Bispecific antibodies that specifically bind c-kit and vegf and uses thereof

Abstract

The present application relates to a bispecific antibody specifically binding to c-Kit and VEGF, and more particularly, to a bispecific antibody capable of binding to c-Kit and VEGF with high affinity, thereby being used for inhibiting or neutralizing the activity or activation of c-Kit and VEGF, a nucleic acid molecule encoding the same, a vector comprising the same, a host cell, a method of preparing the same, a pharmaceutical composition for the prevention or treatment of a neovascular disease comprising the same as an active ingredient, a method of preventing or treating a neovascular disease using the same, and a kit for detecting c-Kit and / or VEGF or diagnosing a neovascular disease comprising the same.

Description

Technical Field

[0001] This application claims priority to Korean Patent Application No. 10-2024-0005633, filed on January 12, 2024, the entire contents of which are disclosed in the specification and drawings of which are incorporated herein by reference.

[0002] This invention relates to a bispecific antibody that specifically binds to c-Kit and VEGF, and more specifically, to a bispecific antibody capable of binding to c-Kit and VEGF with high affinity, thereby inhibiting or neutralizing or activating the activity of c-Kit and VEGF; a nucleic acid molecule encoding the bispecific antibody; a vector containing the nucleic acid molecule; a host cell; a method for preparing the bispecific antibody; a pharmaceutical composition containing the bispecific antibody as an active ingredient for the prevention or treatment of angiogenesis; a method for the prevention or treatment of angiogenesis using the bispecific antibody; and a kit for the detection or diagnosis of angiogenesis using c-Kit and / or VEGF containing the bispecific antibody. Background Technology

[0003] Angiogenesis is the process by which existing microvessels form new capillaries. It typically occurs only during embryonic development, wound healing, and cyclical changes in the female reproductive system, and rarely occurs under other normal conditions. However, when angiogenesis cannot be autonomously regulated and continues to grow pathologically, it can lead to a variety of diseases. Diseases associated with angiogenesis include hemangiomas, angiofibromas, vascular malformations, and cardiovascular diseases such as atherosclerosis, vascular adhesions, and edematous sclerosis. Ophthalmic diseases caused by angiogenesis include age-related macular degeneration, corneal transplant angiogenesis, neovascular glaucoma, diabetic retinopathy, corneal diseases caused by neovascularization, macular degeneration, pterygium, retinal degeneration, posterior lens fibrosis, and granular conjunctivitis. Furthermore, it includes chronic inflammatory diseases such as arthritis, psoriasis, telangiectasia, pyogenic granuloma, seborrheic dermatitis, and acne. The growth and metastasis of cancer cells inevitably depend on angiogenesis.

[0004] VEGF is a potent pro-angiogenic factor, and its expression is increased in various cancer cells. VEGF binds to VEGF receptors on the surface of vascular endothelial cells, thereby activating tyrosine kinases and inducing angiogenesis, playing a crucial role in cancer cell growth and metastasis. Therefore, VEGF inhibitors, by blocking angiogenesis, are used to treat various cancers and macular degeneration. Currently, recombinant antibody anticancer drugs include bevacizumab, macular degeneration treatments include ranibizumab and aflibercept, and kinase inhibitors include sunitinib and sorafenib. However, a large number of patients do not respond to these VEGF inhibitors; therefore, the development of various bispecific antibodies targeting this problem is underway.

[0005] On the other hand, under hypoxia, the expression of c-KIT in endothelial cells increases, and endothelial angiogenesis induced by SCF (stem cell factor) increases. This type of pathological angiogenesis is a major cause of blindness in various ophthalmic diseases such as wet macular degeneration and diabetic retinopathy. Under hypoxia, the expression of stem cell factor (SCF), c-KIT, and hypoxia-inducible factor-1α (HIF-1α) increases. At this time, stem cell factor (SCF) stabilizes hypoxia-inducible factor-1α, thereby prolonging the hypoxic state. When the hypoxic state persists, it will again cause a vicious cycle of increased vascular endothelial growth factor / its receptor (VEGF / VEGFR). Therefore, by blocking stem cell factor (SCF) signaling with c-KIT inhibitors, hypoxia-inducible factor-1α can be inhibited, thereby removing the factors that stimulate angiogenesis. Therefore, the inventors have developed an anti-c-Kit antibody that specifically binds to c-KIT and its excellent angiogenesis inhibitory effect has been demonstrated (Patent Document 1).

[0006] Against the above background, the inventors developed a bispecific antibody that specifically binds to c-Kit and VEGF, and confirmed that the antibody can be used as a therapeutic agent for angiogenesis diseases, especially macular degeneration, thus completing the present invention.

[0007] Existing technical documents

[0008] Patent documents

[0009] (Patent Document 1) Korean Patent Publication No. 10-2020-0040407 Summary of the Invention

[0010] The technical problem that the invention aims to solve

[0011] The purpose of this invention is to provide a bispecific antibody that specifically binds to c-Kit and VEGF.

[0012] Another object of the present invention is to provide a nucleic acid molecule encoding the bispecific antibody.

[0013] Another object of the present invention is to provide a vector containing the nucleic acid molecule and a host cell containing the vector.

[0014] Another object of the present invention is to provide a method for preparing bispecific antibodies that specifically bind to c-Kit and VEGF using the host cells.

[0015] Another object of the present invention is to provide a pharmaceutical composition comprising, as an active ingredient, a bispecific antibody that specifically binds to c-Kit and VEGF, for the prevention or treatment of angiogenesis diseases.

[0016] Another object of the present invention is to provide a method for the prevention or treatment of angiogenesis diseases using bispecific antibodies that specifically bind to c-Kit and VEGF as described above.

[0017] Another object of the present invention is to provide the use of the aforementioned bispecific antibody that specifically binds to c-Kit and VEGF in the preparation of a medicament for the prevention or treatment of angiogenesis diseases.

[0018] Another object of the present invention is to provide a kit that utilizes a bispecific antibody that specifically binds to c-Kit and VEGF for the detection of c-Kit and / or VEGF or the diagnosis of angiogenesis diseases.

[0019] However, the technical problems to be solved by the present invention are not limited to the above-mentioned contents, and other problems not mentioned can be clearly understood by those skilled in the art from the following description.

[0020] means for solving problems

[0021] To address the aforementioned technical problems, the present invention provides a bispecific antibody that specifically binds c-Kit and VEGF, comprising the following (a) and (b): (a) a first antigen-binding site that specifically binds c-Kit, including a heavy chain variable region comprising a heavy chain complementarity-determining region 1 (CDR1) containing the amino acid sequence of sequence number 2, a heavy chain complementarity-determining region 2 (CDR2) containing the amino acid sequence of sequence number 4, and a heavy chain complementarity-determining region 3 (CDR3) containing the amino acid sequence of sequence number 6; and a light chain variable region comprising a light chain complementarity-determining region 1 (CDR1) containing the amino acid sequence of sequence number 13, a light chain complementarity-determining region 2 (CDR2) containing the amino acid sequence of sequence number 15, and a light chain complementarity-determining region 3 (CDR3) containing the amino acid sequence of sequence number 17; and (b) a second antigen-binding site that specifically binds VEGF, wherein the second antigen-binding site is formed by the fusion of a soluble extracellular domain of VEGF and an Fc domain of IgG.

[0022] In this invention, the first antigen binding site may include a heavy chain variable region and a light chain variable region. The heavy chain variable region contains an amino acid sequence that has more than 90% sequence homology with the amino acid sequence of sequence number 27, and the light chain variable region contains an amino acid sequence that has more than 90% sequence homology with the amino acid sequence of sequence number 28.

[0023] In this invention, the soluble extracellular domain may include the immunoglobulin-like domain 2 of the first VEGF receptor and the immunoglobulin-like domain 3 of the second VEGF receptor.

[0024] In this invention, the second antigen binding site may include the amino acid sequence of sequence number 21.

[0025] In this invention, the second antigen binding site can be connected to the C-terminus or N-terminus of the heavy chain of the first antigen binding site via a peptide linker; or, the second antigen binding site can be connected to the C-terminus of the light chain of the first antigen binding site via a peptide linker.

[0026] In this invention, the peptide linker may be GGGGG (serial number 20), GGGGGG (serial number 67), GGGGGGG (serial number 68), GGGGGGGG (serial number 23), GGGGGGGGG (serial number 69), or GGGGGGGGGG (serial number 70).

[0027] In this invention, the Fc domain of the first antigen binding site may include: (a) wild-type IgG Fc;

[0028] (b) Fc variants containing amino acid substitutions of L234A and L235E;

[0029] (c) Fc variants containing substitutions for amino acids L235A and G237A; or

[0030] (d) Fc variants containing amino acid substitutions of L234A, L235A, I253A, H310A, P329G, and H435A;

[0031] The residues can be numbered according to the EU index of Kabat.

[0032] In this invention, the bispecific antibody can be a tetravalent antibody.

[0033] In this invention, the first antigen binding site can be a full-length bivalent antibody, and the second antigen binding site can be composed of two VEGF antagonists, wherein the VEGF antagonist is formed by fusing the soluble extracellular domain of VEGF with the Fc domain of IgG.

[0034] In addition, the present invention provides a nucleic acid molecule encoding a bispecific antibody that specifically binds to the aforementioned c-Kit and VEGF.

[0035] Furthermore, the present invention provides a recombinant vector comprising the aforementioned nucleic acid molecules and a transformant comprising the recombinant vector.

[0036] Furthermore, the present invention provides a method for preparing a bispecific antibody that specifically binds to c-Kit and VEGF, comprising the following steps (a) and (b): (a) culturing the aforementioned transformant; and (b) recovering the bispecific antibody that specifically binds to c-Kit and VEGF from the culture.

[0037] In addition, the present invention provides a pharmaceutical composition comprising the aforementioned bispecific antibody that specifically binds to c-Kit and VEGF as an active ingredient for the prevention or treatment of angiogenesis diseases.

[0038] Furthermore, the present invention provides a method for the prevention or treatment of angiogenesis diseases, comprising the step of administering a bispecific antibody that specifically binds c-Kit and VEGF to an individual in need at a therapeutically effective amount.

[0039] In addition, the present invention provides a bispecific antibody that specifically binds to c-Kit and VEGF, which is used for the prevention or treatment of angiogenesis diseases.

[0040] Furthermore, the present invention provides the use of the aforementioned bispecific antibody that specifically binds to c-Kit and VEGF in the preparation of a medicament for the prevention or treatment of angiogenesis diseases.

[0041] In this invention, the angiogenesis disease can 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 adhesion, 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, osteomyelitis, sepsis, and autoimmune diseases.

[0042] In this invention, the cancer can be selected from the group consisting of bone cancer, lung cancer, head cancer, neck cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, gastric cancer, liver cancer, pancreatic cancer, skin cancer, melanoma of the skin or eye, rectal cancer, perianal cancer, colon cancer, uterine cancer, breast cancer, ovarian cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine gland cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumors, central nervous system lymphoma, spinal cord tumors, glioblastoma, brainstem glioma, and pituitary adenoma.

[0043] In this invention, the ocular vascular disease can 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 laminar separation.

[0044] In addition, the present invention provides a kit comprising a bispecific antibody that specifically binds to c-Kit and VEGF, for the detection of c-Kit and / or VEGF or the diagnosis of angiogenesis diseases.

[0045] The effects of the invention

[0046] The bispecific antibody of this invention, which specifically binds to c-Kit and VEGF, binds to c-Kit and / or VEGF with high affinity, thereby effectively inhibiting venous endothelial barrier disruption induced by hypoxia or inflammation, stem cell factor (SCF)-dependent c-Kit signaling, and the secretion of various cytokines (IL-6, VEGF, Ang-2, IL-8, and MCP-1). Therefore, the bispecific antibody of this invention can significantly inhibit abnormal or excessive angiogenesis, and can thus be used to treat various angiogenesis diseases. Attached Figure Description

[0047] Figure 1 This is a schematic diagram illustrating the structure of a bispecific candidate antibody A that specifically binds to c-Kit and VEGF according to the present invention.

[0048] Figure 2 To demonstrate the results of evaluating antibody-dependent cell-mediated cytotoxicity changes based on Fc variant type.

[0049] Figures 3a to 3c To demonstrate the bispecific candidate antibody B that specifically binds to c-Kit and VEGF according to the present invention ( Figure 3a Candidate antibody C () Figure 3b ) and candidate antibody B-1 ( Figure 3c A schematic diagram of the structure of ).

[0050] Figure 4a and Figure 4b To demonstrate the bispecific candidate antibody D that specifically binds to c-Kit and VEGF according to the present invention ( Figure 4a ) and candidate antibody E ( Figure 4b A schematic diagram of the structure of ).

[0051] Figure 5a and Figure 5b To demonstrate the binding ability of the bispecific candidate antibody according to the present invention to c-Kit and VEGF, as confirmed by ELISA.

[0052] Figure 6a and Figure 6b To demonstrate the results of surface plasmon resonance confirmation of the binding affinity of the bispecific candidate antibody according to the present invention for c-Kit and VEGF to c-Kit and VEGF.

[0053] Figure 7 To demonstrate the results of DSF confirmation of the thermostability of the bispecific candidate antibody that specifically binds to c-Kit and VEGF according to the present invention.

[0054] Figures 8a to 8c A graph illustrating the inhibitory effect on vascular permeability after treatment with the bispecific candidate antibody that specifically binds c-Kit and VEGF according to the present invention in hypoxic or inflammation-induced HUVECs ( Figure 8a and Figure 8b ***P<0.001, ****P<0.0001 vs. control; #P<0.05, ###P<0.001 and ####P<0.0001 vs. 1%O2 or LPS; ns, no significant difference (n=3); Figure 8c***P<0.001, ****P<0.0001 vs. Control; ##P<0.005, ####P<0.0001 vs. 1% O2 or LPS; †P<0.05, ††††P<0.0001 vs. all concentrations under 1% O2 or LPS conditions; ns, no significant difference).

[0055] Figures 9a to 9d The figure illustrates the SCF-dependent c-Kit signal transduction inhibition effect after treatment with the bispecific candidate antibody that specifically binds c-Kit and VEGF according to the present invention in hypoxic or inflammation-induced HRMEC or LAD2.

[0056] Figure 10a and Figure 10b The graph illustrates the inhibitory effect of treatment with the bispecific candidate antibody A that specifically binds c-Kit and VEGF according to the present invention on cytokines (IL-8, MCP-1, IL-6) in HRMEC or ARPE-19 cells under hypoxic or inflammation-induced conditions (P<0.01, *P<0.001 and P<0.0001 vs. control; #P<0.01, ##P<0.005 and ###P<0.001 vs. 1% O2 or LPS).

[0057] Figures 11a to 11r The figure shows the inhibitory effect of treatment with candidate antibody B on cytokines (VEGF, IL-6, IL-8, MCP-1, and Ang-2) in HRMEC or ARPE-19 cells under hypoxic or inflammation-induced conditions (P<0.01, *P<0.001 and P<0.0001 vs. control; #P<0.01, ##P<0.005, ###P<0.001 and ####P<0.0001 vs. 1% O2 or LPS treatment; ns, no significant difference).

[0058] Figures 12a to 12i The figure shows the inhibitory effect of treatment with candidate antibody C or candidate antibody E on the secretion of cytokines (IL-6, VEGF, Ang-2, IL-8, and MCP-1) in HRMEC or ARPE-19 cells under hypoxic or inflammation-induced conditions (***P<0.001, ****P<0.0001 vs. control; #P<0.05, ##P<0.005, ###P<0.001 and ####P<0.0001 vs. 1% O2 or LPS; ns, no significant difference).

