Endo-S2 mutant as a glycosynthase, for the production of glycoproteins and for glycosylation of glycoproteins

Abstract

To synthesize glycoproteins.SOLUTION: The present invention provides recombinant Endo-S2 mutants (named Endo-S2 glycosynthases) that exhibit reduced hydrolysis activity and increased transglycosylation activity for the synthesis of glycoproteins in which a desired sugar chain is added to a fucosylated or nonfucosylated GlcNAc-IgG acceptor. As such, the present invention allows the synthesis and remodeling of therapeutic antibodies, thereby providing certain biological activities, such as, prolonged half-life time in vivo, less immunogenicity, enhanced in vivo activity, increased targeting ability, and / or the ability to deliver a therapeutic agent.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] GOVERNMENT RIGHTS IN THE INVENTION This invention was made with government support under grant numbers R01 GM096973 and R01 GM080374 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to co-pending U.S. Provisional Patent Application No. 62 / 279,087, filed on January 15, 2016, the content of which is incorporated herein by reference for all purposes.

[0003] BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to glycoprotein synthesis, and more specifically to the use of recombinant mutant Endo S2, which is an Endo-β-N-acetylglucosaminidase derived from Streptococcus pyogenes serotype 49 strain NZ131, having transglycosylation activity and limited hydrolysis activity, thereby enabling efficient glycosylation remodeling of antibodies. Background Art

[0004] Description of Related Art Monoclonal antibodies (mAbs) represent a major class of therapeutic proteins used to treat cancer, inflammatory disorders, and infectious diseases [1-3]. Compelling experimental data have shown that glycosylation can significantly affect antibody stability, biological function, and overall therapeutic efficacy [4-7]. For example, core fucosylation of Fc glycans can significantly reduce antibody-dependent cytotoxicity (ADCC), and antibodies with low core fucosylation content have shown improved therapeutic efficacy in anticancer treatment [8, 9]. On the other hand, terminal α-2,6-sialylated Fc glycoforms, which are trace components in intravenous immunoglobulins (IVIGs), have been reported to be involved in the anti-inflammatory activity of IVIGs, as demonstrated in animal models [10-13]. However, natural and recombinant antibodies usually arise as heterogeneous glycoforms, which are difficult to separate for further investigation of the antibody structure-activity relationship. Furthermore, in the case of the majority of anti-cancer mAbs on the market that rely on ADCC as the primary mechanism of therapeutic effect, the most active non-fucosylated glycoforms usually exist as a minority proportion in this heterogeneous mixture [8, 9]. Therefore, methods that can produce antibodies with structurally clearly defined homogeneous glycoforms are highly desirable for both functional studies and the development of better antibody-based therapies. In parallel with attempts to control glycosylation through manipulation of the host glycosylation pathway [14-20], chemoenzymatic glycosylation reconstitution methods, including endoglycosidase-catalyzed deglycosylation and subsequent transglycosylation of intact antibodies, have emerged as promising approaches for obtaining homogeneous antibody glycoforms

[21] . It has been shown that Fc glycans of recombinant IgG-Fc domains can be reconstituted by enzymatic deglycosylation-transglycosylation steps catalyzed by suitable endoglycosidases such as Endo-A and Endo-D without requiring protein denaturation [22-24]. In 2012, the first examples of intact therapeutic monoclonal antibodies and glycosylated reconstitution of IVIG were made possible by the discovery of a glycosynthase variant of Endo-S, an endoglycosidase derived from Streptococcus pyogenes.

[25] In this approach, Heterogeneous Fc glycans of monoclonal antibodies such as rituximab are removed by deglycosylation catalyzed by Endo S to obtain an antibody protein backbone that carries only α1,6-fucosylated GlcNAc acceptors at the glycosylation site. Then, the desired N-glycan is transferred to the GlcNAc acceptor in a site-specific and stereospecific manner using an Endo-S glycosynthase mutant (Endo S-D233A or D233Q) to reconstitute a homogeneous glycoform of the antibody. Several research groups have used Endo-S glycosynthase mutants to synthesize various homogeneous glycoforms of antibodies for structural and functional studies [26-31]. Endo-S glycosynthase was able to transfer branched complex and modified Man3GlcNAc cores, but this mutant showed only slight activity in transferring high-mannose N-glycans. In recent years, glycosynthase mutants derived from Endo-F3, another GH18 family endoglycosidase, have been generated

[32] . Endo-F3 glycosynthases such as the D165A mutant were able to transfer tribranched N-glycans to the Fc domain of intact antibodies, but it was found that they required an α1,6-fucosylated GlcNAc moiety as a transglycosylation acceptor and could not transfer to a non-fucosylated GlcNAc acceptor. These studies have demonstrated that currently available glycosynthases still have limitations due to substrate specificity and that their transglycosylation efficiencies also differ. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] Efforts to broaden the scope of glycosylation reconstitution strategies by understanding the shortcomings of other variants have been described herein, and herein, Endo-S2, an endoglycosidase from serotype M49 Streptococcus pyogenes, has been highlighted [33, 34]. Endo-S2 exhibits only 37% sequence identity with Endo-S from the same bacterium and shows broader glycan substrate specificity in Fc deglycosylation compared to Endo-S

[34] . The data suggested that wild-type Endo-S2 can hydrolyze various types of N-glycans derived from the antibody Fc domain compared to Endo-S. However, it is unknown whether Endo-S2 has transglycosylation activity, and if so, whether Endo-S2 glycosynthase can be generated by site-directed mutagenesis.

[0006] Therefore, it would be beneficial to evaluate whether an efficient glycosynthase can be generated from Endo-S2, and whether this glycosynthase potentially possesses broad substrate specificity in transglycosylation compared to those already reported. [Means for solving the problem]

[0007] Summary of the Invention The present invention provides a recombinant Endo-S2 variant (named Endo-S2 glycosynthase) exhibiting reduced hydrolytic activity and increased transglycosylation activity for the synthesis of glycoproteins in which a desired glycan is attached to a fucosylated GlcNAc-IgG acceptor or a non-fucosylated GlcNAc-IgG acceptor. Thus, the present invention enables the synthesis and reconstitution of therapeutic antibodies, thereby providing specific biological activities (e.g., extended in vivo half-life, lower immunogenicity, enhanced in vivo activity, increased targeting ability, and / or ability to deliver therapeutic agents). This variant Endo-S2 glycosynthase enables the reconstitution of therapeutic antibodies and their Fc fragments with an increased number of diverse glycans (e.g., high-mannose and hybrid glycans), which cannot be achieved with previously disclosed Endo-S variants.

[0008] In one embodiment, the present invention relates to a mutant of endo-β-N-acetylglucosaminidase of the strain NZ131 of serotype M49 of Streptococcus pyogenes. (Endo-S2)(SEQ ID NO: 1) provides transglycosylation activity, and this variant has at least 85% homology to endo-β-N-acetylglucosaminidase, exhibiting transglycosylation activity with both fucosylated and non-fucosylated GlcNAc-IgG acceptors. This endoglycosidase enables the collective transfer of oligosaccharides (in the form of activated sugar oxazolines) to fucosylated or non-fucosylated GlcNAc-IgG to form novel glycoforms of IgG.

[0009] In another embodiment, the present invention provides Endo-S2 mutant proteins exhibiting significantly enhanced transglycosylation efficiency and reduced or suppressed product hydrolysis activity compared to wild-type Endo S2. The mutants preferably include site-directed mutations such as mutations at Asp-184. Examples of these mutant proteins include, but are not limited to, D184A (SEQ ID NO: 2), D184N (SEQ ID NO: 3), D184Q (SEQ ID NO: 4), D184R (SEQ ID NO: 5), D184C (SEQ ID NO: 6), D184M (SEQ ID NO: 7), D184E (SEQ ID NO: 8), D184G (SEQ ID NO: 9), D184H (SEQ ID NO: 10), D184I (SEQ ID NO: 11), D184L (SEQ ID NO: 12), D184K (SEQ ID NO: 13), D184F (SEQ ID NO: 14), D184P (SEQ ID NO: 15), D184S (SEQ ID NO: 16), D184T (SEQ ID NO: 17); D184W (SEQ ID NO: 18), D184Y (SEQ ID NO: 19), D184V (SEQ ID NO: 20), or fragments thereof that contain a catalytic domain and exhibit increased transglycosylation and decreased hydrolysis compared to the wild-type Endo-S2 protein. Preferably, examples of these mutant proteins include D184M (SEQ ID NO: 7), D184E (SEQ ID NO: 8), and D184Q (SEQ ID NO: 4).

[0010] In particular, any Endo-S2 fragment and Endo-S2 domain carrying a mutation at the D184 site are included in the present invention, and any such domain and fragment can be fused to other proteins, including but not limited to CPD, Fc, and MBP. Mutations in the catalytic domain and specific sites are important for enzyme activity; therefore, other sites on this enzyme (e.g., terminals) can be modified or cleaved without significantly affecting glycosynthase activity.

[0011] In a further embodiment, the present invention relates to a chemoenzymatic method for preparing a homogeneous fucosylated glycoform or non-fucosylated glycoform of an IgG antibody, a. To provide an acceptor selected from the group consisting of core-fucosylated GlcNAc-IgG and non-fucosylated GlcNAc-IgG or the corresponding IgG-Fc fragment, and bS pyogenes (S. pyogenes) Endo-S2 Asp-184 mutant protein The process involves reacting the acceptor with a donor substrate containing an activated oligosaccharide moiety in the presence of a substance or fragment thereof containing a catalytic domain and exhibiting increased transglycosylation and decreased hydrolytic activity compared to the wild-type Endo-S2 enzyme, thereby transferring the activated oligosaccharide moiety to the acceptor and obtaining a homogeneous fucosylated glycoprotein or a non-fucosylated glycoprotein. This invention provides a chemical enzymatic method that includes the following:

[0012] In a further embodiment, the present invention relates to a method for preparing a corefucosylated IgG fragment or corefucosylated IgG-Fc fragment having a predetermined oligosaccharide moiety, a. To provide a corefucosylated acceptor protein containing a corefucosylated N-acetylglucosamine (GlcNAc) residue linked to asparagine, and b. In the presence of the endoglycosidase-S2 184 mutant protein or a fragment thereof containing a catalytic domain and exhibiting increased transglycosylation and decreased hydrolytic activity compared to the wild-type Endo-S2 enzyme, this core fucosylation acceptor The method involves enzymatically reacting a protein with an activated oligosaccharide donor, the activated oligosaccharide donor carrying an oligosaccharide moiety containing a predetermined number and type of sugar residues, the oligosaccharide moiety being covalently linked to an acceptor protein, thereby preparing a core-fucosylated IgG fragment or core-fucosylated IgG-Fc fragment having the predetermined oligosaccharide moiety. This provides a method that includes [something].

[0013] In yet another embodiment, the present invention provides an activated oligosaccharide moiety (e.g., glycan or oligosaccharide oxazoline, glycosyl fluoride, glycosyl azide or aryl glycoside) as a donor substrate for the synthesis of a homogeneous core fucosylated glycoprotein or non-fucosylated glycoprotein. Preferably, the activated oligosaccharide moiety is an oligosaccharide oxazoline.

[0014] In a further embodiment, the present invention relates to a chemical enzymatic method for preparing a homogeneous fucosylated glycoprotein or a non-fucosylated glycoprotein, a. To provide acceptors selected from core-fucosylated or non-fucosylated GlcNAc-IgG fragments or IgG-Fc fragments, and bS pyogenes Endo-S Asp-184 mutant protein The present invention relates to a method comprising reacting an acceptor with a donor substrate in the presence of a fragment thereof that contains a catalytic domain and exhibits increased transglycosylation and decreased hydrolytic activity compared to the wild-type Endo-S2 enzyme, wherein the donor substrate comprises a predetermined number and type of sugar residues and a specific linkage type, thereby yielding a homogeneous fucosylated glycoprotein or a non-fucosylated glycoprotein.

[0015] In particular, the S. pyogenes Endo-S Asp-184 mutant protein The quality or fragment may contain additional mutations in the amino acid residues, but it is important that it carries the D184 mutation.

[0016] In one embodiment, the fucosylated GlcNAc-containing protein is the alpha-1-6-fucosyl-GlcNAc-protein.

