NZ722918B2 - PCSK9 Vaccines - Google Patents

Abstract

The present invention relates to a vaccine capable to induce production of antibodies directed to PCSK9 in vivo.

Description

PCSK9 Vaccines The t invention relates to immunogenic peptides capable to induce the formation of antibodies directed to Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9).

Vascular disorders such as hypercholesterolemia, atherosclerosis, coronary heart disease and stoke are one of the main cause of death worldwide and elevated levels of Low y Lipoprotein cholesterol (LDLc) are playing key role in their pathogenesis. Thus, LDLc management is a very important element for a successful treatment of dyslipidemia and atherosclerosis.

The discovery of PCSK9 in 2003 and its identification as the third factor, among LDLR and ApoB-100, involved in the development of the Autosomal Dominant Hypercholesterolemia (ADH), brought a new inside into the mechanism of the ADH disease development. Additionally, cs studies in humans med the connection between LDLc levels, PCSK9 and the onset of coronary heart disease. Link between PCSK9 and elevated LDLc has been also observed in different animal models.

PCSK9 is mainly sed in the liver, intestine and kidney and is also found secreted in the blood stream. It directly interacts with the low density lipoprotein (LDL) receptor (LDLR), and the formed complex is subsequently alized. By binding LDLR, PCSK9 promotes the or ation and results in plasma LDLc elevation. "Gain of function ons" (GOF) in the PCSK9 gene enhance its interaction with the LDLR, leading to markedly higher LDLc levels, subsequent hypercholesterolemia and predisposition to atherosclerosis.

Whereas, PCSK9 "Loss of function mutations" (LOF) ons are linked to reduced risk for coronary heart disease (CHD).

Interestingly, two cases of healthy women having loss of functional PCSK9 and very low LDLc (~ 14mg/dL) highlighted PCSK9 as non-essential and as promising target for lowering LDLc levels in the blood stream.

PCSK9 xpression in wild type mice reduces significantly the hepatic LDLR n, despite the stable mRNA levels, leading to increased circulating LDLc. In comparison, overexpression of PCSK9 in LDLR -/- mice, does not influence the levels of LDLc, confirming the dependence of PCSK9 on LDLR for playing a role in the LDL catabolism. And as expected, PCSK9-/- mice showed a 2.8 fold se in the levels of LDLR and reduction of LDLc compared to wild type animals. Finally, PCSK9 secretion in the circulation was abrogated, upon inactivation of PCSK9 in the liver , confirming the liver as the major organ responsible for the PCSK9 secretion.

Thus, PCSK9 plays a crucial role in LDL catabolism though the direct actions on LDLR. Inhibition of PCSK9 turns to be beneficial for the LDLc levels. Therefore, anti-PCSK9 therapies are promising approach in terms of beneficial modulation of LDLc levels.

PCSK9, also known as neural apoptosis-regulated convertase 1 (NARC-1), is a secreted proteinase K-like subtilase and member of the mammalian proprotein convertase (PC) family. It is synthesized as a ~72kDa proprotein and in order to be functional, the precursor CSK9) is sed for autocatalysis. This results in the formation of a product (aa31- 152) that binds non-covalently tight in 1:1 complex formation with the PCSK9 fragment (aa153-692) in this mature form (~60- 63kDa) PCSK9 is proceeded towards the secretory pathway.

Additionally to this mature PCSK9 form, another truncated (furin cleaved) form of PCSK9 has been observed in many cell lines and blood plasma. Human and mouse plasma contains the two forms of PCSK9, mature (aa153-692) and its truncated version (aa218-692) and in mouse plasma the truncated form can represent up to ~ 50% of the total plasma PCSK9. This furin cleaved truncated PCSK9 is a t of the cleavage of mature PCSK9 (aa153-692) at the site aa218-219, therefore termed as furin/PC5/6A cleavage site. Upon such cleavage a uent PCSK9 truncated fragment (aa219-692) (so called furin d PCSK9) is .

Recent data confirmed the ability of both forms: mature PCSK9 (aa153-692) and furin cleaved PCSK9 (aa219-aa692) to bind the low density lipoprotein receptor (LDLR) and thus regulate its level and therefore LDLc levels.

In the s of binding to LDLR, PCSK9 catalytic domain interacts with LDLR through ent sites. As a result of this binding PCSK9 is positioning itself with the EGF (B) domain of the LDLR in a structural conformation which again strengthens and optimizes this ction through a second site known as region where the furin/PC5/6A cleavage site (aa218-219) is positioned.

Thus, therapeutics targeting the furin/PC5/6A cleavage PCSK9 site (aa218-219) among their ability to abrogate the proper positioning of PCSK9 towards LDLR would be able to block the action of furin on PCSK9. Such eutics will in parallel abrogate the indirect PCSK9/LDLR interaction (positioning), and will inhibit the production of a truncated LDLR binding active form of PCSK9 (aa219-692). This would lead to cial increase of LDLR and therefore beneficial lowering of plasma LDLc.

Clinical trials throughout the last 25 years confirmed the clear benefit in treating cardiovascular diseases and lowering ating LDLc levels by using 3-hydroxy-3 methyl-glutaryl coenzyme A reductase inhibitors (Statins). Statins are acting by inhibiting the hepatic cholesterol biosynthesis leading to subsequent increase in the Sterol-Regulatory Element Binding Protein (hereafter called SREBP). SREBP is a regulator of genes involved in the lipid homeostasis, such as LDLR. And the raised SREBP leads to increase LDLR protein levels and subsequent increase uptake of LDLc from the circulation.

Statins are the most common used y against dyslipidemia. But despite their efficacy, the treatment with s is quite often linked to adverse effects such as raised liver enzymes, muscle pain and myositis. In addition, significant number of statin d patients fails to reach their goals in term of beneficial LDLc management and some of them are even statin intolerant. Interestingly, by acting on the SREBP statins not only increase the LDLR but also elevate the PCSK9 expression, leading to counteracting pharmacologic effect.

Thus, a ation of statins together with an anti-PCSK9 therapy was considered as promising approach for the LDLc management with potency of istic/additive effect in comparison to individual treatment.

And considerably the anti-PCSK9 therapies became even more attractive potential future LDLc modulator. That is suitable not only as a monotherapy, but also as a novel adjuvant therapy for most recommended and used current therapies such as s or other substances such as fibrates or nicotinic acid. Meanwhile, l different gies to suppress the synthesis or function of PCSK9 have been established. During the last decade approaches for inhibiting the synthesis by gene silencing with antisense-oligonucleotides (ASO), locked nucleic acid antisenseoligonucleotides (LNO-ASO) and siRNA have been quite actively developed. In addition, the ASO technology for inhibition of apolipoprotein-B has been successfully applied and recently approved by the Food and Drug Administration (FDA). In fact, ation of siRNA against PCSK9 in monkeys (Macaca ularis) led to a significant reduction of total cholesterol. In general, the outcome from the different methods for PCSK9 gene silencing is controversial and apparently s on the specificity of the approach. Two al phases I using siRNA and LNA-oligonucleotide faced some challenges, and were premature termination for uncertain reasons. However, on the other side a third clinical trial Phase I with inhibiting PCSK9 by siRNA was successfully terminated.

Other promising therapeutic approaches for inhibition of PCSK9-LDLR interaction by mimetic peptides and adnectins have been designed. But despite the ent possibilities for inhibition, one of the most advanced approaches for ion of LDLc by modulating PCSK9 are anti-PCSK9 monoclonal dies.

To date, many clinical studies evaluating anti-PCSK9 onal antibodies are currently ongoing. In Phase I clinical trials with healthy ts, a single dose of anti-PCSK9 monoclonal antibody uced intravenous or subcutaneously was able to reduce the LDLc levels up to 67%. Moreover, the same mAB applied subcutaneously bi-weekly or in 4 weeks interval in subjects on statin treatment ded a reduction of LDLc up to 81%. In addition, a phase II clinical trial with the same monoclonal antibody (bi-weekly treatment) in statin intolerant patients lowered the LDLc in the range of 41-66%. Those studies, confirmed that anti-PCSK9 therapy is efficient not only in healthy ts, but also in statin treated and statin intolerant population. Furthermore, based on the positive outcome from the finalized clinical trials Phase III clinical trials to evaluate the effect of the mAB anti-PCSK9 therapy on patients with history of a prior myocardial infarction or stroke, coronary risk s and coronary syndrome were performed and recently reported as meeting co-primary endpoints.

However, one major issue of the PCSK9 monoclonal antibody therapies is the lack of a long-term persistent LDLc management. 2011/02757 A2, WO 2011/117401 A1, WO 2012/59573 A1 WO 2013/037889 A2 and WO 2013/148284 A1 disclose vaccines with antigenic PCSK9 peptides. Luo et al. (J. Lipid Res. 50(2009): 1581-1588) discloses function and bution of circulating human PCSK9 expressed extrahepatically in transgenic mice.

An object of the present invention is to provide means and methods for reducing LDLc in an individual; it is a specific object of the present invention to provide new antigenic PCSK9 peptides as vaccines with improved antigenic potential and being efficient for reduction of cholesterol in vaccinated individuals.

The present invention concerns specific immunopotentiating ts (amino acid exchanges and optional truncations) of PCSK9 fragment consisting of amino acid residues 209 to 222 SEQ ID No.1.

The peptides of the present invention are so-called VARIOTOPE®s i.e. amino acid variations of the original, native sequence of the peptide PEEDGTRFHRQASK D.No.1). VARIOTOPE®s have an amino acid sequence which is different from the original protein/peptide sequence from which they are derived. The VARIOTOPE®s according to the t invention are considered as foreign by the immune system and ore do not need to break self-tolerance.

