Hybrid molecule comprising an antibody fc portion and at least one peptide binding to a self-reactive lymphocyte involved in autoimmune vasculitis, and uses thereof

Abstract

The invention relates to a hybrid molecule comprising at least one antibody Fc portion covalently bound to at least one peptide binding to a self-reactive lymphocyte involved in autoimmune vasculitis, to the uses of such a hybrid molecule, and to a method for producing same.

Description

Title: Hybrid molecule comprising an antibody Fc fragment 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 antibody Fc fragment covalently linked to at least one peptide binding to an autoreactive lymphocyte involved in autoimmune vasculitis, the uses of such a hybrid molecule, as well as its production method. Said autoreactive lymphocyte receptor is a membrane receptor on the surface of a B lymphocyte (BCR) or on the surface of a T lymphocyte (TCR). Background to the invention

[0002] Autoimmune vasculitis is a rare disease characterized by inflammation of the walls of the body's small vessels, particularly 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: polymorphonuclear 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 (eg 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(1):147-59. doi: 10.1046 / j.1523-1755.2001.00475.x. PMID: 11135067). These autoantibodies therefore represent a therapeutic target of choice.

[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 the small nuclear ribonucleoprotein SmD1.

[0006] Autoimmune vasculitis (or "ANCA-positive vasculitis" or ANCA-associated vasculitis) mainly includes three diseases: granulomatosis with polyangiitis (sometimes called Wegener's disease), 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) that results in tissue ischemia. Eosinophilic granulomatosis with polyangiitis is defined as a systemic vasculitis of small vessels that is also characterized by asthma, blood and tissue eosinophilia, and micropolyangiitis is defined as a systemic inflammatory necrotizing vasculitis that primarily affects small vessels of multiple organs (small arteries, arterioles, capillaries, venules).

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

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

[0009] The present invention is based on the Inventors' research showing that it is possible to target self-reactive 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 self-reactive 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 a self-reactive lymphocyte involved in autoimmune vasculitis and (ii) a human immunoglobulin Fc fragment.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 fixation of the Fc fragment on the 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 that themselves secrete said autoantibodies and / or autoreactive T lymphocytes that are sources of pro-inflammatory cytokines, the hybrid molecules of the invention thus aim to eliminate these “pathogenic” autoantibodies from the patients’ bodies. Statement 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 antibody Fc fragment 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. The scheme of such a construction is presented in Figure 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 antibody Fc fragment and said peptide.

[0014] According to the invention, an antibody "Fc fragment" means the constant region of an immunoglobulin excluding the first immunoglobulin constant region domain (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 can be polar or non-polar.

[0016] According to the invention, a "spacer" is a linking agent that makes it possible to covalently link an antibody Fc fragment to said peptide binding to an autoreactive lymphocyte involved in autoimmune vasculitis, while distancing said Fc fragment from said peptide (thus reducing any possible steric hindrance). It may be any molecule, and in particular a peptide or a polypeptide. Preferably, the spacer does not modify the physicochemical properties of the hybrid molecule.

[0017] The presence of at least one spacer is advantageous: it facilitates the independent accessibility of the two partners of the hybrid molecule (the Fc fragment is more easily accessible to bind to the Fc receptors, just as said peptide is more easily accessible to bind to auto-reactive lymphocytes), and / or stabilizes the hybrid molecule, and / or increases 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. According to 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 itself being covalently linked to said peptide. According to 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 itself being covalently linked to a second spacer and the second spacer is itself covalently linked to said peptide. The bond 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 binding to an autoreactive lymphocyte involved in autoimmune vasculitis. This means that the Fc fragment may be linked to one or two peptides. Indeed, the Fc fragment comprises two monomers, and the Fc fragment may thus be covalently linked to a peptide on only one of the two monomers, or the Fc fragment may be covalently linked to a peptide on each monomer. Preferably, when two peptides are linked on 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 may 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, ie; 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 term "self-reactive lymphocyte receptor involved in autoimmune vasculitis" means a receptor on the surface of a T lymphocyte and / or a B lymphocyte that recognizes a self-peptide involved in the activation of cells leading to autoimmune vasculitis by inappropriate production of autoantibodies or pro-inflammatory cytokines. More particularly, said lymphocyte receptor is a B lymphocyte receptor (BCR) and / or a T lymphocyte receptor (TCR). The binding of the peptide to the BCR and / or the TCR is thus involved in the development of autoimmune vasculitis. For example, and according to one embodiment, the self-peptide involved may comprise all or part of proteinase 3. and / or an "autoantibody responsible for autoimmune vasculitis" means an "anti-neutrophil cytoplasmic autoantibody (ANCA).Typically, the binding of these ACPNs to their target leads to the activation of neutrophils with increased adhesion to the vascular endothelium, production of oxygen free radicals and release of enzymes that cause tissue damage.

