An immune cell comprising an Fc receptor on its surface, and onto which is grafted a hybrid molecule comprising an Fc fragment of antibody and at least one citrullinated peptide derived from fibrin, and its uses

Abstract

The invention relates to an immune cell comprising at least one Fc receptor on its surface, characterized in that a hybrid molecule is grafted onto the Fc receptor, said hybrid molecule comprising at least one Fc fragment of antibody covalently linked to at least one fibrin-derived peptide comprising one or more citrullyl residues. The present invention also relates to the uses of such a grafted cell, as well as its production method. (no figure)

Description

Title of the invention: Immune cell comprising an Fc receptor on its surface, and onto which is grafted a hybrid molecule comprising an Fc fragment of antibody and at least one ci-trullinated peptide derived from fibrin, and its uses

[0001] The present invention relates to an immune cell comprising at least one Fc receptor on its surface, characterized in that a hybrid molecule is grafted at the Fc receptor, said 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 present invention also relates to the uses of such a grafted cell, as well as its production method. Background to the invention

[0002] 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.

[0003] The presence of class G autoantibodies directed against citrullinated proteins and called anti-citrullinated protein autoantibodies (ACPA), is highly specific for rheumatoid arthritis. Sera from patients that contain ACPA, lymphocyte cells that express them on their membrane and the 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.

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

[0005] More specifically, the immunodominant epitopes recognized by ACPA on the α and β polypeptide chains of fibrin have been characterized and published, 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 α36-50Cit (as represented in SEQ ID NO: 5), α171-185Cit (as represented in SEQ ID NO: 6), α501-515Cit (as represented in SEQ ID NO: 14), α621-635Cit (as represented in SEQ ID NO: 18) and β360-74Cit (as represented in SEQ ID NO: 19). Sera from ACPA-positive patients recognize a or more of these five peptides.

[0006] Secreted into the 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, in turn fixing rheumatoid factors, other autoantibodies associated with rheumatoid arthritis and also secreted by local plasma cells, triggers a cascade of pro-inflammatory events.

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

[0008] To date, there is no curative treatment for rheumatoid arthritis. Treatments are aimed only at treating flare-ups and / or preventing their occurrence.

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

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

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

[0012] The present invention is based on the inventors' results showing that it is possible to target B lymphocyte clones expressing ACPAs on their surface and to eliminate them using an "armed" immune cell, i.e. a cell expressing an Fc receptor on its surface and onto which is grafted a hybrid molecule comprising (i) at least one fibrin-derived peptide having at least one citrullyl residue and (ii) a human immunoglobulin Fc fragment. The hybrid molecule is linked to the Fc receptor by means of its 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 of the immune cells expressing at least one Fc receptor.Such cells include macrophages (ACPA-positive B lymphocytes are thus eliminated by phagocytosis) and / or NK cells (ACPA-positive B lymphocytes are thus eliminated by antibody-dependent cell-mediated cytotoxicity - ADCC).

[0013] By targeting B lymphocyte clones expressing ACPAs, the grafted immune cells of the invention thus aim to eliminate ACPAs from the patients' bodies. Statement of the invention

[0014] Immune cell grafted with a hybrid molecule according to the invention

[0015] In a first aspect, the invention relates to an immune cell comprising at least one Fc receptor on its surface, characterized in that a hybrid molecule is grafted at the Fc receptor, said hybrid molecule comprising at least one antibody Fc fragment covalently linked to at least one fibrin-derived peptide having at least one citrullyl residue, a spacer being optionally present between said Fc fragment and said peptide. The scheme of such a construction is presented in [Fig.l].

[0016] According to the invention, an “immune cell” is a cell of the innate or adaptive immune system which expresses at least one Fc receptor on its surface. According to a preferred embodiment, the immune cell is chosen from an NK cell or a macrophage.

[0017] According to the invention, a “hybrid molecule” means a molecule having at least two components of a different nature, in this case the antibody Fc fragment and said peptide.

[0018] According to the invention, an "Fc receptor" means the transmembrane protein which reacts with an Fc fragment, and which contributes to the protective functions of the immune system. The Fc receptor means the Fcgamma receptor, neonatal Fcgamma, Fcalpha receptor and Fcepsilon receptor. According to one embodiment, the Fc receptor is an Fcgamma receptor, in particular FcyRIII (CD16).

[0019] According to the invention, a “hybrid molecule is grafted at the Fc receptor” means that the immune cell expresses at least one Fc receptor on its surface and that a hybrid molecule as defined here is linked to the Fc receptor via an Fc fragment. In the case where the immune cell expresses several Fc receptors on its surface, several hybrid molecules can thus be grafted onto the immune cell, i.e. one hybrid molecule per receptor.

[0020] 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).

[0021] According to the invention, the expression "covalently linked" means a bond covalent, that is, a chemical bond in which two atoms share two electrons. Said covalent bond can be polar or non-polar.

[0022] According to the invention, a "spacer" is a linking agent which makes it possible to covalently link 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). For example, it may be a molecule or a peptide. Preferably, the spacer does not modify the physicochemical properties of the hybrid molecule.

[0023] The presence of at least one spacer is advantageous: it makes it possible to facilitate the independent accessibility of the two partners of the hybrid molecule (the Fc fragment is more easily accessible to bind to the Fc receptor, just as said peptide is more easily accessible to bind to ACPA-positive B lymphocytes), and / or to stabilize the hybrid molecule, and / or increase the solubility of the hybrid molecule.

[0024] According to one embodiment, the hybrid molecule grafted onto the immune cell according to the invention may comprise one or more spacers. Preferably, the hybrid molecule comprises one or two spacers. According to one embodiment, when at least one spacer is present in said hybrid molecule of the invention, said Fc fragment is covalently linked to a spacer, said spacer itself being covalently linked to said peptide. According to another embodiment, when at least two spacers are present in said hybrid molecule of the invention, said Fc fragment is covalently linked to a first spacer, said first spacer itself being covalently linked to a second spacer and the second spacer is itself covalently linked to said peptide. The bond between the Fc fragment and the peptide may therefore be direct, or indirect in the presence of spacers.

[0025] According to one embodiment, the hybrid molecule grafted onto the immune cell 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 to the Fc fragment, the two peptides are identical, for example two peptides of SEQ ID NO: 18 or SEQ ID NO: 19.

[0026] According to one embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100.

[0027] 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.

[0028] According to one embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, said peptide is derived from all or part of the sequence of the α or [3] chain of a vertebrate fibrin, by substitution of at least one arginyl residue with a citrullyl residue. Preferably, said peptide is derived from a sequence of at least 5 consecutive amino acids of the α chain (in particular represented by SEQ ID NO: 27) or [3] 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.

[0029] According to one embodiment, in said hybrid molecule grafted onto the immune cell 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.

[0030] According to one embodiment, in said hybrid molecule grafted onto the im- cell according to the invention, the peptide is linear.

