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 on March 1, 2024, entitled "Novel Endo S2 Mutant Enzyme", and Chinese Patent Application No. CN2025101927037, filed on February 20, 2025, entitled "Novel Endo S2 Mutant Enzyme", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates to the fields of glycochemistry and enzymology, and more specifically to the deglycosylation of N-glycans of peptides or proteins using a novel recombinant Endo S2 mutant enzyme. The modified proteins, such as deglycosylated antibodies, can be used for the production of glycoform homogenization antibodies or the synthesis of antibody-drug conjugates with glycosite targeting. Background Technology

[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, thus stimulating an immune response. Antibodies have become a treatment for a variety of diseases, including cancer, autoimmune diseases, and infectious diseases. Therefore, antibody drugs are experiencing rapid growth and represent the fastest-growing category of therapeutic drugs in the market.

[0004] A typical IgG antibody consists of four peptide chains, including two light chains and two heavy chains. These four chains form three distinct domains: two variable Fab domains and one constant Fc domain. The Fab domain is involved in antigen recognition, while the Fc domain interacts with the Fcγ receptor (FcγR) and other effector proteins (such as complement), thereby activating effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Almost all therapeutic antibodies possess an N-glycan modification at position 297 (N297) of the asparagine in their homodimer Fc domain. This N-glycan shares a common pentasaccharide core composed of the sequence GlcNAc-GlcNAc-Man-(Man)2. The decorative antennas attached to the (Man)2 within this core vary greatly in composition and number, resulting in over 37 different glycoforms. Based on the composition of these decorative antennas, they can be categorized into high-mannose (HM), complex (CT), and hybrid (Hy) types. The composition of the N-glycan affects the conformation of the antibody's Fc region, thereby regulating antibody stability, effector function, immunogenicity, antigen affinity, pharmacokinetics, and aggregation. For example, a lack of fucosylation in the antibody core enhances the antibody's binding affinity to the FcγRIIIa receptor, resulting in a stronger ADCC effect; in highly mannose-sylated antibodies, mannose binds to the desialylate glycoprotein receptor, leading to increased clearance and a shortened half-life. Commercially available antibodies are typically present as mixtures of glycoforms, which is detrimental to the homogeneity of therapeutic activity. Therefore, the need to develop methods for producing antibodies with well-defined structures and homogeneous glycoforms is urgent. A common strategy is glycoengineering based on cell culture, but the produced glycoforms are limited and impurities still exist. Another common strategy is in vitro chemoenzymatic glycoform remodeling. This involves first using glycosidases (ENGases) to cleave all N-glycans, retaining only the first GlcNAc or Fucα1,6-GlcNAc at antibody N297, which is essentially deglycosylation of the natural or recombinant antibody. Then, glycosynthesizers are used to transfer the structurally defined glycan oxazoline to the GlcNAc of the deglycosylated antibody to form a β-1,4 glycosidic bond, thus completing the glycoform remodeling.

[0005] Furthermore, the conserved glycosylation site at N297 of the antibody can serve as a conjugation site for cytotoxins, leading to the development of site-specific glycosylation techniques. In the GlycoConnect conjugation technique, the antibody is first deglycosylated with an endonuclease to generate a GlcNAc-receptor. Then, a glycan linker with an azide group is introduced using β-1,4-galactosyltransferase. Finally, the cytotoxin conjugation is completed using the reaction of azide with cyclooctyne. The DisacLink technique follows a similar procedure: the antibody is first deglycosylated with an endonuclease, then a glycan linker is introduced using a glycosynergist, and finally, the cytotoxin is conjugated using a bioorthogonal reaction.

[0006] The aforementioned glycoside endonucleases, also known as β-N-acetylglucosinolate endonucleases, are glycoside hydrolases that act on β-1,4-glycosidic bonds, including Endo A, Endo D, Endo F3, Endo S, and Endo S2, among others. Current modifications to glycoside endonucleases primarily focus on obtaining high-performance glycosynthetic enzyme mutations, neglecting their hydrolytic activity. Both antibody glycoform homogenization and site-directed ADC conjugation require the hydrolysis of N-glycans by glycoside endonucleases to prepare deglycosylated antibodies. Compared to other glycoside endonucleases, Endo S2 exhibits broader substrate specificity, capable of recognizing complex glycans, high-mannose glycans, hybrid glycans, and glycans without core fucose modification, making it a highly promising glycoside endonuclease. Therefore, obtaining Endo S2 mutants with significantly enhanced hydrolytic activity is beneficial for antibody glycoform homogenization and site-directed ADC conjugation.

