Methods of making glycoprotein-drug conjugates

Abstract

The present invention provides a method for modifying a glycoprotein. The present invention also provides a method for manufacturing a glycoprotein-drug conjugate, and the conjugate produced thereby.

Description

[0001] This application is a divisional application of the invention patent application filed on December 29, 2017, with application number 201780081469.4 and entitled "Method for preparing glycoprotein-drug conjugate". Technical Field

[0002] This invention relates to a method for modifying a glycoprotein to include one or more trimannosyl cores. The invention also relates to glycoprotein-payload conjugates comprising the glycoprotein of this invention and a associated payload. Background Technology

[0003] Therapeutic protein drugs are widely used clinically, and due to their high value, high specificity, and low toxicity, some of the world's largest pharmaceutical companies are dedicated to developing such drugs for clinical trials. Most of these therapeutic proteins are monoclonal antibodies. Although some patients are satisfied with their clinical results, clinical trial data indicate that the therapeutic efficacy of some of these antibodies still needs improvement, especially in cancer treatment. To overcome this shortcoming, scientists have begun to focus on modifying these clinical antibodies through various technologies to enhance their efficacy in cancer therapy. Among these technologies, antibody-drug conjugates (ADCs) have attracted considerable attention due to their friendliness to chemistry, manufacturing, and control (CMC), user-friendliness, and lower side effects. To date, four therapeutic antibody-drug conjugates are available on the market, including Mylotarg. ® Adcetris ® Besponsa ® and Kadcyla ®Many other drugs are under development (Kubizek F1, Eggenreich B1, Spadiut O1. Protein Pept Lett. 2017, 24(8):686-695; Fischer E., Roger Schibli R. Antibodies 2015, 4, 197-224; Sapra, P., Hooper, A., O'Donnell, C. and Gerber, H.-P. Expert Opinion on Investigative Drugs 2011, 20, 1131-1180; Flygare, J., Pillow, T. and Aristoff, P., Chemical Biology and Drug Design (Chem. Biol. Drug Des.) 2013, 81, 113-121; Panowski, S.; Bhakta, S.; Raab, H.; Polakis, P. and Junutula, JR Site-specific antibody drug conjugates for cancer therapy. Monoclonal Antibodies (MAbs) 2013, 6, 34-45).

[0004] In these clinical ADCs, Kadcyla and Mylotarg are formed by random coupling of the amino group of a lysine residue with a reward or linker group, while Adcetris... ®(brentuximab vedotin (cAC10-vcMMAE, SGN-35) is a chimeric anti-CD30 monoclonal antibody, which incorporates the variable heavy chain and light chain regions of the mouse anti-CD30 antibody AC10. An average of 4 (2-8) MMAE molecules are coupled to the SGN-30 structure. The coupling sites of the MMAEs are random -SH groups on cysteine ​​residues generated through the mild reduction of interchain disulfide bonds.) The linker group consists of a thiol-reactive maleic anhydride hexanoyl spacer, a dipeptide valine-citrulline linker, and a PABC spacer (Francco JA, Cerveny CG, Meyer DL, Mixan BJ, Klussman K, Chace DF, Rejniak SX et al. cAC10-vcMMAE. Blood 2003, 4, 1458-65). While the aforementioned techniques facilitate conjugation of the loading or linker group to the antibody, both techniques struggle to control the drug-to-antibody ratio (DAR) of the conjugate due to the multiple lysine sequences and the problem of optimal reduction of cysteine ​​residues in the antibody. These phenomena often lead to heterogeneity in antibody products and induce CMC problems. Some literature even points out that this type of first-generation non-site-specific ADC has drawbacks such as PK and immunogenicity.

[0005] To address these shortcomings of first-generation ADCs, site-specific ADC platforms have been developed, including SMART tags, non-natural amino acids (any tyrosine), therapeutic sortases, and thio-bridges. As expected, these technologies can produce homogeneous ADC products by engineering specific sites or domains in the parent antibody. For example, thio-bridge technology links the linker group and the payload to a partially reduced disulfide bond of the antibody. SMART tags are a technique that mutates adjacent sequences of the antibody to substrate sequences of bacterial oxidases. The resulting product, along with formaldehyde, is used as the linker group to the payload. As anticipated, these second-generation ADC technologies can produce ADC products with unique DAR and high homogeneity. However, due to mutations in natural antibodies, ADC products may have pharmacokinetic and immunogenicity issues. Currently, conjugation of the payload to the antibody via N-glycosylation has attracted considerable attention due to the successful development of antibody glycosylation engineering.

[0006] All naturally occurring IgG and recombinant antibodies possess an amino acid asparagine (Asn297) at position 297 in each of the CH2 constant regions of the heavy chain, which is an N-glycosylation site. Through glycosylation and post-modification in mammalian cells, two biphasic glycan moieties are formed via N-glycosylation on IgG, each moiety consisting essentially of at least seven sugar moieties having the following formula:

[0007] ,

[0008] Among them, the first GlcNAc (GlcNAc) 1 ) are respectively bound to the Asn297 of the antibody and the second GlcNAc (GlcNAc 2 ), and optionally bonded to trehalose (Fuc); GlcNAc 2 Further bonding to the first mannose (Man) 1 ); second and third mannose (Mann) 2 and Man 3 ) respectively bonded to Man 1 The α-1,3 and α-1,6 positions; and the other two GlcNAc sugars (GlcNAc 3 and GlcNAc 4 ) respectively bonded to Man 2 and Man 3 The β-1,2 position. Antibodies containing the glycan moiety and trehalose are denoted as G0F; however, when the trehalose moiety is absent, the antibody is G0. (T. Shantha Raju monoclonal antibody. May 1, 2012; 4(3): 385-391). When GlcNAc 3 or GlcNAc 4 When bound to an additional galactose, the antibody is designated as a G1F / G1 antibody. When the terminal GlcNAc sugars in the glycan moiety of the antibody are bound to two additional galactoses respectively, the antibody is designated as a G2F / G2 antibody. Antibodies produced by mammalian cells generally include G0F (greater than about 40%), G1F (about 30%-40%), and G2F (less than 1%), as well as very small amounts of G1F / G1 and G2F / G2 bound to sialic acid.

[0009] Because engineering the branch sites in N297 glycans can maintain structural integrity and produce some functional diversity (such as antibody ADCC, half-life, and CDC), several N297 glycosylation-engineered ADC platforms have been developed, and some of these products are in clinical trials. WO 2014 / 164534 A2, WO 2014 / 065661 A1, WO 2015 / 032899 A1, WO 2015 / 057064 A1, WO 2015 / 157446 A1, US 8716033 B2, US 7416858 B2, EP2753752 B3 and a review article (Bioconjug Chem.; Nov 18, 2015; 26(11):2070-5) have disclosed many modified glycan moieties for antibody-drug conjugation. However, these techniques cannot adequately control the drug-antibody ratio (DAR) in antibody-drug conjugates (ADCs) and cannot achieve payload diversity. Therefore, there is a need in the art to control the drug-antibody ratio of ADCs and improve payload diversity. This invention meets this need and provides other benefits. Summary of the Invention

[0010] One aspect of the present invention provides a method for manufacturing a glycoprotein-loaded conjugate comprising the structure of formula (1):

[0011] .

[0012] Another aspect of the present invention provides a method for manufacturing a glycoprotein-loaded conjugate comprising the structure of formula (5):

[0013] .

[0014] Another aspect of the present invention provides a method for manufacturing a glycoprotein-loaded A / B conjugate comprising the structure of formula (7):

[0015] .

[0016] Another aspect of the present invention provides glycoprotein-receptor conjugates that can be obtained by the method of the present invention. Attached Figure Description

[0017] Figure 1 This demonstrates a one-step and sequential strategy for manufacturing trimannosyl antibody-drug conjugates.

[0018] Figure 2 The results of reduced mass chromatographic analysis of Example 1 are shown. The results indicate that treatment with β1,4-galactosidase and neuraminidase produced G0F / G0 type Herceptin.

[0019] Figure 3 The results of reduced mass chromatographic analysis for Example 2 are shown. The results indicate that G0F / G0 type hepatoprotective antibody is converted to a trimannosyl core antibody via N-acetylglucosaminease S.

[0020] Figure 4 The results of reduced mass chromatographic analysis of Example 3 are shown. The results show that GlcNAC is coupled to one arm of the terminal mannose at each point of the trimannose-based chromatographic assay via MGAT-1.

[0021] Figure 5 The results of reduced mass chromatographic analysis of Example 4 are shown. The results show that trimannosylhexamine is converted to G0 / G0F type hexamine via MGAT-2 and MGAT-1.

[0022] Figure 6 The results of the reduced mass chromatographic analysis of Example 6 are shown. The results show that MGAT-1 couples UDP-GlcNAz to one arm of the terminal mannose at each of the trimannosylhexaenoic acid sites.

