Novel Endo S2 mutant enzyme

Abstract

A novel Endo S2 mutant enzyme is provided, which shows significantly enhanced transglycosylation activity and retains most of hydrolytic activity, and can be used for glycosyl modification and sugar chain reconstitution of polypeptides or proteins with N-glycans, such as antibodies.

Description

Novel Endo S2 mutant enzyme

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on November 10, 2023, with application number CN2023115016001 and invention name “Novel Endo S2 mutant enzyme”, and the Chinese patent application filed with the China Patent Office on October 31, 2024, with application number CN202411548065X and invention name “Novel Endo S2 mutant enzyme”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present disclosure relates to the fields of carbohydrate chemistry and enzymology, and in particular to the use of a recombinant novel Endo S2 mutant enzyme to perform sugar chain modification on polypeptides or proteins. The modified proteins, such as antibodies, can be used for site-specific carbohydrate conjugation in antibody-drug conjugates. Background Art

[0003] Antibody-drug conjugates (ADCs) use linkers to attach cytotoxins to antibodies. Leveraging the antibody's specific targeting of tumor-associated antigens and the efficient loading capacity of the attached cytotoxin, ADCs achieve targeted delivery and enrichment of the cytotoxin, leading to precise tumor cell killing. Therefore, compared with traditional chemotherapy drugs, ADCs offer fewer side effects, a broader therapeutic effect, and a higher therapeutic index, making them one of the fastest-growing drugs in recent years. Traditional ADC conjugation methods utilize lysine (Lys) or cysteine ​​(Cys) residues, which are abundant and well-exposed in antibodies, to link the cytotoxin. This random conjugation approach can lead to ADC heterogeneity, i.e., uneven cytotoxin conjugation sites and number of conjugated molecules, negatively impacting pharmacokinetics, tolerability, efficacy, and other parameters. Subsequent developments in site-specific conjugation technologies, such as specific amino acid modification (THIOMAB technology), non-natural amino acid insertion, sugar site-specific conjugation, and enzyme-catalyzed conjugation, each have their own advantages and disadvantages.

[0004] A typical IgG antibody is composed of two light chains and two heavy chains. The four chains form three different domains, including two variable Fab domains (used to recognize and bind to antigens) and a constant Fc domain (which binds to FcγR on the surface of receptor cells and mediates the interaction between antibodies and host cells). Almost all therapeutic antibody homodimers have an N-glycan modification at asparagine 297 (N297) in the Fc domain. This conserved glycosylation site can serve as a conjugation site for cytotoxins, and based on this, sugar-specific conjugation technology has been developed. The advantage of sugar-specific conjugation technology is that the N297 glycan site in the Fc domain is far away from the Fab domain, avoiding the risk of reduced antigen binding ability of the conjugated antibody. At the same time, the conjugation method does not require the design and modification of the antibody's amino acid sequence, which also reduces the risk of immunogenicity.

[0005] Endoglycosidases, also known as endo-β-N-acetylglucosaminidase, are glycoside hydrolases that act on β-1,4-glycosidic bonds. In recent years, they have become a research focus for antibody glycoengineering. They have also been found to possess transglycosylation activity and can be used in ADC site-specific sugar conjugation techniques. Endoglycosidases are available from a wide range of sources, including Endo S from Streptococcus pyogenes, Endo F1, Endo F2, and Endo F3 from Flavobacterium meningosepticum, and Endo S2 from serotype M49 of Streptococcus pyogenes. Among them, Endo S2 has a broader substrate specificity and can recognize and act on complex glycans (Endo S cannot), high-mannose glycans (Endo S cannot), complex multi-antennary glycans (Endo F1 cannot), core non-fucosylated glycans (Endo F3 cannot), and core fucosylated glycans. Therefore, Endo S2 has great potential in the field of sugar-directed conjugation to prepare ADCs. However, Endo S2 belongs to the GH18 family of glycosidases and exhibits more glycoside hydrolysis activity and lower transglycosylation activity, making it unsuitable for use in ADC production. Therefore, there is an urgent need to obtain Endo S2 mutants with significantly enhanced transglycosylation activity while retaining most of the hydrolysis activity, so as to facilitate the one-step method for antibody glycosylation remodeling and sugar-directed conjugation to prepare ADC drugs.

[0006] SUMMARY OF THE INVENTION

[0007] The present disclosure provides a recombinant Endo S2 mutant enzyme that exhibits enhanced transglycosylation activity while retaining a substantial portion of its hydrolytic activity. It is capable of glycosylation modification of polypeptides or proteins containing N-glycans, including glycoform hydrolysis and sugar chain reconstruction. Therefore, it is suitable for one-step site-specific glycoconjugation of antibodies or their Fc fragments, such as for the synthesis of ADC drugs or antibody-containing detection drugs. The prepared ADC drugs have the function of delivering therapeutic agents such as cytotoxins, and have a stronger killing effect on target cells.

[0008] The recombinant Endo S2 mutant enzyme disclosed herein is derived from the endo-β-N-acetylglucosaminidase (Endo S2) of Streptococcus pyogenes serotype M49 strain NZ131. The sequence of the wild-type enzyme is shown in SEQ ID NO: 1. According to the literature (Tiezheng Li et al, Glycosynthase mutants of endoglycosidase S2 show potent transglycosylation activity and remarkably relaxed substrate specificity for antibody glycosylation remodeling, 2018-12-22), Endo S2 can satisfy its hydrolysis and transglycosylation activities while retaining the consecutive amino acids from positions 38 to 819 of SEQ ID NO: 1. That is, positions 38 to 819 of SEQ ID NO: 1 are known active fragments of Endo S2. Various truncated fragments of the enzyme that contain positions 38 to 819 of SEQ ID NO: 1 and still possess hydrolysis and transglycosylation activities are within the scope of the present disclosure.

[0009] The Endo S2 mutant enzyme disclosed herein is preferably produced by site-directed mutagenesis in the sequence of SEQ ID NO: 1, or an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1, or in the contiguous amino acid sequence comprising positions 38 to 819 of SEQ ID NO: 1, and has mutations at one or two of the following amino acid positions: Y70, F106, G140, R176, D179, Q250, Y252, S285, E288, Y339, preferably R176 and / or D179.