[0059] Figures 13a to 13iThe figure shows the comparison of the inhibitory effects of candidate antibody C alone or in combination with aflibercept and anti-c-Kit antibody on cytokines (IL-6, VEGF, Ang-2, IL-8, and MCP-1) in hypoxic or inflammation-induced HRMEC or ARPE-19 cells (***P<0.001, ****P<0.0001 vs. control; #P<0.05, ###P<0.001, ####P<0.0001 vs. 1% O2 or LPS; †††P<0.001, ††††P<0.0001 vs. concentrations under 1% O2 or LPS conditions; ns, no significant difference).

[0060] Figure 14a and Figure 14b The figure shows a comparison of the inhibitory effects of candidate antibody C alone or in combination with aflibercept and anti-c-Kit antibody on vascular permeability in mice induced to develop choroidal angiogenesis.

[0061] Figure 14c This figure shows a comparison of the reduction in choroidal neovascularization lesions after treatment with candidate antibody C alone or in combination with aflibercept and anti-c-Kit antibody in mice induced to develop choroidal neovascularization.

[0062] Figure 15a The figure shows the comparison of the inhibitory effects of candidate antibody A alone at different concentrations (0.5, 1 and 2 μg / μL / eye, respectively) and aflibercept alone at different concentrations (2 and 20 μg / μL / eye, respectively) on vascular permeability in mice induced to develop choroidal angiogenesis (*P<0.05, **P<0.01 and ****P<0.0001 vs. G2).

[0063] Figure 15b The figure shows the comparison of the reduction in choroidal neovascularization lesions after treatment with candidate antibody A alone at different concentrations (0.5, 1, and 2 μg / μL / eye, respectively) and aflibercept alone at different concentrations (2 and 20 μg / μL / eye, respectively) in mice induced to develop choroidal neovascularization (***P<0.001 and ****P<0.0001 vs. G2). Detailed Implementation

[0064] The present invention will now be described in further detail.

[0065] All technical terms used in this invention, unless otherwise defined, shall be used in the manner commonly understood by one of ordinary skill in the art to which this invention pertains. Furthermore, preferred methods or samples are described in this specification, but similar or equivalent content is also included within the scope of this invention.

[0066] In this invention, the term "antibody" is used in the broadest sense to include various antibody structures, such as, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (provided they exhibit the desired antigen-binding activity).

[0067] In this invention, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting the population are identical to each other and / or bind to the same epitopes, but may contain variant antibodies containing naturally occurring mutations or variations generated during the preparation of the monoclonal antibody, and such variations are usually present in small amounts. Unlike polyclonal antibody formulations, which typically contain different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation acts against a single determinant on the antigen.

[0068] In this paper, the term "monospecific" antibody refers to an antibody having one or more binding sites, where each binding site binds to the same epitope of the same antigen. The term "bispecific" refers to an antibody that can specifically bind to at least two different antigenic determinants, such as two binding sites formed by pairing antibody heavy chain variable domain (VH) and antibody light chain variable domain (VL) to bind to different antigens or different epitopes on the same antigen. The aforementioned bispecific antibodies are in a 2+2 form (containing two binding sites targeting a first and a second antigen or epitope). Typically, bispecific antibodies contain two antigen-binding sites, each specifically targeting a different antigenic determinant.

[0069] In this invention, the term "binding valence" refers to the presence of the stated number of binding sites in an antigen-binding molecule. Therefore, the terms "bivalent," "tetravalent," and "hexavalent" respectively indicate the presence of two, four, and six binding sites in the antigen-binding molecule. The bispecific antibody according to this invention can be "tetravalent."

[0070] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably in this document to refer to antibodies with a structure substantially similar to that of natural antibodies. "Native antibodies" are naturally occurring immunoglobulin molecules with multiple structures. For example, wild-type IgG antibodies are heterotetrameric glycoproteins with a molecular weight of approximately 150,000 daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain includes a variable region (VH), also known as the heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), called the heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain includes a variable region (VL), also known as the light chain variable domain, followed by the light chain constant domain (CL), called the light chain constant region. Antibody heavy chains can be classified into five types: α (IgA), δ (IgD), ε (IgE), γ (IgG), and μ (IgM). Some of these can be further classified into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Antibody light chains can be classified into one of two types, κ (kappa) or λ (lambda), based on the amino acid sequence of their constant domains.

[0071] As described above, variable regions enable antibodies to selectively recognize and specifically bind to epitopes on antigens. That is, variable regions defining three-dimensional antigen-binding sites are formed by combining the VL and VH domains of the antibody, or subsets of complementarity-determining regions (CDRs). In this type of quaternary antibody structure, antigen-binding sites are formed at the ends of each arm of the Y-shape. 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, such as when a particular immunoglobulin molecule is derived from a camelid species, or is modified based on camelid immunoglobulins, the complete immunoglobulin molecule may consist only of heavy chains and lack light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

[0072] 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).

[0073] In natural antibodies, the six complementarity determining regions (CDRs) present in each antigen-binding domain are short, discontinuous amino acid sequences that specifically localize to form the antigen-binding domain due to the antibody's three-dimensional conformation in an aqueous environment. Within the antigen-binding domain, the remaining amino acids belong to regions called "framework regions," exhibiting low intermolecular variability. Framework regions primarily adopt a β-sheet conformation, while CDRs form linking loops and, in some cases, part of the β-sheet structure. Therefore, the framework regions form a scaffold through interchain non-covalent interactions to orient the CDRs correctly. The antigen-binding domain formed by the localized CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface facilitates non-covalent binding between the antibody and the corresponding epitope. For any corresponding heavy or light chain variable region, the amino acids containing the CDR and framework regions can be readily identified by those skilled in the art because they have been clearly 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. Mol. Biol., 196: 901-917 (1987)).

[0074] Where two or more definitions exist for terms used and / or permitted in this field, all such meanings are intended to be included unless the terms used herein expressly indicate otherwise. As a specific example, the term “complementarity-determining region (CDR)” is used to describe discontinuous antigen-binding sites found within the variable regions of heavy and light chain polypeptides. These specific regions have been 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. According to the CDR definitions of Kabat and Chothia, overlaps or subsets of amino acid residues are included when comparing them. Nevertheless, the scope of the definition of a CDR used to refer to an antibody or a variant thereof is intended to be encompassed within the scope of the terms defined and used herein. The appropriate amino acid residues containing a CDR as defined by the aforementioned cited references are shown for comparison in Table 1 below. The exact number of residues containing a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine whether a residue belongs to a specific CDR by considering the amino acid sequence of the variable region of an antibody.

[0075] Table 1

[0076] CDR-H1 31-35 26-32 CDR-H1 50-65 52-58 CDR-H1 95-102 95-102 CDR-L1 24-34 26-32 CDR-L2 50-56 50-52 CDR-L3 89-97 91-96

[0077] Kabat et al. also defined a numbering system applicable to variable domain sequences of any antibody. Those skilled in the art can explicitly 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 Kabat et al., US Dept. of Health and Human Services, *Sequence of Proteins of Immunological Interest* (1983). The antibodies disclosed herein can be derived from any animal source, including birds and mammals. Preferably, the antibodies are derived from humans, mice, donkeys, rabbits, goats, guinea pigs, camels, alpacas, horses, or chickens.

[0078] As used herein, the term "heavy chain constant region" refers to the amino acid sequence containing the heavy chain of immunoglobulins. As will be understood by those skilled in the art as described above, the heavy chain constant region can be modified, thereby altering its amino acid sequence derived from native immunoglobulin molecules.

[0079] The heavy chain constant region of the antibodies disclosed herein can originate from different immunoglobulin molecules. In this invention, the immunoglobulin in the heavy chain constant region of the antibody can be designated as one of five types, as described above: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM). Furthermore, based on the amino acid composition of the hinge region and the number and position of the heavy chain disulfide bonds, immunoglobulins of the same type can be classified into different subclasses (isotypes). For example, when the heavy chain constant region of the antibody of this invention is γ (IgG), its subclass (isotype) can be further classified as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), or γ4 (IgG4); when the heavy chain constant region of the antibody of this invention is α (IgA), its subclass (isotype) can be further classified as α1 (IgA1) or α2 (IgA2).

[0080] As used herein, the term "light chain constant region" refers to an amino acid sequence comprising an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant κ domain or a constant λ domain.

[0081] A "light chain-heavy chain pair" refers to an aggregate of light and heavy chains that can form dimers through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0082] An "antibody fragment" or "antigen-binding fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody and is capable of binding to the antigen bound by the complete antibody. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab', F(ab')2, xFab, scFab, dsFv, Fv, scFv, scFv-Fc, scFab-Fc, diabody, minibody, scAb, dAb, half-IgG, or combinations thereof. The term "Fab" used in Fab, Fab', F(ab')2, xFab, and scFab can include conventional Fab fragments as well as chimeric Fab-like domains described in PCT / CN2018 / 106766 (Wuxibody). Furthermore, the aforementioned antibody fragments can be derived from any mammal, including but not limited to humans, mice, rats, camels, or rabbits. The functional portion of an antibody, such as one or more CDRs as described herein, can be covalently linked to another protein or small molecule compound, thereby enabling its use as a targeted therapeutic agent against a specific target. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein capable of binding to a specific antigen and forming a complex, thereby functioning like an antibody.

[0083] Antibody fragments can be prepared using a variety of techniques, including but not limited to proteolytic digestion of intact antibodies as described herein, and production via recombinant host cells such as E. coli or phages.

[0084] Papain digestion of the intact antibody produces two identical antigen-binding fragments, referred to as the “Fab” fragment. This fragment contains variable domains of both the heavy and light chains, as well as a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. Therefore, the term “Fab fragment” as used herein refers to the light chain fragment containing the VL domain and constant domain (CL) of the light chain, and the antibody fragment containing the VH domain and first constant domain (CH1) of the heavy chain. The Fab’ fragment differs from the Fab fragment in that it has several residues added to the carboxyl terminus of the CH1 domain of the heavy chain, derived from the antibody hinge region, including one or more cysteine ​​residues. Fab’-SH refers to the Fab’ fragment where the cysteine ​​residues of the constant domain have free thiol groups. Pepsin treatment produces the F(ab’)2 fragment, which has two antigen-binding sites (two Fab fragments) and a portion of the Fc region. In this paper, the “F(ab')2 segment” as described above comprises two light chains and two heavy chains. The two heavy chains contain a variable region, CH1, and a partially constant region located between the CH1 and CH2 domains, thereby forming an intrachain disulfide bond between the two heavy chains. Therefore, the F(ab')2 segment consists of two Fab' segments, which are interconnected by a disulfide bond between them.

[0085] The terms "cross-Fab fragment," "xFab fragment," or "crossover Fab fragment" refer to Fab fragments in which the variable or constant regions of the heavy and light chains are exchanged. Cross-Fab molecules have two distinct chain compositions and can be included in the bispecific antibodies of this invention: On one hand, when the variable regions of the Fab heavy and light chains are exchanged, the cross-Fab molecule comprises a peptide chain composed of a light chain variable domain (VL) and a heavy chain constant domain (CH1), and a peptide chain composed of a heavy chain variable domain (VH) and a light chain constant domain (CL). Such cross-Fab molecules are also called CrossFab (VLVH). On the other hand, when the constant regions of the Fab heavy and light chains are exchanged, the cross-Fab molecule comprises a peptide chain composed of a heavy chain variable domain (VH) and a light chain constant domain (CL), and a peptide chain composed of a light chain variable domain (VL) and a heavy chain constant domain (CH1). Such cross-Fab molecules are also known as CrossFab (CLCH1).

[0086] A "single-chain Fab fragment" or "scFab" is a polypeptide composed of 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. The antibody domains and the linker have one of the following sequences in the N-terminal to C-terminal direction: 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. 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 native disulfide bond between the CL and CH1 domains. Furthermore, these single-chain Fab molecules can be further stabilized by introducing cysteine ​​residues (e.g., at position 44 of the heavy chain variable region and position 100 of the light chain variable region, according to the Kabat number) to form interchain disulfide bonds.

[0087] A “crossover single-chain Fab fragment” or “x-scFab” is a polypeptide composed of 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 domain and the linker have one of the following sequences in the N-terminal to C-terminal direction: (a) VH-CL-linker-VL-CH1 and (b) VL-CH1-linker-VH-CL; the VH and VL together form an antigen-binding domain that specifically binds to the antigen, and the linker is a polypeptide having at least 30 amino acids. Furthermore, these x-scFab molecules can be further stabilized by introducing cysteine ​​residues (e.g., at position 44 of the heavy chain variable region and position 100 of the light chain variable region, according to Kabat numbering) to form interchain disulfide bonds.

[0088] "Fv region" refers to an antibody containing variable regions of both the heavy and light chains but not the constant region. scFv refers to an Fv linked by a flexible linker. scFv-Fc refers to a structure with an Fc linked to an scFv. A divalent antibody (diabody) comprises two scFv molecules. "Single-chain variable fragment" or "scFv" refers to a fusion protein of the heavy chain variable region (VH) and light chain variable region (VL) of an immunoglobulin. In some embodiments, the VH and VL are linked by a short linker peptide having 10 to about 25 amino acids. The linker may be glycine-rich to provide flexibility and serine or threonine-rich to improve solubility, and may link the N-terminus of the VH to the C-terminus of the VL, or vice versa. Despite the removal of the constant region and the introduction of a linker, this type of protein retains the specificity of the original immunoglobulin. scFv molecules are known in the art, for example, as described in U.S. Patent No. 5,892,019.

[0089] "Short-chain antibody (scAb)" refers to an antibody comprising a single polypeptide chain, including a heavy chain variable region and a light chain variable region linked by flexible linkers, and comprising a variable region of the heavy chain or a constant region of the light chain. Examples of short-chain antibodies include, for example, U.S. Patent No. 5,260,203, which is incorporated herein by reference.

[0090] "Domain antibody (dAb)" refers to an immunoglobulin fragment containing only the heavy chain variable region or the light chain variable region that has immunological function. In one embodiment, two or more VH domains are covalently linked by peptide linkers to form a bivalent domain antibody. The two VH domains of this bivalent domain antibody may be the same or target different antigens.

[0091] In this invention, the term "full-length IgG" is defined as containing essentially complete IgG, but not necessarily possessing all the functions of complete IgG. To avoid ambiguity, full-length IgG comprises two heavy chains and two light chains. Each chain contains a constant region (C) and a variable region (V), which can be divided into domains such as CH1, CH2, CH3, VH, and CL, VL. IgG antibodies bind to antigens through the variable region domain contained in the Fab portion, and after binding, interact with cells and molecules of the immune system through the constant region domain, primarily 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. Full-length antibodies according to the invention may comprise IgG molecules with mutations that provide the desired properties. Such mutations should not result in the deletion of a majority of any region. However, IgG molecules lacking one or more amino acid residues are also included in the term "full-length IgG" without substantially altering the binding properties of the generated IgG molecule. For example, the IgG molecule preferably lacks 1 to 10 amino acid residues in a non-CDR region, wherein the loss of these amino acids is not essential for the binding specificity of IgG.

[0092] As used herein, the term "antigen-binding domain" or "antigen-binding site" refers to a portion of an antibody or antibody fragment that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding domain" refers to a portion of an antibody containing a region that specifically binds all or part of an antigen and is complementary to it. When the antigen is large, the antibody or antibody fragment may bind only a specific portion of the antigen, called an epitope. An antigen-binding domain may, for example, be provided by one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain. In one embodiment, the antigen-binding domain may block or partially block its function by binding to its antigen. Antigen-binding domains that specifically bind to c-Kit and VEGF include antibodies and fragments thereof as further defined herein. Furthermore, the antigen-binding domain may also include scaffold-based antigen-binding proteins, such as binding domains based on designed repeating proteins or designed repeating domains (see, for example, WO 2002 / 020565).

