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

Abstract

The subject of the present invention is a novel conjugate of general formula (I) according to claim 1. The conjugate of the present invention comprises 1 / at least one mannose 6-phosphate (M6P) analogue targeting the cation-independent mannose 6-phosphate receptor (RM6P-CI) and 2 / an antibody or antibody fragment capable of binding to a specific target antigen or target molecule. The present invention also relates to a method for producing said conjugates and their medical use, such as therapeutic or diagnostic applications. The conjugate of formula (I) of the present invention is particularly advantageous for use as a drug, in particular in a therapeutic treatment method consisting of administering a therapeutic antibody or a therapeutic antibody fragment.

Description

[Technical field]

[0001] The present invention falls in the field of therapeutic chemistry. The present invention more particularly relates to bifunctional compounds comprising 1 / at least one mannose 6-phosphate (M6P) analogue targeting the cation-independent mannose 6-phosphate receptor (CI-M6PR) and 2 / an antibody or antibody fragment allowing the binding of a specific target antigen or target molecule. The present invention also relates to a method for preparing said bifunctional compounds and their medical use in the therapeutic or diagnostic field. [Background technology]

[0002] Most therapeutic molecules that target disease-related proteins neutralize the targets mentioned above, thereby blocking the pathophysiological functions of those targets. However, therapeutic molecules often have little effect on the expression or degradation levels of targets. In addition, there are still many proteins that have not yet been pharmacologically targeted, and whose dysregulation and accumulation cause pathological conditions.

[0003] In recent years, targeted degradation of proteins involved in various diseases has emerged as a key strategy to achieve therapeutic efficacy. For targeted degradation of proteins by proteasome activation, degradation-inducing drugs such as proteolysis-targeting chimeras are used. However, the design of these chimeras is complicated and has limited cell permeability. Moreover, since the ubiquitin-proteasome system is intracellular, the use of such chimeras is not applicable to extracellular proteins.

[0004] The physiological pathway for protein degradation is represented by the endolysosomal pathway. Lysosomes are the main degradative compartment of eukaryotic cells. In addition to proteasomes, lysosomes can also degrade molecules other than proteins, such as glycosaminoglycans, oligosaccharides and lipids. Therefore, molecules have been developed that can target pathological proteins to lysosomes for degradation.

[0005] The cation-independent mannose 6-phosphate receptor (CI-M6PR), a 300 kDa transmembrane glycoprotein, is the main trafficking and sorting pathway to lysosomes for lysosomal enzymes carrying the mannose 6-phosphate (M6P) signal. CI-M6PR also mediates endocytosis of M6P-containing extracellular ligands (Gary-Bobo et al., 2007). M6P-containing proteins that are internalized by CI-M6PR-dependent trafficking, which are distinct from lysosomal enzymes, include granzyme B, a protease involved in apoptosis induced by cytotoxic T cells, herpes simplex virus (HSV), and leukemia inhibitory factor, a multifunctional protein that plays an important role in the formation of neurons, platelets, and bone. Thus, a prerequisite for proteins to reach lysosomes is the presence of M6P. However, M6P can be dephosphorylated by serum phosphatases and lose its ability to bind to CI-M6PR (El Cheikh K. et al., 2016). Thus, some lysosomal enzymes have been modified to greatly increase the number of M6P residues, but these residues are still degraded by phosphatases (Zhu Y., et al., 2009).

[0006] M6P analogs that are not degraded by phosphatases have been synthesized in which the phosphate group is replaced by other groups that can be recognized by CI-M6PR. M6P analogs refer to compounds that are slightly different from the natural M6P compound but have high binding ability to CI-M6PR. M6P analogs have been proven effective in enhancing the therapeutic potential of lysosomal enzymes produced in baculovirus / insect cell expression systems without M6P signals (El Cheikh K. et al., 2016) and even lysosomal enzymes that already carry M6P (Basile I. et al., 2018).

[0007] Documents EP 2448 600 B1 and EP 3 350 192 B1 describe M6P analogues, called AMFA ("Analogs of Mannose 6-Phosphate Functionalized at the Anomeric Position"), which present 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, have low immunogenicity and can be easily grafted onto lysosomal enzymes. These analogues can also be adjusted, depending on the type of linkage desired, both in terms of the length and structure of the spacer arm, as well as in terms of the terminal reactive group of the spacer arm. In these documents, M6P analogues are more particularly used to internalize lysosomal enzymes into the lysosomes of cells, so that said enzymes can perform their physiological role.

[0008] Document PCT / US2019 / 067228 describes bifunctional molecules obtained by grafting a polypeptide onto an antibody, said polypeptide comprising several M6P analogs ranging from 20 to 90. The addition of this polypeptide to an antibody proved to be effective in enhancing the M6P receptor-mediated internalization of antigens into cultured cells. The fate of the antibody coupled to the polypeptide after cell transfer was not analyzed.

[0009] Document PCT / IB2021 / 050922 describes bifunctional compounds in which M6P analogues (M6Pa) are coupled to specific antagonist ligands for the factors PCSK9 and FHR3 in order to reduce the extracellular concentration of these factors. After cellular internalization of the M6Pa-antagonist complexed with the targeted factor, degradation in lysosomes occurs. Coupling of M6Pa analogues to antibodies is not considered in this document, and the fate of the M6Pa-antagonist after cellular transfer is not analyzed.

[0010] The present 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 recycle out of the cell after the initial cell entry via the CI-M6PR pathway. This unique and unexpected property allows one to envisage multiple reuse of the compounds of the invention by the cell, in particular to reduce / reduce the concentration of soluble or membrane extracellular antigens. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] EP 2 448 600 B1 [Patent Document 2] EP 3 350 192 B1 [Patent Document 3] PCT / US2019 / 067228 [Patent Document 4] PCT / IB2021 / 050922 Summary of the Invention [Means for solving the problem]

[0012] According to a first aspect, the object of the present invention is to provide a compound of general formula (I)

[0013] [ka]

[0014] (In the formula: X's are each independently H (hydrogen), Na (sodium), Li (lithium), K (potassium) or NH 4 (ammonium) with Z representing the phosphonic acid group -CH 2 -P(O)(OZ) 2 or carboxylic acid group -CH 2 -C(O)(OZ), n is an integer ranging from 0 to 2; L1 is (O-CH 2 -CH 2 ) n L for the group 1 * indicates the attachment point of Y 1 L for 1 *-ON=**, with ** indicating the connection point of

[0015] [ka]

[0016] is a group selected from the group comprising n 1 is an integer ranging from 1 to 20, preferably from 1 to 10, Y 1 represents an antibody Y, or an antibody fragment Y comprising at least one antigen-binding domain, The above Y 1 , L 1 and 1 The conjugate is characterized in that it has two covalent bonds.

[0017] According to a second aspect, the object of the present invention is a process for preparing a conjugate of formula (I) as defined above.

[0018] According to a third aspect, the present invention relates to a conjugate of formula (I) for medical use in the therapeutic or diagnostic field. The present invention particularly relates to a conjugate of formula (I) for use in a method of therapeutic treatment consisting of administering a therapeutic antibody or a therapeutic antibody fragment.

[0019] Further features, details and advantages will become apparent from the following detailed description and analysis of the accompanying drawings. [Brief description of the drawings]

