Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
  • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • 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
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/975—Kit
• • 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
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/811—Test for named disease, body condition or organ function
• • 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
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/82—Hepatitis associated antigens and antibodies
• • 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
  • Y10S530/868—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof involving autoimmunity, allergy, immediate hypersensitivity, delayed hypersensitivity, immunosuppression, or immunotolerance

Definitions

• the present invention relates to peptide fragments from human cytochrome P450 IID6 (formerly called cytochrome P450 dbl), to anti-peptide fragment antibodies and to their applications in the diagnosis of autoimmune hepatitis and more. particularly in the differential diagnosis between autoimmune hepatitis and other chronic hepatitis of viral origin, such as hepatitis C or hepatitis B.
• Autoimmune hepatitis is an inflammatory disease that progresses to cirrhosis and liver failure, which usually responds to immunosuppressive therapy and is characterized by the presence of high autoantibody levels non-organ specific.
• Two subgroups have been defined, depending on the autoantibody present in the serum: anti-smooth muscle antibody (anti-SMA) and anti-liver-kidney-microsome antibody (anti-LKM), hereinafter called anti-LKM antibodies.
• the antigen recognized by anti-LKM antibodies is a protein with a molecular weight of 50 kDa, present at a relatively high concentration in the endoplasmic reticulum.
• cytochrome P450 IID6 is the protein recognized in the human liver by the anti-LKM antibody.
• the sera are considered positive for anti-LKM antibodies, when they react to a minimum dilution of 1: 100 with the cytoplasm of hepatocytes and renal tabules near, while no color is obtained at the level of the distal tubules.
• this indirect immunofluorescence method has the major drawback of being insensitive and thus of causing false negatives which can direct the clinician towards a bad diagnosis and thus disorient the latter with respect to the clinical signs observed. .
• the present invention has therefore set itself the aim of providing peptide fragments derived from human cytochrome P450, which better meet the needs of practice than the reagents of the prior art, in particular by allowing the development of highly specific diagnostic tests as well as the preparation of antibodies (polyclonal and / or monoclonal) for therapeutic and / or diagnostic purposes, also highly specific with respect to at least one immuno-dominant epitope of auto hepatitis -immune.
• the subject of the present invention is a peptide fragment of the human cytochrome P450, characterized in that it contains at least one immunodominant epitope of cytochrome P450 IID6, in that it consists of an amino acid sequence which comprises between 3 and 70 amino acids and in that it specifically binds with the anti-LKM autoantibodies produced during autoimmune hepatitis.
• peptide fragment includes not only the sequences comprising a fragment of the human cytochrome P450 IID6, but also those which differ only by substitution, detection, the addition of a small number of amino acids, provided that the sequences thus modified have a binding specificity with anti-LKM autoantibodies equivalent to that of the abovementioned fragments.
• a - a first set of fragments comprising at least one fragment of the sequence of cytochrome P450 IID6 comprised between amino acid 239 and amino acid 278, and comprising major antigenic site of cytochrome P450 and in particular:
• a peptide fragment characterized in that it consists of 39 amino acids, the sequence of which has the following formula I: Lys-Val-Leu-Arg-Phe-Gln-Lys-Ala-Phe-Leu-Thr-Gln-Leu -Asp- Glu-Leu-Leu-Thr-Glu-His-Arg-Met-Thr-Trp-Asp-Pro-Ala-Gln- Pro-Pro-Arg-Asp-Leu-Thr-Glu-Ala-Phe-Leu -Ala (I), which sequence corresponds to amino acids 239-278 of human cytochrome P450 IID6.
• peptide fragment characterized in that it consists of 18 amino acids, the sequence of which has the following formula II:
• a peptide fragment characterized in that it consists of three amino acids, the sequence of which has the following formula IV:
• Thr-Trp-Asp which sequence corresponds to amino acids 261, 262, and 263 of human cytochrome P450 IID6.
• b - a second set of fragments, comprising at least one fragment of the sequence of cytochrome P450 IID6 comprised between amino acid 282 and amino acid 351, and in particular:
• a peptide fragment characterized in that it consists of 70 amino acids, the sequence of which has the following formula V:
• a peptide fragment characterized in that it consists of 13 amino acids, the sequence of which has the following formula VI: Ala-Lys-Gly-Asn-Pro-Glu-Ser-Ser-Phe-Asn-Asp-Glu-Asn
• Va which sequence corresponds to amino acids 321-351 of cytochrome P450 IID6.
• c - a third set of fragments, comprising at least one fragment of the sequence of cytochrome P450 IID6 comprised between amino acid 349 and amino acid 389, and in particular:
• a peptide fragment characterized in that it consists of 41 amino acids, the sequence of which has the following formula VIII-:
• a peptide fragment characterized in that it consists of 17 amino acids, the sequence of which has the following formula IX: Gly-Met-Thr-His-Met-Thr-Ser-Arg-Asp-Ile-Gly-Val-Gln -Gly- Phe-Arg-Ile (IX), which sequence corresponds to amino acids 373-389 of human cytochrome P450 IID6.