[0093] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope," referring to a site on a polypeptide macromolecule (e.g., a conformational structure consisting of a continuous amino acid fragment or different regions of non-continuous amino acids) that can be bound by an antigen-binding moiety to form an antigen-binding moiety-antigen complex. Useful antigenic determinants may be present, for example, on the surface of tumor cells, virus-infected cells, other diseased cells, immune cells, serum-free blood, and / or the extracellular matrix (ECM). In this document, a protein useful as an antigen, unless otherwise stated, can be any naturally occurring form of protein derived from any vertebrate source, including primates (e.g., humans) and rodents (e.g., mice and rats). In a particular embodiment, the antigen is a human or mouse-derived protein. When referring to a particular protein herein, the term includes not only "full-length" unprocessed proteins but also all forms of proteins processed in cells. The term also includes natural variants of proteins, such as splice variants or allelic variants.

[0094] "Specific binding" means that the binding is selective for the antigen and can be distinguished from non-target or non-specific interactions. The ability of an antibody or antibody fragment to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or other techniques known to those skilled in the art, such as surface plasmon resonance (SPR) technology (analyzed by BIAcore instruments) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and conventional binding analysis (Heeley, Endocr Res 28, 217-229 (2002)).

[0095] "Affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen) at a single binding site. Unless otherwise stated, "binding affinity" as used herein refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its ligand Y is typically expressed as a dissociation constant (Kd), which is the ratio of the dissociation rate constant to the binding rate constants (koff and kon, respectively). Therefore, equivalent affinities can include different rate constants, provided that the ratio of the rate constants remains the same. Affinity can be determined using conventional methods known in the art, including those described herein. One specific method for determining affinity is surface plasmon resonance (SPR). According to one embodiment of the present invention, the bispecific antibody of the present invention has a dissociation constant (KD) of ≤1 μM, ≤100 nm, ≤10 nm, ≤1 nm, ≤0.1 nm, ≤0.01 nm or ≤0.001 nm (e.g., below 10⁻⁷ M, e.g., 10⁻⁷ M to 10⁻¹³ M, e.g., 10⁻⁹ M to 10⁻¹³ M).

[0096] The term "high affinity" antibody refers to an antibody with a KD of less than 10⁻⁹ M, preferably less than 10⁻¹⁰ M, against the target antigen.

[0097] The terms “bispecific antibody containing a first antigen-binding site that specifically binds to c-Kit and a second antigen-binding site that specifically binds to VEGF”, “bispecific antibody that specifically binds to c-Kit and VEGF”, and “bispecific antigen-binding molecule that is specific to c-Kit and VEGF” are used interchangeably in this specification and refer to a bispecific antibody that can bind to c-Kit and VEGF with an affinity sufficient for the diagnosis and / or treatment of c-Kit and VEGF.

[0098] The term "c-Kit" used in this invention belongs to class III of receptor tyrosine kinase (RTK) and is also known as the receptor for SCF.

[0099] As one of the targets of the angiogenesis inhibitors, c-Kit belongs to class III of receptor tyrosine kinase (RTK) and is a receptor for SCF (Stem Cell Factor), which plays an important role in hematopoiesis.

[0100] The term "anti-c-Kit antibody" as used in this 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.

[0101] "Anti-c-Kit antibody" and "antibody containing an antigen-binding site for c-Kit" refer to antibodies capable of binding to c-Kit, particularly c-Kit peptides, with an affinity sufficient for diagnosis and / or treatment targeting c-Kit. According to one embodiment, the anti-c-Kit antibody binds to unrelated non-c-Kit proteins to less than about 10% of its binding to c-Kit, as determined, for example, by radioimmunoassay (RIA) or flow cytometry (FACS), or by surface plasmon resonance analysis using a biosensor system such as the Biacore (registered trademark) system. For example, antibodies binding to human c-Kit have a KD value of ≤1 μM, ≤100 nm, ≤10 nm, ≤1 nm, ≤0.1 nm, ≤0.01 nm, or ≤0.001 nm (e.g., below 10⁻⁸ M, e.g., 10⁻⁸ M to 10⁻¹³ M, e.g., 10⁻⁹ M to 10⁻¹³ M).

[0102] In this invention, a “VEGF antagonist” refers to any molecule capable of blocking, reducing, or interfering with the normal biological activity of VEGF or its receptor. The VEGF antagonist includes molecules that interfere with the interaction between VEGF and its natural VEGF receptor, such as molecules that prevent or otherwise interfere with the interaction between VEGF and its receptor by binding to VEGF or its 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”). The VEGF receptor-based chimeric molecule comprises a chimeric polypeptide containing two or more immunoglobulin (Ig)-like domains derived from VEGF receptors such as VEGFR1 (also known as Flt1) and / or VEGFR2 (also known as Flk1 or KDR), and may also contain a polymerizing domain (e.g., an Fc domain capable of polymerizing (e.g., dimerizing) two or more chimeric polypeptides). An exemplary VEGF receptor-based chimeric molecule is VEGFR1R2-FcΔC1(a) or Flt1D2.Flk1D3.FcΔC1 [also known as aflibercept (trade names: Eylea, Zaltrap)], which contains or is composed of the amino acid sequence of sequence number 21.

[0103] "VEGF antagonist" and "antibody containing an antigen-binding site for VEGF" refer to an antagonist or antibody capable of binding to VEGF, particularly VEGF peptides, with an affinity sufficient for VEGF-targeted diagnosis and / or treatment. According to one embodiment, the VEGF antagonist or antibody binds to unrelated non-VEGF proteins to less than about 10% of its binding to VEGF, as determined, for example, by radioimmunoassay (RIA) or flow cytometry (FACS), or by surface plasmon resonance analysis using a biosensor system such as the Biacore (registered trademark) system. For example, an antibody binding to human VEGF has a KD value of ≤1 μM, ≤100 nm, ≤10 nm, ≤1 nm, ≤0.1 nm, ≤0.01 nm, or ≤0.001 nm (e.g., below 10⁻⁸ M, e.g., 10⁻⁸ M to 10⁻¹³ M, e.g., 10⁻⁹ M to 10⁻¹³ M).

[0104] The term "mouse antibody" refers to an antibody having a variable region, the framework region and CDR region of which are derived from mouse germline immunoglobulin sequences. Furthermore, when the antibody contains a constant region, that constant region is also derived from mouse germline immunoglobulin sequences. The mouse antibodies of this disclosure may contain amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro, or through somatic mutations in vivo).

[0105] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remaining heavy chain and / or light chain originates from different sources or species.

[0106] An antibody's "class" refers to the type of constant structural domains or regions possessed by the antibody heavy chain. Antibodies are classified into five main classes: IgA, IgD, IgE, IgG, and IgM. Some of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant structural domains of the heavy chain corresponding to each immunoglobulin class are called α, δ, ε, γ, and μ, respectively.

[0107] "Humanized antibody" refers to a chimeric antibody containing amino acid residues derived from a non-human HVR and amino acid residues derived from a human FR. In some embodiments, the humanized antibody will contain at least one, typically two, substantially complete variable domains, wherein all or substantially all of the HVR (e.g., CDR) corresponds to the HVR of the non-human antibody, and all or substantially all of the FR corresponds to the FR of the human antibody.

[0108] The humanized antibody may also selectively include at least a portion of a constant region derived from a human antibody. An antibody, such as a non-human antibody, in its "humanized form" refers to an antibody that has undergone humanization. Another form of "humanized antibody" as included in this invention is one in which the constant region is further modified or altered relative to the constant region of the original antibody, particularly in relation to C1q binding and / or Fc receptor (FcR) binding, to produce the properties of this invention.

[0109] "Human antibody" refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human antibody using a human antibody library or other human antibody encoding sequences. This definition of human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0110] In this document, the term "Fc domain" or "Fc region" is used to define the C-terminal region of the immunoglobulin heavy chain, which includes at least a portion of the constant region. The term includes both native sequence Fc regions and variant Fc regions.

[0111] The term "linker" refers to a peptide containing 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 linker spaces the light chain variable domain and the heavy chain variable domain sufficiently apart so that the respective variable domains can fold into suitable secondary and tertiary structures. A suitable peptide linker sequence can be selected based on factors such as (a) the ability to form a flexible extended conformation; (b) the ability not to form secondary structures that interact with epitopes; and (c) the absence of hydrophobic or charged residues that can react with epitopes. Preferred peptide linkers contain Gly, Glu, Asn, Lys, Ser, and Pro residues. Other neutral amino acids such as Thr and Ala may also be included in the linker sequence. The linker sequence can consist of 1 to 50 amino acid residues, preferably 10 to 20 amino acid residues. For example, suitable non-immunogenic linker peptides may include, for example, 5 to 10 consecutive glycine (G) residues, but are not limited thereto.

[0112] The "percentage (%) amino acid sequence identity" relative to a reference polypeptide sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence after sequence alignment and, where necessary, the introduction of gaps to achieve the maximum percentage of sequence identity, without taking conserved substitutions into account. Alignments used to determine the percentage of amino acid sequence identity can be performed in various ways in the art, such as using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA package. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm required to achieve maximum alignment across the entire length range of the sequences being compared. However, for the purposes of this document, the % amino acid sequence identity value is generated using the BLOSUM50 alignment matrix and generated by the ggsearch program in FASTA package version 36.3.8c or later. The FASTA package is documented 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, sequences can be compared using a public server accessible from http: / / fasta.bioch.virginia.edu / fasta_www2 / index.cgi, and the ggsearch (global protein:protein) program with default options (BLOSUM50; open: -10; ext: -2; Ktup=2) can be used to perform global alignment instead of local alignment. The percentage (%) of amino acid sequence identity is provided in the output alignment header.

[0113] The term "polypeptide" as used herein is intended to include both the singular and plural forms of "polypeptide," referring 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 group of chains consisting of two or more amino acids, and is not limited to a specific length. Therefore, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to two or more amino acid chains or groups are included within the definition of "polypeptide," and the term "polypeptide" may be used interchangeably with or in place of the aforementioned terms. The term "polypeptide" is also intended to include post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modifications by non-natural amino acids. Polypeptides may be derived from natural biological sources or produced through recombinant technologies, but are not necessarily translated from a specific nucleotide sequence. They can be produced in any manner, including chemical synthesis. The term "polypeptide" also includes variants and derivatives of polypeptides. Furthermore, a "polypeptide fragment" refers to a polypeptide that, compared to a full-length protein, has a deletion of the N-terminal amino acid sequence, a deletion of the C-terminal amino acid sequence, and / or an internal deletion. These fragments may also contain amino acids that have been modified compared to the full-length protein. In one embodiment, the length of the fragment can be from about 5 to 900 amino acids, 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. For the purposes of this invention, useful polypeptide fragments include immunologically functional fragments containing an antigen-binding domain of an antibody. For antibodies that specifically bind to c-Kit and VEGF, such useful fragments include, but are not limited to, CDR sequences containing one, two, or three heavy or light chains, or all or part of an antibody chain containing a variable or constant region of a heavy or light chain.

[0114] As used herein, a “variant” of a polypeptide, such as an antigen-binding fragment, protein, or antibody, refers to a polypeptide with one or more amino acid residues inserted, deleted, added, and / or substituted compared to another polypeptide sequence, and includes fusion polypeptides. Furthermore, protein variants can be altered by protease cleavage, phosphorylation, or other post-translational modifications while retaining the biological activity of the antibodies disclosed herein, such as specific binding to c-Kit and VEGF and their biological activity. Such variants may have approximately 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the antibodies or antigen-binding fragments disclosed herein.

[0115] The term “recombinant” as used herein in relation to polypeptides or polynucleotides refers to a form of polypeptide or polynucleotide that does not exist in nature, and non-limiting examples include molecules formed by combining polynucleotides or polypeptides that do not normally exist simultaneously.

[0116] "Homology," "identity," or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing comparable positions in each sequence. When a position in a compared sequence is occupied by the same base or amino acid, the molecule is homologous at that position. The degree of homology between sequences is determined by the number of identical or homologous positions shared between the sequences. An "unrelated" or "non-homologous" sequence refers to a sequence that shares less than 40% identity, preferably less than 25%, with a sequence in this disclosure.

[0117] "Sequence identity" refers to the fact that when two sequences are compared and aligned, the bases (or amino acids) are identical within that percentage range.

[0118] The term "polynucleotide" refers to an isolated nucleic acid molecule or structure, such as messenger RNA (mRNA), viral RNA, or plasmid DNA (pDNA). Polynucleotides can contain conventional phosphodiester bonds or non-conventional bonds (such as amide bonds, as found in peptide nucleic acids (PNA)). The term "nucleic acid molecule" refers to any one or more nucleic acid fragments, such as DNA or RNA fragments, present in a polynucleotide.

[0119] "Isolated" nucleic acid molecules or polynucleotides refer to nucleic acid molecules, DNA, or RNA derived from their natural environment. For example, recombinant polynucleotides encoding polypeptides contained in a vector are considered isolated in this invention. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or polynucleotides purified (partially or substantially purified) in solution. Isolated polynucleotides generally include polynucleotide molecules present in the cells containing the polynucleotide molecule, but said polynucleotide molecules are present in an extrachromosomal form or located at a chromosomal location different from the natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts according to the invention, as well as positive and negative strand forms and double strand forms. Isolated polynucleotides or nucleic acids according to the invention also include molecules produced by synthesis. Furthermore, polynucleotides or nucleic acids may be or contain regulatory elements, such as promoters, ribosome binding sites, or transcription terminators. The term "isolated" as used herein also means that when nucleic acids or peptides are produced by recombinant DNA technology, there is essentially no cellular material, viral material, or culture medium, or when produced by chemical synthesis, there is essentially no chemical precursor or other chemical substance. The term "isolated" is also used to refer to cells or peptides isolated from other cellular proteins or tissues. Isolated peptides include purified peptides and recombinant peptides.

[0120] The term "expression cassette" refers to a recombinant or synthetically produced polynucleotide that enables transcription of a specific nucleic acid in a target cell using a series of specific nucleic acid elements. Recombinant expression cassettes can be inserted into plasmids, chromosomes, mitochondrial DNA, plasmid DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector includes, in particular, the nucleotide sequence to be transcribed and a promoter. In one embodiment, the expression cassette contains a polynucleotide sequence encoding the bispecific antibody of the present invention.

[0121] The term "vector" or "expression vector" refers to a DNA molecule used to introduce and express a specific gene operatively linked to it in a cell. This term includes not only vectors that function as self-replicating nucleic acid structures but also vectors that integrate and are introduced into the host cell genome. The expression vector of the present invention comprises an expression cassette. The expression vector enables the transcription of large amounts of stable mRNA. When the expression vector is present in a cell, ribonucleic acid molecules or proteins encoded by the gene can be produced through cellular transcription and / or translation mechanisms. In one embodiment, the expression vector of the present invention comprises an expression cassette containing a polynucleotide sequence encoding the bispecific antibody of the present invention.

