Classifications

• • 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
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9

Definitions

• PEPTIDE EPITOPES RECOGNIZED BY ANTIFILAGGRIN AUTOANTIBODIES PRESENT IN THE SERUM OF PATIENTS WITH RHUMATOID POLYARTHRITIS.
• the present invention relates to new preparations of antigens specifically recognized by auto-antibodies specific for rheumatoid arthritis.
• RA Rheumatoid arthritis
• RA Rheumatoid arthritis
• serum of affected patients contains autoantibodies, some of which are specific, and can constitute a marker of this disease, allowing its diagnosis even at early stages.
• Research has therefore been carried out with a view to identifying antigens recognized by these antibodies, in order to obtain purified preparations which can be used in conventional techniques of immunological diagnosis.
• the inventors obtained, from human and murine squamous epithelia, preparations of antigens related to filaggrin and to profilaggrin, recognized specifically by the antibodies present in the serum of patients suffering from rheumatoid arthritis, and showed that the "antibodies antikeratins "were in fact anti-filaggrin autoantibodies (hereinafter referred to as" AAF ").
• Application EP 0 511 116 describes these antigenic preparations, and their use for the diagnosis of rheumatoid arthritis.
• Filaggrins are a family of proteins which has been identified in various species, notably in humans, rats, mice, guinea pigs, in the case of squamous keratinizing epithelia [for a review on filaggrins, cf. DALE et al. [The Keratinocyte Handbook, Cambridge University Press, pp 323-350, (1994)]. They derive from the dephosphorylation and proteolysis of a precursor, profilaggrin, which consists essentially of repeating domains of filaggrin separated by peptide segments between domains.
• the gene coding for profilaggrin consists of repeated subunits, each of which codes for a filaggrin molecule, separated by portions coding for the interdomain peptide segments. All the repeating units coding for each of the human filaggrins have the same length (972 base pairs in humans); however, in humans, there are significant variations (10-15%) in sequence from one subunit to another. While most are conservative, some of these variations induce changes in amino acids and in some cases changes in the electrical charge of the protein. Thus human filaggrins form, independently of post-transcriptional modifications, a heterogeneous population of molecules of similar size but of different sequences and charges (pHi equal to 8.3 ⁇ 1.1) [GAN et al., Bioche. 29, p. 9432-9440 (1990)].
• Profilaggrin is a high molecular weight protein (approximately 400,000 in humans) soluble in the presence of high concentrations of salts or urea. It has a high content of basic amino acids (arginine and histidine), as well as glycine, serine and glutamic acid. It is poor in non-polar amino acids and contains neither methionine, cysteine, nor tryptophan. It is strongly phosphorylated on Serine residues, which gives it an isoelectric point close to neutrality.
• Filaggrins resulting from dephosphorylation and from cleavage of profilaggrin are basic proteins whose amino acid content is similar to that of profilaggrins. They participate in the organization of the keratin filaments, and undergo a progressive maturation during which the arginine residues, basic, are converted into citrulline residues, neutral, under the action of peptidylarginine deiminase [HARDING CR and SCOTT IR, J Mol. Biol. 170, p. 651-673 (1983)]. This leads to a reduction in their affinity for the keratins from which they are detached; they are then completely degraded under the action of various proteases.
• the properties of filaggrins and profilaggrins have been particularly well studied in rats, mice and humans.
• the size of profilaggrin varies, depending on the species, from 300 to 400 kD and that of filaggrins from 27 to 64 kD.
• Filaggrins also exhibit great inter- and intra-specific variability in their sequence. However, this variability does not affect their functional properties, their overall amino acid composition, and their biochemical properties. Likewise, the tissue localizations of profilaggrin and filaggrins are identical in the different mammals studied.
• the inventors sought to reproduce in vi tro, from recombinant filaggrin, these post-translational modifications, in order to determine which were liable to influence the antigenicity of filaggrin.
• This work resulted in the obtaining of artificial antigens recognized specifically by the FAAs present in the serum of RA patients, and constituted by recombinant or synthetic polypeptides, derived from the sequence of filaggrin, or portions of that -ci, by substitution of at least one arginine residue by a citrulline residue.
• filamentaggrin unit means a polypeptide whose sequence is that of the translation product of any of the subunits coding for a filaggrin domain of the gene for human profilaggrin or of another species, or else is a consensus sequence, theoretical sequence obtained from the sequences of the filaggrin domains.
