not limited to, radioimmunoassay (RIA), biolayer interferometry (BLI), MSD analysis, surface plasmon resonance (SPR), or flow cytometry.

[0023] The anti-endoglin antibody or antigen-binding fragment of the present invention may comprise a heavy chain variable region comprising a heavy chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 17, as shown in Table 1 below; a heavy chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 36; and a heavy chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 37 to 57.

[0024] The anti-endoglin antibody or antigen-binding fragment of the present invention may comprise a light chain variable region comprising a light chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 58 to 76, as shown in Table 1 below; a light chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 77 to 90; and a light chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 91 to 112.

[0025] The anti-endoglin antibody or antigen-binding fragment of the present invention may comprise a heavy chain variable region comprising a heavy chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 17, a heavy chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 36, and a heavy chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 37 to 57; and a light chain variable region comprising a light chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 58 to 76, a light chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 77 to 90, and a light chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 91 to 112.

[0026] The anti-endoglin antibody or antigen-binding fragment of the present invention may comprise at least one antibody or antigen-binding fragment selected from the following A-1) to A-25):

[0027] A-1) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 18, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 37; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 58, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 77, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 91;

[0028] A-2) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 18, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 37; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 59, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 77, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 92;

[0029] A-3) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 19, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 38; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 60, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 78, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 93;

[0030] A-4) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 20, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 39; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 61, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 79, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 94;

[0031] A-5) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 20, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 39; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 62, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 80, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 95;

[0032] A-6) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 21, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 40; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 63, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 81, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 96;

[0033] A-7) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 22, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 41; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 64, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 82, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 97;

[0034] A-8) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 23, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 42; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 65, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 98;

[0035] A-9) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 6, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 43; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 66, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 84, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 99;

[0036] A-10) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 6, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 43; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 67, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 85, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 100;

[0037] A-11) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 7, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 25, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 44; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 68, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 86, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 101;

[0038] A-12) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 8, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 26, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 45; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 69, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 79, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 102;

[0039] A-13) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 9, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 46; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 62, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 80, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 103;

[0040] A-14) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 28, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 47; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 70, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 87, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 104;

[0041] A-15) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 20, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 48; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 71, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 88, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 105;

[0042] A-16) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 11, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 29, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 49; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 106;

[0043] A-17) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 8, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 26, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 45; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 89, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 107;

[0044] A-18) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 12, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 25, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 50; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 89, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 108;

[0045] A-19) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 30, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 51; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 73, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 90, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 109;

[0046] A-20) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 14, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 31, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 52; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 89, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 108;

[0047] A-21) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 9, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 32, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 53; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 74, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 90, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 109;

[0048] A-22) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 15, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 33, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 54; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 75, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 80, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 110;

[0049] A-23) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 16, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 34, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 55; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 89, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 108;

[0050] A-24) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 56; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111; and

[0051] A-25) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 17, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 36, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 57; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 76, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 112.

[0052]

[0053] The anti-endoglin antibody or antigen-binding fragment of the present invention may comprise at least one heavy chain variable region selected from the group consisting of SEQ ID NOs: 113 to 133 as shown in Table 2 below.

[0054] The anti-endoglin antibody or antigen-binding fragment of the present invention may comprise at least one light chain variable region selected from the group consisting of SEQ ID NOs: 134 to 158 as shown in Table 2 below.

[0055] The anti-endoglin antibody or antigen-binding fragment of the present invention may comprise at least one heavy chain variable region selected from the group consisting of SEQ ID NOs: 113 to 133; and at least one light chain variable region selected from the group consisting of SEQ ID NOs: 134 to 158.

[0056] The anti-endoglin antibody or antigen-binding fragment of the present invention may comprise at least one antibody or antigen-binding fragment selected from the following B-1) to B-25):

[0057] B-1) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 113 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 134;

[0058] B-2) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 113 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 135;

[0059] B-3) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 114 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 136;

[0060] B-4) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 115 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 137;

[0061] B-5) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 115 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 138;

[0062] B-6) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 116 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 139;

[0063] B-7) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 117 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 140;

[0064] B-8) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 118 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 141;

[0065] B-9) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 119 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 142;

[0066] B-10) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 119 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 143;

[0067] B-11) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 120 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 144;

[0068] B-12) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 121 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 145;

[0069] B-13) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 122 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 146;

[0070] B-14) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 123 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 147;

[0071] B-15) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 124 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 148;

[0072] B-16) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 125 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 149;

[0073] B-17) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 121 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 150;

[0074] B-18) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 126 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 151;

[0075] B-19) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 127 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 152;

[0076] B-20) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 128 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 153;

[0077] B-21) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 129 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 154;

[0078] B-22) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 130 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 155;

[0079] B-23) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 131 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 156;

[0080] B-24) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 132 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 157; and

[0081] B-25) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 133 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 158.

[0082]

[0083] The antibody or antigen-binding fragment of the present invention may further comprise a linker between the heavy chain variable region and the light chain variable region. Here, the linker may be a peptide linker and may have a length of about 10-25 aa. For example, it may include a hydrophilic amino acid such as glycine and / or serine, but is not limited thereto. Specifically, in the present invention, the linker may include, for example, (GS)n, (GGS)n, (GSGGS)n, or (GnS)m (n, m are each 1 to 10), preferably GGGGS, and more preferably GGGGSGGGGSGGGGS represented by SEQ ID NO: 159, but is not limited thereto.

[0084] The antibody provided in the present invention may be a monoclonal antibody.

[0085] The antigen-binding fragment provided in the present invention may be a scFv (single-chain variable fragment) that specifically recognizes and / or binds to endoglin.

[0086] The anti-endoglin scFv of the present invention may comprise a heavy chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 17; a heavy chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 36; a heavy chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 37 to 57; a light chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 58 to 76; a light chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 77 to 90; and a light chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 91 to 112.

[0087] The anti-endoglin scFv of the present invention may comprise at least one of the following scFvs:

[0088] A-1) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 18, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 37; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 58, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 77, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 91;

[0089] A-2) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 18, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 37; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 59, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 77, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 92;

[0090] A-3) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 19, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 38; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 60, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 78, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 93;

[0091] A-4) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 20, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 39; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 61, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 79, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 94;

[0092] A-5) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 20, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 39; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 62, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 80, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 95;

[0093] A-6) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 21, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 40; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 63, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 81, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 96;

[0094] A-7) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 22, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 41; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 64, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 82, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 97;

[0095] A-8) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 23, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 42; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 65, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 98;

[0096] A-9) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 6, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 43; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 66, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 84, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 99;

[0097] A-10) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 6, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 43; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 67, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 85, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 100;

[0098] A-11) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 7, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 25, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 44; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 68, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 86, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 101;

[0099] A-12) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 8, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 26, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 45; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 69, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 79, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 102;

[0100] A-13) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 9, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 46; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 62, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 80, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 103;

[0101] A-14) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 28, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 47; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 70, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 87, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 104;

[0102] A-15) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 20, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 48; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 71, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 88, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 105;

[0103] A-16) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 11, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 29, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 49; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 106;

[0104] A-17) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 8, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 26, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 45; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 89, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 107;

[0105] A-18) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 12, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 25, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 50; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 89, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 108;

[0106] A-19) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 30, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 51; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 73, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 90, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 109;

[0107] A-20) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 14, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 31, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 52; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 89, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 108;

[0108] A-21) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 9, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 32, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 53; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 74, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 90, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 109;

[0109] A-22) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 15, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 33, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 54; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 75, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 80, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 110;

[0110] A-23) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 16, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 34, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 55; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 89, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 108;

[0111] A-24) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 56; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111; and

[0112] A-25) Anti-endoglin scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 17, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 36, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 57; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 76, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 112.

[0113] The anti-endoglin scFv of the present invention may comprise a heavy chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 113 to 133; and a light chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 134 to 158.

[0114] The anti-endoglin scFv of the present invention may comprise at least one of the following scFvs:

[0115] B-1) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 113 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 134;

[0116] B-2) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 113 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 135;

[0117] B-3) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 114 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 136;

[0118] B-4) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 115 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 137;

[0119] B-5) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 115 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 138;

[0120] B-6) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 116 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 139;

[0121] B-7) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 117 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 140;

[0122] B-8) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 118 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 141;

[0123] B-9) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 119 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 142;

[0124] B-10) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 119 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 143;

[0125] B-11) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 120 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 144;

[0126] B-12) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 121 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 145;

[0127] B-13) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 122 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 146;

[0128] B-14) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 123 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 147;

[0129] B-15) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 124 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 148;

[0130] B-16) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 125 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 149;

[0131] B-17) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 121 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 150;

[0132] B-18) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 126 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 151;

[0133] B-19) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 127 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 152;

[0134] B-20) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 128 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 153;

[0135] B-21) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 129 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 154;

[0136] B-22) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 130 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 155;

[0137] B-23) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 131 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 156;

[0138] B-24) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 132 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 157; and

[0139] B-25) Anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 133 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 158.

[0140] In the anti-endoglin scFv of the present invention, the heavy chain variable region and the light chain variable region may be linked to each other without a linker or via a peptide linker.

[0141] In the anti-endoglin scFv of the present invention, the binding order of the heavy chain variable region and the light chain variable region is not particularly limited and may be linked in any order. As a non-limiting example, the scFv may be linked in the order of the light chain variable region and the heavy chain variable region from the N-terminus to the C-terminus, or may be linked in the order of the heavy chain variable region and the light chain variable region. In addition, when the heavy chain variable region and the light chain variable region are linked via a peptide linker, the scFv may be linked in the order of the light chain variable region, the peptide linker, and the heavy chain variable region from the N-terminus to the C-terminus, or may be linked in the order of the heavy chain variable region, the peptide linker, and the light chain variable region.

[0142] In the anti-endoglin scFv of the present invention, the heavy chain variable region and the light chain variable region can be linked by a peptide linker comprising the amino acid sequence represented by SEQ ID NO: 159.

[0143] The amino acid sequence of the endoglin-binding scFv of the present invention may include any one amino acid sequence selected from SEQ ID NOs: 208 to 232, as exemplified in Table 3 below.

[0144]

[0145] The antibody or antigen-binding fragment of the present invention may include not only the sequence of the anti-endoglin antibody or antigen-binding fragment of the present invention described herein, but also biological equivalents thereof, as long as it can specifically recognize or bind to endoglin. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody or antigen-binding fragment. Such amino acid mutations are made based on the relative similarity of amino acid side-chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side-chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine can be considered biologically functional equivalents.

[0146] Considering the mutations having the above-described biological equivalent activity, the antibody or antigen-binding fragment of the present invention or the nucleic acid molecule encoding them is interpreted to also include a sequence showing substantial identity with the sequence described in the sequence number. The substantial identity means a sequence showing at least 90% homology, most preferably at least 95% homology, 96% or more, 97% or more, 98% or more, or 99% or more homology when the sequence of the present invention is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are known in the art. NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI, etc., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet. BLAST is available at www.ncbi.nlm.nih.gov / BLAST / . Instructions for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov / BLAST / blast_ help.html.

[0147] Based on this, the antibody or antigen-binding fragment of the present invention may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology to the specified sequences or the entirety of the sequences described in the specification. Such homology may be determined by sequence comparison and / or alignment using methods known in the art. For example, the percent sequence homology of the nucleic acids or proteins of the present invention may be determined using sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection.

[0148] The binding affinity of the anti-endoglin antibody or antigen-binding fragment provided in the present invention to endoglin is 10 -5 M to 10 -12 M range. For example, the binding affinity of an anti-endoglin antibody or antigen-binding fragment to endoglin is 10 -6 M to 10 -12 M, 10 -7 M to 10 -12 M, 10 -8 M to 10 -12 M, 10 -9 M to 10 -12 M, 10 -5 M to 10 -11 M, 10 -6 M to 10 -11 M, 10 -7 M to 10 -11 M, 10 -8 M to 10 -11 M, 10 -9 M to 10 -11 M, 10 -10 M to 10 -11 M, 10 -5 M to 10 -10 M, 10 -6 M to 10 -10 M, 10 -7 M to 10 -10 M, 10 -8 M to 10 -10 M, 10 -9 M to 10 -10 M, 10 -5 M to 10 -9 M, 10 -6 M to 10 -9 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -9 M, 10 -5 M to 10 -8 M, 10 -6 M to 10 -8 M, 10 -7 M to 10 -8 M, 10 -5M to 10 -7 M, 10 -6 M to 10 -7 M or 10 -5 M to 10 -6 M may be, but is not limited to, this.

[0149]

[0150] humanized antibody or antigen-binding fragment thereof

[0151] The present invention provides an anti-endoglin humanized antibody or an antigen-binding fragment thereof.

[0152] The humanized antibody provided in the present invention can specifically recognize endoglin or bind specifically to endoglin, and can have low immunogenicity even when administered to the human body.

[0153] The humanized antibody or antigen-binding fragment thereof of the present invention was produced based on a mouse antibody comprising a heavy chain variable region and a light chain variable region having the amino acid sequences shown in Tables 1 to 3 above, and in particular, was produced based on a mouse antibody of the ENG24 clone having the sequence information shown in Table 4 below.

[0154] Antibody type classification sequence information (5'->3') (underline corresponds to CDR) Sequence number ENG24VLEVKLEESGGGLVQPGGSMKLSCVVSGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYGTHYAESVKGRFTISRDDSKSSVYLQMNNLKTEDTGIYYCTDWSRHFDVWGAGTTVTVSS132VHQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPPHFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSYPPTFGAGTKLELK157

[0155] The sequence of the heavy chain variable region of the humanized antibody of the present invention or antigen-binding fragment thereof may have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence represented by SEQ ID NO: 132. Furthermore, the sequence of the light chain variable region of the humanized antibody of the present invention or antigen-binding fragment thereof may have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence represented by SEQ ID NO: 157.

[0156] The anti-endoglin humanized antibody of the present invention or an antigen-binding fragment thereof may comprise a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13 as shown in Table 5 below; a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35; and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 56, 233, 234, or 235.

[0157] In addition, the anti-endoglin humanized antibody or antigen-binding fragment of the present invention may comprise a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72 as shown in Table 5 below; a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83; and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111.

[0158] The anti-endoglin humanized antibody or antigen-binding fragment of the present invention may comprise a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 56, 233, 234, or 235, and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111.

[0159] The anti-endoglin humanized antibody or antigen-binding fragment of the present invention may comprise at least one humanized antibody or antigen-binding fragment selected from the following C-1) to C-3):

[0160] C-1) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 56; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111;

[0161] C-2) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111;

[0162] C-3) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 234; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111; and

[0163] C-4) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 235; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111.

[0164]

[0165] The anti-endoglin binding humanized antibody of the present invention or an antigen-binding fragment thereof may comprise a heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236 as shown in Table 6 below; a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 237, 238 or 239; a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240 or 241; and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242.

[0166] In addition, the anti-endoglin humanized antibody of the present invention or an antigen-binding fragment thereof may include a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243 as shown in Table 6 below; a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244 or 245; a light chain FR3 comprising an amino acid sequence represented by any one of SEQ ID NOs: 246 to 254; and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255.

[0167] The anti-endoglin humanized antibody of the present invention or an antigen-binding fragment thereof may comprise a heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 237, 238 or 239, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240 or 241 and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242, and a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244 or 245, a light chain FR3 comprising an amino acid sequence represented by any one of SEQ ID NOs: 246 to 254 and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255.

[0168]

[0169] The anti-endoglin humanized antibody or antigen-binding fragment of the present invention may comprise at least one humanized antibody or antigen-binding fragment selected from the following D-1) to D-13):

[0170] D-1) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 237, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 56, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 246, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0171] D-2) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 247, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0172] D-3) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 246, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0173] D-4) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 245, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 247, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0174] D-5) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 239, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 248, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0175] D-6) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 241, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 234, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 248, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0176] D-7) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 241, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 248, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0177] D-8) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 235, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 245, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 249, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0178] D-9) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 239, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 235, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 250, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0179] D-10) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 251, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0180] D-11) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 252, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0181] D-12) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 235, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; and a humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 245, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 253, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255; and

[0182] D-13) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 241, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 235, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 245, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 254, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255.

[0183] Additionally, the anti-endoglin humanized antibody or antigen-binding fragment of the present invention may comprise at least one heavy chain variable region selected from the group consisting of SEQ ID NOs: 256 to 262 and 325 as shown in Table 7 below.

[0184] The anti-endoglin humanized antibody or antigen-binding fragment of the present invention may comprise at least one light chain variable region selected from the group consisting of SEQ ID NOs: 263 to 272 as shown in Table 7 below.

