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

Abstract

The present invention relates to novel conjugates having a general formula (I) as defined in claim 1. The conjugates of the invention comprise 1) at least one mannose 6-phosphate (M6P) analog targeting the cation-independent mannose 6-phosphate receptor (RM6P-CI) and 2) an antibody or antibody fragment for binding to a specific target antigen or target molecule. The invention also relates to the method for preparing said conjugates and their medical use, whether therapeutic or diagnostic. The conjugates of formula (I) of the invention are particularly of interest for use as a medicinal product, especially in a therapeutic treatment method that involves administering a therapeutic antibody or a therapeutic antibody fragment.

Description

Title of the invention: Bifunctional compounds targeting the cation-independent mannose 6-phosphate receptor technical field

[0001] The present invention relates to the field of medicinal chemistry. More particularly, it relates to bifunctional compounds comprising 1) at least one mannose 6-phosphate (M6P) analog targeting the cation-independent mannose 6-phosphate receptor (RM6P-CI) and 2) an antibody or antibody fragment for binding to a specific target antigen or target molecule. The invention also relates to the process for preparing said bifunctional compounds and their medical use, whether therapeutic or diagnostic. Previous technique

[0002] Most therapeutic molecules targeting disease-associated proteins neutralize the target in question, thereby blocking its pathophysiological function. However, these therapeutic molecules often have little effect on the expression or degradation levels of the target. Moreover, there are still many proteins that are not yet pharmacologically targeted and whose dysregulation 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 by proteasome activation, degradation-inducing drugs are used, such as chimeras targeting proteolysis. However, the design of these chimeras is complex and cell permeability is limited. Moreover, since the ubiquitin-proteasome system is intracellular, the use of these chimeras cannot be applied to extracellular proteins.

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

[0005] The cation-independent mannose 6-phosphate receptor (RM6P-CI), a 300 kDa transmembrane glycoprotein, represents the main transport and sorting pathway for lysosomal enzymes carrying the mannose 6-phosphate (M6P) signal to lysosomes. RM6P-CI also plays a role as a mediator of endocytosis of Extracellular ligands containing M6P (Gary-Bobo et al., 2007). M6P-containing proteins, which differ from lysosomal enzymes and are internalized via 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 condition for proteins to reach the lysosome is therefore the presence of M6P. However, M6P can be dephosphorylated by serum phosphatases, thereby losing its ability to bind to RM6P-CI (El Cheikh K. et al., 2016). Lysosomal enzymes have thus been modified to significantly 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 allowing recognition by RM6P-CI, without being degraded by phosphatases. M6P analogs refer to a compound somewhat different from the natural M6P compound but which exhibits a high binding capacity to RM6P-CI. M6P analogs have proven effective in enhancing the therapeutic potential of a lysosomal enzyme produced in a baculovirus / insect cell expression system lacking 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 analogs that offer several advantages for glycoprotein functionalization, particularly for the functionalization of lysosomal enzymes. Indeed, the M6P analogs described in these documents are small, have low immunogenicity, and can be easily grafted onto the lysosomal enzyme. Furthermore, these analogs are customizable, both in terms of the length and structure of the spacer arm and in terms of the terminal reactive group of the spacer arm, depending on the type of binding desired. In these documents, the M6P analogs are used more specifically to internalize lysosomal enzymes into the cell's lysosome, so that these enzymes can perform their physiological function.

[0008] 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 to the antibody proved effective in increasing the internalization of antigens into cultured cells via M6P receptors.

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

[0010] The inventors have now developed new bifunctional compounds including at least one M6P analogue covalently linked to an antibody. These bifunctional compounds exhibit the surprising and unexpected property of being able to be recycled outside the cell after their initial cellular entry via the RM6P-CI pathway. This original and unexpected property allows for the possibility of multiple reuses of the compound of the invention by cells, particularly for reducing / decreasing the concentration of soluble or membrane-bound 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):

[0012] [Chem.l] (I) 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=**,

[0013] [Chem. 2]

[0014] [Chem.3]

[0015] [Chem.4] 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 a fragment of antibody Y comprising at least one antigen-binding domain, said Yi forming no covalent bond(s) with Lp

[0016] According to a second aspect, the present invention relates to a method for preparing a conjugate of formula (I) as defined above.

[0017] According to a third aspect, the present invention relates to a conjugate of formula (I) for medical use, whether diagnostic or therapeutic. It relates in particular to a conjugate of formula (I) for use in a therapeutic treatment method consisting of administering a therapeutic antibody or a fragment of a therapeutic antibody. Brief description of the drawings

[0018] Other features, details and advantages will become apparent upon reading the detailed description below and analyzing the accompanying drawings, in which: Fig. 1

[0019] [Fig. 1] illustrates the extracellular recycling of the formula conjugates (I) of the invention. The M6P analogue is represented by the abbreviation AMFA. The formula conjugate (I) can be called "AMFA-Antibody". The antibody is represented by its Y shape, AMFA by a small sphere, and the antigen by a small cloud shape. Fig. 2

[0020] [Fig.2] illustrates the general synthesis scheme of the conjugates of formula (I) in which ni is equal to 1, depending on the meaning of the reactive chemical group L of the analogue of M6P of general formula (II). Figure 2, point 1, illustrates the reaction between the oxyamine group of the M6P analog of formula (II) and the carbonyl group (-CHO) of antibody Y. Antibody Y is represented by Yi'-CHO, where Yf represents antibody Y without its functional group L', which is equal to -CHO. Analog (II) is designated 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 group of the M6P analog of formula (II) and the amine group (-NH2) of antibody Y. Antibody Y is represented by Yi'-NH2, with Y / representing antibody Y without its functional group L', which is -NH2. Analogues (II) are designated 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 analog of formula (II) with the thiol (-SH) function of antibody Y. Antibody Y is represented by Yi'-SH, with Yf representing antibody Y without its functional group L' equal to -SH. Analogues (II) are designated 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 antibody Y. The (II) analogues are designated 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

[0021] [Fig.3] illustrates the process of preparing intermediate 3: 2,3,4-tri-O-trimethylsilyl-αD-mannopyranoside of 2-bromoethyl, which will serve as a precursor for the synthesis of AMFAs of formula (II). Fig. 4

[0022] [Fig.4] illustrates the process for preparing compound AMFA1 corresponding to the formula general (II) from compound 3. Fig. 5

[0023] [Fig.5] illustrates the process of preparing compound AMFA2 corresponding to the general formula (II) from compound 3. Fig. 6

[0024] [Fig.6] illustrates the process for preparing the AMFA3 and AMFA5 compounds corresponding to the general formula (II) from compound 6. Fig. 7

[0025] [Fig.7] illustrates the process for preparing the compounds AMF A4 and AMFA6 corresponding to the general formula (II) from compound 1. Fig. 8

[0026] [Fig.8] illustrates the process for preparing the AMFA7 and AMFA8 compounds corresponding to the general formula (II) from compound 16. Fig. 9

[0027] [Fig.9] presents the results obtained by SDS-PAGE electrophoresis and Western blots (WB) for the antibodies mAbl, mAb2, mAb3, and mAb4, alone or in combination with AMFA1 (mAbl-AMFA1, mAb2-AMFA1, mAb3-AMFA1, and mAb4-AMFA1). Antibodies before and after coupling with AMFA1 are detected either by Coomassie blue staining of the proteins or by recognition with anti-human IgG antibodies. AMFA1 coupled to antibodies is detected by anti-AMFA1 rabbit polyclonal serum (represented by "anti-AMFA1" in the figure). Fig. 10

