Novel Endo-β-N-Acetylglucosaminidase and Uses thereof

Abstract

The present invention provides a method for preparing site-specific and quantitative antibody-drug conjugates based on non-natural sugar-engineered antibodies or sugar chain modifications using two novel endo-β-N-acetylglucosaminidases and said two enzymes and disaccharide linkers, and uses thereof.

Description

Technology Field

[0001] This application claims priority to the Chinese patent application filed with the Chinese Intellectual Property Office on January 10, 2024, with application number CN2024100352818 and title of invention “Novel endo-β-N-acetylglucosaminidase and uses thereof,” and the Chinese patent application filed with the Chinese Intellectual Property Office on January 3, 2025, with application number CN2025100132263 and title of invention “Novel endo-β-N-acetylglucosaminidase and uses thereof,” the entire contents of which are incorporated by reference into this application.

[0002] The present disclosure relates to the fields of pharmacochemistry and biotechnological drugs, specifically to two novel endo-β-N-acetylglucosaminidases and novel uses thereof. Background Technology

[0003] Antibodies are a type of glycoprotein belonging to the immunoglobulin superfamily, and antibody drugs are currently widely used to treat various diseases. Natural IgG antibodies consist of two light chains and two heavy chains, which are connected to each other by disulfide bonds. Antibodies possess a conserved Fc domain, and a naturally conserved N-glycosylation site (N297) is present at position 297 of this domain. Generally, therapeutic antibody drugs are obtained through recombinant expression. Monoclonal antibodies produced through recombinant expression using animal-derived cells (e.g., CHO cells) as hosts generally possess a double-antennae complex glycan modification. Glycosylation modifications of antibodies have a significant effect on antibody effector activity (including antibody-dependent cytotoxicity and complement-dependent cytotoxicity).

[0004] In addition to naturally occurring biological functions, various technologies have been derived to produce antibody-based drug conjugates utilizing these conserved modification sites. Antibody-based drug conjugates are a type of biotechnological drug that achieves the concentration of an effector payload at a target site by loading an effector payload using an antibody as a targeting carrier. Possible effector payloads include cytotoxins, radioisotopes, oligonucleotides, immunomodulators, polypeptides, or protein fragments.

[0005] Antibody-Drug Conjugates (ADCs) are a type of biotechnological drug composed of an antibody, a cytotoxin, and a linker. ADCs combine the targeting ability of antibodies with the killing ability of cytotoxins, and control the release of cytotoxins through the linker to achieve the goal of targeted cell death in tumor cells. Currently, commercially available ADCs primarily rely on lysine (e.g., Trastuzumab emtansine) or cysteine ​​(e.g., Enfortumab vedotin) that are naturally present in antibodies. These ADCs, produced using natural amino acid random conjugation methods, have disadvantages such as low homogeneity, instability of the conjugation site, resulting in low drug safety and a narrow therapeutic range. Currently, various methods exist for manufacturing site-specific ADCs, including exogenous cysteine ​​insertion technology, non-natural amino acid insertion technology, enzyme-catalyzed conjugation technology, and glycan-based site-specific conjugation technology.

[0006] Endo-β-N-acetylglucosaminidase (ENGase) is a type of endoglycosidase with hydrolytic activity capable of hydrolyzing sugar chains bound to antibodies in vitro. Based on this, Laixi Wang et al. implemented glyco-engineered modification of antibodies in vitro by using ENGase mutants (e.g., Endo S D233Q, Endo S2 D184M) to transfer a biantenna-type oxazoline substrate containing natural or bioorthogonal reactive groups to the N-glycan terminus. Subsequently, antibody-based drug conjugates can be prepared using the introduced bioorthogonal reactive groups. Furthermore, the research teams of Huang Wei and Laixi Wang reported methods to prepare glyco-engineered antibodies and ADCs, respectively, using ENGase with disaccharide linkers as substrates.

[0007] The ENGase family includes several members, among whom Endo S and Endo S2 possess hydrolytic and disaccharide linker transfer activities, but their broader substrate specificity has not yet been discussed. Endo Si exhibits significant hydrolytic activity, and its mutant possesses transfer activity to a double-antenna type oxazoline substrate. However, it has not been reported whether Endo Si possesses disaccharide linker transfer activity. Therefore, the substrate specificity of ENGase family members toward disaccharide linkers still needs to be explored, and the development of superior ENGases for industrial production is urgent.

[0008] Based on the development of superior endo-β-N-acetylglucosaminoses, the present disclosure discovered and tested endo-β-N-acetylglucosaminoses with broader substrate specificity and superior translocation efficiency. Site-specific introduction of disaccharide linkers is possible through highly efficient enzyme-catalyzed reactions, enabling the glyco-engineered modification of antibody molecules. Bioorthogonal reactive groups introduced by disaccharide linkers enable the highly efficient production of antibody-based drug conjugates, which are simple to operate and easy to industrialize. The resulting antibody-based drug conjugates have good drug development potential and can be used for the treatment of tumors, inflammation, infectious diseases, or other immune diseases.

[0009] In the present disclosure Streptococcus equi A novel endo-β-N-acetylglucosaminidase derived from subsp. zooepidemicus Sz105 was discovered and named Endo Se2. The enzyme has an amino acid sequence represented by SEQ ID NO: 1, where positions 1 through 36 of SEQ ID NO: 1 are signal peptides and positions 37 through 1011 are the full-length sequence of the mature enzyme. Surprisingly, the enzyme exhibited excellent glycan hydrolysis and / or glycan translocation activity.

[0010] In addition, the present disclosure Streptococcus iniae We discovered another endo-β-N-acetylglucosaminidase derived from Endo Si, whose amino acid sequence is denoted by SEQ ID NO: 2, where positions 1 through 33 are the signal peptide and positions 34 through 928 are the full-length sequence of the mature enzyme. Surprisingly, this enzyme also exhibits excellent glycan hydrolysis and / or glycan transfer activity, as well as significant disaccharide linker transfer activity.

[0011] Based on the discovery of the two enzymes mentioned above, the present disclosure provides a method for reconstructing sugar chains for polypeptides or proteins, and

[0012] a) A step of introducing Endo Se2 or Endo Si;

[0013] b) a step of introducing a polypeptide or protein containing at least one N-acetylglucosamine or core fucosylated N-acetylglucosamine;

[0014] c) a step of providing a disaccharide linker or a disaccharide conjugate; and

[0015] d) a step of transferring a disaccharide linker or a disaccharide conjugate using the aforementioned Endo Se2 or Endo Si into the aforementioned polypeptide or protein according to step (b) to provide a novel polypeptide or protein with modified sugar chains.

[0016] In some embodiments, the polypeptide or protein is an antibody or a protein comprising the Fc region of an antibody.

[0017] In some embodiments, the polypeptide or protein containing at least one N-acetylglucosamine or core fucosylated N-acetylglucosamine is obtained by hydrolysis, recombinant expression, or chemical synthesis of an N-glycan-containing polypeptide or protein using an endoglycosidase having endo-β-N-acetylglucosaminidase activity.

[0018] In some embodiments, the N-glycan is a natural or non-natural complex, gomannose, or hybrid N-glycan.

[0019] In some embodiments, endoglycosidases having endo-β-N-acetylglucosaminidase activity include Endo Se2, Endo Si, Endo S, Endo S2, Endo F3, and mutants thereof.

[0020] In some embodiments, the N-glycan-containing polypeptide or protein implements sugar chain reorganization in one step without purification by introducing Endo Se2 or Endo Si.

[0021] In some embodiments, the N-glycan-containing polypeptide or protein implements sugar chain reorganization in one step without purification by simultaneously introducing Endo Se2 or Endo Si and one or more other endoglycosidases.

[0022] In some embodiments, the disaccharide linker comprises at least one oxazolinated or thiazolinated monosaccharide, or a monosaccharide modified / modified with a bioorthogonal functional group.

[0023] In some embodiments, the disaccharide linker described above is

[0024]

[0025]

[0026]

[0027] Selected from.

