Bispecific antibody specifically binding to c-kit and VEGF, and use thereof

Abstract

The present invention relates to a bispecific antibody specifically binding to c-Kit and VEGF and, more specifically, to: a bispecific antibody capable of inhibiting or neutralizing the activity or activation of c-Kit and VEGF by binding to c-Kit and VEGF with high affinity; a nucleic acid molecule encoding the bispecific antibody; a vector comprising the nucleic acid molecule; a method for preparing the bispecific antibody; a pharmaceutical composition for preventing or treating angiogenic diseases, the composition including the bispecific antibody as an active ingredient; a method for preventing or treating angiogenic diseases using the bispecific antibody; and a c-Kit and / or VEGF detection or angiogenic disease diagnosis kit comprising the bispecific antibody.

Description

Bispecific antibodies specifically binding to C-KIT and VEGF and uses thereof

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2024-0005633, filed January 12, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a bispecific antibody that specifically binds to c-Kit and VEGF, and more particularly, to a bispecific antibody that can bind to c-Kit and VEGF with high affinity and inhibit or neutralize the activity or activation of c-Kit and VEGF, a nucleic acid molecule encoding the bispecific antibody, a vector comprising the nucleic acid molecule, a host cell, a method for producing the bispecific antibody, a pharmaceutical composition for preventing or treating angiogenic diseases comprising the bispecific antibody as an active ingredient, a method for preventing or treating angiogenic diseases using the bispecific antibody, and a kit for detecting c-Kit and / or VEGF or diagnosing angiogenic diseases comprising the bispecific antibody.

[0003] Angiogenesis is the process by which new capillaries form from pre-existing microvessels. Normally, it occurs only during embryonic development, wound healing, and cyclical changes in the female reproductive system, and rarely occurs under other conditions. However, when angiogenesis is not regulated autonomously and continues to grow pathologically, it can lead to various diseases. Diseases related to angiogenesis include hemangiomas, angiofibromas, vascular malformations, and cardiovascular diseases such as arteriosclerosis, vascular adhesions, and edematous sclerosis. Ophthalmic diseases caused by angiogenesis include age-related macular degeneration, corneal graft neovascularization, neovascular glaucoma, diabetic retinopathy, corneal diseases caused by neovascularization, macular degeneration, pterygium, retinal degeneration, retrolental fibroplasia, and granular conjunctivitis. Chronic inflammatory diseases such as arthritis, and dermatological diseases such as psoriasis, telangiectasia, pyogenic granuloma, seborrheic dermatitis, and acne. The growth and metastasis of cancer cells are inevitably dependent on angiogenesis.

[0004] VEGF is a potent angiogenesis promoter whose expression is increased in various cancer cells. VEGF binds to VEGF receptors on the surface of vascular endothelial cells, activating tyrosine kinase to induce angiogenesis, thereby playing a crucial role in cancer cell growth and metastasis. Therefore, VEGF inhibitors block angiogenesis and are used to treat various cancers and macular degeneration. Currently, recombinant antibody drugs include anticancer agents such as bevacizumab, macular degeneration treatments such as ranibizumab and aflibercept, and kinase inhibitors such as sunitinib and sorafenib. However, many patients are refractory to these VEGF inhibitors, and the development of various bispecific antibodies to address this issue is ongoing.

[0005] Meanwhile, when hypoxia occurs, the expression of c-KIT increases in endothelial cells, and the production of new blood vessels by endothelial cells increases due to stem cell factor (SCF). This pathological angiogenesis is a major cause of blindness in various eye diseases such as wet macular degeneration and diabetic retinopathy. When hypoxia occurs, the expression of HIF-1α (hypoxia inducible factor-1α) increases along with SCF and c-KIT, and SCF stabilizes HIF-1α, prolonging hypoxia. If hypoxia persists, a vicious cycle occurs in which VEGF / VEGFR increases again, so blocking the SCF signal with a c-KIT inhibitor can suppress HIF-1α and remove the factor that stimulates angiogenesis. Accordingly, the present inventors developed an anti-c-Kit antibody that specifically binds to c-Kit and demonstrated its excellent angiogenesis inhibitory effect (Patent Document 1).

[0006] Against this backdrop, the present inventors developed a bispecific antibody that specifically binds to c-Kit and VEGF, confirmed that the antibody can serve as a therapeutic agent for angiogenic diseases, particularly macular degeneration, and completed the present invention.

[0007] [Prior Art Literature]

[0008] [Patent Document]

[0009] (Patent Document 0001) Republic of Korea Publication Patent No. 10-2020-0040407

[0010] An object of the present invention is to provide a bispecific antibody that specifically binds to c-Kit and VEGF.

[0011] Another object of the present invention is to provide a nucleic acid molecule encoding the bispecific antibody.

[0012] Another object of the present invention is to provide a vector comprising the nucleic acid molecule and a host cell comprising the vector.

[0013] Another object of the present invention is to provide a method for producing a bispecific antibody that specifically binds to c-Kit and VEGF using the host cell.

[0014] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating angiogenic diseases, comprising a bispecific antibody that specifically binds to the aforementioned c-Kit and VEGF as an active ingredient.

[0015] Another object of the present invention is to provide a method for preventing or treating angiogenic diseases using a bispecific antibody that specifically binds to the aforementioned c-Kit and VEGF.

[0016] Another object of the present invention is to provide a use of the bispecific antibody specifically binding to c-Kit and VEGF described above for the manufacture of a drug for preventing or treating angiogenic diseases.

[0017] Another object of the present invention is to provide a kit for detecting c-Kit and / or VEGF or diagnosing angiogenic diseases using a bispecific antibody that specifically binds to c-Kit and VEGF.

[0018] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0019] To solve the above-described problem, the present invention provides a bispecific antibody that specifically binds to c-Kit and VEGF, comprising the following (a) and (b): (a) a first antigen binding site that specifically binds to c-Kit, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and (b) a second antigen binding site that specifically binds to VEGF, wherein the soluble extracellular domain of VEGF and the Fc domain of IgG are fused.

[0020] In the present invention, the first antigen binding site may include a heavy chain variable region including an amino acid sequence having 90% or more sequence homology with the amino acid sequence of SEQ ID NO: 27 and a light chain variable region including an amino acid sequence having 90% or more sequence homology with the amino acid sequence of SEQ ID NO: 28.

[0021] In the present invention, the soluble extracellular domain may include immunoglobulin-like domain 2 of the first VEGF receptor and immunoglobulin-like domain 3 of the second VEGF receptor.

[0022] In the present invention, the second antigen binding site may include the amino acid sequence of SEQ ID NO: 21.

[0023] In the present invention, the second antigen binding site may be linked to the heavy chain C-terminus or N-terminus of the first antigen binding site by a peptide linker; or the second antigen binding site may be linked to the light chain C-terminus of the first antigen binding site by a peptide linker.

[0024] In the present invention, the peptide linker may be GGGGG (SEQ ID NO: 20), GGGGGG (SEQ ID NO: 67), GGGGGGG (SEQ ID NO: 68), GGGGGGGG (SEQ ID NO: 23), GGGGGGGGG (SEQ ID NO: 69), or GGGGGGGGGG (SEQ ID NO: 70).

[0025] In the present invention, the Fc domain of the first antigen binding site may comprise: (a) a wild-type IgG Fc; (b) an Fc variant comprising amino acid substitutions L234A and L235E; (c) an Fc variant comprising amino acid substitutions L235A and G237A; or (d) an Fc variant comprising amino acid substitutions L234A, L235A I253A, H310A, P329G, and H435A, wherein the residues may be numbered according to the EU index of Kabat.

[0026] In the present invention, the bispecific antibody may be a tetravalent antibody.

[0027] In the present invention, the first antigen binding site may be a full-length bivalent antibody, and the second antigen binding site may be composed of two VEGF antagonists in which the soluble extracellular domain of VEGF and the Fc domain of IgG are fused.

[0028] The present invention also provides a nucleic acid molecule encoding a bispecific antibody that specifically binds to the aforementioned c-Kit and VEGF.

[0029] In addition, the present invention provides a recombinant vector comprising the above-described nucleic acid molecule and a transformant comprising the recombinant vector.

[0030] Additionally, the present invention provides a method for producing a bispecific antibody that specifically binds to c-Kit and VEGF, comprising the following steps (a) and (b): (a) culturing the transformant described above; and (b) recovering the bispecific antibody that specifically binds to c-Kit and VEGF from the culture.

[0031] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating angiogenic diseases, comprising a bispecific antibody that specifically binds to the aforementioned c-Kit and VEGF as an active ingredient.

[0032] In addition, the present invention provides a method for preventing or treating angiogenic diseases, comprising administering to a subject in need thereof a therapeutically effective amount of a bispecific antibody that specifically binds to the aforementioned c-Kit and VEGF.

[0033] Additionally, the present invention provides a bispecific antibody that specifically binds to the aforementioned c-Kit and VEGF for use in the prevention or treatment of angiogenic diseases.

[0034] The present invention also provides the use of a bispecific antibody specifically binding to c-Kit and VEGF as described above for the manufacture of a medicament for the prevention or treatment of angiogenic diseases.

[0035] In the present invention, the angiogenic disease may be selected from the group consisting of cancer, leukemia, ocular vascular disease, rheumatoid arthritis, psoriasis, chronic wounds, chronic inflammation, hemangioma, angiofibroma, vascular malformation, arteriosclerosis, vascular adhesions, vasculitis, pyogenic granuloma, bullous disease, pulmonary hypertension, asthma, nasal polyps, infectious diseases, inflammatory bowel disease, periodontal disease, peritoneal adhesions, endometrium, uterine bleeding, ovarian cysts, osteomyelitis, osteosarcoma, sepsis, and autoimmune diseases.

[0036] In the present invention, the cancer may be selected from the group consisting of bone cancer, lung cancer, head and neck cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, stomach cancer, liver cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, rectal cancer, anal cancer, colon cancer, uterine cancer, breast cancer, ovarian cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, central nervous system lymphoma, spinal cord tumor, glioblastoma, brainstem glioma, and pituitary adenoma.

[0037] In the present invention, the ocular vascular disease may be selected from the group consisting of diabetic retinopathy, macular degeneration, age-related macular degeneration, glaucoma, glaucomatous retinitis pigmentosa, choroidal neovascularization, retinopathy of prematurity, corneal dystrophy, and retinal detachment.

[0038] Additionally, the present invention provides a kit for detecting c-Kit and / or VEGF or diagnosing angiogenic diseases, comprising a bispecific antibody that specifically binds to c-Kit and VEGF.

[0039] The bispecific antibody according to the present invention, which specifically binds to c-Kit and VEGF, binds to c-Kit and / or VEGF with high affinity and has an excellent effect of inhibiting hypoxia- or inflammation-induced venous endothelial barrier destruction, SCF-dependent c-Kit signaling, and secretion of various cytokines (IL-6, VEGF, Ang-2, IL-8, and MCP-1). Accordingly, the bispecific antibody according to the present invention can be utilized for the treatment of various angiogenic diseases by significantly inhibiting the formation of abnormal or excessive neovascularization.

[0040] Figure 1 is a schematic diagram showing the structure of a bispecific candidate antibody A that specifically binds to c-Kit and VEGF according to the present invention.

[0041] Figure 2 shows the results of evaluating changes in antibody-dependent cell-mediated cytotoxicity according to the type of Fc variant.

[0042] Figures 3a to 3c are schematic diagrams showing the structures of bispecific candidate antibodies B (Figure 3a), C (Figure 3b), and B-1 (Figure 3c) that specifically bind to c-Kit and VEGF according to the present invention.

[0043] Figures 4a and 4b are schematic diagrams showing the structures of bispecific candidate antibodies D (Figure 4a) and E (Figure 4b) that specifically bind to c-Kit and VEGF according to the present invention.

[0044] Figures 5a and 5b show the results of ELISA to confirm the binding ability of a bispecific candidate antibody that specifically binds to c-Kit and VEGF according to the present invention to c-Kit and VEGF.

[0045] Figures 6a and 6b show the results of confirming the binding affinity of a bispecific candidate antibody that specifically binds to c-Kit and VEGF according to the present invention by surface plasmon resonance.

[0046] Figure 7 shows the results of confirming the thermal stability of a bispecific candidate antibody that specifically binds to c-Kit and VEGF according to the present invention using DSF.

[0047] Figures 8a to 8c show the inhibitory effect of vascular permeability upon treatment with a bispecific candidate antibody specifically binding to c-Kit and VEGF according to the present invention in HUVECs induced under hypoxic conditions or inflammation (Figures 8a and 8b: ***P<0.001, ****P<0.0001 vs. vehicle; #P<0.05, ###P<0.001 and ####P<0.0001 vs. 1% O2 or LPS; ns, no significance. (n=3), Figure 8c: ***P<0.001, ****P<0.0001 vs. vehicle; ##P<0.005, ####P<0.0001 vs. 1% O2 or LPS; †P<0.05, ††††P<0.0001 vs. 1% O2 or LPS, respectively). concentration; ns, not significant).

[0048] Figures 9a to 9d illustrate the inhibitory effect of SCF-dependent c-Kit signaling upon treatment with a bispecific candidate antibody specifically binding to c-Kit and VEGF according to the present invention in hypoxic or inflammation-induced HRMEC or LAD2.

[0049] Figures 10a and 10b show the cytokine (IL-8, MCP-1, IL-6) inhibitory effect of treatment with the bispecific candidate antibody A that specifically binds to c-Kit and VEGF according to the present invention in hypoxic or inflammation-induced HRMEC or ARPE-19 cells (**P<0.01, ***P<0.001, and ****P<0.0001 vs. vehicle; #P<0.01, ##P<0.005, and ###P<0.001 vs. 1% O2 or LPS).

[0050] Figures 11a to 11r show the cytokine (VEGF, IL-6, IL-8, MCP-1, and Ang-2) inhibitory effects of candidate antibody B treatment in hypoxic or inflammation-induced HRMEC or ARPE-19 cells (**P<0.01, ***P<0.001, and ****P<0.0001 vs. vehicle; #P<0.01, ##P<0.005, ###P<0.001, and ####P<0.0001 vs. 1% O2 or LPS treatment; ns, not significant).

[0051] Figures 12a to 12i show the inhibitory effect of candidate antibody C or candidate antibody E on cytokine (IL-6, VEGF, Ang-2, IL-8, and MCP-1) secretion in hypoxic or inflammation-induced HRMEC or ARPE-19 cells (***P<0.001, ****P<0.0001 vs. vehicle; #P<0.05, ##P<0.005, ###P<0.001, and ####P<0.0001 vs. 1% O2 or LPS; ns, not significant).

[0052] Figures 13a to 13i show the comparative cytokine (IL-6, VEGF, Ang-2, IL-8, and MCP-1) inhibitory effects of candidate antibody C alone or in combination with anti-c-Kit antibody in hypoxic or inflammation-induced HRMEC or ARPE-19 cells (***P<0.001, ****P<0.0001 vs. vehicle; #P<0.05, ###P<0.001, ####P<0.0001 vs. 1% O2 or LPS; †††P<0.001, ††††P<0.0001 vs. each concentration in 1% O2 or LPS; ns, not significant).

[0053] Figures 14a and 14b show the comparative effects of candidate antibody C alone or in combination with anti-c-Kit antibody on vascular permeability in mice with induced choroidal neovascularization.

