Method for preparing α-2,6-sialylated immunoglobulin using plasma cells isolated from humans

Abstract

The present invention relates to a method for producing α-2,6-sialylated immunoglobulin using plasma cells isolated from humans. The present invention makes it possible to mass produce α-2,6-sialylated immunoglobulin (2,6 IG) without using human blood, and it was discovered that α-2,6-sialylated immunoglobulin (2,6 IG) produced by the present invention can increase the level of IL-10, which is representative of an immunosuppressive cytokine produced by DC-SIGN stimulation. Thus, the method according to the present invention can be effectively used as an immunoglobulin substitute capable of replacing IVIG.

Description

Method for producing α-2,6-sialylated immunoglobulin using plasma cells isolated from humans

[0001] The present invention was made under the support of the Ministry of Science and ICT of the Republic of Korea under the task identification number 2710062735 and detailed task number NR069505. The task management specialized organization of the said task is the National Research Foundation of Korea, the research project name is "Individual Basic Research (MSIT) (R&D)", the research project name is "Study on Development of Anti-Inflammatory Antibody Treatment Using ST6GAL1 Genetically Modified Myeloma Cell Line", the task performing organization is Konkuk University, and the research period is 2024.03.01-2025.02.28.

[0002] In addition, the present invention was made under the support of the Ministry of Science and ICT of the Republic of Korea under the task identification number 2710003346 and detailed task number 00280626, and the task management specialized organization of the said task is the National Research Foundation of Korea, the research project name is "Individual Basic Research (Ministry of Science and ICT)", the research project name is "Evaluation of the expression of sialylated immunoglobulin in pregnant women with preeclampsia and proof of therapeutic effect in an animal model of preeclampsia", the task performing organization is Konkuk University Global Industry-Academic Cooperation Foundation, and the research period is 2024.03.01-2025.02.28.

[0003] In addition, the present invention was made under the support of the Ministry of Science and ICT of the Republic of Korea under the task identification number 2710001532 and detailed task number 00213334, and the task management specialized organization of the said task is the National Research Foundation of Korea, the research project name is "Individual Basic Research (Ministry of Science and ICT)", the research project name is "Analysis of Maintenance of Immune Tolerance through Placental Transfer of Sialylated Immunoglobulin of Fetal Fc-Receptor and IL-10 Receptor of Trophoblast Cells", the task performing organization is Konkuk University Global Industry-Academic Cooperation Foundation, and the research period is 2024.03.01-2025.02.28.

[0004] This patent application claims priority to Republic of Korea Patent Application No. 10-2024-0092054, filed with the Korean Intellectual Property Office on July 11, 2025, the disclosure of which is incorporated herein by reference.

[0005] The present invention relates to a method for producing α-2,6-sialylated immunoglobulin using plasma cells isolated from humans.

[0006]

[0007] Intravenous Immunoglobulins (IVIG), also known as gamma globulin or intravenous globulin, are highly purified blood products made from a large pool of human plasma donors. The potent immunosuppressive effects of IVIG were recognized in 1981, and in 2004, 4 million grams (g) of IVIG, worth $5 million, were used in the United States to treat various autoimmune diseases. Currently, IVIG is widely used in the treatment of various autoimmune diseases, including autoimmune arthritis, as well as various immune diseases, including immune skin diseases, cancer-related diseases, immune blood disorders, and innate immune diseases.

[0008] Each unit, or “lot,” of IVIG requires the plasma of approximately 1,000 to 10,000 people. Therefore, securing healthy human blood is crucial for IVIG production, and safely and stably purified IVIG is an expensive therapeutic immunoglobulin (Ig or IG) that costs patients hundreds of thousands to tens of millions of won. Specifically, the unit price of IVIG ranges from $100 to $350 per gram depending on the manufacturer, so for a 60-kg adult, the price of IVIG alone would reach $6,000 for treatment at a concentration of 1g / kg. Furthermore, because healthy human blood is essential for producing IVIG from human blood, a shortage in blood supply could lead to uncertainty in the supply of IVIG. Furthermore, as described above, because each unit, or “lot,” of IVIG contains IVIG obtained from plasma from approximately 1,000 to 10,000 people, there is the problem of being unable to precisely control the product quality. Moreover, the process of isolating and purifying IVIG from human blood is extremely complex, requiring significant costs and technology for production. Consequently, the government imposes strict regulations on the use of IVIG.

[0009] Asparagine 297th amino acid of the CH2 domain of the immunoglobulin (Ig) Fc region can be glycosylated in more than 30 different forms. Among these, Ig with sialic acid attached to the α 2,6 position is called α-2,6-sialylated immunoglobulin (2,6 IG). Among IVIG, α-2,6-sialylated immunoglobulin (2,6 IG) exists in less than 5% of all IVIG.

[0010]

[0011] The present inventors, noting that the production of IVIG requires a lot of cost and blood, and that mass production of α-2,6-sialylated immunoglobulin (2,6 IG) requires even more cost and blood, have made extensive research efforts to develop a method for mass production of α-2,6-sialylated immunoglobulin (2,6 IG) without human blood. As a result, when the ST6GAL1 (β-galactoside α-2,6-sialyltransferase 1) gene is transduced into plasma cells isolated from humans and cultured, α-2,6-sialylated immunoglobulin (2,6 IG) can be mass-produced without human blood, and the produced α-2,6-sialylated immunoglobulin (2,6 IG) can increase IL-10, one of the representative immunosuppressive cytokines produced by stimulation of DC-SIGN, thereby completing the present invention.

[0012] Accordingly, the purpose of the present invention is to provide a method for producing α-2,6-sialylated immunoglobulin.

[0013]

[0014] According to one aspect of the present invention, the present invention provides a method for producing α-2,6-sialylated immunoglobulin.

[0015] In one embodiment of the present invention, the manufacturing method comprises the following steps:

[0016] (a) A step of transducing the ST6GAL1 (β-galactoside α-2,6-sialyltransferase 1) gene into plasma cells isolated from a human;

[0017] (b) a step of isolating plasma cells expressing the ST6GAL1 gene; and

[0018] (c) A step of producing α 2,6 sialylated immunoglobulin by culturing plasma cells expressing the isolated ST6GAL1 gene.

[0019]

[0020] The present invention is described step by step below.

[0021]

[0022] Step (a): Step of transducing the ST6GAL1 (β-galactoside α-2,6-sialyltransferase 1) gene into plasma cells isolated from humans.

[0023] This step involves transducing the ST6GAL1 (β-galactoside α-2,6-sialyltransferase 1) gene, an enzyme that can bind sialic acid of antibodies, into plasma cells isolated from humans, thereby inducing sialylation of antibodies produced by the plasma cells.

[0024] As used herein, the term "ST6GAL1 (β-galactoside α-2,6-sialyltransferase 1)" refers to the enzyme encoded by the ST6GAL1 gene. The protein encoded by this gene is a type II membrane protein that catalyzes the transfer of sialic acid from CMP-sialic acid to galactose-containing substrates. Normally found in the Golgi apparatus, but the encoded protein, which can be proteolytically processed into a soluble form, is involved in the production of cell-surface carbohydrate determinants and the differentiation antigens HB-6, CDw75, and CD76. ST6GAL1 transcripts are found in the suprabasal endothelial cells of the mesenteric lymph nodes and Peyer's patches in mice, where they may be involved in B cell homing to Peyer's patches. ST6GAL1 expression has also been shown to be upregulated in several types of cancer and is known to play a role in cancer survival and metastasis.

[0025] As used herein, the term "plasma cell" (also known as plasma B cell or effector B cell) refers to a white blood cell that originates from B cells in lymphoid organs. When presented with an antigen, plasma cells secrete large amounts of proteins called antibodies. These antibodies are transported from the plasma cell to the target antigen (foreign substance) site through the plasma and lymphatic systems, where they initiate neutralization or destruction. B cells differentiate into plasma cells, which produce antibody molecules modeled after receptors on precursor B cells.

