WO2012161372A1 - Double-target antibody fused with a peptide and use of same - Google Patents

Abstract

Disclosed are a double-target antibody, derived from a human monoclonal antibody, that effectively suppresses a signal transduction mechanism for angiogenesis and the propagation of cancer cells by simultaneously neutralizing VEGFR-2 and c-Met, which are both involved in angiogenesis, and a composition comprising said antibody for suppressing angiogenesis and for the treatment of cancer. The double-target antibody according to the present invention simultaneously neutralizes two targets involved in the angiogenesis and propagation of cancer cells. Therefore, the double-target antibody has a good neutralizing property when compared to a conventional single target antibody and is very effective in the treatment of cancer. The present inventor expects better effects from manufacturing the double-target antibody according to the present invention than from a treatment using a single target antibody.

Description

Dual target antibody fused to a peptide and its use.

The present invention relates to a dual target antibody in which the water-soluble peptide that binds to c-Met is fused to the N-terminus of an antibody that binds to VEGFR-2, and its use. More specifically, the present invention provides a dual target antibody capable of simultaneously neutralizing VEGFR-2 and c-Met, in which a water-soluble antagonist peptide that binds to c-Met is fused to the light chain N-terminus of a neutralizing antibody that binds to VEGFR-2. And a DNA encoding the dual target antibody, a recombinant expression vector comprising the DNA, a host cell transformed with the recombinant expression vector, and a method for producing a dual target antibody by culturing the host cell, and the dual target antibody. It relates to a pharmaceutical composition.

Angiogenesis, known as an important factor for solid tumor formation, is a mechanism in which new blood vessels are formed from existing blood vessels by the growth, division, and migration of endothelial cells, and is a normal growth process including healing of a wound and a woman's menstrual cycle. Play an important role in (Risau, Nature , 386: 671, 1997), as well as abnormally excessive angiogenesis, tumor growth and metastasis, age-related macular degeneration (ARMD), It is known to play a decisive role in diseases such as diabetic retinopathy, psoriasis, rheumatoid arthritis, and chronic inflammation (Carmeliet and Jain, Nature , 407: 249, 2000).

1971 Since J. Folkman has hypothesized that tumor growth and metastasis is angiogenesis-dependent, and therefore, anti-angiogenesis-focused therapies may be new therapies for solid cancer, Related work has drawn the attention of many researchers (Ferrara and Kerbel, Nature , 435: 967, 2005). The process of angiogenesis is determined by the overall balance of angiogenic factors and angiogenesis inhibitors, and is a complex, sequential process. Looking at the process, first, various angiogenic factors, including Vasicular Endothelial Growth Factor (VEGF) secreted from tumors or wounded tissues, bind to the corresponding receptors of existing vascular endothelial cells. By activating vascular endothelial cells to increase the permeability of vascular endothelial cells, by secreting proteolytic enzymes such as matrix metalloproteinase (MMP), it breaks down the basement membrane and extracellular matrix around vascular endothelial cells, Endothelial cells move away from existing capillaries and proliferate toward tissues that secrete angiogenic factors. The migrated and proliferated vascular endothelial cells form a vascular tube structure, and finally, blood vessels that are structural supports of vascular endothelial cells. As pericyte enters, stable and mature angiogenesis occurs It is good.

In the present invention, VEGF / VEGFR and HGF / c-Met signaling mechanisms were considered as potential targets in order to simultaneously inhibit tumor proliferation and metastasis in addition to suppression of angiogenesis derived from tumors for the development of antibodies for anticancer treatment. VEGF, which is known to have a significant effect on most stages of angiogenesis, is widely secreted in the hypoxia region of the tumor tissue area. VEGF was founded in 1989 by Dr. Genentech. Protein isolation, purification and cDNA cloning were done by the N. Ferrara group (Leung et al ., Science , 246: 1306, 1989). VEGF, also called VEGF-A, is known to have four isotypes (VEGF ₁₂₁ , VEGF ₁₆₅ , VEGF ₁₈₉ , VEGF ₂₀₆ ) to date, and VEGF ₁₆₅ is reported to be the most abundant in all human tissues except the placenta. (Tisher et al ., J. Biol. Chem. , 266: 11947, 1991).

VEGF binds to its receptors, VEGFR-1 and VEGFR-2 with very high affinity, but transmits its signals mainly through VEGFR-2, thereby regulating mechanisms related to angiogenesis such as proliferation and migration of vascular endothelial cells. It is known to induce. For this reason, VEGF and VEGFR-2 have been the main targets for inhibiting VEGF-induced angiogenesis, and a number of articles have dealt with them (Ellis and Hicklin, Nature Rev. Cancer , 8: 579, 2008; Youssoufian et al ., Clin. Cancer Res. , 13: 5544s, 2007). For example, Genentec's Avastin is a humanized antibody that targets VEGF-A (Ferrara et al ., Biochem. Biophy. Res. Comm. , 333: 328, 2005), and metastatic colorectal cancer in 2004. cancer, non-small cell lung cancer in 2006, Her-2 negative metastatic breast cancer in 2008, and kidney and brain tumors in 2009 Each of them is commercially available with US FDA approval and is currently in clinical trials with various solid carcinomas to expand its indications. In addition, Lucentis, which was released by the same company, cuts only Fab fragments from Avastin in order to prevent excessive angiogenesis around the macula, which is a major aspect of senile macular degeneration, and to improve its permeability when injected into the retina. As a manufactured antibody (Eter et al. , Biodrgus , 20: 167, 2006) and a therapeutic drug for wet age-related macular degeneration (wet-ARMD), the US FDA approval in 2006. Another therapeutic antibody that targets VEGF is Regeneron's VEGF-trap (Holash et al. , PNAS , 99: 11393, 2002). It is a water-soluble decoy receptor, a fusion of the second immunoglobulin domain of VEGFR-1 and the third immunoglobulin domain of VEGFR-2 to human Fc, which has not yet received US FDA approval, but has been metastatic breast cancer and metastatic. Lung cancer, metastatic colorectal cancer, and hormone refractory prostate cancer are currently in Phase III.

Angiogenesis-inhibiting antibodies targeting VEGF receptor VEGFR-2 include IMC-1121B (EP 1916001A2), UCP CDP-791 (PCT / GB02 / 04619), and TTAC developed by the researchers. -0001 (see Korean Patent No. 10-0883430 and International Application No. PCT / KR07 / 003077). IMC-1121B is a monoclonal antibody selected from a fully human Fab library and is currently in Phase III stage for metastatic breast and gastric cancer. UCB's CDP-791 is a humanized antibody, currently undergoing Phase II in the form of PEGylated Di-Fab for non-small cell lung cancer. Because the antibody does not have an Fc, antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity cannot be expected. Finally, TTAC-0001 (Tanibirumab), developed by our team and currently being studied at preclinical stage, is a monoclonal antibody selected from a full human ScFv phage library, which targets VEGFR-2 and at the same time flk- from mouse or rat. It is the only antibody that is responsive to 1 (VEGFR-2 homologue), which is one of the important features that distinguishes it from IMC-1121B (PCT / KR07 / 003077). In particular, cross-species cross reactivity shown by TTAC-0001 enables the study of animal disease models, inducing transitional research and progressing the development of anticancer agents for specific cancers in the future. It can help you finish easily.

On the other hand, HGF / c-Met signaling mechanism, which is simultaneously monitored by VEGF / VEGFR-2 signaling as an anti-cancer therapeutic target, is overexpressed in various cancer tissues and tumor cells, resulting in tumor cell growth and metastasis. It is well known for its great contribution.

The mesenchymal-epithelial transition factor (C-Met) was first cloned from osteosarcoma-derived cancer cells by Cooper et al. (Cooper et al., Nature, 311: 29, 1984). This protein is a cell surface receptor and is an oncogene of the tyrosine kinase family, an alpha (a) subunit consisting of only 50 kD extracellular domain, and extracellular and cell membrane permeation. , A total of 145 kD beta (β) subunits consisting of the tyrosine kinase domain and tyrosine motifs involved in phosphorylation (Dean et al., Nature, 4; 318 (6044): 385, 1985; Park et al., PNAS , 84 (18): 6379, 1987, Maggiora et al., J. Cell Physiol., 173: 183, 1997).

Many researchers have also found that hepatocyte growth factor / scatter factor (HGF / SF) must bind to the c-Met extracellular domain in the beta subunit of the two subunits in order for the tyrosine kinase domain to be activated. It has been reported that the 1349th and 1356th tyrosine of the c-Met, which is very important, can be phosphorylated to activate tyrosine kinase domains (Bottaro et al., Science, 15; 251 (4995): 802, 1991; Cooper, Oncogene, 7 1): 3, 1992, Maggiora et al., J. Cell Physiol, 173: 183, 1997; Ponzetto et al., Cell, 77 (2): 261 , 1994; Maina et al., Cell, 87: 531, 1996). Giordano et al., On the other hand, reported that c-Met's tyrosine kinase activity can be stimulated through the interaction of plexin B1 (PLXNB1) with semaphorin 4D (SEMA4D) receptors in addition to HGF as the only ligand of c-Met. Giordano et al. , Nat. Cell Biol., 4 (9): 720, 2002.

