JP2002512939A - Citrulline-containing synthetic peptide recognized by serum of rheumatoid arthritis as a tool for diagnosis and treatment - Google Patents

Abstract

  (57) [Summary] A method for the production of certain peptides containing citrulline residues constituting the immunogenic determinants of antibodies present in serum from patients with rheumatoid arthritis, wherein the presence of at least one citrulline is Essential for reaction with antibodies. The present invention also relates to a method for producing the antibody and the use of the peptide for diagnosis and treatment of rheumatoid arthritis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to certain citrulline-containing peptides, which constitute immunogenic determinants present in sera from patients with rheumatoid arthritis, wherein the presence of at least one aitrulline lacks a reaction with the antibody For peptides that cannot be used. The present invention also relates to a method for producing the peptide and use of the peptide for diagnosis and treatment of rheumatoid arthritis and related diseases. The present invention also relates to novel filaggrin alleles. Rheumatoid arthritis (RA) is a major and devastating joint disease of virtually systemic, unknown etiology. It affects 1% of the population, with a male to female ratio of 2
: 3. In terms of morbidity, the most important feature of RA is joint erosion, which leads to pain, deformity and possibly severe disability. The life expectancy of patients with severe forms of the disease is reduced by 10 years. RA has all the features of autoimmune diseases, including the presence of various autoantibodies in the serum of patients and the potential to increase disease due to the migration of pathogenic T cells in animal models. The classification of the disease may be suspected on the grounds that borderline morphology is fairly common; moreover, joint inflammation is not limited to RA and other non-inflammatory diseases such as osteoarthritis, reactive arthritis and gout. -It also happens in autoimmune diseases.

Because early diagnosis allows for tailored treatments that can greatly improve the quality of life of RA patients, it is very unlikely that rheumatologists will have reliable diagnostic criteria for their treatment. is important. Serologic support for such diagnosis is poorly established and is mainly based on the presence of rheumatoid factor (RF). Positive Waller-Rose (Waal
er-Rose) or latex-fixed RF tests have definitive values and relate to diseases with more severe consequences. However, a significant number of RA patients are RF seronegative,
RF, on the other hand, is also present in other rheumatic diseases, including Sjogren's syndrome and systemic lupus erythematosus, in certain chronic bacterial and acute viral infections, in certain parasitic and chronic inflammatory diseases, and in control sera from healthy people. (Waaler et al., 1964; Chen et al., 1987). This rather low specificity of RF
-Requires additional testing of specific antibodies. The prevalence of the above-defined antinuclear antibodies such as SSA, RA33 and RNP in RA is low, and their clinical use for the diagnosis of RA is so far limited (Hassfeld et al., 1993). Conversely, both antinuclear perinuclear factor (APF) and anti-keratin antibody (AKA) have proven useful in this regard. APF is an IgG antibody in serum from RA patients that causes a typical immunofluorescent staining pattern in human buccal mucosal cells (Nienhuis and Mandema
, 1964). Several studies have addressed the diagnostic utility of this test (Westgees
Janssens et al., 1988; Vivino and Maul, 1989; Youinou et al., 1992; Fel.
tkamp et al., 1993). High specificity of 80-90% of APF for RA and 5
The good sensitivity of 0-99% makes it a really suitable diagnostic tool. Its value is particularly evident in about one-third of RA patients without RF activity (Westgee
St et al., 1987; Nesher et al., 1992). The second specificity found in the serum of RA patients is Y
AKA, an autoantibody first described by Oung et al. (1979), produces a fluorescent staining pattern with the keratinized layer of rat esophageal epithelium. Despite the lack of biochemical characterization of its target, the antibody was inferred to be an anti-keratin antibody on the suspicion that cytokeratin constitutes a major component of the stratum corneum. These antibodies are present in 36-59% of RA sera and have been found to be very specific for RA (Johsnon et al., 1981; Miossec et al., 1982; Hajiroussou et al., 1985; Vinc
ent et al., 1989). Like APF, they are mainly of the IgG class (Johnson et al.,
1981; Kataaha et al., 1985; Vincent et al., 1980), both are detected in synovial fluid of RA patients (Youinou et al., 1985; Kirstein et al., 1989; Vivino and Maul, 1990).

[0003] The human epithelial protein filaggrin was recently identified by convincing evidence as one of the major targets in reactivity with AKA and APF autoantibodies (Simon
1993; Sebbag et al., 1995). Initial studies showed that the neutral / acidic form of human filaggrin reacted with 75% of the 48 RA serogroups on Western blots (Simon et al., 1993). In more recent data (Vincent et al., 1997), 279 RA
It showed a diagnostic sensitivity of more than 50% for the detection of anti-filaggrin with a matching specificity of 95% within the 492 serogroups including serum. Profilaggrin, a precursor of filaggrin, is histidine-rich ± 400 kDa
Is an insoluble protein. It consists of 10 to 12 filaggrin repeats of 324 amino acids separated by a heptamer linker sequence. Highly phosphorylated polyproteins are stored in keratohyalin granules in the epithelial granular layer. In the terminal differentiation of these cells, profilaggrin is dephosphorylated to a 37 kDa functional basic filaggrin molecule and proteolytically processed by removal of the linker (Resing et al., 1989; 1993; Gan et al., 1990). ). In low-grade keratinocytes, these units are involved in the aggregation of keratin intermediate filaments,
Promotes the formation of intermolecular disulfide bonds, resulting in an intercellular fiber matrix of keratinocytes (Dale et al., 1978; Lynley and Dale, 1983; Harding and Scott, 1983; Ma
ck et al., 1993). After filament aggregation, basic arginine residues are converted to citrulline by peptidylarginine deiminase, resulting in a lower affinity molecule for cytokeratin (Harding and Scott, 1983). Finally, filaggrin is completely proteolyzed to free amino acids, urocanic acid and carboxylic pyrrolidonic acid, which are responsible for maintaining optimal levels of moisture and absorbing UV light
(Scott et al., 1982).

The filaggrin units arranged in tandem are very polymorphic: there are considerable variants in the individual amino acid sequences, and differences of up to 15% between different human filaggrin molecules have been observed. (McLinley-Grant et al., 1989; Gan et al., 1990; M
arkova et al., 1993). Although most variants can accommodate a single base change, many also involve a change in charge. Since the human profilaggrin gene system is still polymorphic in size due to allelic differences between individuals in the number of repeats (10, 11 or 12), there may be 24 different filaggrin molecules in some individuals (McLinley-Grant et al., 1989; Presland et al., 1992). Amino acid variants, along with dephosphorylation events, are responsible for the apparent heterogeneity observed in the two-dimensional gel; conversion of arginine to citrulline is an additional cause of acidification of the molecule. The use of natural filaggrin isolated from human tissues is rather impractical due to the limited class of variability, limited availability of starting materials for troublesome preliminary isolation and purification, and therefore synthetic peptides and Recombinant proteins have been tested to develop diagnostic test kits for RA. Recombinant filaggrin was expressed in eukaryotic COS cell lines. However, this approach did not seem to be successful because none of the four cloned proteins reacted with the serum of APF-positive patients.

[0005] It is an object of the present invention to provide a peptide having high reactivity to antibodies present in serum from rheumatoid arthritis patients. Another object of the present invention is to provide a method for obtaining said peptide. Another object of the present invention is to provide a method for generating an antibody specifically reactive with the peptide. It is another object of the present invention to provide a method that is specifically reactive with the above-described antibodies, thereby producing an anti-idiotypic antibody resembling the peptide. Another object of the present invention is to provide a pharmaceutical composition containing these peptides for treatment or diagnosis. Another object of the present invention is to provide a diagnostic kit for rheumatoid arthritis. Another object of the present invention is to provide novel filaggrin alleles and their corresponding amino acid sequences. It is another object of the present invention to provide a bioassay for identifying compounds that modulate the interaction between an autoantigen and a rheumatoid arthritis-specific autoantibody. Another object of the present invention is to provide modulators, compositions comprising modulators or combinations of modulators identified by the bioassays described above.

[0006] All objects of the invention fulfill the following embodiments of the invention. Immunodominant epitopes of filaggrin were identified, all containing the unusual amino acid citrulline. Synthetic peptides were produced and proved to be useful in the diagnosis of RA. Another specificity was found by probing human rheumatoid arthritis serum on two-dimensional blots containing placental extracts. Immunoreactive spots were identified and peptides from human vimentin, cytokeratin 1 and cytokeratin 9 were recovered in addition to other non-identified peptides. All of these proteins belong to the same family of intermediate filament proteins. Citrullinated forms of vimentin and cytokeratin have been previously described to occur in vivo (Senshu et al., 1992; Senshu et al., 1995; Senshu et al., 1996).
Appear to constitute a specific target for autoantibodies present in rheumatoid arthritis serum. According to its main embodiment, the present invention provides that the invention provides that at least one arginine is replaced by citrulline, wherein the presence of the citrulline is important for the reaction between the peptide and the antibody, the antibody being present in serum from patients with rheumatoid arthritis. It relates to peptides comprising less than 50 amino acids, including fragments of filaggrin variants or fragments of intermediate filament proteins, which are capable of reacting with the antibodies present.

According to a preferred embodiment, the present invention relates to a peptide according to claim 2. According to a further embodiment, the present invention also relates to peptides and / or chemical structures comprising any of the aforementioned peptides fused to a linker molecule. The present invention also relates to a peptide comprising and / or consisting of at least two tandem repeats of any of the aforementioned peptides or a branched peptide comprising at least one of the aforementioned peptides. According to a more particular embodiment, the invention also relates to classical chemical synthesis in which at least one arginine residue is replaced by citrulline at some step during the chemical synthesis.
It also relates to a method for producing any of the above-mentioned peptides. The invention also provides for the production of any one of the preceding peptides, wherein the primary amino acid sequence is produced by classical chemical synthesis, and after chemical synthesis, the arginine residue is induced into citrulline by incubation with peptidylarginine deiminase. It also concerns how to do it. The present invention also relates to a method of producing any of the above-described peptides, comprising the steps of: (i) directing a suitable cell host to express and / or secrete the peptide or protein containing the peptide; Transformation with a recombinant vector into which a polynucleic acid containing a sequence encoding the peptide has been inserted under the control of appropriate regulatory elements, (ii) allowing expression of the protein or the peptide, Culturing the transformed cell host under conditions that allow for partial or optimal induction of the arginine residue into a citrulline residue, (iii)
) Harvesting the peptide. The present invention also relates to a method of producing any of the above-described peptides, comprising the steps of: (i) providing a suitable cellular host with the peptide or protein containing the peptide being expressed, and / or Transformation with a recombinant vector into which a polynucleic acid containing a sequence encoding the peptide has been inserted under the control of an appropriate regulatory element so as to be secreted, and (ii) conditions enabling expression of the protein or the peptide. Culturing said transformed cell host, (iii) harvesting said protein or peptide, and (iv) directing arginine residues of said protein or peptide to citrulline residues. According to a more particular embodiment, the present invention also relates to a method according to the preceding, wherein said host cell is a bacterial host or yeast or any other eukaryotic host cell, preferably transformed by a recombinant baculovirus. Regarding one.

