WO2010057242A2 - Vaccine - Google Patents

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Publication number
WO2010057242A2
WO2010057242A2 PCT/AT2009/000452 AT2009000452W WO2010057242A2 WO 2010057242 A2 WO2010057242 A2 WO 2010057242A2 AT 2009000452 W AT2009000452 W AT 2009000452W WO 2010057242 A2 WO2010057242 A2 WO 2010057242A2
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Prior art keywords
seq id
peptide
according
vaccine
characterized
Prior art date
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PCT/AT2009/000452
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German (de)
French (fr)
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WO2010057242A3 (en )
Inventor
Sylvia Brunner
Pola Linzmayer-Hirt
Walter Schmidt
Bettina Wanko
Gabriele Winsauer
Christina Woess
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Affiris Ag
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/0005Vertebrate antigens
    • A61K39/0012Lipids; Lipoproteins
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/48Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424Serine endopeptidases (3.4.21)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505Inorganic adjuvants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/60Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031Proteins
    • A61K2039/6081Albumin; Keyhole limpet haemocyanin [KLH]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Abstract

The present invention relates to a vaccine, comprising at least one peptide derived from the amino acid sequence TLGTNFGRCVDLFAPGEDII- GASSDCSTCFVSQSGTSQAAAHVAGIA (SEQ ID No. 1) and having a minimum length of 7 amino acids, wherein the peptide also comprises the amino acid sequence DIIGA (SEQ ID No. 2) and/or CFVSQSG (SEQ ID NO. 3) of SEQ ID No. 1.

Description

vaccine

The present invention relates to a vaccine for the treatment or prevention of health problems that can be caused by atherosclerosis, particularly stroke, cardiovascular disease (which can lead to heart attack), and diseases of peripheral vessels ( "peripheral vas- cular disease").

Disease due to atherosclerosis, especially diseases of the coronary vessels are, with 50% the most common cause of death in the Western world. Atherosclerosis is characterized inter alia by deposition of lipid particles in large arteries. In a multi-step process that continues for many years to decades, atherosclerosis leads to constriction of the vessels concerned. Depending on the location of the vessels cardiovascular disease (heart attack), stroke and peripheral vascular disease may be the result. A major cause of atherosclerosis through lifestyle (primarily lack of exercise, high-fat diet, smoking) and / or genetic factors related problems with fat metabolism, ie high total cholesterol or in particular to high levels of LDLc ( "low density lipoprotein cholesterol") in combination with low HDLc values ​​( "high density lipoprotein cholesterol").

High LDLc values ​​directly correlate with the incidence of cardiovascular disease. The most common form of hereditary high LDLc levels is the autosomal dominant hybrid percholesterinämie (ADH). Thus related three genes: the LDL receptor (LDLR), apolipoprotein B-100 (the ligand for the LDLR) and the Proteinkonvertase PCSK9 ( "proprotein convertase subtilisin kexin 9").

The family of secretory proprotein (PC) includes seven specific for basic amino acids subtilisin-like serine proteases ( "subtilisin-like serine proteina- ses w), namely PCl / 3, PC2, furin, PC4, PC5 / 6, PACE4 and PC7 , Also includes two additional PC to the cutting of non-basic amino acids, namely SKI-1 / S1P and PCSK9 (also NARC-I ( "neural apoptosis-regulated convertase 1") called).

With the exception of the PC4 convertases in the brain and peripheral organs are expressed. They have different functions radio, for example for the production of neuropeptides, growth factors, cytokines, receptors, cell adhesion and cell migration, growth and differentiation of progenitor cells. For several convertases is known to play a role in diseases such as cancer, or viral infections. Conditionally off of PC5 / 6 mice results in malformations and bone defects in embryos, presumably due to ausbleibendes processing of the growth differentiation factor 11 ( "growth differentiating factor tion 11") is due.

Some members of the family of the convertases affect lipid metabolism:

- SKI-1 / S1P activates the synthesis of cholesterol and fatty acids and the LDL receptor (LDLR)

- Furin, PC5 and PACE4 inactivate endothelial lipase and lipoprotein lipase

- PCSK9 inactivates the low density lipoprotein receptor (LDLR).

