WO2016099091A1 - Pegylated vegf trap and preparation method thereof - Google Patents

Abstract

The present invention relates to a pegylated VEGF trap and a preparation method thereof. The present invention relates to a SEG-VEGF trap having an improved half-life and anti-cancer effect due to pegylation after a thiol group is introducecd to the c-terminal of the VEGF trap, and a preparation method thereof. The pegylated VEGF trap according to the present invention is useful as an anti-cancer therapeutic agent having an increased half-life as well as an improved anti-cancer effect, compared to existing VEGF traps, through the substitution of the c-terminal with a cysteine having a thiol group, followed by a site-specific pegylation.

Description

PEGylated VEGF Trap and Method for Making the Same

The present invention relates to a pegylated VEGF trap and a method for preparing the same, and relates to a SEG-VEGF trap and a method for preparing the same, wherein the thiol group is introduced into the C-terminus of the VEGF trap and then PEGylated to enhance half-life and anticancer effect.

In order for cancer cells to proliferate and grow, new blood vessels that supply oxygen and nutrients are needed. Several factors are involved in the formation of new blood vessels, but the most important regulator is known as vascular endothelial growth factor (VEGF) (Ferrara and Davis-Smyth, Endocrine Rev. 18: 4). -25, 1997; Ferrara et al., J. Mol . Med . 77: 527-543, 1999). In mammals there are currently five types of VEGFs (VEGF-A, VEGF-B, VEGF-C, VEGF-D, PLGF) identified, three known as VEGF receptors (VEGFR) -1, -2, -3 Binds to receptors. Target gene inactivation studies in mice have revealed that VEGF is a necessary factor in the early stages of angiogenesis, while VEGF molecules are upregulated in tumor cells and their receptors are upregulated in tumor infiltrating vascular endothelial cells, but VEGF and its receptors As it was found that the expression of was maintained low in normal cells not involved in angiogenesis (Brown et al., Cancer Res . 53: 4727-4735, 1993; Mattern et al., Brit. J. Cancer . 73: 931-934, 1996), VEGF, which promotes new blood vessel formation, is drawing attention as a target for the treatment and prevention of cancer.

Recently, new anti-cancer therapies have been developed that block the production of blood vessels that feed cancer cells rather than the cancer cells themselves (Siemeister et al. Cancer Metastasis Rev. 17: 241-248, 1998). It has been shown to inhibit the growth of various human tumor cell lines in nude mice (Warren et al., J. Clin . Invest . 95: 1789-1797, 1995; Borgstrom et al. Cancer Res. 56: 4032-4039, 1996; Melnyk et al. Cancer Res . 56: 921-924, 1996). As humanized antibodies to VEGF, soluble VEGF-specific fusion protein antagonists called “VEGF traps” have been disclosed (Kim et al., Proc . Natl . Acad . Sci . USA 99: 11399-404, 2002; Holash et al. , Proc . Natl. Acad . Sci . USA 99: 11393-8, 2002), in fact Bevacizumab (Avastin) is commercially available. However, when such a protein is administered for therapeutic purposes, if the half-life of the body is short in blood and tissues, there is a problem in that the sustainability and stability of the drug are lowered in vivo.

To address this problem, attempts have been made to conjugate polyethylene glycol (PEG), a biocompatible polymer, to protein or peptide materials in recent decades (G.Pasut & FM Veronese Prog . Polym . Sci . 32, 933, 2007; FM Veronese et al., Biomaterials 22, 405.2001; P.Bailon et al., Pharm. Sci . Tech. Today 1: 352, 1998). Polyethylene glycol (PEG) is a polymer that has excellent biocompatibility that does not cause an immune response in vivo, and is one of synthetic polymers approved by the US Food and Drug Administration (FDA). The polyethylene glycol (PEG) is used in the body through the stealth effect of polyethylene glycol. By exhibiting the effect of inhibiting degradation by protein enzymes, it ultimately increases the half-life and stability in vivo.

However, when polyethylene glycol is used to improve the stability and persistence of protein drugs, polyethylene glycol reacts with a plurality of binding sites of protein drugs, resulting in a heterogeneous mixture of multi-conjugates. There is a problem of being -PEGylated species. On the other hand, even if a single molecule of polyethylene glycol conjugated (mono-PEGylated) conjugates are prepared by strictly adjusting the conditions, the biological or in vivo activity of the drug is significantly affected by the binding sites. In order to solve this problem, most therapeutic proteins do not have an active site located adjacent to the N-terminal. Thus, PEG is attached to the N-terminus of the protein or peptide to initiate the PEGylation reaction. Attempts have been made to minimize protein degradation, the biggest drawback. We also use a PEG derivative in which a compound having a catechol is bound to site-specific mono-PEG (mono-PEGylated) to the N-terminal primary amine of the protein or peptide. ) Was conjugated to the conjugate (Korea Patent 10-1042965).

