Novel endo s2 mutant enzyme

Abstract

Provided is a novel Endo S2 mutant enzyme. The mutant enzyme exhibits significantly enhanced glycoside hydrolysis activity and can be used for deglycosylation of polypeptides or proteins having N-glycans, thereby enabling the preparation of receptors for subsequent glycoengineering.

Description

Novel Endo S2 mutant enzyme

[0001] This application claims priority to Chinese patent application No. CN2024102388547, filed with the Patent Office of China on March 1, 2024, with application number CN2024102388547 and invention name “Novel Endo S2 mutant enzyme”, and Chinese patent application No. CN2025101927037, filed with the Patent Office of China on February 20, 2025, with invention name “Novel Endo S2 mutant enzyme”, the entire contents of which are incorporated into this application by reference. Technical Field

[0002] The present disclosure relates to the fields of glycochemistry and enzymology, and in particular to the use of a recombinant novel Endo S2 mutant enzyme to deglycosylate the N-glycans of polypeptides or proteins. The modified proteins, such as deglycosylated antibodies, can be used for the production of glycoform-homogeneous antibodies or the synthesis of sugar-targeted antibody-based conjugate drugs. Background Art

[0003] Antibodies, also known as immunoglobulins (Ig), are important mediators of homeostasis and host defense. They recognize and internalize antigens by specifically binding to them and interacting with receptors on cells of the innate immune system, thereby stimulating immune responses. Antibodies have become established therapeutics for a wide range of diseases, including cancer, autoimmune diseases, and infectious diseases. Consequently, antibody-based drugs are booming and represent the fastest-expanding therapeutic drug category.

[0004] A typical IgG antibody is composed of four peptide chains: two light chains and two heavy chains. These four chains form three distinct domains: two variable Fab domains and a constant Fc domain. The Fab region is involved in antigen recognition, while the Fc region interacts with Fcγ receptors (FcγRs) and other effector proteins, such as complement, thereby activating effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The Fc domain of nearly all therapeutic antibodies contains an N-glycan modification at asparagine 297 (N297). These N-glycans share a common pentasaccharide core composed of the GlcNAc-GlcNAc-Man-(Man)2 sequence. The composition and number of the (Man)2-linked decorative antennae within the pentasaccharide core vary, resulting in over 37 different glycoforms. These glycoforms are categorized by the composition of the decorative antennae into high-mannose (HM), complex (CT), and hybrid (Hy). The composition of the N-glycans influences the conformation of the antibody Fc region, thereby regulating properties such as antibody stability, effector function, immunogenicity, antigen affinity, pharmacokinetics, and aggregation. For example, a lack of fucosylation in the antibody core enhances binding to the FcγRIIIa receptor, resulting in a stronger ADCC response. Mannoses in highly mannosylated antibodies bind to asialoglycoprotein receptors, leading to increased clearance and a shortened half-life. Commercially available antibodies usually exist in the form of a mixture of glycoforms, which is not conducive to the homogenization of therapeutic activity. Therefore, there is an urgent need to develop methods for producing antibodies with clear structures and uniform glycoforms. A common strategy is glycoengineering regulation based on cell culture, but the glycoforms produced are limited, and impure glycoforms still exist. Another common strategy is in vitro chemical enzymatic glycoform remodeling, which first uses endoglycosidase (ENGase) to cut all N-glycans, retaining only the first GlcNAc or Fucα1,6-GlcNAc at N297 of the antibody, that is, deglycosylation of natural or recombinant antibodies, and then using glycosynthase to transfer the structurally clear glycan oxazoline to the GlcNAc of the deglycosylated antibody to form a β-1,4 glycosidic bond to complete the glycoform remodeling.

