HYBRID MOLECULE WITH AN ANTIBODY FC FRAGMENT AND AT LEAST ONE CITRULLINATED PEPTIDE FROM FIBRIN AND USES THEREOF

Description

[0001] The present invention relates to a hybrid molecule comprising at least one Fc fragment of antibody covalently linked to at least one fibrin-derived peptide comprising one or more citrullyl residue(s), the uses of such a hybrid molecule, and its production process. Context of the invention

[0002] Rheumatoid arthritis is the most common inflammatory rheumatic disease, and also the most common autoimmune disease. The disease is characterized by chronic inflammation of the synovial joints, leading to irreversible joint destruction.

[0003] The presence of class G autoantibodies directed against citrullinated proteins, known as anti-citrullinated protein autoantibodies (ACPA), is highly specific for rheumatoid arthritis. Patient sera containing ACPA, lymphocytes expressing them on their membranes, and the patients themselves are said to be 'ACPA-positive'. Several studies have demonstrated that these ACPA are central to the autoimmune reactions characteristic of rheumatoid arthritis and thus represent a promising therapeutic target.

[0004] The antigenic targets of ACPAs have been characterized. They are specifically directed against deiminated or citrullinated forms of the α and β polypeptide chains of fibrin, a protein abundant in inflammatory synovial tissue. This citrullination corresponds to the enzymatic deimination of the arginyl residues of the polypeptide chains, under the action of peptidyl-arginine deiminases (PADs).

[0005] More specifically, the immunodominant epitopes recognized by ACPA on the α and β polypeptide chains of fibrin have been characterized and published, notably in applications PCT / FR00 / 01857 or PCT / FR2007 / 000758. The five citrullinated peptides carrying the immunodominant epitopes are more specifically the peptides named α36-50Cit (as represented in SEQ ID NO: 5), α171-185Cit (as represented in SEQ ID NO: 6), α501-515Cit (as represented in SEQ ID NO: 14), α621-635Cit (as represented in SEQ ID NO: 18) and β60-74Cit (as represented in SEQ ID NO: 19). Sera from ACPA-positive patients recognize one or more of these five peptides.

[0006] Secreted into the rheumatoid synovial tissue by local plasma cells, ACPAs are present in high concentrations, close to their main target, citrullinated fibrin, which is also abundant in the interstitial deposits. The binding of ACPAs to these deposits, and thus the formation of immobilized immune complexes, which in turn bind rheumatoid factors—other autoantibodies associated with rheumatoid arthritis and also secreted by local plasma cells—triggers a cascade of pro-inflammatory events.

[0007] The stimulation of macrophage cells by these immune macro-complexes, mainly via their membrane Fcgamma receptors, leads them to secrete pro-inflammatory cytokines and in particular TNF-alpha which has been identified as the main cytokine responsible for rheumatoid inflammation.

[0008] Currently, there is no cure for rheumatoid arthritis. Treatments are only aimed at managing flare-ups and / or preventing their occurrence.

[0009] One object of the present invention is thus to provide a treatment for rheumatoid arthritis.

[0010] ACPAs are oligoclonal and therefore secreted by only a few plasma cell clones, themselves resulting from the differentiation of a few B lymphocyte clones.

[0011] In the case of rheumatoid arthritis, clones of B lymphocytes express on their membrane immunoglobulins carrying the ACPA specificity (these are ACPA-positive B lymphocytes), while the plasma cells resulting from the differentiation of ACPA-positive B lymphocyte clones (these are ACPA-positive plasma cells) secrete these same ACPAs in abundance into their microenvironment.

[0012] The present invention is based on the Inventors' findings that it is possible to target and eliminate B lymphocyte clones expressing ACPA on their surface using a hybrid molecule comprising (i) at least one fibrin-derived peptide having at least one citrullyl residue and (ii) an Fc fragment of human immunoglobulin. These hybrid molecules will specifically target ACPA-positive B lymphocytes (using the fibrin-derived peptide having at least one citrullyl residue, which is recognized by said ACPA), which will then be eliminated, after binding of the Fc fragment 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.

[0013] By targeting B lymphocyte clones expressing ACPAs and cells that differentiate into plasma cells that themselves secrete ACPAs, the hybrid molecules of the invention aim to eliminate ACPAs from the patients' bodies. Description of the invention Hybrid molecule according to the invention

[0014] 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 fibrin-derived peptide having at least one citrullyl residue, at least one spacer being optionally present between said Fc fragment and said peptide, said peptide being recognized by anti-citrullinated protein autoantibodies and being derived from all or part of the α or β chain sequence of a vertebrate fibrin, by substitution of at least one arginyl residue with a citrullyl residue. The diagram of such a construct is shown in Figure 1 .

[0015] According to the invention, a "hybrid molecule" is understood to be a molecule having at least two components of a different nature, in this case the Fc fragment of antibody and said peptide.

[0016] 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).

[0017] 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.

[0018] According to the invention, a "spacer" is a linker that covalently binds an Fc fragment of an antibody to a fibrin-derived peptide having at least one citrullyl residue, 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. Preferably, the spacer does not alter the physicochemical properties of the hybrid molecule.

[0019] 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 to bind to Fc receptors, as is the peptide said, which is more easily accessible to bind to ACPA-positive B lymphocytes), and / or stabilizes the hybrid molecule, and / or increases the solubility of the hybrid molecule.

[0020] According to one embodiment, the hybrid molecule of 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.

[0021] According to one embodiment, the hybrid molecule according to the invention may comprise at least one fibrin-derived peptide having at least one citrullyl residue. This means that the Fc fragment can be linked to one or two peptides. Indeed, the Fc fragment comprises two monomers, and the Fc fragment can thus be covalently linked to a peptide on only one of the two monomers, or the Fc fragment can be covalently linked to a peptide on each monomer. Preferably, when two peptides are linked to the Fc fragment, the two peptides are identical, for example, two peptides of SEQ ID NO: 18 or SEQ ID NO: 19.

[0022] In one embodiment, said spacer is a polymer containing one or more repeating units containing the ether group. In a particular embodiment, said spacer is 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. According to the invention, said polyethylene glycol can be functionalized, for example, with an amine group (PEGn-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.

[0023] According to the invention, the expression "fibrin-derived peptide having at least one citrullyl residue" means a fibrin or fibrinogen molecule in which at least one arginyl residue has been substituted by a citrullyl residue. A "fibrin-derived peptide having at least one citrullyl residue" according to the invention means a peptide recognized by ACPAs and may also be called a "citrullinated peptide." Such peptides can be obtained from natural, recombinant, or synthetic fragments of fibrin or fibrinogen. Such peptides can also be directly synthesized. The amino acids constituting the peptide can be of the L or D series, preferably of the L series. This substitution can, for example, be carried out by an enzymatic deimination step under the action of peptidyl-arginine deiminases (PADs). Such a peptide can also be obtained by directly incorporating one or more citrullyl residues into the synthesized peptide.A peptide according to the invention binds to ACPAs, and the binding between said fibrin-derived peptide having at least one citrullyl residue and an ACPA can be verified using an ELISA test or as described in the publication Sebbag M, Moinard N, Auger I, Clavel C, Arnaud J, Nogueira L, Roudier J, Serre G. Epitopes of human fibrin recognized by the rheumatoid arthritis-specific autoantibodies to citrullinated proteins. Eur J Immunol 36:2250-2263, 2006.

[0024] According to the invention, the peptide is derived from all or part of the α or β chain sequence of a vertebrate fibrin by substituting at least one arginyl residue with a citrullyl residue. Preferably, the peptide is derived from a sequence of at least five consecutive amino acids from the α chain (in particular represented by SEQ ID NO: 27) or β chain (in particular represented by SEQ ID NO: 28) of a vertebrate fibrin. More particularly, the vertebrate fibrin is a mammalian fibrin, preferably human.

[0025] 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 5 consecutive amino acids, and even more preferably between 5 and 25 amino acids. According to the invention, "between 5 and 25" means all the values: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25. Preferably, said peptide has a size of between 10 and 20 amino acids, more particularly 15 amino acids.

[0026] According to one embodiment, in said hybrid molecule according to the invention, the peptide is linear.

[0027] According to one embodiment, in said hybrid molecule according to the invention, the peptide can be modified to improve its reactivity towards ACPAs. By way of example, peptides can be cyclized, or they 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 embodiment, in said hybrid molecule according to the invention, the terminal carboxyl (COOH) group of said peptide is replaced by a carboxamide (CONH2) group. Preferably, in the peptide of SEQ ID NO: 12, the terminal carboxyl (COOH) group of said peptide is replaced by a carboxamide (CONH2) group.As an example, the peptide β60-74Cit (as represented in SEQ ID NO: 19) advantageously exhibits such a carboxamide function.

[0028] 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. In a further particular embodiment, in said hybrid molecule according to the invention, the terminal amine (NH₂) group of said peptide is acetylated. Preferably, in the peptide of SEQ ID NO: 1, the terminal amine group of said peptide is acetylated. By way of example, the peptide α621-635Cit (as shown in SEQ ID NO: 18) advantageously has such an acetyl group on its terminal amine (NH₂).

[0029] 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.

[0030] According to one embodiment, in said hybrid molecule according to the invention, said peptide comprises at least one citrullyl residue, and is selected from the group consisting of: a) a peptide defined by the sequence X1 PAPPPISGGGYX2 AX3 (SEQ ID NO: 1) in which X1, X2, and X3 each represent a citrullyl residue or an arginyl residue, and at least one of the residues X1, X2, or X3 is a citrullyl residue; b) a peptide defined by the sequence GPX1 VVEX2 HQSACKDS (SEQ ID NO: 2) in which X1 and X2 each represent a citrullyl residue or an arginyl residue, and at least one of the residues X1 or X2 is a citrullyl residue; (c) a peptide defined by the sequence SGIGTLDGFX 1 HX 2 HPD (SEQ ID NO: 3) in which X 1 and X 2 each represent a citrullyl residue or an arginyl residue, and at least one of the residues X 1 or X 2 is a citrullyl residue; (d) a peptide defined by the sequence VDIDIKIX 1 SCX 2 GSCS (SEQ ID NO: 4) in which X 1 and X 2 each represent a citrullyl residue or an arginyl residue, and at least one of the residues X 1 or X 2 is a citrullyl residue;e) a peptide defined by the sequence X1 GHAKSX2 PVX3 GIHTS (SEQ ID NO: 12) in which X1, X2, and X3 each represent a citrullyl or arginyl residue, and at least one of the residues X1, X2, or X3 is a citrullyl residue; f) a peptide comprising at least 5 consecutive amino acids, including at least one citrullyl residue, from one of the peptides a) to e) above.

[0031] According to a particular embodiment, said peptide comprises at least one citrullyl residue, and is selected from the group consisting of: a peptide defined by the sequence SEQ ID NO: 1 in which at least one residue selected from X1 or X2 or X3 is a citrullyl residue, or a peptide comprising a fragment of at least 5 consecutive amino acids from said sequence containing said citrullyl residue(s); a peptide defined by the sequence SEQ ID NO: 2 in which at least X1 or X2 is a citrullyl residue, or a peptide comprising a fragment of at least 5 consecutive amino acids from said sequence containing said citrullyl residue(s); a peptide defined by the sequence SEQ ID NO: 3 in which at least X1 or X2 is a citrullyl residue, or a peptide comprising a fragment of at least 5 consecutive amino acids from said sequence containing said citrullyl residue(s);a peptide defined by the sequence SEQ ID NO: 4 in which at least X1 or X2 is a citrullyl residue, or a peptide comprising a fragment of at least 5 consecutive amino acids from said sequence containing said citrullyl residue(s); a peptide defined by the sequence SEQ ID NO: 12 in which at least one residue selected from X1 or X2 or X3 is a citrullyl residue, or a peptide comprising a fragment of at least 5 consecutive amino acids from said sequence containing said citrullyl residue(s).

