Bisected trimannose-oligosaccharides, bisected N-glycans comprising said trimannose core, and methods of obtaining same

Abstract

The present invention relates to a reactive compound made from a modified trimannose-oligosaccharide, to a process for the preparation thereof and to the use thereof for improving the characteristics of a first ligand of interest, especially in therapeutic or diagnostic applications. In particular, the present invention relates to a reactive compound comprising a tri-mannose core having formula (I) wherein the Man1, Man2 and Man3 moieties are respectively a first, second and third mannose, R1 is a linking group enabling the GlcNAc group to bind or bind to a first ligand of interest, R2 is a linking group enabling the GlcNAc group to bind to a second ligand of interest, and R3 is a linking group enabling the GlcNAc group to bind to a third ligand of interest. R2 and R3 are monosaccharides or derivatives thereof optionally attached to one or more additional sugars, and wherein GlcNAz is a non-natural N-azidoacetylglucosamine group capable of binding or binding to a second ligand of interest different from the first ligand of interest. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a reactive compound made of a modified trimannose-oligosaccharide capable of binding or binding to one or more target ligands, a method for its preparation and its use for improving the characteristics of such target ligands, in particular in therapeutic or diagnostic applications. Background Art

[0002] The structural modification of the target ligand is mostly based on the reactivity of carboxyl, amino, hydroxyl or thiol groups, which can be simultaneously and advantageously present repeatedly in the target ligand (e.g., protein, sugar, lipid). This leads to structural heterogeneity in terms of product number (molecules with one or more modifications) and isomerism (differences in the spatial arrangement of modifications).

[0003] In these examples of ligands of interest, known (monoclonal or polyclonal) antibodies are functionalized with one or more groups or molecules (be they molecular probes, drugs or fluorophores) via coupling schemes involving cysteine ​​or lysine residues naturally present on the polypeptide backbone.

[0004] These modifications generally confer new properties to these modified antibodies, such as improved function or improved reactivity, which can be used for diagnostic or therapeutic purposes. Effective and useful molecular modifications can consist of random modifications. However, these modifications may result in reduced binding / functional properties compared to the unmodified precursor, because a large portion of different structures are generated by the procedure of carrying modifications (group additions) close to functional sites (e.g., antigen-binding fragments of antibodies).

[0005] However, the known random protocols often result in heterogeneous antibody batches in terms of the amount and spatial distribution of the payload in the processed antibodies. In addition, the resulting antibodies are known to have lower activity when compared to their unmodified counterparts, since such random functionalization can occur on the variable regions of these antibodies, which include the functional sites responsible for antigen recognition.

[0006] On the other hand, site-specific modifications can lead to higher structural homogeneity, higher biological activity and easier further functionalization than those derived from random. The obtained modified molecules therefore become 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 at the whole organism or cellular level prior to biological production of the molecule) or ex vivo / ex-vitro (e.g., chemical manipulation of purified molecules). While in vivo / in vitro strategies are ideal solutions for specific cases and individual products, ex vivo / ex-vitro strategies are much more widely applicable (regardless of the case) and are less demanding in terms of time and economic resources.

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

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

[0010] Prior art

[0011] It is known that an N-glycosylation site exists at asparagine at position 297 (Asn297) in the heavy chain of a naturally occurring antibody, and two biantenna-shaped glycan moieties made of seven sugars are formed at this glycosylation site of the antibody.

[0012] The following publications describe a chemoenzymatic approach to preparing antibody-drug conjugates by targeting native glycosylation sites present within antibodies, which requires enzymatic labeling of native glycans with azide-carrying N-azidoacetylgalactosamine (GalNAz) by the addition of endo-β-N-acetylglucosaminidase: Van Geel, R. et al. (Bioconjug. Chem. 26, 2233-2242 (2015)), and Cai, X. & Janda, KDA (Tetrahedron Lett. 56, 3172-3175 (2015)).

[0013] It is known that terminal galactose increases cytotoxic reactions due to the complement system. Antibody glycosylation and its effects on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc fusion proteins are known, and bisecting GlcNAc can increase affinity for FcγRIIIa, thereby increasing antibody-dependent cellular cytotoxicity (ADCC). (Claudia Ferrara et al. Biotechnol. Bioeng. [Biotechnology and Bioengineering] 93, 851-861 (2006)).

[0014] For antibodies, it is known that the degree of labeling (DOL) plays an important role; indeed, an antibody with an overly important DOL will be eliminated faster and more likely to aggregate because it is more hydrophobic. (Publications: Agarwal, P. & Bertozzi, CR Bioconjug. Chem. 26, 176-192 (2015) and Van Geel, R. et al. (2015)).

[0015] It has been proposed in the prior art to engineer modified branch sites on antibodies to generate new functional diversity of modified antibodies. The N-glycosylation site on the antibody is located on asparagine position 297 (Asn 297 residue), which is very far from the variable region of the antibody and is therefore unlikely to interfere with antibody activity due to its paratope.

[0016] The presence of only two N-glycosylation sites means that the amount of payload that can be added to the glycan can be effectively controlled, thus allowing for better ratio regulation and homogeneity.

[0017] The following authors disclose modified bisected trimannosyl oligosaccharides having non-natural monosaccharides but obtained by chemical synthesis: Unverzagt C. et al. (Tetrahedron letters, Vol. 41, No. 23, (2000)), Eller S. et al. (Tetrahedron Letters, Vol. 51, No. 19 (2010)) and Schuberth R et al. (Tetrahedron Letters, Vol. 46, No. 24 (2005)).

[0018] The following publications disclose the native structure of bisected trimannosyl oligosaccharides, in which the bisected mannose includes a GlcNac residue that plays an important role in the properties of the resulting N-glycans: Nakano M. et al. (Molecular & Cellular Proteomics, Vol. 18, No. 10, pp. 2044-2057 (2019)) and Ferrara M et al. (PNAS, pp. 12669-12674 (2011)).

[0019] US11,085,062-B2 patent, European patent EP2305314-B1 ( Ratiopharm )), European Patent EP 1987068-B1 (Life Technologies), International Patent Application WO2014 / 065661 ( Synaffix ) and international patent application WO2016 / 170186( Genovese (Genovis) ) discloses a method for obtaining a conjugate between an antibody and a modifying group.

[0020] However, in these patent applications, the modifying groups are not added to the bisecting positions of the trimannosyl core (bisecting mannose or Man 1 ), because through Genovese The glycan modification performed was trimmed to the first GlcNac using the addition of GalNAz, with a degree of labeling (DOL) of 2, from Thermofisher The glycan modification of describes the trimming of glycans to G0 by the addition of GalNAz, with a degree of labeling (DOL) of 4, while (Sinafox) describes glycan modification using the addition of GalNAz to trim glycans to the first GlcNac with a degree of labeling (DOL) of 2.

[0021] Purpose of the Invention

[0022] The present invention aims to provide novel reactive compounds that can be used for modification of target ligands, especially (first) target biomolecules, wherein the novel reactive compounds can effectively form conjugates between the first ligand and the reactive compound and optionally at least one additional second target ligand.