[0031] According to one embodiment, in said hybrid molecule grafted onto the immune cell 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 chained in a sequence inverse to that of the peptide to be reproduced), or of the retro-inverso type (the amino acids are of the D type instead of the natural L series and are chained in a sequence inverse to that of the peptide to be reproduced). According to an even more particular embodiment, in said hybrid molecule according to the invention, the terminal carboxyl function (COOH) of said peptide is replaced by a car-boxamide function (CONH2). Preferably, in the peptide of SEQ ID NO: 12, the terminal carboxyl function (COOH) of said peptide is replaced by a car-boxamide function (CONH2).For example, the peptide [360-74Cit (as represented in SEQ ID NO: 19) advantageously has such a carboxamide function.

[0032] According to another embodiment, in said hybrid molecule grafted onto the immune cell 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 a terminal amine function (NH2).

[0033] According to one embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, the amine and carboxyl functions of the peptide may be in the form of the salt corresponding to the acid or the base.

[0034] According to one embodiment, in said hybrid molecule grafted onto the immune cell 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 XiPAPPPISGGGYX2AX3 (SEQ ID NO: 1) in which Xb, X2, and X3 each represent a citrullyl residue or an arginyl residue, and at least one of the residues XI or X2 or X3 is a citrullyl residue; b) a peptide defined by the sequence GPXiVVEX2HQSACKDS (SEQ ID NO: 2) in which Xi and X2 each represent a citrullyl residue or an arginyl residue, and at least one of the residues Xi or X2 is a citrullyl residue; c) a peptide defined by the sequence SGIGTLDGFXiHX2HPD (SEQ ID NO: 3) in which Xi and X2 each represent a citrullyl residue or an arginyl residue, and at least one of the residues Xi or X2 is a citrullyl residue; d) a peptide defined by the sequence VDIDIKIXiSCX2GSCS (SEQ ID NO: 4) in wherein Xi and X2 each represent a citrullyl residue or an arginyl residue, and at least one of residues Xi or X2 is a citrullyl residue; e) a peptide defined by the sequence XiGHAKSX2PVX3GIHTS (SEQ ID NO: 12) in which Xb X2 and X3 each represent a citrullyl residue or an arginyl residue, and at least one of the residues Xi 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.

[0035] According to a particular embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, said peptide comprises at least one citrullyl residue, and is chosen from the group consisting of: - a peptide defined by the sequence SEQ ID NO: 1 in which 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); - 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); - a peptide defined by the sequence SEQ ID NO: 3 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); - a peptide defined by the sequence SEQ ID NO: 4 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); - 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).

[0036] According to an even more particular embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, said peptide comprises at least one citrullyl residue, and is chosen from the group consisting of: - a peptide defined by the sequence SEQ ID NO: 1 in which Xb, X2, and X3 are citrullyl residues, or a peptide of at least 15 amino acids comprising said sequence (this is a peptide of SEQ ID NO: 19); - a peptide defined by the sequence SEQ ID NO: 2 in which Xi and X2 are citrullyl residues, or a peptide comprising a fragment of at least 5 consecutive amino acids of said sequence containing said citrullyl residues (this is a peptide of SEQ ID NO: 5); - a peptide defined by the sequence SEQ ID NO: 3 in which Xi and X2 are citrullyl residues, or a peptide comprising a fragment of at least 5 consecutive amino acids of said sequence containing said citrullyl residues (this is a peptide of SEQ ID NO: 14); - a peptide defined by the sequence SEQ ID NO: 4 in which Xi and X2 are citrullyl residues, or a peptide comprising a fragment of at least 5 consecutive amino acids of said sequence containing said citrullyl residues (this is a peptide of SEQ ID NO: 6); - a peptide defined by the sequence SEQ ID NO: 12 in which Xb 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).

[0037] According to an even more particular embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, said peptide is chosen from the group consisting of: SEQ ID NO: 5 (a36-50cit38,42), SEQ ID NO: 6 (a171-185cit78, i8i), SEQ ID NO: 7 (a183-197cit186>190), SEQ ID NO: 8 (a246-260cit258), SEQ ID NO: 9 (a259-273cit263>271), SEQ ID NO: 10 (a366-380cit367), SEQ ID NO: 11 (a396-410cit 404), SEQ ID NO: 13 (a411-425cit425), SEQ ID NO: 14 (a501-515cit510,5i2), SEQ ID NO: 15 (a546-560cit547), SEQ ID NO: 16 (a561-575cit573), SEQ ID NO: 17 (a588-602cit591), SEQ ID NO: 18 (a621-635cit621>627>630), SEQ ID NO: 19 (|360-74cit 6o,72,74), SEQ ID NO: 20 (|3210-224cit224), SEQ ID NO: 21 ([328l-295cit285>294), SEQ ID NO: 22 (|3420-434cit42i) and SEQ ID NO: 23 (|3433-447cit436,445), more particularly chosen in the group consisting of: SEQ ID NO: 5 (a36-50cit38,42), SEQ ID NO: 6 (al71-185cit178,181), SEQ ID NO: 14 (a501-515cit510j5i2),SEQ ID NO: 18 (a621-635cit62ij627>630) and SEQ ID NO: 19 ([360-74cit60 72 74), even more particularly chosen from SEQ ID NO: 18 (a621-635cit62ij627>630) and SEQ ID NO: 19 ([360-74cit6o>72>74) • ,

[0038] According to one embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, said Fc fragment is a human Fc fragment, in particular of IgG, more particularly of IgGl. The IgGl may correspond to any allotypic variant, for example Glm3 or nGlml. By way of example, the Fc fragment of IgGl is represented by SEQ ID NO: 24, SEQ ID NO: 25 (Fc+Qtag) or SEQ ID NO: 26 (Fc+Qtag bis).

[0039] According to one embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, said Fc fragment is wild type or mutated. The mutation(s) may aim to increase the plasma half-life, decrease it, or modify the effector functions of the Fc fragment. According to an even more particular embodiment, said mutated Fc fragment comprises at least the following mutations: - L234A and L235A (LALA), or - L234A, L235A and P329G (LALAPG), or - G236A, S239D and I332E (GASDIE), or - G236A, S239D, A330L and I332E (GASDALIE), or - S239D, H268F, S324T and I332E (SDHFSTIE or SDH), the numbering being indicated in the sequence of a human IgGl according to the EU index. Such mutations are notably described in the article Bruhns and Jônsson, Immunol Rev. 2015 Nov;268(l):25-51. Preferably, when the hybrid molecule is used in therapy, said mutated Fc fragment comprises at least the mutations GASDIE, GASDALIE, or SDH.

[0040] According to one embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, said Fc fragment has a fucosylation level of between 0% and 100% of the glycosylated forms. According to the invention, “between 0% and 100%” represents all integer values ​​between 0 and 100, ie; 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%. 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.

[0041] 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 one embodiment, in said hybrid molecule grafted onto the immune cell 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 and / or several amino acids constituting the Fc fragment can be. involved in a covalent bond with said peptide.

