# Patent

## Title
Citrulline peptides derived from fibrin and recognized by rheumatoid arthritis specific autoantibodies, and the use thereof

## Abstract

The invention relates to novel citrulline peptides derived from fibrin α and β chains which are recognizable by specific citrulline antiprotein autoantibodies (AAPC) of a rheumatoid arthritis (PR) and to the use thereof for detecting the presence of said specific PR AAPC in a biological sample.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 11/577,674, filed Oct. 2, 2008, which is a 35 U.S.C. 371 national phase application of international application PCT/FR2005/002736 filed Nov. 3, 2005, which claim priority to French Application No. 04 11782, filed Nov. 4, 2004, French Application No. 04 13711, filed Dec. 22, 2004 and French Application No. 05 08422, filed Aug. 8, 2005, which are hereby incorporated herein in their entirety by reference.


FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to novel citrullinated peptides recognized by rheumatoid arthritis-specific autoantibodies.
Rheumatoid arthritis (hereinafter abbreviated to “RA”) is the most common of the chronic inflammatory forms of rheumatism. It is an autoimmune disease and the serum from affected patients contains autoantibodies, some of which are specific and may constitute a marker for this disease, allowing it to be diagnosed even at very early stages. Research studies have therefore been carried out for the purpose of identifying antigens recognized by these antibodies, in order to obtain purified preparations of the latter that can be used in conventional techniques of immunological diagnosis.
It has been shown that RA-specific autoantibodies recognize various isoelectric variants of (pro)filaggrin (for review, cf., for example, SERRE and VINCENT, In: Autoantibodies, PETER and SHOENFELD Eds, Elsevier Science Publishers, 271-276, 1996). These autoantibodies have for this reason been called: “anti-filaggrin autoantibodies (AFAs)”. Application EP 0 511 116 describes the purification and characterization of antigens of the filaggrin family, recognized by these antibodies, and their use for the diagnosis of rheumatoid arthritis.
Subsequently, the filaggrin epitopes identified by AFAs were identified as regions of the filaggrin molecule carrying citrullyl residues, resulting from the conversion of arginyl residues by a peptidylarginine deiminase (Girbal-Neuhauser E et al., J. Immunol, 162, 585-94, 1999; Schellekens G A et al., J Clin Invest, 101, 273-81, 1998). The analysis of various synthetic peptides derived from the sequence of human filaggrin has shown that deimination (citrullination) is necessary in order to form the epitopes recognized by AFAs, which are today also called anti-citrullinated protein autoantibodies (ACPAs). It is this name that will be used in the disclosure which follows.
It has also been observed that the environment of the citrullyl residues also plays an important role (Girbal-Neuhauser E, 1999, mentioned above; Schellekens G A, 1998, mentioned above; Union A et al., Arthritis Rheum, 46, 1185-95, 2002); some amino acids that are “permissive” with respect to binding of the antibody, and the nature of which probably modifies the antibody binding affinity, have been identified. Among these amino acids, -gly-, -ser-, -his-, -thr- and -gln- residues are most commonly found in the immediate environment of the citrullyl residues of citrullinated “filaggrin” peptides which, to date, have been identified as carrying epitopes recognized by ACPAs (Sebbag M et al., Rev Rhum, 68, 106, 2001).
A large number of citrullinated peptides specifically recognized by ACPAs, and that can be used for the diagnosis of RA, have been obtained from filaggrin. However, it has also been observed that, although strictly specific RA, the reactivity of these various citrullinated peptides with respect to ACPAs is heterogeneous, different peptides being recognized by sera derived from different individuals. This implies that, in order to obtain a diagnostic reagent capable of identifying the presence of ACPAs in a large population, it is necessary to combine several different peptides.
In parallel, it has been shown that ACPAs are secreted by synovial tissue plasma cells (Masson-Bessière C et al., Clin Exp Immunol, 119, 544-52, 2000) and are specifically directed against citrullinated forms of fibrin α- and β-chains present in this tissue (Masson-Bessiere C et al., J Immunol, 166, 4177-84, 2001).
The inventors have undertaken, with the aim of more thoroughly characterizing the epitopes recognized by ACPAs, to identify those presented by fibrin α- and β-chains. For this purpose, they have evaluated the reactivity of sera containing ACPAs with respect to citrullinated synthetic peptides derived from the sequence of the α-chain and from the sequence of the β-chain of fibrin. Given the known heterogeneity of the reaction profiles of citrullinated peptides derived from filaggrin, with ACPAs, the inventors used mixtures of sera selected so as to contain ACPAs representing the various reactivity profiles observed in the case of these filaggrin-derived peptides, in order to detect all the fibrin peptides that may be recognized by ACPAs.
The peptides tested were obtained from the sequence of the α-chain and from the sequence of the β-chain of fibrin. In total, 72 peptides, including 41 derived from the fibrin α-chain and 31 derived from its β-chain, were selected. Among the 72 citrullinated peptides analyzed, the inventors identified 13 peptides derived from the sequence of the α-chain and 5 peptides derived from that of the α-chain of fibrin as reacting significantly with one and/or the other of the mixtures of sera tested, and therefore as carrying epitopes recognized by some of the ACPAs present in this of these mixture(s).
The inventors subsequently individually analyzed each of the 18 reactive peptides identified with each of the sera constituting the mixtures tested. This analysis confirmed, for most of the peptides tested, the large interindividual variability in ACPA specificity previously observed in the case of filaggrin-derived citrullinated peptides.
Furthermore, it allowed the inventors to identify peptides which, unexpectedly, have a spectrum of reactivity with ACPAs that is much broader than those reported up until now in the case of filaggrin-derived citrullinated peptides. Indeed, the inventors have identified 5 citrullinated peptides (4 peptides derived from the fibrin α-chain and 1 derived from the fibrin α-chain) which are each individually recognized by at least 40% of the sera analyzed, and therefore appear to carry major epitopes. Among these peptides, two are recognized by the majority of the sera, and also exhibit complementary reactivity profiles encompassing all the sera analyzed. Each of these sera in fact recognizes one and/or the other of these peptides. This suggests that these two peptides carry structural motifs representative of a very large majority of the various motifs recognized by ACPAs.
SUMMARY OF THE INVENTION
A subject of the present invention is an isolated peptide recognized by anti-citrullinated protein autoantibodies (ACPAs) present in the serum of patients suffering from rheumatoid arthritis, characterized in that it comprises at least one citrullyl residue, and in that it is chosen from the group consisting of:
a) a peptide defined by the sequence X1PAPPPISGGGYX2AX3 (SEQ ID NO: 1) in which X1 and X2 each represent a citrullyl residue or an arginyl residue, and at least one of the residues X1 and X2 is a citrullyl residue;
b) a peptide defined by the sequence GPX1VVEX2HQSACKDS (SEQ ID NO: 2) in which X1 and X2 each represent a citrullyl residue or an arginyl residue, and at least one of the residues X1 and X2 is a citrullyl residue;
c) a peptide defined by the sequence SGIGTLDGFX1HX2HPD (SEQ ID NO: 3) in which X1 and X2 each represent a citrullyl residue or an arginyl residue, and at least one of the residues X1 and X2 is a citrullyl residue;
d) a peptide defined by the sequence VDIDIKIX1SCX2GSCS (SEQ ID NO: 4) in which X1 and X2 each represent a citrullyl residue or an arginyl residue, and at least one of the residues X1 and X2 is a citrullyl residue;
e) a peptide defined by the sequence X1GHAKSX2PVX3GIHTS (SEQ ID NO: 12) in which X1, X2 and X3 each represent a citrullyl residue or an arginyl residue, and at least one of the residues X1, X2 and X3 is a citrullyl residue;
f) a peptide comprising at least 5 consecutive amino acids, preferably at least 7 consecutive amino acids, and advantageously from 8 to 14 consecutive amino acids, including at least one citrullyl residue, of one of the peptides a) to e) above.



