PEPTIDE VACCINES USABLE FOR HYPERCHOLESTEROLEMIA RELATED DISEASES

Abstract

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

Claims

1. Vaccine comprising at least one peptide consisting of 9 to 25 amino acid residues having the amino acid sequence TABLE-US-00005 (SEQ ID No. 1) X.sub.1X.sub.2X.sub.3WX.sub.4X.sub.5X.sub.6RX.sub.7(X.sub.8).sub.m(X.sub.9).sub.n, wherein X.sub.1 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, X.sub.2 is an amino acid residue selected from the group of uncharged amino acid residues, preferably selected from the group of isoleucine, valine, glycine, glutamine and alanine, more preferably isoleucine, valine, glutamine and alanine. X.sub.3 is an amino acid residue selected from the group of uncharged amino acid residues, preferably selected from the group consisting of proline, threonine, alanine and valine, more preferably proline, X.sub.4 is an amino acid residue selected from the group consisting of asparagine, serine, alanine, glutamine and aspartic acid, X.sub.5 is an amino acid residue selected from the group consisting of leucine, glycine, alanine, tyrosine, aspartic acid, phenylalanine and valine, preferably leucine, X.sub.6 is an amino acid residue selected from the group of hydrophilic, negatively charged amino acid residue, preferably an amino acid residue selected from the group consisting of glutamic acid and aspartic acid, X.sub.7 is an amino acid residue selected from the group of uncharged amino acid residues, preferably selected from the group consisting of isoleucine, leucine, alanine and threonine, X.sub.8 is an amino acid residue selected from the group of uncharged amino acid residues selected from the group consisting of threonine, leucine, glutamine, alanine and serine, X.sub.9 is X.sub.10X.sub.11X.sub.12 or a C-terminal truncated fragment thereof consisting of 1 or 2 amino acid residues, X.sub.10 is any amino acid residue, preferably an amino acid residue selected from the group of uncharged amino acid residues, preferably selected from the group consisting of proline, alanine and serine, X.sub.11 is any amino acid residue, preferably an amino acid residue selected from the group of uncharged amino acid residues, preferably selected from the group consisting of proline, alanine, valine, threonine and asparagine, X.sub.12 is any amino acid residue, preferably an amino acid residue selected from the group consisting of arginine, alanine, lysine, serine and leucine, m is 0 or 1, n is 0 or 1, and SEQ ID No. 1 is not SIPWNLERITPPR or a C-terminal truncated fragment thereof, wherein said at least one peptide is coupled or fused to a pharmaceutically acceptable carrier.

2. Vaccine according to claim 1, wherein X.sub.1 is valine, serine or alanine, preferably valine.

3. Vaccine according to claim 1, wherein X.sub.2 is valine, isoleucine or glutamine, preferably valine.

4. Vaccine according to claim 1, wherein X.sub.4 is serine or asparagine, preferably serine.

5. Vaccine according to claim 1, wherein X.sub.6 is glutamic acid.

6. Vaccine according to claim 1, wherein X.sub.7 is leucine, isoleucine or threonine, preferably threonine.

7. Vaccine according to claim 1, wherein X.sub.8 is glutamine, threonine or leucine.

8. Vaccine according to claim 1, wherein the at least one peptide is selected from the group consisting of TIPWNLERIT, VIPWNLERIT, AIPWNLERIT, SVPWNLERLT, SQPWNLERIT, SAPWNLERIT, SGPWNLERITPPR, SITWNLERIT, SIAWNLERIT, SIVWNLERITPPR, SIVWNLERIT, SIPWALERIT, SIPWQLERITPPR, SIPWDLERITPPR, SIPWNAERIT, SIPWNGERIT, SIPWNYERIT, SIPWNDERIT, SIPWNVERITPPR, SIPWNVERITPPR, SIPWNLDRITPPR, SIPWNFERIT, SIPWNVERIT, SIPWNLDRIT, SIPWNLERLT, SIPWNLERTT, SIPWNLERLT, SIPWNLERIL, SQPWNLERIQ, SIPWNLERIA, SIPWNLERITPPR, SIPWNLERIS, SIPWNLERITAPR, SIPWNLERITSPR, SIPWNLERITAPR, SIPWNLERITPVR, SIPWNLERITPPR, SIPWNLERITPNR, SIPWNLERITPPA, SIPWNLERITPPK, SIPWNLERITPPS, SIPWNLERITPPL, VIPWNLERLT, VIPWNLERTT, VIPWNLERIL, VIPWNLERILPPR, VIPWNLERIQ, SVPWNLERLT, SVPWNLERTT, SVPWNLERIL, SVPWNLERIQ, SVPWNLERIQPPR, SIPWSLERTTPPR, SIPWNLERLT, SQPWNLERIL, SQPWNLERIQ, SIPWSLERLT, SIPWSLERTT, SIPWSLERIL, SVPWNLERIQ, VQPWSLERLL, SQPWSLERTL, VQPWNLERLQ, VQPWSLERLL and SVPWSLERLT.

