WO2008151847A1

WO2008151847A1 - Autoantibody binding peptides and their use for the treatment of vascular diseases

Info

Publication number: WO2008151847A1
Application number: PCT/EP2008/005044
Authority: WO
Inventors: Gerd Wallukat; Thomas Harrer; Dandel Michael
Original assignee: Max-Delbrück-Centrum für Molekulare Medizin; Deutsches Herzzentrum Berlin
Priority date: 2007-06-13
Filing date: 2008-06-13
Publication date: 2008-12-18

Abstract

The application relates to peptides and nucleic acid molecules encoding peptides which interact with autoantibodies associated with coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma, to the peptides themselves, to a pharmaceutical composition comprising said nucleic acid molecules and peptides, and to the use of said peptides - especially in apheresis - for the treatment of coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma. The peptides of the application are minimally capable of binding the endothelin-1-receptor and for selected diseases additionally the protease-activated- receptor 1, 2 or 3 or the alphal-adrenergic-receptor.

Description

Autoantibody binding peptides and their use for the treatment of vascular diseases

Description

The invention relates to peptides, nucleic acids encoding for such peptides and the use of the peptides and nucleic acids in detection and treatment of diseases relating to the group of vascular diseases.

The invention relates more specifically to peptides being epitopes for antibodies and nucleic acid molecules encoding peptides which interact with autoantibodies associated with vascular diseases to the peptides themselves, to a pharmaceutical composition comprising said nucleic acid molecules and peptides, and to the use of said peptides - especially in apheresis - for the treatment of vascular diseases.

An important regulatory mechanism enabling the organism to cope successfully with microorganisms, and parasites is to provide cellular and humoral substances, such as antibodies. Above all, the function of antibodies is based on the capability of dis- tinguishing between "foreign" and "self. That is, the organism itself produces antibodies which are designed in such a way that endogenous structures such as tissues or organs are not attacked, but bind to bacteria, parasites or other exogenic components, thus initiating or supporting immunological reactions.

During biochemical development of an antibody response in the organism, somatic hypermutations of antibody-producing B cells occur during T cell-dependent immune response. Such mutations selectively involve immunoglobulin genes, being essential to the formation of precursor cells of antibody-forming cells with high affinity. It should be noted in this context that such mutation is a normal and essentially non- pathogenic process. However, mutations may also give rise to the formation of highly affine autoantibodies, thus doing damage to the organism.

Autoantibodies are closely associated with the development of autoimmune diseases or causally responsible for such diseases. Antibody-mediated autoimmune diseases also include diseases such as rheumatism or lupus erythematosus. Numerous autoimmune diseases, being highly inconvenient to affected persons, relate to important biochemical cycles in the organism. Coming from this state of the art, the object of the present invention is to provide means and methods for the treatment of autoantibody related diseases, especially vascular diseases.

The present invention is based on the discovery that in patients suffering from coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma are autoantibodies present binding to the Endothelin-I-Receptor (ETA). For coronary arterial occlusion, angina pectoris and Raynaud syndrome / scleroderma are additionally autoantibod- ies present binding the Protease-Activated-Receptor (PAR) 1 , 2 and/or 3. Finally, patients suffering from pulmonary hypertension or occlusion of peripheral arterial vessels the α1-Adrenergic-Receptor is bound by autoantibodies.

Consequently the present invention provides a peptide as part of an epitope binding to an autoantibody, whereas the peptide comprises at least one of the following amino acid sequences

a) MLNATSK, SKFMEFY, EQHKTCM, MLNATSKSKFMEFY, or

b) KSTANLM, YFEMFKS, MCTKHQE , YFEMFKSKSTANLM or

c) a combination of one or more sequences of a) and b).

Such a peptide is able to bind to the ETA. Additionally a peptide of the present in- vention comprises whether the amino acid sequences chosen from the group APEDET₁ WKEPAP or PPDERF, or from the group ITTCHDVL, LNITTCHD or PALNITTC. Such peptides are able to bind additionally the PAR 1 , 2 or 3 or the α1- Adrenergic-Receptor.

In another aspect, the invention provides a peptide characterized by an epitope for autoantibodies directed against endothelin-l receptor, protease-activated receptor 1 , 2 or 3 and/or α1 -adrenergic receptor.

A peptide according to the present invention is characterized in that is selected from the group consisting of: a) a peptide comprising at least one of the amino acid sequences mentioned above; b) a peptide consisting of an amino acid sequence having sufficient homology to be functionally analogous to an amino acid sequence in accordance with a);

c) a peptide of an amino acid sequence a) or b) which is modified by deletions, additions, substitutions, translocations, inversions and/or insertions and functionally analogous to an amino acid sequence in accordance with a) or b);

d) a peptide of amino acid sequence a), b) or c) which is modified by branch or extension with the same or another peptide of amino acid sequence a), b) or c) to form a homooligomeric or heterooli- gomeric peptide.

Such peptides are further characterized in that the amino acid sequence specified under b) has at least 40% homology to any of the amino acid sequences specified under a). Especially an amino acid sequence specified under b) has at least 60%, preferably 70%, more preferably 80%, especially preferably 90% homology to any of the amino acid sequences specified under a). Another objective of the present invention is the use of the peptides of the invention as medical active substances. This includes the us of the peptides for preparation of a medicament for the treatment of patients suffering from coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma. As previously mentioned the peptides bind specifically to autoantibodies occurring in patients suffering from the listed diseases.

Furthermore the peptides of the invention are intended for being immobilized and/or fixed to magnetic, paramagnetic and/or non magnetic nanoparticles or being bound to a solid phase.

The present invention includes peptides that are inear and branched as well as in cyclic form, whereas the peptide ring closure is effected through disulfide bridging when two cysteines are present, or through amide cyclization, which is optionally effected through side chains, through the C and N termini or through a combination of these three possibilities. Additionally chemical modifications of the peptides are intended like additional amino groups, amides, acetyl groups, biotin groups, markers, spacers and/or linkers.

A further objective of the present invention is an isolated nucleic acid molecule selected from the group comprising: a) a nucleic acid molecule comprising a nucleotide sequence which encodes at least one peptide selected from the group consisting of peptides of claim 1 , 2 or 3;

b) a nucleic acid molecule which is complementary to a nucleotide sequence in accordance with a);

c) a nucleic acid molecule which undergoes hybridization with a nucleotide sequence of a) or b) under stringent conditions;

d) a nucleic acid molecule comprising a nucleotide sequence having sufficient homology to be functionally analogous to a nucleotide sequence of a), b) or c);

e) a nucleic acid molecule which, as a consequence of the genetic code, is degenerated into a nucleotide sequence of a) through d); and

f) a nucleic acid molecule of a nucleotide sequence of a) through e) which is modified by deletions, additions, substitutions, transloca- tions, inversions and/or insertions and functionally analogous to a nucleotide sequence of a) through e).

Accordingly, the invention involves the surprising teaching that the nucleic acid molecules of the invention, as well as the peptides encoded by same, can be used for diagnosis and therapy of vascular diseases like coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma. More specifically, the biological structures, preferably peptides, encoded by said nucleic acid molecules can be used in various forms in the prophylaxis, diagnosis, therapy, follow-up and/or aftercare of diseases like coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma. In a preferred embodiment of the invention the nucleic acid molecule which has sufficient homology to be functionally analogous to the nucleic acid sequence in accordance with a), b) and/or c) has at least 40% homology. In the meaning of the invention the term "to be functionally analogous to the above-mentioned nucleotide se- quences or to the sequences hybridizing with said nucleotide sequences" means that the homologues exhibit a behavior in diseases of coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma., such that reliable and effective use thereof is possible in diagnosis and/or therapy of such diseases or pathogenic conditions as- sociated with said diseases. Functionally analogous sequences in the meaning of the invention are all those sequences which can be identified as equally effective by a person skilled in the art, using routine tests.