[0010] In some preferred embodiments, the Endo S2 mutant enzymes disclosed herein have mutations at one or two of the following amino acid positions: Y70, F106, G140, R176, D179, Q250, Y252, S285, E288, Y339, preferably R176 and / or D179, in an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 1.

[0011] Furthermore, in some preferred embodiments, the mutation is selected from at least one of the following: Y70F, F106W, F106Y, G140N, R176Y, D179A, Q250Y, Q250F, Y252F, S285D, S285A, E288F, E288R, Y339F, R176Y / D179A. More preferably, the mutation is R176Y and / or D179A. Domains and fragments comprising any of the aforementioned Endo S2 mutant enzymes are encompassed by the present disclosure, wherein any such domains and fragments can be fused to other proteins, including but not limited to CPD, Fc, MBP, etc. The mutant proteins can be modified or truncated, including but not limited to the N-terminus and C-terminus. In some embodiments, the sequence before at least one amino acid between M1 and E37 of the mutant can be deleted by cleavage from the N-terminus, and / or the sequence after at least one amino acid between Y820 and D843 of the mutant can be deleted by cleavage from the C-terminus.

[0012] Based on the functions of the aforementioned Endo S2 mutant enzyme disclosed herein, it can be used for glycosylation modification of polypeptides or proteins containing N-glycans. More specifically, it can be used to reshape the glycoforms of antibodies, remodel the sugar chains of glycoproteins, or prepare detection drugs or ADC drugs containing antibodies.

[0013] The present disclosure provides a method for remodeling the sugar chains of a polypeptide or protein containing N-glycans using a one-step method, comprising the following steps:

[0014] (a) introducing the Endo S2 mutant enzyme disclosed herein;

[0015] (b) introducing a polypeptide or protein containing N-glycan as a substrate;

[0016] (c) providing a sugar chain linker donor; and

[0017] (d) using the Endo S2 mutant enzyme to glycoside hydrolyze the polypeptide or protein containing N-glycans and transfer the sugar chain linker donor to the glycoside hydrolysis product to provide a new polypeptide or protein with sugar chain modification;

[0018] The one-step method does not contain a purification step for the glycoside hydrolysis product.

[0019] The one-step method utilizes the characteristics of the Endo S2 mutant enzyme disclosed herein that has both glycoside hydrolysis and transglycosylation activities. In the same reaction step, deglycosylation and transglycosylation are simultaneously completed. This can omit the step of further purification of the glycoside hydrolysis product after glycoside hydrolysis, making the operation more convenient and efficient in industry.

[0020] At the same time, the present disclosure also provides a method for remodeling the sugar chains of polypeptides or proteins containing N-glycans using a two-step process, wherein enzymes are introduced twice to perform glycoside hydrolysis and glycosylation, respectively, wherein the Endo S2 mutant enzyme of the present disclosure is used in at least one of the glycoside hydrolysis and glycosylation steps. The method comprises the following steps:

[0021] (a) introducing the Endo S2 mutant enzyme disclosed herein or other applicable endoglycoside hydrolase;

[0022] (b) introducing a polypeptide or protein containing N-glycan as a substrate, and performing glycoside hydrolysis under the action of the enzyme in step (a), thereby providing a glycoside hydrolysis product as an acceptor for the next step of converting the new glycosyl group;

[0023] (c) providing a sugar chain linker donor; and

[0024] (d) transferring the sugar chain linker donor to the acceptor using the Endo S2 mutant enzyme or other suitable glycosyltransferase to generate a new polypeptide or protein with sugar chain modification;

[0025] Optionally, a purification step of the glycoside hydrolysis product is further included between steps (b) and (c).

[0026] The two-step method first uses an endoglycoside hydrolase to complete a deglycosylation reaction (hydrolysis). The Endo S2 mutant enzyme disclosed herein or other suitable enzymes can be used for this step. Next, a transglycosylase is used to perform a transglycosylation reaction. The Endo S2 mutant enzyme disclosed herein or other suitable enzymes can be used for this step. The Endo S2 mutant enzyme is used in at least one of the steps: glycoside hydrolysis or neoglycosylation. Furthermore, after the deglycosylation step, the glycoside hydrolysis product can be purified as needed.

[0027] Furthermore, the polypeptide or protein containing N-glycans includes but is not limited to naturally occurring antibodies, recombinant antibodies, Fc fragments of antibodies or sialic acid glycopeptides.

[0028] Furthermore, other applicable endoglycoside hydrolases include but are not limited to the following wild-type enzymes and mutants thereof: Endo S, Endo S2, Endo F1, Endo F2, Endo F3, Endo A, Endo D, and Endo M.

[0029] Furthermore, the other applicable glycosyltransferases include but are not limited to the following wild-type enzymes and their mutants: Endo S, Endo S2, Endo F1, Endo F2, Endo F3, Endo A, Endo D, and Endo M.

[0030] Furthermore, the sugar chain linker donor includes but is not limited to natural N-glycan oxazoline or synthetic glycan oxazoline, preferably, di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, deca- or undela-glycan oxazoline and its specific derivatives are used as donors for transglycosylation, particularly preferably disaccharide oxazoline. The natural N-glycan oxazoline comprises complex glycan oxazoline, high mannose glycan oxazoline or hybrid oxazoline. In some embodiments, the selective derivatization type of the sugar chain linker includes but is not limited to glycosyl fluoride, glycosyl azide modification or aryl glycan, and its tag portion includes but is not limited to therapeutic drugs, toxins, fluorescent probes, biotin, lipids, antigen fragments and analogs thereof, PEG substances, genes and analogs thereof, RNA and analogs thereof, DNA and analogs thereof, polypeptides, proteins and analogs thereof, enzymes, enzyme substrates, enzyme inhibitors, enzyme regulators or antibody fragments. In some embodiments, the sugar chain linker has another modification method, which can be azidation, aldehydeation, alkynylation, sulfhydrylation, hydroxylation, carboxylation, phosphorylation, sialylation, albumination, farnesylation or conjugation to a polymer.

[0031] Furthermore, after the glycoside hydrolysis of the polypeptide or protein containing N-glycan, the glycoside hydrolysis product has N-acetylglucosamine or core fucosylated N-acetylglucosamine remaining on the peptide chain as a reaction site for the next transglycosylation step.