[0028] [Fig. 10] presents the affinity curves respectively of the mAbl antibody or of the conjugated mAbl-AMFAl (A) and the antibody mAb2 or the conjugated mAb2-AMFAl (B) for their target molecule, namely the human TNF-alpha antigen. Fig. 11

[0029] [Fig. 11] presents the data from the flow cytometry analysis of the internalization of a non-specific control IgG antibody, the mAbl antibody and the mAbl-AMFAl conjugate with a fluorescent anti-human IgG secondary antibody (Alexa647) in rhabdomyosarcoma cells (top graph), T lymphocyte-like cells (middle graph) and breast cancer cells (bottom graph). Fig. 12

[0030] [Fig. 12] presents the data from the cell fluorescence assay of cells breast cancer (top) and rhabdomyosarcoma cells (bottom), treated with: - a non-specific control IgG antibody, the mAbl antibody and the mAbl-AMFAl conjugate with a fluorescent anti-human IgG secondary antibody (Alexa647) (breast cancer cells), - the mAb3 antibody and the mAb3-AMFAl conjugate with a fluorescent anti-human IgG secondary antibody (Alexa647) (rhabdomyosarcoma cells). This fluorescence assay allows for the definition of cellular internalization of the antibodies alone and of the conjugate of the invention. ** p < 0.01 mAbl-AMFAl versus mAbl (Student's t test). Fig. 13

[0031] [Fig.13] shows the internalization of the Ab antibody or the Ab-AMFAl conjugate Detected by confocal imaging on rhabdomyosarcoma cells (right) and breast cancer cells (left). Nuclei are labeled with Hoechst 33342. Labeling of Ab or Ab-AMFAl complexed with a fluorescent anti-human IgG secondary antibody (Alexa647) is indicated by arrows. The two bottom images show the case where excess M6P was added to the culture medium to saturate the RM6P-CI binding sites on the cells. Fig. 14

[0032] [Fig. 14] presents the data from the analysis of mouse plasma samples mice were injected with the mAbl or mAb2 antibodies, as well as with the corresponding mAbl-AMFAl or mAb2-AMFAl conjugates. Antibody concentrations in the plasma are shown as a function of time after injection. The top graph (A) represents mice treated with mAbl or mAbl-AMFAl, and the bottom graph (B) represents mice treated with mAb2 or mAb2-AMFAl. Fig. 15

[0033] [Fig. 15] shows the detection by Western blot of mAbl antibodies and conjugates mAbl-AMFAl present in mouse plasma 21 days after intravenous (IV) injection of 4 mg / kg of mAbl and mAbl-AMFAl. A control of injected mAbl-AMFAl is added for comparison. Antibodies are detected by recognition with anti-human IgG antibodies, and the presence of antibody-coupled AMFAs is detected by anti-AMFAl rabbit serum (represented by "anti-AMFA" in the figure). Fig. 16

[0034] [Fig. 16] shows the cellular entry of human TNF-alpha into T lymphocytes humans treated with 5 ng / mL of human TNF-alpha and 20 ng / mL of mAbl or mAbl-AMFAl, for 1 h, 5 h and 17 h. The cell lysates were analyzed by Western blot. Intracellular concentrations of human TNF-alpha were quantified as a function of time (hours) after treatment. ** p<0.01 mAbl-AMFAl + TNF-alpha versus mAbl + TNF-alpha (Student’s t-test). Fig. 17

[0035] [Fig. 17] shows neutralization by the mAbl antibody or the conjugate mAbl-AMFAl 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 mAbl-AMFAl versus mAbl (Student’s t-test). Fig. 18

[0036] [Fig. 18] shows the reversal of the mitogenic effect of VEGF in HUVEC cells treated with 50 ng / mL of human VEGF and with different concentrations of mAb3 or mAb3-AMFAl for 72 h. Fig. 19

[0037] [Fig. 19] shows the degradation of the HER2 protein (ERBB2, Human) by an immunocytochemistry method of proteins (In Cell ELIS A) in SK-BR-3 breast cancer cells treated with 10 or 100 nM of mAb4 or mAb4-AMFAl for 24 h. Fig. 20

[0038] [Fig.20] shows the recycling capacity of the mAbl antibody and the conjugate mAbl-AMFAl in Jurkat cells. Each antibody / conjugate is incubated for 5 h with the cells, then the culture medium is replaced with antibody-free medium. Cellular release of the internalized antibody or conjugate is measured at 1 h and 5 h. Detailed description

[0039] Formula conjugates (I)

[0040] As indicated, the present invention relates to a conjugate of formula (I) as defined above. For the purposes of this invention, "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 formula (I) defined above, the M6P analog corresponds to the formula delimited by the large parenthesis. The integer ni indicates the number of M6P analog(s) bound to the antibody or antibody fragment. Indeed, according to the invention, ni M6P analog(s) can be covalently bound to an antibody or antibody fragment comprising at least one antigen-binding domain. These ni M6P analogs are bound to the antibody or antibody fragment via the Lb radical. As already indicated, an analogue of M6P is understood to be a compound that differs from natural M6P but has a high binding capacity to RM6P-Cl. Throughout the application, M6P refers to mannose 6-phosphate and RM6P-CI refers to the 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 binding capacity to RM6P-CI (via the M6P analogue), and a binding capacity to a specific target antigen or target molecule (via the antibody or antibody fragment). The specific target antigen or target molecule refers to a membrane or extracellular molecule of therapeutic or diagnostic interest, in particular a molecule whose overexpression induces a pathological state or is associated with a pathological condition or whose expression is implicated 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 so that there is no longer a pathological state. The term "extracellular or membrane target molecules" refers to 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 new bifunctional compounds of the invention make it possible more particularly to reduce / decrease the concentrations of a soluble extracellular or membrane antigen in an individual, so that the individual no longer presents a pathological state.

[0041] 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 what follows, for the sake of simplicity, the term antibody Y shall mean either the entire antibody Y, 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. The Yi antibody refers to the Y antibody when the latter is covalently linked to the M6P analog via the Lh 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, Yf represents antibody Y without its functional group L'. It is the functional group L' of antibody Y which will react with a functional group or a reactive function carried by the analogue of M6P in order to form a covalent bond between antibody Y and the analogue of M6P and thus obtain the conjugate of formula (I) of the invention. In this 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). An antibody is any type of antibody, of any isotype, from any species. This includes therapeutic or diagnostic antibodies targeting a molecule of therapeutic or diagnostic interest, such as anti-TNFα, anti-VEGF, anti-HER2, and anti-GFP antibodies. Antibodies can be heavy or light chain antibodies, single-chain antibodies, dimeric or multimeric antibodies, and bispecific or multispecific antibodies. Antibodies can also be immunoglobulins of the IgG type, including subtypes IgG1, IgG2, IgG3, or IgG4, and types IgE, IgD, IgA, or IgM. Antibody includes monoclonal antibodies, humanized or fully human antibodies, and chimeric antibodies from different species (human, mouse, rat, rabbit, goat, camelids, chicken, etc.), including human-mouse chimeric antibodies.Antibody fragments include, in particular, 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-domain VhH (nanobody) antibodies and V-NARs). Antibody-derived molecules include fusion proteins comprising an antibody-binding domain to the target molecule, notably in the form of Fc fusion proteins; and antibodies or fusion proteins having multiple binding domains corresponding to several target molecules, i.e., polyspecific antibodies or fusion proteins.The antibody can also be modified to give it a particular property in addition to its recognition of the target molecule, notably 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 allowing 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 usable in clinical practice (radiology, radiography, scintigraphy, MRI, endoscopy, CT scan, etc.).