The gene for the human PCSK9 protein is located on chromosome Ip32.3. The gene is about 22 kilo base pairs in length and encodes a 692 amino acids (aa) long glycoprotein. The expression of PCSK9 is "sterol regulatory element binding proteins" (SREBPs) and statins regulated, which is the case with other important in cholesterol metabolism genes. PCSK9 is expressed mainly in the liver, but also in the small intestine and kidney and secreted. The 74 kDa pro-protein is processed autocatalytically in the endoplasmic reticulum and causes both the approximately 60 kDa protein.

PCSK9 is made

- a 30 AS long signal peptide

- the propeptide or inhibitorisehen domain (aa 31-152)

- the "subtilisin-like catalytic domain" (AS 153-452) and

- the C-terminal domain ( "cysteine-rich unique C-terminal domain", CRD, AS 453-692).

PCSK9 plays a crucial role in Cholesterinstoff- change because it directly regulates the amount of existing LDLR in the liver cells. The LDLR is a localized in the plasma membrane glycoprotein that cholesterol LDLc particles removed by endocytosis from the plasma. The binding of PCSK9 leads for receiving the LDLR in the liver cells and then to its degradation in lysosomes in contrast to recycling cell surface direction, that would occur without binding of PCSK9.

In addition PCSK9 appears to have an effect on the receptors for APOE (ApoER2) and VLDL ( "Very Low Density Lipoprotein").

In in vivo experiments in mice it was shown that the amount of PCSK9 sufficient in human plasma in order to reduce the amount of LDLR on the liver cells. In addition, some naturally occurring mutations in the PCSK9 gene have been identified in recent years, resulting in increased ( "gain-of-function mutations") or reduced ( "loss-of-function mutations") activity of the protein.

lead both in humans and in mice "gain of FUNC tion mutations" to decreased levels of LDLR in the liver. This causes large amounts of LDLc in plasma (Hypercholesterinä- mie), indicating a predisposition to atherosclerotic disease of the coronary arteries (coronary artery disease , CHD) causes.

The over-expression of PCSK9 in mice leads to higher LDLc levels. The Administration of recombinantly produced PCSK9 protein is reduced in the mouse model, the number of LDL receptors dramatically.

"Loss-of-function mutations" cause greater amounts of LDLR, resulting in lower LDLc values. This Hypoeholesteri- Naemie proves protection against diseases of the coronary vessels.

"Nonsense" mutations are common in some populations, for example, have about 2% of African Americans one known of two mutations that lead to a reduction in LDLc levels by -30%. In Caucasians a "missense" mutation is which lowers the LDLc to ~ 15%. These lower LDLc levels lead to a significantly reduced incidence of CHD.

PCSK9- "knock-out" mice have more LDLR on their liver cells, and reduced levels of LDLc in the plasma. The inhibition of PCSK9 leads to a significant reduction of total cholesterol and LDLc in mice that are set to high-fat food. At the same the amount of LDLR in the liver is doubled.

There are no negative effects of a lack of PCSK9 known., Congenital homozygous "knock-out" of PCSK9 leads people to extremely low levels of LDLc with no apparent side effects. Inhibition of PCSK9, for example by antisense oligonucleotides "small molecule inhibitors' or antibodies therefore represents an attractive target. This should the effects of atherosclerosis, especially cardiovascular disease can be reduced. This therapeutic approach is both as a sole therapy or in combination with, for example, statins (which the PCSK9 protein, upregulate) or with drugs that affect the HDLc levels conceivable.

It is an object to provide means of the present invention, which are suitable to reduce the content of PCKS9 and consequently LDLc.

The present invention relates to a vaccine comprising at least one of the amino acid sequence TLGTNFGRCVDLFAPGEDII- GASSDCSTCFVSQSGTSQAAAHVAGIA (SEQ ID NO. 1) derived peptide having a length of at least 7 amino acids, said peptide having the amino acid sequence DIIGA (SEQ ID NO. 2) and / or CFVSQSG (SEQ ID no. 3) of SEQ ID NO. 1 encompassed.

It has been found that peptides having at least 7 amino acid residues, which are derived from SEQ ID Nos. 1 and either SEQ ID NO. 2 or SEQ ID NO. 3 include, in a position in a mammal, and in a human the formation directed antibodies to induce against PCSK9. The antibodies formed by the administration are able to bind to a portion of PCKS9, which is responsible for the binding of PCKS9 to the LDL receptor. This is to prevent the PCSK9-mediated degradation of LDLR and the amount of LDLc be reduced, which in turn means that disease, the result of an elevated LDLc levels (eg, cardiovascular disease) can be treated or that treated prophylactically could be.