[0042] According to a preferred embodiment, when at least one spacer is present in said hybrid molecule grafted onto the immune cell according to the invention, this makes it possible to bind 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 grafted onto the immune cell according to the invention, this can also make it possible to bind 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 grafted onto the immune cell according to the invention comprises at least two spacers: a first spacer which makes it possible to bind the Fc fragment to an azide or to an alkyne and a second spacer which makes it possible to bind the peptide to an azide (when the Fc fragment is bound to an alkyne) or to an alkyne (when the Fc fragment is bound to an azide).

[0043] According to one embodiment according to the invention, in said hybrid molecule grafted onto the immune cell according to the invention, said Fc fragment: - is coupled to at least one azide or an alkyne, such as a cyclooctyne, and in particular DBCO, or - is linked to at least one spacer which is itself coupled to an azide or an alkyne, such as a cyclooctyne, and in particular DBCO, and said peptide is either coupled to an azide or an alkyne, such as a cyclooctyne, and in particular DBCO, or linked to a spacer which is itself coupled to an azide or an alkyne, such as a cyclooctyne, and in particular DBCO, the covalent bond between said Fc fragment and said peptide, optionally in the presence of one or more spacers, being created between the azide and the alkyne.

[0044] According to an embodiment according to the invention, in said hybrid molecule grafted onto the immune cell according to the invention, - the Fc fragment is coupled to at least one azide and said peptide is coupled to an alkyne, such as a cyclooctyne, and in particular DBCO, or - the Fc fragment is coupled to at least one alkyne, such as a cyclooctyne, and in particular DBCO, and said peptide is coupled to an azide, the covalent bond between said Fc fragment and said peptide being created between the azide and the alkyne.

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

[0046] According to one embodiment, in the molecules described above which are grafted onto the immune cell, more particularly in paragraphs

[0042] to

[0044] , the spacer used is preferably a PEGn spacer, n representing more particularly 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.

[0047] According to a particularly preferred embodiment, in the molecules described above which are grafted onto the immune cell, more particularly in paragraphs

[0043] to

[0045] , the peptide is derived from human fibrin or fibrinogen and the Fc fragment is a human Fc, preferably human IgGl.

[0048] 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 hydrazoid 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 CnH2n2, and which are characterized by the presence of at least one triple bond. Preferably the alkyne is a cyclooctyne, even more preferably dibenzocyclooctyne (DBCO).

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

[0050] 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 concerning enzymatic conjugation, see, for example, patent application US20160361434 or US20170313787, or the publication Ohtsuka et al., Bioscience, Biotechnology, and Biochemistry Volume 64, 2000 - Issue 12, Comparison of Substrate Specificities of Transglutaminases Using Synthetic Peptides as Acyl donors. The substrate of transglutaminase is for example a peptide 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 an isolated cysteine ​​or one participating in a disulfide bridge after reduction thereof and for example a maleimide. An example of such a conjugation is shown in [Fig.7]. In this example, the Fc fragment comprises a Qtag peptide and said Fc fragment is linked to a spacer (itself coupled to an azide), thanks to the action of transglutaminase which will create a covalent bond between the glutamyl residue of the Qtag and the NH2 group carried by the PEGn spacer.

[0051] 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".

[0052] According to the invention, the hybrid molecule is grafted onto the immune cell thanks to the bond between the Fc receptor and the Fc fragment.

[0053] According to one embodiment of the invention, said hybrid molecule grafted onto the immune cell according to the invention is represented by: - 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 (|36O-74cit6o,72,74) or SEQ ID NO: 18 (a621-635cit62i,627,63()X - 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 linked in a covalent to a cyclooctyne which is coupled to a peptide represented by SEQ ID NO: 19 (p60-74cit60,72,74) or SEQ ID NO: 18 (a621-635cit621j627j630), - 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 (|36O-74cit 60,72,74) or SEQ ID NO: 18 (a621-635cit621j627>63o), - 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 (|36O-74cit 60,72,74) or SEQ ID NO: 18 (a621-635cit621>627j63o), - 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 (|360-74cit60,72,74) or SEQ ID NO: 18 (a621-635cit 621,627,63o)» - 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 (|360-74cit60,72,74) or SEQ ID NO: 18 (a621-635cit 621,627,63()), - 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 (P60-74cit60,72,74) or SEQ ID NO: 18 (a621-635cit621j627j630), - 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 coupled to a PEGn spacer which is itself linked to a peptide represented by SEQ ID NO: 19 ([360-74cit60j72>74) or SEQ ID NO: 18 (a621-635cit62ij627j630), - 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 ([>60-74citM)72 74) or SEQ ID NO: 18 (a621-635cit62ij627j630), - 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 (|360-74cit60,72,74) or SEQ ID NO: 18 (a621-635cit 621,627,63()), - 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 ([360-74cit60j72>74) or SEQ ID NO: 18 (a621-635cit62i,627,63oX - 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 (|36O-74cit 60,72,74) or SEQ ID NO: 18 (a621-635citœij627,630), - an Fc fragment comprising the mutations G236A, S239D, A330L and I332E which is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne which is coupled to a peptide represented by SEQ ID NO: 19 (p60-74cit60,72,74) or SEQ ID NO: 18 (a621-635cit621j627>630), n preferably representing an integer between 1 and 10, more particularly 1, 2, 3, 4 or 8.

[0054] According to one embodiment according to the invention, said hybrid molecule is represented by: - 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 (|360-74cit60,72,74) or SEQ ID NO: 18 (a621-635cit62ij627>630), - 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 (p60-74cit60,72,74) or SEQ ID NO: 18 (a621-635cit621>627>630), - 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 (|360-74cit 60,72,74) or SEQ ID NO: 18 (a621-635cit621>627j63o), - 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 (p60-74cit60,72,74) or SEQ ID NO: 18 (a621-635cit621>627>630), - 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 coupled to a PEGn spacer which is itself linked to a peptide represented by SEQ ID NO: 19 (|360-74cit60,72,74) or SEQ ID NO: 18 (a621-635cit62i,627,63()), n preferably representing an integer between 1 and 10, more particularly 1, 2, 3, 4 or 8.

[0055] According to an even more particular embodiment according to the invention, said hybrid molecule is represented by: - 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 (|360-74cit60,72,74), - 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 ([360-7401^0,72,74), - 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 (|360-74cit 60,72,74), - 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 (|360-74cit60,72,74) or SEQ ID NO: 18 (a621-635cit62i,627,63o), - 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 coupled to a PEGn spacer which is itself linked to a peptide represented by SEQ ID NO: 19 (|360-74cit60,72,74) 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.

[0056] Use of the hybrid molecules according to the invention

[0057] In a second aspect, the present invention also relates to an immune cell onto which a hybrid molecule as defined above is grafted, for its use as a medicament.

[0058] More particularly, according to the invention, said immune cells onto which the hybrid molecules are grafted 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 the cells making it possible to destroy “ACPA-positive” B lymphocytes, immune cells expressing an Fc receptor, thanks to its Fc fragment, a natural ligand for Fc receptors (for example Fcgamma 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”).

