Note: Descriptions are shown in the official language in which they were submitted.
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Synthetic peptides containing citrulline recognized by rheumatoid
arthritis sera as tools for diagnosis and treatment.
The present invention relates to certain peptides containing citruliine and
that
constitute immunogenic determinants of antibodies present in sera from
patients
with rheumatoid arthritis and wherein the presence of at least one citrulline
is a
prerequisite for reacting with said antibodies. The invention also relates to
the a
method of producing said peptides and the use of said peptides for diagnosis
and
treatment of rheumatoid arthritis and related diseases. The present invention
also
relates to new filaggrin alleles.
Rheumatoid arthritis (RA) is a major crippling joint disease which is systemic
in nature and of unknown aetiology. It affects 1 % of the population, with a
male
to female ratio of 2:3. In terms of morbidity, the most important feature of
RA is
joint erosion which leads to pain, deformity and in some cases, severe
disability.
Life expectations in patients with a severe form of the disease are reduced by
up
to 10 years. RA has all the features of an autoimmune disease, including the
presence of a variety of autoantibodies in patients' sera and the capacity to
induce
illness by transfer of pathogenic T cells in animal models. The classification
of the
disease can be challenged on the grounds that borderline forms are very
common;
furthermore inflammation of the joints is not only restricted to RA, but
occurs also
in other non-autoimmune diseases such as osteoarthritis, reactive arthritis
and gout.
As an early diagnosis allows an adjusted treatment which can highly improve
life quality of RA patients, it is of great importance for rheumatologists to
have
reliable diagnostic criteria to their disposal. The diagnosis of RA is
initially based on
clinical manifestations. Serological support for such a diagnosis is not very
well
established and is based mainly on the presence of rheumatoid factors (RF). A
positive Waaler-Rose or latex fixation RF test has a predictive value and is
related
to disease with a more severe outcome. However, a substantial number of RA
patients are RF seronegative, while on the other hand, RF is also present in
other
rheumatic diseases including Sjogren's syndrome and systemic lupus
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erythematosus, in some chronic bacterial and acute viral infections, in
certain
parasitic diseases and chronic inflammatory diseases, and has furthermore been
demonstrated in control sera from healthy persons (Walter et al., 1964; Chen
et al.,
1987). This rather low specificity of RF necessitates additional testing for a
second
RA-specific antibody. The prevalence of previously defined antinuclear
antibodies
such as SSA, RA33 and RNP in RA is low and their clinical utility for the
diagnosis
of RA has so far been limited (Hassfeld et al., 1993). In contrast, both the
antiperinuclear factor (APF) and antikeratin antibodies (AKA) proved to be
helpful
in this respect. APF are IgG antibodies in sera from RA patients, provoking a
typical
immunofluorescence staining pattern on human buccal mucosa cells (Nienhuis and
Mandema, 1964). Several studies dealt with the diagnostic usefulness of this
test
(Westgeest et al., 1987; Janssens et al., 1988; Vivino and Maul, 1989; Youinou
et al., 1992; Feltkamp et al., 1993). The high specificity of APF for RA of 80-
90%
and a good sensitivity of 50-99% make it indeed a suitable diagnostic tool.
lts value
is especially obvious in about one third of the RA patients who do not possess
RF
activity (Westgeest et al., 1987; Nesher et al., 1992). A second specificity
found
in sera of RA patients are the AKA, autoantibodies that were first described
by
Young et al. ( 19791 and that give rise to a fluorescent staining pattern with
the
keratinized layer of rat oesophageal epithelium. Despite the lack of
biochemical
characterization of their target, the antibodies were infered to be
antikeratin
antibodies on the disputable ground that cytokeratins constitute the major
component of the stratum corneum. These antibodies are present in 36-59% of
the
RA sera and have been found to be highly specific for RA (Johnson et al.,
1981;
Miossec et al., 1982; Hajiroussou et al., 1985; Vincent et al., 1989). Like
APF,
they are predominantly of the IgG class (Johnson et al., 1981; Kataaha et al.,
1985; Vincent et al., 1990) and they both can be detected in synovial fluid of
patients with RA (Youinou et al., 1985; Kirstein et al., 1989; Vivino and
Maul,
1990).
The human epidermal protein fiiaggrin was recently identified with convincing
evidence as one of the major targets for reactivity with AKA and APF
autoantibodies (Simon et al., 1993; Sebbag et al., 1995). In the initial
study, the
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neutral/acidic form of human filaggrin was demonstrated to react with 75% of a
group of 48 RA sera in Western blot (Simon et al., 1993). More recent data
(Vincent et al., 1997) showed a diagnostic sensitivity of more than 50% for
antifilaggrin detection with a corresponding specificity of 95% within a group
of
492 sera which included 279 RA sera.
Profilaggrin, the precursor of filaggrin is a histidine-rich, insoluble
protein of
t 400-kDa. It consists of 10-12 filaggrin repeats of 324 amino acids that are
separated by a heptamer linker sequence. The highly phosphorylated polyprotein
is
stored in the keratohyalin granules of the granular layer of the epidermis.
Upon
terminal differentiation of these cells, profilaggrin is dephosphorylated and
proteolytically processed by excision of the Pinker into functional basic
filaggrin
molecules of 37-kDa (Resing et al., 1989; 1993; Gan et al., 1990). In the
lower
cornified cells, these units are involved in the aggregation of keratin
intermediate
filaments, facilitating formation of intermolecular disulfide bonds and
yielding the
intracellular fibrous matrix of. the cornified cells (Dale et al., 1978;
Lynley and Dale,
1983; Handing and Scott, 1983; Mack et al., 1993). After filament aggregation,
basic arginine residues are converted to citrulline by a peptidylarginine
deiminase,
which results in a lower affinity of the molecule for cytokeratins (Handing
and Scott,
1983). Finally, filaggrin is completely proteolysed into free amino acids,
urocanic
acid and carboxylic pyrrolidone acid, which play a role in maintaining an
optimal
level of moisture and absorbing UV light (Scott et al., 1982).
The tandernly arranged filaggrin units are highly polymorphic: there exists a
considerable variation in amino acid sequence across individuals and also
between
the different filaggrin molecules of one person, where up to 15% of
differences
have been noted (McKinley-Grant et al., 1989; Gan et al., 1990; Markova et
al.;
1993). Most variations are attributable to single-base changes, but many also
involve changes in charge. The human profilaggrin gene system is even
polymorphic
in size due to allelic differences between individuals in the number of
repeats (10,
11 or 12), so that in one person up to 24 different filaggrin molecules can
occur
(McKinley-Grant et al., 1989; Presland et al., 1992). The amino acid
variations,
together with the dephosphorylation events are responsable for the marked
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heterogeneity observed on two-dimensional gels; the conversion of arginine to
citrulline is an additional cause of acidification of the molecules.
As the use of natural filaggrin isolated from human tissues is rather
unpractical because of batch variability, the laborious preparative isolation
and the
restriction on the availability of starting material for purification,
synthetic peptides
and recombinant protein were tested in order to develop a diagnostic test kit
for
RA. Recombinant filaggrin was expressed in the eukaryotic COS cell system.
However, this approach seemed not succesful as none of the four cloned
proteins
reacted with APF positive patient sera.
It is an airn of the present invention to provide peptides which have a high
reactivity for antibodies present in sera from patients with rheumatoid
arthritis.
Another aim of the present invention is to provide methods for obtaining said
peptides.
Another aim of the present invention is to provide methods of raising
antibodies specifically reactive with said peptides.
Another aim of the present invention is to provide methods of raising anti-
idiotype antibodies specifically reactive with the afore mentioned antibodies,
thereby mimicking said peptides.
Another aim of the present invention is to provide a pharmaceutical
composition comprising these peptides, for therapy or diagnosis.
Another aim of the present invention is to provide a diagnostic kit for
rheumatoid arthritis.
Another aim of the present invention is to provide new filaggrin alleles and
their corresponding amino acid sequences.
Another aim of the present invention is to provide a bioassay for identifying
compounds which modulate the interaction between an autoantigen and a
rheumatoid arthritis specific autoantibody.
Another aim of the present invention is to provide a modulator, a composition
containing a modulator, or a combination of modulators identified by the
bioassay
as described above.
