Glycosynthase mutants for antibody-drug conjugate engineering

Abstract

The present disclosure relates to novel glycosynthase enzymes for antibody or antibody-drug conjugates (ADCs) engineering. The enzyme mutants, termed EndoSc-D233Q / E350N and EndoSc-D233Q / E350Q, contain the glycan conjugation and / or modification activity at the conserved N297 glycosylation site of Fc region of an exemplary antibody. It has been demonstrated that the glycosynthase activities of EndoSc-D233Q / E350N and EndoSc- D233Q / E350Q can be applied to various mAbs targeting different receptors. It has been found that both mAb-GlcNAc and mAb-GlucNAc(Fuc) were suitable substrates for both EndoSc mutants.

Description

PATENT Attorney Docket No.: G3004-02000PCT GLYCOSYNTHASE MUTANTS FOR ANTIBODY-DRUG CONJUGATE ENGINEERING CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of U.S. Provisional Patent Application No. 63 / 652,473, filed on May 28, 2024. The entirety of the aforementioned application is incorporated herein by reference. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 27, 2025, is named “G3004-02000PCT_20250527_SeqListing.xml” and is 6,927 bytes in size. FIELD

[0003] The present disclosure relates to glycosynthase enzymes for homogeneous antibody engineering / remodeling. EndoSc mutant contains the glycan conjugation enzymatic activity at the conserved N297 glycosylation site of the Fc region. The present disclosure demonstrated that glycosynthase activities of EndoSc mutants can be applied on various monoclonal antibodies (mAbs) which target different receptors. Both mAb-GlcNAc and mAb- GlucNAc(Fuc) were suitable substrates for transglycosylation with EndoSc mutants, it also demonstrated that glycosynthase activities of EndoSc mutants can be applied on antibody-drug conjugates (ADCs) compositions. Furthermore, both cytotoxicity and animal studies indicated that ADC prepared by EndoSc mutant can significantly decrease cell viability and tumor volume, respectively. BACKGROUND

[0004] Therapeutic monoclonal antibodies (mAbs) have been developed for treating many diseases, such as cancer, autoimmune, and infectious (1-3). For cancer therapy, several commercial mAbs have been found on the market. Those mAbs recognize particular antigens on tumor cell surface and enhance cell apoptosis by different mechanisms, such as turning on antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) or blocking signal pathway. HER2 receptor is the most prominent biomarker overexpressed on breast cancer, which enables Roche Company to develop two related mAbs, Herceptin (trastuzumab) and Perjeta (pertuzumab). EGFR receptor is also a well-known target for mAb drug development. For example, Vectibix (panitumumab) and Erbitux (cetuximab)PATENT Attorney Docket No.: G3004-02000PCT have been produced by Amgen and Merck, respectively, targeting metastatic colorectal cancer therapy. In addition, Rituxan (rituximab, Roche) and Arzerra (ofatumumab, GSK) designed to recognize CD20 receptor are commonly used to treat non-Hodgkin’s B-cell lymphomas and chronic lymphocytic leukemia cancer. Erbitux, Rituxan and Arzerra, kill cancer cells majorly through cytotoxicity in terms of ADCC and CDC. Moreover, several mAbs have been developed to block the function of protein-protein interaction, such as Humira (Adalimumab, AbbVie) blocks the TNF-α receptor mediate signal pathway for autoimmune disease, rheumatoid arthritis; Keytruda (pembrolizumab, Merck) blocks PD1 receptor to destroy the protective mechanism of cancer cells that treats metastatic melanoma. Compare with small molecular drugs, mAbs are more specific to the target cells and have relatively lower side- effect to the patients. These two important features have become the powerful tool against various diseases treatment.

[0005] Antibodies have molecular weight of ~150 kDa composed of two heavy chains (~50 kDa) and two light chains (~25 kDa), which form three domains separated by flexible hinge region. Two Fab domains contain variable complementarity-determining region (CDR) for identifying antigen. One Fc domain is a constant region with N-glycan for mediation of ADCC and CDC cytotoxicity (4). The amino acid N297 at the Fc domain is a conserved N- glycosylation site which connects with heterogeneous glycan types, such as biantennary (G0F, G1F, G2F, G0, G1 and G2 complex type) and triantennary (high-mannose and hybrid types), while expressed in various cell systems. The X-ray structure analysis results indicated that the core fucose of Fc glycan obstructed the particular carbohydrate-carbohydrate interactions between Fc and FcγRIIIa and, as a result, decreased the binding constant for approximately hundred folds (5) and, as a result, reduced cell killing efficiency. The most common cell system used in industry is CHO cell. mAbs produced from CHO cells generally contain glycans compositions predominately in the form of G0F, G1F and G2F. These glycan forms limit the activity of mAbs due to the reduced ADCC binding efficiency caused by fucose. Although modified CHO cell systems have been available by FUT8 (α-1,6-fucosyltransferase 8) gene knock-out (6, 7) or up-regulation of bisecting GlcNAc (N-acetylglycosamine) transferase GnT- III (8) to reduce the glycan complicity of mAbs, to find a better and more general method to obtain the desired N-glycan on mAbs is still in great interests. In addition, it has also reported that removing the Fc glycan will result in losing the ADCC activity (9). According to the above observations, mAbs cytotoxicity can be effectively controlled by the N-glycan type attached to the Fc region.PATENT Attorney Docket No.: G3004-02000PCT

[0006] Enzymatic modification of Fc region is a solution to establish homogeneous mAbs. Few years ago, Lai-Xi Wang and coworkers have tried the chemoenzymatic remodeling by removing the glycan mixture and conjugating homogeneous glycans (9). Several endo-β-N- acetylglucosaminidases (ENGases) have been reported to remove glycan mixture on mAbs. For instance, EndoLL (10), EndoD (11), EndoH (12), and EndoM (13) are able to hydrolyze the glycan with high mannose or terminal mannose types. EndoS (14) and EndoSd (15) has the ability to hydrolyze non-fucosylated and fucosylated N-glycans on the Fc domain, but not high- mannose types. So far, no signal endoglycosidase could completely hydrolyze all glycan types on mAbs. The EndoS crystal structure was solved recently, which revealed five functional domains (16), in which the endoglycosidase domain is highly conserved with rigid β-barrel structure that is suitable for site mutation studies. One the other hand, glycosynthases for antibody Fc were also reported. EndoD-N322Q (17) and EndoM-N175Q (18) only transferred short chain complex-type N-glycan to Fc. EndoF3-D165Q (19) only transferred glycan to the fucosylated Fc domain. EndoS-D233Q (10) enables to conjugate various bi-antennary complex type, whereas EndoS2-D184M (20) has wild substrates including complex, high-mannose and hybrid types.