[0059] According to a particular embodiment, the invention relates to an immune cell onto which is grafted 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-Sjögren 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.

[0060] According to one embodiment, the invention also relates to a pharmaceutical composition comprising an immune cell onto which a hybrid molecule, as defined above, is grafted, in combination with a pharmaceutically acceptable vehicle.

[0061] According to the invention, "a pharmaceutically acceptable vehicle" means any formulation making the composition suitable for administration to a patient, in any galenic form.

[0062] As indicated above, the present invention aims to destroy “ACPA-positive” B lymphocytes. However, “ACPA-positive” B lymphocytes which have differentiated into ACPA-secreting plasma cells are no longer targeted by the hybrid molecules previously described. According to a particular embodiment, the present invention thus relates to an immune cell as previously described, for its use as a medicament, in combination with a hybrid molecule (this second hybrid molecule not being grafted onto the immune cell). Said second hybrid molecule comprises 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 a combination therapy makes it possible to specifically target and destroy ACPA-positive plasma cell and B lymphocyte clones, and thus to eliminate ACPA from the patients' bodies. The antibody used in this second hybrid molecule is preferably a monoclonal antibody.

[0063] According to a particular embodiment, in this second hybrid molecule, the fragment means a portion of antibody which carries the binding site to the antigen. 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, etc. 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.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 marker being expressed on the surface of plasma cells.

[0064] 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 CD 138.

[0065] According to this particular embodiment of the invention, the grafted immune cell and the second hybrid molecule are administered simultaneously, separately or spread out over time.

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

[0067] According to another embodiment, the invention thus relates to the in vitro or ex vivo use of an immune cell comprising at least one Fc receptor on its surface onto which a hybrid molecule is grafted, said 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 mainly at the Qtag level (if present) or at the lysine level.

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

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

[0070] According to one embodiment, the invention thus relates to a method for producing an immune cell grafted with a hybrid molecule as defined previously, comprising the following steps: - (i) obtaining an azide coupled to an Fc fragment or obtaining an alkyne coupled to an Fc fragment, - (ii) obtaining an alkyne coupled to a peptide or obtaining an azide coupled to a peptide, - (iii) realization of the covalent bond between the azide and the alkyne, - (iv) bringing into contact an immune cell comprising at least one Fc receptor on its surface and a hybrid molecule as obtained in step (iii), - step (i) can be carried out before or after step (ii), or concurrently.

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

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

[0073] The sequences of the invention are represented in Table 1 below.

[0074] [Tables 1] Sequence number / Sequence name if applicable Sequence SEQ ID NO: 1 x1papppisgggyx2ax3 SEQ ID NO: 2 GPX|VVEX2HQSACKDS SEQ ID NO: 3 sgigtldgfx,hx2hpd SEQ ID NO: 4 VDIDIKIX^CXzGSCS SEQ ID NO: 12 x1ghaksx2pvx3gihts SEQ ID NO: 5 / a36-50cit38, 42 GPXVVEXHQSACKDS, X representing a citrullyl residue SEQ ID NO: 6 / al71-185citi78, 181 VDIDIKIXSCXGSCS, X representing a citrullyl residue SEQ ID NO: 7 / al83-197citi86> 190 SCSXALAXEVDLKDY, X representing a citrullyl residue SEQ ID NO: 8 / a246-260cit 258 PEWKALTDMPQMXME, X representing a citrullyl residue SEQ ID NO: 9 / a259-273cit263> 271 MELEXPGGNEITXGG, X representing a citrullyl residue SEQ ID NO: EXGSAGHWTSESSVS, X representing a citrullyl residue 10 / a366-380cit367 SEQ ID NO: 11 / a396-410cit404 DSPGSGNAXPNNPDW, X representing a citrullyl residue SEQ ID NO: 13 / a411-425cit425 GTFEEVSGNVSPGTX, X representing a citrullyl residue SEQ ID NO: 14 / a5Ol-515cit5io, 512 SGIGTLDGFXHXHPD, X representing a citrullyl residue SEQ ID NO: 15 / a546-560cit547 SXGSESGIFTNTKES, X representing a citrullyl residue SEQ ID NO: 16 / a561-575cit573 SSHHPGIAEFPSXGK, X representing a citrullyl residue SEQ ID NO: 17 / a588-602cit59i SYNXGDSTFESKSYK, X representing a citrullyl residue SEQ ID NO: 18 / 0621-63501621, 627,630 XGHAKSXPVXGIHTS, X representing a citrullyl residue SEQ ID NO: 19 / (360-74cit 60,72,74 XPAPPPISGGGYXAX, X representing a citrullyl residue SEQ ID NO: 20 / [3210-224cit224 QKLESDVSAQMEYCX, X representing a citrullyl residue SEQ ID NO: 21 / p281-295cit285, 294 VIQNXQDGSVDFGXK, X representing a citrullyl residue SEQ ID NO : 22 / p420-434cit42i PXKQCSKEDGGGWWY, X représentant un résidu citrullyl SEQ ID NO : 23 / [3433-447cit436, 445 WYNXCHAANPNGXYY, X représentant un résidu citrullyl SEQ ID NO : 24 / Fragment Fc APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ ID NO : 25 / Fragment Fc + Qtag LLQGARSDATHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK SEQ ID NO : 26 / Fragment Fc + Qtag bis AHGHGHGLLQGARSDATHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO :27 / Fibrin a-chain GPRVVERHQSACKDSDWPFCSDEDWNYKCPSGCRMKGLIDE VNQDFTNRINKLKNSLFEYQKNNKDSHSLTTNIMEILRGDFSS ANNRDNTYNRVSEDLRSRIEVLKRKVIEKVQHIQLLQKNVRA QLVDMKRLEVDIDIKIRSCRGSCSRALAREVDLKDYEDQQKQ LEQVIAKDLLPSRDRQHLPLIKMKPVPDLVPGNFKSQLQKVPP EWKALTDMPQMRMELERPGGNEITRGGSTSYGTGSETESPRN PSSAGSWNSGSSGPGSTGNRNPGSSGTGGTATWKPGSSGPGST GSWNSGSSGTGSTGNQNPGSPRPGSTGTWNPGSSERGSAGHW TSESSVSGSTGQWHSESGSFRPDSPGSGNARPNNPDWGTFEEV SGNVSPGTRREYHTEKLVTSKGDKELRTGKEKVTSGSTTTRR SCSKTVTKTVIGPDGHKEVTKEVVTSEDGSDCPEAMDLGTLSG IGTLDGFRHRHPDEAAFFDTASTGKTFPGFFSPMLGEFVSETES RGSESGIFTNTKESSSHHPGIAEFPSRGKSSSYSKQFTSSTSYNR GDSTFESKSYKMADEAGSEADHEGTHSTKRGHAKSRPVRGIH TS SEQ ID NO : 28 / Chaîne [3 de fibrine GHRPLDKKREEAPSLRPAPPPISGGGYRARPAKAAATQKKVER KAPDAGGCLHADPDLGVLCPTGCQLQEALLQQERPIRNSVDE LNNNVEAVSQTSSSSFQYMYLLKDLWQKRQKQVKDNENVVN EYSSELEKHQLYIDETVNSNIPTNLRVLRSILENLRSKIQKLESD VSAQMEYCRTPCTVSCNIPVVSGKECEEIIRKGGETSEMYLIQP DSSVKPYRVYCDMNTENGGWTVIQNRQDGSVDFGRKWDPY KQGFGNVATNTDGKNYCGLPGEYWLGNDKISQLTRMGPTEL LIEMEDWKGDKVKAHYGGFTVQNEANKYQISVNKYRGTAGN ALMDGASQLMGENRTMTIHNGMFFSTYDRDNDGWLTSDPRK QCSKEDGGGWWYNRCHAANPNGRYYWGGQYTWDMAKHG TDDGVVWMNWKGSWYSMRKMSMKIRPFFPQQ SEQ ID NO : 29 (Exemple de Qtag) LLQG SEQ ID NO : 30 Chaîne a de fibrinogène MFSMRIVCLVLSVVGTAWTADSGEGDFLAEGGGVRGPRVVE RHQSACKDSDWPFCSDEDWNYKCPSGCRMKGLIDEVNQDFT NRINKLKNSLFEYQKNNKDSHSLTTNIMEILRGDFSSANNRDN TYNRVSEDLRSRIEVLKRKVIEKVQHIQLLQKNVRAQLVDMK RLEVDIKIRSCRGSCSRALAREVDLKDYEDQQKQLEQVIAK DLLPSRDRQHLPLIKMKPVPDLVPGNFKSQLQKVPPEWKALTDMPQMRMELERPGGNEITRGGSTSYGTGSETESPRNPSSAGSW NSGSSGPGSTGNRNPGSSGTGGTATWKPGSSGPGSTGSWNSGS SGTGSTGNQNPGSPRPGSTGTWNPGSSERGSAGHWTSESSVSG STGQWHSESGSFRPDSPGSGNARPNNPDWGTFEEVSGNVSPGT RREYHTEKLVTSKGDKELRGKEKVTSGSTTTTRRSCSKTVTK TVIGPDGHKEVTKEVVTSEDGSDCPEAMDLGTLSGIGTLDGFR HRHPDEAAFFDTASTGKTFPGFFSPMLGEFVSETESRGSESGIF ​​TNTKESSSHHPGIAEFPSRGKSSSYSKQFTSSTSYNRGDSTFESK SYKMADEAGSEADHEGTHSTKRGHAKSRPVRGIHTS SEQ ID NO : 31 Chaine [3 de fibrinogène MKRMVSWSFHKLKTMKHLLLLLLCVFLVKSQGVNDNEEGFF SARGHRPLDKKREEAPSLRPAPPPISGGGYRARPAKAAATQKK VERKAPDAGGCLHADPDLGVLCPTGCQLQEALLQQERPIRNS VDELNNNVEAVSQTSSSSFQYMYLLKDLWQKRQKQVKDNEN VVNEYSSELEKHQLYIDETVNSNIPTNLRVLRSILENLRSKIQKL ESDVSAQMEYCRTPCTVSCNIPVVSGKECEEIIRKGGETSEMYL IQPDSSVKPYRVYCDMNTENGGWTVIQNRQDGSVDFGRKWD PYKQGFGNVATNTDGKNYCGLPGEYWLGNDKISQLTRMGPT ELLIEMEDWKGDKVKAHYGGFTVQNEANKYQISVNKYRGTA GNALMDGASQLMGENRTMTIHNGMFFSTYDRDNDGWLTSDP RKQCSKEDGGGWWYNRCHAANPNGRYYWGGQYTWDMAK HGTDDGVVWMNWKGSWYSMRKMSMKIRPFFPQQ

