Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention relates to certain peptides containing citrulline and that constitute immunogenic determinants of antibodies present in sera from patients with rheumatoid arthritis and wherein the presence of at least one citrulline is a prerequisite for reacting with said antibodies.
• the invention also relates to the a method of producing said peptides and the use of said peptides for diagnosis and treatment of rheumatoid arthritis and related diseases.
• the present invention also relates to new filaggrin alleles.
• RA Rheumatoid arthritis
• RA has all the features of an autoimmune disease, including the presence of a variety of autoantibodies in patients' sera and the capacity to induce illness by transfer of pathogenic T cells in animal models.
• the classification of the disease can be challenged on the grounds that borderline forms are very common; furthermore inflammation of the joints is not only restricted to RA, but occurs also in other non-autoimmune diseases such as osteoarthritis, reactive arthritis and gout.
• RA rheumatologists
• the diagnosis of RA is initially based on clinical manifestations. Serological support for such a diagnosis is not very well established and is based mainly on the presence of rheumatoid factors (RF).
• RF rheumatoid factors
• RA patients are RF seronegative, while on the other hand, RF is also present in other rheumatic diseases including Sj ⁇ gren's syndrome and systemic lupus erythematosus, in some chronic bacterial and acute viral infections, in certain parasitic diseases and chronic inflammatory diseases, and has furthermore been demonstrated in control sera from healthy persons (Waller et al., 1 964; Chen et al., 1 987). This rather low specificity of RF necessitates additional testing for a second RA-specific antibody.
• APF antiperinuclear factor
• AKA antikeratin antibodies
• a second specificity found in sera of RA patients are the AKA, autoantibodies that were first described by Young et al. ( 1 979) and that give rise to a fluorescent staining pattern with the keratinized layer of rat oesophageal epithelium.
• the antibodies were infered to be antikeratin antibodies on the disputable ground that cytokeratins constitute the major component of the stratum corneum.
• the human epidermal protein filaggrin was recently identified with convincing evidence as one of the major targets for reactivity with AKA and APF autoantibodies (Simon et al., 1 993; Sebbag et al., 1 995) .
• the neutral/acidic form of human filaggrin was demonstrated to react with 75% of a group of 48 RA sera in Western blot (Simon et al., 1 993). More recent data (Vincent et al., 1 997) showed a diagnostic sensitivity of more than 50% for antifilaggrin detection with a corresponding specificity of 95% within a group of 492 sera which included 279 RA sera.
• Profilaggrin the precursor of filaggrin is a histidine-rich, insoluble protein of ⁇ 400-kDa. It consists of 1 0-1 2 filaggrin repeats of 324 amino acids that are separated by a heptamer linker sequence. The highly phosphorylated polyprotein is stored in the keratohyalin granules of the granular layer of the epidermis. Upon terminal differentiation of these cells, profilaggrin is dephosphorylated and proteolytically processed by excision of the linker into functional basic filaggrin molecules of 37-kDa (Resing et al., 1 989; 1 993; Gan et al., 1 990).
• these units are involved in the aggregation of keratin intermediate filaments, facilitating formation of intermolecular disulfide bonds and yielding the intracellular fibrous matrix of the cornified cells (Dale et al., 1 978; Lynley and Dale,
• tandemly arranged filaggrin units are highly polymorphic: there exists a considerable variation in amino acid sequence across individuals and also between the different filaggrin molecules of one person, where up to 1 5% of differences have been noted (McKinley-Grant et al., 1 989; Gan et al., 1 990; Markova et al.,
• Another aim of the present invention is to provide methods for obtaining said peptides.
• Another aim of the present invention is to provide methods of raising antibodies specifically reactive with said peptides.
• Another aim of the present invention is to provide methods of raising anti- idiotype antibodies specifically reactive with the afore mentioned antibodies, thereby mimicking said peptides.
• Another aim of the present invention is to provide a pharmaceutical composition comprising these peptides, for therapy or diagnosis.
• Another aim of the present invention is to provide a diagnostic kit for rheumatoid arthritis.
• Another aim of the present invention is to provide a modulator, a composition containing a modulator, or a combination of modulators identified by the bioassay as described above.
• the immunodominant epitopes of filaggrin were identified, which all contained the unusual amino acid citrulline. Synthetic peptides were generated and proved useful for diagnosis of RA. By probing human rheumatoid arthritis sera onto 2-D blots containing placental extracts another specificity was found. The immunoreactive spots were identified and peptides from human vimentin, cytokeratin 1 and cytokeratin 9 were retrieved in addition to other non-identified peptides. These proteins all belong to the same family of intermediate filament proteins.
• the present invention relates to peptides containing less than 50 amino acids, comprising fragments of a filaggrin variant or fragments of intermediate filament proteins, wherein at least one arginine is substituted by citrulline, and that are able to react with antibodies, wherein the presence of said citrulline is crucial for reaction between said peptide and said antibodies, and wherein said antibodies are present in sera from patients with rheumatoid arthritis.
• the present invention relates to peptides as presented in claim 2.
• the present invention also relates to a peptide and/or chemical structure comprising any of the above mentioned peptides, fused to a linker molecule.
• the present invention also relates to peptides comprising and/or consisting of tandem repeats of at least two of any of the above mentioned peptides, or branched peptides that comprises at least one of the above mentioned peptides.
• the present invention also relates to a method for producing any of the above mentioned peptides, by classical chemical synthesis, wherein at least one arginine residue is substituted by citrulline, at certain steps during the chemical synthesis.
• the present invention also relates to a method for producing any of the above mentioned peptides, wherein the primary amino acid sequence is produced by classical chemical synthesis, and wherein said arginine residue is derivatized towards citrulline after chemical synthesis by incubation with peptidylarginine deiminase.
• the present invention also relates to a method for producing any of the above mentioned peptides comprising the following steps: (i) transforming an appropriate cellular host with a recombinant vector in which a polynucleic acid is inserted comprising the sequence that codes for said peptide under the control of the appropriate regulatory elements such that said peptide or a protein comprising said peptide is expressed and/or secreted, (ii) culturing said transformed cellular host under conditions allowing expression of said protein or peptide and allowing a partial or optimal derivatization of said arginines present in said peptide, towards citrulline residues, and (iii) harvesting said peptide.
