WO2016060297A1 - Dual-target antibody having binding ability to vegfr-2 and c-met - Google Patents

Abstract

The present invention relates to a dual-target antibody having a binding ability to vascular endothelial growth factor receptor-2 (VEGFR-2) and mesenchymal epithelial transition factor (c-Met) and, more specifically, to a dual-target antibody having a binding ability to VEGFR-2 and c-Met, a method for producing the dual-target antibody, and a pharmaceutical composition containing the dual-target antibody, wherein the dual-target antibody comprises a VEGFR-2 neutralizing antibody, which has a light chain variable region with an amino acid sequence represented by SEQ ID NO: 1 and a heavy chain variable region represented by SEQ ID NO: 2, and a C-met neutralizing peptide represented by SEQ ID NO: 3 or 4, which is linked to the terminal of the light chain variable region or the heavy chain variable region of the VEGFR-2 neutralizing antibody. The dual-target antibody according to the present invention simultaneously neutralizes two targets involved in angiogenesis and cancer cell proliferation, and thus exhibits an excellent neutralizing ability compared with existing single target antibodies and is very effective in the treatment of cancer. In addition, it can be expected that c-Met neutralizing active domains, which were not previously easy to produce, can be mass-produced.

Description

Double Target Antibody Binding to VEGFR-2 and c-Met

The present invention relates to a double target antibody having binding to VEGFR-2 and c-Met, and more particularly to a light chain variable region having an amino acid sequence represented by SEQ ID NO: 1 and a heavy chain variable region represented by SEQ ID NO: 2 Branched c-Met (Mesenchymal Epithelial Transition factor) neutralizing peptide represented by SEQ ID NO: 3 or SEQ ID NO: 4 at the end of the light chain variable region or the heavy chain variable region of the VEGFR-2 (Vasicular Endothelial Growth Factor Receptoer-2) neutralizing antibody The present invention relates to a dual target antibody having binding to VEGFR-2 and c-Met bound thereto, a method for preparing the dual target antibody, and a pharmaceutical composition comprising the dual target antibody.

Angiogenesis-inhibiting antibodies targeting the VEGF receptor, VEGFR-2, are identified and developed from Dyax's native phage display library, Imclone's IMC-1121B (EP1916001A2) and UCB's CDP-791. (PCT / GB02 / 04619), and Tanibirumab (TTAC-0001, Tanibirumab, Korean Patent No. 883,430 and WO 2008/153237) developed by the researchers. IMC-1121B is a monoclonal antibody selected from a complete human Fab library, and currently undergoes phase 3 clinical trials for metastatic breast and gastric cancer and combined with paclitaxel in patients with gastric cancer, resulting in overall survival and tumor progression survival. progression-free survival). UCB's CDP-791 is a humanized antibody that is a first-line treatment for the treatment of non-small cell lung cancer in the form of PEGylated Di-Fab. It showed improvement in. Because the antibody does not have an Fc, antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity cannot be expected.

Tanivirumab, developed by the present inventors and currently studied in the first stage of clinical trials without toxicity problems, is a monoclonal antibody selected from the complete human ScFv phage library of Paxinsin, which targets VEGFR-2 and simultaneously mouse or rat. It is the only antibody that is responsive to the derived flk-1 (VEGFR-2 homologue), which is one of the important characteristics that distinguishes it from IMC-1121B from Imclones (WO 2008/153237). In particular, the cross-species cross reactivity shown by the Tanibirumap has allowed the study of animal disease models.

In preclinical animal studies, target proteins are inhibited in the treatment of anti-angiogenesis, but the compensatory pathway, ie, alternative neovascular stimulatory signals, is activated, contributing to tumor survival and proliferation. Cancer therapeutic antibodies that block the / VEGFR pathway have been reported to activate drug resistance by activating c-Met upon prolonged administration (Bergers and Hanahan, Nat. Rev. Cancer, 8: 592, 2008). In addition, when GBM growth progressed after the Avastin administration, the survival rate after craniotomy was lower than that of the Avastin non-administered group, so there is a report that there should be a separate consideration for surgical complications when treating Avastin. (Clark et al, Neurosurgery , 70: 361, 2011).

On the other hand, HGF / c-Met signaling mechanism is well known to contribute greatly to the growth and metastasis of tumor cells by overexpression in various cancer tissues and tumor cells.

Mesenchymal-epithelial transition factor (C-Met) is a cell-surface receptor, an oncogene of the receptor tyrosine kinase family, and consists of only 50 kD extracellular domains. It has been found to consist of a total of 145 kD beta (β) subunits consisting of subunits and extracellular, cell membrane permeation, tyrosine kinase domains and phosphorylation related tyrosine motifs (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). Inappropriate expression of c-Met or HGF is associated with several types of malignant tumors and has been reported to have a poor prognosis when overexpressed (www.vai.org/met, Eder et al., Clin. Cancer Res. , 15: 2207, 2009).

Many researchers also believe that in order for the tyrosine kinase domain to be activated, hepatocyte growth factor / scatter factor (HGF / SF) must bind to the c-Met extracellular domain in the beta subunit of the two subunits. It has been reported that the 1349th and 1356th tyrosine of c-Met, which is very important for the mechanism, 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).

C-Met responds to HGF and stimulates various signaling pathways through phosphorylation of c-Met to enhance mitogenesis and cell motility of cancer cells and vascular cells, inhibit cell death, It promotes the formation and transformation of cancer by inducing invasion and metastasis into the 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). 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 the 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), colorectal cancer (Hiscox et al., Cancer Invest ., 15: 513, 1997), etc. As c-Met is activated or overexpressed, it promotes the cancer process. Several methods of inhibiting the activation of c-Met have been developed as promising anti-cancer treatment strategies, such as small molecule compounds designed to prevent 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), which interfere with the phosphorylation of c-Met, downstream of signal transduction However, the 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 that neutralize 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 also 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 real anticancer effects in vitro or in vivo, they can be combined with existing chemotherapy. It is considered to be useful. In addition, gefitinib and erlotinib (EGFR) kinase inhibitors, gefitinib and erlotinib, are often used as effective cancer drugs, but drug resistance is often caused by intensive amplification of c-Met receptors. (Engelman et al., Science , 316 (5827): 1039, 2007), therefore, anti-cancer effects are expected to be amplified by incorporating c-Met inhibitors.

Antibodies currently targeting HGF include AMG-102 (Rilotumumab, Burgess et al., Mol. Cancer Ther ., 9: 400, 2010), which binds to the SPH domain of Amgen, AV-299 from AVEO Pharmaceuticals (Ficlatuzumab). ) Is currently under clinical development, and only one arm of c-Met antibody, which was originally bivalent in order to suppress the activation mode of the antibody itself, is used as an antibody targeting c-Met. MetMab (Onartuzumab, PRO143966) developed as a monovalent (3) breast cancer that lacks both the estrogen receptor (ER), the progesterone receptor (PR) and Her2 / neu (EGFR-2 receptor) (Triple negative breast cancer) and NSCLC (non-small cell lung cancer) are undergoing phase 2 clinical trials, and Lilly LA-480 (LY2875358) is undergoing phase 1 clinical trials.