[0023] A preferred object of the present invention is to obtain reactive compounds that can be effectively used to produce new conjugates, wherein the reactive compound is a linked structure that improves the binding of two, preferably two different target ligands to form the conjugate.

[0024] A specific object of the present invention is to obtain such reactive compounds and the conjugates obtained therefrom in order to improve the characteristics of the first target molecule while overcoming one or more disadvantages of the prior art.

[0025] More preferably, the object of the present invention is to obtain such a combination between an element of a solid support and a suitable ligand (preferably a biomolecule) that can be used for cell culture on this solid support, especially animal cell culture, including human cell culture, or for the screening and recovery of one or more new target ligands.

[0026] In particular, a preferred object of the present invention is to obtain reactive compounds for obtaining new conjugates comprising the addition of one or more groups (such as drugs) and characterized by a higher drug loading on this first target ligand (such as antibodies etc.), but without the disadvantages of the conjugates of the prior art, in particular in terms of homogeneity, reproducibility and solubility.

[0027] A more preferred object of the present invention is to obtain a conjugate,

[0028] The conjugate comprises a first target ligand, which is an antibody or the like, and is characterized by having a low degree of labeling (DOL) close to 2 when modified by the addition of a reactive compound of the present disclosure and an added second target ligand.

[0029] Another object of the present invention is to obtain a process for the efficient production of active compounds, in particular a simplified process, which comprises fewer process steps and is less complex or less time-consuming than prior art processes for obtaining similar conjugates.

[0030] A further object of the present invention is to obtain an improved conjugate comprising or made of a first target molecule (such as a glycoprotein, preferably an antibody, etc.) and connected to one or more drugs or markers via a reactive compound, in particular a conjugate improved by adding reactive groups fixed by the reactive compounds of the present invention and exhibiting improved characteristics when used in the field of diagnosis and in the prevention and / or treatment of plant, animal and human diseases, in particular cancer. Summary of the invention

[0031] A first aspect of the present invention relates to a reactive compound comprising a trimannosyl core having the formula (1),

[0032]

[0033] in

[0034] -Man 1 , Man 2 and Man 3They are the first mannose, the second mannose and the third mannose parts,

[0035] -R1 is a linking group that enables the GlcNAc group to bind or to the first ligand of interest,

[0036] -R2 is a monosaccharide or a derivative thereof,

[0037] - R3 is a monosaccharide or a derivative thereof, and

[0038] The -GlcNAz group is a non-natural N-azidoacetylglucosamine group that is capable of binding or attaching to a ligand, which is a second ligand of interest.

[0039] According to the present invention, 2 and Man 3 Same, Man 1 It is a mannose moiety, but is located at the bisecting position of the trimannose core.

[0040] Advantageously, in the reactive compound according to the invention, the two ligands are different.

[0041] According to the present invention, R1 is a linking group (linking the reactive compound to the first target ligand) preferably selected from the group consisting of monosaccharides or their derivatives and disaccharides or their derivatives, and optionally, this linking group further comprises the following components linked to these monosaccharides: one or more additional (substituted or unsubstituted and / or branched or unbranched) polyethylene glycol (PEG) (such as (OC2H4)n chain, where n is an integer, preferably between 1 and 20, more preferably between 1 and 10 or between 1 and 5), one or more additional alkyl groups, one or more additional aryl groups (5 to 12 carbons, containing or not containing one or more heteroatoms), one or more additional alkenyl groups, one or more additional olefin groups, one or more additional alkyne groups or one or more additional alkynyl groups.

[0042] Advantageously, R1 forms an N-acetylglucosamine-N-acetylglucosamine group (GlcNAc-GlcNAc group) with the first N-acetylglucosamine group. This GlcNAc-GlcNAc group may be further glycosylated, for example further fucosylated to enable binding or binding to the first target ligand.

[0043] Optionally, R2 and / or R3 are linked to one or more additional sugars to form a sugar chain.

[0044] According to a preferred embodiment of the present invention, R2 and / or R3 may also, like R1, contain one or more additional (substituted or unsubstituted and / or branched or unbranched) polyethylene glycol (PEG) (such as (OC2H4)n chain, wherein n is an integer, preferably an integer between 1 and 20, more preferably an integer between 1 and 10 or an integer between 1 and 5), one or more alkyl groups, one or more additional aryl groups (5 to 12 carbons, containing or not containing one or more heteroatoms), one or more additional alkenyl groups, one or more additional olefin groups, one or more additional alkyne groups or one or more additional alkenyl groups.

[0045] Preferably, one or more terminal groups of R2 and / or R3 contain or consist of one or more N-azidoacetylglucosamine (GlcNAz) groups, which are capable of binding or binding to one or more additional ligands, which are the third target ligand and, optionally, the fourth target ligand.

[0046] Another aspect of the invention relates to a conjugate formed between at least a first target ligand and a second target ligand, and optionally formed by a third target and a fourth target ligand or a third target and a fourth target ligand, wherein the target ligands are bound by a reactive compound according to the invention. Advantageously, in this conjugate, all ligands are different.

[0047] In the conjugate according to the present invention, the first ligand is a biomolecule selected from the group consisting of proteins, peptides, nucleic acid molecules (DNA or RNA sequences, such as probes), sugars (such as monosaccharides or polysaccharides), lipids or mixtures thereof.

[0048] Advantageously, the first molecule of the conjugate according to the invention is a protein selected from the group consisting of: an antigen structure or an antibody, etc., and also includes a monoclonal antibody, a polyclonal antibody, an antibody hypervariable part, a nanobody, an alphabody, a microbody, an affytin, a fymomer, an affiline, an affimer or a mixture of two or more thereof.

[0049] Advantageously, the second ligand is a material selected from the group consisting of: a drug, a label, a solid support (eg made of glass, polymer or metal structure), a macromolecule, a carrier molecule, a water-soluble polymer or a mixture thereof.

[0050] The water-soluble polymer may be polyethylene glycol (PEG), preferably having a degree of polymerization comprised between 1 and about 20.000 and / or having a PEG having a molecular weight comprised between 1 KDa and 100 KDa, more preferably between 2 KDa and 50 KDa, even more preferably between 5 KDa and 25 KDa, such as a PEG between 10 KDa and 20 KDa. It is known that such water-soluble polymers such as PEG, when added to biomolecules such as antibodies, etc., can be used to reduce immunogenicity and / or extend the clearance time for circulation of a first target modified material or molecule (such as such an antibody).

[0051] The present invention also relates to a pharmaceutical composition comprising a suitable or appropriate pharmaceutical carrier or diluent and the reactive compound and / or conjugate according to the present invention.

[0052] Another aspect of the invention relates to a diagnostic or screening kit comprising a reactive compound and / or a conjugate according to the invention and optionally means for generating a signal from the label and possibly instructions.

[0053] The present invention also relates to a material, such as a solid support (microbeads, strips or plates) fixed to the conjugate or compound of the present invention, which is used to screen and recover other target molecules by binding between the target molecule and the solid support (microbeads, strips or plates). The present invention also relates to a support (i.e., microbeads, strips, plates) fixed to the conjugate or compound of the present invention, which is used for cell culture, especially animal cell (including human cell) culture.

