Hybrid molecule comprising an Fc fragment of antibody and at least one peptide binding to an autoreactive lymphocyte involved in autoimmune vasculitis, and its uses

Abstract

The invention relates to a hybrid molecule comprising at least one Fc fragment of an antibody covalently linked to at least one peptide that binds to an autoreactive lymphocyte involved in autoimmune vasculitis, the uses of such a hybrid molecule, and its production process. (no figure)

Description

Title of the invention: Hybrid molecule comprising an Fc fragment of antibody and at least one peptide binding to an autoreactive lymphocyte involved in autoimmune vasculitis, and its uses

[0001] The present invention relates to a hybrid molecule comprising at least one Fc fragment of an antibody covalently linked to at least one peptide that binds to a self-reactive lymphocyte involved in autoimmune vasculitis, the uses of such a hybrid molecule, and its production process. Said self-reactive lymphocyte receptor is a membrane receptor on the surface of a B lymphocyte (BCR) or on the surface of a T lymphocyte (TCR). Context of the invention

[0002] Autoimmune vasculitis are rare diseases characterized by inflammation of the walls of small blood vessels in the body, notably linked to the production of autoantibodies by the body and / or pro-inflammatory cytokines secreted by T lymphocytes.

[0003] These autoantibodies are directed in particular against certain white blood cells: neutrophils and can therefore sometimes be called ANCA (anti-neutrophil cytoplasmic antibodies).

[0004] These autoantibodies are often used to establish the diagnosis of autoimmune vasculitis and their role in the pathogenesis of the disease has been studied (e.g. Bruner BF, Vista ES, Wynn DM, Harley JB, James JA. Anti-neutrophil cytoplasmic antibodies target sequential functional proteinase 3 epitopes in the sera of patients with Wegener's granulomatosis. Clin Exp Immunol. 2010 Nov; 162(2):262-70. doi: 10.1111 / j. 1365-2249.2010.04251.x. PMID: 21077276; PMCID: PMC2996593; or Van Der Geld YM, Simpelaar A, Van Der Zee R, Tervaert JW, Stegeman CA, Limburg PC, Kallenberg CG). Antineutrophil cytoplasmic antibodies to proteinase 3 in Wegener's granulomatosis: epitope analysis using synthetic peptides. Kidney Int. 2001 Jan;59(l): 147-59. doi: 10.1046 / j.1523-1755.2001.00475.x. PMID: 11135067). These autoantibodies therefore represent a promising therapeutic target.

[0005] The antigenic targets of these autoantibodies involved in autoimmune vasculitis have been characterized and can be found in the IEDB database, Immune Epitope Database and analysis resource, http: / / www.iedb.org / home_v3.php. These autoantibodies are specifically directed against proteinase 3 (PR3), or specifically against myeloperoxidase (MPO), or against small nuclear ribonucleoprotein SmDl (“small nuclear ribonucleoprotein SmDl”). cleoprotein").

[0006] Autoimmune vasculitis (or "ANCA-positive vasculitis" or ANCA-associated vasculitis) primarily encompasses three diseases: granulomatosis with polyangiitis (sometimes called Wegener's granulomatosis), eosinophilic granulomatosis with polyangiitis (sometimes called Churg-Strauss syndrome), and micropolyangiitis. Granulomatosis with polyangiitis is characterized by inflammatory necrosis of small and medium-sized vessels (capillaries, venules, and arterioles), leading to tissue ischemia. Eosinophilic granulomatosis with polyangiitis is defined as a systemic small vessel vasculitis which is also characterized by asthma, blood and tissue eosinophilia, and micropolyangiitis is defined as a systemic necrotizing inflammatory vasculitis which mainly affects small caliber vessels of multiple organs (small arteries, arterioles, capillaries, venules).

[0007] To date, there is no specific treatment for these autoimmune vasculitides. Treatments generally consist of administering corticosteroids, or even immunosuppressant drugs, which can cause serious side effects. Anti-CD20 antibodies can also be used, but these are not specific to the B lymphocytes that produce the autoreactive antibodies and have no effect on autoreactive T lymphocytes.

[0008] One object of the present invention is thus to provide a more targeted treatment for autoimmune vasculitis. By targeting the B cells that produce the autoreactive antibodies (autoantibodies) associated with autoimmune vasculitis, and / or the autoreactive T lymphocytes that are sources of pro-inflammatory cytokines, the present invention aims to provide a more specific treatment.

[0009] The present invention is based on the Inventors' research showing that it is possible to target autoreactive lymphocyte receptors that recognize self-peptides involved in the development of autoimmune vasculitis. More particularly, the present invention is based on the Inventors' research showing that it is possible to target B lymphocyte clones expressing lymphocyte receptors involved in autoimmune vasculitis and / or autoreactive T lymphocytes (by binding a self-peptide involved in the development of autoimmune vasculitis to the TCR), and to eliminate them using a hybrid molecule comprising (i) at least one peptide binding to an autoreactive lymphocyte involved in autoimmune vasculitis and (ii) an Fc fragment of human immunoglobulin.These hybrid molecules will specifically target B lymphocyte clones expressing autoantibodies responsible for autoimmune vasculitis and / or autoreactive T lymphocytes (using said peptide, which is recognized by said B and / or T receptors expressed by said B and / or T lymphocytes). which will then be eliminated, after the Fc fragment is fixed to Fc receptors, by macrophages (via phagocytosis) and / or NK cells (via antibody-dependent cell-mediated cytotoxicity - ADCC), and / or by activation of the complement cascade.

[0010] By targeting B lymphocyte clones expressing autoantibodies responsible for autoimmune vasculitis and cells that differentiate into plasma cells which themselves secrete said autoantibodies and / or autoreactive T lymphocytes that are sources of pro-inflammatory cytokines, the hybrid molecules of the invention aim to eliminate these "pathogenic" autoantibodies from the patients' bodies. Description of the invention

[0011] Hybrid molecule according to the invention

[0012] In a first aspect, the invention relates to a hybrid molecule comprising at least one Fc fragment of an antibody covalently linked to at least one peptide binding to a self-reactive lymphocyte involved in autoimmune vasculitis, at least one spacer being optionally present between said Fc fragment and said peptide. The diagram of such a construct is shown in [Fig. 1].

[0013] According to the invention, a "hybrid molecule" means a molecule having at least two components of a different nature, in this case the Fc fragment of antibody and said peptide.

[0014] According to the invention, an antibody “Fc fragment” is understood to be the constant region of an immunoglobulin excluding the first constant region domain of the immunoglobulin (i.e., CH1-CL). Thus, the Fc fragment refers to a homodimer, each monomer comprising the last two constant domains of IgA, IgD, IgG (i.e., CH2 and CH3), or the last three constant domains of IgE and IgM (i.e., CH2, CH3, and CH4).

[0015] According to the invention, the expression "covalently bonded" means a covalent bond, that is to say, a chemical bond in which two atoms share two electrons. Said covalent bond may be polar or nonpolar.