Peptides which are derived from SEQ ID Nos. 1, but not SEQ ID NO. 2 or SEQ ID NO. 3 include, surprisingly, the inventive effects or not adequately show.

The peptide according to the invention containing both SEQ ID NO. 2 and SEQ ID NO. Includes 3, the amino acid residues located between the two sequences and derived from SEQ ID NO. 1 on also.

The term "the amino acid sequence TLGTNFGRCVDLFAPGEDII- GASSDCSTCFVSQSGTSQAAAHVAGIA (SEQ ID NO. 1) derived peptide" refers to peptide fragments of SEQ ID Nos. 1, which have a length of at least 7 amino acid residues. These peptides may also have a minimum length of 8, 9, 10 comprise 11 or 12 amino acids. on the other hand, preferably at most 47, 45, 43, 40 or 35 include peptides of the invention amino acids. Preferred peptides have a length of 8 to 20, more preferably 9 to 17, in particular 10 to 15, amino acids.

According to a particularly preferred embodiment of the present invention, the peptide is selected from the group consisting of IIGASSDCSTCFVSQSG (SEQ ID NO. 5), DLFAPGEDIIGASSDC (SEQ ID NO. 6), STCFVSQSGTSQAAAH (SEQ ID NO. 7), LFAPGEDIIGASSDC (SEQ ID NO. ((8), FAPGEDIIGASSDC SEQ ID NO. 9), APGEDIIGASSDC SEQ ID NO. 10), PGEDIIGAC (SEQ ID NO. 11), GEDIIGASSDC (SEQ ID. No. 12), EDIIGASSDC (SEQ ID NO. 13), DIIGASSDC (SEQ ID NO. 14), STCFVSQSGTSQAAA (SEQ ID NO. 15), STCFVSQSGTSQAA (SEQ ID NO. 16), STCFVSQSGTSQA (SEQ ID NO. 17), STCFVSQSGTSQ (SEQ ID NO. 18), STCFVSQSGTS (SEQ ID NO. (((19), STCFVSQSGT SEQ ID NO. 20), STCFVSQSG SEQ ID NO. 21), TCFVSQSGT SEQ ID NO. 22) and CFVSQSG (SEQ ID NO. 23). In particular, the peptide STCFVSQSGTSQAAAH (SEQ ID NO. 7) and its C-terminal deletion derivatives having at least 9 amino acids (SEQ ID NO. 21) have been found to be particularly suitable.

The peptide according to the invention preferably has at the C-terminus and / or at the N-terminus to a cysteine ​​residue, a cysteine ​​residue may for example be added also at the N- and / or C-terminus. Cysteine ​​residues can be used, for example for cyclization of the peptides. In addition, further substances may be coupled to the SH-groups of the cysteine ​​residues, preferably located at the N- or C-terminal cysteine ​​residues. The cysteine ​​residue can be a naturally occurring cysteine ​​residue at this point or else be attached at the N- or C-terminus of a entstamme deriving from the native sequence sequence. but it is also possible, of course, be coupled via an internal cysteine.

According to a preferred embodiment of the present invention, the peptide of the invention is coupled to a pharmaceutically acceptable carrier, preferably to KLH (Keyhole Lim pet hemocyanin (keyhole limpet hemocyanin) (for example NHS-PEO4-maleimides or others those skilled in the Ge ¬ Bidding known suitable linkers)), tetanus toxoid, albumin binding protein, serum albumin, a dendrimer (MAP; Biol Chem 358:.. 581), peptide linkers (or flanking regions) as well as the adjuvant substances described in Singh et al. Nat. Biotech. 17 (1999): 1075-1081 (specifically those in table 1 of this document), and O 'Hagan et al. , Nature Reviews Drug Discovery 2 (9) (2003): are disclosed 727-735 (in particular the endogenous immuno-potentiating compounds and delivery systems described therein), or mixtures thereof.

