N-azidoacetylglucosamine (glcnaz)-derived sugar oxazolines as enzyme substrates for site-specific antibody bioconjugation

Abstract

The present disclosure relates to site-specific modification of antibodies at their Fc glycan site and subsequent conjugation with drugs and other entities to produce structurally well-defined antibody conjugates. The method is based on the discovery that N-azidoacetylglucosamine GlcNAz-derived sugar oxazolines can serve as substrates of endoglycosidases, such as Endo S2 and Endo S for enzymatic Fc glycan remodeling to site-specifically introduce an azide-tag in an antibody in a single step, followed by a click reaction to form site-specific antibody-drug conjugates and antibody-ligand conjugates

Description

N-AZmOACETYLGLUCOSAMINE (GIcNAz)-DERIVED SUGAR OXAZOLINES ASENZYME SUBSTRATES FOR SITE-SPECIFIC ANTIBODY BIOCONJUGATIONThis application claims the benefit of Serial No. 63 / 520,091 filed on August 17, 2023, the entire contents of which are incorporated herein.GOVERNMENT SUPPORT STATEMENTThis invention was made with government support under R01 AI155716 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0001] The present disclosure relates to site-specific modification of antibodies at their Fc glycan site and subsequent conjugation with drugs and other entities to produce structurally well-defined antibody conjugates. The method is based on the discovery that certain endoglycosidases have the ability to both deglycosylate an antibody and to recognize N-azidoacetylglucosamine (GlcNAz)-derived sugar oxazolines substrates for transglycosylation on antibodies without hydrolysis of the resulting products. The azide-tagged antibodies can be used for producing structurally well-defined antibody-drug conjugates with well-defined antibody-drug ratios and can also be applied to produce antibody conjugates with other molecular entities including fluorescent labels and various ligands.BACKGROUND

[0002] Therapeutic antibodies are an important class of biologies that have been used for the treatment of many challenging diseases, such as cancer, autoimmune disorders, and inflammatory diseases. Over a dozen new ADCs were approved by the US Food and Drug Administration (FDA) within the past 10 years for the treatment of cancers and many more are at various stages of preclinical and clinical development.2-3Featured by their high specificity and affinity, the monoclonal antibodies (mAbs) have fewer off-target side effects as compared to small-molecule pharmaceuticals, thus in addition to being used as therapeutic agents, mAbs also provide a promising platform for targeted delivery of small molecules. Antibody with drugconjugates (ADCs) that combine the specificity of antibodies and the high potency of drugs, hold great promise for targeted cell killing. Accordingly, methods arc desired for easily and efficiently generating said antibody with drug conjugates.SUMMARY

[0003] The present disclosure provides a new class of GlcNAz-derived sugar oxazolines that were found to serve as substrates for endoglycosidases, such as Endo S2 and Endo S, for antibody Fc glycan remodeling. The disclosure provides a protocol for one-pot site-specific introduction of azide groups into an antibody. The azide-tagged antibodies can be used for producing structurally well-defined antibody-drug conjugates through, for example, the use of click chemistry reactions, and can be also applied to produce antibody conjugates with other entities including fluorescent labels and various ligands.

[0004] This disclosure is based on results from testing of a series of oxaozline modified disaccharide substrates. It was found that enzyme Endo S2 was sensitive to the direct modification of the methyl group of the sugar oxazoline, as most of the modifications resulted in significant reduction of substrate activities. However, it was found that for the Endo S2 enzyme specific azide-modified sugar oxazolines, e.g., GlcNAz-derived sugar oxazolines, were found to serve as excellent substrates for enzymatic transfer for use in Fc glycan remodeling. In comparison with previously reported azide sugars, the N-azidoacetylglucosamine (GlcNAz)- derived sugar oxazolines are straightforward to synthesize, and the resulting azide-tagged antibodies are readily available for site-specific conjugation with drugs and other molecular moieties through click reactions. Thus, this disclosure provides a new and simple, "one-pot" glycan remodeling method that combines the antibody deglycosylation and transglycosylation reactions in one reactor without the need to separate the deglycosylation intermediate and the enzyme. The present disclosure provides for the synthesis of IgG antibodies and Fc fragments thereof, wherein a desired sugar chain is added to a core fucosylated or nonfucosylated GlcNAc- acceptor, including a fucosylated or nonfucosylated GlcNAc-IgG acceptor. As such, the present disclosure allows for the synthesis and remodeling of therapeutic antibodies and Fc fragments thereof to provide for certain biological activities, such as, prolonged half-life time in vivo, less immunogenicity, enhanced in vivo activity, increased targeting ability, and / or ability to deliver a therapeutic agent.

[0005] In one aspect a one-pot chemoenzymatic remodeling method is provided for both dcglycosylation and transglycosylation reactions in a onc-pot manner to provide an azido-tagged antibody comprising the steps of: (a) providing a single reactor, container, column, or pot; (b) introducing a single endoglycosidase, having both deglycosylation and transglycosylation activity; (c) introducing an antibody for deglycosylation by the single endoglycosidase thereby providing a deglycosylated intermediate that contains at least one N-acetylglucos amine (GlcNAc) or core-fucosylated N-acetylglucosamine (Fucal,6GlcNAc) acceptor; (d) providing a GlcNAz-derived sugar oxazoline ; and (e) transglycosylating the GlcNAz-derived sugar oxazoline to the N-acetylglucosamine (GlcNAc) or core-fucosylated N-acetylglucosamine (Fucal,6GlcNAc) acceptor by the single endoglycosidase to provide the azido-tagged antibody.

[0006] In another aspect a one-pot chemoenzymatic remodeling method is provided for both deglycosylation and transglycosylation reactions in a one-pot manner to provide an azido-tagged antibody comprising the steps of: (a) providing a single reactor, container, column, or pot; (b) introducing an endoglycosidase having deglycosylation activity; (c) introducing an antibody for deglycosylation by an endoglycosidase such as Endo S thereby providing a deglycosylated intermediate that contains at least one N-acetylglucosamine (GlcNAc) or core-fucosylated N- acetylglucosamine (Fucal,6GlcNAc) acceptor; (d) providing an GlcNAz-derived sugar oxazoline and a second endoglycosidase having transglycosylation activity; and (e) transglycosylating the GlcNAz-derived sugar oxazolines to the N-acetylglucosamine (GlcNAc) or core-fucosylated N-acetylglucosamine (Fucal,6GlcNAc) acceptor by a second endoglycosidase such s Endo S2 to provide the azido-tagged antibody.

[0007] In the practice of the provided remodeling methods, disaccharide oxazolines carrying an azide on the oxazoline side chain, i.e., GlcNAz-derived sugar oxazolines, may be used as substrates in the transglycosylation step. Such disaccharide ozazolines include, for example, N- azidoacetylglucosamine-derived disaccharide oxazolines (See FIGS. 11-14).

[0008] In an embodiment, the antibody molecules for modification (azido-tagging) can be any antibody characterized by the presence of one or more glycans that can act as substrates for deglycosylation activity through an endoglycosidase mediated reaction. Such antibodies, include for example, those antibodies having high specificity and affinity to antigens of interest. Such antigens include those expressed on a target cell of interest. In a non-limiting embodiment, the antigens are expressed on target cancer cells. In another embodiment, the antibodies bind toantigens, such as extracellular and membrane-associated proteins for use in targeted lysosomal degradation. Antibodies may also bind to targeted pathogen antigens. Such antigens include, for example, those associated with viral, bacterial, fungal and parasitic infections of a subject.

[0009] In an embodiment, the resulting azido-tagged antibodies are further modified by conjugation to ligands to provide an antibody-conjugate. In an embodiment, the azido-tagged antibody may be modified with conjugation to other ligands by a click chemistry reaction. In one aspect, the antibody-conjugate comprises a ligand (or tag, used interchangeably herein) selected from drugs, toxins, labels, proteins, small molecules, thio, biotin, and fluorescent label. Such tags include, for example, a therapeutic agent or drug such as for treating cancer, viral infections, substances that activates receptors on the cell plasma membrane, agents that affects intracellular chemistry, agents that affects cellular physics, genes, gene analogs, RNA, RNA analogs, DNA, DNA analogs, amino acid sequences of surface receptors such as CCR5 or CD4, antigenic structure having affinity for a specific antibody; amino acid sequences of receptor ligands such as gpl20, gp41 or gpl60, receptor antagonists, receptor blockers, enzymes, enzyme substrates, enzyme inhibitors, enzyme modulators, therapeutic proteins, protein analogs, metabolites, metabolite analogs, oligonucleotides, oligonucleotide analogs, antigens, antigen analogs, antibodies or fragments thereof, antibody analogs, an antibody different from the modified antibody which is reactive to another receptor bacteria, viruses, inorganic ions, metal ions, metal clusters, polymers, fluorescent compounds and any combinations thereof. In an embodiment, such tags may also be added to the azido modified antibody using click chemistry reactions which are well known to those skilled in the art.