[0042] In the conjugate of formula (I) as defined above, Yi can more particularly be represented by the L2-Y'i group in which Y represents the antibody Y bound to Li via the L2 radical, with L2 chosen from the group comprising =CH-, -NH- and -S-, said Yi thus being represented by: =CH-Y'1 ,-NH-Y5! et-S-Y'p The L2 radical is more specifically derived from the transformation of the functional group L' carried by the antibody when the latter forms a covalent bond with the M6P analogue.

[0043] According to an advantageous embodiment of the invention, the conjugate of formula (I) is characterized in that ni is an integer equal to 1, and in that said conjugate is chosen from the group comprising:

[0044] [Chem.5] x.

[0045] [Chem.6]

[0046] [Chem.7]

[0047] [Chem. 8] with X, n and Y' i as defined above.

[0048] According to yet another advantageous embodiment, the conjugate of the invention is characterized in that Yi represents a therapeutic or diagnostic antibody Y.

[0049] 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.

[0050] According to yet 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 the IgG type, in particular of subtype IgG1, IgG2, IgG3 or IgG4, or an immunoglobulin of the IgE, IgD, IgA or IgM type.

[0051] 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 affinity IC5o towards RM6P-CI ranging from 104 M to 109 M, and preferably ranging from 105 M to 108 M. This affinity can for example be measured by a method of competition towards another ligand of RM6P-CI such as pentamannose 6-phosphate (according to Basile I. et al., 2018).

[0052] The conjugates of the invention are very interesting in that they allow cellular internalization of a target molecule such as 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 [Fig. 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. The antigen is then directed to the lysosome, while the AMFA-Antibody conjugate unexpectedly exits the cell. The conjugate thus released from the cell can then be used to bind a new extracellular or membrane antigen. This recycling capability was unexpected because previous examples in the literature using M6P analogs to internalize lysosomal enzymes (see, for example, EP 2 448 600 Bl) indicated the presence of these bifunctional compounds (including enzyme-bound M6P analogs) 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 the possible extracellular recycling of bifunctional molecules. Thus, in all these studies, bifunctional compounds were described as being present and / or degraded in lysosomes with their specific target. In the present In an unexpected way, with formula (I) conjugates, only the target molecule is degraded in the lysosomes while the conjugates are advantageously recycled into the extracellular space.

[0053] The conjugate of formula (I) is further characterized in that it exhibits at least one of the following properties: - it is capable of penetrating the cell's endosome via its binding with RM6P-CI to deliver a soluble or membrane-bound extracellular antigen; - it is capable of exiting the endosome of the cell after depositing the soluble extracellular or membrane antigen; - it allows the degradation in the lysosome of soluble extracellular or membrane antigens recognized by Yi after their cellular internalization; - it is recycled into the extracellular environment after its cellular internalization; According to a particularly advantageous embodiment, the conjugate of the invention exhibits each of the properties defined above.

[0054] The originality of the conjugates of the invention stems in particular from the fact that they do not destroy the antibody's natural ability to exit the cell when needed. Indeed, the coupling of M6P analogs with the antibody is carried out in such a way as not to create obstruction at the level of the antibody's reactive functions, such as, for example, at the level of the portion of the antibody interacting with specific antibody receptors, such as neonatal FcRn. This has the effect of not altering the extracellular recycling capabilities of the antibody when it is covalently bound to the M6P analogs described in this application.

[0055] Method for preparing conjugates of formula (I)

[0056] According to another aspect, the present invention relates to a method for preparing a conjugate of formula (I) as defined above, characterized in that the following reaction is performed: - 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)

[0057] [Chem.9] (II), in which X, n and ni are as defined above, L is a reactive chemical group selected from the group comprising -O-NH2,

[0058] [Chem. 10]

[0059] [Chem. 11] And

[0060] [Chem. 12] said group L of compound (II) forming neither covalent bond(s) with nor functional group(s) carried by said antibody Y or fragment of antibody Y.

[0061] The compound of formula (II) is an analog 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. The aforementioned radical L2 is more specifically formed by the transformation of the functional group L' carried by the antibody when the latter forms a covalent bond with the M6P analog.

[0062] In what follows, for the sake of simplification, the L groups of the analogue of M6P as defined above are respectively named, in order of appearance, "oxy-amine", "squarate", "maleimide" and "group comprising a car-bonylacrylic function".

[0063] Specific examples of analogues of formula (II) M6P are more particularly represented by the abbreviation "AMFA" in the present application.

[0064]

[0065] According to an advantageous embodiment of the process of the invention, compound (II) is chosen from the group comprising: [Chem. 13]

[0066]

[0067]

[0068] AMFA1, [Chem. 14] AMFA2, [Chem. 15] AMFA3, [Chem. 16] AMFA4,

[0069] [Chem. 17] AMFA5,

[0070] [Chem. 18] AMFA6,

[0071] [Chem. 19] AMFA7,

[0072] [Chem.20] AMFA8.

[0073] According to an advantageous embodiment of the process of the invention, the use of AMFA1 to AMFA8 described above allows for a particularly interesting and efficient coupling with an antibody such as described above.

[0074] 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 aforementioned antibody is more specifically that located at the Fc region of the antibody. As an example of the L group of the AMFA compound, we can cite the oxy-amine group.

[0075] According to another embodiment of the invention, the L group of the AMFA compound will bind at the level of an appropriate amino acid residue of the peptide chain / part of the antibody. As an example of a suitable amino acid residue, one could cite an amine group from a lysine or a thiol group from a cysteine. Examples of L groups in the AMFA compound include a squarate group, a maleimide group, or a carbonylacrylic group.

[0076] 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 this application.

[0077] Fig. 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 analogue of M6P of general formula (II). As indicated and shown in [Fig. 2], point 1 / , the carbonyl group (-CHO) of antibody Y reacts with the oxyamine group of compound AMFA with formula (II). This coupling mechanism occurs more specifically at an oligosaccharide chain in the glycosidic region located on the Fc region of the antibody. This coupling method is particularly advantageous in that it does not create congestion at the level of the other reactive functions of the antibody (such as the regions interacting with the antibody receptors), which advantageously allows the antibody to preserve its natural properties of recycling to the outside of the cell and thus allows the conjugates of the invention to be recycled outside the cell. Regarding the coupling mode of the AMFA compound of formula (II) comprising the squarate group (see [Fig.2] point 2 / ), the latter intervenes at the level of a lysine residue of the peptide part of the antibody, namely more particularly at the level of the amine function. Regarding the coupling mode of the AMFA compound of formula (II) with a maleimide group (see [Fig.2] point 3 / ), the latter acts at the level of a cysteine ​​residue of the peptide part of the antibody, namely more particularly at the level of the thiol function. Regarding the coupling mode of the AMFA compound of formula (II) comprising a carbonylacrylic function (see [Fig.2] point 4 / ), the latter acts at the level of a cysteine ​​residue of the peptide part of the antibody, namely more particularly at the level of the thiol function. These coupling modes are particularly advantageous because they do not create or create little obstruction at the level of the other reactive functions of the antibody (such as 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.

[0078] Use of conjugates (I) in a therapeutic or diagnostic treatment method

[0079] The new bifunctional compounds make it possible to reduce / decrease the concentrations of soluble or membrane-bound extracellular antigens in an individual, said reduction / decrease being achieved by cellular endocytosis modulated by RM6P-CI. This property makes them particularly interesting for therapeutic use.