[0122] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells (including their progeny) in which exogenous nucleic acids have been introduced. Host cells include "transformants" and "transformed cells," which include the initial transformed cell and its derived progeny, without limitation on the number of passages. The nucleic acid content of the progeny may not be exactly the same as that of the parent cells, but may contain mutations. Mutant progeny with the same function or biological activity as those screened or selected from the original transformed cells are also included herein. Host cells can be any type of cell system, as long as they can be used to produce the antibodies or bispecific antibodies of the present invention. Host cells include cultured cells, such as mammalian cultured cells, such as HEK cells, CHO cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, as well as yeast cells, insect cells and plant cells, and include cells in transgenic animals, transgenic plants or cultured plant or animal tissues.

[0123] The first aspect of the present invention relates to a bispecific antibody that specifically binds to c-Kit and VEGF.

[0124] The bispecific antibody according to the present invention, which specifically binds to c-Kit and VEGF, has the property of binding to c-Kit and VEGF with high affinity, and may include the following (a) and (b):

[0125] (a) A first antigen-binding site that specifically binds to c-Kit, comprising a heavy chain variable region including a heavy chain complementarity-determining region 1 (CDR1) containing the amino acid sequence of sequence number 2, a heavy chain complementarity-determining region 2 (CDR2) containing the amino acid sequence of sequence number 4, and a heavy chain complementarity-determining region 3 (CDR3) containing the amino acid sequence of sequence number 6; and a light chain variable region including a light chain complementarity-determining region 1 (CDR1) containing the amino acid sequence of sequence number 13, a light chain complementarity-determining region 2 (CDR2) containing the amino acid sequence of sequence number 15, and a light chain complementarity-determining region 3 (CDR3) containing the amino acid sequence of sequence number 17; and (b) a second antigen-binding site that specifically binds to VEGF, wherein the soluble extracellular domain of VEGF is fused with the Fc domain of IgG.

[0126] According to one embodiment of the present invention, the first antigen binding site of the bispecific antibody may include a light chain variable region and a heavy chain variable region, wherein the light chain variable region includes LCDR1, LCDR2 and LCDR3 composed of each amino acid sequence in (a) above, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 composed of each amino acid sequence in (a) above.

[0127] In this invention, the first antigen-binding site may include a heavy chain variable region and a light chain variable region. The heavy chain variable region contains an amino acid sequence having more than 90% sequence homology with the amino acid sequence of sequence number 27, and the light chain variable region contains an amino acid sequence having more than 90% sequence homology with the amino acid sequence of sequence number 28. Specifically, according to an embodiment of the present invention, the first antigen-binding site may include a heavy chain variable region, which contains an amino acid sequence having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology with the amino acid sequence of sequence number 27, and may also include a light chain variable region, which contains an amino acid sequence having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology with the amino acid sequence of sequence number 28.

[0128] The amino acid sequences of sequence numbers 27 and 28 are as follows:

[0129] QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGTNKDYTDSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDWAEAFDMWGQGTTVTVSS (Serial Number 27)

[0130] DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTITFGQGTRLEIK (Serial Number 28)

[0131] In this invention, the soluble extracellular domain may comprise an immunoglobulin-like domain 2 of a first VEGF receptor and an 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 invention, the VEGF antagonist may comprise or consist of the amino acid sequence of sequence number 21.

[0132] In this invention, the second antigen-binding site can be linked to the C-terminus or N-terminus of the heavy chain of the first antigen-binding site via a peptide linker; or, the second antigen-binding site can be linked to the C-terminus of the light chain of the first antigen-binding site via a peptide linker. According to a specific embodiment of the invention, the antibody whose second antigen-binding site is linked to the C-terminus of the heavy chain of the first antigen-binding site via a peptide linker is designated as "candidate antibody A," "candidate antibody B," "candidate antibody B-1," and "candidate antibody C"; the antibody whose second antigen-binding site is linked to the N-terminus of the heavy chain of the first antigen-binding site via a peptide linker is designated as "candidate antibody E"; and the antibody whose second antigen-binding site is linked to the C-terminus of the light chain of the first antigen-binding site via a peptide linker is designated as "candidate antibody D."

[0133] In this invention, the peptide linker connecting the first antigen binding site and the second antigen binding site can be composed of 5 to 10 glycine (Gly) linked together, and can be sequence number 20 (G5), sequence number 67 (G6), sequence number 68 (G7), sequence number 23 (G8), sequence number 69 (G9) or sequence number 70 (G10).

[0134] The Fc domain of the bispecific antibody of the present invention can be modified to reduce Fc receptor binding and / or effector function.

[0135] According to one embodiment of the present invention, one or more amino acid modifications can be introduced into the Fc region of the antibody provided by the present invention to prepare an Fc region variant. The Fc region variant may be contained in a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3 or IgG4) having amino acid modifications (e.g., substitutions) at one or more amino acid sites.

[0136] Specifically, the Fc domain of a bispecific antibody containing a first antigen-binding site that specifically binds to c-Kit and a second antigen-binding site that specifically binds to VEGF may contain one or more amino acid substitutions to reduce its binding to Fc receptors, particularly Fcγ receptors. For example, the Fc domain may be an Fc variant containing L234A and L235E amino acid substitutions; or a human IgG1 subclass of an Fc variant containing L234A, L235A, I253A, H310A, P329G, and H435A amino acid substitutions.

[0137] The Fc domain endows the bispecific antibody of the present invention with favorable pharmacokinetic properties, including a longer serum half-life, thereby facilitating good enrichment in target tissues and a favorable tissue-to-blood distribution ratio. However, the above-mentioned properties may also make the bispecific antibody of the present invention more likely to induce unintended targeting of cells expressing the Fc receptor compared to preferred antigen-containing cells. According to one embodiment of the present invention, the Fc domain of the bispecific antibody exhibits reduced binding affinity and / or reduced effector function to the Fc receptor compared to the wild-type IgG Fc domain, especially the IgG1 Fc domain. Preferably, the Fc domain can be the wild-type IgG1 Fc domain or a variant thereof.

[0138] According to a specific embodiment of the present invention, the Fc domain has a binding affinity to the Fc receptor of less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5%, compared to the wild-type IgG1 Fc domain (or the bispecific antigen-binding molecule of the present invention containing the wild-type IgG1 Fc domain), and / or its effector function is less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5%, compared to the wild-type IgG1 Fc domain. In one embodiment, the Fc domain substantially does not bind to the Fc receptor and / or does not induce effector function. 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 activated Fc receptor. In yet another embodiment, the Fc receptor is an activated 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 yet another embodiment, the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to the wild-type IgG1 Fc domain.

[0139] According to one embodiment of the present invention, the Fc domain can be modified to have a reduced binding affinity for the Fc receptor and / or a reduced effector function compared to the unmodified Fc domain (wild-type Fc). In one embodiment, the Fc domain of the bispecific antibody of the present invention may contain one or more amino acid mutations to reduce the binding affinity of the Fc domain to the Fc receptor and / or the effector function. Typically, the same one or more amino acid mutations are present in the two subunits of the Fc domain. The amino acid mutations can reduce the binding affinity of the Fc domain to the Fc receptor. For example, the amino acid mutations can reduce the binding affinity of the Fc domain to the Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold, or make the Fc domain essentially non-binding to the Fc receptor, thereby having no binding affinity. According to a specific embodiment of the present invention, the bispecific antibody of the present invention containing the modified Fc domain has a binding affinity for the Fc receptor-activated Fc receptor that is particularly less than 10%, and more particularly less than 5%, compared to the bispecific antibody of the present invention containing the unmodified 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 activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, preferably human FcγRIIIa. In one embodiment, the binding affinity to the complement component, specifically the binding affinity to C1q, may also be reduced. In another embodiment, the binding affinity to the neonatal Fc receptor (FcRn) may be reduced. The substantially similar binding to FcRn, i.e., maintaining the binding affinity of the Fc domain to the receptor, is achieved when the binding affinity of the Fc domain to FcRn reaches approximately 70% or more of the unmodified form. The Fc domain, or the bispecific antibody of the present invention containing the Fc domain, can exhibit approximately 80% and approximately 90% or more of the above-described affinity. In some embodiments, the Fc domain of the bispecific antigen-binding molecule of the present invention is modified to have reduced effector function compared to the unmodified 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 phagocytosis (ADCP), reduced cytokine secretion, reduced antigen uptake mediated by immune complexes mediated by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signal-induced apoptosis, reduced dendritic cell maturation, or reduced T cell initiation.

[0140] Antibodies with effector-reducing functions may include antibodies in which one or more of the residues 234, 235, 237, 253, 310, 329, and 435 of the Fc region are substituted. According to a specific embodiment of the present invention, the Fc domain may contain amino acid substitutions of L234A, L235A, and P329G (“LALAPG”), L234A, L235A, P329G, I253A, H310A, and H435A (“LALAPG-AAA”), L235A and G237A (“LAGA”), or amino acid substitutions of L234A and L235E (“LALE”).

[0141] In this invention, an exemplary bispecific antibody having a wild-type Fc domain can be candidate antibody A or candidate antibody D, but is not limited thereto. The heavy chain of the first antigen-binding site of candidate antibody A or candidate antibody D having a wild-type Fc domain may contain or be composed of the amino acid sequence of sequence number 29, and the light chain of the first antigen-binding site may contain or be composed of the amino acid sequence of sequence number 30.

[0142] The amino acid sequences of sequence numbers 29 and 30 are as follows:

[0143] QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGTNKDYTDSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDWAEAFDMWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Serial Number 29)

[0144] DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTITFGQGTRLEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Serial Number 30)

[0145] To reduce effector function, the Fc domain of the bispecific antibody according to the present invention may contain amino acid substitutions as defined above. In this invention, an exemplary bispecific antibody incorporating LALAPG into the Fc domain may be candidate antibody B or candidate antibody E, but is not limited thereto. The heavy chain of candidate antibody B or candidate antibody E incorporating LALAPG into the Fc domain may contain or be composed of the amino acid sequence of sequence number 31, and the light chain of the first antigen-binding site may contain or be composed of the amino acid sequence of sequence number 30.

[0146] The amino acid sequence of sequence number 31 is as follows.

[0147] QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGTNKDYTDSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDWAEAFDMWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Serial Number 31)

[0148] In this invention, the exemplary bispecific antibody incorporating LALE into the Fc domain can be candidate antibody C, but is not limited thereto. The heavy chain of candidate antibody C incorporating LALE into the Fc domain may contain or be composed of the amino acid sequence of sequence number 32, and the light chain of the first antigen binding site may contain or be composed of the amino acid sequence of sequence number 30.

[0149] The amino acid sequence of sequence number 32 is as follows.

[0150] QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGTNKDYTDSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDWAEAFDMWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Serial Number 32)

[0151] In this invention, an exemplary bispecific antibody incorporating LALAPG-AAA into the Fc domain can be candidate antibody B-1, but is not limited thereto. The heavy chain of candidate antibody B-1 incorporating LALAPG-AAA into the Fc domain may contain or be composed of the amino acid sequence of sequence number 33, and the light chain of the first antigen binding site may contain or be composed of the amino acid sequence of sequence number 30.

[0152] The amino acid sequence of sequence number 33 is as follows.

[0153] QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGTNKDYTDSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDWAEAFDMWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALGAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGK (Serial Number 33)

[0154] Binding to the Fc receptor can be readily measured, for example, by enzyme-linked immunosorbent assay (ELISA) or by surface plasmon resonance (SPR) using standard equipment such as BIAcore instruments (GE Healthcare), and the Fc receptor itself can be obtained through recombinant expression. The binding affinity of the Fc domain or cell-activating bispecific antibodies containing the Fc domain to the Fc receptor can be evaluated using cell lines known to express a specific Fc receptor, such as cell lines expressing the human NK cell FcγRIIIa receptor. The effector function of the Fc domain or the bispecific antibodies of the present invention containing the Fc domain can be determined using methods known in the art.

[0155] The bispecific antibody according to the present invention can be a tetravalent antibody having four binding sites. Preferably, the first antigen-binding site of the bispecific antibody according to the present invention can be a full-length bivalent antibody, and the second antigen-binding site can be composed of two VEGF antagonists, wherein the VEGF antagonists are formed by the 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 2+2 binding titer.

[0156] Faricimab (trade name: Vabysmo), marketed as a treatment for macular degeneration, is a 2 (1+1) valent antibody in the form of CrossMab targeting Ang-2 and VEGF. Accordingly, in one embodiment of the present invention, the first antigen binding site and the second antigen binding site are linked using the "knob-into-hole" technique. Two 2(1+1) valent antibodies [Fab-VEGFR-Fc(KH) and Fab-VEGFR-Fc(HK)] are prepared by linking anti-c-Kit antibody with VEGFR. A 2(1+1) valent antibody formed by linking c-Kit scFv with a VEGFR trap through a peptide linker (G4S or G8) is also prepared. The purity, stability (HMW and LMW generation), thermal stability, and binding ability to c-Kit and VEGF are evaluated. However, these antibodies show the detection of endotoxins, low stability (HMW and LMW generation), low thermal stability, and low antigen-antibody binding ability, confirming that they are not suitable as bispecific antibodies.

[0157] In a specific embodiment of the present invention, the antibody in the form of Fab-VEGFR-Fc(KH) prepared above is named "comparative antibody" and is used to compare its efficacy with the bispecific antibody of the present invention.

[0158] The “knob-into-hole” technique is described, for example, in U.S. Patent Nos. 5,731,168 and 7,695,936, in references [Ridgway et al., Prot Eng 9, 617-621 (1996)] and [Carter, JImmunol Meth 248, 7-15 (2001)]. Typically, this method involves introducing a knot into the contact interface of a first polypeptide, allowing it to insert into a corresponding cavity, thereby promoting the formation of heterodimers and inhibiting the formation of homodimers, while simultaneously introducing a corresponding cavity into the contact interface of a second polypeptide. The knot is constructed by replacing a smaller amino acid side chain in the contact interface 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 knot is formed in the contact interface of the second polypeptide by replacing the larger amino acid side chain with a smaller side chain (e.g., alanine or threonine). The protrusions and cavities can be prepared by altering the nucleic acid encoding the polypeptide, for example, through site-specific mutagenesis or peptide synthesis.

[0159] In this invention, to improve the binding affinity and / or other biological properties of the bispecific antibody, the amino acid sequence of the antibody can be altered. Such alterations may include, for example, the deletion, insertion, and / or substitution of amino acid residues in the antibody's amino acid sequence. These amino acid variations can be based on the relative similarity of amino acid side-chain substituents, such as hydrophobicity, hydrophilicity, charge, and size. Analysis of the size, shape, and type of the amino acid side-chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on the above considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functionally equivalent.

[0160] The substitution of amino acids in proteins without altering their overall molecular activity is known in the art (H. Neurath, RL Hill, *The Proteins*, Academic Press, New York, 1979). The most common substitutions include those between amino acid residues such as Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0161] Considering the aforementioned variations with biologically equivalent activity, the bispecific antibody and the nucleic acid molecule encoding it of the present invention should be interpreted as also including sequences with substantial identity to the sequences listed in the sequence listing. Substantial identity means that when the sequences of the present invention are aligned with any other sequences as far as possible, and the aligned sequences are analyzed using algorithms commonly used in the art, the sequences show at least 60% identity, preferably 70%, more preferably 80%, and most preferably at least 90% identity.

[0162] 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 with respect to the amino acid sequence defined above.

[0163] A second aspect of the invention relates to a nucleic acid molecule encoding the bispecific antibody of the invention as described above or a fragment thereof, a vector containing the nucleic acid molecule, and a host cell containing the vector.

[0164] 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 a fragment thereof may comprise or consist of a nucleotide sequence encoding the amino acid sequences shown in sequence numbers 2, 4, 6, 13, 15, 17, 21, 27, 28, 29, 30, 31, 32 and 33.

