Glycan linkers for glycan site-specific antibody-drug conjugates and preparation thereof

Abstract

The present disclosure relates to a glycan linker and a method for preparing a glycoengineered antibody conjugated with payloads. Also provided herein is an antibody conjugate prepared using the glycoengineered antibody and a preparation method thereof. The present disclosure further provides antibody-drug conjugates (ADCs) targeting several antigens (e.g., TROP2, HER2, Nectin-4, etc.) and pharmaceutical compositions comprising said ADCs. Also described herein are methods of using the ADCs for treatment of diseases such as cancers.

Description

GLYCAN LINKERS FOR GLYCAN SITE-SPECIFIC ANTIBODY-DRUG CONJUGATES AND PREPARATION THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of U. S. Provisional Patent Applications No. 63 / 725,551 (filed on November 27, 2024). The entirety of the aforementioned application is incorporated herein by reference.FIELD

[0002] The present disclosure relates to glycan linkers, glycoengineered antibodies and antibody conjugates utilizing the glycan linkers, and preparation methods and uses thereof.BACKGROUND OF THE INVENTION

[0003] A great deal of interest in cancer therapies is focused on the use of monoclonal antibodies (mAbs) for the targeted delivery of cytotoxic agents to cancer cells. The design of antibody-drug conjugates (ADCs), by attaching a drug to an antibody, typically via a linker, involves consideration of a variety of factors. These factors include the types of antibodies and linkers for the conjugation of drugs and the conjugation mechanisms. ADCs developed in the early stage were generally prepared by random coupling of drugs, leading to poor in vivo stability and therapeutic efficacy.

[0004] Currently, site-specific coupling technologies are more widely adopted to produce sitespecific ADCs, such as glycan site-specific ADCs. The glycan site-specific ADCs can be prepared by glycan site-specific coupling technologies, which allow cytotoxic agents to be conjugated with an antibody through linkers extending from the glycosylation site in the Fc domain of antibody. The glycan site-specific coupling methods mainly include enzymatic glycan remodeling using glycosyltransferases and endoglycosidases, enabling removing the N-glycan mixtures on antibodies and transferring homogeneous glycan linkers to the antibody Fc region to generate homogeneous glycoengineered antibodies for subsequent drug conjugation. Accordingly, there is an urgent need for novel glycan linkers that permits high drug conjugation efficiency.SUMMARY OF THE INVENTION

[0005] The present disclosure concerns glycan linkers specifically designed for preparing homogeneous antibody conjugates (such as ADCs), the glycoengineered antibodies modified with the glycan linkers, and the antibody conjugates prepared with the glycoengineered antibodies, as well as methods for preparing homogeneous antibody conjugates through deglycosylation andtransglycosylation reactions utilizing the glycoengineered antibodies and glycosynthases and variants thereof.

[0006] In one aspect, the present disclosure provides a glycan linker represented by Formula (II):Oxa-G IcN Ac-Q-(G)n-(N 6u5Ac)m“L^ -(X-(Y-Z)8)^In Formula II, G represents a glycan moiety connected to an N-acetylglucosamine oxazoline (Oxa-GlcNAc) through Q, wherein the glycan moiety is a linear or branched chain of saccharides selected from the group consisting of galactose (Gal), N-acetyl-glucosamine (GlcNAc), N-acetylneuraminic acid (Neu5Ac), glucose (Glc), mannose (Man), fucose (Fuc), and derivatives thereof;Q is selected from the group consisting of O, S, Se, CH2, NH, or NRP, wherein Rpis a protecting group;Li is connected between -COOH of Neu5Ac and X and selected from:X includes 1 -200 atoms, and is selected from the group consisting of linear or branch alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl network with amino, amide, carbamate, ether, ester, sulfonamide functional group;Y is a linear alkyl or heteroalkyl chain having 1-100 carbon atoms;Z is selected from -N3, dibenzocyclooctyne (DBCO), trans-cyclooctene (TCO) or a ketone group; a and b are independently 1, 2, 3 or 4;m is a number of Neu5Ac units branching from G, and is an integer from 1 to 10; andn is an integer and selected from 1 to 20.

[0007] In another aspect, the glycan linker has a structure of the following formula (III);R-'I a-linkagewherein R1is -H or N-acetylglucosamine attached via a [3-1,4 linkage, and R2and R3are same or different and are independently selected from the group consisting of:04 03 £4 02 04 03 04 £2a3 / 6 04 03 04 02 a3 / 6 04 03 04 02A Fucose N-acetylneuraminic acid V Galactose ® Mannose ■ N-acetylglucosamine. wherein the glycosidic bond is an oc-glycosidic bond or an [3-glycosidic bond.

[0008] In another aspect, the present disclosure provides a method for preparing a glycoengineered antibody, including conjugating an antibody with the glycan linker disclosed herein in the presence of a glycosynthase to obtain the glycoengineered antibody, wherein the antibody has a fucosylated or non-fucosylated N-acetylglucosamine linked to an asparagine residue (e.g., N297 of human IgG heavy chain constant region) of the antibody. The conjugation between the antibody and the glycan linker disclosed herein is accomplished through deglycosylation and transglycosylation reactions catalyzed by one or more glycosynthases and variants thereof. In certain embodiments, the glycosynthase variants are EndoSd-D232M and EndoSz-D234M. Exemplary EndoSd-D232M and EndoSz-D234M are as described in PCT patent publication W02020006176A1, the content of which is incorporated herein by reference in its entirety.

[0009] In another aspect, the present disclosure provides a method for preparing an antibody conjugate, including reacting a glycoengineered antibody disclosed herein with a linker-payload to obtain the antibody conjugate, wherein the linker-payload includes a biorthogonal group, such as a bicyclononyne (BCN) group or a dibenzocyclooctyne (DBCO) group. The glycoengineered antibody being modified to carry the glycan linker disclosed herein can conjugated with the linkerpayload by click chemistry. This method offers a unique glycan site-specific antibody conjugate (e.g., a glycan site-specific ADC) platform that is distinct from conventional ones by high product homogeneity. Therefore, site-specific ADCs with homogeneous DAR (drug-to-antibody ratio) can be prepared readily. These glycan site-specific ADCs have favorable manufacturing, quality control, and in vivo pharmacokinetic profiles.

[0010] In another aspect, the present disclosure provides a pharmaceutical composition, including the antibody conjugate described herein and a pharmaceutically acceptable carrier.

[0011] In another aspect, the present disclosure provides a method for treating a disease such as cancers, infectious diseases, nervous system disorders, inflammatory disorders, or immune disorders, including administering to a subject in need thereof an effective amount of the antibody conjugate described herein, wherein the antibody conjugate is selected from a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitory agent, a protein degrader, a peptide, a radionuclide, a hormone, an anti-viral agent, an anti-bacterial agent, or an immunoregulatory agent.

[0012] In certain embodiments, the disease is characterized by expressing CD44, DLL3, CEACAM-5, CEACAM-6, ROR1, NaPi2b, CLDN18.2, CLDN1, CLDN2, CDH6, CDH17, B7-H3, MUC-1, PD-1, PD-L1, CTLA-4, VEGF, BCMA, PSMA, CGRP, Tfrl, TNF alpha, tissue factor, folate receptor alpha, cMET, HER3, EGFR, HER2, TROP2, Nectin-4, CD326, CDH1, NCAM1, TEM1, SSEA-4, MUC4, MUC5AC, MUC16, GPC3, GPC2, FGFR1, FGFR2, FGFR3, PDGFR-a, PDGFR-b, IGF-1R, AXL, LAG-3, TIM-3, TIGIT, VISTA, B7-H4, CD 19, CD20, CD22, CD24, CD38, CD47, CD33, CD70, CD123, CD133, ITGB1, ITGB5, ITGB6, ITGB8, CAIX, NRP1, NRP2, GD2, GD3, MSLN, EphA2, EphB4, c-KIT, ASCT2, IL 1 RAP, Ly6E, PTK7, uPARAP, CRLF2, SEZ6, TM4SF1, STEAP1, STEAP2, CADM1, CDCP1, GCC or LIV-1, and other antigens. In certain embodiments, the disease is a cancer selected from the group consisting of sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, head-and-neck cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervix cancer, endometrial cancer, ovarian cancer, testicular cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer, prostate cancer, thyroid cancer, and oral cancer.

[0013] The details of one or more embodiments of the invention are set forth in the description below. The features or advantages of the present invention will be apparent from the detailed description of preferred embodiments and the accompanying drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figures 1A-1C. The SDS-PAGE results verifying mAb-(NSCT-di-N3)2 or mAb-(NSCT-tetra-N3)2 production. FIG. 1A shows the SDS-PAGE analysis of R4702 antibody (anti-TROP2 mAb) and its glycoengineered products; FIG. 1A shows the SDS-PAGE analysis of TX05 antibody (anti-HER2 mAb) and its glycoengineered products, FIG. IB shows the SDS-PAGE analysis of R4702 antibody (anti-TROP2 mAb) and its glycoengineered products, and FIG. 1Cshows the SDS-PAGE analysis of 10K06 antibody (anti-Nectin 4 mAh) and its glycoengineered products.

[0015] Figures 2A-2F. The CE-SDS results verifying mAb-(NSCT-di-N3)2 or mAb-(NSCT-tetra-Ns)2 production. FIGs. 2A-2B shows the CE-SDS electropherograms for the TX05 antibody (anti-HER2 mAb). FIGs. 2C-2D shows the CE-SDS electropherograms for the R4702 antibody (anti-TROP2 mAb) and its glycoengineered products; FIGs. 2E-2F shows the CE-SDS electropherograms for the 10K06 antibody (anti-Nectin 4 mAb) and its glycoengineered products.

[0016] Figures 3A-3C. The schematic diagram illustrating the process of preparing glycan sitespecific antibody conjugates; FIG.3B shows an exemplary glycan site-specific ADC with a DAR (drug-to-antibody ratio) of four; and FIG. 3C shows an exemplary glycan site-specific ADC with a DAR of eight.

[0017] Figure 4. The cytotoxicity of exemplary R4702 DAR2 ADC (R4702-hexasaccharide-N3 conjugated with BCN-GGVA-Hydra-PAB-PEG24-Exatecan) in pancreatic adenocarcinoma cells BxPC-3.

[0018] Figure 5. The cytotoxicity of exemplary R4702 ADCs with different bioorthogonal groups in pancreatic adenocarcinoma cells BxPC-3.

[0019] Figures 6A-6D. Tumor inhibition comparison between OBI-902 and Dato-Dxd. FIG.6A: NCI-N87 human gastric cancer cell-derived xenograft; FIG. 6B: DLD-1 human colorectal cancer cell-derived xenograft; FIG.6C: HP AC human PDAC (pancreatic ductal adenocarcinoma) cell-derived xenograft; FIG. 6D: TFK-1 human Cholangiocarcinoma cell-derived xenograft.

[0020] Figures 7A-7B. The in-vitro cytotoxicity IC50 values of anti-Nectin-4 ADCs in human colorectal cancer cell line (DLD-1; FIG. 7A) and human hypopharyngeal squamous cancer cell line (FaDu; FIG. 7B).

[0021] Figures 8A-8C. The in-vivo efficacy assay of anti-Nectin-4 ADCs in Nectin-4 overexpression PC-3 human prostate cancer cell-derived xenograft in BLAB / c nude mice (FIG.8 A), Nectin-4 middle expressed FaDu human head and neck cancer cell-derived xenograft in BLAB / c nude mice (FIG. 8B) and Nectin-4 low expressed TFK-1 human cholangiocarcinoma cell-derived xenograft in BLAB / c nude mice (FIG. 8C).DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Abbreviations

[0022] ACN: acetonitrile; ADC: Antibody-drug conjugate; BCN: Bicyclononyne; bsADC: bispecific ADC; CDR: complementarity-determining region; CE-SDS: capillary electrophoresis-sodium dodecyl sulfate; DAR: drug-to-antibody ratio; DBCO: Dibenzocyclooctyne; DL: drug-linker compound; DMSO: dimethyl sulfoxide; FA: formic acid; GlcNAc: N-acetylglycosamine; HC: heavy chain; HIC: hydrophilic interaction chromatography; HRMS: high resolution mass spectrometry; LC: light chain; mAh: monoclonal antibody; Neu5Ac: N-acetylneuraminic acid; NSCT: sialylated complex type N-glycan; PAB: Para-aminobenzyl alcohol; PBS: phosphate buffered saline; TFA: trifluoroacetic acid; T785: 1-(4-aminobutyl)-2-butylimidazo[4,5-c]quinolin-4-amine; PSar20: polysarcosine 20; MMAE: monomethyl auristatin E; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; TCO: trans-cyclooctene.Definitions

[0023] As used herein, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise.

[0024] As used herein, the term “glycan” refers to a polysaccharide, oligosaccharide or monosaccharide. Glycans can be monomers or polymers of sugar residues and have a linear or branched structure. A glycan may include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2’ -fluoro ribose, 2’ -deoxyribose, phosphomannose, 6’ sulfo N-acetylglucosamine, etc.).

[0025] As used herein, the terms “N-glycan” and “N-linked glycan” are used interchangeably and refer to an N-linked oligosaccharide attached via an N-acetylglucosamine (GlcNAc) to the amide nitrogen of an asparagine residue in a protein or an antibody.

[0026] As used herein, the terms “glycosylation pattern” and “glycosylation profile” are used interchangeably and refer to the characteristic “fingerprint” of the N-glycan species on a glycoprotein or antibody. The glycosylation profile can be obtained by collecting a N-glycan species released from a glycoprotein through enzymatic digestion or chemical hydrolysis, and then analyzing the carbohydrate structure, for example, LC-HPLC, or MALDI-TOF MS, and the like.

[0027] As used herein, the term “antigen” is defined as any substance capable of eliciting an immune response.

[0028] As used herein, the term “epitope” is defined as the parts of an antigen molecule which contact the antigen binding site of an antibody or a T cell receptor.

[0029] As used herein, the term “antigen specific” refers to a property of a cell population such that supply of a particular antigen, or a fragment of the antigen, results in specific cell proliferation.

[0030] As used herein, the term “specific binding,” refers to the interaction between binding pairs (e.g., an antibody and an antigen). In various instances, specifically binding can be embodied by an affinity constant of about 10'6moles / liter, about 1 O'7moles / liter, or about 10'8moles / liter, or less.

[0031] The phrase “substantially similar,” “substantially the same”, “equivalent”, or “substantially equivalent”, as used herein, denotes a sufficiently high degree of similarity between two numeric values (for example, one associated with a molecule and the other associated with a reference / comparator molecule) such that one of skill in the art would consider the difference between the two values to be of little or no biological and / or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values, anti-viral effects, etc.). The difference between said two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and / or less than about 10% as a function of the value for the reference / comparator molecule.

[0032] A “disorder” is any condition that would benefit from treatment with an ADC of the present disclosure. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include cancer.

[0033] The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0034] The term “tumor” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein.

[0035] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More particular examples of such cancers include lung cancer, breast cancer, head-and-neck cancer, esophagus cancer, stomach cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervix cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer and oral cancer.

[0036] As used herein, “treatment” refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing or decreasing inflammation and / or tissue / organ damage, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0037] An “individual” or a “subject” is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs, and horses), primates, mice and rats. In certain embodiments, the vertebrate is a human.

[0038] A “combination therapy” refers to a combination of an amount of an ADC and an amount of other biological or chemical drugs that when administered together (either as coadministration and / or co-formulation), either sequentially or simultaneously, on the same or different days during a treatment cycle, have a synergistic effect that is therapeutically effective and more than therapeutically additive.

[0039] The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. Cytotoxic agents include radionuclides such as radioactive isotopes (e.g.,211At,1311,1251,90Y,186Re,188Re,153Sm,212Bi,32P,60Co, and radioactive isotopes of lutetium- 177, strontium-89 and samarium (153Sm) ), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogs and derivatives thereof.

[0040] The term “photodynamic therapy (PDT)’, sometimes called photochemotherapy, is a form of phototherapy involving light and a photosensitizing chemical substance, used in conjunction with molecular oxygen to elicit cell death (phototoxicity). It is used clinically to treat a wide range of medical conditions, including wet age-related macular degeneration, psoriasis, atherosclerosis and has shown some efficacy in anti-viral treatments, including herpes. It also treats malignant cancers including head and neck, lung, bladder, skin and prostate cancer (Wang, SS et al. Cancer Journal. 8 (2): 154-63. 2002). The “photodynamic therapeutic agent” is selected from Photofrin, Laserphyrin, Aminolevulinic acid (ALA), Silicon Phthalocyanine Pc 4, m-tetrahydroxyphenylchlorin (mTHPC), chlorin e6 (Ce6), Allumera, Levulan, Foscan, Metvix, Hexvix, Photochlor, Photosens, Photrex, Lumacan, Visonac, Amphinex, Verteporfin, Purlytin, ATMPn, Zinc phthalocyanine (ZnPc), Protoporphyrin IX (PpIX), Pyropheophorbidea (PPa) or Pheophorbide a (PhA).

[0041] A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), mertansine (also called DM1), anthracy cline, pyrrolobenzodiazepine, a-amanitin, tubulysin, benzodiazepine, erlotinib (TARCEVA®), Genentech / OSI Pharm.), bortezomib (VELCADE®, Millenium Pharm.), fulvestrant (FASLODEX®, Astrazeneca), sunitinib (SUTENT®, SU11248, Pfizer), letrozole (FEMARA®), Novartis), imatinib mesylate (GLEEVEC®, Novartis), PTK787 / ZK 222584 (Novartis), oxaliplatin (ELOXATIN®, Sanofi), leucovorin,rapamycin (Sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), lonafarnib (SARAS AR®, SCH 66336), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs.), and gefitinib (IRESSA®, Astrazeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and rnethylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone), camptothecin (including the synthetic analogue topotecan), bryostatin, callystatin, CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues), cryptophycins (particularly cryptophycin 1 and cryptophycin 8), dolastatin, duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1), eleutherobin, pancratistatin, sarcodictyin, spongistatin, nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall), dynemicin, including dynemicin A; aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elformithine, elliptinium acetate, epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids such as maytansine and ansamitocins; mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet,pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2''-trichlorotriethylamine, trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine), urethan, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (“Ara-C”), cyclophosphamide, thiotepa, taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N. J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil, GEMZAR® gemcitabine, 6-thioguanine, mercaptopurine, methotrexate, platinum analogs such as cisplatin and carboplatin; vinblastine, platinum, etoposide (VP- 16), ifosfamide, mitoxantrone, vincristine, NAVELBINE® vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO), retinoids such as retinoic acid, capecitabine (XELODA®, Roche), and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0042] The term “prodrug” as used herein refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to its parent form and is capable of being enzymatically activated or converted into the more active parent form. Examples of prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified pro drugs, glycosylated prodrugs, [3-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine, or other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic drug. Examples of cytotoxic drugs that can be derivatized into a prodrug form for use in ADCs include, but are not limited to, those chemotherapeutic agents described above.

[0043] The phrase “pharmaceutically acceptable salt,” as used herein, refers to pharmaceutically acceptable organic or inorganic salts of a cytotoxic agent or an ADC. Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., l, T-methylene-bis-(2-hydroxy-3 -naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion maybe any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterion.

[0044] The term “pharmaceutically acceptable solvate” refers to an association of one or more solvent molecules,a cytotoxic agent or an ADC. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.Glycan linkers, glycoengineered antibodies, and antibody conjugates

[0045] The present disclosure provides an antibody conjugate that can carry one or more payloads through linkers attached to one or more specific glycosylation sites in an antibody or an antigen-binding fragment thereof. The antibody conjugate may be prepared by conjugating a linker-payload with a glycoengineered antibody or an antigen-binding fragment thereof. The glycoengineered antibody can be obtained by coupling a glycan linker disclosed herein with an antibody having a fucosylated or non-fucosylated N-acetylglucosamine (GlcNAc) linked to an asparagine residue of the antibody. Depending on the nature of the linker-payload, which may be a drug-linker compound (DL) that includes a drug unit as the payload or a probe-linker that includes a probe unit, the antibody conjugate may be an antibody-drug conjugate or an antibodyprobe conjugate.

[0046] The term “payload” as used herein refers to a molecule that is to be carried and delivered by an antibody or an antigen-binding fragment thereof. The term “drug unit” refers to a drug molecule (e.g., a cytotoxic agent), an inhibitor of an enzyme, a ligand of a receptor, a pharmaceutically acceptable salt, or a prodrug thereof. The term “probe unit” refers to a probe molecule that assists the visualization of target cells or tissues or a body part of a subject.

