EP0804484A1

EP0804484A1 - Antigenic polypeptide sequence of factor viii, fragments and/or epitopes thereof

Info

Publication number: EP0804484A1
Application number: EP95927598A
Authority: EP
Inventors: Ruth Laub; Mario Di Giambattista
Original assignee: Croix-Rouge de Belgique
Current assignee: CAF - DCF CVBA - SCRL
Priority date: 1994-07-14
Filing date: 1995-07-14
Publication date: 1997-11-05
Also published as: US20030147900A1; WO1996002572A2; US6866848B2; JP2006316062A; CA2195126A1; WO1996002572A3; JPH10506610A; BE1008491A3

Abstract

The present invention relates to an antigenic polypeptide sequence of factor VIII, comprised between the Glutamic Acid 1649 and Asparagine 2019, preferably comprised between Arginine 1652 and Arginine 1917 of the polypeptide sequence of factor VIII, or comprised between Alanine 108 and Methionine 355, or comprised between Aspartic Acid 403 and Aspartic Acid 725, or comprised between Lysine 2085 and Lysine 2249.

Description

ANTIGENIUS POLYPEPTIDE SEQUENCE OF FACTOR VIII.

FRAGMENTS AND / OR EPITOPES THEREOF.

Object, dg the invention,

The present invention relates to the antigenic polypeptide sequence of factor VIII, fragments, epitopes thereof and the strong parts of these epitopes, the inhibitors directed against this sequence, its fragments, its epitopes and / or the strong parts of these epitopes. , as well as the anti-inhibitors directed against said inhibitors. The present invention also relates to a pharmaceutical composition and a diagnostic device comprising the above-mentioned molecules.

Technological background underlying the invention.

Recently, factor preparations have been made available in sufficient quantities for hemophiliacs

VIII purified from large plasma pools by ion exchange chromatography, or very recently by immunoaffinity.

Various preparations of FVIII obtained by genetic engineering are currently in development or in clinical study. These FVIII are either whole molecules or deleted (Bihoreau (1992)).

FVIII is a glycoprotein cofactor for plasma coagulation and acts on the activation of factor X (FX). The characterization of FVIII and its mechanism of action is made more difficult by its low concentration in plasma, size heterogeneity, and its extreme sensitivity to enzymatic degradation. This reaction includes the proteolysis of FX into active factor X (FXa = Stuart factor) and involves a complex (Tenase complex) comprising the enzyme (activated FIX or FIXa), a cofactor (activated FVIII or FVIIIa), calcium ions and phospholipids.

FVIII is such a complex protein that, although the gene sequence has been known since 1984 (Verhar et al., Nature 312, pp. 317-342 (1984)), the complete structure of plasma FVIII has not yet been established. (almost 50% of the protein has only been sequenced) as well as the precise structure of the carbohydrates. The DNA sequence has been accepted as defining the primary sequence of FVIII (rare exception to guidelines prescribed by the FDA for therapeutic products derived from biotechnology).

However, subtle differences between plasma FVIII and recombinant FVIII have been established: glycosylation, plasma half-life after infusion, .... FVIII is mainly synthesized in hepatocytes. It has been cloned into mammalian, insect and yeast cells (Webb et al., 1993). These glycoproteins produced by biotechnological processes may have differences in the structure and composition of sugars compared to the natural protein. The cDNA of FVIII has also been expressed in transgenic sheep (Halter et al., 1993).

CDNA codes for a polypeptide of 2351 amino acids including the signal peptide of 19 amino acids cleaved in the endoplasmic reticulum. Post-translational modifications take place in the Golgi apparatus: glycosylation of serines and threonmes and addition of sulphate ions to tyrosine residues. After maturation, the protein is then secreted in the plasma, in the form of 2 chains 210 kDa (there 1648 residues) and 80 Da (from 1649 to 2332 residues) united by a divalent ion, and the lightest of which is associated non-covalently to von Willebrand Factor (vWf) by its N-terminal end (1 molecule vWf per molecule of FVIII). In plasma, this complex is stabilized by hydrophobic and hydrophilic bonds in the presence of 50 times more vWf. The latter would inhibit the binding of FVIII to phospholipids. The fact that FVIII binds to platelets has been established, however the presence of specific receptors expressed on the surface of the platelets has not been clearly demonstrated (Neshei et al., 1993). After its fixation on membrane phospholipids, it would unmask binding sites of high affinity for FIXa (Bardelle et al., 1993).

The FVIII is made up of three structural domains A, B and C (Kaufman RJ, 1992; Bihoreau et al., 1992) organized in Al: A2: B: A3: C1: C2 (Figure 1). Domains A have more than 40% homology and are also homologous to ceruloplasmin. There is also 30% homology between the A domain of factor V and FVIII. Domain C intervenes twice and would be able to bind glycoconjugates and phospholipids with negative net charge (Kemball-Cook and Barrowcliffe (1992); Fay, PJ, 1993)). It presents a homology with lectins capable of binding to negatively charged phospholipids. It is at this level that the platelet binding site has been located (domain C2) (Foster et al., (1990)). Domain B, which represents more than 40% of the mass of FVIII, has no known specific activity but could play a subtle role in the regulation of FVIII by protecting it, for example, from the action of thro bine. It has no known homology with other proteins.

It has 19 of the 25 glycosylation sites identified for FVIII. The comparison of the amino acid sequences between human and porcine FVIII reveals major differences in this region B. However, porcine FVIII is used effectively to treat hemophiliacs with inhibitors. These observations led to the construction of a FVIII gene deleted in the coding part of this region B and which allows the production of a Recombinant summer FVIII intended for the treatment of hemophilia. By immunopuπfication, different forms of active FVIII have been isolated which have in common a light chain of 80 kDa and whose heavy chain can have a molecular weight between 210 and 90 kDa. These forms would result from the progressive degradation of the C-termmal end of the heavy chain. The bond of the two chains is non-covalent and results via a bond of a divalent metal ion (Me ++) between the residues responsible for the domains Al and A3. After formation of the activated complex (50-45 kDa) (heavy chain with accessible A2 domain) and 70 kDa (light chain), a mactivation phase is observed probably following prolonged contact with thrombin and dissociation of the 50 kDa fragments. and 45 kDa. FVIIIa is also inactivated by activated protein C (APC) after proteolysis of the heavy chain. This mactivation is accelerated if the FVIIIa is fixed on a phospholipic surface. This "down-regulation" of the activity of FVIIIa would depend on phosphorylation by a platelet enzyme (Kalafatis et al, (1990)). Most of the epitopes recognized by the various murine monoclonals isolated to date do not seem to be located in the "functional sites" of FVIII. Some epitopes recognized by antibodies having an effect on the activity of FVIII (inhibition of the chromogenic test and / or clottmg) have been identified.