[0032] According to a further particular embodiment, in said hybrid molecule according to the invention, said peptide comprises at least one citrullyl residue, and is chosen from the group consisting of: a peptide defined by the sequence SEQ ID NO: 1 in which X1, X2, and X3 are citrullyl residues, or a peptide of at least 15 amino acids comprising said sequence (this is a peptide of SEQ ID NO: 19); a peptide defined by the sequence SEQ ID NO: 2 in which X1 and X2 are citrullyl residues, or a peptide comprising a fragment of at least 5 consecutive amino acids of said sequence containing said citrullyl residues (this is a peptide of SEQ ID NO: 5); a peptide defined by the sequence SEQ ID NO: 3 in which X1 and X2 are citrullyl residues, or a peptide comprising a fragment of at least 5 consecutive amino acids of said sequence containing said citrullyl residues (this is a peptide of SEQ ID NO: 14);a peptide defined by the sequence SEQ ID NO: 4 in which X1 and X2 are citrullyl residues, or a peptide comprising a fragment of at least 5 consecutive amino acids of said sequence containing said citrullyl residues (this is a peptide of SEQ ID NO: 6); a peptide defined by the sequence SEQ ID NO: 12 in which X1, X2 and X3 are citrullyl residues, or a peptide comprising a fragment of at least 10 consecutive amino acids of said sequence containing said citrullyl residues (this is a peptide of SEQ ID NO: 18).

[0033] 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: 5 (α36-50cit 38,42), SEQ ID NO: 6 (α171-185cit 178,181), SEQ ID NO: 7 (α183-197cit 186,190), SEQ ID NO: 8 (α246-260cit 258), SEQ ID NO: 9 (α259-273cit 263,271), SEQ ID NO: 10 (α366-380cit 367), SEQ ID NO: 11 (α396-410cit 404), SEQ ID NO: 13 (α411-425cit 425), SEQ ID NO: 14 (α501-515cit 510,512), SEQ ID NO: 15 (α546-560cit 547), SEQ ID NO: 16 (α561-575cit 573), SEQ ID NO: 17 (α588-602cit 591), SEQ ID NO: 18 (α621-635cit 621,627,630), SEQ ID NO: 19 (β60-74cit 60,72,74), SEQ ID NO: 20 (β210-224cit 224), SEQ ID NO: 21 (β281-295cit 285,294), SEQ ID NO: 22 (β420-434cit 421) and SEQ ID NO: 23 (β433-447cit 436,445 ), more particularly chosen from the group consisting of: SEQ ID NO: 5 (α36-50cit 38,42 ), SEQ ID NO: 6 (α171-185cit 178,181 ), SEQ ID NO: 14 (α501-515cit 510,512 ),SEQ ID NO: 18 (α621-635cit 621,627,630) and SEQ ID NO: 19 (β60-74cit 60,72,74), even more specifically chosen from among SEQ ID NO: 18 (α621-635cit 621,627,630) and SEQ ID NO: 19 (β60-74cit 60,72,74).

[0034] 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 IgG1. The IgG1 can correspond to any allotypic variant, for example G1m3 or nG1m1. By way of example, the Fc fragment of IgG1 is represented by SEQ ID NO: 24, SEQ ID NO: 25 (Fc+Qtag) or SEQ ID NO: 26 (Fc+Qtag bis).

[0035] 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 described in particular in the article Bruhns and Jönsson, Immunol Rev. 2015 Nov;268(1):25-51 Preferably, when the hybrid molecule is used in therapy, said mutated Fc fragment includes at least the GASDIE, GASDALIE, or SDH mutations.

[0036] 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 ranging from 0% to 60% of the glycosylated forms, specifically 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.

[0037] 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 the hybrid molecule according to the invention, the covalent bond is located between the C-terminal end of the Fc fragment and the N-terminal end of the peptide, or between the N-terminal end of the Fc fragment and the N-terminal end of the 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, particularly 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 formed 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.

[0038] 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. In 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. In 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).

[0039] 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 DBCO, or 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 Fc fragment and said peptide, possibly in the presence of one or more spacers, being created between the azide and the alkyne.

[0040] 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.

[0041] 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.

[0042] According to one embodiment, in the molecules described above, particularly from page 8, line 21 to page 9, line 17, the spacer used is preferably a PEGn spacer, where n represents more particularly an integer between 1 and 10. When the hybrid molecule according to the invention comprises at least two PEGn spacers, the values ​​n of the two spacers may be identical or different. For example, the first spacer may be a PEG 2 and the second spacer may be a PEG 3 or PEG 4.

[0043] According to a particularly preferred embodiment, in the molecules described above, more particularly from page 8, line 21 to page 9, line 17, the peptide is derived from human fibrin or fibrinogen and the Fc fragment is a human Fc, preferably from human IgG1.

[0044] 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. The azide is understood to be the salts of hydrazotic acid (HN3), or organic azides in which one of the nitrogen atoms is covalently bonded to a carbon atom of an organic compound (for example, methyl azide (CH3N3)). Preferably, the azide is represented by the formula N3. The alkyne is understood to be molecules having the general formula CnH2n-2, and which are characterized by the presence of at least one triple bond. Preferably, the alkyne is a cyclooctyne, and even more preferably dibenzocyclooctyne (DBCO).

[0045] The Fc fragment, the peptide, and optionally the 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.

[0046] More specifically, a conjugation technique can be either enzymatic or chemical. Enzymatic conjugation, for example, involves conjugation using a transglutaminase, which 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 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 containing a glutamyl residue (a Qtag), such as represented by SEQ ID NO: 29 (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 Figure 1. Figure 9 . In this example, the Fc fragment includes 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.

[0047] According to the invention, the term "coupled" or "molecular coupling" refers to the establishment of a covalent bond, whereby 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, for 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."

[0048] According to one embodiment of the invention, said hybrid molecule is represented by: 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 which is coupled to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74) or SEQ ID NO: 18 (α621-635cit 621,627,630), an Fc fragment comprising the S239D, H268F, S324T and I332E mutations that is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne which is coupled to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74) or SEQ ID NO: 18 (α621-635cit 621,627,630 ), an Fc fragment comprising the L234A, L235A and P329G mutations which is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne which is coupled to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74 ) or SEQ ID NO: 18 (α621-635cit 621,627,630 ),a wild-type Fc fragment that is linked to at least one PEGn spacer which is itself coupled to a cyclooctyne covalently linked to an azide coupled to a PEGn spacer itself linked to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74) or SEQ ID NO: 18 (α621-635cit 621,627,630), an Fc fragment comprising the mutations S239D, H268F, S324T and I332E that is linked to at least one PEGn spacer which is itself coupled to a cyclooctyne covalently linked to an azide coupled to a PEGn spacer itself linked to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74) or SEQ ID NO: 18 (α621-635cit 621,627,630 ), an Fc fragment comprising the L234A, L235A and P329G mutations which is linked to at least one PEGn spacer which is itself coupled to a cyclooctyne covalently linked to an azide coupled to a PEGn spacer itself linked to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74 ) or SEQ ID NO: 18 (α621-635cit 621,627,630 ),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 coupled to a PEGn spacer which is itself linked to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74) or SEQ ID NO: 18 (α621-635cit 621,627,630), an Fc fragment comprising the mutations S239D, H268F, S324T and I332E that is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne coupled to a PEGn spacer which is itself linked to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74) or SEQ ID NO: 18 (α621-635cit 621,627,630 ), an Fc fragment comprising the L234A, L235A and P329G mutations which is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne coupled to a PEGn spacer which is itself linked to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74 ) or SEQ ID NO: 18 (α621-635cit 621,627,630 ),an Fc fragment comprising the G236A, S239D, and I332E mutations, which is linked to at least one PEGn spacer that is itself coupled to an azide covalently linked to a cyclooctyne coupled to a PEGn spacer that is itself linked to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74) or SEQ ID NO: 18 (α621-635cit 621,627,630), an Fc fragment comprising the G236A, S239D, A330L, and I332E mutations, which is linked to at least one PEGn spacer that is itself coupled to an azide covalently linked to a cyclooctyne coupled to a PEGn spacer that is itself linked to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74) or SEQ ID NO: 18 (α621-635cit 621,627,630), an Fc fragment comprising the mutations G236A, S239D and 1332E which is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne which is coupled to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74) or SEQ ID NO: 18 (α621-635cit 621,627,630),an Fc fragment comprising the mutations G236A, S239D, A330L and I332E which is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne which is coupled to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74) or SEQ ID NO: 18 (α621-635cit 621,627,630), , n preferably representing an integer between 1 and 10, more particularly 1, 2, 3, 4 or 8.

[0049] According to one embodiment of the invention, said hybrid molecule is represented by: 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 which is coupled to a peptide represented by SEQ ID NO: 19 (B60-74cit 60,72,74) or SEQ ID NO: 18 (α621-635cit 621,627,630), an Fc fragment comprising the mutations S239D, H268F, S324T and I332E that is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne which is coupled to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74) or SEQ ID NO: 18 (α621-635cit 621,627,630), an Fc fragment comprising the L234A, L235A and P329G mutations which is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne which is coupled to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74) or SEQ ID NO: 18 (α621-635cit 621,627,630),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 coupled to a PEGn spacer which is itself linked to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74) or SEQ ID NO: 18 (α621-635cit 621,627,630), an Fc fragment comprising the mutations S239D, H268F, S324T and I332E that is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne coupled to a PEGn spacer which is itself linked to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74) or SEQ ID NO: 18 (α621-635cit 621,627,630), , n preferably representing an integer between 1 and 10, more particularly 1, 2, 3, 4 or 8.

[0050] According to a more particular embodiment of the invention, said hybrid molecule is represented by: 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 which is coupled to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74), an Fc fragment comprising the mutations S239D, H268F, S324T, and I332E that is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne which is coupled to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74), an Fc fragment comprising the mutations L234A, L235A, and P329G that is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne which is coupled to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74),a wild-type Fc fragment that is linked to a PEGn spacer which is itself coupled to at least one azide covalently linked to a cyclooctyne coupled to a PEGn spacer which is itself linked to a peptide represented by SEQ ID NO: 19 (B60-74cit 60,72,74) or SEQ ID NO: 18 (α621-635cit 621,627,630), an Fc fragment comprising the mutations S239D, H268F, S324T and I332E that is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne coupled to a PEGn spacer which is itself linked to a peptide represented by SEQ ID NO: 19 (β60-74cit 60,72,74) or SEQ ID NO: 18 (α621-635cit 621,627,630), , n preferably representing an integer between 1 and 10, more particularly 1, 2, 3, 4 or 8. Use of hybrid molecules according to the invention

[0051] In a second aspect, the present invention also relates to a hybrid molecule as defined above, for its use as a drug.

[0052] More specifically, according to the invention, said hybrid molecules are intended to target and lyse, within the patient's body, via ADCC and / or phagocytosis and / or complement activation, all ACPA-positive B cells (i.e., B lymphocytes that express anti-citrullinated protein (ACPA) autoantibodies on their surface). Indeed, the hybrid molecule according to the invention binds to ACPA-positive B lymphocytes through a fibrin-derived peptide having at least one citrullyl residue: this is the epitope target of said ACPA. The hybrid molecule according to the invention also binds to cells, thereby destroying ACPA-positive B lymphocytes, through its Fc fragment, a natural ligand of Fc receptors (for example, Fc-gamma receptor (FcγR) if the Fc fragment is derived from an IgG), present notably on the surface of macrophages but also of NK ("Natural Killer") cells.

[0053] According to a particular embodiment, the invention relates to a hybrid molecule, as previously defined, for use in the treatment of autoimmune diseases associated with the production of anti-citrullinated protein autoantibodies, in particular Sjögren's syndrome and rheumatoid arthritis. In this embodiment, severe forms of said autoimmune diseases associated with the production of anti-citrullinated protein autoantibodies are targeted, as well as various so-called "borderline" forms with other chronic arthritis, such as psoriatic arthritis or systemic lupus erythematosus.

[0054] 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.

[0055] 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.

[0056] As stated above, the present invention aims to destroy ACPA-positive B lymphocytes. However, ACPA-positive B lymphocytes that have differentiated into ACPA-secreting plasma cells are no longer targeted by the previously described hybrid molecules. In one particular embodiment, the present invention thus relates to a hybrid molecule as previously described, for use as a drug, in combination with a second hybrid molecule, said second hybrid molecule comprising at least one fibrin-derived peptide having at least one citrullyl residue, said peptide being covalently linked to at least one antibody or at least one antibody fragment, said antibody or fragment being capable of binding to CD38 and / or CD138, one or more spacers optionally being present between said peptide and said antibody or fragment.Such combination therapy allows for the specific targeting and destruction of ACPA-positive plasma and B lymphocyte clones, thereby eliminating ACPA from the patient's body. The antibody used is preferably a monoclonal antibody.

