Fusion protein containing improved anti-VEGFR2 (KDR) antibody and its uses

Abstract

The present invention relates to a fusion protein containing an improved anti-VEGFR2 (KDR) antibody or an antigen-binding fragment thereof with improved properties and its uses. Specifically, the present invention relates to a fusion protein in which a DLL4 (Delta-like ligand 4) binding protein domain is bound to the end of an anti-VEGFR2 (KDR) antibody in which the heavy chain FR (framework regoin) of a human antibody that specifically binds to VEGFR2 / KDR is replaced from VH1 to VH3 and the light chain FR (framework regoin) is replaced from VL1 to VK1, and the affinity to the antigen is increased by inducing mutations, a composition for diagnosing or treating angiogenesis-related diseases, a composition for preventing or treating tumors or cancers, and a composition for co-administration with a therapeutic agent other than the fusion protein, which contains the fusion protein.

Description

[Technical field]

[0001] The present invention relates to a fusion protein containing an improved anti-VEGFR2 (KDR) antibody or an antigen-binding fragment thereof with improved properties and its uses. Specifically, the present invention relates to a fusion protein in which a DLL4 (Delta-like ligand 4) binding protein domain is bound to the end of an anti-VEGFR2 (KDR) antibody in which the heavy chain FR (framework regoin) of a human antibody that specifically binds to VEGFR2 / KDR is replaced from VH1 to VH3 and the light chain FR (framework regoin) is replaced from VL1 to VK1, and the affinity to the antigen is increased by inducing mutations, a composition for diagnosing or treating a disease associated with angiogenesis, a composition for preventing or treating a tumor or cancer, and a composition for administering in combination with a therapeutic agent other than the fusion protein, which contains the fusion protein.

[0002] [Background technology]

[0003] VEGFR2 / KDR is the primary angiogenic receptor that binds VEGF isoforms A, C, D, and E and is important for the mitogenic, angiogenic, and permeability-enhancing effects of VEGF as well as endothelial cell differentiation. Anti-VEGFR2 antibodies can prevent all known VEGF isoforms from binding to VEGFR2 / KDR and initiating signaling. Also, because tumors secrete higher numbers of VEGF while the number of receptors remains relatively constant, targeting the receptor increases the probability of completely inhibiting signaling even in the presence of very high levels of VEGF isoforms.

[0004] Angiogenesis means the generation of new blood vessels from existing blood vessels by the growth, division, migration, etc. of endothelial cells, and does not occur in adults except in special cases such as wound healing and female reproductive cycle. However, excessive angiogenesis has been reported in diseases such as tumor growth and metastasis, age-related macular denaturation, rheumatoid arthritis, diabetic retinopathy, psoriasis, and chronic inflammation (Cameliet and Jain, Nature, 407:249, 2000), and for this reason, many efforts are being made to treat diseases, especially tumors, using angiogenesis inhibitors.

[0005] In 1971, Dr. J. Folkman proposed that tumor growth and metastasis are dependent on angiogenesis, and therefore that anti-angiogenesis-focused therapy could be a new therapeutic agent for solid cancers. Since then, research into the inhibition of excessive angiogenesis has attracted the interest of many researchers (Ferrara and Kerbel, Nature, 435:967, 2005). The progression of angiogenesis is determined by the overall balance of angiogenesis-inducing factors and angiogenesis-inhibiting factors, and proceeds through a complex and sequential process consisting of multiple steps. The process is as follows: First, various angiogenesis-inducing factors, including vascular endothelial growth factor (VEGF), secreted from tumors and damaged tissues, bind to the corresponding receptors of existing vascular endothelial cells in the vicinity, activating the vascular endothelial cells, increasing their permeability, and secreting proteolytic enzymes such as matrix metalloproteinase (MMP), which break down the basement membrane and extracellular matrix around the vascular endothelial cells, causing the vascular endothelial cells to leave the existing capillaries and migrate and proliferate toward the tissues that secrete angiogenesis-inducing factors. The migrated and proliferated vascular endothelial cells form an intravascular tube structure, and finally, stable and mature blood vessels are formed as pericytes, which are the structural support base for vascular endothelial cells, flow in.

[0006] DLL4 is one of the main factors responsible for normal vascular tube formation in the above angiogenesis process. DLL4 is specifically induced by VEGF and acts on the Notch receptor to appropriately regulate angiogenic sprouting. Blocking DLL4 causes loss of lateral inhibition in cells at the end of angiogenesis sites, resulting in excessive sprouting. As a result, excessively abundant but unproductive angiogenic responses are promoted, and oxygen perfusion is impaired, inducing hypoxia around the cancer. In experimental cancer models, activation of DLL4 signaling improved angiogenesis, while inhibition inhibited cancer growth (Lobov IB et al., Proc Natl Acad Sci US A. 2007 Feb 27; 104(9): 3219-24).

[0007] DLL4 is one of the ligands for the Notch receptor, and it is known that there are currently four types of Notch receptors (Notch 1-4) and five types of Notch ligands (Jagged-1, Jagged-2, DLL1, DLL3, DLL4) in mammals. In the case of the Notch signaling system, it begins when the Notch ligand of one cell binds to the Notch receptor of another cell, but it can only be activated by direct interaction between different cells (Bray SJ, Nat Rev Mol Cell Biol., 7(9):678, 2006).

[0008] When a Notch ligand binds to the Notch receptor, first, ADAM metalloprotease is activated to cleave the proximal site on the outer side of the cell membrane of the Notch receptor, and then gamma-secretase complex is activated to cleave the proximal site on the inner side of the cell membrane of the Notch receptor, releasing NICD (Notch Intracellular Domain), which then migrates to the nucleus and binds to RBPJ / CSL transcription factor to induce the expression of Notch target genes such as basic helix-loop-helix proteins such as Hes and Hey. Thus, the Notch signaling system determines the fate of the cell, such as proliferation / differentiation / apoptosis, depending on the situation it is in, and also plays an important role in maintaining normal stem cells and cancer stem cells.

[0009] Basically, all Notch receptors can bind to all Notch ligands, but these various combinations of binding are selectively regulated by the microenvironment in which the cells are located. For example, DLL4 is strongly expressed in endothelial cells during angiogenesis during fetal development, and binds to Notch1 and Notch4 expressed in surrounding endothelial cells, among which DLL4-Notch1 binding is exclusively the most important (Yan M, Vasc Cell, 2011), and this binding promotes angiogenesis. This fact has been well investigated by gene deficiency experiments, etc. (Duarte et al., Genes Dev, 2004; Gale et al., PNAS, 2004; Krebs et al., Genes Dev, 2004).

[0010] Therefore, by inhibiting DLL4-Notch1 binding, angiogenesis is suppressed, which allows the treatment of various diseases such as tumors. It has already been demonstrated that in cancer treatment, when VEGF is suppressed using Avastin (bevacizumab) or the like, angiogenesis is suppressed, and as tumor perfusion decreases, tumor size decreases, whereas when DLL4 is targeted and binding to Notch1 expressed in surrounding cells is inhibited, blood vessels are hypersprouted but cannot function normally, which reduces tumor perfusion and ultimately reduces tumor size (Thurston et al, Nat Rev Cancer, 7(5):327,2007).

[0011] Therefore, the present researchers attempted to effectively inhibit neovascularization by preparing a dual-targeting antibody in which Notch1, in particular the DLL4-binding site of Notch1 in the present invention, was fused to an existing antibody.

[0012] Factors involved in angiogenesis include vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor (TGFb), and fibroblast growth factor (FGF). Among these, endothelial growth factor is an endothelial cell-specific factor that is directly involved in the growth, differentiation, and migration of endothelial cells. There are four different isoforms (VEGF165, VEGF121, VEGF189, and VEGF206), and VEGF165 is the most abundant isoform in all human tissues except the placenta (Tisher et al., J Biol Chem, 266:11947, 1991).