[0017] In another aspect, the present invention provides a method for remodeling an IgG glycoprotein or an IgG-Fc glycoprotein with an oligosaccharide having a predetermined oligosaccharide component with a defined number and type of sugar residues and a specific linkage type, wherein a. providing a fucosylated glycoprotein substrate comprising an Fc N-glycan; b. treating the fucosylated glycoprotein substrate with an endoenzyme to hydrolyze the bond between two core GlcNAc residues in the N-glycan to obtain a core fucosylated or non-fucosylated GlcNAc-IgG fragment or IgG-Fc fragment, and c. linking the oligosaccharide to the Asn-linked GlcNAc moiety in the presence of an Endo-S2 mutant or a fragment thereof having an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 20, wherein the fragment comprises a catalytic domain and exhibits increased transglycosylation and decreased hydrolysis activity compared to wild-type Endo-S2 enzyme (SEQ ID NO: 1), whereby the predetermined oligosaccharide component is added relates to a method comprising

[0018] In a further aspect, the present invention provides a method for remodeling a fucosylated or non-fucosylated IgG fragment or IgG-Fc fragment with an oligosaccharide having a predetermined oligosaccharide component with a defined number and type of sugar residues and a specific linkage type, wherein a. fuco obtained from a natural or recombinant source carrying heterogeneous N-glycans providing a silylated or non-fucosylated GlcNAc-IgG fragment or IgG-Fc fragment; b. treating a natural or recombinant IgG fragment or IgG-Fc fragment with an endoenzyme (a wild-type endoglycosidase or a mutant endoglycosidase having efficient hydrolysis activity), wherein the bond between the two GlcNAc residues located closest to the peptide domain is hydrolyzed, thereby forming a deglycosylated protein carrying a core fucosylated or non-fucosylated GlcNAc-IgG fragment or IgG-Fc fragment, and c. attaching a predetermined oligosaccharide to a GlcNAc residue, wherein said attachment is catalyzed by transglycosylation by a fragment that comprises a catalytic domain and exhibits increased transglycosylation and decreased hydrolysis activity compared to a wild-type Endo-S2 enzyme, said fragment being an S. pyogenes Endo-S2 Asp-184 mutant enzyme or a fragment thereof, thereby reconstituting a natural beta-1,4-glycosidic bond and adding the predetermined oligosaccharide component relates to a method comprising the above steps.

[0019] Applicable oligosaccharide oxazolines include, but are not limited to, high-mannose type, hybrid type, sialoglycan oxazoline, complex type N-glycans, and selectively modified derivatives thereof (for example, those having a specific tag). Preferably, disaccharide oxazoline, trisaccharide oxazoline, tetrasaccharide oxazoline, pentasaccharide oxazoline, hexasaccharide oxazoline, heptasaccharide oxazoline, octasaccharide oxazoline, nonasaccharide oxazoline, decasaccharide oxazoline or undecasaccharide oxazoline is used as a donor substrate for highly efficient chemoenzymatic synthesis of homogeneous core-fucosylated or non-fucosylated IgG antibodies and IgG-Fc fragments.

[0020] In yet another aspect, the present invention provides a method for synthesizing a modified antibody or a fragment thereof, comprising: a. using a naturally occurring IgG antibody or a recombinant antibody, or an Fc domain carrying an Fc N-glycan as a precursor, b. Deglycosylation of the Fc domain to form a GlcNAc acceptor is performed using an endoglycosidase such as wild Endo-S2, wherein the GlcNAc portion is located in the Fc region of the antibody, and the GlcNAc portion is either core-fucosylated or not fucosylated, and c. Endo-S2 mutants: Transglycosylation of the GlcNAc portion of an antibody by an oligosaccharide oxazoline or sialoglycan oxazoline having a predetermined number of sugar residues, under the catalytic action of an enzyme selected from the group consisting of SEQ ID NOs: 2 to 20 or fragments thereof that contain a catalytic domain and exhibit increased transglycosylation and decreased hydrolytic activity compared to the wild-type Endo-S2 enzyme, thereby forming a modified antibody having a predetermined number of sugars. Regarding methods including

[0021] In yet another aspect, the present invention relates to a method for reconstituting an intravenous immunoglobulin (IVIG) exhibiting an Fc-sialylated glycoform, a. To provide IVIG supporting Fc N-glycan, b. In order to form a GlcNAc-acceptor, the Fc N-glycan is deglycosylated using an endoglycosidase such as Endo-S, and this GlcNAc-acceptor is located in the Fc region of IVIG, and this GlcNAc-acceptor is either fucosylated or unfucosylated, and c. To form sialyzed IVIG, the Endo-S2 mutants SEQ ID NOs. 2 to 20 or their fragments, which contain a catalytic domain and exhibit increased transglycosylation and decreased hydrolytic activity compared to the wild-type Endo-S2 enzyme. Transglycosylation of the GlcNAc acceptor with a sialoglycan oxazoline having a predetermined number of sugar residues under the catalytic action of an enzyme selected from the group. This provides a method that includes [something].

[0022] Another aspect of the present invention provides an IVIG preparation comprising a composition containing at least 90% homogeneous sialylated Fc glycoform to increase anti-inflammatory activity, wherein the sialylated Fc glycoform is synthesized using the Streptococcus pyogenes Endo-S2 Asp-184 mutant in combination with a sialoglycan oxazoline having a GlcNAc moiety located in the Fc region of deglycosylated IVIG and a predetermined number of sugar residues.

[0023] In a further embodiment, the present invention relates to a method for synthesizing a homogeneous core-fucosylated or non-fucosylated IgG antibody or IgG-Fc fragment, a. To provide natural or recombinant IgG antibody or IgG-Fc fragment, wherein the recombinant IgG or IgG-Fc is produced from typical protein expression systems, including but not limited to yeast, insect, plant, and any mammalian expression system. b. Remove N-glycans using an enzyme selected from the group consisting of Endo-H, Endo-A, Endo-S, Endo S2(WT), and / or Endo-F3 to form core-fucosylated GlcNAc-containing proteins or non-fucosylated GlcNAc-containing proteins. c. To provide sugar oxazolines or sialogrican oxazolines having a desired oligosaccharide component containing a specified number and type of sugar residues in the chain, and d. Enzymatically transglycosylating a fucosylated GlcNAc-containing protein or a non-fucosylated GlcNAc-containing protein with a sugar oxazoline having a desired number of sugar residues or a siaroglycan oxazoline having a desired number of sugar residues and sialic acid residues, using an endoglycosidase selected from the group consisting of Streptococcus pyogenes Endo-S2 Asp-184 mutant enzyme or fragments thereof that exhibit increased transglycosylation and decreased hydrolytic activity compared to the wild-type Endo-S2 enzyme, thereby forming a uniform core-fucosylated or non-fucosylated IgG antibody or IgG-Fc fragment having an extension of a desired number of sugar residues and / or sialic acid. Regarding methods including

[0024] Oligosaccharide oxazolines or sialoglycan oxazolines having a predetermined oligosaccharide component having a predetermined number and type of sugar residues are expected to further include additional parts or tags, such as: for example, therapeutic agents or drugs for treating cancer, HIV or other viruses; substances that activate receptors on the cell plasma membrane; drugs that affect intracellular chemistry; drugs that affect cellular physicophysics; genes; gene analogues; RNA; RNA analogues; DNA; DNA analogues; amino acid sequences of surface receptors (e.g., CCR5 or CD4); and certain antibodies. Antigenic structures having affinity for receptor ligands (e.g., gp120, gp41, or gp160), amino acid sequences of receptor ligands (e.g., gp120, gp41, or gp160), receptor antagonists, receptor blockers, enzymes, enzyme substrates, enzyme inhibitors, enzyme modifiers, therapeutic proteins, protein analogs, metabolites, metabolite analogs, oligonucleotides, oligonucleotide analogs, antigens, antigen analogs, antibodies or fragments thereof, antibody analogs, antibodies that react with another receptor, modified antibodies and antibodies different from other antibodies, bacteria, viruses, inorganic ions, metal ions, metal clusters, polymers, fluorescent compounds, and any combination thereof.

[0025] In this way, the present invention provides a delivery device for delivering a drug or therapeutic agent having biological activity for treating a certain condition, comprising a predetermined sugar chain or sialoglycan, The present invention further provides a delivery device comprising a reconstituted IgG or reconstituted IgG-Fc fragment having a therapeutic agent or drug attached to a terminal sugar residue or sialic acid.

[0026] The present invention envisions the modification of HIV-related monoclonal antibodies (including, but not limited to, 17b, 48d, A32, C11, 2G12, F240, IgG1b12, 19e, X5, TNX-355, and F91, all of which are commercially available).

[0027] Further antibodies associated with cancer or other diseases may also be reconstituted for individual adaptation to specific receptors, thereby increasing their biological activity. Examples of such monoclonal antibodies include, but are not limited to, cetuximab, rituximab, muromonab-CD3, absiximab, daclizumab, basiliximab, palivizumab, infliximab, trastuzumab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab, omalizumab, tocitumomab, I-131 tocitumomab, efalizumab, bevacizumab, panitumumab, pertuzumab, natalizumab, etanercept, IGN101 (Aphton), borosiximab ((Biogen Idec and PDL BioPharm)), anti-CD80 mAb (Biogen Idec), anti-CD23 mAb (Biogen Idel), CAT-3888 (Cambridge Antibody Technology), CDP-791 (Imclone), eraptuzumab (Immunomedics), MDX-010 (Medarex and BMS), MDX-060 (Medarex), MDX-070 (Medarex) , matsuzumab (Merck), CP-675,206 (Pfizer), CAL (Roche), SGN-30 (Seattle Genetics), zanorimumab (Serono and Genmab), adecatumumab (Sereno), olegobomab (United Therapeutics), nimotuzumab (YM Bioscience), ABT-874 (Abbott Laboratories), denosumab (Amgen), AM 108 (Amgen), AMG 714 (Amgen) fontlizumab (Biogen Idec and PDL BioPharm), daclizumab (Biogent Idec) (and PDL BioPharm), golimumab (Centocor and Schering-Plough), CNTO 1275 (Centocor), ocrelizumab (Genetech and Roche), HuMax-CD20 (Genmab), belimumab Br (HGS and GSK), epratuzumab (Immunomedics), MLN1202 (Millennium Pharmaceuticals), vizilizumab (PDL BioPharm), tocilizumab (Roche), ocrerlizumab (Roche), certolizumab pegol (UCB, formerly Celltech), eculizumab (Alexion Pharmaceuticals), pexerizumab (Alexion Pharmaceuticals and Procter & Gamble), absiximab (Centocor), ranibizimumab (Genetech), mepolizumab (GSK), TNX-355 (Tanox) or MYO-029 (Wyeth).

[0028] A further aspect of the present invention is a method for reconstituting an antibody that initially contains heterogeneous sugar chains, a. Removing heterogeneous sugar chains from an antibody using endoglycosidase, leaving a single fucosylated GlcNAc moiety or a non-fucosylated GlcNAc moiety connected to the original glycosylation site, and b. To obtain a tagged antibody, a core oligosaccharide or siaroglycan oxazoline having at least one tag is transferred to a fucosylated GlcNAc moiety or a non-fucosylated GlcNAc moiety by transglycosylation catalyzed by an endoglycosidase, wherein the endoglycosidase is selected from the group consisting of Endo-S2 mutants SEQ ID NOs. 2 to 20 or fragments thereof that contain a catalytic domain and exhibit increased transglycosylation and reduced hydrolytic activity compared to the wild-type Endo-S2 enzyme. Regarding methods including

[0029] This tag portion may include, but is not limited to, antigens, therapeutic agents for cancer or HIV, toxins, fluorescent probes, biotin, PEG species, lipids, or nucleotides.

[0030] In yet another aspect, the present invention relates to an antibody-drug conjugate (i.e., ADC), This invention provides an antibody-drug conjugate that can be modified in accordance with the present invention and is designed as a targeted therapy for the treatment of people with cancer. Specifically, the ADC comprises two parts: a monoclonal antibody and a small amount of a very potent cytotoxic drug conjugated to this antibody. When the antibody of the ADC binds to a specific receptor on the surface of a target cell, this conjugation is broken, and the ADC releases a lethal toxin into the cell. The specific monoclonal antibody is described later herein and is also a potentially cytotoxic drug. Thus, the present invention provides a modified antibody that further comprises additional parts such as a therapeutic agent for treating cancer (e.g., chemokines and / or cytokines), thereby forming an antibody-drug conjugate (ADC).

[0031] In another embodiment, the present invention provides a composition comprising at least one Streptococcus pyogenes Endo-S2 mutant selected from the group consisting of D184M (SEQ ID NO: 7) and D184Q (SEQ ID NO: 4).

[0032] In yet another aspect, the present invention provides substantially homogeneous preparations of core-fucosylated antibodies or non-fucosylated antibodies or Fc fragments thereof having a predetermined oligosaccharide moiety, which are substantially homogeneous preparations produced by any of the methods described above. Compositions comprising such homogeneous preparations are also provided.

[0033] In another embodiment, the present invention provides a treatment method using a reconstituted antibody having a desired glycosylation state and / or sialylated form in an amount sufficient to modulate biological activity in a subject being treated.

[0034] In a further embodiment, the present invention provides a kit comprising at least one Streptococcus pyogenes Endo-S2 mutant selected from the group consisting of SEQ ID NOs: 2 to 20 (preferably D184M (SEQ ID NO: 7), D184E (SEQ ID NO: 8), and D184Q (SEQ ID NO: 4)).

