Immunogenic Compounds

Abstract

An antigenic peptide comprises the structure X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-P-X.sub.9-X.sub.10-X.sub.11-X.sub.12 and is derived from amino acids 113-124 of alpha synuclein. Ac-cording to the structure: P is proline; X.sub.1 is L, K, A or S, wherein L is leucine, K is lysine, A is alanine and S is serine; X.sub.2 is E or S, wherein E and S are as defined above; X.sub.3 is D, E, K, N, A or S, wherein N is asparagine, D is aspartic acid and D, E, K, A and S are as defined above; X.sub.4 is M, A, S, L or K, wherein M is methionine and A, S, L and K are as defined above; X.sub.5 is P or A as defined above; X.sub.6 is V, A or S, wherein V is valine and A and S are as defined above; X.sub.7 is D or S as defined above; X.sub.9 is D or A as defined above; X.sub.10 is N, S or A, wherein N, S and A are as defined above; X.sub.11 is E, A or S, wherein E, A and S are as defined above; X.sub.12 is present or not and, if present, is A, K, V, S, or G wherein G is glycine and A, K, V and S are as defined above. The structure comprises at least one mutation compared to the wild type L-E-D-M-P-V-D-P-D-N-E-A sequence. The peptide does not comprise the dipeptide Y-E immediately following X12, wherein Y is tyrosine and E is as defined above. The peptides are conjugated to a suitable carrier and useful in treating synucleinopathies.

Claims

1. An antigenic peptide comprising, consisting essentially of or consisting of the structure:
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-P-X.sub.9-X.sub.10-X.sub.11-X.sub.12, wherein: P is proline; X.sub.1 is L, K, A or S, wherein L is leucine, K is lysine, A is alanine and S is serine; X.sub.2 is E or S, wherein E is glutamic acid and S is as defined above; X.sub.3 is D, E, K, N, A or S, wherein N is asparagine, D is aspartic acid and E, K, A and S are as defined above; X.sub.4 is M, A, S, L or K, wherein M is methionine and A, S, L and K are as defined above; X.sub.5 is P or A as defined above; X.sub.6 is V, A or S, wherein V is valine and A and S are as defined above; X.sub.7 is D or S as defined above; X.sub.9 is D or A as defined above; X.sub.10 is N, S or A, wherein N, S and A are as defined above; X.sub.11 is E, A or S, wherein E, A and S are as defined above; X.sub.12 is present or not and, if present, is A, K, V, S, or G wherein G is glycine and A, K, V and S are as defined above, with the proviso that X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-P-X.sub.9-X.sub.10-X.sub.11-X.sub.12 is not L-E-D-M-P-V-D-P-D-N-E-A, and which comprises between 1 and 5 amino acid differences compared with the amino acid sequence L-E-D-M-P-V-D-P-D-N-E-A, and further wherein the peptide does not comprise the dipeptide Y-E immediately following X.sub.12, wherein Y is tyrosine and E is as defined above.

2. The antigenic peptide of claim 1 which comprises between 1 and 4 amino acid differences compared with the amino acid sequence L-E-D-M-P-V-D-P-D-N-E-A, preferably between 1 and 3 amino acid differences and most preferably 2 amino acid differences.

3. The antigenic peptide of claim 1 or 2 which comprises amino acid differences compared with the amino acid sequence L-E-D-M-P-V-D-P-D-N-E-A at one or more positions selected from X.sub.1, X.sub.3, X.sub.4 and X.sub.12.

4. The antigenic peptide of any preceding claim comprising, consisting essentially of or consisting of the structure
X.sub.a-X.sub.b-X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-P-X.sub.9-X.sub.10-X.sub.11-X.sub.12, wherein: X.sub.a is present or not and, if present, is G, wherein G is as defined in claim 1; X.sub.b is G, wherein G is as defined above; and X.sub.1-X.sub.12 are as defined in claim 1.

5. The antigenic peptide of any preceding claim, which is 11-20 amino acids in length, preferably 12-14 amino acids in length.

6. The antigenic peptide of any preceding claim, which further comprises a terminal cysteine residue, preferably an N-terminal cysteine residue.

7. The antigenic peptide of any preceding claim, wherein X.sub.1 is L, S, A, or K, X.sub.2 is E or S, X.sub.3 is S, D, E, A, K or N, X.sub.4 is M, X.sub.5 is P; X.sub.6 is V; X.sub.7 is D; X.sub.9 is D; X.sub.10 is N, and/or X.sub.12 is A, S, K or V, preferably wherein X.sub.1 is L or K, X.sub.2 is E, X.sub.3 is S, D, E, K or A, X.sub.4 is M, X.sub.10 is N, and/or X.sub.12 is A, S or K, especially wherein X.sub.1 is L or K, X.sub.3 is D, K or S and X.sub.12 is A.

8. The antigenic peptide of any preceding claim which is selected from the group consisting of AEDMPVDPDNEA, KESMPVDPDNEA, LESMPVDPDNEA, LESMPVDPDNES, SEDMPVDPDNEA, SEKMPVDPDNEA LEEMPVDPDNEA, SESMPVDPDNEA, LEDMPVDPDNES, LEAMPVDPDNEA, LEDMPVDPDNEK, LEDMPVDPDNEV, LEKMPVDPDNEK, LSDMPVDPDNEA, LEKMPVDPDNEA, LEKMPVDPDNES, LENMPVDPDNEA, KESMPVDPDNEK and KEDMPVDPDNEA, preferably SEDMPVDPDNEA, SEKMPVDPDNEA, LEEMPVDPDNEA, LEKMPVDPDNEK, LESMPVDPDNEA, LESMPVDPDNES, KESMPVDPDNEA, KEDMPVDPDNEA, LEKMPVDPDNES, LEKMPVDPDNEA and LESMPVDPDNES, especially LEKMPVDPDNEA, KESMPVDPDNEK, KESMPVDPDNEA and KEDMPVDPDNEA.