• a peptide fragment characterized in that it consists of 20 amino acids, including the following formula X sequence: Glu-Lys-Pro-Tyr-Pro-Glu-His-Phe-Leu-Asp-Ala-Gln-Gly- His-Phe-Val-Lys-Pro-Glu-Ala (X), which sequence corresponds to amino acids 410-429 of human cytochrome P450 IID6.
• These peptides can in particular be prepared by synthesis, in particular by the Merrifield method.
• the present invention also relates to anti-human cytochrome P450 antibodies, characterized in that they are constituted by anti-peptide fragment antibodies as described in the invention.
• said antibodies consist of polyclonal antibodies.
• polyclonal antibodies are advantageously obtained by immunization of a suitable mammal, in particular the rabbit with a peptide in accordance with the invention, optionally coupled to a suitable protein, such as BSA (bovine serum albumin) or KLH (keyhole limpet hemocyanin ).
• BSA bovine serum albumin
• KLH keyhole limpet hemocyanin
• they consist of monoclonal antibodies specific for a peptide fragment derived from human cytochrome P450 IID6 as defined above.
• the present invention also relates to a munological reagent i, usable for the detection, diagnosis and monitoring of autoimmune hepatitis, charac ⁇ terized in that it is chosen from the group which comprises the peptide fragments and the anti-peptide antibodies according to the invention or a fragment thereof.
• fragments of formulas II, III and IV as defined above are used as diagnostic reagents, they cause a positive response in all patients suffering from autoimmune hepatitis and the fragments of formulas Va, IX and X cause a positive response in most patients with autoimmune hepatitis.
• the present invention also relates to a method for detecting and / or diagnosing autoimmune hepatitis, characterized in that it consists in detecting the autoantibodies possibly present in a biological fluid such as blood, by putting in the presence of said biological fluid with at least one appropriate immunological reagent according to the invention, to which the anti-LKM auto-antibodies bind, if such antibodies are present in the biological sample to be checked, the reading of the result being revealed by an appropriate means, in particular RIA, EIA or flow cytometry.
• the immunological reagent is a peptide fragment
• said biological fluid is brought into contact with a pool of peptides in accordance with the invention.
• said pool comprises at least 4 different pep ⁇ tides.
• the first peptide comprises a peptide fragment chosen from the group consisting of the fragments of formulas I, II, III or IV; the second peptide comprises a peptide fragment of formula IX, the third peptide comprises a peptide fragment of formula X and the fourth peptide comprises a peptide fragment of formula Va.
• the reagents and the detection method in accordance with the invention have the advantage of allowing a differential diagnosis between autoimmune hepatitis and other hepatitis of viral origin.
• the present invention also relates to agents for therapeutic and / or preventive purposes, characterized in that they consist of, or comprise as active ingredient, a peptide in accordance with the invention and / or its fragments, alone or conjugated or re ⁇ combined or associated with other substances.
• the present invention also relates to agents for therapeutic and / or preventive purposes, charac ⁇ terized in that they consist of, or comprise as active ingredient, antipeptide antibodies in accordance with the invention, and / or their fragments, alone or conjugated or recombined or associated with other substances.
• the present invention further relates to a diagnostic kit or kit for detecting, diagnosing or monitoring autoimmune hepatitis, characterized in that it comprises: - an appropriate solid support suitably coated at least one ligand chosen from the group which comprises the peptides in accordance with the invention, the antibodies in accordance with the invention, the F (ab) * 2 fragments and the F ab 'fragments of said antibodies; - at least one bottle containing conjugates chosen from the group which includes the enzyme conjugates appropriate-appropriate anti-Ig antibodies, the appropriate enzyme conjugates-antibody Fc fragment of human Ig and the appropriate enzyme-anti-peptide conjugates according to the invention; - appropriate quantities or doses of an appropriate developer substance.
• the patient's anti-LKM antibodies bind to the peptides according to the invention, previously fixed to the solid support and suitable enzyme-anti ⁇ body anti-human Ig antibodies or suitable appropriate enzyme-antibody Fc fragment conjugates of human Ig are introduced, bind with the patient's Ig and are then revealed appropriately.
• suitable enzyme-anti ⁇ body anti-human Ig antibodies or suitable appropriate enzyme-antibody Fc fragment conjugates of human Ig are introduced, bind with the patient's Ig and are then revealed appropriately.
• appropriate enzyme-anti-peptide antibody conjugates according to the invention compete with the patient's antibodies to bind to the solid support coated with peptides or antibodies according to the invention. 'invention.
• LKMHC5 is a cDNA clone in the phage ⁇ GT-11, coding for the human cytochrome P450 IID6 (GUEGUEN M. et al. 1989, cited reference).
• the EcoRI-PstI fragment d'-cDNA (1007 bp) is subcloned in the plasmid EX-627, which allows the expression of a fusion protein ( ⁇ -galactosidase / P450 IID6) in the bacteria E. coli pop 2136.
• the map of restriction of cytochrome P450 IID6 is obtained from GenBank and used to create different constructs.
