Improved anti-VEGFR2 (KDR) antibodies and their uses

Abstract

The present invention relates to an improved anti-VEGFR2 (KDR) antibody or an antigen-binding fragment thereof that exhibits improved properties and uses thereof. Specifically, the present invention relates to an anti-VEGFR2 (KDR) antibody or an antigen-binding fragment thereof that has been mutated by replacing the heavy chain FR (framework regoin) of a human antibody that specifically binds to VEGFR2 / KDR from VH1 to VH3 and replacing the light chain FR (framework regoin) from VL1 to VK1 to increase affinity for the antigen, a composition for diagnosing or treating a disease associated with angiogenesis, a composition for preventing or treating a tumor or cancer, and a composition for administering in combination with a therapeutic agent other than the antibody, which comprises the antibody or the antigen-binding fragment thereof.

Description

[Technical Field]

[0001] The present invention relates to an improved anti-VEGFR2 (KDR) antibody or antigen-binding fragment thereof with improved properties and uses thereof. Specifically, the present invention relates to an anti-VEGFR2 (KDR) antibody or antigen-binding fragment thereof, which has been mutated by substituting the heavy chain framework FR (FR) from VH1 to VH3 and the light chain framework FR (FR) from VL1 to VK1 of a human antibody that specifically binds to VEGFR2 / KDR, thereby increasing affinity for the antigen, as well as compositions containing the antibody or antigen-binding fragment for diagnosing or treating diseases associated with angiogenesis, preventing or treating tumors or cancer, and compositions for administering in combination with therapeutic agents other than the antibody. [Background technology]

[0002] VEGFR2 / KDR is the primary angiogenic receptor, binds VEGF isoforms A, C, D, and E, and is important for the mitogenic, angiogenic, and permeability-enhancing effects of VEGF as well as endothelial cell differentiation. Anti-VEGFR2 antibodies have been shown to bind all known VEGF isoforms. It can bind to VEGFR2 / KDR and prevent it from initiating signaling, and because tumors secrete higher amounts of VEGF while the number of receptors remains relatively constant, targeting the receptor increases the probability of completely suppressing signaling even in the presence of very high levels of VEGF isoforms.

[0003] Angiogenesis refers to the generation of new blood vessels from existing blood vessels through the growth, division, migration, etc. of endothelial cells. Angiogenesis does not occur in adults except for special cases such as wound healing and the female reproductive cycle. However, excessive angiogenesis has been reported in diseases such as tumor growth and metastasis, age-related macular degeneration, rheumatoid arthritis, diabetic retinopathy, psoriasis, and chronic inflammation (Cameliet and Jain, Nature, 407:249, 2000). For this reason, many efforts have been made to treat diseases, especially tumors, using angiogenesis inhibitors.

[0004] Factors involved in angiogenesis include vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor (TGFb), and fibroblast growth factor (FGF). Of these, endothelial growth factor (EGF) exists as an endothelial cell-specific factor that is directly involved in the growth, differentiation, and migration of endothelial cells. There are four different isoforms of VEGF (VEGF165, VEGF121, VEGF189, and VEGF206), and VEGF165 is the most abundant isoform in all human tissues except the placenta (Tisher et al., J Biol Chem, 266: 11947, 1991).

[0005] Endothelial growth factor (VEGF) regulates the formation of new blood vessels in situ from the differentiation of endothelial precursors (angioblasts) and is expressed in embryonic tissues (Breier et al., Development (Camb), 114:521, 1992), macrophages, and proliferating epithelial keratinocytes during wound healing (Brown et al., J. Exp. Med., 176:1375, 1992), and may be responsible for tissue edema associated with inflammation (Ferrara et al., Endocr. Rev., 13:18, 1992). In situ hybridization studies have demonstrated that VEGF is highly expressed in numerous human tumor lines, including glioblastoma multiforme, hemangioblastoma, central nervous system neoplasms, and AIDS-associated Kaposi's sarcoma (Plate et al., Nature 359:845, 1992; Plate et al., Cancer Res. 53:5822, 1993; Berkman et al., J. Clin. Invest. 91:153, 1993; Nakamura et al., AIDS Weekly. 13(1), 1992). High levels of VEGF have also been observed in hypoxia-induced angiogenesis (Shweiki et al., Nature 359:843, 1992).

[0006] The biological functions of VEGF are mediated through VEGF receptors, which have high affinity for VEGF and are selectively expressed in endothelial cells during embryogenesis (Millauer et al., Cell, 72:835, 1993) and tumorigenesis. VEGF receptors (VEGFRs) are generally class III receptor tyrosine kinases characterized by multiple, typically five or seven, immunoglobulin-like loops in their amino-terminal extracellular receptor ligand-binding domain (Kaipainen et al., J. Exp. Med., 178:2027, 1993). The other two domains contain a carboxy-terminal intracellular catalytic domain and a transmembrane domain interrupted by the insertion of a variable-length hydrophilic interkinase sequence called the kinase insert domain (Terman et al., Oncogene, 6:1677, 1991). VEGFRs include the fms-like tyrosine kinase receptor (flt-1), or VEGFR-1 (Shibuya et al., Oncogene, 5:519, 1990; WO 92 / 14248; Terman et al., Oncogene, 6:1677, 1991), and the kinase insert domain-containing receptor / fetal liver kinase (KDR / flk-1), or VEGFR-2 (Matthews et al., Proc. Natl. Acad. Sci. USA, 88:9026, 1991), although other receptors, such as neuropilin-1 and neuropilin-2, can also bind VEGF. Another tyrosine kinase receptor, VEGFR-3 (flt-4), binds to the VEGF homologs VEGF-C and VEGF-D and plays an important role in lymphatic vessel development.

[0007] High levels of Flk-1 are expressed by endothelial cells infiltrating gliomas (Plate et al., Nature 359:845, 1992). Flk-1 levels are specifically upregulated by VEGF produced by human glioblastomas (Plate et al., Cancer Res. 53:5822, 1993). The high expression of Flk-1 in glioblastoma-associated endothelial cells (GAECs) indicates that receptor activity is likely induced during tumorigenesis, since Flk-1 transcripts are barely detectable in normal brain endothelial cells. Such upregulation occurs only in vascular endothelial cells in close proximity to the tumor. Blockade of VEGF activity with a neutralizing anti-VEGF monoclonal antibody (mAb) suppresses the growth of human tumor xenografts in nude mice (Kim et al., Nature 362:841, 1993), indicating a direct role for VEGF in tumor-associated angiogenesis.

[0008] VEGF ligands are upregulated in tumor cells, and their receptors are upregulated in tumor-infiltrating vascular endothelial cells, but expression of VEGF ligands and their receptors is low in normal cells that are not involved in angiogenesis. Therefore, these normal cells block the interaction between VEGF and its receptor, resulting in the suppression of angiogenesis and therefore the absence of tumor growth.

[0009] High levels of VEGFR-2 are expressed by glioma-infiltrating endothelial cells and are specifically upregulated by VEGF produced by human glioblastomas (Plate et al., Nature, 359:845, 1992; Plate et al., Cancer Res., 53:5822, 1993). Because VEGFR-2 transcripts are barely detectable in normal brain endothelial cells, the high expression of VEGFR-2 in glioblastoma-associated endothelial cells (GAECs) indicates that receptor activity is induced during tumorigenesis.