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
Having thus described embodiments of the invention in general terms, reference will be made to that accompanying drawings wherein:
FIG. 1A illustrates SEQ ID NO: 10, which is that of a signal sequence; and
FIG. 1B illustrates SEQ ID NO: 11, which is that of a signal sequence.



DETAILED DESCRIPTION
The peptides in accordance with the invention are at least 5 amino acids in size, preferably from 5 to 25 amino acids in size, and entirely preferably from 10 to 20 amino acids in size.
According to a preferred embodiment of a peptide in accordance with the invention, it is chosen from:

    
    
        a peptide defined by the sequence SEQ ID NO: 1 in which at least 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;
        a peptide defined by the sequence SEQ ID NO: 2 in which at least 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;
        a peptide defined by the sequence SEQ ID NO: 3 in which at least 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;
        a peptide defined by the sequence SEQ ID NO: 4 in which at least X1 is a citrullyl residue, or a peptide comprising a fragment of at least 5 consecutive amino acids of said sequence containing said citrullyl residue;
        a peptide defined by the sequence SEQ ID NO: 12 in which at least 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.
    
    


Particularly preferably, a peptide in accordance with the invention is chosen from:

    
    
        a peptide defined by the sequence SEQ ID NO: 1 in which X1, X2 and X3 are citrullyl residues, or a peptide of at least 16 amino acids comprising said sequence;
        a peptide defined by the sequence SEQ ID NO: 2 in which X1 and X2 are citrullyl residues, or a peptide comprising a fragment of at least 5 consecutive amino acids of said sequence containing said citrullyl residues;
        a peptide defined by the sequence SEQ ID NO: 3 in which X1 and X2 are citrullyl residues, or a peptide comprising a fragment of at least 5 consecutive amino acids of said sequence containing said citrullyl residues;
        a peptide defined by the sequence SEQ ID NO: 4 in which X1 and X2 are citrullyl residues, or a peptide comprising a fragment of at least 5 consecutive amino acids of said sequence containing said citrullyl residues;
        a peptide defined by the sequence SEQ ID NO: 12 in which X1, X2 and X3 are citrullyl residues, or a peptide comprising a fragment of at least 10 consecutive amino acids of said sequence containing said citrullyl residues.
    
