EP1789081A2

EP1789081A2 - Treatment of atherosclerosis

Info

Publication number: EP1789081A2
Application number: EP05789506A
Authority: EP
Inventors: Frank Mattner; Walter Schmidt
Original assignee: Affiris Forschungs und Entwicklungs GmbH
Current assignee: Affiris Forschungs und Entwicklungs GmbH
Priority date: 2004-09-13
Filing date: 2005-09-08
Publication date: 2007-05-30
Also published as: KR20070054206A; CA2580261A1; WO2006029982A2; CN101018564A; JP2008512427A; AU2005284133A1; TW200608990A; WO2006029982A3; AT500835B1; US20090104211A1; AT500835A1

Abstract

The invention relates to the use of a compound comprising the following amino acid sequence X1X2X3X4X5X6X7X8, whereby X1 is an amino acid other than C, X2 is an amino acid other than C, X3 is an amino acid other than C, X4 is an amino acid other than C, X5 is an amino acid other than C, X6 is not present or is an amino acid other than C, X7 is not present or is an amino acid other than C, X8 is not present or is an amino acid other than C and X1X2X3X4X5X6X7X8 is not or does not comprise a 5-mer, 6-mer, 7-mer or 8-mer polypeptide fragment of cholesterine ester transport protein (CETP) or a CETP epitope, whereby the compound has a binding for an antibody specific for the natural CETP glycoprotein, for the production of a means for the prevention and treatment of atherosclerosis, atherosclerosis risk diseases and atherosclerosis consequential diseases.

Description

Treatment of atherosclerosis

The invention relates to the prevention and treatment of atherosclerosis, atherosclerosis risk diseases and atherosclerosis sequelae.

Atherosclerotic secondary diseases, such as peripheral arterial occlusive disease, coronary heart disease and apoplectic cerebral insult, continue to be among the leading causes of death in the United States, Europe and much of Asia. According to Virchow's opinion, the lipid deposits in the arterial wall were changes due to blood lipids, which according to his consideration arise by transduction of the lipids and complex formation with acid mucopolysaccharides. By this "injury" of the arteries he explained the accumulation of lipids and the development of atherosclerotic lesions in the intima and medial of the arteries.Today's generally accepted state of knowledge is that developed by Ross in 1973 and modified in 1986 and 1993. " Ross considers the development of atherosclerosis to be a chronic progressive inflammation of the arterial wall characterized by a complex interaction of growth factors, cytokines, and cell interaction, as well as the integration of Virchow's lipid hypothesis The "injury" of the endothelium, according to the "response-to-injury" hypothesis, represents the initial event of the disease, which leads to an endothelial dysfunction, which is a chain of cellular dysfunction Interacting triggers in the formation of atherosclerotic lesions k As risk factors which favor such an "injury", exogenous and endogenous influences are statistically significantly correlated with atherosclerosis. Among the most important of these endothelial damaging factors are, for example, elevated and modified LDL, Lp (a), arterial hypertension, diabetes mellitus and hyperhomocysteinemia. Since the endothelium is not a rigid, but rather an extremely dynamic barrier, in the course of endothelial dysfunction, in addition to a permeability increase for lipoproteins, there are a large number of molecular changes that affect the interaction of monocytes, T lymphocytes and endothelial cells significantly influence be¬. The expression of endothelial adhesion molecules of the E-, L- and P-selectin type, integrins, ICAM-I, VCAM-I and platelet-endothelial-cell-adhaesionmolecule-1 leads to the lumen attachment of monocytes and T-lymphocytes. The subsequent migration of the leukocytes via the endothelium is mediated by MCP-1, interleukin-8, PDGF, M-CSF and osteopontin. In the intima local macrophages and monocytes are able to take over the so-called scavenger receptor, the invaded LDL particles and deposit them as vacuoles of cholesterol esters in the cytoplasm. The foam cells produced in this way mainly accumulate in groups in the area of the vessel intima and form the "fatty streak" lesions that already occur in childhood. LDLs are low-density lipoproteins and are caused by catabolic effects of lipolytic enzymes from triglyceride-rich VLDL particles. In addition to the damaging properties on endothelial cells and smooth muscle cells of the media, LDL moreover acts chemotactically on monocytes and is capable of increasing the expression of MCSF and MCP-1 of endothelial cells via gene amplification. In contrast to LDL, HDL is capable of resuming cholesterol esters of loaded macrophages with the formation of apolipoprotein E with the formation of so-called HDLc complexes. These particles loaded with cholesterol esters can bind to hepatocytes or adrenocortical cells by interaction of SR-B1 receptors and release cholesterol for the production of bile acids or steroids. This mechanism is referred to as "reverse cholesterol transport" and illustrates the protective function of HDL. Activated macrophages can present antigens via HLA-DR, thereby activating CD4 and CD8 lymphocytes, which, in turn, stimulate the secretion of cytokines, such as IFN-gamma and TNF-alpha, and thereby contribute to the enhancement of the inflammatory response. As the disease progresses, smooth muscle cells of the media enter the inflammatory area of the intima. As a result, the intermediate lesion is formed at this stage. Starting from the intermediate lesion, the advanced and complicated lesion develops in the course, which is characterized morphologically by a necrosis nucleus, cell detritus and a lumen-side collagen-rich fibrinous cap. If the number of cells and the proportion of lipids increases continuously, endothelial lacerations and exposure of surfaces having thrombotic properties occur. The attachment and activation of platelets at these tears results in a release of granules which contain cytokines, growth factors and thrombin. Proteolytic enzymes of the macrophages are responsible for the thinning of the fibrinous cap, which ultimately leads to rupture of the plaque with consecutive thrombosis and stenosis of the vessel, and acute ischemia of the end stream.