[0022] The term "autoreactive lymphocyte involved in autoimmune vasculitis" means a lymphocyte expressing on its surface a receptor that recognizes a self-peptide involving the activation of cells leading to autoimmune vasculitis by production inappropriate levels of autoantibodies or pro-inflammatory cytokines. Preferably, it is 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 SmD1 (anti-SmD1). 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). According to an even more preferred embodiment, an autoantibody according to the invention is understood to be an autoantibody directed against proteinase 3 (anti-PR3), and the autoantibodies are anti-neutrophil cytoplasm autoantibodies such as anti-PR3 (c-ANCA for cytoplasmic-ANCA).

[0024] The term "autoimmune vasculitis" refers specifically to necrotizing inflammation of small vessels, with little or no immune complex deposits, 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 SmD1 (anti-SmD1).

[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 the small nuclear ribonucleoprotein SmD1, preferably a peptide comprising all or part of the polypeptide sequence of proteinase 3.

[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" means a peptide comprising all or part of the polypeptide sequence of myeloperoxidase. Preferably, said polypeptide sequence of myeloperoxidase 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 SmD1. Preferably, said polypeptide sequence of said SmD1 is represented by SEQ ID NO: 30.

[0029] In one embodiment, a "peptide binding to an autoreactive lymphocyte involved in autoimmune vasculitis" means a peptide recognized by an autoantibody directed against proteinase 3, against myeloperoxidase and / or against small ribonucleoprotein nuclear SmD1 . Such peptides may be obtained from fragments of proteinase, myeloperoxidase and / or small nuclear ribonucleoprotein SmD1 , whether these fragments are natural, recombinant or synthetic. Such peptides may 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 small nuclear ribonucleoprotein SmD1 , 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 ANCA 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 the small nuclear ribonucleoprotein SmD1, and said proteinase 3, myeloperoxidase and / or SmD1 is of mammalian origin and is preferably of human origin. 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 or all or part of the polypeptide sequence of the small nuclear ribonucleoprotein SmD1, and said proteinase 3, myeloperoxidase or SmD1 is of mammalian origin 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 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 so as to improve its reactivity with respect to autoantibodies. For example, the peptides can be cyclized, the peptides can be of the retro type (the L series amino acids are chained in a sequence inverse 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 chained in a sequence inverse to that of the peptide to be reproduced). According to an even more in particular, in said hybrid molecule according to the invention, the terminal carboxyl function (COOH) of said peptide is replaced by a carboxamide function (CONH2).

[0034] According to another embodiment, in said hybrid molecule according to the invention, the peptide can be modified so as to facilitate its synthesis and / or improve its stability, for example by alkylation. According to an even more particular embodiment, in said hybrid molecule according to the invention, the terminal amine function (NH2) 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 to the base.

[0036] According to an even more particular embodiment, in said hybrid molecule according to the invention, said peptide is chosen 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 : 19 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 : 38 SEQ ID NO : 40, SEQ ID NO : 41 , SEQ ID NO : 42, SEQ ID NO : 43, SEQ ID NO : 43 44, SEQ ID NO : 45, SEQ ID NO : 46, SEQ ID NO : 47, SEQ ID NO : 48, SEQ ID NO : 49, SEQ ID NO : 49 NO : 50 and SEQ ID NO : 51.

[0037] According to a further embodiment, in said hybrid molecule according to the invention, said peptide is selected from the group consisting of: SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55.

[0038] 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, SEQ ID NO : 51 , SEQ ID NO : 52, SEQ ID NO : 53, SEQ ID NO : 54 et SEQ ID NO : 55.

[0039] According to an even more particular embodiment, in said hybrid molecule according to the invention, said peptide is chosen 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).

[0040] According to an even more particular embodiment, in said hybrid molecule according to the invention, said peptide is chosen 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, SEQ ID NO: 35, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55 (PR3).

[0041] According to an even 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 (SmD1).

