Catalytic anti-factor VIII allo-antibodies

Abstract

ates to a method of determining the presence of catalytic anti-Factor VIII allo-antibodies capable of degrading Factor VIII in a mammal, and of characterising the cleavage sites in said Factor VIII molecule by said catalytic anti-Factor VIII allo-antibodies. It also relates to an anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor; and to a pharmaceutical composition comprising said catalytic anti-Factor VIII allo-antibodies which are capable of degrading Factor VIII and which originate from said method of determination; and further to a pharmaceutical composition comprising said anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor. Finally, the present invention relates to the application in therapeutics of said anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor, of a pharmaceutical composition comprising said catalytic anti-Factor VIII allo-antibodies which are capable of degrading Factor VIII and which originate from said method of determination, and of a pharmaceutical composition comprising said anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor.

Description

FIELD OF THE INVENTION

[0001]The present invention relates to a method of determining the presence of catalytic anti-Factor VIII allo-antibodies capable of degrading Factor VIII in a mammal, and of characterising the cleavage sites in said Factor VIII molecule by said catalytic anti-Factor VIII allo-antibodies.

[0002]The present invention also relates to an anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor.

[0003]The present invention further relates to a pharmaceutical composition comprising said catalytic anti-Factor VIII allo-antibodies which are capable of degrading Factor VIII and which originate from said method of determination, and to a pharmaceutical composition comprising said anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor.

[0004]Finally, the present invention relates to the application in therapeutics of said anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor, of a pharmaceutical composition comprising said catalytic anti-Factor VIII allo-antibodies which are capable of degrading Factor VIII and which originate from said method of determination, and of a pharmaceutical composition comprising said anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor.

BACKGROUND TO THE INVENTION

[0005]Haemophilia A is an X chromosome-linked recessive disorder resulting in defective or deficient Factor VIII molecules, which, in its severe form, is a life-threatening and crippling haemorrhagic disease.

[0006]Infusion of homologous Factor VIII to patients with severe haemophilia A results, in 25% of the cases, in the emergence of anti-Factor VIII allo-antibodies (Ehrenforth, S., Kreuz, W., Scharrer, I., Linde, R., Funk, M., Gungor, T., Krackhardt, B. and Kornhuber, B., <<Incidence of development of factor VIII and factor IX inhibitors in haemophiliacs>>, Lancet, 1992, 339: 594-598), that inhibit Factor VIII procoagulant activity by steric hindrance of the interaction of Factor VIII either with stabilising molecules (Saenko, E. L., Shima, M., Rajalakshmi, K. J. and Scandella, D., <<A role for the C2 domain of factor VIII in binding to von Willebrand factor>>, J. Biol. Chem., 1994, 269: 11601-11605; and Saenko, E. L., Shima, M., Gilbert, G. E., and Scandella, D., <<Slowed release of thrombin-cleaved factor VIII from von Willebrand factor by a monoclonal and a human antibody is a novel mechanism for factor VIII inhibition>>, J. Biol. Chem., 1996, 271: 27424-27431), with molecules essential for its activity (Arai, M., Scandella, D., and Hoyer, L. W., <<Molecular basis of factor VIII inhibition by human antibodies: Antibodies that bind to the factor VIII light chain prevent the interaction of factor VIII with phospholipid>>, J. Clin. Invest., 1989, 83: 1978-1984; and Zhong, D., Saenko, E. L., Shima, M., Felch, M. and Scandella, D., <<Some human inhibitor antibodies interfere with factor VIII binding to Factor IX>>, Blood, 1998, 92: 136-142), or with activating molecules (Lubahn, B. C., Ware, J., Stafford, D. W., and Reiser, H. M., <<Identification of a FVIII epitope recognized by a human hemophilic inhibitor>>, Blood, 1989, 73: 497-499; and Neuenschwander, P. F., and Jesty, J., <<Thrombin-activated and factor Xa-activated human factor VIII: differences in cofactor activity and decay rate>>, Arc. Biochem. Biophys., 1992, 296: 426-434).

SUMMARY OF THE INVENTION

[0007]In an entirely surprising way, a discovery has been made by the Applicants of a degradation of Factor VIII by allo-antibodies of two high responder patients with severe haemophilia A, demonstrating a heretofore unknown mechanism by which Factor VIII inhibitors may prevent the pro-coagulant function of Factor VIII.

[0008]The Applicant's discovery of catalytic anti-Factor VIII allo-antibodies is to the best of his knowledge the first report on the emergence of catalytic antibodies that are INDUCED upon treatment of patients with Factor VIII. It was heretofore considered very surprising, even absurd or unbelievable, that antibodies are formed, in the presence of Factor VIII, which would actually render the Factor VIII molecule inactive through catalytic hydrolysis (proteolysis). However, the catalytic antibodies reported so far, are all auto-antibodies found in the course of a disease process or in physiological conditions. Thus, induced antibodies are called ALLO-antibodies, the origin of which is clearly different from the origin of AUTO-antibodies in any auto-immune disease.

[0009]The calculated average Km and apparent Vmax for the reaction of anti-Factor VIII antibodies of one of the patients were 9.46±5.62 μM and 85±60 fmol.min^-1, respectively. The kinetic parameters of Factor VIII hydrolysis suggest a functional role for the catalytic immune response in the inactivation of Factor VIII in vivo.

[0010]The characterisation of anti-Factor VIII allo-antibodies as site-specific proteases hence provide new approaches to the treatment of diseases of a patient who possess anti-Factor VIII allo-antibodies.

[0011]Thus, according to a first aspect, the present invention provides a method of determining the presence of catalytic anti-Factor VIII allo-antibodies capable of degrading Factor VIII in a mammal, characterised in that it comprises: [0012]i) isolating the plasma from a sample of blood taken from said mammal, [0013]ii) isolating anti-Factor VIII allo-antibodies from said plasma; [0014]iii) placing said anti-Factor VIII allo-antibodies in contact with Factor VIII for a period of time sufficient to permit any degradation of said Factor VIII by said anti-Factor VIII allo-antibodies; and [0015]iv) determining, after said period of time, whether said Factor VIII has effectively been degraded by said anti-Factor VIII allo-antibodies.

