WO2004055043A1 - Epitopic peptides of the thyroperoxidase enzyme, compositions containing same and uses thereof - Google Patents

Abstract

The invention concerns purified peptides of 5 to 20 amino acids identified by antibodies directed against human thyroperoxydase, said peptides constituting discontinuous epitopes derived from the discontinuous immunodominant region of human thyroperoxydase consisting of four separate TPO regions belonging to the MPO and CCP homologous domains. The invention also concerns antibodies directed against said peptides and the uses of said peptides and antibodies for detecting antibodies against human thyroperoxidase.

Description

 EPITOPIC PEPTIDES OF THE THYROPEROXYDASE ENZYME, THE COMPOSITIONS CONTAINING THEM AND THEIR APPLICATIONS.

The present invention belongs to the field of immunology, in particular to the field of autoimmune pathologies and in particular to autoimmune thyroid diseases linked to the appearance of autoantibodies directed against the enzyme thyroperoxidase. The assay of anti-TPO autoantibodies is essential for the diagnosis of these autoimmune thyroid pathologies and the titration by immunological method of these anti-TPO autoantibodies constitutes an important progress. Indeed, numerous studies have shown that anti-TPO autoantibodies are capable of inducing the destruction of the thyroid gland by activating an antibody-dependent cytoxicity.

Human thyroperoxidase (TPO), first described as the microsomal thyroid antigen (Ag) (1), is an enzyme linked to the membrane expressing itself at the apical pole of thyrocytes (2). TPO generates the functional form of thyroglobulin by iodization and coupling of tyrosine residues (3). During autoimmune thyroid disease (MAIT), TPO represents a major target for the immune system (4,5), leading to high titres of specific anti-TPO autoantibodies (aAc) present in sera from patients suffering from Hashimoto's and Graves' diseases. In addition to their role as early and early diagnostic markers of MAIT, anti-TPO aAc also act as effector molecules either by modulating the presentation of Ag to T cells, or as mediators of the destruction of the thyroid as a consequence of activation of antibody-dependent complement or cell cytotoxicity (6-12). Alignment studies and structural homologies have shown that TPO is formed by three distinct domains: a homologous domain of myeloperoxidase (MPO), a homologous domain of the complement control protein. (CCP), and an EGF factor homologous domain, from the N-terminus to the C-terminus (13). Although the structure of each domain has been partially elucidated by three-dimensional modeling (13-16), and low-resolution crystals have been obtained (17,18), the complete three-dimensional structure of TPO remains unknown. The flexibility observed for the pivotal regions probably makes it difficult to pinpoint each area in relation to the others (15).

These observations show a highly complex structure of TPO, which explains why the anti-TPO aAc present in the sera of patients suffering from MAIT preferentially recognize discontinuous epitopes of TPO (22).

Different approaches have been used to determine the epitopic regions recognized by the anti-TPO aAc present in patient sera and to identify the critical amino acids involved in these interactions: (i) competition between mouse mAbs or rabbit polyclonal antisera and human anti-TPO aAc (13,23-25) for binding to TPO, (ii) analysis of human anti-TPO aAc binding to recombinant and / or truncated Ag (15,16,26-30 ), (iii) generation of TPO fragments by enzymatic hydrolysis followed by analysis of their binding to anti-TPO aAc (31,32), and (iv) expression by eukaryotic cells of chimeric MPO-TPO proteins obtained by mutagenesis directed and analysis of their binding to anti-TPO aAc (33-35). These approaches have demonstrated a limited response to specific anti-TPO aAc, which mainly recognize a discontinuous immunodominant region (RID). By competition between anti-TPO mAbs present in patient sera and a pool of anti-TPO mAbs, RID has been divided into two nested domains named A and B (25). Recently, Guo et al. (23), using the same pool of murine antibodies and fragments (Fabs) anti-TPO human monoclonal antibodies as competitors, defined the A and B domains of RID with a reverse nomenclature. This second nomenclature, defined using human Fabs, will be used below. RID was only partially characterized, with no clear direct relationship between the regions identified. The authors hypothesize that most of the techniques commonly used to map the linear epitopes of monoclonal antibodies are inadequate to precisely define a discontinuous epitope on molecules like TPO, which have a highly organized architecture.

The research carried out in the context of the invention is based on the combination of two technological approaches already used to define highly structured epitopes (36-39). First, peptides were selected for their ability to mimic natural epitopes by screening libraries of peptides presented on the surface of phages. Second, the sequence alignments of the selected mimotopes, with the primary Ag sequence, and by repositioning of these peptides on the three-dimensional structure of Ag, allowed the identification of potential conformational epitopes.

This strategy was used to define the epitope recognized by the anti-TPO recombinant Ig13 IgG1 aAc, aAc recognizing the RID of TPO. This recombinant anti-TPO T13 aAc was obtained from a library of antibodies presented on the surface of phages, constructed from B cells which infiltrate the thyroid of patients with Graves' disease (40, 41). Four distinct regions of TPO belonging to the homologous domains of MPO and CCP define the discontinuous RID, recognized by recombinant human aAc T13 but also by anti-TPO aAc present in the sera of patients suffering from Graves' disease and Hashimoto's.

The RID recognized by anti-TPO aAc, which is one of the major autoantigens (aAg) in MAIT, has not yet been fully defined. Using the phage technique having peptides on their surface, the Applicant has identified three critical motifs, LxPExD, QSYP, and Ex (E / D) PPV, in the 12 selected mimotopes, which interact directly with the anti recombinant human aAc -TPO named T13.

The alignment of the mimotope sequences with the TPO molecule, as well as the analysis of the binding of the T13 aAc on the TPO mutants expressed in CHO cells, showed that the regions 353-363, 377-386, and 713- 720 of the MPO homologous domain and region 766-775 of the CCP domain are a part of the RID recognized by the T13 recombinant aAc. In addition, the Applicant has demonstrated that these regions are involved in the binding of anti-TPO aAc present in the sera of patients suffering from autoimmune diseases of Hashimoto and Basedow.

The purpose of the present invention is precisely to offer new means of diagnosis and therapy of MAIT. Indeed, the identification of RID now makes it possible to improve the diagnosis of MAIT by the use of 1) an engineered "miniTPO", replacing TPO in anti-TPO aAc assay kits from patients with MAIT and 2) by the definition of recombinant human anti-TPO aAc capable of competing with the anti-TPO aAc of patients and 3) the use of recombinant human anti-TPO aAc serving as internal standards. On the other hand, the use of mimotope peptides in therapeutics would block unwanted autoimmune responses.

The invention therefore firstly relates to a purified peptide of 5 to 20 amino acids recognized by the autoantibodies directed against human thyroperoxidase, said peptide defining a discontinuous epitope derived from the discontinuous immunodominant region of human thyroperoxidase consisting of four distinct regions of TPO belonging to the homologous domains of MPO and CCP. The invention relates very particularly to peptides comprising one at least one of the three critical units of the following formulas: LxPExD (SEQ ID NO.17), QSYP (SEQ ID NO.18) and Ex (E / D) PPV (SEQ ID NO.19) where x represents any amino acid: LxPExD (SEQ ID NO.17)

QSYP (SEQ ID NO.17)

ExEPPV or ExDPPV (SEQ ID NO.18), said peptides defining a discontinuous epitope derived from the RID of the TPOh consisting of four distinct regions of TPO belonging to the homologous domains of MPO and CCP.

