Bifunctional compounds targeting the cation-independent mannose 6-phosphate receptor

Abstract

The subject matter of the present invention is new conjugates of general formula (I) as defined in claim 1. The conjugates of the invention comprise 1 / at least one mannose 6-phosphate (M6P) analogue which targets the cation-independent mannose 6-phosphate receptor (RM6P-CI) and 2 / an antibody or antibody fragment which makes it possible to bind a specific target antigen or a target molecule. The invention also relates to the method for producing said conjugates and to the medical use thereof, such as therapeutic or diagnostic use. The conjugates of formula (I) of the invention are particularly advantageous for use as a medicament, in particular in a therapeutic treatment method which consists in administering a therapeutic antibody or a therapeutic antibody fragment.

Description

Description Title: Bifunctional compounds targeting the cation-independent mannose 6-phosphate receptor Technical field

[0001] The present invention relates to the field of therapeutic chemistry. It relates more particularly to bifunctional compounds comprising 1 / at least one mannose 6-phosphate (M6P) analogue targeting the cation-independent mannose 6-phosphate receptor (CM6P-CI) and 2 / an antibody or antibody fragment for binding a specific target antigen or a target molecule. The invention also relates to the process for preparing said bifunctional compounds and their medical use, of a therapeutic or diagnostic type. Prior art

[0002] Most therapeutic molecules targeting disease-associated proteins neutralize the target in question, thus blocking the pathophysiological function of their target. However, therapeutic molecules often have little effect on the target's expression or degradation levels. Moreover, there are still many proteins that have not yet been pharmacologically targeted and whose deregulation and accumulation cause a pathological state.

[0003] In recent years, targeted degradation of proteins involved in various diseases has emerged as a key strategy for achieving therapeutic efficacy. For targeted protein degradation through proteasome activation, degradation-inducing drugs are used, such as proteolysis-targeting chimeras. However, the design of these chimeras is complex and cell permeability is limited. Moreover, since the ubiquitin-proteasome system is intracellular, the use of said chimeras cannot be applied to extracellular proteins.

[0004] A physiological pathway for protein degradation is the endolysosomal pathway. Lysosomes are the main degradation compartments in eukaryotic cells. In addition to the proteasome, lysosomes can also degrade molecules other than proteins, such as glycosaminoglycans, oligosaccharides, and lipids. Molecules capable of directing pathological proteins to the lysosome for degradation have therefore been developed.

[0005] The cation-independent mannose 6-phosphate receptor (CM6P-CI), a 300 kDa transmembrane glycoprotein, represents the major transport and sorting pathway for lysosomal enzymes carrying the mannose 6-phosphate (M6P) signal to the lysosomes. RM6P-CI also mediates the endocytosis of extracellular ligands containing M6P (Gary-Bobo et al., 2007). M6P-containing proteins, which differ from lysosomal enzymes and are internalized by RM6P-CI-dependent transport, include granzyme B, a protease involved in cytotoxic T-cell-induced apoptosis, herpes simplex virus (HSV), and even leukemia inhibitory factor, a multifunctional protein that plays an important role in the formation of neurons, platelets, and bones. The prerequisite for proteins to reach the lysosome is therefore the presence of M6P. However, M6P can be dephosphorylated due to serum phosphatases, thus losing its ability to bind RM6P-CI (El Cheikh K. et al., 2016).Lysosomal enzymes have thus been modified to considerably increase the number of M6P residues, although these residues are still degraded by phosphatases (Zhu Y., et al., 2009).

[0006] M6P analogs have been synthesized in which the phosphate is replaced by other groups that allow recognition by RM6P-CI, without being degraded by phosphatases. M6P analogs refer to a compound that is slightly different from the natural M6P compound but has a high binding capacity to RM6P-CI. M6P analogs have been shown to be effective in improving the therapeutic potential of a lysosomal enzyme produced in a baculovirus / insect cell expression system that lacks the M6P signal (El Cheikh K. et al., 2016) and even of a lysosomal enzyme that already carries M6P (Basile I. et al., 2018).

[0007] Documents EP 2 448 600 B1 and EP 3 350 192 B1 described M6P analogues, called AMFA ("Synthetic Analogues of Mannose 6-phosphate Functionalized in the Anomerous Position"), which have several advantages for the functionalization of glycoproteins, in particular for the functionalization of lysosomal enzymes. Indeed, the M6P analogues described in these documents are small in size, are not very immunogenic and can be easily grafted onto the lysosomal enzyme. These analogues are also modular, on the one hand in terms of the length and structure of the spacer arm and on the other hand in terms of the terminal reactive group of the spacer arm depending on the type of bond desired. In these documents, the M6P analogues are more particularly used to internalize lysosomal enzymes into the lysosome of the cell, so that said enzymes can play their physiological role.

[0008] Document PCT / US2019 / 067228 described bifunctional molecules obtained by grafting a polypeptide onto an antibody, said polypeptide comprising a number of M6P analogues ranging from 20 to 90. The addition of this polypeptide onto the antibody has been shown to be effective in increasing the internalization of antigens into cells by culture via M6P receptors. The fate of antibodies coupled to the polypeptide after their cellular entry is not analyzed.

[0009] PCT / IB2021 / 050922 describes bifunctional compounds in which M6P analogs (M6Pa) have been coupled to specific antagonist ligands of PCSK9 and FHR3 factors in order to decrease the extracellular concentrations of these factors. Cellular internalization of M6Pa-antagonists complexed with the targeted factors is followed by degradation in lysosomes. Coupling of M6Pa analogs to antibodies is not considered in this document and the fate of M6Pa-antagonists after their cellular entry is not analyzed.

[0010] The inventors have now developed novel bifunctional compounds comprising at least one M6P analogue covalently linked to an antibody. These bifunctional compounds have the surprising and unexpected property of being able to be recycled to the outside of the cell after their first cellular entry via the RM6P-CI pathway. This original and unexpected property makes it possible to envisage multiple reuse of the compound of the invention by cells, in particular to reduce / decrease the concentration of soluble or membrane extracellular antigens. Summary

[0011] According to a first aspect, the present invention relates to a conjugate characterized in that it has the following general formula (I): [Cheml] in which: X is a phosphonate group -CH2-P(O)(OZ)2 or carboxylate group -CH2-C(O)(OZ) with Z independently representing H (hydrogen), Na (sodium), Li (lithium), K (potassium) or NH4 (ammonium); n is an integer ranging from 0 to 2; Li is a radical chosen from the group including *-ON=**, [Chem2] [Chem3] with * indicating the point of attachment of Li to the (O-CH2-CH2)n group and ** indicating the point of attachment of Li to Yi; ni is an integer ranging from 1 to 20, and preferably from 1 to 10, Yi represents an antibody Y or an antibody fragment Y comprising at least one antigen-binding domain, said Yi forming covalent bond(s) with Li.

[0012] According to a second aspect, the present invention relates to a process for preparing a conjugate of formula (I) as defined above.

[0013] According to a third aspect, the present invention relates to a conjugate of formula (I) for medical use, of a diagnostic or therapeutic type. It relates in particular to a conjugate of formula (I) for use in a therapeutic treatment method which consists of administering a therapeutic antibody or a therapeutic antibody fragment. Brief description of the drawings

[0014] Other features, details and advantages will become apparent upon reading the detailed description below, and upon analyzing the attached drawings, in which: Fig. 1

[0015] [Fig. 1] illustrates the extracellular recycling of the conjugates of formula (I) of the invention. The M6P analogue is represented by the acronym AMFA. The conjugate of formula (I) can be named “AM FA-Antibody”. The antibody is represented by its Y shape, the AMFA by a small ball, and the antigen by a small cloud shape. Fig. 2

[0016] [Fig. 2] illustrates the general synthesis scheme of conjugates of formula (I) in which is equal to 1, depending on the meaning of the reactive chemical group L of the M6P analogue of general formula (II). Figure 2 point 1 / illustrates the reaction between the oxy-amine function of the M6P analogue of formula (II) with the carbonyl function (-CHO) of the antibody Y. The antibody Y is represented by Yi'-CHO, Y representing the antibody Y without its functional group L' equal to -CHO. The analogue (II) is called AMFA1 when X is a phosphonate group and n is equal to 0 and AMFA2 when X is a carboxylate group and n is equal to 0. Figure 2 point 2 / illustrates the reaction between the squarate function of the M6P analogue of formula (II) with the amine function (-NH2) of the antibody Y. The antibody Y is represented by YI'-NH2, Y representing the antibody Y without its functional group L' equal to -NH2. The analogues (II) are called AMFA3 when X is a phosphonate group and n is equal to 0, and AMFA4 when X is a carboxylate group and n is equal to 0. Figure 2 point 3 / illustrates the reaction between the maleimide function of the M6P analogue of formula (II) with the thiol function (-SH) of the antibody Y. The antibody Y is represented by Yi '- SH, Yi ' representing the antibody Y without its functional group L' equal to -SH. The analogues (II) are called AMFA5 when X is a phosphonate group and n is equal to 0 and AMFA6 when X is a carboxylate group and n is equal to 0. Figure 2 point 4 / illustrates the reaction between the carbonylacrylic function of the M6P analogue of formula (II) with the thiol function of the antibody Y. The analogues (II) are called AMFA7 when X is a phosphonate group and n is equal to 0 and AMFA8 when X is a carboxylate group and n is equal to 0. Fig. 3

[0017] [Fig. 3] illustrates the process for the preparation of intermediate 3: 2,3,4-tri-O-trimethylsilyl-oD-mannopyranoside 2-bromoethyl, which will serve as a precursor for the synthesis of AMFA of formula (II). Fig. 4

[0018] [Fig. 4] illustrates the process for preparing the compound AMFA1 having the general formula (II) from compound 3. Fig. 5