    


Citrullinated peptides in accordance with the invention can, for example, be obtained from natural, recombinant or synthetic fragments of fibrin or of fibrinogen, through the action of peptidylarginine deiminase (PAD); they can also be obtained by peptide synthesis, by directly incorporating one or more citrulline residues into the peptide synthesized.
Citrullinated peptides in accordance with the invention also encompass derivatives of the peptides SEQ ID NOS: 1 to 4 and 12, or fragments thereof, defined above, said derivatives carrying modifications intended to improve recognition thereof by ACPAs: by way of examples of such derivatives, mention will be made of: cyclized peptides; retro-type peptides, in which L-amino acids are linked together according to a sequence that is the reverse of that of the peptide to be reproduced; retro-inverso-type peptides, consisting of amino acids of the D series (instead of the amino acids of the L series of natural peptides) linked together according to a sequence that is the reverse of that of the peptide to be reproduced.
Very advantageously, the peptides are peptides in which the terminal carboxyl function (COOH) is replaced with a carboxamide function (CONH2). In this case, particularly preferred peptides are those in which the C-terminal residue is a citrullyl residue, the carboxyl function of which is replaced with a carboxamide function.
A subject of the present invention is also any peptide recognized by ACPAs present in the serum of patients suffering from rheumatoid arthritis, and containing, at its C-terminal end, a citrullyl residue, the carboxyl function of which is replaced with a carboxamide function. Advantageously, said peptide contains at least one other citrullyl residue in its sequence. Preferably, said peptide comprises from 5 to 25 amino acids.
The replacement of the carboxyl function of the C-terminal citrulline residue with a carboxamide function can make it possible to increase the reactivity of the peptide with the ACPAs, or, where appropriate, to render reactive, with ACPAs, peptides which are not naturally reactive.
Citrullinated peptides in accordance with the present invention also encompass derivatives of the peptides of SEQ ID NOS: 1 to 4 and 12, or fragments thereof, as defined above, said derivatives carrying modifications intended to facilitate their synthesis and/or to improve their stability. By way of example of such derivatives, mention will be made of the peptides that include amino acids, the carboxyl groups of which are esterified or converted to amide groups and/or amino acids, an amino group of which is alkylated, for example methylated or acetylated. The amine and carboxyl groups of the peptides may be present in the form of the salt corresponding to the base or to the acid.
From the citrullinated peptides described above, it is also possible to obtain mimotope peptides comprising at least one citrullyl residue (citrullinated mimotope peptide).
These mimotope peptides can be obtained by screening libraries of citrullinated peptides, the sequences of which are defined based on those of the peptides SEQ ID NOS: 1, 2, 3, 4 and 12 of the present invention, used in this context as “model peptides”.
Preferably, these peptide libraries are prepared by synthesizing various peptides of between 10 and 20, preferably between 12 and 17, amino acids, in particular 15 amino acids in size. Each of these peptides conserves at least 2, preferably at least 4, advantageously at least 6, particularly preferably at least 8, and very advantageously at least 10 amino acids, including at least one citrullyl residue, of the sequence of the selected model peptide, at the same positions as on said model peptide, the other positions being variable.
These libraries can advantageously be screened as described in the examples hereinafter, and in particular using sera representative of various ACPA reactivity profiles, as defined in Example 1 hereinafter.
Methods for synthesizing mimotope peptides are well known in themselves. Reference will, for example, be made to Chapter 6 of “Chemical approaches to the synthesis of peptides and proteins”, Paul Lloyd-Williams, Fernando Albericio and Ernest Giralt, CRC Press New York, 1997, “Peptide libraries”, pages 237-270.
These citrullinated mimotope peptides can be used, alone or in combination with other citrullinated peptides, and preferably in combination with at least one of the other peptides of the invention, in a diagnostic test for RA, for recognizing ACPAs.
A subject of the present invention is also the use of the peptides in accordance with the invention, as defined above, for detecting the presence of ACPAs in a biological sample, in the context of the in vitro diagnosis of RA.
A subject of the present invention is thus antigenic compositions that can be used for detecting the presence of ACPAs in a biological sample in the context of the in vitro diagnosis of RA, which compositions are characterized in that they comprise at least one peptide in accordance with the invention.
Compositions in accordance with the invention can associate with one another various peptides chosen from the peptides in accordance with the invention, or can associate one or more peptides in accordance with the invention with one or more citrullinated peptides derived, in particular, from filaggrin.
According to a preferred embodiment of an antigenic composition in accordance with the invention, it comprises at least one peptide of sequence SEQ ID NO: 1, and at least one peptide of sequence SEQ ID NO: 2, as defined above.
This composition has a very broad spectrum of reactivity, and can also make it possible to detect RA at an early stage.
Advantageously, a composition in accordance with the invention can also comprise a peptide of sequence SEQ ID NO: 3 and/or a peptide of sequence SEQ ID NO: 4 and/or a peptide of sequence SEQ ID NO: 12, as defined above.
The compositions in accordance with the invention can, where appropriate, be in the form of multiple-peptide compositions, in which the constitutive peptides are associated with one another or with a carrier molecule, generally by covalent bonding. By way of example, mention will be made of the multiple antigen peptides (MAPs) described in particular by TAM (Proc. Natl. Acad. Sci. U.S.A., 85, 5409-13, 1988).
A subject of the present invention is also a method for detecting the presence of ACPAs in a biological sample, in the context of the in vitro diagnosis of RA, which method is characterized in that it comprises:

    
    
        bringing said biological sample into contact with at least one peptide or one antigenic composition in accordance with the invention, as defined above, under conditions that allow the formation of an antigen/antibody complex with the ACPAs possibly present in said sample;
        detecting, by any appropriate means, the antigen/antibody complex possibly formed.
    
    


This method of detection can be carried out by means of a kit comprising at least one peptide or one antigenic composition according to the invention and, where appropriate, buffers and reagents suitable for making up a reaction medium that allows the formation of an antigen/antibody complex, and/or means for detecting said antigen/antibody complex.
Advantageously, said kit comprises a peptide or an antigenic composition according to the invention, immobilized on a solid substrate. By way of nonlimiting examples of solid substrates that can be used, mention will be made of microtitration plates, pipette tips of the VIDAS® device (sold by BIOMERIEUX), beads, microbeads or microparticles, strips, etc.
Said kit can also comprise reference samples, such as one or more negative serum or sera and one or more positive serum or sera.
A subject of the present invention is also a method for detecting the presence of ACPAs in a biological sample, in the context of the in vitro diagnosis of RA, which method is characterized in that it comprises:

    
    
        providing a first citrullinated peptide capable of competing, for the binding to said ACPAs, with a peptide in accordance with the invention, as defined above;
        bringing said first peptide into contact with said biological sample, under conditions that allow the formation of an antigen/antibody complex with the ACPAs possibly present in said sample;
        detecting, by any appropriate means, the antigen/antibody complex possibly formed.
    
    


In particular, the first citrullinated peptide capable of competing with a peptide in accordance with the invention is a citrullinated mimotope peptide that can be obtained as described above.
The present invention will be understood more clearly from the further description which follows, which makes reference to examples that illustrate the identification of the peptides in accordance with the invention and the demonstration of their reactivity profile with respect to ACPAs.
EXAMPLE 1
Demonstration of the Heterogeneity of the Reaction of Acpas with Citrullinated Peptides Derived from Filaggrin, and Obtaining of Mixtures of Sera Representative of the Various Reactivity Profiles
90 sera, exhibiting ACPAs that can be detected both by ELISA and by immunoblotting on human fibrinogen deiminated in vitro (Masson-Bessière C, 2001, mentioned above; Nogueira L et al., Arthritis Res, 4, 90, 2002) but also by indirect immunofluoresence on frozen sections of rat esophagus (Vincent C et al., Ann Rheum Dis, 48, 712-22, 1989) and by immunoblotting on human epidermal filaggrin (Vincent C et al., J Rheumatol, 25, 838-46, 1998), were used. The reactivity of these 90 multipositive sera was analyzed by ELISA with regard to 5 different citrullinated peptides, the reactivity of which with respect to ACPAs had been previously established. These peptides are the following:














E12D
ESSRDGSXHPRSHD
(SEQ ID NO: 5)


 


T12E
TGSSTGGXQGSHHE
(SEQ ID NO: 6)


 


E12H
EQSADSSXHSGSGH
(SEQ ID NO: 7)


 


cfc6
SHQESTXGXSRGRSGRSGS
(SEQ ID NO: 8)


 


cf48-65-4
TIHAHPGSXXGGRHGYHH
(SEQ ID NO: 9)





(X denotes a citrullyl residue)






The peptides E12D, T12E and E12H were described by Girbal-Neuhauser, E. et al. (1999), the peptides cfc6 and cf48-65-4 were described by Schellekens, G. et al. (1998).
The analysis by ELISA was carried out according to the protocol described by Girbal-Neuhauser, E. et al. (1999).
This analysis made it possible to identify 12 reactivity profiles with regard to the 5 peptides.
These profiles are summarized in Table I below.














TABLE I







Profile
E12D
E12H
T12E
cfc6
cf48-65-4























1
+
+
+

+



2
+
+
+



3
+
+

+



4
+
+



5
+



6
+



+



7

+



8

+
+

+



9


+



10



+
+



11



+



12




+










In order to be as representative as possible of the various ACPA reactivity profiles, mixtures, hereinafter referred to as mixtures: “A” and “B”, were each formed by mixing, in equal parts, 10 sera representing various profiles of reactivity on “filaggrin” peptides.
The composition of these two mixtures is given in Table II below.









