Bifurcated trimannose-oligosaccharide, bifurcated N-glycan containing the trimannose core, and method for obtaining these

Abstract

The present invention relates to a reactive compound made from a modified trimannose-oligosaccharide, to its preparation method, and in particular to its use for improving the properties of a first target ligand in therapeutic or diagnostic applications. Specifically, the present invention provides a compound of formula 1 (wherein Man 1 , Man 2 , and Man 3 moieties are the first, second, and third mannoses respectively, R1 is a linking group capable of binding or already binding a GlcNAc group to the first target ligand, R2 and R3 are monosaccharides or derivatives thereof optionally linked to one or more additional sugars, and wherein GlcNAz is a non-natural N-azidoacetylglucosamine group capable of binding or already binding to a second target ligand different from the first target ligand), and relates to a reactive compound comprising a trimannose core. JPEG2025519204000004.jpg28170

Description

Technical Field

[0001] The present invention relates to the field of biotechnology and to reactive compounds made from modified trimannose-oligosaccharides capable of binding or bound to one or more ligands of interest, to methods for their preparation, and in particular to their use for improving the properties of said ligands of interest in therapeutic or diagnostic applications.

Background Art

[0002] Structural modifications of ligands of interest are mostly based on the reactivity of carboxyl, amino, hydroxyl, or thiol groups that may be present simultaneously and, advantageously, repeatedly in the ligand of interest (e.g., protein, sugar, lipid). This results in structural heterogeneity with respect to the number and isomers (differences in the spatial arrangement of the modifications) of the product (the molecule with one or more modifications).

[0003] Among these examples of ligands of interest, (monoclonal or polyclonal) antibodies are known to be functionalized with one or more groups or molecules that are molecular probes, drugs, or fluorophores via conjugation protocols that use cysteine or lysine residues naturally present in the polypeptide backbone.

[0004] These modifications typically confer new properties, such as improved function or improved reactivity useful for diagnostic or therapeutic purposes, on these modified antibodies. Efficient and useful modification of molecules may lie in stochastic modification. However, these modifications can cause a significant proportion of different structures produced by procedures that result in modifications (addition of groups) close to the functional site (e.g., the antigen-binding fragment of an antibody), which may reduce the binding / function properties compared to the unmodified precursor.

[0005] However, among the antibodies to be treated, known probabilistic protocols typically result in antibody batches that are heterogeneous with respect to the number and spatial distribution of payloads. Furthermore, the resulting antibodies are known to have low activity when compared to their unmodified counterparts, since this probabilistic functionalization can occur in the variable regions of these antibodies, which include the functional sites responsible for antigen recognition.

[0006] On the other hand, site-specific modifications may result in higher structural homogeneity, greater biological activity, and easier further functionalization than modifications resulting from probabilistic ones. As a result, the resulting modified molecules are more suitable candidates for both diagnostic and therapeutic purposes.

[0007] Site-specific modifications can be induced in vivo / in vitro (e.g., by genetic manipulation of the whole organism or at the cellular level before biogenesis of the molecule) or ex vivo / ex vitro (e.g., chemical manipulation of the purified molecule). While the in vivo / in vitro strategy is the ideal solution for specific cases and individual products, the ex vivo / ex vitro strategy is applicable quite widely (case-independent) and does not have such stringent requirements regarding timing and economic resources.

[0008] Among the modified molecules, therapeutic proteins such as antibodies and the like are currently widely used in diagnostic or therapeutic applications. In particular, antibody-drug conjugates can greatly benefit from improvements in terms of efficiency, production purification, and / or reduction of side effects.

[0009] Therefore, there is a need to provide novel simplified methods and novel reactive compounds that can be proposed to modify the biological structure of a target ligand, especially for diagnostic or therapeutic applications, in order to confer novel properties such as function or reactivity.

[0010] Prior Art There is an N-glycosylation site in the heavy chain of a naturally occurring antibody (at asparagine 297 (Asn297)), and it is known that two two-antennary glycan moieties made up of seven sugars are formed at this glycosylation site of the antibody.

[0011] Publications by Van Geel, R. et al. (Bioconjug. Chem. 26, 2233 - 2242 (2015)) and Cai, X. & Janda, K. D. A (Tetrahedron Lett. 56, 3172 - 3175 (2015)) describe chemoenzymatic approaches for preparing antibody-drug conjugates via targeted glycosylation sites present in antibodies that require native glycan enzymatic labeling with N-azidoacetylgalactosamine (GalNAz) bearing an azide by the addition of endo-β-N-acetylglucosaminidase.

[0012] Terminal galactose is known to increase cytotoxic reactions due to the complement system. Antibody glycosylation and its influence on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc fusion proteins are known, and bisecting GlcNAc can increase the affinity for FcγRIIIa and thus antibody-dependent cell cytotoxicity (ADCC). (Claudia Ferrara, et al. Biotechnol. Bioeng. 93, 851 - 861 (2006).

[0013] In the case of antibodies, the degree of labeling (DOL) plays an important role; indeed, antibodies with too high a DOL are known to be eliminated more rapidly and are more likely to aggregate due to being more hydrophobic. (Publications of Agarwal, P. & Bertozzi, C. R. Bioconjug. Chem. 26, 176 - 192 (2015). & Van Geel, R. et al. (2015)).

[0014] The design of modified branching sites on antibodies to create new functional diversities of modified antibodies has been proposed in the prior art. Since the N-glycosylation site on an antibody is located at asparagine 297 (Asn 297 residue), which is quite far from the variable region of the antibody, the possibility of disturbing the antibody activity due to its paratope is low.

[0015] There are only two N-glycosylation sites, which means that the number of payloads that can be added to the glycan can be efficiently controlled, thereby enabling better ratio regulation and uniformity.

[0016] The authors Unverzagt C. et al (Tetrahedron letters, vol. 41, N°23, (2000)), Eller S. et al (Tetrahedron Letters, Vol. 51, N°19 (2010)), and Schuberth R; et al (Tetrahedron Letters, Vol. 46, N°24 (2005)) disclose modified bisecting trimannose-oligosaccharides with non-natural monosaccharides that cannot be obtained by chemical synthesis.

[0017] Publications by Nakano M. et al (Molecular & Cellular Proteomics vol18, N°10, pages 2044 - 2057 (2019)) and Ferrara M et al (P.N.A.S. pages 12669 - 12674 (2011)) disclose the native structure of bisecting trimannose-oligosaccharides (where the bisecting mannose contains a GlcNac residue that plays an important role in the properties of the resulting N-glycan).

[0018] U.S. Patent No. 11,085,062-B2, European Patent No. 2305314-B1 (Ratiopharm), European Patent No. 1987068-B1 (Life technologies), International Patent Application No. 2014 / 065661 Pamphlet (Synaffix), and International Patent Application No. 2016 / 170186 Pamphlet (Genovis) disclose methods for obtaining a complex of an antibody and a modifying group.

[0019] However, in these patent applications, the modifying group is not added at the bisection position of the trimannose core (bisecting mannose or Man 1 ). This is because the glycan modification by Genovis's Glyclick® truncates up to the first GlcNac along with the addition of GalNAz at a degree of labeling (DOL) of 2, the glycan modification of Thermofisher's SiteClick® shows glycan truncation up to G0 via the addition of GalNAz at a degree of labeling (DOL) of 4, and the glycan modification of GlycoConnect® (Synaffix) shows glycan truncation up to the first GlcNac along with the addition of GalNAz at a degree of labeling (DOL) of 2.