[0007] The use of Antibody-Drug Conjugates (ADCs) for the local delivery of cytotoxic or cytostatic drugs to kill or inhibit tumor cells in the treatment of cancer allows targeted delivery of the drug moiety to tumors, and intracellular accumulation therein, while systemic administration of the unconjugated cytotoxic or cytostatic drugs may result in unacceptable levels of toxicity to normal cells as well as to the tumor cells sought to be eliminated. Both polyclonal antibodies and monoclonal antibodies have been reported as useful in these strategies. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands. SUMMARY OF INVENTION

[0008] The present disclosure relates to endoglycosidase mutants derived from Streptococcus that possess / display improved enzymatic activities for the synthesis of glycoproteins and / or glycopeptides comprising a broad range of well-defined N-glycans of high mannose, hybrid, and complex types. In particular, some embodiments of present disclosure also relate to using the endoglycosidase mutants for glycan remodeling of therapeutic antibodies to generate homogenous glycan compositions at the Fc-domain to improve their functions.PATENT Attorney Docket No.: G3004-02000PCT

[0009] In this study, we identified an enzyme, EndoSc, with glycosynthase activity, that is capable of producing homogeneous monoclonal antibodies (mAbs), which can be used to manufacture antibody conjugates. It is believed that the EndoSc from Streptococcus canis (WP_093998831.1) exhibited glycosynthase activity. We generated several EndoSc mutants per multiple sequence alignment. The results showed that the mutated enzymes have glycosynthase activity to conjugate a bi-antennary complex-type glycan to antibodies while the antibodies are not limited to certain immunoglobulin types or antigen-binding specificities. Here, we demonstrated the conjugation using R4702 (an anti-TROP2 antibody) as an example.

[0010] In one aspect, the present disclosure provides a glycosynthase mutant, wherein the mutant has at least about 90%, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence and / or structural homology thereto and exhibits improved transglycosylation activity on both fucosylated and non-fucosylated GlcNAc acceptors against a broad range of N-glycans of high mannose, hybrid, and complex types, wherein the mutant enables transfer of an activated oligosaccharide donors to a fucosylated and non-fucosylated GlcNAc acceptor, thereby generating glycopeptides, glycoproteins, or therapeutic antibodies with a homogenous glycoform. In another aspect, the glycoprotein acceptor is a glycopeptide, a glycoprotein, an antibody or an antigen-binding fragment.

[0011] Some examples of the EndoSc mutants of the present disclosures are listed in Table 1:

[0012] Table 1. Exemplary EndoSc mutants of the Present Disclosure.

[0013] In certain embodiments, the antibody is R4702, an anti-TROP2 monoclonal antibody. R4702 is as described in PCT Publication No. WO2022222992A1, the content of which is incorporated herein by reference in its entirety.

[0014] In certain embodiments, the antibody is an anti-TROP2 antibody selected from a group consisting of hRS7, Hu2G10, hu4D3, MAAP-9001a, Pr1E11, R4702, datopotamb, and sacituzumab.PATENT Attorney Docket No.: G3004-02000PCT

[0015] In another aspect, a method for preparing an engineered glycoprotein and antibody-drug conjugate (ADC) using the glycosynthase mutants is provided.

[0016] In another aspect, a population of homogeneous antibody prepared by using the glycosynthase mutants is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1. Summarizes aspects of the overall process of homogeneous platform. (A) The mAbs were heterogeneous with glycan mixtures which were removed by wild type EndoSc and α-fucosidase to generate mAb-GlcNAc. Then the EndoSc-D233Q / E350Q was used to conjugate glycan-oxazoline and produce homogeneous mAbs. (B) In the glycan cleavage step, only wild type EndoSc enzyme is used to generate mAbs-GlcNAc(Fuc) and will be produces to homogeneous-mAb-glycan(Fuc) after conjugation with EndoSc- D233Q / E350Q. (C) The exemplary picture of biantennary glycans.

[0018] Figure 2. The deglycosylation study results with different ratio of EndoSc-WT enzyme and R4702 Ab.

[0019] Figure 3. The transglycosylation activity investigation of EndoSc-WT, EndoSc-D233Q (single residue mutant), EndoSc-D233Q / E350N, EndoSc-D233Q / E350Q (Mutations at two residues) and EndoSc-D233Q / Q303A / E350N, EndoSc- D233Q / Q303A / E350Q (Mutations at three residues) on R4702-GlcNAc(Fuc) of NSCT-2.

[0020] Figure 4. The reaction time investigation of EndoSc-D233Q / E350Q transglycosylation activity with 15 eq NSCT-2 on R4702-GlcNAc(Fuc).

[0021] Figure 5. The buffer system and pH investigation of EndoSc-D233Q / E350Q transglycosylation activity on R4702-GlcNAc(Fuc).

[0022] Figure 6. Sequence alignment of EndoS, EndoS2 and EndoSc enzymes.

[0023] Figure 7. The transglycosylation activity investigation of diverse EndoSc mutations at single residues on R4702-GlcNAc(Fuc).

[0024] Figure 8. (A) Representative SDS-PAGE results of R4702-GlcNAc(Fuc) and R4702-(NSCT-di-N3)2. (B)(C) Representative CE-SDS chromatography results of R4702- GlcNAc(Fuc) and R4702-(NSCT-di-N3)2. (D) Overlay electropherogram results of R4702, R4702-GlcNAc(Fuc) and R4702-(NSCT-diN3).

[0025] Figure 9. Hydrophobic interaction chromatography (HIC) result and CE-SDS data of several ADCs. (A) HIC result of R4702 DAR4 ADC. (B) CE-SDS data of mAb- GlcNAc(Fuc) and mAb-(NSCT-di-N3)2of diverse antibodies and ADCs.

[0026] Figure 10. Cytotoxicity data of R4702 DAR4 ADC with BxPC-3 cell line.PATENT Attorney Docket No.: G3004-02000PCT

[0027] Figure 11. Animal study data of R4702 DAR4 ADC with NCI-H1975 xenograft model.

[0028] Figure 12. Hydrolytic activity of EndoSc and its mutant on glycopeptides (SGPs). (A) SGP hydrolysis ratio of wild type EndoSc (B) SGP hydrolysis ratio of EndoSc S280A mutant. DETAILED DESCRIPTION OF THE INVENTION

[0029] N-glycosylation is one of the most complex post-translational modifications that often results in a remarkable heterogeneity of glycan structures including high mannose, hybrid, and complex types, depending on the recombinant protein expression systems and organisms. Commercially available therapeutic antibodies typically exist as a mixture of glycoforms that are not optimal for their respective therapeutic activities. Recently, glycoengineering has been gaining attention for its effect in modifying Fc glycosylation for improving efficacy.

[0030] A typical IgG consists of two antigen-binding fragments (Fabs), which are connected via a flexible region to a constant region (Fc). The Fab domains are responsible for antigen recognition while the N-glycan at Asn297 of Fc domain interact with respective Fcγ receptors (such as FcγRIIIa and FcγRIIb) on effector cells and C1q component of the complements that activate the effector functions, including antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Almost all therapeutic antibodies are N-glycosylated on each of the homodimeric Fc domains at the conserved asparagine residue (N297). These N-linked glycans result in more than 30 different glycoforms and are typical biantennary complex type with considerable structural heterogeneity, in which the core heptasaccharide can be differentially decorated with core fucose (Fuc), bisecting N- acetylglucosamine (GlcNAc), terminal galactose (Gal), and terminal sialic acid (Sia). The composition of N-glycans could influence the Fc domain conformation, therefore, modulating the antibody’s stability, pharmacokinetic profile, immunogenicity, effector functions, antibody-mediated inflammation, and complement activation. For example, the absence of the core fucose, as well as the attachment of a bisecting GlcNAc moiety, dramatically enhances the affinity of antibody for the FcγIIIa receptor (FcγRIIIa) on effector cells, resulting in more effective elimination of the target. In addition, the terminal a-2,6-sialylated glycan, which is a minor component of antibodies and the intravenous immunoglobulin (IVIG), is an optimized structure that enhances the anti-inflammatory properties.PATENT Attorney Docket No.: G3004-02000PCT

[0031] Endoglycosidases are a family of at least 18 glycoside hydrolase (GH) from the Streptococcus pyogenes and have recently become the point of attention for glycoengineering of therapeutic antibodies. These enzymes can catalyze the hydrolysis of the β-1, 4 linkage between the two N-acetylglucosamines (GlcNAcs) in the core of the N-linked glycan of human IgG. Additionally, the enzymes remove complex type glycans at IgG Fc domain.