[0047] The antibody conjugate disclosed herein may be represented by Formula (I):Ab’-(L-D)c(I);wherein Ab’ is a glycoengineered antibody or an antigen-binding fragment thereof capable of binding to one or more of tumor-associated antigens or cell-surface receptors;L is a linker connecting D and Ab’ and comprising a protease-cleavable peptide moiety, a glycosidase-cleavable sugar moiety, a pH sensitive moiety, or a hydrolysable moiety;D is a payload and selected from a chemotherapeutic agent, a toxin, a cytokine, a growth inhibitory agent, a protein degrader, a peptide, a radionuclide, a hormone, an anti-viral agent, an anti-bacterial agent, an immunoregulatory agent, a fluorophore, a dye, or a contrast agent; andc is a drug-to-antibody ratio (DAR) ranging from 1 to 128.In certain embodiments, the symbol c of Formula (I) means the drug-to-antibody ratio (DAR) ranging from 1 to 2, 1 to 4, 1 to 8, 1 to 16, 1 to 32, 1 to 64, or about 1 to 128.

[0048] Specifically, the glycoengineered antibody in Formula (I) is an antibody modified to incorporate a glycan derivative as a glycan linker for subsequent conjugation with a linker-payload. Generally, the glycan linker has a structure of Formula (II):In Formula II, G represents a glycan moiety connected to an N-acetylglucosamine oxazoline (Oxa-GlcNAc) through Q, wherein the glycan moiety is a linear or branched chain of saccharides selected from the group consisting of galactose (Gal), N-acetyl-glucosamine (GlcNAc), N- acetylneuraminic acid (Neu5Ac), glucose (Glc), mannose (Man), fucose (Fuc), and derivatives thereof;Q is selected from the group consisting of O, S, Se, CH2, NH, or NRP, wherein Rpis a protecting group;Li is connected between -COOH of Neu5Ac and X and selected from:O, O O, \H 9 H 9 *X includes 1 -200 atoms, and is selected from the group consisting of linear or branch alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl network with amino, amide, carbamate, ether, ester, sulfonamide functional group;Y a linear alkyl or heteroalkyl chain having 1-100 carbon atoms;Z is selected from -N3, dibenzocyclooctyne (DBCO), trans-cyclooctene (TCO) or a ketone group;a and b are independently 1, 2, 3 or 4;m is a number of Neu5Ac units branching from G, and is an integer from 1 to 10; andn is an integer and selected from 1 to 20.

[0049] The glycan moiety G in Formula (II) may be linear or branched. In other embodiments, each of the two terminal saccharide residues of G is linked to the X-Y-Z in the glycan linker of the following formula (III):The two X-Y-Z portions in this structure may be identical or different. For example, X, Y, or Z may be identical or different; or the number a of the Y-Z fragment attached to each X may be identical or different. Furthermore, a is an integer and selected from 1 to 4.

[0050] For example, the glycan moiety G in Formula (II) may have the structure of Formula (IV):AcHN(IV);The symbol ~ means the connection with Neu5Ac and symbol ★ means the connection with Q.

[0051] For example, the glycan moiety G in Formula (II) may have the structure of Formula (V):OH?■ •o?t°HHQ-F~'HO'^-TYO' / ACHN--Z-T** / m HO-JHO NHAcThe symbol ~ means the connection with Neu5Ac and symbolmeans the connection with Q.

[0052] In certain embodiments, the glycan moiety G may have the structure of Formula (VI) or Formula (VII):NHAc (VI), orThe symbol ~ means the connection with Neu5Ac and symbol ★ means the connection with Q.

[0053] In certain embodiments, the glycan linker has the structure of Formula (VIII) (also termed NSCT-2):

[0054] In certain embodiments, the glycan linker has the structure of Formula (IX) (also termed NSCT-4):(IX).

[0055] In certain embodiments, the glycan linker has the structure of Formula (X) (also termed NSCT-14):

[0056] In certain embodiments, the glycan linker has the structure of Formula (XI) (also termed NSCT-16):(XI).

[0057] In certain embodiments, the glycan linker has the structure of Formula (XII) (also termed NSCT-6):(XII).

[0058] In certain embodiments, the glycan linker has the structure of Formula (XIII) (also termed NSCT-8):o o(XIII).

[0059] In certain embodiments, the glycan linker has the structure of Formula (XIV) (also termed NSCT-10):

[0060] In certain embodiments, the glycan linker has the structure of Formula (XV) (also termed NSCT-12):

[0061] The present disclosure also provides an antibody conjugate represented by Formula (XVI):(Fuc)dGlcNAc — GlcNAc — Q~(G)^(Neu5Ac)m-Lr(X-(¥-Z’‘LrDyb—jc(XVI); wherein Ab is an antibody or an antigen-binding fragment thereof;GlcNAc is N-acetylglucosamine;Fuc is fucose;Q, G, Li, X, Y, a, b, m and n are as defined with respect to the glycan linker of Formula (II);Z’ is independently selected from a triazole or imine linkage;d is 0 or 1;c is an integer ranging from 1 to 128;L? is a linker connecting D and Z’ and comprising a protease-cleavable peptide moiety, a glycosidase-cleavable sugar moiety, a pH sensitive moiety, or a hydrolysable moiety;D is a payload selected from a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitory agent, a protein degrader, a peptide, a radionuclide, a hormone, an anti-viral agent, an anti-bacterial agent, an immunoregulatory agent, a fluorophore, a dye, or a contrast agent.

[0062] In certain embodiments, the symbol c of Formula (XVI) means the drug-to-antibody ratio (DAR) ranging from 1 to 2, 1 to 4, 1 to 8, 1 to 16, 1 to 32, 1 to 64, or about 1 to 128.

[0063] In certain embodiments, the antibody or antigen-binding fragment thereof of the ADC is monospecific or multispecific. In certain embodiments, the antibody or antigen-bindingfragment thereof is capable of specifically binding to CD44, DLL3, CEACAM-5, CEACAM-6, R0R1, NaPi2b, CLDN18.2, CLDN1, CLDN2, CDH6, CDH17, B7-H3, MUC-1, PD-1, PD-L1, CTLA-4, VEGF, BCMA, PSMA, CGRP, Tfrl, TNF alpha, tissue factor, folate receptor alpha, cMET, HER3, EGFR, HER2, TR0P2, Nectin-4, CD326, CDH1, NCAM1, TEM1, SSEA-4, MUC4, MUC5AC, MUC16, GPC3, GPC2, FGFR1, FGFR2, FGFR3, PDGFR-a, PDGFR-b, IGF-1R, AXL, LAG-3, TIM-3, TIGIT, VISTA, B7-H4, CD 19, CD20, CD22, CD24, CD38, CD47, CD33, CD70, CD123, CD133, ITGB1, ITGB5, ITGB6, ITGB8, CAIX, NRP1, NRP2, GD2, GD3, MSLN, EphA2, EphB4, c-KIT, ASCT2, IL 1 RAP, Ly6E, PTK7, uPARAP, CRLF2, SEZ6, TM4SF1, STEAP1, STEAP2, CADM1, CDCP1, GCC or LIV-1. In one preferred embodiment, the antibody or antigen-binding fragment thereof is bispecific to HER2 and TR0P2. In another embodiment, the antibody or antigen-binding fragment thereof is bispecific to c-MET and HER3. In another embodiment, the antibody or antigen-binding fragment thereof is bispecific to EGFR and HER3. In another embodiment, the antibody or antigen-binding fragment thereof is bispecific to EGFR and c-Met.

[0064] In certain embodiments, the payload is a cytotoxic agent selected from pyrrolobenzodiazepine compounds or derivatives thereof (e.g., PBD), auristatin compounds or derivatives thereof (e.g., MMAE, MMAF), maytansinoid compounds or derivatives thereof (e.g., maytansine, DM1, DM4, DM21), duocarmycin or derivatives thereof, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors or derivatives thereof, tubulysin compounds or derivatives thereof, enediyne compounds or derivatives thereof (e.g., calicheamicin), anthracycline compounds or derivatives thereof (e.g., doxorubicin), pyrrole-based kinesin spindle protein (KSP) inhibitors or derivatives thereof, cryptophycin compounds or derivatives thereof (e.g., cryptophycin 52), drug efflux pump inhibitors or derivatives thereof, sandramycin or derivatives thereof, amanitin compounds or derivatives thereof, or camptothecin compounds or derivatives thereof (e.g., SN-38, belotecan, exatecan, deruxtecan).

[0065] In certain embodiments, the linker-payload is used to couple with the glycoengineered antibody, and the coupling leads to an antibody conjugate of Formula (XVI) with L2-D having a structure of Formula (XVII-1) or (XVII-2):F (XVII-1);(XVII-2); wherein z is an integer ranging from 6 to 48.

[0066] In certain embodiments, the linker-payload is used to couple with the glycoengineered antibody, and the coupling leads to an antibody conjugate of Formula (XVI) with L2-D having a structure of Formula (XVIII- 1) or (XVIII-2):F(XVIII- 1);(XVIII-2);wherein z is an integer ranging from 6 to 48.Preparation of glycoengineered antibodies and antibody conjugates

[0067] The present disclosure provides a method for preparing a glycoengineered antibody, including conjugating an antibody with the glycan linker disclosed herein to obtain the glycoengineered antibody, wherein the antibody has a fucosylated or non-fucosylated N-acetylglucosamine linked to an asparagine residue of the antibody.

[0068] The conjugation of the antibody with fucosylated or non-fucosylated N-acetylglucosamine with the glycan linker is carried out usually in the presence of a glycosynthase. In some embodiments, the glycosynthase includes EndoSd-D232M and EndoSz-D234M.

[0069] The present disclosure further provides a method for preparing an antibody conjugate, including reacting the glycoengineered antibody disclosed herein with a linker-payload to obtain the antibody conjugate, wherein the glycoengineered antibody includes a fucosylated or non-fucosylated N-acetylglucosamine at an asparagine residue coupled to the glycan linker disclosed herein, and the linker-payload includes a biorthogonal group for conjugation with the glycan linker.

[0070] In certain embodiments, the biorthogonal group is selected from a dibenzocyclooctyne (DBCO) group, a bicyclononyne (BCN) group, a alkyne group, a maleimide group, a a, [3-unsaturated carbonyl group, a sulfonyl pyrimidine group, a 4-dibenzocyclooctynol (DIBO) group, a aza-dibenzocyclooctynes (DIBAC) group, a tetrazine group, a tetrazole group, a norbornene group, a cyclooctyne group, a methylcyclopropene group, an aminooxy group, a hydrazine group, an isocyanide group, an isocyanopropanoate group, a phosphine-containing thioester group, a phosphine phenolic ester group, or an alpha-halo carbonyl group.Therapeutic Applications

[0071] The present disclosure further provides a method for killing or inhibiting the proliferation of tumor cells or cancer cells, including contacting the cells with an effective amount of the ADC disclosed herein.

[0072] The present disclosure also provides a method for treating a disease, including administering to a subject in need thereof an effective amount of the ADC disclosed herein or a pharmaceutical composition that includes one or more of the ADCs described herein. By varying the characteristics of the ADC (such as the type of the antibody or the payload), the disease to be treated may be cancer, infectious diseases (including viral and bacterial infections), nervous system disorders, inflammatory disorders, or autoimmune disorders.

[0073] In some embodiments, the subject (e.g., a human patient) in need of the treatment is diagnosed with, suspected of having, or at risk for cancer. Examples of the cancer include, but are not limited to, lung cancer, breast cancer, head-and-neck cancer, esophagus cancer, stomachcancer, bladder cancer, pancreatic cancer, colorectal cancer, cervix cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer and oral cancer.

[0074] The treatment results in reduction of tumor size, elimination of malignant cells, prevention of metastasis, prevention of relapse, reduction or killing of disseminated cancer, prolongation of survival and / or prolongation of time to tumor cancer progression.

[0075] In some embodiments, the method for treatment further includes administering an additional therapy to said subject prior to, during or subsequent to said administering of the ADCs. In some embodiments, the additional therapy is treatment with a chemotherapeutic agent. In some embodiments, the additional therapy is radiation therapy, photodynamic therapy, chemotherapy, immunotherapy, targeted therapy, or hormone therapy.

[0076] The methods for treating cancers described herein are particularly advantageous in treating and preventing early stage tumors, thereby preventing progression to the more advanced stages resulting in a reduction in the morbidity and mortality associated with advanced cancer. The methods are also advantageous in preventing the recurrence of a tumor or the regrowth of a tumor, for example, a dormant tumor that persists after removal of the primary tumor, or in reducing or preventing the occurrence of a tumor.

[0077] The subject to be treated by the treatment methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice, or rats. A human subject who needs the treatment may be a human patient having, at risk for, or suspected of having cancer, which include, but are not limited to, lung cancer, breast cancer, head-and-neck cancer, esophagus cancer, stomach cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervix cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, or oral cancer. A subject having cancer can be identified by medical examination.

[0078] The phrase “an effective amount” refers to the amount of each active agent or a pharmaceutical composition required to achieve the desired therapeutic result (“therapeutically effective amount”) or the desired prophylactic result (“prophylactically effective amount”), either alone or in combination with one or more other active agents. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of theindividual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0079] As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents to a subject, who has a disease such as cancer, a symptom of cancer, or a predisposition toward cancer, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect cancer, the symptom of cancer, or the predisposition toward cancer, or to delay the development or progression of cancer. “Development” or “progression” of cancer means initial manifestations and / or ensuing progression of cancer. Development of cancer can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein, “onset” or “occurrence” of cancer includes initial onset and / or recurrence.Applications in imaging

[0080] The present disclosure further provides a method of selecting a subject for a cancer therapy by imaging, including:(a) administering to the subject an effective amount of the antibody conjugate disclosed herein where the payload is the probe unit, wherein the probe unit is an imaging agent selected from a fluorophore, a dye, a contrast agent, or a radionuclide;(b) detecting visually or instrumentally a reporting signal of the imaging agent in the subject; and (c) identifying the subject as suitable for the cancer therapy when the reporting signal is detected.

[0081] In certain embodiments, the subject is diagnosed with, suspected of having, or at risk for cancer.

[0082] In certain embodiments, the method further comprises detecting metastasis of a cancer.Articles of Manufacture

[0083] In another embodiment, an article of manufacture, or “kit”, containing ADC and materials useful for the treatment of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, or blister pack. The containers may be formed from a variety of materials such as glass or plastic. The container holds an ADC composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable bya hypodermic injection needle). At least one active agent in the composition is an ADC. The label or package insert indicates that the composition is used for treating the condition of choice, such as cancer, infection, nervous system, inflammatory, or autoimmune disorders.

[0084] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.EXAMPLESExample 1. Preparation of NSCT derivates1-1. Preparation ofNSCT-2 (Oxazoline-NSCT-di-Ns)

[0085] NSCT-1 (235 mg, 0.097 mmol; purchased from Glytech, Inc. Catalog No. GT-25261; HPLC purity > 90%) and triethylamine (605 pL, 0.44 mmol) were dissolved in water (10 mL) and cooled to 0 °C. 2-chloro- 1,3 -dimethyl- lH-benzimidazol-3-ium chloride aqueous solution (1 M, 1.44 mL) was added slowly and the resulting mixture was stirred at 0 to 5 °C for four hours. NaOH solution was added (0.01 M, 1 mL) and the resulting mixture was concentrated under reduced pressure. After most of the triethylamine was evaporated, the residual mixture was purified by Sephadex® g-15 column. Using 0.01 M NaOH as eluent to stabilize NSCT-2. Fractions with desired product were combined and freeze-dried to afford NSCT-2 (170 mg) as a white solid. 1H NMR (D2O): 8 6.10 (d, J = 7.26 Hz, 1H, Hl of oxazoline), 5.24 (s, 1H, Hl of GlcNAc), 4.97 (s, 1H, Hl of GlcNAc), 4.76 (s, 1H, Hl of Man), 4.64-4.60 (m, 2H, Hl oftwo Gal), 4.46 (s, 1H, Hl of Neu5Ac), 4.45 (s, 1H, Hl of Neu5Ac), 4.40 (s, 1H, H3 of [3-form Man), 4.18 (d, J = 21 Hz, 4H, H2 of two Man and two GlcNAc), 3.99-3.48 (m), 2.71 (dd, JI = 12.9 Hz, J2 = 4.2 Hz, 2H, H3eq of two Neu5Ac), 2.15-2.02 (m, 15H), 1.58 (dd, JI = J2 = 12.2 Hz, 2H, H3ax of two Neu5Ac).\C1H2O THF TEA O°C1-2. Preparation of NSCT-3 (NSCT-tetra-Ns)

[0086] NSCT (CAS No. 58902-60-2, 384 mg, 0.19 mmol; purchased from Glytech, Inc.; HPLC purity > 90%), amino linker-3 (560 mg, 1.14 mmol) and benzotriazol- 1-yl-oxy -tripyrrolidino-phosphonium hexafluorophosphate (CAS No. 128625-52-5, 593 mg, 1.14 mmol) were mixed with DMSO (4 mL). Triethanolamine (CAS No. 102-71-6, 506 pL, 3.8 mmol) was added, and the mixture was heated at 40 °C overnight. Added water (10 mL) and washed with DCM (60 mL) twice. The aqueous layer was collected and concentrated under reduced pressure. The residual mixture was purified by C-18 preparative HPLC (eluent: ACN / Water). Fractions with the desired product were combined and freeze-dried to afford NSCT-3 (339 mg) as a white solid. 1H NMR (D2O): 85.22 (d, J= 3.3 Hz, 1H, Hl of GlcNAc), 5.14 (s, 1H, Hl of Man), 4.95 (s, 1H, Hl of Man), 4.73 (br, 1H, Hl of Man), 4.61 (d, J= 7.3 Hz, 2H, Hl of two GlcNAc), 4.45 (d, J= 7.9 Hz, 2H, Hl of two Gal), 4.27 (s, 1H, H2 of [3-form Man), 4.20 (s, 1H, H2 of a-form Man), 4.13 (s, 1H, H2 of a-form Man), 3.99-3.48 (m), 2.80-2.76 (m, 4H, CH2 next to carbonyl on amino linker), 2.70 (dd,Jl= 12.9 Hz, J2= 4.5 Hz, 2H, H3eq of two Neu5Ac), 2.10-2.00 (m, 15H, five NHAc), 1.85 (dd, J1=J2= 12.3 Hz, 2H, H3ax oftwo Neu5Ac). MASS [M+3Na]3+1011.08.NSCT-31-3. Preparation of NSCT-4 (Oxazoline-NSCT-tetra-Ns)

[0087] NSCT-3 (339 mg, 0.114 mmol) and triethylamine (715 pL, 5.12 mmol) were dissolved in water (3.7 mL) and cooled to 0 °C. 2-chloro- 1,3 -dimethyl- lH-benzimidazol-3-ium chloride aqueous solution (1 M, 1.71 mL) was added slowly and the resulting mixture was stirred at -2 to 0 °C for four hours. NaOH solution was added (0.01 M, 1 mL) and the resulting mixture was concentrated under reduced pressure. After most of the triethylamine was evaporated, the residual mixture was purified by Sephadex® G-25 column. 0.0 IM NaOH as eluent is used to stabilize the product. Fractions with the desired product were combined and freeze-dried to afford NSCT-4 (324 mg) as a white solid. 1H NMR (D2O): 8 6.10 (d, J= 7.4 Hz, 1H, Hl of oxazoline), 5.14 (s, 1H, Hl of Man), 4.97 (s, 1H, Hl of Man), 4.76 (s, 1H, Hl of Man), 4.63-4.60 (m, 2H, Hl oftwo GlcNAc), 4.45 (d, J= 7.5 Hz, 2H, Hl of two Gal), 4.40 (s, 1H, H3 of oxa-GlcNAc), 4.20-4.16 (br, 4H, H2 of three Man and H2 of oxa-GlcNAc), 3.99-3.48 (m), 2.80-2.76 (m, 4H, CH2 next to carbonyl on amino linker), 2.70 (dd, Jl= 12.2 Hz, J2= 3.6 Hz, 2H, H3eq of two Neu5Ac), 2.08-2.02 (m, 15H, five NHAc), 1.85 (dd, JI =J2= 12.4 Hz, 2H, H3ax of two Neu5Ac).NSCT-3oNSCT-41-4. Preparation of amino linker-3

[0088] Fmoc-P-Ala-OH (CAS No. 35737-10-1, 382 mg, 1.23 mmol) and HATU (CAS No.148893-10-1, 544 mg, 1.43 mmol), and NH-Bis(PEG3-azide) amino linker-2 (CAS No. 1258939- 39-7, 500 mg, 1.19 mmol) were dissolved in DMF (5 mL) and cooled to 0 °C. N-methyl morpholine (393 pL, 3.57 mmol) was added slowly. The resulting solution was stirred at room temperature for twelve hours.