[0032] Furthermore, the present disclosure also provides antibodies and ADC drugs produced using the sugar chain engineering method.

[0033] In some embodiments, the present disclosure provides a polynucleotide encoding the Endo S2 mutant enzyme described in the present disclosure.

[0034] In some embodiments, the present disclosure provides a vector comprising the polynucleotide described in the present disclosure.

[0035] In some embodiments, the present disclosure provides a host cell transformed with the vector described in the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG1 is a schematic diagram of a recombinant plasmid for protein fusion expression of Endo S2, CPD protein, and His-tag.

[0037] Figure 2 shows ESI-MS analysis of deglycosylation treatment of mAb 1. (A) ESI-MS of the heavy chain of mAb 1 (after deconvolution); (B) ESI-MS of the deglycosylated heavy chain of mAb 1 (after deconvolution).

[0038] FIG3 shows the results of the glycoside hydrolysis activity assay of wild-type Endo S2 and its mutants.

[0039] Figure 4 shows the structure of an azide-modified disaccharide linker oxazoline.

[0040] Figure 5 shows ESI-MS analysis of core fucosylated and non-fucosylated GlcNAc-mAb 1 transglycosylation treatments. (A) ESI-MS of the heavy chain of GlcNAc-mAb 1 (after deconvolution); (B) ESI-MS of the heavy chain of transglycosylated mAb 1 (after deconvolution).

[0041] FIG6 shows the results of the transglycosylation activity assay of wild-type Endo S2 and its mutants.

[0042] FIG7 shows the test results of the glycoside hydrolysis and transglycosylation activities of wild-type Endo S2 and its preferred mutants on mAb 2.

[0043] FIG8 shows the accumulation data of transglycosylation products of mAb 1 by preferred Endo S2 mutants.

[0044] FIG9 shows the results of the transglycosylation reaction of the preferred Endo S2 mutants with mAb 1 for 20 h.

[0045] Figure 10 shows the results of Endo S2 preferred mutants directly shaping the glycoforms of intact antibody 1. (A) Control, ESI-MS of the heavy chain of intact mAb 1 (after deconvolution); (B) ESI-MS of the heavy chain of mAb 1 treated with mutant T138Q (after deconvolution); (C) ESI-MS of the heavy chain of mAb 1 treated with mutant D184M (after deconvolution); (D) ESI-MS of the heavy chain of mAb 1 treated with mutant R176Y (after deconvolution); (E) ESI-MS of the heavy chain of mAb 1 treated with mutant D179A (after deconvolution).

[0046] Figure 11 shows the results of Endo S2 preferred mutants directly shaping the glycoforms of intact mAb 2. (A) Control, ESI-MS of the heavy chain of intact mAb 2 (after deconvolution); (B) ESI-MS of the heavy chain of mAb 2 treated with mutant T138Q (after deconvolution); (C) ESI-MS of the heavy chain of mAb 2 treated with mutant D184M (after deconvolution); (D) ESI-MS of the heavy chain of mAb 2 treated with mutant R176Y (after deconvolution); (E) ESI-MS of the heavy chain of mAb 2 treated with mutant D179A (after deconvolution).

[0047] Specific implementation methods

[0048] Unless otherwise defined, the present disclosure uses conventional techniques in immunology, molecular biology, microbiology, cell biology, genetic engineering, and protein engineering, and the academic terms used in the present disclosure have the same meanings as commonly understood by those skilled in the art. All publications and patent documents mentioned in the present disclosure are to be regarded as recommendations of persons having ordinary knowledge in the relevant technical fields.

[0049] As used herein, the three-letter and one-letter codes for amino acids are as described in J. Biol. Chem, 243, p3558 (1968).

[0050] The mutant enzyme disclosed herein is obtained by recombinant DNA technology. The terms "mutant" or "mutant enzyme" can be used interchangeably, both referring to the mutated recombinant enzyme, which is still a protein molecule in chemical nature and has sequence characteristics that are different from the wild-type enzyme.

[0051] The annotation of amino acids contained in the molecules of the present disclosure conforms to the custom in the art, and the position of the mutation is indicated by the single-letter symbol of the wild-type amino acid and its number, for example, Arg at position 176 is referred to as "R176". Mutations are indicated by the single-letter symbol of the wild-type amino acid, its number, and the single-letter symbol of the mutated amino acid, for example, a mutation replacing Arg at position 176 with Tyr is referred to as "R176Y", and if a mutant has multiple mutations, multiple mutations are indicated using a separator " / ".

[0052] In the present disclosure, Endo S2 mutant R176 is a mutant consisting of a sequence in which Arg at position 176 of the wild-type Endo S2 as shown in SEQ ID NO: 1 is substituted with another natural amino acid. Endo S2 mutant D179 is a mutant consisting of a sequence in which Asp at position 179 of the wild-type Endo S2 as shown in SEQ ID NO: 1 is substituted with another natural amino acid. Endo S2 mutant R176 / D179 is a mutant consisting of a sequence in which Arg at position 176 of the wild-type Endo S2 as shown in SEQ ID NO: 1 is substituted with another natural amino acid and Asp at position 179 is substituted with another natural amino acid.

[0053] In the present disclosure, Endo S2 mutant R176Y is a mutant consisting of a sequence in which Arg at position 176 of the wild-type Endo S2 as shown in SEQ ID NO: 1 is substituted with Tyr. Endo S2 mutant D179A is a mutant consisting of a sequence in which Asp at position 179 of the wild-type Endo S2 as shown in SEQ ID NO: 1 is substituted with Ala. Endo S2 mutant R176 / D179 is a mutant consisting of a sequence in which Arg at position 176 of the wild-type Endo S2 as shown in SEQ ID NO: 1 is substituted with Tyr and Asp at position 179 is substituted with Ala.

[0054] The mutants disclosed herein do not need to have the full-length sequence; as long as they retain regions important for the transglycosylation activity of EndoS2, they are within the scope of the present disclosure. The known active fragment of Endo S2 is the amino acid sequence from positions 38 to 819 of SEQ ID NO: 1. On this basis, recombinant proteins comprising the fragment and appropriately adding, for example, a signal peptide sequence, a purification tag (e.g., a His-tag), a linker sequence (e.g., GGGS), or other functional components to its N-terminus or C-terminus to form a new fusion protein that does not affect the activity of Endo S2 are all within the scope of protection of the present disclosure.

