Methods for modifying glycoproteins

Abstract

The present disclosure provides methods for modifying glycoproteins. The present disclosure also provides methods for producing glycoprotein-payload conjugates, the conjugates produced thereby, and uses thereof.

Description

[Background technology]

[0001] This application claims the benefit of priority under 35 USC § 119(e) of U.S. Provisional Patent Application No. 63 / 477,672, filed December 29, 2022, the contents of which are incorporated by reference in their entirety as if fully set forth herein. [Technical Field]

[0002] The present disclosure relates to methods for conjugating a payload of interest to glycoproteins, including antibodies and their derivatives, to produce glycoprotein-payload conjugates.

[0003] Therapeutic protein drugs have the advantages of high specificity and low toxicity and have been widely used in clinical practice since 1990. More than 80% of them are monoclonal antibodies. Although improved clinical outcomes have been reported in some patients, some clinical trials have ended disappointingly due to unsatisfactory therapeutic effects, especially in cancer treatment.

[0004] To improve the therapeutic efficacy of cancer treatment, scientists are focusing on modifying clinically approved monoclonal antibodies using technologies such as bispecific antibodies, antibody-drug conjugates (ADCs), and CAR-T. Among these technologies, ADCs have attracted more attention due to their high specificity and efficacy. To date, 11 therapeutic antibody-drug conjugates are available in clinical trials, including Mylotarg, Adcetris, Besponsa, Kadcyla, Polivy, Padcev, Enhertu, Trodelvy, Blenrep, Zynlonta, and Aidexi, with many more in development.

[0005] In most of these early clinical ADCs, the payload is randomly conjugated to lysine sites (e.g., Kadcyla and Mylotarg) or cysteine ​​sites (e.g., Adcetris). For example, in Adcetris, an average of four (2-8) monomethyl auristatin E (MMAE) molecules (a synthetic antitumor drug) are randomly conjugated to the brentuximab antibody scaffold via the -SH groups of cysteine ​​residues through mild disulfide bond reduction. Linker components include a thiol-reactive maleimidocaproyl spacer, a dipeptide valine-citrulline linker, and a PABC spacer (Francisco JA, Cerveny CG, Meyer DL, Mixan BJ, Klussman K, Chace DF, Rejniak SX et al., cAC10-vcMMAE, Blood 2003, 4, 1458-65). These approaches allow for easy conjugation of payloads or linkers to antibodies due to the presence of multiple lysine and cysteine ​​residues in antibodies. However, the drug-antibody ratio (DAR) is difficult to control, resulting in a lack of uniformity in ADC products and issues with pharmacokinetic (PK) properties and immunogenicity. These ADCs also face challenges in chemical manufacturing and control (CMC).

[0006] In recent years, site-specific conjugation technologies, such as engineered cysteines, unnatural amino acid (UAA) engineering, enzyme-assisted ligation (e.g., smart tags (SMART-Tag) and bacterial transglutaminase), short peptide tags, and native cysteine ​​re-bridging (e.g., thio-bridge) have been developed to address these shortcomings of first-generation ADCs. These technologies can generate homogeneous ADC products by engineering specific sites on the parent antibody component. For example, thio-bridge technology links the linker and payload to a partially reduced disulfide bond in the antibody. Meanwhile, smart tags generate ADCs by mutating the adjacent antibody sequence to a substrate sequence for bacterial oxidase. The resulting formaldehyde product in the antibody is used as the linker-payload linkage site. As expected, these second-generation ADC technologies can generate homogeneous ADC products with unique DARs. However, the altered antibody sequence can result in undesirable immunogenicity. Furthermore, these ADC products still do not meet PK requirements.

[0007] To address these issues and improve the therapeutic efficacy of ADCs, glycoengineering has attracted the attention of developers, offering a new approach to ADC platform development. Antibody-drug conjugation via glycoengineering offers several advantages, including site specificity, diversity, reduced side effects, and high yields. Patent documents such as US 9504758 B2, US 9580511 B2, WO 2015 / 032899 A1, US 11085062 B2, US 8716033 B2, and US 7416858 B2, as well as review articles (Bioconjug Chem.; 2015 Nov. 18; 26(11):2070-5), disclose various modified glycan moieties for antibody-drug conjugation. However, in these publications, the drug-to-antibody ratio (DAR) and payload diversity of the antibody-drug conjugates disclosed are still limited, and therefore there is an unmet need in the art to increase the drug-to-antibody ratio and payload diversity of ADCs. Summary of the Invention

[0008] The present disclosure relates to methods for conjugating a payload of interest to glycoproteins, including antibodies and derivatives thereof, to produce glycoprotein-payload conjugates.

[0009] The native glycan form of a monoclonal antibody contains the following structure: GlcNAc attached to Asn297 of the antibody 1 , the first mannose (Man 1 ) bound to GlcNAc 2 , Man 1 The second and third mannose (Man 2 and Man 3 ), and Man 2 and Man 3 Two more GlcNAc sugars (GlcNAc 3 and GlcNAc 4 ) Antibodies with such glycan moieties are designated as G0F, whereas if the fucose moiety is absent, the antibody is designated as G0. (T. Shantha Raju MAbs. 2012 May 1;4(3):385-391) GlcNAc 3 and GlcNAc 4 When one of the GlcNAc sugars is conjugated to an additional galactose sugar (Gal), the antibody is referred to as a G1F / G1 antibody. When two GlcNAc sugars on the glycan portion of the antibody are each conjugated to two additional galactose sugars (Gal), the antibody is referred to as a G2F or G2 antibody. Antibodies produced by mammalian cells typically contain G0F (more than 40%), G1F (approximately 30%-40%), G2F (less than 1%), and very small amounts of G1F and G2F conjugated to sialic acid (SA). [ka]

[0010] The present disclosure provides a compound of the formula: GlcNAc-(Q) 0-8The present invention relates to a method for preparing glycoproteins having a core internal mannose (Man) substituent at a compound of formula -C' in the presence of a suitable catalyst, wherein the catalyst is an acetylglucosaminyltransferase such as β-1,4-mannosylglycoprotein 4-β-N-acetylglucosaminyltransferase, mannosyl(α-1,3-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase, and mannosyl(α-1,6-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase. Q is selected from alkylene, alkenylene, or polyethylene glycol; and C' is or contains a reactive group selected from an azide group, a keto group, an alkynyl group, a thiol group, a halogen, a sulfonyloxy group, a halogenated acetamide group, a mercaptoacetamide group, a sulfonylated hydroxyacetamide group, a cyclopropenyl group, a transcyclooctene group, a cycloalkyne group, a tetrazinyl group, a maleimide group, a cyclononyne moiety, and a cyclooctyne moiety.

[0011] The present disclosure also relates to a method for preparing an antibody conjugate comprising reacting a modified antibody according to the present invention with a linker-payload conjugate, said linker-payload conjugate comprising a reactive group G' and one or more payloads of interest, wherein said G' is GlcNAc-(Q) 0-8 -C' is a reactive group capable of reacting with C' of -C' or contains such a reactive group.

[0012] One aspect of the present disclosure provides a method for preparing a glycoprotein conjugate comprising the structure of formula (1): [ka] (1) where Pr is a glycoprotein, L is a linker, D is a payload of interest, x is 1, 2, 3, or 4, y is 1 or 2, z1 is 0 or 1, z2 is 0 or 1, r is a positive integer from 1 to 20, p is an integer from 0 to 8, Q is an alkylene, alkenylene, or polyethylene glycol, and CG is a linking group obtained by the reaction of C' with G', with the proviso that when z1 is 1, GlcNAc 3 is present, and galactose (Gal) and sialic acid (SA) are not bound to it. When z2 is 1, GlcNAc 4 is present, and no galactose (Gal) or sialic acid (SA) is attached to it.