[0016]According to an embodiment of step ii) of the method of the present invention, said anti-Factor VIII allo-antibodies are isolated from said plasma by combining them with said Factor VIII, said Factor VIII being preferably coupled to a matrix. Advantageously, in step ii), said anti-Factor VIII allo-antibodies are isolated by affinity chromatography. Preferably, in step ii), said affinity chromatography comprises the use of a Sepharose matrix, preferably activated with cyanogen bromide.

[0017]According to an embodiment of step iii) of the method of the present invention, said Factor VIII is labelled with a labelling agent, preferably a radio-labelling agent, such as ¹²⁵I in particular. Advantageously, in step iii), said Factor VIII is placed in contact with the anti-Factor VIII allo-antibodies for a period of time of between about 0.5 and about 30 hours, preferably about 10 hours, at a temperature of about 15 to about 40° C., preferably 38° C.

[0018]According to an embodiment of step iv) of the method of the present invention, the determination of whether said Factor VIII has effectively been degraded by said anti-Factor VIII allo-antibodies is carried out by a determination comprising a separation technique, such as gel electrophoresis, such as SDS PAGE in particular, or gel filtration, such as fast protein liquid chromatography gel filtration in particular, and a visualisation technique, such as autoradiography in particular.

[0019]In accordance with a further embodiment of the method of the present invention, said method is characterised in that it further comprises: [0020]v) characterising the site(s) in said Factor VIII molecule cleaved by said anti-Factor VIII allo-antibodies.

[0021]According to an embodiment of step v) of the method of the present invention, said characterisation is carried out by placing said Factor VIII in contact with said anti-Factor VIII allo-antibodies capable of degrading Factor VIII, separating and then sequencing the fragments of Factor VIII resulting therefrom. Advantageously, said separation is carried out using a technique such as gel electrophoresis, such as SDS PAGE in particular, or gel filtration. Said sequencing is advantageously carried out using a technique such as N-terminal sequencing, such as by using an automatic protein microsequencer in particular. By using the said sequencing, the following scissile bonds are located: Arg³⁷²-Ser³⁷³, located between the A1 and A2 domains, Tyr.sup.1680-Asp.sup.1681, located on the N-terminus of the A3 domain, and Glu.sup.1794-Asp.sup.1795 located within the A3 domain of the Factor VIII molecule.

[0022]According to a second aspect, therefore, the present invention provides an amino acid sequence:

TABLE-US-00001 Ser Val Ala Lys Lys His Pro;

an amino acid sequence:

TABLE-US-00002 Asp Glu Asp Glu Asn Gln Ser; and

an amino acid sequence:

TABLE-US-00003 Asp Gln Arg Gln Gly Ala Glu.

[0023]The present invention also extends to variants or analogues of this or any other sequence of Factor VIII which are capable of inhibiting any site in the Factor VIII molecule which is susceptible to being lysed by an anti-Factor VIII allo-antibody. Within the context of the present invention, such a variant can be, for example, a peptide or non-peptide analogue of an amino acid sequence described supra which inhibits any site in the Factor VIII molecule which is susceptible to being lysed by an anti-Factor VIII allo-antibody. Such a variant can be, for example, a variant of the sequence which is either shorter by a few amino acids, at the N-terminal, the C-terminal, or both termini, for example, or longer by a few amino acids (it being possible to obtain such variants by chemical synthesis or by enzymatic digestion of the naturally occurring molecule), so long as the variant inhibits any site in the Factor VIII molecule which is susceptible to being lysed by an anti-Factor VIII allo-antibody.

[0024]Hence, according to a third aspect, the present invention provides an anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor. Advantageously, this inhibitor is characterised in that it comprises a protease inhibitor. Examples of protease inhibitors that can be used as anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitors within the context of the present invention, without being limited thereto, are fluorophosphate-type inhibitors, such as DFP for example, or sulphonyl fluoride-type inhibitors, such as PMSF or AEBSF (4-(2-aminoethyl)benzenesulphonyl fluoride hydrochloride (notably marketed by Roche Diagnostics GmbH, Mannheim, Germany, under the trademark Pefabloc®)), for example. More particularly, this inhibitor is characterised in that said inhibitor inhibits cleavage of the scissile bonds: Arg³⁷²-Ser³⁷³, located between the A1 domains, Tyr.sup.1680-Asp.sup.1681, located on the N-terminus of the A3 domain, and Glu.sup.1794-Asp.sup.1795 located within the A3 domain of the Factor VIII molecule. More preferably still, this inhibitor is characterised in that it comprises a peptide or non-peptide analogue of the amino acid sequence:

TABLE-US-00004 Ser Val Ala Lys Lys His Pro;

a peptide or non-peptide analogue of the amino acid sequence:

TABLE-US-00005 Asp Glu Asp Glu Asn Gln Ser; or

a peptide or non-peptide analogue of the amino acid sequence:

TABLE-US-00006 Asp Gln Arg Gln Gly Ala Glu.

[0025]The Factor VIII degradation inhibitors as defined supra, as well as their addition salts, in particular their pharmaceutically acceptable addition salts, have a very valuable pharmacological profile in that they possess neutralising activity towards anti-Factor VIII allo-antibodies.

[0026]These properties justify their application in therapeutics and the invention further relates, by way of drugs, to the Factor VIII degradation inhibitors above, as well as their addition salts, in particular their pharmaceutically acceptable addition salts.

[0027]They will therefore be particularly indicated in the treatment of diseases of, inter alia, haemophilic nature, more particularly diseases involving coagulation defects due to Factor VIII insufficiency.

[0028]An example of their use which may be mentioned is the treatment of high responder patients with diseases such as mild or severe haemophilia A, for example (in the case in which catalytic antibodies are found in these patients), on the one hand, and/or, on the other hand, patients suffering from auto-immune diseases for example (in the case in which catalytic antibodies are found in these patients).

[0029]Thus, according to a fourth principal aspect, the present invention provides a solution to a long-felt need through a pharmaceutical composition characterised in that it comprises a pharmaceutically effective amount of at least one anti-Factor VIII allo-antibody capable of degrading Factor VIII, as defined supra, notably as obtainable from the method described supra, or one of its pharmaceutically acceptable addition salts incorporated in a pharmaceutically acceptable excipient, vehicle or carrier.

[0030]Further, according to a fifth principal aspect, the present invention provides a pharmaceutical composition characterised in that it comprises a pharmaceutically effective amount of at least one Factor VIII degradation inhibitor, as defined supra, or one of its pharmaceutically acceptable addition salts incorporated in a pharmaceutically acceptable excipient, vehicle or carrier.