• the present invention also relates to a method for producing any of the above mentioned peptides comprising the following steps: (i) transforming an appropriate cellular host with a recombinant vector in which a polynucleic acid is inserted comprising the sequence that codes for said peptide under the control of the appropriate regulatory elements, such that said peptide or a protein comprising said peptide is expressed and/or secreted, (ii) culturing said transformed cellular host under conditions allowing expression of said protein or said peptide, (iii) harvesting said protein or said peptide, and (iv) derivatizing arginine residues of said protein or said peptide towards citrulline residues.
• the present invention also relates to any of the above mentioned methods, wherein said host cell is a bacterial host or yeast or any other eukaryotic host cell which is preferably transformed with a recombinant baculovirus.
• the present invention also relates to an antibody being specifically reactive with said peptides or intermediate filament proteins that contain citrulline residues, and with said antibody being preferably a monoclonal antibody.
• the present invention also relates to an anti-idiotype antibody raised upon immunization with any antibody as defined above, with said anti- idiotype antibody being specifically reactive with said antibody, thereby mimicking any of the above mentioned peptides, and with said antibody being preferably a monoclonal antibody.
• the present invention also relates to an immunotoxin molecule comprising and/or consisting of a cell recognition molecule being a peptide as defined above, or an antibody as defined above, covalently bound to a toxin molecule or active fragment thereof.
• the present invention relates to any of the above mentioned peptides or antibodies or immunotoxine molecules or intermediate filament proteins or a composition thereof for use as a medicament.
• Said use can have the purpose of a medicament for treatment or of a diagnosticum for rheumatoid arthritis.
• the present invention also relates to a treatment for autoimmune diseases by inducing a state of systemic hyporesponsiveness to the auto- antigen after oral administration of any of the above mentioned peptides or antibodies or immunotoxine molecules or intermediate filament proteins or a composition thereof, thereby preventing the pathogenic production of anti-self antibodies.
• the present invention also relates to a diagnostic kit for use in detecting rheumatoid arthritis, wherein said kit comprises at least one of the above mentioned peptides or proteins or antibodies, and with said peptide, proteins or antibody being possibly bound to a solid support. More preferably said kit is comprising a range of said peptides or said antibodies, possibly in combination with other epitopes that can characterize auto-immune disease, wherein said peptides, proteins and/or antibodies are attached to specific locations on a solid substrate. More preferably said solid support is a membrane strip and said polypeptides are coupled to the membrane in the form of parallel lines.
• the present invention relates to those peptide fragments of natural filaggrin variants that react with antibodies characteristically present in sera of patients with rheumatoid arthritis and that are further characterized by a post-translational modification, more preferably a derivatization of arginine towards citrulline.
• the presence of at least one citrulline residue is a prerequisite for recognition by antibodies that are specifically present in sera of patients with rheumatoid arthritis.
• Synthetic peptides were generated wherein arginine residues were substituted by citrulline, thus mimicking the epitopes of natural filaggrin variants. These peptides proved useful for diagnosis of rheumatoid arthritis.
• the present invention relates to peptides which immunologically mimic the immunogenic determinants of self proteins recognized by the immune system in patients suffering from rheumatoid arthritis.
• citrulline can be sufficient for specific recognition by some antibodies present in sera of patients with rheumatoid arthritis.
• 'peptide' refers to a polymer of amino acids and does not refer to a specific length of the product; thus, oligopeptides, polypeptides and proteins are included within the definition of 'peptide'. This term also does not exclude post-expression modifications of the peptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, peptides containing one or more analogues of an amino acid (including, for example, unnatural amino acids, PNA, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
• peptide containing less than 50 amino acids this should be interpreted in a broad sense, as a means of circumscribing an essentially truncated version of the entire immunoreactive protein that still comprises the highly reactive domain characterized by the presence of citrulline residues.
• These peptides have a length of preferably 40, 30, 25, 20 or less amino acids.
• 'immunogenic determinant is meant, those chemical groupings comprising a primary amino acid sequence, and secondary modifications of the amino acid residues in a certain three-dimensional arrangement, that together determine the specific reactivity of the entire antigen for a raised antibody.
• Such antibody can also recognize different chemical groupings, which are then termed to 'immunologically mimic' the immunogenic determinant.
• crossreaction also refers to the reaction of one antigen with antibodies developed against another antigen or against antibodies that are found in sera from patients with different diseases.
• the present invention relates to those peptides or proteins that contain citrulline residues, wherein the presence of said citrulline residues is crucial for high-affinity interaction with antibodies that are characteristically present in sera of patients with rheumatoid arthritis.
• the present invention relates to a peptide that is characterized by the amino acid sequence HSASQDGQDT1RGHPGSS or,
• the present invention relates to peptides comprising a sequence of less than 50 amino acids of any variant of vimentin, cytokeratin 1 or cytokeratin 9, comprising at least one citrulline residue, and wherein the presence of said citrulline is crucial for reacting with antibodies that are present in sera from patients with rheumatoid arthritis.
• the present invention also relates to molecular structures in which at least part represents a peptide or antibody as defined above.
• Such molecular structures can result from fusion of peptides of the present invention with peptides and/or proteins and/or other molecules that are further characterized in that they -specifically interact with other peptides and/or proteins and/or molecular structures, enabling tagging and/or binding of the fused polypeptide and/or protein to specific tissue- or cell types or that allow for purification of said molecular structures due to the presence of for instance 4, or 5 or 6 consecutive histidine residues, or
• cytotoxic to T-cells and/or B-cells such as cholera toxin, or -allow for labelling by means of a radioactive or fluorescent or immunogold or enzymatic marker.
• tandem repeats or branched peptides of the antigens can increase the amount of immobilized antigens presented to the antibodies and thereby increase the sensitivity of the assay.
• the sensitivity can be increased exponentially when the immobilized antigens are used together with a specific concentration of such antigens in a soluble form, thereby inducing the formation of crosslinked antigen- immunoprecipitates.
• a second advantage relates to therapy.
• the deposition of self- antigen autoimmune complexes in various tissues is an important step towards the acquisition of a pathological condition. It is generally accepted that the main cause of said deposition is the insufficient blood clearance by the liver of the antigen- immune complexes due to the small size of said complexes. Administration of tandem repeats or branched forms of said peptides could increase the size of the formed antigen-immune complexes, and thereby increases the clearance and thus decreases the deposition of said complexes.
