KR20130127366A - Double-targeting protein comprising igg antibody specific to vegf and protein specific to dll4 and use thereof - Google Patents

Abstract

The present invention relates to a double target protein in which an antibody specifically binding to VEGF in IgG form and a protein specifically binding to DLL4 are linked by a linker, and specifically, by imparting a disulfide bond inside DLL4 of the double target protein, A dual target protein having improved physical properties, a polynucleotide encoding the dual target protein, an expression vector comprising the polynucleotide, a transformant into which the vector is introduced, a method of preparing the dual target protein, and the dual target protein It relates to a composition for treating cancer, and a method of treating cancer using the composition.

Description

Double-targeting protein comprising IgG antibody specific to VEGF and protein specific to DLL4 and use A} that binds to IgG-binding antibody and DNA-binding protein

The present invention relates to a double target protein in which an antibody specifically binding to VEGF in IgG form and a protein specifically binding to DLL4 are linked by a linker, and specifically, by imparting a disulfide bond inside DLL4 of the double target protein, A dual target protein having improved physical properties, a polynucleotide encoding the dual target protein, an expression vector comprising the polynucleotide, a transformant into which the vector is introduced, a method of preparing the dual target protein, and the dual target protein It relates to a composition for treating cancer, and a method of treating cancer using the composition.

Angiogenesis refers to the process in which new blood vessels are formed in existing blood vessels through growth and division of vascular endothelial cells, and is highly related to normal human defense and physiological phenomena such as wound healing and inflammatory reactions, and early developmental processes. Plays an important role. However, abnormally excessive neovascularization induces tumor growth and provides nutrition and oxygen to cancer cells to allow metastasis of cancer cells. In addition to cancer, it is known to play a decisive role in diseases including other inflammatory reactions (Carmeliet and Jain, Nature, 407: 249, 2000).

The most important ligand in this neovascularization process is VEGF (Vascular endothelial growth factor), which is widely secreted in cancer cells. VEGF was purified by Genentech in 1989 for cDNA cloning and protein purification (Leung et al., Science, 246: 1306,1989). VEGF has a vascular endothelial growth factor receptor-1 (VEGFR-1) and a vascular endothelial growth factor receptor-2 (VEGFR-2) as receptors. Among these receptors, VEGFR-2, rather than VEGFR-1, is known to induce mechanisms related to angiogenesis. Therefore, the development of drugs to suppress the signaling process of VEGF has been made (Ellis and Hicklin, Nature Rev. Cancer, 8: 579, 2008; Youssoufian et al., Clin. Cancer Res., 13: 5544s, 2007). In particular, Genentech's Avastin (Bevacizumab) is already on the market with US FDA approval and is still in clinical trials with indications extended to solid carcinoma.

Notch signaling pathways are cell-to-cell communication systems used for many biological processes such as cell differentiation, proliferation and homeostasis. DLL4 (delta like ligand 4) binds Notch-1 and Notch-4 overexpressed in the vascular endothelium (Shutter et al., Genes Develop., 14: 1313, 2000). It has been reported that DLL4 binds to Notch present in endothelial cells and inhibits angiogenesis, and this result has been demonstrated as a report that the antibody that is a DLL4 antigen inhibits the growth of cancer cells. In other words, the DLL4 antibody acts as an opposite phenomenon to VEGF-Avastin in angiogenesis, but has anti-cancer effects and can be used as a cancer treatment (Blood. 2006 Feb 1; 107 (3): 931-939).

Dual-target antibodies targeting two targets based on IgG (Immnunoglobulin G) have been developed by several companies. The development of such double target antibodies has been developed in various forms, especially the light chian, but the heavy chain is modified by binding to other antigens, and then the amino acid of the CH3 domain of Fc is modified. Several double-target antibodies called 'Knobs and Holes', designed to be modified to form heterodimeric forms have been reported (Merchant et al., Nat. Biotechnol., 16: 677, 1998).

In addition to 'Knobs and Holes' type double target antibodies, scFv (Single-chain variable fragment) is expressed in the constant region of heavy and light chains of IgG and expressed in scFv-IgG form. They have the problems of low productivity and poor physical properties. In addition, diabody-type dual target antibodies have also been developed (Lu et al., J. Biol. Chem., 280: 19665, 2005), but this also has the disadvantage of low stability.

As such, several types of dual target antibodies reported so far have been published. However, depending on the purpose of use, each has shown the functional advantages and disadvantages.

Against this background, the present inventors have made diligent efforts to develop a dual target protein that has good productivity and physical properties but also exhibits excellent anticancer effects in vivo. As a result, the present inventors have suppressed DLL4 into which antibodies and disulfide bonds, which inhibit VEGF in IgG form, have been introduced. The double-targeted protein bound to scFv to improve not only the physical properties, but also showed excellent anti-cancer effect in vivo to complete the present invention.

One object of the present invention is a double target in which an antibody specifically binding to Vascular endothelial growth factor (VEGF) in the form of IgG (Immunoglobulin G) and a protein specifically binding to DLL4 (Delta-like ligand 4) are linked. To provide protein.

Another object of the present invention is to provide a polynucleotide encoding the dual target protein, an expression vector comprising the polynucleotide, or a transformant into which the vector is introduced.

It is another object of the present invention to provide a method for preparing the dual target protein.

Another object of the present invention is to provide a composition for treating cancer comprising the dual target protein and a pharmaceutically acceptable carrier.