[0042] According to an even more particular embodiment, in said hybrid molecule according to the invention, said peptide is chosen 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).

[0043] According to one embodiment, in said hybrid molecule according to the invention, said Fc fragment is a human Fc fragment, in particular of IgG, more particularly of IgGI. The IgGI can correspond to any allotypic variant, for example G1 m3 or nG1 m17. By way of example, the Fc fragment of IgGI is represented by SEQ ID NO: 24, SEQ ID NO: 25 (Fc + Qtag) or SEQ ID NO: 26 (Fc + Qtag bis).

[0044] 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 an even more particular 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 being indicated in the sequence of a human IgG1 according to the EU index. Such mutations are notably described in the article Bruhns and Jonsson, Immunol Rev. 2015 Nov;268(1):25-51. Preferably, when the hybrid molecule is used in therapy, said mutated Fc fragment comprises at least the mutations GASDIE, GASDALIE, or SDH.

[0045] According to one embodiment, in said hybrid molecule according to the invention, said Fc fragment has a fucosylation level 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, ie; 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%. A low fucosylation of the Fc fragment causes a strong ADCC response. This is why, according to a particular embodiment, said Fc fragment has a fucosylation level of between 0% and 60% of the glycosylated forms, in particular 50%, 40%, 30%, 20%, 10% or 0%. According to the invention, the fucosylation level is defined as the average proportion of fucose carried by the Fc fragment, relative to the maximum quantity of fucose that an Fc fragment can carry.

[0046] 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. According to 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. According to one embodiment, when a spacer is present, the spacer can be linked to the Fc fragment via its N- or C-terminal end. According to 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 N-terminal.Alternatively, said covalent bond between said Fc fragment and said 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 said peptide.

[0047] According to a preferred embodiment, when at least one spacer is present in said hybrid molecule of the invention, it makes it possible to link the Fc fragment to an azide or to an alkyne which will itself be involved in the covalent bond with said peptide. According to one embodiment, when at least one spacer is present in said hybrid molecule of the invention, it can also make it possible to link the peptide to an alkyne or to 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 which makes it possible to link the Fc fragment to an azide or to an alkyne and a second spacer which makes it possible to link the peptide 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).

[0048] According to one embodiment according to the invention, in said hybrid molecule according to the invention, said Fc fragment: - 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 to 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 DBCO, - either linked to a spacer which is itself coupled to an azide or to an alkyne, such as a cyclooctyne, and in particular DBCO, the covalent bond between said Fc fragment and said peptide, optionally in the presence of one or more spacers, being created between the azide and the alkyne.

[0049] According to one embodiment according to 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 Fc fragment and said peptide being created between the azide and the alkyne.

[0050] According to one embodiment according to 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 Fc fragment and said peptide, in the presence of one or more spacers, being created between the azide and the alkyne.

[0051] According to the invention, the creation of the covalent bond between the azide and the alkyne corresponds to a step called "click chemistry", the N3 part of the azide reacting with an alkyne. Azide means salts of hydrazonic acid HN3, or organic azides in which one of the nitrogen atoms is covalently bonded to a carbon atom of an organic compound (e.g. example methyl azide CH3N3). Preferably the azide is represented by the formula N3. Alkyne refers to molecules having the general formula C n H2n-2, and which are characterized by the presence of at least one triple bond. Preferably the alkyne is a cyclooctyne, even more preferably dibenzocyclooctyne (DBCO).

[0052] 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 can also be used to covalently link the Fc fragment to the spacer and / or the peptide to the spacer.

[0053] More specifically, a conjugation technique means enzymatic conjugation or chemical conjugation. An enzymatic conjugation means, for example, conjugation using a transglutaminase that catalyzes the formation of covalent bonds between free amino groups and glutamine or lysine residues or using a transpeptidase such as sortase. For further information on enzymatic conjugation, see, for example, patent applications 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 comprising a glutamyl residue (a Qtag), as represented by SEQ ID NO: 27 (LLQG).A chemical conjugation is understood, for example, to be a covalent bond between an isolated cysteine ​​or one participating in a disulfide bridge after reduction thereof and, for example, a maleimide. An example of such a conjugation is shown in Figure 4. In this example, the Fc fragment comprises a Qtag peptide and said Fc fragment is linked to a spacer (itself coupled to an azide), thanks to the action of transglutaminase which will create a covalent bond between the glutamyl residue of the Qtag and the NH2 group carried by the PEGn spacer.