[0020] [Figure 1]1 shows the extracellular recycling of the conjugate of formula (I) of the present invention. The M6P analogue is represented by the acronym AMFA. The conjugate of formula (I) may be referred to as "AMFA-antibody". The antibody is represented by its Y shape, the AMFA is represented by a small ball, and the antigen is represented by a small cloud shape. [Diagram 2] Figure 2 shows a general synthesis scheme for conjugates of formula (I) with n1 equal to 1, depending on the significance of the reactive chemical group L of the M6P analog of general formula (II). Figure 2 point 1 / shows the reaction between the oxy-amine function of the M6P analog of formula (II) and the carbonyl function (-CHO) of antibody Y. Antibody Y is represented by Y1'-CHO, Y1' representing the antibody Y without its functional group L' equal to -CHO. Analog (II) is called AMFA1 when X is a phosphonic acid group and n is equal to 0, and AMFA2 when X is a carboxylic acid group and n is equal to 0. Figure 2 point 2 / shows the reaction between the squaric acid function of the M6P analog of formula (II) and the amine function (-NH2) of antibody Y. Antibody Y is represented by Y1'-NH2, Y1' representing the antibody Y without its functional group L' equal to -NH2. Analogue (II) is called AMFA3 when X is a phosphonic acid group and n is equal to 0, and AMFA4 when X is a carboxylic acid group and n is equal to 0. Figure 2 point 3 / Reaction between the maleimide functional group of the M6P analogue of formula (II) and the thiol functional group (-SH) of antibody Y. Antibody Y is represented by Y1'-SH, Y1' represents antibody Y without its functional group L' equal to -SH. Analogue (II) is called AMFA5 when X is a phosphonic acid group and n is equal to 0, and AMFA6 when X is a carboxylic acid group and n is equal to 0. Figure 2 point 4 / Reaction between the carbonylacryl functional group of the M6P analogue of formula (II) and the thiol functional group of antibody Y. Analogue (II) is called AMFA7 when X is a phosphonic acid group and n is equal to 0, and AMFA8 when X is a carboxylic acid group and n is equal to 0. [Diagram 3]FIG. 1 shows a method for preparing intermediate 3: 2-bromoethyl 2,3,4-tri-O-trimethylsilyl-α-D-mannopyranoside, which serves as a precursor for the synthesis of AMFA of formula (II). [Figure 4] FIG. 1 shows a method for preparing compound AMFA1 of general formula (II) from compound 3. [Diagram 5] FIG. 1 shows a method for preparing compound AMFA2 of general formula (II) from compound 3. [Figure 6] FIG. 1 shows a method for preparing compounds of general formula (II), AMFA3 and AMFA5, from compound 6. [Figure 7] FIG. 1 shows a method for preparing compounds of general formula (II), AMFA4 and AMFA6, from compound 1. [Figure 8] FIG. 1 shows a method for preparing compounds of general formula (II), AMFA7 and AMFA8, from compounds 16 and 21, respectively. [Figure 9] Figure 1 shows results obtained by SDS-PAGE electrophoresis and Western blot (WB) for mAb1, mAb2, mAb3 and mAb4 antibodies alone or in combination with AMFA1 (mAb1-AMFA1, mAb2-AMFA1, mAb3-AMFA1 and mAb4-AMFA1). The antibodies before and after coupling to AMFA1 are detected either by protein staining with Coomassie Blue or by recognition with an anti-human IgG antibody. AMFA1 coupled to the antibody is detected with a polyclonal rabbit anti-AMFA1 serum (represented as "anti-AMFA" in the figure). Treatment of mAb1-AMFA1 with endonuclease H (PNGase) is also described with detection by an anti-AMFA antibody. [Figure 10]Graph showing affinity curves of mAb1 antibody or mAb1-AMFA1 conjugate (A), mAb2 antibody or mAb2-AMFA1 conjugate (B), mAb3 antibody or mAb3-AMFA1 conjugate (C), and mAb4 antibody or mAb4-AMFA1 conjugate (D) to their target molecules, namely human TNF-alpha (A and B), VEGF (C), and HER2 (D) antigens, respectively. [Figure 11] (A) Data from flow cytometry analysis of the internalization of non-specific control IgG antibody, mAb1 antibody and mAb1-AMFA1 conjugate with fluorescent anti-human IgG secondary antibody (Alexa647) in rhabdomyosarcoma cells (top graph), type 7 T-lymphocyte cells (middle graph) and breast cancer cells (bottom graph). (B, C) 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 vs. mAb1 from three experiments (Student's T-test). *p<0.05, mAb4-AMFA1 vs. mAb4 from six microscopic fields (Student's T-test). [Figure 12] Graph showing cytofluorescence assay data from breast cancer cells (A) and rhabdomyosarcoma cells (B) treated with: a non-specific control IgG antibody, mAb1 antibody and mAb1-AMFA1 conjugate (breast cancer cells) (A), with a fluorescent anti-human IgG secondary antibody (Alexa647), and with mAb3 antibody and mAb3-AMFA1 conjugate (rhabdomyosarcoma cells) (B), with a fluorescent anti-human IgG secondary antibody (Alexa647). This fluorescence assay makes it possible to define the cellular internalization of the antibody alone and of the conjugate of the invention. **p<0.01, mAb1-AMFA1 vs. mAb1 (Student's T-test). [Figure 13](A) Images showing internalization of Ab antibodies or Ab-AMFA1 conjugates in rhabdomyosarcoma cells (right) and breast cancer cells (left) detected by confocal imaging. 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 two pictures relate to cases where an excess of M6P was added to the culture medium to saturate the CI-M6PR binding sites of the cells. (B, C) Internalization of mAb1 antibody and mAb1-AMFA1 conjugates in Jurkat T lymphocytes (B) and reversal of this internalization by addition of an excess of AMFA1. **p<0.01, mAb1-AMFA1 vs. mAb1 (Student's T-test) n=6. [Figure 14] Graphs showing data from analysis of plasma samples from mice injected with mAb1 or mAb2 antibodies and the corresponding mAb1-AMFA1 or mAb2-AMFA1 conjugates. Antibody concentrations in plasma are shown 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) is for mice treated with mAb2 or mAb2-AMFA1. [Figure 15] 1 shows images of detection by Western blot of mAb1 antibody and mAb1-AMFA1 conjugate present in mouse plasma 21 days after intravenous (iv) injection of 4 mg / kg mAb1 and mAb1-AMFA1. A control of injected mAb1-AMFA1 is added for comparison. The antibody is detected by recognition with anti-human IgG antibody, and the presence of AMFA coupled to the antibody is detected by anti-AMFA1 rabbit serum (represented as "anti-AMFA" in the figure). [Figure 16](A) Graph showing cellular transfer 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, 5 and 17 hours. Cell lysates were analyzed by Western blot. Intracellular concentrations of human TNF-alpha were quantified as a function of time after treatment (hours). **p<0.01, mAb1-AMFA1+TNF-alpha vs. mAb1+TNF-alpha (Student's T-test). (B) Graph showing TNF-alpha concentrations corresponding to various treatment times measured in the culture medium of the cells shown in (A). [Figure 17] Graph showing neutralization of the anti-proliferative effect of human TNF-alpha by mAb1 antibody or mAb1-AMFA1 conjugate on mouse fibroblast-like cells treated with 5 ng / mL human TNF-alpha. **p<0.01, mAb1-AMFA1 vs. mAb1 (Student's T-test). [Figure 18] FIG. 1 is a graph showing reversal of the mitogenic effect of VEGF in HUVEC cells treated with 50 ng / mL human VEGF and various concentrations of mAb3 or mAb3-AMFA1 for 72 hours. [Figure 19] (A) Degradation of HER2 protein (human ERBB2) in SK-BR-3 breast cancer cells treated with 10 or 100 nM mAb4 or mAb4-AMFA1 for 24 hours by protein immunocytochemistry (intracellular ELISA). (B) Degradation of HER2 in BT474 breast cancer cells treated with 5 nM mAb4 or mAb4-AMFA1 for 5 hours. *p<0.01, mAb4-AMFA1 vs. mAb4 (Student's T-test) (n=2). [Figure 20](A) Graph showing recirculation ability of mAb1 antibody and mAb1-AMFA1 conjugate in Jurkat T lymphocyte cells. Each antibody / conjugate is incubated with cells for 5 hours and then culture medium is replaced with antibody-free medium. Cellular export of internalized antibody or conjugate is measured at 1 hour and 5 hours (n=2). (B) Graph showing cellular export of mAb1 antibody and mAb1-AMFA1 conjugate 10 minutes and 30 minutes after 30 minutes of internalization in Jurkat T lymphocytes (n=4). *p<0.05, mAb1-AMFA1 vs. mAb1 (Student's T-test). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Conjugates of formula (I) As indicated, the subject of the present invention is a conjugate of formula (I) as defined above. By "conjugate" in the sense of the present invention is intended a compound comprising two moieties linked together by a covalent bond. The first moiety of the conjugate is at least one M6P analogue and the second moiety of the conjugate is an antibody or an antibody fragment comprising at least one antigen-binding domain.

[0022] In formula (I) defined above, the M6P analogue corresponds to the formula delimited by the large brackets: 1 indicates the number of M6P analogs bound to the antibody or antibody fragment. Indeed, according to the present invention, n 1 The M6P analogues may be covalently linked to an antibody or antibody fragment comprising at least one antigen-binding domain. 1 The M6P analogs are 1 The antibody or antibody fragment is linked to the antibody via a group.

[0023] As already shown, M6P analogs are compounds that are different from natural M6P but have high binding ability to CI-M6PR.

[0024] Throughout this application, M6P refers to mannose 6-phosphate and CI-M6PR refers to the cation-independent mannose 6-phosphate receptor.

[0025] The conjugates of the invention are bifunctional compounds in the sense that they have two binding capacities, namely the ability to bind to CI-M6PR (via an M6P analogue) and the ability to bind to a specific target antigen or molecule (via an antibody or antibody fragment).

[0026] A 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 state or is associated with a disease condition, or whose expression is involved in a pathological disorder.

[0027] In particular, the novel bifunctional compounds of the present invention make it possible to reduce / reduce the concentration of extracellular or membrane-targeted molecules in an individual, said reduction being achieved by cellular endocytosis regulated by CI-M6PR, so that this reduction / reduction in concentration is sufficient to eliminate the pathological condition.

[0028] As used herein, "extracellular or membrane targeted molecule" refers to a molecule of therapeutic or diagnostic interest, particularly a molecule whose overexpression induces or is associated with a pathological condition, or whose expression is involved in a pathological disorder.

[0029] In particular, the novel bifunctional compounds of the present invention make it possible to reduce / reduce the concentration of soluble extracellular or membrane antigens in an individual, so that the individual does not exhibit a pathological condition.

[0030] In the general formula (I) of the conjugate of the present invention, Y 1 is defined as denoting an antibody Y or an antibody fragment Y comprising at least one antigen-binding domain. In the following, for the sake of simplicity, an antibody Y is understood to mean both the whole antibody Y and an antibody fragment Y comprising at least one antigen-binding domain or a molecule derived from an antibody comprising at least one of said antigen-binding domains.