[0010] VEGF is expressed at high levels in various types of tumors (Plate et al., Nature, 359:8435, 1992; Dvorak et al., J. Exp. Med., 174:1275, 1991), and newly formed capillaries converge around VEGF-producing tumor cells (Plate et al., Nature, 359:8435, 1992). VEGF expression is strongly upregulated under hypoxic conditions, as is associated with rapidly growing tumors (Shweiki et al., Nature, 359:843, 1992). Neutralization of VEGF with antibodies such as Avastin (Ferrara et al., Nat Rev Drug Discov., 3(5):391, 2004; Avery et al., Ophthalmology, 113(3):363, 2006) is a clinically approved treatment for inhibiting cancer or other angiogenic diseases such as AMD. However, resistance to VEGF blockade has also been found when combined with chemotherapy. This resistance may be associated with remodeled vasculature and increased expression of other angiogenic factors. Thus, there remains a need in the art for improved compositions and methods for inhibiting cancer and other diseases associated with angiogenesis.

[0011] Although both tyrosine kinase inhibitors (TKIs) and anti-KDR antibodies can suppress KDR-mediated angiogenesis, antibody approaches have advantages over TKIs. In contrast to TKIs, anti-KDR antibodies are more specific KDR targeting agents (i.e., anti-KDR antibodies do not inhibit other VEGF receptors). Due to their high specificity, anti-KDR antibodies can limit or avoid off-target effects and toxicity caused by less specific TKIs (Witte et al., Cancer Metastasis Rev., Jun;17(2):155, 1998).

[0012] Unlike Avastin, which binds only to one ligand (VEGF-A), anti-KDR antibodies are expected to prevent all known VEGFs from binding to VEGFR2 / KDR. Anti-KDR antibodies may have a more potent inhibitory effect on tumor angiogenesis than simply blocking VEGF-A. Treatment with anti-KDR antibodies may be effective in cases of Avastin resistance.

[0013] Under these technical backgrounds, the inventors of the present application have improved conventional antibodies that specifically bind to VEGFR2 / KDR by substituting the framework region FR (framework region) of the heavy chain variable region from VH1 to VH3 and the framework region FR (framework region) of the light chain from VL1 to VK1, thereby inducing mutations in the CDR3 regions of the heavy and light chains, thereby producing novel antibodies with increased affinity for the antigen, and have completed the present invention. Summary of the Invention [Problem to be solved by the invention]

[0014] An object of the present invention is to provide an improved antibody or antigen-binding fragment thereof that has improved binding affinity to VEGFR2 / KDR. Another object of the present invention is to provide a nucleic acid encoding said antibody or antigen-binding fragment thereof. Another object of the present invention is to provide a vector containing the nucleic acid, a transformed cell containing the vector, and a method for producing the same.

[0015] Another object of the present invention is to provide a composition for preventing or treating angiogenic diseases, comprising the antibody or antigen-binding fragment thereof. Another object of the present invention is to provide a method for preventing or treating angiogenic diseases, comprising administering the antibody or antigen-binding fragment thereof to an individual. Another object of the present invention is to provide a use of the antibody or antigen-binding fragment thereof in the manufacture of a composition for preventing or treating angiogenic diseases. It is yet another object of the present invention to provide a composition for diagnosing angiogenic diseases, which comprises the antibody or its antigen-binding fragment.

[0016] Another object of the present invention is to provide a composition for preventing or treating tumors or cancer, comprising the antibody or antigen-binding fragment thereof. Another object of the present invention is to provide a method for preventing or treating cancer, comprising administering the antibody or antigen-binding fragment thereof to an individual. Another object of the present invention is to provide a use of the antibody or antigen-binding fragment thereof in the manufacture of a composition for preventing or treating cancer. It is yet another object of the present invention to provide a composition for co-administration with other therapeutic agents, which comprises the antibody or antigen-binding fragment thereof. [Means for solving the problem]

[0017] To achieve the above object, the present invention provides a heavy chain CDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19 and 25; a heavy chain CDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20 and 26; and a heavy chain variable region comprising a heavy chain CDR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 51, 52, and 53; and a light chain CDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22 and 28; a light chain CDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23 and 29; and

[0018] An antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR is provided, which comprises a light chain variable region comprising a light chain CDR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, and 30.

[0019] The present invention also provides an antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR, comprising a heavy chain variable region comprising a sequence having 80% or more homology to a sequence selected from the group consisting of SEQ ID NOs: 32, 36, 40, 44, and 48.

[0020] The present invention also provides an antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR, comprising a light chain variable region comprising a sequence having 80% or more homology to a sequence selected from the group consisting of SEQ ID NOs: 34, 38, 42, 46, and 50.

[0021] The present invention also provides an antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR, comprising a heavy chain variable region comprising a sequence having 80% or more identity to a sequence selected from the group consisting of SEQ ID NOs: 32, 36, 40, 44, and 48, and a light chain variable region comprising a sequence having 80% or more identity to a sequence selected from the group consisting of SEQ ID NOs: 34, 38, 42, 46, and 50. The present invention also provides a nucleic acid encoding the antibody or antigen-binding fragment thereof. The present invention also provides a vector comprising the nucleic acid. The present invention also provides a transformed cell containing the vector.

[0022] The present invention also provides a method for producing the antibody or antigen-binding fragment thereof, comprising the steps of: (a) culturing the cells; and (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells. The present invention also provides a composition for preventing or treating angiogenic diseases, comprising the antibody or antigen-binding fragment thereof. The present invention also provides a composition for diagnosing angiogenic diseases, comprising the antibody or antigen-binding fragment thereof. The present invention also provides a composition for preventing or treating tumors or cancer, comprising the antibody or antigen-binding fragment thereof. The present invention also provides compositions comprising the antibody or antigen-binding fragment thereof for co-administration with other therapeutic agents. [Brief explanation of the drawings]

[0023] [Figure 1] This sequence is a grafted version of the heavy chain CDR sequence of 6A6. [Figure 2] This sequence is a graft of the light chain CDR sequence of 6A6. [Figure 3] 1 shows the results of ELISA evaluating the binding of transiently expressed and purified anti-Ang2 antibodies to human and mouse Ang2 and Ang1. [Figure 4] The results show the ability of the selected anti-Ang2 antibodies to neutralize human and mouse Ang2 / Tie2 binding. DETAILED DESCRIPTION OF THE INVENTION

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein is that which is well known and commonly used in the art.

[0025] The present invention relates to an improved fully humanized antibody that neutralizes vascular endothelial growth factor receptors, and in particular to an improved human monoclonal antibody (registered patent 10-0883430) that neutralizes vascular endothelial growth factor receptors, and uses thereof.

[0026] The inventors of this application have developed a unique antibody, derived from a fully human antibody library, that targets VEGFR-2 / KDR while simultaneously reacting with mouse and rat flk-1 (a VEGFR-2 homologue). Clinical trials have demonstrated the superiority of this antibody, but the antibody protein's binding affinity to the antigen is relatively low and its in vivo half-life is short. By improving this antibody, its efficacy as an immunotherapy for cancer and angiogenesis-related diseases is expected to be enhanced.

[0027] To improve on existing antibodies that specifically bind to VEGFR2 / KDR, the heavy chain variable region FR (framework region) was substituted from VH1 to VH3, and the light chain FR (framework region) was substituted from VL1 to VK1, and mutations were introduced into the CDR3 regions of the heavy and light chains to produce novel antibodies with increased affinity for antigens. As a result, the inventors developed antibodies with higher binding avidity and better intracellular efficacy than existing antibodies that specifically bind to VEGFR2 / KDR, and confirmed that the antibodies can serve as targeted immunotherapy agents for cancer or therapeutic agents for angiogenesis-related diseases.

[0028] The improved antibodies were screened as follows: First, the CDR and framework (FR) sequences of 6A6 were identified, and then the heavy chain CDR sequence was grafted onto the human germline VH3 sequence and the light chain CDR sequence was grafted onto the human germline VK1 sequence using CDR grafting technology.