[0032] Embodiments of the present disclosure relate to selected mutants of glycosynthase that show remarkable transglycosylation activities to transfer a broad range of N-glycans of high mannose, hybrid or complex types, from activated oligosaccharide oxazolines to fucosylated or non-fucosylated GlcNAc-peptides, proteins, or IgGs with little or negligible product hydrolysis. The novel glycosynthase acts with a surprisingly high efficiency to provide homogeneous glycosylated glycopeptides, glycoproteins, therapeutic antibodies and their Fc fragments thereof, having various defined glycoforms. Still further, embodiments of the present disclosure may provide glycoengineered antibodies with enhancement of their effector functions, such as FcγIIIA bindings and antibody dependent cell mediated cytotoxicity (ADCC) etc., as well as pharmacological properties. Embodiments of the present disclosure also allow for rapid investigation of effects of diverse Fc glycosylations of therapeutic antibodies on their effector functions.

[0033] In accordance with embodiments of the present disclosure, a novel glycosynthase comprises an amino acid sequence of SEQ ID NO.2 or SEQ ID NO.3. These mutants show unexpectedly improved tranglycosylation activities and reduced hydrolyzing activities. Therefore, they can efficiently transfer activated oligosaccharide donors to core GlcNAc-acceptors, which may be fucosylated or non-fucosylated.

[0034] In accordance with certain embodiments, a glycosynthase may have a sequence identity of at least about 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99%) to SEQ ID No.2 or SEQ ID NO.3 and exhibits a desired transglycosylation activity, or fragment thereof having the transglycosylation activity. Definitions

[0035] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise.

[0036] As used herein, the term “glycan” refers to a polysaccharide, oligosaccharide or monosaccharide. Glycans can be monomers or polymers of sugar residues and can be linear or branched. A glycan may include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-PATENT Attorney Docket No.: G3004-02000PCT acetyl neuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2’-fluororibose, 2’-deoxyribose, phosphomannose, 6’ sulfo N- acetylglucosamine, etc.).

[0037] As used herein, the terms “fucose,” “core fucose,” and “core fucose residue” are used interchangeably and refer to a fucose in α-1,6-position linked to the N-acetylglucosamine.

[0038] As used herein, the terms “N-glycan”, “N-linked glycan”, “N-linked glycosylation”, “Fc glycan” and “Fc glycosylation” are used interchangeably and refer to an N-linked oligosaccharide attached by an N-acetylglucosamine (GlcNAc) linked to the amide nitrogen of an asparagine residue in a Fc-containing polypeptide. The term “Fc-containing polypeptide” refers to a polypeptide, such as an antibody, which comprises an Fc region.

[0039] As used herein, the term “glycosylation pattern,” “glycosylation profile,” and ”glycoform” are used interchangeably and refer to the characteristic “fingerprint” of the N-glycan species that can be released from a glycoprotein or antibody, either enzymatically or chemically, and then analyzed for their carbohydrate structure, for example, using LC-HPLC, or MALDI-TOF MS, and the like. See, for example, the review in Current Analytical Chemistry, Vol.1, No.1 (2005), pp.28-57; herein incorporated by reference in its entirety.

[0040] As used herein, the term “glycoengineered Fc” when used herein refers to N- glycan on the Fc region has been altered or engineered either enzymatically or chemically. The term “Fc glycoengineering” as used herein refers to the enzymatic or chemical process used to make the glycoengineered Fc.

[0041] The terms “homogeneous”, “uniform”, “uniformly” and “homogeneity” in the context of a glycosylation profile of Fc region are used interchangeably and are intended to mean a single glycosylation pattern represented by one desired N-glycan species, with little or no trace amount of precursor N-glycan.

[0042] Table 2. Listed the four classes of amino acids.PATENT Attorney Docket No.: G3004-02000PCT

[0043] As used herein, the terms “monoclonal antibody”, “immunoglobulin”, and “immunoglobulin molecule” are used interchangeably and might include IgG.

[0044] The term “effector function” as used herein refers to a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Exemplary “effector functions” include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor; BCR), etc. Such effector functions can be assessed using various assays known in the art.

[0045] As used herein, the term “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are absolutely required for such killing. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo.

[0046] As used herein, “isolated” means that a subject protein or polypeptide (1) is free of at least some other proteins or polypeptides with which it would typically be found in nature, (2) is essentially free of other proteins or polypeptides from the same source, e.g., from the same species, (3) is expressed by a cell from a different species or an engineered cell that does not exist in nature, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a protein or polypeptide with which the "isolated protein" or “isolated polypeptide” may be associated in nature, (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such an isolated protein or polypeptide can be encoded by genomic DNA, cDNA, mRNA or other RNA, of may be of synthetic origin according to any of a number of well-known chemistries for artificial peptide and protein synthesis, or any combination thereof. In certain embodiments, the isolated protein or polypeptide is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).PATENT Attorney Docket No.: G3004-02000PCT

[0047] The complex N-linked oligosaccharide on each CH2 domain of IgGs is crucial for the structure of the Fc region and thus the interaction with the Fc receptors. The oligosaccharide chain at IgG-Fc domain contains several N-Acetyl-Glucosamine (GlcNAc) and mannose (Man) residues, and galactose (Gal) and fucose (Fuc) residues as well as sialic acid (Sia or NANA for N-acetylneuraminic acid). A GlcNAc, with or without al-6 Fuc, is attached to the Asn297. A GlcNAcpi-4 is attached to this first GlcNAc. A manβ1-4 is then found, to which two Manα1-6 and Manα1-3 arms are attached. Both arms contain an additional GlcNAcβ1-2 to which a Galβ1-4 can be attached or not. Thus, the carbohydrate chain can contain 0, 1 or 2 galactose residues, defining G0, G1, and G2 glycoforms, respectively. Further variations occur, including the presence of a bisecting GlcNAcβ1-4 and the capping of one or both of the terminal galactose residues with a sialic acid or even a Galɑ1-3 residue. The enzymatic cleavage of the Fc-glycan with Endoglycosidases causes the Fc region to deform, and thus, dramatically decrease in IgGs binding to Fcγ receptors. Despite their 37% sequence identity, both EndoS and EndoS2 catalyze the hydrolysis of the β-1,4 linkage between the two N-acetylglucosamines (GlcNAcs) in the core of the N-linked glycan of human IgG. However, in addition to complex types of glycans, EndoS2 hydrolyze hybrid and oligomannose structures to a greater extent compared with EndoS.

[0048] Since the introduction of first antibody therapy in the 1980s, there are more than 240 therapeutic antibodies in clinical trials and the field is steadily expanding. The role of the IgG-Fc glycans on antibody functions has gained attention in the growing field of monoclonal therapeutic antibodies. Therefore, to improve the efficacy of the therapeutic antibodies, the major focus is turning towards the engineering the Fc-glycan that specifically interact with selected Fcγ receptors. The recent improvements in biotechnology tools to control the Fc- glycosylation states of IgG facilitate the development of therapeutic antibodies with predefined glycoforms. Accordingly, the glycosynthase of present disclosure is a great advancement in the field for glyco-engineering of peptides, proteins, and antibodies of interest to attach a broad range of N-glycans of high mannose, hybrid, and complex types for functional and structural studies.