However, since antibody sites such as VEGF traps have an active site at the N-terminus, existing methods of PEGylating the N-terminus hardly show their inherent efficacy.

Accordingly, the present inventors have diligently tried to develop a VEGF trap drug having an enhanced anticancer effect. As a result, the C-terminal position specificity was introduced by introducing a thiol (-SH) group to which the catechol group reacts better than the amine. A red PEGylation method was devised, and the PEG-catechol was conjugated to the C-terminus of the VEGF trap to be PEGylated to confirm that the half-life and anticancer effect were increased, and thus the present invention was completed.

Summary of the Invention

An object of the present invention is to provide a VEGF trap protein having a thiol (-SH) group introduced at the C-terminus and a PEGylated VEGF trap protein conjugated with a polyethylene glycol (PEG-CT) having a catechol bound to the thiol group of the protein. To provide.

Another object of the present invention to provide an antiangiogenic composition or anticancer composition comprising the PEGylated VEGF trap protein as an active ingredient.

Still another object of the present invention is to provide a polynucleotide encoding the VEGF trap protein, an expression vector comprising the polynucleotide, and a hematopoietic cell comprising the expression vector.

Another object of the present invention to provide a method for producing the VEGF trap protein and a PEGylation method.

In order to achieve the above object, the present invention provides a VEGF trap protein having a thiol (-SH) group introduced at the C-terminus.

The present invention also provides a PEGylated VEGF trap protein by conjugating polyethylene glycol (PEG-CT) in which catechol is bound to a thiol group of the VEGF trap protein.

The present invention also provides a composition for inhibiting angiogenesis comprising the PEGylated VEGF trap protein as an active ingredient.

The present invention also provides a method for inhibiting angiogenesis, comprising administering an angiogenesis inhibiting composition comprising the PEGylated VEGF trap protein as an active ingredient.

The present invention also provides a use of the composition for inhibiting angiogenesis comprising the PEGylated VEGF trap protein as an active ingredient for inhibiting angiogenesis.

The present invention also provides an anticancer composition comprising the PEGylated VEGF trap protein as an active ingredient.

The present invention also provides a method for inhibiting cancer, comprising the step of administering an anticancer composition comprising the PEGylated VEGF trap protein as an active ingredient.

The present invention also provides the use of a cancer-containing composition containing the PEGylated VEGF trap protein as an active ingredient for cancer inhibition.

The present invention also provides a polynucleotide encoding the VEGF trap protein.

The present invention also provides an expression vector comprising the polynucleotide.

The present invention also provides a transformed cell comprising the expression vector.

The present invention also provides a method for producing VEGF trap protein, characterized in that the cells are cultured.

The present invention also provides a method for PEGylation of VEGF trap protein, characterized in that the conjugated VEGF trap protein and catechol-bound polyethylene glycol (PEG-CT) prepared by the above method.

1 is a schematic showing PEGylation of the VEGF trap variant protein C-terminus.

2 shows the structure of the VEGF trap protein.

Figure 3 shows the PCR product of the VEGF trap variant protein (Lane 1: 1kb DNA ladder, Lane 2: VEGF-Trap (Cys # 1), Lane 3: VEGF-Trap (Cys # 2), Lane 4: VEGF -Trap (Cys # 3)).

4 is a schematic representation of restriction map of VEGF trap.

5 shows the results of confirming the nucleotide sequence of the VEGF trap.

FIG. 6 shows a vector (Cys # 1-VEGF-Trap) into which a nucleic acid molecule encoding a mutant in which the C-terminal first position of the VEGF trap is substituted with cysteine is introduced.

7 shows a vector (Cys # 2-VEGF-Trap) into which a nucleic acid molecule encoding a mutant whose C-terminal second position of the VEGF trap is substituted with cysteine is introduced.

8 shows a vector (Cys # 3-VEGF-Trap) into which a nucleic acid molecule encoding a mutant whose C-terminal third position of the VEGF trap is substituted with cysteine.

9 is a schematic of cell line development using VEGF traps.

10 is an ELISA schematic for identifying VEGF trap recombinant protein.

FIG. 11 shows the result of comparing the growth rate in the adhered state (ad-CHO) and the suspended state (SFM-CHO) of cells into which each VEGF trap variant was introduced.

12 shows the result of measuring the amount of recombinant protein expression in the suspended state (SFM-CHO) of the cells in which the VEGF trap variants are introduced.

FIG. 13 is a result of comparing recombinant protein production in suspended cells (SFM-CHO) into which VEGF trap variants were introduced (a: Cys # 1-VEGF-Trap, b: Cys # 2-VEGF-Trap, c) : Cys # 3-VEGF-Trap).

FIG. 14 shows the expression levels of VEGF trap variants (Cys # 1, Cys # 2, Cys # 3-VEGF-Trap) recombinant proteins of suspension culture adaptation (a: viable cell number, b: cell survival rate, c: amount of VEGF trap recombinant protein).

