Hybrid molecule comprising an antibody fc fragment and at least one fibrin-derived citrullinated peptide, and uses thereof

Abstract

The invention relates to a hybrid molecule comprising at least one antibody Fc fragment covalently bound to at least one fibrin-derived peptide having at least one citrullyl residue, the uses of such a hybrid molecule, and the method for producing same.

Description

[0001] Hybrid molecule comprising an antibody Fc fragment and at least one citrullinated peptide derived from fibrin, and its uses

[0002] The present invention relates to a hybrid molecule comprising at least one antibody Fc fragment covalently linked to at least one fibrin-derived peptide comprising one or more citrullyl residue(s), the uses of such a hybrid molecule, as well as its production method.

[0003] Background to the invention

[0004] Rheumatoid arthritis is the most common inflammatory rheumatism or arthritis, but also the most common autoimmune disease. The disease is characterized by chronic inflammation of the synovial joints leading to irreversible joint destruction.

[0005] The presence of class G autoantibodies directed against citrullinated proteins, called anti-citrullinated protein autoantibodies (ACPAs), is highly specific for rheumatoid arthritis. Sera from patients containing ACPAs, lymphocyte cells that express them on their membranes, and patients themselves are said to be 'ACPA-positive'. Several studies have demonstrated that these ACPAs are at the heart of the autoimmune reactions specific to rheumatoid disease and thus represent a therapeutic target of choice.

[0006] The antigenic targets of ACPAs have been characterized. In particular, they are specifically directed against deiminated or citrullinated forms of the a and b polypeptide chains of fibrin, a protein abundant in inflammatory synovial tissue. This citrullination corresponds to the enzymatic deimination of arginyl residues of the polypeptide chains, under the action of peptidyl-arginine deiminases (PADs).

[0007] More specifically, the immunodominant epitopes recognized by ACPA on the a and b polypeptide chains of fibrin have been characterized and published, in particular in applications PCT / FR00 / 01857 or PCT / FR2007 / 000758. The five citrullinated peptides carrying the immunodominant epitopes are more particularly the peptides named a36-50Cit (as represented in SEQ ID NO: 5), a171-185Cit (as represented in SEQ ID NO: 6), a501-515Cit (as represented in SEQ ID NO: 14), a621-635Cit (as represented in SEQ ID NO: 18) and b60-74OK (as represented in SEQ ID NO: 19). Sera from ACPA-positive patients recognize one or more of these five peptides.

[0008] Secreted into rheumatoid synovial tissue by local plasma cells, ACPAs present a high concentration there, close to their main target, citrullinated fibrin, which is also abundant there in the form of interstitial deposits. The fixation of ACPAs on these deposits and therefore the formation of immobilized immune complexes, which in turn fix rheumatoid factors, other autoantibodies associated with rheumatoid arthritis and also secreted by local plasma cells, triggers a cascade of pro-inflammatory events. 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.

[0009] Currently, there is no cure for rheumatoid arthritis. Treatments are only aimed at treating flare-ups and / or preventing their onset.

[0010] An object of the present invention is thus to provide a treatment for rheumatoid arthritis.

[0011] ACPAs are oligoclonal and therefore secreted by only a few plasma cell clones, themselves resulting from the differentiation of a few B lymphocyte clones.

[0012] In rheumatoid arthritis, B lymphocyte clones express immunoglobulins carrying ACPA specificity on their membrane (these are ACPA-positive B lymphocytes), while 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.

[0013] The present invention is based on the inventors' results showing that it is possible to target B lymphocyte clones expressing ACPAs on their surface and to eliminate them using a hybrid molecule comprising (i) at least one fibrin-derived peptide having at least one citrullyl residue and (ii) a human immunoglobulin Fc fragment. 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 ACPAs) which will then be eliminated, after fixation of the Fc fragment on the Fc receptors, by macrophages (via phagocytosis) and / or NK cells (via antibody-dependent cell-mediated cytotoxicity - ADCC), and / or by activation of the complement cascade.

[0014] By targeting B lymphocyte clones expressing ACPA and cells that differentiate into plasma cells that themselves secrete ACPA, the hybrid molecules of the invention thus aim to eliminate ACPA from the patients' bodies.

[0015] Statement of the invention

[0016] Hybrid molecule according to the invention

[0017] In a first aspect, the invention relates to 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. The scheme of such a construction is presented in Figure 1.

[0018] According to the invention, a “hybrid molecule” means a molecule having at least two components of a different nature, in this case the Fc fragment of the antibody and said peptide.

[0019] According to the invention, an antibody "Fc fragment" means the constant region of an immunoglobulin excluding the first immunoglobulin constant region domain (i.e. CH1-CL). Thus, the Fc fragment refers to a homodimer, each monomer comprising the last two constant domains of IgA, IgD, IgG (i.e. CH2 and CH3), or the last three constant domains of IgE and IgM (i.e. CH2, CH3 and CH4).

[0020] According to the invention, the expression "covalently bonded" means a covalent bond, that is to say a chemical bond in which two atoms share two electrons. Said covalent bond can be polar or non-polar.

[0021] According to the invention, a "spacer" is a binding agent which makes it possible to covalently bind an antibody Fc fragment to said fibrin-derived peptide having at least one citrullyl residue, while distancing said Fc fragment from said peptide (thus reducing any possible steric hindrance). It may be any molecule, and in particular a peptide. Preferably, the spacer does not modify the physicochemical properties of the hybrid molecule.

[0022] The presence of at least one spacer is advantageous: it facilitates the independent accessibility of the two partners of the hybrid molecule (the Fc fragment is more easily accessible to bind to Fc receptors, just as said peptide 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.

[0023] According to one embodiment, the hybrid molecule according to the invention may comprise one or more spacers. Preferably, the hybrid molecule comprises one or two spacers. According to one embodiment, when at least one spacer is present in said hybrid molecule of the invention, said Fc fragment is covalently linked to a spacer, said spacer itself being covalently linked to said peptide. According to another embodiment, when at least two spacers are present in said hybrid molecule of the invention, said Fc fragment is covalently linked to a first spacer, said first spacer itself being covalently linked to a second spacer and the second spacer itself is covalently linked to said peptide. The bond between the Fc fragment and the peptide may therefore be direct, or indirect in the presence of spacers.

[0024] 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 may be linked to one or two peptides. Indeed, the Fc fragment comprises two monomers, and the Fc fragment may thus be covalently linked to a peptide on only one of the two monomers, or the Fc fragment may be covalently linked to a peptide on each monomer. Preferably, when two peptides are linked on the Fc fragment, the two peptides are identical, for example two peptides of SEQ ID NO: 18 or SEQ ID NO: 19.

[0025] According to one embodiment, said spacer is a polymer containing one or more repeating units containing the ether group. According to a particular embodiment, said spacer is polyethylene glycol of formula 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 may 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, ie; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,

[0026] 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,

[0027] 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,

[0028] 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100.

[0029] 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 may be obtained from natural, recombinant or synthetic fibrin or fibrinogen fragments. Such peptides may also be directly synthesized. The amino acids constituting the peptide may be of the L or D series, preferably of the L series. Said substitution may for example be carried out by an enzymatic deimination step under the action of peptidyl-arginine deiminases (PAD). 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.

[0030] According to one embodiment, in said hybrid molecule according to the invention, said peptide is derived from all or part of the sequence of the a or b chain of a vertebrate fibrin, by substitution of at least one arginyl residue by a citrullyl residue. Preferably, said peptide is derived from a sequence of at least 5 consecutive amino acids of the a chain (in particular represented by SEQ ID NO: 27) or b chain (in particular represented by SEQ ID NO: 28), of a vertebrate fibrin. Even more particularly, said vertebrate fibrin is a mammalian fibrin, preferably human.

[0031] According to one embodiment, in said hybrid molecule according to the invention, the peptide has a size of at least 2 consecutive amino acids, 3 consecutive amino acids, 4 consecutive amino acids, preferably 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.

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

[0033] According to one embodiment, in said hybrid molecule according to the invention, the peptide can be modified so as to improve its reactivity with respect to ACPAs. For example, the peptides can be cyclized, the peptides can be of the retro type (the L series amino acids are linked in a sequence inverse to that of the peptide to be reproduced), or of the retro-inverso type (the amino acids are of the D type instead of the natural L series and are linked in a sequence inverse to that of the peptide to be reproduced). According to an even more particular embodiment, in said hybrid molecule according to the invention, the terminal carboxyl function (COOH) of said peptide is replaced by a carboxamide function (CONH2). Preferably, in the peptide of SEQ ID NO: 12, the terminal carboxyl function (COOH) of said peptide is replaced by a carboxamide function (CONH2).For example, the peptide b60-74OK (as represented in SEQ ID NO: 19) advantageously exhibits such a carboxamide function.

[0034] According to another embodiment, in said hybrid molecule according to the invention, the peptide can be modified so as to facilitate its synthesis and / or improve its stability, for example by alkylation. According to an even more particular embodiment, in said hybrid molecule according to the invention, the terminal amine function (NH2) of said peptide is acetylated. Preferably, in the peptide of SEQ ID NO: 1, the terminal amine function of said peptide is acetylated. By way of example, the peptide a621-635Cit (as represented in SEQ ID NO: 18) advantageously has such an acetyl function on its terminal amine (NH2).

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

[0036] According to one 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) a peptide defined by the sequence X1PAPPPISGGGYX2AX3 (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 or X2 or X3 is a citrullyl residue; b) a peptide defined by the sequence GPX1VVEX2HQSACKDS (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 SGIGTLDGFX1HX2HPD (SEQ ID NO: 3) 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;d) a peptide defined by the sequence VDIDIKIX1SCX2GSCS (SEQ ID NO: 4) 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; e) a peptide defined by the sequence X1GHAKSX2PVX3GIHTS (SEQ ID NO: 12) in which X1, X2 and X3 each represent a citrullyl residue or an arginyl residue, and at least one of the residues X1 or X2 or X3 is a citrullyl residue; f) a peptide comprising at least 5 consecutive amino acids, including at least one citrullyl residue, of one of the peptides a) to e) above.;

[0037] According to a particular embodiment, said peptide comprises at least one citrullyl residue, and is chosen from the group consisting of:

[0038] - a peptide defined by the sequence SEQ ID NO: 1 in which at least one residue chosen from Xi or X2 or X3 is a citrullyl residue, or a peptide comprising a fragment of at least 5 consecutive amino acids of said sequence containing said citrullyl residue(s);

[0039] - a peptide defined by the sequence SEQ ID NO: 2 in which at least Xi or X2 is a citrullyl residue, or a peptide comprising a fragment of at least 5 consecutive amino acids of said sequence containing said citrullyl residue(s);

[0040] - 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 of said sequence containing said citrullyl residue(s);

[0041] - 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 of said sequence containing said citrullyl residue(s);

[0042] - a peptide defined by the sequence SEQ ID NO: 12 in which at least one residue chosen from Xi or X2 or X3 is a citrullyl residue, or a peptide comprising a fragment of at least 5 consecutive amino acids of said sequence containing said citrullyl residue(s).

[0043] According to an even more 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:

[0044] - 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);

[0045] - 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);

[0046] - 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);

[0047] - 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);

[0048] - 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).