[0054] According to the invention, the term "coupled" or "molecular coupling" means the establishment of a covalent bond, thus the Fc fragment and / or the peptide and / or the spacer is covalently bonded to an alkyne or an azide. The term "bonded" also means a covalent bond. Thus, by way of example, the expression "the Fc fragment is coupled to an azide and said peptide is bonded to a spacer, itself coupled to an alkyne, such as a cyclooctyne, and in particular DBCO" can also be read "the Fc fragment is covalently bonded to an azide and said peptide is covalently bonded to a spacer, said spacer itself being covalently bonded to an alkyne, such as a cyclooctyne, and in particular DBCO".

[0055] Use of the hybrid molecules according to the invention

[0056] In a second aspect, the present invention also relates to a hybrid molecule as defined above, for its use as a medicament.

[0057] More particularly, according to the invention, said hybrid molecules are intended to target and lyse in the body of patients, 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 the 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 the TCRs recognizing at least one of the peptides according to the invention. Indeed, the hybrid molecule according to the invention binds to these cells thanks to 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 auto-reactive lymphocyte receptors involved in autoimmune vasculitis thanks to its Fc fragment, a natural ligand for 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 NK cells (“Natural Killers”).

[0058] According to a particular embodiment, the invention relates to a hybrid molecule, as defined above, for its 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 its use in the treatment of granulomatosis with polyangiitis.

[0059] 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: 5, SEQ ID NO: 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 : 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 the purposes of its translational use autoimmune, preferably granulomatosis with polyangiitis.

[0060] According to a particular embodiment, the invention relates to a hybrid molecule as defined above and comprising a peptide chosen 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, 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, SEQ ID NO : 51 , SEQ ID NO : 52, SEQ ID NO : 53, SEQ ID NO : 54 et SEQ ID NO : 55, pour son utilisation dans le traitement d’une vascularite auto-immune.

[0061] According to a particular embodiment, the invention relates to a hybrid molecule as defined above and comprising a peptide chosen from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 31, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55, for its use in the treatment of autoimmune vasculitis. According to a particular embodiment, the invention relates to a hybrid molecule as defined above and comprising a peptide chosen from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 31, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55, for its use in the treatment of granulomatosis with polyangiitis (sometimes called Wegener's disease), eosinophilic granulomatosis with polyangiitis (sometimes called Churg and Strauss syndrome) or micropolyangiitis.

[0062] According to one embodiment, the invention also relates to a pharmaceutical composition comprising a hybrid molecule according to any one of the preceding claims, in combination with a pharmaceutically acceptable vehicle.

[0063] According to the invention, “a pharmaceutically acceptable vehicle” means any formulation making the composition suitable for administration to a patient, in any galenic form.

[0064] 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.

[0065] The invention also relates to the use of a hybrid molecule according to the invention for the preparation of a medicament intended for the treatment of autoimmune vasculitis, in particular granulomatosis with polyangiitis.

[0066] According to another embodiment, the hybrid molecule according to the invention can be coupled to at least one radioisotope or at least one fluorochrome, such as A488 or A647. Such molecules can advantageously be used as molecular tracer tools.

[0067] According to another embodiment, the invention thus relates to the in vitro or ex vivo use of a hybrid molecule comprising at least one antibody Fc fragment 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, 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 cells NK, as well as to analyze the reactivity of macrophages and NK cells to the fixation of the hybrids followed by their bridging by the autoantibodies.... The radioisotopes and / or fluorochromes are preferably coupled to the Fc fragment, even more particularly at the level of the Qtag (if present) or at the level of the lysines.

[0068] Process for producing hybrid molecules according to the invention

[0069] In another aspect, the invention also relates to a method for obtaining a hybrid molecule as defined above.

[0070] According to one embodiment, the invention thus relates to a method for producing a hybrid molecule as defined previously, 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) realization of the covalent bond between the azide and the alkyne, - step (i) can be carried out before or after step (ii), or concurrently.

[0071] According to one embodiment, the invention thus relates to a method for producing a hybrid molecule as defined previously, comprising the following steps: - (i) coupling of at least one azide and one Fc fragment, optionally in the presence of at least one spacer, or coupling of at least one alkyne and at least one Fc fragment, optionally in the presence of a spacer, - (ii) coupling of an alkyne and a peptide, optionally in the presence of a spacer, or coupling of an azide and a peptide, optionally in the presence of a spacer, - (iii) realization of the covalent bond between the azide and the alkyne, - step (i) can be carried out before or after step (ii), or concurrently.