15 is a schematic diagram of a cell mass culture process using a Stirred tank reactor and a perfusion method.

16 is a schematic diagram showing recombinant protein production of suspended cells using a spinner flask.

17 is a result of measuring the recombinant protein expression amount by culturing suspended cells in which Cys # 1-VEGF trap variants were introduced in a spinner flask for 8 days (a: protein expression amount, b: protein production capacity).

FIG. 18 shows the results of measuring the expression level of Cys # 1-VEGF trap recombinant protein per batch in a culture system in a spinner flask.

19 shows the results of SDS-PAGE comparing VEGF traps with VEGF trap variants.

20 is a schematic diagram showing the production of VEGF trap cell line through the limiting dilution culture system.

Figure 21 shows the results of marginal dilution culture of Cys # 1-VEGF trap cell line.

22 shows the selection of monoclonal Cys # 1-VEGF trap cell lines via limiting dilution.

FIG. 23 shows the results of culturing monoclonal with high protein expression in a monoclonal Cys # 1-VEGF trap cell line in serum-free medium.

FIG. 24 shows the expression levels of monoclonal Cys # 1-VEGF trap (A3, B1, C2) recombinant proteins in suspension culture adaptation in a limiting dilution culture system (a: viable cell number, b: cell viability, c: VEGF) Trap recombinant protein amount).

25 shows the results of comparing the protein expression of Cys # 1-VEGF trap monoclonal A3 and B1.

FIG. 26 shows a comparison of hVEGF-A 165 binding capacity of Avastin (positive control), monoclonal B1-VEGF trap, and pool system VEGF trap.

Fig. 27 shows the result of synthesizing the SEG-VEGF trap, which was PEGylated by conjugating PEG-catechol to the mutation site of the VEGF trap.

Figure 28 shows the MALDI-TOF results of the SEG-VEGF trap.

29 shows the result of comparing the half lives.

30 is a result of comparing tumor volumes after drug administration.

Figure 31 is the result of comparing the measurement of red blood cells (Hematocrit).

Detailed description of the invention and preferred Embodiment

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.

In the present invention, the C-terminus of the VEGF trap protein is substituted with cysteine to prepare a VEGF trap protein into which a thiol group is introduced, and the VEGF trap protein is conjugated by gluing catechol-linked polyethylene glycol (PEG-CT). Pegylated and increased half-life and good anticancer effect of pegylated VEGF trap protein.

Accordingly, the present invention relates to a VEGF trap protein having a thiol (-SH) group introduced at the C-terminus in a consistent point.

In the present invention, the introduction of a thiol (-SH) group at the C-terminus is preferably to replace the C-terminal amino acid of the VEGF trap protein with Cysteine, more preferably the position of the cysteine The C-terminal amino acid is any one selected from the group consisting of the last 1 to 5, most preferably the position of the cysteine is the last first C-terminal amino acid, but is not limited thereto.

In another aspect, the present invention relates to a PEGylated VEGF trap protein obtained by conjugating polyethylene glycol (PEG-CT) to which a catechol is bound to a thiol group of the VEGF trap protein.

In the present invention, the PEG-CT is preferably conjugated to the C-terminus of the VEGF trap protein, more preferably a single conjugated to the C-terminal cysteine of the VEGF trap protein, but is not limited thereto.

In another aspect, the present invention relates to a composition for inhibiting angiogenesis and an anticancer composition containing a PEGylated VEGF trap protein as an active ingredient.

As used herein, the term "anticancer" includes "prophylaxis" and "treatment", where "prevention" means any action in which cancer is inhibited or delayed by administration of a composition comprising the antibody protein of the present invention, " By "treatment" is meant any action by which administration of a composition comprising an antibody protein of the invention improves or advantageously alters the symptoms of cancer.

The cancer or carcinoma which can be treated with the composition of the present invention is not particularly limited and includes solid and hematological cancers. Preferably gastric cancer, breast cancer, lung cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, biliary tract Cancer, testicular cancer, rectal cancer, head and neck cancer, cervical cancer, ureter cancer, osteosarcoma, neuroblastoma, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma or glial mass. More preferably include all cancers in which VEGF is expressed.

Compositions containing pegylated VEGF trap proteins of the invention may further comprise a suitable carrier, excipient or diluent according to conventional methods. Carriers, excipients and diluents that may be included in the composition comprising the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, specialty, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

Compositions containing pegylated VEGF trap protein of the present invention may be prepared according to conventional methods for powders, pills, granules, capsules, suspensions, solvents, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, lyophilizers and suppositories. It may have any one formulation selected from the group consisting of.

When formulated, diluents or excipients such as fillers, weights, binders, wetting agents, disintegrating agents and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose ( It is prepared by mixing sucrose or lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 60, cacao butter, laurin butter, glycerol gelatin and the like can be used.