[0089] The reaction was monitored by reversed-phase C-18 TLC, indicating a complete conversion (eluent: ACN / water= 6 / 4, Rf value= 0.25). Add Diethylamine (CAS NO. 109-89-7) 1.5 mL to the intermediate solution and stir at room temperature for two hours. The reaction was monitored by SiO2TLC, indicating a complete conversion (eluent: DCM / MeOH / NH4OH= 90 / 10 / 1, Rf value= 0.3). After being concentrated under reduced pressure to evaporate most of the DMF, the residual mixture was purified by silica gel chromatography (eluent: DCM / MeOH / NH4OH= 920 / 80 / 8). Fractions with the desired product were combined,concentrated, and then freeze-dried to afford amino linker-3 (580 mg, 1.18 mmol) as a light-yellow oil. 1. 1H NMR 600 Hz, (CDC13): 8 3.66-3.38 (m, 28H, PEGs), 3.37 (t, J= 5.0 Hz, 4H, CH2 next to N), 2.96 (t, J= 5.6 Hz, 2H, CH2 next to NH2), 2.54 (t, J= 6.0 Hz, 2H, CH2 next to carbonyl), 1.94 (Br, NH2). Mass: [M+l]+= 491.30.O HATU NMM DMF - ► 2) Diethylamineamino linker-2 amino linker-31-5. Preparation of 2,3-disialyloctasaccharide

[0090] Combined 2 mg of NSCT and H2O in a total reaction volume of 35 pL, then add 5 pL of GlycoBuffer 1 (10X, 5 mM CaCh, 50 mM sodium acetate pH 5.5) to make a 50 pL total reaction volume. Added 10 pL (200 U) u2-3,6,8,9 Neuraminidase A (Catalog: P0722, New England Biolabs) to cleave the terminal ot-Neu5Ac from NSCT and incubated at 37 °C for sixteen hours. To a 75 pL, pH 9.0 solution of Tris buffer, corresponding intermediate octasaccharide (2 mg, 1.36 pmol), Neu5Ac (0.7 mg, 1.36 pmol), ATP (1.35 pg, 0.15 pmol), CTP (0.135 pg, 1.5 pmol), phosphoenolpyruvate (PEP, 1.035 mg, 3 pmol, monopotassium salt), cytidine monophosphate kinase (CMK, 0.025 U), CMP-sialic acid synthetases (NmCSS, 0.06 U), pyruvate kinase (PK, 0.02 U), inorganic pyrophosphatase (PPA, 0.02 U). and u-2,3-sialyltransferase (JT-FAJ-16, 0.075 U) were added. The glycosylation reaction was monitored by TLC until completion. After removing the proteins by MeOH and centrifugation, the residue was purified by column chromatography (WelFlash HILIC amide cartridge 25g, ACN / 15 mM ammonium formate= 85 / 15 to 65 / 35) to give product as a white solid.1H NMR (D2O, 600 MHz) 85.21 (d, 0.6H, J= 3.0 Hz), 5.12 (s, 1H), 4.92 (s, 1H), 4.79 (1H, overlapped with solvent signal), 4.72 (0.4H, overlapped with solvent signal), 4.58 (d, 2H, J= 7.8 Hz), 4.54 (d, 2H, J= 7.7 Hz), 4.28-4.23 (m, 1H), 4.21-4.17 (m, 1H), 4.15-4.08 (m, 3H), 4.06-3.43 (m, 57H), 2.75 (dd, 2H, J= 12.3, 4.5 Hz), 2.11=1.99 (m, 15H), 1.80 (pt, 2H, J= 12.3 Hz).OH HO2,3-disialyloctasaccharide1-6. Preparation of NSCT-5

[0091] NSCT (50.0 mg, 0.0247 mmol; purchased from Glytech Inc.) dissolved in DMF (2.0 mL) with DBCO-NH2(41.0 mg, 0.148 mmol), PyBOP (257.0 mg, 0.494 mmol) and DIPEA (63.8 mg, 0.494 mmol) was added. This mixture was stirred under room temperature for 20 hours. After the reaction was completed, the reaction mixture was extracted with DCM / H2O. The H2O layer was collected and lyophilized to afford crude product. The crude product was purified by reverse phase chromatography (eluent: ACN / Water). The pure fractions were combined and lyophilized to obtainNSCT-5 (41.5 mg, 66.2% yield). MS calculate for [C112H153N9O57 + 2Na+] / 2: 1290.9573, found: 1290.9553. 'HNMR (D2O): 8 7.69 (t, 2H), 7.52-7.59 (m, 12H), 7.48 (t, 2H), 7.43 (t, 2H), 7.37 (t, 2H), 5.12 (t, 4H), 4.59 (t, 4H), 4.43 (dd, 2H), 4.28 (s, 1H), 4.21 (s, 1H), 4.13 (s, 1H), 4.05-3.47(m), 3.38-3.46 (m, 2H), 3.35-3.23 (m, 2H), 3.21-3.12 (m, 2H) 2.58-2.42 (m, 3H), 2.09-2.00 (m), 1.76 (td, 1H).1-7. Preparation of NSCT-6 (Oxazoline-NSCT-DBCO)

[0092] A method similar to that described in NSCT-2 preparation was applied to obtain the oxazoline product NSCT-6 from NSCT-5.OH AcHN 1-8. Preparation ofNSCT-7 (NSCT-di-Ns-di-ketone)

[0093] Amino linker-11 (92.7 mg, 0.160 mmol, 4.0 equiv.) was dissolved in anhydrous DMF (1.6 mL) in a round bottom flask, followed by addition of NSCT (CAS No. 58902-60-2, 83.2 mg, 0.04 mmol, 1.0 equiv.; purchased from Glytech, Inc.), benzotriazol- 1 -yl-oxy-tripyrrolidino-phosphonium hexafluorophosphate (also termed ByPOB, CAS No. 128625-52-5, 209 mg, 0.402 mmol, 10.0 equiv.), and N, N-diisopropylethylamine (also termed DIPEA, CAS No. 7087-68-5, 72 pL, 0.402 mmol, 10.0 equiv.). The reaction mixture was stirred at room temperature for three days.After this time HPLC-UV analysis indicated the completion of reaction. The mixture was purified by Cl 8 silica gel column chromatography (25% H₂O in ACN) to give NSCT-7 as a white solid (44 mg, 34%). HRMS (ESI) m / z calcd for C126H220N17O73 [M+3H]3+1046.80; found 1046.56.amino linker-! 1 PyBOP, DIPEA DMFrt, 3 daysNSCT-71-9. Preparation ofNSCT-8 (Oxazoline-NSCT-di-Ns-di-ketone)

[0094] To a solution of NSCT-7 (11.1 mg, 3.5 pmol) in water were successively added Na? CO3 (43.4 mg) and 2-chloro- 1,3 -dimethyl- lH-benzimidazol-3-ium chloride (also termed CDMBI, CAS No. 37091-73-9, 27 mg) at 0 °C. The mixture was stirred at 0 °C for two hours. After this time HPLC-UV analysis indicated the completion of reaction. The mixture was then subjected to gel filtration chromatography on a Sephadex G-10 column. The product was eluted with an aqueous solution of NaOH (0.01 M). Pure fractions containing the product were combined and lyophilized to afford NSCT-8 as a white foam (47 mg). HRMS (ESI) m / z calcd for C126H218N17O72 [A / +3H]3+1040.80; found 1040.50.NSCT-7- CDMBI NajCOs H2O0°C, 2 ho INSCT-81-10. Preparation of amino linker-4

[0095] Azido-PEG4-OH (CAS No. 86770-87-4, 3.33 g, 15.19 mmol, 1.0 equiv.) was dissolved in anhydrous CH2Q2 (40 mL) in a round bottom flask in ice bath, followed by addition of p-Toluenesulfonyl chloride (4.34 g, 22.78 mmol, 1.5 equiv.), triethylamine (3.18 mL, 22.78 mmol, 1.5 equiv.), and 4-Dimethylaminopyridine (185 mg, 1.52 mmol, 0.01 equiv.). The mixture was stirred at room temperature for 20 hours. The reaction mixture was diluted with CH2Q2 (300 mL) and washed with brine (100 mL). The organic layer was dried over anhydrous MgSCM, fdtered, and concentrated by evaporation. The concentrated crude was purified by MPLC to afford compound amino linker-4 (4.43 g, 77%) as pale-yellow oil. TLC (EtOAc / hexanes = 50%, v / v, CAM and UV) R / = 0.3O O TsCI O OHO— O— — N3Et3N) DMAp" TSO— — O— — N3CH2CI2 amino linker-4rt, 20 h 77%1-11. Preparation of amino linker-5

[0096] Azido-PEG4-OH (CAS No. 86770-87-4, 2.64 g, 12 mmol) and tri ethylamine (2 mL, 14.4 mmol) were dissolved in CH2O2 (25 mL) and cooled to 0 °C. Methanesulfonyl chloride (1.08 mL, 13.8 mmol) in CH2Q2 (4 mL) was added slowly through an addition funnel. The resulting solution was stirred at room temperature for twelve hours. The reaction was monitored by SiO2 TLC, indicating a complete conversion (eluent: CH2C12 / MeOH=95 / 5, Ry =0.7). The reaction was quenched with water and extracted with DCM three times. The organics were combined, washedwith brine, dried over MgSCM, filtrated and concentrated. The resulting oil was dissolved in methylamine in methanol (9.8N, 12 mL), sealed, and heated at 60 oC for 12 hours. The reaction was monitored by SiCh TLC, indicating a complete conversion (eluent: CH2Ch / MeOH / NH4OH= 95 / 5 / 0.5, R / =0.2). After being concentrated under reduced pressure to evaporate most of the methylamine, the residual mixture was purified by silica gel chromatography (eluent: CH2Ch / MeOH / NH4OH=95 / 5 / 0.5). Fractions with the desired product were combined and concentrated to afford amino linker-5 (10.7 g, 2.48 mmol) as a light-yellow oil. 1H NMR 600 Hz, (CDC13): 53.72 (t, J = 5.1 Hz, 2H, CH2 next to N3), 3.69-3.67 (m, 10H, PEGs), 3.43 (t, J = 5.0 Hz, 4H, CH2 next to O), 2.95 (t, J = 5.1 Hz, 2H, CH2 next to NH2), 2.58 (s, Me), 1.94 (Br, NH2).1)MsCI, Et / l, o o CH2Ci2. rt.12hH amino linker-5 2)MeNH2in MeOH60,3C, 2 h1-12. Preparation of amino linker-6

[0097] Amino linker-5 (1.53 g, 6.59 mmol), di-tert-butyl dicarbonate (2.18 g, 10 mmol), and sodium bicarbonate (0.84 g, 10 mmol) in THF (15 mL) was stirred at room temperature for twelve hours. The reaction was monitored by SiCh TLC, indicating a complete conversion (eluent: CH2C12 / MeOH=98 / 2, Rf value=0.8). Sodium bicarbonate was removed by filtration and the organics were concentrated. The residual mixture was purified by silica gel chromatography (eluent: CH2C12 / MeOH=98 / 2). Fractions with the desired product were combined and concentrated to afford intermediate (1.88 g, 5.65 mmol) as a colorless oil.

[0098] The intermediate was dissolved in dry THF (6 mL) and cooled to 0 °C. Triphenylphosphine (1.66 g, 6.33 mmol) in dry THF (10 mL) was added slowly through an addition funnel. The resulting solution was stirred at room temperature for twenty hours with a drying tube. The reaction was monitored by SiCb TLC, indicating a complete consumption of starting material (eluent: CH2C12 / MeOH=98 / 2, the spot with Rf value=0.8 disappeared). Water (5 mL) was added, and the mixture was stirred at room temperature for 45 minutes then heated at 60 °C for two hours. Solid was removed by filtration, washed with CH2Q2, the organics were combined and concentrated. Toluene (20 mL) and water (20 mL) were added and washed with toluene three times. The aqueous layer was extracted with DCM three times. The organics were combined, washed with brine, dried over MgSCfi. filtrated and concentrated to afford amino linker-6 (1.2 g, 3.90 mmol) as a colorless oil. 1HNMR 600 Hz, (CDCI3): 5 3.66-3.61 (m, 10H, PEGs), 3.51 (t, J = 5.2 Hz, 2H, CH2 next to carbamate), 3.39 (br, 2H, CH2 next to O), 2.91 (s, 3H, Me),2.86 (br, 2H, CH2 next to NH2), 1.45 (s, 9H, Boc). HRMS (ESI) m / z calcd for C14H31N2O5 [A / +H]1307.2155; found 307.2227.1) BooO, NaHCO3,THF>, 12h2) PPh,, THF, amino linkei-5 rt, 20h amino linker-^3%3) 60°C, HJ, 2h1-13. Preparation of amino linker-7

[0099] Amino linker-4 (155 mg, 0.416 mmol, 1.0 equiv.) was dissolved in anhydrous DMF (1 mL) in a round bottom flask under N2 atmosphere followed by addition of K2CO3 (143 mg, 1.034 mmol, 2.5 equiv.). Amino linker-6 (180 mg, 0.499 mmol, 1.2 equiv.) was dissolved in anhydrous DMF (3 mL) and then transferred to round bottom flask. The reaction mixture was stirred at 80 °C for 20 hours under N2 atmosphere. The reaction mixture was diluted with CH2O2 (300 mL) and washed with brine (100 mL). DMF was removed in vacuo. The residue was dissolved in CH2Q2 (80 mL) and washed with saturated NaHCOs (aq.) (25 mL) twice. The organic layer was dried over anhydrous MgSCfi, filtered, and concentrated by evaporation. The concentrated crude was purified by MPLC to afford compound amino linker-7 (110 mg, 52%) as pale oil. TLC (MeOH / CH2C12= 15%, v / v, CAM, ninhydrin and UV) R / =0.5.- - amino linker-4 KSCO3amino linker-tii DMF 59% anino linker-780 °C, 20 h 1-14. Preparation of amino linker-8

[0100] Amino linker-7 (95.3 mg, 0.188 mmol, 1.0 equiv.) was dissolved in in anhydrous DMF (1.8 mL) in a round bottom flask under N2 atmosphere, followed by addition of Fmoc-[3-Ala-OH (88 mg, 0.281 mmol, 1.5 equiv.), HATU (107 mg, 0.281 mmol, 1.5 equiv.), and N-Methylmorpholine (62 pL, 0.563 mmol, 3.0 equiv.). The mixture was stirred at room temperature for twenty-four hours. The reaction mixture was diluted with CH2Q2 (75 mL) and washed with brine (10 mL) twice. The organic layer was dried over anhydrous MgSCfi, filtered, and concentrated by evaporation. The concentrated crude was purified by MPLC to afford compound amino linker-8 (168.6 mg, 98%) as pale oil. TLC (MeOH / CH2C12=15%, v / v, CAM, ninhydrin and UV) R / = 0.6.Fmoc^Beta-AlaNHFmocDMF rt, 24harrino liiiker-7 anino linker-852% 98%1-15. Preparation of amino linker-9

[0101] Amino linker-8 (332.6 mg, 0.415 mmol, 1.0 equiv.) was dissolved in a solution TFA / CH2Q2 (4 mL, l / 4=v / v) in a round bottom flask. The reaction mixture was stirred at room temperature for 2.5 hours. TFA and CH2Q2 were removed in vacuo to give a pale oil. Amino linker-9 was directly used in the next step without further purification. TLC (MeOH / CH2O2 = 15%, v / v, CAM, ninhydrin and UV) R / = 0.5... JX... A...,..-xBoc'N- O ” "hl NHFmocXc.ZO. JN OwarrhiMiiikei-v58%20% TF A. (v / v)CH.-CI-.rt, 2 h"anino linker-^1-16. Preparation of amino linker- 10

[0102] Amino linker-9 was dissolved in anhydrous DMF (4 mL) in a round bottom flask under N2 atmosphere, followed by addition of levulinic acid (72 mg, 0.623 mmol, 1.5 equiv.), HATU (237 mg, 0.623 mmol, 1.5 equiv.), and A'-Methylmorpholine (137 pL, 1.245 mmol, 3.0 equiv.). The reaction mixture was stirred at room temperature for seventeen hours. An extra amount of N-Methylmorpholine (137 pL, 1.245 mmol, 3.0 equiv.) was added. After 3 hours, acetic acid (178 pL, 3.115 mmol, 7.5 equiv.) was added to quench the reaction. DMF was removed in vacuo. The residue was dissolved in CHCI3 (40 mL) and washed with saturated NaHCCL (aq.) (20 mL) twice. The organic layer was dried over anhydrous MgSCM, fdtered, and concentrated by evaporation. The concentrated crude was purified by MPLC to afford compound amino linker- 10 (299 mg, 90%) as pale oil. TLC (MeOH / CH2C12=15%, v / v, CAM, ninhydrin and UV) R / =0.55; HRMS (ESI) m / z calcd for C40H59N6O11 [Vf+H]+799.4236; found 799.4239.Levulinicacid NHFmoc HATU. NMM DMFrt, 20 h anino linker-3,1111110 linker-1090% (2 steps)1-17. Preparation of amino linker- 11

[0103] Amino linker-10 (215.9 mg, 0.270 mmol, 1.0 equiv.) was dissolved in a solution Et? NH / DMF (4 mL, l / 4=v / v). The mixture was stirred at room temperature for twenty hours. DMF was removed in vacuo. The residue was purified by flash column chromatography (MeOH / CFhCh = 1%— >5%— >8%— >10%, v / v, with 1% EtsN) to afford amino linker-11 (125.5 mg, 80%) as colorless oil. TLC (MeOH / CH2C12=15%, v / v, CAM, ninhydrin and UV) R / =0.3; HRMS (ESI) m / z calcd for C25H48N6O9 [A / +H]+577.3556; found 577.3571.p O■w'N.0. 0 -O-NJk NHFmoc. Et *-. NH *•O.-0.., O... J DMFN< 0 rt, 20 hartino linker-1090% (2 steps)O OV 'N' "0 N'NH2O k. JN30a ni no linker-1180%1-18. Preparation of amino linker- 12

[0104] N3-PEG3-NH2 (1.2 g, 5.50 mmol, 1.0 equiv.) was dissolved in in anhydrous CH2Q2 (40 mL) in a round bottom flask under N2 atmosphere, followed by addition of BOC2O (1.79 g, 8.20 mmol, 1.5 equiv.) and triethylamine (1.15 mL, 8.20 mmol, 1.5 equiv.). The mixture was stirred at room temperature for eighteen hours. The residue was diluted with CH2O2 and washed with IN HC1 twice. The organic layer was dried over anhydrous MgSCU, fdtered, and concentrated by evaporation. The concentrated crude was purified by MPLC to afford compound amino linker- 12 (1.55 g, 89%) as transparent oil. TLC (EA / Hexane=50%, v / v, CAM, ninhydrin and UV) Rf= 0.25. HRMS (ESI) m / z calcd for Ci3H26N4NaO5[A / +Na]1341.1795; found 341.1821.> O zZx. O'SacHH" A" IT EtA CM Ofst. n amino toker- 288%1-19. Preparation of amino linker- 13

[0105] Amino linker-12 (586 mg, 1.840 mmol) was dissolved in a solution MeOH (40 mL) in a round bottom flask followed by addition of Pd / C (1.32 g). The flask was sealed and stirred under a H? atmosphere at room temperature for three hours. Pd / C was removed by celite, and the filtrate was concentrated by evaporation. The residue was transparent oil and directly used for next step. HRMS (ESI) m / z of amino linker-13 calcd for C13H29N2O5 [ +H]+ 293.2071; found 293.2085.Hj- 0. 0 >:FWCMeQHammo 0nW~12 3 h wiw 6nk«ni31-20. Preparation of amino linker- 14