[0008] According to a preferred embodiment, the invention also relates to an antibody, wherein said antibody is preferably a monoclonal antibody and which is specifically reactive with said peptide or intermediate filament protein comprising a citrulline residue. The present invention also relates to anti-idiotype antibodies generated by immunization with the above antibodies, wherein the anti-idiotype antibodies specifically react with the antibodies, thereby mimicking any of the above peptides. , Preferably a monoclonal antibody. According to a more particular embodiment, the present invention also relates to an immunotoxic molecule comprising and / or consisting of a cell recognition molecule which is the above-mentioned peptide or the above-mentioned antibody covalently linked to a toxic molecule or an active fragment thereof. Related to According to a further embodiment, the present invention relates to any of the above-mentioned peptides or antibodies or immunotoxic molecules or intermediate filament proteins or compositions thereof for use as a medicament. The use has purpose as treatment or diagnosis of rheumatoid arthritis. The present invention also relates to a method of reducing systemic reactivity to autoantibodies following oral administration of any of the above-described peptides or antibodies or immunotoxic molecules or intermediate filament proteins or compositions thereof, thereby reducing anti-autologous activity. It also relates to the treatment of autoimmune diseases by preventing the pathogenic production of antibodies. The present invention also relates to a diagnostic kit for detecting rheumatoid arthritis, said kit comprising at least one of the above described peptides or proteins or antibodies, wherein said peptides, proteins or antibodies are bound to a solid support Have been. More preferably, the kit comprises certain peptides or antibodies described above, possibly together with antigens constituting immunogenic determinants of other autoimmune diseases, wherein the peptides, antibodies and / or proteins are on a solid substrate. It binds to a specific position. More preferably, the solid support is a piece of membrane and the polypeptide is coupled to the membrane in parallel lines. Alternatively, the particular peptide or protein described above is not bound to a solid support, but is provided in a binding solution and used as a competitor, or present in serum from a patient with an autoimmune disease other than rheumatoid arthritis. Should be understood to be used to block other antibodies, thereby reducing or eliminating possible cross-reactivity and / or non-specific binding.

[0009] The epitope mapping technique identified an immunodominant epitope of filaggrin that occurs in patients with rheumatoid arthritis (see Example 1). These epitopes are further characterized by the presence of citrulline residues resulting from the induction of arginine residues. The presence of the citrulline residue is essential for recognition by antibodies present in serum from rheumatoid arthritis. According to its main embodiment, the present invention relates to peptide fragments of these native filaggrin variants, wherein said fragments react with antibodies characteristically present in the sera of patients with rheumatoid arthritis and are further characterized by post-translational modifications. And more preferably characterized by the induction of arginine to citrulline. The presence of at least one citrulline is essential for recognition by antibodies specifically present in the sera of rheumatoid arthritis patients. A synthetic peptide is obtained in which the arginine residue has been replaced by citrulline, thus mimicking the epitope of the native filaggrin variant. These peptides have proven useful in the diagnosis of rheumatoid arthritis. According to another embodiment, the present invention relates to peptides that immunologically mimic immunogenic determinants of self-proteins recognized by the immune system of patients suffering from rheumatoid arthritis. Thus, the presence of one citrulline is expected to be sufficient for specific recognition by certain antibodies present in the sera of rheumatoid arthritis patients.

As used herein and in the claims, the term “peptide” refers to a polymer of amino acids, regardless of the length of the product; thus, oligopeptides, polypeptides and proteins are defined as “peptides”. Included in the definition. The term also does not exclude post-expression modifications of the peptide, for example, glycosylation, acetylation, phosphorylation, and the like. For example, peptides containing one or more amino acid analogs (including, for example, unusual amino acids, PNAs, etc.), polypeptides having substituted bonds, and naturally occurring as well as non-naturally occurring art-known polypeptides. Polypeptides with other modifications are also included within the definition. The expression "peptide containing less than 50 amino acids" is used whenever the terminology is used, as an essential part of the entire immunoreactive protein which still contains a highly reactive domain characterized by the presence of citrulline residues. It should be interpreted broadly to encompass truncated versions. These peptides preferably have a length of 40, 30, 25, 20 or less amino acids.

“Immunogenic determinants” are defined as primary amino acid sequences that determine the specific reactivity of whole antigens against the generated antibodies, as well as chemical modifications involving secondary modifications of specific three-dimensionally arranged amino acid residues. Means a group. Such antibodies may also be 'immunologically mimic'
It also recognizes different chemical groups, called immunogenic determinants, called If a secondary modification of the peptide is said to be 'necessary' or 'critical' or 'indispensable' for reaction with the antibody, then due to the absence of the secondary modification,
The dissociation constant of the interaction with the antibody is at least two orders of magnitude higher, preferably three orders of magnitude higher, more preferably the dissociation constant of the interaction between the antibody and the peptide in which the secondary modification is present. Would result in a four orders of magnitude higher peptide. The term "cross-reactivity" also refers to the reaction between an antibody and a specific antigen that occurs against another antigen or against antibodies found in sera from patients with different diseases.

According to a more particular embodiment, the present invention provides a method for treating a citrulline residue, wherein the citrulline residue is important for high affinity interaction with an antibody characteristically present in the serum of a rheumatoid arthritis patient. These peptides or proteins comprising: In a more specific embodiment, the present invention relates to the amino acid sequence HSASQDGQDTIRGHPGSS or HSGIGHGQASSAVRDSGHRGYS or DSGHRGYSGSQASDNEGH or HSTSQEGQDTIHGHRGS or GGQGSRHQQAR or QGSRHQQARDSSRHSTSQEGQDTIHGHRGS or QGSRHQSGDSG Mimics the major immunogenic determinants of filaggrin. ] Is characterized by the following.

In a more specific embodiment, the present invention relates to a vimentin, a site comprising at least one citrulline residue, wherein the presence of citrulline is important for reacting with antibodies present in serum from rheumatoid arthritis patients. A peptide comprising less than 50 amino acid sequences of either keratin 1 or cytokeratin 9 variants. The invention also relates to a molecular structure which represents in a minimal part a peptide or antibody as defined above. Such molecular structures can be formed by specifically interacting with other peptide and / or protein and / or molecular structures that allow the fusion polypeptide and / or protein to be added and / or bound to specific tissues or cell types. It may be further characterized, or, for example, the presence of four or five or six consecutive histidine residues may result in purification of the molecule, or T-cells and / or B, such as cholera toxin. -Arising from the fusion of a peptide of the invention with peptides and / or proteins and / or other molecules which are cytotoxic to cells or which can be labeled by means of radioactive or fluorescent or immunogold or enzyme markers.

[0014] In some cases, it is desirable to bond two or more peptides to one peptide structure or to synthesize a peptide having a branched chain. One advantage of this arrangement is known in the art and relates to diagnostics. If the antigen is used in an assay to detect the antibody present, peptides with tandem repeats or branches of the antigen will increase the amount of immobilized antigen presented to the antibody, thereby increasing the sensitivity of the assay. To rise. Sensitivity increases exponentially when the immobilized antigen is used together with a specific concentration of a soluble form of such antigen, thereby inducing the formation of cross-reacted antigen-immunoprecipitates. The second advantage relates to treatment. Deposition of autoantigen autoimmune complexes in various tissues is an important step towards understanding pathological conditions. It is generally accepted that the major cause of the deposit is poor liver blood clearance of the antigen-immune complex as a result of the small size of the complex. Administration of a tandem repeat or branched form of the peptide can increase the size of the antigen-immune complex formed, thereby increasing clearance and thus reducing deposition of the complex Will.

[0015] The present invention also relates to cyclized forms of the peptides, the advantages of which are known in the art, and the present invention further relates to linear peptides in a more randomly coiled form. And also increases the affinity of conformationally constrained peptides in other cases. One of ordinary skill in the art would be able to conservatively as well as conservatively modulate the resulting negative features of the claimed peptides, such as rapid degradation, solubility, cytotoxic effects, and the like.
One could design conservative amino acid substituents, or substituents with non-natural amino acids, and the like. Generally, they are less than 35% of the sequence. Such peptides also include peptides having a linkage that is replaced, as well as other art-known modifications, both naturally occurring and non-naturally occurring. If the filaggrin peptide of the invention is highly polymorphic, one or more amino acids may be altered to better mimic different epitopes or to be recognized by antibodies in serum from rheumatoid arthritis patients. It is desirable to change.

According to another embodiment, the present invention also relates to a novel, isolated, cloned, sequenced and characterized by the DNA sequence shown in FIG. 6 and the amino acid sequence shown in FIG. It also relates to allelic variants. The present invention also relates to any analog of the peptide of the present invention. The term "analog" as used herein or in the claims encompasses a protein or peptide having substantially the same amino acid residue sequence as the sequence specifically set forth herein, wherein One or more residues are conservatively substituted with biologically equivalent residues. Examples of conservative substituents are the replacement of a hydrophobic residue such as isoleucine, valine, leucine or methionine with another, the replacement of a water-soluble residue such as between arginine and lysine, between glutamine and asparagine, between glycine and serine. Includes substitution for another, substitution of one basic residue such as lysine, arginine or histidine for another or substitution of an acidic residue for another such as aspartic acid or glutamic acid. Examples of possible mutations according to the invention are found in Table 1. The term "conservative substitution" also includes the use of chemically derived residues in place of non-conservative residues. However, the resulting protein or peptide is biologically equivalent to the protein or peptide of the present invention.