[0055] In the amino acid sequence of the mutant disclosed herein, one to several amino acids may be substituted, deleted, inserted, and / or added at positions other than the positions required for the following mutations, to the extent that the enzyme activity is not affected, wherein the amino acid sequence preferably has at least 95% sequence identity with SEQ ID NO: 1. Any position may be selected for such amino acid alteration as long as it does not affect the enzyme activity, but the position is preferably a position other than amino acid numbers 38 to 819 of SEQ ID NO: 1. In the present disclosure, the term "several" refers to 20 or fewer, preferably 10 or fewer, more preferably 5 or fewer, and most preferably 4, 3, 2, or 1.

[0056] As used herein, "identity" refers to the percentage of identical amino acid residues in the candidate sequence and the reference polypeptide sequence, after the sequences are aligned (and, if necessary, introduced into gaps) to obtain maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required for obtaining maximum alignment over the full length of the compared sequences.

[0057] When used in this disclosure, a reference without a specific number can mean one or more. When used in the claims of this disclosure, a reference without a specific number when used in conjunction with the word "comprising" can mean one or more. When used in this disclosure, "another" can mean at least a second or more.

[0058] As used herein, "sugar" refers to oxidized or unoxidized carbohydrate-containing molecules, including but not limited to monosaccharides, disaccharides, trisaccharides, oligosaccharides, or polysaccharides, including, for example, the L-isomer or D-isomer of 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 thereof. Sugar also refers to such molecules produced naturally, recombinantly, synthetically, and / or semisynthetically.

[0059] As used herein, a "sugar linker," as an activated donor molecule for glycosylation, can be a synthetic glycan oxazoline, such as an oligosaccharide with an activated reducing end, preferably an oligosaccharide molecule with an oxazoline structure; or a natural N-glycan oxazoline. The sugar linker can also be chemically modified, for example, by introducing functional groups through azidation, alkynylation, aldehyde formation, or sulfhydration, thereby facilitating the loading of cytotoxins to produce ADC-conjugated antibodies.

[0060] As used herein, "antibody," "monoclonal antibody," or "IgG" refers to a molecule that contains an antigen binding site that specifically binds an antigen or an Fc region that binds a cellular receptor, and are used interchangeably.

[0061] As used herein, "highly pure" refers to a protein of interest that is separated from contaminants that naturally accompany it or that are produced or used in the process of obtaining the protein of interest. Generally, a protein of interest is considered highly pure if the grayscale of the target protein band after gel electrophoresis separation accounts for at least 90% of the total grayscale of all bands. Preferably, in certain embodiments, the grayscale of the target protein accounts for at least 95%, and most preferably, at least 98%, of the total grayscale of all bands.

[0062] As used herein, "substantial hydrolysis activity" means that, with the glycoside hydrolysis activity of wild-type Endo S2 as 100%, the hydrolysis activity of the Endo S2 mutant measured under the same conditions is at least 50%, preferably at least 60%, more preferably at least 70%, and most preferably at least 80% of the hydrolysis activity of wild-type Endo S2.

[0063] Core-fucosylated and non-fucosylated glycoproteins are important classes of molecules that play key roles in many biological events, such as tumor metastasis, cell adhesion, pathogen infection, and immune responses. Natural and recombinant fucosylated and non-fucosylated glycoproteins are typically produced as mixtures of glycoforms that differ only in the structure of the side-chain oligosaccharides.

[0064] Core fucosylated and non-fucosylated derivatized sugar chain-conjugated antibodies are produced according to the methods described herein. The functional groups introduced by derivatization include but are not limited to azide groups, aldehyde groups, alkyne groups, and thiol groups. The above groups can be introduced into cytotoxins through chemical reactions to produce ADC-conjugated antibodies.

[0065] The present disclosure further provides a recombinant gene encoding the aforementioned EndoS2 mutant, a gene construct comprising the recombinant gene, such as a plasmid or expression vector, a host cell transformed with the gene construct, and a method for producing the mutant of the present disclosure, comprising the step of collecting the mutant of the present disclosure from a culture of the host cell. The recombinant gene, gene construct, host cell, etc. can be prepared based on the amino acid sequence of the mutant of the present disclosure using known genetic engineering techniques.

[0066] Host cells transformed by introducing genes encoding mutants of the present invention can be cultured under appropriate conditions according to the type of cells (cells commonly used for protein production, such as animal cells, plant cells, Escherichia coli, yeast, etc. can be appropriately selected), and mutants of the present invention can be collected from the culture. The collection of mutants is carried out by appropriately combining conventional purification techniques based on the physical properties of the protein. In order to facilitate collection, a gene construct can be designed to express the mutant in the form of a tag peptide such as GST, which is pre-linked to the mutant so that it is possible to collect it using affinity with the tag peptide. The tag peptide can be removed after purification, but when it has no effect on the enzymatic activity of the mutant, the mutant with the tag peptide linked thereto can be used for reactions such as sugar reconstruction. The mutants of the present invention include such an amino acid sequence that contains a tag peptide linked thereto.

[0067] The features and advantages of the present disclosure are more fully demonstrated by the following non-limiting examples. Example

[0068] Materials and methods

[0069] The monoclonal antibodies 1 and 2 used in this disclosure are antibodies produced by the applicant. The azide-modified disaccharide linker LacNAc oxazoline (N3-LacNAc-Oxa) was purchased from Wuhan Tangzhi Pharmaceutical Co., Ltd. The remaining reagents, unless otherwise specified, were conventional reagents produced by Merck, Sigma, Sinopharm Group, etc. The fillers, chromatographic columns, and instruments used in this disclosure include: Cytiva Ni Sepharose Excel filler, Cytive HiLoad TM 26 / 600Superdex TM 200pg chromatographic column, Acquity I-Class / RDa (Waters) liquid chromatography-mass spectrometry, ACCQUITY UPLC BEH PROTEIN C4 column (Waters, 1.7μm, 2.1mm×50mm) chromatographic column. Site-directed mutagenesis, expression, and purification of recombinant Endo S2 wild-type enzyme and its mutant enzymes