[0080] According to another aspect of the invention, a pharmaceutical composition is proposed, 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 amount in the composition of the invention. A "therapeutically effective amount" refers to a dosage sufficient to produce a desired result, for example, an amount sufficient to achieve beneficial or desired therapeutic (including preventive) outcomes, such as reducing the level of a substance whose extracellular or membrane overexpression is responsible for a disease or is implicated in a pathological condition. An effective amount may require one or more administrations.

[0081] According to yet another aspect, the invention relates to a pharmaceutical composition comprising: - a therapeutically effective amount of the formula conjugate (I), - at least one other therapeutically active agent, - possibly at least one pharmaceutically acceptable excipient.

[0082] The present invention also relates to a conjugate of formula (I) as defined above or a pharmaceutical composition as defined above, for medical use, diagnostic or therapeutic type.

[0083] In this regard, the invention relates more particularly to a conjugate of formula (I) as defined above or a pharmaceutical composition as defined above, for use as a medicinal product.

[0084] The invention further 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 fragment of a therapeutic antibody. As already indicated, the extracellular recycling capacity gives the conjugates of the invention particularly advantageous properties in the context of their use to treat pathologies whose treatment consists of administering a therapeutic antibody or a fragment of a therapeutic antibody. Indeed, administering a conjugate of formula (I) allows for the use of a lower dose of therapeutic antibody than if the therapeutic antibody were used alone (without being covalently linked to an M6P analog). This is because the therapeutic antibody, when linked to an M6P analog, exhibits improved therapeutic efficacy compared to the antibody used alone. Thus, at equal doses of antibody, the antibody linked to the M6P analog 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 to reduce the side effects related to antibody treatment.

[0085] Examples of pathologies involving the administration of antibodies may be cited from the group including cancer, inflammatory diseases, autoimmune diseases, blood diseases, ophthalmological diseases, viral infections, allergic diseases and rare diseases.

[0086] The antibodies more particularly tested in the invention in coupling with AMFAs are in particular anti-TNF-alpha, anti-VEGF and anti-HER2 antibodies. Anti-TNF-alpha antibodies are notably used to treat chronic inflammatory bowel diseases, Crohn's disease, ulcerative colitis, and autoimmune diseases affecting the joints such as rheumatoid arthritis, axial spondyloarthritis / ankylosing spondylitis, as well as psoriatic arthritis and psoriasis. The anti-VEGF antibody is notably used clinically to treat neovascularization and tumor growth in many types of cancer, as well as ocular vascular disorders including age-related neovascular macular degeneration. As for the anti-HER2 antibody, it is notably used for the treatment of certain cancers, particularly breast cancers overexpressing the HER2 receptor.

[0087] The invention further 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 formula conjugate (I) covalently linked to an imaging molecule via the Yi antibody can allow the localization of 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 RM6P-CI expression.

[0088] The conjugate of the invention or the pharmaceutical composition can be formulated in various 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 liquid form, lyophilized form, or a liquid form reconstituted from a lyophilized form, where the lyophilized preparation must be reconstituted with a sterile solution before administration. The conjugates of the invention may be formulated into preparations for subcutaneous, intravenous, or intravitreal injection by dissolving, suspending, or emulsifying them in a solvent.

[0089] According to an advantageous embodiment, the invention relates to the conjugate of formula (I) or the pharmaceutical composition for a 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

[0090] 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 analogs 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 have been coupled with antibodies called mAbl, mAb2, mAb3, mAb4 and Ab (see below).

[0091] Example 1: Synthesis of AMFA1 to AMFA8 of general formula (II)

[0092] Preparation of intermediate 3: 2,3,4-tri-O-trimethylsilyl-α-D-mannopyranoside of 2-bromoethvle The process for preparing intermediate 3, which will serve as a precursor for the synthesis of AMFA, is illustrated in [Fig.3], the conditions and reagents of which for steps (i) to (iii) are as follows: (i) 2-bromoethanol, triethylamine, CH2C12, 3.5 h, 80°C; (ii) TMSC1, Et3N, CH2C12, 20 h, TA; (iii) K2CO3, MeOH, 15 min, 0°C. The starting compound is D-mannose onto 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. Position 6 of intermediate 2 was selectively deprotected by potassium carbonate in catalytic amounts in methanol to lead to compound 3 with a yield of 60% over two steps (see [Fig.3]).

[0093] Preparation of compound AMFA1 The process for preparing compound AMFA1 from compound 3 is illustrated in [Fig. 4], the conditions and reagents of which for steps (i) to (vi) are as follows: (i) 0.3 M Dess-Martin per-iodinane, CH2C12, TA, 1 h; (ii) tetraethyl methylene diphosphonate, NaH, THF, 1 h, TA; (iii) Pd / C, Et3SiH, MeOH, 2 h, TA; (iv) N-hydroxyphthalimide, NaH, DMF, 22 h, 60°C; (v) TMSC1, Et3N, CH2C12, 15.5 h, TA; (vi)-a) TMSC1, Nal, CH3CN, 50 min, 35°C; (vi)-b) N2H4.H2O, MeOH, 15.5 h, TA. 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 various AMFAs of formula (II). To synthesize compound AMFA1, aldehyde 4 is reacted with tetraethyl methylene diphosphonate, previously deprotonated with sodium hydride, to form compound 5 in a 76% yield over two steps. In a catalytic hydrogenation step, in the presence of palladium on carbon and in which hydrogen is generated in situ by the progressive addition of triethylsilane, the double bond is reduced and the trimethylsilyl groups are hydrolyzed. This yields compound 6, without purification, in a quantitative yield. The bromine atom is then substituted with N-hydroxyphthalimide to form intermediate 7 in a 61% yield. The alcohol functional groups are then protected with trimethylsilyl chloride to lead to compound 8 in a quantitative yield. The intermediate 8 thus obtained, without purification, is then reacted with trimethylsilyl chloride and sodium iodide to form bis(trimethylsilyl) phosphonate via a Rabinowitz-type reaction. This intermediate is then converted to phosphonic acid by the action of hydrazine monohydrate in methanol, which also displaces the trimethylsilyl groups present on the secondary alcohols of the sugar and reacts with phthalimide to unmask the functional group. amine. In this way, in a single step, the AMFA1 compound is obtained, 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 ([Fig.4]).

[0094] Preparation of compound AMFA2 The process for preparing compound AMFA2 from compound 3 is illustrated in [Fig. 5], the conditions and reagents of steps (i) to (iv) being as follows: (i)-a) 0.3 M Dess-Martin per-iodinane, CH2C12, TA, 1 h; (i)-b) Triethyl phosphonoacetate, NaH, THF, 20 min, TA, 73% (2 steps); (ii)-a) Pd / C, Et3SiH, MeOH, 2 h, TA; (ii)-b) 1 N NaOH, THF / H2O, 2 h, TA; (iii) α-hydroxyphthalimide, NaH, DMF, 20 h, 60°C; (iv) N2H4.H2O, MeOH, 15.5 h, TA. The compound AMFA2 is synthesized following the synthetic routes described in [Fig. 5]. One of the main steps in the synthesis of this saccharide ligand was the introduction of the carboxylate group onto the side chain of the mannose fragments. Similar to previous syntheses, this functionalization is achieved by a Dess-Martin oxidation of intermediate 3 at position 6 followed by a Horner-Wadsworth-Emmons reaction using triethyl phosphonacetate. In this way, the unsaturated carboxylate 10 is obtained. After the steps of reducing the double bond and hydrolyzing trimethylsilyl protecting groups on the hydroxyl functions, 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 unprotected compound AMFA2 ([Fig.5]).