[0019] [Fig. 5] illustrates the process for preparing the compound AMFA2 having the general formula (II) from compound 3. Fig. 6

[0020] [Fig. 6] illustrates the process for preparing compounds AMFA3 and AMFA5 having general formula (II) from compound 6. Fig. 7

[0021] [Fig. 7] illustrates the process of preparation of compounds AMFA4 and AMFA6 corresponding to the general formula (II) from compound 1. Fig. 8

[0022] [Fig. 8] illustrates the process of preparation of compounds AMFA7 and AMFA8 having general formula (II) from compound 16 and 21 respectively. Fig. 9

[0023] [Fig. 9] presents the results obtained by SDS-PAGE electrophoresis and Western blots (WB) for the antibodies mAb1, mAb2, mAb3 and mAb4, alone or associated with AMFA1 (mAb1-AMFA1, mAb2-AMFA1, mAb3-AMFA1 and mAb4-AMFA1). The antibodies before and after coupling with AMFAI are detected either by staining the proteins with Coomassie blue, or by recognition with anti-human IgG antibodies. The AMFA1 coupled to the antibodies are detected by a rabbit polyclonal anti-AMFA1 serum (represented by "anti-AMFA" in the figure). The treatment of mAb1-AMFA1 by endonuclease H (PNGase) is also described with detection by an anti-AMFA antibody. Fig. 10

[0024] [Fig. 10] shows the affinity curves of the mAb1 antibody or the mAb1-AMFA1 conjugate (A), the mAb2 antibody or the mAb2-AMFA1 conjugate (B), the mAb3 antibody or the mAb3-AMFA1 conjugate (C), and the mAb4 antibody or the mAb4-AMFA1 conjugate (D) for their target molecules, namely the human TNF-alpha antigens (A and B), VEGF (C), and HER2 (D). Fig. 11

[0025] [Fig. 11] (A) shows data from flow cytometry analysis of internalization of a nonspecific control IgG antibody, mAb1 antibody, and mAb1-AMFA1 conjugate with a fluorescent anti-human IgG secondary antibody (Alexa647) in rhabdomyosarcoma cells (top graph), type T lymphocytes (middle graph) and breast cancer cells (bottom graph). [Fig. 11] (B, C) shows the entry of mAb1 antibody and mAb1-AMFA1 conjugate and mAb4 antibody and mAb4-AMFA1 conjugate pre-coupled to Alexa647 into T lymphocytes (B) and SKOV3 cells (C), respectively. “ p < 0.01 mAb1-AMFA1 versus mAb1 (Student test) from 3 experiments. * p < 0.05 mAb4-AMFA1 versus mAb4 (Student test) from 6 microscopic fields. Fig. 12

[0026] [Fig. 12] shows the data from the cell fluorescence assay of breast cancer cells (A) and rhabdomyosarcoma cells (B), treated with: - a non-specific control IgG antibody, the mAb1 antibody and the mAb1-AMFA1 conjugate with a fluorescent anti-human IgG secondary antibody (Alexa647) (breast cancer cells) (A), - mAb3 antibody and mAb3-AMFA1 conjugate with a fluorescent anti-human IgG secondary antibody (Alexa647) (rhabdomyosarcoma cells) (B). This fluorescence assay makes it possible to define the cellular internalization of the antibodies alone and of the conjugate of the invention. ** p < 0.01 mAb1 -AMFA1 versus mAb1 (Student's t test). Fig. 13

[0027] [Fig. 13] (A) shows the internalization of Ab or Ab-AMFA1 conjugate detected by confocal imaging on rhabdomyosarcoma cells (right) and on breast cancer cells (left). Nuclei are labeled with Hoechst 33342 dye. Labeling of Ab or Ab-AMFA1 complexed with a fluorescent anti-human IgG secondary antibody (Alexa647) is indicated by arrows. The bottom 2 pictures are for the case where excess M6P was added to the culture medium to saturate the RM6P-CI binding sites of the cells. [Fig. 13] (B, C) shows the internalization of mAb1 and mAb1-AMFA1 conjugate in Jurkat T cells (B) and the reversion of this internalization by the addition of excess AMFAI. ** p < 0.01 mAb1 -AMFA1 versus mAb1 (Student test) n = 6. Fig. 14

[0028] [Fig. 14] shows data from the analysis of plasma samples from mice injected with mAb1 or mAb2 antibodies and with the corresponding mAb1-AMFA1 or mAb2-AMFA1 conjugates. The concentration of antibodies in plasma is given as a function of time after injection. The top graph (A) is for mice treated with mAb1 or mAb1-AMFA1 and the bottom graph (B) that of mice treated with mAb2 or mAb2-AMFA1. Fig. 15

[0029] [Fig. 15] shows the Western blot detection of mAb1 antibodies and mAb1-AMFA1 conjugates present in the plasma of mice at 21 days after intravenous (iv) injection of 4 mg / kg of mAb1 and mAb1-AMFA1. A control of the injected mAb1-AMFA1 is added for comparison. The antibodies are detected by recognition with anti-human IgG antibodies and the presence of AMFA coupled to the antibodies is detected by an anti-AMFA1 rabbit serum (represented by “anti-AMFA” in the figure). Fig. 16

[0030] [Fig. 16] (A) shows the cellular entry of human TNF-alpha into human T lymphocytes treated with 5 ng / mL human TNF-alpha and 20 ng / mL mAb1 or mAb1 - AMFA1, for 1 h, 5 h and 17 h. Cell lysates were analyzed by Western blotting. Intracellular concentrations of human TNF-alpha were quantified as a function of time (hours) after treatment. ** p<0.01 mAb1 -AMFA1 + TNF-alpha versus mAb1 + TNF-alpha (Student's t-test). [Fig. 16] (B) shows the TNF-alpha concentrations measured in the culture media of the cells indicated in (A) and corresponding to the different treatment times. Fig. 17

[0031] [Fig. 17] shows the neutralization by mAb1 antibody or mAb1-AMFA1 conjugate of the anti-proliferative effect of human TNF-alpha on murine fibroblast-like cells treated with 5 ng / mL of human TNF-alpha. ** p<0.01 mAb1 -AMFA1 versus mAb1 (Student's t test). Fig. 18

[0032] [Fig. 18] shows the reversion of the mitogenic effect of VEGF in HUVEC cells treated with 50 ng / mL of human VEGF and different concentrations of mAb3 or mAb3-AMFA1 for 72 h. Fig. 19

[0033] [Fig. 19] (A) shows the degradation of HER2 (ERBB2, Human) protein by protein immunocytochemistry (In Cell ELISA) in SK-BR-3 breast cancer cells treated with 10 or 100 nM mAb4 or mAb4-AMFA1 for 24 h. [Fig. 19] (B) shows the degradation of HER2 in BT474 breast cancer cells treated with 5 nM mAb4 or mAb4-AMFA1 for 5 h. * p<0.01 mAb4-AMFA1 versus mAb4 (Student's t-test) (n = 2). Fig. 20

[0034] [Fig. 20] (A) shows the recycling capacity of mAb1 antibody and mAb1-AMFA1 conjugate in Jurkat T cells. Each antibody / conjugate was incubated for 5 h with the cells and then the culture medium was replaced with medium without antibody. The cellular egress of the internalized antibody or conjugate was measured at 1 h and 5 h (n = 2). [Fig. 20] (B) shows the cellular egress of mAb1 antibody and mAb1-AMFA1 conjugate at 10 min and 30 min after 30 min of internalization in Jurkat T cells (n = 4). * p<0.05 mAb1 -AMFA1 versus mAb1 (Student's t test). Detailed description

[0035] Conjugates of formula (I)

[0036] As indicated, the subject of the present invention is a conjugate of formula (I) as defined above. For the purposes of the invention, the term "conjugate" means a compound comprising two parts linked together by a covalent bond. The first part of the conjugate represents at least one M6P analog while the second part of the conjugate represents an antibody or antibody fragment comprising at least one antigen-binding domain. In the formula (I) defined above, the M6P analogue corresponds to the formula delimited by the large parenthesis. The integer indicates the number of M6P analogue(s) bound to the antibody or antibody fragment. Indeed, according to the invention, M6P analogue(s) may be covalently bound to an antibody or antibody fragment comprising at least one antigen-binding domain. These M6P analogues are bound to the antibody or antibody fragment via the Li radical. As already indicated, an M6P analogue is understood to mean a compound which differs from natural M6P but which has a high binding capacity to RM6P-CI. Throughout the application, M6P denotes mannose 6-phosphate and RM6P-CI denotes cation-independent mannose 6-phosphate receptor. The conjugate of the invention is a bifunctional compound in that it exhibits a dual binding capacity, namely a capacity to bind to RM6P-CI (via the M6P analog), and a capacity to bind to a specific target antigen or target molecule (via the antibody or antibody fragment). Specific target antigen or target molecule refers to a membrane or extracellular molecule of therapeutic or diagnostic interest, particularly a molecule whose overexpression induces a pathological condition or is associated with a pathological condition or whose expression is involved in a pathological disorder. The new bifunctional compounds of the invention make it possible in particular to reduce / decrease the concentrations of extracellular or membrane target molecules in an individual, said reduction being achieved by cellular endocytosis modulated by RM6P-CI, so that this reduction / decrease in concentrations is sufficient for there to no longer be a pathological state. “Extracellular or membrane target molecules” means molecules of therapeutic or diagnostic interest, in particular molecules whose overexpression induces a pathological state or is associated with a pathological condition or whose expression is involved in a pathological disorder. The novel bifunctional compounds of the invention more particularly make it possible to reduce / decrease the concentrations of a soluble or membrane extracellular antigen in an individual, so that the individual no longer presents a pathological state.