[0165] According to a specific embodiment of the present invention, the nucleotide sequences encoding the amino acid sequences of sequence numbers 2, 4, 6, 13, 15 and 17 may contain or be composed of the nucleotide sequences of sequence numbers 35, 37, 39, 46, 48 and 50, respectively, but are not limited thereto.

[0166] According to a specific embodiment of the present invention, the nucleotide sequence encoding the amino acid sequence of sequence number 21 may include or be composed of the nucleotide sequence of sequence number 54, but is not limited thereto.

[0167] According to a specific embodiment of the present invention, the nucleotide sequences encoding the amino acid sequences of sequence numbers 27 and 28 may sequentially include or be composed of the nucleotide sequences of sequence numbers 60 and 61, but are not limited thereto.

[0168] The nucleotide sequences of sequence numbers 60 and 61 are as follows.

[0169] CAGGGTGCAGCTGGTGGAATCTGGTGGCGGAGTTGTGCAGCCTGGCAGATCCCTGAGACTGTCTTGTGCCGCCTCCGGCTTCACCTTCTCCAGATACGGAATGCACTGGGTCCGACAGGCCCCTGGCAAAGGATTGGAATGGGTCGCCGTGATTTGGTACGACGGCACCAACAAGGACTAC ACCGACTCTGTGCGGGGCAGATTCACCATCTCTCGGGACAACTCCAAGAACACCCTGTACCTGCGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAGAGAGGATTGGGCCGAAGCCTTCGATATGTGGGGCCAGGGCACAACCGTGACCGTGTCCTCT (Serial No. 60)

[0170] GACATCGTGATGACCCAGTCTCCACTGAGCCTGCCTGTGACACCTGGCGAGCCTGCTTCCATCTCCTGCAGATCCTCAGTCCTGCTGCACTCCAACGGCTACAACTACCTGGACTGGTATCTGCAGAAGCCCGGCCAGTCTCCTCAGCTGCTGATCTACCTGGGCTC CAACAGAGCTTCTGGCGTGCCCGATAGATTCTCCGGCTCTGGCTCTGGCACCGACTTCACCCTGAAGATCTCCAGAGTGGAAGCCGAGGACGTGGGCGTGTACTACTGTATGCAGGCCCTGCAGACCATCACCTTCGGCCAGGGAACCAGACTGGAAATCAAG (Serial No. 61)

[0171] According to a specific embodiment of the present invention, the nucleotide sequences encoding the amino acid sequences of sequence numbers 29, 30, 31, 32 and 33 may contain or be composed of the nucleotide sequences of sequence numbers 62, 63, 64, 65 and 66, but are not limited thereto.

[0172] The nucleotide sequences of sequence numbers 62, 63, 64, 65 and 66 are as follows.

[0173] The nucleic acid molecule of the present invention should be interpreted as also including a nucleotide sequence that has substantial identity with the above-described nucleotide sequence. Substantial identity means that when the nucleotide sequence of the present invention is arranged with any other sequence as far as possible, and the arranged sequence is analyzed using algorithms commonly used in the art, the nucleotide sequence shows at least 80% identity, preferably at least 90%.

[0174] In one embodiment, the nucleic acid molecule may comprise a nucleic acid molecule having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity relative to the nucleotide sequence defined above.

[0175] In this invention, the vector includes plasmid vectors, phage vectors, Cosmed vectors, and viral vectors, such as phage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors, preferably plasmid vectors.

[0176] The carrier system of the present invention can be constructed by various methods known in the art, the specific methods of which are described in Sambrook et al. (2001), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, which is incorporated herein by reference.

[0177] The vectors of this invention can typically be constructed as vectors for cloning or vectors for expression. Furthermore, the vectors of this invention can be constructed using prokaryotic or eukaryotic cells as hosts.

[0178] When the vector of the present invention is an expression vector and a prokaryotic cell is used as the host, it typically contains a strong promoter capable of 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 translation initiation, and a transcription / translation termination sequence. When using Escherichia coli (E. coli) (e.g., BL21, HB101, DH5α, etc.) as the host cell, the promoter and operator regions of the E. coli tryptophan biosynthesis pathway (Yanofsky, C. (1984), J. Bacteriol., 158:1018-1024) and the left-handed promoter of phage λ (pLλ promoter, Herskowitz, I. and Hagen, D. (1980), Ann. Rev. Genet., 14:399-445) can be used as regulatory regions.

[0179] The vectors that can be used in this invention can be prepared by modifying plasmids commonly used in the art (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.), phage particles (e.g., pComb3X), bacteriophages (e.g., λgt4·λB, λ-Charon, λΔz1 and M13, etc.) or viruses (e.g., SV40, etc.).

[0180] When the vector of the present invention is an expression vector and a eukaryotic cell is used as the host, promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and HSV tk promoter) can be used, and generally contain a polyadenylated sequence as a transcription termination sequence.

[0181] The vector of the present invention can be fused with other sequences as needed to facilitate the purification of the amino terminus of the protein expressed therefrom. The fusion sequence may be, for example, glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6×His (hexahistidine; Quiagen, USA), but is not limited thereto.

[0182] The expression vector of the present invention may contain antibiotic resistance genes commonly used in the art as selection markers, such as resistance genes to ampicillin, gentamicin, carbendazim, chloramphenicol, streptomycin, kanamycin, G418 (Geneticin), neomycin, and tetracycline.

[0183] The vector used to express the bispecific antibody of the present invention can be a vector system in which the first antigen binding site and the second antigen binding site are linked by a peptide linker and expressed in the same vector, or it can be a system in which the first antigen binding site and the second antigen binding site are expressed in different vectors.

[0184] According to one embodiment of the present invention, the host cell is a cell transformed by the above-described vector. The host cell capable of stably and continuously cloning and expressing the vector of the present invention can be any host cell known in the art, such as strains of Bacillus spp. including Escherichia coli, Bacillus subtilis, and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, or Staphylococcus (e.g., Staphylococcus carnosus), but is not limited thereto.

[0185] The applicable eukaryotic host cells for the vector may include multicellular fungi such as Aspergillus spp. and Neurospora crassa from the Phylum Ascomycota, as well as single-celled fungi such as Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces. In addition, other lower eukaryotic organisms, higher eukaryotic organisms such as insect-derived cells, and cells derived from plants or mammals may also be used.

[0186] In this invention, the term "transfection" refers to the introduction of a target gene into a host cell using the recombinant vector of this invention, and has the same meaning as "transformation." Therefore, "transfection" and / or "transformation" of host cells includes any method of introducing nucleic acids into an organism, cell, tissue, or organ, and can be performed using appropriate standard techniques known in the art, depending on the host cell. 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, liposome transfection reagents, and drying / inhibition-mediated transformation methods.

[0187] A third aspect of the present invention relates to a method for preparing a bispecific antibody that specifically binds to c-Kit and VEGF, comprising the steps of (a) and (b): (a) culturing the transformant; and (b) recovering the bispecific antibody that specifically binds to c-Kit and VEGF from the culture.

[0188] The cultivation of transformants for antibody production can be carried out using appropriate culture media and conditions known in the art. The above-described culture process can be appropriately adjusted by those skilled in the art based on the selected strain. Cell culture can be classified into suspension culture and adherent culture according to the cell growth mode, and further classified into batch culture, fed-batch culture, and continuous culture according to the culture method. The culture medium used for culture should appropriately meet the requirements of the specific strain.

[0189] Culture media for animal cell culture include a variety of carbon sources, nitrogen sources, and trace elements. Examples of usable carbon sources include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; lipids 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 usable nitrogen sources include organic nitrogen sources such as peptone, yeast extract, meat broth, malt extract, corn soaking solution (CSL), and soybean flour, as well as 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 culture medium may also contain potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and their corresponding sodium salts as phosphorus sources. Additionally, it may contain metal salts such as magnesium sulfate or ferric sulfate. Furthermore, it may contain amino acids, vitamins, and appropriate precursors.

[0190] During cultivation, the pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid in an appropriate manner. Furthermore, defoamers such as polyethylene glycol esters of fatty acids can be used to suppress foam formation during cultivation. Additionally, to maintain the aerobic state of the culture, oxygen or oxygen-containing gas (e.g., air) can be introduced into the culture. The culture temperature is typically between 20°C and 45°C, preferably between 25°C and 40°C.

[0191] Antibodies obtained by culturing transformants can be used in their unpurified state, or they can be further purified to high purity using various conventional methods, such as dialysis, salting out, and chromatography. Among these, chromatography is the most commonly used method, and the appropriate column type and sequence can be selected from ion exchange chromatography, gel filtration chromatography (size exclusion chromatography), and affinity chromatography, depending on the characteristics of the antibody and the culture method.

[0192] A fourth aspect of the present invention relates to a pharmaceutical composition for the prevention or treatment of angiogenesis diseases utilizing the above-described bispecific antibody.

[0193] Specifically, the present invention provides a bispecific antibody that specifically binds to c-Kit and VEGF according to the first aspect described above, and a pharmaceutical composition containing the antibody as an active ingredient for the prevention or treatment of angiogenesis diseases.

[0194] Furthermore, the present invention also provides a method for the prevention or treatment of angiogenesis diseases, comprising administering to an individual in need of the treatment a therapeutically effective amount of a bispecific antibody that specifically binds to c-Kit and VEGF according to the first aspect described above.

[0195] Furthermore, the present invention also provides the use of a bispecific antibody that specifically binds to c-Kit and VEGF according to the first aspect described above in the preparation of a medicament for the prevention or treatment of angiogenesis diseases.

[0196] The term "angiogenic diseases" refers to diseases related to the occurrence or progression of angiogenesis. Any disease that can be treated with the bispecific antibody of this invention is included within the scope of angiogenic diseases. For example, the term "angiogenic diseases" may be selected from the group including cancer, leukemia, ocular vascular diseases, rheumatoid arthritis, psoriasis, chronic wounds, chronic inflammation, hemangiomas, angiofibromas, vascular malformations, atherosclerosis, vascular adhesions, vasculitis, pyogenic granulomas, bullous diseases, pulmonary hypertension, asthma, nasal polyps, infectious diseases, inflammatory bowel disease, periodontal disease, peritoneal adhesions, endometrial-associated diseases, uterine bleeding, ovarian cysts, osteomyelitis, bone diseases, sepsis, and autoimmune diseases, but is not limited thereto.

[0197] In this invention, the cancer may be selected from the group consisting of bone cancer, lung cancer, head cancer, neck cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, gastric cancer, liver cancer, pancreatic cancer, skin cancer, melanoma of the skin or eye, rectal cancer, perianal cancer, colon cancer, uterine cancer, breast cancer, ovarian cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine gland cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumors, central nervous system lymphoma, spinal cord tumors, glioblastoma, brainstem glioma, and pituitary adenoma, but is not limited thereto.

[0198] In this invention, the ocular vascular disease may be selected from the group including diabetic retinopathy, macular degeneration, age-related macular degeneration, glaucoma, glaucomatous retinitis pigmentosa, choroidal neovascularization, retinopathy of prematurity, corneal dystrophy, and retinal laminar separation, but is not limited thereto.

[0199] The pharmaceutical compositions according to the present invention may contain only the bispecific antibody, or may further contain one or more pharmaceutically acceptable carriers, excipients or diluents.

[0200] The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. The oral administration carrier may include lactose, starch, cellulose derivatives, magnesium stearate, and stearic acid, etc. Furthermore, the parenteral administration carrier may include water, suitable oils, physiological saline, aqueous glucose, and ethylene 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, methylparaben, or propylparaben, and chlorobutanol. In addition, other pharmaceutically acceptable carriers known in the art may be used (Remington's Pharmaceutical Sciences, 19th edition, Mack Publishing Company, Easton, PA, 1995).

[0201] The pharmaceutical compositions of the present invention can be administered to mammals, including humans, in any manner. For example, they can be administered orally or via enteral routes. As enteral routes, administration can be intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, percutaneous, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual, or rectal administration, but is not limited thereto. For example, the pharmaceutical compositions of the present invention can be prepared as injectable dosage forms and administered by pricking the skin with a 30-gauge fine needle, or by direct application to the skin.

[0202] The pharmaceutical compositions of the present invention can be prepared into oral or enteral formulations according to the above-described routes of administration. For oral formulations, the compositions of the present invention can be prepared into powders, granules, tablets, pills, sugar-coated tablets, capsules, solutions, gels, slurries, pastes, and suspensions using methods known in the art. For example, the active ingredient can be mixed with a solid excipient, pulverized, and then processed into a granular mixture after adding appropriate excipients to prepare tablets or sugar-coated tablets. Examples of excipients include sugars such as lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; starches such as corn starch, wheat starch, rice starch, and potato starch; celluloses such as cellulose, methylcellulose, sodium carboxymethylcellulose, and hydroxypropyl methylcellulose; and fillers such as gelatin and polyvinylpyrrolidone. In addition, cross-linked polyvinylpyrrolidone, agar, alginate, or sodium alginate can be added as disintegrants when necessary. Furthermore, the pharmaceutical compositions of the present invention may further comprise anti-caking agents, lubricants, wetting agents, fragrances, emulsifiers, and preservatives. For parenteral formulations, they can be prepared using methods known in the art as injections, creams, emulsions, topical ointments, oils, humectants, gels, aerosols, and nasal inhalers. The above dosage forms are described in all literature generally known in the field of pharmaceutics (Remington's Pharmaceutical Science, 15th edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour).

[0203] The total effective amount of the pharmaceutical composition of the present invention can be administered to the patient as a single dose or over a longer period of time using a fractionated treatment protocol with multiple doses. The content of the active ingredient in the pharmaceutical composition of the present invention can be adjusted according to the severity of the disease symptoms. For example, the daily dosage of the pharmaceutical composition of the present invention can be from 0.0001 to 100 mg / kg. However, the dosage of the pharmaceutical composition of the present invention can be appropriately determined by those skilled in the art based on the effective dosage for the patient, taking into account factors such as route of administration, frequency of administration, and the patient's age, weight, health status, sex, disease severity, diet, and excretion rate. The dosage form, route of administration, and method of administration of the pharmaceutical composition according to the present invention are not particularly limited as long as they achieve the effects of the present invention.

[0204] Furthermore, the pharmaceutical compositions of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents. When administered in combination with other therapeutic agents, the compositions of the present invention can be administered simultaneously, separately, or sequentially with the other therapeutic agents. These other therapeutic agents can be substances known to have therapeutic or ameliorative effects on neovascularization-related diseases, and include other antitumor therapies besides drug therapy, such as radiotherapy.

[0205] When the pharmaceutical composition of the present invention is administered in combination with other therapeutic agents, the bispecific antibody contained in the composition of the present invention and the other therapeutic agents can be prepared separately in different containers or together in the same container.

[0206] In this 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). The term does not indicate a specific age or sex. Therefore, it is intended to include females or males, adults and newborns, and fetuses. "Patient" refers to an individual suffering from a disease or disorder. The term patient includes both human and veterinary subjects.

[0207] 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.

[0208] The kit for detecting c-Kit and / or VEGF according to the present invention can be applied to biological samples for the diagnosis of angiogenesis diseases. Therefore, the present invention provides a diagnostic kit for angiogenesis diseases comprising the above-described bispecific antibodies, and a method for diagnosing angiogenesis diseases using the above-described bispecific antibodies.