9. Vaccine according to claim 1, wherein the at least one peptide is selected from the group consisting of SIPWSLERITPPR, SIPWSLERTT, SIPWSLERITPPR, VIPWNLERIL, VIPWNLERILPPR, SVPWNLERIQ and SVPWNLERIQPPR.

10. Vaccine according to claim 1, 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.

11. Vaccine according to claim 1, wherein the pharmaceutically acceptable carrier is a protein carrier.

12. Vaccine according to claim 11, wherein the protein carrier is selected from the group consisting of keyhole limpet haemocyanin (KLH), tetanus toxoid (TT), protein D or diphtheria toxin (DT), preferably a mutated diphtheria toxin, more preferably CRM197.

13. Vaccine according to claim 1, wherein the compound is formulated with an adjuvant, preferably adsorbed to Alhydrogel.

14. Vaccine according to claim 1 to be used in a method for treating and/or preventing disorders caused by hyperlipidemia, hypercholesterolemia and/or atherosclerosis, preferably cardiovascular diseases, stroke or peripheral vascular diseases.

15. Peptide consisting of an amino acid sequence X.sub.1X.sub.2X.sub.3WX.sub.4X.sub.5X.sub.6RX.sub.7(X.sub.8).sub.m(X.sub.9).sub.n (SEQ ID No. 1) as defined in claim 1.

Description

[0116] FIGS. 1A and 1B Binding ELISA

[0117] FIGS. 1A and 1B show detection of PCSK9 mimotopes sequences by mouse sera containing polyclonal antibodies specific for human PCSK9 aa 153-162 or aa 153-165

[0118] A) Mimotopes containing single exchanges

[0119] B) Mimotopes containing multiple exchanges

[0120] The graphs display median titers from all mimotopes compared to the irrelevant negative control. As a positive control the original human PCSK9 sequence is included: aa 153-162 or aa 153-165. Mimotopes with values over 1:50.000 are considered as good candidates.

[0121] FIGS. 2A and 2B Competition ELISA

[0122] FIGS. 2A and 2B show the ability of PCSK9 mimotopes to compete with the original PCSK9 sequence (aa 153-162 or aa 153-165) for binding polyclonal antibodies in mouse sera raised specifically against the latter.

[0123] All mimotopes are compared to the negative group (0% competition by an irrelevant peptide). As a positive control for each competition ELISA the original PCSK9 sequences were used (aa 153-162 or aa 153-165).

[0124] A) Mimotopes containing single exchanges

[0125] B) Mimotopes containing multiple exchanges

[0126] The graphs display the ability (in percent) of the mimotopes to compete with the original PCSK9 sequence (aa 153-162 or aa 153-165) for binding polyclonal antibodies raised against the latter.

[0127] Mimotopes with competition ability over 20% are considered as good candidates.

[0128] FIG. 3 Protein ELISA

[0129] FIG. 3 shows comparison of the median titers (n=5 mice/group) against human PCSK9 protein induced by the indicated sequences. The data reveal the ability of the mimotopes to induce antibodies cross-reacting with human PCSK9 protein. Antibodies raised by irrelevant peptide do not recognize the protein.

[0130] FIG. 4 Total Cholesterol (% Compared to Control Group)

[0131] FIG. 4 shows comparison of the mean (n=5 mice/group) total cholesterol levels of mice immunized with mimotopes in comparison to a control group immunized with irrelevant peptide. The data reveal the ability of the mimotopes to substantially lower the levels of total cholesterol in mice up to approximately 30%.