[0057] According to a particular embodiment, in this second hybrid molecule, the fragment is understood to be a portion of an antibody comprising an antigen-binding site or a variable region of the antibody. Such antibody fragments are, for example, Fab, F(ab')2, Fv, dsFv, scFv, single-chain fragments such as isolated VH or VL fragments, camelid VHHs, cartilaginous fish VNARs, or multispecific antibodies composed of different fragments, such as bispecific antibodies, or "nanobodies", "diabodies", "triabodies", "tetrabodies", or tandem scFvs, ... These fragments are well known to those skilled in the art. Further information concerning these fragments and constructs is described, for example, in international patent application WO2017137579, Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883, or Nelson, mAbs 2:1, 77-83; January / February 2010.Preferably the fragment is a Fab or F(ab')2.

[0058] According to one particular embodiment, in this second hybrid molecule, said antibody or fragment F(ab')2 is a bispecific antibody or fragment F(ab')2 directed against CD38 and another plasma cell target. Alternatively, in this second hybrid molecule, said antibody or fragment F(ab')2 is a bispecific antibody or fragment F(ab')2 directed against CD138 and another plasma cell target. According to the invention, "another plasma cell target" means any molecule expressed on the surface of plasma cells.

[0059] According to another particular embodiment, in this second hybrid molecule said antibody or fragment F(ab') 2 is a bispecific antibody or fragment F(ab') 2 directed against CD38 and CD138.

[0060] According to this particular embodiment of the invention, the first and second hybrid molecules are administered simultaneously, separately or spread out over time.

[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 thus relates to the use in vitro Or ex vivoof a hybrid molecule comprising at least one Fc fragment of an antibody covalently linked to at least one fibrin-derived peptide having at least one citrullyl residue, with at least one spacer optionally present between said Fc fragment and said peptide, as a molecular tool. Such constructs can be used, in particular, to analyze the binding of hybrid molecules to ACPAs and 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 ACPAs. Radioisotopes and / or fluorochromes are preferably coupled to the Fc fragment, more specifically to the Qtag (if present) or to lysines. Method for producing hybrid molecules according to the invention

[0063] In another aspect, the invention also relates to a process for obtaining a hybrid molecule as defined above.

[0064] 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) establishing the covalent bond between the azide and the alkyne, step (i) being able to be carried out before or after step (ii), or concomitantly.

[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) 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) formation of the covalent bond between the azide and the alkyne, step (i) being able to be carried out before or after step (ii), or concomitantly.

[0066] 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, 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) 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.

[0067] The sequences of the invention are represented in Table 1 below. [Table 1]. Summary table of the sequences of the invention Sequence number / Sequence name if applicable Sequence SEQ ID NO: 1 X 1 PAPPPISGGGYX 2 AX 3 SEQ ID NO: 2 GPX 1 VVEX 2 HQSACKDS SEQ ID NO: 3 SGIGTLDGFX 1 HX 2 HPD SEQ ID NO: 4 VDIDIKIX 1 SCX 2 GSCS SEQ ID NO: 12 X 1 GHAKSX 2 PVX 3 GIHTS SEQ ID NO: 5 / α36-50cit 38.42 GPXVVEXHQSACKDS, X representing a citrullyl residue SEQ ID NO: 6 / α171-185cit 178.181 VDIDIKIXSCXGSCS, X representing a citrullyl residue SEQ ID NO: 7 / α183-197cit 186.190 SCSXALAXEVDLKDY, X representing a citrullyl residue SEQ ID NO: 8 / α246-260cit 258 PEWKALTDMPQMXME, X representing a citrullyl residue SEQ ID NO: 9 / α259-273cit 263.271 MELEXPGGNEITXGG, X representing a citrullyl residue SEQ ID NO: 10 / α366-380cit 367 EXGSAGHWTSESSVS, X representing a citrullyl residue SEQ ID NO: 11 / α396-410cit 404 DSPGSGNAXPNNPDW, X representing a citrullyl residue SEQ ID NO: 13 / α411-425cit 425 GTFEEVSGNVSPGTX, X representing a citrullyl residue SEQ ID NO: 14 / α501-515cit 510,512 SGIGTLDGFXHXHPD, X representing a citrullyl residue SEQ ID NO: 15 / a546-560cit 547 SXGSESGIFTNTKES, X representing a citrullyl residue SEQ ID NO: 16 / α561-575cit 573 SSHHPGIAEFPSXGK, X representing a citrullyl residue SEQ ID NO: 17 / α588-602cit 591 SYNXGDSTFESKSYK, X representing a citrullyl residue SEQ ID NO: 18 / α621-635cit 621,627,630 XGHAKSXPVXGIHTS, X representing a citrullyl residue SEQ ID NO: 19 / B60-74cit 60,72,74 XPAPPPISGGGYXAX, X representing a citrullyl residue SEQ ID NO: 20 / β210-224cit 224 QKLESDVSAQMEYCX, X representing a citrullyl residue SEQ ID NO: 21 / β281-295cit 285.294 VIQNXQDGSVDFGXK, X representing a citrullyl residue SEQ ID NO: 22 / β420-434cit 421 PXKQCSKEDGGGWWY, X representing a citrullyl residue SEQ ID NO: 23 / β433-447cit 436,445 WYNXCHAANPNGXYY, X representing a citrullyl residue SEQ ID NO: 24 / Fragment Fc SEQ ID NO: 25 / Fragment Fc + Qtag SEQ ID NO: 26 / Fragment Fc + Qtag bis SEQ ID NO: 27 / α chain of fibrin SEQ ID NO: 28 / β chain of fibrin SEQ ID NO: 29 (Example of Qtag) LLQG SEQ ID NO: 30 α chain of fibrinogen SEQ ID NO: 31 β chain of fibrinogen

[0068] Other features, details and advantages of the invention will become apparent from the accompanying Figures and examples which illustrate the invention and are in no way intended to limit it.

[0069] In the exemplified hybrid molecules, it is the N-terminal end of the Fc fragment that is linked to a PEGn spacer (except in the case indicated, Cter-PEG (see the Figure 4)), and it is always the N-terminal end of said peptide that is bound to cycloctyn DBCO or to a second PEGn spacer as appropriate. Furthermore, in the exemplified hybrid molecules, in the PEGn spacer bound to the Fc fragment, n most specifically represents 3 or 4, and in the PEGn spacer bound to the peptide, the PEGn spacer represents 3 or 8, although any other value of n, particularly between 1 and 10, can be used.

[0070] In the examples below, an "armed or pre-armed" cell is understood to be a cell to which a hybrid molecule according to the invention is attached. Thus, the hybrid molecule is bound to the cell's Fc receptor via its Fc fragment. Brief description of the Figures