9. The antigenic peptide of any preceding claim which comprises, consists essentially of or consists of the amino acid sequence KESMPVDPDNEA, GKESMPVDPDNEA, GGKESMPVDPDNEA or CGGKESMPVDPDNEA.

10. An immunogenic compound comprising the antigenic peptide of any preceding claim and a carrier comprising T-cell epitopes attached to the antigenic peptide.

11. The immunogenic compound of claim 10, wherein the carrier comprising T-cell epitopes is attached to the antigenic peptide via a linker.

12. The immunogenic compound of claim 10 or 11 wherein the carrier comprising T-cell epitopes is attached at the N terminal end of the antigenic peptide.

13. The immunogenic compound according to claim 12, wherein the carrier protein is selected from the group consisting of Keyhole Limpet Hemocyanin (KLH), tetanus toxoid, heat-labile enterotoxin (LT), cholera toxin (CT), diphtheria toxin (DT) and variants thereof, especially CRM197, tetanus toxoids (TT), mutant toxins, albumin-binding proteins, and bovine serum albumin, preferably CRM197.

14. A pharmaceutical preparation comprising an antigenic peptide according to any one of claims 1 to 9 or an immunogenic compound according to any one of claims 10 to 13 and a pharmaceutically acceptable excipient, preferably in the form of a vaccine composition.

15. The pharmaceutical preparation according to claim 14, further comprising an adjuvant.

16. The pharmaceutical preparation according to claim 15, wherein the adjuvant is selected from the group consisting of MF59 aluminium phosphate, calcium phosphate, cytokines (e.g., IL-2, IL-12, GM-CSF), saponins (e.g., QS21), MDP derivatives, CpG oligos, IC31, LPS, MPLA, polyphosphazenes, and aluminium hydroxide, or mixtures thereof; especially with aluminium hydroxide as adjuvant.

17. The pharmaceutical preparation according to claim 15 or 16, wherein the antigenic peptide is contained in an amount from 0.1 ng to 10 mg, preferably 10 ng to 1 mg, in particular 100 ng to 100 μg.

18. The pharmaceutical preparation according to any one of claims 14 to 17, wherein the preparation is formulated for parenteral administration, such as subcutaneous, intradermal, intravenous or intramuscular administration.

19. The antigenic peptide according to any one of claims 1 to 9, immunogenic compound according to any one of claims 10 to 13 or pharmaceutical preparation according to any one of claims 14 to 18, for use as a medicament.

20. The antigenic peptide according to any one of claims 1 to 9, immunogenic compound according to any one of claims 10 to 13 or pharmaceutical preparation according to any one of claims 14 to 18, for use as in the treatment or prevention of a synucleinopathy.

21. Use of the antigenic peptide according to any one of claims 1 to 9, immunogenic compound according to any one of claims 10 to 13 or pharmaceutical preparation according to any one of claims 14 to 18, for the manufacture of a medicament for the treatment or prevention of a synucleinopathy.

22. A method for the treatment or prevention of a synucleinopathy comprising administering an effective amount of the antigenic peptide according to any one of claims 1 to 9, immunogenic compound according to any one of claims 10 to 13 or pharmaceutical preparation according to any one of claims 14 to 18, to a subject in need thereof.

23. The use according to any one of claim 20 or 21 or method according to claim 22 wherein the synucleinopathy is a primary synucleinopathy or a concomitant pathology.

24. The use or method according to claim 23 wherein the primary synucleinopathy is selected from the group consisting of Parkinson's disease (sporadic, familial with alpha-synuclein mutations, familial with mutations other than alpha-synuclein, pure autonomic failure and Lewy body dysphagia), Lewy Body dementia (LBD; including dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), or Diffuse Lewy Body Disease, multiple system atrophy (Shy-Drager syndrome, striatonigral degeneration and olivopontocerebellar atrophy).

25. The use or method according to claim 23 wherein the concomitant pathology is selected from the group consisting of sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2 or other mutations, familial British dementia, inclusion-body myositis, traumatic brain injury, chronic traumatic encephalopathy, dementia pugilistica, tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, Frontotemporal dementia with Parkinsonism linked to chromosome 17 and Niemann-Pick type C1 disease), Down syndrome, Creutzfeldt-Jakob disease, Huntington's disease, motor neuron disease, amyotrophic lateral sclerosis (sporadic, familial and ALS-dementia complex of Guam), neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type 1 (Hallervorden-Spatz syndrome), prion diseases, Gerstmann-Straussler-Scheinker disease, ataxia telangiectasia, Meige's syndrome, subacute sclerosing panencephalitis, Gaucher disease, Krabbe disease as well as other lysosomal storage disorders (including Kufor-Rakeb syndrome and Sanfilippo syndrome), or rapid eye movement (REM) sleep behavior disorder.

26. The use according to any one of claim 20 or 21 or method according to claim 22 wherein the synucleinopathy is selected from the group consisting of Lewy Body Disorders (LBDs), especially Parkinson's Disease (PD), Parkinson's Disease with Dementia (PDD) and Dementia with Lewy Bodies (DLB), as well as Multiple System Atrophy (MSA) or Neurodegeneration with Brain Iron Accumulation type I (NBIA Type I).

27. The use or method according to any one of claims 21 to 26 wherein a human subject is treated.

Description

[0175] The invention is further described by the following examples and the figures, yet without being limited thereto.

[0176] FIG. 1: Schematic time-course of the experiments. Injections are represented by arrows and blood sampling by drops. PP: Pre-plasma, EP: End-plasma. Pn: Plasma n. Wn: Week n of the experiment.