[0029] CDR grafting generally involves isolating DNA encoding the variable region sequences of a mouse monoclonal antibody, obtaining the CDR sequences through gene cloning, and then grafting the CDR sequences into a matched human antibody sequence in silico (Hou S, et al. Humanization of an anti-CD34 monoclonal antibody by complementarity-determining region grafting based on computer-assisted molecular modeling. J Biochem. 2008;144(1):115-20; and Kashmiri SV, De Pascalis R, Gonzales NR, Schlom J. SDR grafting—a new approach to antibody humanization. Methods. 2005;36(1):25-34). Using this method, the 6A6 framework, which contained human germline VH1 / VL1, was replaced with human germline VH3 / VK1.

[0030] To improve the affinity of the substituted antibody, a library was constructed by introducing mutations into the CDR3 base sequences of the light and heavy chains, and antibodies were screened using recombinant KDR D1-D3-Fc fusion proteins using phage display.

[0031] Based on this, in one aspect, the present invention provides a heavy chain CDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19 and 25; a heavy chain CDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20 and 26; and a heavy chain variable region comprising a heavy chain CDR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 51, 52, and 53; and a light chain CDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22 and 28; a light chain CDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23 and 29; and The present invention relates to an antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR, which comprises a light chain variable region comprising a light chain CDR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24 and 30.

[0032] As used herein, the term "antibody" refers to an anti-VEGFR2 / KDR antibody that specifically binds to VEGFR2 / KDR. The scope of the present invention includes not only intact antibody forms that specifically bind to VEGFR2 / KDR, but also antigen-binding fragments of such antibody molecules.

[0033] Intact antibodies have two full-length light chains and two full-length heavy chains, with each light chain linked to a heavy chain by a disulfide bond. Heavy chain constant regions are of gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, with subclasses of gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). Light chain constant regions are of kappa (κ) and lambda (λ) types.

[0034] Antigen-binding fragments of antibodies or antibody fragments refer to fragments that retain antigen-binding function and include Fab, F(ab'), F(ab')2, and Fv. Among antibody fragments, Fab has a structure comprising light and heavy chain variable regions, a light chain constant region, and the first heavy chain constant region (CH1), and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are produced when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. Fv is the smallest antibody fragment that comprises only the heavy chain variable region and the light chain variable region. In a two-chain Fv, the heavy chain variable region and the light chain variable region are linked non-covalently, while in a single-chain Fv (scFv), the heavy chain variable region and the light chain variable region are linked covalently via a peptide linker or directly at the C-terminus, and can form a dimer structure similar to that of a two-chain Fv. Such antibody fragments can be obtained using protease hydrolases (for example, Fab fragments can be obtained by restrictive cleavage of a whole antibody with papain, or F(ab')2 fragments can be obtained by cleavage with pepsin), or can be produced through genetic recombination techniques.

[0035] In one embodiment, the antibody of the present invention is in the form of an Fv (e.g., scFv) or a whole antibody. The heavy chain constant region can be selected from any one of the isotypes: gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε). For example, the constant region can be gamma 1 (IgG1), gamma 3 (IgG3), or gamma 4 (IgG4). The light chain constant region can be kappa or lambda.

[0036] As used herein, the term "heavy chain" refers to a full-length heavy chain and fragments thereof that includes a variable region domain VH and three constant region domains CH1, CH2, and CH3, each of which contains an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen. As used herein, the term "light chain" refers to a full-length light chain and fragments thereof that includes a variable region domain VL and constant region domain CL, each of which contains an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen.

[0037] Antibodies of the present invention include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of the foregoing antibodies.

[0038] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0039] "Epitope" refers to a determinant on a protein to which an antibody can specifically bind. Epitopes are usually composed of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0040] The "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Often, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody), e.g., mouse, rat, rabbit, or non-human primate, retaining the desired specificity, affinity, and capacity.

[0041] The "human antibody" refers to a molecule derived from human immunoglobulin, in which all amino acid sequences constituting the antibody, including the complementarity determining regions and structural regions, are entirely composed of human immunoglobulin.

[0042] These include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remaining chain(s) are identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies that exhibit the desired biological activity.

[0043] As used herein, "antibody variable region" refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0044] "Complementarity determining region" (CDR; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable region that are necessary for antigen binding. Each variable region typically has three CDR regions, identified as CDR1, CDR2, and CDR3. The present invention includes a heavy chain variable region comprising the heavy chain CDR3 of SEQ ID NO:1 and a light chain variable region comprising the light chain CDR3 of SEQ ID NO:2.

[0045] In the present invention, the antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR comprises a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, and a heavy chain CDR3 of SEQ ID NO: 3; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6; a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 8, and a heavy chain CDR3 of SEQ ID NO: 9; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 10, a light chain CDR2 of SEQ ID NO: 11, and a light chain CDR3 of SEQ ID NO: 12; a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 13, a heavy chain CDR2 of SEQ ID NO: 14, and a heavy chain CDR3 of SEQ ID NO: 15; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 16, a light chain CDR2 of SEQ ID NO: 17, and a light chain CDR3 of SEQ ID NO: 18; a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 19, a heavy chain CDR2 of SEQ ID NO: 20, and a heavy chain CDR3 of SEQ ID NO: 21; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 22, a light chain CDR2 of SEQ ID NO: 23, and a light chain CDR3 of SEQ ID NO: 24; a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 25, a heavy chain CDR2 of SEQ ID NO: 26, and a heavy chain CDR3 of SEQ ID NO: 27; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 28, a light chain CDR2 of SEQ ID NO: 29, and a light chain CDR3 of SEQ ID NO: 30; a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 13, a heavy chain CDR2 of SEQ ID NO: 14, and a heavy chain CDR3 of SEQ ID NO: 51; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 16, a light chain CDR2 of SEQ ID NO: 17, and a light chain CDR3 of SEQ ID NO: 18; a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 13, a heavy chain CDR2 of SEQ ID NO: 14, and a heavy chain CDR3 of SEQ ID NO: 52; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 16, a light chain CDR2 of SEQ ID NO: 17, and a light chain CDR3 of SEQ ID NO: 18; or It may comprise a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 13, a heavy chain CDR2 of SEQ ID NO: 14, and a heavy chain CDR3 of SEQ ID NO: 53, and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 16, a light chain CDR2 of SEQ ID NO: 17, and a light chain CDR3 of SEQ ID NO: 18.

[0046] "Framework regions" (FR) are those variable region residues other than the CDR residues. Each variable region typically has four FRs, identified as FR1, FR2, FR3 and FR4.

[0047] An "Fv" fragment is an antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable region in a tight, essentially covalent association, e.g., scFv.

[0048] The "Fab" fragment contains the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. F(ab')2 antibody fragments typically comprise a pair of Fab fragments covalently linked near their carboxy termini by hinge cysteines between them.

[0049] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, but these domains are present in a single polypeptide chain. The Fv polypeptide can further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0050] VEGFR2 / KDR antibodies can comprise single chain or double chain. Functionally, the binding affinity of VEGFR2 / KDR antibodies is 10 -5 M~10 -12For example, the binding affinity of VEGFR2 / KDR antibodies is in the range of 10 -6 M~10 -12 M, 10 -7 M~10 -12 M, 10 -8 M~10 -12 M, 10 -9 M~10 -12 M, 10 -5 M~10 -11 M, 10 -6 M~10 -11 M, 10 -7 M~10 -11 M, 10 -8 M~10 -11 M, 10 -9 M~10 -11 M, 10 -10 M~10 -11 M, 10 -5 M~10 -10 M, 10 -6 M~10 -10 M, 10 -7 M~10 -10 M, 10 -8 M~10 -10 M, 10 -9 M~10 -10 M, 10 -5 M~10 -9 M, 10 -6 M~10 -9 M, 10 -7 M~10 -9 M, 10 -8 M~10 -9 M, 10 -5 M~10 -8 M, 10 -6 M~10 -8 M, 10 -7 M~10 -8 M, 10 -5 M~10 -7 M, 10 -6 M~10 -7 M or 10 -5 M~10 -6 I am M.