Preferred dosages of the present invention vary depending on the condition and weight of the patient, the severity of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered in 0.001 ~ 100 mg / kg preferably 0.01 ~ 10 mg / kg per day. Administration may be administered once a day or may be divided orally. The dosage does not limit the scope of the invention in any aspect. In the present invention, "administration" means introducing a predetermined substance into a subject by any suitable method, and administration of the composition containing the antibody protein of the present invention is administered by any general route as long as it can reach the target tissue. Can be. Preferably, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention or treatment of cancer.

In another aspect, the present invention provides a method for inhibiting angiogenesis and pegylated VEGF trap protein comprising the step of administering an angiogenesis inhibiting composition comprising a PEGylated VEGF trap protein as an active ingredient It relates to a method for inhibiting cancer comprising the step of administering an anticancer composition comprising a.

In yet another aspect, the present invention provides a composition for inhibiting angiogenesis, wherein the composition for inhibiting angiogenesis comprising the PEGylated VEGF trap protein as an active ingredient and a cancer composition comprising the PEGylated VEGF trap protein as an active ingredient. It is related to the use used for suppression.

In another aspect, the present invention relates to a polynucleotide encoding a VEGF trap protein, an expression vector comprising the polynucleotide, and a transgenic cell comprising the expression vector.

In the present invention, it is preferable that the polynucleotide is substituted with any one sequence selected from the group consisting of SEQ ID NOs: 7 to 9, but is not limited thereto.

In the present invention, the cells are preferably animal cells, plant cells, yeast, E. coli, insect cells or animal cells, more preferably E. coli, but is not limited thereto. In addition, the animal cells are preferably Chinese hamster ovary (CHO) cells, but is not limited thereto.

The vector system of the present invention can be constructed through various methods known in the art, specific methods of which are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001). As used herein, the term "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. Vectors can be plasmids, phage particles or simply potential genomic inserts. Once transformed into a suitable 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 currently the most commonly used form of vectors, plasmids and vectors 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 limiting enzyme cleavage site may be present, synthetic oligonucleotide adapters or linkers according to conventional methods can be used to easily lignate vectors and foreign DNA. When the vector of the present invention is an expression vector and the prokaryotic cell is a host, a strong promoter capable of promoting transcription (for example, the tac promoter, lac Promoter, lac UV5 promoter, lpp promoter, pL ^λ promoter, pR ^λ promoter, rac 5 promoter, amp promoter, rec A promoter, SP6 promoter, trp promoter and T7 promoter, etc.), ribosomal binding sites for initiation of translation and transcription / detox termination sequences. When E. coli is used as a host cell, the promoter and operator sites of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol . 158: 1018-1024, 1984) and the phage leftward promoter (pL ^λ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet . 14: 399-445, 1980) can be used as regulatory sites. Vectors that can be used in the present invention include plasmids often used in the art (eg pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19, pET, etc.), phage (eg λgt4λB, λ-Charon, λΔz1 and M13). Or the like (eg, SV40, etc.). On the other hand, when the vector of the present invention is an expression vector and the eukaryotic cell is a host, a promoter derived from the genome of the mammalian cell (e.g., a metallothionine promoter) or a promoter derived from a mammalian virus (e.g., adeno) Late viral promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV) can be used and generally have a polyadenylation sequence as a transcription termination sequence. The expression vector of the present invention, as an optional marker, may include antibiotic resistance genes commonly used in the art, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neo There are genes resistant to mycin and tetracycline.

According to a preferred embodiment of the present invention, the vector of the present invention is preferably pCMV, but is not limited thereto.

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

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 gene of interest are included in one recombinant vector containing both a bacterial selection marker and a replication start point.

Host cells transformed with the recombinant vectors described above constitute another aspect of the present invention. As used herein, the term “transformation” means that DNA is introduced into a host such 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.

The transgenic cells of the present invention may use any host cell known in the art, and are preferably, but are not limited to, animal cells, plant cells, yeast, E. coli or insect cells. For example, E. coli JM109, E. coli BL21 (DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, and the like can be used as E. coli . Strains of the genus Bacillus, such as Tilis, Bacillus thuringiensis, and enterobacteria and strains such as Salmonella typhimurium, Serratia marsonsons and various Pseudomonas species. In addition, when transforming a vector of the present invention to eukaryotic cells, as host cells, yeast (Saccharomyce cerevisiae), insect cells and animal cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293 , HepG2, 3T3, RIN and MDCK cell lines) and the like can be used.

In another aspect, the present invention, the step of repeat passage of the cells in a medium containing methotrexate (MTX; methotrexate); Performing suspension adaptation to make the cultured attached cells suspended; And a method for producing a cell line for producing a VEGF trap protein, and a cell line for producing a VEGF trap protein produced by the method.

In another aspect, the present invention relates to a method for producing a VEGF trap protein, comprising culturing a transgenic cell comprising a cell line or an expression vector producing the VEGF trap protein.