[0132] FIGS. 5A and 5B Total Cholesterol (TC) Levels Upon Vaccination

[0133] FIGS. 5A and 5B show total cholesterol (TC) levels upon vaccination with original PCSK9 sequence SIPWNLERIT and novel sequence SIPWSLERIT (direct comparison).

[0134] FIGS. 6A and 6B Total Cholesterol (TC) Levels Upon Immunization

[0135] FIGS. 6A and 6B show total cholesterol (TC) levels upon immunization with vaccines containing the novel sequences SIPWSLERIT, VIPWNLERIL and SVPWNLERIQ.

EXAMPLES

Materials and Methods

Vaccine

[0136] The peptides were conjugated via the heterobifunctional linker GMBS (4-Maleimidobutyric acid N-hydroxysuccinimide ester) to KLH (Keyhole Limpet Hemocyanin).

Animal Experiments

[0137] 5 Balb/c mice were subcutaneously 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 experiments was 8 to 10 weeks.

[0138] Mice were injected four times in 2 week intervals with 15 μg of net peptide coupled to KLH and adsorbed to Alhydrogel as adjuvant in a volume of 1 ml in total.

[0139] Blood was taken approximately 2 weeks after the final injection.

Competition ELISA

[0140] 20 μg of each indicated peptide was incubated with sera from mice injected with original PCSK9 sequence (aa 153-162 or aa 153-165: SEQ ID No. 2). Sera were then incubated on ELISA plates coated with the original PCSK9 sequence (aa 153-162 or aa 153-165: SEQ ID No. 2). Detection was performed with anti-mouse IgG antibodies, ABTS was used as a chromogenic substrate and optical density (OD) was measured at 405 nm.

[0141] Mimotopes diminishing >20% subsequent binding of the polyclonal antibodies from the sera to the original human PCSK9 epitope (aa 153-162 or 153-165: SEQ ID No.3) coated on the ELISA plates were considered as competing mimotopes and as good candidates.

Binding ELISA

[0142] Mimotopes were coated on ELISA plates at a concentration of 1 μmol/mL. After blocking unspecific binding (1% BSA in PBS) appropriate dilutions of sera from mice injected with the original human PCSK9 sequence aa 153-162 or aa 153-165 (SEQ ID No.3) were added and incubated for approximately 1 hour. Subsequently, detection with anti-mouse IgG antibodies was performed. ABTS was used as substrate. OD measurements were performed at 405 nm and titers were defined as the dilution of the serum where 50% of the ODmax is reached.

Protein ELISA

[0143] To determine the immunogenicity of the vaccines, 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 were added to the wells, serially diluted 1:2 fold and incubated for approximately 1 hour. Bound antibodies were detected by incubation with anti-mouse IgG antibod, ABTS was added as substrate and the OD at 405 nm was measured. As negative control sera from the control group injected with an irrelevant peptide 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

[0144] Total cholesterol was measured with the LabAssay™ Cholesterol Kit (Wake).

Results

Example 1

[0145] List of sequences and median antibody titers against original human PCSK9 sequence (See FIGS. 1A and 1B) and competition capacity (in percent) of the mimotopes, compared to the negative control group (See FIGS. 2A and 2B). In order to facilitate coupling of the peptides to KLH the peptides may comprise a cysteine residue as a linker at the C-terminus.