[0031]These compositions can be administered by the buccal, rectal, parenteral, transdermal, ocular, nasal or auricular route, for example.

[0032]These compositions can be solid or liquid and can be presented in the pharmaceutical forms commonly used in human medicine, such as, for example, simple or coated tablets, gelatine capsules, granules, suppositories, injectable preparations, transdermal systems, eye drops, aerosols and sprays, and ear drops. They are prepared by the customary methods. The active principle, which consists of a pharmaceutically effective amount of at least one Factor VIII degradation inhibitor as defined supra, or one of its pharmaceutically acceptable addition salts can be incorporated therein together with excipients normally employed in pharmaceutical compositions, such as talc, gum Arabic, lactose, starch, magnesium stearate, polyvidone, cellulose derivatives, cocoa butter, semi-synthetic glycerides, aqueous or non-aqueous vehicles, fats of animal or vegetable origin, glycols, various wetting agents, dispersants or emulsifiers, silicone gels, certain polymers or copolymers, preservatives, flavourings and colours. The preferred pharmaceutical form is an injectable form.

[0033]The invention also covers a pharmaceutical composition with neutralising activity which can be used especially as a favourable treatment of diseases such as haemophilia A with production of anti-Factor VIII allo-antibodies; autoimmune diseases with anti-Factor VIII allo-antibodies (in case catalytic antibodies are found in these patients) in particular, said composition being characterised in that it comprises a pharmaceutically effective amount of at least one Factor VIII degradation inhibitor above, or one of its pharmaceutically acceptable addition salts incorporated in a pharmaceutically acceptable excipient, vehicle or carrier.

[0034]The invention also covers a method of therapeutic treatment of a mammal suffering from a pathology resulting from the level of Factor VIII in the blood thereof, characterised in that a therapeutically effective amount of at least one Factor VIII degradation inhibitor as defined supra or one of its pharmaceutically acceptable addition salts is administered to the said mammal.

[0035]This method affords especially a favourable treatment of diseases of haemophilic nature, in particular a pathology resulting from a lack of Factor VIII in the blood thereof.

[0036]The invention also covers a pharmaceutical composition with anti-thrombotic activity which can be used especially as a favourable treatment of diseases such as thrombosis in particular, said composition being characterised in that it comprises a pharmaceutically effective amount of at least one anti-Factor VIII allo-antibody capable of degrading Factor VIII, notably as obtainable from the method described above, or one of its pharmaceutically acceptable addition salts incorporated in a pharmaceutically acceptable excipient, vehicle or carrier.

[0037]The invention also covers a method of therapeutic treatment of mammals, characterised in that a therapeutically effective amount of at least one anti-Factor VIII allo-antibody as defined supra or one of its pharmaceutically acceptable addition salts is administered to the said mammal.

[0038]This method affords especially a favourable treatment of diseases of thrombotic nature, in particular said pathology resulting from the presence of an excess of Factor VIII in the blood thereof.

[0039]In human and animal therapeutics, the anti-Factor VIII allo-antibodies or the Factor VIII degradation inhibitors as defined supra can be administered by themselves or in association with a physiologically acceptable excipient, in any form, in particular orally in the form of gelatine capsules or tablets, or parenterally in the form of injectable solutions. It is possible to envisage other forms of administration such as suppositories, ointments, creams, gels or aerosol preparations.

[0040]Within the context of the present invention, the following terms are used:

catalytic anti-Factor VIII allo-antibodies, which is understood as meaning antibodies directed to Factor VIII endowed with a catalytic activity induced in haemophilia A patients upon transfusion with therapeutic preparations of Factor VIII;Factor VIII, which is understood as meaning a co-enzyme of Factor IX in the enzymatic cleavage of Factor X during the blood coagulation process;degradation of Factor VIII, which is understood as meaning the generation of fragments from Factor VIII that do not appear due to a spontaneous hydrolysis, or due to hydrolysis by physiologically cleaving enzymes, i.e. thrombin, activated Factor IX, activated Factor X, and activated protein C;anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor, which is understood as meaning any peptide, belonging or not to the Factor VIII sequence, or protease inhibitor that are capable of specifically neutralising the hydrolysing activity of anti-Factor VIII catalytic antibodies;

DESCRIPTION OF A PREFERRED EMBODIMENT OF THE PRESENT INVENTION

[0041]Human recombinant Factor VIII was radio-labelled with ¹²⁵I. Anti-Factor VIII allo-antibodies were affinity-purified from the plasma of three haemophilia patients with inhibitor on a Sepharose matrix to which immunopurified human Factor VIII had been coupled. Affinity-purified anti-Factor VIII antibodies of patients Bor, Che and Wal inhibited Factor VIII pro-coagulant activity up to 57.0, 64.0 and 43.0 BU/mg of IgG, respectively.

[0042]Co-incubation of labelled Factor VIII with the anti-Factor VIII allo-antibodies resulted, in the case of two patients out of three, in the proteolysis of the molecule. The specificity of the hydrolysis on the antibody combining sites of anti-Factor VIII allo-antibodies of the IgG isotype was demonstrated. Co-incubation of [¹²⁵I]-Factor VIII with affinity-purified anti-Factor VIII IgG of patients Bor and Wal in the presence of the protease inhibitors aprotinin (0.15 μM), E-64 (28 μM), EDTA (1.3 μM), leupeptin (10 μM), and pepstatin (10 μM) did not result in inhibition of proteolytic activity.

[0043]The Applicants have characterised the major cleavage sites for catalytic IgG in the Factor VIII molecule, to be as follows: Arg³⁷²-Ser³⁷³, located between the A1 and A2 domains of Factor VIII; Tyr.sup.1680-Asp.sup.1681, located on the N-terminus of the A3 domain; and Glu.sup.1794-Asp.sup.1795 located within the A3 domain.

[0044]The time and dose-dependency of the hydrolysis of Factor VIII by anti-Factor VIII allo-antibodies has been demonstrated. In particular, hydrolysis was observed under conditions where anti-Factor VIII IgG and Factor VIII were co-incubated at molar ratios that were 80- to 9500-fold lower than those expected to be present in patients' plasma, suggesting that hydrolysis is a mechanism of Factor VIII inactivation by the patients' allo-antibodies in vivo.