[0075] Summary table of the sequences of the invention

[0076] Other characteristics, details and advantages of the invention will appear on reading the attached Figures and the examples which illustrate the invention and are in no way intended to limit it.

[0077] 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 [Fig.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 represents more particularly 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. Brief description of the Figures Fig.l

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

[0079] A spacer (which is optional) is shown between the antibody Fc fragment and the fibrin-derived peptide having a citrullyl residue (referred to as "citrullinated peptide"). Fig. 2

[0080] [Fig.2] represents a B lymphocyte expressing trans-ACPA on its surface membranes linked to a hybrid molecule according to the invention, said hybrid molecule itself being linked by the Fc fragment to an NK cell.

[0081] The NK cell will thus be able to destroy the B lymphocyte by ADCC. More specifically, this figure shows a B cell (or B lymphocyte) which expresses on its surface 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 FcyRIIIa (CD 16a) 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; FcγR, Fc-gamma Receptor; NK cell, Natural Killer cell). Fig. 3

[0082] [Fig.3] represents a B lymphocyte expressing on its surface trans-membrane ACPAs linked to a hybrid molecule according to the invention, said hybrid molecule itself being linked by the Fc fragment to a macrophage.

[0083] The macrophage will thus 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 to different FcyRs expressed on the surface of a macrophage will activate ADP, i.e. phagocytosis, and induce the specific destruction of ACPA-positive B lymphocytes. (ACPA, Anti-Citrullinated Protein Au-toantibodies; ADP, Antibody-Dependent Phagocytosis; BCR, B-Cell Receptor; FcyR, Fc-gamma Receptor). Fig. 4

[0084] [Fig.4] represents the reactivity of several hybrid molecules including the peptide of SEQ ID NO: 19 ([>60-74cit(,072.74) with respect to ACPA.

[0085] [360-74 represents the peptide of SEQ ID NO: 19. Fc-WT-[360-74 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 ([36O-74eit6o.72.74). Fc-LALA-[360-74 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-[360-74 represents an Fc fragment comprising the mutations S239D, H268F, S324T and I332E which is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne DBCO coupled to a peptide represented by SEQ ID NO: 19.Fc-WT-PEG-[360-74 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 coupled to a PEGn spacer which is itself linked to a peptide represented by SEQ ID NO: 19 (|36O-74cit6o,72,74)- Fc-SDH-PEG-[360-74 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, itself linked to a peptide represented by SEQ ID NO: 19 ([360-74cit60,72,74)- Fc-SDH(Cter)-PEG-[360-74 corresponds to Fc-SDH-PEG-[360-74, at . the difference being that in the Fc-SDH(Cter)-PEG-[360-74] hybrid, the first PEGn is attached at the C-terminal end of the Fc fragment, unlike the other examples in which the first PEGn is attached at the N-terminal end. n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8. Fig. 5

[0086] [Fig.5] represents the reactivity of a hybrid molecule comprising the peptide of SEQ ID NO: 18 (a621-635cit62i,627,63o) with respect to ACPA.

