KR101796050B1 - Method for increasing sialic acid of erythropoietin using Mgat1, Mgat4 - Google Patents

Abstract

The present invention relates to a method for increasing sialic acid content of erythropoietin and erythropoietin having an increased sialic acid content prepared using the same. According to the method of overexpressing Mgat1 and Mgat4 genes of the present invention, erythropoietin with increased sialic acid can be produced effectively, thereby providing erythropoietin having better in vivo persistence.

Description

Methods for increasing the sialic acid content of erythropoietin using the Mgat 1 and Mgat 4 genes {Methods for increasing sialic acid of erythropoietin using Mgat 1, Mgat 4}

The present invention relates to a method for increasing sialic acid content of erythropoietin and erythropoietin having an increased sialic acid content prepared using the same.

Sialic acid (Sia, NeuAc, NeuGc) is a generic term for the acyl derivatives of neuraminic acid (Neu), first isolated from mucin of the bovine salivary gland by Blix in 1936, It is composed of 9 carbons and is known as an acidic sugar with a COOH group. 23 kinds of sialic acid have been reported due to the difference in substituents, and these are known to exhibit a distribution specific to species or tissues. In higher animals, sialic acid is linked to α-glycosidic bonds by the action of enzymes, which are specific for glycoproteins, glycolipids, Gal, GlcNAc, GalNAc and sialic acid of the oligosaccharide sugar chain, respectively.

Since the sialic acid of the complex carbohydrate sugar chain is located at the distal end of the sugar chain structure existing on the surface of the cell membrane, it is expected that it will be directly involved in the contact between the cell and the extracellular environment, and the lifespan of blood cells or glycoprotein Which is known to be shortened by the removal of the catalyst. As an example, when sialic acid in erythrocyte membrane is removed, galactose is exposed to the cell surface and binds to a receptor lectin that specifically binds to galactose on the surface of Kupffer cells to induce receptor mediated receptor -mediated endocytosis and the sialic acid-removed asialo glycoprotein was also bound by the lectin on the surface of the hepatocyte and removed from the circulatory system in a similar pathway as the red blood cells. In addition, a glycoprotein having a sialo-linkage such as alpha-antitrypsin, cholinesterase, chorionic gonadotropin, CTLA4Ig, Factor VIII, gamma- Gamma-glutamyltransferase, granulocyte colony-stimulating factor (G-CSF) and luteinizing hormone (LH) have been shown to inhibit sialic acid- , The half-life of glycoprotein was remarkably increased.

When the glycoprotein is manufactured in the form of an oligosaccharide with the optimal oligosaccharide as described above, it is possible to produce a drug having excellent performance in which the therapeutic efficacy and persistence of the body are improved and the immune side effect is reduced, .

Among the glycoproteins having sialo-linkage, erythropoietin (EPO) is a glycoprotein hormone that induces erythropoiesis. Recombinant EPO is used as an anemia treatment agent. The wild-type EPO has three N-linked glycans and one O-linked sugar chain. Up to four sialic acids can be bound to one N-linked sugar chain and up to two sialic acids can be bonded to one O- A molecule of EPO can bind a total of 14 sialic acids. When sialic acid is bound to the sugar chain, it prevents binding of the glycosyltransferase to the desialyl present in the liver, thereby preventing EPO from degrading in the liver. In the case of EPO, when the sialic acid was added to the four antenna structures (tetra-antennary), the activity in the body was higher and the persistence of the body was longer than that of the bi-antennary in which two GlcNAc were connected. That is, the higher the sialic acid content, the longer persistence in the body of EPO appears to be due to the reduced rate of elimination in the blood due to the weakening of the binding between EPO and EPO receptors. In order to increase the persistence of EPO, there have been various attempts such as Fc fusion and pegylation of albumin or antibody, but the addition of two N-linked sugar chains to increase sialic acid darbepoetin -alfa).

As such, since the sialic acid content plays an important role in the persistence of EPO in the body, there is a desperate need to increase the sialic acid content of EPO in order to use more effective drugs.

The inventors of the present invention conducted a study to increase the sialic acid content of EPO and found that by overexpressing the Mgat1 and Mgat4 genes in albumin-EPO (Alb-EPO) producing cells, the sialic acid content of EPO was significantly increased And the present invention has been completed.

Therefore, the object of the present invention is to provide an EPO having an increased sialic acid content by controlling the overexpression ratio of Mgat1 and Mgat4 genes.

The present invention provides a vector for producing erythropoietin (EPO) comprising Mgat1 and / or Mgat4 gene.

The present invention also provides a transformed cell into which said vector has been introduced.