[0013] The natural glycan format of a monoclonal antibody having the following structure of formula (1-1) is: [ka] (1-1) The glycoconjugate can be converted to a glycoprotein conjugate having the structure of formula (1) through a glycoengineering process, where UDP-GlcNAc-(Q) is first reacted with β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase (MGAT3; GnT-3; EC 2.4.1.144), optionally with mannosyl(α-1,3-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase (MGAT1; GnT-1; EC: 2.4.1.101), and optionally with mannosyl(α-1,6-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase (MGAT2; GnT-2; EC 2.4.1.143). 0-8 -C' molecule to form GlcNAc-(Q) 0-8 -C' molecule to Man 1 and Man as needed 2 and Man 3to form a glycoprotein containing a glycan of formula (1-2), where C' is or contains a reactive group selected from an azide group, a keto group, an alkynyl group, a thiol group, a halogen, a sulfonyloxy group, a halogenated acetamide group, a mercaptoacetamide group, a sulfonylated hydroxyacetamide group, a cyclopropenyl group, a transcyclooctene group, a cycloalkyne group, a tetrazinyl group, a maleimide group, a cyclononyne moiety, and a cyclooctyne moiety. [ka] (1-2) Next, the glycoprotein containing the glycan of formula (1-2) is treated with G'-L(D) r to form a glycoprotein payload conjugate comprising the structure of formula (1) by reacting the glycoprotein payload conjugate with one or more linker-payload conjugates comprising the structure of formula (1).

[0014] In some preferred embodiments, the linker comprises the structure L1-L2-L3, where L1 is optional and, if present, is a spacer that may be a linear or branched PEG chain having 1 to 10 PEG units, an alkylene, cycloalkylene, alkenylene, cycloalkenylene, alkynylene, arylene, heteroarylene, alkeneoxy, acyl, alkylamine, or arylamine group having 2 to 20 carbon atoms. L2 is a cleavable or non-cleavable linker that may be a thioether linker, maleimidocaproyl linker, disulfide-containing linker, acid labile linker, photolabile linker, peptidase labile linker, esterase labile linker, phosphatase labile linker, β-glucuronide linker, β-glucuronidase labile linker, β-galactosidase labile linker, or sulfatase labile linker. L3 is optional and, if present, is a PAB.

[0015] In some preferred embodiments, the payload is a therapeutic agent selected from antimetabolites, alkylating agents, alkylating mimetics, DNA minor groove alkylators, anthracyclines, antibiotics, calicheamicins, antimitotic agents, topoisomerase inhibitors, proteasome inhibitors, and radioisotopes, e.g., the therapeutic agent is selected from exatecan and MMAE. Alternatively, the payload is a label selected from a fluorescent label, a chromophore label, an electron-dense label, a chemiluminescent label, a radioactive label, an enzymatic label, or a positron emitter.

[0016] In some preferred embodiments, before carrying out step (i), β-N-acetylglucosaminidase may be added to remove GlcNAc from the glycoprotein of formula (1-1).

[0017] The present invention also relates to a glycoprotein payload conjugate comprising the structure of formula (1) as defined above. In some preferred embodiments, the glycoprotein payload conjugate comprises the structure of formula (2): [ka] (2)

[0018] In some preferred embodiments, the glycoprotein payload conjugate comprises the structure of formula (3): [ka] (3) Here, z1 and z2 are both 1, or z1 is 1 and z2 is 0.

[0019] In some preferred embodiments, the glycoprotein payload conjugate comprises the structure of formula (3-1): [ka] (3-1)

[0020] In some preferred embodiments, the glycoprotein payload conjugate comprises the structure of formula (3-2): [ka] (3-2)

[0021] The present invention also relates to a method for treating cancer, comprising administering to a subject in need thereof an effective amount of a glycoprotein payload conjugate comprising the structure of formula (1), formula (2), or formula (3) as defined above.

[0022] The present invention also relates to the use of a glycoprotein payload conjugate comprising a structure of formula (1), formula (2) or formula (3) as defined above for the manufacture of a medicament for the treatment of cancer.

[0023] The present invention also relates to a glycoprotein payload conjugate comprising a structure of formula (1), formula (2) or formula (3) as defined above for treating cancer in a subject in need thereof.

[0024] In some preferred embodiments, the cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, ocular melanoma, gastric cancer, head and neck cancer, kidney cancer, leukemia, lung cancer, lymphoma, melanoma, oral and oropharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, and uterine cancer, for example, the cancer may be breast cancer and / or stomach cancer. [Brief explanation of the drawings]

[0025] The present disclosure will be more readily understood by reference to the following description taken in conjunction with the accompanying drawings, in which: [Figure 1] FIG. 1 shows reduced mass chromatography of Herceptin-2Az produced by transferring GlcNAz to Herceptin antibody using GnT-3. [Figure 2]FIG. 1 shows the LC-MS spectrum of herceptin-2 exatecan of the present disclosure produced by conjugating 2 exatecan to herceptin-2Az. [Figure 3] FIG. 1 shows the LC-MS spectrum of Herceptin-2MMAE of the present disclosure, produced by conjugating 2MMAE to Herceptin-2Az. [Figure 4] FIG. 1 shows the LC-MS spectrum of herceptin-6 exatecan of the present disclosure produced by conjugating 6 exatecan to herceptin-6Az. [Figure 5] FIG. 1 shows the LC-MS spectrum of Herceptin-6MMAE of the present disclosure, produced by conjugating 6MMAE to Herceptin-6Az. [Figure 6] FIG. 1 shows the inhibitory effects of Herceptin-2MMAE and Herceptin-6MMAE on cell viability of breast cancer cell line BT-474. [Figure 7] FIG. 1 shows the inhibitory effects of herceptin-2 exatecan and herceptin-6 exatecan on cell viability of breast cancer cell line BT-474. [Figure 8] FIG. 1 shows the inhibitory effects of Herceptin-2MMAE and Herceptin-6MMAE on cell viability of gastric cancer cell line NCI-N87. [Figure 9] FIG. 1 shows the inhibitory effects of herceptin-2 exatecan and herceptin-6 exatecan on cell viability of gastric cancer cell line NCI-N87. DETAILED DESCRIPTION OF THE INVENTION

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.Although any similar or equivalent methods and materials as those described herein can be used to carry out or test this disclosure, preferred methods and materials are described herein.All publications and patent documents specifically mentioned herein are incorporated by reference for all purposes, including the description and disclosure of chemicals, cell lines, vectors, animals, instruments, statistical analysis and methodology reported in the publications that may be used in connection with the present invention.All references cited herein should be interpreted as indicating the level of skill in the art.

[0027] Abbreviations used in this disclosure are listed below for reference: JPEG2026501620000010.jpg137149

[0028] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Similarly, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" can be used interchangeably.

[0029] Ranges are often expressed herein as from "about" one particular value to "about" another particular value. When such a range is expressed, an embodiment includes a range from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, using the term "about," it will be understood that the particular value forms another embodiment. Further, it will be understood that the endpoints of each range are significant both relative to the other endpoint and independently of the other endpoint. As used herein, the term "about" refers to ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, or ±0.25%.

