Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-09-22
2002-12-31
Chan, Christina Y. (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S358000, C424S185100, C536S023100, C536S023500
Reexamination Certificate
active
06500923
ABSTRACT:
I. FIELD OF THE INVENTION
The present invention relates to a dermatomyositis-specific auto-antigen, a DNA encoding it and a process for the preparation thereof as well as its use.
II. BACKGROUND OF THE INVENTION
Autoimmune diseases distinguish themselves by the occurrence of autoantibodies, i.e., antibodies directed against constituents of the own organism. Autoantibodies may induce damage of the organism, an organ or part of an organ thus triggering partially serious life-threatening diseases. The origination of such a disease is due to differing pathogenic mechanisms such as neutralization of antigens, e.g., hormones, blocking or stimulation of a receptor for biological active substances, e.g., autoantibodies active against the receptor of the thyroid-stimulating hormone in the case of hyperthyreosis, binding to certain cell or tissue structures accompanied by the induction of a complement-mediated inflammation, e g., glomerulonephritis, antiautobodies active against glomerulus basement membranes. Tissues damage may also be induced by cell-mediated mechanisms (autoantibody-dependent cellular cytotoxicity) or by localized and systemic immune complex deposits after the binding of the autoantibodies to soluble antigens.
In addition to such obviously directly damaging autoantibodies, a plurality of autoantibodies active against blood, cell or tissue constituents occur in man, to which a tissue-damaging part cannot be assigned yet clearly by now. The group of rheumatic diseases, particularly the inflammatory rheumatic diseases to which the collagen diseases are attributed, is characterized by the occurrence of numerous autoantibodies. They react, e.g., with antigens of the cell nucleus such as double-stranded DNA, single-stranded DNA, RNA, histones, non-histone proteins, ribonucleoproteins, chromosome-associated antigens, e.g., centromeres or spindle apparatus, or with antigens which are expressed only in certain phases of the cell cycle, e.g., cycline.
The above autoantibodies are found in the case of diseases such as lupus erythematodes, Sjögren's syndrome, mixed connective tissue disease, polymyositis, dermatosclerosis, CREST syndrome, Wegener's granulomatosis and dermatomyositis. They are usually detected by reaction with nuclear extracts from thymocytes of calves or rabbits. Due to this, a differential diagnosis of these diseases, particularly of dermatomyositis, is, however, not possible. But such a diagnosis is a precondition for the selection of treatment.
Therefore, it is the object of the present invention to provide means by which dermatomyositis can be detected by way of differential diagnosis.
III. SUMMARY OF THE INVENTION
The present invention relates to a dermatomyositis-specific auto-antigen, a DNA encoding it and a process for the preparation thereof as well as its use.
IV. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1
shows the cloning and composition of the 218 kD Mi-2 cDNA. The upper portion of the illustration shows a restriction map with a scale given in kb. The thick line corresponds to the open reading frame including restriction sites outlined by arrows (H=Hind III; B=BamHI; E=EcoRI; C=ClaI; S=SpHI). The horizontal lines represent isolated clones; the clones sequenced in both mixtures are marked by dots. The two small clones in the lower left-hand corner were obtained according to the RACE protocol. Frohmann et al., 1988,
Proc. Natl. Acad Sci. USA.
85:8998-9002). 1: nt 649-4891; 2: nt 0-958; 3: nt 153-4902; 4: nt 1931-4881; 5: nt 2204-4888; 6: nt 3224-4888; 7: nt 3620-4888; 8: nt 3666-6155; 9: nt 3967-6155; 11: nt 178-127; 12: nt 168-127.
FIGS. 2A-D
collectively show the nucleotide sequence (SEQ ID NO: 1) and coding amino acid sequence (SEQ ID NO: 2) of the 218 kD Mi-2 cDNA. The framed sequences show the seven helicase-specific motifs (I, IA, II, III, IV, V and VI). The four underlined regions contain 11 potential nuclear target sequences ten of which are localized in three N-terminal regions (region a contains 3 motifs; region b includes 4 motifs and region C comprises 3 motifs). The dotted line (nt 490-520) shows an accumulation of glutamic acid and asparaginic acid residues which might interact electrostatically with chromatin (histones).