[0072] According to another embodiment, the invention thus relates to a method for producing a hybrid molecule as defined previously, comprising the following steps: - (i) coupling of at least one azide to each monomer of the Fc fragment, optionally in the presence of at least one spacer, or coupling of at least one alkyne to each monomer of the Fc fragment, optionally in the presence of a spacer, - (ii) coupling of an alkyne and a peptide, optionally in the presence of a spacer, or coupling of an azide and a peptide, optionally in the presence of a spacer, - (iii) creation of the covalent bond(s) between the azide and the alkyne, - step (i) can be carried out before or after step (ii), or concurrently.

[0073] 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 optionally being separated by a polypeptide linker. In such a case, the peptide of the hybrid molecule is unmodified (e.g., non-cyclized, non-retro, non-retro-inverso), and may be oriented N-terminally or C-terminally. The peptide may be C-terminal or N-terminal to the Fc fragment, with the cDNA including its nucleotide coding sequence upstream or downstream of the Fc cDNA.

[0074] The sequences of the invention are shown in Table 1 below.

[0075] [Table 1] ;00076] [Table 1]. Summary table of the sequences of the invention

[0077] Other characteristics, details and advantages of the invention will appear on reading the attached Figures and the examples which illustrate the invention and are not intended in any way to limit it. Brief description of the Figures Fig. 1

[0078] [Fig. 1] represents the diagram of an example of a hybrid molecule according to the invention.

[0079] A spacer (which is optional) is shown between the antibody Fc fragment and the peptide binding to an autoreactive lymphocyte involved in autoimmune vasculitis (referred to as the “peptide according to the invention”). Fig. 2

[0080] [Fig. 2] represents a B lymphocyte expressing on its surface BCR / transmembrane autoantibodies linked to a hybrid molecule according to the invention, said hybrid molecule itself being linked by the Fc fragment to an NK cell.

[0081] 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) which expresses on its surface a BCR (the ANCA autoantibody) 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 to 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).

[0082] 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. 3

[0083] [Fig. 3] represents a B lymphocyte expressing on its surface transmembrane BCRs linked to a hybrid molecule according to the invention, said hybrid molecule itself being linked by the Fc fragment to a macrophage.

[0084] The macrophage will then be able to destroy the B lymphocyte by phagocytosis. More specifically, this Figure shows B cells (or B lymphocytes) which express on their surface a BCR (the ANCA autoantibody) and which interact specifically with peptides according to the invention carried by the hybrid molecules. The engagement of the Fc fragments carried by these same hybrid molecules to 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).

[0085] 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

[0086] [Fig. 4] represents a method of manufacturing a hybrid molecule according to the invention, comprising the scheme of derivatization and conjugation of the Fc fragments. Fig. 5

[0087] [Fig. 5] represents the reactivity of sera from patients with micropolyangiitis in the presence of peptides. The reactivity of sera in the presence of peptide 80 is presented in Figure 5A, the reactivity of sera in the presence of peptide 81 is presented in Figure 5B, the reactivity of sera in the presence of peptide 82 is presented in Figures 5C and 5H, the reactivity of sera in the presence of peptide 83 is presented in Figure 5D, the reactivity of sera in the presence of peptide 84 is presented in Figures 5E and 5I, the reactivity of sera in the presence of peptide 85 is presented in Figure 5F, and the reactivity of sera in the presence of peptide 86 is presented in Figure 5G. On the graphs, the ordinate represents the OD measured at 450 nm and the abscissa represents the tested serum. The serum is diluted 1 / 25. Fig. 6

[0088] [Fig. 6] represents the reactivity of sera from patients with Wegener's disease in the presence of peptides. The reactivity of sera in the presence of peptide 80 is presented in Figure 6A, the reactivity of sera in the presence of peptide 81 is presented in Figure 6B, the reactivity of sera in the presence of peptide 82 is presented in Figure 6C, the reactivity of sera in the presence of peptide 83 is presented in Figure 6D, the reactivity of sera in the presence of peptide 84 is presented in Figure 6E, the reactivity of sera in the presence of peptide 85 is presented in Figures 6F and 6H, and the reactivity of sera in the presence of peptide 86 is presented in Figure 6G. In the graphs, the ordinate represents the OD measured at 450 nm and the abscissa represents the tested serum. The serum is diluted 1 / 25. Fig. 7