[0185] The anti-endoglin humanized antibody or antigen-binding fragment of the present invention may comprise at least one heavy chain variable region selected from the group consisting of SEQ ID NOs: 256 to 262 and 325; and at least one light chain variable region selected from the group consisting of SEQ ID NOs: 263 to 272.

[0186] The anti-endoglin humanized antibody or antigen-binding fragment of the present invention may comprise at least one humanized antibody or antigen-binding fragment selected from the following E-1) to E-13):

[0187] E-1) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 256 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 263;

[0188] E-2) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 264;

[0189] E-3) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 263;

[0190] E-4) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 265;

[0191] E-5) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 325 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 266;

[0192] E-6) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 258 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 266;

[0193] E-7) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 259 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 266;

[0194] E-8) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 260 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 267;

[0195] E-9) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 261 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 268;

[0196] E-10) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 269;

[0197] E-11) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 270;

[0198] E-12) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 260 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 271; and

[0199] E-13) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 262 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 272.

[0200]

[0201] The anti-endoglin humanized antibody or antigen-binding fragment of the present invention may further comprise a linker between the heavy chain variable region and the light chain variable region. Here, the linker may be a peptide linker and may have a length of about 10-25 aa. For example, it may include a hydrophilic amino acid such as glycine and / or serine, but is not limited thereto. Specifically, in the present invention, the linker may include, for example, (GS)n, (GGS)n, (GSGGS)n or (GnS)m (wherein n and m are each 1 to 10), preferably GGGGS, and more preferably GGGGSGGGGSGGGGS represented by SEQ ID NO: 159, but is not limited thereto. The antigen-binding fragment provided in the present invention may be a humanized scFv (single chain variable fragment) that specifically recognizes and / or binds to endoglin.

[0202] The anti-endoglin humanized scFv of the present invention may comprise a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 56, 233, 234, or 235, and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111.

[0203] The anti-endoglin humanized scFv of the present invention may comprise at least one of the following scFvs:

[0204] C-1) A humanized scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 56; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111;

[0205] C-2) A humanized scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111;

[0206] C-3) A humanized scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 234; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111; and

[0207] C-4) A humanized scFv comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 235; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111.

[0208] The anti-endoglin humanized scFv of the present invention may include at least one humanized scFv selected from among the following D-1) to D-13):

[0209] D-1) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 237, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 56, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized scFv comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 246, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0210] D-2) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized scFv comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 247, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0211] D-3) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized scFv comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 246, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0212] D-4) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized scFv comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 245, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 247, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0213] D-5) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 239, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized scFv comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 248, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0214] D-6) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 241, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 234, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized scFv comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 248, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0215] D-7) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 241, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized scFv comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 248, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0216] D-8) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 235, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized scFv comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 245, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 249, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0217] D-9) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 239, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 235, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized scFv comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 250, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0218] D-10) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized scFv comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 251, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0219] D-11) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized scFv comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 252, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0220] D-12) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 235, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; and a humanized scFv comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 245, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 253, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255; and

[0221] D-13) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 241, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 235, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized scFv comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 245, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 254, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255.

[0222] The anti-endoglin humanized scFv of the present invention may comprise at least one heavy chain variable region selected from the group consisting of SEQ ID NOs: 256 to 262; and at least one light chain variable region selected from the group consisting of SEQ ID NOs: 263 to 272.

[0223] The anti-endoglin humanized scFv of the present invention may comprise at least one humanized scFv selected from among E-1) to E-13) below:

[0224] E-1) A humanized scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 256 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 263;

[0225] E-2) A humanized scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 264;

[0226] E-3) A humanized scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 263;

[0227] E-4) A humanized scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 265;

[0228] E-5) A humanized scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 325 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 266;

[0229] E-6) A humanized scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 258 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 266;

[0230] E-7) A humanized scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 259 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 266;

[0231] E-8) A humanized scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 260 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 267;

[0232] E-9) A humanized scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 261 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 268;

[0233] E-10) A humanized scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 269;

[0234] E-11) A humanized scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 270;

[0235] E-12) A humanized scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 260 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 271; and

[0236] E-13) A humanized scFv comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 262 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 272.

[0237] In the anti-endoglin scFv of the present invention, the heavy chain variable region and the light chain variable region may be linked to each other without a linker or via a peptide linker.

[0238] In the anti-endoglin scFv of the present invention, the binding order of the heavy chain variable region and the light chain variable region is not particularly limited and may be linked in any order. As a non-limiting example, the scFv may be linked in the order of the light chain variable region and the heavy chain variable region from the N-terminus to the C-terminus, or may be linked in the order of the heavy chain variable region and the light chain variable region. In addition, when the heavy chain variable region and the light chain variable region are linked via a peptide linker, the scFv may be linked in the order of the light chain variable region, the peptide linker, and the heavy chain variable region from the N-terminus to the C-terminus, or may be linked in the order of the heavy chain variable region, the peptide linker, and the light chain variable region.

[0239] In the anti-endoglin humanized scFv of the present invention, the heavy chain variable region and the light chain variable region can be linked by a peptide linker comprising the amino acid sequence represented by SEQ ID NO: 159.

[0240] The anti-endoglin humanized scFv of the present invention may comprise at least one humanized scFv selected from the group consisting of SEQ ID NOs: 273 to 285.

[0241]

[0242] The humanized antibody or antigen-binding fragment thereof of the present invention may include not only the sequence of the anti-endoglin humanized antibody or antigen-binding fragment of the present invention described herein, but also a biological equivalent thereof, within the scope of specifically recognizing endoglin. Regarding biological equivalents, description thereof is omitted hereinafter to avoid overlapping with the description above and undue complexity of the specification. The binding affinity of the anti-endoglin humanized antibody or antigen-binding fragment provided in the present invention to endoglin is 10 -5 M to 10 -12 M range. For example, the binding affinity of anti-endoglin humanized antibody or antigen-binding fragment to endoglin is 10 -5 M to 10 -12 M, 10 -6 M to 10 -12 M, 10 -7 M to 10 -12 M, 10 -8 M to 10 -12 M, 10 -9 M to 10 -12 M, 10 -5 M to 10 -11 M, 10 -6 M to 10 -11 M, 10 -7 M to 10 -11 M, 10 -8 M to 10 -11 M, 10 -9 M to 10 -11 M, 10 -10 M to 10 -11 M, 10 -5 M to 10 -10 M, 10 -6 M to 10 -10 M, 10 -7 M to 10 -10 M, 10 -8 M to 10 -10 M, 10 -9 M to 10 -10 M, 10 -5M to 10 -9 M, 10 -6 M to 10 -9 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -9 M, 10 -5 M to 10 -8 M, 10 -6 M to 10 -8 M, 10 -7 M to 10 -8 M, 10 -5 M to 10 -7 M, 10 -6 M to 10 -7 M or 10 -5 M to 10 -6 M may be, but is not limited to, this.

[0243] The anti-endoglin humanized antibody or antigen-binding fragment provided in the present invention may have a binding affinity for the endoglin antigen that is equivalent to or higher than that of the previously defined mouse antibody or antigen-binding fragment derived therefrom (e.g., scFv).

[0244]

[0245] According to another embodiment of the present invention, the present invention relates to a nucleic acid molecule encoding an antibody or antigen-binding fragment provided by the present invention.

[0246] As used herein, the term "nucleic acid" encompasses DNA (gDNA and cDNA) and RNA molecules. Nucleotides, the basic structural units of nucleic acids, include not only natural nucleotides but also analogues with modified sugar or base moieties. The sequences of nucleic acids encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

[0247] In the present invention, the DNA encoding the antibody or antigen-binding fragment can be easily isolated or synthesized using conventional molecular biological techniques (for example, by using an oligonucleotide probe that can specifically bind to DNA encoding the antibody and the heavy and light chains), and the nucleic acid is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or further expression.

[0248]

[0249] According to another embodiment, the present invention relates to a recombinant expression vector comprising a nucleic acid molecule provided in the present invention.

[0250] In the present invention, the above "vector" refers to a recombinant vector that can express a target protein when transfected into a suitable host cell, and a genetic construct that includes essential regulatory elements operably linked to allow the gene insert to be expressed. Here, the term "operably linked" means that a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein are functionally linked to perform a general function. The operably linked vector can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be easily performed using enzymes generally known in the art.

[0251] In the present invention, various types of vectors, such as nanoparticles, plasmids, viruses, and cosmids, can be used as recombinant expression vectors for inserting the foreign gene. The type of recombinant vector is not particularly limited as long as it has the function of expressing the desired gene and producing the desired protein in various host cells of prokaryotic and eukaryotic cells. Specifically, a vector that has a promoter that exhibits strong activity and a strong expression ability while being capable of mass-producing a foreign protein in a form similar to that in the natural state can be used.

[0252] A variety of gene delivery vehicles are known in the art, including both viral and non-viral (e.g., naked DNA, plasmid) vectors. Viral vectors suitable for gene delivery are known to those skilled in the art. Non-limiting examples of the viral vectors may include retroviral vectors (e.g., derived from Moloney murine leukemia virus vector (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenovirus (Ad) vectors including replication competent, replication deficient and emasculated forms thereof, adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus vectors, herpes virus vectors, varicella virus vectors, Harvey murine sarcoma virus vectors, murine mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, poliovirus vectors, vesicular stomatitis virus vectors, Maraba virus vectors and group B adenovirus enadenotuxireb vectors.

[0253] Non-viral vectors for gene transfer include naked DNA, plasmids, transposons, and mRNA. Non-limiting examples include pKK plasmid (Clonetech), pUC plasmid, pET plasmid (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), and pMAL plasmid (New England Biolabs, Beverly, Mass.).

[0254] The vector of the present invention can be introduced into many suitable host cells using methods disclosed or cited herein or otherwise known to those skilled in the art.

[0255] Suitable expression vectors of the present invention may include, in addition to expression regulatory elements such as a promoter, initiation codon, termination codon, polyadenylation signal, or enhancer, a nucleotide sequence encoding a signal peptide for membrane targeting or secretion. The initiation and termination codons are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein, and must be functional in a subject when the genetic construct is administered, and must be in frame with the coding sequence.

[0256] The term "promoter," as used herein, refers to any sequence that regulates the expression of a coding sequence, such as a gene. A promoter may be, for example, constitutive, inducible, repressible, or tissue-specific. A promoter is a control sequence, a region of a polynucleotide sequence where the initiation and rate of transcription are controlled. Non-limiting examples of the promoter in the present invention include Rous sarcoma virus (RSV) LTR promoter (optionally with RSV enhancer), cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, U6 promoter, EF1alpha short form (EFS) promoter, human polypeptide chain elongation factor (EF1a) promoter, P5 promoter, Ubc promoter, CAG promoter, TRE promoter, UAS promoter, Ac5 promoter, polyhedrin promoter, CaMKIIa promoter, Gal1 promoter, TEF1 promoter, GDS promoter, ADH1 promoter, CaMV35S promoter, ubiquitin (Ubi) Promoters such as ubiquitin C (UbiC), H1 promoter, U6 promoter, alpha-1-antitrypsin promoter, spleen focus-forming virus (SFFV) promoter, etc. may be included.

[0257] Additionally, in the present invention, the promoter may be coupled to an enhancer to increase transcription efficiency. Non-limiting examples of the enhancer include, but are not limited to, an RSV enhancer, a CMV enhancer, or an α-fetoprotein MERII enhancer. The vector of the present invention may also be fused with other sequences to facilitate the purification of antibodies expressed therefrom. Examples of the fused sequences include, but are not limited to, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA).

[0258] The vector of the present invention includes an antibiotic resistance gene commonly used in the art as a selection marker, and may include, but is not limited to, resistance genes for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0259]

[0260] According to another embodiment of the present invention, the expression vector provided in the present invention relates to a transfected host cell.

[0261] The term "host cell" as used herein includes an individual cell or cell culture that can be or has been the recipient of a vector(s) for incorporation of a polypeptide insert. Host cells include progeny of a single host cell, which progeny may not necessarily be completely identical (morphologically or in genomic DNA complement) to the original parent cell due to natural, accidental, or intentional mutation. Host cells include cells that have been transfected in vivo with the polypeptide(s) of the present disclosure.

[0262] In the present invention, the host cell may include a mammalian, plant, insect, fungal or cellular cell, and for example, bacterial cells such as Escherichia coli, Streptomyces, and Salmonella Typhimurium; fungal cells such as yeast cells and Pichia pastoris; insect cells such as Drozophylla and Spodoptera Sf9 cells; animal cells such as CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bow melanoma cells, HT-1080, BHK (Baby Hamster Kidney cells), HEK (Human Embryonic Kidney cells), or PERC.6 (human retinal cells); Alternatively, the host cell line may be a plant cell, but is not limited thereto, and any cell known to those skilled in the art that can be used as a host cell line may be used.

[0263] In the present invention, the transfection method is any method for injecting the desired vector into the host cell, and any known method capable of injecting a vector into a host cell can be included, and for example, a method using CaCl2, electroporation, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment, and virus-based transfection can be used, but is not limited thereto.

[0264]

[0265] According to another embodiment, the present invention relates to a method for producing an antibody or antigen-binding fragment that specifically binds to endoglin, comprising: a step of culturing the host cell to produce an antibody or antigen-binding fragment; and a step of isolating and purifying the produced antibody or antigen-binding fragment.

[0266] In the present invention, the host cells can be cultured in various media. Any commercially available media can be used as culture media. Any other essential supplements known to those skilled in the art may be included at appropriate concentrations. Culture conditions, such as temperature, pH, etc., are already used with host cells selected for expression and will be readily apparent to those skilled in the art.

[0267] In the present invention, the antibody or antigen-binding fragment thereof can be recovered by removing impurities, for example, by centrifugation or ultrafiltration, and purifying the resultant product using, for example, affinity chromatography. Additional purification techniques, for example, anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, etc., can be used.

[0268]

[0269] According to another embodiment of the present invention, there is provided a dual or multispecific antibody comprising an antibody or antigen-binding fragment provided by the present invention.

[0270] The term "bispecific antibody" as used herein means an antibody having binding ability or antagonism to more than one target, and means an antibody in which antibodies having binding ability or antagonism to two different targets are combined, or an antibody in which an antibody having binding ability to one target and a substance having antagonism to another target are combined.

[0271] The term "multispecific antibody" as used herein refers to an antibody having binding specificities for at least three different antigens. Multispecific antibodies may include antibodies that target more than three specific targets, such as trispecific antibodies, tetraspecific antibodies, or antibodies that target more than one target.

[0272] In the present invention, antibodies belonging to bispecific or multispecific antibodies can be classified into scFv-based antibodies, Fab-based antibodies, and IgG-based antibodies. In the case of bispecific or multispecific antibodies, since two or more signals can be simultaneously suppressed or amplified, they can be more effective than cases where a single signal is suppressed / amplified. In addition, compared to cases where each signal is treated with a separate signal inhibitor, lower dosage administration is possible, and two or more signals can be suppressed / amplified in the same time and space.

[0273] Methods for producing bispecific or multispecific antibodies are widely known. Traditionally, recombinant production of bispecific antibodies relies on the co-expression of two or more immunoglobulin heavy chain / light chain pairs, where the two or more heavy chains have different specificities.

[0274] In the present invention, in the case of a bispecific or multispecific antibody based on scFv, the VL and VH of different scFvs can be combined with each other to produce a hybrid scFv in a heterodimeric form to produce a diabody, different scFvs can be linked to each other to produce a tendem ScFv, CH1 and CL of Fab can be expressed at the ends of each scFv to produce a heterodimeric miniantibody, and some amino acids of the CH3 domain, which is a homodimeric domain of Fc, can be substituted to change it into a heterodimeric structure in the form of a 'knob into hole', and these changed CH3 domains can be expressed at the ends of different scFvs to produce a minibody in the form of a heterodimeric scFv.

[0275] In the present invention, in the case of a bispecific or multispecific antibody based on Fab, individual Fabs for specific antigens can be combined with each other using a disulfide bond or a mediator to produce a heterodimeric Fab form, and by expressing scFvs for different antigens at the terminals of the heavy or light chains of a specific Fab, it can be produced to have two antigen-binding valencies, or by providing a hinge region between the Fab and scFv, it can be produced to have four antigen-binding valencies in a homodimeric form. In addition, a dual-target bibody in which the antigen-binding valencies are made three by fusing scFvs for different antigens to the light and heavy chain terminals of the Fab, and a tri-target bibody in which the antigen-binding valencies are made three by fusing different scFvs to the light and heavy chain terminals of the Fab, respectively, can be obtained by chemically conjugating three different Fabs.

