Mimotopes of alpha-synuclein and vaccines thereof for the treatment of neurodegenerative disorders

Abstract

The present invention relates to the use of at least one compound comprising the amino acid sequence TABLE-US-00001 (Formula1) (SEQIDNO:57) (X.sub.1).sub.nX.sub.2X.sub.3PVX.sub.4X.sub.5X.sub.6(X.sub.7).sub.m, wherein X.sub.1 is any amino acid residue, X.sub.2 is an amino acid residue selected from the group consisting of aspartic acid (D) and glutamic acid (E), X.sub.3 is any amino acid residue, X.sub.4 is any amino acid residue, X.sub.5 is an amino acid residue selected from the group consisting of proline (P) and alanine (A), X.sub.6 is an amino acid residue selected from the group consisting of aspartic acid (D) and glutamic acid (E), X.sub.7 is any amino acid residue, n and m, independently, are 0 or an integer of more than 0, and wherein the amino acid sequence according to Formula I is not identical with, or does not comprise the 8-mer polypeptide fragment of alpha-synuclein having the amino acid sequence DMPVDPDN (SEQ ID NO: 1), said compound having a binding capacity to an antibody which is specific for an epitope of alpha-synuclein comprising the amino acid sequence DMPVDPDN (SEQ ID NO: 1) for producing a medicament for preventing and/or treating synucleinopathies.

Claims

1. A method of reducing aggregates of -synuclein in a subject having a disease selected from the group consisting of Lewy Body Disorders (LBDs), Parkinson's Disease (PD), Parkinson's Disease with Dementia (PDD), Dementia with Lewy Bodies (DLB), Multiple System Atrophy (MSA), and Neurodegeneration with Brain Iron Accumulation type I (NBIA Type I), comprising administering, to a subject in need thereof, a compound comprising a peptide having the amino acid sequence of XDQPVLPD (SEQ ID NO: 59) wherein X is cysteine or not present, wherein the compound induces an antibody that has specific binding capacity for an epitope of alpha-synuclein comprising the amino acid sequence DMPVDPDN (SEQ ID NO: 1), and wherein the antibody has no reactivity to beta-synuclein or lower immune reactivity to beta-synuclein compared to alpha synuclein epitope comprising DMPVDPDN (SEQ ID NO:1).

2. The method according to claim 1, wherein the compound is coupled to a pharmaceutically acceptable carrier.

3. The method according to claim 1, wherein the compound is formulated for intravenous, subcutaneous, intradermal or intramuscular administration.

4. The method according to claim 1, wherein the compound is formulated with an adjuvant.

5. The method according to claim 1, wherein the compound is administered in the form of a medicament, wherein the medicament comprises the compound in an amount of from 0.1 ng to 10 mg based on the total weight of the medicament.

6. The method according to claim 2, wherein the compound is coupled to KLH (Keyhole Limpet Hemocyanin).

7. The method according to claim 4, wherein the compound is formulated with aluminium hydroxide.

8. The method according to claim 1, wherein the disease is Lewy Body Disorders (LBDs).

9. The method according to claim 1, wherein the disease is Parkinson's Disease (PD).

10. The method according to claim 1, wherein the disease is Parkinson's Disease with Dementia (PDD).

11. The method according to claim 1, wherein the disease is Dementia with Lewy Bodies (DLB).

12. The method according to claim 1, wherein the disease is Multiple System Atrophy (MSA).

13. The method according to claim 1, wherein the disease is Neurodegeneration with Brain Iron Accumulation type I (NBIA Type I).

Description

(1) The present invention is further illustrated in the following figures and examples, however, without being restricted thereto.

(2) FIG. 1 shows detection of alpha-synuclein specific epitopes by ELISA using a monoclonal specific for human alpha-synuclein at position 115-122.

(3) The peptides p4446 (alpha-synuclein), p4449 and p4448 (human epitopes) are detected by the antibody. The negative control peptides p4447 (beta-synuclein) and p4450, p4451 (mouse epitopes) are not detected. The irrelevant peptide p1252 does not show binding in the ELISA assay. Data are presented in a linear scale.