[0031] Antibody Y refers to a free antibody, meaning that antibody Y is not bound to an M6P analogue.

[0032] Antibody Y 1 , L 1 It refers to antibody Y when covalently attached to an M6P analogue via a group.

[0033] Free antibody Y is Y 1 '-L', where L' represents a functional or reactive functional group carried by antibody Y. In other words, Y 1 represents antibody Y without its functional group L'.

[0034] It is the functional group L' of the antibody Y that reacts with a functional group or reactive functional group carried by the M6P analogue to form a covalent bond between the antibody Y and the M6P analogue, thus obtaining a conjugate of formula (I) of the invention.

[0035] In this application, the terms "functional group," "reactive functional group," or "reactive chemical group" may be used interchangeably. Each of these terms designates a group carried by an antibody (or carried by an M6P analog) that can react with a group carried by an M6P analog (or carried by an antibody).

[0036] The antibody is any kind of antibody of any isotype, from any species. It is a therapeutic or diagnostic antibody that targets a molecule of therapeutic or diagnostic interest, such as anti-TNFα, anti-VEGF, anti-HER2 and anti-GFP antibodies. The antibody is a heavy and light chain antibody, a single chain antibody, a dimer or multimer, a bispecific or multispecific antibody. The antibody is an immunoglobulin of the IgG type, in particular of the lgG1, lgG2, lgG3 or lgG4 subtype, of the IgE, IgD, IgA or IgM type.

[0037] "Antibody" includes monoclonal antibodies, humanized or fully human antibodies, and chimeric antibodies from various species (human, mouse, rat, rabbit, goat, camelid, chicken, etc.), including human-mouse chimeric antibodies. Antibody fragments include Fab, Fab', F(ab') 2 , Fv, scFv, Fabc, or Fab fragments containing a portion of the Fc region, and single chain antibody fragments (VFv, scFv, Fabc, or Fab fragments containing a portion of the Fc region) derived from camelid or shark immunoglobulins. H H single domain antibodies (nanobodies and V-NARs). Molecules derived from antibodies include fusion proteins, especially in the form of Fc fusion proteins, that contain the binding domain of an antibody for a target molecule; antibodies or fusion proteins with several binding domains corresponding to several target molecules, i.e. multispecific antibodies or fusion proteins. In addition to their recognition of the target molecule, the antibodies may be further modified to give them specific properties, especially by conjugating them to therapeutically active molecules, such as cytotoxic molecules or cell growth inhibitors, protease inhibitors, which are elements that increase the half-life of the antibody in the patient's blood, or imaging molecules that allow the antibody and / or the target molecule to be detected in the patient by various techniques used in clinical practice (radiology, X-ray examination, scintigraphy, MRI, endoscopy, scanners, etc.).

[0038] In the conjugate of formula (I) defined above, Y 1 For more details, see L 2 -Y' 1 group, wherein Y' 1 is selected from the group consisting of ═CH—, —NH—, and —S— 2 Group L with 2 Via L 1 represents an antibody Y linked to 1 is:=CH-Y' 1 , -NH-Y' 1 and -SY' 1 It can be represented by:

[0039] More specifically, L 2The group results from the conversion of the functional group L' carried by the antibody when L' forms a covalent bond with an M6P analogue.

[0040] According to an advantageous embodiment of the invention, the conjugate of formula (I) is 1 is an integer equal to 1, and the conjugate is:

[0041] [ka]

[0042] X, n and Y' as defined above, 1 The compound is characterized in that it is selected from the group consisting of:

[0043] According to yet another advantageous embodiment, the conjugate of the invention comprises Y 1 represents a therapeutic or diagnostic antibody Y.

[0044] According to a particularly advantageous embodiment, the conjugate of the invention is more particularly 1 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.

[0045] According to a further particularly advantageous embodiment, the conjugate of the invention is more particularly 1 represents an antibody Y which is an immunoglobulin of the IgG type, in particular of the lgG1, lgG2, lgG3 or lgG4 subtype, or an immunoglobulin of the IgE, IgD, IgA or IgM type.

[0046] According to yet another embodiment of the present invention, the conjugate of formula (I) as defined above is -4 M~10 -9 M range, preferably 10 -5 M~10 -8 M range, IC for CI-M6PR 50It may also be characterized by having an affinity, which can be measured, for example, by a competitive method for another ligand of CI-M6PR, such as pentamannose 6-phosphate (Basile I. et al., 2018).

[0047] The conjugates of the invention are of great interest in that they allow cellular internalization of target molecules, such as antigens, via the endolysosomal pathway, which internalization is advantageously followed by extracellular release of the conjugates of the invention, thus allowing a new cycle of capture of the target molecule by the conjugates of the invention.

[0048] The extracellular recycling of the conjugates of the invention is shown in Figure 1. The antigen-bound AMFA-antibody conjugates of the invention import into cell endosomes via CI-M6PR, depositing the antigen. The antigen is then directed to lysosomes, whereas the AMFA-antibody conjugate unexpectedly exits the cell. The conjugates thus released from the cell are available to bind new extracellular or membrane antigens.

[0049] This recycling ability was unexpected, since previous examples in the literature using M6P analogues to internalize lysosomal enzymes (see, for example, EP 2 448 600 B1) demonstrated the presence of these bifunctional compounds (containing M6P analogues linked to enzymes) in the endolysosomal pathway without extracellular recycling.

[0050] Similarly, in PCT / US2019 / 067228, bifunctional molecules linked to antigens are presented in lysosomes. This document does not provide information about possible extracellular recycling of bifunctional molecules. Thus, in all these studies, bifunctional compounds are described as being present and / or degraded in lysosomes together with their specific targets. In the present invention, contrary to expectations, the conjugate of formula (I) allows only target molecules to be degraded in lysosomes, while the conjugate is advantageously recycled in the extracellular space.

[0051] The conjugates of formula (I) are further characterized in that they have at least one of the following properties: - capable of importing into cellular endosomes via its linkage with CI-M6PR to carry soluble extracellular or membrane antigens; - can leave cellular endosomes after depositing soluble extracellular or membrane antigens; - After internalization, Y 1 enable the degradation in lysosomes of soluble or membranous extracellular antigens recognized by - After cell internalization, they are recycled in the extracellular medium.

[0052] In particularly advantageous embodiments, the conjugates of the invention exhibit each of the properties defined above.

[0053] The uniqueness of the conjugates of the present invention is that they do not destroy the normal ability of antibodies to leave cells when necessary.Indeed, the coupling of M6P analogues to antibodies is carried out in such a way that it does not create steric hindrance at the level of the reactive functional groups of the antibody, for example at the level of the part of the antibody that interacts with a specific antibody receptor, for example neonatal FcRn.This has the effect of not changing the extracellular recirculation ability of the antibody when covalently linked to the M6P analogues described herein.

[0054] Methods for the preparation of conjugates of formula (I) According to another aspect, the object of the present invention is a process for preparing a conjugate of formula (I) as defined above, comprising: - General formula (II)

[0055] [ka]

[0056] (Wherein, X, n and n 1 is as previously defined, L is -O-NH 2 ,

[0057] [ka]

[0058] is a reactive chemical group selected from the group comprising: The L group of compound (II) is n carried by the antibody Y or antibody fragment Y. 1 functional groups and n 1 (forming n covalent bonds) 1 With compounds - an antibody Y or an antibody fragment Y comprising at least one antigen-binding domain The method is characterized by:

[0059] The compounds of formula (II) are analogs of M6P. It is the reactive chemical group L carried by the M6P analog of formula (II) that reacts with the functional group L' of the antibody Y to form a covalent bond between the antibody Y and the M6P analog, thus obtaining the conjugate of formula (I) of the invention. 2 The group more particularly originates from the transformation of the functional group L' carried by the antibody when the latter forms a covalent bond with the M6P analogue. Compounds of formula (II) are particularly interesting due to the simplicity of their spacer arm. As used herein, a "spacer arm" refers to a moiety that is located after the oxygen carried by the anomeric carbon of the cyclic structure of the M6P analogue and that contains a reactive chemical group L, i.e. the moiety (CH 2 ) 2 (O-CH 2 -CH 2 ) n Implies -L.

[0060] In fact, the simplicity of the spacer arm structure contributes to the advantage of the conjugate of formula (I) of the present invention.This simplicity of the spacer arm makes it easy to synthesize the compound of formula (II) and then achieve a particularly interesting and effective coupling with the antibody.Therefore, the synthesis of the conjugate of formula (I) is efficient and easy to carry out.

[0061] For simplicity, in what follows, the L groups of the M6P analogues defined above will be referred to as "oxy-amine", "squaric acid group", "maleimide" and "group containing a carbonyl acryl functionality", respectively, in the order of their appearance.

[0062] A particular example of an M6P analogue of formula (II) is more particularly represented in the present application by the acronym "AMFA."

[0063] According to an advantageous embodiment of the process of the invention, compound (II) is

[0064] [ka]

[0065] [ka]

[0066] The compound is selected from the group consisting of:

[0067] According to an advantageous embodiment of the method of the invention, the use of the previously described AMFA1 to AMFA8 allows a particularly interesting and effective coupling with the previously described antibodies. Moreover, as already indicated, the compounds of formula (II), more particularly the previously described AMFA1 to AMFA8, are simple in terms of their chemical structure, in particular due to the simplicity of their spacer arms. These compounds are easy to synthesize and then easy to couple with antibodies.