It is another object of the present invention to provide a method of treating cancer using the composition.

As one embodiment for achieving the above object, the present invention specifically binds to antibodies and DLL4 (Delta-like ligand 4) specifically binding to Vascular endothelial growth factor (VEGF) in the form of IgG (Immunoglobulin G) To provide a dual target protein linked by a linker.

As used herein, the term "double-target protein" refers to a protein capable of binding two different kinds of antigens (target proteins), and does not exist naturally, and refers to a form prepared by genetic engineering or any method. Can mean. For the purposes of the present invention, the dual target protein may bind to VEGF overexpressed in cancer cells and DLL4 expressed in endothelial cells. The dual target protein may be in the form of an antibody. Dual-target proteins of the invention can be used interchangeably with dual-target antibodies. Preferably, the dual target protein of the present invention may have VEGF and DLL4 as antigens. The form of the double-target protein of the present invention is preferably a double-target protein in which an antibody specifically binding to VEGF in IgG form and a protein specifically binding to DLL4 are linked by a linker, and the structure thereof is schematically illustrated in FIG. As shown.

As used herein, the term “antibody” refers to a protein molecule that acts as a receptor for an antigen so as to specifically recognize an antigen including an immunoglobulin molecule that is immunologically reactive with a specific antigen. Polyclonal antibody, monoclonal antibody And antibody fragments. The term also includes forms produced by genetic engineering such as chimeric antibodies (eg humanized antibodies) and heterologous antibodies (eg dual target antibodies). The protein molecule is Y-shaped and consists of two identical light chains and two identical heavy chains, each of which comprises variable and fixed regions. These light and heavy chains are structured by disulfide bonds located in the hinge region. The heavy and light chain variable regions bind to form an binding site with the antigen, and may be an antibody having an isotype of IgA, IgD, IgE, IgG, IgT, IgY and IgM. Preferably the antibody that specifically binds to VEGF of the present invention may be in IgG form.

The term "antibody that specifically binds to VEGF" of the present invention includes any antibody that specifically binds to VEGF as an antigen in a wide range of cancer cells, preferably Bevacizu, a therapeutic antibody that targets VEGF. It may be Mab (Bevacizumab), but is not limited thereto. VEGF is a ligand that plays an important role in angiogenesis. When it is suppressed, angiogenesis is not achieved and cancer can be treated. Bevacizumab is a therapeutic antibody that can be stably used as approved by the US FDA as Avastin of Genentech. The antibody specifically binding to VEGF may include a heavy chain amino acid sequence defined in SEQ ID NO: 5 and a light chain amino acid sequence defined in SEQ ID NO: 6, but may specifically bind to VEGF to exhibit a cancer therapeutic effect. Sequences of proteins that can be included are included without limitation.

Since the antibody specifically binding to VEGF, which is a component of the dual target protein of the present invention, specifically binds to VEGF overexpressed in cancer cells, the dual target protein of the present invention can be concentrated on cancer cells expressing VEGF. In combination with VEGF, it may have anticancer activity by itself.

Antibodies that specifically bind to VEGF and antibodies that specifically bind to DLL4 in the dual target protein of the present invention retain their specific binding, and in particular, because they can simultaneously inhibit two targets (antigens), It may be more effective than inhibiting in combination with the target and may simultaneously inhibit two signals.

The term "antibody fragment" in the present invention encompasses an antigen-binding form of an antibody, including fragments having antigen binding ability, for example, Fab ', F (ab') 2, Fab, Fv and rIgG. The term also refers to a single-chain variavle fragment (scFv) and includes bivalent or bispecific molecules, diabodies, tribodies and tetrabodies.

As used herein, the term "protein that specifically binds to DLL4" refers to a protein that specifically binds to DLL4 and inhibits the growth of cancer, thereby exhibiting a cancer therapeutic effect, and binds to DLL4 with high affinity and DLL4 activity. It can play a role in neutralizing. The protein specifically binding to DLL4 may block DLL4 binding to Notch receptors, and may inhibit signaling by DLL4. The protein that specifically binds to DLL4 may be in the form of scFv. The protein specifically binding to DLL4 may include a heavy chain amino acid sequence defined in SEQ ID NO: 3 and a light chain amino acid sequence defined in SEQ ID NO: 2, but may specifically bind to DLL4 to exhibit a cancer therapeutic effect. Sequences of proteins that can be included are included without limitation.

As used herein, the term "single-chain variavle fragment" (scFv) refers to a minimum antibody fragment having a complete antigen-recognition and -binding site, comprising the V _H and V _L domains of an antibody, wherein the domain is a single Present in the polypeptide chain.

The protein that specifically binds to DLL4 may preferably have disulfide bonds inside scFv, and may have stability by improving physical properties by disulfide bonds. Preferably, at least one amino acid position is selected within the V _H and V _L framework residues of the scFv and substituted with cysteine to have a disulfide bond. More preferably, amino acid 44 of the scFv heavy chain and amino acid 100 of the light chain, which are proteins that specifically bind to DLL4, may be substituted with cysteine, respectively. The amino acid position number uses the Kabat numbering scheme. According to one embodiment of the present invention, the yield of the intact double-target protein was increased from 62.5% to 82.6% to confirm that the physical properties were improved over the double-target protein (WT) without disulfide bonds (FIG. 4), It was confirmed that the affinity or neutralization effect was not reduced by the improvement of such physical properties (FIGS. 5 to 7). In addition, as a result of the pharmacokinetic experiment, it was confirmed through animal experiment that the dual target protein of the present invention in which disulfide bond was introduced was higher in blood stability than the dual target protein in which disulfide bond was not introduced (FIGS. 8 and 9). These results indicate that the dual-target protein of the present invention has improved physical properties and yields, and the dual-target protein mass produced by the increased yield has similar affinity and neutralization effect as the dual-target protein without cysteine. The increased stability in the blood supports the use in cancer treatment.