(4) FIG. 2 shows a detection of mimotopes by ELISA using a monoclonal specific for human alpha-synuclein at position 115-122. Data for two mimotopes (p4553, p4557) are displayed. The peptide p4557 shows weaker binding than the original peptide p4448. The peptide p4553 shows strong binding to the detection antibody. The irrelevant peptide p1253 does not show any binding in the ELISA assay as expected. Both mimotopes induce titers >1/20000 upon vaccination of mice and are considered as strong binders.

(5) FIG. 3 shows detection of competition of mimotopes by ELISA using a monoclonal specific for human alpha-synuclein at position 115-122.

(6) Values depicted are measured by ELISA using 40 g peptide in the inhibition assay. The irrelevant peptide p1253 and the mimotope p4492 do not show competition compared to original peptide p4448. Mimotopes p4490 and p4491 show similar competition as the original peptide p4448. Competition is calculated by comparing OD in ELISA at 40 g peptide concentration to the original epitope. All mimotopes are compared to this reference resulting in a competition index. Values around 1 indicate high inhibiting capacity. Peptides with a competition index above 5 are rated as non competing.

(7) FIG. 4 shows an immune response against injected peptide and an irrelevant peptide.

(8) A) Sera of immunized mice show high titers against their injected peptides (original epitope (p4448) and mimotopes (p4456, p4466 and p4467 respectively) after 4 vaccinations. Titers measured in ELISA are around 1:10.000 (OD half-max), data are presented in a logarithmic scale. As positive control for the ELISA an alpha-synuclein specific monoclonal antibody was used (CTRL pos).

(9) B) The same sera of immunized mice fail to detect an irrelevant peptide (p1253). Titers measured in ELISA are below 1:100 (OD half-max), more than 100 times lower than a signal from a monoclonal antibody specific for the irrelevant peptide (CTRL pos). As negative control no primary antibody is used. Data are presented in a linear scale.

(10) FIG. 5 shows an immune response against synucleins following repeated mimotope immunizations.

(11) A) Pooled sera of all animals within the respective groups show antibody titers against p4448, a peptide located in the C-terminal part of alpha-synuclein. Data are presented in a logarithmic scale.

(12) B) Pooled sera of immunized mice (p4448, p4457 and p4463) show titers against alpha-synuclein after 4 vaccinations. pooled sera of immunized mice (p4466 and p4467) do not detect alpha nor beta-synuclein (Titers measured in ELISA are much less than 1:100 half-max). Pooled sera of mice immunized with the original epitope (p4448) detect alpha and beta-synuclein. Titers in ELISA, which are less than 1:100 half-max are indicated by an asterisk, corresponding to values close to background. Most of the mimotopes tested induce antibodies that do not cross react with beta-synuclein. Data are presented in a logarithmic scale.

(13) FIG. 6A shows a positive control stain using a commercially available antibody specifically detecting human a-syn. In 6B the same antibody has been used to stain non-transgenic mouse brain of the same area which fails to detect any a-syn positive tissue as this animal is not expressing human a-syn. In 6C a specific a-syn staining similar to the staining present in 6A is elicited by a mimotope induced serum (p4498 induced serum). A-syn positive staining in the murine hippocampus is characterized by the speckled staining patterns as shown in 6A and 6C. Arrows indicate three examples for such a-syn positive inclusions in 6A and 6C respectively.

EXAMPLES

(14) An antibody which may be used for the mimotope identification according to the present invention detects the human alpha-synuclein-derived amino acid sequence DMPVDPDN (=original epitope, SEQ ID No. 1) and full length human alpha-synuclein. It does not recognize human beta-synuclein. The antibody may be a monoclonal or polyclonal antibody preparation or any antibody part or derivative thereof and binds specifically to the DMPVDPDN epitope (SEQ ID NO: 1) of human alpha-synuclein, i. e. it does bind to peptide and full length protein but does not bind to human beta-synuclein.

(15) The mimotopes are identified and further characterised with such monoclonal antibodies (detecting a sequence within amino acids 115-122 of the human alpha-synuclein protein) and peptide libraries.