Different risk factors are held responsible for the development of atherosclerotic lesions. Particular importance is attached to hyperlipoproteinemia, arterial hypertension and nicotine abuse. A disease which starts with an excessive increase in total and LDL cholesterol is familial hypercholesterolemia. It is one of the most common monogenic inherited metabolic diseases. The moderate heterozygous form occurs with a frequency of 1: 500, the homozygous form with 1: 1 million significantly less. The causes of familial hypercholesterolemia are mutations in the LDL receptor gene on the short arm of chromosome 19. These mutations can be deletions, insertions or point mutations. The characteristic finding of the lipoproteins in familial hypercholesterolemia is an increase in the total and LDL cholesterol at mostly normal triglyceride and VLDL concentrations. The HDL is often humiliated. Phenotypically there is a type IIa hyperlipoproteinemia according to Fredrikson. The total echo is increased by two to three times in the heterozygous form and by five to six times in the homozygous form. Clinically, familial hyperchoesteremia is manifested by early coronary sclerosis. As a rule, in heterozygous men, the first symptoms of CHD occur between the ages of 30 and 40, and in women, on average, only 10 years later. 50% of the affected men die before the age of 50 from the consequences of their coronary sclerosis. Decreased HDL concentrations are responsible for the rapid progression of atherosclerosis in addition to the massive increase in LDL levels. Also on extracardiac vessels, such as the aorta, the carotids and peripheral arteries, can Atherosclerotic changes manifest. In the ho- mozygous form of the disease, the coronary artery develops already in early childhood. The first myocardial infarction often occurs before the age of 10 and those affected usually die before the age of 20. The development of xanthomas depends on the level of serum cholesterol and the duration of the disease. About 75% of heterozygous people over 20 years of age have tendinous xanthomas. Homozygotes have almost 100% skin and tendon xanthomas. Lipid deposits may also occur on the eyelid and in the cornea (xanthelasma, Arcus lipoides). However, they are not a specific sign of hypercholesterolemia since they are also found in normal cholesterol levels. In addition, acute arthritids and tendosynovitides frequently occur in FH. The individual lipoproteins differ in terms of their size and density, since they contain different amounts of lipids and proteins, so-called apoproteins. The density increases with increasing protein and decreasing lipid content. Due to their different density, they can be separated by ultrafiltration into different fractions. This is the basis for the classification of the lipoproteins into the main groups: chylomicrons, very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), high-density lipoproteins (HDL), Lipoprotein (a) (Lp (a)). The lipoproteins with a high atherogenic potential include, in particular, the LDL, the Lp (a) and the VLDL. LDL have a density of about d = l, 006-1, 063 g / ml. At the core are esterified cholesterol molecules. This highly hydrophobic core is surrounded by a shell of phospholipids, unesterified cholesterol, and a single Apo BlOO molecule. In addition, apoprotein E is found on the surface of the LDL particles. The function of LDL is to transport cholesterol to peripheral tissues, where it, through which apoprotein B-100 mediates, is taken up into the cells via the LDL receptor. In large epidemiological studies such as the Framingham Study, the Multiple Risk Factor Intervention Trial and the Withehall Study, a positive correlation between the level of serum cholesterol and the incidence of coronary heart disease has been demonstrated. LDL cholesterol levels above 160 mg / dl represent a high cardiovascular risk. In addition to the level of LDL cholesterol plays in the assessment the risk profile for cardiovascular diseases, the level of vascular protective HDL cholesterol also plays an important role. Values below 35 mg / dl are associated with increased risk. VLDLs are low-density lipoproteins (d = 0, 94-1, 006 g / ml) and have a high triglyceride content. Essentially VLDL contain apoprotein C, and in smaller proportions the apoproteins B-100 and E. In contrast to chylomicrons, VLDL do not consist of dietary lipids, but are synthesized from endogenously formed triglycerides in the liver and secreted into the circulation. As with chylomicrons, the triglycerides are hydrolyzed by the apoprotein C-II activated lipoprotein lipase, and the free fatty acids are delivered to muscle and adipose tissue. The remaining cholesterol-rich "VLDL-Remnants" are called intermediate density lipoproteins because of the higher density The lipoprotein (a) (Lp (a)) has a density of 1.05-1.12 g / ml and is similar in composition to LDL In addition to the apoprotein B-100, the protein portion consists of the apoprotein (a) which is characteristic for the Lp (a) .The physiology and function of the Lp (a) are still very poorly understood high sequence homology to plasminogen, it is believed that Lp (a) promotes both the formation of thrombi at atherosclerotic plaques and has an atherogenic effect.Lp (a) is found in atherosclerotic lesions together with apoprotein B. Retrospective studies have shown an association between elevated Lp (a) and CHD, and meta-analysis of numerous prospective studies has shown that Lp (a) is an independent risk factor for CHD Values between 15 - 35 mg / dl apply. Lp (a) can not be influenced dietary or medicinal up to now. Therapeutic measures are therefore limited to the reduction of other risk factors. In particular, a decrease in LDL cholesterol appears to lower the cardiovascular risk of Lp (a). Significant pathophysiological significance in the pathogenesis of atherosclerosis moreover possess coagulation factors. Epidemiological findings indicate a correlation between the fibrinogen concentration in plasma and the development of coronary heart disease and, above all, myocardial infarction. Increased fibrinogen levels (> 300 mg / dl) proved to be an independent indicator in this context and a risk factor for cardiovascular diseases. But also high concentrations of tissue plasminogen activator inhibitor tPA-I are associated with the onset of CHD. The relationship between hypertriglyceridemia and coronary risk varies depending on the cause of blood fat elevation. Despite the discussion of whether triglycerides are considered an independent risk factor, it is undisputed that they play an important role in the pathogenesis of coronary heart disease. The incidence of disease is highest in patients who have a high LDL cholesterol and a high triglyceride level.