C-Met interacts with other signaling systems such as EGFR, semaphorin 4D receptor, transforming growth factor-β (TGFβ), WNT, tetraspanins kangai 1 (KAI1 or CD82), and CD151 to help cells grow, differentiate, regenerate, and cancer. Influence (Gherardi, Nat. Rev. Cancer, 12:89, 2012). In addition, c-Met is well known to be activated by HGF, contributing to angiogenesis by itself, and to stimulate angiogenesis through interaction with VEGFR-2, a major receptor for angiogenesis. have. In this case, when c-Met was simultaneously inhibited by targeting with VEGFR-2, it was observed that tumor growth was synergistically inhibited in human xenograft models (Zhang et al., IDrugs , 13: 112, 2010).

HGF, the only ligand of C-Met, consists of N-terminal domain, four Kringle (K1 ~ K4) domains, and C-terminal serine proteinase homology (similar domain of serine protease), NK1 (Chirgadze et al., Nature Struct. Biol. , 6:72, 1999; Ultsch et al., Structur e 6: 1383, 1998), NK2 (Tolbert et al., Proc. Natl Acad. Sci. USA, 107 : 13264, 2010), in the form of a complex of the SPH fragment and the SPH domain (Kirchhofer et al., J. Biol. Chem. , 279: 39915, 2004) and the SEMA domain of Met's extracellular domain (Stamos et al., EMBO J. , 23: 2325, 2004) crystal structure was analyzed. On the other hand, when analyzing the structure of the complex form of InlB (Internalin B) and c-Met, an important protein for internalization of Listeria monocytogenes when infecting hepatocytes or macrophages, 4 of c-Met The first IgG-like domain (IgG1, IgG2, IgG3, IgG4) was shown to bind to the IgG1 domain (Niemann et al., Cell 130: 235, 2007; Ferraris et al., J. Mol. Biol ., 395). : 522, 2010).

HGF is a complex, unstable form of protein that consists of several domains and has a structural and functional relationship with plasma protein, a plasma cleavage precursor, plasminogen. Structural rearrangements occur, and HGF-Met signaling must be made before activated HGF is produced (Gherardi et al. Proc. Natl. Acad. Sci. USA , 103: 4046, 2006). NK1, a splicing variant of HGF, activates c-Met very weakly compared to HGF, but is enhanced by heparan-sulfate proteoglycan (HSPG) (Schwall et al., J. Cell. Biol., 133: 709 , 1996).

Through small angle X-ray scattering (SAXS) analysis and several X-ray crystallographic analyzes, the minimal structure through which c-Met complexes signal is centered on the ligand dimer and outward on both sides of the Met receptor. Was found to be an array of 2: 2 phases (Gherardi et al. Proc. Natl. Acad. Sci. USA , 103: 4046, 2006).

Three types of serine proteinases, HGFA (HGF activator), matriptase (ST14) and hepsin, are involved in HGF cleavage and activation. Matriptase (ST14) and hepsin express c-Met. Although expressed in cells, HGFA is a water-soluble factor, cleaved by thrombin, and serves to connect HGF / c-Met signals involved in blood coagulation and tissue regeneration (Shimomura et al., J. Biol. Chem). 268: 22927, 1993). In addition, HGF activation is regulated by at least HAI1 (HGF activator inhibitor 1; SPINT1), HAI2 (HGF activator inhibitor 2; SPINT2), two inhibitors (inhibitors), such as increased matriptase or hepsin expression, or HAI1 The decrease in the expression level of HAI2 has been studied as a target of cancer treatment in terms of lowering metastatic activity of cancer cells (Morris et al., Cancer Res ., 65: 4598, 2005).

CBL, a ubiquitin E3 ligase as another c-Met inhibitor, recognizes Tyr1003, a c-Met motif located near the cell membrane, and has a ring finger domain that binds to E2 ligase. Ultimately, c-Met degradation is induced, which causes Tyr1003 motifs to be removed or mutations that cause c-Met to be recycled without degradation (Peschard et al., Mol. Cell, 8). : 995, 2001).

Several variants of HGF acted as antagonists of c-Met. The most commonly expressed variant of NK2 partially activates c-Met but ultimately performs antagonism (Otsuka et al., Mol.Cell. Biol ., 20: 2055, 2000), NK2 activates c-Met by opening the "closed" monomer conformation of the N-terminal domain and the second Kringle domain (K2). (Tolbert et al., Proc. Natl Acad. Sci. USA, 107: 13264, 2010). In addition, NK4 is well known as an antagonist of HGF, but its expression and production are not available (Nakamura et al., Anticancer Agents Med. Chem. , 10:36, 2010). Efforts have been made to inhibit c-Met activation using HGF variants such as NK1, NK2, and NK4, but these variants have shown poor results in terms of protein expression and stability.

In particular, NK1 is the smallest form of functional HGF, which is unstable, with difficulty in protein expression (Tolbert et al., Proc. Natl. Acad. Sci. USA , 104: 14592, 2007; Lokker et al., EMBO J., 11: 2503, 1992). For this reason, a group at Stanford University has directed NK1 through direct error (error prone polymerase chain reaction) to increase the expression rate of NK1 by 40 times and increase the melting temperature (Tm, melting temperature). Attempts have been made to improve the protein stability by increasing the temperature above ℃ to be used as a therapeutic protein (Douglas et al., Proc. Natl. Acad. Sci. USA, 108: 13035, 2011).

As such, c-Met targeting may be a good treatment for resistance to antiangiogenic therapy (Comoglio et al., Nat. Rev. Drug. Discov ., 7: 504, 2008). Inhibition of c-Met activity In the case of restoring the activity of VEGFR-2 antagonist (You et al., Cancer Res. 71: 4758, 2011), the treatment that simultaneously inhibits the activity of VEGFR-2 and c-Met is an anti-neovascular inhibitor. It is thought that the therapeutic efficiency of tumors can be improved.

As such, recent antibody studies, in addition to developing antibodies that are functional against a single target, are known as bispecific or multi-specific antibodies that can simultaneously take two or more targets. antibodies have been actively 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 these antibodies has been commercialized with FDA approval yet, but research and development is being conducted 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. As described above, various academic reports on double- or multi-target antibodies and preparation methods thereof have been reported. Although these antibodies have functional advantages and disadvantages according to their morphological characteristics according to the purpose of use, there is still a need for the development of effective new therapeutic double- and multi-target antibodies for treating cancer. 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.