All these aims of the present invention are met by the following embodiments
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of the present invention.
The immunodominant epitopes of filaggrin were identified, which all
contained the unusual amino acid citrulline. Synthetic peptides were generated
and
proved useful for diagnosis of RA. By probing human rheumatoid arthritis sera
onto
5 2-D blots containing placental extracts another specificity was found. The
immunoreactive spots were identified and peptides from human vimentin,
cytokeratin 1 and cytokeratin 9 were retrieved in addition to other non-
identified
peptides. These proteins all belong to the same family of intermediate
filament
proteins. As citrullinated forms of vimentin and cytokeratins are already
described
to occur in vivo (Senshu et al., 1992; Senshu et al., 1995; Senshu et al.,
1996),
it is likely that they constitute specific targets for autoantibodies present
in
rheumatoid arthritis sera.
According to its main embodiment the present invention relates to peptides
containing less than 50 amino acids, comprising fragments of a filaggrin
variant or
fragments of intermediate filament proteins, wherein at least one arginine is
substituted by citrulline, and that are able to react with antibodies, wherein
the
presence of said citrulline is crucial for reaction between said peptide and
said
antibodies, and wherein said antibodies are present in sera from patients with
rheumatoid arthritis.
According to a preferred embodiment the present invention relates to
peptides as presented in claim 2.
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
andlor 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.
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 at least one arginine residue is substituted by
citruliine,
at certain steps during the chemical synthesis. The present invention also
relates
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to a method for producing any of the above mentioned peptides, wherein the
primary amino acid sequence is produced by classical chemical synthesis, and
wherein said arginine residue is derivatized towards citrulline after chemical
synthesis by incubation with peptidylarginine deiminase. The present invention
also
relates to a method for producing any of the above mentioned peptides
comprising
the following steps: (i) transforming an appropriate cellular host with a
recombinant
vector in which a polynucleic acid is inserted comprising the sequence that
codes
for said peptide under the control of the appropriate regulatory elements such
that
said peptide or a protein comprising said peptide is expressed and/or
secreted, (ii)
culturing said transformed cellular host under conditions allowing expression
of said
protein or peptide and allowing a partial or optimal derivatization of said
arginines
present in said peptide, towards citrulline residues, and (iii) harvesting
said peptide.
The present invention also relates to a method for producing any of the above
mentioned peptides comprising the following steps: (i) transforming an
appropriate
cellular host with a recombinant vector in which a polynucleic acid is
inserted
comprising the sequence that codes for said peptide under the control of the
appropriate regulatory elements, such that said peptide or a protein
comprising said
peptide is expressed and/or secreted, (ii) culturing said transformed cellular
host
under conditions allowing expression of said protein or said peptide, (iii)
harvesting
said protein or said peptide, and (iv) derivatizing arginine residues of said
protein or
said peptide towards citrulline residues. 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.
According to a preferred embodiment the present invention also relates to
an antibody being specifically reactive with said peptides or intermediate
filament
proteins that contain citrulline residues, and with said antibody being
preferably a
monoclonal antibody. The present invention also relates to an anti-idiotype
antibody
raised upon immunization with any antibody as defined above, with said anti-
idiotype antibody being specifically reactive with said antibody, thereby
mimicking
any of the above mentioned peptides, and with said antibody being preferably a
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monoclonal antibody.
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.
According to a further embodiment the present invention relates to any of the
above mentioned peptides or antibodies or immunotoxine molecules or
intermediate
filament proteins or a composition thereof for use as a medicament. Said use
can
have the purpose of a medicament for treatment or of a diagnosticum for
rheumatoid arthritis. The present invention also relates to a treatment for
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 intermediate filament proteins or a
composition thereof, thereby preventing the pathogenic production of anti-self
antibodies. The present invention also relates to a diagnostic kit for use in
detecting
rheumatoid arthritis, wherein said kit comprises at least one of the above
mentioned
peptides yr proteins or antibodies, and with said peptide, proteins or
antibody being
possibly bound to a solid support. More preferably said kit is comprising a
range of
said peptides or said antibodies, possibly in combination with other epitopes
that
can characterize auto-immune disease, wherein said peptides, proteins and/or
antibodies are attached to specific locations on a solid substrate. More
preferably
said solid support is a membrane strip and said polypeptides are coupled to
the
membrane in the form of parallel lines. It has to be understood that certain
peptides,proteins 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 rheumatoid arthritis, thereby decreasing or
eliminating possible cross-reaction andlor aspecific binding.
By means of epitope mapping, the immunodominant epitopes of filaggrin
as occuring in patients with rheumatoid arthritis were identified (see example
1 ).
These epitopes are further characterized by the presence of citrulline
residues which
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result from derivatization of arginine residues. The presence of said
citrulline
residues is a prerequisite for recognition by antibodies that are present in
sera from
rheumatoid arthritis. According to its main embodiment, the present invention
relates to those peptide fragments of natural filaggrin variants that react
with
antibodies characteristically present in sera of patients with rheumatoid
arthritis and
that are further characterized by a post-translational modification, more
preferably
a derivatization of arginine towards citrulline.The presence of at least one
citrulline
residue is a prerequisite for recognition by antibodies that are specifically
present
in sera of patients with rheumatoid arthritis.
Synthetic peptides were generated wherein arginine residues were
substituted by citrulline, thus mimicking the epitopes of natural filaggrin
variants.
These peptides proved useful for diagnosis of rheumatoid arthritis. According
to
another embodiment the present invention relates to peptides which
immunologicaliy mimic the immunogenic determinants of self proteins recognized
by the immune system in patients suffering from rheumatoid arthritis.
It is therefore anticipated that the presence of one citrulline can be
sufficient for
specific recognition by some antibodies present in sera of patients with
rheumatoid
arthritis.
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 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.
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 the entire immunoreactive protein that still
comprises the highly reactive domain characterized by the presence of
citrulline
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residues. These peptides have a Length of preferably 40, 30, 25, 20 or less
amino
acids.
With 'irnmunogenic 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.
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.
The term 'crossreaction' also refers to the reaction of one antigen with
antibodies developed against another antigen or against antibodies that are
found
in sera from patients with different diseases.
According to a more specific embodiment the present invention relates to
those peptides or proteins that contain citrulline residues, wherein the
presence of
said citrulline residues is crucial for high-affinity interaction with
antibodies that are
characteristically present in sera of patients with rheumatoid arthritis.
In a more specific embodiment, the present invention relates to a peptide that
is characterized by the amino acid sequence
HSASQDGQDTIRGHPGSS or,
HSGIGHGQASSAVRDSGHRGYS or,
DSGHRGYSGSQASDNEGH or,
HSTSQEGC1DTIHGHRGS or,
GGQGSRHQaAR or,
QGSRHQQARDSSRHSTSQEGQDTIHGHRGS or,
QGSRHQ(lARDSSRHSASQDGQDTIRGHPGSS or,
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HSGIGHGQASSAVRDSGHRGYSGSO.ASDNEGH or,
wherein at least one and preferably each arginine is derivatized towards
citrulline
residues, thereby mimicking the main immunogenic determinant of filaggrin.
In a more specific embodiment the present invention relates to peptides
5 comprising a sequence of less than 50 amino acids of any variant of
vimentin,
cytokeratin 1 or cytokeratin 9, comprising at least one citrulline residue,
and
wherein the presence of said citruliine is crucial for reacting with
antibodies that are
present in sera from patients with rheumatoid arthritis.
The present invention also relates to molecular structures in which at least
10 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 andlor proteins and/or molecular
structures, enabling tagging and/or binding of the fused polypeptide and/or
protein
to specific tissue- ar 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
-are cytotoxic to T-cells andlor B-cells such as cholera toxin, or
-allow for labelling by means of a radioactive or fluorescent or immunogold or
enzymatic marker.
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
antigens are used in an assay in order to detect the antibodies present,
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
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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
S formed antigen-immune complexes, and thereby increases the clearance and
thus
decreases the deposition of said complexes.
The present invention also relates to circularized forms of said peptides, the
advantage being well known in the art, and relating to an increased affinity
of a
conformationally constraint peptide as compared with the more randomly coiled
farms of linear peptides.