Summary of the Invention

Problems to be Solved by the Invention

[0020] The present invention aims to provide a novel reactive compound that can be used for the modification of a ligand, particularly a (first) biological molecule of interest. Here, this novel reactive compound can efficiently form a complex of a first ligand and, optionally, at least one additional second ligand of interest with the reactive compound.

[0021] A preferred object of the present invention is to obtain a reactive compound that can be efficiently used for the generation of a novel complex. Here, this reactive compound is a linkage structure that improves the binding of two, preferably two different, target ligands, thereby forming this complex.

[0022] A specific object of the present invention is to obtain such reactive compounds and the complexes obtained therefrom in order to improve the properties of the first molecule of interest, simultaneously with one or more of the drawbacks of the prior art.

[0023] More preferably, the object of the present invention is to be used for cell culture including human cell culture, particularly for animal cells, on a solid support, or for screening and recovering one or more novel ligands of interest. It is to obtain such a binding between an element of the solid support and a suitable ligand, preferably a biomolecule.

[0024] In particular, a preferred object of the present invention includes the addition of one or more groups such as drugs, without the drawbacks of prior art complexes particularly regarding uniformity, reproducibility, and solubility, and higher drug loading on this first ligand such as antibodies and the like. It is to obtain a reactive compound for obtaining a novel complex.

[0025] A more preferred object of the present invention is to include a first target ligand such as an antibody when modified by the addition of the reactive compound of the present disclosure and the added second target ligand, and to have a low degree of labeling (DOL) close to 2. It is to obtain a complex.

[0026] Another object of the present invention is an efficient method for generating a reactive compound, particularly a simplified method, that includes fewer process steps and is less complex or requires less time than prior art methods used to obtain similar complexes.

[0027] A further object of the present invention is an improved complex which contains or is made of a first molecule of interest, such as a glycoprotein, preferably an antibody, and is linked to one or more drugs or labels by a reactive compound, in particular an improved complex which is improved by the addition of a reactive group immobilized by the reactive compound of the present invention and which exhibits improved properties when applied in the diagnosis, and also in the prevention and / or treatment, of diseases in plants, animals and humans, in particular cancer.

Means for Solving the Problems

[0028] A first aspect of the present invention is of the formula (1)

Chemical formula

[0029] According to the present invention, Man 1 is a mannose moiety like Man 2 and Man 3 and is also a bifurcated position of the trimannose core.

[0030] Advantageously, in the reactive compounds according to the present invention, the two ligands are different.

[0031] According to the present invention, R1 is a linking group (for linking a reactive compound to a first target ligand) preferably selected from the group consisting of monosaccharides or derivatives thereof, and disaccharides or derivatives thereof. Optionally, this linking group may further include one or more additional (substituted or unsubstituted and / or branched or unbranched) polyethylene glycols (PEGs) linked to these monosaccharides, such as (OC2H4)n chains (where n is an integer preferably including 1 to 20, more preferably 1 to 10, or 1 to 5), another additional alkyl group, one or more additional aryl groups (5 to 12 carbons, with or without one or more heteroatoms), one or more additional alkenyl groups, one or more additional alkene groups, one or more additional alkyne groups, or one or more additional alkynyl groups.

[0032] Conveniently, R1 forms a first N-acetylglucosamine (GlcNAc) group and an N-acetylglucosamine-N-acetylglucosamine group (GlcNAc-GlcNAc group). This GlcNAc-GlcNAc group can be further glycosylated, for example, further fucosylated, to be capable of binding to or bound to the first target ligand.

[0033] Optionally, R2 and / or R3 are linked to one or more additional sugars to form a sugar chain.

[0034] According to a preferred embodiment of the present invention, R2 and / or R3 can also include, similar to R1, one or more additional (substituted or unsubstituted and / or branched or unbranched) polyethylene glycols (PEGs), such as (OC2H4)n chains (where n is an integer preferably including 1 to 20, more preferably an integer including 1 to 10, or an integer including 1 to 5), one or more alkyl groups, another additional aryl group of 5 to 12 carbons (with or without one or more heteroatoms), one or more additional alkenyl groups, one or more additional alkene groups, one or more additional alkyne groups, or one or more additional alkynyl groups.

[0035] Preferably, the terminal groups of R2 and / or R3 are capable of binding to or are bound to one or more additional ligands which are the third target ligand and optionally the fourth target ligand and contain or exist as N - azidoacetylglucosamine (GlcNAz) groups.

[0036] Another aspect of the invention relates to a complex formed between at least a first target ligand and a second target ligand, optionally with a third and fourth target ligand or the third and fourth target ligands being formed (wherein the target ligands are bound by a reactive compound according to the invention), wherein advantageously, in this complex, all ligands are different.

[0037] In the complex according to the invention, the first ligand is a biological molecule selected from the group consisting of proteins, peptides, nucleic acid molecules (DNA or RNA sequences, such as probes, etc.), sugars, such as monosaccharides or polysaccharides, lipids or mixtures thereof.

[0038] Advantageously, the first molecule of the complex according to the invention is a protein selected from the group consisting of antigenic structures or antibodies and the like (including monoclonal antibodies, polyclonal antibodies, antibody hypervariable regions, nanobodies, alphabodies, microbodies, affytins, fymomers, affilines, affimers, or mixtures of two or more thereof).

[0039] Advantageously, the second ligand is a material selected from the group consisting of drugs, labels, solid supports (e.g., made of glass, polymer, or metal structures), macromolecules, carrier molecules, water - soluble polymers, or mixtures thereof.

[0040] Water-soluble polymers include polyethylene glycol (PEG), preferably having a degree of polymerization of from 1 to about 20,000 and / or having a molecular weight of from 1 kDa to 100 kDa, more preferably from 2 kDa to 50 kDa, even more preferably from 5 kDa to 25 kDa, such as PEG having a molecular weight of from 10 kDa to 20 kDa. Such water-soluble polymers, such as PEG, are known to be useful for reducing immunogenicity and / or extending the clearance time with respect to the circulation of a first modified material or molecule of interest (such as this antibody) when added to biological molecules such as antibodies and the like.

[0041] The present invention also relates to a pharmaceutical composition comprising a suitable or appropriate pharmaceutical carrier or diluent and a reactive compound and / or complex according to the present invention.

[0042] Another aspect of the present invention relates to a diagnostic or screening kit comprising a reactive compound and / or complex according to the present invention, optionally means for producing a signal from a label and optionally instructions.

[0043] The present invention also relates to materials such as solid supports (beads, strips, plates) immobilized on a complex or compound of the present invention for screening and recovering other molecules of interest via the binding of a molecule of interest to a solid support (beads, strips, plates). The present invention also relates to supports (i.e., beads, strips, plates) immobilized on a complex or compound of the present invention for cell culture, particularly for animal cells including human cells.

[0044] A further aspect of the present invention relates to a method for producing a reactive compound according to the present invention. Advantageously, this production method comprises - a trimannose core comprising three linked mannose (Man 1 , Man 2 , and Man 3 ) moieties, or these three linked mannose (Man 1 , Man 2, and Man 3 Selecting a first material or a first molecule (such as an antibody) of interest that already contains one or more of these trimannose cores having a - A sufficient amount of uridine diphosphate N-azidoacetylglucosamine (UDP-GlcNAz) as a sugar donor substrate, and the first mannose Man of the trimannose core 1 Adding to the trimannose core a sufficient amount of MGAT3 glycosyltransferase (also called Gnt-III or β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase) that acts as an enzyme for adding a terminal N-azidoacetylglucosamine (GlcNAz) group to the part, including any similar enzyme or any variant enzyme of this enzyme that exhibits a nucleotide sequence identity higher than 80%, higher than 90%, higher than 95%, or higher than 98% - 99% with the wild-type MGAT3 enzyme and has a high similarity or nucleotide identity with the MGAT3 glycosyltransferase - Purifying, preferably by one or more steps of chromatography, thereby obtaining a reactive compound or a first ligand (antibody) bound to a reactive compound capable of binding or already bound to a second ligand - Optionally, adding a second ligand to the added N-azidoacetylglucosamine (GlcNAz) group of the reactive compound This is a chemo-enzymatic method.

