US20020068303A1 - Antigenic polypeptide sequences of factor VIII, and fragments and/or epitopes of these sequences - Google Patents

Antigenic polypeptide sequences of factor VIII, and fragments and/or epitopes of these sequences Download PDF

Info

Publication number
US20020068303A1
US20020068303A1 US09/853,080 US85308001A US2002068303A1 US 20020068303 A1 US20020068303 A1 US 20020068303A1 US 85308001 A US85308001 A US 85308001A US 2002068303 A1 US2002068303 A1 US 2002068303A1
Authority
US
United States
Prior art keywords
epitope
ser
asp
inclusive
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US09/853,080
Other languages
English (en)
Inventor
Ruth Laub
Mario Di Giambattista
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
DEPARTEMENT CENTRAL DE FRACTIONNEMENT de la CROIX-ROUGE SCRL
Original Assignee
DEPARTEMENT CENTRAL DE FRACTIONNEMENT de la CROIX-ROUGE SCRL
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from BE9400666A external-priority patent/BE1008491A3/fr
Application filed by DEPARTEMENT CENTRAL DE FRACTIONNEMENT de la CROIX-ROUGE SCRL filed Critical DEPARTEMENT CENTRAL DE FRACTIONNEMENT de la CROIX-ROUGE SCRL
Priority to US09/853,080 priority Critical patent/US20020068303A1/en
Assigned to DEPARTEMENT CENTRAL DE FRACTIONNEMENT DE LA CROIX-ROUGE SCRL reassignment DEPARTEMENT CENTRAL DE FRACTIONNEMENT DE LA CROIX-ROUGE SCRL ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DI GIAMBATTISTA, MARIO, LAUB, RUTH
Priority to JP2002587603A priority patent/JP4500494B2/ja
Priority to AT02732243T priority patent/ATE525470T1/de
Priority to CA2446390A priority patent/CA2446390C/en
Priority to PCT/BE2002/000070 priority patent/WO2002090542A2/en
Priority to EP02732243A priority patent/EP1399556B1/en
Priority to ES02732243T priority patent/ES2372811T3/es
Publication of US20020068303A1 publication Critical patent/US20020068303A1/en
Priority to US11/267,631 priority patent/US7981865B2/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/745—Assays involving non-enzymic blood coagulation factors
    • G01N2333/755—Factors VIII, e.g. factor VIII C [AHF], factor VIII Ag [VWF]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/806—Antigenic peptides or proteins