In addition, the vaccine composition with an adjuvant, preferably a low soluble aluminum composition, in particular aluminum hydroxide, are formulated. Of course, also adjuvants like MF59 aluminum phosphate, calcium phosphate, cytokines (eg, IL-2, IL-12, GM-CSF), saponins (such as QS21), MDP derivatives, CpG oligos, LPS, MPL, polyphosphazenes, emulsions can (for example, Freund's adjuvant, SAF), liposomes, virosomes, Iscome, cochleates, PLG microparticles, poloxamer particles, virus-like particles, heat-unstable enterotoxin (LT), cholera toxin (CT), mutant toxins (eg, LTK63 and LTR72) , Mikroparti- angle and / or polymerized liposomes may be used.

Other suitable adjuvants may, for example, Purcell W et al. (Nature Reviews Drug Discovery 6 (2007): 404-414), in particular Box 2, be removed.

The compound of the present invention is preferably bound to the carrier or adjuvant via a linker, which is selected from the group consisting of NHS-poly (ethylene oxide) (PEO) (for example NHS-maleimide PEθ 4). The conjugation chemistry (for example, via heterobifunctional compounds such as GMBS and of course also others, such as those described in "Bioconjugate Techniques", Greg T. Hermanson) in this context can be selected from reactions known to the skilled artisan. The covalent Conjugation is preferably above the (the) N- or C-terminal (n) cysteine ​​residue (s) in place.

According to a preferred embodiment of the present invention, the peptide for intradermal, subcutaneous or intramuscular administration is formulated in humans.

A vaccine comprising the peptide of the present invention can be applied to any suitable application means, for example, id, iv, ip, im, intranasal, oral, subcutaneous, etc., and can be administered by any suitable delivery device (O 'Hagan et al., Nature Reviews Drug Discovery 2 (9) (2003): 727-735). The peptide of the present invention is particularly preferred for intravenous, subcutaneous, intradermal or intramuscular administration formulated (see. For example "Handbook of Pharmaceutical Manufac- turing Formulations", Sarfaraz Niazi, CRC Press Inc, 2004).

Typically, the vaccine containing the peptide according to the invention in an amount of 0.1 ng to 10 mg, preferably 10 ng to 1 mg, especially 100 ng to 100 ug, or alternatively, for example, 100 fmol to 10 micromol, preferably 10 pmol to 1 micromol, and in particular 100 pmol to 100 nmol. Typically, the vaccine may also contain auxiliary substances, such as buffers, stabilizers, etc., included.

According to a preferred embodiment of invention is the contemporary vaccine for the treatment and / or prevention of cardiovascular diseases and other disorders caused by atherosclerosis, especially cardiovascular disease, strokes and peripheral vascular diseases (see Pschyrembel, Klinisches Wörterbuch, 261.) suitable.

Another aspect of the present patent application relates to a peptide as defined above.

The present invention relates not only to a vaccine of the type described above but also the peptides contained therein.

The peptides of the invention are useful for the treatment and / or prevention of diseases caused by atherosclerosis diseases, especially stroke, cardiovascular diseases, and diseases of peripheral vessels.

Another aspect of the present invention relates to an isolated antibody directed against a peptide of the invention.

Instead of the inventive peptides and antibodies can be used that are able to bind to these peptides. Such antibodies not only bind to the peptides but also to the existing in the body PCSK9 protein.

The antibodies according to the present invention are preferably monoclonal antibodies. Such antibodies, which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique which allows for the production of antibody molecules. These include, for example, the hybridoma technique of Kohler and Milstein (Na- ture 256 (1975):.. 495-497 and US Pat No. 4,376,110)., The human B-cell hybridoma technique (Kosbor et al, Immunology Today 4 (1983): 72; Coie et al, Proc Natl Acad Sci USA 80 (1983):...... 2026-2030) and the EBV-hybridoma technique (Coie et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., (1985): 77-96). Such antibodies may be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and any subclass are the same. The mAb of this invention producing hybridoma can be grown in vitro or in vivo. Of course it is also possible to produce monoclonal antibodies by rekom- binanter technologies in eukaryotic, yeast, insect and plant cells and in plants. These expression systems and methods for the isolation of these antibodies from the cells are well known in the field. Particularly preferred according to the invention, humanized monoclonal antibody or functional parts thereof (Fab, single chain antibodies, etc., which are able to recognize the peptides of the invention), respectively.