[0037] In the general formula (I) of the conjugate of the invention, Yi has been defined as representing an antibody Y or a fragment of antibody Y comprising at least one antigen-binding domain. In the following, for the sake of simplification, antibody Y is understood to mean both the entire antibody Y and a fragment of antibody Y comprising at least one antigen-binding domain or an antibody-derived molecule comprising at least one of said antigen-binding domains. Antibody Y refers to the free antibody, meaning that it does not form a bond with the M6P analogue. Antibody Yi refers to antibody Y when the latter is covalently linked to the M6P analogue via the Li radical. Free antibody Y can be represented by Yi'-L', with L' representing a functional group or reactive function carried by antibody Y. In other words, Y represents antibody Y without its functional group L'. It is the functional group L' of the antibody Y which will react with a functional group or a reactive function carried by the M6P analogue in order to form a covalent bond between the antibody Y and the M6P analogue and thus obtain the conjugate of formula (I) of the invention. In the present application, the terms "functional group", "reactive function" or "reactive chemical group" may be used interchangeably. Each of these terms designates a group carried by the antibody (or carried by the M6P analogue) capable of reacting with a group carried by the M6P analogue (or carried by the antibody). Antibody is any type of antibody, of any isotype, from any species. It includes a therapeutic or diagnostic antibody targeting a molecule of therapeutic or diagnostic interest, such as an anti-TNFo antibody, anti- VEGF, anti-HER2 and anti-GFP. The antibody is in particular a heavy and light chain antibody, a single chain antibody, a dimeric or multimeric antibody, bispecific or multispecific. The antibody is in particular an immunoglobulin of the IgG type, in particular of the IgG1, IgG2, IgG3 or IgG4 subtype, of the IgE, IgD, IgA or IgM type. The antibody includes monoclonal antibodies, humanized or fully human antibodies and chimeric antibodies from different species (human, mouse, rat, rabbit, goat, camelid, chicken, etc.), in particular human-mouse chimeric antibodies. Antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fabc, or Fab fragments comprising a portion of the Fc region and single-chain antibody fragments derived from camelid or shark immunoglobulins (single-chain V-domain antibodies) HH (nanobodies) and V-NAR). Antibody-derived molecules include fusion proteins comprising a binding domain of an antibody to the target molecule, in particular in the form of an Fc fusion protein; antibodies or fusion proteins having several binding domains corresponding to several target molecules, i.e., polyspecific antibodies or fusion proteins.The antibody may further be modified to give it a particular property in addition to its recognition of the target molecule, in particular by conjugating the antibody to a therapeutically active molecule such as a cytotoxic molecule or a cell growth inhibitor, to a protease inhibitor, to an element making it possible to increase the half-life of the antibody in the patient's blood, or to an imaging molecule allowing the detection of the antibody and / or the target molecule in the patient by the various techniques that can be used clinically (radiology, radiography, scintigraphy, MRI, endoscopy, scanner, etc.).

[0038] In the conjugate of formula (I) as defined above, Yi can more particularly be represented by the group L2-Y'I in which Y'i represents the antibody Y linked to Li via the radical L2 with L2 chosen from the group comprising =CH-, -NH- and -S-, said Yi thus being able to be represented by: =CH-Y'i, -NH-Y'i and -S-Y'i. The L2 radical is more particularly derived from the transformation of the functional group L' carried by the antibody when the latter forms a covalent bond with the M6P analogue.

[0039] According to an advantageous embodiment of the invention, the conjugate of formula (I) is characterized in that is an integer equal to 1, and in that said conjugate is chosen from the group comprising: [Chem5] with X, n and Y'i as defined above.

[0040] According to yet another advantageous embodiment, the conjugate of the invention is characterized in that Yi represents a therapeutic or diagnostic antibody Y.

[0041] According to a particularly advantageous embodiment, the conjugate of the invention is more particularly characterized in that Yi represents an antibody Y selected from the group comprising monoclonal antibodies, chimeric antibodies, human or humanized antibodies, heavy and light chain antibodies, single chain antibodies, bispecific or multispecific antibodies and nanobodies.

[0042] According to another particularly advantageous embodiment, the conjugate of the invention is more particularly characterized in that Yi represents an antibody Y which is an immunoglobulin of type IgG, in particular of subtype IgG 1, IgG2, IgG3 or IgG4, or an immunoglobulin of type IgE, IgD, IgA or IgM.

[0043] According to yet another embodiment of the invention, the conjugate of formula (I) as defined above can also be characterized in that it has an IC5o affinity with respect to RM6P-CI ranging from 10 -4 M to 10 -9 M, and preferably ranging from 10 -5 M to 10 -8 M. This affinity can for example be measured by a competition method towards another ligand of RM6P-CI such as pentamannose 6-phosphate (according to Basile I. et al., 2018).

[0044] The conjugates of the invention are very interesting in that they allow cellular internalization of a target molecule such as for example an antigen via the endo-lysosomal pathway, this internalization being advantageously followed by the extracellular release of the conjugates of the invention, which thus allows the realization of a new cycle of capture of the target molecule by the conjugate of the invention. The extracellular recycling of the conjugates of the invention is illustrated in Figure 1. The AMFA-Antibody conjugate of the invention, bound to an Antigen, enters the endosome of the cell via the RM6P-CI to deposit the antigen there. The antigen is then directed towards the lysosome while the AMFA-Antibody conjugate unexpectedly leaves the cell. The conjugate thus left the cell can be used again to fix a new extracellular or membrane antigen. This recycling capacity was unexpected because previous literature examples using M6P analogues to internalize lysosomal enzymes (see e.g. EP 2 448 600 B1) indicated the presence of these bifunctional compounds (comprising M6P analogues linked to enzymes) in the endo-lysosomal pathway without extracellular recycling. Similarly, in document PCT / US2019 / 067228, bifunctional molecules associated with antigens are presented in lysosomes. This document does not provide information on possible extracellular recycling of bifunctional molecules. Thus, in all these studies the bifunctional compounds were described to be present and / or degraded in lysosomes with their specific target. In the present invention, unexpectedly, with the conjugates of formula (I), only the target molecule is degraded in lysosomes while the conjugates are advantageously recycled in the extracellular space.

[0045] The conjugate of formula (I) is further characterized in that it has at least one of the following properties: - it is capable of penetrating the cell's endosome via its binding with RM6P-CI to carry a soluble or membrane extracellular antigen; - it is capable of exiting the cell's endosome after having deposited the soluble or membrane extracellular antigen there; - it allows the degradation in the lysosome of soluble or membrane extracellular antigens recognized by Yi after their cellular internalization; - it is recycled in the extracellular environment after its cellular internalization; According to a particularly advantageous embodiment, the conjugate of the invention has each of the properties defined above.

[0046] The originality of the conjugates of the invention comes in particular from the fact that they do not destroy the natural capacity of the antibody to be able to leave the cell when it needs to. Indeed, the coupling of M6P analogues with the antibody is carried out in such a way as not to create any hindrance at the level of the reactive functions of the antibody, such as for example at the level of the part of the antibody interacting with specific receptors of the antibodies, such as for example the neonatal FcRn. This has the effect of not modifying the extracellular recycling capacities of the antibody when it is covalently linked to the M6P analogues described in the present application.

[0047] Process for the preparation of conjugates of formula (I)

[0048] According to another aspect, the present invention relates to a process for preparing a conjugate of formula (I) as defined above, characterized in that the following are reacted: - an antibody Y or an antibody fragment Y comprising at least one antigen-binding domain with - nor compound(s) corresponding to the general formula (II) [Chem9] (II), in which X, n and are as defined above, L is a reactive chemical group selected from the group consisting of -O-NH2, [Chem 10] said group L of compound (II) forming covalent bond(s) with functional group(s) carried by said antibody Y or antibody fragment Y.

[0049] The compound of formula (II) is an analogue of M6P. It is the reactive chemical group L carried by the analogue of M6P of formula (II) which will react with the functional group L' of the antibody Y in order to form a covalent bond between the antibody Y and the analogue of M6P and thus obtain the conjugate of formula (I) of the invention. The radical L2 previously mentioned is more particularly derived from the transformation of the functional group L' carried by the antibody when the latter forms a covalent bond with the analogue of M6P. Compounds of formula (II) are particularly interesting because of the simplicity of their spacer arm. The term "spacer arm" refers to the part located after the oxygen carried by the anomeric carbon of the cyclic structure of the M6P analogue and which includes the reactive chemical group L, namely the part (CH2)2(O-CH2-CH2)nL. Indeed, the simplicity of the structure of the spacer arm contributes to the advantages presented by the conjugates of formula (I) of the invention. This simplicity of the spacer arm makes it possible in particular to easily synthesize the compounds of formula (II) and then to carry out a particularly interesting and effective coupling with an antibody. The synthesis of the conjugates of formula (I) is thus efficient and easy to implement.

[0050] In the following, for the sake of simplification, the L groups of the M6P analogue as defined above are respectively referred to, in order of appearance, as “oxy- amine”, “squarate”, “maleimide” and “group comprising a carbonylacrylic function”.

[0051] Specific examples of M6P analogues of formula (II) are more particularly represented by the acronym "AMFA" in the present application.

[0052] According to an advantageous embodiment of the process of the invention, the compound (II) is chosen from the group comprising: [Chem 13] AMFA1, [Chem 14] AMFA2, [Chem 15] AMFA3, [Chem16] AMFA4, [Chem 17] AMFA5, [Chem 18] AMFA6, [Chem 19] AMFA7, [Chem20] AMFA8.

[0053] According to an advantageous embodiment of the method of the invention, the use of the AMFA1 to AMFA8 described above makes it possible to carry out a particularly interesting and effective coupling with an antibody as described above. In addition, as already indicated, the compounds of formula (II), and more particularly the AMFA1 to AMFA8 described above, are simple in terms of their chemical structure, in particular due to the simplicity of their spacer arm. These compounds are easy to synthesize and then easy to couple with the antibody.