[0016] According to the invention, a "spacer" is a linker that covalently binds an Fc fragment of an antibody to a peptide that binds to an autoreactive lymphocyte involved in autoimmune vasculitis, while simultaneously separating the Fc fragment from the peptide (thus reducing potential steric hindrance). The spacer can be any molecule, and in particular a peptide or a polypeptide. Preferably, the spacer does not alter the physicochemical properties of the hybrid molecule.

[0017] The presence of at least one spacer is advantageous: it facilitates independent accessibility of the two partners of the hybrid molecule (the Fc fragment is more easily accessible for binding to Fc receptors, as is said peptide is more easily accessible to bind to autoreactive lymphocytes), and / or to stabilize the hybrid molecule, and / or to increase the solubility of the hybrid molecule.

[0018] According to one embodiment, the hybrid molecule according to the invention may comprise one or more spacers. Preferably, the hybrid molecule comprises one or two spacers. In one embodiment, when at least one spacer is present in said hybrid molecule of the invention, said Fc fragment is covalently linked to a spacer, said spacer being itself covalently linked to said peptide. In another embodiment, when at least two spacers are present in said hybrid molecule of the invention, said Fc fragment is covalently linked to a first spacer, said first spacer being itself covalently linked to a second spacer, and the second spacer is itself covalently linked to said peptide. The link between the Fc fragment and the peptide can therefore be direct, or indirect in the presence of spacers.

[0019] According to one embodiment, the hybrid molecule according to the invention may comprise at least one peptide that binds to a self-reactive lymphocyte involved in autoimmune vasculitis. This means that the Fc fragment may be bound to one or two peptides. Indeed, the Fc fragment comprises two monomers, and the Fc fragment may thus be covalently bound to a peptide on only one of the two monomers, or the Fc fragment may be covalently bound to a peptide on each monomer. Preferably, when two peptides are bound to the Fc fragment, the two peptides are identical.

[0020] According to one embodiment, said spacer is a polymer containing one or more repeating units containing the ether group. According to a particular embodiment, said spacer is polyethylene glycol of formula PEG-n, in which n represents an integer between 1 and 100, preferably between 1 and 10, and in particular 1, 2, 3, 4, or 8. According to the invention, said polyethylene glycol can be functionalized, for example, with an amine group (PEG-n-amine such as PEG-NH2). According to the invention, "an integer between 1 and 100" represents all integer values ​​between 1 and 100, i.e.; 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100.

[0021] The expression "auto-reactive lymphocyte receptor involved in autoimmune vasculitis" refers to a receptor on the surface of a T lymphocyte and / or a B lymphocyte that recognizes a self-peptide involving cell activation leading to autoimmune vasculitis through inappropriate production of autoantibodies or pro-inflammatory cytokines. More specifically, said A lymphocyte receptor is a B-cell receptor (BCR) and / or a T-cell receptor (TCR). The binding of the peptide to the BCR and / or TCR is thus implicated in the development of autoimmune vasculitis. For example, in one embodiment, the self-peptide involved may include all or part of proteinase 3. An "autoantibody responsible for autoimmune vasculitis" is understood to be an "anti-neutrophil cytoplasmic autoantibody (ANCA)." Typically, the binding of these ANCAs to their target leads to neutrophil activation with increased adhesion to the vascular endothelium, production of reactive oxygen species, and release of enzymes that cause tissue damage.

[0022] The expression "autoreactive lymphocyte involved in autoimmune vasculitis" refers to a lymphocyte expressing on its surface a receptor that recognizes a self-peptide, leading to cell activation and resulting in autoimmune vasculitis through inappropriate production of autoantibodies or pro-inflammatory cytokines. Preferably, this refers to a T lymphocyte and / or a B lymphocyte.

[0023] According to a preferred embodiment, an autoantibody according to the invention is understood to be an autoantibody directed against proteinase 3 (anti-PR3), an autoantibody directed against myeloperoxidase (anti-MPO), and / or an autoantibody directed against the small nuclear ribonucleoprotein SmDl (anti-SmDl). Preferably, the autoantibodies are anti-neutrophil cytoplasmic autoantibodies such as anti-PR3 (c-ANCA for cytoplasmic-ANCA) or anti-MPO (p-ANCA for perinuclear-ANCA).

[0024] The expression "autoimmune vasculitis" refers more particularly to necrotizing inflammation of small vessels, with little or no deposits of immune complexes, and the frequent presence of circulating antibodies directed against proteinase 3 (PR3) and / or against myeloperoxidase (anti-MPO) and / or against the small nuclear ribonucleoprotein SmDl (anti-SmDl).

[0025] According to one embodiment, the expression "peptide binding to an autoreactive lymphocyte involved in autoimmune vasculitis" means a peptide comprising all or part of the polypeptide sequence of proteinase 3, all or part of the polypeptide sequence of myeloperoxidase and / or all or part of the polypeptide sequence of small nuclear ribonucleoprotein SmDl.

[0026] According to one embodiment, the expression "peptide binding to an autoreactive lymphocyte involved in autoimmune vasculitis" means a peptide comprising all or part of the polypeptide sequence of proteinase 3. Preferably, said polypeptide sequence of proteinase 3 is represented by SEQ ID NO: 28.

[0027] According to one embodiment, the expression "peptide binding to an autoreactive lymphocyte involved in autoimmune vasculitis" refers to a peptide comprising all or part of the myeloperoxidase polypeptide sequence. Preferably, said myeloperoxidase polypeptide sequence is represented by SEQ ID NO: 29.

[0028] According to one embodiment, the expression "peptide binding to an autoreactive lymphocyte involved in autoimmune vasculitis" means a peptide comprising all or part of the polypeptide sequence of the small nuclear ribonucleoprotein SmDl. Preferably, said polypeptide sequence of said SmDl is represented by SEQ ID NO: 30.

[0029] According to one embodiment, a "peptide binding to an autoreactive lymphocyte involved in autoimmune vasculitis" means a peptide recognized by an autoantibody directed against proteinase 3, myeloperoxidase, and / or small nuclear ribonucleoprotein SmDl. Such peptides can be obtained from fragments of proteinase, myeloperoxidase, and / or small nuclear ribonucleoprotein SmDl, whether these fragments are natural, recombinant, or synthetic. Such peptides can also be directly synthesized. The amino acids constituting the peptide may be of the L or D series, preferably of the L series. A peptide according to the invention binds to an autoantibody directed against proteinase 3, against myeloperoxidase, and / or against the small nuclear ribonucleoprotein SmDl, and the binding between said peptide and the autoantibody may, for example, be verified using an ELISA test. See also, for example, Csernok, E., Moosig, F. Current and emerging techniques for ANC A detection in vasculitis. Nat Rev Rheumatol 10, 494-501 (2014). https: / / doi.org / 10.1038 / nrrheum.2014.78. .

[0030] According to one embodiment, in said hybrid molecule according to the invention, said peptide comprises all or part of the polypeptide sequence of proteinase 3, all or part of the polypeptide sequence of myeloperoxidase and / or all or part of the polypeptide sequence of small nuclear ribonucleoprotein SmDl, and said proteinase 3, myeloperoxidase and / or SmDl is derived from mammals and is preferably of human origin.