• the present invention also relates to circularized forms of said peptides, the advantage being well known in the art, and relating to an increased affinity of a conformationally constraint peptide as compared with the more randomly coiled forms of linear peptides.
• peptides In order to accommodate for eventual negative characteristics of the claimed peptides, such as rapid degradation, solubility, cytotoxic effects and so on, the skilled person will be able to design conservative as well as non-conservative amino acid substitutions, or substitutions with non-natural amino acids, etc... These will generally account for less than 35 percent of a specific sequence.
• Such peptides also include peptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. It may be desirable in cases where the filaggrin peptides of the present invention are highly polymorphic, to vary one or more of the amino acids so as to better mimic the different epitopes, or as recognized by antibodies in sera from patients with rheumatoid arthritis.
• the present invention also relates to the new allelic variants that were isolated , cloned and sequenced, characterized by the DNA sequence as presented in figure 6 and the amino acid sequence as presented in figure 2.
• the present invention also relates to any analogs of the peptides of the present invention.
• analog as used throughout the specification or claims to describe the proteins or peptides of the present invention, includes any protein or peptide having an amino acid residue sequence substantially identical to a sequence specifically shown herein in which one or more residues have been conservatively substituted with a biologically equivalent residue.
• conservative substitutions include the substitution of hydrophobic residue such as isoleucine, valine, leucine or methionine for another, the substitution of one hydrophilic residue for another such as between arginine and lysine, between glutamine and asparagines, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. Examples of allowable mutations according to the present invention can be found in Table 1 .
• “Chemical derivative” refers to a protein or peptide having one or more residues chemically derivatized by reaction of a functional side group or peptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
• Examples of such derivatized molecules include but are not limited to, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloracetyl groups or formyl groups.
• Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides.
• Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives.
• the imidazole nitrogen of histidine may be derivatized to form N-imbenzylhistidine.
• chemical derivatives are those proteins or peptides which contain one or more naturally- occurring amino acid derivatives of the twenty standard amino acids. For examples : 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.
• the peptides of the present invention also include any protein or peptide having one or more additions and/or deletions or residues relative to the sequence of a peptide whose sequence is shown herein, as long as the peptide is biologically equivalent to the proteins or peptides of the invention.
• Amino acids Synonymous groups
• linker arm by which the peptide can conveniently be attached to a carrier.
• the linker arm will be at least one amino acid and may be as many as 60 amino acids but will most frequently be
• the peptides of the invention can be prepared by classical chemical synthesis.
• the synthesis can be carried out in homogeneous solution or in solid phase.
• the synthesis technique in homogeneous solution which can be used is the one described by Houbenweyl in the book entitled “Methode der organischen chemie” (Method of organic chemistry) edited by E. Wunsh, vol. 1 5-1 et II. THIEME, Stuttgart 1 974.
• the polypeptides of the invention can also be prepared in solid phase according to the methods described by Atherton and Shepard in their book entitled “Solid phase peptide synthesis” (IRL Press, Oxford, 1 989).
• the forms of the claimed peptides can be obtained by substituting the citrulline residues for the original arginine derivatives during the classical chemical synthesis, or by contacting the peptides after synthesis with a peptidylarginine deiminase of any eukaryotic origin.
• polypeptides according to this invention can also be prepared by means of recombinant DNA techniques as described by Maniatis et al., Molecular Cloning: A Laboratory Manual, New York, Cold Spring Harbor Laboratory, 1 982) by insertion of a polynucleic acid sequence encoding the claimed peptides or part of the claimed peptides in an appropriate vector and transforming a suitable host with said vector.
• This recombinant expression vector comprises a polynucleic acid or a part thereof as defined above, operably linked to prokaryotic, eukaryotic or viral transcription and translation control elements.
• this sequence can be operably linked with sequences that allow for secretion of the claimed peptides.
• the term 'vector' may comprise a plasmid, a cosmid, a phage or a virus or a transgenic organism. Particularly useful may be BCG or adenoviral vectors, as well as avipox recombinant viruses.
• the recombinant peptides can be derivatized in vitro, by contacting the expressed and/or secreted peptides with a of any eukaryotic origin, or in vivo by choosing the appropriate host, like yeast, or any eukaryotic cell, and more preferably by using the baculovirus transformation system, or by coexpressing said peptides with recombinant peptidylarginine deiminase.
• any of the known purification methods for recombinant peptides can be used for the production of the recombinant peptides of the present invention.
• the present invention also relates to a recombinant expression vector comprising a polynucleic acid or a part thereof as defined above, operably linked to prokaryotic, eukaryotic or viral transcription and translation control elements.
• said recombinant vector will comprise a vector sequence, an appropriate prokaryotic, eukaryotic or viral promoter sequence followed by a nucleotide sequence encoding a peptide as defined above, with said recombinant vector allowing the expression and/or secretion of any one of the polypeptides as defined above in a prokaryotic, or eukaryotic host or in living mammals when injected as naked DNA.
• any of the known purification methods for recombinant proteins may be used for the production of the recombinant polypeptides of the present invention.
• vector may comprise a plasmid, a cosmid, a phage, or a virus or a transgenic animal. Particularly useful for vaccine development may be BCG or adenoviral vectors, as well as avipox recombinant viruses.
• the present invention also relates to a method for the production of a recombinant polypeptide as defined above, comprising: transformation of an appropriate cellular host with a recombinant vector, in which a polynucleic acid or a part thereof according to as defined above has been inserted under the control of appropriate regulatory elements, culturing said transformed cellular host under conditions enabling the expression and/or secretion of said insert, and, harvesting said polypeptide.
• recombinantly expressed used within the context of the present invention refers to the fact that the proteins of the present invention are produced by recombinant expression methods be it in prokaryotes, or lower or higher eukaryotes as discussed in detail below.
• lower eukaryote refers to host cells such as yeast, fungi and the like. Lower eukaryotes are generally (but not necessarily) unicellular. Preferred lower eukaryotes are yeasts, particularly species within Saccharomyces. Schizosaccharomvces, Kluveromyces, Pichia (e.g. Pichia pastoris) , Hansenula (e.g.
• Saccharomyces cerevisiae, S_. carlsbergensis and K. lactis are the most commonly used yeast hosts, and are convenient fungal hosts.