The double target protein may be a double target protein in which an antibody specifically binding to VEGF and a protein specifically binding to DLL4 are linked by a linker, and preferably the C- of the Fc region of the antibody specifically binding to VEGF. Proteins that specifically bind to the terminal and DLL4 can be linked by a linker.

As used herein, the term "linker" basically means two different fusion partners (e.g., a biological polymer, etc.) to hydrogen bonds, electrostatic interactions, van der Waals forces, disulfide bonds, salt bridges, It refers to a linker that can be linked using hydrophobic interactions, covalent bonds, etc., preferably to at least one disulfide bond under physiological conditions or other standard peptide conditions (eg, peptide purification conditions, peptide storage conditions). A hinge that can have at least one cysteine that can participate and, in addition to simply connecting each of the fusion partners, serves to provide a certain amount of spacing between the fusion partners or to provide flexibility or rigidity to the fusions. It can also play the role of). The linker may be a non-peptide linker or a peptide linker, and includes all directly linked by peptide bonds, disulfide bonds and the like. In the present invention, the linker is not particularly limited thereto. Preferably, the linker encodes a polypeptide capable of linking an antibody specifically binding to VEGF and a protein specifically binding to DLL4 or an antibody specifically binding to the VEGF. It may be a polypeptide encoded by a polynucleotide capable of linking a gene to a gene encoding a protein that specifically binds to DLL4, more preferably C- of the Fc region of the antibody that specifically binds to the VEGF It may be a peptide linker capable of linking the terminal and the N-terminus of a protein that specifically binds to DLL4, and more preferably, may be a peptide linker consisting of an amino acid sequence of the form repeated three times the GGGGS motif. The GGGGS motif may be repeated 1 to 10 times, most preferably composed of the amino acid sequence encoded by the following amino acid sequence of SEQ ID NO: 1 or polynucleotide sequence of SEQ ID NO: 4.

Linker peptide: GGGGSGGGGSGGGGS (SEQ ID NO: 1)

Linker Polynucleotide: GGTGGAGGTGGCAGCGGTGGTGGCGGCAGTCCCGGTGGCGGCTCC (SEQ ID NO: 4)

As used herein, the term "non-peptide linker" refers to a biocompatible linker in which two or more repeating units are linked, and the repeating units are linked to each other through any covalent bonds, not peptide bonds.

Non-peptide linkers usable in the present invention include polyethylene glycol (PEG) homopolymers, polypropylene glycol homopolymers, ethylene glycol-propylene glycol copolymers, polyoxy ethylated polyols, polyvinyl alcohols, polysaccharides, dextran , Biodegradable polymers such as polyvinyl ethyl ether, lipid polymers, chitin, hyaluronic acid or combinations thereof. Preferably, it is a polyethylene glycol homopolymer. Derivatives thereof known in the art and derivatives which can be easily prepared at the technical level in the art are included in the scope of the present invention. More preferably, it is a polyethylene glycol homopolymer having a molecular weight of 1 to 5 kDa, most preferably about 3.4 kDa, and is bound to the amine group of the polypeptide amino terminal in the form of bifunctional aldehyde at both ends, It is possible to link an antibody that specifically binds to a protein that specifically binds to DLL4. In particular, having a reactor of reactive aldehyde groups at both ends is effective for minimizing nonspecific reactions.

Sites directly or indirectly linked through the linker are not particularly limited thereto, and may be Fc moieties, Fab ′, F (ab ′) 2, Fab, Fv, and the like. The dual target protein is not particularly limited thereto, but a form in which all or a portion of an antibody specifically binding to VEGF and all or a portion of a protein specifically binding to DLL4 are linked; Alternatively, all or a portion of the antibody specifically binding to VEGF and all or a portion of the protein specifically binding to DLL4 may be linked by a peptide linker.

In addition, all or part of the heavy chain of the antibody that specifically binds to VEGF and all or part of a protein that specifically binds to DLL4 is linked to the peptide linker; All or a portion of the light chain of the antibody that specifically binds to VEGF and all or a portion of the protein that specifically binds to DLL4 are linked by peptide linkers; Or a combination thereof.

According to one embodiment of the present invention, the inventors insert a polynucleotide encoding a double-target protein linking the heavy chain region C-terminus of Avastin in IgG form and DLL4 binding protein in scFv form with a linker of SEQ ID NO: 1 into a vector. One or more amino acid positions in the V _H and V _L framework residues of the scFv of the DLL4 binding protein fused to the C-terminus of the heavy chain region C-terminus of Avastin are substituted with cysteine and introduced into the animal cell to introduce an Avastin-DLL4 binding double target. Isolation of the protein confirmed the expression and purity confirmed that the improved physical properties (Fig. 3 and 4). In addition, it was confirmed that the Avastin-DLL4 binding dual target protein specifically binds to the target VEGF and DLL4 (FIG. 5), and the neutralizing effect on DLL4 (FIG. 6), and improved physical properties due to disulfide binding. It was confirmed that there is no change such as reducing affinity or neutralizing ability by (Fig. 7). In addition, it was confirmed that the stability of blood was increased through pharmacokinetic experiments in animals (FIGS. 8 and 9).