[0031] According to one embodiment, in said hybrid molecule according to the invention, the peptide has a size of at least 2 consecutive amino acids, 3 consecutive amino acids, 4 consecutive amino acids, preferably at least 5 consecutive amino acids. According to one embodiment, said peptide has a size of between 5 and 70 amino acids. According to the invention, "between 5 and 70" means all the values: 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70. According to one embodiment, in said hybrid molecule according to the invention, the peptide has a size of between 5 and 15 consecutive amino acids, preferably between 5 and 10.

[0032] According to one embodiment, in said hybrid molecule according to the invention, the peptide is linear.

[0033] According to one embodiment, in said hybrid molecule according to the invention, the peptide can be modified to improve its reactivity towards autoantibodies. By way of example, peptides can be cyclized, peptides can be of the retro-type (the L-series amino acids are linked in a reverse sequence to that of the peptide to be reproduced), or of the retro-inverso type (the amino acids are of the D-type instead of the natural L series and are linked in a reverse sequence to that of the peptide to be reproduced). According to a further particular embodiment, in said hybrid molecule according to the invention, the terminal carboxyl (COOH) group of said peptide is replaced by a carboxamide (CONH2) group.

[0034] According to another embodiment, in said hybrid molecule according to the invention, the peptide can be modified to facilitate its synthesis and / or improve its stability, for example by alkylation. According to a further particular embodiment, in said hybrid molecule according to the invention, the terminal amine (NH2) group of said peptide is acetylated.

[0035] According to one embodiment, in said hybrid molecule according to the invention, the amine and carboxyl functions of the peptide can be in the form of the salt corresponding to the acid or the base.

[0036] Selon un mode de réalisation encore plus particulier, dans ladite molécule hybride selon l’invention, ledit peptide est choisi dans le groupe constitué par : SEQ ID NO : 1, SEQ ID NO : 2, SEQ ID NO : 3, SEQ ID NO : 4, SEQ ID NO : 5, SEQ ID NO : 6, SEQ ID NO : 7, SEQ ID NO : 8, SEQ ID NO : 9, SEQ ID NO : 10, SEQ ID NO : 11, SEQ ID NO : 12, SEQ ID NO : 13, SEQ ID NO : 14, SEQ ID NO : 15, SEQ ID NO : 16, SEQ ID NO : 17, SEQ ID NO : 18, SEQ ID NO : 19, SEQ ID NO : 20, SEQ ID NO : 21, SEQ ID NO : 22, SEQ ID NO : 23, SEQ ID NO : 31, SEQ ID NO : 32, SEQ ID NO: 33, SEQ ID NO : 34, SEQ ID NO : 35, SEQ ID NO : 36, SEQ ID NO : 37, SEQ ID NO : 38, SEQ ID NO : 39, SEQ ID NO : 40, SEQ ID NO : 41, SEQ ID NO : 42, SEQ ID NO : 43, SEQ ID NO : 44, SEQ ID NO : 45, SEQ ID NO : 46, SEQ ID NO : 47, SEQ ID NO : 48, SEQ ID NO : 49, SEQ ID NO : 50 et SEQ ID NO : 51.

[0037] According to a further particular embodiment, in said hybrid molecule according to the invention, said peptide is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35 (PR3).

[0038] According to a further more particular embodiment, in said hybrid molecule according to the invention, said peptide is chosen from the group consisting of: SEQ ID NO: 36 and SEQ ID NO: 37 (SmDl).

[0039] According to a further more particular embodiment, in said hybrid molecule according to the invention, said peptide is selected from the group consisting of: SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51 (MPO).

[0040] According to one embodiment, in said hybrid molecule according to the invention, said fragment Fc is a human Fc fragment, in particular of IgG, more particularly of IgGl. The IgGl can correspond to any allotypic variant, for example Glm3 or nGlml7. By way of example, the Fc fragment of IgGl is represented by SEQ ID NO: 24, SEQ ID NO: 25(Fc+Qtag) or SEQ ID NO: 26 (Fc+Qtag bis).

[0041] According to one embodiment, in said hybrid molecule according to the invention, said Fc fragment is wild-type or mutated. The mutation(s) may aim to increase the plasma half-life, decrease it, or modify the effector functions of the Fc fragment. According to a further specific embodiment, said mutated Fc fragment comprises at least the following mutations: - L234A and L235A (LALA), or - L234A, L235A and P329G (LALAPG), or - G236A, S239D and I332E (GASDIE), or - G236A, S239D, A330L and I332E (GASDALIE), or - S239D, H268F, S324T and I332E (SDHFSTIE or SDH), The numbering is indicated in the sequence of a human IgG1 according to the EU index. Such mutations are notably described in the article by Bruhns and Jönsson, Immunol Rev. 2015 Nov;268(l):25-51. Preferably, when the hybrid molecule is used in therapy, the mutated Fc fragment includes at least the GASDIE, GASDALIE, or SDH mutations.

[0042] According to one embodiment, in said hybrid molecule according to the invention, said fragment Fc exhibits a fucosylation rate of between 0% and 100% of the glycosylated forms. According to the invention, "between 0% and 100%" represents all integer values ​​between 0 and 100, i.e., 0, 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%. Low fucosylation of the Fc fragment elicits a strong ADCC response. Therefore, according to a particular embodiment, said Fc fragment exhibits a fucosylation rate between 0% and 60% of the glycosylated forms, in particular 50%, 40%, 30%, 20%, 10% or 0%. According to the invention, the fucosylation rate is defined as the average proportion of fucose carried by the Fc fragment, relative to the maximum amount of fucose that an Fc fragment can carry.

[0043] The Fc fragment and the peptide each have N- and C-terminal ends. The Fc fragment can therefore be linked via its N- or C-terminal end to the N- or C-terminal end of the peptide. In a preferred embodiment, in said hybrid molecule according to the invention, said covalent bond is located between the C-terminal end of said Fc fragment and the N-terminal end of said peptide, or between the N-terminal end of said Fc fragment and the N-terminal end of said peptide. In one embodiment, when a spacer is present, the spacer can be linked to the Fc fragment via its N- or C-terminal end. In another embodiment, when two spacers are present, the first spacer can be linked to the Fc fragment via its N- or C-terminal end, and the second spacer can be linked to the peptide via its N- or C-terminal end, in particular its N-terminal end.Alternatively, the covalent bond between the Fc fragment and the peptide (optionally in the presence of one or more spacers) can be created on all or part of the Fc fragment. According to the invention, "all or part of the Fc fragment" means that different amino acids constituting the Fc fragment can be involved in a covalent bond with the peptide.

[0044] According to a preferred embodiment, when at least one spacer is present in said hybrid molecule of the invention, it allows the Fc fragment to be linked to an azide or an alkyne, which will itself be involved in the covalent bond with said peptide. According to another embodiment, when at least one spacer is present in said hybrid molecule of the invention, it can also allow the peptide to be linked to an alkyne or an azide, which will itself be involved in the covalent bond with the Fc fragment. According to a preferred embodiment, the hybrid molecule according to the invention comprises at least two spacers: a first spacer that allows the Fc fragment to be linked to an azide or an alkyne, and a second spacer that allows the peptide to be linked to an azide (when the Fc fragment is linked to an alkyne) or to an alkyne (when the Fc fragment is linked to an azide).