[0007] Invention Overview

[0008] This disclosure provides a recombinant Endo S2 mutant enzyme that exhibits enhanced hydrolytic activity, enabling it to cleave N-glycans in polypeptides or proteins containing N-glycans. Therefore, it is suitable for the preparation of deglycosylated antibodies in antibody glycoform homogenization and site-directed glycoconjugation.

[0009] The recombinant Endo S2 mutant enzyme disclosed herein is derived from the β-N-acetylglucosinolate endonuclease of serotype M49 Streptococcus pyogenes strain NZ131. The wild-type enzyme sequence 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 activity by retaining the continuous amino acids from position 38 to position 819 of SEQ ID NO: 1. That is, positions 38 to 819 of SEQ ID NO: 1 are the known active fragment of Endo S2. Various truncated fragments of enzymes containing positions 38 to 819 of SEQ ID NO: 1 and still possessing hydrolytic activity are within the scope of this disclosure.

[0010] The Endo S2 mutant enzyme disclosed herein is preferably produced by site-directed mutagenesis, with a mutation at one amino acid position 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 a continuous amino acid sequence containing positions 38 to 819 of SEQ ID NO: 1: N142.

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

[0012] Domains and fragments containing any of the aforementioned Endo S2 mutant enzymes are included in this disclosure, wherein any such domains and fragments may be fused with other proteins, including but not limited to CPD, Fc, MBP, etc. The mutant protein may be modified or truncated, including but not limited to the N-terminus and 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 aforementioned function of the Endo S2 mutant enzyme disclosed herein, it can be used for glycosylation modification of peptides or proteins containing N-glycans, such as for deglycosylation treatment in remodeling antibody glycoforms, or for deglycosylation in glycan remodeling of glycoproteins. More specifically, it can be used to produce intermediate products for antibody glycoform homogenization or site-directed glycan conjugation—deglycosylated antibodies, which can be further used in the production of antibody-based conjugated drugs.

[0014] This disclosure provides a method for remodeling the glycans of peptides or proteins containing N-glycans, comprising the following steps:

[0015] (a) Introducing the Endo S2 mutant enzyme of this disclosure;

[0016] (b) Introducing peptides or proteins containing N-glycans as substrates; and

[0017] (c) Using the Endo S2 mutant enzyme, glycoside hydrolyze the peptide or protein containing N-glycan to provide a new peptide or protein with the N-glycan removed and only the first GlcNAc or Fucα1,6-GlcNAc retained.

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

[0019] Furthermore, the polypeptides or proteins containing N-glycans include, but are not limited to, naturally occurring antibodies, recombinant antibodies, Fc fragments of antibodies, or sialic acid glycopeptides.

[0020] In some embodiments, this disclosure provides a polynucleotide that encodes the Endo S2 mutant enzyme described herein.

[0021] In some embodiments, this disclosure provides a carrier comprising the polynucleotides described herein.

[0022] In some embodiments, this disclosure provides a host cell that is transformed using the vector described in this disclosure.

[0023] The Endo S2 mutant enzyme disclosed herein possesses significantly enhanced glycoside hydrolysis activity, enabling efficient deglycosylation of peptides or proteins, with lower operating costs and higher efficiency in industrial applications. Attached Figure Description

[0024] Figure 1 is a schematic diagram of the expression plasmid of the recombinant fusion protein of Endo S2 with CPD protein and His-tag.

[0025] Figure 2 shows the ESI-MS analysis of the deglycosylated monoclonal antibody 1. (A) ESI-MS of the heavy chain of monoclonal antibody 1 (after deconvolution); (B) ESI-MS of the heavy chain of deglycosylated monoclonal antibody 1 (after deconvolution).

[0026] Figure 3 shows the results of detecting the glycoside hydrolysis activity of wild-type Endo S2 and its mutant against monoclonal antibody 1.

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

[0028] Figure 5 shows the results of detecting the glycoside hydrolysis activity of wild-type Endo S2 and its mutant against monoclonal antibody 2.

[0029] Figure 6 shows the ESI-MS analysis of the transglycosylated monoclonal antibody 1. (A) ESI-MS of the heavy chains of GlcNAc-monoclonal antibody 1 and Fucα1,6-GlcNAc-monoclonal antibody 1 (after deconvolution); (B) ESI-MS of the heavy chains of transglycosylated monoclonal antibody 1 (after deconvolution).