[0035] Other aspects, features, and embodiments of the present invention will become more fully apparent from the following disclosure and the appended claims. [Brief explanation of the drawing]

[0036] [Figure 1] This figure shows the typical structures of an IgG antibody and an Fc N-glycan. a) Alpha-skeleton structure of human IgG showing the functional region (PDB code 1HZH); b) Structure of a full-length bifurcated complex N-glycan connected to Asn-297 in the Fc domain. [Figure 2] This figure shows the sequence alignment of Endo S2 (SEQ ID NO: 1) and Endo S (SEQ ID NO: 21). Aspartic acid residues (D233 in Endo-S and D184 in Endo-S2), which are important for promoting oxazolinium ion formation during hydrolysis, and common catalytic acid / base residues (E235 in Endo-S and E186 in Endo-S2) are marked. [Figure 3]This figure shows the schemes for glycosylation reconstitution of fucosylated rituximab into homogeneous complex glycoforms, high-mannose glycoforms, and hybrid glycoforms. [Figure 4] This figure shows the SDS-PAGE and ESI-MS analyses of glycosylation reconstitution of rituximab. (A) SDS-PAGE analysis; lane 1, commercially available rituximab; lane 2, EndoS2-deglycosylated rituximab (1); lane 3, transglycosylation product of complex glycoform (5) from an EndoS2-D184Q catalytic reaction between (1) and sialo-complex glycan oxazoline (2); lane 4, transglycosylation product of Man9GlcNAc (6) from an EndoS2-D184A catalytic reaction of (1) and (3); Lane 5, transglycosylation product of sialohybrid glycoform (7) from EndoS2-D184A catalyzed reaction between deglycosylated rituximab (1) and hybrid glycan oxazoline (4); (B) ESI-MS of light chain of commercial rituximab (after deconvolution); (C) ESI-MS of heavy chain of commercial rituximab (after deconvolution); (D) ESI-MS of deglycosylated rituximab (1); (E) ESI-MS of transglycosylated complex product (5); (F) ESI-MS of transglycosylated Man9 product (6); (G) ESI-MS of transglycosylated sialohybrid product (7). [Figure 5] This figure shows the enzymatic reconstitution schemes for rituximab into non-fucosylated homogeneous complexes, high-mannose and hybrid glycoforms. [Figure 6]This figure shows the SDS-PAGE and ESI-MS analysis of the glycosylation of rituximab to a non-fucosylated G2 glycoform. (A) SDS-PAGE analysis: Lane 1, commercially available rituximab; Lane 2, EndoS2 deglycosylated rituximab (1); Lane 3, defucosylated product (8); Lane 4, glycosylated complex glycoform from an EndoS2-D184N catalytic reaction (9); Lane 5, glycosylated Man9 glycoform from an EndoS2-D184N catalytic reaction (10); Lane 6, glycosylated sialohybrid glycoform from an EndoS2-D184A catalytic reaction (11) (B) ESI-MS of the heavy chain of defucosylated rituximab (8) (after deconvolution). (C) ESI-MS of the heavy chain of glycosylated complex rituximab (9). (D) ESI-MS of the heavy chain of glycosylated Man9 rituximab (10). (E) ESI-MS of the heavy chain of glycosylated sialohybrid rituximab (11). [Figure 7] This graph compares the transglycosylation activity of fucosylated rituximab with alanine, asparagine, and glutamine mutations in the EndoS2 D184 complex glycan oxazoline. [Figure 8] This figure schematically illustrates the hydrolysis and transglycosylation of Endo-S2 and its variants using rituximab as a substrate. It shows Fuc α1,6GlcNAc-rituximab, a deglycosylated rituximab supporting a core fucosylated GlcNAc moiety at the glycosylation site; SCT-rituximab; and a sialic complex glycoform of rituximab. [Figure 9]This figure shows the evaluation of the substrate specificity of Endo-S2 mutants for various glycans (HM, CT, and hybrid types). GlcNAc-rituximab, this rituximab glycoform carries only the first GlcNAc moiety at the Fc glycosylation site; Fuc α1,6GlcNAc-rituximab, a deglycosylated rituximab that carries the core fucosylated GlcNAc moiety at the glycosylation site; SCT-rituximab, a sialyl complex glycoform of rituximab; HM-rituximab, a high-mannose glycoform of rituximab; Hyb-rituximab, a hybrid glycoform of rituximab. [Figure 10] This graph shows the ESI-MS analysis of glycosylation reconstitution of rituximab using Endo-S2 D184M. A) ESI-MS of the heavy chain of commercially available rituximab (after deconvolution); B) ESI-MS of the heavy chain of Fuc α1,6GlcNAc-rituximab (2); C and D) ESI-MS of the heavy and light chains of the transglycosylation product (3) (SCT-rituximab), respectively; E and F) ESI-MS of the heavy and light chains of the transglycosylation product (8) (HM-rituximab); G and H) ESI-MS of the heavy and light chains of the transglycosylation product (6) (Hyb-rituximab); I and J) ESI-MS of the heavy and light chains of the transglycosylation product (11) (non-fucosylated rituximab). [Figure 11] This graph shows a comparison of the transglycosylation efficiency of various types of glycans using the Endo-S2 D184M mutant. Transglycosylation reactions were performed under the catalytic action of Endo-S2 D184M (0.05 mg / ml), using deglycosylated rituximab (2) as the acceptor and various types of glycan oxazolines as donor substrates. The donor-to-acceptor molar ratio was 20:1. The dataset shown in this figure is representative of two independent experiments. [Figure 12]This graph compares transglycosylation by SCT using Endo-S2 D184Q, Endo-S2 D184M, and Endo-S D233Q. Transglycosylation was performed under the catalytic action of various endoglycosidase mutants at a fixed concentration of 0.05 mg / ml, using deglycosylated rituximab (2) as the acceptor and SCT glycan oxazoline (4) as the donor substrate. The donor-to-acceptor molar ratio was 20:1. The dataset shown in this figure is representative of two independent experiments. [Figure 13] This figure shows the glycosylation reconstitution of Herceptin (trastuzumab) using a pair of Endo-S2 enzymes (Endo-S2 WT and Endo-S2 D184M mutant), with A) ESI-MS of the heavy chain of commercially available trastuzumab (Herceptin); B) ESI-MS of the heavy chain of deglycosylated Herceptin (12); C) ESI-MS of the heavy chain of transglycosylation product 13 (S2G2F-trastuzumab); D) ESI-MS of the light chain of transglycosylation product (13); E) ESI-MS of the heavy chain of transglycosylation product (13) after PNGase F-catalyzed deglycosylation. [Figure 14] This figure shows the Fc glycosylation of IVIG to improve its anti-inflammatory activity. [Modes for carrying out the invention]

[0037] Detailed description of the invention This invention provides novel glycosynthase EndoS2 Asp184 variants that exhibit remarkable transglycosylation efficiency, enabling the transfer of complex N-glycans, high-mannose N-glycans, and hybrid N-glycans from activated glycan oxazolines to deglycosylated intact antibodies without hydrolysis of the product. Herein, we have found that the glycosynthase EndoS2 Asp184 variants efficiently act on both the core fucosylated and non-fucosylated GlcNAc-Fc domains of intact antibodies to generate various specified IgG glycoforms. As described herein, Endo-S2 possesses potent transglycosylation activity, and systematic site-directed mutagenesis has led to the discovery of several glycosynthase variants, such as D184M and D184Q, that exhibit remarkable transglycosylation activity without apparent product hydrolysis activity. Furthermore, this specification reveals that Endo-S2 glycosynthase exhibited significantly relaxed substrate specificity, enabling the transfer of three major types of N-glycans (complex, high-mannose, and hybrid) for antibody glycosylation reconstitution. Moreover, as further described herein, we found that Endo-S2 glycosynthase variants were generally far more active for transglycosylation than Endo-S variants. Highly efficient glycosylation reconstitution of two therapeutic monoclonal antibodies (rituximab and trastuzumab (Herceptin)) is described. Furthermore, antibodies and intravenous immunoglobulins were transformed into Fc-completely sialylated glycoforms with increased anti-inflammatory activity. In addition, the present invention provides homogeneous non-fucosylated glycoforms with enhanced FcγIIIa receptor binding activity and increased ADCC activity, as well as azido-tagged glycoforms that can be further transformed into other glycoforms.

[0038] The implementation of this invention, unless otherwise indicated, utilizes prior arts in immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the scope of the art. For example, Sambrook, et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel, et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RI Freshney, ed. See (1987).

[0039] It is understood that the embodiments of the present invention described herein include embodiments that "consist of" and / or "essentially consist of."

[0040] definition When used in the specification of this application, "a" or "an" may mean one or more. When used in the claims of this application, when used in conjunction with the word "including," the word "a" or "an" may mean one or more. When used herein, "another" may mean at least one or more.

[0041] As used herein, “biological activity” refers to pharmacological or pharmacokinetic properties, such as molecular affinity or the resulting biochemical or physiological effects, receptor affinity or the resulting biochemical or physiological effects, non-receptor affinity or the resulting biochemical or physiological effects, efficacy, bioavailability, absorption, distribution, metabolism, or excretion.

[0042] As used herein, “sugar” refers to a carbohydrate-containing molecule, whether oxidized or unoxidized, and includes, but is not limited to, monosaccharides, disaccharides, trisaccharides, oligosaccharides, or polysaccharides, e.g., N-acetylglucosamine, mannose, galactose, N-acetylneuraminic acid (sialic acid), glucose, fructose, fucose, sorbose, rhamnose, mannoheptulose, N-acetylgalactosamine, dihydroxyacetone, xylose, xylulose, arabinose, glyceraldehyde, sucrose, lactose, maltose, trehalose, cellobiose, or any combination of these L-isomers or D-isomers. Sugar further refers to such molecules produced naturally, recombinantly, synthetically, and / or semi-synthetically.

[0043] As used herein, “homogeneous” refers to a core fucosylated glycoprotein or a non-fucosylated glycoprotein in which the oligosaccharide component comprises at least 75%, more preferably at least 90%, and most preferably at least 95% of the same number and type of sugar residues.

[0044] As used herein, the terms "protein" or "glycoprotein" are interchangeable with the terms peptide and glycopeptide.

[0045] As used herein, “homology” refers to an amino acid sequence having substantial identity or similarity between two polypeptides and having at least 85%, more preferably at least 95%, similarity to a reference polypeptide. For polypeptides, the length of comparison required to obtain the above percentage of homology between sequences is generally at least 25 amino acids, or at least 50 amino acids, more likely at least 100 amino acids, most likely 200 amino acids or more. Substantially identical or homologous polypeptides include additions, truncations, internal deletions or insertions, conservative and conservative substitutions, or other modifications placed in amino acid sequence positions that do not disrupt the function of the endoglycosidase. Those skilled in the art will recognize a number of amino acids that can be modified or substituted with other chemically similar residues without substantially altering their activity.

[0046] As used herein, “modulate” means an increase or decrease in the “biological activity” as defined above, when comparing the glycosylated antibody of the present invention with the non-glycosylated antibody.

[0047] As used herein, “immunoglobulin molecule” or “antibody” refers to a molecule that contains an antigen-binding site that specifically binds to an antigen or an Fc region that binds to a cell receptor. Structurally, the simplest naturally occurring antibody (e.g., IgG) consists of four polypeptide chains (two heavy (H) chains and two light (L) chains) interconnected by disulfide bonds. Natural immunoglobulins represent a large family of molecules, including several types of molecules (e.g., IgD, IgG, IgA, IgM, and IgE). The term also encompasses hybrid antibodies, or modified antibodies, and their fragments (including, but not limited to, Fab and Fc fragments).

[0048] Antibodies can be fragmented using conventional techniques as described herein, and these fragments can be screened for utility in the same manner as described for the whole antibody. Fab fragments of immunoglobulin molecules are multimeric proteins comprising immunoactive portions of the immunoglobulin molecule, specifically linked to each other and capable of binding to antigens, consisting of immunoglobulin heavy chains and immunoglobulin light chains. Fab and Fc fragments can be prepared by proteolytic digestion of substantially intact immunoglobulin molecules with papain using methods known in the art. However, Fab or Fc fragments may also be prepared by expressing desired portions of immunoglobulin heavy and light chains in suitable host cells using methods known in the art.

[0049] Where used herein with respect to antibodies, “substantially pure” means that the antibody is separated from any contaminants that accompany it in its natural state or contaminants that arise or are used in the process of obtaining the antibody. The term further includes desired products having a single glycosylation state, whether this state involves glycosylation at one or more sites. Typically, an antibody is substantially pure if it constitutes at least 60% by weight of the antibody in the preparation. For example, the antibody in the preparation is at least about 75% by weight of the desired antibody, at least about 80% by weight in certain embodiments, about 85% by weight in certain embodiments, at least about 90% by weight in certain embodiments, at least about 95% by weight in certain embodiments, and most preferably at least about 99% by weight. Substantially pure antibodies include those produced naturally, by recombination, or by synthesis.