[0177] FIG. 2: Comparison of aSyn cross-reacting antibodies induced by p4456 and p4572 or by the corresponding native aSyn epitope of different length. (A) Induced antibody concentrations against aSyn protein from end-plasma of all individual mice are presented. Columns represent mean values with SEM. Extreme outliers (beyond 3× the IQR) have been removed from the graph (B) Relative position of the injected peptides along the amino-acid sequence of the native aSyn sequence. p9524 was used as scaffold for further development of AFFITOPE® s and is highlighted in gray.

[0178] FIG. 3: Immunogenicity of peptides (delivered as peptide-carrier protein conjugates) with aSyn sequence aa113-124 with single alanine (A) or serine (B) exchanges. Group median concentrations of anti-aSyn antibodies in the end-plasma derived from individual immunized mice were determined and set in relation to the concentration of anti-aSyn antibodies elicited by vaccination with p9524, which was set as 100%. Sequences of the injected peptides are shown in Table 3 and 4.

[0179] FIG. 4: Immunogenicity of peptides (delivered as peptide-carrier protein conjugates) with aSyn sequence aa113-124 with single amino acid exchanges at position 1 or 3 (A) and 4 or 12 (B). Group median concentrations of anti-aSyn antibodies in the end-plasma derived from individual immunized mice were determined and set in relation to the concentration of anti-aSyn antibodies elicited by vaccination with p9524, which was set as 100%. Sequences of the injected peptides are shown in Table 7 and 8.

[0180] FIG. 5: Immunogenicity to aSyn induced by aSyn sequence aa.sub.113-124 with double serine and other amino acid exchanges. Group median concentrations of anti-aSyn antibodies in the end-plasma derived from individual immunized mice were determined and set in relation to the concentration of anti-aSyn antibodies elicited by vaccination with p9524, which was set as 100%. Sequences of the injected peptides are shown in Table 11.

[0181] FIG. 6: Immunogenicity to aSyn induced by aSyn sequences aa.sub.113-124 and N-terminally prolonged sequences. Group median concentrations of anti-aSyn antibodies in the end-plasma derived from individual immunized mice were determined and set in relation to the concentration of anti-aSyn antibodies elicited by vaccination with p10074, which was set as 100%. Sequences of the injected peptides are shown in Table 13.

[0182] FIG. 7: Immunogenicity to aSyn induced by aSyn sequences aa.sub.113-124, N-terminally prolonged sequences, and by the aSyn sequence aa.sub.115-121 p4456. Group median concentrations of anti-aSyn antibodies in the end-plasma derived from individual immunized mice were determined and set in relation to the concentration of anti-aSyn antibodies elicited by vaccination with p4456, which was set as 100%. Sequences of the injected peptides are shown in Table 13.

[0183] FIG. 8: Immunogenicity of aSyn targeting sequences p10033 and p10118 and C-terminally truncated sequences thereof. (A) Group median immunogenicity of anti-aSyn antibodies in the end-plasma derived from individual immunized mice were determined and set in relation to the concentration of anti-aSyn antibodies elicited by vaccination with p10033, which was set as 100%. (B) Group median immunogenicity of anti-aSyn antibodies in the end-plasma derived from individual immunized mice were determined and set in relation to the concentration of anti-aSyn antibodies elicited by vaccination with p10118, which was set as 100%. Sequences of the injected peptides are shown in Table 16.

[0184] FIG. 9: IHC staining of post mortem human DLB brains by AFFITOPE® candidate-induced antibodies. The antibodies used in each panel are induced by the peptide shown on top of the respective panel. Underlined letters indicate amino acids that are different from the native sequence. Size bars indicate 50 μM in the main pictures and 10 μM in the smaller box in the lower right corner focusing on single Lewy Bodies (LBs).

[0185] FIG. 10: Preferential binding to the oligomeric and toxic aSyn species vs the monomeric form (BiaCore data). Sensograms of the stability binding of AFFITOPE® candidate-induced antibodies and monoclonal antibodies LB509 and 28A7 to the oligomeric (red curve) or monomeric aSyn (green curve) species. The blue lines represent the negative control (binding to HBS buffer only). X-axis: running time (sec), y-axis: relative binding response units.

[0186] FIG. 11: Competition ELISA showing concentration-dependent inhibition of the binding of AFFITOPE® candidate-specific antibodies to aSyn monomers, oligomers, and filaments. Purified antibodies from AFFITOPE® candidate-immunized mice were pre-incubated with increasing amounts of different aSyn-species and then tested for binding to plate bound aSyn-oligomers. Inhibition of binding by monomeric aSyn is shown in red curves, inhibition by fibrillar aSyn in blue curves, and inhibition by oligomers in green curves. X-axis: decadic logarithm of aSyn-species concentration (ng/ml), y-axis: OD 405 value measured with each aSyn species and concentration.

EXAMPLES

[0187] Identification of aSyn-AFFITOPE® s

[0188] Given the potential toxicity of various aSyn aggregates, therapeutic approaches for synucleinopathies might involve reducing the levels or accumulation of intracellular and extracellular aSyn. The fact that oligomeric aSyn is prone to be secreted into the intercellular space and is able to move from one affected neuron or (e.g. in the case of MSA) oligodendrocyte to a neigh-boring neuron or neuroglial cell in a prion-like fashion (Lee et al., 2008, Lashuel et al., 2013, Bengoa-Vergniory et al., 2017, Bernis et al., 2015) opens an avenue for a therapeutic approach such as AFFIRIS specific active immunotherapy (SAIT), that targets aSyn-transmission in a long-term manner. The AFFITOPE® s PD01 and PD03—short synthetic peptides mimicking defined regions of aSyn—have been tested as compounds for SAIT in clinical Phase I trials in PD—PD01A (NCT01568099; Volc et al., 2020) and PD03A (NCT02267434) —and in multiple system atrophy (MSA) patients (PD01 and PD03) (NCT02270489). The agents were shown to be well tolerated and elicited aSyn-specific antibodies with a preference for oligomeric aSyn.