Overexpressing Mgat1 and Mgat4; The method comprising the steps of: providing an erythropoietin production method comprising:

The present invention also relates to a method for the treatment of cancer, comprising: overexpressing Mgat1 and Mgat4; Lt; RTI ID = 0.0 > erythropoietin. ≪ / RTI >

The present invention also provides a method for producing an albumin-erythropoietin-producing host cell, comprising the steps of: transforming an expression vector containing Mgat1 and / or Mgat4 gene into an albumin-erythropoietin producing host cell; Wherein the sialic acid content of the erythropoietin is increased.

According to the method for regulating overexpression of Mgat1 and Mgat4 genes of the present invention, erythropoietin with increased sialic acid can be efficiently produced, and erythropoietin having better in-vivo persistence can be provided.

Fig. 1 is a schematic diagram showing a pcDNA3.1 / Mgat1 vector and a pcDNA3.1 / Mgat4 vector.
Fig. 2 shows the results of confirming the introduction of Mgat1 and Mgat4. Fig.
FIG. 3 is a graph showing the results of confirming cell viability and cell density upon introduction of Mgat1 and Mgat4 in transformed CHO (Chinese hamster ovary) cells.
FIG. 4 is a graph showing the results of confirming changes in the production amount of Alb-EPO in Mgat1 and Mgat4-introduced cell lines.
FIG. 5 is a graph showing the increase in expression levels of Mgat1 and Mgat4 in transformed CHO cells. FIG.
FIG. 6 is a graph showing changes in sialic acid content according to the introduction of Mgat1 and Mgat4 genes.
FIG. 7 is a graph showing the results of confirming an increase in the production of erythropoietin with four antenna structures in the HG4 cell line. FIG.
FIG. 8 is a graph showing the results of confirming the increase of 2-, 3-, and 4-antenna structure erythropoietin in HG4 cells.

The present invention provides a vector for producing erythropoietin (EPO) comprising Mgat1 and Mgat4 genes.

Since the vector for producing erythropoietin containing the Mgat1 and Mgat4 genes can increase the production amount of erythropoietin of 2 to 4 antenna structure by up-regulating the expression of Mgat1 and Mgat4 gene in the transfected cells, the erythropoietin- Can be effectively produced.

In the present invention, " erythropoietin (EPO) " is a humoral factor that acts on hematopoietic tissues in an animal to promote erythropoiesis. This substance is an acidic glycoprotein with a molecular weight of 30,000 to 40,000 and acts on the bone marrow hepatocytes to participate in total agar differentiation of hepatocytes. Usually, there is little in the blood, but anemia or hypoxia (for example, when ascending to high acid) can be detected in urine and increased in the blood.

It is preferable that the erythropoietin produced in the present invention is Alb-EPO bound with albumin, and Alb-EPO is preferable to the production of erythropoietin having higher efficacy than Darbepoetin-alpha, which has improved performance than the existing erythropoietin .

In the present invention, " sialic acid " refers to a generic term for a compound in which an amino group or a hydroxy group is substituted with respect to a specific 9-carbon naphthalene acid to which an amino group or a carboxy group is bonded. (Neu5AC) or N-glycollyl neuraminic acid (Neu5G), which is most abundant in nature, and more preferably N-acetylneuraminic acid have.

The vector of the present invention may be a vector containing a Mgat1 or Mgat4 gene, and more preferably a vector containing a Mgat1 or Mgat4 gene, respectively.

The vector of the present invention may have SV40 poly A signal sequence and may contain a stronger promoter to further increase the expression level of Mgat1 or Mgat4 gene. It may also contain an antibiotic resistance gene for selection, and the antibiotic may include a neomycin, ampicillin, or giocin resistance gene. An embodiment of the present invention discloses the pcDNA3.1 / Mgat1 vector and the pcDNA3.1 / Mgat4 vector shown in Fig.

Mannosyl (alpha-1,3-) -glycoprotein beta-1,2-N-acetylglucosaminyltransferase and Mgat4 (Mannosyl (Alpha-1,3-) -Glycoprotein Beta-1,4- Gene is a gene expressing N-acetylglucosaminyltransferase-I (GnT-I, EC 2.4.1.101) and N-acetylglucosaminyltransferase-IV (GnT-IV, EC 2.4.1.145), respectively.

The erythropoietin produced by the vector for producing erythropoietin containing the Mgat1 and Mgat4 genes of the present invention includes all the erythropoietin forms produced in the host cell through transformation and is characterized in that it is erythropoietin having 2 to 4 antenna structures can do. That is, when erythropoietin is produced in a host cell by controlling overexpression of Mgat1 and Mgat4 genes, erythropoietin having 2 to 4 antenna structures can be mass-produced as compared with the control.