[0106] Amino linker- 13 (538 mg, 1.84 mmol, 1.0 equiv.) was dissolved in in anhydrous DMF (30 mL) in a round bottom flask under N2 atmosphere, followed by addition of levulinic acid (320 mg, 2.76 mmol, 1.5 equiv.), HATU (1.05 g, 2.76 mmol, 1.5 equiv.), and N-Methylmorpholine (600 pL, 5.52 mmol, 3.0 equiv.). The mixture was stirred at room temperature for 18 hours. DMF was removed by evaporation. The residue was diluted with CH2Q2 (75 mL) and washed with IN HC1 (10 mL) twice. The organic layer was dried over anhydrous MgSCM, filtered, and concentrated by evaporation. The concentrated crude was purified by MPLC to afford compound amino linker-14 (560 mg, 78% in two steps) as transparent oil. TLC (MeOH / CH2C12=10%, v / v, CAM, ninhydrin and UV) Rf=0.55. HRMS (ESI) m / z calcd for Ci8H34N2NaO7[A / +Na]1413.2258; found 413.2271.> WxW H x0amteiMker 131-21. Preparation of amino linker- 15

[0107] Amino linker-14 (200 mg, 0.512 mmol) was dissolved in a solution TFA / CH2O2 (11 mL, l / 10=v / v) in a round bottom flask. The reaction mixture was stirred at room temperature for three hours. TFA and CH2Q2 was removed in vacuo to give a pale oil. Amino linker-15 directly used for next step without further purification. TLC (MeOH / CH2C12=15%, v / v, CAM, ninhydrin and UV) Rf=0.4. HRMS (ESI) m / z calcd for C13H27N2O5 [A / +H]1291.1914; found 291.1924.'W fWiP amkw iinkoM4 SS! &>&!( ■■$<»quant.1-22. Preparation of amino linker- 16

[0108] Boc-PEGs-COOH (240 mg, 0.75 mmol, 1.0 equiv.) and HATU (570 mg, 1.5 mmol, 2.0 equiv.) were dissolved in DMF (24 mL). DIPEA (290 mg, 2.25 mmol, 3.0 equiv.) was then added dropwise to the reaction mixture. The solution was degassed and purged with N2 three times at 25±2 °C. After stirring for thirty minutes, amino linker-2 (340 mg, 0.83 mmol, 1.1 equiv.) was added, and the reaction continued for an additional two hours. The resulting mixture was purified by Cl 8 column chromatography. The desired fractions were combined and concentrated under reduced pressure to afford amino linker-16 as a purified product (389 mg, 72% yield).oHATH, DIPEA, DMOBocHN, -cr X Ck z-x. „O..,.--x’O'I,.0,.,0,.O' 'M3amino linko E1-23. Preparation of amino linker- 17

[0109] Amino linker- 16 (200 mg, 0.28 mmol, 1. Oequiv.) was dissolved in EA (4 mL) in a round bottom flask followed by addition of Pd / C 10% (10 mg). The flask was sealed and stirred under a H? atmosphere at room temperature for two hours. Pd / C was removed by celite, and the filtrate was concentrated by evaporation to afford amino linker-17 (160 mg, 85%).a HgBocH N... CL „ Ox-'vO'10% Pd / C " N. EA, rt aminoX) Ovamino linker-171-24. Preparation of amino linker- 18

[0110] Amino linker-17 (100 mg, 0.15 mmol, 1.0 equiv.) was dissolved in DMF (1.5 mL) in a round bottom flask followed by addition of TCO-OSu (59.8 mg, 0.23 mmol, 1.5 equiv.) and DIPEA (38.5 mg, 0.30 mmol, 2.0 equiv.). The reaction mixture was stirred at room temperature for two hours. The resulting mixture was purified by Cl 8 column chromatography. The desired fractions were combined and concentrated under reduced pressure to afford amino linker- 18 as a purified product (118 mg, 81%).o TCO-OSu DIPEA. DMF. rtamino!ihkeM7BotHN1-25. Preparation of amino linker- 19

[0111] Amino linker- 18 (100 mg, 0.10 mmol, 1.0 equiv.) was dissolved in MeOH in a round bottom flask, oxalyl chloride (39 mg, 0.30 mmol, 3.0 equiv.) then added dropwise to the reaction mixture. The solution was degassed and purged with N2 three times at 25±2 °C, stirred for eighteen hours. The resulting mixture was purified by Cl 8 column chromatography. The desired fractions were combined and concentrated under reduced pressure to afford amino linker- 19 as a purified product (69 mg, 75%). MS calcd for [C43H?8N40i4+H]+875.55, found 875.74.p q i BocHN.,.-'x ^x., O, X,x, OXx-v, zv,. O„ / x A ovOvN O ■■■' ■■■ ■ « 0 \ f k A ° " '' r \ Oxalyl chloride amino itoker-W M<sQH. -ft1-26. Preparation of amino linker-20

[0112] To a solution of amino linker-25 (107 mg, 0.199 mmol) in CH2Q2 (1.15 mL) were successively added amino linker-3 (440 mg, 0.897 mmol) and Et3N (234 pL, 1.35 mmol). Themixture was stirred at room temperature overnight. The residue was diluted with CH2Q2 and washed with saturated sodium bicarbonate solution. The organic layer was then washed with brine, dried with MgSCM, and concentrated. The residue was purified by CC (3% MeOH in CH2O2, silica gel) to give amino linker-20 as a colorless syrup (229 mg, 94%).NMR (CDCI3, 600 MHz) 8 5.73 (br s, 2H), 4.23-4.07 (m, 4H), 3.72-3.50 (m, 56H), 3.49-3.41 (m, 8H), 3.41-3.37 (m, 4H), 2.62 (t, 4H), 2.62 (t, 4H), 1.44 (s, 9H).A-NO, 9 r 9oA O O O8P£A;CHgCI. i Ti' 'O' " Ns -S< JCNS o I O..0 rt. ars., 94% ( | U 0- - H. o0- Amine linker 3 Amine linker 25Amine linker 201-27. Preparation of amino linker-21

[0113] Amino linker-20 (54.6 mg, 0.0441 mmol) was treated with a 3:1 (v / v) solution of CH2Q2 / TFA (450 pL) at room temperature for 5.5 hours. The reaction was quenched with saturated sodium bicarbonate solution and extracted with CH2Q2. The organic layer was then washed with brine, dried with MgSCM, and concentrated to get the crude product amino linker-21 (46.6 mg) for the next step without further purification. HRMS ealed for [C44H83Ni? Oi8+H]+1138.6180, found 1138.6179.25% TFA in CH. Xi;:.Amino linker 20Amino linker 211-28. Preparation of amino linker-22

[0114] To a solution of crude amino linker-21 (46.6 mg) and Fmoc-[3-Ala-OH (19.1 mg, 0.0614 mmol) in DMF (1.00 mL) were successively added HATU (23.3 mg, 0.0614 mmol) and NMM (13.5 pL, 0.123 mmol). The mixture was stirred at room temperature for two hours. The mixture was diluted with CH2O2 and washed with saturated sodium bicarbonate solution. The organic layer was then washed with brine, dried with MgSCM, and concentrated. The residue was purified by CC (3% MeOH in CH2O2, silica gel) to give amino linker-22 as a colorless syrup (49.5 mg, 76% over two steps). HRMS calcd for [C62H9sNisO2i+Na]+1453.7054, found 1453.7085.HATU. NMM, DMF, 2h2 stepsAmino linker 21FmocKN.Amino linker 221-29. Preparation of amino linker-23

[0115] Amino linker-22 (49.5 mg, 0.0346 mmol) in CH2CI2 was treated with piperidine (11.1 pL, 0.113 mmol) at room temperature for five hours. The reaction was quenched with acetic acid, concentrated, and purified by CC (20% MeOH in CH2Q2, silica gel) to get amino linker-23 (32.1 mg, 77%) as a colorless syrup. HRMS calcd for [C47Hs8NisOi9+H]+1209.6551, found 1209.6558.psperidin®, CHgClj Amino Sinker 22 rt, 5h, 77%Amino Sinker 231-30. Preparation ofNSCT-9 (NSCT-di-ketone)

[0116] Amino linker-15 (0.52 mmol, 4.0 equiv.) was dissolved in anhydrous DMF (10 mL) in a round bottom flask, followed by addition of disialyoctasaccharide (253.3 mg, 0125 mmol, 1.0 equiv.), PyBOP (666 mg, 1.28 mmol, 10.0 equiv.), and DIPEA (229 pL, 1.28 mmol, 10.0 equiv.). The reaction mixture was stirred at room temperature for three days. The mixture was purified by Cl 8 silica gel column chromatography (25% H₂O in ACN) to give NSCT-9 as a white solid. HRMS (ESI) m / z calcd for Cio2Hi73N9Na2065[. V / +2Na]121305.0147; found 1305.0184.H IA 3 dAys^SCT-S1-31. Preparation ofNSCT-10 (Oxazoline-NSCT-di-ketone)

[0117] To a solution of NSCT-9 (110 mg, 0.043 mmol) in water (8.6 mL) were successively added Na? CO3 (535 mg) and 2-chloro- 1,3 -dimethyl- lH-benzimidazol-3-ium chloride (CDMBI, 328 mg) at 0 °C. The mixture was stirred at 0 °C for two hours. Ultracentrifugation to remove white precipitation. The fdtrate was then subjected to gel fdtration chromatography on a Sephadex G-10 column. The product was eluted with an aqueous solution of NaOH (0.01 M). Pure fractions containing the product were combined and lyophilized to afford NSCT- 10 as a white foam. HRMS (ESI) m / z calcd for C126H218N17O72 [Tf+2H-2H2O]2+1256.0174; found 1256.4906.HSCI-101-32. Preparation of NSCT-13 (NSCT-hexa-N3)

[0118] To a solution of NSCT (14.4 mg, 7.15 pmol) and amino linker-24 (30.6 mg, 28.6 pmol) in DMSO (450 pL) were successively added DIPEA (12.5 pL, 71.5 pmol) and benzotriazol- 1-yl-oxy-tripyrrolidino-phosphonium hexafluorophosphate (PyBOP, 37.2 mg, 71.5 pmol). The resulting mixture was stirred at room temperature for twenty-four hours and quenched with water. The residue was washed by CH2Q2, concentrated, and purified by reverse phase silica gel column chromatography (70% MeOH in water, C18 silica gel) to afford NSCT-13 as a white fluffy solid (11.5 mg, 39%). HRMS calcd for [Ci62H287N3iO9i+4Na]4+1053.7088, found 1053.7104.HO OH HO., ^© \.. J pH i AcHM-..H° < HC-, O' VP - OH AcHN:NSCT1-33. Preparation ofNSCT-14 (Oxazoline-NSCT-hexa-N3)

[0119] To a solution of NSCT-13 (2.25 mg, 0.545 pmol) in water (20 pL) were successively added Et3N (3.42 pL, 24.5 pmol) and 2-chloro- 1,3 -dimethyl- lH-benzimidazol-3-ium chloride (CDMBI, 2.73 pL of a IM solution, 2.73 pmol) at 0 °C. The mixture was stirred at 0 °C for fourhours. After this time HPLC-UV analysis indicated the completion of reaction. The mixture was then fdtered, diluted in 0.1% NH4OH(aq), and analyzed by HR-MS. HRMS calcd for [CI62H285N3109O+4H]4+1027.2247, found 1027.2247..NsHQ OH ACHN~Z*£. H HO' HO / ° HO— - x O"NT U%O -■V- O o HO ••V-’S- - O HO OH AcHN H HN 0.. N,,;. O HO"\ O'" O HO" V" O O O ' HO-*” o.o. °\ QH N' i H \ HO HO HO-'-\ O V' i —^3 \.! pH \ HO ACHN-2^27' " X HO -y HO HO HO— x • o HO - HO-^-^S'-’OH AcHN NSCT-141-34. Preparation ofNSCT-15 (NSCT-octa-Ns)

[0120] To a solution of NSCT (12.3 mg, 6.08 pmol) and amino linker-23 (29.4 mg, 24.3 pmol) in DMSO (500 pL) were successively added DIPEA (10.6 pL, 60.8 pmol) and benzotriazol- 1-yl- oxy-tripyrrolidino-phosphonium hexafluorophosphate (PyBOP, 31.6 mg, 60.8 pmol). The resulting mixture was stirred at room temperature for twenty-four hours and quenched with water. The residue was purified by reverse phase silica gel column chromatography (70% MeOH in water,C18 silica gel) to afford NSCT-15 as a white fluffy solid (18.7 mg, 70%). HRMS calcd for [C17OH297N4I093+4H]4+1101.2521, found 1101.2527.1-35. Preparation ofNSCT-16 (Oxazoline-NSCT-octa-Ns)

[0121] To a solution of NSCT-15 (2.5 mg, 0.568 pmol) in water (22.5 pL) were successively added Et3N (3.56 pL, 25.6 pmol) and 2-chloro- 1,3 -dimethyl- lH-benzimidazol-3-ium chloride (CDMBI, 2.84. L of a IM solution, 2.84 pmol) at 0 °C. The mixture was stirred at 0 °C for four hours. After this time HPLC-UV analysis indicated the completion of reaction. The mixture was then fdtered, diluted in 0.1% NH4OH(aq), and analyzed by HR-MS. HRMS calcd for [Ci7oH295N4i092+4H]4+1096.7495, found 1096.7507.N ' O' 0' HO OH x; ° PH ] ACHNT HO HOl-C-oOH NSCT-161-36. Preparation ofNSCT-11 (NSCT-tetra-TCO)

[0122] NSCT (100 mg, 0.049 mmol, 1.0 equiv.) and amino linker-19 (87 mg, 0.099 mmol, 2.0 equiv.) were dissolved in DMSO (2 mL) at room temperature. PyBOP (77 mg, 0.15mmol, 3.0 equiv) and TEA (23 mg, 0.22 mmol, 4.5 equiv.) were then added dropwise to the reaction mixture. The solution was degassed and purged with N2 three times at 25±2 °C. The reaction mixture was stirring for three hours. The resulting mixture was purified by Cl 8 column chromatography. The desired fractions were combined and concentrated under reduced pressure to afford NSCT-11 as a purified product (121 mg, 63% yield).1-37. Preparation ofNSCT-12 (Oxazoline-NSCT-tetra-TCO)

[0123] To a solution of NSCT-11 (100 mg, 25.8 pmol) in water were successively added EtsN (130.8 pL, 940 pmol) and 2-chloro- 1,3 -dimethyl- lH-benzimidazol-3-ium chloride (CDMBI, 105 pL of a 1 M solution) at 0 °C. The mixture was stirred at 0 °C for two hours. After this time HPLC-UV analysis indicated the completion of reaction. The mixture was then subjected to gel filtration chromatography on a Sephadex G-10 column. The product was eluted with an aqueous solution of NaOH (0.01 M). Pure fractions containing the product were combined and lyophilized to afford NSCT-12 as a white foam (65 mg, 65%). MS (ESI) m / z calcd for C168H285N15O84 [A / +H]13857.85.Example 2. Synthesis of single-arm saccharide derivatives2-1. Scheme of tetra-saccharide formation

[0124] A mixture of donor G-Cl-0039 (639 mg, 0.967 mmol, 1.2 equiv., manufactured by OBI Pharma, Inc.), acceptor compound 1 (669 mg, 0.820 mmol, 1.0 equiv.), and activated 4 A molecular sieves (1.93 g) in anhydrous CH2Cl2 (32 mL) was stirred at room temperature under an N2 atmosphere for 30 minutes. It was then cooled to -78 °C, and NIS (256 mg, 1.178 mmol, 1.44 equiv.) was added. After stirring at -78 °C for 10 minutes, TfOH (16 pL, 0.164 mmol, 0.2 equiv.) was added, and the reaction mixture was gradually warmed up to -50 °C. After stirring for four hours, the mixture was quenched with triethylamine (50 pL, 0.68 mmol, 0.8 equiv.). The mixturewas filtered and concentrated in vacuo. The residue was diluted with CH2Cl2 (50 mL) and washed with saturated NaHCO3(aq) (30 mL), finally saturated NaS2O3(aq) (30 mL). Organic layer was dried over anhydrous MgSO4 and concentrated by evaporation. The concentrated crude was purified by MPLC to afford compound 2 (951 mg, 86%) as white solid. TLC (EtOAc / hexanes= 30%, v / v, CAM, anisaldehyde and UV) R / = 0.5; HRMS (ESI) m / z calculated for C81H81N5NaO16 [M+Na]+1374.5509; found 1374.5454.G-C1-0039 NIS, TfOH MS AW-300rCH2CI2-78 °C to -50 °C4 h

[0125] Compound 2 (429 mg, 0.317 mmol, 1.0 equiv.) was dissolved in a solution MeOH / CH2Ch (12 / 6, 2 / 1 = v / v) in a round bottom flask followed by addition ofNaOMe (137 mg, 2.536 mmol, 8.0 equiv.). The mixture was stirred at room temperature for eighteen hours. The reaction mixture was diluted with CH2Cl2 (50 mL) and washed with IN HCl(aq.) (40 mL) twice. Organic layer was dried over anhydrous MgSO4, filtered, and concentrated by evaporation. The concentrated crude was purified by flash column chromatography (EtOAc / hexanes = 10% -^20% — 30%, v / v) to afford compound 3 (369 mg, 93%) as white solid. TLC (EtOAc / hexanes = 30%, v / v, CAM, anisaldehyde and UV) R / = 0.3.NaOMe MeOH: CH2CI22:1 rt, 18 h

[0126] A mixture of donor G-Cl-0040 (436 mg, 0.596 mmol, 1.2 equiv., manufactured by OBI Pharma, Inc.), acceptor compound 1 (620 mg, 0.496 mmol, 1.0 equiv.), and activated 4 A molecular sieves (1.2 g) in anhydrous CH2Cl2 (20 mL) was stirred at room temperature under an N2 atmosphere for 30 minutes. It was then cooled to -78 °C, and NIS (161 mg, 0.715 mmol, 1.44 equiv.) was added. After stirring at -78 °C for 10 minutes, TfOH (8.8 pL, 0.099 mmol, 0.2 equiv.) was added, and the reaction mixture was gradually warmed up to -50 °C. After stirring for three hours, the mixture was quenched with tri ethylamine (20 pL, 0.15 mmol, 0.3 equiv.). The mixture was filtered and concentrated in vacuo. The residue was diluted with CH2Cl2 (40 mL) and washed with saturated NaHCO3(aq) (20 mL), and then saturated Na2S2O3(aq) (20 mL). Organic layer wasdried over anhydrous MgSO4, filtered, and concentrated by evaporation. The concentrated crude was purified by MPLC to afford compound 4 (813 mg, 88%) as white solid. TLC (EtOAc / hexanes= 30%, v / v, CAM, anisaldehyde and UV) R / = 0.5; HRMS (ESI) m / z calculated for C104H107Cl3N4Na2O21 [M+2Na]2+949.3139; found 949.3201.TrocHN STol G-C1-0040 N IS, TfOH MS AW-300 CH2CI2-78 °C to -50 °C TrocHN

[0127] Compound 4 (669 mg, 0.360 mmol, 1.0 equiv.) was dissolved in a solution AcCh (682 pL, 7.214 mmol, 20 equiv.), AcOH (682 pL), and THF (7 mL) in a round bottom flask followed by addition of Zn powder (1340 mg). The mixture was stirred at room temperature for twenty hours. Zn powder was removed by celite and concentrated by evaporation. The residue was purified by flash column chromatography (EtOAc / hexanes= 40% -^45% — 50%, v / v) to afford compound 5 (479 mg, 76%) as white solid. TLC (EtOAc / hexanes= 50%, v / v, CAM, anisaldehyde and UV) R / = 0.3; HRMS (ESI) m / z calculated for C105H... [A / +2Na]12891.3771; found 891.3785.AC2O, Zinc AcOH THF rt, 20 h

[0128] Compound 5 (218 mg, 0.125 mmol) was dissolved in a solution MeOH / H2O (12 mL, 2 / 1= v / v) in a round bottom flask followed by addition of Pd(OH)2 / C (400 mg). The flask was sealed and stirred under a H2 atmosphere at room temperature for twenty hours. Pd(OH)2 / C was removed by celite and concentrated by evaporation. The residue was purified by G-10 column chromatography to afford compound 6 (90 mg, 96%) as white solid. TLC (Butanol / Acetic acid / H2O = 5 / 3 / 2, v / v, anisaldehyde and CAM) Ry = 0.3; HRMS (ESI) m / z calculated for C28H49N2O21 [M+H]+749.2822; found 749.2840.NHAc6