TABLE II





Serum
Profile
Mixture

















97.0459
1
A


97.0388
3


97.1436
4


97.0169
6


97.0530
7


97.0311
8


97.0506
9


97.0468
10


97.0796
11


97.0907
12


97.1715
1
B


97.0524
1


97.0323
2


97.0794
4


95.0256
5


97.1795
5


97.1474
9


97.0244
10


97.1548
11


97.1210
12









The two mixtures A and B together are therefore representative of the heterogeneity of specificity of the ACPA+ sera.
EXAMPLE 2
Identification of Citrullinated Peptides Derived From Fibrin α- and β-Chains, that React with the ACPAs
The peptides tested were obtained from the sequence of the fibrin α-chain and from the sequence of the fibrin β-chain [portion corresponding respectively to residues 36-635 and 45-491 of the A(α) (reference NP Acession: NP 068657) and B(β) (reference SWISSPROT FIBS_HUMAN Prim. Accession: P02675) chains of fibrinogen]. The sequences of residues 1 to 635 and 1 to 491 of the fibrinogen A(α) and B(β) chains are also respectively represented in the attached sequence listing under the numbers SEQ ID NO: 10 and SEQ ID NO: 11, and in FIGS. 1A and B. The sequences indicated in bold in FIG. 1 are those of the signal sequences of the proteins followed by those of their fibrinopeptides (A and B, respectively).
Each fibrin α- or β-chain was segmented into contiguous sequences of 15 amino acids, and all the peptides comprising at least one arginyl residue were selected. In the case of the peptides for which the arginyl residue was located at the NH2— or COON-terminal end, a second series of peptides of 15 amino acids, overlapping the first, so as to recenter the terminal arginyl residue in the sequence, was selected. In addition, a peptide corresponding to residues 621-629 of the fibrin α-chain was synthesized. In total, 72 peptides, including 41 derived from the fibrin α-chain and 31 derived from its β-chain, were selected. For each of the peptides, the form with arginyl residue(s) (native form) and the form where all the arginyl residues were substituted with citrullyl residues (citrullinated form) were synthesized according to the solid-phase method of Merrifield, with a purity ≧60% [company NeoMPS (Strasbourg, France)].
The list of the citrullinated peptides selected is given in Table III below:







TABLE III







A. First series:





α-chain










α36-50Cit38,42
α171-185Cit178,181
α351-365Cit353
α456-470Cit458,459


α66-80Cit58
α186-200Cit186,190
α366-380Cit367
α501-515Cit510,512


α81-95Cit84
α216-230Cit216,218
α381-395Cit394
α546-560Cit547


α111-125Cit114,123
α246-260Cit258
α396-410Cit404
α561-575Cit573


α126-140Cit129,135,137
α261-275Cit263,271
α411-425Cit425
α591-605Cit591


α141-155Cit143
α276-290Cit287
α426-440Cit426
α621-629Cit621,627


α156-170Cit160,168
α306-320Cit308
α441-455Cit443
α621-635Cit621,627,630







β-chain










β45-59Cit47,53
β195-209Cit196,199,208
β330-344Cit334
β435-449Cit436,445


β60-74Cit60,72,74a
β210-224Cit224
β375-389Cit376
β465-479Cit478


β75-89Cit87
β240-254Cit246
β390-404Cit395
β480-491Cit485


β120-134Cit121,124
β255-269Cit267
β405-419Cit410


β150-164Cit158
β285-299Cit286,284
β420-434Cit421










B. Second series:





α-chain










α138-152Cit143
α300-314Cit308
α438-452Cit443
α615-629Cit621,627


α183-197Cit186,190
α347-361Cit353
α455-469Cit458,459


α213-227Cit216,218
α363-377Cit367
α542-556Cit547


α259-273Cit263,271
α420-434Cit425,426
α588-602Cit591







β-chain










β50-64Cit53,60
β202-216Cit206
β281-295Cit285,294
β474-488Cit478,485


β116-130Cit121,124
β215-229Cit224
β373-387Cit376


β188-202Cit196,199
β219-233Cit224
β416-430Cit421


β193-207Cit195,199,206
β236-250Cit246
β433-447Cit436,445





aThis peptide was synthesized with the carboxyl function (COOH) of the C-terminal citrullyl residue either in “free” form (COOH), or in amidated form (carboxamide function: CONH2).