Definitions

  • the present invention relates to the antigenic polypeptide sequences of factor VIII, to fragments and epitopes of these sequences and to the major parts of these epitopes, to the inhibitors which are directed against these sequences, its fragments, its epitopes and/or major parts of these epitopes, and to anti-inhibitors which are directed against the said inhibitors.
  • the present invention also relates to a pharmaceutical composition and to a diagnostic device comprising at least one of the above mentioned molecules.
  • FVIII is a large multi-domain protein of 2,332 amino acids made up of three structural domains, A, B and C which are arranged in the order A1:a1:A2:a2:B:a3:A3:C1:C2.
  • the A domains possess more than 40% homology and are also homologous to ceruloplasmin (for recent review, see Pratt (2000) and Saenko (1999)). 30% homology also exists between the A domains of factor V and FVIII.
  • the C domain occurs twice and is reported to be able to bind glyco-conjugates and phospholipids having a net negative charge. It exhibits homology with lectins which are able to bind to negatively charged phospholipids.
  • the platelet attachment site has been located in this region (C2 domain) (Foster et al., (1990)).
  • antigenic determinants consist of fragments 351-365 (A1 domain-heavy chain), 713-740 (A2 domain), 1670-1684 (A3 domain-light chain) (NH 2 end of the light chain) or else 2303-2332 (C2 domain-light chain) (Foster C, (1990)), fragments 701-750, 1663-1689, 330-472, 1694-1782 (EP-0 202 853), 322-740 and 2170-2322.
  • the U.S. Pat. No. 5,744,446 describes an hybrid human/animal Factor VIII having a sequence of amino acids selected from the group of the A2 domain fragments 373-540, 373-508, 445-508, 484-508, 404-508, 489-508 and 484-489, with corresponding sequences of porcine or murine Factor VIII, said hybrid being used for the treatment of Factor VIII deficiencies.
  • Self proteins or derived peptides may elicit an immune response if presented to CD4 T cells at inflammatory sites by professional antigen presenting cells.
  • FVIII domains were recognized: A3 domain was recognized more strongly and frequently and each domain forms several epitopes.
  • FVIII FVIII
  • PCC activated PCC
  • FVIIa FVIIa
  • porcine FVIII may be used to achieve haemostasis in patients with antibodies that do not substantially crossreact with porcine FVIII before or during the treatment (Lollar, 2000).
  • a potential alternative approach to inhibit the production of inhibitors is blockade of the T cell/B cell collaboration mediated by through receptor ligand binding signal events (Ewenstein et al, 2000). Preliminary clinical trials were performed using a humanized mouse monoclonal antibody to human T cell CD40 ligand (CD 154).
  • a profitable strategy for reducing the level of inhibitors has consisted in subjecting patients to an extracorporeal circulation to enable solid-phase absorption of the total IgG.
  • the immunoabsorbant could be sepharose-bound staphylococcal protein A or sepharose-bound polyclonal sheep antibodies to total human immunoglobulin (Knobf and Derfler, 1999).
  • the foreign proteins protein A, sheep anti-human Ig
  • ICH Topic Q5A, Directive 92/79/EC ICH Topic Q5A, Directive 92/79/EC
  • IVIG polyvalent intravenous immunoglobulins
  • the present invention aims to obtain antigenic polypeptide sequences of factor VIII, fragments and epitopes of these sequences, whose purpose is to improve the diagnosis and/or therapy (including prevention) of immune disorders (in particular those induced by inhibitors of FVIII and inhibitors of FVIII, especially inhibitors of the binding of the von Willebrand factor (vWf), to the FIX and/or to membrane phospholipids (PL)), and which allows a screening between non-inhibitory and inhibitory anti-FVIII allo- or auto-antibodies (allo- or auto-immunoglobulins).
  • vWf von Willebrand factor
  • PL membrane phospholipids
  • Another aim of the invention is to obtain inhibitors which exhibit an immunoaffinity with these antigenic polypeptide sequences, fragments and/or epitopes, as well as to obtain anti-inhibitors, in particular antibodies or (T)cell receptors, which are directed against the above-mentioned said inhibitors and whose purpose is to improve the diagnosis and/or therapy (or prevention) of immune disorders.
  • a further aim of the invention is to obtain said molecules at high purity, in industrial level, without contaminants (viruses, prions, . . . ) and according to the GMP practices in the field of therapy and diagnostics (ICH topic QSA, Directive 92/79/EC, etc.).
  • An antigenic polypeptide sequence which is the polypeptide sequence of factor VIII.
  • An antigenic polypeptide sequence which lacks the following fragments: alanine 322-serine 750, leucine 1655-arginine 1689, lysine 1694-proline 1782 and possibly the fragment aspartic acid 2170-tyrosine 2332.
  • An antigenic fragment of the sequence according to paragraph 1 or 2 which is selected from the group consisting of the polypeptide sequences Al, A2, A3 or C of factor VIII.
  • epitope arginine 1648 to tyrosine 1664 inclusive defined by the following sequence:
  • epitope threonine 1739 to tyrosine 1748 inclusive defined by the following sequence:
  • epitope asparagine 1777 to phenylalanine 1785 inclusive defined by the following sequence:
  • epitope glutamic acid 1794 to tyrosine 1815 inclusive defined by the following sequence:
  • epitope methionine 1823 to aspartic acid 1831 defined by the following sequence:
  • epitope glutamic acid 1885 to phenylalanine 1891 inclusive defined by the following sequence:
  • epitope glutamic acid 1885 to alanine 1901 inclusive defined by the following sequence:
  • epitope aspartic acid 1909 to arginine 1917 inclusive defined by the following sequence:
  • epitope glutamic acid 181 to leucine 192 inclusive defined by the following sequence:
  • epitope aspartic acid 203 to alanine 227 inclusive defined by the following sequence:
  • epitope aspartic acid 327 to methionine 355 inclusive defined by the following sequence:
  • epitope aspartic acid 403 to lysine 425 inclusive defined by the following sequence:
  • epitope valine 517 to arginine 527 inclusive defined by the following sequence:
  • epitope tyrosine 555 to glutamine 565 inclusive defined by the following sequence:
  • epitope histidine 693 to glycine 701 inclusive defined by the following sequence:
  • epitope serine 710 to aspartic acid 725 inclusive defined by the following sequence:
  • epitope leucine 730 to serine 741 inclusive defined by the following sequence:
  • epitope serine 817 to serine 830 inclusive defined by the following sequence:
  • epitope isoleucine 2081 to serine 2095 inclusive defined by the following sequence
  • epitope tyrosine 2105 to glycine 2121 inclusive defined by the following sequence:
  • epitope asparagine acid 2128 to asparagine acid 2138 inclusive defined by the following sequence:
  • epitope histidine 2152 to arginine 2163 inclusive defined by the following sequence:
  • epitope serine 2181 to asparagine acid 2198 inclusive defined by the following sequence:
  • epitope serine 2204 to glutamine 2222 inclusive defined by the following sequence:
  • epitope glutamine 2235 to leucine 2251 inclusive defined by the following sequence:
  • epitope glycine 2242 to leucine 2251 inclusive defined by the following sequence:
  • epitope leucine 2273 to serine 2289 inclusive defined by the following sequence:
  • epitope proline 2292 to tyrosine 2305 inclusive defined by the following sequence:
  • epitope glutamic acid 2322 to tyrosine 2332 inclusive defined by the following sequence:
  • a conformational epitope which contains at least two different epitopes according to any one of the preceding paragraphs 8, 10 and 12.
  • a complex comprising a carrier protein or a carrier peptide linked to an element which is selected from the group consisting of the fragment and/or the epitope according to any one of the paragraphs 6 to 14.
  • a pharmaceutical composition which comprises an adequate pharmaceutical carrier and at least one element selected from the group consisting of the sequence, the fragment, the epitope, the pool, the complex, the recombinant factor VIII or the inhibitor and/or the anti-inhibitor according to any one of the preceding paragraphs.
  • a diagnostic and/or purification device which comprises at least one element which is selected from the group consisting of the sequence, the fragment, the epitope, the pool, the complex, the inhibitor and/or the anti-inhibitor according to any one of the preceding paragraphs.
  • a method for a therapeutic treatment and/or prevention of an immune disorder in mammal wherein the pharmaceutical composition according to paragraph 22 is administered to the mammal patient presently or potentially having said immune disorder, in an amount effective to treat and/or prevent said immune disorder.
  • a method for a therapeutic treatment and/or prevention of an immune disorder in a mammal patient wherein a physiological fluid such as serum obtained from said mammal patient is put into the chromatography column of paragraph 25 in order to allow a binding with the inhibitors of factor VIII present in said serum with the sequence, the fragment, the epitope, the pool and/or the complex according to any of the preceding paragraphs 1 to 15, wherein the physiological liquid is eluted from said chromatography column and the physiological liquid from which the inhibitors of factor VIII have been removed is reinjected to the patient.
  • a physiological fluid such as serum obtained from said mammal patient is put into the chromatography column of paragraph 25 in order to allow a binding with the inhibitors of factor VIII present in said serum with the sequence, the fragment, the epitope, the pool and/or the complex according to any of the preceding paragraphs 1 to 15, wherein the physiological liquid is eluted from said chromatography column and the physiological liquid from which the inhibitors of factor VIII have been removed is reinjected to the patient.