Said discontinuous epitope is derived from the discontinuous immunodominant region of human thyroperoxidase consisting of regions 353-363 (SEQ ID NO.l), 377-386 (SEQ ID NO.2), and 713-720 (SEQ ID NO.3) of the homologous domain of myeloperoxidase (MPO) and the region 766-775 (SEQ ID NO.4) of the homologous domain of the complement control protein (CCP).

The invention relates more particularly to the 12 mimotope peptides whose sequences are identified by the numbers SEQ ID N °: 5 to SEQ ID No. 12 in the attached sequence list:

KLLPEDDESRTYHTV (SEQ ID NO.5)

KLFPEEDEMRTETQR (SEQ ID NO 6) RLAPEPDDPITPMTK (SEQ ID NO.7)

ELNPEPDTEVFPMTF (SEQ ID NO 8)

TQSYPPPPA RAASR (SEQ ID NO.9)

QLSPESDYDDHGMRY (SEQ ID NO.10)

SQSYPEPARGSVPMP (SEQ ID NO.11) GQSYPPRPDTSLHVT (SEQ ID NO.12)

KNSRQSYPEPAPVYH (SEQ ID NO.13)

ENEPP TTESKLQS (SEQ ID NO.14) ESDPPVASYQWRLIN (SEQ ID NO.15) ESVMQSYPPHLQIPG (SEQ ID NO.16)

By combining the phage peptide technique with the alignment of mimotope sequences on the TPO molecule, the Applicant has precisely identified three regions (353-363, 377-386, and 713-720) belonging to the homologous domain of the DFO and a region (766-775) located on the homologous domain of the CCP which is a part of the RID. Also the RID which includes these four regions which are crucial for the binding of anti-TPOh aAc was located on a 3D model of TPOh. This RID is recognized (i) by human recombinant aAc T13 obtained using a recombinant human aAc bank constructed from B cells infiltrating the thyroid of patients with Graves' disease (40,41) and (ii) by anti-TPO aAc present in the serum of patients suffering from Graves' and Hashimoto's thyroid diseases.

In addition, this demonstration was obtained using the full-length aAg TPO expressed by eukaryotic cells. Knowing that the anti-TPO aAc present in the sera of patients are directed against this RID, such a result makes it possible to improve the understanding of the recognition of TPO by the immune system during MAIT. Also, the synthesis of a constrained "mini-TPO" which mimics the RID identified by the Applicant presents an important diagnostic value for detecting the specific aAc of TPO.

Consequently, the invention also relates to a method for detecting anti-TPO aAc in a sample of an individual characterized in that it comprises: a) bringing said sample into contact with at least one peptide defined above, in conditions allowing the formation of an immunological complex, b) the detection of the formation of an immunological complex between an anti-TPO aAc and said peptide for detect the presence of anti-TPO aAc in the sample of the individual.

The sample can be any biological fluid from a subject likely to contain the aAc.

The method according to the invention is advantageous for the diagnosis of autoimmune pathologies more particularly thyroid. The invention therefore also relates to a kit for carrying out the above method comprising:

- an adequate amount of at least one peptide defined above to fix anti-TPO aAc in a sample,

one or more reagents for the detection of the immunological complexes formed between said peptide and the anti-TPO aAc,

- optionally, one or more standard reagent such as recombinant human antibodies of reference.

The subject of the invention is also an anti-antibody

Human TPO directed against a peptide above. The invention also relates to recombinant human antibodies directed against TPO. These are human recombinant antibodies obtained using combinatorial banks of antibodies constructed by la Demandresse from B lymphocytes infiltrating the thyroid of patients suffering from Graves' disease. These antibodies are prepared by techniques known to those skilled in the art in accordance with the description of the methods below used in the context of the invention. The antibodies according to the invention are useful in particular as a reference for the method of detecting anti-TPO aAc.

The invention also relates to pharmaceutical compositions comprising as active agent at least one peptide or an antibody as defined above. These compositions are useful for the prevention and treatment of Autoimmune Diseases and more particularly Thyroid and / or Thyroid Cancers.

The invention also relates to a method for preparing mimetic peptides of the anti-TPO aAc binding site on TPO using the peptides and antibodies defined above. These mimetic peptides are useful for the preparation of pharmaceutical compositions intended for the treatment or prevention of MAIT. Indeed, these mimetic peptides are capable of blocking the presentation of Ag by the anti-TPO aAc attached to the membrane of B cells (6,7), leading to the autoimmune response of T cells in MAIT and to blocking the cytotoxic activity of aAc in patients with MAIT, these two mechanisms resulting in the destruction of the thyroid.

In the compositions according to the invention, the peptides can be in soluble form (L-amino acid, D-amino acid or retro-inverso peptide) or in the form of fusion proteins or also presented by a carrier molecule.

The dose of peptide will be chosen to have a lasting therapeutic effect, in accordance with the present invention and while minimizing the harmful physiological effects, which will be determined by the medical profession. The administration of said peptides (alone or in combination) can be done by intramuscular, subcutaneous, intradermal or oral injection according to the indications described by pharmaceutical procedures.

Other advantages and characteristics of the invention will appear from the following examples where reference will be made to the appended figures in which: - Figure 1 illustrates the purity and specificity tests of the anti-TPO T13 aAc. Purity and specificity have been studied as described in the Methods. (A) Each sample (2μg of proteins) was deposited on two 8% polyacrylamide gels in the presence of SDS. The first was stained with brilliant Coomassie blue R-250 (lines 1 and 2) and the second was analyzed by Western Blot (lines 3 and 4). Lanes 1 and 3 show the baculovirus supernatants before purification. Lines 2 and 4 show the aAc T13 after purification (black arrow). The molecular masses (MW) in kDa are indicated on the left of the figure. (B) The specificity of aAc T13 was analyzed by ELISA. The binding of aAc T13 (open circles) and human polyclonal IgG _x as a control (closed triangles) on TPO are also shown.

- Figure 2 shows the results of the analysis of specific T13 mimotopes by the Spot technique.

FIG. 2A illustrates the immunoreactivity of the peptides with the aAc T13 studied by the Spot technique. The reactivity of aAc T13 complexed with a secondary antibody or the reactivity of the secondary antibody alone are shown on the left (see Methods).

FIG. 2B shows the identification, by alanine replacement, of the critical residues of the mimotopes. Sets of alanine analogs from each of the peptide sequences previously found to be reactive with aAc T13 were prepared and tested by the Spot technique. The legend "amino acid replaced" indicates which residue has been replaced by alanine (or glycine when the natural residue is alanine). The specific reactivity of aAc T13 on the Spot membrane is represented by a at the top of each histogram, and the percentage of binding of aAc T13 on each analog in comparison with the wild type sequence is shown by the histograms. Only one mimotope representative of each family has been illustrated. The underlined amino acids represent the critical residues observed for several peptides belonging to each family. - Figure 3 shows the sequence alignment between the mimotopes and the sequence of human TPO. The alignments were obtained as described in the Methods. The critical reasons for the specific mimotopes of aAc T13 are underlined. The identical residues between the mimotopes and the primary sequence of human TPO are represented in bold letters. - Figure 4 shows the location of the mutated regions on a three-dimensional ribbon diagram of the structure of human TPO.

Figure 4A is a modeling of the structure of human TPO showing the homologous domains of MPO, CCP, and EGF, reproduced with permission (13). This representation corresponds to the juxtaposition of the three-dimensional model of each domain. The mutations produced by "directed" mutagenesis are shown, positions 353-363 (1), 377-386 (2), 506-514 (3), 713-720 (4), 737-740 (5), and 766 -778 (6) The flexibility of the hinge regions is represented by a white arrow.