The cholinester transfer protein (CETP) is a stable plasma glycoprotein responsible for the transfer of neutral lipids and phospholipids between lipoproteins, which downregulates the plasma concentration of HDL. The inhibition of CETP lipid transfer activity has already been proposed as a therapeutic approach to increase HDL plasma levels. There are a variety of reasons suggesting that the absence of plasma CETP activity should lead to an increase in HDL levels. Thus, CETP lowers the HDL concentration by transferring cholesterol esters from HDL to LDL and VLDL. Transient inhibition of CETP with anti-CETP monoclonal antibodies, antisense oligonucleotides or CETP inhibitors, led to an increase in HDL levels in animal experiments in rabbits and hamsters. Permanent CETP inhibition with antisense oligonucleotides increased the HDL levels and thus led to a reduction in atherosclerotic damage in a rabbit animal model of atherosclerosis. Patients with familial hypercholesterolemia have twice the CETP plasma levels in the heterozygous gene defect as healthy people, while the homozygous genetic defect is even threefold.

No. 5,512,548 and WO 93/011782 describe polypeptides and their analogs which are capable of inhibiting CETP, which catalyzes the transfer of cholesterol esters of HDL to VLDL and LDL, and therefore has anti-atherosclerotic activity when administered to a patient. According to these documents, such a CETP polypeptide inhibitor is derived from apolipoprotein CI from various sources, in particular N-terminal fragments up to amino acid 36 identified as CETP inhibitors.

Also disclosed in US 5,880,095 A is a CETP binding peptide which is capable of inhibiting the activity of CETP in a subject. The CETP-inhibitory protein comprises an N-terminal fragment of the porcine apolipo-protein C-III.

US 2004/0087481 and US Pat. No. 6,410,022 B1 disclose peptides which are obtained by the induction of a CETP-specific immune response for the treatment and prevention of cardiovascular diseases, such as e.g. Atherosclerosis, can be used. These peptides include a T-helper cell epitope that is not derived from CETP and at least one B-cell epitope derived from and directly derived from CETP. The T helper cell epitope is preferably derived from the tetanus toxoid and is covalently bound to at least one B cell epitope of CETP. By using a T helper cell epitope foreign to the organism, it is possible to induce in the body of an individual antibodies directed against that part of the peptide consisting of at least one CETP B cell epitope.

In the recent past, there have already been proposals for a vaccine approach to CETP. For example, rabbits have been treated with a vaccine containing as antigen the peptide of CETP responsible for the cholesterol ester transfer. The immunized rabbits had decreased CETP activity and altered lipoprotein levels with increased HDL and decreased LDL levels. In addition, the treated animals of the atherosclerosis model also showed reduced atherosclerotic lesions compared to control animals.

At the end of last year, the results of a phase II clinical study published by the American biotech company Avant with the CETi-I vaccine were published (BioCentury Extra For Wednesday, October 22, 2003). In this Phase II study, as in the previous Phase I study, a very good safety profile without any demonstrated that there are no side effects to be expected from an anti-CETP inoculation approach. In terms of efficacy, however, the Avant vaccine was disappointing as it did not result in increased HDL levels significantly better than those achieved with placebo treatment.

The problem with the CETi-I vaccine is that it uses a body antigen. The human immune system is tole¬ rant against endogenous structures, since it is vital for the vast majority of the body's own molecules, unlike CETP, that no autoantibodies are formed. Thus, it was the task of the CETi-I vaccine to break the body's own tolerance, which obviously was not sufficient for him.

Thus, it is the object of the present invention to provide an antigen for an anti-CETP vaccine which is selected to be considered foreign by the immune system and therefore need not break any self-tolerance.

The present invention therefore provides a CETP mimotope for these purposes. The CETP mimotopes according to the present invention are preferably antigenic polypeptides which are completely different in their amino acid sequence from the amino acid sequence of CETP or fragments of CETP. The mimotope according to the invention may comprise one or more non-natural amino acids (ie not from the "classic" amino acids) or may be composed entirely of such non-naturally occurring amino acids Inducible antigens may be composed of D- or L-amino acids or combinations of DL-amino acids, and optionally modified by further modifications, ring-closures or derivatizations Suitable antigens inducing anti-CETP antibodies can be made available from peptide libraries Preferably, these peptides are at least 5 amino acids long, more preferably at least 8 amino acids, with preferred lengths up to 11, preferably up to 14 or 20 amino acids can extend. However, according to the invention, even longer peptides can readily be used as anti-CETP antibody-inducing antigens.