[0023] Figure 7A The results of the reduced mass chromatographic analysis in Example 7 are shown; while Figure 7B The results of the intact mass chromatographic analysis of Example 7 are shown. The results show that MGAT-1 and MGAT-2 couple UDP-GlcNAz to trimannosylhexamethine to produce G0F / G0 type hexamethine, in which four azido groups are in the terminal N-acetylglucosamine.

[0024] Figure 7A and Figure 7B The results of the complete mass chromatographic analysis of Example 7 are shown. The results show that MGAT-1 and MGAT-2 couple UDP-GlcNAz to trimannosylhexamethine to produce G0F / G0 type hexamethine, in which four azido groups are in the terminal N-acetylglucosamine.

[0025] Figure 8 The results of the reduced-mass chromatographic analysis of Example 8 are shown. The results demonstrate that trimannosylhexamethonium is not a substrate for conjugating GlcNAz to MGAT-2.

[0026] Figure 9A The results of the reduced mass chromatographic analysis in Example 9 are shown; while Figure 9B The results of the complete mass chromatographic analysis of Example 9 are shown. The results of the figure show that DBCO-(PEG)4-DM1 is coupled with trimannosylhexamethonium-4GlcNAz via a click chemistry reaction to produce a hexamethonium ADC with DAR4.

[0027] Figure 10 The results of the reduced mass chromatographic analysis of Example 10 are shown. The results of the figure show that the first drug is coupled to each arm of the heavy chain of the trimannosyl-2GlcNAz anticancer antibody via MGAT-1 and DBCO-(PEG)4-DM1.

[0028] Figure 11 The results of the reduced-mass chromatographic analysis of Example 11 are shown. The results of the figure show that the second GlcNAz couples α-6-mannose to the arms of the heavy chain of the trimannosylhexamine-2 (GlcNAc-triazole-DBCO-(PEG)4-DM1) ADC via MGAT-2. This indicates that MGAT-2 is a substrate-flexible enzyme and converts UDP-GlcNAz into a large-functional antibody, such as trimannosylhexamine-2 (GlcNAc-triazole-DBCO-(PEG)4-DM1), producing an intermediate for the dual-loaded ADC product.

[0029] Figure 12 The results of the complete mass chromatographic analysis of Example 12 are shown. The results in the figure show that the addition of DBCO-(PEG) to the intermediate trimannosylhexamethonol-2GlcNAz-2 (GlcNAc-triazole-DBCO-(PEG)4-DM1) generated from the product of Example 11... 12 -MMAE, producing a DAR4 ADC product with one MMAE and one DM1 on each arm of the antibody.

[0030] Figure 13 The results of the full-scale chromatographic analysis of Example 13 are shown. The results show that by adding DBCO-MMAF to the intermediate trimannosylhexamethonol-2GlcNAz-2 (GlcNAc-triazole-DBCO-(PEG)4-DM1) generated from the product of Example 11, a DAR4 ADC hexamethonol product with one MMAF and one DM1 on each arm of the antibody was produced.

[0031] Figure 14 The binding ELISA of Kadcyla and trimannosylhexapine-4 (GlcNAc-triazole-DBCO-(PEG)4-DM1) was shown as described in Example 14. The results indicated that there was no significant difference in Kd between Kadcyla and the product of trimannosylhexapine-4 (GlcNAc-triazole-DBCO-(PEG)4-DM1).

[0032] Figure 15A and Figure 15BThe results of reduced-mass chromatographic analysis of the trimannosyl core trastuzumab antibody in Example 16 are shown. The results show that trimannosyl trastuzumab and trimannosyl anti-TMCC3 were generated through mammalian cell lines.

[0033] Figure 16A , Figure 16B and Figure 16C The results of reduced mass chromatographic analysis and whole mass chromatographic analysis of Example 17 are shown. The results in the figure show that the mammalian cells that produced trimannosyltrastuzumab produced trimannosyltrastuzumab-4 (GlcNAc-triazole-DBCO-(PEG)4-DM1). Detailed Implementation

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing this invention, preferred methods and materials are described hereafter. All disclosures and patents specifically mentioned herein are incorporated herein by reference for all purposes, including descriptions and publications of chemicals, cell lines, vectors, animals, instruments, statistical analyses, and methods reported in the disclosures that may be used in conjunction with this invention. All references cited in this specification are considered as indications of the level of skill in the art to which this invention pertains.

[0035] abbreviation

[0036]

[0037] It should be noted that, as used herein and in the appended claims, the singular forms “a (a / an)” and “the” include a plural of indicators unless the context clearly specifies otherwise. Similarly, the terms “a (a or an),” “one or more,” and “at least one” are used interchangeably herein. It should also be noted that the terms “comprising,” “including,” and “having” are used interchangeably.

[0038] Typically, ranges are expressed herein as from “about” a particular value and / or to “about” another particular value. When such ranges are expressed, embodiments include ranges from one particular value and / or to another particular value. Similarly, when values ​​are expressed as approximate values ​​using the term “about,” it should be understood that the particular value forms another embodiment. It will be further understood that each endpoint of a range is meaningful whether it is related to or unrelated to the other endpoint. As used herein, the term “about” means ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, or ±0.25%.

[0039] When referring to formulation components, the term "pharmaceutical" is intended to cover not only the specific molecular entity but also its pharmaceutically acceptable analogues, including (but not limited to) salts, esters, amides, prodrugs, conjugates, active metabolites, and other such derivatives, analogues, and related compounds.

[0040] As used herein, the general term “sugar” refers to monosaccharides such as glucose (Glc), galactose (Gal), mannose (Man), and trehalose (Fuc), as well as monosaccharide derivatives such as amino sugars and sugar acids, such as glucosamine (GlcN), galactosamine (Galn), N-acetylglucosamine (GlcNAc), N-azidoacetylglucosamine (GlcNAZ), N-acetylglucosamine (GlaNAc), N-acetylneuraminic acid (NeuNAc), N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA), and iduronic acid (IdoA).

[0041] As used herein, the term "protein" can include polypeptides having a natural amino acid sequence, as well as variants and modified forms, regardless of their origin or mode of preparation. A protein having a natural amino acid sequence is a protein having the same amino acid sequence as obtained from nature. Such natural sequence proteins can be isolated from nature or prepared using standard recombinant and / or synthetic methods. Natural sequence proteins explicitly encompass naturally occurring truncated or soluble forms, naturally occurring variant forms (e.g., alternating splice forms), naturally occurring allelic variants, and forms including those with post-translational modifications. Natural sequence proteins include proteins following post-translational modifications of some amino acid residues, such as glycosylation, phosphorylation, or other modifications.

[0042] As used herein, the term "glycoprotein" refers to a protein comprising one or more monosaccharide or oligosaccharide chains covalently bonded to the protein. Glycans may be attached to a hydroxyl group of the protein (O-linked glycosyl group), such as a hydroxyl group attached to serine, threonine, tyrosine, hydroxylysine, or hydroxyproline; or to an amide group on a protein such as aspartic acid or arginine (N-glycoprotein); or to a carbon group on a protein such as tryptophan (C-glycoprotein). Glycoproteins may contain more than one glycan, may contain combinations of one or more monosaccharides and one or more oligosaccharides, and may contain combinations of N-linked, O-linked, and C-linked glycans. Examples of glycoproteins include ligands specific to cell surface antigens, prostate-specific membrane antigens, Candida antarctica lipase, gp41, gp120, erythropoietin (EPO), antifreeze proteins, and antibodies.

[0043] Antibodies are proteins produced by the immune system that recognize and bind to specific antigens. The term "antibody" is used in its broadest sense and particularly includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and double-chain and single-chain antibodies. The term "antibody" is also intended to include human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to cancer antigens. The term "antibody" is intended to include whole antibodies, as well as antibody fragments, such as antibody Fab fragments, F(ab')2, Fv fragments, or Fc fragments from lysed antibodies, scFv-Fc fragments, microantibodies, bifunctional antibodies, or scFv. Furthermore, the term includes genetically engineered derivatives of antibodies. Antibodies, antibody fragments, and genetically engineered antibodies can be obtained by methods known in the art. Suitable commercially available antibodies include (but are not limited to) abciximab, rituximab, basiliximab, palivizumab, infliximab, trastuzumab, alemtuzumab, adalimumab, tositumomab-1131, cetuximab, and ibrituximab. Tiuxetan), omalizumab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, pegylated certolizumabpegol, golimumab, canakinumab, catumaxomab, ustekinumab, tocilizumab, ofatumumab, denosumab, belimumab, ipilimumab, and bentuximab.

