U.S. patent application number 10/056407 was filed with the patent office on 2002-11-07 for methylated, smd homologous peptides, reactive with the antibodies from sera of living beings affected with systemic lupus erythematosus.
This patent application is currently assigned to INNOGENETICS N.V.. Invention is credited to Luhrmann, Reinhard Georg, Meheus, Lydie, Raymackers, Joseph, Union, Ann.
Application Number | 20020165355 10/056407 |
Document ID | / |
Family ID | 8231033 |
Filed Date | 2002-11-07 |
United States Patent
Application |
20020165355 |
Kind Code |
A1 |
Meheus, Lydie ; et
al. |
November 7, 2002 |
Methylated, SmD homologous peptides, reactive with the antibodies
from sera of living beings affected with systemic lupus
erythematosus
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, and
diseases in which Epstein-Barr virus has been implicated.
Inventors: |
Meheus, Lydie; (Merelbeke,
BE) ; Luhrmann, Reinhard Georg; (Marburg, DE)
; Union, Ann; (Aalter, BE) ; Raymackers,
Joseph; (Eke, BE) |
Correspondence
Address: |
Patricia A. Kammerer
HOWREY SIMON ARNOLD & WHITE, LLP
750 Bering Drive
Houston
TX
77057-2198
US
|
Assignee: |
INNOGENETICS N.V.
|
Family ID: |
8231033 |
Appl. No.: |
10/056407 |
Filed: |
January 24, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10056407 |
Jan 24, 2002 |
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09297981 |
May 10, 1999 |
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6362007 |
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09297981 |
May 10, 1999 |
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PCT/EP98/05518 |
Aug 31, 1998 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
C07K 7/08 20130101; C07K
14/4713 20130101; C07K 14/525 20130101; A61P 37/00 20180101; C07K
14/001 20130101; C07K 2319/00 20130101; A61P 29/00 20180101; A61K
38/00 20130101; A61P 35/00 20180101 |
Class at
Publication: |
530/350 |
International
Class: |
C07K 001/00; C07K
014/00; C07K 017/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 29, 1997 |
EP |
97870127.4 |
Claims
1) Peptide containing less than 50 amino acids, comprising at least
one dimer of the type XG, wherein X stands for a N.sup.G-mono- or
N.sup.G-N.sup.G-dimethylated arginine, that is able to react with
antibodies and 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
infection, or certain cancers which are related to infection with
Epstein-Barr virus, such as Burkitt's lymphoma or nasopharyngeal
carcinoma.
2) Peptide according to claim 1 comprising the amino acid
sequence
2 GXGXGXGXGXGXGXGXGXG (SEQ ID NO 1) or, AXGXGXGMGXG (SEQ ID NO 2)
or, KAQVAAXGXGXGMGXGN (SEQ ID NO 3) or,
DVEPKVKSKKREAVAGXGXGXGXGXGXGXGXGXGGPRR (SEQ ID NO 4) or,
DNHGXGXGXGXGXGGG (SEQ ID NO 5) or, GGXGXGGSGGXGXGG (SEQ ID NO 6)
or, ERAXGXGXGXGE (SEQ ID NO 7) or,
GGQQDXGGXGXGGGGGYNXSSGGYEPXGXGGGXGGXGGMGGSDXGG (SEQ ID NO 8) or,
GGQQDXGGXGXGGGGGYN (SEQ ID NO 9) or, SGGYEPXGXGGGXGGXGGMGGSDXGG
(SEQ ID NO 10) or, DFNXGGGNGXGGXGXGG (SEQ ID NO 11) or,
DFNXGGGNGXGGXGXGGPMGXGGYGGGGS (SEQ ID NO 12) or,
GDDXXGXGGYDXGGYXGXGGDXGGFXGGXGGGDXGGFG (SEQ ID NO 13) or,
GDDXXGXGGYDXGG (SEQ ID NO 14) or, GGYXGXGGDXGGFXGGXGGGDXG- GFG (SEQ
ID NO 15) or,
an analog of said peptides comprising conservative amino acid
substitutions.
3) Peptide and/or chemical structure comprising any of the peptides
according to claims 1 or 2, fused to a linker molecule.
4) Circularized peptide that comprises at least one of the peptides
according to any of the claims 1 to 3.
5) Peptide comprising and/or consisting of tandem repeats of at
least two of any of the peptides of claims 1 to 4.
6) Branched peptide that comprises at least one of the peptides
according to any of the claims 1 to 5.
7) Method for producing a peptide according to any of claims 1 to
6, by classical chemical synthesis, wherein methylated arginines
are substituted for unmethylated arginine residues during the
chemical synthesis.
8) Method for producing a peptide according to any of claims 1 to
6, wherein the primary amino acid sequence is produced by classical
chemical synthesis, and wherein the arginine residues that precede
glycine residues are subsequently methylated by contacting said
peptide with a protein arginine methyltransferase.
9) Method for producing a peptide of any of claims 1 to 6
comprising the following steps: transforming an appropriate
cellular host with a recombinant vector in which a polynucleic acid
is inserted comprising the sequence that codes for said peptide
under the control of the appropriate regulatory elements such that
said peptide or a protein comprising said peptide is expressed
and/or secreted, culturing said transformed cellular host under
conditions allowing expression of said protein or peptide and
allowing a partial or optimal methylation of the arginines present
in said peptide, harvesting said peptide.
10) Method for producing a peptide of any of claims 1 to 6
comprising the following steps: transforming an appropriate
cellular host with a recombinant vector in which a polynucleic acid
is inserted comprising the sequence that codes for said peptide
under the control of the appropriate regulatory elements, such that
said peptide or a protein comprising said peptide is expressed
and/or secreted, culturing said transformed cellular host under
conditions allowing expression of said protein or said peptide,
harvesting said protein or said peptide, methylating arginine
residues of said protein or said peptide by contacting with a
protein arginine methyltransferase.
11) Method according to any of claims 9 or 10, wherein said host
cell is a bacterial host or yeast or any other eukaryotic host cell
which is preferably transformed with a recombinant baculovirus.
12) An antibody raised upon immunization with a peptide according
to any of the claims 1 to 6, with said antibody being specifically
reactive with the methylated forms of said peptide, and with said
antibody being preferably a monoclonal antibody.
13) Anti-idiotype antibody raised upon immunization with an
antibody according to claim 12, with said anti-idiotype antibody
being specifically reactive with the antibody of claim 12, thereby
mimicking the methylated form of a peptide according to any of
claims 1 to 6, and with said antibody being preferably a monoclonal
antibody.
14) An immunotoxin molecule comprising and/or consisting of cell
recognition molecule being a peptide of any of claims 1 to 6, or an
antibody according to any of the claims 12 or 13, covalently bound
to a toxin molecule or active fragment thereof.
15) A peptide according to any of the claims 1 to 6 or an antibody
according to any of claims 12 or 13 or an immunotoxin molecule
according to claim 14 or a composition thereof for use as a
medicament.
16) Use of a peptide according to any of claims 1 to 6 or an
antibody according to any of claims 12 or 13 or an immunotoxin
molecule according to claim 14 or a composition thereof for the
preparation of a medicament or of a diagnosticum for auto-immune
diseases such as: systemic lupus erythematosus, discoid lupus
erythematosus, scleroderma, dermatomyositis, rheumatoid arthritis,
Sjogren's syndrome. or for diseases in which Epstein-Barr can be
implicated such as: Burkitt's lymphoma, nasopharyngeal carcinoma,
infectious, recurrent or chronic mononucleosis.
17) Use of a polypeptide according to claim 1 to 6 or a composition
thereof for the preparation of a medicament to treat auto-immune
diseases by increasing the size of antigen-immune complexes,
thereby improving the clearance of the formed immune complexes.
18) Use of a polypeptide according to claim 1 to 6 or a composition
thereof for the preparation of a medicament for oral administration
to treat auto-immune diseases by inducing a state of systemic
hyporesponsiveness to the said polypeptide (`Oral tolerance`).
19) A diagnostic kit for use in detecting auto-immune diseases such
as: systemic lupus erythematosus, discoid lupus erythematosus,
scleroderma, dermatomyositis, rheumatoid arthritis, Sjogren's
syndrome, or for detecting diseases in which Epstein-Barr can be
implicated such as: Burkitt's lymphoma, nasopharyngeal carcinoma,
Hodgkin's disease, infectious, recurrent or chronic mononucleosis,
said kit comprising at least one peptide according to any of claims
1 to 6, or an antibody according to claims 12 or 13, with said
peptide or antibody being possibly bound to a solid support.
20) A diagnostic kit according to claim 19, said kit comprising a
range of peptides according to any of claims 1 to 6 or of
antibodies according to claims 12 or 13, possibly in combination
with native methylated SmD1 or SmD3 and recombinant unmethylated
SmD1 or SmD3, wherein said peptides are attached to specific
locations on a solid substrate.
21) A diagnostic kit according to claim 19 or 20, wherein said
solid support is a membrane strip and said polypeptides are coupled
to the membrane in the form of parallel lines, natural SmD (1, 2 or
3) or in vitro dimethylated SmD (1, 2 or 3) unmethylated SmD
expressed in E. coli (1, 2 or 3) peptide of any of claims 1 to
6.
22) A diagnostic kit according to any of claims 19 to 21 wherein
certain peptides are not attached to a solid support but are
provided in the binding solution to be used as competitors and/or
to block other antibodies that are present in sera from patients
with autoimmune disease other than SLE, thereby decreasing or
eliminating possible cross-reaction and/or aspecific binding.
Description
[0001] 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
[0002] 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).
[0003] 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 Sjogren'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 scleroderma, dermatomyositis, rheumatoid
arthritis, Sjogren's syndrome, in patients treated with certain
drugs, or in patients suffering from infectious mononucleosis,
liver disease, malaria etc.
[0004] 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.
[0005] 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).
[0006] 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.
[0007] 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-aal 19)
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).sub.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, Sjogren's syndrome). The .beta.-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
.beta.-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.
[0008] 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.
[0009] 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.
[0010] Another aim of the present invention is to provide methods
for obtaining said peptides.
[0011] Another aim of the present invention is to provide methods
of raising antibodies specifically reactive with peptides of said
peptides, thereby mimicking said peptides.
[0012] Another aim of the present invention is to provide methods
of raising anti-idiotype antibodies specifically reactive with the
afore mentioned antibodies.
[0013] Another aim of the present invention is to provide a
pharmaceutical composition consisting of these peptides, for
therapy or diagnosis.
[0014] Another aim of the present invention is to provide a
diagnostic kit for systemic lupus erythematosus.
[0015] All these aims of the present invention are met by the
following embodiments of the present invention.
[0016] 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.
[0017] According to a further embodiment the present invention also
relates to a peptide and/or chemical structure comprising any of
the above mentioned peptides, fused to a linker molecule. The
present invention also relates to peptides comprising and/or
consisting of tandem repeats of at least two of any of the above
mentioned peptides, or branched peptides that comprises at least
one of the above mentioned peptides.