[0054] According to a particularly advantageous embodiment of the invention, the L group of the AMFA compound will bind at the level of a carbonyl function previously generated on an oligosaccharide chain / part of the glycosidic part of the antibody. The glycosidic part of the antibody mentioned above is more specifically that located at the level of the Fc region of the antibody. As an example of the L group of the AMFA compound, we can cite the oxy-amine group.

[0055] According to another embodiment of the invention, the L group of the AMFA compound will bind at an appropriate amino acid residue of the peptide chain / portion of the antibody. Examples of suitable amino acid residues include an amine group from lysine or a thiol group from cysteine. Examples of the L group of the AMFA compound include a squarate group, a maleimide group, or a carbonylacrylic group.

[0056] The terms "chain" or "part" used to refer to an oligosaccharide chain or an oligosaccharide part of the antibody, or to refer to the peptide chain or the peptide part of the antibody may be used interchangeably in the present application.

[0057] Figure 2 illustrates the general synthesis scheme of the conjugates of the invention according to the respective definitions of the reactive chemical group L of the M6P analogue of general formula (II). As indicated and shown in Figure 2, point 1 / , the carbonyl function (-CHO) of the antibody Y reacts with the oxy-amine function of the AMFA compound of formula (II). This coupling mode occurs more particularly at the level of an oligosaccharide chain of the glycosidic part located on the Fc part of the antibody. This coupling method is particularly advantageous in that it does not create any hindrance to the other reactive functions of the antibody (such as, for example, the regions interacting with the antibody receptors), which advantageously allows the antibody to preserve its natural recycling properties towards the outside of the cell and thus allows the conjugates of the invention to be recycled outside the cell. Concerning the coupling mode of the AMFA compound of formula (II) comprising the squarate group (see figure 2 point 2 / ), the latter occurs at a lysine residue of the peptide part of the antibody, namely more particularly at the amine function. Concerning the coupling mode of the AMFA compound of formula (II) with a maleimide group (see figure 2 point 3 / ), the latter occurs at a cysteine ​​residue of the peptide part of the antibody, namely more particularly at the thiol function. Concerning the coupling mode of the AMFA compound of formula (II) comprising a carbonylacrylic function (see figure 2 point 4 / ), the latter occurs at a cysteine ​​residue of the peptide part of the antibody, namely more particularly at the thiol function. These coupling modes are particularly advantageous because they do not create or create little hindrance at the level of the other reactive functions of the antibody (such as for example the FcRn receptors of the antibody), which advantageously allows the antibody to preserve its natural properties and thus the conjugates of the invention to be able to be recycled outside the cell.

[0058] Use of conjugates (I) in a method of therapeutic or diagnostic treatment

[0059] The new bifunctional compounds make it possible to reduce / decrease the concentrations of soluble or membrane extracellular antigens in an individual, said reduction / decrease being carried out by cellular endocytosis modulated by RM6P-Cl. This property thus makes them particularly interesting for therapeutic use.

[0060] According to another aspect of the invention, there is provided a pharmaceutical composition characterized in that it comprises: - a conjugate as defined above, - at least one pharmaceutically acceptable excipient. The conjugate of the invention of formula (I) is present in a therapeutically effective in the composition of the invention. A "therapeutically effective amount" means a dosage sufficient to produce a desired result, for example, an amount sufficient to achieve beneficial or desired therapeutic (including preventive) results, such as a reduction in the level of an element whose extracellular or membrane expression or overexpression is responsible for a pathology or is involved in a pathological condition. An effective amount may require one or more administrations.

[0061] According to yet another aspect, the invention relates to a pharmaceutical composition comprising: - a therapeutically effective amount of the conjugate of formula (I), - at least one other therapeutically active agent, - possibly at least one pharmaceutically acceptable excipient.

[0062] The present invention also relates to a conjugate of formula (I) as defined above or a pharmaceutical composition as defined above, for medical use, of a diagnostic or therapeutic type.

[0063] In this regard, the invention more particularly relates to a conjugate of formula (I) as defined above or a pharmaceutical composition as defined above, for use as a medicament.

[0064] The invention also relates to a conjugate of formula (I) as defined above or a pharmaceutical composition as defined above, for use in a therapeutic treatment method which consists of administering a therapeutic antibody or a therapeutic antibody fragment. As already indicated, the extracellular recycling capacity gives the conjugates of the invention particularly advantageous properties in the context of their use for treating pathologies whose treatment consists of administering a therapeutic antibody or a therapeutic antibody fragment. Indeed, the administration of a conjugate of formula (I) allows a lower dose of therapeutic antibody to be used than if the therapeutic antibody were used alone (without being covalently linked to an M6P analogue). Indeed, the therapeutic antibody, when linked to an M6P analogue, has improved therapeutic efficacy compared to the antibody used alone. Thus, at equal doses of antibody, the antibody linked to the M6P analogue is more effective than when used alone. According to an advantageous embodiment of the invention, the use of a conjugate of formula (I) makes it possible to reduce the doses of antibodies usually used in a therapeutic treatment method involving the administration of antibodies, and thus reducing the side effects associated with antibody treatment.

[0065] Examples of pathologies involving the administration of antibodies include those chosen from the group comprising cancer, inflammatory diseases, autoimmune diseases, blood diseases, ophthalmological diseases, viral infections, allergic diseases and rare diseases.

[0066] The antibodies more particularly tested in the invention in coupling with AMFA are in particular anti-TNF-alpha, anti-VEGF and anti-HER2 antibodies. Anti-TNF-alpha antibodies are used to treat inflammatory bowel disease, Crohn's disease, ulcerative colitis, and autoimmune diseases affecting the joints such as rheumatoid arthritis, axial spondyloarthritis / ankylosing spondylitis, psoriatic arthritis, and psoriasis. The anti-VEGF antibody is used clinically to treat neovascularization and tumor growth in many types of cancers, as well as ocular vascular disorders including neovascular age-related macular degeneration. The anti-HER2 antibody is used in particular to treat certain cancers, particularly breast cancers overexpressing the HER2 receptor.

[0067] The invention also relates to a conjugate of formula (I) as defined above or a pharmaceutical composition as defined above, for use in a diagnostic method. Thus, a conjugate of formula (I) covalently linked to an imaging molecule via the Yi antibody can help locate pathological lesions such as tumors or metastases, or other pathological areas to be treated. Examples of diagnostic methods include the diagnosis of diseases or conditions associated with increased expression of RM6P-CI.

[0068] The conjugate of the invention or the pharmaceutical composition can be formulated in different pharmaceutical formulations (solid, semi-solid, liquid or gaseous form), such as tablets, capsules, powders, granules, ointments, solutions, injections, inhalations and aerosols. The pharmaceutical formulation may be in a liquid form, a lyophilized form, or a liquid form reconstituted from a lyophilized form, where the lyophilized preparation must be reconstituted with a sterile solution prior to administration. The conjugates of the invention may be formulated into preparations for injection in cutaneous, intravenous or intravitreal by dissolving, suspending or emulsifying them in a solvent.

[0069] According to an advantageous embodiment, the subject of the invention is the conjugate of formula (I) or the pharmaceutical composition for use as defined above, characterized in that the conjugate or the pharmaceutical composition is in a form suitable for administration by parenteral, intravenous or subcutaneous route. Examples

[0070] The synthesis of the conjugates (I) of the invention and the study of their biological effects is described in detail in the examples below which refer to figures 3 to 20. The analogues of M6P of general formula (II) synthesized in the examples below are respectively named AMFA1, AMFA2, AMFA3, AMFA4, AMFA5, AMFA6, AMFA7 and AMFA8. Some of these AMFAs were coupled with antibodies called mAb1, mAb2, mAb3, mAb4 and Ab (see below).

[0071] Example 1: Synthesis of AMFA1 to AMFA8 of general formula (II)

[0072] Preparation of Intermediate 3: 2,3,4-tri-O-trimethylsilyl-aD-mannoDyranoside de 2-bromoéthyle The process for preparing intermediate 3, which will serve as a precursor for the synthesis of AMFA, is illustrated in Figure 3, the conditions and reagents of steps (i) to (iii) being as follows: (i) 2-bromoethanol, triethylamine, CH2CI2, 3.5 h, 80°C; (ii) TMSCI, Et3N, CH2CI2, 20 h, RT; (iii) K2CO3, MeOH, 15 min, 0°C. The starting compound is D-mannose to which 2-bromoethanol is introduced in the anomeric position using previously reactivated Amberlite IR120 resin to form compound 1. Compound 1 was persilylated with trimethylsilyl chloride in the presence of triethylamine to form intermediate 2. The 6-position of intermediate 2 was selectively deprotected by potassium carbonate in catalytic quantity in methanol to give compound 3 in a yield of 60% over two steps (see Figure 3).