In another advantageous embodiment of the invention the nucleic acid molecule has at least 60%, preferably 70%, more preferably 80%, especially preferably 90% homology to the nucleic acid molecule in accordance with d).

In another preferred embodiment of the invention the nucleic acid molecule is a genomic DNA, a cDNA and/or an RNA.

The invention also relates to a vector comprising a nucleic acid molecule of the invention and, in addition, to a host cell comprising said vector.

In a preferred fashion the invention also relates to a peptide encoded by a nucleic acid molecule of the invention and by the preferred functionally analogous nucleic acid molecules. Surprisingly, the peptide of the invention can be used in the diagnosis and/or therapy of diseases associated with vascular diseases. More specifically, the peptides of the invention are bound by autoantibodies in patients suffering from coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma.

That is, the invention relates to all peptides encoded by the nucleic acid molecules of the invention, preferably those having at least 60%, preferably 70%, more preferably 80%, especially preferably 90% homology to the nucleic acid molecule in accordance with d). Obviously, said functionally analogous nucleic acid molecules can be modified by deletion, addition, substitution, translocation, inversion and/or insertion in such a way that the peptides encoded by same interact with autoantibodies associated with coronary arterial occlusion, angina pectoris, pulmonary hy- pertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma.

By virtue of the teaching of the invention, a person skilled in the art will be capable of generating other equivalent peptides functionally analogous to peptides having the sequence of claim 1 , preferred MLNATSK, SKFMEFY, EQHKTCM, MLNATSKSKFMEFY, APEDET, WKEPAP, PPDERF, ITTCHDVL, LNITTCHD or PALNITTC. Of course, it is also possible to use peptides comprising the above- mentioned structures. Obviously, however, the peptides (as a substance, molecule or a mixture of substances) of the invention would not be selected in a way so as to completely represent the naturally occurring human endothelin-l receptor, protease activated receptors 1 and 2 and/or α1 -adrenergic receptor. With reference to the methods of the invention and the uses of the invention, it can be advantageous to use endothelin-1 receptor, protease-activated receptor 1 , 2 or 3 and α1 -adrenergic receptor or parts thereof as specific ligands for removing significant portions of the immunoglobulin from plasma taken from a patient, suffering from coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma. This means that the substance claims refer to specific parts of the receptor (the peptides) and the use or method claims refer to the use of the whole receptor for the treatment of coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma. A person skilled in the art knows the definition of a ligand of the invention. Ligands or specific ligands in the context of the invention are means which interact specifically with immunoglobulins, preferred autoantibodies, more preferred autoantibodies directed against endothelin-1 receptor, protease-activated receptor 1 , 2 or 3 and α1 -adrenergic receptor, most preferred against the second extracellular loop of said receptors. In another preferred embodiment of the invention, the ligands or specific ligands consist of the peptides of the invention.

In another preferred embodiment, the invention relates to the use of a specific ligand for human immunoglobulin in the manufacture of a column having said ligand coupled thereto for the treatment of a patient suffering from coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma, said treatment comprising passing plasma of the patient over the column under conditions which effect the binding of said specific ligand to immunoglobulin in the patient's plasma, thereby removing a significant portion of the immunoglobulin from the patient's plasma and reinfusing the plasma to the patient. In the context of the invention, the term "significant portion" is directed to an amount of autoantibodies whose removal from the plasma leads to an improvement of the patient's condition or to the non-appearance of a further aggravation. This improvement of the patient's condition relates to at least one of the aspects of coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma, known to a person skilled in the art. In a preferred embodiment of the invention, the specific ligand is the endothelin-l receptor, protease activated receptors 1 and 2 and/or α1 -adrenergic receptor or the peptides of the invention. These aspects of the invention are based on the conclusion made by the inventors, that coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma is caused by autoantibodies, especially by agonistic autoantibodies.

The invention also encompasses the use of a specific ligand in the manufacture of a column for extracorporal removal of autoantibodies directed against coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma structures by removing immonglobu- lins of any or all classes and subclasses, for the treatment of coronary arterial occlu- sion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma. Such removal can be accomplished by using any specific ligands for human immunoglobulin coupled to the IA column. Such ligands include polyclonal and monoclonal anti-human immunoglobulin antibodies, fragments of such antibodies (FAB1 , FAB2), recombinant antibodies or pro- teins, synthesized peptides, Protein A and Protein G.

Another aspect of the invention is therefore also the essentially unspecific removal of immunoglobulins from plasma taken from a patient suffering from coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arte- rial vessels, Raynaud syndrome and scleroderma and reinfusing said plasma to the patient. The reinfused plasma may also contain those immunoglobulins which induce a positive effect in the context of the patient's immune system.

The invention also encompasses the use of more specific ligands in the manufac- ture of a column for extracorporal removal of autoantibodies which are directed against the second loop of the endothelin-l receptor, second loop of protease activated receptors 1 and 2 and/or second loop of α1 -adrenergic receptor. This means that the removal of all antibodies or the removal of all autoantibodies or the removal of all autoantibodies directed against the second loop of the endothelin-l receptor, second loop of protease activated receptors 1 and 2 and/or second loop of α1- adrenergic receptor is part of the invention.

Consequently, the peptides in the meaning of the invention can be designed in such a way and may include such a number of additional amino acids, spacers or other structures that they are suitable for interaction with the antibodies, and preferably in such a way that they represent an epitope for the latter.

Accordingly, the peptides of the invention are not restricted to the sequences MLNATSK, SKFMEFY, EQHKTCM, MLNATSKSKFMEFY, APEDET, WKEPAP₁ PPDERF, ITTCHDVL, LNITTCHD or PALNITTC, but rather, the peptides are preferably antibody epitopes essentially including the above-mentioned or functionally analogous sequences, so that they represent and characterize the epitope in such a way that the antibodies would undergo specific interaction with them. Therefore, under particular conditions the terms "epitope" and "peptide" may also be used synonymously.

Furthermore, it is possible to replace single amino acids or groups of amino acids without adversely affecting the activity of the peptides with respect to accomplishing the object of the present invention. For replacement of such amino acids, reference is made to appropriate standard textbooks of biochemistry and genetics. Various ways of preparing peptides have been disclosed in the prior art. Peptides designed starting from the peptides of the invention using such methods are included in the teaching of the invention. For example, one way of generating functionally analogous peptides has been described in PNAS USA 1998, Oct. 13, 95, 12179-84; WO 99/6293 and/or WO 02/38592; and the above teachings are hereby incorporated in the disclosure of the invention.

A second way of generating peptide sequences starting from the peptides of invention is the subtitutional analysis (MoI. Diversity 2004, 8, 281-290; J. of Immunol. Methods 2002, 267, 37-51). The substitutional analysis is carried out by systematical replacement of each amino acid by all amino acids/building blocks of your interest. The activity of these peptides/constructs shows the ability of those substitutions. The resulting sequences could have improved properties i.e. higher stability and/or lower hydrophicity). Another method to generate peptide structure anologues is the synthesis of retro- inverso peptides. Using this method the L-amino acids of the peptides of invention were substituted by D-amino acids with simultaneously inversion of the direction of the peptide bounds. This method is published e.g. in Biochemistry 2001 , Apr. 19, 40, 5720-27; Biopolymers (Peptide Science) 2005, Feb. 23, 80, 67-84. The generated peptides are structurally analogue to the peptides of the invention. But due to the use of D-amino acids, these peptides are stable against proteolytic degradation. One example of such peptide sequence is the sequence: naycniG. This sequence is the retro-inverso sequence of a peptide of the invention, in which several amino ac- ids were replaced by other amino acids with similar properties. The small letters indicate D-amino acids. Glycine (G) doesn't exist in two different D- or L-structures.

That is, all peptides, peptide fragments or structures comprising peptides generated using the methods mentioned above - starting from the peptides of the invention - are peptides of the invention, provided they accomplish the object of the invention and, in particular, interact with the pathogenic autoantibodies. For example, these autoantibodies - via dimerization - can be agonistic autoantibodies activating receptors. Some preferred receptors will be described below.