[0051] The present invention relates to an antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR, which comprises a heavy chain variable region comprising a sequence having 80% or more homology to a sequence selected from the group consisting of SEQ ID NOs: 32, 36, 40, 44, and 48.

[0052] The present invention also relates to an antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR, comprising a light chain variable region comprising a sequence having 80% or more homology to a sequence selected from the group consisting of SEQ ID NOs: 34, 38, 42, 46, and 50.

[0053] The present invention also relates to an antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR, comprising a heavy chain variable region comprising a sequence having 80% or more identity to a sequence selected from the group consisting of SEQ ID NOs: 32, 36, 40, 44, and 48, and a light chain variable region comprising a sequence having 80% or more identity to a sequence selected from the group consisting of SEQ ID NOs: 34, 38, 42, 46, and 50.

[0054] The homology can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology compared to the entire sequence of the claimed SEQ ID NO.

[0055] The VEGFR2 / KDR-binding antibody or antigen-binding fragment thereof may comprise a heavy chain variable region selected from the group consisting of SEQ ID NOs: 32, 36, 40, 44, and 48. The VEGFR2 / KDR-binding antibody or antigen-binding fragment thereof may comprise a light chain variable region selected from the group consisting of SEQ ID NOs: 34, 38, 42, 46, and 50. In a specific embodiment of the present invention, a heavy chain variable region of SEQ ID NO: 32 and a light chain variable region of SEQ ID NO: 34; a heavy chain variable region of SEQ ID NO: 36 and a light chain variable region of SEQ ID NO: 38; a heavy chain variable region of SEQ ID NO: 40 and a light chain variable region of SEQ ID NO: 42; a heavy chain variable region of SEQ ID NO: 44 and a light chain variable region of SEQ ID NO: 46; or It may comprise a heavy chain variable region of SEQ ID NO:48 and a light chain variable region of SEQ ID NO:50.

[0056] "Phage display" is a technique in which mutant polypeptides are displayed on the surface of phages, e.g., filamentous phage particles, as fusion proteins with at least a portion of a coat protein. The utility of phage display lies in the fact that large libraries of randomized protein variants can be targeted and sequences that bind with high affinity to target antigens can be rapidly and efficiently sorted. Displaying peptide and protein libraries on phage has been used to screen millions of polypeptides to find those with specific binding properties.

[0057] Phage display technology has provided a powerful tool for generating and selecting novel proteins that bind to specific ligands (e.g., antigens). Using phage display technology, large libraries of protein variants can be generated and sequences that bind with high affinity to target antigens can be rapidly sorted. Nucleic acids encoding mutant polypeptides are fused to nucleic acid sequences encoding viral coat proteins, such as the gene III protein or gene VIII protein. Monovalent phage display systems have been developed in which nucleic acid sequences encoding proteins or polypeptides are fused to nucleic acid sequences encoding portions of the gene III protein. In monovalent phage display systems, the gene fusions are expressed at low levels, while the wild-type gene III protein is also expressed, maintaining particle infectivity.

[0058] Demonstrating expression of peptides on the surface of filamentous phage and expression of functional antibody fragments in the periplasm of E. coli is key to developing antibody phage display libraries. Libraries of antibodies or antigen-binding polypeptides have been produced in a number of ways, for example, by altering a single gene by inserting random DNA sequences or by cloning related gene sequences. Libraries can be screened for expression of antibodies or antigen-binding proteins with desired characteristics.

[0059] Phage display technology offers several advantages over conventional hybridoma and recombinant methods for producing antibodies with desired characteristics. These techniques allow for the generation of large antibody libraries with diverse sequences in a short period of time without the use of animals. Hybridoma production and humanized antibody production can require several months of production time. Furthermore, because no immunization is required, phage antibody libraries can generate antibodies against antigens with low toxicity or antigenicity. Phage antibody libraries can also be used to generate and identify novel therapeutic antibodies.

[0060] Techniques are available for generating human antibodies from immunized, non-immunized humans, germline sequence, or naive B cell Ig repertoires using phage display libraries. Various lymphoid tissues can be used to generate naive or non-immunized antigen-binding libraries.

[0061] Techniques that can identify and isolate high-affinity antibodies from phage display libraries are important for isolating novel antibodies for therapeutic use. Isolating high-affinity antibodies from libraries can depend on the size of the library, production efficiency in bacterial cells, and library diversity. Library size can be reduced by improper folding of the antibody or antigen-binding protein and inefficient production due to the presence of stop codons. Expression in bacterial cells can be suppressed if the antibody or antigen-binding domain does not fold properly. Expression can be improved by alternating residues at the variable / invariant interface or selected CDR residues. The sequence of the scaffold region is one factor in providing proper folding when generating antibody phage libraries from bacterial cells.

[0062] In high-affinity antibody isolation, it is important to generate a diverse library of antibodies or antigen-binding proteins. The CDR3 region has been shown to frequently participate in antigen binding. The CDR3 region on the heavy chain is highly diverse in size, sequence, and structural conformation, and can therefore be used to generate diverse libraries.

[0063] Diversity can also be generated by randomizing the CDR regions of the variable heavy and light chains using all 20 amino acids at each position, which may generate a greater variety of variant antibody sequences and increase the chances of identifying novel antibodies.

[0064] The antibodies or antibody fragments of the present invention can include not only the sequences of the anti-VEGFR2 / KDR antibodies of the present invention described herein, but also biological equivalents thereof, as long as they can specifically recognize VEGFR2 / KDR. For example, additional changes can 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, deletion, insertion, and / or substitution of residues in the amino acid sequence of the antibody. Such amino acid mutations are made based on the relative similarity of amino acid side chain substitutions, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substitutions 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 are considered to be biologically functional equivalents.

[0065] Considering the aforementioned biologically equivalent mutations, the antibodies of the present invention or nucleic acid molecules encoding them are also understood to include sequences that exhibit substantial identity to the sequences set forth in SEQ ID NOs. The term "substantial identity" refers to sequences that exhibit at least 90% homology, most preferably at least 95%, 96% or more, 97% or more, 98% or more, or 99% or more when the sequences are aligned to maximize correspondence with any other sequence and analyzed using algorithms commonly used in the art. Alignment methods for sequence comparison are well known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NCBI and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet. BLAST can be accessed at www.ncbi.nlm.nih.gov / BLAST / . Methods for sequence homology comparison using this program can be found at www.ncbi.nlm.nih.gov / BLAST / blast_help.html.

[0066] Based on this, the antibodies or antigen-binding fragments of the present invention can have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology to the sequences explicitly set forth herein or to the entirety of the sequences. Such homology can be determined by sequence comparison and / or alignment using methods known in the art. For example, percent sequence identity of the nucleic acids or proteins of the present invention can be determined using sequence comparison algorithms (e.g., BLAST or BLAST 2.0), manual alignment, or visual inspection. In another aspect, the present invention relates to a nucleic acid encoding the antibody or antigen-binding fragment thereof.

[0067] The nucleic acid can be selected from the group consisting of SEQ ID NOs: 31, 35, 39, 43, 47, which encode heavy chain variable regions. The nucleic acid can be selected from the group consisting of SEQ ID NOs: 33, 37, 41, 45, 49, which encode light chain variable regions.