[0054] Figure 14c shows the comparative effect of reducing choroidal neovascular lesions in mice with induced choroidal neovascularization by treatment with candidate antibody C alone or in combination with Ilia and anti-c-Kit antibodies.

[0055] Figure 15a shows a comparison of the inhibitory effects on vascular permeability according to treatment with candidate antibody A alone at different concentrations (0.5, 1, and 2 μg / μL / eye, respectively) and treatment with Aylea (aflibercept) alone at different concentrations (2 and 20 μg / μL / eye, respectively) in mice with induced choroidal neovascularization (* P<0.05, ** P<0.01, and **** P<0.0001 vs. G2).

[0056] Figure 15b shows the comparison of the effects of treatment with candidate antibody A alone at different concentrations (0.5, 1, and 2 μg / μL / eye, respectively) and treatment with Aylea (aflibercept) alone at different concentrations (2 and 20 μg / μL / eye, respectively) on reducing choroidal neovascularization lesions in mice induced with choroidal neovascularization (*** P<0.001 and **** P<0.0001 vs. G2).

[0057] Hereinafter, the present invention will be described in more detail.

[0058] All technical terms used in this invention, unless otherwise defined, have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of this invention.

[0059] The term "antibody" in the present invention is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (so long as they exhibit the desired antigen-binding activity).

[0060] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, with the possible exception of variant antibodies, including those containing naturally occurring mutations or those arising during the production of the monoclonal antibody preparation, which mutations are generally present in minor amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0061] As used herein, the term "monospecific" antibody refers to an antibody having more than one binding site, each of which binds to the same epitope on the same antigen. The term "bispecific" means that the antibody can specifically bind to at least two distinct antigenic determinants, e.g., two binding sites formed by a pair of antibody heavy chain variable domains (VH) and antibody light chain variable domains (VL), each of which binds to a different antigen or a different epitope on the same antigen. Such bispecific antibodies are in a 2+2 format (containing two binding sites for a first and a second antigen or epitope). Typically, a bispecific antibody contains two antigen binding sites, each of which is specific for a different antigenic determinant.

[0062] As used herein, the term "binding" refers to the presence of a specified number of binding sites in an antigen-binding molecule. Similarly, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two, four, and six binding sites, respectively, in an antigen-binding molecule. A bispecific antibody according to the present invention may be "tetravalent."

[0063] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to antibodies having a structure substantially similar to that of native antibodies. "Native antibodies" refer to naturally occurring immunoglobulin molecules with a variety of structures. For example, native IgG-class antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two light chains and two heavy chains disulfide-bonded. From N-terminus to C-terminus, each heavy chain has a variable region (VH), called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), called heavy chain constant regions. Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), called the variable light domain or light chain variable domain, followed by a light chain constant domain (CL), called the light chain constant region. The heavy chains of antibodies can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further classified into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of antibodies can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0064] As described above, the variable region allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the variable region defines a three-dimensional antigen-binding site by combining the VL domain and VH domain, or a subset of the complementarity determining regions (CDRs) of the antibody. This quaternary antibody structure forms the antigen-binding site that is located at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs on each VH and VL chain (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3). In some cases, for example, when a given immunoglobulin molecule is derived from a camelid species or is engineered based on a camelid immunoglobulin, the complete immunoglobulin molecule may be composed solely of heavy chains, without a light chain. See, for example, Hamers-Casterman et al., Nature 363: 446-448 (1993).

[0065] The terms "CDR-H", "HCDR", and "CDRH" are used interchangeably herein to refer to the VH chain of a CDR (e.g., CDR-H1, HCDR1, and CDRH1 refer to the VH1 of a CDR). The terms "CDR-L", "LCDR", and "CDRL" are used interchangeably herein to refer to the VL chain of a CDR (e.g., CDR-L1, LCDR1, and CDRL1 refer to the VL1 of a CDR).

[0066] In naturally occurring antibodies, six "complementarity determining regions", or "CDRs," present in each antigen-binding domain are short, non-contiguous amino acid sequences that are specifically positioned to form the antigen-binding domain as the antibody adopts a three-dimensional conformation in an aqueous environment. The remaining amino acids in the antigen-binding domain, referred to as the "framework" region, exhibit less intermolecular variability. The framework region primarily adopts a β-sheet conformation, and the CDRs form loops that connect them, and in some cases, form part of the β-sheet structure. Thus, the framework region functions to form a scaffold that positions the CDRs in the correct orientation by interchain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to epitopes on an immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDR and framework regions, respectively, for any given heavy or light chain variable region can be readily identified by those skilled in the art, as these are precisely defined (see www.bioinf.org.uk: Dr. Andrew CR Martin's Group; "Sequences of Proteins of Immunological Interest," Kabat, E., et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol., 196: 901-917 (1987)).

[0067] Where there are two or more definitions for a term used and / or accepted in the art, the definition of the term as used herein is intended to encompass all such meanings unless explicitly stated to the contrary. As a specific example, the term "complementarity determining region" ("CDR") is used to describe the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These specific regions are described in Kabat et al., US Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., J. Mol. Biol. 196: 901-917 (1987), the entire contents of which are incorporated herein by reference. The CDR definitions according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. Nonetheless, the application of the definitions to refer to CDRs of an antibody or variant thereof is intended to be within the scope of the terms defined and used herein. Appropriate amino acid residues comprising the CDRs defined by each of the references cited above are provided in Table 1 below for comparison. The exact number of residues comprising a particular CDR will vary depending on the sequence and size of the CDR. One of skill in the art can routinely determine whether a residue comprises a particular CDR by considering the variable region amino acid sequence of the antibody.

[0068] KabatchotiaCDR-H131-3526-32CDR-H150-6552-58CDR-H195-10295-102CDR-L124-3426-32CDR-L250-5650-52CDR-L389-9791-96

[0069] Kabat et al. also defined a numbering system for variable domain sequences applicable to any antibody. One skilled in the art can unambiguously assign the "Kabat numbering" system to any variable domain sequence without relying on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth in Kabat et al., US Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The antibodies disclosed herein may be derived from any animal source, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken.

[0070] As used herein, the term "heavy chain constant region" encompasses an amino acid sequence derived from an immunoglobulin heavy chain. As described above, those skilled in the art will appreciate that the heavy chain constant region can be modified to vary its amino acid sequence from that of a naturally occurring immunoglobulin molecule.

[0071] The heavy chain constant region of the antibodies disclosed herein may be derived from different immunoglobulin molecules. The heavy chain constant region of the antibodies according to the present invention may be designated as one of the five types of immunoglobulins, referred to as α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), as described above. Furthermore, immunoglobulins of the same type may be classified into different subclasses (isotypes) depending on the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, if the heavy chain constant region of the antibody of the present invention is γ (IgG), the subclass (isotype) can be further classified into γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), or γ4 (IgG4), and if the heavy chain constant region of the antibody of the present invention is α (IgA), the subclass (isotype) can be further classified into α1 (IgA1) or α2 (IgA2).

[0072] As used herein, the term "light chain constant region" comprises an amino acid sequence derived from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain.

[0073] A "light chain-heavy chain pair" refers to a collection of light and heavy chains that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0074] "Antibody fragment" or "antigen-binding fragment" refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab', F(ab')2, xFab, scFab, dsFv, Fv, scFv-Fc, scFab-Fc, diabodies, minibodies, scAbs, dAbs, half-IgGs or combinations thereof. The term "Fab" as used in Fab, Fab', F(ab')2, xFab and scFabs can include conventional Fab fragments and chimeric Fab-like domains as described in PCT / CN2018 / 106766 (Wuxibody). Additionally, it can be derived from any mammal, including but not limited to a human, mouse, rat, camelid, or rabbit. A functional portion of an antibody, such as one or more of the CDRs described herein, can be covalently linked to a second protein or small molecule compound, thereby enabling it to be used as a targeted therapeutic for a specific target. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen and forming a complex.

[0075] Antibody fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies as described herein, as well as production in recombinant host cells, such as E. coli or phage.

[0076] Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments, which contain the heavy and light chain variable domains and the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Therefore, as used herein, the term "Fab fragment" refers to a light chain fragment comprising the VL domain and the constant domain (CL) of the light chain, and an antibody fragment comprising the VH domain and the first constant domain (CH1) of the heavy chain. A Fab' fragment differs from a Fab fragment in that it adds several residues to the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residue(s) of the constant domains have a free thiol group. Pepsin treatment produces an F(ab')2 fragment, which has two antigen-binding sites (two Fab fragments) and part of the Fc region. As used herein, the "F(ab')2 fragment" comprises, as described above, two light chains and two heavy chains comprising a variable region, CH1, and a portion of a constant region between the CH1 and CH2 domains, thereby forming an intrachain disulfide bond between the two heavy chains. Accordingly, the F(ab')2 fragment is composed of two Fab' fragments, and the two Fab' fragments are linked to each other by a disulfide bond therebetween.

[0077] The term "cross-Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which the variable or constant regions of the heavy and light chains are exchanged. Two different chain compositions of crossover Fab molecules are possible and are included in the bispecific antibodies of the invention: on the one hand, the variable regions of the Fab heavy and light chains are exchanged, i.e. the crossover Fab molecule comprises a peptide chain composed of a light chain variable region (VL) and a heavy chain constant region (CH1), and a peptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). Such a crossover Fab molecule is also called CrossFab (VLVH). In contrast, when the constant regions of the Fab heavy and light chains are exchanged, the crossover Fab molecule comprises a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL), and a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1). Such a crossover Fab molecule is also called CrossFab(CLCH1).

[0078] A "single chain Fab fragment" or "scFab" is a polypeptide comprising an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domains and the linker have one of the following sequences from N-terminus to C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; and wherein the linker is a polypeptide having at least 30 amino acids, preferably 32 to 50 amino acids. The single chain Fab fragment is stabilized by a natural disulfide bond between the CL domain and the CH1 domain. Additionally, these single-chain Fab molecules can be further stabilized by the creation of interchain disulfide bonds through insertion of cysteine ​​residues (e.g., at position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

[0079] A "crossover single chain Fab fragment" or "x-scFab" is a polypeptide comprising an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domains and the linker have one of the following sequences from N-terminus to C-terminus: (a) VH-CL-linker-VLCH1 and (b) VL-CH1-linker-VH-CL; wherein the VH and VL together form an antigen-binding domain that specifically binds to an antigen, and wherein the linker is a polypeptide having at least 30 amino acids. Additionally, these x-scFab molecules can be further stabilized by formation of an interchain disulfide bond through insertion of a cysteine ​​residue (e.g., at position 44 in the variable heavy chain and position 100 in the variable light chain according to the Kabat numbering).

[0080] An "Fv region" is an antibody that includes the variable regions of each heavy and light chain, but not the constant regions. An scFv is an Fv linked by a flexible linker. An scFv-Fc is an Fc linked to an scFv. A diabody comprises two molecules of scFv. A "single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. In some embodiments, the regions are linked to a short linker peptide having from 10 to about 25 amino acids. The linker may be glycine-rich for flexibility and serine or threonine-rich for solubility, and may link the N-terminus of the VH to the C-terminus of the VL, or vice versa. These proteins retain the specificity of the native immunoglobulin despite the deletion of the constant region and the introduction of a linker. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0081] A "short-chain antibody (scAb)" is a single polypeptide chain comprising one heavy chain variable region or one light chain constant region, with the heavy and light chain variable regions connected by a flexible linker. For single-chain antibodies, see, for example, U.S. Patent No. 5,260,203, which is incorporated herein by reference.

[0082] A "domain antibody (dAb)" is an immunologically functional immunoglobulin fragment comprising only the variable region of a heavy chain or the variable region of a light chain. In one embodiment, two or more VH regions are covalently linked by a peptide linker to form a bivalent domain antibody. The two VH regions of such a bivalent domain antibody may target the same or different antigens.

[0083] The term "full-length IgG" according to the present invention is defined as comprising essentially complete IgG, but does not necessarily have all the functions of a complete IgG. For the avoidance of doubt, a full-length IgG contains two heavy chains and two light chains. Each chain contains constant (C) and variable (V) regions, which can be divided into domains designated CH1, CH2, CH3, VH, and CL, VL. IgG antibodies bind to antigens through the variable region domains contained in the Fab portion, and after binding, can interact with cells and molecules of the immune system through the constant domains, mostly through the Fc portion. The terms 'variable region domain', 'variable region', 'variable domain', 'VH / VL pair', 'VH / VL', 'Fab portion', 'Fab arm', 'Fab' or 'arm' are used interchangeably herein. A full-length antibody according to the present invention includes an IgG molecule that may have mutations that provide the desired characteristics. Such mutations must not result in the deletion of a significant portion of any region. However, an IgG molecule in which one or more amino acid residues are deleted without substantially altering the binding properties of the resulting IgG molecule is included within the term "full-length IgG". For example, such an IgG molecule may have one or more deletions of 1 to 10 amino acid residues, preferably in a non-CDR region, wherein the deletion of the amino acid is not essential for the binding specificity of the IgG.

[0084] As used herein, the term "antigen binding domain" or "antigen-binding site" refers to the portion of an antibody or antibody fragment that specifically binds to an antigenic determinant. More specifically, the term "antigen binding domain" refers to the portion of an antibody that specifically binds to and is complementary to part or all of an antigen. When the antigen is large, the antibody or antibody fragment may bind only to a specific portion of the antigen, which portion is called an epitope. The antigen binding domain may be provided, for example, by one or more variable domains (also referred to as variable regions). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In one embodiment, the antigen binding domain can bind to its antigen and block or partially block its function. Antigen binding domains that specifically bind to c-Kit and VEGF include antibodies and fragments thereof as further defined herein. Additionally, the antigen binding domain may comprise a scaffold antigen binding protein, e.g., a binding domain based on a designed repeat protein or a designed repeat domain (see e.g. WO 2002 / 020565).

[0085] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a contiguous stretch of amino acids or a conformational configuration comprised of different regions of non-contiguous amino acids) to which an antigen-binding moiety binds to form an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, the surface of virus-infected cells, the surface of other diseased cells, the surface of immune cells, in serum-free blood, and / or in the extracellular matrix (ECM). Unless otherwise specified, a protein useful as an antigen herein can be any native form of a protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In a particular embodiment, the antigen is a human or mouse protein. When referring to a specific protein herein, the term encompasses the "full-length," unprocessed protein, as well as all forms of the protein produced by processing in cells. The term also encompasses naturally occurring variants of the protein, such as splice variants or allelic variants.

[0086] "Specific binding" means that binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody or antibody fragment to bind a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

[0087] "Affinity" or "binding affinity" refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise specified, "binding affinity" refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (Kd), which is the ratio of the dissociation rate constant and the association rate constant (koff and kon, respectively). Thus, equivalent affinities can include different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by routine methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR). According to one embodiment of the present invention, the bispecific antibody of the present invention has an IC of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10-7 M or less, for example 10 -7 M to 10 -13 M, for example 10 -9 M to 10 -13 It has a dissociation constant (KD) of M).

[0088] The term "high affinity" of an antibody refers to its ability to bind to its target antigen at a rate of 10 -9 M or less, preferably 10 -10 Refers to antibodies with a KD of M or less.