[0045] Optionally, in the method of the present invention, this first ligand is an antibody that has been pre-modified with one or more of its sugar chains through the addition of a sufficient amount of a mixture of, for example, neuraminidase (NAM), galactosidase (GO), and N-acetylglucosaminidase (NAG).

[0046] The last aspect of the present invention relates to a method for generating a complex according to the present invention. Advantageously, this method comprises the binding of the reactive compound obtained by the method described above to a second ligand and optionally to the first ligand described above.

[0047] Advantageously, in the method according to the present invention, this binding is achieved by adding a sufficient amount of the second ligand to the reactive compound, and the method further comprises a subsequent purification step of the resulting complex, preferably by one or more steps of chromatography.

Brief Description of the Drawings

[0048]

Figure 1-2

Figure 3

Figure 4

[0049] List of Definitions and Abbreviations 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 belongs. All publications and patent documents mentioned herein are incorporated by reference and may be used in connection with the present invention. The following abbreviations are represented by the explanations in parentheses: ACN (acetonitrile), BGG (bovine γ-globulin), CCD (charge-coupled device), CHO (Chinese hamster ovary), DAR (drug-antibody ratio), ELISA (enzyme-linked immunosorbent assay), ESI (electrospray ionization), FPLC (fast protein liquid chromatography), GalT (galactosyltransferase), GNT-III (β-1,4-mannosyl-glycoprotein-4-β-N-acetylglucosaminyltransferase), GO (galactosidase), HEK (human embryonic kidney), HILIC (hydrophilic interaction liquid chromatography), HPLC (high-pressure liquid chromatography), HRMS (high-resolution mass spectrometry), LC (liquid chromatography), MGAT3 (β-1,4-mannosyl-glycoprotein-4-β-N-acetylglucosaminyltransferase), MS (mass spectrometry), NAG (N-acetylglucosaminidase), NAM (neuraminidase), PAGE (polyacrylamide gel electrophoresis), PBS (phosphate-buffered saline), SDS (sodium dodecyl sulfate), SEC (size exclusion chromatography), SPE (solid-phase extraction), SPI (soybean protein isolate), TLC (thin-layer chromatography), TMB (3,3’,5,5’-tetramethylbenzidine), TRIS (tris(hydroxymethyl)aminomethane), and UDP (uridine diphosphate).

[0050] As used herein, the general terms "sugar" or "glycan" refer to natural or unnatural sugars or glycans, including glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), fructose (Fru), xylose (Xyl), N-acetylneuraminic acid (Neu5Ac), and N-glycolylneuraminic acid (Neu5Gc), and derivatives thereof (such as glucosamine (GlcN), galactosamine (Gain), N-acetyl-glucosamine (GlcNAc), N-azidoacetylglucosamine (GlcNAZ), N-acetylgalactosamine (GlaNAc), (tri)mannose, N-glycan, and azidomannose).

[0051] The term "trimannose core" refers to a tri-mannosyl core as represented by the following formula (1), in which two mannose (Man 2 and Man 3 ) moieties are linked to a first mannose (Man 2 and Man 3 ) moiety at a bifurcated position between two other mannose (Man 1 ) moieties via an α-1,3 glycosidic bond or an α-1,6 glycosidic bond, and the three connected mannose (Man 1 , Man 2 , and Man 3 ) moieties are included.

[0052] Examples of the first target ligand and the second target ligand are biological molecules or materials. The term "material" can be added as a ligand to the reactive compound of the present invention and is a solid support that is bound to or can bind to one or more of moieties R 1、 R 2、 and / or R3 (or via one or more), and is also bound to or can bind to a GlcNAz (non-natural N-azidoacetylglucosamine) group.

[0053] The term "biological molecule" refers to biological molecules that can be synthesized or purified and have favorable target-directed properties. Preferably, the biological molecule is capable of binding to or is bound to other biological molecules such as proteins and peptides that target other similar biological molecules such as antigens or receptors. Alternatively, and even more preferably, these "biological molecules" are selected from the group consisting of sugars (including polysaccharides), nucleic acids (DNA or RNA sequences, or specific nucleic acid probes of hundreds of nucleotides capable of binding to complementary sequences), lipids, or mixtures of two or more of these. and the like), lipids, or mixtures of two or more of these.

[0054] The term "protein" includes polypeptide or peptide molecules having an amino acid sequence, including natural amino acid sequences, as well as variants and modified forms regardless of their origin or method of preparation. These proteins may optionally be modified by glycosylation (by addition of N-linked, O-linked, and / or C-linked glycans), and / or by addition of groups to form phosphorylated proteins. Preferably, these proteins are therapeutic moieties and / or target-directed moieties such as antibodies and the like, cytokines, growth factors, hormones, interferons, blood coagulation proteins, erythropoietin (EPO), G-coupled receptors (GPCRs) or molecules capable of binding to these receptors, enzymes such as glucosaminyltransferase, galactosidase, neuraminidase, N-acetylglucosaminidase, mannosidase, mannosyltransferase, urokinase, and DNAse.

[0055] The term "antibodies and the like" includes monoclonal chimeric antibodies or polyclonal antibodies (especially those obtained from homo- or hetero-hybridomas or from serum), and immunoglobulins or specific fragments of these antibodies, such as the hypervariable portion of the antibody or the so-called immunoglobulin variable domain (IVD) (including the complementarity-determining regions (CDRs) of the antibody, preferably Fab fragments, such as F(ab) fragments, F(ab’)2 fragments, Fv fragments, Fc fragments, and scFv-Fc fragments), and also proteinaceous, target-directed molecules that exhibit the same specificity and target-directed properties as the antibody. Preferably, the proteinaceous target-directed antibodies and the like are selected from the group consisting of bispecific antibodies, mini-antibodies, humanized antibodies, pegolyzated antibodies, nanobodies, alphabodies, microbodies, affitins, fimos, affilins, affimers, or mixtures of two or more thereof.

[0056] Antibodies are proteins produced by the immune system and are capable of recognizing and binding to specific antigens. Due to their sensitivity and specificity properties, antibodies are often used as molecular tools for investigative and diagnostic purposes, as well as for targeting specific sites (i.e., epitopes) in therapeutic or prophylactic methods.

[0057] A preferred example of a first ligand that is a biological material of interest for forming the complex of the present invention. This is because antibodies often present the trimannose core of the reactive compounds of the present invention. This arrangement enables one of ordinary skill in the art to directly incorporate an N-azidoacetylglucosamine group at the R 1 position of the first mannose (Man 4 ) moiety as described above.

[0058] Preferred examples of antibodies are those that may target tumor cells and can be used against cancer. These molecules can be produced by various types of prokaryotic or eukaryotic cells, preferably CHO or HEK293 cells, and can also be modified to express certain types of antibodies (i.e., immunoglobulin-type species) that target specific antigens or specific epitopes of antigens.