[0068] Nucleic acids encoding the antibodies or antigen-binding fragments thereof of the present invention can be isolated and the antibodies or antigen-binding fragments thereof can be recombinantly produced. The nucleic acids are isolated and inserted into replicable vectors for further cloning (DNA amplification) or for further expression. Accordingly, in another aspect, the present invention relates to vectors containing the nucleic acids.

[0069] The term "nucleic acid" encompasses DNA (gDNA and cDNA) and RNA molecules, and nucleotides, the basic building blocks of nucleic acids, include not only natural nucleotides but also analogs in which the sugar or base moiety is modified. The nucleic acid sequences encoding the heavy and light chain variable regions of the present invention can be modified. Such modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0070] DNA encoding the antibody is readily isolated or synthesized using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to DNA encoding the antibody heavy and light chains). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0071] As used herein, the term "vector" refers to a means for expressing a gene of interest in a host cell, and includes plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, and viral vectors such as adeno-associated virus vectors. In the vector, the nucleic acid encoding the antibody is operably linked to a promoter.

[0072] "Operably linked" refers to a functional connection between a nucleic acid expression control sequence (e.g., a promoter, signal sequence, or an array of binding sites for transcriptional regulators) and another nucleic acid sequence, such that the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0073] When a prokaryotic cell is used as the host, it generally contains a strong promoter capable of driving transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for the initiation of translation, and a transcription / translation termination sequence. Furthermore, for example, when eukaryotic cells are used as hosts, promoters derived from mammalian cell genomes (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, and these generally have a polyadenylation sequence as a transcription termination sequence.

[0074] In some cases, the vector can be fused with other sequences to facilitate purification of the antibody expressed therefrom, such as glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA).

[0075] The vectors contain antibiotic resistance genes commonly used in the art as selection markers, such as genes for resistance to ampicillin, gentamicin, cavenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0076] In another aspect, the present invention relates to a cell transformed with the aforementioned vector. The cells used to produce the antibodies of the present invention may be, but are not limited to, prokaryotic, yeast, or higher eukaryotic cells.

[0077] Prokaryotic host cells such as strains of the genus Bacillus, such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus) can be used.

[0078] However, of greatest interest are animal cells, examples of useful host cell lines may be, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, HEK293, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0079] In yet another aspect, the present invention relates to a method for producing the antibody or antigen-binding fragment thereof, comprising the steps of: (a) culturing the cells; and (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.

[0080] The cells can be cultured in a variety of media. Commercially available media can be used as the culture medium without limitation. Any other necessary supplements known to those skilled in the art can also be included at appropriate concentrations. The culture conditions, such as temperature, pH, etc., will be those already used with the host cell selected for expression and will be apparent to those skilled in the art.

[0081] The antibody or antigen-binding fragment thereof can be recovered by removing impurities, for example, by centrifugation or ultrafiltration, and the resulting product can be purified using, for example, affinity chromatography. Additional purification techniques, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, etc., can also be used.

[0082] The antibody may be an IgG or a fragment containing the variable region, i.e., ScFv or Fab. The heavy chain variable region may be IgG1, IgG2, IgG3, or IgG4. In still another aspect, the present invention relates to a composition for preventing or treating angiogenic diseases, which comprises the antibody or an antigen-binding fragment thereof as an active ingredient.

[0083] The term "angiogenesis" refers to the formation or growth of new blood vessels from pre-existing blood vessels, and "angiogenesis-related disease" refers to a disease associated with the development or progression of angiogenesis. Diseases that can be treated with the antibody are not limited and may be included in the scope of angiogenesis-related diseases.Examples of angiogenesis-related diseases include cancer, metastasis, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, macular degeneration, neovascular glaucoma, erythrosis, proliferative retinopathy, psoriasis, hemophilic arthritis, capillary formation in atherosclerotic plaques, keloids, wound granulation, vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune diseases, and Crohn's disease. These conditions include, but are not limited to, arterial disease, restenosis, atherosclerosis, intestinal adhesions, cat scratch disease, ulcer, liver cirrhosis, nephritis, diabetic nephropathy, diabetic foot ulcers, chronic kidney failure, diabetes mellitus, inflammatory diseases, idiopathic pulmonary fibrosis, sepsis, acute respiratory distress syndrome, and neurodegenerative diseases.

[0084] Furthermore, the cancers include esophageal cancer, stomach cancer, large intestine cancer, rectal cancer, oral cancer, pharynx cancer, larynx cancer, lung cancer, colon cancer, breast cancer, uterine cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testis cancer, bladder cancer, renal cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, The cancer is selected from the group consisting of, but not limited to, lymphoma, lymphoma, and multiple myeloid blood cancer.

[0085] As used herein, the term "prevention" or "prophylaxis" refers to any measure of administering an antibody or composition of the invention to prevent or delay the onset of a disease of interest. The term "treatment" or "therapy" refers to any measure of administering an antibody or composition of the invention to improve or reverse the symptoms of a disease of interest.

[0086] The present invention may be, for example, a pharmaceutical composition for preventing or treating tumors or cancer, comprising (a) a pharmaceutically effective amount of an antibody against VEGFR2 / KDR or an antigen-binding fragment thereof according to the present invention; and (b) a pharmaceutically acceptable carrier. The present invention may also be a method for preventing or treating tumors or cancer, comprising administering the antibody or antigen-binding fragment thereof to a patient with tumor or cancer. The present invention may further be a use of the antibody or antigen-binding fragment thereof for interfering with the VEGFR2 / KDR mechanism and thereby preventing or treating tumors or cancer.

[0087] Tumors or cancers, which are diseases to which the compositions are applicable, typically include tumors or cancers that overexpress VEGF or VEGFR2 / KDR, and tumors or cancers that do not overexpress VEGF or VEGFR2 / KDR. Non-limiting examples of tumors or cancers preferred for treatment include melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate carcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic carcinomas. Furthermore, the present invention includes refractory or recurrent cancers that can be treated using the antibodies of the present invention.

[0088] For example, the present invention provides a method for treating an angiogenic disease, comprising administering a therapeutically effective amount of the anti-VEGFR2 / KDR antibody or antigen-binding fragment thereof to a patient in need of angiogenesis inhibition. The treatment of an angiogenic disease may further comprise identifying the patient in need of angiogenesis inhibition prior to the administering step. In another example, the present invention provides a method for preventing and / or treating a VEGFR2 / KDR-related disease, comprising administering a therapeutically effective amount of the anti-VEGFR2 / KDR antibody or antigen-binding fragment thereof to a patient in need of VEGFR2 / KDR-related disease prevention and / or treatment. The preventive and / or therapeutic method may further comprise identifying the patient in need of VEGFR2 / KDR-related disease prevention and / or treatment prior to the administering step.

[0089] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, which is one commonly used in drug formulations and may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. The pharmaceutical composition may also further comprise one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives commonly used in the manufacture of pharmaceutical compositions.

[0090] An effective amount of the pharmaceutical composition, antibody, or antigen-binding fragment thereof can be administered orally or parenterally. Parenteral administration can be by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration. Since proteins or peptides are digested during oral administration, oral compositions can be formulated to coat the active agent or protect it from degradation in the stomach. In addition, the compositions can be administered by any device that can deliver the active agent to target cells.