[0030] Figure 7 shows the results of detecting the transglycosylation activity of wild-type Endo S2 and mutant N142W against deglycosylated monoclonal antibody 1.

[0031] Specific implementation methods

[0032] Unless otherwise defined, conventional techniques in immunology, molecular biology, microbiology, cell biology, genetic engineering, and protein engineering are used in this disclosure, and academic terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. All publications and patent documents mentioned in this disclosure are to be regarded as hints to those of ordinary skill in the art.

[0033] The three-letter and single-letter codes for amino acids used in this article 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" are used interchangeably and both refer to the mutated recombinant enzyme. Its chemical nature is still a protein molecule, and it has sequence characteristics that distinguish it from wild-type enzymes.

[0035] The annotation of amino acids contained in the molecules disclosed herein follows the conventions of the art, and the position of mutation is indicated by the single-character symbol and number of the wild-type amino acid, for example, Asn at position 142 is referred to as "N142". Mutations are indicated by the single-character symbol and number of the wild-type amino acid and the single-character symbol of the mutated amino acid, for example, a mutation in which Asn at position 142 is replaced by Trp is referred to as "N142W", and if the mutant has multiple mutations, the multiple mutations are indicated by the separator " / ".

[0036] In this 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 replaced by Trp.

[0037] The mutants disclosed herein need not have the full-length sequence. As long as the regions important for the hydrolysis and / or transglycosylation activity of EndoS2 are retained, they are within the scope of this disclosure. The known active fragment of EndoS2 is the amino acid sequence from position 38 to 819 of SEQ ID NO:1. Based on this, recombinant proteins that contain the fragment and appropriately add, for example, a signal peptide sequence, a purification tag (e.g., His-tag), a linker sequence (e.g., GGGS), or other functional components (e.g., CPD for solubilization) to their N-terminus or C-terminus to form new fusion proteins without affecting the activity of EndoS2 are all within the scope of protection of this disclosure.

[0038] 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 required mutation positions described below, without affecting enzyme activity. Preferably, the amino acid sequence 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 enzyme activity, but the position is preferably other than amino acid numbers 38 to 819 of SEQ ID NO: 1. In this disclosure, the term "several" means 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 a candidate sequence to those in a reference polypeptide sequence after aligning the sequences (and, where necessary, introducing gaps) to obtain the maximum percentage of sequence identity, without considering any conserved 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 the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0040] When used in this disclosure, a designation without a specific quantity can mean one or more. When used in conjunction with the word "comprising," a designation without a specific quantity 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 cell receptor, and these terms are used interchangeably. The term “wild-type antibody” as used in this disclosure typically refers to an antibody that is naturally occurring or generated through recombinant expression and has an N-glycosylation site (N-glycan). For example, all antibodies with an N297 site in their Fc region fall under the category 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 the immunoglobulin heavy chain, specifically the two polypeptide chains that form a dimer containing the self-linking C-terminal constant region of the immunoglobulin heavy chain. This 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 amino acid residues in the Fc region or constant region are numbered according to 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 well-defined, and the sequences of all types of native and variant Fc regions are known in the art.

[0043] The term "deglycosylated antibody" in this disclosure refers to an antibody formed by the action of glycoside hydrolases on wild-type antibodies, containing one N-acetylglucosamine (GlcNAc) or a core fucosylated N-acetylglucosamine (Fucα1,6-GlcNAc).

[0044] The term "β-N-acetylglucosinolate endonuclease" in this disclosure refers to a class of enzymes (EC 3.2.1.96) produced by a range of organisms that possess glycoside endonuclease activity. These enzymes typically belong to glycoside hydrolases 18 or 85. Some β-N-acetylglucosinolate endonucleases known in the art, such as Endo S and Endo S2, are described in WO2022 / 050300. These enzymes may also possess disaccharide linker transfer activity; known enzymes with this activity include Endo S, Endo S2, Endo F3, and their mutants, as described in WO2022 / 226420.