[0068] According to a particularly advantageous embodiment of the invention, the group L of the AMFA compound is linked to a preformed carbonyl function of an oligosaccharide chain / moiety of a glycoside moiety of an antibody, said glycoside moiety being more particularly a moiety located in the Fc region of the antibody.

[0069] An example of a group L in an AMFA compound is an oxy-amine group.

[0070] According to another embodiment of the invention, the group L of the AMFA compound is attached to a suitable amino acid residue of a peptide chain / moiety of an antibody.

[0071] Examples of suitable amino acid residues include the amine group derived from lysine, or the thiol group derived from cysteine.

[0072] Squaric acid, maleimide or carbonylacryl groups are examples of groups L in the AMFA compounds.

[0073] The terms "chain" or "moiety" used to refer to oligosaccharide chains or oligosaccharide portions of an antibody or peptide chains or peptide portions of an antibody may be used interchangeably in this application.

[0074] FIG. 2 shows a general synthetic scheme for the conjugates of the invention based on the respective definitions for the reactive chemical group L of the M6P analogues of general formula (II).

[0075] As shown in Figure 2, at point 1 / , the carbonyl function (-CHO) of antibody Y reacts with the oxy-amine function of the AMFA compound of formula (II). More specifically, this coupling mode takes place at the level of the oligosaccharide chain of the glycoside moiety located in the Fc portion of the antibody.

[0076] This mode of coupling is particularly advantageous in that it does not create any disturbances at the level of other reactive functional groups of the antibody (such as, for example, the regions of the antibody that interact with receptors), which advantageously allows the antibody to retain its natural recycling properties outside the cell, and therefore allows the conjugates of the invention to be recycled outside the cell.

[0077] As regards the coupling mode of the AMFA compounds of formula (II) containing a squaric acid group (see FIG. 2 point 2 / ), the latter is interposed at the level of the lysine residues of the peptide part of the antibody, i.e. more particularly at the level of the amine functions.

[0078] As regards the coupling of the AMFA compound of formula (II) bearing a maleimide group (see FIG. 2 point 3 / ), the latter takes place at a cysteine ​​residue, more particularly at a thiol function, of the peptide part of the antibody.

[0079] As regards the coupling mode of the AMFA compounds of formula (II) containing a carbonylacryl function (see Figure 2 point 4 / ), this takes place at the level of the cysteine ​​residues of the peptide part of the antibody, i.e. more particularly at the level of the thiol functions.

[0080] These coupling modes are particularly advantageous since they create little or no disturbance at the level of other reactive functionalities of the antibody (e.g. the antibody binding site for the FcRn receptor), which advantageously allows the antibody to retain its native properties and therefore allows the conjugates of the invention to be recycled extracellularly.

[0081] Use of the conjugate (I) in a therapeutic treatment or diagnostic method The novel bifunctional compounds are capable of reducing / decreasing the concentration of soluble extracellular or membrane antigens in an individual, said reduction / decrement being achieved by CI-M6PR regulated cellular endocytosis. This property makes the novel bifunctional compounds particularly interesting for therapeutic use.

[0082] According to another aspect of the present invention: - a conjugate as defined above, at least one pharma- ceutically acceptable excipient; A pharmaceutical composition is proposed, which comprises:

[0083] The inventive conjugate of formula (I) is present in the inventive composition in a therapeutically effective amount.

[0084] By "therapeutically effective amount" we mean a dosage sufficient to produce a desired result, e.g., an amount sufficient to obtain a beneficial or desired therapeutic (including prophylactic) result, e.g., a reduction in the level of a component whose extracellular or membrane expression or overexpression causes or contributes to a pathology. An effective amount may require one or more administrations.

[0085] According to yet another aspect, the present invention provides a method for producing a method of making a semiconductor device comprising: a therapeutically effective amount of a conjugate of formula (I), at least one other therapeutically active agent, - optionally at least one pharma- ceutically acceptable excipient; The present invention relates to a pharmaceutical composition comprising:

[0086] 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 diagnostic or therapeutic type.

[0087] In this respect, the invention relates more particularly to a conjugate of formula (I) as defined above, or to a pharmaceutical composition as defined above, for use as a medicament.

[0088] A further object of the present invention is a conjugate of formula (I) as defined above, or a pharmaceutical composition as defined above, for use in a method of therapeutic treatment comprising administering a therapeutic antibody or a therapeutic antibody fragment.

[0089] As already indicated, the ability to recycle extracellularly confers particularly advantageous properties to the conjugates of the invention when used to treat pathologies where therapy consists of administering a therapeutic antibody or antibody fragment.

[0090] In fact, administration of the conjugate of formula (I) allows for the use of smaller doses of therapeutic antibody than when the therapeutic antibody is used alone (not covalently linked to the M6P analogue).In fact, linking a therapeutic antibody to an M6P analogue improves its therapeutic efficacy compared to the antibody used alone.At the same dose of antibody, the antibody linked to the M6P analogue is more effective than when used alone.

[0091] According to an advantageous embodiment of the invention, the use of the conjugates of formula (I) makes it possible to reduce the antibody doses normally used in therapeutic treatment methods involving the administration of antibodies, thus reducing the side effects associated with antibody therapy.

[0092] Examples of pathologies involving administration of an antibody include pathologies selected from the group including cancer, inflammatory diseases, autoimmune diseases, hematological diseases, ophthalmic diseases, viral infections, allergic diseases and rare diseases.

[0093] Antibodies that have been tested in more detail in the present invention in coupling with AMFA include anti-TNF-alpha, anti-VEGF and anti-HER2 antibodies. Anti-TNF-alpha antibodies are used to treat chronic inflammatory bowel disease, Crohn's disease, ulcerative colitis, and autoimmune diseases that affect the joints, such as rheumatoid arthritis, axial spondyloarthritis / ankylosing spondylitis, psoriatic arthritis and psoriasis.

[0094] Anti-VEGF antibodies are used clinically to treat angiogenesis and tumor growth in many types of cancer, as well as ocular vascular disorders, including neovascular age-related macular degeneration. Anti-HER2 antibodies are used, among others, in the treatment of certain cancers, especially breast cancers that overexpress the HER2 receptor.

[0095] A further object of the present invention is a conjugate of formula (I) as defined above, or a pharmaceutical composition as defined above, for use in a diagnostic method.

[0096] Therefore, antibody Y 1Conjugates of formula (I) covalently linked to an imaging molecule via can be used to localize 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 CI-M6PR expression.

[0097] The conjugate or pharmaceutical composition of the present invention may be formulated into various pharmaceutical preparations (solid, semi-solid, liquid or gas form), such as tablets, capsules, powders, granules, ointments, liquids, injections, inhalants and aerosols.The pharmaceutical preparation may be in liquid form, lyophilized form, or liquid form reconstituted from lyophilized form, where the lyophilized preparation must be reconstituted with a sterile solution before administration.The conjugate of the present invention may be formulated into a preparation for subcutaneous, intravenous or intravitreal injection by dissolving, suspending or emulsifying in a solvent.

[0098] According to an advantageous embodiment, the invention relates to a conjugate of formula (I) or a pharmaceutical composition, for the use defined above, characterized in that it is in a form suitable for parenteral, intravenous or subcutaneous administration. EXAMPLES

[0099] The synthesis of the conjugates (I) of the present invention and studies on their biological effects are described in detail in the following examples with reference to Figures 3 to 20. The M6P analogs of general formula (II) synthesized in the following examples are named AMFA1, AMFA2, AMFA3, AMFA4, AMFA5, AMFA6, AMFA7 and AMFA8, respectively.

[0100] Some of these AMFAs are coupled to antibodies designated mAb1, mAb2, mAb3, mAb4 and Ab (see below).

[0101] Example 1 Synthesis of AMFA1 to AMFA8 of general formula (II) Intermediate 3: Preparation of 2-bromoethyl-2,3,4-tri-O-trimethylsilyl-α-D-mannopyranoside The method for preparing intermediate 3 used as a precursor for AMFA synthesis is shown in FIG. 3, and the conditions and reagents for steps (i)-(iii) are as follows: (i) 2-Bromoethanol, triethylamine, CH 2 Cl 2 , 3.5 hours, 80℃; (ii) TMSCI, Et3N, CH 2 Cl 2 , 20 hours, RT; (iii)K 2 CO 3 , MeOH, 15 min, 0 °C.

[0102] The starting compound was D-mannose in which 2-bromoethanol was introduced at the anomeric position using Amberlite IR120 resin previously reactivated 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 with catalytic amount of potassium carbonate in methanol to lead to compound 3 in 60% yield over two steps (see FIG. 3).