[0013] Endothelial growth factor (VEGF) regulates the formation of new blood vessels in situ from the differentiation of endothelial precursors (vascular stem cells), is expressed in embryonic tissues (Breier et al., Development (Camb), 114:521, 1992), macrophages, and proliferating epithelial keratinocytes during wound healing (Brown et al., J. Exp. Med., 176:1375, 1992), and may be responsible for tissue edema associated with inflammation (Ferrara et al., Endocr. Rev., 13:18, 1992). In situ hybridization studies have demonstrated that VEGF is highly expressed in numerous human tumor lines, including glioblastoma multiforme, hemangioblastoma, central nervous system neoplasms, and AIDS-associated Kaposi's sarcoma (Plate et al., Nature 359:845, 1992; Plate et al., Cancer Res. 53:5822, 1993; Berkman et al., J. Clin. Invest. 91:153, 1993; Nakamura et al., AIDS Weekly. 13(1), 1992). High levels of VEGF have also been observed following hypoxia-induced angiogenesis (Shweiki et al., Nature 359:843, 1992).

[0014] The biological functions of VEGF are mediated by VEGF receptors, which have high affinity for VEGF and are selectively expressed in endothelial cells during embryogenesis (Millauer et al., Cell, 72:835, 1993) and tumorigenesis. In general, VEGF receptors (VEGFRs) are class III receptor tyrosine kinases characterized by multiple, generally five or seven, immunoglobulin-like loops in their amino-terminal extracellular receptor ligand-binding domain (Kaipainen et al., J. Exp. Med., 178:2027, 1993). The other two sites contain a carboxy-terminal intracellular catalytic domain and a transmembrane domain interrupted by the insertion of a hydrophilic interkinase sequence of variable length, called the kinase insert domain (Terman et al., Oncogene, 6:1677, 1991). VEGFRs include the fms-like tyrosine kinase receptor (flt-1), or VEGFR-1 (Shibuya et al., Oncogene, 5:519, 1990; WO92 / 14248; Terman et al., Oncogene, 6:1677, 1991) and the kinase insert domain-containing receptor / embryokinase (KDR / flk-1), or VEGFR-2 (Matthews et al., Proc. Natl. Acad. Sci. USA, 88:9026, 1991), although other receptors such as neuropilin-1 and neuropilin-2 can also bind VEGF. Another tyrosine kinase receptor, VEGFR-3 (flt-4), binds the VEGF homologs VEGF-C and VEGF-D and plays an important role in lymphatic development.

[0015] High levels of Flk-1 are expressed by endothelial cells infiltrating glioblastomas (Plate et al., Nature 359:845, 1992). Flk-1 levels are specifically upregulated by VEGF produced by human glioblastomas (Plate et al., Cancer Res. 53:5822, 1993). The fact that Flk-1 is expressed at high levels in glioblastoma terminal endothelial cells (GAECs) indicates that Flk-1 transcripts are barely detectable in normal brain endothelial cells and that receptor activity is probably induced during tumorigenesis. Such upregulation occurs only in vascular endothelial cells in close proximity to the tumor. Blockade of VEGF activity with a neutralizing anti-VEGF monoclonal antibody (mAb) inhibits the growth of human tumor xenografts in nude mice (Kim et al., Nature 362:841, 1993), indicating a direct role for VEGF in tumor-associated angiogenesis.

[0016] VEGF ligands are upregulated in tumor cells and their receptors are upregulated in tumor-infiltrating vascular endothelial cells, whereas the expression of VEGF ligands and their receptors is low in normal cells not involved in angiogenesis, and therefore such normal cells block the interaction between VEGF and its receptors, suppressing angiogenesis and thus preventing tumor growth.

[0017] High levels of VEGFR-2 are expressed by endothelial cells infiltrating gliomas and are specifically upregulated by VEGF produced by human glioblastomas (Plate et al., Nature, 359:845, 1992; Plate et al., Cancer Res., 53:5822, 1993). Because VEGFR-2 transcripts are barely detectable in normal brain endothelial cells, the expression of high levels of VEGFR-2 in glioblastoma-associated endothelial cells (GAECs) indicates that receptor activity is induced during tumorigenesis.

[0018] VEGF is expressed at high levels in a variety of tumors (Plate et al., Nature, 359:8435, 1992; Dvorak et al., J. Exp. Med., 174:1275, 1991), and new capillaries congregate around VEGF-producing tumor cells (Plate et al., Nature, 359:8435, 1992). VEGF expression is strongly upregulated under hypoxic conditions, such as those associated with fast-growing tumors (Shweiki et al., Nature, 359:843, 1992). VEGF neutralization with antibodies such as Avastin (Ferrara et al., Nat Rev Drug Discov., 3(5):391, 2004; Avery et al., Ophthalmology, 113(3):363, 2006) is a clinically approved therapy for inhibiting cancer or other angiogenic diseases such as AMD. However, resistance to VEGF blockade has been found, even when combined with chemotherapy. This resistance may be associated with a remodeled vasculature and increased expression of other angiogenic factors. Thus, there remains a need in the art for improved compositions and methods for inhibiting cancer and other diseases associated with angiogenesis.

[0019] Although both tyrosine kinase inhibitors (TKIs) and anti-KDRP antibodies can inhibit KDR-mediated angiogenesis, antibody approach has advantages over TKIs. In contrast to TKIs, anti-KDR antibodies are more specific KDR targeting agents (i.e., anti-KDR antibodies do not inhibit other VEGF receptors). Due to such high specificity, anti-KDR antibodies can limit and / or avoid off-target effects and toxicity caused by less specific TKIs (Witte et al., Cancer Metastasis Rev., Jun;17(2):155,1998).

[0020] Unlike Avastin, which binds only one ligand (VEGF-A), anti-KDR antibodies are expected to prevent all known VEGFs from binding to VEGFR2 / KDR. Anti-KDR antibodies may have a stronger inhibitory effect on tumor angiogenesis than simply blocking VEGF-A. Anti-KDR antibody therapy may be effective in Avastin resistance.

[0021] Against this technical background, the inventors of the present application have produced a novel antibody with increased affinity for the antigen by replacing the FR (framework receptor) of the heavy chain variable region from VH1 to VH3 and the light chain FR (framework receptor) from VL1 to VK1 in order to improve an existing antibody that specifically binds to VEGFR2 / KDR, and by inducing mutations in the CDR3 regions of the heavy and light chains. They also fused the DLL4 binding site of Notch1 to the end of the antibody to produce a fusion protein, thereby completing the present invention.

[0022]

[0023] Summary of the Invention [Problem to be solved by the invention]

[0024] An object of the present invention is to provide a fusion protein in which a DLL4-binding protein domain is fused to the end of an antibody having improved binding ability to VEGFR2 / KDR.

[0025] Another object of the present invention is to provide a nucleic acid encoding the fusion protein.

[0026] Another object of the present invention is to provide a vector containing the nucleic acid, a transformed cell containing the vector, and a method for producing the same.

[0027] It is still another object of the present invention to provide a composition for preventing or treating angiogenic diseases, comprising the fusion protein. It is also an object of the present invention to provide a method for preventing or treating angiogenic diseases, comprising administering the fusion protein to an individual. It is also an object of the present invention to provide a use of the fusion protein for the manufacture of a composition for preventing or treating angiogenic diseases.

[0028] It is still another object of the present invention to provide a composition for diagnosing angiogenic diseases, comprising the fusion protein. It is also an object of the present invention to provide a method for diagnosing angiogenic diseases, comprising administering the fusion protein to an individual or treating a sample with the fusion protein. It is also an object of the present invention to provide a use of the fusion protein for the manufacture of a composition for diagnosing angiogenic diseases.

[0029] It is still another object of the present invention to provide a composition for preventing or treating tumors or cancer, comprising the fusion protein. It is also an object of the present invention to provide a method for preventing or treating tumors or cancer, comprising the step of administering the fusion protein to an individual. It is also an object of the present invention to provide a use of the fusion protein for the manufacture of a composition for preventing or treating tumors or cancer.