[0049] In one embodiment, the activated oligosaccharide is a glycan oxazoline comprising diverse N-glycans of high mannose, hybrid, and complex types having a structure of the formula:PATENT Attorney Docket No.: G3004-02000PCT; wherein, R1is –H or N-acetyl glucosamine attached via β-1, 4 linkage and R2and R3are same or different and are independently selected from the group consisting of:.

[0050] In another aspect, the present disclosure provides exemplary glycosynthase for transglycosylation at core fucosylated or non-fucosylated GlcNAc-acceptor, wherein the core fucosylated or non-fucosylated GlcNAc-acceptor comprises core fucosylated or non- fucosylated GlcNAc-peptides, proteins, or IgG Fc domain or fragment thereof.

[0051] In further aspect, the present disclosure provides a composition of fucosylated or non-fucosylated glyco-engineered antibodies or antigen binding fragments thereof comprising IgG molecules having a homogenous glycoform, wherein the N-glycan of the glycoform is of high mannose, hybrid, and complex types and is selected from the group consisting of:;PATENT Attorney Docket No.: G3004-02000PCT wherein R1is –H or N-acetyl glucosamine attached via β-1, 4 linkage and R2and R3are same or different and are independently selected from the group consisting of:.

[0052] In another aspect, the present disclosure provides a glycoengineered antibody with unexpectedly improved effector functions, such as, binding to FcγIIIA, ADCC and regulating immune response, as compared to a non-modified antibody.

[0053] Another aspect of the present disclosure features a pharmaceutical composition comprising a population of glyco-engineered antibodies described herein and a pharmaceutically acceptable carrier for cancer treatment.

[0054] The present disclosure discloses a glycosynthase mutant that shows excellent transglycosylation activities with a broad range of N-glycans of high mannose, hybrid, and complex types.

[0055] In preferred embodiments, N-glycans of high mannose, hybrid, and complex types are in an active oxazoline form or its derivative thereof.

[0056] In some embodiments, the high mannose type N-glycan described herein is selected from group consisting of Man3GlcNAc, Man5GlcNAc, Man6GlcNAc, Man7GlcNAc, Man8GlcNAc, and Man9GlcNAc. In preferred embodiments, the high mannose type N-glycan is Man5GlcNAc.

[0057] In some embodiments, the hybrid type N-glycan described herein comprises at least one α-2,6- or α-2,3 terminal sialic acid on the alpha-1,3 arm thereof, wherein the alpha- 1,6 arm contains trimannose residues.PATENT Attorney Docket No.: G3004-02000PCT

[0058] In some embodiments, the hybrid type N-glycan described herein comprises at least one terminal galactose on the alpha-1,3 arm thereof, wherein the alpha-1,6 arm contains the trimannose residues.

[0059] In some embodiments, the hybrid type N-glycan described herein comprises at least one terminal GlcNAc on the alpha-1,3 arm thereof, wherein the alpha-1,6 arm contains trimannose residues.

[0060] In some embodiments, the complex type of glycan described herein can be a bi-, tri-, or tetra-antennary complex type.

[0061] In some embodiments, the bi-antennary complex type N-glycan described herein comprises at least one α-2,6 or α-2,3 terminal sialic acid. In preferred embodiments, the N-glycan comprises two α-2,6 and / or α-2,3 terminal sialic acids.

[0062] In some embodiments, the bi-antennary complex type N-glycan described herein comprises at least one terminal galactose or GlcNAc. In preferred embodiments, the N- glycan comprises two terminal galactose and / or GlcNAc.

[0063] In some embodiments, the bi-antennary complex type N-glycan described herein comprises at least one alpha-1,2-fucose. In preferred embodiments, the N-glycan comprises two alpha-1,2-fucoses.

[0064] In some embodiments, the bi-antennary complex type N-glycan described herein comprises at least one alpha-1,3-fucose. In preferred embodiments, the N-glycan comprises two alpha-1,3-fucose.

[0065] In some embodiments, the bi-antennary complex type N-glycan described herein comprises bisecting GlcNAc.

[0066] In some embodiments, the bi-antennary complex type N-glycan described herein comprises at least one LacNAc repeat unit. In preferred embodiments, the N-glycan comprises two LacNAc repeat units.

[0067] In some embodiments, the tri-antennary complex type N-glycan described herein comprises at least one α-2,6 or α-2,3 terminal sialic acid. In preferred embodiments, the N-glycan comprises three α-2-6 and / or α-2,3 terminal sialic acids.

[0068] In some embodiments, the tri-antennary complex type N-glycan described herein comprises at least one terminal galactose or GlcNAc. In preferred embodiments, the N- glycan comprises three terminal galactose and / or GlcNAc.

[0069] In some embodiments, the complex type of glycan can be biantennary or triantennary, comprising asymmetric antennae on either the alpha-1,3 or alpha-1,6 arm.PATENT Attorney Docket No.: G3004-02000PCT

[0070] In some embodiments, the hybrid biantennary complex type N-glycan or the hybrid triantennary complex type N-glycan described herein comprises an α-2,6 or α-2,3 terminal sialic acid. In other embodiments, the hybrid biantennary complex type N-glycan or the hybrid triantennary complex type N-glycan comprises an α-2,6 terminal sialic acid.

[0071] In another aspect, the present disclosure provides an engineered bioconjugate, comprising an engineered glycoprotein and a drug moiety. In some embodiments, wherein the engineered glycoprotein further comprises a triazole moiety, a DBCO-derived moiety, a BCN- derived moiety, or a maleimide-derived moiety, which connects the activated oligosaccharide to the glycoprotein acceptor.

[0072] In certain embodiments, the drug moiety may be a chemotherapeutic agent, a toxin, a cytokine, a growth inhibitory agent, a protein degrader, a peptide, a radionuclide, a hormone, an anti-viral agent, an anti-bacterial agent, or an immunoregulatory agent. In certain embodiments, the chemotherapeutic agent is a topoisomerase inhibitor, including a topoisomerase I inhibitor and a topoisomerase II inhibitor. In certain embodiments, the chemotherapeutic agent is a type I topoisomerase inhibitor, which is camptothecin (CPT) or non-camptothecins, selected from irinotecan, topotecan, camptothecin, rubitecan, MLN576, exatecan, belotecan, seconeolitsine, SN-38, Genz-644282, betulinic acid, β-lapachone, karenitecin, gimatecan, namitecan, edotecarin, SW044248, LMP744, T-2513, podocarpusflavone A, indimitecan, lurtotecan, TP3011 or 10-hydroxycamptothecin. EXAMPLES

[0073] Embodiments of the invention will be further illustrated with the following specific examples. One skilled in the art would appreciate that these specific examples are for illustration only and that other modifications and variations are possible without departing from the scope of the invention. For example, the enzyme mutants of the invention may be used to glycoengineer any glycoproteins or glycopeptides, including antibodies. The specific examples described herein use anti-TROP2 antibodies. However, one skilled in the art would appreciate that other glycoproteins or antibodies may also be used in a similar manner. Materials

[0074] Monoclonal anti-TROP2 antibody, R4702 was produced according to our previously procedure disclosed in PCT patent publication (WO2022222992A1).