[0189] In the course of the present invention a second-generation of aSyn-targeting AFFITOPE® s were developed, which induced higher titers and cross-reactivity to aSyn protein, than seen for PD01 and PD03, respectively. Furthermore, the focus of selection was also on the ability of the induced antibodies to discriminate between the aggregated and toxic aSyn species (oligomeric aSyn) and the monomeric aSyn protein. Thus, the binding of AFFITOPE®-induced antibodies to the overrepresented monomeric aSyn species in the periphery is supposed to be of minor extent, however, the binding to the toxic oligomeric and underrepresented aSyn species in the CNS and the periphery would occur preferably.

[0190] To identify highly potential AFFITOPE® s the aSyn epitope aa.sub.113-124 was targeted. Well-defined selection criteria were applied such as high immunogenicity, high cross-reactivity to aSyn native epitope, and binding of induced antibodies with high selectivity towards oligomeric and fibrillar toxic aSyn species (oligomer binding>fibril binding).

[0191] The selection strategy consisted of several steps: (i) epitope finding within the C-terminus of aSyn target protein in order to identify a highly immunogenic and suitable epitope, (ii) alanine scan in order to detect positions along the native target sequence that can be exchanged in order to enhance immunogenicity and cross-reactivity, (iii) serine scans in order to detect positions along the native target sequence that can be exchanged in order to enhance immunogenicity and cross-reactivity, (iv) double serine exchanges along the native target sequence, (v) other than Ala or Ser amino acid exchanges able to improve immunogenicity and cross-reactivity towards aSyn, and (vi) removal of one amino acid at the C-terminus from two selected AFFITOPE® sequences.

Immunization Schedule of all In Vivo Experiments

[0192] In all experiments described herein with BALB/c, mice received 3 injections with AFFITOPE® s or native aSyn epitope sequences (10 μg net peptide per injection), in biweekly intervals (FIG. 1).

Epitope Finding within the C-Terminus of the aSyn Target Protein (aSyn-28 In Vivo Experiment)

[0193] Epitope screening for highly immunogenic sequences along the C-terminal region of the aSyn protein has been performed. For this purpose, differently long amino acid stretches varying between 7 and 13 amino acids were used for immunogenicity studies in wt BALB/c mice (Table 1, FIG. 1). In parallel, previously selected AFFITOPE® s by AFFiRiS p4456 and p4572 were included for comparison. Plasmas from all individual animals were collected two weeks after the third immunization and subsequently analyzed by ELISA in order to determine the titers against the injected peptide and aSyn protein as well as the concentrations of aSyn reacting antibodies. The peptide p9524 corresponding to aSyn aa.sub.113-124 sequence induced the highest amount of aSyn-specific antibodies (FIG. 2, Table 2). Based on these findings this sequence was selected as native target sequence for further AFFITOPE® candidate selection. The two other peptides p9964 and p9556 which elicited also high anti-aSyn titers were excluded from further development. The peptide p9964 includes the amino acids Y.sub.125 and E.sub.126 and peptide fragments derived from p9964 are predicted by in silico analyses to bind with high affinity to different allelic variants of MHCI and thus be potential cytotoxic T cell epitopes (www.syfpeithi.de). The peptide p9556 was not chosen for further development because it does not cover the potentially pathology-related calpain cleavage site aSyn L.sub.113/E.sub.114. Table 2 summarizes the titers to aSyn found in AFFITOPE® induced mouse plasmas.

Table 1: Setup of experiments aSyn-28. The table shows the drug product, the sequence of the peptide in the drug product and the corresponding sequence ID numbers.

TABLE-US-00001 TABLE 1 Seq Additional ID Group Drug Product Sequence information 1 1 p4456-CRM197 C-DQPVLPD aSyn115-121 (M.fwdarw.Q, Aluminium hydroxide D.fwdarw.L) 2 2 p4572-CRM197 C-YDRPVQPDR aSyn114-122 (E.fwdarw.Y, Aluminium hydroxide M.fwdarw.R, D.fwdarw.Q, N.fwdarw.R) 3 3 p9964-CRM197- C-EDMPVDPDNEAYE aSyn114-126 Aluminium hydroxide 4 4 p9524-CRM197 C-LEDMPVDPDNEA aSyn113-124 Aluminium hydroxide 5 5 p9556-CRM197 C-DMPVDPDNEA aSyn115-124 Aluminium hydroxide 6 6 p9557-CRM197 C-EDMPVDPDNE aSyn114-123 Aluminium hydroxide 7 7 p9663-CRM197 C-VDPDNEAYE aSyn118-126 Aluminium hydroxide
Table 2: aSyn-28, key results including titers, induced antibody concentration, and cross-reactivity. All parameters were evaluated in single mice and values refer to medians. The concentration of anti-aSyn antibodies in the plasma of immunized mice was extrapolated from a reference curve generated with the mAb LB509.