For the preparation of such CETP mimotopes (ie anti-CETP anti-body-inducing antigens), of course, phage libraries, peptide libraries, e.g. produced by combinatorial chemistry or obtained by means of high throughput screening techniques for a wide variety of structures. Display: A Laboratory Manual by Carlos F. Barbas (Editor), et al; Willat's WG, Phage display: practicalities and prospects. Plant Mol Biol. 2002 Dec; 50 (6): 837-54 (http: //www.microcollections .de / showpublications .php #).

Furthermore, according to the invention it is also possible to use anti-CETP antibody-inducing antigens based on nucleic acids ("aptamers"), whereby these also have very different

(Oligonucleotide) libraries (eg with 2-180 nucleic acid residues) can be found (eg Burgstaller et al., Curr Opin, Drug Discov. Dev 5 (5) (2002), 690-700, Famulok et al. Acc. Chem. Res. 33 (2000), 591-599; Mayer et al., PNAS 98

(2001), 4961-4965, and many more). The nucleic acid backbone can be provided in the case of anti-CETP antibody-inducing antigens on a nucleic acid base, for example by the natural phophordiester compounds, but also by phosphorotioates or combinations or chemical variations (eg as PNA), whereby the bases used according to the invention are, in particular, U , T, A, C, G, H and mC can be used. The 2 'residues of the nucleotides which can be used according to the present invention are preferably H, OH, F, Cl, NH ₂ , O-methyl, O-ethyl, O-propyl or O-butyl, the nucleic acids also can still be modifi¬ ed differently, that is, for example, with protective groups, as they are commonly used in oligonucleotide synthesis, provided. Anti-CETP antibody-inducing antigens based on aptamers are therefore likewise preferred antigens inducing anti-CETP antibodies in the context of the present invention.

In another aspect, the present invention relates the use of a compound comprising the following amino acid sequence wherein

Xi is an amino acid other than C, X2 is an amino acid other than C, X3 is an amino acid except C, X4 is an amino acid other than C, X5 is an amino acid other than C,

Xe is absent or an amino acid other than C, X7 is absent or an amino acid other than C, Xe is absent or an amino acid other than C, and X1X2X3X4X5X6X7X8 is not a 5-mer, 6-mer, 7-mer or 8-mer polypeptide or is a peptide fragment of the cholesterol ester transport protein (CETP) or a CETP epitope, wherein the compound has a binding ability to an antibody which is specific for the natural CETP glycoprotein, for the preparation of a preventive agent and treatment of atherosclerosis, atherosclerosis-risk diseases and atherosclerosis-sequelae.

Particularly preferred compounds are specific mimotopes for the CETP epitopes known per se, in particular for the epitopes represented by the amino acids 131-142, 451-476, 184-260, 261-331, 332-366, 367-409 and 410-450 of the CETP amino acid sequence, in particular FGFPEHLLVDFLQSLS or CDSGRVRTDAPD.

CEPT total sequence (unprocessed precursor):

10 20 30 40 50 60

I I I I I

MLAATVLTLA LLGNAHACSK GTSHEAGIVC RITKPALLVL NHETAKVIQT AFQRASYPDI

70 80 90 100 110 120 I I I I I

TGEKAMMLLG QVKYGLHNIQ ISHLSIASSQ VELVEAKSID VSIQNVSWF KGTLKYGYTT

130 140 150 160 170 180 I I I I I

AWWLGIDQSI DFEIDSAIDL QINTQLTCDS GRVRTDAPDC YLSFHKLLLH LQGEREPGWI

190 200 210 220 230 240 I I I I I

KQLFTNFISF TLKLVLKGQI CKEINVISNI MADFVQTRAA SILSDGDIGV DISLTGDPVI

250 260 270 280 290 300 I I I I I

TASYLESHHK GHFIYKNVSE DLPLPTFSPT LLGDSRMLYF WFSERVFHSL AKVAFQDGRL

310 320 330 340 350 360 I I I I I

MLSLMGDEFK AVLETWGFNT NQEIFQEWG GFPSQAQVTV HCLKMPKISC QNKGWVNSS

370 380 390 400 410 420 I I I I I

VMVKFLFPRP DQQHSVAYTF EEDIVTTVQA SYSKKKLFLS LLDFQITPKT VSNLTESSSE

430 440 450 460 470 480 I I I I I

SIQSFLQSMI TAVGIPEVMS RLEWFTALM NSKGVSLFDI INPEIITRDG FLLLQMDFGF

490 I PEHLLVDFLQ SLS

The compound of the invention (mimotope) has a preferred length of 5 to 15 amino acids. This compound may be in the vaccine in isolated (peptide) form or may be coupled or complexed to other molecules, such as pharmaceutical carriers or polypeptide, lipid or carbohydrate structures. The mimotopes according to the invention preferably have a (minimum) length of between 5 and 15, 6 and 12 amino acid residues, specifically between 9 and 11. However, the mimotopes can be (covalently or noncovalently) coupled to unspecific linkers or carriers, in particular Peptide linker or protein carrier. Furthermore, the peptide linkers or protein carriers may consist of or contain T cell helper epitopes.