[0044] Various expression systems, including prokaryotic and eukaryotic expression systems, can be used to generate antibodies. In some embodiments, the expression system is a mammalian cell expression system, such as a fusion tumor; or a CHO cell expression system. Many such systems are widely available from commercial suppliers. In antibodies containing V H and V L In the example of the area, V H and V LThe region can be expressed using a single vector, such as a bicistronic expression unit; or expressed under the control of different promoters. In other embodiments, V H and V L Regions can be expressed using individual carriers. As described in this paper, V... H and V L The region may optionally contain methionine at the N-terminus.

[0045] Genes encoding the heavy and light chains of relevant antibodies can be cloned from cells; for example, genes encoding monoclonal antibodies can be cloned from fusion tumors and used to produce recombinant monoclonal antibodies. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be obtained from fusion tumors or plasma cells. Random combinations of heavy and light chain gene products generate large pools of antibodies with different antigen specificities.

[0046] The techniques used to manufacture single-chain antibodies or recombinant antibodies (US Patent Nos. 4,946,778 and 4,816,567) are applicable to the manufacture of antibodies against the peptides of the present invention. In addition, humanized antibodies or human antibodies can be expressed by gene transfection of mice or other organisms such as other mammals (see, for example, U.S. Patent 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 Lombberg and Huszar, International Review of Immunology 13:65-93 (1995).

[0047] As used in this article, "GlcNAc" 1 “GlcNAc” 2 “GlcNAc” 3 "and "GlcNAc 4 "These represent GlcNAc sugars at different locations in the tactile glycan region."

[0048] As used in this article, " "" indicates a trimannosyl structure containing three mannoses, where the first mannose (Mann) 1 ) is linked to GlcNAc sugar; while the second and third mannose (Man) 2 and Man 3 ) are linked to Man via α-1,3 and α-1,6 glycosidic bonds, respectively. 1 .

[0049] As used in this article, "-(Fuc)" 0-1 "This indicates that trehalose is present arbitrarily, and when present, there is only one trehalose.

[0050] As used in this article, "-(CH2)" 0-8 -” indicates that -CH2- may or may not be present, and when present, it can independently be 1, 2, 3, 4, 5, 6, 7 or 8 -CH2- groups.

[0051] One aspect of the present invention provides a method for manufacturing a glycoprotein-loaded conjugate comprising the structure of formula (1):

[0052] ,

[0053] The method includes the following steps:

[0054] (i) To make a glycoprotein containing a polysaccharide having formula (2)

[0055]

[0056] Reaction with β-N-acetylglucosaminease produces a modified glycoprotein containing a trimannosyl core of formula (3).

[0057] ;

[0058] (ii) To make the modified glycoprotein containing the trimannosyl core of formula (3) with UDP-GlcNAc-(CH2) 0-8 -R reacts in the presence of mannosyl (α-1,3-)-glycoprotein β-1,2-N-acetylglucosamine aminotransferase and mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine aminotransferase, where R is an azide, keto, or aldehyde, resulting in two GlcNAc-(CH2) groups. 0-8 -R sugars are bonded to Man. 2 and Man 3 The β-1,2 positions of each, thereby forming the glycan moiety of formula (4).

[0059] ;

[0060] as well as

[0061] (iii) React two coupling agent-linking groups-loaded with the glycan portion of formula (4), wherein the two coupling agent-linking groups-loaded are the same or different, to produce a glycoprotein-loaded conjugate containing the structure of formula (1).

[0062] In some embodiments, the two payloads are different, and the payloads may be randomly attached to any of the four Man-GlcNAc structures in the glycoprotein.

[0063] Another aspect of the present invention provides a method for manufacturing a glycoprotein-loaded conjugate comprising the structure of formula (5):

[0064] ,

[0065] The method includes the following steps:

[0066] (i) To make a modified glycoprotein containing a trimannosyl core as defined in formula (3) above, and UDP-GlcNAc-(CH2). 0-8 -R reacts in the presence of mannosyl (α-1,3-)-glycoprotein β-1,2-N-acetylglucosamine aminotransferase, where R is an azide, keto, or aldehyde, resulting in GlcNAc-(CH2). 0-8 -R sugar bonds to Man 2 At the β-1,2 position, and thereby form a glycoprotein containing the glycan moiety of formula (6).

[0067] ;

[0068] as well as

[0069] (ii) React the coupling agent-linking group-loaded glycoprotein containing the glycan moiety of formula (6) to produce a glycoprotein-loaded conjugate containing the structure of formula (5).

[0070] In some embodiments, in order to precisely control the attachment location of the payload, a glycoprotein-payload A / B conjugate comprising the structure of formula (7) can be generated by the following steps:

[0071]

[0072] (i) To make a modified glycoprotein containing a trimannosyl core as defined in formula (3) above, and UDP-GlcNAc-(CH2). 0-8 -R reacts in the presence of mannosyl (α-1,3-)-glycoprotein β-1,2-N-acetylglucosamine aminotransferase, where R is an azide, keto, or aldehyde, resulting in GlcNAc-(CH2). 0-8 -R sugar bonds to Man 2The β-1,2 positions are used to form a glycoprotein containing a glycan moiety as defined above in formula (6);

[0073] (ii) React the coupling agent-linking group-loaded A with a glycoprotein containing the glycan moiety of formula (6) to produce a glycoprotein-loaded A conjugate containing the structure of formula (8).

[0074] ;

[0075] (iii) Make a glycoprotein-loaded A conjugate containing the structure of formula (8) with UDP-GlcNAc-(CH2). 0-8 -R reacts in the presence of mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine aminotransferase, where R is an azide, keto, or aldehyde, resulting in GlcNAc-(CH2). 0-8 -R sugar bonds to Man 3 At the β-1,2 position, thereby forming a glycoprotein containing a glycan-loaded A moiety having formula (9).

[0076] ;

[0077] as well as

[0078] (iv) React the coupling agent-linking group-loaded B with a glycoprotein containing a glycan-loaded A moiety having formula (9) to produce a glycoprotein-loaded A / B conjugate containing the structure of formula (7).

[0079] The payload A may be the same as or different from the payload B.

[0080] The glycoproteins used herein can be obtained, for example, through solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant manufacturing. Regarding recombinant manufacturing, one or more polynucleotides encoding the glycoprotein are isolated and inserted into a vector for further selection and / or expression in host cells. Such polynucleotides can be readily isolated and sequenced using known procedures. Expression vectors containing the coding sequence of the glycoprotein can be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / gene recombination. See, for example, the techniques described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (NY) (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, New York (1989).

[0081] As used herein, β-N-acetylglucosaminease represents a family of glycosidases that catalyze the hydrolysis of β-N-acetylglucosamine residues from oligosaccharides. Many β-N-acetylglucosamineases have been found to catalyze the hydrolysis of various types of β-glycosidic bonds. In a preferred embodiment, the β-N-acetylglucosaminease may be an exoglycosidase capable of hydrolyzing the β1-2 bond between the terminal acetylglucosamine residue and the N-glycan in a glycoprotein. Exo-β-N-acetylglucosaminease variants can be obtained from various sources, such as Streptococcus spp. (…). Streptococcus spp .) and Guan Dao Dou ( Canavalia ensiformis ).

[0082] According to the present invention, the mannosyl (α-1,3-)-glycoprotein β-1,2-N-acetylglucosamine aminotransferase (MGAT1; GnT-I; EC: 2.4.1.101) transfers N-acetyl-D-glucosamine from UDP-GlcNAc to terminal mannose, which is linked to another sugar moiety or glycan via an α1-3 glycosidic bond. The bond between GlcNAc transferred by MGAT1 and α3-mannose is a β1-2 glycosidic bond. MGAT1 has been found to be widely expressed in eukaryotes because it is an essential enzyme for the biosynthesis of heterozygous and complex N-glycans in the Golgi apparatus.

[0083] According to the present invention, the mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine aminotransferase (MGAT2; GnT-II; EC 2.4.1.143) transfers N-acetyl-D-glucosamine from UDP-GlcNAc to terminal mannose, which is linked to another sugar moiety or glycan via an α1-6 glycosidic bond. The bond between GlcNAc transferred by MGATII and α6-mannose is a β1-2 glycosidic bond. MGAT2 has been found to be widely expressed in eukaryotes because it is an essential enzyme for the biosynthesis of complex N-glycans in high matrix organisms.