[0087] a621-635 represents the peptide of SEQ ID NO: 18. Fc-WT-PEG-a621-635 re has 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-SDH-PEG-a621-635 represents an Fc fragment comprising the mutations S239D, H268F, 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. Fig. 6

[0088] [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.

[0089] FcWT-PEG-a621-635 represents a wild-type Fc fragment that is linked to at minus 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, H268F, S324T and I332E, which is linked to at least one PEGn spacer which is itself coupled to an azide covalently linked to a cyclooctyne DBCO coupled to a PEGn spacer which is itself linked to a peptide represented by SEQ ID NO: 18. n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8. Fig. 7

[0090] [Fig.7] represents a method of manufacturing a hybrid molecule according to the invention, comprising the scheme for derivation and conjugation of the Fc fragments. Fig. 8

[0091] [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.

[0092] The results of the 6 experiments presented in Example 6 are shown (N=6). The P value is as follows: * < 0.05 ** < 0.01 *** < 0.001. Fig. 9

[0093] [Fig.9] represents the lysis by human NK cells of cells of a line transduced malignant human plasmablastic B cell 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.

[0094] Nahn6 represents the case where only Nahn6 cells were incubated; Nalm6+NK the case where Nalm6 cells were incubated with NK cells; WTaCIT the case where the WTaCIT hybrid was incubated with Nahn6 cells and NK cells; WTaArg the case where the WTaArg hybrid was incubated with Nalm6 cells and NK cells; SDHaCIT the case where the SDHaCIT hybrid was incubated with Nalm6 cells and NK cells and SDHaArg the case where the SDHaArg hybrid was incubated with Nalm6 cells and NK cells.

[0095] Examples

[0096] Example 1: Example of production of a hybrid molecule according to the invention, and of an immune cell grafted with such a hybrid molecule

[0097] The hybrid molecule described here comprises the following construction: an Fc fragment covalently linked to a PEGn, itself coupled to an azide, said azide being covalently linked to an alkyne, which is itself linked to a spacer coupled to a citrullinated peptide. Such a molecule can be read: Fc-PEGn-N3-DBCO-PEGn-ci-trullinated peptide. The two PEGns can be identical or different (for example the first PEGn is PEG3 and the second PEGn is PEG2).

[0098] 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).

[0099] 2. Bringing NH2-PEGn-N3 into contact 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. 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.

[0100] 3. Synthesis of a Cys-PEGn-citrullinated peptide (i.e. a spacer coupled to a peptide with one end and having a free cysteine ​​at the other end). DBCO is then coupled to the Cys-PEGn-citrullinated peptide at the cysteine, and a DBCO-PEGn-citrullinated peptide is thus obtained.

[0101] 4. Realization of the “click”: coupling between the N3 and the DBCO. The DBCO- PEGn-citrullinated peptide is brought into contact with 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.

[0102] 5. Obtaining the hybrid molecule: Fc-PEGn-N3-DBCO-PEGn-citrullinated peptide. This embodiment is illustrated in [Fig.7].

[0103] 6. An immune cell grafted with such a hybrid molecule is then obtained by bringing into contact an immune cell expressing at least one Fc receptor on the surface and a hybrid molecule. Thanks to the Fc receptor / Fc fragment bond, the hybrid molecule and the immune cell are grafted.

[0104] Similarly, an Fc-PEGn-DBCO and an N3-PEGn-citrullinated peptide can be obtained, then coupled to obtain Fc-PEGn-DBCO-N3-PEGn-citrullinated peptide.

[0105] Similarly, an Fc-PEGn-DBCO and a citrullinated N3-peptide can be obtained, or an Fc-PEGn-N3 and a citrullinated DBCO-peptide, then coupled to obtain respectively Fc-PEGn-DBCO-N3-citrullinated peptide or Fc-PEGn-N3-DBCO-citrullinated peptide.

[0106] Example 2: Reactivity of ACPAs purified by affinity chromatography on citrullinated peptides and eluted at pH3 (pH3 fraction), with respect to the hybrid molecules according to the invention

[0107] 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).

[0108] a. Examples with hybrid molecules containing the peptide [360-74cit

[0109] ELISA microtiter plate wells were coated by passive adsorption using 100 μL of a solution containing either the peptide [360-74cit (SEQ ID NO: 19, also referred to as [360-74), or a hybrid molecule Fc-WT-[360-74cit, Fc-LALA-[360-74cit, Fc-SDH-[360-74cit, Fc-WT-PEG-[360-74cit, Fc-SDH-PEG-[360-74cit or Fc-SDH(Cter)-PEG-[360-74cit. 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 (|360-74arg, 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 ACPA were incubated at 1, 0.5 or 0.25 pg / mL, diluted in PBS buffer 2% BSA 2M NaCl for 1 h at 4°C. After washing, the reactivity of the ACPA was detected using a secondary antibody anti-human IgG Fab diluted 1 / 2500 in PBS buffer 2% BSA. The results are expressed in ADO (Delta-Optical Density), corresponding to the OD obtained with the peptide [360-74cit or with the hybrid molecules constructed with the peptide [360-74cit, subtracted respectively from the OD obtained with the peptide [360-74arg or with the corresponding hybrid molecules constructed with the peptide [360-74arg.

[0110] The results are presented in [Fig.4]. They show a dose-dependent reactivity of the ACPA purified on peptide [360-74cit, with respect to the hybrid molecules Fc-WT-[360-74cit, Fc-LALA-[360-74cit, Fc-SDH-[360-74cit, Fc-WT-PEG-[360-74cit, Fc-SDH-PEG-[360-74cit or Fc-SDH(Cter)-PEG-[360-74cit. They show that included in the different hybrids, the citrullinated peptides remain perfectly reactive with the ACPA.

[0111] b. Examples with hybrid molecules containing the peptide a621-635cit

[0112] ELISA plate wells were coated using 100 pL of a solution containing the peptide a621-635cit (SEQ ID NO: 18, also called a621-635) or a hybrid molecule Fc-WT-PEG-a621-635cit or Fc-SDH-PEG-a621-635cit, at a concentration of 5 pg / 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 h at 4°C. After washing, the purified ACPA were incubated at 4, 2 and 1 pg / mL diluted in PBS 2% BSA 2M NaCl buffer for 1 h at 4°C.After washing, ACPA reactivity was detected using a secondary antibody anti-Fab of human IgG diluted 1 / 2500 in PBS buffer 2% BSA. The results are expressed as ADO (Delta-Optical Density), corresponding to the OD obtained with the peptide a621-635cit 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.

[0113] The results are presented in [Fig.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.

[0114] 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

[0115] The ACPA used here is a recombinant monoclonal human ACPA ((clone 022014CCP14CFCT2H06, known 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 Ci-trulline-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 accession number MH629700.1.