TABLE-US-00002 TABLE 2 Titer against Titer against Ab conc. to Seq ID Group injected peptide aSyn aSyn [μg/ml] 1 1 98101 22951 97.0 2 2 114544 12449 49.5 3 3 62282 88308 375.1 4 4 90418 91136 420.9 5 5 78491 90089 376.9 6 6 33753 29295 117.5 7 7 93592 30084 129.7
Alanine-Scan Along the aSyn113-124 Epitope (aSyn-30) and Serine-Scan (aSyn-31)

[0194] As a next step, each amino acid position of the native aSyn.sub.113-124 sequence was exchanged either by an alanine or a serine in order to identify substitutable positions in order to maintain or enhance immunogenicity (Tables 3 and 4). BALB/c mice were injected three times with the respective AFFITOPE® s and two weeks after the third and last injection, plasma of each individual mouse was collected and analyzed by ELISA in order to determine the AFFITOPE®-induced titers and aSyn antibody concentrations. An alanine scan revealed that the Leu at position 1 (p9988) and Asp at position 3 (p9990) along the aSyn 113-124aa (p9524) sequence are not essential, and on the contrary, their exchange to Ala leads to higher cross-reactivity to aSyn (FIG. 3A). The serine scan i.e. exchanges of each native occurring amino acids along the aSyn 113-124 sequence with serine (FIG. 3B) revealed a higher cross-reactivity to aSyn by amino acid exchanges at position 1 (p9999), 2 (p10000), 3 (p10001), and 12 (p10010) to Ser. Tables 5 and 6 summarize the induced titers relative to the titers induced by the WT sequence against aSyn present in the plasma of immunized mice.

Table 3: Setup of experiment aSyn-30. The table shows the different treatment groups, the respective drug product in use, the sequence of the peptide in the drug product, and the corresponding sequence ID numbers.

TABLE-US-00003 TABLE 3 Seq ID Group Drug Product Sequence  4  1 p9524-CRM197 Aluminum hydroxide C-LEDMPVDPDNEA  8  2 p9988-CRM197 Aluminum hydroxide C-AEDMPVDPDNEA  9  3 p9989-CRM197 Aluminum hydroxide C-LADMPVDPDNEA 10  4 p9990-CRM197 Aluminum hydroxide C-LEAMPVDPDNEA 11  5 p9991-CRM197 Aluminum hydroxide C-LEDAPVDPDNEA 12  6 p9992-CRM197 Aluminum hydroxide C-LEDMAVDPDNEA 13  7 p9993-CRM197 Aluminum hydroxide C-LEDMPADPDNEA 14  8 p9994-CRM197 Aluminum hydroxide C-LEDMPVAPDNEA 15  9 p9995-CRM197 Aluminum hydroxide C-LEDMPVDADNEA 16 10 p9996-CRM197 Aluminum hydroxide C-LEDMPVDPANEA 17 11 p9997-CRM197 Aluminum hydroxide C-LEDMPVDPDAEA 18 12 p9998-CRM197 Aluminum hydroxide C-LEDMPVDPDNAA
Table 4: Setup of the experiment aSyn-31. The table shows the different treatment groups, the respective drug product in use, the sequence of the peptide in the drug product and the corresponding sequence ID numbers.

TABLE-US-00004 TABLE 4 Seq ID Group Drug Product Sequence  4  1 p9524-CRM197 Aluminum hydroxide C-LEDMPVDPDNEA 19  2 p9999-CRM197 Aluminum hydroxide C-SEDMPVDPDNEA 20  3 p10000-CRM197 Aluminum hydroxide C-LSDMPVDPDNEA 21  4 p10001-CRM197 Aluminum hydroxide C-LESMPVDPDNEA 22  5 p10002-CRM197 Aluminum hydroxide C-LEDSPVDPDNEA 23  6 p10003-CRM197 Aluminum hydroxide C-LEDMSVDPDNEA 24  7 p10004-CRM197 Aluminum hydroxide C-LEDMPSDPDNEA 25  8 p10005-CRM197 Aluminum hydroxide C-LEDMPVSPDNEA 26  9 p10006-CRM197 Aluminum hydroxide C-LEDMPVDSDNEA 27 10 p10007-CRM197 Aluminum hydroxide C-LEDMPVDPSNEA 28 11 p10008-CRM197 Aluminum hydroxide C-LEDMPVDPDSEA 29 12 p10009-CRM197 Aluminum hydroxide C-LEDMPVDPDNSA 30 13 p10010-CRM197 Aluminum hydroxide C-LEDMPVDPDNES
Table 5: aSyn-30, key results. The titers to aSyn evaluated in single mice and values represent medians relative to the median obtained with p9524.

TABLE-US-00005 TABLE 5 Seq ID Group Titer relative to p9524 (%)  4 1 100.0  8 2 391.8  9 3 56.2 10 4 581.0 11 5 170.0 12 6 215.5 13 7 117.0 14 8 63.9 15 9 50.1 16 10 127.7 17 11 90.4 18 12 114.4
Table 6: aSyn-31, key results. The titers to aSyn were evaluated in single mice and values represent medians relative to the median obtained with p9524.

TABLE-US-00006 TABLE 6 Seq ID Group Titer relative to p9524 (%)  4 1 100.0 19 2 699.1 20 3 300.5 21 4 1161.7 22 5 105.0 23 6 19.5 24 7 183.9 25 8 115.2 26 9 21.2 27 10 49.7 28 11 124.6 29 12 107.3 30 13 251.3
Amino Acid Exchanges on Positions 1, 3, 4, and 12 Along the aSyn aa113-124 Epitope Sequence (In Vivo Experiments aSyn-32 and aSyn-33)

