JP7523789B2 - Anti-inflammatory nonfucosylated immunoglobulin preparation and method for producing same - Google Patents

Description

The present invention relates to an anti-inflammatory nonfucosylated immunoglobulin preparation and a method for producing the same.

IVIG is one of the few treatment options for autoimmune diseases such as Kawasaki disease, idiopathic thrombocytopenic purpura, and Guillain-Barré syndrome. IVIG is produced by fractionating IgG from the plasma of several thousand healthy individuals, purifying it, and concentrating it. The therapeutic effect is in the IgG-Fc portion (Non-Patent Documents 1 and 2), and it has been reported that the Asn297-linked glycan of IgG-Fc is necessary (Non-Patent Document 2), but the mechanism of the anti-inflammatory effect is unclear. The glycan of IgG-Fc is essential for the expression of IgG effector functions such as Fcγ receptors and complement activation, and removal of the glycan significantly impairs antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (Non-Patent Document 3). The IgG-Fc glycan shows high heterogeneity with fucose, galactose, sialic acid, etc. linked to a core structure consisting of seven sugars (Figure 1). It has been reported that the monosaccharides linked to these non-reducing ends are associated with various biological activities (Non-Patent Document 4).

It has been shown that fucose removal enhances ADCC by about 100-fold (Non-Patent Documents 5 and 6), and non-fucosylated monoclonal antibodies (Mogamulizumab, Obinutuzumab) have already been used clinically for adult T-cell leukemia and CD20-positive follicular lymphoma. Identification of the active ingredient in IVIG that has an anti-inflammatory effect in autoimmune diseases and elucidation of its mechanism of action are also necessary for the development of new antibody drugs to replace IVIG, for which demand will continue to grow.

Debre, M., et al., Infusion of Fc gamma fragments for treatment of children with acute immune thrombocytopenic purpura. Lancet, 1993. 342(8877): p. 945-9. Schwab, I. and F. Nimmerjahn, Intravenous immunoglobulin therapy: how does IgG modulate the immune system? Nat Rev Immunol, 2013. 13(3): p. 176-89. Mimura, Y., et al., Glycosylation engineering of therapeutic IgG antibodies: challenges for the safety, functionality and efficacy. Protein Cell, 2018. 9(1): p. 47-62. Zhang, P., et al., Challenges of glycosylation analysis and control: an integrated approach to producing optimal and consistent therapeutic drugs. Drug Discov Today, 2016. 21(5): p. 740-65. Shinkawa, T., et al., The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J Biol Chem, 2003. 278 (5): p.3466-73. Shields, R.L., et al., Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity. J Biol Chem, 2002. 277(30): p. 26733-40.

The present invention was made in consideration of these problems, and aims to provide a new therapeutic agent for inflammatory diseases such as autoimmune diseases.

The anti-inflammatory nonfucosylated immunoglobulin preparation of the present invention is characterized in that the IgG antibody contained therein is a human serum IgG antibody in which the glycan shown below is bound to asparagine 297 (Asn297) in the Fc portion, where S is sialic acid, G is galactose, N is N-acetylglucosamine, and M is mannose.

The anti-inflammatory nonfucosylated immunoglobulin preparation of the present invention is characterized in that the IgG antibody contained therein is a human serum IgG antibody in which the glycan shown below is bound to asparagine 297 (Asn297) in the Fc portion, where G is galactose, N is N-acetylglucosamine, and M is mannose.

The method for producing an anti-inflammatory nonfucosylated immunoglobulin preparation according to the present invention is characterized by comprising a glycan removal step in which the glycan bound to the asparagine 297 (Asn297) residue in the Fc portion of a human serum IgG antibody is cleaved at the chitobiose core using endoglycosidase S (Endo S) to remove all but the N-acetylglucosamine bound to the Asn297 residue and the fucose bound to the N-acetylglucosamine, a fucose removal step in which the fucose bound to the N-acetylglucosamine is removed using α-L-fucosidase (AlfC), and a transfer step in which an oxazoline glycan (SG-Ox) prepared from a sialyl glycopeptide is transferred to the N-acetylglucosamine bound to the asparagine 297 (Asn297) residue in the Fc portion of the human serum IgG antibody using glycosynthase (Endo S D233Q).

