Novel endo-beta-n-acetylglucosaminidases and use thereof

Abstract

Provided are two novel endo-beta-N-acetylglucosaminidases, as well as a method for using the two with a disaccharide linker to prepare a non-natural sugar-engineered antibody, or a fixed-point quantitative antibody drug conjugate based on glycosyl modification, and a use.

Description

Novel beta-N-acetylglucosaminyl endoenzyme and its use

[0001] This application claims priority to the Chinese patent application filed with the Patent Office of China on January 10, 2024, with application number CN2024100352818 and invention name “Novel beta-N-acetylglucosaminyl endoenzyme and its use”, and the Chinese patent application filed with the Patent Office of China on January 3, 2025, with application number CN2025100132263 and invention name “Novel beta-N-acetylglucosaminyl endoenzyme and its use”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present disclosure relates to the fields of medicinal chemistry and biotechnology drugs, and in particular to two novel β-N-acetylglucosaminidases and novel uses thereof. Background Art

[0003] Antibodies are a type of glycoprotein in the immunoglobulin superfamily. Currently, antibody drugs are widely used in the treatment of multiple diseases. Natural IgG antibodies contain two light chains and two heavy chains, which are interconnected by disulfide bonds. Antibodies have a conserved Fc region, and there is a natural and conserved N-glycosylation site (N297) at position 297 of this domain. Generally, antibody drugs used for treatment are obtained through recombinant expression. Monoclonal antibodies produced by recombinant expression using animal-derived cells as hosts (such as CHO cells) usually have a biantennary complex sugar chain modification. Glycosylation modification of antibodies can have a significant effect on the effector activity of antibodies (including antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity).

[0004] In addition to its naturally occurring biological functions, this conserved modification site has been exploited to develop a variety of technologies for producing antibody-based drug conjugates. Antibody-based drug conjugates are a class of biopharmaceuticals that utilize antibodies as targeting carriers to carry effector payloads, thereby enriching the effector payload in the targeted region. These payloads can include cytotoxins, radioisotopes, oligonucleotides, immunomodulators, peptides, or protein fragments.

[0005] Antibody-Drug Conjugates (ADCs) are a class of biopharmaceuticals composed of antibodies, cytotoxins, and linkers. ADCs combine the targeting capabilities of antibodies with the killing ability of cytotoxins, controlling the release of cytotoxins through linkers to achieve the purpose of targeted killing of tumor cells. Currently marketed ADCs mainly rely on naturally occurring lysine (such as Trastuzumab emtansine) or cysteine ​​(such as Enfortumab vedotin) in antibodies. This type of ADC produced using random conjugation of natural amino acids has poor uniformity and poor stability of the conjugation sites, resulting in poor drug safety and a narrow therapeutic window. Currently, there are a variety of methods that can be used to prepare site-specific ADCs, including exogenous cysteine ​​insertion technology, non-natural amino acid insertion technology, enzyme-catalyzed conjugation technology, and sugar-specific site conjugation technology.

[0006] Beta-N-acetylglucosaminyl endoglycosidase (ENGase) is a type of endoglycosidase with hydrolytic activity that can hydrolyze sugar chains on antibodies in vitro. On this basis, Laixi Wang et al. used mutants of ENGase (such as Endo S D233Q, Endo S2 D184M) to transfer natural or biantennary oxazoline substrates with bioorthogonal reaction groups to the N-sugar terminus, thereby realizing in vitro glycoengineering of antibodies. The subsequent use of the introduced bioorthogonal reaction groups can realize the preparation of antibody-based conjugated drugs. In addition, Huang Wei's team and Laixi Wang's team respectively reported the use of ENGase to prepare glycoengineered antibodies and ADCs using disaccharide linkers as substrates.

[0007] The ENGase family comprises multiple members, among which Endo S and Endo S2 possess both hydrolytic and disaccharide linker transfer activities, but their broader substrate specificity remains unexplored. Endo Si exhibits significant hydrolytic activity, and its mutants possess transfer activity toward biantennary oxazoline substrates. However, there are no reports demonstrating whether Endo Si possesses disaccharide linker transfer activity. Therefore, the disaccharide linker substrate specificity of ENGase family members remains to be explored, and the development of more potent ENGases remains an urgent need for industrial production.

[0008] SUMMARY OF THE INVENTION

[0009] Based on the development of a superior beta-N-acetylglucosaminyl endonuclease, the present invention has discovered and tested two beta-N-acetylglucosaminyl endonucleases with broader substrate specificity and better transfer efficiency. Through efficient enzyme-catalyzed reactions, it is possible to achieve site-specific introduction of disaccharide linkers, thereby realizing glycoengineering of antibody molecules. The bioorthogonal reactive groups introduced by the disaccharide linkers can achieve efficient preparation of antibody-based conjugated drugs, which are simple to operate and easy to industrialize. The resulting antibody-based conjugated drugs have good drugability and can be used to treat tumors, inflammation, infectious diseases or other immune diseases.

[0010] The present disclosure discloses a novel endo-beta-N-acetylglucosaminylase from Streptococcus equi subsp. zooepidemicus Sz105, named Endo Se2. The enzyme has the amino acid sequence set forth in SEQ ID NO:1, wherein positions 1-36 are a signal peptide and positions 37-1011 are the full-length sequence of the mature enzyme. Surprisingly, the enzyme exhibits excellent sugar chain hydrolysis and / or sugar chain transfer activity.

[0011] Furthermore, the present disclosure discloses another endo-beta-N-acetylglucosaminidase, Endo Si, from Streptococcus iniae, whose amino acid sequence is shown in SEQ ID NO: 2, wherein positions 1-33 are a signal peptide and positions 34-928 are the full-length sequence of the mature enzyme. Surprisingly, this enzyme also exhibits excellent sugar chain hydrolysis and / or sugar chain transfer activity, and has significant disaccharide linker transfer activity.

[0012] Based on the discovery of the above two enzymes, the present disclosure provides a method for remodeling the sugar chains of a polypeptide or protein, comprising the following steps:

[0013] a) Introducing Endo Se2 or Endo Si;

[0014] b) introducing a polypeptide or protein containing at least one N-acetylglucosamine or core-fucosylated N-acetylglucosamine;

[0015] c) providing a disaccharide linker or a disaccharide conjugate; and

[0016] d) using the Endo Se2 or Endo Si to transfer the disaccharide linker or disaccharide conjugate to the polypeptide or protein in step (b) to provide a new polypeptide or protein with modified sugar chains.

[0017] In some embodiments, the polypeptide or protein is an antibody or a protein comprising the Fc region of an antibody.

[0018] In some embodiments, the polypeptide or protein containing at least one N-acetylglucosamine or core-fucosylated N-acetylglucosamine is obtained by hydrolyzing a polypeptide or protein containing N-glycans with an endoglycosidase having beta-N-acetylglucosamine endoside activity, or by recombinant expression, or by chemical synthesis.

[0019] In some embodiments, the N-glycans are natural or non-natural complex, high mannose, or hybrid N-glycans.