[0195] Since exchanges of Leu at position 1, Asp at 3, Met at 4 and Ala at 12 into Ala and/or Ser of the native aSyn.sub.113-124 sequence induced high aSyn specific antibody concentrations (FIG. 3), these positions are promising candidates for modifications leading to the design of AFFITOPES®. As a next step these positions were exchanged by amino acids with different properties (e.g. differently charged amino acids or amino acids with different polarity) in order to define favorable or less favorable exchanges with regard to immunogenicity and aSyn cross-reactivity (Tables 7 and 8). For comparison, a group injected with the native aSyn.sub.113-124 (p9524) was included. The plasmas of immunized mice were analyzed by ELISA and antibody titers relative to the titers elicited with the native sequence against aSyn were determined (FIG. 4A, B). Exchanges to Lys at both positions 1 and 3 resulted in the induction of higher amounts of aSyn-specific Abs (p10029 and p10033), whereas exchanges to Trp dramatically reduced aSyn specific antibody production (p10026 and p10031) (FIG. 4A). In addition, the Asp to Glu exchange at position 3 resulted in higher cross-reactivity to aSyn in comparison to the native aSyn sequence (FIG. 4A). Met at position 4 and Ala at position 12 were exchanged with a similar strategy in experiment aSyn-33 (Table 8) as shown for position 1 and 3 in experiment aSyn-32. Here again, exchanges to Lys or Val at position 12 (p10045 or p10042) resulted in higher anti-aSyn antibody titers, whereas exchange to Trp at either position 4 or 12 reduced the cross-reactivity to aSyn (FIG. 4B). Tables 9 and 10 summarize the induced titers relative to the titers induced by the WT sequence against aSyn present in the plasma of immunized mice.

Table 7: Setup of experiment aSyn-32. The table shows the different treatment groups, the respective drug product in use, the sequence of the peptide in the drug product and the corresponding sequence ID numbers.

TABLE-US-00007 TABLE 7 Seq ID Group Drug Product Sequence  4  1 p9524-CRM197 Aluminum hydroxide C-LEDMPVDPDNEA 31  2 p10026-CRM197 Aluminum hydroxide C-WEDMPVDPDNEA 32  3 p10027-CRM197 Aluminum hydroxide C-IEDMPVDPDNEA 33  4 p10028-CRM197 Aluminum hydroxide C-NEDMPVDPDNEA 34  5 p10029-CRM197 Aluminum hydroxide C-KEDMPVDPDNEA 35  6 p10030-CRM197 Aluminum hydroxide C-DEDMPVDPDNEA 36  7 p10031-CRM197 Aluminum hydroxide C-LEWMPVDPDNEA 37  8 p10032-CRM197 Aluminum hydroxide C-LEEMPVDPDNEA 38  9 p10033-CRM197 Aluminum hydroxide C-LEKMPVDPDNEA 39 10 p10034-CRM197 Aluminum hydroxide C-LELMPVDPDNEA 40 11 p10035-CRM197 Aluminum hydroxide C-LENMPVDPDNEA
Table 8: Setup of experiment aSyn-33. The table shows the different treatment groups, the respective drug product in use, the sequence of the peptide in the drug product and the corresponding sequence ID numbers.

TABLE-US-00008 TABLE 8 Seq ID Group Drug Product Sequence  4  1 p9524-CRM197 Aluminum hydroxide C-LEDMPVDPDNEA 41  2 p10036-CRM197 Aluminum hydroxide C-LEDWPVDPDNEA 42  3 p10037-CRM197 Aluminum hydroxide C-LEDLPVDPDNEA 43  4 p10038-CRM197 Aluminum hydroxide C-LEDNPVDPDNEA 44  5 p10039-CRM197 Aluminum hydroxide C-LEDDPVDPDNEA 45  6 p10040-CRM197 Aluminum hydroxide C-LEDKPVDPDNEA 46  7 p10041-CRM197 Aluminum hydroxide C-LEDMPVDPDNEW 47  8 p10042-CRM197 Aluminum hydroxide C-LEDMPVDPDNEV 48  9 p10043-CRM197 Aluminum hydroxide C-LEDMPVDPDNEG 49 10 p10044-CRM197 Aluminum hydroxide C-LEDMPVDPDNED 50 11 p10045-CRM197 Aluminum hydroxide C-LEDMPVDPDNEK
Table 9: aSyn-32, key results. The titers to aSyn were evaluated in single mice and values represent medians relative to the median obtained with p9524.

TABLE-US-00009 TABLE 9 Seq ID Group Titer relative to p9524 (%)  4 1 100.0 31 2 41.9 32 3 57.7 33 4 42.5 34 5 128.9 35 6 33.1 36 7 30.9 37 8 338.4 38 9 137.2 39 10 41.4 40 11 128.2
Table 10: aSyn-33, key results. The titers to aSyn were evaluated in single mice and values represent medians relative to the median obtained with p9524.

TABLE-US-00010 TABLE 10 Seq ID Group Titer relative to p9524 (%)  4 1 100.0 41 2 13.6 42 3 84.6 43 4 41.5 44 5 15.2 45 6 160.8 46 7 22.2 47 8 225.5 48 9 129.5 49 10 42.2 50 11 270.8
Double Serine-Exchanges Along the aSyn aa113-124 Epitope Sequence (In Vivo Experiment aSyn-37)

[0196] VARIOTOPES® with additional serine exchanges along the aSyn.sub.113-124 native sequence (p9524) were designed and tested for their immunogenicity in wt BALB/c mice (Table 11, FIG. 5). Two weeks after the third and last injection, plasma of each individual mouse was collected and analyzed by ELISA in order to determine the AFFITOPE®-induced titers and aSyn antibody concentrations. The native aSyn sequence was injected for direct comparison purposes (Group 1). Double Ser exchanges at positions 3 and 12 (p10074), as well as at positions 1 and 3 (p10075), were able to further enhance the titers of aSyn-specific antibody when compared to the native target sequence (FIG. 5). Double serine exchanges at position 1 and 4 as well as position 4 and 12 did not increase the immunogenicity towards aSyn (FIG. 5). Table 12 summarizes the induced titers relative to the titers induced by the WT sequence against aSyn present in the plasma of immunized mice.

Table 11: Setup of experiment aSyn-37. The table shows the different treatment groups, the respective drug product in use, the sequence of the peptide in the drug product and the corresponding sequence ID numbers.