[0028] In some embodiments, the disaccharide conjugate further comprises, in addition to comprising an oxazolinated or thiazolinated monosaccharide, a toxin, a small molecule drug, a near-infrared or fluorescent probe, a polypeptide, RNA and related drug, a radioisotope label, a contrast agent, and an MRI imaging agent; optionally, may or may not comprise a cleavable linker, a non-cleavable linker, or a combination thereof. Herein, the small molecule drug is preferably selected from maytansine, DM-1, DM-4, ​​MMAE, MMAF, Auristatin 0101, SN-38, Dxd, exatecan, duocarmycin, amanitin, PBD series, VP-16, camptothecin, paclitaxel, docetaxel, anthracyclines, and derivatives of said compounds, or the small molecule drug is a radiotherapeutic agent.

[0029] In some embodiments, the above-described disaccharide conjugate is selected from the following structures:

[0030]

[0031] .

[0032] The present disclosure further provides uses for Endo Se2 or Endo Si used to transfer disaccharide linkers or disaccharide conjugates in sugar chain modification.

[0033] The present disclosure enables the production of homogenized glycan-modified antibodies through the above-described glycosylation modification method. Endo Se2 or Endo Si exhibit superior transfer efficiency and broader substrate specificity compared to existing technologies. The discovery and application of Endo Se2 and Endo Si not only provide superior available tool enzymes for the structural development of disaccharide linkers but also offer superior choices for the industrial production of disaccharide-linker-based glycosylation modification antibodies and antibody-based drug conjugates derived therefrom. Brief explanation of the drawing

[0034] Figure 1 shows the results of measuring the transglycosylation activity of different endoglycosidases for compound G0. Figure 2 shows the results of measuring the transglycosylation activity of different endoglycosidases for compound G1. Figure 3 shows the results of measuring the transglycosylation activity of different endoglycosidases for compound G2. Figure 4 shows the efficacy of a low-dose site-specific glycoconjugated ADC in an NCI-N87 xenograft model. Figure 5 shows the efficacy of a high-dose site-specific glycoconjugate ADC in an NCI-N87 xenograft model. Specific details for implementing the invention

[0035] Each publication, patent, or patent application is referenced by reference to the same extent as specific numerical values ​​specifically and individually indicated herein.

[0036] 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 and may be modified. It should also be understood that the terms used herein are intended to describe 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 generally understood by those skilled in the art to which the present disclosure pertains.

[0037] Some embodiments disclosed herein include numerical ranges, and some aspects of the disclosure may be described through ranges. Unless otherwise stated, numerical ranges or descriptions through ranges should be understood for the sake of brevity and convenience only and should not be interpreted as a strict limitation on the scope of the disclosure. Accordingly, descriptions through ranges should be deemed to specifically disclose all possible sub-ranges and all possible specific numerical values ​​within such ranges as if they were explicitly described in the disclosure. The principles set forth above apply equally regardless of the width of the numerical range. In the case of descriptions through ranges, the range includes both endpoints of the range.

[0038] In this disclosure, the term “antibody” includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primateized antibodies, human antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, polyvalent antibodies, etc., insofar as they exhibit the desired biological activity. The term “immunoglobulin” may be used interchangeably with “antibody” in this specification. As used in this specification, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies.

[0039] The term “antibody fragment” comprises at least a portion of a complete antibody. As used herein, the “fragment” of an antibody molecule comprises an “antigen-binding fragment” of an antibody, and the term “antigen-binding fragment” refers to a polypeptide fragment that specifically binds to or reacts with a selected antigen or its epitope within an immunoglobulin or antibody, or a fusion protein product further derived from such fragment, such as a single-chain antibody, an extracellular binding domain of a chimeric antigen receptor, etc. Exemplary antibody fragments or their antigen-binding fragments 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), bispecific antibodies or multispecific antibodies formed by antibody fragments.

[0040] The term “multispecific antibody” refers to a novel antibody construct that binds to two or more different sites and / or targets, formed by functionally linking an antibody or antibody fragment to one or more other binding molecules (including the antibody or antibody fragment or other molecules having binding ability) (e.g., chemical coupling, gene fusion, non-covalent bonding, or other methods). Thus, a “bispecific antibody” (or referred to as a “bispecific antigen-binding molecule” or “bispecific antibody”) specifically refers to an antibody construct that is specific to two different antigens and / or epitopes. Generally, a bispecific antibody or a multispecific antibody comprises at least two different antigen (or epitope) binding domains.

[0041] The term “sugar” refers to a molecule containing carbohydrates, whether oxidized or unoxidized, and includes, but is not limited to, monosaccharides, disaccharides, trisaccharides, oligosaccharides, or polysaccharides. Sugar is also used herein to refer to the carbohydrate portion of a sugar conjugate, such as glycoproteins, glycolipids, glycopeptides, glycoproteins, peptidoglycans, lipopolysaccharides, or proteoglycans. The term “sugar chain” may be used interchangeably with “sugar” herein.

[0042] As used in the present disclosure, the “glycan linker” is an activated donor molecule for sugar conjugation and may be a synthetic oxazoline or thiazolin-containing sugar (e.g., an oligosaccharide with an activated reduction end, preferably an oligosaccharide molecule having an oxazoline structure); or may be a natural N-glycan oxazoline. The glycan linker may also be chemically modified to introduce functional groups, for example, through azide, alkynylation, aldehydeation, thiolation, etc. The term “disaccharide linker” is a glycan linker composed of at least two monosaccharide units, preferably the 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 comprises a payload such as a cytotoxin, and optionally may further comprise a linker between the disaccharide linker and the payload.

[0043] Core fucosylated and non-fucosylated glycoproteins are an important class of molecules that play a key role 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 branched oligosaccharides.

[0044] The term “antibody-based drug conjugate” in this disclosure collectively refers to all conjugates formed by covalently bonding a polypeptide / protein targeting specific cells to a payload. The polypeptide / protein targeting specific cells may be an antibody or an antigen-binding fragment thereof, e.g., a monoclonal antibody, a bispecific antibody, a polyclonal antibody, etc.; the payload may be a cytotoxin, a small molecule drug, a near-infrared or fluorescent probe, a polypeptide, RNA and related drug, a radioisotope label, a contrast agent, an MRI imaging agent, etc.; and the formed covalent conjugate may be used for treatment or detection. The term “antibody-drug conjugate” in this disclosure refers to all conjugates formed by covalently bonding a polypeptide / protein targeting specific cells to a cytotoxin.

[0045] The term “endo-β-N-acetylglucosaminodase” in this disclosure refers to an enzyme having endoglycosidase activity produced by various organisms (EC 3.2.1.96), which generally belongs to glycoside hydrolyzing enzyme series 18 or 85, and some endo-β-N-acetylglucosaminodases known in the art, such as Endo S, Endo S2, Endo F3, Endo A, Endo D, Endo Si, etc., refer to WO2022 / 050300. These enzymes may also simultaneously possess disaccharide linker transfer activity, and enzymes known in the art having such activity, such as Endo S, Endo S2, Endo F3, and their mutants, refer to WO2022 / 226420. The term “” may be used interchangeably with “endo-β-N-acetylglucosaminodase” in this specification.

[0046] As used in this disclosure, “wild-type antibody” generally refers to a naturally occurring or recombinant antibody having an N-glycosylated site (N-glycan), for example, all antibodies having an N297 site in the Fc region fall into the category of “wild-type antibody,” and the Fc region is derived from IgG1, IgG2, IgG3, or IgG4.

[0047] The term “deglycosylated antibody” in the present disclosure refers to an antibody containing one N-acetylglucosamine or core-fucosylated N-acetylglucosamine formed from a wild-type antibody by the action of a glycoside hydrolase, or an antibody containing one N-acetylglucosamine or core-fucosylated N-acetylglucosamine that is directly recombinantly expressed in specific cells or prepared by chemical synthesis.

[0048] The term “glyco-engineered antibody” in this disclosure collectively refers to an engineered antibody that achieves homogenization of glyco-chain modifications in vitro using glyco-chain modification technology. The glyco-engineered antibodies of this disclosure are obtained by transferring a natural or non-natural glyco-chain linker to a deglycosylated antibody under the catalytic action of endo-β-N-acetylglucosaminidase. The glyco-chain modification of the glyco-engineered antibody may be a natural or non-natural modification and may include a bioorthogonal reactive group.