[0073] Preparation of AMFA1 compound The process for preparing compound AMFA1 from compound 3 is illustrated in Figure 4, the conditions and reagents of steps (i) to (vi) being as follows: (i) 0.3 M Dess-Martin periodinane, CH2Cl2, RT, 1 h; (ii) tetraethyl methylenediphosphonate, NaH, THF, 1 h, RT; (iii) Pd / C, Et3SiH, MeOH, 2 h, RT. (iv) / V-hydroxyphthalimide, NaH, DMF, 22 h, 60°C; (v) TMSCl, Et3N, CH2Cl2, 15.5 h, RT; (vi)-a) TMSCl, NaI, CH3CN, 50 min, 35°C; (vi)-b) N2H4.H2O, MeOH, 15.5 h, RT. Alcohol 3, obtained as described above, is oxidized to aldehyde using Dess-Martin periodinane to form compound 4. This key intermediate will allow access to the different AMFAs of formula (II). To synthesize the compound AMFA1, aldehyde 4 is reacted with tetraethyl methylenediphosphonate, previously deprotonated with sodium hydride, to form compound 5 with a yield of 76% over two steps. During a catalytic hydrogenation step, in the presence of palladium on carbon and for which hydrogen is generated in situ by progressive addition of triethylsilane, the double bond is reduced and the trimethylsilyl groups are hydrolyzed. Compound 6 is thus obtained, without purification, with a quantitative yield. The bromine atom is then substituted with / V-hydroxyphthalimide to form intermediate 7 with a yield of 61%. The alcohol functions are then protected with trimethylsilyl chloride to give compound 8 with a quantitative yield.Intermediate 8 thus obtained, without purification, is then reacted with trimethylsilyl chloride and sodium iodide to form the bis(trimethylsilyl) phosphonate by a Rabinowitz-type reaction. This intermediate is then converted into phosphonic acid by the action of hydrazine monohydrate in methanol, which will also displace the trimethylsilyl groups present on the secondary alcohols of the sugar and react with the phthalimide in order to unmask the amine function. In a single step, we thus obtain the compound AMFA1 deprotected on the phosphonate part, on the sugar alcohols and carrying the aminooxy function in the anomeric position with a yield of 85% over two steps (Figure 4).

[0074] Preparation of AMFA2 compound The process for preparing compound AMFA2 from compound 3 is illustrated in Figure 5, the conditions and reagents of steps (i) to (iv) being as follows: (i)-a) 0.3 M Dess-Martin periodinane, CH2CI2, RT, 1 h; (i)-b) Triethyl phosphonoacetate, NaH, THF, 20 min, RT, 73% (2 steps); (ii)-a) Pd / C, Et3SiH, MeOH, 2 h, RT; (ii)-b) 1 N NaOH, THF / H2O, 2 h, RT; (iii) / V-hydroxyphthalimide, NaH, DMF, 20 h, 60°C; (iv) N2H4.H2O, MeOH, 15.5 h, RT. The AMFA2 compound is synthesized following the synthetic routes described in Figure 5. One of the main steps in the synthesis of this saccharide ligand was the introduction of the carboxylate function on the side chain of the mannose fragments. In the same way as the previous syntheses, this functionalization is carried out by a Dess-Martin oxidation of intermediate 3 in position 6 followed by a Horner-Wadsworth-Emmons reaction using the triethyl phosphonacetate anion. In this way, the unsaturated carboxylate 10 is obtained. After the steps of reduction of the double bond and hydrolysis of trimethylsilyl protecting groups on the hydroxyl functions, the The carboxylate function was saponified to give intermediate 11. The bromine is then substituted by hydroxyphthalimide to give intermediate 12. Unmasking of the amine function by hydrazinolysis leads to the fully deprotected AMFA2 compound (Figure 5).

[0075] Preparation of AMFA3 and AMFA5 compounds The process for preparing compounds AMFA3 and AMFA5 from compound 6 is illustrated in Figure 6, the conditions and reagents of steps (i) to (v) being as follows: (i) 0.3 M Dess-Martin periodinane, CH2Cl2, RT, 1 h; (ii) tetraethyl methylenediphosphonate, NaH, THF, 1 h, RT; (iii) Pd / C, EtsSiH, MeOH, 2 h, RT. (iv) potassium phthalimide, DMF, 22 h, 60°C; (v) TMSCl, Et3N, CH2Cl2, 15.5 h, RT; (vi)-a) TMSCl, NaI, CHsCN, 50 min, 35°C; (vi)-b) N2H4.H2O, MeOH, 15.5 h, RT; (vii) Diethyl squarate, Et3N, EtOH / H2O, 30 min; (viii) / V-(methoxycarbonyl)maleimide, Saturated NaHCOs solution, 30 min, 0°C. To synthesize AMFA3 and AMFA5, the bromine atom of compound 6 is substituted with potassium phthalimide to form intermediate 14 with a yield of 74%. The alcohol functions are then protected with trimethylsilyl chloride to give compound 15 with a quantitative yield. Intermediate 15 thus obtained, without purification, is then reacted with trimethylsilyl chloride and sodium iodide to form the bis(trimethylsilyl) phosphonate by a Rabinowitz-type reaction. This intermediate is then converted into phosphonic acid by the action of hydrazine monohydrate in methanol, which will also displace the trimethylsilyl groups present on the secondary alcohols of the sugar and react with the phthalimide to unmask the amine function.In a single step, we thus obtain compound 16 deprotected on the phosphonate part, on the alcohols of the two sugars and carrying the amine function in the anomeric position with a yield of 65% over two steps. Compound 16 is then reacted either with diethyl squarate to form the product AMFA3 with a yield of 50% or with / V-(methoxycarbonyl)maleimide to form the compound AMFA5 with a yield of 80% (figure 6).

[0076] Preparation of AMFA4 and AMFA6 compounds The process for preparing compounds AMFA4 and AMFA6 from compound 1 is illustrated in Figure 7, the conditions and reagents of steps (i) to (v) are as follows: (i) NaN3, DMF, 20 h, RT; TMSCI, Et3N, CH2CI2, 20 h, RT; K2CO3cat, MeOH, 50 min, 0°C, 38% (3 steps); (ii)-a) Dess-Martin periodinane, CH2CI2, 3 h, RT, (ii)-b) Triethyl phosphonoacetate, NaH, THF, 2 h, RT, 73% (2 steps); (iii)-a) 0.5 N HCl, THF, 30 min, TA, (iii)-b) 0.1 N NaOH, 30 min, TA, 99%, (iii)-c) H2, Pd / C, EtOH / H2O, 1.5 h, TA, 99%; (iv) diethyl squarate, EtOH / H2O, 40 min, RT, 34%; (v) / V-(methoxycarbonyl)maleimide, Solution saturated NaHCOs, 30 min, 0°C. AMFA4 and AMFA6 are synthesized following the synthetic routes described in Figure 7. One of the main steps in the synthesis of these saccharide ligands was the introduction of the carboxylate function on the side chain of the mannose fragments. This functionalization is achieved by a Dess-Martin oxidation of alcohol 19 at position 6 followed by a Horner-Wadsworth-Emmons reaction using the triethyl phosphonacetate anion. In this way, the unsaturated carboxylate 20 is obtained. Another critical step was the functionalization of the aglycone fragments with diethyl squarate or maleimide. This reaction was carried out on the fully deprotected compound 21 to provide AMFA4 and AMFA6 respectively (Figure 7).

[0077] Preparation of AMFA7 and AMFA8 compounds The process for preparing compound AMFA7 from compound 16, and that for preparing compound AMFA8 from compound 21 are respectively illustrated in Figure 8. The conditions and reagents of step (i) are as follows: (i) 3-benzoylacrylic acid, N-methylmorpholine, isobutyl chloroformate, DMF, 1 h, -10°C at RT. To prepare AMFA7 and AMFA8, the same procedures used in the preparation of AMFA3 and AMFA4 are followed, except for the last step. Indeed, the last step consists of the reaction between the amines of the respective intermediates 16 and 21 and 3-benzoylacrylic acid activated by isobutyl chloroformate in the presence of / V-methylmorpholine (Figure 8).

[0078] Example 2: Synthesis of conjugates of general formula (I)

[0079] AMFA engineering was validated on five different antibodies that were coupled to AMFA of formula (II): - two antibodies directed against tumor necrosis factor alpha (TNF-alpha), called infliximab and adalimumab, and referred to respectively in the application as mAb1 and mAb2; - an antibody directed against the vascular endothelial growth factor (VEGF), called bevacizumab, referred to as mAb3; - an antibody directed against the human epidermal growth factor receptor 2 (HER2), called trastuzumab, referred to as mAb4; and - an antibody directed against the green fluorescent protein (GFP), called Ab.

[0080] The grafting of AMFA, for these non-limiting examples, can be carried out at the level: - of the oligosaccharide chain of the glycosidic part of the recombinant antibody (see points 1 / to 5 / below) with AMFAI, or - of the amine function of the lysine residues of the peptide part of the recombinant antibody (see point 6 / below) with AMFA4.

[0081] 1 / Grafting of AMFAI onto the oligosaccharide chains of the glycoside part of a chimeric anti-TNF-alpha antibody The coupling of AMFAI was tested on a commercial mouse-human chimeric monoclonal antibody directed against human TNF-alpha, infliximab, defined as mAb1. A conjugate of formula (I) is formed which may indifferently be represented in the application by "mAb1 -AMFA1" or "AMFA1 -mAb1". This conjugate comprises a part (mAb1) which binds to a specific target (such as an extracellular or membrane antigen) and a part (AMFA1) which binds to RM6P-CI. The mAb1 antibody was chosen as a model in the present application because it has oligosaccharide chains on the constant regions that are very similar to those produced in humans. The coupling between AMFAI and the mAb1 antibody is carried out according to the protocol described below. In a first step, the mAb1 antibody is oxidized. Specifically, the mAb1 antibody and 1 mM sodium metaperiodate (NalO4) solution are reacted in 25.5 mM phosphate buffer pH 6.25 for 30 min at 4°C in the dark. Glycerol (20 μL / mL) is added and incubated for 10 min at 4°C to stop the reaction. Low molecular weight compounds are removed using a PD-10 desalting column pre-packed with Sephadex G-25 resin. In a second step, AMFAI is added to the previously obtained oxidized antibody solution and the mixture is left to react under stirring for 2 h at 37°C. Finally, the samples are dialyzed overnight against a phosphate buffer. The first step of the above protocol leads to the oxidation of the oligosaccharide chains of the mAb1 antibody and generates aldehyde groups that interact covalently with the oxy-amine groups of AMFAI to form an oxime bond. In addition, only the oligosaccharide chains of the mAb1 antibody are remodeled. Indeed, the peptide part of the mAb1 antibody is not affected by the conditions used for the grafting of AMFAI.