[0103] Preparation of compound AMFA1 The method for preparing compound AMFA1 from compound 3 is shown in FIG. 4, and the conditions and reagents for steps (i) to (vi) are as follows: (i) Dess-Martin periodinane 0.3 M, CH 2 Cl 2 , RT, 1 h; (ii) tetraethyl methylenediphosphonate, NaH, THF, 1 h, RT; (iii) Pd / C, Et 3 SiH, MeOH, 2 h, RT. (iv) N-hydroxyphthalimide, NaH, DMF, 22 h, 60 °C; (v) TMSCl, Et 3 N, C.H. 2 Cl 2 , 15.5 h, RT; (vi)-a) TMSCI, NaI, CH 3 CN, 50 min, 35°C; (vi)-b) N 2 H4 .H 2 O, MeOH, 15.5 h, RT.

[0104] The alcohol 3 obtained as described above is oxidized to the aldehyde using Dess-Martin periodinane to form compound 4. This key intermediate gives access to various AMFAs of formula (II).

[0105] To synthesize compound AMFA1, aldehyde 4 is reacted with tetraethyl methylenediphosphonate, previously deprotonated with sodium hydride, to form compound 5 in 76% yield over two steps. In a catalytic hydrogenation step, in the presence of palladium on charcoal, whereby hydrogen is generated in situ by incremental addition of triethylsilane, the double bond is reduced, and the trimethylsilyl group is hydrolyzed. Compound 6 is thus obtained in quantitative yield without purification. The bromine atom is then replaced with N-hydroxyphthalimide to form intermediate 7 in 61% yield. The alcohol function is then protected with trimethylsilyl chloride to give compound 8 in quantitative yield. The resulting crude intermediate 8 is then reacted with trimethylsilyl chloride and sodium iodide to form bis(trimethylsilyl)phosphonate by a Rabinowitz-type reaction. This intermediate is then converted to a phosphonic acid by the action of hydrazine monohydrate in methanol, which further displaces the trimethylsilyl group present on the secondary alcohol of the sugar and reacts with phthalimide to expose the amine function. In a single step, we thus obtain the compound AMFA1, in which the phosphonic acid moiety of the sugar alcohol has been deprotected and which carries an aminooxy function at the anomeric position, in 85% yield over two steps (Figure 4).

[0106] Preparation of compound AMFA2 The method for preparing compound AMFA2 from compound 3 is shown in FIG. 5, and the conditions and reagents for steps (i) to (iv) are as follows: (i)-a) Dess-Martin periodinane 0.3M, CH 2 Cl2 , RT, 1 h; (i)-b) triethyl phosphonoacetate, NaH, THF, 20 min, RT, 73% (2 steps); (ii)-a) Pd / C, Et 3 SiH, MeOH, 2 hours, RT; (ii)-b) NaOH 1N, THF / H 2 O, 2 h, RT; (iii) N-hydroxyphthalimide, NaH, DMF, 20 h, 60 °C; (iv) N 2 H 4 .H 2 O, MeOH, 15.5 h, RT.

[0107] The AMFA2 compound is synthesized according to the synthetic route described in Figure 5. One of the main steps in the synthesis of this sugar ligand was the introduction of a carboxylic acid function in the side chain of the mannose fragment. As in the previous synthesis, this functionalization is achieved by Dess-Martin oxidation at the 6-position of intermediate 3, followed by a Horner-Wadsworth-Emmons reaction using triethyl phosphonoacetate anion. In this way, the unsaturated carboxylic acid ester 10 is obtained. After reduction of the double bond and hydrolysis of the trimethylsilyl protecting group at the hydroxyl function, the functional carboxylic acid is saponified to give intermediate 11. The bromine is then replaced by hydroxyphthalimide to give intermediate 12. Exposure of the amine function by hydrazinolysis leads to the fully deprotected compound AMFA2 (Figure 5).

[0108] Preparation of compounds AMFA3 and AMFA5 The method for preparing compounds AMFA3 and AMFA5 from compound 6 is shown in FIG. 6, and the conditions and reagents for steps (i) to (v) are as follows: (i) Dess-Martin periodinane 0.3 M, CH 2 Cl 2 , RT, 1 h; (ii) tetraethyl methylenediphosphonate, NaH, THF, 1 h, RT; (iii) Pd / C, Et 3 SiH, MeOH, 2 h, RT. (iv) Potassium phthalimide, DMF, 22 h, 60° C.; (v) TMSCl, Et 3 N, C.H. 2 Cl 2, 15.5 h, RT; (vi)-a) TMSCI, NaI, CH 3 CN, 50 min, 35°C; (vi)-b) N 2 H 4 .H 2 (vii) diethyl squarate, Et 3 N, EtOH / H 2 HO, 30 min; (viii) N-(methoxycarbonyl)maleimide, NaHCO 3 Saturated solution, 30 min, 0°C.

[0109] To synthesize compounds AMFA3 and AMFA5, the bromine atom of compound 6 is replaced by potassium phthalimide to form intermediate 14 in 74% yield. The alcohol function is then protected by trimethylsilyl chloride to give compound 15 in quantitative yield. The intermediate 15 thus obtained is then reacted, without purification, with trimethylsilyl chloride and sodium iodide to form bis(trimethylsilyl)phosphonate by a Rabinowitz-type reaction. This intermediate is then converted to a phosphonic acid by the action of hydrazine monohydrate in methanol, which further displaces the trimethylsilyl group present in the secondary alcohol of the sugar and reacts with phthalimide to expose the amine function. In a single step, the phosphonic acid moieties in both sugar alcohols are deprotected, and compound 16, bearing an amine function in the anomeric position, is obtained in 65% yield over two steps. Compound 16 was then reacted with diethyl squarate to produce product AMFA3 in 50% yield or with N-(methoxycarbonyl)maleimide to form compound AMFA5 in 80% yield (Figure 6).

[0110] Preparation of compounds AMFA4 and AMFA6 The method for the preparation of compounds AMFA4 and AMFA6 from compound 1 is shown in FIG. 7, and the conditions and reagents for steps (i) to (v) are as follows: (i) NaN 3 , DMF, 20 h, RT; TMSCl, Et 3 N, C.H. 2 Cl2 , 20 hours, TA;K 2 CO 3 cat, MeOH, 50 min, 0° C., 38% (3 steps); (ii)-a) Dess-Martin periodinane, CH 2 Cl 2 , 3 h, RT, (ii)-b) triethyl phosphonoacetate, NaH, THF, 2 h, RT, 73% (2 steps); (iii)-a) HCl 0.5N, THF, 30 min, RT, (iii)-b) NaOH 0.1N, 30 min, RT, 99%, (iii)-c) H 2 , Pd / C, EtOH / H 2 HO, 1.5 h, RT, 99%; (iv) diethyl squarate, EtOH / H 2 HO, 40 min, RT, 34%; (v) N-(methoxycarbonyl)maleimide, NaHCO 3 Saturated solution, 30 min, 0°C.

[0111] Compounds AMFA4 and AMFA6 were synthesized according to the synthetic route described in Figure 7. One of the main steps in the synthesis of these sugar ligands was the introduction of a carboxylic acid functionality at the side chain of the mannose moiety. This functionalization was achieved by Dess-Martin oxidation at the 6-position of alcohol 19, followed by a Horner-Wadsworth-Emmons reaction using triethyl phosphonoacetate anion. In this way, unsaturated carboxylic ester 20 is obtained. Another key step was the functionalization of the aglycone fragment with diethyl squarate or maleimide. This reaction was carried out on the fully deprotected compound 21 to give AMFA4 and AMFA6, respectively (Figure 7).

[0112] Preparation of compounds AMFA7 and AMFA8 The methods for preparing compound AMFA7 from compound 16 and compound AMFA8 from compound 21, respectively, are shown in FIG. 8. The conditions and reagents for step (i) are as follows: (i) 3-benzoylacrylic acid, N-methylmorpholine, isobutyl chloroformate, DMF, 1 h, −10° C. to RT. To prepare compounds AMFA7 and AMFA8, the same operating procedure used in the preparation of compounds AMFA3 and AMFA4 was followed, except for the last step. In fact, the last step consists of the reaction between the amine of each intermediate 16 and 21 and 3-benzoylacrylic acid activated by isobutyl chloroformate in the presence of N-methylmorpholine (FIG. 8).

[0113] Example 2 Synthesis of conjugates of general formula (I) The AMFA design was tested for five different antibodies coupled to the AMFA of formula (II): - two antibodies directed against tumor necrosis factor alpha (TNF-alpha), called infliximab and adalimumab, referred to in the present application as mAb1 and mAb2, respectively; - an antibody directed against vascular endothelial growth factor (VEGF), called bevacizumab and designated mAb3; - an antibody against human epidermal growth factor receptor 2 (HER2), called trastuzumab and designated mAb4; and -Antibodies against green fluorescent protein (GFP), called Ab.

[0114] In these non-limiting examples, the implantation of AMFAs is: - AMFA1 and the oligosaccharide chains of the glycosidic moiety of a recombinant antibody (see points 1 to 5 below), or - AMFA4 and the amine functional groups of lysine residues of the peptide moiety of the recombinant antibody (see point 6 / below) The method may be carried out in

[0115] 1 / Grafting of AMFA1 onto the oligosaccharide chains of the glycoside moiety of a chimeric anti-TNF-alpha antibody AMFA1 coupling was tested on infliximab, a commercially available mouse-human chimeric monoclonal antibody against human TNF-alpha, defined as mAb1. A conjugate of formula (I) is formed, which may be interchangeably referred to as "mAb1-AMFA1" or "AMFA1-mAb1" in this application. This conjugate comprises a portion (mAb1) that binds to a specific target (e.g., an extracellular or membrane antigen) and a portion (AMFA1) that binds to CI-M6PR. The mAb1 antibody was chosen as a model in this application because it has oligosaccharide chains in the constant region, which are very similar to those produced in humans. The coupling between AMFA1 and mAb1 antibodies is performed according to the protocol described below.