[0089] The terms "bispecific antibody comprising a first antigen binding site that specifically binds c-Kit and a second antigen binding site that specifically binds VEGF", "bispecific antibody that specifically binds c-Kit and VEGF", and "bispecific antigen binding molecule specific for c-Kit and VEGF" are used interchangeably herein and refer to a bispecific antibody capable of binding c-Kit and VEGF with sufficient affinity to render the antibody useful as a diagnostic and / or therapeutic agent in targeting c-Kit and VEGF.

[0090] The term "c-Kit" used in the present invention belongs to class III of receptor tyrosine kinase (RTK) and is also known as a receptor for SCF.

[0091] c-Kit, one of the targets of the above angiogenesis inhibitor, belongs to class III of receptor tyrosine kinase (RTK) and is a receptor for Stem Cell Factor (SCF), which plays an important role in hematopoiesis.

[0092] The term "anti-c-Kit antibody" as used in the present invention refers to an antibody that specifically binds to c-Kit. Specifically, the anti-c-Kit antibody specifically binds to domain II of c-Kit, thereby inhibiting or neutralizing the activity or activation of c-Kit.

[0093] "Anti-c-Kit antibody" and "antibody comprising an antigen binding site that binds c-Kit" refer to an antibody capable of binding c-Kit, particularly a c-Kit polypeptide, with sufficient affinity to render the antibody useful as a diagnostic and / or therapeutic agent in targeting c-Kit. In one embodiment, the extent of binding of the anti-c-Kit antibody to an unrelated, non-c-Kit protein is less than about 10% of the binding of the antibody to c-Kit, as measured, for example, by radioimmunoassay (RIA) or flow cytometry (FACS) or by surface plasmon resonance analysis using a biosensor system such as the Biacore® system. By way of example, antibodies that bind to human c-Kit have a concentration of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, for example 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M) has a KD value of binding affinity for binding to human c-Kit.

[0094] As used herein, "VEGF antagonist" means any molecule that blocks, reduces, or interferes with the normal biological activity of vascular endothelial growth factor (VEGF) or a VEGF receptor. VEGF antagonists include molecules that interfere with the interaction between VEGF and a native VEGF receptor, for example, molecules that bind to VEGF or a VEGF receptor and prevent or otherwise interfere with the interaction between VEGF and the VEGF receptor. Specific examples of VEGF antagonists include anti-VEGF antibodies (e.g., bevacizumab [AVASTIN®]), anti-VEGF receptor antibodies (e.g., anti-VEGFR1 antibodies, anti-VEGFR2 antibodies, etc.), and VEGF receptor-based chimeric molecules (also referred to herein as "VEGF-traps"). VEGF receptor-based chimeric molecules include chimeric polypeptides comprising two or more immunoglobulin (Ig)-like domains of a VEGF receptor, such as VEGFR1 (also referred to as Flt1) and / or VEGFR2 (also referred to as Flk1 or KDR), and which may also contain a multimerization domain (e.g., an Fc domain that allows multimerization [e.g., dimerization] of two or more chimeric polypeptides). Exemplary VEGF receptor-based chimeric molecules are VEGFR1R2-FcΔC1(a) or Flt1D2.Flk1D3.FcΔC1 (also known as aflibercept (trade names: Eylea, Zaltrap)) comprising or consisting of the amino acid sequence of SEQ ID NO: 21.

[0095] "VEGF antagonist" and "antibody comprising an antigen binding site that binds VEGF" refer to an antibody capable of binding VEGF, particularly a VEGF polypeptide, with sufficient affinity to render the antagonist or antibody useful as a diagnostic and / or therapeutic agent in the targeting of VEGF. In one embodiment, the extent of binding of the VEGF antagonist or antibody to an unrelated, non-VEGF protein is less than about 10% of the binding of the antagonist or antibody to VEGF, as measured, for example, by radioimmunoassay (RIA) or flow cytometry (FACS) or by surface plasmon resonance analysis using a biosensor system such as the Biacore® system. By way of example, antibodies that bind to human VEGF have a concentration of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, for example 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M) has a KD value of binding affinity for human VEGF binding.

[0096] The term "mouse" antibody is intended to encompass antibodies having variable regions in which both the framework and CDR regions are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from mouse germline immunoglobulin sequences. The mouse antibodies of the present disclosure may comprise amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by in vivo complementary somatic mutagenesis).

[0097] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remainder of the heavy and / or light chain is derived from a different source or species.

[0098] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the various classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0099] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.

[0100] A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been further modified or altered from the constant region of the original antibody to produce the properties of the present invention, particularly with respect to C1q binding and / or Fc receptor (FcR) binding.

[0101] A "human" antibody is one that has an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or other human antibody-coding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0102] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

[0103] The term "linker" refers to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids. Any peptide linker known in the art can be used as the linker. The peptide linker separates the light chain variable domain and the heavy chain variable domain by a sufficient distance to allow each variable domain to fold into the appropriate secondary and tertiary structures. The sequence of a suitable peptide linker can be selected by considering the following factors: (a) the ability to have a flexible extended conformation; (b) the ability to not create secondary structures that interact with the epitope; and (c) the absence of hydrophobic residues or charged residues that can react with the epitope. Preferred peptide linkers include Gly, Glu, Asn, Lys, Ser, and Pro residues. Other neutral amino acids, such as Thr and Ala, can also be included in the linker sequence. The linker sequence may consist of 1-50 amino acid residues, preferably 10-20 amino acid residues. For example, a suitable non-immunogenic linker peptide may include, but is not limited to, 5-10 consecutive glycines (G).

[0104] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage by which the amino acid residues in the candidate sequence are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA program package. One skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithm necessary to achieve maximum alignment over the entire length of the compared sequences. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program in the FASTA package, version 36.3.8c or later, in conjunction with a BLOSUM50 comparison matrix. The FASTA program package is described in WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis," PNAS 85:2444-2448; WR Pearson (1996), "Effective Protein Sequence Comparison," Meth. Enzymol. 266:227-258; and Pearson et al. (1997) Genomics 46:24-36, and is publicly available at http: / fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml. Alternatively, see http: / fasta.bioch.virginia.edu / fasta_www2 / index.You can compare sequences using a public server accessible from cgi, and use the ggsearch(global protein:protein) program with default options (BLOSUM50; open: -10; ext: -2; Ktup=2) to perform a global alignment rather than a local one. The percent amino acid identity (%) is provided in the output alignment header.

[0105] As used herein, the term "polypeptide" is intended to encompass not only a singular "polypeptide" but also a plural "polypeptides," and refers to a molecule comprising monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids are encompassed within the definition of "polypeptide," and the term "polypeptide" may be used in place of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to refer to the product of post-expression modification of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources or produced by recombinant techniques, but are not necessarily translated from a designated nucleic acid sequence. They may be produced by any means, including chemical synthesis. The term "polypeptide" also encompasses variants and derivatives of polypeptides. Furthermore, "polypeptide fragment" refers to a polypeptide having a deletion of the amino-terminal amino acid sequence, a deletion of the carboxyl-terminal amino acid sequence, and / or an internal deletion, compared to the full-length protein. Such fragments may also contain modified amino acids compared to the full-length protein.In one embodiment, the fragment can be about 5 to 900 amino acids in length, for example at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or more amino acids in length. For the purposes of the present invention, useful polypeptide fragments include immunologically functional fragments of antibodies comprising an antigen-binding domain. For antibodies that specifically bind to c-Kit and VEGF, such useful fragments include, but are not limited to, all or part of an antibody chain comprising one, two or three heavy or light chain CDR sequences, or a variable or constant region of a heavy or light chain.

[0106] As used herein, a "variant" of a polypeptide, e.g., an antigen-binding fragment, protein, or antibody, is a polypeptide having one or more amino acid residues inserted, deleted, added, and / or substituted as compared to another polypeptide sequence, including fusion polypeptides. Protein variants also include those that have been modified by proteolytic enzyme cleavage, phosphorylation, or other post-translational modifications, yet retain the biological activity of an antibody disclosed herein, e.g., specific binding to c-Kit and VEGF, and biological activity. Variants can be about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% identical to the sequence of an antibody or antigen-binding fragment thereof disclosed herein.

[0107] As used herein, the term "recombinant" with respect to a polypeptide or polynucleotide means a form of a polypeptide or polynucleotide that does not exist in nature, a non-limiting example of which can be formed by combining polynucleotides or polypeptides that do not normally exist together.

[0108] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of positions shared by the sequences that are identical or homologous. An "unrelated" or "non-homologous" sequence shares less than 40% identity, and preferably less than 25% identity, with one of the sequences of the present disclosure.

[0109] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a given percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence means that when two sequences are aligned, the bases (or amino acids) are the same by that percentage.

[0110] The term "polynucleotide" refers to an isolated nucleic acid molecule or structure, such as messenger RNA (mRNA), virally derived RNA, or plasmid DNA (pDNA). A polynucleotide may comprise conventional phosphodiester linkages or non-conventional linkages (e.g., amide linkages, such as those found in peptide nucleic acids (PNA). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0111] An "isolated" nucleic acid molecule or polynucleotide refers to a nucleic acid molecule, DNA, or RNA, obtained from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of the present invention. Additional examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or polynucleotides purified (partially or substantially) in solution. An isolated polynucleotide generally includes a polynucleotide molecule contained in a cell containing the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location different from the natural chromosomal location. Isolated RNA molecules include the in vivo or in vitro RNA transcripts of the present invention, as well as positive and negative strand forms, and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present invention further include synthetically produced molecules. Additionally, the polynucleotide or nucleic acid may be or include regulatory elements such as a promoter, ribosome binding site, or transcription terminator. The term "isolated" as used herein also refers to a nucleic acid or peptide being substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. The term "isolated" is also used herein to refer to a cell or polypeptide being separated from other cellular proteins or tissues. Isolated polypeptides are meant to include both purified polypeptides and recombinant polypeptides.

[0112] The term "expression cassette" refers to a polynucleotide produced recombinantly or synthetically using a series of specified nucleic acid elements that enable transcription of a specific nucleic acid in a target cell. A recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plasmid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector comprises, among other things, the nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette comprises a polynucleotide sequence encoding a bispecific antibody of the invention.

[0113] The term "vector" or "expression vector" refers to a DNA molecule used to introduce and express a specific gene operably linked to a cell. The term encompasses vectors as self-replicating nucleic acid structures as well as vectors introduced by integration into the genome of a host cell. The expression vector of the present invention comprises an expression cassette. The expression vector enables the transcription of large quantities of stable mRNA. When the expression vector is present within a cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and / or translation machinery. In one embodiment, the expression vector of the present invention comprises an expression cassette comprising a polynucleotide sequence encoding a bispecific antibody of the present invention.

[0114] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced (including the progeny of such cells). Host cells include "transformants" and "transformed cells," which include primary transformed cells and their derived progeny, regardless of the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as that selected or selected from the originally transformed cell are included herein. A host cell is any type of cell system that can be used to produce an antibody or bispecific antibody of the invention. Host cells include cultured cells, for example mammalian cultured cells such as HEK cells, CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, including cells contained within transgenic animals, transgenic plants or cultured plant or animal tissues.

[0115] A first aspect of the present invention relates to a bispecific antibody specifically directed to c-Kit and VEGF.

[0116] The bispecific antibody specifically binding to c-Kit and VEGF according to the present invention has the property of binding to c-Kit and VEGF with high affinity, and may include the following (a) and (b):

[0117] (a) a first antigen binding site that specifically binds c-Kit, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and (b) a second antigen binding site that specifically binds VEGF, wherein the soluble extracellular domain of VEGF and the Fc domain of IgG are fused.

[0118] The first antigen binding site of the bispecific antibody according to one embodiment of the present invention may include a light chain variable region comprising LCDR1, LCDR2, and LCDR3 each consisting of the amino acid sequences described in (a) above, and a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 each consisting of the amino acid sequences described in (a) above.

[0119] In the present invention, the first antigen binding site may include a heavy chain variable region comprising an amino acid sequence having 90% or more sequence homology with the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising an amino acid sequence having 90% or more sequence homology with the amino acid sequence of SEQ ID NO: 28. Specifically, the first antigen binding site according to one embodiment of the present invention may include a heavy chain variable region comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence homology with the amino acid sequence of SEQ ID NO: 27, and a light chain variable region comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence homology with the amino acid sequence of SEQ ID NO: 28.

[0120] The amino acid sequences of the above sequence numbers 27 and 28 are as follows.

[0121] QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGTNKDYTDSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDWAEAFDMWGQGTTVTVSS (SEQ ID NO: 27)

[0122] DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTITFGQGTRLEIK (SEQ ID NO: 28)

[0123] In the present invention, the soluble extracellular domain may comprise immunoglobulin-like domain 2 of a first VEGF receptor and immunoglobulin-like domain 3 of a second VEGF receptor. Preferably, the second antigen binding site may be a VEGF antagonist known as aflibercept (trade name: Eylea, Zaltrap). According to a specific embodiment of the present invention, the VEGF antagonist may comprise or consist of an amino acid of SEQ ID NO: 21.

[0124] In the present invention, the second antigen binding site may be bound to the heavy chain C-terminus or N-terminus of the first antigen binding site by a peptide linker; or the second antigen binding site may be bound to the light chain C-terminus of the first antigen binding site by a peptide linker. According to a specific embodiment of the present invention, antibodies in which the second antigen binding site is bound to the heavy chain C-terminus of the first antigen binding site by a peptide linker are 'candidate antibody A', 'candidate antibody B', 'candidate antibody B-1', and 'candidate antibody C', antibodies in which the second antigen binding site is bound to the heavy chain N-terminus of the first antigen binding site by a peptide linker are 'candidate antibody E', and antibodies in which the second antigen binding site is bound to the light chain C-terminus of the first antigen binding site by a peptide linker are 'candidate antibody D'.

[0125] In the present invention, the peptide linker connecting the first antigen binding site and the second antigen binding site may be a peptide linker having 5 to 10 glycines (Gly) connected in series, and is designated as SEQ ID NO: 20 (G5), SEQ ID NO: 67 (G6), SEQ ID NO: 68 (G7), SEQ ID NO: 23 (G8), SEQ ID NO: 69 (G9), or SEQ ID NO: 70 (G10), respectively.

[0126] The bispecific antibodies of the present invention may have their Fc domains modified to reduce Fc receptor binding and / or effector function.

[0127] According to one embodiment of the present invention, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein to produce an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0128] Specifically, the Fc domain of the bispecific antibody comprising a first antigen-binding site that specifically binds c-Kit and a second antigen-binding site that specifically binds VEGF can comprise one or more amino acid substitutions that reduce binding to an Fc receptor, particularly an Fcγ receptor. For example, the Fc domain is of the human IgG1 subclass, comprising an Fc variant comprising amino acid substitutions L234A and L235E; or an Fc variant comprising amino acid substitutions L234A, L235A I253A, H310A, P329G, and H435A.

[0129] The Fc domain confers favorable pharmacodynamic properties to the bispecific antibody of the present invention, including a long serum half-life that contributes to good accumulation in target tissues and a favorable tissue-to-blood distribution ratio. However, this may also lead to undesirable targeting of the bispecific antibody of the present invention to cells expressing Fc receptors rather than to desired antigen-bearing cells. The Fc domain of the bispecific antibody according to one embodiment of the present invention exhibits reduced binding affinity for Fc receptors and / or reduced effector function compared to a native IgG Fc domain, particularly an IgG1 Fc domain. Preferably, the Fc domain may be a wild-type IgG1 Fc domain or a variant thereof.