[0129] To a solution of Compound 6 (13 mg, 17.4 pmol) in water were successively added EtsN (109 pL, 783 pmol) and 2-chloro- 1,3 -dimethyl- lH-benzimidazol-3-ium chloride (CDMBI, 87 pL of a IM solution) at 0 °C. The mixture was stirred at 0 °C for two hours. After this time HPLC-UV analysis indicated the completion of reaction. The mixture was then subjected to gel filtration chromatography on a Sephadex G-10 column. The product was eluted with an aqueous solution of NaOH (0.01 M). Pure fractions containing the product were combined and lyophilized to afford compound 7 as a white foam (9 mg, 71%).1H NMR (D2O, 600 MHz) 8 6.09 (d, 1H, J= 7.3 Hz), 5.12 (s, 1H), 4.73 (s, 1H), 4.55 (d, 1H, J= 8.4 Hz), 4.39-4.36 (m, 1H), 4.21-4.17 (m, 2H), 4.14 (d, 1H, J= 2.9 Hz), 3.97-3.88 (m, 4H), 3.80-3.59 (m, 10H), 3.57-3.48 (m, 2H), 3.46-3.38 (m, 4H), 2.07 (d, 3H, J= 1.1 Hz), 2.05 (s, 3H).CDMBIEt3N H20 0 °C, 2 h NHAc2-2. Scheme of hexa-saccharide formation

[0130] To a 1 mL solution containing Compound 6 (1.5 mg, 2 μmol), ATP (11 μg, 0.01 μmol), UTP (27 pg, 0.025 pmol), galactose (0.72 mg, 2 pmol), phosphoenolpyruvate (1.82 mg, 4.4 pmol, monopotassium salt), and MgCh (final 10 mM) were added the enzymes galactokinase (GalK, 0.02 U), UDP-sugar pyrophosphorylase (AtUSP, 0.06 U), ul,4-galactosyltransferase (Sigma-Aldrich, 0.75 U), pyruvate kinase (PK, 0.08 U), and inorganic pyrophosphatase (PPA, 0.08 U). The final volume of the mixture was adjusted to 1 mL, and the reaction was carried out at room temperature over the course of twenty-four hours with the pH controlled at 8.6. The reaction was monitored by thin layer chromatography (5:3:2 butanol / acetate / water). To the corresponding penta-saccharide mixture (11 mg, 15 pmol), Neu5Ac (0.42 mg, 15 pmol), ATP (0.82 pg, 0.17 pmol), CTP (0.04 pg, 1.7 pmol), phosphoenolpyruvate (PEP, 0.31 mg, 10 pmol, monopotassium salt), cytidine monophosphate kinase (CMK, 0.167 U), CMP-sialic acid synthetases (CSS, 0.4 U)and a-2,6-sialyltransferase (SHIZ-145, 0.5 U) were added. The reaction was carried out at 37 °C for six hours with the pH controlled at pH 9.0 solution of Tris buffer. The glycosylation reaction was monitored by TLC until completion. The reaction was stopped by the treatment of MeOH, and precipitated protein was removed by centrifugation at 20,000g for five minutes. The supernatant was concentrated and purified by silica gel column chromatography (25% H2O in ACN) to give Compound 8 as a white solid (14.3 mg, 81% over two steps).1H NMR (D2O, 600 MHz) δ5.21 (d, 0.6H, J= 3.2 Hz), 5.15 (s, 1H), 4.79 (1H, overlapped with solvent signal), 4.72 (d, 0.4H, J= 7.7 Hz), 4.61 (d, 1H, J= 7.6 Hz), 4.45 (s, 1H, J= 7.9 Hz), 4.25 (dd, 1H, J= 7.1, 3.2 Hz), 4.22-4.19 (m, 1H), 4.04-3.45 (m, 35H), 2.67 (dd, 1H, J= 12.4, 4.7 Hz), 2.07 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 1.72 (pt, 1H, J= 12.4 Hz). HRMS calculated for [C45H75N3O34+Na]+1224.4130, found 1224.4096.UDP-Gal, GalK CMP-Neu5Ac MBP-AtUSP NmCSS, PPA, PK PK, PPA, NmLetB CMK, SHIZ-145HO OHOH[01311 Following the same procedure as described for the preparation of Compound 7, Compound 9 was obtained as a white foam (9.8 mg, 98%) from Compound 8.1H NMR (D2O, 600 MHz) 8 6.09 (d, 1H, J= 7.3 Hz), 5.14 (s, 1H), 4.74 (s, 1H), 4.60 (d, 1H, J= 8.0 Hz), 4.44 (d, 1H, J= 7.9 Hz), 4.38 (dd, 1H, J= 3.0, 1.7 Hz), 4.22-4.17 (m, 2H), 4.15 (d, 1H, J= 3.1 Hz), 4.03-3.49 (m, 31H), 3.46-3.39 (m, 2H), 2.67 (dd, 1H, J= 12.4, 4.7 Hz), 2.07 (s, 6H), 2.03 (s, 3H), 1.72 (pt, 1H,. / = 12.4 Hz).CDMBIEt3N H2O 0 °C, 2 h2-3. Preparation of oxazoline-hexasaccharide-Ns

[0132] Step 1. Synthesis of hexasaccharide-Ns (Compound 10)

[0133] To a solution of Compound 8 (23.6 mg, 19.6 pmol) and H2N-PEG3-N3 (12.9 mg, 58.9 pmol) in DMSO (270 pL) were successively added DIPEA (20.6 pL, 118 pmol) and benzotriazol-1-yl-oxy-tripyrrolidino-phosphonium hexafluorophosphate (PyBOP, 61.3 mg, 118 pmol). The resulting mixture was stirred at room temperature for 4 hours and quenched with water. The residue was washed by CH2O2, concentrated, and purified by reverse phase silica gel column chromatography (15% MeOH in water, C18 silica gel) to afford hexasaccharide-N3 (Compound 10) as a white fluffy solid (20.6 mg, 75%).1H NMR (D2O, 600 MHz) δ5.21 (d, 0.6H, J = 3.1 Hz), 5.15 (s, 1H), 4.79 (1H, overlapped with solvent signal), 4.72 (m, 0.4H), 4.60 (d, 1H, J = 7.3 Hz), 4.45 (d, 1H, J = 7.9 Hz), 4.25 (dd, 1H, J= 7.1, 3.2 Hz), 4.22 - 4.19 (m, 1H), 4.02 - 3.45 (m, 51H), 2.71 (dd, 1H, J = 12.8, 4.3 Hz), 2.07 (s, 3H), 2.04 (s, 6H), 1.85 (pt, 1H, J = 12.3 Hz). HRMS calcd for [C53H9iN7O36+2Na]2+723.7645, found 723.7669., OH HO NHAc H2N-PEG3-N3. PyBOP -: —: - DMSO. DIPEA, rt, 4h, 75% 8 step 1

[0134] Step 2. Synthesis of oxazoline-hexasaccharide-N3 (Compound 11)

[0135] To a solution of Compound 10 (12.5 mg, 8.9 pmol) in water (60 pL) were successively added Et3N (56 pL, 402 pmol) and 2-chloro- 1,3 -dimethyl- lH-benzimidazol-3-ium chloride (CDMBI, 44.5 pL of a IM solution, 44.5 pmol) at 0 °C. The mixture was stirred at 0 °C for 4 hours.After this time HPLC-UV analysis indicated the completion of reaction. The mixture was then subjected to gel filtration chromatography on a Sephadex G-10 column. The product was eluted with an aqueous solution of NaOH (0.01 M). Pure fractions containing the product were combined and lyophilized to afford oxazoline-hexasaccharide-Ns (Compound 11) as a white fluffy solid (12.2 mg, quantitative yield).1H NMR (D2O, 600 MHz) δ 6.09 (d, 1H, J = 7.3 Hz), 5.13 (s, 1H), 4.73 (s, 1H), 4.59 (d, 1H, J= 8.0 Hz), 4.44 (d, 1H, J = 7.9 Hz), 4.38 - 4.35 (m, 1H), 4.21 - 4.17 (m, 2H), 4.14 (d, 1H, J = 2.9 Hz), 4.00 - 3.38 (m, 49H), 2.70 (dd, 1H, J= 12.9, 4.6 Hz), 2.07 (d, 3H, J = 1.7 Hz), 2.06 (s, 3H), 2.04 (s, 3H), 1.83 (pt, 1H, J = 12.9 Hz).step 2 2-4. Scheme of tri-saccharide formation

[0136] Step 1. Preparation of Compound 12

[0137] A solution of G-Cl-0016 (2.0 g, 5.01 mmol, 1.0 equiv) in dry dichloromethane (40 mL) was treated with tri ethylamine (6.13 mL, 44.1 mmol, 8.8 equiv) and 4-(dimethylamino) pyridine (DMAP, 61 mg, 0.50 mmol, 0.1 equiv) under a nitrogen atmosphere at room temperature. Acetic anhydride (3.78 mL, 40.1 mmol, 8.0 equiv) was added dropwise, and the reaction mixture was stirred for 16 hours at room temperature. Without workup, the reaction was cooled to 0 °C, and acetyl chloride (0.71 mL, 20.0 mmol, 4.0 equiv) was added dropwise. The mixture was stirred for 1 hour at 0 °C, then quenched with saturated aqueous NaHCO3. The organic layer was separated, washed with water and brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate=1:1) to afford compound 12 as a colorless solid. (1.96 g, 81% yield). MS (ESI) m / z 590.9 [M + Na]+.OAr AcO„0AcCOOMe 1) Ac2O, TEA, DMAP SPh 2) AcCI, TEACH2CI2, 0 °C G-C1-0016 12

[0138] Step 2. Preparation of Compound 13

[0139] A flame-dried round-bottom flask equipped with a magnetic stir bar was charged with donor G-Cl-0013 (2.07 g, 3.59 mmol, 1.2 equiv), acceptor G-Cl-0041 (1.43 g, 3.01 mmol, 1.0 equiv), and activated 4 A molecular sieves (MS AW-500, 3.5 g) under a nitrogen atmosphere. The mixture was co-evacuated and back-filled with nitrogen three times, then suspended in anhydrous CH2Cl2 (70 mL, 20 mL g'1). To this mixture was added N-iodosuccinimide (NIS, 0.95 g, 4.21 mmol, 1.4 equiv) in one portion, and the suspension was stirred at room temperature for 2 hours. The reaction mixture was then cooled to -78 °C, and trifluoromethanesulfonic acid (TfOH, 53.1 pL, 0.60 mmol, 0.2 equiv) was added dropwise. The reaction was maintained between -78 °C and -50 °C for 4 hours, and progress was monitored by TLC (hexane / ethyl acetate=2:l). Upon completion, the reaction was quenched with triethylamine (0.5 mL) and allowed to warm to room temperature. The mixture was diluted with CH2Cl2 and filtered through a Celite pad to remove the molecular sieves. The filtrate was washed successively with 10% aqueous Na2S2O3, saturated NaHCOs, water, and brine, then dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane / ethyl acetate=4:1) to afford compound 13 as a colorless foam. MS (ESI) m / z 964.9 [M+Na]+.Ph NIS, TfOH / MS AW-500 VO CH2C!2-78 °C to -50 °CG-C1-0013 G-C1-0041 4 h 13

[0140] Step 3. Preparation of Compound 14

[0141] A flame-dried round-bottom flask equipped with a magnetic stir bar was charged with compound 12 (199 mg, 0.211 mmol, 1.0 equiv) and anhydrous CH2Cl2 (4.0 mL, 20 mL g'1) under a nitrogen atmosphere. The reaction mixture was cooled to 0 °C, and BH3 THF (1 M in THF, 2.11 mL, 2.11 mmol, 10.0 equiv) was added dropwise, followed by trimethylsilyl trifluoromethanesulfonate (TMSOTf, 7 pL, 0.049 mmol, 0.2 equiv). The reaction mixture was stirred at 0 °C for 18 hours, and progress was monitored by TLC (hexane / ethyl acetate=2:l). Uponcompletion, the reaction was quenched with triethylamine (0.5 mL) and stirred for 10 minutes at 0 °C. The mixture was diluted with CH2Cl2 and filtered through a pad of Celite to remove any solids. The filtrate was washed successively with water and brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (hexane / ethyl acetate=2:l) to afford the compound 14 as a colorless foam (128 mg, 62% yield).OH BH3-THF BnO. OBn TMSOTf AcO THF TrocHN 0 ’C

[0142] Step 4. Preparation of Compound 15

[0143] A flame-dried round-bottom flask equipped with a magnetic stir bar was charged with donor compound 12 (43.2 mg, 0.076 mmol, 1.2 equiv), acceptor compound 14 (60.1 mg, 0.063 mmol, 1.0 equiv), and activated 4 A molecular sieves (MS AW-500, 200 mg) under a nitrogen atmosphere. The mixture was co-evacuated and back-filled with nitrogen three times, then suspended in anhydrous CH2Cl2 (3.0 mL). N-Iodosuccinimide (NIS, 17.15 mg, 0.077 mmol, 1.4 equiv) was added in one portion, and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was then cooled to -78 °C, and trifluoromethanesulfonic acid (TfOH, 1.1 pL, 0.013 mmol, 0.2 equiv) was added dropwise. The mixture was stirred at -78 °C to -50 °C for 4 hours, and the progress was monitored by TLC (hexane / ethyl acetate=l:1). Upon completion, the reaction was quenched with triethylamine (0.2 mL) and allowed to warm to room temperature. The mixture was diluted with CH2Cl2 and filtered through a pad of Celite to remove the molecular sieves. The filtrate was washed successively with 10% aqueous Na2S2O3, saturated NaHCCL, water, and brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (hexane / ethyl acetate=1:1) to afford compound 15 as a colorless foam (68 mg, 72% yield).UAC OH NIS, TfOHAc0U pAc MS AW-500SPh 4.A— oo 78 °C to -50 °C 12 14 4 hOBn

[0144] Step 5. Preparation of Compound 16

[0145] A solution of the compound 15 (68 mg, 0.0485 mmol, MW=1401.63 g mol'1, 1.0 equiv) in anhydrous THF (0.7 mL, 10 mL g'1) was prepared under a nitrogen atmosphere. To this solution were added acetic anhydride (91 pL, 0.97 mmol, 20 equiv) and acetic acid (91 pL, 1 mL per mL of AC2O). The mixture was stirred at room temperature for 5 minutes, followed by the addition of zinc powder (136 mg, 2 g per g of substrate) in one portion. The reaction mixture was stirred at room temperature for 20 hours, and the progress was monitored by TLC (hexane / ethyl acetate= 1:2). After completion, the reaction mixture was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (hexane / ethyl acetate=1:2) to afford compound 16 as a colorless foam (39 mg, 63% yield).16

[0146] Step 6. Preparation of Compound 17

[0147] A solution of the compound 16 (30 mg, 1.0 equiv) in MeOH / H2O (1:1, 1.0 mL) was prepared under a nitrogen atmosphere. To the mixture was added 3 M LiOH (125.1 mg, ca. 4.17 mmol, large excess) in one portion, and the reaction was stirred at room temperature for 18 hours. Reaction progress was monitored by TLC (CH2C12 / MeOH=15:l and 4:1). After completion, the reaction mixture was concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (CH2C12 / MeOH=15:l — 4:1) to afford compound 17 as a colorless solid.AcO 3 M LiOH MeOH: H2O 1:1 rt. 18 h

[0148] Step 7. Preparation of Compound 18

[0149] A flame-dried round-bottom flask equipped with a magnetic stir bar was charged with the compound 17 (11 mg) and Pd(OH)2 / C (33 mg) under a nitrogen atmosphere. The mixture was dissolved in MeOH (0.5 mL) and H₂O (0.5 mL), then evacuated and back-fdled with hydrogen (balloon) three times. The reaction was stirred under a hydrogen atmosphere at room temperature for twenty-five hours. After completion (monitored by TLC), the reaction mixture was filtered through filter paper to remove the catalyst, and the filtrate was concentrated under reduced pressure. The crude product was purified by Sephadex G-10 gel filtration chromatography (H2O as eluent) to afford compound 18 as a colorless foam. MS (ESI-) m / z 714.2 [M - H]'.H2, PdKOHh MeOH: H2O rt, 1 d17

[0150] Step 8. Preparation of Compound 19

[0151] To a solution of compound 18 (3.8 mg, 5.3 pmol) and H2N-PEG3-N3 (5.8 mg, 26.5 pmol) in DMF (380 pL) were successively added DIPEA (4.62 pL, 26.5 pmol) and benzotriazol- 1-yl-oxy-tripyrrolidino-phosphonium hexafluorophosphate (PyBOP, 13.8 mg, 26.5 pmol). The resulting mixture was stirred at room temperature for sixteen hours. After completion (monitored by TLC), the solvent was removed by rotavapor. The residue solid was washed by diethyl ether (3 mL, three times) and dichloromethane (3 mL, three times). The solid was recovered by MeOH and dried overnight to afford compound 19 (7.8) mg as off-white solid without further purification.

[0152] Step 9. Preparation of Compound 20

[0153] To a solution of compound 19 (2.1 mg, 2.25 pmol) in water (15 pL) were successively added Et3N (14.1 pL, 0.1 mmol) and 2-chloro- 1,3 -dimethyl- lH-benzimidazol-3-ium chloride (CDMBI, 11.25 pL of a IM solution, 11.3 pmol) at 0 °C. The mixture was stirred at 0 °C for four hours. HPLC-UV analysis indicated the completion of reaction. The crude product was purified by Sephadex G-10 gel filtration chromatography (H2O as eluent) to afford compound 20.Example 3. Deglycosylation, transglycosylation and purification of monoclonal antibodies 3-1. Deglycosylation of mAbs by EndoSz-D234M

[0154] Three monoclonal antibodies were used to illustrate the process to obtain glycoengineered antibodies. R4702 is an anti-TROP2 monoclonal antibody obtained from Biosion Inc.. TX05 is an anti-HER2 monoclonal antibody obtained from Tanvex BioPharma Inc. 10K06 is an anti-Nectin 4 monoclonal antibody developed by OBI Pharma Inc.. R4702 and TX05 was deglycosylated with EndoSz-D234M in 50 mM Tris pH 7.2 at 37 °C for 24-49 hours. Only the antibodies with high mannose N-glycan modification were further supplied with EndoH and incubated at 25 °C overnight to remove glycans completely and produced mAb-GlcNAc(Fuc).10K06 was deglycosylated with EndoSz-D234M at a weight ratio of 1:250 (enzyme: mAbs) and 10U Endo H / mg mAb in 100 mM Sodium phosphate, pH 7.0 at 37 °C for 35-42 hours to remove glycans and generate 10k06-GlcNAc(Fuc). The complete cleavage of FcN-glycans were analyzed by SDS-PAGE and CE-SDS.

[0155] The EndoSz-D234M was applied to other mAbs for further deglycosylation and tranglycosylation investigation. For the deglycosylation studies, the mAbs were incubated with EndoSz-D234M at a weight ratio of 1:30 (EndoSz-D234M: mAbs). All the mAb-GlcNAc(Fuc) from different mAbs reached to >90% by EndoSz-D234M cleavage. For the tranglycosylation investigation with NSCT-2 andNSCT-4, 20 or 38 equivalents ofNSCT-2 orNSCT-4 were added to mAb-GlcNAc(Fuc) for incubation with EndoSz-D234M at 37 °C for two hours. Table 1 showed that the yields of mAb-(NSCT-di-N3)2 was R4702-(NSCT-di-N3)2: 95.61% and TX05-(NSCT-di- NS)2: 96.7%, analyzed by CE-SDS. Furthermore, the yields of mAb-(NSCT-tetra-N3)2 were 10K06-(NSCT-tetra-N3)2: 95.5%. These data indicate that EndoSz-D234M can be applied to various mAbs.

[0156] Table 1. Deglycosylation and transglycosylation of R4702, TX05 and 10K06 mAbs mAh R4782 7X05 10K06 Form mAb- mAh- mAb- mAb- mAb- GleNActFuc) (NSCT-di-N;}- Gfc! W{Fuc) GfcNAcfFuc} (NSCPtetra-MJj HC 3.58% t.85% 1.0;3-2. Transglycosylation of mAb-GlcNAc(Fuc)

[0157] R4702-GlcNAc(Fuc) and TX05-GlcNAc(Fuc) was incubated with 20-38 equivalents of NSCT-2 at 37 °C for two hours to generate mAb-(NSCT-di-N3)2. 10K06-GlcNAc(Fuc) was incubated with 8-10 equivalents of NSCT-4 at 15 °C for eight hours to generate 10K06-(NSCT-tetra-Ns)2. The transglycosylation efficiency was monitored by SDS-PAGE and CE-SDS.3-3. Purification of mAb-(NSCT-di-N3)2 / mAb-(NSCT-tetra-N3)2

[0158] Sodium chloride was added into the transglycosylation mixture to reach a final concentration of 3M and then applied to PBS and 3M NaCl pre-equilibrated HiTrap Phenyl HP (Cytiva). The non-bound contaminations were washed by 5CV of equilibration buffer (PBS and 3M NaCl). R4702-(NSCT-di-N3)2 and TX05-(NSCT-di-N3)2 was eluted with a 30-100% elution buffer (Sodium phosphate 20 mM and 20% IPA pH 7.2) in 20CV linear gradient. The eluted fractions were applied to a prepacked column, HiTrap Protein A HP (Cytiva). The impurities were washed by two steps pH gradient, 100 mM Sodium citrate pH 6.0 and pH 5.5, with 5CV in each step. 50 mM Sodium citrate pH 3.5 was employed to elute bound antibody. The eluted fractions were immediately neutralized with 1 M Tris-HCl pH 9.0 to natural pH and change buffer to 20 mM Sodium Acetate pH 5.0 with Amicon centrifugation membrane (30 kDa cutoff, Millipore). The purified R4702-(NSCT-di-N3)2 and TX05-(NSCT-di-N3)2 was stored at -80°C.