[0095] Preparation of AMFA3 and AMFA5 compounds The process for preparing compounds AMFA3 and AMFA5 from compound 6 is illustrated in [Fig. 6], the conditions and reagents of which for steps (i) to (v) are as follows: (i) 0.3 M Dess-Martin periodinane, CH2C12, TA, 1 h; (ii) tetraethyl methylene diphosphonate, NaH, THF, 1 h, TA; (iii) Pd / C, Et3SiH, MeOH, 2 h, TA; (iv) potassium phthalimide, DMF, 22 h, 60°C; (v) TMSC1, Et3N, CH2C12, 15.5 h, TA; (vi)-a) TMSC1, Nal, CH3CN, 50 min, 35°C; (vi)-b) N2H4.H2O, MeOH, 15.5 h, TA; (vii) Diethyl squarate, Et3N, EtOH / H2O, 30 min; (viii) N-(methoxycarbonyl)maleimide, Saturated NaHCO3 solution, 30 min, 0°C. To synthesize compounds AMFA3 and AMFA5, the bromine atom of compound 6 is substituted with potassium phthalimide to form intermediate 14 in a 74% yield. The alcohol groups are then protected with trimethylsilyl chloride to lead to compound 15 in a quantitative yield. Intermediate 15, obtained without purification, is then reacted with trimethylsilyl chloride and sodium iodide to form bis(trimethylsilyl) phosphonate via a Rabinowitz-type reaction. This intermediate is then Converted to phosphonic acid by the action of hydrazine monohydrate in methanol, which also displaces the trimethylsilyl groups present on the secondary alcohols of the sugar and reacts with phthalimide to unmask the amine function. Thus, in a single step, compound 16 is obtained, deprotected on the phosphonate portion, on the alcohols of both sugars, and with 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 product AMFA3 in a 50% yield or with A-(methoxycarbonyl)maleimide to form compound AMFA5 in an 80% yield ([Fig. 6]).

[0096] Preparation of the compounds AMF A4 and AMFA6 The process for preparing the compounds AMF A4 and AMFA6 from compound 1 is illustrated in [Fig.7], the conditions and reagents of steps (i) to (v) of which are as follows: (i) NaN3, DMF, 20 h, TA; TMSC1, Et3N, CH2C12, 20 h, TA; K2CO3 cat, MeOH, 50 min, 0°C, 38% (3 steps); (ii)-a) Dess-Martin periodinane, CH2C12, 3 h, TA, (ii)-b) Triethyl phosphonoacetate, NaH, THF, 2 h, TA, 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) A-(methoxycarbonyl)maleimide, Saturated NaHCO3 solution, 30 min, 0°C. The compounds AMF A4 and AMFA6 were synthesized following the synthetic routes described in [Fig. 7]. One of the main steps in the synthesis of these saccharide ligands was the introduction of the carboxylate group onto the side chain of the mannose fragments. This functionalization was achieved by a Dess-Martin oxidation of alcohol 19 at position 6 followed by a Homer-Wadsworth-Emmons reaction using triethyl phosphonacetate. In this way, the unsaturated carboxylate 20 was 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 ([Fig. 7]).

[0097] Preparation of AMFA7 and AMFA8 compounds The process for preparing compound AMFA7 from compound 16, and that of compound AMFA8 from compound 21 are respectively illustrated in [Fig.8]. The conditions and reagents of step (i) are as follows: (i) 3-benzoylacrylic acid, A-methylmorpholine, isobutyl chloroformate, DMF, 1 h, -10°C at TA. To prepare compounds AMFA7 and AMFA8, the same procedures used in the preparation of compounds AMFA3 and AMFA4 are followed, except for the last step. This last step consists of the reaction between the amines of intermediates 16 and 21, respectively, and chloro-activated 3-benzoylacrylic acid. isobutyl formate in the presence of V-methylmorpholinc ([Fig.8]).

[0098] Example 2: Synthesis of conjugates of general formula (I)

[0099] The engineering of AMFAs has been validated on five different antibodies which have been coupled to AMFAs 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 mAbl and mAb2; - an antibody directed against vascular endothelial growth factor (VEGF), called bevacizumab, designated mAb3; - an antibody directed against the human epidermal growth factor receptor 2 (HER2), called trastuzumab, designated mAb4; and - an antibody directed against the green fluorescent protein (GFP), called Ab.

[0100] The grafting of AMFAs, for these non-limiting examples, can be carried out at the level of: - of the oligosaccharide chain of the glycosidic portion of the recombinant antibody (see points 1 to 5 below) with AMFAl, or - of the amine function of the lysine residue of the peptide part of the recombinant antibody (see point 6 / below) with AMF A4.

[0101] 1 / Grafting of AMFAl onto the oligosaccharide chains of the glycine portion coside of a chimeric anti-TNF-alpha antibody The coupling of AMFAl was tested on a commercial chimeric mouse-human monoclonal antibody directed against human TNF-alpha, infliximab, defined as mAbl. A conjugate of formula (I) is formed which may be represented interchangeably in the application as "mAbl-AMFA 1" or "AMFA 1-mAbl". This conjugate comprises a portion (mAbl) that binds to a specific target (such as an extracellular or membrane antigen) and a portion (AMFA1) that binds to RM6P-CI. The mAbl antibody was chosen as the model in this application because it has oligosaccharide chains on constant regions that are very similar to those produced in humans. The coupling between AMFAl and the mAbl antibody is carried out according to the protocol described below. In a first step, the mAbl antibody is oxidized. Specifically, the mAbl antibody and a 1 mM sodium meta-periodate (NaIO4) solution are reacted in 25.5 mM phosphate buffer, pH 6.25, for 30 min at 4°C in the dark. Glycerol (20 pL / mL) is added and the mixture is incubated for 10 min at 4°C. stop the reaction. Low molecular weight compounds are removed using a PD-10 desalting column pre-filled with Sephadex G-25 resin. In a second step, AMFAl is added to the previously obtained oxidized antibody solution, and the mixture is allowed to react under stirring for 2 hours 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 mAbl antibody and generates aldehyde groups that interact covalently with the oxyamine groups of AMFAl to form an oxime bond. Furthermore, only the oligosaccharide chains of the mAbl antibody are remodeled. Indeed, the peptide portion of the mAbl antibody is not affected by the conditions used for AMFAl grafting.

[0102] 2 / Grafting of AMFAl onto the oligosaccharide chains of the glycine portion coside 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 AMFAl onto the oligosaccharide chains of the glycosidic part of the mAb2 antibody. A conjugate of formula (I) is formed, which may be represented interchangeably in the application as "mAb2-AMFAl" or "AMFAl-mAb2". This conjugate comprises a portion (mAb2) that binds to a specific target (such as an extracellular or membrane antigen) and a portion (AMFA1) that binds to RM6P-CI.

[0103] 3 / Grafting of AMFAl onto the oligosaccharide chains of the glycine portion coside 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 AMFAl onto the oligosaccharide chains of the mAb3 antibody. A conjugate of formula (I) is formed, which may be represented interchangeably in the application as "mAb3-AMFAl" or "AMFAl-mAb3". This conjugate comprises a portion (mAb3) that binds to a specific target (such as an extracellular or membrane antigen) and a portion (AMFA1) that binds to RM6P-CI.

[0104] 4 / Grafting of AMFAl onto the oligosaccharide chains of the glycine portion coside 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 AMFAl onto the oligosaccharide chains of the mAb4 antibody. A conjugate of formula (I) is formed, which may be represented interchangeably in the application as "mAb4-AMFAl" or "AMFAl-mAb4". This conjugate includes a portion (mAb4) that binds to a specific target (such as an extracellular antigen or membrane) and a part (AMFA1) that binds to RM6P-CI.