In the present invention, the culture is carried out using any one or more selected from the group consisting of a stirred tank reactor, a spinner flask, a limiting dilution culture system, and a disposable bioreactor. Preferably, but not limited thereto.

In another aspect, the present invention relates to a method for PEGylation of a VEGF trap protein, comprising conjugating VEGF trap protein and catechol-bound polyethylene glycol (PEG-CT).

In the PEGylated VEGF trap protein of the present invention, the PEGylation method of binding the PEG-CT can be carried out in a manner contrary to the art (MJ Roberts et al., Advanced Drug Delivery Reviews ; 54: 459476, 2002 Francesco et al., Veronese Biomaterials ; 22: 405-417, 2001).

PEG-CT bound to the VEGF trap protein of the present invention preferably has a molecular weight of 5-100 kDa, more preferably 30 kDa, but is not limited thereto.

As used herein, the term “angiogenesis” refers to a cellular phenomenon in which vascular endothelial cells proliferate and reorganize to form new blood vessels from existing vascular networks. Such angiogenesis involves angiogenesis factors that promote angiogenesis, endothelial cell growth, vascular stability and angiogenesis. The angiogenesis factors include, for example, the VEGF and VEGF family, placental growth factor (PIGF), members of the platelet-derived growth factor (PDGF) family, fibroblast growth factor family (FGF), TIE ligands (angiopoietin), Ephrin, Del-1, fibroblast growth factor (acidic (aFGF) and basic (bFGF)), follistatin, granulocyte colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF) / scattering factor (SF), Interleukin-8 (IL-8), leptin, midkine, placental growth factor, platelet-derived endothelial growth factor (PD-ECGF), platelet-derived growth factor, especially PDGF-BB or PDGFR-beta, playotropin (PTN ), Progranulin, prolipin, transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), vascular endothelial growth factor ( VEGF) / vascular permeation factor (VPF) and the like, but is not particularly limited thereto.

As used herein, the term “angiogenesis inhibitor” refers to a low molecular weight substance, polynucleotide, polypeptide, isolated protein, recombinant protein, antibody, or that directly or indirectly inhibits angiogenesis, angiogenesis, or undesirable vascular permeability. It means conjugates or fusion proteins thereof. In addition, the angiogenesis inhibitors include substances that bind angiogenesis factors or receptors thereof and block angiogenic activity. For example, angiogenesis inhibitors can include antibodies to angiogenesis agents or other antagonists, such as antibodies to VEGF-A or VEGF-A receptors (eg, KDR receptors or Flt-1 receptors), VEGF-traps, Angiopoietin 2 is included.

Preferably, the antibody protein according to the present invention uses a VEGF trap as an angiogenesis inhibitor. In the present invention, VEGF trap means a multi-binding protein capable of binding to VEGF and means a substance useful for treating VEGF-related conditions and diseases which are enhanced, alleviated, or inhibited by elimination, inhibition or reduction of VEGF. The VEGF trap according to the present invention is preferably a PEGylated VEGF trap protein obtained by conjugating catechol-bound polyethylene glycol (PEG-CT) to a VEGF trap protein having a thiol (-SH) group introduced at the C-terminus. However, the present invention is not limited thereto.

Example

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

Example 1: VEGF Trap Biobetter Protein Design

The VEGF trap was designed as a VEGF trap bettor protein that can increase process yield due to high PEGylation efficiency (FIG. 1). Since an antibody protein such as a VEGF trap has an active site at the N-terminus, the C-terminus was mutated in order to introduce a C-terminal position-specific PEGylation method rather than the conventional method of PEGylating the N-terminus.

1-1: C-terminal position specific mutation of VEGF trap

To introduce a thiol group at the C-terminus of the VEGF trap and to react well with the catechol group, an amino acid located at the C-terminus was replaced with a cysteine having a thiol group. After designing the optimized codons with system biotechnology, polymerase chain reaction (PCR) was performed. Three primer sets (SEQ ID NO: 1/2; SEQ ID NO: 1/3; SEQ ID NO: 1/4) prepared according to the position of cysteine were reacted under the conditions of Tables 1 and 2.

primer

Forward: 5'-CAAAAGCTGGAGCTCCACC-3 '(SEQ ID NO: 1)

Reverse:

Cys # 1 5'-TCTCCCTGTCTCCGGGTtgtCTCGAGCATGCA-3 '(SEQ ID NO: 2)

Cys # 2 5'-GCCTCTCCCTGTCTCCGtgtAAACTCGAGCATGCA-3 '(SEQ ID NO: 3)

Cys # 3 5'-GAAGAGCCTCTCCCTGTCTtgcGGTAAACTCGAGCATGCA-3 '(SEQ ID NO: 4)

1 μl of the PCR product obtained by PCR was electrophoresed at 130V for 20 minutes on a 1.5% agarose gel to optimize PCR conditions and confirm the molecular weight of VEGF trap about 1.4 kb (FIG. 3).