[0209] The kit of the present invention can be prepared for various immunoassays or immunostaining. These immunoassays or immunostainings include, but are not limited to: enzyme-linked immunosorbent assay (ELISA), immunofluorescence, Western blot, immunohistochemistry staining, flow cytometry, immunocytochemistry, radioimmunoassay (RIA), immunoprecipitation assay, and protein chip analysis.

[0210] In addition to bispecific antibodies against c-Kit and VEGF, the kit of the present invention may further include tools or reagents known in the art for immunological analysis.

[0211] Tools or reagents used for immunological analysis may include suitable carriers or supports, labels capable of generating detectable signals, solvents, detergents, and stabilizers. Suitable carriers include, for example (but are not limited to): when the label is an enzyme, substrates for measuring enzyme activity, suitable buffer solutions, secondary antibodies labeled with chromogenic enzymes or fluorescent substances, chromogenic substrates, and reaction terminators.

[0212] The bispecific antibodies against c-Kit and VEGF included in the kit of this invention are preferably immobilized on suitable carriers or supports using various methods disclosed in the literature. Examples of suitable carriers or supports include PBS, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluoropolymers, agarose, cellulose, nitrocellulose, dextran, Sephadex, Sepharose, liposomes, carboxymethyl cellulose, polyacrylamide, polystyrene, reaction plates, filter paper, ion exchange resins, plastic films, plastic tubes, polyamine-methyl vinyl ether-maleic acid copolymers, amino acid copolymers, ethylene-maleic acid copolymers, nylon, metals, glass, glass microbeads, or magnetic particles. Other solid matrices include cell culture plates, ELISA plates, test tubes, and polymer membranes. The supports can be of any form, such as spherical (microbeads), cylindrical (test tube or well wall), or planar (sheets, test strips).

[0213] Labels capable of generating detectable signals can be used for qualitative or quantitative detection of antigen-antibody complex formation. Examples include enzymes, fluorescent substances, ligands, luminescent materials, microparticles, redox molecules, and radioactive isotopes. Examples of enzymes include β-glucuronidase, β-D-glucosidase, urease, peroxidases (such as horseradish peroxidase), alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, invertase, and luciferase. Examples of fluorescent substances include luciferin, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, and luciferin isothiocyanate. Examples of ligands include biotin derivatives. Examples of luminescent materials include acridine esters and luciferin. As microparticles, colloidal gold, colored latex, etc., can be used; as redox molecules, ferrocene, ruthenium complexes, bipyridyl salts, quinones, Ti ions, Cs ions, diimine, 1,4-benzoquinone, hydroquinone, etc., can be used. As radioactive isotopes, [the following can be used]. 3 H, 14 C 32 P, 35S, 36 Cl、 51 Cr 57 Co、 58 Co、 59 Fe、 90 Y、 125 I, 131 I, 186 Re, etc. However, this is not limited to the examples above; any substance that can be used in methods applicable to immunological analysis may be employed.

[0214] For example, when horseradish peroxidase (HRP) is chosen as the enzyme label, a solution containing 3-amino-9-ethylcarbazole, 5-aminosalicylic acid, 4-chloro-1-naphthol, o-phenylenediamine, 2,2'-azidobis(3-ethylbenzothiazoline-6-sulfonic acid), 3,3-diaminobenzidine, 3,3',5,5'-tetramethylbenzidine, o-anisidine, or 3,3-dimethoxybenzidine can be used as the substrate. Furthermore, when alkaline phosphatase is chosen as the enzyme label, a solution containing 5-bromo-4-chloro-3-indolyl phosphate, nitroblue tetrazolium, or p-nitrophenyl phosphate can be used as the substrate. Additionally, when β-D-galactosidase is chosen as the enzyme label, a solution containing o-nitrophenyl-β-D-galactopyranoside or 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside can be used as the substrate. In addition, various enzymes and enzyme chromogenic substrates known in the art can be used.

[0215] According to one embodiment of the present invention, the c-Kit and / or VEGF detection method can detect c-Kit and / or VEGF proteins in biological samples or mammals, including humans. Specifically, the c-Kit and / or VEGF detection method according to the present invention may include the step of detecting c-Kit and / or VEGF antigen-antibody complexes using the above-described bispecific antibodies.

[0216] In this invention, the sample is a biological sample, such as tissue, cells, whole blood, serum, plasma, tissue biopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatant, lysed eukaryotic cells, and bacterial expression systems, but not limited thereto. For example, the biological sample may be derived from mammals such as humans suffering from c-Kit and / or VEGF overexpression-related diseases or c-Kit and / or VEGF-related diseases, or from animal models of c-Kit and / or VEGF protein overexpression or c-Kit and / or VEGF-related diseases, but not limited thereto. These biological samples, in a treated or untreated state, can be reacted with the bispecific antibody of this invention to confirm the presence or absence of c-Kit and VEGF proteins.

[0217] The mammals mentioned include, but are not limited to, livestock (such as cattle, sheep, cats, dogs, and horses), primates (such as humans, non-human primates such as monkeys), rabbits, and rodents (such as mice and rats).

[0218] Implementing the embodiments of the present invention

[0219] The present invention will be described in more detail below through embodiments. However, the following embodiments are only for illustrating the present invention and do not limit the scope of the present invention.

[0220]

Preparation Example 1

[0221] Construction of bispecific candidate antibodies that specifically bind to c-Kit and VEGF

[0222] 1-1. Construction of the anti-c-Kit antibody expression vector

[0223] Based on the antibody sequence confirmed in Patent Document 1, a signal peptide sequence was added to its front end before gene synthesis (contracted to Bioneer). The synthesized antibody gene was cloned into the PhiC31 vector (SBI, FC600A-1-SBI). The cloning steps are as follows: First, the synthesized antibody gene and the PhiC31 vector were prepared with restriction endonucleases NotI (New England Biolabs, R3189S) and EcoRV (New England Biolabs, R3195S), respectively, and 10× buffer (10× Cutsmart Buffer) to a total reaction volume of 50 μL, and reacted in a 37°C incubator for 5 hours. Subsequently, electrophoresis was performed on a 1% agarose gel. After confirming the formation of a band at the expected size and position, the band was excised and purified using a purification kit (MN, Macherey-Nagel). The two isolated products were mixed with T4 ligase (New England Biolabs, M0202S) and 10× buffer (10× T4 DNA Ligase Buffer) to prepare a 20 μL reaction system, and reacted at 25°C for 2 hours. The ligated vector was transformed into E. coli DH5α competent cells (RBC, RH718) and plated on LB agar plates containing kanamycin, and cultured at 37°C for 16 hours. After obtaining single colonies, the colonies were cultured, and DNA was extracted using a Mini-prep kit (Qiagen, 27106). The obtained DNA was digested with the same restriction endonuclease, and the gene insertion into the vector was confirmed by electrophoresis based on the size of the digested bands. Final analysis was then performed by sequencing (contracted to Bioneer) to obtain the vector expressing the anti-c-Kit antibody.

[0224] 1-2. Cloning and expression of bispecific candidate antibodies that specifically bind to c-Kit and VEGF

[0225] Based on the constructed vector expressing the anti-c-Kit antibody, the antibody heavy chain, peptide linker G5 (serial number 20), and VEGFR sequence were cloned via gene synthesis (contracted to Bioneer). The cloning steps were as follows: First, to remove the invariant region of the original vector, restriction endonucleases NheI (New England Biolabs, R3131S) and EcoRV (New England Biolabs, R3195S) were mixed with 10× buffer (10× Cutsmart Buffer) to a total reaction volume of 50 μL, and incubated at 37°C for 5 hours. Subsequently, the cleaved bands were confirmed by electrophoresis on a 1% agarose gel, and the cleaved vector was separated using a purification kit (MN, Macherey-Nagel). Using the same method, the antibody heavy chain, linker, and VEGFR gene obtained through gene synthesis were cleaved with restriction endonucleases NheI and EcoRV, and then separated and purified to obtain the insert fragment. To ligate the digested vectors with the insert fragments, T4 ligase (New England Biolabs, M0202S) and 10× buffer (10×T4 DNA Ligase Buffer) were added to each gene to a total volume of 20 μL, and the reaction was carried out at 25°C for 2 hours. The ligated vectors were then transformed into *E. coli* DH5α competent cells (RBC, RH718) and plated on LB agar plates containing kanamycin. The plates were incubated at 37°C for 16 hours to confirm the formation of single colonies. Mini-prep (Qiagen, 27106) was performed on the single colonies to obtain DNA. The obtained DNA was digested with restriction endonucleases, and the insertion of the gene into the vector was confirmed by electrophoresis based on the size of the cleavage bands. This was subsequently verified by sequencing (contracted to Bioneer).

[0226] The constructed vector was cultured in 3 mL LB medium containing antibiotics for 8 hours, then transferred to 250 mL LB medium containing the same antibiotics and cultured for another 16 hours. Plasmid DNA was extracted using the Maxi-prep kit (MN, Macherey-Nagel, 740414.50), filtered through a 0.22 μm PES filter, and introduced into cells as follows.

[0227] One day before transfection, ExpiCHO cells (Gibco) were seeded in ExpiCHO expression medium (Gibco, A2910002) and adjusted to a concentration of 3 × 10⁻⁶. 6 ~4×10 6 Live cells / mL, cultured for 1 day at 8% CO2, 37℃, and 120 rpm. On the day of DNA transfection, the cells will grow to 7 × 10⁻⁶ cells / mL. 6 ~1×10 7 Cells with a viable cell count / mL and a viability greater than 95% were diluted to 6 × 10⁶ cells / mL with fresh culture medium. 6 Live cells / mL were prepared for use. When transfecting the prepared ExpiCHO cells, the ExpiFectamine CHO transfection kit (Gibco, A29129) and OptiPRO SFM medium (Gibco, 12309019) were used, and DNA was transfected at a concentration of 1 μg / mL. After transfection, feed was added on day 1, and the culture conditions were adjusted to 5% CO2, 32℃, and 120 rpm. Feed was added again on day 5, and the cells were cultured for another 10 to 12 days to complete expression production.

[0228] To obtain the culture medium after production, the culture medium was transferred to a centrifuge container and centrifuged at 4°C and 8000 rpm for 30 minutes. The supernatant was then mixed with diatomaceous earth and filtered through a 0.2 μm PES filter membrane to remove suspended solids, thus obtaining the culture medium for subsequent purification.

[0229] Purification was performed using affinity chromatography (Protein A) resin. The column was first equilibrated with 1×PBS, then the recovered culture medium was loaded onto the column. After loading, the column was washed with equilibration buffer, followed by an intermediate wash with 20 mM sodium citrate, 5% sorbitol, and pH 5.0 buffer. After washing, elution was performed with 20 mM sodium citrate, 5% sorbitol, and pH 3.5 buffer. The eluent was neutralized to pH 7.0 by adding 1 M Tris-HCl (pH 9.0).

[0230] For further purification, ion exchange chromatography (cation exchange) resin was used. The column was first equilibrated with equilibration buffer [20 mM potassium phosphate, pH 7.0], and then the neutralized eluent was loaded onto the column. After loading, the column was washed with equilibration buffer and eluted with 20 mM potassium phosphate, pH 8.0 buffer. Subsequently, the eluent was buffer-displaced and concentrated with 20 mM L-histidine, 9% sucrose, pH 6.0, and its suitability for subsequent activity analysis was confirmed by purity analysis (SEC-HPLC) and endotoxin analysis. The finally confirmed antibody was stored at -80°C before use.

[0231] The resulting bispecific antibody is in the form of a VEGFR linked to the C-terminus of the IgG heavy chain of the anti-c-Kit antibody via a G5 peptide linker. The structure of the prepared bispecific antibody is shown below. Figure 1 As shown in table a, it is named "Candidate Antibody A". The amino acid sequence of Candidate Antibody A and its encoded nucleotide sequence information are listed in Tables 2 and 3, respectively.

[0232] Table 2

[0233]

[0234] Table 3

[0235]

[0236]

Example 1

[0237] Fc Engineering

[0238] 1-1. Introducing multiple Fc mutations

[0239] The peptide linker connecting the anti-c-Kit antibody to VEGFR was replaced with a G8 peptide linker instead of the G5 peptide linker used in the preparation example, and mutations were introduced into the Fc region to reduce Fc receptor binding and / or effector function. Fc mutation types that can reduce FcγRIIIa (CD16a) binding affinity to below 10% are shown in Table 4. The above four Fc mutations were introduced into the Fc region to prepare bispecific antibodies. For comparison, bispecific antibodies with a G8 peptide linker and wild-type Fc were also prepared. The preparation methods for these bispecific antibodies were the same as in Preparation Example 1.

[0240] Table 4

[0241] FcγIII binding force relative to wild type 3% 8% 8% 1.5%

[0242] 1-2. Confirm Fc receptor binding affinity

[0243] The five bispecific antibodies prepared (i.e., bispecific antibodies with G8 peptide linkers and wild-type Fc, LALAPGFc mutant, LAGA Fc mutant, LALE Fc mutant or LD Fc mutant respectively) were screened by the following SPR analysis to identify the Fc mutant with the best function in reducing immune effector function.

[0244] To confirm the binding affinity of the five bispecific antibodies listed in Table 8 for the Fc receptor, SPR analysis was performed. 20× HBS-EP+ buffer was diluted with deionized water to 1× as the run buffer, and 10 N NaOH was diluted with deionized water to 5 mM to prepare the regeneration solution (5 mM NaOH). Immobilization and binding analysis were performed using 1× HBS-EP+ buffer at 25°C. The ligand and analyte were designated as the Fc receptor and antibody, respectively.

[0245] The ligand immobilization method employed amine coupling. An EDC / NHS mixture was injected into both the sample and reference flow cells for 420 seconds to activate the sensor chip surface. The Fc receptor protein, diluted with 10 mM sodium acetate solution, was injected only into the sample flow cell, allowing the protein to be covalently immobilized onto the sensor chip surface. The reference flow cell served as a control group for correcting non-specific signals, and the protein immobilization step was omitted. Once an appropriate immobilization level was reached, ethanolamine was injected into both the sample and reference flow cells for 420 seconds to inactivate any remaining activation sites.

[0246] The general settings and operating conditions are shown in Table 5.

[0247] Table 5

[0248] Running conditions in human c-Kit analysis

[0249] Immobilization method Covalent fixation using amine coupling activation EDC / NHS mixture, lasting 420 seconds Deactivation Ethanolamine, lasting 420 seconds fixative solution 10mM sodium acetate, pH 5.5 Flow rate 10uL / min concentration 1ug / mL Run buffer HBS-EP+

[0250] To obtain data for affinity analysis, binding analysis was performed. Different concentrations of analyte were flowed through sample and reference flow cells, and binding and dissociation signals were measured in real time. The injection sequence was from low to high concentration, with each concentration injected as an independent cycle. One cycle included: a baseline phase where only running buffer was flowed before analyte injection; a binding phase where analyte injection and binding to the ligand; a dissociation phase where analyte injection was stopped and only running buffer was flowed to allow dissociation of the ligand-analyte complex; and a regeneration phase where regeneration solution was injected to restore the ligand to its original state.

[0251] The analyte concentrations and operating conditions for each ligand are shown in Tables 6 and 7.

[0252] Table 6

[0253] Analyte concentration

[0254] Human FcγRI 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200 nm Human FcγRIIa (167 His) 15.625, 31.25, 62.5, 125, 250, 500, 1000, 2000 nm Human FcγRIIIa (176 Val) 15.625, 31.25, 62.5, 125, 250, 500, 1000, 2000 nm Human FcγRIIIb (NA1) 62.5, 125, 250, 500, 1000, 2000 nm Human FcγRIIIb (NA2) 62.5, 125, 250, 500, 1000, 2000 nM Human FcRn (pH 6.0) 62.5, 125, 250, 500, 1000, 2000 nM Human FcRn (pH 7.4) 62.5, 125, 250, 500, 1000, 2000 nm

[0255] Table 7

[0256] Run configuration

[0257] dissociation time 120 seconds, 300 seconds for FcγRI analysis. Flow rate 30 μL / min Regeneration solution and injection time 5mM NaOH, 10 seconds

[0258] The collected data was evaluated using Biacore Insight Evaluation Software.