TABLE-US-00011 TABLE 11 Seq ID Group Drug Product Sequence  4 1 p9524-CRM197 Aluminum hydroxide C-LEDMPVDPDNEA 51 2 p10074-CRM197 Aluminum hydroxide C-LESMPVDPDNES 52 3 p10075-CRM197 Aluminum hydroxide C-SESMPVDPDNEA 53 4 p10076-CRM197 Aluminum hydroxide C-LESSPVDPDNEA 54 5 p10077-CRM197 Aluminum hydroxide C-SEDMPVDPDNES 55 6 p10078-CRM197 Aluminum hydroxide C-LEDSPVDPDNES 56 7 p10079-CRM197 Aluminum hydroxide C-SEDSPVDPDNEA
Table 12: aSyn-37, key results. The titers to aSyn were evaluated in single mice and values represent medians relative to the median obtained with p9524.

TABLE-US-00012 TABLE 12 Seq ID Group Titer relative to p9524 (%)  4 1 100.0 51 2 622.3 52 3 397.0 53 4 145.9 54 5 188.8 55 6 110.5 56 7 101.3
Amino Acid Exchanges with Ser and Lys on Position 1, 3, and 12 Along the aSyn aa113-124 Epitope Sequence (In Vivo Experiment aSyn-44)

[0197] The exchanges to Ser and Lys at the positions 1, 3, and 12 of the native aSyn.sub.113-124 sequence have been shown to be favorable to induce high anti-aSyn antibody concentrations (FIGS. 3B, 4 and 5). In the immunogenicity study presented herein combinations of serine and lysine exchanges along the aSyn.sub.113-124 epitope were tested in wt BALB/c mice (aSyn-44 experiment, Table 13). In addition, linker amino acids have been added to different peptides. In parallel, the previously selected AFFITOPE® s candidate p4456 was included in this experiment for direct comparison. Two weeks after the third and last injection plasma of each individual mouse was collected and analyzed by ELISA in order to determine the AFFITOPE®-induced titers and aSyn antibody concentrations. The AFFITOPE® candidates tested in group 2-7 were able to induce higher titers of anti-aSyn antibodies when compared to the highly successful AFFITOPE® p10074 presented in FIG. 5 (FIG. 6). Especially, AFFITOPE® p10118 revealed to induce the highest titers against aSyn. Table 14 summarizes the induced titers relative to the titers induced by p10074 against aSyn present in the plasma of immunized mice.

[0198] In order to directly compare the immunogenicity of the newly selected AFFITOPE® sequences (group 2-7) to the peptide p4456 previously selected at AFFiRiS, the group median concentrations of anti-aSyn antibodies induced by these AFFITOPE® candidates were set in relation to the group median anti-aSyn antibody concentration elicited by p4456 (FIG. 7). All new AFFITOPE® candidates tested induced significantly higher aSyn specific titres compared to p4456 (FIG. 7). Table 15 summarizes the induced titers relative to the titers induced by the AFFITOPE® sequence p4456 against aSyn present in the plasma of immunized mice.

Table 13: Setup of experiment aSyn-44. The table shows the different treatment groups, the respective drug product in use, the sequence of the peptide in the drug product and the corresponding sequence ID numbers.

TABLE-US-00013 TABLE 13 Seq ID Group Drug Product Sequence 51 1 p10074-CRM197 Aluminum hydroxide C-LESMPVDPDNES 57 2 p10114-CRM197 Aluminum hydroxide CGG-KEDMPVDPDNEA 58 3 p10115-CRM197 Aluminum hydroxide C-LEKMPVDPDNES 59 4 p10116-CRM197 Aluminum hydroxide C-SEKMPVDPDNEA 60 5 p10117-CRM197 Aluminum hydroxide C-LEKMPVDPDNEK 61 6 p10118-CRM197 Aluminum hydroxide CGG-KESMPVDPDNEA 62 7 p10119-CRM197 Aluminum hydroxide CGG-KESMPVDPDNEK  1 8 p4456-CRM197 Aluminum hydroxide C-DQPVLPD
Table 14: aSyn-44, key results. The titers to aSyn were evaluated in single mice and values represent medians relative to the median obtained with p10074 set to 100%.

TABLE-US-00014 TABLE 14 Seq ID Group Titer relative to p10074 (%) 51 1 100.0 57 2 160.1 58 3 141.6 59 4 143.4 60 5 258.2 61 6 295.1 62 7 205.5
Table 25: The titers to aSyn were evaluated in single mice and values represent group medians relative to the group median obtained with p4456 set to 100%.

TABLE-US-00015 TABLE 15 Seq ID Group Titer relative to p4456 (%)  1 8 100.0 57 2 432.5 58 3 382.5 59 4 387.3 60 5 697.5 61 6 797.1 62 7 555.2
C-Terminal Truncation of the Two Selected AFFITOPE® Sequences p10033 and p10118 by Removing the Ala at Position 12

[0199] AFFITOPE® sequences p10033 and especially p10118 have been shown to induce high anti-aSyn antibody concentrations (FIG. 4A and FIG. 6, respectively). In the immunogenicity studies presented herein the C-terminal Ala at position X12 was removed from AFFITOPE® sequences p10033 and p10118 in order to test whether C-terminal truncation has an influence on the immunogenicity and aSyn cross-reactivity of designed peptides. wt BALB/c mice where immunized in independent experiments with either p10033, p10118, p10166 or p10167. Table 16 lists the peptide sequences. Two weeks after the third injection plasma of each individual mouse was collected and analyzed by ELISA in order to determine the AFFITOPE®-induced titers and aSyn antibody reactivity. The truncated AFFITOPE® candidates tested were able to induce titers of anti-aSyn antibodies that, although lower than those generated using the AFFITOPE® sequences incorporating the Ala at position X.sub.12 (FIG. 8), were still higher than those obtained using p4456. Table 17 summarizes the induced titers relative to the titers induced by either by the AFFITOPE® sequence p10033 (A) or by the AFFITOPE® sequence p10118 (B) against aSyn present in the plasma of immunized mice.