The present invention relates to a vaccine, vaccine composition or composition comprising at least one peptide ting of a VARIOTOPE® peptide or a peptide fragment thereof, derived from the PCSK9 nt consisting of amino acid residues 209 to 222 (SEQ ID No.1).

The present invention therefore provides a vaccine comprising at least one peptide consisting of 9 to 25 amino acid residues, said peptide being a variant of the e PEEDGTRFHRQASK D.No.1) with an increased immunogenicity in mammals, especially , ed to PEEDGTRFHRQASK (SEQ.ID.No.1) and wherein said variant is characterised by at least one and at most four amino acid exchanges compared to PEEDGTRFHRQASK (SEQ.ID.No.1).

Preferably, the vaccine according to the present invention comprises at least one peptide consisting of 9 to 25 amino acid residues, said e having or sing the amino acid sequence GX6RFX9X10X11X12X13X14, (SEQ ID No. 46), X1 is an amino acid residue selected from the group consisting of lysine, threonine, alanine and proline, preferably alanine or e, X2 is glutamine or aspartic acid, preferably ic acid, X6 is threonine or serine, X9 is an amino acid residue selected from the group consisting of histidine, alanine and serine X10 is an amino acid residue selected from the group consisting of arginine, alanine, glutamine, lysine, methionine, proline and serine, preferably arginine, serine or alanine, X11 is an amino acid e selected from the group consisting of glutamine, e, glutamic acid, lysine, threonine, and arginine, preferably glutamine, lysine, arginine, and threonine, X12 is an amino acid residue selected from the group consisting of alanine, serine and threonine, preferably alanine or serine, X13 is an amino acid residue selected from the group consisting of serine, alanine, and asparagine, ably serine, X14 is an amino acid e selected from the group consisting of lysine, alanine, arginine, leucine, serine, threonine, and valine, preferably lysine or serine, or a fragment of SEQ.ID.No.46 having at least 9 consecutive amino acid residues, and wherein SEQ ID No. 46 is not PEEDGTRFHRQASK (SEQ.ID.No.1) or an N- or C-terminally truncated fragment thereof.

According to a preferred embodiment of the present invention, the vaccine comprise a peptide that consists or comprises an amino acid sequence selected from the group ting of AEEDGTRFHRQASK, PEEDGTRFARQASK, PEEDGTRFHAQASK, PEEDGTRFHRAASK, PEEDGTRFHRQAAK, PEEDGTRFHRQASA, TEEDGTRFHRQASK, PQEDGTRFHRQASK, PEEDGSRFHRQASK, RFHQQASK, PEEDGTRFHKQASK, PEEDGTRFHMQASK, PEEDGTRFHREASK, PEEDGTRFHRRASK, PEEDGTRFHRKASK, PEEDGTRFHRQSSK, PEEDGTRFHRQANK, PEEDGTRFHRQASR, PEEDGTRFHRQASL, KEEDGTRFHRQASK, PEEDGTRFSRQASK, PEEDGTRFHPQASK, PEEDGTRFHSQASK, PEEDGTRFHRTASK, PEEDGTRFHRQTSK, PEEDGTRFHRQASS, PEEDGTRFHRQAST, PEEDGTRFHRQASV, PEEDGSRFHKQASK, PEEDGSRFHMQASK, PEEDGSRFHRRASK, and PEEDGSRFHRQATK ; preferably AEEDGTRFHRQASK, PEEDGTRFARQASK, PEEDGTRFHAQASK, PEEDGTRFHRAASK, PEEDGTRFHRQASA, TEEDGTRFHRQASK, RFHRQASK, PEEDGTRFHRRASK, PEEDGTRFHRKASK, PEEDGTRFHRQSSK, RFSRQASK, PEEDGTRFHPQASK, PEEDGTRFHSQASK, PEEDGTRFHRTASK, RFHRQTSK, PEEDGTRFHRQASS, PEEDGTRFHRQASV, PEEDGSRFHKQASK, PEEDGSRFHRRASK, and PEEDGSRFHRQATK; especially AEEDGTRFHRQASK, PEEDGTRFHAQASK, PQEDGTRFHRQASK, PEEDGTRFHRRASK PEEDGTRFHRKASK, PEEDGTRFHRQSSK, PEEDGTRFSRQASK, RFHSQASK, PEEDGTRFHRTASK, PEEDGTRFHRQTSK, PEEDGTRFHRQASS, RFHKQASK and PEEDGSRFHRRASK.

The peptides provided with the present invention are immunopotentiating variants of the native PCSK9 amino acid sequence PEEDGTRFHRQASK (SEQ ID No.1). The peptides according to the present ion have been ntly designed and selected to provide an improved immune response against PCSK9 under careful consideration of self-tolerance issues usually connected to vaccines relying on native PCSK9 sequences.

The peptides according to the present invention have amino acid variations ed to the native sequence PEEDGTRFHRQASK (SEQ ID No.1). According to a preferred ment, the number of variations (exchanged amino acid es) does not exceed 3 amino acid residues. ably, the peptide according to the present ion has only one or two amino acid exchanges. It is sing that with such few amino acid exchanges, immunopotentiating peptide variations could be provided, especially with regard to their other beneficial property, namely that these novel peptides do not have to break selftolerance.

Preferred peptides according to the present invention have an increased immunogenicity, compared to the peptide PEEDGTRFHRQASK (SEQ.ID.No.1), of at least 50 %, preferably at least 100 %, especially at least 200%, as evidenced in a serum ELISA, for example as evidenced by the example below.

According to a preferred embodiment, the peptides in the vaccine of the present invention have an increased ability to reduce total cholesterol , compared to the peptide PEEDGTRFHRQASK (SEQ.ID.No.1), of at least 3 %, preferably at least 5 %, especially at least 10%, as evidenced in a serum cholesterol test (in absolute figures, the untreated comparison being 100%), for example as evidenced by the example below. For at least some of the peptides ing to the present invention, such enhanced total cholesterol ng levels compared to the native sequence has been experimentally med in a scientifically accepted terol model.

Accordingly, the t according to the present invention is preferably ed from the group consisting of AEEDGTRFHRQASK (SEQ.ID.No.2), TEEDGTRFHRQASK (SEQ.ID.No.9), RFHRQASK (SEQ.ID.No.10), PEEDGTRFHRRASK (SEQ.ID.No.17), PEEDGTRFHRKASK (SEQ.ID.No.18), PEEDGTRFHRQASR (SEQ.ID.No.23), and RFHRTASK (SEQ.ID.No.36). Although it is already surprising that a variant peptide of the native sequence has an increased genicity, compared to the native sequence peptide PEEDGTRFHRQASK (SEQ.ID.No.1), it was even more surprising that such peptides can also – in a scientifically accepted cholesterol model – reduce total cholesterol more efficient than the corresponding native sequence.

The peptides contained in the vaccines according to the present invention are preferably coupled or fused to a pharmaceutically acceptable carrier, preferably a r protein, especially a protein comprising at least one T cell epitope.

The administration of a vaccine according to the present invention allows treating or ting pathological conditions linked to PCSK9 and its role in diseases such as dyslipidemia, hyperlipidemia, hypercholesterolemia and/or atherosclerosis.

The peptides of the present ion are variants (amino acid exchanges and optional truncations) of the PCSK9 fragment having amino acid sequence PEEDGTRFHRQASK (SEQ ID No.1) and consist of 9 to 25 amino acid residues, preferably 9 to 20 amino acid residues, especially 9 to 15 amino acid residues.

Specifically preferred peptides according to the present invention consist of 10, 11, 12, 13, 14 or 15 amino acid residues, preferably 13 or 14 amino acid residues, especially 14 amino acid residues. Although PCSK9 VARIOTOPE® peptides according to the present invention are preferably used in a form where the peptides t of the sequences given (e.g. SEQ ID Nos. 2-46; optionally with a linker attached to its N- or C-terminus, especially at its inus; said linker being or containing preferably a ne e, especially a cysteine residue at the C-terminus); however, it is also possible to use shortened or longer sequences (see e.g.: it is possible to delete a single amino acid from the N- or C- terminus and arrive at a virtually comparable peptide vaccine with respect to immunogenicity or TC reduction. Preferably such deletion can be made at the C-terminus. In some instances, also longer deletions (i.e. two or more amino acids) may be possible (see specifically: WO 37889 A1, additional experiments provided for IPRP (Figures 5/6)). On the other hand, it is also possible to add amino acid residues N-or C-terminally (preferably C-terminally) without significant change in the properties of these peptides with respect to the present invention (see e.g. Amar et al., AHA presentation 18960 (2014)).

Addition of amino acid es at the N- or C-terminus are preferably additions of r amino acid residues lly occurring at this (added) site, i.e. providing the native sequence extension in such VARIOTOPE®s.

The vaccine of the present invention comprise at least one, at least 2, or at least 3, of the peptides defined herein and allows the active immunization of a mammal, in particular a human individual, where neutralizing antibodies to the PCSK9 are induced by vaccination with derived fragments, especially when coupled or fused to a peptide or polypeptide or a carrier protein (as a T cell epitope comprising molecule).

The peptide/carrier combination is mostly important since peptides of the present invention usually do not have the capacity to induce relevant s of dies when injected without coupling.

Thus the vaccine may comprise a combination of two or more peptides as disclosed herein. However, it is also le that the vaccine of the present invention comprises next to one or more peptides linked to SEQ ID No. 1 and as defined herein also other peptides such as mimotopes (i.e. mutants of PCSK9 fragments ; EP12182241) or fragments of PCSK9 (see e.g. WO 2013/037889).