[0050] As used herein, “therapeutic amount” means an amount that results in improvement or treatment of the symptoms of a disease or condition.

[0051] Antigens useful for tagging modified fucosylated glycoproteins or modified non-fucosylated glycoproteins (more preferably antibodies or fragments thereof) of the present invention may be exogenous antigens, endogenous antigens, fragments thereof, or variants having the same functional activity.

[0052] As used herein, “endogenous antigen” refers to a protein or part thereof that is naturally present in the cells or tissues of a recipient animal (e.g., a cellular protein, an immunomodulator, or a therapeutic agent).

[0053] As used herein, “exogenous antigen” refers to a protein or fragment thereof that is exogenous to the cells or tissues of a recipient animal, and includes, but is not limited to, viral proteins, parasitic proteins, immunomodulators, or therapeutic agents.

[0054] This exogenous antigen may be a protein derived from a viral pathogen or a parasitic pathogen, or an antigenic fragment thereof.

[0055] Alternatively, this exogenous antigen may be encoded by a synthetic gene, which may be constructed using conventional recombinant DNA methods, and this synthetic gene may express antigens or parts thereof derived from viral pathogens and parasitic pathogens. These pathogens may be infectious in humans, livestock, or wild animal hosts.

[0056] This exogenous antigen may be any molecule expressed by any viral or parasitic pathogen before or during their invasion, colonization, or replication into these animal hosts.

[0057] Examples of viral pathogens from which viral antigens originate include, but are not limited to, orthomyxoviruses, such as influenza virus (Taxonomy ID: 5977). 1); Retroviruses, such as RSV, HTLV-1 (Taxonomy ID: 39015) and HTLV-II (Taxonomy ID: 11909); herpesviruses, such as EBV (Taxonomy ID: 10295), CMV (Taxonomy ID: 10358), or herpes simplex virus. Rus (ATCC#:VR-1487); lentiviruses, e.g., HIV-1 (Taxonomy ID:12721) and HIV-2 (Taxonomy ID:11709); rhabdoviruses, e.g., rabies; picornoviruses, e.g., poliovirus (Taxonomy ID:12080); Xviruses, such as vaccinia (Taxonomy ID: 10245); rotavirus (Taxonomy ID: 10912); and parvoviruses, such as adeno-associated virus 1 (Taxonomy ID: 85106).

[0058] Examples of viral antigens include, but are not limited to, the following: human immunodeficiency virus antigens (Nef) (National Institute of Allergy and Infectious Disease HIV Repository catalog #183; GenBank registration #AF238278), Gag, Env (National Institute of Allergy and Infectious Disease HIV Repository catalog #2433; GenBank registration #U39362), and Tat (National Institute of Allergy and Infectious Disease HIV Repository catalog #827; GenBank registration #M13). 137), Rev (National Institute of Allergy and Infectious Disease HIV Repository catalog #2088; GenBank registration #L14572), Pol (National Institute of Allergy and Infectious Disease HIV Repository catalog #238; GenBank registration #AJ237568), and T cell epitopes and B cell epitopes of gp120; hepatitis B surface antigen (GenBank registration #AF043578); rotavirus antigens, e.g., VP4 (GenBank registration #AJ293721) and VP7 (GenBank registration #AY003871); influenza virus antigens, e.g., hemagglutinin (GenBank registration #AJ404627); nucleoprotein (GenBank registration #AJ289872); and herpes simplex virus antigens, e.g., thymidine kinase (GenBank registration #AB047378).

[0059] The following are examples of bacterial pathogens from which bacterial antigens originate, but are not limited to these: Mycobacterium spp., Helicobacter pylori, Salmonella spp., Shigella spp., Escherichia coli, Rickettsia spp., Listeria spp., Legionella pneumoniae, Pseudomonas spp., Vibrio species (Vibrio spp.) and Borellia burgdorferi ).

[0060] Examples of protective antigens for bacterial pathogens include: enterotoxigenic Escherichia coli (E. coli) Cellular antigens, e.g., CFA / I ciliary antigen and non-toxic B subunit of the thermolabile toxin; partactin of Bordetella pertussis, adenylyl cyclase hemolysin of B. pertussis, Clostridium tetanus ni) Tetanus toxin fragment C, Borellia burgdorferi OspA, Rickettsia prowazekii and Rickettsia Protective paracrystalline-surface-layer protein of Rickettsia typhi, listeriolisin (also known as "Llo" and "Hly") of Listeria monocytogenes, and / or superoxide dismutase (also known as "SOD" and "p60"). The receptor-binding domains of the urease of Helicobacter pylori, and the lethal toxin and / or protective antigen of Bacillus anthrax.

[0061] Examples of antigens from biological weapons or pathogens include, but are not limited to, smallpox, anthrax, tularemia, plague, listeria, brucellosis, hepatitis, vaccinia, mycobacteria, coxsackievirus, tuberculosis, malaria, ehrlichiosis, and bacterial meningitis.

[0062] Parasitic pathogens from which parasitic antigens originate include, but are not limited to, the following: Plasmodium species (e.g., Plasmodium falciparum) (ATCC#:30145); Trypanosome species (e.g., Trypanosoma cruzi) (ATCC#:50797). Giardia species (e.g., Giardia intestinalis) (ATCC#:30888D); Boophilus species (e.g., Boophilus); Babesia species (e.g., Babesia microti) (ATCC# :30221); Entamoeba species (Entamoeba spp.), e.g., Entamoeba histolytica (ATCC#:30015); Eimeria species (Eimeria spp.), e.g. Eimeria maxima (ATCC#40357); Leishmania spp. (Taxonomy ID:38568); Schistosome spp. For example, Schistosoma mansoni (GenBank registration #AZ301) 495); Brugia spp., e.g., Brugia malayi (GenBank registration #BE352806); Fascida spp., e.g., liver fluke (Fasciola hepatica) (GenBank registration #AF286903); Dirofilaria Species (Dirofilaria spp.), for example, Dirofilaria immitis (GenBank registration #AF008300); filamentous insect species (Wuchereria spp.), for example Wuchereria bancrofti (GenBank registration #AF250996); and Onchocerea species (Onchocerea spp); for example, Onchocerca volvulus (GenBank registration #BE588251).

[0063] Examples of parasitic antigens include, but are not limited to, the following: pre-intraerythrostatic antigens of Plasmodium species (P. falciparum) (GenBank registration #M22982) and Plasmodium vivax (GenBank registration) Circumsporozoite antigen of #M20670; liver of Plasmodium species (Plasmodium spp.) Organ-phase antigens, e.g., hepatic-phase antigen 1 (referred to as LSA-1; GenBank registration #AF086802); merozoite-phase antigens of Plasmodium species (Plasmodium spp.); e.g., merozoite-phase antigens Zoit surface antigen-1 (also known as MSA-1 or MSP-1; GenBank registration #AF199410); surface antigen of Entamoeba histolytica, e.g., Gala Ctose-specific lectin (GenBank registration #M59850) or serine-rich Entamoeba histolytica protein; surface proteins of Leishmania species, e.g., 63kDa glycoprotein (gp63) or 46kDa glycoprotein (gp46) of forest-type tropical Leishmania (Leishmania major) (GenBank registration #Y00647); paramyosin of Brugia malayi (GenBank registration #U77590); manso The trio of Schistosoma mansoni (GenBank registration #W06781) Thric acid isomerase; secreted globin-like protein of the nematode *Trichostrongylus colubriformis* (GenBank registration #M63263); glutathione-S-transfer protein of *Fasciola hepatica* (GenBank registration #M77682) Schistosoma bovis (GenBank registration #M77682) ); Japanese schistosomiasis (S. japonicum) (GenBank registration #U58012); and KLH(B) of Schistosoma bovis and Japanese schistosomiasis (S. japonicum) (See above for details on ashir, etc.)

[0064] Examples of tumor-specific antigens include prostate-specific antigen (PSA), TAG-72, and CEA; human tyrosinase (GenBank registry #M27160); and tyrosinase-related proteins. Examples include TRP (also known as TRP; GenBank registration #AJ132933) and tumor-specific peptide antigens.

[0065] Examples of transplanted antigens include CD3 molecules on T cells, histocompatibility antigens such as HLA A, HLA B, HLA C, HLA DR, and HLA.

[0066] Examples of autoimmune antigens include: IAS beta chain (GenBank registration #D88762), useful in therapeutic vaccines for autoimmune encephalomyelitis; glutamate decarboxylase (GenBank registration #NM013445), useful in therapeutic vaccines for insulin-dependent type 1 diabetes; thyrotropin receptor (TSHr) (GenBank registration #NM000369), useful in therapeutic vaccines for Graves' disease; and tyrosinase-related protein 1 (GenBank registration #NM000550), useful in therapeutic vaccines for vitiligo.

[0067] HIV drugs that may be used in the construction of tagged antibodies or this fragment include, but are not limited to, antiviral drugs such as nucleoside RT inhibitors, CCR5 inhibitors / antagonists, viral entry inhibitors, and functional analogues thereof. Specifically, antiviral drugs may include: nucleoside RT inhibitors such as zidovudine (ZDV, AZT), lamivudine (3TC), stabudine (d4T), didanosine (ddl), zalcitabine (ddC), abacavir (ABC), emiribin (FTC), and tenofovir (TDF). ), Delaviradine (DLV), Efavirenz (EFV), Nevirapine (NVP), Saquinavir (SQV), Ritonavir (RTV), Indinavir (IDV), Nelfinavir (NFV), Amprenavir (APV), Lopinavir (LPV), Atazanavir, Combivir (ZDV / 3TC), Kaletra (RTV / LPV), Trizivir (ZDV / 3TC / ABC).

[0068] CCR5 inhibitors / antagonists, e.g., SCH-C, SCH-D, PRO 140, TAK 779, TAK-220, RANTES analogs, AK602, UK-427, 857, monoclonal antibodies; and viral entry inhibitors, e.g., Fuzeon (T-20) (enfvirtide), NB-2, NB-64, T-649, T-1249, SCH-C, SCH-D, PRO 140, TAK 779, TAK-220, RANTES analogs, AK602, UK-427, 857; and functional analogues or equivalents thereof.

[0069] Many different fucosylated and non-fucosylated glycoproteins are expected to be modified according to the methods of the present invention or used as therapeutic agents for coupling to terminal sugars, and such therapeutic agents include, but are not limited to, adrenocorticotropic hormone (ACTH); renocorticotropic hormone derivatives (e.g., ebiratide). Angiotensin; Angiotensin II; Asparaginase; Atrial natriuretic Peptides; atrial sodium diuretic peptide; bacitracin; beta-endorphin; blood coagulation factors VII, VIII and IX; blood thymic factor (FTS); blood thymic factor derivatives; bombesin; bone morphogenetic factor (BMP); bone morphogenetic protein; bradykinin; cerulein; calcitonin gene-related polypeptide (CGRP); calcitonin; CCK-8; cell growth factors (e.g., EGF; TGF-alpha; TGF-beta; PDGF; acidic FGF; basic FGF); cerulein; chemokines; cholecystokinin; cholecystokinin-8; cholecystokinin Nin-pancreozymin (CCK-PZ); colistin; colony-stimulating factors (e.g., CSF; GCSF; GMSCF; MCSF); corticotropin-releasing factor (CRF); cytokines; desmopressin; dinorphin; dipeptides; dismutase; dynorphin; eledoisin; endorphin; endothelin; endothelin antagonist peptide; endothelin (endotherin); enkephalin; enkephalin derivatives; epidermal growth factor (EGF); erythropoietin (EPO); follicle-stimulating hormone (FSH); gala Nin; gastric suppressor polypeptide; gastrin-releasing polypeptide (GRP); gastrin; G-CSF; glucagon; glutathione peroxidase; glutathione-peroxidase; gonadotropins (e.g., human placental gonadotropin and its alpha and beta subunits); gramicidin; gramicidin group, growth factor (EGF), growth hormone-releasing factor (GRF); growth hormone group; hormone-releasing hormone (LHRH); human atrial natriuretic polypeptide (h-ANP); human placental lactogen; i Insulin; insulin-like growth factor (IGF-I; IGF-II); interferon; interferons (e.g., alpha-interferons, beta-interferons, and gamma-interferons); interleukins (e.g., 1;2;3;4;5;6;7;8;9;10;11, and 12); intestinal polypeptide (VIP); kallikrein; kyotorphin; lurivelin; luteinizing hormone (LH); luteinizing hormone-releasing hormone (LH-RH); lysozyme chloride; melanocyte-stimulating hormone (MSH);Melanin-releasing cell-stimulating hormone; melittin; motilin; muramil; muramil dipeptide; nerve growth factor (NGF); neurotrophic factors (e.g., NT-3; NT-4; CNTF; GDNF; BDNF); neuropeptide Y; neurotensin; oxytocin; pancreastatin; pancreatic polypeptide; pancreozymin; parathyroid hormone (PTH); pentagastrin; polypeptide YY; pituitary adenylcyclase-activating polypeptide (PACAP); platelet-derived growth factor; polymyxin B; prolactin; protein synthesis-stimulating polypeptide; PTH-related protein; relaxin; renin; secretin; serum thymic factor; somatomedin; somatostatin derivatives; superoxide dismutase; tuftosin; tetragastrin; thrombopoietin (TPO); thymic humoral factor (THF); thymopoietin; thymosin; thymostimulin; thyroid hormone-releasing hormone; thyroid-stimulating hormone (TSH); thyroid-stimulating hormone; Hormone-releasing hormone (TRH); trypsin; tuftosin; tumor growth factor (TGF-alpha); tumor necrosis factor (TNF); tyrosidine; urogastron; urokinase; vasoactive intestinal polypeptide; and vasopressin.