HGF, known as a ligand of C-Met, is a growth hormone with multiple functions, stimulates various signaling pathways through c-Met to enhance cell mitogenesis and cell motility, inhibit cell death, Promote the transformation, including invasion and metastasis by angiogenesis and extracellular matrix (ECM) (Jeffers et al. , J. Mol. Med., 74: 505) , 1996; Amicone et al., EMBO J., 16: 495, 1997; Matsumoto and Nakamura, Biochem.Biophys.Res.Comm ., 239: 639, 1997; Corps et al., Int. J. Cancer, 73: 151, 1997). C-Met itself is usually expressed from a variety of epithelial and mesenchymal derived cells, while HGF is known to be expressed only in cells derived from mesoderm (Rong et al., Cancer Res. , 53 (22): 5355 (1993), c-Met was found to be activated by HGF (paracrine) derived from other cells or tissues as well as by HGF (autocrine) produced in the same cell (Ferracini et al., Oncogene , 10). (4): 739, 1995; Tsarfaty et al., Science , 263 (5143): 98, 1994). In particular, glioma (gliomas, Koochekpour et al., Cancer Res., 57: 5391, 1997), breast cancer (Nagy et al., Surg. Oncol., 5: 15,1996; Tuck et al., Am. J. Pathol., 148: 225, 1996), pancreatic cancer (Ebert et al., Cancer Res., 54: 5775, 1994), pleural mesotheliomas, Tolpay et al., J. Cancer Res. Clin.Oncol ., 124 : 291, 1998); Klominek et al. Intl. J. Cancer, 76: 240, 1998) c-Met and HGF are reported to be overexpressed in various cancer tissues and cells at the same time, but the development of cancer is also progressed through overexpression of c-Met regardless of HGF. Are commonly observed, liver cancer (hepatocellular carcinoma, Suzuki et al., Hepatology, 20 (5): 1231, 1996), gastric cancer (Taniguchi et al. , Cancer, 82: 2112-2122 (1998), lung cancer) et al. , J. Pathol., 180: 389, 1996), kidney cancer (Natali et al. , Intl. J. Cancer, 69: 212, 1996), ovarian cancer (Nagy et al., J. Surg. Oncol , 60:95, 1995) and colorectal cancer (Hiscox et al. , Cancer Invest., 15: 513, 1997).

As such, when c-Met is activated or overexpressed, the cancerous process is promoted, various methods of inhibiting the activation of c-Met have been developed as promising anticancer treatment strategies. One example is low molecular weight compounds designed to block the binding of ATP (adenosine triphosphate) to c-Met. These low molecular weight compounds include K252a from Fermentek biotechnology, SU11274 from Sugen, PHA-665752 from Pfiza (Morotti et al.,Oncogene, 21 (32): 4885, 2002; Berthou et al.,Oncogene, 23 (31): 5387, 2004; Pfizer, Christensen et al.,Cancer Res.,63 (21): 7345, 2003), they are designed to interfere with the phosphorylation of c-Met, preventing the downstream proteins of signal transduction from being activated. However, a disadvantage of these low molecular weight compounds is that they cannot specifically inhibit only phosphorylation by c-Met.

In addition, a second method of neutralizing HGF / c-Met signaling mechanism is a method of inhibiting the binding of c-Met and its ligand, HGF. Such methods include missing HGF fragments (Matsumoto & Nakamura, Cancer Sci. , 94 (4): 321, 2003) or antibodies neutralizing HGF (Cao et al. , PNAS., 98 (13): 7443, 2001; Kim et al. , Clin. Cancer Res., 12: 1292, 2006; Burgess et al. , Cancer Res. , 66 (3): 1721, 2006) or bind c-Met with a stronger affinity than the original HGF, but c And HGF precursors that do not activate -Met (pro-HGF, Mazzone et al. , J. Clin. Invest. , 114 (10): 1418, 2004). In addition, by using a phage display method and a panning technique (peptide) sequence that can inhibit the activity of c-Met (peptide) by selecting the peptide, the peptide is used to prevent c-Met activation ( Kim et al. , Biochem Biophys Res Commun. , 354: 115, 2007). Selected peptides are also applied to molecular imaging to search for tissues or organs in vivo that c-Met is overexpressed (Cao et al. , Clin. Cancer Res. , 13 (20); 6049, 2007). They have the limitation of inhibiting only HGF-dependent c-Met activation, but since they show actual anticancer effects in the laboratory ( in vitro ) or in vivo ( in vivo ), they can be combined with existing chemotherapy. It is considered to be useful. Currently, the epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib are used as effective cancer treatments, but drug resistance often develops, which has been reported to be due to intensive amplification of the c-Met receptor. (Engelman et al ., Science, 316 (5827): 1039, 2007), therefore, anti-cancer effects are expected to be amplified by incorporating c-Met inhibitors.

As such, recent market trends in the market for antibodies, in addition to developing antibodies that are functional against a single target, are also known as bispecific or multi-target antibodies that can simultaneously take two or more targets. Specific antibodies are actively being studied (Van Spriel et al. , Immunol. Today , 21: 391, 2000; Kufer et al. , Trend in Biotechnol. , 22: 238, 2004; Marvin and Zhu, Curr.Opin Drug Discovery Dev. , 9: 184, 2006). None of the antibodies in this class have been commercialized with FDA approval, but research and development is ongoing at the laboratory and clinical level with continued interest and potential. Antibodies belonging to this class can be broadly classified into 1) ScFv based antibodies, 2) Fab based antibodies, 3) IgG based antibodies, and the like.

First, for multi-target antibodies based on ScFv, there is a diabody of heterodimeric forms of hybrid ScFv obtained by combining V _L and V _H of different ScFv's with each other (Holliger et al., Proc. Natl.Acad. Sci. USA , 90: 6444, 1993). However, this type of antibody has a disadvantage of poor stability due to the weak binding force of the heterodimer. In addition, tendem ScFv obtained by linking different ScFv to each other (Kipriyanov et al. , J. Mol. Biol. , 293: 41, 1999; Robinson et al. , Brit. J. Cancer , 99: 1415, 2008), different ScFv Heterodimeric ScFv (De Kruif and Logtenberg, J. Biol. Chem. , 271: 7630, 1996) obtained by expressing Jun and Fos having mutual binding ability at the ends, respectively, and the Fab C _H1 and C _L at the ends of each ScFv Heterodimeric miniantibodies (Muller et al. , FEBS lett. , 432: 45, 1998) obtained from expression, and some amino acids in the C _H3 domain, a homodimeric domain of Fc, are substituted for a knob into hole heterodimeric structure. To produce heterodimeric ScFv forms of minibody by expressing these altered C _H3 domains at different ScFv ends (Merchant et al. , Nat. Biotechnol. , 16: 677). 1998). Among them, Micromet's BiTEs (Bispecific T-cell Engagers) technology is a technology for the production of dual target antibodies in the form of Tendem ScFv, by linking the anti-CD3 ScFv that activates T-cells with a specific linker to the tumor specific antigen, It has a mechanism of action by attracting T-cells to tumor cells to selectively remove only tumor cells. Currently, five BiTEs antibodies have been developed and are undergoing phase 1 and phase 2 trials for various diseases. In addition, various analogs based on ScFv have been reported academically (Kipriyanov and Le Gall, Curr. Opin. Drug Discovery Dev. , 7: 233, 2004), and triple target or tetrabody using triabody ( Quadruple target ScFv using tetrabody has also been reported in the academic world (Hudson and Kortt, J. Immunol. Methods , 231: 177, 1999).

Second, for Fab-based multitarget antibodies, heterodimeric Fabs (Brennan et al. , Science , 229: 81, 1985; Kostelny) obtained by combining individual Fabs for a specific antigen with one another using disulfide bonds or mediators et al. , J. Immunol. , 148: 1547, 1992). On the other hand, by expressing ScFv for different antigens at the ends of the heavy or light chain of a specific Fab, two antigen valencys are made (Schoonjans et al. , J. Immunol. , 165: 7050, 2000; Lu et al. .., J. Immunol Methods, 267 : 213, 2002), by placing the hinge region (hinge region) between the Fab and ScFv, can also reported dual targeting antibody made to have a valence of four antigens in homodimeric form ( Coloma and Morrison, Nat. Biotechnol. , 15: 159, 1997). In addition, by fusing the ScFv for different antigens at the light and heavy chain ends of the Fab, a double-target bibody that has three binding values for the antigen, and a different ScFv at the light and heavy chain ends of the Fab are respectively fused. Triple-target bibodies that have a valence of three for each have also been reported in the academic world (Schoonjans et al. , J. Immunol. , 165: 7050, 2000), a simple form obtained by chemically conjugating three different Fabs Triple target antibody F (ab ') _{3 has} also been reported (Tutt et al. , J. Immunol., 147: 60, 1991).

