HK1170248A - Immunogenes peptide - Google Patents

Description

Err1:Expecting ',' delimiter: line 1 column 335 (char 334)

Err1:Expecting ',' delimiter: line 1 column 850 (char 849)

Err1:Expecting ',' delimiter: line 1 column 398 (char 397)

Err1:Expecting ',' delimiter: line 1 column 192 (char 191)

Err1:Expecting ',' delimiter: line 1 column 678 (char 677)

Some members of the convertases family also influence 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 lipasePCSK9 mediates the breakdown of the low density lipoprotein receptor (LDLR).

Err1:Expecting ',' delimiter: line 1 column 258 (char 257)

Err1:Expecting ',' delimiter: line 1 column 154 (char 153)

PCSK9 plays a critical role in cholesterol metabolism by directly regulating the amount of LDLR present on liver cells. LDLR is a plasma membrane-located glycoprotein that removes cholesterol-rich LDLc particles from plasma by endocytosis. PCSK9 binding leads to LDLR being absorbed into liver cells and subsequently degraded in lysosomes, as opposed to recycling towards the cell surface which would occur without PCSK9 binding.

Err1:Expecting ',' delimiter: line 1 column 130 (char 129)

Err1:Expecting ',' delimiter: line 1 column 327 (char 326)

Err1:Expecting ',' delimiter: line 1 column 88 (char 87)

Over-expression of PCSK9 in mice leads to higher LDLc levels, and administration of recombinant PCSK9 protein also dramatically reduces the number of LDL receptors in the mouse model.

Err1:Expecting ',' delimiter: line 1 column 43 (char 42)

Err1:Expecting ',' delimiter: line 1 column 43 (char 42)

Err1:Expecting ',' delimiter: line 1 column 49 (char 48)

Err1:Expecting ',' delimiter: line 1 column 145 (char 144)

The present invention is intended to provide a means of reducing the LDLR binding activity of PCSK9 and, consequently, the LDLc content.

The present invention relates to a peptide consisting of 10 to 30 amino acid residues derived from the amino acid sequence SIPWNLERITPPRYRADEYQPPDGSLVEV (SEQ ID No 1) where the peptide includes the amino acid sequence SIPWNLERIT (SEQ ID No 9).

A peptide with the amino acid sequence described above has been shown to induce the production of antibodies directed against PCSK9 when administered to an individual, significantly reducing the interaction between PCSK9 and LDL receptor as the antibodies produced bind to PCSK9, preventing PCSK9-mediated degradation of LDLR and reducing the amount of LDLc, which in turn allows the treatment or prevention of diseases caused by elevated LDLc levels (e. g. cardiovascular disease).

Err1:Expecting ',' delimiter: line 1 column 159 (char 158)

The peptide of the invention includes at least the first 10 amino acid residues of SEQ ID No 1, since this particular region of PCSK9 is capable of inducing PCSK9 specific antibodies, which are capable of specifically inhibiting the interaction between PCSK9 and LDL receptor, when administered to an individual. It has been experimentally demonstrated that a peptide must include at least the amino acid sequence SIPWNLERIT in order to exhibit the properties of the invention. Already the omission of the C-terminal threonine of SEQ ID No 9 results in a peptide that is insufficiently capable of inducing the formation of antibodies that are responsible for the invention.

The peptides of the invention can be produced by methods which have been sufficiently described in the state of the art. The peptides can be both recombinantly synthesized in corresponding expression systems and chemically synthesized. As suitable expression systems, eukaryotic (e.g. yeast, especially Pichia pastoris) and prokaryotic systems (e.g. bacteria, especially E. coli), which are well known to the professional, are suitable. Peptides can also be chemically synthesized by numerous standardized methods, whereby an individual synthesis strategy must be developed for each individual peptide.