Accordingly, the present invention provides a vector for producing erythropoietin having 2 to 4 antenna structures including Mgat1 and / or Mgat4 gene.

The antenna structure specifies the structure produced by the connection of GlcNAc, and when two, three or four GlcNAc are connected, two (bi), three (tri-), and four (tetra-) May be referred to as erythropoietin having an < RTI ID = 0.0 > antennary. ≪ / RTI >

As described above, erythropoietin having 2 to 4 antenna structures can be characterized as erythropoietin having an increased sialic acid content by additionally providing a site capable of binding sialic acid at its terminal.

The vector for producing erythropoietin may be a vector for producing erythropoietin containing both a vector containing Mgat1 or Mgat4 gene and a single vector containing Mgat1 and Mgat4 all at once.

Thus, the present invention provides a vector for producing erythropoietin with an increased sialic acid content comprising the genes of Mgat1 and / or Mgat4.

The present invention also provides a transformed cell into which the vector for producing erythropoietin is introduced.

The transformed cell of the present invention may be any one selected from the group consisting of mammalian cells, yeast cells and insect cells, and in particular, it may be an erythropoietin production host cell, more preferably an albumin-erythropoietin production host cell.

Cells that can be used as transformed cells include, but are not limited to, various host cells which can be widely used in the art, such as BHK (baby hamster kidney) cells or CHO cells , And these cells show only a terminal sialic acid of an α-2,3-linked form because there is no α-2,6-sialyltransferase in their glycosylation system, and a complex such as human EPO Complex type glycoprotein and has a merit that the production rate of N-glycorhylenamic acid is low or rarely produced among the sialic acid formed at the end of the produced complex type sugar.

In particular, when albumin-erythropoietin (Alb-EPO) production host cells are used, erythropoietin having an increased body persistence can be produced by increasing an antenna as compared with the existing sugar chain-increased multifunctional alpha-alpha. The Alb-EPO production host cell may include, without limitation, a host cell known in the art capable of producing the Alb-EPO production host cell, and may be manufactured or purchased. Preferably, the Alb-EPO production host cell is disclosed in Korean Patent Application No. 10-2007-0114360 The cell line described can be used.

Mgat1 and Mgat4 of the present invention play an important role in forming an antenna of a glycoprotein. Because the Mgat1 gene affects the expression of Mgat4 depending on the expression level, changes in expression of the two genes may be effective in increasing the antenna structure, and controlling the overexpression ratio of these two genes is an important and essential factor in increasing the antenna structure do. More specifically, when Mgat1 is introduced at a higher ratio than Mgat4, it may not be effective in achieving the objective of increasing the sialic acid content. Accordingly, the present invention discloses a method of maximizing sialic acid through a method of regulating the overexpression ratio of two genes. More specifically, it is preferable that the transformed cells are introduced at a higher rate than the Mgat1 gene in a higher ratio, The Mgat1 gene and the Mgat4 gene are introduced at a ratio of 1: 1 to 1:30, preferably 1: 1 to 1:15, more preferably 1: 1 to 1:10, most preferably 1: 4 .

In the present invention, a cell capable of producing erythropoietin having increased sialic acid content as described above was deposited on Nov. 17, 2015 with the GenBank of Korea Research Institute of Bioscience and Biotechnology (52, Eun-dong, Yuseong-gu, Daejeon, Korea), and its deposit number is KCTC18426P .

In still another aspect of the present invention, there is provided a method for producing a recombinant vector comprising: overexpressing Mgat1 and Mgat4; The method comprising the steps of: providing an erythropoietin production method comprising:

In still another aspect of the present invention, there is provided a method for producing a recombinant vector comprising: overexpressing Mgat1 and Mgat4; Lt; RTI ID = 0.0 > erythropoietin. ≪ / RTI >

The above method increases the production of erythropoietin having four antenna structures through overexpression of Mgat1 and Mgat4, thereby increasing the sialic acid content in erythropoietin, thereby mass-producing erythropoietin with improved half-life and persistence in body .

Accordingly, the present invention provides a method for producing an albumin-erythropoietin-producing host cell, comprising: transforming an expression vector containing Mgat1 and / or Mgat4 gene into an albumin-erythropoietin producing host cell; Wherein the sialic acid content of the erythropoietin is increased.

The present invention also provides a method for producing erythropoietin, wherein the erythropoietin has an increased sialic acid content, which is 2 to 4 antenna structure erythropoietin.