For example, the peptide sequences can be a natural component of the s second loop of the endothelin-l receptor, second loop of protease activated receptors 1 and 2 and/or second loop of α1 -adrenergic receptor. Surprisingly, the autoantibodies specifically interact with the second loop of the endothelin-l receptor, second loop of protease activated receptors 1 and 2 and/or second loop of α1 -adrenergic receptor. Accordingly, the invention also relates to the surprising teaching that the major epitope of the autoantibodies associated with diseases like coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma are peptides having the amino acid sequences of claim 1. Accordingly, the autoantibodies can be antibodies acting as agonistic autoantibodies, i.e., these autoantibodies are capable of activating the corresponding receptors by binding thereto. As a result of such receptor activation, various diseases or pathogenic changes can be induced and/or accompanied. For example, it is possible that activation of receptors modulates the production of messenger molecules. However, the autoantibodies may also damage receptors of cells which are responsible for essentially normal blood flow in limbs. In contrast to the effect of agonistic autoantibodies, the function of attacking and damaging the corresponding target by chronic stressing - usually observed with antibodies - is the predominant one in non-agonistic autoantibodies. However, this does not exclude activation or deactivation of the receptors.

In a preferred embodiment of the invention the peptide additionally comprises amino groups, amides, acetyl groups, biotin groups, markers, spacers and/or linkers.

By virtue of such structures, the peptide can be used with advantage in various fields of diagnosis and therapy of autoimmune diseases like coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial ves- sels, Raynaud syndrome and scleroderma. Various ways of modifying peptides for various applications are well-known to those skilled in the art. If, for example, the peptides are envisaged to be administered as a drug, the structure of the peptides has to be changed in an specific fashion, as is well-known to those skilled in the art. However, the peptides may also be bound to the supporting material of an affinity column in order to be used in the purification of body fluids, especially blood; binding of the peptides to a matrix requires specific structural modifications of the peptides of the invention, and such modifications are also well-known to those skilled in the art or can be determined using routine tests.

These amino acids are representing gene encoded peptides with similar properties as well as retro-inverso peptides with D-amino acids (small letters) with similar properties, available by biochemical or chemical peptide synthesis.

As is well-known to those skilled in the art, some amino acids have analogous phys- icochemical properties so that these amino acids advantageously can be replaced by each other. For example, these include the group of nonpolar (hydrophobic) amino acids (a) glycine, alanine, valine, leucine and/or isoleucine; or the hydroxy amino acids (b) serine, threonine and/or tyrosine, the amides of amino dicarboxylic acids (c) asparagine and glutamine, the amino dicarboxylic acids (d) aspartic acid and glutamic acid; the basic amino acids (e) lysine, arginine and/or ornithine as well as the group of aromatic amino acids (f) phenylalanine, tyrosine and/or tryptophan. Another aspect is the replacement of amino acids by structural similar amino acids. For example this is the case in the group with a β-functional group (g) cysteine, methionine, serine, α- aminobutyric acid and selenocysteine as well as the turn- inducing group (h) praline, 1-amino-2-carboxy cyclohexane, pipecolic acid and an ortho-aminobenzoic acid. Amino acids within one and the same group (a-h) can be replaced with one another. In all cases, peptide sequences will have a sufficient homology to be an analogous to an amino acid sequence of the peptides of the in- vention. Furthermore, the amino acids can be replaced by modified amino acids or specific enantiomers.

It is obviously known to a person skilled in the art that it is possible to provide func- tionally analogous means for the peptides and nucleic acids of the invention. In the meaning of the invention, "functionally analogous means" are means which are functionally equivalent or equivalent to the peptides and nucleic acids of the invention. In order to check whether a sequence is functionally equivalent (= equivalent) to the peptides or nucleic acids of the invention, one must check whether the functionally analogous/equivalent sequence of the peptide or of the nucleic acid produces essentially the same function in essentially the same way with essentially the same outcome. That is, the outcome produced with the peptides or nucleic acids of the invention may also be produced with the functionally analogous/equivalent means. A person skilled in the art can therefore easily and quickly verify whether a functionally analogous/equivalent sequence provided by them is covered by the invention. The functionally analogous sequence is covered by the invention if it may be used for solving the problem of the invention and if the solving of the problem of the invention can be carried out in essentially the same way as is claimed by the invention. While the invention has been described in terms of preferred embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions and changes may be made without departing from the spirit thereof. Accordingly, it is intended that the scope of the present invention be limited solely by the scope of the claims of the invention, including equivalents thereof. Therefore, as will be apparent to those skilled in the art to which the invention is addressed, the present invention may be embodied in forms other than those specifically disclosed without departing from the spirit or essential characteristics of the invention. The particular embodiments of the present invention as described are therefore to be considered in all respects as illustrative and not restrictive. The scope of the present invention is as set forth in the appended claims rather than being limited to the examples contained in the foregoing description.

It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the claims of the invention define the scope of the invention and that the method and apparatus within the scope of these claims and their equivalents be covered thereby. Those skilled in the art will therefore understand all those molecules as equivalent which he may apply without this having an effect on the solution of the problem of the invention. They know that the claimed sequences must not be modified by changes, substitutions or deletions in a way that they cannot be used for solving the problem of the invention.

In another preferred embodiment the peptide comprises a linker and/or a spacer selected from the group comprising α-aminocarboxylic acids as well as homo- and heterooligomers thereof, α,ω-aminocarboxylic acids and branched homo- or hete- rooligomers thereof, other aliphatic and/or aromatic amino acids, as well as linear and branched homo- or heterooligomers (peptides); amino-oligoalkoxyalkylamines; maleinimidocarboxylic 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, as well as 4- (oligoalkoxybenzyl)phenyl or 4-(oligoalkoxybenzyl)phenoxy derivatives; trityl derivatives; benzyloxyaryl or benzyloxyalkyl derivatives; xanthen-3-yloxyalkyl derivatives; (4-alkylphenyl)- or ω-(4-alkylphenoxy)alkanoic acid derivatives; oligoalkylphenoxyal- kyl or oligoalkoxyphenoxyalkyl derivatives; carbamate derivatives; amines; trialkyl- silyl or dialkylalkoxysilyl derivatives; aliphatic or aromatic mono- or oligomercapto derivatives; alkyl or aryl derivatives and/or combinations thereof; other possible structures have been described in EP 1 214 350 which hereby is incorporated in the disclosure of the invention. The following sequence is an example: GLN- Cys(Maleinimidopropionic acid)-TYAN. In this sequence a peptide of the invention several amino acids were replaced by other with similar properties as described above. Additionally, the SH group of the cysteine reacts with the double bound of the maleinimide group to form a covalent bound to a linker molecule. This linkage is could couple to another molecule or a solid phase.

Also claimed of the invention are solid phases for affinity chromatography or solid phase extraction constisting of organic, inorganic, synthetic polymers or of mixed polymers, preferably cross-linked agarose, cellulose, silica gel, polyamide and polyvinyl alcohols, which are optionally chemically activated, with peptides of the inven- tion immobilized on the surface of the solid phase.

In the solid phase of the invention, the peptides are bound to the solid support phase preferably covalently or by adsorption. In another preferred embodiment of the solid phases of the invention, the peptides are distanced from the support surface by linkers/spacers.

The invention relates to a method for the adsorption of immunoglobulins to a solid phase, wherein the peptides of the invention are bound to a solid phase usual in affinity chromatography or to mobile solid phases, and the immunoglobulin- containing samples to be adsorbed are contacted, in particular, with the solid phases of the invention.

Preferably, the method for the adsorption of immunoglobulins is performed on a solid phase, wherein the peptides of the invention are bound to a solid phase usual in affinity chromatography or to mobile solid phases, and the immunoglobulin- containing samples to be adsorbed are anticoagulant-treated human blood plasma or human whole blood and are contacted with the solid phases of the invention.