According to a preferred embodiment of the present invention, the peptide has the amino acid sequence number SIPWNLERITPPRADEYQNLDGGSLVEV (SEQ ID No 1), SIPWNLERITPPRADEYQNLPPDGGSLVE (SEQ ID No 13), SIPWNLERITPPRADEYQNLPPDGGSLV (SEQ ID No 14), SIPWNLERITPRADEYQNLDGGSL (SEQ ID No 15), SIPWNLERITPPRADEYQNLDGGS (SEQ ID No 16), SIPWNLERITPPRADEYQNLDSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSEQSE

The peptide of the invention preferably has a cysteine residue at the C-terminus and/or the N-terminus. Cysteine residues can be used, for example, to cyclize the peptides. Furthermore, other substances can be coupled to the SH groups of the cysteine residues, preferably to the N- or C-terminal cysteine residues. The cysteine residue can be a naturally occurring cysteine at this site or attached at the N- or C-terminus to a sequence derived from the native sequence.

The peptide of the invention may be used in a preferred embodiment in a procedure for the treatment and/or prevention of disorders or diseases caused by atherosclerosis, in particular cardiovascular disease, stroke or peripheral vascular disease.

In a preferred embodiment of the present invention, the peptide is administered to a patient in amounts of 0.1 ng to 10 mg, preferably 1 μg to 500 μg.

A further aspect of the present invention concerns a vaccine comprising a peptide according to the present invention.

The peptides of the invention are particularly suitable for use in a vaccine formulation. The vaccine of the invention may include not only one peptide of the invention but also at least two, three, four, five, six or ten different peptides selected from the group consisting of SIPWNLERITPPRYRADEYQPPDGGSLVEV (SEQ ID No 1), SIPWNLERITPPRYRADEYQPPDGGLVE (SEQ ID No 13), SIPWNLERITPPRYRADEYQPPDGNLV (SEQ ID No 14), SIPWNLERITPPRYRADEYQQSEPPDGLS (SEQ ID No 15), SIPWNLERITPPRYRADYQSEPPRYRADEYQQS (SEQ ID No 16), SIPWNLERITQS (SEQ ID No 25), SIPWNLERITPPRADEYQS (SEQ ID No 25), SIPWNLERITPPRADEYQS (SEQ ID No 20), ID (SEQ ID No 28), ID (SEQ ID (SEQ ID No 20), ID (SEQ ID (SEQ ID No 25), ID (SEQ ID (SEQ ID No 20), ID (SEQ ID (SEQ ID (SEQ ID No 28), ID (SEQ ID (SEQ ID)), ID (S ID (S ID (S), ID (S), ID (S), ID (S), ID (S), ID (S), ID (S), ID (S), ID (S), ID (S), ID (S), ID (S), ID (S), ID (S), ID (S), ID (S), ID (S), ID (S), ID (S (S), ID (S), ID (S), ID (S), ID (S), ID (S), ID (S), ID (S), ID (S), ID (S), ID (S), ID (), ID (), ID (), ID (), ID (), (), (S (), (), (), (), (), (), (), (), (), (), (), (), (), (), (), (), (), (), (), (), (), (), (), (

Err1:Expecting ',' delimiter: line 1 column 142 (char 141)

Other suitable and preferred carriers are tetanus toxoid, albumin binding protein, serum albumin, a dendrimer (MAP; Biol., Chem. 358: 581), peptide linkers (or flanking regions) and adjuvants, as described in Singh et al., Nat. Biotech. 17 (1999): 1075-1081 (in particular those in Table 1 of this document) and O'Hagan et al., Nature Reviews Drug Discovery 2 (9) (2003): 727-735 (in particular the endogenous immune-boosting compounds and delivery systems described) or mixtures thereof.

In addition, the vaccine of the invention may be formulated with an adjuvant, preferably a slightly soluble aluminium compound, in particular aluminium hydroxide, and of course adjuvants such as MF59, aluminium phosphate, calcium phosphate, cytokines (e.g. IL-2, IL-12, GM-CSF), saponins (e.g. QS21), MDP derivatives, CpG oligos, LPS, MPL, Lipopeptide'Sylvia Br1], polyphosphates, emulsions (e.g. Friendly Adjuvant, SAF), liposomes, virosomes, LTLT, Iscome, cochleates, PLG micro-particles, polymerase xylem, lipoproteins, cholera-like substances (Klorophosphor, LTR/T), mutated toxins (CT63), and paroxetins (LTR/T72), and mutated toxins.

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

Err1:Expecting ',' delimiter: line 1 column 379 (char 378)

The peptide is formulated for intradermal, subcutaneous or intramuscular administration according to preferred embodiment.