[0054] Another aspect of the present invention relates to a method for producing a reactive compound according to the present invention. Advantageously, this production method is a chemoenzymatic method comprising the following steps:

[0055] -Select the sugar containing three linked mannose (Man 1 , Man 2 and Man 3 ) portion, or already contains one or more mannoses having three such linkages (Man 1 , Man 2 and Man 3 ) portion of a first target material or a first target molecule (such as an antibody),

[0056] - Add sufficient uridine diphosphate N-azidoacetylglucosamine (UDP-GlcNAz) as a donor substrate and sufficient MGAT3 glycosyltransferase (also known as Gnt-III or β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase) as an enzyme to the trimannosyl core to form a glycosyltransferase at the first mannose of the trimannosyl core. 1 The MGAT3 glycosyltransferase comprises any similar enzyme or any variant of this enzyme, which exhibits high similarity or nucleotide identity with the wild-type MGAT3 enzyme, preferably with a nucleotide sequence identity of more than 80%, more than 90%, more than 95% or more than 98%-99% with the MGAT3 glycosyltransferase, and

[0057] - purifying, preferably by one or more chromatography steps, so as to obtain the reactive compound, or the first ligand (antibody) bound to the reactive compound capable of binding or binding to the second ligand, and

[0058] - Possibly adding a second ligand to the reactive compound at the added N-azidoacetylglucosamine (GlcNAz) group.

[0059] Optionally, in the methods of the invention, the first ligand is an antibody that has previously undergone modification of one or more of its sugar chains, for example by adding a sufficient mixture of neuraminidase (NAM), galactosidase (GO) and acetylglucosaminidase (NAG).

[0060] A final aspect of the invention relates to a method for producing a conjugate according to the invention. Advantageously, this method comprises combining the reactive compound obtained by the method described above with a second ligand and, possibly, with the first ligand described above.

[0061] Advantageously, in the method according to the invention, the 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 conjugate obtained, preferably by one or more chromatography steps. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figures 1 and 2 show the structures of preferred examples of active compounds according to the present invention, wherein M represents the first target ligand. In the left part of the figure, according to the symbolic nomenclature of the ligand (which is a polysaccharide) (SNFG, Glycobiology [Glycobiology] 29: 620-624, 2019), a legend for assigning the monosaccharide structure to a combination of geometric elements is shown. The general structure of the product

[0063] Figure 3Represents a UPLC-FLR chromatogram showing the detection of reactive compounds according to the present invention after treatment with RapiFluor glycans and analysis by HRMS.

[0064] Figure 4 The MaxEnt1 background-subtracted deconvoluted spectrum of F(6)A2BGlcNAz (left) shows a major peak with a molecular weight of 150014 Da, corresponding to the reactive conjugate of the invention.

[0065] Definitions and List of Abbreviations

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. All publications and patent documents mentioned herein are incorporated by reference and may be used in conjunction with the present invention. The following abbreviations appear in the attached specification: ACN (acetonitrile), BGG (bovine gamma globulin), CCD (charge coupled device), CHO (Chinese hamster ovary), DAR (drug to 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 (soy protein isolate), TLC (thin layer chromatography), TMB (3,3',5,5'-tetramethylbenzidine), TRIS (tris(hydroxymethyl)aminomethane), and UDP (uridine diphosphate).

[0067] The general term "sugar" or "glycan" as used herein refers to natural or non-natural sugars or polysaccharides, including glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), fructose (Fru), xylose (Xyl), N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), and their derivatives such as glucosamine (GlcN), galactosamine (Gain), N-acetyl-glucosamine (GlcNAc), N-azidoacetylglucosamine (GlcNAZ), N-acetylgalactosamine (GlaNAc), (tri)mannose, N-glycans and azidomannose.

[0068] The term "trimannosyl core" refers to a polysaccharide containing three linked mannose sugars (Man 1 , Man 2 and Man 3 ) part, as shown in the following formula (1), wherein two mannose (Man 2 and Man 3 ) part is connected to the first mannose (Man 1 ) moiety, the first mannose moiety being located between two additional mannose (Man 2 and Man 3 ) parts.

[0069] Examples of the first target ligand and the second target ligand are biomolecules or materials. The term "material" means a solid support that can be added as a ligand to the reactive compound of the present invention and that is bound or capable of binding to (or through) one or more of the moieties R1, R2 and / or R3 and to the GlcNAz (non-natural N-azidoacetylglucosamine) group.

[0070] The term "biomolecule" refers to a biomolecule that may be synthetic or purified and has favorable targeting properties. Preferably, the biomolecule is capable of binding or binding to other biomolecules (such as proteins and peptides) that target other similar biomolecules (such as antigens or receptors). Alternatively, and also preferably, these "biomolecules" are selected from the group consisting of: carbohydrates (including polysaccharides), nucleic acids (such as DNA or RNA sequences or specific nucleic acid probes of hundreds of nucleotides that can bind to complementary sequences), lipids, or mixtures of two or more thereof.

[0071] The term "protein" includes polypeptides or peptide molecules having an amino acid sequence, having a natural amino acid sequence, and variants and modified forms, regardless of their source or preparation method. These proteins may be modified by adding groups to form glycosylated proteins, by adding N-linked, O-linked and / or C-linked polysaccharides and / or phosphorylated proteins. Preferably, these proteins are therapeutic moieties and / or targeting moieties, such as antibodies, cytokines, growth factors, hormones, interferons, coagulation proteins, erythropoietin (EPO), G-coupled receptors (GPCRs) or molecules capable of binding to these receptors, enzymes (such as glucosamine transferases, galactosidases, neuraminidase, acetylglucosaminidase, mannosidase, mannosyltransferase, urokinase and DNA enzymes).

[0072] The term "antibodies, etc." refers to proteins, including monoclonal chimeric antibodies or polyclonal antibodies (especially those obtained from the serum of homotypic or heterotypic hybridomas), as well as immunoglobulins or specific fragments of these antibodies, such as one or more hypervariable parts of antibodies or so-called immunoglobulin variable domains (IVDs), including complementary determining regions (CDRs) of antibodies, preferably Fab fragments, such as F(ab) fragments, F(ab')2 fragments, Fv fragments, Fc fragments and scFv-Fc fragments, and similar targeting molecules that exhibit the same specificity and targeting properties of antibodies. Preferably, proteins such as targeting antibodies are selected from the group consisting of: diabodies, miniantibodies, humanized antibodies, pegolized antibodies, nanoantibodies, alpha bodies, miniantibodies, avidins, fymomers, avidins, avidin polymers or mixtures of two or more thereof.

[0073] Antibodies are proteins produced by the immune system and capable of recognizing and binding to specific antigens. Due to their sensitive and specific properties, they are often used as molecular tools for research and diagnostic purposes, but also for targeting specific sites (i.e., epitopes) in therapy or prevention.

[0074] Antibodies are preferred examples of first ligands and are target biomaterials for forming the conjugates of the present invention, as they often present a trimannosyl core of the reactive compound of the present invention. This configuration allows the skilled person to construct a first mannose (Man 1 ) part 4 The N-azidoacetylglucosamine group is directly incorporated at the position.