[0018] According to a more specific embodiment the present
invention also relates to a method for producing any of the above
mentioned peptides, by classical chemical synthesis, wherein
methylated arginines are substituted for unmethylated arginine
residues at certain steps during the chemical synthesis. The
present invention also relates to a method for producing any of the
above mentioned peptides, wherein the primary amino acid sequence
is produced by classical chemical synthesis, and wherein the
arginine residues that precede glycine residues are subsequently
methylated by contacting said peptides with a protein arginine
methyltransferase. The present invention also relates to a method
for producing any of the above mentioned peptides comprising the
following steps: (i) transforming an appropriate cellular host with
a recombinant vector in which a polynucleic acid is inserted
comprising the sequence that codes for said peptide under the
control of the appropriate regulatory elements such that said
peptide or a protein comprising said peptide is expressed and/or
secreted, (ii) culturing said transformed cellular host under
conditions allowing expression of said protein or peptide and
allowing a partial or optimal methylation of the arginines present
in said peptide, and (iii) harvesting said peptide. The present
invention also relates to a method for producing any of the above
mentioned peptides comprising the following steps: (i) transforming
an appropriate cellular host with a recombinant vector in which a
polynucleic acid is inserted comprising the sequence that codes for
said peptide under the control of the appropriate regulatory
elements, such that said peptide or a protein comprising said
peptide is expressed and/or secreted, (ii) culturing said
transformed cellular host under conditions allowing expression of
said protein or said peptide, (iii) harvesting said protein or said
peptide, and (iv) methylating arginine residues of said protein or
said peptide by contacting with a protein arginine
methyltransferase. According to a more specific embodiment the
present invention also relates to any of the above mentioned
methods, wherein said host cell is a bacterial host or yeast or any
other eukaryotic host cell which is preferably transformed with a
recombinant baculovirus.
[0019] According to a preferred embodiment the present invention
also relates to an antibody raised upon immunization with any of
the above mentioned peptides, with said antibody being specifically
reactive with the methylated forms of said peptide, and with said
antibody being preferably a monoclonal antibody. The present
invention also relates to an anti-idiotype antibody raised upon
immunization with any antibody as defined above, with said
anti-idiotype antibody being specifically reactive with said
antibody, thereby mimicking the methylated form of any above
mentioned peptide, and with said antibody being preferably a
monoclonal antibody.
[0020] According to a more specific embodiment the present
invention also relates to an immunotoxin molecule comprising and/or
consisting of a cell recognition molecule being a peptide as
defined above, or an antibody as defined above, covalently bound to
a toxin molecule or active fragment thereof.
[0021] According to a further embodiment the present invention
relates to any of the above mentioned peptides or antibodies or
immunotoxine molecules or a composition thereof for use as a
medicament. Said use can have the purpose of a medicament for
treatment or of a diagnosticum for any of the following auto-immune
diseases: systemic lupus erythematosus, discoid lupus
erythematosus, scleroderma, dermatomyositis, rheumatoid arthritis,
Sjogren's syndrome, or for diseases in which Epstein-Barr virus can
be implicated such as Burkitt's lymphoma or nasopharyngeal
carcinoma, or infectious, recurrent or chronic mononucleosis. More
specifically, the present invention relates to a treatment for
auto-immune diseases by increasing the size of antigen-immune
complexes, thereby improving the clearance of the formed immune
complexes. The present invention also relates to a treatment for
auto-immune diseases by inducing a state of systemic
hyporesponsiveness to the auto-antigen after oral administration of
any of the above mentioned peptides or antibodies or immunotoxine
molecules or a composition thereof, thereby preventing the
pathogenic production of anti-self antibodies like anti-Sm
antibodies or anti-DNA antibodies. The present invention also
relates to a diagnostic kit for use in detecting any of the afore
mentioned diseases, wherein said kit comprises at least one of the
above mentioned peptides or antibodies, and with said peptide or
antibody being possibly bound to a solid support. More preferably
said kit is comprising a range of said peptides or said antibodies,
possibly in combination with native methylated SmD1 or SmD3 or Sm69
and recombinant unmethylated SmD1 or SmD3 or Sm69, wherein said
peptides are attached to specific locations on a solid substrate.
More preferably said solid support is a membrane strip and said
polypeptides are coupled to the membrane in the form of parallel
lines. It has to be understood that certain peptides, or antibodies
as defined above, alternatively, are not attached to a solid
support but are provided in the binding solution to be used as
competitors and/or to block other antibodies that are present in
sera from patients with autoimmune diseases other than SLE, thereby
decreasing or eliminating possible cross-reaction and/or aspecific
binding.
[0022] We have demonstrated for the first time that well defined
secondary modifications (mostly N.sup.G,N.sup.G-dimethylarginine)
are present on the Arg residues of the C-terminal peptide, that are
followed by a glycine residue. Moreover, we have raised evidence
that the C-terminal peptide can only show an immunoreactivity
almost identical to the immunoreactivity of natural SmD, if these
arginine residues are methylated. These dimethylarginines present
on the nine Arg positions of the C-terminus, have been demonstrated
for the first time in the natural SmD1 molecule. In SmD2 no
dimethylarginine was retrieved while in the C-terminus of SmD3 the
four RG motifs in the C-terminus again were found to be
dimethylated.
[0023] The amino acid N.sup.G,N.sup.G-dimethylarginine is the
result of a post-translational modification which seems to occur
predominantly in RNA binding proteins (Najbauer, 1993). These
nuclear proteins are enzymatically modified by a nuclear protein
methylase I ( S-adenosyl-methionine: protein-arginine
N-methyltransferase, E.C.2.1.1.23; Rajpurohit, et al.,1994). The
structural specificity of this enzyme seems to be an arginine
containing peptide with glycine in the C-flanking position as was
shown by substrate evaluation with synthetic peptides (Rawal,
1995). Nevertheless, in the same study it was demonstrated that the
entire molecule also plays an important though thus far unknown
role in the methylation process. Interestingly, this cellular
methylation process can be mimicked in vitro with purified
methylasel as was illustrated with recombinant heterogeneous
nuclear RNP protein A1 (Rajpurohit,et al.1994)
[0024] From our results, we thus can conclude that in SmD
immunoreactivity, at least 2 epitopes are involved. One of the
epitopes is apparently present in the recombinant SmD1 molecule and
can not be assigned to a linear epitope (epitope mapping of E. coli
recombinant SmD 1, data not shown). This is in agreement with the
discontinuous epitope described by Rokeach et al. (1992b). The
epitope localized at the C-terminus both by epitope mapping with E.
coli fusion fragments (Rokeach et al.,1992b; Rivkin et al.,1994)
and synthetic peptides (Sabbatini et al.,1993a; James et al.,1994)
could well be explained by the work of Rivkin et al. (1994). The
latter group has demonstrated that a dipeptide repeat
(Gly-Arg).sub.9 is recognized by 35% of sera from SLE patients but
also by 15% of sera from other autoimmune diseases. This result is
in contrast with the high SLE specificity of the anti-Sm
antibodies. Indeed, the specificity of the unmodified C-terminal
SmD1 peptide has not been thoroughly investigated by Rokeach
(1992b) nor by James et al.(1994). Only Sabbatini (1993a) described
a certain disease specificity for the C-terminal synthetic peptide.
On one hand, Rivkin has shown that out of 32 sera positive for the
(Gly-Arg).sub.9 peptide, only 6 sera are positive with native SmD.
On the other hand, of the positive SmD sera identified on Western
blot, only half of them are reactive with the unmodified C-terminal
peptide (Rivkin: 4/8; Rokeach 9/19; Sabbatini: 5/9). Based on these
results it can be concluded that immunoreactivity of the unmodified
C-terminal peptide does not well correlate with natural SmD and is
less SLE specific than natural SmD. In contrast, our results show
that 15 out of 17 SmD positive sera are immunoreactive with the
dimethylated C-terminal peptide while only one serum reacts with
the unmodified C-terminal peptide. The 2 sera that do not recognize
the dimethylated C-terminal peptide are immunoreactive with the
total recombinant SmD and are apparently monospecific for the
discontinuous epitope.
[0025] In conclusion, natural SmD1 contains nine dimethylated
arginines at the C-terminus and this modification plays a crucial
role in the SLE specific immunoreactivity of the SmD antigen.
[0026] According to its main embodiment the present invention
relates to peptides that contain arginine residues that are
immediately followed by a glycine residue, and wherein at least one
arginine residue is methylated or dimethylated at one terminal
aminogroup of the guanidino-group of the arginine residue, and
wherein this methylation is a prerequisite for the peptide to be
recognized by antibodies that characterize certain diseases.
Antibodies that are specifically reacting with this type of
peptides can be found in sera from patients with systemic lupus
erythematosus or related autoimmune diseases such as discoid lupus
erythematosus, or patients with infectious mononucleosis, or
recurrent or chronic mononucleosis, or that suffer from diseases in
which Epstein-Barr virus has been implicated such as nasopharyngeal
carcinoma and Burkitt's lymphoma.
[0027] Peptides are described which immunologically mimic the
immunogenic determinants of self proteins recognized by the immune
system in patients suffering from lupus erythematosus. A crucial
aspect of such peptides is the fact that arginines followed by a
glycine are methylated. One peptide (SmD1) has been demonstrated to
contain a stretch of 9 consecutive arginine-glycine residues,
wherein each arginine is methylated and wherein this methylation is
necessary for specific recognition by antibodies present in sera of
patients with lupus erythematosus. A second peptide (SmD3) has been
demonstrated to contain isolated arginine-glycine residues, wherein
the arginine is methylated. Also, a third peptide (Sm69) has been
demonstrated to contain several domains characterised by several
arginine-glycine residues, wherein the-arginine is dimethylated. It
is therefore anticipated that the presence of one dimethylated
arginine, followed by a glycine can be sufficient for specific
recognition by some antibodies present in sera of patients with
lupus erythematosus. The invention therefore relates to those
peptides wherein at least one arginine residue is followed by a
glycine, wherein the arginine residue is methylated, and wherein
this methylation is necessary for specific recognition by
antibodies.
[0028] The term `peptide` as used throughout the specification and
claims refers to a polymer of amino acids and does not refer to a
specific length of the product; thus, oligopeptides, polypeptides
and proteins are included within the definition of `peptide`. This
term also does not refer to or exclude post-expression
modifications of the peptide, for example, glycosylations,
acetylations, phosphorylations and the like. Included within the
definition are, for example, peptides containing one or more
analogues of an amino acid (including, for example, unnatural amino
acids, PNA, etc.), polypeptides with substituted linkages, as well
as other modifications known in the art, both naturally occurring
and non-naturally occurring.
[0029] Whenever the expression "peptide containing less than 50
amino acids" is used, this should be interpreted in a broad sense,
as a means of circumscribing an essentially truncated version of
entire immunoreactive proteins that still comprises the highly
reactive domain characterized by the presence of methylated
arginine residues. These peptides have a length of preferably 40,
30, 25, 20 or less amino acids. The present invention also relates
to peptides having a length of 50, 60 or more amino acids without
comprising the full length of the native protein. It is for
practical purpose of peptide synthesis that peptides containing
less than 50 amino acids are defined.
[0030] With `immunogenic determinant` is meant, those chemical
groupings comprising a primary amino acid sequence, and secondary
modifications of the amino acid residues in a certain
three-dimensional arrangement, that together determine the specific
reactivity of the entire antigen for a raised antibody. Such
antibody can also recognize different chemical groupings, which are
then termed to `immunologically mimic` the immunogenic
determinant.