[0005] In addition, the conserved glycosylation site at position N297 of the antibody can be used as a coupling site for cytotoxins, and based on this, a sugar-specific coupling technology has been developed. In the GlycoConnect coupling technology, the antibody is first deglycosylated with an endoglycosidase to produce a GlcNAc-receptor, and then β-1,4-galactosyltransferase is used to introduce a sugar chain linker with an azide group, and finally the azide and cyclooctyne reaction are used to complete the coupling of the cytotoxin. The DisacLink technology also has similar steps. The antibody is first deglycosylated with an endoglycosidase, and then the sugar chain linker is introduced with a sugar synthase, and finally the cytotoxin is coupled using a bioorthogonal reaction.

[0006] The aforementioned endoglycosidases, also known as β-N-acetylglucosaminidases, are glycoside hydrolases that act on β-1,4-glycosidic bonds. These include Endo A, Endo D, Endo F3, Endo S, and Endo S2. Currently, research on endoglycosidases focuses on obtaining high-performance glycosynthase mutants, while neglecting their hydrolase activity. Both antibody glycoform homogenization and ADC site-specific conjugation require the use of endoglycosidases to hydrolyze N-glycans and prepare deglycosylated antibodies. Compared to other endoglycosidases, Endo S2 exhibits broader substrate specificity, recognizing complex glycans, high-mannose glycans, hybrid glycans, and glycans without core fucose modifications, making it a highly promising endoglycosidase. Therefore, obtaining Endo S2 mutants with significantly enhanced hydrolytic activity would be beneficial for antibody glycoform homogenization and site-specific conjugation.

[0007] SUMMARY OF THE INVENTION

[0008] The present disclosure provides a recombinant Endo S2 mutant enzyme, which exhibits enhanced hydrolytic activity and is capable of cleaving N-glycans from polypeptides or proteins containing N-glycans. Therefore, it is suitable for the preparation of deglycosylated antibodies in antibody glycoform homogenization and sugar site-specific conjugation.

[0009] The recombinant Endo S2 mutant enzyme disclosed herein is derived from the β-N-acetylglucosaminidase 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 meet its activity requirements by 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 have hydrolytic activity are within the scope of the present disclosure.

[0010] The Endo S2 mutant enzyme disclosed herein preferably comprises a mutation at one of the following amino acid positions 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, using site-directed mutagenesis: N142.

[0011] Furthermore, in some preferred embodiments, the mutation is selected from the following: N142W.

[0012] Domains and fragments comprising any of the aforementioned Endo S2 mutant enzymes are encompassed by the present disclosure, wherein any such domains and fragments may be fused to other proteins, including, but not limited to, CPD, Fc, MBP, and the like. The mutant proteins may be modified or truncated, including, but not limited to, the N-terminus and the C-terminus. In some embodiments, the sequence preceding at least one amino acid between M1 and E37 of the mutant may be deleted by cleavage from the N-terminus, and / or the sequence following at least one amino acid between Y820 and D843 of the mutant may be deleted by cleavage from the C-terminus.

[0013] Based on the functions of the aforementioned Endo S2 mutant enzyme disclosed herein, it can be used for glycomodification of N-glycan-containing polypeptides or proteins, such as deglycosylation in reshaping antibody glycoforms or deglycosylation in sugar chain remodeling of glycoproteins. More specifically, it can be used to produce deglycosylated antibodies—intermediates for homogenizing antibody glycoforms or site-specific sugar conjugation. These deglycosylated antibodies can then be used in the production of antibody-based drug conjugates.

[0014] The present disclosure provides a method for remodeling the sugar chains of a polypeptide or protein containing N-glycans, comprising the following steps:

[0015] (a) introducing the Endo S2 mutant enzyme disclosed herein;

[0016] (b) introducing a polypeptide or protein containing N-glycan as a substrate; and

[0017] (c) using the Endo S2 mutant enzyme to perform glycoside hydrolysis on a polypeptide or protein containing N-glycans to provide a new polypeptide or protein with the N-glycans removed and only the first GlcNAc or Fucα1,6-GlcNAc remaining.

[0018] Furthermore, the N-glycans include high mannose glycans, complex glycans and hybrid glycans.

[0019] 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.

[0020] In some embodiments, the present disclosure provides a polynucleotide encoding the Endo S2 mutant enzyme described in the present disclosure.