The method of the invention is suitable, in particular, for the adsorption of immunoglobulins from immunoglobulin-containing samples which contain auto-antibodies related to autoimmune diseases, especially rheumatoid diseases, multiple sclerosis, myasthenia gravis and other auto-antibody-mediated diseases.

The method of the invention is advantageously performed with a device of the invention for removing immunoglobulins from immunoglobulin-containing samples on solid phases, wherein the device contains a solid phase of the invention, and means for the entry of immunoglobulin-containing samples are provided.

The invention also relates to the use of the peptides of the invention, the solid phases of the invention and the devices of the invention for removing immunoglobulins from immunoglobulin-containing samples.

Of another particularly preferred embodiment of the invention, the peptide is selected from the group comprising:

a) a peptide essentially containing the amino acid sequence of claim 1 ;

b) a peptide comprising an amino acid sequence having sufficient homology to be functionally analogous to an amino acid sequence in accordance with a); c) a peptide of an amino acid sequence a) or b) which is modified by deletions, additions, substitutions, translocations, inversions and/or insertions and functionally analogous to an amino acid sequence in accordance with a) or b).

d) a peptide of amino acid sequence a), b) or c) which is modified by branch or extension with the same or another peptide of amino acid sequence a), b) or c) to form a homooligomeric or heterooligomeric peptide. The coupling could result by a direct bond to amino acids of the sequences or by an indirect bond to amino acids of the sequences using a linker or spacer.

In another preferred embodiment of the invention the peptide is used or employed as a therapeutic active substance. In the meaning of the invention, use as a therapeutic active substance means use of the peptide in the entire field of medicine.

In another particularly preferred embodiment of the invention the peptide is bound by specific antibodies of patients with coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma, especially by autoantibodies. At a defined quantity ratio of peptide and autoantibody, which is well-known to those skilled in the art, autoantibody-peptide complexes will form which, for example, undergo precipitation or exhibit specific reaction behavior in a way so as to allow elimination or reduction of the autoantibodies.

In another preferred embodiment of the invention the peptide, in particular, is immobilized. In the meaning of the invention, immobilization is understood to involve various methods and techniques to fix the peptides on specific carriers, e.g. of WO 99/56126 or WO 02/26292. For example, immobilization can serve to stabilize the peptides so that their activity would not be reduced or adversely modified by biological, chemical or physical exposure, especially during storage or in single-batch use. Immobilization of the peptides allows repeated use under technical or clinical routine conditions; furthermore, a sample - preferably blood components - can be reacted with at least one of the peptides of the invention in a continuous fashion. In particular, this can be achieved by means of various immobilization techniques, with binding of the peptides to other peptides or molecules or to a carrier proceeding in such a way that the three-dimensional structure - particularly in the active center mediat- ing the interaction with the autoantibodies - of the corresponding molecules, especially of said peptides, would not be changed. Advantageously, there is no loss in specificity to the autoantibodies of the patient as a result of such immobilization. In the meaning of the invention, three basic methods can be used for immobilization:

(i) Crosslinking: in crosslinking, the peptides are fixed to one another without adversely affecting their activity. Advantageously, they are no longer soluble as a result of such crosslinking.

(ii) Binding to a carrier: binding to a carrier proceeds via adsorption, ionic binding or covalent binding, for example. Such binding may also take place inside microbial cells or liposomes or other membranous, closed or open structures. Advantageously, the peptides are not adversely affected by such fixing. For example, multiple or continuous use of carrier-bound peptides is possible with advantage in clinics in diagnosis or therapy.

(iii) Inclusion: inclusion in the meaning of the invention especially is inclusion in a semipermeable membrane in the form of gels, fibrils or fibers. Advantageously, encapsulated peptides are separated from the surrounding sample solution by a semipermeable membrane in such a way that interaction with the autoantibodies or fragments thereof still is possible.

Various methods are available for immobilization, such as adsorption on an inert or electrically charged inorganic or organic carrier. For example, such carriers can be porous gels, aluminum oxide, bentonite, agarose, starch, nylon or polyacrylamide. Immobilization proceeds via physical binding forces, frequently involving hydrophobic interactions and ionic binding. Advantageously, such methods are easy to handle, having little influence on the conformation of the peptides. Advantageously, binding can be improved as a result of electrostatic binding forces between the charged groups of the peptides and the carrier, e.g. by using ion exchangers such as Sephadex.

Another method is covalent binding to carrier materials. In addition, the carriers may have reactive groups forming homopolar bonds with amino acid side chains. Suit- able functional side chain groups in peptides are carboxy, hydroxy and hydrosulfide groups and especially the terminal amino groups of lysines. Aromatic groups offer the possibility of diazo coupling. The surface of microscopic porous glass particles can be activated by treatment with silanes and subsequently coated with peptides. For example, hydroxy groups of natural polymers can be activated with bromo- cyanogen and subsequently coupled with peptides. Advantageously, a large number of peptides can undergo direct covalent binding with polyacrylamide resins. Inclusion in three-dimensional networks involves inclusion of the peptides in ionotropic gels or other structures well-known to those skilled in the art. More specifically, the pores of the matrix are such in nature that the peptides are retained, allowing interaction with the target molecules. In crosslinking, the peptides are converted into polymer aggregates by crosslinking with bifunctional agents. Such structures are gelatinous, easily deformable and, in particular, suitable for use in various reactors. By adding other inactive components such as gelatin in crosslinking, advantageous improvement of mechanical and binding properties is possible. In microencapsulation, the reaction volume of the peptides is restricted by means of membranes. For example, microencapsulation can be carried out in the form of an interfacial polymerization. Owing to the immobilization during microencapsulation, the peptides are made insoluble and thus reusable. In the meaning of the invention, immobilized peptides are all those peptides being in a condition that allows reuse thereof. Restricting the mobility and solubility of the peptides by chemical, biological or physical means advantageously results in lower process cost, particularly when eliminating autoantibodies from blood components.

In another preferred embodiment of the invention the peptide is bound to a solid phase. Binding the peptide to the solid phase may proceed via a spacer. All those chemical compounds can be used as spacers which have the structural and functional preconditions suitable for the function of a spacer, provided they do not modify the binding behavior in such a way that binding of the autoantibody with the peptide would be adversely affected.

The invention also relates to recognition molecules directed against the agonistic autoantibodies which interact with the peptides of the invention. These agonistic autoantibodies are directed against the endothelin-l receptor, protease activated receptors 1 and 2 and/or α1 -adrenergic receptor. The interaction or reaction or binding of the autoantibody is a specific antigen-antibody-interaction. In a preferred em- bodiement the recognition molecules are antibodies, antisense constructs and/or chelating agents and/or aptamers or Spiegelmers. The recognition molecules of the invention can be antibodies directed against the autoantibodies inducing e.g. diseases like coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma; it is also possible that the recognition molecule is a peptidomimetic. To understand what a Spiegelmer is, it is important to first explain the concept of aptamers. An aptamer is a nucleic acid structure that can bind to a target molecule conceptually similar to an antibody that recognizes an antigen (Ellington & Szostak, 1990). Based on the SELEX (Systematic Evolution of Ligands by Exponential enrichment) process (Tuerk & Gold, 1990), aptamers can be identified from huge combinatorial nucleic acids libraries of up to > 1015 different sequences. The sequences in the library are comprised of a central randomized region flanked by fixed sequences that permit amplification by polymerase chain reactions (PCR). The library size depends on the length of the random portion of the molecule, creating a diverse universe of mole- cules ready for screening. The aptamers capable of binding to a target are then selected by incubating the libraries with the immobilized target, washing extensively, then eluting the bound aptamer. After amplification by PCR, these binding molecules are re-selected, eluted and amplified. By repeating this procedure multiple times, and eluting bound aptamers under progressively more stringent conditions, the molecules with greatest affinity and specificity are selected.