[0045] According to an embodiment of the invention, in said hybrid molecule according to the invention, said fragment Fc: - is coupled to at least one azide or an alkyne, such as a cyclooctyne, and in particular DBCO, or - is linked to at least one spacer which is itself coupled to an azide or an alkyne, such as a cyclooctyne, and in particular DBCO, and said peptide is: - either coupled to an azide or an alkyne, such as a cyclooctyne, and in particular the DBCO, - either linked to a spacer which is itself coupled to an azide or an alkyne, such as a cyclooctyne, and in particular DBCO, the covalent bond between said fragment Fc and said peptide, possibly in the presence of one or more spacers, being created between the azide and the alkyne.

[0046] According to an embodiment of the invention, in said hybrid molecule according to the invention, - the Fc fragment is coupled to at least one azide and said peptide is coupled to an alkyne, such as a cyclooctyne, and in particular DBCO, or - the Fc fragment is coupled to at least one alkyne, such as a cyclooctyne, and in particular DBCO, and said peptide is coupled to an azide, the covalent bond between said fragment Fc and said peptide being created between the azide and the alkyne.

[0047] According to an embodiment of the invention, in said hybrid molecule according to the invention: - the Fc fragment is coupled to at least one azide and said peptide is linked to a spacer, itself coupled to an alkyne, such as a cyclooctyne, and in particular DBCO or - the Fc fragment is coupled to at least one alkyne, such as a cyclooctyne, and in particular DBCO, and said peptide is linked to a spacer, itself coupled to an azide or - the Fc fragment is linked to at least one spacer, itself coupled to an azide, and said peptide is coupled to an alkyne, such as a cyclooctyne, and in particular DBCO or - the Fc fragment is linked to at least one spacer, itself coupled to an alkyne, such as a cyclooctyne, and in particular DBCO, and said peptide is coupled to an azide or - the Fc fragment is linked to at least one spacer, itself coupled to an azide, and said peptide is linked to a spacer, itself coupled to an alkyne, such as a cyclooctyne, and in particular DBCO or - the Fc fragment is linked to at least one spacer, itself coupled to an alkyne, such as a cyclooctyne, and in particular DBCO, and said peptide is linked to a spacer, itself coupled to an azide, the covalent bond between said fragment Fc and said peptide, in the presence of one or more spacers, being created between the azide and the alkyne.

[0048] According to the invention, the formation of the covalent bond between the azide and the alkyne corresponds to a so-called "click chemistry" step, the N3 portion of the azide reacting with an alkyne. Azide refers to salts of hydrozotic acid (HN3), or to organic azides in which one of the nitrogen atoms is covalently bonded with A carbon atom of an organic compound (for example, methyl azide CH3N3). Preferably, azide is represented by the formula N3. An alkyne is understood to be a molecule with the general formula CnH2n2, characterized by the presence of at least one triple bond. Preferably, an alkyne is a cyclooctyne, and even more preferably, a dibenzocyclooctyne (DBCO).

[0049] The Fc fragment, said peptide, and optionally said spacer(s) are coupled to the alkyne or azide by any conventionally used molecular coupling technique (such as conjugation). Any technique may also be used to covalently link the Fc fragment to the spacer and / or the peptide to the spacer.

[0050] More specifically, a conjugation technique means either enzymatic conjugation or chemical conjugation. Enzymatic conjugation means, for example, conjugation using a transglutaminase that catalyzes the formation of covalent bonds between free amine groups and glutamine or lysine residues, or using a transpeptidase such as sortase. For further information concerning enzymatic conjugation, see, for example, patent application US20160361434 or US20170313787, or the publication Ohtsuka et al., Bioscience, Biotechnology, and Biochemistry, Volume 64, 2000 - Issue 12, Comparison of Substrate Specificities of Transglutaminases Using Synthetic Peptides as Acyl Donors. The substrate of transglutaminase is for example a peptide containing a glutamyl residue (a Qtag), such as represented by SEQ ID NO: 27 (LLQG).A chemical conjugation is understood, for example, as a covalent bond between an isolated cysteine ​​or a cysteine ​​participating in a disulfide bridge after its reduction, and, for example, a maleimide. An example of such a conjugation is shown in [Fig. 4]. In this example, the Fc fragment comprises a Qtag peptide, and this Fc fragment is linked to a spacer (itself coupled to an azide), through the action of transglutaminase, which creates a covalent bond between the glutamyl residue of the Qtag and the NH2 group attached to the PEGn spacer.

[0051] According to the invention, the term "coupled" or "molecular coupling" refers to the establishment of a covalent bond, such that the Fc fragment and / or the peptide and / or the spacer is covalently linked to an alkyne or an azide. The term "linked" also refers to a covalent bond. Thus, by way of example, the expression "the Fc fragment is coupled to an azide and said peptide is linked to a spacer, itself coupled to an alkyne, such as a cyclooctyne, and in particular DBCO" can also be read as "the Fc fragment is covalently linked to an azide and said peptide is covalently linked to a spacer, said spacer being itself covalently linked to an alkyne, such as a cyclooctyne, and in particular DBCO."

[0052] Use of hybrid molecules according to the invention

[0053] In a second aspect, the present invention also relates to a molecule hybrid as defined above, for its use as a medicine.

[0054] More particularly, according to the invention, said hybrid molecules are intended to target and lyse, in the patient's body, by ADCC and / or phagocytosis and / or complement activation, all cells expressing autoreactive lymphocyte receptors in the context of autoimmune vasculitis: namely, B cells (lymphocytes) expressing on their surface BCRs recognizing at least one of the peptides of the invention (autoantibodies responsible for autoimmune vasculitis, binding to at least one peptide according to the invention) and T lymphocytes expressing on their surface TCRs recognizing at least one of the peptides according to the invention. Indeed, the hybrid molecule according to the invention binds to these cells via the peptide: it is the epitope target of said autoreactive lymphocyte receptor.The hybrid molecule according to the invention also binds to cells, enabling the destruction of B and / or T lymphocytes expressing on their surface the autoreactive lymphocyte receptors involved in autoimmune vasculitis thanks to its Fc fragment, a natural ligand of Fc receptors (for example Fc-gamma receptor (FcyR) if the Fc fragment is derived from an IgG), present in particular on the surface of macrophages but also of NK cells (“Natural Killers”).

[0055] According to a particular embodiment, the invention relates to a hybrid molecule, as defined above, for use in the treatment of autoimmune vasculitis, in particular granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, and micropolyangiitis. According to a preferred embodiment, the invention relates to a hybrid molecule, as defined above, for use in the treatment of granulomatosis with polyangiitis.