• the endonucleases used are: EcoRI, Apal, BstEII, Saul, Aval, StuI, PstI, EcoNI and PpuMI; the specific restriction sites are represented in FIG. 1.
• the protocols used for the restriction and the ligation are those described in SAMBROOK et al. (Molecular cloning, Laboratory Manual, 1989, Cold Spring Harbor Laboratory).
• the different constructions obtained, designated by letters, correspond to different fragments of cytochrome P450 as follows:
• the sign * indicates an antigenic site recognized by the sera of all the patients tested and the sign indicates other antigenic sites recognized by a certain number of patients.
• the clone LKMCl is a clone of cDNA in the phage ⁇ GT-11, coding for the cytochrome P450 db2 of rats.
• the EcoRI-BamHI fragment of LKMCl 800 bp is cloned in the plasmid pEX-627 and transfected into E. coli pop 2136. This makes it possible to test the peptide corresponding to the 5 ′ region of the gene (coding for amino acid 1 to amino acid 125).
• the complete P450 IID6 protein contains 495 amino acids.
• the LKMHC5 EcoRI / PSTI clone as defined above, which codes for amino acids 125 to 458, allows the analysis of the C-terminal part of the molecule, while the N-terminal region is analyzed in use.
• an EcoRI / BamHILKMC1 subclone as defined above, and which codes for the amino acid sequence 1-266 of human cytochrome P450 IID6.
• a third epitope is located between amino acids 307 and 349.
• the presence of multiple epitopes on an autoantigen is in favor of the hypothesis which considers that the autoimmune response is polyclonal. Similar results have been obtained with other autoantigen / autoantibody systems and these have been interpreted as the absence of a random mutation as the mechanism for the production of autoantibodies and support the fact that the autoimmune response is induced by the antigen.
• fusion proteins from cDNA constructs are described by STANLEY (Nucleic Acids Research, 1983, 11, 4077). Samples of 100 ⁇ l of culture, for each fusion protein, are subjected to electrophoresis on polyacrylamide-SDS 8-20% gel, transferred to nitrocellulose and analyzed by the immunoblot technique.
• the first antibodies used for the immunoblot method are:
• the second antibody is either a goat anti-human IgG peroxidase-conjugate, or a peroxidase-IgG conjugate anti-goat mouse (Biosys), at a dilution of 1/1000.
• the rat liver and human liver microsomes and the ⁇ -galactosidase, expressed by the plasmid pEX-627 not comprising the P450 DNA insert, are included on the same gels as positive controls and negative control, respectively. . c) synthesis of peptides.
• the synthesis of the solid phase peptide is carried out using an Applied Biosystems device.
• the peptides are synthesized on a p-benzyloxybenzylalcohol resin, using 9-fluorenylmethoxycarbonyl
• the major antigenic site located between amino acids 239 and 273 was also analyzed using three synthetic peptides which cover the region between amino acids 241 and 281 as visible in Table I above.
• One of the peptides covering the 254-271 region is recognized by all the LKM positive sera when tested in ELISA and in immunoblotting, while the other three peptides are not (Va, recognized by 8 patients / 15; IX, recognized by 1 patient / 15; X, recognized by 2 patients / 15).
• the tripeptide Thr-Trp-Asp represents an essential part of the epitope.
• the different peptides and in particular pep ⁇ tides 254-271 (peptide of formula II), 264-281 and 410-429 (peptide of formula X), are coupled to BSA via their free NH2-terminal group, using glutaraldehyde (see in particular E. HARLO and D. LANE, 1988, Antibodies, A laboratory manual, Cold ' Spring Harbor Laboratory).
• a number of peptides (241-260, 383-399, 392-417 and peptide of formula X) carry an additional N-terminal cysteine residue which allows coupling to BSA via the sulfhydryl group of cysteine, using an ester of m-maleimidobenzoyl-N-hydroxysuccinimide.
• EXAMPLE 2 ELISA test. Peptides are redissolved in a buffer
• the anti-human goat IgG antibody conjugated with alkaline phosphatase is used as the second antibody, at a dilution of 1/1000.
• the test is developed by adding 100 ⁇ l of p-nitrophenylphosphate at a concentration of 1 g / ml, in 0.05 M NaC0 3 buffer, pH 9.8 and 0.001 M MgCl 2.
• the results read after 30 minutes d incubation at room temperature are considered positive when they are higher by a factor 2 compared to the control serum.
• FIG. 3 which comprises the dilutions on the abscissa and the optical densities on the ordinate, illustrates an ELISA test carried out on sera from 10 patients (curves 1 to 10), with a peptide comprising at least a fragment of the sequence 239 as antigen -273 (peptides of formulas I, II, III or IV); the curve -x- corresponds to the upper limit of the normal.

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• 1991
  • 1991-06-17 FR FR9107363A patent/FR2677653A1/fr active Granted
• 1992
  • 1992-06-16 EP EP92912543A patent/EP0589993B1/fr not_active Expired - Lifetime
  • 1992-06-16 WO PCT/FR1992/000539 patent/WO1992022656A1/fr not_active Ceased
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