[0010] As such, the present disclosure further provides a delivery device for delivering a drug or therapeutic agent having biological activity to treat a condition, the delivery device comprising: a remodeled IgG or IgG-Fc fragment having a predetermined sugar chain, and a therapeutic agent or drug attached to the terminal sugar residue. Antibodies related to cancer or other diseases may be remodeled for individual fit to certain receptors thereby increasing biological activity.

[0011] In another embodiment, the provided delivery device is designed to deliver a detectable label or marker to a targeted antigen for diagnostic and prognostic uses. Said a delivery device comprises a remodeled antibody or fragment thereof, e.g., an IgG or IgG-Fc fragment, having a predetermined sugar chain, and the detectable label or marker attached to the remodeled antibody. A "detectable label" or a "marker" refers to a composition that is detectable byspectroscopic, photochemical, biochemical, immunochemical, radioactive or chemical means. For example, a useful label includes32P,35S, fluorescent dyes, clcctron-dcnsc reagents, enzymes (e.g., enzymes that are generally used in ELISA), biotin- streptavidin, digoxigenin, hapten, proteins or nucleic acid molecules with a sequence complementary to a target. The detectable label often generates a measurable signal, e.g., a radioactive signal, a color signal or a fluorescent signal, which is usable to quantify an amount of the detectable moiety that binds to the target antigen. Quantification of the signal may be accomplished by, for example, scintillation counting, density gauge, flow cell analysis, ELISA, or direct analysis by mass spectroscopy. Those skilled in the art are familiar with techniques and detection means for a label compound of interest. These techniques and methods are conventional and well known in the art.

[0012] In yet another aspect, the present disclosure provides a substantially homogeneous preparation of core fucosylate or nonfucosylated antibody(ies) or Fc fragment thereof having a predetermined azido-oligosaccharide moiety, wherein the substantially homogeneous preparation is produced by any of the aforementioned single pot methods disclosed herein. Also provided are compositions comprising such homogeneous preparations.

[0013] The present disclosure provides efficient methods for producing homogeneous and sitespecific antibody-drug conjugates with well-defined antibody-drug ratios (DARs). The disclosed one-pot modeling method provides an efficient method for producing antibody-drug conjugates (ADCs) using any given antibody, linker, and payload.

[0014] In an embodiment, a method is provided of treating a subject wherein said treatment typically comprises administering to the subject, of an effective amount of a remodeled antibody generated using the one-pot chemoenzymatic remodeling methods disclosed herein.

[0015] In further embodiments, pharmaceutical compositions comprising a remodeled antibody generated using the one-pot chemoenzymatic remodeling methods disclosed herein, and a pharmaceutical acceptable carrier are provided. The antibodies exhibit properties for use as therapeutic agents, e.g., in the treatment of cancer for example.

[0016] In yet another embodiment, kits comprising a remodeled antibody are provided. Such kits contain, in addition to the remodeled antibody, materials useful for the treatment of diseases, or for diagnosis of a disease as described herein. The kits may comprise one or more of the following components: a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and / or diagnosing 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 by a hypodermic injection needle).

[0017] Other aspects, features and embodiments of the present disclsoure will be more fully apparent from the ensuing disclosure and appended claims.BRIEF DESCRIPTION OF FIGURES

[0018] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of nonlimiting example, with reference to the accompanying drawings. With specific reference to the drawings, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the disclosure.

[0019] FIG 1A-B. Sugar oxazolines as donor substrate in Endo S / S2 (WT / mutants) catalyzed IgG Fc glycan remodeling and functionalization. FIG 1A. Previous work in which all the sugar oxazolines carrying modifications at the non-reducing sugar moieties; FIG IB. this work, in which the modifications are carried out directly on the 5-membered heterocyclic ring.

[0020] FIG. 2. Synthesis of LacNAc oxazoline derivatives with modifications at the N-acetyl group.

[0021] FIG. 3. One-pot IgG glycoengineering with oxazoline modified LacNAc derivatives. Transglycosylation conditions: Herceptin, 500 pg; oxazoline, 20 equiv / site, Endo S2 WT, 1 / 200; 100 mM phosphate buffer, pH 7.0, 25 pL; 25 °C, 3h; Herceptin, 500 pg; oxazoline, 20 equiv / site, Endo S2 WT, 1 / 50; 100 mM phosphate buffer, pH 7.0, 25 pL; 25 °C, 3 h.

[0022] FIG 4. Synthesis of GlcBl,4-GlcNAz oxazoline 11.

[0023] FIG. 5A-B. One-pot IgG glycoengineering with GlcBl,4-GlcNAz oxazoline (11). FIG. 5A the one-enzyme two-step (enzymatic deglycosylation and simultaneous disaccharide transfer) Fc-glycan remodeling scheme; FIG. 5B. the comparison of enzymatic transfer efficiency with LacNAz-ox (3b) and GlcBl,4-GlcNAz-ox (11). Reaction conditions: Trasturumab, 500 pg (30 mg / mL); oxazoline, 20 equiv / site; Endo S2 WT, 1 / 200 (w / w); 100 mM phosphate buffer, pH 7.0, 30 °C.

[0024] FIG. 6. Synthesis of Homogeneous ADCs through Click Reaction.

[0025] FIG. 7. LC-ESI-MS analysis of the antibody-drug conjugates (13, 14, 15 and 16).Deconvoluted mass of Fc domains released by IdeS treatment. Asterisked peaks indicate the ion fragments derived from the Fc domain.

[0026] FIG. 8A-B. Cytotoxicity assays of the antibody-drug conjugates with the SK-BR-3 (HER2 overexpression) and the T47D (HER2 low expression) cancer cell lines. All assays were performed in triplicate.

[0027] FIG. 9A-C. One-pot IgG glycoengineering with ManBl,4-GlcNAz oxazoline (20). FIG.9A. Synthesis of ManBl,4-GlcNAz oxazoline 20. FIG. 9B. The one-enzyme two-step (enzymatic deglycosylation and simultaneous disaccharide transfer) Fc-glycan remodeling scheme; FIG.9C. the comparison of enzymatic transfer efficiency of ManBl,4-GlcNAz-ox (20) with LacNAz- ox (3b) and GlcBl,4-GlcNAz-ox (11). Reaction conditions: Trasturumab, 500 pg (30 mg / mL); oxazoline, 20 equiv / site; Endo S2 WT, 1 / 200 (w / w); 100 mM phosphate buffer, pH 7.0, 30 °C.

[0028] FIG. 10A-C. Synthesis and Cytotoxicity assays of Homogeneous ADC (22) fromManBl,4-GlcNAz remodeled antibody (21). FIG. 10A. Synthesis of Homogeneous ADC through Click Reaction. FIG. 10B. LC-ESI-MS analysis of the antibody-drug conjugate (22).Deconvoluted mass of Fc domains released by IdeS treatment. Asterisked peaks indicate the ion fragments derived from the Fc domain. FIG. 10C. Cytotoxicity assays of the antibody-drug conjugate (22) with the SK-BR-3 (HER2 overexpression) and the T47D (HER2 low expression) cancer cell lines. All assays were performed in triplicate.

[0029] FIG. 11A-B. 11A. Group I. GlcBl,4GlcNAc based azido-functionalized oxazolines. FIG. 11B. Examples.

[0030] FIG. 12A-B 12A Group II. GalBl,4GlcNAc based azido-functionalized oxazolines. FIG. 12B. Examples.

[0031] FIG.13A-B. 13A. Group III. ManBl,4GlcNAc based azido-functionalized oxazolines.FIG. 13B. Examples.

[0032] FIG. 14A-B. 14A. Group IV. Oligosaccharide Bl,4GlcNAc based azido-functionalized oxazolines. FIG. 14B. Examples.DETAILED DESCRIPTION

[0033] Disclosed herein are modified antibodies prepared via glycan engineering. The disclosurerelates to the Fc region of an antibody molecule, wherein the Fc region is specifically glycosylated with GlcNAz-dcrivcd sugar oxazolincs carrying an azide on the oxazilinc side chain that increase the efficacy and stability of the Fc region, and the antibody or antibody fragment comprising the Fc region. In some embodiments the specifically glycosylated Fc fragment comprises a monoclonal antibody, preferably a human or humanized monoclonal antibody. Methods for generating such Fc glycosylated antibodies or antibody fragments by glycan engineering are disclosed herein

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by those skill in the art to which the present disclsoure belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed methods and compositions. All publications and patents specifically mentioned herein are incorporated by reference for all purposes including describing and disclosing the chemicals, cell lines, vectors, animals, instruments, statistical analysis and methodologies which are reported in the publications which might be used in connection with the subject matter of the present disclosure.