[0276] In the case of IgG-based bispecific or multispecific antibodies in the present invention, a method for producing bispecific antibodies by producing hybrid hybridomas, also known as quadromas, by crossbreeding mouse and rat hybridomas again has been known by Trion Pharma. In addition, bispecific antibodies can be produced in the so-called 'Holes and Knob' form, which is produced in a heterodimeric form by modifying some amino acids in the CH3 homodimeric domain of Fc for different heavy chains while sharing the light chain portion. In addition to the heterodimeric form of bispecific antibodies, two different scFvs can be fused and expressed in the constant domains of the light and heavy chains of IgG instead of the variable domains, thereby producing (scFv)4-IgG in a homodimeric form. In addition, ImClone reported the production of a bispecific antibody by fusing only a single variable domain for mouse platelet-derived growth factor receptor-α to the light chain amino terminus of IMC-1C11, a chimeric monoclonal antibody against human VEGFR-2. In addition, antibodies with multiple antigen-binding affinities for CD20 can be produced through the so-called 'dock and lock (DNL)' method using the dimerization and docking domain (DDD) of the R subunit of protein kinase A (PKA) and the anchoring domain of PKA.

[0277] In the present invention, a wide variety of recombinant antibody formats have been developed, for example, bivalent, trivalent, or tetravalent bispecific or multispecific antibodies. For example, it also includes bivalent, trivalent, or tetravalent antibodies described in International Patent Application Publication Nos. WO2001 / 077342, WO2009 / 080251, WO2009 / 080252, WO2009 / 080253, WO2009 / 080254, WO2010 / 112193, WO2010 / 115589, WO2010 / 136172, WO2010 / 145792, WO2010 / 145793, and WO2011 / 117330. An antibody being bivalent, trivalent, or tetravalent indicates that two or more binding domains, three or more binding domains, or four or more binding domains are present in the antibody molecule, respectively.

[0278] The bispecific or multispecific antibody according to the present invention may specifically comprise the anti-endoglin antibody or antigen-binding fragment thereof in the form of a complete IgG antibody or a fragment thereof, for example, a single-chain Fv, a VH domain and / or a VL domain, a Fab or (Fab)2. In addition, the antibody targeting the endoglin may comprise an antibody binding to a target other than the antibody targeting the endoglin, for example, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, An antibody targeting one or more selected from the group consisting of MARCO, specifically an IgG complete antibody or fragment thereof, for example in the form of a single chain Fv, VH domain and / or VL domain, Fab or (Fab)2.

[0279] The bispecific or multispecific antibodies according to the present invention may provide additional binding specificities induced or mediated by other targets in addition to endoglin.

[0280] For example, the bispecific antibody according to the present invention binds to endoglin and PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD3, CD20, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, MARCO, FcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, NKG2D, CD3, CD16a, CD19, CD33, CD22, It can simultaneously target one or more selected from the group consisting of CD79A, CD79B, EDAR, GFRA1, MRP4, RET, Steap1, and TenB2.

[0281] For example, the multispecific antibody according to the present invention binds to endoglin and PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD3, CD20, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, MARCO, FcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, NKG2D, CD3, CD16a, CD19, CD33, CD22, It can simultaneously target two or more selected from the group consisting of CD79A, CD79B, EDAR, GFRA1, MRP4, RET, Steap1, and TenB2.

[0282]

[0283] According to another embodiment of the present invention, there is provided an immune cell engaging bispecific or multispecific antibody comprising an antigen binding fragment (scFv) provided by the present invention and at least one scFv that binds to an immune cell activating antigen.

[0284] In the present invention, a cytolytic synapse between cytotoxic T cells or NK cells and cancer target cells is temporarily induced through the immune cell engaging bispecific or multispecific antibody, thereby causing the release of toxic substances.

[0285] In the present invention, the T cell activation antigen may be, but is not limited to, CD3, TCRα, TCRβ, TCRγ, TCRξ, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2 or CD226.

[0286] In the present invention, the NK cell activating antigen may be, but is not limited to, NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (eg, CD16a, CD16b), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E or CD160.

[0287] Other immune cell engagers are specifically described in U.S. Patent Application Publication No. 2017 / 0368169, which may be incorporated herein by reference.

[0288] Specifically, the immune cell engaging bispecific or multispecific antibody comprises a tandem scFv and can bind to the following antigen and a surface antigen on a cancer cell. The surface antigen on the cancer cell is endoglin, which is targeted by the antibody according to the present invention.

[0289] In the present invention, the immune cell engaging bispecific or multispecific antibody may include a structure in the form of, for example, VL(endoglin)-VH(endoglin)-VH(CD3 or CD16A)-VL(CD3 or CD16A), VH(endoglin)-VL(endoglin)-VH(CD3 or CD16A)-VL(CD3 or CD16A), VH(CD3 or CD16A)-VL(CD3 or CD16A)-VH(endoglin)-VL(endoglin), or VH(CD3 or CD16A)-VL(CD3 or CD16A)-VL(endoglin)-VH(endoglin).

[0290] The present invention may further include a linker between the heavy chain variable region and the light chain variable region. The linker may be a peptide linker and have a length of about 10-25 amino acids. For example, the linker may include, but is not limited to, hydrophilic amino acids such as glycine and / or serine.

[0291] Specifically, in the present invention, the linker may include, for example, (GS)n, (GGS)n, (GSGGS)n or (GnS)m (n and m are each 1 to 10), preferably GGGGS, and more preferably GGGGSGGGGSGGGGS represented by SEQ ID NO: 159, but is not limited thereto.

[0292] Examples of the immune cell-engaging bispecific or multispecific antibodies in the present invention include blinatumomab (Amgen), which binds to CD3 and CD19; solitomab (Amgen), which binds to CD3 and EpCAM; MEDI 565 (MedImmune, Amgen), which binds to CD3 and CEA; and BAY2010112 (Bayer, Amgen), which binds to CD3 and PSMA. Exemplary DARTs include MGD006 (Macrogenics), which binds to CD3 and CD123; and MGD007 (Macrogenics), which binds to CD3 and gpA33. Exemplary TandAbs may include AFM11 (Affimed Therapeutics), which binds to CD3 and CD19; and AFM13 (Affimed Therapeutics), which binds to CD30 and CD16A.

[0293]

[0294] immunoconjugate

[0295] According to another embodiment of the present invention, the present invention relates to an immunoconjugate in which an antibody or antigen-binding fragment provided by the present invention is combined with a cytotoxic agent.

[0296] In the present invention, the cytotoxic agent may be at least one selected from the group consisting of a chemotherapeutic agent or a chemotherapeutic drug, a growth inhibitor, a toxin (e.g., a protein toxin, an enzymatically active toxin or fragment thereof derived from bacteria, fungi, plants or animals), and a radioactive isotope.

[0297] As an example of implementation, the immune conjugate provided by the present invention may be an antibody-drug conjugate (ADC) in which the antibody or antigen-binding fragment is bound to a drug.

[0298] The term "antibody-drug conjugate (ADC)" used herein refers to a form in which a drug and an antibody are chemically linked without reducing the biological activity of the antibody and drug. In the present invention, the antibody-drug conjugate refers to a form in which a drug is bound to an amino acid residue at the N-terminus of the heavy chain and / or light chain of an antibody, specifically, a form in which a drug is bound to an α-amine group at the N-terminus of the heavy chain and / or light chain of an antibody.

[0299] In the present invention, the antibody or antigen-binding fragment may be bound to a drug via a linker. The linker serves as a linking moiety between the antibody and the drug. It must be cleavable under intracellular conditions, i.e., enable the drug to be released from the antibody in the intracellular environment. Reflecting the antibody's long half-life, the antibody must be stable in systemic circulation, and the linker-drug binding must not affect the stability or pharmacokinetics of the antibody.

[0300] In the present invention, the linker may include, for example, a cleavable linker or a non-cleavable linker. In the case of a cleavable linker, like a peptide linker, it can be cleaved by an intracellular peptidase or protease enzyme, such as a lysosomal or endosomal protease, and in the case of a non-cleavable linker, for example, a thioether linker, the drug can be released after the antibody is non-selectively degraded by intracellular hydrolysis.

[0301] In the present invention, the cleavable linker may include a peptide linker. The peptide linker has a length of at least two amino acids. For example, it may include a Val-Cit, Val-Ala, or Val-Cit dipeptide, or Phe-Leu or Gly-Phe-Leu-Gly. Examples of linkers are specifically described in International Patent Application Publication No. WO2004 / 010957, which may be incorporated herein by reference.

[0302] In the present invention, the antibody-drug conjugate binds to the antigen of the target cancer cell, forming an ADC-antigen complex, which is then internalized into the cancer cell via the endosome-lysosome pathway. In this case, the intracellular release of the cytotoxic drug is controlled by the internal environment of the endosome / lysosome.

[0303] In the present invention, the cleavable linker is pH-sensitive and may be susceptible to hydrolysis at a specific pH value. Generally, a pH-sensitive linker indicates that it can be hydrolyzed under acidic conditions. For example, it may be an acid-labile linker that can be hydrolyzed in lysosomes, such as a hydrazone, a semicarbazone, a thiosemicarbazone, a cis-aconitic amide, an orthoester, an acetal, a ketal, etc.

[0304] In the present invention, the linker may be cleaved under reducing conditions, and for example, a disulfide linker may correspond to this. Various disulfide bonds may be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene). These disulfide linkers can be cleaved by disulfide exchange with the thiol of intracellular glutathione.

[0305] In the present invention, the drug and / or drug-linker may be randomly conjugated via lysine of the antibody, or conjugated via cysteine ​​exposed when the disulfide bond chain is reduced. In some cases, the linker-drug may be conjugated via cysteine ​​present in a genetically engineered tag, for example, a peptide or a protein. The genetically engineered tag, for example, a peptide or a protein, may include an amino acid motif that can be recognized by, for example, an isoprenoid transferase. The peptide or protein may have a deletion at the carboxyl terminus of the peptide or protein, or may have an addition via covalent bonding of a spacer unit to the carboxyl (C) terminus of the peptide or protein.

[0306] In the present invention, the peptide or protein may be directly covalently linked to the amino acid motif or may be covalently linked to a spacer unit to be linked to the amino acid motif. The amino acid spacer unit is composed of 1 to 20 amino acids, and among them, a glycine unit is preferred.

[0307] In the present invention, the linker may include a beta-glucuronide linker that is recognized and hydrolyzed by beta-glucuronidase, which is present in large numbers in lysosomes or is overexpressed in some tumor cells. Unlike peptide linkers, it has a high hydrophilicity, and thus has the advantage of increasing the solubility of the antibody-drug complex when bound to a drug with a high hydrophobicity. In this regard, the beta-glucuronide linker disclosed in International Patent Application Publication No. WO2015 / 182984, for example, a beta-glucuronide linker including a self-immolative group, may be used, and the above document is incorporated by reference.

[0308] In the present invention, the linker may be, for example, a non-cleavable linker, and the drug is released through a single step of antibody hydrolysis within the cell, producing, for example, an amino acid-linker-drug conjugate. This type of linker may be a thioether group or maleimidocaproyl group, and may maintain stability in the blood.

[0309] In the present invention, when the disulfide bond chain of the antibody or antigen-binding fragment is reduced, the linker-drug can be bound by introducing a random linkage or an antibody-terminal binding peptide having the sequence GGGGGGGCVIM through a cysteine ​​exposed therein.

[0310] In the present invention, the drug is a pharmacologically effective agent that can be bound to an antibody, and may be, specifically, a chemotherapeutic agent, a toxin, microRNA (miRNA), siRNA, shRNA, or a radioactive isotope. The chemotherapeutic agent may be, for example, a cytotoxic agent or an immunosuppressant. Specifically, it may include a microtubulin inhibitor, a mitotic inhibitor, a topoisomerase inhibitor, or a chemotherapeutic agent that can function as a DNA intercalator. In addition, it may include a drug for treating cancer, inflammation, or immune diseases. For example, it may include an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an anthelmintic agent, or a combination thereof.

[0311] In the present invention, the anticancer agent is, for example, maytansinoid, auristatin (including MMAE, MMAF), aminopterin, actinomycin, bleomycin, talisomycin, camptothecin, N8-acetyl spermidine, 1-(2 chloroethyl)-1,2-dimethyl sulfonyl hydrazide, esperamycin, etoposide, 6-mercaptopurine, dolastatin, trichothecene, calicheamicin, taxol, taxane, paclitaxel, docetaxel, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, duocarmycin, L-asparaginase, mercaptopurine, Thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustard, cytoxan, etoposide, 5-fluorouracil, bischloroethylnitrosourea, irinotecan, camptothecin, bleomycin, idarubicin, daunorubicin, Dactinomycin, plicamycin, mitoxantrone, asparaginase, vinorelbine, chlorambucil, melphalan, carmustine, lomustine,Busulfan, treosulfan, decarbazine, etoposide, teniposide, topotecan, 9-aminocamptothecin, crisnatol, mitomycin C, trimetrexate, mycophenolic acid, tiazofurin, ribavirin, EICAR (5-ethynyl-1-beta-Dribofuranosylimidazole-4-carboxamide), hydroxyurea, defoxamine, fluxuridine, doxifluridine, raltitrexed, Cytarabine (ara C), cytosine arabinoside, fludarabine, tamoxifen, raloxifene, megestrol, goserelin, leuprolide acetate, flutamide, bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4, demethoxy-hypocrellin A, interferon-α, interferon-γ, tumor necrosis factor, Gemcitabine, Velcade, Revamid, Thalamide, Lovastatin,It may be at least one selected from the group consisting of 1-methyl-4-phenylpyridinium ion, staurosporine, actinomycin D, dactinomycin, bleomycin A2, bleomycin B2, peplomycin, epirubicin, pirarubicin, zorubicin, mitoxantrone, verapamil, and thapsigargin, nucleases, and toxins of bacterial, animal, or plant origin, but is not limited thereto.

[0312] In the present invention, the anti-inflammatory agent may include, but is not limited to, a steroid preparation that reduces inflammation or swelling by binding to a glucocorticoid receptor, a non-steroidal anti-inflammatory drug (NSAID) that relieves pain by responding to cyclic oxygenase (COX) that synthesizes prostaglandins that cause inflammation, or an immunospecific anti-inflammatory drug (ImSAID) that alters the activation and transport of inflammatory cells.

[0313] In the present invention, the immune disease treatment agent may include, but is not limited to, azathioprine, chlorambucil, cyclophosphamide, cyclosporine, mycophenolate, azathioprine, or methotrexate.

[0314] In the present invention, in some cases, the drug may include one or more nucleophilic groups selected from the group consisting of, but not limited to, amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups that can react to form a covalent bond with an electrophilic group on a linker and a linker reagent.

[0315]

[0316] chimeric antigen receptor (CAR)

[0317] According to another embodiment of the present invention, there is provided a chimeric antigen receptor (CAR) comprising an antigen binding fragment provided by the present invention.

[0318] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an engineered receptor comprising an extracellular antigen-binding domain and an intracellular signaling domain. Although the most common type of CAR comprises a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to the transmembrane and intracellular domains of a T cell coreceptor, such as the CD3ζ chain, the invention described herein is not limited to these domains. Rather, as used herein, "chimeric antigen receptor" or "CAR" refers to any receptor engineered to express any intracellular signaling molecule and an extracellular antigen-binding domain fused or linked thereto.

[0319] In the present invention, the binding domain may include an antigen binding fragment provided in the present invention, preferably scFv.

[0320] As an example, in the present invention, the binding domain comprises a heavy chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 113 to 133; and a light chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 134 to 158, wherein the heavy chain variable region and the light chain variable region may be an anti-endoglin scFv comprising an amino acid sequence represented by SEQ ID NO: 159, but are not limited thereto.

[0321] As another example, in the present invention, the binding domain may be an anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 113 to 133; and a light chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 134 to 158, wherein the heavy chain variable region and the light chain variable region are connected by a peptide linker comprising an amino acid sequence represented by SEQ ID NO: 159, but is not limited thereto.

[0322] As another example, in the present invention, the binding domain may be an anti-endoglin scFv comprising any one amino acid sequence selected from SEQ ID NOs: 208 to 232, but is not limited thereto.

[0323] As another example, the binding domain in the present invention may be an anti-endoglin scFv comprising a heavy chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 256 to 262; and a light chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 263 to 272, but is not limited thereto.