[0026] In one embodiment of the present invention, the plasma cells are human myeloma-derived cells. The human myeloma-derived cells include previously established human myeloma-derived cells, cells obtained from human myeloma patients, or immortalized plasma cell lines based on normal human plasma cells.

[0027] The term "myeloma," also known as "multiple myeloma," is a blood cancer caused by the proliferation of malignant plasma cells. This disease occurs when normal plasma cells become abnormally transformed and proliferate uncontrolled. Plasma cells derived from myeloma can produce excessive antibodies, including monoclonal antibodies. The present inventors sought to utilize this characteristic of myeloma-derived plasma cells in the production of α2,6-sialylated immunoglobulins.

[0028] In one embodiment of the present invention, the plasma cells secrete whole immunoglobulins, light chain immunoglobulins, or a combination thereof.

[0029] In one embodiment of the present invention, the immunoglobulin is in the IgG form.

[0030] In one embodiment of the present invention, the myeloma cells producing the complete immunoglobulin include ACB-885, ACB-1085, AD3, AMO1, delta-47, DP-6, EJM, FR4, GM2132, HL407, ILKM2, ILKM3, JIM-1, JJN-1, JJN-2, JJN-3, Karpas 620, Karpas 707, KAS-6 / 1, KHM-1A, KHM-1B, KHM-11, KMS-5, KMS-11, KMS-18, KP-6, KPMM2, L363, LB-831, LB-832, LB 84-1, LOPPA-1, LP-1, MEF-1, MM.1, MM5.1, MM-A1, MM-C1, These include, but are not limited to, MM-M1, MM-Y1, MT3, NCI-H929, NCI-H929, OCI-My5, OH-2, OPM-1, OPM-2, PCM6, RPMI 8226, U-1957, U-1958, U-2030, U266B1, UCD-HL461, UMJF-2, UTMC-2, XG-1, XG-2, XG-4, XG-6, XG-7, XG-8, XG-9, KMM-1, IM-9, MM.1S, U266B1, SKO-007, etc.

[0031] In another embodiment of the present invention, the myeloma cells producing the light chain immunoglobulin include, but are not limited to, ILKM3, JJN-2, Karpas 620, Karpas 707, KMM-1, KMS-11, KMS-18, MEF-1, MM5.1, MM5.2, MM-S1, NOP-2, OCI-My5, OPM-1, OPM-2, RPMI8226, SK-MM-1, SK-MM-2, U-1996, etc.

[0032] In one embodiment of the present invention, the ST6GAL1 (β-galactoside α-2,6-sialyltransferase 1) gene is of human origin.

[0033] In a specific embodiment of the present invention, the protein encoded by the ST6GAL1 gene may include the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

[0034] In a specific embodiment of the present invention, the ST6GAL1 gene may be composed of the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto. The protein and gene are accessible under NCBI Reference Sequence No. "NM_173216.2".

[0035] In one embodiment of the present invention, the sialic acid is N-acetylneuraminic acid (Neu5Ac).

[0036] The sialic acid mainly present in the human body is N-acetylneuraminic acid (Neu5Ac), and in mammals other than humans, sialylation proceeds using N-glycolylneuraminic acid (Neu5Gc). Since antibodies that recognize Neu5Gc as an antigen exist in the human body, when a recombinant protein to which Neu5Gc is added as sialic acid is injected into the human body, an adverse reaction may be induced (NatBiotech. 2010.28: 863-867). Therefore, the human myeloma cell-derived immunoglobulin of the present invention has the advantageous characteristic of producing an immunoglobulin that maintains specificity for antigens and biological properties without the risk of immune side effects in humans because sialylation occurs using Neu5Ac.

[0037] In one embodiment of the present invention, the term "transduction" refers to the process of transferring genes to bacteria or other cells. This process is typically mediated by a vector, through which genetic material is exchanged between bacteria and cells. Transduction can occur naturally or be artificially manipulated in a laboratory.

[0038] As for the above transduction method, if the host cell is a eukaryotic cell, the vector can be injected into the host cell by microinjection (Capecchi, MR, Cell, 22:479(1980)), calcium phosphate precipitation (Graham, FL et al., Virology, 52:456(1973)), electroporation (Neumann, E. et al., EMBO J., 1:841(1982)), liposome-mediated transfection (Wong, TK et al., Gene, 10:87(1980)), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5:1188-1190(1985)), and gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572(1990)). Can be.

[0039] The term "vector" as used herein refers to a means for expressing a target gene in a host cell, including but not limited to plasmid vectors; cosmid vectors; and viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors.

[0040] According to one embodiment of the present invention, the nucleic acid molecule encoding ST6GAL1 in the vector of the present invention is operatively linked to a promoter.

[0041] As used herein, the term "operatively linked" refers to a functional association between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, whereby the regulatory sequence regulates transcription and / or translation of the other nucleic acid sequence.

[0042] The recombinant vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

[0043] The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.

[0044] For example, when the vector of the present invention is an expression vector and uses a eukaryotic cell as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter, beta-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, promoter of Epstein-Barr virus (EBV), and promoter of Rous sarcoma virus (RSV)) can be used, and generally has a polyadenylation sequence as a transcription termination sequence.

[0045] The vector of the present invention may be fused with other sequences to facilitate purification of antibodies expressed therefrom. Examples of such fusion sequences include glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Qiagen, USA).

[0046] Meanwhile, the vector of the present invention includes an antibiotic resistance gene commonly used in the art as a selectable marker, for example, a resistance gene for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, puromycin, neomycin, hygromycin, and tetracycline.

[0047] In another embodiment of the present invention, the vector of the present invention may include, but is not limited to, histidinol dehydrogenase (hisD) and guanine phosphosribosyltransferase (Gpt) as selectable markers.

[0048] Optionally, the vector may additionally carry genes encoding reporter molecules (e.g., luciferase and glucuronidase).

[0049] In a specific embodiment of the present invention, the transduction is performed by a process of infecting plasma cells isolated from the human with a viral vector into which the ST6GAL1 (β-galactoside α-2,6-sialyltransferase 1) gene is inserted.

[0050] In one embodiment of the present invention, the vector may include a fluorescent protein gene. Examples of the fluorescent protein include, but are not limited to, GFP (Green Fluorescent Protein), eGFP (Enhanced GFP), YFP (Yellow Fluorescent Protein), CFP (Cyan Fluorescent Protein), RFP (Red Fluorescent Protein), mCherry, mOrange, mCerulean, DsRed, Venus, tGFP (turboGFP), AmCyan, ZsGreen, mKO (monomeric Kusabira-Orange), and mRuby.

[0051] In a specific embodiment of the present invention, the viral vector expresses a fluorescent protein gene together with the inserted ST6GAL1 gene in the transduced cell.

[0052] In a specific embodiment of the present invention, the vector of the present invention is characterized by including a gene expressing a fluorescent protein, eGFR, together with the ST6GAL1 gene.

[0053]

[0054] Step (b): Isolating plasma cells expressing the ST6GAL1 gene

[0055] This step is a step for isolating only plasma cells expressing the ST6GAL1 gene transduced in the above step (a).

[0056] Whether the ST6GAL1 gene has been transduced into the above-mentioned plasma cells can be confirmed by the expression of the above-mentioned selection marker, reporter molecule, or fluorescent protein.

[0057] For example, if the vector used for transduction of the present invention contains a hygromycin resistance gene, hygromycin is added to the culture medium of the transfected cells. Hygromycin allows only cells successfully transfected with the vector to survive, and non-transfected cells are killed by hygromycin.