[0084] In some embodiments, the reaction between a glycoprotein comprising a polysaccharide having formula (2) and β-N-acetylglucosaminease is carried out in a mammalian cell culture. In the mammalian cell culture, a mammalian cell line is cultured in a medium under conditions suitable for the expression of the glycoprotein and β-N-acetylglucosaminease, the mammalian cell line comprising: a first polynucleotide encoding a glycoprotein comprising a polysaccharide having formula (2) and a second polynucleotide encoding β-N-acetylglucosaminease. Examples of mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7), human embryonic kidney (293 or 293T cells), baby hamster kidney cell (BHK), mouse sertoli cell (TM4 cells), monkey kidney cell (CV1), African green monkey kidney cell (VERO-76), human cervical cancer cell (HELA), canine kidney cell (MDCK), buffalo rat hepatocyte (BRL 3A), human lung cell (W138), human hepatocyte (Hep G2), mouse mammary tumor cell (MMT 060562), TRI cells, MRC 5 cells, FS4 cells, Chinese hamster ovary (CHO) cells, and myeloma cell lines such as YO, NSO, P3X63, and Sp2 / 0.

[0085] In some embodiments of the present invention, the glycoprotein is an antibody or a fragment thereof. The antibody or fragment thereof may be an antibody Fab fragment, F(ab')2, Fv fragment, or Fc fragment, scFv-Fc fragment, microantibody, bifunctional antibody, or scFv derived from a lysed antibody. In a preferred embodiment, the antibody is trastuzumab, anti-TMCC3 antibody, or anti-TMCC3 antibody.

[0086] In one embodiment of the present invention, when R is an azide group (is this the normal name?) and the coupling agent is an alkynyl group, the coupling agent-linking group-reward is loaded with -GlcNAc-(CH2). 0-8The -R group reacts via a click reaction to form -GlcNAc-(CH2). 0-8 -Linking group-reward loading (Angewandte Chemie International Edition. 40 (11): 2004-2021; and Australian Journal of Chemistry. 60 (6): 384-395). In another embodiment, when R is a ketone or aldehyde and the coupling agent is an amino group, the coupling agent-linking group-reward loading is combined with -GlcNAc-(CH2). 0-8 The -R group reacts via reductive amination to form -GlcNAc-(CH2). 0-8 -Linking group-reward loading (J. Org. Chem., 2010, 75, 5470-5477; and Synthesis, 2011, 490-496). In another embodiment, when R is a ketone or aldehyde and the coupling agent is β-arylethylamino, the coupling agent-linking group-reward loading is combined with -GlcNAc-(CH2). 0-8 The -R group reacts via the Pictet-Spengler reaction to form -GlcNAc-(CH2). 0-8 -Linking group-reward (Bioconjugate Chem., 2013, 24 (6), pp. 846-851).

[0087] In some embodiments, when a glycoprotein-load conjugate is used to treat an individual's disease, the load may be a therapeutic agent. The therapeutic agent may be a cell inhibitor or cytotoxic agent, or an isotope chelator carrying a corresponding radioactive isotope. Examples of cell inhibitors or cytotoxic agents include (but are not limited to) antimetabolites such as fluorouracil (5-FU), fluorouridine (5-FUdR), methotrexate, folate, hydroxyurea, thioguanine (6-TG), mercaptopurine (6-MP), cytarabine, pentostatin, fludarabine phosphate, and cladribine. (2-CDA), asparagine, gemcitabine, capecitabine, azathioprine, cytosine methotrexate, trimethoprim, pyrimethamine, or pemetrexed; alkylating agents (e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, dacarbazine, mitomycin C). C) Cyclophosphamide, mechlorethamine, uramustine, dibromomannitol, tetranitrate, procarbazine, altretamine, mitozolomide, or temozolomide; alkylating agents (e.g., cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, or triplatin); DNA minor groove alkylating agents (e.g., duocarmycin, such as CC-1065, and any analogues or derivatives thereof; pyrrolobenzodiazapene or any analogues or derivatives thereof);Anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, or valrubicin); antibiotics (e.g., dactinomycin, bleomycin, mithramycin, antramycin, streptozotocin, gramicidin D, mitomycin (e.g., mitomycin C)); calicheamicin; antimitotic agents (including, for example, maytansinoids (e.g., DM1, DM3, and DM4), oligristatins (including, for example, monomethyloligristatin E (MMAE) and monomethyloligristatin F)). MMAF, dolastatin, cryptophycin, vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinorelbine), taxanes (e.g., paclitaxel, docetaxel, or novel taxanes), tubulysin, and colchicine); topoisomerase inhibitors. (e.g., irinotecan, topotecan, camptothecin, etoposide, teniposide, amsacrine, or mitoxantrone)); HDAC inhibitors (e.g., vorinostat, romidepsin, chidamide, panobinostat, or belinostat)); proteasome inhibitors (e.g., ... ); and radioactive isotopes, such as At; 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi212 or 213 P 32 Radioactive isotopes of Lu, including Lu 177 Examples of isotope chelating agents include (but are not limited to) ethylenediaminetetraacetic acid (EDTA), diethylenetriamine-N,N,N',N",N"-pentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), 1,4,7,10-tetra(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane (THP), triethylenetetramine-N,N,N',N",N"',N"'-hexaacetic acid (TTHA), 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetra(methylenephosphonate) (DOTP), and mercaptoacetyltriglycine (MAG3).

[0088] In some embodiments, when detection is performed using a glycoprotein-loaded conjugate, the load may be a label. Labels include (but are not limited to) labels or portions that are directly detected (such as fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and portions that are indirectly detected, such as enzymes or ligands, through enzymatic reactions or molecular interactions. Exemplary labels include (but are not limited to) the radioisotope P. 32 C 14 I 125 H 3 and I 131 ; fluorophores, such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone; luciferases, such as firefly luciferase and bacterial luciferase; luciferin; 2,3-dihydrophthalazinedione; horseradish peroxidase (HRP); alkaline phosphatase; β-galactosidase; glucoamylase; lysozyme; sugar oxidases, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase; heterocyclic oxidases, such as uricase and xanthine oxidase, coupled with enzymes that oxidize dye precursors using hydrogen peroxide (such as HRP, lactoperoxidase, or microperoxidase); biotin / avidin; spin labeling; phage labeling; stable free radicals and similar labels. In another embodiment, the label is a positron emitter. Positron emitters include (but are not limited to) Ga 68 F 18 Cu 64 Y 86 ,Br 76Zr 89 and I 124 .

[0089] In some embodiments, the linking group has a functional group capable of reacting with an electrophilic group present on the glycoprotein. Examples of such electrophilic groups include (but are not limited to) aldehydes and ketone carbonyl groups. In some embodiments, the heteroatom of the reactive functional group of the linking group can react with the electrophilic group on the glycoprotein and form a covalent bond with the glycoprotein unit. Non-limiting examples of such reactive functional groups include (but are not limited to) acylhydrazides, oximes, amino groups, hydrazine, thiosemicarbazone, hydrazide carboxylates, and aryl acylhydrazides.

[0090] In some embodiments, the coupling agent has functional groups capable of reacting with electrophilic groups present on the glycoprotein. Examples of such electrophilic groups include (but are not limited to) azide groups, aldehydes, and ketones. In some embodiments, the heteroatoms of the reactive functional groups of the coupling agent can react with the electrophilic groups on the glycoprotein and form covalent bonds with the glycoprotein units. Non-limiting examples of such reactive functional groups include (but are not limited to) alkynes, dibenzocyclooctylene, acylhydrazides, oximes, amino groups, hydrazides, thioureas, hydrazide carboxylates, and aryl acylhydrazides.

[0091] In some embodiments, the linking group has a functional group capable of linking the coupling agent and the loaded substance. Examples of such linking groups include (but are not limited to) non-cleavable linking groups and cleavable linking groups. In some embodiments, non-cleavable linking groups include (but are not limited to) straight-chain or branched alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, alkoxy, acyl, alkylamino, or arylamino groups having 2 to 20 carbon atoms. In some embodiments, cleavable linking groups include (but are not limited to) linking groups containing disulfide bonds, acid-labile linking groups, light-labile linking groups, peptidase-labile linking groups, and esterase-labile linking groups.

[0092] The following examples illustrate certain embodiments of the present invention, but should not be construed as limiting the scope of the invention. The scope of the invention includes, for example, the following: Figure 1 The diagram shows a one-step or sequential method for generating homogeneous ADCs with one or two loadings from trimannosyl core antibodies.

[0093] Example 1. Preparation of Heleping antibody using β1,4-galactosidase and neuraminidase

[0094] To remove the galactose and sialic acid fractions of the N-glycan from the zigapascal antibody (Roche Inc.), 10 mg of zigapascal antibody was treated at 37°C with 20 μl of β1,4-galactosidase (NEB, P0745L, 8 units / μl) and 5 μl of α2-3,6,8-neuronodilase (NEB, P0720L, 50 units / μl) in 1×glucose buffer (NEB, total volume 1 ml) for 24 hours. An additional 10 μl of β1,4-galactosidase (NEB, P0745L, 8 units / μl) was added to the reaction mixture, and the reaction was continued at 37°C for another 24 hours to obtain G0F / G0 antibody samples. The antibody samples were purified using rProtein ASepharose Fast Flow (GE Healthcare, 17-1279-02). After purification, the antibody samples were subjected to reduced-mass chromatographic analysis. Figure 2 The results shown reveal that the majority of the antibody in the sample was G0F (a heavy chain with a molecular weight of 50,600 Da) and only a small amount was G0 (trehalose-free; a heavy chain with a molecular weight of 50,451 Da).