These antigenic determinants consist of fragments 351 - 365 (domain Al - heavy chain), 713 - 740 (domain A2), 1670 - 1684 (domain A3 - light chain) (NH2 terminal of the light chain) or 2303 - 2332 ( domain C2 - light chain) (Foster C, (1990)), the fragments 701 - 750 (Ware et al. (1989)), 1673 - 1689 (Leyte et al. (1989)), 330 - 472, 1694-1782 (EP-0 202 853), 322 - 740 and 2170 - 2322 (Scandella et al. (1992)).

The antibodies recognize these various sites, respectively interfere with the activation of FVIII, the binding of vWf or the binding of phospholipids. Other antibodies, which are not inhibitors of conventional in vitro activity tests, can act on the action of FVIII with the other constituents of the coagulation cascade by binding to sites on the molecule which are very far from the active sites. These, thus modified, can interfere with the natural folding of FVIII, altering some of its properties ("allosteric model").

These "mapping" experiments use peptides synthesized by fragments of FVIII genes cloned into E coii, and allowing only an approximate idea of the location of the antigenic determinants recognized by these monoclonals. Indeed, the size of the identified fragments ranges from 30 to 100 amino acids.

Currently, to unequivocally identify the antigenic sites of a protein, it must be crystallized and analyzed using X-rays. Unfortunately, no data are available for FVIII, whose high molecular weight is a major handicap for crystallization.

The antigenic regions coincide with the hydrophilic character of these regions: the more the oligopeptide sequence is exposed to the external medium (located on the surface), the more this part will be likely to be recognized in an immunization reaction. On the other hand, the hydrophobic parts, generally located inside the protein, are considered to be not very antigenic.

Currently, a concept which predominates in hemophiliac patients, clinicians and fractionators, is the availability of a purified FVIII, devoid of any pathogenic plasma contaminant and of side effects. However, whether after immunopurification using murine monoclonals, or after obtaining by genetic recombination in mammalian cells, highly purified FVIII is extremely unstable for reasons which are not apparent. To be able to stabilize it, significant amounts of human plasma albumin are added during the purification so that the activity specific final is of the order of 2-3 U / mg protein. The same is true for rFVIII coexpressed with factor von illebrand, a natural stabilizer, in CHO cells. These data seem to suggest an influence of the purification steps on the FVIII molecule, which can interfere with its natural folding, introduce more or less stable conformational changes and reveal potential new epitopes after infusion in the patient.

One of the serious complications presents in 5 to 50% according to the authors (L] ung et al. (1992); Sultan et al., (1992); Lorenzo et al. (1992)) hemophiliacs who receive multiple therapeutic infusions of FVIII is the appearance of antibodies (inhibitors) which inactivate FVIII and make any subsequent injection of FVIII ineffective. The spontaneous appearance of autoantibodies with pathological anti-FVIII activity is rare in non-hemophiliacs (prevalence: 10 ^"5 ) and is reported in elderly individuals, manifesting immunological or postpartum disorders (Kessler ( 1991), Hultin (1991)). A multicenter study carried out on 3435 hemophiliac patients shows that all age groups are concerned, including patients under the age of 5. The majority (82%) have a very high response ( > 10 BU) (Sultan et al. (1992)). These anti-FVIII drugs are essentially composed of IgG, of the IgG4 type, but IgG2 (Gilles et al. (1993) b) IgA and IgM have also been described ( Lottenburg et al. (1987). They react weakly with heterologous FVIII molecules purified from other mammals (Bennett, B et al. (1972)). At the present time, the causes which induce in certain hemophiliacs are unknown. appearance of inhibitors. If there is an association between the severity of he deletion of the gene and the development of an immune response no longer recognizing FVIII as a self protein, this association has only been demonstrated in a minority of patients. No specific susceptibility of the host, linked to genetic markers, could be demonstrated, for example a privileged association with certain determinants of the MHC class II complex (Hoyer (1991)), no doubt, because all the epitopes of FVIII, recognized by the specific antibodies have not yet been determined. It also seems that the different methods of preparing FVIII could influence its structure, its physicochemical properties or its natural micro-environment (Vermeylen, J and Peerlinck (1991); Gomperts, et al. (1992); Peerlinck et al. (1993)). Barrowcliffe et al. (1983) have shown that phospholipids protect the procoagulant activity from inactivation by specific human antibodies. The presence of natural anti-FVIII antibodies in 17% of healthy donors (screening carried out on 500 plasma donations), without pathological manifestation, demonstrates the importance of better understanding the three-dimensional structural aspect of a physiological FVIII (Ciavarella and Schiavoni (1992)).

The transfusion studied on mixed cultures of lymphocytes, in animal models and during clinical trials, has shown a modification of the immunomodulation in the transfused, inducing alloimmunization and also a down-regulation of certain immune functions. It manifests as suppressor cells, anti-idiotype antibodies, or a decrease in NK cells. Everything happens as if we were inducing a certain degree of tolerance. These effects can be reversed by infusion of interleukin-2

(IL-2) (Triulzi et al., 1990). In vitro, an inhibitory effect on the secretion of IL-2 as well as the proliferation of peripheral blood mononuclear cells, are obtained in the presence of cryoprecipitate or poorly purified preparations of FVIII (0.5 to 10 U / mg proteins) (Madhok and al., 1991; adhwa, M et al., 1992). These effects are not observed in the presence of rFVIIl or FVIII purified by immunoaffinity. This last preparation would have an activating effect on T cells

(Madhok et al., 1991). However, these data cannot be directly extrapolated to an in vivo situation.

There is no experimental model to establish a prognosis as to the immunogenicity or the immunomodulatory effect of the preparations of FVIII or the susceptibility of the host before their clinical administration. This model becomes an absolute necessity in view of the increase in the frequency of appearance of anti-FVIII antibodies during current clinical trials using FVIII preparations of very high specific activity obtained either by immunopurification or by DNA engineering techniques. (Seremetis et al. (1991)). In addition, Aledorf (1993) shows that when using these two types of preparations in virgin subjects, not previously transfused (PUPS), we observe a prevalence of inhibitors of up to 27%. State of the art.

Patients who develop an anti-FVIII immune response find themselves in a serious situation which requires the use of heavy, aggressive and excessively expensive means. One of the most used techniques is to drown the body by regularly injecting very high doses of FVIII (100 to 200 U / kg / day) (Ewing et al. (1988)) in combination with a concentrated Prothrombin complex (FEIBA) (Bonn protocol), which effectively reduces the level of inhibitors in the blood (Sultan et al., 1986). In addition, this type of treatment must be continued for a very long time (Lian et al., 1989). Trials using lower doses of FVIII have had some success in patients with significantly lower levels of anti-FVIII antibodies (Gruppo, (1991)).

An alternative route is the use of FVIII from a non-human species such as that of pigs, not neutralized by the patient's anti-FVIIIs and allowing hemostasis. A multicenter study has shown the benefits of such a treatment but has also revealed anti-porcine FVIII antibodies (Lozier

(1993); Moreau et al. (1993); Hay and Bolton-Maggs (1991);

Clyne et al. (1992)). Activated factor VIII, obtained by recombinant DNA, has also been used as an alternative coagulation pathway in patients with inhibitors (Ingerslev et al. (1991)).