FIG. 4B is a modeling representing the possible folding of the homologous domain of the CCP onto the homologous domain of DFO (the homologous domain of the EGF is not shown to simplify the figure). A white arrow indicates the possible movement of the homologous domain of the CCP to form the immunodominant fixation surface virtually represented by three light gray stained lines. The junction between the homologous domains of MPO and CCP is shown by the white stained lines. The distance (in A) between regions 377-387 and 713-720 is shown. The model was adjusted using the public software Swiss-PDB viewer 3.7b2 available at the URL address: www. ExPASy. ch / spdbv. - Figure 5 shows the results of the analyzes by

Western Blot and ELISA which prove that aAc T13 recognizes the homologous domains of MPO and CCP.

Figure 5A shows Western blots with native or denatured membrane proteins made as indicated in the Methods. Monoclonal antibodies Acm 47 (10 μg / ml), Acm 6F5 (10 μg / ml), aAc T13 (10 μg / ml), and polyclonal rabbit antisera (1: 300) were used and identified on the right of the figure. The specific bands of TPO are shown by black arrows and the molecular weights in kDa are indicated on the left. FIG. 5B illustrates the competitive ELISA experiments carried out with murine monoclonal antibodies Acm 47, Acm 6F5, and mAb 2C2 directed against digoxigenin (control), as competitors for the binding of aAc T13 to TPO ( see Methods). - Figure 6 illustrates that the binding of anti-mAbs

TPO and patient sera on membrane proteins is affected by TPO mutations. The effects of TPO mutations on the binding of anti-TPO mAbs or patient sera were evaluated by ELISA.

Figure 6A shows the results obtained with aAc T13 or Acm 6F5. (b) Sera from patients with Hashimoto's thyroiditis [patient 1 (- • -), patient 2

(-B-), patient 3 (—àr-), patient 4 (), and patient 5 (-X—)], Graves' disease [patient 6 (O), patient 7 (-Eh), patient 8 ( -er ~) / patient 9 (-0-), and patient 10 ( ^• X-)], the other autoimmune disorders were tested for their capacity to fix the membrane proteins extracted from stable or type transfected CHO cells wild (see Methods). The results are given in absorbance after subtracting the values of the background noise corresponding to the binding of the sera to the membrane proteins of the non-transfected CHO cells and are expressed as the average of the duplicated values +/- SD.

I - Methods.

1) Patient sera. The sera of five patients with Graves' disease, those of five patients with Hashimoto's thyroiditis, and those of four healthy donors were obtained (Guy de Chauliac Hospital, Montpellier, France). The anti-TPO aAc titers were determined by radioimmunoassay using an AB-CT TPO kit (Cis bio, Gif sur Yvette, France). The patient's sera were then characterized for the presence of anti-TSH receptor AAs by a test with a radio-labeled receptor using the TRAK human kit (BRAHMS, Hennigsdorf, Germany) and for their T3 levels. , T4 and TSH. As a control, eight sera from patients suffering from other autoimmune diseases, two from type 1 diabetes (T1D), four from systemic lupus erythematosus (SLE), one from vasculitis and one from rheumatoid arthritis (RA).

2) Material.

Purified TPOh (purity greater than 95%) from the thyroid glands was used. The plasmid encoding complete human TPO was provided by the Cedars-Sinai Medical Center, Los Angeles, California, USA. The anti-TPO Acm 47 mAbs were supplied by INSERM 555, Marseille, France. (42). The anti-TPO Acm 6F5 mAbs and the anti-TPO polyclonal rabbit sera were respectively obtained from Hy Test Ltd (Turku, Finland) and Abcys S.A. (Paris, France).

3) Cloning, expression and purification of human T13 aAc. The scFv fragment of the recombinant human anti-TPO antibody T13 was selected from a library of antibodies presented by phages obtained from B cells extracted from thyroid tissues of patients with Graves' disease (40) . AAc T13 was expressed as whole IgG1 immunoglobulin using the baculovirus / insect cell system as described (41,43-44). IgGl T13 was purified by affinity chromatography on a protein G column and the concentration was determined by absorbance at 280 nm (E ° ' ^1% = 1.40).

4) Purity, specificity, and kinetic parameters of aAc T13. Purity was analyzed by electrophoresis on 8% acrylamide gel in the presence of SDS (SDS-PAGE) under non-reducing conditions, followed by staining with Coomassie brilliant blue or transfer to a PVDF membrane ( Novex, San Diego, California, USA) and revealed by a specific anti-Fc antibody of human IgG conjugated to peroxidase (Sigma-Aldrich, St. Louis, Missouri, USA) diluted 1: 1000, using a system ECL detection (Amersham Pharmacia Biotech, Piscataway, New Jersey, USA). Then, the specificity of the aAc was determined by ELISA. The microtiter plate wells were covered with 9 nM TPO (lμg / ml) in 100 mM NaHC0 ₃ , pH 9, overnight at 4 ° C. The wells were washed with 0.05% Tween 20 in PBS (PBS-T), pH 7.3 and saturated with 2% milk powder in PBS-T for one hour at 37 ° C. After washing, the aAc T13, or the purified human IgG1 (Sigma) used as a control, was incubated with 1% milk powder in PBS-T (incubation buffer) for 1 h 30 at 37 ° C. . After washing, an anti-human IgG antibody conjugated with alkaline phosphatase (Sigma, diluted 1: 1000) was added to the incubation buffer, and the wells were incubated for 1 h at 37 ° C. After three washes, the reactivity was revealed with 4 mg nitrophenyl phosphate 1 mg / ml in 0.5 M diethanolamine / HCl, for 1 h at 37 ° C. The kinetic parameters of aAc T13 were obtained by real-time analysis as previously described (41). AAc T13, expressed as a scFv fragment or as an intact IgG1 immunoglobulin, inhibits between 7.3 and 100% of TPO binding in serum samples from patients with MAIT who have been tested (40,41).

5) Selection and enrichment (Panning) followed by screening of the peptides presented by phages.

Four phage libraries (LX-8 / f88.4, X _ls /f88.4, X ₈ CX _{8 /} f88.4 and X _{30 /} f88.4) in which the peptides were expressed at the N-terminus of the pV II protein and presented on the surface of the filamentous bacteriophage M13, were obtained from Simon Fraser University, Burnaby, Canada (45). The specific phages of the aAc T13 were selected by a panning method as previously described (46). The phage-peptides specific for aAc T13 were eluted (i) by means of an acid elution, with 3 ml of a solution: 0.1 M HCl, 0.1 M glycine, BSA 1 mg / ml , pH 2.2, for 30 minutes at room temperature and then neutralized by adding 300 μl of 5 M Tris-HCl, pH 9.2, (ii) by competition with a 180nM TPO solution (20 μg / ml) in 5 ml of NaCl / Tris (50 mM Tris base, 150 mM NaCl, 0.05% Tween 20, pH 7.5) for 2 hours at 37 ° C. The phage-peptides were amplified after each round of panning using a K91 cell culture of Es ch eri ch iacoli growing exponentially and the T13-specific phage-peptides were cloned and characterized by ELISA as described (46 ). The clones having given an absorbance more than twice greater than the basic level were selected.