[0089] [Fig. 7] represents the reactivity of sera from patients with Churg-Strauss syndrome in the presence of peptides. The reactivity of sera in the presence of peptide 80 is presented in Figure 7A, the reactivity of sera in the presence of peptide 81 is presented in Figure 7B, the The reactivity of sera in the presence of peptide 82 is shown in Figure 7C, the reactivity of sera in the presence of peptide 83 is shown in Figure 7D, the reactivity of sera in the presence of peptide 84 is shown in Figure 7E, the reactivity of sera in the presence of peptide 85 is shown in Figure 7F, and the reactivity of sera in the presence of peptide 86 is shown in Figure 7G. On the graphs, the ordinate represents the OD measured at 450 nm and the abscissa represents the serum tested. The serum is diluted 1 / 25. Fig. 8

[0090] [Fig. 8] represents the reactivity of sera from healthy patients in the presence of peptides. The reactivity of HD6 serum in the presence of peptides 80-86 is shown in Figure 8A, the reactivity of HD7 serum in the presence of peptides 80-86 is shown in Figure 8B, the reactivity of HD8 serum in the presence of peptides 80-86 is shown in Figure 8C, the reactivity of HD9 serum in the presence of peptides 80-86 is shown in Figure 8D, and the reactivity of HD10 serum in the presence of peptides 80-86 is shown in Figure 8E. On the graphs, the ordinate represents the OD measured at 450 nm and the abscissa represents the tested peptide. The serum is diluted 1 / 25.

[0091] Examples

[0092] Example 1: Example of production of a hybrid molecule according to the invention

[0093] The hybrid molecule described here comprises the following construction: 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: Fc-PEGn-Ns-DBCO-PEGn-peptide. The two PEGns can be identical or different (for example the first PEGn is PEG3 and the second PEGn is PEG2).

[0094] 1. Synthesis of an NH2-PEGn-N3 (i.e. a spacer coupled to an azide at one end and having a free amine function at another end).

[0095] 2. Bringing the NFL-PEGn-Ns together with an Fc fragment having a Qtag and a transglutaminase, preferably for 16 hours at 37°C. This so-called "derivatization" step is, for example, carried out with 20 times more moles of NFL-PEGn-Ns than moles of Fc fragment. The transglutaminase is used, for example at a level of 15U / pmol per Qtag present (on one or both monomers). Optionally, desalting can be carried out to remove the excess spacer not bound to the Fc fragment at the end of the step. An Fc-PEGn-Ns is thus obtained. If the Fc fragment has 2 Qtags (one carried on each monomer), then the Fc fragment can carry two PEGn-N3.

[0096] 3. Synthesis of a Cys-PEGn-peptide (i.e. a spacer linked to a peptide at one end and having 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.

[0097] 4. Realization of the “click”: coupling between N3 and DBCO. The DBCO-PEGn-peptide is placed in the presence of Fc-PEGn-Ns with, preferably, 10 times more moles of DBCO-PEGn-peptide than moles of Fc-PEGn-Ns. The click reaction takes place in particular at room temperature and is almost complete after 4 hours.

[0098] 5. Obtaining the hybrid molecule: Fc-PEGn-Ns-DBCO-PEGn-peptide. This embodiment is illustrated in Figure 4.