Example 1: Generation of Monoclonal Antibodies to Specifically Detect Original Human Alpha-Synuclein Epitope C-DMPVDPDN (SEQ ID NO: 159) and Human Alpha-Synuclein but not Human Beta-Synuclein

(16) A monoclonal antibody derived from the fusion AFFiRiS 3: Balb/c mice were immunized with original alpha-synuclein epitope C-DMPVDPDN (SEQ ID NO: 159) coupled to BTG (bovine Thyroglobulin) and CFA (complete Freund's adjuvant; first injection) as well as IFA (incomplete Freund's adjuvant; 3 booster injections) as adjuvant. DMPVDPDN-peptide-specific (DMPVDPDN disclosed as SEQ ID NO: 1), antibody-producing hybridomas are detected by ELISA (DMPVDPDN-peptide-coated ELISA plates (DMPVDPDN disclosed as SEQ ID NO: 1)). Human alpha-synuclein (recombinant protein) is used as positive control peptide: hybridomas recognizing the recombinant protein immobilised on ELISA plates are included because they are binding both peptide and full length alpha-synuclein specifically. Human beta-synuclein (recombinant protein) is used as negative control peptide: hybridomas recognizing both recombinant proteins immobilised on ELISA plates are excluded because they do not distinguish between the two different synuclein proteins.

(17) The Hybridoma clone (AFFiRiS3/9 (internal name A509; IgG1) was analysed for specific detection of the natural human alpha-synuclein epitope DMPVDPDN (SEQ ID NO: 1). A509 recognizes the injected epitope as well as full length alpha-synuclein protein (recombinant protein; obtained from rPeptide, Bogart, Ga., USA) in ELISA. It however does not detect beta-synuclein protein (recombinant protein, obtained from rPeptide, Bogart, Ga., USA) in ELISA. Furthermore, the A509 antibodies do not detect the peptide encoding the mouse variant of alpha-synuclein. Similar results can be obtained with commercially available mAB clones (i.e. alpha-synuclein (LB509) Monoclonal Antibody Catalog Number SIG-39725; Covance (Princton, N.J., USA)).

Example 2: Phage Display, In Vitro Binding and Inhibition ELISA

(18) Phage Display libraries used in this example were: Ph.D. 7: New England BioLabs E8102L (linear 7mer library) and Ph.D. 12: New England BioLabs E8111L (linear 12mer library) Phage Display was done according to manufacturer's protocol.

(19) After 2 or 3 subsequent rounds of panning, single phage clones were picked and phage supernatants were subjected to ELISA on plates coated with the antibody that was used for the panning procedure. Phage clones that were positive in this ELISA (strong signal for the target, but no signal for unspecific control) were sequenced. From DNA sequences, peptide sequences were deduced. These peptides were synthesized and characterised in binding and inhibition ELISA. To some peptides additional AA were attached to the C-terminus. Additionally, some novel mimotopes were created by combining sequence information from mimotopes identified in the screen. Both groups containing newly designed mimotopes were used to support the identification of a consensus sequence for a mimotope vaccination.

(20) 1. In Vitro Binding Assay (ELISA)

(21) Peptides derived from Phage Display as well as C-terminally prolonged variants thereof were coupled to BSA and bound to ELISA plates (1 M; as indicated in the respective figures) and subsequently incubated with the monoclonal antibody that was used for the screening procedure to analyse binding capacity of identified peptides.

(22) 2. In Vitro Inhibition Assay (ELISA)

(23) Different amounts of peptides (concentrations ranging from 40 g to 0.3 g (serial dilutions), as indicated in the respective figures) derived from Phage Display were incubated with the monoclonal antibody that was used for the screening procedure. Peptides diminishing subsequent binding of the antibody to the original human alpha-synuclein epitope (amino acids: 115-122 of human alpha-synuclein protein) coated on ELISA plates were considered as inhibiting in this assay.