[0058] Additionally, PCR, Western blot, flow cytometry (FACS, MACS), etc. can be used to confirm the expression of the inserted target gene (ST6GAL1) through the above transduction.

[0059] The above PCR can confirm the presence of transduction through amplification of the ST6GAL1 gene, Western blot can confirm the expression of the ST6GAL1 protein, and flow cytometry can confirm the expression of a fluorescent protein (e.g., eGFP) inserted together with ST6GAL1.

[0060]

[0061] Step (c): A step of producing α 2,6 sialylated immunoglobulin by culturing plasma cells expressing the isolated ST6GAL1 gene.

[0062] This step is a step for producing the 2,6-sialylated immunoglobulin aimed at by the present invention by culturing plasma cells expressing the ST6GAL1 gene separated in the above step (b).

[0063] The plasma cells of the present invention secrete a specific type of immunoglobulin, and the ST6GAL1 enzyme inserted by transduction induces sialylation of the expression of these immunoglobulins, thereby secreting α-2,6-sialylated immunoglobulin.

[0064] In one embodiment of the present invention, the α-2,6-sialylation occurs at asparagine in IgG of human immunoglobulin. More specifically, it occurs at asparagine present in the CH2 region of IgG of human immunoglobulin. Most specifically, it occurs at asparagine, which is the 297th amino acid present in the CH2 region of IgG of human immunoglobulin, but is not limited thereto.

[0065] In one embodiment of the present invention, whether sialylation has occurred in the manufactured α-2,6-sialylated immunoglobulin can be confirmed by SNA lectin immunoblotting.

[0066] Sambucus nigra agglutinin (SNA) lectin immunoblotting is used to detect sialylated glycoproteins. SNA lectin binds specifically to galactose or N-acetylgalactosamine, which are linked to sialic acid by an α(2-6) linkage. This principle can be used to detect sialylation of antibodies.

[0067] The principle of SNA lectin immunoblotting utilizes the property that SNA lectin specifically binds to α(2-6)-linked sialic acids. SNA lectin binds to α(2-6)-linked sialic acids on the surface of antibodies, and the bound SNA lectin can be detected using biotinylated SNA, streptavidin-HRP, or directly HRP-conjugated SNA. Since the SNA lectin can visualize the location of sialylated glycoproteins using the ECL (chemiluminescence) technique, this method can be used to confirm the presence of sialylation in a specific antibody.

[0068] SNA lectin has little nonspecific binding to other glycoproteins and is specific for a specific linkage form of sialic acid (α(2-6) linkage), so it can confirm specific sialylation, and the use of chemiluminescence techniques allows detection of even very low concentrations of sialylated proteins, making SNA lectin immunoblotting a powerful tool for accurately confirming sialylation of antibodies.

[0069]

[0070] In one embodiment of the present invention, the culture of the isolated plasma cells expressing the ST6GAL1 gene in step (c) includes in vitro cell culture or in vivo cell culture of an animal other than a human.

[0071] In vitro cell culture is performed by culturing transduced plasma cells, and when the plasma cells secrete α-2,6-sialylated immunoglobulin, α-2,6-sialylated immunoglobulin can be obtained from the culture medium.

[0072] In a specific embodiment of the present invention, the in vivo cell culture includes a method of culturing plasma cells by inoculating them into the peritoneal cavity of an animal other than a human. The animal may be, but is not limited to, a mouse.

[0073] For example, immunoglobulins can be mass-produced using hybridoma cells via intraperitoneal injection into mice. This process involves tumor formation within the peritoneal cavity and the accumulation of antibodies in the ascites fluid of the mouse.

[0074] In brief, mice (e.g., BALB / c mice) are prepared and treated with Pristane to create a tumor environment in the mouse peritoneum to facilitate cell growth. Next, the isolated myeloma-derived plasma cells transduced with the ST6GAL1 gene are collected and suspended in saline. The prepared cell suspension is injected into the mouse peritoneum. After cell inoculation, the mice are observed for 1-3 weeks. Once intraperitoneal tumors have formed and ascites has formed, the mice are anesthetized and, under sterile conditions, a needle is inserted into the peritoneal cavity to collect ascites. The collected ascites is centrifuged to remove cell debris and debris, and the immunoglobulins in the supernatant are collected. Protein A / G column chromatography, etc., is used to purify the immunoglobulins from the collected ascites.

[0075] This method allows the production of large quantities of α-2,6-sialylated immunoglobulin.

[0076]

[0077] Optionally, (d) a step of purifying the manufactured α-2,6-sialylated immunoglobulin.

[0078] In one embodiment of the present invention, the purification uses, but is not limited to, Protein L beads, Protein A beads, Protein G beads, affinity chromatography, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, or a combination thereof.

[0079] In one embodiment of the present invention, the principle of purifying antibodies using Protein L beads utilizes the property of Protein L binding to a specific region of an antibody. Specifically, Protein L interacts with and binds to the light chain (kappa light chain) of immunoglobulins. This property can be utilized to selectively purify antibodies. Because Protein L binds to a specific type of light chain (kappa light chain), nonspecific binding with other types of proteins or antibodies is reduced. Furthermore, Protein L can be used to purify antibodies from various animal species, and is particularly useful for isolating antibodies or immunoglobulins with kappa light chains from humans, mice, and rabbits. However, Protein L is not effective for purifying lambda-type antibodies.

[0080] In another embodiment of the present invention, the purification may utilize Protein A or Protein G Beads.

[0081] The above Protein A mainly binds to the Fc portion of IgG and works well in various species including humans, rabbits, and mice. It can also bind to antibodies with some subtypes of lambda light chains.

[0082] The above Protein G binds to the Fc portion of IgG similarly to Protein A, but has broader applicability, encompassing a wider range of species and subclasses.

[0083] In another embodiment of the present invention, the purification may utilize Protein G / L beads. Protein G / L is a mixture of Protein G and Protein L, which binds to both the Fc and Fab regions and exhibits high binding affinity for various antibody types. It is particularly useful for purifying both antibodies with lambda light chains and antibodies with kappa light chains.

[0084] In another embodiment of the present invention, the purification may utilize Anti-Lambda Light Chain Specific Antibody Affinity Chromatography. Lambda Specific Antibody is a method of purifying antibodies using antibodies specific for lambda light chains, and antibodies having lambda light chains can be selectively captured using beads bound to the antibodies.

[0085] In another embodiment of the present invention, the purification may utilize affinity chromatography. Affinity chromatography is a method for highly specifically separating and purifying biomolecules (e.g., proteins, enzymes, antibodies, etc.) by utilizing the interaction between a specific ligand and a molecule. When using affinity chromatography to separate and purify immunoglobulins such as those of the present invention, agarose or sepharose beads are mainly used as a matrix, and a ligand that specifically binds to the antibody is bound to the matrix. In a specific embodiment of the present invention, the affinity chromatography can be usefully used to specifically separate antibodies having a lambda light chain. In a more specific embodiment, the affinity chromatography may use epoxide-activated agarose, but is not limited thereto.

[0086] In another embodiment of the present invention, the purification may utilize ion exchange chromatography (IEC). Ion exchange chromatography is a purification method that utilizes differences in protein charge and can separate various proteins, including antibodies with lambda light chains. Specifically, anion exchange captures negatively charged proteins, and cation exchange captures positively charged proteins.

[0087] In another embodiment of the present invention, the purification may utilize size exclusion chromatography (SEC). Size exclusion chromatography is a method for separating proteins based on molecular size and shape, and can separate antibodies with lambda light chains from other proteins and impurities.

[0088] In another embodiment of the present invention, the purification may utilize hydrophobic interaction chromatography (HIC). Hydrophobic interaction chromatography is a separation method that utilizes the hydrophobic properties of proteins, and can be purified using the hydrophobic portion of an antibody.