FIG. 3
shows a Northern blot analysis of the 218 kD Mi-2 gene expression in HEp-2 cells. RNA samples were separated in accordance with their size by means of formaldehyde agarose gel electrophoresis and hybridized with
32
P-labeled Mi-2 cDNA subject to a high degree of stringency. Lane A: 5 &mgr;g total RNA of HEp-2 cells; lane B: 0.3 &mgr;g HEp-2 cells poly(A)
−
RNA. The size of the bands in the autoradiograph corresponds to about 6.8 kb.
FIG. 4
shows a computer-assisted analysis of functional domains of the 218 kD Mi-2 protein. NT: potential nuclear target sequences; CB: potential chromatin-binding region; I-VI: helicase-specific domains. The thick horizontal line in the lower portion of the illustration shows a recombinant protein (rMi-2) which was used for immunological studies. A scale indicating the positions of the amino acids is plotted on the top.
FIG. 5
shows the localization of the 218 kD Mi-2 gene with fluorescence-in situ hybridization (FISH) at the short arm of the human chromosome 12 (12p 13) (arrows).
FIG. 6
shows immunoblots of SDS-PAGE-separated (A) recombinant Mi-2 polypeptide (rMi-2) and (B) nuclear proteins of HEp-2 cells which reacted with human sera and rabbit sera and affinity-purified anti-rMi-2-Ig. Lanes A1, B1: human non-reactive control serum. Lanes A2, B2: human anti-Mi-2-positive serum No. 1 (TABLE I) recognizes the rMi-2 with 55 kD (lane A2) as well as the natural 235 kD protein (lane B2) and an additional second natural nuclear protein having 80 kD. Lanes A3, B3: affinity-purified antibodies of serum No. 1 (human anti rMi-2-Ig) react with both the rMi-2 used for affinity purification and the natural 235 kD nuclear protein. The specificity of the antibody preparation is demonstrated in exemplary fashion by the absent reaction with the 80 kD protein. Lane A4, B4: non-reactive rabbit pre-immune serum. Lanes A5 and B5: following immunization with rMi-2, the rabbit serum recognizes the recombinant antigen (rMi-2) and the natural 235 kD protein. Lanes A6 and B6: affinity-purified rabbit antibodies (rabbit anti-rMi-2-Ig) also react with both proteins, the recombinant antigen and the natural 235 kD protein. Lane C (control): core proteins of HEp-2 cells react with a plurality of human antibodies.
FIG. 7A
shows immunoblots of nuclear (N), mitochondrial (Mt), microsomal (Ms) and cytoplasmic (S-100, S) fractions (80 &mgr;g protein/cm) of HEp-2 cells which reacted with affinity-purified rabbit-anti-rMi-2 antibodies. The 218 kD Mi-2 protein was found almost exclusively in the nuclear fraction at M
r
=235 kD. A very small portion of immunoreactive protein can be recognized in the S-100 supernatant. The evaluation of the blot scan yield the following distribution of the 218 kD Mi-2 protein: 97.5% in the nucleus
FIG. 7E
, 0% each in the mitochondrial and microsomal fractions and 2.5% in the S-100 fraction FIG.
7
C.
FIG. 8A
shows immunoprecipitations of
35
S-methionine-labeled proteins of HEp-2 cell lysates separated by SDS-PAGE with anti-Mi-2 sera (P2-P12) of DM patients and healthy control persons (C1, C2). b1=protein A sepharose alone. All patient sera precipitated a 235 kD protein and furthermore additional proteins which are marked by asterisks on the right-hand side and have individually differing patterns; the molecular weights are indicated additionally.
FIG. 8B
shows an immunoprecipitation of proteins of a HEp-2 cell lysate with the DM patient serum No. 1 (P1), with rMi-2 protein affinity-purified antibodies of serum No. 1 (P1 alg), affinity-purified rabbit antibodies (Ra alg), rabbit-IgG (Ra Ig), rabbit immune serum (Ra IS) and rabbit pre-immune serum (Ra pIS). Affinity-purified anti-rMi-2 antibodies precipitated the same 235 kD protein as the patient-anti-Mi-2-positive serum.
FIG. 8C
shows immunoprecipitations of
3
Renz Manfred
Seelig Hans Peter
Chan Christina Y.
Huynh Phuong N.
Pennie & Edmonds LLP
Privates Institut fur Immunologie und Molekulargenetik GmbH
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