In order to accommodate for eventual negative characteristics of the claimed
peptides, such as rapid degradation, solubility, cytotoxic effects and so on,
the
skilled person will be able to design conservative as well as non-conservative
amino
acid substitutions, or substitutions with non-natural amino acids, etc...
These will
generally account for less than 35 percent of a specific sequence. Such
peptides
also include peptides with substituted linkages, as well as other
modifications
known in the art, both naturally occurring and non-naturally occurring. It may
be
desirable in cases where the filaggrin peptides of the present invention are
highly
polymorphic, to vary one or more of the amino acids so as to better mimic the
different epitopes, or as recognized by antibodies in sera from patients with
rheumatoid arthritis.
According to another embodiment the present invention also relates to the
new allelic variants that were isolated , cloned and sequenced, characterized
by the
DNA sequence as presented in figure 6 and the amino acid sequence as presented
in figure 2. The present invention also relates to any analogs of the peptides
of the
present invention.
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
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substitutions include the substitution of hydrophobic residue such as
isoleucine,
valine, leucine or methionine for another, the substitution of one hydrophilic
residue
for another such as between arginine and lysine, between glutamine and
asparagines, between glycine and serine, the substitution of one basic residue
such
as lysine, arginine or histidine for another, or the substitution of one
acidic residue,
such as aspartic acid or glutamic acid for another. Examples of allowable
mutations
according to the present invention can be found in Table 1.
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.
"Chemical derivative" refers to a protein or peptide having one or more
residues chemically derivatized by reaction of a functional side group or
peptides
with substituted linkages, as well as other modifications known in the art,
both
naturally occurring and non-naturally occurring. Examples of such derivatized
molecules, include but are not limited to, those molecules in which free amino
groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl
groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloracetyl groups or
formyl groups. Free carboxyl groups may be derivatized to form salts, methyl
and
ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may
be
derivatized tv form O-acyl or 0-alkyl derivatives. The imidazote 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.
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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) . VaI, Met, Ile, Tyr, Phe, Leu, Val
Gly (G) Gly, Ala, Thr, Pro, Ser
IIe (1) Ile, Met, Leu, Phe, Val, lle, 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
T 1 Overview of the amino acid substitutions which could form the
basis of analogs (muteins) as defined above.
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
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14
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, giutamic 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.
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 forms of the claimed peptides can be
obtained by substituting the citruiline residues for the original arginine
derivatives
during the classical chemical synthesis, or by contacting the peptides after
synthesis with a peptidylarginine deiminase of any eukaryotic origin.
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'
CA 02309534 2000-OS-15
WO 99128344 ' PCT/EP98107714
may comprise a plasmid, a cosmid, a phage or a virus or a transgenic organism.
Particularly useful may be BCG or adenoviral vectors, as well as avipox
recombinant
viruses.
The recombinant peptides can be derivatized in vitro, by contacting the
5 expressed and/or secreted peptides with a of any eukaryotic origin, or in
vivo by
choosing the appropriate host, like yeast, or any eukaryotic cell, and more
preferably by using the baculovirus transformation system, or by coexpressing
said
peptides with recombinant peptidylarginine deiminase.
Also any of the known purification methods for recombinant peptides can be
10 used for the production of the recombinant peptides of the present
invention.
The present invention also relates to a recombinant expression vector
comprising a polynucleic acid or a part thereof as defined above, operably
linked to
prokaryotic, eukaryotic or viral transcription and translation control
elements.
In general, said recombinant vector will comprise a vector sequence, an
15 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.
Also any of the known purification methods for recombinant proteins may
be used for the production of the recombinant polypeptides of the present
invention.
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.
The present invention also relates to a method for the production of a
recombinant polypeptide as defined above, comprising:
- transformation of an appropriate cellular host with a recombinant vector, in
which a polynucleic acid or a part thereof according to as defined above has
been inserted under the control of appropriate regulatory elements,
- culturing said transformed cellular host under conditions enabling the
CA 02309534 2000-OS-15
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16
expression and/or secretion of said insert, and,
harvesting said polypeptide.
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.
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, .ISluveromyces, P_ichia (e.g. is ' Qr~), Hansenula (e.g.
Hansenula polymor h~a), Yarowia, Sc~waniomyces, Schizosaccharomyces,
Zygosaccharomyces and the like. Saccharomyces cereyisiae, S. carlsbergensis
and
_K. ct' are the most commonly used yeast hosts, and are convenient fungal
hosts.
The term "prokaryotes" refers to hosts such as E.coli, Lactobacillus,
Lactococcus, Salmonella, Stre~~tococcus, Bacillus subtilis or Stre~ tots
omyces. Also
these hosts are contemplated within the present invention.
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.
~podoptera
r i a). 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.
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.
CA 02309534 2000-OS-15
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17
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 cefis 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.
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.
The term "vector" is a replicon further comprising sequences providing
replication and/or expression of a desired open reading frame.
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.
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.
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
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18
conditions compatible with the control sequences.
The polynucleic acids encoding the peptides of the present invention and
inserted into the vector sequence may be attached to a signal sequence. Said
signal
sequence may be that from any source, e.g. the IgG or tissue plasminogen
activator
(tpa) leader sequence for expression in mammalian cells, or the a-mating
factor
sequence for expression into yeast cells.
A variety of vectors may be used to obtain the peptides of the present
invention. Lower eukaryotes such as yeasts and glycosylation mutant strains
are
typically transformed with piasmids, or are transformed with a recombinant
virus.
The vectors may replicate within the host independently, or may integrate into
the
host cell genome.
Higher eukaryotes may be transformed with vectors, or may be infected with
a recombinant virus, for example a recombinant vaccinia virus. Techniques and
vectors for the insertion of foreign DNA into vaccinia virus are well known in
the
art, and utilize, for example homologous recombination. A wide variety of
viral
promoter sequences, possibly terminator sequences and poly(A)-addition
sequences, possibly enhancer sequences and possibly amplification sequences,
all
required for the mammalian expression, are available in the art. Vaccinia is
particularly preferred since vaccinia halts the expression of host cell
proteins.
Vaccinia is also very much preferred since it allows the expression of f.i.
peptides
of the present invention in cells or individuals which are immunized with the
live
recombinant vaccinia virus. For vaccination of humans the avipox and Ankara
Modified Virus (AMV) are particularly useful vectors.
Also known are insect expression transfer vectors derived from baculovirus
Autogr~.pha 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
posttranslational modification recognized by insect cells are known in the
art.
CA 02309534 2000-OS-15
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19
The present invention also relates to a host cell transformed with a
recombinant vector as defined above.
The present invention also relates to antibodies that are specifically raised
against the peptides of the present invention, preferably against those
peptides
wherein the arginines are derivatized towards citrulline. These antibodies may
be
poiyclonal 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 is derivatized towards citrulline.
Pharmaceutically acceptable carriers include any carrier that does not itself
induce
the production of antibodies harmful to the individual receiving the
composition.
Suitable carriers are typically large, slowly metabolized macromolecules such
as
proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric
amino
acids, amino acid copolymers; and inactive virus particles. Such carriers are
well
known to those of ordinary skill in the art.
Preferred adjuvants to enhance effectiveness of the composition include, but
are not limited to : aluminim hydroxide (alum), N-acetyl-muramyl-L-threonyl-D-
isoglutamine (thr-MDP) as found in U.S. Patent No. 4,606,918, N-acetyl-
normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-
isoglutaminyl-L-aianine-2-( 1 '-2'-dipalmitoyl-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% squalenelTween 80 emulsion. Any of the 3
components MPL, TDM or CWS may also be used alone or combined 2 by 2.
Additionally, adjuvants such as Stimulon (Cambridge Bioscience, Worcester, MA)
or SAF-1 (Syntax) 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.
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.
CA 02309534 2000-OS-15
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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
5 proteins may also be incorporated into Immune Stimulating Complexes together
with saponins, for example Quil A (ISCOMS).
Immunogenic compositions used to raise antibodies comprise a 'sufficient
amount' or 'an immunologically effective amount' of the peptides of the
present
invention, as well as any other of the above mentioned components, as needed.
10 '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.),
15 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 Ng/dose, more particularly from 0.1 to 100 Ng/dose.
20 The immunogenic compositions are conventionally administered parenterally,
typically by injection, for example, subcutaneously or intramuscularly.