[0035] As used herein, the term "glycan" refers to a polysaccharide, or oligosaccharide. Glycan is also used herein to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide or a proteoglycan.

[0036] The term "antibody" (Ab) as used herein includes monoclonal antibodies, polyclonal antibodies, multi- specific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. A "human antibody" as used herein refers to an antibody naturally existing in humans, a functional fragment thereof, or a humanized antibody, i.e., a genetically engineered antibody a portion of which (e.g., a frame region or the Fc region) derives from a naturally occurring human antibody.

[0037] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0038] A "mammal" for purposes of treatment, refers to any mammal, including humans,domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.

[0039] "Treating" or "treatment" or "alleviation" refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully "treated" if, after receiving a therapeutic amount of an antibody according to the methods of the present disclosure, relief to some extent from one or more of the symptoms associated with the specific pathologic condition or disorder; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

[0040] The term "therapeutically effective amount" refers to an amount of an antibody or a drug effective to "treat" a disease or disorder in a subject or mammal. See preceding definition of "treating." "Chronic" administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. "Intermittent" administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature. Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0041] " Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™’ polyethylene glycol (PEG), and PLURONICS™

[0042] 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. The term is intended to include radioactive isotopes, chemotherapeutic agents, e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below.

[0043] A "growth inhibitory agent" when used herein refers to a compound or composition which inhibits growth of a cell, either in vitro or in vivo. Examples of growth inhibitory agents include agents that block cell cycle progression, such as agents that induce G1 arrest and biphase arrest. Such inhibitory agents include, for example, the vinca alkaloids (vincristine, vinorelbine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, bleomycin, tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.

[0044] As used herein “N-azidoacetylglucosamine (GlcNAz)-derived sugar oxazoline” is used interchangeably with “GlcNAz-derived sugar oxazoline.”

[0045] As used herein Endo S includes the endoglycosidase from Streptococcus pyogenes and Endo S2 includes the endoglycosidase from serotype M49 group A Streptococcus.

[0046] The present disclosure provides for the synthesis of IgG antibodies, or Fc fragments thereof, wherein a desired sugar chain carrying an azide, i.e. a GlcNAz-derived sugar moiety, is added to a core fucosylated or nonfucosylated GlcNAc- acceptor, including fucosylated or nonfucosylated GlcNAc-IgG acceptor. As such, the present disclosure allows for the synthesis and remodeling of therapeutic antibodies, or Fc fragments thereof, to provide for certain biological activities, such as, prolonged half-life time in vivo, less immunogenicity, enhanced in vivo activity, increased targeting ability, and / or ability to deliver a therapeutic agent.

[0047] In one aspect a one-pot chemoenzymatic remodeling method is provided for both deglycosylation and transglycosylation reactions in a one-pot manner to provide an azide tagged antibody (FIG.1B). In one aspect a one-pot chemoenzymatic remodeling method is provided for both deglycosylation and transglycosylation reactions in a one-pot manner to provide an azido-tagged antibody comprising the steps of: (a) providing a single reactor, container, column, or pot; (b) introducing a single cndoglycosidasc, having both dcglycosylation and transglycosylation activity; (c) introducing an antibody for deglycosylation by the single endoglycosidase thereby providing a deglycosylated intermediate that contains at least one N- acetylglucosamine (GlcNAc) or core-fucosylated N-acetylglucosamine (Fucal,6GlcNAc) acceptor; (d) providing a GlcNAz-derived sugar oxazoline; and (e) transglycosylating the GlcNAz-derived sugar oxazoline to the N-acetylglucosamine (GlcNAc) or core-fucosylated N- acetylglucosamine (Fucal,6GlcNAc) acceptor by the single endoglycosidase to provide the azido-tagged antibody.

[0048] In another aspect a one-pot chemoenzymatic remodeling method is provided for both deglycosylation and transglycosylation reactions in a one-pot manner to provide an azido-tagged antibody comprising the steps of: (a) providing a single reactor, container, column, or pot; (b) introducing an endoglycosidase, having deglycosylation activity; (c) introducing an antibody for deglycosylation by the endoglycosidase thereby providing a deglycosylated intermediate that contains at least one N-acetylglucosamine (GlcNAc) or core-fucosylated N-acetylglucosamine (Fucal,6GlcNAc) acceptor; (d) providing a GlcNAz-derived sugar oxazoline and a second endoglycosidase, having transglycosylation activity; and (e) transglycosylating the GlcNAz- derived sugar oxazoline to the N-acetylglucosamine (GlcNAc) or core-fucosylated N- acetylglucosamine (Fucal,6GlcNAc) acceptor by the second endoglycosidase to provide the azido-tagged antibody.

[0049] In one aspect, for the disclosed one-pot remodeling methods an endoglycosidase that has both deglycosylation and transglycosylation ability without hydrolytic activity toward the resulting transglycosylation product is utilized. In a non-limiting embodiment, the endoglycosidases may be selected from the group consisting of wild type Endo S, wild type Endo S2 and Endo F3. Such enzymes are well known in the ail and commercially available.

[0050] Said endoglycosidases include, but are not limited to, endoglycosidases from Streptococcus pyogenes, including Endo S2, wherein the endoglycosidases enable the transfer of an oligosaccharide (in the form of an GlcNAz-derived sugar oxazoline) en bloc to a fucosylated or nonfucosylated GlcNAc-IgG (or an Fc fragment thereof) to form a new glycoform of IgG (or an Fc fragment thereof).

[0051] Accordingly, the present disclosure provides for use in the provided one-pot method of aGlcNAz-derived sugar oxazoline, as well as its selectively modified derivatives, to provide an azide tagged antibody. Such GlcNAz-dcrivcd sugar oxazolincs carrying an azide on the oxazilinc side chain are utilized as donor substrates for an efficient chemoenzymatic synthesis of homogeneous core fucosylated or nonfucosylated IgG antibodies and IgG-Fc fragments.

[0052] In the practice of the provided remodeling methods, a number of GlcNAz-derived sugar oxazolines may be used as substrates in the transglycosylation step (See, FIGS. 9-14). In an embodiment, the sugar oxazolines carrying an azide on the oxaziline side chain, for use in the above methods, is selected from the group consisting of the following oxazoline structures.

[0053] In one embodiment, the GlcNAz-derived sugar oxazoline has a glucose core as the second sugar moiety (Group I), and has the following general structure:In another embodiment, the GlcNAz-derived sugar oxazoline has a galactose core as the second sugar moiety (Group II), and has the following general structure:In another embodiment, the GlcNAz-derived sugar oxazoline has a mannose core as the second sugar moiety (Group III), and has the following general structure:In yet another embodiment, the GlcNAz-derived sugar oxazoline carries an oligosaccharide structure at the non-reducing terminus (Group IV), and has the following general structure:

[0054] In another embodiment, the azide tagged antibody produced using the disclosed methods may serve as a substrate for a click chemistry step for conjugation of a drug or ligand of interest to the antibody. Accordingly, in an embodiment, the azido-tagged antibody may be modified with conjugation to ligands or tags of interest by a click chemistry reaction. Such click chemistry reactions are well known to those skilled in the art and provide a controlled reaction medium for generation of antibodies with desired ligand density and spatial arrangement. For example, a ring- strained cyclooctyne moiety-containing drug or ligand to the azide-tagged antibody via ring- strained cycloaddition reaction to provide homogeneous antibody-drug conjugates or antibodyligand conjugates.

[0055] Accordingly, provided herein are antibody-conjugates wherein the the antibody carries selected molecular entities site-specifically linked at the Fc glycan site. Said antibody-conjugates comprise appropriate molecular entities selected from the group consisting of drugs, toxins, labels, proteins, small molecules, thio, biotin and fluorescent label. Said antibody-drug conjugate includes, for example, a conjugate wherein the molecular entity is a drug and the drug to antibody ratio is 2 to 12. The additional moiety may include, for example, a therapeutic agent or drug such as for treating cancer, viral infections, substances that activates receptors on the cell plasma membrane, agents that affects intracellular chemistry, agents that affects cellular physics, genes, gene analogs, RNA, RNA analogs, DNA, DNA analogs, amino acid sequences of surface receptors such as CCR5 or CD4, antigenic structure having affinity for a specific antibody; amino acid sequences of receptor ligands such as gpl20, gp41 or gpl60, receptor antagonists, receptor blockers, enzymes, enzyme substrates, enzyme inhibitors, enzyme modulators,therapeutic proteins, protein analogs, metabolites, metabolite analogs, oligonucleotides, oligonucleotide analogs, antigens, antigen analogs, antibodies or fragments thereof, antibody analogs, an antibody different from the modified antibody which is reactive to another receptor bacteria, viruses, inorganic ions, metal ions, metal clusters, polymers, fluorescent compounds and any combinations thereof.