[0031] When secondary modifications of a peptide are said to be
`necessary` or `crucial`, or to `be a prerequisite` for reacting
with an antibody, the absence of said secondary modifications will
result in a peptide of which the dissociation constant for
interaction with said antibody will be at least two orders of
magnitude higher than the dissociation constant for the interaction
between said antibody and the peptide wherein the secondary
modifications are present, preferably three orders of magnitude
higher, and more preferably four orders of magnitude higher.
[0032] The term `crossreaction` refers to the reaction of one
antigen with antibodies developed against another antigen or
antibodies that are found in sera from patients with different
diseases.
[0033] The term `methylated arginine` as used throughout the
ensuing specification and claims is used in a broad sense and
refers any methylated form. More preferably the term methylated
refers to a dimethylated form of arginine in which one amino-group
of the guanidino group of the arginine residue is substituted with
one or two methyl groups. The term `methylation` refers to the
process that results in said forms of arginine.
[0034] It has to be understood that the peptides of the present
invention are not limited to include only those peptides that are
recognized by antibodies specific for systemic lupus erythematosus,
but also relate to those peptides that react with antibodies
associated with related autoimmune diseases such as discoid lupus
erythematosus and scleroderma or peptides that are related to
infection with Epstein-Barr virus such as infectious, recurrent, or
chronic mononucleosis. Several autoimmune diseases are related to
systemic lupus erythematosus such as discoid lupus erythematosus,
scleroderma. The antibodies that are present in sera of patients
with these autoimmune diseases tend to recognize self-antigens,
that are often localized in the nucleus and present in all tissues.
As a result, similar or analogous epitopes are often recognized,
thereby leading to crossreactivity, and difficult diagnosis.
[0035] The few antibodies present in sera from patients with
systemic lupus erythematosus that are also able to react with
recombinant and thus unmethylated C-terminal parts of SmD1, were
shown to cross-react with the EBNA-1 Epstein-Barr virus associated
nuclear antigen, of which the primary sequence displays homology
with the primary sequence of SmD1 (Sabbatini et al.,1993b). EBNA-1
is the only protein which is consistently expressed during latency
of Epstein-Barr virus. The extreme persistence of Epstein-Barr
virus during latency can be explained by the very low
immunogenicity of this viral protein. EBNA-1 most importantly
contains six arginine residues which are followed by a glycine. It
can therefore be anticipated that the frequency of finding specific
antibodies in sera from patients with mononucleosis (infected with
Epstein-Barr virus) or from people with recurrent, or chronic
mononucleosis or with a history of mononucleosis, will increase,
when antigens (such as EBNA-1) are used wherein the arginine
residues that precede a glycine residue are methylated, as we have
shown to be the case with the antibodies directed against SmD-1 in
patients with lupus erythematosus.
[0036] According to a more specific embodiment the present
invention relates to those peptides that contain methylated
arginine residues that are preceding glycine residues, wherein said
methylation is crucial for high-affinity interaction with
antibodies that are found in sera from patients with autoimmune
diseases such as systemic lupus erythematosus, or that are infected
with Epstein Barr virus.
[0037] The present invention also relates to those peptides wherein
the arginine-glycine doublets are repeated, at least once, and more
preferably 3 or 4 or 5 or 6 or 7 or 8 times, and even more
preferably 9 times, as is the case with the natural antinuclear
antigen SmD-1, and wherein at least one arginine residue that
precedes a glycine residue is methyfated, preferably dimethylated,
with said methylation being necessary for specific reaction with
antibodies, for instance with antibodies found in sera from
patients with SLE.
[0038] In a more specific embodiment, the present invention relates
to a peptide that is characterized by the amino acid sequence
GRGRGRGRGRGRGRGRGRG (SEQ ID NO 16) wherein at least one and
preferably each arginine is methylated, preferably dimethylated and
even more preferably dimethylated in an asymmetric way, thereby
mimicking the main immunogenic determinant of the C-terminal part
of antinuclear antigen SmD1.
[0039] In a more specific embodiment, the present invention relates
to a peptide that is characterized by the amino acid sequence
DVEPKVKSKKREAVAGRG RGRGRGRGRGRGRGRGGPRR (SEQ ID NO 17) wherein at
least one and preferably each arginine that precedes a glycine, is
methylated, preferably dimethylated and even more preferably
dimethylated in an asymmetric way, thereby mimicking the main
immunogenic determinant of the C-terminal part of antinuclear
antigen SmD 1.
[0040] In a more specific embodiment, the present invention relates
to a peptide that is characterized by the amino acid sequence
ARGRGRGMGRG (SEQ ID NO 18) wherein at least one and preferably each
arginine is methylated, preferably dimethylated and even more
preferably dimethylated in an asymmetric way, thereby mimicking the
main immunogenic determinant of the C-terminal part of antinuclear
antigen SmD3.
[0041] In a more specific embodiment, the present invention also
relates to a peptide that is characterized by the amino acid
sequence KAQVAARGRGRGMGRGNIFQKRR (SEQ ID NO 19) wherein at least
one and preferably each arginine that precedes a glycine is
methylated, preferably dimethylated and even more prefeably
dimethylated in an asymmetric way, thereby mimicking the main
immunogenic determinant and its borders of the C-terminal part of
antinuclear antigen SmD3.
[0042] According to a more specific embodiment, the present
invention also relates to a peptide that comprises or consists of
by the amino acid sequence GGQQDR
GGRGRGGGGGYNRSSGGYEPRGRGGGRGGRGGMGGSDRGG (SEQ ID NO 20) or
GGQQDRGGRGRGGGGGYN (SEQ ID NO 21) or SGGYEPRGRGGGRGGRGGM GGSDRGG
(SEQ ID NO 22) or DFNRGGGNGRGGRGRGG (SEQ ID NO 23) or
DFNRGGGNGRGGRGRGGPMGRGGY- GGGGS (SEQ ID NO 24) or GDDRRGR
GGYDRGGYRGRGGDRGGFRGGRGGGDRGGFG (SEQ ID NO 25) or GDDRRGRGGYDRGG
(SEQ ID NO 26) or GGYRGRGGDRGGFRGGRGGGDRGGFG (SEQ ID NO 27) wherein
at least one and preferably each arginine that precedes a glycine
is methylated, preferably dimethylated and even more preferably
dimethylated in an asymmetric way, thereby mimicking the main
immunogenic determinant and its borders of the C-terminal part of
antinuclear antigen Sm69.
[0043] According to a more specific embodiment, the present
invention relates to a peptide that comprises or consists of -the
amino acid sequence DNHGRGRGRGRGRGGG (SEQ DI NO 28) or
GGRGRGGSGGRGRGG (SEQ DI NO 29) or ERARGRGRGRGE (SEQ ID NO 30)
wherein at least one and preferably each arginine that precedes a
glycine is methylated, preferably dimethylated and even more
preferably dimethylated in an asymmetric way, thereby mimicking the
Epstein-Barr virus nuclear antigen 1.
[0044] The present invention also relates to molecular structures
in which at least part represents a peptide or antibody as defined
above. Such molecular structures can result from fusion of peptides
of the present invention with peptides and/or proteins and/or other
molecules that are further characterized in that they specifically
interact with other peptides and/or proteins and/or molecular
structures, enabling tagging and/or binding of the fused
polypeptide and/or protein to specific tissue- or cell types or
that allow for purification of said molecular structures due to the
presence of for instance 4, or 5 or 6 consecutive histidine
residues, or
[0045] are cytotoxic to T-cells and/or B-cells such as cholera
toxin, or
[0046] allow for labelling by means of a radioactive or fluorescent
or immunogold or enzymatic marker.
[0047] It may also be desirable in certain instances to join two or
more peptides together in one peptide structure, or to create
branched peptides. One advantage of this arrangement is well known
in the art and relates to diagnosis. When antibodies are used in an
assay in order to detect the present antigens, tandem repeats or
branched peptides of the antigens can increase the amount of
immobilized antigens presented to the antibodies and thereby
increase the sensitivity of the assay. The sensitivity can be
increased exponentially when the immobilized antigens are used
together with a specific concentration of such antigens in a
soluble form, thereby inducing the formation of crosslinked
antigen-immunoprecipitates. A second advantage relates to therapy.
The deposition of self-antigen autoimmune complexes in various
tissues is an important step towards the acquisition of a
pathological condition. It is generally accepted that the main
cause of said deposition is the insufficient blood clearance by the
liver of the antigen-immune complexes due to the small size of said
complexes. Administration of tandem repeats or branched forms of
said peptides could increase the size of the formed antigen-immune
complexes, and thereby increase the clearance and thus decrease the
deposition of said complexes.
[0048] The present invention also relates to circularized forms of
said peptides, the advantage being well known in the art, and
relating to an increased affinity of a conformationally constraint
peptide as compared with the more randomly coiled forms of linear
peptides.
[0049] In order to accommodate for eventual negative
characteristics of the claimed peptides, such as rapid degradation,
solubility, cytotoxic effects and so on, the skilled person will be
able to design conservative as well as non-conservative amino acid
substitutions, or substitutions with non-natural amino acids, PNA
etc. . . These will generally account for less than 35 percent of a
specific sequence. Such peptides also include peptides with
substituted linkages, as well as other modifications known in the
art, both naturally occurring and non-naturally occurring. It may
be desirable in cases where the SmD peptides or other antigenic
peptides of the present invention are highly polymorphic, to vary
one or more of the amino acids so as to better mimic the different
epitopes of several viral strains, or as recognized by antibodies
in sera from patients with SLE or other autoimmune diseases.
[0050] The present invention also relates to any analogs of the
peptides of the present invention.
[0051] The term "analog" as used throughout the specification or
claims to describe the proteins or peptides of the present
invention, includes any protein or peptide having an amino acid
residue sequence substantially identical to a sequence specifically
shown herein in which one or more residues have been conservatively
substituted with a biologically equivalent residue. Examples of
conservative substitutions include the substitution of hydrophobic
residue such as isoleucine, valine, leucine or methionine for
another, the substitution of one hydrophilic residue for another
such as between arginine and lysine, between glutamine and
asparagines, between glycine and serine, the substitution of one
basic residue such as lysine, arginine or histidine for another, or
the substitution of one acidic residue, such as aspartic acid or
glutamic acid for another. Examples of allowable mutations
according to the present invention can be found in Table 4.
1TABLE 4 Overview of the amino acid substitutions which could form
the basis of analogs (muteins) as defined above. Amino acids
Synonymous groups Ser (S) Ser, Thr, Gly, Asn Arg (R) Arg, His, Lys,
Glu, Gln Leu (L) Leu; Ile, Met, Phe, Val, Tyr Pro (P) Pro, Ala,
Thr, Gly Thr (T) Thr, Pro, Ser, Ala, Gly, His, Gln Ala (A) Ala,
Pro, Gly, Thr Val (V) Val, Met, Ile, Tyr, Phe, Leu, Val Gly (G)
Gly, Ala, Thr, Pro, Ser Ile (I) Ile, Met, Leu, Phe, Val, Ile, Tyr
Phe (F) Phe, Met, Tyr, Ile, Leu, Trp, Val Tyr (Y) Tyr, Phe, Trp,
Met, Ile, Val, Leu Cys (C) Cys, Ser, Thr, Met His (H) His, Gln,
Arg, Lys, Glu, Thr Gln (Q) Gln, Glu, His, Lys, Asn, Thr, Arg Asn
(N) Asn, Asp, Ser, Gln Lys (K) Lys, Arg, Glu, Gln, His Asp (D) Asp,
Asn, Glu, Gln Glu (E) Glu, Gln, Asp, Lys, Asn, His, Arg Met (M)
Met, Ile, Leu, Phe, Val
[0052] The phrase "conservative substitution" also includes the use
of a chemically derivatized residue in place of a non-derivatized
residue provided that the resulting protein or peptide is
biologically equivalent to the protein or peptide of the
invention.