[0095] Example 2. He'aiping converted into a trimannosyl core antibody.

[0096] 10 mg of G0F / G0 Healopin antibody from Example 1 was treated at 37°C with 20 μl of β-N-acetylglucosaminease S (NEB, P0744L, 4 units / μl) in 1×glucose buffer 1 (NEB, total volume 1 ml) for 24 hours. 10 μl of β-N-acetylglucosaminease S (NEB, P0744L, 4 units / μl) was added to the reaction mixture, and the reaction was continued at 37°C for another 24 hours to obtain a digested antibody sample. The digested antibody sample was purified using rProtein A Sepharose Fast Flow (GE Healthcare, 17-1279-02). After purification, the antibody sample was analyzed by reduced-mass chromatography. Figure 3 The results shown reveal that a trimannosyl core hemodialysis antibody with a heavy chain of 50,194 Da was obtained, and almost all G0F and G0 hemodialysis antibodies were converted into trimannosyl core antibodies. This indicates that β-N-acetylglucosaminease S can efficiently convert G0F and G0 antibodies into antibodies with a trimannosyl core.

[0097]

[0098] Example 3. GlcNAc and α-3-mannose were coupled to one arm of each heavy chain of the trimannosyl core hepatoprotective antibody via mannosyl (α-1,3-)-glycoprotein β-1,2-N-acetylglucosamine aminotransferase (MGAT-1; GnT-1).

[0099] The trimannosyl core hepatoprotective antibody (40 μg) from Example 2 was incubated with UDP-GlcNAc (final concentration 2.5 mM) (Sigma, U4375) in 80 μl of 1× buffer SP (25 mM 4-morpholinoethanesulfonic acid (MES), 10 mM MnCl2, pH 6.5) at 37°C for 16 h in the presence of MGAT-1 (0.15 μg; R&D, 8334-GT). The product was analyzed by reduced-mass chromatographic analysis. Figure 4 The results shown indicate that, compared to the trimannosyl core antibody with a heavy chain of 50,195 Da, an antibody product with an additional GlcNAc (molecular weight 203 Da) and a molecular weight of 50,398 Da was obtained. This demonstrates that MGAT-1 transfers only one N-acetylglucosamine to its substrate protein.

[0100]

[0101] Example 4. The trimannosyl-based anti-cancer antibody was converted to G0F / G0 anti-cancer antibody using MGAT-1 and mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine aminotransferase (MGAT-2; GnT-2).

[0102] The trimannosyl core hepatoprotective antibody (40 μg) and UDP-GlcNAc (final concentration 2.5 mM) (Sigma, U4375) from Example 2 were incubated in 80 μl of 1× buffer SP (25 mM MES, 10 mM MnCl2, pH 6.5) at 37°C for 16 h in the presence of MGAT-1 (0.15 μg) and MGAT-2 (0.1 μg). After incubation, the reaction products were analyzed by reduced-mass chromatography. Figure 5 The results shown indicate that, compared to a trimannosyl core hepatoprotective antibody with a heavy chain of 50,194 Da, a G0F antibody product with a small amount of G0 was obtained, whose heavy chain additionally contains two GlcNAc (203 Da × 2) with a total molecular weight of 50,600 Da. These results suggest that trimannosyl core antibodies can be converted into G0 / G0F antibodies by combining MGAT-1, MGAT-2, and N-acetylglucosamine.

[0103]

[0104] Example 5. Trimannosyl core anticancer antibody is not a substrate of MGAT-2.

[0105] MGAT-2 only transfers GlcNAc sugar from UDP-GlcNAc to α(1,6)mannose in the trimannosyl core if the trimannosyl core already has a GlcNAc sugar linked to α(1,3)mannose. In other words, if there is no GlcNAc sugar linked to α(1,3)mannose in the trimannosyl core, MGAT-2 will not be able to transfer GlcNAc sugar to α(1,3)mannose or α(1,6)mannose. To confirm these observations, trimannosyl core hepatoprotective antibody (40 μg) from Example 2 was incubated with UDP-GlcNAc (2.5 mM) in 80 μl of 1× buffer SP (25 mM MES, 10 mM MnCl2, pH 6.5) at 37°C for 16 h in the presence of MGAT-2 (0.1 μg). The product was analyzed by reduced mass chromatography. In mass spectrometry, the molecular weight of the trimannosyl core antibody showed no significant change (data not shown). This result indicates that the trimannosyl core is not a substrate of MGAT-2, and that MGAT-2 requires the conversion product of MGAT-1 to generate G0F / G0 type antibodies.

[0106]

[0107] Example 6. GlcNAz was conjugated to the terminal α-3-mannose of one arm of each heavy chain of the trimannosyl core hepatopanol antibody via MGAT-1.

[0108] The trimannosyl core hepatoprotective antibody (40 μg) from Example 2 was incubated with UDP-GlcNAz (1 mM) (R&D, ES104-100) in 80 μl of 1× buffer SP (20 mM Tris, 10 mM MnCl2, pH 6.5) at 37°C for 16 hours in the presence of MGAT-1 (0.25 μg; R&D, 8334-GT). The product was analyzed by reduced-mass chromatographic analysis. Figure 6 The results shown indicate that, compared to the trimannosyl core hecaramin antibody with a heavy chain of 50,195 Da, an antibody product with an additional GlcNAz (molecular weight 244 Da) and a molecular weight of 50,438 Da was obtained. This result suggests that UDP-GlcNAz is one of the substrates of MGAT-1, and that GlcNAz can be linked via MGAT-1 to the α-3-mannose in each arm of the heavy chain of the trimannosyl core hecaramin antibody, forming the trimannosyl hecaramin-2GlcNAz antibody.

[0109]

[0110] Example 7. Conjugation of UDP-GlcNAz to trimannosyl core hepatopanol antibody via MGAT-1 and MGAT-2 to produce trimannosyl hepatopanol-4GlcNAz.

[0111] The trimannosyl core hepatoprotective antibody (2 mg) from Example 2 was incubated with UDP-GlcNAz (1 mM) in 800 μl of 1× buffer SP (25 mM MES, 10 mM MnCl2, pH 6.5) at 37°C for 16 hours in the presence of rabbit MGAT-1 (25 μg) and rat MGAT-2 (10 μg). After incubation, the reaction products were analyzed by reduced mass chromatography and intact mass chromatography, respectively. Figure 7A The reduced-mass chromatographic results shown indicate that, compared to the trimannosyl core hecarbaz antibody with a heavy chain of 50,194 Da, a trimannosyl hecarbaz-4GlcNAz antibody product was obtained, in which the heavy chain contains two GlcNAz molecules (molecular weight 244 Da × 2 = 488) with each heavy chain having a molecular weight of 50,680 Da. This result indicates that GlcNAz is coupled to the α-3-mannose and α-6-mannose of each heavy chain of the trimannosyl core hecarbaz antibody via MGAT-1 and MGAT-2. This result was further confirmed by whole-mass chromatography. Figure 7B The results shown indicate that, compared to the 147,237 Da fully trimannosyl core hecarbaz antibody, a G0F trimannosyl hecarbaz-4GlcNAz antibody product was obtained, containing four GlcNAz molecules (molecular weight 244 Da × 4 = 976 Da) with a molecular weight of 148,213 Da. Our results further demonstrate that UDP-GlcNAz is one of the substrates for MGAT-1 and MGAT-2, and that we were able to successfully synthesize the intermediate tetraazido antibody using our one-step hypothesis.

[0112]

[0113] Example 8. Trimannosyl core anti-cancer antibody is not a substrate for MGAT-2 conjugated with GlcNAz.

[0114] The trimannosyl core hepatoprotective antibody (40 μg) from Example 2 was incubated with UDP-GlcNAz (1 mM) (R&DES104-100) in 80 μl of 1× buffer SP (25 mM MES, 10 mM MnCl2, pH 6.5) in the presence of rat MGAT-2 (0.25 μg) at 37°C for 16 hours. The product was analyzed by reduced mass chromatography. Figure 8 The results showed that the molecular weight of the trimannosyl core antibody did not change significantly in the mass spectrometry, indicating that, as in Example 5, the trimannosyl antibody is not a substrate of MGAT-2.

[0115]

[0116] Example 9. Conjugation of trimannosylhexamethonium-4GlcNAz antibody with DBCO-(PEG)4-DM1 to generate hexamethonium ADC with DAR4.