[0049] According to an even more particular embodiment, in said hybrid molecule according to the invention, said peptide is chosen from the group consisting of: SEQ ID NO: 5 (a36-50cit38, 42 ), SEQ ID NO: 6 (a 171 -185citi7s, isi), SEQ ID NO: 7 (a183-197citi 86, 190), SEQ ID NO: 8 (a246- (p433-447cit436,445), more particularly chosen from the group consisting of: SEQ ID NO: 5 (a36- 50cit 38 ,42), SEQ ID NO: 6 (a171 -185citi 78 ,isi), SEQ ID NO: 14 (a501-515citsio,5i2), SEQ ID NO: 18 (a621-635cit62i, 627,630) and SEQ ID NO: 19 (p60-74cit6o,72,74), even more particularly chosen from SEQ ID NO: 18 (a621-635cit62i,627,63o) and SEQ ID NO: 19 (360-74cit6o,72,74).

[0050] According to one embodiment, in said hybrid molecule according to the invention, said Fc fragment is a human Fc fragment, in particular of IgG, more particularly of IgGI. The IgGI can correspond to any allotypic variant, for example G1 m3 or nG1m1. By way of example, the Fc fragment of IgGI is represented by SEQ ID NO: 24, SEQ ID NO: 25 (Fc+Qtag) or SEQ ID NO: 26 (Fc+Qtag bis).

[0051] According to one embodiment, in said hybrid molecule according to the invention, said Fc fragment is wild type or mutated. The mutation(s) may aim to increase the plasma half-life, decrease it, or modify the effector functions of the Fc fragment. According to an even more particular embodiment, said mutated Fc fragment comprises at least the following mutations:

[0052] - L234A and L235A (LALA), or

[0053] - L234A, L235A and P329G (LALAPG), or

[0054] - G236A, S239D and I332E (GASDIE), or

[0055] - G236A, S239D, A330L and I332E (GASDALIE), or

[0056] - S239D, H268F, S324T and I332E (SDHFSTIE or SDH), the numbering being indicated in the sequence of a human IgG 1 according to the EU index. Such mutations are notably described in the article Bruhns and Jonsson, Immunol Rev. 2015 Nov;268(1):25-51. Preferably, when the hybrid molecule is used in therapy, said mutated Fc fragment comprises at least the mutations GASDIE, GASDALIE, or SDH.

[0057] According to one embodiment, in said hybrid molecule according to the invention, said Fc fragment has a fucosylation level of between 0% and 100% of the glycosylated forms. According to the invention, “between 0% and 100%” represents all integer values ​​between 0 and 100, ie; 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,

[0058] 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,

[0059] 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,

[0060] 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%. A low fucosylation of the Fc fragment causes a strong ADCC response. This is why, according to a particular embodiment, said Fc fragment has a fucosylation level of between 0% and 60% of the glycosylated forms, in particular 50%, 40%, 30%, 20%, 10% or 0%. According to the invention, the fucosylation level is defined as the average proportion of fucose carried by the Fc fragment, relative to the maximum quantity of fucose that an Fc fragment can carry.

[0061] The Fc fragment and the peptide each have N- and C-terminal ends. The Fc fragment can therefore be linked via its N- or C-terminal end to the N- or C-terminal end of the peptide. According to a preferred embodiment, in said hybrid molecule according to the invention, said covalent bond is located between the C-terminal end of said Fc fragment and the N-terminal end of said peptide, or between the N-terminal end of said Fc fragment and the N-terminal end of said peptide. According to one embodiment, when a spacer is present, the spacer can be linked to the Fc fragment via its N- or C-terminal end. According to another embodiment, when two spacers are present, the first spacer can be linked to the Fc fragment via its N- or C-terminal end and the second spacer can be linked to the peptide via its N- or C-terminal end, in particular N-terminal.Alternatively, said covalent bond between said Fc fragment and said peptide (optionally in the presence of one or more spacers) can be created on all or part of the Fc fragment. According to the invention “all or part of the Fc fragment” means that different amino acids constituting the Fc fragment can be involved in a covalent bond with said peptide.

[0062] According to a preferred embodiment, when at least one spacer is present in said hybrid molecule of the invention, it makes it possible to link the Fc fragment to an azide or to an alkyne which will itself be involved in the covalent bond with said peptide. According to one embodiment, when at least one spacer is present in said hybrid molecule of the invention, it can also make it possible to link the peptide to an alkyne or to an azide which will itself be involved in the covalent bond with the Fc fragment. According to a preferred embodiment, the hybrid molecule according to the invention comprises at least two spacers: a first spacer which makes it possible to link the Fc fragment to an azide or to an alkyne and a second spacer which makes it possible to link the peptide to an azide (when the Fc fragment is linked to an alkyne) or to an alkyne (when the Fc fragment is linked to an azide).

[0063] According to one embodiment according to the invention, in said hybrid molecule according to the invention, said Fc fragment:

[0064] - is coupled to at least one azide or an alkyne, such as a cyclooctyne, and in particular DBCO, or

[0065] - is linked to at least one spacer which is itself coupled to an azide or to an alkyne, such as a cyclooctyne, and in particular DBCO, and said peptide is:

[0066] - either coupled to an azide or an alkyne, such as a cyclooctyne, and in particular DBCO,

[0067] - either linked to a spacer which is itself coupled to an azide or to an alkyne, such as a cyclooctyne, and in particular DBCO, the covalent bond between said Fc fragment and said peptide, optionally in the presence of one or more spacers, being created between the azide and the alkyne.

[0068] According to one embodiment according to the invention, in said hybrid molecule according to the invention,

[0069] - 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

[0070] - the Fc fragment is coupled to at least one alkyne, such as a cyclooctyne, and in particular DBCO, and said peptide is coupled to an azide, the covalent bond between said Fc fragment and said peptide being created between the azide and the alkyne. According to one embodiment according to the invention, in said hybrid molecule according to the invention:

[0071] - 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

[0072] - 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

[0073] - 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

[0074] - 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

[0075] - 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

[0076] - the Fc fragment is linked to at least one spacer, itself coupled to an alkyne, such as a cyclooctyne, and in particular DBCO, and said peptide is linked to a spacer, itself coupled to an azide, the covalent bond between said Fc fragment and said peptide, in the presence of one or more spacers, being created between the azide and the alkyne.

[0077] According to one embodiment, in the molecules described above, more particularly from page 8, line 21 to page 9, line 17, the spacer used is preferably a PEGn spacer, n more particularly representing an integer between 1 and 10. When the hybrid molecule according to the invention comprises at least two PEGn spacers, the n of the two spacers may be identical or different. For example, the first spacer may be a PEG2 and the second spacer may be a PEG3 or PEG4.

[0078] 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 human IgGI.

[0079] According to the invention, the creation of the covalent bond between the azide and the alkyne corresponds to a step called "click chemistry", the N3 part of the azide reacting with an alkyne. Azide means salts of hydrazonic acid HN3, or organic azides in which one of the nitrogen atoms is covalently bonded to a carbon atom of an organic compound (for example methyl azide CH3N3). Preferably, the azide is represented by the formula N3. Alkyne means molecules having the general formula C n H2n-2, and which are characterized by the presence of at least one triple bond. Preferably the alkyne is a cyclooctyne, even more preferably dibenzocyclooctyne (DBCO).

[0080] The Fc fragment, said peptide and optionally said spacer(s), are coupled to the alkyne or azide by any conventionally used molecular coupling technique (such as conjugation). Any technique may also be used to covalently link the Fc fragment to the spacer and / or the peptide to the spacer. More particularly, a conjugation technique means an enzymatic conjugation or a chemical conjugation. An enzymatic conjugation means, for example, a 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 application US20160361434 or US20170313787, or the publication Ohtsuka et al., Bioscience, Biotechnology, and Biochemistry Volume 64, 2000

[0081] - Issue 12, Comparison of Substrate Specificities of Transglutaminases Using Synthetic Peptides as Acyl Donors. The substrate of the transglutaminase is for example a peptide comprising a glutamyl residue (a Qtag), as represented by SEQ ID NO: 29 (LLQG). A chemical conjugation is understood for example to be a covalent bond between a cysteine ​​isolated or participating in a disulfide bridge after reduction thereof and for example a maleimide. An example of such a conjugation is represented in Figure 9. In this example, the Fc fragment comprises a Qtag peptide and said Fc fragment is linked to a spacer (itself coupled to an azide), thanks to the action of the transglutaminase which will create a covalent bond between the glutamyl residue of the Qtag and the NH2 group carried by the PEGn spacer.

[0082] According to the invention, the term "coupled" or "molecular coupling" means the establishment of a covalent bond, thus the Fc fragment and / or the peptide and / or the spacer is covalently bonded to an alkyne or an azide. The term "bonded" also means a covalent bond. Thus, by way of example, the expression "the Fc fragment is coupled to an azide and said peptide is bonded to a spacer, itself coupled to an alkyne, such as a cyclooctyne, and in particular DBCO" can also be read "the Fc fragment is covalently bonded to an azide and said peptide is covalently bonded to a spacer, said spacer itself being covalently bonded to an alkyne, such as a cyclooctyne, and in particular DBCO".

[0083] According to one embodiment of the invention, said hybrid molecule is represented by:

[0084] - 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 which is coupled to a peptide represented by SEQ ID NO: 19 (b60-74aΐ6o,72,74) or SEQ ID NO: 18 (a621-635cit62i, 627,630),

[0085] - 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 which is coupled to a peptide represented by SEQ ID NO: 19 (360-74cit6o,72,74) or SEQ ID NO: 18 (a621 -635cit62i,627,630),

[0086] - an Fc fragment comprising the mutations L234A, L235A and P329G 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 ^60-74cit6o,72,74) or SEQ ID NO: 18 (a621 -635cit62i ,627,630),

[0087] - a wild-type Fc fragment 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 (p60-74cit6o,72,74) or SEQ ID NO: 18 (a621-

[0088] 635CÏt621 ,627,63q) ,

[0089] - 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 a cyclooctyne covalently linked to an azide coupled to a PEGn spacer itself linked to a peptide represented by SEQ ID NO: 19 (bdq- 74cit6o,72,74) or SEQ ID NO: 18 (a621-635cit62i,627,63o),

[0090] - an Fc fragment comprising the mutations L234A, L235A and P329G 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 (bdq- 74cit6o,72,74) or SEQ ID NO: 18 (a621-635cit62i,627,63o),

[0091] - 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 coupled to a PEGn spacer which is itself linked to a peptide represented by SEQ ID NO: 19 (b60-74oK6o,7 2,74 ) or SEQ ID NO: 18 (a621 -635cit62i, 627,630),

[0092] - an Fc fragment comprising the mutations S239D, FI268F, S324T and I332E 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 (a60-74citæ,72,74) or SEQ ID NO: 18 (a621-635cit62i,627,63o),

[0093] - an Fc fragment comprising the mutations L234A, L235A and P329G 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 (bdq- 74cit6o,72,74) or SEQ ID NO: 18 (a621-635cit62i,627,63o),

[0094] - an Fc fragment comprising the mutations G236A, S239D and I332E, which is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne coupled to a PEGn spacer which is itself linked to a peptide represented by SEQ ID NO: 19 (a60-74citæ,72,74) or SEQ ID NO: 18 (a621-635cit62i,627,63o),