[0070] Fucosylated and non-fucosylated glycoproteins are important classes of biomolecules that play crucial roles in many biological events, including cell adhesion, tumor metastasis, pathogen infection, and immune responses. As already shown herein, a major problem in the study of the structure and function of fucosylated or non-fucosylated glycoproteins is the structural microheterogeneity of these proteins. Natural and recombinant fucosylated or non-fucosylated glycoproteins are generally pendent oligosaccharides. It is produced as a mixture of glycofoams that differ only in their structure.

[0071] The reconstituted glycoprotein (e.g., antibody) is subjected to any further necessary or desirable procedures to introduce additional functional groups or tags, including but not limited to glycosyl transfer and selective ligation (e.g., click chemistry, Staudinger reaction, etc.). This allows for structural modifications. The functional group can be any suitable type, including but not limited to toxins, specific antigens (e.g., alpha-Gal), radioactive species, photoactive species, PEG, etc. This glycoprotein can be catalytically reacted by a "click chemistry" cycloaddition reaction between its azide functional group and an alkyne having the desired functional group moiety. These azide and alkyne functional groups can be switched between their respective ligation components, and the glycoprotein can be functionalized with an alkynyl functional group and reacted with an azide-functionalized compound containing the desired moiety. It will also be recognized that other ligation pairs can be devised for click chemistry reactions.

[0072] Fucosylated and non-fucosylated glycoproteins produced according to the methods described herein may be used for diagnostic and therapeutic purposes. Approximately two-thirds of therapeutic proteins currently in use on the market and / or in clinical trials are glycoproteins. However, the structural heterogeneity of various glycoforms of natural and recombinant glycoproteins presents a major obstacle in the development of glycoprotein-based drugs because different glycoforms may have different biological activities, and controlling glycosylation to a homogeneous glycoform is extremely difficult during expression. The previous discovery of the transglycosylation activity of a certain class of endoglycosidases represents a major advance in the field of glycosylation manipulation that enhances the therapeutic and diagnostic potential of glycoproteins, and the Endo-S2 variant of the present invention can transglycosylate fucosylated and non-fucosylated natural and recombinant glycoproteins without the negative consequence of hydrolysis.

[0073] The features and advantages of the present invention are more fully illustrated in the following non-limiting embodiments. [Examples]

[0074] Examples Generation of Endo-S2 glycosynthase mutants, and use of these mutants for glycosylation reconstitution of the intact monoclonal antibody rituximab. Glycosynthases have already been produced from several GH85 endoglycosidases (ENGases), such as EndoA, EndoM, EndoD, and the GH18 endoglycosidase EndoS, by site-directed mutagenesis of key asparagine (Asn) residues in the GH85 family or aspartic acid (Asp) residues in the GH18 family, which are involved in promoting the formation of oxazolinium ion intermediates during hydrolysis [36, 38]. Endo-S2 is an endoglycosidase belonging to the glycoside hydrolase family 18 (GH18)

[33] , and is part of the same GH family as EndoS, EndoF1, EndoF2, and EndoF3, which have recently been found to have transglycosylation activity. Based on the premise that hydrolysis catalyzed by EndoS2 also proceeds via a substrate-assisted mechanism involved in the formation of an oxazolinium ion intermediate, as demonstrated by other GH18 endoglycosidases such as EndoS, we created a latent glycosynthase derived from Endo-S2 by identifying and mutating residues involved in promoting oxazolinium ion formation. Previous structural and mutagenesis studies of Endo-S have shown that the aspartic acid residue at position 233 (D233) is involved in promoting oxazoline formation and that the E235 residue is a general acid / base for catalytic hydrolysis [47, 48]. As shown in Figure 2, from the sequence alignment of EndoS2 and EndoS (Figure 2), we identified two important residues in EndoS for catalytic activity: the D184 residue involved in promoting oxazolinium ion formation (corresponding to D233 in EndoS) and the E186 residue as a general acid / base residue in glycan hydrolysis (identical to E235 in EndoS). Therefore, assuming that D184 is an important residue that promotes the formation of oxazolinium ion intermediates in hydrolysis via a substrate-assisted mechanism, 19 specific mutants D184A~Y (SEQ ID NOs. 2~20) were generated by site-directed mutagenesis of Endo-S2 (SEQ ID NO. 1). These mutants and wild-type Endo-S2 were then subjected to CP The D-fusion protein was expressed in Escherichia coli at high yields (20-30 mg / L) and purified by Ni-NTA affinity chromatography.

[0075] The deglycosylation and potential transglycosylation activities of the enzyme were investigated using rituximab, a therapeutic monoclonal antibody, as a model mAb. As is evident in Figure 4C, the main Fc glycans of commercially available rituximab are core-fucosylated branched-type oligosaccharides carrying 0-2 galactose moieties, named G0F glycoform, G1F glycoform, and G2F glycoform, respectively. Treatment of rituximab with EndoS2-CPD fusion protein (referred to herein as wild-type EndoS2 or EndoS2) rapidly deglycosylated rituximab with a fucosylated GlcNAc disaccharide moiety (Fucα1,6GlcNAc) at the glycosylation site (N297). This result supports the remarkable Fc glycan hydrolysis activity of wild-type EndoS2 to intact IgG and suggests its usefulness in the first step of mAb glycosylation reconstitution. Next, as shown in Figure 3, the transglycosylation potential of the Endo-S2 mutant was investigated using deglycosylated rituximab as an acceptor and complex glycan oxazolines, high-mannose glycan oxazolines, and hybrid glycan oxazolines as donor substrates. As shown in Figure 4, the glycosylation reconstitution process was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-mass spectrometry (LC-MS). The heavy and light chains of rituximab appeared at approximately 50 kDa and 25 kDa, respectively, under reducing conditions (lane 1 in Figure 4A). After deglycosylation by wild-type EndoS2, the heavy chain appeared as a single band at approximately 48 kDa, suggesting the removal of two N-glycans (each derived from the heavy chain) in rituximab (lane 2 in Figure 4A). Incubation of deglycosylated rituximab (1) and sialo-complex glycan oxazoline (2) (donor / acceptor, 20:1 molar ratio) with the mutant EndoS2-D184Q yielded a transglycosylation product (5) (Figure 4E). The heavy chain of this transglycosylation product appeared as a single band approximately 2 kDa larger than the band of deglycosylated rituximab (1) (Figure 4A, lane 3).These results suggest that a novel N-glycan was attached to each of the Fc heavy chains. Interestingly, essentially quantitative transglycosylation of the Fc domain of the intact antibody was achieved within one hour of incubation.

[0076] The characteristics of this transglycosylation were further clarified by LC-MS analysis. The heavy and light chains of rituximab were separated under LC-MS conditions. Deconvolution of the light chain MS data yielded a mass of 23039 (Figure 4B), which was in agreement with the calculated mass of the rituximab light chain (M=23042Da)

[47] . As shown in Figure 4C, deconvolution of the heavy chain MS data yielded three distinct m / z species 50508, 50669, and 50829, which were in good agreement with the theoretical masses of the heavy chain glycoform: G0F, M=50515Da; G1F, M=50677Da; and G2F, M=50839Da, respectively

[47] . As shown in graph c of Figure 4D, deconvoluted electron-spray ionized mass spectrometry (ESI-MS) of the heavy chain of deglycosylated rituximab (1) showed a single peak at 49411, which was in good agreement with the heavy chain supporting the Fucα1,6GlcNAc disaccharide moiety (calculated, M=49420Da). As shown in the graph of Figure 4E, after glycosylation reconstruction, a single peak at 51414 was observed, with an additional 2003Da from the heavy chain of the transglycosylation product by complex glycan (5) to the deglycosylated heavy chain of rituximab. This result indicates the siaroglycan linkage from the corresponding sugar oxazoline (2) to the heavy chain.

[0077] In addition to sialylated complex N-glycan oxazolines (2), the EndoS2 variant is equally efficient in using high-mannose Man9GlcNAc core oxazolines (3) and sialohybrid oxazolines (4) for rituximab glycosylation, respectively. Corresponding homogeneous glycoforms (6) and (7) are formed (Figures 4F and 4G). As shown in Figure 4F, the deconvolution ESI-MS of the heavy chain of the transglycosylation product (6) showed a single species at 51074, which was in good agreement with the calculated molecular weight (M=51082Da) of the rituximab heavy chain supporting Man9GlcNAc2 glycan. Similarly, as shown in Figure 4G, the deconvolution ESI-MS of the heavy chain of the transglycosylation product (7) showed a single species at 51080, which was in good agreement with the calculated molecular weight (M=50190Da) of the rituximab heavy chain supporting N3Man3GlcNAc2 glycan. The results described herein represent the first report of glycosylation reconstitution of intact IgG monoclonal antibodies by a highly efficient deglycosylation-reglycosylation protocol enabled by the combined use of EndoS2 and EndoS2-based glycosynthase, resulting in the unified transfer of full-size native high-mannose (Man9) and sialohybrid N-glycans to the Fc domain. After completion of transglycosylation, the product could be purified by simple protein A affinity column chromatography to obtain a well-defined, homogeneous glycoform. In particular, glycosylation of intact rituximab by transfer of branched complex N-glycans to the Fc domain using EndoS / EndoS-based glycosynthase has been reported. However, this system is inefficient for the transfer of high-mannose and hybrid glycans to antibodies. The development of the EndoS2 / EndoS2-glycosynthase system has significantly expanded the range of glycan-specification for chemienzymatic glycosylation of antibodies.

[0078] Glycan manipulation of rituximab to generate non-fucosylated complex glycoforms, high-mannose glycoforms, and hybrid glycoforms. Non-fucosylated IgG glycoforms are preferred for anticancer treatment because mAbs containing low fucose-content Fc N-glycans have already been shown to enhance ADCC activity in vitro and enhance anticancer effects in vivo, particularly in patients carrying the low-affinity F158 allele of the FcγIIIa receptor [8, 39, 40, 49]. However, α-fucosidase cannot be used to remove α1,6-fucose from intact rituximab. The α-1,6-fucose portion may be blocked by the Fc domain and / or complex N-glycans, thus preventing access by α-fucosidase. We theorized that rituximab in the Fuc(α1,6)GlcNAc glycoform obtained as a result of deglycosylation may be accessible by α-fucosidase. In practice, after deglycosylation by EndoS2, overnight incubation with α1,6-fucosidase derived from Lactobacillus casei is performed. By vaising, most of the α-1,6-fucose moiety was removed to obtain GlcNAc-containing rituximab (8). This result was confirmed by LC-MS (Figure 6B). Next, the glycosynthases EndoS2-D184A, EndoS-D184Q, or EndoS-D184N were also demonstrated to be efficient in recognizing unfucosylated GlcNAc in (8) for transglycosylation of complex (2), high-mannose (3), or hybrid (4) glycoforms by oxazolines, for generating relatively homogeneous unfucosylated complex (9), high-mannose (10), and hybrid (11) glycoforms through essentially quantitative conversion (Figure 5). As shown in Figure 6, the identity and purity of the glycosylation products (9, 10, 11) were confirmed by SDS-PAGE and LC-MS analysis. Defucosylated rituximab (8) showed a major peak at 49266 (Figure 6B), supporting fucose removal (calculated value for the heavy chain of GlcNAc-rituximab, M=49274Da). Deconvoluted ESI-MS of the heavy chain of the transglycosylation product of complex glycans (9) showed 51268 as the major species (Figure 6C), which was in good agreement with the calculated molecular weight (M=51276Da) of the rituximab heavy chain carrying the sialylated branched complex N-glycan Sia2Gal2GlcNAc2Man3GlcNAc2. Similarly, LC-MS analysis of the heavy chains of the transglycosylation products of high-mannose (10) and hybrid (11) glycans showed 50930 (Figure 6D) and 50939 (Figure 6E). These emerged as the main species, and their calculated molecular weights (50936 Da and 50944 Da) were in good agreement. In the transfer of hybrid glycan oxazolines, approximately 33% of the starting material remained untransferred. Optimization of reaction conditions (increasing the EndoS2 mutant concentration, adding more oxazolines, etc.) should allow the reaction to complete. Fucosylated rituximab (1) appears to be a preferred acceptor compared to unfucosylated rituximab (8). In comparative studies, we also found that mutants D184A, D184N, and D184Q exhibited faster transglycosylation to fucosylated GlcNAc-rituximab (1) compared to the unfucosylated acceptor (8) (data not shown). In summary, these experimental results revealed a complex enzymatic approach for producing homogeneous (or relatively homogeneous) complex, high-mannose, and hybrid glycoforms from commercially available monoclonal antibodies. The resulting non-fucosylated rituximabs are expected to acquire improved ADCC and CDC effector functions, as suggested by previous studies [8, 42, 49].