[0053] "Chemical derivative" refers to a protein or peptide having
one or more residues chemically derivatized by reaction of a
functional side group or peptides with substituted linkages, as
well as other modifications known in the art, both naturally
occurring and non-naturally occurring. Examples of such derivatized
molecules, include but are not limited to, those molecules in which
free amino groups have been derivatized to form amine
hydrochlorides, p-toluene suifonyl groups, carbobenzoxy groups,
t-butyloxycarbonyl groups, chloracetyl groups or formyl groups.
Free carboxyl groups may be derivatized to form salts, methyl and
ethyl esters or other types of esters or hydrazides. Free hydroxyl
groups may be derivatized to form O-acyl or O-alkyl derivatives.
The imidazole nitrogen of histidine may be derivatized to form
N-imbenzylhistidine. Also included as chemical derivatives are
those proteins or peptides which contain one or more
naturally-occurring amino acid derivatives of the twenty standard
amino acids. For examples: 4-hydroxyproline may be substituted for
proline; 5-hydroxylysine may be substituted for lysine;
3-methylhistidine may be substituted for histidine; homoserine may
be substituted for serine; and ornithine may be substituted for
lysine. The peptides of the present invention also include any
protein or peptide having one or more additions and/or deletions or
residues relative to the sequence of a peptide whose sequence is
shown herein, as long as the peptide is biologically equivalent to
the proteins or peptides of the invention.
[0054] Furthermore, additional amino acids or chemical groups may
be added to the amino- or carboxyl terminus for the purpose of
creating a "linker arm" by which the peptide can conveniently be
attached to a carrier. The linker arm will be at least one amino
acid and may be as many as 60 amino acids but will most frequently
be 1 to 10 amino acids. The nature of the attachment to a solid
phase or carrier can be non-covalent as well as covalent. Possible
arrangements of this nature are well described in the art. Natural
amino acids such as histidine, cysteine, lysine, tyrosine, glutamic
acid, or aspartic acid may be added to either the amino- or
carboxyl terminus to provide functional groups for coupling to a
solid phase or a carrier. However, other chemical groups such as,
for example, biotin and thioglycolic acid, may be added to the
termini which will endow the peptides with desired chemical or
physical properties. The termini of the peptides may also be
modified, for example, by N-terminal acetylation or terminal
carboxy-amidation. In each instance, the peptide will preferably be
as small as possible while still maintaining substantially all of
the sensitivity of the larger peptide.
[0055] The peptides of the invention, and particularly the
fragments, can be prepared by classical chemical synthesis. The
synthesis can be carried out in homogeneous solution or in solid
phase. For instance, the synthesis technique in homogeneous
solution which can be used is the one described by Houbenweyl in
the book entitled "Methode der organischen chemie" (Method of
organic chemistry) edited by E. Wunsh, vol. 15-I et II. THIEME,
Stuttgart 1974. The polypeptides of the invention can also be
prepared in solid phase according to the methods described by
Atherton and Shepard in their book entitled "Solid phase peptide
synthesis" (IRL Press, Oxford, 1989). The methylated forms of the
claimed peptides can be obtained by substituting the methylated
arginine derivatives for the normal arginine derivatives during the
classical chemical synthesis, or by contacting the unmethylated
peptides after synthesis with a protein arginine methyl transferase
enzyme of any eukaryotic origin.
[0056] The polypeptides according to this invention can also be
prepared by means of recombinant DNA techniques as described, by
Maniatis et al., Molecular Cloning: A Laboratory Manual, New York,
Cold Spring Harbor Laboratory, 1982) by insertion of a polynucleic
acid sequence encoding the claimed peptides or part of the claimed
peptides in an appropriate vector and transforming a suitable host
with said vector. This recombinant expression vector comprises a
polynucleic acid or a part thereof as defined above, operably
linked to prokaryotic, eukaryotic or viral transcription and
translation control elements. In addition this sequence can be
operably linked with sequences that allow for secretion of the
claimed peptides. The term `vector` may comprise a plasmid, a
cosmid, a phage or a virus or a transgenic organism. Particularly
useful may be BCG or adenoviral vectors, as well as avipox
recombinant viruses.
[0057] The recombinant peptides can be methylated in vitro, by
contacting the expressed and/or secreted peptides with a protein
arginine methyl transferase of any eukaryotic origin, or in vivo by
choosing the appropriate host, like yeast, or any eukaryotic cell,
and more preferably by using the baculovirus transformation
system.
[0058] The present invention does not exclude the option of using
additional proteins like the BTG1 and TIS21 proteins which have
been demonstrated to be essential for methylation in vivo (as a
co-expressed protein) or to be required for optimal methylation in
vitro (Lin et al., 1996), or any other proteins, peptides or
chemical substances that can optimize the level of methylation.
[0059] Also any of the known purification methods for recombinant
peptides can be used for the production of the recombinant peptides
of the present invention.
[0060] The present invention also relates to a recombinant
expression vector comprising a polynucleic acid or a part thereof
as defined above, operably linked to prokaryotic, eukaryotic or
viral transcription and translation control elements.
[0061] In general, said recombinant vector will comprise a vector
sequence, an appropriate prokaryotic, eukaryotic or viral promoter
sequence followed by a nucleotide sequence encoding a peptide as
defined above, with said recombinant vector allowing the expression
and/or secretion of any one of the polypeptides as defined above in
a prokaryotic, or eukaryotic host or in living mammals when
injected as naked DNA.
[0062] Also any of the known purification methods for recombinant
proteins may be used for the production of the recombinant
polypeptides of the present invention.
[0063] The term "vector" may comprise a plasmid, a cosmid, a phage,
or a virus or a transgenic animal. Particularly useful for vaccine
development may be BCG or adenoviral vectors, as well as avipox
recombinant viruses.
[0064] The present invention also relates to a method for the
production of a recombinant polypeptide as defined above,
comprising:
[0065] transformation of an appropriate cellular host with a
recombinant vector, in which a polynucleic acid or a part thereof
according to as defined above has been inserted under the control
of appropriate regulatory elements,
[0066] culturing said transformed cellular host under conditions
enabling the expression and/or secretion of said insert, and,
[0067] harvesting said polypeptide.
[0068] The term "recombinantly expressed" used within the context
of the present invention refers to the fact that the proteins of
the present invention are produced by recombinant expression
methods be it in prokaryotes, or lower or higher eukaryotes as
discussed in detail below.
[0069] The term "lower eukaryote" refers to host cells such as
yeast, fungi and the like. Lower eukaryotes are generally (but not
necessarily) unicellular. Preferred lower eukaryotes are yeasts,
particularly species within Saccharomyces, Schizosaccharomyces,
Kluveromyces, Pichia (e.g. Pichia pastoris), Hansenula (e.g.
Hansenula polymorpha), Yarowia, Schwaniomyces, Schizosaccharomyces,
Zygosaccharomyces and the like. Saccharomyces cerevisiae, S.
carlsbergensis and K. lactis are the most commonly used yeast
hosts, and are convenient fungal hosts.
[0070] The term "prokaryotes" refers to hosts such as E. coli,
Lactobacillus, Lactococcus, Salmonella, Streptococcus, Bacillus
subtilis or Streptomyces. Also these hosts are contemplated within
the present invention.
[0071] The term "higher eukaryote" refers to host cells derived
from higher animals, such as mammals, reptiles, insects, and the
like. Presently preferred higher eukaryote host cells are derived
from Chinese hamster (e.g. CHO), monkey (e.g. COS and Vero cells),
baby hamster kidney (BHK), pig kidney (PK15), rabbit kidney 13
cells (RK13), the human osteosarcoma cell line 143 B, the human
cell line HeLa and human hepatoma cell lines like Hep G2, and
insect cell lines (e.g. Spodoptera frugiperda). The host cells may
be provided in suspension or flask cultures, tissue cultures, organ
cultures and the like. Alternatively the host cells may also be
transgenic animals.
[0072] The term "recombinant polynucleotide" or "nucleic acid"
intends a polynucleotide or nucleic acid of genomic, cDNA,
semisynthetic, or synthetic origin which, by virtue of its origin
or manipulation (1) is not associated with all or a portion of a
polynucleotide with which it is associated in nature, (2) is linked
to a polynucleotide other than that to which it is linked in
nature, or (3) does not occur in nature.
[0073] The term "recombinant host cells", "host cells", "cells",
"cell lines", "cell cultures", and other such terms denoting
microorganisms or higher eukaryotic cell lines cultured as
unicellular entities refer to cells which can be or have been, used
as recipients for a recombinant vector or other transfer
polynucleotide, and include the progeny of the original cell which
has been transfected. It is understood that the progeny of a single
parental cell may not necessarily be completely identical in
morphology or in genomic or total DNA complement as the original
parent, due to natural, accidental, or deliberate mutation.
[0074] The term "replicon" is any genetic element, e.g., a plasmid,
a chromosome, a virus, a cosmid, etc., that behaves as an
autonomous unit of polynucleotide replication within a cell; i.e.,
capable of replication under its own control.
[0075] The term "vector" is a replicon further comprising sequences
providing replication and/or expression of a desired open reading
frame.
[0076] The term "control sequence" refers to polynucleotide
sequences which are necessary to effect the expression of coding
sequences to which they are ligated. The nature of such control
sequences differs depending upon the host organism; in prokaryotes,
such control sequences generally include promoter, ribosomal
binding site, splicing sites and terminators; in eukaryotes,
generally, such control sequences include promoters, splicing
sites, terminators and, in some instances, enhancers. The term
"control sequences" is intended to include, at a minimum, all
components whose presence is necessary for expression, and may also
include additional components whose presence is advantageous, for
example, leader sequences which govern secretion.
[0077] The term "promoter" is a nucleotide sequence which is
comprised of consensus sequences which allow the binding of RNA
polymerase to the DNA template in a manner such that mRNA
production initiates at the normal transcription initiation site
for the adjacent structural gene.
[0078] 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 a coding sequence is ligated in such
a way that expression of the coding sequence is achieved under
conditions compatible with the control sequences.
[0079] The polynucleic acids encoding the peptides of the present
invention and inserted into the vector sequence may be attached to
a signal sequence. Said signal sequence may be that from any
source, e.g. the IgG or tissue plasminogen activator (tpa) leader
sequence for expression in mammalian cells, or the .alpha.-mating
factor sequence for expression into yeast cells.
[0080] A variety of vectors may be used to obtain the peptides of
the present invention. Lower eukaryotes such as yeasts and
glycosylation mutant strains are typically transformed with
plasmids, or are transformed with a recombinant virus. The vectors
may replicate within the host independently, or may integrate into
the host cell genome.
