Note: Descriptions are shown in the official language in which they were submitted.
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Peptides of the ATE receptor and their use in preeclarnpsia and malign
hypertension
The invention relates to peptides of the ATE receptor and their use and
subsequent
products in antigenic and immunogenic agents and test kits, in particular for
the
elimination of specifically binding, cell-physiologically active, pathological
antibodies
in preeclampsia and for their diagnostic proof. In addition, the invention
relates to a
process for the proof of anti-ATi receptor antibodies in biological fluids.
The immune system is an essential component part of all animal life. In
mammals, it
is particularly used as a defence of micro-organisms, for tissue regeneration
and for
destruction of tumour cells. In classical immunology, a distinction is made
between a
cellular and a humoral immune defence. This means two distinguishable systems
which nevertheless cooperate with one another, all told portraying the immune
sys-
tem.
There exist a series of diseases which are termed as auto-immune diseases. In
such
diseases, the immune system in the persons involved frequently works against
itself.
The predominantly cell-mediated auto-immune diseases include Multiple
Sclerosis
and Type I diabetes. A second groups is formed by the antibody-mediated auto-
im-
mune diseases. For example, this includes rheumatism or also the less frequent
auto-immune diseases such as myasthenia gravis or lupus erymathematodes.
The pathogenesis of most auto-immune diseases is unknown. There are various hy-
potheses and models of how to explain the origin of auto-immune diseases. For
ex-
ample, one explanatory model portrays the antigeniclmolecular mimicry. In
this, it is
presupposed that micro-organisms, e.g. viruses or parasites, equip themselves
with
certain molecules which are not recognised by the host's own immune system and
avoid it. However, if they are recognised as being external and antibodies are
in-
duced and produced against them, these antibodies also recognise similar body-
in-
herent structures.
Part of the nature of auto-immune diseases and auto-antibodies is that they
bind
onto body-inherent cells and tissue. In this, either the cellular immune
system and
the complementary system are activated, thus triggering pathogenic reactions
in the
tissue in situ - e.g. chronic inflammations -, or there is a pathological
dysfunction of
the cells to which the auto-antibodies have bound.
CA 02363999 2001-08-29
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A classic example of this is dilatative cardiomyopathy. In this auto-immune
disease,
the organism incorrectly forms auto-antibodies which bind to a defined epitope
of the
f~1-adrenergenic receptor. These auto-antibodies generate an increase in the
pulsa-
tion rate in biological tests on rat cardiomyocytes in a cell culture (these
cells have a
practically identical f31-adrenergenic receptor on the surface). We speak of a
phar-
maco-active effect of the auto-antibodies, similar to that of adrenaline.
Dilative cardiomyopathy is an auto-immune disease which, if not treated, leads
to
major impairment of the cardiac output by reduction of the pumping performance
with a simultaneous expansion of the heart muscle tissue by infiltrates. But
if the an-
tibodies are removed from the blood in the early stages of the disease by
blood-
washing, there is a regeneration of the heart muscle in the course of a year
and a
drastic improvement of the heart muscle performance, almost reaching the
figures
for healthy people again.
Apparently, the regeneration of the heart muscle can thus be initiated by the
elimina-
tion of the pathological antibodies from the blood's circulation - and that is
all that
happens in the removal of the overall immunoglobulin.
The situation in preeclampsia is similar.
Preeclampsia is a pregnancy-specific form of high pressure and is one of the
most
important causes of maternal mortality during pregnancy and in the course of
birth.
Preeclampsia has even greater importance for the fate of the fruit, i.e. it is
responsi-
ble for prematurity, retardation of growth and perinatal mortality.
Although a great deal of knowledge has been obtained in the past few years,
the
causes of this clinical picture have yet to be clarified. The only causal
therapy is
premature termination of the pregnancy. However, if the symptoms of the
disease
occur at an early stage, i.e. particularly before the 20~" week of pregnancy,
this is
hardly compatible with a healthy survival of the child. On the other hand,
each day of
extension of a pregnancy can improve the child's chances of survival in this
critical
phase. The best preconditions for achieving this objective are provided by
early rec-
ognition (diagnosis) of the development of a preeclampsia and monitoring and
treatment methods on this basis (immunoglobulin adsorption)
Therefore, the task of the invention entailed finding substances which enable
the
proof of pathological antibodies in preeclampsia and malign hypertension and
' ~ ' '' ' CA 02363999 2001-08-29
3
provide corresponding systems therefor. A further task entails enabling the
elimination of such antibodies from the blood.
The invention is implemented according to the claims, the sub-claims being pre-
ferred variants.
The invention is based on first-time proof that patients with preeclampsia
manifest
specific antibodies against blood-pressure-effective angiotensin-ATE-
receptors.
These antibodies did not occur in women with normal pregnancies, likewise not
with
pregnant women with chronic hypertension, i.e. hypertension independent of the
pregnancy. The angiotensin-II-ATE-receptor antibodies observed lead to an
activation of the ATE-receptor, which is probably also responsible for
dangerous
increases of blood pressure and an acute deterioration of the supply of blood
to vital
organs of mother and child.
In patients with this disease, an immunoglobulin fraction can be isolated from
the '
plasma, containing auto-antibodies which bind onto the angiotensin-1 receptor
and
activate the cell via it. If peptides of the ATE receptor portraying the point
of binding
for the antibodies are added to the cell culture system - in vitro -, the
pathological
effect of the auto-antibodies can be annulled. Similar things are possible by
using
peptides with analog functions, preferably with the amino-acid sequence
AFHYESQ,
AVHYQSN, SHFYQTR, GYYFDTN or ENTNIT.
The surprising thing is that the same epitope structures eliminate the anti-
bodies re-
sponsible for the pathological effect from the blood plasma of the patients
when they
are bound to a solid phase.
An essential part of the invention is thus the provision of amino-acid
sequences in
the form of peptides which recognise, bind and eliminate the pathological auto-
anti-
bodies from the plasma of patients with preeclampsia.
Serum samples of patients with preeclampsia contain auto-antibodies which are
directed against the angiotensin-II-ATE receptor sub-type. In a bioassay,
these anti-
bodies develop a positively chronotropic effect. This effect is inhibited like
that of an-
giotensin II by the sub-type-selective AT1 receptor blocker Losastan. Alpha
and beta-
adrenergenic antagonists and the AT2 receptor blocker PD 123319 had no
influence.
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4
It was surprisingly established that the antibodies recognise an epitope on
the sec-
ond extra-cellular loop of the ATE receptor and that they can be neutralised
and affin-
ity-chromatographically cleaned with the help of peptides corresponding to
this loop.
The epitope is characterised by the amino-acid sequence AFHYESQ. Further, func-
tion-analog peptides with the amino-acid sequence AVHYQ~SN, SHFYQTR,
GYYFDTN or ENTNIT are part of the scope of the invention.
Thus, the object of the invention is peptides containing the epitope of the
AT1 re-
ceptor binding physiologically active auto-antibodies, preferably comprising 5
to 10
amino-acids and their variants, which can form a epitope and bind auto-
antibodies
occurring in preeclampsia.
Peptides partly or totally containing SEQ ID no. 1 AFHYESQ are preferred.
The peptides are synthesised or produced in gene technology according to
methods
known per se by the set-up of the amino-acids.
Antibodies according to the invention aimed against the epitope of the ATE
receptor
are characterised by the fact that they recognise these peptides. Preferably,
they
recognise the peptide of SEQ ID no. 1 or its variants. Further antibodies
recognise
the peptides with the amino-acid sequence AVHYQSN, SHFYQTR, GYYFDTN or
ENTNIT. They are produced with methods known per se by immunisation of small
mammals or immunisation of spleen cells in vitro with the peptides according
to the
invention.
The antibodies are used in various bio-assays, immunological detection systems
and
ELISA test systems.
Further, the invention relates to antigenic agents for detection of
preeclampsia, con-
taining at least one peptide according to the invention, preferably the
peptide of SEQ
ID no. 1, or also peptides with the amino-acid sequence AVHYQSN, SHFYQTR,
GYYFDTN or ENTNIT. They react with the specific antibodies against blood-pres-
sure-effective angiotensin-ATE receptors occurring in preeclampsia. If need
be, the
antigenic agents are bound to various carriers, such as activated sepharose,
cellu-
lose or polystyrene carriers.
A further use of the peptides according to the invention entails immunogenic
agents.
These contain at least one peptide, preferably the peptide of SEQ ID no. 1, or
also
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peptides with the amino-acid sequence AVHYQSN, SHFYQTR, GYYFDTN or
ENTNIT, which induce the production of antibodies capable of recognising auto-
anti-
gens in preeclampsia.
In addition, a test kit for detection of anti-AT, receptor antibodies to prove
pre-
eclampsia is provided by the invention.
The test kit entails
- at least one peptide according to the invention, if need be bound to a solid
phase
- a buffer
- a specific conjugate plus enzyme
- a washing solution
- the substrate solution for detection of the enzyme reaction
- and a stop solution
The bio-assay entails
- spontaneously pulsing neo-natal cardiomyocytes in primary culture or
- cardiomyocytes differentiated from undifferentiated embryonal stem cells
- in one culture medium
Thanks to the development of the new test kits on the basis of the peptides
accord-
ing to the invention, the proof of preeclampsia and assessments of the
sequence
can be done quickly and simply.
The invention further relates to a process of detection of anti-ATE receptor
antibodies
in biological fluids. The sample to be examined is put into contact with at
least one
peptide according to the invention or with a combination of these peptides
with a car-
rier material under conditions permitting an antigen-antibody reaction.
Detection is
then done by means of chemical or physical methods known per se.
The anti-ATE receptor antibodies were detected in all serums of patients with
pre-
eclampsia examined up to now. The antibodies appear after the 20t" week of
preg-
nancy and disappear relatively quickly after labour. The anti-ATE receptor
antibodies
were not detected in normal pregnancies or in pregnant sufferers from
hypertension.
As the antibodies behave like the agonist angiotensin II in in vitro tests,
these anti-
bodies play a role in pathogenesis or preeclampsia. As they can be detected in
all
CA 02363999 2001-08-29
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6
preeclampsia serums examined, they are of importance as diagnostic markers.
Cultivated neo-natal rat heart cells were used as a bioassay. These cells
develop a
rhythmic spontaneous pulsation and react to an angiotensin-II stimulation with
an in-
crease of the beat frequency.
Detection of these AT, receptor antibodies according to the invention is used
both for
early recognition of preeclampsia and also as a basis for new therapeutic
methods.
Thus, the object of the invention is also therapeutic agents against
preeclampsia
containing these peptides, as the removal of the angiotensin-ATE receptor
antibodies
from maternal blood (for example by means of specific or inspecific immuno-
adsorp-
tion) leads to an improvement of the clinical image or can at least prevent a
progres-
sion, which is connected with a reduction of the maternal risk and in
particular with a
distinct improvement of the chances of the child surviving.
The specific immunoglobulin adsorption is done on a column on which there are
peptides in which at least the antibody-binding sequence AFHYESQ is contained
(preferably containing the second extra-cellular loop of the ATE receptor or
sequence
I D no. 1 ).
The inspecific immunoglobulin adsorption is done on a column preferably
containing
sheep's or chicken's antibodies against the human immunoglobulin or protein A
or
C1q.
With this adsorber, all the Ig of the blood plasma, also the auto-antibodies
directed
against the ATE receptor, are bound and eliminated, making use of a suitable
appa-
ratus known to experts.
The invention described with the example of preeclampsia can equally be used
for
some cases of malign hypertension in which an auto-antibody recognising the
same
epitope (the same sequence) is also found.
The invention is to be explained below in more detail with some examples of em-
bodiments.
Fig. 1:
Effect of loops I - III of the ATE receptor on the cell-contracting activity
of the auto-
antibodies (contained in the y-globulin fraction isolated from the serum of
preeclamp-
sia patients)
The Y-globulin fraction of the serum of preeclampsia patients increases the
beat fre-
quency of the heart muscle cells by 22 ~ x beats per minute (fictive). If the
y-globulin
fractions are pre-incubated with peptides portraying these parts of the ATE
receptor
corresponding to loops I - III and the antibodies are subsequently added to
the cell
test system, the loop II peptides inhibit the antibody effect on the cells.
Fig. 2:
Epitope analysis of loop II of the ATE receptor - effect of amino-acid
sequences from
loop II on the auto-antibody-mediated cell stimulations
The y-globulin fraction of the serum of preeclampsia patients increases the
beat fre-
quency of the heart muscle cells. The amino acid sequence AFHYESQ from loop II
inhibits the effect of the auto-antibodies, but not, on the other hand, the
sequence
areas from other parts of loop II.
CA 02363999 2001-08-29
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3. Bioassay to detect the antibodies
To identify and characterise the ATE receptor antibodies, a sensitive bioassay
was
used. Spontaneously pulsing cardiomyocytes reacting to an angiotensin-II
stimula-
tion with an increase of the beat frequency were used. This positively
chronotropic
effect was blocked by the selective antagonist Losartan. The incubation of
these
cells with the anti-ATE receptor auto-antibody also led to an increase of the
pulsation
rate, which was stopped by Losartan. Further, this agonistic effect was
neutralised
by a peptide corresponding to the second extra-cellular loop of the ATE
receptor.
In order to identify the epitopes of the auto-antibodies on the second extra-
cellular
loops of the angiotensin II ATE receptor, an attempt was made to neutralise
the anti-
AT~ receptor auto-antibodies with short overlapping peptides. It was seen that
two
epitopes existed on this extra-cellular loop of the ATE receptor of
hypertension suffer-
ers, these being in a position to annul the effect of the antibody. These were
the
epitopes ENTNIT and AFHYESQ. In preeclampsia patients, the agonistic effect of
the antibodies achieved via the AT, receptor was only neutralised by the
peptide
AFHYESQ. This epitope has a special importance in this disease, as it was
identified
in all the patients examined. The function-analog peptides SHFYQTR and
GYYFDTN were also in a position to neutralise the antibodies, Tab. I.
Tab. I Patients' antibodies, pre-treated with peptide
Patient Antibody + Peptide
No. 1:40 AFHYESQ SHFYQTR GYYFDTN
1 19.6~1.90 1.2~0.84 0.811.16 2.410.72
2 20.0~1.90 1.611.24 0.810.80 2.811.20