[0089] Through the various methods described above, one skilled in the art can effectively purify antibodies or immunoglobulins having kappa light chains or lambda light chains.

[0090]

[0091] According to another aspect of the present invention, the present invention provides an α-2,6-sialylated immunoglobulin produced in a human-derived cell line.

[0092] The above “sialylated immunoglobulin” refers to an immunoglobulin having one or more sialic acid residues at the end of a sugar chain.

[0093] In one embodiment of the present invention, the Fc region of the immunoglobulin comprises a CH2 domain of IgG, and the sialic acid is α-2,6-linked to the terminal galactose of an N-glycan bound to the Asn297 residue of the CH2 domain. In addition, the sialic acid is N-acetylneuraminic acid (Neu5Ac).

[0094] In one embodiment of the present invention, the N-glycan comprises a complex-type biantennary glycan. The term "complex-type biantennary glycan" refers to a complex-type N-glycan structure having two branches, and generally comprises two branches composed of GlcNAc-Gal-Sialic acid.

[0095] The above “human-derived cell line” includes a cell line that expresses a human-derived gene or has a human-type glycosylation pattern, and is specifically a cell line that produces antibodies, such as a cell line derived from myeloma.

[0096] Additionally, in one embodiment of the present invention, the immunoglobulin is in the IgG form.

[0097] “IgG” is an immunoglobulin that forms the central part of the antibody response in mammals, and includes subtypes of IgG1, IgG2, IgG3, and IgG4.

[0098] In a specific embodiment of the present invention, the immunoglobulin is of the IgG2 type. IgG2 type antibodies are a subtype that generally exhibits relatively low effector function.

[0099] The α-2,6-sialylated immunoglobulin of the present invention is an IgG type antibody protein expressed in a human-derived cell line, and may be produced by the method for producing α-2,6-sialylated immunoglobulin according to one embodiment of the present invention described above.

[0100]

[0101] The features and advantages of the present invention are summarized as follows:

[0102] The present invention provides a method for producing α-2,6-sialylated immunoglobulin.

[0103] The present invention can produce α-2,6-sialylated immunoglobulin (2,6 IG) in large quantities without human blood, and the α-2,6-sialylated immunoglobulin (2,6 IG) produced by the present invention can increase IL-10, one of the representative immunosuppressive cytokines produced by stimulation of DC-SIGN. Therefore, the method of the present invention can be usefully used as an immunoglobulin substitute that can replace IVIG.

[0104]

[0105] Figure 1 is a diagram showing a vector in which the eGFP control lentivirus was purchased to introduce the hST9GAL1 gene into a myeloma cell line and the human ST6GAL1 gene was cloned.

[0106] Figures 2a to 2c are diagrams showing the process of producing an IM-9 ST6GAL1+ cell line and the results of Western blot and FACS analysis to confirm transduction.

[0107] Figure 3a shows IM-9 ST6GAL1, an ST6GAL1-overexpressing myeloma cell line. To determine whether 2,6-sialylated human IgG was increased within the cells, IM-9 WT cells and IM-9 ST6GAL1 This diagram shows the results of making whole cell lysates and confirming the increase in the expression level of ST6GAL1 and sialylated IgG.

[0108] Figure 3b shows IM-9 ST6GAL1, an ST6GAL1-overexpressing myeloma cell line. This diagram shows the results of SNA lectin blotting after Protein L bead immunoprecipitation to determine whether IgG secreted by cells into the culture medium was sialylated.

[0109] Figure 4a is a diagram showing the IgG separation and purification process using a Protein L bead column.

[0110] Figure 4b is a diagram showing the purity of IgG according to IgG separation and purification using a Protein L bead column.

[0111] Figure 4c is a diagram showing the results of SNA lectin immunoblotting to confirm sialylation of separated and purified IM-9 WT IG and IM-9 2,6 IG.

[0112] Figure 5 is a diagram comparing the glycosylation characteristics of commercially available total IVIG, purified 2,6-SA-IVIG, and 2,6-SA-IVIG secreted by ST6GAL1-overexpressing myeloma cell lines.

[0113] Figure 6 is a diagram comparing the glycosylation characteristics of 2,6-SA-IVIG secreted by ST6GAL1-overexpressing myeloma cell lines by production batch.

[0114] Figures 7a to 7f show human ST6GAL1 gene transduced LP-1 cell line (LP-1 ST6GAL1+ ) This is a diagram showing the purity and sialylation of the manufactured and purified LP-1 WT immunoglobulin G (LP-1 WT IG) and LP-1 α 2,6 sialylated immunoglobulin G (LP-1 2,6 IG).

[0115] Figure 8 is a diagram showing the results of comparative analysis of signals for various sugar components for each isolated and purified immunoglobulin to compare the characteristics of glycosylation forms between GC tIVIG, GC 2,6 IVIG, IM-9 WT IG, IM-9 2,6 IG, LP-1 WT IG and LP-1 2,6 IG.

[0116] Figures 9a to 9d are diagrams comparing the expression level of STLAGAL1 (9a), purity of Ig (9b), production amount of Ig (9c), and glycosylation type (9d) of IM-9 WT and IM-9 ST6GAL1 to determine the stable production ability of α 2,6 sialylated IM-9 2,6 from the ST6GAL1-overexpressing IM-9 myeloma cell line.

[0117] Figures 10a to 10e are diagrams confirming the increase of DC-SIGN by stimulation with IM-9 WT and 2,6 huIgG in various DC-SIGN-expressing cells (mouse DCEK: Figure 10a, normal HBC: Figures 10b-c, preeclampsia HBC: Figures 10d-10e).

[0118] Figures 11a to 11d are diagrams confirming the increase in IL-10 by stimulation with IM-9 WT and 2,6 huIgG in various DC-SIGN expressing cells (mouse DCEK: Figure 11a, normal HBC: Figure 11b, preeclampsia HBC: Figures 11c-11d).

[0119] Figure 12 is a diagram showing the expression of anti-inflammatory cytokines IL-10 and IL-10 receptors A and B after treatment with substances such as Human Serum Albumin (HSA), GC IVIG, GC 2,6 IVIG, IM-9 WT and IM-9 2,6 to compare whether each substance induces an anti-inflammatory effect in a chorionic BeWo cell line derived from human placenta.

[0120]

[0121] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and 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.

[0122]

[0123] Example

[0124]

[0125] Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight) % for solid / solid, (weight / volume) % for solid / liquid, and (volume / volume) % for liquid / liquid, unless otherwise noted.

[0126]

[0127] Example 1: Culturing of myeloma cell lines for immunoglobulin production

[0128] The present inventors have obtained two myeloma cell lines (IM-9, LP-1) that secrete human immunoglobulin type G (IgG) to produce complete immunoglobulins with increased sialylation. The IM-9 cell line is a multiple myeloma cell line established from peripheral blood B lymphoblasts of a woman with multiple myeloma. The IM-9 cell line is known to secrete IgG2 (H+L), and its light chain type is known to be kappa chain. The present inventors purchased the IM-9 cell line from the Korea Cell Line Bank and used RPMI-1640 medium with 10% FBS for its culture. The known huIgG secretion capacity of the IM-9 cell line is 2.4 μg / 24 hrs / 1x10 6 It is cells.

[0129] The LP-1 cell line was established from the peripheral blood of a 56-year-old woman with multiple myeloma (IgG, EBV negative) with leukemic transformation (refractory, terminal) in 1986. It secretes IgG (H+L) and its light chain type is known to be lambda light chain. The present inventors purchased the LP-1 cell line from Creative Bioarray and used it for its culture in RPMI-1640 medium supplemented with 10% FBS or in 80-90% Iscove's MDM medium supplemented with 10-20% FBS. The known huIgG secretion capacity of the LP-1 cell line is 50 μg / 24hr / 1X10 6cells (with 5 μg / ml transferrin + 5 μg / ml porcine insulin).