[0081] Higher eukaryotes may be transformed with vectors, or may be
infected with a recombinant virus, for example a recombinant
vaccinia virus. Techniques and vectors for the insertion of foreign
DNA into vaccinia virus are well known in the art, and utilize, for
example homologous recombination. A wide variety of viral promoter
sequences, possibly terminator sequences and poly(A)-addition
sequences, possibly enhancer sequences and possibly amplification
sequences, all required for the mammalian expression, are available
in the art. Vaccinia is particularly preferred since vaccinia halts
the expression of host cell proteins. Vaccinia is also very much
preferred since it allows the expression of f.i. peptides of the
present invention in cells or individuals which are immunized with
the live recombinant vaccinia virus. For vaccination of humans the
avipox and Ankara Modified Virus (AMV) are particularly useful
vectors.
[0082] Also known are insect expression transfer vectors derived
from baculovirus Autographa californica nuclear polyhedrosis virus
(AcNPV), which is a helper-independent viral expression vector.
Expression vectors derived from this system usually use the strong
viral polyhedrin gene promoter to drive the expression of
heterologous genes. Different vectors as well as methods for the
introduction of heterologous DNA into the desired site of
baculovirus are available to the man skilled in the art for
baculovirus expression. Also different signals for
posttransiational modification recognized by insect cells are known
in the art.
[0083] The present invention also relates to a host cell
transformed with a recombinant vector as defined above.
[0084] The present invention also relates to antibodies that are
specifically raised against the peptides of the present invention,
preferably against those peptides wherein the arginines that
precede a glycine residue are methylated. These antibodies may be
polyclonal or monoclonal. To prepare antibodies a host animal is
immunized using the peptides of the present invention in a
pharmaceutically acceptable carrier, wherein at least one of the
arginines that precede a glycine residue of said peptides is
methylated. Pharmaceutically acceptable carriers include any
carrier that does not itself induce the production of antibodies
harmful to the individual receiving the composition. Suitable
carriers are typically large, slowly metabolized macromolecules
such as proteins, polysaccharides, polylactic acids, polyglycolic
acids, polymeric amino acids, amino acid copolymers; and inactive
virus particles. Such carriers are well known to those of ordinary
skill in the art.
[0085] Preferred adjuvants to enhance effectiveness of the
composition include, but are not limited to: aluminim hydroxide
(alum), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) as
found in U.S. Pat. No. 4,606,918,
N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-di-
palmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE)
and RIBI, which contains three components extracted from bacteria,
monophosphoryl lipid A, trehalose dimycolate, and cell wall
skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. Any of
the 3 components MPL, TDM or CWS may also be used alone or combined
2 by 2. Additionally, adjuvants such as Stimulon (Cambridge
Bioscience, Worcester, Mass.) or SAF-1 (Syntex) may be used.
Further, Complete Freund's Adjuvant (CFA) and Incomplete Freund's
Adjuvant (IFA) may be used for non-human applications and research
purposes.
[0086] The immunogenic compositions typically will contain
pharmaceutically acceptable vehicles, such as water, saline,
glycerol, ethanol, etc. Additionally, auxiliary substances, such as
wetting or emulsifying agents, pH buffering substances,
preservatives, and the like, may be included in such vehicles.
[0087] Typically, the immunogenic compositions are prepared as
injectables, either as liquid solutions or suspensions; solid forms
suitable for solution in, or suspension in, liquid vehicles prior
to injection may also be prepared. The preparation also may be
emulsified or encapsulated in liposomes for enhanced adjuvant
effect. The proteins may also be incorporated into Immune
Stimulating Complexes together with saponins, for example Quil A
(ISCOMS).
[0088] Immunogenic compositions used to raise antibodies comprise a
`sufficient amount` or `an immunologically effective amount` of the
peptides of the present invention, as well as any other of the
above mentioned components, as needed. `Immunologically effective
amount`, means that the administration of that amount to an
individual, either in a single dose or as part of a series, is
effective to provoke an immune response and to raise antibodies, as
defined above. This amount varies depending upon the health and
physical condition of the individual, the taxonomic group of the
individual to be treated (e.g. nonhuman primate, primate, rabbit,
etc.), the capacity of the individual's immune system to synthesize
antibodies, the immunogenicity of the antigenic peptide, and its
mode of administration, and other relevant factors. It is expected
that the amount will fall in a relatively broad range that can be
determined through routine trials. Usually, the amount will vary
from 0.01 to 1000 .mu.g/dose, more particularly from 0.1 to 100
.mu.g/dose.
[0089] The immunogenic compositions are conventionally administered
parenterally, typically by injection, for example, subcutaneously
or intramuscularly. Additional formulations suitable for other
methods of administration include oral formulations and
suppositories. Dosage treatment may be a single dose schedule or a
multiple dose schedule. The vaccine may be administered in
conjunction with other immunoregulatory agents.
[0090] The host serum or plasma is collected following an
appropriate time interval to provide a composition comprising
antibodies reactive with the peptides of the present invention. The
gamma globulin fraction or the IgG antibodies can be obtained, for
example, by use of saturated ammonium sulfate or DEAE Sephadex, or
other techniques known to those skilled in the art. The antibodies
are substantially free of many of the adverse side effects which
may be associated with other anti-viral agents such as drugs, for
the treatment of infectious, chronic, or recurrent mononucleosis.
Such antibodies may also be used to diagnose certain diseases, such
as Burkitt's lymphoma, wherein Epstein-Barr virus has been
implicated.
[0091] The term `immunogenic` refers to the ability of a substance
to cause a humoral and/or cellular response, whether alone or when
linked to a carrier, in the presence or absence of an adjuvant.
[0092] The antibodies of the claimed invention may also be
monoclonals that are prepared with said antibody being specifically
reactive with any of said peptides, and with said antibody being
preferably a monoclonal antibody.
[0093] The monoclonal antibodies of the invention can be produced
by any hybridoma liable to be formed according to classical methods
from splenic cells of an animal, particularly from a mouse or rat,
immunized against the claimed peptides of the present invention on
the one hand, and of cells of a myeloma cell line on the other
hand, and to be selected by the ability of the hybridoma to produce
the monoclonal antibodies recognizing the methylated forms of the
peptides which has been initially used for the immunization of the
animals.
[0094] The antibodies involved in the invention can be labelled by
an appropriate label of the enzymatic, fluorescent, or radioactive
type.
[0095] The monoclonal antibodies according to this preferred
embodiment of the invention may be humanized versions of mouse
monoclonal antibodies made by means of recombinant DNA technology,
departing from parts of mouse and/or human genomic DNA sequences
coding for H and L chains or from cDNA clones coding for H and L
chains.
[0096] Alternatively the monoclonal antibodies according to this
preferred embodiment of the invention may be human monoclonal
antibodies. These antibodies according to the present embodiment of
the invention can also be derived from human peripheral blood
lymphocytes of patients with SLE or any other autoimmune disease or
with infectious, or recurrent or chronic mononucleosis. Such human
monoclonal antibodies are prepared, for instance, by means of human
peripheral blood lymphocytes (PBL) repopulation of severe combined
immune deficiency (SCID) mice (for recent review, see Duchosal et
al. 1992) or by screening Epstein Barr-virus-transformed
lymphocytes of infected or vaccinated individuals for the presence
of reactive B-cells by means of the antigens of the present
invention.
[0097] The present invention also relates to the anti-idiotype
antibodies that are raised upon immunization with an antibody as
defined above and that specifically react with said antibodies,
thereby mimicking the peptides of the present invention. The
methods for production of monoclonal anti-idiotype antibodies,
which are well known in the art, have been described, for instance,
by Gheuens et MaceFarlin (1982).
[0098] The present invention also relates to truncated versions or
single chain versions of the antibodies and anti-idiotype
antibodies as defined above, that have retained their original
specificity for reacting with the antigens.
[0099] The present invention also relates to proteins or peptides
that mimic the antibodies as defined above such as microproteins as
can be obtained by phage display or the highly variable domain of a
recombinant antibody as obtained by screening upon repertoire
cloning.
[0100] The present invention also relates to a method for detecting
antibodies that specifically react with the peptides or
anti-idiotype antibodies of the present invention, present in a
biological sample, comprising:
[0101] (i) contacting the biological sample to be analysed for the
presence of said antibodies with a peptide or anti-idiotype
antibody as defined above,
[0102] (ii) detecting the immunological complex formed between said
antibodies and said peptide or anti-idiotype antibody.
[0103] The present invention also relates to a reverse method for
detecting the peptides and/or the anti-idiotype antibodies of the
present invention with antibodies present in a biological sample
that specifically react with methylated forms of said peptides
and/or anti-idiotype antibodies that mimic such peptides,
comprising:
[0104] (i) contacting the biological sample to be analysed for the
presence of said peptides or anti-idiotype antibodies with the
antibodies as defined above,
[0105] (ii) detecting the immunological complex formed between said
antibodies and said peptide or anti-idiotype antibody.
[0106] The methods as defined above, can be used in the diagnosis
of autoimmune diseases such as systemic lupus erythematosus,
discoid lupus erythematosus, scleroderma, dermatomyositis,
rheumatoid arthritis, Sjogren's syndrome, or diseases in which
Epstein-Barr virus can be implicated such as infectious, recurrent
or chronic mononucleosis, or Burkitt's lymphoma, or nasopharyngeal
carcinoma, or Hodgkin's disease, or of certain cancers such as
Ewing sarcoma, or malignant melanoma of soft tissue.
[0107] According to a specific embodiment, the present invention
relates to the development of a diagnostic technique that allows
differentiation between those autoimmune diseases in which the
characteristic antibodies often crossreact with the same antigen,
thus resulting in difficult and slow diagnosis. Such diagnostic
technique can be obtained by the simultaneous use of several
antigens, methylated and unmethylated, and at least two epitopes, a
methylated and a non-methylated form of any of the claimed peptides
and/or anti-idiotype antibodies of the present invention.
[0108] The present invention also relates to a diagnostic kit for
use in detecting the presence of said antibodies, said kit
comprising at least one peptide or anti-idiotype antibody or
microprotein as defined above, with said peptide or anti-idiotype
antibody or microprotein being preferably bound to a solid
support.
[0109] The present invention also relates to a diagnostic kit for
determining the type of autoimmune disease or the type of infection
or to characterize certain cancers, said kit comprising at least
one peptide or anti-idiotype antibody or microprotein as defined
above, with said peptide or anti-idiotype antibody or microprotein
being preferably bound to a solid support.
[0110] The present invention also relates to a diagnostic kit as
defined above, said kit comprising a range of said peptides and/or
anti-idiotype antibodies or microprotein which are attached to
specific locations on a solid substrate.
[0111] The present invention also relates to a diagnostic kit as
defined above, wherein said solid support is a membrane strip and
said peptides and/or anti-idiotype antibodies or microproteins are
coupled to the membrane in the form of parallel lines.
[0112] The immunoassay methods according to the present invention
may utilize for instance single or specific oligomeric antigens,
dimeric antigens, as well as combinations of single or specific
oligomeric antigens. The peptides of the present invention may be
employed in virtually any assay format that employs a known antigen
to detect antibodies that characterize a certain disease or
infection. A common feature of all of these assays is that the
antigenic peptide or anti-idiotype antibody or microprotein is
contacted with the body component suspected of containing the
antibodies under conditions that permit the antigen to bind to any
such antibody present in the component. Such conditions will
typically be physiologic temperature, pH and ionic strength using
an excess of antigen. The incubation of the antigen with the
specimen is followed by detection of immune complexes comprised of
the antigen.