[0130] According to a specific embodiment of the present invention, the Fc domain exhibits a binding affinity for an Fc receptor of less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5%, compared to a native IgG1 Fc domain (or a bispecific antigen binding molecule of the present invention comprising a native IgG1 Fc domain), and / or an effector function of less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5%, compared to a native IgG1 Fc domain. In one embodiment, the Fc domain does not substantially bind to and / or induce an effector function of an Fc receptor. In one embodiment, the Fc receptor is an Fcγ receptor. In another embodiment, the Fc receptor is a human Fc receptor. In yet another embodiment, the Fc receptor is an activating Fc receptor. In another embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, preferably human FcγRIIIa. In one embodiment, the effector function is one or more of CDC, ADCC, ADCP, and cytokine secretion. In another embodiment, the effector function is ADCC. In another embodiment, the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to a native IgG1 Fc domain.

[0131] According to one embodiment of the present invention, the Fc domain can be engineered to have reduced binding affinity and / or reduced effector function for an Fc receptor, compared to a non-engineered Fc domain (wild-type Fc). In one embodiment, the Fc domain of the bispecific antibody of the present invention can comprise one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain for an Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. The amino acid mutations can reduce the binding affinity of the Fc domain for an Fc receptor. For example, the amino acid mutations can reduce the binding affinity of the Fc domain for an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold, or can eliminate binding affinity such that the Fc domain does not bind to an Fc receptor at all. In a specific embodiment, the bispecific antibody of the invention comprising an engineered Fc domain may exhibit a binding affinity for an Fc receptor of less than 20%, particularly less than 10%, and more particularly less than 5%, compared to a bispecific antibody of the invention comprising a non-engineered Fc domain. In one embodiment, the Fc receptor is an Fcγ receptor. In another embodiment, the Fc receptor is a human Fc receptor. In yet another embodiment, the Fc receptor is an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI or FcγRIIa, preferably human FcγRIIIa. In one embodiment, the binding affinity for complement components, particularly binding affinity for C1q, may also be reduced. In another embodiment, the binding affinity for neonatal Fc receptor (FcRn) may be reduced.Substantially similar binding to FcRn, i.e., preservation of the binding affinity of the Fc domain for the receptor, is achieved when the Fc domain exhibits a binding affinity for FcRn that is greater than about 70% of that of the unengineered form of the Fc domain. The Fc domain, or a bispecific antibody of the invention comprising the Fc domain, can exhibit such affinities of greater than about 80% and even greater than about 90%. In some embodiments, the Fc domain of the bispecific antigen binding molecule of the invention is engineered to have reduced effector function compared to the unengineered Fc domain. The reduced effector function may include, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced dendritic cell maturation, or reduced T cell priming.

[0132] Antibodies with reduced effector function can comprise those having substitutions at one or more of Fc region residues 234, 235, 237, 253, 310, 329, and 435. In a specific embodiment, the Fc domain can comprise amino acid substitutions L234A, L235A, and P329G (“LALAPG”), L234A, L235A, P329G, I253A, H310A, and H435A (“LALAPG-AAA”), L235A and G237A (“LAGA”), or amino acid substitutions L234A and L235E (“LALE”).

[0133] In the present invention, an exemplary bispecific antibody having a wild-type Fc domain may be, but is not limited to, candidate antibody A or candidate antibody D. The heavy chain of the first antigen-binding portion of candidate antibody A or candidate antibody D having a wild-type Fc domain may comprise or consist of the amino acid sequence of SEQ ID NO: 29, and the light chain of the first antigen-binding portion may comprise or consist of the amino acid sequence of SEQ ID NO: 30.

[0134] The amino acid sequences of the above sequence numbers 29 and 30 are as follows.

[0135] QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGTNKDYTDSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDWAEAFDMWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 29)

[0136] DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTITFGQGTRLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 30)

[0137] For reduced effector function, the Fc domain of the bispecific antibody according to the invention may include amino acid substitutions as defined above. In the present invention, an exemplary bispecific antibody having LALAPG introduced into the Fc domain may be, but is not limited to, candidate antibody B or candidate antibody E. The heavy chain of candidate antibody B or candidate antibody E having LALAPG introduced into the Fc domain may comprise or consist of the amino acid sequence of SEQ ID NO: 31, and the light chain of the first antigen-binding site may comprise or consist of the amino acid sequence of SEQ ID NO: 30.

[0138] The amino acid sequence of the above sequence number 31 is as follows.

[0139] QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGTNKDYTDSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDWAEAFDMWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 31)

[0140] In the present invention, an exemplary bispecific antibody having LALE introduced into the Fc domain may be, but is not limited to, candidate antibody C. The heavy chain of candidate antibody C having LALE introduced into the Fc domain may include or consist of the amino acid sequence of SEQ ID NO: 32, and the light chain of the first antigen binding site may include or consist of the amino acid sequence of SEQ ID NO: 30.

[0141] The amino acid sequence of the above sequence number 32 is as follows.

[0142] QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGTNKDYTDSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDWAEAFDMWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 32)

[0143] In the present invention, an exemplary bispecific antibody having LALAPG-AAA introduced into the Fc domain may be, but is not limited to, candidate antibody B-1. The heavy chain of candidate antibody B-1 having LALAPG-AAA introduced into the Fc domain may include or consist of the amino acid sequence of SEQ ID NO: 33, and the light chain of the first antigen-binding site may include or consist of the amino acid sequence of SEQ ID NO: 30.

[0144] The amino acid sequence of the above sequence number 33 is as follows.

[0145] QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGTNKDYTDSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDWAEAFDMWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALGAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGK (SEQ ID NO: 33)

[0146] Binding to Fc receptors can be readily measured, for example, by surface plasmon resonance (SPR) using standard equipment such as an ELISA or BIAcore instrument (GE Healthcare), and the Fc receptors themselves can be obtained by recombinant expression. The binding affinity of an Fc domain or a cell-activating bispecific antibody comprising an Fc domain to an Fc receptor can be assessed using a cell line known to express a specific Fc receptor, such as the human NK cell-expressed FcγIIIa receptor. The effector function of the Fc domain or a bispecific antibody of the invention comprising an Fc domain can be measured by methods known in the art.

[0147] The bispecific antibody according to the present invention may be a tetravalent antibody having four binding sites. The bispecific antibody according to the present invention is a bispecific antibody having four binding sites. Preferably, the first antigen-binding site of the bispecific antibody according to the present invention may be a full-length bivalent antibody, and the second antigen-binding site may be composed of two VEGF antagonists, each of which is a fusion of the soluble extracellular domain of VEGF and the Fc domain of IgG. Therefore, the bispecific antibody according to the present invention has a valency of 2+2.

[0148] Faricimab (brand name: Vabysmo), which is marketed as a treatment for macular degeneration, is a 2(1+1) antibody that binds Ang-2 and VEGF CrossMab. Accordingly, in one embodiment of the present invention, two types of 2(1+1)valent antibodies [Fab-VEGFR-Fc(KH) and Fab-VEGFR-Fc(HK)] were produced by linking the first antigen binding site and the second antigen binding site to an anti-c-Kit antibody and VEGFR using the "knob-into-hole" technology, and one type of 2(1+1)valent antibody in which the c-Kit scFv and the VEGFR trap were linked with a peptide linker (G4S or G8) was produced, and their purity, stability (HMW and LMW production), thermostability, and binding affinity to c-Kit and VEGF were evaluated. However, these antibodies showed endotoxin detection, low stability (HMW and LMW production), low thermostability, and low antigen-antibody binding affinity, and were confirmed to be unsuitable as bispecific antibodies.

[0149] In a specific embodiment of the present invention, the antibody in the form of Fab-VEGFR-Fc(KH) produced above was named a “comparative antibody” and was used for comparison of efficacy with the bispecific antibody according to the present invention.

[0150] The "knob-into-hole" technique is described, for example, in U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a knob ("knob") into the contact surface of a first polypeptide and a corresponding cavity ("hole") into the contact surface of a second polypeptide such that the knob is positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The knob is constructed by replacing a small amino acid side chain from the contact surface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensating cavity of the same or similar size as the knob is formed by replacing the large amino acid side chain with the large amino acid side chain. The protrusions and cavities are created at the contact surface of the second polypeptide by replacing the side chains with smaller ones (e.g., alanine or threonine). The protrusions and cavities can be produced by modifying the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0151] In the present invention, the bispecific antibody may have its amino acid sequence altered to improve its binding affinity and / or other biological properties. Such alterations include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody. These amino acid mutations are made based on the relative similarity of the 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 are similar in size; and phenylalanine, tryptophan, and tyrosine are similar in shape. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0152] Amino acid exchanges in proteins that do not alter the overall activity of the molecule are well known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly.

[0153] Considering the mutations having the above-described biological equivalent activity, the bispecific antibodies of the present invention and the nucleic acid molecules encoding them are interpreted to also include sequences that exhibit substantial identity with the sequences listed in the sequence listing. The substantial identity refers to a sequence that exhibits at least 60% identity, more preferably 70% identity, even more preferably 80% identity, and most preferably at least 90% identity when the sequences of the present invention and any other sequences are aligned to the greatest extent possible and the aligned sequences are analyzed using an algorithm commonly used in the art.

[0154] According to one embodiment of the present invention, the bispecific antibody may comprise a peptide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence defined above.

[0155] A second aspect of the present invention relates to a nucleic acid molecule encoding the aforementioned bispecific antibody or fragment thereof according to the present invention, a vector comprising the nucleic acid molecule, and a host cell comprising the vector.

[0156] The nucleic acid molecule of the present invention is an isolated nucleic acid molecule. In one embodiment of the present invention, the nucleic acid molecule encoding the bispecific antibody or fragment thereof may comprise or consist of a nucleotide sequence encoding an amino acid sequence of SEQ ID NOs: 2, 4, 6, 13, 15, 17, 21, 27, 28, 29, 30, 31, 32, and 33.

[0157] According to a specific embodiment of the present invention, the nucleotide sequence encoding the amino acid sequences of SEQ ID NOs: 2, 4, 6, 13, 15 and 17, respectively, may include or consist of the nucleotide sequences of SEQ ID NOs: 35, 37, 39, 46, 48 and 50 in that order, but is not limited thereto.

[0158] According to a specific embodiment of the present invention, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 21 may include or consist of the nucleotide sequence of SEQ ID NO: 54, but is not limited thereto.

[0159] According to a specific embodiment of the present invention, the nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 27 and 28, respectively, may include or consist of the nucleotide sequences of SEQ ID NOs: 60 and 61, in that order, but are not limited thereto.

[0160] The nucleotide sequences of the above sequence numbers 60 and 61 are as follows.

[0161] CAGGTGCAGCTGGTGGAATCTGGTGGCGGAGTTGTGCAGCCTGGCAGATCCCTGAGACTGTCTTGTGCCGCCTCCGGCTTCACCTTCTCAGATACGGAATGCACTGGGTCCGACAGGCCCCTGGCAAAGGATTGGAATGGGTCGCCGTGATTTGGTACGACGGCACCAACAAGGAC TACACCGACTCTGTGCGGGGCAGATTCACCATCTCTCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAGAGAGGATTGGGCCGAAGCCTTCGATATGTGGGGGCCAGGGCACAACCGTGACCGTGTCCTCT (SEQ ID NO: 60)

[0162] GACATCGTGATGACCCAGTCTCCACTGAGCCTGCCTGTGACACCTGGCGAGCCTGCTTCCATCTCCTGCAGATCCTCTCAGTCCCTGCTGCACTCCAACGGCTACAACTACCTGGACTGGTATCTGCAGAAGCCCGGCCAGTCTCCTCAGCTGCTGATCTACCTGG GCTCCAACAGAGCTTCTGGCGTGCCCGATAGATTCTCCGGCTCTGGCTCTGGCACCGACTTCACCCTGAAGATCTCCAGAGTGGAAGCCGAGGACGTGGGCGTGTACTACTGTATGCAGGCCCTGCAGACCATCACCTTCGGCCAGGGAACCAGACTGGAAATCAAG (SEQ ID NO: 61)

[0163] According to a specific embodiment of the present invention, the nucleotide sequence encoding the amino acid sequences of SEQ ID NOs: 29, 30, 31, 32 and 33, respectively, may include or consist of the nucleotide sequences of SEQ ID NOs: 62, 63, 64, 65 and 66 in that order, but is not limited thereto.

[0164] The nucleotide sequences of the above sequence numbers 62, 63, 64, 65 and 66 are as follows.

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171] The nucleic acid molecule of the present invention is also interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence described above. The substantial identity refers to a nucleotide sequence that exhibits at least 80% identity, more preferably at least 90% identity, when the nucleotide sequence of the present invention is arranged to correspond as much as possible to any other sequence and the arranged sequence is analyzed using an algorithm commonly used in the art.

[0172] In one embodiment, the nucleic acid molecule can comprise a nucleic acid molecule having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence defined above.

[0173] In the present invention, the vector includes a plasmid vector; a phagemid vector; a cosmid vector; and a viral vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector, and preferably a plasmid vector.

[0174] The vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al. (2001), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, which is incorporated herein by reference.

[0175] The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.

[0176] When the vector of the present invention is an expression vector and uses a prokaryotic cell as a host, it generally includes a strong promoter capable of initiating transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. When E. coli (e.g., BL21, HB101, DH5α, etc.) is used as a host cell, E. The promoter and operator regions of the tryptophan biosynthetic pathway of B. coli (Yanofsky, C. (1984), J. Bacteriol., 158:1018-1024) and the left-hand promoter of phage λ (pLλ promoter, Herskowitz, I. and Hagen, D. (1980), Ann. Rev. Genet., 14:399-445) can be used as regulatory regions.

[0177] Vectors that can be used in the present invention can be produced by manipulating plasmids (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series (pET28a, pET21a, etc.) and pUC19, etc.), phagemids (e.g., pComb3X), phages (e.g., λgt4·λB, λ-Charon, λΔz1 and M13, etc.) or viruses (e.g., SV40, etc.) that are frequently used in the art.

[0178] When the vector of the present invention is an expression vector and uses a eukaryotic cell as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and HSV late promoter) can be used, and generally has a polyadenylation sequence as a transcription termination sequence.

[0179] The vector of the present invention may be fused with other sequences as needed to facilitate purification of the amino-terminal protein of the protein expressed therefrom. The sequences to be fused 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).

[0180] The expression vector of the present invention may include an antibiotic resistance gene commonly used in the art as a selectable marker, for example, a resistance gene for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0181] The vector expressing the bispecific antibody of the present invention may be a vector system in which the first antigen binding site and the second antigen binding site are expressed in a single vector in a form in which they are linked by a peptide linker, or a system in which the first antigen binding site and the second antigen binding site are expressed in separate vectors.

[0182] A host cell according to one embodiment of the present invention is a cell transformed with the vector described above. Any host cell known in the art that can stably and continuously clone and express the vector of the present invention may be used, and examples thereof include, but are not limited to, prokaryotic host cells such as Bacillus spp. such as Escherichia coli, Bacillus subtilis and B. thuringensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis or Staphylococcus (e.g., Staphylococcus carnosus).