[0056] In an embodiment, the ligand may include one or more high-affinity M6P oligosaccharide ligands. Such remodeled antibody molecules may be used for targeting degradation of extracellular and membrane associated proteins through conjugation of high affinity mannose-6-phosphate (M6P) glycan ligands.

[0057] In an embodiment, the antibody for remodeling can be any antibody, or antibody fragment, characterized by the presence of one or more glycans that can act as substrates for deglycosylation activity through a endoglycosidases mediated reaction. Such antibodies, include for example, those antibodies having high specificity and affinity to antigens expressed on a target cell. In a non-limiting embodiment, the antigens are expressed on target cancer cells.

[0058] In an embodiment, the antibodies for use in the disclosed methods herein can be prepared from a commercially available therapeutic antibody (e.g., ReoPro™ (abeiximab), RITUXAN™ (rituximab), ZENAPAX® (daclizumab), Simulect® (basiliximab), SYNAGIS™ (palivizumab), REMICADE ® (infliximab), Herceptin® (trastuzumab), MYLOTARG® (gemtuzumab ozogamicin), Campath® (alemtuzumab), Zevalin® (ibritumomab tiuxetan), HUMIRA® (adalimumab), XOLAIR® (omalizumab), BEXXAR (tositumomab), RAPTIVA (efalizumab), ERBITUX® (cetuximab), Avastin® (bevacizumab), TYSABRI® (natalizumab), human or humanized antibodies produced via a conventional method, preferably those undergoing clinical trials.

[0059] Other monoclonal antibodies suitable for use in the methods provided herein include, but are not limited to cetuximab, rituximab, muromonab-CD3, abeiximab, daclizumab, basiliximab, palivizumab, infliximab, trastuzumab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab, omalizumab, tositumomab, tositumomab, efalizumab, bevacizumab, panitumumab, pertuzumab, natalizumab, etanercept, IGN101®, volociximab, Anti-CD80 Mab, Anti-CD23 Mab, CAT-3888.RTM., CDP-791®, eraptuzumab, MDX-010®, MDX-060®., MDX-07® matuzumab, CP-675.degree., 206®., CAL® SGN-30, zanolimumab, Adecatumumab®. adecatumumab, oregovomab, nimotuzumab, ABT-874® (briakinumab),denosumab, AM 108®, AMG 714®., fontolizumab, dcaclizumab, golimumab, CNTO 1275® (ustckinumab), ocrclizumab, HumaxCD20®. (ofatumumab), bclimumab, cpratuzumab, MLN1202®, visilizumab, tocilizumab, ocrerlizumab, certolizumab pegol, eculizumab, pexelizumab, abciximab, ranibizimumab, mepolizumab, TNX-355® and MYO-029® (stamulumab).

[0060] The present disclosure also envisions modifying monoclonal antibodies associated with pathogenic infections such as, for example, bacterial, fungal, parasitic or viral infections. In a specific embodiment, the antibodies may be HIV antibodies including, but not limited to 17b, 48d, A32, Cl l, 2G12, F240, IgGlbl2, 19e, X5, TNX-355 and F91, all of which are commercially available.

[0061] As such, the present disclosure provides a delivery device for delivering a drug or therapeutic agent having biological activity to treat a disease or disorder, the delivery device comprising: a remodeled IgG or IgG-Fc fragment having a predetermined sugar chain or sialoglycan and a therapeutic agent or drug attached to the terminal sugar residue or sialic acid. Further antibodies related to cancer or other diseases may also be remodeled for individual fit to certain receptors thereby increasing biological activity.

[0062] In yet another aspect, the present disclosure provides a substantially homogeneous preparation of core fucosylate or nonfucosylated antibody or Fc fragment thereof having a predetermined azido-oligosaccharide moiety, wherein the substantially homogeneous preparation is produced by any of the aforementioned methods. Also provided are compositions comprising such homogeneous preparations.

[0063] In further embodiments, pharmaceutical compositions comprising a remodeled antibody generated using the one-pot chemoenzymatic remodeling methods disclosed herein and a pharmaceutical acceptable carrier are provided. The antibodies exhibit properties for use as therapeutic agents, e.g. in the treatment of cancer in a subject for example. Such pharmaceutical compositions comprise a therapeutically effective amount of one or more of a remodeled antibody dissolved or dispersed in a pharmaceutically acceptable carrier. The preparation of a pharmaceutical composition that contains at one or more a remodeled antibody and optionally an additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. For human administration, it will be understood that preparations should meetsterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards or corresponding authorities in other countries. Preferred compositions arc lyophilized formulations or aqueous solutions.

[0064] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g. antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.

[0065] The composition may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for such routes of administration as injection. A remodeled antibody (and any additional therapeutic agent) can be administered by any method or any combination of methods as would be known to one of skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference). Parenteral administration, in particular intravenous injection, is most commonly used for administering protein or polypeptide molecules such as a remodeled antibody.

[0066] Any of the remodeled antibodies derived using the one-pot methods disclosed herein, may be used in therapeutic methods as described herein. "Treating" or "treatment" or "alleviation" refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully "treated" if, after receiving a therapeutic amount of an antibody according to the methods of the present disclosure, relief to some extent from one or more of the symptoms associated with the specific pathologic condition or disorder; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routineprocedures familiar to a physician. The term "therapeutically effective amount" refers to an amount of an antibody or a drug effective to "treat" a disease or disorder in a subject or mammal.

[0067] For use in the therapeutic methods described herein, the remodeled antibody is formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular subject being treated, the clinical condition of the subject, the cause of the disease or condition, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners or those of skill in the art.

[0068] For the treatments, the appropriate dosage of the remodeled antibodies (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the body weight of the patient, the severity and course of the disease, whether the remodeled antibody is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the remodeled antibody, and the discretion of the attending physician. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0069] In another embodiment, the provided delivery device is designed to deliver a detectable label or marker to a targeted antigen for diagnostic and prognostic uses. Said a delivery device comprises a remodeled IgG or IgG-Fc fragment having a predetermined sugar chain and the detectable label or marker attached to the remodeled antibody. A "detectable label" or a "marker" refers to a composition that is detectable by spectroscopic, photochemical, biochemical, immunochemical, radioactive or chemical means. For example, a useful label includes32P,35S, fluorescent dyes, electron-dense reagents, enzymes (e.g., enzymes that are generally used in ELISA), biotin-streptavidin, digoxigenin, hapten, proteins or nucleic acid molecules with a sequence complementary to a target. The detectable label often generates a measurable signal, e.g., a radioactive signal, a color signal or a fluorescent signal, which is usable to quantify an amount of the detectable moiety that binds to the target antigen.Quantification of the signal may be accomplished by, for example, scintillation counting, density gauge, flow cell analysis, ELISA, or direct analysis by mass spectroscopy. Those skilled in theart are familiar with techniques and detection means for a label compound of interest. These techniques and methods arc conventional and well known in the art.

[0070] In another aspect of the embodiment, an article of manufacture (e.g., a kit) containing materials useful for the treatment or diagnosis of diseases or disorders as 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, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and / or diagnosing 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 by a hypodermic injection needle). The label or package insert indicates that the composition is used for treating the condition of choice. The article of manufacture may comprise a container with a composition contained therein, wherein the composition comprises a remodeled antibody

[0071] Kits in certain embodiments may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the kit may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0072] The subject matter of the present disclosure includes the information and references presented herein. Such references are hereby incorporated by reference each in their respective entirety, except for any statement contradictory to the express disclosure herein, except for subject matter disclaimers or disavowals, and except to the extent that the incorporated material is inconsistent with the express disclosure herein, in which case the express language of this disclosure controls. Incorporation or citation of any such reference shall not be considered an admission by the applicant that the incorporated material is prior ait to the present disclosure or considered material to patentability of the present disclosure.