  • FIG. 1 This figure depicts the hydrophilicity, flexibility and accessibility graph of the A3 sequence of Factor VIII, renumbered 1 to 371 amino acids (surface value for each amino acid).
  • FIG. 2 a represents the elution profile related to the purification of human anti-SEQ. ID NO: 32 antibodies by affinity chromatography in peptide-Sepharose column.
  • Cohn fraction II+III solution 50 mL was loaded onto the column (1 mL gel) at a flow rate of 1 mL/min. The separation of specific antibodies was performed as described in the Examples.
  • the arrow indicates the position of specific human anti-SEQ. ID NO: 32.
  • the clotting activity of FVIII ( 2 b ) was measured as described in Examples in the presence of increasing amount of anti-SEQ. ID NO: 32.
  • the of FVIII activity (FVIII activity in the presence of antibody/FVIII activity in the absence of antibody) * 100.
  • FIG. 3 This figure represents the human anti-peptide antibody immunoreactions with FVIII polypeptides after Western Blotting (panel A from left to right: human antibodies HAP1 through HAP4, specific for different FVIII epitope sequences found in the FVIII HC—see also Table 2 and panel B: human antibodies specific for the P5 peptide and the FVIII LC sequences, P7, P8, and P9—see also Table 2).
  • the RAP9 lane shows the reactivity of FVIII polypeptieds towards purified rabbit antibodies specific for the peptide sequence Arg 1797 -Tyr 1815 (see also Table 2).
  • FIG. 4 This figure represents the ELISA reactivity of the four inhibitor plasmas with different peptide sequences. Inhibitors present in four patient plasmas were analyzed by ELISA test using as coated antigens the different selected FVIII epitopes synthetic peptides, as indicated by the ordinate.
  • the present invention relates to the antigenic polypeptide sequences of factor VIII and/or fragments of these sequences, as described by Verhar et al. (1984), the disclosure of which is incorporated herein by reference in its entirety.
  • polypeptide sequence of factor VIII is understood to be the natural human or animal sequence, which may be glycosylated and which has been obtained by purification from pools of plasma, in particular cryoprecipitate, by synthesis and/or by genetic manipulation (sequence from which portions which are not involved in the mechanism of blood coagulation may have been deleted) of factor VIII.
  • the present invention relates, in particular, to the antigenic polypeptide sequences of factor VIII which lacks the fragments comprised between alanine 322-serine 750, leucine 1655-arginine 1689 and lysine 1694-proline 1782, and possibly also the fragments comprised between aspartic acid 2170 and tyrosine 2332.
  • the present invention relates, in particular, to the antigenic polypeptide sequences A1, A2, A3 and C (C1 and C2) of factor VIII.
  • a first embodiment of the invention relates to the antigenic polypeptide sequence A3 of factor VIII, and to fragments and/or epitopes of this sequence.
  • the said sequence contains the fragments glutamic acid 1649 to histidine 2031 inclusive, arginine 1652 to arginine 1917 inclusive or arginine 1803 to arginine 1917 inclusive, of the polypeptide sequence of factor VIII as published by Verhar et al. (1984) and Toole et al. (1984).
  • the fragments of the said sequence are arginine 1648 to arginine 1696 inclusive, threonine 1739 to aspartic acid 1831 inclusive or glutamic acid 1885 to arginine 1917 inclusive.
  • the fragments, epitopes and major parts thereof are preferably polypeptidic sequences made of at least 7 amino acids of the FVIII polypeptidic sequence.
  • the invention also relates to the sequence epitopes of these fragments, in particular:
  • epitope arginine 1648 to tyrosine 1664 inclusive defined by the following sequence:
  • epitope aspartic acid 1681 to arginine 1696 (P8) inclusive defined by the following sequence:
  • epitope threonine 1739 to tyrosine 1748 inclusive defined by the following sequence:
  • epitope asparagine 1777 to phenylalanine 1785 inclusive defined by the following sequence:
  • epitope glutamic acid 1794 to tyrosine 1815 inclusive defined by the following sequence:
  • epitope methionine 1823 to aspartic acid 1831 inclusive defined by the following sequence:
  • epitope glutamic acid 1885 to alanine 1901 inclusive defined by the following sequence:
  • epitope aspartic acid 1909 to arginine 1917 inclusive defined by the following sequence:
  • epitope comprised between serine 2018 and histidine 2031 inclusive, defined by the following sequence:
  • the said sequences, specific fragments and epitopes exhibit an antigenic characteristic which is illustrated by Table 1.
  • Another preferred embodiment of the invention relates to antigenic polypeptide sequence Al of factor VIII, fragments and/or epitopes of this sequence.
  • the fragments of the said sequence are alanine 108 to methionine 355 inclusive, preferably alanine 108 to alanine 227 inclusive.
  • the invention also relates to the sequence epitopes of these fragments, in particular:
  • epitope alanine 108 to valine 128 inclusive defined by the following sequence:
  • epitope glutamic acid 181 to leucine 192 inclusive defined by the following sequence:
  • epitope aspartic acid 203 to alanine 227 inclusive defined by the following sequence:
  • epitope aspartic acid 327 to methionine 355 inclusive defined by the following sequence:
  • Another preferred embodiment of the invention relates to the antigenic polypeptide sequence A2 of factor VIII, fragments and/or epitopes of this sequence.
  • the fragments of the said sequence are aspartic acid 403 to serine 840 inclusive, preferably histidine 693 to aspartic acid 725 inclusive.
  • the invention also relates to the sequence epitopes of these fragments, in particular:
  • epitope aspartic acid 403 to lysine 425 inclusive defined by the following sequence:
  • epitope histidine 693 to glycine 701 inclusive defined by the following sequence:
  • epitope serine 710 to aspartic acid 725 inclusive defined by the following sequence (P4):
  • a final preferred embodiment of the invention relates to the antigenic polypeptide sequence C of factor VIII, and fragments and/or epitopes of this sequence.
  • the fragments of the said sequence are histidine 2082 to lysine 2251 inclusive or leucine 2273 to tyrosine 2332 inclusive, preferably lysine 2085 to glycine 2121 inclusive and serine 2181 to leucine 2251 inclusive.
  • the invention also relates to the sequence epitopes of these fragments, in particular:
  • epitope isoleucine 2081 to serine 2095 inclusive defined by the following sequence:
  • epitope tyrosine 2105 to glycine 2121 inclusive defined by the following sequence:
  • epitope asparagine 2128 to asparagine 2138 inclusive defined by the following sequence:
  • epitope histidine 2152 to arginine 2163 inclusive defined by the following sequence:
  • epitope serine 2181 to asparagine 2198 inclusive defined by the following sequence:
  • epitope glycine 2242 to leucine 2251 inclusive defined by the following sequence:
  • epitope isoleucine 2262 to glutamine 2270 inclusive defined by the following sequence:
  • epitope proline 2292 to tyrosine 2305 inclusive defined by the following sequence (P15):
  • the invention also relates to the major parts of the said epitopes or the said fragments.
  • Said epitopes can be deleted from one or more terminal amino acids, preferably from one, two or three amino acids, or can be replaced by one or more amino acids that present the same characteristic of hydrophilicity, flexibility and accessibility.
  • epitopes according to the invention are comprised in major determinants of human inhibitors epitopes or several factors binding sites or binding sites of known monoclonal antibodies, especially the portion C2 that is known to be the binding site of the monoclonal antibody Mas531P or the binding site ESH8 as well as phospholipids, Factor Xa or the von Willebrand factor binding site.
  • the specific epitopes according to the invention or their major parts are preferred selected portions of said binding sites or may include a possible overlapping with said binding sites.
  • the epitopes according to the invention are more specific portions of known epitopes. Therefore, an artificial epitope could be easily obtained by synthesis and the specific above-described fragments can be deleted from non-epitopic portions such as the fragment described in a C2 fragment (amino acids phenylalanine 2196 to tryptophan 2203 inclusive and amino acids valine 2222 to phenylalanine 2234 inclusive, or the sequence leucine 2252 to threonine 2272 inclusive or the amino acids phenylalanine 2290 to threonine 2291 inclusive as well as the amino acids leucine 2306 to methionine 2321 inclusive).
  • Another aspect of the present invention is related to a modified (recombinant or transgenic) FVIII, possibly obtained by genetic engineering, and deleted from one or more of the above-identified fragments, epitopes or major parts of said epitopes and/or said fragments.
  • said FVIII still allows the binding of coagulation factor(s), but will be less immunogenic and will not induce or induce less the formation of inhibitors directed against said modified FVIII or natural FVIII.
  • said polypeptide sequences, fragments or epitopes are also independently immunogenic (that is to say they are immunogenic even without being complexed with a protein of large size such as BSA, KLH haemocyanin, etc.), and preferably exhibit an immunoaffinity within inhibitors of factor VIII, such as anti-factor VIII antibodies, and/or exhibit an immunoaffinity for the receptors of the T lymphocytes and possibly B lymphocytes.