[0070] The cDNA encoding the amino acid sequence of Endo S2 derived from serotype M49 Streptococcus pyogenes NZ131 strain was fused with the cDNA encoding the cysteine ​​protease domain (CPD) of Vibrio cholerae MARTX toxin by PCR amplification, and a His-tag protein (10×His) was connected at the C-terminus. The two proteins were inserted between the SalI and NotI regions of the multiple cloning site of the vector plasmid pET-22b(+). The codons for the mutant amino acids were introduced using primers containing mutations. Recombinant plasmids containing the nucleic acid sequences of wild-type Endo S2 and its mutants were produced by Escherichia coli DH5α strains and transformed into Escherichia coli BL21 (DE3) cells. The EndoS2-CPD-10×His fusion protein (hereinafter referred to as Endo S2 fusion protein) was expressed by adding isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.25 mM and culturing overnight at 20°C. The cells were collected by centrifugation, resuspended in lysis buffer (20 mM PB, 500 mM NaCl, pH 7.5), and then disrupted by an ultrasonic cell disruptor (Wuxi Jereian Instrument Equipment Co., Ltd.). After centrifugation, the supernatant of the cell lysate was collected and the Endo S2 fusion protein was purified using Ni Sepharose Excel filler (Cytiva). The cells were concentrated using an Amicon centrifugal filter (30 kDa, Millipore) and further passed through HiLoad TM 26 / 600Superdex TM Purification was performed by size exclusion using a 200 pg column (Cytiva). Fractions containing the Endo S2 fusion protein were concentrated using Amicon centrifugal filters (30 kDa, Millipore) and stored in storage buffer (20 mM PB, pH 7.5). Protein purity was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel imaging using a Gel Doc EZ Imager (Bio-RAD) and Image Lab scanning software. Protein concentration was quantified using a spectrophotometer (Nano-300).

[0071] Liquid chromatography-mass spectrometry (LC-ESI-MS) of IgG

[0072] LC-MS analysis was performed on an Acquity I-Class / RDa (Waters). To analyze the antibody heavy chain, the IgG antibody was treated with 20 mM TCEP and heated at 37°C for 30 minutes, then analyzed using an ACCQUITY UPLC BEH PROTEIN C4 column (1.7 μm, 2.1 mm × 50 mm) with a 20%-50% linear gradient of MeCN containing 0.1% formic acid at a flow rate of 0.3 mL / min over 15 minutes. The raw data were deconvoluted using UNIFI (Waters).

[0073] Preparation of GlcNAc-mAb 1 and GlcNAc-mAb 2

[0074] Intact mAb 1 was dissolved in 20 mM phosphate, 150 mM NaCl, pH 8.0 buffer (10 mg / mL), and wild-type Endo S2 was added to a final concentration of 0.4 mg / mL. The mixture was incubated at 37°C for 3 hours. LC-MS analysis demonstrated complete cleavage of N-glycans from the heavy chain, and deglycosylated mAb 1 was purified by Protein A chromatography. LC-MS data revealed: m / z: 49,354 ± 2 Da (deconvoluted data), indicating the heavy chain of non-fucosylated mAb 1 (i.e., GlcNAc-mAb 1); m / z: 49,500 ± 2 Da (deconvoluted data), indicating the heavy chain of core-fucosylated mAb 1 (i.e., Fucα1,6-GlcNAc-mAb 1).

[0075] The preparation method of GlcNAc-mAb 2 was the same as above. LC-MS data: m / z: 49285±2Da (deconvoluted data), indicating the heavy chain of non-fucosylated mAb 2 (i.e., GlcNAc-mAb 2); m / z: 49431±2Da (deconvoluted data), indicating the heavy chain of core-fucosylated mAb 2 (i.e., Fucα1,6-GlcNAc-mAb 2).

[0076] Determination of glycoside hydrolysis activity of wild-type Endo S2 and its mutants

[0077] The glycoside hydrolysis activity of wild-type Endo S2 and its mutants was measured in 20 mM phosphate buffer (pH 7.5) at 37°C using 10 mg / mL intact mAb (e.g., mAb 1 or mAb 2) as a substrate. The reaction mixture was terminated by adding a final concentration of 0.1% formic acid and then analyzed by reducing LC-MS. After deconvolution of the heavy chain ESI-MS raw data and integration of the corresponding MS peaks, the relative amounts of substrate (mAb 1) and glycoside hydrolysis product (deglycosylated mAb 1) were quantified to calculate changes in the glycoside hydrolysis activity of wild-type Endo S2 and its mutants.

[0078] Determination of transglycosylation activity of Endo S2 wild type and its mutants

[0079] 10 mg / mL of deglycosylated mAb (e.g., GlcNAc-mAb 1 or GlcNAc-mAb 2 containing core fucosylation and non-fucosylation), N3-LacNAc-Oxa (3.33 mM, 50 equivalents) were incubated with an appropriate amount of wild-type Endo S2 or its mutants at 30°C in 20 mM phosphate buffer (pH 7.5), terminated by the addition of formic acid to a final concentration of 0.1%, and analyzed by reducing LC-MS.

[0080] Accumulation of transglycosylation products in Endo S2 mutants

[0081] Deglycosylated mAb 1 (10 mg / mL, containing core fucosylated and non-fucosylated GlcNAc-mAb 1), N3-LacNAc-Oxa (3.33 mM, 50 equivalents), and 200 nM Endo S2 mutant were mixed in 20 mM phosphate buffer (pH 7.5) at 30°C. Aliquots of the reaction mixture were taken at the same time as the gradient, terminated by the addition of formic acid to a final concentration of 0.1%, and then analyzed by reducing LC-MS.

[0082] Endo S2 mutants directly transglycosylate intact antibodies

[0083] N3-LacNAc-Oxa, intact mAb (e.g., mAb 1 or mAb 2), and Endo S2 mutant were added to 20 mM phosphate buffer (pH 7.5) to final concentrations of 3.33 mM, 10 mg / mL, and 0.2 mg / mL, respectively. After incubation at 30°C for 3 hours, the reaction was terminated by addition of 0.1% formic acid. A control group without endoglycosidase was also established to eliminate interference. These samples were analyzed by reduction LC-MS.