“Chemical derivative” refers to a protein or peptide having one or more residues chemically induced by the reaction of a functional side chain, a peptide having a substituted bond, and known in the art. As well as other naturally occurring repair forms. Examples of such derived molecules are amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butoxyloxycarbonyl groups, chloroacetyl groups or formyl groups in which a free amino acid group has been derived. But not limited to these molecules. Free carboxyl groups can be derived to form salts, methyl and ethyl esters or other types of esters, or hydrazides. Free hydroxyl groups can be derived to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine can be induced to form N-imibenzyl histidine. Also, naturally occurring amino acid derivatives of the 20 standard amino acids
These proteins or peptides containing one or more are also included in the chemical derivatives. For example, 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; ornithine can be substituted for lysine. The peptides of the invention may also have one or more additions of residues to the sequence of the peptides set forth herein and / or as long as the peptide is biologically equivalent to the protein or peptide of the invention. Includes any protein or peptide with a deletion.

[Table 1]

In addition, additional amino acids or chemical groups can be added to the amino or carboxyl terminus to create a “linker arm” to which the peptide can be conveniently attached to a carrier. The linker arm is at least one amino acid, and may be as many as 60 amino acids, but will most often be from 1 to 10 amino acids.
The nature of attachment to the solid phase or carrier may be non-covalent or covalent.
Possible arrangements of this nature are well described in the art. Naturally occurring amino acids, such as histidine, cysteine, lysine, tyrosine, glutamic acid, or aspartic acid, can be added to either the amino or carboxyl terminus to provide a functional group that couples to a solid phase or carrier. However, other chemical groups, such as biotin and thioglycolic acid, which add desirable chemical or physical properties to the peptide are added to the termini. The termini of the peptides can also be modified, for example, by N-terminal acetylation or terminal carboxy-amidation. In each case, the peptide will preferably be as small as possible, while retaining substantially the overall sensitivity of the larger peptide.

The peptides and especially fragments of the present invention can be prepared by classical chemical synthesis. The synthesis is performed in a homogeneous solution or solid phase. For example, a synthesis technique in a homogeneous solution that can be used is described in "Me, published by E. Wush, vol. 15-I et II. THIEME, Stuttgart 1974.
thode der organischen chemi "(method of organic chemistry) as described by Houbenweyl. The polypeptides of the invention are also described in" Solid phase peptide synthesis "(IRL Press, Oxford , 1989) in a solid phase by the method described by Atherton and Shepard in a book titled The claimed peptide form is obtained by substitution of the original arginine derivative with a citrulline residue in classical chemical synthesis or by contacting the synthesized peptide with any eukaryotic peptidylarginine deiminase.

[0020] The polypeptides according to the invention can also be prepared as described in Maniatis et al., Molecular Cloning: A Labo
ratory Manual, New York, Cold Spring Harbor Laboratory, 1982), by a method of recombinant DNA technology, into a suitable vector, a claimed nucleic acid or a polynucleic acid encoding a portion of the claimed peptide. It can be produced by inserting a sequence and transforming an appropriate host with the vector. The recombinant expression vector comprises a polynucleic acid as defined above or a portion thereof, operably linked to prokaryotic, eukaryotic or viral transcription and translation control elements. In addition, this sequence can be operably linked to a sequence that allows for secretion of the claimed peptide. The term 'vector' includes a plasmid, cosmid, phage or virus or transgenic organism. In particular, BCG or adenovirus vectors and avipox recombinant viruses are useful. Recombinant peptides can be obtained from any prokaryotic species, either in vitro by contacting the expressed and / or secreted peptide, or in vivo by selecting an appropriate host, such as yeast or any eukaryotic cell. (in vi
vo), and more preferably by using a baculovirus transformation system or by co-expressing the peptide with a recombinant peptidylarginine deiminase. Also, any of the known purification methods for recombinant peptides can be used to produce the recombinant peptides of the present invention. The invention also relates to a recombinant expression vector comprising a polynucleic acid as defined above or a part thereof operably linked to prokaryotic, eukaryotic or viral transcription and translation control elements. Generally, the recombinant vector, when injected as naked DNA,
A vector sequence followed by a nucleotide sequence encoding the peptide, a suitable prokaryotic, eukaryotic or viral promoter sequence, wherein the recombinant vector is a prokaryotic or eukaryotic host or a polypeptide as defined above in a living mammal. Which allows expression and / or secretion of any one of In addition, any known method for purifying a recombinant protein can be used for producing the recombinant polypeptide of the present invention. The term 'vector' includes a plasmid, cosmid, phage or virus or transgenic animal. In particular, BCG or adenovirus vectors and avipox recombinant viruses are useful for vaccine development.

The present invention also relates to transforming a suitable cell host with a recombinant vector in which a polynucleic acid as defined above or a part thereof has been inserted under the control of suitable control elements, expression and / or secretion of said insert. And culturing the transformed cell host under conditions that allow for the harvesting of the polypeptide.

The term “recombinantly expressed” as used within the context of the present invention,
The recombinant expression method of the protein of the present invention as described in detail in
It refers to the fact that it is produced in nuclear cells or lower or higher eukaryotic cells. The term "lower eukaryotic cells" refers to host cells such as yeast, fungi and the like. True lower
Nuclear cells are generally (but not necessarily) single cells. Yeast, especially Saccharomyces (Sac charomyces ), Shizo Saccharomyces (Schizosaccharomyces), Kulveromyces
(Kluveromyces), Picia (Pichia) (For example, Piscia pastoris (Pichia pastor is) ), Hansenula (Hansenula ) (E.g., Hansenula polymorpha (Hansenula  polymorpha )), Yarrowia (Yarowia), Schwanio Myces (Schwaniomyces),
Zosaccharomyces (Schizosaccharomyces,), Chigosaccharomyces (Zygosacch aromyces ) Are preferred lower eukaryotic cells. Saccharomyces
Cerevisiae (Saccharomyces cerevisiae), Es Karsbergenesis (S.carls bergensis ) And Kay Lactis (K.lactis) Is the most commonly used yeast inn
It is the main and simple fungal host. The term "prokaryotic cell" is called E. coli (E.coli), Lactobacillus (Lactobacillus)
, Lactococcus (Lactococcus), Salmonella (Salmonella), Streptococcus
(Streptococcus), Bacillus Sacillus (Bacillus subtilis) Or strep
Tomises (Streptomyces)Host. These hosts are also those within the present invention.
And intended. The term “higher eukaryotic cells” is derived from higher animals such as mammals, reptiles and insects
Means the host cell. Recently, a preferred higher eukaryotic host cell has been Chinese Hams.
(Eg, CHO), monkeys (eg, COS and Vero cells), neonates
Hamster kidney (BHK), pig kidney (PK15), rabbit kidney 13 cells (RK13)
, Human osteosarcoma cell line 143B, human cell lines HeLa and Hep G2
Human hepatoma cell lines and insect cell lines (eg, Spodoptera frangipelda (S podoptera frugiperda )). Host cells can be suspension or flask cultures
, Tissue culture, organ culture and the like. Alternatively, the host cell
It may be a transgenic animal.

The term “recombinant polynucleotide” or “nucleic acid”, depending on its source or operation, may (1) not be associated with all or a portion of a naturally occurring polynucleotide,
2) linked to a polynucleotide other than the one linked naturally, or (3)
) Polynucleotides or nucleic acids of non-naturally occurring genomic, cDNA, semi-synthetic or synthetic species are contemplated. “Recombinant host cell,” “host cell,” “cell,” “cell line,” “cell culture,” and other such terms refer to other microorganisms or higher eukaryotic cell lines that are cultured as single cells. A cell that can or has been used as a receptor for a vector or other transfer polynucleotide, including the progeny of the original cell to be transduced. The progeny of a single parent cell are not necessarily completely identical to the original parent in polymorphisms or in genomic or total DNA due to natural, accidental, or deliberate mutations. The term “replicon” acts as an autonomous unit of polynucleotide replication in a cell; ie, capable of replicating under its own control, eg, a plasmid, chromosome,
Any genetic element such as a virus or cosmid. The term "vector" is a replicon that further contains sequences that provide for the replication and / or expression of the desired open reading frame. The term "control sequences" refers to the polynucleotide sequences necessary to effect the expression of a linked coding sequence. The nature of such control sequences will vary depending on the host organism; in prokaryotes, such control sequences generally include a promoter, a ribosome binding site, a splicing site, and a terminator;
Such control sequences include a promoter, a splicing site, a terminator,
In some cases, includes enhancers. The term "control sequences" is intended to include the minimum of all components necessary for expression, and also to include other additional components whose presence is free, such as leader sequences that govern secretion. Including. The term "promoter" is a nucleotide sequence that includes a consensus sequence that binds RNA polymerase to a DNA template such that in a certain fashion, mRNA production begins at the normal transcription start site of the adjacent structural gene. The expression "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 the coding sequence is ligated such that expression of the coding sequence occurs under conditions compatible with the control sequences.

A polynucleic acid encoding a peptide of the invention and inserted into a vector sequence is attached to a signal sequence. The signal sequence may be from any source, for example, an IgG or tissue plasminogen activator (tpa) leader sequence for expression in mammalian cells or an α-mating factor sequence for expression in yeast cells. A variety of vectors can be used to obtain the peptides of the present invention. Lower eukaryotic cells such as yeast and glycosylation mutants are generally transformed with a plasmid or transduced with a recombinant virus. Vectors can replicate independently in the host or can be integrated into the host cell genome. Higher eukaryotic cells may be transformed with the vector or with a recombinant virus, eg, a recombinant vaccinia virus. Techniques and vectors for inserting foreign DNA into vaccinia virus are known in the art and utilize, for example, homologous recombination. All sequences necessary for mammalian expression are available in the art, including a wide variety of viral promoter sequences, or terminator sequences and poly (A) -addition sequences, or enhancer sequences and / or amplification sequences. Vaccinia is particularly preferred because vaccinia stops expression of host cell proteins. Vaccinia is also highly preferred because it allows expression of the fi peptide of the invention in cells or individuals immunized with live recombinant vaccinia virus. Avipox and Ankara Modified Virus (AMV) are particularly useful vectors for human vaccination. Also, baculoviruses, which are helper-independent virus expression vectors, and Autographa californica nuclear polynucleosis virus (A
Insect transfer vectors derived from cNPV) are also known. Expression vectors from this system typically use the strong viral polyhedrin gene promoter to drive expression of the heterologous gene. Different vectors and methods for introducing heterologous DNA into a desired site of a baculovirus are also available to those of skill in the art of baculovirus expression. Different signals of post-translational modifications recognized by insect cells are also known in the art. The invention also relates to a host cell transformed with a recombinant vector as defined above.