[0030] It is still another object of the present invention to provide a composition for co-administration with other therapeutic agents, comprising the fusion protein. It is also an object of the present invention to provide a method for co-administration with other therapeutic agents, comprising the step of administering the fusion protein to an individual. It is also an object of the present invention to provide a use of the fusion protein for the manufacture of a composition for co-administration with other therapeutic agents. [Means for solving the problem]

[0031] In order to achieve the above object, the present invention provides a fusion protein comprising an antibody or an antigen-binding fragment thereof that binds to VEGFR2 / KDR; and a DLL4 (Delta-like ligand 4) binding protein domain,

[0032] The antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR provides a fusion protein comprising a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, and a heavy chain CDR3 of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6.

[0033] The present invention also provides a nucleic acid encoding the fusion protein.

[0034] The present invention also provides a vector comprising the nucleic acid.

[0035] The present invention also provides a transformed cell containing the vector.

[0036] The present invention also provides a method for producing the fusion protein, comprising: (a) culturing the cell to produce the fusion protein; and (b) recovering the produced fusion protein.

[0037] The present invention also provides a composition for preventing or treating angiogenic diseases, comprising the fusion protein.

[0038] The present invention also provides a composition for diagnosing angiogenic diseases, comprising the fusion protein.

[0039] The present invention also provides a composition for preventing or treating tumors or cancer, comprising the fusion protein.

[0040] The present invention also provides a composition comprising the fusion protein for co-administration with other therapeutic agents.

[0041] [Brief description of the drawings]

[0042] [Figure 1] This sequence is a graft of the heavy chain CDR sequence of 6A6.

[0043] [Diagram 2] This sequence is a graft of the light chain CDR sequence of 6A6.

[0044] [Figure 3A-3B] The binding strength of the fusion protein thus produced was confirmed by SPR.

[0045] [Figure 4] The ability to simultaneously bind to two types of antigens was confirmed by SPR.

[0046] [Diagram 5] This result confirmed that the proliferation of HUVEC cells can be suppressed by growth regulators.

[0047] [Figure 6] These results confirm the inhibitory effect of antibodies on VEGFR-2 phosphorylation in vascular endothelial cells.

[0048] [Figure 7] This shows the results of confirming the binding ability of the fusion protein in vascular endothelial cells using FACS.

[0049] [Figure 8] The binding ability of the fusion protein to hDLL4 expressed on the cell surface was confirmed using FACS.

[0050]

[0051] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein is one that is well known and commonly used in the art.

[0053] The present invention relates to an antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR; and a fusion protein comprising a DLL4 binding protein domain, wherein the antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR comprises a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, and a heavy chain CDR3 of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6.

[0054] In the fusion protein of the present invention, the antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR is an improved version of a fully humanized antibody that neutralizes vascular endothelial growth factor receptor, particularly an improved version of a human monoclonal antibody (Korean Patent Registration No. 10-0883430) that neutralizes vascular endothelial growth factor receptor.

[0055] The inventors of the present application have improved the only antibody that was invented from a fully human antibody library and targets VEGFR-2 / KDR while also reacting with mouse or rat flk-1 (VEGFR-2 homologue). The superiority of this antibody has been proven through clinical trials, but the antibody protein has a somewhat low binding affinity to the antigen and a short half-life in the body. By improving this, it is expected that the efficacy as an immune anticancer agent and efficacy in angiogenesis-related diseases will be improved.

[0056] In order to improve an existing antibody that specifically binds to VEGFR2 / KDR, a novel antibody with increased affinity to an antigen was produced by substituting the FR (framework receptor) of the heavy chain variable region from VH1 to VH3, and substituting the light chain FR (framework receptor) from VL1 to VK1, and inducing mutations in the CDR3 regions of the heavy and light chains. As a result, the present inventors developed an antibody that has higher binding strength and better intracellular efficacy than existing antibodies that specifically bind to VEGFR2 / KDR, and confirmed that the antibody can function as a targeted immune anticancer agent or a therapeutic agent for angiogenesis-related diseases.

[0057] The improved antibodies were screened as follows: First, the CDR region sequences and framework (FR) sequences of 6A6 were confirmed, and the heavy chain CDR sequence was grafted to human germline VH3 sequence and the light chain CDR sequence was grafted to germline VK1 sequence using a CDR grafting technique.

[0058] CDR grafting is generally a method of isolating DNA encoding the variable region sequence of a mouse monoclonal antibody, obtaining the CDR sequence by gene cloning, and then grafting the CDR sequence into an appropriate human antibody sequence in silico (Hou S, et al. Humanization of an anti-CD34 monoclonal antibody by complementarity-determining region grafting based on computer-assisted molecular modelling. J Biochem. 2008; 144(1): 115-20; and Kashmiri SV, De Pascalis R, Gonzales NR, Schlom J. SDR grafting--a new approach to antibody humanization. Methods. 2005; 36(1): 25-34). Using the above method, the framework of 6A6 with germline VH1 / VL1 was replaced with germline VH3 / VK1.

[0059] In order to improve the affinity of the substituted antibody, a library was constructed by inducing mutations in the CDR3 base sequences of the light and heavy chains, and the antibody was screened using recombinant KDR D1-D3-Fc fusion protein. The antibody screening was performed using phage display.

[0060] Based on this, the present invention includes an antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR, comprising a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, and a heavy chain CDR3 of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6.

[0061] The fusion protein of the invention may specifically bind to each of VEGFR2 / KDR and DLL4. "Bispecific" or "dual specific" refers to a property of a binding protein that can specifically bind to two different targets and modulate the activity of the targets, which may be produced, for example, by an antibody or protein, or fragment thereof, that specifically binds to each target, and that possesses two distinct antigen-binding arms (arms: specificity for two targets) and is monovalent for each antigen that binds to it.

[0062] As used herein, the term "antibody" refers to an anti-VEGFR2 / KDR antibody that specifically binds to VEGFR2 / KDR. The scope of the present invention includes intact antibody forms that specifically bind to VEGFR2 / KDR as well as antigen-binding fragments of said antibody molecules.

[0063] Intact antibodies have a structure with two full-length light chains and two full-length heavy chains, with each light chain linked to a heavy chain by a disulfide bond. Heavy chain constant regions are of the gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, with gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2) subclasses. Light chain constant regions are of the kappa (κ) and lambda (λ) types.

[0064] The antigen-binding fragment of an antibody or antibody fragment means a fragment having an antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv. Among antibody fragments, Fab has a structure having a light chain and a heavy chain variable region, a light chain constant region, and the first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibody is generated by disulfide bonds formed between cysteine ​​residues in the hinge region of Fab'. Fv is the smallest antibody fragment having only a heavy chain variable region and a light chain variable region. In a two-chain Fv, the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, and in a single-chain Fv (scFv), the heavy chain variable region and the light chain variable region are generally linked by a covalent bond via a peptide linker or directly at the C-terminus, and can form a dimer-like structure like a two-chain Fv. Such antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restrictively cleaving a whole antibody with papain, and F(ab')2 fragments can be obtained by cleavage with pepsin), or can be produced by genetic recombination techniques.

[0065] In one embodiment, the antibodies of the present invention are in Fv format (e.g., scFv) or in whole antibody format. The heavy chain constant region may be selected from any one of the gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε) isotypes. For example, the constant region may be gamma 1 (IgG1), gamma 3 (IgG3) or gamma 4 (IgG4). The light chain constant region may be of the kappa or lambda type.

[0066] As used herein, the term "heavy chain" refers to any full-length heavy chain or fragment thereof that includes a variable region domain VH that contains an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and three constant region domains CH1, CH2, and CH3. As used herein, the term "light chain" refers to any full-length light chain or fragment thereof that includes a variable region domain VL that contains an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and a constant region domain CL.

[0067] Antibodies of the present invention include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFV), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFV), and anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of these antibodies.

[0068] The monoclonal antibody refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0069] "Epitope" refers to a protein determinant to which an antibody can specifically bind. Epitopes are usually composed of chemically active surface groupings of molecules such as amino acids or sugar side chains and generally have specific three-dimensional structural features as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0070] The "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate that retains the desired specificity, affinity, and capacity.