[0030] When referring to components of a therapeutic formulation, the term used (e.g., "agent") is intended to include not only the specific molecular entity but also pharmaceutically acceptable analogs thereof, including, but not limited to, salts, esters, amides, prodrugs, conjugates, active metabolites and other such derivatives, analogs and related compounds.

[0031] The general term "sugar" as used herein refers to monosaccharides (e.g., glucose (Glc), galactose (Gal), mannose (Man), and fucose (Fuc)), as well as derivatives of monosaccharides (such as amino sugars and sugar acids, e.g., glucosamine (GlcN), galactosamine (GalN), N-acetylglucosamine (GlcNAc), N-azidoacetylglucosamine (GlcNAz), N-acetylgalactosamine (GlaNAc), N-acetylneuraminic acid (NeuNAc), N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA), and iduronic acid (IdoA)).

[0032] As used herein, the term "protein" includes polypeptides having native amino acid sequences as well as variants and modified forms, regardless of their origin or method of preparation. A protein having a native amino acid sequence is a protein having the same amino acid sequence as obtained from nature. Such native sequence proteins can be isolated from nature or prepared using standard recombinant and / or synthetic methods. Native sequence proteins specifically include naturally occurring truncated or soluble forms, naturally occurring variant forms (e.g., alternatively spliced ​​forms), naturally occurring allelic variants, and forms containing post-translational modifications. Native sequence proteins include proteins that have undergone post-translational modifications, such as glycosylation, phosphorylation, or other modifications of some amino acid residues.

[0033] As used herein, the term "glycoprotein" refers to a protein containing one or more monosaccharide or oligosaccharide chains covalently attached to the protein. Glycans may be attached to hydroxyl groups (O-linked glycosyl) of the protein (e.g., the hydroxyl groups of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline), amides (N-glycosyl) on the protein (e.g., asparagine, arginine), or carbons (C-glycosyl) on the protein (e.g., tryptophan). A glycoprotein may contain one or more glycans, a combination of one or more monosaccharide and one or more oligosaccharide glycans, or a combination of N-linked, O-linked, and C-linked glycans. Examples of glycoproteins include ligands specific for cell surface antigens, prostate-specific membrane antigen, Candida antarctica lipase, gp41, gp120, erythropoietin (EPO), antifreeze proteins, and antibodies.

[0034] Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. The term "antibody" is used herein in the broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (such as bispecific antibodies), antibody fragments, as well as double-chain and single-chain antibodies. The term "antibody" as used herein also includes human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to cancer antigens. The term "antibody" includes not only whole antibodies but also antibody fragments, such as antibody Fab fragments, F(ab')2, truncated antibody Fv or Fc fragments, scFv-Fc fragments, minibodies, diabodies, or scFv. Furthermore, the term also includes genetically engineered derivatives of antibodies. Antibodies, antibody fragments, and genetically engineered antibodies can be obtained by methods known in the art. Examples of commercially available antibodies include, but are not limited to, abciximab, rituximab, basiliximab, palivizumab, infliximab, trastuzumab, alemtuzumab, adalimumab, tositumomab-1131, cetuximab, ibrituximab tiuxetan, omalizumab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, certolizumab pegol, golimumab, canakinumab, catumaxomab, ustekinumab, tocilizumab, ofatumumab, denosumab, belimumab, ipilimumab, and brentuximab.

[0035] Antibodies can be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems. In some embodiments, the expression system is a mammalian cell expression system, such as a hybridoma, or a CHO cell expression system. Many such systems are widely available from commercial suppliers. In embodiments in which an antibody comprises both heavy chain (VH) and light chain (VL) variable domains, the VH and VL regions can be expressed using a single vector, e.g., a dicistronic expression unit, or under the control of different promoters. In other embodiments, the VH and VL regions can be expressed using separate vectors. The VH or VL regions described herein may optionally include an N-terminal methionine.

[0036] Genes encoding the heavy and light chains of an antibody of interest can be cloned from cells; for example, genes encoding a monoclonal antibody can be cloned from a hybridoma and used to generate recombinant monoclonal antibodies. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be generated from hybridomas or plasma cells. Random combination of heavy and light chain gene products generates antibodies with many different antigen specificities.

[0037] Techniques for producing single chain antibodies or recombinant antibodies (US Pat. No. 4,946,778, US Pat. No. 4,816,567) can be adapted to produce antibodies to polypeptides of the present disclosure. Also, transgenic mice or other organisms, such as other mammals, can be used to express humanized or human antibodies (see, e.g., U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016; Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); and See Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0038] Naturally occurring IgG and recombinant antibodies have an N-glycosylation site at amino acid asparagine 297 (Asn297) within each CH2 constant region of the IgG1 heavy chain. Upon glycosylation and post-translational modification in mammalian cells, N-glycosylation on IgG results in the formation of two diantennary glycan moieties, each consisting of at least seven sugar moieties, GlcNAc and Man, with the structure essentially shown in formula (4). [ka] (4)

[0039] The glycan moiety shown in formula (4) is "GlcNAc bound to Asn297 of the antibody." 1 ", followed by the first mannose (Man 1 ) bound to GlcNAc 2 ", "Man 1The second and third mannose (Man 2 and Man 3 )" and "Man 2 and Man 3 Two more GlcNAc sugars (GlcNAc 3 and GlcNAc 4 )" sequence. Antibodies with such glycan moieties are designated as G0, but are composed of GlcNAc 1 If there is a fucose moiety attached to GlcNAc, the antibody is GOF (T. Shantha Raju MAbs. 2012 May 1;4(3):385-391). 3 and GlcNAc 4 If one of the GlcNAc sugars on the glycan of the antibody is bound to an additional galactose sugar, the antibody is referred to as a G1 / G1F antibody. If both GlcNAc sugars on the glycan of the antibody are each bound to two additional galactose sugars, the antibody is referred to as a G2 or G2F antibody. Antibodies produced by mammalian cells typically contain G0F (greater than 40%), G1F (approximately 30%-40%), G2F (less than 1%), and very small amounts of G1F and G2F bound to sialic acid.

[0040] Antibody glycoengineering, which involves modifying the branching site of the N297 glycan, can enhance therapeutic properties such as antibody-dependent cell-mediated cytotoxicity (ADCC), half-life, and the chemical manufacturing and management (CMC) of antibodies, while preserving their structure and generating functional diversity.

[0041] One aspect of the present invention provides a method for producing a glycoprotein conjugate comprising the structure of formula (1): [ka] (1) The production method includes the following steps (i) and (ii). (i) reacting a glycoprotein having the structure of formula (1-1) with UDP-GlcNAc-(Q) in the presence of β-1,4-mannosylglycoprotein 4-β-N-acetylglucosaminyltransferase, and optionally mannosyl(α-1,3-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase, and optionally mannosyl(α-1,6-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase; 0-8 -C' molecule, [ka] (1-1) GlcNAc-(Q) 0-8 -C' molecule, Man 1 and Man who responds to needs 2 and Man 3 to form a glycoprotein comprising the glycan of formula (1-2), and [ka] (1-2) (ii) The modified glycoprotein (1-2) is converted to G'-L(D) r forming a glycoprotein-payload conjugate comprising the structure of formula (1) by reacting with one or more linker-payload conjugates comprising the structure of formula (1).

[0042] In formula (1), CG is a linking group obtained by the reaction of C' with G'. The term "linking group" refers to a structural element that connects one part of a compound to another part of the same compound. In formula (1), CG links the antibody to the payload, possibly via Q and a linker L (if present).