The peptides of the t invention can be chemically synthesized by methods which are well known in the art. Of course, it is also possible to produce the peptides of the present invention using recombinant methods. The peptides can also be produced in microorganisms such as bacteria, yeast or fungi, in eukaryotic cells such as mammalian or insect cells, or in a recombinant virus vector such as adenovirus, poxvirus, herpes virus, i forest virus, baculovirus, bacteriophage, sindbis virus or Sendai virus. Suitable bacteria for producing the peptides include E. coli, B. subtilis or any other ium that is capable of expressing such peptides. Suitable yeast cells for expressing the peptides of the present ion include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida, Pichia is or any other yeast capable of sing peptides. Corresponding means and methods are well known in the art. Also methods for isolating and purifying recombinantly produced peptides are well known in the art and include e.g. gel filtration, affinity chromatography, ion ge chromatography etc.

The peptides according to the present invention are able to induce antibodies that specifically bind to human PCSK9 and inhibit the PCSK9-mediated degradation of LDLR.

To facilitate isolation of the peptides of the present invention, fusion ptides may be made wherein the peptides are translationally fused (covalently ) to a heterologous polypeptide which enables isolation by affinity chromatography.

Typical heterologous polypeptides are g (e.g. His6; 6 histidine residues), GST-Tag (Glutathione-S-transferase) etc.

The fusion ptide tates not only the purification of the peptides but can also prevent the degradation of the peptides during the purification steps. If it is desired to remove the heterologous ptide after purification the fusion polypeptide may comprise a cleavage site at the junction between the peptide and the heterologous polypeptide. The cleavage site may consist of an amino acid sequence that is cleaved with an enzyme specific for the amino acid sequence at the site (e.g. proteases).

The vaccine and peptides of the present invention can be administered to any kind of mammal including humans. It is however preferred to administer the vaccine and peptides of the present invention to humans. ing to the preferred ment of the present invention at least one peptide sed in the vaccine of the present invention is ed from the group consisting of Seq 2: AEEDGTRFHRQASK, Seq 4: PEEDGTRFARQASK, Seq 5: PEEDGTRFHAQASK, Seq 6: PEEDGTRFHRAASK, Seq 7: PEEDGTRFHRQAAK, Seq 8: PEEDGTRFHRQASA, Seq 9: TEEDGTRFHRQASK, Seq 10: PQEDGTRFHRQASK, Seq 12: PEEDGSRFHRQASK, Seq 13: PEEDGTRFHQQASK, Seq 14: RFHKQASK, Seq 15: PEEDGTRFHMQASK, Seq 16: PEEDGTRFHREASK, Seq 17: PEEDGTRFHRRASK, Seq 18: PEEDGTRFHRKASK, Seq 19: PEEDGTRFHRQSSK, , Seq 21: PEEDGTRFHRQANK, , Seq 23: PEEDGTRFHRQASR, Seq 24: PEEDGTRFHRQASL, Seq 25: RFHRQASK, Seq 29: PEEDGTRFSRQASK, Seq 34: PEEDGTRFHPQASK, Seq 35: PEEDGTRFHSQASK, Seq 36: PEEDGTRFHRTASK, Seq 37: PEEDGTRFHRQTSK, Seq 38: PEEDGTRFHRQASS, Seq 39: PEEDGTRFHRQAST, Seq 40: PEEDGTRFHRQASV, Seq 41: PEEDGSRFHKQASK, Seq 42: PEEDGSRFHMQASK, Seq 43: PEEDGSRFHRRASK, Seq 44: PEEDGSRFHRQATK; and fragments thereof having a length of at least 9 amino acids.

Following peptides that do not have the capacity to elicit a humoral immune response targeting PCSK9, highlight that amino acid exchanges have to be accurately selected to result in immunogenic peptides inducing antibodies efficiently blocking PCSK9 function: Seq 26: PEWDGTRFHRQASK, Seq 28: GFHRQASK, Seq 32: PEEDGTRFGRQASK. ing to a ularly preferred embodiment of the present invention the at least one peptide comprised in the vaccine of the present invention is selected from the group consisting of Seq 2: AEEDGTRFHRQASK, Seq 4: PEEDGTRFARQASK, Seq 5: PEEDGTRFHAQASK, Seq 6: PEEDGTRFHRAASK, Seq 7: PEEDGTRFHRQAAK, Seq 8: PEEDGTRFHRQASA, Seq 9: TEEDGTRFHRQASK, Seq 10: PQEDGTRFHRQASK, Seq 12: PEEDGSRFHRQASK, Seq 13: PEEDGTRFHQQASK, Seq 14: PEEDGTRFHKQASK, Seq 15: PEEDGTRFHMQASK, Seq 16: PEEDGTRFHREASK, Seq 17: PEEDGTRFHRRASK, Seq 18: RFHRKASK, Seq 19: PEEDGTRFHRQSSK, Seq 21: PEEDGTRFHRQANK, Seq 23: PEEDGTRFHRQASR, Seq 24: PEEDGTRFHRQASL, Seq 25: KEEDGTRFHRQASK, Seq 29: PEEDGTRFSRQASK, Seq 34: PEEDGTRFHPQASK, Seq 35: PEEDGTRFHSQASK, Seq 36: PEEDGTRFHRTASK, Seq 37: PEEDGTRFHRQTSK, Seq 38: PEEDGTRFHRQASS, Seq 39: PEEDGTRFHRQAST, Seq 40: PEEDGTRFHRQASV, Seq 41: PEEDGSRFHKQASK, Seq 42: PEEDGSRFHMQASK, Seq 43: PEEDGSRFHRRASK, Seq 44: PEEDGSRFHRQATK.

Table A: Amino acid residues Amino acid Three letter code One letter code alanine ala A arginine arg R asparagine asn N ic acid asp D ne cys C glutamic acid glu E glutamine gln Q glycine gly G histidine his H cine ile I e leu L lysine lys K methionine met M alanine phe F proline pro P serine ser S threonine thr T phan trp W tyrosine tyr Y valine val V According to a particularly preferred embodiment at least one peptide comprised in the vaccine of the present invention comprises at its N- and/or C-terminus at least one cysteine residue bound directly or via a spacer sequence thereto.

This cysteine residue may serve as a reactive group in order to bind the peptide to another molecule or a carrier. For instance, this group may be used to bind the peptide to a carrier protein. The cysteine residue can be bound directly to the peptides of the present invention or via a spacer sequence.

The spacer sequence comprises preferably at least one, preferably at least two, more ably at least three, even more preferably at least four, and optionally a maximum of ten, preferably a minimum of five small lar amino acid residues such as glycines.

It is, however, clear that such cysteine residue or other amino acid linkers (such as, e.g. CG-, CGG-, -GC, -GGC, etc.) are not to be regarded as (N- or C-terminal) ges or variations of the native PCSK9 e epitope PEEDGTRFHRQASK (SEQ.ID.No.1) or fragments thereof, but additions to the epitope sequence eliciting the specific antibody response in the vaccinated individual.

According to a preferred embodiment of the present invention the vaccine according to the present invention comprises a protein r, preferably a protein carrier selected from the group consisting of keyhole limpet haemocyanin (KLH), CRM (preferably CRM197), tetanus toxoid (TT), diphtheria toxin (DT), protein D or any other protein or e containing helper T- cell epitopes.

According to the present ion the e is preferably coupled or fused to a pharmaceutically acceptable carrier, preferably KLH (Keyhole Limpet Hemocyanin), CRM, tetanus toxoid, albumin-binding protein, bovine serum albumin, a dendrimer, peptide linkers (or flanking regions) as well as the adjuvant substances described in Singh et al. (Singh et al., Nat.

Biotech. 17, (1999): 1075 -1081 (in ular those in Table 1 of that document)), and n et al.(O'Hagan and Valiante, Nature Reviews, Drug Discovery 2 (9); ( 2003): 727 -735 (in ular the endogenous immuno-potentiating compounds and delivery systems described therein)), or mixtures thereof. The conjugation chemistry (e.g. via heterobifunctional compounds such as GMBS and of course also others as described in "Bioconjugate Techniques", Greg T. Hermanson) in this context can be selected from reactions known to the skilled man in the atively it is also le to fuse the at least one peptide of the present invention to a protein carrier by methods known in the art. Such proteins comprise a peptide as described herein together with an unrelated immunogenic protein.

Preferably the immunogenic protein is capable of eliciting a recall response. Examples of such ns include tetanus, ulosis, hepatitis proteins and n D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91/18926). Preferably a protein D derivative is used which ses approximately the first third of the protein (e.g., the first N-terminal 100-110 amino acids) and which may be lipidated. Another carrier which may be used to provide fusion proteins may be the protein known as LYTA, or a n thereof (preferably a C-terminal portion). LYTA is derived from Streptococcus pneumoniae, which synthesizes an yl-L- alanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 43; :265-292). LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. Within a red embodiment, a repeat portion of LYTA may be incorporated into a fusion protein. A repeat portion is found in the C-terminal region starting at residue 178. A particularly preferred repeat portion incorporates residues 188-305.

According to a preferred embodiment of the present invention the peptide is formulated with an adjuvant, preferably adsorbed to aluminium hydroxide (Alhydrogel, Al(OH)3).

The vaccine according to the present invention may be formulated with an adjuvant, preferably a low e aluminum composition, in particular aluminum hydroxide. Of course, also nts like MF59, um phosphate, calcium phosphate, cytokines (e.g. IL-2, IL-12, GM-CSF), saponins (e.g. QS21), MDP derivatives, CpG oligonucleotides, LPS, MPL, polyphosphazenes, emulsions (e.g. Freund's, SAF), liposomes, virosomes, iscoms, cochleates, PLG microparticles, mer particles, virus-like particles, heat-labile enterotoxin (LT), cholera toxin (CT), mutant toxins (e.g. LTK63 and LTR72), microparticles and/or polymerized liposomes may be used.