[0075] Preferred examples of antibodies are antibodies that can target tumor cells and can be used for anticancer. These molecules can be produced by different types of prokaryotic or eukaryotic cells, preferably CHO or HEK293 cells, and can be modified to express antibody types (i.e. immunoglobulin types) that target specific antigens or specific epitopes of antigens.

[0076] Preferred examples of proteins are tumor necrosis factor alpha receptor / IgG fusion peptide, chimeric anti-glycoprotein IIb / IIIa antibody, chimeric anti-Her2 antibody, chimeric anti-respiratory syncytial virus antibody, chimeric anti-CD20 antibody, chimeric anti-tumor necrosis factor antibody, anti-RSV antibody, anti-IL2 antibody and anti-CEA antibody.

[0077] Preferred examples of antibodies are those that target epidermal growth factor receptor (EGFR) and are used to treat or prevent colorectal cancer, head and neck cancer, (CTX) (trade name ), and for the treatment or prevention of certain types of breast cancer and targeting the HER2 receptor (Product name ).

[0078] Other examples of proteins or molecules that can be modified by adding the reactive compounds of the invention are glucosaminyltransferase, GnT 1, GnT2, GnT3, MGAT3, β-galactosidase, neuraminidase, acetylglucosaminidase, mannosidase, mannosyltransferase, Alg 2, UDP-GlcNAz, GDP-ManAz...

[0079] The term "solid support" refers to any support made of any solid material but preferably selected from the material consisting of: a pair of binding elements, a metal support, a polysaccharide support (such as a gel), a glass support, a ceramic material or a polymeric support (such as a homopolymer and a copolymer), a complex, or a combination of two or more thereof. The solid support can have any shape or form, such as microbeads, including magnetic microbeads, strips, plates or mixtures thereof. The solid support is preferably made from the material selected from the group consisting of: glass, polymeric structures, such as latex particles or microbeads, metals or metal alloys, such as metal particles or metal microbeads, including magnetic particles or magnetic microbeads, silicon particles or silicon microbeads.

[0080] The solid support can be modified to include a linking group, one or more additional (substituted or unsubstituted and / or branched or unbranched) polyethylene glycol (PEG) (such as an (OC2H4)n chain, where n is an integer, preferably comprised between 1 and 20, more preferably comprised between 1 and 10 or comprised between 1 and 5), one or more additional alkyl groups, one or more additional aryl groups (5 to 12 carbons, including or not including one or more heteroatoms), one or more additional alkenyl groups, one or more additional olefin groups, one or more additional alkyne groups or one or more additional alkynyl groups to bind to a target material, in particular a target biological material, such as a protein.

[0081] The term "reactive compound" means a chemical compound consisting of a sugar (here in particular of Man represented in Formula 1) 1 , Man 2 and Man 3 The invention relates to a conjugate comprising a trimannosyl core (prepared with a trimannosyl core) and capable of binding to a first target material or a solid support to form a conjugate and improve the characteristics of the first target material or generate new binding possibilities for the first target material or its new binding possibilities with target molecules that can be bound, especially allowing the generated conjugate to bind to one or more other target molecules, such as new antigenic structures.

[0082] The terms "conjugate" and "glyco-conjugate" refer to a first target material or molecule (such as a protein or an element of a solid support) that is advantageously linked to a reactive compound by covalent bonding and optionally linked to at least one other second target material or molecule, which is advantageously different from the first target material or molecule.

[0083] The term "label" refers to a chemical or biochemical group that can be easily detected by its molecular size and / or is capable of generating a detectable signal, such as a fluorescent label, preferably a fluorophore (including but not limited to cyanine-like and fluorescein-like fluorophores), a chromophore label, an electron-dense label, a chemiluminescent label, a radioactive label, an enzyme label (preferably including but not limited to the following enzymes: HRP, AP and β-lactamase), a positron emitter, a metal particle (preferably colloidal gold) that can react with silver particles to produce a colorimetrically detectable label, or a first member of a binding pair.

[0084] Preferably, the binding pair is made of a commercially available or synthetically obtainable binding pair derivative, preferably a biotin molecule or an iminobiotin molecule as the first member of the binding pair and capable of specifically binding to the second member of the binding pair, the second member being avidin or streptavidin or an antibody-hapten pair (including but not limited to digoxigenin, dinitrophenol, FAM...).

[0085] According to the present invention, the term "drug" means a therapeutic molecule that can cure or kill target cells, preferably target human cells (such as tumor cells). These drugs are cell inhibitors or cytotoxic agents well known to technicians. Preferably, this drug is selected from the group consisting of: maytansine compounds (derived from the macrolide maytansine from the genus Maytenus), auristatins (derived from the tail sea urinary peptide), calicheamicins (enediyene antibiotics from the bacteria Micromonospora echinospora), antimetabolites (such as but not limited to fluorouracil, floxuridine, methotrexate, leucovorin, hydroxyurea, thioguanine, mercaptopurine, cytarabine, pentostatin, fludarabine phosphate, cladribine, asparaginase, gemcitabine, capecitabine, azathioprine, cytosine methotrexate, trimethoprim, pyrimethamine or pyrimethamine. methylprednisolone), alkylating agents (such as but not limited to melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozotocin, dacarbazine, mitomycin C, cyclophosphamide, nitrogen mustard, uramustine, dibromomannitol, tetranitrate, procarbazine, hexamethylmelamine, mitozolamide or temozolomide), anthracyclines (such as but not limited to daunorubicin, doxorubicin, epirubicin cin, idarubicin or valrubicin), antibiotics (such as but not limited to daunomycin, bleomycin, mithramycin, anthramycin, streptomycin or gramicidin D), calicheamicins, antimitotic agents, dolastatins, nostocs, vinca alkaloids (such as but not limited to vincristine, vinblastine, vindesine or vinorelbine), taxanes (such as but not limited to paclitaxel or docetaxel), topoisomerase inhibitors (such as but not limited to irinotecan, topotecan, camptothecin, etoposide, teniposide, amsacrine or mitoxantrone), proteasome inhibitors (such as but not limited to peptidyl boronic acid), HDAC inhibitors (such as but not limited to vorinostat, romidepsin, cedanilide, panobinostat or belinostat), radioactive isotopes, radioactive agents, salts of these molecules or mixtures of two or more thereof.

[0086] According to the present invention, the drug is any suitable cytotoxic compound, such as auristatin E, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracinedione, mitoxantrone, mithramycin, actinomycin D, procaine, tetracaine, lidocaine, propranolol, puromycin, immunotoxins (such as endotoxin A of Pseudomonas), and salts, analogs or homologs of these molecules.

[0087] Such a conjugate between a drug and an antibody is called an "antibody-drug conjugate" or ADC. ADCs are generally characterized by improved therapeutic efficacy and / or lower toxicity compared to native drugs. Examples of ADCs known in the art include ADCs used in leukemia cancer therapy. For anti-lymphoma Trastuzumab for breast cancer For breast cancer of Gemtuzumab And Anshili

[0088] Furthermore, the medicament according to the invention may be selected from the group consisting of antimicrobial molecules (ie in particular antiparasitic, such as trypanosoma species or plasmodium species), antibacterial molecules or antiviral molecules.