The present invention also relates to antibodies specifically raised against the peptides of the present invention, preferably against peptides in which arginine is induced into citrulline. These antibodies may be polyclonal or monoclonal. To prepare antibodies, a host animal is immunized with a peptide of the present invention in a pharmaceutically acceptable carrier, wherein at least one arginine has been converted to citrulline. Pharmaceutically acceptable carriers include any carrier that does not itself induce the production of harmful antibodies in 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 known to those skilled in the art.

Preferred adjuvants that enhance the effect of the composition are US Pat. No. 4,606,9
No. 18, aluminum hydroxide (alum), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP) ), N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2- (1 ′
-2'-dialuminitoyl-sn-glycero-3-hydroxyphosphoryloxy)
-RIBI containing three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL + TDM + CWS) in ethylamine (MTP-PE) and 2% squalene / Tween 80 emulsion
Including, but not limited to. Any of the three components MPL, TDM or CWS can also be used alone or in a 2x2 combination. In addition, Stimulon (S
Adjuvants such as timulon) (Cambridge Bioscience, Worcester, MA) or SAF1 (syntex) can also be used. In addition, Freund's complete adjuvant (CF
A) and Freund's Incomplete Adjuvant (IFA) can be used for non-human applications and research purposes. Generally, the immunological composition will include a vehicle such as pharmaceutically acceptable water, saline, glycerol, ethanol and the like. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances, preservatives and the like can be included in such vehicles. Typically, immunological compositions are prepared as injectables as liquid solutions or suspensions, or as solid forms suitable for solution or suspension in liquid carriers prior to injection. It can be emulsified or encapsulated in liposomes to achieve an enhanced adjuvant effect. The protein is also incorporated into an immunostimulating complex (ISOCOMS) with, for example, Quil A, a saponin.

The immunogenic composition used to procure the antibodies comprises a 'sufficient amount' or an 'immunologically effective amount' of the peptide of the invention and optionally any of the other components described above. An “immunologically effective amount” means that one or several separate administrations to an individual are effective to elicit an immune response and raise antibodies as defined above. This amount depends on the health and physical condition of the individual, the taxonomic group of the individual being treated (eg, non-human primates, primates, rabbits, etc.), the ability of the individual's immune system to synthesize antibodies, It will vary depending on the immunogenicity and mode of administration and other related factors. It is expected that doses will fall in a relatively broad range that can be determined through routine trials. Usually the amount is 0.01 to 100 μg / dose,
More specifically, 0.1 to 100 μg / dose. The immunogenic composition is conventionally administered parenterally, typically by injection, for example, either subcutaneously or intramuscularly. Further formulations suitable for other modes of administration include oral formulations and suppositories. The regimen may be a single dose schedule or a multiple dose schedule. The vaccine can be administered with other immunomodulators.

[0028] Host serum or plasma is collected after a suitable time interval to provide a composition comprising an antibody reactive with a peptide of the invention. For example, saturated ammonium sulfate or D
The use of EAE Sephadex, or other techniques known to those skilled in the art, provides gamma globulin factor or IgG antibodies. Antibodies do not substantially suffer from many of the adverse side effects associated with other antiviral agents, such as drugs against infectious, chronic or recurrent mononucleosis. Such antibodies can also be used to diagnose certain diseases, such as Burkitt's lymphoma, associated with Epstein-Barr virus.

The term “immunogenic” refers to the ability of a substance to cause a humoral and / or cellular response, whether alone or linked to a carrier, in the presence or absence of an adjuvant. The antibodies of the claimed invention may also be monoclonal antibodies that specifically react with any of the peptides, preferably the antibodies are monoclonal antibodies. The monoclonal antibodies of the invention are, on the one hand, immunized against the claimed peptide of the invention and, on the other hand, an animal, in particular a mouse or a mouse, immunized against the claimed peptide on cells of the myeloma cell line. Hybridomas are also produced by hybridomas that can be formed by classical methods from spleen cells from rats, and hybridomas are selected for their ability to produce monoclonal antibodies that recognize the citrullinated form of the peptide that was originally used for immunization of the animals. The antibodies according to the present invention can be labeled with a suitable label of the enzymatic, fluorescent or radioactive type. Monoclonal antibodies according to this preferred embodiment of the invention may comprise a portion of mouse and / or human genomic DNA sequences encoding heavy and light chains, or heavy and light chains.
Starting from a cDNA clone encoding the chain, it may be a humanized version of a mouse monoclonal antibody by means of recombinant DNA technology. Alternatively, a monoclonal antibody according to this preferred embodiment of the invention may be a human monoclonal antibody. These antibodies according to this embodiment of the invention may also be derived from human peripheral vascular lymphocytes of rheumatoid arthritis patients. Such human monoclonal antibodies can be obtained, for example, by the method of human peripheral vascular lymphocyte (PBL) repopulation of severe combined immunodeficiency (SCID) mice (see recent review, Duchosal et al., 1992) or of the present invention. It is prepared by screening individuals vaccinated with the antigen for the presence of reactive B cells.

The present invention also relates to anti-idiotype antibodies which are produced upon immunization with an antibody as defined above, react specifically with said antibody and thus mimic the peptide of the present invention. The present invention also relates to truncated or single chain versions of the antibodies and / or anti-idiotype antibodies as defined above, which retain the original specificity of reacting with the antigen. The present invention also relates to microproteins (
It also relates to proteins or peptides that mimic the antibody as defined above, such as the very hypervariable sites of a recombinant protein obtained by screening by microprotein or repertoire cloning.

The present invention also relates to a method of detecting an antibody present in a biological sample and specifically reacting with a peptide or anti-idiotype antibody of the invention, comprising: (i) Contacting a biological sample to be analyzed for the presence of the above peptide or anti-idiotype antibody, (ii) an immunological complex formed between the antibody and the peptide or anti-idiotype antibody Detecting.

The present invention also relates to a reverse method for detecting a peptide and / or anti-idiotype antibody of the invention, said method comprising an anti-idiotype antibody mimicking said peptide and / or such a peptide Using an antibody present in a biological sample that specifically reacts with: (i) the biological sample to be analyzed for the presence of the peptide or anti-idiotype antibody Contacting with an antibody; (ii) detecting an immunological complex formed between the antibody and the peptide or anti-idiotype antibody. The method as defined above can be used for the diagnosis of rheumatoid arthritis.

By virtue of the obvious embodiments, the present invention relates to a diagnostic technique which allows the distinction between these autoimmune diseases in which the characteristic antibodies often cross-react with the same antigen, thus resulting in difficult and slow diagnosis. About the development of. Such diagnostic techniques are obtained by the simultaneous use of several antigens and / or anti-idiotype antibodies of the invention.

The present invention also relates to a diagnostic kit for use in detecting the presence of said antibody, said kit preferably comprising said peptide or anti-idiotype antibody or microprotein bound to a solid support. It comprises at least one of the above defined peptides or anti-idiotype antibodies or microproteins. The present invention also relates to a diagnostic kit for determining the type of autoimmune disease, said kit preferably comprising a peptide as defined above together with said peptide or anti-idiotype antibody or microprotein bound to a solid support. Or at least one of an anti-idiotype antibody or a microprotein. The invention also relates to a diagnostic kit as defined above, said kit comprising a range of said peptide and / or anti-idiotype antibody or microprotein attached to a specific location on a solid substrate. The present invention also relates to a diagnostic kit as defined above, wherein said solid support is a piece of membrane and said peptide and / or anti-idiotype antibody or microprotein is coupled to the membrane in the form of parallel lines.

The immunoassay method according to the invention can utilize, for example, single or oligomeric antigens, dimeric antigens as well as combinations of single or specific oligomeric antigens.
The peptides of the invention can be used in virtually any assay format using known antigens to detect antibodies that characterize a disease or infection. A common feature of all of these assays is that, under conditions that bind the antigen to any such antibodies present in the component, an antigenic peptide or anti-idiotype antibody or microprotein is expressed in the body suspected of containing the antibody. Contacting the ingredients. Such conditions generally involve the use of excess antigen and will be at physiological temperature, pH and ionic strength. Incubation of the specimen with the antigen is followed by detection of the immune complex containing the antigen. The design of immunoassays is subject to many variations, and many formats are known in the art. Protocols can use, for example, solid supports or immunoprecipitates. Most assays involve the use of labeled antibodies or peptides;
The label may be, for example, an enzymatic, fluorescent, chemiluminescent, radioactive or staining molecule. Assays that amplify the signal from the immune complex are also known; examples are assays utilizing biotin and avidin or streptavidin and enzyme-labeled mediated immunoassays, such as ELISA assays. The immunoassay may be in a heterogeneous or homogeneous format and may be of no standard or competitive type limitation. In a heterologous format, the peptide or anti-idiotype antibody or microprotein is generally bound to a solid matrix or support after incubation to facilitate separation of the sample from the peptide or anti-idiotype antibody or microprotein. Examples of solid supports that can be used include nitrocellulose (e.g., in a membrane or microtiter well form), polyvinyl chloride (e.g., in a sheet or microtiter well), polystyrene latex (e.g., beads, or microtiter plates, polyvinylidene fluoride). (Known as Imunolon ^™ ), diazotized paper, nylon membranes, activated beads and protein A beads, such as Dynatech (Dynatech).
(Tech) Imunolon ^™ 1 or Imunolon ^™ 2 microtiter plates or 0.25 inch polystyrene beads (Precision Plastic Ball) can be used in heterogeneous formats. Typically, the solid support containing the antigenic peptide or anti-idiotype antibody or microprotein is washed after separating it from the sample prior to detecting the bound antibody. Standard and competitive formats are also known in the art.