Suitable adjuvants are commercially available as, for example, AS01B, AS02A, AS15, AS-2 and derivatives thereof (GlaxoSmithKline, Philadelphia, PA); CWS, TDM, Leif, aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of ed tyrosine; acylated sugars; ically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable pheres; monophosphoryl lipid A and quil A.

Cytokines, such as GM-CSF or interleukin-2, -7 or -12 may also be used as adjuvants.

Preferred adjuvants for use in eliciting a predominantly pe response include, for e, a combination of monophosphoryl lipid A, preferably 3-O-deacylated monophosphoryl lipid A (3D-MPL), optionally with an aluminum salt (see, for example, Ribi et al., Immunology and Immunopharmacology of Bacterial Endotoxins, Plenum Publ. Corp., NY, : 407-419; GB 2122204B; GB 2220211; and US 4,912,094). A preferred form of 3D-MPL is an on having a small particle size less than 0.2 mm in er, and its method of manufacture is disclosed in WO 94/21292. Aqueous formulations comprising monophosphoryl lipid A and a surfactant have been described in WO 98/43670. Exemplified preferred adjuvants include AS01B (MPL and QS21 in a liposome formulation), 3D-MPL and QS21 in a liposome formulation, AS02A (MPL and QS21 and an oil-in-water emulsion), 3D-MPL and QS21 and an oil-in-water emulsion, and AS 15. MPL adjuvants are disclosed e.g. in US 4,436,727; US 4,877,611; US 4,866,034 and US 4,912,094. ntaining oligonucleotides (in which the CpG dinucleotide is ylated) also induce a predominantly Th1 response. CpG is an abbreviation for cytosine-guanosine dinucleotide motifs present in DNA. Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488, US 6,008,200 and US 5,856,462. Immunostimulatory DNA ces are also described, for example, by Sato et al., Science 273; (1996):352. CpG when formulated into vaccines is generally administered in free solution together with free n (WO 96/02555; McCluskie and Davis, supra) or covalently conjugated to an antigen (WO 98/16247), or formulated with a carrier such as aluminium hydroxide ((Hepatitis surface antigen) Davis et al., supra; Brazolot-Millan et al., PNAS USA, 95(26), (1998):15553-8). CpG is known in the art as being an adjuvant that can be administered by both systemic and mucosal routes (WO 96/02555, EP 0 468 520, Davis et al., J.lmmunol, 160(2), (1998):870-876; McCluskie and Davis, J.Immunol., 161(9), (1998):4463-6).

Another preferred adjuvant is a saponin or saponin mimetics or derivatives, preferably QS21 a Biopharmaceuticals Inc.), which may be used alone or in combination with other adjuvants. For example, an ed system es the combination of a osphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL as described in WO 94/00153, or a less genic composition where the QS21 is quenched with cholesterol as described in WO 39. Other preferred formulations comprise an oil-in-water on and tocopherol. A particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210. onal saponin adjuvants of use in the present invention include QS7 ibed in WO 96/33739 and WO 96/11711) and QS17 (described in US 5,057,540 and EP 0 362 279 B1).

Other preferred adjuvants include Montanide ISA 720 (Seppic, France), SAF (Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2, AS2', AS2, SBAS-4, or SBAS6, available from GlaxoSmithKline), Detox (Corixa), RC-529 (Corixa, on, MT) and other aminoalkyl glucosaminide 4-phosphates (AGPs). Further example adjuvants include synthetic MPL and adjuvants based on Shiga toxin B subunit (see WO 2005/112991). It is particularly red to use aluminium hydroxide as adjuvant.

The vaccine of the present invention may be administered by any suitable route known for es, ably subcutaneously, intramuscularly, intradermally, or intravenously. ing on the route of administration, the ment may comprise respective carriers, adjuvants, and/or excipients.

A vaccine which comprises a peptide of the present invention and the ceutically acceptable carrier may be administered by any suitable mode of application, e.g. intradermally (i.d.), intraperitoneally , intramuscularly (i.m.), intranasally, orally, subcutaneously (s.c.), etc. and in any suitable delivery device (O'Hagan et al., Nature Reviews, Drug Discovery 2 (9), (2003), 727-735). The peptides of the present invention are preferably formulated for intradermal, subcutaneous or intramuscular administration. Means and s for obtaining respective formulations are known to the person skilled in the According to a preferred embodiment of the present invention the vaccine is used for the treatment and/or prevention of disorders caused by hyperlipidemia, hypercholesterolemia and/or atherosclerosis, preferably cardiovascular diseases, stroke or peripheral vascular diseases and other diseases linked to PCSK9, e.g. stic diseases, such as melanoma and liver cancer metastasis (Sun et al, Neoplasia, 14(12) 2012, 1122-1131) in particular in s, preferably in humans.

As outlined, the peptides of the present invention are able to induce the formation of antibodies which are able to bind specifically PCSK9. The interaction of the antibodies with PCSK9 leads to the increase of low density lipoprotein receptor in liver hepatocytes in vivo, increased plasma cholesterol uptake and subsequent reduction of the plasma LDL terol levels and thus the overall cholesterol levels.

In particular, the present invention relates to antibodies able to bind the aa209-222 PCSK9 region and negatively influence the LDLR interaction and ore beneficially reduce plasma cholesterol. Those antibodies also block the cleavage of the mature PCSK9 -692) protein by furin and therefore inhibit the production of LDLR binding truncated form of PCSK9 -692). Additionally, the peptides of the present invention are able to induce the formation of dies which are able to bind PCSK9 in the region 209-222 and inhibit the process of furin cleavage of the mature PCSK9.

The disease associated with atherosclerosis is preferably selected from the group consisting of peripheral arterial occlusive disease, coronary heart disease, apoplectic cerebral insultus and stroke.

The terms "diseases associated with hyperlipidemia, hypercholesterolemia and/or atherosclerosis" and "disorders caused by hyperlipidemia, hypercholesterolemia and/or atherosclerosis" refer to diseases which are a consequence of hyperlipidemia, hypercholesterolemia and atherosclerosis. These es include among others peripheral arterial ive disease, coronary heart disease and apoplectic al insultus (see e.g. Steinberg, D. J Lipid Res 5):179-190 and Steinberg, D. J Lipid Res 47(2006):1339-1351). According to a preferred embodiment of the present invention the peptides of the present invention are administered to a mammal or an individual in an amount of 0.1 ng to 10 mg, preferably of 0.5 to 500 µg, more preferably 1 to 100 µg, per immunization. In a preferred embodiment these amounts refer to all peptides (if more than one peptide is used in the vaccine) present in the vaccine. In r preferred embodiment these amounts refer to each single fragment present in the e. It is of course possible to provide a vaccine in which the peptides are present in different or equal amounts. However, the peptides of the present invention may alternatively be administered to a mammal or an individual in an amount of 0.1 ng to 10 mg, preferably ng to 1 mg, in particular 100 ng to 300 µg/kg body .

The amount of peptides that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.

The dose of the vaccine may vary ing to factors such as the disease state, age, sex and weight of the mammal or individual, and the ability of antibody to elicit a desired response in the dual. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally d as indicated by the exigencies of the eutic situation. The dose of the vaccine may also be varied to provide optimum preventative dose response depending upon the circumstances. For instance, the peptides and vaccine of the present invention may be administered to an individual at intervals of several days, one or two weeks or even months or years depending always on the level of antibodies directed to PCSK9.

In a preferred embodiment of the present invention the peptide/vaccine is applied between 2 and 10, preferably between 2 and 7, and most preferably up to 5. This number of immunizations may lead to a basic immunisation. In a particularly preferred embodiment the time interval between the subsequent vaccinations is chosen to be between 2 weeks and 5 years, preferably between 1 month and up to 3 years, more preferably between 2 months and 1.5 years. An exemplified vaccination schedule may comprise 3 to 4 initial vaccinations over a period of 6 to 8 weeks and up to 6 months, preferably ed with further administrations after such initial ations. Thereafter the vaccination may e.g. be repeated every two to ten years. The repeated administration of the e/vaccine of the present invention may maximize the final effect of a eutic vaccination.

The vaccine of the t invention may also comprise antigens d from other proteins. For examples proteins involved in the regulation of the LDL and/or HDL levels within a human body. For instance, the PCSK9 fragments of the present invention may be ed with epitopes derived from human CETP n. The vaccine of the present invention may also comprise antigens derived from a different epitope of the PCSK9 protein.

The vaccine of the present invention may also comprise antigens derived from other proteins suitable for the ent of hyperlipidemia, hypercholesterolemia and/or atherosclerosis, preferably cardiovascular diseases, stroke or peripheral vascular diseases.

Typically, the vaccine contains the peptides of the present invention in an amount of 0.5 to 500 µg, preferably 1 to 100 µg and atively from 0.1 ng to 10 mg, preferably 10 ng to 1 mg, in ular 100 ng to 100 µg, or, alternatively, e.g. 100 fmol to 10 µmol, preferably 10 pmol to 1 µmol, in particular 100 pmol to 100 nmol. lly, the vaccine may also contain auxiliary nces, e.g. buffers, stabilizers etc.

Yet another aspect of the present invention relates to a method for treating an individual suffering or at risk to suffer from atherosclerosis or a disease ated with atherosclerosis in the course of which a peptide or vaccine according to the present invention is administered to said individual.