[0021] In some embodiments, the present disclosure provides a vector comprising the polynucleotide described in the present disclosure.

[0022] In some embodiments, the present disclosure provides a host cell transformed with the vector described in the present disclosure.

[0023] The Endo S2 mutant enzyme disclosed herein has significantly enhanced glycoside hydrolysis activity and can efficiently complete the deglycosylation treatment of polypeptides or proteins, resulting in lower operating costs and higher efficiency in the industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG1 is a schematic diagram of the expression plasmid of the recombinant fusion protein of Endo S2, CPD protein and His-tag.

[0025] 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).

[0026] FIG3 shows the results of the detection of the glycoside hydrolysis activity of wild-type Endo S2 and its mutants against mAb 1.

[0027] Figure 4 shows the ESI-MS analysis of the deglycosylation treatment of mAb 2. (A) ESI-MS of the heavy chain of mAb 2 (after deconvolution); (B) ESI-MS of the deglycosylated heavy chain of mAb 2 (after deconvolution).

[0028] FIG5 shows the results of the detection of the glycoside hydrolysis activity of wild-type Endo S2 and its mutants against mAb 2.

[0029] Figure 6 shows ESI-MS analysis of transglycosylation of deglycosylated mAb 1. (A) ESI-MS of the heavy chain of GlcNAc-mAb 1 and Fucα1,6-GlcNAc-mAb 1 (after deconvolution); (B) ESI-MS of the heavy chain of transglycosylated mAb 1 (after deconvolution).

[0030] FIG7 shows the results of detecting the transglycosylation activity of wild-type Endo S2 and mutant N142W on deglycosylated mAb 1.

[0031] Specific implementation methods

[0032] 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 those having ordinary knowledge in the art.

[0033] As used herein, the three-letter and one-letter codes for amino acids are as described in J. Biol. Chem, 243, p3558 (1968).

[0034] 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.

[0035] The annotation of amino acids contained in the molecules of the present disclosure conforms to the customary practice 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, Asn at position 142 is referred to as "N142". 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 Asn at position 142 with Trp is referred to as "N142W", and if a mutant has multiple mutations, the multiple mutations are indicated using a separator " / ".

[0036] In the present disclosure, the Endo S2 mutant N142W is a mutant consisting of a sequence in which Asn at position 142 of the wild-type Endo S2 as shown in SEQ ID NO: 1 is substituted with Trp.

[0037] The mutants disclosed herein do not need to have the full-length sequence; as long as they retain regions important for the hydrolysis and / or transglycolysis 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 such fragments 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 (e.g., a CPD for promoting solubility) to their N- or C-termini 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.

[0038] In the amino acid sequences of the mutants 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 they do not affect enzyme activity. Preferably, the amino acid sequence has at least 95% sequence identity with SEQ ID NO: 1. Any position can be selected for such amino acid alterations as long as they do not affect enzyme activity, but such positions are preferably other than amino acids 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.

[0039] 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.

[0040] 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.

[0041] As used herein, "antibody," "monoclonal antibody," or "IgG" refers to a molecule containing an antigen-binding site that specifically binds to an antigen or an Fc region that binds to a cellular receptor, and the terms are used interchangeably. As used herein, "wild-type antibody" typically refers to an antibody that has N-glycosylation sites (N-glycans) in a naturally occurring or recombinantly expressed manner. For example, all antibodies with an N297 site in the Fc region fall within the scope of "wild-type antibodies," and the Fc region is derived from IgG1, IgG2, IgG3, or IgG4.

[0042] The term "Fc" is used herein to define the C-terminal region of an immunoglobulin heavy chain, i.e., the two polypeptide chains that form a dimer and comprise the C-terminal constant region of an immunoglobulin heavy chain that is capable of stabilizing its own association. The term includes native sequence Fc regions and variant Fc regions. The IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is in accordance with the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Therefore, the numbering position of N297 is clear and the sequences of various native sequence Fc regions and variant Fc regions are known in the art.