[0159] The transglycosylation mixture was applied to the pre-equilibrated PrismA column (Cytiva). The non-bound contaminations were washed by 3CV of 3 M NaCl PBS, pH 7.2 buffer, 5CV of 100 mMpH 6.0 Citrate Buffer, and 5CV of 100 mMpH 5.5 Citrate Buffer. 10K06-(NSCT-trtra-N3)2 was eluted with 5CV of 50 mM pH 3.0 Citrate Buffer with 150 mM NaCl and the eluted fractions were immediately neutralized with 1 M Tris-HCl pH 9.0 to natural pH. The neutralized sample was diluted to reduce conductivity and concentration, then activated carbon was added at a 3:1 weight ratio relative to the antibody (AC= 3:1 w / w). Stirred the mixture and filtered after two hours to collect the clarified sample. The purified 10K06-(NSCT-tetra-N3)2 was concentrated with 7 volumes of storage buffer (20 mM Sodium Acetate, pH 5.0) using a 30 kDa cutoff cassette (PES, Sartorius). The purified 10K06-(NSCT-tetra-N3)2 was also stored at -80 °C.Example 4. Synthesis of drug-linker compound4-1. Preparation of BCN-GGVA-Hydra-PAB-PEG24-Exatecan (also termed DL-1)

[0160] Step l:A solution of H-PAB-5 (57.0 mg, 0.0289 mmol) in CH2O2 (1.2 mL) was cooled to 0 °C. TFA (285.0 pL) was added dropwise. The reaction mixture was stirred at 0-4 °C for four hours. After the reaction was completed, the reaction mixture was diluted with MeOH (2 mL). The solutionwas concentrated in the vacuo at 30-35 °C and then azeotroped with Toluene (5 mL x 3) to remove excess TFA. The residue was dried in high vacuum to generate crude deprotected intermediate. The prepared crude intermediate, Boc-Gly-OH (5.1 mg, 0.0289 mmol) and HATU (13.2 mg, 0.0347 mmol) were dissolved in anhydrous DMF (0.58 mL). EtsN (12.1 pL, 0.0868 mmol) was added. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 40.4 mg of H-PAB-7 with 68.9% yield.

[0161] Table 2. HPLC condition (Column: YMC-Actus Trait Cl 8250 x 20 mm, 5 pm, 12 nm) Gradient:Time Flow (ml / min) H2O ACN0 min 10 90% 10%1 min 20 80% 20%5 min 20 60% 40%25 min 20 50% 50%28 min 20 10% 90%33 min 20 10% 90%

[0162] Step 2:A solution of H-PAB-7 (38.5 mg, 0.0190 mmol) in CH2O2 (0.77 mL) was cooled to 0 °C. TFA (192.5 piL) was added dropwise. The reaction mixture was stirred at 0-4 °C for four hours. After the reaction was completed, the reaction mixture was diluted with MeOH (2 mL). The solution was concentrated in the vacuo at 30-35 °C and then azeotroped with Toluene (5 mL x 3) to remove excess TFA. The residue was dried in high vacuum to generate crude deprotected intermediate. The prepared crude intermediate, Boc-Gly-OH (3.4 mg, 0.0190 mmol) and HATU (8.7 mg, 0.0228mmol) were dissolved in anhydrous DMF (0.38 mL). Et3N (7.9 pL, 0.0570 mmol) was added. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 28.7 mg of H-PAB-8 with 72.6% yield.

[0163] Table 3. HPLC condition (Column: YMC-Actus Trait Cl 8250 x 20 mm, 5 pm, 12 nm) Gradient:Time Flow (ml / min) H2O ACN0 min 10 90% 10%1 min 20 70% 30%3 min 20 55% 45%4 min 20 54% 46%6 min 20 53% 47%8 min 20 52% 48%10 min 20 52% 48%14 min 20 50% 50%15 min 20 46% 54%16 min 20 42% 58%17 min 20 38% 62%17.1 min 20 10% 90%22 min 20 10% 90%W, if Step 2

[0164] Step 3:A solution of H-PAB-8 (25.0 mg, 0.0120 mmol) in CH2O2 (0.50 mL) was cooled to 0 °C. TFA (125.0 pL) was added dropwise. The reaction mixture was stirred at 0 °C for four hours. After the reaction was completed, the reaction mixture was diluted with MeOH (5 mL) and then concentrated in the vacuo at 30-35 °C and then azeotroped with Toluene (5 mL x 3) to remove excess TFA. The resulting material was dried in high vacuum to obtain crude deprotectedintermediate. A solution of crude intermediate (0.0120 mmol) in anhydrous DMF (0.24 mL), BCN-OSu (3.5 mg, 0.0120 mmol) and EtsN (5.1 pL, 0.0360 mmol) were added sequentially. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 15.7 mg of DL-1 with 60.9% yield.

[0165] Table 4. HPLC condition (Column: YMC-Actus Trait Cl 8250 x 20 mm, 5 pm, 12 nm)Gradient:Time Flow (ml / min) H2O ACN0 min 10 90% 10%1 min 20 70% 30%3 min 20 55% 45%4 min 20 54% 46%6 min 20 53% 47%8 min 20 52% 48%10 min 20 52% 48%14 min 20 50% 50%15 min 20 46% 54%16 min 20 42% 58%17 min 20 38% 62%17.1 min 20 10% 90%22 min 20 10% 90%5S, G. ft Step 34-2. Preparation of BCN-GGVA-Hydra-PAB-PEG24-MMAE (also termed DL-2) and BCN- GGVA-Hydra-PAB-PEG24-T785 (also termed DL-3)

[0166] Step l:A solution of N-DT-0024 (50.0 mg, 0.065 mmol) and payload (MMAE or T785, 1.1 eq) in anhydrous DMF (0.65 mL), DIPEA (33.4 pL, 0.192 mmol) was added slowly. The resulting mixture was stirred at room temperature until N-DT-0024 was consumed. After reaction was completed, the reaction mixture was added by dropwise into a stirring TBME (6.5 mL) to get precipitate. The solids were collected by fdtration and followed with high vacuum drying to obtain crude HL-1 (X=MMAE with 75.6% yield) or HL-2 (X=T785 with 79.1% yield). This crude product was used in next step without further purification.

[0167] Step 2:A stirring suspension of HL-1 or HL-2 (83.4 mg) in DCM (1.46 mL) was cooled to 0 °C. TFA (0.63 mL) was added at 0 °C slowly. The reaction mixture was stirred at 0-4 °C for 20-24 hours. After reaction was completed, the reaction mixture was added by dropwise into a stirring TBME (20.9 mL) to get precipitate. The solids were collected by fdtration and followed with high vacuum drying to obtain crude intermediate. This crude product was used in next step without further purification.

[0168] The intermediate and BCN-OSu (1 eq) were dissolved in anhydrous DMF (0.05 M). EtsN (3 eq) was added into the reaction solution at room temperature. The reaction mixture was stirred at room temperature overnight. After reaction was completed, the reaction mixture was added by dropwise into a stirring TBME (10 times amount of intermediate) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude BCN intermediate. This crude product was used in next step without further purification.

[0169] The BCN intermediate (1 eq), NH2-PEG24-Ome (2 eq) and HATH (1.2 eq) were dissolved in anhydrous DMF (0.05 M). NMM (5 eq) was added. The reaction mixture was stirred at room temperature overnight. After reaction was completed, the resulting mixture was purified with preparative HPLC to obtain DL-2 or DL-3.

[0170] Table 5. HPLC condition (Column: YMC-Actus Trait Cl 8250 x 20 mm, 5 pm, 12 nm) Gradient:Time Flow (ml / min) H2O ACN0 min 10 90% 10%1 min 20 90% 10%5 min 20 80% 20%20 min 20 60% 40%25 min 20 10% 90%30 min 20 10% 90%DL-2 was obtained with 26.0% yield. MS (ESI) m / z found [(M+H+NH4 / 2]+, 1229.810Ci2oH2ioNi204o2+, required 1229.738.DL-3 was obtained with 27.7% yield. HRMS (ESI) m / z found [(M+2H) / 2]+, 1018.0743 C99H165N1 IO332+, required 1018.0785.payload DIPEA, DMFN-DT-0024 Step 1(1) 30% TFA in CH2CI2, 0-4 °C (2) BCN-OSu, Et3N, DMF, rt (3) HATU, NMM, NH2-PEG24-OMe, DMF HL-1, X= MMAE Step 2 HL-2, X= T785DL-2: X= MMAE DL-3: X= T7854-3. Preparation of BCN-GGVA-Hydra-PAB-PEG24-Eribulin (also termed DL-4)

[0171] Step l:

[0172] Fmoc-VA-PAB(COOtBu)-OH (202.8 mg, 0.329 mmol) in anhydrous CH2O2 (3.8 mL) was cooled to 0 °C. After five minutes, TFA (1.27 mL) was added slowly. The reaction mixture was stirred at 0-4 °C overnight. After reaction was completed, the reaction mixture was added by dropwise into a stirring TBME (50.0 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude intermediate (75.71 mg ). The previous intermediate (75.71 mg, 0.135 mmol), NH2-PEG24-OMe (161.9 mg, 0.149 mmol) andHATU (61.7 mg, 0.162 mmol) were dissolved in anhydrous DMF (1.35 mL). NMM (44.8 pL, 0.406 mmol) was added and the reaction mixture was stirred at room temperature for eight hours. After the reaction completed, the reaction mixture was concentrated in vacuo at 30-35 °C, the residue was purifiedby flash silica gel column (CH2Ch / MeOH = 12 / 1 to 9 / 1) to obtain 212.7 mg of HL-3 with 39.6% yield.

[0173] Step 2:

[0174] HL-3 (22.57 mg, 0.014 mmol) and Bis(4-nitrophenyl) carbonate (8.42 mg, 0.028 mmol) in anhydrous CH2O2 (0.28 mL) was cooled to 0-4 °C. 2,6-lutidine (2.4 pL, 0.021 mmol) and DIPEA (2.4 pL, 0.014 mmol) were added sequentially at 0-4 °C. The resulting mixture was stirred at 0-4 °C for 20-24 hours. Et2NH (1.4 pL, 0.014 mmol) was added slowly at 0-4 °C to quench excess Bis(4-nitrophenyl) carbonate. Anhydrous DMF (0.55 mL), Eribulin (15.2 mg, 0.021 mmol), DIPEA (7.2 pL, 0.042 mmol) were added into previous reaction solution at 0-4 °C. The reaction mixture was stirred at 0-4 °C overnight. After reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 7.54 mg of HL-4 with 22.8% yield.

[0175] Table 6. HPLC condition (Column: YMC-Actus Trait Cl 8250 x 20 mm, 5 pm, 12 nm) Gradient:Time Flow (ml / min) H2O ACN0 min 10 90% 10%1 min 20 80% 20%15 min 20 50% 50%25 min 20 10% 90%35 min 20 10% 90%

[0176] Step 3:

[0177] HL-4 (7.54 mg, 0.003 mmol) was dissolved in Q Ch / MeOH (226.0 pL, 1 / 1) and then cooled to 0 °C. Et2NH (45.2 pL) was added and the reaction mixture was stirred at 0-4 °C overnight. After the reaction completed, the reaction mixture was concentrated in vacuo at 30-35 °C, and azeotroped with Toluene (1 mL x 3) to remove excess Et2NH. The crude compound was high vacuum drying to obtain crude intermediate (8.2 mg). The intermediate (8.2 mg), Fmoc-GG-OH (1.61 mg, 0.004 mmol) and HATH (2.16 mg, 0.005 mmol) were dissolved in anhydrous DMF (0.2 mL). NMM (1.25 pL, 0.011 mmol) was added at room temperature. The resulting mixture was stirred at room temperature overnight. After the reaction completed, the reaction mixture was concentrated in vacuo at 30-35 °C, the residue was purified by flash silica gel column (CH2Ch / MeOH = 100 / 0 to 87 / 13) to obtain Fmoc-GG-intermediate. Fmoc-GG-intermediate was dissolved in CFLCb / MeOH (0.6 mL, 1 / 1) and then cooled to 0 °C. Et2NH (0.13 mL) was added and the reaction mixture was stirred at 0-4 °C for 20-24 hours. After the reaction completed, the reaction mixture was concentrated in vacuo at 30-35 °C, and azeotroped with Toluene (2 mL x 3) to remove excess Et? NH. The crude compound was high vacuum drying to obtain crude intermediate. The intermediate (19.3 mg), BCN-OSu (2.51 mg, 0.008 mmol) were dissolved inanhydrous DMF 0.17 mL. Et.iN (3.61 pL, 0.026 mmol) was added. The resulting mixture was stirred at room temperature overnight. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 2.85 mg of DL-3 with 37.4% yield. HRMS (ESI) m / z found [(M+2H) / 2]+, 1227.1713 Ci2iHi99N7O442+, required 1227.1697.

[0178] Table 7. HPLC condition (Column: YMC-Actus Trait Cl 8250 x 20 mm, 5 pm, 12 nm) Gradient:Time Flow (ml / min) H2O ACN0 min 10 90% 10%1 min 20 80% 20%15 min 20 50% 50%25 min 20 10% 90%35 min 20 10% 90%O^O'Bu (1) 25% TFA in CH2CI2, 0-4 °C (2) NH2-PEG24-OMe, HATU, NMM, DMFStep 1 Fmoc-VA-PAB(COOtBu)-OH2,6-lutidine, DIPEA, CH2CI20-4 °C, then Et2NH (2) Eribulin, DIPEA, DMF, 0-4 °C Step 2 HL-3(1) 20% Et2NH, CH2Cl2 / MeOH (1:1), 0-4 °C (2) Fmoc-GG-OH, HATU, NMM, DMF(3) 20% Et2NH, CH2CI2 / MeOH (1:1), 0-4 °C (4) BCN-OSu, Et3N, DMF Step 34-4. Preparation of DBCO-GGVA-Hydra-PAB-PEG24-Exatecan (also termed DL-5)

[0179] Step 1:DBCO-acid (265.7 mg, 0.87 mmol), tert-butyl 2-(2-aminoacetamido)acetate (163.8 mg, 0.87 mmol) and HATH (363.9 mg, 0.96 mmol) were dissolved in CH2O2 (8.7 mL) under room temperature. NMM (287.0 pL, 2.61 mmol) was added. After the addition, the resulting mixture was stirred at room temperature for 16-18 hours. The reaction solution was concentrated in vacuo at 30-35 °C, and the residue was purified by flash silica gel column (CH2Q2 / EA = 1 / 2 to 1 / 4) to obtain 342.2 mg of DBCO-4 with 82.7% yield.TL A. H2$ Y *•'" QrBa 0 2^2^®i®s~ ' W ’ CHsC!jBSCO-acid DSCCM Step 1

[0180] Step 2:A solution of DBCO-4 (340.0 mg, 0.71 mmol) in CH2O2 (6.8 mL) was cooled to 0 °C. TFA (1.7 mL) was added dropwise. The reaction mixture was warmed to room temperature (rt) slowly and then stirred for two hours. After the reaction was completed, the reaction mixture was concentrated in the vacuo at 30-35 °C and then azeotroped with Toluene (5 mL x 3) to remove excess TFA. The resulting mixture was purified with preparative HPLC to obtain 168.4 mg of DBCO-5 with 56.1% yield.

[0181] Table 8. HPLC condition (Column: YMC-Actus Trait Cl 8250 x 20 mm, 5 pm, 12 nm) Gradient:Time Flow (ml / min) H2O ACN0 min 20 90% 10% 2 min 20 80% 20% 10 min 20 60% 40% 20 min 20 60% 40% 25 min 20 10% 90% 27 min 20 10% 90%oicp z

[0182] Step 3:DBCO-5 (3.98 mg, 0.0095 mmol), H-PAB-6 (17.8 mg, 0.0095 mmol) andHATU (3.97 mg, 0.0104 mmol) were dissolved in anhydrous DMF (0.32 mL). Et3N (4.0 pL, 0.0285 mmol) was added. The reaction mixture was stirred at room temperature for four hours. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 10.7 mg of DL-7 with 49.6% yield.

[0183] Table 9. HPLC condition (Column: YMC-Actus Trait Cl 8250 x 20 mm, 5 pm, 12 nm) Gradient:Time Flow (ml / min) H2O ACN0 min 10 90% 10%1 min 20 80% 20%5 min 20 60% 40%15 min 20 45% 55%20 min 20 10% 90%25 min 20 10% 90%H. ABJStep 3Example 5. ADC preparation and analysis (ADC-1 to ADC-3)5-1. R4702-DAR4-ADC (ADC-1) preparation

[0184] BCN-GGVA-Hydra-PAB-PEG24-Exatecan (also termed as DL-1) (6-8 eq) was dissolved in DMSO (1 mg DL-1 / 50 pL DMSO) and then added slowly to R4702-(NSCT-4Ns)2 solution (antibody concentration 5 mg / mL in 20 mM NaOAc, pH 5.0) and shook at 25 °C for 16-24 hours. After the conjugation was completed, the crude ADC-1 was further purified by using Spectrum® Hollow Fiber Filter Modules (buffer: 20 mM NaOAc, pH 5.0) to afford ADC-1. ADC-1 was adjusted to around 10 mg / mL and sterilized by passing through ProMax™ Syringe Filter (PVDF, 0.22 pm). The drug-to-antibody ratio (DAR) value of final ADC-1 is determined by hydrophilic interaction chromatography (HIC).5-2. TX05-DAR4-ADC (ADC-2) preparation

[0185] BCN-GGVA-Hydra-PAB-PEG24-Exatecan (also termed as DL-1) 25.55 mg was dissolved in 1100 pL DMSO to form a solution. The solution (970 pL) was added slowly to TX05-(NSCT-4Ns)2 solution (antibody; concentration 5.16 mg / mL in 20 mM NaOAc, pH 5.0) and shook at 25 °C for sixteen hours. After the conjugation was completed, the participation was removed by filtration through a 0.22 pM Millipore Express® disposable vacuum filtration system. The crude ADC-2 was purified by hollow fiber (buffer: 20 mM NaOAc, pH 5.0). After washing with 1500 mL buffer, the ADC-2 was collected and exchanged the buffer to storage buffer (20 mM NaOAc, pH 5.0). The concentration of ADC-2 was adjusted to 4.97 mg / mL and sterilized by passing through ProMax™ Syringe Filter (PVDF, 0.22 um). Finally, 20.9 mL of ADC-2 was produced. The drug-to-antibody ratio (DAR) value of final ADC-2 is determined by hydrophilic interaction chromatography (HIC).5-3. 10K06-DAR8-ADC (ADC-3) preparation

[0186] BCN-GGVA-Hydra-PAB-PEG24-Exatecan (also termed as DL-1) was dissolved in DMSO to form a solution. The solution was added slowly to 10K06-(NSCT-tetra-N3)2 in 20 mM NaOAc, pH 5.0 buffer and stirred at 25 °C for 16 -24 hours. After conjugation, the crude ADC-3 was purified using a 30 kDa cut-off cassette, with a 40-volume buffer exchange (10 mM Histidine,pH 6.2). The concentration of ADC-3 was adjusted to 10 mg / mL and sterilized by passing through Millex-GP Syringe Filter (PES, 0.22 um). The drug-to-antibody ratio (DAR) value of final ADC-3 is determined by hydrophilic interaction chromatography (HIC).5-4. ADC analysis

[0187] The CE-SDS analysis was conducted under reducing condition. Beckman Coulter PA800Plus system equipped with a UV photodiode array detector (220 nm wavelength employed) was used in this test. A bare fused-silica capillary (50 m ID x 30 cm total length) with the 20 cm effective capillary separation length was rinsed with 0.1 M NaOH, 0.1 M HC1 and SDS gel buffer prior to injection. Electrokinetic injection mode was applied at -5 kV for 20 seconds in reverse polarity and followed by applying a -15 kV voltage for capillary separation. The total separation time was 35 minutes. 60 pg of Test article was sampled and diluted in 120pL sample buffer which was 1% SDS in diluted PBS, pH 7.0. Then diluted sample was mixed with 5 pL of 2ME, and 2 pL of 10 kDa internal standard, followed by incubation at 65 °C for 10 minutes. Finally, the sample was cooled down at room temperature for CE-SDS analysis.