Aptamers have binding characteristics similar to peptides or antibodies, with affinities in the low nanomolar to the picomolar range. However, there are several drawbacks to aptamers as useful therapeutic products. As relatively small molecules, aptamers demonstrate circulating half-lives in vivo in the order of minutes. This situation can be addressed by attaching large inert molecules to aptamers (e.g. polyethylene glycol) to reduce their elimination via the kidney and increase their presence in the circulation. Finally, aptamers, as natural nucleic acid polymers, are prone to rapid degradation by nucleases that are present in all tissues in the body. To overcome this latter problem, methods of creating nuclease-resistant molecules that retained the binding capabilities of aptamers needed to be created. The answer to this latter problem was solved by the discovery and development of Spiegelmers (Klussmann et al., 1996; Vater & Klussmann, 2003). These molecules, which are biostable aptamers, have all of the diversity characteristics of aptamers but possess a structure that prevents enzymatic degradation. While aptamers are created from the natural D-nucleotides, which are recognized by the nucleic acid degrading enzymes, a molecule synthesized as the mirror image L-oligonucleotide will not be degraded by any nuclease since there are no such enzymes in the body capable of interacting with these unnatural molecules. As such, the amplification methods used to create large aptamer libraries cannot be employed to synthesize the L-nucleic acid-containing molecules. However, in the state of the art there are known methods used to form high affinity L-oligonucleotide aptamers that possess outstanding binding affinities and functionality. These molecules are termed "Spiegelmer" from the German "Spiegel" or mirror. Based on the simple concept that if an aptamer binds its natural target, the mirror image of the aptamer will identically bind the mirror image of the natural target. If the process of aptamer selection is carried out against the mirror image target, an aptamer against this unnatural mirror image will be obtained. The corresponding mirror image nucleic acid (L-oligonucleotide) of this aptamer, or the Spiegelmer, should - and will - now bind to the natural target ligand with similar binding characteristics as the aptamer itself. More important, this Spiegelmer is now resistant to nuclease degradation. Spiegelmers possess the high affinity binding characteristics of the best aptamers and antibodies in the low nanomolar and pico- molar range, while defying enzymatic degradation that severely limits the utility of aptamers. Data indicate that Spiegelmers are stable in human plasma for over 60 hours at 37⁰C, while non-modified RNA aptamers are degraded in seconds under the same conditions. Results in animals indicate that a similar stability can be expected in vivo as well. Spiegelmers should not be confused with antisense RNAs in that they do not directly interfere with protein synthesis of their target molecules. They are designed to bind specifically to extracellular molecules, either a receptor or its ligand, similar to the behavior of a monoclonal antibody, aptamer or peptide. However, recent findings demonstrate that Spiegelmers are capable of entering cells and interfering with intracellular processes as well. Finally, Spiegelmers appear to be non-immunogenic, even under the most inductive conditions for antibody formation in rabbits. This situation is of critical importance if Spiegelmer therapy is to be used repeatedly to treat chronic diseases. It is therefore preferred in the context of the present invention, to detect substances which bind or react or interact with the agonistic autoantibodies against the endothelin-l receptor, protease activated recep- tors 1 and 2 and/or α1 -adrenergic receptor. These Spiegelmers can be used as peptidomimetic. It is also preferred to detect substances which block Spiegelmers that block the antigen binding site of the agonistic autoantibodies.

Also claimed by the invention are RNAi-pharmaceuticals for blocking the plasma cells which produce the agonistic autoantibodies directed against the endothelin-l receptor, protease activated receptors 1 and 2 and/or α1 -adrenergic receptor.

The invention also relates to a pharmaceutical composition comprising the inventive nucleic acid molecules, vectors, host cells, peptides and/or recognition molecules, optionally together with a pharmaceutically tolerable carrier. In particular, the pharmaceutical composition can be used as a drug. To this end, it is possible, for example, to modify the peptides by means of cyclization or other procedures well-known to those skilled in the art such that destruction thereof by endogenous peptide- degrading structures, e.g. serum proteases, is prevented. By using the peptides or recognition molecules of the invention, in vivo or ex vivo neutralization of autoantibodies is possible. In in vivo neutralization, the drugs are administered directly to the patient; in ex vivo neutralization, the blood is conducted out of the body e.g. via a loop, e.g. in the form of a tube circulation, subsequently contacted with the drug and, following neutralization of the autoantibodies, resupplied into the organism, i.e., the patient. Regarded as drugs in the meaning of the invention are compositions for therapeutic and prophylactic purposes, as well as pharmaceutical compositions usable as diagnostic agents.

Of the invention, drugs or pharmaceutical compositions - used in a synonymous fashion herein - are substances and formulations of substances intended to cure, alleviate or avoid diseases, illness, physical defects or pathological affection by application on or in the human body. Of the invention, medical adjuvants are sub- stances used as active ingredients in the production of drugs. Pharmaceutical- technical adjuvants serve to suitably formulate the drug or pharmaceutical composition and, if required during the production process only, can even be removed thereafter, or they can be part of the pharmaceutical composition as pharmaceutically tolerable carriers. Examples of pharmaceutically tolerable carriers will be given below. Drug formulation or formulation of the pharmaceutical composition is optionally effected in combination with a pharmaceutically tolerable carrier and/or diluent. Examples of suitable pharmaceutically tolerable carriers are well-known to those skilled in the art and comprise e.g. phosphate-buffered saline, water, emulsions such as oil/water emulsions, various types of detergents, sterile solutions, and so forth. Drugs or pharmaceutical compositions comprising such carriers can be formulated by means of well-known conventional methods. These drugs or pharmaceutical compositions can be administered to an individual at a suitable dose, e.g. in a range of from 0,01 μg to 10 g of peptides per day and patient. Doses of from 1 μg to 1 g are preferred. Preferred is administration of doses as small in number and as low as possible, preferably a single dose. Administration can be effected on various routes, e.g. intravenous, intraperitoneal, intrarectal, intragastrointestinal, intranodal, intramuscular, local, but also subcutaneous, intradermal or on the skin or via mucosa. Administration of nucleic acids encoding the peptide of the invention can also be effected in the form of a gene therapy, e.g. via viral vectors. The kind of dosage and route of administration can be determined by the attending physician of clinical factors. As is familiar to those skilled in the art, the kind of dosage will depend on various factors such as size, body surface, age, sex, or general health condition of the patient, but also on the particular agent being administered, the time period and type of administration, and on other medications possibly administered in parallel. Those skilled in the art will also be familiar with the fact that the concentration of autoantibodies can be diagnosed first, using the peptides of the invention, in order to determine the required concentration of drug.

More specifically, the pharmaceutical compositions or drugs comprise a pharmacological substance which includes one or more peptides or recognition molecules of the invention, or/and nucleic acid molecules encoding same, in a suitable solution or administration form. Administration thereof can be effected either alone or together with appropriate adjuvants described in connection with drugs or pharmaceutical compositions, or in combination with one or more adjuvants, e.g. QS-21 , GPI-0100 or other saponines, water-oil emulsions such as Montanide adjuvants, polylysine, polyarginine compounds, DNA compounds such as CpG, Detox, bacterial vaccines such as typhoid vaccines or BCG vaccines, salts such as calcium phosphates, and/or other suitable material enhancing the effect, preferably immunostimulatory molecules such as interleukins, e.g. IL-2, IL-12, IL-4 and/or growth factors such as GM-CSF. They are mixed with the peptides or recognition molecules of the invention of well-known methods and administered in suitable formulations and dosages. Formulations, dosages and suitable components are well-known to those skilled in the art.

Obviously, the pharmaceutical composition or drug can also be a combination of two or more of the inventive pharmaceutical compositions or drugs, as well as a combination with other drugs, such as antibody therapies, chemotherapies or radiothera- pies, suitably administered or applied at the same time or separately in time. The production of the drugs or pharmaceutical compositions proceeds of per se known methods.

