Chemistry: analytical and immunological testing – For preexisting immune complex or auto-immune disease
Reexamination Certificate
1999-05-10
2002-03-26
Wortman, Donna C. (Department: 1645)
Chemistry: analytical and immunological testing
For preexisting immune complex or auto-immune disease
C436S518000, C530S324000
Reexamination Certificate
active
06362007
ABSTRACT:
The present invention relates to a method of producing certain peptides containing methylated arginines that are followed by a glycine residue and that constitute immunogenic determinants of antibodies present in sera from patients with systemic lupus erythematosus, or Epstein-Barr virus and wherein the methylation is a prerequisite for reacting with said antibodies. The invention also relates to the use of said peptides for diagnosis and treatment of systemic lupus erythematosus and related diseases, diseases in which Epstein-Barr virus has been implicated.
BACKGROUND OF THE INVENTION
Systemic lupus erythematosus is an autoimmune disease, in which the patient develops antibodies that react with many tissues of his own body. Dominant antibodies are directed against components of the cell nucleus, with epitopes that may be found in DNA, and in proteins that constitute small ribonucleoprotein particles (snRNPs).
The first laboratory test ever devised for this disease was the LE (lupus erythematosus) cell test. This test has to be repeated many times, before it results in a positive reaction in about 90% of the people with systemic lupus erythematosus. Also, the LE cell test is not specific for lupus, and can be positive in up to 20% of the people with rheumatoid arthritis, in some patients with other rheumatic conditions like Sjögren's syndrome or scleroderma, in patients with liver disease, and in persons taking drugs such as hydralazine and procainamide. The ANA test, which detects antibodies against nuclear antigens, is more specific for lupus than the LE test, and is positive in many patients that suffer from systemic lupus erythematosus. As with the LE test, a positive ANA is not diagnostic for lupus since the test may also be positive in people with scieroderma, dermatomyositis, rheumatoid arthritis, Sjögren's syndrome, in patients treated with certain drugs, or in patients suffering from infectious mononucleosis, liver disease, malaria etc. For these reasons and because the summed tests are expensive, new tests have been developed which are very helpful in the diagnosis of SLE. These include the anti-DNA antibody test, the anti-Sm antibody test, the anti-RNP antibody test, the anti-Ro antibody test, and tests which measure serum complement levels. Often, correct diagnosis will depend on the interpretation of many separate tests and symptoms.
The Sm antigen is a complex macromolecular structure consisting of 8 proteins (B, B′, D1, D2, D3, E, F, G) associated with the U series of small RNA molecules. SmBB′ and SmD are considered as the major antigenic components of the complex (for review see S. O. Hoch, 1989). However, SmBB′ shows cross reactivity with the anti-RNP antibodies, consequently SmD is regarded as the most specific autoantigen for Sm (W. J. van Venrooij et al, 1991).
The SmD cDNA has been isolated from a human B-lymphocyte library with synthetic oligonucleotide probes, designed on the basis of the N-terminal sequence of SmD (Rokeach et al., 1988). Subsequently, it was shown that the in vitro transcription product could be immunoprecipitated by anti-Sm IgG. The D protein has since been characterized either as a doublet designated D and D′ (Andersen et al., 1990) or as three polypeptides designated D1 (16 kDa), D2 (16.5 kDa) and D3 (18 kDa) (Lehmeier et al., 1990), D1 being identical with the SmD cloned by Rokeach et al.(1988). The sequence of D2 and D3 is substantially different from D1.