Example 3: In Vivo Testing of Mimotopes: Analysis of Immunogenicity and Crossreactivity

(24) 1. In Vivo Testing of Mimotopes

(25) Inhibiting as well as non-inhibiting peptides were coupled to KLH and injected into mice (wildtype C57/B16 mice; subcutaneous injection into the flank) together with an appropriate adjuvant (aluminium hydroxide). Animals were vaccinated 4-6 times in biweekly intervals and sera were taken biweekly as well. Titers to injected peptides as well as to an irrelevant peptide were determined with every serum. Titers against the recombinant human alpha-synuclein protein and recombinant human beta-synuclein were determined starting with Serum 3 respectively. Pooled sera were tested against the original human alpha-synuclein epitope (aa115-122). In general sera were analysed by reaction against peptides coupled to Bovine Serum Albumin (BSA) and recombinant full length proteins which were immobilised on ELISA plates. Titers were determined using anti mouse IgG specific antibodies. For detailed results see FIGS. 4 and 5.

(26) 2. In Situ Testing of Mimotopes

(27) Selected sera eliciting an a-syn cross reactivity were also tested for the ability to detect human a-syn on mouse brain sections in situ. For detailed results see FIG. 6.

(28) 3. Results

(29) 3.1. Identification of an Alpha-Synuclein Specific mAB:

(30) FIG. 1 depicts the characterisation of the alpha-synuclein specific monoclonal antibody AFFiRiS3/9 (internal name A509; IgG1) derived from fusion AFFiRiS 3.

(31) 3.2. Screening with Alpha-Synuclein Specific mAB:

(32) 3.2.1. Phage Display Library Ph.D. 7 and 12

(33) 3.2.1.1. Screening with Monoclonal Antibody Directed Against DMPVDPDN (SEQ ID NO: 1)

(34) 51 sequences were identified by screening PhD 7 and PhD12 phage display libraries in this screen: Table 1 summarises the peptides identified and their binding capacity as compared to the original epitope.

(35) TABLE-US-00004 TABLE1 alpha-synucleinmimotopesbindingtothe parentalantibody Internal SEQ Peptide ID Binding number No. Sequence Capacity p4456 2 CDQPVLPD 3 p4457 3 CDMPVLPD 3 p4458 4 CDSPVLPD 3 p4460 5 CDSPVWAE 1 p4461 6 CDTPVLAE 1 p4462 7 CDQPVLPDN 3 p4463 8 CDMPVLPDN 3 p4464 9 CDSPVLPDN 3 p4465 10 CDQPVTAEN 3 p4466 11 CDSPVWAEN 3 p4467 12 CDTPVLAEN 3 p4484 13 CHDRPVTPD 3 p4485 14 CDRPVTPD 3 P4486 15 CDNPVHPE 1 p4487 16 CDVPVLPD 3 p4488 17 CDTPVYPD 3 p4489 18 CDTPVIPD 3 p4490 19 CHDRPVTPDN 3 p4491 20 CDRPVTPDN 3 p4492 21 CDNPVHPEN 3 p4493 22 CDVPVLPDN 3 p4494 23 CDTPVYPDN 3 p4495 24 CDTPVIPDN 3 p4496 25 CDQPVLPDG 3 p4497 26 CDMPVLPDG 3 p4498 27 CDSPVLPDG 3 p4499 28 CDSPVWAEG 3 p4553 29 CDRPVAPEG 3 p4554 30 CDHPVHPDS 3 p4555 31 CDMPVSPDR 3 p4556 32 CDSPVPPDD 3 p4557 33 CDQPVYPDI 3 p4558 34 CDRPVYPDI 3 p4559 35 CDHPVTPDR 1 p4560 36 CEYPVYPES 3 p4561 37 CDTPVLPDS 3 p4562 38 CDMPVTPDT 3 p4563 39 CDAPVTPDT 3 p4564 40 CDSPVVPDN 3 p4566 41 CDLPVTPDR 3 p4567 42 CDSPVHPDT 3 p4568 43 CDAPVRPDS 3 p4569 44 CDMPVWPDG 3 p4570 45 CDAPVYPDG 3 p4571 46 CDRPVQPDR 3 p4572 47 CYDRPVQPDR 3 p4635 48 CDMPVDPEN 3 p4636 49 CDMPVDADN 3 p4640 50 DQPVLPDC 3 p4641 51 DMPVLPDC 3 P4648 52 CEMPVDPDN 3

(36) Legend to Table 1: the binding capacity is coded by the following binding code: 1:X describes the dilution factor of the parental AB.