[0116] ELISA plate wells were coated with 100 pL of a solution containing either the hybrid molecule Fc-WT-PEG-a621-635cit or Fc-SDH-PEG-a621-635cit, used at a concentration of 5 pg / mL in PBS (Phosphate Buffered Saline) buffer incubated overnight at 4°C. The hybrid molecules constructed with a non-citrullinated peptide (a621-635arg) 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 2% BSA 2M NaCl buffer for 1 h at 4°C. After washing, their reactivities were detected using a secondary anti-human IgG Fab antibody diluted 1 / 2500 in PBS 2% BSA buffer.The results are expressed in ADO (Delta-Optical Density), corresponding to the OD obtained with the hybrid molecules constructed with the peptide a621-635cit subtracted from the OD obtained with the hybrid molecules constructed with the negative control peptide a621-635arg.

[0117] The hybrid molecules Fc-WT-PEG-a621-635cit and Fc-SDH-PEG-a621-635cit are identical to those used in Example 2.

[0118] The results are presented in [Fig.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.

[0119] Example 4: Fixation of hybrid molecules on macrophages at 2h and 20h, at physiological temperature

[0120] Human macrophages, differentiated in vitro in the presence of M-CSF (100 ng / mL) from CD 14+ 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-[360-74Cit (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-[360-74Cit (an Fc fragment comprising 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-DBCO-[360-74Cit (an Fc fragment comprising 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 fixation of the hybrid molecules to the surface of the macrophages is demonstrated and quantified by cytofluorimetry (FACS Canto II) after incubation of the macrophages with an anti-Fc antibody coupled to FITC, used 1 / 1000.In the constructs, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8. .

[0121] The results are presented in Tables 2 and 3 below. “NM” and “ANTI FC” respectively represent the well containing the macrophages incubated without antibody or with the anti-Fc antibody alone.

[0122] [Tables2] 2h Macrophages Ratio NM 177 / ANTI FC 285 / WT 808 2.8 LALAPG 502 1.8 SDH 1330 4.7

[0123] Results at 2 a.m.

[0124] [Tables3] 8 p.m. Macrophages Ratio NM 213 / ANTI FC 275 / WT 1003 3.6 LALAPG 404 1.5 SDH 1457 5.3

[0125] Results at 8 p.m.

[0126] The results presented in Tables 2 and 3 show that the hybrid molecules FcWT-N3-DBCO-[360-74Cit and FcSDHFSTIE-N3-DBCO-[360-74Cit at the physiological temperature of 37°C, bind to the macrophage membrane from the 2nd hour and are still present 20 hours later. These results also show that the binding of the hybrid FcSDHFSTIE-N3-DBCO-[360-74Cit is higher than that of the wild-type FcWT-N3-DBCO-[360-74Cit.

[0127] Example 5: Phagocytosis induced by the pôOcitrullinated peptide Fc-SDH hybrid when B lymphocytes are armed via their FcyRIIb (CD32b) with purified ACPA.

[0128] 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 [360-74cit (SEQ ID NO: 19) at a concentration of 5 μg / mL for 30 min at 37°C. Subsequently, the cells were washed and then placed in the presence of the 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-[360-74Arg (which corresponds to the construct FcSDHFSTIE-N3-DBCO-PEG3-[360-74Cit in which the peptide [360-74 is not citrullinated). This construct is called NCIT in Table 4 below). The cells were placed in the presence of the hybrids at 10 μg / 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. .

[0129] The results are presented in Table 4 according to four lines which correspond to four independent experiments,

[0130] [Tables4] % Phagocytosis % Population B NCIT CIT % (ACIT-NCI T) NCIT CIT % (ACIT-NCI T) 9.28 17.8 +91% 46.1 32.9 -40% 16.2 20.4 +26% 40.5 30.5 -33% 3.42 5 +46% 77.1 66.7 - 16% 6.28 8.95 +42% 77.3 69.3 - 12%

[0131] Percentage of phagocytosis and percentage of B lymphocytes

[0132] 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 the hybrid FcSDHFSTIE-N3-DBCO-PEG3-[360-74Cit (CIT) versus FcSDHFSTIE-N3-DBCO-PEG3-[360-74Arg (NCIT).

[0133] 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.

[0134] 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 hour at 37°C with recombinant human monoclonal ACPA 2H06 whose Fc is mutated SDH, at a concentration of 10 µg / mL. Such antibodies can be obtained as in Example 3. In parallel, macrophages labeled with red PKH-26 were 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 μg / 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 constructs, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8.

[0135] 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 (ACIT-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.

[0136] [Tables5] % Phagocytosis NCIT CIT % (ACIT-NCIT) 4.3 7.0 +63% 15.8 19.2 +21% 4.6 7.3 +58% 12.8 18.2 +42% 16.9 21.4 + 27% 15.7 20.1 + 28% % Population B NCIT CIT % (ACIT-NCIT) 66.6 61.2 - 8% 78.7 76.6 -3% 67.6 57.2 - 15% 54.2 47.7 - 12% 44.5 37 - 17% 43.8 38.5 - 12%

[0137] Percentage of phagocytosis and percentage of B lymphocytes

[0138] The results show that the 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 decreased when macrophages were armed with Fc-WT-PEG-a621-635cit (CIT) versus Fc-WT-PEG-a621-635arg (NCIT) hybrids. The results are also shown in Figure 8A and 8B.

[0139] Example 7: Stability of the binding of Fc-SDH A488 and Fc-SDH p60 citrullinated peptide hybrids, on NK cells after 30 min, 24 h and 48 h, at physiological temperature

[0140] The 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-[360-74cit-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.

[0141] 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.

[0142] [Tableauxô] MFI RATIO NM 562 / FcSDHFSTIE-N3-DBCO-PEG 3-[360-74cit-lysineA488 4588 8.163 FcSDHFSTIE-N3-DBCO-A488 2973 5.289

[0143] Results in 30 minutes

[0144] [Tables7] MFI RATIO NM 558 / FcSDHFSTIE-N3-DBCO-PEG 3-[360-74cit-lysineA488 4091 7.324 FcSDHFSTIE-N3-DBCO-A488 2353 4.212

[0145] Results at 24 hours

[0146] [Tables8] MFI RATIO NM 494 / FcSDHFSTIE-N3-DBCO-PEG 3-[360-74cit-lysineA488 4336 8.77 FcSDHFSTIE-N3-DBCO-A488 1710 3.460

[0147] Results at 48 hours

[0148] The results presented in Tables 6 to 8 show that FcSDHFSTIE-N3-DBCO-A488 and FcSDHFSTIE-N3-DBCO-PEG3-[360-74cit-lysine A488 remain attached to the membrane of NK cells for at least up to 48 hours at physiological temperature and that the attachment of the peptide to the Fc fragment does not prevent its interaction with the Fc gamma receptors of NK cells.

[0149] Example 8: 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.

[0150] The NK cells were incubated for 30 min at 37°C either in the presence of the FcSDHFSTIE-N3-DBCO-A647 fragment (an Fc fragment comprising the 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 FcSDHFSTIE-N3-DBCO-A647 fragment 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.

[0151] The results are presented in Table 9.

