EP1570272A2

EP1570272A2 - Identification of agonistic autoantibodies

Info

Publication number: EP1570272A2
Application number: EP03788849A
Authority: EP
Inventors: Gerd Wallukat
Original assignee: Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Current assignee: Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Priority date: 2002-11-29
Filing date: 2003-11-28
Publication date: 2005-09-07
Also published as: AU2003293004A1; US20060263835A1; WO2004051280A2; US7741050B2; WO2004051280A3

Abstract

The invention relates to a method for detecting disease-associated autoantibodies, which recognize extracellular structures of G protein-coupled receptors, and to the use of peptides, which comprise these loops or fragments thereof, for treating autoimmune diseases.

Description

Determination of agonistic autoantibodies

description

The invention relates to a method of detecting disease-associated autoantibodies that bind loops of G protein-coupled receptors and to the use of peptides comprising these loops or fragments thereof for the treatment of autoimmune diseases.

The immune system of multicellular organisms is based on the distinction between "self" and "not self". Due to its great diversity, the "self" and "not self" differentiating immune system has a large repertoire of specificities expressed particularly by T and B cells. By means of complicated mechanisms, the immune system is able to distinguish between "self" and "not self", with which the body can protect itself, in particular, against the consequences of so-called autoimmunity. That is, the humoral and cellular components of the immune system of an organism are so pronounced that they are not directed against the organism itself. Within the immune system, however, it can lead to a variety of disorders, especially the mechanisms of self-detection can be limited or completely eliminated. Therefore, there are a number of diseases that can be triggered by autoantibodies or autoreactive T cells. One of the first diseases involving autoantibodies to a specific organ Hashimoto's thyroiditis was found. It is a thyroid disorder that occurs mainly in middle-aged women and leads to the development of a goiter and hypofunction of the thyroid gland. Unless the disease is treated, it completely destroys and shrinks the organ. In addition to autoantibodies that are directed exclusively against an organ, there are numerous autoantibodies that can be directed against multiple organs or tissue types.

Depending on the orientation of the autoantibodies, organ-specific and non-organ-specific autoimmune diseases can be distinguished. Typical organ-specific autoimmune diseases include, for example, premature menopause, juvenile diabetes, male infertility, pernicious anemia, or Addison's disease. Non-organ specific autoimmune diseases include, for example, rheumatoid arthritis, dermatomyositis, scleroderma or mixed connective tissue diseases and others. The target organs of the organ-specific disease are often the thyroid, the adrenals, the stomach and the pancreas. The non-organ-specific diseases are summarized under the term of the so-called rheumatic type and concern the skin, the kidneys, the joints and the muscles. For the diagnosis and treatment of autoimmune diseases, only few, insufficient Nerfahren are known. With the well-known laboratory routines, such as ELISA or other well-established diagnostic procedures, such as mass screening under the laboratory It has not been possible to detect the autoantibodies in the serum of a patient which are often present only in low concentrations. In the case of organ-specific diseases, treatment is usually carried out by establishing the metabolic balance; For example, in hypothyroidism, the missing thyroid hormone is substituted with thyroxine, and in the case of thyrotoxicosis, anti-metabolites of the hormone can be given. In the case of pernicious anemia, a depot of vitamin B _{12 may} be administered parenterally, and in myasthenia gravis cholinesterase inhibitors may be given. If a complete loss of function of the organ has occurred, for example, an organ transfer or an implantation of a prosthesis are possible. Such therapy methods are not suitable for organ-specific autoimmune diseases, since, for example, a whole group of organs, such as the skin, kidney, joints and muscles, would have to be substituted in a patient, whereby only the substitution of the joints, the muscles and / or or the skin is nearly impossible. Another way of treating autoimmune diseases is the ability to bind, complex and subsequently eliminate the disease-inducing autoantibodies. However, such methods can be successfully used in diagnosis or therapy only if a target, agent or structure is known with which the autoantibodies interact so that they can be detected or eliminated with the aid of the agent or the target. For numerous autoimmune diseases, such as those associated with autoantibodies Hypertension, pre-eclampsia, humoral kidney rejection or Chagas' cardiomyopathy are not known, and diagnostic methods such as bioassays are difficult to handle and therefore unsuitable for laboratory routine of angiotensin II ATI receptor autoantibodies.

The object of the invention was therefore to provide means, devices and Nerfahren for the diagnosis of autoantibodies and therapies of autoimmune diseases, which allow a simple, efficient and safe detection or treatment and do not have the disadvantages mentioned.

The invention solves this technical problem by providing a method for detecting disease-associated autoantibodies directed against G protein-coupled receptors, the method comprising the steps of:

a) contacting body fluid with a denaturing agent,

b) bringing the precipitated fraction into contact with a peptide comprising in particular a biotin which comprises a partial sequence of the first and / or second loop of the receptor, resulting in a mixture,

c) incubation of the mixture with a particular avidin or streptavidin-coated carrier, d) washing the materials of the wearer,

e) incubation of the carriers with anti-IgG subclasses, wherein the anti-IgG antibody is labeled and

f) carrying out a detection, in particular an enzyme or color reaction.

The invention thus relates to the surprising teaching that it is possible with the aid of an enzyme or color reaction, for example in an ELISA common in laboratory routines, to detect disease-associated autoantibodies which are directed in particular against G protein-coupled receptors. This applies in particular to autoantibodies associated with the diseases of dilated cardiomyopathy, Chagas' cardiomyopathy, myocarditis, preeclampsia, humoral kidney rejection, hypertension, essential hypertension, refractory hypertension, pulmonary hypertension, psoriasis and / or The antibodies of agonistic autoantibodies are preferred, with the aid of an enzyme-linked immunoassay according to the invention being particularly simple, safe and effectively detectable Such diseases, in particular the antibodies associated with them, have hitherto been possible only with complicated indirect tests, such as For example, angiotensin II ATI receptor autoantibodies associated with, for example, preeclampsia have been safely cultured by only contacting the respective sera Cardiomyocytes, especially newborn rats, detected via the modification of the beat frequency. For this, it was necessary to culture the cardiomyocytes of newborn rats and to bring them into contact with the serum of the patients, whereby by detecting the increase of beats per minute, antibodies to the ATI receptor could be detected in patients with preeclampsia. The method according to the invention provides an immunoassay with which numerous autoantibodies directed against G protein-coupled receptors can be detected. These autoantibodies are preferably an anti-beta-adrenergic receptor autoantibody, an anti-muscarinic M2 receptor autoantibody, an angiotensin II ATI receptor autoantibody, an alpha adrenergic receptor autoantibody, and autoantibodies are directed against endothelin IA, PAR-1, PAR-2 and / or PAR-3. The autoantibodies can be in particular agonistic autoantibodies. Of course, the antibodies may also be inhibitory antibodies, for example in allergic asthma (interaction with the 3rd loop). A particular streptavidin-coated carrier is a carrier which is preferably coated with streptavidin or avidin. Particularly preferred are from 6 to 2, in particular 4 subunits existing proteins M _R about 40,000 to 80,000, especially 60000 or 66,000, isoelectric point near the neutral point, such as streptavidin or avidin, which has a high affinity (eg K _D = 10 ^"15 M ^"1 ) to other, in particular by choline inactivating compounds, preferably biotin show. It is known to the person skilled in the art that a streptavidin in particular Coated carrier is advantageous if associated with this operable structure with biotin or an equivalent. If it is determined by experiments that binding without biotin / streptavidin / avidin association or binding is sufficient for detection, the peptide or the carrier need not be linked to these excipients (biotin and streptavidin / avidin).