[0059] Preferred examples of proteins are tumor necrosis factor α receptor / IgG fusion peptide, chimeric anti-glycoprotein IIb / IIIa antibody, chimeric anti-Her2 antibody, chimeric anti-RSV antibody, chimeric anti-CD20 antibody, chimeric anti-tumor necrosis factor antibody, anti-RSV antibody, anti-IL2 antibody, and anti-CEA antibody.

[0060] Preferred examples of antibodies are cetuximab (registered trademark) (CTX) (commercially available as Erbitux (registered trademark)), which targets the epidermal growth factor receptor (EGFR) and is used for the treatment or prevention of colorectal and head and neck cancers, and trastuzumab (registered trademark) (commercially available as Herceptin (registered trademark)), which targets the HER2 receptor and is used for the treatment or prevention of certain types of breast cancer.

[0061] Other examples of proteins or molecules that may be modified by the addition of the reactive compounds of the present invention are glucosaminyltransferase, GnT1, GnT2, GnT3, MGAT3, galactosidase, neuraminidase, N-acetylglucosaminidase, mannosidase, mannosyltransferase, Alg 2, UDP-GlcNAz, GDP-ManAz,....

[0062] The term "solid support" refers to any support made from any solid material, preferably, however, a material selected from the group consisting of binding pair elements, metal supports, polysaccharide supports (such as gels), glass supports, ceramic materials or polymer supports, such as homopolymers and copolymers, composites, or combinations of two or more of these. This solid support can have any shape or form, such as beads (including magnetic beads), strips, plates, or mixtures thereof. The solid support is preferably made from a material selected from the group consisting of glass, polymeric structures such as latex particles or beads, metals or metal alloys, such as metal particles or metal beads (including magnetic particles or magnetic beads), silicon particles or silicon beads.

[0063] The solid support can be modified to include a linking group, one or more additional (substituted or unsubstituted and / or branched or unbranched) polyethylene glycols (PEGs), such as (OC2H4)n chains (where n is an integer preferably including 1 to 20, more preferably including 1 to 10, or 1 to 5), one or more additional alkyl groups, another additional aryl group (with 5 to 12 carbons, with or without one or more heteroatoms), one or more additional alkenyl groups, one or more additional alkene groups, one or more additional alkyne groups, or one or more additional alkynyl groups, so as to be able to bind to a target material, particularly a target biological molecule such as a protein.

[0064] The term "reactive compound" means a compound made of sugars that binds to a target first material or solid support to form a complex, which improves the properties of the target first material, or creates new binding possibilities for the target first material, or targets molecules to which it can bind, particularly a compound that can bind the created complex to one or more other target molecules such as a new antigen structure (in this specification, particularly the tri-mannose core made of Man 1 , Man 2 , and Man 3 ).

[0065] The terms "complex" and "gluco-complex" refer to a first material or molecule of interest, such as an element of a protein or a solid support, which is preferably linked by a covalent bond to a reactive compound, optionally to at least one other second material or molecule of interest (which is preferably different from the first material or molecule of interest).

[0066] The term "label" refers to a chemical or biochemical group that can be easily detected via its molecular size and / or is capable of producing a detectable signal, such as a fluorescent label, preferably a fluorophore (including but not limited to cyanines and fluoresceins), a chromophore label, an electron density label, a chemiluminescent label, a radioactive label, an enzyme label (preferably an enzyme including but not limited to HRP, AP, and β-lactamase), a positron emitter, a metal particle, preferably a silver particle or a gold colloid capable of reacting with a member of a first binding pair to create a colorimetrically detectable label.

[0067] Preferably, the binding pair is made from a derivative of the binding pair, which is preferably available commercially or by synthesis, and is preferably a member of the first binding pair and is capable of specifically binding to a second member of the binding pair (avidin or streptavidin, or an antibody-hapten conjugate (including but not limited to digoxigenin, dinitrophenol, FAM...)), and is made from a biotin molecule or an iminobiotin molecule.

[0068] For the purposes of the present invention, the term "drug" means a therapeutic molecule capable of curing or killing target cells, preferably target human cells such as tumor cells. These drugs are cytostatic or cytotoxic agents well known to those skilled in the art. Preferably, the drug is a maytansinoid (a macrolide maytacin derived from a plant of the genus Maytenus), an auristatin (obtained from a dolastatin peptide), a calicheamicin (an enediyene antibiotic derived from the bacterium Micromonospora echinospora), an antimetabolite, for example, but not limited to, fluorouracil, floxuridine, methotrexate, leucovorin, hydroxyurea, thioguanine, mercaptopurine, cytarabine, pentostatin, fludarabine phosphate, cladribine, asparaginase, gemcitabine, capecitibine, azathioprine, cytosine methotrexate, trimethoprim, pyrimethamine, or pemetrexed, an alkylating agent, for example, but not limited to, melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, dacarbazine, mitomycin C, cyclophosphamide, mechlorethamine, uramustine, dibromomanitol, tetranitrate, procarbazine, altretamine, mitozolomide, or temozolomide, an anthracycline, for example, but not limited to, daunorubicin, doxorubicin, epirubicin, idarubicin, or valrubicin, an antibiotic, for example, but not limited to, dactinomycin, bleomycin, mithramycin, anthramycin, streptomycin, or gramicidin D, calicheamicin, a mitotic inhibitor, dolastatin, cryptophycin, a vinca alkaloid, for example, but not limited to, vincristine, vinblastine, vindesine,Or a taxane such as virorelbine, for example, but not limited to, paclitaxel, or a topoisomerase inhibitor such as docetaxel, for example, but not limited to, irinotecan, topotecan, camptothecin, etoposide, teniposide, amsacrine, or mitoxantrone, a proteasome inhibitor such as, for example, but not limited to, peptidyl boronic acid, an HDAC inhibitor such as, for example, but not limited to, vorinostat, romidepsin, tidamide, panobinostat, or belinostat, a radioisotope, a radiopharmaceutical, a salt of these molecules, or a mixture of two or more of these.

[0069] According to the present invention, the drug is any suitable cytotoxic compound, for example, auristatin E, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblatine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracinedione, mitoxantrone, mithramycin, actinomycin D, procaine, tetracaine, lidocaine, propranolol, puromycin, immunotoxin, for example, endotoxin A of pseudomonasor, and salts, analogs, or homologs of these molecules.

[0070] Such a complex of a drug and an antibody is called an "antibody-drug conjugate" or ADC. ADCs typically feature a greater therapeutic effect and / or lower toxicity than natural drugs. Examples of ADCs known in the art include gemtuzumab ozogamicin (registered trademark) (used in leukemia and cancer treatment), inotuzumab ozogamicin (registered trademark) (used against lymphoma), trastuzumab emtansine (registered trademark) (used against breast cancer), Kadcyla (registered trademark) obtained from trastuzumab (registered trademark) (used to treat breast cancer), Mylotarg (registered trademark), Besponsa (registered trademark), and Adcetris (registered trademark).

[0071] Furthermore, according to the present invention, the drug can be selected from the group consisting of antimicrobial molecules (i.e., especially those against parasites such as species of the genus Trypanosomae or Plasmodium), antibacterial molecules, or antiviral molecules.

[0072] The term "radiopharmaceutical" includes any effective radioactive molecule used to diagnose and / or destroy cells, tissues, or organs affected by a disease, especially a part of a tumor, or a tumor, or an entire tumor cell or tissue.

[0073] Preferably, the radiopharmaceutical is selected from the group consisting of indium-111, yttrium-90, phosphorus-32, bismuth-212, iodine-131, iodine-123, cobalt-60, technetium, lutetium-177, or a mixture thereof.