[0043] C' is or contains a reactive group selected from an azide group, a keto group, an alkynyl group, a thiol group, a halogen, a sulfonyloxy group, a halogenated acetamide group, a mercaptoacetamide group, a sulfonylated hydroxyacetamide group, a cyclopropenyl group, a transcyclooctene group, a cycloalkyne group, a tetrazinyl group, a cyclononyne moiety, and a cyclooctyne moiety.

[0044] In addition, the G' is GlcNAc-(Q) 0-8 -G' is or contains a reactive group capable of reacting with C' of -C'. In preferred embodiments, G' contains a terminal azide, an alkyne, a cyclononyne moiety, or a cyclooctyne moiety, and G' is different from C'.

[0045] For example, the cyclononyne moiety may be bicyclononyne (BCN) and the cyclooctyne moiety may be selected from the group consisting of azadibenzocyclooctyne (DIBAC / DBCO), dibenzocyclooctyne (DIBO), and sulfonylated dibenzocyclooctyne (s-DIBO).

[0046] As will be appreciated by those skilled in the art, the nature of the linking group will depend on the type of reaction between C' and G'. For example, if C' is an azide and the conjugator G' is an alkynyl, the linker payload can be converted by a click reaction to -GlcNAc-(Q) 0-8 Reacts with molecules containing a -C' group to form -GlcNAc-(Q) 0-8 (Angewandte Chemie International Edition. 40(11):2004-2021; and Australian Journal of Chemistry. 60(6):384-395). In another embodiment, when C' is a ketone or aldehyde group and G' is an amino group, the linker payload can be formed by reductive amination to give -GlcNAc-(Q) 0-8 Reacts with molecules containing a -C' group to form -GlcNAc-(Q) 0-8(J. Org. Chem., 2010, 75, 5470-5477; and Synthesis, 2011, 490-496). In a further embodiment, when C' is a ketone or aldehyde group and G' is β-arylethylamino, the linker payload is formed by the Pictet-Spengler reaction to give -GlcNAc-(Q) 0-8 Reacts with molecules containing a -C' group to form -GlcNAc-(Q) 0-8 -CG-linker payload (Bioconjugate Chem., 2013, 24(6), pp846-851). Further suitable combinations of C' and G', as well as the nature of the resulting linking group CG, are known to those skilled in the art.

[0047] In formula (1), Q is alkylene, alkenylene, or polyethylene glycol, and Q may or may not be present. Furthermore, when Q is present, p may independently be 1, 2, 3, 4, 5, 6, 7, or 8.

[0048] In formula (1), "-(Fuc) 0-1 " represents an optionally present fucose sugar, and if present, only one fucose sugar moiety is present. Similarly, "-(Gal) 0-1 " represents an optionally present galactose sugar, and if present, only one galactose sugar moiety is present. "-(SA) 0-1 " represents an optionally present sialic acid, and if present, only one sialic acid moiety is present. 0-1 " represents an optionally present GlcNAc, and if present, only one GlcNAc moiety is present.

[0049] In formula (1), "-[(Q) p -CG-L(D) r ] z1 " represents the linker payload, which is optionally present and 3 It is conjugated to "-[(Q) p -CG-L(D)r ] z1 If " is present, one "-[(Q) p -CG-L(D) r ]" and GlcNAc 3 There are no galactose sugars or sialic acids in the α-[(Q) p -CG-L(D) r ] z2 " represents the linker payload, which is optionally present and 4 It is conjugated to "-[(Q) p -CG-L(D) r ] z2 If " is present, one "-[(Q) p -CG-L(D) r ]" and GlcNAc 4 There are no galactose sugars or sialic acids in "-[(Q) p -CG-L(D) r ] z1 " and "-[(Q) p -CG-L(D) r ] z2 " may both be present, may not be present, or "-[(Q) p -CG-L(D) r ] z1 " may be present, and is represented by the following formulas (2), (3-1) and (3-2). [ka] (2) [ka] (3-1) and [ka] (3-2)

[0050] In formula (1), r is a positive integer in the range of 1 to 20, and for example, r may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0051] In formula (1), x is a positive integer in the range of 1 to 4; for example, x is 1, 2, 3, or 4.

[0052] In formula (1), y is 1 or 2.

[0053] In some embodiments, the glycoprotein payload conjugate comprising the structure of formula (1) has the structure of formula (2): [ka] (2)

[0054] In another embodiment, the glycoprotein payload conjugate comprising the structure of formula (1) has the structure of formula (3): [ka] (3) Here, z1 and z2 are both 1, or z1 is 1 and z2 is 0.

[0055] The glycoproteins used in the present invention can be obtained, for example, by solid-phase peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production. In the case of recombinant production, one or more polynucleotides encoding the glycoprotein are isolated and inserted into a vector for further cloning and / or expression in a host cell. Such polynucleotides can be easily isolated and sequenced using conventional procedures. Methods well known to those skilled in the art can be used to construct expression vectors containing glycoprotein-coding sequences. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, NY (1989).

[0056] As used herein, β-N-acetylglucosaminidase belongs to a family of glycosidases that catalyze the hydrolysis of β-N-acetylglucosamine residues from oligosaccharides. Many β-N-acetylglucosaminidases are known to have broad hydrolysis capabilities, catalyzing multiple types of β-glycosidic linkages. In this disclosure, β-N-acetylglucosaminidase is defined as any exoglycosidase that can hydrolyze, but is not limited to, the β1-2 bond between the terminal acetylglucosamine residue and the N-glycan of a glycoprotein. Exo-β-N-acetylglucosaminidases are widely expressed in multiple species, including Streptococcus spp. and jack bean (Canavalia ensiformis), and are routinely used to identify terminal GlcNAc from N-glycoproteins.

[0057] According to the present invention, mannosyl(α-1,3-)glycoprotein β-1,2-N-acetylglucosaminyltransferase (MGAT1; GnT-1; EC:2.4.1.101) transfers N-acetyl-D-glucosamine from UDP-GlcNAc to a terminal mannose linked to another sugar moiety or glycan via an α-1,3 glycosidic linkage. The linkage between the GlcNAc and α-3 mannose transferred by MGAT1 is a β1,2 glycosidic linkage. MGAT1 has been found to be ubiquitously expressed in eukaryotes because it is an essential enzyme for the biosynthesis of hybrid and complex N-glycans in the Golgi apparatus.

[0058] According to the present invention, mannosyl(α-1,6-)glycoprotein β-1,2-N-acetylglucosaminyltransferase (MGAT2; GnT-2; EC:2.4.1.143) transfers N-acetyl-D-glucosamine from UDP-GlcNAc to a terminal mannose linked to another sugar moiety or glycan via an α-1-6 glycosidic linkage. The linkage between the GlcNAc and α-6 mannose transferred by MGAT2 is a β-1-2 glycosidic linkage. MGAT2 has been found to be ubiquitously expressed in eukaryotes because it is an enzyme essential for the biosynthesis of complex N-glycans in the Golgi apparatus.

[0059] As used herein, β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase (MGAT3; GnT-3; EC 2.4.1.144) transfers GlcNAc from UDP-GlcNAc to mannose attached to another sugar moiety or glycan via a glycosidic bond. MGAT3 is an essential enzyme for the biosynthesis of hybrid and complex N-glycans in the Golgi apparatus and has therefore been found to be ubiquitously expressed in eukaryotes.