For the purposes of the invention, the peptide is a molecule which consists essentially of amino acids. Peptides within the meaning of the invention are also structures which comprise more than 50 or 100 amino acids and can therefore also be referred to as proteins. Accordingly, peptides and proteins are used synonymously in connection with the invention. Of course, the peptides may include other structures such as lipids or carbohydrates but also artificial or natural amino acid or non-amino acid building blocks.

The biotin-comprising peptide or protein may also be combined with a tag other than biotin. A tag within the meaning of the invention is a protein tag or a peptide or another structure which combines with the peptide or protein, for example, is fused. The peptide can - as already stated - for example, be biotinylated and thus have biotin as a tag. The person skilled in the art is familiar with further tag structures or tags from catalogs and standard works in biochemistry. Preferred tags are His tag, Flag tag, Strep tag, T7 tag (11 N-terminal amino acids of the T7GenlO protein), S tag, CBP (calmodulin-binding peptide), MBP (maltose-binding-peptide), Neb, protein A, GST-tag, PinPoint-tag, thioredoxin, PET (cellulose-binding-domain), PMal (maltose-binding-domain) and / or biotin tag. This tag has a high affinity for an anti-tag substance or an anti-tag on or on the carrier. Preferred anti-tags are streptavidin, glutathione, biotin, nickel-NTA, cellulose, amylose, thiobond, avidin, and / or immunoglobulin. Day / anti-tag pairs are known to the person skilled in the art, ie the selection of the tag determines the structure of the anti-tag, without the need for an inventive selection by a person skilled in the art, or if he first has to solve a technical problem when carrying out the teaching according to the invention. When the peptide is biotinylated and thus has biotin as tag, the anti-tag structure is streptavidin, ie a streptavidin-coated carrier. Further preferred

Tag / anti-tag pairs are: Protein A / immunoglobulin, GST

(Glutathione-S-transferase) / glutathione, pin-point (in vivo

Biotinylation) / avidin, thioredoxin / thiobond, PET (cellulose-binding domain) / cellulose and / or PMal (maltose-binding domain) / amylose and others. Preferred anti-tag-coated carriers are selected from the group comprising streptavidin, glutathione, biotin, nickel-NTA, cellulose, amylose, thiobond, avidin and / or immunoglobulin coated carriers.

The loops in the sense of the invention may be extracellular structures with which functional, agonistic or antagonistic autoantibodies interact, recognize or bind them. In a particular embodiment of the invention, the denaturing agent is ammonium sulfate. Of course, however, any less structurally-altering denaturant can be used, such as alcohol in the alcohol precipitation. Advantageously, it is possible with ammonium sulfate or alcohol to precipitate body fluids, such as serum, in particular to precipitate fractionated. Thus, for example, antibodies, in particular autoantibodies, can be separated from other constituents of the body fluid. The denaturing of the body fluid is carried out in particular so that the separated components can be returned by means of methods known in the art again in a substantially native state or in a state that allows their detection. Of course, any denaturant known to those skilled in the art other than ammonium sulfate, or any other structurally non-modifying denaturant, will be suitable for the precipitation of body fluids.

In another preferred embodiment of the invention, the carrier is a magnetic particle or ELISA plate or other structure suitable for incubation of the mixture. Magnetic particles allow in particular the separation of the bound mixture by means of current or a magnetic charge. The use of ELISA plates preferably allows the use of laboratory routines or standardized equipment, since ELISA plates, in particular 96-well microtiter plates, in Clinical and basic research can be used as a standard.

In a further preferred embodiment of the invention, the autoantibody is against a betal adrenergic receptor, a muscarinic M2 receptor, an angiotensin II ATI receptor, an alpha adrenergic receptor, an endothelin IA receptor, a PAR-1, PAR -2 and / or PAR-3. Preferably, the receptors are G protein-coupled receptors.

In a particularly preferred embodiment of the invention, the autoantibodies directed against the betal adrenergic receptor are associated with dilated cardiomyopathy, Chagas 'cardiomyopathy or myocarditis, the autoantibodies directed against the M2 muscarinic receptor with dilated cardiomyopathy or Chagas' cardiomyopathy, the autoantibodies directed against the angiotensin II AT1 receptor with preeclampsia, humoral renal rejection or malignant hypertension, the autoantibodies directed against the alpha adrenergic receptor with essential hypertension, refractory hypertension, pulmonary hypertension or psoriasis and / or autoantibodies directed against endothelin IA, PAR-1, PAR-2 and / or PAR-3 are associated with Raynaud's syndrome. Advantageously, these are autoimmune diseases in which the disease-associated autoantibodies are directed against certain extracellular structures of the G protein-coupled receptor. Such autoimmune diseases are difficult with previous means of laboratory routine they can not be diagnosed at all and, furthermore, they can only be treated with considerable, partly surgical effort - for example heart transplantation or implantation of cardiac support systems - in particular by substituting individual tissues, organ areas or complete organs with prostheses or other organs, for example living or dead patients become.

In a very particular embodiment of the invention, in the detection of dilated cardiomyopathy, myocarditis, essential hypertension, refractory hypertension, pulmonary hypertension or psoriasis, a sequence, preferably the peptide, which contains a sequence or partial sequence of the first and / or In the case of Chagas' cardiomyopathy, dilated cardiomyopathy, humoral kidney rejection and Raynaud's syndrome, the peptide comprising a sequence or partial sequence of the second loop of the receptor is advantageously used or the second loop or only the second loop or peptides comprising portions or fragments of the first and / or second loop or only the second loop, for the detection or therapy of said diseases Disclosure of the relationship between autoantibodies, the loop and the respective autoimmune disease Given various possibilities to diagnose the autoimmune diseases mentioned, predict, treat, post-treat or undergo during the course of treatment of a follow-up of the respective healing process. The loops or the peptides comprising portions of the loops are preferably modified.

It is known to the person skilled in the art that individual amino acids have analogous physicochemical properties, which advantageously lead to these amino acids being able to be exchanged with one another. These include, for example, the group of amino acids (a) glycine, alanine, valine, leucine and / or isoleucine; or the amino acids (b) serine and threonine, the amino acids (c) asparagine and glutamine, the amino acids (d) aspartic acid and glutamic acid; the amino acids (e) lysine and arginine and the group of aromatic amino acids (f) phenylalanine, tyrosine and / or tryptophan. Amino acids within one and the same group (a-f) can be interchanged. Furthermore, it is possible that amino acids are replaced by modified amino acids or specific enantiomers. Further modifications are possible according to the teaching of WO99 / 62933 or WO02 / 38592.

Various ways of preparing peptides are disclosed in the prior art. Peptides which are designed starting from the peptides according to the invention with such methods are covered by the teaching according to the invention. One way of generating functionally analogous peptides is, for example, in PNAS USA 1998, Oct. 13; 9521: 12179-84, WO 99/6293 and / or WO 02/38592; these teachings are included in the disclosure of the invention. That is to say, all peptides, peptide fragments or structures comprising peptides obtained by the method according to the invention Peptides were generated - are peptides in the context of the invention, provided that they achieve the object of the invention, in particular interacting with the disease-causing autoantibodies. These autoantibodies may be, for example, agonistic autoantibodies which activate receptors or inhibitory antibodies. -