Another aspect of the present invention relates to the use of a peptide of the invention or antibody for the manufacture of a medicament for the treatment and / or prevention of diseases caused by atherosclerosis disturbances, in particular stroke, cardiovascular diseases, and diseases of peripheral vessels.

The present invention is further illustrated in the following figures and examples, without however being limited thereto.

Fig. 1 shows the detection of PCK9-specific antibodies in mice after administration of a peptide having the amino acid sequence SEQ ID NO. 6 by ELISA.

Fig. 2 shows the detection of PCK9-specific antibodies in mice following administration of a peptide having the amino acid sequence SEQ ID NO. 27 by ELISA.

FIGS. 3 to 9 show the detection of PCK9-specific antibodies in mice after administration of a peptide having the amino acid sequences SEQ ID NO. 8 to 14 by ELISA.

Fig. 10 shows the detection of PCK9-specific antibodies in mice following administration of a peptide having the amino acid sequence SEQ ID NO. 24 by ELISA.

Fig. 11 shows the detection of PCK9-specific antibodies in mice following administration of a peptide having the amino acid sequence SEQ ID NO. 7 by ELISA. Figs. 12 to 20 show the detection of PCK9-specific antibodies in mice following administration of a peptide having the amino acid sequence SEQ ID Nos. 15 to 23 by means of Edisa.

Fig. 21 shows the detection of PCK9-specific antibodies in mice following administration of a peptide having the amino acid sequence SEQ ID NO. 25 by ELISA.

Fig. 22 shows an ELISA in which purified mouse monoclonal antibodies that were generated after immunization with SEQ ID NO. 7, (the immunized peptide sequence as well as truncations of this peptide Seq. ID NO. 21, 22, 23 and 25) were bound.

Samples: purified monoclonal antibody detection: α-mouse IgG

In the table, the ELISA raw data of Figure 22 are shown. the binding of monoclonal anti-PCSK9 antibody Care was tested on various peptides to determine the binding site of the antibody in more detail.

No signal (background): less than 0.2 OD

Weak signal: to 0.4 OD

Strong signal means: OD 0.4 to 1.2

Strong signal: OD higher than 1.2

An antibody should preferably not only bind to the C-terminus, therefore, all antibodies that recognize the peptides tested, suitable in principle.

Figure imgf000010_0001

. (: Ng / ml 500 Start) detection: Figure 23 shows the titration of monoclonal antibodies of Figure 22 (immunization with p4918 = SEQ ID NO. 7) samples. Monoclonal antibody α mouse IgG in the table are the ELISA raw data of the Figure 23 is illustrated. the signal strengths of defined amounts of monoclonal anti-PCSK9- antibodies to recombinantly produced PCSK9 protein were compared in ELISA, with the signal strengths refer to a dilution of 15.6 ng / ml antibody.

Weak signal: to 0.4 OD

Strong signal means: OD 0.4 to 1.2 Strong signal: OD higher than 1.2

thereby as high as possible signals are preferred (about OD 1.2 in an antibody dilution of 15.6 ng / ml).

Figure imgf000011_0001

FIGS. 24 and 25 show the detection of antibodies to the administered peptides (STCFVSQSGT-C, IIGASSDCSTCFVSQS, C- IIGASSDCSTCFVSQS and IIGASSDCSTCFVSQS-C) (in mice after injection of the peptides by peptide ELISA to measure the antibody titer Fig. 24 ) and the detection of PCSK9 specific antibodies by PCSK9 protein-ELISA (in these sera were to the sera diluted 1: 400; Figure 25): 100 and 1..

EXAMPLES:

Example 1:

1. Methods:

Manufacture vaccines:

The peptides were coupled to KLH via GMBS 4-maleimidobutyric acid N-hydroxysuccinimide ester. 30 micrograms of these conjugates (the amount refers onto the peptide) were mixed with aluminum hydroxide (Alum final concentration 0.2%) were mixed. The buffer used was PBS.

Immunization:

Balb / c or C57B1 / 6 mice were immunized subcutaneously (in the flank). Number of injections 2 to 4 times. The interval between immunizations was 1 week to 6 weeks. The injection volume per mouse was between 200 .mu.l and 1 ml. 2, weeks after immunization blood was taken.