[0071] The "human antibody" refers to a molecule derived from human immunoglobulin, in which all of the amino acid sequences constituting the antibody, including the complementarity determining regions and structural regions, are composed of human immunoglobulin.

[0072] Also included are "chimeric" antibodies (immunoglobulins) in which portions of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remaining chains are identical to or homologous to corresponding sequences in antibodies from yet another species or belonging to yet another antibody class or subclass, as well as fragments of such antibodies that exhibit the desired biological activity.

[0073] As used herein, "antibody variable region" refers to the light and heavy chain portions of an antibody molecule that contain the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0074] "Complementarity determining region (CDR; i.e., CDR1, CDR2, and CDR3)" refers to the amino acid residues of an antibody variable region that are necessary for antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2, and CDR3. The present invention includes a heavy chain variable region comprising a heavy chain CDR3 of SEQ ID NO:3 and a light chain variable region comprising a light chain CDR3 of SEQ ID NO:6.

[0075] In the present invention, the antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR and DLL4 may comprise a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, and a heavy chain CDR3 of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6.

[0076] "Framework regions (FR)" are those variable region residues other than the CDR residues. Each variable region typically has four FRs, identified as FR1, FR2, FR3 and FR4.

[0077] An "Fv" fragment is an antibody fragment that contains a complete antigen recognition and binding site. Such a region consists of a dimer of one heavy- and one light-chain variable domain in tight, essentially covalent association, e.g., scFv.

[0078] A "Fab" fragment contains the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. F(ab')2 antibody fragments generally comprise a pair of Fab fragments covalently linked near their carboxy termini by hinge cysteines between them.

[0079] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, but these domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0080] VEGFR2 / KDR antibodies may comprise single chains or double chains. Functionally, the binding affinity of VEGFR2 / KDR antibodies is within the range of 10 -5 M~10 -12 For example, the binding affinity of VEGFR2 / KDR antibodies is in the range of 10 -6 M~10 -12 M, 10 -7 M~10 -12 M, 10 -8 M~10 -12 M, 10-9 M~10 -12 M, 10 -5 M~10 -11 M, 10 -6 M~10 -11 M, 10 -7 M~10 -11 M, 10 -8 M~10 -11 M, 10 -9 M~10 -11 M, 10 -10 M~10 -11 M, 10 -5 M~10 -10 M, 10 -6 M~10 -10 M, 10 -7 M~10 -10 M, 10 -8 M~10 -10 M, 10 -9 M~10 -10 M, 10 -5 M~10 -9 M, 10 -6 M~10 -9 M, 10 -7 M~10 -9 M, 10 -8 M~10 -9 M, 10 -5 M~10 -8 M, 10 -6 M~10 -8 M, 10 -7 M~10 -8 M, 10 -5 M~10 -7 M, 10 -6 M~10 -7 M or 10 -5 M~10 -6 It's M.

[0081] The present invention includes an antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR, comprising a heavy chain variable region comprising a sequence having 80% or more homology to the sequence of SEQ ID NO:8.

[0082] The present invention also includes an antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR, comprising a light chain variable region comprising a sequence having 80% or more homology to the sequence of SEQ ID NO:10.

[0083] The present invention also includes an antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR, comprising a heavy chain variable region comprising a sequence having 80% or more homology to the sequence of SEQ ID NO:8, and a light chain variable region comprising a sequence having 80% or more homology to the sequence of SEQ ID NO:10.

[0084] The homology may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology compared to the entire sequence of the claimed SEQ ID NO.

[0085] The present invention includes not only the sequences of the fusion proteins described in the specification, but also their biological equivalents. For example, additional modifications can be made to the amino acid sequence to further improve the binding affinity and / or other biological properties of the fusion protein. Such modifications include, for example, deletion, insertion and / or substitution of amino acid sequence residues. Such amino acid mutations are made based on the relative similarity of the amino acid side chain substitutes, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape and type of amino acid side chain substitutes shows that arginine, lysine and histidine are all positively charged residues; alanine, glycine and serine have similar sizes; and phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

[0086] Considering the above-mentioned biologically equivalent mutations, the fusion protein of the present invention or the nucleic acid molecule encoding the same is also understood to include sequences that show substantial identity with the sequences described in SEQ ID NO. The above-mentioned substantial identity means a sequence that shows at least 90% homology, most preferably at least 95% homology, 96% or more, 97% or more, 98% or more, or 99% or more homology when the above-mentioned sequence of the present invention is aligned with any other sequence as close as possible and the aligned sequences are analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are well known in the art. NCBI BLAST (Basic Local Alignment Search Tool) is accessible from NCBI, etc., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet. BLSAT can be accessed at www.ncbi.nlm.nih.gov / BLAST / . Instructions for sequence homology comparison using this program can be found at www.ncbi.nlm.nih.gov / BLAST / blast_help.html.

[0087] On this basis, the fusion proteins of the invention may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the sequences explicitly set forth herein or to the entirety of the sequences set forth herein. Such homology may be determined by sequence comparison and / or alignment by methods known in the art. For example, percent sequence homology of the nucleic acids or proteins of the invention may be determined using sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection.

[0088] The DLL4-binding protein domain is a protein domain that binds to DLL4, and includes the DLL4-binding site of Notch1. For example, it may include the EGF-like domain of human Notch1, and may include the sequence of SEQ ID NO:18.

[0089] The DLL4 binding protein domain may be bound to the end of an antibody that binds to VEGFR2 / KDR, for example the light chain end, specifically the light chain N-terminus.

[0090] Korean Patent Registration No. 10-1569083 describes a DIG-KN substance containing VEGFR2 / KDR and DLL4 binding protein domains, but the present invention includes an anti-VEGFR2 (KDR) antibody in which the heavy chain FR (framework region) of a human antibody that specifically binds to VEGFR2 / KDR has been replaced from VH1 to VH3, and the light chain FR (framework region) has been replaced from VL1 to VK1, thereby inducing mutations to increase affinity for the antigen, and is different in that a DLL4 binding protein domain (Notch1 EGF-like domain) is bound to the N-terminus of the light chain of the anti-VEGFR2 (KDR) antibody.

[0091] The VEGFR2 / KDR-binding antibody or antigen-binding fragment thereof and the DLL4-binding protein domain may be linked via a linker. The linker may be a peptide linker and may be about 10 -25 It may have any aa length, and may include, for example and without limitation, hydrophilic amino acids such as glycine and / or serine.

[0092] Specifically, the linker may include, for example, (GS)n, (GGS)n, (GSGGS)n, or (GnS)m (n and m are each 1 to 10), but the linker may be, for example, (GnS)m (n and m are each 1 to 10).

[0093] In another aspect, the present invention relates to a nucleic acid encoding the fusion protein.

[0094] The nucleic acid may comprise the sequence of SEQ ID NO: 7, which encodes the heavy chain variable region. The nucleic acid may comprise the sequence of SEQ ID NO: 9, which encodes the light chain variable region. The nucleic acid may comprise the sequence of SEQ ID NO: 17, which encodes the DLL4 binding protein domain.

[0095] The nucleic acid encoding the fusion protein of the invention can be isolated and recombinantly produced. The nucleic acid can be isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for further expression. Based on this, the invention relates in a further aspect to a vector comprising said nucleic acid.

[0096] The term "nucleic acid" refers to a nucleic acid that includes DNA (gDNA and cDNA) and RNA molecules, and includes not only natural nucleotides but also analogues in which the sugar or base moiety is modified. The sequence of the nucleic acid encoding the fusion protein of the present invention may be modified. The modification includes addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0097] The DNA is readily isolated or synthesized using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to DNA encoding the heavy and light chains of the antibody). Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0098] The term "vector" as used herein refers to a means for expressing a gene of interest in a host cell, and includes plasmid vectors, cosmid vectors, viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors, etc. In the vector, the nucleic acid encoding the fusion protein is operably linked to a promoter.