The method for producing an anti-inflammatory nonfucosylated immunoglobulin preparation according to the present invention is characterized by comprising a glycan removal step in which the glycan bound to the asparagine 297 (Asn297) residue in the Fc portion of a human serum IgG antibody is cleaved at the chitobiose core using endoglycosidase S (Endo S) to remove all but the N-acetylglucosamine bound to the Asn297 residue and the fucose bound to the N-acetylglucosamine, a fucose removal step in which the fucose bound to the N-acetylglucosamine is removed using α-L-fucosidase (AlfC), and a transfer step in which an oxazoline glycan (GG-Ox) prepared from a galactosyl glycopeptide is transferred to the N-acetylglucosamine bound to the asparagine 297 (Asn297) residue in the Fc portion of the human serum IgG antibody using glycosynthase (Endo S D233Q).

The present invention provides a new therapeutic agent for inflammatory diseases such as autoimmune diseases.

FIG. 1 is a diagram illustrating the heterogeneity of IgG-Fc sugar chains. This figure explains the difference in the killing activity of NK cells between fucosylated serum IgG (S2F), in which fucose is present in the glycan of the Fc portion of the antibody, and non-fucosylated serum IgG (S2), in which fucose is not present in the glycan of the Fc portion of the antibody. This is a photograph of SDS-PAGE of fucosylated serum IgG (S2F), in which fucose is present in the glycans of the Fc portion of the antibody, and non-fucosylated serum IgG (S2), in which fucose is not present in the glycans of the Fc portion of the antibody. FIG. 1 shows the process by which oxazolinyl glycan (SG-Ox) is produced from a glycan released from a sialylglycopeptide by Endo S. FIG. 2 is a diagram showing by high performance liquid chromatography that fucose is removed from IgG(S2) of the anti-inflammatory nonfucosylated immunoglobulin preparation of this example. FIG. 1 shows that normal serum IgG (95% fucosylated IgG) suppresses ADCC in a concentration-dependent manner. FIG. 1 shows that nonfucosylated serum IgG (S2) exhibits a more pronounced inhibitory effect on antibody-dependent cellular cytotoxicity (ADCC) than fucosylated serum IgG (S2F). FIG. 1 is a photograph showing the results of SDS electrophoresis of fucosylated serum IgG (S2F), non-fucosylated serum IgG (S2), and galactosyl-non-fucosylated serum IgG (G2). FIG. 1 shows the process by which an oxazolinyl glycan (GG-Ox) is produced from a glycan released from a galactosyl glycopeptide by Endo S. FIG. 1 shows, by high performance liquid chromatography, that fucose has been removed from IgG(S2) and IgG(G2) of the anti-inflammatory nonfucosylated immunoglobulin preparation of the present invention. FIG. 1 shows that normal serum IgG (95% fucosylated IgG) suppresses ADCC in a concentration-dependent manner. FIG. 1 shows that nonfucosylated serum IgG (G2) exhibits a significant inhibitory effect on antibody-dependent cellular cytotoxicity (ADCC) compared with fucosylated serum IgG (S2F) and nonfucosylated serum IgG (S2).

The following describes in detail an embodiment of the present invention with reference to the attached drawings. However, this embodiment is intended to facilitate understanding of the principles of the present invention, and the scope of the present invention is not limited to the embodiment described below. Other embodiments in which a person skilled in the art appropriately replaces the configuration of the embodiment described below are also included in the scope of the present invention.

The anti-inflammatory nonfucosylated immunoglobulin preparation according to this embodiment is characterized in that the IgG antibody contained therein is a human serum IgG antibody in which the glycan shown below is bound to asparagine 297 (Asn297) in the Fc portion, where S is sialic acid, G is galactose, N is N-acetylglucosamine, and M is mannose.