Third, in the case of IgG-based multi-target antibodies, Trion Pharma hybridizes mice and let hybridomas to produce hybrid hybridomas, also known as quadromas, that produce double-target antibodies. Got it. The company's dual-target antibody Ertumaxomab (antigen: Her-2 / neu, CD3) is currently entering Phase II for metastatic breast cancer (Kiewe and Thiel, Expert Opin. Investig.Drugs , 17: 1553, 2008), Another dual-target antibody, Catumaxomab (antigen: EpCAM, CD3), received EU approval for malignant ascites in 2009 and is currently entering Phase II for gastric and ovarian cancer (Shen and Zhu, Curr). Opin.Mol.Ther. , 10: 273, 2008). However, these antibodies are not able to rule out human anti-mouse antibody (HAMA) or human anti-rat antibody (HARA) response due to repeated administration. In addition, Pfiza's dual-target antibody CVX-241, manufactured using CovX-body technology, is capable of chemical reaction using angipoietin-2 (Ang-2) and VEGF-derived peptides using specific linkers. It was developed as an antibody that neutralizes Ang-2 / Tie-2 and VEGF / VEGFR-2 signaling mechanisms, which are the major mechanisms related to angiogenesis by binding to specific antibodies, and is currently used for phase 1/2 dose escalation study (DES). ) In the case of CVX241, production costs are expected to increase as the process is increased in that the final product is recovered through a number of chemical reactions in the production of biopharmaceuticals, and the active dose is required to be over 30 mg / Kg. In this regard, there is room for improvement.

On the other hand, although it is far from being commercialized, many studies are being conducted at the academic level. For example, the light chains are shared, and so-called Holes made by modifying some amino acids of the C _H3 homodimeric domain of Fc for different heavy chains. and Knob-type dual target antibodies have been reported (Merchant et al. , Nat. Biotechnol. , 16: 677, 1998). In addition to the heterotarget antibody of the heterotype, two different ScFvs were expressed in the homodimeric form (ScFv) ₄ -IgG (antigens: EGFR, IGF-1R) by fusion expression of two different ScFvs into the constant domain instead of the variable domain of the light and heavy chains of IgG, respectively. Cases have also been reported (Lu et al. , J. Biol. Chem. , 279: 2856, 2004). However, the low productivity of this antibody has emerged as a problem. Therefore, the same research group (ScFv) supplemented with _4- IgG produced a di-diabody that improved productivity for the same target. The possibility was confirmed (Lu et al. , J. Biol. Chem. , 280: 19665, 2005). However, this type of antibody did not overcome the disadvantage of poor stability, which is a problem of diabodies themselves. In addition, Shen et al. Is based on the IMC-1C11, a chimeric monoclonal antibody against human VEGFR-2, and the mouse platelet-derived growth factor receptor-alpha (PDGFR-a) at the light chain amino terminus of the antibody. The dual target antibody was prepared by fusing only a single variable domain for the same (Shen et al. , J. Biol. Chem. , 281: 10706, 2006; Shen et al. , J. Immunol. Methods , 318: 65, 2007). Recently Rossi et al. A number of antigen-binding molecules for CD20 can be identified by a method called so-called dock and lock (DNL) using dimerization and docking domain (DDD) of protein kinase A (PKA) R subunit and anchoring domain of PKA. Genie has demonstrated antibodies (Rossi et al. , Proc. Natl. Acad. Sci. USA , 103: 6841, 2006; Rossi et al. , Cancer Res. , 68: 8384, 2008) and such techniques in the same research group A dual target antibody has been reported based on (Chang et al. , Clin. Cancer Res. , 13: 5586, 2007). Antibodies using the DNL method are easy to apply, have a modular form, and can be variously combined, and have excellent in vivo stability, but may be degraded by proteolytic enzymes in vivo and may have immunogenicity-related problems. It is known that.

As described above, there have been various academic reports on the double-target or multi-target antibody and its manufacturing method, and the antibodies have functional advantages and disadvantages according to their morphological characteristics according to the purpose of use, but they are effective and new therapeutic agents for treating cancer. The development of double- and multi-target antibodies is still needed. In particular, it is an important question whether an antibody prepared for a dual target or a multi-target properly exhibits its function. In this case, it is understood that the selection for the co-targeting antigen is very important.

Therefore, the present inventors simultaneously neutralize VEGFR-2 and c-Met, which are receptors closely involved in the angiogenesis mechanism and post-war processes related to tumor growth and metastasis, in order to develop a dual target antibody for anticancer treatment through angiogenesis inhibition. As a result of intensive efforts to prepare a double target antibody, TTAC-0001, a complete human anti-KDR antibody, developed and filed by the present inventors (Korean Patent No. 10-0883430 and International Application No. PCT / KR07 / 003077). As a skeleton, a double-target antibody (PIG-KM) was prepared by linking a peptide showing antagonistic action against c-Met with a specific linker sequence, and the double-target antibody simultaneously produced VEGFR-2 and c-Met. The present invention was completed by confirming that the cells can be neutralized and show comparable or better anti-cancer effects at the cellular level compared to VEGFR-2 single target antibody or c-Met antagonist peptide. .

One object of the present invention is a double soluble peptide that is fused to the light chain N-terminus of the vascular endothelial growth factor receptor neutralizing antibody, which can inhibit the proliferation and migration of cancer cells by the phosphorylation of c-Met with anti-angiogenic effects. To provide a target antibody.

Another object of the present invention is to provide a DNA encoding the double target antibody.

Still another object of the present invention is to provide a recombinant expression vector comprising the DNA.

Still another object of the present invention is to provide a host cell transformed with the recombinant expression vector.

Still another object of the present invention is to provide a method of producing a double target antibody by culturing the host cell.

Still another object of the present invention is to provide a pharmaceutical composition comprising the double target antibody.

In order to achieve the above object, the present invention provides a novel target double target antibody in which a water-soluble peptide is fused to the light chain N-terminus of the vascular endothelial growth factor receptor neutralizing antibody.

Dual target antibodies of the present invention include, but are not limited to, neoplastic cells, cancer stromal cells, tumor associate endothelial cells, tumor associated endothelial progenitors cells, tumor associated circulating endothelial cells, circulating tumor cells, cancer stem cells, and the like.

More specifically, the antigen for the dual target antibody of the present invention is Vascular Endothelial Growth Factor Receptor-1 (VEGFR-1), Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2), Vascular Endothelial Growth Factor Receptor 3 ), FLT3 (FMS-like Tyrosine Kinase 3), CSF1R (Colony Stimulating Factor 1 Receptor), RET (Rearranged during Transfection), c-Met (Mesenchymal-Epithelial Transition Factor), EGFR (Epidermal Growth Factor Receptor), Her2 / neu (Human Epidermal Growth Factor Receptor 2), HER3 (Human Epidermal Growth Factor Receptor 3), HER4 (Human Epidermal Growth Factor Receptor 4), Fibroblast Growth Factor Receptors (FGFRs), Insulin-like Growth Factors, PDGFR (Platelet-Derived Growth Factor Receptors), c-KIT, BCR, Integrin (Integrin), MMPs (Matrix Metalloproteinases) and other proteins that can be expressed on the cell surface, but is not limited thereto.

In the present invention, the dual target antibody may be a 'polyclonal' or 'monoclonal' antibody, but a monoclonal antibody is more preferable. Monoclonal antibodies refer to antibodies obtained from a substantially homogeneous population of antibodies, ie the individual antibodies that make up this population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, in contrast to polyclonal antibodies that include different antibodies to different epitopes, each monoclonal antibody is directed against a single epitope on the antigen. Monoclonal should not be construed to mean that it requires the production of antibodies in any particular way. For example, monoclonal antibodies useful in the present invention are prepared by the hybridoma method described in Kohler et al., Nature, 256: 495 (1975), or by recombinant DNA method [see US Pat. No. 4,816,567]. can do. In addition, monoclonal antibodies are described, eg, in Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol., 222: 581-597 (1991), can also be isolated from phage antibody libraries.

It is preferable that the dual target antibody of this invention is a "humanized antibody." Humanized antibody means an antibody composed of amino acid sequences derived in part or in whole from human antibody germlines by altering the sequence of an antibody having a non-human complementarity determining region (CDR). Moreover, it is especially preferable that the antibody of this invention is a "human antibody." Human antibodies are antibodies having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or an antibody produced using any of the techniques for making a human antibody. Human antibodies can be prepared using various techniques known in the art.

The vascular endothelial growth factor receptor neutralizing antibody of the present invention is a 6A6-scFv (TTAC-0001), which is a complete human anti-KDR antibody previously filed in Korean Patent Registration No. 10-0883430 and International Application No. PCT / KR07 / 003077, And all variants in which the light chain sequences of 6A6-IgG and 6A6-scFv are mutated by the light chain shuffling method. All the contents including the manufacturing method and effects of TTAC-0001 to TTAC-0019 disclosed in the Republic of Korea Patent No. 10-0883430 and International Application No. PCT / KR07 / 003077 are incorporated herein by reference.