[0073] The following examples are included to demonstrate preferred embodiments of the present disclosure. It should be appreciated by those of skill in the ail that the techniques disclosed in the examples which follow represent techniques discovered by the inventor tofunction well in the practice of embodiments of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.EXAMPLESMaterial and Methods

[0074] General Methods. Chemicals, reagents, and solvents were purchased from Sigma-Aldrich and / or TCI, and used as received unless otherwise specified. Monoclonal antibody Herceptin was purchased from RelDrug Inc. (Hillsborough, NJ). Payload MMAE was purchased from MedChemExpress (Monmouth Junction, NJ). Thin-layer chromatography (TLC) was performed on silica gel 60-F254 on glass plates (Merck) and stained with p-anisaldehyde. Flash chromatography was performed on Isolera One system with ZIP KP- Sil columns (Biotage). NMR spectra were recorded on a 600 MHz spectrometer (Bruker, Tokyo, Japan) with CDCb or D2O as the solvent. MALDI-TOF analysis was performed using a Bruker UltrafleXtreme (UTX) mass spectrometer in positive reflectron mode with DHB (ACN / H2O 1 / 1) as the matrix. Analytical reverse-phase HPLC was performed on a Waters Alliance® e2695 HPLC system equipped with a dual absorbance 2489 UV / Vis detector. Separations were performed using a C18 column (YMCTriart C18, 4.6 x 250 mm, 5 pm). ESI-MS spectra were obtained using a Waters SQ Detector 2 single quadrupole mass spectrometer. Preparative RP-HPLC was performed on a Waters 600 HPLC system equipped with a dual absorbance UV detector using a C18 column (Waters-Symmetry Prep C18, 19 x 300 mm, 7 pm) at a flow-rate of 10 mL / min, or a C18 column (Waters XBridge, Prep Shield, 10 x 250 mm, 5 pm) at a flow-rate of 4 mL / min. LC-ESI-MS analysis was performed on an Ultimate 3000 HPLC system coupled to an Exactive Plus Orbitrap mass spectrometer (Thermo Fischer Scientific) with a C4 (whole antibody, gradient, 5-95% aq MeCN containing 0.1% FA for 6 min, 0.4 mL / min) or C8 (IdeS digestion, gradient, 25-35% aq MeCN containing 0.1% FA for 6 min, 0.4 mL / min) column. Deconvolution data were transformed with MagTran software.

[0075] Synthesis of LacNAz oxazoline (3b). NaHCO3 (IM aq. solution, 0.33 mL, 0.33 mmol) was added to a cold solution (0°C) of lacto s amine HC1 (50 mg, 0.13 mmol) in 1 mL water. The reaction mixture was stirred at 0 °C for 5 min before azidoacetic acid NHS ester (32mg, 0.16 mmol) in 0.5 mL MeCN was added. The resulting mixture was further stirred for 2 hours until MALDI analysis showed completion. The reaction mixture was purified by RP- HPLC to give LacNAz (2b, 42 mg, 75%) as a white powder after lyophilization. To a solution of LacNAz (18 mg, 42 pmol) in water (1.8 mL) was added triethylamine (59 pL, 0.42 mmol) and 2-chloro-l,3- dimethylimidazolinium chloride (DMC) (36 mg, 0.21 mmol) at 0 °C. The reaction mixture was stirred on an ice bath for 2 h. The glycan oxazoline product was purified by gel filtration on a Bio-Gel P-2 column eluting with 0.1% triethylamine. The carbohydrate containing fractions were pooled and lyophilized to give LacNAz oxazoline (3b, 14.5 mg, 84%) as a white powder. 1H NMR (600 MHz, D2O) 8 6.17 (d, J= 7.3 Hz, 1H, H-l ), 4.44 (dd, J= 3.1, 1.9 Hz, 1H, H-3), 4.39 (d, J= 7.8 Hz, 1H, H-l'), 4.28 -4.24 (m, 1H, H-2), 4.17 (ddd, J= 42.0, 16.5, 1.8 Hz, 2H, N3-CH2), 3.87 (d, J= 3.0 Hz, 1H, H-4'), 3.83 -3.62 (m, 6H, H-4, H-5', H-6, H-6'), 3.59 (dd, J= 10.0, 3.4 Hz, 1H, H-3'), 3.51 - 3.42 (m, 2H, H-2', H-5).LC NMR (150 MHz, D2O) 8 165.1 (N=C), 104.2(C-l'), 100.4 (C-l), 77.5 (C-4), 74.7 (C-5'), 72.0 (C-3'),70.8 (C-5), 70.3 (C-2'), 68.5 (C-3), 68.1 (C-4'), 64.4 (C-2), 61.1, 60.6 (C-6, 6'), 45.6 (N3-CH2).

[0076] Synthesis of Glc|31,4-GlcNAz oxazoline (11). A mixture of acceptor 5 (1 .0 g, 2.01 mmol), donor 6 (1.24 g, 2.52 mmol), and 4 A molecular sieves (2 g) in anhydrous CH2CI2 (30 mL) was stirred under an N2 atmosphere for 30 min and then cooled to -10°C. TMSOTf (38 pL, 0.21 mmol) was added to the mixture. The reaction mixture was stirred at -10 °C for 30 min, then warmed up to rt and further stirred for 30 min, at the end of which time TLC (silica, 1:2 EtOAc-hexanes) showed it was complete. The reaction mixture was quenched with Et3N (30 pL) and filtrated. The filtrate was evaporated in vacuo to give a residue, which was purified by silica gel column chromatography (silica, EtOAc-hexanes, 1:5 — 1:2) to give compound 7 (1.44 g, 85%) as a colorless syrup. MeONa (7 mg, 0.13 mmol) was added in one portion to a solution of 7 (1.00 g, 1.24 mmol) in MeOH (15 mL) at room temperature. The reaction mixture was stirred at room temperature overnight. TLC (silica, 3: 1 EtOAc-hexanes) showed the reaction was complete. Then acidic ion-exchange resin (Amberlite IR- 120 (H+), Alfa Aesar) was added to neutralize the reaction mixture. The mixture was filtered. To the filtrate was added Pd / C (15 mg) in one portion. The reaction was then stirred under an atmosphere of hydrogen (1 atm) at room temperature for 4 h. The reaction mixture was filtered through a pad of Celite using MeOH (5 mL) as the eluent and the resulting filtrate was concentrated to a volume of 5 mL. EtiO (50 mL) was added to the solution to precipitate out crude product, which was further dissolved in 5 mL MeOH and precipitateagain by adding EtiO (50 mL). The precipitate was dried to give 9 (0.42 g, 90% over two steps) as a white powder. NaHCQ3 (1 M aq. solution, 0.33 mL, 0.33 mmol) was added to a cold solution (0 °C) of GlcBl,4-glucosamineHCl (9, 50 mg, 0.13 mmol) in 1 mL water. The reaction mixture was stirred at O °C for 5 min before azidoacetic acid NHS ester (32 mg, 0.16 mmol) in 0.5 mL MeCN was added. The resulting mixture was further stirred for 2 hours until MALDI analysis showed completion. The reaction mixture was purified by RP-HPLC to give GlcBl,4-GlcNAz (10, 46 mg, 82%) as a white powder after lyophilization. To a solution of GlcBl,4-GlcNAz (12 mg, 42 u mol ) in water (1.2 mL) was added triethylamine (39 pL, 0.28 mmol) and2-chloro-l,3- dimethylimidazolinium chloride (DMC) (24 mg, 0.14 mmol) at 0 °C. The reaction mixture was stirred on an ice bath for 2 h. The glycan oxazoline product was purified by gel filtration on a Bio-Gel P-2 column eluting with 0.1% triethylamine. The carbohydrate containing fractions were pooled and lyophilized to give GlcBl,4-GlcNAz oxazoline (11,9.9 mg, 86%) as a white powder. 1H NMR (600 MHz, D2O) 86.24 (d, J= 7.3 Hz, 1H, H-l), 4.52 (d,J=7.9 Hz, 1H, H-l'), 4.49 (dd, J= 3.0, 1.9 Hz, 1H, H-3), 4.35 - 4.30 (m, 1H, H-2), 4.23 (ddd, J= 50.9, 16.5, 1.8 Hz, 2H, N3-CH2), 3.95 (dd, J= 12.4, 2.2 Hz, 1H, H-6'), 3.85 (dd, J= 12.4, 2.5 Hz, 1H, H- 6), 3.80 - 3.74 (m, 2H, H-6', H-4), 3.72 (dd, J= 12.4, 6.3 Hz, 1H, H-6), 3.55 - 3.46 (m, 3H, H-3', H- 5, H-5'), 3.45 - 3.39 (m, 1H, H-4'), 3.31 (dd, J= 9.5, 8.0 Hz, 1H, H-2'). EC NMR (150 MHz, D2O) 8 165.0,103.6, 100.4, 77.7, 75.4, 75.1, 72.6, 70.7, 69.0, 68.3, 64.5, 61.2, 60.1, 45.6.

[0077] General procedure for transglycosylation of Trasturumab with sugar oxazolines catalyzed by WT. The transglycosylation of Trasturumab was carried out at a 0.5 mg scale to screen library of sugar oxazoline substrates. Generally, to a solution of wild-type Trasturumab (0.5 mg, 20 mg / mLfinal concentration) and sugar oxazolines (3a-3j, 20 equiv per reaction site) in 100 mM phosphate buffer (pH= Use LC-ESI-MS to monitor the progress of the transglycosylation reaction. The functionalized Trasturumab were purified by protein A affinity chromatography, buffer exchanged and centrifuge concentrated in 100 mM phosphate buffer. The concentration of the final functionalized antibody product was measured using Nanodrop).