• these epitopes comprise a tripeptide motif centered on a citrulline residue, specifically present on citrulline peptides derived from the sequences SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, and absent from the sequence SEQ ID NO: 4.
• the present invention relates to a peptide constituting an epitope recognized by anti-filaggrin autoantibodies present in the serum of patients suffering from rheumatoid arthritis, characterized in that said epitope comprises a tripeptide motif centered on a citrulline residue, specifically present on at least one of the citrullinated peptides derived from the sequences SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6.
• said peptide comprises at least one pentapeptide motif centered on a citrulline residue, present on at least one of the citrulline peptides derived from the sequences SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6.
• said peptide comprises the tripeptide motif Ser-Cit-His, in which Cit represents a citrulline residue.
• the peptides in accordance with the invention allow the preparation of artificial antigens recognized specifically by the anti-filaggrin autoantibodies present in the serum of patients suffering from rheumatoid arthritis. These artificial antigens are also part of the object of the present invention.
• Artificial antigens according to the invention comprise at least one peptide epitope centered on a citrulline residue, as defined above. They are for example constituted by peptides of at least 5 amino acids, preferably at least 10 amino acids, and advantageously at least 14 amino acids.
• They may be peptides consisting of at least one fragment of at least one of the citrullinated peptides derived from the sequences SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, or containing at least one such fragment.
• These peptides can comprise several citrullinated epitopes specifically recognized by AAF, of identical or different sequences.
• peptide as used in the present Application means in particular protein or protein fragment, oligopeptide, extract, separated or substantially isolated or synthesized, in particular those obtained by chemical synthesis or by expression in a recombinant organism; any peptide in the sequence of which one or more amino acids of the L series are replaced by an amino acid of the D series, and vice versa; any peptide of which at least one of the CO-NH bonds, and advantageously all of the CO-NH bonds of the peptide chain is (are) replaced by one (of) NH-CO bonds; any peptide of which at least one of the CO-NH bonds and advantageously all of the CO-NH bonds is or are replaced by one or more NH-CO bonds, the chirality of each aminoacyl residue, whether either involved or not in one or more CO-NH bonds mentioned above, being either conserved or reversed with respect to the aminoacyl residues constituting a reference peptide, these compounds being also designated immunoretroids, a mimotope, etc.
• Antigens according to the invention can for example be obtained by the action of PAD (peptidyl arginine deiminase) on proteins or peptides natural, recombinant, or synthetic, comprising arginine residues, and in particular comprising at least one arginine residue constituting the center of a tripeptide motif identical to those present in the sequences SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6; they can also be obtained by peptide synthesis, by directly incorporating one or more citrulline residues, and preferably one or more epitopes comprising a citrulline residue, as defined above, in the synthesized peptide.
• PAD peptidyl arginine deiminase
• the present invention also relates to the use of the antigens in accordance with the invention, as defined above, for the in vi tro diagnosis of RA.
• the present invention encompasses in particular antigenic compositions for diagnosing the presence of RA-specific autoantibodies in a biological sample, which compositions are characterized in that they contain at least one antigen according to the invention, optionally labeled and / or conjugated with a carrier molecule.
• the present invention also relates to a method for detecting RA specific class G autoantibodies in a biological sample, which method is characterized in that it comprises:
• This detection method can be implemented using a kit comprising at least one antigen according to the invention, as well as buffers and reagents suitable for constituting a medium. reaction allowing the formation of an antigen / antibody complex, and / or means of detection of said antigen / antibody complex.
• Said kit may also include, where appropriate, reference samples, such as one or more negative serum (s) and one or more positive serum (s).
• reference samples such as one or more negative serum (s) and one or more positive serum (s).
• EXAMPLE 1 IN VITRO DEIMINATION OF RECOMBINANT FILAGGRIN BY PEPTIDYL ARGININE DEIMINASE (P.A.D.).
• Recombinant filaggrin is produced according to the following protocol:
• a DNA fragment coding for a filaggrin unit is amplified by PCR, from human genomic DNA (RAJI cells: ATCC CCL86) using the following 2 primers: 5 'primer:
• the amplification product is cloned into the SmaI site of the vector pUC19.
• the recombinant clones are selected, checking the presence of a 972 bp insert obtained after digestion with SacI and Xbal.