[0130]

[0131] Example 2: Generation of ST6GAL1-overexpressing myeloma cell lines that produce α 2,6-sialylated immunoglobulins.

[0132] In order to increase sialylation of complete human immunoglobulin secreted from a myeloma cell line, the present inventors produced a myeloma cell line (Cell ST6GAL1+) that overexpressed ST6GAL1, an α-2,6 silalyltransferase enzyme gene that induces α-2,6 sialylation at position 297 of asparagine in the CH2 domain of human IgG, through the following process.

[0133] First, to introduce the hST9GAL1 gene into the above-mentioned myeloma cell line, an eGFP control lentivirus (VB160109-10005) was purchased, and the lentiviral vector construct for stable transfection of ST6GAL1 used a lentivirus (pLV[Exp]-EGFP-CMV>hST6GAL1) containing human ST6GAL1 ([NM_173216.2]) as an ORF, eGFP as a marker, and a hygromycin resistance gene (Fig. 1). An eGFP expression lentiviral system (pLV[Exp]-EGFP:T2A:Hygro-EF1A) was used as a positive control. The lentiviral vector was cloned by VectorBuilder and packaged into VB Ultra stable lentivirus as a host cloning strain.

[0134] The provided ST6GAL1 lentiviral vector and eGFP expression lentiviral vector (positive control) (10 8transducing units / ml) were infected with 20 μl of the above-mentioned myeloma cell line, and the medium was replaced the next day. To select myeloma cells into which the human ST6GAL1 gene was introduced, the cells were cultured in FBS 10% + RPMI 1640 media containing hygromycin B (50 μg / ml) to kill untransfected cells, thereby selecting only transfected myeloma cells (Fig. 2a).

[0135] Next, to confirm whether the selected myeloma cells were successfully transfected with the ST6GAL1 gene, whole cell lysates of IM-9 original myeloma cell line (IM-9 WT) cells and ST6GAL1 transfected IM-9 cell line (IM-9 ST6GAL1+) were prepared, and overexpression of α 2,6 sialyltransferase and EGFP was confirmed by Western blot analysis after SDS-PAGE (Fig. 2b).

[0136] In addition, to confirm whether the ST6GAL1 gene was successfully transfected in living cells, the fluorescence of eGFP co-transfected with ST6GAL1 as a transfection marker was confirmed by FACS analysis. As a result of confirming the IM-9 WT group, the positive control group expressing only eGFP (IM-9 eGFP+), and the experimental group expressing eGFP and ST6GAL1 together (IM-9 ST6GAL1+) cells, IM-9 eGFP+ and Only IM-9 ST6GAL1+ cells expressed eGFP at very high levels, and the expression was maintained for several months (Fig. 2c).

[0137]

[0138] Example 3: Confirmation of increased production, secretion, and sialylation of secreted 2,6-sialylated immunoglobulin G (IM-9 2,6 IG) in ST6GAL1-overexpressing myeloma cell lines.

[0139] The present inventors have developed a ST6GAL1-overexpressing myeloma cell line, IM-9 ST6GAL1 To determine whether 2,6-sialylated human IgG at Asn297 was increased within the cells, IM-9 WT cells and IM-9 ST6GAL1 Whole cell lysates were prepared and the expression of ST6GAL1 and the increase in sialylated IgG were confirmed.

[0140] As shown in Fig. 3a, the expression of ST6GAL1 was increased only in IM-9 ST6GAL1 cells, and as a result, an increase in sialylation in the heavy chain of human IgG in IM-9 ST6GAL1 cells bound to SNA lectin was observed.

[0141] In addition, to isolate IgG contained in IM-9 ST6GAL1 cell culture medium, an immunoprecipitation experiment was performed using Protein L beads that specifically bind only to the kappa light chain of the 50-fold concentrated cell culture medium. The bovine IgG in the 10% FBS contained in the culture medium was removed during this process because its light chain consists only of the lambda chain. After Protein L bead immunoprecipitation, human IgG was confirmed to be normal, and SNA lectin blotting confirmed that sialylation was increased in proteins of the same size as IgG.

[0142] When comparing the commercially available Green Cross IVIG (GC total Intravenous immunoglobulin, GC tIVIG) preparation with Green Cross 2,6 IVIG (GC 2,6 IVIG), which was purified by column method using SNA lectin to isolate only sialylated IgG, the sialylation signal of 2,6 IVIG was observed to be slightly larger in the size of the heavy chain than that of general IgG, which was consistent with the results of previous studies. However, it was confirmed that the IM-9 2,6 IG of the present invention showed an increase in the SNA signal but no significant difference in the size of the heavy chain (Fig. 3b).

[0143]

[0144] Example 4: Establishment of a method for separating and purifying IM-9 WT IG and IM-9 2,6 IG, and confirmation of increased sialylation.

[0145] The present inventors have developed a ST6GAL1-overexpressing myeloma cell line, IM-9 ST6GAL1 In order to selectively isolate only human IgG from the cell culture medium, the culture medium cultured with 5% FBS was concentrated 50-fold and purified twice using a column instead of immunoprecipitation with Protein L beads (Fig. 4a).

[0146] After SDS-PAGE of the purified solution and column washing solution and silver staining, it was confirmed that only IM-9 WT IG was accurately separated without contamination with other proteins when purified twice with Protein L beads. When purified once, it was confirmed that bovine serum albumin (BSA), the protein most abundant in FBS, was detected as is after passing the column purification process at a concentration so high (Fig. 4b).

[0147] The sialylation of the isolated and purified IM-9 WT IG and IM-9 2,6 IG was confirmed by SNA lectin immunoblotting, and it was confirmed that sialylation was increased in IM-9 2,6 IG, similar to the results in cells (Fig. 4c).

[0148]

[0149] Example 5: Comparative analysis of glycosylation of commercial total IVIG, purified 2,6-SA-IVIG, and 2,6-SA-IVIG secreted by ST6GAL1-overexpressing myeloma cell lines.

[0150] The glycosylation types of IM-9 WT IG and IM-9 2,6 IG separated and purified in Example 4 above were compared with Green Cross GC 2,6 IVIG separated and purified by column method using SNA lectin from commercially available GC tIVIG (Green Cross total IVIG).

[0151]

[0152] <GC 2,6 IVIG의 당화 특성>

[0153] As a result, as shown in Figures 3b and 4c, the sialylation of GC 2,6 IVIG mainly increased in the upper band size of the heavy chain of GC tIVIG, and the sialylation signal was clearly confirmed in the light chain as well (Figure 5, second column from the left).

[0154] A similar pattern to sialylation was also observed in fucosylation identified by AAL lectin (Fig. 5, third column from the left).

[0155] In the case of alpha-mannosylation and alpha-glucosylation confirmed by CON A lectin, when combined with the results in the 6th column from the left, glucosylation was observed slightly more intensely in the heavy chain than in the tIVIG heavy chain (Fig. 5, 4th column from the left).

[0156] In the case of a 2,3 sialylation confirmed by MAL2, it was not found at all in GC tIVIG and GC 2,6 IVIG (Fig. 5, fifth column from the left).

[0157] Alpha-mannosylation and galactosylation, as identified by NPL / NPA lectin, were very weakly detected in GC tIVIG but were not detected in GC 2,6 IVIG (Fig. 5, 6th column from the left).

[0158]

[0159] <IM-9 2,6 IG의 당화 특성>

[0160] On the other hand, in the case of IM-9 2,6 IG of the present invention, unlike GC 2,6 IVIG, it was confirmed that the sialylation signal was increased compared to IM-9 WT IG although the size was the same (Fig. 5, second column from the left).