[0045] The term "antibody-drug conjugate" in this disclosure broadly refers to any conjugate formed by covalently linking a peptide / protein targeting a specific cell to a payload. The peptide / protein targeting the specific cell can be an antibody or its antigen-binding fragment, such as a monoclonal antibody, bispecific antibody, or polyclonal antibody; the payload can be a cytotoxin, a small molecule drug, a near-infrared or fluorescent probe, a peptide, RNA and related drugs, a radiolabeled isotope, a contrast agent, or a magnetic resonance imaging agent; the resulting covalent conjugate can be used for treatment or detection. "Antibody-drug conjugate" (ADC) refers to any conjugate formed by covalently linking a peptide / protein targeting a specific cell to a cytotoxin.

[0046] As used herein, "high purity" refers to the separation of the target protein from contaminants that accompany it in its native state or from contaminants generated or used during the preparation of the target protein. Typically, the target protein is considered high purity when the grayscale of the target protein band after gel electrophoresis accounts for at least 90% of the total grayscale of all bands. Preferably, in some embodiments, the grayscale of the target protein accounts for at least 95% of the total grayscale of all bands, and most preferably at least 98%.

[0047] This disclosure further provides a recombinant gene encoding the aforementioned EndoS2 mutant, a gene construct containing the recombinant gene such as a plasmid or expression vector, a host cell transformed with the gene construct, and a method for generating the mutant of this disclosure, which includes the step of collecting the mutant of this 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 this disclosure using known genetic engineering techniques.

[0048] Host cells transformed by introducing a gene encoding a mutant of this disclosure can be cultured under appropriate conditions according to cell type (cells commonly used for protein production, such as animal cells, plant cells, *E. coli*, yeast, etc., can be appropriately selected), and mutants of this disclosure can be collected from the culture. Collection of mutants is carried out by appropriately combining conventional purification techniques based on the physical properties of the protein. To facilitate collection, gene constructs can be designed to express the mutant in the form of a tagged peptide, such as GST, which is pre-linked to the mutant to make collection possible using the affinity for the tagged peptide. The tagged peptide can be removed after purification, but when it has no effect on the enzymatic activity of the mutant, the mutant with the tagged peptide linked thereto can be used for reactions such as sugar remodeling. Mutants of this disclosure comprise amino acid sequences containing the tagged peptide linked thereto.

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

[0050] Materials and methods

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

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

[0053] By PCR amplification, cDNA encoding the Endo S2 amino acid sequence of serotype M49 Streptococcus pyogenes strain NZ131 was fused with cDNA encoding the cysteine ​​protease domain (CPD) of Vibrio cholerae MARTX toxin, and a His-tag protein (10×His) was attached to the C-terminus. The two cDNAs were then inserted between SalI and NotI in the multiple cloning site region of the vector plasmid pET-22b(+). A codon containing the mutant amino acid was introduced using primers containing the mutant. Recombinant plasmids containing the nucleic acid sequences of wild-type Endo S2 and its mutant were produced using *E. coli* DH5α strain and transformed into *E. coli* BL21(DE3) cells. Expression of the Endo S2-CPD-10×His fusion protein (hereinafter referred to as Endo S2 fusion protein) was achieved by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.25 mM and incubating overnight at 20°C. Cells were collected by centrifugation, resuspended in lysis buffer (20 mM PB, 500 mM NaCl, pH 7.5), and then disrupted using an ultrasonic cell disruptor (Wuxi Jerean Instrument Equipment Co., Ltd.). The cell lysate supernatant was collected after centrifugation and purified using Ni Sepharose Excel packing material (Cytiva). The protein was concentrated using an Amicon centrifuge filter (30 kDa, Millipore) and further processed through a HiLoad filter. TM 26 / 600 Superdex TM Purification was performed using a 200 pg column (Cytiva) via size exclusion. The fraction containing the Endo S2 fusion protein was concentrated using an Amicon centrifugal filter (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 (Gel Doc EZ Imager, Bio-RAD) using 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) column. For antibody heavy chain analysis, IgG antibodies were treated with 20 mM TCEP and heated at 37 °C for 30 min, followed by analysis using an ACCQUITYUPLC 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 min. Raw data were deconvolved using UNIFI (Waters).

[0056] Determination of glycoside hydrolysis activity of wild-type Endo S2 and its mutant against monoclonal antibody 1

[0057] The glycoside hydrolytic activity of wild-type Endo S2 and its mutants was determined at 37 °C using intact monoclonal antibody 1 (10 mg / mL) as a substrate in a 20 mM phosphate buffer (pH 7.5) system. The reaction mixture was terminated by adding formic acid to a final concentration of 0.1%, and then analyzed by reductive LC-MS. After deconvolving the raw heavy chain ESI-MS data and integrating the corresponding MS peaks, the relative amounts of the substrate (monoclonal antibody 1) and the glycoside hydrolysis product (deglycosylated monoclonal antibody 1) were quantified to calculate the changes in glycoside hydrolytic activity of wild-type Endo S2 and its mutants.