[0020] In some embodiments, the endoglycosidase having endo-beta-N-acetylglucosaminidase activity includes Endo Se2, Endo Si, Endo S, Endo S2, Endo F3, and mutants thereof.

[0021] In some embodiments, polypeptides or proteins containing N-glycans can be remodeled in one step by introducing Endo Se2 or Endo Si without purification.

[0022] In some embodiments, polypeptides or proteins containing N-glycans can be remodeled in one step by simultaneously introducing Endo Se2 or Endo Si and one or more other endoglycosidases without purification.

[0023] In some embodiments, the disaccharide linker comprises at least one monosaccharide that has been oxazolined or thiazolined, or modified / engineered with a bioorthogonal functional group.

[0024] In some embodiments, the disaccharide linker is selected from the following structures:

[0025] In some embodiments, the disaccharide conjugate comprises, in addition to an oxazoline or thiazoline monosaccharide, a toxin, a small molecule drug, a near-infrared or fluorescent probe, a polypeptide, RNA and related drugs, a radioisotope label, a contrast agent, and a magnetic resonance imaging agent, and optionally, a cleavable linker, a non-cleavable linker, or a combination thereof. The small molecule drug is preferably selected from maytansine, DM-1, DM-4, ​​MMAE, MMAF, Auristatin 0101, SN-38, Dxd, exitecan, duocarmycin, amanitin, PBDs, VP-16, camptothecin, paclitaxel, docetaxel, anthracyclines, and derivatives of the above compounds, or the small molecule drug is a radioactive therapeutic.

[0026] In some embodiments, the disaccharide conjugate is selected from the following structures:

[0027] The present disclosure also provides the use of Endo Se2 or Endo Si for transferring disaccharide linkers or disaccharide conjugates in glycosylation engineering.

[0028] The present disclosure discloses that homogenized sugar chain-modified antibodies can be prepared through the above-mentioned glycoengineering method. Compared with existing technologies, Endo Se2 or Endo Si demonstrates better transfer efficiency and broader substrate specificity. The discovery and application of Endo Se2 and Endo Si provide higher-quality available tool enzymes for the structural development of disaccharide linkers, and also provide better options for the industrial production of glycoengineered antibodies based on disaccharide linkers and the antibody-based conjugate drugs derived therefrom. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 shows the transglycosylation activity assay of compound G0 using different endoglycosidases.

[0030] Figure 2 shows the transglycosylation activity assay of compound G1 using different endoglycosidases.

[0031] FIG3 shows the transglycosylation activity assay of compound G2 using different endoglycosidases.

[0032] Figure 4 shows the efficacy of low-dose sugar-site-conjugated ADC in the NCI-N87 xenograft tumor model.

[0033] Figure 5 shows the efficacy of high-dose sugar-specifically conjugated ADC in the NCI-N87 xenograft tumor model. DETAILED DESCRIPTION

[0034] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0035] Before describing the present disclosure in detail below, it should be understood that the present disclosure is not limited to the specific methodologies, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs.

[0036] Certain embodiments disclosed herein include numerical ranges, and certain aspects of the present disclosure may be described in terms of ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions in terms of ranges are intended for simplicity and convenience and should not be considered as strict limitations on the scope of the present disclosure. Therefore, descriptions in terms of ranges should be considered to specifically disclose all possible subranges and all possible specific numerical points within the range, as these subranges and numerical points have been clearly stated herein. Regardless of the width of the numerical value, the above principles apply equally. When describing in terms of ranges, the range includes the endpoints of the range.

[0037] In the present disclosure, the term "antibody" includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primatized antibodies, human antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, etc., as long as they exhibit the desired biological activity. The term "immunoglobulin" can be used interchangeably with "antibody" herein. The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population.

[0038] The term "antibody fragment" comprises at least a portion of a complete antibody. As used herein, a "fragment" of an antibody molecule includes an "antigen-binding fragment" of an antibody, and the term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that specifically binds or reacts with a selected antigen or its antigenic epitope, or a fusion protein product further derived from this fragment, such as a single-chain antibody, an extracellular binding region in a chimeric antigen receptor, etc. Exemplary antibody fragments or antigen-binding fragments thereof include, but are not limited to, variable light chain fragments (VL), variable heavy chain fragments (VH), Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single domain antibodies, linear antibodies, single-chain antibodies (scFv), and bispecific antibodies or multispecific antibodies formed by antibody fragments.

[0039] The term "multispecific antibody" refers to the formation of a new antibody construct that binds to two or more different sites and / or targets by functionally linking an antibody or antibody fragment (e.g., chemical coupling, gene fusion, non-covalent binding or other methods) to one or more other binding molecules (including antibodies or antibody fragments or other molecules with binding ability). Therefore, a "bispecific antibody" (or "bispecific antigen-binding molecule" or "bi-antibody") specifically refers to an antibody construct that is specific for two different antigens and / or epitopes. Typically, a bispecific antibody or multispecific antibody includes at least two different antigen (or epitope) binding domains.

[0040] The term "sugar" refers to a polysaccharide or oxidized or unoxidized carbohydrate-containing molecule, including but not limited to a monosaccharide, disaccharide, trisaccharide, oligosaccharide, or polysaccharide. Sugar is also used herein to refer to the carbohydrate portion of a sugar-containing conjugate, such as a glycoprotein, glycolipid, glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide, or proteoglycan. The term "sugar chain" is used interchangeably with "sugar" herein.

[0041] As used in the present disclosure, "sugar chain linker" as an activated donor molecule for glycosyl coupling can be a synthetic sugar containing oxazoline or thiazoline, such as an oligosaccharide with an activated reducing end, preferably an oligosaccharide molecule with an oxazoline structure; it can also be a natural N-glycan oxazoline. The sugar chain linker can also be chemically modified, for example, by introducing functional groups through azidation, alkynylation, aldehyde formation, sulfhydration, etc. The term "disaccharide linker" is a sugar chain linker composed of at least two monosaccharide units, preferably two monosaccharide units are connected by a glycosidic bond or a thioether bond. The term "disaccharide conjugate" is based on the disaccharide linker and further includes a payload such as a cytotoxin, and optionally also includes a linker between the disaccharide linker and the payload.

[0042] Core-fucosylated and non-fucosylated glycoproteins are important classes of molecules that play key roles in many biological events, such as tumor metastasis, cell adhesion, pathogen infection, and immune responses. Natural and recombinant fucosylated and non-fucosylated glycoproteins are typically produced as mixtures of glycoforms that differ only in the structure of the side-chain oligosaccharides.

[0043] The term "antibody-based drug conjugate" disclosed herein refers to all conjugates formed by covalently linking a polypeptide / protein targeting a specific cell to a payload. The polypeptide / protein targeting a specific cell can be an antibody or an antigen-binding fragment thereof, such as a monoclonal antibody, a bispecific antibody, or a polyclonal antibody; the payload can be a cytotoxin, a small molecule drug, a near-infrared or fluorescent probe, a polypeptide, RNA and related drugs, a radioisotope label, a contrast agent, and a magnetic resonance imaging agent; the covalent conjugate formed can be used for treatment or detection. The term "antibody-drug conjugate" disclosed herein refers to all conjugates formed by covalently linking a polypeptide / protein targeting a specific cell to a cytotoxin.