[0324] As another example, in the present invention, the binding domain comprises a heavy chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 256 to 262; and a light chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 263 to 272, wherein the heavy chain variable region and the light chain variable region may be an anti-endoglin scFv comprising an amino acid sequence represented by SEQ ID NO: 159, but are not limited thereto.

[0325] As another example, in the present invention, the binding domain may be an anti-endoglin scFv comprising any one amino acid sequence selected from SEQ ID NOs: 273 to 285, but is not limited thereto.

[0326] The chimeric antigen receptor of the present invention may further comprise at least one of a hinge region (or spacer) and a signal transduction domain.

[0327] In the present invention, the hinge region is a portion that connects the antigen binding domain and the transmembrane domain, also called a 'spacer', and has the purpose of extending the antigen binding domain from the T cell membrane or NK cell membrane.

[0328] In the present invention, the hinge region may be obtained from any suitable sequence from any genus, including, for example, human or a part thereof, or may include a hinge region of a human protein, including all or part of CD8, CD28, 4-1BB, OX40, CD3 zeta (ζ) chain, T cell receptor α or β chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, or combinations thereof, but is not limited thereto.

[0329] Additionally, in the present invention, the hinge region may include, but is not limited to, one selected from immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, and IgD).

[0330] In the present invention, the hinge region may be a CD8 hinge region including an amino acid sequence represented by SEQ ID NO: 161, but is not limited thereto.

[0331] In the present invention, the signal transduction domain refers to a portion of a chimeric antigen receptor that is found or engineered to be found within a T cell. In the present invention, the signal transduction domain may or may not include a transmembrane domain that serves to anchor the chimeric antigen receptor to the plasma membrane of the T cell.

[0332] In the present invention, the transmembrane domain and the signal transduction domain may be derived from the same protein (e.g., CD3ζ), or the transmembrane domain and the signal transduction domain may be derived from different proteins (e.g., the transmembrane domain of CD28 and the intracellular signal transduction domain of the CD3ζ molecule, or vice versa).

[0333] In the present invention, the transmembrane domain comprises a hydrophobic polypeptide that spans the cell membrane. In particular, the transmembrane domain can span from one side of the cell membrane (extracellular) to the other side of the cell membrane (intracellular or cytoplasmic).

[0334] In the present invention, the transmembrane domain may be in the form of an alpha helix, a beta barrel, or a combination thereof. Furthermore, in the present invention, the transmembrane domain may comprise a multi-component protein having multiple transmembrane segments, each of which is in the form of an alpha helix, a beta sheet, or a combination thereof.

[0335] In the present invention, the transmembrane domain may include, but is not limited to, for example, T cell receptor α or β chain, all or part of CD3 zeta (ζ) chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, or combinations thereof.

[0336] In the present invention, the transmembrane domain may be, for example, an artificially designed polypeptide comprising primarily hydrophobic residues such as leucine and valine. In one embodiment of the present invention, a triad of phenylalanine, tryptophan, and valine may be found at each end of the synthetic transmembrane domain.

[0337] In the present invention, the transmembrane domain may be a CD8 transmembrane domain comprising an amino acid sequence represented by SEQ ID NO: 162, but is not limited thereto.

[0338] In the present invention, at least one costimulatory domain may be further included between the transmembrane domain and the signal transduction domain. In the present invention, the costimulatory domain is a site through which a costimulatory signal is transmitted, and may play a role in transmitting a signal so that CAR-T cells or CAR-NK cells that recognize a specific antigen bound to the antigen binding domain cause an immune response, promote self-proliferation, and increase the duration of their retention in the body.

[0339] In the present invention, the costimulatory domain may include, but is not limited to, a functional signaling domain derived from a polypeptide comprising a ligand that binds to 4-1BB (CD137); OX40; CD27; CD28; CD30; CD40; PD-1; CD2; CD7; CD258; natural killer group 2 member C (NKG2C); natural killer group 2 member (NKG2D); B7-H3; CD83; ICAM-1; LFA-1 (CD11a / CD18) or ICOS; an active fragment thereof; a functional derivative thereof; or a combination thereof.

[0340] In the present invention, the co-stimulatory domain may be 4-1BB including an amino acid sequence represented by SEQ ID NO: 163, but is not limited thereto.

[0341] In the present invention, the signal transduction domain may comprise a polypeptide that provides activation of an immune cell to stimulate or activate at least some aspect of an immune cell signal transduction pathway.

[0342] In the present invention, the signal transduction domain may include, but is not limited to, a functional signal transduction domain derived from a polypeptide comprising all or part of CD3 zeta (ζ), common FcR gamma (FcER1G), FcgammaRIIIa, FcRbeta (Fc epsilon lip), CD3gamma, CD3delta, CD3epsilon, CD79a, CD79b, DNAX-activating protein 10 (DAP10), DNAX-activating protein 12 (DAP12), an active fragment thereof, a functional derivative thereof, or a combination thereof, and such signal transduction domains are known in the art.

[0343] In the present invention, the signal transduction domain may be CD3 zeta (ζ) comprising an amino acid sequence represented by SEQ ID NO: 164, but is not limited thereto.

[0344] Additionally, the chimeric antigen receptor of the present invention may further comprise a signal peptide.

[0345] In the present invention, the "signal peptide" comprises a peptide sequence, which is a type of peptide of any secreted or transmembrane protein, and can direct the transport of the chimeric antigen receptor of the present invention to the cell membrane and cell surface, and provide precise localization of the chimeric antigen receptor of the present invention. In particular, in the present invention, the signal peptide supports the chimeric antigen receptor of the present invention to the cell membrane, in which the extracellular portion of the chimeric antigen receptor is displayed on the cell surface, the transmembrane portion spans the plasma membrane, and the active domain is in the cytoplasmic portion, or inside the cell.

[0346] In the present invention, the signal peptide may be used without limitation as long as it is a peptide having the above function, but for example, it may be an n-terminal CD8α signal peptide including an amino acid sequence represented by SEQ ID NO: 160, but is not limited thereto.

[0347] FIG. 7 schematically illustrates the structure of a chimeric antigen receptor according to one embodiment of the present invention, which may be composed of or include an endoglin binding domain (scFv according to the present invention (VL-linker-VH))-CD8 hinge-CD8 transmembrane domain-4-1BB-CD3ζ, or may be composed of or include a signal peptide-endoglin binding domain (scFv according to the present invention)-CD8 hinge-CD8 transmembrane domain-4-1BB-CD3ζ.

[0348] As an example, the chimeric antigen receptor provided in the present invention may be composed of or include an amino acid sequence represented by any one of SEQ ID NOs: 168 to 192, but is not limited thereto.

[0349] As another example, the chimeric antigen receptor provided in the present invention may be composed of or include an amino acid sequence represented by any one of SEQ ID NOs: 286 to 298, but is not limited thereto.

[0350] As another example, the chimeric antigen receptor provided in the present invention may further include a sequence in which a CD8α signal peptide (n-terminal CD8α signal peptide) including an amino acid sequence represented by SEQ ID NO: 160 is linked to the N-terminus of an amino acid sequence represented by any one of SEQ ID NOs: 168 to 192 and 286 to 298. The chimeric antigen receptor to which a signal peptide is linked in the present invention may be composed of or include an amino acid sequence represented by any one of SEQ ID NOs: 193 to 199 and 299 to 311, but is not limited thereto.

[0351]

[0352] According to another embodiment of the present invention, the present invention relates to a nucleic acid molecule encoding a chimeric antigen receptor according to the present invention.

[0353] In the present invention, the nucleic acid molecule encoding the chimeric antigen receptor is readily prepared from the amino acid sequence of the specified chimeric antigen receptor by any conventional method. The base sequence encoding the amino acid sequence can be obtained for each domain from the aforementioned NCBI RefSeq ID or GenBenk accession number, and the nucleic acid of the present disclosure can be produced using standard molecular biological and / or chemical procedures. For example, a polynucleotide based on the base sequence can be synthesized, and the nucleic acid molecule of the present disclosure can be produced by combining DNA fragments obtained from a cDNA library using polymerase chain reaction (PCR).

[0354] The nucleic acid molecule of the present invention may be, but is not limited to, a gene or part of an expression or cloning cassette.

[0355]

[0356] According to another embodiment of the present invention, there is provided an expression cassette comprising a nucleic acid molecule provided by the present invention.

[0357] The expression cassette of the present invention may include expression control sequences such as a promoter, enhancer, polyadenylation signal, transcription terminator or internal ribosome entry site (IRES) capable of regulating the expression of the nucleic acid molecule of the present invention.

[0358] In the present invention, the promoter may include, but is not limited to, for example, the SFFV promoter, the elongation factor 1α (EF 1a) promoter, or the CAG (chicken beta-actin promoter with CMV enhancer) promoter.

[0359] In the present invention, the promoter may be an SFFV promoter including a base sequence represented by SEQ ID NO: 167, but is not limited thereto.

[0360] The expression cassette of the present invention further comprises a gene sequence encoding a surrogate marker useful for monitoring its expression, for example a cell surface marker (e.g., a truncated cell surface marker), which can be used to confirm transduction or manipulation of cells expressing the receptor.

[0361] Exemplary marker genes in the present invention may include, but are not limited to, base sequences encoding truncated epidermal growth factor receptor (EGFRt), prostate-specific membrane antigen (PSMA), CD34, nerve growth factor receptor (NGFR), or variant forms thereof.

[0362] In addition, in the present invention, the marker gene may include, but is not limited to, a base sequence encoding a fluorescent protein, for example, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), for example, super-fold GFP (sfGFP), red fluorescent protein (RFP), for example, tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP) and yellow fluorescent protein (YFP), and species variants, monomer variants and codon-optimized and / or enhanced variants of the fluorescent protein.

[0363] In addition, in the present invention, the marker gene is or includes an enzyme, for example, luciferase, lacZ gene derived from Escherichia coli, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyl transferase (CAT). Exemplary luminescent reporter genes may include, but are not limited to, a base sequence encoding luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or a variant thereof.

[0364] In addition, in the present invention, the marker gene is a gene encoding a polypeptide that confers resistance to an exogenous agent or drug, for example, a puromycin resistance gene, a hygromycin resistance gene, a blasticidin resistance gene, a neomycin resistance gene, a geneticin resistance gene, or a zeocin resistance gene, or a modified form thereof, or may include the same, but is not limited thereto.

[0365] As an example, in the present invention, the marker gene may include a base sequence encoding GFP represented by SEQ ID NO: 166.

[0366] Additionally, in the present invention, one end of the marker gene and the polynucleotide of the present invention may be linked by a polynucleotide encoding a cleavable peptide.

[0367] In the present invention, the cleavable peptide corresponds to a self-cleavage site and may be, but is not limited to, P2A, F2A, E2A, T2A peptides or a combination thereof.

[0368] As an example, the gene encoding the cleavable peptide in the present invention may include a base sequence encoding P2A represented by SEQ ID NO: 165.

[0369]

[0370] According to another embodiment of the present invention, there is provided a recombinant expression vector comprising an expression cassette provided by the present invention.

[0371] In the present invention, the description of the recombinant expression vector overlaps with that described above for the antibody or antigen-binding fragment, and thus the description thereof is omitted below.

[0372]

[0373] According to another embodiment of the present invention, the present invention relates to an immune effector cell transduced with a recombinant expression vector provided by the present invention.

[0374] In the present invention, the immune effector cells may be allogeneic or autologous immune cells. Furthermore, the immune effector cells in the present invention may be derived from, but are not limited to, T cells, dendritic cells, killer dendritic cells, mast cells, NK cells, NKT cells, macrophages, monocytes, B cells, and stem cells.

[0375] The term "natural killer cells (NK cells)" as used herein is defined as large granular lymphocytes (LGL), which constitute three types of cells that differentiate from common lymphoid progenitor-producing B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus and enter the circulation. In the present invention, the NK cells may include any type of NK cell without limitation, and may include, but are not limited to, cultured NK cells, such as primary NK cells, NK cells from a cultured NK cell line, or NK cells obtained from a mammal. When the NK cells are obtained from a mammal, the NK cells may be obtained from a number of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The NK cells may be concentrated or purified. The NK cells may preferably be human NK cells (e.g., isolated from a human). NK cell lines are available from, for example, the American Type Culture Collection (ATCC), and include, for example, NK-92 cells (ATCC CRL-2407), NK92MI cells (ATCC CRL-2408), or derivatives thereof.

[0376]

[0377] According to another embodiment of the present invention, the present invention relates to a polypeptide expressed from an immune effector cell provided by the present invention.

[0378] As an example, the polypeptide expressed in the immune effector cell provided in the present invention may be composed of or include an amino acid sequence represented by any one of SEQ ID NOs: 168 to 192, but is not limited thereto.

[0379] As another example, the polypeptide expressed in the immune effector cells provided in the present invention may be composed of or include an amino acid sequence represented by any one of SEQ ID NOs: 286 to 298, but is not limited thereto.

[0380]

[0381] therapeutic uses

[0382] According to another embodiment of the present invention, there is provided a pharmaceutical composition for preventing, improving or treating an endoglin-related disease, comprising as an active ingredient an antibody or antigen-binding fragment provided by the present invention; an immunoconjugate; a chimeric antigen receptor; or an immune effector cell expressing a chimeric antigen receptor.

[0383] In the present invention, the endoglin-related disease may be an angiogenesis-related disease.

[0384] The term "angiogenesis" is used herein to encompass all aspects of blood vessel maintenance and development. Therefore, angiogenesis encompasses not only the maintenance and regulation of existing vasculature and small blood vessels, but also the formation of new capillaries, leading to neovascularization. Angiogenesis is a complex process involving a series of sequential steps, including endothelial cell-mediated degradation of the vascular basement membrane and interstitial matrix, endothelial cell migration, endothelial cell proliferation, and endothelial cell-mediated capillary loop formation. Angiogenesis encompasses the growth and / or development of new blood vessels (also called neovascularization), the dilatation of small blood vessels, excessive or prolonged vessel growth, and the maintenance of existing vasculature. Endoglin is known to be involved in the regulation of angiogenesis and is thought to be involved in multiple biochemical pathways involved in the induction of angiogenesis (Duff et al., FASEB J., 17:984-992 (2003); Bernabeu et al., J. Cell. Biochem., 102(6):1375-1388 (2007)).

[0385] The term "angiogenesis-related disease" is used herein to refer to a specific pathological process in humans in which angiogenesis is abnormally prolonged. This further encompasses angiogenic diseases and conditions, such as diseases and conditions related to, caused by, or associated with angiogenesis. Non-limiting examples of such diseases include various forms of ocular diseases characterized by angiogenesis / neovascularization (e.g., macular degeneration, diabetic retinopathy), diabetic nephropathy, chronic inflammatory diseases (e.g., IBD), rheumatoid arthritis, osteoarthritis, and various forms of cancer and metastasis. The antibodies and antigen-binding fragments thereof disclosed herein can be used to treat angiogenesis-related diseases by binding to endoglin and inhibiting angiogenesis.

[0386] The term "cancer" as used herein refers to or represents a physiological condition typically characterized by uncontrolled cell growth in mammals. The cancer to be prevented, improved, or treated in the present invention may be a solid tumor formed by a mass of abnormally grown cells in a solid organ, and may include, but is not limited to, gastric cancer, liver cancer, glioma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, renal cell carcinoma, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, or lung cancer, depending on the location of the solid organ.

[0387] The term "ocular disease characterized by neovascularization" is used herein for purposes of this application and the claims to mean any ocular disease caused by or leading to increased neovascularization within any part of the eye, including the retina, cornea, pupil, iris, vitreous humor, or aqueous humor. Such diseases may include, for example, age-related macular degeneration, diabetic retinopathy, non-diabetic retinopathy, choroidal neovascularization (CNV), and subretinal neovascularization.

[0388] In the present invention, the term "prevention" may include, without limitation, any action that can block, suppress or delay symptoms caused by an endoglin-related disease by using the composition of the present invention.

[0389] In the present invention, the terms “improvement” and “treatment” may include, without limitation, any act that can improve or benefit symptoms caused by an endoglin-related disease by using the composition of the present invention.

[0390] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, capsules, tablets, aqueous suspensions, etc., external preparations, suppositories, and sterile injection solutions, according to conventional methods, but is not limited thereto. Preferably, the pharmaceutical composition may be formulated for intravenous administration or inhalation; or for use as an injection, but is not limited thereto.