Each selected and purified clone was then verified by ELISA for its ability to be inhibited by soluble TPO at a concentration of 90 nM (10 μg / ml). The wells of the microtitration plates were covered with aAc T13 at a concentration 13 nM in 100 mM NaHCO ₃ , pH9, overnight at 4 ° C. The wells were washed with 0.05% Tween 20 in PBS (PBS-T), and then milk powder (2%) in PBS-T was added for 1 h at 37 ° C. After saturation and four washes, a 1:10 dilution of the purified phages of each clone was co-incubated with soluble TPO for 1 h 30 at 37 ° C. After washing, an anti-M13 antibody conjugated to peroxidase (Amersham Pharmacia Biotech) diluted 1: 5000 with milk powder (1%) in PBS-T) was added for 1 h at 37 ° C. Three washes were performed and the bound residual phages were revealed with a solution of 2-phenylenediamine (4 mg / ml) containing 0.03% (vol / vol) of hydrogen peroxide in 0.1 M citrate buffer, pH 5.0. After 20 minutes, the reaction was stopped by adding 50 μl of 2 M H ₂ S0 ₄ to each well and the resulting absorbance was measured at 490 nm. The phages selected, whose binding to aAc T13 was inhibited by more than 15%, were sequenced for the identification of the peptide sequence. As a control, the effect of the solution containing 90 nM of soluble thyroglobulin (60 μg / ml) on the binding to aAc T13 of these selected phages was tested.

6) Synthesis of mimotopes, successive mutation into alanine of each amino acid (alanine scanning), and immunoassay on peptides fixed on a cellulose membrane.

The general protocol for the synthesis of peptides in parallel with Spot has been described previously (47). Twelve pentadecapeptides corresponding to the selected specific mimotopes of aAc T13 and their fifteen alanine analogs, were synthesized by the Spot method. The peptides attached to the membrane were tested by co-incubation of a solution of aAc T13 (0.1 μM) with a specific anti-human Fc antibody conjugated to peroxidase (Sigma) diluted to 1: 1000 for 1 30 hrs at 37 ° C. After three washes, the complex was revealed using the ECL detection system (Amersham Pharmacia Biotech) on a sensitive film, and the intensities of the spots were evaluated by the Scionlmage software. The membrane was reused after a regeneration cycle. 7) Alignments of sequence and molecular modeling.

Each amino acid sequence corresponding to an immunoreactive mimotope was aligned with the other selected peptides or with the sequence of human TPO using the Multalin multiple sequence alignment program (48). The sequence homologies between the specific mimotopes of aAc T13 and human TPO were located on a three-dimensional model of TPO, using the atomic coordinates provided by Hobby et al. (13).

8) Site-directed mutagenesis and stable expression of mutant wild-type (wt) and mutant TPO DNA. A restriction site of KpnI was added by polymerase chain reaction (PCR) just after the stop codon of the cDNA of complete human TPO. The wild-type TPO (wt) cDNA was cloned using the Hind III and Kpn I sites in the expression vector pcDNA5 / FRT of the Flp-In system (Invitrogen, San Diego, California, USA). The mutants were constructed by overlapping PCR extension as previously described (49). The substitution mutations were carried out as follows: The sequence ³⁵³ HARLRDSGRAY ³⁶³ (SEQ ID NO.l) of wild TPO (wt) has been replaced by the sequence ³⁵³ NQAFQANGAAL ³⁶³ (SEQ ID NO.20) in TPO ^{353 " 363} mutant, wild TPO (wt) ³⁷⁷ PEPGI PGETR ³⁸⁶ (SEQ ID NO .2) by ³⁷⁷ LLTNRLGRIG ³⁸⁶ (SEQ ID NO. 21) TPO ^{377 "386} ', wild TPO (wt) ⁵⁰⁶ ASFQEHPDL ⁵¹⁴ (SEQ ID NO .22) by ⁵⁰⁶ NRYLPMEPN ⁵¹⁴ (SEQ ID NO. 23) TPO ^{506 "514} , wild TPO (wt) ⁷¹³ KFPEDFES ⁷²⁰ (SEQ ID NO. 24) by ⁷¹³ SYARLAVN ⁷²⁰ (SEQ ID NO. 25) TPO ^{713" 720} , wild TPO (wt) ⁷³⁷ PQDD ⁷⁴⁰ (SEQ ID NO. 26) by ⁷³⁷ ALAA ⁷⁴⁰ (SEQ ID NO. 27) TPO ^{737 "740} , wild TPO (wt) ^76S YSCRHGYELQ ⁷⁷⁵ (SEQ ID NO. 4) by ^7S6 LTCEGGFRIS ⁷⁷⁵ (SEQ ID NO.28) TPO ^{766 "775} .

All sequences were verified by the dideoxynucleotide termination method (50). The Flp-In system was used to generate stable isogenic mammalian line cells expressing wild-type (wt) and mutated TPOs. CHO cells (ATCC CCL61) were cultured according to routine techniques in DMEM / F-12 medium containing 10% fetal calf serum (FCS). The stable transfectants were obtained as recommended by the manufacturer. The CHO host cell line was constructed by transfection with 0.5 μg of the vector pFRT / lacZeo and selection in a growth medium containing 500 μg / ml of zeocine ™ (Cayla, Toulouse, France). The zeocin ™ resistant clones were screened by identifying those which contain a single integrated FRT site and which express β-galactosidase at a high level. A single clone was selected and amplified for other studies. The generation of wild TPO (wt) or mutated was obtained by cotransfection, in the zeocin ™ resistant CHO host cell line selected, of the plasmid pcDNA5 / FRT resistant to hygromycin carrying each form of TPO and of the plasmid pOG44 constitutively expressing the Flp recombinase. Two days later, the cells were cultured in a medium supplemented with hygromycin B 300 μg / ml (Life Technologies, Carlsbad, California, USA). The clones of interest were selected for their resistance to hygromycin, their sensitivity to zeocin ™, and the absence of β-galactosidase activity.

9) Extraction of the membrane protein.

The transfected stable CHO cells were washed three times with PBS and placed at 4 ° C. The membrane proteins were extracted as follows: after centrifugation at 1000 rpm for 5 min at 4 ° C, the membrane proteins were solubilized by adding 500 μl of lysis buffer [50 mM Tris / HCl, pH7, 3; 150 mM NaCl; 5 mM EDTA; 10% glycerol; 1% Triton X100 and a tablet of a mixture of protease inhibitors per 10 ml of lysis buffer (Roche diagnostics GmbH, Manheim, Germany)] and were incubated for 30 min in ice. After centrifugation at 13,000 rpm for 30 min at 4 ° C, the supernatants containing the membrane proteins were collected and stored at -80 ° C. Protein concentrations were evaluated using the BCA protein test reagent (Pierce, Helsingborg, Sweden).

10) Analysis by Western Blot and competition experiments. About 20 μg of membrane proteins were mixed in a protein buffer (0.2 mM Tris / HCl, pH 6.8; 50% glycerol; 1% SDS; and 0.1% bromophenol blue) for the conditions native, supplemented with 10% DTT, 10% 2-mercaptoethanol and heated for 5 min for denaturing conditions. Each sample was loaded into an individual well and underwent electrophoresis in a polyacrylamide gel (9%) in presence of SDS. Proteins were transferred to a nitrocellulose membrane, which was saturated with milk powder (5%) in PBS-T for 10 min at room temperature, the membranes were incubated with diluted peroxidase conjugated secondary antibodies. in an incubation buffer for 1 h at 37 ° C. After three washes, the signal was detected by ECL chemiluminescence (Amersham Pharmacia Biotech) on a sensitive film.