[0084] Example 1: Preparation of recombinant proteins of wild-type Endo S2 and Endo S2 mutants

[0085] Endo-β-N-acetylglucosaminidase (ENGase) (EC 3.2.1.96) can be basically divided into GH85 and GH18 families, which follow basically the same catalytic mechanism. The GH85 endoglycosidase includes Endo A and Endo M, and the GH18 endoglycosidase includes Endo S, as well as Endo F1, Endo F2, and Endo F3 from Flavobacterium meningosepticum, and Endo S2 from serotype M49 Streptococcus pyogenes. These GH18 endoglycosidases are widely known for their efficient hydrolysis of asparagine-bonded bibranched glycans at position 297 of IgG antibodies to produce GlcNAc-Ig, among which Endo S2 has attracted much attention due to its wide substrate specificity. These enzymes not only have glycoside hydrolysis activity but also transglycosylation activity, and can be used as potential advantageous enzymes for sugar site-directed coupling technology. Based on the catalytic mechanisms of Endo S, Endo F1, Endo F2, and Endo F3, Endo S2's catalytic process is hypothesized to involve a substrate-assisted mechanism that forms an oxazolinium ion intermediate. Specifically, the aspartic acid (Asp) at position 184 of Endo S2 acts as a general acid to protonate the glycosidic oxygen. The 2-acetamide group of GlcNAc acts as a nucleophile to displace the leaving group at the anomeric center, leading to the formation of an oxazolinium ion intermediate. The catalytic carboxylate residue of the glutamic acid (Glu) side chain at position 186 then acts as a general base to activate a water molecule or glycosyl donor. Finally, the oxazolinium intermediate reacts with the activated donor to undergo glycoside hydrolysis or transglycosylation. The substrate binding mode of Endo S2 has also been demonstrated by crystal structures, including those with high mannan (PDB ID: 6MDV) and complex biantennary glycans (PDB ID: 6MDS).

[0086] Based on the above catalytic mechanism and substrate binding mode, in order to explore the transglycosylation effect of Endo S2, the present disclosure selected residues around the catalytic active site and residues interacting with the substrate as entry points for site-directed mutagenesis at positions Y70, F106, G140, R176, D179, Q250, Y252, S285, E288, and Y339, including but not limited to Y70F, F106W, F106Y, G140N, R176Y, D179A, Q250Y, Q250F, Y252F, S285D, S285A, E288F, E288R, Y339F, and R176Y / D179A. The above mutation positions are represented by the sequence corresponding to SEQ ID NO: 1. Wild-type Endo S2 and its mutants are recombinantly expressed in Escherichia coli (E. coli) by fusing CPD protein to the C-terminus and adding a His-tag protein (see Figure 1). The recombinant protein, referred to as Endo S2 fusion protein, can be purified using Ni Sepharose Excel affinity medium. Fractions containing the target protein can be further purified using a HiLoad™ 26 / 600 Superdex™ 200pg column to obtain highly pure Endo S2 fusion protein.

[0087] Example 2: Detection of glycoside hydrolysis activity of wild-type Endo S2 and its mutants

[0088] The glycoside hydrolysis activity of wild-type Endo S2 and its mutants was assayed using mAb 1 as a substrate. The primary Fc glycan of mAb 1 is a core-fucosylated, bibranched complex oligosaccharide bearing 0, 1, or 2 galactose moieties, designated as G0F, G1F, and G2F glycoforms, respectively. mAb 1 was deglycosylated using wild-type Endo S2 fusion protein. The deglycosylation process and results were analyzed by reducing LC-MS, yielding completely deglycosylated mAb 1. As shown in Figure 2, the ESI-MS deconvolution data of the heavy chain of mAb 1 in Figure 2A shows four major different m / z species, namely 50448±2, 50595±2, 50757±2, and 50919±2, corresponding to the G0 (core non-fucosylated and carrying 0 galactose), G0F, G1F, and G2F glycoforms of the heavy chain, respectively. Figure 2B shows the deglycosylation results of mAb 1. The ESI-MS deconvolution data of the heavy chain show two species at 49354±2Da and 49500±2Da, which match the GlcNAc glycoform and the fucosylated glycoform, respectively. These results confirm the excellent glycoside hydrolysis activity of wild-type Endo S2 on intact IgG and suggest that it can be used for the first step of antibody sugar site-directed conjugation - antibody deglycosylation.

[0089] The glycoside hydrolysis activities of wild-type Endo S2 and its mutants were measured using intact mAb 1 as a substrate (including both core-fucosylated and non-fucosylated intact mAb 1). As shown in Figure 3, compared to wild-type Endo S2, mutants Y70F, F106Y, G140N, R176Y, D179A, and R176Y / D179A retained most of their glycoside hydrolysis activity and could also be used for deglycosylated antibody preparation. In contrast, mutants F106W, Q250Y, Q250F, Y252F, S285D, S285A, E288F, E288R, and Y339F exhibited significantly reduced glycoside hydrolysis activity, with E288F and E288R, in particular, having almost completely lost their glycoside hydrolysis activity.

[0090] Example 3: Detection of transglycosylation activity of wild-type Endo S2 and its mutants

[0091] The transglycosylation activity of wild-type Endo S2 and its mutants was assayed using core-fucosylated and non-fucosylated GlcNAc-mAb 1 as acceptors and the azide-modified LacNAc disaccharide linker oxazoline (N3-LacNAc-Oxa, structure shown in Figure 4) as donors. Transglycosylation data analysis of the wild-type Endo S2 fusion protein is shown in Figure 5 . Figure 5A shows the results for deglycosylated mAb 1. The deconvoluted ESI-MS data for the heavy chain revealed two species at 49354±2Da and 49500±2Da, representing the GlcNAc glycoform and the fucosylated glycoform, respectively. The ESI-MS data in Figure 5B show two additional species at 49776±2Da and 49922±2Da, respectively. This result matches well with the molecular weight of the N3-LacNAc-Oxa transglycosylation linker (after azide degradation and protonation under acidic conditions).