[0056] According to a particular embodiment, the invention relates to a hybrid molecule as previously defined and comprising a peptide selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: SEQ ID: SEQ ID: 7, SEQ ID NO : 8, SEQ ID NO : 9, SEQ ID NO : 10, SEQ ID NO : 11, SEQ ID NO : 12, SEQ ID NO : 13, SEQ ID NO : 14, SEQ ID NO : 15, SEQ ID NO : 16, SEQ ID NO : 17, SEQ ID NO : 18, SEQ ID NO : 19, SEQ ID NO : 19 SEQ ID NO : 21, SEQ ID NO : 22, SEQ ID NO : 23, SEQ ID NO : 31, SEQ ID NO : 32, SEQ ID NO: 33, SEQ ID NO : 34, SEQ ID NO : 35, SEQ ID NO : 36, SEQ ID NO : 37, SEQ ID NO : 38, SEQ ID NO : 39, SEQ ID NO : 39 SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51, for its use in a translational vessel autoimmune, preferably granulomatosis with polyangiitis.

[0057] According to one embodiment, the invention also relates to a pharmaceutical composition maceutic comprising a hybrid molecule according to any one of the preceding claims, in combination with a pharmaceutically acceptable vehicle.

[0058] According to the invention, "a pharmaceutically acceptable vehicle" means any formulation that makes the composition suitable for administration to a patient, in any pharmaceutical form.

[0059] The present invention also relates to a method of treating autoimmune vasculitis, in particular granulomatosis with polyangiitis, comprising administering a therapeutically effective amount of a hybrid molecule according to the invention.

[0060] The invention also relates to the use of a hybrid molecule according to the invention for the preparation of a drug intended for the treatment of autoimmune vasculitis, in particular granulomatosis with polyangiitis.

[0061] According to another embodiment, the hybrid molecule according to the invention can be coupled to at least one radioisotope or to at least one fluorochrome, such as A488 or A647. Such molecules can advantageously be used as molecular tracer tools.

[0062] According to another embodiment, the invention relates to the in vitro or ex vivo use of a hybrid molecule comprising at least one Fc fragment of an antibody covalently linked to at least one peptide that binds to a self-reactive lymphocyte involved in autoimmune vasculitis, optionally at least one spacer being present between said Fc fragment and said peptide, as a molecular tool. Such constructs can, in particular, be used to analyze the binding of hybrid molecules to autoantibodies and to Fc receptors of macrophages and NK cells, as well as to analyze the reactivity of macrophages and NK cells to the binding of the hybrids followed by their bridging by autoantibodies.... The radioisotopes and / or fluorochromes are preferably coupled to the Fc fragment, more particularly at the level of the Qtag (if present) or at the level of the lysines.

[0063] Method for producing hybrid molecules according to the invention

[0064] In another aspect, the invention also relates to a process for obtaining a hybrid molecule as defined above.

[0065] According to one embodiment, the invention thus relates to a process for producing a hybrid molecule as defined above, comprising the following steps: - (i) obtaining an azide coupled to an Fc fragment or obtaining an alkyne coupled to an Fc fragment, - (ii) obtaining an alkyne coupled to a peptide or obtaining an azide coupled to a peptide, - (iii) formation of the covalent bond between the azide and the alkyne, - step (i) can be carried out before or after step (ii), or concomitantly.

[0066] According to one embodiment, the invention thus relates to a process for producing a hybrid molecule as defined above, comprising the following steps: - (i) coupling of at least one azide and one Fc fragment, possibly in the presence of at least one spacer, or coupling of at least one alkyne and at least one Fc fragment, possibly in the presence of a spacer, - (ii) coupling of an alkyne and a peptide, possibly in the presence of a spacer, or coupling of an azide and a peptide, possibly in the presence of a spacer, - (iii) formation of the covalent bond between the azide and the alkyne, - step (i) can be carried out before or after step (ii), or concurrently.

[0067] According to another embodiment, the invention thus relates to a process for producing a hybrid molecule as defined above, comprising the following steps: - (i) coupling of at least one azide to each monomer of the Fc fragment, possibly in the presence of at least one spacer, or coupling of at least one alkyne to each monomer of the Fc fragment, possibly in the presence of a spacer, - (ii) coupling of an alkyne and a peptide, possibly in the presence of a spacer, or coupling of an azide and a peptide, possibly in the presence of a spacer, - (iii) formation of the covalent bond(s) between the azide and the alkyne, - step (i) being able to be carried out before or after step (ii), or concomitantly.

[0068] According to one embodiment, the hybrid molecules according to the invention can also be obtained by bioproduction methods and encoded by recombinant DNA encoding the peptide according to the invention and the Fc fragment, said peptide and said Fc fragment being optionally separated by a polypeptide linker. In such a case, the peptide of the hybrid molecule is unmodified (e.g., uncyclized, non-retro, non-retro-inverso), and may be N-terminally or C-terminally oriented. The peptide may be C-terminal or N-terminal to the Fc fragment, the cDNA including its nucleotide coding sequence upstream or downstream of the Fc cDNA.

[0069] The sequences of the invention are represented in Table 1 below.