Recently, a successful strategy (Nilsson et al. (1990)) to reduce the level of inhibitors has consisted in subjecting the patients to an extracorporeal circulation to allow the solid phase absorption of total IgG on Protein A while at the same time dealing with cytostatic agents like cyclophosphamide.

The infusion of polyvalent intravenous immunoglobulins (IVIG) combined or not with unosuppressive therapy has proved to be relatively effective, the reason for this efficacy not yet being well established. Different hypotheses have been put forward involving feedback inhibition of IgG synthesis, stimulation of their clearance, activation of T suppressors (Bloom (1992)). An interesting explanation is that these commercial intravenous immunoglobulins would contain antibodies capable of reacting with neutralizing anti-FVIII antibodies in their variable part (idiotypes). This anti-idiotype activity would be specific to each donor and could be synergistic in a pool of IgG (Dietrich et al. (1992)).

Unfortunately, none of these approaches has proven satisfactory in terms of safety, efficiency and cost. 9utg of the invention.

The present invention aims to obtain an antigenic polypeptide sequence of factor VIII, fragments and epitopes thereof, intended to improve the diagnosis and / or therapy of immune disorders, in particular those induced by FVIII inhibitors, the inhibitors of the binding of FVIII to von Willebrand factor (vWf) and / or to membrane phospholipids (PL).

Another object of the invention is to obtain inhibitors having immunoaffinity with this antigenic polypeptide sequence, its fragments and / or its epitopes, also intended to improve the diagnosis and / or the therapy. immune disorders.

A further aim is to obtain anti-inhibitors, in particular antibodies, directed against said aforementioned inhibitors, intended to improve the diagnosis and / or therapy of immune disorders. Brief description of the figures. FIG. 1 schematically represents the polypeptide sequence of factor VIII. FIG. 2 represents the hydrophilicity graph of the A3 sequence of factor VIII renumbered from 1 to

371 amino acids (area value for each amino acid). FIG. 3 represents the flexibility graph of this A3 sequence of factor VIII. FIG. 4 represents the accessibility graph of this A3 sequence of factor VIII. FIG. 5 represents the general graph showing the sum of the values defined in graphs 2 to 4. FIG. 6 represents the detection of anti-factor VIII antibodies in the sera of mice by an ELISA test. Characteristic elements of the invention.

The present invention relates to the antigenic polypeptide sequence of factor VIII and / or fragments thereof, as described by Verhar et al. (Nature, vol. 312, p 339 (1984)).

The term "factor VIII polypeptide sequence" means the natural human or animal sequence, optionally glycosylated, obtained by purification of plasma pools, in particular Cohn fraction I, by synthesis and / or genetic engineering (that is to say also say a possibly deleted sequence of the portions not involved in the blood coagulation mechanism) of factor VIII. The present invention relates in particular to the factor VIII antigenic polypeptide sequence lacking fragments Alanine 322 - Serine 750, Leucme 1655 - Arginine 1689, Lysine 1694 - Prolme 1782 and Aspartic Acid 2170 - Tyrcsme 2332.

The present invention relates in particular to the antigenic polypeptide sequences A1, A2, A3, and C (Cl and C2) of factor VIII.

In the remainder of the text, the amino acids will be represented by their three-letter abbreviation or by the symbol of a single letter as identified in the table below.

Alanine Ala A Leucme Leu L

Arginine Arg R Lysine Lys K

Asparagine Asn N Methionine Met M

Asp D Phenylalanme Phe F Aspartic Acid

Cysteine Cys C Proline Pro P

Glutamme Gin Q Serine Ser S

Acid Glu E Threonine Thr T Glutamic

Wisteria Gly G Tryptophan Trp w

Histidine His H Tyrosine Tyr Y

Isoleucme Ile I Valme Val V

A first embodiment of the invention relates to the A3 antigenic polypeptide sequence of factor VIII, fragments and / or epitopes thereof. Said sequence is between Glutamic Acid 1649 and Asparagine 2019, preferably between Arginine 1652 and Arginine 1917 or between Arginine 1803 and Arginine 1917, of the polypeptide sequence of factor VIII such as published by Verhar et al. (Nature, vol. 312, p 339 (1984)) and Toole et al. (Nature, vol. 312, pp. 342-347 (1984)).

Preferably, the fragments of said sequence are between Arg ine 1652 and Arginme 1696, preferably between Arginine 1652 and Argmine 1689, between Threonine 1739 and Aspartic Acid 1831 or between Acid Glutamic 1885 and Arginine 1917.

The invention also relates to the sequence epitopes of these fragments, in particular:

- the epitope between Arginine 1652 and Tyrosine 1664 defined by the following sequence:

SEQ ID NO: l:

Arg Thr Thr Leu Gin Ser Asp Gin Glu Glu Ile Asp Tyr 1 5 10

the epitope comprised between Aspartic Acid 1681 and Arginine 1696 defined by the following sequence: SEQ ID NO: 2:

Asp Glu Asp Glu Asn Gin Ser Pro Arg Ser Phe Gin Lys Lys Thr Arg 1 5 10 15

- the epitope between Threonine 1739 and Tyrosine 1748 defined by the following sequence:

SEQ ID NO: 3:

Thr Asp Gly Ser Phe Thr Gin Pro Leu Tyr 1 5 10

- the epitope between Asparagine 1777 and Phenylalanine 1785 defined by the following sequence:

SEQ ID NO: 4:

Asn Gin Ala Ser Arg Pro Tyr Ser Phe 1 5

- the epitope between Glutamic Acid 1794 and Tyrosine 1815 defined by the following sequence:

SEQ ID NO: 5: Glu Asp Gin Arg Gin Gly Ala Glu Pro Arg Lys Asn Phe Val Lys Pro 1 5 10 15

Asn Glu Thr Lys Thr Tyr 20 the epitope comprised between Methionine 1823 and Aspartic Acid 1831 defined by the following sequence: SEQ ID NO: 6:

Met Ala Pro Thr Lys Asp Glu Phe Asp 1 5

- the epitope between Glutamic Acid 1885 and Phenylalanine 1891 defined by the following sequence:

SEQ ID NO: 7: Glu Thr Lys Ser Trp Tyr Phe 1 5

- the epitope between Glutamic Acid 1893 and Alanine 1901 defined by the following sequence: SEQ ID NO: 8:

Glu Asn Met Glu Arg Asn Cys Arg Ala 1 5

- the epitope comprised between Aspartic Acid 1909 and Arginine 1917 defined by the following sequence:

SEQ ID NO: 9:

Asp Pro Thr Phe Lys Glu Asn Tyr Arg 1 5

Advantageously, said sequence, its specific fragments and its epitopes exhibit an antigenic characteristic illustrated by FIGS. 2 to 5 appended.