[0049] The antibody used in the embodiments of the present disclosure is a wild-type antibody containing an Fc region, the sequence of said Fc region is indicated, for example, by SEQ ID NO: 5, and pertuzumab is a representative antibody.

[0050] In this disclosure, antibodies, glycans modified antibodies, ADC purification, packing materials used for characterization, chromatography columns, and instruments include the following: 5 mL cOmplete His-Tag Purification Column (Roche), HiLoad TM 26 / 600 Superdex TM200 prep grade chromatography column (Cytiva), AmMag TM Protein A Magnetic Beads (GenScript), SDA030 Protein Purification System (Sepure), Acquity I-Class / RDa (Waters) Liquid Chromatography-Mass Spectrometer, Arc Premier High Performance Liquid Chromatography Instrument, ACCQUITY UPLC BEH PROTEIN C4 (Waters, 1.7 μm, 2.1 mm × 50 mm) Chromatography Column, TSKgel G3000SWXL (7.8 mm × 30 cm, 5 μm) SEC Chromatography Column, TSKgel Butyl-NPR (4.6 mm × 10 cm, 2.5 μm) HIC Chromatography Column.

[0051] In the present disclosure, the linker-payloads DBCO-GGFG-Dxd and TCO-PEG4-GGFG-Dxd were purchased from Shanghai Tekanbio Pharm-Tech Co., Ltd.; the linker-payloads DBCO-PEG4-GGFG-Dxd, DBCO-PEG4-VC-PAB-MMAE, and N3-PEG4-VC-PAB-MMAE were purchased from MedChemExpress LLC. In the present disclosure, trastuzumab was purchased from Sanyou Biopharmaceuticals (Shanghai) Co., Ltd. In the present disclosure, the glycan linker was synthesized by Wuhan GLYCOGENE Pharmaceutical Co., Ltd. Other compounds and reagents not otherwise specified were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0052] General manufacturing example

[0053] General Procedure 1: Method for Manufacturing Sugar Engineering Antibodies

[0054] Wild-type antibody, prepared disaccharide linker, and endoglycosidase (Endo Si or Endo Se2) were mixed to concentrations of 10 mg / mL, 2 mM (30 equivalents of antibody concentration), and 0.4 mg / mL, respectively, and the pH of the reaction system was adjusted to 7.4. After incubation at 25°C for 12 hours, the required non-natural glycosyl-engineered antibody was obtained through Protein A purification.

[0055] General Procedure 2: Method for manufacturing a site-specific ADC based on an azide-modified glyco-engineered antibody

[0056] The prepared azide-modified glycosyl-engine antibody and cycloalkyne-based (e.g., DBCO) linker-payload were mixed to concentrations of 5 mg / mL and 0.33 mM, respectively, and the pH of the reaction system was adjusted to 7.4. The mixture was incubated overnight at 25°C, and after confirming the conversion to a product by LC-MS, the required site-specific glycosyl-conjugated ADC was obtained through Protein A purification or ultrafiltration.

[0057] General Procedure 3: Method for manufacturing site-specific ADCs based on cycloalkynyl group modified sugar-engineered antibodies

[0058] The prepared cycloalkynyl group-modified glycosyl-engineered antibody and azide-based (N3) linker-payload were mixed to concentrations of 5 mg / mL and 0.33 mM, respectively, and the pH of the reaction system was adjusted to 7.4. The mixture was incubated overnight at 25°C, and after confirming the conversion to a product by LC-MS, the required site-specific glycosyl-conjugated ADC was obtained through Protein A purification or ultrafiltration.

[0059] General Procedure 4: Method for Manufacturing Site-Specific ADCs Based on Tetrazine Modified Sugar-Engineered Antibodies

[0060] The prepared tetrazine group-modified glycosyl-engineered antibody and trans-cyclooctene (TCO)-based linker-payload were mixed to concentrations of 5 mg / mL and 0.33 mM, respectively, and the pH of the reaction system was adjusted to 7.4. The mixture was incubated overnight at 25°C, and after confirming the conversion to a product by LC-MS, the required site-specific glycosyl-conjugated ADC was obtained through Protein A purification or ultrafiltration.

[0061] General Procedure 5: Method for Manufacturing Site-Specific Glycoconjugated ADCs by One-Step Method

[0062] A disaccharide linker (i.e., a disaccharide conjugate) bound to the prepared payload, a wild-type antibody, and endoglycosidase (Endo Si or Endo Se2) were mixed to sequentially set concentrations of 0.4 mM, 5 mg / mL, and 0.4 mg / mL, respectively; the pH of the reaction system was adjusted to 7.4 and incubated overnight at 25°C; after confirming the conversion to a product by LC-MS, site-specific glycoconjugated ADCs were obtained through Protein A purification or ultrafiltration.

[0063] General Procedure 6: Method for manufacturing a dual payload site-specific ADC based on an azide and tetrazine group modified glyco-engine antibody

[0064] The prepared azide and tetrazine group modified glycosyl-engine antibodies, cycloalkyne-based (e.g., DBCO) linker-payloads, and TCO-based linker-payloads were mixed to concentrations of 5 mg / mL, 0.33 mM, and 0.33 mM, respectively, and the pH of the reaction system was adjusted to 7.4 and incubated overnight at 25°C. After confirming the conversion to a product by LC-MS, site-specific glycosyl-conjugated ADCs were obtained through Protein A purification or ultrafiltration.

[0065] General Procedure 7: Method for Manufacturing Site-Specific ADCs Based on Linear Alkynyl Group Modified Sugar-Engineered Antibodies

[0066] Copper sulfate, tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), and sodium ascorbate were pre-mixed to concentrations of 20 mM, 100 mM, and 150 mM, respectively, and used as catalysts. The prepared linear alkynyl group-modified glyco-engineered antibody, azide-based linker-payload, and catalyst were mixed to sequentially achieve concentrations of 5 mg / mL, 0.5 mM, 0.67 mM (copper sulfate), 3.33 mM (THPTA), and 5 mM (sodium ascorbate), respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated overnight at 25°C. After confirming the conversion to a product by LC-MS, site-specific glyco-conjugated ADCs were obtained through Protein A purification or ultrafiltration.

[0067] A specific process for manufacturing a sugar-engineered antibody and a site-specific sugar-conjugated ADC using the manufacturing method of the present disclosure is described below through specific examples.

[0068] Example 1: Recombinant Expression and Purification of Enzymes

[0069] The coding genes for the following four enzymes were cloned into the pET22b vector (GenScript). The four enzymes are, respectively Streptococcus equi Endo Se2 derived from subsp. zooepidemicus Sz105, the amino acid sequence being SEQ ID NO: 1, positions 37 through 1011; Streptococcus iniae Endo Si of the origin, the amino acid sequence of which is SEQ ID NO: 2, numbers 34 to 928; Streptococcus pyogenes Endo S2 derived from NZ131 (serotype M49), its amino acid sequence being SEQ ID NO: 3, 38 to 843, Streptococcus pyogenes Endo S of the origin, and its amino acid sequence is SEQ ID NO: 4, numbers 37 to 995.

[0070] A plasmid containing the target gene was transformed into E. coli BL21(DE3), plated on 2×YT solid medium containing 100 μg / mL ampicillin, and incubated overnight at 37°C. A single colony was taken and inoculated into 4 mL of 2×YT liquid medium containing 100 μg / mL ampicillin, and incubated overnight. 4 mL of the bacterial culture was inoculated into 1 L of 2×YT broth medium containing 100 μg / mL ampicillin, and the OD 600 Incubation was performed at 37°C until 0.8–1.0 was reached. Next, 0.4 mM isopropyl β-thiogalactopyranoside (IPTG) was added to the incubation medium, and protein overexpression was induced by incubation at 20°C. After 16 hours, bacterial cells were obtained by centrifugation. B-PER TM The cell pellet was lysed using Bacterial Protein Extraction Reagent (Thermo) according to the manufacturer's instructions. 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 samples were concentrated using an Amicon centrifuge filter (30 kDa, Millipore) and further processed by HiLoad TM 26 / 600 Superdex TMThe samples were purified by size exclusion by passing them through a 200 prep grade chromatography column (Cytiva). Fractions containing Endo S, Endo S2, Endo Si, and Endo Se2 fusion proteins were each concentrated using an Amicon centrifuge filter (30 kDa, Millipore) and stored in storage buffer (20 mM PB, pH 7.5). Protein purity was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a gel imager (Gel Doc EZ Imager, Bio-RAD), and Image Lab analysis software, and protein concentration was quantified using a spectrophotometer (Nano-300).