Accordingly, the present inventors have made diligent efforts to develop a dual target antibody having a cancer treatment effect by inhibiting angiogenesis. Thus, the present inventors prepared an IgG-based dual target antibody capable of simultaneously neutralizing VEGFR-2 and c-Met. The present invention has been completed by confirming that the dual target antibody shows a comparable or better anticancer effect at the cellular level compared to the VEGFR-2 single target antibody or c-Met antagonist domain.

Summary of the Invention

It is an object of the present invention to provide an IgG-based dual target antibody capable of simultaneously neutralizing VEGFR-2 and c-Met.

It is another object of the present invention to provide a DNA encoding the dual target antibody and a recombinant vector containing the DNA.

Still another object of the present invention is to provide a host cell transformed with a recombinant vector containing the DNA encoding the dual target antibody.

Another object of the invention (a) culturing the host cell transformed with the recombinant vector to produce a dual target antibody having binding to VEGFR-2 and c-Met; And (b) obtaining a dual target antibody having binding to the generated VEGFR-2 and c-Met, the method for preparing a dual target antibody having binding to VEGFR-2 and c-Met. To provide.

Still another object of the present invention is to provide a pharmaceutical composition for inhibiting angiogenesis, including the double target antibody.

Another object of the present invention to provide a pharmaceutical composition for treating cancer comprising the double target antibody.

In order to achieve the above object, the present invention is a VEGFR-2 (Vasicular Endothelial Growth Factor Receptoer-2) neutralizing antibody having a light chain variable region having an amino acid sequence represented by SEQ ID NO: 1 and a heavy chain variable region represented by SEQ ID NO: 2 Has binding to VEGFR-2 and c-Met to which a c-Met (Mesenchymal Epithelial Transition factor) neutralizing peptide represented by SEQ ID NO: 3 or SEQ ID NO: 4 is bound to the end of the light chain variable region or the heavy chain variable region. Provide dual target antibodies.

The present invention also provides a DNA encoding the dual target antibody and a recombinant vector containing the DNA.

The present invention also provides a host cell transformed with a recombinant vector containing the DNA encoding the dual target antibody.

In addition, the present invention comprises the steps of (a) culturing the host cell transformed with the recombinant vector to generate a dual target antibody having binding to VEGFR-2 and c-Met; And (b) obtaining a dual target antibody having binding to the generated VEGFR-2 and c-Met, the method for preparing a dual target antibody having binding to VEGFR-2 and c-Met. to provide.

In addition, the present invention provides a pharmaceutical composition for inhibiting angiogenesis comprising the double target antibody.

The present invention also provides a pharmaceutical composition for treating cancer comprising the double target antibody.

Figure 1 shows that the production of the vector pPMC-102 according to the present invention using 293T cells, the expression rate of PMC-102 is very low by Western blot and ELISA (a and b), pPMC-102FC in 293T cells After optional expression and purification, the production and purification of the dual target antibody PMC-102FC was confirmed by SDS-PAGE.

Figure 2 shows the nucleotide sequence of the NK1 domain of HGF and its variants that bind to c-Met.

3 is a schematic diagram of the entire pPMC-102FC including the heavy chain (b) the nucleotide sequence of the light chain portion of the PMC-102FC obtained by performing codon optimization when constructing the vector pPMC-102FC according to the present invention. a) is shown.

Figure 4 shows the binding capacity and affinity for VEGFR-2 and human and mouse c-Met of the dual target antibody according to the present invention (a) and co-binding capacity (b) for VEGFR-2 and human and mouse c-Met BiaCore The analysis results are shown.

Figure 5 shows the results of proliferation assay in the BxPC3 cell line (a) of the dual target antibody PMC-102 in accordance with the present invention, and in the KP-4 cell lines (b and c) of PMC-102FC.

Figure 6 shows the results of the proliferation assay (proliferation assay) for HUVEC of the dual target antibody PMC-102FC according to the present invention.

Figure 7 shows the results of Western blot analysis of the phenomenon that the activation of c-Met is inhibited, the activation of Erk is inhibited when the dual target antibody PMC-102FC according to the present invention treated KP-4.

Figure 8 shows the results of Western blot analysis of a phenomenon in which the activation of VEGFR-2 is inhibited and the activation of Erk is inhibited when the dual target antibody according to the present invention is treated with HUVEC.

Detailed Description of the Invention and Specific Embodiments

Unless defined otherwise, 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. In general, the nomenclature used herein is well known and commonly used in the art.

The present invention consistently, the light chain variable region of the VEGFR-2 (Vasicular Endothelial Growth Factor Receptoer-2) neutralizing antibody having a light chain variable region having an amino acid sequence represented by SEQ ID NO: 1 and a heavy chain variable region represented by SEQ ID NO: 2 Dual-target antibody having binding to VEGFR-2 and c-Met to which a c-Met (Mesenchymal Epithelial Transition factor) neutralizing peptide represented by SEQ ID NO: 3 or SEQ ID NO: 4 at the end of the region or the heavy chain variable region is bound It is about.

In the present invention, in addition to the inhibition of angiogenesis derived from tumors for the development of antibodies for anticancer treatment, VEGF / VEGFR and HGF / c-Met signaling mechanisms were targeted to simultaneously inhibit tumor proliferation and metastasis.

In the present invention, Tanibirumab (TTAC-0001, Tanibirumab, Republic of Korea Patent No. 883,430 and WO 2008/153237), which forms the skeleton of the dual target antibody, targets VEGFR-2 and at the same time flk derived from mouse or rat. Reactive to -1 and the second target c-Met targeting domain is also designed to target human and mouse at the same time, enabling the study of animal disease model using cross-reactivity between different species, Induce transitional research and develop anticancer drugs for specific cancers in the future to make related research easier.

In the present invention, the VEGFR-2 neutralizing antibody may be characterized in that the scFV (Single Chain Fragment Variable) or IgG, light chain variable region or heavy chain variable region of the c-Met neutralizing peptide and the VEGFR-2 neutralizing antibody May be connected via a linker.

The c-Met neutralizing peptide may be connected to the N-terminus or C-terminus of the light chain variable region or heavy chain variable region of the VEGFR-2 neutralizing antibody.

In one embodiment of the present invention, pPMC-102.Lgt is prepared by linking NK1 (amino acids 28 to 210: SEQ ID NO: 3) domains to the amino terminus of the light chain sequence (SEQ ID NO: 1) of Tanivirumab with a linker. Together with pIgGLD-6A6Hvy, a heavy chain sequence vector of Tanivirumab, 293T cells were co-transduced to express a dual target antibody that simultaneously inhibits the activity of VEGFR-2 and C-Met.