Another preferred embodiment of the invention relates to the A1 antigenic polypeptide sequence of factor VIII, fragments and / or epitopes thereof.

Preferably, the fragments of said sequence are between Alanine 108 and Methionine 355, preferably between Alanine 108 and Glutamine 228.

The invention also relates to the sequence epitopes of these fragments, in particular: - the epitope between Alanine 108 and Val e 128 defined by the following sequence: SEQ ID NO: 10:

Ala Ser Glu Gly Ala Glu Tyr Asp Asp Gin Thr Ser Gin Arg Glu Lys 1 5 10 15

Glu Asp Asp Lys Val 20

- the epitope between Glutamic Acid 181 and Leucme 192 defined by the following sequence: SEQ ID NO: 11:

Glu Gly Ser Leu Ala Lys Glu Lys Thr Gin Thr Leu 1 5 10

the epitope comprised between Aspartic Acid 203 and Glutamine 218 defined by the following sequence: SEQ ID NO: 12:

Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gin 1 5 10 15 - the epitope between Aspartic Acid 327 and Methionine 355 defined by the following sequence: SEQ ID NO: 13:

Asp Ser Cys Pro Glu Glu Pro Gin Leu Arg Met Lys Asn Asn Glu Glu 1 5 10 15 Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met

20 25

Another preferred embodiment of the invention relates to the A2 antigenic polypeptide sequence of factor VIII, fragments and / or epitopes thereof. Preferably, the fragments of said sequence are between the Aspartic Acid 403 and the Aspartic Acid 725, preferably between the Histidme 693 and the Aspartic Acid 725.

The invention also relates to the epitopes of the sequence of these fragments, in particular: the epitope comprised between Aspartic Acid 403 and Lysine 425 defined by the following sequence:

SEQ ID NO: 14:

Asp Asp Arg Ser Tyr Lys Ser Gin Tyr Leu Asn Asn Gly Pro Gin Arg 1 5 10 15

Ile Gly Arg Lys Tyr Lys Lys

20

- the epitope between Valine 517 and Arginine 527 defined by the following sequence:

SEQ ID NO: 15:

Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg 1 5 10

- the epitope between Histidme 693 and the

Glycine 701 defined by the following sequence: SEQ ID NO: 16:

His Asn Ser Asp Phe Arg Asn Arg Gly 1 5

- the epitope between Serine 710 and Aspartic Acid 725 defined by the following sequence:

SEQ ID NO: 17:

Ser Cys Asp Lys Asn Thr Gly Asp Tyr Try Gly Asp Ser Tyr Glu Asp 1 5 10 15

A final preferred embodiment of the invention relates to the factor VIII antigenic polypeptide sequence C, fragments and / or epitopes thereof. Preferably, the fragments of said sequence are between Lysine 2085 and Lysine 2249, preferably between Lysine 2085 and Glycine 2121.

The invention also relates to the epitopes of the sequence of these fragments, in particular: - the epitope between Lysine 2085 and Phenylalanine 2093 defined by the following sequence:

SEQ ID NO: 18:

Lys Thr Gin Gly Ala Arg Gin Lys Phe 1 5

the epitope comprised between Aspartic Acid 2108 and Glycine 2121 defined by the following sequence:

SEQ ID NO: 19: Asp Gly Lys Lys Trp Gin Thr Tyr Arg Gly Asn Ser Thr Gly 1 5 10

- the epitope between Glycine 2242 and Lysine 2249 defined by the following sequence: SEQ ID NO: 20:

Gly Val Thr Thr Gin Gly Val Lys 1 5

The invention also relates to the strong parts of said epitopes or of said fragments, that is to say the sequence portions of said epitopes comprising the amino acids Tyrosine and Histidme, which unexpectedly exhibit a particularly high affinity towards factor VIII inhibitors. Preferably, these strong parts comprise said amino acid Tyrosine or Histidine linked to at least two other identical or different amino acids.

These sequences, these fragments and these epitopes, in particular the strong parts of the epitopes or of the fragments, are characterized in a particularly advantageous manner by a high hydrophilicity as described by Parker, Guo and Hodges (Biochemistry 25, pp 5425-5432 (1986) ), significant flexibility as described by Karplus and Schultz (Naturwissenschaften 72, p 212 (1985)) and significant accessibility as described by Janin (Nature 277, pp 491-492 (1979)) (see Figures 2 to 5) .

These fragments and these epitopes are in particular exposed on the surface of the factor VIII protein and have a marked antigenic characteristic.

Advantageously, said polypeptide sequence, its fragments, its epitopes and / or these strong parts of said fragments or said epitopes are also independently immunogenic (that is to say that they are immunogenic even without being complexed with a large protein such as BSA, hemocyanin, ...), and preferably exhibit immunoaffinity with factor VIII inhibitors, such as anti-factor VIII antibodies and / or exhibit immunoaffinity for T and / or B lymphocyte receptors .

This sequence, these fragments, these epitopes and / or the strong parts of said fragments or said epitopes cause an immune reaction (synthesis of antibodies) when they are injected into a rabbit. These characteristics are particularly important for the epitopes SEQ ID NO: 2 and SEQ ID NO: 5 which comprise relatively "long" amino acid sequences of 16 and 22 amino acids, respectively.

These sequences are therefore characterized by a high immunogenicity with respect to monoclonal and polyclonal antibodies.

However, these sequences are short enough to be easily obtained by synthesis.

For example, the peptides Asp 1681 - Arg 1696 and Asp 327 - Met 355 were synthesized to demonstrate anti-factor VIII antibodies in the sera of mice by an ELISA test.

The free peptides (not coupled to a vector protein) were injected into two BALB / C mice according to the following scheme:

- day 0 100 μg of peptide emulsified in adjuvant of

Incomplete freunds are injected intramuscularly.

- days 7, 14, 21 and 28: immunization with 50 μg of peptide. A blood sample is taken every day before the injection. Polystyrene microtiter plates (NUNC) are saturated with a preparation of plasma factor diluted using 40 IU / ml. 50 μl of increasing dilutions (1/60, 1/300 and 1/600 with mouse antisera are added to the wells. After incubation and washing, the presence of anti-factor VIII antibodies is demonstrated by the addition of 50 μl of a 1/5000 dilution of a rabbit anti-mouse IgG antibody labeled with biotin. After incubation and washing, the wells are incubated with 50 μl of avidin peroxidase (1/2500) and washed, and finally, 100 ml of OPD are added to the wells. The optical density is measured at 490 mm. The results of the ELISAs are shown in the appended FIG. 6 (EXA, EX2 and a BLC sample which serves as blank). The present invention also relates to conformational epitopes comprising at least two different fragments of said sequence, at least two epitopes of sequence and / or at least two strong parts of said epitopes or said different fragments according to the invention and identified above. of two or several different portions of a polypeptide sequence located close to each other during the folding of the protein into its tertiary or quaternary structure.