Over the years, several research groups have reported on the use of recombinant SmD and of SmD derived peptides and have published conflictory data. Rokeach et al. (1992a) expressed SmD1 in
E. coli
and in
S. cerivisiae
, but in contrast to the reactivity of natural SmD from HeLa cells, most of the patient anti-SmD sera bound recombinant SmD1 at a level not significantly higher than normal human sera. Nevertheless, the same group (Rokeach et al., 1992b) has performed epitope mapping based on multiple fusions between the TrpE gene and fragments of the SmD coding sequence, expressed in
E. coli
. Two patterns of anti-Sm reactivity emerged: discontinuous epitopes were found scattered over the full-length antigen, and a dominant epitope was located at the C-terminus, from amino acid 87 to 119 (Rokeach et al., 1992b). Using synthetic peptides, Barakat et al. (1990) showed that the N-terminus (peptide 1-20) and peptide 44-67 could be used as a valuable probe for SLE diagnosis although their results did not match the anti-SmD reactivity obtained by the traditional assay (patent EP-B-0491014). Using a similar strategy, Sabbatini et al. (1993a) have identified a dominant epitope in the C-terminal region of SmD1 (aa95-aa119) confirming the results of Rokeach et al. (1992b), but opposing the results obtained by Barakat et al. (1990). The most recent work on epitope mapping of SmD1 by means of synthetic peptides (James et al. 1994) showed that 8 of 9 SmD positive sera (precipitin positive) are reactive with the sequence spanning octapeptides 92-112. An additional epitope, clearly reactive with 7 of 9 SmD positive sera was located in the region of amino acid 82-90. Finally, a SmD-like epitope was recently identified by Rivkin et al. (1994) and consists of a (Gly-Arg)
9
dipeptide repeat (homology with the C-terminus). In contrast to the SLE specificity of anti-Sm antibodies, the defined epitope is also recognized by patients with other autoimmune diseases (rheumatoid arthritis, scleroderma, Sjögren's syndrome). The &bgr;galactosidase fusion protein in
E. coli
of the above mentioned epitope was reactive with 35% of the SLE sera, but only 6 out of these 32 positive sera were reactive with the native SmD protein indicating that the fusion protein is less specific than the native SmD protein. Vice versa, only four of eight SmD sera reacted with the fusion protein. It should be noted however, that SmD was also expressed as a full-size &bgr;-galactosidase fusion protein in
E. coli
(Wagatsuma et al. 1993), but that this recombinant SmD antigen was not recognized by patient sera, although all sera recognized the natural Sm 16 kDa antigen on Western blot.
In conclusion, none of the described synthetic peptides nor the entire recombinant protein or parts of the molecule result in an immunoreactivity identical with the reactivity obtained with natural SmD.
SUMMARY OF THE INVENTION
It is an aim of the present invention to provide peptides which have a high reactivity for antibodies present in sera from patients with systemic lupus erythematosus.
Another aim of the present invention is to provide methods for obtaining said peptides.
Another aim of the present invention is to provide methods of raising antibodies specifically reactive with peptides of said peptides, thereby mimicking said peptides.
Another aim of the present invention is to provide methods of raising anti-idiotype antibodies specifically reactive with the afore mentioned antibodies.
Another aim of the present invention is to provide a pharmaceutical composition consisting of these peptides, for therapy or diagnosis.
Another aim of the present invention is to provide a diagnostic kit for systemic lupus erythematosus.
All these aims of the present invention are met by the following embodiments of the present invention.
According to its main embodiment the present invention relates to peptides containing less than 50 amino acids, comprising at least one dimer of the type XG, wherein X stands for a methylated arginine residue, and that are able to react with antibodies, with said methylation being crucial for the reaction between said peptide and said antibodies, and wherein said antibodies are present in sera from patients with systemic lupus erythematosus, or infectious, recurrent or chronic mononucleosis, or certain cancers which are related to infection with Epstein-Barr virus, such as Burkitt's lymphoma or nasopharyngeal carcinoma.
According to a further embodiment the present invention also relates to a peptide and/or chemical structure comprising any of the above mentioned peptides, fus
Lührmann Reinhard Georg
Meheus Lydie
Raymackers Joseph
Union Ann
Howrey Simon Arnold & White , LLP
Innogenetics N.V.
Wortman Donna C.
Zeman Robert A.
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