The TTAC-0001 antibody is an IgG-type scFv (Single Chain Fragment Variable) antibody having a light chain variable region represented by the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 20. In addition, the TTAC-0002 to TTAC-0019 antibodies each have a light chain variable region represented by the amino acid sequence of SEQ ID NO: 2 to SEQ ID NO: 19, and has a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 20.

The term water-soluble peptide used in the present invention refers to a peptide having less than 20 amino acids exhibiting water-soluble properties that can be dissolved in water as a protein that specifically binds to a receptor present on a cell surface, particularly on a cell surface. Of the present invention The water soluble peptide may preferably be pep1 described in SEQ ID NO: 21 or pep2 described in SEQ ID NO: 22, and may have an antagonistic effect of binding to c-Met and inhibiting c-Met (pep1: KSLSRHDHIHHH, pep2 : YLFSVHWPPLKA).

The N-terminus or C-terminus of the heavy or light chain of the vascular endothelial growth factor receptor neutralizing antibody of the present invention and the water-soluble peptide may be linked through a linker. In the present invention, the 'linker' refers to a peptide fragment that connects two portions of the fusion protein. Linkers suitable for the present invention include peptides having 5 to 25 amino acids, preferably 10 to 20 amino acids, more preferably 10 to 15 amino acids. The linker in the present invention may preferably be a linker set forth in SEQ ID NO: 23 (linker: GGGGSGGGGSGS).

In the dual target antibody of the present invention, the vascular endothelial growth factor receptor neutralizing antibody and the water soluble peptide play their respective roles. In addition, since the dual target antibody can simultaneously suppress or amplify two signals, it can be more effective than suppressing / amplifying a single signal, compared to the case of treating each signal with a respective signal inhibitor. In addition, low-dose administration is possible, and there is an advantage of suppressing / amplifying two signals at the same time and space.

Preferably, the double target antibody is a vascular endothelial growth factor receptor neutralizing antibody comprising a light chain variable region represented by the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 19 and a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 20 Binding to VEGFR-2 (Vasicular Endothelial Growth Factor Receptor-2) and c-Met (Mesenchymal-Epithelial Transition factor) linked to the light chain N-terminus of the water-soluble peptide represented by SEQ ID NO: 21 or SEQ ID NO: 22 It may be a dual target antibody with specificity. More preferably, the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 22 is displayed at the N-terminus of the light chain of the vascular endothelial growth factor receptor neutralizing antibody (TTAC-0001) having the light chain variable region represented by the amino acid sequence of SEQ ID NO: 1 It may be a double target antibody to which the water-soluble peptide is linked.

In addition, the light chain or heavy chain of the vascular endothelial growth factor receptor neutralizing antibody and the water-soluble peptide or linker constituting the double target antibody of the present invention are represented by SEQ ID NOs: 1 to 23, as long as they do not affect the functional properties of the encoded protein. It may have an amino acid sequence or DNA sequence having at least about 80%, at least 90%, or at least 95% homology with the amino acid sequence and the DNA sequence encoding it.

Also, preferably, the base sequence encoding the amino acid sequence constituting the double target antibody may be codon optimized. Codon optimization refers to the substitution of bases with codons with high expression in host cells or animals in order to increase the expression rate of DNA. When a DNA is transcribed and translated into a protein in a host or animal, a codon with high affinity exists between the codons that command the amino acid, and the amino acid or protein encoded by the nucleic acid by substituting these highly preferred codons. To increase the expression efficiency of. For the purposes of the present invention, codon optimization of the light chain or heavy chain sequence of the VEGFR-2 binding antibody constituting the double target antibody, the base sequence of the linker and the c-Met binding receptor peptide is performed as a therapeutic antibody by increasing the expression rate in cells or in animals. You will be able to maximize the effect.

In a specific embodiment of the present invention, the dual target antibody (PIG-KMs) was completed by fusing a peptide that binds c-Met through a specific linker to the light chain amino terminal region of the antibody based on TTAC-0001 ( 1 to 3), the binding capacity and affinity of the dual target antibody PIG-KMs to VEGFR-2 and c-Met were analyzed using BIAcore (see Figure 4), Protein A affinity column, SP-sepharose Purifying only purified antibody with purity of 95% or more through FPLC using column and size exclusion column (refer to FIGS. 5 and 6), and analyzing the cell migration to PIG-KM, inhibiting the mobility of HUVEC cells by HGF. In contrast to the TTAC-0001-treated group, PIG-KM was found to effectively inhibit the mobility of HUVEC cells derived from VEGF and HGF, respectively, and effectively inhibited the mobility of HUVEC cells induced by VEGF and HGF. Number that was confirmed (see Figs. 7 and 8). In addition, as a result of cell proliferation assay for HUVEC cells, it was confirmed that the PIG-KM dual target antibody of the present invention could inhibit the viability of HUVEC cells caused by VEGF or HGF, respectively. It was confirmed that the proliferation rate of HUVEC cells can also be effectively inhibited (see FIG. 9). In addition, as a result of cell wound healing assay for primary cultured HUVEC cells, PIG-KM treated group inhibited HUVEC proliferation and migration as compared with TTAC-0001, which had a lot of proliferation and migration, resulting in wound healing of HUVEC cells. It was confirmed that there is an inhibitory effect (see FIG. 10), and furthermore, the PIG-KM of the present invention was able to effectively inhibit the signaling mechanism of c-Met induced by HGF (see FIG. 11).

Therefore, the dual target antibody of the present invention has binding specificity to VEGFR-2 and c-Met, and has an effect of neutralizing VEGFR-2 and c-Met simultaneously. Angiogenesis by migration and proliferation of vascular endothelial cells Given that these VEGF / VEGFR-2 (KDR) signaling mechanisms are primarily involved, and that cancer cell proliferation and metastasis are largely caused by HGF / c-Met signaling mechanisms, these two targets may be Signaling mechanisms can be neutralized at the same time, which is expected to be very effective as a cancer treatment strategy. In particular, under efficient angiogenesis conditions with VEGF and HGF, intracellular crosstalk from c-Met and KDR can lead to cytoskeleton, migration and morphogenesis. Because it can promote the metastasis process (Sulpice et al., Biol. Cell , 101: 525, 2009), it is significant to inhibit the two tyrosine kinase receptors with one antibody. .

In addition, the binding of each having specific binding specificity to an antigen target does not necessarily result in a bitarget antibody having a bispecific effect of neutralizing a desired target at the same time, but it was confirmed that the present invention has an excellent bispecific effect. In that sense, it can be said that the meaning can be used in the drug for cancer treatment. While the unpredictability of the preparation of such double-target antibodies is not fully understood, as the dual-target antibodies take the form deviated from the conventional IgG form, the efficiency of being degraded or secreted into cells may be reduced, and the purification of the produced antibodies is easy. Or the stability of the final product may differ from the existing antibodies.

In another aspect, the present invention provides DNA encoding the dual target antibody of the present invention described above. The DNA includes all sequences having at least about 80%, at least 90%, or at least 95% homology with the DNA sequence, as long as it does not affect the functional properties of the encoded protein as described above, It may have a codon optimized sequence to increase the expression rate.

Preferably, the DNA encoding the dual target antibody may be one described as SEQ ID NO: 24 or SEQ ID NO: 25 (see FIGS. 1 and 2). The DNA sequence is a fusion of the three ends of the nucleic acid sequence encoding the linker to the 5 'end of the nucleic acid sequence encoding the light chain of the neutralizing antibody, and the nucleic acid sequence of the nucleic acid sequence encoding the water-soluble peptide at the five ends of the nucleic acid sequence encoding the linker It can be produced by fusing the 3 'end.

In one embodiment, the nucleic acid sequence encoding a dual target antibody fused through a linker can be obtained by designing the primer to include the nucleic acid sequences of the peptide and the linker and then performing PCR. A recombinant expression plasmid was prepared by inserting the double-target antibody coding gene prepared above into a vector, and then introducing the plasmid into a host cell to prepare a transfectant or transformant, and amplifying and culturing the cell. The target antibody can be separated and purified to obtain the desired double target antibody.

In another aspect, the present invention provides a recombinant expression vector comprising a DNA encoding the dual target antibody. Preferably the vector may be a recombinant expression vector having a cleavage map of pIgG-pep1 / 6A6lgt or pIgG-pep2 / 6A6lgt shown in FIG. 3.

As used herein, the term "recombinant expression vector" refers to a gene construct, which is an expression vector capable of expressing a protein of interest in a suitable host cell, comprising an essential regulatory element operably linked to express a gene insert.