[0099] "Operably linked" refers to the functional association of a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) with another nucleic acid sequence, whereby the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0100] When a prokaryotic cell is used as the host, it generally contains a strong promoter capable of driving transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for the initiation of translation, and a transcription / translation termination sequence. Furthermore, for example, when a eukaryotic cell is used as the host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter), or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) may be used, which generally has a polyadenylation sequence as a transcription termination sequence.

[0101] In some cases, the vector may be fused to other sequences to facilitate purification of the fusion protein expressed from the vector, such as glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA).

[0102] The vectors contain antibiotic resistance genes commonly used in the art as selection markers, such as genes for resistance to ampicillin, gentamicin, cabenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

[0103] In yet another aspect, the present invention relates to a cell transformed with the above-mentioned vector. The cells used to produce the fusion protein of the present invention may be, but are not limited to, prokaryotic, yeast or higher eukaryotic cells.

[0104] Prokaryotic host cells such as Escherichia coli, strains of Bacillus, such as Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus) can be used.

[0105] However, of greatest interest are animal cells, and examples of useful host cell lines may be, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0106] In yet another aspect, the present invention relates to a method for producing the fusion protein, comprising: (a) culturing the cells; and (b) recovering the fusion protein from the cultured cells.

[0107] The cells can be cultured in a variety of media. Any commercially available medium can be used as the culture medium. Any other necessary supplements known to those skilled in the art may be included at appropriate concentrations. The culture conditions, such as temperature, pH, etc., will be used with the host cell selected for expression and will be apparent to those skilled in the art.

[0108] The fusion protein can be recovered by, for example, centrifugation or ultrafiltration to remove impurities, and the resultant can be purified, for example, by affinity chromatography. Additional purification techniques, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, etc., may be used.

[0109] The antibody contained in the fusion protein may be IgG or a fragment containing the variable region, i.e., ScFv or Fab, and the variable region of the heavy chain may be IgG1, IgG2, IgG3, or IgG4.

[0110] In still another aspect, the present invention relates to a composition for preventing or treating angiogenic diseases, comprising the fusion protein as an active ingredient.

[0111] The angiogenesis means the formation or growth of new blood vessels from pre-existing blood vessels, and the "angiogenesis-related disease" means a disease associated with the occurrence or progression of angiogenesis. If the disease can be treated by the fusion protein, the disease may be included in the scope of the angiogenesis-related disease without limitation.Examples of angiogenesis-related diseases include cancer, metastasis, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, macular degeneration, neovascular glaucoma, erythrosis, proliferative retinopathy, psoriasis, hemophilic arthritis, capillary formation in atherosclerotic plaques, keloids, wound granulation, vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune diseases, Crohn's disease, and the like. Disease, restenosis, atherosclerosis, intestinal adhesions, cat scratch disease, ulcers, liver cirrhosis, nephritis, diabetic nephropathy, diabetic foot ulcers, chronic kidney failure, diabetes mellitus, inflammatory diseases, idiopathic pulmonary fibrosis, sepsis, acute respiratory distress syndrome, and neurodegenerative diseases.

[0112] The cancers include esophageal cancer, stomach cancer, large intestine cancer, rectal cancer, oral cancer, pharynx cancer, larynx cancer, lung cancer, colon cancer, breast cancer, uterine cervical cancer, and endometrial cancer. cancer), ovarian cancer, prostate cancer, testis cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer cancer), leukemia, Hodgkin's lymphoma The cancer is selected from the group consisting of, but not limited to, lymphoma, lymphoma, and multiple myeloid blood cancer.

[0113] As used herein, the term "prevention or prophylaxis" refers to any measure in which a fusion protein of the invention is administered to prevent or delay the onset of a disease of interest. The term "treatment or therapy" refers to any measure in which a fusion protein of the invention is administered to improve or ameliorate the symptoms of a disease of interest.

[0114] The present invention may be, for example, a pharmaceutical composition for preventing or treating tumors or cancer, comprising (a) a pharma- ceutical effective amount of the fusion protein of the present invention; and (b) a pharma- ceutical acceptable carrier. The present invention may also be a method for preventing or treating tumors or cancer, comprising administering the fusion protein to a patient with tumor or cancer. The present invention may further be a use of the fusion protein for interfering with a mechanism and thereby preventing or treating tumors or cancer.

[0115] Non-limiting examples of tumors or cancers suitable for treatment include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate carcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioblastoma, leukemia, lymphoma, and other neoplastic carcinomas. Additionally, the present invention includes refractory or recurrent cancers that can be treated using the fusion proteins.

[0116] The present invention provides, for example, a method for treating an angiogenic disease, comprising administering a therapeutically effective amount of the fusion protein to a patient in need of angiogenesis inhibition. The treatment of an angiogenic disease may further comprise identifying a patient in need of angiogenesis inhibition prior to the administration step. In yet another example, a method for preventing and / or treating an angiogenic disease is provided, comprising administering a therapeutically effective amount of the fusion protein to a patient. The method for preventing and / or treating an angiogenic disease may further comprise identifying a patient in need of angiogenesis inhibition prior to the administration step.

[0117] The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier, which may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc., which are generally used in the formulation of drugs, but are not limited thereto. The pharmaceutical composition may further comprise one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives, which are generally used in the manufacture of pharmaceutical compositions.

[0118] The pharmaceutical composition or the effective amount of the angiogenic disease can be administered orally or parenterally. Parenteral administration can be by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration. Since proteins or peptides are digested during oral administration, oral compositions can be formulated to protect the active agent from degradation in the stomach, such as by coating the active agent. The composition can also be administered by any device capable of transferring the active agent to the target cells.

[0119] The composition of the present invention is administered in a pharma- ceutical effective amount. As used herein, the term "pharma-ceutical effective amount" refers to an amount of a pharmaceutical composition sufficient to treat a disease at a reasonable benefit / risk ratio applicable to all medical treatments. The effective amount varies depending on various factors, including the severity of the disease to be treated, the age and sex of the patient, the type of disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration, the rate of excretion, the duration of treatment, co-administration of drugs, and other parameters well known in the art. The composition of the present invention may be administered alone or in combination with other therapies. In such cases, it may be administered in sequence or simultaneously with the conventional therapies. The composition may also be administered in a single dose or in multiple doses. When all of these factors are taken into consideration, it is important to administer the minimum amount sufficient to obtain the maximum effect without side effects, and this dosage can be easily determined by a person skilled in the art. The dosage of the pharmaceutical composition of the present invention is not particularly limited, but may vary depending on various factors, including the health condition and weight of the patient, the severity of the disease, the type of drug, the route of administration, and the time of administration. The compositions may be administered in a single dose or in multiple doses daily to mammals, including rats, mice, livestock, humans, etc., via any typically accepted route, such as orally, rectally, intravenously, subcutaneously, intrauterinely, or intracerebrovascularly.

[0120] The content of the fusion protein in the pharmaceutical composition may be variously formulated depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration interval, administration route, excretion rate and reaction sensitivity. For example, the daily dose of the fusion protein may be in the range of 0.001 to 1000 mg / kg, specifically 0.01 to 100 mg / kg, more specifically 0.1 to 50 mg / kg, and even more specifically 0.1 to 20 mg / kg, but is not limited thereto. The daily dose may be formulated as a single preparation in the form of a unit dose, or may be appropriately divided and formulated, or may be prepared in a multi-dose container.

[0121] The pharmaceutical composition may be administered in combination with other drugs, such as other therapeutic agents for angiogenesis inhibition-related diseases, and the dosage, administration method and type of other drugs may be appropriately prescribed depending on the condition of the patient.

[0122] The pharmaceutical composition may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be formulated in the form of an extract, powder, powder, granule, tablet or capsule, etc., and may further contain a dispersing agent or stabilizer for formulation.