Additional
formulations suitable for other methods of administration include oral
formulations
and suppositories. Dosage treatment may be a single dose schedule or a
multiple
dose schedule. The vaccine may be administered in conjunction with other
immunoregulatory agents.
The host serum or plasma is collected following an appropriate time interval
to provide a composition comprising antibodies reactive with the peptides of
the
present invention. The gamma globulin fraction or the IgG antibodies can be
obtained, for example, by use of saturated ammonium sulfate or DEAF Sephadex,
or other techniques known to those skilled in the art. The antibodies are
substantially free of many of the adverse side effects which rnay be
associated with
CA 02309534 2000-OS-15
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21
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.
S The term 'immunogenic' refers to the ability of a substance to cause a
humoral and/or cellular response, whether alone or when linked to a carrier,
in the
presence or absence of an adjuvant.
The antibodies of the claimed invention may also be monoclonals that are
prepared with said antibody being specifically reactive with any of said
peptides,
and with said antibody being preferably a monoclonal antibody.
The monoclonal antibodies of the invention can be produced by any
hybridoma liable to be formed according to classical methods from splenic
cells of
an animal, particularly from a mouse or rat, immunized against the claimed
peptides
of the present invention on the one hand, and of cells of a myeloma cell line
on the
other hand, and to be selected by the ability of the hybridoma to produce the
monoclonal antibodies recognizing the citrullinated forms of the peptides
which has
been initially used for the immunization of the animals.
The antibodies involved in the invention can be labelled by an appropriate
label of the enzymatic, fluorescent, or radioactive type.
The monoclonal antibodies according to this preferred embodiment of the
invention may be humanized versions of mouse monoclonal antibodies made by
means of recombinant DNA technology, departing from parts of mouse and/or
human genomic DNA sequences coding for H and L chains or from cDNA clones
coding for H and L chains.
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 rheumatoid
arthritis. Such human monoclonal antibodies are prepared, for instance, by
means
of human peripheral blood lymphocytes (PBL) repopulation of severe combined
immune deficiency (SCID) mice (for recent review, see Duchosal et al. 1992) or
by
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22
screening vaccinated individuals for the presence of reactive B-cells by means
of
the antigens of the present invention.
The present invention also relates to the anti-idiotype antibodies that are
raised upon immunization with an antibody as defined above and that
specifically
react with said antibodies, thereby mimicking the peptides of the present
invention.
The present invention also relates to truncated versions or single chain
versions of the antibodies and anti-idiotype antibodies as defined above, that
have
retained their original specificity for reacting with the antigens.
The present invention also relates to proteins or peptides that mimic the
antibodies as defined above such as microproteins as can be obtained by phage
display or the highly variable domain of a recombinant antibody as obtained by
screening upon repertoire cloning.
The present invention also relates to a method for detecting antibodies that
specifically react with the peptides or anti-idiotype antibodies of the
present
invention, present in a biological sample, comprising:
(i) contacting the biological sample to be analysed for the presence of said
antibodies with a peptide or anti-idiotype antibody as defined above,
(ii) detecting the immunological complex formed between said antibodies and
said
peptide or anti-idiotype antibody.
The present invention also relates to a reverse method for detecting the
peptides and/or the anti-idiotype antibodies of the present invention with
antibodies
present in a biological sample that specifically react with said peptides
and/or anti-
idiotype antibodies that mimic such peptides, comprising:
(i) contacting the biological sample to be analysed for the presence of said
peptides
or anti-idiotype antibodies with the antibodies as defined above,
(ii) detecting the immunological complex formed between said antibodies and
said
peptide or anti-idiotype antibody.
The methods as defined above, can be used in the diagnosis of rheumatoid
arthritis.
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
CA 02309534 2000-OS-15
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23
the same antigen, thus resulting in difficult and slow diagnosis. Such
diagnostic
technique can be obtained by the simultaneous use of several antigens and/or
anti-
idiotype antibodies of the present invention.
The present invention also relates to a diagnostic kit for use in detecting
the
presence of said antibodies, said kit comprising at least one peptide or anti-
idiotype
antibody or microprotein as defined above, with said peptide or anti-idiotype
antibody or microprotein being preferably bound to a solid support.
The present invention also relates to a diagnostic kit for determining the
type
of autoimmune disease, said kit comprising at least one peptide or anti-
idiotype
antibody or microprotein as defined above, with said peptide or anti-idiotype
antibody or microprotein being preferably bound to a solid support.
The present invention also relates to a diagnostic kit as defined above, said
kit comprising a range of said peptides andlor anti-idiotype antibodies or
microprotein which are attached to specific locations on a solid substrate.
The present invention also relates to a diagnostic kit as defined above,
wherein said solid support is a membrane strip and said peptides and/or anti-
idiotype antibodies or microproteins are coupled to the membrane in the form
of
parallel lines.
The immunoassay methods according to the present invention may utilize for
instance single or specific oligomeric antigens, dimeric antigens, as well as
combinations of single or specific oligomeric antigens. The peptides of the
present
invention may be employed in virtually any assay format that employs a known
antigen to detect antibodies that characterize a certain disease or infection.
A
common feature of all of these assays is that the antigenic peptide or anti-
idiotype
antibody or microprotein is contacted with the body component suspected of
containing the antibodies under conditions that permit the antigen to bind to
any
such antibody present in the component. Such conditions will typically be
physiologic temperature, pH and ionic strength using an excess of antigen. The
incubation of the antigen with the specimen is followed by detection of immune
complexes comprised of the antigen.
Design of the immunoassays is subject to a great deal of variation, and many
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24
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.
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 ImmunolonT""),
diazotized paper, nylon membranes, activated beads, and Protein A beads. For
example, Dynatech lmmunolonT"" 1 or ImmunolonT"" 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.
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 rheumatoid
arthritis in a
standard format, the amount of rheumatoid arthritis antibodies in the antibody-
antigen complexes is directly monitored. This may be accomplished by
determining
whether a second type of labelled anti-xenogenetic (e.g. anti-human)
antibodies
which recognize an epitope on the first type of rheumatoid arthritis-
antibodies will
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WO 99/28344 PCTIEP98107714
bind due to complex formation. In a competitive format, the amount of
rheumatoid
arthritis-antibodies in the sample is deduced by monitoring the competitive
effect
on the binding of a known amount of labelled antibody (or other competing
ligand)
in the complex. The detection of rheumatoid arthritis-antibodies for diagnosis
of
5 rheumatoid arthritis is used as an illustration. Wherever the term "
rheumatoid
arthritis-antibodies" is used throughout the specification, this should not be
considered as limitative. 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.
10 Complexes formed comprising rheumatoid arthritis-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
rheumatoid arthritis-antibodies in the complex may be detected using a
conjugate
of anti-xenogenetic Ig complexed with a label (e.g. an enzyme label).
15 In an immunoprecipitation or agglutination assay format the reaction
between
the rheumatoid arthritis-antigens and the rheumatoid arthritis-antibody forms
a
network that precipitates from the solution or suspension and forms a visible
layer
or film of precipitate. If no rheumatoid arthritis-antibody is present in the
test
specimen, no visible precipitate is formed.
20 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
25 body component, if any, causes the RBCs coated with the purified antigen to
agglutinate.
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.
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WO 99128344 PCTIEP98/07714
26
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 andlor negative), labelled antibody when the assay
format
requires the same and signal generating reagents (e.g. enzyme substrate) if
the label
does not generate a signal directly. The antigenic peptide or anti-idiotype
antibody
may be already bound to a solid matrix or separate with reagents for binding
it to
the matrix. Instructions (e.g. written, tape, CD-ROM, etc.) for carrying out
the
assay usually will be included in the kit.
The solid phase selected can include polymeric or glass beads, nitrocellulose,
microparticles, microwells of a ,reaction tray, test tubes and magnetic beads.
The
signal generating compound can include an enzyme, a luminescent compound, a
chromogen, a radioactive element and a chemiluminescent compound. Examples of
enzymes include alkaline phosphatase, horseradish peroxidase and beta-
galactosidase. 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 Rabbit Ig or anti-human IgG, preferably
aggregated, to form a mixture, and incubating the mixture for a time and under
conditions sufficient to form a reaction mixture product substantially free of
rheumatoid factor-like substance.