[0095] - 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 coupled to a PEGn spacer which is itself linked to a peptide represented by SEQ ID NO: 19 (a60-74citæ,72,74) or SEQ ID NO: 18 (a621-635cit62i,627,63o),

[0096] - an Fc fragment comprising the mutations G236A, S239D and I332E which is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne which is coupled to a peptide represented by SEQ ID NO: 19 (b60-74oK6o,7 2,74) or SEQ ID NO: 18 (a621 -635cit62i, 627,630),

[0097] - 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 (b60-74oK6o,7 2,74 ) or SEQ ID NO: 18 (a621 -635cit62i ,627,630), n preferably representing an integer between 1 and 10, more particularly 1, 2, 3, 4 or 8. According to one embodiment according to the invention, said hybrid molecule is represented by:

[0098] - 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 which is coupled to a peptide represented by SEQ ID NO: 19 (b60-74aΐ6o,72,74) or SEQ ID NO: 18 (a621-635cit62i,627,63o),

[0099] - an Fc fragment comprising the mutations S239D, FI268F, S324T 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 (b60-74oK6o,72,74) or SEQ ID NO: 18 (a621 -635cit62i,627,630),

[0100] - an Fc fragment comprising the mutations L234A, L235A and P329G 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 (b60-74oK6o,7 2,74 ) or SEQ ID NO: 18 (a621 -635cit62i, 627,630),

[0101] - 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 coupled to a PEGn spacer which is itself linked to a peptide represented by SEQ ID NO: 19 (b60-74oK6o,7 2,74 ) or SEQ ID NO: 18 (a621 -635cit62i, 627,630),

[0102] - an Fc fragment comprising the mutations S239D, FI268F, S324T and I332E 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 (b60-74oK6o,72,74) or SEQ ID NO: 18 (a621-635cit62i, 627,630), n preferably representing an integer between 1 and 10, more particularly 1, 2, 3, 4 or 8.

[0103] According to an even more particular embodiment according to the invention, said hybrid molecule is represented by:

[0104] - 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 which is coupled to a peptide represented by SEQ ID NO: 19 (60-74cifeo,72,74),

[0105] - an Fc fragment comprising the mutations S239D, FI268F, S324T 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 ^60-74cit6o,7 2,74 ),

[0106] - an Fc fragment comprising the mutations L234A, L235A and P329G 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 ^60-74cit6o,72,74),

[0107] - a wild-type Fc fragment which 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 ^60-74cit6o,7 2,74 ) or SEQ ID NO: 18 (a621-

[0108] 635CÏt621 ,627,63q) ,

[0109] - an Fc fragment comprising the mutations S239D, FI268F, S324T and I332E 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 (360-74cifeo,72,74) or SEQ ID NO: 18 (a621-635cit62i,627,63o), n preferably representing an integer between 1 and 10, more particularly 1, 2, 3, 4 or 8.

[0110] Use of the hybrid molecules according to the invention

[0111] In a second aspect, the present invention also relates to a hybrid molecule as defined above, for its use as a medicament.

[0112] More particularly, according to the invention, said hybrid molecules are intended to target and lyse in the body of patients, by ADCC and / or phagocytosis and / or complement activation, all “ACPA-positive” B cells (i.e. B lymphocytes which express on their surface the anti-citrullinated protein autoantibodies (ACPA)). Indeed, the hybrid molecule according to the invention binds to “ACPA-positive” B lymphocytes thanks to the 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 making it possible to destroy “ACPA-positive” B lymphocytes thanks to its Fc fragment, a natural ligand for Fc receptors (for example Fc-gamma receptor (FcyR) if the Fc fragment is derived from an IgG), present in particular on the surface of macrophages but also NK cells (“Natural Killers”).

[0113] According to a particular embodiment, the invention relates to a hybrid molecule, as defined above, for its use in the treatment of autoimmune diseases associated with the production of anti-citrullinated protein autoantibodies, in particular Gougerot-Sjogren syndrome and rheumatoid arthritis. In this embodiment, the severe forms of said autoimmune diseases associated with the production of anti-citrullinated protein autoantibodies are targeted, as are various so-called "borderline" forms with other chronic arthritis, such as psoriatic arthritis or systemic lupus erythematosus.

[0114] 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.

[0115] According to the invention, “a pharmaceutically acceptable vehicle” means any formulation making the composition suitable for administration to a patient, in any galenic form.

[0116] As indicated 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. According to a particular embodiment, the present invention thus relates to a hybrid molecule as previously described, for its use as a medicament, 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 being optionally present between said peptide and said antibody or said fragment.Such combination therapy can specifically target and destroy ACPA-positive plasma cell and B lymphocyte clones, thereby eliminating ACPA from the patient's body. The antibody used is preferably a monoclonal antibody.

[0117] According to a particular embodiment, in this second hybrid molecule, the fragment is understood to be a portion of 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 constructions is for example described in international 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.

[0118] According to a particular embodiment, in this second hybrid molecule said antibody or F(ab')2 fragment is a bispecific antibody or F(ab')2 fragment directed against CD38 and another plasma cell target. Alternatively, in this second hybrid molecule said antibody or F(ab')2 fragment is a bispecific antibody or F(ab')2 fragment directed against CD138 and another plasma cell target. According to the invention, an "other plasma cell target" means any molecule being expressed on the surface of plasma cells.

[0119] According to another particular embodiment, in this second hybrid molecule said antibody or F(ab')2 fragment is a bispecific antibody or F(ab')2 fragment directed against CD38 and CD138.

[0120] According to this particular embodiment of the invention, the first and second hybrid molecules are administered simultaneously, separately or spread out over time.

[0121] According to another embodiment, the hybrid molecule according to the invention can be coupled to at least one radioisotope or at least one fluorochrome, such as A488 or A647. Such molecules can advantageously be used as molecular tracer tools.

[0122] According to another embodiment, the invention thus relates to the in vitro or ex vivo use of a hybrid molecule comprising at least one antibody Fc fragment covalently linked to at least one fibrin-derived peptide having at least one citrullyl residue, at least one spacer being optionally present between said Fc fragment and said peptide, as a molecular tool. Such constructs can in particular be used to analyze the binding of the hybrid molecules to ACPAs and to the 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 the bridging of these by the ACPAs.... The radioisotopes and / or fluorochromes are preferably coupled to the Fc fragment, even more particularly at the level of the Qtag (if present) or at the level of the lysines.

[0123] Method for producing hybrid molecules according to the invention In another aspect, the invention also relates to a method for obtaining a hybrid molecule as defined above.

[0124] According to one embodiment, the invention thus relates to a method for producing a hybrid molecule as defined previously, comprising the following steps:

[0125] - (i) obtaining an azide coupled to an Fc fragment or obtaining an alkyne coupled to an Fc fragment,

[0126] - (ii) obtaining an alkyne coupled to a peptide or obtaining an azide coupled to a peptide,

[0127] - (iii) realization of the covalent bond between the azide and the alkyne,

[0128] - step (i) can be carried out before or after step (ii), or concurrently.

[0129] According to one embodiment, the invention thus relates to a method for producing a hybrid molecule as defined previously, comprising the following steps:

[0130] - (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,

[0131] - (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,

[0132] - (iii) realization of the covalent bond between the azide and the alkyne,

[0133] - step (i) can be carried out before or after step (ii), or concurrently.

[0134] According to another embodiment, the invention thus relates to a method 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,

[0135] - (ii) coupling of an alkyne and a peptide, optionally in the presence of a spacer, or coupling of an azide and a peptide, optionally in the presence of a spacer, - (iii) creation of the covalent bond(s) between the azide and the alkyne,

[0136] - step (i) can be carried out before or after step (ii), or concurrently.

[0137] The sequences of the invention are shown in Table 1 below.

[0138] [Table 1]. Summary table of the sequences of the invention

[0139] Other characteristics, details and advantages of the invention will appear on reading the appended Figures and the examples which illustrate the invention and are not intended in any way to limit it. In the exemplified hybrid molecules, it is the N-terminal end of the Fc fragment which is linked to a PEGn spacer (except in the indicated case Cter-PEG (see Figure 4)), and it is always the N-terminal end of said peptide which is linked to the cycloctyne DBCO or to a second PEGn spacer where appropriate. Furthermore, in the exemplified hybrid molecules, in the PEGn spacer linked to the Fc fragment, n more particularly represents 3 or 4, and in the PEGn spacer linked to the peptide, the PEGn spacer represents 3 or 8, although any other value of n, in particular between 1 and 10, can be used.

[0140] In the examples below, an “armed or pre-armed” cell means a cell to which a hybrid molecule according to the invention is attached. Thus, the hybrid molecule is linked to the cell’s Fc receptor by means of its Fc fragment.

[0141] Brief description of Figures Fig. 1

[0142] [Fig. 1] represents the diagram of an example of a hybrid molecule according to the invention.

[0143] A spacer (which is optional) is shown between the antibody Fc fragment and the fibrin-derived peptide having a citrullyl residue (referred to as "citrullinated peptide").

[0144] Fig. 2

[0145] [Fig. 2] represents a B lymphocyte expressing on its surface transmembrane ACPAs linked to a hybrid molecule according to the invention, said hybrid molecule itself being linked by the Fc fragment to an NK cell.

[0146] The NK cell will then be able to destroy the B lymphocyte by ADCC. More specifically, this figure shows a B cell (or B lymphocyte) which expresses on its surface an ACPA-type BCR and which interacts specifically with the citrullinated peptide carried by the hybrid molecule. The engagement of the Fc fragment carried by the same hybrid molecule with FcγRIIIa (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 R, Fc-gamma Receptor; NK cell, Natural Killer cell).

[0147] Fig. 3

[0148] [Fig. 3] represents a B lymphocyte expressing on its surface transmembrane ACPAs linked to a hybrid molecule according to the invention, said hybrid molecule itself being linked by the Fc fragment to a macrophage.

[0149] The macrophage will then be able to destroy the B lymphocyte by phagocytosis. More specifically, this Figure shows B cells (or B lymphocytes) which express on their surface an ACPA-type BCR and which interact specifically with citrullinated peptides carried by the hybrid molecules. The engagement of the Fc fragments carried by these same hybrid molecules with different FcγRs 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-Citrullated Protein Autoantibodies; ADP, Antibody-Dependent Phagocytosis; BCR, B-Cell Receptor; Fc7R, Fc-gamma Receptor).