The invention also relates to a kit comprising the nucleic acid molecule, the vector, the host cell, the peptide and/or the recognition molecule, optionally together with instructions or information as to the pharmaceutical provision or the procedure of therapeutical treatment. For example, the information can be an instruction leaflet or other medium providing the user with information in which therapeutical procedure the above-mentioned substances should be used. In particular, the instruction leaflet includes detailed and/or important information about the therapeutical treatment. Obviously, the information need not necessarily be in the form of an instruction leaflet, and the information may also be imparted via the Internet. The invention also relates to an apparatus for chromatography, which includes the peptides of the invention. In a preferred embodiment the peptides are bound to a solid phase or an apparatus which catches from blood or plasma extracellular loop 2 peptides of the human β2 receptor or antibody binding peptido mimetica fixed to nanoparticles, magnetic or paramagnetic particles or microspheres of cellulosis and other polymeric matrix molecules.

In another preferred embodiment the peptides are bound to a solid phase within the chromatography system.

In another preferred embodiment, anti-human immunoglobulin coupled columns are used for the removal of immunoglobulin from the plasma or blood of a patient suffering from coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma. Because of the conclusion reached by the inventors that coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma is caused by antibodies, especially by autoantibodies, the treatment of coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma may also be effected by providing columns which remove antibodies or autoantibodies irrespectively from their specificity from the plasma or blood of a patient suffering from coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma. In a preferred embodiment of the invention, the antibodies or autoantibodies which are not associ- ated with coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma can be reinfused to the patient.

In particular, the apparatus of the invention (this may be an apparatus which fea- tures a specific ligand for human immunoglobulins, for example endothelin-l receptor, protease activated receptors 1 and 2 and/or α1 -adrenergic receptor or the second loops of the receptors) can be used to eliminate the autoantibodies from fluids of a patient or to neutralize the autoantibodies. This method is known to those skilled in the art under the term of immunoadsorption and/or plasma apheresis ther- apy. With the aid of immunoadsorption, immunoglobulins are removed from the blood or serum of a coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma patient. Advantageously, this immunoadsorption treatment can be con- ducted in a stationary and ambulant fashion. It can be envisaged that the apparatus, particularly the so-called adsorber, is part of an extracorporeal blood circulation. To this end, blood is taken continuously or discontinuously from a patient's major vessel, particularly from an arm vein, and separated into single components, such as cellular and humoral components, using filtration or centrifugation. In particular, one essential blood component obtained in this fashion is blood plasma. Advantageously, the blood plasma can be passed through the apparatus of the invention and, following adsorption of the autoantibodies, returned into the patient, particularly through another vein of arms or legs, together with previously separated blood com- ponents, especially cellular components. It can also be envisaged that the specific ligand for the human immunoglobulin or the peptides are immobilized on a Sepha- rose matrix. The matrix can be placed in a container having a volume of e.g. 10 to 400 ml. Thereafter, the blood plasma of the patient can be passed over the matrix where the autoantibodies will be bound, thus allowing elimination thereof from the blood plasma. Those skilled in the art will be familiar with various ways of providing such solid phase-fixed peptides or a specific ligand for immunoglobulin, e.g. in the form of (i) regeneratable adsorption columns, (ii) double columns and (iii) disposable columns. The diverse wash and elution solutions, permitting high efficiency of treatment, can easily be determined by a person skilled in the art by using routine tests. By providing the teaching of the invention, particularly the specific ligand for the human immunoglobulin or the peptides of the invention, various ways of employing the specific ligand for the human immunoglobulin or the peptides in vivo, ex vivo and in vitro in prophylaxis, diagnosis, therapy and aftercare of coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma-induced, autoantibody-mediated diseases are disclosed to a person skilled in the art.

The invention also relates to a method for the treatment of coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial ves- sels, Raynaud syndrome and scleroderma by binding and/or removing autoantibodies by means of peptides or specific ligands for human immunoglobulin/auto- immunoglobulin of the invention bound to a solid phase.

In a preferred embodiment of the invention the autoantibodies are directed against human endothelin-l receptor, protease activated receptors 1 and 2 and/or α1- adrenergic receptor. The invention also relates to the use of the nucleic acid molecules of the invention, the host cells of the invention, the vector of the invention, the peptides of the invention, the recognition molecules of the invention, the pharmaceutical composition of the invention, the kit of the invention and the apparatus of the invention in the pro- phylaxis, diagnosis, therapy, follow-up and/or aftercare of coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma.

The invention also relates to the use of the nucleic acid molecules of the invention, the host cells of the invention, the vector of the invention, the peptides of the invention, the recognition molecules of the invention, the pharmaceutical composition of the invention, the kit of the invention and the apparatus of the invention in the production of a drug for the treatment of coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syn- drome and scleroderma.

In a preferred embodiment of the invention the coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma are a primary open angle coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma (POAG), a normal tension coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma (NTG), an acute angle-closure coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of pe- ripheral arterial vessels, Raynaud syndrome and scleroderma, a primary congenital coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma and/or a secondary coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma.

Of the invention, another type, acute angle-closure coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma, is characterized by an acute rise in the intraocular pressure. This occurs in susceptible eyes when the pupil dilates and blocks the flow of fluid through it, leading to the peripheral iris blocking the trabecular meshwork. Acute angle-closure coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma can cause pain and reduced visual acuity (blurred vision), and may lead to irreversible visual loss within a short time. This is an ocular emergency requiring immediate treatment. Many people with coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma experience halos around bright lights as well as the loss of sight characterized by the disease.

In the meaning of the invention, primary congenital coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma or buphthalmos is a rare genetic disease affecting infants. Newborns present with enlarged globes and clouded corneas. It is thought that reduced trabecular permeability is the cause of increased intraocular pressure. Surgery is the treatment.

In the meaning of the invention, secondary coronary arterial occlusion, angina pec- toris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma occurs as a complication of various medical conditions such as eye surgery, advanced cataracts, eye injuries, some eye tumors, uveitis, diabetes or use of corticosteroid drugs.

The invention also relates to the use of a specific ligand for human immunoglobulin or the inventive peptides and nucleic acids or host cells, the pharmaceutical composition of the invention, the kit of the invention and/or the apparatus of the invention in drug screening. For example, drug screening may comprise the identification of substances, particularly peptides, proteins, carbohydrates and/or lipids, which inter- act with autoantibodies directed against the endothelin-l receptor, protease activated receptors 1 and 2 and/or of α1 -adrenergic receptor.

Interaction, for example, can be binding to said peptides, but also activation or inhibition of or by peptides. Accordingly, a drug can be e.g. a structure binding to the autoantibodies or - in another embodiment of the invention - to the peptide structures in the body of a patient, and consequently to the loops, thus competing with the autoantibodies for a binding site. By virtue of the disclosure of the teaching of the invention, especially the disclosure with respect to the connection of disease and binding site of the autoantibodies, a person skilled in the art will be capable of screening various drugs. Drug screening based on disclosed targets is part of the general knowledge of a person skilled in the art and is effected using routine tests; reference is made to the corresponding standard textbooks of molecular biology and pharmacology. The invention also relates to a method for the treatment of an autoimmune disease by binding and/or removal of autoantibodies by means of the inventive peptides bound to a solid phase. The autoantibodies are bound, complexed and/or neutral- ized on the solid phase by means of the peptides bound to the solid phase.

In a preferred embodiment of the invention, autoantibodies directed against endo- thelin-l receptor, protease activated receptors 1 and 2 and/or α1 -adrenergic receptor are bound, complexed and/or neutralized by means of the above-mentioned inven- tive materials and products, apparatus, particularly the chromatography apparatus, and peptides. For example, the peptides can be used in the detection of autoantibodies in serums of patients, using an ELISA or other immunological detection methods well-known to those skilled in the art. For detection, it can be advantageous, for example, when the autoantibodies are bound by biotinylated or otherwise coupled peptides and separated by streptavidin-coupled supports such as magnetic particles or plates. Such a method has been described in DE 102 56 897.9 which hereby is incorporated in the disclosure of the present application. More specifically, the separated autoantibodies are detected using igG subtype-specific labelled antibodies. In the event of a primary open angle coronary arterial occlusion, angina pec- toris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma, the antibodies are detected using particularly IgGβ subtype-specific labelled antibodies.