[0183] Suitable eukaryotic host cells for the above vector include multicellular fungi such as Aspergillus spp. and Neurospora crassa belonging to the Phylum Ascomycota, unicellular fungi including yeasts such as Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces, other lower eukaryotic cells, higher eukaryotic cells such as insect-derived cells, and cells derived from plants or mammals.

[0184] In the present invention, the term "transfection" refers to introducing a desired gene into a host cell using the recombinant vector of the present invention, and is used with the same meaning as "transformation." Therefore, "transfection" and / or "transformation" into a host cell includes any method for introducing a nucleic acid into an organism, cell, tissue, or organ, and can be performed by selecting an appropriate standard technique depending on the host cell as is known in the art. Such methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, stirring using silicon carbide fibers, Agrobacterium-mediated transformation, PEG, dextran sulfate, lipofectamine, and desiccation / inhibition-mediated transformation methods.

[0185] A third aspect of the present invention relates to a method for producing a bispecific antibody that specifically binds to c-Kit and VEGF, comprising the following steps (a) and (b): (a) culturing the transformant described above; and (b) recovering the bispecific antibody that specifically binds to c-Kit and VEGF from the culture.

[0186] Transformants for antibody production can be cultured using appropriate media and culture conditions known in the art. These culture processes can be easily adjusted by those skilled in the art for the selected strain. Cell culture can be categorized into suspension and adherent cultures based on cell growth patterns, and batch, fed-batch, and continuous culture methods based on culture method. The culture medium used must adequately meet the requirements of the specific strain.

[0187] The medium used for culturing animal cells contains various carbon sources, nitrogen sources, and trace element components. Examples of carbon sources that can be used include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These carbon sources can be used alone or in combination. Examples of nitrogen sources that can be used include organic nitrogen sources such as peptone, yeast extract, meat juice, malt extract, corn steep liquor (CSL), and soybean meal; and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. These nitrogen sources can be used alone or in combination. The above-mentioned medium may contain, as personnel, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the corresponding sodium-containing salt. It may also contain a metal salt such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, and suitable precursors may be included.

[0188] During cultivation, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be appropriately added to the culture to adjust the pH of the culture. Furthermore, foaming can be suppressed during cultivation using antifoaming agents such as fatty acid polyglycol esters. Furthermore, oxygen or an oxygen-containing gas (e.g., air) is injected into the culture to maintain an aerobic state. The culture temperature is usually between 20°C and 45°C, preferably between 25°C and 40°C.

[0189] Antibodies obtained by culturing transformants can be used in an unpurified state, or can be further purified to high purity using various conventional methods, such as dialysis, salt precipitation, and chromatography. Among these, the method using chromatography is the most commonly used, and the type and order of columns can be selected from ion exchange chromatography, size exclusion chromatography, and affinity chromatography, depending on the characteristics of the antibody, the culture method, etc.

[0190] The fourth aspect of the present invention relates to a pharmaceutical composition for preventing or treating angiogenic diseases using the aforementioned bispecific antibody.

[0191] Specifically, the present invention provides a c-Kit and VEGF bispecific antibody according to the first aspect for use in preventing or treating angiogenic diseases and a pharmaceutical composition for preventing or treating angiogenic diseases comprising the same as an active ingredient.

[0192] In addition, the present invention provides a method for preventing or treating angiogenic diseases, comprising administering a therapeutically effective amount of a c-Kit and VEGF bispecific antibody according to the first aspect to a subject in need thereof.

[0193] Furthermore, the present invention provides the use of a c-Kit and VEGF bispecific antibody according to the first aspect for the manufacture of a medicament for the prevention or treatment of angiogenic diseases.

[0194] The above angiogenic disease refers to a disease related to the occurrence or progression of angiogenesis. Any disease that can be treated with the bispecific antibody according to the present invention may be included in the scope of angiogenic diseases. For example, the above angiogenic disease may be selected from the group consisting of cancer, leukemia, ocular vascular disease, rheumatoid arthritis, psoriasis, chronic wounds, chronic inflammation, hemangioma, angiofibroma, vascular malformation, arteriosclerosis, vascular adhesions, vasculitis, pyogenic granuloma, bullous disease, pulmonary hypertension, asthma, nasal polyps, infectious diseases, inflammatory bowel disease, periodontal disease, peritoneal adhesions, endometrium, uterine bleeding, ovarian cysts, osteomyelitis, osteosarcoma, sepsis, and autoimmune diseases, but is not limited thereto.

[0195] In the present invention, the cancer may be selected from the group consisting of bone cancer, lung cancer, head cancer, cervical cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, stomach cancer, liver cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, rectal cancer, anal cancer, colon cancer, uterine cancer, breast cancer, ovarian cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, central nervous system lymphoma, spinal cord tumor, glioblastoma, brainstem glioma, and pituitary adenoma, but is not limited thereto.

[0196] In the present invention, the ocular vascular disease may be selected from the group consisting of diabetic retinopathy, macular degeneration, age-related macular degeneration, glaucoma, glaucomatous retinitis pigmentosa, choroidal neovascularization, retinopathy of prematurity, corneal dystrophy, and retinal detachment, but is not limited thereto.

[0197] The pharmaceutical composition according to the present invention may contain the bispecific antibody alone, or may additionally contain one or more pharmaceutically acceptable carriers, excipients or diluents.

[0198] The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. In addition, carriers for parenteral administration may include water, suitable oils, saline solution, aqueous glucose, glycol, etc., and may further include stabilizers and preservatives. Examples of stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Examples of preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Other pharmaceutically acceptable carriers known in the art may be used (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0199] The pharmaceutical composition of the present invention can be administered to mammals, including humans, by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration. For example, the pharmaceutical composition of the present invention can be prepared in an injectable formulation and administered by lightly pricking the skin with a 30-gauge thin injection needle, or by directly applying it to the skin.

[0200] The pharmaceutical composition of the present invention can be formulated into a preparation for oral administration or parenteral administration according to the administration route as described above. In the case of a preparation for oral administration, the composition of the present invention can be formulated into powder, granules, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc. using methods known in the art. For example, an oral preparation can be obtained by mixing an active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and then processing it into a granular mixture to obtain a tablet or sugar-coated tablet. Examples of excipients may include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; starches including corn starch, wheat starch, rice starch, and potato starch; cellulosics including cellulose, methyl cellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; and fillers such as gelatin and polyvinylpyrrolidone. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as disintegrants, if desired. In addition, the pharmaceutical composition of the present invention may further include anticoagulants, lubricants, wetting agents, flavorings, emulsifiers, and preservatives. For parenteral administration, preparations can be formulated into injections, creams, lotions, topical ointments, oils, moisturizers, gels, aerosols, and nasal inhalants using methods known in the art. These formulations are described in a generally well-known reference in pharmaceutical chemistry (Remington's Pharmaceutical Science, 15th Edition, 1975 Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour).

[0201] The total effective amount of the pharmaceutical 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 pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease symptoms. For example, the daily dosage of the pharmaceutical composition of the present invention may be 0.0001 to 100 mg / kg. However, the dosage of the pharmaceutical composition of the present invention takes into account various factors such as the administration route and number of treatments as well as the patient's age, weight, health status, sex, severity of the disease, diet, and excretion rate, and a person having ordinary skill in the art will be able to determine an appropriate effective dosage for the patient. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, and administration method as long as it exhibits the effects of the present invention.

[0202] Additionally, the pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent. When administered in combination with another therapeutic agent, the composition of the present invention and the other therapeutic agent may be administered simultaneously, separately, or sequentially. The other therapeutic agent may be a substance already known to have a therapeutic or ameliorating effect on angiogenic diseases, and includes all anticancer therapies other than drug therapy, such as radiation therapy.

[0203] When the pharmaceutical composition of the present invention is administered in combination with another therapeutic agent, the bispecific antibody and the other therapeutic agent contained in the composition of the present invention may be formulated separately in separate containers or formulated together in the same container.

[0204] In the present invention, the term "subject" includes, but is not limited to, any animal (e.g., human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig, or rodent). This term does not indicate a specific age or gender. Therefore, it is intended to include female / female or male / male, adult / adult and newborn subjects, as well as fetuses. A patient refers to a subject suffering from a disease or disorder. The term patient includes human and veterinary subjects.

[0205] The fifth aspect of the present invention relates to a kit for detecting c-Kit and / or VEGF comprising the above-described bispecific antibody and a method for detecting c-Kit and / or VEGF using the above-described bispecific antibody.

[0206] The c-Kit and / or VEGF detection kit according to the present invention can be applied to biological samples for the purpose of diagnosing angiogenic diseases. Accordingly, the present invention provides a kit for diagnosing angiogenic diseases comprising the aforementioned bispecific antibody and a method for diagnosing angiogenic diseases using the aforementioned bispecific antibody.

[0207] The kit of the present invention can be manufactured to be suitable for various immunoassays or immunostaining. The immunoassays or immunostaining include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, Western blotting, immunohistochemistry staining, flow cytometry, immunocytochemistry, radioimmunoassay (RIA), immunoprecipitation assay, radioimmunoassay (RIA), and protein chips.

[0208] The kit of the present invention may further comprise, in addition to the bispecific antibody for c-Kit and VEGF, tools or reagents known in the art for use in immunological analysis.

[0209] Tools or reagents used in immunological analysis may include suitable carriers or supports, labels capable of generating detectable signals, solubilizers, detergents, and stabilizers. Suitable carriers may also include, but are not limited to, a substrate capable of measuring enzyme activity when the label is an enzyme, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or fluorescent substance, a chromogenic substrate, and a reaction stopper.

[0210] The bispecific antibodies against c-Kit and VEGF included in the kit of the present invention can preferably be immobilized on a suitable carrier or support using various methods as disclosed in the literature, and examples of suitable carriers or supports include PBS, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, agarose, cellulose, nitrocellulose, dextran, sephadex, sepharose, liposome, carboxymethyl cellulose, polyacrylamide, polyesterine, gabbro, filter paper, ion exchange resin, plastic film, plastic tube, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, metal, glass, glass beads, or magnetic particles. Other solid substrates include cell culture plates, ELISA plates, tubes, and polymeric membranes. The support may have any possible shape, for example spherical (bead), cylindrical (inside a test tube or well), planar (sheet, test strip).

[0211] Labels capable of generating a detectable signal enable qualitative or quantitative measurement of the formation of antigen-antibody complexes, and examples of such labels include enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioactive isotopes. Enzymes that can be used include β-glucuronidase, β-D-glucosidase, urease, peroxidase (such as horseradish peroxidase), alkaline phosphatase, acetylcholinesterase, glycose oxidase, hexokinase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, invertase, and luciferase. Fluorescent substances that can be used include fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, and fluorsine isothiocyanate. Ligands include biotin derivatives, and luminescent substances include acridinium esters and luciferin. Microparticles include colloidal gold and colored latex, and redox molecules include ferrocene, ruthenium complexes, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, and hydroquinone. Radioisotopes include 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, 186 Re, etc. However, in addition to those exemplified above, any that can be used in immunological analysis can be used.

[0212] As an enzyme chromogenic substrate, for example, when horseradish peroxidase (HRP) is selected as an enzyme label, a solution containing 3-amino-9-ethylcarbazole, 5-aminosalicylic acid, 4-chloro-1-naphthol, o-phenylenediamine, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), 3,3-diaminobenzidine, 3,3',5,5'-tetramethylbenzidine, o-dianisidine, or 3,3-dimethoxybenzidine can be used as a substrate. In addition, when alkaline phosphatase is selected as an enzyme label, a solution containing 5-bromo-4-chloro-3-indolyl phosphate, nitroblue tetrazolium, or p-nitrophenyl phosphate can be used as a substrate. Additionally, when β-D-galactosidase is selected as an enzyme marker, a solution containing o-nitrophenyl-β-D-galactoside or 5-bromo-4-chloro-3-indole-β-D-galactopyranoside can be used as a substrate. In addition, various enzymes and enzyme chromogenic substrates known in the art can be used.

[0213] A method for detecting c-Kit and / or VEGF according to one embodiment of the present invention may detect c-Kit and / or VEGF proteins in a biological sample or a mammal, including a human. Specifically, the method for detecting c-Kit and / or VEGF according to the present invention may include a step of detecting a c-Kit and / or VEGF antigen-antibody complex using the above-described bispecific antibody.

[0214] In the present invention, the sample is a biological sample, and may include, but is not limited to, tissue, cell, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph node, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic cells, and bacterial expression systems. For example, the biological sample may be isolated from a mammal, including a human, having a disease in which c-Kit and / or VEGF is overexpressed or a disease related to c-Kit and / or VEGF, or may be isolated from an animal model that overexpresses c-Kit and / or VEGF protein or an animal model of a disease related to c-Kit and / or VEGF, but is not limited thereto. These biological samples may be reacted with the bispecific antibody of the present invention, with or without manipulation, to confirm the presence or absence of c-Kit and VEGF proteins.

[0215] The mammals include, but are not limited to, livestock (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans, non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0216] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0217] [Manufacturing Example 1]

[0218] Development of a bispecific candidate antibody that specifically binds to c-KIT and VEGF

[0219] 1-1. Production of a vector expressing anti-c-Kit antibodies

[0220] Based on the antibody sequence identified in Patent Document 1, a leader peptide sequence for secretion outside of cells was added and gene synthesis (commissioned by Bioneer) was performed. The synthesized antibody gene was cloned into the PhiC31 vector (SBI, FC600A-1-SBI). For cloning, the synthesized antibody gene and PhiC31 vector were each mixed with restriction enzymes NotI (New England Biolabs, R3189S) and EcoRV (New England Biolabs, R3195S) and 10X buffer (10X Cutsmart Buffer) to make a total volume of 50 ㎕, and the mixture was reacted in a 37℃ incubator for 5 hours. After spreading on a 1% agarose gel and confirming that a band was generated at the expected size, the band was cut and separated using a purification kit (MN, Macherey-Nagel). The two separated products were mixed with T4 ligase (New England Biolabs, M0202S) and 10X buffer (10X T4 DNA Ligase Buffer) in 20 ㎕ reaction mixture and reacted at 25℃ for 2 hours. The ligated vector was transformed into E. coli DH5a competent cells (RBC, RH718), spread on kanamycin LB agar plates, and reacted at 37℃ for 16 hours. After obtaining a single colony, the colony was cultured and DNA was obtained using a Mini-prep (Qiagen, 27106) kit. The obtained DNA was digested with the same restriction enzyme, and the insertion of the gene into the vector was confirmed by the size of the digested band through electrophoresis, and then sequencing (commissioned by Bioneer) was used for final analysis, and a vector expressing the anti-c-Kit antibody was obtained.

[0221] 1-2. Cloning and expression of a bispecific candidate antibody that specifically binds to c-Kit and VEGF.