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

[0045] The "wild-type antibody" used in this disclosure typically refers to an antibody with an N-glycosylation site (N-glycan) that exists naturally or is produced after recombinant expression. For example, all antibodies with an N297 site in the Fc segment fall into the category of "wild-type antibodies", and the Fc segment is derived from IgG1, IgG2, IgG3 or IgG4.

[0046] The term "deglycosylated antibody" disclosed herein refers to an antibody containing one N-acetylglucosamine or core-fucosylated N-acetylglucosamine formed by a wild-type antibody under the action of glycoside hydrolase, or refers to an antibody containing one N-acetylglucosamine or core-fucosylated N-acetylglucosamine produced by direct recombinant expression in specific cells or by chemical synthesis.

[0047] The term "glycoengineered antibody" as used herein broadly refers to engineered antibodies that utilize in vitro glycan modification techniques to achieve uniform glycan modifications. Glycoengineered antibodies herein are obtained by transferring natural or non-natural glycan linkers to deglycosylated antibodies under the catalysis of endo-beta-N-acetylglucosaminidase. The glycan modifications of glycoengineered antibodies can be natural or non-natural, and may also carry bioorthogonal reactive groups.

[0048] The antibody used in the embodiments of the present disclosure is a wild-type antibody comprising an Fc segment, and the Fc segment sequence is, for example, as shown in SEQ ID NO: 5, such as Pertuzumab.

[0049] In this disclosure, the fillers, chromatographic columns, and instruments used for purification and property determination of antibodies, sugar chain-modified antibodies, and ADCs include: 5 mL cOmplete His-Tag Purification Column (Roche), HiLoad TM26 / 600Superdex TM 200 prep grade chromatography column (Cytive), AmMag TM Protein A Magnetic Beads (GenScript), SDA030 protein purification system (Sepure), Acquity I-Class / RDa (Waters) liquid chromatography-mass spectrometry, Arc Premier high-performance liquid chromatograph, ACCQUITY UPLC BEH PROTEIN C4 (Waters, 1.7 μm, 2.1 mm × 50 mm) column, TSKgel G3000SWXL (7.8 mm × 30 cm, 5 μm) SEC column, TSKgel Butyl-NPR (4.6 mm × 10 cm, 2.5 μm) HIC column.

[0050] In this disclosure, linker-payloads DBCO-GGFG-Dxd and TCO-PEG4-GGFG-Dxd were purchased from Shanghai Xiyao Pharmaceutical Technology Co., Ltd.; linker-payloads DBCO-PEG4-GGFG-Dxd, DBCO-PEG4-VC-PAB-MMAE, and N3-PEG4-VC-PAB-MMAE were purchased from MedChemExpress LLC. In this disclosure, trastuzumab was purchased from Sanyou Biopharmaceuticals (Shanghai) Co., Ltd. In this disclosure, sugar chain linkers were commissioned for synthesis at Wuhan Tangzhi Pharmaceutical Co., Ltd. Other compounds and reagents, unless otherwise specified, were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0051] General Preparation Example

[0052] General Operation 1: Preparation of Glycoengineered Antibodies

[0053] The wild-type antibody, the prepared disaccharide linker, and endoglycosidase (Endo Si or Endo Se2) were mixed to a concentration of 10 mg / mL, 2 mM (30 times the equivalent of the antibody concentration), and 0.4 mg / mL, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated at 25°C for 12 hours. The desired non-natural glycoengineered antibody was then purified with protein A.

[0054] General Operation 2: Preparation of Site-Specific ADCs Based on Azide-Modified Glycoengineered Antibodies

[0055] The prepared azide-modified glycoengineered antibody and cycloalkyne (e.g., DBCO) linker-payload were mixed to adjust the concentrations to 5 mg / mL and 0.33 mM, respectively. The pH of the reaction system was adjusted to 7.4 and incubated overnight at 25°C. After LC-MS confirmation of conversion to product, the desired sugar-specifically conjugated ADC was obtained by protein A purification or ultrafiltration.

[0056] General Operation 3: Preparation of Site-Specific ADCs Based on Cycloalkyne-Modified Glycoengineered Antibodies

[0057] The prepared cycloalkynyl-modified sugar-engineered antibody and azide (N3) linker-payload were mixed to adjust the concentrations to 5 mg / mL and 0.33 mM, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated at 25°C overnight. After LC-MS confirmation of conversion to product, the desired sugar-specifically conjugated ADC was obtained by protein A purification or ultrafiltration.

[0058] General Operation 4: Preparation of Site-Specific ADCs Based on Tetrazine-Modified Glycoengineered Antibodies

[0059] The prepared tetrazine-modified glycoengineered antibody and trans-cyclooctene (TCO)-type linker-payload were mixed to adjust the concentrations to 5 mg / mL and 0.33 mM, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated at 25°C overnight. After LC-MS confirmation of conversion to product, the desired sugar-specifically conjugated ADC was obtained by protein A purification or ultrafiltration.

[0060] General Operation 5: One-step preparation of sugar-specific site-conjugated ADC

[0061] The prepared loaded disaccharide linker (i.e., disaccharide conjugate), wild-type antibody, and endoglycosidase (Endo Si or Endo Se2) were mixed to adjust the concentrations to 0.4 mM, 5 mg / mL, and 0.4 mg / mL, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated at 25°C overnight. After LC-MS confirmation of conversion to product, the desired sugar-specifically conjugated ADC was obtained by protein A purification or ultrafiltration.

[0062] General Operation 6: Preparation of Dual-Loaded Site-Directed ADCs Based on Azide- and Tetrazine-Modified Glycoengineered Antibodies

[0063] The prepared azide- and tetrazine-modified glycoengineered antibodies, cycloalkyne (e.g., DBCO) linker-payload, and TCO linker-payload were mixed to adjust the concentrations to 5 mg / mL, 0.33 mM, and 0.33 mM, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated at 25°C overnight. After LC-MS confirmation of conversion to product, the desired sugar-specifically conjugated ADC was obtained by protein A purification or ultrafiltration.

[0064] General Operation 7: Preparation of Site-Specific ADCs Based on Linear Alkyne-Modified Glycoengineered Antibodies

[0065] Copper sulfate, tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), and sodium ascorbate were premixed to concentrations of 20mM, 100mM, and 150mM, respectively, for use as a catalyst. The prepared linear alkyne-modified glycoengineered antibody, the azide linker-payload, and the catalyst were mixed to concentrations of 5mg / mL, 0.5mM, 0.67mM (copper sulfate), 3.33mM (THPTA), and 5mM (sodium ascorbate), respectively. The reaction system was adjusted to pH 7.4 and incubated overnight at 25°C. After LC-MS confirmation of conversion to product, the desired sugar-specifically conjugated ADC was purified by protein A or ultrafiltration.