[0089] The term "radioactive agent" includes any effective radioactive molecule for use in diagnosis and / or for destroying cells, tissues or organs affected by a disease, in particular a part of a tumor or tumor or tumor cells or whole tissue.

[0090] Preferably, the radioactive agent 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 mixtures thereof.

[0091] The term "disease" refers to a health state of an animal in which the health of the animal (including humans) is declining. Preferably, the disease is caused by cells (such as tumor cells and tissues) that have hyperproliferative characteristics and / or have deleterious effects on animal and human health.

[0092] The term "chemo-enzymatic method" refers to a method for producing a reactive compound or conjugate according to the invention, wherein the method involves one or more enzymatic reactions. Such enzymatic reactions are usually specific in terms of chemical activity, target region and / or stereospecificity, which ensures the structural homogeneity of the product obtained.

[0093] The term "enzyme MGAT3" refers to the enzyme encompassed by the present invention but presenting the first mannose Man 1 The invention relates to any enzyme having the same activity / ability to add a terminal N-azidoacetylglucosamine (GlcNAz) group to the MGAT3 enzyme, which has less than 100%, preferably less than 90%, less than 80% but more than 70% sequence identity with the wild-type MGAT3 enzyme sequence.

[0094] "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conservative amino acid substitutes to 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, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer [Primers for Sequence Analysis], Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied Math. [Journal of the Society for Industrial and Applied Mathematics], 48:1073 (1988).

[0095] Preferred methods for determining identity are designed to give the maximum match between two or more sequences tested. Methods for determining identity and similarity are incorporated into 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 [Nucleic Acids Research] 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN and FASTA (Altschul, SF et al., J. Mol. Biol. [Molecular Biology] 215: 403-410 (1990)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLMNIH 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;

[0096] The present invention will be further described in the following detailed description and examples with reference to the accompanying drawings, both of which are presented as non-limiting preferred embodiments of the present invention. DETAILED DESCRIPTION

[0097] The present invention relates to a novel reactive compound made of an oligosaccharide structure based on a trimannosyl core (also called a trimannosyl core according to the general structure of formula 1 as described above) carrying one or more novel "clickable" units.

[0098] This new configuration contains non-natural monosaccharides at the bisecting position of the trimannosyl core, which is the Man 1 Partly, the non-natural monosaccharide is N-azidoacetylglucosamine (GlcNAz), which is capable of binding or binding to a suitable first ligand (preferably a first target material or a first target molecule).

[0099] The inventors have found that such a new configuration is very advantageous because it allows the creation of new "clickable" units that limit undesirable consequences such as multiple isomers and reduced solubility during and after their preparation. These new units allow obtaining the binding of a ligand at this position, which ligand is a target material (solid support) or molecule.

[0100] In addition, the compounds of the invention obtained present lower modifications than those found in the prior art and modified in a random manner or by a site-selective manner as described in the prior art. In particular, the compounds obtained present two modification sites and a degree of labeling that may be 0 to 2, which ensures effective structural homogeneity that can be used for therapeutic applications.

[0101] Furthermore, if the conjugate obtained is a modified antibody, the site-selective modification of the product ensures a better immunoreactivity fraction (IRF), which means that due to the use of site-selective modification, a lower percentage of antibodies may lose their activity due to this modification.

[0102] Another advantage of the obtained compounds of the invention is the minor modification of any protein to which the compounds are combined to form the conjugates of the invention, and in particular the glycan distribution of the modified protein leads to better solubility.

[0103] As shown in Formula 1 and the accompanying drawings, the reactive compound obtained resembles a simplified scorpion, wherein the scorpion mouth is advantageously a bisected mannose (or Man 1 )part.

[0104] Advantageously, two additional mannose Man 2 and Man 3 Parts are modified similarly, such as adding additional ligands (which are target materials or molecules). These additional ligands (which are target materials or target molecules) can be different or the same, and can be the same as the ligands (which are second ligands) added above and present in the scorpion's mouth, which are second target materials or second target molecules.

[0105] According to the present invention, the first target ligand is advantageously an antibody present at the end of the scorpion's tail, for example an antibody bound to a trimannose core having Formula 1 via R1, a further added second ligand (being a second target material or a second target molecule) is in the scorpion's mouth, and a third target ligand and a fourth target ligand are optionally present at the end of one or both claws of the scorpion in this simplified diagram.

[0106] The ligand or ligands added to the reactive compound of the invention are advantageously obtained by so-called "click chemistry".

[0107] Unlike classical random coupling protocols using organic solvents and toxic reagents (carbodiimides, benzotriazoles, olefin thiols, etc. ...), the present invention is advantageously based on one or more enzymatic reactions occurring in one or more aqueous buffers and in the presence of a (bio)catalyst, which is less energy intensive. Even if some reagents have to be produced by organic synthesis, the present invention tends to be a more sustainable protocol.

[0108] The enzyme of choice is advantageously MGAT3 glycosyltransferase (also known as GnT-III or β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase), as the enzyme for the first mannose (Man 1 ) portion, optionally using a cofactor that promotes the enzymatic reaction (such as sufficient MnCl2, TRIS buffer, etc.).

[0109] Example

[0110] Example 1: Production of a conjugate according to the invention

[0111] Step 1: Buffer exchange

[0112] After 4 buffer exchanges over a 16 h time span, the antibody (1 equivalent, Ci 22 μM, 3.3 mg·mL-1, Cf 6.47 μM, 0.97 mg·mL-1, 50 μg) was dialyzed (Mini D-Tube from Merck Millipore) TM The buffer was exchanged into TRIS buffer (100 mM, pH = 7) using a dialyzer (ref. cat. no. 71504-3).

[0113] Step 2: Enzymatic modification.

[0114] UDP-GlcNAz (25 equiv., Ci 0.1 M, Cf 0.16 mM, 5.4 μg), enzyme MGAT3 (0.31 equiv., Ci 6.9 μM, 0.4 mg·mL-1, Cf 0.8 μM, 0.12 mg·mL-1, 6 μg) and MnCl2 (204 equiv., Ci 0.1 M, Cf 1.3 mM, 8.3 μg) were then added to the buffer-exchanged material, and the mixture was incubated at 37°C overnight.

[0115] Step 3: Conjugate separation

[0116] To confirm the presence of the conjugate, ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was used.

[0117] The UHPLC-HRMS analyses reported below were performed by using an Acquity liquid chromatograph equipped with different columns and coupled to a Xevo G2-XS QTof quadrupole time-of-flight system (Waters) with an electrospray ionization (ESI) source.

[0118] Prior to UHPLC-HRMS analysis, the protein containing the conjugate was first diluted to 0.1 mg mL-1 with HPLC-grade water in a final volume of 100 μl.