In another preferred embodiment of the invention, it is preferred that the autoantibodies associated with the dilated cardiomyopathy be contacted with the peptide comprising a sequence or partial sequence of the first or second loop of the betal adrenergic receptor associated with the autoantibodies associated with Chagas' cardiomyopathy contacting the peptide comprising a sequence or partial sequence of the second loop of the betal adrenergic receptor, - the autoantibodies associated with myocarditis are brought into contact with the peptide comprising a sequence or partial sequence of the first or second loop of the betal adrenergic receptor in that the autoantibody associated with the dilated cardiomyopathy is brought into contact with the peptide comprising a sequence or partial sequence of the second loop of the muscarinic M2 receptor associated with the Chagas cardiomyopathy associated autoantibody with the peptide comprising a sequence he partial sequence of the second loop of the muscarinic M2 receptor are brought into contact the autoantibodies associated with the pre-eclampsia are brought into contact with the peptide comprising a sequence or partial sequence of the second loop of the angiotensin II ATI receptor,

the autoantibodies associated with the humoral kidney rejection are contacted with the peptide comprising a sequence or partial sequence of the second loop of the angiotensin II ATI receptor, the autoantibodies associated with the malignant hypertension with the peptide comprising a sequence or partial sequence of the second loop of the Angiotensin II ATI receptor, the autoantibodies associated with essential hypertension are brought into contact with the peptide comprising a sequence or partial sequence of the first or second loop of the alpha adrenergic receptor,

the autoantibodies associated with refractory hypertension are contacted with the peptide comprising a sequence or partial sequence of the first or second loop of the alpha adrenergic receptor, the autoantibodies associated with the pulmonary hypertension with the peptide comprising a sequence or partial sequence of the autoantibody first and / or second loops of the alpha adrenergic receptor, the psoriasis-associated autoantibody having the peptide comprising a sequence or subsequence of the first and / or second loops of the Alphal-adrenergic receptor are brought into contact and / or brought associated with Raynaud's syndrome Autoanti- body with the peptide comprising a sequence or ^'partial sequence of the second loop of the endothelin IA, PAR-1 and / or PAR-2 in contact become. ^•

In a further preferred embodiment of the invention, the IgG subclasses IgG1, IgG2, IgG3 and / or IgG4 are subclasses. Advantageously, specific subclasses are used to easily and effectively detect disease-associated autoantibodies. As a result, in contrast to the methods without the use of specific subclasses, a simple and reliable detection of autoantibodies is feasible. Surprisingly, the subclasses can be assigned to certain clinical pictures. Thus, essentially no mixture of IG sublases is associated with a disease, which is surprising in that, as subclasses are generated by switch and other biochemical mechanisms, different subclasses are generated.

In a further advantageous embodiment it is preferred that - in dilated cardiomyopathy, the IgG3 and / or IgG4 subclasses are used when the peptide comprises a sequence or partial sequence of the first loop and the IgG1 subclass, when the peptide is a sequence or partial sequence of the includes second loops, - Chagas' cardiomyopathy uses the IgG1, IgG2, IgG3 and / or IgG4 subclasses,

in myocarditis, the IgG3 and / or IgG4 subclasses are used if the peptide comprises a sequence or partial sequence of the first loop and the IgG1 subclass if the peptide comprises a sequence or partial sequence of the second loop; the IgG3 subclass is used in preeclampsia, - the IgGl and IgG3 subclasses are used in humoral kidney rejection,

in the case of malignant hypertension, the IgG1 and / or IgG3 subclasses are used, in the case of essential hypertension the IgG1 and / or IgG3 subclasses are used if the peptide comprises a sequence or partial sequence of the first loop and the IgG2 subclass is used, if the peptide a sequence or partial sequence of the second loop, - in the case of refractory hypertension, the IgG1 and / or IgG3 subclasses are used, if the peptide comprises a sequence or partial sequence of the first loop and the IgG2 subclass is used, if the peptide comprises a sequence or partial sequence of the includes second loops,

- the IgGl, IgG2 and / or IgG3 subclasses are used in pulmonary hypertension,

- in psoriasis the IgGl, IgG2, IgG3 and / or IgG4 subclasses are used and / or - in which Raynaud's syndrome the IgGl subclass is used. Advantageously, an IgG subclass or subclasses can be provided by this embodiment of the invention for a specific autoimmune disease or for the autoantibodies, with which autoantibodies can be easily and safely. can be detected.

In a further preferred embodiment of the invention, the autoantibodies are concentrated or purified before detection using methods known to the person skilled in the art.

In a particular embodiment of the invention, the method for concentrating or purifying the autoantibodies comprises the following steps: a) obtaining an IgG fraction from body fluid, b) contacting the obtained IgG fraction with a peptide which is a partial sequence of a first or second loops of a G protein-coupled receptor to obtain a mixture, c) incubating the mixture with a carrier which is washed and concentrated, and d) eluting the autoantibodies from the concentrated carrier.

Expediently, a very reliable diagnosis or detection of the autoantibodies becomes possible by the purification or concentration.

In a particular embodiment of the invention, the peptide which contains the sequence or a partial sequence of the comprises first and / or second loops selected from the group comprising:

EYGSFF, SFFCEL, ARRCYND, PKCCDF, AESDE, CYIQFF, EDGECY, VRTVEDGECYIQFFSNAAVTFGTAI, AFHYESQ, ENTNIT, FWAFGR, GRAFCDV, ITEEAGY, ERFCGI, GRIFCD and / or ITTCHDVL.

In particular, these peptides can be assigned as follows (see also Tab. 3):

DCM I loop EYGSFF, SFFCEL II DCM II loop ARRCYND, PKCCDF my myocardium. I loop ARRCYND, PKCCDF my myocardium. II loop ARRCYND, PKCCDF ß Chagas II loop AESDE musc. M2 DCM II loop CYIQFF, EDGECY musc. M2 Chagas II loop VRTVEDGECYIQFFSNAAVTFGTAI

ATI Praekl. II loop AFHYESQ ATI humor.

Kidney II loop ENTNIT, AFHYESQ ATI malign. Hyp II loop ENTNIT, AFHYESQ αlA essent. Hyp I loop FWAFGR, GRAFCDV αlA essent. Hyp II loop ITEEAGY and ERFCGI αlA pulm. Hyp I loop FWAFGR, GRAFCDV αlA pulm. Hyp II loop ITEEAGY and ERFCGI αlA refract. Hyp I loop FWAFGR, GRAFCDV αlA refract. Hyp II loop ITEEAGY and ERFCGI αlB Psoriasis I loop GRIFCD αlA Psoriasis I loop GRAFCDV

PAR-1 and PAR-2 II loop ITTCHDVL Endothelin IA; Raynaud's Syn.

It is of course known to the person skilled in the art that, because of the disclosure of certain sequence sections of the loop, he can also use sequences which flank the sequences mentioned in the naturally occurring loop. Furthermore, it is known to the person skilled in the art that it can modify the sequences by deletions, substitutions, additions, insertions or other biochemical or biophysical processes in such a way that individual parameters of them - such as, for example, their action as a diagnostic or therapeutic agent - can be improved.

In a further preferred embodiment of the invention, in particular the peptide is immobilized. For the purposes of the invention, immobilization means various procedures and techniques for fixing the peptides on certain carriers. The immobilization can serve, for example, to stabilize the peptides, as a result of which their activity is not reduced or adversely modified in particular during storage or in the case of a single batch batch by biological, chemical or physical agents. Immobilization of the peptides allows repeated use under routine technical or clinical conditions; Furthermore, a sample - preferably blood constituents - can be continuously reacted with at least one of the peptides according to the invention. This can be achieved, in particular, by various immobilization techniques, the binding of the peptides to other peptides or molecules or to a carrier being carried out in such a way that the three-dimensional structure, especially at the center, which mediates the interaction with the autoantibodies, the corresponding molecules, in particular the peptides, is not changed. Advantageously, the specificity to the autoantibodies of the patients is not lost by the immobilization. For the purposes of the invention, three basic methods of immobilization can be used:

(i) Crosslinking: In crosslinking, the peptides are fixed with each other without adversely affecting their activity. They are advantageously no longer soluble by the crosslinking.