(37) TABLE-US-00005 binding code OD halfmax 1:X 0 no binding :0 1 weak binding :<5000 2 medium binding :5000-20000 3 binding as original epitope (strong :20000-128000 binding)

(38) 3.3. In Vitro Characterisation of Mimotopes Identified in Screening Phage Display Libraries with a Monoclonal Antibody Directed Against Alpha-Synuclein:

(39) FIGS. 2 and 3 show representative examples for binding and inhibition assays used to characterise mimotopes in vitro. Data obtained are summarised in Tables 1 and 2 respectively.

(40) From the 51 sequences presented 29 sequences inhibit binding of the alpha-synuclein specific monoclonal antibody in in vitro competition experiments: Additional 22 sequences were identified that do not inhibit binding of monoclonal antibody in in vitro competition experiments but still retain binding capacity to the parental antibody:

(41) TABLE-US-00006 TABLE2 Alpha-synucleinmimotopesidentifiedin thisinventiongivingpositiveresultsin inhibitionassays Internal SEQ Peptide ID number No. Sequence Inhibition p4462 7 CDQPVLPDN 2 p4463 8 CDMPVLPDN 2 p4464 9 CDSPVLPDN 2 p4490 19 CHDRPVTPDN 2 p4491 20 CDRPVTPDN 1 p4493 22 CDVPVLPDN 2 p4494 23 CDTPVYPDN 2 p4495 24 CDTPVIPDN 1 p4496 25 CDQPVLPDG 1 p4497 26 CDMPVLPDG 1 p4498 27 CDSPVLPDG 1 p4554 30 CDHPVHPDS 1 p4555 31 CDMPVSPDR 1 p4557 33 CDQPVYPDI 1 p4558 34 CDRPVYPDI 2 p4559 35 CDHPVTPDR 1 p4561 37 CDTPVLPDS 2 p4562 38 CDMPVTPDT 2 p4563 39 CDAPVTPDT 1 p4564 40 CDSPVVPDN 1 p4566 41 CDLPVTPDR 1 p4567 42 CDSPVHPDT 1 p4569 44 CDMPVWPDG 1 p4570 45 CDAPVYPDG 1 p4571 46 CDRPVQPDR 1 p4572 47 CYDRPVQPDR 1 p4640 50 DQPVLPDC 2 p4641 51 DMPVLPDC 2 P4648 52 CEMPVDPDN 1

(42) Legend to Table 2: the inhibition capacity is coded by the following code:

(43) Weak inhibition means more peptide is required to lower AB binding than with the original epitope; strong inhibition means similar peptide amounts are required for mimotope and original epitope for lowering AB binding. Mimotopes are compared to the original peptide as standard. OD at 40 g peptide used in the assay is used to calculate the competition capacity compared to original peptide.

(44) TABLE-US-00007 competition code 0 no inhibition (OD of 40 g peptide above 5 times of original peptide) 1 Weaker than original epitope (OD of 40 g peptide below 5 times of original peptide) 2 strong inhibition (as original epitope; OD of 40 g peptide below 2 times of original peptide)

(45) TABLE-US-00008 TABLE3 Non-Mimotopepeptidesandproteins: Internal SEQ Peptide ID number No. Sequence 1 DMPVDPDN p4446 Humanalpha-syn(Full length;NCBIAcc.No. NP_000336) p4447 Humanbeta-syn(Full length;NCBIAcc.No. NP_001001502) p4448 53 CDMPVDPDN p4449 54 DMPVDPDNC p4450 55 CDMPVDPGS p4451 56 DMPVDPGSC

(46) 3.4. In Vivo Characterisation of Mimotopes Identified in Screening Phage Display Libraries with a Monoclonal Antibody Directed Against Alpha-Synuclein:

(47) Female C57/B16 mice, 5-6 mice per group, were subcutaneously immunized with 30 g peptide coupled to KLH. Control groups were administered p4448-KLH conjugate. As adjuvant alum was used (always 1 mg per mouse). The peptides administered were all able to bind to monoclonal antibodies specifically binding aa115-122 of human alpha-synuclein although some of the peptides did not inhibit the binding of the original epitope to its parental antibody in vitro (in an in vitro inhibition assay). The in vitro ELISA assay to determine the antibody titer was performed with sera of single mice or pooled sera (see FIG. 5) after each vaccination in a two week interval (see FIGS. 4 and 5, respectively). The wells of the ELISA plate were coated with mimotope-BSA conjugate and an irrelevant peptide-BSA conjugate (negative control). The positive control was performed by reaction of the parental antibody with the respective mimotope-BSA conjugate. The detection was performed with anti-mouse IgG. Additionally, recombinant proteins were immobilised on ELISA plates and sera reacted accordingly.