[0391] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may include a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a colorant, a fragrance, etc. for oral administration. In the case of injections, a buffer, a preservative, an analgesic, a solubilizer, an isotonic agent, a stabilizer, etc. may be mixed and used. In the case of topical administration, a base, an excipient, a lubricant, a preservative, etc. may be used. The dosage form of the pharmaceutical composition of the present invention may be prepared in various ways by mixing it with the pharmaceutically acceptable carrier described above. For example, the composition may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. for oral administration. In the case of injections, the composition may be prepared in the form of unit dose ampoules or multiple doses. In addition, the composition may be formulated as a solution, suspension, tablet, capsule, sustained-release preparation, etc.

[0392] Examples of carriers, excipients, and diluents suitable for the formulation of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may be additionally included.

[0393] The routes of administration of the pharmaceutical composition of the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. It can be administered orally or parenterally, and is preferably administered orally, but is not limited thereto.

[0394] The parenteral route of the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0395] The pharmaceutical composition of the present invention may vary depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, dosage form, administration time, administration route, excretion rate, drug combination, and severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, form of medicine, administration route, and period, but may be appropriately selected by those skilled in the art, and may be administered at 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. Administration may be administered once a day or divided into several times. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, a sugar-coated tablet, a capsule, a liquid, a gel, a syrup, a slurry, or a suspension.

[0396]

[0397] According to another embodiment of the present invention, there is provided a method for preventing, improving or treating an endoglin-related disease, comprising administering a pharmaceutically effective amount of a composition of the present invention to a subject in need of administration.

[0398] In the present invention, the subject is an subject that has developed or is suspected of developing an endoglin-related disease (particularly a disease caused by overexpression of endoglin), and the subject suspected of developing the disease means all animals including humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs that have developed or can develop the disease, but subjects that can be treated with the effective substance provided in the present invention are included without limitation.

[0399] The term "pharmaceutically effective amount" as used herein is an amount sufficient to stop or alleviate the physiological effects of a subject or patient caused by an endoglin-related disease. An appropriate effective amount may be determined by a treating physician within the scope of sound medical judgment, and may be administered once or in several divided doses. However, for the purpose of the present invention, it is preferable to apply a specific therapeutically effective amount for a specific patient differently depending on various factors such as the type and degree of response to be achieved, whether other agents are used in some cases, the composition containing the specific effective ingredient, the patient's age, weight, general health condition, sex, and diet, the time of administration, the route of administration, the number of administrations, the secretion rate of the composition containing the effective ingredient, the treatment period, drugs used together or concurrently with the specific composition, and similar factors well known in the medical field.

[0400] The total effective amount of the composition of the present invention can be administered to a patient as a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. The composition of the present invention may vary in the content of the active ingredient depending on the severity of the disease. Specifically, a preferred total dose of the composition of the present invention may be about 0.0001 mg to 500 mg per kg of patient body weight per day. However, since the dosage of the composition is determined by taking into consideration various factors such as the route of administration of the pharmaceutical composition and the number of treatments, as well as the patient's age, weight, health status, sex, severity of the disease, diet, and excretion rate, a person having ordinary skill in the art will be able to determine an appropriate effective dosage for a specific use of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration, or method of administration, as long as it exhibits the effects of the present invention.

[0401] In addition, the method for preventing, improving or treating the endoglin-related disease in the present invention may be a combination therapy further comprising administering a compound or substance having therapeutic activity against one or more diseases.

[0402] In the present invention, the term "combination" should be understood to refer to simultaneous, separate, or sequential administration. If the administration is sequential or separate, the interval between administrations of the secondary components should be such that the beneficial effects of the combination are not lost.

[0403]

[0404] Diagnostic purposes

[0405] According to another embodiment of the present invention, there is provided a composition for diagnosing an endoglin-related disease, comprising an antibody or antigen-binding fragment provided by the present invention; or a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment.

[0406] Endoglin detected by the antibody or antigen-binding fragment of the present invention is known to be expressed at high levels in proliferating vascular endothelial cells (Burrows et al, 1995, Clin. Cancer Res. 1:1623-1634). Therefore, in the present invention, by detecting endoglin by the antibody or antigen-binding fragment, it can be used as a diagnostic marker for diagnosing angiogenesis-related diseases, such as cancer or ocular diseases characterized by neovascularization, evaluating the disease progression status and prognosis before and after treatment.

[0407] The antibodies or antigen-binding fragments of the present invention can be used in any known analytical methods, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays.

[0408] The antibody or antigen-binding fragment of the present invention may be labeled with a label that: (i) provides a detectable signal; (ii) reacts with a first or second label, for example, a second label to modify a detectable signal provided by Fluorescence Resonance Energy Transfer (FRET); (iii) affects mobility, for example, electrophoretic mobility, by charge, hydrophobicity, shape, or other physical parameters, or (iv) provides a capture moiety, such as an affinity, antibody / antigen, or ion complex. Suitable labels include fluorescent labels, luminescent labels, chromophore labels, radioactive labels, isotope labels, isobaric labels, enzymatic labels, particulate labels, in particular metal particle labels, magnetic particle labels, polymer particle labels, small organic molecules such as biotin, receptor ligands or binding molecules such as cell adhesion proteins or lectins, label-sequences comprising nucleic acid and / or amino acid residues that are detectable by use of a binding agent, etc.

[0409] In the present invention, the specific type of the label is not particularly limited, but examples thereof include enzymes that utilize hydrogen peroxide to oxidize a dye precursor, such as HRP, lactoperoxidase or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, radioisotopes 32P, 14C, 125I, 3H and 131I linked to stable free radicals, etc., fluorophores, such as rare earth chelates or fluorescein and derivatives thereof, rhodamine and derivatives thereof, dansyl, umbelliferone, luciferases, such as firefly luciferase and bacterial luciferase (US Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, Glucoamylase, lysozyme, saccharide oxidases such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, but are not limited thereto.

[0410]

[0411] According to another embodiment of the present invention, there is provided a diagnostic kit for an endoglin-related disease comprising the diagnostic composition of the present invention.

[0412] In the present invention, the "kit" refers to a tool that can evaluate the expression level of a biomarker by labeling a probe or antibody that specifically binds to a biomarker component with a detectable label. It includes not only direct labeling of a detectable substance related to a probe or antibody through reaction with a substrate, but also indirect labeling in which a chromogenic label is conjugated through reactivity with another directly labeled reagent. It may include a chromogenic substrate solution, a washing solution, and other solutions that will react with the label for chromogenic reaction, and may be manufactured including the reagent components used. In the present invention, the kit may be a kit that includes the essential elements required for performing RT-PCR, and may include, in addition to each primer pair specific for a marker gene, a test tube, a reaction buffer, deoxynucleotides (dNTPs), Taq polymerase, reverse transcriptase, DNase, RNase inhibitor, sterile water, etc. In addition, the kit may be a kit for detecting genes for cancer diagnosis that includes the essential elements required for performing a DNA chip. The DNA chip kit comprises a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof. The kit of the present invention is not limited thereto, as long as it is known in the art.

[0413] In the present invention, the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

[0414] The kit of the present invention may further comprise one or more other component compositions, solutions, or devices suitable for the analysis method. For example, the kit of the present invention may further comprise essential elements necessary for performing a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit comprises a pair of primers specific for a gene encoding a marker protein. The primers are nucleotides having a sequence specific for the nucleic acid sequence of the gene, and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also comprise a primer specific for the nucleic acid sequence of a control gene. In addition, the reverse transcription polymerase reaction kit may comprise a test tube or other appropriate container, a reaction buffer (with various pH and magnesium concentrations), deoxynucleotides (dNTPs), an enzyme such as Taq polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC-water, sterile water, etc.

[0415] Additionally, the diagnostic kit of the present invention may include essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotides corresponding to a gene or fragment thereof are attached, and reagents, preparations, enzymes, etc. for producing a fluorescently labeled probe. The substrate may also include cDNA or oligonucleotides corresponding to a control gene or fragment thereof.

[0416] Additionally, the diagnostic kit of the present invention may include essential components necessary for performing an ELISA. The ELISA kit includes an antibody specific for the protein. The antibody is an antibody with high specificity and affinity for the marker protein and little cross-reactivity with other proteins, and may be a monoclonal antibody, polyclonal antibody, or recombinant antibody. The ELISA kit may also include an antibody specific for a control protein. In addition, the ELISA kit may include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated to antibodies), and their substrates or other substances capable of binding to antibodies.

[0417] In the present invention, a fixative for the antigen-antibody binding reaction may be a nitrocellulose membrane, a PVDF membrane, a well plate synthesized from polyvinyl resin or polystyrene resin, a glass slide glass, etc., but is not limited thereto.

[0418] In the present invention, the washing solution preferably contains a phosphate buffer solution, NaCl, and Tween 20, and a buffer solution (PBST) composed of 0.02 M phosphate buffer solution, 0.13 M NaCl, and 0.05% Tween 20 is more preferred. After the antigen-antibody binding reaction, the washing solution reacts the secondary antibody with the antigen-antibody complex, and then an appropriate amount is added to the fixative and washed 3 to 6 times. The reaction stopping solution can preferably be a sulfuric acid solution (H2SO4).

[0419]

[0420] According to another embodiment of the present invention, there is provided a method for providing information for diagnosing an endoglin-related disease, comprising the step of contacting a biological sample isolated from a target subject with an antibody or antigen-binding fragment provided in the present invention; or a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment.

[0421] In the present invention, the subject is an subject that has developed or is suspected of developing an endoglin-related disease (particularly a disease caused by overexpression of endoglin), and the subject suspected of developing the disease means all animals including humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs that have developed or can develop the disease, but any subject that can be diagnosed with the disease using the effective substance provided in the present invention is included without limitation.

[0422] In the present invention, the biological sample is whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint It may include, but is not limited to, joint aspirate, organ secretions, cells, cell extracts, or cerebrospinal fluid.

[0423] The information providing method of the present invention may include a step of measuring the expression level of endoglin protein in the biological sample using the antibody or antigen-binding fragment provided in the present invention.

[0424] In the present invention, measurement of the expression level of the protein can be performed by Western blotting, enzyme linked immunosorbent assay (ELISA), competitive binding assay, or a similar method.

[0425] The information providing method of the present invention may include a step of predicting that an endoglin-related disease has developed or is likely to develop if the expression level of the endoglin protein measured for the biological sample is increased compared to a control group. Here, the control group may be, but is not limited to, the expression level of endoglin in a healthy normal control group.

[0426] The antibody or antigen-binding fragment that specifically binds to endoglin provided in the present invention exhibits excellent affinity and binding ability to endoglin, and therefore, an antibody-drug conjugate, bispecific antibody, or chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment can be developed, and further, the antibody or antigen-binding fragment according to the present invention can be usefully applied to the treatment or diagnosis of endoglin-related diseases, such as cancer.

[0427] Figure 1 shows the results of ELISA analysis of changes in antibody titer according to the number of weeks for each mouse after mouse immunization in Example 1.

[0428] Figures 2a to 2c show the results of scFv expression and purification screened in mammalian cells (HEK293F) in Example 1.

[0429] Figures 3a and 3b show the results of expression and purification of scFv screened in E. coli in Example 1.

[0430] Figure 4 shows the results of analyzing the binding ability of the anti-endoglin scFv according to the present invention to the endoglin antigen in Example 1 using ELISA.

[0431] Figure 5 shows the results of flow cytometry analysis of the binding ability of the anti-endoglin scFv according to the present invention to endoglin-nonexpressing K562 cells in Example 1.

[0432] Figure 6 shows the results of flow cytometry analysis of the binding ability of anti-endoglin scFv according to the present invention to endoglin-expressing K562 cells in Example 1.

[0433] FIG. 7 illustrates a construction construct of an endoglin-specific chimeric antigen receptor according to one embodiment of the present invention.

[0434] Figure 8 shows the results of flow cytometry analysis of NK92 cells expressing an endoglin-specific CAR according to the present invention in Example 2.

[0435] Figure 9 shows the results of analyzing the death rate of pancreatic cancer cells after co-culture of endoglin-overexpressing pancreatic cancer cells (Panc1) and endoglin-specific CAR-NK92 cells according to the present invention in Example 2.

[0436] Figure 10 shows the results of analyzing the death rate of pancreatic cancer cells after co-culture of endoglin-overexpressing K562 cells and endoglin-specific CAR-NK92 cells according to the present invention in Example 2.

[0437] Figure 11 shows the results of analyzing the death rate of pancreatic cancer cells after co-culture of endoglin-overexpressing K562 cells and endoglin-specific CAR-NK92 cells according to the present invention in Example 2.

[0438] Figures 12a to 12c show the results of flow cytometry analysis of changes in the expression level of the CD107a marker after co-culture of endoglin-overexpressing pancreatic cancer cells (Aspc1-ENG) and endoglin-specific CAR-NK92 cells according to the present invention in Example 2.

[0439] Figures 13a to 13c show the results of flow cytometry analysis of changes in the expression level of IFN-gamma after co-culture of endoglin-overexpressing pancreatic cancer cells (Aspc1-ENG) and endoglin-specific CAR-NK92 cells according to the present invention in Example 2.

[0440] Figure 14 shows the results of flow cytometry analysis of primary NK cells expressing an endoglin-specific CAR according to the present invention in Example 3.

[0441] Figure 15 shows the results of fluorescence image analysis of live cells expressing endoglin through fluorescence staining after co-culturing endoglin-specific CAR-pNK cells according to the present invention with pancreatic cancer fibroblasts (CAF16) derived from a pancreatic cancer patient with high endoglin expression or human pancreatic stellate cells (hPaSC) with low endoglin expression in Example 3.

[0442] Figure 16 shows the results of analyzing the change in fluorescence intensity in living cells after co-culturing endoglin-specific CAR-pNK cells according to the present invention with pancreatic cancer fibroblasts (CAF16) derived from a pancreatic cancer patient with high endoglin expression or human pancreatic stellate cells (hPaSC) with low endoglin expression in Example 3.

[0443] Figures 17a and 17b show the results of flow cytometry analysis of changes in the expression level of the CD107a marker after co-culturing endoglin-specific CAR-pNK cells according to the present invention with pancreatic cancer fibroblasts (CAF16) derived from a pancreatic cancer patient with high endoglin expression or human pancreatic stellate cells (hPaSC) with low endoglin expression in Example 3.

[0444] Figures 18 and 19 show the results of expression and purification of anti-endoglin humanized scFv screened in E. coli in Example 4.

[0445] Figure 20 shows the results of flow cytometry analysis of the binding ability of the anti-endoglin humanized scFv according to the present invention to endoglin-nonexpressing K562 cells in Example 5.

[0446] Figure 21 shows the results of flow cytometry analysis of the binding ability of anti-endoglin humanized scFv according to the present invention to endoglin-expressing K562 cells in Example 5.

[0447] Figure 22 shows the results of ELISA analysis of the binding ability of the anti-endoglin humanized scFv according to the present invention to the endoglin antigen in Example 6.

[0448] Figure 23 shows the results of analyzing the death rate of pancreatic cancer cells after co-culturing CAR-NK92 cells according to the present invention with pancreatic cancer cells in Example 7.

[0449] Figure 24 shows the results of analyzing the death rate of pancreatic cancer cells after co-culturing CAR-NK92 cells according to the present invention with pancreatic cancer cells in Example 7.

[0450] According to one embodiment of the present invention, it relates to an antibody or antigen-binding fragment that specifically binds to Endoglin.

[0451] The anti-endoglin antibody or antigen-binding fragment of the present invention may comprise a heavy chain variable region comprising a heavy chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 17, a heavy chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 36, and a heavy chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 37 to 57; and a light chain variable region comprising a light chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 58 to 76, a light chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 77 to 90, and a light chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 91 to 112.

[0452] The anti-endoglin antibody or antigen-binding fragment of the present invention may comprise at least one antibody or antigen-binding fragment selected from the following A-1) to A-25):

[0453] A-1) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 18, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 37; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 58, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 77, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 91;

[0454] A-2) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 18, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 37; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 59, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 77, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 92;

[0455] A-3) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 19, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 38; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 60, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 78, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 93;

[0456] A-4) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 20, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 39; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 61, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 79, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 94;

[0457] A-5) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 20, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 39; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 62, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 80, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 95;

[0458] A-6) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 21, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 40; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 63, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 81, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 96;

[0459] A-7) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 22, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 41; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 64, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 82, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 97;

[0460] A-8) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 23, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 42; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 65, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 98;

[0461] A-9) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 6, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 43; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 66, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 84, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 99;

[0462] A-10) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 6, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 43; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 67, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 85, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 100;

[0463] A-11) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 7, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 25, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 44; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 68, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 86, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 101;

[0464] A-12) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 8, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 26, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 45; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 69, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 79, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 102;

[0465] A-13) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 9, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 46; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 62, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 80, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 103;

[0466] A-14) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 28, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 47; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 70, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 87, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 104;

[0467] A-15) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 20, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 48; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 71, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 88, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 105;

[0468] A-16) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 11, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 29, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 49; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 106;

[0469] A-17) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 8, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 26, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 45; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 89, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 107;

[0470] A-18) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 12, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 25, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 50; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 89, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 108;

[0471] A-19) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 30, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 51; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 73, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 90, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 109;

[0472] A-20) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 14, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 31, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 52; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 89, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 108;

[0473] A-21) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 9, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 32, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 53; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 74, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 90, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 109;

[0474] A-22) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 15, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 33, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 54; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 75, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 80, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 110;

[0475] A-23) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 16, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 34, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 55; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 89, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 108;

[0476] A-24) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 56; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111; and

[0477] A-25) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 17, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 36, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 57; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 76, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 112.