(ii) Binding to a Carrier: Binding to a carrier occurs, for example, by adsorption, ionic bonding or covalent bonding. This can also take place within microbial cells or liposomes or other membrane-containing closed or open structures. The peptides are advantageously not affected by the fixation in their activity. The peptides can advantageously be used repeatedly or continuously in the hospital in diagnosis or therapy carrier-bound.

(iii) Inclusion: The inclusion is within the meaning of the invention. in particular to a semipermeable membrane in the form of gels, fibrils or fibers. Encapsulated peptides are separated by a semipermeable membrane by the surrounding sample solution so that they can advantageously interact with the autoantibodies or with fragments of them. For immobilization, various methods are available, such as adsorption to an inert or electrically charged inorganic or organic carrier. Such supports may be, for example, porous gels, alumina, concrete, agarose, starch, nylon or polyacrylamide. The immobilization takes place here by physical binding forces, often involving hydrophobic interactions and ionic bonds. Such methods are advantageously easy to handle and have little effect on the conformation of the peptides.Through electrostatic binding forces between the charged groups of the peptides and the support, the binding can be advantageously improved, for example by the use of ion exchangers, especially Sephadex.

Another method is covalent bonding to support materials. The carriers may have reactive groups that undergo homopolar bonds with amino acid side chains. Suitable groups in peptides are carboxy, hydroxy and sulfide groups and especially the terminal amino groups of lysines. Aromatic groups offer the possibility for diazo couplings. The surface of microscopic porous glass particles can be activated by treatment with silanes and then reacted with peptides. For example, hydroxy groups of natural polymers can be activated with bromocyane and then coupled with peptides. With polyacrylamide resins, many peptides can advantageously undergo direct covalent bonds. When included in three-dimensional networks, the peptides are included in ionotropic gels or other structures known to those skilled in the art. The pores of the matrix are particularly so to ensure that the peptides are retained and that interaction with the target molecules is possible. In cross-linking, the peptides are converted to polymeric aggregates by cross-linking with bifunctional agents. Such structures are gelatinous and easily deformable and particularly suitable for use in various reactors. By adding other inactive components such as gelatin in crosslinking, the mechanical and bonding properties can be advantageously improved. In microencapsulation, the reaction space of the peptides is limited by means of membranes. The microencapsulation can be carried out, for example, as interfacial polymerization. Immobilization in microencapsulation renders the peptides insoluble and thus reusable. For the purposes of the invention, immobilized peptides are all peptides that are in a state that allows their reuse. The limitation of the mobility and the solubility of the peptides by chemical, biological or physical means advantageously leads to low process costs, in particular in the elimination of autoantibodies from blood constituents.

In a further preferred embodiment of the invention, the peptide is bound to a solid phase. The binding of the peptide to the solid phase can take place via a spacer. As a spacer, it is possible to use all chemical compounds which have the appropriate structural and functional requirements for the function of the spacer, as long as they do not modify the bonding behavior in this way, ^■ that binding of the autoantibody with the peptide is adversely affected.

In a particularly preferred embodiment of the invention, the peptide comprises amino groups, amides, acetyl groups, biotin groups, markers, spacers, linkers, GKK and / or SGKK. Such structures advantageously allow use of the peptides in apheresis therapy.

In a particularly preferred embodiment of the invention, the linker and / or the spacer comprises -aminocarboxylic acids and their homo- and hetero-oligomers; α, ω-aminocarboxylic acids and their branched homo- or hetero-oligomers; other amino acids and the linear and branched homo- or hetero-oligomers; Amino oligoalkoxyalkylamines; Maleimidocarboxylic acid derivatives; Oligomers of alkylamines; .4-alkylphenyl derivatives; 4-oligoalkoxyphenyl or 4-oligoalkoxyphenoxy derivatives; 4-Oligoalkylmercaptophenyl- or 4-Oligoalkylmercapto- phenoxy derivatives; 4-Oligoalkylaminphenyl- or 4-Oligo- alkylaminyphenoxy derivatives; (Oligoalkylbenzyl) phenyl or 4-oligoalkylbenzyl) phenoxy derivatives and 4-oligoalkoxybenzyl) phenyl or 4-oligoalkoxybenzyl) phenoxy derivatives; Trityl derivatives; Benzyloxyaryl or benzyloxyalkyl derivatives; Xanthene-3-yl-oxyalkyl derivatives; (4-alkylphenyl) - or ω- (4-alkylphenoxy) -alkanoic acid derivatives; Oligoalkyl phenoxyalkyl or oligoalkoxy phenoxyalkyl derivatives; Carbamate derivatives; amines; Trialkylsilyl or dialkylalkoxysilyl derivatives; Alkyl or aryl derivatives and / or combinations thereof. In a further particularly preferred embodiment of the invention, the immobilized peptides are modified by deletion, addition, substitution, translocation, inversion and / or insertion.

The invention also relates to a peptide selected from the group comprising EYGSFF and / or SFFCEL (DCM, 1st loop); ARRCYND and / or PKCCDF (DCM, 2nd loop); AESDE (Chagas, 2nd loop), CYIQFF and / or EDGECY (DCM, 2nd loop); VRTVEDGECYIQFFSNAAVTFGTAI (Chagas, 2nd loop), AFHYESQ

(Preeclampsia, 2nd loop), ENTNIT and / or AFHYESQ (humoral

Nienabstossung, malignant hypertension, 2nd loop); FWAFGR and / or GRAFCDV (essential hypertension, 1st loop),

ITEEAGY and / or ERFCGI (essential hypertension, 2nd loop), GRIFCD, GRAFCDV (psariasis, 1st loop) and / or ITTCHDVL for use as a medicinal agent. In the case of pulmonary and refractory hypertension, the statements on essential hypertension apply to the 1st and 2nd loop. The use as a therapeutic agent in the context of the invention means the use of the peptide or peptides in the entire field of medicine, preferably for the diagnosis and therapy of autoimmune diseases.

The person skilled in the art is aware that, on account of the disclosed peptides, he can generate further functionally analogous peptides as a diagnostic and / or therapeutic agent. These functionally analogous peptides are covered by the teaching of the invention. In particular, the dissertations PNAS USA 1998, Oct. 13; 9521: 12179-84, WO 99/6293 and / or WO 02/38592, which are incorporated in the disclosure of the teaching of the invention. In a preferred embodiment of the invention, the peptide is bound by autoantibodies of patients with one of the following diseases: dilated cardiomyopathy, Chagas' cardiomyopathy, myocarditis, preeclampsia, humoral renal rejection, malignant hypertension, essential hypertension, refractory hypertension, pulmonary hypertension, psoriasis and Raynaud's Disease The skilled artisan can provide routine diagnostic and therapeutic procedures from this disclosure.

The invention also relates to recognition molecules which are directed against the peptide according to the invention. Preferably, the recognition molecules are antibodies, antisense constructs and / or a chelator. The recognition molecules according to the invention may be antibodies directed against autoantibodies which in particular induce the following diseases: dilated cardiomyopathy, Chagas' cardiomyopathy, myocarditis, preeclampsia, humoral renal rejection, malignant hypertension, essential hypertension, refractory hypertension, pulmonary hypertension, psoriasis and / or Raynaud's syndrome.