  • Said sequences are unexpectedly characterized by substantial immunogenicity towards monoclonal and polyclonal antibodies, but are sufficiently short to be readily and advantageously obtained by synthesis.
  • the present invention also relates to the conformational epitopes which comprise at least two different fragments of said sequence, at least two sequence epitopes and/or at least two major parts of said epitopes or said different fragments according to the invention and above identified.
  • the conformational epitopes are made up of two or more different portions of a polypeptide sequence, which portions are located in proximity to each other when the protein is folded in its tertiary or quaternary structure.
  • These epitopes are capable of being “recognized” (that is to say of exhibiting an immunoaffinity), preferably simultaneously, with inhibitors of factor VIII, in particular B and T lymphocytes (by way of the major histocompatibility locus (MHC I and/or II)) and/or anti-factor VIII antibodies (Scandella et al. (2000); Reding et al. (2000)).
  • the said sequence, said fragments, said epitopes and/or the major parts of said epitopes or said fragments are complexed with a carrier protein or a carrier peptide, such as BSA, or KLH haemocyanin, as to form a complex exhibiting a more powerful immunogenicity.
  • a carrier protein or a carrier peptide such as BSA, or KLH haemocyanin
  • the present invention is also related to a pool of more than three of said fragments, epitopes or major parts of said epitopes having advantageously important antigenic and/or immunogenic properties and which may be used advantageously in a diagnostic or therapeutic method or device such as a kit or a dialysis column, etc. allowing an efficient, preferably complete, screening and characterization of the major (if not all) known inhibitors directed against factor VIII by (human) patients.
  • Another aspect of the present invention relates to an inhibitor of factor VIII which exhibits an immunoaffinity with antigenic polypeptide sequences according to the present invention, with fragments and epitopes of said sequences, with the major parts of said epitopes or said fragments and/or with the complex according to the invention.
  • An inhibitor is understood to mean any biological molecule or cell (such as a T-lymphocyte) binding to said FVIII and capable of giving rise to immune disorders (characterized by humoral immune response and/or cellular immune response against said FVIII).
  • such an inhibitor can be an anti-factor VIII monoclonal or polyclonal antibody or antibody fragment (such as the hypervariable Fab portion of the said antibody) which inactivates the said factor VIII and/or which inhibits the binding of factor VIII to the von Willebrand factor and/or to membrane phospholipids.
  • an anti-factor VIII monoclonal or polyclonal antibody or antibody fragment such as the hypervariable Fab portion of the said antibody
  • the said inhibitors are synthesized by a “chimeric” animal which comprises a human immune system, such as an hu-SCID mouse or transgenic mouse producing human antibodies or other antibodies production technologies as phage display technology or immortalized B-cells, by EPV in particular.
  • a human immune system such as an hu-SCID mouse or transgenic mouse producing human antibodies or other antibodies production technologies as phage display technology or immortalized B-cells, by EPV in particular.
  • Another aspect of the invention relates to an anti-inhibitor which is directed against the said previously described factor VIII inhibitor.
  • An anti-inhibitor which is directed against the factor VIII inhibitor is understood to mean any chemical or biological molecule, a cell and/or a cell fragment (receptor) which is capable of interfering with the said inhibitor in such a way as to ensure its inactivation or avoid or reduce its binding to the factor VIII.
  • such an anti-inhibitor is an anti-anti-factor VIII idiotype (monoclonal or polyclonal) antibody or antibody fragment, natural or obtained by genetic engineering.
  • Another aspect of the invention relates to a pharmaceutical composition which comprises an adequate pharmaceutical carrier or a diluent and an element selected from the group consisting of said antigenic polypeptide sequence of factor VIII, fragments and epitopes of this sequence or a pool thereof, an inhibitor of factor VIII which is directed against them, an anti-inhibitor which is directed against the said inhibitor, and/or a mixture of these.
  • the type and amount of adequate pharmaceutical carrier or diluent (and possibly adjuvant or excipient) present in said pharmaceutical composition may vary according to the method of administration and is possibly combined an adjuvant in order to improve therapeutical properties of the pharmaceutical composition according to the invention or to reduce its possible side effects.
  • Suitable pharmaceutical acceptable carriers used in the pharmaceutical composition according to the invention are well known by the person skilled in the art and are selected according to the methods generally applied by pharmacists and may include solid, liquid or gaseous non-toxic pharmaceutically acceptable carriers.
  • the percentage of active product/pharmaceutical acceptable carrier may vary within very large ranges only limited by the tolerance and the possible side effects on patients (including humans), and by frequency and/or mode of administration.
  • a diagnostic and/or purification device such as a diagnostic kit, an affinity filter, or a chromatography column which comprises an element which is selected from the group consisting of these antigenic polypeptide sequences, fragments and epitopes and/or major parts of said epitopes or said fragments, the complex according to the invention or a pool thereof, an inhibitor which is directed against them, an anti-inhibitor which is directed against said inhibitor, and/or a mixture of these.
  • said device comprises a pool of said epitopes which allow a screening of patients and may detect the most important inhibitors present in said patients and which allow a positive test with enough specificity and sensibility.
  • the purification device can therefore consist of a chromatography column which comprises these sequences of factor VIII, fragments and epitopes and/or major parts of said fragments or epitopes, attached to the solid phase of the chromatography column.
  • a physiological liquid (such as serum), which is derived from a patient and which comprises inhibitors of factor VIII pass through this chromatography column, with said inhibitors (for example antibodies) becoming attached specifically to said factor VIII sequences, fragments, epitopes or said major parts or a pool thereof. Following elution, it is possible to collect said inhibitors by causing them to react with anti-inhibitors (anti-anti-factor VIII idiotype antibodies).
  • the present invention is also related to a method of treatment (ex vivo treatment) of a patient suffering from a pathology induced by inhibitors to the factor VIII which comprises the steps of extracting said physiological liquid (blood or serum) from the patient, obtaining its reaction upon a solid support binding the factor VIII fragments, epitopes or a pool thereof according to the invention and reinjecting said physiological liquid to the patient after the removing of the inhibitors having fixed said factor VIII fragments, epitopes, majors parts or a pool thereof.
  • a method of treatment ex vivo treatment of a patient suffering from a pathology induced by inhibitors to the factor VIII which comprises the steps of extracting said physiological liquid (blood or serum) from the patient, obtaining its reaction upon a solid support binding the factor VIII fragments, epitopes or a pool thereof according to the invention and reinjecting said physiological liquid to the patient after the removing of the inhibitors having fixed said factor VIII fragments, epitopes, majors parts or a pool thereof.
  • a final aspect of the invention relates to the use of the pharmaceutical composition according to the invention for preparing a medicament used for preventing and/or treating immune disorders, in particular those induced by inhibitors of factor VIII, inhibitors of the binding of factor VIII to the factor IX and/or the factor X and/or the von Willebrand factor (vWF) and/or inhibitors of the binding of factor VIII to membrane phospholipids.
  • immune disorders in particular those induced by inhibitors of factor VIII, inhibitors of the binding of factor VIII to the factor IX and/or the factor X and/or the von Willebrand factor (vWF) and/or inhibitors of the binding of factor VIII to membrane phospholipids.
  • vWF von Willebrand factor
  • Reagents MAS530p (Harlan-Seralab, Indianapolis, Ind.) is a mouse monoclonal antibody specific for the 44-kDa A2 domain of the factor VIII heavy chain. Biotin-labeled rabbit IgG anti-mouse IgG was purchased from Dakopatts (Copenhagen, Denmark).
  • Biotin-labeled goat IgG anti-human IgG and biotin-labeled mouse IgG anti-rabbit IgG were obtained from Sigma Chemicals (St Louis, Mich.), purified a-thrombin (3000 IU/mg), streptavidin-peroxidase conjugate, ovalbumin (OVA), bovine serum albumin (BSA), keyhole limpet haemocyanin (KLH), and o-phenylenediamine (OPD) were purchased from Sigma Chemicals (St. Louis, Mich.). Casein was obtained from Merck (Darmstadt, Germany). 4-chloro-1-naphtol and biotinylated molecular weight markers were obtained from Bio-Rad Laboratories (Hercules, Calif.). Freund's adjuvant was from Difco (Detroit, Mich.).
  • Plasma FVIII was a solvent/detergent-treated FVIII concentrate (100 IU/mg protein) purified by ion exchange chromatography (FVIII Conc. SD, CAF-DCF-Red Cross, Brussels, Belgium). Albumin-free recombinant FVIII (rFVIII) was obtained from Hyland (Glendale, Calif.).