Competitive ELISA experiments were performed as follows. The wells were covered with 9nM TPO (lμg / ml) in 100 mM Na HC0 ₃ , pH 9, overnight at 4 ° C. Then they were washed and blocked with 2% BSA in PBS-T (saturation buffer), for 1 h at 37 ° C. After three washes, 3 nM mAb T13 (at a dilution giving an absorbance of 1.5 to 490 nm) were incubated with or without decreasing concentrations of inhibitor in a saturation buffer for 1 h 30 at 37 ° C. gentle agitation. The wells were washed and an anti-human IgG antibody conjugated to peroxidase diluted 1: 2000 in saturation buffer was added for 1 h at 37 ° C. Three washes were carried out then the aAc T13 fixed to TPO was detected by adding a solution of 2-phenylenediamine 4 mg / ml containing 0.03% (vol / vol) of hydrogen peroxide in a citrate buffer 0.1 M, pH 5.0. The reaction was stopped with 2 M H ₂ S0 ₄ and the resulting absorbance was measured at 49Onm.

11) ELISA with monoclonal antibodies (mAbs) and human sera. The wells were covered with 20 μg / ml of membrane proteins in PBS overnight at 4 ° C. The plates were washed with PBS-T and blocked with saturation buffer, for 1 h at 37 ° C. After three washes, the mAbs or the patient serum were incubated in a saturation buffer for 1 h 30 at 37 ° C. The wells were washed and the anti-mouse or anti-human IgG antibody conjugated to peroxidase (HRP) was added, the antibody being diluted to 1: 2000 in a saturation buffer, for 1 h at 37 ° C. Three washes were performed and the reactivity was revealed as described above.

II - Results.

1) Characterization of the T13 aAc expressed in the form of complete IgG-i in insect cells.

The anti-TPO a13 Ac13 was first produced as a human scFv fragment (40). For the present study, we prepared the complete human IgG _x T13 using the baculovirus / insect cell system and we purified it by affinity chromatography on protein-G. The purity was analyzed on acrylamide gel (10%) in the presence of SDS, stained with brilliant Coomassie blue under non-reducing conditions (Figure 1A, left frame). A 150 kDa band corresponding to 1 IgG _x T13 was visualized and the purity was estimated to be greater than 95%. The identity of the band was confirmed by Western Blot, under non-reducing conditions, with a specific anti-Fc antibody of human IgG conjugated to peroxidase (FIG. 1A).

Table 1 below describes the sera of patients with autoimmune thyroid disease.

Table 1

^A : The values were obtained by radioimmunoassay and standardized for the WHO / National Institute for Biological Standards and Control 66/387 (see Methods). ^B : The values were obtained by a radio receiver test using the DYNOtest TRAK kit for human use and standardized for WHO 90/762 (see Methods). ^c ND: Not determined.

About 5 mg of purified aAc T13 was obtained per liter of culture supernatant of insect cells sf9. The specificity of the binding of aAc T13 to human TPO (TPOh) was determined by ELISA. As shown in Figure 1B, aAc T13 specifically binds to TPOh in a dose-dependent manner, while human polyclonal IgG _x used as a control does not bind. In addition, no binding of aAc T13 for thyroglobulin was observed (data not shown).

The kinetic parameters of aAc T13 were evaluated by real-time analysis, using BIACORE 2000 as described above (41), by injecting three concentrations of purified TPOh on aAc T13 or on an irrelevant antibody (Table 2) . Table 2 below shows the kinetic parameters of aAc T13 as assessed by real-time analysis.

Table 2

^A : The binding of purified human TPO to monoclonal antibodies was measured by resonance units (RU) as described (41). The UK values have been corrected by subtracting the baseline. ND ^B : Not determined. NM ^C : Not measurable.

AAc T13 was found to show a strong affinity for TPOh (K _D = 0.9 nM, average of three experiments), whereas TPOh did not bind the antibody to 1 evan t.

2) Selection of mimotopes fixed to aAc T13 by the phage technique.

AAc T13 has already been shown to strongly inhibit the binding of TPO to aAc present in the sera of patients with MAIT (40,41). In addition, it has been shown that aAc T13 does not recognize a linear epitope (unpublished data), which suggests the conformational nature of this dominant epitope. To answer this question, biopannings were carried out with a mixture of four phage banks having peptides of 8, 15, 17 or 30 amino acids on their surface, in the aim of obtaining specific mimotopes selected by aAc T13 and which mimic the conformational epitope. A hundred phages were eluted with a pH 2.2 buffer after three rounds of panning on the aAc T13 and 100 other phages after 4 rounds of panning for elution by competition. These 200 phages were isolated and then purified.

Table 3 below represents the sequences of peptides selected by phage libraries having peptides on their surface.

Table 3

^A : Clones with the same sequence. ^B : Absorbance corresponding to the fixation of each clone to the aAc T13. ^c : The binding of each clone to aAc T13 was inhibited by soluble TPOh or thyroglobulin at 90 nM; only clones showing an inhibition greater than 15% with TPOh are indicated; no inhibition was observed with thyroglobulin as an inhibitor. : When a clone has been represented at least twice, the values of absorbance and percentage of inhibition are given as mean value +/- SD.

As shown in Table 3, twelve specific mimotopes of aAc T13, six obtained by acid elution and six obtained by competition, were then selected according to (i) their rate of binding to aAc T13 by ELISA and (ii) their ability to compete with soluble TPOh as an inhibitor for binding to aAc T13; only mimotopes with at least 15% inhibition have been shown. This inhibition was classified from 19% to 55% for the selected mimotopes. The binding of these mimotopes was not inhibited by thyroglobulin, used as a control protein

(data not shown). Only the peptides derived from the X _{ls /} f88.4 library were obtained, suggesting that the conformation of the 15-mer peptides leads to better recognition by aAc T13. On the other hand, similar experiments were carried out with another human anti-TPO aAc and allowed the selection of peptides from the libraries X _{15 /} f88.4, X ₈ CX _{8 /} f88.4 and X ₃₀ / f88. 4 (unpublished results). Except for the mimotope RLAPEPDDPITPPMTK (Table 3), each peptide sequence was obtained individually.

3) Analysis of the specific mimotopes of aAc T13 by sequence alignment and Spot technique.

The specific mimotopes of aAc T13 were aligned with each other and grouped into three families according to their sequence homology (Table 4).

Table 4 below shows the classification of mimotopes by sequence homology.

Table 4

Families 1, 2 and 3 contain mimotopes carrying the homologous motifs which are LxPExD, (S / T) x ₂ QSYPxP, and E x ₂ PPV x ₆ L respectively (where x represents any amino acid). However, the peptides KNSRQSYPEPAPVYH, QLSPESDYDDHGMRY, and SQSYPEPARGSVPMP have shown similarities found in families 1 and 2. More generally, mimotopes often have a proline-rich sequence representing between 6.6 and 27% of their residues. Acid residues were strongly represented in family 1 sequences (between 6.6 and 33% of their residues) and to a lesser extent in family 3 (between 13.3 and 20% of their residues).