The method of producing erythropoietin with increased sialic acid content of the present invention can be used in parallel with various known methods for increasing the sialic acid content, for example, by maintaining the sialic acid precursor at a high level in the cell, A high sialic acid content can be achieved.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example  One. Mgat1  And Mgat4  Preparation of vector for overexpression

The Mgat gene is an important gene that forms an antenna in the glycan structure of erythropoietin (EPO) and can provide a site for sialic acid binding at the terminal. Therefore, a vector capable of overexpressing the Mgat1 and Mgat4 genes was prepared to increase sialic acid binding at the end of Alb-EPO bound to albumin (Alb). Each of the pcDNA3.1 / Mgat1 vector and pcDNA3.1 / The Mgat4 vector is shown in Fig. FIG. 2 shows the results of confirming whether or not Mgat1 and Mgat4 were normally introduced into an expression vector to be used for Mgat1 and Mgat4 over-expressing cell lines.

As shown in Fig. 2, it was confirmed that Mgat1 and Mgat4 were successfully introduced into the pcDNA3.1 / Mgat1 vector and the pcDNA3.1 / Mgat4 vector, respectively, and this was used in the subsequent experiments.

Example  2. Mgat1 , Mgat4  Introduction CHO  Preparation of cells and their properties

2.1 Mgat1 , Mgat4  Introduction CHO  Production of cells

Transformed CHO cells were prepared using the vector prepared in Example 1. As CHO cells, Alb-EPO-producing cells were used and Mgat1 and Mgat4 genes were introduced. Overexpression of the two genes was performed by adding 37.5 μg of Freestyle MAX Reagent and 600 μL of OptiPRO-SFM to a solution of pcDNA3.1 / Mgat1 and pcDNA3.1 / Mgat4 vector in total of 37.5 μg in OptiPRO-SFM (600 μL) -lipid complexes. The resulting complex was reacted for 20 minutes and then introduced into CHO cells at a concentration of 1 × 10 ⁶ cells / mL. 4 mM glutamine was added to 30 mL of ProCHO 5 medium and the suspension was cultured at 125 rpm. Transgenic CHO cells transfected with transgenic antibiotics were screened and a transformed cell line was constructed from cells grown from one cell by performing limiting dilution to obtain a single clone. The ratios of the introduced Mgat1 and Mgat4 genes were changed as shown in Table 1 below.

[Table 1]

2.2 Mgat1 , Mgat4  Introduction CHO  Cell proliferation and Alb -PEPO production confirmation

Each transformant was inoculated with 125-mL Erlenmeyer flask at a concentration of 3 x 10 ⁵ cells / mL, and 4 mM glutamine was added to 20 mL of ProCHO5 medium, followed by suspension culture at 100 rpm. Changes in Alb-EPO production in G1, G4, G1, 4, HG1 and HG4 cell lines were examined by analyzing cell density and survival rate to determine whether the introduction of Mgat1 and Mgat4 was toxic in the transfected CHO cells. Were observed for 8 days. The results are shown in Fig. 3 and Fig.

As shown in FIG. 3, the cell lines into which Mgat1 and Mgat4 were introduced showed no significant difference in cell survival rate and cell density compared to the control group in which the above genes were not introduced, that is, they did not show toxicity to the cells. Also, as shown in Fig. 4, it was confirmed that the production amounts of Alb-EPO were similar to those of the control group in Mgat1 and Mgat4-introduced cell lines. Based on the above results, it was confirmed that the introduction of Mgat1 and Mgat4 did not cause toxicity to CHO cells and did not adversely affect the yield of Alb-EPO.

2.3. Mgat1 , Mgat4  Identification of gene expression and sialic acid content by transfection ratio

The expression levels of Mgat1 and Mgat4 gene were confirmed in the respective transformed CHO cells into which the Mgat1 and Mgat4 genes were introduced, and the results are shown in FIG.

As shown in FIG. 5, the expression levels of Mgat1 and Mgat4 genes were increased in each cell into which the gene was introduced, as compared with the control. More specifically, in Mg1-introduced G1, G1,4, HG1, and HG4, Mgat1 expression was significantly increased compared with the control, and Mgat4 gene expression was significantly increased in G4, G1,4, HG1 and HG4 containing Mgat4 , Mgat4 expression was also increased in G1 group which only Mgat1 was introduced, and Mgat4 gene expression was highest in HG4 group.