[0478] The anti-endoglin antibody or antigen-binding fragment of the present invention may comprise at least one heavy chain variable region selected from the group consisting of SEQ ID NOs: 113 to 133; and at least one light chain variable region selected from the group consisting of SEQ ID NOs: 134 to 158.

[0479] The anti-endoglin antibody or antigen-binding fragment of the present invention may comprise at least one antibody or antigen-binding fragment selected from the following B-1) to B-25):

[0480] B-1) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 113 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 134;

[0481] B-2) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 113 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 135;

[0482] B-3) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 114 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 136;

[0483] B-4) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 115 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 137;

[0484] B-5) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 115 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 138;

[0485] B-6) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 116 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 139;

[0486] B-7) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 117 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 140;

[0487] B-8) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 118 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 141;

[0488] B-9) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 119 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 142;

[0489] B-10) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 119 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 143;

[0490] B-11) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 120 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 144;

[0491] B-12) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 121 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 145;

[0492] B-13) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 122 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 146;

[0493] B-14) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 123 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 147;

[0494] B-15) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 124 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 148;

[0495] B-16) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 125 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 149;

[0496] B-17) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 121 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 150;

[0497] B-18) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 126 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 151;

[0498] B-19) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 127 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 152;

[0499] B-20) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 128 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 153;

[0500] B-21) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 129 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 154;

[0501] B-22) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 130 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 155;

[0502] B-23) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 131 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 156;

[0503] B-24) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 132 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 157; and

[0504] B-25) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 133 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 158.

[0505] The antibody or antigen-binding fragment of the present invention may further comprise a linker between the heavy chain variable region and the light chain variable region, preferably GGGGSGGGGSGGGGS represented by SEQ ID NO: 159, but is not limited thereto.

[0506] In the present invention, the antibody includes a chimeric antibody, a human antibody, a humanized antibody, a bivalent, a bispecific molecule, a minibody, a domain antibody, a bispecific or multispecific antibody, an immune cell engaging bispecific or multispecific antibody, an antibody mimetic, a unibody, a diabody, a triabody, or a tetrabody.

[0507] The antigen-binding fragment provided in the present invention may be a scFv (single-chain variable fragment) that specifically recognizes and / or binds to endoglin.

[0508] The amino acid sequence of the endoglin-binding scFv of the present invention may include any one amino acid sequence selected from SEQ ID NOs: 208 to 230.

[0509] The present invention provides an anti-endoglin humanized antibody or an antigen-binding fragment thereof.

[0510] The anti-endoglin humanized antibody or antigen-binding fragment of the present invention may comprise a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 56, 233, 234, or 235, and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111.

[0511] The anti-endoglin humanized antibody or antigen-binding fragment of the present invention may comprise at least one humanized antibody or antigen-binding fragment selected from the following C-1) to C-3):

[0512] C-1) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 56; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111;

[0513] C-2) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111;

[0514] C-3) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 234; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111; and

[0515] C-4) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 235; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111.

[0516] The anti-endoglin humanized antibody or antigen-binding fragment of the present invention may comprise at least one humanized antibody or antigen-binding fragment selected from the following D-1) to D-13):

[0517] D-1) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 237, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 56, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 246, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0518] D-2) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 247, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0519] D-3) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 246, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0520] D-4) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 245, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 247, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0521] D-5) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 239, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 248, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0522] D-6) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 241, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 234, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 248, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0523] D-7) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 241, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 248, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0524] D-8) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 235, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 245, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 249, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0525] D-9) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 239, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 235, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 250, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0526] D-10) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 251, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0527] D-11) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 252, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255;

[0528] D-12) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 240, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 235, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; and a humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 245, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 253, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255; and

[0529] D-13) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 236, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 238, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 241, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 235, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 242; A humanized antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 245, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 254, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 255.

[0530] The anti-endoglin humanized antibody or antigen-binding fragment of the present invention may comprise at least one heavy chain variable region selected from the group consisting of SEQ ID NOs: 256 to 262 and 325; and at least one light chain variable region selected from the group consisting of SEQ ID NOs: 263 to 272.

[0531] The anti-endoglin humanized antibody or antigen-binding fragment of the present invention may comprise at least one humanized antibody or antigen-binding fragment selected from the following E-1) to E-13):

[0532] E-1) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 256 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 263;

[0533] E-2) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 264;

[0534] E-3) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 263;

[0535] E-4) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 265;

[0536] E-5) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 325 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 266;

[0537] E-6) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 258 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 266;

[0538] E-7) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 259 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 266;

[0539] E-8) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 260 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 267;

[0540] E-9) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 261 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 268;

[0541] E-10) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 269;

[0542] E-11) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 270;

[0543] E-12) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 260 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 271; and

[0544] E-13) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 262 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 272.

[0545] The anti-endoglin humanized antigen-binding fragment provided in the present invention may be a humanized scFv that specifically recognizes and / or binds to endoglin, and specifically may include at least one selected from the group consisting of SEQ ID NOs: 273 to 285.

[0546] According to another embodiment of the present invention, the present invention relates to a nucleic acid molecule encoding an antibody or antigen-binding fragment provided by the present invention.

[0547] According to another embodiment, the present invention relates to a recombinant expression vector comprising a nucleic acid molecule provided in the present invention.

[0548] According to another embodiment of the present invention, the expression vector provided in the present invention relates to a transfected host cell.

[0549] According to another embodiment of the present invention, there is provided a dual or multispecific antibody comprising an antibody or antigen-binding fragment provided by the present invention.

[0550] According to another embodiment of the present invention, there is provided an immune cell engaging bispecific or multispecific antibody comprising an antigen binding fragment (scFv) provided by the present invention and at least one scFv that binds to an immune cell activating antigen.

[0551] According to another embodiment of the present invention, the present invention relates to an immunoconjugate in which an antibody or antigen-binding fragment provided by the present invention is combined with a cytotoxic agent.

[0552] According to another embodiment of the present invention, there is provided a chimeric antigen receptor (CAR) comprising an antigen binding fragment provided by the present invention.

[0553] A schematic diagram of the structure of a chimeric antigen receptor according to one embodiment of the present invention may be composed of or include an endoglin binding domain (scFv according to the present invention (VL-linker-VH))-CD8 hinge-CD8 transmembrane domain-4-1BB-CD3ζ, or may be composed of or include a signal peptide-endoglin binding domain (scFv according to the present invention)-CD8 hinge-CD8 transmembrane domain-4-1BB-CD3ζ.

[0554] As an example, the chimeric antigen receptor provided in the present invention may be composed of or include an amino acid sequence represented by any one of SEQ ID NOs: 168 to 192, but is not limited thereto.

[0555] As another example, the chimeric antigen receptor provided in the present invention may be composed of or include an amino acid sequence represented by any one of SEQ ID NOs: 286 to 298, but is not limited thereto.

[0556] According to another embodiment of the present invention, there is provided an immune effector cell genetically engineered to express a chimeric antigen receptor provided by the present invention.

[0557] According to another embodiment of the present invention, there is provided a pharmaceutical composition for preventing, improving or treating an endoglin-related disease, comprising as an active ingredient an antibody or antigen-binding fragment provided by the present invention; an immunoconjugate; a chimeric antigen receptor; or an immune effector cell expressing a chimeric antigen receptor.

[0558] According to another embodiment of the present invention, there is provided a composition for diagnosing an endoglin-related disease, comprising an antibody or antigen-binding fragment provided by the present invention; or a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment.

[0559] Here, the endoglin-related disease may be an angiogenesis-related disease.

[0560] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0561]

[0562] Example

[0563]

[0564] [Example 1] Screening and binding ability evaluation of endoglin-specific antibodies

[0565] 1. Mouse immunization

[0566] To obtain immunized mice necessary for the development of endoglin-specific antibodies, the endoglin antigen represented by SEQ ID NO: 207 and Imject TM Alum Adjuvant (Thermo Scientific TM ) were mixed and injected intraperitoneally into 6-week-old male Balb / c mice. The antigen prepared at a concentration of 0.5 ug / ul and Alum Adjuvant were mixed 600 ㎕ each in a 1:1 ratio to make an emulsion, and the final immune concentration of 200 ㎕ / dose / 1 time of antigen was injected intraperitoneally into the mouse. The immunization reaction was performed four times in total at two-week intervals using the same method as the initial immunization method.

[0567] To determine whether mice were immune, tail blood samples were collected weekly to obtain serum, and the increase in antibody titer against the immunogen was confirmed by ELISA. Specifically, each well of a 96-well ELISA plate was coated with endoglin-his antigen overnight at 4°C, and then 3% BSA in PBS was added and incubated for 1 hour at room temperature. Mouse serum was collected at 10 -3After serial dilution of 1 / 10 from the dilution ratio, the cells were incubated at room temperature for 1 hour. After washing three times with PBST (tween20 0.05%), anti-mouse IgG HRP was treated at a dilution ratio of 20,000:1 and incubated at room temperature for 1 hour. After washing three times with PBST (tween20 0.05%), TMB was treated and incubated at room temperature for 20 minutes. After treatment with 2 M H2SO4, the absorbance was measured at 450 nm. As a result, as shown in Figure 1, the antibody titer increased weekly in all six mice, indicating that immunity was sufficiently induced.

[0568] 2. Single cell clone screening

[0569] After euthanizing the immunized mice, the isolated spleen and bone marrow cells were enriched using a plasma cell sorting kit (Miltenyi Biotec, catalog 130-092-530, lot 5190121409). These cells were then transferred to the chip using a microfluidic system of the Beacon platform (Berkeley Lights). Single cells were isolated and added to individual chambers of the chip using optoelectronic positioning (OEP) technology. After incubating the single B cells for a certain period of time (20 minutes), multiplexed assays can be performed on the chip: not only can the analysis of specific binding of antigen to antibody be completed based on magnetic beads, but also the binding of antibody to antigen on the cell surface can be detected using cell lines that overexpress the antigen. If the cells can secrete antigen-specific antibodies, a fluorescent signal will be generated and detected by the device. At this stage, the detection assay can be flexibly adjusted as needed. For further sequencing, analysis, and expression, single-positive cells were exported by OEP technology and transferred to a 96-well plate containing cell lysate (Qiagen, catalog 1031586, lot 163013365) and mineral oil (Sigma, catalog M5904-500ML, lot MKBZ6778V). A total of 25 antibodies of ENG1-25 were obtained.

[0570] The heavy chain variable region and light chain variable region and CDR sequence information of the initial antibodies obtained through single cell clone screening are shown in Table 9 below. However, the sequence information at this time was presented based on the IMGT / DomainGapAlign web program (https: / www.ncbi.nlm.nih.gov / igblast / igblast.cgi).

[0571]

[0572] 3. Expression and purification of antibodies (1)

[0573] The gene encoding the anti-endoglin scFv shown in Table 10 was inserted into the pcDNA3.4 vector (Invitrogen) using the Gibson assembly technique.

[0574] Transfections were performed for overexpressed recombinant proteins. 1 μg of mammalian expression vector and 81 vL of ExpiFectamine per ml of culture volume TM 293 Transfection Kit (Gibco) in Opti-MEM equivalent to 1 / 17 of the cell culture volume TM (Gibco) and left at room temperature for 15 minutes. Mammal cells HEK293F (3X10) used for protein overexpression system 6 The above mixture was added to cells / ml, Invitrogen) and incubated for 6 days Freestyle TM In 293 expression medium (Invitrogen), it was cultured under conditions of 37°C, 8% CO2, and 135 rpm agitation.

[0575] The supernatant of the cell culture was collected, and affinity gel chromatography using Ni-NTA resin was used to purify the scFv protein. The purified protein was run on a 12% Bis-Tris gradient gel electrophoresis. The protein size and yield were confirmed by Coomassie Brilliant Blue staining.

[0576]

[0577] Clone OD280nmMWCo-efficient valueActual concentration(mg / ml)Volume after purificationTotal yield2934(ENG3)0.12828.9kDa1.9740.065250uL16.25ug / 30mL = (0.54ug / mL) = (0.54mg / L)14119(ENG11)0.41829.3kDa1.9820.211250uL52.75ug / 30mL = (1.76ug / mL) = (1.76mg / L)17361(ENG12)1.32129.2kDa1.5600.8471.5mL1.27mg / 30mL = (42.3ug / mL) = (42.3mg / L)8781(ENG7)2.2728.6kDa1.8041.261.2mL1.5mg / 30mL = (50ug / mL) = (50mg / L)10782-1(ENG9)0.12328.9kDa1.9270.064600uL38.4ug / 30mL = (1.28ug / mL) = (1.28mg / L)10782-2(ENG10)0.04428.9kDa2.0650.021350uL5.25ug / 30mL = (0.175ug / mL) = (0.175mg / L)

[0578] Clone OD280nmMWCo-efficient valueActual concentration(mg / ml)Volume after purificationTotal Yield 431-1(ENG1)0.056529.1kDa2.2550.025300uL7.5ug / 30mL=(0.25ug / mL)=( 0.25mg / L)431-2(ENG2)0.29829.2kDa2.1960.136310uL42.16ug / 30mL=(1.40 ug / mL)=(1.40mg / L)7313-1(ENG4)0.16529.5kDa2.1050.0784350uL27.44ug / 30mL=(0.914ug / mL)=(0.914mg / L)7313-2(ENG5)0.15628.9kDa2.0100.077635 0uL27.16ug / 30mL=(0.905 / mL)=(0.905mg / L)19176(ENG13)0.08829.1kDa1.9 260.0457350uL16.0ug / 30mL=(0.53ug / mL)=(0.53mg / L)8866(ENG8)0.10527. 4kDa2.5410.0413250uL10.3ug / 30mL=(0.34ug / mL)=(0.34mg / L)8718(ENG6)0 .18329.7kDa1.9720.09281050uL97.44ug / 30mL=(3.248ug / mL)=(3.248ug / mL)

[0579] As a result, it was confirmed that scFv having a molecular weight of about 28-29 kDa was purified, as shown in Tables 11 and 12 and Figures 2a to 2c.

[0580] 4. Antibody expression and purification (2)

[0581] Using primers for the previously selected anti-endoglin scFv, a PCR product was produced that inserted the overlapping part with the pMopac12 vector. The PCR product (insert) and the pMoapc12 vector were combined using the Gibson assembly technique to insert the insert gene into the vector. After transformation with JudeI, the product was plated on an LB plate supplemented with chloramphenicol and cultured at 37°C for 16 h. The following day, colonies were collected and inoculated into LB media supplemented with chloramphenicol and cultured at 37°C for 16 h. The following day, the plasmid was isolated, restricted with SfiI, and electrophoresed on a 1% agarose gel to confirm the insertion band.

[0582] The cloned anti-endoglin scFv antibody clone was transformed into BL21(DE3) Escherichia coli. The transformants were cultured in LB medium containing chloramphenicol until the OD value was 0.6, and expression was induced by treatment with 0.5 mM IPTG at 25°C. The cells obtained by centrifugation of the culture solution were suspended in a 100 mM Tris, 0.75 M sucrose, pH 7.5 buffer, and osmotic shock was applied by adding 1 mM EDTA, followed by centrifugation to obtain the supernatant. To purify only the induced expressed protein, a resin with an affinity for Ni-NTA was used, and the purified protein was confirmed by SDS-PAGE analysis.