[0075] Monoclonal anti-Nectin4 antibody, A904 was produced by OBI Pharma, Inc.PATENT Attorney Docket No.: G3004-02000PCT

[0076] The commercial antibodies: Adalimumab, Rituximab, Trastuzumab, Panitumumab and Pertuzumab. Examples Example 1. Molecular cloning, overexpression and purification of EndoSc-WT and EndoSc mutants

[0077] The enzyme EndoSc from Streptococcus canis (WP_093998831.1) were used for this study. To enhance transglycosylation activity we aligned the EndoSc protein sequence to EndoS-D233Q and found the relative position is D233. We decided to mutate the relative position D to Q. By the similar sequence alignment method with EndoS the mutant EndoSc- D233Q / E350Q was selected for investigation. Therefore, the gene encoding amino acids 20- 1065 of EndoSc-WT and EndoSc-D233Q / E350Q mutants were synthesized and sub-cloned into pET-21b(+) with 5`-NdeI and 3`-XhoI restriction sites.

[0078] For purification purpose, we inserted additional ten histidines at both N- terminal and C-terminal of enzyme sequence for affinity Ni-NTA column. Plasmids were transformed into BL21 (DE3) and cultured at 37 ºC in TB medium containing ampicillin antibiotic (100 μg / mL). The proteins were induced by 0.5 mM isopropyl-β-D- thiogalactopyranoside (IPTG) while the cell density OD600reached 0.6 ~ 0.8. And the cultivation temperature was shifted to 16 ˚C and cultivated overnight. The cells were harvested at 4 ºC by centrifugation (BACKMAN / JLA-8.1, 6000 g) for 12 minutes. The cell pellet was resuspension with wash buffer containing 50 mM sodium phosphate, 100 mM NaCl, 10 mM imidazole, pH 7.5 (10 mL buffer / 1g cell pellet) for the homogenizer (NanoLyzer N-10) to break the cell. After 60 minutes / 12,000g (BACKMAN / JA-10) centrifugation at 4 ºC and discard pellet. The resin was loaded onto an open column and washed non-bound protein with wash buffer until the concentration of non-bound protein was less than 1 mg / mL (defined by Bradford assay, Thermo). The bound protein was eluted with elute buffer containing 50 mM sodium phosphate, 100 mM NaCl, 500 mM imidazole, pH 7.5. The eluted fraction was dialysis to a storage buffer containing 50 mM sodium phosphate, 25 mM NaCl, pH 7.5 and concentrated using 30 kDa PES centrifuge filter. The final samples were assayed by SDS-PAGE and Braford for detecting MW and concentrates, respectively. Example 2. Deglycosylation of R4702 antibody by EndoSc-WT to generate R4702- GlcNAc(Fuc)

[0079] The R4702 monoclonal antibody (15 mg / mL) were incubated with EndoSc-WT in a 50 mM MOPS buffer, pH 6.7 at 37 °C. The complete cleavage of Fc N-glycans wasPATENT Attorney Docket No.: G3004-02000PCT analyzed by 4-12 % gradient SDS-PAGE. The percentage of deglycosylation was calculated by integration of SDS-PAGE with ImageJ program. Figure 2 showed the deglycosylation study of R4702. The study demonstrated that the complete removal of glycans could be successfully accomplished within a concise 16 hour. Notably, this achievement holds true even when varying the ratios of mAb to enzyme, with the examined 1000:1 (w / w) ratio showing effectiveness with around 96 %. Example 3. Preparation of NSCT-2 (Oxazoline-NSCT-N3)

[0080] NSCT-1 (235 mg, 0.097 mmol; purchased from Glytech, Inc. Cat. No. GT- 25261; HPLC purity > 90%) and triethylamine (605 µL, 0.44 mmol) were dissolved in water (10 mL) and cooled to 0oC.2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride aqueous solution (1 M, 1.44 mL) was added slowly and the resulting mixture was stirred at 0 to 5oC for four hours. NaOH solution was added (0.01 M, 1 mL) and the resulting mixture was concentrated under reduced pressure. After most of the triethylamine was evaporated, the residual mixture was purified by Sephadex®g-15 column. Using 0.01 M NaOH as eluent to stabilize NSCT-2. Fractions with desired product were combined and freeze-dried to afford NSCT-2 (170 mg) as a white solid.1H NMR (D2O): δ 6.10 (d, J=7.26 Hz, 1H, H1 of oxazoline), 5.24 (s, 1H, H1 of GluNAc), 4.97 (s, 1H, H1 of GluNAc), 4.76 (s, 1H, H1 of Man), 4.64-4.60 (m, 2H, H1 of two Gal), 4.46 (s, 1H, H1 of Neu5Ac), 4.45 (s, 1H, H1 of Neu5Ac), 4.40 (s, 1H, H3 of β-form Man), 4.18 (d, J=21 Hz, 4H, H2 of two Man and two GluNAc), 3.99-3.48 (m), 2.71 (dd, J1=12.9 Hz, J2=4.2 Hz, 2H, H3eq of two Neu5Ac), 2.15-2.02 (m, 15H), 1.58 (dd, J1=J2=12.2 Hz, 2H, H3ax of two Neu5Ac).PATENT Attorney Docket No.: G3004-02000PCTExample 4. Tansglycosylation of biantennary glycan to mAb-GlcNAc(Fuc) by EndoSc mutants

[0081] R4702-GlcNAc(Fuc) monoclonal antibody (10 mg / mL) was incubated with NSCT-2 and EndoSc to generate R4702-(NSCT)2 / R4702-(NSCT-di-N3)2. The transglycosylation activity studies with 15 eq NSCT-2 by using EndoSc D233Q, D233Q / E350N, D233Q / E350Q, D233Q / Q303A / E350N, D233Q / Q303A / E350Q. The reaction was performed at 15 C for 3.5 hours, 50 mM MOPS, pH 6.7 buffer and enzyme : R4702- GlcNAc(Fuc) = 1 : 60 (w / w). The reactions were stopped by 70 °C for 10 minutes and analyzed by SDS-PAGE. The percentage of transglycosylation was calculated by integration of SDS- PAGE with ImageJ program.

[0082] Figure 3 showed the investigation of the wild type, mutations at single residue, mutations at two residues and mutations at three residues of EndoSc transglycosylation activities on R4702-GlcNAc(Fuc) with 15 equivalents of NSCT-2. The result indicated that EndoSc D233Q / E350N and EndoSc D233Q / E350Q mutants were the most potential enzyme for transglycosylation reaction. However, EndoSc-D233Q / Q303A / E350N and EndoSc- D233Q / Q303A / E350Q mutants only had very low transglycosylation activities.

[0083] Figure 4 showed the reaction time investigation of EndoSc-D233Q / E350Q transglycosylation activity on R4702-GlcNAc(Fuc). The data showed the transglycosylation percentage of 15eq NSCT-2 maintained comparable amount (> 90 %) during the time frame from 4 to 10 hours, which indicated the stability property of R4702-(NSCT-di-N3)2. The reactions were performed at 15 °C, 50mM MOPS, pH 6.7 and EndoSc-D233Q / E350Q : R4702- GlcNAc(Fuc)=1 : 60 (w / w).

[0084] Figure 5 showed the pH investigation and buffer screening of EndoSc- D233Q / E350Q transglycosylation activity on R4702-GlcNAc(Fuc) with 15 eq NSCT-2. The reactions were performed at 15 °C for 5 hours and EndoSc-D233Q / E350Q : R4702-PATENT Attorney Docket No.: G3004-02000PCT GlcNAc(Fuc)=1 : 60 (w / w). The results clearly showed EndoSc D233Q / E350Q have the best transglycosylation activity with NSCT-2 and 50 mM MOPS, pH 6.7 buffer. The results demonstrated that the EndoSc-D233Q / E350Q had transglycosylaiton activities for mAb with artificial compounds (NSCT-2) at the pH range between 6.5 to 7.2 with 50 mM MOPS, NaPi, PIPES and Tris buffers.