TABLE-US-00004 % Competition Median Titer compared to Binding ELISA the related Seq ID Sequence (ODmax/2) control group 1 X.sub.1X.sub.2X.sub.3WX.sub.4X.sub.5X.sub.6RX.sub.7(X.sub.8).sub.m(X.sub.9).sub.n 2 SIPWNLERITPPR 3 ERTKSDPGAQHREF (negative 0 control) 4 ITELSRWPNI (negative 0 control) 5 TIPWNLERIT 198,000 52 6 VIPWNLERIT 152,000 51 7 AIPWNLERIT 199,000 83 8 SVPWNLERIT 197,000 62 9 SQPWNLERIT 149,000 30 10 SAPWNLERIT 131,000 76 11 SGPWNLERITPPR 81,000 54 12 SITWNLERIT 124,000 62 13 SIAWNLERIT 147,000 78 14 SIVWNLERITPPR 146,000 78 15 SIPFNLERIT 42,000 15 16 SIPANLERIT 22,000 8 17 SIPGNLERIT 21,000 0 18 SIPHNLERIT 23,000 0 19 SIPDNLERIT 23,000 3 20 SIPSNLERIT 22,000 7 21 SIPWSLERIT 147,000 73 22 SIPWALERIT 157,000 75 23 SIPWQLERITPPR 224,000 79 24 SIPWDLERITPPR 231,000 74 25 SIPWNAERIT 156,000 73 26 SIPWNGERIT 56,000 52 27 SIPWNYERIT 115,000 76 28 SIPWNDERIT 131,000 71 29 SIPWNFERITPPR 186,000 79 30 SIPWNVERITPPR 197,000 78 31 SIPWNLARIT 34,000 13 32 SIPWNLDRITPPR 151,000 53 33 SIPWNLEKIT 59,000 19 34 SIPWNLEAIT 54,000 15 35 SIPIPINLEDIT 48,000 13 36 SIPIPINLERLT 192,000 76 37 SIPIPINLERTT 177,000 73 38 SIPIPINLERAT 149,000 77 39 SIPIPINLERIL 168,000 56 40 SIPWNLERIQ 181,000 64 41 SIPWNLERIA 189,000 80 42 SIPWNLERISPPR 219,000 79 43 SIPWNLERITAPR 226,000 70 44 SIPWNLERITSPR 153,000 68 45 SIPWNLERITPAR 184,000 74 46 SIPWNLERITPVIR 265,000 75 47 SIPWNLERITPTR 155,000 73 48 SIPWNLERITPNR 186,000 70 49 SIPWNLERITPPA 163,000 65 50 SIPWNLERITPPK 158,000 66 51 SIPWNLERITPPS 149,000 66 52 SIPWNLERITPPL 139,000 66 53 VIPWNLERLT 191,000 54 54 VIPWNLERTT 162,000 69 55 VIPWNLERIL 173,000 49 56 VIPWNLERIQ 166,000 47 57 SVPWNLERLT 154,000 57 58 SVPWNLERTT 116,000 73 59 SVPWNLERIL 188,000 59 60 SVPWNLERIQ 222,000 61 61 SQPIAINLERLT 200,000 69 62 SQPIAINLERIL 196,000 69 63 SQPIAINLERIQ 167,000 66 64 SIPIAISLERLT 164,000 46 65 SIPIAISLERTT 147,000 64 66 SIPWSLERIL 199,000 46 67 SIPWSLERIQ 223,000 44 68 VQPWSLERTL 95,000 48 69 SQPWSLERTL 98,000 53 70 SVPINNLEKLT 47,000 11 71 VQPINNLERLQ 184,000 72 72 VQPIAISLERLL 166,000 66 73 SVPWSLERLT 251,000 74

[0146] Example 2

Unexpected Effect of The Peptides According to The Present Invention

[0147] In a further experiment, the functionality of the antibodies raised upon immunization with the peptide vaccines according to the present invention is investigated. In the present experiment, the highly significant functionality of the antibodies raised upon immunization with vaccines containing the novel sequences (SEQ ID No.21 SIPWSLERIT, SEQ ID No. 55 VIPWNLERIL and SEQ ID No. 60 SVPWNLERIQ) is demonstrated in comparison to the antibodies raised upon vaccination with vaccines containing the original PCSK9 sequence (SIPWNLERIT) and a negative control sequence—all coupled to the same carrier KLH (FIGS. 5A and 5B and FIGS. 6A and 6B).

[0148] For this purpose mice have been immunized with HPLC purified peptides coupled to KLH and formulated with an adjuvant (Alum) as vaccines (Sequence-KLH/Alum). In the first experiment, a direct comparison between the original PCSK9 sequence and the novel sequence SIPWSLERIT has been performed. Here, mice have been immunized with irrelevant peptide vaccine (irrelevant peptide-KLH/Alum) as a negative control group, original PCSK9 sequence vaccine (SIPWNLERIT- KLH/Alum) and the novel sequence vaccine (SIPWSLERIT-KLH/Alum). The latter group (SIPWSLERIT-KLH/Alum) has been used not only to compare its functionality of the induced antibodies to the functionality of the antibodies generated using the original PCSK9 sequence vaccine (SIPWNLERIT-KLH/Alum), but also as a bridging group between different experiments. As it is shown, both vaccines - the original PCSK9 sequence vaccine (SIPWNLERIT- KLH/Alum) and the novel sequence vaccine (SIPWSLERIT-KLH/Alum) are able to strongly reduce the levels of total cholesterol in comparison to the negative control group (FIGS. 5A and 5B). However interestingly, upon vaccination with the novel sequence vaccine (SIPWSLERIT-KLH/Alum), mice show a significant reduction of total cholesterol, compared not only to the negative control group but in fact, compared also to the group injected with vaccines containing the original PCSK9 sequence SIPWNLERIT (FIGS. 5A and 5B).