The invention also relates to a pharmaceutical composition comprising the peptides and / or the recognition molecules optionally with a pharmaceutically acceptable carrier. The pharmaceutical composition can be used in particular as a pharmaceutical. For this purpose, it is possible, for example, to modify the peptides by cyclization or other methods known to the person skilled in the art in such a way that they are metabolized by the body's own peptide-degrading Structures, such as can not be destroyed. By using the peptides or recognition molecules according to the invention, it is possible to neutralize the autoantibodies in or ex vivo or in vitro. In vitro neutralization is advantageous, for example, in the study of autoimmune diseases in tissue or cell cultures. In an in vivo neutralization, the drugs are administered directly to the patient, for example, in an ex vivo neutralization, the blood is passed out of the body via a loop, for example in the form of a hose circuit, following contact with the drug and after completed neutralization of autoantibodies back into the organism, especially the human patient, attributed. For the purposes of the invention, medicaments include both those pharmaceutical compositions which are used for the therapeutic and prophylactic purposes and those pharmaceutical compositions which can be used as a diagnostic.

Medicaments or pharmaceutical compositions used interchangeably herein are, according to the invention, substances and preparations of substances which are intended to heal, alleviate or prevent diseases, suffering, bodily injury or pathological complaints by application to or in the human body. According to the invention, medical adjuvants are substances which are used for production as active ingredients of medicaments. Pharmaceutical-technical excipients serve for the suitable formulation of the pharmaceutical or pharmaceutical composition and can even, if needed only during the manufacturing process, are subsequently removed or may be part of the pharmaceutical composition as pharmaceutically acceptable carriers. The drug formulation or formulation of the pharmaceutical composition is optionally in combination with a pharmaceutically acceptable carrier and / or diluent. Examples of suitable pharmaceutically acceptable carriers are known to those skilled in the art and include, for example, phosphate buffered saline, water, emulsions such as oil / water emulsions, various types of detergents, sterile solutions, etc., pharmaceuticals or pharmaceutical compositions containing them Carriers may be formulated by known conventional methods. These drugs or pharmaceutical compositions may be administered to an individual in a suitable dose, for example in the range of from 1 μg to 10 g of peptides per day per patient. Doses of 1 mg to 1 g are preferred. Preference is given to administering as few and as low doses as possible, and more preferably a single dose. Administration may be by various routes, for example, intravenous, intraperitoneal, intrarectal, intra gastrointestinal, intranodal, intramuscular, local, but also subcutaneous, intradermal or on the skin or mucous membranes. The administration of nucleic acids coding for the peptide according to the invention can also be carried out in the form of gene therapies, for example via viral vectors. The nature of the dosage and route of administration may be determined by the attending physician according to the clinical factors. It is known to the person skilled in the art that the type of dosage depends on various factors, such as the size, the body surface, the age, the sex or the general health of the patient, but also on the particular agent administered, the duration and mode of administration and of other medications that may be administered in parallel. The person skilled in the art can be guided by the usual standard values and by special teachings, for example the teaching of EP 1 085 955, which is included in the disclosure of the invention. Furthermore, it is known to the person skilled in the art that he can first diagnose the concentration of the autoantibodies with the peptides according to the invention in order to determine the necessary concentration of the medicament.

In particular, the pharmaceutical compositions or the medicament comprise a pharmacological substance which contains one or more peptides or recognition molecules according to the invention or / and nucleic acid molecules coding for them in a suitable solution or administration form. These may either be used alone with the corresponding auxiliaries described under medicaments or pharmaceutical compositions or in combination with one or more adjuvants, for example QS-21, GPI-OlOO or other saponins, water-oil emulsions such as montanides, adjuvants, polylysine, polyarginine compounds , DNA compounds such as CpG, detox, bacterial vaccines such as typhoid or BCG vaccines, salts such as calcium phosphates and / or other suitable agent for enhancing the effect of administration; preferably immune stimulatory molecules, such as interleukins, for example IL-2, IL-12, IL-4 and / or growth factors, for example GM-CSF. These are mixed in known methods with the peptides or recognition molecules according to the invention and administered in a suitable formulation and dosage. Formulations, dosages and suitable components are known to the person skilled in the art.

The pharmaceutical composition or the medicament may, of course, also be a combination of two or more of the pharmaceutical compositions or medicaments according to the invention, as well as a combination with other medicaments, such as antibody therapies, chemotherapies or radiotherapies, administered in a suitable manner together or separately; be applied. The preparation of the pharmaceutical or pharmaceutical compositions is carried out according to known methods.

The invention also relates to a kit comprising the peptide according to the invention, the recognition molecules according to the invention and / or the pharmaceutical composition according to the invention, optionally with an instruction for combining the contents of the kit and / or for providing a formulation for a recipient and an algorithm of administering the Formulation, that is, in which dose or at which time intervals a single component of the kit is administered to a patient. However, the recipient according to the invention may also be a cell or tissue in vivo, ex vivo or in vitro. The information may be, for example, a Leaflets, but also to act on information that the user can call by phone or via the Internet. In particular, the algorithm of administering the formulation includes instructions for the diagnostic and / or therapeutic method of treating a patient. These can be one-stage as well as multi-stage Nerfahren, as well as those that are performed in the absence or presence of a doctor. That is, the therapy schema or information about it is preferably a component of the kit.

The invention also relates to a chromatographic apparatus comprising the peptides of the invention.

In a preferred embodiment, the peptides within the chromatography system are bound, for example, to a solid phase.

The device according to the invention can be used in particular to eliminate the autoantibodies from the fluids of a patient or to neutralize the autoantibodies. This method is known to the person skilled in the art by the term immunoadsorption or apheresis therapy. Immunoadsorption removes immunoglobulins from the patient's blood. Advantageously, this immunoadsorbent treatment can be performed inpatient and outpatient. It can be provided that the device, in particular the so-called adsorber, is part of an extracorporeal blood circulation. Here, the patient from a larger body vessel, especially an arm vein, continuously or discontinuously blood removed and separated by filtration or centrifugation into individual components, such as the cellular and the humoral components. An essential component of the blood that is thereby obtained is, in particular, blood plasma. The blood plasma can advantageously be passed through the device according to the invention and returned to the patient after adsorption of the autoantibodies together with the previously separated blood constituents, in particular the cellular constituents, in particular by another arm or leg vein. It may also be provided that the peptides are immobilized on a Sepharose matrix. This matrix can be placed in a container having a volume of 10 to 400 ml. The patient's blood plasma can then be passed through this matrix, whereby the autoantibodies can bind and thus be eliminated from the blood plasma. The person skilled in various ways known to provide such solid phase-fixed peptides, for example in the form of (i) adsorptive adsorp- tion columns, in the form of (ii) double columns as well as in the form of (iii) one-time columns. The various rinsing and elution solutions which allow a high efficiency of the treatment can easily be determined by the skilled person by routine experimentation. By providing the teaching according to the invention, in particular the peptides according to the invention, various possibilities are disclosed to the person skilled in the art, in vivo, ex vivo and in vitro, for the prophylaxis, diagnosis, therapy as well as for the follow-up treatment of dilated cardiomyopathy, the Chagas cardiomyopathy. Myopathy, myocarditis, preeclampsia, humoral kidney rejection, malignant hypertension, essential Hypertension, refractory hypertension, pulmonary hypertension, psoriasis, and / or Raynaud's syndrome. The person skilled in further embodiments of WO 02/38592, EP 1 214, 350 and WO 99/56126 are known, which are included in the disclosure of the teaching of the invention.

The invention also relates to the use of the peptides according to the invention, the pharmaceutical composition according to the invention, the kit according to the invention and / or the device according to the invention for the prophylaxis, diagnosis, therapy, follow-up and / or after-treatment of autoimmune diseases selected from the group comprising dilated cardiomyopathy Chagas' cardiomyopathy, myocarditis, preeclampsia, humoral kidney rejection, malignant hypertension, essential hypertension, refractory hypertension, pulmonary hypertension, psoriasis, and / or Raynaud's syndrome.