[0092] The transglycosylation activity results are shown in Figure 6. Mutants R176Y and D179A exhibited significantly increased transglycosylation activity. Mutants G140N, Q250F, Y339F, and R176Y / D179A showed little difference in transglycosylation activity compared to the wild type. However, mutants Y70F, F106W, F106Y, Q250Y, Y252F, S285D, S285A, E288F, and E288R lost most of their transglycosylation activity. Combined with the glycoside hydrolysis activity test results in Example 2, it was surprisingly found that mutants R176Y and D179A retained most of their glycoside hydrolysis activity while exhibiting significantly enhanced transglycosylation activity.

[0093] At the same time, the present disclosure also measured the glycoside hydrolysis activity and transglycosylation activity of the preferred mutants R176Y and D179A on monoclonal antibody 2, as shown in Figure 7. Figure 7A shows the glycoside hydrolysis activity of the preferred mutants. The hydrolysis activity of R176Y is comparable to that of the wild type, and the hydrolysis activity of D179A is slightly increased, indicating that the mutants R176Y and D179A retain at least most of the hydrolysis activity; Figure 7B shows the transglycosylation activity of the mutants. Compared with the wild type, R176Y and D179A have significantly increased transglycosylation activity on monoclonal antibody 2. In summary, mutants R176Y and D179A retain most of the glycoside hydrolysis activity in the activity detection of monoclonal antibody 1 and monoclonal antibody 2, and at the same time have significantly increased transglycosylation activity, indicating that the preferred mutants disclosed in the present disclosure have universality for antibody substrates.

[0094] Subsequently, the preferred mutants R176Y and D179A were subjected to a transglycosylation product accumulation experiment on monoclonal antibody 1. The results are shown in Figure 8. Both mutants R176Y and D179A can efficiently complete the accumulation of transglycosylation products, and more than 80% of the antibody has been glycosylated in 4 hours. At the same time, the sample that reacted for 20 hours still had 100% transglycosylated antibody 1 (Figure 9), indicating that the hydrolysis activity retained by R176Y and D179A does not affect the stability of the antibody after glycoform shaping, and can achieve the generation of 100% transglycosylated antibody products. Therefore, the mutants R176Y and D179A, which have both glycoside hydrolysis activity and transglycosylation activity, do not affect their application in the field of transglycosylation, and can complete the two steps of antibody sugar site-specific coupling-antibody deglycosylation and sugar chain linker coupling.

[0095] Example 4: One-step glycoform shaping of intact antibodies using wild-type Endo S2 and its mutants

[0096] In the above-mentioned embodiment, the glycoform shaping of monoclonal antibody 1 is divided into two steps. First, the antibody is deglycosylated using Endo S2 wild type or its mutants, and then N3-LacNAc-Oxa is coupled using glycoside hydrolase catalysis. The above two-step method can prepare antibodies with bioorthogonal reactive groups for ADC drugs, but the deglycosylated antibody must be purified after deglycosylation, which is complicated and cumbersome. Therefore, the R176Y and D179A mutants screened in this disclosure were used to attempt to complete the glycoform shaping of the intact monoclonal antibody in a one-step method (i.e., antibody deglycosylation and sugar chain linker coupling are completed in one step). At the same time, the best known glycoside synthases, Endo S2 T138Q mutants and D184M mutants, were used as controls for the one-step method (T138Q mutants and D184M mutants are described in patent applications CN110234341A and CN109071630A).

[0097] The results of one-step glycoform remodeling of intact mAb 1 are shown in FIG10 . Figure 10A shows the control group without endoglycosidase. The ESI-MS deconvolution data of the heavy chain of mAb 1 showed that all antibodies in the system were native glycoforms, namely G0 (50448±2Da), G0F (50595±2Da), G1F (50756±2Da), and G2F (50918±2Da). In the experimental system with Endo S2 T138Q (Figure 10B) and D184M (Figure 10C), the main substances were m / z 49352±2Da and 49499±2Da, corresponding to deglycosylated GlcNAc-mAb 1 and Fucα1,6-GlcNAc-mAb 1, accounting for more than 88%. The glycoform remodeling product GlcNAc-(N3-LacNAc)-mAb 1 (m / z 49777±2Da corresponds to azide shedding under acidic conditions, m / z 49801±2Da corresponding to azide not shed) and Fucα1,6-GlcNAc-(N3-LacNAc)-mAb1 (m / z 49922±2D corresponding to azide shed under acidic conditions, m / z 49946±2Da corresponds to azide not being shed) accounts for less than 12%; while in the experimental systems of the mutants R176Y ( Figure 10D ) and D179A ( Figure 10E ) disclosed herein, the ESI-MS spectra are basically m / z 49801±2Da, 49922±2Da, and 49946±2Da substances, corresponding to the molecular weights of GlcNAc-(N3-LacNAc)-mAb 1 (azide not shed), Fucα1,6-GlcNAc-(N3-LacNAc)-mAb 1 (azide shed under acidic conditions), and Fucα1,6-GlcNAc-(N3-LacNAc)-mAb 1 (azide not shed), respectively. mAb 1 basically (greater than 95%) completed the coupling of the disaccharide linker and the coupling was uniform (DAR=2).

[0098] The results of the one-step glycoform remodeling of intact mAb 2 are shown in Figure 11. Figure 11A shows the control group without endoglycosidase. The ESI-MS deconvolution data of mAb 2 heavy chain showed that all antibodies in the system were original glycoforms, namely G0 (50379±2Da), G0F (50525±2Da) and G1F (50687±2Da) glycoforms; in the Endo S2 T138Q mutant (Figure 11B) and D184M mutant (Figure 11C) test systems, deglycosylated mAb 2, including GlcNAc-mAb 2 (m / z 49285±2Da) and Fucα1,6-GlcNAc-mAb2 (m / z 49431±2Da), accounting for more than 84%; while in the R176Y mutant (Figure 11D) and D179A mutant (Figure 11E) systems disclosed in the present invention, more than 97% are glycoform-remodeled antibodies and are evenly coupled (DAR=2), including GlcNAc-(N3-LacNAc)-mAb2 (m / z 49707±2Da corresponding to azide shedding under acidic conditions; m / z 49371±2Da corresponding to azide not shedding) and Fucα1,6-GlcNAc-(N3-LacNAc)-mAb2 (m / z 49852±2Da corresponding to azide shedding under acidic conditions; m / z 49877±2Da corresponding to azide not shedding).