[0091] The compositions of the present invention are administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to a sufficient amount of a pharmaceutical composition for the treatment of a disease at a reasonable benefit / risk ratio applicable to all medical treatments. The effective amount will vary depending on various factors, including the severity of the disease to be treated, the patient's age and sex, the type of disease, the activity of the drug, sensitivity to the drug, the time of administration, the route of administration, the excretion rate, the duration of treatment, co-administration of other drugs, and other parameters known in the art. The compositions of the present invention can be administered alone or in combination with other therapies. In such cases, they can be administered sequentially or simultaneously with conventional therapies. The compositions can also be administered in a single dose or in multiple divided doses. Taking these factors into consideration, it is important to administer the minimum amount sufficient to achieve maximum efficacy without side effects, and this dosage can be easily determined by a person skilled in the art. The dosage of the pharmaceutical compositions of the present invention is not particularly limited, and will vary depending on various factors, including the patient's health condition and weight, the severity of the disease, the type of drug, the route and time of administration. The compositions can be administered in one or more doses daily to mammals, including rats, mice, livestock, humans, etc., by any typically accepted route, such as orally, rectally, intravenously, subcutaneously, intrauterinely, or intracerebrovascularly.

[0092] The amount of anti-VEGFR2 / KDR antibody or antigen-binding fragment thereof in the pharmaceutical composition can be varied depending on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, diet, administration time, administration interval, administration route, excretion rate, and reaction sensitivity. For example, the daily dose of the anti-VEGFR2 / KDR antibody or antigen-binding fragment thereof may be in the range of 0.001 to 1000 mg / kg, specifically 0.01 to 100 mg / kg, more specifically 0.1 to 50 mg / kg, and even more specifically 0.1 to 20 mg / kg, but is not limited thereto. The daily dose can be formulated as a single formulation in the form of a unit dose, or can be appropriately divided into formulations or packaged in a multi-capacity container.

[0093] The pharmaceutical composition can be administered in combination with other drugs, such as other therapeutic agents for angiogenesis inhibitor-related diseases, and the dosage, administration method, and type of other drugs can be appropriately prescribed depending on the condition of the patient.

[0094] The pharmaceutical composition may be formulated in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or in the form of an extract, acid, powder, granules, tablets or capsules, etc., and may further contain a dispersant or stabilizer for formulation.

[0095] In particular, pharmaceutical compositions containing the anti-VEGFR2 / KDR antibody or its antigen-binding fragment can be formulated as immunoliposomes because they contain the antibody or antigen-binding fragment. Antibody-containing liposomes can be prepared according to methods well known in the art. The immunoliposomes can be prepared by reverse-phase evaporation using a lipid composition containing phosphatidylcholine, cholesterol, and polyethylene glycol-derivatized phosphatidylethanolamine (Patent Publication No. 10-2015-0089329). For example, the Fab' fragment of an antibody can be conjugated to liposomes via a disulfide exchange reaction.

[0096] Meanwhile, because the anti-VEGFR2 / KDR antibody or antigen-binding fragment thereof specifically binds to VEGFR2 / KDR, it can be used to determine the presence or absence of VEGFR2 / KDR activation and / or hyperproduction. Accordingly, another embodiment of the present invention provides a pharmaceutical composition for diagnosing a VEGFR2 / KDR inhibitor-associated disease, comprising the anti-VEGFR2 / KDR antibody or antigen-binding fragment thereof. In yet another embodiment, the present invention provides a diagnostic method or a method for providing information for diagnosis, comprising the steps of treating a biological sample obtained from a patient with the anti-VEGFR2 / KDR antibody or antigen-binding fragment thereof; determining whether or not an antigen-antibody reaction is present; and, if an antigen-antibody reaction is detected, diagnosing the patient as having symptoms of VEGFR2 / KDR activation and / or hyperproduction or a VEGFR2 / KDR activation and / or hyperproduction-associated disease. The biological sample may be selected from the group consisting of cells, tissues, body fluids, etc., obtained from the patient.

[0097] The step of determining whether or not there is an antigen-antibody reaction can be performed by various methods known in the art, such as, but not limited to, conventional enzyme reactions, fluorescence, luminescence, and / or radiation detection, including immunochromatography, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzymeimmunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), Western blotting, etc.

[0098] Cancer can be diagnosed by analyzing the intensity of the final signal obtained by the immunoassay process. That is, when the protein of the marker of the present invention is highly expressed in a biological sample and the signal is stronger than that of a normal biological sample (e.g., normal stomach tissue, blood, plasma, or serum), cancer is diagnosed.

[0099] In another aspect, the present invention relates to a cancer diagnostic kit comprising the cancer diagnostic composition. The kit according to the present invention can diagnose cancer by analyzing a signal generated by the reaction of a sample with an antibody. In this case, the signal may include, but is not limited to, an enzyme bound to the antibody, such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, luciferase, or cytochrome P450. In this case, when alkaline phosphatase is used as the enzyme, the substrate for the enzyme may be bromochloroindolyl phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1-phosphate, or enhanced chromatin (ECF). When horseradish peroxidase is used, substrates that can be used include, but are not limited to, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resorufin benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di[3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD), and naphthol / pyronin; when glucose oxidase is used, substrates that can be used include, but are not limited to, t-NBT (nitroblue tetrazolium), and m-PMS (phenzaine methosulfate).

[0100] The kit according to the present invention may also contain a label that generates a detectable signal, and the label may include, but is not limited to, chemical substances (e.g., biotin), enzymes (alkaline phosphatase, β-galactosidase, horseradish peroxidase, and cytochrome P450), radioactive substances (e.g., C14, I125, P32, and S35), fluorescent substances (e.g., fluorescein), luminescent substances, chemiluminescent substances, and FRET (fluorescence resonance energy transfer). Measurement of the activity or signal of an enzyme used for diagnosing angiogenesis can be carried out according to various methods known in the art. The patient to be administered with or diagnosed with the pharmaceutical composition may be a mammal, including humans, primates including monkeys, and rodents including mice and rats.

[0101] The VEGFR2 / KDR-related disease may be cancer; cancer metastasis; eye diseases such as retinopathy of prematurity, macular degeneration (e.g., age-related macular degeneration), diabetic retinopathy, and neovascular glaucoma; asthma; rheumatoid arthritis; psoriasis; inflammatory diseases such as pneumonia and chronic inflammation; cardiovascular diseases such as hypertension and arteriosclerosis, or sepsis. The cancer may overexpress VEGFR2 / KDR and may be a solid cancer or a blood cancer, including, but not limited to, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, skin cancer, cutaneous or intraocular melanoma, rectal cancer, perianal cancer, esophageal cancer, small intestine cancer, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, head and neck cancer, brain tumor, osteosarcoma, etc. The cancer may be a primary cancer or a metastatic cancer.

[0102] The antibody of the present invention can also be used in combination with other antibodies, biologically active agents, or substances for various purposes. In this respect, the present invention relates to a composition comprising the antibody or its antigen-binding fragment for combined administration with other therapeutic agents for angiogenic diseases.

[0103] The other therapeutic agent for angiogenic diseases may include an anti-angiogenic drug, an anti-inflammatory drug, and / or an anti-cancer drug, thereby overcoming resistance to each other and enhancing efficacy.

[0104] When the compositions according to the present invention are administered in combination with other therapeutic agents for angiogenesis disorders, the anti-VEGFR2 / KDR antibody or its antigen-binding fragment and the other therapeutic agents for angiogenesis disorders can be administered sequentially or simultaneously. For example, an anti-angiogenic drug, an anti-inflammatory drug, and / or an anti-cancer drug can be administered to a subject, and then a composition containing the anti-VEGFR2 / KDR antibody or its antigen-binding fragment as an active ingredient can be administered to the subject, or the composition can be administered to a subject, and then the anti-angiogenic drug, the anti-inflammatory drug, and / or the anti-cancer drug can be administered to the subject. Optionally, the composition can be administered to a subject simultaneously with the anti-angiogenic drug, the anti-inflammatory drug, and / or the anti-cancer drug. [Example]

[0105] The present invention will be described in more detail below through examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention.