[0116] In the first step, the mAb1 antibody is oxidized. Specifically, the mAb1 antibody and 1 mM sodium metaperiodate (NaIO 4 ) is reacted in 25.5 mM phosphate buffer pH 6.25 for 30 min at 4° C. in the dark. The reaction is stopped by adding glycerol (20 μL / mL) and incubating for 10 min at 4° C. Low molecular weight compounds are removed using a PD-10 desalting column pre-filled with Sephadex G-25 resin.

[0117] In the second step, AMFA1 is added to the previously obtained oxidized antibody solution and the mixture is left to react for 2 hours at 37°C under stirring. Finally, the sample is dialyzed overnight against phosphate buffer. The first step of the above protocol results in the oxidation of the oligosaccharide chains of the mAb1 antibody, generating aldehyde groups that covalently interact with the oxy-amine groups of AMFA1 to form oxime bonds. Furthermore, only the oligosaccharide chains of the mAb1 antibody are reconstructed. In fact, the peptide portion of the mAb1 is not affected by the conditions used for AMFA1 grafting.

[0118] 2 / Grafting of AMFA1 onto the oligosaccharide chains of the glycoside portion of a humanized anti-TNF-alpha antibody The anti-TNF-alpha antibody, adalimumab, defined as mAb2, was tested and the same procedure described in point 1 / above was carried out to graft AMFA1 onto the oligosaccharide chains of the glycoside moiety of the mAb2 antibody. A conjugate of formula (I) is formed, which may be referred to interchangeably in the present application as "mAb2-AMFA1" or "AMFA1-mAb2". This conjugate comprises a moiety (mAb2) that binds to a specific target (e.g. an extracellular or membrane antigen) and a moiety (AMFA1) that binds to CI-M6PR.

[0119] 3 / Grafting of AMFA1 onto the oligosaccharide chain of the glycoside portion of a humanized anti-VEGF antibody The anti-VEGF antibody bevacizumab, defined as mAb3, was tested and the same procedure described in point 1 / above was carried out to graft AMFA1 onto the oligosaccharide chains of the mAb3 antibody. A conjugate of formula (I) is formed, which may be referred to interchangeably in the present application as "mAb3-AMFA1" or "AMFA1-mAb3". This conjugate comprises a moiety (mAb3) that binds to a specific target (e.g. an extracellular or membrane antigen) and a moiety (AMFA1) that binds to CI-M6PR.

[0120] 4 / Grafting of AMFA1 onto the oligosaccharide chain of the glycoside portion of a humanized anti-HER2 antibody The anti-HER2 antibody, trastuzumab, defined as mAb4, was tested and the same procedure described in point 1 / above was carried out to graft AMFA1 onto the oligosaccharide chains of the mAb4 antibody. A conjugate of formula (I), which may be interchangeably represented in the present application by "mAb4-AMFA1" or "AMFA1-mAb4", was formed. This conjugate comprises a moiety (mAb4) that binds to a specific target (e.g. an extracellular or membrane antigen) and a moiety (AMFA1) that binds to CI-M6PR.

[0121] 5 / Grafting of AMFA1 onto the oligosaccharide chain of the glycoside moiety of anti-GFP antibody Anti-GFP antibodies, defined as Ab, were tested and the same procedure as described in point 1 / above was carried out to graft AMFA1 onto the oligosaccharide chains of the Ab antibody. A conjugate of formula (I) is formed, which may be interchangeably referred to as "Ab-AMFA1" or "AMFA1-Ab". This conjugate comprises a moiety (Ab) that binds to a specific target (e.g. an extracellular or membrane antigen) and a moiety (AMFA1) that binds to CI-M6PR.

[0122] 6 / Grafting of AMFA4 to the amine functional groups of lysine residues in the peptide portion of a humanized anti-VEGF antibody Coupling between AMFA4 and mAb3 antibody (bevacizumab) is carried out at the level of the mAb3 peptide chain, in particular at the lysine residue, according to the protocol described below. The mAb3 antibody solution and AMFA4 are reacted in phosphate buffer for 24 hours at 37° C. with gentle stirring. The sample is dialyzed overnight against phosphate buffer to form a conjugate of formula (I), which can be represented as either "mAb3-AMFA4" or "AMFA4-mAb3". This conjugate comprises a portion (mAb3) that binds to a specific target (e.g., an extracellular or membrane antigen) and a portion (AMFA4) that binds to CI-M6PR.

[0123] Example 3 Studies on the conjugates (I) and their biological properties 1 / A study of the number of AMFA1 or AMFA4 bound to mAb1, mAb2, mAb3 or mAb4 antibody molecules 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 according to the method adapted and described in Zhou, Q et al. 2013 to quantify the number of AMFA1 bound to the antibody molecule.

[0124] The molecular weight of each mAb1, mAb2, mAb3 and mAb4 antibody was measured before and after coupling with AMFA1 or AMFA4. Samples and standards were diluted 1:5 with 0.1% aqueous formic acid, then 1:1 with saturated sinapinic acid solution in 50% acetonitrile / 0.1% TFA. 1 μL (microliter) of the mixture was applied to the target. Samples, corresponding standards and BSA calibration controls were analyzed in duplicate. To obtain the conjugates of the present invention, the number of AMFA1 or AMFA4 grafted to mAb1, mAb2, mAb3 or mAb4 antibodies was calculated based on the difference between the molecular weight of the "AMFA-antibody" conjugate and its corresponding control "antibody", and then the difference was divided by the molecular weight of each AMFA.

[0125] Table 1 below shows the MALDI-TOF analysis data for the various batches of AMFA1-mAb1, AMFA1-mAb2, AMFA1-mAb3 and AMFA1-mAb4 conjugates obtained in Example 2 (points 1 / to 4 / ).

[0126] [Table 1]

[0127] Table 2 below shows the MALDI-TOF analysis data for the different batches of AMFA4-mAb3 conjugate obtained in Example 2 (point 6 / ).

[0128] [Table 2]

[0129] Findings and conclusions The increase in molecular weight after grafting of AMFA1 or AMFA4 is an indication of the number of AMFA coupled per antibody. The reproducibility of the grafting reaction is indicated by the small standard deviation of the number of grafted AMFA between the various assays. The data in Tables 1 and 2 show that AMFA1 and AMFA4 can be easily grafted to mAb1, mAb2, mAb3 or mAb4 with a high level of reproducibility, and the number of AMFA1 or AMFA4 grafted to each antibody can be controlled by adjusting the reaction conditions. Decreasing the concentration of the oxidizing agent, sodium metaperiodate, results in a decrease in the grafting rate of AMFA1. The grafting of the AMFA of the present invention is reproducible and easily controlled by the reaction conditions.

[0130] 2 / A study of the major structural modifications associated with grafting AMFA1 onto mAb1, mAb2, mAb3, or mAb4 antibodies mAb1, mAb2, mAb3 and mAb4 antibodies alone or conjugated to AMFA1 (mAb1-AMFA1, mAb2-AMFA1, mAb3-AMFA1 and mAb4-AMFA1 (mAb-AMFA conjugates)) were analyzed by native and denaturing SDS-PAGE. Coomassie blue staining revealed one band (under native conditions) or two bands (under denaturing conditions) of the same molecular weight for all antibodies before and after coupling with AMFA1. Binding of AMFA1 to the antibodies did not induce visible degradation or significant changes in molecular weight of the antibody heavy or light chains due to the small number of grafted analogs. The mAb-AMFA conjugates showed unchanged immunoreactivity against a commercial secondary anti-human IgG antibody in Western blots compared to the mAbs. Hybridization with rabbit anti-AMFA1 antibody (indicated as "anti-AMFA" in Figure 9) confirmed the presence of AMFA1 in the heavy chain of the antibody linked to AMFA1 (following the protocol described in Basile I. et al., 2018). The results obtained are shown in Figure 9.

[0131] Western blot analysis of various antibodies shows that oxidation does not change the overall structure of the antibody, as shown by the detection of AMFA1 with rabbit anti-AMFA1 antibody, and that AMFA1 coupling is effective for the heavy chain of each mAb1, mAb2, mAb3 or mAb4 antibody. In conclusion, grafting AMFA1 onto mAb1, mAb2, mAb3 or mAb4 antibody does not alter the integrity of the antibody. Furthermore, the detection of AMFA1 on the heavy chain of mAb1, mAb2, mAb3 and mAb4 antibodies is consistent with grafting only onto the oligosaccharide chain carried by the heavy chain. Treatment with PNGase for hydrolysis of the N-glycosylated chains allows the release of AMFA1 from mAb1-AMFA1, indicating the grafting specificity for the oligosaccharide chains.