[0149] Subsequently, a second experiment has been performed wherein mice have been immunized with irrelevant peptide as a negative control group and all the three novel sequences SIPWSLERIT, VIPWNLERIL and SVPWNLERIQ, all coupled to KLH and formulated as vaccines. As it has been already mentioned above, the mice immunized with the novel sequence (SIPWSLERIT-KLH/Alum) have been used as a bridging group between the experiments. In fact, the bridging group injected with the novel sequence (SIPWSLERIT-KLH/Alum) is behaving in a similar way in both experiments (FIGS. 5A, 5B, 6A and 6B) and most importantly, all three groups immunized with vaccines containing one of the sequences SIPWSLERIT, VIPWNLERIL or SVPWNLERIQ behave in a very similar way in terms of reduction of plasma total cholesterol levels (FIGS. 6A and 6B).

[0150] Thus, the present data confirm the significant efficiency of the vaccines according to the present invention, especially the peptide vaccines containing the sequences SEQ ID No.21 SIPWSLERIT, SEQ ID No. 55 VIPWNLERIL and SEQ ID No. 60 SVPWNLERIQ, to induce functional antibodies that are able not only strongly to decrease the levels of plasma TC compared to the irrelevant negative control, but also to significantly reduce the total cholesterol levels in comparison to a vaccine containing the original PCSK9 sequence (SIPWNLERIT-KLH/Alum).

[0151] FIG. 5A shows the plasma mean values in mg/dl upon immunization with vaccine containing the novel sequence SIPWSLERIT, compared to immunization with vaccine containing the original PCSK9 sequence SIPWNLERIT and to the irrelevant (negative) control group (SEQ ID No. 2 ERTKSDPGAQHREF). The highly significant reduction of the TC levels in the group immunized with vaccine containing the novel sequence SIPWSLERIT, compared on one side to vaccine containing the original PCSK9 sequence SIPWNLERIT and on the other side to the negative control group, is evident from this figure. FIG. 5B provides the % decrease of TC of the groups, compared to the irrelevant (negative) control group. Specific attention should be given to the −20% reduction of TC upon vaccination with the original PCSK9 sequence (SIPWNLERIT-KLH/Alum), compared to the irrelevant negative control and the very strong up to 35-40% reduction in TC levels upon vaccination with the novel sequence (SIPWSLERIT-KLH/Alum).

[0152] FIG. 6A shows the plasma mean values in mg/dl upon immunization with vaccines containing the novel sequences SIPWSLERIT, VIPWNLERIL and SVPWNLERIQ, compared to the irrelevant (negative) control group (SEQ ID No. 2 ERTKSDPGAQHREF). The highly significant reduction of the TC levels in all three groups immunized with the novel sequences in comparison to the negative control group, is evident from this figure. FIG. 6B provides the decrease of TC of the groups injected with SIPWSLERIT, VIPWNLERIL and SVPWNLERIQ, compared to the irrelevant (negative) control group. Specific attention should be given to the significantly strong—up to 40% reduction of TC upon vaccination with all three novel sequences SIPWSLERIT, VIPWNLERIL and SVPWNLERIQ. In fact, the group immunized with vaccine containing the novel sequence SIPWSLERIT (bridging group between experiments) has behaved in a similar way in both experiments (see FIGS. 5A and 5B), confirming the significantly higher functionality of the antibodies generated upon immunizations with vaccines containing the novel sequences SIPWSLERIT, VIPWNLERIL and SVPWNLERIQ, in comparison to vaccines containing the original PCSK9 sequence SIPWNLERIT.

[0153] From these data it is evident that the vaccines according to the present invention, especially the peptide vaccines comprising the sequences SEQ ID No.21 SIPWSLERIT, SEQ ID No. 55 VIPWNLERIL and SEQ ID No. 60 SVPWNLERIQ show an unexpected and surprisingly advantageous effect compared to the vaccines presented in the prior art.