Preferably, the pharmaceutically acceptable carrier is KLH, nustoxoid, albumin binding protein, bovine serum albumin, a dendrimer (MAP, Biol Chem 358: 581), and the adjuvant substances described in Singh et al. , Nat. Biotech. 17 (1999), 1075-1081

(especially those in Table 1 of this document), and O'Hagan et al., Nature Reviews, Drug Discovery 2 (9) (2003), 727-735

(in particular the endogenous immunopotentiating compounds described therein and the delivery systems), or mixtures thereof. In addition, the vaccine composition may contain aluminum hydroxide.

A vaccine comprising the instant compound (mimotope) and the pharmaceutically acceptable carrier may be administered in any suitable manner, e.g. i.V., i.p., i.m., intranasally, orally, subcutaneously, etc. and in any suitable delivery device (O'Hagan et al., Nature Reviews, Drug Discovery 2 (9), (2003), 727-735). Typically, the vaccine will contain the compound of the invention in an amount of 0.1 ng to 10 mg, preferably 10 ng to 1 mg, especially 100 ng to 100 μg or, alternatively, e.g. 100 μmol to 10 μmol, preferably 10 pmol to 1 μmol, in particular 100 pmol to 100 nmol. The vaccine may also contain typical adjuvants, e.g. Buffers, stabilizers, etc. included.

Especially for the vaccine preparation according to the invention for the prevention and treatment of atherosclerosis, atherosclerosis risk disorders and atherosclerosis-sequelae, molecules have been found which contain a peptide which has a binding ability to an antibody which is suitable for natural CETP Glycoprotein is specific, and under the general formula

wherein

Xi is any amino acid or not present, preferably

A, L, I or absent is, provided that Xi is absent, Xe is present,

X 2 is D, G, A, N, L, V, Q or I, in particular L, V, Q or I,

X 3 is H, P, K or R, in particular K or R, X4 is any amino acid (other than C), in particular W, N, S, G, H, Y, D or E,

X5 is H, S, T, P, K or R, in particular K or R, Xe is absent or N, F, H, L, V or I, in particular L, V or I,

X7 does not exist or W, L, V, I, F, N, P or G, in particular P or G,

Xe is absent or any amino acid other than C falls. These molecules are preferably peptides which include or consist of the general peptide sequence described herein as part of a larger peptide molecule. Particularly preferred are one or more peptides selected from the group consisting of ALKNKLP, ALKSKIP, AVKGKLP, ALKHKIP, ALK-HKVP, ALKNKIP, ALKGKIP, ALKYKLP, ALKDKLP, ALKDKVP, AAQKDKVP, LKLHHGTPFQFN, SLPPDHWSLPVQ, QQQLGRDTFLHL or TNHWPNIQDIGG.

In likewise advantageous peptides, the above formula is defined as follows (always of course with the proviso of speci¬ fic binding ability to CETP / CETP fragment): Xi is A, L or I, in particular A, X ₂ is L, V, Q or I, X ₃ is K or R,

X4 is any amino acid (except C), in particular N, S, G, H, Y, D or E,

Xe is absent or L, V or I, X7 is absent or P or G, Xe is absent or any amino acid except C.

According to a further aspect, the present invention relates to a method for isolating a compound which binds to an antibody which is specific for the natural CETP or a CETP fragment, comprising the following steps: - Providing one Peptide Compound Library, encompassing peptides containing the following amino acid sequence:

in which Xi is an amino acid except C, X2 is an amino acid other than C,

X3 is an amino acid other than C,

X4 is an amino acid other than C,

X5 is an amino acid other than C,

Xe is absent or an amino acid other than C,

X7 is absent or an amino acid is other than C,

Xe is absent or an amino acid other than C, and X1X2X3X4X5X6X7X8 is not or comprises a 5-mer, 6-mer, 7-mer, or 8-mer polypeptide fragment of the cholesterol ester transport protein (CETP) or a CETP epitope;

- bringing this peptide library into contact with this antibody, and

Isolation of those members of the peptide library which bind to these antibodies.

Such a process has proven successful in obtaining the CETP mimotopes according to the invention. Antibodies specific for the natural CETP or a CETP fragment have been extensively described in the art or commercially available (e.g., US 6,410,022 or 6,555,113.

Preferably, these peptides are expressed in the library mentioned in individualized i. provided in isolated form, in particular immobilized on a solid surface, such as e.g. with MULTIPIN ™ peptide technology. The library may also be provided as a peptide mixture, and the antibody-peptide complexes may be isolated after anti-body binding. Alternatively, the antibody may be immobilized, and the peptide library (in suspenion or solution) is then contacted with the immobilized antibodies.

Preferably, the screening antibodies (or the members of the peptide library) comprise a suitable marker which makes it possible to detect or isolate the antibody or the antibody: peptide complex when bound to a peptide of the library. Suitable marker systems (including biotinylation, fluorescence, radioactivity, magnetic markers, color-developing markers, secondary antibodies) are readily apparent to those skilled in the art. available.