[0036] In a homogeneous format, the specimen is incubated in combination with the antigen in solution. For example, any antigen-antibody or anti-idiotype antibody formed
-May be under conditions under which the antibody or microprotein-antibody complex precipitates. Both standard and competitive formats for these assays are known in the art. For example, to characterize rheumatoid arthritis in a standard format, the amount of rheumatoid arthritis antibodies in the antibody-antigen complex is directly monitored. This is a labeled anti-xenogenetic (xenogeneti) that recognizes the first type of epitope of rheumatoid arthritis-antibodies.
c) can be accomplished by determining whether a second type of (eg, anti-human) antibody binds by complex formation. In a competitive format, the amount of rheumatoid arthritis-antibody in a sample is predicted by monitoring the competitive effect on binding of a labeled antibody (or other competing ligand) in a known amount of the complex. The detection of rheumatoid arthritis-antibodies for the diagnosis of rheumatoid arthritis is used as an illustrative example. The term "rheumatoid arthritis-antibody" should not be considered limiting in any case as used herein. Similarly, other autoimmune diseases are diagnosed by detection of other antibodies, and mononucleosis is diagnosed by detection of anti-Epstein-Barr virus antibodies. The complex formed containing the rheumatoid arthritis-antibody (or, in the case of a competition assay, the amount of competing antibody) can be detected by format and by any of a number of known techniques. For example, unlabeled rheumatoid arthritis antibody in the conjugate can be labeled (
For example, it can be detected using a conjugate of an anti-xenogenetic Ig conjugated to an enzyme label). In an immunoprecipitation or agglutination assay format, the reaction between rheumatoid arthritis-antigen and rheumatoid arthritis-antibody forms a network that precipitates from solution or suspension, forming a visible layer or film of sediment. If no rheumatoid arthritis-antibody is present in the test specimen, no visible sediment is formed.

Recently, there are three specific types of particle agglutination (PA) assays. These assays are used to detect antibodies to various antigens when coated on a support. One type of this assay uses an antigen (which passively adsorbs to red blood cells (RBC)).
Or an antibody) sensitized red blood cells. The addition of a specific antigen antibody present in the body component causes the RBC coated with the purified antigen, if present, to aggregate. To exclude potential non-specific reactions in the hemagglutination assay, PA
Two artificial carriers can be used instead of the RBCs therein. Latex particles are the most common of these. However, gelatin particles can also be used. Assays utilizing either of these carriers are based on passive aggregation of particles coated with purified antigen. The antigenic peptides of the invention will generally be packaged in kits for use in these immunoassays. Kits usually contain, in separate containers, the antigenic peptide or anti-idiotype antibody, the control antibody formulation (positive and / or negative), if the label does not directly signal, the assay format is the same and the reagents that produce the signal (e.g., (Enzyme substrate), if necessary, include a labeled antibody. The antigenic peptide or anti-idiotype antibody may already be bound to the solid matrix, or may be separate with the reagent that binds it to the matrix. Instructions for running the assay (
Documents, tapes, CD-ROMs) will typically be included in the kit. The solid phase selected can include polymeric or glass beads, nitrocellulose, microparticles, reaction vessel microwells, test tubes, and magnetic beads.
Compounds that produce a signal can include enzymes, fluorescent compounds, chromogens, radioactive elements, and chemiluminescent compounds. Examples of enzymes include alkaline phosphatase, horseradish peroxidase and beta-galactosidase. Examples of enhancer compounds include biotin, anti-biotin and avidin. In order to inhibit the effect of the rheumatoid factor-like substance, the sample is subjected to conditions sufficient to inhibit the effect of the rheumatoid factor-like substance. These conditions allow the specimen to be consumed in a certain amount, for example,
Preferably, the mixture is contacted with the rabbit or anti-human IgG to be aggregated to form a mixture, and for a time and under conditions sufficient to form a reaction mixture product substantially free of rheumatoid factor-like substances. Incubating.

The invention particularly relates to an immunoassay format in which some peptides of the invention are coupled in parallel to a membrane. This assay format has the particular advantage that it allows the distinction between separate autoimmune diseases. In another embodiment, the present invention provides a method comprising: i) contacting a rheumatoid arthritis-specific autoantibody with any of the above peptides or intermediate filament proteins or a combination thereof; Measuring binding to any of the filament proteins or combinations thereof ii) contacting the compound or combination of compounds and the rheumatoid arthritis-specific autoantibody simultaneously or sequentially with any of the above peptides or intermediate filament proteins or combinations thereof Chronic Determining the binding of a rheumatoid arthritis-specific autoantibody to any of the above peptides or intermediate filament proteins or combinations thereof, iii) by comparing the results of i) and ii) Measuring the modulation of the binding of a rheumatoid arthritis-specific autoantibody to any of the above peptides or intermediate filament proteins or a combination thereof, induced by the compound or combination of compounds, including autoantigen and rheumatoid arthritis specific Bioassays that identify compounds that modulate binding between specific autoantibodies.

In a further embodiment, the present invention relates to modulators for the interaction between an autoantigen and this antibody specific for rheumatoid arthritis, and the present invention further relates to a method for the production of said modulator, said modulator comprising Identified by the assay). The term "compound" as used herein is to be understood in a broad sense and refers to proteins, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules, or hosts for vaccination. It may be an antibody generated by itself. As used herein, the term "binding" indicates that the above-described peptide physically binds and interacts with the antibody. The binding of the peptide to the antibody was determined by
Any method or assay known in the art, such as ISA and RIA type assays or competitive assays, can be demonstrated (see, eg, the Examples section and recent protocols in immunology). As used herein, the term "modulation" or "modulate" refers to the upregulation of binding between a peptide and an anti-HCV antibody (ie, for example, activating or enhancing to activate Or promote)
And down-regulation (ie, inhibition or suppression, eg, by antagonism, reduction, or inhibition). The term "modulator", as used herein, refers to the aforementioned modulatory ability of the aforementioned compound.

The term “peptide-like substance” as used herein refers to molecules that can be produced and mimic these residues of the peptide that modulate the interaction of the above-described peptide with an antibody. For example, a non-hydrolyzable peptide analog of such a residue is a benzodiazepine (Freidinger et al. In Peptides: Chemistry and Biology, GR Marsha
ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., Huffman et al. in Peptides: Chemistry and Biology, GRM).
arshall ed., ESCOM Publisher: Leiden, Netherlands, 1988),
PNA, substituted gamma-lactam ring (Garvey et al. In Peptides: Chemistry and
Biology, GR Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988
), Ketomethylene pseudopeptide (Ewenson et al. (1986) J Med Chem 29: 295
: and Ewenson et al. in Peptides: Structure and Function (Proceedings of
the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill.,
1985), β-turn dipeptide core (Nagai et al. (1985) Tetrahedron Lett
26: 647; and Sato et al. (1986) J Chem Soc Perkin Trans 1: 1231), and bami
noalcohols (Gordon et al. (1985) Biochem Biophys Res Commun, 126: 419; an
d Dann et al. (1986) Biochem Biophys Res Commun 134: 71).

[0041]