In another embodiment of the present invention, by producing a M15 domain having a mutation in the eight codons in NK1, and removing the unnecessary sequence for the heavy chain sequence of Tanivirumab, and performing codon optimization, improved expression ability Eggplant was prepared a dual target antibody, PMC-102FC that simultaneously inhibits the activity of VEGFR-2 and C-Met.

As a result of confirming the binding ability, dissociation constant (Kd, dissociation constant) and co-binding of the dual target antibody PMC-102FC to human VEGFR-2 and human and mouse c-Met of the double-target antibody according to the present invention, It was confirmed that PMC-102FC showed a similar binding capacity, while it was confirmed that only PMC-102FC had binding capacity in human and mouse c-Met (FIG. 4A). In addition, it was confirmed that in the case of Tanivirumab, a positive control for VEGFR-2, only binds to VEGFR-2, and in the case of Fc-M15, a positive control for c-Met, it binds only to c-Met (FIG. 4B). As a result of analyzing the affinity for each batch, it was confirmed that the high affinity for each of VEGFR-2 and c-Met of Tanivirumab, PMC-102FC (Fig. 4a), the double-target antibody PMC-102FC of the present invention is VEGFR Simultaneous binding to -2 and c-Met was shown (Fig. 4b).

In another embodiment of the present invention, the change in the proliferative capacity of the cancer cells BxPC3 and KP4 after treatment with the dual target antibody PMC-102FC was analyzed, and as a result, when the PMC-102 is treated with 10 μg / ml in BxPC3, the proliferation of the cells effectively Was inhibited (FIG. 5A). In addition, KP4, an autocrine cell line of HGF, was also used to inhibit the proliferation of cells compared to the Tanibirumab, a skeletal antibody, and the inhibitory effect was similar to that of the c-Met inhibitor (SantaCruz, USA) (FIG. 5B). And FIG. 5C).

In another embodiment of the present invention, after the dual target antibody PMC-102FC treatment, the proliferative capacity of vascular endothelial cells (HUVEC) was confirmed, and as a result, the dual target antibody PMC-102FC inhibited the proliferative capacity of HUVEC cells caused by VEGF. It was inhibited similarly to the chain Tanibimap, and also blocked the effects of HGF at the same time, it was confirmed that even when the VEGF and HGF treatment at the same time can more strongly inhibit the proliferative capacity of HUVEC than Tanibimap (Figure 6a and 6b) ).

In another embodiment of the present invention it was confirmed that PMC-102FC can effectively inhibit c-Met phosphorylation in KP4 cells, which are pancreatic cancer cells (Fig. 7), PMC-102FC phosphorylation of VEGFR-2 and c-Met At the same time it was confirmed that can be effectively inhibited (Fig. 8).

In another aspect, the present invention relates to a DNA encoding the dual target antibody and a recombinant vector containing the DNA.

In the present invention, the DNA encoding the double-target antibody is a DNA encoding the light chain variable region of the VEGFR-2 neutralizing antibody represented by SEQ ID NO: 6 and the fragment to which the c-Met neutralizing peptide is bound, and the VEGFR- represented by SEQ ID NO: 7 It may be characterized by comprising a DNA encoding a heavy chain variable region of the two neutralizing antibodies.

In the present invention, "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. Vectors can be plasmids, phage particles or simply potential genomic inserts. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors. Typical plasmid vectors that can be used for this purpose include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure comprising an antibiotic resistance gene and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA.

After ligation, the vector should be transformed into the appropriate host cell. Transformation is described by Sambrook, et. al., easily achieved using the calcium chloride method described in section 1.82 of supra . Alternatively, electroporation (Neumann, et. Al., EMBO J. , 1: 841, 1982) can also be used for transformation of these cells.

Nucleic acids are "operably linked" when placed in a functional relationship with other nucleic acid sequences. This may be genes and regulatory sequence (s) linked in such a way as to enable gene expression when appropriate molecules (eg, transcriptional activating proteins) bind to regulatory sequence (s). For example, the DNA for a pre-sequence or secretion leader is operably linked to the DNA for the polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, "operably linked" means that the linked DNA sequence is in contact, and in the case of a secretory leader, is in contact and present within the reading frame. However, enhancers do not need to touch. Linking of these sequences is performed by ligation (linking) at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used. As is well known in the art, to raise the expression level of a transfected gene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host. Preferably, the expression control sequence and the corresponding gene will be included in one recombinant vector containing the bacterial selection marker and the replication origin. If the host cell is a eukaryotic cell, the recombinant vector must further comprise an expression marker useful in the eukaryotic expression host.

Host cells transformed with the recombinant vectors described above constitute another aspect of the present invention. As used herein, the term “transformation” means introducing DNA into a host so that the DNA is replicable as an extrachromosomal factor or by chromosomal integration.

Of course, it should be understood that not all vectors function equally well to express the DNA sequences of the present invention. Likewise not all hosts function equally for the same expression system. However, those skilled in the art can make appropriate choices among various vectors, expression control sequences and hosts without departing from the scope of the present invention without undue experimental burden. For example, in selecting a vector, the host must be considered, since the vector must be replicated in it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered.

In another aspect, the present invention relates to a host cell transformed with a recombinant vector containing the DNA encoding the dual target antibody.

In another aspect, the present invention is (a) culturing a host cell transformed with the DNA encoding the dual target antibody and the recombinant vector containing the DNA to have a double binding to VEGFR-2 and c-Met Generating a target antibody; And (b) obtaining a dual target antibody having binding to the generated VEGFR-2 and c-Met, the method for producing a dual target antibody having binding to VEGFR-2 and c-Met. It is about.

The double-target antibody of the present invention can be used as a pharmaceutical, as a pharmaceutical composition for the purpose of preventing and / or treating diseases and cancers caused by excessive angiogenesis. Antibodies of the invention can be formulated and administered orally or parenterally. The formulation containing the antibody of the present invention can be safely administered to a human or animal as a pharmaceutical composition.

In another aspect, the present invention relates to a pharmaceutical composition for inhibiting angiogenesis comprising the double target antibody and a method for inhibiting angiogenesis using the double target antibody.

The antihypertensive composition of the present invention is a tumor growth and metastasis caused by excessive angiogenesis (metastasis), age-related macular degeneration (ARMD), diabetic retinopathy, psoriasis It can be used for the treatment of diseases such as psoriasis, rheumatoid arthritis, chronic inflammation.

In another aspect, the present invention relates to a pharmaceutical composition for treating cancer comprising the double target antibody and a method for treating cancer using the double target antibody.

In the present invention, the cancer is composed of stomach 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 It may be characterized in that it is selected from the group.

Carriers used in the pharmaceutical compositions of the present invention include pharmaceutically acceptable carriers, adjuvants and vehicles and are collectively referred to as pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the invention include, but are not limited to, ion exchange, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer materials (eg, Various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal Silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substrates, polyethylene glycols, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene-polyoxypropylene-blocking polymers, polyethylene glycols and wool, and the like.