[0105] 5 / Grafting of AMFAl onto the oligosaccharide chains of the glycine portion coside 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 AMFAl onto the oligosaccharide chains of the Ab antibody. A conjugate of formula (I) is formed, which can be represented interchangeably as "Ab-AMFAl" or "AMFAl-Ab". This conjugate comprises a portion (Ab) that binds to a specific target (such as an extracellular or membrane antigen) and a portion (AMFA1) that binds to RM6P-CI.

[0106] 6 / Grafting of AMF A4 onto the amine function of lysine residues of the part peptide of a humanized anti-VEGF antibody The coupling between AMF A4 and the mAb3 antibody (bevacizumab) is carried out at the level of the mAb3 peptide chain, in particular at the level of the lysine residues according to the protocol described below. The mAb3 antibody solution and AMF A4 are reacted in phosphate buffer 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 can be represented interchangeably as "mAb3-AMFA4" or "AMFA4-mAb3". This conjugate comprises a portion (mAb3) that binds to a specific target (such as an extracellular or membrane antigen) and a portion (AMFA4) that binds to RM6P-CI.

[0107] Example 3: Study of conjugates (I) and their biological properties

[0108] 1 / Study of the number of AMFAl or AMFA4 bound to an mAbl antibody molecule. mAb2 or mAb3 The conjugates AMFAl-mAbl, AMFAl-mAb2, AMFAl-mAb3 and AMFA4-mAb3 were analyzed by MALDLTOF mass spectrometry using a RapifleX mass spectrometer (Bruker) in linear mode, in order to quantify the number of AMFAl bound to an antibody molecule, according to the method adapted and described in Zhou, Q et al. 2013. The molecular weights of each antibody, mAbl, mAb2, and mAb3, were measured before and after conjugation with AMF1 or AMF4. 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 metric liter (qL) of this mixture was applied to a target. Samples, corresponding references, and the BSA calibration control were analyzed in duplicate. Two-point calibration was performed with [M+H]+ ions and BSA dimer ions. The number of AMFA1 or AMFA4 grafted to the mAbl, mAb2 or mAb3 antibodies 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 "Antibody" control, then dividing the difference by the molecular weight of each AMFA.

[0109] Table 1 below presents the MALDLTOF analysis data of different batches of AMFAl-mAbl, AMFAl-mAb2 and AMFAl-mAb3 conjugates as obtained in Example 2 (points 1 / to 3 / ).

[0110] [Tables 1] Sodium Metaperiodate Batch Mean AMFA count relative to Antibody ± SD (standard deviation) (4 to 7 assessments) AMFAl-mAb 1 1.2 1 mM 3.3 +0.01 AMFAl-mAb 1 2.3 0.2 mM 1+0.1 AMFAl-mAb 2 1.2 1 mM 2.8 +0.3 AMFAl-mAb 3 1.2 1 mM 5.8+1

[0111] Table 2 below presents the MALDLTOF analysis data of different batches of AMFA4-mAb3 conjugates as obtained in Example 2 (point 6 / ).

[0112] [Tableaux2] Batch Coupling Method Average AMFA Count vs Antibody + DS (4 evaluations) AMFA4-mAb 3 1.2 Lysine-squarate 1.9 + 0.7

[0113] Remarks and conclusion The increase in molecular weights after AMF1 or AMF4 grafting indicates the number of AMFAs coupled per antibody. The reproducibility of the grafting reaction is indicated by the small standard deviation of the number of AMFAs grafted between different trials. The data in Tables 1 and 2 demonstrate that AMF1 and AMF4 can be easily grafted onto the mAbl, mAb2, or mAb3 antibody, with high reproducibility standards, and where the number of AMFAl 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 AMFAl. The grafting of the AMFAs of the invention is reproducible and easily controllable according to the reaction conditions.

[0114] 2 / Study of the structural modifications associated with the grafting of AMFAl onto the mAbl, mAb2, mAb3 or mAb4 antibodies The antibodies mAbl, mAb2, mAb3, and mAb4, alone or conjugated to AMFA1 (mAbl-AMFA1, mAb2-AMFA1, mAb3-AMFA1, and mAb4-AMFA1 (the mAb-AMFA1 conjugates)), were analyzed by 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. AMF1 binding to 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 small number of grafted analogs. The immunoreactivity of the mAb-AMFA conjugates to a second commercial anti-human IgG antibody was unchanged by Western blot compared to the mAbs. Hybridization with a rabbit anti-AMFA1 antibody (represented by "anti-AMFA" in [Fig. 9]) confirmed the presence of AMFA1 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 [Fig. 9]. Western blot analyses of the different antibodies demonstrate, on the one hand, that oxidation does not alter the overall structure of the antibody and, on the other hand, that the coupling of AMFA1 is effective on the heavy chains of each antibody (mAbl, mAb2, mAb3, or mAb4), as evidenced by the detection of AMFA1 by the rabbit anti-AMFA1 antibody. In conclusion, the grafting of AMFA1 to the mAbl, mAb2, mAb3, or mAb4 antibodies does not modify the integrity of the antibody. Furthermore, the detection of AMFA1 on the heavy chains of the mAbl, mAb2, and mAb3 antibodies is consistent with grafting only onto the oligosaccharide chains attached to the heavy chains.

[0115] 3 / Study of the capacity of the conjugates mAbl-AMFAl and mAb2-AMFAl to bind to RM6P-CI The affinity tests of the conjugates of the invention mAbl-AMFAl and mAb2-AMFAl for RM6P-CI were carried out using biotinylated RM6P-CI (Basile et al, 2018). The affinity of the conjugates mAbl-AMFAl and mAb2-AMFAl for RM6P-CI is understood to mean the ability of the conjugates mAbl-AMFAl and mAb2-AMFAl to bind to RM6P-CI. In brief, RM6P-CI, purified on a phosphomannan-sepharose affinity column, was biotinylated with biotin A-hydroxysuccinimide. The binding of biotinylated RM6P-CI (RM6P-CLb) to pentamannose 6-phosphate (PMP) previously adsorbed onto a microtiter plate was prevented by the competition of increasing concentrations of the conjugates mAbl-AMFAl or mAb2-AMFAl. The RM6P-CLb bound to the adsorbed PMP was then determined using streptavidin coupled to peroxidase and its substrate, o-phenylenediamine dihydrochloride (OPD), by optical density measurements. The value 100% corresponds to the concentration of RM6P-CLb bound to the adsorbed PMP in the absence of a competitor. Table 3 below presents the binding assay data for natural M6P to RM6P-CLb and for the mAbl-AMFAl and mAb2-AMFAl conjugates to RM6P-CLb. The conjugates studied comprise a portion (mAbl or mAb2) that targets a target molecule (antigen) and a portion (AMFAl) that targets RM6P-CLb. The affinity (expressed as the concentration required for 50% binding inhibition, IC50) was determined by a competitive assay with the pentamannose 6-phosphate ligand.

[0116] [Tables3] Ligand Affinity for RM6P-CLb (IC50) M6P 2.105M mAbl-AMFAl 6.107M mAb2-AMFAl 1.7107M

[0117] Remarks and conclusion While the binding capacity of the antibody alone mAbl or mAb2 to RM6P-CI is close to zero (less than 10% binding over a concentration range of 5.108-2.106 M), Table 3 shows that the mAbl-AMFAl or mAb2-AMFAl 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 specifically, the grafting of AMFAl onto mAbl and mAb2 respectively allows the conjugates of the invention mAbl-AMFA and mAb2-AMFAAl to gain affinity for RM6P-CI.