The anti-inflammatory nonfucosylated immunoglobulin preparation according to this embodiment is characterized in that the IgG antibody contained therein is a human serum IgG antibody in which the glycan shown below is bound to asparagine 297 (Asn297) in the Fc portion, where G is galactose, N is N-acetylglucosamine, and M is mannose.

Immunoglobulins include, but are not limited to, antibodies and antibody fragments (scFv, Fab, Fc, F(ab')2) and other genetically engineered portions of antibodies. Immunoglobulins, or antibodies, are a group of glycoproteins present in the serum and tissue fluids of all mammals. IgG (Immunoglobulin G) accounts for approximately 75-85% of immunoglobulins in normal human serum. Glycosylation plays an important role in maintaining the overall three-dimensional structure of IgG, and there are 16 types of neutral glycans. The glycan structure of IgG is composed of a very heterogeneous mixture of glycans, but the relative ratio of the 16 types is almost constant in healthy individuals. It is known that the glycans of myeloma and rheumatoid arthritis patients show very specific relative ratios.

Antibody-dependent cellular cytotoxicity (ADCC) is a targeted cytotoxic activity induced in an antibody-dependent manner when an antibody bound to a target cell binds to an Fc receptor on effector cells such as natural killer cells (NK cells) and macrophages. ADCC activity is considered to be one of the most important factors in the manifestation of the efficacy of antibody drugs.

Two N-glycoside-linked glycans are bound to the Fc region of one IgG antibody molecule. The N-glycoside-linked glycans of this antibody are complex-type biantennary glycans with a mannosyl-chitobiose core structure as the basic structure, and there is diversity in the presence or absence of galactose and sialic acid at the non-reducing end and in the presence or absence of fucose at the reducing end.

Removal of fucose residues from the N-acetylglucosamine at the reducing end of the N-glycosidically linked complex glycan bound to the antibody Fc region increases the affinity for the Fcγ receptor IIIa. In this specification, nonfucosylated IgG having sialic acid at the end can be expressed as IgG(S2) (or simply referred to as nonfucosylated IgG), and nonfucosylated IgG having galactose at the end can be expressed as IgG(G2) (or can simply be referred to as galactosyl nonfucosylated IgG).

IgG(S2) has a structure in which the glycan shown below is bound to asparagine 297 (Asn297) in the Fc portion. Here, S is sialic acid, G is galactose, N is N-acetylglucosamine, and M is mannose.

IgG(G2) has a structure in which the glycan shown below is bound to asparagine 297 (Asn297) in the Fc portion. Here, G is galactose, N is N-acetylglucosamine, and M is mannose.

The expression "the IgG antibodies contained in the immunoglobulin preparation are composed of human serum IgG antibodies in which a specific sugar chain is bound to asparagine 297 (Asn297) in the Fc portion" means that 95% to 100% of the IgG antibodies contained in the immunoglobulin preparation are human serum IgG antibodies in which a specific sugar chain is bound to asparagine 297 (Asn297) in the Fc portion, preferably 98% to 100%, and most preferably 100%.

Therefore, an anti-inflammatory nonfucosylated immunoglobulin preparation (where S is sialic acid, G is galactose, N is N-acetylglucosamine, and M is mannose) in which the IgG antibody contained therein is a human serum IgG antibody in which the glycan shown below is bound to asparagine 297 (Asn297) in the Fc portion means that the human serum IgG antibody in which the glycan shown below is bound to asparagine 297 (Asn297) in the Fc portion is contained in 95% to 100%, preferably 98% to 100%, and most preferably 100% of the IgG antibody contained in the immunoglobulin preparation.

An anti-inflammatory nonfucosylated immunoglobulin preparation (where G is galactose, N is N-acetylglucosamine, and M is mannose) in which the IgG antibody contained therein is a human serum IgG antibody in which the glycan shown below is bound to asparagine 297 (Asn297) in the Fc portion means that 95% to 100%, preferably 98% to 100%, and most preferably 100% of the IgG antibody contained in the immunoglobulin preparation is a human serum IgG antibody in which the glycan shown below is bound to asparagine 297 (Asn297) in the Fc portion.