[0150] Fig. 4

[0151] [Fig. 4] represents the reactivity of several hybrid molecules comprising the peptide of SEQ ID NO: 19 (P60-74citeo, 72,74) with respect to ACPA. b60-74 represents the peptide of SEQ ID NO: 19. Fc-WT^60-74 represents 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 peptide represented by SEQ ID NO: 19 (b60-74aί6o,72,74). Fc-LALA^60-74 represents an Fc fragment comprising the mutations L234A, L235A and P329G 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^60-74 represents an Fc fragment comprising the mutations S239D, FI268F, 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 peptide represented by SEQ ID NO: 19.Fc-WT-PEG^60-74 represents a wild-type Fc fragment that is linked to at least one PEGn spacer that is itself coupled to an azide covalently linked to a cyclooctyne DBCO coupled to a PEGn spacer that is itself linked to a peptide represented by SEQ ID NO: 19 (b60-74aί6o,72,74). Fc-SDH-PEG^60-74 represents an Fc fragment comprising the mutations S239D, FI268F, S324T and I332E that is linked to at least one PEGn spacer that is itself coupled to an azide covalently linked to a cyclooctyne DBCO that is coupled to a PEGn spacer, itself linked to a peptide represented by SEQ ID NO: 19 (b60-74dt6o,72,74). Fc-SDH(Cter)-PEG^60-74 corresponds to Fc-SDH-PEG^60-74, except that in the Fc-SDH(Cter)-PEG^60-74 hybrid, the first PEGn is attached at the C-terminus of the Fc fragment, unlike the other examples in which the first PEGn is attached at the N-terminus.n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8.

[0152] Fig. 5

[0153] [Fig. 5] represents the reactivity of a hybrid molecule comprising the peptide of SEQ ID NO: 18 (a621-635cit 62i,027,030) vis-à-vis the ACPA. a621-635 represents the peptide of SEQ ID NO: 18. Fc-WT-PEG-a621-635 represents a wild-type (WT) Fc fragment that is linked to at least one PEGn spacer that is itself coupled to an azide covalently linked to a cyclooctyne DBCO coupled to a PEGn spacer that is itself linked to a peptide represented by SEQ ID NO: 18. Fc-SDFI-PEG-a621-635 represents an Fc fragment comprising the mutations S239D, FI268F, S324T and I332E that is linked to at least one PEGn spacer that is itself coupled to an azide covalently linked to a cyclooctyne DBCO that is coupled to a PEGn spacer that 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.

[0154] Fig. 6

[0155] [Fig. 6] represents the reactivity of a recombinant human monoclonal ACPA (clone 2H06), comprising an unmodified Fc or modified with SDH-type mutations, with respect to hybrid molecules comprising the peptide of SEQ ID NO: 18.

[0156] FcWT-PEG-a621-635 represents 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 coupled to a PEGn spacer which is itself linked to a peptide represented by SEQ ID NO: 18. FcSDH-PEG-a621-635 represents an Fc fragment comprising the mutations S239D, FI268F, 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.

[0157] Fig. 7

[0158] [Fig. 7] represents a method of manufacturing a hybrid molecule according to the invention, comprising the scheme of derivatization and conjugation of the Fc fragments.

[0159] Fig. 8

[0160] [Fig. 8] represents the evolution of the percentage of phagocytosis and B lymphocytes in the presence of hybrid molecules Fc-WT-PEG-a621-635arg and Fc-WT-PEG-a621-635cit.

[0161] 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 .

[0162] Fig. 9

[0163] [Fig. 9] represents the lysis by human NK cells of cells of a transduced human malignant lymphoblastic B cell line expressing on its membrane a recombinant monoclonal ACPA, in the presence of the hybrids FcWT-N3-DBCO-PEG3-a621-635Cit and FcSDH-N3-DBCO-PEG3-a621-635Cit.

[0164] Nalm6 represents the case where only Nalm6 cells were incubated; Nalm6+NK the case where Nalm6 cells were incubated with NK cells; WTaCIT the case where the hybrid

[0165] WTaCIT was incubated with Nalm6 cells and NK cells; WTaArg in the case where the hybrid

[0166] WTaArg was incubated with Nalm6 cells and NK cells; SDFIaCIT in the case where the hybrid

[0167] SDHaCIT was incubated with Nalm6 cells and NK cells and SDHaArg in case the hybrid

[0168] SDHaArg was incubated with Nalm6 cells and NK cells.

[0169] The transduced human malignant lymphoblastic B cell line expressing a recombinant monoclonal ACPA on its membrane corresponds to the Nalm6 line as described in Example 11 and Figure 10. The monoclonal antibody is clone 022014CCP14CFCT2H06, known as 2H06.

[0170] Fig. 10

[0171] [Fig. 10] represents the membrane expression of the monoclonal ACPA 2H06 by the B lymphoblastic cell line Nalm6 after transduction (Nalm62H06).

[0172] A- Flow cytometry analysis of the transduction efficiency of the Nalm6 B cell line for the expression of the monoclonal ACPA 2H06 at its membrane, thanks to a double labeling by an anti-Fab IgG antibody and a tetramer of the peptide a621-635cit (SEQ ID NO: 18). B- Flow cytometry analysis of the enrichment in transduced Nalm6 2H06 cells, following cell sorting carried out using an anti-Fab antibody. C- Flow cytometry analysis of the interaction between the membrane ACPA 2H06 expressed by the transduced Nalm6 cell line and the Fc-WT-PEG-a621-635cit (cit) or Fc-WT-PEG-a621-635arg (arg) hybrids, coupled to the fluorochrome A647. Nalm6 NT: non-transduced control Nalm6 cell line. Fc-WT-PEG-a621-635 represents a wild-type (WT) Fc fragment 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.Fc-WT-PEG-a621-635arg represents a wild-type (WT) Fc fragment 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 in which the X residues are not citrullinated (i.e. they are arginyl residues).

[0173] Nalm6 cells expressing the monoclonal ACPA 2H06 correspond to the transduced Nalm6 line mentioned in the examples.

[0174] Fig. 11

[0175] [Fig. 11] represents the phagocytosis of cells of the B line transduced Nalm6 ACPA+ 2H06 by macrophages pre-armed with Fc-peptide a621-635 hybrids.

[0176] A- Figure shows the results of a flow cytometry experiment where cell populations are represented as scatter plots. T0 represents the result at time 0, after contacting Nalm6 target cells and macrophages. T2 represents the result at time 2h, after 2h of contacting Nalm6 target cells and macrophages, in the absence of hybrid molecules (spontaneous phagocytosis). All other cytometry diagrams represent the results obtained after 2h of contacting Nalm6 target cells and macrophages armed with Fc-PEG-a621-635cit (citrullinated) hybrids or control Fc-PEG-a621-635arg (non-citrullinated) hybrids.The hybrids are identical to those described in Figure 10, except that different types of Fc fragments were used: a wild-type (WT) Fc fragment, a GASDIE Fc fragment (presenting the mutations G236A, S239D and I332E), or an SDH Fc fragment (presenting the mutations S239D, H268F, S324T and I332E). The cells showing double fluorescence (PKH-67 green / PKH-26 red) correspond to macrophages that have phagocytosed target cells. The cells showing single PKH-26 fluorescence correspond to residual Nalm6 target cells, i.e., cells not phagocytosed after 2 hours in the presence of macrophages (% target population). In each quadrant of the cytometry diagrams, the different cell populations are expressed as a percentage of the total cell population.B- Figures show as histograms, for each experimental condition described in A, the mean % of double-positive cells and the mean % of cells in the target population, calculated from the results of 3 experiments. The histograms in black represent the control conditions. The histograms in white represent the conditions in the presence of Fc-PEG-a621-635cit hybrid. The p-value is as follows: * p< 0.05 ; ** p< 0.01 .

[0177] Fig. 12

[0178] [Fig. 12] represents the phagocytosis of Nalm6 ACPA+ 2H06 B cell line by pre-armed macrophages in the presence of Fc-peptide a621-635 hybrids at various concentrations. The Figure shows the results of a flow cytometry experiment performed after phagocytosis of Nalm6 2H06 B cells by pre-armed macrophages by incubation in the presence of Fc-peptide a621-635 hybrids at various concentrations. A- The Figure shows in the form of histograms, for each experimental condition, the % of double-positive cells. B- The Figure shows the % of the residual target Nalm6 population, i.e. not phagocytosed. The dark histograms represent the control conditions obtained in the presence of Fc-PEG-a621-635arg hybrids, non-citrullinated. The light histograms represent the conditions in the presence of Fc-PEG-a621-635cit hybrid. The hybrid concentrations used were between 5 pg / ml and 5 ng / ml.The hybrids are identical to those described in Figure 11, except that a hybrid molecule with a GASDALIE Fc fragment (comprising the mutations G236A, S239D, A330L and I332E) was also tested.

[0179] Fig. 13

[0180] [Fig. 13] represents the confirmation of the phagocytosis process of the transduced Nalm6 ACPA+ 2H06 line by macrophages pre-armed with FcWT-PEG-peptide a621-635 hybrids.

[0181] These Figures show that the double-positive cells observed by cytometry in the previous experiments correspond well to the phagocytosis of Nalm6 target cells by macrophages. The four left panels show flow cytometry results obtained after placing Nalm6 2H06 cells labeled with pHRodo SE and macrophages armed with Fc-WT-PEG-a621-635 hybrids in the presence or absence of cytochalasin D, a phagocytosis inhibitor. The two right panels show the cell populations at the end of the experiment, observed by optical microscopy after staining with MGG. The hybrids are identical to those described in Figure 10.

[0182] Fig. 14

[0183] [Fig. 14] represents the phagocytosis of the transduced Nalm6 ACPA+ 2H06 line by macrophages pre-armed with Fc-peptide a621-635 hybrids or incubated in the presence of Fc-peptide a621-635 hybrids.

[0184] 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, just after contact between the Nalm6 target cells and macrophages. T2 represents the result at the 2nd hour after contact between the Nalm6 target cells and macrophages, in the absence of hybrid molecules (spontaneous phagocytosis). All other cytometry diagrams present the results obtained after 2 hours of contact between Nalm6 target cells and macrophages, either pre-armed with the FcWT-PEG-a621-635 or FcGASDIE-PEG-a621-635 hybrids, citrullinated or not, or incubated with the WT-PEG-a621-635 or FcGASDIE-PEG-a621-635 hybrids, citrullinated or not, solubilized in the incubation medium. The hybrids are identical to those described in Figure 11.

[0185] Cells showing double fluorescence (PKH-67 green / PKH-26 red) correspond to macrophages that have phagocytosed Nalm6 target B cells (% double positives). Cells showing single PKH-67 fluorescence correspond to residual Nalm6 target B cells, i.e., cells not phagocytosed after 2 hours in the presence of macrophages. In each quadrant of the cytometry diagrams, the different cell populations are expressed as a percentage of the total cell population.

[0186] Fig. 15

[0187] [Fig. 15] represents the phagocytosis of the transduced Nalm6 ACPA+ 2H06 line by macrophages incubated in the presence of Fc-PEG-peptide a621-635 hybrids and, or not, in the presence of human IgG at physiological serum concentration.

[0188] The Figure shows the average results of four (n=4) independent identical experiments of phagocytosis of cells of the transduced Nalm6 ACPA+ 2H06 line by macrophages, incubated in the presence of Fc-PEG-peptide a621-635 hybrids, and in the presence, or absence, of human IgG at physiological serum concentration. The Figure shows for each experimental condition, the average residual percentage of the target cell population 2h after contact between macrophages and citrullinated (cit) or non-citrullinated (arg) Fc-PEG-a621-635 hybrids, soluble at a concentration of 1 pg / mL, in the presence (gray histograms) or absence (black histograms), of competing human IgG. The value of "p" is as follows: * p < 0.05 ; ** p < 0.01 . The hybrids are identical to those described in Figure 11.