[0119] Calibration was performed using Leu-Enkephalin (m / z 555.26). Data were processed using the Waters Corporation, Milford, MA, USA. Use the MaxEnt1 setting on The merged raw spectra were deconvoluted. At this point, 10 μL of the sample diluted in DMF / ACN solution was injected into the AQUITY UPLC Glycan BEH Amide, 1.7 μm, 2.1 x 150 mm column (Waters Corporation, reference number 186004742).

[0120] The running conditions used were as follows: mobile phase A was 50 mM ammonium formate solution (pH 4.4), and mobile phase B was 100% acetonitrile.

[0121] The gradient conditions are as follows:

[0122] 0 to 35 min flow rate 0.4 mL·min-1 25% A, 35 to 36.5 min flow rate 0.4 mL·min-1 46% A, 36.5 to 43.1 min flow rate 0.2 mL·min-1 100% A, 43.1 to 47.6 min flow rate 0.2 mL·min-1 25% A, and finally 47.6 to 55 min flow rate 0.4 mL·min-1 25% A.

[0123] The settings used were as follows: FLR wavelength EX 265 / EM 425 nm, FLR sampling rate 2 Hz. Source temperature = 120°C; gas flow rate = 800 L·h-1; capillary voltage = 3 kV; cone voltage = 80 V; desolvation temperature = 350°C; positive ion mode; scan mode = positive sensitivity; scanned mass range = 500 to 2,000 m / z. Mass range calibration (Nal, 6.7 μM, 1 μg·mL-1) m / z included between 500 and 2,000.

[0124] N-glycan release and functionalization were performed using the Waters GlycoWorks RapiFluor-MS (Waters Corporation, Ref. No. 176003713) and coupled to the AQUITY FLR (fluorescence) for detection of the product after its release. This was achieved by adapting UHPLC-HRMS (λex=265nm, λem=425nm).

[0125] More specifically, the antibody carrying the compound (13.3 μM, 2 mg mL-1, 7.5 μL, 15 μg) was dissolved with anionic surfactant RapiGest (Waters, reference number 186001861, 5% w:v in Glycoworks fast buffer, 6 μL) and HRMS grade water (15.3 μL). The solution was mixed by gently pipetting and then heated at 90 ° C for 3 min to induce protein denaturation. The solution was cooled for 3 min, and then PNGase F (fast PNGase F, 1.2 μL) was added and incubated at 50 ° C for 5 min. The sample was cooled at RT for 3 min before adding the labeling solution (12 μL, 0.16 M, 68.7 mg mL-1 in DMF).

[0126] The mixture was homogenized by pipetting and after incubation for 5 min at RT, it was diluted with 358 μL of ACN for subsequent separation by HILIC SPE step.

[0127] HILIC SPE Steps: GlycoWorks HILIC μelution Adjust with HPLC grade water (200 μL / well) and then equilibrate with water / ACN (15:85 (vol:vol), 200 μL).

[0128] The samples were loaded onto the wells (400 μL), then washed with a solution of formic acid / water / ACN (1:9:90 (vol:vol), 2 x 600 μL) and finally eluted with Glycoworks SPE elution buffer (200 mM ammonium acetate in 5% acetonitrile, 3 x 30 μL).

[0129] These eluates (90 μL) were further The samples were then diluted in sample diluent (DMF / ACN, 30:70, 310 μL). The samples were then processed by HRMS or first stored at 4°C for at least 3 days or at -80°C for long term storage.

[0130] Figure 1b The conjugation to a non-natural glucosamine derivative of a bisected trisaccharide core is depicted in the figure, such as in the modification of G0(F) glycan (CAS No. 84825-26-3), which represents the core form found in glycoproteins and antibodies in most mammalian species and in therapeutic proteins. The conjugation of one or more of the mannose residues results in the modification of the pentasaccharide portion (M3) of the N-glycan core depicted in the figure.

[0131] The inventors have been able to add suitable reactive ligands such as molecules (biotin), drugs (ozogamicin, emtansine) or labels (fluorophores, including but not limited to cyanine-like and fluorescein-like molecules) to the trisaccharide core (Man 1 The invention relates to conjugates of the present invention, which are made of antibodies linked to these reactive ligands by various classical conjugate reactions, and the inventors have tested these novel antibody conjugates in therapeutic or diagnostic applications. These transformations have been achieved according to known "click chemistry" procedures in the presence of copper or in the "copper-free" case, such as those reported by Van Geel and coworkers (Bioconjugate Chem. 2015, 26, 11, 2233-2242, 2015 https: / / doi.org / 10.1021 / acs.bioconjchem.5b00224), or Thompson and coworkers (ACS Med Chem Lett. 2016 November 10: 7(11): 1005-1008), among others.

[0132] The inventors have been able to add suitable inorganic nanoparticles (such as gold, iron, silicon) and organic nanoparticles (such as poly(lactide-co-glycolide) (PLGA), poly(methyl methacrylate) (PMMA), poly(ethylene glycol) (PEG)) to the trisaccharide core (Man) of the obtained compound of the invention. 1 The invention relates to the conjugates of non-natural glucosamine derivatives (terminal N-azidoacetylglucosamine (GlcNAz) groups) of the reactive compounds (partial or first mannose) to form conjugates made of antibodies linked to these reactive compounds by various classical conjugate reactions, and the inventors have tested these new protein-nanoparticle conjugates in therapeutic or diagnostic detection applications. These transformations have been achieved according to known procedures of "click chemistry" between azide and alkyne groups, such as those reported in: Finetti et al., Langmuir 2016, 32, 29, 7435-7441, 2016.

[0133] The inventors have been able to add suitable biological particles (such as viral particles, liposomes and exosomes) to the trisaccharide core (Man 1The invention relates to the conjugation of unnatural glucosamine derivatives (terminal N-azidoacetylglucosamine (GlcNAz) groups) to form conjugates made from antibodies attached to these biologics by various classical conjugate reactions, and the inventors have tested these novel antibody conjugates in therapeutic or diagnostic detection applications. These transformations have been achieved 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 viral 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).

[0134] The inventors have also realized the connection between these reactive ligands (polymers, drugs and markers) and additional mannose Man immobilized on the obtained compound. 2 and Man 3 The invention also provides a method for further combining the linking groups (non-natural terminal N-azidoacetylglucosamine (GlcNAz) groups) on the second mannose and third mannose moieties to improve the properties of the conjugates obtained by the invention. Based on the bioconjugation scheme reported by Adumeau (Mol. Pharm. [Sub-Pharmaceuticals], 2018, 15, 892-898), these "bifunctional" derivatives consist of a drug installed at the R4 position and a tracer (e.g., Zr) attached to the R2 or R3 position. 89 ) is composed of or consists of a tracer installed on R4 and a drug located on R2 or R3.