  • Plasma fraction immunoglobulins Cohn Fraction II+III was obtained from large plasma pool from 4,800 unpaid donors, after precipitation in the presence of increasing ethanol concentration. This fraction contains all Ig classes and subclasses. IgG composition was determined by nephelometry. The relative percentage of each subclass was 63,7; 30,1; 3,4 and 2,8 for IgG1, IgG2, IgG3 and IgG4 respectively (average values for 3 different batches of FII+III).
  • Factor VIII activity was determined in a one-stage clotting assay adapted for use on the Coagulometer KC4A (Sigma Diagnostics). The assay uses severe hemophilia A plasma (Organon Teknika, Cambridge, UK) and APPT reagent from Instrumentation Laboratory (Warrington, UK). Potencies were calculated relative to the 5 th International Standard FVIII concentrate 88/640 (5.4 IU/ml) (NIBSC, Potters Bar, UK). FVIII-inhibitory activity was measured in purified rabbit and human IgG preparations according to the modified Bethesda assay. Briefly, affinity-purified IgGs were serially diluted and incubated for 1 h in the presence of FVIII concentrate 88/640 (1 IU/ml) at 37° C. The residual FVIII activity was measured as described above.
  • samples were dialyzed 3 times against 5 volumes of TE buffer (20 mM Tris-HCl pH 7.2, 150 mM NaCl and 0.02% NaN 3 ) and loaded onto the column at a flow rate of 1 ml/min.
  • the column was sequentially washed at 2 ml/min with 50 ml TE buffer and 30 ml TE containing 1 M NaCl.
  • the material was eluted (1 ml/min) with 5 ml of 0.1 M citric acid pH 2.5 and directly recovered in 5 ml of IM Tris-HCl, pH 9.0.
  • Samples were finally dialyzed versus 10 volumes of equilibration buffer and concentrated on Centriprep-30 (Amicon, Beverly, Mass.). Ig recovery was determined by the Bio-Rad protein assay.
  • Results are summarized in Table 1 which concerns the characterization of rabbit anti-FVIII-peptide antisera and recovered affinity-purified of immunoglobulins.
  • Sixteen synthetic peptides (from 10 to 20 amino acids) were selected in the A, B, Cl and C2 domains. After conjugation with ovalbumin, the OVA-peptide conjugates were injected into rabbits and FVIII anti-peptide antisera RAP1 to RAP16 were studied. More precisely, two rabbits were immunized with each FVIII-peptide-ovalbumin preparation.
  • RAP1 to RAP16 were prepared and assayed in an ELISA (column c, Table 1) using rFVIII or FVIII-peptide-KLH as the antigen.
  • ELISA titer is expressed as the negative log of the reciprocal of the serum dilution giving 50% binding.
  • the immunoglobulins were then purified by chromatography on peptide-bound Sepharose.
  • the FVIII domain recognized by the anti-FVIII peptide Ig after immunoblotting is shown in (column d, Table 1) and Ig protein recoveries (column e, Table 1) were measured using immunoglobulins as the standard.
  • the inhibitory activity, expressed in BU/mg protein, was determined in a FVIII neutralizing activity assay (column f, Table 1).
  • FVIII anti-peptide antisera The reactivity of FVIII anti-peptide antisera was measured by an ELISA using, as antigen, either the different corresponding FVIII-peptide coupled to KLH protein or purified rFVIII.
  • the binding reaction of each anti-FVIII-peptide antiserum was specific both for the FVIII peptide used to elicit the immune response in rabbit and for rFVIII (see Table 1).
  • the specific rabbit IgG were purified by affinity chromatography on peptide-Sepharose as described under Methods. When FVIII-neutralizing activity was measured in a one-stage clotting assay, significant inhibition was found with two rabbit IgG purified preparations: RAP2, corresponding to IgG specific for SEQ ID No. 14 and RAP7 specific for SEQ ID NO.:1.
  • rFVIII and the rFVIII fragments obtained after treatment with thrombin were resolved by SDS-PAGE and analyzed by western blotting with different preparations of rabbit IgGs (RAP1 to RAP17 Igs).
  • Antisera RAP1 and RAP2 reacted with the 50-kDa A1-domain fragment; RAP3 and RAP4 bound to the 44-kDa fragment (domain A2); RAP5 (specific for the B domain) bound to the high-molecular-weight FVIII heavy chain (about 200-kDa).
  • RAP7, RAP8, and RAP9 reacted with the 80-kDa light-chain doublet.
  • RAP9 and RAP12 to RAP17 antibodies also detected the 72-kDa FVIII light-chain fragment.
  • each reactive antiserum showed a strong reaction with the corresponding FVIII fragment containing the selected linear epitope. No reaction was detectable in the gels between RAP6 or RAP10 and the HC or LC FVIII fragments.
  • Table 2 concerns the characterization of human anti-FVIII antibodies from Cohn fraction II+III of healthy individuals.
  • FIG. 2 shows the chromatographic profile obtained with SEQ ID 32, a sequence found in C2 domain. Table 2 summarizes the results obtained with 17 epitopic sequences selected in each FVIII domain (Al, A2, A3, B, Cl and C2).
  • IgG isotype distribution in the human purified antibody preparations was found to be quite heterogeneous. Interestingly, 40 to 79% of the recovered IgGs belonged to the IgG2 subclass. In most preparations, IgG4 appeared to be over-represented (up to 25%).
  • FIG. 3 shows the immunoreaction of high-molecular-weight FVIII ( ⁇ 92-kDa) with four human antibody preparations, purified on Sepharose coupled to FVIII peptide SEQ ID NO.: 11 (Ser 109 -Lys 127 ), SEQ ID NO.: 14 (Cys 329 -Asp 348 ), SEQ ID NO.: 15 (Tyr 407 -Lys 425 ) or SEQ ID NO.: 19 (Cys 711 -Asp 725 ).
  • the 50-kDa FVIII fragment (domain A1) was recognized by human antibodies purified on Ser 109 -Lys 127 or Cys 329 -Asp 348 -Sepharose and the 44-kDa FVIII fragment (A2) by immunoglobulins purified on Tyr 407 -Lys 425 and Cys 711 -Asp 725 -Sepharose.
  • the lack of reactivity of the anti-(Ser 817 -Ser 830 ) immunoglobulin preparation (HAP5) with the FVIII fragments confirms that this epitope is located in the amino-terminal end of domain B (FIG. 3).
  • the selected peptides were used in ELISA experiment to determine the anti-FVIII antibody specificity's present in hemophilia A plasmas.
  • the peptides were coated on microplate (25 ⁇ g/ml in PBS buffer during 16 h at 4° C.).
  • a ⁇ fraction (1/10) ⁇ to ⁇ fraction (1/1000) ⁇ dilution of plasma patient in Tris-casein buffer was reacted with the coated peptide for 2 h at 37° C.
  • the bound human IgG was measured as described in Methods.
  • Control samples were plasma pools of healthy donors.
  • FIG. 4 shows the results obtained with the plasma of 4 hemophilia A patients.
  • the optical densities are corrected average values (OD patient-OD normal plasma pool) of two independent experiments.
  • Epitope Arg 1652 -Tyr 1664 and the adjacent major binding site vWF are located in the acidic light-chain peptide a3. As shown by western blotting, a3 is released from the A3 domain after thrombin treatment, preventing further binding of anti-(Arg 1652 -Tyr 1664 ) Ig to activated FVIII. Similar results have been reported by Shima et al (1991), who described the FVIII sequence Asp 1663 -Ser 1669 as a binding site of rabbit polyclonal antibodies neutralizing FVIII activity.
  • Epitope Cys 329 -Asp 348 overlapped the acidic Asp 348 -Lys 362 sequence (in al) described as adjacent to the activated protein C (Arg 336 ) and thrombin (Arg 372 ) cleavage sites. It is the target of human hemophilic inhibitors.
  • Anti-(Asp 348 -Lys 362 ) antibodies may interfere with proteolysis or with the FX interaction site (Met 337 -Arg 372 ) (Saenko et al., 1999 and Scandella et al., 2000).
  • FVIII-neutralizing activity was measured in all 13 Ig preparations. Seven human Ig preparations displayed inhibition of procoagulant activity, these being specific for amino-acid residues Cys 711 -Asp 725 , Tyr 1681 -Arg 1696 and Arg 1797 -Tyr 1815 respectively.
  • the Cys 711 -Asp 725 sequence contains sulfated tyrosines at Tyr 718 , Tyr 719 , and Tyr 723 , and overlaps with the FVIII HC region Lys 713 -Arg 740 described as promoting both activation and HC proteolysis.
  • the additional sulfated groups may be required for proper interaction with thrombin or another component as in the FX-activating complex.
  • Peptide P8 (Tyr 1681 -Arg 1696 ) (FVIII LC) includes the sequence Glu 1684 -Arg 1689 already described by Shima et al, 1991. It contains the thrombin activation site Arg 1689 -Ser 1690 .
  • P4 (Cys 711 -Asp 725 ) is also included in the Asp 712 -Ala 736 sequence detected by analysis of the patient antibody repertoire by gene phage display technology. It is proposed as a possible additional inhibitor in patients (van den Brink et al, 2000).
  • Peptide P9 (Arg 1797 -Tyr 1815 ) contains the FXa binding site (see below).
  • FVIII epitope sequences help to determine the contribution of patient polyclonal anti-FVIII Igs to overall inhibitory and regulatory activity. They could also be used to monitor the usual switch in anti-FVIII specificity in a patient during treatment. Said characterization of FVIII epitopes and a model of their locations on the folded molecule improves the treatment of inhibitors in both hemophilic and non-hemophilic patients (detection, follow-up, therapeutic use of FVIII epitope peptides . . . ).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Urology & Nephrology (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Biophysics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biomedical Technology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Rheumatology (AREA)
  • Epidemiology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Mycology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Diabetes (AREA)
  • Peptides Or Proteins (AREA)
US09/853,080 1994-07-14 2001-05-09 Antigenic polypeptide sequences of factor VIII, and fragments and/or epitopes of these sequences Abandoned US20020068303A1 (en)