[0043] The term "deglycosylated antibody" disclosed herein refers to an antibody containing one N-acetylglucosamine (GlcNAc) or core fucosylated N-acetylglucosamine (Fucα1,6-GlcNAc) formed by a wild-type antibody under the action of glycoside hydrolase.

[0044] The term "β-N-acetylglucosaminyl endoenzyme" disclosed herein refers to a class of enzymes (EC 3.2.1.96) with endoglycosidic activity produced by a series of organisms, generally belonging to glycoside hydrolase family 18 or 85, some β-N-acetylglucosaminyl endoenzymes known in the art, such as Endo S, Endo S2, etc., see those described in WO2022 / 050300. Such enzymes may also have disaccharide linker transfer activity at the same time, and enzymes with such activity known in the art, such as Endo S, Endo S2, Endo F3 and their mutants, etc., see those described in WO2022 / 226420.

[0045] The term "antibody-based drug conjugate" disclosed herein refers to a conjugate formed by covalently linking all polypeptides / proteins targeting specific cells to a payload. The polypeptide / protein targeting specific cells may be an antibody or an antigen-binding fragment thereof, such as a monoclonal antibody, a bispecific antibody, or a polyclonal antibody; the payload may be a cytotoxin, a small molecule drug, a near-infrared or fluorescent probe, a polypeptide, RNA and related drugs, a radioisotope label, a contrast agent, and a magnetic resonance imaging agent; the covalent conjugate formed may be used for treatment or detection. "Antibody-drug conjugate" (ADC) refers to a conjugate formed by covalently linking all polypeptides / proteins targeting specific cells to a cytotoxin.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] The features and advantages of the present disclosure are more fully demonstrated by the following non-limiting examples. Example

[0050] Materials and methods

[0051] The monoclonal antibodies 1 and 2 used in the present disclosure are wild-type antibodies produced by the applicant, both containing an Fc segment at the N297 site, the sequence of which is typically shown in SEQ ID NO: 2, that is, both antibodies can be used as substrates for glycoside hydrolysis by the Endo S2 mutant enzyme disclosed in the present disclosure; the azide-modified disaccharide linker LacNAc oxazoline (N3-LacNAc-Oxa) was purchased from Wuhan Tangzhi Pharmaceutical Co., Ltd., and the remaining reagents, unless otherwise specified, are conventional reagents produced by Merck, Sigma, Sinopharm Group, etc. The fillers, chromatographic columns, and instruments used in the present disclosure include: Cytiva Ni Sepharose Excel filler, Cytive HiLoad TM 26 / 600Superdex TM 200 pg chromatographic column, Acquity I-Class / RDa (Waters) liquid chromatography-mass spectrometry, ACCQUITY UPLC BEH PROTEIN C4 column (Waters, 1.7 μm, 2.1 mm×50 mm) chromatographic column.

[0052] Gene cloning, protein expression and purification of recombinant EndoS2 wild-type enzyme and its mutant enzymes

[0053] 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 SalI and NotI in the multiple cloning site region 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-beta-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).

[0054] Liquid chromatography-mass spectrometry (LC-ESI-MS) of IgG

[0055] 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 was deconvoluted using UNIFI (Waters).

[0056] Determination of glycoside hydrolysis activity of wild-type Endo S2 and its mutants against mAb 1

[0057] 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 intact mAb 1 (10 mg / mL) as a substrate. The reaction mixture was terminated by the addition of a final concentration of 0.1% formic acid and 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 products (deglycosylated mAb 1) were quantified to calculate changes in the glycoside hydrolysis activity of wild-type Endo S2 and its mutants.

[0058] Determination of glycoside hydrolysis activity of wild-type Endo S2 and its mutants against mAb 2

[0059] Monoclonal antibody 2 (10 mg / mL) was incubated with an appropriate amount of wild-type Endo S2 or its mutants at 37°C in 20 mM phosphate buffer (pH 7.5) for glycoside hydrolysis reaction, which was terminated by adding formic acid at a final concentration of 0.1% and analyzed by reducing LC-MS.