1-2: Cloning of VEGF Trap Mutations

To clone the VEGF Trap PCR product into the pCMV vector, it was digested with NotI and XhoI restriction enzymes present in both the VEGF Trap PCR product and the pCMV vector. 4 is a map of restriction enzyme cleavage site of the VEGF trap. The cleavage reaction conditions are shown in Table 3, and the VEGF trap PCR product and the pCMV vector were reacted at 37 ° C. for 2 hours and purified by PCR purification kit (Intron).

To the purified cloning product (VEGF trap and pCMV vector) was added 3 μl T4 ligase buffer (solgent) and 1 μl T4 ligase and reacted overnight at 16 ° C. to prepare pCMV-VEGF trap vector (FIG. 6). , 7, 8).

The 10 μl pCMV-VEGF trap vector was mixed with E. coli (c2984 competent cells) for 30 minutes on ice, then subjected to heat shock for 45 seconds in a 42 ℃ constant temperature water bath, and then stored for 2 minutes on ice. Transformation was performed by the method. 1 ml of SOC (super optimal broth with catabolite repression) medium was added to the reaction and incubated for 1 hour in a 37 ° C. incubator, and then 100 μl was plated on LB agar medium containing ampicillin antibiotics and incubated for 24 hours in a 37 ° C. incubator.

1-3: Identification of VEGF Trap Variants

In order to confirm that the pCMV-VEGF trap vector was transformed into Escherichia coli, 16 colonies generated in each LB plate were subjected to colony PCR using the primers of SEQ ID NOs: 5/6 under the conditions of Table 4 and Table 5. .

primer

Forward: 5'-ATCAGCGAGCTCTAGCATTTAG-3 '(SEQ ID NO: 5)

Reverse: 5'-ATCCGCTAGCGATTACGC-3 '(SEQ ID NO: 6)

PCR products obtained by performing colony PCR were electrophoresed with agarose gel to optimize PCR conditions and identify a pCMV-VEGF trap with a molecular weight of about 8.5 kb.

Each colony was extracted using a plasmid extraction kit (Intron), and then sequenced (Marcrogen). As a result, three mutant Cys # 1-VEGF traps, Cys # 2-VEGF traps and Cys # 3-VEGF traps were obtained in which the C-terminus of the VEGF trap was substituted with cysteine. The Cys # 1-VEGF trap is the CCGGGTAAA region at the C-terminus of the VEGF trap with CCGGGT TGT (SEQ ID NO: 7), the Cys # 2-VEGF trap with CCG TGT AAA (SEQ ID NO: 8), and the Cys # 3-VEGF trap is Substituted with TGC GGTAAA (SEQ ID NO: 9) (FIG. 5).

Example 2: VEGF Trap Expression Cell Line Development

To develop cell lines using the three pCMV-VEGF traps obtained in Example 1, CHO-DG44 cells were used.

2-1: Stabilized Cell Line Development

CHO-DG44 cells were passaged at 1 × 10 ⁵ cells / ml in T-25 flasks in Iscoves Modified Dulbeccos medium (IMDM) containing 10% dialyzed FBS and 1 × hypoxanthine-thymidine, one day before transfection. . Transfection was performed using a Lipofectamine 2000 kit (Invitrogen), and 0.8 μg of each pCMV-VEGF trap vector and 20 μl of lipofectamine were mixed in 0.5 ml of Opti-MEM and reacted at room temperature for 20 minutes. After the reaction, the cells were incubated at 37 ° C for 24 hours in IMDM medium containing 10% FBS, hypoxanthine-thymidine, G418 (600 µg / ml), and 5 nM Methotrexate (MTX). 9).

In order to prepare a stabilized cell line, the cell was centrifuged at the fourth passage after three passages and the supernatant and the precipitate were separated and stored at -70 ° C. At the fifth passage, G418 (600 µg / ml) and Methotrexate (MTX), which gradually increased the concentration at 5 nM, were added to the medium, followed by incubation. The supernatant and precipitate were separated by centrifugation and stored at -70 ° C.

As a result of confirming the most stabilized cell line by SDS-PAGE and Western blot, the most stabilized cell line was obtained when incubated with 1 μM MTX.

2-2: Choosing High Productivity Cell Lines

For mass culture of cell lines, suspension adaptation was carried out to make the adhered cells into suspended cells. Inoculate serum-free medium at the concentrations of 5 × 10 ⁵ cells / mL, 3 × 10 ⁵ cells / mL, and 2 × 10 ⁵ cells / mL, and finally culture the cells at 2 × 10 ⁵ cells / mL Adaptation was carried out to 1 × 10 ⁶ cells / mL on day 3. In addition, the cell viability and protein production capacity was confirmed to determine the time for the cells to adapt to the inoculation concentration of 2 × 10 ⁵ cells / mL in serum-free medium. Production capacity was performed until the cell viability was reduced to less than 50%, analyzed by sandwich ELISA (Fig. 10).