[0058] Determination of glycoside hydrolysis activity of wild-type Endo S2 and its mutant against monoclonal antibody 2

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

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

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

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

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

[0064] To investigate the glycoside hydrolysis of Endo S2, this disclosure uses residues surrounding the catalytically active sites D184 and E186, as well as residues interacting with the substrate, as entry points. Site-directed mutagenesis is performed 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 above-mentioned mutation positions correspond to the sequence of SEQ ID NO: 1. Wild-type Endo S2 and its mutants were recombinantly expressed in Escherichia coli (E. coli) by fusing CPD protein at the C-terminus and adding His-tag protein (see Figure 1). The recombinant protein is referred to as Endo S2 fusion protein. It can be purified by Ni Sepharose Excel affinity packing material. The component containing the target protein can be further purified by HiLoad™ 26 / 600 Superdex™ 200 pg column to obtain high-purity Endo S2 fusion protein.

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

[0066] Monoclonal antibody 1 was used as a substrate to detect the glycoside hydrolytic activity of wild-type Endo S2 and its mutants. The main Fc glycan of monoclonal antibody 1 is a bibranched complex oligosaccharide with a core fucosylated core, carrying 0, 1, or 2 galactose moieties, designated as G0F, G1F, and G2F glycoforms, respectively. A small portion of monoclonal antibody 1 also contains a G0 glycoform without a core fucosylation and without lactose. Monoclonal antibody 1 was deglycosylated using wild-type Endo S2 fusion protein. The deglycosylation process and results were detected by reducing LC-MS to calibrate different glycoforms and deglycosylated monoclonal antibody 1.

[0067] The deglycosylation detection results of monoclonal antibody 1 are shown in Figure 2. Figure 2(A) is the original heavy chain mass spectrometry detection of monoclonal antibody 1. After ESI-MS deconvolution, there are four main peaks, corresponding to four main different m / z substances, namely 50448±1, 50595±1, 50757±1 and 50919±1 Da, which correspond to the G0, G0F, G1F and G2F glycoforms, respectively. Figure 2(B) is the detection result of deglycosylation of monoclonal antibody 1 by wild-type Endo S2 fusion protein. The heavy chain ESI-MS deconvolution data added two substances at 49354±1 Da and 49500±1 Da, which matched the deglycosylated GlcNAc glycoform and the fucylized GlcNAc (Fucα1,6-GlcNAc) glycoform, respectively. The above results confirm that wild-type Endo S2 can perform glycosylation on N-glycans in the intact IgG Fc region. Therefore, Endo S2 can be used as the first step in antibody glycoform homogenization or site-directed glycosylation—antibody deglycosylation.

[0068] Using intact monoclonal antibody 1 as a substrate (including both core fucosylated and non-fucosylated intact monoclonal antibody 1), the glycoside hydrolysis activities of wild-type Endo S2 fusion protein and its mutants were determined. As shown in Figure 3 and Table 1, compared with wild-type Endo S2, the glycoside hydrolysis activity of mutant N142W was significantly increased to 3.58 times that of WT; mutant E57W retained most of the glycoside hydrolysis activity; the remaining mutants K85L, I185T, D226N, and W297Y had significantly reduced glycoside hydrolysis activity, especially A259N, A291R, and D299Y, which essentially or directly lost glycoside hydrolysis activity.

[0069] Example 3: Detection of glycosylation activity of wild-type Endo S2 and its mutant against monoclonal antibody 2