【Example】

 Example 1 Epitope Mapping of Filaggrin 1.1 Serum Human serum was obtained from the Department of Rheumatology at Ghent University Hospital (Belgium).
. A total of 265 sera were included, of which 75 were ARA diagnostic criteria for RA
(Arnett et al., 1987), and 155 sera were tested for APF fluorescence (De Keyser et al.).
al., in press), 98 were positive in the Western blot.
Naturally, it reacted with filaggrin, 80 were APF negative and 16 were from healthy controls
there were. 1.2 Preparation of human filaggrin Native filaggrin was isolated by the protocol of Simon et al. (1993).
It was purified from freshly obtained skin after wall arthroplasty. 5 mM skin pieces at 56 ° C
Epidermis separated from dermis by incubating in PBS containing EDTA
did. The material was dried and stored at -20 ° C until use. Epidermis cut into small pieces
To 40 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM
 EDTA, 0.5% NP-40, 0.1% sodium azide, 0.1 mM P
MSF (0.2 ml / cm^Two)) And stirred at 4 ° C. overnight. Homo
The dinate was centrifuged at 15,000 xg for 15 minutes and the extracted tan in the supernatant was
The protein was precipitated overnight at -20 ° C by adding 5 volumes of absolute ethanol.
I let you go. After centrifugation at 10,000 xg for 15 minutes, the protein pellet is
Air dried and subsequently resuspended in water. This partially purified protein preparation
Refers to filaggrin used in the above. The amount of protein is determined by following the BSA standard curve.
Bradford, modified by Peterson (1983)
Bradford) protein assay. 1.3 Immunodetection of anti-filaggrin antibody in human serum Crude filaggrin preparation using Bio-Rad minigel device
Was subjected to 10% Tricine SDS-PAGE. Under standard conditions, 2 μg protein
The quality / cm was loaded into a large slot and electrophoresed. Then, 10% methanol
Gel in nitrocellulose for 40 minutes in 10 mM CAPS, pH 11.0
The membrane was electroblotted. Blots were washed with PBS, 0.05% Tween 20, 1
% Gelatin, cut into 3 mm sides, diluted 1/100 overnight in PB
Probed with human serum in S, 0.05% Tween 20, 0.1% gelatin. No.
Anti-human IgG-AP conjugate (Sigma, St Louis, MI) as 1/1
000 dilution; visualized with NBT / BCIP chromogenic substrate. 1.4 Two-dimensional electrophoresis of filaggrin
Lip pH 3-102-D (Immobiline DryeStrips pH 3-10) (Pharmacia Biote
ch, Uppsala, Sweden) and 10% Tricine SDS-PAGE in two dimensions.
Separation by two-dimensional electrophoresis. Load 300 μg protein into the prepared gel.
And stained with Coomassie R-250. Anti-filaggrin mA
Comma-shaped filaggrin by immune reaction with b (BTI, Stoughton, MA)
Spots were identified. The pI of this heterologous protein is 6.7-8.5 (on the gel
7.1-8.5), while a form having a molecular weight of 35-68 kDa was detected.
The more acidic isoform showed the highest mass. 1.5 Electroelution of filaggrin The large filaggrin spot was cut from the gel and cut into three parts: i) pI7.1-
7.5 acidic fraction, ii) neutral fraction of pI 7.5-8.1, i
ii) Separated into basic fractions with pI 8.1-8.5. Each fraction is
, 50 mM (NH_Four) HCO_ThreeHunka using 0.1% SDS
Electroelution was performed by the method of piller et al. (1983). Koenigsberg and Hende
Coomassie by ion-pair extraction described by rson (1983)
And SDS were removed. Vacuum-dried protein pellets for further analysis
The sample was redissolved in an appropriate buffer. 1.6 Peptide mapping 40 μl of ± 100 μg of purified and electroeluted filaggrin fraction
100 mM (NH_Four) HCO_ThreeDissolved in 10% acetonitrile, pH 8.0
And trypsin (1/40 enzyme / substrate ratio). Incubate at 37 ° C overnight
After incubation, the digest was stored at -20 ° C before use. 140B Solvent
Using the Revali System (Solvent Delivery System) (ABI, Foster City, CA)
Reversed phase on a C-4 Vydac column (2.1 x 250 mm, Hesperia, CA)
Peptides are separated by HPLC and purified in 0.1% trifluoroacetic acid (TFA).
Eluted with an 8-70% linear gradient of 70% acetonitrile. 1000
Detected at 214 nm by S diode array detector,
The peptide was recovered manually. 1.7 Dot spot analysis for microsequencing 90% of each peak fraction was vacuum dried, followed by a small amount of 10% acetonitrile.
Toril, 50 mM NaCO_ThreeResuspended in buffer, pH 9.5. Immunoreactive epi
Immunodyne ABC membrane probed with human serum to specify tope
Domestic peptide (Immunodyne ABC membranes) (Pall BioSupport, UK)
To spot. First, block the membrane with PBS, 0.5% casein,
0.5% casein, 0.1% Triton X705, 10 mM MgCl_Two・ 6H_Two
Incubated overnight with serum diluted 1/50 in O. PBS, 0.0
After washing with 5% Tween 20, anti-human IgG (Promega) diluted in PBS, 0
. 1% casein, 0.2% Triton X705 was added for 1.5 hours. Substrate for coloring membrane
100 mM NaCl containing NBT / BCIP, 100 mM Tris-HCl,
pH 9.8, 50 mM MgCl_Two・ 6H_TwoDeveloped in O substrate buffer. 0.2N H _Two SO_FourThe reaction was stopped by the addition of. The remaining 10% of the immunoreactive fraction was microsequenced (microseq
uencing). Therefore, fractions are directly pulsed-lipid model.
On a 477A sequencer (pulsed-liquid model 477A Sequencer)
-On-line 120 phenyl thiodation analyzer
thiohydantoin analyzer (ABI)).

[0042] 1.8 Results acidic filaggrin acidic filaggrin trypsin digestion of each fraction (Fig. 1A) on ABC membrane (ABC membranes), were dotted spots in triplicate. Four APF-positive sera reactive with human filaggrin on Western blots (IG24395, IG3
5247 and IG24183 / 24184 pool) and one APF negative control serum were used for the immune reaction. Fractions T2, T9, T16 and T66 show a weak reaction with IG24395 serum, whereas the series from T30 to T44 showed a clear positive reaction, especially the T34 and T38 peptide fractions. The APF serum pool also scored positive with T34 and T38, while only IG35247 was reactive with T4. The APF negative control sera was clearly unreactive with the peptide fraction. The following amino acid sequence was recovered in each fraction. T2: R ↓ AGHGHSADSSR T4: R ↓ QGS R HQQAR R ↓ AGHGHSADSSR R ↓ HGSHHQQSADSSR T9: R ↓ HSQVGQGESSGPR T16 R ↓ HSASQDGQDTI R GHPG T33: R ↓ HSASQDGQDTI R GH R ↓ HSGIGHGQASSAVR T34: R ↓ HSASQDGQDTI T35: R ↓ HSGIGHGQASSAVR R ↓ DSGH R GYSGSQASDNEGH R ↓ HSTSQEGQDTIHG
H R GS R ↓ HSASQDGQDTI R GHPG T38; T39: Fraction T35 identical peptide T40, T41: Fraction T34 and identical peptide T66: signal was recovered. R represents the amino acid citrulline and was found at a specific retention time different from that of arginine. Further evidence of the presence of citrulline was i) the fact that the sequence did not cleave at the specific position expected, if any, and ii) the arginine residues were not sequenced. A similar method to natural filaggrin was performed on natural filaggrin (FIG. 1B), and the dot spots were
F serum was incubated with IG24395 and IG24184 and the same negative control serum used for acidic filaggrin mapping. T28, T65, T
81 and the series from T30 to T40 (especially T32, T35 and T40)
36) reacted positively with APF serum IG24395. Other sera are T4, T28,
It showed a weak reaction with T45 and T81. One fraction (T48) showed non-specific reactivity with all sera including negative control sera. The following sequence results were collected. T32 , T36 , T37 , T48 and T65 did not produce reliable amino acid sequence signals. T35: R ↓ HSGIGHGQASSAVR R ↓ DSGH R GYSGSQASDNEGH R ↓ HSTSQEGQDTIHGH R GS RvHSASQDGQDTI R GHPG R ↓ GYSGSQASDNEGHSE natural filaggrin citrulline in - the form and without form with modified 2
The fifth peptide was clearly derived from the second peptide, with the considerable possibility that one was always present. These results indicate that the same four peptides identified in the acidic T35 fraction were recovered. Second mapping synthetic peptide IGP1155, 1156, 1157 or 1158 of native filaggrin (Example 2
A mixture of two other sera of IG35038 / 35041, both of which were negative, was used to map native filaggrin from another skin source to the peptide. This resulted in specific reactivity with fractions T28, T31, T34 and T35, while APF-negative sera did not show any reaction. Sequencing results: T28 , T31 , T34 did not produce a sequenceable signal. T35 : R ↓ NDEQSGDGSR An overview of the sequenced immunoreactive portion of the filaggrin molecule is shown in FIG.

Example 2 Reactivity of Synthetic Peptide in Line Immunoassay (LIA) System 2.1 Synthetic Peptide The amino acid sequencing results described in Example 1 were used for the production of synthetic peptide. Some citrulline-containing peptides have synthesized un-modified counterparts to arginine to compare their respective reactivities. The following peptides were produced. IGP1155 IGP1179 HSASQDGQDTI R GHPGSS HSASQDGQDTIRGHPGSS IGP1156 IGP1180 HSGIGHGQASSAVRDSGH R GYS HSGIGHGQASSAVRDSGHRGYS IGP1157 IGP1181 DSGH R GYSGSQASDNEGH DSGHRGYSGSQASDNEGH IGP1158 IGP1182 HSTSQEGQDTIHGH R GS HSTSQEGQDTIHGHRGS IGP1249 GGQGS R HQQAR IGP1250 IGP1251 GGAGHGHSADSSR GGHGSHHQQSADSSR IGP1252 IGP1326 GGNDEQSGDGSRHSGS SRHSQVGQGESSGPR R represents citrulline. 2.2 Line immunoassay analysis Streptavidin-conjugated peptide was applied directly onto a plastic lined nylon membrane. The piece to be blocked was 1/100 in 1 ml PBS, 0.5% casein, 0.1% Triton X705, 10 mM MgCl ₂ .6H ₂ O.
Overnight with human serum diluted to 1. After washing with PBS, 0.05% Tween 20, goat anti-human IgG-AP conjugate (Promega) diluted with PBS, 0.1% casein, 0.2% Triton X705 was added for 1 hour 30 minutes.
Pieces were prepared with 100 mM NaCl, 100 mM Tri containing chromogenic substrate NBT / BCIP.
It was developed with s-HCl, pH 9.8, 50 mM MgCl ₂ .6H ₂ O substrate buffer. After 30 min, by addition of 0.2 N H ₂ SO _4, the reaction was stopped. 2.3 Results The serogroup of 107 individuals from patients meeting the ARA criteria for RA was 2.1.
The reactivity with the 13 synthetic peptides listed in Table 1 was tested in the LIA system. IGP1155, 1156, 11 with a combined sensitivity of 48% in this group
Reactivity was observed with 57, 1158 and 1249 (Table 2). Taking into account that only sera reacted with human native filaggrin on Western blots, each synthetic peptide reached significantly higher sensitivity compared to filaggrin-negative RA sera. Similar findings were obtained when splitting between APF positive and negative sera. 5
Control sera from one healthy human, 61 SLE and 62 osteoarthritis showed relatively insignificant reactivity with the peptide. In all groups, IPG1156 and IPG1158 appear to be the most reactive peptides, while the immune response to IPG1157 was always very faint and did not add any value in that it increased the sensitivity of RA diagnosis.

[Table 2] Table 3 shows the relationship between filaggrin positive on Western blot and reactivity with the synthetic peptide in the RA group. Statistical analysis revealed a modest association between the two reactivity patterns (95% Cl 0.41-0.73, kappa value 0.57; 78.5).
% Match coefficient). Non-modified counterparts IGP1179, IGP1180, IGP1181
And when tested with IGP1182, the citrulline-containing peptide IGP11
Positive LIA signals found at 55, IGP1156, IGP1157 and IGP1158 were not recovered (FIG. 3). Only for two sera, a weak color development of all non-modified peptides was obtained, which would be considered a non-specific background staining associated with a particular serum. These results suggest that the presence of citrulline is essential for immunoreactivity, and that this unusual amino acid constitutes a key epitope of anti-filaggrin antibodies.

Table 3 : Association between the reactivity of anti-filaggrin positive and LIA with the five synthetic peptides IGP1155, 1156, 157, 1158 and 1249 on Western blots.