The library must be constructed to exclude the naturally occurring CETP sequences since vaccination with this sequence is clearly excluded from this invention.

Another suitable technique for isolating the epitopes according to the present invention is screening in phage peptide libraries, such as e.g. in WO 03/020750.

The present invention also relates to a vaccine for the prevention and treatment of atherosclerosis, atherosclerosis risk diseases and atherosclerosis secondary diseases, comprising an antigen which contains at least one peptide selected from the group consisting of ALKNKLP, ALKSKIP, AVKGKLP, ALKHKIP, ALK-HKVP, ALKNKIP, ALKGKIP, ALKYKLP, ALKDKLP, ALKDKVP, AAQKDKVP, LKLHHGTPFQFN, SLPPDHWSLPVQ, QQQLGRDTFLHL or TNHWPNIQDIGG. These peptides are, in addition to the other peptides made available with the present invention, specifically suitable for use in the preparation of a pharmaceutical composition, in particular for atherosclerosis vaccines. These sequences are purely artificial CETP mimotopes. The peptides may be - for vaccination purposes - (covalently or noncovalently) coupled to suitable carriers and may be used as peptide compounds or complexes together with other compounds or moieties, e.g. Adjuvants, peptides or protein carriers, etc., and administered in a suitable manner (as described, for example, in O'Hagan et al., Nature Reviews, Drug Discovery 2 (9) (2003), 727-735).

Finally, the present invention also relates to the use of a CETP mimotope for the preparation of an agent for the prevention and treatment of atherosclerosis, atherosclerosis-risk diseases and atherosclerosis-related diseases. The CETP mimotope according to the invention may have a peptide structure (such as the library peptides screened according to the invention) or (eg as aptamers) other structures (for example based on nucleic acids). It is only important that they have an affinity for anti-bodies against the natural CETP, about those the natural sequences correspond (at least 50% of the binding affinity) but do not include "self-structures".

The invention will be explained in more detail with reference to the following example, but without being limited thereto.

Example:

There is a pronounced inverse relationship between the plasma concentration of cholesterol in high-density lipoproteins (HDLs) and the development of coronary heart disease (CHD) (1). Thus, the risk for CHD is higher as the HDLs become less. Although 33% of patients with CHD have low plasma levels of HDLs, there is currently no effective therapy for increasing the plasma concentration of HDLs. Diet and moderate balance sport are ineffective (2), statins achieve only a small, 5-7% increase in HDL (3), and niacin has side effects and compliance profiles that limit its use (4).

Inhibition of CETP activity has been proposed as a therapeutic approach to increasing plasma HDL levels (5). CETP is a plasma glycoprotein that facilitates the transfer of neutral lipids and phospholipids between lipoproteins and regulates the concentration of plasma HDL (6). Inhibition of CETP activity is expected to increase plasma HDL levels for several reasons. CETP lowers HDL levels by shifting cholesteryl esters from HDLs to VLDLs and LDLs (5). Transient inhibition of CETP in rabbits and hamsters by monoclonal antibodies (7, 8), small molecules (9), or antisense oligonucleotides (10) causes HDL enhancement. Long-lasting CETP inhibition with antisense nucleotides increased plasma HDL and decreased atherosclerotic lesions in a rabbit atherosclerosis model (11). CETP transgenic mice (12) and rats (13) show reduced plasma HDL. People with reduced CETP activity have elevated plasma HDL (14).

Recently, a vaccine approach has been proposed (15). Rabbits were immunized with a human CETP-derived peptide containing a CETP region necessary for neutral lipid transduction. fer function is crucial. Vaccinated rabbits had a reduced CETP activity and an altered lipoprotein profile with a lower LDL and higher HDL concentration. Furthermore, it was found that rabbits vaccinated with CETP had smaller atherosclerotic lesions than control animals.

The problem with the anti-CETP vaccine approach discussed above is that the vaccine formulation has a self-peptide and therefore must break the natural tolerance to self-antigens. The invention describes a CETP mimotope which can be used for vaccination: The mimotop is intended to induce the production of antibodies against CETP. The CETP mimotope has no self-sequence and therefore does not need to break tolerance. Thus, the induction of an anti-CETP antibody response is greatly facilitated. The mimotope is identified with a monoclonal antibody (rnAb) and (commercially available) peptide libraries (e.g., 16). A monoclonal anti-CETP antibody is used which neutralizes CETP activity (17). This rnAb detects a sequence within the C-terminal 26 amino acids of CETP necessary for neutral lipid transfer activity (18).

CETP is a 476 amino acid glycoprotein. The following regions within the protein have been described as being immunogenic: amino acids 132-142 (19) amino acids 451-476 (20, 21) amino acids 184-260 (22) amino acids 261-331 (22) amino acids 332-366 (22) Amino Acids 367-409 (22) Amino Acids 410-450 (22)

Inhibitory as well as non-inhibitory antibodies which detect the above enumerated regions within CETP can be used to detect mimotopes.

The sequences

A monoclonal antibody responsible for mimotope identification is used, detects the CETP-derived amino acid sequence FGFPEHLLVDFLQSLS (= original epitope).

The mimotope has a preferred length of 5 to 15 amino acids. Two different libraries are used in ELISA tests to define the mimotope sequences.