[0071] Example 2 Study of the transfer activity of Endo Se2 and Endo Si on various disaccharide linkers

[0072] A wild-type antibody containing the Fc region of SEQ ID NO: 5 was dissolved in a buffer of 20 mM phosphate, 150 mM NaCl, and pH 7.4 to a final antibody concentration of 10 mg / mL, wild-type Endo S2 with a final concentration of 0.4 mg / mL was added and hydrolyzed by incubation overnight at 37°C, and purified with Protein A magnetic beads to obtain a deglycosylated antibody containing one N-acetylglucosamine or core-fucosylated N-acetylglucosamine.

[0073]

[0074] 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 mixed to sequentially achieve concentrations of 10 mg / mL, 1.67 mM (25 equivalents 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 set to 25°C. 4 μL of samples were collected at 0.5, 1, 2, 3, and 6 hours. The reaction was terminated by adding 36 μL of PBS solution containing 0.1% formic acid, and the results were analyzed by reducing LC-MS. The results are shown in Figures 1 to 3 and Table 1.

[0075] For compound G0, at 1 hour, the glycosyl transfer efficiency of Endo Se2 and Endo Si was similar and significantly superior compared to Endo S2 and Endo S; at 6 hours, the glycosyl transfer efficiency of Endo Se2, Endo Si and Endo S2 was similar and significantly superior compared to Endo S (Fig. 1, Table 1).

[0076] For compound G1, at 1 hour, the Endo Si glycosyl transfer efficiency was superior to Endo Se2 and significantly superior to Endo S2. The transfer efficiency of Endo S for compound G1 was relatively low; at 6 hours, the Endo Se2 and Endo Si glycosyl transfer efficiencies were similar and superior to Endo S2 (Fig. 2, Table 1).

[0077] For compound G2, at 1 hour, the glycosyl transfer efficiency of Endo Si and Endo Se2 was similar and significantly superior to that of Endo S2. The transfer efficiency of Endo S for compound G2 was relatively low; at 6 hours, the glycosyl transfer efficiency of Endo Se2 and Endo Si was similar and superior to that of Endo S2 (Fig. 3, Table 1).

[0078] Transglycosylation activity of different endoglycosidases disaccharide linker Endoglycosidase Glycosyl transfer efficiency (%) 0.5 h 1 h 2 h 3 h 6 h G0 Endo Se2 96.6 97.9 98.0 97.2 96.2 Endo Si 89.3 97.1 97.8 97.8 97.6 Endo S2 65.7 82.7 92.4 94.8 97.3 Endo S 15.7 26.2 36.6 43.7 56.4 G1 Endo Se2 64.8 82.1 92.4 94.5 94.7 Endo Si 93.2 95.2 95.5 95.5 95.5 Endo S2 33.1 49.2 67.7 77.3 87.5 Endo S 5.6 9.3 13.0 15.7 21.1 G2 Endo Se2 92.2 92.9 92.8 92.7 97.2 Endo Si 93.4 93.4 93.3 93.1 93.8 Endo S2 22.2 36.2 51.9 61.0 82.7 Endo S 7.0 12.3 17.9 22.1 30.5

[0079] Example 3 Study on the substrate specificity of endoglycosidases such as Endo Se2 and Endo Si to various disaccharide linkers

[0080] Wild-type pertuzumab, a disaccharide linker, and wild-type endoglycosidase (i.e., Endo S or Endo S2 or Endo Si or Endo Se2) were mixed to sequentially achieve concentrations of 10 mg / mL, 1.67 mM (25 equivalents 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 set to 25°C. After 6 hours, 4 μL of the sample was collected, and 36 μL of PBS solution containing 0.1% formic acid was added to terminate the reaction, followed by analysis by LC-MS.

[0081] The structure of each type of disaccharide linker is as shown below.

[0082]

[0083] Transglycosylation activity of different endoglycosidases Glycosyl transfer efficiency (%) enzyme Endo Se2 Endo Si Endo S2 Endo S G0 >95 >95 >95 56.4 G1 94.7 >95 87.5 21.1 G2 >95 93.8 82.7 30.5 G4b 16.1 38.4 13.9 17.8 G7 76.3 90.6 36.3 13 G9 36.3 >95 4.7 7.6 G10 13.1 10.1 8.1 17.4 G13 75.3 51.8 6.9 0 G20 89 77.7 89.8 56.7 G21 >95 >95 72.6 29.4 G22 >95 68.2 7.7 0 G23 56.7 93.7 6.0 2.6 G24 88.4 60.8 29.9 11.7 G25 22.5 21.2 23.6 15.9 G27 34.4 >95 10.2 5.8 G28b 94.9 >95 55 13.6 G32 >95 >95 25 10.8 G33 >95 >95 35.3 24.1 G35 80.1 >95 7.8 6.6 G36 80.1 >95 >95 81.4 G37 94.2 51.3 11.7 11.0 G38 17.7 60.7 6.7 5.1 G39 34.1 >95 23.5 20.9 G40 >95 >95 79.9 33.2 G42 59.4 >95 13.1 9.7 G44 >95 >95 52.9 11.7 G45 53.1 15.1 6.9 0 G46 14.9 21.1 14.0 14.8

[0084] As shown in Table 2, for different disaccharide linker compounds, Endo Se2, Endo Si, Endo S2, and Endo S exhibited different substrate specificities and glycosyl transfer efficiencies. Endo Se2 and Endo Si have broader substrate specificities compared to Endo S2 and Endo S, and the optimal endoglycosidase for each substrate is generally Endo Se2 or Endo Si.

[0085] Example 4 Preparation of a wild-type antibody-based sugar-engineered antibody

[0086] Preparation Example 1 Preparation of the sugar engineering antibody Ab-G1

[0087] The non-natural sugar-engineered antibody Ab-G1 was obtained from compound G1 and the wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the sugar-engineered antibody Ab-G1 is 49893.48 Da.

[0088] Preparation Example 2 Preparation of Sugar Engineering Antibody Ab-G2

[0089] The non-natural sugar-engineered antibody Ab-G2 was obtained from compound G2 and the wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the sugar-engineered antibody Ab-G2 is 49902.46 Da.

[0090] Preparation Example 3: Preparation of the sugar engineering antibody Ab-G4b

[0091] The non-natural sugar-engineered antibody Ab-G4b was obtained from compound G4b and wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the sugar-engineered antibody Ab-G4b is 49789.06 Da.

[0092] Preparation Example 4 Preparation of the sugar engineering antibody Ab-G7

[0093] The non-natural glycosylated antibody Ab-G7 was obtained from compound G7 and the wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the glycosylated antibody Ab-G7 is 49810.12 Da.

[0094] Preparation Example 5 Preparation of the sugar engineering antibody Ab-G9

[0095] The non-natural sugar-engineered antibody Ab-G9 was obtained from compound G9 and the wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the sugar-engineered antibody Ab-G9 is 50154.80 Da.

[0096] Preparation Example 6 Preparation of the sugar engineering antibody Ab-G10

[0097] The non-natural glycosylated antibody Ab-G10 was obtained from compound G10 and wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the glycosylated antibody Ab-G10 is 50081.15 Da.

[0098] Preparation Example 7 Preparation of the sugar engineering antibody Ab-G13

[0099] The non-natural glycosylated antibody Ab-G13 was obtained from compound G13 and wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the glycosylated antibody Ab-G13 is 49807.70 Da.

[0100] Preparation Example 8 Preparation of the sugar engineering antibody Ab-G20

[0101] The non-natural glycosylated antibody Ab-G20 was obtained from compound G20 and wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the glycosylated antibody Ab-G20 is 49836.03 Da.

[0102] Preparation Example 9 Preparation of the sugar engineering antibody Ab-G21

[0103] The non-natural sugar-engineered antibody Ab-G21 was obtained from compound G21 and the wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the sugar-engineered antibody Ab-G21 is 49835.47 Da.