The invention also relates to the use of the peptides according to the invention, the pharmaceutical composition according to the invention, the kit according to the invention and / or the device according to the invention for the production of a medicament for the treatment of autoimmune diseases selected from the group comprising dilated cardiomyopathy, Chagas' cardiomyopathy, myocarditis, preeclampsia, humoral kidney rejection, malignant hypertension, essential hypertension, refractory hypertension, pulmonary hypertension, psoriasis, and / or Raynaud's syndrome. The invention also relates to the use of the peptides according to the invention, the pharmaceutical composition according to the invention, the kit according to the invention and / or the device according to the invention for the screening of medicinal products. The screening of drugs may, for example, include the identification of substances, in particular peptides, proteins, carbohydrates and / or lipids, which interact with the peptides. An interaction may for example be a binding to these peptides or else an activation or inhibition of or by the mentioned peptides. Accordingly, a drug could, for example, be a structure which binds to the peptides in the body of a patient and accordingly to the corresponding loops and thus competes for a binding site with the autoantibodies present there. By disclosing the teaching of the invention, in particular on the disclosure of the relationship between disease and the binding site of the autoantibodies, the

Professional screen various drugs. The screening of drugs based on disclosed targets is well within the skill of the artisan and is made through routine experimentation, with reference to the corresponding standard works of molecular biology and pharmacology.

The invention also relates to a method of treating an autoimmune disease selected from the group comprising dilated cardiomyopathy, Chagas' cardiomyopathy, myocarditis, pre-eclampsia, humoral renal rejection, malignant hypertension, essential hypertension, refractory hypertension, pulmonary hypertension, Psoriasis, Raynaud's syndrome Binding and / or removal of autoantibodies by means of peptides bound to a solid phase according to the invention. By bound to the solid phase peptides, the autoantibodies are bound, complexed and / or neutralized on the solid phase.

In a particular embodiment of the treatment method, it is preferred that the autoantibodies in the dilated cardiomyopathy against betal adrenergic receptors, in Chagas' cardiomyopathy against beta-adrenergic receptors, in myocarditis against beta-adrenergic receptors, in dilated cardiomyopathy against muscarinerge M2 receptors, in Chagas' cardiomyopathy against muscarinic M2 receptors, in preeclampsia for angiotensin II ATI receptors, in humoral renal rejection against angiotensin II AT1 receptors, in malignant hypertension to angiotensin II ATI receptors, in the essential Hypertension to alpha adrenergic receptors, to refractory hypertension to alpha adrenergic receptors, to pulmonary hypertension to alpha adrenergic receptors, to psoriasis to alpha adrenergic receptors, autoantibodies to endothelin IA in Raynaud's disease , PAR-1 and / or PAR-2.

In the following, the invention will be explained in more detail by means of an example, without being limited to this example. example

Determination of angiotensin II AT- _L receptor autoantibodies

Spontaneous beating, cultured cardiomyocytes of newborn rats are a very useful model for studying the effects of autoantibodies.

Research on ß _{j ^} -adrenoceptor autoantibodies has already been reported by Wallukat et al. , 2001, reported. This report is about angiotensin II AT _x receptor autoantibodies in preeclamptic women. Pre-eclampsia is a disorder characterized by an increase in blood pressure that can lead to the death of mother and fetus. Dechend et al. , 2000, were able to demonstrate the manifestation of agonistic antibodies against angiotensin AT _x receptors, which are common in preeclamptic women. Activation of the AT ^ receptor by agonistic autoantibodies accounts for many of the pathophysiological features of preeclampsia. The findings of Wallukat et al. , 1999, show that immunoglobulin fractions and affinity-purified antibodies from pre-eclamptic women can stimulate the AT ^ receptor of cultured cardiomyocytes. The addition of losartan (1 μM) reduces the beats per minute. Neutralization experiments have shown that the IgG subclass 3 is responsible for the increase of the beat rate. In accordance with these findings, an enzyme-linked immunoassay was developed for the determination of angiotensin II AT _{j ^} receptor autoantibodies (anti-AT _{j ^} -AAB).

First, peptide solutions corresponding to the amino acid sequence of the second loop of the human A ^ receptor (Sm 1986/1, 100 μg / ml) were incubated with anti-AT- _L- ABA (1: 1;

Vol. / Vol.) For 1 hour at 4 ° C incubated. Anti-AT- _L -AABs were prepared by ammonium sulfate precipitation from waste fluids at birth (blood and isotonic saline). These samples were more concentrated than serum samples.

Second, this mixture was incubated with washed streptavidin-coated magnetic particles for 1 hour at 4 ° C.

Third, to separate the IgG / peptide mixture, the magnetic particles were washed three times with washing buffer (20 M potassium phosphate buffer, 0.15 M NaCl, pH 7.5). The separation or washing can be carried out easily with a magnetic onzentrationsapparat (Dynal). Unspecific binding sites were blocked with 1% bovine serum albumin in wash buffer.

Fourth, the magnetic particles were incubated with a solution of horseradish peroxidase-labeled antibodies to human IgG3 (1: 200, 1 hour, room temperature). Fifth, the particles were treated with a standardized ready to use solution of TMB (tetramethylbenzidine) for 30 minutes at room temperature in the dark. The color reactions (blue-green) were stopped with 0.1N HCl (yellow-orange). The optical densities were measured in a microplate reader (Anthos HTII) at 492 nm (reference filter 620 nm). The results are summarized in Table 1.

The same peptide of the human AT ^ receptor (Sm 1986/1) was used for purifying anti-AT- _L -AABs. IgG solutions were mixed with peptide solution (100 μg / ml, 1: 1, v / v) and incubated for one hour at 4 ° C. The three times washed streptavidin-coated magnetic particles were added (300 μl). The particles were collected with the magnetic concentrator. The supernatants were carefully separated and stored in ice. The magnetic particles were washed three times and eluted with 3M potassium thiocyanate solution for 15 minutes at room temperature. After magnetic concentration, the solutions were carefully separated and dialyzed against NaCl (0.9%) in phosphate buffered solution along with the first supernatant. After 5 replacements within 3 days, the protein content was determined by optical density (280 nm). The chronotropic effect of supernatant and eluate on primary cultured neonatal rat cardiomyocytes (bioassay) was recorded using an imaging computer system (IMAGOQANT). Table 2 shows the reproducibility of the purification process. Six of the six cleaned showed the increase in beat rate / min (> 24.4). By contrast, cultures treated with supernatant caused no or only moderate changes in the beat frequency (<10.0).

The process of coimmunoprecipitation of the A ^ receptor was similar to that of the (Wallukat, 2001). The differences are: lysed membranes of transfected CHO cells (Couchon, 1997) were used for coimmunoprecipitation. The lysed membranes should be freshly prepared. The proteins were identified by means of an antibody to a peptide having the sequence of the N-terminal portion of the AT ^ receptor produced in rabbits (N10, 1: 100, Santa Cruz), and Western blot and ECL. System with anti-rabbit IgG peroxidase conjugates (1: 10,000, Sigma) detected.

Figure 1 shows the results of the Western blot. A gang

(Molecular weight> 40.0 kDa) could be transfected with the aid of internal positive samples (lysed membranes

CHO cells and human placenta tissue). In earlier experiments (Neichel, unpublished data), this band could be blocked by the peptides used to make the N10 antibodies. This band was absent in pure Sepharose samples and in the supernatants of the purification experiments.

The results show the usefulness of the IMAGOQUANT computer imaging system in demonstrating the increase in Beats per minute due to AT ^ AABs in patients with preeclampsia. The enzyme-linked immunoassay should be tested with sera from pre-eclampsia patients and healthy donors. The purified AT ^ AABs can be used to further study the pathogenesis of preeclampsia.