In the present invention, "operably linked" means that the nucleic acid expression control sequence and the nucleic acid sequence encoding the protein of interest is functionally linked to perform a general function. Operative linkage with recombinant vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be facilitated using enzymes commonly known in the art. have

Suitable expression vectors of the invention may include signal sequences for membrane targeting or secretion in addition to expression control elements such as promoters, initiation codons, termination codons, polyadenylation signals and enhancers. Initiation and termination codons are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein and must be functional in the subject and be in frame with the coding sequence when the gene construct is administered. Generic promoters can be either constitutive or inducible. Prokaryotic cells include, but are not limited to, lac, tac, T3 and T7 promoters. Eukaryotic cells include monkey virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV), for example the long terminal repeat (LTR) promoter of HIV, moronivirus, cytomegalovirus (CMV) ), Epstein bar virus (EBV), Loose sacoma virus (RSV) promoters, as well as promoters derived from human heroglobin, human muscle creatine, human metallothionein.

The expression vector may comprise a selectable marker for selecting a host cell containing the vector. The selection marker is for selecting cells transformed with the vector, and markers conferring a selectable phenotype such as drug resistance, nutritional requirements, resistance to cytotoxic agents or expression of surface proteins can be used. Since only cells expressing a selection marker survive in an environment treated with a selective agent, transformed cells can be selected. In addition, when the vector is a replicable expression vector, the vector may include a replication origin, which is a specific nucleic acid sequence from which replication is initiated.

As a recombinant expression vector for inserting a foreign gene, various forms of vectors such as plasmids, viruses, and cosmids can be used. The type of recombinant vector is not particularly limited as long as it functions to express a desired gene and to produce a desired protein in various host cells of prokaryotic and eukaryotic cells, but has a promoter with strong activity and strong expression, similar to natural state. Vectors that can produce large amounts of foreign protein in form are preferred.

In another aspect, the present invention provides a host cell transformed with the recombinant vector.

In the present invention, the host cell used for dual target antibody expression may be a prokaryotic or eukaryotic cell. In addition, a host having a high DNA introduction efficiency and a high expression efficiency of the introduced DNA is usually used. Well-known eukaryotic and prokaryotic hosts such as E. coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such as Spodoprera pruiferferda (SF9), Chinese hamster ovary cells (CHO) and mice Animal cells such as cells, COS1, COS7, human embryonic kidney cells, African green monkey cells such as BSC 1, BSC 40 and BMT 10, and other tissue cultured human cells. Is an example.

In the present invention, a wide variety of expression host / vector combinations can be used to express dual target antibodies. Suitable expression vectors for eukaryotic hosts include, for example, SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus and retrovirus. Expression vectors that can be used in bacterial hosts include bacterial plasmids such as pBluescript, pGEX2T, pUC, pCR1, pBR322, pMB9 and derivatives thereof, plasmids with a wider host range such as RP4, and a wide variety of phages such as gt10 and 11, NM989 Phage DNA, which may be exemplified as a phage lamda derivative, and other DNA phages such as M13 and filamentary single stranded DNA phage. Useful expression vectors for yeast cells are 2μ plasmids and derivatives thereof.

Transformation of the recombinant expression vector into a host cell may include, for example, DEAE-dextran mediated transfection, electroporation, transduction, calcium phosphate transfection. ), Cationic lipid-mediated transfection, scrape loading and infection.

In another aspect, the present invention provides a method for producing a dual target antibody by culturing the host cell described above, and separating the dual target antibody from the culture thereof.

In the present invention, the host cell may be cultured by small or large-scale fermentation, shake flask culture in a laboratory or industrial fermenter performed under conditions that allow the expression and / or isolation of the appropriate medium and the dual target antibody. The cultivation is carried out in a suitable culture medium containing carbon, nitrogen sources and inorganic salts using known techniques. Suitable media are commercially available and can be made, for example, according to the components described in the catalog of the American Type Culture Collection (ATCC) and the like and their composition ratios.

Dual-target antibodies from such cultures can be isolated by methods known in the art. For example, dual target antibodies can be isolated from the culture by conventional methods, including but not limited to centrifugation, filtration, extraction, spray drying, evaporation or precipitation. Furthermore, dual target antibodies are known in the art, including chromatography (eg, ion exchange, affinity, hydrophobicity and size exclusion), electrophoresis, fractional solubility (eg, ammonium sulfate precipitation), SDS-PAGE or extraction. It can be purified through various methods. Preferably, the dual target antibody of the present invention can be further purified using Fast Protein Liquid Chromatography (FPLC) with a Protein A affinity column, SP-Sepharose column and size exclusion chromatography.

In another aspect, the present invention provides a pharmaceutical composition for inhibiting angiogenesis or cancer, comprising the dual target antibody described above. As described above, since the dual target antibody of the present invention has an inhibitory effect on angiogenesis, it can be used to treat diseases related to angiogenesis.

In the present invention, 'angiogenesis related disease' includes cancer, age-related macular degeneration, rheumatoid arthritis, diabetic retinopathy, psoriasis and chronic inflammation. inflammation).

The dual target antibody of the present invention may have a cancer therapeutic function by inhibiting the activity of antigen molecules related to the occurrence, growth, and metastasis of cancer by overexpression in cancer cells by administration to a patient.

In the present invention, the cancer is gastric cancer, liver cancer, lung cancer, thyroid cancer, breast cancer, cervical cancer, colon cancer, pancreatic cancer, rectal cancer, colorectal cancer, prostate cancer, kidney cancer, melanoma, prostate cancer, bone metastasis cancer, ovarian cancer, etc. It includes, but is not limited to, solid and blood cancers.

The dual target antibodies of the invention can be administered to cancer patients in an amount sufficient to prevent, inhibit or reduce the progression of the tumor, for example, the growth, invasion, metastasis and / or recurrence of the tumor for therapeutic treatment. A suitable amount to achieve this goal is defined as a therapeutically effective dose. The amount effective for this use will depend on the severity of the disease and the general state of the patient's own immune system.

In addition, the compositions of the invention can be administered by any route suitable for the particular molecule. The compositions of the present invention may be used directly or by systemic (eg, parenteral or oral) to any animal, including humans, directly (eg, by injection, subcutaneous injection or topical administration to a tissue location) using any suitable means. May be provided). The compositions according to the invention may be parenterally, such as intravenous, subcutaneous, eye, abdominal, intramuscular, oral, rectal, vaginal, orbital, intracranial, spinal cord, intraventricular, intratracheal, intramural, intraoral, intranasal, or aerosol. When provided by administration, the composition preferably comprises a fluid suspension or solution portion that is aqueous or physiologically compatible. Therefore, the carrier or excipient must be physiologically acceptable and, in addition to delivering the desired composition to the patient, must not adversely affect the electrolyte and / or volume balance of the patient.

The pharmaceutical composition containing the dual target antibody of the present invention may be formulated for oral administration such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, sterile injectable solutions, suppositories, and transdermal administrations according to conventional methods. Can be formulated and used. Carriers, excipients and diluents which may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose , Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. If necessary, it is formulated with diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like.

The dual target antibodies of the present invention can be formulated in solid or liquid formulations for oral administration. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which include at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like. It is formulated by mixing. In addition, liquid preparations for oral administration include suspensions, solutions, emulsions, syrups, and the like, which include various excipients in addition to conventionally used inert diluents (e.g., purified water, ethanol, liquid paraffin), For example, wetting agents, sweetening agents, fragrances, preservatives and the like can be included.

The composition according to the invention can be used in combination or in combination with other therapeutic agents associated with the disease. Synergy may be present when treating a tumor, including a human tumor, using the dual target antibody of the invention in combination with a chemotherapeutic agent, radiation, additional receptor antagonist or a combination thereof. As one example, the pharmaceutical composition may further include a substance that binds an angiogenic factor or a receptor thereof and blocks angiogenic activity. For example, antibodies to VEGF-A or VEGF-A receptors (eg, KDR receptors or Flt-1 receptors) that are different from the present invention, VEGF traps, anti-PDGFR inhibitors, such as imatinib mesylate Gleevec ^™ ). In addition, natural angiogenesis inhibitors include angiostatin, endostatin, and the like. In addition, bevacizumab (trade name Avastin, sunit nib, sorafenib, lenalidomide, etc. can be included.

Thus, the dual target antibodies of the present invention can be used in vivo and in vitro for research, prophylaxis or treatment methods well known in the art. Of course, it will be apparent to those skilled in the art that the principles of the invention disclosed herein may be varied and that such modifications are included within the scope of the invention.

The present invention is to neutralize VEGFR-2 and c-Met involved in angiogenesis at the same time, to effectively inhibit the signaling mechanisms involved in angiogenesis and cancer cell proliferation, containing a dual target antibody derived from a human monoclonal antibody and the antibody Provided are compositions for inhibiting angiogenesis and treating cancer. The dual target antibody according to the present invention simultaneously neutralizes two targets involved in angiogenesis and cancer cell proliferation, thereby exhibiting superior neutralizing ability as compared to the conventional single target antibody, and is very effective in treating cancer. In addition, for the two targets that are correlated with each other, by producing a double-target antibody of the present invention can be expected to be superior to the benefit of the single-target antibody treatment.

Figure 1 shows the DNA sequence and function of the gene inserted into the pIgG-pep1 / 6A6lgt vector.