[0104] Preparation Example 10 Preparation of the sugar engineering antibody Ab-G22

[0105] The non-natural glycosylated antibody Ab-G22 was obtained from compound G22 and wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the glycosylated antibody Ab-G22 is 49835.38 Da.

[0106] Preparation Example 11 Preparation of the sugar engineering antibody Ab-G23

[0107] The non-natural glycosylated antibody Ab-G23 was obtained from compound G23 and wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the glycosylated antibody Ab-G23 is 49835.18 Da.

[0108] Preparation Example 12 Preparation of the sugar engineering antibody Ab-G24

[0109] The non-natural sugar-engineered antibody Ab-G24 was obtained from compound G24 and wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the sugar-engineered antibody Ab-G24 is 50064.00 Da.

[0110] Preparation Example 13 Preparation of the sugar engineering antibody Ab-G25

[0111] The non-natural glycosylated antibody Ab-G25 was obtained from compound G25 and wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the glycosylated antibody Ab-G25 is 49893.33 Da.

[0112] Preparation Example 14 Preparation of the sugar engineering antibody Ab-G27

[0113] The non-natural glycosylated antibody Ab-G27 was obtained from compound G27 and wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the glycosylated antibody Ab-G27 is 50024.69 Da.

[0114] Preparation Example 15 Preparation of the sugar engineering antibody Ab-G28b

[0115] The non-natural sugar-engineered antibody Ab-G28b was obtained from compound G28b and wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the sugar-engineered antibody Ab-G28b is 50175.80 Da.

[0116] Preparation Example 16 Preparation of the sugar engineering antibody Ab-G32

[0117] The non-natural glycosylated antibody Ab-G32 was obtained from compound G32 and wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the glycosylated antibody Ab-G32 is 49844.75 Da.

[0118] Preparation Example 17 Preparation of the sugar engineering antibody Ab-G33

[0119] The non-natural glycosylated antibody Ab-G33 was obtained from compound G33 and wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the glycosylated antibody Ab-G33 is 50047.05 Da.

[0120] Preparation Example 18 Preparation of the patented antibody Ab-G35

[0121] The non-natural glycosylated antibody Ab-G35 was obtained from compound G35 and wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the glycosylated antibody Ab-G35 is 49886.09 Da.

[0122] Preparation Example 19 Preparation of the sugar engineering antibody Ab-G36

[0123] The non-natural glycosylated antibody Ab-G36 was obtained from compound G36 and the wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the glycosylated antibody Ab-G36 is 49891.14 Da.

[0124] Preparation Example 20 Preparation of the sugar engineering antibody Ab-G37

[0125] The non-natural glycosylated antibody Ab-G37 was obtained from compound G37 and the wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the glycosylated antibody Ab-G37 is 49904.60 Da.

[0126] Preparation Example 21 Preparation of the sugar engineering antibody Ab-G38

[0127] The non-natural glycosylated antibody Ab-G38 was obtained from compound G38 and wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the glycosylated antibody Ab-G38 is 50018.20 Da.

[0128] Preparation Example 22 Preparation of the sugar engineering antibody Ab-G39

[0129] The non-natural sugar-engineered antibody Ab-G39 was obtained from compound G39 and wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the sugar-engineered antibody Ab-G39 is 50032.24 Da.

[0130] Preparation Example 23 Preparation of the sugar engineering antibody Ab-G40

[0131] The non-natural glycosylated antibody Ab-G40 was obtained from compound G40 and wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the glycosylated antibody Ab-G40 is 49833.19 Da.

[0132] Preparation Example 24 Preparation of the sugar engineering antibody Ab-G42

[0133] The non-natural sugar-engineered antibody Ab-G42 was obtained from compound G42 and wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the sugar-engineered antibody Ab-G42 is 50046.26 Da.

[0134] Preparation Example 25 Preparation of the sugar engineering antibody Ab-G44

[0135] The non-natural sugar-engineered antibody Ab-G44 was obtained from compound G44 and wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the sugar-engineered antibody Ab-G44 is 50624.49 Da.

[0136] Preparation Example 26 Preparation of the sugar engineering antibody Ab-G45

[0137] The non-natural sugar-engineered antibody Ab-G45 was obtained from compound G45 and the wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the sugar-engineered antibody Ab-G45 is 50405.15 Da.

[0138] Preparation Example 27 Preparation of the sugar engineering antibody Ab-G46

[0139] The non-natural sugar-engineered antibody Ab-G46 was obtained from compound G46 and the wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the sugar-engineered antibody Ab-G46 is 49865.77 Da.

[0140] Preparation Example 28 Preparation of the sugar engineering antibody Ab-G47

[0141] The non-natural glycosylated antibody Ab-G47 was obtained from compound G47 and the wild-type antibody pertuzumab according to general procedure 1. The actual value after HRMS deconvolution for the heavy chain of the glycosylated antibody Ab-G47 is 49866.18 Da.

[0142] Example 5 Preparation of a wild-type antibody-based site-specific glycosylconjugated antibody-drug conjugate

[0143] Preparation Example 29 Preparation of site-specific glycoconjugated ADC Ab-G1-DXd

[0144]

[0145] Ab-G1-DXd was obtained from compound DBCO-GGFG-Dxd and the non-natural sugar-engineered antibody Ab-G1 according to general procedure 2, and the measured value after HRMS deconvolution for the heavy chain is 51021.77 Da.

[0146] Preparation Example 30 Preparation of site-specific glycoconjugated ADC Ab-G2-DXd

[0147] Ab-G2-DXd was obtained from compound DBCO-GGFG-Dxd and the non-natural sugar-engineered antibody Ab-G2 according to general procedure 2, and the measured value after HRMS deconvolution for the heavy chain is 52158.93 Da.

[0148] Preparation Example 31 Preparation of Site-Specific Glycoconjugated ADC Ab-G4b-MMAE

[0149]

[0150] Ab-G4b-MMAE was obtained from compound N3-PEG4-VC-PAB-MMAE and the non-natural sugar-engineered antibody Ab-G4b according to general procedure 7, and the measured value after HRMS deconvolution for the heavy chain is 51185.38 Da.

[0151] Preparation Example 32 Preparation of site-specific glycoconjugated ADC Ab-G7-DXd

[0152] Ab-G7-DXd was obtained from compound DBCO-GGFG-Dxd and the non-natural sugar-engineered antibody Ab-G7 according to general procedure 2, and the measured value after HRMS deconvolution for the heavy chain is 50964.74 Da.

[0153] Preparation Example 33 Preparation of Site-Specific Glycoconjugated ADC Ab-G9-MMAE

[0154] Ab-G9-MMAE was obtained from compound N3-PEG4-VC-PAB-MMAE and the non-natural sugar-engineered antibody Ab-G9 according to general procedure 3, and the measured value after HRMS deconvolution for the heavy chain is 51551.58 Da.

[0155] Preparation Example 34 Preparation of Site-Specific Glycoconjugated ADC Ab-G10-MMAE

[0156] Ab-G10-MMAE was obtained from compound N3-PEG4-VC-PAB-MMAE and the non-natural sugar-engineered antibody Ab-G10 according to general procedure 3, and the measured value after HRMS deconvolution for the heavy chain is 51478.22 Da.

[0157] Preparation Example 35 Preparation of site-specific glycoconjugated ADC Ab-G13-DXd

[0158] Ab-G13-DXd was obtained from compound DBCO-GGFG-Dxd and the non-natural sugar-engineered antibody Ab-G13 according to general procedure 2, and the measured value after HRMS deconvolution for the heavy chain is 50978.76 Da.

[0159] Preparation Example 36 Preparation of site-specific glycoconjugated ADC Ab-G20-DXd

[0160] Ab-G20-DXd was obtained from compound DBCO-GGFG-Dxd and the non-natural sugar-engineered antibody Ab-G20 according to general procedure 2, and the measured value after HRMS deconvolution for the heavy chain is 50963.63 Da.

[0161] Preparation Example 37 Preparation of site-specific glycoconjugated ADC Ab-G21-DXd

[0162] Ab-G21-DXd was obtained from compound DBCO-GGFG-Dxd and the non-natural sugar-engineered antibody Ab-G21 according to general procedure 2, and the measured value after HRMS deconvolution for the heavy chain is 50963.46 Da.