Table 1

Measurement of AT _{j ^} autoantibodies using an enzyme-linked immunoassay

IgG n Optical density (OD, 492 nm) range

Table 2

Influence of supernatants and eluates of the magnetic particles on the beat frequency of cultured cardiomyocytes of newborn rats

+ SA from the mean

Table 3

Autoantibodies to G protein-coupled receptors

Information on the epitopes and IgG subclass

Antibody disease epitope IgGS b class against Rez.

Betal-Adren. dilat. Cardiomyopathy 1. loop IgG3 u. IgG4

2nd loop IgGl

Betal-Adren. Chagas Cardiomyopathy 2. loop Betal-adrenal myocarditis 1. loop IgG3 and IgG4

2nd loop igGi muskarin. M2 dilat. Cardiomyopathy 2. loop igGi muscarin. M2 Chagas Cardiomyopathy 2. loop Ang. II ATI preeclampsia 2. loop IgG3 Ang. II ATI humoral kidney rejection 2. loop IgGl and IgG3 Ang. II ATI malignant hypertension 2. loop IgGl and IgG3 alpha adrenergic essential hypertension 1. loop IgG1 and IgG3

2. loop IgG2

Alphal Adren. refractory hypertension 1. loop IgGl u. IgG3

2. loop IgG2

Alphal Adren. pulmonary hypertension 1. loop IgG 1 u. IgG3 alpha adrenes. Psoriasis 1. loop IgG 2

2nd loop

PAR-1 u. PAR-2 Raynaud's syndrome 2. loop IgGl endothelin IA Legend

Figure 1: Western blot of coimmunoprecipitation of the angiotensin AT ^ receptor

Lane 1 Protein A / Sepharose; 2 pre-eclamptic patient D. without cleaning; 3 KSCN eluate; 4 supernatant; 5 lysed CHO membranes; 6 lysed placental tissue.

Claims

claims

A method for detecting disease-associated autoantibodies directed against G protein-coupled receptors, characterized in that the method comprises the steps of: a) contacting body fluid with a denaturing agent, b) contacting Bringing the precipitated fraction with a biotin-comprising peptide comprising a sequence or partial sequence of a first and / or second loop of the receptor to form a mixture, c) incubating the mixture with an avidin or streptavidin-coated carrier, d) washing the materials the carrier, e) incubation of the carriers with anti-IgG antibody subclasses, wherein the anti-IgG antibody is labeled and f) performing an enzyme or color reaction.

2. Nerfahren according to claim 1, characterized in that the denaturing agent is ammonium sulfate and / or alcohol.

3. The method according to claim 1, characterized in that the carrier is a magnetic particle or an ELISA plate.

, A method according to claim 1 or 2, characterized in that the autoantibodies to a betal adrenergic receptor, a muscarinic M2 receptor, an angiotensin II ATI receptor, an alpha adrenergic receptor, an endothelin IA receptor, a PAR-1, PAR-2 and / or PAR-3 are directed.

5. The method according to any one of claims 1 to 4, characterized in that the directed against the beta-1-adrenergic receptor autoantibodies with dilated cardiomyopathy, Chagas' cardiomyopathy and / or myocarditis, which against the muscarinic M2 receptor with the autoantibodies and / or Chagas' cardiomyopathy, the autoantibodies directed against the angiotensin II ATI receptor, with pre-eclampsia, humoral renal rejection and / or malignant hypertension, autoantibodies directed against the alpha adrenergic receptor and essential hypertension , refractory hypertension, pulmonary hypertension and / or psoriasis and / or autoantibodies directed against endothelin IA receptor, PAR-1, PAR-2 and / or PAR-3 are associated with Raynaud's syndrome.

6. The method according to any one of the preceding claims, characterized in that in the detection of dilated cardiomyopathy, myocarditis, essential hypertension, refractory hypertension, pulmonary hypertension and / or psoriasis, the peptide is used which comprises a sequence or partial sequence of the first and / or second loop and in Chagas' cardiomyopathy, dilated cardiomyopathy, humoral renal rejection, and / or Raynaud's syndrome involves the peptide comprising a sequence or partial sequence of the second loop.

7. The method according to any one of the preceding claims, characterized in that - the autoantibodies associated with the dilated cardiomyopathy are brought into contact with the first and / or second loop of the betal adrenergic receptor,

the autoantibodies associated with Chagas' cardiomyopathy are contacted with the peptide comprising a sequence or partial sequence of the second loop of the betal adrenergic receptor; the autoantibody associated with the myocarditis with the peptide comprising a sequence or partial sequence of the first and / or second Loops of the beta-adrenergic receptor are brought into contact

the autoantibodies associated with the dilated cardiomyopathy are brought into contact with the peptide comprising a sequence or partial sequence of the second loop of the muscarinic M2 receptor, the autoantibodies associated with Chagas' cardiomyopathy are contacted with the peptide comprising a sequence or partial sequence of the second loop of the M2 muscarinic receptor, the autoantibodies associated with the pre-eclampsia with the peptide comprising a sequence or partial sequence of the second loop of the angiotensin II AT1 receptor, the autoantibodies associated with the humoral kidney rejection are contacted with the peptide comprising a sequence or partial sequence of the second loop of the angiotensin II ATI receptor, the autoantibodies associated with the malignant hypertension comprising the peptide Sequence or partial sequence of the second loop of the angiotensin II ATI receptor are brought into contact, which are associated with the essential hypertension autoantibody associated with the peptide comprising a sequence or partial sequence of the first and / or second loop of the alpha adrenergic receptor, the autoantibodies associated with refractory hypertension are contacted with the peptide comprising a sequence or partial sequence of the first and / or second loop of the alpha adrenergic receptor which comprises autoantibodies associated with pulmonary hypertension with the peptide comprising a sequence or partial sequence of the autoantibody first and / or second loops of the alpha adrenergic receptor are brought into contact, associating the psoriasis-associated autoantibodies with the peptide comprising a sequence or subsequence of the first and / or second loop of the alpha adrenergic receptor,

- The autoantibodies associated with Raynaud's syndrome are contacted with the peptide comprising a sequence or partial sequence of the first and / or second loop of the endothelin IA receptor, PAR-1, PAR-2 and / or PAR-3.

8. The method according to any one of the preceding claims, characterized in that

the IgG subclasses are IgG1, IgG2, IgG3 and / or IgG4 subclasses.

9. The method according to any one of the preceding claims, characterized in that

in the case of dilated cardiomyopathy, the IgG3 and / or IgG4 subclasses are used, if the

Peptide comprises a sequence or partial sequence of the first loop, and / or the IgG1 subclass, if the peptide comprises a sequence or partial sequence of the second loop, - Chagas' cardiomyopathy uses the IgG1, IgG2, IgG3 and / or IgG4 subclasses,

in the case of myocarditis, the IgG3 and / or IgG4 subclasses are used if the peptide comprises a sequence or partial sequence of the first loop and / or the IgG1 subclass, if the peptide comprises a sequence or partial sequence of the second loop,

- the IgG3 subclass is used in preeclampsia, - the IgGl and IgG3 subclass is used in humoral kidney rejection,

- in the case of malignant hypertension the IgGl and / or IgG3 subclass is used, in the case of essential hypertension the IgGl and / or IgG3 subclasses are used, if the peptide comprises a sequence or partial sequence of the first loop and / or the IgG2 subclass is used, if the peptide comprises a sequence or partial sequence of the second loop, - the refractory hypertension uses the IgG1 and / or IgG3 subclasses, if the peptide comprises a sequence or partial sequence of the first loop and / or the IgG2 subclass is used, if the peptide comprises a sequence or subsequence of the second loop,

- the IgGl, IgG2, IgG3 and / or IgG4 subclasses are used in pulmonary hypertension,

- in psoriasis the IgGl, IgG2, IgG3 and / or IgG4 subclasses are used and / or - in which Raynaud's syndrome the IgGl subclass is used.