[0079] Comparison of transglycosylation activity of various EndoS2 D184 mutants The activity of EndoS2-D184A, D184N, and D184Q in transferring sialoni-branched complex glycan oxazoline (2) to fucosylated GlcNAc-rituximab (1) was compared. Under identical reaction conditions, the asparagine (N) mutant transferred over 30% of the oxazoline to the acceptor in 5 minutes, while the alanine (A) and glutamine (Q) mutants transferred less than 20% (Figure 7). The N mutant appears to be more active than the A and Q mutants in the transfer of complex oxazoline.

[0080] Cloning, expression, and characterization of Endo-S2 The cDNA sequence encoding Endo-S2 (sequence number 44-843 of sequence number 1) (the GenBank registry number for the Endo-S2 gene (ndoS2) is ACI61688) was cloned into the pET22b-CPD vector. This vector has the cysteine ​​protea of ​​Vibrio cholerae MARTX toxin at the C-terminus of the expressed protein. The zedimain (CPD) and a 10× histidine tag are added

[35] (SEQ ID NO: 22). It has recently been reported that high levels of saturable expression of Endo-F3 and its variants can be achieved using this vector

[32] . Alternatively, the cDNA sequence encoding Endo-S2 may include (amino acids 1 and 44-843 of SEQ ID NO: 1). Following a similar method, Endo-S2 was successfully expressed in Escherichia coli (E. coli) and immobilized metal ions were added. The enzyme was easily purified using infinity chromatography (IMAC) to obtain a soluble enzyme in a yield of over 20 mg / L. Recombinant Endo-S2 exhibited high hydrolytic activity, as demonstrated by the rapid deglycosylation of commercially available rituximab, and this hydrolytic activity was monitored by LC-MS analysis.

[0081] Generation of glycosynthase mutants from Endo-S2 Site-directed mutagenesis at key residues involved in promoting the formation of oxazolinium ion intermediates during substrate-assisted hydrolysis generated glycosynthase mutants derived from both the GH85 and GH18 families of endoglycosidases. These glycosynthase mutants include key asparagine residues for GH85 family endoglycosidases Endo-A (Asn171)

[36] , Endo-M (Asn175)[37,38], and Endo-D (Asn322)

[24] , or key aspartic acid residues for GH18 family endoglycosidases Endo-S (Asp233)

[25] and Endo-F3 (Asp-165)

[32] . From the sequence alignment of Endo-S2 and Endo-S, it was determined that Asp-184 of Endo-S2 is essential for promoting oxazolinium ion formation during hydrolysis. It was determined that this corresponds to residue 233 (Figure 2). To efficiently generate glycosynthase mutants from Endo-S2, Asp-184 was systematically replaced with 19 other native amino acid locus residues (residues 44-843) in cleavage-type expression proteins using site-directed mutagenesis. In particular, it is assumed that non-native proteins may also be used in this substitution. Using the same method demonstrated for the wild-type enzyme, the resulting 19 D184 mutants were expressed as soluble proteins in the pET22bCPD vector and purified using immobilized metal ion affinity chromatography. Expression of the mutant enzymes showed yields (15-20 mg / L) comparable to those of the wild-type enzyme.

[0082] In particular, it is assumed that non-natural proteins may also be used in this substitution. Examples of non-natural amino acids that may be used by the translation system include: non-natural analogs of tyrosine amino acids; non-natural analogs of glutamine amino acids; non-natural analogs of phenylalanine amino acids; non-natural analogs of serine amino acids; non-natural analogs of threonine amino acids; alkyl, aryl, acyl, azide, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thio acid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino-substituted amino acids, or any combination thereof.

[0083] Comparative study on the hydrolysis and transglycosylation activity of 19 mutants and WT (Washington Threat) strains. The hydrolytic activity against Fc N-glycans and the transglycosylation activity by glycan oxazolines in intact antibodies were evaluated according to the scheme shown in Figure 8. These results indicate that most mutants at the D184 residue resulted in significantly reduced or completely reduced hydrolytic activity against Fc N-glycans. Of these, the D184F, D184H, D184K, D184R, and D184W mutants completely lacked hydrolytic activity, while several other mutants (e.g., D184C, D184E, D184G, D184N, D184S, D184Y) still retained significant hydrolytic activity (Table 1). On the other hand, the evaluation of transglycosylation showed that almost all mutants possessed transglycosylation activity when using a deglycosylated rituximab as the acceptor and a branched complex glycan oxazoline as the donor substrate, but the activity varied significantly among the various mutants (Figure 8, Table 2). In particular, we found that D184C, D184M, D184G, D184E, D184Y, D184S, and D184A were the most active mutants. However, D184C, D184G, D184E, D184Y, D184S, and D184A also showed significantly residual hydrolytic activity. The most interesting mutant was D184M (which retained only slight hydrolytic activity but showed very high transglycosylation activity (second only to D184C)), and is one of the optimal glycosynthase mutants to select for glycosylation remodeling.

[0084] Endo-S2 glycosynthase exhibits remarkably broad substrate specificity in transglycosylation. The transglycosylation activity of the Endo-S2 D184 mutant was investigated using rituximab, a therapeutic monoclonal antibody, as a model. The main Fc glycans of commercially available rituximab are core-fucosylated branched-complex oligosaccharides carrying 0-2 galactose moieties, named G0F glycoform, G1F glycoform, and G2F glycoform, respectively. A typical glycosylation reconstitution approach is shown in Figure 9, and the reaction products were evaluated by LC-MS analysis (Figure 10). Treatment of rituximab (1) with wild-type Endo-S2 completely converts rituximab, as demonstrated by the conversion of the glycoform mixture (G0F, G1F, and G2F) found in commercially available rituximab (Figure 10A) to the Fucα1,6GlcNAc-glycoform (2) (Figure 10B). Deglycosylated Fucα1,6GlcNAc-rituximab (2) was purified from WT endoglycosidase, and the glycan was released by protein A affinity chromatography and used as an acceptor in the transglycosylation reaction. We discovered that EndoS2 D184M can efficiently transfer sialylated branched complex (SCT) N-glycans from the corresponding glycan oxazoline (4) to the Fucα1,6GlcNAc-rituximab acceptor (2) to form the S2G2F glycoform (3) of rituximab. This reaction could be easily completed with 20 molar equivalents (i.e., 10 molar equivalents per monomeric Fc domain) of glycan oxazoline. Since almost no starting material was detected, the reaction yield was estimated to be over 95% by LC-MS analysis, which confirmed the completion of transglycosylation. LC-MS analysis of the transglycosylation product (3) supporting SCT N-glycan revealed that the heavy chain of (3) appears as a single type at 51412 (deconvolution data), which indicates that each of the SCT The calculated molecular weight (M=51421Da) for the heavy chain supporting the N-glycan (containing core fucose) is in good agreement with the calculated value (Figure 10C).

[0085] In addition to the complex N-glycan, the specificity of Endo-S2 was further tested with high-mannose (HM) Man9GlcNAc oxazoline (7) and sialohybrid (Hyb) Neu5AcGalGlcNAcMan5GlcNAc oxazoline (5) as donor substrates in the transglycosylation reaction. This reaction formed the corresponding homogeneous glycoforms (8) and (6), respectively (Figure 10E, Figure 10G). Deconvoluted ESI-MS of the heavy chain of the transglycosylation product (8) showed a single species at 51074, as shown in Figure 10E, which was in good agreement with the calculated molecular weight (M=51081Da) of the rituximab heavy chain supporting Man9GlcNAc2 glycan. Similarly, deconvoluted ESI-MS of the heavy chain of the transglycosylation product (6) showed a single species at 51082, as shown in Figure 10G, which was in good agreement with the calculated molecular weight (M=51090Da) of the rituximab heavy chain carrying Neu5AcGalGlcNAcMan5GlcNAc2 glycan. Under the same conditions, previously reported Endo-S mutants, such as the D233A and D233Q mutants of Endo-S

[25] , showed only slight transglycosylation activity with high-mannose and hybrid N-glycans, but this mutant was found to be able to efficiently transfer branched complex N-glycans. In addition, the recently reported Endo-F3 D165A mutant

[32] was unable to transfer high-mannose or hybrid N-glycans, but was able to act on branched and tribranched complex sugars. Therefore, these Endo-S2-derived variants represent the first glycosynthases capable of efficiently transferring high-mannose and hybrid N-glycans to corefucosylated GlcNAc acceptors in intact antibodies. It should be noted that while Endo-A variants (N171A and N171Q) were capable of transferring high-mannose N-glycans to the GlcNAcFc domain, they were unable to utilize corefucosylated GlcNAc-Fc as acceptors [22, 23, 36].These studies also demonstrate that the combined use of wild-type Endo-S2 and Endo-S2 glycosynthase variants provides a particularly efficient glycosylation reconstitution approach to various homogeneous glycoforms of antibodies starting from a single precursor.

[0086] Endo-S2-based glycosylation reconstruction for the preparation of non-fucosylated glycoforms Non-fucosylated IgG glycoforms are preferred for anticancer treatment because mAbs with low fucose content and Fc glycosylation have already been shown to enhance ADCC activity in vitro and enhance anticancer effects in vivo, especially in patients carrying the low-affinity F158 allele of the FcγIIIa receptor [8, 9, 39, 40]. To test whether Endo-S2 can glycosylate non-fucosylated IgG, Fucα1,6GlcNAc-rituximab (2) was used to test Lactobacillus casei (Lactobacillus casei) recombinant α1,6-fucosidase

[41] is incubated together. We baited the molecule to obtain GlcNAc-rituximab (9) lacking core fucose. Transglycosylation of GlcNAc-rituximab (9) catalyzed by Endo-S2 D184M was performed with sialo-branched complex (CT) N-glycan oxazoline (10). We found that the D184M mutant efficiently transfers the N-glycan to the GlcNAc acceptor in the antibody, resulting in a completely galactosylated and non-fucosylated glycoform (11) with essentially quantitative conversion. Deconvoluted ESI-MS of the heavy chain of the transglycosylation product (11) showed a single species at 50684, as shown in Figure 10I, which was in good agreement with the calculated molecular weight (M=50693Da) of the rituximab heavy chain carrying a completely galactosylated, bifurcated complex N-glycan lacking core fucose. In addition to confirmation of site-specific glycosylation of the heavy chain by LC-MS analysis combined with enzymatic transformation, the light chains of the transglycosylation products (3, 6, 8, 11) also appeared as a single type at 23034, which is consistent with the calculated molecular weight (M=23039Da) of the light chain of unmodified rituximab (Figures 10D, 10F, 10H, 10J). These results indicate that no non-enzymatic modifications occurred in the heavy and light chains during the Endo-S2-catalyzed glycosylation reconstitution process, except for the linkage of transferable N-glycans at the GlcNAc acceptor in the Fc domain. It should be noted that rituximab in its fully galactosylated and unfucosylated glycoforms (11) has already been shown to have at least 20-fold enhanced affinity for the FcγIIIA receptor compared to commercially available rituximab, which is a manifestation of significantly enhanced ADCC

[25] .

[0087] Comparison of transglycosylation efficiency to various N-glycan substrates by the Endo-S2 D184M mutant. To further elucidate the characteristics of Endo-S2 D184M's N-glycan substrate selection, three parallel transglycosylation reactions were performed with complex (SCT), high-mannose (HM), and hybrid (Hyb) N-glycan oxazolines, respectively. The progress of the reactions was monitored by LC-MS analysis of reaction aliquots taken at multiple time points, and the results are summarized in Figure 11. Under identical conditions, the transglycosylation reaction with SCT-oxazoline (4) was completed within 20 minutes to produce S2G2F-rituximab (3), while transglycosylation with HM-oxazoline (7) and Hyb-oxazoline (5) was very slow. These results suggest that Endo-S2 D184M prefers complex-type N-glycans over high-mannose and hybrid-type N-glycans, despite the significantly relaxed N-glycan specificity.