[0163] Preparation Example 38 Preparation of site-specific glycoconjugated ADC Ab-G22-DXd

[0164] Ab-G22-DXd was obtained from compound DBCO-GGFG-Dxd and the non-natural sugar-engineered antibody Ab-G22 according to general procedure 2, and the measured value after HRMS deconvolution for the heavy chain is 52117.98 Da.

[0165] Preparation Example 39 Preparation of site-specific glycoconjugated ADC Ab-G23-DXd

[0166] Ab-G23-DXd was obtained from compound DBCO-GGFG-Dxd and the non-natural sugar-engineered antibody Ab-G23 according to general procedure 2, and the measured value after HRMS deconvolution for the heavy chain is 52116.81 Da.

[0167] Preparation Example 40 Preparation of site-specific glycoconjugated ADC Ab-G24-DXd

[0168] Ab-G24-DXd was obtained from compound DBCO-GGFG-Dxd and the non-natural sugar-engineered antibody Ab-G24 according to general procedure 2, and the measured value after HRMS deconvolution for the heavy chain is 52320.37 Da.

[0169] Preparation Example 41 Preparation of site-specific glycoconjugated ADC Ab-G25-DXd

[0170] Ab-G25-DXd was obtained from compound DBCO-GGFG-Dxd and the non-natural sugar-engineered antibody Ab-G25 according to general procedure 2, and the measured value after HRMS deconvolution for the heavy chain is 52174.43 Da.

[0171] Preparation Example 42 Preparation of site-specific glycoconjugated ADC Ab-G27-DXd

[0172]

[0173] Ab-G27-DXd was obtained from compound TCO-PEG4-GGFG-Dxd and the non-natural sugar-engineered antibody Ab-G27 according to general procedure 4, and the measured value after HRMS deconvolution for the heavy chain is 51234.41 Da.

[0174] Preparation Example 43 Preparation of site-specific glycoconjugated ADC Ab-G28b-DXd

[0175] Ab-G28b-DXd was obtained from compound DBCO-GGFG-Dxd and the non-natural sugar-engineered antibody Ab-G28b according to general procedure 2, and the measured value after HRMS deconvolution for the heavy chain is 52432.16 Da.

[0176] Preparation Example 44 Preparation of site-specific glycoconjugated ADC Ab-G32-DXd

[0177] Ab-G32-DXd was obtained from compound DBCO-GGFG-Dxd and the non-natural sugar-engineered antibody Ab-G32 according to general procedure 2, and the measured value after HRMS deconvolution for the heavy chain is 52101.64 Da.

[0178] Preparation Example 45 Preparation of site-specific glycoconjugated ADC Ab-G33-DXd

[0179] Ab-G33-DXd was obtained from compound DBCO-GGFG-Dxd and the non-natural sugar-engineered antibody Ab-G33 according to general procedure 2, and the measured value after HRMS deconvolution for the heavy chain is 52304.05 Da.

[0180] Preparation Example 46 Preparation of site-specific glycoconjugated ADC Ab-G35-DXd

[0181] Ab-G35-DXd was obtained from compound DBCO-GGFG-Dxd and the non-natural sugar-engineered antibody Ab-G35 according to general procedure 2, and the actual value after HRMS deconvolution for the heavy chain is 53271.19 Da.

[0182] Preparation Example 47 Preparation of site-specific glycoconjugated ADC Ab-G36-DXd

[0183] Ab-G36-DXd was obtained from compound DBCO-GGFG-Dxd and the non-natural sugar-engineered antibody Ab-G36 according to general procedure 2, and the measured value after HRMS deconvolution for the heavy chain is 51018.64 Da.

[0184] Preparation Example 48 Preparation of site-specific glycoconjugated ADC Ab-G37-DXd

[0185] Ab-G37-DXd was obtained from compound DBCO-GGFG-Dxd and the non-natural sugar-engineered antibody Ab-G37 according to general procedure 2, and the measured value after HRMS deconvolution for the heavy chain is 51032.43 Da.

[0186] Preparation Example 49 Preparation of Site-Specific Glycoconjugated ADC Ab-G38-DXd-MMAE

[0187]

[0188] Ab-G38-DXd-MMAE was obtained from compounds TCO-PEG4-GGFG-Dxd, DBCO-PEG4-VC-PAB-MMAE and the non-natural sugar-engineered antibody Ab-G38 according to general procedure 6, and the measured value after HRMS deconvolution for the heavy chain is 52887.29 Da.

[0189] Preparation Example 50 Preparation of Site-Specific Glycoconjugated ADC Ab-G39-DXd-MMAE

[0190] Ab-G39-DXd-MMAE was obtained from compounds TCO-PEG4-GGFG-Dxd, DBCO-PEG4-VC-PAB-MMAE and the non-natural sugar-engineered antibody Ab-G39 according to general procedure 6, and the measured value after HRMS deconvolution for the heavy chain is 52900.70 Da.

[0191] Preparation Example 51 Preparation of site-specific glycoconjugated ADC Ab-G40-DXd

[0192] Ab-G40-DXd was obtained from compound DBCO-GGFG-Dxd and the non-natural sugar-engineered antibody Ab-G40 according to general procedure 2, and the measured value after HRMS deconvolution for the heavy chain is 50961.38 Da.

[0193] Preparation Example 52 Preparation of Site-Specific Glycoconjugated ADC Ab-G42-DXd-MMAE

[0194] Ab-G42-DXd-MMAE was obtained from compounds TCO-PEG4-GGFG-Dxd, DBCO-PEG4-VC-PAB-MMAE and the non-natural sugar-engineered antibody Ab-G42 according to general procedure 6, and the measured value after HRMS deconvolution for the heavy chain is 52914.89 Da.

[0195] Preparation Example 53 Preparation of Site-Specific Glycoconjugated ADC Ab-G44-DXd-MMAE

[0196] Ab-G44-DXd-MMAE was obtained from compounds TCO-PEG4-GGFG-Dxd, DBCO-PEG4-VC-PAB-MMAE and the non-natural sugar-engineered antibody Ab-G44 according to general procedure 6, and the measured value after HRMS deconvolution for the heavy chain is 53492.60 Da.

[0197] Preparation Example 54 Preparation of Site-Specific Glycoconjugated ADC Ab-G45-DXd-MMAE

[0198] Ab-G45-DXd-MMAE was obtained from compounds TCO-PEG4-GGFG-Dxd, DBCO-PEG4-VC-PAB-MMAE and the non-natural sugar-engineered antibody Ab-G45 according to general procedure 6, and the measured value after HRMS deconvolution for the heavy chain is 53300.04 Da.

[0199] Preparation Example 55 Preparation of site-specific glycoconjugated ADC Ab-G46-DXd

[0200] Ab-G46-DXd was obtained from compound DBCO-GGFG-Dxd and the non-natural sugar-engineered antibody Ab-G46 according to general procedure 2, and the measured value after HRMS deconvolution for the heavy chain is 52174.17 Da.

[0201] Preparation Example 56 Preparation of site-specific glycoconjugated ADC Ab-G47-DXd

[0202] Ab-G47-DXd was obtained from compound DBCO-GGFG-Dxd and the non-natural sugar-engineered antibody Ab-G47 according to general procedure 2, and the measured value after HRMS deconvolution for the heavy chain is 52173.90 Da.

[0203] Preparation Example 57 Site-specific glycoconjugated ADC Ab-G40-DBCO-PEG 4 -Manufacture of VC-PAB-MMAE

[0204]

[0205] Ab-G40-DBCO-PEG4-VC-PAB-MMAE was obtained from the wild-type antibody pertuzumab and the disaccharide conjugate G40-DBCO-PEG4-VC-PAB-MMAE according to general procedure 5, and the actual value after HRMS deconvolution for the heavy chain is 51491.63 Da.