These epitopes are likely to be "recognized" (that is to say to have an immunoaffinity), preferably simultaneously, with inhibitors of factor VIII, in particular B lymphocytes (via the major locus of histoco patibility

(MHC I and / or II)) and / or anti-factor VIII antibodies

(Scandella et al., Blood 76, p 437 (1990)). Preferably, said sequence, said fragments, said epitopes and / or the strong parts of said epitopes or said fragments are complexed with a carrier protein or a carrier peptide, such as BSA or hemocyanin, so as to form a complex having a stronger immunogenicity. 19

Another aspect of the present invention relates to a factor VIII inhibitor having immunoaffinity with the antigenic polypeptide sequence according to the present invention, with fragments, epitopes thereof, strong parts of said epitopes or said fragments and / or the complex. according to the invention.

The term “inhibitor” is intended to mean any biological molecule or cell intervening with and / or against factor VIII, and capable of creating immune disorders. In particular, such an inhibitor can be a monoclonal or polyclonal antibody (gamma globulin) or an anti-factor VIII antibody fragment (such as the hypervariable Fab portion of said antibody), which inactivates said factor VIII and / or which inhibits the binding of factor VIII to von Willebrand factor and / or to membrane phospholipids.

Advantageously, said inhibitors are synthesized by a "chimeric" animal comprising a human immune system such as a SCID-hu mouse producing human antibodies. SCID (severe combined immunodeficient) mice are mice with a deficiency in functional B and T lymphocytes due to a dysfunction in the recombination of the genes responsible for antigenic receptors. The immune systems of SCID mice can be reconstituted with human immunocompetent cells from fetal organs or peripheral blood (Mosler et al. (1988) ^•

Once reconstituted, these SCID-hu mice produce human antibodies either spontaneously,: Said after immunization. It seems that there is no dramatic cross-reactivity between human and murine factors VIII (Kessler, 1991).

Peripheral blood lymphocytes are taken from several types of donors: non-hemophiliac volunteers, hemophiliacs lacking inhibitors detectable by conventional methods, hemophiliacs with a high rate inhibitors as well as donors producing auto¬ antibodies.

This model is used in two types of work.

First, the reconstruction of mice is obtained with cells from a single donor, and after checking the reproducibility of the system, the antigenicity of several preparations of factor VIII can be compared.

In addition, this model makes it possible to obtain and study an anti-factor VIII response at the clonal level. The study of the specific monoclonal response of B cells is very important, because it precisely makes it possible to identify the sequential and conformational epitopes of factor VIII. B cells are cultured cloned in the presence or not of anti-CD40, from the spleen of mice producing anti-factor VIII, or else transformed in the presence of the EBV virus. Anti-CD40 antibodies recognize a membrane antigen and activate B cells in the presence of a line of fibroblasts (Banchereau et al. (1991)). It is therefore possible to envisage the use of these immunodominant epitopes as a possible target of immunotherapy.

The determination of MHC class I and class II markers carried by the clones of B lymphocytes makes it possible to analyze the immune response of anti-factor VIII at the genetic level, and thus to follow the recognition by specific T cells. This is also a great method to see if there is a risk factor associated with this condition.

BALB / C mice chosen for the preparation of anti-factor VIII mAbs are injected three times beforehand, 2 weeks apart with a solution of recombinant factor VIII (rFVIII). This type of preparation has the advantage of containing a factor VIII of high purity at a high concentration with a minimum of contaminating proteins. Four days after the last injection, the splenocytes are fused with cells of a mouse myeloma (SP207) (van Snick and Coulie (1982)). The selection of hybridomas producing anti-factor VIII antibodies are carried out by the ELISA technique using polystyrene plates on which rFVIII has previously been insolubilized. Supernatants of hybridomas containing anti-factor VIII antibodies are cloned by the limiting dilution technique, then cultured in vitro.

The antibodies are purified by chromatography from these supernatants.

The quantification, the determination of the light chain (k or 1) and the subclass (IgGl, IgG2a, IgG2b, IgG3) of anti-factor VIII mAbs are carried out by the ELISA technique.

The determination of the epitopes recognized on the factor VIII molecule is carried out by the immunoblot technique with native factor VIII solutions or after enzymatic cleavage with thrombin.

The capacity of the anti-factor VIII mAbs produced to inhibit function is evaluated, for each of these, by a coagulation method (Bethesda method) (Kasper et al.

(1975)) as well as by a chromogenic method based on the ability of activated factor X by the combination of factor VIII and activated factor IX, to transform a colorless substrate into a colored substrate (Svendsen et al. (1984 )).

The cell lines producing human monoclonal anti-factor VIII antibodies are derived from human B lymphocytes taken from the abdominal cavity of SCID mice immunized with different batches of factor VIII after reconstitution of the animal immune system with human lymphocytes. The B lymphocytes are cultured in the presence of fibroblastic cells expressing a receptor for the Fc portion of the immunoglobulins to which a monoclonal anti-CD40 antibody is attached. These cells, activated by the polymerization of the CD40 receptor, are then infected and immortalized by the Epstein-Barr virus (Kozbor, (1981)). The cell lines producing the desired antibodies can then be subcloned. Another aspect of the invention relates to an anti¬ inhibitor characterized in that it is directed against said factor VIII inhibitor previously described.

The term anti-inhibitor directed against the factor VIII inhibitor means any biological molecule and / or cell capable of interfering with said inhibitor so as to ensure its inactivation.

Preferably, such an anti-inhibitor is an antibody (monoclonal or polyclonal), or a fragment of anti-idiotypic anti-factor VIII antibody.

Advantageously, these anti-inhibitors directed against factor VIII inhibitors are synthesized by a "chimeric" animal exhibiting a human immune system such as a SCID-hu mouse. Only mice which produce less than 10 μg / ml of residual immunoglobulins are used for the experiments.

The model is developed using peripheral white blood cells from volunteers immunized against tetanus.

Reconstitution is carried out with a single i.p. from 15 to 20,106 mononuclear cells of human origin. These cells are obtained after centrifugation in a Ficoll-Hypaque gradient of peripheral blood (approximately 200 ml). Twelve to twenty mice can be reconstructed from a single donor. The production of human immunoglobulins is measured as a function of time.

Anti-idiotypic anti-factor VIII antibodies are purified from a starting plasma pool, which is made up of voluntary donations from at least 7200 donors to increase the probability of finding anti-idiotypic antibodies, by immunoaffinity by means of human anti-factor VIII antibodies covalently attached to a Sepharose column or by an Fc part to a protein G column. After fractionation, by the Cohn-Oncley method, two rich Fr II and Fr III fractions in IgG are obtained. They will serve as a starting preparation for the purification of anti-idiotypic antibodies. These monoclonal antibodies will be obtained from B cells collected from hemophiliac patients. These cells have previously proliferated in SCID mice and have been transformed into secreting cell cultures by the EBV virus. The use of these human monoclonal antibodies makes it possible to avoid the introduction of non-human proteins into the therapeutic preparations. These preparations are evaluated for their effectiveness in neutralizing inhibitors from as many hemophiliac patients as possible, by in-depth immunochemical analyzes. Several steps of physical viral inactivation (treatment by UVCF radiation), thermal and / or chemical (for example by solvent-detergent) are introduced into the purification process in order to ensure the greatest possible viral security.