[0118] 4 / Study of the impact of AMFAl grafting on antibody binding affinity mAbl or mAb2 to the target molecule, namely the human TNF-alpha antigen. The affinity of mAbl and mAb2 antibodies alone, and of mAbl-AMFAl conjugates and mAb2-AMFAl, for human TNF-alpha (target molecule) was evaluated using an ELISA test to determine binding to human TNF-alpha. The "affinity" of the antibody or conjugate for the human TNF-alpha antigen refers to the binding capacity of said antibody or conjugate for said antigen. In short, commercial human TNF-alpha was adsorbed onto a microwell plate and incubated with mAbl, mAb2, mAbl-AMFAl, and mAb2-AMFAl, respectively. The level of mAbl or mAb2 antibodies or conjugates bound to the target of interest is determined by an anti-human IgG antibody coupled to horseradish peroxidase and OPD substrate using optical density measurements. Figure 10 shows that grafting AMFal onto mAbl or mAb2 antibodies does not affect their affinity for the target antigen, human TNF-alpha. The mAbl-AMFAl or mAb2-AMFAl conjugates of the invention exhibit an affinity for human TNF-alpha comparable to that of the mAbl and mAb2 antibodies alone.

[0119] 5 / Study of the stability of the mAbl-AMFAl conjugate of the invention mAbl-AMFAl conjugates from different lots 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 AMFAl was still bound to the mAbl antibody. The number of AMFAl molecules bound to the mAbl antibody was determined on day 0 and day 12 at pH 5 and 7. The results are shown in Table 4 below.

[0120] [Tables4] pH Incubation time Mean number of AMFAs relative to the Antibody ± SD (2 to 4 assessments) AMFAl-mAbl 7 0 days 3.3 + 0.1 AMFAl-mAbl 5 12 days 2.0 + 0.9 AMFAl-mAbl 7 12 days 3.0 + 0.1

[0121] Remarks and conclusion Table 4 shows that the number of AMFAl cells grafted onto the mAbl antibody is stable at both different pH levels, even after 12 days. Therefore, the AMFAl-mAbl conjugates are stable over time and at different pH levels.

[0122] 6 / Study of the cellular internalization of mAb3-AMFAl conjugates. and Ab-AMFAl Cellular internalization of the antibodies and conjugates of the invention has been observed in rhabdomyosarcoma cells and T-lymphocyte-type cells and breast cancer cells. Data were obtained by flow cytometry and total cell fluorescence assays. Rhabdomyosarcoma cells, T-lymphocyte-like cells, and breast cancer cells were treated for 24 h or 48 h with the anti-human IgG fluorescent secondary antibody Alexa647 (Figures 11 and 12). Before incubation with cells, the mAbl antibody, the mAbl-AMFAl conjugate, and a non-specific 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 (Fig. 11). Data from Fig. 11 show significantly higher fluorescence (7.9-fold, 11.3-fold, and 11.8-fold higher compared to the control) in cells treated with mAbl-AMFAl. Fig. 11 demonstrates that AMFal enhances cellular uptake of the mAbl-AMFAl conjugate compared to mAbl alone. Grafting AMFal to the mAbl antibody significantly increases cellular internalization of the antibodies. These results were confirmed by internalization experiments of mAbl, mAbl-AMFAl, and mAb3 and mAb3-AMFAl, performed using a total cell fluorescence assay ([Fig. 12]). Rhabdomyosarcoma and breast cancer cells were treated for 24 h under the same conditions as previously described. [Fig. 12] shows that higher fluorescence is observed in cells treated with mAbl-AMFAl versus mAbl (breast cancer cells) and with mAb3-AMFAl versus mAb3 (rhabdomyosarcoma cells). Grafting AMFal to the mAbl or mAb3 antibody significantly increases cellular internalization of the antibodies. These results were confirmed by confocal microscopy on live rhabdomyosarcoma and breast cancer cells ([Fig. 13]). The cells were pre-incubated with 200 nM of the Ab antibody or the Ab-AMFAl conjugate (see example 2, point 5) and 400 nM of green fluorescent protein for 24 h. Higher fluorescence was observed with Ab-AMFAl versus Ab alone in both cell types (fluorescent cell vesicles indicated by arrows in [Fig. 13]). The addition of an excess of M6P (10 mM) to the cell culture medium to saturate the RM6P-CI binding sites showed that Ab-AMFAl internalization into cells was significantly reduced in both cell types. This experiment demonstrates that the majority of Ab-AMFAl internalization was mediated by RM6P-CI. More broadly, these results demonstrate that grafting AMFAs onto antibodies induces increased cellular internalization via the RM6P-CI-mediated transport pathway. mAb2-AMFAl of the invention

[0124] The effect of grafting AMFAl on mAbl and mAb2 antibodies targeting TNF-alpha was studied by comparing the pharmacokinetic profile of mAbl or mAb2 with that of the corresponding mAbl-AMFAl or mAb2-AMFAl conjugates. The mAbl or mAb2 antibodies, as well as their corresponding mAbl-AMFAl or mAb2-AMFAl 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. 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 post-injection were collected in heparin-containing tubes and centrifuged at 3000 g for 15 min. Plasma was stored at -20 °C until analysis. Mice were sacrificed 21 days post-injection. No adverse clinical events were observed in the four treatment groups.

[0125] Plasma concentrations of mAbl, mAb2, mAbl-AMFAl, and mAb2-AMFAl were measured using ELISA techniques. Briefly, recombinant human TNF-alpha was adsorbed onto a Maxisorp microwell plate and recognized by mAbl, mAb2, mAbl-AMFAl, or mAb2-AMFAl present in the plasma samples. The therapeutic antibody was detected by an HRP-conjugated anti-human IgG antibody specific to the Fc fragment, which was then quantified by colorimetric assay with the OPD substrate and absorbance reading at 450 nm.

[0126] The results obtained are shown in [Fig. 14]. The pharmacokinetic profiles of mAbl compared to mAbl-AMFAl (A) and of mAb2 compared to mAb2-AMFAl (B) detected in plasma samples from treated animals, expressed as a percentage of the 4-hour values, are similar. No significant difference in antibody half-life is observed with the grafting of AMFAl onto mAbl or mAb2. Therefore, grafting AMFAl onto the antibodies does not alter their plasma stability. In conclusion, grafting AMFAl onto mAbl and mAb2 antibodies targeting the TNF-alpha antigen does not alter the pharmacokinetic profile of the antibodies.

[0127] 8 / Study of the in vivo stability of the mAbl-AMFAl conjugate of the invention The in vivo stability of the binding of AMFAl to the therapeutic antibody mAbl was evaluated by analyzing the presence of AMFAl in plasma samples collected at 21 days from mice treated with mAbl-AMFAl or mAbl as described in point 7 / above. mAbl or mAbl-AMFAl 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 mAbl-AMFAl diluted in phosphate buffer were also incubated. After washing, the bound antibodies were analyzed by Western blot for the presence of AMFAl using a polyclonal anti-AMFAl antibody (represented by "anti-AMFA" in [Fig. 15]) and with an anti-human IgG antibody to control the amount of mAbl or mAbl-AMFAl present at 21 days. Figure 15 provides evidence that AMFAl is still conjugated to the therapeutic antibody mAbl at 21 days. Figure 15 shows that both mAbl and mAbl-AMFAl are detected in the plasma of treated mice after 21 days at a quantity similar to that of the 50 ng mAbl-AMFAl control, as confirmed by hybridization with anti-IgG. The majority of AMFA1s are still linked to mAbl, the intensity of the band signal being comparable to that of the mAbl-AMFAl used as a control.