[0074] The term "disease" refers to the health state of an animal (here, the health of this animal including humans is deteriorating). Preferably, this disease is caused by cells having hyperproliferative properties and / or harmful effects on the health of animals and humans, such as tumor cells and tissues.

[0075] The term "chemical-enzymatic method" refers to a method for producing a reactive compound or complex according to the present invention, the production method of which comprises one or more enzymatic reactions. Such enzymatic reactions are usually specific with respect to chemo-activity, target region, and / or stereospecificity, thereby ensuring the structural homogeneity of the resulting product.

[0076] The term "enzyme MGAT3" refers to any enzyme encompassed by the present invention that has a sequence identity of less than 100%, preferably less than 90%, less than 80% but more than 70% with the wild-type MGAT3 enzyme sequence, except that it exhibits the same activity / capability of adding a terminal N-azidoacetylglucosamine (GlcNAz) group to the first mannose Man of the trimannose core of formula 1. 1

[0077] The "similarity" of two amino acid sequences is determined by comparing the amino acid sequence of a polypeptide and its conserved amino acid substitutions with the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods including, but not limited to, those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).

[0078] Preferred methods for determining identity are designed to maximize the fit between two or more sequences being tested. Methods for determining identity and similarity are embodied in publicly available computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, for example, the GCG program package (Devereux, J., et al, Nucleic Acids Research 12(1):387(1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S.F. et al, J. Mol. Biol. 215:403 - 410(1990)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al, J. Mol. Biol. 215:403 - 410(1990)). The well-known Smith Waterman algorithm can also be used to determine identity.

[0079] The present invention will be further described in the following detailed description of the invention and in the examples that comply with the included figures, both of which are shown as non-limiting preferred embodiments of the invention.

Modes for Carrying Out the Invention

[0080] The present invention relates to novel reactive compounds made from oligosaccharide structures based on the trimannose core (also called tri-mannosyl core) having one or more novel "clickable" units according to the general structure of Formula 1 as described above.

[0081] This novel configuration is Man in Formula 1 1It contains a non-natural monosaccharide that is N-azidoacetylglucosamine (GlcNAz) which can bind to or is bound to a suitable first ligand, preferably a first material or first molecule of interest, at the bifurcation position of the trimannose core which is a part.

[0082] The inventors have found that such novel configurations are very advantageous since they give rise to novel "click-reactive" units that limit unwanted consequences such as multiple isomers and reduced solubility during and after their preparation. These novel units enable the binding of a ligand which is the material (solid support) or molecule of interest at this position.

[0083] Furthermore, the obtained compounds of the present invention exhibit lower modification than the products found in the prior art and the products modified in a probabilistic manner as described in the prior art or via a site-selective manner. Specifically, the obtained compounds exhibit two modification sites that guarantee efficient structural homogenization useful for therapeutic applications and an estimated labeling degree of 0 to 2.

[0084] Furthermore, if the obtained complex is a modified antibody, the site-selective modification of this product guarantees a better immunoreactive fraction (IRF), which means that thanks to the use of site-selective modification, the proportion of antibodies that may lose their activity due to this modification is low.

[0085] Another advantage of the obtained compounds of the present invention is the minor modification of any protein bound to this compound for forming the complex of the present invention, and in particular, the glycan profile of the modified protein that results in better solubility.

[0086] As shown in Formula 1 and the drawings, the obtained reactive compounds are similar to a schematic scorpion, where the mouth of the scorpion is, conveniently, the bisecting mannose (or Man 1 ) moiety modified as described above.

[0087] Conveniently, the other two mannoses, Man 2 and Man 3 moieties can likewise be modified, for example, to add further ligands which are the material or molecule of interest. These further ligands which are the material or molecule of interest may be different or the same, and may be the same as the added ligands mentioned above, which are the second ligand which is the second material or second molecule of interest present at the mouth of the scorpion.

[0088] According to the present invention, the first ligand of interest is advantageously an antibody present at the end of the scorpion's tail, which is bound to the trimannose core of formula 1 by, for example, R1, and the further second ligand to be added is the second material or second molecule of interest present at the mouth of the scorpion, and the third ligand of interest and the fourth ligand of interest are optionally present at one or both ends of one of the blades of this schematic pair of scorpion pincers.

[0089] The addition of ligands to the reactive compounds of the present invention is advantageously obtained by so-called "click chemistry".

[0090] Unlike classical stochastic coupling protocols that use organic solvents and toxic reagents (carbodiimide, benzitriazole, alkenethiol, etc.), this invention is advantageously based on one or more enzyme reactions that occur in aqueous buffer and in the presence of (bio)catalysts, consuming less energy. Even if some reagents have to be produced by organic synthesis, this invention tends to be a more sustainable protocol.

[0091] The enzyme selected is advantageously accompanied, optionally, by cofactors (e.g., a sufficient amount of MnCl2, TRIS buffer,...) that facilitate this enzyme reaction, and is the first mannose (Man) of the trimannose core of formula 1. 1)MGAT3 glycosyltransferase (also called GnT-III or β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase), as an enzyme for adding a terminal N-azidoacetylglucosamine (GlcNAz) group to a portion.

Example

[0092] Example 1: Generation of the complex according to the present invention Step 1: Buffer exchange The buffer of the antibody (1 equivalent, Ci 22 μM, 3.3 mg·mL-1, Cf 6.47 μM, 0.97 mg·mL-1, 50 μg) was exchanged into TRIS buffer (100 mM, pH = 7) by dialysis (Mini D-Tube (trademark) dialyzer from Merck Millipore, Ref#71504-3) during 4 buffer exchanges in 16 hours.

[0093] Step 2: Enzyme modification. Subsequently, UDP-GlcNAz (25 equivalents, Ci 0.1 M, Cf 0.16 mM, 5.4 μg), enzyme MGAT3 (0.31 equivalent, Ci 6.9 μM, 0.4 mg·mL-1, Cf 0.8 μM, 0.12 mg·mL-1, 6 μg), and MnCl2 (204 equivalents, Ci 0.1 M, Cf 1.3 mM, 8.3 μg) were added to the buffer-exchanged material, and the mixture was incubated overnight at 37 °C.

[0094] Step 3: Complex isolation To prove the presence of the complex, ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) is used.

[0095] The UHPLC-HRMS analysis reported below was performed by using an Acquity liquid chromatograph connected to an Xevo G2-XS QT of a quadrupole time-of-flight system (Waters) equipped with different columns and having an electrospray ionization (ESI) source.

[0096] The protein containing the complex was first diluted to 0.1 mg mL-1 with HPLC-grade water and brought to a final volume of 100 μL before UHPLC-HRMS analysis.

[0097] Calibration was performed using Leu-enkephalin (m / z 555.26). The data was processed using UNIFI® software (Waters, Milford, MA, USA), and the inverse deconvolution of the combined raw spectra was performed using Masslynx® on UNIFI® with MaxEnt1 settings. Here, 10 μL of the sample diluted with a DMF / ACN solution was injected into an AQUITY UPLC Glycan BEH Amide, 130 Å, 1.7 μm, 2.1×150 mm column (Waters, Ref#186004742) heated to 60 °C.

[0098] The mobile phase conditions used were as follows: mobile phase A was a 50 mM ammonium formate solution, pH 4.4, and mobile phase B was 100% acetonitrile.

[0099] The gradient conditions were as follows: 0 - 35 min flow rate 0.4 mL·min-1 25% A, 35 - 36.5 min flow rate 0.4 mL·min-1 46% A, 36.5 - 43.1 flow rate 0.2 mL·min-1 100% A, 43.1 - 47.6 min flow rate 0.2 mL·min-1 25% A, and finally 47.6 - 55 min flow rate 0.4 mL·min-1 25% A.