[0099] Similarly, an Fc-PEGn-DBCO and an Ns-PEGn-peptide can be obtained, then coupled to obtain Fc-PEGn-DBCO-Ns-PEGn-peptide. [000100] Similarly, an Fc-PEGn-DBCO and an Ns-peptide can be obtained, or an Fc-PEGn-Ns and a DBCO-peptide, then coupled to obtain respectively Fc-PEGn-DBCO-Ns-peptide or Fc-PEGn-Ns-DBCO-peptide. [000101] Example 2: Reactivity of sera from patients suffering from autoimmune vasculitis with respect to the hybrid molecules according to the invention [000102] The sera used are sera from patients suffering from autoimmune vasculitis, more particularly granulomatosis with polyangiitis, which contain specific autoantibodies. [000103] 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, 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 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 : 38 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 , only one molecule hybrid according to the invention, wherein the Fc fragment is wild-type (WT) or the Fc fragment comprises 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 that is itself coupled to an azide covalently linked to a cyclooctyne DBCO coupled to a PEGn spacer that 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, "FcWT-LALAPG-SEQ ID NO: 1-23 / 31-51" represents an Fc fragment comprising the mutations L234A, L235A and P329G that is linked to at least one PEGn spacer that is itself coupled to an azide covalently linked to a cyclooctyne DBCO coupled to a PEGn spacer that 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, which 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 SEQ ID NO: 1 to SEQ ID NO: 23 or SEQ ID NO: 31 to SEQ ID NO: 51. In the constructs, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8. [000104] 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 random sequence (scramble) were used at the same concentration as negative controls. After washing in PBS Tween 0.05% 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 buffer + 5% milk for 1 h at room temperature. After washing, the reactivity of the sera was detected using an anti-human IgG Fab detection antibody coupled to HRP diluted 1 / 2500 in PBS buffer 5% milk 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 on the spectrophotometer at 450nm. [000105] The results are expressed in A DO (Delta-Optical Density), corresponding to the DO obtained with a hybrid peptide scramble or with the hybrid molecule constructed with said peptide. [000106] The results show a dose-dependent reactivity of the sera on the peptides with respect to the hybrid molecules. They show that included in the different hybrids, the peptides remain perfectly reactive to the sera comprising the autoantibodies. [0001071 Example 3: Kinetics of fixation of hybrid molecules on macrophages, at physiological temperature [000108] 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 / MI. The hybrid molecules are, for example, those described in [Example 2. [000109] The hybrid molecules are incubated for 2h, 8h, 16h, 24h and 48h at 37°C. The fixation of the hybrid molecules to the surface of the 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. [000110] “NM” and “ANTI FC” represent respectively the well containing the macrophages incubated without antibody or with the anti-Fc antibody alone. “Wt”, “LALAPG” and “GASDIE” respectively represent the well containing the hybrid molecule with the wild Fc fragment or with the LALAPG mutation or with the GASDIE mutation. [000111] The results show that the hybrid molecules at the physiological temperature of 37°C, bind to the membrane of macrophages. These results also show that the binding of the hybrid comprising an Fc fragment comprising the GASDIE mutation is higher than that of the wild-type form. r000112l 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 [000113] Freshly purified human B lymphocytes from peripheral blood of healthy donors by CD19+ magnetic sorting were labeled with green PKH-67 (Paul Karl Horan / fluorescent lipid marker) and then incubated with sera from patients suffering from autoimmune vasculitis at a concentration of 5 pg / mL for 30 min at 37°C. Subsequently, the cells were washed and then placed in the presence of the hybrid according to the invention comprising a wild-type Fc fragment (Wt) or comprising the GASDIE mutations. The hybrid molecules are, for example, those described in Example 2. The cells were placed in the presence of the hybrids at 10 pg / mL for 30 min at 37°C. After washing, the cells were placed in contact with macrophages (labeled red PKH-26), at a rate of 1 B lymphocyte for 1 macrophage, for 2 h at 37°C. The cells were then detached and analyzed by flow cytometry.Cells showing double 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. [000114] The results obtained confirm that the phagocytosis activity is increased in the presence of the hybrids (increase in phagocytosis associated with a decrease in the B lymphocyte population). Indeed, a significant increase in the percentage of phagocytosis, always associated with a significant decrease in the B lymphocyte population, was observed when the B lymphocytes were covered with the hybrid. [000115] Example 5: Stability of the fixation of Fc-GASDIE A488 and Fc-GASDIE peptide hybrids, on NK cells after 30 min, 24 h and 48 h, at physiological temperature [000116] The 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 mutations G236A, S239D and I332E which is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a DBCO, the molecule being labeled with the fluorochrome A488) or FcGASDIE-N3-DBCO-PEG3-peptide-lysineA488 (an Fc fragment comprising the mutations G236A, S239D and I332E which is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a DBCO