[0161] In the case of fucosylation, a-mannosylation and a-glucosylation, the degree of intensity was reduced compared to GC tIVIG and GC 2,6 IVIG, but there was no difference in degree between IM-9 WT IG and IM-9 2,6 IG, unlike GC 2,6 IVIG (Fig. 5, 3rd and 4th columns from the left).

[0162] For a-2,3 sialylation, it was not found in IM-9 WT IG and IM-9 2,6 IG, as in GC tIVIG and GC 2,6 IVIG (Fig. 5, fifth column from the left).

[0163] a For mannosylation and galactosylation, no mannosylation was found in GC 2,6 IVIG, but the same amount, although very weak, was observed in IM-9 WT IG and IM-9 2,6 IG (Fig. 5, 6th column from the left).

[0164] Therefore, an increase in sialylation was commonly observed between GC 2,6 IVIG and IM-9 2,6 IG. However, for the remaining investigated glycations except for 2,6 sialylation, changes in increase or decrease were observed in GC 2,6 IVIG, but in the case of IM-9 2,6 IG of the present invention, the same amount as IM-9 WT IG was maintained. This means that when sialylated IVIG is separated from the entire IVIG, there is a difference in glycation characteristics, but when 2,6 IG is manufactured by increasing sialylation in myeloma as in the present invention, there is no difference in glycation characteristics other than sialylation from wild type IG.

[0165]

[0166] Example 6: Confirmation of material stability through analysis of differences in sialylation between production batches of 2,6-SA-IVIG secreted by ST6GAL1-overexpressing myeloma cell lines.

[0167] In the case of GC tIVIG, which is commercially available and used in patients, various adjuvants are included in the dilution to stabilize the glycation of IVIG immunoglobulins. However, the final isolated and purified IM-9 WT IG and IM-9 2,6 IG of the present invention were collected in various concentrations in PBS without the addition of any adjuvant components and then stored in a refrigerator at 4°C. The glycation of IM-9 WT IG and IM-9 2,6 IG stored under these harsh conditions for more than 6 months and the glycation of newly isolated immunoglobulins were analyzed using the same method as in Example 5 and FIG. 5 to determine whether there were any changes.

[0168] As shown in Fig. 6, it was confirmed that in the case of IM-9 WT IG and IM-9 2,6 IG of the present invention, there was no change at all in the saccharification characteristics even after storage at 4°C for more than 6 months. From these results, it was confirmed that both IM-9 WT IG and IM-9 2,6 IG had very stable protein structures and saccharification structures.

[0169]

[0170] Example 7: Construction of human ST6GAL1 gene-transduced LP-1 cell line (LP-1 ST6GAL1+) and production of LP-1 WT immunoglobulin G (LP-1 WT IG) and LP-1 α 2,6 sialylated immunoglobulin G (LP-1 2,6 IG).

[0171] IM-9 myeloma cells produce and secrete a complete human immunoglobulin with a kappa light chain. The present inventors secured LP-1 myeloma cells that produce and secrete a complete human immunoglobulin with a lambda light chain, which is different from IM-9, and created LP-1 ST6GAL1+ myeloma cells by inserting the human ST6GAL1 gene into LP-1 WT myeloma cells using the same method as in the IM-9 cell line, and confirmed that the expression of eGFP and a 2,6-sialylated enzyme increased in these cells (Figs. 7a and 7b).

[0172] Additionally, LP-1 WT and LP-1 ST6GAL1+ We further demonstrated that a 2,6-sialylated enzyme was highly abundant along with human immunoglobulin within myeloma cells (Fig. 7c), and LP-1 ST6GAL1+ In the case of human immunoglobulins present in myeloma cells, it was confirmed that sialylation was significantly increased compared to human immunoglobulins in LP-1 WT myeloma cells (Fig. 7d).

[0173] In addition, LP-1 WT and LP-1 ST6GAL1+ myeloma cells were cultured in large quantities using the same amount of culture medium, and the culture medium was collected. Human immunoglobulins were isolated and purified from each culture medium, and then separated on an SDS-PAGE gel and stained with silver. As a result, very interestingly, LP-1 WT IG was hardly present in the culture medium of LP-1 WT myeloma cells, but LP-1 ST6GAL1+ We confirmed the distinct presence of LP-1 2,6 IG in myeloma cell cultures (Fig. 7e). This unexpected and important result demonstrates that overexpressing α 2,6 sialylation in primary myeloma cells not only increases sialylation of immunoglobulins but also enhances their production capacity.

[0174] Human immunoglobulins were finally confirmed to be human immunoglobulins using a human immunoglobulin antibody for each immunoglobulin purified from LP-1 WT and LP-1 ST6GAL1+ myeloma cells, and staining with SNA lectin for the confirmed human immunoglobulins confirmed that a 2,6 sialylation signal was distinct (Fig. 7f).

[0175] Through this, it was further elucidated that when human immunoglobulin-secreting myeloma cells are used, wild-type immunoglobulins and immunoglobulins with increased a 2,6 sialylation can be produced in the same manner as when IM-9 myeloma cells are used to produce IM-9 WT IG and IM-9 2,6 IG.

[0176] Myeloma cell lines with diverse characteristics are stored in cell line banks worldwide, and unfortunately, a significant number of new blood cancer cases are diagnosed each year. Therefore, a continuous supply of novel immunoglobulin-producing myeloma cell lines is available. Therefore, the ability to produce immunoglobulins with different native or a2,6-sialylated levels from various myeloma cell lines allows the establishment of production lines capable of producing customized immunosuppressive immunoglobulins for various autoimmune diseases.

[0177]

[0178] Example 8: Comparison of Glycosylation Forms Among GC tIVIG, GC 2,6 IVIG, IM-9 WT IG, IM-9 2,6 IG, LP-1 WT IG, and LP-1 2,6 IG

[0179] In order to compare the characteristics of glycosylation forms between GC tIVIG, GC 2,6 IVIG, IM-9 WT IG, IM-9 2,6 IG, LP-1 WT IG and LP-1 2,6 IG, the inventors compared and analyzed signals for various sugar components using the lectins of Fig. 8 for each isolated and purified immunoglobulin.

[0180] For LP-1 WT IG, direct comparative analysis of glycation with LP-1 2,6 IG was not possible due to the low production volume. However, since the amounts of immunoglobulins in IM-9 2,6 IG and LP-1 2,6 IG were confirmed to be identical (Fig. 8, 1st column), signals for various sugar components were compared and analyzed using the lectins in Fig. 8.

[0181] As a result, it was confirmed that a 2,6 sialylation, fucosylation, a mannosylation, a glucosylation, and galactosylation were all increased in LP-1 2,6 IG compared to IM-9 2,6 IG (Fig. 8, 2nd, 3rd, 4th, and 6th columns, respectively). From the above results, it is expected that DC-SIGN will be able to strongly recognize LP-1 2,6 IG because a 2,6 sialylation also increased in LP-1 2,6 IG similar to IM-9 2,6 IG, and it is expected that the development of customized treatments for various inflammatory and autoimmune diseases will be possible.

[0182] From the above results, it was confirmed that the intact immunoglobulins secreted from IM-9 and LP-1 myeloma cells not only differ in light chain conformation but also in their glycosylation patterns. Therefore, if intact immunoglobulins and immunoglobulins enhanced with specific glycosylation components are produced from various myeloma cells and their in vivo mechanisms of action are studied, it will be possible to induce different immune responses, thereby enabling the production of customized human monoclonal immunoglobulins for various diseases.