[0100] The settings used were as follows: FLR wavelength EX 265 / EM 425 nm, FLR sampling rate 2 Hz, source temperature = 120 °C; gas flow = 800 L·h-1; capillary voltage = 3 kV; cone voltage = 80 V; desolvation temperature = 350 °C; positive ion mode; scan mode = positive sensitivity; mass range scanned = 500 - 2,000 m / z. Mass range calibration (NaI, 6.7 μM, 1 μg·mL-1) m / z includes 500 - 2,000.

[0101] N-glycan release and functionalization were achieved by using the Waters kit GlycoWorks RapiFluor-MS N-Glycan kit (registered trademark) (Waters, Ref# 176003713) and adapting UHPLC-HRMS by connecting to an AQUITY FLR (fluorescence) detector (registered trademark) (λex = 265 nm, λem = 425 nm) used to detect the product after its release.

[0102] More specifically, a compound having an antibody (13.3 μM, 2 mg·mL-1, 7.5 μL, 15 μg) was solubilized with an anionic surfactant RapiGest (Waters, Ref# 186001861, 5% w:v Glycoworks Rapid Buffer, in 6 μL) and HRMS grade water (15.3 μL). This solution was mixed by gentle pipetting and then heated at 90 °C for 3 minutes to induce protein denaturation. The solution was cooled for 3 minutes and then PNGase F was added (Rapid PNGase F, 1.2 μL) and incubated at 50 °C for 5 minutes. The sample was cooled at room temperature for 3 minutes and then a labeling solution (12 μL, 0.16 M, in 68.7 mg·mL-1 DMF) was added.

[0103] This mixture was homogenized by pipetting and after a 5-minute incubation at room temperature, it was diluted with 358 μL of ACN for subsequent separation by HILIC SPE step.

[0104] HILIC SPE step: The GlycoWorks HILIC μelution plate (registered trademark) was conditioned with HPLC grade water (200 μL / well) and then equilibrated with water / ACN (15:85 vol:vol, 200 μL).

[0105] The sample was loaded into a well (400 μL), then washed with a solution of formic acid / water / ACN (1:9:90 vol:vol, 2 × 600 μL), and finally eluted with Glycoworks SPE Elution buffer (200 mM ammonium acetate in 5% acetonitrile, 3 × 30 μL).

[0106] These eluates (90 μL) were further diluted with GlycoWorks® Sample Diluent (DMF / ACN, 30:70, 310 μL). The sample was then processed by HRMS or stored initially at 4 °C for at least 3 days or at -80 °C for long-term storage.

[0107] For example, the attachment on a non-natural glucosamine derivative bisecting trisaccharide core in the modification of G0(F) glycan (CAS No. 84825-26-3) is depicted in Figure 1b. This corresponds to the core form found in glycoproteins and antibodies in most mammalian species and in therapeutic proteins. Attachment of one or more mannose residues results in modification of the N-glycan core pentasaccharide moiety (M3) depicted in the drawing.

[0108] The inventors have the trisaccharide core (Man of the obtained compounds of the present invention 1The terminal N - azidoacetylglucosamine (GlcNAz) group that bisects the partial or first mannose) is added with suitable reactive ligands such as a molecule (biotin), a drug (ozogamicin, emtansine), or a label (including but not limited to cyanines and fluoresceins, fluorophores), etc., and complexes made of antibodies linked to these reactive ligands can be formed by various classical complex reactions, and these novel antibody complexes have been tested for their therapeutic or diagnostic uses. These transformations are realized according to known procedures of "click chemistry" in the presence of copper or "copper - free", such as those reported by Van Geel and co - workers (Bioconjugate Chem. 2015, 26, 11, 2233 - 2242, 2015 https: / / doi.org / 10.1021 / acs.bioconjchem.5b00224) or Thompson and co - workers (ACS Med Chem Lett. 2016 Nov 10;7(11):1005 - 1008).

[0109] The inventors bisect the trisaccharide core (Man 1 of the obtained compounds of the present invention with a non - natural glucosamine derivative (terminal N - azidoacetylglucosamine (GlcNAz) group) that bisects the partial or first mannose), and add suitable inorganic nanoparticles (such as gold, iron, silica, etc.) and organic nanoparticles (e.g., poly(lactide - co - glycolide), PLGA, poly(methyl methacrylate), PMMA, poly(ethylene glycol), PEG), and complexes made of antibodies linked to these reactive compounds can be formed by various classical complex reactions, and these novel protein - nanoparticle complexes have been tested for their therapeutic or diagnostic detection uses. These transformations are realized according to known procedures of "click chemistry" between azide and alkyne groups, such as those reported by Finetti et.al, Langmuir 2016, 32, 29, 7435 - 7441, 2016.

[0110] The inventors added suitable biological particles (such as virus particles, liposomes, and exosomes) to a non-natural glucosamine derivative (terminal N-azidoacetylglucosamine (GlcNAz) group) that bisects the trisaccharide core (Man 1 moiety or the first mannose) of the compounds obtained in the present invention, and were able to form complexes made of antibodies linked to these biopharmaceuticals through various classical conjugation reactions. These antibody complexes have been tested for their therapeutic or diagnostic detection applications. These transformations are realized according to known procedures of "click chemistry" between azide and alkyne groups, such as those reported by Park et.al, (Bioconjug Chem. 2020 May 20;31(5):1408-1416, for virus particles), Smyth et al. (Bioconjugate Chem. 2014, 25, 10, 1777-1784, for exosomes), and Gai et al. (Polym.Chem., 2020, 11, 527-540, for liposomes).

[0111] To improve the properties of the complexes obtained in the present invention, additional reactions were also realized by the inventors, including further binding of these reactive ligands (macromolecules, drugs, and labels) to the linking groups (non-natural terminal N-azidoacetylglucosamine (GlcNAz) groups) fixed to the other mannose Man 2 and Man 3 moieties (as the second mannose and the third mannose) of the obtained compounds. These "bifunctional" derivatives are composed of one drug introduced at the R4 position and one tracer (e.g., Zr 89 ) linked to the R2 or R3 position, or a tracer introduced at the R4 position and a drug located at R2 or R3, based on the bioconjugation protocol reported by Adumeau (Mol.Pharm., 2018, 15, 892-898).

[0112] The reactive compounds according to the present invention enable the modulation of biological functions and site-selective modification at two levels of structural hierarchy: At the oligosaccharide level: The presence of non-natural units determines its chemical reactivity (including but not limited to bioconjugation) by the click chemistry protocol described above. Thus, derivatives showing a bifurcated trimannose core modulate several biological functions, among others, fertility, cancer, and neurodegenerative diseases. The reactive compounds according to the present invention can be applied as an alternative to current technological solutions in the field of dynamic in vivo imaging (Bertozzi et al. Proceedings of the National Academy of Sciences 104.43 (2007): 16793 - 16797), and also for the production of glycoconjugates (major constituents of mammalian cells formed via covalent bonds between carbohydrates and other biomolecules such as proteins and lipids and often expressed on the cell surface) (Shivatare et al., Chem. Rev. 2022, 122, 20, 15603 - 15671). At the macromolecule level: This compound enables new functions such as solubilization (Ma et al. Front. Chem., 23 July 2020, 8, 2020), the reactivity described above, and biological functions described below, in spatially defined regions of a target molecule or material (e.g., the sequence Asn - X - Ser / Thr, where X can be any amino acid other than proline) that may be naturally or artificially exposed, which can be part of a biologically occurring structure including, but not limited to, N - and O - glycans. This product is intended for industrial use and provides solutions for scientific research, such as the following:

[0113] The biological functions and reactivity of proteins, peptides, glycol - peptides, glycolipids, nucleic acids located inside, on, or outside the cell membrane (e.g., cell membrane proteins exposed to the product). Examples thereof are: constructing azide - labeled cell surfaces (Bertozzi et al. Methods in Enzymology 362, 2003, 249), metabolic labeling of glycans with azide sugars and subsequent glycan profiling and visualization (Bertozzi et al. Nat Protoc. 2007;2(11):2930 - 44), alteration of glycan permissibility by carbohydrate - processing enzymes (Liu et al. ACS Cent Sci. 2022 May 25;8(5):656 - 662), in vivo cell - based analysis of glycosynthase activity (Agrawal et al. ACS Chem. Biol. 2021, 16, 11, 2490 - 2501), modification of glycosaminoglycans (among several uses, particularly) (Bioorthogonal chemistry, Nature Reviews Methods Primers vol.1, 30, 2021).