The route of administration of the pharmaceutical composition according to the present invention is not limited thereto, but is oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Sublingual or rectal.

Oral and parenteral administration is preferred. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intramuscular, intrasternal, intradural, intralesional and intracranial injection or infusion techniques.

Pharmaceutical compositions may be in the form of sterile injectable preparations as sterile injectable aqueous or oily suspensions. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (eg, Tween 80) and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions (eg, solutions in 1,3-butanediol) in nontoxic parenterally acceptable diluents or solvents. Vehicles and solvents that may be used that are acceptable include mannitol, water, ring gel solution, and isotonic sodium chloride solution. In addition, sterile nonvolatile oils are conventionally employed as a solvent or suspending medium. For this purpose, any non-irritating oil may be used including synthetic mono or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in injection formulations as well as pharmaceutically acceptable natural oils (eg olive oil or castor oil), especially their polyoxyethylated ones.

The pharmaceutical compositions of the present invention may be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, and aqueous suspensions and solutions. For oral tablets, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also typically added. Useful diluents for oral administration in a capsule form include lactose and dried corn starch. The active ingredient is combined with emulsifiers and suspending agents when the aqueous suspension is administered orally. If desired, sweetening and / or flavoring and / or coloring agents may be added.

The compounds of the present invention can be used in combination with conventional anti-inflammatory agents or in combination with matrix metalloprotease inhibitors, lipoxygenase inhibitors and inhibitors of cytokines other than IL-1. The compounds of the present invention also provide immunomodulators (eg, bropyrimin, anti-human alpha interferon antibodies, IL-2, GM-CSF, methionine enkephalins, interferons to prevent or combat IL-1 mediated disease symptoms such as inflammation). Alpha, diethyldithiocarbamate, tumor necrosis factor, naltrexone and rEPO) or prostaglandin. When the compounds of the present invention are administered in combination with other therapeutic agents, they may be administered sequentially or simultaneously to the patient. Alternatively, the pharmaceutical composition according to the present invention may be made by mixing the compound of formula (1) with other therapeutic or prophylactic agents described above.

An effective amount for a pharmaceutical composition of the present invention may be determined by various methods including the activity, age, weight, general health, sex, diet, time of administration, route of administration, rate of release, drug combination and severity of the particular disease to be prevented or treated. It will be appreciated that the factors may vary. The pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

In a preferred embodiment, for parenteral administration, the pharmaceutical composition of the present invention can be prepared in an aqueous solution. Preferably, a physically suitable buffer such as Hanks' solution, Ringer's solution, or physically buffered saline may be used. Aqueous injection suspensions may add a substrate that can increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. In addition, suspensions of the active ingredient may be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or carriers include fatty acids such as sesame oil or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Polycationic amino polymers can also be used as carriers. Optionally, the suspension may use a suitable stabilizer or agent to increase the solubility of the compound and to prepare a high concentration of solution.

Antibodies in pharmaceutical compositions adsorb and are unstable in glass containers such as vials, syringes, and the like, and are easily deactivated by various physicochemical factors such as heat, pH, and humidity. Thus, stabilizers, pH adjusters, buffers, solubilizers, surfactants and the like are added to formulate into stable forms. Examples of the stabilizer include amino acids such as glycine and alanine, sugars such as dextran 40 and mannose, sugar alcohols such as sorbitol, mannitol, and xyltol, and two or more kinds thereof may be used in combination. The amount of these stabilizers added is preferably 0.01 to 100 times, in particular 0.1 to 10 times the weight of the antibody. By adding these stabilizers, the storage stability of the liquid preparation or the lyophilized preparation can be improved. Examples of the buffer include phosphate buffers and citric acid buffers. The buffer adjusts the pH of the aqueous solution after redissolution of the liquid or lyophilized formulation and contributes to the stability and solubility of the antibody. As the addition amount of the buffer, for example, the amount of the liquid preparation or the freeze-dried preparation is preferably 1 to 10 mM with respect to the amount after re-dissolution. Polysorbate 20, pulluronic F-68, polyethyleneglycol, etc. can be used as surfactant, Especially preferably, polysorbate 80 is mentioned, You may use together 2 or more types.

As described above, polymer proteins such as antibodies are easily adsorbed to glass, resin, and the like, which are the materials of the container. Therefore, by adding a surfactant, adsorption of the antibody into the container after re-dissolution of the liquid or lyophilized agent can be prevented. have. As the addition amount of the surfactant, it is preferable to add 0.001 to 1.0% to the weight of water after re-dissolution of the liquid formulation or the lyophilized formulation. Although the above stabilizer, buffer, or adsorption agent can be added to the preparation of the antibody of the present invention, the osmotic pressure ratio that can be used as the osmotic pressure ratio is preferably 1 to 2, especially when used as a medical or animal medicine injection. Osmotic pressure ratio can be prepared by increasing or decreasing sodium chloride at the time of chemical | medicalization. The antibody content in the preparation can be appropriately adjusted according to the applied disease application route, and the dose of the humanized antibody to human is determined by the affinity of the antibody for human protein NSP, that is, the dissociation constant for human protein NSP (Kd value). Depends on the drug, the drug can be expressed even if the dose to the human being is reduced by the high affinity (low Kd value).

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Particularly, in the following examples, only succinic acid-producing microorganisms of the genus Maniamia are exemplified by specific vectors and host cells in order to remove the genes according to the present invention. What can be obtained will be apparent to those of ordinary skill in the art.

Example 1 Cloning of NK1

Human HGF domain-associated DNA that binds to C-Met and inhibits its activity was extracted and used through conventional molecular and biological methods using RT-PCR.

PCR was performed under the following conditions to amplify only the NK1 domain using the extracted DNA: 94 ° C. 4 min, (94 ° C., 45 sec / 50 ° C., 45 sec / 72 ° C., 1 min) 25 times, 72 ° C 7 minutes, 4 ° C. The reactant composition used was as follows: primers SP-SBs with BstXI restriction enzyme recognition site and reverse primer NK1HisSal-As with SalI site and 6XHis sequence, respectively 2 μl (10 pmole / μl), human HGF cDNA as template DNA. 1 μl (100 ng / μl), i-Max ™ II Taq 2.5 U as polymerase (Intron ＃ 25261, South Korea), 5 μl of 10 × buffer, 2 μl of dNTP (2.5 mM each), 37.5 μl of distilled water. The product obtained by PCR was confirmed by electrophoresis through a 1% agarose gel, cleaved with BstXI and SalI, and then a faint band of less than 600 bp was detected by HiYieldTM Gel / PCR DNA Extraction Kit (RBC Bioscience ＃ YDF300, Taiwan), and cloned the cloned expression vector of pcDNA3 (invitrogen) to the fragment cut with Bst XI and Xho I and cloned the NK1 expression vector pSP.NK1.His that was correctly cloned through sequencing. Obtained.