[0078] One-step functionalization of WT Trasturumab with LacNAz oxazoline at 10 mg scale (4b). The transglycosylation of Trasturumab with LacNAz oxazoline (3b) was then scaled up to 10 mg. To a solution ofwild-type Trasturumab (10.0 mg, 25 mg / mL final concentration) and LacNAz oxazoline (3b, 1.3 mg, 25 equiv per reaction site) in 100 mM phosphate buffer (pH = 7.0) was added wild-type Endo S2 (50 pg, 0.5% of the antibody, w / w). The mixture wasincubated at 30 °C. LC-ESI-MS monitoring indicated complete glycosylation within 2 h. The functionalized Trasturumab was purified by protein A affinity chromatography to give 4b (8.1 mg as measured using Nanodrop). LC-ESI-MS: ealed for IdeS treated Fc of Trasturumab-GNF- LacNAz (4b), M = 24,540 Da; found (m / z), 24,541 Da (deconvolution data).

[0079] One-step functionalization of wild-type Trasturumab with GlcBl,4-GlcNAz oxazoline (12). To a solution of wild-type Trasturumab (10.0 mg, 25 mg / mL final concentration) and GlcBl,4-GlcNAz oxazoline (11, 1.1 mg, 20 equiv per reaction site) in 100 mM phosphate buffer (pH = 7.0) was added wild- type Endo S2 (50 pg, 0.5% of the antibody, w / w). The mixture was incubated at 30 °C. LC-ESI-MS monitoring indicated the complete glycosylation within I h. The functionalized Trasturumab was purified by protein A affinity chromatography to give 12 (8.5 mg as measured using Nanodrop). LC-ESI- MS: ealed for IdeS treated Fc of Trasturumab-GNF-GlcB4GlcNAz (12), M = 24,540 Da; found (m / z), 24,542 Da (deconvolution data).

[0080] Preparation of antibody-drug conjugates 13 and 14 with DAR2. A solution of azide- tagged antibody 4b or 12 (1 mg, 6.8 nmol) and the DBCO-PEG5-VC-PAB-MMAE (230 pg, 20 eq) in a phosphate buffer (50 mM, pH 7.2) containing 30% dimethyl sulfoxide (DMSO) (final volume, 0.5 mL) was incubated at room temperature. The reaction mixture was shielded from light and gently vortexed. The reaction was monitored by LC-ESI-MS analysis. After 6 h, the click reaction was complete as indicated by LC-ESI-MS. The mixture was then diluted with phosphate buffer (5 mL, 50 mM, pH 7.2) and filtered using a 0.22 pm syringe filter to remove most of the unreacted hydrophobic payload. The filtrate was purified by protein A chromatography to give ADC 13 (0.84 mg, 82%) and 14 (0.87 mg, 85%), respectively. ESI-MS of 13: ealed for IdeS treated Fc of 13, M = 26,242 Da; found (m / z), 26,243 Da (deconvolution data). ESI-MS of 14: ealed for IdeS treated Fc of 14, M = 26,242 Da; found (m / z), 26,244 Da (deconvolution data).

[0081] Preparation of antibody-drug conjugates 15 and 16 with DAR4. A solution of azide- tagged antibody 4b or 12 (1 mg, 6.8 nmol) and the DBCO-PEG5-bis-VC-PAB-MMAE (430 pg, 20 eq) in a phosphate buffer (50 mM, pH 7.2) containing 50% dimethyl sulfoxide (DMSO) (final volume, 1 mL) was incubated at room temperature. The reaction mixture was shielded from light and gently vortexed. The reaction was monitored by LC-ESI-MS analysis. After 18 h, the click reaction was complete as indicated by LC-ESI-MS. The mixture was then diluted with phosphatebuffer (5 mL, 50 mM, pH 7.2) and filtered using a 0.22 pm syringe filter to remove most of the unrcactcd hydrophobic payload. The filtrate was purified by protein A chromatography to give ADC 15 (0.73 mg, 70%) and 16 (0.79 mg, 76%), respectively. ESI-MS of 15: ealed for IdeS treated Fc of 15, M = 27,755 Da; found (m / z), 27,757 Da (deconvolution data). ESI-MS of 16: ealed for IdeS treated Fc of 16, M = 27,755 Da; found (m / z), 27,757 Da (deconvolution data).

[0082] Synthesis of Man|31,4-GlcNAz oxazoline (20). A mixture of donor 17 (7.20 g, 12.6 mmol), BSP (3.30 g, 15.8 mmol), TTBP (7.84 g, 31.5 mmol), and activated 4 A molecular sieves (10 g) in CH2C12 (100 mL) was stirred for 30 min at -60 °C under an argon atmosphere. Then TI'oO (2.65 mL, 15.8 mmol) was added at this temperature. After 5 min, a solution of acceptor 5 (5.00 g, 10.5 mmol) in CH2C12 (50 mL) was added and the mixture was stirred at -60°C for 3 h. The mixture was filtered through a Celite pad. The filtrate was poured into saturated NaHCCL and extracted with CH2C12. The organic layer was washed with brine, dried over Na2SO4, and concentrated. The residue was subjected to silica gel column chromatography (hexanes / EtOAc, 8: 1-4: 1) to afford 18 (6.49 g, 66%) as a white solid. To a solution of 18 (500 mg) in MeOH (10 mL) containing 1.1 eq HC1 was added Pd / C (100 mg) in one portion. The reaction was then stirred under an atmosphere of hydrogen (1 atm) at room temperature for 4 h. The reaction mixture was filtered through a pad of Celite using MeOH (10 mL) as the eluent and the resulting filtrate was concentrated to a volume of 2 mL. Et20 (50 mL) was added to the solution to precipitate out crude product (189 white powder). NaHCCM 1 M aq. solution, 0.58 mL, 0.58 mmol) was added to a cold solution (0 °C) of crude ManBl,4-glucosamineHCl (80 mg, 0.23 mmol) in 1 mL water. The reaction mixture was stirred at 0 °C for 5 min before azidoacetic acid NHS ester 55.7 mg, 0.28 mmol) in 0.5 mL MeCN was added. The resulting mixture was further stirred for 2 hours until MALDI analysis showed completion. The reaction mixture was purified by RP- HPLC to give ManBl,4-GlcNAz (19, 55.8 mg, 58% over two steps) as white powder after lyophilization. To a solution of 19 (10 mg, 24 pmol) in water (1.0 mL) was added triethylamine (66 u L, 0.48 mmol) and 2- chloro-1,3- dimethylimidazolinium chloride (DMC) (40 mg, 0.24 mmol) at 0 °C. The reaction mixture was stirred on an ice bath for 2 h. The glycan oxazoline product was purified by gel filtration on a Bio-Gel P-2 column eluting with 0.1% triethylamine. The carbohydrate containing fractions were pooled and lyophilized to give ManBl,4-GlcNAz oxazoline (20, 8.1 mg, 85%) as a white powder.1H NMR (600 MHz, D2O) 6 6.16 (d, 7 = 7.3 Hz, 1H), 4.69 (s, 1H), 4.39 (dd, 7= 3.1, 1.9 Hz, 1H), 4.26 - 4.22 (m, 1H), 4.17 (d, 7 1.7 Hz, 1H), 3.97 - 3.94 (m, 1H), 3.85 (dd, 7 = 12.2, 2.2 Hz, 1H), 3.78 - 3.66 (m, 3H), 3.62 (dd, 7 = 12.5, 6.3 Hz, 1H), 3.58 (dd, 7 = 9.6, 3.2 Hz, 1H), 3.52 (t, 7 = 9.6 Hz, 1H), 3.43- 3.39 (m, 1H), 3.37 - 3.30 (m, 1H).13C NMR (150 MHz, D2O) 8 165.2, 100.6, 100.4, 76.7, 75.9, 72.3, 70.8, 69.9, 68.6, 66.3, 64.6, 61.2, 60.6.

[0083] One-step functionalization of wild-type Trasturumab with ManBl,4-GlcNAz oxazoline (21). To a solution of wild-type Trasturumab (10.0 mg, 25 mg / mL final concentration) and ManBl,4-GlcNAz oxazoline (20, 1.1 mg, 20 equiv per reaction site) in 100 mM phosphate buffer (pH=7.0) was added wild- type Endo S2 (50 pg, 0.5% of the antibody, w / w). The mixture was incubated at 30 °C. LC-ESI-MS monitoring indicated complete glycosylation within 10 min. The functionalized Trasturumab was purified by protein A affinity chromatography to give 21 (8.6 mg as measured using Nanodrop). LC-ESI- MS: calcd for IdeS treated Fc of Trasturumab-GNF-ManB4GlcNAz (21), M = 24,540 Da; found (m / z), 24,542 Da (deconvolution data).