[0088] Comparison of transglycosylation efficiency of typical Endo-S2 and Endo-S mutants. Endo-S glycosynthase mutants D233Q and D233A, which efficiently transglycosylate rituximab with complex N-glycan oxazolines, have already been generated

[25] . More recently, the EndoS D233 mutant has been used to generate homogeneous monoclonal antibodies for structural and functional studies [26-31]. To compare the transglycosylation efficiency of glycosynthase mutants derived from Endo-S2 and Endo-S, the Endo-S D233Q mutant, its equivalent Endo-S2 D184Q mutant, and the Endo-S2 D184M mutant were selected to catalyze three parallel transglycosylation reactions. The time course of the transglycosylation reaction was monitored by LC-MS analysis and summarized in Figure 12. Under reaction conditions, the Endo-S2 D184M mutant showed remarkably potent transglycosylation activity, with glycan transfer completed within 10 minutes. Other Endo-S2 mutants were also able to smoothly transfer glycans, completing the process within one hour. However, the corresponding Endo-S mutant (D233Q) was very inefficient, achieving only about 10% transglycosylation in one hour under the same conditions (Figure 12). This has been demonstrated in other experiments. As such, achieving the same level of transglycosylation as catalyzed by the Endo-S2 D184Q mutant required an additional (10-fold) amount of Endo-S D233Q mutant and a large excess of glycan oxazoline, and the Endo-S2 D184M mutant was far more efficient than the D184Q mutant. These studies suggest that the newly discovered Endo-S2 D184 mutant is superior to previously reported Endo-S mutants for antibody glycosylation reconstitution in both reaction efficiency and substrate diversity.

[0089] Glycosylation reconstitution of trastuzumab (Herceptin) using a set of Endo-S2 enzymes To demonstrate that the observed enzymatic properties of Endo-S2 and its variants are generally applicable to antibody glycosylation reconstitution, we performed glycosylation reconstitution of trastuzumab (Herceptin), another monoclonal antibody widely used in the treatment of breast cancer. Glycosylation reconstitution was evaluated by the synthesis of sialyzed glycoforms of trastuzumab (Figure 13). Treatment of trastuzumab with wild-type Endo-S2 completely deglycosylated it, as demonstrated by the conversion of the glycoform mixture (G0F, G1F, and G2F) found in commercially available trastuzumab (Figure 13A) to the Fucα1,6GlcNAc-glycoform (12) (Figure 13B). Deglycosylated trastuzumab was purified from endoglycosidase, and the glycan was released by protein A affinity chromatography and used as an acceptor in the transglycosylation reaction. Incubation of Fucα1,6GlcNAc-trastuzumab (12) with the donor substrate SCT-oxazoline (4) in the presence of the Endo-S2 D184M mutant rapidly converted deglycosylated trastuzumab (12) to fully glycosylated trastuzumab (13). This reaction was essentially quantitative, yielding a single transglycosylation product (13). LC-MS analysis of this transglycosylation product (13) revealed that the heavy chain of (13) appeared as a single species at 51500 Da (deconvolution data) (Figure 13C), indicating the linkage of a single sialylated N-glycan on the heavy chain. On the other hand, the light chain of this transglycosylation product (13) appeared as a single species at 23438, which closely matches the light chain of trastuzumab without any additional modifications (Figure 13D).

[0090] As implied in a recent publication

[30] , to confirm that the N-glycan specifically attaches to the Asn-297 N-glycosylation site of the Fc domain instead of other sites on the polypeptide backbone that may occur by non-enzymatic reactions, this transglycosylation product (13) was treated with PNGase F and the protein moiety was examined by mass spectrometry. PNGase F was highly specific and could only release the N-glycan when it attached to the Asn side chain in the N-glycosylamide linkage at a conserved glycosylation site in the N-glycoprotein. LC-MS analysis of the heavy chain of the transglycosylation product (13) treated with PNGase F yielded a single species of 49150 (deconvolution data), which corresponds to the polypeptide backbone of the heavy chain without any additional modifications (Figure 13E). In summary, these results clearly demonstrate that a single sialylated bibranched N-glycan is conjugated to the antibody heavy chain, and that the intact N-glycan is linked at a conserved N-glycosylation site without any non-enzymatic glycation of the antibody. Highly efficient transformation catalyzed by the Endo-S2 glycosynthase variant allowed for a significant reduction in reaction time and the use of much less excess glycan oxazoline to achieve quantitative conversion.

[0091] This specification describes the discovery of a novel class of glycosynthases derived from the endoglycosidase (Endo-S2) of the Streptococcus pyogenes M49 serotype, which has not been previously reported. Compared to the glycosynthases derived from Endo-A, Endo-M, Endo-S, and Endo-F3, the mutants exhibited broad substrate characteristics and much potent transglycosylation activity for antibody glycosylation reconstitution. These findings were made possible by systematic mutagenesis at the critical residue D184, coupled with a comparative analysis of the hydrolytic and transglycosylation activities of the 19 resulting mutants. The experimental data also revealed significant differences in both hydrolytic and transglycosylation activity among the 19 mutants (Tables 1 and 2), which would have been difficult to predict without this comparative study. Several notable mutants were identified, such as the D184M and D184Q mutants, which exhibit high transglycosylation activity for glycosylation reconstitution but retain only slight hydrolytic activity.

[0092] A comparison of the hydrolytic and transglycosylating activities of these mutants reveals several interesting characteristics. Firstly, most of the mutants that showed high transglycosylating activity, such as the D184C, D184G, D184E, D184Y, D184S, and D184A mutants, also possessed relatively high residual hydrolytic activity. The exception was the D184M mutant, which showed remarkable transglycosylating activity but retained a small amount of residual hydrolytic activity, making it the most efficient glycosynthase for glycosylation reconstitution. Secondly, most of the mutants in which the D184 residue was replaced with an amino acid having a positively charged side chain (K, R, H) or a bulky hydrophobic side chain (I, L, F, W) showed very low activity in both transglycosylation and hydrolysis. Interestingly, however, the D184Y mutant retained the highest hydrolytic activity among all the mutants and also possessed relatively high transglycosylation activity.

[0093] Another significant finding is the much broader substrate specificity of Endo-S2-derived glycosynthase variants compared to previously reported glycosynthase substrate specificity. Two notable Endo-S2 glycosynthase variants identified, D184M and D184Q, were found to be able to efficiently transfer all three major types of N-glycans, including high-mannose, complex, and hybrid types, in antibody glycosylation reconstitution. In addition, these Endo-S2 glycosynthases were able to recognize both core-fucosylated GlcNAc and non-fucosylated GlcNAc moieties in the Fc domain as transglycosylation acceptors. These findings significantly expand the scope of glycosylation reconstitution strategies. For example, previously reported Endo-S and Endo-F3 are specific to complex N-glycans and cannot efficiently transfer high-mannose and hybrid N-glycans, and Endo-F3 is efficient only for core-fucosylated GlcNAc acceptors

[32] . Direct comparison of transglycosylation activity between typical Endo-S2 glycosynthase mutants and Endo-S glycosynthase mutants reveals that Endo-S2 mutants are generally far more active than their corresponding Endo-S mutants. Initial rate estimates suggest that the Endo-S2 D184Q mutant is at least 10-fold more active than the Endo-S D233Q mutant, and the best Endo-S2 mutant, D184M, is estimated to be 100-fold superior to the Endo-S D233Q mutant in the glycosylation of deglycosylated rituximab (Figure 12). Finally, in addition to rituximab reconstitution, the highly efficient glycosylation reconstitution of trastuzumab (Herceptin) by using a pair of Endo-S2 (wild-type and D184M mutant) enzymes to produce a single, homogeneous glycoform without side effects (Figure 13) demonstrates the capabilities of the newly discovered glycosynthase. These highly efficient glycosynthases, which also exhibit significantly relaxed substrate specificity, are expected to find broad applications in the production of various homogeneous glycoforms of antibodies for structural and functional studies, as well as in the more effective development of antibody-based therapies.

[0094] Figure 14 illustrates the use of the present invention to produce a desired homogeneous glycoform of an antibody. IVIG is widely used in the treatment of rheumatoid arthritis, but usually requires high doses (e.g., 1-1.5 g / kg). However, as shown in Figure 14, a homogeneous glycoform offers the advantages of reduced dose, enhanced potency, and reduced side effects. Such effects are attributed to reconstituted IVIG with over 95% sialylation due to the use of the EndoS2 variant of the present invention. Such improvements in glycosylation are applicable to glycoprotein hormones, cytokines (IL-2, interferon, etc.), and enzyme replacement therapy (lysosomal storage diseases).

[0095] Experimental Procedure Materials - The monoclonal antibodies rituximab and trastuzumab (Herceptin) were products of Genentech Inc., (South San Francisco, CA). Sialo-conjugated oxazoline And siaroglycan complex oxazolines were synthesized according to previously reported procedures

[42] . High-mannose (HM) glycans (Man9GlcNAc) were prepared from soybean flour according to previously described procedures

[43] . The synthesis of hybrid (Hyb) glycans (Neu5AcGalGlcNAcMan5GlcNAc) was achieved by sequential enzymatic glycosylation of Man5GlcNAc under the catalytic action of β-1,2-GlcNAc transferase (GnT1)

[44] , β-1,4-galactosyltransferase

[45] and α-2,6-sialyltransferase

[46]

[43] . HM glycan oxazolines and hybrid glycan oxazolines were synthesized according to previously described one-pot transformation procedures

[42] . Endo-S D233Q derived from Streptococcus pyogenes was overexpressed and purified according to the inventors' previous procedure

[25] .

[0096] Site-directed mutagenesis, expression, and purification of recombinant Endo-S2 The cDNA encoding amino acids 44-843 of Endo-S2 from Streptococcus pyogenes NZ131 (serotype M49) was amplified by PCR and cloned into a pCPDLasso vector (pET22b-CPD derivative) (35), and the CPD (cysteine) of Vibrio cholerae MARTX toxin was extracted. The sequences of the protease domain and histidine tag are described in SEQ ID NO: 22. For saturation mutagenesis of the Asp-184 residue, the forward primer 5'-CGTAAATTCGTGCTCAATNNNAATATCTAGTCCATCGACACCACGATCAGTT-3' (SEQ ID NO: 23) and the reverse primer 5'-AACTGATCGTGGTGTCGATGGACTAGATATTNNNATTGAGCACGAATTTACG-3' (SEQ ID NO: 24) were used. Mutations were confirmed by DNA sequencing. Plasmids containing the mutated Endo-S2 gene were used to transform *E. coli* BL21 (DE3). To simultaneously produce 20 Endo-S2 D184 variants, the transformants were cultured in 20 mL of 2×YT broth medium supplemented with 100 μg / mL carbenicillin. The cultures were grown at 37°C until the cells reached an OD600 of 0.8–1.0. Then, 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the cultures to induce protein overproduction at 20°C. After 24 hours, the cells were collected by centrifugation. The cell pellet was subjected to bacterial cell lysing according to the manufacturer's instructions. Dissolved in Buffer (Gold Biotechnology, Inc.). 10x histidine (His10) Tagged EndoS2 / CPD fusion proteins were purified using NiNTA Spin Columns (Qiagen). The purified EndoS2 proteins were then subjected to Amicon ultrafiltration (10kDa, Millipore). Desalting was performed in PBS (pH 7.4) using centrifugal diafiltration. The purity of this protein was confirmed by SDS-PAGE, and the concentration was measured using absorbance at 280 nm with NanoDrop 2000c. For large-scale purification of selected Endo-S2 variants, 1 L of culture medium was used. Cell lysates were applied to a HisTrap HP column (GE) and washed with PBS containing 0.5 M NaCl and 20 mM imidazole (pH 7.4). His-tagged proteins were eluted using a gradient of 0–250 mM imidazole in PBS buffer. The eluted fraction containing Endo-S2 protein was pooled, concentrated, and further purified by size exclusion chromatography using a HiPrep 16 / 60 Sephacryl S-200 HR column (GE). did.

[0097] Liquid chromatography-mass spectrometry (L-ESI-MS) of IgG LC-MS analysis was performed using Exactive Plus Orbitrap (Thermo Scientific). For standard antibodies, this analysis was performed within 9 minutes at a flow rate of 0.4 ml / min using a Waters XBridge™ BEH300 C4 column (3.5 μm, 2.1 × 50 mm) with a linear gradient of 5–90% MeCN containing 0.1% formic acid. For analysis of the light and heavy chains of antibodies, IgG antibodies were treated with 50 mM TCEP, heated at 37°C for 20 minutes, and then subjected to LC-MS analysis using an Agilent Poroshell 300SB-C8 column (5 μm, 75 × 1 mm). This analysis was performed within 6 minutes at a flow rate of 0.40 mL / min with elution at 60°C with a linear gradient of 25–35% MeCN containing 0.1% formic acid. LC-MS analysis of PNGase F-treated antibody glycoforms was performed using the same method, but included a 3-hour incubation with PNGase F before TCEP treatment. The raw data was deconvolved using MagTran (Amgen).