[0060] In some embodiments, the reaction between a glycoprotein comprising a glycan having formula (4) and β-N-acetylglucosaminidase is carried out in a mammalian cell culture, wherein a mammalian cell line comprising a first polynucleotide encoding a glycoprotein comprising a glycan having formula (4) and a second polynucleotide encoding the β-N-acetylglucosaminidase is incubated in a medium under conditions suitable for expression of the glycoprotein and the β-N-acetylglucosaminidase. Examples of mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7), human embryonic kidney (293 or 293T) cells, baby hamster kidney (BHK) cells, mouse Sertoli cells (TM4), monkey kidney (CV1), African green monkey kidney (VERO-76) cells, human cervical carcinoma (HELA) cells, canine kidney (MDCK) cells, buffalo rat liver (BRL3A) cells, human lung (W138) cells, human liver (HepG2) cells, mouse mammary tumor (MMT060562) cells, TRI cells, MRC5 cells, FS4 cells, Chinese hamster ovary (CHO) cells, and myeloma cell lines such as YO, NS0, P3X63, and Sp2 / 0.

[0061] In some embodiments, when the glycoprotein-payload conjugate is used to treat a disease of interest, the payload may be a therapeutic agent, which may be a cytostatic agent, a cytotoxic agent, or an isotope chelator containing a corresponding radioisotope. Examples of cytostatic or cytotoxic drugs include antimetabolites (e.g., fluorouracil (5-FU), floxuridine (5-FudR), methotrexate, leucovorin, hydroxyurea, thioguanine (6-TG), mercaptopurine (6-MP), cytarabine, pentostatin, fludarabine phosphate, cladribine (2-CDA), asparaginase, gemcitabine, 25riethylenediamine, azathioprine, cytosine methotrexate, trimethoprim, pyrimethamine, or pemetrexed); alkylating agents (e.g., simephalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, dacarbazine, mitomycin C, cyclophosphamide, mechlorethamine, uramustine, dibromomannitol, tetracycline ... nitrates, procarbazine, altretamine, mitozolomide, or temozolomide; alkylating analogs (e.g., cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, or triplatin); DNA minor groove alkylating agents (e.g., duocarmycins such as CC-1065, and analogs or derivatives thereof; pyrrolobenzodiazepines, or analogs or derivatives thereof); anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, or valrubicin); antibiotics (e.g., dactinomycin, bleomycin, mithramycin, anthramycin, streptozotocin, gramicidin D, mitomycins (e.g., mitomycin C); calicheamicin; antimitotic agents (e.g., maytansinoids (DM1, DM3, DM4, etc.));auristatins (e.g., including monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF)), dolastatins, cryptophycins, vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinorelbine), taxanes (e.g., paclitaxel, docetaxel, or novel taxanes), tubulysins, colchicine); topoisomerase inhibitors (e.g., exatecan, irinotecan, topotecan, camptothecin, etoposide, teniposide, amsacrine, mitoxantrone); HDAC inhibitors (e.g., vorinostat, romidepsin, chidamide, 25riethylenet, or belinostat); proteasome inhibitors (e.g., peptidylboronic acid); as well as At; 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 or Bi 213 , P 32 Radioactive isotopes such as Lu 177 Examples of isotope chelators include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriamine-N,N,N',N",N"-pentaacetate (DTPA), 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetate (DOTA), 1,4,7,10-tetrakis(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane (THP), 26-triethylenetetramine-N,N,N',N",N"',N"'-hexaacetate (TTHA), 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetrakis(methylenephosphonate) (DOTP), and mercaptoacetyltriglycine (MAG3).

[0062] In some embodiments, when the glycoprotein-payload conjugate is used for detection, the payload may be a label. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent, chromophore, electron-dense, chemiluminescent, radioactive labels) as well as moieties such as enzymes or ligands that are indirectly detectable, e.g., through enzymatic reactions or molecular interactions. Examples of labels include the radioisotope P 32 , C 14 , I 125 , H 3 , I 131 Examples of suitable labels include, but are not limited to, rare earth chelates and fluorophores such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases (e.g., firefly luciferase and bacterial luciferase), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase) in combination with enzymes that use hydrogen peroxide to oxidize dye precursors (e.g., HRP, lactoperoxidase, or microperoxidase), biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like. In another embodiment, the label is a positron emitter. The positron emitter is Ga 68 , F 18 , Cu 64 , Y 86 , Br 76 , Zr 89 , I 124 These include, but are not limited to:

[0063] As used herein, L is a linker that can be used to connect the linking group CG to a therapeutic agent or label. In some embodiments, L can include one or more of L1, L2, and L3. L1, L2, and L3 can each be present or absent. In some embodiments, all three linking units are present. In some embodiments, L includes the structure L1-L2-L3.

[0064] The linker L1 is a spacer that connects the reactive moiety G' or the linking group CG to L2 (if present) or the payload. A "spacer" or spacer moiety is defined herein as a moiety that spacers (i.e., provides distance) between and covalently connects two (or more) different moieties. Spacers that can be used in the linker are known in the art. For example, the spacer used herein can be selected from the group consisting of a linear or branched PEG chain having 1 to 10 PEG units, an alkylene, cycloalkylene, alkenylene, cycloalkenylene, alkynylene, arylene, heteroarylene, alkenoxy, acyl, alkylamine, and arylamine group having 2 to 20 carbon atoms.

[0065] In one embodiment, the term "PEG chain" refers to a PEG chain of the formula: -[CH2CH2O] n - (where n is 1 to 10). For example, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the term "PEG1-10" refers to a polyethylene glycol moiety having 1 to 10 PEG units. In one embodiment, the term "branched polyethylene glycol" refers to a polymer having the formula: -[CH2CHO] n - means that at least one H on is replaced with one or more substituents such as polyethylene glycol to form a branched structure.

[0066] Furthermore, L2 is a cleavable or non-cleavable linker. Non-cleavable linkers include, but are not limited to, thioether linkers and maleimidocaproyl linkers. Cleavable linkers include, but are not limited to, disulfide-containing linkers, acid-labile linkers, photolabile linkers, peptidase-labile linkers, esterase-labile linkers, phosphatase-labile linkers, β-glucuronide linkers, β-glucuronidase-labile linkers, β-galactosidase-labile linkers, and sulfatase-labile linkers. Examples of L2 include peptide-aminobenzylcarbamate linkers, such as maleimidocaproyl-L-phenylalanine-L-lysine-p-aminobenzylcarbamate and maleimidocaproyl-L-valine-L-citrulline-p-aminobenzylcarbamate (vc); N-succinimidyl 3-(2-pyridyldithio)propionate (also known as N-succinimidyl 4-(2-pyridyldithio)pentanoate or SPP); 4-succinimidyl-oxycarbonyl-2-methyl-2-(2-pyridyldithio)-toluene (SMPT); N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP); N-succinimidyl 4-(2-pyridyldithio)butyrate (SPDB); 2-iminothiolane; S-acetylsuccinic anhydride; disulfide benzyl Carbamates; Carbonates; Hydrazone Linkers; N-(α-Maleimidoacetoxy)succinimide ester; N-[4-(p-Azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (AMAS); N-[b-Maleimidopropyloxy]succinimide ester (BMPS); [Ne-Maleimidocaproyloxy]succinimide ester (EMCS); N-[g-Maleimidobutyryloxy]succinimide ester (GMBS); Succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxy-[6-amidocaproic acid] (LC-SMCC); Succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate (LC-SPDP); m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS);N-Succinimidyl [4-iodoacetyl]aminobenzoate (SIAB); Succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC); N-Succinimidyl 3-[2-pyridyldithio]-propionamide (SPDP); [Ne-maleimidocaproyloxy]sulfosuccinimide ester (Sulfo-EMCS); N-[g-maleimidobutyryloxy]sulfosuccinimide ester (Sulfo-GMBS); 4-Sulfosuccinimidyl-6-methyl Sulfosuccinimidyl 6-(3'-[2-pyridyldithio]propionamido)hexanoate (Sulfo-LC-SMPT); Sulfosuccinimidyl 6-(3'-[2-pyridyldithio]propionamido)hexanoate (Sulfo-LC-SPDP); m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester (Sulfo-MBS); N-Sulfosuccinimidyl [4-iodoacetyl]aminobenzoate (Sulfo-SIAB); Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1 -carboxylate (Sulfo-SMCC); sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate (Sulfo-SMPB); ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester) (EGS); disuccinimidyl tartrate (DST); 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); diethylenetriaminepentaacetic acid (DTPA); thiourea linker; glutamic acid-valine-citrulline; valine-alanine; glutamic acid Examples of linkers include, but are not limited to, valine-alanine; valine-lysine; valine-lysine-glycine; alanine-alanine; Gly-Gly-Phe-Gly; Gly-Gly-Gly; β-glucuronide; β-galactoside; pyrophosphate; phosphate; BrAc-Gly-Glu; CL2A; D-leucine-alanine-glutamic acid (DLAE); furin cleavable linker; L-Ala-D-Ala-L-Ala; ortho-hydroxy-protected aryl sulfate (OHPAS); and Val-Ser(GlcA).