• prokaryotes refers to hosts such as E.coli. Lactobacillus. Lactococcus, Salmonella, Streptococcus, Bacillus subtilis or Streptomyces. Also these hosts are contemplated within the present invention.
• higher eukaryote refers to host cells derived from higher animals, such as mammals, reptiles, insects, and the like.
• Presently preferred higher eukaryote host cells are derived from Chinese hamster (e.g. CHO), monkey (e.g. COS and Vero cells), baby hamster kidney (BHK), pig kidney (PK1 5), rabbit kidney
• the host cells may be provided in suspension or flask cultures, tissue cultures, organ cultures and the like. Alternatively the host cells may also be transgenic animals.
• polynucleotide or “nucleic acid” intends a polynucleotide or nucleic acid of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation : ( 1 ) is not associated with all or a portion of a polynucleotide with which it is associated in nature, (2) is linked to a polynucleotide other than that to which it is linked in nature, or (3) does not occur in nature.
• recombinant host cells refer to cells which can be or have been, used as recipients for a recombinant vector or other transfer polynucleotide, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
• telome any genetic element, e.g., a plasmid, a chromosome, a virus, a cosmid, etc., that behaves as an autonomous unit of polynucleotide replication within a cell; i.e., capable of replication under its own control.
• vector is a replicon further comprising sequences providing replication and/or expression of a desired open reading frame.
• control sequence refers to polynucleotide sequences which are necessary to effect the expression of coding sequences to which they are ligated.
• control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, splicing sites and terminators; in eukaryotes, generally, such control sequences include promoters, splicing sites, terminators and, in some instances, enhancers.
• control sequences is intended to include, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, for example, leader sequences which govern secretion.
• promoter is a nucleotide sequence which is comprised of consensus sequences which allow the binding of RNA polymerase to the DNA template in a manner such that mRNA production initiates at the normal transcription initiation site for the adjacent structural gene.
• operably linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
• a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
• the polynucleic acids encoding the peptides of the present invention and inserted into the vector sequence may be attached to a signal sequence.
• Said signal sequence may be that from any source, e.g. the IgG or tissue plasminogen activator (tpa) leader sequence for expression in mammalian cells, or the ⁇ -mating factor sequence for expression into yeast cells.
• a variety of vectors may be used to obtain the peptides of the present invention.
• Lower eukaryotes such as yeasts and glycosylation mutant strains are typically transformed with plasmids, or are transformed with a recombinant virus.
• the vectors may replicate within the host independently, or may integrate into the host cell genome.
• Higher eukaryotes may be transformed with vectors, or may be infected with a recombinant virus, for example a recombinant vaccinia virus.
• Techniques and vectors for the insertion of foreign DNA into vaccinia virus are well known in the art, and utilize, for example homologous recombination.
• a wide variety of viral promoter sequences, possibly terminator sequences and poly(A)-addition sequences, possibly enhancer sequences and possibly amplification sequences, all required for the mammalian expression, are available in the art.
• Vaccinia is particularly preferred since vaccinia halts the expression of host cell proteins.
• Vaccinia is also very much preferred since it allows the expression of f.i. peptides of the present invention in cells or individuals which are immunized with the live recombinant vaccinia virus.
• AMV Ankara Modified Virus
• insect expression transfer vectors derived from baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), which is a helper- independent viral expression vector.
• AcNPV Autographa californica nuclear polyhedrosis virus
• Expression vectors derived from this system usually use the strong viral polyhedrin gene promoter to drive the expression of heterologous genes.
• Different vectors as well as methods for the introduction of heterologous DNA into the desired site of baculovirus are available to the man skilled in the art for baculovirus expression.
• signals for posttranslational modification recognized by insect cells are known in the art.
• the present invention also relates to a host cell transformed with a recombinant vector as defined above.
• the present invention also relates to antibodies that are specifically raised against the peptides of the present invention, preferably against those peptides wherein the arginines are derivatized towards citrulline. These antibodies may be polyclonal or monoclonal.
• a host animal is immunized using the peptides of the present invention in a pharmaceutically acceptable carrier, wherein at least one of the arginines is derivatized towards citrulline.
• Pharmaceutically acceptable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.
• Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers; and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.
• Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to : aluminim hydroxide (alum), N-acetyl-muramyl-L-threonyl-D- isoglutamine (thr-MDP) as found in U.S. Patent No.
• N-acetyl- normuramyl-L-alanyl-D-isoglutamine (nor-MDP)
• N-acetylmuramyl-L-alanyl-D- isoglutaminyl-L-alanine-2-( 1 '-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)- ethylamine (MTP-PE)
• RIBI which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton (MPL + TDM + CWS) in a 2% squalene/Tween 80 emulsion.
• any of the 3 components MPL, TDM or CWS may also be used alone or combined 2 by 2. Additionally, adjuvants such as Stimulon (Cambridge Bioscience, Worcester, MA) or SAF-1 (Syntex) may be used. Further, Complete Freund's Adjuvant (CFA) and
• IFA Incomplete Freund's Adjuvant
• the immunogenic compositions typically will contain pharmaceutically acceptable vehicles, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, preservatives, and the like, may be included in such vehicles.
• the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
• the preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect.
• the proteins may also be incorporated into Immune Stimulating Complexes together with saponins, for example Quil A (ISCOMS).
• Immunogenic compositions used to raise antibodies comprise a 'sufficient amount' or 'an immunologically effective amount' of the peptides of the present invention, as well as any other of the above mentioned components, as needed.
• 'Immunologically effective amount' means that the administration of that amount to an individual, either in a single dose or as part of a series, is effective to provoke an immune response and to raise antibodies, as defined above. This amount varies depending upon the health and physical condition of the individual, the taxonomic group of the individual to be treated (e.g. nonhuman primate, primate, rabbit, etc.), the capacity of the individual's immune system to synthesize antibodies, the immunogenicity of the antigenic peptide, and its mode of administration, and other relevant factors.
• the amount will fall in a relatively broad range that can be determined through routine trials. Usually, the amount will vary from 0.01 to 1000 ⁇ g/dose, more particularly from 0.1 to 100 ⁇ g/dose.
• the immunogenic compositions are conventionally administered parenterally, typically by injection, for example, subcutaneously or intramuscularly. Additional formulations suitable for other methods of administration include oral formulations and suppositories. Dosage treatment may be a single dose schedule or a multiple dose schedule.