On the other hand, fucosylated IgG can be expressed as IgG(S2F), which has a structure in which the glycan shown below is bound to asparagine 297 (Asn297) in the Fc portion. Here, S is sialic acid, G is galactose, N is N-acetylglucosamine, M is mannose, and F is fucose.

As shown in Figure 2, fucosylated IgG cannot inhibit the interaction between autoantibody-antigen complexes and Fcγ receptor IIIa on NK cells, whereas non-fucosylated IgG (non-fucosylated serum IgG shown in B of Figure 2 is IgG(S2) or IgG(G2)) strongly binds to Fcγ receptor IIIa on NK cells and inhibits the killing action of NK cells, which is thought to result in the suppression of inflammation.

When the immunoglobulin preparation of the present invention is a liquid preparation, it is used as is or after dilution with an appropriate solvent (e.g., distilled water for injection, physiological saline, glucose solution, etc.), and when it is a dry preparation, the immunoglobulin solution is lyophilized and then dissolved in an appropriate solvent (e.g., distilled water for injection, etc.) at the time of use.

The immunoglobulin preparation of the present invention is usually administered by injection, and intravenous administration is particularly preferred. The standard dosage of the immunoglobulin preparation of the present invention is 50 to 1,000 mg of immunoglobulin per kg of body weight per day, administered intravenously for one to several days in a row, but the dosage can be increased or decreased depending on symptoms, sex, body weight, etc.

The immunoglobulin solution of the present invention may contain pharmacologically acceptable additives (e.g., carriers, excipients, diluents, etc.), stabilizers, or pharma- ceutical components commonly used in pharmaceuticals, to the extent that this does not contradict the object of the present invention.

Examples of stabilizers include monosaccharides such as glucose, disaccharides such as saccharose and maltose, sugar alcohols such as mannitol and sorbitol, neutral salts such as sodium chloride, amino acids such as glycine, polyethylene glycol, polyoxyethylene-polyoxypropylene copolymers (Pluronic (registered trademark)), polyoxyethylene sorbitan fatty acid esters (Tween), and other non-ionic surfactants, and are preferably added at about 1 to 10 w/v%.

Diseases for which the immunoglobulin preparation of the present invention is administered include, but are not limited to, Kawasaki disease, idiopathic thrombocytopenic purpura, Guillain-Barré syndrome, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, pemphigus, pemphigoid, myasthenia gravis, ANCA-associated vasculitis, chronic inflammatory demyelinating polyneuropathy, autoimmune neutropenia, autoimmune hemolytic anemia, autoimmune acquired coagulation factor VIII deficiency, stiff-person syndrome, multifocal neuropathy, Behçet's disease, ulcerative colitis, Crohn's disease, Reiter's arthritis, polymyositis, dermatomyositis, localized scleroderma, systemic scleroderma, Sjogren's syndrome, antiglomerular basement membrane nephritis, primary sclerosing cholangitis, primary antiphospholipid syndrome, toxic epidermal necrolysis, graft-versus-host disease, and sepsis.

Antibody drugs in which fucose has been removed from the glycan of the Fc portion of a monoclonal IgG antibody have been developed for the purpose of enhancing antibody-dependent cellular cytotoxicity (ADCC) (e.g., Mogamulizumab (trade name PoteligeoTM)). Conventional nonfucosylated antibodies are antigen-specific monoclonal antibodies produced from mammalian host cells in which glycan-related genes have been engineered, and are intended for use in the treatment of malignant tumors. On the other hand, the immunoglobulin of the present invention is a nonspecific IgG antibody derived from serum in which the fucose has been enzymatically removed and then a uniform glycan has been transferred by a chemical enzyme reaction, and is intended for use in the treatment of autoimmune diseases. In other words, the immunoglobulin preparation of the present invention is used for the purpose of inhibiting undesirable immune responses caused by autoantibodies.