[0222] Based on the vector expressing the anti-c-Kit antibody manufactured above, cloning was performed to link the heavy chain of the antibody, peptide linker G5 (SEQ ID NO: 20), and VEGFR sequence using gene synthesis (commissioned by Bioneer). For cloning, first, to remove the constant region of the existing vector, restriction enzymes NheI (New England Biolabs, R3131S) and EcoRV (New England Biolabs, R3195S) were mixed with 10X buffer (10X Cutsmart Buffer) to make a total reaction volume of 50 ㎕, and the reaction was performed in an incubator at 37℃ for 5 hours. The cut band was confirmed through electrophoresis on a 1% agarose gel, and the cut vector was isolated using a purification kit (MN, Macherey-Nagel). In the same way, the antibody heavy chain, linker, and VEGFR genes synthesized using gene synthesis were also digested with restriction enzymes NheI and EcoRV, isolated, and purified to secure the inserts. To ligate the cleaved vector and the inserts, T4 ligase (New England Biolabs, M0202S) and 10X buffer (10X T4 DNA Ligase Buffer) were added to each gene to make a total of 20 ㎕, and the mixture was incubated at 25°C for 2 hours. The ligated vector was transformed into E. coli DH5a competent cells (RBC, RH718), and the cells were plated on kanamycin LB agar plates and incubated at 37°C for 16 hours to confirm the formation of single colonies. DNA was secured by performing Mini-prep (Qiagen, 27106) using single colonies. The secured DNA was cut with a restriction enzyme, and the gene was inserted into the vector by confirming the size of the cut band through electrophoresis. Afterwards, it was verified again using sequencing (commissioned by Bioneer).

[0223] The vector prepared above was cultured in 3 ml LB medium supplemented with antibiotics for 8 hours, then increased in amount to 250 ml LB medium supplemented with the same antibiotics and cultured for 16 hours. Plasmid DNA was extracted using a Maxi-prep (MN, Macherey-Nagel, 740414.50) kit, filtered through a 0.22 μm PES filter, and then introduced into cells as follows.

[0224] The day before transfection, ExpiCHO (Gibco) cells were cultured in ExpiCHO Expression Medium (Gibco, A2910002) at a concentration of 3 x 10E6 to 4 x 10E6 viable cells / mL for 1 day at 8% CO2, 37°C, and 120 rpm. On the day of DNA transfection, 7 x 10E6 to 10 x 10 6 Viable cells / mL, viability is 6 x 10 cells grown to 95% or more using fresh medium. 6 It was prepared by diluting to viable cells / mL. For transfection of prepared ExpiCHO cells, ExpiFectamine CHO transfection kit (Gibco, A29129) and Optipro SFM (Gibco, 12309019) were used, and DNA transfection was performed at a concentration of 1 μg / mL. After transfection, supplements were added on the 1st day and the culture conditions were changed to 5% CO2, 32°C, 120 rpm, and then supplements were added again on the 5th day and cultured for 10 to 12 days to complete production.

[0225] To secure the culture solution after production was completed, the culture solution was transferred to a centrifugation-only container, centrifuged at 4℃, 8000 rpm for 30 minutes, the supernatant was mixed with diatomaceous earth, and filtered through a 0.2 um PES filter to secure the culture solution excluding floating matter, and the subsequent purification process was performed.

[0226] Purification was performed using affinity chromatography (Protein A) resin, and the column was equilibrated with equilibration buffer (1X PBS). The recovered culture medium was injected into the column. After injection, the column was washed using equilibration buffer, and an intermediate wash was performed using 20 mM sodium citrate, 5% sorbitol, pH 5.0 buffer. After washing, the column was eluted using 20 mM sodium citrate, 5% sorbitol, pH 3.5 buffer. The eluate was neutralized to pH 7.0 by adding 1 M Tris-HCl, pH 9.0.

[0227] If further purification was required, it was purified using ion exchange chromatography resin. After equilibration using equilibration buffer [20 mM potassium phosphate, pH 7.0], the neutralized eluate was loaded onto the column. After loading, it was washed using equilibration buffer and eluted using 20 mM potassium phosphate, pH 8.0 buffer. The eluate was then buffer exchanged with 20 mM L-histidine, 9% sucrose, pH 6.0, and concentrated. The purity was analyzed (SEC-HPLC) and endotoxin assays were performed to confirm its suitability for subsequent activity analysis. The finally confirmed antibody was stored at -80℃ until use.

[0228] The resulting bispecific antibody is in the form of a VEGFR linked to the heavy chain C-terminus of an anti-c-Kit IgG antibody via a G5 peptide linker. The structure of the produced bispecific antibody is shown in Figure 1a and was designated "candidate antibody A." The amino acid sequence and the nucleotide sequence encoding candidate antibody A are shown in Tables 2 and 3, respectively.

[0229]

[0230]

[0231] [Example 1]

[0232] FC Engineering

[0233] 1-1. Introduction of various Fc mutations

[0234] The peptide linker connecting the anti-c-Kit antibody and VEGFR was replaced with a G8 peptide linker in the above manufacturing example, and mutations were introduced into the Fc to reduce Fc receptor binding and / or effector function. The types of Fc mutations that reduce the binding affinity for FcγRIIIa (CD16a) to less than 10% are shown in Table 4. Bispecific antibodies were produced by inserting the above four types of Fc mutations into the Fc region, and for comparison, bispecific antibodies having a G8 peptide linker and a wild-type Fc were also produced. The method for producing these bispecific antibodies is the same as in Manufacturing Example 1.

[0235] Fc mutation type LALAP GALAGAL A LEL D FcγIII binding affinity 3% 8% 8% 1.5% compared to wild type

[0236] 1-2. Confirmation of binding affinity for Fc receptors

[0237] SPR was performed on the five types of bispecific antibodies produced (i.e., bispecific antibodies having a G8 peptide linker and a wild-type Fc, a LALAPG Fc variant, a LAGA Fc variant, a LALE Fc variant, or a LD Fc variant) as follows to select the Fc variant most optimized for reducing immune effector function.

[0238] SPR was performed to determine the binding affinity of the five bispecific antibodies in Table 8 to the Fc receptor. A running buffer was prepared by diluting 20X HBS-EP+ buffer to 1X with deionized water, and a regeneration solution (5 mM NaOH) was prepared by diluting 10 N NaOH to 5 mM with deionized water. Immobilization and binding assays were performed at 25°C using 1X HBS-EP+ buffer as the running buffer. The ligand and analyte were set as Fc receptor and antibody, respectively.

[0239] Amine coupling was used as a ligand immobilization method. The sensor chip surface was activated by injecting an EDC / NHS mixture into the sample and reference flow cells for 420 s. A diluted Fc receptor protein in 10 mM sodium acetate solution was injected only into the sample flow cell, allowing covalent binding of the protein to the sensor chip surface. The reference flow cell served as a control to compensate for nonspecific signals, and the protein immobilization process was omitted. Once an appropriate level of immobilization was achieved, ethanolamine was injected into the sample and reference flow cells for 420 s to inactivate any remaining active sites.

[0240] The general settings and running configuration are shown in Table 5.

[0241] Running configuration in human c-Kit assay Sensor chip Series S CM5 Immobilization method Covalent immobilization using amine coupling Activation EDC / NHS mixture, 420 s Deactivation Ethanolamine, 420 s Immobilization solution 10 mM sodium acetate, pH 5.5 Flow rate 10 uL / min Concentration 1 ug / mL Running buffer HBS-EP+

[0242] A binding analysis was performed to collect data to be used for affinity analysis. The binding and dissociation signals were measured in real time while various concentrations of analyte were flowed through the sample and reference flow cells. The injection order was from lowest to highest concentration, and each concentration was injected as an independent cycle. One cycle consists of a baseline period in which only the running buffer was flowed before the analyte injection, a binding period in which the analyte was injected and bound to the ligand, a dissociation period in which the analyte injection was stopped, only the running buffer flowed, and the ligand-analyte complex dissociated, and a regeneration period in which a regeneration solution was injected to return the ligand to its original state.

[0243] The analyte concentrations and running configurations for each ligand are shown in Tables 6 and 7.

[0244] Analyte ConcentrationLigandAnalyte Concentration RangeHuman FcγRI1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200 nMHuman FcγRIIa (167 His)15.625, 31.25, 62.5, 125, 250, 500, 1000, 2000 nMHuman FcγRIIIa (176 Val)15.625, 31.25, 62.5, 125, 250, 500, 1000, 2000 nMHuman FcγRIIIb (NA1)62.5, 125, 250, 500, 1000, 2000 nMHuman FcγRIIIb (NA2)62.5, 125, 250, 500, 1000, 2000 nMHuman FcRn (at pH 6.0)62.5, 125, 250, 500, 1000, 2000 nMHuman FcRn (at pH 7.4)62.5, 125, 250, 500, 1000, 2000 nM

[0245] Running configuration: Binding time 120 s, Dissociation time 120 s, 300 s for FcγRI analysis, Flow rate 30 uL / min, Regeneration solution and injection time 5 mM NaOH, 10 s

[0246] Biacore Insight Evaluation Software was used to evaluate the collected data.

[0247] The steady-state affinity method was used to calculate the binding affinity (KD). In the case of FcγRI, the steady-state affinity method is not suitable, so the binding rate constant ( ) and dissociation rate constant ( ) was calculated, and the binding affinity (KD) was calculated as the ratio of the dissociation rate and the association rate. If the binding reaction signal was too low or non-specific binding occurred, the binding affinity (KD) could not be accurately determined, so only the binding was determined.

[0248] As confirmed in Table 8, when LALAPG, LAGA, and LALE Fc mutations were introduced, the binding affinity between the Fc fragment expressed on immune cells and the Fcγ receptor was lowered. However, contrary to what was expected in Table 4, when the LD Fc mutation was introduced, the binding affinity to FcγIII was increased compared to the wild-type Fc, indicating that it is not a suitable Fc mutant.

[0249] Wild-type LALAPGLAGALALELDKD(M)FcγR1a1.48x10 -8 NBNBNBNBFcγR2a_167H1.26x10 -6 (NSB)NBNBNB5.78x10 -6 (NSB)FcγR3a_167V4.18x10 -7 NBNBNB2.36x10 -6 (NSB)FcγR2bNBNBNBNBNBBFcγR3b_NA17.18x10 -6 NBNBNB1.09x10 -1 (NSB)FcγR3b_NA25.44x10 -6 NBNBNB1.58x10 -2 (NSB)FcRn(pH 6.0)2.06x10 -6 9.21x10 -7 1.21x10-6 1.09x10 -6 8.11x10 -7 FcRn(pH 7.4)1.56x10 -2 (Weak) 2.45x10 -2 (Weak)1.28x10 -5 4.27x10 -4 1.38x10 -2 (weakness)

[0250] Next, antibody-dependent cellular cytotoxicity (ADCC) was tested in peripheral blood mononuclear cells, which contain immune cells in human blood, to confirm whether the mutations did not actually induce immune effectors.

[0251] To measure antibody-dependent cellular cytotoxicity (ADCC), experiments were conducted using 96-well cell culture plates. GIST-430 / 654 cells, the target cells, were seeded at 6 x 10 per well. 3 , were inoculated in a final volume of 100 μl and cultured overnight. Before starting the ADCC assay, the supernatant was removed and 1.5 X 10 human peripheral blood mononuclear cells (hPBMC; Lonza_CC-2704), which are effector cells, were seeded per well. 5, and added in a total volume of 50 μl to adjust the effector:target ratio to 25:1. Each bispecific antibody was diluted with PBS to 20 μg / ml (2x the treatment concentration) and serially diluted to 10-fold concentrations in a dilution plate. 50 μl of antibody was transferred to the cell culture plate containing target and effector cells and incubated at 37°C for 6 hours. After 6 hours of incubation, 100 μl of HiBiT extracellular reagent (Promega, N2421) was added to each well and incubated at room temperature for approximately 10 minutes. After that, luminescence was measured using the GloMax® Navigator system. The measured results were graphed and compared with the negative control (untreated group).

[0252] As shown in Fig. 2, cytotoxicity was confirmed to occur in the group treated with the wild-type Fc, while no cytotoxicity was observed in the groups treated with the mutants LALAPG, LAGA, and LALE, respectively. On the other hand, although cytotoxicity was reduced compared to the wild-type, the LD Fc mutants were still confirmed to have toxicity. Trastuzumab was used as a positive control. Therefore, it was confirmed that the LALAPG, LAGA, and LALE Fc mutants significantly reduced ADCC compared to the wild-type Fc, and these three Fc mutants were the optimal mutants.

[0253] According to the above results, the bispecific antibodies linked by the G8 peptide linker and each introducing LALAPG and LALE Fc mutations were named 'candidate antibody B' (LALAPG) and 'candidate antibody C' (LALE), respectively, and the structure and sequence information of these bispecific antibodies are shown in Figures 3a to 3b and Tables 9 to 12, respectively.

[0254]

[0255]

[0256]

[0257]

[0258] 1-3. Additional introduction of I253A, H310A, and H435A mutations

[0259] A bispecific antibody was constructed by introducing additional mutations I253A, H310A, and H435A into the above candidate antibody B, and this was designated 'candidate antibody B-1'. The binding affinity of candidate antibody B-1 to human FcRn (neonatal Fc receptor) was evaluated by SPR analysis. As confirmed in Table 13, no significant interaction between antibody B-1 and human FcRn was observed at pH 6.0, and no KD value was measured at pH 7.4.

[0260] Binding AffinityLigandpHAnalyteKD (M)Rmax (RU)Human FCGRT & B2M heterodimer protein6.0Antibody B8.66E-0789.9Antibody B-1N.D*Anti-c-Kit antibody1.99E-0646.17.4Antibody BN.D*Antibody B-1N.D*Anti-c-Kit antibodyND*

[0261] ND*: Not measured in low response units (RU).

[0262] Since the absence of binding to FcRn results in a short half-life in serum, the results of this example suggest that additional introduction of the I253A, H310A, and H435A mutations is expected to reduce systemic side effects that may be induced by simultaneously targeting c-Kit and VEGF in the blood. The structure and sequence information of candidate antibody B-1 are shown in Fig. 3c and Tables 14 and 15, respectively.

[0263]

[0264]

[0265] [Example 2]

[0266] Production of various types of bispecific antibodies according to the binding site of VEGFR

[0267] To produce a bispecific antibody in which VEGFR is linked to the C-terminus of the light chain of a c-Kit antibody, gene synthesis (commissioned by Bioneer) was performed based on the vector expressing the c-Kit antibody produced in Manufacturing Example 1-1 to clone the light chain of the antibody, the linker, and the VEGFR sequence. Thereafter, the cloning and expression of the antibody were performed in the same manner as in Manufacturing Example 1-2, and the purification process was performed. The structure of the obtained antibody is shown in Fig. 4a, and it was named 'candidate antibody D'. The sequence information of candidate antibody D is identical to that of candidate antibody A.

[0268] Next, in order to produce a bispecific antibody in which VEGFR is linked to the heavy chain N-terminus of a c-Kit antibody via a G8 peptide linker and a LALAPG mutation is introduced into the Fc, cloning was performed using gene synthesis (commissioned by Bioneer) based on the vector expressing the c-Kit antibody produced in Manufacturing Example 1-1 to link the heavy chain of the antibody containing the mutation, the linker, and the VEGFR sequence. Thereafter, the cloning and expression of the antibody were performed in the same manner as in Manufacturing Example 1-2, and the purification process was performed. The structure of the obtained antibody is shown in Fig. 4b, and it was named 'candidate antibody E'. The sequence information of candidate antibody E is the same as that of candidate antibody B.