SP-SBs Primer: NNN CCA GCG GTG TGG GCC ACC ATG GGC TGG TCC TAC ATC (SEQ ID NO: 8)

NK1HisSal-As Primer: NNN GTC GAG CTA GTG ATG GTG ATG GTG ATG TTC AAC TTC TGA ACA CT (SEQ ID NO: 9)

The present inventors have applied for and registered pIgGLD-6A6Lgt described in Tanivirumab (TTAC-0001, Korean Patent No. 10-0883430 and International Application No. PCT / KR07 / 003077) as backbone vectors, and the other of pIgGLD-6A6Lgt NK1 (amino acids 28-210 of hHGF: SEQ ID NO: 3) domains at the amino terminus of the nibirumap light chain sequence (SEQ ID NO: 1) with the [G4S] X2 linker (amino acid sequence; sggggsggggsgs), using PCR, pPMC- 102. Lgt was produced.

Co-transduction of 293T cells with pIgGLD-6A6Hvy, a heavy chain sequence vector of Tanivirumab, induced random expression, and the culture was obtained to confirm its expression through SDS-PAGE and Western blotting on human IgG. (FIG. 1A).

At this time, transduction was performed using lipofectamine ™ 2000 (Invitrogen ＃ 11668-019, USA), and inoculated with 2 × 10 ⁶ 293T cells per well in a 6-well plate containing DMEM medium (Welgene, South Korea). A humidified CO ₂ (5%) incubator was used to stand at 37 ° C for 24 hours, and the cells were densely cultured to have a cell density of 90% or more.

3 μg of the recombinant vectors (2 μg each of pPMC-102.Lgt and pIgGLD-6A6Hvy) and 6 μl of lipofectamin ™ 2000 were diluted in 250 μl of serum-free OptiMEM medium and allowed to stand at room temperature for 5 minutes. The DNA and lipofectamin ™ 2000 dilutions were mixed and allowed to react at room temperature for 20 minutes to form the DNA-lipofectamin ™ 2000 complex. After removing the existing medium from the cultured cells, 500 μl of DNA-lipofectamin ™ 2000 complex and 500 μl of serum-free OptiMEM medium were added to each well and incubated for 6 hours in a CO ₂ incubator at 37 ° C. After adding 2 ml of Freestyle 293 medium 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 generally used in the art, and the samples used were as follows: 10% 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).

IgG expression concentration in the medium obtained by the arbitrary expression was confirmed using ELISA. For this purpose, 100 μl of 2 μg / ml goat anti-human IgG (Fc) (Pierce, USA) was added to the 96-well plate as a primary antibody, and the coating was completed by standing at 4 ° C. for 12 hours. After discarding the remaining solution in the well, 200 μl each of the 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 μl of the cell culture solution obtained through 293T transduction was added thereto 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 μl each at room temperature for 1 hour. Reacted for a while. After washing each well three times with washing buffer again, 100 μl of TMB substrate reagent (BD biosciences, USA) was added and reacted for 5-10 minutes, followed by 50 μl of 2N sulfuric acid (H ₂ SO ₄ ) solution. The color reaction was stopped by the addition. Using a microplate reader (Tecan, Switzerland) to obtain the OD _450-650nm value was compared with the standard results and the results were shown in Figure 1b.

Example 2 Codon Adaptation and pPMC-102FC Construction of Tanivirumab Sequences

Through quantitative experiments, the expression rate of PMC-102 was found to be very low (FIG. 4B). NK1 itself is known as an unstable protein, and since its expression is poor, it was judged that its expression rate is low even when it is connected to the amino terminal of the light chain. Accordingly, NK1 variants (M15) were prepared (Douglas et al., Proc. Natl. Acad. Sci. USA, 108: 13035, 2011), which improved the expression rate and stability (FIG. 2).

That is, pSP.NK1.His prepared in Example 1 was used as a template, and PCR and cloning were performed using the primers described below until M15 having a mutation in 8 codons as shown in FIG. . Thus, pSP.M15.His was prepared. Using wild-type NK1 gene sequence, mutations were carried out through multiple steps of PCR, and six histidines (6X His) were inserted immediately before the stop codon, thereby expressing the expression vector pSP.M15.His. Was prepared, and the sequence of the M15 fragment is shown in SEQ ID NO: 4.

M1KS: TGA AGA TAG AAA CCA AAA AA (SEQ ID NO: 10)

M1KAS: TTT TTT GGT TTC TAT CTT CA (SEQ ID NO: 11)

M2KS: CAA GAA AAA GAT GCC TCT G (SEQ ID NO: 12)

M2KAS: CAG AGG CAT CTT TTT CTT G (SEQ ID NO.:13)

M345KS: ATT GGT AAT GGA CGC AGC TAC AGG GGA ACA G (SEQ ID NO: 14)

M345KAS: GCT GCG TCC ATT ACC AAT GAT GCA TTT TCT AAT GT (SEQ ID NO: 15)

M67KS: ATC GGG GTG AAG ACC TAC GGG AAA ACT (SEQ ID NO: 16)

M67KAS: AGT TTT CCC GTA GGT CTT CAC CCC GAT (SEQ ID NO: 17)

M8KS: TCA CAA GCG ATC CAG AGG T (SEQ ID NO.:18)

M8KAS: ACC TCT GGA TCG CTT GTG A (SEQ ID NO.:19)

The mutant M15 of NK1 thus prepared was synthesized through codon adaptation (Invitrogen, USA), and the above-mentioned [G4S] X2 linker was linked to the carboxy terminus of the light chain sequence of Tanivirumab, which was also obtained through codon adaptation. Tanivirumab light chain-M15 was amplified using the PCR method. During PCR amplification, the restriction enzyme Asc I was injected into the N-terminal primer and Hpa I was injected into the C-terminal primer, and then cleaved. The PCR was inserted into the Asc I and Hpa I cleavage sites of the dual expression vector based on pcDNA3. Subsequently, the heavy chain sequence obtained through codon adaptation of Tanibirumab was digested by inserting restriction enzyme Bam HI into the N-terminal primer and Nhe I into the C-terminal primer, and then cutting the Tanibirumab light chain. The pPMC-102FC of FIG. 3 was completed by inserting the BamHI and Nhe I cleavage sites of the dual expression vector already containing the -M15 sequence. The base sequences of the heavy and light chain portions of the completed vector are set forth in SEQ ID NOs: 6 and 7.

When the pPMC-102FC prepared above was transfected and expressed in 293T by the method exemplified in Example 1, and the culture was obtained and quantified by ELISA, a significantly larger amount of PMC-102FC was produced than PMC-102. It was confirmed (Fig. 1b). Accordingly, while maintaining the properties of antagonizing VEGFR-2 in the gene sequence and amino acid sequence, it is designed to remove unnecessary sequences, and commissioned to Invitrogen-GeneArt to codon optimization (codon optimization) The heavy chain sequence of SEQ ID NO: 7 was synthesized.