[0106] Example 1. CDR grafting The CDR sequence of 6A6 was confirmed using Kabat numbering and the IMGT program, and then cloned to transplant it into the human germline VH3 / VK1. The columns are labeled and the light chain CDR-grafted sequences are labeled in FIG.

[0107] Example 2. Mutation and selection for affinity enhancement The CDR-grafted antibody was optimized to improve its affinity. Using soft-randomization, which preserves 70% of the original DNA sequences of the light and heavy chain CDR3s, primers were created to introduce random mutations into the light and heavy chain CDR3s. DNA fragments encoding the mutated light and heavy chain variable regions were isolated through PCR using these primers. These DNA fragments were then substituted with the light and heavy chain variable regions of scFv phage phagemids to generate a light chain CDR3 mutant scFv phage library and a heavy chain CDR3 mutant scFv phage DNA library, respectively.

[0108] The mutant scFv phage DNA library was purified with phenol-chloroform and then transformed into the E. coli strain XL-1 Blue using electroporation. After confirming the diversity through transformation efficiency analysis and DNA sequence analysis, the cells were cultured at a 500 ml scale to induce phage expression, and a light and heavy chain CDR3 mutant scFv phage library was prepared using the PEG precipitation method.

[0109] Biopanning was performed using each mutant scFv phage library. 100 ml of antigen (KDR1-3 domian-Fc) was added to each well of a 96-well immunoplate at 2 mg / ml and incubated overnight at 4°C. The next day, the antigen-coated plate was washed three times with PBST (0.1% Tween 20) and then 200 ml of 2% BSA blocking buffer was added and incubated at room temperature for 2 hours. 50 ml of XL1-Blue stock was added to 2 ml of 2x YT-TET (tetracycline 10 μg / ml) growth medium and incubated at 37°C and 200 rpm for approximately 2 hours. An additional 13 ml was then added and the OD was measured. 600The wells were incubated until the OD reached 0.5. After 2 hours of blocking, the wells were washed three times with 1X PBST (0.1% Tween 20). Equal volumes of the mutant library and 4% BSA were mixed and added to each washed well, 200 ml of which was then rocked at room temperature for 30 minutes and then incubated for 2 hours. After the phage library reaction was complete, the supernatant was discarded and the wells were washed five times with 0.1% PBST and five times with PBS. 100 ml of 100 mM TEA (trimethylamine) was added to each well and shaken at room temperature for 10 minutes. After 10 minutes, 50 ml of 1 M Tris (pH 7.5) was added to each well and mixed. The supernatant was analyzed by OD 600 The resulting solution was added to 10 ml of XL1-blue with a pH of 0.5 and allowed to infect for 30 minutes at 37°C. After infection, 100 ml was used as the output titer, and the remaining solution was centrifuged at 6,000 rpm for 10 minutes. The supernatant was discarded, and the precipitate was spread onto a large square plate (CM 34 μg / ml + 1% glucose) and incubated overnight at 37°C. The remaining 100 ml for the output titer was diluted 1 / 10, 1 / 100, and 1 / 1000, spread onto CM plates, and incubated overnight at 37°C. The next day, the colonies grown on the square plate were placed in 50 ml of 2x YT medium, scraped using a loop, and centrifuged at 6000 rpm for 10 minutes. The supernatant was discarded, and the precipitate was used to prepare a primary panning stock. 100 ml of 2x YT growth medium (CM 34 μg / ml + 1% Glucose) was placed in a 500 ml Erlenmeyer flask, and the OD was measured. 600 The cells were added so that the OD was 0.2 and grown at 200 rpm at 37°C until the OD reached 0.5. 600After culturing the cells until the RI value reached 0.5, helper phage (M13KO7 mutant) was added at a 20x cell volume. The helper phage was added and the cells were infected at 37°C for 30 minutes, followed by centrifugation at 6000 rpm for 10 minutes. The supernatant was discarded, and the cells were replaced with 100 ml of 2xYT medium (CM 34 μg / ml + Kansas 70 μg / ml + 1 mM IPTG + 5 mM MgCl2) and incubated overnight at 200 rpm at 30°C. The next day, the grown cells were centrifuged at 7000 rpm for 10 minutes and then centrifuged again in the same manner. The collected supernatant was added to 1 / 5 (v / v) of the supernatant in 20% PEG / 2.5 mM NaCl and precipitated on ice for 1 hour. After precipitation, the cells were centrifuged at 9000 rpm for 1 hour. The supernatant was discarded, and the precipitate was dissolved in 5 ml of PBS, filtered through a 0.45 mm filter, and stored at 4°C before being used in the next panning step. This process was repeated 3-4 times, and the antibodies that bind to the antigen were confirmed by ELISA. Then, in the selection process, the dissociation rate constant K of the scFv was used as a quantitative evaluation index of the ability to maintain binding. dis was measured.

[0110] Example 2. Selection of antibodies with high affinity for antigens (off-rate screening) The antigen binding strength of the selected antibodies was measured using Octet (Fortebio). To do this, the KDR1-3 domains were immobilized on a biosensor, and then candidate antibodies expressed in the form of scFv were added and allowed to bind, after which the dissociation rate constants were measured. The amino acid sequences (Tables 2 and 3) and dissociation rate constant measurement results (Table 1) for the five selected optimized clones are shown in the figure. The CDR sequences shown were analyzed using the IMGT database. [Table 1] [Table 2] [Table 3] JPEG0007770551000004.jpg155163JPEG0007770551000005.jpg154162JPEG0007770551000006.jpg154163JPEG0007770551000007.jpg115128

[0111] Example 3. Antibody Expression The selected scFv phages were converted to IgG form using molecular biology techniques. Phagemids were extracted from the selected E. coli clones, and the variable regions were amplified using PCR. The amplified heavy chain variable region was inserted into an expression vector containing a heavy chain constant region (Invivogen, pfusess-hchg1), and the amplified light chain variable region was inserted into an expression vector containing a light chain constant region (Invivogen, pfuse2ss-hclk), completing the DNA cloning into IgG form.

[0112] For transient expression of IgG, the Expi293F expression system kit (Thermo Fisher Scientific, US) was used. Expi293 cells included in the kit were cultured in suspension in the kit's dedicated medium at 37°C in a 5% CO2 environment on an orbital shaker at 125 rpm. 3 x 10 cells were cultured every 3 days. 5 Subculture was performed to obtain cells / ml, and the expression vector was introduced at a concentration of 3 x 10 6 The cells were then used after adjusting the cell number to 100 cells / ml. Gene transfection was performed using the dedicated reagent Expifectamine. Lipid-DNA complexes containing 1 mg of expression vector DNA per ml of cell suspension and 2.7 μl of Expifectamine were prepared and added to the cell suspension. 16-18 hours after transfection, Enhancer 1 / 2 was added to induce expression. After incubation for 3-4 days under the same conditions, the cells were centrifuged to obtain the IgG-containing supernatant.

[0113] Example 4. Antibody purification The supernatant was loaded onto a Protein A column (GE Healthcare) and IgG purified by affinity chromatography. The column was equilibrated with 20 mM Tris-HCl, 50 mM NaCl, and 5 mM EDTA (pH 7.0), after which the supernatant was loaded and washed with 50 mM Tris-HCl, 500 mM NaCl, 5 mM EDTA, and 0.2% polysorbate 20 (pH 7.0). The column was then eluted with 50 mM NaCl and 0.1 M glycine-HCl (pH 3.5) and neutralized with 1 M Tris. The eluted protein was dialyzed with a 10,000 MWCO Spectra / Pore dialysis membrane (Spectrum Labs, US) and the solvent was exchanged with PBS. The desired concentration was then concentrated using Vivaspin (Satorius, DE), aliquoted, and stored at -80°C.