[0583] Clone OD280nmMW(kDa)Extinction coefficient valuePurityConcentration(mg / ml)Concentration considering purity(mg / mL)Volume after purificationTotal yield#2934(ENG3)0.71628.6kDa1.82286.6%0.3930.340250uL85ug / 500mL, 0.170mg / L#8718(ENG6)0.22929.3kDa1.992>99%0.1150.115250uL28.75ug / 500mL, 0.0316mg / L#8781(ENG7)1.48228.3kDa1.82247.9%0.8130.389250uL97.25ug / 500mL, 0.204mg / L

[0584] Clone OD280nmMW(kDa)Extinction coefficient valuePurityConcentration(mg / ml)Concentration considering purity(mg / mL)Volume after purificationTotal yield185(ENG15)0.05828.22.50180.0%0.0230.0184300uL5.52ug / 500mL, 0.011mg / L1793(ENG18)2.10927.92.278>99%0.9260.926900uL833.4ug / 500mL, 1.67mg / L2122(ENG19)2.52528.32.020>99%1.251.254000uL5000ug / 500mL, 10mg / L4296(ENG20)0.07028.02.41389.2%0.0290.0259300uL7.77ug / 500mL, 0.0155mg / L5577(ENG21)0.09428.92.19975.2%0.0430.0323300uL9.69ug / 500mL, 0.0194mg / L19806(ENG16)0.34028.92.165>99%0.1570.157500uL78.5ug / 500mL, 0.157mg / L

[0585] As a result, as shown in Tables 13 and 14 and Figures 3a and 3b, it was confirmed that scFv having a molecular weight of approximately 28-29 kDa was synthesized.

[0586] 5. ELISA-based antigen binding ability evaluation

[0587] The binding ability of each purified scFv antibody to the endoglin antigen was analyzed based on the ELISA method. First, anti-endoglin scFv antibody was coated on an ELISA plate at a concentration of 2 μg / mL at 4°C overnight. The plate was blocked with 3% PBS BSA for 1 hour. Biotinylated endoglin-his protein was serially diluted 1 / 5 from 1 μM and bound to the anti-endoglin scFv coated on the ELISA plate at room temperature for 1 hour. Each well was washed three times with PBST (tween 20 0.05%) washing buffer, and then HRP enzyme-conjugated streptavidin (Streptavidin-HRP; abcam) was diluted 40,000:1 to detect the biotin-labeled antibody and incubated at room temperature for 1 hour. Each well was washed three times with PBST (tween20 0.05%) washing buffer, treated with TMB, and incubated at room temperature for 20 minutes. After treatment with 2 M H2SO4, the absorbance was measured at 450 nm.

[0588] Clone # (anti Endoglin scFv) IC 50 (nM)2934(ENG3)30.1±9.5817361(ENG12)6.65±1.168781(ENG7)12.6±16.88718(ENG6)28.9±6.03185(ENG15)645±21717 93(ENG16)527±1054296(ENG18)2084±9955577(ENG19)2684±255919806(ENG14)6557±195217341(ENG24)458±19.7431-2 (ENG2)1027±5347313-2 (ENG5)405±52.010782-1 (ENG9)421±23710782-2 (ENG10)498±25.014119 (ENG11)8.40±0.94019176 (ENG13)514±61.0

[0589] As a result, as shown in Table 15 and FIG. 4, it was confirmed that the anti-endoglin scFv antibodies manufactured according to the present invention had excellent binding ability to the endoglin antigen.

[0590] 6. Evaluation of antigen binding capacity based on endoglin-expressing cells

[0591] The binding ability of each purified scFv antibody to the endoglin antigen was analyzed using cells expressing endoglin. Specifically, endoglin-positive K562 cells and endoglin-negative K562 cells were prepared and seeded in 96-well U-bottom plates at 5 x 10 per well. 4 The cells were seeded with 100 μg of the cell number. After washing with PBSF (1% FBS) washing buffer, anti-endoglin scFv according to the present invention was serially diluted 1 / 4 from 1 μM and incubated at 4°C for 1 hour. The cells were washed with PBSF (1% FBS) washing buffer. Afterwards, anti-HISx6-FITC was treated at a ratio of 250:1 and incubated at 4°C for 1 hour. After washing with PBSF (1% FBS), FACS analysis was performed.

[0592] As a result, as shown in FIGS. 5 and 6, it was confirmed that the anti-endoglin scFv antibodies manufactured according to the present invention did not bind to endoglin-negative K562 cells, but selectively bound only to endoglin-positive K562 cells.

[0593]

[0594] [Example 2] Production of endoglin-specific CAR-NK92 cells and evaluation of their anticancer activity.

[0595] 1. Design of an endoglin-specific chimeric antigen receptor expression cassette

[0596] In order to design a chimeric antigen receptor comprising an anti-endoglin scFv (VL-linker-VH), CD8 hinge, CD8 transmembrane domain, 4-1BB, and CD3 zeta signaling domain according to the present invention as shown in FIG. 7, a polynucleotide encoding an anti-endoglin scFv (VL-linker-VH), CD8 hinge, CD8 transmembrane domain, 4-1BB, and CD3 zeta signaling domain according to the present invention (base sequence encoding amino acids of SEQ ID NOs: 170, 172, 173, 174, 187, 188, and 191) was constructed as an expression cassette and inserted into a lentiviral transfer vector. At this time, a polynucleotide encoding a CD8 signal peptide was inserted at the 5' end, and a polynucleotide encoding P2A represented by SEQ ID NO: 165 and a polynucleotide encoding green fluorescent protein (GFP) represented by SEQ ID NO: 166 were inserted at the 3' end. At this time, the recombinant gene was placed under the control of the SFFV single promoter (SEQ ID NO: 167). The amino acid sequence encoded by the polynucleotide is shown in Table 16 below. However, in the following experiments, a positive control was produced by replacing the anti-endoglin scFv with the scFv of the 9H10 antibody (Creativebiolabs, CAT#: TAB-1577CL) or TRC105 (carotuximab) antibody.

[0597]

[0598] 2. Transduction of NK cells with lentivirus

[0599] Natural killer cells were used as immune effector cells genetically engineered to express the endoglin-specific chimeric antigen receptor. The NK-92 cell line was purchased from ATCC, and the 293T cell line, the cell line for lentivirus transduction, was also purchased from ATCC. NK-92 cells and the lentivirus-transduced NK-92 cell lines described below were all cultured in a medium containing 100 μg / mL streptomycin, 100 units / mL penicillin, 20% fetal bovine serum (FBS), 10% MEM vitamin solution, 1X 2-mercaptoethanol, and 200 IU rhIL-2. The 293T cell line was cultured in a medium containing 100 μg / mL streptomycin, 100 units / mL penicillin, and 10% fetal bovine serum (FBS). All cell cultures were cultured in an environment maintained at 37°C with 5% CO2 (95% air).

[0600] Physical titer of the virus (using One-Wash Lentivirus Titer Kit) 5 x 10 8 To obtain lentiviruses with TU / mL or more, 1.2 x 10 6293T cells were cultured in 60 mm culture dishes for 24 hours and then transfected with 5.5 μg of the previously prepared lentiviral transfer vector, 3.7 μg of the packaging vector (pMD2G and psPAX2 (Addgene)), and 1.8 μg of the envelope vector (BaEV) using Lipofectamine 3000 transfection reagent. After transfection, the lentivirus produced in 293T cells was released from the cells and existed in the cell culture medium in the form of virus particles. Therefore, 48 or 72 hours after transfection, only the cell culture medium was removed from the culture plate and concentrated 100-fold with the lenti-X concentrator to obtain lentiviral particles in the form of pellets. After that, 1 x 10 6 The NK-92 cell line and concentrated viral supernatant were added to MOI 100, and 8 μg / mL of polybrene was added to induce transduction.

[0601] As a result, as shown in Fig. 8, it was confirmed that the endoglin-targeting CAR according to the present invention was stably expressed in NK92 cells.

[0602] 3. Confirmation of anticancer cytotoxicity of CAR-NK92 cells (1)

[0603] To confirm the anticancer cytotoxicity of endoglin-specific CAR-NK92 cells against a pancreatic cancer cell line (Panc-1), previously prepared CAR-NK92 cells and endoglin-overexpressing Panc1 pancreatic cancer cells (Panc-1-ENG-luc cells) were co-cultured at a ratio of 0.5:1 in NK92 medium containing rhIL2 (200 IU / ml) for 24 h. After removing the medium, the cells were washed with DPBS, RIPA buffer was added, and incubated at 37°C for 20 min. After centrifugation at 4000 rpm for 15 min at room temperature, the supernatant was transferred to a new tube, and 50 μl of cancer lysate and 50 μl of Nanoglo luciferase assay substrate (Promega, N1120) were added, followed by incubation for 3 min. Bioluminescence was then measured.

[0604] As a result, as shown in Fig. 9, it was confirmed that all endoglin-targeting CAR-expressing NK92 cells according to the present invention exhibited toxicity against pancreatic cancer cells.

[0605] 4. Confirmation of anticancer cytotoxicity of CAR-NK92 cells (2)

[0606] To determine the anticancer cytotoxicity of endoglin-specific CAR-NK92 cells against endoglin-overexpressing K562 cell lines, 0.2 M endoglin-overexpressing K562 cells (K562-ENG cells) were seeded in a 96-well plate, and then 0.8 M of the previously prepared endoglin-specific CAR-NK92 cells were seeded together with 100 μl of NK 92 medium containing rhIL2 (200 IU / ml), and incubated for 4 hours. NK92 cells were transferred to a 12x75 mm round-bottom test tube, 2 ml of dPBS was added, resuspended, and centrifuged at 1350 rpm for 3 minutes. The supernatant was discarded and the experiment was repeated once under the same conditions, and then fluorochrome-labeled antibody (CD56-APC, eBioscience, 17-0566-42) diluted in 100 μl PBS together with live / dead fixable dye (eBioscience, 65-0866-14) was added and resuspended. After incubation for 20 min at 4°C in a darkroom, 2 ml of dPBS was added, resuspended, and centrifuged at 1350 rpm for 3 min. The supernatant was discarded and the experiment was repeated once under the same conditions, and the cells resuspended in 0.5 ml of dPBS were analyzed using flow cytometry.

[0607] As a result, as shown in FIGS. 10 and 11, it was confirmed that the endoglin-targeting CAR-expressing NK92 cells according to the present invention have killing activity against endoglin-overexpressing K562 cells.

[0608] 5. Confirmation of CAR-NK92 cell activation in the presence of cancer cells

[0609] In order to evaluate whether NK92 cells expressing an endoglin-specific CAR were activated in the presence of a pancreatic cancer cell line (Aspc1-ENG) overexpressing luciferase gene and endoglin, the endoglin-overexpressing pancreatic cancer cell line (Aspc1-ENG) was co-cultured with endoglin-specific CAR-NK92 cells using the same method as in 4. above, and the expression of CD107a, which can indirectly confirm granule release, and IFN gamma, which can confirm CAR-NK cell activity, were confirmed using flow cytometry.

[0610] As a result, as shown in FIGS. 12a to 12c and 13a to 13c, in the case of wild-type NK92 cells, there was no change in the expression levels of CD107a and IFN gamma after co-culture with pancreatic cancer cells (Aspc1-ENG), but in the case of the endoglin-specific CAR-NK92 cells according to the present invention, the expression levels of CD107a and IFN gamma significantly increased after co-culture with pancreatic cancer cells (Aspc1-ENG), indicating that the endoglin-specific CAR-expressing NK92 cells according to the present invention were activated by the pancreatic cancer cell line (Aspc1-ENG).

[0611]

[0612] [Example 3] Production of endoglin-specific CAR-pNK cells and evaluation of their anticancer activity.

[0613] 1. Production of endoglin-specific CAR-expressing primary NK cells

[0614] Endoglin-specific CAR-NK cells were produced in the same manner as in Example 2, but primary NK cells derived from whole blood obtained from a healthy donor were used as natural killer cells into which the CAR was to be introduced.

[0615] 2. Evaluation of transduction efficiency

[0616] In order to evaluate the endoglin-specific CAR expression efficiency in primary NK cells in the above 1., the lentiviral transduction efficiency was evaluated using the following experimental method. Specifically, 1 M of the cells obtained in the above 1. were transferred to a 12 x 75 mm round-bottomed test tube, 2 ml of dPBS was added, resuspended, and centrifuged at 1350 rpm for 3 minutes. The supernatant was discarded and repeated 2-4 times, and then biotin-ENG protein (1 ug per 1 M cells) diluted in 100 μl PBS together with a live / dead fixative dye (eBioscience, 65-0866-14) was added and resuspended. After incubation for 20 minutes at 4°C in a darkroom, 2 ml of dPBS was added, resuspended, and centrifuged at 1350 rpm for 3 minutes. The supernatant was discarded and the mixture was repeated 8–10 times. After adding a fluorochrome-labeled antibody (CD56-APC, eBioscience, 17-0566-42) diluted in 100 μl of PBS, the mixture was resuspended. After incubating in a dark room at 4°C for 20 minutes, 2 ml of dPBS was added, resuspended, and centrifuged at 1350 rpm for 3 minutes. The supernatant was discarded and the mixture was repeated once under the same conditions. The cells were resuspended in 0.5 ml of dPBS and analyzed using flow cytometry.

[0617] As a result, as shown in Fig. 14, it was confirmed that the endoglin-specific CAR according to the present invention was stably expressed in primary NK cells.

[0618] 3. Confirmation of anticancer cytotoxicity of CAR-pNK

[0619] To confirm the anticancer cytotoxicity of endoglin-specific CAR-pNK cells, we evaluated their cell killing activity against pancreatic cancer fibroblasts (CAF16) derived from pancreatic cancer patients with high endoglin expression and human pancreatic stellate cells (hPaSC) with relatively low endoglin expression. Specifically, 0.02 M pancreatic cancer fibroblasts (CAF16) or human pancreatic stellate cells (hPaSC) were seeded in 96-well plates and incubated for 24 h. The cells were stained with DiO (Invitrogen, V22886), and 0.02 M of the previously prepared endoglin-specific CAR-pNK cells were seeded together with 100 μl of PBNK medium containing rhIL2 (200 IU / ml), and incubated for 20 h. Afterwards, fluorescence images were captured using a LionheartFX (BioTek, Germany) automated live-cell microscope, and fluorescence intensity was measured.

[0620] As a result, as shown in FIGS. 15 and 16, it was confirmed that the endoglin-specific CAR-pNK cells according to the present invention had a higher cell killing ability against CAF16 cells, in which endoglin is highly expressed, compared to hPaSCs, in which endoglin is relatively lowly expressed.

[0621] 4. Confirmation of CAR-pNK cell activation in the presence of cancer cells

[0622] To evaluate whether CAR-pNK cells are activated in the presence of endoglin-overexpressing cancer fibroblasts, pancreatic cancer patient-derived cancer fibroblasts (CAF16) with high endoglin expression and human pancreatic stellate cells (hPaSC) with relatively low endoglin expression were co-cultured using the same method as described in 2 above, and the expression of CD107a, which can indirectly confirm granule release, was confirmed using flow cytometry.

[0623] As a result, as shown in Figures 17a and 17b, in the case of endoglin-specific CAR-pNK cells, the CD107a expression level was significantly increased after co-culture with pancreatic cancer fibroblasts (CAF16) derived from a pancreatic cancer patient, indicating that the endoglin-specific CAR-expressing pNK cells according to the present invention were activated by endoglin-expressing cancer fibroblasts.

[0624]

[0625] [Example 4] Induction of endoglin-specific humanized antibodies

[0626] The mouse antibody ENG 24 clone was selected for humanization design. Human germline antibody framework regions that could be used for mouse antibody humanization were screened using the CDR grafting method. By screening for the human germline antibody or its subtype with the highest sequence identity to the amino acid sequence of the mouse antibody variable region, the mouse antibody CDR was inserted into the screened framework region (HENG24-WT). However, since direct grafting of the CDR from a mouse antibody results in a human receptor with low binding affinity, it is desirable to mutate specific residues in the structure to mouse residues at certain positions. Therefore, by back-mutating residues in the framework region and / or CDR region, more candidate antibodies were obtained (HENG24-5, HENG24-10, HENG24-12, HENG24-16, HENG24-20, HENG24-21, HENG24-24, HENG24-25, HENG24-26, HENG24-29, HENG24-37, HENG24-39). A total of 13 humanized antibodies were obtained for the mouse antibody ENG 24 clone. The amino acid sequences of the heavy chain variable region and light chain variable region of the humanized antibodies are as follows. However, the sequence information at this time is presented based on the IMGT / DomainGapAlign web program (https: / www.ncbi.nlm.nih.gov / igblast / igblast.cgi).