[0132] 3 / Study on the ability of mAb1-AMFA1 and mAb2-AMFA1 conjugates to bind to CI-M6PR Affinity assays of the conjugates mAb1-AMFA1 and mAb2-AMFA1 of the present invention for CI-M6PR were carried out using biotinylated CI-M6PR (Basile et al., 2018). As used herein, the affinity of the mAb1-AMFA1 and mAb2-AMFA1 conjugates for CI-M6PR refers to the ability of the mAb1-AMFA1 and mAb2-AMFA1 conjugates to bind to CI-M6PR. Briefly, CI-M6PR purified on a phosphomannan-Sepharose affinity column was biotinylated with biotin N-hydroxysuccinimide. Binding of biotinylated CI-M6PR (b-CI-M6PR) to pentamannose hexaphosphate (PMP) pre-adsorbed on a microtiter plate was competitively inhibited with increasing concentrations of mAb1-AMFA1 or mAb2-AMFA1 conjugates. The b-CI-M6PR bound to the adsorbed PMP was then determined by optical density measurements using streptavidin conjugated to peroxidase and the peroxidase substrate, o-phenylenediamine dihydrochloride (OPD). The 100% value corresponds to the concentration of b-CI-M6PR bound to the adsorbed PMP in the absence of competitor.

[0133] Table 3 below presents data from binding assays for native M6P to b-CI-M6PR and for the mAb1-AMFA1 and mAb2-AMFA1 conjugates to b-CI-M6PR. The conjugates studied contain a moiety (mAb1 or mAb2) that targets the target molecule (antigen) and a moiety (AMFA1) that targets b-CI-M6PR. Affinity (expressed as the concentration required for 50% inhibition of binding, IC 50 ) was determined by a competition assay using the pentamannose 6-phosphate ligand.

[0134] [Table 3]

[0135] Findings and conclusions The binding ability of mAb1 or mAb2 antibody alone to CI-M6PR was close to zero (concentration range 5.10 8 ~2.10 6 While the binding affinity of M6P to CI-M6PR is less than 10% at M, Table 3 shows that the mAb1-AMFA1 or mAb2-AMFA1 conjugates of the invention have a binding affinity to CI-M6PR that is greater than the affinity of native M6P to CI-M6PR. The conjugates of the invention thus acquire the ability to bind to CI-M6PR. In particular, the grafting of AMFA1 onto mAb1 and mAb2, respectively, allows the mAb1-AMFA1 and mAb2-AMFA1 conjugates of the invention to have increased affinity to CI-M6PR.

[0136] 4 / A study on the effect of AMFA1 grafting on the binding affinity of mAb1 to mAb4 antibodies to their target molecules, i.e., the corresponding antigens (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 to target antigens (human TNF-alpha, VEGF or HER2) was evaluated using an ELISA assay to determine antigen binding. By "affinity" of an antibody or conjugate to an antigen, it is intended the ability of said antibody or conjugate to bind to said antigen. Briefly, antigens (commercially available human TNF-alpha, VEGF or HER2) were 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.

[0137] The level of antibody or conjugate bound to the target of interest is determined using optical density measurements with an anti-human IgG antibody coupled to horseradish peroxidase and an OPD substrate. Figure 10 shows that grafting AMFA1 onto mAb1, mAb2, mAb3 or mAb4 antibodies does not affect their affinity for the target antigen (human TNF-alpha, VEGF or HER2). The conjugates of the present invention have affinities for their antigens that are comparable to the affinity of the antibodies alone.

[0138] 5 / Stability studies on the mAb1-AMFA1 conjugate of the present invention mAb1-AMFA1 conjugates from different batches were incubated in acetate buffer (pH 5) or phosphate buffer (pH 7) for up to 12 days and then analyzed by MALDI-TOF to determine whether AMFA1 was still bound to the mAb1 antibody. The number of AMFA1 grafted to the mAb1 antibody was determined at pH 5 and 7 on days 0 and 12. The results obtained are shown in Table 4 below.

[0139] [Table 4]

[0140] Findings and conclusions Table 4 shows that the number of AMFA1 grafted onto the mAb1 antibody is stable at two different pH levels even after 12 days. Thus, the AMFA1-mAb1 conjugate is stable over time and at various pH levels.

[0141] 6 / Study on cell internalization of mAb1-AMFA1, mAb3-AMFA1 and Ab-AMFA1 conjugates The cellular internalization of the antibody and the conjugate of the present invention was observed in rhabdomyosarcoma cells, T-lymphocyte cells and breast cancer cells. Data were obtained by flow cytometry and whole cell fluorescence assay. Rhabdomyosarcoma cells, T-lymphocyte cells and breast cancer cells were treated with a complex containing mAb1-AMFA1 and secondary fluorescent anti-human IgG antibody Alexa647 for 24 hours or 48 hours (Figure 11(A) and Figure 12(A)). Before incubation with cells, mAb1 antibody, mAb1-AMFA1 conjugate and non-specific control human IgG were incubated with Alexa647 anti-human IgG fluorescent secondary antibody. Then, the fluorescence in cells was analyzed by flow cytometry (Figure 11(A)).

[0142] The data in Figure 11(A) show significantly greater fluorescence in cells treated with mAb1-AMFA1 (7.9, 11.3 and 11.8 times greater than the control). Figure 11(B,C) shows the transfer of mAb1 and mAb1-AMFA1, and mAb4 and mAb4-AMFA1 pre-coupled to Alexa647, into T lymphocytes and SKOV3 ovarian cancer cells, respectively. Figure 11 shows that AMFA1 enhances the cellular uptake of mAb1-AMFA conjugates compared to mAb1 alone. Addition of AMFA1 to the mAb1 antibody significantly increased the cellular internalization of the antibody, and these results were confirmed by whole-cell fluorescence internalization experiments using mAb1, mAb1-AMFA1, mAb3 and mAb3-AMFA1 (Figure 12). Rhabdomyosarcoma and breast cancer cells were treated for 24 hours under the same conditions as described above. Figure 12 shows that greater fluorescence is observed in cells treated with mAb1-AMFA1 (breast cancer cells) versus mAb1, and in cells treated with mAb3-AMFA1 (rhabdomyosarcoma cells) versus mAb3. The engraftment of AMFA1 on mAb1 or mAb3 antibodies significantly increased cellular internalization of the antibodies.

[0143] These results were confirmed by confocal microscopy on live rhabdomyosarcoma and breast cancer cells (Figure 13(A)). Cells were preincubated with 200 nM Ab antibody or Ab-AMFA1 conjugate (see Example 2, point 5 / ) and 400 nM green fluorescent protein for 24 hours. In both cell types, greater fluorescence was observed with Ab-AMFA1 versus Ab alone (fluorescent cell vesicles indicated by arrows in Figure 13). Addition of excess M6P (10 mM) to the cell culture medium to saturate CI-M6PR binding sites was shown to significantly reduce internalization of Ab-AMFA1 into cells in both cell types. In a separate experiment, mAb1 and mAb1-AMFA1 were coupled to Alexa647 fluorophores to measure their internalization in Jurkat cells (T lymphocytes). The increased internalization of mAb1-AMFA1 compared to mAb1, and the reversal of mAb-AMFA1 import by the addition of 30 mM AMFA1 excess, are described and quantified in Figure 13 (B, C). More broadly, these results indicate that grafting AMFA onto antibodies induces increased cellular internalization via the CI-M6PR-mediated transport pathway.

[0144] 7 / Study on the pharmacokinetic profile of the mAb1-AMFA1 and mAb2-AMFA1 conjugates of the present invention The effect of AMFA1 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. 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 saline solution. Mice were weighed before injection and throughout the experiment to monitor their health. Blood samples taken 4 hours and 1, 2, 3, 7, 10, 14, 17 and 21 days after injection were collected in tubes containing heparin, centrifuged at 3,000 g for 15 minutes, and plasma was stored at -20°C until measurement. Mice were sacrificed 21 days after injection. No adverse clinical events were observed in any of the four treatment groups.

[0145] The plasma concentrations of mAb1, mAb2, mAb1-AMFA1 and mAb2-AMFA1 were measured using ELISA technique. Briefly, recombinant human TNF-alpha was adsorbed to Maxisorp microwell plates and recognized by mAb1, mAb2, mAb1-AMFA1 or mAb2-AMFA1 present in plasma samples. The therapeutic antibodies were detected using HRP-conjugated anti-human IgG antibodies specific for the Fc fragment, which were then quantified by a colorimetric assay using OPD substrate and absorbance measurement at 450 nm. The results obtained are shown in FIG. 14. The pharmacokinetic profiles of mAb1 compared to mAb1-AMFA1 (A) and mAb2 compared to mAb2-AMFA1 (B), detected in plasma samples from treated animals and expressed as a percentage of the value at 4 hours, are similar. No significant differences in antibody half-life were observed upon engraftment of AMFA1 onto mAb1 or mAb2. In conclusion, grafting AMFA1 onto the mAb1 and mAb2 antibodies targeting the TNF-alpha antigen does not alter the pharmacokinetic profile of the antibodies.