In another aspect, the present invention provides a polynucleotide encoding the dual target protein, an expression vector comprising the polynucleotide, and a transformant into which the expression vector is introduced.

Expression vectors comprising the polynucleotide encoding the dual target protein provided in the present invention is not particularly limited, mammalian cells (eg, human, monkey, rabbit, rat, hamster, mouse cells, etc.), plant cells May be a vector capable of replicating and / or expressing the polynucleotide in eukaryotic or prokaryotic cells, including yeast cells, insect cells or bacterial cells (e.g., E. coli, etc.), preferably in the host cell It may be a vector operably linked to an appropriate promoter for expression of the polynucleotide, and may comprise a vector comprising at least one selection marker, more preferably phage, plasmid, cosmid, mini-chromosome, virus, retroviral vector It may be a form in which the polynucleotide is introduced.

The transformant introduced with the expression vector provided by the present invention is not particularly limited thereto, but the bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium transformed by introducing the expression vector; Yeast cells; Fungal cells such as Pichia pastoris; Insect cells such as Drosophila and Spodoptera Sf9 cells; Animal cells such as CHO, COS, NSO, 293T, and Bowmanella cells; Or plant cells. According to one embodiment of the present invention, CHO-S cells were used as host cells.

As used herein, the term "introduction" refers to a method of delivering a vector comprising a polynucleotide encoding a dual target protein to a host cell. Such introduction may be accomplished by methods such as calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposomal fusion, lipofectamine and protoplast fusion Can be carried out by various methods known in the art. In addition, transduction refers to the delivery of the desired product into cells using viral particles by means of infection. In addition, vectors can be introduced into host cells by gene bombardment or the like. Introduction in the present invention can be used in combination with transformation.

In another aspect, the present invention provides a method for preparing the dual target protein.

Preferably (a) obtaining a polynucleotide encoding an antibody specifically binding to VEGF and a polynucleotide encoding a protein binding to DLL4; (b) 3'-terminus of the polynucleotide encoding the Fc region of the polynucleotide encoding the antibody specifically binding to VEGF obtained in step (a) and 5 of the polynucleotide encoding the protein binding to DLL4 Linking the '-terminus with a linker to obtain a polynucleotide encoding a dual target protein; (c) preparing an expression vector by cloning the polynucleotide encoding the dual target protein of step (b); (d) culturing the transformant by introducing the expression vector of step (c) into a host cell; And (e) may be a method comprising the step of recovering the dual target protein from the transformant of step (d). Step (a) or step (b) may further comprise the step of replacing any amino acid of the protein that binds DLL4 with cysteine.

Preferably (a) obtaining a polynucleotide encoding an antibody specifically binding to VEGF and a polynucleotide encoding a protein binding to DLL4; (b) cloning the polynucleotide of step (a) to prepare an expression vector; (c) culturing the transformant by introducing the expression vector of step (b) into a host cell; And (d) obtaining an antibody and a protein binding to DLL4 specifically binding to VEGF from the transformant of step (c), to the C-terminus and DLL4 of the Fc region of the antibody specifically binding to VEGF. It may be a method comprising the step of linking the N-terminus of the binding protein with a linker. Step (a) or step (b) may further comprise the step of replacing any amino acid of the protein that binds DLL4 with cysteine.

The dual target protein of the present invention can be prepared by any of the above known recombinant means, and the antibody can be introduced into an appropriate host cell and recovered from the culture of the transformant.

Preferably, the dual target protein can be separated by known separation methods, such as conventional immunoglobulin purification procedures such as protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. It is appropriately separated from the culture medium by, but is not limited thereto.

In another aspect, the present invention provides a composition for treating cancer comprising the dual target protein and a pharmaceutically acceptable carrier.

In the present invention, the term "cancer" includes, without limitation, the type of cancer, for example esophageal cancer, stomach cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovary Cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, lymphoma, or multiple myeloma hematoma cancer.

As used herein, the term "treatment" means any action that improves or advantageously changes the symptoms caused by cancer by administration of the composition.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate the organism and does not interfere with the biological activity and properties of the administered compound. Examples of the pharmaceutical carrier that is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

According to an embodiment of the present invention, the dual target protein of the present invention binds to both VEGF and DLL4 (FIG. 5), and can be neutralized DLL4 (FIG. 6) to be used as an active ingredient for cancer treatment compositions. Confirmed that it can.

In another aspect, the present invention provides a method for treating cancer using a pharmaceutical composition comprising the dual target protein.

The dual target protein may be a method for treating cancer, comprising administering a pharmaceutical composition further comprising a pharmaceutically acceptable carrier to an individual with or suspected of having cancer, and which may be used as a carrier and The cancer is the same as described above. The subject includes mammals, birds, etc., including cattle, pigs, sheep, chickens, dogs, humans and the like, and the subject to which the cancer is treated by administration of the composition of the present invention is not limited.

Here, the composition may be administered in single or multiple doses in a pharmaceutically effective amount. At this time, the composition may be administered in the form of a liquid preparation, a powder, an aerosol, a capsule, an intravaginal tablet, a capsule, or a suppository. Routes of administration include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, endothelial administration, oral administration, topical administration, intranasal administration, pulmonary administration, rectal administration, and the like. However, upon oral administration, the peptide will be digested and the oral composition should be formulated to coat the active agent or protect it from degradation from above. In addition, the pharmaceutical composition may be administered by any device in which the active agent may migrate to the target cell.