[0117] In Example 7, a trimannosyl antibody was generated, with one of four GlcNAz molecules attached to each of the four terminal mannose groups. To fulfill the one-step hypothesis of this invention, a toxicant was coupled to trimannosylhexamethonium-4GlcNAz using DBCO-(PEG)4-DM1, resulting in an ADC with DAR4. A click chemistry reaction was initiated overnight at 25°C by slowly adding 5 μL of DBCO-(PEG)4-DM1 (10 mM in DMSO) to 50 μL of buffer (25 mM MES; pH 6.5) containing 5 mg / mL trimannosylhexamethonium-4GlcNAz antibody obtained from Example 7. Following the reaction, the antibody product was purified by Amicon Ultra-15 centrifugation and filtration to obtain the trimannosylhexamethonium-4 (GlcNAc-triazole-DBCO-(PEG)4-DM1) ADC. The product was analyzed by reduced-mass chromatography. Figure 9A The published results showed that, compared to the parental trimannosyl antibody with four GlcNAz, the heavy chain of the reaction product trimannosylhexamethonium-4 (GlcNAc-triazole-DBCO-(PEG)4-DM1) had a molecular weight of 53,509 Da. This indicates that two DBCO-(PEG)4-DM1 molecules (molecular weight 1,413 Da × 2 = 2,826) were coupled to each heavy chain of the antibody. This result was further confirmed by complete mass chromatography analysis. Figure 9BThe results disclosed show that, compared to the trimannosylhexamethonium-4GlcNAz antibody with a molecular weight of approximately 148,224 Da, a trimannosylhexamethonium-4(GlcNAc-triazole-DBCO-(PEG)4-DM1) ADC product was obtained, containing four DBCO-(PEG)4-DM1 molecules (molecular weight 1,413 Da × 4 = 5,652 Da) with a molecular weight of 153,876 Da. The results of Examples 7 and 9 indicate that the ADC-4DM1 product was generated from the trimannosyl antibody via a click chemistry reaction of MGAT-1, MGAT-2, and GlcNAz with DBCO-(PEG)4-DM1. Therefore, we have successfully rationalized our one-step hypothesis for ADC generation in this invention.

[0118]

[0119] Example 10. Coupling of the first reward to the terminal GlcNAz in each arm of the heavy chain of trimannosylhexamethonium-2GlcNAz via DBCO-(PEG)4-DM1

[0120] Examples 4 through 9 demonstrate the feasibility of the one-step method used in this invention for generating site-specific ADCs with homogeneous DAR4. Further research is conducted based on these successful results to demonstrate… Figure 1 The sequential method is shown. To synthesize the first intermediate, DBCO-(PEG)4-DM1 was used to couple the loading agent, thereby linking the terminal GlcNAz of the heavy chain of the reactant antibody. 14 μL of DBCO-(PEG)4-DM1 (10 mM in DMSO) was slowly added to 350 μL of buffer (25 mM MES; pH 6.5) containing 2 mg / mL trimannosylhexamethonium-2GlcNAz antibody obtained from Example 6. The reaction mixture was stirred overnight at 25°C under argon atmosphere to carry out the click chemistry reaction. After the reaction, the antibody product was filtered through an Amicon Ultra-15 centrifuge to obtain the trimannosylhexamethonium-2(GlcNAc-triazole-DBCO-(PEG)4-DM1) intermediate. The product was analyzed by reduced mass chromatography. Figure 10 The results shown indicate that, compared to the trimannosylhexamethonium-2GlcNAz antibody, the heavy chain of the 51,852 Da trimannosylhexamethonium-2(GlcNAc-triazole-DBCO-(PEG)4-DM1) intermediate contains an additional 1414 Da DBCO-(PEG)4-DM1 molecule on one arm.

[0121]

[0122] Example 11. Coupling of the second GlcNAz to the terminal α-6-mannose in each arm of the heavy chain of the trimannosylhexamethonol-2 (GlcNAc-triazole-DBCO-(PEG)4-DM1) ADC via MGAT-2.

[0123] Trimannosylhexamethonium-2GlcNAz-2 (GlcNAc-triazole-DBCO-(PEG)4-DM1) and UDP-GlcNAz (1 mM) (R&D ES104-100) obtained from Example 10 were incubated in 500 μl of 1× buffer (25 mM MES, 10 mM MnCl2, pH 6.5) in the presence of rat MGAT-2 (15 μg) at 37°C for 16 hours. After the reaction, the antibody product was filtered through an Amicon Ultra-15 centrifuge to obtain trimannosylhexamethonium-2GlcNAz-2 (GlcNAc-triazole-DBCO-(PEG)4-DM1). The product was analyzed by reduced mass chromatography. Figure 11 The results shown indicate that, compared to the parental trimannosylhexamethonium-2 (GlcNAc-triazole-DBCO-(PEG)4-DM1), the heavy chain of trimannosylhexamethonium-2GlcNAz-2 (GlcNAc-triazole-DBCO-(PEG)4-DM1) each contains an additional GlcNAz molecule (MW=244), with a molecular weight of 52,097 Da. This result suggests that MGAT-2 is a highly substrate-flexible enzyme that converts UDP-GlcNAz into large-functionalized antibodies, such as trimannosylhexamethonium-2 (GlcNAc-triazole-DBCO-(PEG)4-DM1), producing intermediates for dual-loaded ADC products.

[0124]

[0125] Example 12. DAR4 ADC product containing one MMAE and one DM1 on each arm of the constructed antibody.

[0126] In Example 11, a trimannosylhexamethonol-2GlcNAz-2(GlcNAc-triazole-DBCO-(PEG)4-DM1) intermediate was generated. To complete... Figure 1 The sequential method of the present invention shown uses DBCO-(PEG). 12 -MMAE coupled the toxicity loading to trimannosylhexamethonium-2GlcNAz 2 (GlcNAc-triazole-DBCO-(PEG)4-DM1) and produced an ADC with DAR4 and both loadings.

[0127] Slowly add 3.8 μL of DBCO-(PEG) to 76 μL of 1× buffer (25 mM MES, pH 6.5) containing 2.5 mg / mL trimannosylhexamethonium-2GlcNAz-2 (GlcNAc-triazole-DBCO-(PEG)4-DM1) ADC obtained from Example 11. 12 -MMAE (10 mM, in DMSO). The reaction mixture was stirred overnight at 25°C under argon atmosphere to carry out the click chemistry reaction. After the reaction, the antibody product was filtered through an Amicon Ultra-15 centrifuge to obtain trimannosylhexamethonol-2(GlcNAc-triazole-DBCO-(PEG)4-DM1)-2(GlcNAc-triazole-DBCO-(PEG)). 12 -MMAE) ADC. After purification, the product was then subjected to complete mass chromatographic analysis. For example... Figure 12 The results shown indicate that, compared to the parental trimannosylhexamethine-2GlcNAz-2(GlcNAc-triazole-DBCO-(PEG)4-DM1) with a molecular weight of 151,050 kD, the obtained trimannosylhexamethine-2GlcNAz-2(GlcNAc-triazole-DBCO-(PEG)4-DM1) is... 12 The -MMAE) ADC contains two additional DBCO-(PEG) molecules. 12 -MMAE molecules (MW = 1648 × 2 = 3,296) with a molecular weight of 154,345 Da. This result indicates that the method of the present invention can precisely control two different reward loadings (e.g., DBCO-PEG4-DM1 and DBCO-(PEG)). 12 The coupling of MMAE with trimannose-based omega-3-amine (MMAE) and trimannose-based omega-3-amine.

[0128]

[0129] Example 13. A DAR4 ADC product containing one MMAF and one DM1 on each arm of the constructed antibody.

[0130] 3.8 μL of DBCO-MMAF (10 mM in DMSO) was slowly added to 76 μL of 1× buffer (25 mM MES, pH 6.5) containing 2.5 mg / mL trimannosylhexamethonium-2GlcNAz-2(GlcNAc-triazole-DBCO-(PEG)4-DM1) ADC obtained from Example 11. The reaction mixture was stirred overnight at 25°C under argon atmosphere to carry out the click chemistry reaction. After the reaction, the antibody product was filtered through an Amicon Ultra-15 centrifuge to obtain the trimannosylhexamethonium-2(GlcNAc-triazole-DBCO-(PEG)4-DM1)-2(GlcNAc-triazole-DBCO-MMAF) ADC. The product was subjected to complete mass chromatographic analysis. Figure 13 The results shown indicate that, compared to the parental trimannosylhexamethonium-2GlcNAz-2(GlcNAc-triazole-DBCO-(PEG)4-DM1) ADC (MW = 151,050), the obtained trimannosylhexamethonium-2(GlcNAc-triazole-DBCO-(PEG)4-DM1)-2(GlcNAc-triazole-DBCO-MMAF) ADC contains two additional DBCO-MMAF molecules with a molecular weight of 2,038 Da, resulting in a total molecular weight of 153,082 Da. This result demonstrates that the method of the present invention can precisely control the loading of different reward species (e.g., DBCO-(PEG)4-DM1, DBCO-(PEG)4-DM1, etc.). 12 The conjugation of MMAE and DBCO-MMAF with trimannose-based omega-3-amine.