• the vaccine may be administered in conjunction with other immunoregulatory agents.
• the host serum or plasma is collected following an appropriate time interval to provide a composition comprising antibodies reactive with the peptides of the present invention.
• the gamma globulin fraction or the IgG antibodies can be obtained, for example, by use of saturated ammonium sulfate or DEAE Sephadex, or other techniques known to those skilled in the art.
• the antibodies are substantially free of many of the adverse side effects which may be associated with other anti-viral agents such as drugs, for the treatment of infectious, chronic, or recurrent mononucleosis.
• Such antibodies may also be used to diagnose certain diseases, such as Burkitt's lymphoma, wherein Epstein-Barr virus has been implicated.
• the term 'immunogenic' refers to the ability of a substance to cause a humoral and/or cellular response, whether alone or when linked to a carrier, in the presence or absence of an adjuvant.
• the antibodies of the claimed invention may also be monoclonals that are prepared with said antibody being specifically reactive with any of said peptides, and with said antibody being preferably a monoclonal antibody.
• the monoclonal antibodies of the invention can be produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly from a mouse or rat, immunized against the claimed peptides of the present invention on the one hand, and of cells of a myeloma cell line on the other hand, and to be selected by the ability of the hybridoma to produce the monoclonal antibodies recognizing the citrullinated forms of the peptides which has been initially used for the immunization of the animals.
• the antibodies involved in the invention can be labelled by an appropriate label of the enzymatic, fluorescent, or radioactive type.
• the monoclonal antibodies according to this preferred embodiment of the invention may be humanized versions of mouse monoclonal antibodies made by means of recombinant DNA technology, departing from parts of mouse and/or human genomic DNA sequences coding for H and L chains or from cDNA clones coding for H and L chains.
• the monoclonal antibodies according to this preferred embodiment of the invention may be human monoclonal antibodies.
• These antibodies according to the present embodiment of the invention can also be derived from human peripheral blood lymphocytes of patients with rheumatoid arthritis.
• Such human monoclonal antibodies are prepared, for instance, by means of human peripheral blood lymphocytes (PBL) repopulation of severe combined immune deficiency (SCID) mice (for recent review, see Duchosal et al. 1 992) or by screening vaccinated individuals for the presence of reactive B-cells by means of the antigens of the present invention.
• PBL peripheral blood lymphocytes
• SCID severe combined immune deficiency
• the present invention also relates to the anti-idiotype antibodies that are raised upon immunization with an antibody as defined above and that specifically react with said antibodies, thereby mimicking the peptides of the present invention.
• the present invention also relates to truncated versions or single chain versions of the antibodies and anti-idiotype antibodies as defined above, that have retained their original specificity for reacting with the antigens.
• the present invention also relates to proteins or peptides that mimic the antibodies as defined above such as microproteins as can be obtained by phage display or the highly variable domain of a recombinant antibody as obtained by screening upon repertoire cloning.
• the present invention also relates to a method for detecting antibodies that specifically react with the peptides or anti-idiotype antibodies of the present invention, present in a biological sample, comprising:
• the present invention also relates to a reverse method for detecting the peptides and/or the anti-idiotype antibodies of the present invention with antibodies present in a biological sample that specifically react with said peptides and/or anti- idiotype antibodies that mimic such peptides, comprising:
• the methods as defined above can be used in the diagnosis of rheumatoid arthritis.
• the present invention relates to the development of a diagnostic technique that allows differentiation between those autoimmune diseases in which the characteristic antibodies often crossreact with the same antigen, thus resulting in difficult and slow diagnosis.
• diagnostic technique can be obtained by the simultaneous use of several antigens and/or anti- idiotype antibodies of the present invention.
• the present invention also relates to a diagnostic kit for use in detecting the presence of said antibodies, said kit comprising at least one peptide or anti-idiotype antibody or microprotein as defined above, with said peptide or anti-idiotype antibody or microprotein being preferably bound to a solid support.
• the present invention also relates to a diagnostic kit for determining the type of autoimmune disease, said kit comprising at least one peptide or anti-idiotype antibody or microprotein as defined above, with said peptide or anti-idiotype antibody or microprotein being preferably bound to a solid support.
• the present invention also relates to a diagnostic kit as defined above, said kit comprising a range of said peptides and/or anti-idiotype antibodies or microprotein which are attached to specific locations on a solid substrate.
• the present invention also relates to a diagnostic kit as defined above, wherein said solid support is a membrane strip and said peptides and/or anti- idiotype antibodies or microproteins are coupled to the membrane in the form of parallel lines.
• the immunoassay methods according to the present invention may utilize for instance single or specific oligomeric antigens, dimeric antigens, as well as combinations of single or specific oligomeric antigens.
• the peptides of the present invention may be employed in virtually any assay format that employs a known antigen to detect antibodies that characterize a certain disease or infection.
• a common feature of all of these assays is that the antigenic peptide or anti-idiotype antibody or microprotein is contacted with the body component suspected of containing the antibodies under conditions that permit the antigen to bind to any such antibody present in the component. Such conditions will typically be physiologic temperature, pH and ionic strength using an excess of antigen.
• the incubation of the antigen with the specimen is followed by detection of immune complexes comprised of the antigen.
• Protocols may, for example, use solid supports, or immunoprecipitation.
• Most assays involve the use of labelled antibody or peptide; the labels may be, for example, enzymatic, fluorescent, chemiluminescent, radioactive, or dye molecules.
• Assays which amplify the signals from the immune complex are also known; examples of which are assays which utilize biotin and avidin or streptavidin, and enzyme-labelled and mediated immunoassays, such as EL1SA assays.
• the immunoassay may be, without limitation, in a heterogeneous or in a homogeneous format, and of a standard or competitive type.
• the peptide or anti-idiotype antibody or microprotein is typically bound to a solid matrix or support to facilitate separation of the sample from the peptide or anti-idiotype antibody or microprotein after incubation.
• solid supports examples include nitrocellulose (e.g., in membrane or microtiter well form), polyvinyl chloride (e.g., in sheets or microtiter wells), polystyrene latex (e.g., in beads or microtiter plates, polyvinylidene fluoride (known as ImmunolonTM), diazotized paper, nylon membranes, activated beads, and Protein A beads.