[0123] In particular, the pharmaceutical composition containing the fusion protein can be formulated as an immunoliposome since it contains the fusion protein. The liposome containing the fusion protein may be prepared by a method well known in the art. The immunoliposome is a lipid composition containing phosphatidylcholine, cholesterol, and polyethylene glycol-derivatized phosphatidylethanolamine, and may be prepared by a reverse phase evaporation method (Korean Patent Publication No. 10-2015-0089329). For example, an antibody Fab' fragment may be conjugated to the liposome by a disulfide exchange reaction.

[0124] The fusion protein of the present invention may be used in combination with other pharmaceutical preparations, antibodies, or biologically active agents, or with substances for various purposes. In this respect, the present invention relates to a composition for combined administration with other therapeutic agents for angiogenesis diseases, comprising the fusion protein.

[0125] The other therapeutic agent for angiogenic diseases may include an antiangiogenic drug, an anti-inflammatory drug, and / or an anticancer drug, thereby overcoming the resistance of each other and enhancing the efficacy.

[0126] In the composition according to the present invention, when it is administered in combination with other therapeutic agents for angiogenesis diseases, the fusion protein and the other therapeutic agents for angiogenesis diseases may be administered sequentially or simultaneously. For example, an anti-angiogenic drug, an anti-inflammatory drug, and / or an anti-cancer drug is administered to a subject, and then a composition containing the fusion protein as an active ingredient is administered to the subject, or the composition is administered to a subject, and then an anti-angiogenic drug, an anti-inflammatory drug, and / or an anti-cancer drug is administered to the subject. In some cases, the composition may be administered to a subject simultaneously with the anti-angiogenic drug, the anti-inflammatory drug, and / or the anti-cancer drug.

[0127]

[0128] Working Example

[0129] The present invention will be described in more detail below with reference to examples. It will be apparent to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0130]

[0131] Example 1. CDR grafting

[0132] The 6A6 CDR sequence was confirmed using Kabat numbering and the IMGT program, and then cloned to transplant it into human germline VH3 / VK1. The heavy chain CDR-grafted sequence is shown in FIG. 1, and the light chain CDR-grafted sequence is shown in FIG. 2.

[0133]

[0134] Example 2. Mutations and selection for affinity enhancement

[0135] Optimization was performed to improve the affinity of the CDR-grafted antibody. Primers were prepared to introduce random mutations into the light chain CDR3 and heavy chain CDR3 using a soft-randomization method that randomizes the original DNA sequences of the light chain and heavy chain CDR3 while preserving 70% of the original DNA sequences. DNA fragments encoding the light chain variable region and heavy chain variable region with mutations were obtained by PCR using these primers. These DNA fragments were substituted with the light chain variable region of scFv phage and phagemid, and the heavy chain variable region of scFv phage and phagemid, respectively, to prepare a light chain CDR3 mutant scFv phage library and a heavy chain CDR3 mutant scFv phage DNA library, respectively.

[0136] The mutant scFv phage DNA library was purified with phenol-chloroform and then transformed into E. coli strain XL-1 Blue by electroporation. After confirming that diversity was ensured by transformation efficiency analysis and DNA sequence analysis, the phage expression was induced by culturing at a 500 ml scale, and a light chain and heavy chain CDR3 mutant scFv phage library was prepared by PEG precipitation.

[0137] Biopanning was performed using each mutant scFv phage library. 100 μl of antigen (KDR1-3 domian-Fc) was added to each well of a 96-well immunoplate at 2 μg / ml and left at 4°C overnight. The next day, the antigen-coated plate was washed three times with PBST (0.1% Tween 20), and then 200 μl of 2% BSA blocking buffer was added and reacted at room temperature for 2 hours. 50 μl of XL1-Blue stock was added to 2 ml of 2x YT-TET (tetracycline 10 μg / ml) growth medium, and grown at 37°C and 200 rpm for about 2 hours, after which 13 ml was added and the OD 600 The wells were incubated until the OD reached 0.5. After 2 hours of blocking, the wells were washed three times with 1X PBST (0.1% Tween 20). The mutant library was mixed with the same amount of 4% BSA to each washed well, and 200 μl of the mixture was added. The wells were then rocked at room temperature for 30 minutes and reacted for 2 hours. After the phage library reaction was complete, the supernatant was discarded and the wells were washed five times with 0.1% PBST and five times with PBS. Each well was added with 100 μl of 100 mM TEA (trimethylamine), and the wells were shaken at room temperature for 10 minutes. After 10 minutes, 50 μl of 1 M Tris (pH 7.5) was added to each well and mixed. The supernatant was analyzed by OD 600The resulting solution was placed in 10 ml of XL1-blue with a titer of 0.5 and allowed to infect for 30 minutes at 37°C. After infection, 100 μl was used as the output titer and the remaining was centrifuged at 6,000 rpm for 10 minutes. The supernatant was discarded and the precipitate was spread onto a large square plate (CM 34 μg / ml + 1% glucose) and incubated overnight at 37°C. The 100 μl remaining as the output titer was diluted 1 / 10, 1 / 100, and 1 / 1000 and spread onto a CM plate and incubated overnight at 37°C. The next day, the colonies grown on the square plate were collected using a loop after adding 50 ml of 2x YT medium, and then centrifuged at 6000 rpm for 10 minutes. The supernatant was discarded, and the precipitate was used to make a first panning stock. 100 ml of 2x YT culture medium (growth media: CM34 μg / ml + 1% glucose) was placed in a 500 ml Erlenmeyer flask, and the OD 600 Cells were added so that the OD was 0.2, and the cells were incubated at 200 rpm and 37°C. 600 The OD was increased to 0.5. 600 After culturing the cells until the value reached 0.5, helper phage (M13KO7 mutant) was added at 20 times the number of cells. The helper phage was added and infected at 37°C for 30 minutes, and then centrifuged at 6000 rpm for 10 minutes. The supernatant was discarded, and the cells were resuspended in 2xYT medium (CM34μg / ml + Kan.70μg / ml + 1mM IPTG + 5mM MgCl 2) and left overnight at 200 rpm and 30°C. The next day, the grown cells were centrifuged at 7000 rpm for 10 minutes and centrifuged once more in the same manner. The collected supernatant was added with 1 / 5 (v / v) 20% PEG / 2.5m NaCl of the supernatant and precipitated on ice for 1 hour. After precipitation, it was centrifuged at 9000 rpm for 1 hour. The supernatant was discarded, the precipitate was dissolved in 5 ml of PBS, filtered through a 0.45 μm filter, stored at 4°C, and used in the next panning process. This process was repeated 3 to 4 times, and the antibody binding to the antigen was confirmed by ELISA.

[0138] The dissociation rate constant Kdis of scFv was then measured during the selection process as a quantitative evaluation index of the ability to retain binding.

[0139]

[0140] Example 3. Selection of antibodies with high affinity to antigen (off-rate screening)

[0141] The binding strength of the selected antibodies to the antigen was measured using Octet (Fortebio). To do so, KDR 1-3 domains were immobilized on a biosensor, and then candidate antibodies expressed in the form of scFv were added and allowed to bind, after which the dissociation rate constants were measured. The dissociation rate constants for the five selected optimized clones are shown in Table 1 and their amino acid sequences in Tables 2 and 3.

[0142] [Table 1] Dissociation rate constants of antibodies that specifically bind to antigens JPEG2025503728000002.jpg50128

[0143]

[0144] Example 4. Fusion protein production

[0145] Among the scFv phage antibodies selected for the production of fusion proteins, D4, which had the lowest dissociation constant, was converted to IgG form. The IgG form conversion was performed using molecular biology techniques. Phagemids were extracted from the selected E. coli clones, and the variable regions were amplified using PCR techniques. The amplified heavy chain variable region was inserted into an expression vector (Invivogen, pfusess-hchg1) containing a heavy chain constant region, and the amplified light chain variable region was inserted into an expression vector (Invivogen, pfuse2ss-hclk) containing a light chain constant region, completing DNA cloning in IgG form and converting it to IgG1 form. A DLL4 binding domain (Notch1 EGF-like domain) containing a linker (G4S sequence) was genetically synthesized and fused to the N-terminus of the converted light chain constant region. The nucleotide sequences of the antibodies produced are shown in Tables 2 and 3.