[0098] Deglycosylation of rituximab by wild-type Endo-S2 to obtain (Fucα1,6)GlcNAc-rituximab Commercial rituximab in the initial buffer was incubated with wild-type Endo-S2 at 37°C for 1 hour at an antibody-to-enzyme ratio (weight ratio) of 500:1. LC-MS analysis showed complete cleavage of the N-glycan on the heavy chain. Deglycosylated rituximab was purified by protein A chromatography. LC-MS: Calculation of the heavy chain of (Fucα1,6)GlcNAc-rituximab (2), M=49420Da; measured (m / z), 49412 (deconvolution data).

[0099] Defucosylation of (Fucα1,6)GlcNAc-rituximab by bacterial α-fucosidase (Fucα1,6)GlcNAc-rituximab (2) in Tris-HCl buffer solution (50 mM, pH 7.4) was incubated with Lactobacillus casei-derived α-fucosidase AlfC at 37°C in a 50:1 antibody-to-enzyme ratio. After 16 hours of incubation, LC-MS monitoring showed that (Fucα1,6)GlcNAc rituximab (2) was completely defucosylated to produce the product GlcNAc-rituximab (9). This defucosylated rituximab was purified by protein A chromatography. LC-MS: Calculation of the heavy chain of GlcNAc-rituximab (9) supporting the GlcNAc moiety, M=49274Da; measured (m / z), 49265 (deconvolution data).

[0100] Enzyme assay The hydrolytic activity of each Endo-S2 variant (0.1 μg) was assayed at 30°C using pure sialo-complexed (S2G2F) rituximab 3 (10 μg, 7.0 μM) as the substrate in PBS buffer (pH 7.4, 10 μl). Aliquots of each reaction mixture were diluted with 0.1% formic acid to stop the reaction and analyzed by LC-MS. The relative amounts of substrate and hydrolysis product were determined by deconvolution of raw data using MagTran and corresponding M2. Quantification was performed after integration of the S peak. The transglycosylation reaction by synthetic sugar oxazolines was assayed as follows: (Fucα1,6)GlcNAc-rituximab (100 μg, 69 μM) and SCTox (1.38 mM, 20 equivalents) were incubated with 0.1 μg of each Endo-S2 variant in PBS (pH 7.4, 10 μl) at 30°C. This reaction was stopped and analyzed as in the hydrolysis activity assay described above. The experiment was repeated at least twice to ensure consistency in these results.

[0101] Transglycosylation of Fuc1,6 GlcNAc rituximab by Endo-S2 D184M using SCT-ox, Hyb-ox, and HM-ox: Synthesis of 3, 6, and 8. A solution of Fuc1,6GlcNAc-rituximab (1 mg, 69 μM) (2) and SCTox (1.38 mM, 20 equivalents) (4) was incubated with Endo-S2 D184M (5 μg) in 100 μl of 100 mM Tris buffer (pH 7.4) at 30°C for 15 minutes. LC-MS analysis indicated completion of this transglycosylation reaction. Product (3) was purified using protein A chromatography. LC-MS: Calculation of the heavy chain of (3) supporting the fully sialyzed bibranched N-glycan, M=51421Da; measured (m / z), 51412 (deconvolution data).

[0102] A solution of Fuc1,6GlcNAc-rituximab (1 mg, 69 μM) (2) and Hybox (1.38 mM, 20 equivalents) (5) was incubated with Endo-S2 D184M (5 μg) in 100 μl of 100 mM Tris buffer (pH 7.4) at 30°C for 30 minutes. Then, an additional 10 equivalents of Hybox (5) were added, and the reaction was monitored by aliquot LC-MS. When LC-MS analysis indicated near completion of the transglycosylation reaction, the product (6) was purified using protein A chromatography. LC-MS: Calculation of the heavy chain of (5) supporting the sialyzed hybrid N-glycan, M=51090Da; measured (m / z), 51082 (deconvolution data).

[0103] A solution of Fuc1,6GlcNAc-rituximab (1 mg, 69 μM) (2) and HMox (1.38 mM, 20 equivalents) (7) was incubated with Endo-S2 D184M (5 μg) in 100 μl of 100 mM Tris buffer (pH 7.4) at 30°C for 30 minutes. Then, an additional 10 equivalents of HMox (7) were added, and the reaction was monitored by aliquot LC-MS. If LC-MS analysis indicated completion of the transglycosylation reaction, the product (8) was purified using protein A chromatography. LC-MS: Calculation of the heavy chain of (12) supporting the high-mannose type (Man9GlcNAc2) N-glycan, M=51081Da; measured (m / z), 51074 (deconvolution data).

[0104] Synthesis of GlcNAc-rituximab transglycosylation by CT-ox using Endo-S2 D184M:11 Solutions of GlcNAc-rituximab (1 mg, 69 μM) (9) and CTox (1.38 mM, 20 equivalents) (10) were incubated with Endo-S2 D184M (5 μg) in 100 μl of 100 mM Tris buffer (pH 7.4) at 30°C for 30 minutes. LC-MS analysis indicated completion of the transglycosylation reaction. Product (11) was purified using protein A chromatography. LC-MS: Calculation of the heavy chain of (11) supporting the branched N-glycan, M=50693Da; measured (m / z), 50684 (deconvolution data).

[0105] Comparison of transglycosylation activity of Endo-S2 D184M mutants using SCT-oxazoline, HM-oxazoline, and Hyb-oxazoline as donor substrates. In three separate reactions, Fuc1,6GlcNAc-rituximab (0.2 mg, 69 μM) (2) was incubated with SCTox (4), Hyb-ox (5), or HM-ox (7) (1.38 mM, 20 equivalents) in 20 μl of 100 mM Tris buffer (pH 7.4) at 30°C with Endo-S2 D184M (1 μg). Aliquots (0.5 μl) of the reaction were taken at several time points and the reaction was stopped by dilution with 0.1% formic acid. All aliquots were analyzed by LC-MS, and deconvolution data were obtained. Then, the percentage of transglycosylation was calculated.

[0106] Comparison of transglycosylation activity of Endo-S2 D184M mutant, D184Q mutant, and Endo-S D233Q mutant using SCT-oxazoline as a donor substrate. In three separate reactions, Fuc1,6GlcNAc-rituximab (0.2 mg, 69 μM) (2) and SCTox (4) (1.38 mM, 20 equivalents) were incubated with Endo-S2 D184M, Endo-S2 D184Q, or Endo-S D233Q (1 μg) in 20 μl of 100 mM Tris buffer (pH 7.4) at 30°C. Aliquots (0.5 μl) of the reaction were taken at several time points and the reaction was stopped by dilution with 0.1% formic acid. All aliquots were analyzed by LC-MS, and the percentage of transglycosylation was calculated from the deconvolution data.

[0107] Transglycosylation of Fuc1,6GlcNAc trastuzumab by Endo-S2 D184M and SCT-ox. Commercially available trastuzumab (lyophilized powder) was dissolved in water and deglycosylated with wild-type Endo-S2 according to the same procedure as for rituximab. For transglycosylation, a solution of Fuc1,6GlcNAc-trastuzumab (12) (1 mg, 69 μM) and SCTox (4) (1.38 mM, 20 equivalents) was incubated with Endo-S2 D184M (5 μg) in 100 μl of 100 mM Tris buffer (pH 7.4) at 30°C for 15 minutes. LC-MS analysis indicated completion of the transglycosylation reaction. The product (13) was purified using protein A chromatography.

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[0109] [Table 1]

[0110] Table 2

Claims

1. A method for preparing a fucosylated glycoprotein or a non-fucosylated glycoprotein having a predetermined oligosaccharide moiety containing a predetermined number and type of sugar residues, wherein the method is To provide fucosylated acceptor proteins or non-fucosylated acceptor proteins, including fucosylated N-acetylglucosamine (GlcNAc) acceptor protein or non-fucosylated GlcNAc acceptor protein, and The fucosylated acceptor protein or non-fucosylated acceptor protein and the activated oligosaccharide donor are used in Streptococcus pyogenes (serotype M49) Endo-S2 The method involves enzymatically reacting in the presence of an Asp184 mutant enzyme, the Endo-S2 mutant enzyme, wherein the mutant enzyme includes a catalytic domain and has amino acid mutations that show increased transglycosylation efficiency compared to the wild-type Endo-S2 enzyme selected from the group consisting of D184A (SEQ ID NO: 2), D184N (SEQ ID NO: 3), D184Q (SEQ ID NO: 4), D184S (SEQ ID NO: 16), and D184T (SEQ ID NO: 17); the activated oligosaccharide donor includes oxazoline and a predetermined oligosaccharide moiety containing a predetermined number and type of sugar residues; the oligosaccharide moiety is covalently linked to the fucosylated GlcNAc acceptor protein or non-fucosylated GlcNAc acceptor protein by the enzymatic reaction with the Endo-S2 mutant enzyme, thereby preparing the fucosylated glycoprotein or non-fucosylated glycoprotein having the predetermined oligosaccharide moiety. Includes, The fucosylated acceptor protein or non-fucosylated acceptor protein is an antibody, its Fc-containing fragment, or intravenous immunoglobulin (IVIG). The antibody further comprises additional portions selected from the group consisting of therapeutic agents for treating cancer, therapeutic agents for HIV, toxins, antigens, chemokines, and cytokines, for forming a modified antibody. method.

2. The method according to claim 1, wherein the activated oligosaccharide donor is a synthetic oligosaccharide oxazoline or a natural N-glycan oxazoline.

3. The method according to claim 2, wherein the synthetic oligosaccharide oxazoline comprises a high-mannose type, a hybrid type, a sialoglycan oxazoline or a complex type N-glycan, and has disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, heptasaccharides, octasaccharides, xusaccharides, decasaccharides or elevensaccharides.

4. The method according to any one of claims 1 to 3, wherein the activated oligosaccharide donor further comprises an additional biological activator or tag.

5. The method according to claim 4, wherein the additional biological activator or tag is a drug, a toxin, a fluorescent probe, biotin, PEG, a lipid, or a polypeptide.

6. The method according to any one of claims 1 to 5, wherein the fucosylated acceptor protein is alpha-1-6-fucosyl-GlcNAc-protein.

7. The fucosylated glycoproteins or non-fucosylated glycoproteins mentioned above are 17b, 48d, A32, C11, 2G12, F240, IgG1b12, 19e, X5, TNX-355, cetuximab, rituximab, muromonab-CD3, absiximab, daclizumab, basiliximab, palivizumab, infliximab, trastuzumab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab, omalizumab, tositumomab, I-131 tositumomab, efalizumab, bevacizumab, panitumumab, pertuzumab, natalizumab, etanercept, IGN101, borosiximab, and anti-CD80. mAb, anti-CD23 mAb, CAT-3888, CDP-791, epratuzumab, MDX-010, MDX-060, MDX-070, matuzumab, CP-675, 206, CAL, SGN-30, zanorimumab, adecatumumab, olegobomab, nimotuzumab, ABT-874, denosumab, AM-108, AMG-714, fontrizumab, daclizumab, golimumab, CNTO The method according to any one of claims 1 to 6, wherein the antibody is selected from the group consisting of 1275, HuMax-CD20, belimumab, MLN1202, vizilizumab, tocilizumab, ocrelizumab, certolizumab pegol, eculizumab, pexerizumab, absiximab, ranibizumab, mepolizumab, and MYO-029.

8. The method according to any one of claims 1 to 7, wherein the fucosylated acceptor protein or non-fucosylated acceptor protein is formed by removing heterogeneous or undesirable N-glycans with an enzyme selected from the group Endo-H, Endo-F3, Endo-S, Endo-S2, and Endo-A.

9. A use of the method according to any one of claims 1 to 8 for preparing a glycan-reconstituted antibody for delivering a drug having biological activity to treat a certain condition, wherein the reconstituted antibody comprises a recombinant fucosylated antibody or a non-fucosylated antibody having a predetermined number of sugar residues and a therapeutic agent linked to a terminal sugar or sialylated group.

10. The method according to claim 1, wherein the IVIG exhibits an Fc-sialylated glycoform, To provide IVIG supporting FcN-glycan, The method involves deglycosylating the Fc N-glycan using endoglycosidase-S enzyme (Endo-S), endoglycosidase-S2 enzyme (Endo-S2), or endoglycosidase-F3 enzyme (Endo-F3) to form a GlcNAc acceptor, wherein the GlcNAc portion is located in the Fc region of the IVIG, and the GlcNAc acceptor is either fucosylated or unfucosylated, and Under the catalytic action of the Endo-S2 mutant enzyme, which exhibits increased transglycosylation and decreased hydrolytic activity compared to the wild-type Endo-S2 enzyme, the GlcNAc moiety is transglycosylated with a siaroglycan oxazoline having a predetermined number of sugar residues to form sialylated IVIG. A method that further includes this.

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