The method for producing an anti-inflammatory nonfucosylated immunoglobulin preparation according to this embodiment includes a glycan removal step in which the glycan bound to the asparagine 297 (Asn297) residue in the Fc portion of a human serum IgG antibody is cleaved with a chitobiose core to remove all but the N-acetylglucosamine bound to the Asn297 residue and the fucose bound to the N-acetylglucosamine, a fucose removal step in which the fucose bound to the N-acetylglucosamine is removed, and a transfer step in which an oxazolinyl glycan (SG-Ox) prepared from an egg yolk-derived sialylglycopeptide is transferred to the N-acetylglucosamine bound to the asparagine 297 (Asn297) residue in the Fc portion of a human serum IgG antibody using glycosynthase (Endo S D233Q).

The method for producing an anti-inflammatory nonfucosylated immunoglobulin preparation according to the present embodiment is characterized by comprising a glycan removal step of cleaving the glycan bound to the asparagine 297 (Asn297) residue in the Fc portion of a human serum IgG antibody with endoglycosidase S (Endo S) at the chitobiose core to remove N-acetylglucosamine bound to the Asn297 residue and fucose bound to the N-acetylglucosamine, a fucose removal step of removing the fucose bound to the N-acetylglucosamine using α-L-fucosidase (AlfC), and a transfer step of transferring an oxazoline glycan (GG-Ox) prepared from an egg yolk-derived galactosyl glycopeptide to the N-acetylglucosamine bound to the asparagine 297 (Asn297) residue in the Fc portion of the human serum IgG antibody.

The enzyme that cleaves the glycan attached to the asparagine 297 (Asn297) residue in the Fc portion at the chitobiose core is endoglycosidase S (Endo S).

The enzyme that removes fucose bound to N-acetylglucosamine is α-L-fucosidase (AlfC). α-L-fucosidase includes 1,2-α-L-fucosidase, 1,3-α-L-fucosidase, and 1,6-α-L-fucosidase, but it is preferable to use 1,6-α-L-fucosidase here.

Oxazolinyl glycans (SG-Ox or GG-Ox) are prepared by dehydration condensation of the reducing end of sialylglycan.

The enzyme that transfers SG-Ox or GG-Ox to deglycosylated IgG is glycosynthase (Endo S D233Q).

1. Production of anti-inflammatory nonfucosylated immunoglobulin preparations
1a-1. Purification of serum-derived IgG 20 ml of serum prepared from healthy subjects was dialyzed against 0.01 M phosphate buffer (pH 7.0) and then loaded onto a diethylaminoethyl (DEAE)-cellulose anion exchange column (DE52, Whatman Biosystems, Chalfont, St Giles, UK) (1 x 30 cm) equilibrated with the same buffer, and IgG contained in the flow-through fraction was collected. The sugar chain structure of serum IgG was analyzed by high performance liquid chromatography as described below (Figure 5-A), and it was confirmed that more than 95% was fucosylated.

1a-2. Deglycosylation of serum IgG
Endo S was used as an endoglycosidase to cleave the Fc-linked glycan of IgG. The chitobiose core of the glycan was hydrolyzed between the two N-acetylglucosamines, leaving a disaccharide of N-acetylglucosamine and fucose, and deglycosylated. The deglycosylated IgG was purified with Protein G-Sepharose 4 (GE).

1a-3. Defucosylation of serum IgG α-L-fucosidase (AlfC) was used to remove fucose. The DNA sequence of the expression vector for this enzyme was previously reported (SEQ ID NO: 1), and it was expressed in BL21(DE3) Escherichia coli according to the literature. IgG deglycosylated with Endo S was reacted with AlfC in 50 mM Tris-HCl (pH 7.4) at 37°C overnight to remove fucose.