The idiotype specific to human antibodies is analyzed by sequencing the variable part of the molecule. These data "are of extreme importance, because they are of great utility .. both in the diagnosis and the regulation of the production of anti-factor VIII.

Until now, the source of antibodies necessary for the production of antigen-antibody complexes has been autologous, that is to say that the patient himself provided the antibodies. We have recently known that normal individuals with normal levels of circulating factor VIII produce anti-factor VIII antibodies whose activity in plasma is limited by corresponding anti-idiotypic antibodies. Anti-factor VIII antibodies prepared from a pool of gamma globulins can advantageously replace the autologous source.

It is also possible to obtain human B cells transformed with the EBV virus which produce inhibitors for hemophiliac or non-hemophiliac patients. Four lines were thus obtained, one of them recognizing the light chain of factor VIII. SCID mice were repopulated with B cells secreting inhibitors from hemophiliac patients or not. Production is stimulated with the injection of plasma factor VIII and recombinant factor VIII. It is therefore possible to obtain continuous cultures in vitro producing said inhibitors. This technique also makes it possible to obtain a continuous production of anti-idiotypic anti-factor VIII antibodies.

Another aspect of the invention relates to a pharmaceutical composition comprising an element chosen from the group consisting of said factor VIII antigenic polypeptide sequence, fragments, epitopes thereof and / or strong parts of said epitopes or said fragments, a a factor VIII inhibitor directed against them, an anti-inhibitor directed against said inhibitor, and / or a mixture of them.

Another aspect of the invention relates to a diagnostic and / or purification device, such as a diagnostic kit or a chromatography column (as described by Ezzedine et al. (1993)), comprising an element chosen from group consisting of the antigenic polypeptide sequence according to the invention, fragments, epitopes thereof and / or the strong parts of said epitopes or said fragments, the complex according to the invention, an inhibitor directed against them, an anti-inhibitor directed against said inhibitor, and / or a mixture thereof.

The purification device can therefore consist of a chromatography column as described by Ezzedine et al. (1993), comprising the factor VIII sequence, fragments, epitopes thereof and / or the strong parts of said fragments or epitopes attached to the solid phase of the chromatography column.

A physiological liquid (such as serum) from a patient comprising inhibitors of factor VIII is then passed over this chromatography column, said inhibitors (for example antibodies) binding specifically to said factor VIII, said fragments. said epitopes or said strong parts.

After elution, it is possible to collect said inhibitors by reacting them with anti-inhibitors (anti-idiotypic anti-factor VIII antibodies). It is also possible to characterize the anti-idiotypic anti-factor VIII antibodies present in a serum by passing these anti-inhibitors on a chromatography column on which factor VIII inhbiters have been fixed to the solid phase. A final aspect of the invention relates to the use of the pharmaceutical composition according to the invention, for the preparation of a medicament intended for the prevention and / or treatment of immune disorders, in particular those induced by factor VIII inhibitors , von Willebrand factor VIII binding inhibitors (vWF) and / or inhibitors of factor VIII binding to membrane phospholipids.

References ,

Aledort, LM (1993), Sem Hematol 30, 7-9

Bardelle, C, Furie, B, Furie, BC and Gilbert, GE (1993),

J. Biol Che 268, 8815-8824 - Barrowcliffe, TW (1993), Sem Thromb Hemost 19, 73-79

Bennet, B, Ratnoff, OD (1972), Sol Exp Biol Med Process

143, 137-155

Bihoreau, N (1992), M / S 8, 1043-1050

Blomm, AL (1992), haemost 22, 268-275 - Blanchereau, J, de Paoli, P, Vallé, A, Garcia, E and

Rousset, F (1991), Science 251, 70

Cauldfield, MJ and Schaffer, D (1987), J. Immunol 138, 3680

Ciavarella, N and Shiavoni, M (1992), Lancet 339, 1301

Clyne, LP, Levy, A, Stein and McPhedran, P (1992), Thromb and Haemost 68, 475-476

Dietrich, G, Algiman, M, Sultan, Y, Nydegger, EU,

Kazatchkine, MD (1992), Blood 79, 2946-2951

Ehrenforth, S, Kreuz, W, Scharrer, I, Linde, R, Funk, M,

Gungor, T, Krackhardt, B and Kornhuber, B (1992), Lancet 339, 594-598

Elder, B, lakich, D and Gitschier, J (1993), Genomics 16,

374-379

Ewing, NP, Sanders, NL, Dietrich, SL and Kasper, CK (1988),

JAMA 259, 65-68 - Fay, PJ (1993), Thromb Haemost 70, 63-67

Fulcher, CA, Roberts, IZ, Holland, Tous and Zimmerman, Tous (1985), J. Clin Invest 76, 1117-1124

Foster, PA, Fulcher, CA, Houghten, RA and Zimmerman,

TS (1985), Blood 75, 1999-2004 - Gilles, JG, Ar out, J, Vermylen, J and Saint-Rémy, JM (1991), XlVth Int Congress Allerg and Clin Immunol October

13-18

Gilles, JG and Saint-Rémy, JM (1993a), XlVth Congress Int

Soc thromb Haemost July 49 - Gomperts, ED, de Biasi, R and De Vreker, R (1992),

Transfusion Med Rev 1, 44-54 Gruppo, R (1991), Thromb Haemostas 65, 1168

Halter, R, Carnwath, J, Espanon, G, Herrman, D, Lemme, E,

Niemenn, H and Paul, D (1993), Thériogenology 39, 137-149

Hay, CRM and Bolton-Maggs (1991), Transfusion Med Rev V, 145-151

Hedner, U and Tenborn (1985), Thromb Haemostas 54, 776-779

Hultin, MB (1991), Am J. Med 91 (Suppl 5A), 23-27

Hoyer, LW (1991), Am J. Med 91 (Suppl 5A), 405-409

Ingerslev, J, Feldstedt, M and Sindet-Pedersen (1991), Lancet 338, 831-832

Kalafatis, M, Rand, MD, Jenny, RJ, Erlich, YH and Mann, KG

(1993), Blood 81, 704-709

Kaufman, Rj (1992), Transfusion Med Rev VI, 235-246

Kasper, CK, Aledort, LM, Edson, JR, Fratantone, J, Green, D et al (1975), Thrombos Diathes Haemorh 34, 869

Ke ball-Cook, G and Barrowcliffe, TW (1992), Thromb Res 67,

57-71

Kessler, CM (1991), Am J Med 91 (Suppl 5A), 15-19

Kosbor, D and Roder, JC (1981), 127-1275 - Leroy, BL, Lachapelle, JM, Jacquemin, MG and Saint-Rémy,