As a result of confirming the reaction of the VEGF trap recombinant protein with human IgG1 (capture Ab), it was confirmed that the cell number of the three cell lines increased more than 20 times in the suspended state than in the adhered state, and was well adapted to the floating culture (FIG. 11).

As a result of comparing the amount and production of recombinant protein in the suspended state of three cell lines (Cys # 1-VEGF trap, Cys # 2-VEGF trap and Cys # 3-VEGF trap), Cys # 2-VEGF trap The protein expression was lower than that of the fast growth rate, and the Cys # 1-VEGF trap and the Cys # 3-VEGF trap showed similar growth rates, but the protein expression of the Cys # 1-VEGF trap was excellent (FIGS. 12, 13 and 14). Thus, the Cys # 1-VEGF trap cell line was found to express more recombinant protein in a less cellular state compared to other cell lines.

Example 3: Mass Production System of VEGF Trap Biobetter Protein

3-1: Scale-up Process

Cell culture was carried out using Stirred Tank Reactor, which is a kind of batch cell culture method, using a cell line (CHO-DG44 cell) made to be cultured in a suspended state. The maximum capacity is 20L, considering the shape of the baffle for gentle stirring, the round bottom surface for good mixing at low rpm, and the water-jacket for easy temperature control. The major causes of cell damage during mass culture were the formation of eddy and bursting of air bubbles. To reduce cell damage caused by air bubbles, a cell protective agent or shear protective agent (Pluronic F26) was added to the culture medium. In addition, gas sparging was performed to provide oxygen without damaging the cells when culturing mass cells.

Processes such as fed-batch, continuous, and medium perfusion have been developed for the medium supply method, but the perfusion method keeps the cells at a high concentration to increase productivity and easily recover proteins. (FIG. 15), purification of the VEGF trap protein was performed by modifying the Protein A sepharose affinity chromatography method.

3-2: Pool Culture System Using Spinner Flask

When the production capacity of the recombinant protein is low, less contamination and lower cost than the Stirred Tank Reactor, the spinner flask culture system was used to culture the cells collectively, and the recombinant proteins were collected in batches (FIG. 16).

In order to investigate the incubation time when a large amount of recombinant protein is expressed during 8 days of cell line culture, it was confirmed by the sandwich ELISA that the highest amount of protein was expressed on the 5th day of culture (FIG. 17) on the 5th day using a spinner flask. After culturing until protein purification was performed.

In addition, the VEGF trap recombinant protein expressed per batch in the spinner flask culture system was compared by ELISA (FIG. 18), and the molecular weights of the Cys # 1-VEGF trap and the existing VEGF trap in which point mutations were generated were determined using SDS-PAGE. Comparison was made (FIG. 19). Accordingly, it was confirmed that the Cys # 1-VEGF trap and the conventional VEGF trap show similar molecular weights.

3-3: Limit Dilution Culture System

In order to increase the production capacity of the recombinant protein in addition to the pool culture system, the protein production process using a limiting dilution culture system (Fig. 20) to express the protein by culturing cells in a single clone was developed.

Cys # 1-VEGF trap cell lines were cultured using a limiting dilution process to increase the production capacity of Cys # 1-VEGF trap (FIG. 21), and then monoclonal cells were selected to cultivate the Cys # 1-VEGF trap. Recombinant protein production capacity was confirmed using ELISA (Fig. 22).

As a result, monoclonal A3, B1, and C2 having high protein expression in Cys # 1-VEGF trap cell line were selected and cultured in serum-free medium (FIG. 23).

After culturing Cys # 1-VEGF trap monoclonal A3 and B1 for 4 days, the expression level of recombinant protein VEGF trap expressed was compared. Since monoclonal B1 is expressed more than A3, monoclonal B1-VEGF trap was selected. (Figures 24 and 25).

Finally, as a result of comparing hVEGF-A 165 binding strength of Avastin, monoclonal B1-VEGF trap and pool system VEGF trap, it was confirmed that the binding of Avastin was superior to monoclonal B1-VEGF trap (FIG. 26). .

Example 4: PEGylation of VEGF Trap Biobetter Proteins

In order to increase the half-life of the existing VEGF traps, the point mutated VEGF traps prepared in this example were site-specific PEGylated. The VEGF trap variant introduced cysteine with a thiol group to which the catechol group reacted well at the C-terminus, and PEG-CT (catechol) was bound to the C-terminus to pegylate. PEG-CT coupled CT (catechol) to PEG (polyethylene glycol) was PEGylated using the one according to the Republic of Korea Patent 10-1042965.

PEGylated VEGF traps were conjugated to VEGF traps of 30 kDa PEG-CT (catechol) to measure the absorbance of the PEGylated VEGF traps at 220 nm and 280 nm of GPC.