To determine the critical amino acids involved in the recognition of each peptide by aAc T13, each mimotope, each amino acid of which has been successively mutated into alanine (alanine scanning), has been synthesized on a cellulose membrane by the method Spot. Most of the twelve mimotopes reacted strongly with aAc T13. Mimotope 8 which did not show reactivity with aAc T13, and mimotope 3 which showed cross-reaction with secondary antibody (Figure 2B), were eliminated for the following alanine scanning studies. As shown in FIG. 2B, the systematic replacement in alanine of each amino acid and for each mimotope revealed the critical residues for the binding of aAc T13 and showed three different critical motifs, LxPExD, QSYP and Ex (E / D) PPV for families 1, 2 and 3 respectively; these motifs have been correlated with those demonstrated by sequence homology. In addition, the results of alanine scanning have clarified the ambiguity for the allocation of the peptides indicated above, for example, peptides 7 and 12, KNSRQSYPEPAPVYH and SQSYPEPARGSVPMP, have been definitively assigned to family 2, while the peptide 10, QLSPESDYDDHGMRY, belongs to family 1 (Table 4).

4) Location of putative interaction sites of aAc T13 on human TPO.

The amino acid sequences of the specific mimotopes of aAc T13 were aligned with the primary sequence of TPOh in order to identify the regions which contain sequence homologies with TPO. First, the alignment of the critical patterns previously identified by the Spot technique (Figure 2B), with the primary sequence of the TPOh was carried out. Then, the other amino acids of each peptide were aligned according to the best homology with the primary sequence of the TPOh. As shown in Figure 3, this procedure has identified four different regions (residues 356-382, 713-720, 737-747, and 766-775) which showed the best pairing with mimotopes and their critical motifs. Interestingly, both regions carry the amino acids Lys ₇₁₃ and Tyr ₇₇₂ described previously as critical for immunodominance (15,32). Analysis of the three-dimensional model of TPOh showed that these regions were localized on the surface of the globular structure

(Figure 4A) and can form an immunodominant binding surface between the homologous domains of MPO and CCP, due to the flexibility of the hinge region (Figure 4, A and B). As these locations correspond to the putative sites of interaction of aAc T13 on TPO, we produced six mutant TPOs by overlapping PCR extension.

(Table 5) based on sequence alignment.

Table 5 below shows the comparison of the amino acid sequences between wild type and mutated TPOs.

Table 5

^A : The mutants are indicated by the position of the first and last amino acid in the primary sequence of the TPOh.

The three-dimensional model revealed that region 356-382 had two exposed loops around the putative site of interaction; therefore, two separate mutant TPOs were constructed, each showing a mutated region on each separate loop (TPO ^{353 "363} and TPO ^{377" 386} ). As a control, a mutant TPO, carrying a mutated region (residues 506-514) not identified by the mimotope alignment but located near the RID (binding identification site) (Figure 4, A and B) , was also generated. All mutations are carried out according to two rules: (i) when they are different, the amino acids are replaced by those present in the primary sequence of human MPO for the mutants TPO ^{353 "363} , TPO ^{377" 386} , TPO ^{506 " 514} , and TPO ^{713 "720} or by the primary sequence of the CCP domain of the factor H protein for the mutant TPO ^{766" 775} , and (ii) when they are similar, the residues are present in the TPO / MPO sequences and the TPO / factor H sequences, the amino acids are then replaced by uncharged residues (alanine or leucine). For example, the amino acid

Arg ₃₅₅ has been replaced by Ala ₃₅₅ in the mutated protein

(TPO ^{353 "363} ). For the mutant TPO ^737-740 , located on the periphery of the parts of the MPO and CCP modules, only the substitutions of alanine and leucine were used. Finally, the residue Cys _7S8 did not not changed since it can contribute to the complete folding of the TPO molecule by the formation of a disulfide bridge in the homologous domain of the CCP.

5) Expression and analysis of mutated and wild-type TPOs.

To demonstrate that the mutated regions are part of RID, wild type (wt) and chimeric proteins have been expressed in eukaryotic cells using the new Flp-In ™ expression system capable of producing a similar level of different proteins. interest. Through immunofluorescence, it was observed a defect in the transport of the mutant TPO ^{766 "775} in the plasma membrane (data not shown). A similar observation was recently described by Umeki et al. (51) which showed that when Gly ⁷⁷¹ is replaced by Arg ⁷⁷¹ in the genes of TPO of patients, it results from it a defect of localization (accumulation of TPO mutated in the endoplasmic reticulum) and causes a congenital hypothyroidism. To solve this problem, it was decided to extract all the membrane proteins of wild and transfected stable CHO cells, and to study them by Western Blot and ELISA. All the antibodies tested by Western Blot are reactive, under non-reducing conditions, with a protein of approximately HOkDa, corresponding to TPOh ( Figure 5A). The rabbit polyclonal antiserum is able to recognize all mutant TPO whatever the conditions. Mutations in general do not affect the n expression level for most chimeric proteins, although expression of mutated TPO ^{737 "740} is lower than with the other five. This weaker expression was confirmed with all of the mAbs tested. As expected, mAb 47, which recognizes a linear epitope (713-721) (42), does not fix the TPO mutant ^{713 "720} but reacts with all the other mutants under native or denaturing conditions. Surprisingly, the MAb 6F5, like aAc T13, does not react under denaturing conditions with wild-type (wt) or mutated TPO (Figure 5A). Furthermore, only the binding of Acm 6F5 to TPO ^{766 "775} mutant was eliminated under native conditions. These results demonstrate that mAb 6F5 and aAc T13 are directed against two different conformational epitopes which overlap in region 766-775. These observations lead to the conclusion that (i) the folding of the mutant TPO is homologous to that of the wild TPO (wt) and (ii) in the mutated regions, amino acids 766-775 compose only part of the recognized conformational epitope by Acm 6F5. Finally, the reactivities of aAc T13 on native proteins are different depending on the mutants. Strong signals were observed with wild-type TPO (wt) and mutated TPO ^{506 "514} , while the binding of aAc T13 to the mutants TPO ^{353" 363} , TPO ³⁷⁷ " ⁵¹⁴ , and _{T 0} ⁷⁶⁶ - ⁷⁷⁵ was notoriously decreased or no longer existed for the TPO ^{713 "720} mutant. To confirm that the epitope recognized by aAc T13 is localized in both the homologous domains of MPO and CCP, mAbs 47 and 6F5 were used in competition with aAc T13 for binding to TPO in ELISA . As illustrated in FIG. 5B, the mAbs compete in a dose-dependent manner with the aAc T13 and partially inhibit the binding of the aAc T13, the inhibition being 45% and 35% for the mAbs 47 and 6F5 respectively, with a molar excess of 100 for the inhibitor. No inhibition was observed with a control mAb. However, a steric hindrance effect cannot be excluded using the competition method, this is why we studied the interaction of mAb 6F5 and aAc T13 with the membrane proteins of wild TPO (wt ) and mutated by ELISA (Figure 6A). While the AMC 6F5 similarly recognizes wild TPO

(wt) and mutated (except TPO mutated ^7S6 - ⁷⁷⁵ ) ₇ aAc T13 which showed significant and specific binding for

Wild TPO (wt) and, as expected for the TPO ^{506 "514} mutant, shows a significant decrease in binding on other TPO mutations. Mutations 353-363, 377-386, 766-775, partially affected recognition of aAc T13 for TPO, while the amino acid changes of 713-720 completely prevented the binding of aAc T13 to TPO. These results reinforce the observations previously obtained by Western Blot (Figure

5A).

6) Analysis of the recognition of TPO mutated by patient sera.