[0071] Fig. 1 [ Fig. 1 ] represents the diagram of an example of a hybrid molecule according to the invention. A spacer (which is optional) is shown between the Fc fragment of antibody and the fibrin-derived peptide having a citrullyl residue (called a "citrullinated peptide"). Fig. 2 [ Fig. 2 ] represents a B lymphocyte expressing on its surface transmembrane ACPAs linked to a hybrid molecule according to the invention, said hybrid molecule being itself linked by the Fc fragment to an NK cell. The NK cell can thus destroy the B lymphocyte via ADCC. More specifically, this figure shows a B cell (or B lymphocyte) that expresses an ACPA-type BCR on its surface and interacts specifically with the citrullinated peptide carried by the hybrid molecule. The engagement of the Fc fragment carried by the same hybrid molecule with FcyR11a (CD16a) expressed on the surface of an NK cell will activate ADCC and induce the specific destruction of the ACPA-positive B lymphocyte. (ACPA, Anti-Citrullinated Protein Autoantibodies; ADCC: Antibody-Dependent Cell Cytotoxicity; BCR, B-Cell Receptor; Fc γ R, Fc-gamma Receptor; NK cell, Natural Killer cell). Fig. 3 [ Fig. 3 ] represents a B lymphocyte expressing on its surface transmembrane ACPAs linked to a hybrid molecule according to the invention, said hybrid molecule being itself linked by the Fc fragment to a macrophage.The macrophage can then destroy the B lymphocyte by phagocytosis. More specifically, this figure shows B cells (or B lymphocytes) that express an ACPA-type BCR on their surface and interact specifically with citrullinated peptides carried by hybrid molecules. The engagement of the Fc fragments carried by these same hybrid molecules with different FcyRs expressed on the surface of a macrophage will activate ADP, i.e., phagocytosis, and induce the specific destruction of ACPA-positive B lymphocytes. (ACPA, Anti-Citrullinated Protein Autoantibodies; ADP, Antibody-Dependent Phagocytosis; BCR, B-Cell Receptor; Fc γ R, Fc-gamma Receptor). Fig. 4 [ Fig. 4 ] represents the reactivity of several hybrid molecules including the SEQ ID NO peptide: 19 (β60-74cit 60,72,74 ) towards ACPA.β60-74 represents the peptide of SEQ ID NO: 19. Fc-WT-β60-74 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 which is coupled to a peptide represented by SEQ ID NO: 19 (β60-74 cit60,72,74). Fc-LALA-β60-74 represents an Fc fragment comprising the L234A, L235A and P329G mutations which is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne DBCO which is coupled to a peptide represented by SEQ ID NO: 19. Fc-SDH-β60-74 represents an Fc fragment comprising the S239D, H268F, S324T and I332E mutations 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 peptide represented by SEQ ID NO: 19.Fc-WT-PEG-β60-74 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 SEQ ID NO: 19 (β60-74 cit60,72,74). Fc-SDH-PEG-β60-74 represents an Fc fragment comprising the S239D, H268F, S324T, and I332E mutations, 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 SEQ ID NO: 19 (β60-74 cit60,72,74). Fc-SDH(Cter)-PEG-β60-74 corresponds to Fc-SDH-PEG-β60-74, with the difference that in the Fc-SDH(Cter)-PEG-β60-74 hybrid, the first PEGn is attached at the C-terminal end of the Fc fragment, unlike the other examples in which the first PEGn is attached at the N-terminal end.n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8. Fig. 5 [ Fig. 5 ] represents the reactivity of a hybrid molecule comprising the SEQ ID NO peptide : 18 (α621-635cit 621,627,630 ) vis-à-vis the ACPA. α621-635 represents the peptide of SEQ ID NO: 18. Fc-WT-PEG-α621-635 represents a wild-type (WT) 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 SEQ ID NO: 18. Fc-SDH-PEG-α621-635 represents an Fc fragment comprising the mutations S239D, H268F, S324T, 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 SEQ ID NO: 18. n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8. Fig. 6 [ Fig. 6 ] represents the reactivity of a recombinant human monoclonal ACPA (clone 2H06), comprising an unmodified Fc or one modified with SDH-type mutations, towards hybrid molecules comprising the SEQ ID NO peptide : 18.FcWT-PEG-α621-635 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 SEQ ID NO: 18. FcSDH-PEG-α621-635 represents an Fc fragment comprising the S239D, H268F, S324T, and I332E mutations, 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 SEQ ID NO: 18. n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4, or 8. Fig. 7 [ Fig. 7 ] represents a method for manufacturing a hybrid molecule according to the invention, comprising the scheme for derivation and conjugation of the Fc fragments. Fig. 8 [ Fig. 8 ] represents the evolution of the percentage of phagocytosis and B lymphocytes in the presence of hybrid molecules Fc-WT-PEG-α621-635arg and Fc-WT-PEG-α621-635cit. The results of the 6 experiments presented in Example 6 are shown (N=6). The P value is as follows: * < 0.05 ** < 0.01 *** < 0.001. Fig. 9 [ Fig. 9 ] represents the lysis by human NK cells of transduced human malignant lymphoblastic B cell line expressing a recombinant monoclonal ACPA on its membrane, in the presence of FcWT-N3-DBCO-PEG3-α621-635Cit and FcSDH-N3-DBCO-PEG3-α621-635Cit hybrids.Nalm6 represents the case where only Nalm6 cells were incubated; Nalm6+NK the case where Nalm6 cells were incubated with NK cells; WTαCIT the case where the WTαCIT hybrid was incubated with both Nalm6 and NK cells; WTαArg the case where the WTαArg hybrid was incubated with both Nalm6 and NK cells; SDHαCIT the case where the SDHαCIT hybrid was incubated with both Nalm6 and NK cells; and SDHαArg the case where the SDHαArg hybrid was incubated with both Nalm6 and NK cells. The transduced human malignant lymphoblastic B cell line expressing a recombinant monoclonal ACPA on its membrane corresponds to the Nalm6 cell line as described in Example 11 and the Figure 10 The monoclonal antibody is clone 022014CCP14CFCT2H06, also known as 2H06. Fig. 10 [ Fig. 10 ] represents the membrane expression of monoclonal ACPA 2H06 by the Nalm6 B lymphoblastic lineage after transduction (Nalm6 2H06).A- Flow cytometry analysis of the transduction efficiency of the Nalm6 cell line B for the expression of the monoclonal ACPA 2H06 at its membrane, using double labeling with an anti-Fab antibody of IgG and a tetramer of the α621-635cit peptide (SEQ ID NO: 18). B- Flow cytometry analysis of the enrichment in Nalm6 2H06 transduced cells, following cell sorting using an anti-Fab antibody. C- Flow cytometry analysis of the interaction between membrane-bound ACPA 2H06 expressed by the transduced Nalm6 cell line and the Fc-WT-PEG-α621-635cit (cit) or Fc-WT-PEG-α621-635arg (arg) hybrids, coupled to fluorochrome A647. Nalm6 NT: non-transduced control Nalm6 cell line. Fc-WT-PEG-α621-635 represents a wild-type (WT) 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 SEQ ID NO: 18.Fc-WT-PEG-α621-635arg represents a wild-type (WT) Fc fragment 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 SEQ ID NO: 18 in which the X residues are not citrullinated (i.e., they are arginyl residues). Nalm6 cells expressing the monoclonal ACPA 2H06 correspond to the Nalm6 transduced cell line mentioned in the examples. Fig. 11 [ Fig. 11 ] represents the phagocytosis of Nalm6 ACPA+ 2H06 transduced B-lineage cells by macrophages pre-armed with Fc-peptide α621-635 hybrids.A- Figure A shows the results of a flow cytometry experiment where cell populations are represented as scatter plots. T0 represents the result at time 0, after contact between Nalm6 target cells and macrophages. T2 represents the result at time 2h, after 2h of contact between Nalm6 target cells and macrophages, in the absence of hybrid molecules (spontaneous phagocytosis). All other flow cytometry plots represent the results obtained after 2h of contact between Nalm6 target cells and macrophages armed with Fc-PEG-α621-635cit (citrullinated) hybrids or Fc-PEG-α621-635arg (non-citrullinated) control hybrids. The hybrids are identical to those described in Figure 10The only difference is that different types of Fc fragments were used: a wild-type (WT) Fc fragment, a GASDIE Fc fragment (containing the G236A, S239D, and I332E mutations), or an SDH Fc fragment (containing the S239D, H268F, S324T, and I332E mutations). Cells exhibiting dual fluorescence (PKH-67 green / PKH-26 red) correspond to macrophages that phagocytosed target cells. Cells exhibiting single PKH-26 fluorescence correspond to residual Nalm6 target cells, i.e., those not phagocytosed after 2 hours in the presence of macrophages (% target population). In each quadrant of the flow cytometry diagrams, the different cell populations are expressed as a percentage of the total cell population.B- The figures show, in the form of histograms, for each experimental condition described in A, the average percentage of double-positive cells and the average percentage of cells from the target population, calculated from the results of 3 experiments. The black histograms represent the control conditions. The white histograms represent the conditions in the presence of the Fc-PEG-α621-635cit hybrid. The p-value is as follows: * p < 0.05; ** p < 0.01. Fig. 12 [ Fig. 12 ] represents the phagocytosis of B cell line Nalm6 ACPA+ 2H06 by pre-armed macrophages in the presence of Fc-peptide α621-635 hybrids at various concentrations.Figure 1 shows the results of a flow cytometry experiment performed after phagocytosis of Nalm6 2H06 B cells by macrophages pre-armed by incubation in the presence of Fc-α621-635 peptide hybrids at various concentrations. A- Figure 1 shows, in histogram form, the percentage of double-positive cells for each experimental condition. B- Figure 2 shows the percentage of the residual target Nalm6 population, i.e., cells not phagocytosed. The dark histograms represent the control conditions obtained in the presence of non-citrullinated Fc-PEG-α621-635arg hybrids. The light histograms represent the conditions in the presence of the Fc-PEG-α621-635cit hybrid. The hybrid concentrations used ranged from 5 µg / ml to 5 ng / ml. The hybrids are identical to those described in Figure 11 , with the difference that a hybrid molecule with an Fc GASDALIE fragment (including the G236A, S239D, A330L and I332E mutations) was also tested. Fig. 13 [ Fig. 13] represents the confirmation of the phagocytosis process of the transduced Nalm6 ACPA+ 2H06 line by macrophages pre-armed with FcWT-PEG-peptide α621-635 hybrids. These figures show that the double-positive cells observed by flow cytometry in previous experiments correspond to the phagocytosis of Nalm6 target cells by macrophages. The four panels on the left show flow cytometry results obtained after combining pHRodo SE-labeled Nalm6 2H06 cells with macrophages armed with Fc-WT-PEG-α621-635 hybrids, with and without cytochalasin D, a phagocytosis inhibitor. The two panels on the right show the cell populations at the end of the experiment, observed by light microscopy after MGG staining. The hybrids are identical to those described in Figure 10 . Fig. 14 [ Fig. 14 ] represents the phagocytosis of the transduced Nalm6 ACPA+ 2H06 cell line by macrophages pre-armed with Fc-peptide α621-635 hybrids or incubated in the presence of Fc-peptide α621-635 hybrids.The figure shows the results of an experiment analyzed by flow cytometry: the scatter plots correspond to cell populations. T0 represents the result at time 0, immediately after contact between Nalm6 target cells and macrophages. T2 represents the result at 2 hours after contact between Nalm6 target cells and macrophages, in the absence of hybrid molecules (spontaneous phagocytosis). All other flow cytometry diagrams show results obtained after 2 hours of contact between Nalm6 target cells and macrophages, either pre-armed with citrullinated or uncitrullinated FcWT-PEG-α621-635 or FcGASDIE-PEG-α621-635 hybrids, or incubated with citrullinated or uncitrullinated WT-PEG-α621-635 or FcGASDIE-PEG-α621-635 hybrids solubilized in the incubation medium. The hybrids are identical to those described in Figure 11Cells exhibiting dual fluorescence (PKH-67 green / PKH-26 red) correspond to macrophages that have phagocytosed Nalm6 target B cells (% double positives). Cells exhibiting single PKH-67 fluorescence correspond to residual Nalm6 target B cells, i.e., those not phagocytosed after 2 hours in the presence of macrophages. In each quadrant of the flow cytometry diagrams, the different cell populations are expressed as a percentage of the total cell population. Fig. 15 [ Fig. 15 ] represents the phagocytosis of the transduced Nalm6 ACPA+ 2H06 cell line by macrophages incubated in the presence of Fc-PEG-peptide α621-635 hybrids and, or not, in the presence of human IgG at physiological serum concentration.The figure shows the mean results of four (n=4) identical independent experiments of phagocytosis of Nalm6 ACPA+ 2H06 transduced cell lines by macrophages incubated in the presence of Fc-PEG-α621-635 peptide hybrids, and with or without human IgG at physiological serum concentrations. For each experimental condition, the figure shows the mean residual percentage of the target cell population 2 hours after contact with macrophages and citrullinated (cit) or non-citrullinated (arg) Fc-PEG-α621-635 hybrids, soluble at a concentration of 1 µg / mL, in the presence (gray histograms) or absence (black histograms) of competing human IgG. The p-value is as follows: * p < 0.05; ** p < 0.01. The hybrids are identical to those described in Figure 11 . Fig. 16 [ Fig. 16 ] represents the phagocytosis of the transduced Nalm6 ACPA+ 2H06 cell line by human macrophages incubated with Fc-peptide α621-635 hybrids, in the presence, or not, of human IgG at physiological concentration or human serum.The figure shows the results of an experiment analyzed by flow cytometry, where cell populations are represented by scatter plots. T0 corresponds to the result obtained immediately after contact between Nalm6 2H06 target cells and macrophages. T2 represents the result 2 hours after contact between target cells and macrophages in the absence of hybrid molecules (spontaneous phagocytosis). The other diagrams represent the results obtained 2 hours after contact between target cells and macrophages, incubated with citrullinated (cit) or non-citrullinated (arg) FcWT-PEG-α621-635 hybrids, in the presence or absence of human IgG at physiological serum concentration or human serum. The double-positive CFSE+ CD11b+ cells correspond to macrophages that phagocytosed target cells. CFSE+ single-positive cells correspond to residual Nalm6 target cells, not phagocytosed after 2h.In each quadrant of the flow cytometry diagrams, the different cell populations are expressed as a percentage of the total cell population. The hybrids are identical to those described in [reference missing]. Figure 11 . Fig. 17 [ Fig. 17 ] represents the phagocytosis of the transduced Nalm6 ACPA+ 2H06 cell line by human macrophages pre-armed with Fc-peptide α621-635 hybrids, in the presence of the non-transduced Nalm6 cell line.The figure shows the results of a phagocytosis experiment of Nalm6 ACPA+ 2H06 transduced cells by macrophages pre-armed with Fc-peptide α621-635 hybrids, in the presence of untransduced, wild-type Nalm6 cells. The flow cytometry analysis results are shown as scatter plots representing the cell populations. T0 represents the situation at time 0, immediately after contact between Nalm6 2H06 target cells, untransduced Nalm6 NT cells, and macrophages. T2 represents the result after 2 hours in the absence of hybrid molecules (spontaneous basal phagocytosis). All other cytometry diagrams represent the results obtained after 2h in the presence of macrophages armed with FcWT-PEG-α621-635 or FcGASDIE-PEG-α621-635 hybrids, citrullinated (cit) or non-citrullinated (arg).CFSE+ CD19+ double-positive cells correspond to residual Nalm6 2H06 target cells, i.e., those not phagocytosed after 2 hours in the presence of macrophages. CFSE- CD19- double-negative cells correspond to macrophages. CFSE- CD19+ single-positive cells correspond to residual non-transduced (NT) Nalm6 cells, i.e., those not phagocytosed after 2 hours in the presence of macrophages. CFSE+ CD19- single-positive cells correspond to macrophages that have phagocytosed Nalm6 2H06 target cells. In each quadrant of the flow cytometry diagrams, the different cell populations are expressed as a percentage of the total cell population. The hybrids are identical to those described in [reference missing]. Figure 11 . Fig. 18 [ Fig. 18 ] represents specific destruction in vivo of the Nalm6 ACPA+ 2H06 cell line by human NK cells in the presence of Fc-peptide α621-635 hybrids, in SCID / BEIGE immunodeficient mice.Figure A shows the destruction of ACPA+ target cells (Nalm6 2H06 transduced cell line) by human NK cells in the peritoneal cavity of immunodeficient SCID / BEIGE mice, in the presence of citrullinated Fc-α621-635 peptide hybrids. Figure A presents the flow cytometry analysis of Nalm6 2H06 and non-transduced Nalm6 NT cell populations. The observed peaks correspond to target cells (Nalm6 2H06 CTV high < ) or control cells (Nalm6 NT CTV low < ). Figure B presents the overall results obtained for four groups of seven immunodeficient SCID / BEIGE mice. These results are expressed as the ratio of CTV high < / CTV low < fluorescence, corresponding respectively to Nalm6 2H06 cells (CTV high < ) and Nalm6 NT cells (CTV low < ). A decrease in the ratio reflects the disappearance of the Nalm6 2H06 target cells. The "p" value is as follows: * p < 0.05; ** p < 0.01, *** p < 0.001.The hybrids are identical to those described in . Figure 10 . Fig. 19 [ Fig. 19 ] represents specific destruction in vivo of the Nalm6 ACPA+ 2H06 cell line by human macrophages pre-armed with Fc-peptide α621-635 hybrids, in immunodeficient SCID / BEIGE mice. The figure shows the destruction by human macrophages, in the peritoneal cavity of immunodeficient SCID / BEIGE mice, of ACPA+ target cells (Nalm6 cell line transduced 2H06) in the presence of citrullinated Fc-peptide α621-635 hybrids. The figure represents the results obtained for 4 groups of 5 immunodeficient SCID / BEIGE mice. The number of residual Nalm6 2H06 CTV high< target cells for each group at the end of the experiment is shown, indicating the efficiency of phagocytosis. The p-value is as follows: * p < 0.05. The hybrids are identical to those described in Figure 10 . Examples Example 1 : Example of the production of a hybrid molecule according to the invention

[0072] The hybrid molecule described here 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 citrullinated peptide. Such a molecule can be read as: Fc-PEGn-N3-DBCO-PEGn-citrullinated 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). 1. Synthesis of an NH2-PEGn-N3 (i.e., a spacer coupled to an azide at one end and possessing a free amine group at the other). 2. Reaction of the NH2-PEGn-N3 with an Fc fragment possessing a Qtag and transglutaminase, preferably for 16 h at 37°C. This "derivatization" step is preferably carried out with 20 times more moles of NH2-PEGn-N3 than moles of the Fc fragment. Transglutaminase is used at a concentration of 15 U / µmol 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 2Qtags (one on each monomer), then the Fc fragment can carry two PEGn-N 3.3. Synthesis of a citrullinated 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 DBCO, is then coupled to the citrullinated Cys-PEGn-peptide at the cysteine ​​site, resulting in DBCO-PEGn-citrullinated peptide. 4. Performing the "click": coupling between N3 and DBCO. The DBCO-PEGn-citrullinated peptide is reacted with Fc-PEGn-N3, preferably with 10 times more moles of DBCO-PEGn-citrullinated peptide than moles of Fc-PEGn-N3. The click reaction occurs primarily at room temperature and is almost complete after 4 hours. 5. Obtaining the hybrid molecule: Fc-PEGn-N3-DBCO-PEGn-citrullinated peptide. This method is illustrated in Figure 1. Figure 7 .