[0183]

[0184] Example 9: Investigation of the ability of the ST6GAL1-overexpressing IM-9 myeloma cell line to produce stable α 2,6 sialylated IM-9 human IgG (IM-9 2,6)

[0185] When mass-culturing cell lines that produce antibodies, immunoglobulins, and proteins, the following characteristics must be maintained: productivity, genetic stability, product quality, cell line stability, and culture suitability. Productivity refers to the ability to consistently produce large quantities of antibodies, while genetic stability refers to maintaining consistent characteristics over long periods of time without genetic modification. Furthermore, product quality requires that the antibody structure and function be consistent and free of impurities. Cell line stability ensures that characteristics remain unchanged after long-term culture or freezing and thawing. Finally, culture suitability requires that characteristics be maintained even when scaled up from small to large scale.

[0186] In order to confirm the above characteristics, the inventors of the present invention first compared and observed whether there was a change in the amount of ST6GAL expression in IM-9 ST6GAL1 cells passaged 30 times (in 2022) and cell lines cultured for 60 passages long-term (in 2025), since the IM-9 cell line producing IM-9 2.6 overexpresses the ST6GAL1 enzyme that mediates sialylation of immunoglobulin (IM-9 ST6GAL1 cell).

[0187] Since ST6GAL1 was cloned to be co-expressed with GFP in the IM-9 cells, GFP fluorescence was analyzed using FACS. As a result, it was confirmed that ST6GAL1 expression was maintained in IM-9 ST6GAL1 cells even after 60 passages, confirming that genetic stability for ST6GAL1 expression was secured in the IMP-9 cells (Fig. 9a, arrow).

[0188] In addition, IM-9 WT IgG and IM-9 2,6 IgG were isolated and purified from the culture medium of cell lines passaged 30 or 60 times, and it was confirmed through silver staining that the immunoglobulins were isolated and purified with high purity (Fig. 9b). It was additionally confirmed that there was no difference in IgG production capacity according to the number of passages (Fig. 9c, arrow). Through this, it was confirmed that there were no problems with the stability of IM-9 cell lines, IgG productivity, and culture suitability.

[0189] Finally, to determine whether there were differences in glycosylation types between IM-9 WT IgG and IM-9 2,6 IgG produced from cell lines passaged 30 or 60 times, lectin blotting was used to analyze α-2,6 sialylation (SAN), galactosylation and α mannosylation (NPL / NPA), α glucosylation and α mannosylation (CON A), fucosylation (AAL), and α 2,3 sialylation (MAL 2). As a result, it was confirmed that sialylation was increased in IM-9 2,6 IgG produced from cells passaged 60 times compared to IM-9 WT IgG, and there was no difference in the remaining glycosylation types (Fig. 9d, arrow). It was also confirmed that there was no difference in glycosylation types between IM-9 WT and IM-9 2,6 produced from cells passaged 30 times (Fig. 9d).

[0190]

[0191] Example 10: Investigation of the DC-SIGN-increasing effect of IM-9 WT IG and IM-9 2,6 IG in DC-SIGN-expressing cells.

[0192] Since IM-9 WT IG is also basically α 2,6 sialylated and α 2,6 sialylation is greatly increased in IM-9 2,6 IG, we compared the effects on the stimulation of GC total IVIG and GC 2,6 IVIG DC-SIGN.

[0193] When the above substances were reacted with DC-SIGN-overexpressing mouse DCEK fibroblasts (DCEK_DC-SIGN) at a concentration of 25 ug / ml for 24 hours, all of the substances, except GC total IVIG, increased DC-SIGN (Fig. 10a). However, it was confirmed that none of the substances bound to the cells in mouse DCEK_WT fibroblasts (Fig. 10a, left WT).

[0194] Meanwhile, since the DCEK cell line is a mouse cell, in order to predict the response in humans, the same experiment as that shown in Fig. 10a was performed at a concentration of 10 ug / ml using DC-SIGN-expressing Hofbauer cells (normal placental HBCs) isolated from normal human placentas, and then it was confirmed whether human immunoglobulins bound to HBCs. As a result, it was confirmed that all human immunoglobulins bound well to HBCs (Fig. 10b, top).

[0195] In addition, when we confirmed whether the binding of the reacted human immunoglobulin to HBCs increases the expression of DC-SIGN, GC total IVIG did not increase the expression of DC-SIGN, but GC 2,6 IVIG, IM-9 WT IG, and IM-9 2,6 IG were all confirmed to clearly increase the expression of DC-SIGN (Fig. 10b, middle). In addition, GC 2,6 IVIG and IM-9 2,6 with increased sialylation were confirmed to increase DC-SIGN more than IM-9 WT. Therefore, it was additionally confirmed that α-2,6 sialylation of immunoglobulin is important for the reaction of DC-SIGN. These results indicate that IM-9 WT IG and IM-9 2,6 IG can amplify the function of DC-SIGN by increasing its expression through binding to DC-SIGN when delivered to HBCs in the trophoblast tissue of the placenta.

[0196] In addition, to determine which of the two substances, GC 2,6 IVIG and IM-9 2,6 IG, increases DC-SIGN more effectively, normal HBCs were treated with the two substances for 6, 12, 24, and 36 hours, and then the binding of the two substances and the increase in DC-SIGN were examined. As a result, GC 2,6 IVIG showed increased binding and increased DC-SIGN from 12 hours (Fig. 10c, left, arrow head), but IM-9 2,6 showed increased binding and DC-SIGN from 6 hours (Fig. 10c, right, arrow). The reason for this result is that GC 2,6 IVIG is a mixture of IgG1, 2, 3, and 4, which takes more time to bind to and stimulate DC-SIGN by immunoglobulin type, while IM-9 2,6 is an IgG2 monoclonal immunoglobulin, which is expected to induce more rapid binding and response.

[0197] In addition, to determine whether GC 2,6 IVIG and IM-9 2,6 can be developed as therapeutic agents for human preeclampsia, HBCs (PE HBCs) were isolated from the placenta of pregnant women with preeclampsia and the same experiment as in Fig. 10b was performed. As a result, all three substances, GC 2,6 IVIG, IM-9 WT IG, and IM-9 2,6 IG, clearly bound to PE HBCs and increased DC-SIGN (Fig. 10d, top and middle). In addition, as in normal HBCs, GC 2,6 IVIG and IM-9 2,6 increased DC-SIGN more in PE HBCs than in IM-9 WT.

[0198] Since binding of substances and an increase in DC-SIGN were confirmed from 6 hours after treatment with IM-9 2,6 in normal HBCs (Fig. 10c, right), the same experiment was performed using PE HBCs. As a result, IM-9 2,6 was confirmed to bind to PE HBCs from 6 hours, but an increase in DC-SIGN was observed from 12 hours (Fig. 10e, top and middle). Thus, the reason why the increase of DC-SIGN by IM-9 2,6 in PE HBCs was slower than in normal HBCs is expected to be because PE HBCs have been exposed to the preeclamptic environment including hypoxic conditions for a long period of time, so the expression and function of DC-SIGN are maintained at a low level each week, making them slow to respond to stimulation by IM-9 2,6.

[0199]

[0200] Example 11: Identification of the IL-10 increase effect by stimulation with IM-9 WT IG and IM-9 2,6 IG in human placental DC-SIGN-expressing HBCs.

[0201] In addition, since the inventors of the present invention confirmed in Example 10 that IM-9 WT and IM-9 2,6 bind to HBCs and stimulate and increase DC-SIGN, they wanted to confirm whether the production of IL-10, one of the representative immunosuppressive cytokines produced by stimulation of DC-SIGN, increases. To this end, as in Figs. 10a-10e, HBCs isolated from human placenta were treated with GC total IVIG, GC 2,6 IVIG, IM-9 WT IG, and IM-9 2,6 IG, respectively, and then an experiment to increase IL-10 in HBCs cells was performed and an analysis for IL-10 was performed.

[0202] As a result, no increase in IL-10 was observed in GC tIVIG and GC 2,6 IVIG, whereas a clear increase in IL-10 was observed in HBCs samples treated with IM-9 WT IG and IM-9 2,6 IG (Fig. 10A).