[0066] The following describes the specific process of preparing glycoengineered antibodies and sugar-specifically conjugated ADCs using the preparation method disclosed herein through specific examples.

[0067] Example 1 Recombinant expression and purification of enzyme

[0068] The following four enzyme coding genes were cloned into the pET22b vector (GenScript). The four enzymes are: Endo Se2 from Streptococcus equi subsp. zooepidemicus Sz105, whose amino acid sequence is 37-1011 of SEQ ID NO: 1; Endo Si from Streptococcus iniae, whose amino acid sequence is 34-928 of SEQ ID NO: 2; Endo S2 from Streptococcus pyogenes NZ131 (serotype M49), whose amino acid sequence is 38-843 of SEQ ID NO: 3; and Endo S from Streptococcus pyogenes, whose amino acid sequence is 37-995 of SEQ ID NO: 4.

[0069] Transform the plasmid containing the target gene into Escherichia coli BL21 (DE3), spread it on a 2×YT solid plate containing 100μg / mL ampicillin, and culture it at 37°C overnight. Pick a single colony and inoculate it into 4mL of 2×YT liquid medium containing 100μg / mL ampicillin and culture it overnight. Pipette 4mL of bacterial liquid into 1L of 2×YT broth medium containing 100μg / mL ampicillin and culture it at 37°C until the OD 600Then, 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the culture and cultured at 20°C to induce protein overexpression. After 16 hours, the cells were harvested by centrifugation. TM Bacterial Protein Extraction Reagent (Thermo) was used to lyse the cell pellet according to the manufacturer's instructions. The recombinant proteins of Endo S, Endo S2, Endo Si, and Endo Se2 were purified using a cOmplete His-Tag Purification Column (Roche) and an SDA030 protein purification system (Sepure). The proteins were concentrated using an Amicon centrifugal filter (30 kDa, Millipore) and further purified using a HiLoad TM 26 / 600Superdex TM Purification was performed by size exclusion using a 200prep grade column (Cytiva). Fractions containing Endo S, Endo S2, Endo Si, and Endo Se2 fusion proteins were concentrated using Amicon centrifugal filters (30 kDa, Millipore) and stored in storage buffer (20 mM PB, pH 7.5). Protein purity was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel imaging using a Gel Doc EZ Imager (Bio-RAD) and Image Lab scanning software. Protein concentration was quantified using a spectrophotometer (Nano-300).

[0070] Example 2 Study on the transfer activity of Endo Se2 and Endo Si on various disaccharide linkers

[0071] A wild-type antibody containing the Fc segment of SEQ ID NO: 5 was dissolved in 20 mM phosphate, 150 mM NaCl, pH 7.4 buffer to a final concentration of 10 mg / mL. Wild-type Endo S2 was added to a final concentration of 0.4 mg / mL for hydrolysis. The antibody was incubated overnight at 37°C and purified using Protein A magnetic beads to obtain a deglycosylated antibody containing one N-acetylglucosamine or core fucosylated N-acetylglucosamine.

[0072] The prepared deglycosylated antibody, disaccharide linker (i.e., compounds G0-G2), and endoglycosidase (i.e., Endo S or Endo S2 or Endo Si or Endo Se2) were prepared at concentrations of 10 mg / mL, 1.67 mM (25 times the equivalent of the antibody concentration), and 0.4 mg / mL, respectively. The pH of the reaction system was adjusted to 7.4, and the reaction temperature was 25°C. 4 μL of sample was removed every 0.5, 1, 2, 3, and 6 h, and the reaction was terminated by adding 36 μL of PBS solution containing 0.1% formic acid. The results were analyzed by reduction LC-MS, and are shown in Figures 1-3 and Table 1.

[0073] For compound G0, at 1 h, the glycosylation transfer efficiency of Endo Se2 and Endo Si was comparable, and significantly better than Endo S2 and Endo S; at 6 h, the glycosylation transfer efficiency of Endo Se2, Endo Si and Endo S2 was comparable, and significantly better than Endo S (Figure 1, Table 1).

[0074] For compound G1, at 1 hour, Endo Si showed better transglycosylation efficiency than Endo Se2 and significantly better than Endo S2. Endo S showed weaker transglycosylation efficiency for compound G1. At 6 hours, Endo Se2 and Endo Si showed comparable transglycosylation efficiency, and Endo S2 showed better transglycosylation efficiency (Figure 2, Table 1).

[0075] For compound G2, at 1 hour, the glycosylation transfer efficiency of Endo Si was comparable to that of Endo Se2, and significantly superior to that of Endo S2. Endo S had a weaker transfer efficiency for compound G2. At 6 hours, the glycosylation transfer efficiency of Endo Se2 was comparable to that of Endo Si, and superior to that of Endo S2 (Figure 3, Table 1).

[0076] Table 1 Transglycosidation activity of different endoglycosidases

[0077] Example 3 Study on the substrate specificity of Endo Se2, Endo Si and other endoglycosidases for various disaccharide linkers

[0078] Wild-type pertuzumab, a disaccharide linker, and a wild-type endoglycosidase (i.e., Endo S, Endo S2, Endo Si, or Endo Se2) were mixed to respective concentrations of 10 mg / mL, 1.67 mM (25 times the equivalent of the antibody concentration), and 0.4 mg / mL, respectively. The pH of the reaction system was adjusted to 7.4 and the reaction temperature was 25°C. After 6 hours, a 4 μL sample was removed and the reaction was terminated by adding 36 μL of PBS solution containing 0.1% formic acid. The sample was then analyzed by LC-MS.

[0079] The structures of various disaccharide linkers are shown below:

[0080] Table 2 Transglycosidation activity of different endoglycosidases

[0081] As can be seen from Table 2, Endo Se2, Endo Si, Endo S2, and Endo S exhibit different substrate specificities and transglycosylation efficiencies for different disaccharide linker compounds. Endo Se2 and Endo Si have broader substrate specificity than Endo S2 and Endo S, and the optimal endoglycosidase for each substrate is usually Endo Se2 or Endo Si.

[0082] Example 4 Preparation of glycoengineered antibodies based on wild-type antibodies

[0083] Preparation Example 1 Preparation of Glycoengineered Antibody Ab-G1

[0084] The non-natural glycoengineered antibody Ab-G1 was obtained by combining compound G1 and the wild-type antibody Pertuzumab via general procedure 1. The HRMS deconvolution value of the heavy chain of the glycoengineered antibody Ab-G1 was 49893.48 Da.

[0085] Preparation Example 2 Preparation of Glycoengineered Antibody Ab-G2

[0086] The non-natural glycoengineered antibody Ab-G2 was obtained by combining compound G2 and the wild-type antibody pertuzumab via general procedure 1. The HRMS deconvolution value of the heavy chain of the glycoengineered antibody Ab-G2 was 49902.46 Da.