Library 1: This 7m library contains peptides with the following sequences (amino acid positions 1 to 7):

Position 1 all natural AS except C (19 possibilities) position 2 all natural AS except C (19 possibilities) position 3 all natural AS except C (19 possibilities) position 4 all natural AS except C (19 possibilities) position 5 all natural AS except C (19 possibilities) position 6 all natural AS except C (19 possibilities) position 7 all natural AS except C (19 possibilities)

The 7-mer peptides ALKNKLP, ALKSKIP, AVKGKLP, ALKHKIP, ALKHKVP, ALKNKIP, ALKGKIP, ALKYKLP, ALKDKLP and ALKDKVP are examples of mimotopes detected by a monoclonal antibody.

Library 2: This 8-mer library contains peptides with the following sequences (amino acid positions 1 to 8):

Position 1 all natural AS except C (19 possibilities) position 2 all natural AS except C (19 possibilities) position 3 all natural AS except C (19 possibilities) position 4 all natural AS except C (19 possibilities) position 5 all natural AS except C (19 possibilities) position 6 all natural AS except C (19 possibilities) position 7 all natural AS except C (19 possibilities) position 8 all natural AS except C (19 possibilities)

The 8mere peptide AAQKDKVP is an example of a monoclonal antibody-detected mimotope.

Another monoclonal antibody used for mimotope identification detects the CETP-derived amino acid sequence CDSGRVRTDAPD (= original epitope).

The mimotope used for vaccination must be administered in immunogenic form, eg coupled to a carrier. references

(1) Gordon et al 1989: "High-density lipoprotein: the clinical implications of recent studies" N Engl J Med 321: 1311

(2) Stefanick et al 1998: "Effects of diet and exercise in men and men with low levels of HDL cholesterol and high levels of LDL cholesterol" N Engl J Med 339: 12

(3) Schonfeld et al. 1998: "Roel of 3-hydroxy-3-methylglutaryl coenzymes A reductase inhibitors (" statins ") in familial combined hyperlipidemia" Am J Cardiol 81: 43B

(4) King et al 1994: "Evaluation of effects of unmodified niacin on fasting and postprandial plasma lipids in normolipidemic with hypoalphalipoproteinemia" Am J Med 97: 323

(5) TaIl 1993: "Plasma cholesteryl ester transfer protein" J Lipid Res 34: 1255

(6) Barter et al 1994: "Cholesteryl ester transfer protein: its role in plasma lipid transport" Clin Exp Pharmacol Physiol 21: 663

(7) Whitlock et al 1989: "Monoclonal antibody inhibition of cholesterol ester transfer protein activity in the rabbit: effects on lipoprotein composition and high density lipoprotein cholesterol ester metabolism" J Clin Invest 84: 129

(8) Gaynor et al 1994: "Inhibition of cholesteryl ester transfer protein activity in hamster's age HDL lipid composition" Atherosclerosis 110: 101

(9) Kothari et al 1997: "Inhibition of cholesteryl ester protein by CGS 25159 and changes in lipoproteins in hamster" Atherosclerosis 128: 59

(10) Sugano et al 1996: "Changes in plasma lipoprotein cholestrol levels by antisense oligodeoxynucleotides against cholesteryl ester transfer protein in cholesterol-fed rabbits" J Biol Chem 271: 19080

(11) Sugano et al. 1998: "Effect of antisense oligonucleotides against cholesteryl ester transfer protein on the development of atherosclerosis in cholesterol-fed rabbits" J Biol Chem 273: 5033

(12) Agellon et al 1991: "Reduced high density lipoprotein cholesterol in human cholesteryl ester transfer protein transgenic mice" J Biol Chem 266: 10796

(13) Herrera et al 1999: "Spontaneous combined hyperlipidemia, coronary heart disease and decreased survival dahl-salt sensitization" Hypertensive rats transgenic for human cholesteryl ester transfer protein "Nat Med 5: 1383

(14) Koizumi et al 1985: "Deficiency of serum cholesteryl-ester transfer protein activity in patients with familial hyperallophalipoproteinaemia" atherosclerosis 58: 175

(15) Rittershaus et al 2000: "Vaccine-induced antibody inhibition CETP activity in vivo and reduced aortic lesions in a rabbit model of atherosclerosis" Arterioscler thromb vase Biol. 20: 2106

(16) Reineke et al. 2002: "Identification of Distinct Antibody Epitopes and Mimotopes from a Peptide Array of 5520 Randomly Generated Sequences" J Immunol. Methods 267: 37

(17) Swenson et al 1989: "Mechanism of cholesteryl ester transfer protein inhibition by a neutralizing monoclonal antibody and mapping of the monoclonal antibody epitope" J Biol Chem 264: 14318

(18) Wang et al 1992: "Identification of a sequence within the C-terminal 26 amino acids of cholesteryl ester transfer protein responsible for binding a neutralizing monoclonal antibody and necessary for neutral lipid transfer activity" J Biol Chem 267: 17487

19) Thomas et al. 1996: "Mouse monoclonal anti-peptide antibodies speeifie for cholesteryl ester transfer protein (CETP)" Hy bridoma 15 (5): 359