[Table 3]

Example 3 Reactivity of Synthetic Peptides in ELISA 3.1 ELISA ELISA The electroeluted human native filaggrin was placed in Maxisorp polystyrene plates at a concentration of 1 or 4 μg / ml in 50 mM carbonate buffer, pH 9.6. Overnight 4
-C. The serum was diluted 1/50 with PBS and 100 μg / m
For 1 hour, either the appropriate peptide or 1 or 10 μg / ml of isolated native filaggrin (positive control) or PBS (negative control) was preincubated for 2 hours. Plates were blocked for 1 hour at 37-C in PBS, 0.1% casein, followed by incubation with serum. 2 hours at 37 ° C. Plates were washed 5 times with PBS, 0.05% Tween 20, and PBS, 0.1% casein, 0%
. Among _{1mM K 3 Fe (CN) 6} , anti - were incubated with human IgG-HRP. 0.1 M Na ₂ HPO ₄ , 0.1 M citric acid, 0.006% H ₂ O ₂ , pH4.
Color development was performed using tetramethylbenzide diluted 1/100 in 3. 2
The reaction was stopped by the addition of NH ₂ SO ₄ and Bio-tek ELISA was performed.
The OD value was measured at 450 nm using a reader. The percentage of inhibition was calculated as follows. (OD without inhibition-OD with inhibition) x 100 / OD without inhibition 3.2 Results Five APF-positive sera with strong immunoreactivity to native filaggrin on blots were added with or without inhibitory filaggrin Was analyzed on filaggrin-coated plates (FIG. 4; Table 4). Upon preincubation at a concentration of 10 μg / ml, the signal dropped to 64-79% and 1 μg / ml (4-
45%) was significantly higher. APF-negative serum IG24805, which was not reactive with filaggrin on Western blots, produced only a background signal in this ELISA and showed no significant inhibition. Synthetic, citrulline-containing peptide IGP1155, 1156, 1157, 11
When 58 or any one of these four peptide mixtures are used as competitors, IGP1155 significantly inhibits two of the four sera and IGP1158 significantly inhibits one at a concentration of 100 μg / ml. Yes, this is LIA
(FIG. 5; Table 4). The peptide mixture was able to inhibit the anti-filaggrin binding of the four sera up to about 37-92%, whereas the APF-negative serum did not.

Example 4: Cloning and expression of human filaggrin Genome D isolated from human lymphocytes as template using PCR technology
Using NA, four candidates for human filaggrin sequences were cloned. The PCR primers were designed as follows: Sense PCR primers: EcoRI 5 'CC GAA TTC GCC ACC ATG GGG TCT TTC CTC TAC CAG GTG 3' Met Gly Ser Phe Leu Tyr Gln Val PCR sense primer was added to the filaggrin linker sequence It was chosen to overlap and designed to introduce a functional start codon (Kozak environment) upstream of the linker sequence (Phe Leu Tyr Gln Val Ser Thr). The linker sequence was included within the filaggrin repeat to be amplified, possibly as it is involved in the correct targeting of the processed protein. An EcoRI restriction site was introduced for subcloning the PCR fragment. Antisense PCR primer Thr Ser Gly His Ser Gly Ser Pro Gly His STOP 3 ' TGC AGA CCT GTA AGT CCT AGA GGG CCC GAG ATC TGG SmaI XbaI

[0047]

[Table 4] The antisense primer is located just upstream of the adjacent filaggrin linker sequence and introduces a translation stop codon TAG. Thus, the filaggrin sequence to be amplified consists of a filaggrin linker followed by an integral filaggrin repeat and three additional amino acids (Pro / Gly / His) as a result of the cloning procedure.
The PCR amplified fragment was used as an EcoRI-XbaI fragment as pBLSK (Stratagene).
The cDNA cloned into the vector and amplified could be sequenced. The four individual clones characterized by sequencing were HB2641, HB2
642, HB2648 and HB2650 (FIG. 6). The cDNA insert was recloned as an EcoRV / Ecl 136II fragment (1030 bp) into the E. coli expression vector pIGRHISA opened with HsiI blunt end opening. The filaggrin protein was expressed in three different E. coli strains as a recombinant filaggrin-His6 fusion protein, resulting in high levels of Coomassie staining expression. His6 fusion protein
The tail allows for convenient purification of the protein using metal-affinity chromatography.

Example 5 Isolation of Vimentin 5.1 Antigen as a Marker for Diagnosis of Rheumatoid Arthritis Placental extracts were prepared as described by Despres et al. (1994).
Placental tissue was stored at -70-C and 50 mM Tris-HCl, pH 7.4, 1
20 mM NaCl, 1.5 mM DTT, 0.02% NaN ₃ , 1 mM PMS
F and 5 μg / ml chymostatin, leupeptin, antipain, pepstatin. After clarification, the homogenate was subjected to DE52 ion exchange chromatography, and the 200, 250 and 300 mM salt elution fractions were further analyzed by one- and two-dimensional gel electrophoresis. 5.2 Two-dimensional gel electrophoresis and blotting methods Proteins present in different chromatographic fractions were separated by two-dimensional gel electrophoresis. Samples were prepared using 7M urea, 2M thiourea, 4% CHAPS
0.5% Triton X-100, 1% Pharmalytes 3-10
, 10 mM DTT, 0.01% orange G in a rehydration buffer. R
By the in-gel rehydration method of abilloud et al. (1997), 50-500 μg of protein was transferred to an IPG4-7 iso-electrofocusing strip (iso-ele
ctrofocusing strips) (Pharmacia). For the second dimension electrophoresis performed by the standard method, the one-dimensional piece was subjected to 10% Laemmli gel or SD.
SDS Gradient 12-14 ExcelGels (Phar
macia). Gel was placed on a nitrocellulose membrane with 10% methanol, 10m
Electroblot in MCAPS, pH 11.0 for 2 hours. Immunodetection with human serum and mAb was performed as described in 1.3. Anti-vimentin mAb was purchased from Sigma and used at a 1/5000 dilution. Anti-calretilurin pAb and anti-PDI mAb were delivered by Affinity BioReagents Inc. (Golden, CO) and used at 1/1000 dilution. 5.3 Protein Identification by MS Analysis Coomassie-stained protein spots were excised from the two-dimensional gel, washed with water and 50% acetonitrile / 1% TFA, and 20 hours at 37 ° C., 10 ml of 25 mM NH ₄ HCO ₃ / In 10% acetonitrile, pH 8.0,
Incubated with 0.08 μg trypsin. The peptide was extracted with 60% acetonitrile / 0.1% TFA, vacuum dried and 10 μl of 30% MeOH / 1
% Formic acid. Approximately 5% of the digestion mixture is applied to Voyager-DE STR (Voyager
-DE STR) (Perseptive Biosystems, Framingham, MA) on DE-MALDI-R
It was subjected to ETOF-MS analysis. The remaining material was purified on Poros R2; the bound peptide was eluted with 4 ml of 60% MeOH / 1% formic acid, 65
% Were used for ESI-MS analysis on a Q-TOF mass spectrometer (Micromass, UK). 5.4 Results 25 human RA sera, 3 osteoarthritis sera and 20 negative controls were probed on 2-D blots containing placental extracts. ± 14 for 14 RA sera
Very strong immunoreactivity with multiple-spot-train and pI 4.5-4.8 located at kDa (variable between 56 and 61 kDa depending on the gel system) was seen (FIG. 7a). ). None of the controls showed any response to this protein. Blots were probed with anti-calretilurin pAb, anti-PDI mAb and anti-vimentin to confirm the location of these major placental proteins. Calretilurin is 56-
Found as an abundant spot of 61 kDa, pI 4.3, while PDI was 5
It was found at 8-64 kDa, pI 4.6-4.8, and thus both agreed with the expected theoretical two-dimensional position. The latter spot was further identified as PDI by tandem MS analysis. The anti-vimentin mAb showed strong reactivity with a number of proteins between 40 and 61 kDa as shown in FIG. 7b. This pattern was derived from different humans and proved to be very reproducible between different groups of purified substances even when tested at different time points. It was revealed that RA-specific reactivity co-localized with the high MW form of vimentin when the blot probed with RA serum was placed on top of the anti-vimentin blot. Insert spots are also MALDI
-Identified by TOF and tandem mass spectrometry. Using both techniques, peptides from human vimentin, cytokeratin 1 and cytokeratin 9 were recovered in addition to other non-identified peptides. All of these proteins belong to the same family of intermediate filament proteins. It has been previously described that the citrullinated forms of vimentin and cytokeratin occur in vivo (Senshu et al.
., 1992; Senshu et al., 1995; Senshu et al., 1996), which appear to constitute targets for autoantibodies present in rheumatoid arthritis serum. Citrullinated proteins have been shown to cause a mobility shift in the higher MW region on SDS-PAGE as compared to the non-citrullinated form (Senshu et al., 1995;
Tarcsa et al., 1996), which could explain the selective immunoreactivity of human sera against the highest MW form of vimetin. Thus, certain intermediate filament protein forms could be used as markers for diagnosing rheumatoid arthritis disease.

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9

[Brief description of the drawings]

FIG. 1A: HPLC profile of tryptic digestion of human native acidic filaggrin. Peptides were purified on C-4 Vydac columns (2.1 x 250 mm, Hesperia, CA).
140B Solvent Delivery System (ABI
, Foster City, CA) and eluted with an 8-70% linear gradient of 70% acetonitrile in 0.1% trifluoroacetic acid (TFA). Detection was performed with a 1000S diode array detector at 214 nm.

FIG. 1B: HPLC profile of tryptic digestion of human native neutral filaggrin. Peptides were prepared on C-4 Vydac columns (2.1 x 250 mm, Hesperia, CA).
140B Solvent Delivery System (ABI,
(Foster City, CA) and eluted with a linear gradient of 70-70% acetonitrile in 0.1% trifluoroacetic acid (TFA) from 8-70%. Detection was performed with a 1000S diode array detector at 214 nm.

FIG. 2. Multiple alignments of the filaggrin protein sequence and an overview of the arranged peptides. HB2641, HB2642, HB2648 and HB2650 clones were isolated by the company; other sequences were retrieved from the literature. Symbol indicating that the position is completely preserved: * 'Symbol indicating that the position is well preserved:'. '

FIG. 3: Reactivity of 26 human RA sera with synthetic peptides by LIA Peptides IGP1155, 1156, 1157 and 1158 all contain citrulline; corresponding relatives without citrulline incorporating IGP1179, 1180, 1181 and 1182 It was a thing. The peptide was applied after conjugation with streptavidin at a concentration of 400 μg / ml. IGP1154 constitutes an unrelated synthetic peptide.