[0082] 2 / Grafting of AMFAI onto the oligosaccharide chains of the glycoside part of a humanized anti-TNF-alpha antibody The anti-TNF-alpha antibody, adalimumab, defined as mAb2, was tested and subjected to the same procedure as described in point 1 / above in order to graft AMFAI onto the oligosaccharide chains of the glycosidic part of the mAb2 antibody. A conjugate of formula (I) is formed, which may indifferently be represented in the application by “mAb2-AMFA1” or “AMFA1-mAb2”. This conjugate comprises a part (mAb2) which binds to a specific target (such as an extracellular or membrane antigen) and a part (AMFA1) which binds to RM6P-CI.

[0083] 3 / Grafting of AMFAI onto the oligosaccharide chains of the glycoside part of a humanized anti-VEGF antibody The anti-VEGF antibody, bevacizumab, defined as mAb3, was tested and subjected to the same procedure as described in point 1 / above in order to graft the AMFAI onto the oligosaccharide chains of the mAb3 antibody. A conjugate of formula (I) is formed, which may indifferently be represented in the application by “mAb3-AMFA1” or “AMFA1-mAb3”. This conjugate comprises a part (mAb3) which binds to a specific target (such as an extracellular or membrane antigen) and a part (AMFA1) which binds to RM6P-CI.

[0084] 4 / Grafting of AMFAI onto the oligosaccharide chains of the glycoside part of a humanized anti-HER2 antibody The anti-HER2 antibody, trastuzumab, defined as mAb4, was tested and subjected to the same procedure as described in point 1 / above in order to graft the AMFAI onto the oligosaccharide chains of the mAb4 antibody. A conjugate of formula (I) is formed, which may indifferently be represented in the application by “mAb4-AMFA1” or “AMFA1-mAb4”. This conjugate comprises a part (mAb4) which binds to a specific target (such as an extracellular or membrane antigen) and a part (AMFA1) which binds to RM6P-CI.

[0085] 5 / Grafting of AMFAI onto the oligosaccharide chains of the glycoside part of an anti-GFP antibody The anti-GFP antibody, defined as Ab, was tested and subjected to the same procedure as described in point 1 / above in order to graft the AMFAI onto the oligosaccharide chains of the Ab antibody. A conjugate of formula (I) is formed, which may be represented interchangeably by “Ab- AMFA1” or “AMFA1-Ab”. This conjugate comprises a part (Ab) which binds to a specific target (such as an extracellular or membrane antigen) and a part (AMFA1) which binds to RM6P-CI.

[0086] 6 / Grafting of TAMFA4 onto the amine function of lysine residues of the peptide part of a humanized anti-VEGF antibody The coupling between TAMFA4 and the antibody mAb3 (bevacizumab) is carried out at the level of the peptide chain of mAb3, in particular at the level of lysine residues according to the protocol described below. The mAb3 and TAMFA4 antibody solution are reacted in phosphate buffer solution for 24 h at 37 °C with gentle stirring. The samples are dialyzed overnight against phosphate buffer. A conjugate of formula (I) is formed, which may be represented interchangeably by “mAb3- AMFA4” or “AMFA4-mAb3”. This conjugate comprises a part (mAb3) which binds to a specific target (such as an extracellular or membrane antigen) and a part (AMFA4) which binds to RM6P-CI.

[0087] Example 3: Study of conjugates (I) and their biological properties

[0088] 1 / Study of the number of AMFAI or AMFA4 bound to an antibody molecule mAb1, mAb2, mAb3 or mAb4 The AMFA1-mAb1, AMFA1-mAb2, AMFA1-mAb3, AMFA1-mAb4 and AMFA4-mAb3 conjugates were analyzed by MALDI-TOF mass spectrometry using a RapifleX mass spectrometer (Bruker) in linear mode, in order to quantify the number of AMFAI bound to an antibody molecule, according to the method adapted and described in Zhou, Q et al. 2013. The molecular weights of each antibody mAb1, mAb2, mAb3, and mAb4 were measured before and after coupling with AMFAI or TAMFA4. Samples and standards were diluted 1:5 in 0.1% formic acid in water, then diluted 1:1 in saturated sinapic acid in 50% acetonitrile / 0.1% TFA. One pL (microliter) of this mixture was applied to a target. Samples, corresponding references, and the BSA calibration control were analyzed in duplicate. A two-point calibration was performed with [M+H] ions + and BSA dimer ions. The number of AMFAI or AMFA4 grafted to the antibodies mAb1, mAb2, mAb3 or mAb4 to obtain the conjugates of the invention was calculated on the basis of the difference in molecular weights between the “AMFA-Antibody” conjugate and its corresponding control “Antibody”, then dividing the difference by the molecular weight of each AMFA.

[0089] Table 1 below presents the data from the MALDI-TOF analysis of different batches of conjugates AMFA1 -mAb1, AMFA1 -mAb2, AMFA1 -mAb3 and AMFA1 -mAb4 as obtained in Example 2 (points 1 / to 4 / ).

[0090] [Table 1]

[0091] Table 2 below presents the data from the MALDI-TOF analysis of different batches of AMFA4-mAb3 conjugates as obtained in Example 2 (point 6 / ).

[0092] [Table 2]

[0093] Remarks and conclusion The increase in molecular weights after grafting of AMFAI or AMFA4 is an indication of the number of AMFAs coupled per antibody. The reproducibility of the grafting reaction is indicated by the low standard deviation of the number of AMFAs grafted between different assays. The data in Tables 1 and 2 demonstrate that AMFAI and TAMFA4 can be readily grafted onto mAb1, mAb2, mAb3, or mAb4 with high reproducibility standards, and that the number of AMFAI or AMFA4 grafted onto each antibody is controlled by modulating the reaction conditions. Lower concentrations of the oxidizing agent, sodium metaperiodate, allow for a lower grafting rate of AMFAI. The grafting of the AMFAs of the invention is reproducible and easily controllable depending on the reaction conditions.

[0094] 2 / Study of the structural modifications associated with the grafting of AMFAI onto the antibodies mAb1, mAb2, mAb3 or mAb4 The antibodies mAb1, mAb2, mAb3 and mAb4 alone or conjugated to AMFAI (mAb1-AMFA1, mAb2-AMFA1, mAb3-AMFA1 and mAb4-AMFA1 (the mAb-AMFA conjugates)) were analyzed on non-denaturing and denaturing SDS-PAGE. Coomassie blue staining revealed a single band (under non-denaturing conditions) or two bands (under denaturing conditions) of the same molecular weight for all antibodies before and after coupling with AMFA1. Binding of AMFAI with the antibody did not induce any visible degradation or significant shift in the molecular weight of the antibody heavy or light chains due to the low number of grafted analogues. The immunoreactivity of the mAb-AMFA conjugates towards a second commercial anti-human IgG antibody was unchanged on Western blot compared to the mAbs. Hybridization with a rabbit anti-AMFA1 antibody (represented by “anti-AMFA” in Figure 9) confirmed the presence of AMFAI on the heavy chain of the antibodies bound to AMFA1 (according to the protocols described in Basile I. et al., 2018). The results obtained are illustrated in Figure 9. Western blot analyses of the different antibodies demonstrate on the one hand that oxidation does not alter the general structure of the antibody and on the other hand that the coupling of AMFAI is effective on the heavy chains of each antibody mAb1, mAb2, mAb3 or mAb4 and efficient as proven by the detection of AMFA1 by the rabbit anti-AMFA1 antibody. In conclusion, the grafting of AMFA1 to the antibodies mAb1, mAb2, mAb3 or mAb4 does not modify the integrity of the antibody. Moreover, the detection of AMFA1 on the heavy chains of the antibodies mAb1, mAb2, mAb3 and mAb4 is in agreement with the grafting only on the oligosaccharide chains carried by the heavy chains. The fact that treatment with PNGase hydrolyzing N-glycosylated chains allows AMFA1 to be detached from mAb1-AMFA1 indicates the specificity of grafting onto oligosaccharide chains.

[0095] 3 / Study of the capacity of the mAb1-AMFA1 and mAb2-AMFA1 conjugates to bind to RM6P-CI Affinity assays of the inventive conjugates mAb1-AMFA1 and mAb2-AMFA1 for RM6P-CI were performed using biotinylated RM6P-CI (Basile et al, 2018). Affinity of mAb1-AMFA1 and mAb2-AMFA1 conjugates for RM6P-CI refers to the ability of mAb1-AMFA1 and mAb2-AMFA1 conjugates to bind to RM6P-CI. Briefly, RM6P-CI, purified on a phosphomannan-sepharose affinity column, was biotinylated by biotin / V-hydroxysuccinimide. The binding of biotinylated RM6P-CI (RM6P-CI-b) to pentamannose 6-phosphate (PMP) previously adsorbed on a microtiter plate was prevented by competition of increasing concentrations of the mAb1-AMFA1 or mAb2-AMFA1 conjugates. The RM6P-CI-b bound to the adsorbed PMP was then determined using peroxidase-coupled streptavidin and its substrate, o-phenylenediamine dihydrochloride (OPD), by optical density measurements. The value 100% corresponds to the concentration of RM6P-CI-b bound to the adsorbed PMP in the absence of competitor. Table 3 below presents the data from the binding assays of natural M6P to RM6P-Cl-b and the conjugates mAb1-AMFA1 and mAb2-AMFA1 to RM6P-CI-b. The conjugates studied include a part (mAb1 or mAb2) that targets a target molecule (antigen) and a part (AMFA1) that targets RM6P-CI-b. The affinity (expressed as the concentration required for 50% inhibition of binding, IC50) was determined by a competitive assay with the ligand pentamannose 6-phosphate.