[0131] In summary, by combining the enzymatic reactions of MGAT-1, MGAT-2, and GlcNAz with the DBCO-loaded chemical reaction, we have rationalized our inventive hypothesis. We are able to generate homogeneous site-specific ADC products with either DAR4 or DAR2 using our one-step method. Furthermore, this invention is also applicable to the sequential synthesis of homogeneous site-specific ADC products with both loads.

[0132]

[0133] Example 14. Binding affinity analysis of trimannosylhexamethonium-4 (GlcNAc-triazole-DBCO-(PEG)4-DM1)

[0134] The trimannosylhexamethonol-4 (GlcNAc-triazole-DBCO-(PEG)4-DM1) ADC was constructed using the method described above. Kadcyla targeting the Her2 / Neu molecule was purchased from Roche Inc. ERBB2-ECD (ebioscience BMS362) (100 ng / well) was added to each well of a NUNC Maxisorp plate, and the plate was incubated overnight at 4°C. The plate was washed with 1× PBS-T (0.1%) to remove uncoated reagents. 3% skim milk was added to the wells of the plate, and the plate was incubated at room temperature for 2 hours. The plate was washed three times with 1× PBS-T (0.1%), dried, and then stored at -20°C for further use. Continuous dilutions from 1×10⁻⁶ were added to the plate. -6 g / mL to 1×10 -12 Individual antibodies were added at g / mL, and the culture plates were incubated at 37°C for 1 hour. Goat anti-human IgG conjugated with horseradish peroxidase (HRP) was added, and the plates were incubated for 1 hour, followed by the addition of 3,3',5,5'-tetramethylbenzidine (TMB). OD405 was read to calculate activity. Each study was repeated three times, and data are presented as mean ± SD. Polyscatter plots were generated using Prism software, employing OD readings and antibody concentrations. Figure 14 The results shown indicate that the curves for the trimannosylhemafen-4 (GlcNAc-triazole-DBCO-(PEG)4-DM1) ADC are almost identical to those for the positive control Kadcyla and trimannosylhemafen-4GlcNAz. The negative control anti-mesothelien showed no binding affinity to the Her2 / Neu molecule. These results suggest that the binding affinity of the trimannosylhemafen-4 (GlcNAc-triazole-DBCO-(PEG)4-DM1) ADC to the Her2 / Neu molecule is not affected by the modifications performed.

[0135] Example 15. Cytotoxic effects of trimannosyl hyaluronidone-4 (GlcNAc-triazole-DBCO-(PEG)4-DM1) ADC

[0136] The Her2 / Neu high-expression cell line SK-BR-3, the Her2 / Neu intermediate-expression cell line HCC-1954, and the Her2 / Neu low-expression cell line MDA-MB-231 were diluted to 10⁻⁶. 6Cells / ml. After adding 100 μL of diluted cell culture to the wells of a 96-well plate, the cells were incubated at 37°C for 24 hours. 80 μL of complete culture medium was added to each well, followed by 20 μL of trimannosylhexamine-4 (GlcNAc-triazole-DBCO-(PEG)4-DM1) ADC at different doses to each well. After incubating the plate at 37°C for 48 hours, 100 μL of CellTiter-Glo® reagent was added to each well. After incubating at room temperature for another 10 minutes, the luminescence (light) of the wells was measured using a luminometer. The IC50 values ​​of trimannosylhexamine-4 (GlcNAc-triazole-DBCO-(PEG)4-DM1) ADC against the Her2 / Neu overexpressing cell line SK-BR-3 and the Her2 / Neu moderately expressing cell line HCC-1954 were 4.7 nM and 14 nM, respectively. The IC50 values ​​also showed that all tested antibodies had no anti-proliferative effect on the Her2 / Neu low-expression cell line MDA-MB-231. This result indicates that, for Kadcyla, the trimannosyl hyaluronidone-4 (GlcNAc-triazole-DBCO-(PEG)4-DM1) ADC is cytotoxic not only to Her2 / Neu high-expression cells but also to Her2 / Neu moderately expressed cells, and that modification of the antibody by the trimannosyl ADC platform does not affect its biological activity.

[0137] Example 16. Production of trimannosyl-core trastuzumab antibody and trimannosyl-core anti-TMC33 antibody from F293 cells.

[0138] Plastomeres pTCAE8.3-exo-Gal containing cDNA encoding β-N-acetylglucosaminease S were constructed and co-transfected into two F293 cell lines to express trimannosyl trastuzumab (anti-Her2 antibody) and trimannosyl anti-transmembrane and coil-coil domain family 3 (TMCC3) antibody, respectively. After cultivation, the supernatants of the two cell cultures were collected and the antibodies contained therein were purified separately using rProtein A Sepharose Fast Flow (GE Healthcare, 17-1279-02). Following purification, the purified antibody samples were analyzed by reduced-mass chromatography. Figure 15A The results shown indicate that, when compared with antibodies isolated from F293 cells transfected only with the trastuzumab gene, the heavy chain of the trastuzumab antibody obtained from the same cells transfected with both β-N-acetylglucosaminease S and the trastuzumab gene exhibited a peak at a molecular weight of 50,195 Da, indicating that the obtained trastuzumab antibody was a trimannosyl core antibody. Similar results were observed in the same cells transfected with the anti-TMCC3 antibody gene. Figure 15BThe results indicate that mass production of trimannosyl antibodies from commercial cell lines is feasible and suitable for industrial CMC amplification.

[0139]

[0140] Example 17. Conjugation of UDP-GlcNAz to cellularly expressed trimannosyl core trastuzumab antibody via MGAT-1 and MGAT-2 to generate trimannosyl trastuzumab-4 (GlcNAc-triazole-DBCO-(PEG)4-DM1) ADC.

[0141] Following the method described in Example 7, 2 mg of trimannosyl core trastuzumab antibody (produced from mammalian cells) obtained from Example 16 and UDP-GlcNAz (1 mM) were incubated in 800 μl of 1× buffer SP (25 mM MES, 10 mM MnCl2, pH 6.5) at 37°C for 16 hours in the presence of rabbit MGAT-1 (25 μg) and rat MGAT-2 (10 μg). After incubation, the reaction products were analyzed by reduced mass chromatography and intact mass chromatography. Figure 16A The reduced-mass chromatographic results shown indicate that, compared to a trimannosyl trastuzumab antibody with a heavy chain having a molecular weight of 50,194 Da, a trimannosyl trastuzumab-4GlcNAz antibody product was obtained, in which the heavy chain contains two additional GlcNAz molecules with a molecular weight of approximately 244 Da × 2 = 488, and each heavy chain has a molecular weight of approximately 50,680 Da. This result suggests that GlcNAz can be coupled to the α-3-mannose and α-6-mannose of each heavy chain of a trimannosyl trastuzumab antibody produced from mammalian cells via MGAT-1 and MGAT-2.

[0142] Following the method described in Example 9, DBCO-(PEG)4-DM1 was slowly added to Tris buffer (pH 7.0) containing the obtained trimannosyltrastuzumab-4GlcNAz antibody at 25°C for a click chemistry reaction lasting 16 hours. After reaction and purification, the product was subjected to reduced mass chromatography and whole mass chromatography. Figure 16B The converted mass chromatographic analysis results were published, showing that the molecular weight of the heavy chain of the product was 53,509 Da. This suggests that two DBCO-(PEG)4-DM1 molecules (molecular weight 1,413 Da × 2 = 2,826) were coupled to the heavy chain of the trimannosyltrastuzumab-4GlcNAz antibody. Figure 16CThe results of complete mass chromatographic analysis further confirmed this finding. This result discloses the yield of a trimannosyl-4 (GlcNAc-triazole-DBCO-(PEG)4-DM1) ADC product containing four DBCO-(PEG)4-DM1 molecules (molecular weight 1,413 Da × 4 = 5,652 Da) with a total molecular weight of approximately 153,868 Da.

[0143] It is expected that those skilled in the art will conceive of numerous modifications and variations of the invention as illustrated in the above exemplary examples. Therefore, the invention should be limited only by the appended claims.