[0045]The Applicants have further investigated the kinetics of antibody-mediated hydrolysis of Factor VIII by incubating anti-Factor VIII IgG of patient Wal with increasing concentrations of unlabeled Factor VIII in the presence of a fixed concentration of [¹²⁵I]-Factor VIII. The curves of the reciprocal of the velocity plotted as a function of the reciprocal of the substrate concentration were linear (r=0.99), suggesting that the reaction conformed to simple Michaelis-Menten kinetics, as already observed for polyclonal catalytic auto-antibodies. The apparent catalytic efficiency, Vmax and rate of hydrolysis of anti-Factor VIII allo-antibodies were calculated in the case of patient Wal. The kinetic parameters of hydrolysis calculated in vitro, suggest that proteolysis may be a mechanism of Factor VIII inactivation by patients' allo-antibodies in vivo.

[0046]The association of Factor VIII with von Willebrand Factor (vWF) increases the catalytic rate of thrombin for Factor VIII, whereas it protects Factor VIII from hydrolysis by activated protein C (APC). The addition of vWF to Factor VIII resulted in partial inhibition of hydrolysis of Factor VIII by anti-Factor VIII IgG, i.e. 36.9%, when purified vWF and Factor VIII were mixed using a wt/wt ratio similar to that present in normal plasma, i.e. 30 μg/ml of vWF versus 300 ng/ml of Factor VIII.

[0047]The identification of anti-Factor VIII allo-antibodies as catalytic antibodies extends the spectrum of catalytic immune responses, in addition to previous reports of hydrolysing antibodies against vasoactive intestinal peptide (VIP) in asthma patients, DNA-hydrolysing antibodies in patients with SL and thyroglobulin-specific catalytic antibodies in patients with autoimmune thyroiditis. This is also the first report to the knowledge of the Applicants of the induction of a catalytic antibody in the human, in response to exogeneous administration of a protein antigen. The kinetic parameters of Factor VIII hydrolysis by anti-Factor VIII IgG exhibiting catalytic properties and the estimated amounts of these antibodies in plasma, suggest a functional role for the catalytic immune response in inactivating Factor VIII in vivo. Within a polyclonal mixture of anti-Factor VIII allo-antibodies which differ in their functional properties, catalytic antibodies may inhibit Factor VIII pro-coagulant activity at faster rates than non-catalysing anti-Factor VIII antibodies. Identification of peptide epitopes that are the targets for proteolytic anti-Factor VIII antibodies may thus be critical for our understanding of the pathophysiology of the Factor VIII inhibitor response. Furthermore, the characterisation of Factor VIII inhibitors as site-specific proteases will provide new approaches to the treatment of patients possessing anti-Factor VIII allo-antibodies.

BRIEF DESCRIPTION OF THE FIGURES

[0048]The invention will be better understood and other objects, characteristics and advantages thereof will become more clearly apparent from the following explanatory description referring to the attached Figures, which are given solely by way of non-limiting Examples illustrating the specificity of the cleavage of Factor VIII by anti-Factor VIII allo-antibodies.

[0049]FIG. 1: Hydrolysis of [¹²⁵I]-Factor VIII by affinity-purified anti-Factor VIII IgG antibodies of haemophilia A patients with inhibitor

[0050]FIG. 1(A):

[0051][125I]-labelled Factor VIII was incubated with affinity-purified anti-Factor VIII IgG of patients Bor (lane Bor), Che (lane Che) and Wal (lane Wal), or with buffer alone (lane 1) for 10 h at 38° C. prior to SDS-PAGE and autoradiography. In two of the three patients (Bor and Wal), incubation of Factor VIII with affinity-purified anti-Factor VIII IgG resulted in hydrolysis of the Factor VIII molecule. In contrast, the migration profile of Factor VIII was unchanged when [¹²⁵I]-labelled Factor VIII was incubated with anti-Factor VIII IgG purified from the plasma of patient Che (lane Che). The migration profile of Factor VIII was also unchanged upon incubation with human monoclonal M061 anti-digoxin IgG (mAb) or with normal unfractionated polyclonal human IgG (Sandoglobulin®, IVIg) that exhibit no inhibitory activity to Factor VIII.

[0052]FIG. 1(B):

[0053]Flow-throughs of the affinity columns were devoid of anti-Factor VIII antibodies as determined by ELISA, and did not hydrolyse [¹²⁵I]-labelled Factor VIII.

[0054]FIG. 1(C):

[0055]Removal of IgG from the acid eluates containing affinity-purified anti-Factor VIII antibodies of patients Wal and Bor by chromatography on protein G, resulted in the loss of their hydrolytic activity to Factor VIII.

[0056]FIG. 2: Size exclusion chromatography of the catalytic activity of anti-Factor VIII antibodies

[0057]FIG. 2(A):

[0058]To further exclude the possibility that the proteolytic activity of the antibodies was due to contaminating proteases, affinity-purified anti-Factor VIII antibodies of patient Wal were treated with 8 M urea and subjected to size exclusion chromatography. A major peak was isolated in fraction 25 that corresponded to IgG as indicated by ELISA. The hydrolysing activity co-eluted with the IgG fraction and that the activity was not detected in fractions in which IgG was not present (e.g., fraction 35).

[0059]FIG. 2(B):

[0060]The major peak that was isolated in fraction 25 corresponded to IgG as indicated by SDS-PAGE of the radio-labelled content of the fraction.

[0061]FIG. 3: Dose- and time-dependency of proteolysis of [¹²⁵I]-Factor VIII by affinity-purified anti-Factor VIII antibodies of haemophilia A patients with inhibitor.

[0062]The kinetics of the hydrolysis of Factor VIII by anti-Factor VIII allo-antibodies of patients Bor and Wal. The rate of hydrolysis of [¹²⁵I]-labelled Factor VIII by anti-Factor VIII IgG of patient Wal was faster than that exhibited by anti-Factor VIII IgG of patient Bor, suggesting either that catalytic antibodies of the patients exhibit different kinetic properties, or, alternatively, that the proportion of catalytic antibodies among the anti-Factor VIII antibodies differ between the patients.