[0073] Similarly, an Fc-PEGn-DBCO and an N 3-PEGn-citrullinated peptide can be obtained and then coupled to obtain Fc-PEGn-DBCO-N 3-PEGn-citrullinated peptide.

[0074] Similarly, an Fc-PEGn-DBCO and an N 3-citrullinated peptide can be obtained, or an Fc-PEGn-N 3 and a DBCO-citrullinated peptide, then coupled to obtain respectively Fc-PEGn-DBCO-N 3-citrullinated peptide or Fc-PEGn-N 3-DBCO-citrullinated peptide. Example 2: Reactivity of ACPA purified by affinity chromatography on citrullinated peptides and eluted at pH 3 (pH 3 fraction), towards the hybrid molecules according to the invention

[0075] The ACPAs (here ACPA, pH3 fraction) used are ACPAs obtained from serum of ACPA-positive rheumatoid arthritis patients. Such ACPAs were obtained by a classic column affinity chromatography method known to those skilled in the art, using as bound antigens one or more of the five immunodominant peptides of the invention (see SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 18 or SEQ ID NO: 19). a. Examples with hybrid molecules containing the β60-74cit peptide

[0076] ELISA microtiter plate wells were coated by passive adsorption with 100 µL of a solution containing either the β60-74cit peptide (SEQ ID NO: 19, also referred to as β60-74), or a hybrid molecule containing Fc-WT-β60-74cit, Fc-LALA-β60-74cit, Fc-SDH-β60-74cit, Fc-WT-PEG-β60-74cit, Fc-SDH-PEG-β60-74cit, or Fc-SDH(Cter)-PEG-β60-74cit. Each solution was used at a concentration of 5 µg / mL in PBS (Phosphate Buffered Saline) and incubated overnight at 4°C. The non-citrullinated peptide and hybrid molecules constructed with the non-citrullinated peptide (β60-74arg, corresponding to a SEQ ID NO: 19 peptide in which the citrullyl residues have been replaced by arginyl residues) were used at the same concentration as negative controls. The wells were then saturated with 2% BSA (Bovine Serum Albumin) for 1 h at 4°C.After washing, the purified ACPAs were incubated at 1, 0.5, or 0.25 µg / mL, diluted in 2% PBS BSA 2M NaCl buffer for 1 h at 4°C. After washing, the reactivity of the ACPAs was detected using a secondary anti-Fab antibody of human IgG diluted 1 / 2500 in 2% PBS BSA buffer. The results are expressed as ΔOD (Delta-Optical Density), corresponding to the OD obtained with the β60-74cit peptide or with the hybrid molecules constructed with the β60-74cit peptide, subtracted from the OD obtained with the β60-74arg peptide or with the corresponding hybrid molecules constructed with the β60-74arg peptide, respectively.

[0077] The results are presented in Figure 4They demonstrate a dose-dependent reactivity of ACPA purified on β60-74cit peptide towards the hybrid molecules Fc-WT-β60-74cit, Fc-LALA-β60-74cit, Fc-SDH-β60-74cit, Fc-WT-PEG-β60-74cit, Fc-SDH-PEG-β60-74cit, or Fc-SDH(Cter)-PEG-β60-74cit. They show that, when included in the different hybrids, the citrullinated peptides remain fully reactive with ACPA. b. Examples with hybrid molecules containing the α621-635cit peptide

[0078] ELISA plate wells were coated with 100 µL of a solution containing the peptide α621-635cit (SEQ ID NO: 18, also referred to as α621-635) or a hybrid molecule Fc-WT-PEG-α621-635cit or Fc-SDH-PEG-α621-635cit, at a concentration of 5 µg / mL in PBS (Phosphate Buffered Saline), incubated overnight at 4°C. The non-citrullinated peptide α621-635arg and the corresponding hybrid molecules constructed with the non-citrullinated peptide (α621-635arg, corresponding to a peptide of SEQ ID NO: 18 in which the citrullyl residues have been replaced by arginyl residues) were used at the same concentration as negative controls. The wells were then saturated with 2% BSA (Bovine Serum Albumin) in PBS buffer for 1 hour at 4°C. After washing, the purified ACPAs were incubated at concentrations of 4, 2, and 1 µg / mL diluted in 2% BSA 2M NaCl PBS buffer for 1 hour at 4°C.After washing, the reactivity of ACPA was detected using a secondary anti-Fab antibody of human IgG diluted 1 / 2500 in 2% BSA PBS buffer. The results are expressed as ΔOD (Delta-Optical Density), corresponding to the OD obtained with the peptide. α 621-635cit or with hybrid molecules constructed with peptide α621-635cit, subtracted respectively from the OD obtained with peptide α621-635arg or with hybrid molecules constructed with peptide α621-635arg.

[0079] The results are presented in Figure 5 They show a strong dose-dependent reactivity of ACPA purified on peptide α621-635cit compared to the hybrid molecules Fc-WT-PEG-α621-635cit and Fc-SDH-PEG-α621-635cit. The reactivity of ACPA towards the hybrid molecules is comparatively stronger, while the epitope density they exhibit is lower than that of the α621-635cit peptide. Example 3 : Reactivity of recombinant monoclonal ACPA 2H06 in wild type - WT) or mutated on their Fc fragment (SDH mutation), with respect to the hybrid molecules according to the invention

[0080] The ACPA used here is a recombinant monoclonal human ACPA (clone 022014CCP14CFCT2H06, also known as 2H06). Such an ACPA can be obtained as described in Titcombe PJ, Wigerblad G., Sippl N., Zhang N., Shmagel AK, Sahiström P., Zhang Y., Barsness LO, Ghodke-Puranik Y., Baharpoor A., ​​et al. Pathogenic Citrulline-Multispecific B Cell Receptor Clades in Rheumatoid Arthritis. Arthritis & Rheumatology 2018, 70 (12), 1933-1945. https: / / doi.org / 10.1002 / art.40590. The VH of ACPA 2H06 has the accession number MH629710.1 under GenBank, and the VL has the number accession MH629700.1.

[0081] ELISA plate wells were coated using 100 µL of a solution containing either the hybrid molecule Fc-WT-PEG- α 621-635cit, i.e., Fc-SDH-PEG- α621-635cit, used at a concentration of 5 µg / mL in PBS (Phosphate Buffered Saline) buffer incubated overnight at 4°C. Hybrid molecules constructed with a non-citrullinated peptide ( α 621-635arg) were used at the same concentration as negative controls. Well saturation was then performed in 2% PBS BSA (Bovine Serum Albumin) buffer for 1 h at 4°C. After washing, the recombinant monoclonal ACPAs 2H06, WT, and SDH were incubated at 40, 20, and 10 µg / mL, diluted in 2% PBS BSA 2M NaCl buffer for 1 h at 4°C. After washing, their reactivities were detected using a 1 / 2500 anti-Fab human IgG secondary antibody diluted in 2% PBS BSA buffer. The results are expressed as ΔOD (Delta Optical Density), corresponding to the OD obtained with the hybrid molecules constructed with the peptide α 621-635cit subtracted from the DO obtained with the hybrid molecules constructed with the negative control peptide α621-635arg.

[0082] The hybrid molecules Fc-WT-PEG-α621-635cit and Fc-SDH-PEG-α621-635cit are identical to those used in Example 2.

[0083] The results are presented in Figure 6 The results show a strong dose-dependent reactivity of monoclonal WT and SDH ACPAs towards Fc-WT-PEG hybrid molecules. α621-635cit and Fc-SDH-PEG-α621-635cit. Example 4 : Binding of hybrid molecules to macrophages at 2h and 20h, at physiological temperature

[0084] Differentiated human macrophages in vitroin the presence of M-CSF (100ng / mL) from CD14+ monocytes isolated from peripheral blood of a healthy subject were incubated at 500.000 cells / well, in the presence of hybrid molecules at 5 µg / mL, FcWT-N3-DBCO-β60-74Cit (a wild-type Fc fragment that is linked to a PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne DBCO which is coupled to a peptide represented by SEQ ID NO: 19, referred to as WT in Tables 2 and 3), FcSDHFSTIE-N3-DBCO-β60-74Cit (an Fc fragment comprising the mutations S239D, H268F, S324T and I332E that is linked to a PEGn spacer which is itself coupled to an azide covalently linked to a DBCO which is coupled to a peptide represented by SEQ ID NO: 19, referred to as SDH in Tables 2 and 3) or FcLALAPG-N3-DBCO-β60-74Cit (an Fc fragment comprising the L234A, L235A and P329G mutations which is linked to a PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne DBCO which is coupled to a peptide represented by SEQ ID NO: 19, named LALAPG in Tables 2 and 3), for 2h or 20h at 37°C.The binding of hybrid molecules to the surface of macrophages is demonstrated and quantified by cytofluorimetry (FACS Canto II) after incubation of macrophages with an anti-Fc antibody coupled to FITC, used 1 / 1000. In the constructs, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8.

[0085] The results are presented in Tables 2 and 3 below. "NM" and "ANTI FC" represent the well containing macrophages incubated without antibodies or with the anti-Fc antibody alone, respectively. [Table 2]. Results at 2 a.m. 2h Macrophages Ratio NM 177 / ANTI FC 285 / WT 808 2.8 LALAPG 502 1.8 SDH 1330 4.7 [Table 3]. Results at 8 p.m. 20h Macrophages Ratio NM 213 / ANTI FC 275 / WT 1003 3.6 LALAPG 404 1.5 SDH 1457 5.3

[0086] The results presented in Tables 2 and 3 show that the hybrid molecules FcWT-N3-DBCO-β60-74Cit and FcSDHFSTIE-N3-DBCO-β60-74Cit, at a physiological temperature of 37°C, bind to the macrophage membrane within 2 hours and are still present 20 hours later. These results also show that the binding of the hybrid FcSDHFSTIE-N3-DBCO-β60-74Cit is greater than that of the wild-type form FcWT-N3-DBCO-β60-74Cit. Example 5 : Phagocytosis induced by the Fc-SDH β60citrullinated peptide hybrid when B lymphocytes are armed via their FcγRIIb (CD32b) with purified ACPA

[0087] 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 purified ACPA (pH3 fraction, obtained as in Example 2) on β60-74cit peptide (SEQ ID NO: 19) at a concentration of 5 µg / mL for 30 min at 37°C. Subsequently, the cells were washed and then placed in the presence of the FcSDHFSTIE-N3-DBCO-PEG3-β60-74Cit hybrid (an Fc fragment comprising the S239D, H268F, S324T and I332E mutations which is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne DBCO which is coupled to a PEGn spacer itself linked to a peptide represented by SEQ ID NO: 19). This construct is referred to as CIT in Table 4 below).The cells were also treated with the FcSDHFSTIE-N3-DBCO-PEG-β60-74Arg hybrid (which corresponds to the FcSDHFSTIE-N3-DBCO-PEG3-β60-74Cit construct in which the β60-74 peptide is not citrullinated). This construct is referred to as NCIT in Table 4 below. The cells were treated 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 phagocytosed lymphocytes. The different cell populations are expressed as a percentage of the total cell population: percentage of phagocytosis and percentage of B lymphocytes.In the constructions, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8.