• nitrocellulose e.g., in membrane or microtiter well form
• polyvinyl chloride e.g., in sheets or microtiter wells
• polystyrene latex e.g., in beads or microtiter plates, polyvinylidene fluoride (known as ImmunolonTM)
• diazotized paper e.g., in beads or microtiter plates
• Dynatech ImmunolonTM 1 or ImmunolonTM 2 microtiter plates or 0.25 inch polystyrene beads (Precision Plastic Ball) can be used
• the test sample is incubated with the combination of antigens in solution.
• it may be under conditions that will precipitate any antigen-antibody or anti-idiotype antibody-antibody or microprotein-antibody complexes which are formed.
• Both standard and competitive formats for these assays are known in the art.
• the amount of rheumatoid arthritis antibodies in the antibody- antigen complexes is directly monitored. This may be accomplished by determining whether a second type of labelled anti-xenogenetic (e.g. anti-human) antibodies which recognize an epitope on the first type of rheumatoid arthritis-antibodies will bind due to complex formation.
• the amount of rheumatoid arthritis-antibodies in the sample is deduced by monitoring the competitive effect on the binding of a known amount of labelled antibody (or other competing ligand) in the complex.
• the detection of rheumatoid arthritis-antibodies for diagnosis of rheumatoid arthritis is used as an illustration. Wherever the term " rheumatoid arthritis-antibodies" is used throughout the specification, this should not be considered as limitative.
• the other autoimmune diseases are diagnosed by detection of other antibodies, and mononucleosis is diagnosed by detection of anti-Epstein-Barr virus antibodies.
• Complexes formed comprising rheumatoid arthritis-antibody are detected by any of a number of known techniques, depending on the format.
• unlabelled rheumatoid arthritis-antibodies in the complex may be detected using a conjugate of anti-xenogenetic Ig complexed with a label (e.g. an enzyme label) .
• a label e.g. an enzyme label
• the reaction between the rheumatoid arthritis-antigens and the rheumatoid arthritis-antibody forms a network that precipitates from the solution or suspension and forms a visible layer or film of precipitate.
• PA particle agglutination
• the antigenic peptides of the present invention will typically be packaged in the form of a kit for use in these immunoassays.
• the kit will normally contain in separate containers the antigenic peptide or anti-idiotype antibody, control antibody formulations (positive and/or negative), labelled antibody when the assay format requires the same and signal generating reagents (e.g. enzyme substrate) if the label does not generate a signal directly.
• the antigenic peptide or anti-idiotype antibody may be already bound to a solid matrix or separate with reagents for binding it to the matrix. Instructions (e.g. written, tape, CD-ROM, etc.) for carrying out the assay usually will be included in the kit.
• the solid phase selected can include polymeric or glass beads, nitrocellulose, microparticles, microwells of a reaction tray, test tubes and magnetic beads.
• the signal generating compound can include an enzyme, a luminescent compound, a chromogen, a radioactive element and a chemiluminescent compound. Examples of enzymes include alkaline phosphatase, horseradish peroxidase and beta- galactosidase.
• enhancer compounds include biotin, anti-biotin and avidin.
• enhancer compounds binding members include biotin, anti- biotin and avidin.
• the test sample is subjected to conditions sufficient to block the effect of rheumatoid factor-like substances. These conditions comprise contacting the test sample with a quantity of for instance Rabbit Ig or anti-human IgG, preferably aggregated, to form a mixture, and incubating the mixture for a time and under conditions sufficient to form a reaction mixture product substantially free of rheumatoid factor-like substance.
• the present invention particularly relates to an immunoassay format in which several peptides of the invention are coupled to a membrane in the form of parallel lines.
• This assay format is particularly advantageous for allowing a discrimination between the separate autoimmune diseases.
• the present invention refers to a bioassay for identifying compounds which modulate the binding between an autoantigen and a rheumatoid arthritis specific autoantibody comprising: i) - contacting rheumatoid arthritis specific autoantibodies with any of the above mentioned peptides or intermediate filament proteins or a combination thereof.
• the present invention refers to a modulator for the interaction between an autoantigen and a rheumatoid arthritis specific autoantibody, and the method for producing said modulator, wherein said modulator is identified by the bioassay described above.
• compound as used herein has to be interpreted in a broad sense and can be proteins, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules, or antibodies which may be generated by the host itself upon vaccination.
• binding indicates that a peptide as described above is physically connected to, and interacts with antibodies. Binding of the peptide to the antibody can be demonstrated by any method or assay known in the art such as binding-, ELISA, and RIA-type of assays or competition assays (eg see Examples section and Current protocols in immunology).
• modulation or “modulate” as used herein refer to both upregulation (i.e., activation or stimulation (e.g., by agonizing or potentiating)) and downregulation (i.e. inhibition or suppression (e.g. by antagonizing, decreasing or inhibiting) of the binding between a peptide and an anti-HCV antibody.
• modulator refer to the ability of a compound as described above to modulate as described above.
• peptidomimetics refers to molecules which can be manufactured and which mimic those residues of peptides which modulate the interaction of the antibody with the peptide as described above.
• non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1 988), azepine (e.g., see Huffman et al. in Peptides: Chemistry and Biology, G. R.
• Fig 1 HPLC profile of tryptic digests of human natural acidic (a) and neutral (b) filaggrin.
• Peptides were separated by reversed-phase HPLC on a C-4 Vydac column (2.1 x 250 mm, Hesperia, CA) using a 140B Solvent Delivery System (ABI, Foster City, CA) and eluted with a 8-70% linear gradient of 70% acetonitrile in 0.1 % trifluoroacetic acid (TFA). Detection occured at 21 4 nm with a 1 000S diode array detector.
• TFA trifluoroacetic acid
• Fig3 Reactivity of 26 human RA sera with synthetic peptides on LIA.
• IGP1 1 54 consisted of an irrelevant synthetic peptide.
• Fig4 Inhibition ELISA using natural filaggrin as inhibitory agent. Plates were coated with purified human natural filaggrin at 1 ⁇ g/ml. Sera were diluted 1 /50 and added to the plate with or without preincubation with natural filaggrin at 1 or 10 ⁇ g/ml. OD values were measured at 450 nm.
• Fig5 Inhibition ELISA using synthetic citrulline-containing filaggrin peptides as inhibitory agents.