Claims

1. A method for preparing glycoprotein-loaded conjugates comprising the structure of formula (1), The method includes the following steps: (i) making a glycoprotein containing a polysaccharide having formula (2) Reaction with β-N-acetylglucosaminease produces a modified glycoprotein containing a trimannosyl core of formula (3). ； (ii) To make the modified glycoprotein containing the trimannosyl core of formula (3) with UDP-GlcNAc-(CH2) 0-8 -R reacts in the presence of mannosyl (α-1,3-)-glycoprotein β-1,2-N-acetylglucosamine aminotransferase and mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine aminotransferase, where R is an azide, keto, or aldehyde, resulting in two GlcNAc-(CH2) groups. 0-8 -R sugars are bonded to Man. 2 and Man 3 Each of the β-1,2 positions, thereby forming the glycan moiety of formula (4). ； as well as (iii) Reacting two coupling agent-linking groups-loaded with the glycan portion of formula (4), wherein the loads of the two coupling agent-linking groups-loaded are the same or different, to produce a glycoprotein-loaded conjugate comprising the structure of formula (1).

2. The method according to claim 1, wherein the glycoprotein comprising the polysaccharide having formula (2) and the β-N-acetylglucosamine enzyme in step (i) are produced by a mammalian cell line.

3. The method according to claim 2, wherein the mammalian cell line is a monkey kidney CV1 strain (COS-7) transformed with SV40, a human embryonic kidney strain (293 or 293T cells), baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells, MRC 5 cells, FS4 cells, Chinese hamster ovary (CHO) cells, or myeloma cell lines.

4. The method of claim 1, wherein the glycoprotein is an antibody or a fragment thereof, such as an antibody Fab fragment, F(ab')2, Fv fragment or Fc fragment, scFv-Fc fragment, microantibody, bifunctional antibody or scFv from a lysed antibody.

5. The method according to claim 1, wherein R is an azide group, the coupling agent is an alkynyl group, and the reaction in step (iii) is a click reaction.

6. The method according to claim 1, wherein R is a ketone or aldehyde, the coupling agent is an amino group, and the reaction in step (iii) is a reductive amination.

7. The method according to claim 1, wherein R is a ketone or aldehyde, the coupling agent is β-arylethylamino, and the reaction in step (iii) is a Pictet-Spengler reaction.

8. The method according to claim 1, wherein the linking group is selected from straight-chain or branched-chain alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, alkoxy, acyl, alkylamino or arylamino, linking groups containing disulfide bonds, acid-unstable linking groups, light-unstable linking groups, peptidase-unstable linking groups and esterase-unstable linking groups having 2 to 20 carbon atoms.

9. The method of claim 1, wherein the receptacle is independently selected from therapeutic agents and markers.

10. The method of claim 9, wherein the therapeutic agent is selected from antimetabolites, alkylating agents, alkylating agents-like agents, DNA minor groove alkylating agents, anthracycline, antibiotics, calicheamicin, antimitotic agents, topoisomerase inhibitors, proteasome inhibitors, and radioisotopes.

11. The method according to claim 9, wherein the label is a fluorescent label, a chromogenic label, an electron-dense label, a chemiluminescent label, a radioactive label, an enzyme label, or a positron emitter.

12. A glycoprotein-loaded conjugate comprising the structure of formula (1) as defined in claim 1.

13. A method for manufacturing a glycoprotein-loaded conjugate comprising the structure of formula (5), ， The method includes the following steps: (i) To react a modified glycoprotein comprising a trimannosyl core as defined in formula (3) of claim 1 with UDP-GlcNAc-(CH2). 0-8 -R reacts in the presence of mannosyl (α-1,3-)-glycoprotein β-1,2-N-acetylglucosamine aminotransferase, where R is an azide, keto, or aldehyde, resulting in GlcNAc-(CH2). 0-8 -R sugar bonds to Man 2 At the β-1,2 position, and thereby form a glycoprotein containing the glycan moiety of formula (6). ； as well as (ii) React the coupling agent-linking group-loaded glycoprotein containing the glycan moiety of formula (6) to produce a glycoprotein-loaded conjugate containing the structure of formula (5).

14. The method of claim 13, wherein the glycoprotein is an antibody or a fragment thereof, such as an antibody Fab fragment, F(ab')2, Fv fragment or Fc fragment, scFv-Fc fragment, microantibody, bifunctional antibody or scFv from a lysed antibody.

15. The method according to claim 13, wherein R is an azide group, the coupling agent is an alkynyl group, and the reaction in step (ii) is a click reaction.

16. The method according to claim 13, wherein R is a ketone or aldehyde, the coupling agent is an amino group, and the reaction in step (ii) is a reductive amination.

17. The method according to claim 13, wherein R is a ketone or aldehyde, the coupling agent is β-arylethylamino, and the reaction in step (ii) is a Pickett-Spengler reaction.

18. The method of claim 13, wherein the linking group is selected from straight-chain or branched-chain alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, alkoxy, acyl, alkylamino or arylamino, linking groups containing disulfide bonds, acid-unstable linking groups, light-unstable linking groups, peptidase-unstable linking groups and esterase-unstable linking groups having 2 to 20 carbon atoms.

19. The method of claim 13, wherein the substance is a therapeutic agent or a label.

20. The method of claim 19, wherein the therapeutic agent is selected from antimetabolites, alkylating agents, alkylating agents-like agents, DNA minor groove alkylating agents, anthracyclines, antibiotics, kazimidic acid, antimitotic agents, topoisomerase inhibitors, proteasome inhibitors, and radioisotopes.

21. The method of claim 19, wherein the label is a fluorescent label, a chromogenic label, an electron-dense label, a chemiluminescent label, a radioactive label, an enzyme label, or a positron emitter.

22. A glycoprotein-loaded conjugate comprising the structure of formula (5) as defined in claim 13.

23. A method for manufacturing a glycoprotein-loaded A / B conjugate containing the structure of formula (7), ， The method includes the following steps: (i) To react a modified glycoprotein comprising a trimannosyl core as defined in formula (3) of claim 1 with UDP-GlcNAc-(CH2). 0-8 -R reacts in the presence of mannosyl (α-1,3-)-glycoprotein β-1,2-N-acetylglucosamine aminotransferase, where R is an azide, keto, or aldehyde, resulting in GlcNAc-(CH2). 0-8 -R sugar bonds to Man 2 The β-1,2 positions are used to form a glycoprotein containing the glycan moiety of formula (6) as defined in claim 13; (ii) React the coupling agent-linking group-loaded A with a glycoprotein containing the glycan moiety of formula (6) to produce a glycoprotein-loaded A conjugate containing the structure of formula (8). ； (iii) Make the glycoprotein-loaded A conjugate containing the structure of formula (8) with UDP-GlcNAc-(CH2). 0-8 -R reacts in the presence of mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine aminotransferase, where R is an azide, keto, or aldehyde, resulting in GlcNAc-(CH2). 0-8 -R sugar bonds to Man 3 At the β-1,2 position, thereby forming a glycoprotein containing a glycan-loaded A moiety having formula (9). ； as well as (iv) React the coupling agent-linking group-loaded B with a glycoprotein containing a glycan-loaded A moiety having formula (9) to produce a glycoprotein-loaded A / B conjugate containing the structure of formula (7). The payload A may be the same as or different from the payload B.

24. The method of claim 23, wherein the glycoprotein is an antibody or a fragment thereof, such as an antibody Fab fragment, F(ab')2, Fv fragment or Fc fragment, scFv-Fc fragment, microantibody, bifunctional antibody or scFv from a lysed antibody.

25. The method according to claim 23, wherein R is an azide group, the coupling agent is an alkynyl group, and the reaction carried out in step (ii) / (iv) is a click reaction.

26. The method according to claim 23, wherein R is a ketone or aldehyde, the coupling agent is an amino group, and the reaction carried out in step (ii) / (iv) is a reductive amination.

27. The method according to claim 23, wherein R is a ketone or aldehyde, the coupling agent is β-arylethylamino, and the reaction carried out in step (ii) / (iv) is a Pickett-Spengler reaction.

28. The method of claim 23, wherein the linking group is selected from straight-chain or branched-chain alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, alkoxy, acyl, alkylamino or arylamino, linking groups containing disulfide bonds, acid-unstable linking groups, light-unstable linking groups, peptidase-unstable linking groups and esterase-unstable linking groups having 2 to 20 carbon atoms.

29. The method of claim 23, wherein the reward A and the reward B are independently selected from therapeutic agents and labels.

30. The method of claim 29, wherein the therapeutic agent is selected from antimetabolites, alkylating agents, alkylating agents-like agents, DNA minor groove alkylating agents, anthracyclines, antibiotics, kazimidic acid, antimitotic agents, topoisomerase inhibitors, proteasome inhibitors, and radioisotopes.

31. The method of claim 29, wherein the label is a fluorescent label, a chromogenic label, an electron-dense label, a chemiluminescent label, a radioactive label, an enzyme label, or a positron emitter.

32. A glycoprotein-loaded A / B conjugate comprising the structure of formula (7) as defined in claim 23.

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