[0088] The results are presented in Table 4 in four rows which correspond to four independent experiments. [Table 4]. Percentage of phagocytosis and percentage of B lymphocytes % Phagocytosis % Population B NCIT CIT % (ΔCIT-NCIT) NCIT CIT % (ΔCIT-NCIT) 9.28 17.8 +91% 46.1 32.9 - 40% 16.2 20.4 +26% 40.5 30.5 - 33% 3.42 5 +46% 77.1 66.7 - 16% 6.28 8.95 +42% 77.3 69.3 - 12%

[0089] The results obtained confirm that phagocytosis activity is increased in the presence of citrullinated hybrids (increased phagocytosis associated with a decrease in the B lymphocyte population). Indeed, in the 4 independent experiments, 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 coated with the FcSDHFSTIE-N3-DBCO-PEG3-β60-74Cit (CIT) hybrid versus FcSDHFSTIE-N3-DBCO-PEG3-β60-74Arg (NCIT) hybrid. Example 6 : Phagocytosis of B lymphocytes loaded with recombinant monoclonal human ACPA 2H06 mutated SDH, by macrophages armed with the Fc-WT-PEG-α621-635cit hybrid

[0090] 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 for 1h at 37°C with recombinant human monoclonal ACPA 2H06 with SDH-mutated Fc, at a concentration of 10 µg / mL. Such antibodies can be obtained as in Example 3. In parallel, macrophages labeled with red PKH-26 were exposed to the hybrids Fc-WT-PEG-α621-635cit (a wild-type Fc fragment 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 which is itself linked to the peptide represented by SEQ ID NO: 18) and Fc-WT-PEG-α621-635arg (this is the hybrid Fc-WT-PEG-α621-635cit, except that the peptide is not citrullinated), at 5 µg / mL for 1 h at 37°C.After washing, the cells were incubated at a ratio of 1 B lymphocyte to 1 macrophage for 2 hours at 37°C. The cells were then detached and analyzed by flow cytometry. The percentage of phagocytosis corresponds to the percentage of cells labeled with the two fluorochromes (PKH-67 green / PKH-26 red). In the constructs, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4, or 8.

[0091] The results are presented in Table 5 in six rows which correspond to five independent experiments. NCIT represents the result with the peptide Fc-WT-PEG-α621-635arg, CIT represents the result with the peptide Fc-WT-PEG-α621-635cit, and (Δ CIT-NCIT) represents the difference between the OD obtained in the presence of Fc-WT-PEG-α621-635cit and the OD obtained in the presence of Fc-WT-PEG-α621-635arg, divided by the OD obtained in the presence of Fc-WT-PEG-α621-635arg. [Table 5]. Percentage of phagocytosis and percentage of B lymphocytes % Phagocytosis NCIT CIT % (ΔCIT-NCIT) 4.3 7.0 +63% 15.8 19.2 +21% 4.6 7.3 +58% 12.8 18.2 +42% 16.9 21.4 + 27% 15,7 20.1 + 28% % Population B NCIT CIT % (ΔCIT-NCIT) 66.6 61.2 - 8% 78.7 76.6 - 3% 67.6 57.2 - 15% 54.2 47.7 - 12% 44.5 37 - 17% 43.8 38.5 - 12%

[0092] The results show that phagocytic activity is increased in the presence of citrullinated hybrids: an increase in phagocytosis (from 21% to 63%), associated with a decrease in the B lymphocyte population (from 3% to 17%). In each of the six experiments, the percentage of double-positives is higher, therefore phagocytosis is increased and the B lymphocyte population decreases when macrophages are armed with Fc-WT-PEG-α621-635cit (CIT) hybrids versus Fc-WT-PEG-α621-635arg (NCIT) hybrids. The results are also presented in Figures 8A and 8B . Example 7 : Stability of binding of Fc-SDH A488 and Fc-SDH β60 citrullinated peptide hybrids to NK cells after 30 min, 24 h, and 48 h at physiological temperature

[0093] NK cells were incubated for 30 min, 24 h, or 48 h at 37°C in the presence of FcSDHFSTIE-N3-DBCO-A488 (an Fc fragment containing the S239D, H268F, S324T, and I332E mutations linked to at least one PEGn spacer, which is itself coupled to an azide covalently linked to a DBCO, the molecule being labeled with fluorochrome A488) or FcSDHFSTIE-N3-DBCO-PEG3-β60-74cit-lysineA488 (an Fc fragment containing the S239D, H268F, S324T, and I332E mutations 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 SEQ ID NO peptide). : 19, the molecule being labeled with the fluorochrome A488, which is linked to the molecule via a lysine residue). 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.

[0094] The results are presented in Tables 6 (30 minutes), 7 (24 hours), and 8 (48 hours) below. NM represents unlabeled NK cells, without antibodies. [Table 6]. Results at 30 minutes MFI RATIO NM 562 / FcSDHFSTIE-N3-DBCO-PEG3-β60-74cit-lysineA488 4588 8,163 FcSDHFSTIE-N3-DBCO-A488 2973 5,289 [Table 7]. 24-hour results MFI RATIO NM 558 / FcSDHFSTIE-N3-DBCO-PEG3-β60-74cit-lysineA488 4091 7,324 FcSDHFSTIE-N3-DBCO-A488 2353 4,212 [Table 8]. Results at 48 hours MFI RATIO NM 494 / FcSDHFSTIE-N3-DBCO-PEG3-β60-74cit-lysineA488 4336 8,77 FcSDHFSTIE-N3-DBCO-A488 1710 3,460

[0095] The results presented in Tables 6 to 8 show that FcSDHFSTIE-N3-DBCO-A488 and FcSDHFSTIE-N3-DBCO-PEG3-β60-74cit-lysine A488 remain bound to the membrane of NK cells for at least 48 hours at physiological temperature and that the binding of the peptide to the Fc fragment does not prevent its interaction with the Fc gamma receptors of NK cells. Example 8 : Binding of various forms of Fc (WT, SDH and LALAPG) labeled with fluorochrome A647 onto NK cells

[0096] NK cells were incubated for 30 min at 37°C in the presence of the FcSDHFSTIE-N3-DBCO-A647 fragment (an Fc fragment containing the S239D, H268F, S324T, and I332E mutations, which is linked to at least one PEGn spacer that is itself coupled to an azide covalently linked to a DBCO; the molecule is labeled with the fluorochrome A647) at 10 µg / mL. This construct is referred to as SDH in Table 9 below. The experiment was also carried out with the following hybrids: FcWT-N3-DBCO-A647 (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 DBCO, the molecule being labeled with the fluorochrome A647), or FcLALAPG-N3-DBCO-A647 (an Fc fragment comprising the L234A, L235A and P329G mutations that 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 A647), at the same concentrations.These two constructs are respectively designated WT and LALAPG in Table 9 below. In the constructs, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4, or 8. The binding of Fc fragments to NK cells was analyzed by cytofluorometry. The results are presented in Table 9. [Table 9]. Results MFI RATIO NEG 212.08 / WT 10 µg / mL 1105.89 5.21 SDH 10 µg / mL 38545.45 181.74 LALAPG 10 µg / mL 450.43 2.12

[0097] The results presented in Table 9 show that the FcWT-N3-DBCO-A647 and FcSDHFSTIE-N3-DBCO-A647 fragments bind to the NK cell membrane. The binding of FcSDHFSTIE-N3-DBCO-A647 is significantly greater than that of FcWT-N3-DBCO-A647. Example 9 : Binding of the Fc-SDH fragment labeled with fluorochrome A647 onto NK cells compared to binding of the same hybrids in the presence of human IgG at increasing concentrations, up to physiological serum concentration

[0098] NK cells were incubated for 30 min at 37°C either in the presence of the FcSDHFSTIE-N3-DBCO-A647 fragment (an Fc fragment comprising the S239D, H268F, S324T and I332E mutations 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 A647) at 10 µg / mL, or in the presence of this same FcSDHFSTIE-N3-DBCO-A647 fragment at 10 µg / mL, co-incubated with human IgG at increasing concentrations (1, 10, 100, 1,000, 10,000 µg / mL). In the constructs, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8. The binding of the FcSDHFSTIE-N3-DBCO-A647 fragment (called SDH) to NK cells was analyzed by cytofluorometry.

[0099] The results are presented in Table 10. [Table 10]. Results MFI RATIO NEG 212.08 / SDH 10 µg / mL 38545.45 181.74 SDH (1µg / mL IgG) 37899.98 178.70 SDH (10µg / mL IgG) 38771.43 182.81 SDH (100µg / mL IgG) 36270.21 171.02 SDH (1000µg / mL IgG) 29526.19 139.22 SDH (10000µg / mL IgG) 20486.26 96.59

[0100] The results show a dose-dependent decrease in the binding of FcSDHFSTIE-N3-DBCO-A647 to NK cells from 100 µg / mL of IgG, but this remains effective and high, even in the presence of an IgG concentration 1,000 times greater than that of the Fc fragment (10,000 µg / mL) which corresponds to the physiological serum concentration of IgG in humans. Example 10: Lysis by human NK cells of cells from a malignant human B cell line with plasmablasts expressing a recombinant monoclonal ACPA on its membrane, in the presence of the hybrids FcWT-N3-DBCO-PEG3-α621-635Cit and FcSDH-N3-DBCO-PEG3-α621-635Cit

[0101] WTαCIT represents the hybrid comprising a wild-type Fc fragment which is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne DBCO which is coupled to a PEGn spacer which is itself linked to the peptide represented by SEQ ID NO: 18; WTαArg corresponds to the WTαCIT hybrid except that the peptide of SEQ ID NO: 18 is not citrullinated; SDHαCIT represents an Fc fragment comprising the mutations S239D, H268F, S324T and I332E which is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne DBCO which is coupled to a PEGn spacer which is itself linked to the peptide represented by SEQ ID NO: 18; SDHαArg corresponds to the SDHαCIT hybrid, with the difference that the peptide in SEQ ID NO: 18 is not citrullinated. In the constructs, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4, or 8.

[0102] The experiment was performed in 96-well round-bottom plates. Nalm6 cell lines (transduced for membrane expression of recombinant human monoclonal ACPA 2H06) were used as target cells. 100,000 cells / well were incubated in the presence of either WTαCit or SDHαCit hybrid molecules at 15 µg / mL and freshly purified NK cells (labeled with purple cell tracker) as effector cells, also at 100,000 cells / well, for 17 h at 37°C. WTαArg and SDHαArg hybrid molecules, used as specificity controls, were incubated under the same conditions. As negative controls, Nalm6 cells were incubated in the absence of hybrids, either alone or in the presence of NK cells. Cell lysis was measured by flow cytometry using the fluorescent probe 7-AAD. The results show the percentage of cells positive for this marker (% of dead cells).(%) (ΔCIT-Arg) represents the difference between the percentages of dead 7-AAD-positive cells in the presence of WT and SDH citrullinated peptide hybrids versus their non-citrullinated homologs, divided by the percentage of 7-AAD-positive cells in the presence of the corresponding non-citrullinated hybrid.

[0103] The results are presented in Figure 9 and in Table 11. [Table 11]. Results % 7-AAD + % (ΔCIT-Arg) Nalm6 8 Nalm6 + NK 45 Nalm6 + NK + WT αCIT 65 + 62 % Nalm6 + NK + WT αArg 40 Nalm6 + NK + SDH αCIT 55 + 83 % Nalm6 + NK + SDH αArg 30

[0104] The results show that citrullinated hybrids (WT and SDH) are able to specifically enhance ADCC lysis of Nalm6 ACPA-positive target cells, induced by NK cells. This increase in mortality is 62% for WT hybrids and 83% for SDH hybrids. Example 11 Membrane expression of monoclonal ACPA 2H06 by the Nalm6 B lymphoblastic cell line after transduction

[0105] A- The human B lymphoblastic cell line "Nalm6" was transduced to induce the expression of recombinant monoclonal ACPA 2H06 at its membrane. Transduction efficiency was analyzed by flow cytometry using dual labeling with an anti-Fab IgG antibody and a fluorescent avidin-fixed biotinylated α621-635cit tetramer. B- Sorting from 70 million Nalm6 cells allowed the recovery of Nalm6 Fab+ cells, from which a pool of 300,000 cells was recultured. After proliferation, the enrichment of the transduced Nalm6 2H06 cell line was confirmed by flow cytometry. C- The Fc-WT-PEG-α621-635cit and arg hybrids were labeled with Alexa Fluor 647 using the Lightning Link kit. The untransduced Nalm6 (NT) and Nalm6 2H06 lines were incubated with the Fc-WT-PEG-α621-635 hybrids. After washing, the membrane fixation of the Fc-WT-PEG-α621-635 hybrids was analyzed by flow cytometry.