1a-4. Preparation of sialylglycan oxazoline (SG-Ox) The glycan of the sialylglycopeptide shown below (10 mg, Tokyo Chemical Industry Co., Ltd.) was cleaved with Endo S in 50 mM phosphate buffer (pH 6.0), and 2-chloro-1,3-dimethylimidazolinium chloride (Tokyo Chemical Industry Co., Ltd.) and triethylamine were added. The mixture was left at 0°C for 1 hour, and SG-Ox was prepared by dehydration condensation reaction of the reducing end.

Figure 4 shows the process for producing oxazoline glycans (SG-Ox) from sialyl glycans released by Endo S. The structure of SG-Ox is shown below.

1a-5. Preparation of nonfucosylated IgG The DNA sequence of the expression vector for glycosynthase Endo S D233Q was reported in the literature (SEQ ID NO: 2) and expressed in BL21(DE3) E. coli. The glycosylation reaction was carried out using SG-Ox by incubating in 50 mM Tris-HCl (pH 7.4) at 37°C for 3 hours. This resulted in the production of an anti-inflammatory nonfucosylated immunoglobulin preparation according to this example. The nonfucosylated IgG according to this example can be referred to as IgG(S2). Both IgG(S2) and its fucosylated IgG, IgG(S2F), were confirmed to have a purity of 95% or more by SDS-PAGE (Figure 3). The glycosylation reaction of SG-Ox to GlcNAc-IgG is shown below.

To confirm the modification of the glycans in the anti-inflammatory nonfucosylated immunoglobulin preparation of this example, IgG(S2) and IgG(S2F) with the replaced glycans were digested with N-glycosidase F to release the glycans, which were then fluorescently labeled with 2-aminobenzamide and analyzed by high performance liquid chromatography (HPLC). IgG(S2F) is a fucosylated IgG according to the comparative example, specifically a human serum IgG in which the glycan shown below is bound to asparagine 297 (Asn297) in the Fc portion.

As shown in Figure 5, the glycans of the control IgG mostly contain fucose, but it was confirmed that fucose was removed from the IgG(S2) of the anti-inflammatory nonfucosylated immunoglobulin preparation of this example.

1b. ADCC Suppression (Anti-Inflammation) Effect of Nonfucosylated Serum IgG The anti-inflammatory effect of the anti-inflammatory nonfucosylated immunoglobulin preparation of this example was confirmed by the following method.

In a preliminary experiment, the effect of normal serum IgG (95% fucosylated IgG, the control IgG shown in Figure 5A) on ADCC was examined using the ADCC Reporter bioassay kit (Promega). Target cells were labeled with a hapten (4-hydroxy-3-iodo-5-nitrophenacetyl) and sensitized with anti-hapten monoclonal IgG antibody (0.001-3 μg/ml) (Figure 6).

The strength of the ADCC activity of anti-hapten IgG is indicated by the anti-hapten IgG concentration (EC50) that can induce 50% of the maximum level of target cell death (cytotoxicity, Figure 6, Y axis). The lower the EC50, the higher the ADCC activity of the specific antibody. When serum IgG was added to the ADCC measurement system at concentrations of 0.01, 0.1, 1, and 8 mg/ml and the EC50 of anti-hapten IgG was compared, the higher the concentration of serum IgG added, the higher the EC50 (shifted to the right on the X axis), indicating that ADCC was suppressed (Figure 6).

On the other hand, to investigate whether there is a difference in the ADCC inhibitory effect between the glycosylated serum IgGs nonfucosylated IgG (S2) and fucosylated IgG (S2F), the ADCC activity of anti-CD20 IgG was compared in their presence (Figure 7). The concentrations of S2 and S2F were 0.1 mg/ml. S2F showed the same EC50 (0.2 μg/ml) as the control and did not show any inhibitory effect (Figure 7). On the other hand, in the presence of S2, the EC50 was >3 μg/ml, which was more than 15 times higher (Figure 7). In other words, S2 exerted an ADCC inhibitory effect more than 15 times that of S2F. Therefore, the use of nonfucosylated IVIG is expected to have an anti-inflammatory effect at a smaller dose than regular IVIG, and it may also have an anti-inflammatory effect in cases where regular IVIG is ineffective.