The nomenclature used is the following: name of origin of the polypeptide chain (α or β) of fibrinogen from which the sequence derives, then position in this sequence of the amino-terminal residue of the peptide—position of the carboxy-terminal residue of the peptide. These positions are numbered relative to the N-terminal end of fibrinogen. The designation Cit indicates that it is a citrullinated form of the peptide. The position of the arginyl residue which is substituted with a citrullyl residue is indicated as an index. Only the citrullinated forms of the peptides are presented.
Each pair of peptides (citrullinated and noncitrullinated) was assayed by ELISA with a mixture, in equal parts, of 10 sera having no ACPAs (control mixture) and with the 2 mixtures A and B described in Example 1.
The peptides were tested after coating of irradiated polystyrene plates (Nunc Maxisorp) in three different buffers (acetate, pH 5.0; PBS, pH 7.4; and carbonate, pH 9.0), so as to optimize the chances of passive binding of the peptides (10 μg/ml) that exhibited very heterogeneous isoelectric points (extending from 4 to 12 for the noncitrullinated forms). Each pair of peptides (native and citrullinated form) was tested on the same plate and a pair of control peptides—citrullinated “filaggrin” peptide cfc6 and its corresponding native peptide cf0 (Schellekens G A, 1998, mentioned above)—was included in each experiment, which made it possible to calculate an inter-assay coefficient of variation and to perform corrections.
After saturation in PBS-2% BSA, the mixtures of sera diluted to 1/50 in PBS 2M NaCl—2% BSA were incubated and then their binding was revealed with peroxidase-labeled anti-human IgG goat IgGs (Southern) diluted to 1/1000 in PBS-2% BSA. All the incubations were carried out for 1 h at 4° C. and were followed by washing in PBS-0.1% Tween. The peroxidase activity was visualized with a solution of ortho-phenylenediamine (2 mg/ml—Sigma) in hydrogen peroxide (0.03%—Sigma). The reaction was stopped after 5 minutes by adding 4M sulfuric acid, and the optical density (OD) at 492 nm was measured by virtue of an automatic spectrophotometer (Multiskan, Thermo Labsystems).
The specific reactivity of the mixtures of sera with regard to the citrullinated peptides corresponded to the difference between the OD obtained with the citrullinated peptide and that obtained with the corresponding native peptide (delta OD). The results correspond to the mean of two determinations. Any citrullinated peptide making it possible to obtain a delta OD of greater than 0.250 for at least one of the two mixtures A and B after coating in at least one of the three buffers, was considered to be reactive.
Among the 72 citrullinated peptides analyzed, 13 peptides derived from the sequence of the fibrin α-chain and 5 peptides derived from that of the β-chain were found to carry epitopes recognized by the ACPAs. Among these peptides, 6 were very reactive (delta OD≧1.5), 8 peptides were moderately reactive (0.5≦delta OD<1.5) and 4 peptides were relatively nonreactive (0.25≦delta OD<0.5). The other citrullinated peptides prove to be barely reactive or nonreactive (0.0≦delta OD<0.25). No reactivity with the control mixture was observed, which makes it possible to identify the reactive peptides as carriers of epitope(s) recognized by the ACPAs.
The results obtained for the 18 reactive peptides are given in Table IV below:










TABLE IV









Mixture
Coating buffer











Peptide
of sera
Acetate
PBS
Carbonate





α36-50Cit38,42
Mixture A
4.00
2.69
3.43



Mixture B
4.27
2.65
2.64


α171-185Cit178,181
Mixture A
0.15
0.29
0.75



Mixture B
0.08
0.21
0.47


α183-197Cit186,190
Mixture A
0.41
1.68
1.36



Mixture B
0.01
0.15
0.07


α246-260Cit258
Mixture A
0.04
0.00
0.00



Mixture B
0.27
0.25
0.33


α259-273Cit263,271
Mixture A
0.14
0.60
0.12



Mixture B
0.21
0.69
0.20


α366-380Cit367
Mixture A
0.28
0.37
0.26



Mixture B
0.00
0.04
0.08


α396-410Cit404
Mixture A
0.04
0.02
0.43



Mixture B
0.15
0.17
0.20


α411-425Cit425
Mixture A
0.09
0.16
0.56



Mixture B
0.38
0.66
0.43


α501-515Cit510,512
Mixture A
0.16
0.92
0.15



Mixture B
0.72
2.60
0.79


α546-560Cit547
Mixture A
0.38
0.74
0.33



Mixture B
0.05
0.15
0.16


α561-575Cit573
Mixture A
0.05
0.30
0.01



Mixture B
0.14
0.52
0.17


α588-602Cit591
Mixture A
0.05
0.16
0.36



Mixture B
0.06
0.33
0.67


α621-635Cit621,627,630
Mixture A
0.25
1.57
1.17



Mixture B
0.18
1.51
1.20


β60-74Cit60,72,74a
Mixture A
1.02
2.06
1.27



Mixture B
2.83
2.69
2.72


β210-224Cit224
Mixture A
0.25
0.29
1.14



Mixture B
0.00
0.60
1.56


β281-295Cit285,294
Mixture A
0.12
0.60
0.61



Mixture B
0.11
0.75
0.71


β420-434Cit421
Mixture A
0.36
0.48
0.44



Mixture B
0.00
0.00
0.00


β433-447Cit435,445
Mixture A
0.41
0.58
0.67



Mixture B
0.00
0.00
0.03





aThe results indicated correspond to those obtained with the form of the peptide having an amidated C-terminal function (carboxamide function: CONH2)