[0114] The need for diagnostic and therapeutic products where optimal bioactivity and control over functionality are key: Examples are related to the products and procedures reported in "The Chemistry of Creating Chemically Programmed Antibodies (cPAbs): Site-Specific Bioconjugation of Small Molecules", Mol. Pharmaceutics 2023, 20, 2, 853 - 874. The biological functions and reactivities of proteins, peptides, glycopeptide - peptides, glycolipids, nucleic acids once administered to biological systems (cells, animals, humans) act inside, on, or outside the cell membrane (e.g., therapeutic proteins); examples are related to the products and procedures reported in "An Overview of Recent Advances in Biomedical Applications of Click Chemistry", Bioconjugate Chem. 2021, 32, 8, 1455 - 1471.

[0115] Example 2: Regulation of Biological Function When the product exhibits click - reactive N - azidoacetylglucosamine (GlcNAz) as a bisecting unit of the trimannose core, this can be used to confer biological function on a molecule or (nano) material (derived from natural or artificial sources) with respect to its native counterpart lacking such modifications, such as the structures reported in the drawings. Such biological functions include, but are not limited to, regulation of neurodegenerative diseases, immune tolerance, IgG function, tumor metastasis, and development, and these are detailed below.

[0116] Regulation of Neurodegenerative Diseases Bisecting GlcNAc modification has been shown to favorably stabilize the BACE1 protein under conditions of oxidative stress (EMBO Mol. Med. 7, 175 - 189, doi:10.15252 / emmm.201404438). An increase in the content of bisecting GlcNAc in the Alzheimer's disease brain may function as an adaptive response to protect the brain from damage caused by further beta-amyloid production (Glycobiology 20, 99 - 106, doi:10.1093 / glycob / cwp152).

[0117] Lack of bisecting GlcNAc on BACE1 induces the transport of this protein to lysosomes and accelerates its degradation, which led to less accumulation of beta-amyloid in Alzheimer's disease (Glycoconj. J. 35, 345 - 351. doi:10.1007 / s10719-018-9829-4). These findings highlight the importance of bisecting GlcNAc modification in the nervous system.

[0118] Regulation of immune tolerance The bisecting GlcNAc structure has been reported to have immunosuppressive functions. For example, K562 cells are readily killed by natural killer (NK) cells; however, after transfection with the gene encoding GlcNAc-T III, K562 cells with more bisecting GlcNAc acquire NK cell resistance (Mol. Hum. Reprod. 3, 501 - 505. doi:10.1093 / molehr / 3.6.501).

[0119] Natural killer (NK) cells are a major type of immune cells found in the human uterus, which indicates that these cells potentially target sperm (Mol. Hum. Reprod. 20, 185 - 199. doi:10.1093 / molehr / gat064). Human sperm have been found to express bisecting GlcNAc structures, which explains why sperm are not killed by the maternal immune system when they enter the female as a foreign substrate and thus support hu-FEDS (Biomed Res. Int. 2019:5397804. doi:10.1155 / 2019 / 5397804).

[0120] Furthermore, abundant bisecting GlcNAc glycans were detected in human syncytiotrophoblasts (STB) and cytotrophoblast cell layers (CTB) (Mol. Cell. Proteomics 15, 1857 - 1866. doi:10.1074 / mcp.M115.055798). It is most likely that the maternal immune system was suppressed due to the presence of bisecting GlcNAc glycans and the fetus benefited from this suppression; the mother would be able to nourish the fetus in her body for several months without rejection (similar to a foreign organ where the father contributes half of its genome).

[0121] A possible mechanism underlying this suppression could be that the glycol - complex interacted with a lectin associated with specific signaling pathways that regulate immune cell function. For example, α - 2,3 - linked sialic acid on soluble CD52 (i.e., a 12 - amino - acid glycoprotein anchored to glycosylphosphatidylinositol) could mediate T - cell suppression by binding to Siglec - 10 (Front. Immunol. 10:1967. doi:10.3389 / fimmu.2019.0196). Bisecting GlcNAc may be able to function in a similar manner to suppress NK cells.

[0122] Regulation of IgG Function and Properties Fc-glycan affects the biological activity of IgG. For example, the absence of fucose in Fc-glycan significantly improves the binding to human FcγR III, and this result is applied to improve the efficacy of therapeutic monoclonal antibodies. The attachment of bisecting GlcNAc to Fc-glycan will induce antibody-dependent cell cytotoxicity (ADCC).

[0123] In another approach, the inventors demonstrated the importance of site-selective modification (GlcNAz at the R4 position) compared to probabilistic modification via IRF calculation. Kinetic ELISA experiments were performed to calculate the immunoreactivity rates of different functionalizations of monoclonal antibodies, including probabilistic biotinylation and site-specific azidation. Using a native antibody with an IRF of 100%, probabilistic functionalization was found to reduce the IRF to 71%, while site-specific modification (R4 = GlcNAz) maintained the IRF at 92%. Here, a protocol using rabbit monoclonal Ab induced against soybean protein (SPI) is described: Two ELISA plates were coated overnight at 4°C, one with different SPI concentrations (Ci 6.1 μM, 0.91 mg·mL-1, 200 μL, 0 - 3.57 - 7.14 - 14 - 20 - 28 - 43 - 57 - 86 - 114 - 171 - 229 10~14 mol / well), for rows A - D, columns 1 - 2 at 0 10~14 mol·L-1, columns 3 - 4 at 3.57 10~14 mol·L-1, columns 5 - 6 at 7.14 10~14 mol·L-1, columns 7 - 8 at 14 10~14 mol·L-1, columns 9 - 10 at 20 10~14 mol·L-1, columns 11 - 12 at 28 10~14 mol·L-1, and for rows E - H, columns 1 - 2 at 43 10~14 mol·L-1, columns 3 - 4 at 57 10~14 mol·L-1, columns 5 - 6 at 86 10~14 mol·L-1, columns 7 - 8 at 114 10~14 mol·L-1, columns 9 - 10 at 171 10~14 mol·L-1, columns 11 - 12 at 229 10~14 mol·L-1, and the other with SaR (6 10~12 mol / well). The wells were aspirated and a saturation solution was added (PBS 1% BSA, 250 μL) for 2 hours at 37°C. The plates were washed 3 times with PBS and then RabmAb was added to the SPI-coated plate (5 10~14 mol / well, 150 μL). The plates were incubated overnight at 4°C, then the supernatant was removed (125 μL) and placed into a plate coated with sheep anti-rabbit. The SPI-coated plate was washed 3 times with PBS and the SaR-coated plate was incubated at 37°C for 2 hours.Both plates were washed three times with PBS and incubated with secondary antibody goat anti-rabbit-HRP (Ci 5 μM, 1 mg·mL-1, Cf 10 nM, 2 μg·mL-1, 150 μL). Color development was carried out with TMB (150 μL) and stopped with H2SO4 (3 M, 50 μL) at different times for each row. Rows A and E were stopped at 0 s, rows B and F at 15 s, rows C and G at 30 s, and rows D and H at 45 s [Moreno, et al. J. Immunol. Methods 476, 5-10 (2020)].