[0070] [Tables 1] Sequence Number / Sequence Name (if applicable) Sequence SEQ ID NO: 1 VVLGAHNV SEQ ID NO: 2 VLGAHNVR SEQ ID NO: 3 LGAHNVRT SEQ ID NO: 4 GAHNVRTQ SEQ ID NO: 5 PHSRPYMA SEQ ID NO: 6 QPHSRPYM SEQ ID NO: 7 HSRPYMAS SEQ ID NO: 8 FLNNYDAE SEQ ID NO: 9 CFGDSGGP SEQ ID NO: 10 MAHRPPSP SEQ ID NO: 11 AHRPPSPA SEQ ID NO: 12 HRPPSPAL SEQ ID NO: 13 AQPHSRPY SEQ ID NO: 14 PGSHFCGG SEQ ID NO : 15 PVPHGTQC SEQ ID NO : 16 FCRPHNI SEQ ID NO : 17 PRRKAGI SEQ ID NO : 18 ATVQLPQ SEQ ID NO : 19 QLPQQDQ SEQ ID NO : 20 PVPHGTQ SEQ ID NO : 21 QPHSRPY SEQ ID NO : 22 RVGAHDP SEQ ID NO : 23 IDSFVIW SEQ ID NO : 24 / Fragment Fc APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ ID NO : 25 / Fragment Fc + Qtag LLQGARSDATHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK SEQ ID NO : 26 / Fragment Fc + Qtag bis AHGHGHGLLQGARSDATHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO : 27 / (Exemple de Qtag) LLQG SEQ ID NO : 28 / Séquence de la PR3 MAHRPPSPALASVLLALLLSGAARAAEIVGGHEAOPHSRPYM ASLOMRGNPGSHFCGGTLIHPSFVLTAAHCLRDIPORLVNVVL GAHNVRTQEPTQQHFSVAQVFLNNYDAENKLNDVLLIQLSSP ANLSASVATVOLPOODOPVPHGTOCLAMGWGRVGAHDPPAO VLOELNVTVVTFFCRPHNICTFVPRRKAGICFGDSGGPLICDGII QGIDSFVIWGCATRLFPDFFTRVALYVDWIRSTLRRVEAKGRP SEQ ID NO : 29 / Séquence de la MPO MGVPFFSSLRCMVDLGPCWAGGLTAEMKLLLALAGLLAILAT PQPSEGAAPAVLGEVDTSLVLSSMEEAKQLVDKAYKERRESIK QRLRSGSASPMELLSYFKQPVAATRTAVRAADYLHVALDLLE RKLRSLWRRPFNVTDVLTPAQLNVLSKSSGCAYQDVGVTCPE QDKYRTITGMCNNRRSPTLGASNRAFVRWLPAEYEDGFSLPY GWTPGVKRNGFPVALARAVSNEIVRFPTDQLTPDQERSLMFM QWGQLLDHDLDFTPEPAARASFVTGVNCETSCVQQPPCFPLKI PPNDPRIKNQADCIPFFRSCPACPGSNITIRNQINALTSFVDASM VYGSEEPLARNLRNMSNQLGLLAVNQRFQDNGRALLPFDNLH DDPCLLTNRSARIPCFLAGDTRSSEMPELTSMHTLLLREHNRL ATELKSLNPRWDGERLYQEARKIVGAMVQIITYRDYLPLVLGP TAMRKYLPTYRSYNDSVDPRIANVFTNAFRYGHTLIQPFMFRL DNRYQPMEPNPRVPLSRVFFASWRVVLEGGIDPILRGLMATPAKLNRQNQIAVDEIRERLFEQVMRIGLDLPALNMQRSRDHGLPG YNAWRRFCGLPQPETVGQLGTVLRNLKLARKLMEQYGTPNNI DIWMGGVSEPLKRKGRVGPLLACIIGTQFRKLRDGDRFWWEN EGVFSMQQRQALAQISLPRIICDNTGITTVSKNNIFMS NSY-PRDFVNCSTLPALNLASWREAS SEQ ID NO: 30 HGTQCLAMGWGRVGAH SEQ ID NO: 33 NICTFVPRRKAGIC SEQ ID NO: 34 SRPYMA SEQ ID NO: 35 ATRLFPDFFTRVALY SEQ ID NO: 36 VEPKVKSKKREAVAGRGRGRGRGRGRGRGGPRR SEQ ID NO: 37 VAGRGRGRGRGRGRGRGRGGPRR SEQ ID NO: 38 MPELTSMHTLLLREH SEQ ID NO: 39 PILRGLMATPAKLNR SEQ ID NO: 40 DHGLPGYNAWR SEQ ID NO: 41 FPTDQLTPDQER SEQ ID NO: 42 IANVFTNAFR SEQ ID NO: 43 IGLDLPALNMQR SEQ ID NO: 44 IPCFLAGDTR SEQ ID NO: 45 LFEQVMR SEQ ID NO: 46 NNIFMSNTYPR SEQ ID NO :47 RKIVGAMVQIITY SEQ ID NO: 48 RSPTLGASNR SEQ ID NO: 49 VFFASWR SEQ ID NO: 50 VVLEGGIDPILR SEQ ID NO: 51 RKIVGAMVQIITYRD

[0071] [Table 1]. Summary table of the sequences of the invention

[0072] Other features, details and advantages of the invention will become apparent from reading the accompanying Figures and examples which illustrate the invention and are in no way intended to limit it. Brief description of the Figures Fig. 1

[0073] [Fig.1] represents the diagram of an example of a hybrid molecule according to the invention.

[0074] A spacer (which is optional) is represented between the Fc fragment of antibody and the peptide binding to a self-reactive lymphocyte involved in autoimmune vasculitis (referred to as "peptide according to the invention"). Fig. 2

[0075] [Fig.2] represents a B lymphocyte expressing BCR / autoantibodies on its surface transmembrane molecules linked to a hybrid molecule according to the invention, said hybrid molecule being itself linked by the Fc fragment to an NK cell.

[0076] The NK cell will thus be able to destroy the B lymphocyte by ADCC. More specifically, this figure shows a B cell (or B lymphocyte) that expresses on its surface a BCR (F autoantibody ANCA) which interacts specifically with the peptide according to the invention carried by the hybrid molecule. The engagement of the Fc fragment carried by the same hybrid molecule with FcyRIIIa (CD16a) expressed on the surface of an NK cell will activate ADCC and induce the specific destruction of the "ANCA-positive" B lymphocyte (which expresses an ANCA). (ADCC, Antibody-Dependent Cell Cytotoxicity; BCR, B-Cell Receptor; FcyR, Fc-gamma Receptor; NK cell, Natural Killer cell).

[0077] Note: a similar representation could be achieved by replacing the B lymphocyte with a T lymphocyte, and the BCR with a TCR (T-Cell Receptor). Fig. 3

[0078] [Fig.3] represents a B lymphocyte expressing transmembrane BCRs on its surface branes linked to a hybrid molecule according to the invention, said hybrid molecule being itself linked by the Fc fragment to a macrophage.

[0079] The macrophage will thus be able to destroy the B lymphocyte by phagocytosis. More specifically, this figure shows B cells (or B lymphocytes) that express a BCR (F autoantibody ANCA) on their surface and that interact specifically with peptides according to the invention carried by the hybrid molecules. The engagement of Fc fragments carried by these same hybrid molecules with different FcyRs expressed on the surface of a macrophage will activate ADCP, i.e. phagocytosis, and induce the specific destruction of "ANCA positive" B lymphocytes. (ADCP, Antibody-Dependent Cellular Phagocytosis; BCR, B-Cell Receptor; FcyR, Fc-gamma Receptor).

[0080] Nb: a similar representation could be achieved by replacing the B lymphocyte with a T lymphocyte, and the BCR with a TCR (T-Cell Receptor). Fig. 4

[0081] [Fig.4] represents a process for manufacturing a hybrid molecule according to the invention, comprising the derivation and conjugation scheme of the Fc fragments.

[0082] Examples

[0083] Example 1: Example of the production of a hybrid molecule according to the invention

[0084] The hybrid molecule described herein comprises the following construct: an Fc fragment covalently linked to at least one PEGn spacer, itself coupled to an azide, said azide being covalently linked to an alkyne, which is itself coupled to a PEGn spacer linked to a peptide according to the invention. Such a molecule can be read as: Fc-PEGn-N3-DBCO-PEGn-peptide. The two PEGn fragments may be identical or different (for example, the first PEGn fragment is PEG3 and the second PEGn fragment is PEG2).

[0085] 1. Synthesis of an NH2-PEGn-N3 (i.e., a spacer coupled to an azide at one end) and possessing a free amine function at the other end).

[0086] 2. In the presence of NH2-PEGn-N3, with an Fc fragment possessing a Qtag, and A transglutaminase is used, preferably for 16 hours at 37°C. This so-called "derivatization" step is performed, for example, with 20 times more moles of NH2-PEGn-N3 than moles of Fc fragment. The transglutaminase is used, for example, at a concentration of 15 U / pmol per Qtag present (on one or both monomers). Optionally, desalting can be performed to remove excess spacer not bound to the Fc fragment at the end of the step. An Fc-PEGn-N3 is thus obtained. If the Fc fragment has two Qtags (one on each monomer), then the Fc fragment can carry two PEGn-N3s.