[0113] Design of the immunoassays is subject to a great deal of
variation, and many formats are known in the art. Protocols may,
for example, use solid supports, or immunoprecipitation. Most
assays involve the use of labelled antibody or peptide; the labels
may be, for example, enzymatic, fluorescent, chemiluminescent,
radioactive, or dye molecules. Assays which amplify the signals
from the immune complex are also known; examples of which are
assays which utilize biotin and avidin or streptavidin, and
enzyme-labelled and mediated immunoassays, such as ELISA
assays.
[0114] The immunoassay may be, without limitation, in a
heterogeneous or in a homogeneous format, and of a standard or
competitive type. In a heterogeneous format, the peptide or
anti-idiotype antibody or microprotein is typically bound to a
solid matrix or support to facilitate separation of the sample from
the peptide or anti-idiotype antibody or microprotein after
incubation. Examples of solid supports that can be used are
nitrocellulose (e.g., in membrane or microtiter well form),
polyvinyl chloride (e.g., in sheets or microtiter wells),
polystyrene latex (e.g., in beads or microtiter plates,
polyvinylidene fluoride (known as Immunolon.TM.), diazotized paper,
nylon membranes, activated beads, and Protein A beads. For example,
Dynatech Immunolon.TM. 1 or Immunolon.TM. 2 microtiter plates or
0.25 inch polystyrene beads (Precision Plastic Ball) can be used in
the heterogeneous format. The solid support containing the
antigenic peptides or anti-idiotype antibodies or microprotein is
typically washed after separating it from the test sample, and
prior to detection of bound antibodies. Both standard and
competitive formats are know in the art.
[0115] In a homogeneous format, the test sample is incubated with
the combination of antigens in solution. For example, it may be
under conditions that will precipitate any antigen-antibody or
anti-idiotype antibody-antibody or microprotein-antibody complexes
which are formed. Both standard and competitive formats for these
assays are known in the art. For instance, to characterize SLE or
systemic lupus erythematosus in a standard format, the amount of
SLE-antibodies in the antibody-antigen complexes is directly
monitored. This may be accomplished by determining whether a second
type of labelled anti-xenogenetic (e.g. anti-human) antibodies
which recognize an epitope on the first type of SLE-antibodies will
bind due to complex formation. In a competitive format, the amount
of SLE-antibodies in the sample is deduced by monitoring the
competitive effect on the binding of a known amount of labelled
antibody (or other competing ligand) in the complex. The detection
of SLE-antibodies for diagnosis of SLE is used as an illustration.
Wherever the term "SLE-antibodies" is used throughout the
specification, this should not be considered as limitative. Like
wise, the other autoimmune diseases are diagnosed by detection of
other antibodies, and mononucleosis is diagnosed by detection of
anti-Epstein-Barr virus antibodies.
[0116] Complexes formed comprising SLE-antibody (or in the case of
competitive assays, the amount of competing antibody) are detected
by any of a number of known techniques, depending on the format.
For example, unlabelled SLE-antibodies in the complex may be
detected using a conjugate of anti-xenogenetic 1 g complexed-with a
labet (e.g. an enzyme-labet).
[0117] In an immunoprecipitation or agglutination assay format the
reaction between the SLE-antigens and the SLE-antibody forms a
network that precipitates from the solution or suspension and forms
a visible layer or film of precipitate. If no SLE-antibody is
present in the test specimen, no visible precipitate is formed.
[0118] Currently, there exist three specific types of particle
agglutination (PA) assays. These assays are used for the detection
of antibodies to various antigens when coated to a support. One
type of this assay is the hemagglutination assay using red blood
cells (RBCs) that are sensitized by passively adsorbing antigen (or
antibody) to the RBC. The addition of specific antigen antibodies
present in the body component, if any, causes the RBCs coated with
the purified antigen to agglutinate.
[0119] To eliminate potential non-specific reactions in the
hemagglutination assay, two artificial carriers may be used instead
of RBC in the PA. The most common of these are latex particles.
However, gelatin particles may also be used. The assays utilizing
either of these carriers are based on passive agglutination of the
particles coated with purified antigens.
[0120] The antigenic peptides of the present invention will
typically be packaged in the form of a kit for use in these
immunoassays. The kit will normally contain in separate containers
the antigenic peptide or anti-idiotype antibody, control antibody
formulations (positive and/or negative), labelled antibody when the
assay format requires the same and signal generating reagents (e.g.
enzyme substrate) if the label does not generate a signal directly.
The antigenic peptide or anti-idiotype antibody may be already
bound to a solid matrix or separate with reagents for binding it to
the matrix. Instructions (e.g. written, tape, CD-ROM, etc.) for
carrying out the assay usually will be included in the kit.
[0121] The solid phase selected can include polymeric or glass
beads, nitrocellulose, microparticles, microwells of a reaction
tray, test tubes and magnetic beads. The signal generating compound
can include an enzyme, a luminescent compound, a chromogen, a
radioactive element and a chemiluminescent compound. Examples of
enzymes include alkaline phosphatase, horseradish peroxidase and
beta-galactosidase. Examples of enhancer compounds include biotin,
anti-biotin and avidin. Examples of enhancer compounds binding
members include biotin, anti-biotin and avidin. In order to block
the effects of rheumatoid factor-like substances, the test sample
is subjected to conditions sufficient to block the effect of
rheumatoid factor-like substances. These conditions comprise
contacting the test sample with a quantity of for instance
anti-human IgG to form a mixture, and incubating the mixture for a
time and under conditions sufficient to form a reaction mixture
product substantially free of rheumatoid factor-like substance.
[0122] The present invention particularly relates to an immunoassay
format in which several peptides of the invention are coupled to a
membrane in the form of parallel lines. This assay format is
particularly advantageous for allowing a discrimination between the
separate autoimmune diseases. The antigens that are immobilized on
the membrane will preferentially be the methylated and unmethylated
form of poly(Arg-Gly), combined with native and thus methylated
SmD1 and/or SmD3 and/or Sm69, and unmethylated, recombinant SmD1
and/or SmD3 and/or Sm69.
FIGURES LEGENDS
[0123] FIG. 1: HPLC profile of the Endo-Lys digest.
[0124] FIG. 2: immunodot of HPLC fractions with 5 patients sera and
1 control serum.
[0125] FIG. 3: Immunodot of the C-terminal peptide (C-term mod) and
without (C-term nt mod) dimethylarginine, and of the recombinant
(baculo SmD, coli SmD) and natural protein (native). Strips were
incubated with a anti-SmD positive serum (+) and a control serum
(-). Total protein staining (Aurodyne) was performed on the third
strip.
[0126] FIG. 4: LIA with modified (dimethyl arginine) C terminal
peptide (fraction 15 from EndoLys-C digest, line 1 on the strip),
and non-modified C terminal peptide (fraction 8 from the EndoLys-C
digest, line 2 on the strip), both applied in equal amounts (60
ng). Additionally, 7, 15 and 30 ng of recombinant SmD1 from
baculovirus- or E. coli-infected insect cells (resp. 4, 5, 6 and 7,
8, 4) as well as 15 and 30 ng of a mixture of gel-purified SmD
(native) were applied to the strips. The total protein staining
(Aurodyne) was performed on the first strip. The strips were
incubated with (A) a panel of anti-SmD positive sera selected by
INNO-LIA ANA from ANF-positive sera, (B) a panel of anti-SmD
positive sera selected by INNO-LIA ANA from a cohort of SLE
patients diagnosed according to the ACR criteria, (C) sera selected
from MCTD patients (control panel) and (D) sera selected from
ANF-negative sera (control panel). No reactivity was observed with
sera from the control panels.
EXAMPLES
Example 1
[0127] Sera
[0128] Sm positive sera were obtained from the Department of
Rheumatology of the University clinic in Ghent, Belgium (Dr. De
Keyser and Dr. Veys). These sera were identified by microgel
diffusion blotting (MDB) using rabbit thymus extract (Zeus, Bayer,
Raritan, USA) as substrate (De Keyser et al., 1990). The
Sm-positivity was defined by a positive immunoreaction at the same
molecular weight position (approx. 14 kDa) as the .alpha.-SmD
reference serum.
Example 2
[0129] Isolation of Native SmD
[0130] The snRNP particles are purified from HeLa nuclear extracts
by immuno affinity chromatography (R. Luhrmann, Marburg,
Germany).
[0131] The snRNP particles are received in 20 mM Hepes/KOH, pH
7.9-250 a 420 mM NaCl-5% glycerol-1.5M MgC.sub.2-0.2 mM EDTA-0.5 mM
DTE-0.5 mM PMSF. SmD is isolated from these particles as described
by Lehmeier et al. (1990) with some modifications. Briefly, in a
first step, snRNPs are concentrated in a centricon concentrator
(30K centripep, Amicon) to a final volume of 5-10 mg/ml.
Subsequently, the snRNPs are dissolved in Laemmi sample buffer,
separated in a preparative 15% Laemmli gel (1 cm thick, 14 cm well;
protein load 3 mg) and stained with Coomassie Brilliant Blue.
[0132] The 14 kDa SmD (containing SmD1, SmD2 and SmD3) band is cut
from the gel, rinsed in water and cut into 1 mm.sup.3 cubes.
Proteins are eluted from the polyacrylamide gel in the BioRad
apparatus according to the manufacture's instructions. Residual SDS
and CoomassieBB are removed from the electroeluted proteins by
ion-pair extraction (precipitation of the dried protein with
aceton/acidic acid/triethylamine/water:85/5/5/5). The pellet is
dissolved in 6M ureum, 0.1 M acetic acid (glacial) and immediately
neutralized with 1.5 M Tris-HCl. Protein concentration is
determined by MicroBCA method (Pierce, USA), an average yield of 80
.mu.g SmD/mg snRNPs is obtained.
Example 3
[0133] Expression of SmD1 as Short mTNF-fusion in E. coli and
Purification of the Fusion Protein.
[0134] The SmD1 coding sequence (357 bp) was isolated from a cDNA
clone bought from Organon Technika as a 367 bp PCR fragment by
using pfu polymerase (Tm: 55.degree. C.). This PCR fragment was cut
with BamHI and XbaI and inserted into the BamHI/XbaI cut expression
vector pIGFH111. This expression vector was transformed to E. coli
expression strain SG4044(pc1857). Induction of this vector/strain
combination at 37.degree. C. showed a strong signal of .+-.18 kDa
on CBB stained gels and on Western blot. Upon localisation analysis
the protein proved to be present in the soluble fraction. No
significant proteolytic breakdown could be observed. Bacterial
cells derived from three liter culture were suspended in lysis
buffer (10 mM Tris- 100 mM KCl pH 6.8) until 3 times the amount of
wet cells. Prior to lysis by French press, .epsilon.-aminocapronic
acid, DTT, and PMSF were added to a final concentration of 25 mM, 1
mM and 2 mM respectively. The cell suspension was forced twice
through the French press and pressure was kept at 14000 psi. Before
centrifugation, the lysate was diluted with lysis buffer (5 times
the wet cell weight) and was centrifuged for 20 minutes at 27,000 g
at 4.degree. C. Guanidine HCl was added to the supernatant to an
end concentration of 4.5 M. The recombinant fusion protein,
containing a His-tag, was purified in a single step by metal
affinity chromatography (Ni-IMAC sepharose). Chromatography was
performed at room temperature. The column was loaded with 1 column
volume of NiCl.sub.2 (5 mg/ml), washed with water and equilibrated
with buffer A (6 M guanidine HCl, 0.1 M sodium phosphate, 0.05%
TritonX100, pH 6.5) . The proteins were loaded on Ni.sup.2+
chelating sepharose (Pharmacia, Sweden; approx. 18 mg protein/ml
gel) and the column was washed with 4 bed volumes of buffer A. SmD1
was eluted with a linear gradient of buffer B (6 M guanidine HCl,
0.1 M sodium phosphate, 0.05% TritonX100, pH 3.5) and the protein
eluted between 70% and 90% buffer B.