A pharmaceutical composition comprising the dual target protein of the present invention is administered in a pharmaceutically effective amount. “Pharmaceutically effective amount” means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and an effective dose level refers to the severity of the disease, the activity of the drug, the age of the patient, Body weight, health, sex, sensitivity to the drug of the patient, time of administration of the composition of the invention used, route of administration and rate of release, duration of treatment, elements including drugs used or combined with the composition of the invention used and other medical fields Can be determined according to well-known factors. In addition, the composition for treating cancer of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

The dual target protein of the present invention can treat cancer by simultaneously binding to VEGF and DLL4, and disulfide bond is introduced by replacing cysteine inside DLL4 to obtain a protein having a more stable structure in the conventional physical properties. Not only does not deviate significantly from the range of existing proteins in the blood, but also increases the stability in the blood, which can be used for the production of more effective dual target proteins and cancer treatment.

1 is a schematic diagram showing the structure of a dual target protein fused with a protein capable of binding Avastin IgG and DLL4.
Figure 2 shows the amino acid sequence of the DLL4 binding protein in the Avastin-DLL4 binding dual target protein inserted into the vector. The box portion shows the linker peptide of SEQ ID NO: 1 connecting the light and heavy chains in the scFv type DLL4 binding protein.
Figure 3 shows the SDS-PAGE results of the purified dual target protein.
4 shows SEC-HPLC results of purified dual target proteins.
Figure 5 shows the results of the analysis of the binding assay for DLL4 by ELISA.
Figure 6 shows the results of the analysis of the neutralizing effect (neutralizing assay) for DLL4 by ELISA.
Figure 7 shows the results of the BIACORE assay of the purified dual target protein.
8 shows the results of pharmacokinetic parameter analysis of purified dual target proteins.
9 is a graphical representation of the results of pharmacokinetic analysis.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example  One: Avastin  ( Avastin ) - DLL4  Preparation of Binding Dual Target Proteins

Genes encoding the heavy and light chains of the Avastin-DLL4 binding double target protein and the DLL4 binding protein were newly designed using Genentech's gene sequence for the purpose of producing Avastin-DLL4 binding double target protein in CHO-S mammalian cell line. It was. Specifically, based on the sequence, the amino acid sequence of SEQ ID NO: 5, the Avastin heavy chain sequence, the amino acid sequence of SEQ ID NO: 6, the Avastin light chain sequence, the amino acid sequence of SEQ ID NO: 7, the DLL4 heavy chain sequence, and the amino acid sequence of SEQ ID NO: 8, the DLL4 light chain sequence Was designed.

Expression of nucleic acids encoding fusion antibodies can be carried out by conventional methods in prokaryotic or eukaryotic cells, and eukaryotic host-vector systems can be performed using a wide range of vectors for different species. As a representative vector for amplifying DNA, a pcDNA3.1 vector was used, and again, a vector capable of expressing an antibody protein, which encodes an antibody polypeptide in a CHO-S mammalian cell line, was obtained through gene recombination.

To design a gene capable of expressing Avastin-DLL4 binding dual target protein, Avastin was first set to IgG type and DLL4 binding protein was set to scFv type. The linker peptide connecting the heavy chain region C-terminus of Avastin to the DLL4 binding protein was linked using GGGGSGGGGSGGGGS (SEQ ID NO: 1). The gene amino acid sequence of the Avastin-DLL4 binding dual target protein inserted into the vector is shown in FIG. 2.

One or more amino acid positions in the V _H and V _L framework residues of the scFv fused at the C-terminus were selected for cysteine substitution in the scFv light and heavy chains of the DLL4 binding protein. At this time, the sequence number of the amino acid was used Kabat numbering system. Specifically, VL43 / VH105 is located at positions 43 and 105, respectively, in the V _L and V _H framework residues, VL100 / VH44 is located at positions 100 and 44, respectively, and VL46 / VH101 are located at positions 46 and 101, respectively, and VL100 / VH101 Denotes the substitution of cysteine at positions 100 and 101, respectively.

For antibody production, floating animal cells transfected with the gene using a polymer that enhances the efficiency of intracellular gene transfer were cultured at 250 ml per bottle in a 500 ml culture Erlenmeyer flask (Corning Costa) and a total of 1 L was obtained. Incubated.

1 L of a mixture of RPMI medium (Invitrogen Corp.) and CHO cell medium containing ultra low IgG fetal bovine serum (Invitrogen Corp.) was cultured in a cell culture medium (Sanyo) for 4 days. Recombinant protein was produced. The cell culture was obtained and subjected to centrifugation to separate the supernatant containing the suspended cells and the secreted recombinant protein and filter once with a 0.22 쨉 m vacuum filter device (Millipore, Maryland, USA).