To place this study in a context of pathological study, the binding of anti-TPO aAc present in the sera of patients suffering from MAIT, other autoimmune disorders and healthy donors was tested by ELISA as was done above with mAb 6F5 and aAc T13 (Figure 6B). While the sera of healthy donors or patients suffering from autoimmune diseases (T1D, SLE, RA, or vasculitis) do not react with any of the clones, all the sera of patients with Hashimoto's or Graves' disease specifically bind TPO wild (wt) in a dose-dependent manner. The control mutations in positions 506-514 do not generally affect the binding of the aAc present in the sera of the patients, while the mutations in the other positions decrease their binding to TPO. Interestingly, these mutants can be classified into two groups, those which weakly affect the binding of anti-TPO ^aAc (TPO ^377-386 ) and those which strongly affect it (TPO ^353-363 , TPO ^{713 "720} , TPO ^{766 "775} ). In contrast to the competition studies between anti-TPO mAbs and polyclonal aAc present in the sera of patients previously described by our group or by other teams (13,14,25,40-41) in which the results could be biased by steric hindrance, the effects of each mutation on the binding of anti-TPO polyclonal aAc present in the sera have the same profile here for a particular mutation. Similar results were obtained by the group of McLachlan and Rapoport (30).

7) Analysis of the recognition of TPO mimotopes by the aAcT13 autoantibody.

The mimotopes corresponding to the sequences represented in the sequence list submitted in the appendix under the n ° SEQ ID n ° 6, SEQ ID n ° 7, Seq ID n ° 10 and SEQ ID n ° 11 were analyzed for their ability to be recognized by the T13 autoantibody. These mimotopes were produced by chemical synthesis then purified by HPLC and biotinylated and tested in soluble form.

These mimotopes are adsorbed in the wells of a 96-well microplate at 10 μg / ml. After saturation,

The T13 autoantibody is incubated in dilution and then revealed with an anti-human γ-specific IgG antibody conjugated to peroxidase.

The results of this analysis are presented in FIG. 7. These results show that the T13 autoantibody binds in a dose-dependent manner to the mimotopes corresponding to the sequences represented in the list of sequences submitted in the appendix under No. SEQ ID No. 6, SEQ ID No. 7 and Seq ID No. 10. The T13 autoantibody does not bind to the control mimotope or to the mimotope of sequence SEQ ID No. 11.

8) Analysis of the recognition of TPO mimotopes by recombinant human antiTPO autoantibodies.

The mimotopes corresponding to the sequences represented in the sequence list submitted in the appendix under the n ° SEQ ID n ° 6, SEQ ID n ° 7, Seq ID n ° 10 and SEQ ID n ° 11 were analyzed for their ability to be recognized by recombinant human anti-TPO autoantibodies.

These mimotopes are adsorbed in the wells of a 96-well microplate at 10 μg / ml. After saturation, the recombinant human anti-TPO autoantibodies are incubated in dilution from 1 μg / ml, then revealed with an anti-human γ-specific IgG antibody conjugated to peroxidase.

The results of this analysis are presented in Figures 8A and 8B. These results show that the different mimotopes are recognized in a dose-dependent manner by the Recombinant human anti-i-TPO autoantibodies produced apart from intra-thyroid B lymphocytes from patients suffering from autoimmune thyroid diseases. Only the anti-TPO human recombinant autoantibody named B4 does not recognize any of the mimotopes tested.

9) Analysis of the recognition of TPO mimotopes by purified IgG from patients with Graves' disease.

The binding of purified IgG from patients with Graves' disease has been tested for their binding to soluble mimotopes. The mimotopes are adsorbed in the wells of a microplate in a 2-by-2 dilution from 50 μg / ml. After a saturation step, the purified immunoglobulins from patients are incubated and then revealed using an anti-human Ig conjugate coupled to peroxidase. Mimotopes 1, 3, 4, 7 (SEQ ID No. 10, SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 11 ° respectively) are recognized in a dose-dependent manner by the immunoglobulins of patients. Mimotope 3 (SEQ ID No. 6) corresponding to the sequence KLFPEEDEMRTETQR is the most highly recognized by the majority of the immunoglobulins of patients tested. These results are presented in Figures 9A and 9B.

III - Discussion.

The three-dimensional structure of the molecule

TPOh has not been resolved by crystallography studies, although low resolution diffracting crystals have been obtained (17,18). However, the analysis of the three-dimensional structure of the TPOh evaluated by homology modeling, reveals a highly complex structure comprising three distinct parts, the homologous domains of MPO, CCP, and EGF (13). This highly organized architecture of the TPO aAg is a plausible explanation for the reactivity of the anti-TPO aAc, which is mainly directed against an RID on the native structure of their aAg (19,53). Preliminary investigations aimed to define RID using polyclonal rabbit antisera directed against polypeptide fragments of TPO (13,24) or mAbs (25) in competition with human aAc. These studies, even if they lead to a better knowledge of RID, have used the antibodies produced by immunization of animals which do not exactly reflect the repertoire of specific human anti-TPO antibodies in MAIT. Other studies (33-35), which have used eukaryotic cells transfected with MPO-TPO chimeras, have not led to the identification of the structure of RID, probably due to the low expression of Ag in clones transfected and / or 1 significant heterogeneity of expression of TPO by the clones. More recently, Guo et al. (30) clearly excluded the homologous domain of EGF as being a part of RID and located this region at the junction between the homologous domains of MPO and CCP but with a wider extension in the MPO part. Although these results are crucial to localize RID globally, so far little data has been reported for the identification of restricted regions on these domains recognized by human antibodies and on the localization in the three-dimensional structure of TPO, the RID.

The phage technique with peptides followed by sequence alignment seems to be the most powerful strategy for positioning discontinuous epitopes on a highly complex structure like TPOh (38, 39, 53). In the present study, this technique was applied to the well-known human anti-TPO T13 aAc, in order to identify peptides capable of mimicking its discontinuous and immunodominant epitope. After performing the alignment of the sequences between the critical peptide motifs and the primary sequence of the TPOh, it was clearly shown by site-directed mutagenesis on the TPOh and the generation (with the new "Flp-In" system) of stable isogenic cells of mammal expressing the same level of wild-type TPO (wt) and of mutated TPO as four restricted regions (regions 353-363, 377-386 and 713-720 in the MPO-like domain and region 766-775 in the CCP- domain like TPOh) define the immunodominant binding surface (RID) on the 3D structure of TPOh. In addition, the comparison of the binding, on wild TPOs

(wt) and mutated, aAc T13 and anti-TPO aAc present in patient sera showed different levels of binding depending on the mutated region. While mutations in region 377-386 weakly affect the binding of human aAc, mutations in regions 353-363, 713-720, and 766-775 greatly reduced their recognition (Figure 6, A and B).

These data underscore the discontinuous nature of RID and provide further insight into the positioning of the homologous domains of DFO and PAC. Indeed, the homologous part of the CCP must be close to the homologous domain of MPO to form the putative interaction surface with the recombinant human aAc T13. As shown in Figure 4B, regions 377-386, 713-720, and 766-775 (constituting the ends of the RID) define a surface aAg. The Ag / Ac interface zones have been identified for other discontinuous epitopes by X-ray crystallography analyzes and NMR studies and have shown that these Ag / Ac interface zones are between 650 A ² and 1000 A ² (54.55). Thus, to form an RID compatible with these zones, the distances between regions 377-386, 713-720, and 766-775 must be fairly close to one another. The folding of the structure of these three domains constituting the TPOh seems to be homologous to that of proteins known as MPO, the CCP part of factor H, and EGF (13), but no information is available concerning the exact folding of one domain with respect to the others. Estienne et al. (15) underlined the high flexibility of the hinge region separating the homologous domains of the CCP and the EGF. It is assumed that similar flexibility exists between the homologous domains of MPO and CCP to form RID. In addition, two hypotheses are proposed to explain this phenomenon: either the homologous domains of MPO and CCP interact directly by interactions of the hydrogen bond or salt bonds type, or the flexibility of the hinge region results from a permanent rearrangement between the three modules and the binding of the aAc, like for example the aAc T13, on at least two domains "freezes" the structure in a stable conformation. If the latter hypothesis is correct, this could explain why it is so difficult to obtain crystals with a high resolution of diffraction from the TPO ectodomain (17,18). The co-crystallization of TPO with a specific Fab, stabilizing the structure, should definitively solve this problem.