[0259] To calculate binding affinity (KD), a steady-state affinity method was employed. For FcγRI, since the steady-state affinity method is not applicable, kinetic analysis was used to calculate the binding rate constant (kon) and dissociation rate constant (koff), and the binding affinity (KD) was calculated as the ratio of the dissociation rate to the binding rate. When the binding reaction signal is too low or non-specific binding occurs, the binding affinity (KD) cannot be accurately determined; only the presence or absence of binding is assessed.

[0260] As shown in Table 8, when the LALAPG, LAGA and LALE Fc mutants are introduced, the binding affinity between the Fc fragments expressed in immune cells and the Fcγ receptor decreases. However, contrary to the expectations in Table 4, when the LD Fc mutant is introduced, its binding ability to FcγIII is increased compared to wild-type Fc. Therefore, it can be determined that it is not a suitable Fc variant.

[0261] Table 8

[0262]

[0263] Next, to confirm whether the immune cells contained in human peripheral blood mononuclear cells (PBMCs) actually fail to produce immune effector function due to the above mutations, an antibody-dependent cellular cytotoxicity (ADCC) assay was performed.

[0264] To determine antibody-dependent cytotoxicity (ADCC), experiments were performed using 96-well cell culture plates. Target cells (GIST-430 / 654 cells) were seeded at 6 × 10³ cells per well, with a final volume of 100 μL, and cultured overnight. Subsequently, before starting ADCC analysis, the supernatant was removed, and effector cells (human peripheral blood mononuclear cells, hPBMCs; Lonza, CC-2704) were added at 1.5 × 10³ cells per well. 5 Target cells were cultured at a total volume of 50 μL to achieve an effector cell:target cell ratio of 25:1. Each bispecific antibody was diluted with PBS at 20 μg / mL (treatment 2-fold concentration) and serially diluted 10-fold in dilution plates. 50 μL of antibody was added to each culture plate containing both target and effector cells, and the plate was 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 the plate was incubated at room temperature for approximately 10 minutes. The luminescence signal was then measured using the GloMax® Navigator system. The results were compared with the negative control group (untreated group) and plotted graphically.

[0265] like Figure 2 As shown, cytotoxicity was observed in the wild-type Fc group, but not in the groups treated with the LALAPG, LAGA, and LALE mutants, respectively. Conversely, while cytotoxicity was reduced compared to wild-type in the LD Fc variants, it still exhibited some toxicity. Trastuzumab was used as a positive control. Therefore, the LALAPG, LAGA, and LALE Fc mutants significantly reduced ADCC activity compared to wild-type Fc, confirming these three Fc variants as the optimal variants.

[0266] Based on the above results, the bispecific antibodies linked by the G8 peptide linker and incorporating the LALAPG and LALE Fc mutants respectively were named "Candidate Antibody B" (LALAPG) and "Candidate Antibody C" (LALE), respectively. The structural and sequence information of these bispecific antibodies is listed below. Figures 3a to 3b And Tables 9 to 12.

[0267] Table 9

[0268]

[0269] Table 10

[0270]

[0271] Table 11

[0272]

[0273] Table 12

[0274]

[0275] 1-3. Further introduction of I253A, H310A and H435A mutations

[0276] Based on the aforementioned candidate antibody B, I253A, H310A, and H435A mutations were introduced to prepare a bispecific antibody, which was named "candidate antibody B-1". The binding affinity of candidate antibody B-1 to the human neonatal Fc receptor (human FcRn) was evaluated using SPR analysis. As shown 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.

[0277] Table 13

[0278] Combining affinity

[0279]

[0280] ND*: The signal could not be detected because the response unit (RU) was too low.

[0281] The loss of FcRn binding leads to a shortened half-life in serum. Therefore, based on the results of the above examples, it can be expected that further introduction of I253A, H310A, and H435A mutations can reduce the systemic side effects that may be caused by simultaneously targeting c-Kit and VEGF in the blood. The structure and sequence information of candidate antibody B-1 are as follows: Figure 3c As shown in Tables 14 and 15.

[0282] Table 14

[0283]

[0284] Table 15

[0285]

[0286]

Example 2

[0287] Preparation of various bispecific antibodies with different VEGFR (VEGFR) linker sites

[0288] To prepare a bispecific antibody with VEGFR linked to the C-terminus of the c-Kit antibody light chain, the c-Kit antibody expression vector constructed in Example 1-1 was used as a basis. The antibody light chain was cloned by gene synthesis (contracted to Bioneer). Subsequently, the cloning, expression, and purification of the antibody were performed according to the method in Example 1-2. The structure of the resulting antibody is shown below. Figure 4a As shown, it was named "Candidate Antibody D". The sequence information of Candidate Antibody D is the same as that of Candidate Antibody A.

[0289] Next, to prepare a bispecific antibody containing the VEGFR receptor linked to the N-terminus of the c-Kit antibody heavy chain via a G8 peptide linker and with the LALAPG mutation introduced into the Fc region, the c-Kit antibody expression vector constructed in Example 1-1 was used as a basis. The mutant antibody heavy chain was cloned by gene synthesis (contracted to Bioneer) by linking the linker and the VEGFR receptor sequence. Subsequently, the cloning, expression, and purification of the antibody were performed according to the method in Example 1-2. The structure of the resulting antibody is shown below. Figure 4b As shown, it was named "Candidate Antibody E". The sequence information of Candidate Antibody E is the same as that of Candidate Antibody B.

[0290] Based on the above results, the information of the six candidate antibodies prepared is summarized in Table 16.

[0291] Table 16

[0292] Molecular form of bispecific antibodies IgG(H)-VEGFR IgG(H)-VEGFR IgG(H)-VEGFR IgG(H)-VEGFR VEGFR-IgG (L) VEGFR-IgG(H) Combined price 2+2 2+2 2+2 2+2 2+2 2+2 Fc Engineering wild type LALAPG LALAPG-AAA LALE wild type LALAPG Connector subtypes G5 G8 G8 G8 G5 G8

[0293] [Example 3]

[0294] Confirming the binding ability of c-Kit and VEGF

[0295] To confirm the target binding ability of the six bispecific antibodies in Table 16, an ELISA experiment 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 sequence numbers 71 and 72, respectively.

[0296]

[0297]

[0298] To coat the c-Kit / VEGF working solution onto the detection plate, add 100 μL of working solution (0.5 μg / mL) to each well, seal with sealing film, and incubate overnight at 2–8°C. Use 5% BSA solution diluted in PBS as blocking buffer, and filter through a 0.22 μm filter. After removing the coating buffer, wash the plate three times with 300 μL / well of 0.05% PBST buffer, then add 300 μL of blocking buffer to each well. Cover the plate with a sealing device and incubate at 25±2°C for 1 hour without shaking.

[0299] Each antibody was diluted to 1 μg / mL with DPBS and serially diluted 2-fold in the dilution plate. After blocking, the solution in the assay plate was removed, 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 sealing film and incubated at 25±2℃ for 1 hour without shaking. After 1 hour, the plate was washed three times with 300 μL / well of 0.05% PBST buffer, and 100 μL of diluted secondary antibody solution (80 ng / mL; final concentration of secondary antibody) was added to each well using a multichannel pipette. The plate was sealed with sealing film and incubated at 25±2℃ for 1 hour without shaking. After the secondary antibody incubation, the solution in the assay plate was removed. Wash the plate three times with 300 μL / well of 0.05% PBST buffer, and add 100 μL of TMB substrate reagent solution to each well using a multichannel pipette. Cover the plate with sealing film and incubate at 25±2℃ for 5 minutes. Add 100 μL of 1N sulfuric acid to each well and read the plate within 10 minutes. Measure the absorbance at 450 nm using a SPECTROstar Nano.

[0300] like Figure 5a and 5b As shown, all six antibodies exhibited concentration-dependent binding to c-Kit and VEGF, and demonstrated excellent binding ability to both c-Kit and VEGF.

[0301] [Example 4]

[0302] Confirmed binding affinity of c-Kit and VEGF

[0303] To confirm the binding affinity of the six bispecific antibodies in Table 9 for c-Kit and VEGF, SPR analysis was performed. 20× HBS-EP+ buffer was diluted with deionized water to a 1× run buffer, and 10 N NaOH was diluted with deionized water to a 10 mM regeneration solution (10 mM NaOH). Immobilization and binding analysis were performed using 1× HBS-EP+ buffer at 25°C.

[0304] In human c-Kit analysis, the c-Kit protein was directly immobilized onto the sensor chip surface via primary amine coupling. Freshly prepared EDC / NHS mixture was injected into the sensor chip surfaces of both the sample and reference flow cells, and activated for 420 seconds. Subsequently, the c-Kit protein was diluted with 10 mM sodium acetate solution and injected into the sample flow cell to achieve an appropriate immobilization level; the reference flow cell served as a blank. After the amine coupling reaction, the remaining activated coupling sites were blocked with ethanolamine for 420 seconds.

[0305] In human VEGF165 analysis, protein A, serving as the capture molecule, is immobilized on the sensor chip surface via primary amine coupling. This amine coupling process is identical to that of c-Kit analysis, except that protein A is immobilized in two channels. Subsequently, diluted antibody or aflibercept (Eylea) is injected only into the sample flow cell, utilizing the affinity of its Fc region to capture the pre-immobilized protein A.

[0306] General settings and operating conditions are shown in Tables 17 to 19.

[0307] Table 17

[0308] General Settings

[0309] Run buffer HBS-EP+ Flow rate 10 μL / min

[0310] Table 18

[0311] Run configuration in human c-Kit analysis

[0312] Fixing method Covalent fixation using amine coupling Flow cell used Fc2 (Fc1 is the reference pool) fixative solution 10mM sodium acetate, pH 5.5 concentration 2μg / mL activation EDC / NHS mixture, 420 seconds Deactivation Ethanolamine, 420 seconds

[0313] Table 19

[0314] Runtime configuration in human VEGF165 analysis

[0315] name Protein A Antibody Fixation method Covalent fixation using amine coupling Capture using affinity for protein A Use a flow cell Fc1, Fc2 Fc2 fixative solution 10mM sodium acetate, pH 4.5 N / A concentration 30μg / mL c-Kit antibody: 4nm; Aflibercept (Eylea): 6nm activation EDC / NHS mixture, 420 seconds N / A Deactivation Ethanolamine, 420 seconds N / A

[0316] Binding analysis was performed at a flow rate of 30 μL / min. All analytes were diluted in run buffer at concentration gradients, and all samples were prepared on ice. After injecting the analyte sample during the binding phase, run buffer was injected to achieve dissociation. The sensor chip surface was regenerated in each cycle by injecting regeneration solution. In c-Kit analysis, 10 mM NaOH was used as the regeneration solution; in VEGF analysis, 10 mM glycine at pH 1.5 was used. Analyte concentrations and operating conditions are shown in Tables 20 and 21.

[0317] Table 20

[0318] Analyte concentration

[0319] c-Kit Antibody 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10nm Human VEGF165 Antibody 0.020, 0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10nm

[0320] Table 21

[0321] Run configuration

[0322] Start loop Twice, using run buffer Blank loop Two cycles per analyte, performed after the concentration gradient. Combined with time c-Kit analysis: 240 seconds, VEGF165 analysis: 120 seconds dissociation time c-Kit analysis: 480 seconds, VEGF165 analysis: 240 seconds Flow rate 30 μL / min

[0323] Data analysis was performed using Biacore Insight Evaluation Software. A 1:1 model was used to fit the overall sensor spectrum curves, and kinetic parameters (combination rate constant [ka] and dissociation rate constant [kd]) were calculated. The KD value (equilibrium dissociation constant) was calculated using the ratio of kd to ka.

[0324] like Figure 6a and 6b As shown, all six antibodies exhibited excellent binding affinity for c-Kit and VEGF.

[0325]

Example 5

[0326] Stability confirmed

[0327] 5-1. Thermal stability

[0328] DSF analysis was performed to assess the thermostability of the six bispecific antibodies listed in Table 16. A PBS (1X) buffer diluted with PBS (20X) buffer was prepared as a negative control, and the stock solutions of each test sample were diluted to 1 mg / mL using PBS (1X), resulting in a total test sample volume of 5 μg for each reaction. Protein Thermal Shift™ dye (1000X) was freshly diluted to 8X using PBS (1X) buffer. The reaction components listed in Table 22 were added to each well of the MicroAmp® Optical 8 strip.

[0329] Table 22

[0330] Reaction components

[0331] Protein Thermal Shift™ Buffer 5.0μL Diluted Protein Thermal Shift™ dye (8X) 2.5μL Diluted test sample (1 mg / mL) 5.0μL PBS (1X) 7.5μL Total volume of each reaction 20μL

[0332] Seal the tubes using a MicroAmp® Optical 8-Cap Strip. Mix the samples thoroughly using a vortex mixer and then centrifuge. Each reaction was prepared four times. Monitor fluorescence readings using the StepOnePlus® Real-Time PCR System and its control software. Perform the protein melting experiment according to the experimental conditions shown in Table 23 below.

[0333] Table 23

[0334] Experimental characteristics

[0335]

[0336] Use Protein Thermal Shift™ software to analyze the melting temperature (Tm). Set the analysis mode as shown in Table 24 below.

[0337] Table 24

[0338] Analysis Pattern

[0339] Analytical methods Boltzmann fitting ROA (Analysis Area) automatic Automatic analysis options Single Tm

[0340] The results were statistically analyzed using GraphPad Prism 10 software through one-way ANOVA and Tukey multiple comparisons.

[0341] Six antibodies were subjected to temperature changes from 25°C to 99°C, and the fluorescence signals generated during protein folding and unfolding were quantitatively analyzed. The results are as follows: Figure 7 As shown. Figure 7 As shown, all six antibodies prepared exhibited excellent thermal stability.

[0342] 5-2. Protein stability after repeated freeze-thaw cycles

[0343] The six bispecific antibodies listed in Table 16 were frozen at -80°C for 16 hours and then thawed at 4°C for 2 hours. This process was repeated as one cycle for a total of 5 cycles. Subsequently, the antibody stock solution was diluted to 1 mg / mL with formulation buffer and analyzed by SEC-HPLC according to the conditions shown in Table 25. The results are shown in Tables 26 to 31.

[0344] Table 25

[0345]

[0346] Table 26

[0347] Candidate antibody A

[0348]

[0349] Table 27

[0350] Candidate antibody B

[0351]

[0352] Table 28

[0353] Candidate antibody B-1

[0354]

[0355] Table 29

[0356] Candidate antibody C

[0357]

[0358] Table 30

[0359] Candidate antibody D

[0360]

[0361] Table 31

[0362] Candidate antibody E

[0363]

[0364] Of the six antibodies prepared, the other five antibodies, excluding candidate antibody A, maintained a monomer content of over 97.5% after repeated freeze-thaw cycles, demonstrating good stability even under drastic temperature changes.