[0087] 3. Synthesis of a Cys-PEGn-peptide (i.e., a spacer bound to a peptide at one end) and possessing a free cysteine ​​at the other end). An alkyne, for example a DBCO, is then coupled to the Cys-PEGn-peptide at the cysteine, and a DBCO-PEGn-peptide is thus obtained.

[0088] 4. Implementation of the "click": coupling between the N3 and the DBCO. The DBCO- PEGn-peptide is reacted with Fc-PEGn-N3, preferably with 10 times more moles of DBCO-PEGn-peptide than moles of Fc-PEGn-N3. The click reaction occurs primarily at room temperature and is almost complete after 4 hours.

[0089] 5. Obtaining the hybrid molecule: Fc-PEGn-N3-DBCO-PEGn-peptide. This method The implementation is illustrated in [Fig.4].

[0090] Similarly, an Fc-PEGn-DBCO and an N3-PEGn-peptide can be obtained, then coupled to obtain Fc-PEGn-DBCO-N3-PEGn-peptide.

[0091] Similarly, an Fc-PEGn-DBCO and an N3-peptide can be obtained, or an Fc-PEGn-N3 and a DBCO-peptide, then coupled to obtain respectively Fc-PEGn-DBCO-N3-peptide or Fc-PEGn-N3-DBCO-peptide.

[0092] Example 2: Reactivity of sera from patients with autoimmune vasculitis to the hybrid molecules according to the invention

[0093] The sera used are sera from patients with autoimmune vasculitis, more particularly granulomatosis with polyangiitis, which contain specific autoantibodies.

[0094] ELISA microtitration plate wells were coated by passive adsorption using 100 pL of a solution containing either a peptide selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: : 7, SEQ ID NO : 8, SEQ ID NO : 9, SEQ ID NO : 10, SEQ ID NO : 11, SEQ ID NO : 12, SEQ ID NO : 13, SEQ ID NO : 14, SEQ ID NO : 15, SEQ ID NO : 16, SEQ ID NO : 17, SEQ ID NO : 18, SEQ ID NO : 18 20, SEQ ID NO : 21, SEQ ID NO : 22, SEQ ID NO : 23, SEQ ID NO : 31, SEQ ID NO : 32, SEQ ID NO: 33, SEQ ID NO : 34, SEQ ID NO : 35, SEQ ID NO : 36, SEQ ID NO : 37, SEQ ID NO : 38, SEQ ID NO : 39, SEQ ID NO : 39 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51, only a hybrid mill. the invention,whose Fc fragment is wild-type (WT) or the Fc fragment includes the GASDIE mutation. Such a hybrid molecule is for example "FcWT-PEG-SEQ ID NO: 1-23 / 31-51", "FcLALAPG-PEG-SEQ ID NO: 1-23 / 31-51" or "FcGASDIE-PEG-SEQ ID NO: 1-23 / 31-51". “FcWT-PEG-SEQ ID NO: 1-23 / 31-51” represents a wild-type Fc fragment that is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne DBCO coupled to a PEGn spacer which is itself linked to a peptide represented by any one of the SEQ ID NO: 1 to SEQ ID NO: 23 or SEQ ID NO: 31 to SEQ ID NO: 51, “FcWT-LALAPG-SEQ ID NO: 1-23 / 31-51” represents an Fc fragment comprising the L234A mutations,L235A and P329G, which is linked to at least one PEGn spacer, which is itself coupled to an azide covalently linked to a cycloclooctyne DBCO coupled to a PEGn spacer, which is itself linked to a peptide represented by any one of SEQ ID NO: 1 to SEQ ID NO: 23 or SEQ ID NO: 31 to SEQ ID NO: 51, and "FcGASDIE-PEG-SEQ ID NO: 1-23 / 31-51" represents an Fc fragment comprising the mutations G236A, S239D, and I332E ...51. NO: , 31 to SEQ ID NO: 51. In the constructions, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8.

[0095] These solutions were each used at a concentration of 5 pg / mL in PBS (Phosphate Buffered Saline) buffer and incubated overnight at 4°C. The selected peptides and the hybrid molecules constructed with said peptides, synthesized in a random (scramble) sequence, were used at the same concentration as negative controls. After washing with 0.05% PBS Tween, the wells were then saturated with 5% milk for 1 h at room temperature. After washing, the sera were incubated in serial dilution in PBS + 5% milk buffer for 1 h at room temperature. After washing, the reactivity of the sera was detected using an anti-Fab human IgG HRP-coupled detection antibody diluted 1 / 2500 in 5% milk PBS buffer, also incubated for 1 h at room temperature. After washing, the presence of autoantibodies recognized by the hybrid molecule is revealed by adding TMB and stopping the enzymatic reaction by adding H2SO4 and reading with a spectrophotometer at 450nm..

[0096] The results are expressed in ADO (Optical Delta Density), corresponding to the OD obtained with a scramble peptide hybrid or with the hybrid molecule constructed with said peptide.

[0097] The results show a dose-dependent reactivity of the sera to the peptides with respect to the hybrid molecules. They show that, when included in the different hybrids, the peptides remain perfectly reactive to sera containing autoantibodies.

[0098] Example 3: Kinetics of binding of hybrid molecules to macrophages, at physiological temperature

[0099] Human macrophages, differentiated in vitro in the presence of M-CSF (100 ng / mL) from CD14+ monocytes isolated from the peripheral blood of a healthy subject, were incubated at 500,000 cells / well in the presence of hybrid molecules at 5 pg / mL. The hybrid molecules are, for example, those described in Example 2.

[0100] The hybrid molecules are incubated for 2h, 8h, 16h, 24h and 48h at 37°C. The binding of the hybrid molecules to the surface of macrophages is demonstrated and quantified by cytofluorimetry (FACS Canto II) after incubation of the macrophages with an anti-Fc antibody coupled to FITC, used 1 / 1000.

[0101] “NM” and “ANTI FC” respectively represent the well containing the ma Crophages incubated without antibodies or with the anti-Fc antibody alone. "Wt", "LALAPG" and "GASDIE" represent respectively the well containing the hybrid molecule with the wild-type Fc fragment or with the LALAPG mutation or with the GASDIE mutation.

[0102] The results show that the hybrid molecules, at the physiological temperature of 37°C, bind to the macrophage membrane. These results also show that the hybrid containing an Fc fragment with the GASDIE mutation binds to the macrophage membrane. is superior to that of the wild form.