(48) For all mimotopes tested in C57/B16 mice, antibodies reacting to the individual injected peptide could be detected after repeated vaccination. Additionally, 2 out of 4 depicted mimotopes (see FIG. 5 and Table 1 respectively) developed antibodies reacting with human alpha-synuclein but not with human beta-synuclein. 2/4 showed no cross reactivity with recombinant proteins. Importantly, the original epitope DMPVDPDN (SEQ ID NO: 1) led to an immune response which did not distinguish between the two recombinant synuclein proteins.

(49) FIGS. 4 and 5 show representative examples for assays used to characterise mimotopes in vivo. FIG. 4 shows an example for in vivo characterisations of the immune response elicited by mimotope vaccination by analysing the immune response against injected peptide and an irrelevant peptide, containing an unrelated sequence. The original epitope p4448, the positive control peptide, and the mimotopes p4456, p4466 and p4467, elicited immune responses against the injected peptide (themselves) but failed to induce an unspecific immune response against an unrelated sequence (p1253).

(50) FIG. 5 shows an example for in vivo characterisations of the immune response elicited by mimotope vaccination against full length alpha-synuclein and beta-synuclein. All vaccines tested in this example mounted a detectable immune response against the original alpha-synuclein epitope 115-122. Nearly all mimotopes and the original epitope tested in this example (exception: p4466 and p4467) furthermore also show reactivity with full length alpha-synuclein. However, the original epitope-induced immune response also detected the full length beta-synuclein protein thus loosing the specificity for alpha-synuclein and the ability to distinguish between the two proteins. In contrast to this finding most of (but not all) the mimotope-induced sera failed to detect beta-synuclein thus preserving the ability to discriminate between the two synuclein proteins and guaranteeing high anti alpha-synuclein specificity of an active immunisation programme, to obtain efficacy and an excellent safety profile.

(51) 3.5. In Situ Testing of Mimotopes

(52) Mimotopes eliciting an a-syn specific immune response can also detect a-syn immunoreactive inclusions in transgenic mouse brain tissue. As depicted in FIG. 6, sera derived from mimotope vaccinated animals are able to stain a-syn positive structures present on mouse brain sections from animals overexpressing human a-syn. Briefly, sera positive for human a-syn reactivity in ELISA have been used for immunohistochemistry (IHC). Paraffin embedded 7 M sections of mouse brain, mounted on Superfrost Plus glass slides, were subjected to IHC. Sections were incubated with sera (dilution 1:100 and 1:400 in PBS) and subsequently stained according to standard protocols for immunohistochemistry using VECTASTAIN ABC Systems, DAB and MOM blocking (all reactions have been performed using commercially available reagents obtained from Vector labs respectively and have been performed according to manufacturer's protocols). Counterstaining was performed with Haematoxylin. Slides were mounted in Entellan and subsequently documented using conventional brightfield microscopy. A monoclonal antibody specific for human a-syn (LB509, Covance) has been used as a positive control for synuclein detection at a final dilution of 1/250.

(53) In FIG. 6A a positive control stain is depicted. In 6B the same antibody has been used on non-transgenic mouse brain of the same area which failed to detect any a-syn positive tissue as this animal is not expressing human a-syn. In 6C a specific a-syn staining similar to the staining present in 6A is elicited by a mimotope induced serum (p4498 induced serum). A-syn positive staining in the murine hippocampus is characterized by the speckled staining patterns as shown in 6A and 6C. Examples for the potential to induce a-syn specific antibodies include but are not limited to vaccines based on p4456, p4498 and p4562 respectively.