• This insert is then subcloned into pUC19.
• the insert resulting from this subcloning is then transferred into the vector pGEX (sold by the company PHARMACIA), between the EcoRI and HindIII sites.
• the expression vector thus obtained expresses, in E. coli, filaggrin in fusion with glutathione-S-transferase (GST), under the control of the prokaryotic promoter Tac.
• the mixture of the 9 fragments is subjected to in vitro elimination by peptidyl arginine deiminase.
• a preparation of rabbit muscle peptidyl arginine deiminase (681 U / ml) sold by TAKARA BIOMED EUROPE is used, according to the protocol recommended by the manufacturer.
• the operating conditions are as follows:
• BSA bovine serum albumin
• PHAST ® -SDS 12.5% gel, PHARMACIA electrophoresis gel
• electrophoresis is carried out with the PHAST-SYSTEM ® device (PHARMACIA), under the conditions recommended by the manufacturer.
• nitrocellulose either with a pool of 5 sera from RA patients, diluted to 1/2000, or with the anti-filaggrin monoclonal antibody AHF2 [SIMON et al. J. Invest. Dermatol. 105, 432, (1995)] at the concentration of 0.2 ⁇ g / ml.
• the antigen / antibody complex is revealed using a secondary antibody coupled to peroxidase, by the ECL technique.
• the peptide of 49 amino acids S-47-S of sequence (code 1 letter): NH 2 -STGHSGSQHSHTTTQGRSDASRGSSGSRSTSRETRDQEQSGDGSRHSGS-COOH corresponding to amino acids 71 to 119 of the sequence of a human filaggrin unit, and comprising 6 arginine residues, and the peptide of 37 amino acids S-35-R of sequence (code 1 letter):
• the peptides S-47-S and S-35-R are represented in the sequence list in the appendix under the respective numbers SEQ ID NO: 3 and SEQ ID NO: 4.
• Peptides E-12-H and E-12-0 were determined by reference to the nucleotide sequences of the human profilaggrin gene described by GAN S.Q et al. [Biochemistry, 29: 9432-9440, (1990)].
• NH 2 -EQSADSSRHSGSGH-COOH comprises 1 arginine residue, and the peptide of 14 amino acids E-12-D of sequence (code 1 letter):
• NH 2 -ESSRDGSRHPRSHD-COOH contains 3 arginine residues.
• the peptides E-12-H and E-12-D are represented in the sequence list in the appendix under the respective numbers SEQ ID NO: 5 and SEQ ID NO: 6.
• citrullinated peptides E-12-H and E-12-D were synthesized directly by incorporation of a citrulline in replacement of an arginine.
• the wells of NUNC MAXISORP microtiter plates were coated respectively with the non-citrullinated and citrullinated peptides E-12-D and E-12-H, diluted to a concentration of 5 ⁇ g / ml in PBS buffer (pH : 7.4) and incubated overnight at 4 ° C (final volume: 100 ⁇ g / well).
• the wells were saturated for 30 minutes at 37 ° C. in PBS-T een 20, 0.05% gelatin 2.5%, 200 ⁇ l / well.
• the negative control serum normal serum was diluted 1/120.
• anti-filaggrin antibodies were diluted in PBS-Tween 20, 0.05% - 0.5% gelatin (PBS TG) so that the final concentrations of anti-filaggrin autoantibody are those indicated in Table I attached.
• Negative control serum, PR sera and anti-filaggrin antibodies were added (final volume: 100 ⁇ l / well) and incubated for 1 hour at 37 ° C and overnight at 4 ° C.
• Goat anti-heavy chain gamma antibodies of human immunoglobulins labeled with peroxidase (marketed by the company SOUTHERN BIOTECHNOLOGIES) were added to each well (dilution in PBSTG: 1/2000, final volume: 100 ⁇ l / well) and subjected incubation for 1 hour at 37 ° C. The revelation was carried out by adding orthophenylenediamine (2 mg / ml, for 10 minutes).

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• 1997
  • 1997-12-30 FR FR9716673A patent/FR2773157B1/fr not_active Expired - Fee Related
• 1998
  • 1998-12-29 AU AU19717/99A patent/AU1971799A/en not_active Abandoned
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  • 1998-12-29 EP EP98964536A patent/EP1042366A1/de not_active Withdrawn
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