In addition, the domain of NK1 targeting c-Met is designed to connect M15, a variant of NK1, to the carboxy terminus of the light chain sequence of Tanivirumab (SEQ ID NO: 1) via a linker, which is also an invitrogen-geneart (Invitrogen). Codon optimization was performed by requesting to -GeneArt). The light chain-M15 hybrid was synthesized to complete the vector pPMC-102FC comprising the light chain-M15 ( Asc I / Hpa I sites) and heavy chain ( Bam HI / Nhe I sites) of pPMC-102FC (FIG. 3). The amino acid sequence of is shown in SEQ ID NO: 5, the nucleotide sequence is shown in SEQ ID NO: 6 and Figure 3b. pPMC-102FC was temporarily expressed in 293T cells by PEI precipitation, yielding about 8 mg / L. Purification of IgG bound to protein A resin yielded 4.4 mg of high purity PMC-102FC. Coomassie blue staining results after reducing and non-reducing SDS-PAGE are shown in FIG. 1C.

The dual-target antibody PMC-102FC obtained through random expression culture was packed with MAbSelect Sure (GE health care, Sweden) for further study, and then purified by FPLC (fast protein liquid chromatography) system. Only bays were secured (FIG. 1C). First, the culture medium was filtered using a 0.45 μm filter, and the medium passed through ultrafiltration (UF) was added to a protien A column stabilized with 20 mM sodium phosphate (pH 7.0) containing 0.1 M NaCl. The protein was washed out using the same buffer, and once again, the protein bound to protein A resin was non-specifically washed using 20 mM sodium phosphate (pH 7.0) buffer solution containing 0.5 M NaCl. 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.

Example 3 Avidity Test of Double Target Antibody PMC-102FC

Binding affinity, affinity and co-binding of PMC-102FC to VEGFR-2 and c-Met

BIACORE® 3000 (GE Healthcare) was used to investigate the binding capacity, dissociation constant (Kd, dissociation constant), and covalent binding of PMC-102FC to human VEGFR-2 and human and mouse c-Met, and CM5 chip. The dissociation constant is a similar value of the Km value and is used as an indicator of the affinity of the enzyme in the enzyme-substrate complex. The lower the value, the higher the affinity between the enzyme and the substrate.

Immobilization of the sample was performed using Amine Coupling Kit (GE Healthcare) 400 mM N-ethyl-N '-(dimethylaminopropyl) Carbodiimide (EDM), 100 mM NHS (N-Hydroxysuccinimide), and 1 M Ethanolamine hydrocholoride (pH 8.5). After dilution with 20 mM sodium hydroxide as the regeneration buffer and 1 × PBS as the immobilization buffer, the assay sample was diluted to 1/40 in 10 mM acetate (GE Healthcare) at pH 5.0. The immobilization range was immobilized at 4000RU (Response Unit). As the adsorption measurement buffer of the assay sample, HBS-EP buffer (GE Healthcare) was used. As antigen, human c-Met (hHGFR; R & D Systems) was measured at concentrations of 7.8 nM, 15.6 nM, 31.3 nM, 62.5 nM, 125 nM and 250 nM, and mouse c-Met (mHGFR; R & D Systems) was measured at concentrations of 0.78 nM, respectively. Dilutions were stepwise at 1.56 nM, 3.13 nM, 6.25 nM, 12.5 nM and 25 nM with HBS-EP buffer to a final volume of 200 μl. Five of the six concentrations were selected and fitted. The regeneration buffer was selected after using the sodium hydroxide hydroxide after the binding step and dissociation step of the 7.8 nM sample in advance, before the actual analysis. The assay flow rate was 30 μl / min, binding time was 60 seconds, dissociation time was 300 seconds, and the affinity of the sample was measured.

As a result, it was confirmed that Tanivirumab and PMC-102FC showed similar binding capacity to VEGFR-2, whereas only PMC-102FC had binding capacity to human and mouse c-Met (FIG. 4A). In addition, it was confirmed that in the case of Tanivirumab, a positive control for VEGFR-2, only binds to VEGFR-2, and in the case of Fc-M15, a positive control for c-Met, it binds only to c-Met (FIG. 4B).

As a result of analyzing the affinity for each batch, it was confirmed that the high affinity for each of VEGFR-2 and c-Met of Tanivirumab, PMC-102FC (Fig. 4a), the double-target antibody PMC-102FC of the present invention is VEGFR Simultaneous binding to -2 and c-Met was shown (Fig. 4b).

Example 4 Analysis of Proliferative Capacity of BxPC3 and KP4 after Treatment with Dual Target Antibody PMC-102FC

After treatment with the dual target antibody PMC-102FC according to the present invention, cell proliferation analysis was performed to confirm the proliferation change of BxPC3 cells (ATCC, USA) and KP4 cells (Riken, Japan). BxPC3 and KP4 were cultured using RPMI-1640 medium (Welgene, Korea) added with 10% fetal bovine serum (Gibco, USA), and cell cultures were incubated in a humidified 5% CO ₂ mixed air in a 37 ° C incubator. . BxPC3 and KP4 cells were cultured in RPMI-1640 medium supplemented with 1% fetal bovine serum for 10 g / ml PMC-102 (basic), PMC-102FC, human IgG, and Tanibirumab, a negative control. 20 min pretreatment with mixed control Fc-M15 and final 2 μM c-Met inhibitor (SantaCruz, USA) followed by 30 ng / ml recombinant human HGF (R & D systems, USA). KP4 treated with antibody and growth factor was incubated for 72 hours at a density of 2 × 10 ³ cells / well in a 96-well plate, and then absorbed at 450 nm by treatment with WST-8 (Dojindo, Japan) for 2-4 hours. By doing this, the cell proliferation ability in each condition was compared.

As a result, when BxPC3 was treated with 10 μg / ml of PMC-102, cell proliferation was effectively inhibited (FIG. 5A). In addition, even when KP4, an autocrine cell line of HGF, was used, cell proliferation was inhibited compared to the Tanibirumab, a skeletal antibody, and the inhibitory effect was similar to that of a c-Met inhibitor (SantaCruz, USA) (FIG. 5B). And FIG. 5C).

Example 5 Analysis of Proliferation Capacity of HUVEC Following Treatment with Dual Target Antibody PMC-102FC

After treatment with the dual target antibody PMC-102FC according to the present invention, cell proliferation analysis was performed to confirm the change in proliferative capacity of vascular endothelial cells (HUVEC) (Lonza, Switzerland).