Priority Applications (8)

Application Number Priority Date Filing Date Title
US09/853,080 US20020068303A1 (en) 1994-07-14 2001-05-09 Antigenic polypeptide sequences of factor VIII, and fragments and/or epitopes of these sequences
ES02732243T ES2372811T3 (es) 2001-05-09 2002-05-06 Epítopos antigénicos del factor viii, inhibidores dirigidos contra dichos epítopos y uso de los mismos.
EP02732243A EP1399556B1 (en) 2001-05-09 2002-05-06 Antigenic epitopes of factor viii, inhibitors directed against said epitopes and use thereof
CA2446390A CA2446390C (en) 2001-05-09 2002-05-06 Antigenic epitopes of factor viii, inhibitors directed against said epitopes and use thereof
AT02732243T ATE525470T1 (de) 2001-05-09 2002-05-06 Antigene epitope des faktors viii, inhibitoren dagegen, und deren verwendungen
JP2002587603A JP4500494B2 (ja) 2001-05-09 2002-05-06 第ｖｉｉｉ因子の抗原エピトープ、当該エピトープに対して向けられたインヒビターおよびそれらの使用
PCT/BE2002/000070 WO2002090542A2 (en) 2001-05-09 2002-05-06 Antigenic epitopes of factor viii, inhibitors directed against said epitopes and use thereof
US11/267,631 US7981865B2 (en) 1994-07-14 2005-11-03 Antigenic fragments of human factor VIII polypeptides