[0067] L3 may be present or absent, and when present, connects L2 (if present), or the reactive moiety G', or the linking group CG to the payload. In a preferred embodiment, L3 is p-aminobenzylcarbamate (PAB).

[0068] The present disclosure also provides a method for treating cancer, comprising administering to a subject in need thereof an effective amount of a glycoprotein payload conjugate comprising the structure of formula (1) as defined above; use of a glycoprotein payload conjugate comprising the structure of formula (1) as defined above for the manufacture of a medicament for treating cancer; and a glycoprotein payload conjugate comprising the structure of formula (1) as defined above for treating cancer in a subject in need thereof.

[0069] In at least one embodiment of the present disclosure, the cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, ocular melanoma, gastric cancer, head and neck cancer, kidney cancer, leukemia, lung cancer, lymphoma, melanoma, oral and oropharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, and uterine cancer, for example, the cancer may be breast cancer and / or stomach cancer.

[0070] The following examples are provided to illustrate certain embodiments of the present disclosure, but should not be construed as limiting the scope of the invention. (Example)

[0071] Exemplary embodiments of the present disclosure are further illustrated in the following examples, which should not be construed as limiting the scope of the disclosure.

[0072] Example 1: Preparation of G0F / G0 Herceptin antibody

[0073] To remove the galactose and sialic acid moieties of the N-glycans from the Herceptin antibody having the structure shown in formula (5), 10 mg of Herceptin antibody (Roche) was treated with 20 μl of β1,4-galactosidase (NEB, P0745L, 8 units / μl) and 5 μl of α2-3,6,8-neuraminidase (NEB, P0720L, 50 units / μl) in 1× GlycoBuffer 1 (NEB, total volume 1 ml) at 37°C for 24 hours. 10 μl of β1,4-galactosidase (NEB, P0745L, 8 units / μl) was further added to the reaction mixture, and the reaction was allowed to proceed for an additional 24 hours at 37°C to obtain a G0F / G0 antibody sample having the structure shown in formula (6). The antibody sample was purified using rProtein A Sepharose Fast Flow (GE Healthcare, 17-1279-02). After purification, the antibody sample was subjected to reduced mass chromatographic analysis. The results showed that the majority of the antibody in the sample was G0F (having a heavy chain with a molecular weight of 50,600 Da), with only a small amount of G0 (having a heavy chain with no fucose sugar and a molecular weight of 50,451 Da). [ka]

[0074] Example 2: Preparation of trimannosyl-co-herceptin antibody

[0075] Ten mg of the G0F / G0 Hercepin antibody (having the structure shown in Formula (6)) from Example 1 was treated with 20 μl of β-N-acetylglucosaminidase S (NEB, P0744L, 4 units / μl) in 1× GlycoBuffer 1 (NEB, total volume 1 ml) at 37°C for 24 hours. Ten μl of β-N-acetylglucosaminidase S (NEB, P0744L, 4 units / μl) was added to the reaction mixture, and the reaction was allowed to proceed for an additional 24 hours at 37°C to obtain a digested antibody sample having the structure shown in Formula (7). The digested antibody sample was purified using rProtein A Sepharose Fast Flow (GE Healthcare, 17-1279-02). After purification, the antibody sample was subjected to reduced mass chromatographic analysis. A trimannosyl-core Herceptin antibody with a heavy chain molecular weight of 50,194 Da was obtained, and almost all of the G0F and G0 Herceptin antibodies were converted to the trimannosyl-core antibody, suggesting that β-N-acetylglucosaminidase S can efficiently convert the G0F and G0 antibodies to antibodies with a trimannosyl core. [ka]

[0076] Example 3: GlcNAz transfer to Herceptin antibody by GnT-3

[0077] MGAT-3 binds UDP-azido-N-acetylglucosamine to the trimannosyl core protein Man 1To confirm this phenomenon in antibodies, trimannosyl-Herceptin (25 μg) obtained in Example 2 and UDP-GlcNAz (purchased from sci-pharmtech, CAS: 1611490-64-2) (16.6 μg) in 10 μl of 1× buffer SP (25 mM MES (4-morpholineethanesulfonic acid), 10 mM MnCl, pH 6.5) were incubated in the presence of GnT-3 (0.75 μg, R&D, 7359-GT) at 37° C. for 18 hours. The protein produced was named Herceptin-2Az, and its structure is shown in formula (8). The protein was subjected to reduced pressure mass chromatography analysis. The results are shown in FIG. 1. [ka]

[0078] Example 4: Transfer of GlcNAz to trimannosyl-co-herceptin antibody by GnT-1, GnT-2 and GnT-3

[0079] Trimannosyl-Herceptin (5 mg) obtained in Example 2 and UDP-GlcNAz (purchased from Sci-Pharmtech, CAS: 1611490-64-2) (2.5 mg) in 800 μl of 1× Buffer SP (25 mM MES, 10 mM MnCl, pH 6.5) were incubated at 37°C for 16 hours in the presence of GnT-1 (Development Center of Biotechnology) (0.2 mg), GnT-2 (Development Center of Biotechnology) (0.05 mg), and GnT-3 (R&D, 7359-GT). After incubation, the resulting protein was named Herceptin-6Az, and its structure is shown in formula (9). The protein was subjected to reduced pressure mass chromatographic analysis. [ka]

[0080] Example 5: Conjugating Linker Payload to Herceptin-2Az

[0081] In this example, two therapeutic agents, exatecan and MMAE, were used as payloads of interest. Two ADCs were prepared by conjugating Herceptin-2Az obtained in Example 3 with payloads as follows:

[0082] DBCO-PEG3-vc-exatecan: 10 equivalents of DBCO-PEG3-vc-exatecan (10 mM DMA solution) was slowly added to the Herceptin-2Az protein solution (0.5 mg antibody, 2.366 mg / mL) obtained in Example 3 in a buffer solution (80 mM sodium citrate, 0.18 M Tris, pH 6.5). The reaction mixture was incubated at 37°C for 18 hours in a shaking incubator. The ADC was purified and concentrated using an Amicon Ultra 15 centrifugal filter device equipped with a 30 kDa NMWL and 25 mM sodium citrate pH 6.5 buffer. The ADC thus obtained was named Herceptin-2 exatecan, and its structure is shown in Formula (10). The concentration of the ADC was estimated by measuring the absorbance at 280 nm and used to calculate the recovery yield: 86%. The drug-antibody ratio (DAR) of the ADC was determined by LC-MS: 1.95. The resulting LC-MS spectrum is shown in Figure 2.