Example 4
[0135] Expression of SmD1 as Short mTNF-Fusion in the Baculoviral
System and Purification of the Fusion Protein
[0136] The cDNA gene coding for the mTNF-His6-hSmD fusion protein
was isolated from the bacterial expression plasmid plGFH111 hSmD
(see example 3) as a 520 bp DraI-XbaI fragment, and inserted in the
BamHI (filled in)-XbaI opened baculo transfer plasmid pVL1393,
resulting in the recombinant transfer plasmid pVLTNFH6hSmD (see
FIG. 2). The fusion gene is here under transcriptional control of
the baculovirus strong polyhedrin promoter. The pVmTNFH6hSmD1
baculo transfervector was used to generate recombinant
mTNF-His6-hSmD1 baculovirus following the baculogold transfection
approach (Pharmingen, San Diego, USA). Infection of Spodoptera
frugiperda cells (Sf9) with the recombinant virus resulted in the
expression of a 18 kDa protein which was recognized on Western blot
by a monoclonal antibody specific for SmD (Progen, Heidelberg,
Germany, data not shown). Using the cell lysate for testing the
specificity of different human sera was not feasible since a high
aspecific background reaction of the human sera with baculoviral
proteins masked possible specific SmD recognition. The SmD fusion
protein was therefore purified by Ni-IMAC purification as described
previously with one adaptation: following french press, the cell
lysate was precipitated and redissolved in buffer A (see example
3).
Example 5
[0137] Sequence and Mass Analysis of Natural, E. coli, and
Baculoviral SmD1
[0138] Natural SmD electroeluted from HeLa nuclear extracts
immobilized on a PVDF membrane in a ProSpin device (Perkin Elmer,
Calif., USA) was subjected to endoLys-C digestion to obtain
detailed sequencing data of internal peptides. The membrane was
incubated with 100 mM Tris pH 8.2, 1% hydrogenated TritonX-100, 1
mM K.sub.3-EDTA, 10% acetonitrile and 0.5 .mu.g enzyme. The
digestion was performed overnight at 37.degree. C. The peptide
mixture was separated on a C4 Vydac HPLC-column (using a gradient
of 10-70% solvent B: 70% acetonitrile/0.1 % TFA) and a flow rate of
0.2 ml/min. The eluted peptide peaks were manually recovered. In
the C-terminal 25-mer peptide of SmD1 nine dimethylarginine
residues were sequenced, only the last two arginines were
unmodified. The position of N.sup.G, N.sup.G-dimethylarginine in
the sequence chromatogram was confirmed by applying the pure
modified amino acid (Sigma, St Louis, USA) as standard. This
modification was absent in recombinant SmD1 from E. Coli (revealed
in the course of sequencing peptides generated by endo-GluC)
implying that the modification, resulting from the action of
methyltransferase, does not occur in E. coli.
[0139] This conclusion was confirmed by mass analysis of E. coli
recombinant SmD1. This protein, eluting in a single peak upon
reversed-phase chromatography, was analysed by electrospray on a
Bio-Q quadrupole mass spectrometer equipped with an electrospray
ion source (Fisons). Ten .mu.l of the sample solution containing 20
pmol in 50% acetonitrile-1% acetic acid was analysed. Calibration
of the scans was performed with 50 pmol horse heart myoglobin. The
sample contained 3 masses: 17,435 Da, 17,305 Da, and 16,992 Da
corresponding respectively to the full size protein, the protein
without the N-terminal methionine, and the protein lacking the
N-terminal Met and the C-terminal Arg-Arg. From these results, it
can be concluded that the purified E. coli recombinant SmD1 is the
intact, unmodified molecule and that the lack of specific
immunoreactivity of the recombinant SmD1 is not due to loss of the
C-terminus.
[0140] Mass analysis of baculoviral recombinant SmD1 showed a
heterogeneous result: one of the major mass peaks (17,297 Da) could
be assigned to the unmodified protein lacking the N-terminal
methionine while within the minor peaks masses of 17,629 and 17,711
could be tentatively assigned to the presence of 7 and 10
dimethylarginines.
Example 6
[0141] Epitope Mapping of Baculo SmD1
[0142] Baculo SmD1 fusion protein was digested with EndoGlu-C as
follows: 300 .mu.g TCA-precipitated protein was dissolved in 50
.mu.l 1100 mM NH.sub.4-acetate buffer pH 4.3.The EndoGlu-C enzyme
(Boehringer, Mannheim, Germany) was added at a ratio of 1/100 and
the mixture was incubated overnight at 26.degree. C. The digest was
subsequently vacuum dried (SpeedVac), redissolved in 0.1% TFA -20%
acetic acid and the peptides were separated on a reversed-phase
HPLC column (C.sub.4-Vydac). Peptide peaks were manually recovered.
A similar approach was followed for EndoLys-C (Boehringer,
Mannheim, Germany) digestion of baculo SmD1 with the following
modifications:the protein is dissolved in 50 pl 100 mM Tris-HCl, pH
8, 10% acetonitrile, 10 mM K.sub.3EDTA, and enzyme is added at a
ratio of 1/120.
[0143] The HPLC fractions were vacuum dried and dissolved in 10%
acetonitrile, 50 mM carbonate buffer pH 9.6. From each fraction 2
.mu.l was spotted on ABC nylon membrane (Pall, N.Y.). After
spotting, the membranes were blocked for 1 hour in 0.5% caseine in
PBS to which 0.1% 0.25 glycine is added. Subsequently, the
membranes are incubated overnight with serum (1/100) in 0.5%
caseine in PBS supplemented with Triton X705 and 2.03 g/L
MgCl.sub.2.6H.sub.2O. The membranes were washed 3 times for 3 min.
in PBS, 0.05% Tween20 and incubated with anti-human IgG (1/8000)
conjugated with alkaline phophatase. The immune reaction was
visualized by adding NBT/BCIP in a 1/500 dilution.
[0144] The endoGlu-C derived fractions were incubated with one
positive serum and one control serum. A strong immunoreaction was
revealed with fraction 17. Sequencing of fraction 15 learned that
this fraction contained the C-terminal peptide in which the RG
motif is dimethylated. Mass analysis of fraction 8 showed that this
fraction contained the C-terminal peptide without the modified
arginines. Analysis of the fractions located between 8 and 17
indicated that these fractions contain the C-terminal peptide in
which the 5 final RG motifs are dimethylated while and the first 4
RG motifs are partially monomethylated. This can be concluded from
a mass difference of 14 between the fractions.
[0145] The EndoLys-C derived fractions (FIG. 1) were incubated
separately with 6 positive sera and one control serum. In 5 out of
6 positive sera, a signal significantly higher than the control
serum was limited to fraction 15 which was identified both by
sequencing and mass analysis as the C-terminal peptide with
dimethylargines. Again, mass analysis indicated that the fractions
8 to 14 correspond to the non-methylated and less methylated forms
of the C-terminal peptide.
[0146] These results were confirmed by isolating selectively the
non-modified and the modified peptide from a preparative endoLys-C
digest of baculo SmD1. In FIG. 1 it can be seen that fraction 8
with the non-modified SmD1 peptide contained less material than
fraction 15 with the dimethylated peptide. It is therefore possible
that the exclusive reactivity of the modified peptide was due to
different amounts of modified and non-modified peptide being
transferred in the dot-blot experiment (FIG. 2). To exclude such
quantitative variations, the peptides were analysed in a dot spot
experiment as described. However, in this experiment equal
quantities (based on BCA protein determination) of both peptides,
modified and non-modified peptide, were applied. For comparison,
the total natural SmD, the total recombinant E. coli and
baculoviral SmD1 were applied in comparable amounts in the
immunodot (FIG. 3). Finally peptides were applied in a line
immunoassay experiment (Polet et al., Clinical Chemistry, 37, 1991)
(FIG. 4). Again equal amounts (60 ng) of modified and non-modified
SmD1 peptides were applied to a nylon membrane. The amount of
peptide bound was visualized by protein colloidal staining
(Aurodye, Amersham, Buckinghamshire, UK; FIG. 4). Additionally, 30,
15 and 7 ng of recombinant SmD1 from E. coli- or
baculovirus-infected insect cells as well as a mixture of
gel-purified SmD1, SmD2 and SmD3 were applied to the strips. These
were then tested with 21 anti-Sm patient sera that were
immunoreactive to a mixture of HeLa SmD1, SmD2 and SmD3. Six (29%)
of these anti-Sm patient sera gave significant signals with the
modified peptide D1, while the non-modified peptide reacted with
none of the 21 tested sera. An independent set of anti-Sm Brazilian
sera (n=93) showed a comparable rate of reactivity with the
modified peptide (4/14 anti-Sm sera; 29%). These experiments
substantiate our hypothesis that there are at least 2 epitopes
involved in the immunoreactivity of natural SmD: one epitope is
present is the total E. coli recombinant SmD molecule while an
additional epitope is located in the C-terminus (90-119) of the
SmD1 molecule. The presence of dimethylarginines in this peptide is
crucial for recognition by patient sera.
References
[0147] Andersen, J., Feeney, R. J. and Zieve, G. W. 1990.
Identification and characterization of the small nuclear
ribonucleoprotein particle D' core protein. Mol. Cell .Biol. 10,
4480-4485
[0148] Barakat, S., Briand, J. -P., Weber, J .-C., Van Regenmortel,
M. H. V. and Muller, S. 1990. Recognition of synthetic peptides of
Sm-D autoantigen by lupus sera. Clin. exp. immunol. 81, 256-262
[0149] De Keyser, F. G., Verbruggen, G., Veys, E. M., Nimmegeers,
J., Schatteman,L., Goethals, K., Vandenbossche, M. 1990, "Microgel
Diffusionblotting" for sensitive detection of antibodies to
extractable nuclear antigens. Clin. Chem. 36, 337-339
[0150] Gheuens, J., MacFarlin, D. 1982. Use of monoclonal
anti-idiotypic antibody to P3-X63Ag8 myeloma protein for analysis
and purification of B lymphocyte hybridoma products. Eur. J.
Immunol. 12, 701-703
[0151] Hoch, S. O. 1989. Application of protein blotting in the
study of autoimmunedisease. In Manual of Biological Markers of
Disease B2.41 -29 Kluwer, Netherlands
[0152] Weiner, H. L. 1997. Oral tolerance for the treatment of
autoimmune diseases. Annu. Rev. Med. 48, 341-351
[0153] James, J. A., Mamula, M. J. and Harley, J. B. 1994.