[0206] Example 6 Preparation of a site-specific glycoconjugated ADC based on a core bifucosylated antibody

[0207] Wild-type pertuzumab was dissolved in a buffer of 20 mM phosphate, 150 mM NaCl, pH 7.4 to obtain a final monoclonal antibody concentration of 10 mg / mL, and wild-type Alfc at a final concentration of 0.5 mg / mL ( Lacticaseibacillus paracasei (derived from, GENBANK registration number is WP_012492118.1) was added and hydrolyzed by incubation overnight at 37°C, and purified with Protein A magnetic beads to obtain a defucosylated antibody (Ab-defuc) containing one N-acetylglucosamine.

[0208] The prepared defucosylated antibody, disaccharide linker, and wild-type endoglycosidase Endo Se2 or Endo Si were added to the same reaction system at concentrations of 10 mg / mL, 1.67 mM (25 equivalents 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 set to 25°C. After 6 hours, 4 μL of the sample was collected, and the reaction was terminated by adding 36 μL of PBS solution containing 0.1% formic acid. The results were then analyzed by LC-MS. The measured values ​​for the heavy chain after HRMS deconvolution are shown in Table 3.

[0209] Corresponding ADCs were prepared from the defucosylated non-natural sugar engineering antibodies Ab-defuc-G7, G20, G21, G23, G33, G35, G40 and the compound DBCO-GGFG-Dxd according to general procedure 2, and the measured values ​​after HRMS deconvolution for the heavy chain are shown in Table 3.

[0210] Corresponding ADCs were prepared from the defucosylated non-natural sugar-engineered antibodies Ab-defuc-G38, G39, G42, G44, G45 and compounds TCO-PEG4-GGFG-Dxd, DBCO-PEG4-VC-PAB-MMAE, respectively, according to general procedure 6, and the measured values ​​after HRMS deconvolution for the heavy chain are shown in Table 3.

[0211] Preparation of core bifucosylated antibody-based site-specific glycoconjugate ADCs antibodies disaccharide linker Antibody molecular weight (Da) after glycosyl transfer Linker-Payload Site-specific glycoconjugated ADC molecular weight (Da) Ab-defuc - 49283.84 - - Ab-defuc G7 49664.23 DBCO-GGFG-Dxd 50818.78 Ab-defuc G20 49690.03 DBCO-GGFG-Dxd 50817.53 Ab-defuc G21 49689.14 DBCO-GGFG-Dxd 50817.49 Ab-defuc G23 49663.11 DBCO-GGFG-Dxd 51969.36 Ab-defuc G33 49901.12 DBCO-GGFG-Dxd 52157.45 Ab-defuc G35 49646.46 DBCO-GGFG-Dxd 53124.32 Ab-defuc G38 49872.52 TCO-PEG4-GGFG-Dxd / DBCO-PEG4-VC-PAB-MMAE 52740.79 Ab-defuc G39 49885.94 TCO-PEG4-GGFG-Dxd / DBCO-PEG4-VC-PAB-MMAE 52754.62 Ab-defuc G40 49687.05 DBCO-GGFG-Dxd 50815.49 Ab-defuc G42 49900.29 TCO-PEG4-GGFG-Dxd / DBCO-PEG4-VC-PAB-MMAE 52768.25 Ab-defuc G44 50477.69 TCO-PEG4-GGFG-Dxd / DBCO-PEG4-VC-PAB-MMAE 53346.16 Ab-defuc G45 50258.23 TCO-PEG4-GGFG-Dxd / DBCO-PEG4-VC-PAB-MMAE 53152.98

[0212] As can be seen from Table 3, the manufacturing method of the ADC provided in the present disclosure can be used to manufacture a core non-fucosylated ADC.

[0213] Example 7: Pharmacological effects of a site-specific glycosylated antibody-drug conjugate

[0214] 1. Preparation of site-specific glycoconjugated ADCs targeting HER2

[0215] (1) Synthesis of Tmab-G7 / G9 / G22 / G23 / G24 / G28b

[0216] Wild-type antibody trastuzumab (Tmab), disaccharide oxazoline (i.e., compounds G7, G9, G22, G23, G24, G28b), and wild-type endoglycosidase Endo Si (for compounds G7, G9, G23, G28b) or Endo Se2 (for compounds G22, G24) were added to the same reaction system to sequential concentrations of 10 mg / mL, 1.67 mM (25 equivalents of antibody concentration), and 0.6 mg / mL, respectively. The pH of the reaction system was adjusted to 7.4, the reaction temperature was set to 25°C, and the reaction was carried out at 800 rpm for 3 hours. The corresponding transglycosylated antibodies were obtained by removing small molecule compounds and glycoside hydrolases through Protein A purification.

[0217] (2) Manufacturing of the corresponding ADC

[0218] The prepared transglycosylated antibodies Tmab-G7 and DBCO-PEG4-VC-PAB-MMAE were placed in the same reaction system at concentrations of 5 mg / mL and 0.33 mM, respectively, and the pH of the reaction system was adjusted to 7.4. The mixture was incubated overnight at 25°C, and the site-specific glycoconjugated ADC, Tmab-G7-MMAE, was obtained by ultrafiltration and analyzed by LC-MS, HIC, and SEC.

[0219] The prepared transglycosylated antibodies Tmab-G9 and N3-PEG4-VC-PAB-MMAE were placed in the same reaction system at concentrations of 5 mg / mL and 0.33 mM, respectively, and the pH of the reaction system was adjusted to 7.4. The mixture was incubated overnight at 25°C, and the site-specific glycoconjugated ADC, Tmab-G9-MMAE, was obtained by ultrafiltration and analyzed by LC-MS, HIC, and SEC.

[0220] The prepared transglycosylated antibodies Tmab-G22, Tmab-G23, Tmab-G24, Tmab-G28b, and DBCO-PEG4-VC-PAB-MMAE were placed in the same reaction system at concentrations of 5 mg / mL and 0.33 mM, respectively, and the pH of the reaction system was adjusted to 7.4. After incubation overnight at 25°C, 8 equivalents of DBCO-PEG4-VC-PAB-MMAE were added, and the total reaction time was 24 hours. The site-specific glycoconjugated ADCs, namely Tmab-G22 / G23 / G24 / G28b-MMAE, were obtained by ultrafiltration and analyzed by LC-MS, HIC, and SEC.

[0221] For antibody-drug conjugates, ADC purity was measured using SEC, and DAR values ​​were measured using hydrophobic chromatography (HIC-UPLC). Sample treatment: Sample concentrations of 1.0–5 mg / ml were filtered through a 0.22 μm filter. Typical detection methods include the following.

[0222] (1) Molecular exclusion chromatography SEC-HPLC

[0223] Sample processing: Sample concentrations 1.0–5 mg / ml, filtered through a 0.22 μm filter; Chromatography 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; Loading weight: 30 μg; SEC chromatography elution method: Isothermal elution.

[0224] (2) Hydrophobic Interaction Chromatography HIC-UPLC

[0225] Chromatography column: TOSOH, HIC TSK Butyl-NPR, 2.5 μm, 4.6 mm × 100 mm; Column temperature: Room temperature; Mobile phase A: 0.05 M PB, 1.2 M Ammonium sulfate, pH 7.0; Mobile phase B: 0.05 M PB, pH 7.0, 20% Isopropanol; Flow rate: 0.5 mL / min; Loading amount: 30 μg; Gradient method: Increase from 0% to 100% over 20 minutes; Detection wavelength: 280 nm & 248 nm.

[0226] Here, PB refers to a sodium phosphate buffer solution with disodium hydrogen phosphate as the main component. Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solutions of different pH values ​​are generally prepared using sodium dihydrogen phosphate and disodium hydrogen phosphate solutions of the same concentration.

[0227] For the ADC products obtained by conjugation using this method, the HIC DAR value and SEC purity were measured using HIC-UPLC and SEC-HPLC, respectively.

[0228] Quality control data for the site-specific glycoconjugate ADC targeting HER2 manufactured above are shown in Table 4.