• Plates were coated with purified human natural filaggrin at 4 ⁇ g/ml.
• Sera were diluted 1 /50 and added to the plate with or without preincubation with the individual peptides (IGP1 1 55, 1 1 56, 1 1 57, 1 1 58) or a mixture of the four peptides (mix), each at 1 00 ⁇ g/ml.
• OD values were measured at 450 nm.
• Fig 7 2-D immunoblots of placental extracts.
• Natural filaggrin was purified from human skin obtained freshly after abdominoplasty according to the protocol of Simon et al. (1 993) .
• the epidermis was separated from the dermis by incubating the skin pieces at 56° C in PBS containing 5 mM EDTA. The material was stored dry at -20°C untill use.
• the epidermis was cut into small pieces which were homogenized in 40 mM Tris-HCI, pH 7.4, 1 50 mM NaCI, 5 mM EDTA, 0.5% NP-40, 0.1 % sodium azide, 0.1 mM PMSF (0.2 ml/cm 2 ) and stirred overnight at 4°C.
• the homogenate was centrifuged at 1 5,000 x g for 1 5 min and the extracted proteins in the supernatant were precipitated overnight at -20 °C by adding 5 volumes of absolute ethanol. After centrifugation for 1 5 min at 1 0,000 x g, the protein pellet was vacuum-dried and subsequently resuspended in water. This partially purified protein preparation is refered to filaggrin used in the present study. The amount of protein was determined by the Bradford protein assay as modified by Peterson (1 983) using BSA standard curves.
• the crude filaggrin preparation was submitted to 10% Tricine SDS-PAGE using the
• Bio-Rad mini-gel apparatus Two ⁇ g protein/cm was loaded in a large slot and electrophoresed under standard conditions. The gel was subsequently electroblotted onto nitrocellulose membrane in 10% methanol, 10 mM CAPS, pH 1 1 .0 during 40 min. The blot was blocked in PBS, 0.05% Tween20, 1 % gelatin and cut into 3 mm strips, which were probed with human sera overnight at a 1 /1 00 dilution in PBS, 0.05% Tween20, 0.1 % gelatin. As a secondary antibody the anti-human IgG-AP conjugate (Sigma, St Louis, Ml) was added at a 1 /1000 dilution; visualization occured with the NBT/BCIP chromogenic substrate.
• the filaggrin material was separated by 2-D electrophoresis using Immobiline DryeStrips pH 3-1 0 (Pharmacia Biotech, Uppsala, Sweden) in the first dimension and 1 0% Tricine SDS-PAGE in the second dimension using standard procedures.
• Preparative gels were loaded with 300 ⁇ g protein and stained with Coomassie R- 250.
• the large comma-shaped filaggrin spot was identified by immunoreaction with the antifilaggrin mAb (BTI, Stoughton, MA) .
• the pi of this heterogeneous protein ranged from 6.7-8.5 (7.1 -8.5 on gel), while molecular weight forms of 35-68 kDa were detected, with the more acidic isoforms representing the highest masses.
• the large filaggrin spot was cut out of the gel and separated into three parts: i) acidic fraction with pi 7.1 -7.5; ii) neutral fraction with pi 7.5-8.1 ; iii) basic fraction with pi 8.1 -8.5.
• Each fraction was electro-eluted by the method of Hunkapiller et al. ( 1 983) using 50 mM (NH 4 )HC0 3 , 0.1 % SDS as elution buffer.
• Coomassie stain and SDS were removed from the eluted proteins by ion pair extraction as described by K ⁇ nigsberg and Henderson (1 983). Vacuum-dried protein pellets were redissolved in the appropriate buffer for further analysis.
• Peptides were separated by reversed-phase HPLC on a C-4 Vydac column (2.1 x 250 mm, Hesperia, CA) using a 1 40B Solvent Delivery System (ABI, Foster City, CA) and eluted with a 8-70% linear gradient of 70% acetonitrile in 0.1 % trifluoroacetic acid (TFA). Detection occured at 21 4 nm with a 1 000S diode array detector and peptides were manually recovered.
• TFA trifluoroacetic acid
• APF serum pool also scored positive with both T34 and T38, while IG35247 was only reactive with T4.
• the APF negative control serum was clearly unreactive with the peptide fractions.
• T38 T39: Same peptides as in fraction T35
• ⁇ represents the amino acid citrulline, which was found at a specific retention time different from that of arginine. Further proove for the presence of citrulline was i) the fact that the sequence was not cleaved at that specific position, which would be expected if arginine was present and ii) that no arginine residue was sequenced.
• T45 and T81 There was one fraction (T48) that showed aspecific reactivity with all sera including the negative control serum.
• T48 There was one fraction (T48) that showed aspecific reactivity with all sera including the negative control serum.
• T32, T36, T37, T48 and T65 yielded no reliable amino acid sequencing signals.
• the fifth peptide was clearly derived from the second peptide, most probably due to the fact that in the neutral filaggrin two forms with and without citrulline- modification do exist. These results indicate that the same four peptides were retrieved as identified in the acidic T35 fraction.
• T28, T31 , T34 yielded no sequencable signals.
• IGP1 1 56 IGP1180
• IGP1158 IGP1182 HSTSQEGQDTIHGHRGS HSTSQEGQDTIHGHRGS
• Streptavidin-complexed peptides were applied directly on a nylon membrane with a plastic backing. Blocked strips were incubated overnight with human sera diluted 1 /1 00 in 1 ml PBS, 0.5% caseine, 0.1 % Triton X705, 1 0 mM MgCI 2 .6H 2 O. After washing with PBS, 0.05% Tween20, goat anti-human IgG-AP conjugated (Promega) diluted in PBS, 0. 1 % caseine, 0.2% Triton X705 was added for 1 h30.
• Strips were developed in 1 00 mM NaCI, 1 00 mM Tris-HCI, pH 9.8, 50 mM MgCI 2 .6H 2 O substrate buffer containing the chromogenic substrate NBT/ BCIP. Reaction was stopped after 30 min by addition of 0.2 N H 2 SO 4 .