Next to the vaccine of the present invention, the individual to be treated may receive also other active ingredients known to influence the LDL and/or HDL levels in humans and mammals such as statins, fibrates, nicotinic acid, cholesterol uptake inhibitor (e.g. ezetimibe), ApoA1 Milano, delipidated HDL, plant sterols. It is particularly preferred to administer to an individual the vaccine of the present invention together (i.e. at the same time, consecutively etc.) with statins. The vaccine of the present invention can also be combined with s like LDL apheresis. LDL apheresis is a form of apheresis to eliminate the cholesterol-containing particle low-density lipoprotein (LDL) from the tream. Typically LDL apheresis works by leading venous blood through a column coated with antibodies to apolipoprotein B (the main n of LDL particles), dextran sulfate or polyacrylate, or by precipitating LDL with heparin at low pH. Respective methods are known to a person skilled in the The term "preventing", as used herein, covers measures not only to prevent the occurrence of disease, such as risk factor reduction, but also to arrest its progress and reduce its consequences once ished.

As used herein, the term "treatment" or grammatical equivalents encompasses the improvement and/or reversal of the symptoms of disease. A compound which causes an improvement in any parameter associated with disease when used in the screening methods of the instant invention may thereby be identified as a therapeutic compound. The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures.

For example, those who may benefit from treatment with compositions and methods of the present invention include those already with a e and/or disorder as well as those in which a e and/or disorder is to be prevented (e.g., using a prophylactic treatment of the present invention).

The present invention is further illustrated by the following e and the figures, however, without being restricted thereto.

Fig. 1 Protein ELISA Figure 1A and 1B show comparison of the mean titers (n=5 mice/group) against human PCSK9 protein d by the indicated sequences.

The data reveal the ability of the selected VARIOTOPEs® to induce higher antibodies titers against human PCSK9 protein in comparison to the native sequence (Seq 1: PEEDGTRFHRQASK).

Fig. 2 % Total terol in comparison to the ve l group set as 100%.

Figure 2A shows ison of the % mean (n=3 mice/group) total cholesterol levels of mice immunized with selected immunogenic OPE®s (Seq 10: PQEDGTRFHRQASK, Seq 17: PEEDGTRFHRRASK, Seq 18: PEEDGTRFHRKASK, Seq 23: PEEDGTRFHRQASR) in comparison to a negative control group immunized with irrelevant peptide and to the native original PCSK9 sequence (Seq 1: PEEDGTRFHRQASK). Note the ability of the VARIOTOPE®s to reduce the total cholesterol levels to similar or even lower levels in comparison to the native sequence.

Figure 2B shows comparison of the % mean (n=5 mice/group) total cholesterol levels of mice immunized with selected immunogenic VARIOTOPE® (Seq 36: RFHRTASK) in comparison to a negative control group immunized with irrelevant peptide and to the native original PCSK9 sequence (Seq 1: RFHRQASK).

Note the ability of the VARIOTOPE® to reduce the total cholesterol levels stronger in comparison to the native sequence.

Figure 2C shows comparison of the % (n=5 mice/group) total cholesterol levels of mice immunized with selected VARIOTOPE® with multiple aa exchanges (Seq 41: PEEDGSRFHKQASK and Seq 44: PEEDGSRFHRQATK) in comparison to a negative control group immunized with irrelevant peptide and to the native original PCSK9 sequence (Seq 1: PEEDGTRFHRQASK). Note the ability of the VARIOTOPE®s to reduce the total cholesterol levels er in comparison to the native sequence.

Fig. 3. Inhibition of furin cleavage and production of truncated furin cleaved PCSK9.

Figure 3 reveals the ability of selected high immunogenic VARIOTOPE®s (Seq 10: PQEDGTRFHRQASK, Seq 17: PEEDGTRFHRRASK, Seq 18: PEEDGTRFHRKASK, Seq 23: PEEDGTRFHRQASR) to induce antibodies that are able to inhibit the furin cleavage of the mature PCSK9 (aa153-692) and thus abrogate the production of truncated PCSK9 (~50kDa product) (aa219-692). The cleavage process is compared to negative control (PCSK9 incubated with plasma from mice injected with irrelevant peptide) and positive controls (huPCSK9 incubated with or without furin).

Figure 4 shows % total terol (TC) in comparison to the negative control group set as 100%.

Figure 5 shows % difference of TC of VARITOPE® treated groups in comparison to the group d with the original sequence set to 0.

Figure 6 shows % difference of TC of VARITOPE® d groups in comparison to the group treated with the original ce set to 0.

Materials and Methods Vaccine: The peptides were conjugated via the heterobifunctional linker GMBS (4-Maleimidobutyric acid N-hydroxysuccinimide ester) to KLH (Keyhole Limpet Hemocyanin).

Animal experiments: BALB/c mice were aneously immunized. Mice had access to food and water ad libitum and were kept under a 12 h light/dark cycle. The age of mice at the beginning of ments was 8 to 10 weeks.

Mice were injected four times in 2 week intervals with 15 µg of net peptide d to KLH and adsorbed to ogel as adjuvant in a volume of 1 ml in total.

Blood was taken approximately 2 weeks after the final injection.

Protein ELISA: To determine the immunogenicity of the vaccines, and thus to identify the amount of PCSK9 specific dies in the plasma of immunized animals, ELISA immunoassay was performed. The ELISA immunoassay generates a signal which can be easily quantified and represents a quantitative e of the amount of vaccine induced PCSK9-specific antibodies. Thus the titers as measured by ELISA correlate directly with the amount ) of target specific antibodies in the plasma sample of treated animals. All plasma samples were collected two weeks after the final immunization and equality treated. In order to have a direct comparison, the tative evaluation by the PCSK9 Protein ELISA immunoassay of the vaccine induced PCSK9-specific antibodies and the comparison to their ve controls (originals sequence and negative control) was performed for all samples simultaneously. For this purpose, ELISA plates were coated with recombinantly expressed human PCSK9 protein.

Unspecific binding was blocked by incubation with blocking buffer (1% BSA in PBS). Appropriate serum dilutions (with a starting dilution of 1:100) were added to the wells, serially diluted 1:2 fold (12 dilution steps) and incubated for approximately 1 hour. Bound antibodies were detected by incubation with anti-mouse IgG antibody, ABTS was added as substrate and the OD at 405 nm was measured. As negative control sera from the control group ed with an irrelevant e were analyzed. The titers were defined as the dilution of the serum where 50% of the ODmax in the assay is reached.

Total Cholesterol assay: All plasma samples were collected two weeks after the final zation and equality treated. Total cholesterol (TC) measurements were med simultaneously for all samples and side by side compared to their related controls (originals sequence and negative control). The simultaneous quantitative measurement of the plasma TC levels in mg/dL was measured by LabAssayTM terol Kit (Wako). In details, upon incubation with the chromogen reagent containing cholesterol esterase, the cholesterol esters in the samples were decomposed into free terol and fatty acids. Subsequently the free cholesterol was oxidized by the cholesterol oxidase, leading to the simultaneously release of hydrogen peroxide. The produced hydrogen peroxide let DAOS and 4-Aminoantipyrin oxidize and condensate quantitatively by peroxidase (HRP), which produced blue pigment. Optical density was measured at 600nm and quantification of the TC was calculated according to a standard curve.

Inhibition of Furin cleavage The furin cleavage reaction was performed with 2 Units Furin (~110ng) (New England Biolabs) in PCSK9 buffer containing 100 mM Hepes Buffer pH 7.5, 5% Triton-X and 1mM CaCl. In details, 4µl mouse plasma from VARIOTOPE®s vaccinated mice was incubated with 250ng biotinylated huPCSK9 (BPS Bioscience) in PCSK9 buffer for 1h at room temperature. Subsequently, 2U Furin g) (New d s) was added to the on solution and incubated over night at RT. The reaction product was analyzed by SDS-PAGE under reducing conditions.

Results: 1. Sequence information and titers against huPCSK9 (ODmax/2): Seq ID Sequence ODmax/2 Seq 1 PEEDGTRFHRQASK 12074 Seq 2 AEEDGTRFHRQASK 36869 Seq 3 PAEDGTRFHRQASK 2774 Seq 4 PEEDGTRFARQASK 21494 Seq 5 RFHAQASK 33442 Seq 6 PEEDGTRFHRAASK 22475 Seq 7 PEEDGTRFHRQAAK 15676 Seq 8 PEEDGTRFHRQASA 23638 Seq 9 TEEDGTRFHRQASK 23944 Seq 10 RFHRQASK 29228 Seq 11 PKEDGTRFHRQASK 6610 Seq 12 RFHRQASK 12636 Seq 13 PEEDGTRFHQQASK 18490 Seq 14 PEEDGTRFHKQASK 17795 Seq 15 PEEDGTRFHMQASK 15556 Seq 16 PEEDGTRFHREASK 18670 Seq 17 PEEDGTRFHRRASK 28996 Seq 18 PEEDGTRFHRKASK 30537 Seq 19 PEEDGTRFHRQSSK 41310 Seq 20 PEEDGTRFHRQATK 10813 Seq 21 PEEDGTRFHRQANK 15303 Seq 22 PEEDGTRFHRQALK 8694 Seq 23 PEEDGTRFHRQASR 14722 Seq 24 PEEDGTRFHRQASL 19471 Seq 25 KEEDGTRFHRQASK 19315 Seq 26 RFHRQASK 840 Seq 27 PEEDKTRFHRQASK 7154 Seq 28 PEEDGTGFHRQASK 0 Seq 29 PEEDGTRFSRQASK 33111 Seq 30 PEEDGTRFTRQASK 9519 Seq 31 PEEDGTRFVRQASK 11850 Seq 32 PEEDGTRFGRQASK 822 Seq 33 PEEDGTRFMRQASK 10651 Seq 34 PEEDGTRFHPQASK 20318 Seq 35 PEEDGTRFHSQASK 29223 Seq 36 PEEDGTRFHRTASK 29984 Seq 37 PEEDGTRFHRQTSK 25826 Seq 38 PEEDGTRFHRQASS 31481 Seq 39 PEEDGTRFHRQAST 13172 Seq 40 PEEDGTRFHRQASV 22678 Seq 41 PEEDGSRFHKQASK 37296 Seq 42 PEEDGSRFHMQASK 18599 Seq 43 PEEDGSRFHRRASK 29435 Seq 44 PEEDGSRFHRQATK 20684 Seq 45 PEEDGSRFHRRATK 11097 Seq 46 X1X2EDGX6RFX9X10X11X12X13X14 These results are also depicted in Fig. 1. According to the present invention, those VARIOPTOPEs® that have the potential to elicit a higher titer against huPCSK9 (e.g. measured as ODmax/2) according to the present example) are regarded as immunopotentiating variants of the native ce PEEDGTRFHRQASK. A preferred VARIOPTOPE® according to the present invention has a pronounced immunopotentiating property (measured e.g. as an ODmax/2 in the present e of above 20000 or as a g this effect to 150%, preferably of doubling of this effect, compared to the native sequence). An even more preferred VARIOPTOPE® according to the present invention has an even more pronounced immunopotentiating property (measured e.g. as an ODmax/2 in the present e of above 25000, preferably of above 30000; or as a tripling of this effect, compared to the native sequence). 2. Total Cholesterol in % compared to the control group set as 100% in mice zed with VARIOTOPE®s with single AA exchange (Seq 10, 17, 18 and 23; Figure 2A and and Seq 36 2B) and multiple exchanges (Seq 41, 44; Figure 2C).