[0146] [Table 2] CDR sequences of selected antibodies JPEG2025503728000003.jpg36134

[0147] [Table 3] Sequences of selected antibodies JPEG2025503728000004.jpg138135

[0148] JPEG2025503728000005.jpg137139

[0149] JPEG2025503728000006.jpg134136

[0150] JPEG2025503728000007.jpg126128

[0151] JPEG2025503728000008.jpg126129

[0152] JPEG2025503728000009.jpg170128

[0153] JPEG2025503728000010.jpg119128

[0154]

[0155] Example 5. Fusion Protein Expression

[0156] For transient expression of IgG forms, the Expi293F expression system kit (Thermo Fisher Scientific, US) was used. Expi293 cells included in the kit were incubated in a dedicated medium at 37°C, 5% CO 2 The suspension culture was performed on an orbital shaker at 125 rpm under ambient conditions. 5 The cells were subcultured at a concentration of 3 × 10 6 The cell number was adjusted to 1000 cells / ml. A dedicated reagent, Expifectamine, was used for gene transfection. A Lipid-DNA complex containing 1μg of expression vector DNA and 2.7μl of Expifectamine per 1ml of cell suspension was prepared and added to the cell suspension. 16-18 hours after transfection, Enhancer 1 / 2 was added to induce expression. After that, the cells were cultured for 3-4 days under the same conditions, and then centrifuged to collect the IgG-containing supernatant.

[0157]

[0158] Example 6. Purification of fusion proteins

[0159] The resulting supernatant was loaded onto a Protein A column (GE Healthcare) and IgG was purified by affinity chromatography. The column was equilibrated with 20 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA (pH 7.0), and then the supernatant was loaded onto the column and washed with 50 mM Tris-HCl, 500 mM NaCl, 5 mM EDTA, 0.2% polysorbate 20 (pH 7.0) solution, followed by elution with 50 mM NaCl, 0.1 M glycine-HCl (pH 3.5) and neutralization with 1 M Tris. The eluted protein was exchanged for PBS by a dialysis process using a MWCO 10,000 spectra / por dialysis membrane (Spectrum Labs, US). The eluted protein was then concentrated to the required concentration using a Vivaspin (Satorius, DE), and stored at -80°C after aliquoting.

[0160]

[0161] Example 7. Binding specificity analysis of fusion proteins

[0162] The binding strength of the selected fusion proteins to the antigen was measured using SPR (Biacore T200, Cytiva). The fusion proteins were captured and analyzed using the antigen protein as an analyte. The fusion proteins were immobilized on the surface of a Protein A chip according to the manufacturer's instructions. The fusion proteins were diluted in PBS-T running buffer at a concentration of 5ug / ml and injected into the sensor chip at a flow rate of 10ul / min to reach approximately 200RU. The antigens were diluted in PBS-T running buffer according to concentration and injected at a flow rate of 30ul / min for 4 minutes, with a dissociation time of 10 minutes, for analysis. To remove the bound fusion proteins every cycle, a regeneration solution (10mM glycine-HCl, pH 1.5) was injected for 1 minute at a flow rate of 30ul / min. The experimental results were calculated using a 1:1 binding model in the BIA evaluation software version 1.0. The binding strengths are shown in Table 4, and the SPR sensorgrams are shown in Figures 3A and 3B.

[0163] [Table 4] Binding strength of fusion proteins that specifically bind to antigens JPEG2025503728000011.jpg46146

[0164]

[0165] Example 8. Confirmation of simultaneous binding ability of fusion proteins to antigens

[0166] The ability of the selected fusion protein to simultaneously bind to two antigens was confirmed using SPR (Biacore T200, Cytiva). The fusion protein was immobilized on the surface of a Protein A chip according to the manufacturer's instructions. The fusion protein was diluted in PBS-T running buffer at a concentration of 5ug / ml onto the sensor chip above to reach approximately 200RU. First, 100nM hKDR-ECD diluted in PBS-T running buffer was injected at a flow rate of 30ul / min and allowed to bind for 120 seconds, and then PBS-T running buffer was run at a flow rate of 30ul / min for 60 seconds. 200nM hDLL4 diluted in PBS-T running buffer without regeneration was injected at a flow rate of 30ul / min and allowed to bind for 120 seconds, and then PBS-T running buffer was run at a flow rate of 30ul / min for 60 seconds. The results of simultaneous binding of both antigens are shown in FIG.

[0167]

[0168] Example 9. Confirmation of the proliferation ability of fusion protein in HUVECs

[0169] To compare the changes in proliferation ability of vascular endothelial cells (Human Umbilical Vein Endothelial Cells, HUVECs) of the prepared fusion proteins, fusion proteins and control antibodies (6A6, 6A6xDLL4, D4, Minod-Fc) were treated with vascular endothelial cells (Human Umbilical Vein Endothelial Cells, HUVECs). 6A6xDLL4 is as shown in Table 5. Minod-Fc is a protein in which Notch1 of SEQ ID NO: 18 is fused with Fc.

[0170] [Table 5] JPEG2025503728000012.jpg46144

[0171] Briefly, HUVEC (LONZA, Cat No. C2519A) cells were cultured on 2% gelatin-coated plates using VascuLife medium complete kit (Lifeline cell technologies, Cat No. #LCT-LL-0005) medium. The medium was maintained at 37°C, 5% CO 2 The HUVECs used in the assay were cells within passage 10, and were cultured at 1x10 cells / well in a 96-well plate in M199 medium (Gibco, Cat No. #11043-023) containing 0.5% heat-inactivated FBS. 4 Each antibody was diluted to 250nM, 50nM, 10nM, or 2nM, applied to the corresponding well of the assay plate, and then incubated for 30 minutes. Then, VEGF165 (R&D systems, Cat No. 293-VE) was applied to each well at a concentration of 100ng / mL. The negative control group (VEGF- / Ab-,) not treated with VEGF or drugs, and the positive control group (VEGF+ / Ab-) treated with VEGF only were incubated at 37°C for 3 days in CO. 2 The cells were cultured in an incubator. Viability was measured by detecting ATP in living cells using a CellTier Glo-Luminescent Cell viability Assay kit (Promega, Cat No. G7571).

[0172] As a result, the D4 comparison group, which is an optimized antibody, and the D4xDLL4 test group, which has a D4 backbone, showed greater inhibition of VEGF165-mediated HUVEC proliferation than the 6A6 comparison group and the 6A6xDLL4 comparison group having a 6A6 backbone, and both of these showed concentration-dependent behavior. The results are shown in Figure 5.

[0173]

[0174] Example 10. Inhibitory effect of fusion protein on VEGFR-2 phosphorylation

[0175] To confirm the ability of the fusion proteins to dephosphorylate KDR (VEGFR-2), the fusion proteins and control antibodies (6A6, 6A6xDLL4, D4, Minod-Fc) were compared by Western blot analysis.