JMR (1992), Dermatology 184: 271

Leyte et al. (1989), Biochem J. 263, 189-194

Lian, ECY, Larcada, AF and Chiu, AZY (1989 ^' ), Ann Int Med

110, 771-778 - Ljung, R, Petrini, P, Lindgren, AC, Tengborn, L and

Nilsson, IM (1992), Lancet 339, 1550

Lorenzo, JL, Garcia, R and Molina, R (1992), Lancet 339,

1551

Lottenburg, R, Kentro, TB and Kitchins, CS (1987), 147, 1077-1081

Lozier, JN, Santagostino, E, Kasper, Ck, Teitel, JM and

Hay, CRM (1993), Sem Hematol 30, 10-21

Madhok, R, Smith, J, Jenkind, A and Lowe, GDO (1991), Br

J Haematol 79, 235-238 Me Cune, LM, Namikawe, R, Kaneshima, H, Schultz, LD,

Lieberman, M and Weissman, IL (1988), Sciences 241, 1632-

1639

Moreau, PH, Staikowsky, F, Laneelle, D, Dellile, F, Simonin, D, Schiffer, C and Laurian, Y (1993), Presse Méd

22, 472-479

Mosler, DE, Gulizia, RJ, Baird, SB and Wilson, DB (1988),

Nature 335, 256-259

Nesheim, ME, Furmaniak-Kazmierczak, E, Henin, CH and Côté, G (1993), Thromb and Haemost 70, 80-86

Nilsson, JM, Berntop, E, Zettervall, 0 and Dahlbàck, B

(1990), Blood 10, 378-383

Peerlinck, K, Arnout, J, Tamise, A, Vannerie, .P, Fondu, P and Vermylen, J (1991), Acta Clin Belg 46, 298-304 - Peerlinck, K, Arnout, J, Gilles, JG, Saint -Rémy, JM and

Vermylen, J (1993a), Thromb Haemost 69, 2, 115-118

Peerlinck, K, Rosendaal, FR and Vermylen, J (1993b), Blood

81, 3332-3335

Scandella, D, de Graaf Mahoney, S, Mattingly, M, Roeder, D, Timmons, L and Fulcher, CA (1985), Proc.Natl.Acad.Sci.

USA 85, 6152-6156

Scandella, D, Timmons, L, Mattingly, M, Trabold, N and

Hoyer, LW (1992), Thromb Haemost 65, 1160

Seremitis, S, Aledort, L, Lusher, M, Hilgartner, M, Mannucci, PM and Mariani, G (1991), Thromb Haemostas 65,

1160

Smith, CIE, Habedi, M, Islam, KB, Johansson, MEB,

Christenson, B and Hammerstrôm, L (1991), Immunol Rev 124,

113-135 - Sul tan, Y, Rossi, F and Kazatchkine, M (1987), Proc. Natl.

Acad .Sci .84, 828- 831

Sultan, Y, White, GC, Aronstam, A, Bosser, C, Brackmann,

HH et al (1986), Nouv Rev Fr Hematol 28, 85-89

Sultan, Y and French Hemophilia Study Group (1992), Thromb Haemost 67, 600-602 Svendsen, L, Brogli, M, Lindeberg, G and Stocker, K (1984), Thro bos Res 34, 457

Triulzi, DJ, Heal, JM and Blu berg, N (1990), In "Transfusion Médecine in the 1990's". Ed. Nance, ST; American Association of Blood Banks; Arlington, Virginia Van Snick, J and Coulie, P (1982), J. Exp Med 155, 219 Vermylen, J and Peerlinck, K (1991), Acta Clin Belg 46, 419-420

Wadhwa, M, Dilger, P, Tubbs, J, Barrowcliffe, T, Mahon, B and Thorpe, R (1992), Br J Haematol 82, 578-583

Ware, J, MacDonald, MJ, Lo, M, de Graaf, S and Fulcher, CA (1992), Blood Coagul Fibrin 3, 703-716

Webb, E, Tkalcevic, S, Hocking, D and Nisbet, I (1993), Biochem Biophys Res Commun 190, 536-543

Claims

CLAIMS.

1. Antigenic polypeptide sequence, characterized in that it is the polypeptide sequence of factor VIII.

2. Antigenic polypeptide sequence, characterized in that it is devoid of the following fragments:

Alanine 322 - Serine 750, Leucine 1655 - Arginine 1689, Lysine 1694 - Proline 1782 and Aspartic Acid 2170 - Tyrosine 2332.

3. Sequence according to claim 1 or 2, characterized in that it is immunogenic.

4. Sequence according to claim 3, characterized in that it has immunoaffinity with factor VIII inhibitors, preferably with anti-factor VIII antibodies.

5. Sequence according to claim 3 or 4 characterized in that it has immunoaffinity for the T and / or B lymphocyte receptors.

6. Antigenic fragment of the sequence according to claim 1 or 2, characterized in that it is chosen from the group consisting of the polypeptide sequences A1, A2, A3 or C of factor VIII.

7. Antigenic fragment of the A3 polypeptide sequence according to claim 6, characterized in that it is chosen from the group consisting of the sequence fragment between Arginine 1652 and Arginine 1696, the sequence fragment between Threonine 1739 and Aspartic Acid 1831 and / or the sequence fragment between Glutamic Acid 1885 and Arginine 1917.

8. A fragment sequence epitope according to claim 7 characterized in that it is chosen from the group consisting of:

- the epitope between Arginine 1652 and the

Tyrosine 1664 defined by the following sequence:

SEQ ID NO: l:

Arg Thr Thr Leu Gin Ser Asp Gin Glu Glu Ile Asp Tyr 1 5 10 - the epitope comprised between Aspartic Acid 1681 and Arginine 1696 defined by the following sequence:

SEQ ID NO: 2:

Asp Glu Asp Glu Asn Gin Ser Pro Arg Ser Phe Gin Lys Lys Thr Arg 1 5 10 15,

SEQ ID NO: 3: Thr Asp Gly Ser Phe Thr Gin Pro Leu Tyr 1 5 10,

- the epitope between Asparagine 1777 and Phenylalanine 1785 defined by the following sequence: SEQ ID NAME:

Asn Gin Ala Ser Arg Pro Tyr Ser Phe 1 5

SEQ ID NO: 5:

Glu Asp Gin Arg Gin Gly Ala Glu Pro Arg Lys Asn Phe Val Lys Pro

1 5 10 15

Asn Glu Thr Lys Thr Tyr 20

- the epitope between Methionine 1823 and Aspartic Acid 1831 defined by the following sequence: SEQ ID NO: 6: Met Ala Pro Thr Lys Asp Glu Phe Asp

1 5

- the epitope between Glutamic Acid 1885 and Phenylalanine 1891 defined by the following sequence: SEQ ID NO: 7:

Glu Thr Lys Ser Trp Tyr Phe 1 5 - the epitope between Glutamic Acid 1893 and Alanine 1901 defined by the following sequence:

SEQ ID NO: 8:

Glu Asn Met Glu Arg Asn Cys Arg Ala 1 5

SEQ ID NO: 9: Asp Pro Thr Phe Lys Glu Asn Try Arg 1 5

9. Antigenic fragment of the polypeptide sequence A1 according to claim 6, characterized in that it is chosen between Alanine 108 and Methionine 355, preferably between Alanine 108 and Glutamine 228.