In addition, MALDI-TOF results showed that the SEG-VEGF trap was synthesized by combining 120 kDa VEGF trap and 30 kDa PEG-CT to have a molecular weight of 150 kDa (FIG. 28).

Example 5: Performance Evaluation of PEGylated VEGF Trap

5-1: Half-Life Test

Half-life was tested by injecting 6 μg of Avastin, conventional VEGF trap, and SEG-VEGF trap into the tail vein of C57BL / 6 mice.

As a result, in the case of SEG-VEGF trap, it was found that the half-life is longer than that of Avastin or VEGF trap (FIG. 29), and the dose can be improved from the half-life. Since Avastin has a half-life of 20 hours and SEG-VEGF traps have a half-life of 40 hours, the dose of Avastin is 1/14 days and SEG-VEGF traps are 1 when compared to Avastin. It can be expected to have a dosage of 24 times / 28 days.

5-2: Drug test

Lewis lung carcinoma (LLC) cells were implanted by subcutaneous injection (SC) in the right flank of C57BL / 6 mice at a concentration of 1.0 × 10 ⁶ cells / mouse. Seven days after tumor cell transplantation, 25 mg / kg of drugs (Avastin, VEGF Trap and SEG-VEGF Trap) were injected subcutaneously in the back of the neck of the mice at two-day intervals. After two weeks, the animal was sacrificed and the tumor size was measured (tumor volume = (4/3) π (length / 2) (width / 2) ((length + width) / 4). By confirming that the tumor size of the treated group was greatly reduced, it was confirmed as an animal model of drug efficacy test (tumor growth inhibition).

Avastin (positive control), conventional VEGF trap, SEG-VEGF trap and Fc protein (negative control) were injected into the set-up animal model, and the efficacy test was performed. The test conditions are shown in Table 6.

As a result of the pharmacological test, it was confirmed that in the experimental animals injected with drugs (Avastin, VEGF trap and SEG-VEGF trap), tumor formation was suppressed as compared with the Fc protein (negative control) group (FIG. In addition, the SEG VEGF trap is more effective than the conventional VEGF trap in suppressing tumor formation, and the SEG VEGF trap was found to have a performance equal to or higher than that of Avastin used as a positive control.

5-3: Hematoctit Test

C57BL / 6 mice were used to intravenously inject 1 mg / kg of drug (Avastin, VEGF Trap, and SEG-VEGF Trap), and then change in hematocrit was measured once every 4 days.

As a result of the SEG-VEGF trap and Avastin, it was confirmed that the activity is maintained in the body until 20 days (Fig. 31).

The PEGylated VEGF trap according to the present invention is useful as an anti-cancer therapeutic agent having an increased half-life and an anticancer effect than the conventional VEGF trap through position-specific PEGylation after replacing the C-terminus with a cysteine having a thiol group.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. 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 (16)

VEGF trap protein having a thiol (-SH) group introduced at the C-terminus. The VEGF trap protein according to claim 1, wherein the C-terminus is substituted with amino acid Cystein containing a thiol group. The VEGF trap protein according to claim 2, wherein the cysteine is any one selected from the group consisting of the first to fifth C-terminal amino acids. The PEGylated VEGF trap protein by conjugating polyethylene glycol (PEG-CT) bonded to a catechol to the thiol group of the protein of any one of claims 1 to 3. 5. The PEGylated VEGF trap protein of claim 4, wherein the PEG-CT is single conjugated to the C-terminal cysteine of the VEGF antibody protein. Angiogenesis inhibition composition comprising the PEGylated VEGF trap protein of claim 4 as an active ingredient. An anticancer composition comprising the PEGylated VEGF trap protein of claim 4 as an active ingredient. A polynucleotide encoding the VEGF trap protein of any one of claims 1 to 3. The polynucleotide according to claim 8, wherein the C-terminus is substituted with any one sequence selected from the group consisting of SEQ ID NOs: 7-9. An expression vector comprising the polynucleotide of any one of claims 8 to 9. A transformed cell comprising the expression vector of claim 10. The transformed cell of claim 11, wherein the cell is at least one selected from the group consisting of animal cells, plant cells, yeast, E. coli, and insect cells. The method of claim 12, wherein the animal cells are any one or more selected from the group consisting of Chinese hamster ovary (CHO) cells, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines Transformed cells. A method for producing a VEGF trap protein, comprising culturing the cells of claim 11. The method of claim 14, wherein the culturing is performed using any one or more selected from the group consisting of a stirred tank reactor, a spinner flask, a limiting dilution culture system, and a disposable bioreactor. Method for producing a culture, characterized in that the culture. Claim 1 to 3 of the VEGF trap protein and the VEGF trap protein prepared by the method of claim 14 any one or more selected from the group consisting of catechol conjugated polyethylene glycol (PEG-CT) Method for PEGylation of VEGF Trap Protein.

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