[0096] [Table 3]

[0097] Remarks and conclusion While the binding capacity of the single antibody mAb1 or mAb2 to RM6P-CI is close to zero (less than 10% binding at a concentration range of 5.10 8 - 2.10 6M), Table 3 shows that the mAb1 -AMFA1 or mAb2-AMFA1 conjugate of the invention has a higher binding affinity for RM6P-CI than that of natural M6P for RM6P-CI. The conjugates of the invention thus acquire the ability to bind to RM6P-CI. More particularly, the grafting of AMFAI, respectively on mAb1 and mAb2, allows the conjugates of the invention mAb1 -AMFA1 and mAb2-AMFA1 to gain affinity for RM6P-CI.

[0098] 4 / Study of the impact of AMFAI grafting on the binding affinity of mAb1 to mAb4 antibodies to the target molecule, namely the corresponding antigen (human TNF-alpha, VEGF or HER2). The affinity of mAb1, mAb2, mAb3, mAb4 antibodies alone, and mAb1-AMFA1, mAb2-AMFA1, mAb3-AMFA1, and mAb4-AMFA1 conjugates for target antigens (human TNF-alpha, VEGF, or HER2) was assessed using an ELISA assay to determine antigen binding. The term "affinity" of the antibody or conjugate for the antigen means the binding capacity of said antibody or conjugate for said antigen. Briefly, the antigen (commercial human TNF-alpha, VEGF or HER2) was adsorbed onto a microwell plate and incubated with the corresponding antibody (mAb1, mAb2, mAb3, mAb4) or conjugate (mAb1-AMFA1, mAb2-AMFA1, mAb3-AMFA1 and mAb4-AMFA1), respectively. The level of antibody or conjugate bound to the target of interest is determined by an anti-human IgG antibody coupled to horseradish peroxidase and the OPD substrate using optical density measurements. It is clear from Figure 10 that grafting AMFAI onto antibodies mAb1, mAb2, mAb3 or mAb4 does not affect their affinity for their target antigen (human TNF-alpha VEGF or HER2). The conjugates of the invention have an affinity for their antigen comparable to that of the antibodies alone.

[0099] 5 / Study of the stability of the mAb1 -AMFA1 conjugate of the invention mAb1-AMFA1 conjugates from different batches were incubated in acetate buffer (pH 5) or phosphate buffer (pH 7) for up to 12 days. The conjugates were then analyzed by MALDI-TOF to determine whether AMFAI was still bound to the mAb1 antibody. The number of AMFAI grafted onto the mAb1 antibody was determined at day 0 and day 12 at pH 5 and 7. The results obtained are shown in Table 4 below.

[0100] [Table 4]

[0101] Remarks and conclusion Table 4 shows that the number of AMFAI grafted onto the mAb1 antibody is stable at both different pHs even after 12 days. The AMFA1-mAb1 conjugates are therefore stable over time and at different pHs.

[0102] 6 / Study of the cellular internalization of the conjugates mAb1-AMFA1, mAb3-AMFA1 and Ab-AMFA1 Cellular internalization of the antibodies and conjugates of the invention was observed in rhabdomyosarcoma cells, T-cell-like cells, and breast cancer cells. Data were obtained by flow cytometry and whole-cell fluorescence assays. Rhabdomyosarcoma cells, T-cell-like cells, and breast cancer cells were treated for 24 h or 48 h with a complex comprising mAb1-AMFA1 and an anti-human IgG Alexa647 fluorescent secondary antibody (Figures 11 (A) and 12 (A)). Prior to incubation with cells, mAb1 antibody, mAb1-AMFA1 conjugate, and a nonspecific human IgG control were pre-incubated with the fluorescent anti-human IgG secondary antibody Alexa647. The fluorescence present in the cells was then analyzed by flow cytometry (Figure 11 (A)). The data in Figure 1 1 (A) show significantly higher fluorescence (7.9-fold, 11.3-fold, and 11.8-fold higher compared to control) in cells treated with mAb1-AMFA1. Figure 1 1 (B, C) presents the entry of mAb1 and mAb1-AMFA1, and mAb4 and mAb4-AMFA1 pre-coupled to Alexa647 into T cells and SKOV3 ovarian cancer cells, respectively. Figure 1 1 demonstrates that AMFAI enhances the cellular uptake of the mAb1-AMFA conjugate compared to mAb1 alone. Grafting AMFAI to mAb1 antibody significantly increases the cellular internalization of antibodies. These results were confirmed by the internalization experiments of mAb1, mAb1-AMFA1 as well as mAb3 and mAb3-AMFA1, performed by a total cell fluorescence assay (Figure 12). Rhabdomyosarcoma and breast cancer cells were treated for 24 h under the same conditions as described previously. Figure 12 shows that higher fluorescence is observed in cells treated with mAb1-AMFA1 versus mAb1 (breast cancer cells) and mAb3-AMFA1 versus mAb3 (rhabdomyosarcoma cells). Grafting AMFAI to mAb1 or mAb3 significantly increases the cellular internalization of antibodies. These results were confirmed by confocal microscopy on live rhabdomyosarcoma and breast cancer cells (Figure 13 (A)). The cells were pre-incubated with 200 nM of the Ab antibody or the Ab-AMFA1 conjugate (see Example 2, point 5 / ) and 400 nM of green fluorescent protein, for 24 h.Higher fluorescence was observed with Ab-AMFA1 versus Ab alone in both cell types (fluorescent cell vesicles marked by arrows in Figure 13). Addition of excess M6P (10 mM) to the cell medium to saturate the RM6P-CI binding sites showed that Ab-AMFA1 internalization into cells is significantly reduced in both cell types. This experiment demonstrates that the majority of Ab-AMFA1 internalization was achieved through RM6P-CI. In another experiment, mAb1 and mAb1-AMFA1 were coupled to the Alexa467 fluorophore to measure their internalization into Jurkat cells (T lymphocytes). The increase in internalization of mAb1-AMFA1 relative to mAb1, and the reversion of mAb-AMFA1 entry by the addition of an excess of 30 mM AMFA1 are depicted and quantified in Figure 13 (B, C).More broadly, these results demonstrate that grafting AMFA onto antibodies induces an increase in cellular internalization via the RM6P-CI-mediated transport pathway.

[0103] 7 / Study of the pharmacokinetic profile of the mAb1-AMFA1 and mAb2-AMFA1 conjugates of the invention The effect of AMFAI grafting on mAb1 and mAb2 antibodies targeting TNF-alpha was studied by comparing the pharmacokinetic profile of mAb1 or mAb2 with that of the corresponding mAb1-AMFA1 or mAb2-AMFA1 conjugates. The mAb1 or mAb2 antibodies and the corresponding mAb1-AMFA1 or mAb2-AMFA1 conjugates were administered to 10-week-old male C57BL / 6J mice as a single intravenous bolus injection of 4 mg / kg diluted in physiological saline. The mice were weighed before injection and throughout the experiment to monitor their health status. Blood samples collected at 4 hours and 1, 2, 3, 7, 10, 14, 17, and 21 days after injection were collected in heparin-containing tubes and centrifuged at 3000 g for 15 min, and the plasma was stored at -20 °C until measurement. The mice were sacrificed 21 days after injection. No adverse clinical events were observed in the four treatment groups. Plasma concentrations of mAb1, mAb2, mAb1-AMFA1, and mAb2-AMFA1 were measured using ELISA techniques. Briefly, recombinant human TNF-alpha was adsorbed onto a Maxisorp microwell plate and was recognized by mAb1, mAb2, mAb1-AMFA1, or mAb2-AMFA1 present in plasma samples. The therapeutic antibody was detected by an HRP-conjugated, Fc-specific anti-human IgG antibody, which was then quantified by a colorimetric assay with OPD substrate and absorbance reading at 450 nm. The results obtained are shown in Figure 14. The pharmacokinetic profiles of mAb1 compared to mAb1-AMFA1 (A) and of mAb2 compared to mAb2-AMFA1 (B) detected in the plasma samples of the treated animals, expressed as a percentage relative to the 4-h values, are similar. No significant difference in antibody half-life is observed with the grafting of AMFAI onto mAb1 or mAb2. The grafting of AMFAI onto the antibodies therefore does not modify their plasma stability. In conclusion, grafting AMFAI onto mAb1 and mAb2 antibodies targeting the TNF-alpha antigen does not alter the pharmacokinetic profile of the antibodies.

[0104] 8 / Study of the in vivo stability of the mAb1-AMFA1 conjugate of the invention The in vivo stability of AMFAI binding to the therapeutic antibody mAb1 was assessed by analyzing the presence of AMFAI in plasma samples collected at 21 days from mice treated with mAb1 -AMFA1 or mAb1 as described in point 7 / above. mAb1 or mAb1-AMFA1 were first isolated from plasma samples by binding for 24 h to an anti-human IgG antibody specific for the Fc fragment previously adsorbed onto a microwell plate. As a loading control, 50 ng of mAb1-AMFA1 diluted in phosphate buffer was also incubated. After washing, the bound antibodies were analyzed by Western blotting for the presence of AMFAI using an antibody polyclonal anti-AMFA1 antibody (represented by “anti-AMFA” in Figure 15) and with an anti-human IgG antibody to control for the amount of mAb1 or mAb1-AMFA1 present at 21 days. Figure 15 provides evidence that AMFAI is still conjugated to the therapeutic antibody mAb1 at 21 days. Figure 15 shows that both mAb1 and mAb1-AMFA1 are detected in the plasma of treated mice after 21 days at an amount similar to that of the 50 ng mAb1-AMFA1 control, as confirmed by hybridization with anti-IgG. The majority of AMFA1 is still bound to mAb1, with the signal intensity of the band being comparable to that of mAb1-AMFA1 used as a control.