[0084] Preparation of antibody-drug conjugates 22 with DAR2. A solution of azide-tagged antibody 21 (1 mg, 6.8 nmol) and the DBCO-PEG5-VC-PAB-MMAE (230 pg, 20 eq) in a phosphate buffer (50 mM, pH 7.2) containing 30% dimethyl sulfoxide (DMSO) (final volume, 0.5 mL) was incubated at room temperature. The reaction mixture was shielded from light and gently vortexed. The reaction was monitored by LC-ESI-MS analysis. After 6 h, the click reaction was complete as indicated by LC-ESI-MS. The mixture was then diluted with phosphate buffer (5 mL, 50 mM, pH 7.2) and filtered using a 0.22 pm syringe filter to remove most of the unreacted hydrophobic payload. The filtrate was purified by protein A chromatography to give ADC 22 (0.77 mg, 75%). ESLMS of 22: calcd for IdeS treated Fc of 22, M = 26,242 Da; found (m / z), 26,245 Da (deconvolution data).

[0085] Cytotoxicity Assay. SKBR3 and T47D (ATCC) cells were planted into 96- well plates (cell number: 10,000 cells per well), and the plates were incubated for 24 hat 37 °C with 5% CO2. The ADC samples were diluted by 3-fold serial dilution with the corresponding medium from 20000 to 0.339 ng / mL (I l concentrations) and then added to the wells in triplicate (150 pL per well) for every single concentration. The cells were cultured at 3 7 °C with 5% CO2 for 3 days before the removal of the medium and addition of Cell Counting Kit-8 (Sigma). The absorbance of formazan released by viable cells was measured at 450 nm usmg a spectrophotometer after incubation at 37 °C with 5% CO2 for 2-3 h, and the background absorption was deducted by 550 nm absorbance. Finally, the cell viability curve and EC50 values were calculated by GraphPad Prism software. For the BT474 cell line, the cells wereplanted into 96-well plates with 4000 cells per well. The plates were incubated for 24 h at 37 °C with 5% CO2. The ADC samples were diluted by 3-fold serial dilution with the corresponding medium from 5000 to 0.085 ng / mL (11 concentrations) and then added to the wells in triplicate (200 pL per well) for every single concentration. The cells were cultured at 37 °C with 5% CO2 for 6 days before the removal of the medium and addition of Cell Counting Kit-8 (Sigma). The absorbance of formazan released by viable cells was measured at 450 nm using a spectrophotometer after incubation at 37 °C with 5% CO2 for 2-3 h, and the background absorption was deducted by 550 nm absorbance. Finally, the cell viability curve and EC50 values were calculated by GraphPad Prism software.RESULTS

[0086] Synthesis of the LacNAz disaccharide derivatives. To test whether endoglycosidases such as Endo S and Endo S2 can tolerate modification at the N-acetyl group for substrate recognition, various derivatives starting from lactosamine were synthesized (FIG. 2). First, starting with commercially available lactosamine, direct N-acylation was performed using free acids or their activated form (either as an acid-NHS ester or acyl chloride) to provide the lactosamine derivatives (2a-2j). Then the oxazoline formation was achieved by treatment of 2a- 2j with 2-chloro-l,3-dimethylimidazolinium chloride (DMC) in the presence of TEA in water to afford the disaccharide oxazoline derivatives (3a-3j) in a single step23(FIG. 2). The products were purified by size exclusion chromatography on a Bio-Gel P-2 column and their identities were confirmed by MS and NMR analysis.

[0087] Test LacNAz substrate with Endo S2 enzyme for antibody Fc glycan remodeling.With the synthetic LacNAc oxazoline derivatives in hand, they were evaluated for their activity for Endo S2 catalyzed Fc glycan remodeling, using trastuzumab (Herceptin), a therapeutic monoclonal antibody used for the treatment of breast cancer, as a model system. First, the transglycosylation efficiency of LacNAc oxazoline (3a) and LacNAz oxazoline (3b) as donor substrates was evaluated. It was found that LacNAz oxazoline (3b) could be efficiently transferred to Herceptin using a catalytic amount of enzyme (1 :200, 0.5%, w / w, enzyme / antibody), with a conversion yield over 95% within 3 h (FIG. 3, 4b), which is comparable with LacNAc oxazoline (>95%, FIG. 3, 4a). Inspired by the promising efficiency of LacNAz oxazoline donor, motivation was provided to screen the LacNAc oxazoline derivatives carrying different substitutes at the acetyl group (3c-3j). Replacement of methyl group withethyl group (3f vs 3a), did not affect the activity significantly, which also observed a conversion yield over 95% within 3 h (Figure 3, 4f). It’s interesting that although Endo S2 could tolerate modification on acetyl group by adding an azido group or one methylene group (3b / 3f vs 3a), compounds carrying an azido group through a longer linker (3i and 3j) were found as poor substrates for Endo S2 (<5% even with pushed conditions). We have observed that N-azido- propionyl lactosamine oxazoline (3h) is prone to elimination reactions during either oxazoline formation reaction or transglycosylation reaction as we detected a side product during the antibody glycan remodeling and characterized the side product was N-acryloyl lactosamine tagged antibody. One interesting observation is that the acryloyl substituted oxazoline (3g) could be transferred on to Herceptin with complete conversion (>95%) when 2% was used within 3 h (FIG. 3, 4g). The fluorinated sugar oxazoline (3c) acted as an excellent substrate. It gave a 75% conversion under the condition (0.5% enzyme, 2 h) and the reaction could be pushed to completion by using 2% of Endo S2 within 2 h. However, it was found that the substitution with a chloro or hydroxyl group (3d and 3e) on the methyl group resulted in significantly reduced activity, giving only 23% and 18% yield, respectively, when 0.5% enzyme was used, and 48% and 37% yield when 2% enzyme was applied within 3 h. These results suggest that Endo S2 was sensitive for introducing a big moiety at the methyl group on the oxazoline ring, but azide and fluorine attachment appeared to be tolerated and the resulting fluorinated and azide-substituted disaccharide oxazolines acted as an excellent substrate for enzymatic transfer.

[0088] Synthesis and test of Glc 1,4GlcNAz substrate with Endo S2 enzyme for antibody Fc glycan remodeling. To extend the scope of N-azidoacetyl modified oxazolines as enzyme substrates, the non-reducing terminus galactose in LacNAz was replaced with a glucose moiety to give the GlcBl,4-GlcNAz oxazoline. A chemical synthesis of this GlcBl,4-GlcNAz oxazoline was shown in FIG 4. Coupling of glycosyl acceptor 5 and glucosyl donor 6 was achieved using TMSOTf as the catalyst to obtain the disaccharide derivative (7). Deacetylation, followed by reduction of 8 afforded glucosyl-Bl,4-glucosamine (9) in excellent yield (90% over two steps). Installation of the azidoacetyl group to 9 followed by oxazoline formation gave the GlcBl,4- GlcNAz oxazoline (11) in excellent yield (FIG. 4).

[0089] Next, the activity of the synthetic GlcBl,4-GlcNAz-ox (11) was tested together with the LacNAz oxazoline (3b) for enzymatic glycan remodeling. Interestingly, it was found that GlcBl,4-GlcNAz-ox was an excellent substrate, which achieved a 98% transglycosylation yield within 30 nun when only 0.5%Endo S2 was used. Under the same condition, the LacNAz-ox (3b) gave about 75% yield (Figure 5). Thus, the presence of a glucose moiety as the second sugar enhanced its substrate activity toward the enzyme. This study identified both LacNAz oxazoline (3b) and GlcBl,4-GlcNAz-ox (11) as a facile donor substrate for antibody Fc glycan remodeling.

[0090] Test of GlcNAz-derived disaccharide oxazolines with Endo S enzyme for antibody Fc glycan remodeling. Synthetic substrates were also tested with Endo S enzyme. It was found that Endo S had much reduced activity on the two disaccharide substrates (3b and 11) In comparison, under the same transglycosylation conditions as described in FIG. 3 , i.e., 30 mg / mL antibody concentration in lx PB (pH 7.0), 20 equiv / site oxazoline and 1 / 200 (w / w) enzyme usage, Endo S gave 35% and 70% yield after 3 h for LacNAz-ox (3b) and GlcBl,4-GlcNAz-ox (11), respectively, while Endo S2 gave over >95% yield. Again, the GlcBl,4-GlcNAz-ox (11) was found to be a more efficient disaccharide oxazoline than the LacNAz oxazoline for both enzymes.

[0091] Synthesis of site-specific ADCs using the azide-tagged antibodies. The preparation ofADCs through click chemistry was next evaluated using the obtained azido-tagged antibodies (4b and 12). The conjugation between 4b / 12 and DBCO-PEG5-VC-PAB-MMAE were carried out in 30% DMSO at rt with a final concentration of the antibody at 2 mg / mL and 20 mol equiv of the payload per click handle being used (FIG. 6). The reaction was monitored by LC- ESLMS (FIG. 7), which indicated the completion of conjugation within 6 h to give ADCs 13 and 14.