[0365]

Example 6

[0366] In vitro bispecific antibody function confirmation

[0367] 6-1. Vascular permeability

[0368] Human umbilical vein endothelial cells (HUVECs; Lonza_C2519A) cultured in EBM medium containing 0.5% FBS were pretreated with specified concentrations of each preparative bispecific antibody for 2 hours. Cells were then further exposed to 100 mM cobalt chloride (CoCl2, under hypoxic conditions) or 100 ng / mL LPS overnight. Endothelial permeability was assessed by measuring the permeability of FITC-dextran in the HUVEC monolayer. Permeability was measured using fluorescein isothiocyanate-dextran, excited at 480 nm and detected at 520 nm.

[0369] Vascular permeability of candidate antibodies B and C was compared with that of aflibercept (Eylea), anti-c-Kit antibody alone, or palicimab; vascular permeability of candidate antibodies C and E was compared with that of anti-c-Kit antibody alone or control antibody. The control antibody was a Fab-VEGFR-Fc (KH) bispecific antibody formed by linking anti-c-Kit antibody IgG (knob) to aflibercept (VEGFR hole) using a "knob-into-hole" technique. Dexamethasone treatment served as a positive control.

[0370] 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 indicates no significance; n=3].

[0371] like Figure 8a As shown, candidate antibody B exhibited significantly better vascular permeability inhibition compared to the combination treatment group of aflibercept (Eylea) and anti-c-Kit antibody and the treatment group of palicimab. Figure 8b As shown, candidate antibodies C and E both exhibited significantly better inhibitory effects on vascular permeability compared to the control antibody treatment group. Furthermore, as... Figure 8c As shown, candidate antibody C exhibited significantly better vascular permeability inhibition compared to the combination treatment group of aflibercept (Eylea) and anti-c-Kit antibody, and showed similar vascular permeability inhibition effect to the palicimab treatment group.

[0372] In summary, candidate antibodies B, C, and E significantly inhibited HUVEC cell permeability in vitro, indicating that the bispecific candidate antibodies of the present invention can inhibit venous endothelial barrier disruption induced by hypoxia or LPS.

[0373] 6-2. Signal transduction blocking

[0374] Human retinal microvascular endothelial cells (HRMEC; Cell Systems, ACBRI-181) or human mast cell line LAD2 (provided by Dr. Kirshenbaum; National Institutes of Health) were pretreated with their respective bispecific antibodies for 30 minutes. After treatment, the cells were stimulated with 100 ng / mL SCF for 5 minutes. Western blot experiments were performed to analyze the expression levels of signal transduction-related proteins in whole-cell lysates.

[0375] Cells were lysed using RIPA buffer containing a mixture of protease inhibitors and phosphatase inhibitors. Proteins were quantified using a BCA protein quantification kit. Western blot samples were prepared, containing adequate protein, NuPAGELDS sample buffer containing a reducing agent, and RIPA buffer, and heated at 70°C for 10 minutes. 30 μg of protein sample was added to each well of an 8% SDS-PAGE gel. Electrophoresis was performed at 60 V for 30 minutes until the gel entered the separating gel. The voltage was then increased to 120 V and electrophoresis was continued for 1 hour. Transfer was performed at 320 mA for 120 minutes [run buffer: 1×Tris-Glycine buffer containing SDS; transfer buffer: 1×Tris-Glycine buffer containing 20% ​​methanol and without SDS]. The membrane was blocked in TBST for 1 hour using SuperBlock blocking buffer. The membrane was then 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 of these primary antibodies was diluted with SuperBlock blocking buffer dissolved in TBST before use. The membrane was washed with TBST for 10 minutes, repeated three times. The membrane was then 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). Each of these secondary antibodies was diluted with 5% skim milk dissolved in TBST before use. The membrane was washed again with TBST for 10 minutes, repeated three times. The membrane was then analyzed using ECL reagent.

[0376] The signal transduction inhibition effects of candidate antibodies B and B-1 were compared with those of the anti-c-Kit antibody treatment group; the signal transduction inhibition effects of candidate antibodies C and E were compared with those of the control antibody treatment group. The control antibody was the same as the antibody used in Example 6-1.

[0377] like Figures 9a to 9d As shown, candidate antibodies B, B-1, C, and E all exhibited effective inhibition of SCF-dependent c-Kit signaling in HRMEC or LAD2 cells.

[0378] 6-3. Cytokine Analysis

[0379] Human retinal microvascular endothelial cells (HRMEC) or ARPE-19 (ATCC, CRL-2302) cells were cultured in medium containing 0.5% FBS and pretreated with their respective bispecific antibodies for 6 hours. The cells were then further exposed to 1% O2 hypoxia or LPS for 12 hours. Cell supernatants were collected, and VEGF, Ang-2, IL-6, IL-8, and MCP-1 were detected by quantitative ELISA.

[0380] The cytokine-inhibiting effects of candidate antibody A were compared with those of the Eylea treatment group; the cytokine-inhibiting effects of candidate antibodies B and C were compared with those of the Eylea, anti-c-Kit antibody treatment group, and palicimab treatment group (dexamethasone treatment group served as a positive control); the cytokine-inhibiting effects of candidate antibodies C and E were compared with those of the control antibody treatment group. The control antibody was the same as that used in Example 6-1.

[0381] 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 indicates no significant difference).

[0382] like Figures 10a to 10b , Figures 11a to 11r and Figures 12a to 12i As shown, candidate antibodies A, B, C, and E all dose-dependently reduced hypoxia- or LPS-induced cytokine levels in HRMEC and ARPE-19 cells in vitro; Figures 11a to 11r and Figures 13a to 13i As shown, candidate antibodies B and C exhibited significantly better cytokine inhibition effects compared to the combination treatment group of aflibercept and anti-c-Kit antibody and the treatment group of palicizumab.

[0383]

Example 7

[0384] Confirming the efficacy of bispecific antibodies in vivo

[0385] 7-1. Laser-induced choroidal angiogenesis Neovascularization (LCNV) and intravitreal injection

[0386] C57BL / 6 mice were intraperitoneally injected with 10 mg / kg xylazine (Rompun®, Bayer Korea) and 100 mg / kg ketamine (Ketamine®, Yuhan) to induce general anesthesia. Local anesthesia was achieved by instilling anesthetic eye drops Alcaine® (proparacaine, Alcon Korea) into the eye, followed by mydriatic eye drops Tropherine Eyedrops® (Alcon Korea) to dilate the pupil. The mice were placed on a stage, and the imaging camera was focused on the fundus using a Micron-IV system (Phoenix, CA). Lubricant was applied to the cornea, and the Micron-IV system lens was brought into contact with the cornea. CNV induction conditions were as follows: wavelength 532 nm, diameter 50 μm, duration 80 ms, power 240 mW. One drop of antibiotic eye drops (Tobrex® (tobramycin); Novartis Korea) was instilled into the eye.

[0387] LCNV was established on day 0 (D0) in 6–8 week old male C57BL / 6 mice to mimic the subretinal neovascularization characteristics of human wet age-related macular degeneration (wAMD). On day 1 (D1) after laser treatment, the mice were treated with various bispecific antibodies via intravitreal injection.

[0388] 7-2. Fluorescein angiography (qFA)

[0389] Lesion permeability was assessed by quantitative fluorescein angiography (qFA) on days 7 and 13 post-laser treatment. Animals were anesthetized by intraperitoneal (IP) injection of ketamine / xylazine and administered 10% sodium fluorescein at a dose of 10 μL / g body weight via IP injection. Fundus imaging of each animal's eye was performed using a Micron IV imaging system when the lesion was filled with fluorescein, and a second imaging was performed 2 minutes later. Fluorescence intensity of individual lesions in each eye was quantitatively analyzed using ImageJ software via an integrated density function. The difference in integrated density between the two imaging sessions following injection was used as a reading for lesion leakage.

[0390] like Figure 14a and 14bAs shown, candidate antibody C exhibited significantly better inhibitory effects on vascular permeability compared to the combination treatment group of aflibercept (Eylea) and anti-c-Kit antibody.

[0391] 7-3. CNV lesions

[0392] On day 14 (D14) after laser treatment, animals were euthanized and their eyes were enucleated, and the choroidal tissue was incised. After incision of the choroid, fluorescein isothiocyanate (FITC) coupled with B4 (IB4) staining was used to visualize the endothelial cells constituting choroidal neovascularization (CNV) lesions before planarization. The CNV lesion area at each Bruch's membrane rupture site was quantified using computer-aided image analysis based on high-resolution digital images of the choroidal planarized samples. The average area of ​​all lesions in each eye was taken and used as a single data point for each eye (two data points per mouse) for statistical analysis.

[0393] like Figure 14c As shown, candidate antibody C showed a statistically significant reduction in CNV lesion area compared to the combination treatment group of aflibercept (Eylea) and anti-c-Kit antibody.

[0394] All qFA and CNV area data were analyzed using one-way ANOVA and Tukey post-hoc tests. Pairwise comparisons were performed between the means, and the statistical hypothesis tests were corrected through multiple comparisons.

[0395] 7-4. Retinal Image Evaluation (FFA / OCT)

[0396] To conduct another efficacy evaluation experiment, immediately after LCNV induction in Example 7-1 above, mice were injected intravitreal with each bispecific antibody at concentration gradients (0.5, 1, or 2 μg / μL per eye). Quantitative fluorescein angiography (qFA) and optical coherence tomography (OCT) were performed on day 10 (D10) following laser treatment. For efficacy comparison, the negative control group of the carrier-treated group received formulation buffer, while the positive control group of the reference drug received aflibercept at 2 or 20 μg / μL per eye.

[0397] The extent of leakage in LCNV lesions was analyzed using corrected total fluorescence (CTF) values ​​calculated based on FFA (fundus fluorescein angiography) images. LCNV lesion volume measurements based on OCT images were performed using the "InSight" and "Image-J" programs.

[0398] like Figure 15a As shown in Table 32, the CTF value of the candidate antibody A administration group was significantly lower than that of the vector-treated negative control group, and no statistically significant difference was observed between different administration doses.

[0399] Table 32

[0400] G1 (Sham) 0±0 G2 (vector-treated negative control group) 1027397±840775 G3 (Aflibercept 2μg / μL / eye) 619330±441069 0.3522 G4 (Aflibercept 20 μg / μL / eye) 587441±586826 0.0321 G5 (Candidate antibody A 0.5 μg / μL / eye) 417531±392373 <0.0001 G6 (candidate antibody A1 μg / μL / eye) 519124±478599 0.0033 G7 (Candidate antibody A 2 μg / μL / eye) 522365±434247 0.0113

[0401] In addition, such as Figure 15b As shown in Table 33, the CNV lesion volume was significantly reduced in the candidate antibody A administration group compared to the vector-treated negative control group.

[0402] Table 33

[0403] G1 (Sham) 0±0 G2 (vector-treated negative control group) 2833216±1407777 G3 (Aflibercept 2μg / μL / eye) 2389355±1439831 >0.9999 G4 (Aflibercept 20 μg / μL / eye) 1413476±679812 0.0002 G5 (Candidate antibody A 0.5 μg / μL / eye) 1432213±1012822 <0.0001 G6 (candidate antibody A1 μg / μL / eye) 1607532±829899 0.0004 G7 (Candidate antibody A 2 μg / μL / eye) 1613292±970409 0.0005

[0404] Data in Tables 32 and 33 are expressed as mean ± standard deviation (mean ± SD) (G1: n=48, G2: n=37, G3: n=37, G4: n=33, G5: n=37, G6: n=43, G7: n=37). Statistical significance was determined using the Kruskal-Wallis multiple comparison test, with G2 (the vector-treated negative control group) as the comparison group.

Claims

1. 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 heavy chain CDR1 containing the amino acid sequence of sequence number 2, heavy chain CDR2 containing the amino acid sequence of sequence number 4, and heavy chain CDR3 containing the amino acid sequence of sequence number 6; and a light chain variable region comprising light chain CDR1 containing the amino acid sequence of sequence number 13, light chain CDR2 containing the amino acid sequence of sequence number 15, and light chain CDR3 containing the amino acid sequence of sequence number 17; and (b) A second antigen-binding site that specifically binds to VEGF, wherein the second antigen-binding site is formed by the fusion of the soluble extracellular domain of VEGF and the Fc domain of IgG.

2. The bispecific antibody that specifically binds to c-Kit and VEGF according to claim 1, wherein the first antigen binding site comprises: The heavy chain variable region and the light chain variable region, wherein the heavy chain variable region contains an amino acid sequence having more than 90% sequence homology with the amino acid sequence of sequence number 27, and the light chain variable region contains an amino acid sequence having more than 90% sequence homology with the amino acid sequence of sequence number 28.

3. The bispecific antibody that specifically binds c-Kit and VEGF according to claim 1, wherein the soluble extracellular domain comprises an immunoglobulin-like domain 2 of a first VEGF receptor and an immunoglobulin-like domain 3 of a second VEGF receptor.

4. The bispecific antibody that specifically binds to c-Kit and VEGF according to claim 1, wherein the second antigen binding site comprises the amino acid sequence of sequence number 21.

5. The bispecific antibody that specifically binds to c-Kit and VEGF according to claim 1, wherein the second antigen-binding site is linked to the C-terminus or N-terminus of the heavy chain of the first antigen-binding site via a peptide linker; or The second antigen binding site is connected to the C-terminus of the light chain of the first antigen binding site via a peptide linker.

6. The bispecific antibody that specifically binds to c-Kit and VEGF according to claim 2, wherein the peptide linker is GGGGG (serial number 20), GGGGGG (serial number 67), GGGGGGG (serial number 68), GGGGGGGG (serial number 23), GGGGGGGGG (serial number 69) or GGGGGGGGGG (serial number 70).

7. The bispecific antibody that specifically binds to c-Kit and VEGF according to claim 1, wherein the Fc domain of the first antigen-binding site comprises: (a) Wild-type IgG Fc; (b) Fc variants containing L234A and L235E amino acid substitutions; (c) Fc variants containing substitutions for L235A and G237A amino acids; or (d) Fc variants containing amino acid substitutions of L234A, L235A, I253A, H310A, P329G, and H435A; The residues are numbered according to the Kabat EU index.

8. The bispecific antibody that specifically binds to c-Kit and VEGF according to claim 1, wherein the bispecific antibody is a tetravalent antibody.

9. The bispecific antibody that specifically binds c-Kit and VEGF according to claim 1, wherein the first antigen binding site is a full-length bivalent antibody, and the second antigen binding site is composed of two VEGF antagonists, wherein the VEGF antagonist is formed by fusing the soluble extracellular domain of VEGF with the Fc domain of IgG.

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 the nucleic acid molecule according to claim 10.

12. A transformant comprising the recombinant vector according to claim 11.

13. A method for preparing a bispecific antibody that specifically binds to c-Kit and VEGF, comprising the following steps (a) and (b): (a) Cultivating the transformant according to claim 12; and (b) Recover bispecific antibodies that specifically bind to c-Kit and VEGF from the culture.

14. A pharmaceutical composition comprising, as an active ingredient, a bispecific antibody that specifically binds to c-Kit and VEGF according to any one of claims 1 to 9, for the prevention or treatment of angiogenesis diseases.

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

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

17. The 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 laminar detachment.

18. A diagnostic kit for angiogenesis diseases, comprising a bispecific antibody that specifically binds to c-Kit and VEGF according to any one of claims 1 to 9.

19. A method for the prevention or treatment of angiogenesis disease, comprising the step of administering a bispecific antibody that specifically binds to c-Kit and VEGF according to any one of claims 1 to 9 to an individual in need of the antibody in a therapeutically effective amount.

20. Use of a bispecific antibody that specifically binds to c-Kit and VEGF for the preparation of drugs for the prevention or treatment of angiogenesis diseases.

Images