[0176] Briefly, HUVEC (LONZA, Cat No. C2519A) cells were cultured in 6-well plates at 8×10 cells using VascuLife medium complete kit (Lifeline cell technology, Cat No. #LCT-LL-0005). 5 The cells were seeded at a concentration of 1000 cells / well and incubated at 37°C, 5% CO 2The cells were cultured for one day under the same conditions. HUVEC cells were treated with each antibody at 125nM or 25nM for 30 minutes, and then treated with VEGF165 (R&D, Cat No. 293-VE) at a concentration of 50ng / mL for 10 minutes in each well. A negative control group (VEGF- / Ab-) not treated with VEGF or drugs and a positive control group (VEGF+ / Ab-) treated with VEGF only were included. The drug-treated HUVECs were washed with phosphate-buffered saline, and then lysis buffer (Thermo Scientific, RIPA buffer and phosphatase inhibitor) was added and cell lysis was performed on ice for 10 minutes. The lysed cells were scraped using a cell scraper and centrifuged at 13,000 rpm at 4℃ for 10 minutes to obtain only the supernatant. The total protein of the obtained supernatant was quantified using the BCA protein quantification method, and 20 μg of each was loaded onto a 4-15% grade SDS-PAGE gel and electrophoresed. After electrophoresis, the gel was transferred to a nitrocellulose membrane (Bio-Rad, Trans-Blot Turbo Transfer System) for Western blot analysis. The nitrocellulose membrane blot was blocked for 1 hour at room temperature with blocking buffer [5% skim milk / TBS-T (0.1% Tween 20, Tris-based saline)]. After washing with TBS-T buffer, the primary antibody (p-VERFR2 (Y1175), Cell Signaling) was diluted 1:1000 and incubated at room temperature for 1 hour, and then washed. The secondary antibody (anti-rabbit-HRP) was diluted 1:5000 and incubated at room temperature for 1 hour. After washing with TBS-T buffer, the cells were treated with enhanced chemiluminescence (ECL) solution (Thermo Fisher Scientific, Cat No. 34096) and reacted, and images were obtained using a Luminescent Image Analyzer (GE Healthcare, Amersham Imager 680).

[0177] The results showed that VEGF165-mediated VEGFR2 phosphorylation was partially inhibited in a concentration-dependent manner in the 6A6 and 6A6xDLL4 control groups, whereas VEGFR2 was almost completely dephosphorylated in the test groups treated with the same amount of the optimized antibodies D4 control group and D4xDLL4. The results are shown in Figure 6.

[0178]

[0179] Example 11. Confirmation of KDR (VEGFR-2) binding ability using FACS

[0180] To examine the binding activity of the fusion proteins to HUVECs, FACS was performed using the fusion proteins and control antibodies (6A6, 6A6xDLL4, D4, and Minod-Fc).

[0181] Briefly, HUVEC (LONZA, Cat No. C2519A) cells were cultured in VascuLife medium complete kit (Lifeline cell technology, Cat No. #LCT-LL-0005) medium on 2% gelatin-coated plates at 37°C and 5% CO. 2 The cells were cultured under the following conditions: 2 × 10 7The cells were prepared at a concentration of 100 μl / ml and dispensed into FACS tubes at 50 μl each. 100 μl each of 5 nM antibodies were mixed into FACS tubes and reacted at 4°C for 30 minutes. After the reaction was completed, 2 mL of FACS buffer was added to each tube and centrifuged at 1500 rpm for 3 minutes. The supernatant was removed, the cells were re-suspended, and 100 μl each of anti-human IgG-PE antibody (1:200, Bethyl, #A80-248PE) was added and reacted at 4°C for 20 minutes. 2 mL of FACS buffer was added to each FACS tube and centrifuged at 1500 rpm for 3 minutes. The separated supernatant was removed, and 200 μl each of FACS buffer was added and suspended, and the binding activity was analyzed using FACS (Beckton Dickinson, Lyric).

[0182] As a result of the test, it was confirmed that 6A6, 6A6xDLL4, D4, and D4xDLL4, which target VEGFR2, all bound to HUVEC, except for Minod-Fc. In addition, it was found that D4 and D4xDLL4 had improved binding activity compared to 6A6 and 6A6xDLL4. The results are shown in Figure 7.

[0183]

[0184] Example 12. Confirmation of binding ability of antibody to hDLL4 expressed on cell surface

[0185] To generate cells expressing DLL4 on the cell surface, HEK293 cells were transformed with pcDNA-hDLL4.

[0186] Briefly, the pcDNA-hDLL4 vector synthesized at GeneArt was transfected into HEK293 cells using lipofectamine. TMThe cells were transfected using 2000 (Invitrogen #1168-019, USA) and selected by adding neomycin (Gibco #10131, 50mg / mL), after which all the growing cells were collected and cultured in a pool state. Then, they were cultured using the limiting dilution method to obtain 23 single colonies, and one cell line that highly overexpressed hDLL4 was selected from the single colonies for the experiment. The selected cells were diluted to 2 x 10 7 The cells were prepared at a concentration of 100 μL / mL and dispensed into FACS tubes at 50 μL each. 100 μL each of fusion proteins and control antibodies (6A6, 6A6xDLL4, D4, D4xDLL4, Minod-Fc) at a concentration of 5 nM were added to the FACS tubes containing the cells, and the tubes were reacted at 4°C for 30 minutes. After the reaction was completed, 2 mL of FACS buffer was added to each tube, and the tubes were centrifuged at 1500 rpm for 3 minutes. The supernatant was removed, the cells were thoroughly suspended, and 100 μL of anti-human IgG-PE antibody (1:200, BioLegend, #A80-248PE) was added to each tube, and the tubes were reacted at 4°C for 20 minutes. After the reaction was completed, 2 mL of FACS buffer was added to each FACS tube, and the tubes were centrifuged at 1500 rpm for 3 minutes. The separated supernatant was removed, and the cells were suspended in 200 ul of FACS buffer each, and the binding activity was analyzed using FACS (Beckton Dickinson, Lyric).

[0187] As a result of the test, only Minod-Fc, 6A6xDLL4, and D4xDLL4, which contain the Minod domain targeting DLL4, bound to HEK293 cells overexpressing DLL-4 (DLL4-overexpressed HEK293), and the fusion proteins 6A6xDLL4 and D4xDLL4 bound better than Minod-Fc alone. 6A6 and D4 did not bind to HEK293 cells overexpressing DLL-4. The results are shown in Figure 8.

[0188] [Industrial Applicability]

[0189] The present invention provides a fusion protein that simultaneously targets VEGFR2 / KDR and DLL4, and contains an antibody with improved affinity compared to existing antibodies targeting VEGFR2 / KDR, thereby providing a more effective combination therapy and single therapy in tumor treatment compared to existing therapeutic agents. It shows improved binding to VEGFR2 / KDR, and can be usefully used in the prevention or treatment of a target cancer / tumor or disease associated with angiogenesis inhibition, and has superior angiogenesis inhibition ability compared to single treatments by simultaneously inhibiting the activity of DLL4 by Notch1.

[0190]

[0191] Although the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that such specific descriptions are merely preferred embodiments and thus limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

[0192] Sequence Listing Free Text

[0193] Attach as electronic file

Claims

1. A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR; and a DLL4 (Delta-like ligand 4) binding protein domain, The antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR is a fusion protein comprising a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, and a heavy chain CDR3 of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO:

6.

2. The fusion protein of claim 1 , wherein the VEGFR2 / KDR-binding antibody comprises a heavy chain variable region of SEQ ID NO:8 and a light chain variable region of SEQ ID NO:

10.

3. The fusion protein of claim 1 , wherein the DLL4 binding protein is Notch1 comprising the sequence of SEQ ID NO:

18.

4. The fusion protein of claim 1 , wherein the DLL4 binding protein domain binds to the N-terminus of a light chain of an antibody that binds to VEGFR2 / KDR.

5. The fusion protein according to claim 1 , wherein the VEGFR2 / KDR-binding antibody or antigen-binding fragment thereof and the DLL4 binding protein domain are linked via a linker.

6. The linker is ｎ S) ｍ (n, m are each 1 to 10).

7. A nucleic acid encoding the fusion protein according to any one of claims 1 to 6.

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

9. A transformed cell comprising the expression vector according to claim 8.

10. A method for producing a fusion protein comprising the steps of: (a) culturing the cell of claim 9 to produce the fusion protein; and (b) recovering the produced fusion protein.

11. A composition for preventing or treating angiogenic diseases, comprising the fusion protein according to any one of claims 1 to 6 as an active ingredient.

12. A composition for diagnosing angiogenic diseases, comprising the fusion protein according to any one of claims 1 to 6 as an active ingredient.

13. A composition for preventing or treating tumors or cancer, comprising the fusion protein according to any one of claims 1 to 6 as an active ingredient.

14. A composition for combined administration with another therapeutic agent, comprising the fusion protein according to any one of claims 1 to 6.

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