2. Preparation of anti-inflammatory galactosyl nonfucosylated immunoglobulin preparations
2a-1. Purification of serum-derived IgG 20 ml of serum prepared from healthy subjects was dialyzed against 0.01 M phosphate buffer (pH 7.0) and then loaded onto a diethylaminoethyl (DEAE)-cellulose anion exchange column (DE52, Whatman Biosystems, Chalfont, St Giles, UK) (1 x 30 cm) equilibrated with the same buffer, and the IgG contained in the flow-through fraction was collected. The purity was confirmed to be 95% or more by SDS-PAGE (Figure 8, lane 1).

2a-2. Glycosylation of serum IgG
Endo S was used as an endoglycosidase to cleave the Fc-linked glycan of IgG. The DNA sequence of the expression vector for Endo S was previously reported and expressed in BL21(DE3) Escherichia coli. Endo S immobilized on cyanogen bromide-activated Sepharose 4B beads was used to hydrolyze the bond between the two N-acetylglucosamines in the chitobiose core of the glycan, leaving a disaccharide of N-acetylglucosamine and fucose for deglycosylation. The deglycosylated IgG was purified with Protein G-Sepharose 4 (GE).

2a-3. Defucosylation of serum IgG α-L-fucosidase (AlfC) was used to remove fucose. The DNA sequence of the expression vector for this enzyme was previously reported (SEQ ID NO: 1), and it was expressed in BL21(DE3) Escherichia coli according to the literature. IgG deglycosylated with Endo S was reacted with AlfC in 50 mM Tris-HCl (pH 7.4) at 37°C overnight to remove fucose.

2a-4. Preparation of galactosylglycan oxazoline (GG-Ox) Sialyl glycopeptide (10 mg, Tokyo Chemical Industry Co., Ltd.) was reacted with sialidase (Roche) and Endo S immobilized on Sepharose in 50 mM phosphate buffer (pH 6.0) overnight at 37°C. The galactosyl glycopeptide shown below was the intermediate, and its sugar chain was cleaved to produce galactosyl glycan. 2-Chloro-1,3-dimethylimidazolinium chloride (Tokyo Chemical Industry Co., Ltd.) and triethylamine were then added, and the mixture was left at 0°C for 1 hour, and GG-Ox was prepared by dehydration condensation reaction of the reducing end. GG-Ox was purified using a cellulose column (Sigma-Aldrich).

Figure 9 shows the process for producing GG-Ox from glycans released by Endo S. The structure of GG-Ox is shown below.

2a-5. Preparation of Galactosyl-Defucosylated IgG The DNA sequence of the expression vector for glycosynthase Endo S D233Q was reported in the literature and expressed in BL21(DE3) Escherichia coli. The glycosyl transfer reaction was performed by incubating Endo S D233Q with 50 mM Tris-HCl (pH 7.4) at 30°C for 4 hours using GG-Ox. This resulted in the production of an anti-inflammatory galactosyl-defucosylated immunoglobulin preparation according to this embodiment. The galactosyl-defucosylated IgG according to this embodiment can be referred to as IgG(G2). To confirm the modification of the glycan, the IgGs (S2, S2F, G2) with the replaced glycan were digested with N-glycosidase F to release the glycan, and then fluorescently labeled with 2-aminobenzamide and analyzed by high performance liquid chromatography (HPLC) (Figure 10). The glycosyl transfer reaction of GG-Ox to GlcNAc-IgG is shown below.