Figure 2A TC in % ve Control 100 PEEDGTRFHRQASK Seq 1 (original sequence) 81 Seq 10 PQEDGTRFHRQASK 78 Seq 17 PEEDGTRFHRRASK 81 Seq 18 PEEDGTRFHRKASK 81 Seq 23 PEEDGTRFHRQASR 70 Figure 2B TC in % Negative Control 100 PEEDGTRFHRQASK Seq 1 (original sequence) 89 Seq 36 PEEDGTRFHRTASK 72 Figure 2C TC in % multiple AA exchanges Negative Control 100 PEEDGTRFHRQASK Seq 1 (original sequence) 81 Seq 41 PEEDGSRFHKQASK 66 Seq 44 PEEDGSRFHRQATK 74 These results are also depicted in Fig. 2. According to the present invention, those VARIOPTOPEs® that have the potential to elicit a comparable reduction of TC ed to the native sequence PEEDGTRFHRQASK are preferred. Of course, an even more preferred VARIOPTOPE® according to the present invention has the y to reduce TC to an even higher amount than the native ce (measured e.g. as a reduction in % TC as measured in the present example of more than 5 %, especially more than 10% (absolute, i.e. compared to the negative control), compared to the native sequence). 3. Western blot analysis reveals the ability of the induced antibodies upon immunization with Seq 10, 17, 18 and 23 to t the formation of ted furin cleaved PCSK9 (a219-692; ~50kDa)(see Fig. 3).

Reduction of total cholesterol (TC) in treated animals Additional experiments were performed in order to r further experimental evidence showing that the capacity of selected VARIOTOPE® vaccine candidates have the ability to reduce total cholesterol (TC) in treated animals to a higher extent than a vaccine containing the corresponding native (original) sequence.

Peptides used for immunizations in the following experiments: Seq 1: RFHRQASK (original native Sequence) Seq 2: AEEDGTRFHRQASK Seq 9: TEEDGTRFHRQASK Seq 10: PQEDGTRFHRQASK Seq 17: PEEDGTRFHRRASK Seq 18: PEEDGTRFHRKASK Seq 23: PEEDGTRFHRQASR Seq 36: PEEDGTRFHRTASK Seq 24: PEEDGTRFHRQASL In order to evaluate the ability of selected VARIOTOPE® vaccine candidates to reduce TC and to compare the magnitude of TC reduction in VARIOTOPE® d animals with the TC reduction in s treated with a vaccine containing the original sequence, 5 to 10 mice per group were injected five times in 2 week als with vaccines containing 1 µg of net peptide. As usual, antigenic peptides were coupled to KLH and adsorbed to 0.2% Alhydrogel as adjuvant in a volume of 1 ml in total. For the described experiments GMP-like material was used. Blood s were taken approximately 2 weeks after the final injection.

In a first experiment, vaccines containing the original sequence and containing the following VARIOTOPE®s were tested: Seq 1: PEEDGTRFHRQASK Seq 9: TEEDGTRFHRQASK Seq 17: PEEDGTRFHRRASK Seq 18: PEEDGTRFHRKASK Seq 23: PEEDGTRFHRQASR In order to include a vaccine known to be able to reduce TC levels in d animals but to be inferior in reducing TC levels compared to the vaccine containing the original sequence the following peptide vaccine was included in this experiment: Seq 24: PEEDGTRFHRQASL As already outlined above e 1A), all vaccine ates are highly immunogenic and have the capacity to induce an antibody response that ively bind to huPCSK9 as well as mouse PCSK9. In order to prove efficacy of the induced antibodies, TC measurements of blood samples derived from individual mice were performed. Figure 4 depicts relative group mean TC values (in %) compared to the control group set as 100%.

In this experiment 5 s per group were immunized. As can be seen, in all vaccine treated groups TC values were significantly reduced in comparison to the control group.

Since the aim of this experiment was to compare VARIOTOPE® treated groups with the group treated with the original native sequence, in Figure 5 the % reduction of TC values of vaccine treated groups compared to the group treated with the al sequence set to 0% is ted.

As depicted in Figure 5 vaccines ning the ces Seq 9: TEEDGTRFHRQASK Seq 17: RFHRRASK Seq 18: PEEDGTRFHRKASK Seq 23: PEEDGTRFHRQASR are 3 to 10% more powerful to reduce TC levels in comparison to the vaccine containing the al sequence. In contrast to this, the vaccine containing the Seq 24: PEEDGTRFHRQASL possesses the ability to reduce TC levels compared to the negative control e 4) but compared to the original peptide containing vaccine the TC levels were higher (+9%).

In a further experiment following VARIOTOPE®s were tested and compared again to Seq 1: Seq 2: AEEDGTRFHRQASK Seq 10: RFHRQASK Seq 36: PEEDGTRFHRTASK In this experiment 10 animals per group were injected five times in 2 week intervals with vaccines containing 1 µg of net peptide. Blood samples were taken again approximately 2 weeks after the final injection. In order to compare TC reduction in these animals directly with the TC values in animals treated with the original sequence % reduction of TC values of VARITOPE® vaccine treated groups compared to the group treated with the original sequence set to 0% is presented in Figure 6.

As shown in Figure 6 all three vaccine candidates tested in this experiment were more powerful in reducing TC levels compared to the vaccine containing the original sequence (-7% to -13%).

Based on the disclosure, the following preferred embodiments can be specifically highlighted: 1. Vaccine comprising at least one peptide consisting of 9 to amino acid residues, said peptide being a variant of the peptide PEEDGTRFHRQASK (SEQ.ID.No.1) with an increased immunogenicity in mammals, especially humans, compared to PEEDGTRFHRQASK D.No.1) and wherein said t is characterised by at least one and at most four amino acid exchanges compared to PEEDGTRFHRQASK (SEQ.ID.No.1), wherein said t is preferably selected from the group consisting of AEEDGTRFHRQASK D.No.2), TEEDGTRFHRQASK (SEQ.ID.No.9), PQEDGTRFHRQASK (SEQ.ID.No.10), RFHRRASK (SEQ.ID.No.17), PEEDGTRFHRKASK (SEQ.ID.No.18), RFHRQASR (SEQ.ID.No.23), and PEEDGTRFHRTASK D.No.36). 2. Vaccine according to embodiment 1, comprising at least one e consisting of 9 to 25 amino acid residues, said e having or comprising the amino acid sequence X1X2EDGX6RFX9X10X11X12X13X14, (SEQ ID No. 46), wherein X1 is an amino acid residue ed from the group consisting of lysine, threonine, alanine and proline, preferably alanine or e, X2 is glutamine or aspartic acid, preferably aspartic acid, X6 is threonine or serine, X9 is an amino acid residue selected from the group consisting of histidine, alanine and serine, X10 is an amino acid residue selected from the group consisting of arginine, alanine, glutamine, lysine, methionine, proline and serine, preferably arginine, serine or alanine, X11 is an amino acid residue selected from the group consisting of glutamine, alanine, glutamic acid, lysine, threonine, and arginine, preferably glutamine, lysine, arginine, and threonine, X12 is an amino acid residue selected from the group consisting of alanine, serine and threonine, ably alanine X13 is an amino acid residue selected from the group consisting of serine, alanine, and asparagine, preferably serine, X14 is an amino acid residue selected from the group consisting of lysine, alanine, arginine, leucine, serine, threonine, and valine, preferably lysine or serine, or a fragment of SEQ.ID.No.46 having at least 9 consecutive amino acid residues, and wherein SEQ ID No. 46 is not RFHRQASK (SEQ.ID.No.1) or a N- or C-terminally truncated fragment thereof. 3. Vaccine according to embodiment 1 or 2, wherein the peptide consists or comprises an amino acid ce selected from the group consisting of AEEDGTRFHRQASK, PEEDGTRFARQASK, RFHAQASK, PEEDGTRFHRAASK, PEEDGTRFHRQAAK, PEEDGTRFHRQASA, TEEDGTRFHRQASK, PQEDGTRFHRQASK, PEEDGSRFHRQASK, PEEDGTRFHQQASK, PEEDGTRFHKQASK, RFHMQASK, PEEDGTRFHREASK, PEEDGTRFHRRASK, PEEDGTRFHRKASK, PEEDGTRFHRQSSK, PEEDGTRFHRQANK, PEEDGTRFHRQASR, RFHRQASL, KEEDGTRFHRQASK, PEEDGTRFSRQASK, PEEDGTRFMRQASK, PEEDGTRFHPQASK, PEEDGTRFHSQASK, PEEDGTRFHRTASK, PEEDGTRFHRQTSK, PEEDGTRFHRQASS, PEEDGTRFHRQAST, PEEDGTRFHRQASV, PEEDGSRFHKQASK, PEEDGSRFHMQASK, PEEDGSRFHRRASK, and RFHRQATK; preferably AEEDGTRFHRQASK, PEEDGTRFARQASK, PEEDGTRFHAQASK, PEEDGTRFHRAASK, PEEDGTRFHRQASA, TEEDGTRFHRQASK, PQEDGTRFHRQASK, PEEDGTRFHRRASK, PEEDGTRFHRKASK, PEEDGTRFHRQSSK, PEEDGTRFSRQASK, PEEDGTRFHPQASK, PEEDGTRFHSQASK, RFHRTASK, PEEDGTRFHRQTSK, PEEDGTRFHRQASS, PEEDGTRFHRQASV, PEEDGSRFHKQASK, PEEDGSRFHRRASK, and PEEDGSRFHRQATK; especially AEEDGTRFHRQASK, PEEDGTRFHAQASK, PQEDGTRFHRQASK, PEEDGTRFHRRASK PEEDGTRFHRKASK, PEEDGTRFHRQSSK, PEEDGTRFSRQASK, PEEDGTRFHSQASK, RFHRTASK, PEEDGTRFHRQTSK, PEEDGTRFHRQASS, RFHKQASK and PEEDGSRFHRRASK. 4. Vaccine according to any one of embodiments 1 to 3, wherein said at least one peptide is coupled or fused to a ceutically acceptable carrier.