HUVEC was cultured using LifeLine medium (Lifefactors (including rhVEGF, rhIGF-1, rh FGF-B, Ascorbic acid, rh EGF, Heparin, FBS, L-Glutamine, Hydrocort) added to Vasculife medium) (LifeLine, USA). , Cell culture was incubated in a 37 ℃ incubator in humidified 5% CO ₂ mixed air conditions. For survival 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). Antibodies of various concentrations were pretreated with cells for 30 min, followed by 20 ng / ml VEGF (R & D systems, USA). After 48 hours of incubation, WST-8 (Dojindo, Japan) was treated for 2 hours and the absorbance at 450 nm was measured to compare cell proliferation capacity under each condition.

As a result, through the cell proliferative assay for HUVEC cells, the dual target antibody PMC-102FC inhibited the proliferative capacity of HUVEC cells caused by VEGF similarly to the parental antibody Tanivirumab, and simultaneously blocked the effects of HGF, resulting in VEGF. In the case of simultaneous treatment with and HGF, it was confirmed that the proliferative capacity of HUVECs could be more strongly inhibited than that of Tanivirumab (FIGS. 6A and 6B).

Example 6 Inhibition of Phosphorylation of Intracellular c-Met or VEGFR-2 by Western Blotting

6-1. Cancer cell specific c-Met phosphorylation inhibition

Western blotting was performed to confirm the inhibition of phosphorylation on c-Met of the dual target antibody PMC-102FC according to the present invention. KP4 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum for 24 hours at a density of 5 × 10 ⁵ cells / well in 6-well plates. Subsequently, KP4 was changed to RPMI-1640 medium containing no fetal bovine serum, followed by incubation for 6 hours, 10 μg / ml or 20 μg / ml PMC-102FC, 10 μg / ml human IgG, and negative control Tanibirumab. The positive control Fc-M15 was pretreated for 30 minutes. Thereafter, 30 ng / ml recombinant human HGF was treated for 15 minutes.

For analysis by western blotting, sample buffer (1% (w / v) SDS, 1 mM Na3VO4, 1X Protease Inhibitor Cocktail) was treated to obtain lysate and quantitated by BCA quantification. Β-mercaptoethanol was added to the quantitative lysate and boiled for 10 minutes. SDS-PAGE and Western blotting were applied according to the methods 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), anti-phospho-Erk antibody, anti-phospho-c-Met antibody (Cell Signaling technology, USA) as primary antibody 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 with anti-β actin antibody (Abfrontier, Korea) and as primary antibody for chemiluminescence rabbit IgG (Santa Cruz Biotechnology, USA).

As a result, it could be confirmed that PMC-102FC can effectively inhibit c-Met phosphorylation in KP4 cells, which are pancreatic cancer cells (FIG. 7). Since phosphorylation of C-Met proceeds dependent on HGF, in the control group (Mock), c-Met phosphorylation was confirmed in HGF-treated KP4 cells. On the other hand, in the 10 μg / ml or 20 μg / ml PMC-102FC treated group, c-Met phosphorylation was not confirmed even in cells treated with HGF.

6-2. Phosphorylation of c-Met and VEGFR-2 in Vascular Endothelial Cells

Inhibition of Intracellular VEGFR-2 Phosphorylation by Western Blotting

Western blotting was performed to confirm the inhibition of phosphorylation on VEGFR-2 of the dual target antibody PMC-102FC according to the present invention. After treatment with the dual target antibody PMC-102FC, Western blotting was performed to confirm the change in cell signaling of vascular endothelial cells. Vascular endothelial cells (HUVEC) cultured for 24 hours were cultured for 6 hours in M199 medium containing 1% fetal bovine serum, followed by 10 μg / ml PMC-102FC antibody, positive control Tanivirumab and Fc-M15, negative control. hIgG antibody was pretreated for 20 minutes. Thereafter, 10 ng / ml VEGF and 30 ng / ml hHGF were treated for 15 minutes, and the cells were lysed and used for western blot.

Example 6-1 was applied mutatis mutandis, but c-Met activation for Fc-M15 as a result of Western blotting using an additional anti-VEGFR-2 antibody (Cell Signaling technology, USA) as a primary antibody. By inhibiting only, it appears to partially inhibit Erk, but does not inhibit the phosphorylation of VEGFR-2, while PMC-102FC can be equivalent to, or more effectively, inhibit the phosphorylation of VEGFR-2 and Erk in the case of PMB-102FC. It could be confirmed (Fig. 8).

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, the effect of mass production of c-Met neutralizing domain, which was not easy to produce in the past, can be expected.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Electronic file attached.

Claims (12)

1. Of the light chain variable region or the heavy chain variable region of VEGFR-2 (Vasicular Endothelial Growth Factor Receptoer-2) neutralizing antibody having a light chain variable region having an amino acid sequence represented by SEQ ID NO: 1 and a heavy chain variable region represented by SEQ ID NO: 2 A dual target antibody having binding to VEGFR-2 and c-Met to which a c-Met (Mesenchymal Epithelial Transition factor) neutralizing peptide represented by SEQ ID NO: 3 or SEQ ID NO: 4 is bound at its terminal.
2. The dual target antibody of claim 1, wherein the VEGFR-2 neutralizing antibody is scFV (Single Chain Fragment Variable) or IgG.
3. The dual target antibody of claim 1, wherein the c-Met neutralizing peptide and the light chain variable region or heavy chain variable region of the VEGFR-2 neutralizing antibody are linked through a linker.
4. The c-Met neutralizing peptide is a dual target antibody, characterized in that connected to the N-terminal or C-terminus of the light chain variable region or heavy chain variable region of the VEGFR-2 neutralizing antibody.
5. DNA encoding the dual target antibody of any one of claims 1-4.
6. The heavy chain of the VEGFR-2 neutralizing antibody represented by SEQ ID NO: 7, wherein the DNA encodes a fragment in which the light chain variable region of the VEGFR-2 neutralizing antibody represented by SEQ ID NO: 6 is bound to the c-Met neutralizing peptide. DNA comprising a DNA encoding the variable region.
7. A recombinant vector containing the DNA of claim 5.
8. A host cell transformed with the recombinant vector of claim 7.
9. A method for preparing a dual target antibody having binding to VEGFR-2 and c-Met, comprising the following steps:

   (a) culturing the host cell of claim 8 to produce a dual target antibody having binding to VEGFR-2 and c-Met; And

   (b) obtaining a dual target antibody having binding to the generated VEGFR-2 and c-Met.
10. A pharmaceutical composition for inhibiting angiogenesis, comprising the dual target antibody of any one of claims 1 to 4.
11. A pharmaceutical composition for treating cancer, comprising the dual target antibody of any one of claims 1 to 4.
12. The method of claim 11, 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, bone metastasis cancer of the prostate cancer, ovarian cancer Pharmaceutical composition, characterized in that selected from the group consisting of.

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