EXAMPLE 3
Reactivity Profile of the Citrullinated Peptides that React with the ACPAs
The 14 peptides carrying the most reactive epitopes (delta OD≧0.5 for one or other of the mixtures of serum (A and B) with at least one of the three coating buffers) were tested independently with the 20 sera constituting the mixtures A and B, in order to evaluate the reactivity profile of each of these peptides. The tests were carried out by ELISA under the same conditions as in Example 2 above, with the exception that, for each pair of peptides, the coating buffer selected was that that had made it possible to obtain the strongest reactivity with respect to this peptide during the screening (the buffer for which the sum of the delta ODs obtained respectively for the serum mixtures A and B was at a maximum). In addition, the dilutions of the sera were adjusted such that they had an equivalent avidity for whole deiminated fibrinogen. Thus, for each serum, the dilution that made it possible to obtain an OD of 1 by ELISA on deiminated fibrinogen was selected (Nogueira L, 2002, mentioned above). The dilutions ranged from 1/20 to 1/2700.
The results are illustrated by Table V below.













TABLE V







Serum
Dilution
β60-74Cit60,72,74a
α36-50Cit36,42
α621-635Cit621,627,630
α501-515Cit510,512
α171-185Cit178,181





97.0459
1/60 
0.71c
3.55


0.29


97.1715
1/300

3.50

1.79


97.0524
1/200

3.39

2.85
0.59


97.0323
1/120
1.73
1.87

3.23


97.0388
1/50 

3.32
2.71
2.88


97.1436
1/150

3.30

2.80


97.0794
1/50 
0.75
3.43

3.30
2.09


95.0256
1/35 
3.48
2.35

2.37


97.1795
1/50 

2.29


0.57


97.0169
1/400
0.31

0.80


97.0530
1/50 

2.97

0.69


97.0311
1/200
1.93
0.71
0.37

1.95


97.0506
1/75 
1.93


0.44


97.1474
1/20 
0.37
1.93
0.59

1.08


97.0468
1/700
1.08

0.81

0.42


97.0244
 1/2700
1.23

0.50


97.1548
1/60 
1.48

3.29

0.71


97.0796
1/500
0.36

0.69


97.0907
1/300
1.17

1.04

0.44


97.1210
1/300
3.33

0.37












Number of
14/20
12/20
10/20
9/20
9/20


positive sera
















Serum
Dilution
β281-295Cit285,294
β210-224Cit224
α183-197Cit186,190b
α561-575Cit573
α546-560Cit547b





97.0459
1/60 
2.22

3.21
0.38


97.1715
1/300


97.0524
1/200
0.25


97.0323
1/120
1.16
0.36


97.0388
1/50 


97.1436
1/150


97.0794
1/50 
0.26


1.95


95.0256
1/35 


97.1795
1/50 

0.49


97.0169
1/400


97.0530
1/50 




0.44


97.0311
1/200

0.46
0.87
0.30


97.0506
1/75 
1.80

0.85


97.1474
1/20 
0.36


0.71


97.0468
1/700


97.0244
 1/2700


97.1548
1/60 



0.53


97.0796
1/500

0.67


1.18


97.0907
1/300

0.33


97.1210
1/300

0.72












Number of
6/20
6/20
3/10
5/20
2/10


positive sera

















Serum
Dilution
β433-447Cit436,445b
α259-273Cit263,271
α588-602Cit591
α411-425Cit425







97.0459
1/60 



97.1715
1/300



97.0524
1/200



97.0323
1/120



97.0388
1/50 
1.14



97.1436
1/150



97.0794
1/50 



95.0256
1/35 



97.1795
1/50 


0.79



97.0169
1/400


1.34



97.0530
1/50 



97.0311
1/200

0.29



97.0506
1/75 



97.1474
1/20 

0.39



97.0468
1/700

0.32



97.0244
 1/2700



97.1548
1/60 



97.0796
1/500
0.71



97.0907
1/300



97.1210
1/300



0.84













Number of
2/10
13/20
2/20
1/20



positive sera







aThe results indicated correspond to those obtained with the form of the peptide having an amidated C-terminal function (carboxamide function: CONH2).



bFor these three peptides, the individual reactivity of the sera of mixture B was not tested since the latter was not reactive.



cThe delta ODs <0.25 are not presented.