ELISA:

Serum or plasma of the immunized mice was tested by ELISA for antibodies against the immunized peptide and antibodies to the PCSK9 protein.

Procedure:

• the microtiter plates were coated with peptide (coupled to BSA) or protein

• the coated wells were incubated with serum or plasma at various dilutions

• the presence of peptide or PCKS9-specific antibodies in serum or plasma was detected using IgG antibody (preferably biotinylated) polyclonal anti-mouse. The bound anti-mouse antibodies were then detected by incubation with streptavidin-HRP ( "horse wheel dish peroxidase") was detected (color reaction, for example, ABTS as substrate)

2 results:

Figure imgf000012_0001

Figure imgf000013_0001

These results (see also Fig. 1 to 23) show that results in the injection of peptides according to the invention for forming PCKS9- specific antibodies. In contrast, the injection of the peptides with the sequences SEQ ID Nos. 24 and 25 showed no formation PCKS9-specific antibodies.

Example 2:

Peptide ELISA (see Fig. 22);

Said peptides were coupled to BSA and coated onto ELISA plates. Next, the monoclonal antibodies were added to the plates. The binding of antibodies to the peptides were biotinylated by standard polyclonal anti-mouse IgG antibody (detection antibody) and subsequent Parbreaktion (streptavidin-HRP ( "horseradish Peroxida- se *); ABTS (2,2 '-Azinobis- (3 -ethylbenzthiazolin-6-sulfonic acid) for the color reaction measured at OD 405 nm) was detected.

After immunizing a mouse with SEQ ID NO. 7 different monoclonal antibodies were generated. These antibodies were tested in tidsequenzen described ELISA for their formation at of SEQ ID NO. 7 or at derived shortened Pep to see if any antibodies against the same amino acids of SEQ ID NO. 7 are directed, or whether some of them for example, bind only at the N- or only at the C-terminus. This ELISA can be used to select for the following experiments antibodies that bind to specific amino acids may or may not bind. It can thus be defined, the binding site of the antibodies on immunized peptide.

All the antibodies tested bind strongly (OD higher than 1.2) of Seq ID No. 7, 21, 22 and 23. Differences only occur upon binding to Seq ID No. 25. No binding (background;.. OD is less than 0, 2) it can be seen in four of the antibodies (U1B1A4, U7F95H11, U10F11D4, U17G6C10), weakly (OD up to 0.4) binds an antibody (U4G5G8), medium strong binding (OD 0.4 to 1.2) (two antibodies U9D8A2, U15E8C2).

It results from this ELISA that for a binding of all antibodies tested for the presence of glutamine (AS No. 9 in SEQ ID NO. 7) seems to be important. The C-terminal amino acids of this glutamine located are obviously irrelevant for the antibody binding. For all the antibodies tested the 7 amino acids of Seq seem. to suffice ID NO. 23 to give a strong signal in the ELISA, that is the two amino acids at the N-terminus of Seq. ID no. 7 also do not seem to be much in this analysis.

PCSK 9 protein ELISA:

The illustrated in Fig. 22 monoclonal mouse anti hu PCSK9 antibodies were in varying amounts (between 7.8 each and 500 ng / ml) applied to a coated with recombinantly produced protein PCSK9 ELISA plate. Detection of bound antibodies was performed with biotinylated anti-mouse IgG and streptavidin-HRP. After addition of the substrate (ABTS), the OD was measured at 405 nm. The signal strength of various antibodies was compared here. The higher the signal, the more the tested antibody binds to the PCSK9 protein. This ELISA can be used to auszuwählen- those antibodies for subsequent experiments that bind strongly as possible to the target protein.

Analysis of the ELISA data:

As a strongly binding antibodies are those designated in this ELISA, which also at a dilution of 15.6 ng / ml OD of greater than 1.2, OD 0.4 to 1.2 is referred to as medium-strong signal, including as weak signal. Preferably, antibodies were used with the highest possible signal in further experiments. Example 3:

Each 8 mice (coupled to KLH and mixed with alum) with the described in the figures 24 and 25 and the sera vaccinated peptides after 1, 2, and 3 injections analyzed by peptide ELISA to measure the antibody titer. The sera with comparable high anti-peptide titers were subsequently by means of PCSK9 protein ELISA tested for PCSK9-specific antibodies to determine the ability of the peptides to induce determine PCSK9-specific antibodies.