Figure 2 shows the DNA sequence and function of the gene inserted into the pIgG-pep2 / 6A6lgt vector.

Figure 3 schematically shows a method for the preparation of the vectors pIgG-pep1 / 6A6lgt and pIgG-pep2 / 6A6lgt according to the present invention.

4 shows that the dual target antibodies PIG-KM1 and PIG-KM2 produced according to the present invention can bind to VEGFR-2 and c-Met, respectively.

Figure 5 shows the results of the selection of high productivity clones through MTX iteration (up to 320 nM) in order to establish a high productivity cell line according to the present invention.

6 shows the SDS-PAGE results of purified PIG-KM1 and PIG-KM2.

Figure 7 graphically shows the SDS-PAGE results of purified PIG-KM1 and PIG-KM2.

Figure 8 shows the results of the migration assay (migration assay) for HUVEC of PIG-KM1 and PIG-KM2 according to the present invention.

Figure 9 shows the results of the proliferation assay (proliferation assay) for HUVEC of PIG-KM1 and PIG-KM2 according to the present invention.

Figure 10 shows the results of wound healing (wound healing assay) for HUVEC of PIG-KM1 according to the present invention.

11 is a result of confirming the phosphorylation inhibition of c-Met by HGF of PIG-KM1 according to the present invention through Western blotting.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

Example 1 Preparation of Expression Vectors for PIG-KMs Production

Peptide-related DNA that binds to C-Met and inhibits its activity has been studied by Dr. Joon-Ho Chung (Kim et al. , Biochem Biophys Res Commun. , 354: 115, 2007) and Brian Cao, Ph.D. et al. , Clin. Cancer Res. , 13 (20); 6049, 2007). Each peptide was named pep1 and pep2, and was linked to the light chain amino terminal region of TTAC-0001 via a specific linker, for which four rounds of PCR reactions were performed. The reaction conditions for the PCR are as follows: 94 4 min, (94 45 sec / 50 45 sec / 72 45 sec), 30 times, 72 7 min, 4 8. NPfu forte 2.5 U (Enzynomics # P425), 10X buffer 5, dNTP 2 (2.5 mM each), distilled water 37.5 as polymerase. Forward PCR F-ksw002 (5'-AGT GGC GGA GGA GGC TCC GGT TCC AAT TTT ATG CTG ACT CAG, SEQ ID NO: 26) and reverse primer R-ksw002 (5'-CAG ATC TTT CCA) CGA GGC TGG CTC CTC, SEQ ID NO: 27) 2 (10 pmole /), pIgGLD6A6lgt (SEQ ID NO: 1, light chain variable sequence of TTAC-0001) 0.5 (200 ng /) was additionally used as template DNA. Primary PCR product 6A6lgt 1 was obtained. The product obtained by PCR was subjected to electrophoresis through 1% agarose gel, and then the DNA band of less than 400 bp using the MEGA-spin ^TM Gel / PCR DNA Extraction Kit (iNtRON Biotechnology Inc. # 17183, South Korea). I separated it.

To obtain a secondary PCR product, 6A6lgt 1 was used as a template under the same reaction conditions, and F-ksw003 (5'-GGA GGC GGA GGT AGT GGC GGA GGA GGC TCC, SEQ ID NO: 28) and R-ksw002 were added to a common PCR reaction product. PCR reaction was performed. The PCR product thus obtained was named 6A6lgt 2, the target band was moved by 1% agarose gel electrophoresis, and separated using a gel extraction kit.

In the third PCR reaction, 6A6lgt 2 was added as a template to the common PCR reaction under the same reaction conditions, and F-pep1 (5'-TCT AGA CAT GAT CAT ATT CAT CAT GGA GGC GGA GGT AGT GGC GGA GGA, SEQ ID NO: 29) PCR was performed by adding F-pep2 (5'-TCT GTT CAT TGG CCA CCA TTG AAA GCT GGA GGC GGA GGT AGT GGC GGA GGA, SEQ ID NO: 30) as a forward primer and R-ksw002 as a reverse primer. , PCR reaction products pep1 / 6A6lgt 1 and pep2 / 6A6lgt 1 were obtained, respectively. The PCR product separated only the target band in the same manner as before. Acquisition of the TTAC-0001 light chain region linked to the C-met antagonist peptide was achieved via a fourth-order PCR reaction. PCR reactions were performed under the same reaction conditions as before, and pep1 / 6A6lgt 1 and pep2 / 6A6lgt 1 were used as templates. The primers used were F-pep1a (5'-CAC TCC AGC GGT GTG GGT TCC AAA TCT TTG TCT AGA CAT GAT CAT ATT C, SEQ ID NO: 31) and F-pep2a (5'-CAC TCC AGC GGT GTG) GGT TCC TAT TTG TTT TCT GTT CAT TGG CCA CCA TTG (SEQ ID NO: 32) and R-ksw002 were used as reverse primers.

The final PCR product was isolated by 1% agarose gel electrophoresis and recovered using a gel extraction kit (named pep1 / 6A6lgt 2 and pep2 / 6A6lgt 2, respectively). Each of the recovered PCR products pep1 / 6A6lgt 2 and pep2 / 6A6lgt 2 was inserted into T-vector using TOPcloner TA cloning kit (Enzymonics # EZ111, South Korea), and then transformed into E. coli DH5 for miniprep and restriction enzyme Bst. After XI treatment, DNA sequencing was requested for a vector containing about 400 bp of target DNA, and the final sequence (SEQ ID NO: 24, SEQ ID NO: 25) was confirmed (see FIGS. 1 and 2).

Light chain expression vector preparation method for the expression of the dual target antibody PIG-KM is as follows (see Fig. 3). First, the TTAC-0001 light chain expression vector, pIgGLD6A6lgt, which the team had, was treated with Bst XI, and electrophoresis separated only the portion that could be used as a vector from the 1% agarose gel. In addition, T-vectors in which the PCR products pep1 / 6A6lgt 2 and pep2 / 6A6lgt 2 were inserted were also treated with Bst XI to isolate only fragments that were cut from the 1% agarose gel via electrophoresis. Thereafter, the separated two fragments were prepared as one completed vector by standing for 4 to 12 hours using T4 DNA ligase (Enzynomics # M001S, South Korea). The prepared vector was transformed through E. coli DH5, and finally confirmed whether pep1 / 6A6lgt 2 and pep2 / 6A6lgt 2 were inserted through miniprep and Bst XI. The identified recombinant vectors were named 'pIgG-pep1 / 6A6lgt and pIgG-pep2 / 6A6lgt'.

Example 2: Production and Identification of PIG-KMs

The completed light chain expression vectors pIgG-pep1 / 6A6lgt and pIgG-pep2 / 6A6lgt and the existing heavy chain expression vector pIgGHD6A6hvy (PCT / KR07 / 003077) were converted to CHO DG-44, a dhfr- deficeint CHO cell. Induction of random expression through co-transduction was confirmed by the expression of SDS-PAGE and Western blotting. Transduction was performed using lipofectamine ^™ 2000 (Invitrogen # 11668-019, USA), the method following the manufacturer's instructions. Briefly, 5 x 10 ⁵ CHO-DG44 cells per well were inoculated into a 6-well plate containing aMEM medium (Welgene, South Korea), followed by 37 to 24 using a CO ₂ (5%) incubator maintained in humidification. By allowing to stand for time, the cells were densely cultured so that the cell density was about 80-90%. Recombinant vectors 3 (pIgGHD6A6hvy and pIgG-pep1 / 6A6lgt or pIgGHD6A6hvy and pIgG-pep2 / 6A6lgt each 1.5) and 6 lipofectamin ^™ 2000 were diluted in 250 serum-free aMEM medium and allowed to stand at room temperature for 5 minutes. A DNA-lipofectamin ^™ 2000 complex was formed by reacting the DNA dilution with lipofectamin ^™ 2000 dilution for 20 minutes at room temperature. After removing the existing medium from the cultured cells, DNA-lipofectamin ^™ 2000 complex 500 and serum-free aMEM medium 500 were added to each well and incubated for 6 hours in a CO ₂ incubator at 37 conditions. After adding 1 ml of aMEM medium containing 20% dialysis fetal calf serum and incubating for 48-72 hours, only the supernatant was isolated and confirmed by SDS-PAGE and Western blotting. SDS-PAGE and Western blotting were applied in a manner commonly used in the art, and the samples used were: 12% SDS-polyacrylamide Gel, PVDF membrane (Millipore # IPVH00010, USA), HRP-conjugated goat anti -human IgG (kappa) antibodies, and HRP-conjugated goat anti-human IgG (Fc) antibodies (Pierce, USA). Antibodies identified through SDS-PAGE and western blotting were named PIG-KM1 and PIG-KM2, respectively.