The present invention particularly relates to an immunoassay format in which
several peptides of the invention are coupled to a membrane in the form of
parallel
lines. This assay format is particularly advantageous for allowing a
discrimination
between the separate autoimmune diseases.
In another embodiment the present invention refers to a bioassay for
identifying compounds which modulate the binding between an autoantigen and a
rheumatoid arthritis specific autoantibody comprising:
i) - contacting rheumatoid arthritis specific autoantibodies with
CA 02309534 2000-OS-15
WO 99128344 PCTIEP98/07714
27
any of the above mentioned peptides or intermediate filament proteins
or a combination thereof.
- determining the binding of rheumatoid arthritis specific
antibodies with any of the above.mentioned peptides or intermediate
filament proteins or a combination thereof.
ii) - contacting a compound or a combination of compounds and
the rheumatoid arthritis specific autoantibodies simultaniosly or one
after the other with any of the above mentioned peptides or
intermediate filament proteins or a combination thereof.
- determining the binding of rheumatoid arthritis specific
antibodies with any of the above mentioned peptides or
intermediate filament proteins or a combination thereof.
iii) - determining the modulation of the binding of rheumatoid
arthritis specific autoantibodies with any of the above mentioned
peptides or intermediate filament proteins or a combination
thereof, induced by the compound or the combination of compounds,
by comparising the results of I) and II).
In a further embodiment the present invention refers to a modulator for the
interaction between an autoantigen and a rheumatoid arthritis specific
autoantibody,
and the method for producing said modulator, wherein said modulator is
identified
by the bioassay described above.
The term "compound" as used herein has to be interpreted in a broad sense and
can be proteins, peptides, polypeptides, peptidomimetics, carbohydrates,
lipids or
other organic or inorganic molecules, or antibodies which may be generated by
the
host itself upon vaccination.
The term "binding" as used herein indicates that a peptide as described above
is
physically connected to, and interacts with antibodies. Binding of the peptide
to the
antibody can be demonstrated by any method or assay known in the art such as
binding-, ELISA, and RIA-type of assays or competition assays (eg see Examples
section and Current protocols in immunology).
CA 02309534 2000-OS-15
WO 99/283~i4 PCT1EP98/07714
28
The terms "modulation" or "modulate" as used herein refer to both upregulation
(i.e., activation or stimulation (e.g., by agonizing or potentiating)) and
downregulation (i.e. inhibition or suppression (e.g. by antagonizing,
decreasing or
inhibiting) of the binding between a peptide and an anti-HCV antibody.
The term "modulator" as used herein refer to the ability of a compound as
described above to modulate as described above.
The term "peptidomimetics" as used herein refers to molecules which can be
manufactured and which mimic those residues of peptides which modulate the
interaction of the antibody with the peptide as described above. For instance,
non-hydrolyzable peptide analogs of such residues can be generated using
benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and
Biology, G. R.
Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see
Huffman et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM
Publisher: Leiden, Netherlands, 1988), PNA, substituted gamma lactam rings
(Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM
Publisher: Leiden, Netherlands, 1988), ketomethylene pseudopeptides (Ewenson
et
al. ( 1986) J Med Chem 29:295: and Ewenson et al. in Peptides: Structure and
Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical
Co. Rockland, III., 1985), [3-turn dipeptide cores (Nagai et al. (1985)
Tetrahedron
Lett
26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1:1231 ), and
~i-aminoalcohols (cordon et al. (1985) Biochem Biophys Res Commun, 126:419;
and Dann et al. (1986) Biochern Biophys Res Commun 134:71 ).
30
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29
Legends to the Figures
,~jg1_: HPLC profile of tryptic digests of human natural acidic (a) and
neutral (b)
filaggrin.
Peptides were separated by reversed-phase HPLC on a C-4 Vydac column (2.1 x
250 mm, Hesperia, CA) using a 140B Solvent Delivery System (ABI, Foster City,
CA) and eluted with a 8-70% linear gradient of 70% acetonitrile in 0.1
trifluoroacetic acid (TFA). Detection occured at 214 nm with a 1000S diode
array
detector.
Fia 2. Multiple alignment of filaggrin protein sequences and overview of the
sequenced peptides. HB2641, HB2642, HB2648 and HB2650 clones were isolated
in house; all other sequences were retrieved from literature.
Character to show that a position is perfectly conserved: ''*'
Character to show that a position is well conserved: '.'
~: Reactivity of 26 human RA sera with synthetic peptides on LIA.
Peptides IGP1 155, 1156, 1 157 and 1 158 all contained citrulline; IGP1 179, 1
180,
1181 and 1182 were the corresponding counterparts without citrulline
incorporated. Peptides were applied after complexing with streptavidin at a
concentration of 400,ug/ml. IGP1 154 consisted of an irrelevant synthetic
peptide.
.F'g~.: Inhibition ELISA using natural fifaggrin as inhibitory agent.
Plates were dated with purified human natural filaggrin at 1 ,ug/ml. Sera were
diluted 1 /50 and added to the plate with or without preincubation with
natural
filaggrin at 1 or 10 Ng/ml. OD values were measured at 450 nm.
Inhibition ELISA using synthetic citrulline-containing filaggrin peptides as
inhibitory agents.
Plates were coated with purified human natural filaggrin at 4 ,ug/ml. Sera
were
CA 02309534 2000-OS-15
WO 99/28344 PCT/EP98/07714
diluted 1 /50 and added to the plate with or without preincubation with the
individual peptides (IGP1 155, 1156, 1157, 1158) or a mixture of the four
peptides
(mix), each at 100 Ng/ml. OD values were measured at 450 nm.
S Fig 6: Multiple sequence alignment of clones HB2641, HB2642, HB2648 and
HB2650 with the HFIL1 sequence retrieved from literature (McKinley-Grant et
al.
1989).
Character to show that a position is perfectly conserved: ' *''
Character to show that a position is well conserved: ''"'
i 7~ 2-D immunoblots of placental extracts.
Two hundred ~g of the 200 mM salt elution fraction of placental extract was
separated by 2-D gelelectrophoresis using IPG 4-7 strips in the first
dimension and
10% laemli gel in the second dimension. Proteins were electro blotted onto
nitrocellulose membranes and blots were probed with a) human rheumatoid
arthritis
serum (diluted 11200) or b) anti-human vimentin mAb (Sigma) at a 1 /5000
dilution.
The arrow indicates the location of the reference PDI-protein, ticked off with
a
needle after Ponceau S total protein staining.
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31
Examples
~.ole 1: Egitog'e mad ig~,gi of filaggrin
1.1 Sera
Human sera were obtained from the Department of Rheumatology of the University
Hospital in Ghent (Belgium). In total, 2fi5 sera were included, of which 75
fulfilled
the ARA criteria for RA (Arnett et al., 1987), 155 sera scored positive in the
APF
fluorescence test (De Keyser et al., in press), 98 reacted with natural
filaggrin on
Western blot, 80 were APF negative and 16 were derived from healthy controls.
1.2 Preparation of human filaggrin
Natural filaggrin was purified from human skin obtained freshly after
abdominoplasty according to the protocol of Simon et al. (1993). The epidermis
was separated from the dermis by incubating the skin pieces at 56 ° C
in PBS
containing 5 mM EDTA. The material was stored dry at -20°C untill use.
The
epidermis was cut into small pieces which were homogenized in 40 mM Tris-HCI,
pH 7.4, 150 mM NaCI, 5 rnM EDTA, 0.5% NP-40, 0.1 % sodium azide, 0.1 mM
PMSF (0.2 ml/cmz) and stirred overnight at 4°C. The homogenate was
centrifuged
at 15,000 x g for 15 min and the extracted proteins in the supernatant were
precipitated overnight at -20°C by adding 5 volumes of absolute
ethanol. After
centrifugation for 15 min at 10,000 x g, the protein pellet was vacuum-dried
and
subsequently resuspended in water. This partially purified protein preparation
is
refered to filaggrin used in the present study. The amount of protein was
determined by the Bradford protein assay as modified by Peterson ( 1983) using
BSA standard curves.