[0135] The reactive compounds according to the invention enable the modulation of biological functions and site-selective modifications at two levels of structural hierarchy:

[0136] At the oligosaccharide level:Derivatives that bisect the trimannose core have been shown to modulate several biological functions: fertility, cancer, neurodegenerative diseases, etc.), while the presence of the non-natural unit determines its chemical reactivity (including but not limited to bioconjugation by the click chemistry scheme described above). The reactive compounds according to the present invention can be used as an alternative to current technical solutions in the field of dynamic in vivo imaging (Bertozzi et al. Proceedings of the National Academy of Sciences [Proceedings of the National Academy of Sciences of the United States] 104.43 (2007): 16793-16797) and for the generation of glycoconjugates (a major component of mammalian cells, which is formed by covalent coupling of carbohydrates to other biomolecules such as proteins and lipids and is generally expressed on the cell surface) (Shivatare et al., Chem. Rev. [Chemical Review] 2022, 122, 20, 15603-15671).

[0137] At the macromolecular level: Such compounds can be part of biologically existing structures, including but not limited to N-glycans and O-glycans, which can be exposed naturally or artificially in spatially defined areas of target molecules or materials (e.g., the sequence Asn-X-Ser / Thr, where X can be any amino acid except proline), thereby achieving new functions such as solubilization (Ma et al., Front. Chem. [Chemistry Frontier], July 23, 2020, 8, 2020), the reactivity described above, and the biological functions described below. Such products are intended for industrial use and provide solutions for scientific research, such as:

[0138] Biological functions and reactivity of proteins, peptides, glycolipids, glycolipids, nucleic acids located inside, on the surface or outside of the cell membrane (e.g. cell membrane proteins that expose products). Examples are: construction of azide-labeled cell surfaces (Bertozzi et al., Methods in Enzymology 362, 2003, 249), metabolic labeling of glycans with azidosugars and subsequent glycan profiling and visualization (Bertozzi et al., Nat Protoc. 2007; 2(11): 2930-44), alteration of glycan acceptance by carbohydrate processing enzymes (Liu et al., ACS Cent Sci. 2022 May 25; 8(5): 656-662), in vivo cell-based glycogen synthase activity assays (Agrawal et al., ACS Chem. Biol. 2021, 16, 11, 2490-2501), modification of glycosaminoglycans, and other uses (Bioorthogonal chemistry, Nature Reviews Methods Primers [Nature Reviews - Methods Primers] Volume 1, 30, 2021).

[0139] The necessity of diagnostic and therapeutic products where optimal bioactivity and control over functionalization are key: Examples relate 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. Biological functions and reactivities of proteins, peptides, glycolipids, glycolipids, nucleic acids (e.g., therapeutic proteins) that act inside, on the surface or outside of the cell membrane once administered to a biological system (cell, animal, human); examples relate 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.

[0140] Example 2: Regulation of biological functions

[0141] When the product displays clickable N-azidoacetylglucosamine (GlcNAz) as a trimannose core aliquot, it can be used to confer biological functions to molecules or (nano) materials (from natural or artificial sources) as their natural counterparts lacking such modifications (such as the structures reported in the figure). Such biological functions include, but are not limited to, regulation of neurodegenerative diseases, immune tolerance, IgG function, tumor metastasis and development, and are described in detail below.

[0142] Regulation of neurodegenerative diseases

[0143] Bisecting GlcNAc modification has been shown to favorably stabilize the BACE1 protein under conditions of oxidative stress (EMBO Mol. Biol. 7, 175-189, doi: 10.15252 / emmm.201404438). Increased bisecting GlcNAc content in AD brains may function as an adaptive response that protects the brain from damage caused by additional β-amyloid production (Glycobiology 20, 99-106, doi: 10.1093 / glycob / cwp152).

[0144] The lack of GlcNAc cleavage of BACE1 directs the transport of this protein to lysosomes and accelerates its degradation, which leads to lower amyloid-β accumulation in AD (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.

[0145] Regulation of immune tolerance

[0146] It has been reported that bisected GlcNAc structures have immunosuppressive functions. For example, K562 cells are easily killed by natural killer (NK) cells; however, after transfection with a gene encoding GlcNAc-T III, K562 cells with more bisected GlcNAc acquired NK cell resistance (Mol. Hum. Reprod. [Molecular Human Reproduction] 3, 501-505. doi: 10.1093 / molehr / 3.6.501).

[0147] Natural killer (NK) cells are the main type of immune cells found in the human uterus, suggesting that they could potentially target sperm (Mol. Hum. Reprod. 20, 185-199. doi: 10.1093 / molehr / gat064). Human sperm were found to express bisecting GlcNAc structures, which explains why sperm are not killed by the maternal immune system when entering the female as a foreign matrix and thus supports hu-FEDS (Biomed Res. Int. 2019: 5397804. doi: 10.1155 / 2019 / 5397804).

[0148] In addition, abundant bisected GlcNAc glycans have been detected in human syncytiotrophoblast (STB) and cytotrophoblast (CTB) (Mol. Cell. Proteomics 15, 1857-1866. doi: 10.1074 / mcp.M115.055798). Most likely, the maternal immune system is suppressed by the presence of bisected GlcNAc glycans, and the fetus benefits from this suppression; the mother can nourish the fetus (similar to a foreign organ, because the father contributes half of the fetus' genome) in her body for several months without rejection.

[0149] The mechanism underlying this inhibition could be that the diol conjugates interact with lectins that are associated with specific signal transduction pathways that regulate immune cell function. For example, α-2,3-linked sialic acid on soluble CD52 (a 12-amino acid glycoprotein anchored to glycosylphosphatidylinositol) can mediate T cell inhibition by binding to siglec-10 (Front. Immunol. 10:1967. doi:10.3389 / fimmu.2019.0196). It is possible that cleaving GlcNAc could inhibit NK cells in a similar manner.

[0150] Regulation of IgG functions and properties

[0151] Fc-glycans have an impact on the biological activity of IgG. For example, the lack of fucose in Fc glycans significantly improves binding to human FcγR III, and this result has been applied to improve the efficacy of therapeutic monoclonal antibodies. Attachment of bisecting GlcNAc to Fc glycans will induce antibody-dependent cell-mediated cytotoxicity (ADCC).

[0152] The inventors demonstrated the importance of site-selective modification (GlcNAz at position R4) compared to random modification in another way via IRF calculations. Kinetic ELISA experiments were performed to calculate the immunoreactivity scores of different functionalizations of monoclonal antibodies (including random biotinylation and site-specific azidation). It was found that random functionalization reduced the native antibody to 71% of the IRF, while the site-specificity on (R4 = GlcNAz) maintained it at 92%. Here the protocol for a rabbit monoclonal Ab against soy protein (SPI) is described: two ELISA plates were coated, 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 to D, columns 1-2 were 0 10-14 mol·L-1, columns 3-4 were 3.57 10-14 mol·L-1, columns 5-6 were 7.14 10-14 mol·L-1, columns 7-8 were 14 10-14 mol·L-1, columns 9-10 were 20 10-14 mol·L-1, columns 11-12 were 28 10-14mol·L-1, for rows E to H, columns 1-2 were 43 10-14mol·L-1, columns 3-4 were 57 10-14mol L-1, columns 5-6 were 86 10-14mol·L-1, columns 7-8 were 114 10-14mol·L-1, columns 9-10 were 171 10-14mol·L-1, columns 11-12 were 229 10-14mol·L-1, and the other was with SaR (6 10-12mol / well) at 4°C overnight. The wells were aspirated and saturated solution (PBS 1% BSA, 250μL) was added at 37°C for 2h. The plate was washed 3 times with PBS, and then RabmAb (5 10-14mol / well, 150μL) was added to the SPI-coated plate. The plates were incubated overnight at 4°C, and then the supernatant (125 μL) was removed and placed in the sheep anti-rabbit coated plate. The SPI coated plate was washed 3 times with PBS, and the SaR coated plate was incubated at 37°C for 2 h. Both plates were washed 3 times with PBS and incubated with the secondary antibody goat anti-rabbit-HRP (Ci 5 μM, 1 mg·mL-1, Cf 10 nM, 2 μg·mL-1, 150 μL). The color was developed with TMB (150 μL) and terminated with H2SO4 (3M, 50 μL) at different times.Rows A and E terminate at 0 s, rows B and F terminate at 15 s, rows C and G terminate at 30 s, and rows D and H terminate at 45 s [Moreno et al., J. Immunol. Methods 476, 5-10 (2020)].