[0188] EndoSz-D234M showed high deglycosylation and transglycosylation activity. R4702 (Anti-TROP2 mAb), TX05 (Anti-HER2 mAb) and 10K06 (Anti-Nectin-4) mAbs pretreated with EndoSz-D234M were used to produce R4702-BCN-GGVA-Hydra-PAB-PEG24-Exatecan ADC (ADC-1), TX05-BCN-GGVA-Hydra-PAB-PEG24-Exatecan (ADC-1) and 10K06-BCN-GGVA-Hydra-PAB-PEG24-Exatecan ADC (ADC-2), as the model of glycan site-specific ADCs. For ADC-1, R4702 mAb were deglycosylated by EndoSz-D234M with additional enzyme EndoH to cleave high mannose glycans. The yield of deglycosylated R4702 with one GlcNAc or potential fucose (termed R4702-GlcNAc(Fuc)) was about 96.42%. Next, R4702-GlcNAc(Fuc) were mixed with 20 equivalents of a modified complex type N-glycan (NSCT-2) at 37 °C for 1.5 hour to generate R4702-(NSCT-di-N3)2. The CE-SDS results indicated that 95.61% R4702-(NSCT-di-N3)2was produced by EndoSz-D234M. For ADC-1, EndoSz-D234M was used to hydrolyze the biantennary hybrid glycan on TX05 mAb to generate 94.5% TX05-GlcNAc(Fuc). In the tranglycosylation step, NSCT-2 was added to a mixture of TX05-GlcNAc(Fuc) and EndoSz-D234M. With 15 equivalents of NSCT-2 to TX05-GlcNAc(Fuc), 96.7% of TX05-(NSCT-di-N3)2was obtained by EndoSz-D234M catalysis at 15 °C for 4.5 hours incubation (FIGs. 2A-B). For ADC-2, EndoSz-D234M was used to hydrolyze the biantennary hybrid glycan on 10K06 mAb to generate 98.2% 10K06-GlcNAc(Fuc). In the tranglycosylation step, NSCT-4 were added to a mixture of 10K06-GlcNAc(Fuc) and EndoSz-D234M. With 15 equivalents of NSCT-4 to 10K06-GlcNAc(Fuc), 95.5% of 10K06-(NSCT-tetra-N3)2was obtained by EndoSz-D234M catalysis at 15 °C for 4.5 hours incubation (FIGs. 2C-D). These data demonstrate that EndoSz-D234M facilitatesthe generation of mAb-(NSCT-di-N3)2 and mAb-(NSCT-terta-N3)2, which were exemplary starting materials to produce glycan site-specific ADCs. The overall process of preparing a glycan sitespecific ADC with high homogeneity is summarized in FIG.3 A. Furthermore, FIG.3B shows an exemplary glycan site-specific ADC with DAR4 and FIG. 3C shows an exemplary glycan sitespecific ADC with DAR8.Example 6. DAR2 ADC (R4702-hexasaccharide-N3 conjugated with BCN-GGVA-Hydra-PAB-PEG24-Exatecan) preparation6-1. Generation of R4702-(hexasaccharide-N3)2 by glycan remodeling

[0189] R4702 (Anti-TROP2 mAb) firstly underwent deglycosylation by treated with EndoSz- D234M (enzyme / mAb ratio of 1 / 60, w / w) and Endo H (enzyme / mAb ratio of 1 / 5000, w / w) in 100 mM MOPS buffer (pH 7.0) at 37 °C for 72 hours. The resulting R4702-GlcNAc(Fuc) was incubated with ten equivalents of Compound 10 at 25 °C for 2.5 hours. Additional ten equivalents of oxazoline were added, and the mixture was further incubated at 15 °C for 2.5 hours. The residue was applied to a protein A column, followed by incubation with activated carbon to give pure R4702-(hexasaccharide-N3)2.6-2. Bioconjugation of R4702-(hexasaccharide-N3)2 with BCN-GGVA-Hydra-PAB-PEG24-Exatecan

[0190] To a solution of R4702-(hexasaccharide-N3)2 in 20 mM NaOAc buffer (pH 5.0), four equivalents of BCN-GGVA-Hydra-PAB-PEG24-Exatecan (DL-1) dissolved in DMSO were added at 25 °C. This mixture was stirred under 25 °C for 16 hours. After the reaction was completed, the crude DAR2 ADC was purified by UF / DF with a 30 kDa cut-off cassette. The concentration of final R4702-DAR2-ADC was adjusted to 10 mg / mL in 20 mM NaOAc buffer (pH 5.0), and sterilized by passing through Millex-GP Syringe Filter (PES, 0.22 um). The drug-to-antibody ratio (DAR) value of final ADC is determined by hydrophobic interaction chromatography (HIC). 6-3. The in-vitro cytotoxicity assay of DAR2 ADC

[0191] Cytotoxicity was determined using CellTiter-Glo™ Luminescent assay reagent (Promega). Human BxPC-3 pancreatic adenocarcinoma cancer cells (ATCC, CRL-1687) were used in this study. The cells were plated in 96-well plates with 4E+03 cells per well and incubated at 37 °C overnight. Different bioorthogonal groups of ADCs were prepared from 30 to 0.005 nM with four-fold serial dilution in cell culture medium (McCoy’s 5A medium contain 10% FBS) and then added to the plates. After incubated at 37 °C for six days, the CellTiter-Glo luciferase assay reagent was added to each well. The plate was placed on an orbital shaker for two minutes to induce cell lysis and then incubated at room temperature for ten minutes before recording theluminescent signals by Luminometer. Luminescence was determined using a microplate luminometer SpectraMax L (Molecular Devices, Sunnyvale, CA). The percentage of the cytotoxicity was calculated by dividing the non-treated cell luminescence minus experimental cell luminescence by the non-treated cell luminescence and multiplying by 100. IC50 was determined by plotting x (Concentration in nM) - y (Cell viability in %) and fitting the data in a 4PL nonlinear regression model by PRISM 6 Software. FIG.4 indicated the IC50 of R4702 DAR2 ADC (R4702-hexasaccharide-Ns conjugated with BCN-GGVA-Hydra-PAB-PEG24-Exatecan) was 0.68 nM, but the IC50 of negative control IgG-ADC was >100 nM. The IC50 data of R4702 DAR2 ADC demonstrate its potency.Example 7. The in-vitro cytotoxicity assay of different linker-payloads (BCN and DBCO) in DAR4 ADC

[0192] Cytotoxicity was determined using CellTiter-Glo™ Luminescent assay reagent (Promega). Human BxPC-3 pancreatic adenocarcinoma cancer cells (ATCC, CRL-1687) were used in this study. The cells were plated in 96-well plates with 8E+03 cells per well and incubated at 37 °C overnight. Different bioorthogonal groups of ADCs were prepared from 30 to 0.005 nM with three-fold serial dilution in cell culture medium (McCoy’s 5 A medium contain 10% FBS) and then added to the plates. After incubated at 37 °C for six days, the CellTiter-Glo luciferase assay reagent was added to each well. The plate was placed on an orbital shaker for two minutes to induce cell lysis and then incubated at room temperature for ten minutes before recording the luminescent signals by Luminometer. Luminescence was determined using a microplate luminometer SpectraMax L (Molecular Devices, Sunnyvale, CA). The percentage of the cytotoxicity was calculated by dividing the non-treated cell luminescence minus experimental cell luminescence by the non-treated cell luminescence and multiplying by 100. IC50 was determined by plotting x (concentration in nM) - y (drug cytotoxicity in %) and fitting the data in a 4PL nonlinear regression model by PRISM 6 Software. FIG. 5 indicated the IC50 of R4702-DBCO-GGVA-Hydra-PAB-PEG24-Exatecan was 0.23 nM, and the IC50 of OBI-902 (R4702-BCN-GGVA-Hydra-PAB-PEG24-Exatecan) was 0.28 nM. The IC50 data for the ADCs with different bioorthogonal groups were similar.Example 8. In-vivo anti-tumor efficacy comparison between OBI-902 (R4702-BCN-GGVA-Hydra-PAB-PEG24-Exatecan) DAR4 ADC and datopotamab deruxtecan (Dato-Dxd)

[0193] 8-1. Measurement of anti-tumor activity inNCI-N87 human gastric cancer cell-derived xenograft in BLAB / c nude mice

[0194] 8-1-1. Test substances and dosing pattern(a) OBI-902: 3 mg / mL(b) Dato-Dxd: 3 mg / mLTable 10. Study Design and samplingVolume ofRoute and Dose of injection Animal Groups injectionadministration (mg / kg) No.(mL / kg)Gl: Vehicle IV - 5 5 G2: OBI-902 IV 3 5 5 G3: Dato-Dxd IV 3 5 5Intravenous injection (IV) ■ Volume of injection is 5 mL / kg

[0195] 8-1-2. Cell line: NCI-N87 (High TROP2 expressed cancer cell)

[0196] 8-1-3. Animal(a) Species: Mus musculus(b) Strain: CAnN. Cg-Foxnlnu / CrlBltw (BALB / c nude)(c) Source: BioLasco Taiwan(d) Sex: Female(e) Age at initiation of study: 7 weeks(f) Body weight range at start of study: 15-25 g(g) Animal grouping: The mice were divided into 3 groups and each group contained 5 mice. A total of 15 mice were involved in study of NCI-N87 CDX model.

[0197] 8-1-4. Equipment and material(a) Biosafety cabinet (NUAIRE / NU-620-400)(b) Electronic balance (CROMTECH / YP30002)(c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line) (d) Vernier (METRGLGGY / EC-9001V)(e) Matrigel (BD / Cat. No.: 356234)

[0198] 8-1-5. Method(a) Establishment of xenograft mouse modelSubcutaneous inoculation of tumor cells: 2.5x106NCI-N87 cells were mixed with the equal volume of matrigel (volume ratio 1:1) (Corning, Cat. No.354248). Subcutaneous injection volume was 100 pL / mouse.(b) Route and administration of test articleThe first dosing day was denoted as Day 1 when average tumor volume reaches 200-250 mm3in NCI-N87 CDX model. All test articles (test item a and b) or reference item (Sodium citrate solution)were intravenously administered to the mice on Day 1. The injection was performed using insulin syringe with the dosage 3 mg / kg for NCI-N87 CDX model, and the injection volume was 5 mL / kg. (c) Tumor growth inhibition rate calculationTumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 - (Ti - T1 ) / (Ci - Cl)] x 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 57). Whereas TI and Cl indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1).(d) Statistical analysisResults were presented as mean and standard error of the mean (mean ± SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t-test. p < 0.05 is considered significance.

[0199] 8-1-6. ResultFIG. 6A indicated the in-vivo efficacy result in NCI-N87 CDX model. The mean±SEM of tumor volume to vehicle group was 1292.48 315.89 mm3on Day 26. Mean±SEM of tumor volume to treated groups were 240.481164.37 to OBI-902 and 944.01482.01 to Dato-Dxd on Day 57. It demonstrated OBI-902 had better inhibition efficacy than Dato-DxD inNCI-N87 gastric cancer.

[0200] 8-2. Measurement of anti -tumor activity in DLD-1 human colorectal cancer cell-derived xenograft in BLAB / c nude mice

[0201] 8-2-1. Test substances and dosing pattern(a) OBI-902: 10 mg / mL(b) Dato-Dxd: 10 mg / mLTable 11. Study Design and samplingRoute and Dose of injection Volume of injection Animal Groupsadministration (mg / kg) (mL / kg) No.Gl: Vehicle IV - 5 5 G2: OBI-902 IV 10 5 5 G3: Dato-Dxd IV 10 5 5Intravenous injection (IV) ■ Volume of injection is 5 mL / kg

[0202] 8-2-2. Cell line: DLD-1 (Middle TROP2 expressed cancer cell)

[0203] 8-2-3. Animal(a) Species: Mus musculus(b) Strain: CAnN. Cg-Foxnlnu / CrlBltw (BALB / c nude)(c) Source: BioLasco Taiwan(d) Sex: Female(e) Age at initiation of study: 7 weeks(f) Body weight range at start of study: 15-25 g(g) Animal grouping: The mice were divided into 3 groups and each group contained 5 mice. A total of 15 mice were involved in study of DLD-1 CDX model.

[0204] 8-2-4. Equipment and material(a) Biosafety cabinet (NUAIRE / NU-620-400)(b) Electronic balance (CRGMTECH / YP30002)(c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line) (d) Vernier (METRGLGGY / EC-9001V)(e) Matrigel (BD / Cat. No.: 356234)

[0205] 8-2-5. Method(a) Establishment of xenograft mouse modelSubcutaneous inoculation of tumor cells: 5x106DLD-1 cells were mixed with the equal volume of matrigel (volume ratio 1:1). Subcutaneous injection volume was 100 pL / mouse.(b) Route and administration of test articleThe first dosing day was denoted as Day 1 when average tumor volume reaches 250-300 mm3in DLD-1 CDX model. All test articles (test item a and b) or reference item (Sodium citrate solution) were intravenously administered to the mice on Day 1. The injection was performed using insulin syringe with the dosage 10 mg / kg for DLD-1 CDX model, and the injection volume was 5 mL / kg. (c) Tumor growth inhibition rate calculationTumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 - (Ti - Tl) / (Ci - Cl)] x 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 56). Whereas TI and Cl indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1).(d) Statistical analysisResults were presented as mean and standard error of the mean (mean ± SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t-test. p < 0.05 is considered significance.

[0206] 8-2-6. Result

[0207] FIG. 6B indicated the in-vivo efficacy result in DLD-1 CRC CDX model. The mean±SEM of tumor volume to vehicle group was 1038.67 309.98 mm3on Day 11. MeanlSEM of tumor volume to treated groups were 329.351302.27 to OBI-902 on Day 56 and 980.901605.56to Dato-Dxd on Day 18. It demonstrated OBI-902 had better inhibition efficacy than Dato-DxD in DLD-1 colorectal cancer.

[0208] 8-3. Measurement of anti -tumor activity in HP AC human PDAC (pancreatic ductal adenocarcinoma) cell-derived xenograft in BLAB / c nude mice

[0209] 8-3-1. Test substances and dosing pattern(a) OBI-902: 5 mg / mL(b) Dato-Dxd: 5 mg / mLTable 12. Study Design and samplingRoute and Dose of injection Volume of injection Animal Groupsadministration (mg / kg) (mL / kg) No.Gl: Vehicle IV - 5 5 G2: OBI-902 IV 5 5 5 G3: Dato-Dxd IV 5 5 5Intravenous injection (IV) ■ Volume of injection is 5 mL / kg

[0210] 8-3-2. Cell line: HP AC (Middle TROP2 expressed cancer cell)

[0211] 8-3-3. Animal(a) Species: Mus musculus(b) Strain: CAnN. Cg-Foxnlnu / CrlBltw (BALB / c nude)(c) Source: BioLasco Taiwan(d) Sex: Female(e) Age at initiation of study: 7 weeks(f) Body weight range at start of study: 15-25 g(g) Animal grouping: The mice were divided into 3 groups and each group contained 5 mice. A total of 15 mice were involved in study of HP AC CDX model.

[0212] 8-3-4. Equipment and material(a) Biosafety cabinet (NUAIRE / NU-620-400)(b) Electronic balance (CROMTECH / YP30002)(c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line) (d) Vernier (METROLOGY / EC-9001V)(e) Matrigel (BD / Cat. No.: 356234)

[0213] 8-3-5. Method(a) Establishment of xenograft mouse modelSubcutaneous inoculation of tumor cells: 3x106HP AC cells were mixed with the equal volume of matrigel (volume ratio 1:1). Subcutaneous injection volume was 100 pL / mouse.(b) Route and administration of test articleThe first dosing day was denoted as Day 1 when average tumor volume reaches 150-200 mm3in HP AC CDX model. All test articles (test item a and b) or reference item (Sodium citrate solution) were intravenously administered to the mice on Day 1. The injection was performed using insulin syringe with the dosage 5 mg / kg for HP AC CDX model, and the injection volume was 5 mL / kg. (c) Tumor growth inhibition rate calculationTumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 - (Ti - Tl) / (Ci - Cl)] x 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 43). Whereas TI and Cl indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1).(d) Statistical analysisResults were presented as mean and standard error of the mean (mean ± SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t-test. p < 0.05 is considered significance.

[0214] 8-3-6. Result

[0215] FIG. 6C indicated the in-vivo efficacy result in HP AC PDAC CDX model. The mean±SEM of tumor volume to vehicle group was 1085.60±586.09 mm3on Day 24. Mean±SEM of tumor volume to treated groups were 426.13±336.02 to OBI-902 and 1064.30±504.94 to Dato-Dxd on Day 43. It demonstrated OBI-902 had better inhibition efficacy than Dato-DxD in HP AC PDAC cancer.

[0216] 8-4. Measurement of anti -tumor activity in TFK-1 human cholangiocarcinoma cell-derived xenograft in BLAB / c nude mice

[0217] 8-4-1. Test substances and dosing pattern(a) OBI-902: 6 mg / mL(b) Dato-Dxd: 6 mg / mLTable 13. Study Design and samplingRoute and Dose of injection Volume of injection Animal Groupsadministration (mg / kg) (mL / kg) No.Gl: Vehicle IV - 5 5 G2: OBI-902 IV 6 5 5 G3: Dato-Dxd IV 6 5 5Intravenous injection (IV) ■ Volume of injection is 5 mL / kg

[0218] 8-4-2. Cell line: TFK-1 (High TROP2 expressed cancer cell)

[0219] 8-4-3. Animal(a) Species: Mus musculus(b) Strain: CAnN. Cg-Foxnlnu / CrlBltw (BALB / c nude)(c) Source: BioLasco Taiwan(d) Sex: Female(e) Age at initiation of study: 7 weeks(f) Body weight range at start of study: 15-25 g(g) Animal grouping: The mice were divided into 3 groups and each group contained 5 mice. A total of 15 mice were involved in study of TFK-1 CDX model.

[0220] 8-4-4. Equipment and material(a) Biosafety cabinet (NUAIRE / NU-620-400)(b) Electronic balance (CROMTECH / YP30002)(c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line) (d) Vernier (METROLOGY / EC-9001V)(e) Matrigel (BD / Cat. No.: 356234)

[0221] 8-4-5. Method(a) Establishment of xenograft mouse modelSubcutaneous inoculation of tumor cells: IxlO7TFK-1 cells were mixed with the equal volume of matrigel (volume ratio 1:1). Subcutaneous injection volume was 200 pL / mouse.(b) Route and administration of test articleThe first dosing day was denoted as Day 1 when average tumor volume reaches 150-200 mm3in TFK-1 CDX model. All test articles (test item a and b) or reference item (Sodium citrate solution) were intravenously administered to the mice on Day 1. The injection was performed using insulin syringe with the dosage 6 mg / kg for TFK-1 CDX model, and the injection volume was 5 mL / kg. (c) Tumor growth inhibition rate calculationTumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 - (Ti - Tl) / (Ci - Cl)] x 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 62). Whereas TI and Cl indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1).(d) Statistical analysisResults were presented as mean and standard error of the mean (mean ± SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t-test. p < 0.05 is considered significance.

[0222] 8-4-6. Result

[0223] FIG. 6D indicated the in-vivo efficacy result in TFK-1 cholangiocarcinoma CDX model. The mean±SEM of tumor volume to vehicle group was 577.37±232.58 mm3on Day 44. Mean±SEM of tumor volume to treated groups were complete regression (0 mm3) to OBI-902 and 254.01±317.99 to Dato-Dxd on Day 62. It demonstrated OBI-902 had better inhibition efficacy than Dato-DxD in TFK-1 cholangiocarcinoma cancer.Example 9. In-vitro cytotoxicity comparison of Anti-nectin-4 ADCs

[0224] Tumor cells (2 x 103cells / well) were seeded in 96 well plate and treated with 10K06-DAR8-ADC (also termed OBI-904) for six days. CellTiter-Glo® Reagent (Cat. G7572, Promega) was prepared by adding CellTiter-Glo® Buffer into lyophilized CellTiter-Glo® Substrate. Reconstituted CellTiter-Glo® Reagent was added into the culture medium with cells at 1:1 ratio after treatment for six days. The plate was placed on an orbital shaker for two minutes to induce cell lysis and then incubated at room temperature for ten minutes before recording the luminescent signals by Luminometer. Luminescence was determined using a microplate luminometer SpectraMax L (Molecular Devices, Sunnyvale, CA). Percentage of the cytotoxicity was calculated through dividing the non-treated cell luminescence minus experimental cell luminescence by the non-treated cell luminescence and multiplying by 100. IC50 was determined by plotting x (concentration in nM) - y (drug cytotoxicity in %) and fitting the data in a 4PL nonlinear regression model by PRISM 6 Software.