[0087] Preparation Example 3 Preparation of Glycoengineered Antibody Ab-G4b

[0088] The non-natural glycoengineered antibody Ab-G4b was obtained by reacting compound G4b with the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G4b after HRMS deconvolution was 49789.06 Da.

[0089] Preparation Example 4 Preparation of Glycoengineered Antibody Ab-G7

[0090] The non-natural glycoengineered antibody Ab-G7 was obtained by combining compound G7 and the wild-type antibody Pertuzumab via general procedure 1. The HRMS deconvolution value of the heavy chain of the glycoengineered antibody Ab-G7 was 49810.12 Da.

[0091] Preparation Example 5 Preparation of Glycoengineered Antibody Ab-G9

[0092] The non-natural glycoengineered antibody Ab-G9 was obtained by combining compound G9 and wild-type antibody Pertuzumab via general procedure 1. The HRMS deconvolution value of the heavy chain of the glycoengineered antibody Ab-G9 was 50154.80 Da.

[0093] Preparation Example 6 Preparation of Glycoengineered Antibody Ab-G10

[0094] The non-natural glycoengineered antibody Ab-G10 was obtained by combining compound G10 and the wild-type antibody Pertuzumab via general procedure 1. The HRMS deconvolution value of the heavy chain of the glycoengineered antibody Ab-G10 was 50081.15 Da.

[0095] Preparation Example 7 Preparation of Glycoengineered Antibody Ab-G13

[0096] The non-natural glycoengineered antibody Ab-G13 was obtained by combining compound G13 and wild-type antibody Pertuzumab via general procedure 1. The HRMS deconvolution value of the heavy chain of the glycoengineered antibody Ab-G13 was 49807.70 Da.

[0097] Preparation Example 8 Preparation of Glycoengineered Antibody Ab-G20

[0098] The non-natural glycoengineered antibody Ab-G20 was obtained by reacting compound G20 and the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G20 after HRMS deconvolution was 49836.03 Da.

[0099] Preparation Example 9 Preparation of Glycoengineered Antibody Ab-G21

[0100] The non-natural glycoengineered antibody Ab-G21 was obtained by reacting compound G21 and the wild-type antibody pertuzumab via general procedure 1. The HRMS deconvolution value of the heavy chain of the glycoengineered antibody Ab-G21 was 49835.47 Da.

[0101] Preparation Example 10 Preparation of Glycoengineered Antibody Ab-G22

[0102] The non-natural glycoengineered antibody Ab-G22 was obtained by reacting compound G22 and the wild-type antibody pertuzumab via general procedure 1. The HRMS deconvolution value of the heavy chain of the glycoengineered antibody Ab-G22 was 49835.38 Da.

[0103] Preparation Example 11 Preparation of Glycoengineered Antibody Ab-G23

[0104] The non-natural glycoengineered antibody Ab-G23 was obtained by reacting compound G23 and the wild-type antibody Pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G23 after HRMS deconvolution was 49835.18 Da.

[0105] Preparation Example 12 Preparation of Glycoengineered Antibody Ab-G24

[0106] The non-natural glycoengineered antibody Ab-G24 was obtained by reacting compound G24 and the wild-type antibody Pertuzumab via General Procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G24 after HRMS deconvolution was 50064.00 Da.

[0107] Preparation Example 13 Preparation of Glycoengineered Antibody Ab-G25

[0108] The non-natural glycoengineered antibody Ab-G25 was obtained by reacting compound G25 with the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G25 after HRMS deconvolution was 49893.33 Da.

[0109] Preparation Example 14 Preparation of Glycoengineered Antibody Ab-G27

[0110] The unnatural glycoengineered antibody Ab-G27 was obtained by reacting compound G27 with the wild-type antibody Pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G27 after HRMS deconvolution was 50024.69 Da.

[0111] Preparation Example 15 Preparation of Glycoengineered Antibody Ab-G28b

[0112] The non-natural glycoengineered antibody Ab-G28b was obtained by reacting compound G28b with the wild-type antibody Pertuzumab via General Procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G28b after HRMS deconvolution was 50175.80 Da.

[0113] Preparation Example 16 Preparation of Glycoengineered Antibody Ab-G32

[0114] The non-natural glycoengineered antibody Ab-G32 was obtained by reacting compound G32 and the wild-type antibody Pertuzumab via General Procedure 1. The HRMS deconvolution value of the heavy chain of the glycoengineered antibody Ab-G32 was 49844.75 Da.

[0115] Preparation Example 17 Preparation of Glycoengineered Antibody Ab-G33

[0116] The non-natural glycoengineered antibody Ab-G33 was obtained by reacting compound G33 with the wild-type antibody Pertuzumab via General Procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G33 after HRMS deconvolution was 50047.05 Da.

[0117] Preparation Example 18 Preparation of Glycoengineered Antibody Ab-G35

[0118] The non-natural glycoengineered antibody Ab-G35 was obtained by reacting compound G35 with the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G35 after HRMS deconvolution was 49886.09 Da.

[0119] Preparation Example 19 Preparation of Glycoengineered Antibody Ab-G36

[0120] The unnatural glycoengineered antibody Ab-G36 was obtained by reacting compound G36 with the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G36 after HRMS deconvolution was 49891.14 Da.

[0121] Preparation Example 20 Preparation of Glycoengineered Antibody Ab-G37

[0122] The unnatural glycoengineered antibody Ab-G37 was obtained by reacting compound G37 with the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G37 after HRMS deconvolution was 49904.60 Da.

[0123] Preparation Example 21 Preparation of Glycoengineered Antibody Ab-G38

[0124] The non-natural glycoengineered antibody Ab-G38 was obtained by reacting compound G38 with the wild-type antibody Pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G38 after HRMS deconvolution was 50018.20 Da.

[0125] Preparation Example 22 Preparation of Glycoengineered Antibody Ab-G39

[0126] The non-natural glycoengineered antibody Ab-G39 was obtained by reacting compound G39 with the wild-type antibody Pertuzumab via General Procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G39 after HRMS deconvolution was 50032.24 Da.

[0127] Preparation Example 23 Preparation of Glycoengineered Antibody Ab-G40

[0128] The non-natural glycoengineered antibody Ab-G40 was obtained by reacting compound G40 and the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G40 after HRMS deconvolution was 49833.19 Da.

[0129] Preparation Example 24 Preparation of Glycoengineered Antibody Ab-G42

[0130] The non-natural glycoengineered antibody Ab-G42 was obtained by reacting compound G42 and the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G42 after HRMS deconvolution was 50046.26 Da.

[0131] Preparation Example 25 Preparation of Glycoengineered Antibody Ab-G44

[0132] The non-natural glycoengineered antibody Ab-G44 was obtained by reacting compound G44 with the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G44 after HRMS deconvolution was 50624.49 Da.

[0133] Preparation Example 26 Preparation of Glycoengineered Antibody Ab-G45

[0134] The non-natural glycoengineered antibody Ab-G45 was obtained by reacting compound G45 with the wild-type antibody Pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G45 after HRMS deconvolution was 50405.15 Da.