[0189] Fig. 16

[0190] [Fig. 16] represents the phagocytosis of the transduced Nalm6 ACPA+ 2H06 line by human macrophages incubated with Fc-peptide a621-635 hybrids, in the presence or absence of human IgG at physiological concentration or human serum.

[0191] 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 contacting the Nalm6 2H06 target cells and macrophages. T2 represents the result 2 hours after contacting the target cells and macrophages in the absence of hybrid molecules (spontaneous phagocytosis). The other diagrams represent the results obtained 2 hours after contacting the target cells and macrophages, incubated with the FcWT-PEG-a621-635 or FcGASDIE-PEG-a621-635 hybrids, citrullinated (cit) or not (arg), and this in the presence or absence of human IgG at physiological serum concentration or human serum. The CFSE+ CD11 b+ double-positive cells correspond to macrophages that have phagocytosed target cells. CFSE+ single-positive cells correspond to residual Nalm6 target cells, not phagocytosed after 2 hours.In each quadrant of the cytometry diagrams, the different cell populations are expressed as a percentage of the total cell population. The hybrids are identical to those described in Figure 11.

[0192] Fig. 17

[0193] [Fig. 17] represents the phagocytosis of the transduced Nalm6 ACPA+ 2H06 cell line by human macrophages pre-armed with the Fc-peptide a621-635 hybrids, in the presence of the non-transduced Nalm6 cell line. The Figure shows the results of an experiment of phagocytosis of cells of the transduced Nalm6 ACPA+ 2H06 cell line by macrophages pre-armed with the Fc-peptide a621-635 hybrids, in the presence of cells of the same Nalm6 cell line, wild type, non-transduced. The results of the flow cytometry analysis appear in the form of scatter plots representing the cell populations. T0 represents the situation at time 0, just after contact between the Nalm6 2H06 target cells, the non-transduced Nalm6 NT cells and the 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 2 hours in the presence of macrophages armed with FcWT-PEG-a621-635 or FcGASDIE-PEG-a621-635 hybrids, citrullinated (cit) or non-citrullinated (arg). The CFSE+ CD19+ double-positive cells correspond to the residual Nalm6 2H06 target cells, i.e., not phagocytosed after 2 hours in the presence of macrophages. The CFSE- CD19- double-negative cells correspond to macrophages. The CFSE- CD19+ single-positive cells correspond to the residual non-transduced (NT) Nalm6 cells, i.e., not phagocytosed after 2 hours in the presence of macrophages. CFSE+ CD19- positive single cells correspond to macrophages that phagocytosed Nalm6 2H06 target cells. In each quadrant of the cytometry diagrams, the different cell populations are expressed as a percentage of the total cell population. The hybrids are identical to those described in Figure 11.

[0194] Fig. 18

[0195] [Fig. 18] represents the specific in vivo destruction of the Nalm6 ACPA+ 2H06 line by human NK cells in the presence of Fc-peptide a621-635 hybrids, in immunodeficient SCID / BEIGE mice.

[0196] The Figure shows the destruction by human NK cells, in the peritoneal cavity of SCID / BEIGE immunodeficient mice, of ACPA+ target cells (transduced Nalm6 line 2H06) in the presence of citrullinated Fc-peptide 0621-635 hybrids. A- The Figure represents the analysis of the populations of non-transduced Nalm6 2H06 and Nalm6 NT cells, in flow cytometry. The peaks observed correspond to the target cells (Nalm6 2H06 CTV h '9 h ) or control cells (Nalm6 NT CTV |0W ). B- The figure represents all the results obtained for four groups of 7 SCID / BEIGE immunodeficient mice. These results are expressed as a ratio of CTV fluorescences h '9h / CTV l0W , corresponding respectively to Nalm6 2H06 cells (CTV h '9 h ) and Nalm6 NT cells (CTV |0W ). A decrease in the ratio indicates the disappearance of 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.

[0197] Fig. 19

[0198] [Fig. 19] represents the specific in vivo destruction of the Nalm6 ACPA+ 2H06 line by human macrophages pre-armed with the Fc-peptide a621-635 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 (transduced Nalm6 line 2H06) in the presence of citrullinated Fc-peptide a621-635 hybrids. The Figure represents the results obtained for 4 groups of 5 immunodeficient SCID / BEIGE mice. The number of Nalm6 2H06 CTV target cells h '9 h residuals for each group at the end of the experiment are presented, it indicates the efficiency of phagocytosis. The "p" value is as follows: * p < 0.05. The hybrids are identical to those described in Figure 10.

[0199] Examples

[0200] Example 1: Example of production of a hybrid molecule according to the invention

[0201] The hybrid molecule described here comprises the following construction: an Fc fragment covalently linked to at least one PEGn spacer, itself coupled to an azide, said azide being covalently linked to an alkyne, which is itself coupled to a PEGn spacer linked to a citrullinated peptide. Such a molecule can be read: Fc-PEGn-lsh-DBCO-PEGn-citrullinated peptide. The two PEGns can be identical or different (for example the first PEGn is PEG3 and the second PEGn is PEG2).

[0202] 1. Synthesis of an NH2-PEGn-N3 (i.e. a spacer coupled to an azide at one end and having a free amine function at another end).

[0203] 2. Bringing together NFE-PEGn-Ish, with an Fc fragment having a Qtag, and a transglutaminase, preferably for 16 hours at 37°C. This so-called "derivatization" step is preferably carried out with 20 times more moles of NH2-PEGn-N3 than moles of Fc fragment. The transglutaminase is used, at a rate of 15U / pmol per Qtag present (on one or both monomers). Optionally, desalting can be carried out to remove the excess spacer not bound to the Fc fragment at the end of the step. An Fc-PEGn-N3 is thus obtained. If the Fc fragment has 2 Qtags (one carried on each monomer), then the Fc fragment can carry two PEGn-N3s.

[0204] 3. Synthesis of a Cys-PEGn-citrullinated peptide (i.e. a spacer linked to a peptide at one end and having a free cysteine ​​at the other end). An alkyne, for example a DBCO, is then coupled to the Cys-PEGn-citrullinated peptide at the cysteine, and a DBCO-PEGn-citrullinated peptide is thus obtained.

[0205] 4. Implementation of the “click”: coupling between N3 and DBCO. The DBCO-PEGn-citrullinated peptide is placed in the presence of Fc-PEGn-N3 with, preferably, 10 times more moles of DBCO-PEGn-citrullinated peptide than moles of Fc-PEGn-N3. The click reaction takes place at room temperature and is almost complete after 4 hours.

[0206] 5. Obtaining the hybrid molecule: Fc-PEGn-N3-DBCO-PEGn-citrullinated peptide. This embodiment is illustrated in Figure 7.

[0207] Similarly, an Fc-PEGn-DBCO and an N3-PEGn-citrullinated peptide can be obtained, and then coupled to obtain Fc-PEGn-DBCO-N3-PEGn-citrullinated peptide. Similarly, an Fc-PEGn-DBCO and an N3-citrullinated peptide can be obtained, or an Fc-PEGn-N3 and a DBCO-citrullinated peptide, and then coupled to obtain Fc-PEGn-DBCO-N3-citrullinated peptide or Fc-PEGn-N3-DBCO-citrullinated peptide, respectively.

[0208] Example 2: Reactivity of ACPA purified by affinity chromatography on citrullinated peptides and eluted at pH3 (pH3 fraction), with respect to the hybrid molecules according to the invention

[0209] The ACPAs (here ACPA, pH3 fraction) used are ACPAs obtained from serum of ACPA-positive rheumatoid arthritis patients. Such ACPAs were obtained by a conventional 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 peptide B60-74cit

[0210] ELISA microtiter plate wells were coated by passive adsorption using 100 μL of a solution containing either the peptide b60-74oK (SEQ ID NO: 19, also referred to as b60-74), or a hybrid molecule Fc-WT^60-74cit, Fc-LALA^60-74cit, Fc-SDH^60-74cit, Fc-WT-PEG^60-74cit, Fc-SDFI-PEG^60-74cit or Fc-SDFI(Cter)-PEG^60-74cit. These solutions were each used at a concentration of 5 μg / mL in PBS (Phosphate Buffered Saline) buffer and incubated overnight at 4°C. The non-citrullinated peptide and hybrid molecules constructed with the non-citrullinated peptide ^60-74arg, corresponding to a peptide of SEQ ID NO: 19 in which the citrullyl residues were 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 pg / mL, diluted in PBS 2% BSA 2M NaCl buffer for 1 h at 4°C. After washing, the reactivity of the ACPAs was detected using a secondary antibody anti-human IgG Fab diluted 1 / 2500 in PBS 2% BSA buffer. The results are expressed as A OD (Delta-Optical Density), corresponding to the OD obtained with the peptide b60-740i! or with the hybrid molecules constructed with the peptide b60-740K, subtracted respectively from the OD obtained with the peptide b60-743^ or with the corresponding hybrid molecules constructed with the peptide b60-743^.

[0211] The results are presented in Figure 4. They show a dose-dependent reactivity of the purified ACPAs on peptide b60-74oK, with respect to the hybrid molecules Fc-WT^60-74cit, Fc-LALA^60-74cit, Fc-SDFI^60-74cit, Fc-WT-PEG^60-74cit, Fc-SDFI-PEG^60-74cit or Fc-SDH(Cter)-PEG^60-74cit. They show that included in the different hybrids, the citrullinated peptides remain perfectly reactive with the ACPAs. b. Examples with hybrid molecules containing the peptide a621-635cit

[0212] ELISA plate wells were coated with 100 μL of a solution containing the peptide a621-635cit (SEQ ID NO: 18, also referred to as a621-635) or a hybrid molecule Fc-WT-PEG-a621-635cit or Fc-SDH-PEG-a621-635cit, at a concentration of 5 μg / mL in PBS (Phosphate Buffered Saline) buffer, incubated overnight at 4°C. The non-citrullinated peptide a621-635arg and the corresponding hybrid molecules constructed with the non-citrullinated peptide (a621-635arg, corresponding to a peptide of SEQ ID NO: 18 in which citrullyl residues were 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 4, 2 and 1 pg / mL diluted in PBS 2% BSA 2M NaCl buffer for 1 hour at 4°C.After washing, ACPA reactivity was detected using a secondary anti-Fab antibody of human IgG diluted 1 / 2500 in PBS 2% BSA buffer. The results are expressed as A OD (Delta-Optical Density), corresponding to the OD obtained with the peptide 621-635cit or with the hybrid molecules constructed with the peptide a621-635cit, subtracted respectively from the OD obtained with the peptide a621-635arg or with the hybrid molecules constructed with the peptide a621-635arg.

[0213] The results are presented in Figure 5. They show a strong dose-dependent reactivity of the purified ACPAs on peptide a621-635cit towards the hybrid molecules Fc-WT-PEG-a621-635cit and Fc-SDH-PEG-a621-635cit. The reactivity of the ACPAs towards the hybrid molecules is comparatively stronger, while the epitope density they present is lower than that of the peptide a621-635cit.

[0214] Example 3: Reactivity of recombinant monoclonal ACPA 2H06 in wild type (WT) or mutated form on their Fc fragment (SDH mutation), with respect to the hybrid molecules according to the invention

[0215] The ACPA used here is a recombinant monoclonal human ACPA (clone 022014CCP14CFCT2H06, referred to as 2H06). Such an ACPA can be obtained as described in Titcombe PJ, Wigerblad G., Sippl N., Zhang N., Shmagel AK, Sahlstrôm 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.