Using the phage technique presenting a peptide, peptides recognized by aAc T13 were selected and the critical motifs [LxPExD, QSYP, and Ex (E / D) PPV] involved in its recognition with mimotopes were identified. Controversial results have sometimes located the Acm47 / C21 epitope (713-721) inside the RID (25,32,56) and others outside this region (23,35,57). The team from McLachlan and Rapoport deduced from their studies, using the recombinant antibody TRI .9, that the TPO Lys ₇₁₃ located inside the RID recognized by human aAc, is at the border of the epitope recognized by mAb 47 / C21, as is also suggested for the region 713-720 identified by the Applicant's investigations. Interestingly, other residues from this region such as Pro ₇₁₅ , Glu _7ιs and Asp ₇₁₇ are aligned with the PexD motif, identified in family 1 mimotopes specific for aAc T13, suggesting that they could contribute to the binding of aAc to TPO. In addition, the residue Tyr ₇₇₂ , recently identified by the Ruf and Carayon group as being critical for immunodominance (15), is included in the region 766-775 which the Applicant has characterized as belonging to RID. The motif QSYP, identified as critical for the binding of aAc T13 to family 2 mimotopes, comprises three residues also found in region 766-775 of the homologous domain of CCP. It is hypothesized that the QSYP motif mimics a motif composed of residue Tyr ₇₇₂ close to Ser _6S7 and Gln ₇₇₅ in the three-dimensional structure of TPO. These residues could be contact amino acids within the immunodominant epitope of aAc. The other region, having a strong contribution, 353-363, also presents residues

(Ser _3s9 , Tyr ₃₆₃ ) which are aligned with the family 2 mimetic motif (QSYP) identified by aAc T13. These observations should offer rational leads to carry out mutagenesis experiments on the TPOh and to identify precisely the amino acids involved in RID, even if we cannot formally exclude that one of these regions containing amino acids essential for folding of RID is not directly involved in the interaction with aAc.

Another previously described region called C2 (16,58), has been limited to region 599-617 on TPO and has been defined by inhibiting human anti-TPO aAc with polyclonal rabbit antisera directed against this peptide (13 ), as a major autoantigenic determinant. Examination of the 3D model of TPO shows that the epitope is located on the opposite site of the homologous domain of MPO with respect to the immunodominant binding surface described by the Applicant. This observation could be explained by the fact that overlapping antigenic domains have been characterized on the TPO molecule. In particular, two overlapping immunodominant domains named A and B (23,25) have been described on the basis of competition between anti-TPO antibodies from patients and murine or human antibodies. It is important to note that domain A defined by mAbs (25) corresponds to domain B defined by human Fabs (23) and vice versa. Since the Applicant used a human anti-TPO aAc in its experiments as did the team of McLachlan and Rapoport (23), it was chosen to use the nomenclature of RID / A and B defined by this group. Consequently, the residue Lys ₇₁₃ is part of the epitope recognized by the antibody TRI .9 specific for RID / B (32), and the region 713-720 belongs to the immunodominant epitope represented by aAc T13. In addition, the antibody Acm 47, which recognizes RID / B, partially competes with human aAc T13 for binding to the TPO molecule (Figure 5B). In addition, non-IGKVl-39 aAc like aAc T13 IGLV1-40 have been described as interacting with RID / B of TPO, while antibodies which use the light chain gene of the germ line IGKV1- 39 have been attributed to the recognition of RID / A (23). Given all of these results, it is possible that the recombinant human aAc T13 recognizes RID / B on the TPO molecule, even if a cross-reaction with domain A due to the nesting of RID / A and B cannot be formally excluded.

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Claims

1) Purified peptide of 5 to 20 amino acids recognized by the autoantibodies directed against human thyroperoxidase, said peptide constituting a discontinuous epitope derived from the discontinuous immunodominant region of human thyroperoxidase consisting of four distinct regions of TPO belonging to the homologous domains DFO and PAC.

2) Peptide comprising according to claim 1, characterized in that it comprises at least one of the three critical units of the following formulas: LxPExD (SEQ ID NO.17), QSYP (SEQ ID NO.18) and Ex ( E / D) PPV (SEQ ID NO.19) where x represents any amino acid.

3) Peptide according to one of claims 1 or 2, characterized in that said discontinuous epitope is derived from the discontinuous immunodominant region of human thyroperoxidase consisting of regions 353-363 (SEQ ID NO.l), 377-386 (SEQ ID NO. 2), and 713-720 (SEQ ID NO. 3) of the myeloperoxidase homologous domain (MPO) and region 766-775 (SEQ ID NO.4) of the homologous domain of the complement control protein ( CCP).

4) Peptide according to one of claims 1 or 2, characterized in that it responds to a sequence chosen from the group comprising the following sequences identified by the numbers SEQ ID NO. 5 to SEQ ID NO. 16 in the attached sequence list:

KLLPEDDESRTYHTV (SEQ ID NO.5)

KLFPEEDEMRTETQR (SEQ ID NO 6)

RLAPEPDDPITPMTK (SEQ ID NO.7)

ELNPEPDTEVFPMTF (SEQ ID NO 8) TQSYPPPPAWRAASR (SEQ ID NO.9)

QLSPESDYDDHGMRY (SEQ ID NO.10)

SQSYPEPARGSVPMP (SEQ ID NO.11) GQSYPPRPDTSLHVT (SEQ ID NO.12)

KNSRQSYPEPAPVYH (SEQ ID NO.13)

ENEPPVWTTESKLQS (SEQ ID NO.14)

ESDPPVASYQWRLIN (SEQ ID NO.15) ESVMQSYPPHLQIPG (SEQ ID NO.16)

5) A method of detecting anti-TPO autoantibodies in a sample of an individual, characterized in that it comprises: a) bringing said sample into contact with at least one peptide according to one of claims 1 to 4, under conditions allowing the formation of an immunological complex, b) the detection of the formation of an immunological complex between an anti-TPO autoantibody and said peptide to detect the presence of anti-TPO autoantibodies in the sample of the individual.

6) A method of diagnosing autoimmune pathologies more particularly thyroid and or thyroid cancers, characterized in that it comprises the implementation of a method according to claim 5.

7) Kit for setting up a method according to one of claims 5 or 6, characterized in that it comprises:

- a sufficient quantity of at least one peptide according to one of claims 1 to 4 to fix anti-TPO autoantibodies in a sample, - one or more reagents for the detection of the immunological complexes formed between said peptide and the autoantibodies TPO,

- optionally, one or more standard reagent such as reference antibodies.

8) An anti-human TPO antibody directed against a peptide according to any one of claims 1 to 4. 9) Pharmaceutical composition comprising as active agent at least one peptide according to one of claims 1 to 4 or an anti-TPO antibody capable of recognizing said peptides according to claim 8.

10) Pharmaceutical composition according to claim 9, characterized in that said peptide is in soluble form or in the form of fusion proteins or also presented by a carrier molecule.