[0106] The results are presented in the Figure 10 and in Table 12.

[0107] Table 12 shows the mean fluorescence intensities (MFI) and the MFI ratio between the condition with fluorescent hybrid and the control condition where cells are not labeled. nm represents unlabeled Nalm6 cells, without fluorescent hybrids. [Table 12]. Results MFI - Nalm6 NT Ratio - Nalm6 NT MFI - Nalm6-2H06 Ratio - Nalm6-2H06 nm 37 37 cit 263 7 10783 290 arg 260 7 263 7

[0108] The results show that after sorting and cell proliferation, 89.1% of Nalm6 cells express an IgG Fab, that of ACPA 2H06, thus validating the success of the transduction and the establishment of the Nalm6 2H06 cell line (B). The results show a strong interaction between the Nalm6 2H06 cell line and the fluorescent cit Fc-WT-PEG-α621-635 hybrid, while no interaction exists with the non-citrullinated Fc-WT-PEG-α621-635 hybrid (C). Dual labeling with the fluorescent cit Fc-WT-PEG-α621-635 hybrid and the fluorescent IgG anti-Fab antibody shows a perfect correlation between the two labels on the Nalm6 2H06 cell line, whereas neither labels the untransduced Nalm6 cell line (A). These results show that the binding of the Fc-WT-PEG-α621-635 cit hybrid is strictly linked to the expression of membrane ACPA 2H06. Example 12 : Phagocytosis of Nalm6 ACPA+ 2H06 transduced B-lineage cells by macrophages pre-armed with Fc-peptide α621-635 hybrids

[0109] Macrophages were labeled with the membrane fluorochrome PKH-67 green and then incubated with Fc-WT-PEG-α621-635, Fc-SDH-PEG-α621-635, or Fc-GASDIE-PEG-α621-635 hybrids, in citrullinated (Cit) or non-citrullinated (Arg) form, at a concentration of 5 µg / ml for 60 min at 37°C. Human B lymphoblastic Nalm6 cell line transduced for the expression of the monoclonal ACPA 2H06 at its membrane were labeled with PKH-26 red (a fluorescent lipid marker) and then incubated with macrophages at a ratio of 2 Nalm6 target cells to 1 macrophage. After 2 hours at 37°C, the macrophages were detached and then the cell populations were analyzed by flow cytometry.

[0110] The results of the Figure 11AThese results show that at T2, in the absence of hybrids, there is an increase in double-positive cells, indicating spontaneous basal phagocytosis of Nalm6 cells. At 2 hours, in the presence of hybrids, regardless of the hybrid used (WT, SDH, or GASDIE), a sharp increase in double-positive cells and a collapse in the number of target cells are observed when macrophages are armed with citrullinated hybrids, but this is absent with the non-citrullinated versions of the hybrids. The percentage of double-positive cells drops from 52% to 14.6% in the presence of the Fc-WT-PEG-α621-635 hybrid, to 24.5% for Fc-SDH-PEG-α621-635, and to as low as 8% for Fc-GASDIE-PEG-α621-635, which is the most effective.

[0111] The synthesis of 3 independent experiments (see Figure 11B ), confirms that the effect is reproducible and significant under all conditions, with an average increase of 70% in double-positive cells and a decrease of at least 50% in Nalm6 target cells. Example 13 Phagocytosis of Nalm6 ACPA+ 2H06 B cell lineage cells by pre-armed macrophages in the presence of Fc-peptide α621-635 hybrids at various concentrations

[0112] Macrophages were labeled with the membrane fluorochrome PKH-67 green and then armed with the hybrids Fc-WT-PEG-α621-635, Fc-SDH-PEG-α621-635, Fc-GASDIE-PEG-α621-635 or Fc-GASDALIE-PEG-α621-635, in citrullinated (Cit) or non-citrullinated (Arg) form, at different concentrations (5µg / ml; 1µg / ml; 100ng / ml; 50ng / ml; 10ng / ml; 5ng / ml) for 60 min at 37°C. Nalm6 B cell line transduced for the expression of the monoclonal ACPA 2H06 were labeled with PKH-26 red (a fluorescent lipid marker) and then incubated with armed macrophages at a ratio of one Nalm6 target cell per macrophage. After 2 hours at 37°C, the macrophages were detached and the cell populations analyzed by flow cytometry.

[0113] The results presented in the Figure 12They show a sharp increase in the percentage of double-positive cells (A) associated with a sharp decrease in the percentage of target cells (B) when macrophages are armed with citrullinated Fc-PEG-α621-635 hybrids, whether WT, SDH, GASDIE, or GASDALIE. Both are absent with non-citrullinated hybrids (Fc-PEG-α621-635 Arg). This specific phagocytosis is correlated with the concentration of Fc-PEG-α621-635 hybrids in the solution used to arm them. The model is particularly robust because phagocytosis is still observed for all citrullinated hybrids up to 50 ng / mL. Due to their increased affinity for FcγR, the Fc-GASDIE-PEG-α621-635Cit and Fc-GASDALIE-PEG-α621-635Cit hybrids are more effective than Fc-WT-PEG-α621-635Cit or Fc-SDH-PEG-α621-635Cit, and remain active beyond 50 ng / ml. Example 14 : Confirmation of the phagocytosis process of the transduced Nalm6 ACPA+ 2H06 cell line by macrophages armed with FcWT-PEG-peptide α621-635 hybrids

[0114] To confirm that the destruction of Nalm6 2H06 B cells results from phagocytosis, Nalm6 2H06 B cells were labeled with a pH-sensitive dye, pHRodoSE, which exhibits low fluorescence intensity at neutral pH but intense fluorescence in acidic environments. The pH within the intracytoplasmic vesicles (lysosomes) where phagocytosed elements are found is acidic. Therefore, non-phagocytosed cells are undetectable, while phagocytosed cells, internalized into the lysosomal compartment, become fluorescent.

[0115] Nalm6 2H06 cells were labeled with pHRodoSE (Thermofisher) at 20 ng / ml. These labeled Nalm6 cells were incubated with macrophages, either armed or unarmed with Fc-WT-PEG-α621-635 Cit or Arg hybrids, at 5 µg / ml for 1 h at 37°C. The cells were then treated or untreated with cytochalasin D for 30 min at 2 µg / ml, followed by 1 µg / ml during phagocytosis. The cells were incubated for 2 h at 37°C, after which the macrophages were detached and the cell populations analyzed by flow cytometry or stained with May-Grünwald-Giemsa (MGG) for morphological analysis by light microscopy.

[0116] The results presented in the Figure 13These results show the appearance of a pHRodoSE-positive population (2nd peak) in the presence of macrophages armed with the Fc-WT-PEG-α621-635 Cit hybrid, a population that is absent when the macrophages are armed with the non-citrullinated version of the hybrids. In the presence of cytochalasin D (a phagocytosis inhibitor), this population does not appear, confirming that it corresponds to a population of macrophages that have specifically phagocytosed Nalm6 2H06 B cells. These results are confirmed by optical microscopy analysis in which numerous phagocytosed Nalm6 2H06 B cells are observed in an intracytoplasmic situation within macrophages when they are armed with citrullinated Fc-WT-PEG-α621-635 hybrids (top panel), whereas this situation is totally absent when the macrophages were armed with the non-citrullinated form of the hybrid. Example 15 : Phagocytosis of the Nalm6 ACPA+ 2H06 transduced cell line by macrophages pre-armed with Fc-peptide α621-635 hybrids or incubated in the presence of Fc-peptide α621-635 hybrids

[0117] Nalm6 2H06 B cell lines were labeled with the membrane fluorochrome PKH-67 green and macrophages with PKH-26 red. Fc-WT-PEG-α621-635 and Fc-GASDIE-PEG-α621-635 hybrids, in citrullinated (Cit) or non-citrullinated (Arg) form, were either incubated for 1 h at 37°C with macrophages at a concentration of 5 µg / ml (arming followed by washing), before being placed in contact with target cells at a ratio of 1 Nalm6 target cell to 1 macrophage for 2 h at 37°C, or incubated at the same concentration in the medium containing Nalm6 target cells and macrophages at the same 1:1 ratio for 2 h at 37°C. The macrophages were then detached and the cell populations analyzed by flow cytometry.

[0118] The results presented in the Figure 14They show a sharp increase in double-positive cells (phagocytosis), associated with a collapse in the Nalm6 2H06 target cell population, when macrophages were either pre-armed or incubated with the citrullinated form of the Fc-WT-PEG-α621-635 or Fc-GASDIE-PEG-α621-635 hybrids. This result is specific, as it was not observed with the non-citrullinated forms of the hybrids. Example 16: Phagocytosis of the Nalm6 ACPA+ 2H06 transduced cell line by macrophages incubated with Fc-PEG-peptide α621-635 hybrids, in the presence or absence of human IgG at physiological serum concentration

[0119] Nalm6 2H06 cell lines were labeled with the fluorescent dye CFSE (CarboxyFluorescein Succinimidyl Ester) and then incubated with macrophages in the presence of Fc-WT-PEG-α621-635 or Fc-GASDIE-PEG-α621-635 hybrids, citrullinated (Cit) or non-citrullinated (Arg), at a concentration of 1 µg / mL, for 2 h at 37°C, with or without competing human IgG at a physiological serum concentration of 10 mg / mL (hybrid-to-IgG concentration ratio: 1 / 10,000). The macrophages were detached and then labeled with the anti-CD11b antibody BV421, and the cell populations were analyzed by flow cytometry.

[0120] The results presented in the Figure 15show that in the absence of competing IgG, the citrullinated forms of the 2 soluble hybrids Fc-WT-PEG-α621-635 and Fc-GASDIE-PEG-α621-635 incubated with the cells, induce almost total and specific phagocytosis because it is absent with the non-citrullinated forms, of the Nalm6 2H06 cell population. The presence of IgG induces a relative but significant inhibition (p<0.01) of spontaneous phagocytosis at T2, a near-total and significant inhibition of phagocytosis induced by the Fc-WT-PEG-α621-635cit hybrid (p<0.05), whereas this inhibition is only partial and not significant with the Fc-GASDIE-PEG-α621-635 hybrid, whose mutated Fc is more affin for macrophage Fc gamma receptors than that of serum IgG. Example 17 Phagocytosis of the Nalm6 ACPA+ 2H06 transduced cell line by human macrophages incubated with Fc-peptide α621-635 hybrids, in the presence or absence of human IgG at physiological concentration or human serum

[0121] Nalm6 2H06 cell lines were labeled with the fluorescent dye CFSE (CarboxyFluorescein Succinimidyl Ester) and then incubated with macrophages in the presence of Fc-WT-PEG-α621-635 or Fc-GASDIE-PEG-α621-635 hybrids in citrullinated (cit) or non-citrullinated (arg) form, at a concentration of 10 µg / mL. The experiment was conducted for 2 hours at 37°C in the presence or absence of competing human IgG at a concentration of 10 mg / mL or a mixture of human sera with the same IgG concentration. The concentration ratio between hybrids and IgG is therefore 1 / 1000. The cells were collected and labeled with an anti-CD11 b BV421 antibody to identify macrophages and then analyzed by flow cytometry.