[0216] ELISA plate wells were coated with 100 μL of a solution containing either the hybrid molecule Fc-WT-PEG- a 621-635cit or Fc-SDH-PEG- a 621-635cit, used at a concentration of 5 μg / mL in PBS (Phosphate Buffered Saline) buffer and incubated overnight at 4°C. Hybrid molecules constructed with a non-citrullinated peptide ( 621-635arg) were used at the same concentration as negative controls. The wells were then saturated in PBS 2% BSA (Bovine Serum Albumin) buffer for 1 h at 4°C. After washing, recombinant monoclonal ACPA 2H06, WT and SDH, were incubated at 40, 20 and 10 pg / mL, diluted in PBS buffer 2% BSA 2M NaCl for 1h at 4°C. After washing, their reactivities were detected using a secondary antibody anti-human IgG Fab diluted 1 / 2500 in PBS buffer 2% BSA.The results are expressed in D OD (Delta-Optical Density), corresponding to the OD obtained with the hybrid molecules constructed with the peptide 621-635cit subtracted from the OD obtained with the hybrid molecules constructed with the negative control peptide a 621-635arg.

[0217] The hybrid molecules Fc-WT-PEG-a621-635cit and Fc-SDH-PEG-a621-635cit are identical to those used in Example 2.

[0218] The results are presented in Figure 6. The results show a strong dose-dependent reactivity of WT and SDH monoclonal ACPAs towards the hybrid molecules Fc-WT-PEG-a621-635cit and Fc-SDH-PEG-a621-635cit. Example 4: Binding of the hybrid molecules on macrophages at 2h and 20h, at physiological temperature

[0219] Human macrophages, differentiated in vitro in the presence of M-CSF (100ng / mL) from CD14+ monocytes isolated from the peripheral blood of a healthy subject were incubated at 500,000 cells / well, in the presence of hybrid molecules at 5 pg / mL, FcWT-N3-DBCO-p60-74Cit (a wild-type Fc fragment 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, referred to as WT in Tables 2 and 3), FcSDHFSTIE-N3-DBCO- 60-74Cit (an Fc fragment comprising the mutations S239D, H268F, S324T and I332E which 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-

[0220] DBCO-p60-74Cit (an Fc fragment comprising the mutations L234A, L235A and P329G 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, called LALAPG in Tables 2 and 3), for 2h or 20h at 37°C. The attachment of the hybrid molecules to the surface of the macrophages is demonstrated and quantified by cytofluorimetry (FACS Canto II) after incubation of the macrophages with an anti-Fc antibody coupled to FITC, used 1 / 1000. In the constructs, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8.

[0221] The results are presented in Tables 2 and 3 below. “NM” and “ANTI FC” represent, respectively, the well containing macrophages incubated without antibody or with the anti-Fc antibody alone.

[0222] [Table 2]. Results at 2 hours

[0223] [Table 3]. Results at 8 p.m.

[0224] The results presented in Tables 2 and 3 show that the hybrid molecules FcWT-N3-DBCO-60-74Cit and FcSDHFSTIE-N3-DBCO^60-74Cit at the physiological temperature of 37°C, bind from the 2 ème hour to the macrophage membrane and are still present there 20 hours later. These results also show that the fixation of the FcSDHFSTIE-N3-DBCO-p60-74Cit hybrid is higher than that of the wild-type FcWT-N3-DBCO-p60-74Cit.

[0225] Example 5: Phagocytosis induced by the Fc-SDH hybrid p60citrullinated peptide when B lymphocytes are armed via their FcγRIIb (CD32b) with purified ACPA

[0226] Freshly purified human B lymphocytes from peripheral blood of healthy donors by CD19+ magnetic sorting were labeled with green PKH-67 (Paul Karl Horan / fluorescent lipid marker) and then incubated with purified ACPA (pH3 fraction, obtained as in Example 2) on peptide b60-74oK (SEQ ID NO: 19) at a concentration of 5 pg / mL for 30 min at 37°C. Subsequently, the cells were washed and then placed in the presence of the hybrid FcSDHFSTIE-N3-DBCO-PEG3-360-74Cit (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 itself linked to a peptide represented by SEQ ID NO: 19). This construction is called CIT in Table 4 below).The cells were also placed in the presence of the hybrid FcSDHFSTIE-N3-DBCO-PEG^60-74Arg (which corresponds to the construct FcSDHFSTIE-N3-DBCO-PEG3^60-74Cit in which the peptide b60-74 is not citrullinated). This construct is called NCIT in Table 4 below). The cells were placed in the presence of the hybrids at 10 pg / mL for 30 min at 37°C. After washing, the cells were placed in contact with macrophages (labeled PKH-26 red), at a rate of 1 B lymphocyte for 1 macrophage, for 2 h at 37°C. The cells were then detached and analyzed by flow cytometry. The cells showing double fluorescence (PKH-67 green / PKH-26 red) correspond to macrophages that have phagocytosed lymphocytes. The different cell populations are expressed as a percentage of the total cell population: percentage of phagocytosis and percentage of B lymphocytes.In the constructs, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8.

[0227] The results are presented in Table 4 in four rows corresponding to four independent experiments.

[0228] [Table 4]. Percentage of phagocytosis and percentage of B lymphocytes

[0229] The results obtained confirm that the phagocytosis activity is increased in the presence of citrullinated hybrids (increase in 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 versus FcSDHFSTIE-N3-DBCO-PEG3-

[0230] Example 6: Phagocytosis of B lymphocytes loaded with recombinant monoclonal human ACPA 2H06 mutated SDH, by macrophages armed with the hybrid Fc-WT-PEG-a621-635cit

[0231] Freshly purified human B lymphocytes from peripheral blood of healthy donors by CD19+ magnetic sorting were labeled with green PKH-67 (Paul Karl Horan / fluorescent lipid marker) and then incubated for 1 h at 37°C with recombinant human monoclonal ACPA 2H06 whose Fc is mutated SDH, at a concentration of 10 pg / mL. Such antibodies can be obtained as in Example 3. In parallel, macrophages labeled with PKH-26 red were placed in the presence of the hybrids Fc-WT-PEG-a621-635cit (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-a621-635arg (this is the hybrid Fc-WT-PEG-a621-635cit, with the difference that the peptide is not citrullinated), at 5 pg / mL for 1 h at 37°C.After washing, the cells were brought into contact, at a rate of 1 B lymphocyte for 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 by the 2 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. The results are presented in Table 5 according to six lines which correspond to five independent experiments. NCIT represents the result with the peptide Fc-WT-PEG-a621-635arg, CIT represents the result with the peptide Fc-WT-PEG-a621-635cit, and ( A CIT-NCIT) represents the difference between the OD obtained in the presence of Fc-WT-PEG-a621-635cit and the OD obtained in the presence of Fc-WT-PEG-a621-635arg, divided by the OD obtained in the presence of Fc-WT-PEG-a621-635arg.

[0232] [Table 5]. Percentage of phagocytosis and percentage of B lymphocytes The results show that phagocytosis activity is increased in the presence of citrullinated hybrids: 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 % of double-positives is higher, therefore phagocytosis is increased and the B population decreases when the macrophages are armed with the Fc-WT-PEG-a621-635cit (CIT) hybrids versus Fc-WT-PEG-a621-635arg (NCIT). The results are also presented in Figure 8A and 8B.

[0233] Example 7: Stability of the binding of Fc-SDH A488 and Fc-SDH peptide b60 citrullinated hybrids, on NK cells after 30 min, 24 h and 48 h, at physiological temperature

[0234] 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 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 DBCO, the molecule being labeled with the fluorochrome A488) or FcSDHFSTIE-N3-DBCO-PEG3^60-74cit-lysineA488 (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 DBCO which is coupled to a PEGn spacer linked to a peptide of SEQ ID NO: 19, the molecule being labeled with the fluorochrome A488 which is linked to the molecule via a lysine). The binding of these 2 fluorescent probes to NK cells was analyzed by cytofluorimetry. In the constructs, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8.The results are presented in the following Tables 6 (30 minutes), 7 (24 hours), and 8 (48 hours). NM represents unlabeled NK cells, without antibodies.

[0235] [Table 6]. Results at 30 minutes

[0236] [Table 7]. Results at 24 hours [Table 8]. Results at 48 hours

[0237] The results presented in Tables 6 to 8 show that FcSDHFSTIE-N3-DBCO-A488 and FcSDHFSTIE-N3-DBCO-PEG3^60-74cit-lysine A488 remain bound to the NK cell membrane for at least up to 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.

[0238] Example 8: Binding on NK cells of the various forms of Fc (WT, SDH and LALAPG) labeled with the fluorochrome A647 The NK cells were incubated for 30 min at 37°C in the presence of the fragment

[0239] FcSDHFSTIE-N3-DBCO-A647 (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 DBCO, the molecule being labeled with the fluorochrome A647) at 10 pg / mL. This construct is referred to as SDH in Table 9 below. The experiment was also performed with the following constructs: FcWT-N3-DBCO-A647 (a wild-type Fc fragment that is linked to at least one PEGn spacer that is itself coupled to an azide covalently linked to a DBCO, the molecule being labeled with the fluorochrome A647), or FcLALAPG-N3-DBCO-A647 (an Fc fragment comprising the mutations L234A, L235A and P329G that is linked to at least one PEGn spacer that is itself coupled to an azide covalently linked to a DBCO, the molecule being labeled with the fluorochrome A647), at the same concentrations. These two constructs are respectively referred to as 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. Analysis of Fc fragment binding to NK cells was performed by cytofluorometry. The results are shown in Table 9.

[0240] [Table 9]. Results

[0241] The results presented in Table 9 show that the fragments FcWT-N3-DBCO-A647, FcSDHFSTIE-N3-DBCO-A647 bind to the membrane of NK cells. The binding of FcSDHFSTIE-N3-DBCO-A647 is much greater than that of FcWT-N3-DBCO-A647.

[0242] Example 9: Binding on NK cells of the Fc-SDH fragment labeled with the fluorochrome A647 compared to the binding of the same hybrids in the presence of human IgG at increasing concentrations, up to the physiological serum concentration. The NK cells were incubated for 30 min at 37°C, i.e. in the presence of the fragment

[0243] FcSDHFSTIE-N3-DBCO-A647 (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 DBCO, the molecule being labeled with the fluorochrome A647) at 10 pg / mL, or in the presence of this same fragment FcSDHFSTIE-N3-DBCO-A647 at 10 pg / mL, co-incubated with human IgG at increasing concentrations (1, 10, 100, 1,000, 10,000 pg / 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.

[0244] The results are presented in Table 10. [Table 10]. Results The results show a dose-dependent decrease in the binding of FcSDHFSTIE-N3-DBCO-A647 to NK cells from 100 pg / mL of IgG but this remains effective and high, even in the presence of an IgG concentration 1,000 times higher than that of the Fc fragment (10,000 pg / mL) which corresponds to the physiological serum concentration of IgG in humans.