[0146] 8 / In vivo stability studies on the mAb1-AMFA1 conjugate of the present invention The in vivo stability of AMFA1 binding to the therapeutic mAb1 antibody was evaluated by analyzing the presence of AMFA1 in plasma samples collected on day 21 from mice treated with mAb1-AMFA1 or mAb1 as previously described in 7 / . mAb1 or mAb1-AMFA1 were first isolated from the plasma samples by 24-hour binding to an anti-human IgG antibody specific for the Fc fragment pre-adsorbed to a microwell plate. As a loading control, 50 ng of mAb1-AMFA1 diluted in phosphate buffer was also incubated. After washing, the bound antibody was analyzed by Western blot for the presence of AMFA1 using a polyclonal anti-AMFA1 antibody (represented as "anti-AMFA" in Figure 15) and an anti-human IgG antibody to control for the amount of mAb1 or mAb1-AMFA1 present on day 21. Figure 15 provides evidence that AMFA1 is still conjugated to the mAb1 therapeutic antibody at day 21. Figure 15 shows that both mAb1 and mAb1-AMFA1 are detected in the plasma of treated mice after 21 days at levels similar to that of the 50 ng mAb1-AMFA1 control, as confirmed by hybridization with anti-human IgG. Most of the AMFA1 is still bound to mAb1, and the signal intensity of the band is comparable to that of mAb1-AMFA1 used as a control.

[0147] 9 / A study on the ability of mAb1-AMFA1 conjugate to increase cellular internalization of human TNF-alpha This study shows that human TNF-alpha antigen is degraded over time after internalization into cells with mAb1-AMFA1 conjugate. Degradation of human TNF-alpha was studied in human T-lymphocyte cells. Cells were 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 blot for intracellular human TNF-alpha. For mAb1-AMFA1 conjugate, the data show an increase in the levels of human TNF-alpha up to 17 h (Figure 16(A)). In contrast, only a slight increase in intracellular human TNF-alpha is observed with mAb1 antibody. The data show that human TNF-alpha is better internalized with mAb1-AMFA1 than with mAb1 antibody. The amount of TNF-alpha measured by Western blot in the culture medium during treatment with mAb1-AMFA1 and mAbs shows that mAb1-AMFA1 causes a greater reduction in TNF-alpha (Figure 16(B)). This significant reduction in the external medium correlates with an increased cellular uptake of TNF-alpha.

[0148] Study of the ability of 10 / mAb1-AMFA1 conjugate to reduce the biological effect of human TNF-alpha This study shows that the mAb1-AMFA1 conjugate not only improves internalization in cells, but also reduces the biological effect to a greater extent than the antibody alone. L929 mouse fibroblast 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 percentage of viable cells after 48 hours of treatment with mAb1 or mAb1-AMFA1 was analyzed by MTT method. Figure 17 shows the neutralizing effect of mAb1 or mAb1-AMFA1 on human TNF-alpha toxicity. mAb1-AMFA1 neutralizes human TNF-alpha toxicity at a much smaller dose (1 / 4-1 / 5) than mAb1. In conclusion, the mAb1-AMFA1 conjugate induces a dose-dependent neutralization of human TNF-alpha-induced cell growth inhibition more effectively than mAb1 alone.

[0149] 11 / Study on the ability of the present mAb3-AMFA1 to reduce the effect of human VEGF on the proliferation of HUVEC cells In this study, the mAb3-AMFA1 conjugate was shown to improve internalization into cells and to reduce the biological effect more than that observed with the mAb3 antibody alone. HUVEC human umbilical vein endothelial cells were treated with 50 ng / mL human VEGF and increasing doses of mAb3 or mAb3-AMFA1. Since human VEGF induces proliferation of HUVEC cells, the viable cell number at 72 hours in cells treated with mAb3 or mAb3-AMFA1 was analyzed by MTT method. Figure 18 shows that the inhibition of the mitogenic effect of VEGF in HUVEC cells is greater with the mAb3-AMFA1 conjugate than with 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 a much smaller dose (1 / 10) than mAb3.

[0150] Study of the ability of the 12 / AMFA1-mAb4 conjugate to reduce the amount of HER2 SK-BR-3 breast cancer cells were treated with 10 or 100 nM mAb4 or mAb4-AMFA1 for 24 hours. The cells were then fixed and the amount of target protein was measured using a colorimetric method based on indirect ELISA. HER2 protein is recognized by a specific antibody, and the Fc part of the antibody is recognized by a secondary antibody coupled to peroxidase (HRP). Figure 19(A) shows the reduction of HER2 protein after treatment with mAb4-AMFA1 compared to mAb4 alone in SK-BR3 cells. Increasing doses of mAb4-AMFA1 cause a greater reduction in cellular HER2 concentration compared to the same dose of mAb4. BT474 breast cancer cells were treated with 5 nM mAb4 or mAb4-AMFA1 for 5 hours. Total HER2 was quantified by Western blot. After 5 hours of treatment with mAb4-AMFA1, the amount of HER2 is significantly reduced, whereas treatment with mAb4 is not effective (Figure 19(B)).

[0151] 13 / Extracellular recycling of AMFA1-mAb1 conjugates after internalization in T lymphocytes The extracellular recycling of the internalized mAb1 antibody and the mAb1-AMFA1 conjugate was shown by an experiment involving 5-hour cellular transfer of the antibody into immortalized T-lymphocyte cells (Jurkat), followed by medium exchange with antibody-free medium, and release of the previously internalized antibody into the culture medium for periods of 1 and 5 hours (Figure 20(A)). In another experiment, the cells are treated with the antibody for 30 minutes, and after medium exchange, the release of the internalized antibody is measured after 10 and 30 minutes (Figure 20(B)). The concentrations of mAb1 and mAb1-AMFA1 were measured using ELISA, as previously described in 7 / . Figure 20 shows that the extracellular recycling of the mAb1-AMFA1 conjugate is comparable to that of mAb1 alone. These results indicate that mAb1-AMFA1 is successfully recycled out of the cell after its significant cellular internalization mediated by CI-M6PR.

[0152] The present disclosure is not limited to the embodiments described above, purely by way of example, but encompasses all variants that may be envisaged by a person skilled in the art in relation to the protection sought.

[0153] [References] TIFF2025507408000013.tif115138

Claims

1. The following general formula (I) 【Chemistry 1】 (In the formula: X is independently H, Na, Li, K or NH 4 With Z representing the phosphonic acid group -CH 2 -P(O)(OZ) 2 or carboxylic acid group -CH 2 -C(O)(OZ), n is an integer ranging from 0 to 2; L 1 is (O-CH 2 -CH 2 ) n L for the group 1 * indicates the attachment point of Y 1 L for 1 *-ON=**, with ** indicating the connection point of 【Chemistry 2】 is a group selected from the group comprising n 1 is an integer ranging from 1 to 20, preferably from 1 to 10, Y 1 represents an antibody Y, or an antibody fragment Y comprising at least one antigen-binding domain, The above Y 1 , L 1 and 1 A conjugate comprising:

2. Y 1 L 2 -Y' 1 group, Y' 1 is selected from the group consisting of ═CH—, —NH—, and —S—; 2 Group L with 2 Via L 1 and thus represents an antibody Y or an antibody fragment Y linked to said Y 1 But, =CH-Y' 1 , -NH-Y' 1 and -SY' 1 2. The conjugate according to claim 1, characterized in that it can be represented by:

3. n 1 is an integer equal to 1, and said conjugate of formula (I) is: 【Chemistry 3】 X, n and Y' as defined in claim 1 or 2, 1 3. The conjugate according to claim 1 or 2, characterized in that it is selected from the group with

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

5. Y 1 5. The conjugate according to claim 4, characterized in that: 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. Y 1 6. The conjugate according to claim 5, characterized in that: represents an antibody Y which is an immunoglobulin of the IgG type, in particular of the lgG1, lgG2, lgG3 or lgG4 subtype, or an immunoglobulin of the IgE, IgD, IgA or IgM type.

7. 10 -4 M to 10 -9 M range, preferably 10 -5 M to 10 -8 IC for the cation-independent mannose 6-phosphate receptor (CI-M6PR) in the range of M 50 A conjugate according to any one of claims 1 to 6, characterized in that it has affinity.

8. A process for preparing a conjugate of formula (I) according to any one of claims 1 to 7, comprising: - an antibody Y, or an antibody fragment Y comprising at least one antigen-binding domain, - General formula (II) 【Chemistry 4】 (Wherein, X, n and n 1 is as defined in claim 1, L is -O-NH 2 、 【Chemistry 5】 is a reactive chemical group selected from the group comprising: The group L of compound (II) is n carried by the antibody Y or antibody fragment Y. 1 functional groups and n 1 n corresponding to forming covalent bonds 1 A method comprising reacting a compound of formula (I) with a compound selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29

9. Compound (II) 【Chemistry 6A】 【Chemistry 6B】 9. The process according to claim 8, characterized in that the compound is selected from the group comprising:

10. - a conjugate according to any one of claims 1 to 7, at least one pharma- ceutically acceptable excipient; A pharmaceutical composition comprising:

11. A conjugate according to any one of claims 1 to 7, or a pharmaceutical composition according to claim 10, for use as a medicament.

12. A conjugate according to any one of claims 1 to 7, or a pharmaceutical composition according to claim 10, for use in a method of therapeutic treatment comprising administering a therapeutic antibody or a therapeutic antibody fragment.

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

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

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