Err1:Expecting ',' delimiter: line 1 column 486 (char 485)

Typically, the vaccine contains the peptide of the invention in an amount of 0.1 ng to 10 mg, preferably 10 ng to 1 mg, in particular 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. Typically, the vaccine may also contain auxiliary substances, e.g. buffers, stabilizers, etc.

The vaccine of the invention may be used in a procedure for the treatment and/or prevention of disorders caused by atherosclerosis, in particular cardiovascular disease, stroke or peripheral vascular disease.

The vaccine may contain, in addition to the peptides described herein, other antigenic molecules capable of inducing the production of corresponding antibodies in an individual. In particular, it is preferable to use peptides and polypeptides which are also capable of inducing the formation of PCSK9 specific antibodies. For example, corresponding peptides are disclosed in WO 2008/125623. The vaccine may thus contain, in addition to one or more of the peptides of the invention, at least one additional peptide (preferably at least two, at least three, at least four, at least five, peptides) or a functional fragment thereof which has essentially the same characteristics with respect to the formation of PCSK9 specific antibodies in an individual such as the following:[Sylvia Peptid]

29	166-177	YRADEYQPPDGG
30	187-202	TSIQSDHREIEGRVMV
31	206-219	ENVPEEDGTRFHRQ
32	231-246	AGVVSGRDAGVAKGAS
33	277-283	VQPVGPL
34	336-349	VGATNAQDQPVTLG
35	368-383	IIGASSDCSTCFVSQS
36	426-439	EAWFPEDQRVLTPN
37	443-458	ALPPSTHGAGWQLFCR
38	459-476	TVWSAHSGPTRMATAIAR
39	486-500	CSSFSRSGKRRGERM
40	557-573	HVLTGCSSHWEVEDLGT
41	577-590	PVLRPRGQPNQCVG
42	636-645	SALPGTSHVL
43	659-677	RDVSTTGSTSEEAVTAVAI

The vaccine or peptides of the invention may be used in the process to reduce or prevent the risk of disease due to, but not limited to, cardiovascular disease, stroke and peripheral vascular disease and lipid metabolism disorders in humans, or to lower LDL cholesterol, therapeutic or prophylactic treatment of atherosclerosis, lipid metabolism disorders and the like.

The present invention is illustrated in more detail by the drawings and examples below, but is not limited to them. Figure 1 shows a PCSK9 LDLR interaction assay: median (n=5) % of inhibition.Figure 2 shows a PCSK9 protein ELISA: median (n=5) antibody titer against human PCSK9.Figure 3 shows a peptide ELISA: median (n=5) antibody titer against the vaccinated peptide.

The following examples: Example 1: Manufacture of vaccines

Err1:Expecting ',' delimiter: line 1 column 370 (char 369) PCSK9-Peptide, die zur Herstellung der Vakzine verwendet wurden (sämtliche Peptide umfassten einen C-terminalen Cystein-Rest)

2	SIPWNLERITPPR
3	IPWNLERITPPR
4	PWNLERITPPR
5	WNLERITPPR
6	NLERITPPR
7	SIPWNLERITPP
8	SIPWNLERITP
9	SIPWNLERIT
10	SIPWNLERI
11	IPWNLERITPP
12	PWNLERITP

Example 2: Immunisation

5 Balb/c mice were immunised subcutaneously. number of injections 2 to 4 times. interval between immunisations 2 weeks. volume of injection 200 μl/ mouse.

Blood samples were taken 2 weeks after the 3rd and 4th vaccinations respectively.

Err1:Expecting ',' delimiter: line 1 column 62 (char 61)

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

The peptide ELISA:

Err1:Expecting ',' delimiter: line 1 column 438 (char 437)

The test chemical is a PCSK9 protein ELISA.