EXAMPLE 4
Comparison of the Reactivity of the Peptide β60-74Cit60,72,74 Depending on Whether Its C-Terminal Function is a Carboxyl (COOH) or a Carboxamide (CONH2)
ELISA plates were coated with the peptide β60-74Cit60,72,74, in which the C-terminal function of the carboxy-terminal citrullyl residue is not amidated (form carrying a terminal COOH function, hereinafter referred to as “nonamidated form”), with the peptide β360-74Cit60,72,74, in which the C-terminal function of the carboxy-terminal citrullyl residue is amidated (form carrying a terminal CONH2 function, hereinafter referred to as “amidated form”) and the noncitrullinated peptide β60-74 (in which the C-terminal function of the carboxy-terminal arginyl residue is not amidated), all diluted in PBS. The serum mixtures A and B described in Example 1 were tested according to the method described in Example 2. As in Example 2, the specific reactivity of the peptide in nonamidated form or in amidated form corresponded to the difference between the OD obtained with these two citrullinated peptides and that obtained with the noncitrullinated peptide β60-74. The reactivity of the amidated form of the peptide appeared to be clearly greater than that of the nonamidated form, which was, however, significant. The results correspond to the mean of two experiments, each comprising two determinations. The results are given in Table VI below.











TABLE VI







Peptide
Serum mixture
Delta OD









β60-74Cit60, 72, 74 nonamidated
Mixture A
0.30




Mixture B
1.16



β60-74Cit60, 72, 74 amidated
Mixture A
2.13




Mixture B
2.74

## Claims

1. A method for detecting the presence of ACPAs in a biological sample comprising:
bringing said biological sample into contact with at least one peptide chosen from the group consisting of:
a peptide consisting of the sequence X1PAPPPISGGGYX2AX3 (SEQ ID NO: 1) in which X1, X2 and X3 each represent a citrullyl residue or an arginyl residue, and at least one of the residues X2 and X3 is a citrullyl residue; and
a peptide of 5 to 25 amino acids comprising a fragment of at least 5 consecutive amino acids of the peptide consisting of SEQ ID NO: 1, including at least X2 or X3 as a citrullyl residue, or

an antigenic composition comprising said at least one peptide under conditions that allow the formation of an antigen/antibody complex with the ACPAs possibly present in said sample;
detecting the antigen/antibody complex possibly formed.

2. A method for detecting the presence of ACPAs in a biological sample comprises:
providing a first citrullinated peptide capable of competing, for the binding to said ACPAs, with a peptide chosen from the group consisting of:
a peptide consisting of the sequence X1PAPPPISGGGYX2AX3 (SEQ ID NO: 1) in which X1, X2 and X3 each represent a citrullyl residue or an arginyl residue, and at least one of the residues X2 and X3 is a citrullyl residue; and
a peptide of 5 to 25 amino acids comprising a fragment of at least 5 consecutive amino acids of the peptide consisting of SEQ ID NO: 1, including at least X2 or X3 as a citrullyl residue;

bringing said first peptide into contact with said biological sample under conditions that allow the formation of an antigen/antibody complex with the ACPAs possibly present in said sample;
detecting the antigen/antibody complex possibly formed.

3. The method of claim 1, wherein said peptide is chosen from the group consisting of a peptide consisting of SEQ ID NO: 1 in which at least X3 is a citrullyl residue; and a peptide of 5 to 25 amino acids comprising a fragment of at least 5 consecutive amino acids of the peptide consisting of SEQ ID NO: 1, including at least X3 as a citrullyl residue.

4. The method of claim 1, wherein said peptide is chosen from the group consisting of a peptide consisting of SEQ ID NO: 1 in which at least X2 and X3 are citrullyl residues; and a peptide of 5 to 25 amino acids comprising a fragment of at least 5 consecutive amino acids of the peptide consisting of SEQ ID NO: 1, including at least X2 and X3 as citrullyl residues.

5. The method of claim 1, wherein said peptide is chosen from the group consisting of a peptide consisting of SEQ ID NO: 1 in which X1, X2 and X3 are citrullyl residues; and a peptide of 16 to 25 amino acids comprising said sequence, including X1, X2 and X3 as citrullyl residues.

6. The method of claim 1, wherein the terminal carboxyl function (COOH) of said peptite is replaced with a carboxamide function (CONH2).

7. The method of claim 2, wherein said peptide is chosen from the group consisting of a peptide consisting of SEQ ID NO: 1 in which at least X3 is a citrullyl residue; and a peptide of 5 to 25 amino acids comprising a fragment of at least 5 consecutive amino acids of the peptide consisting of SEQ ID NO: 1, including at least X3 as a citrullyl residue.

8. The method of claim 2, wherein said peptide is chosen from the group consisting of a peptide consisting of SEQ ID NO: 1 in which at least X2 and X3 are citrullyl residues; and a peptide of 5 to 25 amino acids comprising a fragment of at least 5 consecutive amino acids of the peptide consisting of SEQ ID NO: 1, including at least X2 and X3 as citrullyl residues.

9. The method of claim 1, wherein said peptide is chosen from the group consisting of a peptide consisting of SEQ ID NO: 1 in which X1, X2 and X3 are citrullyl residues; and a peptide of 16 to 25 amino acids comprising said sequence, including X1, X2 and X3 as citrullyl residues.

10. The method of claim 2, wherein the terminal carboxyl function (COOH) of said peptite is replaced with a carboxamide function (CONH2).