[0122] The results presented in the Figure 16These results show that the two citrullinated hybrids, Fc-WT-PEG-α621-635 and Fc-GASDIE-PEG-α621-635, used at a concentration of 10 µg / ml, specifically and very efficiently induce phagocytosis of Nalm6 2H06 cells. Through competition with the hybrids, IgG inhibits phagocytosis of Nalm6 2H06 cells by macrophages, but the inhibitory effect is much stronger for the Fc-WT (wild-type) hybrid than for the GASDIE hybrid (mutated, with a higher affinity for macrophage Fc gamma receptors). Equivalent results are obtained in the presence of human serum, demonstrating that it therefore contains no other inhibitory factors of phagocytosis besides IgG. Example 18: Phagocytosis of the transduced Nalm6 ACPA+ 2H06 cell line by human macrophages pre-armed with Fc-peptide α621-635 hybrids, in the presence of the untransduced Nalm6 cell line

[0123] Nalm6 2H06 cell lines were labeled with the fluorescent dye CFSE (CarboxyFluorescein Succinimidyl Ester) and then mixed with untransduced Nalm6 NT cells in a 1:4 ratio (1 transduced cell to 4 untransduced cells). Macrophages were pre-armed with Fc-WT-PEG-α621-635 or Fc-GASDIE-PEG-α621-635 hybrids, in citrullinated (Cit) or non-citrullinated (Arg) form, by incubation for 1 h at a concentration of 1 g / mL at 37°C. Nalm6 cells were then incubated with macrophages for 2 hours at 37°C. The macrophages were then detached, the total cell population collected, and labeled with an anti-CD19 APC antibody to differentiate between Nalm6 2H06 cells and non-transduced Nalm6 NT cells. The total cell population was then analyzed by flow cytometry.

[0124] To confirm the specificity of phagocytosis towards ACPA+ cells in the presence of citrullinated hybrids, we co-incubated Nalm6 2H06 target cells (ACPA+) and untransduced Nalm6 NT cells (ACPA-). The results presented in the Figure 17 The results show that only Nalm6 cells expressing the membrane-bound ACPA 2H06 (CFSE+CD19+) disappear and are phagocytosed by macrophages when these macrophages have been previously armed with the citrullinated (Cit) form of Fc-WT-PEG-α621-635 or Fc-GASDIE-PEG-α621-635 hybrids. In contrast, the proportion of wild-type, untransduced Nalm6 NT cells that are ACPA- (CD19+CFSE-) remains unchanged under all conditions, confirming that the induced interaction is indeed specific and due to the binding of the citrullinated peptide and ACPA. The same results were obtained for a ratio of 1:9 (1 Nalm6 2H06 cell for every 9 untransduced Nalm6 NT cells). Example 19 : Specific destruction in vivo of the Nalm6 ACPA+ 2H06 cell line by human NK cells in the presence of Fc-peptide α621-635 hybrids, in SCID / BEIGE immunodeficient mice

[0125] Membrane expression of ACPA 2H06 on Nalm6 2H06 target cells was monitored by labeling with an anti-Fab antibody, and the lytic activity (ADCC) of effector human NK cells was verified. in vitro.Nalm6 2H06 target cells were labeled with the fluorescent marker CTV (CellTrace Violet) at a high concentration (5 µM), while untransduced Nalm6 NT cells were labeled with the same marker but at a concentration ten times lower (0.5 µM). By flow cytometry, the highly labeled cells (Nalm6 2H06 CTV high< ) can thus be distinguished from the weakly labeled cells (Nalm6 NT CTV low< ). Immunodeficient SCID / BEIGE mice received 50µg of anti-FcR antibody intraperitoneally (IP) to block FcR in murine peritoneal macrophages and then, 30 minutes later, Nalm6 2H06 target cells and untransduced Nalm6 NT cells were injected IP in the presence, or not, of freshly prepared human NK cells and citrullinated (cit) or uncitrullinated (arg) Fc-WT-PEG-α621-635 hybrids.

[0126] Four groups of 7 SCID / BEIGE mice received the following cell and hybrid combinations via intraperitoneal injection (IP): 1 / 2.10 5< target cells Nalm6-2H06 + 2.10 5< Nalm6 NT cells 2 / 2.10 5< target cells Nalm6-2H06 + 2.10 5< Nalm6 NT cells + 2.10 5< NK cells 3 / 2.10 5< target cells Nalm6-2H06 + 2.10 5< Nalm6 NT cells + 2.10 5< NK cells + 3µg / mouse of Fc-WT-PEG-α621-635 Cit hybrid 4 / 2.10 5< target cells Nalm6-2H06 + 2.10 5< Nalm6 NT cells + 2.10 5< NK cells + 3µg / mouse of Fc-WT-PEG-α621-635 Arg hybrid

[0127] Five hours after injection, the cells were recovered by peritoneal lavage with 2% PBS FBS and then analyzed by flow cytometry after labeling with 7AAD and Annexin (Becton Dickinson kit).

[0128] The results presented in the Figure 18show that in the peritoneal cavity of SCID / BEIGE immunodeficient mice, the Fc-WT-PEG-α621-635 Cit hybrid induces the total and specific destruction of Nalm6 2H06 target cells by NK cells. Indeed, the CTV high peak corresponding to Nalm6 2H06 cells disappears exclusively in the presence of NK cells and the Fc-WT-PEG-α621-635 Cit hybrid.

[0129] This highly significant result (***, p<0.001) provides proof in vivo of the efficacy and specificity of ADCC lysis of ACPA+ cells by human NK cells in the presence of Fc-citrullinated peptide hybrids carrying ACPA target epitopes. Example 20 Specific destruction in vivo of the Nalm6 ACPA+ 2H06 cell line by human macrophages pre-armed with Fc-peptide α621-635 hybrids, in immunodeficient SCID / BEIGE mice

[0130] Membrane expression of ACPA 2H06 on Nalm6 2H06 target cells was monitored by labeling with an anti-Fab antibody. Monocytes were purified from peripheral blood of a healthy donor. Monocyte differentiation into macrophages was induced in the presence of 100 ng / ml of M-CSF, and the phagocytic activity of the resulting macrophages was assessed. in vitro.

[0131] Before intraperitoneal (IP) injection, macrophages were armed with the different citrullinated (cit) or non-citrullinated (arg) Fc-WT-PEG-α621-635 hybrids and Nalm6-2H06 target cells were labeled with the fluorescent marker CTV (CellTrace Violet) at 0.5µM: strongly fluorescent when free (Nalm6 2H06 CTV high< ), these cells exhibit a much weaker fluorescence (Nalm6-2H06 CTV low< ) after phagocytosis by macrophages.

[0132] Four groups of 5 SCID / BEIGE mice received target cells via IP, in the presence or absence of pre-armed fresh macrophages and, or absence, of FcWT-PEG-α621-635 cit or arg hybrids, according to the following combinations: 1 / 2.10 5< Nalm6-2H06 target cells 2 / 2.10 5< Nalm6-2H06 target cells + 2.10 5< unarmed macrophages 3 / 2.10 5< Nalm6-2H06 target cells + 2.10 5< Fc-WT-PEG-α621-635 Cit hybrid pre-armed macrophages 4 / 2.10 5< Nalm6-2H06 target cells 2.10 5< Fc-WT-PEG-α621-635 Arg hybrid pre-armed macrophages

[0133] Two hours after injection, the cells were recovered by peritoneal lavage with 5% FBS PBS, the erythrocytes were removed by gentle lysis and the cells were labeled with an anti-human CD45-FITC antibody and then with 7AAD (Becton Dickinson kit) before flow cytometry analysis.

[0134] The results presented in the Figure 19show that in the peritoneal cavity of SCID / BEIGE immunodeficient mice, macrophages loaded with the Fc-WT-PEG-α621-635 cit hybrid specifically phagocytose Nalm6 2H06 target cells, their decrease compared to the control (Fc-WT-PEG-α621-635 arg hybrid, non-citrullinated) being significant (p<0.05).

[0135] This result provides proof in vivo of the efficiency and specificity of phagocytosis of ACPA+ cells by human macrophages loaded with Fc-citrullinated peptide hybrids carrying the target epitopes of ACPA.

Claims

1. A hybrid molecule comprising at least one antibody Fc fragment covalently linked to at least one fibrin-derived peptide having at least one citrullyl residue, at least one spacer being optionally present between said Fc fragment and said peptide, said peptide being recognized by the anti-citrullinated protein autoantibodies and being derived from all or part of the sequence of the α or β chain of a vertebrate fibrin by substitution of at least one arginyl residue by a citrullyl residue.

2. A hybrid molecule according the preceding claim, said vertebrate fibrin being a mammalian fibrin, more particularly a human fibrin.

3. A hybrid molecule according to any 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, wherein n represents an integer between 1 and 100, preferably between 1 and 10 and in particular 1, 2, 3, 4 or 8.

4. A hybrid molecule according to any one of the preceding claims, wherein said peptide comprises at least one citrullyl residue, and is selected from the group consisting of: a) a peptide defined by the sequence X1PAPPPISGGGYX2AX3 (SEQ ID NO: 1) wherein X1, X2, and X3 each represent a citrullyl residue or an arginyl residue, and at least one of the X1 or X2 or X3 residues is a citrullyl residue; b) a peptide defined by the sequence GPX1VVEX2HQSACKDS (SEQ ID NO: 2) wherein X1 and X2 each represent a citrullyl residue or an arginyl residue, and at least one of the X1 or X2 residues is a citrullyl residue; c) a peptide defined by the sequence SGIGTLDGFX1HX2HPD (SEQ ID NO: 3) wherein X1 and X2 each represents a citrullyl residue or an arginyl residue, and at least one of the X1 or X2 residues is a citrullyl residue; d) a peptide defined by the sequence VDIDIKIX1SCX2GSCS (SEQ ID NO: 4) wherein X1 and X2 each represent a citrullyl residue or an arginyl residue, and at least one of the X1 or X2 residues is a citrullyl residue; e) a peptide defined by the sequence X1GHAKSX2PVX3GIHTS (SEQ ID NO: 12) wherein X1, X2 and X3 each represent a citrullyl residue or an arginyl residue, and at least one of the X1 or X2 or X3 residues is a citrullyl residue; f) a peptide comprising at least 5 consecutive amino acids, including at least one citrullyl residue, from one of the peptides a) to e) above.

5. A hybrid molecule according to any one of claims 1-3, wherein said peptide is selected from the group consisting of: 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: 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 and SEQ ID NO: 23, more particularly selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 18 and SEQ ID NO: 19.

6. A hybrid molecule according to any one of the preceding claims, wherein the said Fc fragment is a human Fc fragment, in particular of IgG, more particularly of IgG1.

7. A hybrid molecule according to any 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 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 bound 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 bound to a spacer, itself coupled to an azide, or - the Fc fragment is bound 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 bound 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 bound to at least one spacer, itself coupled to an azide, and said peptide is bound to a spacer, itself coupled to an alkyne, such as a cyclooctyne, and in particular DBCO, or - the Fc fragment is bound to at least one spacer, itself coupled to an alkyne, such as a cyclooctyne, and in particular DBCO, and said peptide is bound 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. A hybrid molecule according to any one of the preceding claims, said molecule being represented by: - a wild-type Fc fragment that is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a cyclooctyne that is coupled to a peptide represented by SEQ ID NO: 19 or SEQ ID NO: 18, - an Fc fragment comprising the S239D, H268F, S324T and I332E mutations that is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a cyclooctyne that is coupled to a peptide represented by SEQ ID NO: 19 or SEQ ID NO: 18, - an Fc fragment comprising the L234A, L235A and P329G mutations that is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a cyclooctyne that is coupled to a peptide represented by SEQ ID NO: 19 or SEQ ID NO: 18, - a wild-type Fc fragment that is bound to a PEGn spacer that is itself coupled to at least one azide covalently bound to a cyclooctyne coupled to a PEGn spacer that is itself bound to a peptide represented by SEQ ID NO: 19 or SEQ ID NO: 18, - an Fc fragment comprising the S239D, H268F, S324T and I332E mutations that is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a cyclooctyne coupled to a PEGn spacer that is itself bound to a peptide represented by SEQ ID NO: 19 or SEQ ID NO: 18, n preferably representing an integer between 1 and 10, more particularly 1, 2, 3 or 4.

10. A 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 diseases associated with the production of anti-citrullinated protein autoantibodies, in particular Gougerot-Sjögren syndrome and rheumatoid arthritis.

11. A hybrid molecule according to any one of the preceding claims for use as a medicinal product in combination with a second hybrid molecule, said second hybrid molecule comprising at least one fibrin-derived peptide having at least one citrullyl residue, said peptide being covalently bound to at least one antibody or to at least one antibody fragment, said antibody or fragment being capable of binding to CD38 and / or CD138, one or more spacers being optionally present between said peptide and said antibody or said fragment.