For antibody purification, Avastin-DLL4 binding dual target protein was purified from the culture using recombinant Protein-A Sepharose column (Hitrap MabSelect Sure, 5 mL, GE healthcare). Specifically, the filtered culture medium was loaded onto a recombinant protein-A Sepharose column. The column was washed with 20 mM column volume with 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, and impurities were washed with 10 mM column volume of 50 mM Na-citrate, pH 5.0. The antibody was eluted with 5 mM Na-citrate 10 mM NaCl (pH 3.4) and neutralized with 1M Tris-HCl, pH 8.0. Fractions were analyzed by SDS-PAGE and the positive fractions were collected and concentrated in a centrifugal concentrator (Amicon Ultra, 30,000 MWCO, Millipore, Bedford, MD). Buffer exchange and concentration were performed with phosphorylated formulation buffer using the same centrifugal concentrator. Finally, the antibody was sterile filtered with a syringe filter having a pore size of 0.22 mu m and the absorbance (A280) was measured to determine the antibody concentration.

Example  2: term VEGF - DLL4  Identification and improvement of physical properties of dual target antibodies

The expression of the Avastin-DLL4 binding dual target protein product was confirmed by SDS-PAGE. SDS-PAGE was applied according to the methods commonly used in the art, and the samples used were as follows: 4-10% NuPAGE Gel (Invitrogen, USA), 20X MOPS buffer (Biorad, USA), Coomassie blue staining buffer (Biorad, USA).

As a result, expression of the Avastin-DLL4 binding dual target protein product was confirmed (FIG. 3). WT is a form that does not introduce disulfide bonds in dual target proteins.

And the purity of the antibody was measured by SEC-HPLC analysis (Fig. 4). WT is a form that does not introduce disulfide bonds in dual target proteins.

The above results indicate that when the Avastin-DLL4 binding double target protein of the present invention is obtained, the amount of the double target protein incorporating disulfide bond is higher than that of the conventional WT, and the purity thereof is high. . Yield here means the amount of the dual target protein in its intact and pure form.

Example  3: Avastin - DLL4  Of binding double-target proteins Cohesion Assay

A binding assay to determine whether Avastin-DLL4 binding dual target protein can bind VEGF and DLL4 was performed using an enzyme-linked immunosorbent assay (ELISA).

For this purpose, 100 ng of VEGF and DLL4 were respectively dispensed into the wells in 96-well plates, coated at room temperature for 1 day, and then reacted at 37 ° C. for 2 hours using 1% BSA / PBS. After the reaction plate was washed with PBS, the pre-reacted dual-target protein mixture for 1 hour at room temperature was put in wells coated with various concentrations (5 nM ~ 0 nM), and reacted for 2 hours at room temperature. After 2 hours, the cells were washed with PBST, and then HRP-conjugated goat anti-Kappa light chain antibody was added at a dilution of 1: 10000 as a secondary antibody to the antibody, and reacted at 37 ° C. for 1 hour. . Thereafter, the color reaction was induced using a TMB substrate reagent (BD Biosciences # 555214, USA), and 50 µl of 2N sulfuric acid (H ₂ SO ₄ ) solution was added to stop the color reaction. The color reaction was measured at absorbance 450 nm and 650 nm using a microplate reader (Tecan, Switzerland) (FIG. 5). WT is a form that does not introduce disulfide bonds in dual target proteins.

As a result, it was confirmed that the VL100 / VH44 dual target protein was able to bind to both VEGF and DLL4 similarly to the control WT, and that other dual target antibodies could bind to both VEGF and DLL4 (FIG. 5). ).

Example  4: Avastin - DLL4  Neutralizing Effect of Binding Dual Target Proteins Assay

A neutralization assay was performed using ELISA to determine whether Avastin-DLL4 binding dual target protein had a neutralizing effect on DLL4.

For this purpose, 40 ng of Notch1 was dispensed into the wells in 96-well plates and coated at room temperature for one day, and then reacted at 37 ° C. for 2 hours using 1% BSA / PBS. After the reaction plate was washed with PBS, a mixture of the antibody and DLL4 (600 ng / ml) pre-reacted for 1 hour at room temperature was put in wells coated with various concentrations (17 pM to 70 nM), and immediately using PBST. Washed by. Thereafter, HRP-conjugated HisX6 antibody was diluted 1: 700 in order to detect the protein, reacted at 37 ° C. for 2 hours, and then a color reaction was induced using TMB substrate reagent (BD Biosciences # 555214, USA). . Thereafter, 50 µl of 2N sulfuric acid (H ₂ SO ₄ ) solution was added to stop the color reaction. The color reaction was measured at absorbance 450 nm and 650 nm using a microplate reader (Tecan, Switzerland) (FIG. 6). WT is a form that does not introduce disulfide bonds in dual target proteins.

As a result, it was confirmed that the Avastin-DLL4 binding dual target protein can neutralize DLL4 similarly to the control WT (FIG. 6). These results support the anti-cancer activity by supporting that the dual target protein of the present invention can neutralize DLL4.

Example  5: Avastin - DLL4  Of binding double-target proteins Disulfide  Analysis of binding capacity difference by binding

BIACORE assay was performed to investigate the difference in binding ability of the Avastin-DLL4 binding dual target protein according to disulfide binding.