[0245] Example 10: Lysis by human NK cells of cells from a malignant human plasmoblastic B line expressing a recombinant monoclonal ACPA on its membrane, in the presence of the hybrids FcWT-N3-DBCO-PEG3-a621-635Cit and FcSDH-N3-DBCO-PEG3-a621-635Cit

[0246] WTaCIT 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; WTaArg corresponds to the WTaCIT hybrid except that the peptide of SEQ ID NO: 18 is not citrullinated; SDHaCIT 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; and SDHaArg corresponds to the hybrid SDHaCIT except that the peptide of 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.

[0247] The experiment was performed in 96-well round-bottom plates. Nalm6 cells (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 WTaCit or SDHaCit hybrid molecules used at 15 pg / mL and freshly purified NK cells (labeled with cell tracker violet) used as effector cells, also at 100,000 cells / well, for 17 h at 37°C. WTaArg and SDHaArg hybrid molecules, used as specificity controls, were incubated under the same conditions. As negative controls, Nalm6 cells were incubated in the absence of hybrids, 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 (% dead cells).(%) (ACIT-Arg) represents the difference between the percentages of 7-AAD-positive dead cells in the presence of the WT and SDH citrullinated peptide-bearing hybrids versus their non-citrullinated counterparts, divided by the percentage of 7-AAD-positive cells in the presence of the corresponding non-citrullinated hybrid.

[0248] The results are presented in Figure 9 and Table 11.

[0249] [Table 11]. Results

[0250] The results show that citrullinated hybrids (WT and SDH) are able to specifically increase the 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.

[0251] Example 11: Membrane expression of the monoclonal ACPA 2H06 by the B lymphoblastic cell line Nalm6 after transduction A- The human B lymphoblastic cell line "Nalm6" was transduced to induce the expression of the recombinant monoclonal ACPA 2H06 at its membrane. The transduction efficiency was analyzed by flow cytometry using a double labeling using an anti-Fab IgG antibody and a tetramer of the biotinylated a621-635cit peptide bound to fluorescent avidin. B- Sorting carried out from 70 million Nalm6 cells allowed the recovery of Nalm6 Fab+ cells, a pool of 300,000 cells of which was recultured. After proliferation, the enrichment of the transduced line Nalm6 2H06 was confirmed by flow cytometry. C- Fc-WT-PEG-a621-635 cit and arg hybrids were labeled with Alexa Fluor 647 using the “Lightning Link” kit. Non-transduced Nalm6 (NT) and Nalm6 2H06 lines were incubated with Fc-WT-PEG-a621-635 hybrids.After washing, membrane binding of Fc-WT-PEG-a621-635 hybrids was analyzed by flow cytometry.

[0252] The results are presented in Figure 10 and Table 12.

[0253] Table 12 represents the mean fluorescence intensities (MFI) and the ratio of MFI between the condition in the presence of fluorescent hybrid and the control condition where the cells are not labeled. nm represents the unlabeled Nalm6 cells, without fluorescent hybrids.

[0254] [Table 12]. Results The results show that after cell sorting and proliferation, 89.1% of Nalm6 cells express an IgG Fab, that of ACPA 2H06, validating the success of transduction and reestablishment of the Nalm6 2H06 line (B). The results show a strong interaction between the Nalm6 2H06 line and the fluorescent Fc-WT-PEG-a621-635 cit hybrid, while no interaction exists with the non-citrullinated Fc-WT-PEG-a621-635 arg hybrid (C). Double labeling with the fluorescent Fc-WT-PEG-a621-635 cit hybrid and the fluorescent IgG anti-Fab antibody shows a perfect correlation of the two labels on the Nalm6 2H0G line, while neither of them labels the non-transduced Nalm6 line (A). These results show that the binding of the Fc-WT-PEG-a621-635 cit hybrid is strictly linked to the expression of the membrane ACPA 2H06.

[0255] Example 12: Phagocytosis of cells of the transduced B line Nalm6 ACPA+ 2H06 by macrophages pre-armed with Fc-peptide a 621-635 hybrids

[0256] Macrophages were labeled with the membrane fluorochrome PKH-67 green and then incubated with the hybrids Fc-WT-PEG-a621-635, Fc-SDH-PEG-a621-635 or Fc-GASDIE-PEG-a621-635, in citrullinated (Cit) or non-citrullinated (Arg) form, at a concentration of 5 pg / ml for 60 min at 37°C. Cells of the human lymphoblastic B cell line Nalm6 transduced for the expression of the monoclonal ACPA 2FI06 on its membrane, were labeled with PKFI-26 red (fluorescent lipid marker) and then placed in the presence of macrophages at a rate of 2 Nalm6 target cells for 1 macrophage. After 2 hours at 37°C, the macrophages were detached and the cell populations analyzed by flow cytometry.

[0257] The results in Figure 11A show that at T2, in the absence of hybrids, there is an increase in double-positive cells that reflects spontaneous basal phagocytosis of Nalm6 cells. At 2 hours, in the presence of hybrids, regardless of the hybrid used (WT, SDFI or GASDIE), a strong increase in double-positive cells and a collapse in the number of target cells are observed when macrophages are armed with citrullinated hybrids, but absent with non-citrullinated versions of the hybrids. Here we go from 52% of double-positive cells to 14.6% in the presence of the Fc-WT-PEG-a621-635 hybrid, 24.5% for Fc-SDH-PEG-0621-635 and up to 8% for Fc-GASDIE-PEG-a621-635 which is the most effective.

[0258] Synthesis of 3 independent experiments (see Figure 11 B), confirms that the effect is reproducible and significant in all conditions, with an average increase of 70% in double-positive cells and a decrease of at least 50% in Nalm6 target cells.

[0259] Example 13: Phagocytosis of Nalm6 ACPA+ 2H06 B cell line by pre-armed macrophages in the presence of Fc-peptide a621-635 hybrids at various concentrations

[0260] Macrophages were labeled with the membrane fluorochrome PKH-67 green and then armed with the hybrids Fc-WT-PEG-a621-635, Fc-SDH-PEG-a621-635 Fc-GASDIE-PEG-a621-635 or Fc-GASDALIE-PEG-a621-635, in citrullinated (Cit) or non-citrullinated (Arg) form, at different concentrations (5pg / ml; 1 pg / 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 red PKH-26 (fluorescent lipid marker) and then placed in the presence of armed macrophages, at a rate of 1 Nalm6 target cell for 1 macrophage. After 2 hours at 37°C, the macrophages were detached and the cell populations analyzed by flow cytometry.

[0261] The results presented in Figure 12 show a strong increase in the percentage of double-positive cells (A) associated with a strong decrease in the percentage of target cells (B), when macrophages are armed with citrullinated Fc-PEG-a621-635 hybrids, whether WT, SDH, GASDIE or GASDALIE. Both are absent with non-citrullinated hybrids (Fc-PEG-a621-635 Arg). This specific phagocytosis is correlated with the concentration of Fc-PEG-a621-635 hybrids in the solution used to arm them. The model is particularly robust because phagocytosis is still observed for all citrullinated hybrids up to 50ng / mL. Due to their increased affinities towards FcγRs, the Fc-GASDIE-PEG-a621-635Cit and Fc-GASDALIE-PEG-a621-635Cit hybrids are more effective than Fc-WT-PEG-a621-635Cit or Fc-SDH-PEG-a621-635Cit, and remain active beyond 50 ng / ml.

[0262] Example 14: Confirmation of the phagocytosis process of the transduced Nalm6 ACPA+ 2H06 line by macrophages armed with FcWT-PEG-peptide a621-635 hybrids

[0263] To confirm that the destruction of Nalm6 2H06 B cells results from a phagocytosis process, Nalm6 2H06 B cells were labeled with a pH-sensitive dye, pHRodoSE, which has a low fluorescence intensity at neutral pH but emits intense fluorescence in acidic environments. However, the pH in the intracytoplasmic vesicles (lysosomes) where the phagocytosed elements are found is acidic. Non-phagocytosed cells are therefore not detectable, whereas phagocytosed cells, internalized in the lysosomal compartment, become fluorescent.

[0264] Thus, Nalm6 2H06 cells were labeled with pHRodoSE (Thermofisher) at 20ng / ml. The labeled Nalm6 cells were incubated with macrophages armed or not with Fc-WT-PEG-a621-635 Cit or Arg hybrids, at 5pg / ml for 1h at 37°C, treated or not with cytochalasin D for 30 min at 2pg / ml then 1pg / ml during phagocytosis. The cells were incubated for 2h at 37°C, then the macrophages were detached and the cell populations analyzed by flow cytometry or stained with May Grunwald Giemsa (MGG) for morphological analysis by optical microscopy.

[0265] The results presented in Figure 13 show the appearance of a pHRodoSE-positive population (2nd peak) in the presence of macrophages armed with the Fc-WT-PEG-a621-635 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 (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 intra-cytoplasmic situation in macrophages, when they are armed with citrullinated Fc-WT-PEG-a621-635 hybrids (top panel), whereas this situation is totally absent when the macrophages were armed with the non-citrullinated form of the hybrid.

[0266] Example 15: Phagocytosis of the transduced Nalm6 ACPA+ 2H06 line by macrophages pre-armed with Fc-peptide a621-635 hybrids or incubated in the presence of Fc-peptide a621-635 hybrids

[0267] Nalm6 2H06 B cell line was labeled with the green membrane fluorochrome PKH-67 and macrophages with red PKH-26. Fc-WT-PEG-0621-635 and Fc-GASDIE-PEG-a621-635 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 pg / ml (arming followed by washing), before being brought into contact with the target cells according to the ratio 1 Nalm6 target cell for 1 macrophage, for 2 h at 37°C, or incubated at the same concentration in the medium containing the Nalm6 target cells and macrophages according to the same 1 / 1 ratio, for 2 h at 37°C. The macrophages were then detached and the cell populations analyzed by flow cytometry.

[0268] The results presented in Figure 14 show a strong increase in double-positive cells (phagocytosis), associated with a collapse of the Nalm6 2H06 target cell population, when macrophages were either pre-armed or incubated with the Fc-WT-PEG-a621-635 or Fc-GASDIE-PEG-a621-635 hybrids in citrullinated form. This result is specific since it was not found with the non-citrullinated forms of the hybrids.

[0269] Example 16: Phagocytosis of the transduced Nalm6 ACPA+ 2H06 line by macrophages incubated with Fc-PEG-peptide a621-635 hybrids, in the presence or absence of human IgG at physiological serum concentration

[0270] Nalm6 2H06 cells were labeled with the fluorescent dye CFSE (CarboxyFluorescein Succinimidyl Ester) and then incubated with macrophages in the presence of Fc-WT-PEG-a621-635 or Fc-GASDIE-PEG-a621-635 hybrids, in citrullinated (Cit) or non-citrullinated (Arg) form, at a concentration of 1 pg / mL, for 2 hours at 37°C, in the presence or absence of competing human IgG at a physiological serum concentration of 10 mg / mL (concentration ratio between hybrids and IgG: 1 / 10,000). Macrophages were detached and labeled with an anti-CD11 b antibody BV421 and cell populations were analyzed by flow cytometry.