10. The method according to any one of the preceding claims, characterized in that the autoantibodies are concentrated or purified before detection.

11. Method according to the preceding claim, characterized in that the concentration or purification of the autoantibodies comprises the following steps: a) obtaining an IgG fraction from body fluid, b) contacting the obtained IgG fraction with a peptide which a subsequence of a first or second loop of a G-protein coupled receptor, whereby a mixture is recovered, c) incubation of the mixture with a carrier which is washed and concentrated and d) eluting the autoantibodies from the concentrated carrier.

12. Nerfahren according to any one of the preceding claims, characterized in that the peptide comprising the sequence or partial sequences of the first and / or second loop is selected from the group comprising EYGSFF, SFFCEL, ARRCYND, PKCCDF, AESDE, CYIQFF, EDGECY, NRTVEDGECYIQFFSNAANTFGTAI, AFHYESQ, EΝTΝIT, FWAFGR, GRAFCDN, ITEEAGY, ERFCGI, GRIFCD and / or ITTCHDVL.

13. Nerfahren according to the preceding claim, characterized in that the peptide comprises amino groups, amides, acetyl groups, biotin groups, markers, spacers, linkers, GKK and / or SGKK.

4. The method according to the preceding claim, characterized in that the linker and / or the spacer are selected from the group comprising α-aminocarboxylic acids and their homo- and hetero-oligomers; et, ω-aminocarboxylic acids and their branched homo- or hetero-oligomers; other amino acids and the linear and branched homo- or hetero-oligomers; Amino-oligoalkoxy-alkylamines;

Maleimidocarboxylic acid derivatives; Oligomers of alkylamines; 4-alkylphenyl derivatives; 4-oligoalkoxyphenyl or 4-oligoalkoxyphenoxy derivatives; 4-oligoalkylmercaptophenyl or 4-oligoalkylmercaptophenoxy derivatives; 4-oligoalkylaminophenyl or 4-oligoalkylaminophenoxy derivatives; (Oligoalkylbenzyl) phenyl or 4-Oligoalkylbenzyl) -phenoxy derivatives and 4-Oligo- alkoxybenzyl) phenyl or 4-Oligoalkoxybenzyl) - phenoxy derivatives; Trityl derivatives; Benzyloxyaryl or benzyloxyalkyl derivatives; Xanthen-3-yl-oxyalkyl derivatives; (4-alkylphenyl) or ω- (4-alkylphenoxy) alkanoic acid derivatives; Oligoalkyl-phenoxyalkyl or oligoalkoxy-phenoxyalkyl derivatives; Carba at derivatives; amines; Trialkylsilyl or dialkylalkoxysilyl derivatives; Alkyl or aryl derivatives and / or combinations thereof.

15. Nerfahren according to any one of the preceding claims, characterized in that the peptide is modified by deletion, addition, substitution, translocation, inversion and / or insertion.

16. A peptide selected from the group comprising EYGSFF, SFFCEL, ARRCYND, PKCCDF, AESDE, CYIQFF, EDGECY, VRTVEDGECYIQFFSNAAVTFGTAI, AFHYESQ, ENTNIT, FWAFGR, GRAFCDV, ITEEAGY, ERFCGI, GRIFCD and ITTCHDVL. for use as a medicinal agent.

17. Peptide according to the preceding claim, characterized in that the peptide of autoantibodies of patients is bound to one of the following diseases: dilated cardiomyopathy, Chagas' cardiomyopathy, myocarditis, preeclampsia, humoral renal rejection, malignant hypertension, essential hypertension, refractory hypertension, pulmonary Hypertension, psoriasis and / or Raynaud's syndrome.

18. Peptide according to claim 15 or 16, characterized in that the peptide is immobilized.

19. Peptide according to the preceding claim, characterized in that the peptide is bound to a solid phase.

20. Recognition molecule directed against the peptide according to one of claims 16 to 19.

21. Recognition molecule according to the preceding claim, characterized in that it is an antibody, a lectin, an antisense construct and / or a chelator.

22. A pharmaceutical composition comprising a peptide according to any one of claims 16 to 19 and / or a

Recognition molecule according to claim 20 or 21.

23. A kit comprising a peptide according to any one of claims 16 to 19, a recognition molecule according to claim 20 or 21 and / or a pharmaceutical composition according to claim 22, optionally with an instruction for combining the contents of the kit and / or for providing a formulation.

24. Chromatography device comprising peptides according to one of claims 16 to 19 and / or recognition molecules according to claim 20 or 21.

25. Device according to the preceding claim, characterized in that the peptides are bound to a solid phase.

26. Use of the peptides according to any one of claims 16 to 19 and / or recognition molecules according to claim 20 or 21 and / or a pharmaceutical composition according to claim 22 and / or a kit according to claim 23 and / or a device according to claim 24 to 25 for the prophylaxis , Diagnosis, therapy, follow-up and / or follow-up of autoimmune diseases selected from the group comprising dilated cardiomyopathy, Chagas' cardiomyopathy, myocardiopathy tis, pre-eclampsia, humoral kidney rejection, malignant hypertension, essential hypertension, refractory hypertension, pulmonary hypertension, psoriasis and / or Raynaud's syndrome.

27. Use of the peptides according to any one of claims 16 to 19 and / or recognition molecules according to claim 20 or 21 and / or a pharmaceutical composition according to claim 22 and / or a kit according to claim 23 and / or a device according to claim 24 to 25 for the preparation of a medicament for the treatment of autoimmune diseases selected from the group comprising dilated cardiomyopathy, Chagas' cardiomyopathy, myocarditis, preeclampsia, humoral kidney rejection, malignant hypertension, essential hypertension, refractory hypertension, pulmonary hypertension, psoriasis and / or Raynaud's syndrome.

28. Use of the peptides according to any one of claims 16 to 19 and / or recognition molecules according to claim 20 or 21 and / or a pharmaceutical composition according to claim 22 and / or a kit according to claim 23 and / or a device according to claim 24 to 25 for screening of medicines.

29. Use of the peptides according to any one of claims 16 to 19, characterized in that against betal adrenergic receptor, muscarinic M2 receptor, Angeotensin II ATI receptor, alpha adrenergic receptor, endothelin IA, PAR-1, PAR-2 and / or PAR-3-directed autoantibodies are detected, bound, complexed and / or neutralized.

30. A method of treating an autoimmune disease selected from the group comprising dilated cardiomyopathy, Chagas' cardiomyopathy, myocarditis, pre-eclampsia, humoral kidney rejection, malignant hypertension, essential hypertension, refractory hypertension, pulmonary hypertension, psoriasis , Raynaud's syndrome by binding and / or removing autoantibodies by means of solid phase-bound peptides according to any of claims 16 to 19.

31. The method according to the preceding claim, characterized in that the autoantibodies in the dilated cardiomyopathy against beta-adrenergic receptors, in Chagas' cardiomyopathy against beta-1-adrenergic receptors, in the myocarditis against beta-1-adrenergic receptors, in the dilated cardiomyopathy against muscarinic M2 receptors, in Chagas' cardiomyopathy against muscarinic M2 receptors, in preeclampsia against angiotensin II ATI receptors, in humoral renal rejection against angiotensin II ATI receptors, in malignant hypertension to angiotensin II ATI receptors , in essential hypertension to alpha adrenergic receptors, in refractory hypertension to alpha adrenergic receptors, in pulmonary hypertension to alpha adrenergic receptors, in psoriasis against alpha adrenergic receptors, the autoantibodies in Raynaud's disease are directed against endothelin IA, PAR-1, PAR 2 and / or PAR-3.