[0063]FIG. 4: Hydrolysis of [¹²⁵I]-Factor VIII by anti-Factor VIII IgG antibodies in the presence of increasing amounts of cold Factor VIII

[0064]Kinetics of antibody-mediated hydrolysis of Factor VIII by incubating anti-Factor VIII IgG of patient Wal with increasing concentrations of unlabelled Factor VIII in the presence of a fixed concentration of [¹²⁵I]-Factor VIII. The addition of increasing amounts of unlabelled Factor VIII resulted in dose-dependent inhibition of hydrolysis of [¹²⁵I]-Factor VIII by anti-Factor VIII IgG. Saturation of Factor VIII hydrolysis was not attained with the maximum concentration of Factor VIII that was used (i.e. 1.7 μM). The curves of the reciprocal of the velocity plotted as a function of the reciprocal of the substrate concentration were linear (r=0.99), suggesting that the reaction conformed to simple Michaelis-Menten kinetics, as already observed for polyclonal catalytic auto-antibodies.

[0065]FIG. 5: Inhibition of catalytic activity of anti-Factor VIII IgG of patient Wal

[0066]The proteolysis of radio-labelled Factor VIII by the anti-Factor VIII allo-antibodies of patient Wal was inhibited to about 62% when the antibodies and Factor VIII were co-incubated in the presence of Pefabloc® (marketed by Roche Diagnostics GmbH, Mannheim, Germany), indicating the potency of certain serine protease inhibitor to neutralise the catalytic activity of some of the catalytic antibodies.

EXAMPLES

Example I

Affinity-Purification of Anti-Factor VIII Antibodies

[0067]Antibodies were isolated from plasma by ammonium sulphate precipitation. Antibodies reactive with Factor VIII were then affinity-purified on a CNBr-activated Sepharose 4B matrix to which immuno-purified commercial human plasma-derived Factor VIII had been coupled (25000 U/3 g of gel). The flow-throughs of the columns were collected. After extensive washing with PBS pH 7.4, anti-Factor VIII antibodies were eluted using 0.2 M glycine pH 2.8, dialysed against PBS and concentrated with Centriprep. Flow-throughs and eluates were aliquoted and stored at -20° C. until use. F(ab')₂ fragments of anti-Factor VIII antibodies were prepared as previously described.

[0068]The concentration of anti-Factor VIII IgG was 130, 20 and 280 μg per 10 mg of IgG applied to the column in the case of patients Bor, Che and Wal, respectively, (i.e., 143±130 μg/mI of unfractionated plasma), which is in agreement with previous observations.

Example II

Factor VIII-Neutralising Activity

[0069]The Factor VIII-neutralising activity of anti-Factor VIII antibodies was determined by the method of Kasper et al. and expressed as Bethesda units (BU) (ref). BU were defined as the inverse of the concentration of IgG which causes 50% inhibition of Factor VIII procoagulant activity. Residual Factor VIII activity was measured in a one-stage assay by determination of the activated partial thromboplastin time using human plasma depleted of Factor VIII (Behring) as substrate and human placental Pathromtin® (Behring) as activators. Heated plasma or immunopurified anti-Factor VIII IgG to be tested, were incubated with pooled citrated human plasma for 2 h at 37° C. The clotting time of four serial dilutions of a reference plasma pool (Immuno AG, Wien) was compared with the clotting time of three dilutions of each sample to be tested. Dilutions were carried out in Owren-Koller buffer (Diagnostica Stago). Inter-assay variation ranged between 1 and 2.5%.

[0070]Affinity-purified anti-Factor VIII antibodies of patients Bor, Che and Wal inhibited Factor VIII pro-coagulant activity up to 57.0, 64.0 and 43.0 BU/mg of IgG, respectively.

Example III

Assay for Hydrolysis of Factor VIII

[0071]Commercial human recombinant Factor VIII was labelled with ¹²⁵I to a specific activity of 11.6 nCi/μg, by using the iodogen method. [¹²⁵I]-Factor VIII (1.5 to 150 ng) was incubated in 50 μl of 50 mM tris-HCI pH 7.7, 100 mM glycine, 0.025% Tween-20 and 0.02% NaN₃ alone or with 17 to 1667 nM of immuno-purified anti-Factor VIII IgG for 5 min to 10 hours at 38° C. Human monoclonal anti-digoxin IgG M061 (mAb) and normal unfractionated human polyclonal IgG (IVIg, Sandoglobulin®), were used as negative controls. Samples were mixed 1:1 with Laemmli's buffer without mercaptoethanol, and were subjected to SDS electrophoresis without boiling, after loading 20 μl of each sample per lane. Samples were run in parallel on 7.5% and 15% SDS-PAGE under non-reducing conditions, after loading 20 μl of each sample per lane. Migration was performed at room temperature using a mini-PROTEAN II system at 25 mA/gel, until the dye front reached the bottom of the gel. The gels were then dried and protein bands revealed using X-OMAT AR. Following autoradiography, the Factor VIII bands of apparent molecular weight 200 and 300 kDa that are consistently hydrolysed by anti-Factor VIII IgG, were scanned so as to allow for the calculation of the rate of hydrolysis of labelled Factor VIII.

Example IV

Fast Protein Liquid Chromatography Gel Filtration

[0072]A hundred μl aliquot of anti-Factor VIII IgG of patient Wal (740 μg) treated with 8M urea was subjected to gel filtration on a superose-12 column equilibrated with PBS-0.01% azide at a flow rate of 0.2 ml/min. Five hundred μl fractions were collected and assayed for the presence of IgG by sandwich ELISA and for Factor VIII proteolytic activity after ten-fold dilution. The proteins in fraction 25 were radiolabelled with ¹²⁵I and subjected to SDS-PAGE under non-reducing conditions in parallel with normal polyclonal human IgG. The gel was stained with Comassie Blue, and also autoradiographed; both images were then overlaid. A major peak was isolated in fraction 25 that corresponded to IgG as indicated by ELISA and SDS-PAGE of the radiolabelled content of the fraction. The hydrolysing activity co-eluted with the IgG fraction and that the activity was not detected in fractions in which IgG was not present (e.g., fraction 35).