[0135] Preparation Example 27 Preparation of Glycoengineered Antibody Ab-G46

[0136] The unnatural glycoengineered antibody Ab-G46 was obtained by reacting compound G46 with the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G46 after HRMS deconvolution was 49865.77 Da.

[0137] Preparation Example 28 Preparation of Glycoengineered Antibody Ab-G47

[0138] The unnatural glycoengineered antibody Ab-G47 was obtained by reacting compound G47 with the wild-type antibody pertuzumab via general procedure 1. The HRMS deconvolution value of the heavy chain of the glycoengineered antibody Ab-G47 was 49866.18 Da.

[0139] Example 5 Preparation of Sugar-Directed Antibody-Drug Conjugates Based on Wild-Type Antibodies

[0140] Preparation Example 29 Preparation of Sugar-Directed ADC Ab-G1-DXd

[0141] Ab-G1-DXd is obtained by combining compound DBCO-GGFG-Dxd and non-natural glycoengineered antibody Ab-G1 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 51021.77 Da.

[0142] Preparation Example 30 Preparation of Sugar-Directed ADC Ab-G2-DXd

[0143] Ab-G2-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G2 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 52158.93 Da.

[0144] Preparation Example 31 Preparation of Sugar-Directed ADC Ab-G4b-MMAE

[0145] Ab-G4b-MMAE is obtained by combining compound N3-PEG4-VC-PAB-MMAE and non-natural glycoengineered antibody Ab-G4b through general operation seven. The measured value of the heavy chain after HRMS deconvolution is 51185.38 Da.

[0146] Preparation Example 32 Preparation of Sugar-Directed ADC Ab-G7-DXd

[0147] Ab-G7-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G7 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 50964.74 Da.

[0148] Preparation Example 33 Preparation of Sugar-Directed ADC Ab-G9-MMAE

[0149] Ab-G9-MMAE is obtained by combining compound N3-PEG4-VC-PAB-MMAE and non-natural glycoengineered antibody Ab-G9 through general operation three. The measured value of the heavy chain after HRMS deconvolution is 51551.58 Da.

[0150] Preparation Example 34 Preparation of Sugar-Directed ADC Ab-G10-MMAE

[0151] Ab-G10-MMAE is obtained by combining compound N3-PEG4-VC-PAB-MMAE and non-natural glycoengineered antibody Ab-G10 through general operation three. The measured value of the heavy chain after HRMS deconvolution is 51478.22 Da.

[0152] Preparation Example 35 Preparation of Sugar-Directed ADC Ab-G13-DXd

[0153] Ab-G13-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G13 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 50978.76 Da.

[0154] Preparation Example 36 Preparation of Sugar-Directed ADC Ab-G20-DXd

[0155] Ab-G20-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G20 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 50963.63 Da.

[0156] Preparation Example 37 Preparation of Sugar-Directed ADC Ab-G21-DXd

[0157] Ab-G21-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G21 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 50963.46 Da.

[0158] Preparation Example 38 Preparation of Sugar-Directed ADC Ab-G22-DXd

[0159] Ab-G22-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G22 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 52117.98 Da.

[0160] Preparation Example 39 Preparation of Sugar-Directed ADC Ab-G23-DXd

[0161] Ab-G23-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G23 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 52116.81 Da.

[0162] Preparation Example 40 Preparation of Sugar-Directed ADC Ab-G24-DXd

[0163] Ab-G24-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G24 through general procedure 2. The measured value of the heavy chain after HRMS deconvolution is 52320.37 Da.

[0164] Preparation Example 41 Preparation of Sugar-Directed ADC Ab-G25-DXd

[0165] Ab-G25-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G25 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 52174.43 Da.

[0166] Preparation Example 42 Preparation of Sugar-Directed ADC Ab-G27-DXd

[0167] Ab-G27-DXd is obtained by combining the compound TCO-PEG4-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G27 through general operation 4. The measured value of the heavy chain after HRMS deconvolution is 51234.41 Da.

[0168] Preparation Example 43 Preparation of Sugar-Directed ADC Ab-G28b-DXd

[0169] Ab-G28b-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G28b through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 52432.16 Da.

[0170] Preparation Example 44 Preparation of Sugar-Directed ADC Ab-G32-DXd

[0171] Ab-G32-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G32 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 52101.64 Da.

[0172] Preparation Example 45 Preparation of Sugar-Directed ADC Ab-G33-DXd

[0173] Ab-G33-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G33 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 52304.05 Da.

[0174] Preparation Example 46 Preparation of Sugar-Directed ADC Ab-G35-DXd

[0175] Ab-G35-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G35 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 53271.19 Da.

[0176] Preparation Example 47 Preparation of Sugar-Directed ADC Ab-G36-DXd

[0177] Ab-G36-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G36 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 51018.64 Da.

[0178] Preparation Example 48 Preparation of Sugar-Directed ADC Ab-G37-DXd

[0179] Ab-G37-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G37 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 51032.43 Da.

[0180] Preparation Example 49 Preparation of Sugar-Directed ADC Ab-G38-DXd-MMAE

[0181] Ab-G38-DXd-MMAE was obtained by combining the compounds TCO-PEG4-GGFG-Dxd, DBCO-PEG4-VC-PAB-MMAE and the non-natural glycoengineered antibody Ab-G38 through general operation six. The measured value of the heavy chain after HRMS deconvolution was 52887.29 Da.

[0182] Preparation Example 50 Preparation of Sugar-Directed ADC Ab-G39-DXd-MMAE

[0183] Ab-G39-DXd-MMAE was obtained by combining the compounds TCO-PEG4-GGFG-Dxd, DBCO-PEG4-VC-PAB-MMAE and the non-natural glycoengineered antibody Ab-G39 through general operation six. The measured value of the heavy chain after HRMS deconvolution was 52900.70 Da.

[0184] Preparation Example 51 Preparation of Sugar-Directed ADC Ab-G40-DXd

[0185] Ab-G40-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G40 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 50961.38 Da.

[0186] Preparation Example 52 Preparation of Sugar-Directed ADC Ab-G42-DXd-MMAE

[0187] Ab-G42-DXd-MMAE was obtained by combining compounds TCO-PEG4-GGFG-Dxd, DBCO-PEG4-VC-PAB-MMAE and the non-natural glycoengineered antibody Ab-G42 through general operation six. The measured value of the heavy chain after HRMS deconvolution was 52914.89 Da.

[0188] Preparation Example 53 Preparation of Sugar-Directed ADC Ab-G44-DXd-MMAE

[0189] Ab-G44-DXd-MMAE was obtained by combining compounds TCO-PEG4-GGFG-Dxd, DBCO-PEG4-VC-PAB-MMAE and the non-natural glycoengineered antibody Ab-G44 through general operation six. The measured value of the heavy chain after HRMS deconvolution was 53492.60 Da.