[0099] The above data show that the mutants R176Y and D179A can simultaneously complete the two steps of antibody sugar site-specific coupling - antibody deglycosylation and sugar chain linker coupling in a one-step reaction, and can prepare antibodies with bioorthogonal reaction groups more quickly than the currently known best glycosylation synthases - Endo S2 T138Q mutant and D184M mutant, thereby reducing production steps and lowering production costs.

[0100] This disclosure describes mutant enzymes of the endo-β-N-acetylglucosaminidase (Endo S2) derived from serotype M49 Streptococcus pyogenes strain NZ131. These mutant enzymes include the R176Y and / or D179A mutations. Compared to previously reported wild-type Endo S2 and its mutants, these mutant enzymes exhibit enhanced transglycosylation activity while retaining more glycoside hydrolysis activity. Therefore, these mutant enzymes have broader applications in antibody sugar-specific conjugation processes, such as deglycosylated antibody production and sugar linker conjugation.

[0101] Those skilled in the art will appreciate that the above examples are illustrative only, and that various modifications and alterations may be made to the mutant proteins without departing from the scope of the present disclosure, including but not limited to insertions and / or truncations at the N-terminus and / or C-terminus, glycosylation, sialylation, albumination, farnesylation, carboxylation, hydroxylation, phosphorylation, and conjugation to polymers. Importantly, the mutant proteins possess the Endo S2 catalytic domain and include mutations at positions R176 and / or D179. Furthermore, the examples described herein utilize monoclonal antibodies, and those skilled in the art will appreciate that similar methods may be applied to other glycoproteins or antibodies. Furthermore, the examples described herein utilize azide-modified LacNAc disaccharide linkages, and those skilled in the art will appreciate that linker modification methods may also include but are not limited to sulfhydryl, alkyne, and aldehyde modifications, and that sugar chain linkers may be expanded to include di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, deca-, undeca-, or other glycan forms.

Claims

1. An Endo S2 mutant enzyme having the following sequence: (a) SEQ ID NO: 1 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1; or (b) comprising consecutive amino acids from positions 38 to 819 of SEQ ID NO: 1 and having mutations at one or two of the following amino acid positions: R176 and / or D179, and the mutant enzyme has glycoside hydrolysis and / or glycosyl transfer activity.

2. The Endo S2 mutant enzyme according to claim 1, wherein the mutation is selected from at least one of the following: R176Y, D179A.

3. A polynucleotide encoding the Endo S2 mutant enzyme according to claim 1 or 2. A vector comprising the polynucleotide according to claim 3. A host cell transformed with the vector according to claim 4.

6. Use of the Endo S2 mutant enzyme according to claim 1 or 2 in glycosylation modification of polypeptides or proteins containing N-glycans.

7. The use according to claim 6, which is used for reshaping the glycoform of an antibody, or for remodeling the sugar chains of a glycoprotein, or for preparing a detection drug or ADC drug comprising an antibody.

8. A method for remodeling the sugar chains of a polypeptide or protein containing N-glycans using a one-step method, comprising the following steps: (a) introducing the Endo S2 mutant enzyme according to claim 1 or 2; (b) introducing a polypeptide or protein containing N-glycan as a substrate; (c) providing a sugar chain linker donor; and (d) using the Endo S2 mutant enzyme to perform glycosidic hydrolysis on a polypeptide or protein containing N-glycans and transferring a sugar chain linker donor to the glycosidic hydrolysis product to provide a new polypeptide or protein with sugar chain modification; The one-step method does not contain a purification step for the glycoside hydrolysis product.

9. A method for remodeling the sugar chain of a polypeptide or protein containing N-glycans by a two-step method, wherein the enzyme is introduced twice to perform glycoside hydrolysis and transfer of new sugar groups, respectively, wherein: The Endo S2 mutant enzyme according to claim 1 or 2 is used in at least one step of glycoside hydrolysis or glycosyl transfer, and the method comprises the following steps: (a) introducing the Endo S2 mutant enzyme according to claim 1 or 2 or other applicable endoglycoside hydrolase; (b) introducing a polypeptide or protein containing N-glycan as a substrate, and performing glycoside hydrolysis under the action of the enzyme in step (a), thereby providing a glycoside hydrolysis product as an acceptor for the next step of transferring a new glycosyl group; (c) providing a sugar chain linker donor; and (d) transferring the sugar chain linker donor to the acceptor by the Endo S2 mutant enzyme or other suitable glycosyltransferase to generate a new polypeptide or protein with sugar chain modification; Optionally, a purification step of the glycoside hydrolysis product is further included between steps (b) and (c).

10. The method according to claim 8 or 9, wherein the polypeptide or protein containing N-glycans comprises a naturally occurring antibody, a recombinant antibody, an Fc fragment of an antibody, or a sialic acid glycopeptide.

11. The method according to claim 8 or 9, wherein the sugar chain linker donor is a synthetic glycan oxazoline or a natural N-glycan oxazoline.

12. The method of claim 11, wherein the natural N-glycan oxazoline comprises a complex-type glycan oxazoline, a high-mannose glycan oxazoline, or a hybrid oxazoline.

13. The method of claim 11, wherein the synthetic polysaccharide oxazoline is a disaccharide oxazoline, a trisaccharide oxazoline, a tetrasaccharide oxazoline, a pentasaccharide oxazoline, a hexasaccharide oxazoline, a heptasaccharide oxazoline, an octasaccharide oxazoline, a nonasaccharide oxazoline, a decasugar oxazoline or an undecasugar oxazoline.

14. The method according to claim 8 or 9, wherein the sugar chain linker donor further comprises an additional modification method or an additional tag.

15. The method of claim 14, wherein the additional modification method is selected from the group consisting of azidation, aldehydeation, alkynylation, sulfhydrylation, hydroxylation, carboxylation, phosphorylation, sialylation, albumination, farnesylation, or conjugation to a polymer.

16. The method of claim 14, wherein the additional label is selected from a therapeutic drug, a toxin, a fluorescent probe, biotin, a lipid, an antigenic fragment, PEG, RNA, DNA, a polypeptide, a protein, an enzyme, an enzyme substrate, an enzyme inhibitor, an enzyme modulator or an antibody fragment.

17. A sugar chain remodeled antibody and ADC drug thereof, which are prepared by the method according to claims 8-16.

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