[0225] The in vitro efficacy of OBI-904 and commercial anti-Nectin-4 ADCs in human colorectal cancer cell line DLD-1 cells (Cat. No. 60132, Bioresource Collection and Research Center) and human hypopharyngeal squamous cell line FaDu (Cat. No. 60214, Bioresource Collection and Research Center) were evaluated by cytotoxicity assay. The IC50 of ETx-22 was 32 nM and OBI-904 was 143 nM in DLD-1 cancer cell (FIG. 7A). It indicated the IC50 of these two ADCs were better than Padcev®. Furthermore, the IC50 of ETx-22 was 14 nM, OBI-904 was 67 nM, and Padcev® was 153 nM in FaDu cancer cell (FIG. 7B). It also demonstrated OBI-904 is competitive with other commercial anti-Nectin-4 ADCs.Example 10. In-vivo tumor inhibition efficacy comparison of Anti-nectin-4 ADCs

[0226] 10.1. Nectin-4 overexpression PC-3 human prostate cancer cell-derived xenograft in BLAB / c nude mice

[0227] 10.1.1 Test Substances and Dosing Pattern(a) OBI-904: 3 mg / kg(b) EV (Enfortumab Vedotin): 3 mg / kgTable 14. Study Design and samplingRoute and Dose of Volume of Animal Groups administration injection injectionNo. of test item (mg / kg) (mL / kg)Gl: Vehicle IV - 5 4 G2: OBI-904 IV 3 5 4 G3: EV (PADCEV, Enfortumab Vedotin) IV 3 5 4 Intravenous injection (IV) ■Volume of injection is 5 mL / kg

[0228] 10.1.2 Cell line: Nectin-4 overexpression PC-3 cells (High Nectin-4 expressed) (Cat. No. 60122, Bioresource Collection and Research Center)

[0229] 10.1.3 Animal(a) Species: Mus musculus(b) Strain: CAnN. Cg-Fox«7"“ / CrlBltw (BALB / c nude)(c) Source: BioLasco Taiwan(d) Sex: Female(e) Age at initiation of study: seven weeks(f) Body weight range at start of study: 15-25 g(g) Animal Grouping: The mice were divided into three groups and each group contained four mice. A total of twelve mice were involved in the study.

[0230] 10.1.4 Equipment and Material(a) Biosafety cabinet (NUAIRE / NU-620-400)(b) Electronic balance (CROMTECH / YP30002)(c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line)(d) Vernier (METROLOGY / EC-9001V)(e) Matrigel (BD / Cat. No.: 356234)

[0231] 10.1.5 Method(a) Establishment of xenograft mouse modelSubcutaneous inoculation of tumor cells: IxlO7Nectin-4 overexpression PC3 cells were mixed with the equal volume of matrigel (volume ratio 1:1) (Corning, 354248, Lot No.: 0261002). Subcutaneous injection volume was 200 pL / mouse.(b) Route and administration of test article:The first dosing day was denoted as Day 1 when average tumor volume reach 150-200 mm3. All test articles (test item a and b) or reference item (Sodium citrate solution) were intravenouslyadministered to the mice on Day 1. The injection was performed using insulin syringe with the dosage 3 mg / kg and the injection volume was 5 mL / kg.(c) Tumor growth inhibition rate calculationTumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 - (Ti - T1 ) / (Ci - Cl)] x 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 49). Whereas TI and Cl indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1).(d) Statistical analysisResults were presented as mean and standard error of the mean (mean±SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t-test. p < 0.05 is considered significance.

[0232] 10.1.6 Result

[0233] FIG. 8A indicated the in-vivo efficacy result in Nectin-4 overexpression PC3 CDX model. The mean±SEM of tumor volume to vehicle group was 1036.52±145.39 mm3on Day 14. The mean±SEM of tumor volume to treated groups were 168.48±183.98 mm3to OBI-904 on Day 49 and 585.06±467.77 mm3to EV (Enfortumab Vedotin) on Day 43. It demonstrated that the tumor inhibitions efficacy of OBI-904 was higher than EV.

[0234] 10.2. Nectin-4 middle expressed FaDu human head and neck cancer cell-derived xenograft in BLAB / c nude mice

[0235] 10.2.1 Test Substances and Dosing Pattern(a) OBI-904: 6 mg / kg(b) EV (Enfortumab Vedotin): 6 mg / kgTable 15. Study Design and samplingRoute and Dose of Volume of Animal Groups administration injection injectionNo. of test item (mg / kg) (mL / kg)Gl: Vehicle IV - 5 5 G2: OBI-904 IV 6 5 5 G3: EV (PADCEV, Enfortumab Vedotin) IV 6 5 5 Intravenous injection (IV) ■Volume of injection is 5 mL / kg

[0236] 10.2.2 Cell line: FaDu cells (Middle Nectin-4 expressed) (Cat. No. 60214, Bioresource Collection and Research Center)

[0237] 10.2.3 Animal(a) Species: Mus musculus(b) Strain: CAnN. Cg-Fox«7"“ / CrlBltw (BALB / c nude)(c) Source: BioLasco Taiwan(d) Sex: Female(e) Age at initiation of study: seven weeks(f) Body weight range at start of study: 15-25 g(g) Animal Grouping: The mice were divided into three groups and each group contained five mice. A total of fifteen mice were involved in the study.

[0238] 10.2.4 Equipment and Material(a) Biosafety cabinet (NUAIRE / NU-620-400)(b) Electronic balance (CROMTECH / YP30002)(c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line)(d) Vernier (METROLOGY / EC-9001V)(e) Matrigel (BD / Cat. No.: 356234)

[0239] 10.2.5 Method(a) Establishment of xenograft mouse modelSubcutaneous inoculation of tumor cells: 5x106FaDu cells were mixed with the equal volume of matrigel (volume ratio 1:1) (Corning, 354248, Lot No.: 0261002). Subcutaneous injection volume was 100 pL / mouse.(b) Route and administration of test article:The first dosing day was denoted as Day 1 when average tumor volume around 150 mm3. All test articles (test item a and b) or reference item (Sodium citrate solution) were intravenously administered to the mice on Day 1. The injection was performed using insulin syringe with the dosage 6 mg / kg and the injection volume was 5 mL / kg.(c) Tumor growth inhibition rate calculationTumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 - (Ti - Tl) / (Ci - Cl)] x 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 14). Whereas TI and Cl indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1).(d) Statistical analysisResults were presented as mean and standard error of the mean (mean±SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t-test. p < 0.05 is considered significance.

[0240] 10.2.6 Result

[0241] FIG. 8B indicated the in-vivo efficacy result in FaDu head and neck CDX model. The mean± SEM of tumor volume to vehicle group was 1050.83±305.98 mm3on Day 14. The mean±SEM of tumor volume to treated groups were 241.70±193.64 mm3to OBI-904 and 931.14±431.90 mm3to EV (Enfortumab Vedotin). It demonstrated that the tumor inhibitions efficacy of OBI-904 was higher than EV.

[0242] 10.3. Nectin-4 low expressed TFK-1 human cholangiocarcinoma cell-derived xenograft in BLAB / c nude mice

[0243] 10.3.1 Test Substances and Dosing Pattern(a) OBI-904: 8 mg / kg(b) EV (Enfortumab Vedotin): 8 mg / kgTable 16. Study Design and samplingRoute and Dose of Volume of Animal Groups administration injection injectionNo. of test item (mg / kg) (mL / kg)Gl: Vehicle IV - 5 5 G2: OBI-904 IV 8 5 5 G3: EV (PADCEV, Enfortumab Vedotin) IV 8 5 5 Intravenous injection (IV) ■Volume of injection is 5 mL / kg

[0244] 10.3.2 Cell line: TFK-1 cells (Low Nectin-4 expressed) (Cat. No. ACC344, DSMZ)

[0245] 10.3.3 Animal(a) Species: Mus musculus(b) Strain: CAnN. Cg-Fox«7"“ / CrlBltw (BALB / c nude)(c) Source: BioLasco Taiwan(d) Sex: Female(e) Age at initiation of study: seven weeks(f) Body weight range at start of study: 15-25 g(g) Animal Grouping: The mice were divided into three groups and each group contained six mice. A total of eighteen mice were involved in the study.

[0246] 10.3.4 Equipment and Material(a) Biosafety cabinet (NUAIRE / NU-620-400)(b) Electronic balance (CROMTECH / YP30002)(c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line)(d) Vernier (METROLOGY / EC-9001V)(e) Matrigel (BD / Cat. No.: 356234)

[0247] 10.3.5 Method(a) Establishment of xenograft mouse modelSubcutaneous inoculation of tumor cells: IxlO7TFK-1 cells were mixed with the equal volume of matrigel (volume ratio 1:1) (Corning, 354248, Lot No.: 0261002). Subcutaneous injection volume was 200 pL / mouse.(b) Route and administration of test article:The first dosing day was denoted as Day 1 when average tumor volume reach 150-200 mm3. All test articles (test item a and b) or reference item (Sodium citrate solution) were intravenously administered to the mice on Day 1. The injection was performed using insulin syringe with the dosage 8 mg / kg and the injection volume was 5 mL / kg.(c) Tumor growth inhibition rate calculationTumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 - (Ti - Tl) / (Ci - Cl)] x 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 62). Whereas TI and Cl indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1).(d) Statistical analysisResults were presented as mean and standard error of the mean (mean±SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t-test. p < 0.05 is considered significance.

[0248] 10.3.6 Result

[0249] FIG. 8C indicated the in-vivo efficacy result in TFK-1 human cholangiocarcinoma CDX model. The mean±SEM of tumor volume to vehicle group was 916.80±320.79 mm3, 204.62±319.50 mm3to OBI-904 and 669.04±237.85 mm3to EV (Enfortumab Vedotin) on Day 62. It demonstrated that the tumor inhibitions efficacy of OBI-904 was higher than EV.

[0250] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of this invention. Although any compositions, methods, kits, and means for communicating information similar or equivalent to those described herein can be used to practice this invention, the preferred compositions, methods, kits, and means for communicating information are described herein.

[0251] All references cited herein are incorporated herein by reference to the full extent allowed by law. The discussion of those references is intended merely to summarize the assertions madeby their authors. No admission is made that any reference (or a portion of any reference) is relevant prior art. Applicants reserve the right to challenge the accuracy and pertinence of any cited reference.

Claims

Claims1. A glycan linker having a structure represented by Formula (II):wherein:G represents a glycan moiety connected to an N-acetylglucosamine oxazoline (Oxa-GlcNAc) through Q, wherein the glycan moiety is a linear or branched chain of saccharides selected from the group consisting of galactose (Gal), N-acetyl-glucosamine (GlcNAc), N-acetylneuraminic acid (Neu5Ac), glucose (Glc), mannose (Man), fticose (Fuc), and derivatives thereof;Q is selected from the group consisting of O, S, Se, CH2, NH, or NRP, wherein Rpis a protecting group;Li is connected between -COOH of Neu5Ac and X and selected from:X includes 1 -200 atoms, and is selected from the group consisting of linear or branch alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl network with amino, amide, carbamate, ether, ester, sulfonamide fimctional group;Y is a linear alkyl or heteroalkyl chain having 1-100 carbon atoms;Z is selected from -N3, dibenzocyclooctyne (DBCO), trans-cyclooctene (TCO) or a ketone group;a and b are independently 1, 2, 3 or 4;m is a number of Neu5Ac units branching from G, and is an integer from 1 to 10; and n is an integer selected from 1 to 20.The glycan linker according to claim 1, wherein the structure of Formula (II) has a structure of the following Formula (III):wherein a is an integer selected from 1 to 4.The glycan linker according to claim 1, wherein the glycan moiety G in Formula (II) is represented by one of the following Formula:NHAc (VI), orwherein in the above formulas, symbol ~ means the connection with Neu5Ac and symbolmeans the connection with Q.The glycan linker according to claim 1, wherein the glycan linker is represented by one of the following Formula:OH ACHN (VIII),5. The glycan linker according to claim 1, wherein the glycan linker is represented by one of the following Formula:An antibody conjugate having a structure represented by Formula (XVI):(Fuc)d(XVI); wherein:Ab is an antibody or an antigen-binding fragment thereof;GlcNAc is N-acetylglucosamine;Fuc is fucose;G represents a glycan moiety connected to an N-acetylglucosamine oxazoline (Oxa-GlcNAc) through Q, wherein the glycan moiety is a linear or branched chain of saccharides selected from the group consisting of galactose (Gal), N-acetyl-glucosamine (GlcNAc), N-acetylneuraminic acid (Neu5Ac), glucose (Glc), mannose (Man), fucose (Fuc), and derivatives thereof;Q is selected from the group consisting of O, S, Se, CH2, NH, or NRP, wherein Rpis a protecting group;Li is connected between -COOH of Neu5Ac and X and selected from:X includes 1 -200 atoms, and is selected from the group consisting of linear or branch alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl network with amino, amide, carbamate, ether, ester, sulfonamide functional group;Y is a linear alkyl or heteroalkyl chain having 1-100 carbon atoms;a and b are independently 1, 2, 3 or 4;m is a number of Neu5Ac units branching from G, and is an integer from 1 to 10; andn is an integer selected from 1 to 20.Z’ is independently selected from a triazole or imine linkage;d is 0 or 1;c is an integer ranging from 1 to 128;L2 is a linker connecting D and Z’ and comprising a protease-cleavable peptide moiety, a glycosidase-cleavable sugar moiety, a pH sensitive moiety, or a hydrolysable moiety; and D is a payload selected from a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitory agent, a protein degrader, a peptide, a radionuclide, a hormone, an anti-viral agent, an anti-bacterial agent, an immunoregulatory agent, a fluorophore, a dye, or a contrast agent.The antibody conjugate according to claim 6, wherein the antibody or antigen-binding fragment thereof is monospecific or multispecific.The antibody conjugate according to claim 6, wherein the antibody or antigen-binding fragment thereof is capable of binding to CD44, DLL3, CEACAM-5, CEACAM-6, ROR1, NaPi2b, CLDN18.2, CLDN1, CLDN2, CDH6, CDH17, B7-H3, MUC-1, PD-1, PD-L1, CTLA-4, VEGF, BCMA, PSMA, CGRP, Tfr 1, TNF alpha, tissue factor, folate receptor alpha, cMET, HER3, EGFR, HER2, TROP2, Nectin-4, CD326, CDH1, NCAM1, TEM1, SSEA-4, MUC4, MUC5AC, MUC16,GPC3, GPC2, FGFR1, FGFR2, FGFR3, PDGFR-a, PDGFR-b, IGF-1R, AXL, LAG-3, TIM-3, TIGIT, VISTA, B7-H4, CD19, CD20, CD22, CD24, CD38, CD47, CD33, CD70, CD123, CD133, ITGB1, ITGB5, ITGB6, ITGB8, CAIX, NRP1, NRP2, GD2, GD3, MSLN, EphA2, EphB4, c-KIT, ASCT2, IL1RAP, Ly6E, PTK7, uPARAP, CRLF2, SEZ6, TM4SF1, STEAP1, STEAP2, CADM1, CDCP1, GCC or LIV-1.The antibody conjugate according to claim 6, wherein the antibody or antigen-binding fragment thereof is bispecific to HER2 and TR0P2, c-Met and HER3, EGFR and HER3, or EGFR and c-Met.The antibody conjugate according to claim 6, wherein the cytotoxic agent is selected from pyrrolobenzodiazepine compounds or derivatives thereof, auristatin compounds or derivatives thereof, maytansinoid compounds or derivatives thereof, duocarmycin or derivatives thereof, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors or derivatives thereof, tubulysin compounds or derivatives thereof, enediyne compounds or derivatives thereof, anthracycline compounds or derivatives thereof, pyrrole-based kinesin spindle protein (KSP) inhibitors or derivatives thereof, cryptophycin compounds or derivatives thereof, drug efflux pump inhibitors or derivatives thereof, sandramycin or derivatives thereof, amanitin compounds or derivatives thereof, or camptothecin compounds or derivatives thereof.The antibody conjugate according to claim 10, wherein the cytotoxic agent is exatecan, monomethyl auristatin E (MMAE), l-(4-aminobutyl)-2-butylimidazo[4,5-c]quinolin-4-amine (T785), or Eribulin.The antibody conjugate according to claim 6, wherein L2-D has a structure of Formula (XVII-2) or Formula (XVIII-2):(XVII-2), or(XVIII-2); wherein z is an integer ranging from 6 to 48.

13. A pharmaceutical composition comprising the antibody conjugate according to any one of claims 6-12 and a pharmaceutically acceptable carrier.

14. A method for preparing an antibody conjugate, comprising:(a) conjugating an antibody with the glycan linker according to any one of claims 1 -6 to obtain the glycoengineered antibody, wherein the antibody has a fucosylated or non-fucosylated N- acetylglucosamine (GlcNAc) linked to an asparagine residue of the antibody, and(b) reacting the glycoengineered antibody with a linker-payload to obtain an antibody conjugate, wherein the linker-payload comprises a biorthogonal group.

15. The method according to claim 14, wherein the biorthogonal group is selected from a dibenzocyclooctyne (DBCO) group, a bicyclononyne (BCN) group, a alkyne group, a maleimide group, a a, P-unsaturated carbonyl group, a sulfonyl pyrimidine group, a 4-dibenzocyclooctynol (DIBO) group, a aza-dibenzocyclooctynes (DIBAC) group, a tetrazine group, a tetrazole group, a norbornene group, a cyclooctyne group, a methylcyclopropene group, an aminooxy group, a hydrazine group, an isocyanide group, an isocyanopropanoate group, a phosphine-containing thioester group, a phosphine phenolic ester group, or an alpha-halo carbonyl group.

16. The method according to claim 14, wherein the payload is a drug unit and the antibody conjugate is an antibody-drug conjugate (ADC).

17. A method for treating a disease, comprising administering to a subject in need thereof an effective amount of the antibody conjugate according to claim 6, wherein the payload D is selected from a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitory agent, a protein degrader, a peptide, a radionuclide, a hormone, an anti-viral agent, an anti-bacterial agent, or an immunoregulatory agent.

18. The method according to claim 17, wherein the payload D is exatecan, monomethyl auristatin E(MMAE), l-(4-aminobutyl)-2-butylimidazo[4,5-c]quinolin-4-amine (T785), or Eribulin.

19. The method according to claim 17, wherein the disease is characterized by expressing CD44, DLL3, CEACAM-5, CEACAM-6, R0R1, NaPi2b, CLDN18.2, CLDN1, CLDN2, CDH6, CDH17, B7-H3, MUC-1, PD-1, PD-L1, CTLA-4, VEGF, BCMA, PSMA, CGRP, Tfrl, TNF alpha, tissue factor, folate receptor alpha, cMET, HER3, EGFR, HER2, TROP2, Nectin-4, CD326, CDH1, NCAM1, TEM1, SSEA-4, MUC4, MUC5AC, MUC16, GPC3, GPC2, FGFR1, FGFR2, FGFR3, PDGFR-a, PDGFR-b, IGF-1R, AXL, LAG-3, TIM-3, TIGIT, VISTA, B7-H4, CD19, CD20, CD22, CD24, CD38, CD47, CD33, CD70, CD123, CD133, ITGB1, ITGB5, ITGB6, ITGB8, CAIX, NRP1, NRP2, GD2, GD3, MSLN, EphA2, EphB4, c-KIT, ASCT2, IL1RAP, Ly6E, PTK7, uPARAP, CRLF2, SEZ6, TM4SF1, STEAP1, STEAP2, CADM1, CDCP1, GCC orLIV-1.0 The method according to claim 17, wherein the disease is a cancer selected from the group consisting of sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head-and-neck cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervix cancer, endometrial cancer, ovarian cancer, testicular cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer, prostate cancer, thyroid cancer, and oral cancer.

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