[0190] Preparation Example 54 Preparation of Sugar-Directed ADC Ab-G45-DXd-MMAE

[0191] Ab-G45-DXd-MMAE was obtained by combining the compounds TCO-PEG4-GGFG-Dxd, DBCO-PEG4-VC-PAB-MMAE and the non-natural glycoengineered antibody Ab-G45 through general operation six. The measured value of the heavy chain after HRMS deconvolution was 53300.04 Da.

[0192] Preparation Example 55 Preparation of Sugar-Directed ADC Ab-G46-DXd

[0193] Ab-G46-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G46 through general procedure 2. The measured value of the heavy chain after HRMS deconvolution is 52174.17 Da.

[0194] Preparation Example 56 Preparation of Sugar-Directed ADC Ab-G47-DXd

[0195] Ab-G47-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G47 through general procedure 2. The measured value of the heavy chain after HRMS deconvolution is 52173.90 Da.

[0196] Preparation Example 57 Preparation of Sugar-Directed ADC Ab-G40-DBCO-PEG4-VC-PAB-MMAE

[0197] Ab-G40-DBCO-PEG4-VC-PAB-MMAE is a conjugate of wild-type antibody Pertuzumab and disaccharide conjugate G40-DBCO-PEG4-VC-PAB-MMAE obtained by general procedure 5. The measured value of the heavy chain after HRMS deconvolution is 51491.63 Da.

[0198] Example 6 Preparation of Sugar-Directed ADC Based on Non-Core Fucosylated Antibody

[0199] Wild-type pertuzumab was dissolved in 20 mM phosphate, 150 mM NaCl, pH 7.4 buffer to a final concentration of 10 mg / mL. Wild-type Alfc (from Lacticaseibacillus paracasei, GENBANK accession number WP_012492118.1) was added to a final concentration of 0.5 mg / mL for hydrolysis. The mixture was incubated overnight at 37°C and purified using Protein A magnetic beads to obtain a defucosylating antibody (Ab-defuct) containing one N-acetylglucosamine.

[0200] The prepared defucosylated antibody, disaccharide linker, and wild-type endoglycosidase Endo Se2 or Endo Si were placed in the same reaction system at concentrations of 10 mg / mL, 1.67 mM (25 times the antibody concentration), and 0.4 mg / mL, respectively. The reaction pH was adjusted to 7.4 and the reaction temperature was maintained at 25°C. After 6 hours, a 4 μL sample was removed and terminated with 36 μL of PBS containing 0.1% formic acid. The sample was then analyzed by LC-MS. The deconvoluted HRMS values ​​for the heavy chain are shown in Table 3.

[0201] The defucosylating non-natural glycoengineered antibodies Ab-defuc-G7, G20, G21, G23, G33, G35, and G40 and the compound DBCO-GGFG-Dxd were used to obtain the corresponding ADCs through general operation 2. The actual HRMS deconvolution values ​​of the heavy chain are shown in Table 3.

[0202] The defucosylating non-natural glycoengineered antibodies Ab-defuc-G38, G39, G42, G44, and G45 were reacted with the compounds TCO-PEG4-GGFG-Dxd and DBCO-PEG4-VC-PAB-MMAE to obtain the corresponding ADCs, respectively. The measured values ​​of heavy chain HRMS deconvolution are shown in Table 3.

[0203] Table 3 Preparation of sugar-targeted ADCs based on core-free fucosylated antibodies

[0204] As can be seen from Table 3, the ADC preparation method provided in the present disclosure can be used to prepare ADC without core fucosylation.

[0205] Example 7: Efficacy of Sugar-Directed Antibody-Drug Conjugates

[0206] 1. Preparation of HER2-targeted sugar ADC

[0207] (1) Synthesis of Tmab-G7 / G9 / G22 / G23 / G24 / G28b

[0208] The wild-type antibody trastuzumab (Tmab), disaccharide oxazolines (i.e., compounds G7, G9, G22, G23, G24, and G28b), and the wild-type endoglycosidase Endo Si (for compounds G7, G9, G23, and G28b) or Endo Se2 (for compounds G22 and G24) were placed in the same reaction system at concentrations of 10 mg / mL, 1.67 mM (25 times the antibody concentration), and 0.6 mg / mL, respectively. The reaction system pH was adjusted to 7.4, and the reaction temperature was 25°C, at 800 rpm, for 3 hours. Protein A purification was performed to remove small molecules and glycoside hydrolases to obtain the corresponding transglycosylating antibodies.

[0209] (2) Preparation of corresponding ADC

[0210] The prepared transglycosylated antibody Tmab-G7 and DBCO-PEG4-VC-PAB-MMAE were placed in the same reaction system with the concentrations of each component adjusted to 5 mg / mL and 0.33 mM, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated at 25°C overnight. The desired sugar-specifically conjugated ADC, Tmab-G7-MMAE, was obtained by ultrafiltration and analyzed by LC-MS, HIC, and SEC.

[0211] The prepared transglycosylated antibody Tmab-G9 and N3-PEG4-VC-PAB-MMAE were placed in the same reaction system with the concentrations of each adjusted to 5 mg / mL and 0.33 mM, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated at 25°C overnight. The desired sugar-specifically conjugated ADC, Tmab-G9-MMAE, was obtained by ultrafiltration and analyzed by LC-MS, HIC, and SEC.

[0212] The prepared transglycosylated antibodies Tmab-G22, G23, G24, and G28b and DBCO-PEG4-VC-PAB-MMAE were placed in the same reaction system with the concentrations of each at 5 mg / mL and 0.33 mM, respectively. The pH of the reaction system was adjusted to 7.4. After incubation at 25°C overnight, 8 equivalents of DBCO-PEG4-VC-PAB-MMAE were added. The total reaction time was 24 hours. The desired sugar-specific site-conjugated ADC, Tmab-G22 / G23 / G24 / G28b-MMAE, was obtained by ultrafiltration and analyzed by LC-MS, HIC, and SEC.

[0213] The purity of the conjugated drug was determined by SEC and the DAR value was determined by hydrophobic chromatography (HIC-UPLC). Sample preparation: Sample concentration was 1.0-5 mg / ml and filtered through a 0.22 μm filter. Common detection methods include:

[0214] (1) Size Exclusion Chromatography (SEC)-HPLC

[0215] Sample preparation: Sample concentration 1.0-5 mg / ml, filtered through a 0.22 μm filter membrane; chromatographic column: TOSOH, TSKgel G3000SWxL, 5 μm, 7.8 mm × 300 mm; mobile phase: 0.2 M PB, 5-15% isopropanol, pH 7.0, flow rate: 0.5-1 mL / min; detection wavelength: 280 nm & 248 nm; column temperature: room temperature; sample load: 30 μg; SEC chromatography elution method: isocratic elution.