The protein ELISA was performed as the peptide ELISA, except that instead of the peptide-BSA conjugate, recombinant human PCSK9 was immobilized (see Figures 2 and 3 and the following table). Other

2	19.629	15.340	19.984	2.827	38.175	19.629	50.531
3	3.653	7.607	5.707	2.978	10.556	5.707	71.149
4	3.490	10.241	300	3.687	1.008	3.490	75.701
5	500	0	2.554	795	3.492	795	44.409
6	5.562	1.455	2.577	2.236	18.858	2.577	98.149
7	36.853	9.407	16.987	11.310	3.980	11.310	88.883
8	12.427	6.700	11.124	10.561	32.751	11.124	169.855
9	7.777	5.776	41.508	7.419	2.228	7.419	38.970
10	4.767	5.303	24.630	15.053	9.502	9.502	66.643
11	19.701	5.224	5.975	2.497	1.170	5.224	46.047
12	0	0	0	0	0	0	7.6797

All peptides administered were able to induce antibody formation against the same antibodies.

The test chemical is used to determine the concentration of the active substance in the test chemical.

Err1:Expecting ',' delimiter: line 1 column 445 (char 444)After incubation for 30 min at room temperature, 25 μl/well of recombinant human LDLR (Low Density Lipoprotein Receptor, R& D System) diluted to 1.5 μg/ml in a binding buffer were added and incubated with the mice. After incubation for 2 hours at room temperature, all unbound proteins were washed away. LDLR bound to the intercepted PCSK9 was measured with an anti-LDLR antibody (cyclonel anti-LDLR antibody, Abcaminy), followed by a biotriated chicken antibody and a streptavidin anti-RPH conjugate. The drug was detected as a substrate for the peroxidase 3,5-tetrahydramine (3,5-tetrahydramine), which serves as a stop-gap drug at 450 nm.

The % of binding and inhibition were calculated as follows. Serums from 5 control mice were included in each assay. The average of these values was calculated and assumed to be 100% binding (equivalent to 0 % inhibition). The standard deviation of the determined binding was also calculated and multiplied by 2. All values below 2x standard deviation were assumed to be inhibition. In the results shown, this 2x standard deviation is ∼22% (see table below and Figure 1). Other

2	8
3	23	22	9			23
4	26	0	0	18	19	18
5	0	0	0	17	9	0
6	26	12	0	10		12
7	23			5
8				12
9	0
10	6	0	7		13	7
11	16	9	11	22	0	11
12	4	9	0	0	0	0

The results show that the peptide with SEQ ID 2, 7, 8 and 9 in particular are able to inhibit the interaction between PCSK9 and LDL receptor.

Claims (11)

1. A peptide consisting of 10 to 30 amino acid residues derived from the amino acid sequence SIPWNLERITPPRYRADEYQPPDGGSLVEV (SEQ ID No 1) characterised by the peptide containing the amino acid sequence SIPWNLERIT.
2. Peptide as claimed 1, characterised by the peptide having the amino acid sequence SIPWNLERITPPR, SIPWNLERITPP, SIPWNLERITP or SIPWNLERIT.
3. Peptide as claimed by claim 1 or 2, characterised by the presence of a cysteine residue at the C-terminus and/or N-terminus of the peptide.
4. Peptide according to any of claims 1 to 3 for use in a procedure for the treatment and/or prevention of disorders caused by atherosclerosis, in particular cardiovascular disease, stroke or peripheral vascular disease.
5. Peptide of claim 4, characterised by the peptide being administered to a patient in an amount of 0.1 ng to 10 mg, preferably 1 μg to 500 μg.
6. The vaccine contains one peptide according to claims 1 to 3.
7. Vaccination as claimed by claim 6, characterised by the peptide being coupled to a pharmaceutically acceptable carrier, preferably KLH (Keyhole Limpet Haemocyanin).
8. Vaccination claim 6 or 7, characterised by the peptide being formulated for intradermal, subcutaneous or intramuscular administration.
9. Vaccination according to claims 6 to 8 characterised by the peptide being formulated with an adjuvant, preferably aluminium hydroxide.
10. Vaccination according to one of claims 6 to 9, characterised by the presence of the peptide in a dose of 0.1 ng to 10 mg, preferably 10 ng to 1 mg, in particular 100 ng to 100 μg, per dose of vaccine.
11. A vaccine according to one of claims 6 to 10 for use in a procedure for the treatment and/or prevention of disorders caused by atherosclerosis, in particular cardiovascular disease, stroke or peripheral vascular disease.