1.3 Immune detection of antifilaggrin antibodies in human sera
The crude filaggrin preparation was submitted to 10% Tricine SDS-PAGE using
the
Bio-Rad mini-gel apparatus. Two Ng protein/cm was loaded in a large slot and
electrophoresed under standard conditions. The gel was subsequently
electroblotted
onto nitrocellulose membrane in 10% methanol, 10 mM CAPS, pH 1 1.0 during 40
CA 02309534 2000-OS-15
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32
min. The blot was blocked in PBS, 0.05% Tween20, 1 % gelatin and cut into 3 mm
strips, which were probed with human sera overnight at a 1 /100 dilution in
PBS,
0.05% Tween20, 0.1 % gelatin. As a secondary antibody the anti-human IgG-AP
conjugate (Sigma, St Louis, MI) was added at a 1 /1000 dilution; visualization
occured with the NBT/BCIP chromogenic substrate.
1.4 Two-dimensional electrophoresis of filaggrin
The filaggrin material was separated by 2-D electrophoresis using Immobilise
DryeStrips pH 3-10 (Pharmacia Biotech, Uppsala, Sweden) in the first dimension
and 10% Tricine SDS-PAGE in the second dimension using standard procedures.
Preparative gels were loaded with 300 ,ug protein and stained with Coomassie R-
250. The large comma-shaped filaggrin spot was identified by immunoreaction
with
the antifilaggrin mAb (BTI, Stoughton, MA). The pl of this heterogeneous
protein
ranged from 6.7-8.5 (7.1-8.5 on gel), while molecular weight forms of 35-68
kDa
were detected, with the more acidic isoforms representing the highest masses.
1.5 Electro-elution of filaggrin
The large filaggrin spot was cut out of the gel and separated into three
parts: i)
acidic fraction with pl 7.1-7.5; ii) neutral fraction with pl 7.5-8.1; iii)
basic fraction
with pl 8.1-8.5. Each fraction was electro-eluted by the method of Hunkapiller
et
al. (1983) using 50 mM (NH4)HC03, 0.1 % SDS as elution buffer. Coomassie stain
and SDS were removed from the eluted proteins by ion pair extraction as
described
by Konigsberg and Henderson (1983). Vacuum-dried protein pellets were
redissolved in the appropriate buffer for further analysis.
1.6 Peptide mapping
Purified electro-eluted filaggrin fractions of t 100 Ng were dissolved in 40
NI 100
mM (NH4)HC03, pH 8.0, 10% acetonitrile and digested with trypsin (1140 E/S
ratio). After overnight incubation at 37°C the digest was stored at -
20°C before
use.
Peptides were separated by reversed-phase HPLC on a C-4 Vydac column (2.1 x
CA 02309534 2000-OS-15
WO 99128344 PCT/EP981077I4
33
250 mm, Hesperia, CA) using a 140B Solvent Delivery System (ABI, Foster City,
CA) and eluted with a 8-70% linear gradient of 70% acetonitrile in 0.1
trifluoroacetic acid (TFA). Detection occured at 214 nm with a 1000S diode
array
detector and peptides were manually recovered.
1.7 Dot spot analysis and microsequencing
Ninety percent of each peak fraction was vacuum-dried and subsequently
resuspended in a small volume of 10% acetonitrile, 50 mM NaC03 buffer, pH 9.5.
The peptides were dot spotted onto Immunodyne ABC membranes (Pal( BioSupport,
UK), which were probed with human sera in order to assign the immunoreactive
epitopes. First, membranes were blocked with PBS, 0.5% caseine and incubated
overnight with sera 1/50 diluted in PBS, 0.5% caseine, 0.1 % Triton X705, 10
mM
MgC12.6H20. After washing with PBS, 0.05% Tween20, anti-human IgG (Promega)
diluted in PBS, 0.1 % caseine, 0.2% Triton X705 was added for 1 h30. Membranes
were developed in 100 mM NaCI, 100 mM Tris-HCI, pH 9.8, 50 mM MgC12.6H20
substrate buffer containing the chromogenic substrate NBT/BCIP. Reaction was
stopped by addition of 0.2 N H2S04.
The remaining 10% of the immunoreactive fractions was used for
microsequencing.
Therefore, fractions were directly analyzed on a pulsed-liquid model 477A
5equencer equipped with an on-line 120 phenylthiohydantoin analyser (ABI).
1.8 Results
Acidic filaggrin
Each fraction of the tryptic digest of acidic filaggrin (Fig 1 a) was dot
spotted in
triplicate on ABC membranes. Four APF positive sera reacting with human
filaggrin
on Western blot (IG24395, IG35247 and IG24183/24184 pool) and one APF
negative control serum were used for immunoreaction. Fractions T2, T9, T16 and
T66 showed weak reaction with the IG24395 serum, while the series from T30 to
T44, especially T34 and T38 peptide fractions, showed clear positive reaction.
The
APF serum pool also scored positive with both T34 and T38, while IG35247 was
CA 02309534 2000-OS-15
WO 99128344 PCT/EP98/07714
34
only reactive with T4. The APF negative control serum was clearly unreactive
with
the peptide fractions.
Following amino acid sequences were retrieved in each fraction:
I2.:
R1AGHGHSADSSR
L:
R 1 QGSRHQQAR
R1AGHGHSADSSR
R 1 HGSHHQQSADSSR
J9:
R1HSQVGQGESSGPR
R1 HSASQDGQDTI~GHPG
_l"~.:
R1HSASQDGQDTIRGH
R 1 HSGIGHGQASSAVR
R1 HSASQDGC1DT1
RiHSGIGHGQASSAVR
R1DSGH~GYSGSQASDNEGH
R1 HSTSC1EGQDTIHGHRGS
R 1 HSASQDG QDTI RG H PG
T38: T39:
Same peptides as in fraction T35
T40, T41:
Same peptide as in fraction T34
T~:
No signals could be retrieved.
R represents the amino acid citruliine, which was found at a specific
retention time
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WO 99/28344 PCT/EP98107714
different from that of arginine. Further proove for the presence of citrulline
was i)
the fact that the sequence was not cleaved at that specific position, which
would
be expected if arginine was present and ii) that no arginine residue was
sequenced.
S Neutral filaggrin
A similar procedure was carried out for neutral filaggrin (Fig 1 b) and dot
spots were
incubated with APF sera IG24395 and IG24184 and the same negative control
serum as used for the acidic filaggrin mapping. Fractions T28, T65, T81 and
the
series from T30 towards T40 (especially T32, T35 and T36) reacted positively
with
10 one APF serum IG24395. The other serum showed weak reaction with T4, T28,
T45 and T81. There was one fraction (T48) that showed aspecific reactivity
with
all sera including the negative control serum.
Following sequencing results were retrieved:
~, ~, ~7, T~8_ and ~6 , yielded no reliable amino acid sequencing signals.
15 J
R 1 HSGIGHGQASSAVR
R1DSGH$GYSGSQASDNEGH
R 1 HSTSQEGQDTIHGHF~GS
R 1 HSASQDGQDTIRGHPG
20 R1GYSGSCZASDNEGHSE
The fifth peptide was clearly derived from the second peptide, most probably
due
to the fact that in the neutral filaggrin two forms with and without
citrulline-
modification do exist. These results indicate that the same four peptides were
retrieved as identified in the acidic T35 fraction.
S~r~~~d mapl~g of neutral filaggrin
A mix of two other sera IG35038/ 35041 that were both negative on the
synthetic
peptides IGP1155, 1 156, 1 157 or 1 158 (see example 2) was used for mapping
with peptides of neutral filaggrin derived from another skin source. This
yielded
specific reactivity with fractions T28, T31, T34 and T35, while the APF
negative
serum showed no reaction.
CA 02309534 2000-OS-15
WO 99128344 PCTIEP98I07714
36
Sequencing results:
~, ~, ~ yielded no sequencable signals.
~5:
R1NDEQ,SGDGSR
S
An overview of the sequenced immunoreactive parts of filaggrin molecules is
shown in Fig 2.
Example 2' Reactivity of synthetic peptides in a Line Immuno Assay iLIAI
system
2.1 Synthetic peptides
Amino acid sequencing results described in example 1 were used for the
generation
of synthetic peptides. For some citrulline-containing peptides, the non-
modified
counterpart with arginine was synthesized in order to compare their respective
1S reactivities. The following peptides were made.