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
BE9400666A BE1008491A3 (fr) 1994-07-14 1994-07-14 Sequence polypeptidique antigenique du facteur viii, fragments et/ou epitopes de celle-ci.
BE09400666 1994-07-14
US08/765,837 US6866848B2 (en) 1994-07-14 1995-07-14 Antigenic polypetide sequence of factor VIII, fragments and/or epitopes there of
US09/853,080 US20020068303A1 (en) 1994-07-14 2001-05-09 Antigenic polypeptide sequences of factor VIII, and fragments and/or epitopes of these sequences

Related Parent Applications (3)

Application Number Title Priority Date Filing Date
US08/765,837 Continuation-In-Part US6866848B2 (en) 1994-07-14 1995-07-14 Antigenic polypetide sequence of factor VIII, fragments and/or epitopes there of
US08765837 Continuation-In-Part 1995-07-14
PCT/BE1995/000068 Continuation-In-Part WO1996002572A2 (fr) 1994-07-14 1995-07-14 Sequence polypeptidique antigenique du facteur viii, fragments et/ou epitopes de celle-ci

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US11/267,631 Continuation US7981865B2 (en) 1994-07-14 2005-11-03 Antigenic fragments of human factor VIII polypeptides

Publications (1)

Publication Number Publication Date
US20020068303A1 true US20020068303A1 (en) 2002-06-06

Family

ID=25314986

Family Applications (2)

Application Number Title Priority Date Filing Date
US09/853,080 Abandoned US20020068303A1 (en) 1994-07-14 2001-05-09 Antigenic polypeptide sequences of factor VIII, and fragments and/or epitopes of these sequences
US11/267,631 Expired - Fee Related US7981865B2 (en) 1994-07-14 2005-11-03 Antigenic fragments of human factor VIII polypeptides