Sequential autoantigenic determinants of the small nuclear
ribonucleoprotein Sm D shared by human lupus autoantibodies and MRL
Ipr/Ipr antibodies. Clin. Exp. Immunol. 98, 419-426
[0154] Lehmeier, T., Foulaki, K. And Luhrman, R. 1990. Evidence for
three distinct D proteins, which react differentially with anti-Sm
autoantibodies, in the cores of the major snRNPs U1, U2, U4/U6 and
U5. Nucleic Acids Res 18, 6475-6484
[0155] Najbauer, J., Johnson, B. A., Young, A. L. and Aswad, D. W.
1993. Peptides with sequences similar to glycine arginine rich
motifs in proteins interacting with RNA are efficiently recognized
by methyltransferases modifying arginine in numerous proteins. J.
Biol. Chem. 268, 10501-10509
[0156] Rajpurohit, R., Lee, S. O., Park, J. O., Paik, W. K. and
Kim, S. 1994. Enzymatic methylation of recombinant heterogeneous
nuclear RNP protein A1. J. Biol. Chem. 269, 1075-1082
[0157] Rawal, N., Rajpurohit, R., Lischwe, M. A., Williams, K. R.;,
Paik, W. K., Kim, S. 1995. Structural specificity of substrate for
S-Adenosylmethionine:protein arginine N-methyltransferases.
Biochem. Biophys. Acta, 1248, 11-18
[0158] Rivkin, E., Vella, M. J. and Lahita, R. G. 1994. A
heterogeneous immune response to an Sm-D-like epitope by SLE
patients. J. Autoimmun. 7, 119-132
[0159] Rokeach, L. A., Haselby, J. A and Hoch, S. O. 1988.
Molecular cloning of a cDNA encoding the human Sm-D autoantigen.
Proc. Natl. Acad. Sci. USA, 85, 4832-4836
[0160] Rokeach, L. A., Haselby, J. A. and Hoch, S. O. 1992a.
Overproduction of a human OsnRNP)-associated Sm-D autoantigen in
Escherichia coli and Sacoharomyces cerevisiae. Gene 118,
247-253
[0161] Rokeach, L. A., Jannatipour, M., Haselby, J. A. and Hoch, S.
O. 1992b. Mapping of the immunoreactive domains of a small nuclear
ribonucleiprotein-associated Sm-D autoantigen. Clin. Immunol.
Immunopath. 35, 315-324
[0162] Sabbatini, A., Dolcher, M. P., Marchini, B., Bombardieri, S.
And Migliorini, P. 1993a. Mapping of epitopes on the SmD molecule:
the use of multiple antigen poepotides to measure autoantibodies in
systemlic lupus erythematosus. J. Rheumatol. 20,1679-1683
[0163] Sabbatini, A., Bombardieri, S. And Migliorini, P. 1993b.
Autoantibodies from patients with systemic lupus erythematosus bind
a shared sequence of SmD and Epstein-Barr virus-encoded nuclear
antigen EBNA I. Eur. J. Immunol. 23, 1146-1152
[0164] Van Venrooij, W. J., P. Charles and R. N. Maini 1991. The
conscensus workshops for the detection of autoantibodies to
intrzcellular antigens in rheumatic diseases. J. Immunol. Methods,
140, 181-189
[0165] Wagatsuma, M., Asami, N., Miyachi, J., Uchida, S., Watanabe,
H. and Amann, E. 1993. Antibody recognition of the recombinant
human nuclear antigens RNP 70 kD, Sm-A, Sm-B and Sm-D by autoimmune
sera. Mol. Immunol. 30, 1491-1498
Sequence CWU 1
1
30 1 19 PRT Artificial Sequence Synthetic Peptide 1 Gly Xaa Gly Xaa
Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa 1 5 10 15 Gly Xaa
Gly 2 11 PRT Artificial Sequence Synthetic Peptide 2 Ala Xaa Gly
Xaa Gly Xaa Gly Met Gly Xaa Gly 1 5 10 3 17 PRT Artificial Sequence
Synthetic Peptide 3 Lys Ala Gln Val Ala Ala Xaa Gly Xaa Gly Xaa Gly
Met Gly Xaa Gly 1 5 10 15 Asn 4 38 PRT Artificial Sequence
Synthetic Peptide 4 Asp Val Glu Pro Lys Val Lys Ser Lys Lys Arg Glu
Ala Val Ala Gly 1 5 10 15 Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly
Xaa Gly Xaa Gly Xaa Gly 20 25 30 Xaa Gly Gly Pro Arg Arg 35 5 16
PRT Artificial Sequence Synthetic Peptide 5 Asp Asn His Gly Xaa Gly
Xaa Gly Xaa Gly Xaa Gly Xaa Gly Gly Gly 1 5 10 15 6 15 PRT
Artificial Sequence Synthetic Peptide 6 Gly Gly Xaa Gly Xaa Gly Gly
Ser Gly Gly Xaa Gly Xaa Gly Gly 1 5 10 15 7 12 PRT Artificial
Sequence Synthetic Peptide 7 Glu Arg Ala Xaa Gly Xaa Gly Xaa Gly
Xaa Gly Glu 1 5 10 8 46 PRT Artificial Sequence Synthetic Peptide 8
Gly Gly Gln Gln Asp Xaa Gly Gly Xaa Gly Xaa Gly Gly Gly Gly Gly 1 5
10 15 Tyr Asn Xaa Ser Ser Gly Gly Tyr Glu Pro Xaa Gly Xaa Gly Gly
Gly 20 25 30 Xaa Gly Gly Xaa Gly Gly Met Gly Gly Ser Asp Xaa Gly
Gly 35 40 45 9 18 PRT Artificial Sequence Synthetic Peptide 9 Gly
Gly Gln Gln Asp Xaa Gly Gly Xaa Gly Xaa Gly Gly Gly Gly Gly 1 5 10
15 Tyr Asn 10 26 PRT Artificial Sequence Synthetic Peptide 10 Ser
Gly Gly Tyr Glu Pro Xaa Gly Xaa Gly Gly Gly Xaa Gly Gly Xaa 1 5 10
15 Gly Gly Met Gly Gly Ser Asp Xaa Gly Gly 20 25 11 17 PRT
Artificial Sequence Synthetic Peptide 11 Asp Phe Asn Xaa Gly Gly
Gly Asn Gly Xaa Gly Gly Xaa Gly Xaa Gly 1 5 10 15 Gly 12 29 PRT
Artificial Sequence Synthetic Peptide 12 Asp Phe Asn Xaa Gly Gly
Gly Asn Gly Xaa Gly Gly Xaa Gly Xaa Gly 1 5 10 15 Gly Pro Met Gly
Xaa Gly Gly Tyr Gly Gly Gly Gly Ser 20 25 13 38 PRT Artificial
Sequence Synthetic Peptide 13 Gly Asp Asp Xaa Xaa Gly Xaa Gly Gly
Tyr Asp Xaa Gly Gly Tyr Xaa 1 5 10 15 Gly Xaa Gly Gly Asp Xaa Gly
Gly Phe Xaa Gly Gly Xaa Gly Gly Gly 20 25 30 Asp Xaa Gly Gly Phe
Gly 35 14 14 PRT Artificial Sequence Synthetic Peptide 14 Gly Asp
Asp Xaa Xaa Gly Xaa Gly Gly Tyr Asp Xaa Gly Gly 1 5 10 15 26 PRT
Artificial Sequence Synthetic Peptide 15 Gly Gly Tyr Xaa Gly Xaa
Gly Gly Asp Xaa Gly Gly Phe Xaa Gly Gly 1 5 10 15 Xaa Gly Gly Gly
Asp Xaa Gly Gly Phe Gly 20 25 16 19 PRT Artificial Sequence
Synthetic Peptide 16 Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly
Arg Gly Arg Gly Arg 1 5 10 15 Gly Arg Gly 17 38 PRT Artificial
Sequence Synthetic Peptide 17 Asp Val Glu Pro Lys Val Lys Ser Lys
Lys Arg Glu Ala Val Ala Gly 1 5 10 15 Arg Gly Arg Gly Arg Gly Arg
Gly Arg Gly Arg Gly Arg Gly Arg Gly 20 25 30 Arg Gly Gly Pro Arg
Arg 35 18 11 PRT Artificial Sequence Synthetic Peptide 18 Ala Arg
Gly Arg Gly Arg Gly Met Gly Arg Gly 1 5 10 19 23 PRT Artificial
Sequence Synthetic Peptide 19 Lys Ala Gln Val Ala Ala Arg Gly Arg
Gly Arg Gly Met Gly Arg Gly 1 5 10 15 Asn Ile Phe Gln Lys Arg Arg
20 20 46 PRT Artificial Sequence Synthetic Peptide 20 Gly Gly Gln
Gln Asp Arg Gly Gly Arg Gly Arg Gly Gly Gly Gly Gly 1 5 10 15 Tyr
Asn Arg Ser Ser Gly Gly Tyr Glu Pro Arg Gly Arg Gly Gly Gly 20 25
30 Arg Gly Gly Arg Gly Gly Met Gly Gly Ser Asp Arg Gly Gly 35 40 45
21 18 PRT Artificial Sequence Synthetic Peptide 21 Gly Gly Gln Gln
Asp Arg Gly Gly Arg Gly Arg Gly Gly Gly Gly Gly 1 5 10 15 Tyr Asn
22 26 PRT Artificial Sequence Synthetic Peptide 22 Ser Gly Gly Tyr
Glu Pro Arg Gly Arg Gly Gly Gly Arg Gly Gly Arg 1 5 10 15 Gly Gly
Met Gly Gly Ser Asp Arg Gly Gly 20 25 23 17 PRT Artificial Sequence
Synthetic Peptide 23 Asp Phe Asn Arg Gly Gly Gly Asn Gly Arg Gly
Gly Arg Gly Arg Gly 1 5 10 15 Gly 24 29 PRT Artificial Sequence
Synthetic Peptide 24 Asp Phe Asn Arg Gly Gly Gly Asn Gly Arg Gly
Gly Arg Gly Arg Gly 1 5 10 15 Gly Pro Met Gly Arg Gly Gly Tyr Gly
Gly Gly Gly Ser 20 25 25 38 PRT Artificial Sequence Synthetic
Peptide 25 Gly Asp Asp Arg Arg Gly Arg Gly Gly Tyr Asp Arg Gly Gly
Tyr Arg 1 5 10 15 Gly Arg Gly Gly Asp Arg Gly Gly Phe Arg Gly Gly
Arg Gly Gly Gly 20 25 30 Asp Arg Gly Gly Phe Gly 35 26 14 PRT
Artificial Sequence Synthetic Peptide 26 Gly Asp Asp Arg Arg Gly
Arg Gly Gly Tyr Asp Arg Gly Gly 1 5 10 27 26 PRT Artificial
Sequence Synthetic Peptide 27 Gly Gly Tyr Arg Gly Arg Gly Gly Asp
Arg Gly Gly Phe Arg Gly Gly 1 5 10 15 Arg Gly Gly Gly Asp Arg Gly
Gly Phe Gly 20 25 28 16 PRT Artificial Sequence Synthetic Peptide
28 Asp Asn His Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Gly Gly
1 5 10 15 29 15 PRT Artificial Sequence Synthetic Peptide 29 Gly
Gly Arg Gly Arg Gly Gly Ser Gly Gly Arg Gly Arg Gly Gly 1 5 10 15
30 12 PRT Artificial Sequence Synthetic Peptide 30 Glu Arg Ala Arg
Gly Arg Gly Arg Gly Arg Gly Glu 1 5 10
* * * * *