• IGP1 1 55, IGP1 1 56, IGP1 1 57, and IGP1 1 58 were not retrieved when tested with the non-modified counterparts IGP1 1 79, IGP1 1 80, IGP1 1 81 , and IGP1 1 82 (Fig. 3). Only for 2 sera, a weak colouring of all non-modified peptides was obtained, which could be regarded as aspecific background staining related to the particular sera. These results indicate that the presence of citrulline is indispensable for immunoreactivity and that this unusual amino acid constitutes an important epitope for antifilaggrin antibodies.
• Electro-eluted human natural filaggrin was coated in Maxisorp polystyrene plates at a concentration of 1 or 4 ⁇ g/ml in 50 mM carbonate buffer, pH 9.6 overnight at 4°C.
• Sera were diluted 1 /50 in PBS, and preincubated with either the appropriate peptides at 1 00 ⁇ g/ml, with isolated natural filaggrin at 1 or 1 0 ⁇ g/ml (positive control) or with PBS (negative control) for 2 hours.
• the plates were blocked 1 hour with PBS, 0.1 % case ⁇ ne at 37°C and subsequently incubated with the sera. During 2 hours at 37 ° C.
• PCR primers were designed as follows:
• the PCR sense primer was chosen to overlap the filaggrin linker sequence and was designed to introduce a functional initiation codon (Kozak environment) upstream of the linker sequence (Phe Leu Tyr Gin Val Ser Thr) .
• the linker sequence was included in the amplified filaggrin repeat, because it is possibly involved in the correct targetting of the processed protein.
• An EcoRI restriction site was introduced for subcloning of the PCR fragment.
• the antisense PCR primer is the antisense PCR primer
• the antisense primer is located just upstream from the next filaggrin linker sequence and introduces a translation stop codon TAG.
• the amplified filaggrin sequence consists therefore of the filaggrin linker followed by an integral filaggrin repeat and three additional amino acids (Pro/Gly/His) resulting from the cloning strategy.
• the PCR amplified fragments were cloned in a pBLSK
• the four individual clones characterized by sequencing were named HB2641 , HB2642, HB2648 and HB2650 (Fig 6) .
• the cDNA inserts were recloned as EcoRV/Ecl 1 3611 fragments ( 1 030 bp) in the E. coli expression vector pIGRHISA opened with Hsil blunted.
• the filaggrin proteins were expressed as recombinant filaggrin-His ⁇ fusion proteins in three different E. Coli strains, resulting in high Coomassie stainable expression levels.
• the His6 tail of the fusion protein allows easy purification of the protein using metal-affinity chromatography.
• Example 5 Vimentin as marker for diagnosis of rheumatoid arthritis
• Placental tissue stored at -70°C was homogenized in 50 mM Tris-HCI, pH 7.4, 1 20 mM NaCI, 1 .5 mM DTT, 0.02% NaN 3 , 1 mM PMSF, and 5 ⁇ g/ml of chymostatin, leupeptin, antipain, pepstatin. After clarification, the homogenate was subjected to DE52 ion exchange chromatography; the 200, 250 and 300 mM salt elution fractions were further analyzed by 1 - and 2-D gelelectrophoresis.
• Proteins present in the different chromatographic fractions were separated by 2- D gel electrophoresis. Samples were resolved in rehydration buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 0.5% Triton X-1 00, 1 % Pharmalytes 3-10, 1 0 mM DTT, 0.01 % Orange G. Fifty-500 ⁇ g of protein was applied onto IPG4-7 iso-electrofocusing strips (Pharmacia) by the in-gel rehydration method of Rabilloud et al. (1 997). First dimension strips were loaded on 1 0% Laemmli gels or SDS Gradient 1 2-1 4 ExcelGels (Pharmacia) for second dimension running according to standard procedures.
• Coomassie-stained protein spots were cut out from 2-D gels, washed with water and 50% acetonitrile/ 1 % TFA and incubated with 0.08 ⁇ g trypsine in 1 0 ⁇ l 25 mM NH 4 HC0 3 / 1 0% acetonitrile, pH 8.0 during 20 hours at 37 ° C. Peptides were extracted with 60% acetonitrile/ 0.1 % TFA, vacuum-dried and redissolved in 1 0 ⁇ l 30% MeOH/ 1 % formic acid. Approximately 5% of the digest mixture was subjected to DE-MALDI-RETOF-MS analysis on a Voyager-DE STR (Perseptive Biosystems, Framingham, MA).
• Calreticulin was found as an abundant spot of 56-61 kDa, pi 4.3, while PDI was observed at 58-64 kDa with a pi of 4.6-4.8, hence both corresponding to the expected theoretical 2-D positions. The latter spot was further identified as being PDI by tandem MS analysis.
• the anti-vimentin mAb showed strong reactivity as shown in Fig 7b with numerous proteins between 40 and 61 kDa. This pattern proved highly reproducable between different batches of purified material, even when derived from different persons and tested at different points in time. Upon superimposing the blots probed with RA sera on the anti-vimentin blot, it became clear that the RA-specific reactivity colocalized with the high MW form of vimentin.
• the spots of intrest were further identified by MALDI-TOF and tandem mass spectrometry.
• peptides from human vimentin, cytokeratin 1 and cytokeratin 9 were retrieved in addition to other non- identified peptides. These proteins all belong to the same family of intermediate filament proteins.
• citrullinated forms of vimentin and cytokeratins are already described to occur in vivo (Senshu et al., 1 992; Senshu et al., 1 995; Senshu et al., 1 996) , it is likely that they constitute targets for autoantibodies present in rheumatoid arthritis sera.
• Citrullinated proteins have been shown to cause a mobility shift on SDS-PAGE towards higher MW regions in comparison with the non-citrullinated forms (Senshu et al., 1 995; Tarcsa et al., 1 996), which could explain the selective immunoreactivity of the human sera to the highest MW forms of vimentin.
• Specific intermediate filament protein forms could hence be used as marker for diagnosing rheumatological disorders.
• the Sa system a novel antigen-antibody system specific for rheumatoid arthritis.
• Vincent C Serre G., Lapeyre F., Fournie B., Ayrolles C, Fournie A., Soleilhavoup J.-P. (1989) High diagnostic value in rheumatoid arthritis of antibodies to the stratum corneum of rat oesophagus epithelium, so-called 'antikeratin antibodies'. 1 989. Ann. Rheum. Dis., 48: 71 2-722 Vincent C, Serre G., Basiie J.-P., Lestra H.C., Girbal E., Sebbag M.,

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