[0105] 9 / Study of the capacity of the mAb1 -AMFA1 conjugate to increase the cellular internalization of human TNF-alpha It is shown in this study that human TNF-alpha antigen, after being internalized into cells with the mAb1 -AMFA1 conjugate, is degraded over time. The degradation of human TNF-alpha was studied in human T-lymphocyte-like cells. The cells were treated with 5 ng / mL of human TNF-alpha and 20 ng / mL of mAb1 or mAb1 -AMFA1 , for 1 h, 5 h and 17 h. The cell lysates were analyzed by Western blotting for the detection of intracellular human TNF-alpha. For the mAb1 -AMFA1 conjugate, the data show an increase in the level of human TNF-alpha up to 17 h (Figure 16 (A)). In contrast, only a slight increase in intracellular human TNF-alpha is noticed with the mAb1 antibody. Data demonstrate that human TNF-alpha is better internalized by mAb1 -AMFA1 compared to mAb1 antibody.The amounts of TNF-alpha measured by Western Blot in the culture medium during treatment with mAb1 -AMFA1 and mAb show that mAb1 -AMFA1 causes a greater decrease in TNF-alpha (Figure 16 (B)). This strong decrease in the external medium is correlated with the increase in cellular entry of TNF-alpha.

[0106] 10 / Study of the capacity of the mAb1 -AMFA1 conjugate to allow the reduction of the biological effect of human TNF-alpha It is shown in this study that the mAb1-AMFA1 conjugate allows, in addition to better internalization in the cell, a reduction in the biological effect greater than that observed with the antibody alone. Murine L929 fibroblast-like cells were treated with 5 ng / mL human TNF-alpha and increasing doses of mAb1 or mAb1-AMFA1. Since human TNF-alpha induces cytotoxicity in L929 cells, the live cell rate after 48 h of treatment with mAb1 or mAb1-AMFA1 was analyzed by the MTT method. Figure 17 shows the neutralizing effect on human TNF-alpha toxicity of mAb1 or mAb1-AMFA1. mAb1-AMFA1 can neutralize human TNF-alpha toxicity at much lower doses (4 to 5 times) than mAb1. In conclusion, mAb1-AMFA1 conjugates induce a dose-dependent neutralization of human TNF-alpha-induced cell growth inhibition more effectively than mAb1 alone.

[0107] 11 / Study of the capacity of the mAb3-AMFA1 of the invention to allow a reduction in the effect of human VEGF on the growth of HUVEC cells It is shown in this study that the mAb3-AMFA1 conjugate allows, in addition to better internalization in the cell, a reduction in the biological effect greater than that observed with the mAb3 antibody alone. Human umbilical vein endothelial cells (HUVECs) were treated with 50 ng / mL human VEGF and increasing doses of mAb3 or mAb3-AMFA1. Since human VEGF induces HUVEC proliferation, the live cell rate at 72 h on cells treated with mAb3 or mAb3-AMFA1 was analyzed by the MTT method. Figure 18 shows that the inhibition of the mitogenic effect of VEGF in HUVEC cells is greater with the mAb3-AMFA1 conjugate compared to the antibody alone. At low doses, the mAb3-AMFA1 conjugate is twice as effective as the mAb3 antibody alone. The mAb3-AMFA1 conjugate is effective at much lower doses (10 times) than the mAb3.

[0108] 12 / Study of the capacity of AMFA1 -mAb4 conjugates to allow a reduction in the quantity of HER2 SK-BR-3 breast cancer cells were treated with 10 or 100 nM mAb4 or mAb4-AMFA1 for 24 h. Then the cells were fixed and the amount of the target protein was measured by a colorimetric method based on an indirect ELISA. The HER2 protein is recognized by a specific antibody, the Fc portion of which will in turn be recognized by a secondary antibody coupled to peroxidase (HRP). The peroxidase enzyme will catalyze the colorimetric reaction after addition of the substrate. Figure 19 (A) shows the decrease in HER2 protein after treatment with mAb4-AMFA1 compared to mAb4 alone on SK-BR3 cells. Increasing doses of mAb4-AMFA1 allowed a greater decrease in cellular HER2 concentration compared to the same doses of mAb4. BT474 breast cancer cells were treated with 5 nM mAb4 or mAb4-AMFA1 for 5 h. The total amount of HER2 was quantified by Western blotting. After 5 h of treatment with mAb4-AMFA1, the amount of HER2 was significantly decreased while mAb4 treatment was not effective (Figure 19 (B)).

[0109] 13 / Extracellular recycling of AMFA1-mAb1 conjugates after their internalization into T lymphocytes Extracellular recycling of internalized mAb1 and mAb1-AMFA1 conjugate was demonstrated by cellular entry experiments of antibodies for 5 h in immortalized T lymphocyte cells (Jurkat) followed by a change of medium to a medium lacking antibody and a period of 1 and 5 h of release into the culture medium of the previously internalized antibodies (Figure 20 (A)). In another experiment, the cells were treated for 30 min with antibodies and after change of medium, the release of internalized antibodies was measured after 10 and 30 min (Figure 20 (B)). The concentrations of mAb1 and mAb1-AMFA1 were measured using the ELISA technique as described in point 7 / above. Figure 20 shows that the extracellular recycling of mAb1-AMFA1 conjugates is comparable to that of mAb1 alone. These results indicate that mAb1-AMFA1 is normally recycled out of the cell after its strong cellular internalization mediated by RM6P-CI.

[0110] This disclosure is not limited to the examples described above, only as an example, but it encompasses all the variations that a person skilled in the art may envisage within the framework of the protection sought. List of cited documents Patent documents

[0111] For convenience, the following patent documents are cited: - patcitl: EP 2 448 600 B1; - patcit2: EP 3 350 192 B1; - patcit3: .PCT / US2019 / 067228; - patcit4: PCT / IB2021 / 050922. Non-patent literature

[0112] For convenience, the following non-patent elements are cited: - nplcitl : Gary-Bobo et al. Curr. Med. Chem. Rev. 2007, 14, 2945–2953; - nplcit2 : El Cheikh K. et al., Angew. Chem. Int. Head. Rev. 2016, 55, 52016; - nplcit3 : Zhu Y., et al. Mol Ther. Rev. 2009.17, 954–963; - nplcit4 : Basil I. et al., J. Control. Release, 2018, 10, 269,15–23; In: Zhou, Q et al., Bioconjugate Chem. 2013, 24, 12, 2025–2

Claims

Demands

1. Conjugate characterized in that it has the following general formula (I) [Cheml] in which: X is a phosphonate group -CH2-P(O)(OZ)2 or carboxylate group -CH2-C(O)(OZ) with Z independently representing H, Na, Li, K or NH4; n is an integer from 0 to 2; L1 is a radical chosen from the group including *-ON=**, [Chem2] [Chem3] with * indicating the attachment point of Li to the (O-CH2-CH2)n group and ** indicating the attachment point of Li to Yi; ni is an integer from 1 to 20, and preferably from 1 to 10, Yi represents an antibody Y or a fragment of antibody Y comprising at least one antigen-binding domain, said Yi forming covalent bond(s) with Li.

2. Conjugated according to claim 1, characterized in that Yi is represented by the L2-Y'I group in which Y'i represents the antibody Y or fragment of antibody Y linked to Li via the radical L2 with L2 chosen from the group comprising =CH- , -NH- and -S-, said Yi can thus be represented by =CH-Y'i , -NH-Y'i and -S-Y'i.

3. Conjugate according to claim 1 or 2, characterized in that is an integer equal to 1 and in that said conjugate of formula (I) is chosen from the group comprising: [Chem5] [Chem7] with X, n and Y'i as defined in claim 1 or 2.

4. Conjugated according to any one of claims 1 to 3, characterized in that Yi represents a therapeutic or diagnostic antibody Y.

5. Conjugated according to claim 4, characterized in that Yi represents an antibody Y selected from the group comprising monoclonal antibodies, chimeric antibodies, human or humanized antibodies, heavy and light chain antibodies, single chain antibodies, bispecific or multispecific antibodies and nanobodies.

6. Conjugated according to claim 5, characterized in that Yi represents an antibody Y which is an immunoglobulin of the IgG type, in particular of subtype lgG1, lgG2, lgG3 or lgG4, or an immunoglobulin of the IgE, IgD, IgA or IgM type.

7. Conjugated according to any one of claims 1 to 6, characterized in that it has an IC50 affinity for the cation-independent mannose 6-phosphate receptor (RM6P-CI) ranging from 10 -4 M to 10 -9 M, and preferably ranging from 10 -5 M to 10 -8 Mr.

8. A method for preparing a conjugate of formula (I) as defined in any one of claims 1 to 7, characterized in that the following reaction is made: - a Y antibody or a fragment of a Y antibody comprising at least one antigen-binding domain with - nor compound(s) corresponding to the general formula (II) [Chem9] in which X, n and are such as defined in claim 1, L is a reactive chemical group chosen from the group comprising -O-NH2, [Chem 10] [Cheml 1] And [Chem 12] said group L of compound (II) forming covalent bond(s) with functional group(s) carried by said antibody Y or fragment of antibody Y.

9. A preparation method according to claim 8, characterized in that compound (II) is selected from the group comprising [Chem 13] [Chem 17]

10. Pharmaceutical composition characterized in that it comprises: - a conjugate as defined in any one of claims 1 to 7, - at least one pharmaceutically acceptable excipient.

11. Conjugated as defined in any one of claims 1 to 7 or pharmaceutical composition according to claim 10, for use as a medicinal product.

12. Conjugated as defined in any one of claims 1 to 7 or pharmaceutical composition according to claim 10, for use in a therapeutic treatment method which consists of administering a therapeutic antibody or a fragment of a therapeutic antibody.

13. Conjugated as defined in any one of claims 1 to 7 or pharmaceutical composition according to claim 10, for use in a diagnostic method.

14. Conjugate or pharmaceutical composition for use according to any one of claims 11 to 13, characterized in that the conjugate or pharmaceutical composition is in a form suitable for administration by parenteral, intravenous or subcutaneous route.

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