For SPR analysis, T200 was used, and running buffer was HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.15% surfactant P20). Surface preparation was carried out by diluting the ligand hDLL4 to 10 μg / ml in 10 mM sodium acetate pH 4.5 using the surface preparation_target immobilization (condition: 25 ° C., 5 μl / min) of the wizard program. It fixed as much as the target immobilization level. Immobilization consists of two flow paths (Fc, flow cell) in one set. In this experiment, the first flow path was designated as blank and the second flow path was fixed with hDLL4. The first channel is a reference that shows the nonspecific binding and the change by the buffer. The experimental result uses the value of Fc2-Fc1. Avastin IgG-DLL4 scFv and Avastin IgG-DLL4 scFv (disulfide stabilization structure) were converted into molar concentration by diluting with 100 nM in running buffer, and then serially diluted 1/2 for analysis at 5 concentration intervals. It was. The analytical sample was prepared in high purity / concentration so as to have a minimum dilution multiple of 100 or more to minimize the buffer effect. All analyzes were performed in multiples of one sample using the Wizard program, with regeneration steps between all analyzes to ensure that the baseline of the experiment was constant. The results were analyzed by Biaevaluation software 4.0 version. In Fc2-Fc1 and Fc4-Fc3 results, baseline was set to 0, and then buffer injection (analyte 0 nM) was removed from the entire sensorgram. The results were analyzed by binding affinity using the 1: 1 binding model. The analysis items are k _a (M ^-1 s ^-1 ), k _d (s ^-1 ), K _a (1 / M), and K _D (M). k _a is an association constant indicative of affinity and k _d is a dissociation constant indicative of stability. Equilibrium dissociation constant K _D (M) was converted to k _d / k _a (FIG. 7). WT is a form that does not introduce disulfide bonds in dual target proteins.

As a result, it was confirmed that the WT without the disulfide bond was similar in the affinity or neutralization effect of the dual target protein having the disulfide bond introduced with improved physical properties (FIG. 7).

Example  6: Avastin - DLL4  Of binding double-target proteins Disulfide  In rodents by combination Pharmacokinetics  Difference analysis

To determine pharmacokinetics in mice following disulfide binding of Avastin IgG-DLL4 scFv (a form without disulfide bonds in Avastin IgG-DLL4 dual target protein) and Avastin IgG-DLL4 scFv (disulfide stabilization) To this end, 2.5 mg / kg was administered once intraperitoneally to five animal groups of Balb / c magnetic mice (6-7 weeks). Blood collection was performed at different time points, 0, 0.5, 1, 2, 6, 24, 48, 96, 168, 216, 264, 336 hours, based on the time of administration (0 hour). All blood samples were collected from the tail vein by tail cut and the volume of blood was 50 ~ 80 μl. The collected samples were centrifuged for 20 minutes and serum was stored at -70 ° C until dialysis. The concentration of the antibody was measured by ELISA.

For ELISA analysis, 100 ng of VEGF and DLL4 were respectively dispensed into the wells in 96-well plates, coated at 4 ° C. for 14hr, and reacted at 37 ° C. for 2 hours using 1% BSA / PBS. After the reaction plate was washed with PBS, 100 μl of a standard or sample dilution per well was added to the ELISA plate and reacted at room temperature for 2 hours. After 2 hours, the cells were washed with PBST, and then HRP-conjugated goat Anti-Human IgG [F (ab ') 2] antibody was added at a dilution of 1: 10000 as a secondary antibody to the antibody. The reaction was carried out at 1 ° C. for 1 hour. Thereafter, the color reaction was induced using a TMB substrate reagent (BD Biosciences # 555214, USA), and 50 µl of 2N sulfuric acid (H ₂ SO ₄ ) solution was added to stop the color reaction. The color reaction was measured at 450 nm and 650 nm of absorbance using a microplate reader (Tecan, Switzerland).

The results of the samples were calculated from standard curves fitted using a four-parameter logistic curve fit (half-life determinations from the mean plasma concentration curves to concentrations in the X-axis and the mean of the absorbance results in the Y-axis). The 4-parameter equation was used to indirectly determine the dual-target protein content in the sample.All samples, including STDs, proceeded to duplicates and used mean values with CV (%) within 20%, and regression coefficients of STDs ( R2) value was based on 0.98 or more).

As a result of pharmacokinetic parameter analysis, the AUCstin, Cmax, t1 / 2 and tmax of Avastin-IgG DLL4 were 2380.4625 hr * μg / ml, 32.28 μg / ml, 176.7 hr and 2 hr in rhVEGF coated ELISA, and Avastin-IgG DLL4 cc The AUClast, C0, t1 / 2 and tmax of the bond (disulfide stabilized structure) were 1932.8625 hr * μg / ml, 29.83 μg / ml, 172.4 hr and 3 hr, respectively. > 0.05). In the rhDll4 coating ELISA, the AUClast, Cmax, t1 / 2 and tmax of Avastin-DLL4 were 1723.7250 hr * μg / mL, 25.53 μg / mL, 78.9 hr and 2 hr, respectively, and Avastin-IgG DLL4 cc bond (disulfide stabilized structure) AUClast, Cmax, t1 / 2 and tmax were 2218.9688 hr * μg / mL, 41.58 μg / mL, 105.9 hr and 3 hr, respectively. The results of rhDll4 coating ELISA showed no difference in Cmax (p = 0.063619) and AUClast (p = 0.094814) values, but Avastin-IgG DLL4 cc bond showed higher value than Avastin-IgG DLL4. (FIG. 8 and FIG. 9). Therefore, the dual target protein with disulfide bond is thought to enhance the blood stability of the drug. In other words, these results support that the dual target protein incorporating the disulfide bond with improved physical properties of the present invention is more stable in the treatment of cancer than WT without disulfide bond.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> HANWHA CHEMICAL CORPORATION <120> Double-targeting protein comprising IgG antibody specific to VEGF          and protein specific to DLL4 and use <130> PA130301 / KR <150> 10-2012-0051168 <151> 2012-05-14 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 1 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser   1 5 10 15 <210> 2 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> VL of DLL4 <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala              20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile          35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Ser Ser Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Gly Thr Val                  85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg             100 105 <210> 3 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VH of DLL4 <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Asn              20 25 30 Trp Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Tyr Ile Ser Pro Asn Ser Gly Phe Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Asp Asn Phe Gly Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 4 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Linker <400> 4 ggtggaggtg gcagcggtgg tggcggcagt cccggtggcg gctcc 45 <210> 5 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of Avastin <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr              20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe      50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val             100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly         115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly     130 135 140 Thr Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe                 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val             180 185 190 Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val         195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys     210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr                 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val             260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val         275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser     290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala                 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro             340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln         355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala     370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu                 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser             420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser         435 440 445 Leu Ser Pro Gly Lys     450 <210> 6 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Light chain of Avastin <400> 6 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr              20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile          35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Ser Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp                  85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 7 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of DLL4 <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Asn              20 25 30 Trp Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Tyr Ile Ser Pro Asn Ser Gly Phe Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Asp Asn Phe Gly Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Ser Ser Val Phe Pro         115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly     130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln                 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Ser Ser             180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser         195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr     210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg                 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Ser Glu Asp Pro             260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala         275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val     290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr                 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu             340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys         355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser     370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser                 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala             420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 8 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Light chain of DLL4 <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala              20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile          35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Ser Ser Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Gly Thr Val                  85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210