10. A fragment sequence epitope according to claim 9, characterized in that it is chosen from the group consisting of

- the epitope between Alanine 108 and Valine 128 defined by the following sequence:

SEW ID N0: 10:

Ala Ser Glu Gly Ala Glu Try Asp Asp Gin Thr Ser Gin Arg Glu Lys

1 5 10 15

Glu Asp Asp Lys Val 20

- the epitope between Glutamic Acid 181 and Leucine 192 defined by the following sequence:

SEQ ID NO: 11: Glu Gly Ser Leu Ala Lys Glu Lys Thr Gin Thr Leu

1 5 10

Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gin 1 5 10 15 the epitope comprised between Aspartic Acid 327 and Methionine 355 defined by the following sequence:

SEQ ID N0: 13:

Asp Ser Cys Pro Glu Glu Pro Gin Leu Arg Met Lys Asn Asn Glu Glu 1 5 10 15

Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met 20 25

11. The antigenic fragment of the A2 antigenic polypeptide sequence according to claim 6, characterized in that it is comprised between Aspartic Acid 403 and Aspartic Acid 725, preferably between Histidine 693 and Aspartic Acid 725.

12. Fragment sequence epitope according to claim 11, characterized in that it is chosen from the group consisting of:

the epitope comprised between Aspartic Acid 403 and Lysine 425 defined by the following sequence:

SEQ ID NO: 14:

Asp Asp Arg Ser Tyr Lys Ser Gin Tyr Leu Asn Asn Gly Pro Gin Arg 1 5 10 15

Ile Gly Arg Lys Tyr Lys Lys

20

SEQ ID NO: 15:

Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg 1 5 10

- the epitope between Histidine 693 and the

Glycine 701 defined by the following sequence: SEQ ID NO: 16:

His Asn Ser Asp Phe Arg Asn Arg Gly 1 5

- the epitope between Serine 710 and Aspartic Acid 725 defined by the following sequence: SEQ ID NO: 17:

Ser Cys Aso Lys Asn Thr Gly Asp Tyr Try Gly Asp Ser Tyr Glu Asp 1 5 10 15

13. Antigenic fragment of the antigenic polypeptide sequence C according to claim 6, characterized in that it is between Lysine 2085 and Lysine 2249, preferably between Lysine 2085 and Glycine 2121.

14. Fragment sequence epitope according to claim 13, characterized in that it is chosen from the group consisting of:

- the epitope between Lysine 2085 and Phenylalanine 2093 defined by the following sequence:

SEQ ID NO: 18: Lys Thr Gin Gly Ala Arg Gin Lys Phe 1 5

the epitope comprised between Aspartic Acid 2108 and Glycine 2121 defined by the following sequence: SEQ ID NO: 19:

Asp Gly Lys Lys Trp Gin Thr Tyr Arg Gly Asn Ser Thr Gly 1 5 10

- the epitope between Glycine 2242 and Lysine 2249 defined by the following sequence:

SEQ ID NO: 20:

Gly Val Thr Thr Gin Gly Val Lys 1 5

15. Strong part of the fragments and / or epitopes according to any one of the preceding claims 6 to 14, characterized in that it comprises the amino acid Tyrosine and / or the amino acid Histid e linked to at least two other identical or different amino acids.

16. Conformational epitope characterized in that it comprises at least two different fragments, at least two different epitopes and / or at least two different strong parts according to any one of claims previous 6 to 15.

17. Complex comprising a carrier protein or a carrier peptide linked to an element chosen from the group consisting of the sequence, the fragment, the epitope and / or the strong part of an epitope or of a fragment according to any one of the preceding claims.

18. A factor VIII inhibitor characterized in that it has immunoaffinity with the sequence, the fragment, the epitope, the strong part of an epitope or of a fragment and / or the complex according to any one of claims previous.

19. An inhibitor according to claim 18, characterized in that it is an antibody or a fragment of anti-factor VIII antibody.

20. Anti-inhibitor characterized in that it is directed against the factor VIII inhibitor according to claim 18 or 19.

21. Anti-inhibitor according to claim 20, characterized in that it is an antibody or a fragment of anti-idiotypic anti-factor VIII antibody.

22. Pharmaceutical composition characterized in that it comprises at least one element chosen from the group consisting of the sequence, the fragment, the epitope, the strong part, the complex, the inhibitor and / or the anti-inhibitor according to any of the preceding claims.

23. A diagnostic and / or purification device characterized in that it comprises at least one element chosen from the group consisting of the sequence, the fragment, the epitope, the strong part, the complex, the inhibitor and / or 1 anti-inhibitor according to any one of the preceding claims 1 to 21.

24. Device according to claim 23, characterized in that it is a diagnostic kit.

25. Device according to claim 23, characterized in that it is a chromatography column.

26. Use of the pharmaceutical composition according to claim 22 for the preparation of a medicament intended for the prevention and / or therapy of immune disorders.

27. Use according to claim 26, characterized in that the immune disorders are disorders induced by factor VIII inhibitors, inhibitors of factor VIII binding to von Willebrand factor and / or inhibitors of factor VIII binding to membrane phospholipids.

28. A method of therapeutic and / or preventive treatment of immune disorders, characterized in that the pharmaceutical composition according to claim 22 is administered to a patient.

29. A method of therapeutic and / or preventive treatment according to claim 28, characterized in that the immune disorders are disorders induced by factor VIII inhibitors, inhibitors of factor VIII binding to von Willebrand factor and / or inhibitors of factor VIII binding to membrane phospholipids.

30. A method of identifying and obtaining inhibitors and / or anti-inhibitors according to any one of the preceding claims 18 to 21, characterized in that a column of chromatography an element chosen from the group consisting of the sequence, the fragment, the epitope, the strong part and / or the complex according to any one of the preceding claims 1 to 17, in that it is passed over said column chromatography a physiological liquid of a patient comprising inhibitors of factor VIII, in that one elutes and one collects the fraction of the inhibitors of factor VIII having presented an immunoaffinity with at least one element chosen from the group constituted by the sequence , the fragment, the epitope, the strong part and / or the complex according to any one of the preceding claims 1 to 17, and optionally in this said method comprises the following stages: said inhibitors are fixed u factor VIII collected on the solid support of a chromatography column, factor VIII anti¬ inhibitors, such as anti-factor VIII anti-idiotypic antibodies, are passed over said column, eluted and the anti-inhibitors having been presented immunoaffinity with factor VIII inhibitors.