[0269] Information on the six candidate antibodies produced through the above results is shown in Table 16.

[0270] Candidate Antibody A Candidate Antibody B Candidate Antibody B-1 Candidate Antibody C Candidate Antibody D Candidate Antibody E Molecular Form of Bispecific Antibody IgG(H)-VEGFRI gG(H)-VEGFRI gG(H)-VEGFRI gG(H)-VEGFR VEGFR- IgG(L) VEGFR- IgG(H) Binding Valence 2+22+22+22+22+22+2 Fc Engineering Wild Type LALAPG LALAPG-AAALALE Wild Type LALAPG Linker Type G5G8G8G8G5G8

[0271] [Example 3]

[0272] Confirmation of binding ability to c-Kit and VEGF

[0273] To confirm the target binding ability of the six bispecific antibodies in Table 16 above, ELISA was performed. The amino acid sequence information of the c-Kit antigen (Sino Biological, 11996-H08H) and VEGF antigen (Sino Biological, 11066-HNAH) used in the experiment are as shown in SEQ ID NOs: 71 and 72, respectively.

[0274]

[0275]

[0276] To coat the assay plate with c-Kit / VEGF working solution, 100 μl of working solution (0.5 μg / mL) was added per well, the plate was sealed with film, and incubated overnight at 2–8°C. A 5% BSA solution diluted in PBS was prepared as a blocking solution and filtered through a 0.22 μm filter. The coating solution was removed, and the plate was washed three times with 300 μl / well of 0.05% PBST buffer. After that, 300 μl of blocking solution was added per well of the assay plate. The plate was covered with a plate sealer and incubated at 25 ± 2°C for 1 h without shaking.

[0277] Each antibody was diluted to 1 μg / ml in DPBS and prepared as a two-fold serial dilution in a dilution plate. After blocking, the solution was removed from the assay plate, and the plate was washed three times with 300 μl / well of 0.05% PBST buffer. Using a multichannel pipette, 100 μl of antibody was transferred from the dilution plate to the assay plate, the plate was sealed with film, and incubated for 1 hour at 25 ± 2°C without shaking. After 1 hour, the plate was washed three times with 300 μl / well of 0.05% PBST buffer, and 100 μl of the diluted secondary antibody solution (80 ng / mL; final secondary antibody) was added to each well using a multichannel pipette. The plate was sealed with film and incubated for 1 hour at 25 ± 2°C without shaking. After incubation with the secondary antibody, the solution was removed from the assay plate. The plate was washed three times with 300 μl / well of 0.05% PBST buffer, and 100 μl of TMB substrate reagent solution was added to each well using a multichannel pipette. The plate was covered with a film and incubated at 25 ± 2°C for 5 minutes. The plate was read within 10 minutes after adding 100 μl of 1 N sulfuric acid to the wells. The plate was read using a SPECTROstar NaNO at an absorbance of 450 nm.

[0278] As confirmed in Figures 5a and 5b, all six antibodies bound to c-Kit and VEGF in a concentration-dependent manner, and showed excellent binding ability to c-Kit and VEGF, respectively.

[0279] [Example 4]

[0280] Confirmation of binding affinity for c-Kit and VEGF

[0281] SPR was performed to confirm the binding affinity of the six bispecific antibodies in Table 9 above to c-Kit and VEGF. A running buffer was prepared by diluting 20X HBS-EP+ buffer to 1X with deionized water, and a regeneration solution (10 mM NaOH) was prepared by diluting 10 N NaOH to 10 mM with deionized water. Immobilization and binding assays were performed at 25°C using 1X HBS-EP+ buffer as the running buffer.

[0282] For human c-Kit analysis, c-Kit protein was directly immobilized on the sensor chip surface via primary amine coupling. The sensor chip surfaces of the sample and reference flow cells were activated with a freshly mixed EDC / NHS mixture for 420 s. Subsequently, c-Kit protein was diluted in a 10 mM sodium acetate solution and injected into the sample flow cell to achieve an appropriate level of immobilization, while the reference flow cell was set as a blank. After the amine coupling reaction, the remaining active coupling sites were blocked with ethanolamine for 420 s.

[0283] For human VEGF165 analysis, the capture molecule, protein A, was directly immobilized on the sensor chip surface via primary amine coupling. The amine coupling process was identical to the c-Kit assay, except that protein A was immobilized in both channels. Diluted antibodies or Eylea were then injected into the sample flow cell only, and captured by pre-immobilized protein A using affinity for the Fc region.

[0284] General settings and running configurations are shown in Tables 17 to 19.

[0285] General settings: Sensor chip Series S CM5, Running buffer HBS-EP, Flow rate 10 uL / min

[0286] Running configuration in human c-Kit assay. Ligand. Human c-Kit. Attachment method. Covalent immobilization using amine coupling. Flow cell used. Fc 2 (Fc 1 is the reference cell). Fixation solution. 10 mM sodium acetate, pH 5.5. Concentration. 2 ug / mL. Activation. EDC / NHS mixture, for 420 s. Inactivation. Ethanolamine, for 420 s.

[0287] Running configuration in human VEGF165 assay Type of capture molecule Ligand name Protein A Antibody Attachment method Covalent immobilization using amine coupling Capture using affinity for protein A Flow cell used Fc 1, 2 Fc 2 Immobilization solution 10 mM sodium acetate, pH 4.5 N / A Concentration 30 ug / mL c-Kit antibody: 4 nM Eylea: 6 nM Activation EDC / NHS mixture, for 420 sec N / A Deactivation Ethanolamine, for 420 sec N / A

[0288] Binding assays were performed at a flow rate of 30 μL / min. All analytes were serially diluted in running buffer, and all samples were prepared on ice. Analyte samples were injected during the binding step, followed by running buffer for separation. The sensor chip surface was regenerated after each cycle by injecting a regeneration solution. For the c-Kit assay, 10 mM NaOH solution was used as the regeneration solution, and for the VEGF assay, 10 mM glycine, pH 1.5 solution was used. Analyte concentrations and running configurations are shown in Tables 20 and 21.

[0289] Analyte ConcentrationLigandAnalyteAnalyte Concentration RangeHuman c-Kit Antibody0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 nMHuman VEGF165 Antibody0.020, 0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 nM

[0290] Running configuration Assay type Multicycle reaction rate Starting cycles 2 times, using running buffer Blank cycles 2 cycles per analyte, concentration series Post-binding time c-Kit assay: 240 s, VEGF165 assay: 120 s Dissociation time c-Kit assay: 480 s, VEGF165 assay: 240 s Flow rate 30 uL / min

[0291] Evaluations were performed using Biacore Insight Evaluation Software. A 1:1 binding model was used to globally fit the sensorgram curves, and kinetic parameters (association rate constants [kA] and dissociation rate constants [kD]) were calculated. The KD value (equilibrium dissociation constant) was calculated as the ratio of kD to kA.

[0292] As confirmed in Figures 6a and 6b, all six antibodies showed excellent binding affinity to c-Kit and VEGF.

[0293] [Example 5]

[0294] Stability check

[0295] 5-1. Thermal stability

[0296] To evaluate the thermal stability of the six bispecific antibodies in Table 16 above, DSF analysis was performed. A PBS (1X) buffer solution to be used as a negative control diluted in PBS (20X) buffer was prepared, and the stock solution of each test sample was diluted to a concentration of 1 mg / mL using PBS (1X) buffer so that the total amount of test sample for each reaction was 5 ug. Protein Thermal Shift™ Dye (1000X) was freshly diluted to 8X using PBS (1X) buffer. The reaction components described in Table 22 below were added to each well of a MicroAmp® Optical 8-tube strip.

[0297] Reaction Ingredients Ingredient VolumeProtein Thermal Shift™ Buffer5.0 uL Diluted Protein Thermal Shift™ Dye (8X)2.5 uLDiluted Test Sample (1 mg / mL)5.0 uLLPBS (1X)7.5 uLTotal Volume for Each Reaction20 uL

[0298] The tubes were sealed with MicroAmp® Optical 8-Cap Strips. Samples were mixed using a vortex mixer and then centrifuged. Each reaction was prepared in quadruplicate. Fluorescence readings were monitored using a real-time PCR instrument, the StepOnePlus® Real-Time PCR System, and control software. Protein melting reactions were performed with experimental parameters set as described in Table 23 below.

[0299] Experimental Characteristics Field Item Experimental Characteristics Experiment Type Melting Curve Reagent Type Other Ramp Rate Fast Target Characteristics Reporter ROX Quencher None Plate Layout Manual Reference None How to Run Reaction Volume per Well 20 uL Ramp Mode Continuous Thermal Profile Step 1. Temperature: 25.0°C, Time: 2 min Step 2. Temperature: 99.0°C, Time: 2 min Ramp Rate Step 1. 100% Step 2. 1%

[0300] Melting temperature (Tm) analysis was performed using Protein Thermal Shift™ software. The analysis mode was set as shown below and in Table 24.

[0301] Analysis Mode Field Item Analysis Method Boltzmann Fitting ROA (Analysis Area) Automatic Automatic Analysis Options Single Tm

[0302] Results were statistically analyzed using one-way ANOVA and Tukey's multiple comparisons using GraphPad Prism 10 software.

[0303] Six types of antibodies were exposed to temperature changes from 25°C to 99°C, and the fluorescence signals generated as the protein unfolded were calculated and shown in Figure 7. As confirmed in Figure 7, all six types of antibodies produced exhibited excellent thermal stability.

[0304] 5-2. Protein stability after repeated freeze / thaw cycles

[0305] The six bispecific antibodies in Table 16 were frozen at -80°C for 16 hours and then thawed at 4°C for 2 hours. This cycle was repeated five times, making one cycle. The antibody stock solution was then diluted to 1 mg / mL using the formulation buffer, and SEC-HPLC analysis was performed under the conditions in Table 25. The results are shown in Tables 26 to 31.

[0306] Parameter Conditions Column TOSOH TSKgel Super SW3000, 4 μm, 4.6 mm x 30 cm Column and autosampler temperature 30 °C and 4 °C Mobile phase 50 mM potassium phosphate buffer, 150 mM potassium chloride; pH 6.8 Isocratic method Time % Mobile phase 0 1 0 Flow rate 0.35 mL / min Injection volume 10 μL Wavelength 280 nm / Bw: 4 nm Ref 360 nm / Bw: 100 nm 284 nm / Bw: 4 nm Ref 360 nm / Bw: 100 nm

[0307] Candidate Antibody A Cycle 1 RT Area (%) 7.17695.69 Monomer 7.7674.18 LMW 9.6200.13 Cycle 2 RT Area (%) 7.17496.02 Monomer 7.7333.86 LMW 9.6320.12 Cycle 3 RT Area (%) 7.17395.45 Monomer 7.7334.42 LMW 9.6210.13 Cycle 4 RT% Area 7.17095.60 Monomer 7.8174.27 LMW 9.6230.13

[0308] Candidate Antibody B Cycle 1 RT (min) Area (%) 6.5831.41HMW7.12897.55Monomer8.9260.76LMW9.5490.28Cycle 2 RT (min) Area (%) 6.5501.35HMW7.11097.71Monomer8.9260.69LMW9.5400.25Cycle 3 RT (min) Area (%) 6.5331.37HMW7.11897.70Monomer8.9410.69LMW9.5520.25Cycle 4 RT (min) Area (%) 6.5171.27HMW7.11397.82Monomer8.9450.67LMW9.5490.25

[0309] Candidate Antibody B-1 Cycle 1 RT (min) Area (%) 6.4970.20HMW7.22499.80Monomer Cycle 2 RT (min) Area (%) 6.4880.25HMW7.19999.75Monomer Cycle 3 RT (min) Area (%) 6.4930.27HMW7.20599.73Monomer Cycle 4 RT (min) Area (%) 6.4790.25HMW7.20299.75Monomer

[0310] Candidate Antibody C Monomer HMW LMW Cycle RT Area (%) RT Area (%) RT Area (%) 07.20699.846.5570.16N.DN.D16.94999.866.3090.14N.DN.D36.94699.836.2960.17N.DN.D56.95199.836.2900.17N.DN.D

[0311] Candidate Antibody D Cycle 0 RT (min) Area (%) 7.235 100 Monomer Cycle 1 RT (min) Area (%) 6.87 8 100 Monomer Cycle 3 RT (min) Area (%) 6.89 0 100 Monomer Cycle 5 RT (min) Area (%) 6.88 0 100 Monomer

[0312] Candidate Antibody E Cycle 0 RT (min) Area (%) 7.8491.44HMW8.55598.56Monomer Cycle 1 RT (min) Area (%) 7.3501.73HMW7.94898.27Monomer Cycle 3 RT (min) Area (%) 7.3501.79HMW7.94198.21Monomer Cycle 5 RT (min) Area (%) 7.3501.63HMW7.94398.37Monomer

[0313] All five antibodies, except candidate antibody A, were confirmed to be relatively stable even under rapid temperature changes, with monomers remaining at 97.5% or higher even after repeated freeze / thaw cycles.

[0314] [Example 6]

[0315] In vitro confirmation of bispecific antibody efficacy

[0316] 6-1. Vascular permeability

[0317] Human umbilical vein endothelial cells (HUVEC; Lonza_C2519A) cultured in EBM medium containing 0.5% FBS were pretreated with the indicated concentrations of each constructed bispecific antibody for 2 h. The cells were additionally exposed to 100 mM CoCl2 (hypoxia) or 100 ng / ml LPS overnight. Endothelial permeability was determined by measuring the passage of FITC-dextran in HUVEC monolayers. To measure fluorescein isothiocyanate-dextran permeability, excitation was performed at 480 nm and fluorescence was detected at 520 nm.

[0318] The vascular permeability of candidate antibodies B and C was compared with that of Eylea alone, anti-c-Kit antibody alone, or Faricimab-treated groups, and that of candidate antibodies C and E was compared with that of anti-c-Kit antibody alone or a comparator antibody-treated groups. The comparator antibody is a 2(1+1) antibody in the Fab-VEGFR-Fc(KH) format, which links anti-c-Kit IgG (knob) to Eylea (VEGFR trap (hole)) using knob-into-hole technology. The dexamethasone-treated group was used as a positive control.

[0319] Statistical significance was determined using an unpaired t test [***P<0.001, ****P<0.0001 vs. vehicle; #P<0.05, ###P<0.001, and ####P<0.0001 vs. 1% O2 or LPS; ns, not significant. (n=3)].

[0320] As confirmed in Fig. 8a, candidate antibody B showed a significantly superior vascular permeability inhibition effect compared to the combination treatment group of Eylea and anti-c-Kit antibody and the paricimab treatment group, and as confirmed in Fig. 8b, candidate antibodies C and E showed a significantly superior vascular permeability inhibition effect compared to the comparative antibody. In addition, as confirmed in Fig. 8c, candidate antibody C showed a significantly superior vascular permeability inhibition effect compared to the combination treatment group of Eylea and anti-c-Kit antibody, and showed a vascular permeability inhibition effect similar to the paricimab treatment group.

[0321] In summary, candidate antibodies B, C and E were observed to significantly inhibit the effect on paracellular permeability of HUVECs in vitro, suggesting that the bispecific candidate antibodies according to the present invention inhibit hypoxia- or LPS-induced venous endothelial barrier disruption.