Example 3: Determination of affinity and affinity of PIG-KM1 and PIG-KM2

The avidity of the dual target antibodies PIG-KM1 and PIG-KM2 to c-Met and VEGFR-2 was analyzed using BIAcore, and the affinity for each antigen was also measured using the same equipment. The instrument used for the analysis was BIAcore 3000 (GE Healthcare, USA), which used CM5 at a rate of 5 / min for hIgG, TTAC-0001, PIG-KM1, and PIG-KM2 with a signal value of 4,000 RU through the amine coupling method. Immobilized on a chip (GE Healthcare, USA). In this case, all samples used were samples of a state melted in PBS. Thereafter, 500 nM of c-Met-Fc (R & D systems) was flowed on the chip where each ligand was immobilized to induce binding with the ligand. After stabilization by flowing HBS-EP (GE Healthcare, USA) working buffer for 1 minute and 30 seconds, 500 nM of VEGFR-2 ECD (PharmAbcine) was flowed for 5 minutes to induce binding with the ligand again. After binding to the ligand CM5 chip was used for affinity measurement by flowing the regeneration solution (8 mM NaOH) for 5 minutes to remove the analyte bound to the ligand (see Figure 4). As shown in FIG. 4, PIG-KM1 and PIG-KM2 showed binding ability to both c-Met and VEGFR-2, whereas TTAC-0001, a VEGFR-2 single target antibody, bound only to VEGFR-2, and hIgG was found to bind neither c-Met nor VEGFR-2. This demonstrated that the dual target antibodies PIG-KM1 and PIG-KM2 can specifically bind c-Met and VEGFR-2.

For the affinity measurement of PIG-KM1 and PIG-KM2, the CM5 chip used for binding capacity was reused, and c-Met-Fc and VEGFR-2 ECD of various concentrations up to 9.8 nM-156.3 nM were 30 / min. The affinity was calculated by flowing each at a rate of. Through this, PIG-KM1 and PIG-KM2 were confirmed to have a KD value of sub nM for VEGFR-2 and c-Met.

Example 4 Establishment of Cell Lines for PIG-KM1 and PIG-KM2 Production and Separation and Purification of Antibodies

PIG-KMs producing cell lines were constructed via CHO-DG44 ( dhfr- deficeint CHO) cells. The transduction procedure for establishing the recombinant antibody expressing CHO-DG44 cell line is the same as described in Example 2. For selection of transduced CHO-DG44 cells ( dhfr- positive), aMEM medium without hypoxantine-thymidine was used, and 500 / ml G418 (Sigma-aldrich, USA) and 400 / ml zeocine as selection markers. (Invitrogen, USA) was used for primary selection. To obtain monoclonal colonies expressing recombinant antibodies, the primary selected cells were diluted to a density of 10 cells / ml, inoculated in 96-well plates (Nunc, USA), cultured for two weeks, and single colonies formed by dividing from single cells. Isolation to establish parental clones. In order to obtain a high expression cell line, the cells were passaged three to five times in a medium to which methotrexate (MTX) at various concentrations (40 nM, 80 nM, 160 nM, 320 nM) was added, and the expression level was confirmed by ELISA. . To this end, coating was completed by adding 100 ml of goat anti-human IgG (Fc) (Pierce, USA) as a primary antibody to a 96-well plate, and standing at 4 to 12 hours. Then, after discarding the remaining solution in the well, 200 x blocking solution containing 2% skim milk in 1x PBS, and left for 1 hour at 37. After washing each well three times with Washing buffer containing 0.05% Tween-20 in 1x PBS, 100 cells of the culture medium obtained from the antibody-expressing CHO-DG44 cell line were added and reacted at room temperature for 1 hour. After washing each well three times with washing buffer again, dilute HRP-conjugated goat anti-human IgG (kappa) in washing buffer at a ratio of 1: 5,000 in a washing buffer, and add 100 parts each for 1 hour at room temperature. Reacted. After washing each well three times with washing buffer again, 100 TMB substrate reagent (BD biosciences, USA) was added and reacted for 5-10 minutes, followed by 50 2N sulfuric acid (H ₂ SO ₄ ) solution. The color reaction was stopped. OD _{450-650 nm} values were obtained using a microplate reader (Tecan, Switzerland). In the same manner, VEGFR-2 and Tie-2 were used as primary antibodies, and HISA-conjugated goat anti-human IgG (kappa) was used as a secondary antibody to confirm the results. Finally, clones with the highest expression rates at MTX 320 nM were established as high expressing cell lines (see FIG. 5). Cultures of high expressing cell lines were treated with 10% dialysis fetal calf serum (KDR, Korea), 100 units / ml penicillin (Hyclone, USA), 100 / ml streptomycin (Hyclone, USA), and aMEM medium (Welgene, Korea). Cell culture was carried out under humidified 5% CO ₂ mixed air conditions using 37 incubators.

The dual-target antibody PIG-KMs obtained through the culture of high expressing cell lines were purified using Fast Protein Liquid Chromatography (FPLC) with Protein A affinity column, SP-sepharose column and size exclusion chromatography for further study. Only at least% purified antibody was obtained (see FIG. 6). First, the culture medium was separated into cell mass and medium by centrifugation, and PIG-KMs in the separated medium were concentrated through an ultrafiltration membrane (Millipore, USA) having a molecular weight cut-off of 10,000 Da or less. Ultrafiltration medium was first purified using protein A affinity chromatography.

The above process is described in detail. In a protien A column stabilized with 20 mM sodium phosphate (pH 7.0) buffer containing 0.1 M NaCl, ultrafiltration medium was added, and the unbound protein was washed out using the same buffer. 20 mM sodium phosphate (pH 7.0) buffer solution containing NaCl washed non-specifically bound protein A protein. Proteins that specifically bind to Protein-A were eluted using 0.1 M Glycine-Cl (pH 3.5) buffer containing 0.1 M NaCl, and the samples were neutralized to pH 6.0 using 1 M Tris. Then, cation exchange chromatography was performed according to the following procedure to remove contamination of DNA, endotoxin, and protein-A that may remain in the sample separated through the affinity column. First, the sample eluted from the protein-A column is mixed with an equal volume of 20 mM sodium phosphate (pH 6.0) buffer. After stabilizing the SP-sepharose (5 ml, GE healthcare, USA) column with 10 mM sodium phosphate (pH 6.0) buffer containing 50 mM NaCl, samples were added to wash unbound DNA and endotoxin. . The antibody molecules bound to the resin were eluted using pH, salt gradient (50 mM sodium phosphate (pH 7.0), 1 M NaCl). Finally, in order to remove the multimeric antibody, the sample was placed in a Superdex 200 (16 mm x 60 cm, GE healthcare) column stabilized with PBS, followed by size exclusion chromatography. Cell analysis was performed using antibodies that completed the above purification process.

Example 5: Mobility Analysis of HUVECs After PIG-KM1 and PIG-KM2 Treatment

Cultures of HUVEC consisted of 20% fetal bovine serum (Hyclone, USA), penicillin 100 units / ml (Hyclone, USA), streptomycin 100 / ml (Hyclone, USA), fibroblast growth factor (bFGF, basic fibroblast growth factor, Upstate Biotechnology Phenol red-free M199 medium (Invitrogen, USA) with 3 ng / ml and heparin 5 units / ml (Sigma-Aldrich, USA) was used. The cells were cultured in 37 incubators with 5% CO ₂ mixed air.

In order to confirm the mobility (chemotability) inhibition of PIG-KM 1 and PIG-KM 2 against HUVECs, cell mobility analysis was performed. For mobility analysis of HUVEC, 8-pore-sized polycarbonate filters transwell (Corning, USA) was used, and the bottom surface of the filter was coated with 10 gelatin and dried before use. In the lower wells, 20 ng / ml of VEGF and 50 ng / ml of HGF were added to M199 medium containing 1% fetal bovine serum. The vascular endothelial cells cultured at low serum concentration for 6 hours were trypsinized, removed, and then suspended in M199 medium containing 1% fetal bovine serum at a density of 1 × 10 ⁶ cells / ml. The cells pre-treated with antibodies of various concentrations for 30 minutes were evenly sprayed into the upper transwells by 100, and then cultured in a 37 ° C. cell incubator for 4 hours. The cells were stained with crystal violet (Sigma, USA), and the unmigrated cells attached to the top of the filter were removed using a cotton swab, leaving only the migrated cells on the bottom of the filter. In the optical microscope equipped with a digital camera (Olympus, IX71, Japan), the number of cells moved by taking a 100-fold photograph was compared, and 10 images were counted and analyzed for each condition.

As a result, in the negative control group (Null / Mock), the mobility of HUVEC showed basal mobility, but in the positive control group (Mock) to which angiogenic factors VEGF, HGF, or VEGF / HGF were added, It was confirmed that the mobility is greatly increased, and that the effect is more dependent on the addition of HGF (see FIGS. 7 and 8). On the other hand, the TTAC-0001 treatment group that inhibited the VEGF / VEGFR-2 signaling mechanism was found to inhibit the mobility of HUVECs by VEGF, but did not inhibit the mobility of HUVECs by HGF. On the other hand, in the PIG-KM 1 and PIG-KM 2 treatment groups which simultaneously inhibit VEGF / VEGFR-2 and HGF / c-Met signaling mechanisms, the HUVEC was not only added to VEGF or HGF alone but also to VEGF / HGF simultaneous addition conditions. It has been found to effectively inhibit the mobility of. This result is a good example showing that double-target antibodies can act more effectively than single-target antibodies in micro-environment conditions where various growth factors are present.