It was found that the peptide STCFVSQSGT-C induces stronger PCSK9-specific Iiranunantwort as the peptides IIGASSDCSTCFVSQS, C-IIGASSDCSTCFVSQS and IIGASSDCSTCFVSQS-C (24 and 25, see Fig.) Was found. These results show that at least the C-terminal amino acid (G), is necessary of STCFVSQSGT-C to induce PCSK9-specific antibodies. This is also shown in the summary table in Example 1; the injection of SEQ ID NO. 20 (STCFVSQSGT) and SEQ ID NO. 21 (STCFVSQSG) lead to the formation of PCSK9-specific antibody, while SEQ ID NO. 25 (STCFVSQS) does not induce any PCSK9-specific antibody.

Claims

claims:
1. A vaccine comprising at least one of the amino acid sequence TLGTNFGRCVDLFAPGEDIIGASSDCSTCFVSQSGTSQAAAHVAGIA (SEQ ID NO. 1) derived peptide having a length of at least 7 amino acids, said peptide having the amino acid sequence DIIGA (SEQ ID NO. 2) and / or CFVSQSG (SEQ ID NO. 3) encompassed by SEQ ID NO. 1,.
2. A vaccine according to claim 1, characterized in that the peptide is selected from the group consisting of II GASSDCSTCFVSQSG (SEQ ID NO. 5), DLFAPGEDIIGASSDC (SEQ ID NO. 6), STCFVSQSGTSQAAAH (SEQ ID NO. 7), LFAPGEDIIGASSDC (SEQ ID NO. 8), FAPGEDIIGASSDC (SEQ ID NO. 9), APGEDIIGASSDC (SEQ ID NO. 10), PGEDIIGAC (SEQ ID NO. 11), GEDIIGASSDC (SEQ ID. No. 12), EDII- GASSDC (SEQ (ID NO. 13), DIIGASSDC SEQ ID NO. 14), STCFVSQSGTSQAAA (SEQ ID NO. 15), STCFVSQSGTSQAA (SEQ ID NO. 16), STCFVSQSGTSQA (SEQ ID NO. 17), STCFVSQSGTSQ (SEQ ID NO. 18) , STCFVSQSGTS (SEQ ID NO. 19), STCFVSQSGT (SEQ ID NO. 20), STCFVSQSG (SEQ ID NO. 21), TCFVSQSGT (SEQ ID NO. 22) and CFVSQSG (SEQ ID NO. 23).
includes 3. A vaccine according to claim 1 or 2, characterized in that the peptide at the C-terminus and / or at the N-terminus of a cysteine ​​residue.
4. A vaccine according to any one of claims 1 to 3, characterized in that the peptide is coupled to a pharmaceutically acceptable carrier, preferably KLH (keyhole limpet hemocyanin).
5. A vaccine according to any one of claims 1 to 4, characterized in that the peptide is formulated for intradermal, subcutaneous or intramuscular administration.
6. A vaccine according to any one of claims 1 to 5, characterized in that the peptide is formulated with an adjuvant, preferably aluminum hydroxide.
7. A vaccine according to any one of claims 1 to 6, characterized in that the peptide in an amount of 0, 1 ng to 10 mg, preferably 10 ng to 1 mg, especially 100 ng to 100 ug, is included in the vaccine.
8. A vaccine according to any one of claims 1 to 7 for the treatment and / or prevention of diseases caused by atherosclerosis disorders, especially cardiovascular disease, stroke or peripheral vascular diseases.
9. peptide as in any one of claims 1 to 5 defined.
10. A peptide according to claim 9, in particular for treating cardiovascular and / or prevention of diseases caused by atherosclerosis disorders, diseases, strokes or peripheral vascular diseases.
11. An isolated antibody directed against a peptide according to claim 10th
12. Use of a peptide according to claim 9 or an antibody according to claim 11 for the manufacture of a medicament for the treatment and / or prevention of diseases caused by atherosclerosis disorders, especially cardiovascular disease, stroke or peripheral vascular diseases.
PCT/AT2009/000452 2008-11-19 2009-11-19 Vaccine WO2010057242A3 (en)

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