Claims (19)

A double-target protein in which an antibody specifically binding to VEGF (Vascular endothelial growth factor) in the form of IgG (Immunoglobulin G) and a protein specifically binding to DLL4 (Delta-like ligand 4) are linked.
The dual target protein of claim 1, wherein the protein specifically binding to DLL4 is in the form of a single-chain variable fragment (scFv).
The dual target protein according to claim 2, which has a disulfide bond inside scFv, which is a protein that specifically binds to DLL4.
The double target protein of claim 3, wherein the disulfide bond is substituted with cysteine at amino acid 44 of the scFv heavy chain and amino acid 100 of the light chain, respectively, which are proteins that specifically bind to DLL4.
The dual target protein of claim 1, wherein the dual target protein is linked to a C-terminus of the Fc region of the antibody that specifically binds VEGF, and a protein that specifically binds to DLL4 by a linker.
The dual target protein of claim 1, wherein the linker is a non-peptide linker or a peptide linker.
The dual target protein of claim 6, wherein the peptide linker is a peptide of SEQ ID NO: 1.
7. The non-peptide linker of claim 6, wherein the non-peptide linker is a polyethylene glycol homopolymer, a polypropylene glycol homopolymer, ethylene glycol-propylene glycol copolymer, polyoxy ethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl A dual target protein selected from the group consisting of ethers, biodegradable polymers, lipid polymers, chitin, hyaluronic acid and combinations thereof.
The dual target protein of claim 1, wherein the antibody that specifically binds VEGF is Bevacizumab.
The dual target protein of claim 1, wherein the antibody specifically binding to VEGF comprises a heavy chain amino acid sequence defined by SEQ ID NO: 5 and a light chain amino acid sequence defined by SEQ ID NO: 6. 8.
The dual target protein of claim 1, wherein the protein specifically binding to DLL4 comprises a heavy chain amino acid sequence defined by SEQ ID NO: 3 and a light chain amino acid sequence defined by SEQ ID NO: 2. 6.
A polynucleotide encoding the dual target protein of any one of claims 1-11.
An expression vector comprising the polynucleotide of claim 12.
A transformant into which the vector of claim 13 is introduced.
(a) obtaining a polynucleotide encoding an antibody specifically binding to VEGF and a polynucleotide encoding a protein binding to DLL4;
(b) 3'-terminus of the polynucleotide encoding the Fc region of the polynucleotide encoding the antibody specifically binding to VEGF obtained in step (a) and 5 of the polynucleotide encoding the protein binding to DLL4 Linking the '-terminus with a linker to obtain a polynucleotide encoding a dual target protein;
(c) preparing an expression vector by cloning the polynucleotide encoding the dual target protein of step (b);
(d) culturing the transformant by introducing the expression vector of step (c) into a host cell; And
(e) A method for producing the dual target protein of any one of claims 1 to 11, comprising recovering the dual target protein from the transformant of step (d).
(a) obtaining a polynucleotide encoding an antibody specifically binding to VEGF and a polynucleotide encoding a protein binding to DLL4;
(b) cloning the polynucleotide of step (a) to prepare an expression vector;
(c) culturing the transformant by introducing the expression vector of step (b) into a host cell; And
(d) obtaining the antibody specifically binding to VEGF and the protein binding to DLL4 from the transformant of step (c), thereby binding to the C-terminus and DLL4 of the Fc region of the antibody specifically binding to VEGF 12. A method for preparing the dual target protein of any one of claims 1 to 11, comprising linking the N-terminus of the protein to a linker.
17. The method of claim 15 or 16, wherein step (a) or (b) further comprises the step of substituting cysteine for the amino acid of any of the proteins that bind DLL4.
A composition for treating cancer comprising the dual target protein of any one of claims 1 to 11 and a pharmaceutically acceptable carrier.
The method of claim 18, wherein the cancer is esophageal cancer, stomach cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer Cancer liver, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease (Hodgkin) disease, lymphoma, and multiple myeloma hematoma cancer cancer composition is selected from the group consisting of.

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