P1 5 LGP1 179
HSASQDGQDTIRGHPGSS HSASQDGC1DTIRGHPGSS
G P 1 I_G_P 1 L$.Q
HSGIGHGCZASSAVRDSGH~GYS HSGIGHGQASSAVRDSGHRGYS
IGP11.~Z L~~$2
DSGHRGYSGSQASDNEGH DSGHRGYSGSQASDNEGH
2S
1 1 IGP1 182182
HSTSQEGQDTIHGHRGS HSTSQEGQDTIHGHRGS
~124~
GGQGSRHQQAR
CA 02309534 2000-OS-15
WO 99128344 PCT/EP98/07714
37
I P
GGAGHGHSADSSR
I P
GGHGSHHC2QSADSSR
J~,1252
GGNDEQSGDGSRHSGS
I P 2
SRHSQVGQGESSGPR
R represents citrulline
2.2 Line Immuno Assay analysis
Streptavidin-complexed peptides were applied directly on a nylon membrane with
a plastic backing. Blocked strips were incubated overnight with human sera
diluted
1 /100 in 1 ml PBS, 0.5% caseine, 0.1 % Triton X705, 10 mM MgC12.6H20. After
washing with PBS, 0.05% Tween20, goat anti-human IgG-AP conjugated
(Promega) diluted in PBS, 0.1 % caseine, 0.2% Triton X705 was added for 1 h30.
Strips were developed in 100 mM NaCI, 100 mM Tris-HC1, pH 9.8, 50 mM
MgC12.6H20 substrate buffer containing the chromogenic substrate NBT/ BCIP.
Reaction was stopped after 30 min by addition of 0.2 N H2S04.
2.3 Results
A group of 107 individual sera derived from patients who fulfilled the ARA
criteria
for RA were tested in the LIA system for reactivity with the 13 synthetic
peptides
listed in 2.1. Reactivity was noted with IGP1155, 1 156, 1 157, 1 158 and 1249
(Table 2), with a combined sensitivity of 48% in this group. Taking into
account
only the sera that reacted with human natural filaggrin on Western blot,
significantly higher sensitivities were reached for each synthetic peptide,
compared
CA 02309534 2000-OS-15
WO 99128344 PCT/EP98/07714
38
as
.o
v v v ..
O at l~ O O M p ~ p
~
a a r. r~ o ~c o o, .o c~
~ v ~ ~ ~ ~ ~ W M
V ~ .r
.., - O .-~ Ov N M wt N
y y ~n ~r - ~r
V
o
s
C
r. ~.
N O ~ ~ ~' o
C17 , N o O M ~O
n
c ~"~ ,ri N o O
'r
.O H ~O ~ en ~C .~ N ....
c
O
V ~ \ \ ~ \ ~ ~
O ~ M d' o ~O
O ' O O
v ~ ~ a N
~,~"WO v m1 ~t N ~
.- ~
.-~
N N N
' 0
N
y
M
c ~ \ \ \ ~ ~ o
O t~ 00 00 o O ~O ~O r-W
tO .-
~ O ~. ~ U
U
yes..~.~y~ M .-n 'd' n .-arr
~o o i-, ~ ~ v1
t ~ o i n ~ \ ~ bA
O
. v, o~ ,
G
C1 \D ~j M
"' W ~ v
" r v.
.r
H ~' ~ t~ N N M
M N
~ ~ ~
~D ~ ~ O ~ o ~ o
~
r O N ~ M O O t0 ~O
-tt
7
H N t~ V1 O N ..r ~ ....~
N .r N ~
M
00
M
i
-, ~ ~
O
> ~ r'
'
_ w N n r. O -. .-~N II
p U V 00 ~ t0 II
~ 1 1 N n ~ U
w U
z
U
N ~
m
E O
:a
1. rn by _ .,
~ 'i ~ ~ Q
' ~"
f. n d ~ ,
r D .
,
O o G. ~ ~' ~ O
O
p W N
x
'n w w ~ ~ o ~ 0 ~ w
w
U
CA 02309534 2000-OS-15
WO 99/28344 PCT/EP98/07714
39
to the filaggrin negative RA sera. Similar findings were obtained when a
division
was made between APF positive and negative sera. Control sera derived from
51 healthy persons, 61 SLE and 62 osteoarthritis patients showed only minor
reactivities with the peptides.
In all groups, IGP1 156 and 1 158 appeared the best reactive peptides, while
immunoreaction towards IGP1 157 was always very faint and did not add any
value in terms of increasing the sensitivity for RA diagnosis.
The relationship between filaggrin postivity on Western blot and reactivity
with
the synthetic peptides within the RA group is depicted in Table 3. Upon
statistical analysis, a moderate correlation was observed between both
reactivity
patterns (kappa value 0.57 with 95%CI 0.41-0.73; agreement coefficient of
78.5 %y.
The positive LIA signals observed with the citrulline-containing peptides
iGP1 155, IGP1 156, IGP1 157, and IGP1158 were not retrieved when tested
with the non-modified counterparts IGP1 179, IGP1180, iGP1 181, and IGP1 182
(Fig. 3). Only for 2 sera, a weak colouring of all non-modified peptides was
obtained, which could be regarded as aspecific background staining related to
the particular sera.These results indicate that the presence of citrulline is
indispensable for immunoreactivity and that this unusual amino acid
constitutes
an important epitope for antifilaggrin antibodies.
Zable 33: Correlation between anti-filaggrin positivity on Western blot and
reactivity with 5 synthetic peptides IGP1 155, 1156, 157, 1 158 and 1249 on
LIA.
RA sera ~ Peptides combined
I +
+ I 40 12
Filaggrin blot
- I 11 44
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WO 99/Z8344 PCT/EP98/07714
~,xam~le 3' Reactivity of synthetic ~e_ratides in ELISA
3.1 ELISA
Electro-eluted human natural filaggrin was coated in Maxisorp polystyrene
plates
5 at a concentration of 1 or 4 Ng/ml in 50 mM carbonate buffer, pH 9.6
overnight
at 4°C. Sera were diluted 1 /50 in PBS, and preincubated with either
the
appropriate peptides at 100 ,ug/mI, with isolated natural filaggrin at 1 or 10
,uglml (positive control) or with PBS (negative control) for 2 hours. The
plates
were blocked 1 hour with PBS, 0.1 % case'tne at 37°C and subsequently
10 incubated with the sera. During 2 hours at 37 ° C. Plates were
washed 5 times
with PBS, 0.05% Tween 20 and incubated with anti-human IgG-HRP in PBS,
0.1 % caseine, 0.1 mM K3Fe(CN)6. Colour development was performed using
tetramethylbenzidine diluted 1 /100 into 0.1 M NazHP04, 0.1 M citric acid,
0.006% H202, pH 4.3. Reaction was stopped by addition of 2N H2S04 and OD
15 values were measured at 450 nm using a Bio-tek ELISA reader.
The percentage of inhibition was cacufated as folows:
(OD without inhibition - OD with inhibition) x 100
OD without inhibition
3.2 Results
Five APF positive sera showing strong immunoreactivity towards natural
filaggrin
on blot were analyzed on filaggrin-coated plates, with and without inhibitory
filaggrin added (Fig 4; Table 4). Upon preincubation at a concentration of 10
~uglml, the signal dropped with 64-79%, which was significantly higher than
with the use of 1 ,ug/ml (4-45%). The APF negative serum IG24805 not reactive
with filaggrin in Western blot yielded only background signals in this ELISA
and
no significant inhibition could be observed.
When using either one of the synthetic, citrulline-containing peptides IGP1
155,
1 156, 1157, 1158 or a mixture of these four peptides as competitor, each at a
concentration of 100 Ng/ml, 2 out of 4 sera could be significantly inhibited
by
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41
IGP1 155 and 1 serum by IGP1 158, which was in agreement with the LIA results
(Fig 5; Table 4). The peptide mixture was able to inhibit the anti-filaggrin
binding
of the four sera to an extend of 37-92%, while no inhibition was observed with
the APF negative serum.
Example 4: Cloning~~ and expression of human filaggrin
Four candidate human filaggrin sequences were cloned using PCR
technology, using genomic DNA isolated from human lymphocytes as template.
PCR primers were designed as follows:
Sense PCR arimer:
EcoRl
5' CC GAA TTC GCC ACC ATG GGG TCT TTC CTC TAC CAG GTG 3'
Met Glv Ser Phe Ley '~~ Gln Val
The PCR sense primer was chosen to overlap the filaggrin linker sequence and
was designed to introduce a functional initiation codon (Kozak environment)
upstream of the linker sequence (Phe Leu Tyr Gln Val Ser Thr). The linker
sequence was included in the amplified filaggrin repeat, because it is
possibly
involved in the correct targetting of the processed protein. An EcoRl
restriction
site was introduced for subcloning of the PCR fragment.
The antisense PCR primer:
Ser Glv His Ser Gl~ Pro Gly His STOP
3' TGC AGA CCT GTA AGT CCT AGA GGG CCC GAG ATC TGG 5'
SmaI XbaI
CA 02309534 2000-OS-15
WO 99128344 PCT/EP98/07714
42
c~ a y o ~ y
o r. c~o ~ ~ c~
r
c
o
c
:. .c
C IQ M ~ !1' r e- ~f7
C r
~L ~0
0
V
_ '
0 ~ a? M o
0 -
0 ~ 0 0 0 0 0
c
c .o m
0 0 0 0
N = X ON7 ~ ~V ~ GOO M r
C
i
L
~U ~G
pi s ~ 0 0 0 0 0
c c ~r o c~ N c~
'c a c .
o c? j r
+r
c
o W.
0 0 0
fn = r ~ i \ i ~ 0 0
a r' ~ ~ ~ o
:a y t?
+r
a
a~
V t ~ o \ o \ o
~
C 0. O ~ N ~ N O
a
C
C .C ~' o 0
_ C d. CN7 ' M 1 ~ N f~
N
a
O
V ~ M COO cN~7
' '
I~ M 00 M O
C O C7 O O O
C
.'
a7
C7 C
C ~ ~ + + + + + +
:H. Q
C
r-
O ,..
C d
0 ~ + + + + + +
Q
C
d- ~ CO M 00 ~' O lf~ In
M O M ~ In O
O 00
LIB M M M M ~ M N
CA 02309534 2000-OS-15
WO 99IZ8344 PCT/EP98/07714
43
The antisense primer is located just upstream from the next filaggrin linker
sequence and introduces a translation stop codon TAG. The amplified filaggrin
sequence consists therefore of the filaggrin linker followed by an integral
filaggrin repeat and three additional amino acids (Pro/Gly/His) resulting from
the
cloning strategy. The PCR amplified fragments were cloned in a pBLSK
(Stratagene) vector, as EcoRl-Xbal fragment, allowing sequencing of the
ampified cDNA.
The four individual clones characterized by sequencing were named HB2641,
HB2642, HB2648 and HB2650 (Fig 6) . The cDNA inserts were recloned as
EcoRV/Ecl 13611 fragments (1030 bp) in the E. coli expression vector pIGRHISA
opened with Hsil blunted. The filaggrin proteins were expressed as recombinant
filaggrin-His6 fusion proteins in three different E. Coli strains, resulting
in high
Coomassie stainable expression levels. The His6 tail of the fusion protein
allows
easy purification of the protein using metal-affinity chromatography.
Example 5' Vimentin as marker for diagnosis of rheumatoid arthritis
5.1 Isolation of antigen
Placental extracts were prepared as described by Despr~s et al. ( 1994).
Placental tissue stored at -70°C was homogenized in 50 mM Tris-HCI,
pH 7.4,
120 mM NaCI, 1.5 mM DTT, 0.02% NaN3, 1 mM PMSF, and 5 Ng/ml of
chymostatin, leupeptin, antipain, pepstatin. After clarification, the
homogenate
was subjected to DE52 ion exchange chromatography; the 200, 250 and 300
mM salt elution fractions were further analyzed by 1- and 2-D
gelelectrophoresis.
5.2 Two-dimensional gel electrophoresis and blotting procedure
Proteins present in the different chromatographic fractions were separated by
2-
D gel electrophoresis. Samples were resolved in rehydration buffer containing
7
M urea, 2 M thiourea, 4% CHAPS, 0.5% Triton X-100, 1 % Pharmalytes 3-10,
CA 02309534 2000-OS-15
WO 99128344 PCT/EP98/07714
44
mM DTT, 0.01 % Orange G. Fifty-500,ug of protein was applied onto IPG4-7
iso-electrofocusing strips (Pharmacia) by the in-gel rehydration method of
Rabilloud et al. (1997). First dimension strips were loaded on 10% Laemmli
gels
or SDS Gradient 12-14 ExceIGels (Pharmacia) for second dimension running
5 according to standard procedures. Gels were electroblotted onto
nitrocellulose
membranes far 2 hours in 10% methanol, 10 mM CAPS, pH 1 1Ø
Immunodetection with human sera and mAbs was carried out as described under
1.3. Anti-vimentine mAb was purchased from Sigma and used at a 1 !5000
dilution. Anti-calreticulin pAb and anti-PDI mAb were delivered by Affinity
10 BioReagents inc. (Golden, CO), and used at 1 /1000 dilutions.
5.3 Protein identification by MS analysis
Coomassie-stained protein spots were cut out from 2-D gels, washed with water
and 50% acetonitrile/ 1 % TFA and incubated with 0.08 ,ug trypsine in 10 ,ul
25
mM NH4HC03/ 10% acetonitrile, pH 8.0 during 20 hours at 37°C. Peptides
were extracted with 60% acetonitrile/ 0.1 % TFA, vacuum-dried and redissolved
in 10,u1 30% MeOH/ 1 % formic acid. Approximately 5% of the digest mixture
was subjected to DE-MALDI-RETOF-MS analysis on a Voyager-DE STR
(Perseptive Biosystems, Framingham, MA). The remaining material was cleaned
up on PorosR2; bound peptides were eluted in 4,u1 60% MeOH/ 1 % formic acid
of which 65% was used for ESI-MS analysis on a Q-TOF mass spectrometer
(Micromass, UK).
5.4 Results
Twenty five human RA sera, 3 osteoarthritis sera and 20 negative controls were
probed onto 2-D blots containing placental extracts. Very striking
immunoreactivity was observed in 14 RA sera with a multiple-spat-train located
at ~ 60 kDa (variation between 56 and 61 kDa according to the gelsystem) and
a pl of 4.5-4.8 (Fig7a). None of the controls showed any reactivity towards
this
protein. Blots were probed with anti-calreticulin pAb, anti-PDI mAb and anti-
vimentin in order to locate these major placental proteins. Calreticulin was
found
CA 02309534 2000-OS-15
WO 99128344 PCTIEP98/07714
as an abundant spot of 56-61 kDa, pl 4.3, while PDI was observed at 58-64
kDa with a pl of 4.6-4.8, hence both corresponding to the expected theoretical
2-D positions. The latter spot was further identified as being PDI by tandem
MS
analysis.
5 The anti-vimentin mAb showed strong reactivity as shown in Fig 7b with
numerous proteins between 40 and fi 1 kDa. This pattern proved highly
reproducable between different batches of purified material, even when derived
from different persons and tested at different points in time. Upon
superimposing the blots probed with RA sera on the anti-vimentin blot, it
10 became clear that the RA-specific reactivity colocalized with the high MW
form
of vimentin. The spots of intrest were further identified by MAL.DI-TOF and
tandem mass spectrometry. Using both techniques, peptides from human
vimentin, cytokeratin 1 and cytokeratin 9 were retrieved in addition to other
non-
identified peptides. These proteins all belong to the same family of
intermediate
15 filament proteins. As citrullinated forms of vimentin and cytokeratins are
already
described to occur in vivo (Senshu et al., 1992; Senshu et al., 1995; Senshu
et
al., 1996) , it is likely that they constitute targets for autoantibodies
present in
rheumatoid arthritis sera. Citrullinated proteins have been shown to cause a
mobility shift on SDS-PAGE towards higher MW regions in comparison with the
20 non-citrullinated forms (Senshu et al., 1995; Tarcsa et al., 1996), which
could
explain the selective immunoreactivity of the human sera to the highest MW
forms of vimentin. Specific intermediate filament protein forms could hence be
used as marker for diagnosing rheumatological disorders.
30
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WO 99128344 PCT/EP98/07714
46
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