Example V

Analysis of NH₂-Terminal Sequences

[0073]Unlabelled human recombinant Factor VIII sucrose formulation (rDNA-BHK) (300 μg, octocog alfa, Bayer Corporation, Berkeley, Calif.) was treated with the anti-Factor VIII IgG of patient Wal (74 μg) in 1500 μl of 50 mM tris-HCI pH 7.7, 100 mM glycine, 0.025% tween-20 and 0.02% NaN₃ for 24 hours at 38° C. The resultant Factor VIII fragments were run on a 10% SDS-PAGE at 50 mA under non-reducing conditions and transferred for 2 hours at 100 mA on a Hybond-P PVDF membrane (Amersham, Little Chalfont, England) in 10 mM CAPS, 10% ethanol at pH 11.0. After staining with coomassie blue, visible bands were cut and subjected to N-terminal sequencing, using an automatic protein microsequencer Prosize 492 cLC (PE-Applied Biosystems, Foster City, Calif.). The amount of protein sequenced ranged from 0.5 to 2 pmoles, depending on the fragment.

[0074]The major scissile bonds were as follows: Arg³⁷²-Ser³⁷³ (R³⁷²-S³⁷³), located between the A1 and A2 domains of Factor VIII; Tyr.sup.1680-Asp.sup.1681 (Y.sup.1680-D.sup.1681), located in the N-terminus of the A3 domain; and Glu.sup.1794-Asp.sup.1795 (E.sup.1794-D.sup.1795) located within the A3 domain. Multiple site cleavage of Factor VIII by anti-Factor VIII antibodies might originate from individual antibodies with polyspecific catalytic activities or polyclonal populations of antibodies, each exhibiting a unique cleavage site specificity.

TABLE-US-00007 Amino acid sequence Cleavage site Ser Val Ala Lys Lys His Pro Arg³⁷² - Ser³⁷³ (SVAKKHP) (R³⁷²-S³⁷³) Asp Gln Arg Gln Gly Ala Glu Glu.sup.1794-Asp.sup.1795 (DQRQGAE) (E.sup.1794-D.sup.1795) Asp Glu Asp Glu Asn Gln Sr Tyr.sup.1680-Asp.sup.1681 (DEDENQS) (Y.sup.1680-D.sup.1681)

Example VI

Inhibition Studies were Performed Using Pefabloc®, a Generic Inhibitor of Serine Proteases

[0075]Hydrolysis of [¹²⁵I]-Factor VIII by affinity-purified anti-Factor VIII IgG antibodies of haemophilia A patients with inhibitor in the presence of Pefabloc®. [¹²⁵I]-Factor VIII (150 ng) was incubated alone, with 50 μg/ml of immunopurified anti-Factor VIII IgG of patient Wal or in the presence of both anti-Factor VIII IgG and 4 mM of the serine protease inhibitor Pefabloc® (Boehringer) for 5 h at 38° C. Factor VIII was then analysed by 7.5% SDS-PAGE under non-reducing conditions. Following autoradiography, the Factor VIII bands of apparent molecular weight 200 and 300 kDa that are consistently hydrolysed by anti-FVIII IgG, were scanned so as to allow for the calculation of the % of hydrolysis of labelled Factor VIII.

[0076]The proteolysis of radiolabelled Factor VIII by the anti-Factor VIII allo-antibodies of patient Wal was inhibited to about 62% when the antibodies and Factor VIII were co-incubated in the presence of Pefabloc®, indicating the potency of some serine protease inhibitor to neutralise the catalytic activity of some catalytic antibodies.

Further Observation

[0077]Upon screening the purified IgG of TEN high responder patients with haemophilia A using ¹²⁵I-radiolabelled Factor VIII as the target molecule, a change was observed in the migration profile of Factor VIII in the case of six patients. These results substantiate the Applicant's previous observations and indicate that catalytic anti-Factor VIII antibodies are found in about 60% of the patients.