Family Applications After (1)

Application Number Title Priority Date Filing Date
US11/267,631 Expired - Fee Related US7981865B2 (en) 1994-07-14 2005-11-03 Antigenic fragments of human factor VIII polypeptides

Country Status (7)

Country Link
US (2) US20020068303A1 (ja)
EP (1) EP1399556B1 (ja)
JP (1) JP4500494B2 (ja)
AT (1) ATE525470T1 (ja)
CA (1) CA2446390C (ja)
ES (1) ES2372811T3 (ja)
WO (1) WO2002090542A2 (ja)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1495052B9 (en) * 2002-04-18 2009-12-16 MERCK PATENT GmbH Modified factor viii
GB0713187D0 (en) * 2007-07-06 2007-08-15 Cambridge Entpr biomolecule binding ligands
GB0801513D0 (en) * 2008-01-28 2008-03-05 Circassia Ltd Peptides from factor VIII

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4965199A (en) * 1984-04-20 1990-10-23 Genentech, Inc. Preparation of functional human factor VIII in mammalian cells using methotrexate based selection

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4649132A (en) 1983-03-31 1987-03-10 Scripps Clinic And Research Foundation Treatment of Factor VIII inhibitors
KR910006424B1 (ko) 1985-08-21 1991-08-24 인코텍스 비.브이 편성브리프(brief) 제조방법
BE1008491A3 (fr) * 1994-07-14 1996-05-07 Croix Rouge De Belgique Depart Sequence polypeptidique antigenique du facteur viii, fragments et/ou epitopes de celle-ci.
EP0965597A4 (en) * 1996-12-27 2003-01-08 Mochida Pharm Co Ltd CELL MEMBRANE EFFECTIVE DRUGS
EP1051432B1 (en) * 1998-12-08 2007-01-24 Biovation Limited Method for reducing immunogenicity of proteins
US20040096456A1 (en) * 2000-12-01 2004-05-20 Conti-Fine Bianca M Method to treat hemophilia

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4965199A (en) * 1984-04-20 1990-10-23 Genentech, Inc. Preparation of functional human factor VIII in mammalian cells using methotrexate based selection

Also Published As

Publication number Publication date
WO2002090542A8 (en) 2004-02-12
US20060051367A1 (en) 2006-03-09
WO2002090542A2 (en) 2002-11-14
EP1399556A2 (en) 2004-03-24
ES2372811T3 (es) 2012-01-26
CA2446390C (en) 2012-01-17
ATE525470T1 (de) 2011-10-15
CA2446390A1 (en) 2002-11-14
JP4500494B2 (ja) 2010-07-14
US7981865B2 (en) 2011-07-19
JP2004535393A (ja) 2004-11-25
EP1399556B1 (en) 2011-09-21
WO2002090542A3 (en) 2003-12-24

Similar Documents

Publication Publication Date Title
Gilles et al. Anti-factor VIII antibodies of hemophiliac patients are frequently directed towards nonfunctional determinants and do not exhibit isotypic restriction
Gilles et al. Healthy subjects produce both anti-factor VIII and specific anti-idiotypic antibodies.
Gilles et al. Neutralizing antiidiotypic antibodies to factor VIII inhibitors after desensitization in patients with hemophilia A.
Nogami et al. A novel mechanism of factor VIII protection by von Willebrand factor from activated protein C–catalyzed inactivation
AU626255B2 (en) Factor viii binding domain of von willebrand factor
JP2009528285A (ja) ファクターｖｉｉｉのｃ１ドメインに対する阻害抗体の阻害活性を中性化する抗イデオタイプ抗体
JPH0638790A (ja) ファクターｖｉｉｉ：ｃ凝固因子ポリペプチド類に特異的なモノクローナル抗体
JP2006316062A (ja) 因子ｖｉｉｉの抗原ポリペプチド配列、その断片および／またはエピトープ
ES2377128T3 (es) Anticuerpo antiidiotípico que neutraliza la actividad inhibidora de un anticuerpo inhibidor del factor VIII
Gilles et al. Some factor VIII (FVIII) inhibitors recognise a FVIII epitope (s) that is present only on FVIII-vWF complexes
US7981865B2 (en) Antigenic fragments of human factor VIII polypeptides
EP0319315A2 (en) The von Willebrand factor binding domain of factor VIII
Mühle et al. Epitope mapping of polyclonal clotting factor VIII-inhibitory antibodies using phage display
Shima et al. Localization of the binding site for a factor VIII activity neutralizing antibody to amino acid residues Asp1663-Ser1669.
US7582296B2 (en) Anti-idiotypic antibodies against factor VIII inhibitor and uses thereof
Shima Characterization of factor VIII inhibitors
Gharagozlou et al. Epitope specificity of anti-factor VIII antibodies from inhibitor positive acquired and congenital haemophilia A patients using synthetic peptides spanning A and C domains
Di Giambattista et al. Mapping of natural anti-factor VIII antibodies in plasma pools from healthy donors: use of rationally designed synthetic peptides
Di Giambattista et al. In silico prediction of FVIII epitopes recognised by natural autoantibodies in polyvalent immunoglobulin concentrates
Saenko et al. Molecular modifications in factor VIII concentrates produced from different plasma pools
Furlong et al. Monoclonal antibodies to factor VIII: their application in immunoblotting for the visualization of factor VIII in therapeutic concentrates and plasma
Gilles et al. Restricted epitope specificity of anti-FVIII antibodies that appeared during a recent outbreak of inhibitors
MÜHLE et al. and H. SCHNEIDER
Mühle et al. Characterization of Polyclonal Factor VIII-Inhibitory Antibodies

Legal Events

Date Code Title Description
AS Assignment

Owner name: DEPARTEMENT CENTRAL DE FRACTIONNEMENT DE LA CROIX-

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:LAUB, RUTH;DI GIAMBATTISTA, MARIO;REEL/FRAME:012200/0049

Effective date: 20010615

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION