EP1141018A2

EP1141018A2 - Peptides of the at1 receptor and their use for preeclampsia and malign hypertension

Info

Publication number: EP1141018A2
Application number: EP99967916A
Authority: EP
Inventors: Gerd Wallukat; Volker Homuth; Friedrich Luft
Original assignee: Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Current assignee: Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Priority date: 1998-12-24
Filing date: 1999-12-22
Publication date: 2001-10-10
Also published as: AU2429100A; JP2002539075A; WO2000039154A2; WO2000039154A8; CA2363999A1; WO2000039154A3

Abstract

The invention relates to peptides of the AT1 receptor and their use for eliminating specifically binding, cell-physiologically active, pathological antibodies in preeclampsia. The inventive peptides are further used for the diagnosis of preeclampsia. According to the invention, peptides having the sequence AFHYESQ, AVHYQSN, SHFYQTR, GYYFDTN or ENTNIT are preferred.

Description

ATi receptor peptides and their use in preeclampsia and malignant hypertension

The invention relates to peptides of the ATi receptor and their use and their secondary products in antigenic and immunogenic agents or test kits, in particular for the elimination of specifically binding, cell-physiologically active, pathological antibodies in pre-eclampsia and for their diagnostic detection. In addition, the invention relates to a method for the detection of anti-ATi receptor antibodies in biological fluids.

The immune system is an essential component in all animal life. In mammals, it is used in particular to ward off microorganisms, to regenerate tissue and to destroy tumor cells. In classic immunology, a distinction is made between cellular and humoral immune defense. These are two distinguishable, but cooperating systems that ultimately represent the immune system.

There are a number of diseases known as autoimmune diseases. In such diseases, the immune system of those affected is directed against itself. The predominantly cell-mediated autoimmune diseases include multiple sclerosis and type I diabetes. A second group is made up of antibody-mediated autoimmune diseases. They include, for example, rheumatism or the less common autoimmune diseases such as myasthenia gravis or lupus erymathematodes.

The pathogenesis of most autoimmune diseases is unknown. There are various hypotheses and models on how to explain the development of autoimmune diseases. An explanatory model is, for example, the antigenic / molecular mimicry. It is assumed that microorganisms, e.g. B. viruses or parasites equip themselves with certain molecules that are not recognized by the host's own immune system and undermine it. However, if they are recognized as foreign and antibodies are induced and produced against them, these antibodies recognize similar endogenous structures.

It is part of the nature of autoimmune diseases or autoantibodies that they bind to the body's own cells and tissues. Here either the cellular immune system and the complement system can be activated, which then on site pathogenic reactions in the tissue - e.g. B. chronic inflammation - triggers or there is a pathological malfunction of the cells to which the autoantibodies have bound.

A classic example of this is dilated cardiomyopathy. In this autoimmune disease, the organism incorrectly forms autoantibodies that bind to a defined epitope of the ß1-adrenergic receptor. These autoantibodies produce an increase in the pulsation rate in biological tests on rat cardiomyocytes in cell culture (these cells have an almost identical β1-adrenergic receptor on the surface). One speaks here of a pharmacoactive, adrenaline-like effect, the autoantibodies.

Dilated cardiomyopathy is an autoimmune disease that, if left untreated, severely impairs cardiac output by reducing pumping power while expanding cardiac muscle tissue due to infiltrates. However, if the antibodies are removed from the blood of the patient in the preliminary stages of the disease by blood washing, the heart muscle regenerates over the course of a year and the cardiac muscle performance improves drastically, almost returning to the values of healthy people.

Obviously, the elimination of the pathological antibodies from the bloodstream - and nothing else happens when the total immunoglobulins are removed - the regeneration of the heart muscle can be initiated.

The situation is similar with preeclampsia.

Preeclampsia is a high pressure form specific to pregnancy and is one of the most important causes of maternal deaths during pregnancy and during childbirth. Preeclampsia is even more important for the fate of the fruit, meaning that it is responsible for prematurity, growth retardation and perinatal mortality.

Although a lot of knowledge has been gained in recent years, the causes of this clinical picture are still not clear. The only causal therapy is the premature termination of pregnancy. However, if the symptoms of the disease appear early, that is, especially before the 20th week of pregnancy, this is hardly compatible with the child's healthy survival. On the other hand, every day of pregnancy can be extended improve the child's chances of survival during this critical period. The best prerequisites for achieving this goal are the early detection (diagnosis) of the development of preeclampsia and the monitoring and treatment procedures based on it (immunoglobulin adsorption).

The object of the invention was therefore to find substances which enable the detection of pathological antibodies in preeclampsia and malignant hypertension and to provide appropriate systems for this purpose. enable the elimination of such antibodies from the blood.

The invention is implemented according to the claims, the subclaims are preferred variants.

The invention is based on the first proof that patients with preeclampsia have specific antibodies against angiotensin ATi receptors which have an effect on blood pressure. These antibodies did not occur in women with normal pregnancy, nor in pregnant women with chronic hypertension, that is, pregnancy-independent hypertension. The observed angiotensin II ATi receptor antibodies lead to an activation of the ATi receptor, which is probably jointly responsible for dangerous increases in blood pressure and an acute deterioration in blood flow to vital maternal and child organs.

In patients with this disease, an immunoglobulin fraction can be isolated from the plasma, which contains autoantibodies that bind to the angiotensin-1 receptor and thereby activate the cell. If - in vitro - peptides of the AT1 receptor, which represent the binding site for the antibodies, are added to the cell culture system, the pathological effect of the autoantibodies can be eliminated. The same is possible through the use of functionally analogous peptides, preferably with the amino acid sequence AFHYESQ, AVHYQSN, SHFYQTR, GYYFDTN or ENTNIT.

It is surprising that the same epitope structures, when bound to a solid phase, eliminate the antibodies from the patient's blood plasma that are responsible for the pathological effect. An essential part of the invention is therefore the provision of amino acid sequences in the form of peptides which recognize, bind and eliminate pathological autoantibodies from the plasma of patients with pre-eclampsia.

Serum samples from patients with preeclampsia contain autoantibodies directed against the angiotensin II ATi receptor subtype. These antibodies develop a positive chronotropic effect in a bioassay. This effect, like that of angiotensin II, is prevented by the subtype-selective ATi receptor blocker losastan. Alpha and beta adrenergic antagonists and the AT ₂ receptor blocker PD 123319 were without influence.

It was surprisingly found that the antibodies recognize an epitope on the second extracellular loop of the ATi receptor and that they can be neutralized or purified by affinity chromatography using peptides that correspond to this loop. The epitope is characterized by the amino acid sequence AFHYESQ. Furthermore, functionally analogous peptides with the amino acid sequence AVHYQSN, SHFYQTR, GYYFDTN or ENTNIT also belong to the scope of the invention.

The invention thus relates to peptides which contain the physiologically active autoantibody binding epitope of the AT1 receptor, preferably consisting of 5 to 10 amino acids and their variants which form an epitope and can bind autoantibodies which occur in preeclampsia.

Preferred are peptides that contain all or part of SEQ ID No. 1 AFHYESQ.

The peptides are synthesized according to methods known per se by building up the amino acids or are produced by genetic engineering.

Antibodies according to the invention which are directed against the epitope of the ATi receptor are characterized in that they recognize these peptides. They preferably recognize the peptide of SEQ ID No. 1 or its variants. Other antibodies recognize the peptides with the amino acid sequence AVHYQSN, SHFYQTR, GYYFDTN or ENTNIT. They are produced according to methods known per se by immunizing small mammals or immunizing spleen cells in vitro with the peptides according to the invention. O

The antibodies are used in various bio-assays, immunological

Detection systems and ELISA test systems.

Furthermore, the invention relates to antigenic agents for the detection of preeclampsia, which contain 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 angiotensin-AT receptors which are active in preeclampsia. Possibly. the antigenic agents are bound to various carriers, e.g. activated Sepharose, cellulose or polystyrene carrier.

Another use of the peptides according to the invention is in immunogenic agents. These contain at least one peptide, preferably the peptide of SEQ ID No. 1 or also peptides with the amino acid sequence AVHYQSN, SHFYQTR, GYYFDTN or ENTNIT, which induce the production of antibodies which are able to recognize autoantigens in preeclampsia.

The invention also provides a test kit for the determination of anti-ATr receptor antibodies for the detection of preeclampsia.

The test kit includes

- At least one peptide according to the invention optionally bound to a solid phase

- a buffer

- a specific conjugate with enzyme

- a washing solution

- The substrate solution for the detection of the enzyme reaction

- and a stop solution

The bio-assay includes

- spontaneously pulsating neonatal cardiomyocytes in primary culture or

- Cardiomyocytes differentiated from undifferentiated embryonic stem cells

- in a culture medium

By developing the new test kit based on the peptides according to the invention, the detection of pre-eclampsia and progress assessments can be carried out simply and quickly. The invention further relates to a method for the detection of anti-ATi receptor antibodies in biological fluids. The sample to be examined is brought into contact with at least one peptide according to the invention or with a compound of these peptides with a carrier material under conditions which permit an antigen-antibody reaction. The detection is then carried out using known chemical or physical methods.

The anti-ATi receptor antibodies could be detected in all sera examined by patients with preeclampsia. The antibodies appear after the 20th week of pregnancy and disappear relatively quickly after delivery. The anti-ATi receptor antibodies have not been detected in normal pregnancies or in pregnant hypertensives.

Since the antibodies behave like the agonist angiotensin II in in vitro tests, these antibodies play a role in pathogenesis or preeclampsia. Since they are detectable in all pre-eclampsia sera examined, they are important as diagnostic markers.

Cultivated neonatal rat heart cells were used as bioassay. These cells develop a rhythmic spontaneous pulsation and react to angiotensin-Il stimulation with an increase in the stroke rate.

According to the invention, the detection of these A ^ receptor antibodies serves both for the early detection of preeclampsia and as a basis for new types of therapy.

The invention thus also relates to therapeutic agents against preeclampsia which contain these peptides, since the removal of the angiotensin ATi receptor antibodies from the mother's blood (for example by means of specific or non-specific immunoadsorption) leads to an improvement in the clinical picture or at least one Prevent progression, which is associated with a reduction in the maternal risk and in particular with a significant improvement in the child's chances of survival.

The specific immunoglobulin adsorption is carried out on a column on which there are peptides which contain at least the antibody-binding sequence AFHYESQ (which preferably contains the second extracellular loop of the AT receptor or the sequence ID No. 1). The non-specific immunoglobulin adsorption is carried out on a column which preferably contains sheep or chicken antibodies against the human immunoglobulins or protein A or C1 _q .

With this adsorber, all Ig of the blood plasma, including the autoantibodies directed against the AT1 receptor, are bound and eliminated using a suitable apparatus known to the person skilled in the art.

The invention described using the example of preeclampsia is equally applicable to some cases of malignant hypertension in which an autoantibody is found which recognizes the same epitope (the same sequence).

The invention will be explained in more detail below by means of exemplary embodiments.

Fig. 1:

Effect of Loops I - III of the ATi receptor on the cell-contracting activity of the autoantibodies (contained in the γ-globulin fraction isolated from the serum of preeclampsia patients)

The γ-globulin fraction on the serum of preeclampsia patients increases the heart rate of the heart muscle cells by 22 + x beats per minute (fictitious). If the γ-globulin fractions are preincubated with peptides which represent parts of the ATi receptor corresponding to Loops I - III, and the antibodies are subsequently added to the Zeil test system, the Loop II peptides inhibit the antibody effect on the cells.

Fig. 2:

Epitope analysis of Loop II of the ATi receptor - Effect of amino acid sequences from Loop II on the autoantibody-mediated cell stimulation

The γ-globulin fraction on the serum of pre-eclampsia patients increases the beat frequency of the heart muscle cells. The amino acid sequence AFHYESQ from Loop II inhibits the action of the autoantibodies, but not the sequence regions from other parts of Loop II. 3. Bioassay for the detection of the antibodies

A sensitive bioassay was used to identify and characterize the ATi receptor antibodies. Pulsating cardiac myocytes were used spontaneously, which reacted to angiotensin II stimulation with an increase in the stroke rate. This positive chronotropic effect was blocked by the selective antagonist losartan. Incubation of these cells with the anti-ATi receptor autoantibody also led to an increase in the pulsation rate, which was prevented by losartan. Furthermore, this agonistic effect could be neutralized by a peptide that corresponded to the second extracellular loop of the ATi receptor.

In order to identify the epitopes of the autoantibodies on the second extracellular loop of the angiotensin II ATi receptor, attempts were made to neutralize the anti-ATi receptor autoantibodies with short overlapping peptides. It was shown that there are two epitopes on this extracellular loop of the ATi receptor of hypertensives, which were able to cancel the action of the antibody. The epitopes ENTNIT and AFHYESQ were involved. In preeclampsia patients, the agonistic effect of the antibodies, which was achieved via the ATi receptor, was only neutralized by the peptide AFHYESQ. This epitope is of particular importance in this disease because it has been identified in all examined patients. The functionally analogous peptides SHFYQTR and GYYFDTN were also able to neutralize the antibodies Tab. I.