[0152] [Tables9] MFI RATIO NEG 212.08 / SDH 10 pg / mL 38545.45 181.74 SDH (1 pg / mL IgG) 37899.98 178.70 SDH (lOpg / mL IgG) 38771.43 182.81 SDH (lOOpg / mL IgG) 36270.21 171.02 SDH (lOOOpg / mL IgG) 29526.19 139.22 SDH (lOOOOpg / mL IgG) 20486.26 96.59

[0153] Results

[0154] 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.

[0155] Example 9: Lysis by human NK cells of cells from a transduced malignant plasmoblastic human 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.

[0156] 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.

[0157] The experiment was carried out in 96-well round-bottom plates. Cells of the Nalm6 line (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, on the one hand, the hybrid molecules WTaCit or SDHaCit used at 15pg / mL and, on the other hand, freshly purified NK cells (labeled with cell tracker violet) used as effector cells, also at 100,000 cells / well, for 17h at 37°C. The hybrid molecules WTaArg and SDHaArg, used as specificity controls, were incubated under the same conditions. As negative controls, Nalmô cells were incubated in the absence of hybrids, alone or in the presence of NK cells. Cell lysis was measured in flow cytometry by labeling with the fluorescent probe 7-AAD. The results show the percentage of cells positive for this marker (% of 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.

[0158] The results are presented in [Fig.9] and in Table 10.

[0159] [TableauxlO] % 7-AAD + % (ACIT-Arg) Nalmô 8 Nalmô + NK 45 Nalmô + NK + WT aCIT 65 + 62% Nalmô + NK + WT aArg 40 Nalmô + NK + SDH aCIT 55 + 83% Nalmô + NK + SDH aArg 30

[0160] Results

[0161] The results show that the citrullinated hybrids (WT and SDH) are able to specifically increase the ADCC lysis of Nalmô ACPA-positive target cells caused by NK cells. This increase in mortality is 62% for the WT hybrids and 83% for the SDH hybrids.

[0162] Example 10: Binding of Fc (WT, SDH, LALAPG)-pôO Cit peptide hybrids to NK cells does not induce their degranulation

[0163] The hybrids used here are as follows: FcWT-[360-74 represents a wild-type Fc fragment which is linked to a 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 ([360-74cit60j72,74)- FcSDH-[360-74 represents 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 cyclooctyne coupled to a PEGn spacer which is itself linked to a peptide represented by SEQ ID NO: 19 ([360-74cit60j72,74)- FcLALAPG-[360-74 represents 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 coupled to a PEGn spacer which is itself to a peptide represented by SEQ ID NO: 19 (|360-74cit60j72j74). In the constructs, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8.

[0164] The NK cells were incubated for 30 min at 37°C in the presence of the hybrid molecules Fc-WT-[360-74cit, Fc-SDH-[360-74cit, Fc-LALAPG-[360-74cit at 10 pg / mL. After washing, the cells were incubated for 30 min at 4°C in the presence of a fluorescent anti-CD107a antibody (degranulation marker). After washing, analysis of the membrane expression of CD107a was carried out by flow cytometry.

[0165] The results are presented in Table 11.

[0166] [T ables 11] NK Cells Alone Coated with FcWT-[360-74 Coated with FcSDH-[360-74 Coated with FcLALAPG-[360-74 % CD 107a expression 2.5% 1.8% 3% 2.2%

[0167] Results

[0168] The results presented in Table 11 show that the membrane expression of the degranulation marker CD 107a is equivalent (between 1.8 and 3%), whether or not the NK cell is coated with the hybrid molecules according to the invention. The fixation of the hybrid molecules therefore does not induce the degranulation of the NK cells.

Claims

Claims

1. Immune cell comprising at least one Fc receptor on its surface, characterized in that a hybrid molecule is grafted at the Fc receptor, said hybrid molecule comprising at least one antibody Fc fragment covalently linked to a fibrin-derived peptide having at least one citrullyl residue, a spacer being optionally present between said Fc fragment and said citrullinated peptide, and characterized in that the peptide is recognized by anti-citrullinated protein autoantibodies and in that the Fc receptor is the FcyRIII receptor.

2. An immune cell according to claim 1, said cell being selected from an NK cell or a macrophage.

3. Immune cell according to any one of the preceding claims, wherein said peptide of the hybrid molecule is derived from all or part of the sequence of the α or β chain of a vertebrate fibrin, by substitution of at least one arginyl residue by a citrullyl residue, said vertebrate fibrin preferably being a mammalian fibrin, more particularly human.

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

5. UU o. An immune cell according to any preceding claim, wherein said peptide of the hybrid molecule comprises at least one citrullyl residue, and is selected from the group consisting of: a) a peptide defined by the sequence XiPAPPPISGGGYX2AX3 (SEQ ID NO: 1) in which Xb, X2, and X3 each represent a citrullyl residue or an arginyl residue, and at least one of residues Xi or X2 or X3 is a citrullyl residue; b) a peptide defined by the sequence GPXiVVEX2HQSACKDS (SEQ ID NO: 2) in which Xi and X2 each represent a citrullyl residue or an arginyl residue, and at least one of residues Xi or X2 is a citrullyl residue; c) a peptide defined by the sequence SGIGTLDGFXiHX2HPD (SEQ ID NO: 3) in which Xi and X2 each represent a citrullyl residue or an arginyl residue, and at least one of the residues Xi or X2 is a citrullyl residue; d) a peptide defined by the sequence VDIDIKIXiSCX2GSCS (SEQ ID NO: 4) in which Xi and X2 each represent a citrullyl residue or an arginyl residue, and at least one of the residues Xi or X2 is a citrullyl residue; e) a peptide defined by the sequence XiGHAKSX2PVX3GIHTS (SEQ ID NO: 12) in which Xb X2 and X3 each represent a citrullyl residue or an arginyl residue, and at least one of the residues Xi 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.

6. The immune cell of any one of claims 1-4, wherein said peptide of the hybrid molecule is selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, more particularly selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 18 and SEQ ID NO:

19.

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

8. An immune cell according to any one of the preceding claims, wherein said Fc fragment of the hybrid molecule 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 IgGl according to the EU index.

9. An immune cell according to any preceding claim, wherein in said hybrid molecule: - the Fc fragment is coupled to an azide and said peptide is coupled to a alkyne, such as cyclooctyne, and in particular DBCO, or - the Fc fragment is 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 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 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, or - the Fc fragment is linked to a 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 a 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 a 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 a spacer, itself coupled to an alkyne, such as a cyclooctyne, and in particular DBCO, and said peptide is linked to a spacer, itself coupled to an azide, the covalent bond between said Fc fragment and said peptide, optionally in the presence of one or more spacers, being created between the azide and the alkyne.

10. Immune cell according to any one of the preceding claims, for use as a medicament, in particular for use in the treatment of autoimmune diseases associated with the production of anti-citrullinated protein autoantibodies, in particular Gougerot-Sjögren syndrome and Rheumatoid Arthritis.

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