[0060] Determination of transglycosylation activity of wild-type Endo S2 and its mutants

[0061] Intact mAb 1 was dissolved in 20 mM phosphate, pH 7.5 buffer (10 mg / mL), wild-type Endo S2 was added to a final concentration of 0.2 mg / mL, and incubated at 37°C for 1 hour. After LC-MS analysis showed complete cleavage of N-glycans on the heavy chain, the deglycosylated mAb 1 was purified by protein A chromatography.

[0062] Deglycosylated mAb 1 (10 mg / mL), modified activated sugar chain linker N3-LacNAc-Oxa (3.33 mM, 50 equivalents) and appropriate amount of wild-type Endo S2 or its mutants were incubated at 30°C in 20 mM phosphate buffer (pH 7.5). The reaction was terminated by adding formic acid to a final concentration of 0.1% and analyzed by reducing LC-MS.

[0063] Example 1: Preparation of recombinant proteins of wild-type Endo S2 and Endo S2 mutants

[0064] To explore the glycoside hydrolysis activity of Endo S2, the present disclosure selected residues surrounding the catalytic active sites D184 and E186 and residues interacting with the substrate as entry points for site-directed mutagenesis at positions E57, K85, N142, I185, D226, A259, A291, W297, and D299, including but not limited to E57W, K85L, N142W, I185T, D226N, A259N, A291R, W297Y, and D299Y. The aforementioned 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.

[0065] Example 2: Detection of glycoside hydrolysis activity of wild-type Endo S2 and its mutants against mAb 1

[0066] The glycoside hydrolysis activity of wild-type Endo S2 and its mutants was assayed using mAb 1 as a substrate. The major Fc glycans of mAb 1 are core-fucosylated, bibranched complex oligosaccharides bearing 0, 1, or 2 galactose moieties, designated as G0F, G1F, and G2F glycoforms, respectively. A small fraction of the core non-fucosylated G0 glycoform, which does not carry lactose, is also present in mAb 1. mAb 1 was deglycosylated using wild-type Endo S2 fusion protein. The deglycosylation process and results were analyzed by reducing LC-MS, and the different glycoforms and deglycosylated mAb 1 were calibrated.

[0067] The deglycosylation detection results of mAb 1 are shown in Figure 2. Figure 2(A) is the original mass spectrometry detection diagram of mAb 1 heavy chain. After ESI-MS deconvolution, there are four main peaks corresponding to four major different m / z substances, namely 50448±1, 50595±1, 50757±1 and 50919±1Da, which correspond to G0, G0F, G1F and G2F glycoforms, respectively. Figure 2(B) is the deglycosylation detection result of mAb 1 by wild-type Endo S2 fusion protein. The deconvoluted data of heavy chain ESI-MS have two substances added at 49354±1Da and 49500±1Da, which match the deglycosylated GlcNAc glycoform and fucosylated GlcNAc (Fucα1,6-GlcNAc) glycoform, respectively. These results confirm that wild-type Endo S2 can hydrolyze the N-glycans in the intact IgG Fc region. Therefore, Endo S2 can be used for antibody deglycosylation, the first step in antibody glycoform homogenization or sugar-specific conjugation.

[0068] The glycoside hydrolysis activities of wild-type Endo S2 fusion protein 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 and Table 1, the glycoside hydrolysis activity of mutant N142W was significantly increased to 3.58-fold compared to wild-type Endo S2; mutant E57W retained most of its glycoside hydrolysis activity. The remaining mutants K85L, I185T, D226N, and W297Y had significantly reduced glycoside hydrolysis activity, and A259N, A291R, and D299Y, in particular, had little or no glycoside hydrolysis activity.