[0128] 9 / Study of the capacity of the mAbl-AMFAl conjugate to allow degradation cellular human TNF-alpha This study shows that human TNF-alpha antigen, after being internalized into cells with the mAbl-AMFAl conjugate, is degraded over time. The degradation of human TNF-alpha was investigated in human T-lymphocyte-like cells. Cells were treated with 5 ng / mL of human TNF-alpha and 20 ng / mL of either mAbl or mAbl-AMFAl for 1 h, 5 h, and 17 h. Cell lysates were analyzed by Western blot for the detection of intracellular human TNF-alpha. For the mAbl-AMFAl conjugate, the data show an increase in human TNF-alpha levels up to 17 h ([Fig. 16]). In contrast, only a slight increase in intracellular human TNF-alpha was observed with the mAbl antibody. The data demonstrate that human TNF-alpha is better internalized via mAbl-AMFAl compared to the mAbl antibody.

[0129] 10 / Study of the capacity of the mAbl-AMFAl conjugate to allow the reduction of the biological effect of human TNF-alpha This study shows that the mAbl-AMFAl conjugate allows, in addition to better internalization into the cell, a greater reduction in biological effect than that observed with the antibody alone. Murine L929 fibroblast-like cells were treated with 5 ng / mL of human TNF-alpha and increasing doses of mAbl or mAbl-AMFAl. Since human TNF-alpha induces cytotoxicity in L929 cells, the live cell rate after 48 h of treatment with mAbl or mAbl-AMFAl was analyzed by the MTT method. Figure 17 shows the neutralizing effect of mAbl or mAbl-AMFAl on the toxicity of human TNF-alpha. mAbl-AMFAl neutralizes the toxicity of human TNF-alpha at much lower doses (4 to 5 times) than mAbl. In conclusion, mAbl-AMFAl conjugates induce a dose-dependent neutralization of human TNF-alpha-induced cell growth inhibition that is more effective than that of the mAbl antibody alone.

[0130] 11 / Study of the ability of the mAb3-AMFAl of the invention to allow a reduction of the effect of human VEGF on HUVEC cell growth This study shows that the mAb3-AMFAl conjugate allows, in addition to better internalization into the cell, a greater reduction in biological effect than that observed with the mAb3 antibody alone. Human umbilical vein endothelial cells (HUVECs) were treated with 50 ng / mL of human VEGF and increasing doses of mAb3 or mAb3-AMFAl. Since human VEGF induces proliferation of HUVECs, the live cell count at 72 h in cells treated with mAb3 or mAb3-AMFAl 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-AMFAl conjugate compared to the antibody alone. At low doses, the mAb3-AMFAl conjugate is twice as effective as the mAb3 antibody alone. The mAb3-AMFAl conjugate is effective at much lower doses (10 times) than mAb3.

[0131] 12 / Study of the ability of AMFAl-mAb4 conjugates to allow a decrease in the amount of HER2 SK-BR-3 breast cancer cells were treated with 10 or 100 nM of mAb4 or mAb4-AMFAl 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, whose Fc portion is in turn recognized by a peroxidase-coupled secondary antibody (HRP). The peroxidase enzyme catalyzes the colorimetric reaction after the substrate is added. Figure 19 shows the decrease in HER2 protein after treatment with mAb4-AMFAl compared to mAb4 alone. Indeed, after 24 h of treatment with mAb4-AMFAl, the total amount of HER2 is reduced more significantly than with treatment using only mAb4. Increasing doses of mAb4-AMFAl result in a greater decrease in cellular HER2 concentration compared to the same doses of mAb4.

[0132] 13 / Extracellular recycling of AMFAl-mAbl conjugates after their internalization in T lymphocytes Extracellular recycling of the internalized mAbl antibody and mAbl-AMFAl conjugate was demonstrated by antibody entry experiments for 5 h in immortalized T lymphocytes (Jurkat) followed by The release period of previously internalized antibodies into the culture medium was between 1 and 5 hours. The recycling of mAbl and mAbl-AMFAl was analyzed. The concentrations of mAbl and mAbl-AMFAl were measured using the ELIS A technique as described in section 7 above. Figure 20 shows that the extracellular recycling of mAbl-AMFAl conjugates is comparable to that of mAbl alone. These results indicate that mAbl-AMFAl is normally recycled out of the cell after its strong cellular internalization mediated by RM6P-CI.

[0133] This disclosure is not limited to the examples described above, which are merely examples, but encompasses all the variations that a person skilled in the art may consider in the context of the protection sought. List of cited documents Patent documents

[0134] For the avoidance of doubt, 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

[0135] For the avoidance of doubt, the following non-patent elements are cited: - nplcitl: Gary-Bobo et al. Curr. Med. Chem. 2007, 14, 2945-2953; - nplcite2: El Cheikh K. et al., Angew. Chem. Int. Ed. 2016, 55, 52016; - nplcite3: Zhu Y., et al. Mol Ther. 2009,17, 954-963; - nplcit4 : Basile I. et al., J. Control. Release, 2018, 10, 269 ,15-23 ; - nplcit5 : Zhou, Q et al., Bioconjugate Chem. 2013, 24, 12, 2025-2035.

Claims

Claims

1. Conjugate characterized in that it has the following general formula (I): [Chem.l] (I) 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 ranging from 0 to 2; Li is a radical chosen from the group including *-ON=**, [Chem. 2] [Chem. 3] And [Chem. 4] 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 Lb

2. Conjugate according to claim 1, characterized in that Yi is represented by the group L2-Y'i in which Y\ represents the antibody Y or antibody fragment 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.

3. Conjugate characterized in that it is chosen from the group comprising: [Chem. 5] X.. [Chem. 7] \ GH .Y3 V \_gZ i / <3 and [Chem. 8] ​​x.,_ \ »! i to V3X \ -Y'i KG.....,»*^****-aci / XA ,1.....1 " ï '□ with X, n and Y' i as defined in claim 1 or 2.

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

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

6. Conjugate according to claim 5, characterized in that Yi represents an antibody Y which is an immunoglobulin of type IgG, in particular of subtype IgG1, IgG2, IgG3 or IgG4, or an immunoglobulin of type IgE, IgD, IgA or IgM.

7. Conjugate according to any one of claims 1 to 6, characterized in that it has an IC50 affinity with respect to the cation-independent mannose 6-phosphate receptor (RM6P-CI) ranging from 10 4 M to 109 M, and preferably ranging from 105 M to 108 M.

8. Process for the preparation of a conjugate of formula (I) as defined in any one of claims 1 to 7, characterized in that the following are reacted: - an antibody Y or a fragment of antibody Y comprising at least one antigen-binding domain with - nor compound(s) corresponding to the general formula (II) [Chem.9] (II) wherein X, n and ni are as defined in claim 1, L is a reactive chemical group selected from the group consisting of -O-NH2, [Chem. 10] [Chem. 11] And [Chem. 12] said group L of compound (II) forming neither covalent bond(s) with nor functional group(s) carried by said antibody Y or antibody fragment Y.

9. Preparation process according to claim 8, characterized in that the compound (II) is selected from the group comprising [Chem. 13] HO-p>° HO 'V OH HO-"V HO- NHj [Chem. 14] HO' HO OH Olympic Games nh2 [Chem. 15] [Chem. 16] [Chem. 17] [Chem. 18] [Chem. 19] O HO^' HO V.. OH HO'-VÀJ-O 0 o. --. AAH oh and [Chem. 20] ho ., ° OH HO-'VX-JO i ° rp 0 ,¼ J -- N . y H ô

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

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

13. A conjugate as defined in 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 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 parenteral, intravenous or subcutaneous administration. cutaneous.

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