[0203] In addition, when IM-9 WT IG and IM-9 2,6 IG were treated with HBCs isolated from the placenta of pregnant women with preeclampsia, it was confirmed that IL-10 increased within HBCs cells. It was confirmed that both IL-10 monomer and dimer clearly increased with both substances (B in Fig. 10).

[0204] In addition, it was confirmed that DC-SIGN was increased more by IM-9 WT IG in C of Fig. 10, and in parallel, more IL-10 monomer was increased by M-9 WT IG ((Fig. 10 B, middle).

[0205] As a result, no substance increased IL-10 in mouse DCEK_WT (Fig. 11a, left), but in the DCEK_DC-SIGN cell line, GC 2,6 IVIG, IM-9 WT, and IM-9 2,6 all increased IL-10 production (Fig. 11a, right). In addition, it was confirmed that IM-9 WT and IM-9 2,6 increased IL-10 more than GC 2,6 IVIG (Fig. 11a, right, arrow).

[0206]

[0207] Next, since GC 2,6 IVIG and IM-9 2,6 increased DC-SIGN the most in normal HBCs (Fig. 10b, middle), we sought to determine how effectively GC 2,6 IVIG and IM-9 2,6 increased IL-10 production in normal HBCs.

[0208] As a result, GC 2,6 IVIG increased IL-10 from 24 hours after DC-SIGN began to increase (Fig. 11b, left, arrow head), but IM-9 2,6 increased IL-10 from 6 hours after DC-SIGN began to increase (Fig. 11b, right, arrow).

[0209]

[0210] In addition, IL-10 production was analyzed after PE HBCs derived from the placenta of patients with preeclampsia were treated with GC 2,6 IVIG, IM-9 WT, and IM-9 2,6 at a concentration of 10 μg / ml for 24 hours. As a result, all three substances increased IL-10 production, and among them, GC 2,6 IVIG and IM-9 2,6 increased IL-10 production to a greater extent (Fig. 11c, arrow).

[0211] In addition, when PE HBCs were treated with IM-9 2,6 at a concentration of 10 ug / ml for different treatment times and IL-10 production was analyzed, it was confirmed that IL-10 production increased from 6 hours, unlike DC-SIGN, which increased from 12 hours (Fig. 11d, arrow). This result is expected to be because IM-9 2,6 is an IgG2 type monoclonal immunoglobulin, so it stimulates DC-SIGN of HBCs more effectively, and thus even a small amount of IM-9 2,6 binds more effectively to stimulate IL-10 production.

[0212]

[0213] Example 12: Comparative confirmation of the anti-inflammatory effect of IM-9 2,6 compared to GC 2,6 IVIG in human placental-derived chorionic BeWo cell line.

[0214] The BeWo cell line is a choriocarcinoma cell line derived from the human placenta. This cell line exhibits the characteristics of placental trophoblast cells and is widely used in studies of placental development, trophoblast syncytialization, and hormone secretion (e.g., hCG, estradiol, etc.). In particular, BeWo cells can mimic the structural and functional characteristics of the placenta in vitro, and are thus utilized in various studies, including nutrient and drug transport, intercellular signaling, and immune responses (Ref. Am J Physiol Cell Physiol. 2008 Sep 24;295(5):C1445-C1453., doi: 10.1152 / ajpcell.00286.2008). These BeWo cell membranes primarily harbor transferrin receptors, FcRn (IgG Fc receptors), and various forms of progesterone receptors, which play crucial roles in iron ion and IgG transport, as well as progesterone signaling, respectively. Furthermore, preliminary experiments by the inventors confirmed that IL-10, IL-10RA, and IL-10RB are expressed in the BeWo cell line.

[0215] Specifically, the BeWo cell line was treated with Human Serum Albumin (HSA), GC IVIG, GC 2,6 IVIG, IM-9 WT and IM-9 2,6 at a concentration of 10 ug / ml for 24 hours, and the expression of anti-inflammatory cytokine IL-10 and IL-10 receptors A and B was confirmed to compare whether each substance induced an anti-inflammatory effect.

[0216] As a result, GC total IVIG showed no response at all to any of the anti-inflammatory factors identified in this experiment, but GC 2,6 IVIG, IM-9 WT, and IM-9 2,6 all induced a marked anti-inflammatory response, and among them, IM-9 2,6 induced the strongest anti-inflammatory response (Fig. 12).

[0217] Since the trophoblast layer is the part of the chorion in the placenta where the maternal blood and fetal cells directly come into contact, and plays a very important role in the immune regulation function through immunosuppression, the fact that IM-9 2,6 can induce the greatest immunosuppressive effect in the placenta by increasing the ability to produce IL-10 and the expression of its receptors compared to other substances suggests that it is also possible to treat preeclampsia using this immunosuppressive function.

Claims

1. A method for producing α-2,6-sialylated immunoglobulin, comprising the following steps: (a) A step of transducing the ST6GAL1 (β-galactoside α-2,6-sialyltransferase 1) gene into plasma cells isolated from a human; (b) a step of isolating plasma cells expressing the ST6GAL1 gene; and (c) A step of producing α 2,6 sialylated immunoglobulin by culturing plasma cells expressing the isolated ST6GAL1 gene.

2. A manufacturing method according to claim 1, wherein the immunoglobulin is in the form of IgG, IgM, IgE, or IgA.

3. A manufacturing method according to claim 1, wherein the plasma cells are cells derived from human myeloma.

4. A manufacturing method according to claim 1, wherein the plasma cells secrete complete immunoglobulin, light chain immunoglobulin, or a combination thereof.

5. A manufacturing method according to claim 1, wherein the ST6GAL1 (β-galactoside α-2,6-sialyltransferase 1) gene is of human origin.

6. A manufacturing method according to claim 1, wherein the sialic acid is N-acetylneuraminic acid (Neu5Ac).

7. A manufacturing method according to claim 1, wherein the transduction is performed by a process of infecting plasma cells isolated from the human with a viral vector into which the ST6GAL1 (β-galactoside α-2,6-sialyltransferase 1) gene is inserted.

8. A manufacturing method according to claim 7, wherein the viral vector comprises a selectable marker gene capable of confirming transduction.

9. A manufacturing method in accordance with claim 1, wherein the culture of the separated plasma cells expressing the ST6GAL1 gene in step (c) is an in vitro cell culture or an in vivo cell culture of an animal other than a human.

10. A manufacturing method according to claim 9, wherein the in vivo cell culture is performed by inoculating plasma cells into the peritoneal cavity of an animal other than a human.

11. A manufacturing method according to claim 1, further comprising a step of purifying the manufactured α-2,6-sialylated immunoglobulin (d).

12. A manufacturing method according to claim 11, wherein the purification uses Protein L beads, Protein A beads, Protein G beads, affinity chromatography, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, or a combination thereof.

13. α-2,6-sialylated immunoglobulin produced in a human-derived cell line.

14. In claim 13, the Fc region of the immunoglobulin comprises a CH2 domain of IgG, and the sialic acid is α-2,6-linked to the terminal galactose of an N-glycan linked to the Asn297 residue of the CH2 domain, an α-2,6-sialylated immunoglobulin.

15. An α-2,6-sialylated immunoglobulin according to claim 14, wherein the N-glycan comprises a complex-type biantennary glycan.

16. In the 13th paragraph, the α-2,6-sialylated immunoglobulin, wherein the sialic acid is N-acetylneuraminic acid (Neu5Ac).

17. In claim 13, the immunoglobulin is an α-2,6-sialylated immunoglobulin in the form of IgG.

18. In the 13th paragraph, the immunoglobulin is an α-2,6-sialylated immunoglobulin in the form of IgG2.

19. In claim 13, the α-2,6-sialylated immunoglobulin is produced by the production method of any one of claims 1 to 12.

Images