[0069] Example 3: Detection of glycosyl hydrolysis activity of wild-type Endo S2 and its mutants against mAb 2

[0070] To test the universality of Endo S2 and its mutants in hydrolyzing antibody N-glycans, the glycoside hydrolysis activity of wild-type Endo S2 and its mutants was again assayed using another antibody, mAb 2, as a substrate. The reduced LC-MS results for intact and deglycosylated mAb 2 are shown in Figure 4. Figure 4(A) shows the ESI-MS deconvolution of the intact mAb 2 heavy chain, which exhibits three main peaks at 50379±1, 50525±1, and 50687±1 Da, corresponding to the G0, G0F, and G1F glycoforms, respectively. Figure 4(B) shows the deglycosylation analysis of mAb 2. The ESI-MS deconvolution data for the heavy chain show two species at 49285±1 Da and 49431±1 Da, matching the deglycosylated GlcNAc-mAb2 and Fucα1,6-GlcNAc-mAb2 glycoforms, respectively. Furthermore, activity assays of the wild-type Endo S2 fusion protein and its mutants against intact mAb 2 (Figure 5 and Table 1) show that mutant N142W exhibits significantly enhanced hydrolytic activity, 4.69-fold that of WT; mutant E57W retains most glycoside hydrolysis activity; the remaining mutants K85L, I185T, D226N, A259N, and W297Y exhibit significantly reduced glycoside hydrolysis activity, while A291R and D299Y completely lose their glycoside hydrolysis activity. These data are consistent with the activity data for mAb 1 as a substrate (Figure 3), indicating that the significantly enhanced hydrolytic activity of mutant N142W is exhibited across different antibodies. The N142W mutant is universally applicable to mAb substrates and can be used for deglycosylation of various glycoforms.

[0071] Table 1: Comparison of glycoside hydrolysis activities of wild-type Endo S2 and mutant N142W using mAb 1 and mAb 2 as complex glycoform substrates

[0072] Example 4: Detection of transglycosylation activity of wild-type Endo S2 and its mutant N142W on mAb 1

[0073] Deglycosylated mAb 1 was prepared, and the transglycosylation activity of Endo S2 mutant N142W was investigated using the azide-modified disaccharide linker LacNAc-oxazoline (N3-LacNAc-Oxa). The results of the transglycosylation system reduction LC-MS analysis are shown in Figure 6. Figure 6(A) shows deglycosylated mAb 1. The deconvoluted ESI-MS data for the heavy chain show two species at 49354±1Da and 49500±1Da, representing GlcNAc-mAb 1 and Fucα1,6-GlcNAc-mAb 1, respectively. The ESI-MS data in Figure 6(B) show two additional species at 49776±1Da and 49922±1Da, respectively. This result matches well with the molecular weight of the N3-LacNAc-Oxa transglycosylation reaction (after azide degradation and protonation under acidic conditions). Compared with wild-type Endo S2, N142W retained 70.7% of the transglycosylation activity, as shown in Figure 7, indicating that N142W can also be used for glycoform remodeling of deglycosylated antibodies.

[0074] Those skilled in the art will appreciate that the above examples are for illustrative purposes 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 N-terminal and / or C-terminal insertions and / or truncations, protein glycosylation, sialylation, albumination, farnesylation, carboxylation, hydroxylation, phosphorylation, and polymer conjugation. Importantly, the mutant proteins possess the Endo S2 catalytic domain and include a mutation at position N142W. Furthermore, while monoclonal antibodies were used in the examples described herein, those skilled in the art will appreciate that similar methods can be applied to other glycoproteins or antibodies.

Claims

1. An Endo S2 mutant enzyme having the following sequence: (a) SEQ ID NO: 1 or an amino acid sequence that has 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 a mutation at one of the following amino acid positions: N142, 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 N142W.

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 preparing deglycosylated antibodies in reshaping the glycoforms of antibodies, or for deglycosylation in sugar chain remodeling of glycoproteins, or for preparing antibody-based conjugate drugs.

8. A method for remodeling the sugar chains of a polypeptide or protein containing N-glycans, 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; and (c) using the Endo S2 mutant enzyme to perform glycoside hydrolysis on a polypeptide or protein containing N-glycans to provide a new polypeptide or protein with the N-glycans removed and only the first GlcNAc or Fucα1,6-GlcNAc remaining.

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

10. The method of claim 9, wherein the N-glycans comprise high mannose glycans, complex glycans, and hybrid glycans.

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