[0085] Previous reference indicated that some mutant sites, such as EndoS-D233Q and EndoS2-D184M can increase transglycosylation activity for glycan (9) (20). According to the multiple sequence alignment results among EndoS, EndoS2 and EndoSc enzymes (Figure 6), the equivalent positions T182, D233, S280, Q303, E350 and D405 of EndoSc were selected as targets for site-directed mutagenesis investigation. These sites were replaced with different types of amino acids including the characteristics of positive / negative charge, polar and hydrophobic. The transglycosylation activity studies with 15 eq NSCT-2 by using diverse EndoSc mutants were investigated in Figure 7. The reactions were performed at 15 °C for 4 hours, 50 mM MOPS pH 6.7 and EndoSc mutant : R4702-GlcNAc(Fuc)=1 : 60 (w / w). Example 5. Purification of mAb-(NSCT-di-N3)2

[0086] Transglycosylation reaction mixture samples were loaded in prepacked Cytiva HiTrap MabSelect PrismA 5ml column which pre-equilibrated with 1x PBS, pH 7.4 buffer. Non-bound contaminations were washed by three steps pH gradient: 1x PBS, pH 7.4 buffer, 100 mM Sodium Citrate, pH 6.0 buffer and 100 mM Sodium Citrate, pH 5.5 buffer, each step with six times of column volume.50 mM Sodium Citrate, 150 mM NaCl, pH 3.0 buffer was employed to elute bound antibody. The eluted fractions were immediately neutralized to pH 7.4 using 1M Tris, pH 9.0 buffer. Eluted samples were concentrated by 30kDa PES centrifuge filter and exchanged to the final buffer 50 mM MOPS, pH 6.7. All mAb-(NSCT-di-N3)2samples were stored at -80 °C. Example 6. Transglycosylation activity on various antibodies

[0087] Homogeneous platform is a potential process to generate homogeneous mAbs with linker-payloads. Several mAbs were selected for the conjugation investigation with EndoSc D233Q / E350Q, including R4702 Ab, A904 Ab, Adalimumab, Rituximab, Trastuzumab, Panitumumab and Pertuzumab. Reaction condition of deglycosylation was EndoSc-WT : mAb=1 : 1000 (w / w) and EndoH : mAb=1 : 5000 (w / w) for 16 hours at 37 °C with 50 mM MOPS, PH 6.7 buffer. On the other hand, the transglycosylation activity studies with 15 eq NSCT-2 by using EndoSc D233Q / E350Q. The reaction was performed at 15 °C forPATENT Attorney Docket No.: G3004-02000PCT 3.5 hours, 50 mM MOPS, pH 6.7 buffer and EndoSc D233Q / E350Q : mAb-GlcNAc(Fuc)=1 : 60 (w / w). Representative SDS-PAGE results are shown in Figure 8A.

[0088] Reduced capillary electrophoresis was used to determine the efficiency of deglycosylation for EndoSc WT and transglycosylation for EndoSc D233Q / E350Q for antibody applications. 20-30 μg of ADC was taken for this analysis and diluted with PBS Sample Buffer. ADC was denatured by treatment with sodium dodecyl sulphate (SDS), and 2- mercaptoethanol to break the disulfide bonds and enable unfolding for separation of proteins. Sample was incubated at 65°C for 10 min and then transferred to vials for CE-SDS analysis. The CE-SDS analysis was performed by PA800 plus, Sciex, with 30 cm bare fused-silica capillary under 15kV high voltage. The detection wavelength was 220 nm.

[0089] The N-glycan of Mab was cleaved by EndoSc and one GlcNAc was left on Asn to form Mab-GlcNAc. To evaluate the deglycosylation ratio, Mab-GlcNAc(Fuc) was detected by reduced CE. Heavy chain (HC) with GlcNAc(Fuc) (HC-GlcNAc(Fuc) was separated with original HC. The deglycosylation efficiency was higher than 99.5%. HC-GlcNAc(Fuc) was the major form in Mab-GlcNac (Figure 8B). Heavy chain (HC) conjugated with NSCT-diN3 was formed after transglycosylation of Mab-GlcNAc(Fuc) by EndoSc D233Q / E350Q (Figure 8C). The efficiency of transglycosylation of EndoSc D233Q / E350Q is evaluated by the percentage of HC-NSCT-diN3which was higher than 90.0%. Overlay electropherogram results of R4702, R4702-GlcNAc(Fuc) and R4702-(NSCT-diN3) are shown in Figure 8D. More applications for different antibodies by EndoSc D233Q / E350Q are shown in Figure 9B. These findings evaluate that the EndoSc D233Q / E350Q highly and effectively facilitated the specific transfer of glycans on antibody. Example 7. Click reaction of R4702 DAR4 ADC

[0090] N-PM-0022 intermediate (OBI Pharma, Inc.) was dissolved in DMSO to form 20 mg / mL stock solution. Purified mAb-(NSCT-di-N3)2sample was mixed with N-PM-0022. Reaction condition was stirring for 18 hours at 25 °C. After reaction, the sample buffer would be exchanged with 20 mM sodium acetate, pH 5.0 buffer by using 30 kDa PES hollow fiber.

[0091] Hydrophobic interaction chromatography (HIC) is currently considered the reference technique for the analysis of ADCs to determine drug load distribution (DLD) and average drug antibody ratio (DAR) for antibodies. TOSOH TSKgel Butyl-NPR column (2.5 µm, 4.6 mm ID × 3.5 cm L) was applied. ADC was eluted with 1.5 M Ammonium sulfate and 50 mM sodium phosphate pH 7.0 in 15% 2-propanol gradient at a flow rate of 0.8 mL / min. The chromatograms were detected at 280 nm.PATENT Attorney Docket No.: G3004-02000PCT

[0092] DAR0, DAR2, and DAR4 were well separated and detected by HIC. The major DAR species is DAR4 which was more than 90.0%. The DLD results were DAR0 1.01%, DAR2 6.45% and DAR4 92.54% for R4702 DAR4 ADC. HIC chromatography data were shown in Figure 9. Example 8. Cytotoxicity results of R4702 DAR4 ADC with BxPC-3 cell line

[0093] Cytotoxicity was determined using CellTiter-Glo ™ Luminescent assay reagent (Promega). BxPC-3 cell line are used in this study. The cells were plated in 96-well plate with 4000 cells per well and incubated at 37°C overnight. The following day, test samples were prepared from 30 to 0.005 nM with 3-fold serial dilution in cell culture medium and then added to the plates. After incubating at 37°C for 6 days, the CellTiter-Glo luciferase assay reagent was added to each well and mix well. Subsequently, the plate was incubated at an ambient temperature for 10 minutes to stabilize luminescent signal. The luminescence of each well was carried out on a microplate luminescence reader and cell viability was calculated to serve as in-vitro cytotoxicity.