[0322] 6-2. Signal transmission blocking

[0323] Human retinal microvascular endothelial cells (HRMEC; Cell Systems_ ACBRI-181) or human mast cell line LAD2 (a gift from Dr. Kirshenbaum; National Institutes of Health) were pretreated with each bispecific antibody for 30 min, and after treatment, the cells were stimulated with 100 ng / mL SCF for 5 min. Western blot was performed to analyze the expression levels of cell signaling-related proteins in whole cell lysates as follows.

[0324] Cells were lysed with RIPA buffer containing a cocktail of protease and phosphatase inhibitors. Proteins were quantified using the BCA protein assay kit. Western blot samples containing the appropriate amount of protein, NuPAGE LDS sample buffer containing reducing agent, and RIPA buffer were prepared and boiled at 70°C for 10 minutes. 30 μg of protein was loaded onto each well of an 8% SDS-PAGE gel. Gel electrophoresis was performed at 60 V for 30 minutes until the membrane passed through the separating gel. The voltage was then increased to 120 V for 1 hour. Transfer was performed at 320 mA for 120 minutes [Running buffer: 1X Tris-glycine buffer with SDS; Transfer buffer: 1X Tris-glycine buffer (without SDS) containing 20% ​​methanol]. The membrane was blocked with SuperBlock blocking buffer in TBST for 1 hour. The membranes were incubated overnight at 4°C with the following primary antibodies: pho-cKit (1:1000), pho-AKT (1:1000), pho-Erk (1:3000), cKit (1:1000), AKT (1:1000), Erk (1:3000), and β-actin (1:10000). Each primary antibody was diluted in SuperBlock blocking buffer dissolved in TBST. The membranes were washed with TBST for 10 minutes, repeated three times. The membranes were incubated for 1 hour at room temperature with the following secondary antibodies: mouse-anti-goat IgG-HRP (1:5000), mouse anti-rabbit IgG-HRP (1:5000), and goat anti-mouse IgG-HRP (1:5000). The above secondary antibodies were diluted in 5% skimmed milk dissolved in TBST and used. The membrane was washed with TBST for 10 minutes, repeated three times. The membrane was detected using ECL buffer.

[0325] The signal transduction inhibitory effects of candidate antibodies B and B-1 were compared with the anti-c-Kit antibody treatment group, and the signal transduction inhibitory effects of candidate antibodies C and E were compared with the comparative antibody treatment group. The comparative antibody was the same as that used in Example 6-1.

[0326] As confirmed in Figures 9a to 9d, candidate antibodies B, B-1, C, and E were observed to effectively inhibit SCF-dependent c-Kit signaling in HRMEC or LAD2.

[0327] 6-3. Cell cytokine analysis

[0328] HRMEC or ARPE-19 (ATCC: CRL-2302) cells cultured in medium containing 0.5% FBS were pretreated with each bispecific antibody for 6 h. The cells were further exposed to 1% O2 hypoxia or LPS for 12 h. Cell supernatants were harvested and subjected to quantitative ELISA for VEGF, Ang-2, IL-6, IL-8, and MCP-1.

[0329] The cytokine inhibitory effect of candidate antibody A was compared with the Ilia treatment group, the cytokine inhibitory effects of candidate antibodies B and C were compared with the Ilia treatment group, the anti-c-Kit antibody treatment group, and the faricimab treatment group (the dexamethasone treatment group was used as a positive control group), and the cytokine inhibitory effects of candidate antibodies C and E were compared with the comparative antibody treatment group. The comparative antibody was the same as that used in Example 6-1.

[0330] Statistical significance was determined using an unpaired t test (***P<0.001, ****P<0.0001 vs. vehicle; #P<0.05, ##P<0.005, ###P<0.001, and ####P<0.0001 vs. 1% O2 or LPS; ns, not significant).

[0331] As confirmed in FIGS. 10a to 10b, 11a to 11r and 12a to 12i, candidate antibodies A, B, C and E dose-dependently reduced the levels of cytokines induced by hypoxia or LPS in HRMEC and ARPE19 cells in vitro, and as confirmed in FIGS. 11a to 11r and 13a to 13i, candidate antibodies B and C showed significantly superior cytokine suppression effects compared to the combined treatment group of Ilia and anti-c-Kit antibodies and the Faricimab treatment group.

[0332] [Example 7]

[0333] In vivo confirmation of bispecific antibody efficacy

[0334] 7-1. Laser-induced choroidal neovascularization (LCNV) and intravitreal injection

[0335] General anesthesia was induced in C57BL / 6 mice via intraperitoneal injection of 10 mg / kg xylazine (Rompun®, Bayer Korea) and 100 mg / kg ketamine (Ketamine®, Yuhan). Local anesthesia was induced by applying an anesthetic eye drop (Alcaine®, Alcon Korea) to the eye, and pupillary dilation was induced by applying a mydriatic agent (Tropherine Eyedrops®, Alcon Korea). After placing the mouse on the stage, the imaging camera of the Micron-IV system (Micron-IV, Phoenix, CA) was focused on the fundus. Lubricant was applied to the eye, and the lens of the Micron-IV system was placed in contact with the cornea. CNV induction conditions were as follows: wavelength 532 nm, diameter 50 μm, duration 80 ms, and output level 240 mW. A drop of antibiotic (Tobrex®, Novartis Korea) was placed in the eye.

[0336] LCNV was generated on day 0 (D0) of the experiment in 6- to 8-week-old male C57BL / 6 mice, recapitulating the subretinal neovascularization characteristic of human wet age-related macular degeneration (wAMD). One day after laser treatment (D1), each bispecific antibody was administered via intravitreal injection.

[0337] 7-2. Fluorescein Angiography (qFA)

[0338] Lesion permeability was assessed by quantitative fluorescein angiography (qFA) on days 7 and 13 (D7 and D13) after laser administration. Animals were anesthetized with an intraperitoneal injection of ketamine / xylazine, and 10% sodium fluorescein was administered via an intraperitoneal injection at a dose of 10 μL / g body weight. Fluorescent fundus images were taken of one eye of each animal using a Micron IV imaging system during lesion filling with fluorescein and again 2 minutes later. The fluorescence intensity of one lesion per eye was quantified via the integrated density function using ImageJ software. The difference in integrated density between the two hours after injection was reported as a reading of lesion leakage.

[0339] As confirmed in Figures 14a and 14b, candidate antibody C showed a much better effect on vascular permeability compared to the combined treatment with Ilia and anti-c-Kit antibody.

[0340] 7-3. CNV lesions

[0341] Fourteen days (D14) after laser administration, animals were euthanized, eyes were enucleated, and choroidal tissue was dissected. The choroids were dissected and stained with FITC-conjugated isolectin B4 (IB4) to visualize the endothelial cells comprising the CNV lesions before flat-mounting. The area of ​​the CNV lesion at each Bruch's membrane rupture site was quantified using computer-assisted image analysis of high-resolution digital images of these choroidal flat-mounts. The area of ​​all lesions in each eye was averaged and reported as a single data point per eye (two data points per mouse).

[0342] As confirmed in Figure 14c, candidate antibody C showed a statistically significant reduction in CNV lesion area compared to the combined treatment with Ilia and anti-c-Kit antibody.

[0343] All qFA and CNV area measurements were analyzed by one-way ANOVA and Tukey post hoc tests, each mean was compared to all other means, and multiple comparisons were corrected using statistical hypothesis testing.

[0344] 7-4. Retinal Image Evaluation (FFA / OCT)

[0345] To conduct another efficacy evaluation test, each dual-specific antibody was administered into the vitreous of mice at different concentrations (0.5, 1, or 2 μg / μL per eye) immediately after LCNV was induced in Example 7-1, and qFA and optical coherence tomography (OCT) were performed 10 days (D10) after laser administration. For efficacy comparison, the vehicle-treated negative control group was administered formulation buffer, and the reference drug-treated positive control group was administered aflibercept at 2 or 20 μg / μL per eye.

[0346] The extent of leakage in LCNV lesions was analyzed using the Corrected Total Fluorescence (CTF) value based on fundus fluorescein angiography (FFA) images. LCNV lesion volume measurement based on OCT images was performed using the 'InSight' and 'Image-J' programs.

[0347] As confirmed in Fig. 15a and Table 32, the candidate antibody A administration group showed a significantly decreased CTF value compared to the vehicle-treated negative control group, and there was no statistically significant difference according to the dosage.

[0348] Group CTF (pixel) P-value vs G2 G1 (Sham) 0 ± 0 G2 (vehicle-treated negative control) 1027397 ± 840775 G3 (aflibercept 2 μg / μL / eye) 619330 ± 4410690.3522 G4 (aflibercept 20 μg / μL / eye) 587441 ± 5868260.0321 G5 (candidate antibody A 0.5 μg / μL / eye) 417531 ± 392373 < 0.0001 G6 (candidate antibody A 1 μg / μL / eye) 519124 ± 4785990.0033 G7 (candidate antibody A 2 μg / μL / eye) 522365 ± 4342470.0113

[0349] Additionally, as confirmed in Figure 15b and Table 33, the candidate antibody A administration group showed a significant decrease in CNV lesion volume compared to the vehicle-treated negative control group.

[0350] Group volume (μm 3)P-value vs G2G1(Sham)0 ± 0G2(Vehicle-treated vocal control group)2833216 ± 1407777G3(Aflibercept 2 μg / μL / eye)2389355 ± <1439831>0.9999G4(Aflibercept 20 μg / μL / eye)1413476 ± 6798120.0002G5(Candidate antibody A 0.5 μg / μL / eye)1432213 ± 1012822< 0.0001G6(Candidate antibody A 1 μg / μL / eye)1607532 ± 8298990.0004G7(Candidate antibody A 2 μg / μL / eye)1613292 ± 9704090.0005

[0351] The data in Tables 32 and 33 are presented as mean ± S.D (G1: n = 48, G2: n = 37, G3: n = 37, G4: n = 33, G5: n = 37, G6: n = 43 and G7: n = 37). Statistically significant differences were compared with G2 (vehicle-treated vocal control group) through the multiple comparison test of Kruskal-Wallis.

Claims

1. A bispecific antibody specifically binding to c-Kit and VEGF comprising the following (a) and (b): (a) a first antigen binding site that specifically binds c-Kit, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and (b) a second antigen binding site that specifically binds to VEGF, wherein the soluble extracellular domain of VEGF is fused to the Fc domain of IgG.

2. A bispecific antibody that specifically binds to c-KIT and VEGF in the first paragraph, wherein the first antigen-binding site comprises a heavy chain variable region comprising an amino acid sequence having 90% or more sequence homology to the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising an amino acid sequence having 90% or more sequence homology to the amino acid sequence of SEQ ID NO:

28.

3. A bispecific antibody that specifically binds to c-KIT and VEGF, wherein the soluble extracellular domain comprises immunoglobulin-like domain 2 of a first VEGF receptor and immunoglobulin-like domain 3 of a second VEGF receptor, in the first paragraph.

4. A bispecific antibody that specifically binds to c-KIT and VEGF, wherein the second antigen binding site comprises an amino acid sequence of SEQ ID NO: 21 in the first paragraph.

5. In the first paragraph, the second antigen binding site is linked to the heavy chain C-terminus or N-terminus of the first antigen binding site by a peptide linker; or A bispecific antibody that specifically binds to c-Kit and VEGF, wherein the second antigen binding site is linked to the light chain C-terminus of the first antigen binding site via a peptide linker.

6. A bispecific antibody specifically binding to c-Kit and VEGF, wherein the peptide linker in the second paragraph is GGGGG (SEQ ID NO: 20), GGGGGG (SEQ ID NO: 67), GGGGGGG (SEQ ID NO: 68), GGGGGGGG (SEQ ID NO: 23), GGGGGGGGG (SEQ ID NO: 69), or GGGGGGGGGG (SEQ ID NO: 70).

7. In the first paragraph, the Fc domain of the first antigen binding site: (a) Wild-type IgG Fc; (b) Fc variants comprising amino acid substitutions L234A and L235E; (c) an Fc variant comprising amino acid substitutions L235A and G237A; or (d) Fc variants comprising amino acid substitutions L234A, L235A I253A, H310A, P329G and H435A; A bispecific antibody that specifically binds to c-Kit and VEGF, wherein the residues are numbered according to the EU index of Kabat.

8. In the first paragraph, the bispecific antibody is a bispecific antibody that specifically binds to c-Kit and VEGF, which is a tetravalent antibody.

9. A bispecific antibody that specifically binds to c-Kit and VEGF, wherein the first antigen binding portion is a full-length bivalent antibody, and the second antigen binding portion is composed of two VEGF antagonists in which the soluble extracellular domain of VEGF and the Fc domain of IgG are fused.

10. A nucleic acid molecule encoding a bispecific antibody that specifically binds to c-Kit and VEGF according to any one of claims 1 to 9.

11. A recombinant vector comprising a nucleic acid molecule according to Article 10.

12. A transformant comprising a recombinant vector according to Article 11.

13. A method for producing a bispecific antibody specifically binding to c-Kit and VEGF, comprising the following steps (a) and (b): (a) a step of culturing the transformant of clause 12; and (b) a step of recovering a bispecific antibody that specifically binds to c-Kit and VEGF from the culture.

14. A pharmaceutical composition for preventing or treating angiogenic diseases, comprising a bispecific antibody specifically binding to c-Kit and VEGF according to any one of claims 1 to 9 as an active ingredient.

15. A pharmaceutical composition according to claim 14, wherein the angiogenic disease is selected from the group consisting of cancer, leukemia, ocular vascular disease, rheumatoid arthritis, psoriasis, chronic wounds, chronic inflammation, hemangioma, angiofibroma, vascular malformation, arteriosclerosis, vascular adhesions, vasculitis, pyogenic granulomas, bullous diseases, pulmonary hypertension, asthma, nasal polyps, infectious diseases, inflammatory bowel diseases, periodontal diseases, peritoneal adhesions, endometrium, uterine bleeding, ovarian cysts, osteomyelitis, osteophytes, sepsis, and autoimmune diseases.

16. A pharmaceutical composition according to claim 15, wherein the cancer is selected from the group consisting of bone cancer, lung cancer, head and neck cancer, cervical cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, stomach cancer, liver cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, rectal cancer, anal cancer, colon cancer, uterine cancer, breast cancer, ovarian cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, central nervous system lymphoma, spinal cord tumor, glioblastoma, brainstem glioma, and pituitary adenoma.

17. A pharmaceutical composition according to claim 15, wherein the ocular vascular disease is selected from the group consisting of diabetic retinopathy, macular degeneration, age-related macular degeneration, glaucoma, glaucomatous retinitis pigmentosa, choroidal neovascularization, retinopathy of prematurity, corneal dystrophy, and retinal detachment.

18. A kit for diagnosing angiogenic diseases, comprising a bispecific antibody specifically binding to c-Kit and VEGF according to any one of claims 1 to 9.

19. A method for preventing or treating angiogenic diseases, comprising administering to a subject in need thereof a therapeutically effective amount of a bispecific antibody that specifically binds to c-Kit and VEGF according to any one of claims 1 to 9.

20. Use of a bispecific antibody specifically binding to c-Kit and VEGF according to any one of claims 1 to 9 for the manufacture of a medicament for preventing or treating angiogenic diseases.

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