FIG. 4. Inhibition ELISA using natural filaggrin as inhibitor. Plates were coated with 1 μg / ml purified human native filaggrin. Serum was diluted 1/50 and pre-incubated with native filaggrin at 1 or 10 μg / ml and added to the plate. OD value is 450nm
Was measured.

FIG. 5: Inhibition EL using synthetic citrulline-containing filaggrin as an inhibitor
ISA. Plates were coated with 4 μg / ml purified human native filaggrin. The serum was diluted 1/50 and each peptide (IGP115) at 100 μg / ml.
5, 1156, 1157, 1158) or a mixture of four peptides (mix
) With or without pre-incubation. OD
Values were measured at 450 nm.

FIG. 6. HFIL1 sequence retrieved from literature (McKinley-Grant et al. 198)
9) and the multiple sequences of clones HB2641, HB2642, HB2648 and HB2650. Symbol indicating that the position is completely preserved: * 'Symbol indicating that the position is well preserved:'. '

FIG. 7: Two-dimensional immunoblot of placental extract. 200 μg of the 200 mM salt-eluting fraction of the placenta extract was used in one dimension for IPG4
-7 pieces were separated by two-dimensional gel electrophoresis using a 10% laemli gel in two dimensions. Proteins were electroblotted onto nitrocellulose membrane, blots were either a) human rheumatoid arthritis serum (diluted 1/200) or b).
Anti-human vimetin mAb (Sigma) diluted 1/5000 was probed. The arrow indicates the position of the reference PDI-protein, and Ponceau S (Po
nceau S) Needles were examined after total protein staining.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 29/00 101 C07K 1/107 C07K 1/107 16/42 16/42 C12P 21/02 C C12P 21 / 02 G01N 33/564 B G01N 33/564 A61K 37/12 // C12N 15/09 ZNA C12N 15/00 ZNAA (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR) , GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD) , TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, T , TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE , GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Josef Laimackels Belgium, B-99810 Eke, Seidelby Sten No. 10 F-term (reference) 4B024 AA01 AA11 BA31 BA80 CA05 DA02 DA05 DA12 EA04 GA11 HA01 4B064 AG01 CA02 CA10 CA19 CC24 DA03 DA13 4C084 AA02 BA01 BA08 BA BA31 CA18 CA53 NA14 ZA891 ZA961 ZB051 ZB151 4C085 AA 14 AA15 BB14 CC03 DD61 DD62 4H045 AA10 AA11 AA20 AA30 BA16 BA17 BA18 BA19 CA40 DA76 EA22 EA50 FA52 FA72 FA74

Claims (27)

[Claims] 1. A peptide comprising less than 50 amino acid sequences of a native filaggrin variant or an intermediate filament protein variant and comprising at least one citrulline, wherein the presence of said citrulline is from a patient with rheumatoid arthritis Peptides that are important for the reaction with the antibodies present in the serum of the rat. 2. Vimentin or cytokeratin 1 or cytokeratin 9
A peptide comprising less than 50 amino acid sequences of at least one variant and comprising at least one citrulline, wherein the presence of said citrulline is important for the reaction with an antibody present in serum from a patient with rheumatoid arthritis . Wherein the amino acid sequence HSASQDGQDTIXGHPGSS or HSGIGHGQASSAVRDSGHXGYS or DSGHXGYSGSQASDNEGH or HSTSQEGQDTIHGHXGS or GGQGSXHQQAR or QGSXHQQARDSSRHSTSQEGQDTIHGHXGS or QGSXHQQARDSSRHSASQDGQDTIXGHPGSS or HSGIGHGQASSAVRDSGHXGYSGSQASDNEGH or filaggrin analogues of said peptides comprising a characteristic amino acid substitutions allelic variants [wherein, X is citrulline residue Represents ] The peptide according to claim 1, comprising: 4. A peptide and / or chemical structure comprising any one of claims 1 to 3 fused to a linker molecule. A cyclic peptide comprising at least one peptide according to any one of claims 1 to 4. 6. A peptide comprising and / or consisting of at least two tandem repeats of the peptide according to any one of claims 1 to 5. 7. A peptide having a branched chain, comprising at least one peptide according to any one of claims 1 to 6. 8. A method for producing the peptide according to any one of claims 1 to 7 by classical chemical synthesis in which an arginine residue is replaced with a citrulline residue in a specific step during chemical synthesis. 9. The method of claim 1, wherein the primary amino acid sequence is prepared, and then the peptide is contacted with peptidylarginine deiminase to convert arginine residues to citrulline residues by classical chemical synthesis. A method for producing the peptide according to the above. 10. A method for producing the peptide according to any one of claims 1 to 7, wherein the peptide or the protein containing the peptide is expressed and / or secreted. Transforming a suitable cell host with a recombinant vector into which a polynucleic acid containing a sequence encoding the peptide has been inserted under the control of appropriate control elements, enabling expression of the protein or the peptide, and retaining arginine residue. Culturing the transformed cell host under conditions that allow derivatization of the group to a citrulline residue, and harvesting the peptide. 11. A method for producing a peptide according to any one of claims 1 to 7, wherein the method comprises the following steps: wherein the peptide or a protein comprising the peptide is expressed and / or secreted. Transforming a suitable cell host with a recombinant vector into which a polynucleic acid containing a sequence encoding the peptide has been inserted under the control of appropriate control elements, under conditions which allow expression of the protein or the peptide. Culturing said transformed cell host, harvesting said protein or said peptide, derivatizing arginine residues of said protein or said peptide by contacting with peptidyl arginine deiminase. how to. 12. The method according to claim 10, wherein the host cell is a bacterial host or yeast or any other eukaryotic host cell, preferably transformed with the recombinant baculovirus. . 13. An antibody which reacts specifically with the citrulline residue of the peptide form according to any one of claims 1 to 7, or is a citrulline residue of an intermediate filament protein and preferably a monoclonal antibody Antibodies that specifically react with 14. An anti-idiotype antibody which is produced when immunized with the antibody according to claim 13, and specifically reacts with the antibody according to claim 13, thereby comprising: An anti-idiotype antibody, preferably a monoclonal antibody, that mimics the citrulline-containing peptide described in 1. 15. A cell-recognition molecule which is the antibody according to any one of claims 13 and 14, which covalently binds to the peptide according to any one of claims 1 to 7, or a toxic molecule or an active fragment thereof. And / or an immunotoxic molecule comprising such a molecule. 16. The peptide according to any one of claims 1 to 7, or the antibody according to any one of claims 13 or 14, or the immunotoxic molecule according to claim 15, or a molecule thereof, which is used as a medicament. Composition. 17. The peptide according to any one of claims 1 to 7, or the antibody or the antibody according to any one of claims 13 to 14, for the manufacture of a medicament or a diagnostic agent for rheumatoid arthritis. Use of the described immunotoxic molecule or composition thereof. 18. Use of immunization with an intermediate filament protein, preferably vimentin or cytokeratin 1 or cytokeratin 9, or an intermediate filament protein or a composition thereof, for the manufacture of a medicament or diagnostic for rheumatoid arthritis. Use of the antibody produced in the process. 19. A method for preparing a medicament for treating rheumatoid arthritis by increasing the size of an antigen-immune complex and thereby improving the clearance of the immune complex formed thereby. Use of a polypeptide or a composition thereof according to any one of claims 7 to 7. 20. Decreased systemic sensitivity to the polypeptide ('oral tolerance')
Use of the polypeptide according to any one of claims 5 to 7 or a composition thereof for the preparation of a medicament for oral administration for treating rheumatoid arthritis by inducing the above condition. 21. A diagnostic kit for use in detecting an autoimmune disease such as rheumatoid arthritis systemic lupus erythematosus discoid lupus erythematosus scleroderma dermatomyositis Sjögren's syndrome.
16. A peptide, antibody or protein comprising at least one of the peptide of any one of claims 1 to 7, or the antibody of any one of claims 13 or 15, or an intermediate filament protein. Is a kit bound to a solid support. 22. The peptide according to any one of claims 1 to 7 or the peptide according to any one of claims 13 to 15, possibly together with an antigen constituting an immunogenic determinant of another autoimmune disease. 22. A diagnostic kit comprising a range of the antibody or intermediate filament protein of claim 21, wherein the peptide, antibody and / or protein binds to a specific location on a solid substrate. 23. The diagnostic kit according to claim 21, wherein the solid support is a piece of membrane, and the polypeptide is coupled to the membrane in a parallel line. 24. Certain peptides or proteins are not bound to a solid support, but are provided in a binding solution, used as competitors, or present in serum from a patient with an autoimmune disease other than rheumatoid arthritis. 23. A diagnostic kit according to claim 21 or 22, which is used to block other antibodies that reduce or eliminate possible cross-reactivity and / or non-specific binding. 25. A bioassay for identifying a compound that modulates an interaction between an autoantigen and a rheumatoid arthritis-specific autoantibody, comprising: i) a rheumatoid arthritis-specific autoantibody. A peptide or intermediate filament protein according to any one of claims 1 to 7, which is contacted with a peptide or intermediate filament protein according to any one of claims 7 or a combination thereof. Or measuring the binding to a combination thereof. Ii) The peptide or intermediate filament protein according to any one of claims 1 to 7 or a compound or combination of the compound and the rheumatoid arthritis-specific autoantibody simultaneously or sequentially. Contact with combination Rheumatoid arthritis specific Determining the binding of an autoantibody to the peptide or intermediate filament protein of any one of claims 1 to 7 or a combination thereof; iii) comparing the results of i) and ii) with the compound or A method comprising measuring the modulation of the binding of a rheumatoid arthritis-specific autoantibody to a peptide or an intermediate filament protein according to any one of claims 1 to 7 or a combination thereof induced by a combination of compounds. Assay. 26. A modulator, obtained by the method of claim 25, for the interaction between an autoantigen and a rheumatoid arthritis-specific autoantibody. 27. A method for producing the modulator according to claim 26.