[0216] (2) Hydrophobic Interaction Chromatography (HIC)-UPLC

[0217] Chromatographic column: TOSOH, HIC TSK Butyl-NPR, 2.5μm, 4.6mm×100mm; column temperature: room temperature; mobile phase A: 0.05MPB, 1.2M ammonium sulfate, pH 7.0; mobile phase B: 0.05M PB, pH 7.0, 20% isopropanol; flow rate: 0.5mL / min; sample load: 30μg; gradient method: from 0% to 100% in 20 minutes; detection wavelength: 280nm&248nm.

[0218] PB refers to a sodium phosphate buffer solution composed primarily of disodium hydrogen phosphate and sodium dihydrogen phosphate. Disodium hydrogen phosphate and sodium dihydrogen phosphate buffer solutions of varying pH values ​​are typically prepared using sodium dihydrogen phosphate and sodium dihydrogen phosphate solutions of equal concentration.

[0219] The ADC product coupled by this method was tested for HIC DAR value and SEC purity by HIC-UPLC and SEC-HPLC, respectively.

[0220] The quality control data of the sugar-targeted ADC targeting HER2 prepared above are shown in Table 4.

[0221] Table 4 Quality control data of sugar-targeted ADC targeting HER2

[0222] 2. In vitro efficacy of HER2-targeted sugar-directed ADCs

[0223] The purpose of this experiment is to detect the in vitro inhibitory activity of the ADC compounds disclosed herein against SK-BR-3 (human breast cancer cells), NCI-N87 (human gastric cancer cells) and MDA-MB-468 (human breast cancer cells).

[0224] Take tumor cells SK-BR-3 (source: Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), NCI-N87 (source: Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) or MDA-MB-468 (source: Cell Bank of Type Culture Collection Committee, Chinese Academy of Sciences) in the logarithmic growth phase and add them to the cell plate at a number of 2000 cells / well. Place the cell plate in a 37°C, 5% CO2 cell culture incubator and incubate for 12-16 hours. Add 100 μL of sample to each well (starting at 50 μg / mL, 5-fold dilution, 9 concentrations), shake gently and place in the incubator for incubation. After incubation for 144 hours, add 70 μL CellTiter-Glo TM(Promega, Cat. No. G7572) working solution was added, gently shaken to lyse the cells, and the plate was read on a microplate reader. The cell proliferation inhibition rate was calculated as follows: Cell proliferation inhibition rate = (1 - sample well / control well) × 100%. GraphPad Prism 8.0 software was used to plot the logarithm of sample concentration against cytotoxicity % on the ordinate. Nonlinear regression (curve fit) analysis was performed on the data to obtain the IC value of each test sample. 50 The specific results are shown in Table 5.

[0225] Table 5 In vitro anti-tumor activity test of the ADC compounds disclosed herein

[0226] As can be seen from Table 5, the ADC drug in the present disclosure has obvious proliferation inhibitory activity against HER2-positive cells SK-BR-3 and NCI-N87, but has weak proliferation inhibitory activity against HER2-negative cells MDA-MB-468, thereby showing good selectivity.

[0227] 3. In vivo efficacy of HER2-targeted sugar-directed ADCs

[0228] BALB / c Nude mice (purchased from Beijing Weitonglihua) were used as test animals to evaluate the efficacy of anti-HER2 ADC after tail vein injection on nude mice with human gastric cancer cell NCI-N87 transplanted tumors.

[0229] Mice were subcutaneously inoculated with NCI-N87 cells (source: ATCC) (5×10 6 / , with 50% Matrigel), and the tumors grew for 7 days to an average tumor volume of 150 mm 3 The animals were randomly divided into groups according to the tumor volume on day 7 (D7), with 7 animals in each group.

[0230] The mice were injected into the tail vein once a day, and the tumor volume and body weight were measured twice a week and the data were recorded. 28 -T7) / (V 28 -V7)]×100,T 28 T7 and T8 are the tumor volumes of the experimental group on the 28th and 7th day after inoculation, respectively. 28 V and V7 are the tumor volumes of the blank control group (Vehicle, PBS) on day 28 and day 7 after inoculation, respectively. Detailed experimental results at the end of the experiment on day 28 after inoculation are shown in Table 6 and Figures 4-5.

[0231] Table 6 In vivo efficacy evaluation of the ADC compounds disclosed herein

[0232] As can be seen from Table 6, the ADC drug constructed using the disaccharide linker in the present disclosure exhibited excellent anti-tumor activity against the HER2-overexpressing NCI-N87 transplanted tumor, indicating that the enzyme disclosed in the present disclosure has industrial applicability for preparing ADC with sugar chain remodeling.

[0233] The embodiments of the present disclosure described above are merely exemplary, and any person skilled in the art will recognize or be able to determine the equivalents of numerous specific compounds, materials, and operations without requiring undue experimentation. All such equivalents are within the scope of the present disclosure and are encompassed by the claims.

Claims

1. An enzyme having the amino acid sequence shown in SEQ ID NO:1, said enzyme exhibiting glycan hydrolysis and / or glycan transfer activity.

2. A method for glycan remodeling of a polypeptide or protein, comprising the following steps: a) introducing Endo Se2 or Endo Si; b) introducing a polypeptide or protein containing at least one N-acetylglucosamine or core fucosylated N-acetylglucosamine; c) providing a disaccharide linker or disaccharide conjugate; d) transferring the disaccharide linker or disaccharide conjugate to the polypeptide or protein described in step (b) using the said Endo Se2 or Endo Si to provide a new polypeptide or protein with glycan modification; The sequence of the said Endo Se2 is shown in SEQ ID NO:1, and the sequence of the said Endo Si is shown in SEQ ID NO:

2.

3. The method according to claim 2, wherein, The said polypeptide or protein is an antibody or a protein containing the Fc region of an antibody.

4. The method according to claim 2, wherein The said polypeptide or protein containing at least one N-acetylglucosamine or core fucosylated N-acetylglucosamine is obtained by hydrolysis of a polypeptide or protein containing N-glycan by an endoglycosidase or obtained by recombinant expression or obtained by chemical synthesis.

5. The method according to claim 4, wherein, The N-glycan is a natural or non-natural complex-type, high-mannose type, hybrid N-glycan.

6. The method according to claim 4, wherein The endoglycosidase includes Endo Se2, Endo Si, Endo S, Endo S2, Endo F3 and their mutants.

7. The method according to claim 4, wherein The polypeptide or protein containing N-glycan achieves glycan remodeling in one step by introducing Endo Se2 or Endo Si.

8. The method according to claim 4, wherein The polypeptide or protein containing N-glycan achieves glycan remodeling in one step by simultaneously introducing Endo Se2 or Endo Si and another or more endoglycosidases.

9. The method according to claim 2, wherein The disaccharide linker or disaccharide conjugate contains at least one monosaccharide modified by oxazoline or thiazoline or a monosaccharide modified / transformed by a bioorthogonal functional group.

10. The method according to claim 9, wherein the disaccharide linker or disaccharide conjugate is selected from the following structures:

11. Use of Endo Se2 shown in SEQ ID NO:1 or Endo Si shown in SEQ ID NO:2 for transferring a disaccharide linker or disaccharide conjugate in glycan modification.

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