Sequence CWU 1

417PRTHomo sapiens 1Ser Val Ala Lys Lys His Pro1 527PRTHomo sapiens 2Asp Glu Asp Glu Asn Gln Ser1 537PRTHomo sapiens 3Asp Gln Arg Gln Gly Ala Glu1 542332PRTHomo sapiens 4Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr1 5 10 15Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro 50 55 60Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val65 70 75 80Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 115 120 125Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135 140Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser145 150 155 160His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185 190His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 210 215 220Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg225 230 235 240Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His 245 250 255Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu 260 265 270Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile 275 280 285Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly 290 295 300Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met305 310 315 320Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg 325 330 335Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 370 375 380Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu385 390 395 400Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 405 410 415Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425 430Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 435 440 445Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 450 455 460Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile465 470 475 480Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 485 490 495His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys 500 505 510Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 515 520 525Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala 530 535 540Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp545 550 555 560Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 565 570 575Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580 585 590Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 595 600 605Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 610 615 620Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu625 630 635 640Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 645 650 655Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680 685Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 690 695 700Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu705 710 715 720Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 725 730 735Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Phe Ser Thr Arg 740 745 750Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys 755 760 765Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn 770 775 780Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser Pro Thr Pro785 790 795 800His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe 805 810 815Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser 820 825 830Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val 835 840 845Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly 850 855 860Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser865 870 875 880Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala 885 890 895Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His 900 905 910Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro 915 920 925Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp 930 935 940Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp945 950 955 960Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys 965 970 975Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys 980 985 990Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala 995 1000 1005Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser Leu Leu Ile Glu Asn 1010 1015 1020Ser Pro Ser Val Trp Gln Asn Ile Leu Glu Ser Asp Thr Glu Phe Lys1025 1030 1035 1040Lys Val Thr Pro Leu Ile His Asp Arg Met Leu Met Asp Lys Asn Ala 1045 1050 1055Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr Thr Ser Ser Lys 1060 1065 1070Asn Met Glu Met Val Gln Gln Lys Lys Glu Gly Pro Ile Pro Pro Asp 1075 1080 1085Ala Gln Asn Pro Asp Met Ser Phe Phe Lys Met Leu Phe Leu Pro Glu 1090 1095 1100Ser Ala Arg Trp Ile Gln Arg Thr His Gly Lys Asn Ser Leu Asn Ser1105 1110 1115 1120Gly Gln Gly Pro Ser Pro Lys Gln Leu Val Ser Leu Gly Pro Glu Lys 1125 1130 1135Ser Val Glu Gly Gln Asn Phe Leu Ser Glu Lys Asn Lys Val Val Val 1140 1145 1150Gly Lys Gly Glu Phe Thr Lys Asp Val Gly Leu Lys Glu Met Val Phe 1155 1160 1165Pro Ser Ser Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu 1170 1175 1180Asn Asn Thr His Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys1185 1190 1195 1200Lys Glu Thr Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His Thr 1205 1210 1215Val Thr Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr 1220 1225 1230Arg Gln Asn Val Glu Gly Ser Tyr Asp Gly Ala Tyr Ala Pro Val Leu 1235 1240 1245Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys His 1250 1255 1260Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu Gly Leu1265 1270 1275 1280Gly Asn Gln Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys Thr Thr Arg 1285 1290 1295Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr Gln Arg Ser Lys 1300 1305 1310Arg Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu Glu Thr Glu Leu Glu 1315 1320 1325Lys Arg Ile Ile Val Asp Asp Thr Ser Thr Gln Trp Ser Lys Asn Met 1330 1335 1340Lys His Leu Thr Pro Ser Thr Leu Thr Gln Ile Asp Tyr Asn Glu Lys1345 1350 1355 1360Glu Lys Gly Ala Ile Thr Gln Ser Pro Leu Ser Asp Cys Leu Thr Arg 1365 1370 1375Ser His Ser Ile Pro Gln Ala Asn Arg Ser Pro Leu Pro Ile Ala Lys 1380 1385 1390Val Ser Ser Phe Pro Ser Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu 1395 1400 1405Phe Gln Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys 1410 1415 1420Asp Ser Gly Val Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys1425 1430 1435 1440Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln 1445 1450 1455Arg Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr 1460 1465 1470Lys Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr 1475 1480 1485Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gln Lys Asp 1490 1495 1500Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu Asp Leu1505 1510 1515 1520Val Glu Gly Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile Lys Trp Asn 1525 1530 1535Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg Val Ala Thr Glu 1540 1545 1550Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp Pro Leu Ala Trp Asp 1555 1560 1565Asn His Tyr Gly Thr Gln Ile Pro Lys Glu Glu Trp Lys Ser Gln Glu 1570 1575 1580Lys Ser Pro Glu Lys Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser1585 1590 1595 1600Leu Asn Ala Cys Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly 1605 1610 1615Gln Asn Lys Pro Glu Ile Glu Val Thr Trp Ala Lys Gln Gly Arg Thr 1620 1625 1630Glu Arg Leu Cys Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg 1635 1640 1645Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr 1650 1655 1660Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr1665 1670 1675 1680Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg 1685 1690 1695His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser 1700 1705 1710Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro 1715 1720 1725Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr 1730 1735 1740Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly1745 1750 1755 1760Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg 1765 1770 1775Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr 1780 1785 1790Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys 1795 1800 1805Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His Met Ala 1810 1815 1820Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp1825 1830 1835 1840Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu 1845 1850 1855Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr 1860 1865 1870Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser 1875 1880 1885Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn 1890 1895 1900Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala1905 1910 1915 1920Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln 1925 1930 1935Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn 1940 1945 1950Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys 1955 1960 1965Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu 1970 1975 1980Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys1985 1990 1995 2000Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val 2005 2010 2015Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile 2020 2025 2030Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro 2035 2040 2045Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr 2050 2055 2060Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile2065 2070 2075 2080Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu 2085 2090 2095Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp 2100 2105 2110Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly 2115 2120 2125Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile 2130 2135 2140Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser2145 2150 2155 2160Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met 2165 2170 2175Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala 2180 2185 2190Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala 2195 2200 2205Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn 2210 2215 2220Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val2225 2230 2235 2240Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr 2245 2250 2255Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr 2260 2265 2270Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp 2275 2280 2285Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg 2290 2295 2300Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg2305 2310 2315 2320Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr 2325 2330

Claims

1-27. (canceled)

28. An isolated amino acid sequence: TABLE-US-00008 SEQ. ID No. 1: Ser Val Ala Lys Lys His Pro. 1 5

29. An isolated amino acid sequence: TABLE-US-00009 SEQ. ID No. 2: Asp Glu Asp Glu Asn Gln Ser. 1 5

30. An isolated amino acid sequence: TABLE-US-00010 SEQ. ID No. 3: Asp Gln Arg Gln Gly Ala Glu. 1 5

31. A peptide or non-peptide analogue of an amino acid sequence of claim 28, which is capable of inhibiting any site in the Factor VIII molecule which is susceptible to being lysed by an anti-Factor VIII allo-antibody.

32. A peptide or non-peptide analogue of an amino acid sequence of claim 29, which is capable of inhibiting any site in the Factor VIII molecule which is susceptible to being lysed by an anti-Factor VIII allo-antibody.

33. A peptide or non-peptide analogue of an amino acid sequence of claim 30, which is capable of inhibiting any site in the Factor VIII molecule which is susceptible to being lysed by an anti-Factor VIII allo-antibody.

34. A method of neutralising catalytic anti-Factor VIII allo-antibodies comprising using an anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor.

35. The method of claim 34, wherein said inhibitor comprises a protease inhibitor.

36. The method of claim 35, wherein said protease inhibitor is 4-(2-aminoethyl)benzenesulphonyl fluoride hydrochloride.

37. The method of claim 34, wherein said inhibitor inhibits cleavage of the scissile bonds: Arg³⁷²-Ser³⁷³, located between the A1 and A2 domains, Tyr.sup.1680-Asp.sup.1681, located on the N-terminus of the A3 domain, and Glu.sup.1794-Asp.sup.1795 located within the A3 domain of the Factor VIII molecule.

38. The method of claim 34, wherein said inhibitor comprises a peptide or non-peptide analogue of the isolated amino acid sequence: TABLE-US-00011 SEQ. ID No. 1: Ser Val Ala Lys Lys His Pro. 1 5

39. The method of claim 34, wherein said inhibitor comprises a peptide or non-peptide analogue of the isolated amino acid sequence: TABLE-US-00012 SEQ. ID No. 2: Asp Glu Asp Glu Asn Gln Ser. 1 5

40. The method of claim 34, wherein said inhibitor comprises a peptide or non-peptide analogue of the isolated amino acid sequence: TABLE-US-00013 SEQ. ID No. 3: Asp Gln Arg Gln Gly Ala Glu. 1 5

41. A pharmaceutical composition which comprises a pharmaceutically effective amount of a pharmaceutically active ingredient selected from the group consisting of an anti-Factor VIII allo-antibody capable of degrading Factor VIII, and a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable excipient, vehicle or carrier.

42. The pharmaceutical composition of claim 41, wherein said anti-Factor VIII allo-antibody capable of degrading Factor VIII is as obtainable from a method which comprises:i) isolating the plasma from a sample of blood taken from said mammal,ii) isolating anti-Factor VIII allo-antibodies from said plasma;iii) placing said anti-Factor VIII allo-antibodies in contact with Factor VIII for a period of time sufficient to permit any degradation of said Factor VIII by said anti-Factor VIII allo-antibodies; andiv) determining, after said period of time, whether said Factor VIII has been degraded by said anti-Factor VIII allo-antibodies.