[0229] Quality control data for HER2-targeted site-specific glycoconjugate ADCs ADC name Target payload Molecular weight (Da) Drug-Antibody Ratio (HIC) SEC(%) Tmab-G7-MMAE HER2 MMAE 149926.45 1.95 99.29 Tmab-G9-MMAE HER2 MMAE 150041.06 2 98.21 Tmab-G22-MMAE HER2 MMAE 153293.84 3.64 99.61 Tmab-G23-MMAE HER2 MMAE 153293.86 3.84 99.60 Tmab-G24-MMAE HER2 MMAE 153699.92 3.71 99.86 Tmab-G28b-MMAE HER2 MMAE 153922.45 3.7 99.77

[0230] 2. In vitro efficacy of HER2-targeted site-specific glycoconjugate ADCs

[0231] The purpose of this experiment is to measure the in vitro inhibitory activity of the ADC compounds of the present disclosure against SK-BR-3 (human breast adenocarcinoma cells), NCI-N87 (human gastric cancer cells), and MDA-MB-468 (human breast cancer cells).

[0232] Tumor cells in the logarithmic growth phase SK-BR-3 (Source: Cell Resource Center, Shanghai Institute of Life Sciences, Chinese Academy of Sciences), NCI-N87 (Source: Cell Resource Center, Shanghai Institute of Life Sciences, Chinese Academy of Sciences), or MDA-MB-468 (Source: Cell Bank of Type Culture Collection of the Chinese Academy of Sciences) were added to cell plates at a cell count of 2,000 cells per well, and the cell plates were incubated in a 37°C 5% CO2 cell incubator for 12–16 hours. 100 μL of the sample (5-fold dilution starting from 50 μg / mL, 9 concentrations) was added to each well, gently shaken, and incubated in the incubator. After 144 hours of incubation, 70 μL of CellTiter-Glo™ (Product No.: G7572) working solution was added, gently shaken to lyse the cells, and read using a microplate reader. Formula for calculating cell proliferation inhibition rate: Cell proliferation inhibition rate = (1 - Sample well / Control well) × 100%. Using GraphPad Prism 8.0 software, plot the log value of the sample concentration on the horizontal axis and the cytotoxicity% on the vertical axis, and analyze the data using nonlinear regression (curve fit) to determine the IC of each test substance. 50 Values ​​were obtained. The specific results are shown in Table 5.

[0233] Results of measuring the in vitro antitumor activity of the ADC compounds of the present disclosure ADC compounds SK-BR-3(HER2+++) NCI-N87(HER2+++) MDA-MB-468(HER2-) IC 50 (μg / mL) Max inhibition(%) IC 50 (μg / mL) Max inhibition(%) IC 50 (μg / mL) Max inhibition(%) Tmab-G7-MMAE 0.004 92 0.005 81 44 60 Tmab-G9-MMAE 0.003 92 0.003 80 34 76 Tmab-G22-MMAE 0.002 92 0.003 82 35 77 Tmab-G23-MMAE 0.001 92 0.002 84 31 81 Tmab-G24-MMAE 0.001 92 0.002 82 19 84 Tmab-G28b-MMAE 0.002 92 0.003 81 15 86

[0234] As can be seen in Table 5, the ADC drug of the present disclosure exhibited significant proliferation inhibitory activity against HER2-positive cells SK-BR-3 and NCI-N87, while showing excellent selectivity with weak proliferation inhibitory activity against HER2-negative cells MDA-MB-468.

[0235] 3. In vivo efficacy of HER2-targeted site-specific glycoconjugated ADCs

[0236] Using BALB / c Nude mice (purchased from Beijing Weitong Lihua) as test animals, the efficacy of the drug was evaluated in nude mice carrying human gastric cancer cell NCI-N87 xenograft tumors after tail vein injection of an anti-HER2 ADC.

[0237] NCI-N87 cells in the right axilla of a mouse (Source: ATCC) (5×10 6 / animal (containing 50% stromal gel) was inoculated subcutaneously, and tumors grew for 7 days with an average tumor volume of approximately 150 mm³ 3 When reached, animals were randomly grouped according to tumor volume (D7), 7 animals / group.

[0238] It was administered once via tail vein injection, and tumor volume and body weight were measured and data recorded twice a week. Tumor growth inhibition rate TGI(%)=[1-(T 28 -T7) / (V 28 -V7)]×100, where T 28 and T7 are the tumor volumes of the experimental group on days 28 and 7 after inoculation, respectively, and V 28 V7 and V7 are the tumor volumes of the blank control (Vehicle, PBS) on days 28 and 7 after inoculation, respectively. The specific experimental results at the end of the experiment on day 28 after inoculation are shown in Table 6 and Figures 4 and 5.

[0239] Evaluation of the in vivo efficacy of the ADC compounds of the present disclosure ADC compounds Dosage Inhibition rate Tmab-G7-MMAE 4 mpk 68.6% Tmab-G9-MMAE 4 mpk 62.4% Tmab-G22-MMAE 2 mpk 85.3% Tmab-G23-MMAE 2 mpk 82.2% Tmab-G24-MMAE 2 mpk 74.1% Tmab-G28b-MMAE 2 mpk 68.0% Tmab-G7-MMAE 8 mpk 106.5% Tmab-G9-MMAE 8 mpk 90.6% Tmab-G22-MMAE 4 mpk 115.6% Tmab-G23-MMAE 4 mpk 111.5% Tmab-G24-MMAE 4 mpk 110.8% Tmab-G28b-MMAE 4 mpk 96.4%

[0240] As can be seen in Table 6, the ADC drug prepared using a disaccharide linker exhibited excellent antitumor activity against HER2-highly expressing NCI-N87 xenograft tumors, which indicates that using the enzyme of the present disclosure in the preparation of a glycan-reconstructed ADC has industrial feasibility.

[0241] The embodiments of the present disclosure described above are exemplary, and those skilled in the art will be able to recognize or determine numerous equivalents of specific compounds, materials, and processes without special experimentation. All such equivalents are included within the scope of the present disclosure and are included in the claims.

Claims

Claim 1 As an enzyme, having an amino acid sequence represented by SEQ ID NO: 1, said enzyme exhibiting glycan hydrolysis and / or glycan transfer activity. Claim 2 A method for reconstructing a glycosyl chain for a polypeptide or protein, comprising: 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 a disaccharide conjugate; and d) transferring the disaccharide linker or a disaccharide conjugate to the polypeptide or protein according to step (b) using the Endo Se2 or Endo Si to provide a novel polypeptide or protein with a modified glycosyl chain, wherein the sequence of the Endo Se2 is denoted by SEQ ID NO: 1 and the sequence of the Endo Si is denoted by SEQ ID NO:

2. Claim 3 A method for reconstructing sugar chains for a polypeptide or protein, wherein the polypeptide or protein is an antibody or a protein comprising the Fc region of an antibody, in paragraph 2. Claim 4 A method for reconstructing sugar chains for a polypeptide or protein, wherein the polypeptide or protein containing at least one N-acetylglucosamine or core fucosylated N-acetylglucosamine is obtained by hydrolysis of an N-glycan-containing polypeptide or protein using an endoglycosidase, or by recombinant expression or chemical synthesis. Claim 5 In paragraph 4, the N-glycan is a natural or non-natural complex, gomannose, or hybrid N-glycan, and the method for reconstructing the sugar chains for polypeptides or proteins. Claim 6 In claim 4, the endoglycosidase is a method for reconstructing sugar chains for polypeptides or proteins comprising Endo Se2, Endo Si, Endo S, Endo S2, Endo F3 and mutants thereof. Claim 7 A method for reconstructing sugar chains for a polypeptide or protein in one step by introducing Endo Se2 or Endo Si into an N-glycan-containing polypeptide or protein, in accordance with claim 4. Claim 8 A method for reconstructing sugar chains in a polypeptide or protein according to claim 4, wherein sugar chain reconstruction in a single step is achieved by simultaneously introducing Endo Se2 or Endo Si and one or more other endoglycosidases into an N-glycan-containing polypeptide or protein. Claim 9 A method for reconstructing sugar chains for a polypeptide or protein, wherein the disaccharide linker or disaccharide conjugate comprises at least one oxazolinated or thiazolinated monosaccharide or a monosaccharide modified / modified with a bioorthogonal functional group. Claim 10 A method for reconstructing sugar chains for a polypeptide or protein according to claim 9, wherein the disaccharide linker or disaccharide conjugate is selected from the following structures: . Claim 11 Use of Endo Se2 represented by SEQ ID NO: 1 or Endo Si represented by SEQ ID NO: 2 used to transfer disaccharide linkers or disaccharide conjugates in sugar chain modification.

Images