[0627] 클론구분서열정보(5'->3')서열번호HENG24-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

[0628] 클론구분서열정보(5'->3')서열번호HENG24-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

[0629] 클론구분서열정보(5'->3')서열번호HENG24-10HFR1EVQLVESGGGLVQPGGSLRLSCAAS236HCDR1GFTFSNYW13HFR2MNWVRQSPGKGLEWVGE238HCDR2IRLKSNNYGT35HFR3HYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTGVYYC240HCDR3ARWSRHFDV233HFR4WGQGTMVTVSS242VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQSPGKGLEWVGEIRLKSNNYGTHYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTGVYYCARWSRHFDVWGQGTMVTVSS257LFR1DIQLTQSPSFLSASVGDRVTITCRAS243LCDR1SSVSY72LFR2MHWYQQKPGSAPKPLIY244LCDR2ATS83LFR3NLASGVPPRFSGSGSGTEFTLTISSLQAEDAATYYC246LCDR3QQWSSYPPT111LFR4FGQGTKLEIKR255VLDIQLTQSPSFLSASVGDRVTITCRASSSVSYMHWYQQKPGSAPKPLIYATSNLASGVPPRFSGSGSGTEFTLTISSLQAEDAATYYCQQWSSYPPTFGQGTKLEIKR263

[0630] 클론구분서열정보(5'->3')서열번호HENG24-12HFR1EVQLVESGGGLVQPGGSLRLSCAAS236HCDR1GFTFSNYW13HFR2MNWVRQSPGKGLEWVGE238HCDR2IRLKSNNYGT35HFR3HYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTGVYYC240HCDR3ARWSRHFDV233HFR4WGQGTMVTVSS242VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQSPGKGLEWVGEIRLKSNNYGTHYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTGVYYCARWSRHFDVWGQGTMVTVSS257LFR1DIQLTQSPSFLSASVGDRVTITCRAS243LCDR1SSVSY72LFR2MHWYQQKPGNAPKPLIY245LCDR2ATS83LFR3NLASGVPSRFSGSGSGTEFTLTISSLQAEDAATYYC247LCDR3QQWSSYPPT111LFR4FGQGTKLEIKR255VLDIQLTQSPSFLSASVGDRVTITCRASSSVSYMHWYQQKPGNAPKPLIYATSNLASGVPSRFSGSGSGTEFTLTISSLQAEDAATYYCQQWSSYPPTFGQGTKLEIKR265

[0631] 클론구분서열정보(5'->3')서열번호HENG24-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

[0632] 클론구분서열정보(5'->3')서열번호HENG24-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

[0633] 클론구분서열정보(5'->3')서열번호HENG24-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

[0634] 클론구분서열정보(5'->3')서열번호HENG24-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

[0635] 클론구분서열정보(5'->3')서열번호HENG24-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

[0636] 클론구분서열정보(5'->3')서열번호HENG24-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

[0637] 클론구분서열정보(5'->3')서열번호HENG24-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

[0638] 클론구분서열정보(5'->3')서열번호HENG24-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

[0639]

[0640] 클론구분서열정보(5'->3')서열번호HENG24-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

[0641] 3. 인간화 항체의 발현 및 정제

[0642] Using primers for the anti-endoglin humanized scFv shown in Table 30 below, a PCR product was generated that inserted the overlapping part with the pMopac12 vector. The PCR product (insert) and the pMoapc12 vector were combined using the Gibson assembly technique to insert the insert gene into the vector. After transformation with JudeI, the product was plated on an LB plate supplemented with chloramphenicol and cultured at 37°C for 16 hours. The following day, colonies were picked and inoculated into LB media supplemented with chloramphenicol and cultured at 37°C for 16 hours. The following day, colony PCR was performed using primers for the scFv to confirm insertion, and bands were confirmed by electrophoresis on a 1% agarose gel. The cloned anti-endoglin scFv antibody clone was transformed into BL21 (DE3) Escherichia coli host. The transformants were cultured in LB medium containing chloramphenicol until the OD value reached 0.6, and expression was induced by treating with 0.5 mM IPTG at 25°C. The cells obtained by centrifuging the culture solution were suspended in a 100 mM Tris, 0.75 M sucrose pH 7.5 buffer, and osmotic shock was applied by adding 1 mM EDTA. The supernatant was obtained by centrifugation at 12,000 xg for 1 hour. To purify only the induced expressed protein, a resin having an affinity for Ni-NTA was used, and the purified protein was confirmed by SDS-PAGE analysis.

[0643]

[0644] Clone MW (kDa) Extinction coefficient value Purity Purity consideration Concentration (mg / mL) Volume after purification (uL) Yield (ug) Total YieldHENG24-wt28.32.192>99%0.341500170.50.682mg / LHENG24-528.52.1810.352500175.50.702mg / LHENG24-1028.52.1800.07360043.80.175m g / LHENG24-1228.52.1790.242800193.60.774mg / LHENG24-1628.52.1790.12160072.60.290mg / LHENG24-2028.62.1760.215500107.50.430mg / L

[0645] Clone MW (kDa) Extinction coefficient value Purity Considered concentration (mg / mL) Volume after purification (uL) Yield (ug) Total yield HENG24-21 28.5 2.17 7 >99% 0.03 2 3 5 0 11.2 0.04 4 mg / L HENG24-24 28.42 18 4 0.10 0.30 0.30 0.12 0 mg / L HENG24-25 28.42 18 7 0.11 0.34 0.13 8 mg / L HENG24-26 28.5 2.17 70.08430025.20.101mg / LHENG24-2928.52.1790.08530025.50.102mg / LHENG24-3 728.42.1850.0193002.70.023mg / LHENG24-3928.52.1780.15930047.70.191mg / L

[0646] As a result, as shown in Tables 31 and 32 and Figures 18 and 19, it was confirmed that humanized scFv HENG24-5, HENG24-10, HENG24-12, HENG24-16, HENG24-20, HENG24-21, HENG24-24, HENG24-25, HENG24-26, HENG24-29, HENG24-37, HENG24-39 scFv) having a molecular weight of about 28 kDa were purified.

[0647]

[0648] [Example 5] Evaluation of antigen binding capacity based on endoglin-expressing cells

[0649] The binding ability of previously purified anti-endoglin humanized scFvs to endoglin antigen was analyzed using cells expressing endoglin. Specifically, endoglin-positive K562 cells and endoglin-negative K562 cells were prepared and seeded in 96-well U-bottom plates at 5 × 10 per well. 4 The cells were seeded with 10 μg of the same number of cells. After washing with PBSF (1% FBS) washing buffer, anti-endoglin humanized scFv according to the present invention (clone #ENG24, #HENG24-wt, #HENG24-5, #HENG24-10, #HENG24-12, #HENG24-16, #HENG24-20, #HENG24-21, #HENG24-24, #HENG24-25, #HENG24-29, #HENG24-37, #HENG24-39 scFv) was serially diluted 1 / 4 at 1 μM and incubated at 4°C for 1 hour. Washing was performed with PBSF (1% FBS) washing buffer. Afterwards, anti-HISx6-FITC was treated at a ratio of 250:1 and incubated at 4°C for 1 hour. After washing with PBSF (1% FBS), FACS analysis was performed. However, scFv of TRC105 (carotuximab) antibody was used as a control in the experiment.

[0650] Clone IC 50 (nM)ENG246.02±0.18HENG24 wt8.13±0.13HENG24-510.2±0.98HENG24-1027.8±1.24HENG24-128.75±0.50HENG24-1614.5±0.85HENG24-2015.7±0.41TRC105(positive control)7.43±0.07

[0651] As a result, as shown in Table 33 and FIGS. 20 and 21, it was confirmed that the anti-endoglin humanized scFv antibodies manufactured according to the present invention do not bind to endoglin-negative K562 cells, but selectively bind only to endoglin-positive K562 cells.

[0652]

[0653] [Example 6] Evaluation of the Endoglin Binding Ability of Anti-Endoglin Humanized Antibodies

[0654] The binding ability of the purified anti-endoglin humanized scFv antibodies to the endoglin antigen was analyzed using an ELISA method. First, the anti-endoglin humanized scFv antibody was coated on an ELISA plate at a concentration of 2 μg / mL at 4°C overnight. The plate was blocked with 3% PBS BSA for 1 hour. The biotinylated endoglin-his protein was serially diluted 1 / 5 from 1 μM and bound to the anti-endoglin scFv coated on the ELISA plate at room temperature for 1 hour. Each well was washed three times with PBST (tween 20 0.05%) washing buffer, and then HRP enzyme-conjugated streptavidin (Streptavidin-HRP; abcam) was diluted 40,000:1 to detect the biotin-labeled antibody and incubated at room temperature for 1 hour. Each well was washed three times with PBST (tween20 0.05%) washing buffer, treated with TMB, and incubated at room temperature for 20 minutes. After treatment with 2 M H2SO4, the absorbance was measured at 450 nm. However, in the following experiments, the anti-endoglin scFv was used as a positive control, either the scFv of the 9H10 antibody (Creativebiolabs, CAT#: TAB-1577CL) or the scFv of the TRC105 (carotuximab) antibody.

[0655] Clone IC 50(nM)mENG2435.9±4.4HENG24-2437.1±0.98HENG24-2524.1±3.8HENG24-2669.1±58.2HEN G24-2924.2±3.0HENG24-3796.0±69.1HENG24-3959.7±3.29H104.9±0.3TRC10528.7±2.5

[0656] As a result, as shown in Table 34 and Figure 22, it was confirmed that the anti-endoglin humanized scFv antibodies manufactured according to the present invention had excellent binding ability to the endoglin antigen.

[0657]

[0658] [Example 7] Production of endoglin-specific CAR-NK92 cells and evaluation of their anticancer activity.

[0659] 1. Design of an endoglin-specific chimeric antigen receptor expression cassette

[0660] A chimeric antigen receptor was designed in the same manner as in Example 2, but an anti-endoglin humanized scFv was inserted as the anti-endoglin binding domain. Specifically, a polynucleotide encoding the anti-endoglin humanized scFv (VL-linker-VH) according to the present invention, CD8 hinge, CD8 transmembrane domain, 4-1BB, and CD3 zeta signaling domain (base sequence encoding amino acids of SEQ ID NOs: 286-289) was constructed as an expression cassette and inserted into a lentiviral transfer vector. At this time, a polynucleotide encoding a CD8 signal peptide was inserted at the 5' end, and a polynucleotide encoding P2A represented by SEQ ID NO: 165 and a polynucleotide encoding green fluorescent protein (GFP) represented by SEQ ID NO: 166 were inserted at the 3' end. The amino acid sequence of each chimeric antigen receptor is shown in Table 35 below. However, in the following experiments, the anti-endoglin scFv was replaced with the scFv ...

Claims

1. A heavy chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 17; A heavy chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 36; A heavy chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 37 to 57 and 233 to 235; A light chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 58 to 76; A light chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 77 to 90; and An anti-endoglin antibody or antigen-binding fragment comprising a light chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 91 to 112.

2. In paragraph 1, The above anti-endoglin antibody or antigen-binding fragment is an antibody or antigen-binding fragment selected from the following: 1) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 18, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 37; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 58, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 77, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 91; 2) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 18, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 37; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 59, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 77, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 92; 3) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 19, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 38; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 60, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 78, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 93; 4) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 20, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 39; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 61, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 79, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 94; 5) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 20, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 39; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 62, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 80, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 95; 6) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 21, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 40; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 63, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 81, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 96; 7) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 22, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 41; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 64, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 82, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 97; 8) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 23, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 42; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 65, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 98; 9) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 6, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 43; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 66, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 84, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 99; 10) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 6, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 43; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 67, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 85, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 100; 11) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 7, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 25, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 44; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 68, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 86, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 101; 12) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 8, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 26, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 45; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 69, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 79, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 102; 13) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 9, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 46; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 62, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 80, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 103; 14) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 28, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 47; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 70, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 87, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 104; 15) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 20, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 48; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 71, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 88, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 105; 16) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 11, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 29, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 49; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 106; 17) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 8, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 26, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 45; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 89, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 107; 18) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 12, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 25, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 50; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 89, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 108; 19) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 30, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 51; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 73, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 90, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 109; 20) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 14, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 31, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 52; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 89, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 108; 21) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 9, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 32, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 53; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 74, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 90, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 109; 22) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 15, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 33, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 54; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 75, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 80, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 110; 23) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 16, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 34, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 55; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 89, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 108; 24) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 56; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111; 25) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 17, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 36, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 57; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 76, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 112; 26) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 233; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111; 27) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 234; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 111; and 28) A humanized antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 235; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 72, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO:

111.

3. In paragraph 1, The above anti-endoglin antibody or antigen-binding fragment, Comprising at least one heavy chain variable region selected from the group consisting of sequence numbers 113 to 133, 256 to 262 and 325, An anti-endoglin antibody or antigen-binding fragment comprising at least one light chain variable region selected from the group consisting of SEQ ID NOs: 134 to 158 and 263 to 272.

4. In paragraph 1, The above anti-endoglin antibody or antigen-binding fragment is an antibody or antigen-binding fragment selected from the following: 1) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 113 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 134; 2) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 113 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 135; 3) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 114 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 136; 4) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 115 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 137; 5) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 115 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 138; 6) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 116 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 139; 7) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 117 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 140; 8) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 118 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 141; 9) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 119 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 142; 10) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 119 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 143; 11) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 120 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 144; 12) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 121 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 145; 13) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 122 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 146; 14) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 123 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 147; 15) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 124 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 148; 16) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 125 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 149; 17) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 121 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 150; 18) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 126 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 151; 19) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 127 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 152; 20) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 128 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 153; 21) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 129 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 154; 22) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 130 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 155; 23) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 131 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 156; 24) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 132 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 157; 25) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 133 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 158; 26) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 256 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 263; 27) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 264; 28) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 263; 29) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 265; 30) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 325 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 266; 31) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 258 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 266; 32) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 259 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 266; 33) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 260 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 267; 34) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 261 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 268; 35) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 269; 36) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 270; 37) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 260 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 271; and 38) An anti-endoglin antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 262 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO:

272.

5. In paragraph 1, The above anti-endoglin antibody or antigen-binding fragment is an anti-endoglin antibody or antigen-binding fragment comprising an anti-endoglin single chain antibody, Fd, Fab, Fab', F(ab')2, dsFv or scFv.

6. In paragraph 1, The anti-endoglin antibody or antigen-binding fragment thereof comprises a polyclonal antibody, a monoclonal antibody, a whole antibody, a chimeric antibody, a human antibody, a humanized antibody, a bivalent, a bispecific molecule, a minibody, a domain antibody, a bispecific or multispecific antibody, an immune cell engaging bispecific or multispecific antibody, an antibody mimetic, a unibody, a diabody, a triabody or a tetrabody.

7. In paragraph 1, The above anti-endoglin antibody or antigen-binding fragment is an anti-endoglin scFv.

8. In paragraph 7, The anti-endoglin scFv is an anti-endoglin antibody or antigen-binding fragment comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 208 to 230 and 273 to 285.

9. A nucleic acid molecule encoding an anti-endoglin antibody or antigen-binding fragment of any one of claims 1 to 8.

10. A recombinant expression vector comprising the nucleic acid molecule of paragraph 9.

11. A host cell transfected with the expression vector of Article 10.

12. An immunoconjugate comprising an anti-endoglin antibody or antigen-binding fragment of any one of claims 1 to 8 and a cytotoxic agent.

13. A pharmaceutical composition for preventing or treating an endoglin-related disease, comprising an anti-endoglin antibody or antigen-binding fragment of any one of claims 1 to 8 as an active ingredient.

14. In paragraph 13, The above endoglin-related disease is a pharmaceutical composition, which is an eye disease related to neovascularization such as cancer, age-related macular degeneration, diabetic retinopathy, non-diabetic retinopathy, choroidal neovascularization (CNV) or subretinal neovascularization.

15. A composition for diagnosing an endoglin-related disease, comprising an antibody or antigen-binding fragment of any one of claims 1 to 8 as an active ingredient.

16. A chimeric antigen receptor (CAR) comprising an antigen binding domain that specifically binds to endoglin, The above antigen binding domain is, A chimeric antigen receptor comprising an antigen-binding fragment comprising a heavy chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 17; a heavy chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 36; a heavy chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 37 to 57 and 233 to 235; a light chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 58 to 76; a light chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 77 to 90; and a light chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 91 to 112.

17. In paragraph 16, A chimeric antigen receptor comprising an anti-endoglin scFv, wherein the antigen binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 208 to 230 and 273 to 285.

18. An immune effector cell expressing the chimeric antigen receptor of item 17.

19. A pharmaceutical composition for preventing or treating cancer, comprising the immune effector cell of Article 18 as an active ingredient.

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