43. A method of therapeutic treatment of a mammal suffering from a pathology resulting from abnormal level of Factor VIII in the blood thereof, wherein a therapeutically effective amount of a pharmaceutically active ingredient selected from the group consisting of at least one anti-Factor VIII allo-antibody capable of degrading Factor VIII, and a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable excipient, vehicle or carrier, is administered to said mammal.

44. The method of claim 43, wherein said pathology results from the presence of an excess of Factor VIII in the blood thereof.

45. The method of claim 44, wherein said pathology is of thrombotic nature.

46. The method of claim 45, which is a therapeutic treatment of a mammal suffering from thrombosis.

47. A pharmaceutical composition which comprises a pharmaceutically effective amount of a pharmaceutically active ingredient selected from the group consisting of a Factor VIII degradation inhibitor of claim 34, and a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable excipient, vehicle or carrier.

48. A method of therapeutic treatment of a mammal suffering from a pathology resulting from the sub-physiological level of Factor VIII in the blood thereof, wherein a therapeutically effective amount of a pharmaceutically active ingredient selected from the group consisting of at least one Factor VIII degradation inhibitor, and a pharmaceutically acceptable salt thereof, is administered to said mammal.

49. The method of claim 48, wherein said inhibitor comprises a protease inhibitor.

50. The method of claim 49, wherein said protease inhibitor is 4-(2-aminoethyl)benzenesulphonyl fluoride hydrochloride.

51. The method of claim 48, wherein said inhibitor inhibits cleavage of the scissile bonds: Arg³⁷²-Ser³⁷³, located between the A1 and A2 domains, Tyr.sup.1680-Asp.sup.1681, located on the N-terminus of the A3 domain, and Glu.sup.1794-Asp.sup.1795 located within the A3 domain of the Factor VIII molecule.

52. The method of claim 48, which comprises a peptide or non-peptide analogue of the amino acid sequence: TABLE-US-00014 Ser Val Ala Lys Lys His Pro.

53. The method of claim 48, which comprises a peptide or non-peptide analogue of the amino acid sequence: TABLE-US-00015 Asp Glu Asp Glu Asn Gln Ser.

54. The method of claim 48, which comprises a peptide or non-peptide analogue of the amino acid sequence: TABLE-US-00016 Asp Gln Arg Gln Gly Ala Glu.

55. The method of claim 48, wherein said pathology is of haemophilic nature.

56. The method of claim 55, wherein said pathology of haemophilic nature is a disease involving coagulation defects due to Factor VIII insufficiency.

57. The method of claim 55, which is a method of therapeutic treatment of a mammal suffering from haemophilia A.

58. An anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor, which comprises a peptide or non-peptide analogue of the isolated amino acid sequence: TABLE-US-00017 SEQ. ID No. 1: Ser Val Ala Lys Lys His Pro. 1 5

59. An anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor, which comprises a peptide or non-peptide analogue of the isolated amino acid sequence: TABLE-US-00018 SEQ. ID No. 2: Asp Glu Asp Glu Asn Gln Ser. 1 5

60. An anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor, which comprises a peptide or non-peptide analogue of the isolated amino acid sequence: TABLE-US-00019 SEQ. ID No. 3: Asp Gln Arg Gln Gly Ala Glu. 1 5

61. A pharmaceutical composition, which comprises a pharmaceutically effective amount of an anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor.

62. The pharmaceutical composition of claim 61, which comprises a protease inhibitor.

63. The pharmaceutical composition of claim 62, wherein said protease inhibitor is 4-(2-aminoethyl)benzenesulphonyl fluoride hydrochloride.

64. The pharmaceutical composition of claim 61, wherein said inhibitor inhibits cleavage of the scissile bonds: Arg³⁷²-Ser³⁷³, located between the A1 and A2 domains, Tyr.sup.1680-Asp.sup.1681, located on the N-terminus of the A3 domain, and Glu.sup.1794-Asp.sup.1795 located within the A3 domain of the Factor VIII molecule.

65. The pharmaceutical composition of claim 61, which comprises a peptide or non-peptide analogue of the amino acid sequence: TABLE-US-00020 Ser Val Ala Lys Lys His Pro.

66. The pharmaceutical composition of claim 61, which comprises a peptide or non-peptide analogue of the amino acid sequence: TABLE-US-00021 Asp Glu Asp Glu Asn Gln Ser.

67. The pharmaceutical composition of claim 61, which comprises a peptide or non-peptide analogue of the amino acid sequence: TABLE-US-00022 Asp Gln Arg Gln Gly Ala Glu.

68. An isolated anti-Factor VIII allo-antibody, which has a catalytic activity capable of catalysing degradation of Factor VIII.

69. An isolated anti-Factor VIII allo-antibody which is obtainable by a method of determining the presence of anti-Factor VIII allow-antibodies capable of degrading Factor VIII in a mammal, which comprises:i) isolating the plasma from a sample of blood taken from said mammal,ii) isolating anti-Factor VIII allo-antibodies from said plasma;v) placing said anti-Factor VIII allo-antibodies in contact with Factor VIII for a period of time sufficient to permit any degradation of said Factor VIII by said anti-Factor VIII allo-antibodies; andvi) determining, after said period of time, whether said Factor VIII has been degraded by said anti-Factor VIII allo-antibodies.

70. The isolated anti-Factor VIII allo-antibody of claim 68, which cleaves the following scissile bonds in the Factor VIII molecule: Arg³⁷²-Ser³⁷³, located between the A1 and A2 domains, Tyr.sup.1680-Asp.sup.1681, located on the N-terminus of the A3 domain, and Glu.sup.1794-Asp.sup.1795 located within the A3 domain of the Factor VIII molecule.

71. The isolated anti-Factor VIII allo-antibody of claim 69, which cleaves the following scissile bonds in the Factor VIII molecule: Arg³⁷²-Ser³⁷³, located between the A1 and A2 domains, Tyr.sup.1680-Asp.sup.1681, located on the N-terminus of the A3 domain, and Glu.sup.1794-Asp.sup.1795 located within the A3 domain of the Factor VIII molecule.

Images