The invention relates also to the use of a specific ligand for human immunoglobulins in the manufacture of a column coupled to said ligand for the treatment of a patient suffering from coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma, said treatment comprising passing plasma of the patient, over the column under conditions which effect the binding of said specific ligand to immunoglobulin in the pa- tient's plasma, thereby removing a significant portion of the immunoglobulin from the patient's plasma, and reinfusing the plasma to the patient.

Additionally, the invention relates also to the treatment of a patient suffering from coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma, said treatment comprising the steps of: (a) providing a column coupled to a specific ligand for human immunoglobulin,

(b) passing plasma of the patient over the column under conditions which effect the binding of said specific ligand to immunoglobulin in the patient's plasma, thereby removing a significant portion of the immunoglobulin from the patient's plasma, and

(c) reinfusing said plasma to the patient.

In a preferred embodiment, the treatment of a patient is characterized in that the specific ligand is selected from the group consisting of polyclonal anti-human immunoglobulin antibodies, monoclonal anti-human immunoglobulin antibodies, a fragment of such antibodies, recombinant molecules of the antibody idiotype, synthesized peptides, Protein A and Protein G.

In another preferred embodiment of the treatment of a patient, the specific ligand recognizes antibodies directed against endothelin-l receptor, protease activated receptors 1 and 2 and/or α1 -adrenergic receptor. Preferably, the specific ligand is an antigen-mimicking molecule selected from the group consisting of polyclonal and monoclonal antiidiotypic antibodies, fragments of such antibodies, and synthesized peptides.

Preferably, the specific ligand is a synthesized peptide mimicking a sequence of a receptor structure.

In another preferred embodiment of the treatment of a patient, the receptor is the endothelin-l receptor, protease activated receptors 1 and 2 and/or α1 -adrenergic receptor.

Preferably, the autoantibodies are directed against a molecule selected from the group consisting of endothelin-l receptor, protease activated receptors 1 and 2 and/or α1 -adrenergic receptor.

In another preferred embodiment of the treatment of a patient, the invention is characterized by a parallel or subsequent combination with β-blockers or intravenous immunoglobulin.

In the context of the invention is a treatment of a patient suffering from coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma a preferred embodiment of the use of the specific ligand for human immunoglobulin in the manufacture of a column for the treatment of a patient.

The teachings of the present invention are characterised by the following features:

departure from the beaten track a new perception of the problem satisfaction of a long-felt need or want - hitherto all efforts of experts were in vain the simplicity of the solution, which proves inventive action, especially since it replaces a more complex doctrine the development of scientific technology followed another direction the achievement forwards the development - misconceptions among experts about the solution of the according problem (prejudice) technical progress, such as: improvement, increased performance, price- reduction, saving of time, material, work steps, costs or resources that are difficult to obtain, improved reliability, remedy of defects, improved quality, no main- tenance, increased efficiency, better yield, augmentation of technical possibilities, provision of another product, opening of a second way, opening of a new field, first solution for a task, spare product, alternatives, possibility of rationalisation, automation or miniaturisation or enrichment of the pharmaceutical fund special choice; since a certain possibility, the result of which was unforesee- able, was chosen among a great number of possibilities, it is a patentable lucky choice error in citations young field of technology combined invention; a combination of a number of known elements, with a sur- prising effect licensing praise of experts and commercial success

Said advantages are shown especially in the preferential embodiments of the invention. Table 1 : Identified epitopes

The peptides shown in the table are able to bind to antibodies or to neutralize antibodies, preferably autoantibodies. The reverse sequences may also comprise D- amino acids. Epitopes of the muscarinergic M2 receptor (2^nd loop) may also be used for this purpose; as well as the epitopes of Alphal B and Alphai D receptors (1^st and 2^nd loop), since the antibody interacts specifically with the three receptor subtypes.

The sequences shown in line 1 of the table may be used in the treatment of essential, refractory and/or pulmonary hypertension.

The peptides shown in line 2 and 3 of the table may be used in the treatment of preeclampsia, humoral kidney rejection and/or malign hypertension.

The peptides shown in line 4, 5, 6 and 7 may be used in the treatment of Chagas Disease, dilated cardiomyopathy (DCM), myocarditis and/or cardiomyopathy. The peptides of line 4 are preferred in the treatment of Chagas Disease, cardiomyopathy and/or DCM. The peptides of line 5 and/or 7 are preferred in the treatment of DCM. The peptides of line 6 are preferred in the treatment of DCM and/or myocarditis. The peptides shown in line 8 and 9 may be used in the treatment of the following diseases: Raynaud's phenomenon, pulmonary hypertension, scleroderma, angina pectoris, occlusion of arteries and veins, especially of leg veins and of brain arteries.

Claims

1. A peptide as part of an epitope binding to an autoantibody, whereas the peptide comprises at least one of the following amino acid sequences

d) MLNATSK, SKFMEFY, EQHKTCM, MLNATSKSKFMEFY, or

e) KSTANLM, YFEMFKS, MCTKHQE , YFEMFKSKSTANLM or

f) a combination of one or more sequences of a) and b).

2. Peptide of claim 1 , whereas the peptide comprises additionally at least one of the following amino acid sequences APEDET, WKEPAP or PPDERF.

3. Peptide of claim 1 , whereas the peptide comprises additionally at least one of the following amino acid sequences ITTCHDVL, LNITTCHD or PALNITTC.

4. Peptide of claim 1 , characterized by an epitope for autoantibodies directed against endothelin-l receptor,

5. A peptide of claim 2, characterized by an additional epitope for protease- activated receptor 1 , 2 or 3.

6. A peptide of claim 3, characterized by an additional epitope for α1- adrenergic receptor.

7. Peptide of at least on of claims 1 to 6, characterized in that it is selected from the group consisting of: a) a peptide comprising at least one of the amino acid sequences of claim 1 , 2 or 3;

b) a peptide consisting of an amino acid sequence having suffi- cient homology to be functionally analogous to an amino acid sequence in accordance with a);

c) a peptide of an amino acid sequence a) or b) which is modified by deletions, additions, substitutions, translocations, in- versions and/or insertions and functionally analogous to an amino acid sequence in accordance with a) or b);

d) a peptide of amino acid sequence a), b) or c) which is modi- fied by branch or extension with the same or another peptide of amino acid sequence a), b) or c) to form a homooligomeric or heterooligomeric peptide.

8. Peptide of claim 7, characterized in that the amino acid sequence specified under b) has at least 40% homology to any of the amino acid se- quences specified under a).

9. Peptide of claim 7 or 8, characterized in that the amino acid sequence specified under b) has at least 60%, preferably 70%, more preferably 80%, especially preferably 90% homology to any of the amino acid sequences specified under a).

10. Peptide of any of the preceding claims for use as a medical active substance.

11. Peptide of any of the preceding claims 1 to 3, characterized in that the peptide binds to antibodies of patients suffering from coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of periph- eral arterial vessels, Raynaud syndrome and scleroderma.

12. Peptide of any of the preceding claims 1 to 11 , characterized in that the peptide is immobilized and/or fixed to magnetic, paramagnetic and/or non magnetic nanoparticles.

13. Peptide of any of the preceding claims, characterized in that the peptide is bound to a solid phase.

14. Peptide of any of the preceding claims in a linear and branched as well as cyclic form, whereas the peptide ring closure is effected through disulfide bridging when two cysteines are present, or through amide cycliza- tion, which is optionally effected through side chains, through the C and N termini or through a combination of these three possibilities.

15. Peptide of any of the preceding claims, characterized in that said peptide is capable to bind immunoglobulins or antigen-immunoglobulin complexes in biological fluids.

16. Peptides of any of the preceding claims, characterized in that the immunoglobulins are agonistic autoantibodies which interact with the endo- thelin-1 receptor, protease-activated receptor 1 , 2 or 3 and α1- adrenergic receptor.