[0094] Cytotoxicity data are shown in Figure 10. These results indicated that R4702 DAR4 ADC can induce significant cytotoxicity and its half-maximal inhibitory concentration (IC50) was around 0.24 nM. Example 9. Animal study results of R4702 DAR4 ADC with NCI-H1975 xenograft model

[0095] In the TROP2-ADCs synthesized by EndoSc-D233Q / E350Q enzyme efficacy study, which concluded on Day 22. All ADCs were administered as a single dose on Day 1. Tumor volume was monitored twice weekly on Days 0, 4, 7, 11, 14, 18 and 22. Individual and average tumor volumes are recorded, and tumor growth trends are visualized in Figure 11.3 mg / kg SG (Trodelvy)-treated group, one mouse was humanely sacrificed (HS) on Day 18 due to ulceration on the tumor surface. The results demonstrated a significant difference between 3mg / kg R4702 DAR4 ADC which is TROP2-ADCs synthesized with EndoSc-D233Q / E350Q enzyme and 3 mg / kg SG (Trodelvy)-treated group, which showed no anti-tumor activity compared with vehicle control. Throughout the entire study duration, there was no significant body weight loss in all mice. Example 10. Analysis of hydrolase activity of EndoSc and the mutants

[0096] The hydrolase activity of EndoSc and the mutants was analyzed by TLC. Sialylglycopeptide (SGP) was incubated with wild type EndoSc and the S280A mutant in a glycobuffer (5 mM CaCl2, 50 mM Sodium acetate, pH 5.5) at 37 °C for four hours. The reaction samples were spotted onto a 5 cm long TLC Silica gel 60 plate (Cat. No.1055540001, Merck) and separated using 100% isopropanol / 1M ammonium acetate (3:2, v / v) as the solvent.PATENT Attorney Docket No.: G3004-02000PCT The plate was dried and stained by Ceric Ammonium Molybdate Solution (contains H2SO4) (Cat, No. C1794, Tokyo Chemical Industry). The colored spots on the plate were detected using LAS-4000 mini (Fujifilm).

[0097] Figure 12 indicated the wild type of EndoSc was able to hydrolyze the SGP at the hydrolysis ratio (enzyme:SGP) 1:40 after four hours treatment. Futhermore, the EndoSc S280A mutant showed the better hydrolytic activity of SGP (enzyme:SGP=1:80) than wild type.

[0098] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of this invention. Although any compositions, methods, kits, and means for communicating information similar or equivalent to those described herein can be used to practice this invention, the preferred compositions, methods, kits, and means for communicating information are described herein.

[0099] All references cited herein are incorporated herein by reference to the full extent allowed by law. The discussion of those references is intended merely to summarize the assertions made by their authors. No admission is made that any reference (or a portion of any reference) is relevant prior art. Applicants reserve the right to challenge the accuracy and pertinence of any cited reference.PATENT Attorney Docket No.: G3004-02000PCT SEQUENCE LISTING SEQ ID NO 1: Name: Amino acids sequence of EndoSc-WT enzyme (total 1,065 amino acids) Organism: Streptococcus canis (WP_093998831.1) Note: wild type (no mutation) MDKGLLVKRTLGCVCAATVMGAILATHHDSLNTVTAEEKTIQVQKELASIDSLHYLSENSKK EFKEELSKAGQASQKVKEILAKAQQADKQAQALAEMKIPEKIPMKPLHGPLYGGYFRSWHDK TSDPSEKDKVNSMGELPKEVDLAFVFHDWTKDYSLFWKELATKHVPKLNKQGTRVIRTIPWR FLAGGDNSGIAEDASKYPNTPEGNKALAKAIVDEYVYKYNLDGLDVDIERDSIPKVNGEVSD ENLKRSIHVFEEIGKLIGPKGADKSRLFIMDSTYMADKNPLIERGAPYIDLLLVQVYGARGE QGEFQNDTKLVTETPEERWQGYSKYIRPEQYMIGFSFYEERAGSGNLWYDINTRKDEDTANG INTDIAGTRAERYARWQPKTGGVKGGIFSYAVDRDGVAHQPEKVAQQDKRSQMQVDEITDNI FHSDYSVSKALKQVMLKDKSYDLIDEKDFPDKALREAVIAQVGTRKGDLERFNGTLRLDNPA IQSLEGLNKFKKLSQLDLIGLSRITKLDRSVLPANMKSGKDTLETVLETYKKNSKEEPATIP PVSLTISGLTGLKELDLSGFDRETLAGLDAATLTSLEKVDISGNKLDLAPGTENRQIFDVMR STVSNHVGSNEQTVRFDKQKPTGHYPTTYSTTSLRLPVAEGNIDLQSRLLFGTVTNQGTLIN SEADYKAYQNQKIAGHNFVDPDYHYNNFKVSYDNYTLTVTDSTLGTTTDKRLATDKEETYNV DFFSPADKTKAVHTAKVIVGDEKTMMVNLAEGATVIGGSADPVNARKVFDSEIQSNLLTFDN QASIIFEIKDPSLAKYWRLFNDSSKGKDDYIKEAKLEVFTGQLNAEADVKTSLEKSGDWVTV STYSGEEKIYSHSLDNISAKYWRVTVDTKGGNYSWPSLPELQILGYPLPNADAIMKTVTAAK ELSQQKDKFPQQVLGELTAKEAVVEASLNSKLFDTAVINTNVEALKNVVDECLAYDKNKETA FKVTEDYQAAVNRIKAERVTVEEMAQFKDLTEKATSLNSKIEAKLFDRGYDEDLMGLVDKLT HITEALKLFAKPATENT Attorney Docket No.: G3004-02000PCT SEQ ID NO 2: Name: Amino acids sequence of EndoSc-D233Q / E350Q enzyme (total 1,065 amino acids) Organism: Streptococcus canis (WP_093998831.1) Note: two amino acids mutation (D233Q and E350Q) MDKGLLVKRTLGCVCAATVMGAILATHHDSLNTVTAEEKTIQVQKELASIDSLHYLSENSKK EFKEELSKAGQASQKVKEILAKAQQADKQAQALAEMKIPEKIPMKPLHGPLYGGYFRSWHDK TSDPSEKDKVNSMGELPKEVDLAFVFHDWTKDYSLFWKELATKHVPKLNKQGTRVIRTIPWR FLAGGDNSGIAEDASKYPNTPEGNKALAKAIVDEYVYKYNLDGLDVQIERDSIPKVNGEVSD ENLKRSIHVFEEIGKLIGPKGADKSRLFIMDSTYMADKNPLIERGAPYIDLLLVQVYGARGE QGEFQNDTKLVTETPEERWQGYSKYIRPEQYMIGFSFYEQRAGSGNLWYDINTRKDEDTANG INTDIAGTRAERYARWQPKTGGVKGGIFSYAVDRDGVAHQPEKVAQQDKRSQMQVDEITDNI FHSDYSVSKALKQVMLKDKSYDLIDEKDFPDKALREAVIAQVGTRKGDLERFNGTLRLDNPA IQSLEGLNKFKKLSQLDLIGLSRITKLDRSVLPANMKSGKDTLETVLETYKKNSKEEPATIP PVSLTISGLTGLKELDLSGFDRETLAGLDAATLTSLEKVDISGNKLDLAPGTENRQIFDVMR STVSNHVGSNEQTVRFDKQKPTGHYPTTYSTTSLRLPVAEGNIDLQSRLLFGTVTNQGTLIN SEADYKAYQNQKIAGHNFVDPDYHYNNFKVSYDNYTLTVTDSTLGTTTDKRLATDKEETYNV DFFSPADKTKAVHTAKVIVGDEKTMMVNLAEGATVIGGSADPVNARKVFDSEIQSNLLTFDN QASIIFEIKDPSLAKYWRLFNDSSKGKDDYIKEAKLEVFTGQLNAEADVKTSLEKSGDWVTV STYSGEEKIYSHSLDNISAKYWRVTVDTKGGNYSWPSLPELQILGYPLPNADAIMKTVTAAK ELSQQKDKFPQQVLGELTAKEAVVEASLNSKLFDTAVINTNVEALKNVVDECLAYDKNKETA FKVTEDYQAAVNRIKAERVTVEEMAQFKDLTEKATSLNSKIEAKLFDRGYDEDLMGLVDKLT HITEALKLFAKPATENT Attorney Docket No.: G3004-02000PCT SEQ ID NO 3: Name: Amino acids sequence of EndoSc-D233Q / E350N enzyme (total 1,065 amino acids) Organism: Streptococcus canis (WP_093998831.1) Note: two amino acids mutation (D233Q and E350N) MDKGLLVKRTLGCVCAATVMGAILATHHDSLNTVTAEEKTIQVQKELASIDSLHYLSENSKK EFKEELSKAGQASQKVKEILAKAQQADKQAQALAEMKIPEKIPMKPLHGPLYGGYFRSWHDK TSDPSEKDKVNSMGELPKEVDLAFVFHDWTKDYSLFWKELATKHVPKLNKQGTRVIRTIPWR FLAGGDNSGIAEDASKYPNTPEGNKALAKAIVDEYVYKYNLDGLDVQIERDSIPKVNGEVSD ENLKRSIHVFEEIGKLIGPKGADKSRLFIMDSTYMADKNPLIERGAPYIDLLLVQVYGARGE QGEFQNDTKLVTETPEERWQGYSKYIRPEQYMIGFSFYENRAGSGNLWYDINTRKDEDTANG INTDIAGTRAERYARWQPKTGGVKGGIFSYAVDRDGVAHQPEKVAQQDKRSQMQVDEITDNI FHSDYSVSKALKQVMLKDKSYDLIDEKDFPDKALREAVIAQVGTRKGDLERFNGTLRLDNPA IQSLEGLNKFKKLSQLDLIGLSRITKLDRSVLPANMKSGKDTLETVLETYKKNSKEEPATIP PVSLTISGLTGLKELDLSGFDRETLAGLDAATLTSLEKVDISGNKLDLAPGTENRQIFDVMR STVSNHVGSNEQTVRFDKQKPTGHYPTTYSTTSLRLPVAEGNIDLQSRLLFGTVTNQGTLIN SEADYKAYQNQKIAGHNFVDPDYHYNNFKVSYDNYTLTVTDSTLGTTTDKRLATDKEETYNV DFFSPADKTKAVHTAKVIVGDEKTMMVNLAEGATVIGGSADPVNARKVFDSEIQSNLLTFDN QASIIFEIKDPSLAKYWRLFNDSSKGKDDYIKEAKLEVFTGQLNAEADVKTSLEKSGDWVTV STYSGEEKIYSHSLDNISAKYWRVTVDTKGGNYSWPSLPELQILGYPLPNADAIMKTVTAAK ELSQQKDKFPQQVLGELTAKEAVVEASLNSKLFDTAVINTNVEALKNVVDECLAYDKNKETA FKVTEDYQAAVNRIKAERVTVEEMAQFKDLTEKATSLNSKIEAKLFDRGYDEDLMGLVDKLT HITEALKLFAKPATENT Attorney Docket No.: G3004-02000PCT REFERENCES Adams, G. 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Claims