Tab. I patient antibodies pretreated with peptide

Patient antibody + peptide

No. 1:40 AFHYESQ SHFYQTR GYYFDTN

1 19.6 + 1, 90 1, 2 + 0.84 0.8 ± 1, 16 2.4 ± 0.72

2 20.0 + 1, 90 1, 6 ± 1, 24 0.8 + 0.80 2.8 ± 1, 20

Claims

claims

1. peptides of the ATi receptor, preferably consisting of 5 to 30, preferably 5 to 10, amino acids and their variants which form an epitope and can bind autoantibodies which occur in preeclampsia and malignant hypertension.

2. Peptides according to claim 1, characterized in that they are constructed in whole or in part or as a variant of SEQ ID No. 1 AFHYESQ.

3. Peptides according to claim 1, characterized in that they are constructed entirely or partially or as a variant of the functionally analogous peptides with the amino acid sequence AVHYQSN, SHFYQTR, GYYFDTN or ENTNIT.

4. Antibodies which are directed against the epitope of the ATi receptor, characterized in that they recognize the peptides according to claims 1 to 3.

5. Antiköφer according to claim 4, characterized in that they recognize the peptides of SEQ ID No. 1 or functionally analogous peptides with the amino acid sequence AVHYQSN, SHFYQTR, GYYFDTN or ENTNIT or their variants.

6. Use of the whole human ATi receptor or parts thereof, at least containing the amino acid sequence AFHYESQ or parts thereof or of functionally analogous peptides for binding pathological, functionally active autoantibodies, for diagonistic and therapeutic purposes in diseases with a positive antibody status, in particular preeclampsia.

7. Use according to claim 6, characterized in that autoantibody-binding variants and functionally analogous peptides from AFHYESQ are used.

8. Use according to claim 6 and 7. characterized in that recombinantly produced autoantibody-binding receptor parts of the ATi receptor and functionally analogous peptides are used.

9. Use according to claim 6 to 8, characterized in that the amino acid sequence AFHYESQ and functionally analogous peptides or variants containing molecules soluble or solid phase bound for direct or indirect (competitive) antibody detection in body fluids, especially blood.

10. Use according to claim 6 to 9, characterized in that the amino acid sequence AFHYESQ and functionally analogous peptides or variants containing molecules thereof are bound to the solid phase for binding and elimination of the pathological, functionally active autoantibodies in body fluids, in particular blood, that is to say for immunoglobulin adsorption.

11. Use according to claim 6 to 10, characterized in that the amino acid sequence AFHYESQ and functionally analogous peptides or variants containing molecules thereof are bound to the solid phase for binding and elimination of the pathological, functionally active autoantibodies in body fluids, in particular blood, that is to say for immunoglobulin addition.

12. Use according to claim 6 to 11, characterized in that the amino acid sequence AFHYESQ and functionally analogous peptides or variants containing molecules bound to the solid phase for binding and elimination of the pathological, functionally active autoantibodies in body fluids, in particular blood, that is to say for immunoglobulin adsorption in combination with nonspecific (total immunogloblin -binding ligands) can be used.

13. Binding and elimination of the pathological, functionally active autoantibodies in body fluids, in particular blood, by using non-specific adsorbent molecules such as protein A, protein G, anti-human immunoglobulins and total immunogloblin-binding ligands such as amino acids, in particular L-tryptophan or Peptides.

14. Use of peptides containing at least the amino acid sequence AFHYESQ and functionally analogous peptides for the immunization of mammals for the purpose of obtaining poly- and monoclonal antibodies.

15. Use of antibodies directed against the amino acid sequence AFHYESQ and functionally analogous peptides for the immunization of mammals for the purpose of obtaining anti-idiotypic antibodies.

16. Antigenic agent for the detection of preeclampsia or malignant hypertension, characterized in that it contains at least one peptide according to claim 1 to 3, preferably SEQ ID No. 1, contains.

17. Immunogenic agent, characterized in that it contains at least one peptide according to claims 1 to 3, preferably SEQ ID No. 1, which induces the production of antibodies which are capable of recognizing autoantigens in preeclampsia or malignant hypertension .

18. Test kit for the determination of anti-ATi receptor antibodies for the detection of preeclampsia or malignant hypertension, containing at least one peptide according to claims 1 to 3.

19. A method for the detection of anti-ATi receptor antibodies in biological fluids, characterized in that the sample to be examined is brought into contact with at least one peptide of claims 1 to 3 or a compound of these peptides with a carrier material under such conditions, which allow an antigen-antibody reaction and the detection by means of chemical or physical methods known per se.

20. Use of the peptides according to claim 1 to 3 for the preparation of therapeutic agents against preeclampsia or malignant hypertension.