The sialic acid content of the expressed protein in each transformed cell line was also measured. Purification was performed on a HiTrap Blue HP column to determine the content of sialic acid. To the purified sample, 20 μL of 10 mM sodium periodate was added and reacted at 4 ° C. and the reaction was terminated with 100 μL of 50 mM sodium thiosulfate solution. Then, 500 μL of 4 mM ammonium acetate and 400 μL of 100 mM acetoacetanilide were added. After 10 minutes of reaction, sialic acid content was measured at an excitation wavelength of 338 nm and a 471 nm emission wavelength. The results are shown in FIG.

As shown in Fig. 6, the sialic acid content was increased in all the transformed cell lines as compared with the control group, and the increase in sialic acid content was confirmed according to the introduction ratio of Mgat1 and Mgat4. These results show that the introduction ratio of Mgat1 and Mgat4 can be an important factor. As a result, the sialic acid content was further increased when Mgat1 was introduced at a lower ratio than Mgat4, and the HG4 cell line in which Mgat1 and Mgat4 were introduced at a ratio of 1: 4 showed the highest sialic acid content Glycan analysis was performed using the HG4 cell line.

Example  3. Glycann  Through structure analysis Tetra  Confirm antenna structure increase

Since sialic acid content was increased through the introduction of Mgat1 and Mgat4 genes, glycan structure analysis was performed to confirm that this was achieved by increasing the antenna formation in the glycan structure through Mgat1 and Mgat4 gene introduction. More specifically, the glycan structure of the protein expressed in the HG4 cell line with the highest sialic acid content was analyzed by hydrophilic interaction chromatography (HILIC). Purified samples were treated with PNGase F to separate N-glycans and labeled with 2-aminobenzamide (2-AB) fluorescent dye and purified with GlycoClean S cartridge. Fluorescently labeled glycans were analyzed using an HPLC and a fluorescence detector at an excitation wavelength of 330 nm and a 420 nm emission wavelength. For HPLC analysis, TSK gel-Amide 80 column (5 μm, 4.6 × 250 mm) was used and the retention time of the peaks was converted to glucose (GU) as compared to the dextran calibration ladder.

Antenna analysis was also performed to determine whether the production of 2, 3-, 4 antenna structures of erythropoietin was increased in HG4 cells. The ratio of N-glycan antennas in the samples expressed in HG4 cells was determined by the weak anion exchange (WAX) chromatography method. 2-AB labeled Alb-EPO was analyzed by HPLC using TSK gel-DEAE 5PW column (10 μm, 7.5 × 75 mm). These experimental results are shown in FIGS. 7 and 8. FIG.

As shown in FIG. 7, in the HG4 cell line, an increase in fluorescence signal indicating erythropoietin having four antenna structures (tetra-antennary) was confirmed as compared with that of the control group. Thus, introduction of Mgat1 and Mgat4, (Tetraantennary structure) was increased and thus the terminal sialic acid content was increased.

As shown in FIG. 8, it was confirmed that the output of the two antenna structures increased by about 5 times, that of the three antenna structures by about 7 times, and that of the four antenna structures increased by about 7 times.

These results indicate that erythropoietin having 2 to 4 antenna structures can be effectively formed by introducing Mgat1 and Mgat4, thereby increasing the glycan end and effectively producing EPO having increased sialic acid content.

Korea Biotechnology Research Institute KCTC18426P 20151117

Claims (11)

1) and Mannosyl (Alpha-1,3-) -Glycoprotein Beta-1,4-N-Acetylglucosaminyltransferase) gene is 1 : ≪ / RTI > 1 to 1:10, with increased sialic acid content.
The transformed cell for producing erythropoietin (EPO) according to claim 1, wherein the erythropoietin is an antenna of 2 to 4, wherein the sialic acid content is increased.
delete delete The transformed cell of claim 1, wherein the transformed cell is any one selected from the group consisting of mammalian cells, yeast cells, and insect cells.
The transformed cell of claim 1, wherein the transformed cell is an albumin-erythropoietin producing host cell.
delete Overexpressing Mgat1 and Mgat4 in a ratio of 1: 1 to 1:10; ≪ / RTI >
Overexpressing Mgat1 and Mgat4 in a ratio of 1: 1 to 1:10; Lt; RTI ID = 0.0 > erythropoietin. ≪ / RTI >
Transforming an expression vector containing Mgat1 and Mgat4 genes into an albumin-erythropoietin producing host cell; / RTI >
Wherein the Mgat1 and Mgat4 are introduced in a ratio of 1: 1 to 1:10, wherein the sialic acid content is increased.
11. The method of claim 10, wherein the erythropoietin is 2 to 4 antennary erythropoietin, wherein the sialic acid content is increased.

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