. Vaccine according to any one of ments 1 to 4, wherein the at least one peptide comprises at its N- and/or C-terminus at least one cysteine residue bound directly or via a spacer sequence o. 6. Vaccine according to embodiment 4 or 5, wherein the pharmaceutically acceptable carrier is a protein carrier. 7. Vaccine according to embodiment 6, wherein the protein carrier is selected from the group consisting of keyhole limpet haemocyanin (KLH), tetanus toxoid (TT), CRM197, protein D or a diphtheria toxin (DT), preferably a mutated diphtheria toxin, CRM197, or KLH, especially KLH. 8. Vaccine ing to any one of ments 1 to 7, wherein the e is formulated with an adjuvant, preferably with Al(OH)3 rogel). 9. Vaccine according to any one of embodiments 1 to 8, wherein said at least one peptide consists of 9 to 20 amino acid residues, especially 9 to 15 amino acid residues.

. Vaccine according to any one of embodiments 1 to 9, n said at least one peptide consists of 10, 11, 12, 13, 14 or 15 amino acid residues, preferably 13 or 14 amino acid residues, especially 14 amino acid residues. 11. Vaccine according to any one of embodiments 1 to 10, comprising at least 2, at least 3, or at least 4 of said peptides consisting of 9 to 25 amino acid residues. 12. Vaccine according to any one of embodiments 1 to 11, comprising the at least one peptide in an amount of 0.1 ng to 10 mg, preferably of 0.5 to 500 µg, more preferably 1 to 100 µg. 13. e according to any one of embodiments 1 to 12, wherein the peptide has an increased immunogenicity, compared to the peptide PEEDGTRFHRQASK (SEQ.ID.No.1), of at least 50 %, preferably at least 100 %, especially at least 200%, as evidenced in a serum ELISA. 14. Vaccine according to any one of embodiments 1 to 13, wherein the peptide has an increased ability to reduce total cholesterol levels, compared to the peptide PEEDGTRFHRQASK (SEQ.ID.No.1), of at least 3 %, ably at least 5 %, especially at least 10%, as evidenced in a serum cholesterol test.

. Vaccine according to any one of embodiments 1 to 14 for use in a method for treating and/or preventing disorders caused by hyperlipidemia, hypercholesterolemia and/or atherosclerosis, preferably cardiovascular diseases, stroke or eral vascular diseases, or neoplastic diseases, preferably melanoma and liver cancer metastasis linked to PCSK9. 16. P eptide consisting of 9 to 25 amino acid residues, said e being a variant of the e PEEDGTRFHRQASK (SEQ.ID.No.1) with an increased immunogenicity in mammals, especially humans, compared to PEEDGTRFHRQASK (SEQ.ID.No.1) and wherein said variant is characterised by at least one and at most four amino acid exchanges compared to PEEDGTRFHRQASK (SEQ.ID.No.1). 17. Peptide according to ment 16, consisting of 9 to 25 amino acid residues, said peptide having or sing the amino acid sequence X1X2XEDGX6RFX9X10X11X12X13X14, (SEQ ID No. 46), wherein X1 is an amino acid residue selected from the group consisting of lysine, threonine, alanine and proline, preferably alanine or proline, X2 is glutamine or aspartic acid, ably aspartic acid, X6 is threonine or serine, X9 is an amino acid residue selected from the group ting of histidine, alanine and serine X10 is an amino acid residue selected from the group consisting of arginine, alanine, glutamine, lysine, methionine, proline and serine, preferably ne, serine or alanine, X11 is an amino acid residue selected from the group consisting of glutamine, alanine, glutamic acid, , threonine, and arginine, preferably glutamine, lysine, arginine, and threonine, X12 is an amino acid residue selected from the group consisting of alanine, serine and threonine, preferably alanine or serine, X13 is an amino acid residue selected from the group consisting of serine, e, and asparagine, preferably serine, X14 is an amino acid residue selected from the group consisting of lysine, alanine, arginine, leucine, serine, threonine, and valine, preferably lysine or serine, or a fragment of SEQ.ID.No.46 having at least 9 consecutive amino acid es, and n SEQ ID No. 46 is not PEEDGTRFHRQASK (SEQ.ID.No.1) or a N- or C-terminally truncated nt thereof. 18. Peptide according to embodiment 16 or 17, wherein the peptide ts or comprises an amino acid sequence selected from the group ting of AEEDGTRFHRQASK, PEEDGTRFARQASK, PEEDGTRFHAQASK, PEEDGTRFHRAASK, PEEDGTRFHRQAAK, RFHRQASA, TEEDGTRFHRQASK, PQEDGTRFHRQASK, RFHRQASK, PEEDGTRFHQQASK, PEEDGTRFHKQASK, PEEDGTRFHMQASK, PEEDGTRFHREASK, PEEDGTRFHRRASK, PEEDGTRFHRKASK, PEEDGTRFHRQSSK, PEEDGTRFHRQANK, RFHRQASR, PEEDGTRFHRQASL, KEEDGTRFHRQASK, PEEDGTRFSRQASK, PEEDGTRFMRQASK, PEEDGTRFHPQASK, PEEDGTRFHSQASK, PEEDGTRFHRTASK, PEEDGTRFHRQTSK, PEEDGTRFHRQASS, PEEDGTRFHRQAST, PEEDGTRFHRQASV, PEEDGSRFHKQASK, PEEDGSRFHMQASK, PEEDGSRFHRRASK, and PEEDGSRFHRQATK; p referably AEEDGTRFHRQASK, PEEDGTRFARQASK, RFHAQASK, PEEDGTRFHRAASK, PEEDGTRFHRQASA, TEEDGTRFHRQASK, RFHRQASK, PEEDGTRFHRRASK, PEEDGTRFHRKASK, PEEDGTRFHRQSSK, PEEDGTRFSRQASK, PEEDGTRFHPQASK, PEEDGTRFHSQASK, PEEDGTRFHRTASK, PEEDGTRFHRQTSK, PEEDGTRFHRQASS, PEEDGTRFHRQASV, PEEDGSRFHKQASK, PEEDGSRFHRRASK, and PEEDGSRFHRQATK; especially AEEDGTRFHRQASK, PEEDGTRFHAQASK, PQEDGTRFHRQASK, PEEDGTRFHRRASK PEEDGTRFHRKASK, PEEDGTRFHRQSSK, PEEDGTRFSRQASK, PEEDGTRFHSQASK, PEEDGTRFHRTASK, PEEDGTRFHRQTSK, PEEDGTRFHRQASS, PEEDGSRFHKQASK and PEEDGSRFHRRASK. 19. Peptide according to any one of embodiments 16 to 18, wherein the peptide consists of 9 to 20 amino acid es, especially 9 to 15 amino acid residues.

. Peptide according to any one of embodiments 16 to 19, wherein the peptide ts of 10, 11, 12, 13, 14 or 15 amino acid residues, preferably 13 or 14 amino acid residues, especially 14 amino acid residues. 21. Peptide according to any one of embodiments 16 to 20, wherein the peptide has an increased immunogenicity, ed to the peptide PEEDGTRFHRQASK (SEQ.ID.No.1), of at least 50 %, preferably at least 100 %, especially at least 200%, as evidenced in a serum ELISA. 22. Peptide according to any one of embodiments 16 to 21, n the peptide has an sed ability to reduce total cholesterol levels, ed to the peptide PEEDGTRFHRQASK (SEQ.ID.No.1), of at least 3 %, preferably at least 5 %, especially at least 10%, as evidenced in a serum terol test. 23. Method for treatment of patients having or having a risk of developing disorders caused by ipidemia, hypercholesterolemia and/or atherosclerosis, comprising administering to the patient an effective amount of a vaccine according to any one of embodiments 1 to 15. 24. Method according to embodiment 23, wherein the er is selected from the group consisting of cardiovascular diseases, stroke or peripheral vascular diseases.

. Method according to embodiment 23 or 24, wherein preferably subcutaneously, intramuscularly, intradermally, or intravenously. 26. Method according to any one of embodiments 23 to 25, wherein the vaccine administered contains the at least one peptide in an amount of 0.1 ng to 10 mg, preferably of 0.5 to 500 µg, especially 1 to 100 µg. 27. Method according to any one of ments 23 to 26, wherein the vaccine is applied between 2 and 10, preferably between 2 and 7, and most preferably up to 5 times to the patient. 28. Method ing to any one of ments 23 to 27, wherein the vaccine is administered at least twice and wherein the interval of administration is between 2 weeks and 5 years, preferably between 1 month and up to 3 years, more preferably between 2 months and 1.5 years. 29. Method according to any one of embodiments 23 to 28, wherein the vaccine is administered for 3 to 4 initial vaccinations over a period of 6 to 8 weeks and up to 6 months, preferably followed with further administrations after such initial vaccinations. 1. Vaccine comprising at least one peptide, selected from the group consisting of PEEDGTRFHRRASK (SEQ.ID.No.17), PEEDGTRFHRKASK (SEQ.ID.No.18), PEEDGTRFHRTASK (SEQ.ID.No.36), PEEDGSRFHKQASK (SEQ.ID.No.41), and PEEDGSRFHRQATK (SEQ.ID.No.44). 2. Vaccine according to claim 1, wherein said at least one peptide is coupled or fused to a pharmaceutically acceptable carrier. 3. e according to claim 1 or 2, wherein the at least one e comprises at its N- and/or C-terminus at least one cysteine residue bound directly or via a spacer sequence thereto. 4. e according to claim 2 or 3, wherein the pharmaceutically acceptable carrier is a protein carrier.

. Vaccine according to claim 4, wherein the protein carrier is selected from the group consisting of keyhole limpet yanin (KLH), tetanus toxoid (TT), CRM197, protein D or a diphtheria toxin (DT), preferably a mutated diphtheria toxin, CRM197, or KLH, especially KLH. 6. Vaccine according to any one of claims 1 to 5, wherein the vaccine is formulated with an adjuvant, ably with Al(OH)3 (Alhydrogel). 7. Vaccine according to any one of claims 1 to 6 for use in a method for treating and/or preventing disorders caused by hyperlipidemia, holesterolemia and/or atherosclerosis, preferably vascular diseases, stroke or peripheral vascular es, or neoplastic diseases, preferably melanoma and liver cancer metastasis linked to PCSK9. 8. Peptide consisting of an amino acid sequence selected from the group consisting of PEEDGTRFHRRASK (SEQ.ID.No.17), PEEDGTRFHRKASK (SEQ.ID.No.18), PEEDGTRFHRTASK (SEQ.ID.No.36), PEEDGSRFHKQASK (SEQ.ID.No.41), and PEEDGSRFHRQATK (SEQ.ID.No.44). 9. e according to claim 8, for use for treatment of patients having or having a risk of developing disorders caused by hyperlipidemia, hypercholesterolemia and/or atherosclerosis.

. Use of a vaccine comprising at least one peptide selected from the group consisting of PEEDGTRFHRRASK (SEQ.ID.No.17), PEEDGTRFHRKASK (SEQ.ID.No.18), PEEDGTRFHRTASK (SEQ.ID.No.36), PEEDGSRFHKQASK (SEQ.ID.No.41), and PEEDGSRFHRQATK D.No.44), for the manufacture of a medicament for the treatment of a condition associated with high plasma LDL cholesterol levels. 11. Use of a vaccine according to claim 10, wherein said condition is selected from the group consisting of hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular e, stroke, peripheral vascular disease, peripheral al e, occlusive e, coronary heart e, apoplectic cerebral insultus; stic diseases, melanoma, and liver cancer metastasis linked to PCSK9. 12. Use of a peptide consisting of an amino acid sequence selected from the group consisting of PEEDGTRFHRRASK (SEQ.ID.No.17), PEEDGTRFHRKASK (SEQ.ID.No.18), PEEDGTRFHRTASK (SEQ.ID.No.36), PEEDGSRFHKQASK (SEQ.ID.No.41), and PEEDGSRFHRQATK (SEQ.ID.No.44), for the manufacture of a medicament for the treatment of a condition associated with high plasma LDL cholesterol . 13. Use of a peptide ing to claim 12, wherein said condition is selected from the group consisting of hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular disease, stroke, peripheral vascular disease, peripheral arterial disease, occlusive disease, coronary heart disease, ctic cerebral insultus; neoplastic diseases, melanoma, and liver cancer metastasis linked to PCSK9.

Figure 1A Figure 1B Figure 2A Figure 2B Figure 2C Figure 3 Figure 4 Figure 5 Figure 6 SEQUENCE G <110> Affiris AG <120> Vaccine <130> R 67027 <150> EP 14157221.4 <151> 201428 <160> 46 <170> PatentIn version 3.5 <210> 1 <211> 14 <212> PRT <213> cial Sequence <220> <223> Artificial ce, Mimotope <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa is an amino acid residue selected from the group of uncharged amino acid residues, preferably selected from the group consisting of serine, threonine, valine and alanine <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa is an amino acid residue selected from the group of uncharged amino acid es, preferably selected from the group of isoleucine, valine, e, glutamine and alanine, more preferably isoleucine, valine, glutamine and alanine <220> <221> MISC_FEATURE <222> (3)..(3) <223> Xaa is an amino acid e selected from the group of uncharged amino acid residues, preferably selected from the group consisting of proline, threonine, alanine and valine, more preferably proline <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa is an amino acid residue selected from the group consisting of asparagine, serine, alanine, glutamine and aspartic acid <220> <221> MISC_FEATURE Page | 1

Claims (13)

Claims:

1. Vaccine comprising at least one peptide, selected from the group consisting of PEEDGTRFHRRASK (SEQ.ID.No.17), PEEDGTRFHRKASK (SEQ.ID.No.18), PEEDGTRFHRTASK (SEQ.ID.No.36), PEEDGSRFHKQASK (SEQ.ID.No.41), and PEEDGSRFHRQATK (SEQ.ID.No.44).

2. Vaccine according to claim 1, wherein said at least one peptide is coupled or fused to a pharmaceutically acceptable carrier.

3. Vaccine according to claim 1 or 2, wherein the at least one peptide comprises at its N- and/or C-terminus at least one cysteine residue bound directly or via a spacer sequence thereto.

4. Vaccine according to claim 2 or 3, wherein the pharmaceutically acceptable carrier is a protein carrier.

5. Vaccine according to claim 4, wherein the protein carrier is selected from the group consisting of keyhole limpet haemocyanin (KLH), tetanus toxoid (TT), CRM197, protein D or a diphtheria toxin (DT), preferably a mutated diphtheria toxin, CRM197, or KLH, especially KLH.

6. Vaccine according to any one of claims 1 to 5, wherein the vaccine is formulated with an adjuvant, preferably with Al(OH)3 (Alhydrogel).

7. Vaccine according to any one of claims 1 to 6 for use in a method for treating and/or preventing disorders caused by hyperlipidemia, hypercholesterolemia and/or atherosclerosis, preferably cardiovascular diseases, stroke or peripheral vascular diseases, or neoplastic diseases, preferably melanoma and liver cancer metastasis linked to PCSK9.

8. Peptide consisting of an amino acid sequence selected from the group consisting of PEEDGTRFHRRASK (SEQ.ID.No.17), PEEDGTRFHRKASK (SEQ.ID.No.18), PEEDGTRFHRTASK (SEQ.ID.No.36), PEEDGSRFHKQASK (SEQ.ID.No.41), and PEEDGSRFHRQATK (SEQ.ID.No.44). 37

9. Peptide according to claim 8, for use for treatment of patients having or having a risk of developing disorders caused by hyperlipidemia, hypercholesterolemia and/or atherosclerosis.

10. Use of a vaccine comprising at least one peptide selected from the group consisting of PEEDGTRFHRRASK (SEQ.ID.No.17), PEEDGTRFHRKASK (SEQ.ID.No.18), PEEDGTRFHRTASK (SEQ.ID.No.36), PEEDGSRFHKQASK (SEQ.ID.No.41), and PEEDGSRFHRQATK (SEQ.ID.No.44), for the manufacture of a medicament for the treatment of a condition associated with high plasma LDL cholesterol levels.

11. Use of a vaccine according to claim 10, wherein said condition is selected from the group consisting of hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular disease, stroke, peripheral vascular disease, peripheral arterial disease, occlusive disease, coronary heart disease, apoplectic cerebral insultus; neoplastic diseases, melanoma, and liver cancer metastasis linked to PCSK9.

12. Use of a peptide consisting of an amino acid sequence selected from the group consisting of PEEDGTRFHRRASK (SEQ.ID.No.17), PEEDGTRFHRKASK (SEQ.ID.No.18), PEEDGTRFHRTASK (SEQ.ID.No.36), PEEDGSRFHKQASK (SEQ.ID.No.41), and PEEDGSRFHRQATK (SEQ.ID.No.44), for the manufacture of a medicament for the treatment of a condition associated with high plasma LDL cholesterol levels.

13. Use of a peptide according to claim 12, wherein said condition is selected from the group consisting of hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular disease, stroke, peripheral vascular disease, peripheral arterial disease, occlusive disease, coronary heart disease, apoplectic cerebral insultus; neoplastic diseases, melanoma, and liver cancer metastasis linked to PCSK9.