2b. ADCC inhibitory (anti-inflammatory) effect of galactosyl-nonfucosylated immunoglobulin We found that fucose-depleted serum IgG has anti-inflammatory activity, and that this effect is enhanced by having galactose at the terminus. In a preliminary experiment, the effect of the presence of normal serum IgG (IVIG, 95% fucosylated, Fig. 10A) on ADCC was examined using the ADCC Reporter bioassay kit (Promega). Target cells (Raji cells) were sensitized with CD20 antibody (rituximab) (0.001-3 μg/ml, Fig. 11). The strength of ADCC activity is indicated by the antibody concentration (EC50, Fig. 11 X-axis) that can induce 50% of the maximum level of target cell death (cytotoxicity, Fig. 11 Y-axis). The smaller the EC50 value, the stronger the ADCC activity. When serum IgG (IVIG) was added to the ADCC measurement system at concentrations of 0, 0.1, 1, and 10 mg/ml, the EC50 values were compared, being 0.026, 0.032, 0.6, and >2 μg/ml, respectively. This indicates that the higher the IVIG concentration, the higher the EC50 value and the more inhibited ADCC was (Figure 11).

Next, ADCC was measured in the presence of 0.1 mg/ml of glycosylated nonfucosylated IgG (S2), galactosyl nonfucosylated IgG (G2) and fucosylated IgG (S2F) (Figure 12). The EC50 values were 0.023, 0.028, 0.12 and 0.3 μg/ml in the presence of PBS (negative control), S2F, S2 and G2, respectively, and the strongest inhibitory effect was observed in the presence of G2 (Figure 12). This corresponds to a relative ADCC activity of 100, 82, 19.2 and 7.6%. In other words, G2 exerted 11-fold and 2.5-fold greater ADCC inhibitory effect than S2F and S2, respectively. Therefore, the use of galactosyl-nonfucosylated IVIG is expected to have an anti-inflammatory effect at a smaller dose than regular IVIG, and may even be able to exert an anti-inflammatory effect in cases where regular IVIG is ineffective.

It can be used to treat autoimmune diseases.

Sequence numbers 1 and 2: Enzyme

Claims (4)

An anti-inflammatory nonfucosylated intravenous immunoglobulin preparation (wherein S is sialic acid, G is galactose, N is N-acetylglucosamine, and M is mannose), characterized in that the IgG antibody comprises a human serum IgG antibody having the following glycan linked to asparagine 297 (Asn297) in the Fc portion, and is used for the prevention and/or treatment of autoimmune diseases.

The anti-inflammatory nonfucosylated immunoglobulin preparation according to claim 1, wherein the autoimmune disease is any one of Kawasaki disease, idiopathic thrombocytopenic purpura, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, autoimmune neutropenia, autoimmune hemolytic anemia, autoimmune acquired coagulation factor VIII deficiency, multiple sclerosis, myasthenia gravis, stiff-person syndrome, multifocal neuropathy, ANCA-associated vasculitis, rheumatoid arthritis, systemic lupus erythematosus, pemphigus, pemphigoid, Behcet's disease, ulcerative colitis, Crohn's disease, Reiter's arthritis, polymyositis, dermatomyositis, localized scleroderma, systemic sclerosis, Sjogren's syndrome, antiglomerular basement membrane nephritis, primary sclerosing cholangitis, primary antiphospholipid syndrome, toxic epidermal necrolysis, graft-versus-host disease, and sepsis. a glycan removal step in which the glycan bound to the asparagine 297 (Asn297) residue in the Fc portion is cleaved at the chitobiose core using endoglycosidase S (Endo S) on the human serum IgG antibody, thereby removing all but N-acetylglucosamine bound to the Asn297 residue and fucose bound to the N-acetylglucosamine;
a fucose removal step of removing fucose bound to the N-acetylglucosamine using α-L-fucosidase (AlfC);
A method for producing an anti-inflammatory nonfucosylated immunoglobulin preparation for intravenous injection, comprising a transfer step of transferring an oxazoline glycan (SG-Ox) prepared from a sialylglycopeptide to an N-acetylglucosamine bound to the asparagine 297 (Asn297) residue in the Fc portion of a human serum IgG antibody using glycosynthase ( Endo S D233Q). 4. The method for producing an anti-inflammatory nonfucosylated immunoglobulin preparation according to claim 3 , wherein in the fucose removal step, α-L-fucosidase is 1,6-α-L-fucosidase.

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