[0153] ]Regulation of tumor metastasis and development

[0154] Bisecting GlcNAc structures can inhibit hypoxia-induced epithelial-mesenchymal transition in breast cancer (Front. Physiol. [Front of Physiology] 9:210.doi:10.3389 / fphys.2018.00210). However, the underlying mechanism remains unclear. It is speculated that the addition of bisected GlcNAc to key glycoproteins of signal transduction (such as growth factors, integrins and cytokine receptors) has its special signal transduction strength under hypoxia. In fact, observations in non-solid tumors contradict this explanation. GlcNAc-T III is more activated in patients with blast crisis chronic myeloid leukemia (CML-BC) and multiple myeloma (MM) (Biochem. J. [Journal of Biological Chemistry] 331 (Part 3), 733-742.doi:10.1042 / bj3310733).

[0155] When the product displays clickable mannose, it can be used to confer biological functions to molecules or (nano)materials (from natural or artificial sources) as their natural counterparts lacking such modifications (such as the structures reported in the figure).

[0156] These biological functions include but are not limited to the regulation of 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), glycosylation of therapeutically valuable diabetes peptide drugs (Front. Chem., 122021, doi: 10.3389 / fchem.2021.65002), IgG function, etc.

[0157] Example 3: Modulation of Molecular Reactivity

[0158] Once the product is part of a molecule or (nano)material from natural or artificial sources, it exhibits the typical reactivity of the azido group, such as reactions with alkynes and alkenes, phosphine (The Chemistry of the Azido Group ISBN: 978-0-470-77126-6), allowing bioconjugation with all kinds 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.

[0159] Reaction with diagnostic molecules

[0160] In this regard, commercially available or synthetically available derivatives of biotin (e.g., including but not limited to iminobiotin), fluorophores (e.g., including but not limited to cyanine-like and fluorescein-like fluorophores), enzymes (including but not limited to HRP, AP and β-lactamase), and other antibody-hapten conjugates (including but not limited to digoxin, dinitrophenol and FAM) are references. Examples 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.

[0161] Response to molecules of therapeutic interest

[0162] In this regard, commercial or synthetic labels are references, such as derivatives of tracers available for in vivo imaging, including but not limited to chelators such as DOTA, drugs (including but not limited to antitumor compounds such as monomethyl auristatin E, and antimicrobial and antiviral molecules, or macromolecules, including but not limited to PEG, proteins, lipids, nucleic acid sequences. Examples 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 [Chemistry for generating chemically programmed antibodies (cPAbs): site-specific bioconjugation of small molecules],” Mol. Pharmaceutics [Molecular Pharmaceutics] 2023, 20, 2, 853-874, and “Click chemistry: a transformative technology in nuclear medicine,” Nat Protoc [Nature-Experimental Manual] (2023). https: / / doi.org / 10.1038 / s41596-023-00825-8.

Claims

1. A reactive compound comprising a trimannosyl core having formula 1 in -Man 1 , Man 2 and Man 3 The first mannose, the second mannose and the third mannose are respectively -R 1 is a linking group that enables the GlcNAc group to bind or to the first target ligand, -R 2 is a monosaccharide, optionally linked to one or more additional sugars, -R 3 is a monosaccharide, optionally linked to one or more additional sugars, and -GlcNAz is a non-natural N-azidoacetylglucosamine group capable of binding or binding to a second ligand of interest that is different from the first ligand of interest.

2. The reactive compound as claimed in claim 1, wherein R 1 is a linking group selected from the group consisting of monosaccharides and disaccharides.

3. The reactive compound as claimed in claim 2, wherein R 1 is an N-acetylglucosamine (GlcNAc or GlcNAc-GlcNAc group) group, which is optionally further fucosylated.

4. A reactive compound as claimed in any one of the preceding claims, wherein R 2 and / or R 3 It further comprises a terminal GlcNAz (N-azidoacetylglucosamine) group.

5. A conjugate between at least a first target ligand and a second target ligand, wherein the first ligand and the second ligand are different materials or molecules, and wherein the first ligand and the second ligand are bound together by a reactive compound as described in any one of the preceding claims.

6. The conjugate of claim 5, wherein the first ligand is a biomolecule selected from the group consisting of proteins, nucleic acids, carbohydrates, lipids, or a mixture of two or more thereof.

7. conjugate as claimed in claim 6, wherein the protein is selected from the group consisting of: monoclonal antibody, polyclonal antibody, antibody hypervariable part, nanobody, alpha body (alphabody), microbody (microbody), affinity Ding (affytin), fymomer, affiline (affiline), affinity polymer (affimer) or its mixture of two or more.

8. The conjugate according to any one of claims 5 to 7, wherein the second ligand is a material selected from the group consisting of: a macromolecule, a drug or a label.

9. A pharmaceutical composition comprising a suitable pharmaceutical carrier or diluent and the reactive compound as claimed in any one of claims 1 to 5 and / or the conjugate as claimed in any one of claims 5 to 8.

10. A diagnostic kit comprising: the reactive compound according to any one of the preceding claims 1 to 5 and / or the conjugate according to claim 9, and a device for generating a signal from the label.

11. A solid support made of the conjugate as described in any one of claims 5 to 8, wherein the first ligand is a material selected from the group consisting of: microbeads, strips or plates, and wherein the second ligand is a protein.

12. The solid support of claim 11, which is a cell culture solid support.

13. A method for producing a reactive compound as claimed in any one of claims 1 to 4, comprising the following steps - selecting a trimannosyl core or a first target ligand comprising the trimannosyl core; - Add sufficient uridine diphosphate N-azidoacetylglucosamine (UDP-GlcNAz) as a donor substrate and sufficient glycosyltransferase as an enzyme to the trimannosyl core to convert the first mannose Man 1 Adding a terminal N-azidoacetylglucosamine (GlcNAz) group to the moiety; as well as - Purifying the obtained product by chromatography to obtain the reactive compound.

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

15. A method for producing a conjugate as claimed in any one of claims 5 to 8, comprising combining a reactive compound obtained from the method of claim 13 or 14 with the second ligand, and optionally combining a reactive compound obtained from the method of claim 13 with the first ligand.

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