[0070] To verify the universality of Endo S2 and its mutants in the hydrolysis of antibody N-glycans, another antibody—monoclonal antibody 2—was used as a substrate to further detect the glycoside hydrolysis activity of wild-type Endo S2 and its mutants. The LC-MS results of intact monoclonal antibody 2 and deglycosylated monoclonal antibody 2 reduction are shown in Figure 4. Figure 4(A) shows the ESI-MS unconvolution results of the intact monoclonal antibody 2 heavy chain, with 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 detection results of monoclonal antibody 2. The ESI-MS unconvolution data of the heavy chain showed two substances at 49285±1 Da and 49431±1 Da, which matched the glycoforms of the deglycosylated GlcNAc-monoclonal antibody 2 and Fucα1,6-GlcNAc-monoclonal antibody 2, respectively. Furthermore, the activity data of wild-type Endo S2 fusion protein and its mutants against intact monoclonal antibody 2 (Figure 5 and Table 1) show that mutant N142W exhibits significantly enhanced hydrolytic activity, which is 4.69 times that of WT. Mutant E57W retains most of the glycoside hydrolytic activity. The remaining mutants K85L, I185T, D226N, A259N, and W297Y have significantly reduced glycoside hydrolytic activity, while A291R and D299Y directly lose glycoside hydrolytic activity. These data are consistent with the trend of the activity data of monoclonal antibody 1 as a substrate (Figure 3), indicating that the significantly enhanced hydrolytic activity of mutant N142W is shown on different antibodies. The N142W mutant has universality for monoclonal antibody substrates and can be used for deglycosylation treatment of various glycotype antibodies.

[0071] Table 1: Comparison of glycoside hydrolytic activity of wild-type Endo S2 and mutant N142W using monoclonal antibody 1 and monoclonal antibody 2 as complex glycoform substrates.

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

[0073] Deglycosylated monoclonal antibody 1 was prepared, and its transglycosylation activity in the Endo S2 mutant N142W was studied using the azide-modified disaccharide linker LacNAc oxazoline (N3-LacNAc-Oxa). The LC-MS results of the transglycosylation system reduction are shown in Figure 6. Figure 6(A) shows the deglycosylated monoclonal antibody 1. The heavy chain ESI-MS deconvolution data at 49354±1 Da and 49500±1 Da show two substances, namely GlcNAc-monoclonal antibody 1 and Fucα1,6-GlcNAc-monoclonal antibody 1, respectively. Figure 6(B) shows the addition of two substances at 49776±1 Da and 49922±1 Da in the ESI-MS data. This result matches well with the molecular weight of N3-LacNAc-Oxa after transglycosylation (after azide degradation and protonation under acidic conditions). Compared with wild-type Endo S2, N142W retained 70.7% of 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 to which this disclosure pertains should understand that the above embodiments are for illustrative purposes only, and various alterations and modifications can be made to the mutant protein without departing from the scope of this disclosure. These modifications include, but are not limited to, insertion and / or truncation of the N-terminus and / or C-terminus, glycosylation, sialylation, albuminization, farnesylation, carboxylation, hydroxylation, phosphorylation, and conjugation to polymers. Importantly, the mutant protein possesses an Endo S2 catalytic domain and includes a mutation at the N142W position. Furthermore, monoclonal antibodies were used in the embodiments described herein, and those skilled in the art to which this disclosure pertains should understand that similar methods can be used for other glycoproteins or antibodies.

Claims

1. An Endo S2 mutant enzyme having a mutation at one amino acid position on the basis of SEQ ID NO: 1 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1: (a) SEQ ID NO: 1; or (b) comprising the contiguous amino acids from position 38 to 819 of SEQ ID NO: 1, and the mutant enzyme possesses glycoside hydrolyzing and / or glycosyl transferase activity.

2. The Endo S2 mutant enzyme of claim 1, wherein the mutation is N142W.

3. A polynucleotide encoding the Endo S2 mutant enzyme of claim 1 or 2.

4. A vector comprising the polynucleotide of claim 3.

5. A host cell transformed with the vector of claim 4.

6. Use of the Endo S2 mutant enzyme of claim 1 or 2 in the glycoengineering of a polypeptide or protein containing N-glycans.

7. The use of claim 6 for the production of deglycosylated antibodies in reshaping the glycoforms of antibodies, or deglycosylation in the remodeling of the sugar chains of glycoproteins, or in the production of antibody-based conjugated drugs.

8. A method for remodeling the sugar chains of a polypeptide or protein containing N-glycans, comprising the steps of: (a) introducing the Endo S2 mutant enzyme of claim 1 or 2; (b) introducing a polypeptide or protein containing N-glycans as a substrate; and (c) using the Endo S2 mutant enzyme to glycoside hydrolyze the polypeptide or protein containing N-glycans to provide a new polypeptide or protein with N-glycans removed, with only the first GlcNAc remaining, or Fucal,6-GlcNAc.

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 sialylglycopeptide.

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

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