[0083] DBCO-PEG3-vc-MMAE: 15 equivalents of DBCO-PEG3-vc-MMAE (10 mM DMA solution) was slowly added to the Herceptin-2Az protein solution (0.71 mg antibody, 3.449 mg / mL) obtained in Example 3 in a buffer solution (25 mM sodium citrate, pH 6.5). The reaction mixture was incubated at 37°C for 18 hours in a shaking incubator. To remove DBCO-PEG3-vc-MMAE, an Amicon Ultra 15 centrifugal filter device equipped with a 30 kDa NMWL and 25 mM sodium citrate, pH 6.5 buffer was used. The ADC thus obtained was named Herceptin-2MMAE, and its structure is shown in Formula (10). The concentration of the ADC was estimated by measuring the absorbance at 280 nm and used to calculate the recovery yield: 81%. The drug-antibody ratio (DAR) of the ADC was determined by LC-MS: 1.94. The resulting LC-MS spectrum is shown in FIG. [ka]

[0084] Example 6: Conjugating Linker Payload to Herceptin-6Az

[0085] In this example, two therapeutic agents, exatecan and MMAE, were used as payloads of interest. Two ADCs were prepared by conjugating Herceptin-6Az obtained in Example 4 with the payloads as follows:

[0086] DBCO-PEG3-vc-exatecan: 15 equivalents of DBCO-PEG3-vc-exatecan (10 mM DMA solution) was slowly added to a Herceptin-6Az protein solution (0.5 mg antibody, 2.1 mg / mL) obtained in Example 4 in a buffer solution (80 mM sodium citrate, 0.21 M Tris, pH 6.5). The reaction mixture was incubated at 37°C for 18 hours in a shaking incubator. To remove DBCO-PEG3-vc-exatecan, an Amicon Ultra 15 centrifugal filter device equipped with a 30 kDa NMWL and 25 mM sodium citrate pH 6.5 buffer was used. The ADC thus obtained was named Herceptin-6 exatecan, and its structure is shown in Formula (11). The concentration of the ADC was estimated by measuring the absorbance at 280 nm and used to calculate the recovery yield: 67%. The drug-antibody ratio (DAR) of the ADC was determined by LC-MS: 5.99. The resulting LC-MS spectrum is shown in Figure 4.

[0087] DBCO-PEG3-vc-MMAE: 30 equivalents of DBCO-PEG3-vc-MMAE (10 mM DMA solution) was slowly added to a Herceptin-6Az protein solution (1.0 mg antibody, 2.1 mg / mL) obtained in Example 4 in a buffer solution (80 mM sodium citrate, 0.2 M Tris, pH 6.0). The reaction mixture was incubated at 37°C for 18 hours in a shaking incubator. To remove DBCO-PEG3-vc-MMAE, an Amicon Ultra 15 centrifugal filter device equipped with a 30 kDa NMWL and 25 mM sodium citrate pH 6.5 buffer was used. The ADC thus obtained was designated Herceptin-6MMAE, and its structure is shown in Formula (11). The concentration of the ADC was estimated by measuring the absorbance at 280 nm and used to calculate the recovery yield: ~71%. The drug-antibody ratio (DAR) of the ADC was determined by LC-MS: 5.62. The resulting LC-MS spectrum is shown in Figure 5. [ka]

[0088] Example 7: Cytotoxicity of Herceptin ADC in BT-474 and NCI-N87 cells

[0089] In this example, two types of cancer cell lines were used to test the cytotoxicity of the ADCs obtained in Examples 5 and 6 above, respectively.

[0090] BT-474, a HER2-positive human breast cancer cell line, was obtained from ATCC. This cell line was supplied as a powder, reconstituted in 1 L of cell culture-grade water, and cultured in Hybridare Medium (ATCC) supplemented with 1.5 g / L sodium bicarbonate and 10% fetal bovine serum (Gibco). NCI-N87, a HER2-positive human gastric cancer cell line, was obtained from ATCC. This cell line was cultured in RPMI (ATCC) supplemented with 10% fetal bovine serum (Gibco). BT-474 and NCI-N87 cell lines were maintained in a humidified 37°C incubator under a 5% CO2 atmosphere. The day before treatment, cells were harvested and seeded into 96-well plates (BT-474: 2,500 cells per well, NCI-N87: 1,500 cells per well). On day 2, cells were treated with 3-fold serial dilutions of the ADCs obtained in Examples 5 and 6 above. Each treatment was performed with 10 triplicate data points. After 144 hours of treatment, cell viability was assessed using a Cell Titer-Glo kit (Promega) according to the manufacturer's instructions. At the end of the incubation period, luminescence was measured using a SpectraMax i3x multimode detection platform (Molecular Devices). Compound cytotoxicity was assessed relative to cells treated with 0.05% PBS (ADC) or 0.05% DMSO (toxic payload). IC 50 Values ​​were calculated by fitting the adapted survival data to a four-parameter logistic equation using GraphPad Prism 5.0 software. The results are shown in Table 1 and Figures 6-9. [Table 1]

[0091] As shown in Table 1 and Figures 6 to 9, glycoprotein conjugates having specific structures of the present disclosure, whether containing two or six therapeutic agents, exhibit excellent inhibitory effects on cancer cells and can be used in the treatment of cancer.

[0092] It was further found that the level of inhibitory effect of the glycoprotein conjugates of the present disclosure can be adjusted by changing the number of payloads conjugated to the conjugate, indicating that the drug-antibody ratio (DAR) and payload diversity of the glycoprotein conjugates of the present disclosure can be precisely designed according to clinical needs, for example, the number of payloads can be designed according to the severity and type of disease, or the age, condition, and gender of the patient.

[0093] The above detailed description of the embodiments is intended to illustrate the preferred embodiments according to the present disclosure, but is not intended to limit the scope of the present disclosure, and therefore all changes and modifications made by those skilled in the art are intended to fall within the scope of the present disclosure as defined by the appended claims.

Claims

1. 1. A method for preparing a glycoprotein conjugate comprising the structure of formula (1), comprising: 【Chemistry 1】 （１） where: Pr is a glycoprotein, L is a linker, D is the payload of interest, x is 1, 2, 3, or 4; y is 1 or 2; z １ is 0 or 1, z ２ is 0 or 1, r is a positive integer from 1 to 20; p is an integer from 0 to 8; Q is alkylene, alkenylene, or polyethylene glycol; CG is a linking group obtained by reaction of C′ and G′, C' is or contains a reactive group selected from an azide group, a keto group, an alkynyl group, a thiol group, a halogen, a sulfonyloxy group, a halogenated acetamide group, a mercaptoacetamide group, a sulfonylated hydroxyacetamide group, a cyclopropenyl group, a transcyclooctene group, a cycloalkyne group, a tetrazinyl group, a maleimide group, a cyclononyne moiety, and a cyclooctyne moiety; G' is or contains a reactive group capable of reacting with C'; However, z １ is 1, GlcNAc ３ is present, and galactose (Gal) and sialic acid (SA) bound thereto are not present, z ２ is 1, GlcNAc ４ is present, and galactose (Gal) and sialic acid (SA) bound thereto are not present, The method for preparing a glycoprotein conjugate comprises the following steps (i) and (ii): (i) reacting a glycoprotein having the structure of formula (1-1) with UDP-GlcNAc-(Q) in the presence of β-1,4-mannosylglycoprotein 4-β-N-acetylglucosaminyltransferase, and optionally mannosyl(α-1,3-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase, and optionally mannosyl(α-1,6-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase; ０－８ -C' molecule, 【Chemistry 2】 （１－１） GlcNAc-(Q) ０－８ -C' molecule, Man １ and Man as needed ２ and Man ３ to form a glycoprotein comprising the glycan of formula (1-2), and 【Transformation 3】 （１－２） (ii) A glycoprotein containing the glycan of formula (1-2) is subjected to G'-L(D) ｒ forming a glycoprotein-payload conjugate comprising the structure of formula (1) by reacting the glycoprotein-payload conjugate with one or more linker-payload conjugates comprising the structure of formula (1).