which is coupled to a PEGn spacer linked to a peptide according to the invention (represented by any one of 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 linked to the molecule via a lysine). The binding of these 2 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. [000117] Results are presented at 30 minutes, 24 hours and 48 hours following. NM represents unlabeled NK cells, without antibodies. [000118] Example 6: Reactivity of sera with peptides according to the invention [000119] The peptides used herein are the peptides of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 31, SEQ ID NO: 55 and SEQ ID NO: 7, as indicated in Table 1 above. [000120] The peptide represented by SEQ ID NO: 52 corresponds to the peptide named peptide 80 in the examples. The peptide represented by SEQ ID NO: 53 corresponds to the peptide named peptide 81 in the examples. The peptide represented by SEQ ID NO: 54 corresponds to the peptide named peptide 82 in the examples. The peptide represented by SEQ ID NO: 2 corresponds to the peptide named peptide 83 in the examples. The peptide represented by SEQ ID NO: 31 corresponds to the peptide named peptide 84 in the examples. The peptide represented by SEQ ID NO: 55 corresponds to the peptide named peptide 85 in the examples. The peptide represented by SEQ ID NO: 7 corresponds to the peptide named peptide 86 in the examples. [000121] The peptides were synthesized and their reactivity was tested with the serum of different patients. [000122] The sera used here are indicated in Table 2 below. [000123] [Table 2] 00124] [Table 2]. Sera tested [000125] ELISA microtiter plate wells were coated by passive adsorption using 100 pL of a solution containing a peptide represented by SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 31, SEQ ID NO: 55 or SEQ ID NO: 7. [000126] These solutions were each used at a concentration of 10 pg / mL in PBS (Phosphate Buffered Saline) buffer and incubated overnight at 4°C. The next day, the wells were saturated with a blocking buffer with 200 pg / mL per well for 1 h at room temperature. After washing, the sera were incubated, diluted 1 / 25 èmewith a buffer including PBS. They were incubated at 100 pg / mL per well for 2.5 hours at 4°C, with orbital shaking. After washing, the reactivity of the sera was detected using an anti-human IgG Fc detection antibody coupled to HRP, diluted 1 / 5000 in PBS-2% BSA buffer, and also incubated for 1 hour at 4°C with orbital shaking. After washing, the revelation is carried out by adding TMB and stopping the enzymatic reaction by adding sulfuric acid and reading on a spectrophotometer at 450 nm. [000127] The results express the optical density (OD) obtained with each serum and each peptide tested. [000128] The background noise was obtained by following the same protocol described above but without the first peptide coating step. This thus represents the non-specific adhesion of IgG present in the patients' serum, this value being equivalent regardless of the pathology. [000129] The reactivity of peptides 80, 81, 82, 83, 84, 85 and 86 with sera from patients with micropolyangiitis (sera 10, 24, 27, 29, 36 and 45) is shown in Figure 5. [000130] The reactivity of peptides 80, 81, 82, 83, 84, 85 and 86 with sera from patients with Wegener's disease (sera 2, 15, 18 and 36) is shown in Figure 6. [000131] The reactivity of peptides 80, 81, 82, 83, 84, 85 and 86 with sera from patients with Churg-Strauss syndrome (sera 6, 38 and 79) is shown in Figure 7. [000132] The reactivity of peptides 80, 81, 82, 83, 84, 85 and 86 with sera from healthy patients (sera HD6, HD7, HD8, HD9 and HD10) is shown in Figure 8. [000133] The results thus confirm that the sera of healthy donors are not reactive in the presence of the tested peptides. On the contrary, the sera of patients suffering from micropolyangiitis, Wegener's disease or Churg-Strauss syndrome react in the presence of the tested peptides. These results thus illustrate that the peptides according to the invention are recognized by the autoantibodies present in the sera of patients suffering from micropolyangiitis, Wegener's disease or Churg-Strauss syndrome.

Claims

Claims

1. A hybrid molecule comprising at least one antibody Fc fragment 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.

2. Hybrid molecule according to the preceding claim, 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 the small nuclear ribonucleoprotein SmD1.

3. Hybrid molecule according to the preceding claim, in which the sequence is of human origin.

4. Hybrid molecule according to any one of the preceding claims, wherein said spacer is a polymer containing one or more repeating units 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 5] Molécule hybride selon l’une quelconque des revendications précédentes, 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, SEQ ID NO : 51 , SEQ ID NO : 52, SEQ ID NO : 53, SEQ ID NO : 54 et SEQ ID NO :

55.

6. A 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 IgG 1 .

7. A 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 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 IgG1 according to the EU index.

8. A hybrid molecule according to any preceding claim, 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, optionally in the presence of one or more spacers, being created between the azide and the alkyne.

9. Hybrid molecule according to any one of the preceding claims, for its use as a medicament, in particular for its use in the treatment of autoimmune vasculitis.

10. Hybrid molecule for its use according to the preceding claim, for its use in the treatment of granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis or micropolyangiitis.

Images