20) Hesler et al 1988: "Monoclonal antibodies to the Mr 74,000 cholesteryl ester transfer protein neutralize all of the cholesterol ester and triglyceride transfer activities in human plasma J Biol Chem 258: 11751

21) Swenson et al 1989: "Mechanism of cholesteryl ester transfer protein inhibition by a neutralizing monoclonal antibody and mapping of the monoclonal antibody epitope" J Biol Chem 264: 14318

22) Roy et al 1996: "Structure-function relationships of human cholesteryl ester transfer protein: analysis using monoclonal antibodies" J Lipid Res 37:22

Claims

claims:

1. Use of a compound comprising the following amino acid sequence

X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈₁ in which

Xi is an amino acid other than C, X _{2 is} an amino acid other than C, X _{3 is} an amino acid except C, X _{4 is} an amino acid other than C, X _{5 is} an amino acid other than C,

X ₆ is absent or an amino acid other than C, X ₇ is absent or an amino acid other than C, X ₈ is absent or an amino acid other than C, and X _{1 is} X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X _{8 is} not or comprises a 5-mer, 6-mer, 7-mer, or 8-mer polypeptide fragment of the cholesterol ester transport protein (CETP) or a CETP epitope, which compound has a binding ability to an antibody useful for the natural CETP glycoprotein is specific for the manufacture of a compound for the prevention and treatment of atherosclerosis, atherosclerosis-risk diseases and atherosclerosis-sequelae.

2. Use according to claim 1, characterized in that the compound is a CETP mimotope for a CETP epitope selected from the epitopes defined by amino acids 131-142, 451-476, 184-260, 261-331, 332-366, 367 409 or 410-450 of the CETP amino acid sequence, in particular FGFPEHLLVDFLQSLS or CDSGRVRTDAPD.

Use according to claim 1, characterized in that the compound is a polypeptide comprising 5 to 15 amino acid residues.

4. Use according to one of claims 1 to 3, characterized ge indicates that the compound is coupled to a pharmaceutically acceptable carrier, preferably KLH, and optionally aluminum hydroxide.

5. Use according to any one of claims 1 to 4, characterized ge indicates that the compound is contained in an amount of 0.1 ng to 10 mg, preferably 10 ng to 1 mg, in particular 100 ng to 100 micrograms.

6. Use according to one of claims 1 to 5, characterized in that the compound comprises the following peptides: ALKNKLP, ALKSKIP, AVKGKLP, ALKHKIP, ALKHKVP, ALKNKIP, ALKGKIP, ALKYKLP, ALKDKLP, ALKDKVP, AAQKDKVP, LKLHHGTPFQFN, SLPPDHWSLPVQ, QQQLGRDTFLHL or TNHWPNIQDIGG.

7. A method of isolating a compound that binds to an antibody that is specific for the natural CETP or a CETP fragment, comprising the following steps:

- Provision of a peptide compound library, encompassing peptides containing the following amino acid sequence:

wherein

Xi is an amino acid other than C,

X _{2 is} an amino acid other than C,

X _{3 is} an amino acid other than C,

X _{4 is} an amino acid other than C,

X _{5 is} an amino acid except C,

X ₆ is absent or an amino acid other than C,

X ₇ is absent or an amino acid is other than C,

X ₈ or an amino acid other than C does not exist, and wherein X1X2X3X4X5X6X7X8 no 5-mer, 6-mer, 7-mer or 8-mer poly peptide fragment of the Cholesterinestertransport protein (CETP) or a CETP epitope is or comprises .

- bringing this peptide library into contact with this antibody, and

8. The method according to claim 7, characterized in that these peptides are provided in said library in individualized form, in particular immobilized on a solid surface.

9. The method according to claim 7 or 8, characterized in that said antibody comprises a suitable marker, the nen NEN detection or its isolation when binding to a peptide of the library allows.

10. A vaccine for the prevention and treatment of atherosclerosis, atherosclerosis-risk diseases and atherosclerosis-sequelae, comprising an antigen containing at least one peptide having a binding ability to an antibody specific for the natural CETP glycoprotein, and un ¬ ter the general formula

wherein

Xi is any amino acid or not present, preferably

A, L, I, or does not exist, that if Xi, with the proviso not present, X ₆ is present,

X _{2 is} D, G, A, N, L, V, Q or I, in particular L, V, Q or I,

X _{3 is} H, P, K or R, in particular K or R,

X _{4 is} any amino acid (except C), in particular W, N, S, G,

H, Y, D or E,

X _{5 is} H, S, T, P, K or R, in particular K or R,

X ₆ is absent or N, F, H, L, V or I, in particular L, V or I,

X ₇ does not exist or W, L, V, I, F, N, P or G, in particular

P or G, is

X ₈ is absent or any amino acid other than C is included, in particular a peptide selected from

Group ALKNKLP, ALKSKIP, AVKGKLP, ALKHKIP, ALKHKVP, ALKNKIP,

ALKGKIP, ALKYKLP, ALKDKLP, ALKDKVP, AAQKDKVP, LKLHHGTPFQFN, SL-

PPDHWSLPVQ, QQQLGRDTFLHL or TNHWPNIQDIGG.

11. Use of a CETP mimotope for the manufacture of an agent for the prevention and treatment of atherosclerosis, atherosclerosis risk diseases and atherosclerosis complications.