2. The method of claim 1 , wherein the glycoprotein is an antibody or a fragment thereof.

3. 2. The method of claim 1, wherein G' comprises a terminal azide, alkyne, cyclononyne moiety, or cyclooctyne moiety, and G' is different from C'.

4. 4. The method of claim 1 or 3, wherein the cyclononyne moiety is bicyclononyne (BCN) and the cyclooctyne moiety is selected from the group consisting of azadibenzocyclooctyne (DIBAC / DBCO), dibenzocyclooctyne (DIBO), and sulfonylated dibenzocyclooctyne (s-DIBO).

5. The linker is L １ -L ２ -L ３ The structure of L １ is optionally present and, if present, is a spacer, L ２ is a cleavable or non-cleavable linker, L ３ The method of claim 1 , wherein is may or may not be present and, if present, is PAB.

6. L １ is a linear or branched PEG chain having 1 to 10 PEG units, an alkylene, cycloalkylene, alkenylene, cycloalkenylene, alkynylene, arylene, heteroarylene, alkenoxy, acyl, alkylamine, or arylamine group having 2 to 20 carbon atoms; and / or L ２ is a thioether linker, maleimidocaproyl linker, disulfide-containing linker, acid labile linker, photolabile linker, peptidase labile linker, esterase labile linker, phosphatase labile linker, β-glucuronide linker, β-glucuronidase labile linker, β-galactosidase labile linker, or sulfatase labile linker.

7. L ２ are peptide-aminobenzylcarbamate linkers; L-phenylalanine-L-lysine-p-aminobenzylcarbamate and L-valine-L-citrulline-p-aminobenzylcarbamate (vc); N-succinimidyl 3-(2-pyridyldithio)propionate; 4-succinimidyl-oxycarbonyl-2-methyl-2-(2-pyridyldithio)toluene (SMPT); N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP); N-succinimidyl 4-(2-pyridyldithio)butyrate (SPD B); 2-iminothiolane; S-acetylsuccinic anhydride; disulfide benzyl carbamate; carbonate; hydrazone linker; N-(α-maleimidoacetoxy)succinimide ester; N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (AMAS); N-[b-maleimidopropyloxy]succinimide ester (BMPS); [N-e-maleimidocaproyloxy]succinimide ester (EMCS); N-[g-maleimidobutyryloxy]succinimide ester (GM BS); Succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxy-[6-amidocaproic acid] (LC-SMCC); Succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate (LC-SPDP); m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-Succinimidyl[4-iodoacetyl]aminobenzoate (SIAB); Succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC); N-Succinimidyl Sulfo-3-[2-pyridyldithio]propionamide (SPDP); [N-e-maleimidocaproyloxy]sulfosuccinimide ester (Sulfo-EMCS); N-[g-maleimidobutyryloxy]sulfosuccinimide ester (Sulfo-GMBS); 4-sulfosuccinimidyl-6-methyl-a-(2-pyridyldithio)toluamido]hexanoate (Sulfo-LC-SMPT); sulfosuccinimidyl 6-(3'-[2-pyridyldithio]propionamido)hexanoate (Sulfo-LC-SPDP);m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester (Sulfo-MBS); N-Sulfosuccinimidyl [4-iodoacetyl]aminobenzoate (Sulfo-SIAB); Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfo-SMCC); Sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate (Sulfo-SMPB); Ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester) (EGS); Disuccinimidyl tartrate (DST); 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); Diethylene glycol 6. The method of claim 5, wherein the linker is selected from the group consisting of ethylenetriaminepentaacetic acid (DTPA); a thiourea linker; glutamic acid-valine-citrulline; valine-alanine; glutamic acid-valine-alanine; valine-lysine; valine-lysine-glycine; alanine-alanine; Gly-Gly-Phe-Gly; Gly-Gly-Gly; β-glucuronide; β-galactoside; pyrophosphate; phosphate; BrAc-Gly-Glu; CL2A; D-leucine-alanine-glutamic acid (DLAE); a furin cleavable linker; L-Ala-D-Ala-L-Ala; orthohydroxy-protected aryl sulfate (OHPAS); and Val-Ser(GlcA).

8. 2. The method of claim 1, wherein the payload is a therapeutic agent selected from antimetabolites, alkylating agents, alkylating mimetics, DNA minor groove alkylating agents, anthracyclines, antibiotics, calicheamicins, antimitotic agents, topoisomerase inhibitors, proteasome inhibitors, and radioisotopes.

9. 9. The method of claim 8, wherein the therapeutic agent is selected from exatecan and MMAE.

10. 10. The method of claim 1, wherein the payload is a label selected from a fluorescent label, a chromophore label, an electron-dense label, a chemiluminescent label, a radioactive label, an enzymatic label, or a positron emitter.

11. The method of claim 1, further comprising the step of adding β-N-acetylglucosaminidase to remove GlcNAc on the glycoprotein of formula (1-1) before performing step (i).

12. A glycoprotein payload conjugate comprising the structure of formula (1) according to claim 1.

13. 13. The glycoprotein payload conjugate of claim 12, having the following formula (2): 【Chemistry 4】 （２）

14. It has the following formula (3): 【Transformation 5】 （３） Here, z １ and Z ２ are both 1 or z １ is 1 and z ２ 13. The glycoprotein payload conjugate of claim 12, wherein is 0.

15. 13. The glycoprotein payload conjugate of claim 12, wherein the payload is a therapeutic agent selected from antimetabolites, alkylating agents, alkylating mimetics, DNA minor groove alkylating agents, anthracyclines, antibiotics, calicheamicins, antimitotic agents, topoisomerase inhibitors, proteasome inhibitors, and radioisotopes.

16. 16. The glycoprotein payload conjugate of claim 15, wherein the therapeutic agent is selected from exatecan and MMAE.

17. 16. The glycoprotein payload conjugate of claim 15, wherein the payload is a label selected from a fluorescent label, a chromophore label, an electron-dense label, a chemiluminescent label, a radioactive label, an enzymatic label, or a positron emitter.

18. 13. A method of treating cancer, comprising administering to a subject in need thereof an effective amount of the glycoprotein payload conjugate of claim 12.

19. 19. The method of claim 18, wherein the cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, ocular melanoma, gastric cancer, head and neck cancer, kidney cancer, leukemia, lung cancer, lymphoma, melanoma, oral and oropharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer and uterine cancer.

20. 20. The method of claim 19, wherein the cancer is breast cancer and / or gastric cancer.

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