[0103] Example 4: Phagocytosis induced by the Fc-WT hybrid or the Fc-GASDIE hybrid when B lymphocytes are armed via their FcyRIIb (CD32b) in the presence of sera

[0104] Freshly purified human B lymphocytes from peripheral blood of healthy donors by CD19+ magnetic sorting were labeled with PKH-67 green (Paul Karl Horan / fluorescent lipid marker) and then incubated with sera from patients with autoimmune vasculitis at a concentration of 5 µg / mL for 30 min at 37°C. Subsequently, the cells were washed and then incubated with the hybrid according to the invention comprising a wild-type (Wt) Fc fragment or comprising the GASDIE mutations. The hybrid molecules are, for example, those described in Example 2. The cells were incubated with the hybrids at 10 µg / mL for 30 min at 37°C. After washing, the cells were incubated with macrophages (labeled with PKH-26 red), at a ratio of 1 B lymphocyte per macrophage, for 2 h at 37°C. The cells were then detached and analyzed by flow cytometry.Cells exhibiting dual fluorescence (PKH-67 green / PKH-26 red) correspond to macrophages that have phagocytosed lymphocytes. The different cell populations are expressed as a percentage of the total cell population: percentage of phagocytosis and percentage of B lymphocytes.

[0105] The results obtained confirm that phagocytic activity is increased in the presence of the hybrids (increased phagocytosis associated with a decrease in the B lymphocyte population). Indeed, a significant increase in the percentage of phagocytosis, again associated with a significant decrease in the B lymphocyte population, was observed when the B lymphocytes were coated with the hybrid.

[0106] Example 5: Stability of the binding of Fc-GASDIE A488 and Fc-GASDIE peptide hybrids to NK cells after 30 min, 24 h and 48 h at physiological temperature

[0107] NK cells were incubated for 30 min, 24 h, or 48 h at 37°C in the presence of FcGASDIE-N3-DBCO-A488 (an Fc fragment comprising the G236A, S239D, and I332E mutations that is linked to at least one PEGn spacer that is itself coupled to an azide covalently linked to a DBCO, the molecule being labeled with fluorochrome A488) or FcGASDIE-N3-DBCO-PEG3-peptide-lysineA488 (an Fc fragment comprising the G236A, S239D, and I332E mutations that is linked to at least one PEGn spacer that is itself coupled to an azide covalently linked to a DBCO that is coupled to a PEGn spacer linked to a peptide according to the invention (represented by any one of the SEQ ID NO: 1 to SEQ ID NO: 23 or SEQ ID NO: 31 to SEQ ID NO: 51) the molecule being labeled with the fluorochrome A488 which is bound to the molecule via a lysine). The binding of these two fluorescent probes to NK cells was analyzed by cytofluorimetry. In the constructs, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4, or 8.

[0108] The results are presented at 30 minutes, 24 hours and 48 hours. NM represents unlabeled NK cells, without antibodies.

Claims

Demands

1. Hybrid molecule comprising at least one Fc fragment of antibody covalently linked to at least one peptide binding to an autoreactive lymphocyte involved in autoimmune vasculitis, at least one spacer being optionally present between said Fc fragment and said peptide, and wherein said peptide comprises all or part of the polypeptide sequence of proteinase 3, all or part of the polypeptide sequence of myeloperoxidase and / or all or part of the polypeptide sequence of small nuclear ribonucleoprotein SmDl.

2. Hybrid molecule according to the preceding claim, wherein the sequence is of human origin.

3. Hybrid molecule according to any one of the preceding claims, wherein said spacer is a polymer containing one or more repeating motifs containing the ether group, said spacer preferably being polyethylene glycol of formula PEGn, in which n represents an integer between 1 and 100, preferably between 1 and 10 and in particular 1, 2, 3, 4 or 8. [Revendication 4] Molécule hybride selon l’une quelconque des revendications 1-2, dans laquelle ledit peptide est choisi dans le groupe constitué par : SEQ ID NO : 1, SEQ ID NO : 2, SEQ ID NO : 3, SEQ ID NO : 4, SEQ ID NO : 5, SEQ ID NO : 6, SEQ ID NO : 7, SEQ ID NO : 8, SEQ ID NO : 9, SEQ ID NO : 10, SEQ ID NO : 11, SEQ ID NO : 12, SEQ ID NO : 13, SEQ ID NO : 14, SEQ ID NO : 15, SEQ ID NO : 16, SEQ ID NO : 17, SEQ ID NO : 18, SEQ ID NO : 19, SEQ ID NO : 20, SEQ ID NO : 21, SEQ ID NO : 22, SEQ ID NO : 23, SEQ ID NO : 31, SEQ ID NO : 32, SEQ ID NO: 33, SEQ ID NO : 34, SEQ ID NO : 35, SEQ ID NO : 36, SEQ ID NO : 37, SEQ ID NO : 38, SEQ ID NO : 39, SEQ ID NO : 40, SEQ ID NO : 41, SEQ ID NO : 42, SEQ ID NO : 43, SEQ ID NO : 44, SEQ ID NO : 45, SEQ ID NO : 46, SEQ ID NO : 47, SEQ ID NO : 48, SEQ ID NO : 49, SEQ ID NO : 50 et SEQ ID NO :

51.

5. Hybrid molecule according to any one of the preceding claims, wherein said Fc fragment is a human Fc fragment, in particular of IgG, more particularly of IgGl.

6. Hybrid molecule according to any one of the preceding claims, wherein said Fc fragment is wild-type or mutated, said mutated Fc fragment preferably comprising at least the The following mutations: - L234A and L235A, or - L234A, L235A and P329G, or - G236A, S239D and I332E, or - G236A, S239D, A330L and I332E, or - S239D, H268F, S324T and I332E, the numbering being indicated in the sequence of a human IgGl according to the EU index.

7. Hybrid molecule according to any one of the preceding claims, wherein: - the Fc fragment is coupled to at least one azide and said peptide is coupled to an alkyne, such as a cyclooctyne, and in particular DBCO, or - the Fc fragment is coupled to at least one alkyne, such as a cyclooctyne, and in particular DBCO, and said peptide is coupled to an azide, or - the Fc fragment is coupled to at least one azide and said peptide is linked to a spacer itself coupled to an alkyne, such as a cyclooctyne, and in particular DBCO or - the Fc fragment is coupled to at least one alkyne, such as a cyclooctyne, and in particular DBCO, and said peptide is linked to a spacer itself coupled to an azide or - the Fc fragment is linked to at least one spacer, itself coupled to an azide, and said peptide is coupled to an alkyne, such as a cyclooctyne, and in particular DBCO or - the Fc fragment is linked to at least one spacer, itself coupled to an alkyne, such as a cyclooctyne, and in particular DBCO, and said peptide is coupled to an azide or - the Fc fragment is linked to at least one spacer, itself coupled to an azide, and said peptide is linked to a spacer, itself coupled to an alkyne, such as a cyclooctyne, and in particular DBCO or - the Fc fragment is linked to at least one spacer, itself coupled to an alkyne, such as a cyclooctyne, and in particular DBCO, and said peptide is linked to a spacer, itself coupled to an azide, the covalent bond between said Fc fragment and said peptide, possibly in the presence of one or more spacers, being created between the azide and the alkyne.

8. Hybrid molecule according to any one of the preceding claims, for use as a medicinal product, in particular for use in the treatment of autoimmune vasculitis, by ticular a granulomatosis with polyangiitis.

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