[0114] Example 5. Analysis of antibody binding specificity The binding affinity of the selected antibodies to the KDR1-3 domain was measured using the Octet system (Fortebio Inc., US). To this end, anti-VEGFR2 / KDR antibodies were immobilized on a biosensor, and the binding kinetics of human KDR1-3 at various concentrations was measured. The binding rate constant (k a ), dissociation rate constant (k dis ) and the binding constant (K D ) was calculated (Table 4). [Table 4]

[0115] Example 6. Confirmation of antibody neutralizing ability (HUVEC viability assay) To measure the biological activity of the antibody, the following experiment was performed. HUVECs were purchased from Lonza (cat# C2519A) and cultured in 2% gelatin-coated plates using VascuLife VEGF-Mv medium complete kit (Lifeline cell technology, cat# LCT-LL-0005). Culture was performed at 37°C in a 5% CO2 incubator. Cells within passage 10 were used for the assay, and were cultured at 1 x 10 per well in a 96-well plate in M199 medium (Gibco, cat# 11043-023) containing 0.5% heat-inactivated FBS. 4 The assay was performed using 1 x 10 cells per well after pre-treatment with various diluted antibodies in the assay plate. 4 After the cells were added, they were incubated in an incubator for 30 minutes. After 30 minutes of incubation, VEGF165 (R&D, cat# 293-VE) was added at a concentration of 30 ng / mL per well. After 3 days of incubation in a 37°C CO2 incubator, viability was measured. Viability was measured by quantifying ATP in live cells using Promega's CellTier Glo-Luminescent Cell Viability Assay Kit (cat no. G7571) (Figure 3). As shown in Figure 3, the selected antibodies inhibited the effect of VEGF165 at various concentrations.

[0116] Example 7. Analysis of the VEGF165 / VEGFR2 signal suppression effect of antibodies (p-KDR assay) To analyze the inhibitory effect of VEGF / VEGFR2 signaling, HUVEC cells were used. HUVECs were purchased from Lonza (cat# C2519A). After cell culture in VascuLife VEGF-Mv medium complete kit (Lifeline cell technology, cat# LCT-LL-0005), 8 x 10 cells were cultured per well in a 6-well plate. 5The cells were then incubated overnight in a 37°C carbon dioxide-supplied incubator. The cells were treated with antibody at 10 μg / mL for 20 minutes, followed by VEGF165 (R&D, cat# 293-VE) for 10 minutes. A well containing only VEGF165 protein without antibody served as a baseline for signal suppression analysis. After each drug reaction, HUVEC cells were washed twice with PBS and then incubated on ice for 20 minutes in lysis buffer (Thermo Scientific, RIPA buffer & phosphatase inhibitors) to obtain cell extracts. The cells were then centrifuged at 13,000 rpm at 4°C to obtain the supernatant. After quantification using the BCA protein assay, the lysates were loaded onto a 10% SDS-PAGE gel and subjected to electrophoresis. After transfer onto a nitrocellulose membrane (Bio-Rad), the gel was blocked for 1 hour at room temperature with 5% skim milk diluted in 0.1% Tween 20 TBST buffer. Next, the primary antibody (p-VEGFR2 (Y1175); Cell Signaling) was diluted 1:1000 and incubated overnight at 4°C with shaking. The peroxidase-conjugated secondary antibody (Santa Cruz) was diluted 1:5000 and incubated at room temperature for 1 hour. After incubation with ECL solution (Pierce), the target protein bands were visualized. The results confirmed that D4, D3, and A4 inhibited VEGF165-induced phosphorylation more effectively than 6A6 (Figure 4). [Industrial Applicability]

[0117] The present invention provides an antibody with improved affinity compared to conventional antibodies targeting VEGFR2 / KDR, thereby providing combination therapies and monotherapies with superior efficacy compared to conventional therapeutic agents for treating tumors. The anti-VEGFR2 / KDR antibodies or antigen-binding fragments thereof according to the present invention exhibit improved binding to VEGFR2 / KDR and can be useful for the prevention or treatment of target cancers / tumors or angiogenesis inhibitor-related diseases.

[0118] Although certain parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is to be defined by the appended claims and their equivalents.

Claims

1. a heavy chain CDR1 comprising the sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 2, a heavy chain CDR3 comprising the sequence of SEQ ID NO: 3, a light chain CDR1 comprising the sequence of SEQ ID NO: 4, a light chain CDR2 comprising the sequence of YDA, and a light chain CDR3 comprising the sequence of SEQ ID NO: 6; a heavy chain CDR1 comprising the sequence of SEQ ID NO:7, a heavy chain CDR2 comprising the sequence of SEQ ID NO:8, a heavy chain CDR3 comprising the sequence of SEQ ID NO:9, a light chain CDR1 comprising the sequence of SEQ ID NO:10, a light chain CDR2 comprising the sequence of YDA, and a light chain CDR3 comprising the sequence of SEQ ID NO:12; a heavy chain CDR1 comprising the sequence of SEQ ID NO: 13, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 14, a heavy chain CDR3 comprising the sequence of SEQ ID NO: 15, a light chain CDR1 comprising the sequence of SEQ ID NO: 16, a light chain CDR2 comprising the sequence of YDA, and a light chain CDR3 comprising the sequence of SEQ ID NO: 18; a heavy chain CDR1 comprising the sequence of SEQ ID NO: 19, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 20, a heavy chain CDR3 comprising the sequence of SEQ ID NO: 21, a light chain CDR1 comprising the sequence of SEQ ID NO: 22, a light chain CDR2 comprising the sequence of YDA, and a light chain CDR3 comprising the sequence of SEQ ID NO: 24; or a heavy chain CDR1 comprising the sequence of SEQ ID NO: 25, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 26, a heavy chain CDR3 comprising the sequence of SEQ ID NO: 27, a light chain CDR1 comprising the sequence of SEQ ID NO: 28, a light chain CDR2 comprising the sequence of YDA, and a light chain CDR3 comprising the sequence of SEQ ID NO: 30; An antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR, comprising:

2. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region selected from the group consisting of SEQ ID NOs: 32, 36, 40, 44, and 48.

3. The antibody or antigen-binding fragment thereof of claim 1, comprising a light chain variable region selected from the group consisting of SEQ ID NOs: 34, 38, 42, 46, and 50.

4. The antibody or antigen-binding fragment thereof according to claim 1, which binds to VEGFR2 / KDR and comprises a heavy chain variable region comprising an array having 80% or more identity to an array selected from the group consisting of SEQ ID NOs: 32, 36, 40, 44, and 48.

5. The antibody or antigen-binding fragment thereof according to claim 1, which binds to VEGFR2 / KDR and comprises a light chain variable region having an sequence having 80% or more identity to a sequence selected from the group consisting of SEQ ID NOs: 34, 38, 42, 46, and 50.

6. The antibody or antigen-binding fragment thereof according to claim 1, which binds to VEGFR2 / KDR and comprises a heavy chain variable region comprising a sequence having 80% or more identity to a sequence selected from the group consisting of SEQ ID NOs: 32, 36, 40, 44, and 48, and a light chain variable region comprising a sequence having 80% or more identity to a sequence selected from the group consisting of SEQ ID NOs: 34, 38, 42, 46, and 50.

7. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. An expression vector comprising the nucleic acid of claim 7.

9. A transformed cell comprising the expression vector of claim 8.

10. A method for producing an antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR, comprising the steps of: (a) culturing the cells of claim 9; and (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.

11. A composition for preventing or treating angiogenic diseases, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 as an active ingredient.

12. A diagnostic composition for angiogenic diseases, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 as an active ingredient.

13. A composition for preventing or treating tumors or cancers, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 as an active ingredient.

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