[0271] The results presented in Figure 15 show that in the absence of competing IgG, the citrullinated forms of the 2 soluble hybrids Fc-WT-PEG-a621-635 and Fc-GASDIE-PEG-a621-635 incubated with the cells, induce a quasi-total and specific phagocytosis because 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 quasi-total and significant inhibition of phagocytosis induced by the Fc-WT-PEG-a621-635cit hybrid (p<0.05), whereas this inhibition is only partial and not significant with the Fc-GASDIE-PEG-a621-635 hybrid, whose mutated Fc is more affinity for macrophage gamma receptor Fc than that of serum IgG.

[0272] Example 17: Phagocytosis of the transduced Nalm6 ACPA+ 2H06 line by human macrophages incubated with Fc-peptide a621-635 hybrids, in the presence or absence of human IgG at physiological concentration or human serum

[0273] Nalm6 2H06 cell line was labeled with the fluorescent dye CFSE (CarboxyFluorescein Succinimidyl Ester) and then incubated with macrophages in the presence of Fc-WT-PEG-a621-635 or Fc-GASDIE-PEG-a621-635 hybrids in citrullinated (cit) or non-citrullinated (arg) form, at a concentration of 10 pg / mL. The experiment was carried out 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, presenting the same IgG concentration. The concentration ratio between hybrids and IgG is therefore 1 / 1000. Cells were collected and labeled with an anti-CD11b antibody BV421 to identify macrophages and then analyzed by flow cytometry.

[0274] The results presented in Figure 16 show that the two citrullinated Fc-WT-PEG-a621-635 and Fc-GASDIE-PEG-a621-635 hybrids, used at a concentration of 10 pg / ml, specifically and very effectively induce phagocytosis of Nalm6 2H06 cells. By competing with the hybrids, IgG inhibits phagocytosis of Nalm6 2H06 cells by macrophages, but the inhibitory effect is much stronger for the Fc-WT hybrid (wild type) than for the GASDIE hybrid (mutated, more affinity for Fc gamma macrophage receptors). Equivalent results are obtained in the presence of human serum, showing that it therefore does not contain any other factors inhibiting phagocytosis than IgG.

[0275] Example 18: Phagocytosis of the transduced Nalm6 ACPA+ 2H06 line by human macrophages pre-armed with the Fc-peptide a621-635 hybrids, in the presence of the non-transduced Nalm6 line

[0276] Nalm6 2H06 cells were labeled with the fluorescent dye CFSE (CarboxyFluorescein Succinimidyl Ester) and then mixed with non-transduced Nalm6 NT cells at a ratio of ¼ (1 transduced cell to 4 non-transduced cells). Macrophages were pre-armed with Fc-WT-PEG-a621-635 or Fc-GASDIE-PEG-a621-635 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 placed in the presence of macrophages for 2 hours at 37°C, then the macrophages were detached, the total cell population was collected and labeled with an anti-CD19 APC antibody, in order to discriminate between Nalm6 2H06 cells and non-transduced Nalm6 NT cells. The total cell population was then analyzed by flow cytometry.

[0277] To confirm the specificity of phagocytosis towards ACPA+ cells in the presence of citrullinated hybrids, we co-incubated Nalm6 2H06 target cells (ACPA+) and non-transduced Nalm6 NT cells (ACPA-). The results presented in Figure 17 show that only Nalm6 cells expressing membrane ACPA 2H06 (CFSE+CD19+) disappear and are phagocytosed by macrophages when these have been previously armed with Fc-WT-PEG-a621-635 or Fc-GASDIE-PEG-a621-635 hybrids in citrullinated form (Cit). In contrast, the proportion of Nalm6 NT cells, wild type, non-transduced ACPA- (CD19+CFSE-) is unchanged, whatever the 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 Nalm62H06 cell for 9 non-transduced Nalm6 NT cells).

[0278] Example 19: Specific in vivo destruction of the Nalm6 ACPA+ 2H06 line by human NK cells in the presence of Fc-peptide a621-635 hybrids, in SCID / BEIGE immunodeficient mice

[0279] 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 (CelITrace Violet) at a high concentration (5 mM) while non-transduced Nalm6 NT cells were labeled with the same marker but at a tenfold lower concentration (0.5 mM). In flow cytometry, highly labeled cells (Nalm6 2H06 CTV h '9 h ) can thus be distinguished from weakly labeled cells (Nalm6 NT CTV |0W). Immunodeficient SCID / BEIGE mice received 50pg of anti-FcR antibodies intraperitoneally (IP) to block FcRs in murine peritoneal macrophages and then, 30 minutes later, Nalm6 2H06 target cells and non-transduced Nalm6 NT cells were injected IP in the presence or absence of freshly prepared human NK cells and citrullinated (cit) or non-citrullinated (arg) Fc-WT-PEG-a621-635 hybrids.

[0280] Four groups of 7 SCID / BEIGE mice received the following cell and hybrid combinations IP:

[0281] - 1 / 2.10 5 target cells Nalm6-2H06 + 2.10 5 Nalm6 NT cells

[0282] - 212.10 5 target cells Nalm6-2H06 + 2.10 5 Nalm6 NT + 2.10 cells 5 NK cells

[0283] - 3 / 2.10 5 target cells Nalm6-2H06 + 2.10 5 Nalm6 NT + 2.10 cells 5 NK cells +

[0284] 3pg / mouse of hybrid Fc-WT-PEG-a621-635 Cit

[0285] - 4 / 2.10 5 target cells Nalm6-2H06 + 2.10 5 Nalm6 NT + 2.10 cells 5 NK cells + 3pg / mouse of Fc-WT-PEG-a621-635 Arg hybrid

[0286] Five hours after injection, cells were recovered by peritoneal lavage with PBS 2% FCS and then analyzed by flow cytometry after labeling with 7AAD and Annexin (Becton Dickinson kit).

[0287] The results presented in Figure 18 show that in the peritoneal cavity of SCID / BEIGE immunodeficient mice, the Fc-WT-PEG-a621-635 Cit hybrid induces the total and specific destruction of Nalm6 2H06 target cells by NK cells. Indeed, the CTV peak h '9 hcorresponding to Nalm6 2H06 cells disappears exclusively in the presence of NK cells and the Fc-WT-PEG-a621-635 Cit hybrid. This highly significant result (***, p<0.001) provides in vivo proof of the efficacy and specificity of ADCC lysis of ACPA+ cells by human NK cells in the presence of Fc-citrullinated peptide hybrids carrying the ACPA target epitopes.

[0288] Example 20: Specific in vivo destruction of the Nalm6 ACPA+ 2H06 line by human macrophages pre-armed with the Fc-peptide a621-635 hybrids, in SCID / BEIGE immunodeficient mice

[0289] Membrane expression of ACPA 2H06 on Nalm6 2FI06 target cells was monitored by labeling with an anti-Fab antibody. Monocyte purification was performed from peripheral blood of a healthy donor. Monocyte differentiation into macrophages was induced in the presence of 100 ng / ml M-CSF and the phagocytic activity of the resulting macrophages was verified in vitro.

[0290] Before intraperitoneal injection (IP), macrophages were armed with the different citrullinated (cit) or non-citrullinated (arg) Fc-WT-PEG-a621-635 hybrids and the Nalm6-2H06 target cells were labeled with the fluorescent marker CTV (CelITrace Violet) at 0.5mM: strongly fluorescent when free (Nalm6 2H06 CTV h '9 h ), these cells exhibit much weaker fluorescence (Nalm6-2H06 CTV |0W ) after phagocytosis by macrophages.

[0291] Four groups of 5 SCID / BEIGE mice received target cells in IP, in the presence or absence of fresh pre-armed macrophages and, or absence of FcWT-PEG-a621-635 cit or arg hybrids, according to the following combinations:

[0292] - 1 / 2.10 5 Nalm6-2H06 target cells

[0293] 212.10 5 target cells Nalm6-2H06 + 2.10 5 unarmed macrophages 3 / 2.10 5 target cells Nalm6-2H06 + 2.10 5 macrophages pre-armed with hybrid Fc-WT-PEG-0621-635 Cit

[0294] 4 / 2.10 5 Nalm6-2H06 2.10 target cells 5 macrophages pre-armed with hybrid Fc-WT-PEG- 0621-635 Arg

[0295] Two hours after injection, cells were recovered by peritoneal lavage with PBS 5% FCS, erythrocytes were removed by gentle lysis and cells were labeled with an anti-human CD45-FITC antibody and then with 7AAD (Becton Dickinson kit) before flow cytometry analysis.

[0296] The results presented in Figure 19 show that in the peritoneal cavity of SCID / BEIGE immunodeficient mice, macrophages loaded with the Fc-WT-PEG-0621-635 cit hybrid specifically phagocytose Nalm6 2H06 target cells, their decrease compared to the control (Fc-WT-PEG-a621-635 arg hybrid, non-citrullinated) being significant (p<0.05).

[0297] This result provides in vivo evidence of the efficiency and specificity of phagocytosis of ACPA+ cells by human macrophages loaded with Fc-citrullinated peptide hybrids carrying ACPA target epitopes.

Claims

Demands

1. Hybrid molecule comprising at least one Fc fragment of 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.

2. Hybrid molecule according to the preceding claim, wherein said peptide is 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, said vertebrate fibrin preferably being a mammalian fibrin, more particularly human.

3. Hybrid molecule according to any one of the preceding claims, wherein said spacer is a polymer containing one or more repeating motifs containing the ether group, said spacer preferably being polyethylene glycol of formula PEGn, in which n represents an integer between 1 and 100, preferably between 1 and 10 and in particular 1, 2, 3, 4 or 8.

4. 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) in which X1, X2, and X3 each represent a citrullyl residue or an arginyl residue, and at least one of the residues X1 or X2 or X3 is a citrullyl residue; b) a peptide defined by the sequence GPX1VVEX2HQSACKDS (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 SGIGTLDGFX1HX2HPD (SEQ ID NO: 3) 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;d) a peptide defined by the sequence VDIDIKIX1SCX2GSCS (SEQ ID NO: 4) 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; e) a peptide defined by the sequence X1GHAKSX2PVX3GIHTS (SEQ ID NO: 12) in which X1, X2 and X3 each represent a citrullyl residue or an arginyl residue, and at least one of the residues X1 or 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.

5. 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 in 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. Hybrid molecule according to any one of the preceding claims, wherein said Fc fragment is a human Fc fragment, in particular of IgG, more particularly of IgG 1.

7. Hybrid molecule according to any one of the preceding claims, wherein said Fc fragment is wild-type or mutated, said mutated Fc fragment preferably comprising at least the following mutations: - L234A and L235A, or - L234A, L235A and P329G, or - G236A, S239D and I332E, or - G236A, S239D, A330L and I332E, or - S239D, H268F, S324T and I332E, the numbering being indicated in the sequence of a human lgG1 according to the EU index.

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

9. Hybrid molecule according to any one of the preceding claims, said molecule being 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 or SEQ ID NO: 18, - 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 which is coupled to a peptide represented by SEQ ID NO: 19 or SEQ ID NO: 18, - an Fc fragment comprising the mutations L234A, L235A and P329G 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 or SEQ ID NO: 18, - 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 or SEQ ID NO: 18, - an Fc fragment comprising the mutations S239D, FI268F, S324T and I332E 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 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 Sjögren's 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 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 being optionally present between said peptide and said antibody or fragment.