PATENT Attorney Docket No.: G3004-02000PCT CLAIMS What is claimed is:

1. A mutant of glycosynthase comprising: an amino acid sequence of at least 90% sequence homology to the amino acid sequence set forth in the sequence of SEQ ID NO.1, and one or more mutations.

2. The mutant of glycosynthase of claim 1, wherein the one or more mutations are located within residues 172-192, residues 223-243, residues 270-290, residues 293-313, residues 340-360, or residues 395-415 of SEQ ID NO.

1.

3. The mutant of glycosynthase of claim 1, wherein the one or more mutations comprise T182R, D233A, D233M, D233Q, D233S, Q303A, Q303E, Q303S, E350R, E350N, E350Q, D405Q, or a combination thereof.

4. The mutant of glycosynthase of claim 3, wherein the one or more mutations comprise D233Q, E350N, E350Q, or a combination thereof.

5. The mutant of glycosynthase of claim 4, wherein the one or more mutations comprise (1) D233Q and E350N or (2) D233Q and E350Q.

6. The mutant of glycosynthase of claim 5, comprising an amino acid sequence of SEQ ID NO.2 or SEQ ID NO.

3.

7. A method for preparing an engineered glycoprotein, comprises coupling an activated oligosaccharide to a glycoprotein acceptor using the glycosynthase mutant of any one of claims 1 to 6.

8. The method of claim 7, wherein the activated oligosaccharide is a glycan oxazoline and comprises an N-glycan having a structure of the following formula:wherein R1is -H or N-acetyl glucosamine attached via a β-1,4 linkage, and R2and R3are same or different and are independently selected from the group consisting of:PATENT Attorney Docket No.: G3004-02000PCT.

9. The method of claim 7, wherein the glycoprotein acceptor is a glycopeptide, a glycoprotein, an antibody or an antigen-binding fragment thereof.

10. The method of claim 7, wherein the glycoprotein acceptor is a core fucosylated or non- fucosylated GlcNAC-IgG acceptor or an antigen-binding fragment thereof.

11. The method of claim 7, wherein the GlcNAC-IgG acceptor is derived from a monoclonal antibody or a bispecific antibody targeting HER2, Nectin-4, HER3, cMet, CD44, TROP2, CD20, TNF-α, or EGFR.

12. An engineered glycoprotein prepared by the method of any one of claims 7 to 11.

13. A method for preparing an engineered glycopeptide, comprises hydrolyzing a N-glycan of a glycopeptide using the glycosynthase mutant of any one of claims 1 to 6.

14. The method according to claim 13, wherein the glycopeptide is a sialylglycopeptide (SGP).

15. A glycan-engineering enzyme comprising an amino acid sequence of SEQ ID NO.2 or SEQ ID NO.

3.

16. The enzyme of claim 15, wherein the amino acid sequence is derived from Streptococcus canis.

17. An engineered bioconjugate, comprising: an engineered glycoprotein of claim 12 and a drug moiety.

18. The engineered bioconjugate of claim 17, wherein the engineered glycoprotein further comprises a triazole moiety, a DBCO-derived moiety, a BCN-derived moiety, or aPATENT Attorney Docket No.: G3004-02000PCT maleimide-derived moiety, which connects the activated oligosaccharide to the glycoprotein acceptor.

19. The engineered bioconjugate of claim 18, wherein the activated oligosaccharide is coupled with the glycoprotein acceptor at N297 site of a Fc region thereof.

20. The engineered bioconjugate of claim 17, wherein the drug moiety comprises a topoisomerase I inhibitor or a topoisomerase II inhibitor.

21. The engineered bioconjugate of claim 20, wherein the topoisomerase I inhibitor is selected from a group consisting of irinotecan, topotecan, camptothecin, rubitecan, MLN576, exatecan, belotecan, seconeolitsine, SN-38, Genz-644282, betulinic acid, β- lapachone, karenitecin, gimatecan, namitecan, edotecarin, SW044248, LMP744, T- 2513, podocarpusflavone A, indimitecan, lurtotecan, TP3011, and 10- hydroxycamptothecin.

22. A pharmaceutical composition comprising the engineered bioconjugate of any one of claims 17 to 21 and a pharmaceutically acceptable carrier.

23. A method for treating cancer, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 22.

24. The method of claim 23, wherein the subject is a human.

25. The method of claim 23, wherein the cancer is selected from the group consisting of sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head-and-neck cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervix cancer, endometrial cancer, ovarian cancer, testicular cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer, prostate cancer, thyroid cancer, and oral cancer.

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