[0124] Regulation of tumor metastasis and occurrence Bisecting GlcNAc structures were able to inhibit hypoxia-induced epithelial-mesenchymal transition in breast cancer (Front. Physiol. 9:210. doi:10.3389 / fphys.2018.00210). However, the underlying mechanism is still unclear. It is speculated that the addition of bisecting GlcNAc to key glycoproteins in signal transduction, such as growth factors, integrins, and cytokine receptors, has its special signal transduction intensity under hypoxia. In fact, the findings of non-solid tumors contradict this explanation. GlcNAc-T III is more activated in patients with chronic myeloid leukemia in blast crisis (CML-BC) and also in patients with multiple myeloma (MM) (Biochem. J. 331 (Pt 3), 733-742. doi:10.1042 / bj3310733).

[0125] When the product shows click-reactive mannose, this can be used to confer biological functions to molecules or (nano)materials (derived from natural or artificial raw materials) with respect to their natural counterparts lacking such modifications, such as the structures reported in the drawings.

[0126] These biological functions include, but are not limited to, among others, synthetic hormone analogs (Org. Biomol. Chem., 2014, 12, 8142 - 8151, doi:10.1039 / C4OB01208A), binding to HIV and other viruses (J Biol Chem 280:29269 - 29276, DOI:10.1074 / jbc.M504642200), therapeutically valuable diabetes, glycosylation of peptide drugs (Front. Chem., 12 2021, doi:10.3389 / fchem.2021.65002), and regulation of IgG function.

[0127] Example 3: Regulation of Molecular Reactivity Once the product becomes part of a molecule or (nano) material derived from natural or artificial raw materials, this product exhibits typical reactivity of azide groups, such as reactions with alkynes and alkenes, phosphines (The Chemistry of the Azido Group ISBN:978 - 0 - 470 - 77126 - 6), enabling bioconjugation with all classes of molecules of diagnostic and therapeutic interest. Examples are those described in "An Overview of Recent Advances in Biomedical Applications of Click Chemistry", Bioconjugate Chem. 2021, 32, 8, 1455 - 1471.

[0128] Reaction with Molecules of Diagnostic Interest In this regard, derivatives of biotin (e.g., but not limited to, including iminobiotin), fluorophores (e.g., including cyanines and fluoresceins), enzymes (including but not limited to HRP, AP, and β-lactamase), and other antibody-hapten conjugates (including but not limited to digoxigenin, dinitrophenol, and FAM) that are commercially available or obtainable by synthesis are reference molecules. Examples thereof are those reported in "An Overview of Recent Advances in Biomedical Applications of Click Chemistry", Bioconjugate Chem. 2021, 32, 8, 1455-1471, and "Click Chemistry in Biomaterials, Nanomedicine, and Drug Delivery", Biomacromolecules 2016, 17, 1, 1-3.

[0129] Reaction with molecules of therapeutic interest In this regard, commercially available or synthetic labels, such as tracers for in vivo imaging (including, but not limited to, chelating agents such as DOTA), drugs (including, but not limited to, anti-tumor compounds such as monomethyl auristatin E, as well as antimicrobial and antiviral molecules), or macromolecules (including, but not limited to, PEG, proteins, lipids, nucleic acid sequences), available derivatives are reference molecules. Examples thereof are those reported in "An Overview of Recent Advances in Biomedical Applications of Click Chemistry", Bioconjugate Chem. 2021, 32, 8, 1455-1471, and "Click Chemistry in Biomaterials, Nanomedicine, and Drug Delivery", Biomacromolecules 2016, 17, 1, 1-3, and "The Chemistry of Creating Chemically Programmed Antibodies (cPAbs): Site-Specific Bioconjugation of Small Molecules", Mol. Pharmaceutics 2023, 20, 2, 853-874, and "Click chemistry: a transformative technology in nuclear medicine", Nat Protoc (2023). https: / / doi.org / 10.1038 / s41596-023-00825-8.

Claims

1. Formula 1: 【Chemistry 1】 (In the formula, - Man １ Man ２ , and Man ３ The parts are the first mannose, the second mannose, and the third mannose, respectively. -R １ This is a linking group capable of or already having an N-acetylglucosamine (GlcNAc) group attached to a first ligand. -R ２ It is a monosaccharide that is optionally linked to one or more additional sugars, -R ３ It is a monosaccharide that is optionally linked to one or more additional sugars, - N-azidoacetylglucosamine (GlcNAz) is a non-natural N-azidoacetylglucosamine group that is capable of or bound to a second ligand different from the first ligand. A reactive compound containing trimannous core.

2. R １ The reactive compound according to claim 1, wherein the linking group is selected from the group consisting of monosaccharides and disaccharides.

3. R １ The reactive compound according to claim 2, wherein the reactive compound is optionally further fucosylated GlcNAc or GlcNAc-GlcNAc group.

4. R ２ and / or R ３ The reactive compound according to any one of claims 1 to 3, wherein R and / or R further comprises a terminal GlcNAz (N-azidoacetylglucosamine) group.

5. A complex of at least a first ligand and a second ligand, wherein the first ligand and the second ligand are different materials or molecules, and the first ligand and the second ligand are bound together by the reactive compound described in claim 1.

6. The complex according to claim 5, wherein the first ligand is a biological molecule selected from the group consisting of proteins, nucleic acids, sugars, lipids, or mixtures of two or more thereof.

7. The complex according to claim 6, wherein the protein is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, an antibody hypervariable region, a nanobody, an alpha body, a microbody, an affitin, a fimomer, an affin, an affimer, or a mixture of two or more of these.

8. The complex according to any one of claims 5 to 7, wherein the second ligand is a material selected from the group consisting of macromolecules, drugs, or labels.

9. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and the reactive compound described in Claim 1 and / or the complex described in Claim 5.

10. A diagnostic kit comprising the reactive compound described in claim 1 and / or the complex described in claim 5, and means for producing a signal from a label.

11. A solid support made from the complex according to claim 5, wherein the first ligand is a material selected from the group consisting of beads, strips, or plates, and the second ligand is a protein.

12. The solid support according to claim 11, which is a solid support for cell culture.

13. A method for producing the reactive compound described in claim 1, - A step of selecting a trimannoscore or a first ligand containing the trimannoscore, - Uridine diphosphate N-azidoacetylglucosamine (UDP-GlcNAz) as a sugar donor, and the first mannose Man of the aforementioned trimannose score １ The steps include adding glycosyltransferase, an enzyme for attaching terminal N-azidoacetylglucosamine (GlcNAz) groups to a portion of the trimannos core, and - The step of purifying the obtained product by chromatography to obtain the reactive compound. Methods that include...

14. The method according to claim 13, wherein the glycosyltransferase is MGAT3 glycosyltransferase.

15. A method for generating the complex according to claim 5, comprising binding a reactive compound obtained from the method of claim 13 or 14 to the second ligand.

16. The method of claim 15, further comprising binding a reactive compound obtained from the method of claim 13 to the first ligand.

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