[0092] Evaluation of the synthetic ADCs for cancer cell killing.

[0093] After obtaining the ADCs, they were evaluated for their in vitro cytotoxicity using SK-BR-3 (high HER-2 expressing) and T47D (low HER-2 expressing) cells. For the SK-BR-3 cell line, the ADC 13 and 14 demonstrated a dose-dependent killing of the SK-BR-3 cell (FIG. 8A) with ICso at 17.2 ng / mL and 17.0 ng / mL, respectively, which is comparable with the ADC obtained using glycan oxazolines carrying azido groups in the non-reducing terminus of the glycan (13.9-25.6 ng / mL) 17, 19- 20. To generate ADC with a higher drug loading, branched payload (DBCO-PEG5-Bis-VC-P AB- MMAE) was clicked to the azido-tagged antibodies (4b and 12) to obtain ADC 15 and 16 with DAR 4. The in vitro cytotoxicity of the obtained ADC 15 / 16 with SK-BR-3 (high HER-2 expressing) and T47D (low HER-2 expressing) cells were performed. For the SK-BR-3 cell line, 15 and 16 demonstrated a dose-dependent killing of the SK-BR-3 cell (FIG. 8B) with IC50 at 7.7 ng / mL and 7.5 ng / mL,respectively, showing better cell killing than the ADC 13 and 14 with a lower drug loading (DAR 2).

[0094] Synthesis and test of Man£l,4GlcNAz substrate with Endo S2 enzyme for antibody Fc glycan remodeling. To extend the scope of N-azidoacetyl modified oxazolines as enzyme substrates, the non-reducing terminus galactose in LacNAz was replaced with a mannose moiety to give the ManBl,4-GlcNAz oxazoline. A chemical synthesis of this ManBl,4-GlcNAz oxazoline is shown in FIG. 9A. Glycosylation of the monosaccharide acceptor 5 with mannosyl donor 17 afforded the known disaccharides 18 in 66% yield. Hydrogenation of 18 followed by installation of the azidoacetyl group to furnish Manpi,4-GlcNAz (19). Eventually, oxazoline formation gave the Manpi,4-GlcNAz oxazoline (20) in excellent yield.

[0095] Next, the activity of the synthetic ManBl,4-GlcNAz-ox (20) was tested for Endo S2 catalyzed enzymatic antibody glycan remodeling (FIG.9B). It was found that ManBl,4-GlcNAz-ox (20) was much more active than the LacNAz oxazoline (3b) and GlcBl,4-GlcNAz-ox (11), which achieved a 100% transglycosylation yield within 10 min when only 0.5% Endo S2 was used (FIG. 9C).

[0096] Synthesis of site-specific ADC using ManBl,4-GlcNAz-tagged antibodies (21). The preparation of ADC through click chemistry was next evaluated using the obtained azidotagged antibody (20). The conjugation between 20 and DBCO-PEG5-VC-PAB-MMAE were carried out in 30% DMSO at rt with a final concentration of the antibody at 2 mg / mL and 20 mol equiv of the payload per click handle being used (FIG. 10A). The reaction was monitored by LC-ESLMS (FIG. 10B), which indicated the completion of conjugation within 6 h to give ADC 22.

[0097] Evaluation of the synthetic ADC 22 for cancer cell killing.

[0098] After obtaining the ADC (22), it was evaluated for their in vitro cytotoxicity using SK-BR-3 (high HER-2 expressing) and T47D (low HER-2 expressing) cells. For the SK-BR-3 cell line, the ADC 21 demonstrated a dose-dependent killing of the SK-BR-3 cell (FIG. 10C) with ICso at 19.3 ng / mL, which is comparable with the ADCs 13 and 14. No obvious cell killing was observed for low HER-2 expressing cells (T47D).REFERENCES(1) Drago, J. Z.; Modi, S.; Chandarlapaty, S., Unlocking the potential of antibody-drug conjugates for cancer therapy. Nat. Rev. Clin. Oncol. 2021, 18 (6), 327-344.(2) do Pazo, C.; Nawaz, K.; Webster, R. M., The oncology market for antibody-drug conjugates. Nat. Rev. Drug Discov. 2021, 20 (8), 583-584.(3) Dumontet, C.; Reichert, J. M.; Senter, P. D.; Lambert, J. M.; Beck, A., Antibody-drug conjugates come of age in oncology. 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Claims

WHAT IS CLAIMED IS:

1. A one-pot chemoenzymatic remodeling method for both deglycosylation and transglycosylation reactions in a one-pot manner to provide an azido-tagged antibody comprising the steps of: (a) providing a single reactor, container, column, or pot; (b) introducing a single endoglycosidase, or mutant thereof, having both deglycosylation and transglycosylation activity; (c) introducing an antibody for deglycosylation by the single endoglycosidase thereby providing a deglycosylated intermediate that contains at least one N-acetylglucosamine (GlcNAc) or core- fucosylated N-acetylglucosamine (Fucal,6GlcNAc) acceptor; (d) providing GlcNAz-derived sugar oxazoline; and (e) transglycosylating the GlcNAz-derived sugar oxazoline to the N- acetylglucosamine (GlcNAc) or core-fucosylated N-acetylglucosamine (Fucal,6GlcNAc) acceptor by the single endoglycosidase to provide the azido-tagged antibody.

2. The one-pot chemoenzymatic method according to claim 1, wherein the functionalized oxazolines for use as substrates in the transclycosylation step include those having the general structure :wherein : R = N3, OH, SH, F, Cl, Br, I, alkene, alkyne, and other molecular moietiesG =mannose, glucose, galactose, xylose, lactose, maltose, cellobiose, and any other sugar moieties. The glycosylic bond can be in either a or P configuration.

3. The method of claim 1, wherein the GlcNAz-derived sugar oxazoline has a glucose core as the second sugar moiety (Group I), and has the following general structure, where the glucose moiety can be linked to the first sugar moiety in either a- or -glycosidic bond:

4. The method of claim 1, wherein the GlcNAz-derived sugar oxazoline has a galactose core as the second sugar moiety (Group II), and has the following general structure, where the galactose moiety can be linked to the first sugar moiety in either a- or fkglycosidic bond:

5. The method of claim 1 , wherein the GlcNAz-derived sugar oxazoline has a mannose core as the second sugar moiety (Group III), and has the following general structure, where the mannose moiety can be linked to the first sugar moiety in either ex- or P-glycosidic bond:

6. The method of claim 1 , wherin the GlcNAz-derived sugar oxazoline carries an oligosaccharide structure at the non-reducing terminus (Group IV), and has the following general structure:

7. The one-pot chemoenzymatic remodeling method according to claim 1, wherein the deglycosylated intermediate and endoglycosidase do not need to be separate from the reaction in the single reactor, container, column, or pot.

8. The method of claim 1, further comprising the addition of a tag to the azido-tagged antibody or glycoprotein via a click-chemistry conjugation reaction.

9. The method of claim 8 wherein the tag is selected from the group consisting of a therapeutic agent, drug, ligand, azide, biotin, fluorescent probe, a diagnostic reagent, and any other molecular entities.

10. The one-pot Fc glycan remodeling method of claim 1 , wherein the glycan oxazoline comprises multiple molecular linkers attached to a disaccharide core to form a dendrimer.

11. A delivery device for the delivery of a therapeutic agent, drug, ligand, or diagnostic agent, wherein the delivery device comprises the azido tagged antibody or glycoprotein according to claim 1 and wherein a therapeutic agent, drug, ligand, or diagnostic agent are conjugated site- specifically to the antibody.

12. A delivery device for the delivery of a therapeutic agent, wherein the delivery device is a dendrimer according to the method of claim 11 , and wherein the therapeutic agent is attached to at least one of a periphery azido end.

13. An antibody-conjugate comprising the azido-tagged antibody or glycoprotein according to claim 1 or 2, and further comprising the addition of special molecular entities to the azido-tagged antibody or glycoprotein via a click-chemistry conjugation reaction.

14. The antibody-conjugate of claim 13, wherein the special molecular entities are a therapeutic agent, drug, ligand, or diagnostic agent and are site-specifically conjugated to the antibody.

15. The antibody-conjugate of claim 14, wherein the therapeutic agent is selected from the group consisting of antibiotics, analgesics, antibodies; cancer drugs, antivirals, metal chelates, proteins, hormones, and nucleic acids.

16. A method of treating a subject suffering from an ailment, disease, or disorder comprising administration of the antibody-drug conjugate of claim 15.

17. The method of claim 16, wherein the ailment, disease, or disorder is cancer or an infectious disease.

18. A kit comprising the antibody-conjugate of claim 14.

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