Example 6 Analysis of Proliferation Rate of HUVEC After PIG-KM Treatment

For proliferation analysis of vascular endothelial cells, these cells were incubated for 24 hours at a density of 2 × 10 ⁴ cells / well in 24-well plates. Then, after washing twice with M199 medium, and cultured for 6 hours at low serum concentration conditions of M199 medium containing 1% fetal bovine serum (Hyclone, USA). After 30 min pretreatment of cells with various concentrations of TTAC-0001, PIG-KM 1 and PIG-KM 2 antibodies, 20 ng / ml VEGF (R & D systems, USA) and 50 ng / ml HGF (R & D systems, USA) Was treated. After 41 hours of incubation, CCK-8 (Dojindo, Japan) was treated for 2 hours and absorbance at 450 nm wavelength was measured to compare cell viability under each condition.

As a result, the positive control group co-treated with VEGF and HGF (HGF + VEGF / Mock) showed an increase in HUVEC proliferation up to about 70% compared to the non-treated group (null / Mock), and these growth factors were treated with HUVEC. Treatment with TTAC-0001, PIG-KM1 and PIG-KM2 resulted in inhibition of proliferation of HUVECs. Both PIG-KM1 and PIG-KM2 showed about 10% inhibition of HUVEC cell proliferation compared to the single target antibody TTAC-0001, and about 45% of both antibodies compared to the control group (Mock) co-treated with VEGF and HGF. It showed HUVEC cell proliferation inhibitory ability (see FIG. 9). The HUVEC proliferation inhibition rate of TTAC-0001 is relatively indistinguishable from that of the dual target antibodies PIG-KM1 and PIG-KM2. HGF / c-Met signaling mechanism is mainly associated with mobility and tube formation rather than HUVEC proliferation in angiogenesis. Consistent with previous findings reported to be involved in (Yi Xu et al., Mol. Vision, 16: 1982, 2010).

Example 7: c-Met Function Inhibition Analysis by Wound Healing

For proliferation and mobility analysis of vascular endothelial cells, these HUVECs were cultured in 6-well plates for 24 hours at a density of 2 × 10 ⁵ cells / well. When cells grew more than 90% after 24 hours of incubation, a constant thickness wound was made at the bottom of the plate with a 200 pipette tip. Various concentrations of TTAC-0001 and PIG-KM 1 antibodies were pretreated with cells for 30 minutes and treated with 50 ng / ml HGF. After 16 hours of incubation, photographs were taken of how the wounds healed for the first time, and the inhibition was determined by looking at the gap of the wounds.

As a result, in the case of Mock and TTAC-0001, it was confirmed that HUVEC proliferation and mobility due to HGF progressed slightly compared to the PIG-KM1 treatment group (see FIG. 10). These results show that PIG-KM1 interferes with HGF / c-Met signaling mechanisms, thereby inhibiting the proliferation and mobility of HUVECs and consequently slowing wound healing.

Example 8 Intracellular c-Met Phosphorylation Inhibition Assay by Western Blotting

In order to confirm the phosphorylation inhibition of c-Met of the dual target antibody PIG-KM1 of the present invention, immunoprecipitation and western blotting were performed. Vascular endothelial cells cultured for 24 hours were incubated for 6 hours under conditions of M199 medium containing 1% fetal bovine serum, followed by pretreatment with 10 / ml PIG-KMs antibody for 30 minutes. Thereafter, 20 ng / ml VEGF and 100 ng / ml Ang1 were treated for 15 minutes.

For analysis by Western blotting, lysis buffer (1% (w / v) SDS, 10 mM Tris (pH 7.4), 2 mM sodium orthovanadate, 2 mM EGTA, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1 mM) sodium fluoride) was treated to obtain a lysate, which was then boiled and centrifuged at 10,000 g at 4 ° C. for 5 minutes to remove insoluble precipitate. The supernatant was prepared by mixing with SDS sample buffer and boiling for 10 minutes. SDS-PAGE and Western blotting were applied in the same manner as commonly used in the art, and the samples used were as follows: 4-20% SDS-polyacrylamide Gel (BioRad, USA), PVDF membrane (Millipore # IPVH00010, USA ), anti-c-Met antibody (Santa Cruz, USA) and anti-phospho-c-Met antibody (Cell Signaling technology, USA) as primary antibodies for c-Met phosphorylation inhibitory activity assay; HRP-conjugated goat anti-mouse IgG antibody (Santa Cruze Biotechnology, USA) and HRP-conjugated goat anti- as secondary antibodies to bind anti-β actin antibody (Sigma, USA) and primary antibody for chemiluminescence. rabbit IgG (Santa Cruze Biotechnology, USA)

As a result of Western blotting, it was confirmed that PIG-KM1 can effectively inhibit c-Met phosphorylation (see FIG. 11). As shown in FIG. 11, since phosphorylation of c-Met proceeds dependent on HGF, c-Met phosphorylation was confirmed in HGF-treated U-87 MG cells in the control group (Mock). On the other hand, in the PIG-KM1 treatment group, c-Met phosphorylation was not confirmed in UGF-87 MG cells treated with HGF, which may be attributed to the c-Met inhibition function of PIG-KM1.

The dual target antibody according to the present invention can simultaneously neutralize two targets involved in angiogenesis and cancer cell proliferation. Therefore, the present invention not only shows superior neutralizing ability as compared to conventional single target antibodies, but may also contribute to cancer treatment.

Claims (18)

1. A sequence at the N-terminus of the light chain of a vascular endothelial growth factor receptor neutralizing antibody comprising a light chain variable region represented by the amino acid sequence of any one of SEQ ID NOs: 1 to 19 and a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 20 A dual target antibody having binding specificity to Vasicular Endothelial Growth Factor Receptor-2 (VEGFR-2) and Mesenchymal-Epithelial Transition factor (cEG) -2, to which a water-soluble peptide represented by the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 22 is linked.
2. The dual target antibody of claim 1, wherein the vascular endothelial growth factor receptor neutralizing antibody is a scFv (Single Chain Fragment Variable) or an IgG (immunoglobulin g).
3. According to claim 1, wherein the double target antibody is SEQ ID NO: 21 or SEQ ID NO: 21 at the N-terminal end of the light chain of the vascular endothelial growth factor receptor neutralizing antibody (TTAC-0001) having a light chain variable region represented by the amino acid sequence of SEQ ID NO: A dual target antibody linked to a water soluble peptide represented by the amino acid sequence set forth in 22.
4. The dual target antibody of claim 1, wherein the N-terminus of the heavy or light chain of the vascular endothelial growth factor receptor neutralizing antibody and the water-soluble peptide are linked through a linker.
5. The dual target antibody of claim 4, wherein the linker has a nucleotide sequence of SEQ ID NO: 23. 6.
6. The dual target antibody of claim 1, wherein the water soluble peptide binds to c-Met and antagonizes.
7. A DNA encoding a dual target antibody according to any one of claims 1 to 6.
8. 8. The DNA of claim 7, wherein said DNA has a codon optimized sequence to increase expression.
9. 8. The DNA according to claim 7, wherein the DNA consists of the nucleotide sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 25.
10. Recombinant expression vector comprising a DNA according to claim 9.
11. The recombinant expression vector of claim 10, wherein the vector has a cleavage map of pIgG-pep1 / 6A6lgt or pIgG-pep2 / 6A6lgt shown in FIG. 2.
12. A host cell transformed with the recombinant expression vector according to claim 10.
13. A method of preparing a double target antibody by culturing the host cell according to claim 12 and separating the double target antibody from the culture thereof.
14. The method of claim 13, wherein the dual target antibody is further purified using Fast Protein Liquid Chromatography (FPLC) with a Protein A affinity column, SP-Sepharose column and size exclusion chromatography.
15. A pharmaceutical composition for inhibiting angiogenesis comprising the dual target antibody according to any one of claims 1 to 6.
16. The group of claim 15 wherein the pharmaceutical composition comprises endostatin, angiostatin, bevacizumab (tradename Avastin ^™ ), sunitinib, sorafenib, lenalidomide, and VEGF-trap. A pharmaceutical composition further comprising an angiogenesis inhibitor selected from.
17. A pharmaceutical composition for treating cancer comprising the dual target antibody according to any one of claims 1 to 6.
18. The method of claim 17, wherein the cancer is gastric cancer, liver cancer, lung cancer, thyroid cancer, breast cancer, cervical cancer, colon cancer, pancreatic cancer, rectal cancer, colorectal cancer, prostate cancer, kidney cancer, melanoma, prostate cancer, bone metastasis cancer, ovarian cancer Pharmaceutical composition is selected from a variety of solid and hematological cancers.

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