17. Peptide of any of the preceding claims, characterized in that it additionally comprises amino groups, amides, acetyl groups, biotin groups, markers, spacers and/or linkers.

18. An isolated nucleic acid molecule selected from the group comprising: a) a nucleic acid molecule comprising a nucleotide sequence which encodes at least one peptide selected from the group consisting of peptides of claim 1 , 2 or 3;

f) a nucleic acid molecule of a nucleotide sequence of a) through e) which is modified by deletions, additions, substitutions, translocations, inversions and/or insertions and func- tionally analogous to a nucleotide sequence of a) through e).

19. Nucleic acid molecule of claim 18, characterized in that the nucleotide sequence specified under d) has at least 40% homology to any of the nucleotide sequences specified under a) through c).

20. Nucleic acid molecule of claim 18 or 19, characterized in that the nucleo- tide sequence specified under d) has at least 60%, preferably 70%, more preferably 80%, especially preferably 90% homology to any of the nucleotide sequences specified under a) through c).

21. Nucleic acid molecule of any of claims 18 to 20, characterized in that it is a genomic DNA, a cDNA and/or a RNA.

22. A vector comprising a nucleic acid molecule of any of claims 18 to 21.

23. A host cell comprising a vector of claim 22.

24. A peptide, said peptide being encoded by a nucleic acid molecule of any of claims 18 to 21.

25. Solid phases for affinity chromatography or solid-phase extraction con- sisting of organic, inorganic, synthetic polymers or of mixed polymers, preferably cross-linked agarose, cellulose, silica gel, polyamide and polyvinyl alcohols, which are optionally chemically activated, with peptides of at least one of claims 1 to 17 or 24, immobilized on the surface of the solid phase.

26. The solid phases of claim 25, whereas the peptides are bound to the solid support phase covalently or by adsorption.

27. The solid phases of any of claims 25 to 26, whereas the peptides are distanced from the support surface by linkers/spacers.

28. A device for removing immunoglobulins from immunoglobulin-containing samples on solid phases, whereas the device contains a solid phase of any of claims 25 to 27, and means for the entry of immunoglobulin- containing samples are provided.

29. A pharmaceutical composition comprising a nucleic acid molecule of any of claims 18 to 21 , a vector of claim 22, a host cell of claim 23, a peptide of any of claims 1 to 17 or 24 and/or a solid phase of claims 25 to 27, optionally together with a pharmaceutically tolerable carrier.

30. A kit comprising a nucleic acid molecule of any of claims 18 to 21 , a vector of claim 22, a host cell of claim 23, a peptide of any of claims 1 to 17 or 24, a solid phase of claims 25 to 27 and/or a pharmaceutical composi- tion of claim 29, optionally together with instructions for combining the contents of the kit and/or providing a formulation.

31. An apparatus for chromatography, comprising peptides of any of claims 1 to 17 or 24.

32. Apparatus of claim 31 , characterized in that the peptides are bound to a solid phase of claims 25 to 27.

33. Use of a nucleic acid molecule of any of claims 18 to 21 , a vector of claim 22, a host cell of claim 23, a peptide of any of claims 1 to 17 or 24, a solid phase of claims 25 to 27, a pharmaceutical composition of claim 29, a kit of claim 30, an apparatus of claim 31 or 32 in the prophylaxis, diagnosis, therapy, follow-up and/or aftercare of patients suffering from vascular diseases.

34. Use of claim 33, whereas the vascular diseases comprises coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma.

35. Use of a nucleic acid molecule of any of claims 18 to 21 , a vector of claim 22, a host cell of claim 23, a peptide of any of claims 1 to 17 or 24, a solid phase of claims 25 to 27, a pharmaceutical composition of claim 29, a kit of claim 30, an apparatus of claim 31 or 32 in the production or screening of a drug for the treatment of diseases like coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of periph- eral arterial vessels, Raynaud syndrome and scleroderma.

36. Use of one of claims 33 or 34, characterized in that a peptide of any of claims 1 to 17 or 24 is used to detect, bind, complex and/or neutralize autoantibodies directed against endothelin-l receptor, protease-activated receptor 1 , 2 or 3 and/or α1 -adrenergic receptor.

37. Use of a peptide of claim 1 for the treatment of patients suffering from coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma.

38. Use of a peptide of claim 2 for the treatment of patients suffering from pulmonary hypertension and occlusion of peripheral arterial vessels.

39. Use of a peptide of claim 3 for the treatment of patients suffering from coronary arterial occlusion, angina pectoris, Raynaud syndrome and scleroderma.

40. Method for the treatment of vascular diseases by binding and/or removal of autoantibodies by means of peptides of any of claims 1 to 17 or 24 bound to a solid phase.

41. Method of claim 40, whereas the vascular diseases comprises coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma.

42. Method of claim 40 or 41 , characterized in that the autoantibodies are directed against the second loop of the endothelin-l receptor, second loop of protease activated receptors 1 and 2 and/or second loop of α1- adrenergic receptor and/or its polymorphisms.

43. Method of any of the claims 40 to 42,. characterized in that the autoantibodies against endothelin-l receptor are lgG2 and 3 and against protease activated receptors 1 and 2 are IgGI and 3.

44. Use of a specific ligand for human immunoglobulin comprising the se- quence of at least one of the peptides of claims 1 , 2 or 3 in the manufacture of a column coupled to said ligand for the treatment of a patient suffering from vascular diseases, said treatment comprising passing plasma of the patient, over the column under conditions which effect the binding of said specific ligand to immunoglobulin in the patient's plasma, thereby removing a significant portion of the immunoglobulin from the patient's plasma, and reinfusing the plasma to the patient.

45. Use of a specific ligand of claim 44, whereas selective IgGI ₁ 2 and/or 3 apheresis from blood or plasma to treat coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma is used.

46. Use of claim 44 or 45, whereas said specific ligand is selected from the group consisting of polyclonal anti-human immunoglobulin antibodies, monoclonal anti-human immunoglobulin antibodies, a fragment of such antibodies, recombinant molecules of the antibody idiotype, synthesized peptides, Protein A and Protein G and Plasmapheresis and removal of autoantibodies which react with the endothelin-l receptor, protease activated receptors 1 and 2 and/or α1 -adrenergic receptor or all IgG from whole blood.

47. Use of claim 44 to 46, whereas said specific ligand is an antigen- mimicking molecule selected from the group consisting of polyclonal and monoclonal antiidiotypic antibodies, fragments of such antibodies, and synthesized peptides.

48. Use of claim 37 to 40, whereas said specific ligand is an antigen- mimicking molecule of MLNATSK, SKFMEFY, EQHKTCM, MLNATSKSKFMEFY, APEDET, WKEPAP, PPDERF, ITTCHDVL, LNITTCHD or PALNITTC, selected from the group consisting of polyclonal and monoclonal antiidiotypic antibodies, fragments of such anti- bodies, and synthesized peptides.

49. Use of claim 44 to 48, whereas said specific ligand is a synthesized peptide mimicking a sequence of a receptor structure.

50. Use of claim 44 to 49, whereas said receptors are endothelin-l receptor, protease-activated receptor 1 , 2 or 3 and/or α1 -adrenergic receptor.

51. Use of claim 44 to 50, whereas said autoantibodies are directed against a molecule selected from the group consisting of endothelin-l receptor, protease-activated receptor 1 , 2 or 3 and α1 -adrenergic receptor.

52. Use of claim 44 to 51 in parallel or subsequent combination with β- blockers or intravenous immunoglobulin.

53. A method for removing a portion of the immunoglobulin from plasma taken from a patient suffering from coronary arterial occlusion, angina pectoris, pulmonary hypertension, occlusion of peripheral arterial vessels, Raynaud syndrome and scleroderma, the method comprising

(a) providing a column coupled to a specific ligand for human immunoglobulin, which column is as defined in any one of claims 44 to 52; and

(b) passing the plasma over the column under conditions which ef- feet the binding of said specific ligand to immunoglobulin in the plasma.