Table 16: The table shows the different treatment groups, the respective drug product in use, the sequence of the peptide in the drug product and the corresponding sequence ID numbers.

TABLE-US-00016 TABLE 16 Seq ID Drug Product Sequence 38 p10033-CRM197 Aluminum hydroxide C-LEKMPVDPDNEA 63 p10166-CRM197 Aluminum hydroxide C-LEKMPVDPDNE 61 p10118-CRM197 Aluminum hydroxide CGG-KESMPVDPDNEA 64 p10167-CRM197 Aluminum hydroxide CGG-KESMPVDPDNE
Table 17: The titers to aSyn were evaluated in single mice and values represent medians relative to the median obtained with either p10033 or p10118.

TABLE-US-00017 TABLE 17 Seq ID Titer relative to p10033 or p10118 (%) 38 100.0 63 58.8 61 100.0 64 76.2

Target Engagement of Antibodies Induced by Selected AFFITOPE® s

[0200] To investigate whether antibodies induced by AFFITOPE® candidate were able to detect aggregated aSyn in situ, sera of AFFITOPE® candidate-vaccinated wt BALB/c mice were tested by IHC staining on brain sections of human brain tissue derived postmortem of PD/DLB-diseased patients. Lewy bodies are a pathological hallmark in brains of PD/DLB-diseased patients and are mainly enriched with pathogenic, aggregated forms of aSyn (Spillantini et al., 1997).

[0201] Plasma from AFFITOPE® candidate-treated mice detected Lewy Bodies on brain sections of the cortex similar to the control anti-aSyn mAb 28A7 (FIG. 9). Specificity was confirmed by the lack of staining after pre-absorption of the serum with the corresponding peptide moiety of AFFITOPE® candidate (data not shown).

Preferential Binding to aSyn Oligomeric (Toxic) Vs Monomeric aSyn Species

[0202] Next, Abs induced by different AFFITOPE® s were tested for their selective binding to oligomeric aSyn (low MW, soluble aggregates of aSyn, predominantly di- and trimers) over the monomeric species using a SPR-based methodology (FIG. 10). Equal amounts of AFFITOPE® candidate-induced antibodies or monoclonal antibodies were first immobilized on an anti-mouse capture antibody coated chip. Subsequently monomeric and oligomeric aSyn species were applied consecutively, and the differential binding (defined as RU) to aSyn monomer and oligomeric aSyn species was assessed. As a control, two monoclonal antibodies were used: LB509 (Biolegend, San Diego, Calif.) and 28A7 (AFFiRiS AG). Immobilization of LB509 to the chip surface, which does not discriminate between monomeric and oligomeric aSyn, resulted in comparable RU to both aSyn species. The second control antibody, 28A7, which was raised against the peptide p4456, did discriminate between monomeric and oligomeric aSyn species. The AFFITOPE® candidate-induced antibodies showed high selectivity for oligomeric aggregates of aSyn compared to the monomeric form of aSyn (FIG. 10).

[0203] In addition to SPR (BiaCore) analyses, AFFITOPE®-induced Abs have been tested for their preferential binding to the aSyn filaments over the monomeric species of aSyn by inhibition ELISA. In these assays, a constant amount of affinity-purified AFFITOPE®-induced antibodies was preincubated with titrated amounts of monomeric and filamentous aSyn and then transferred to ELISA plates coated with aSyn filaments (for details see M&M). In FIG. 11, the results of two representative AFFITOPE® candidates (p10033 and p10118) are shown. Very good competition was seen with aSyn oligomers followed by aSyn filaments, whereas the competition with the monomeric form of aSyn was only of minor extent (FIG. 11).

[0204] A mAb 28A7 that was known to preferably bind the oligomeric and aggregated forms of aSyn was used as control (FIG. 11C).

[0205] Altogether, the binding data for AFFITOPE® candidate-specific antibodies provide clear evidence of high selectivity of AFFITOPE® candidate-induced antibodies to the toxic, oligomeric aggregates of aSyn, which are considered to be the relevant toxic species that lead to cell death, as opposed to the monomeric form.

Material and Methods

Mice

[0206] BALB/c mice were purchased from Janvier Elevages (Le Genest-Sainte-Isle, F).

[0207] The animals were housed and kept under standard conditions described in the application of the IMP to license its activity as breeders, suppliers and users. The respective permission was granted by the relevant authorities on May 13 2013 with the notification GZ:223633/2013/4.

[0208] Briefly, mice were kept in TECNIPLAST Sealsafe NextIVC Blue Line—Cages (Milano, IT) á five mice. Cages were equipped with enrichment in the form of nesting material and little plastic houses for hiding/playing purposes. The age of the mice at the beginning of the experiment was between 6 and 8 weeks. They were provided with standard diet and acidified water ad libitum and were kept under a 12 hour light/dark cycle.

[0209] All animal experiments were done according to Austrian and European law and were granted permission by the Vienna City Ad-ministration, municipal department 58.

Peptides and Proteins

[0210] Peptides used for immunization were purchased from EMC micro-collections (Tübingen, Germany).

[0211] CRM197 was purchased from Pfenex (San Diego, Calif.).

Manufacturing of Immunogenic Products

[0212] All immunogenic AFFITOPE®-based products used in the described experiments are conjugates of the synthetic AFFITOPE® peptides to the carrier protein CRM197. The conjugation is a directed procedure using the side chain amino groups of lysine residues in CRM197 and the free thiol group of the amino (N)-terminal cysteine in the peptide. For the activation of CRM197, the aqueous CRM197 solution is adjusted to 10 mM phosphate buffered saline (PBS) and is then gently shaken with the bifunctional linker 4-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS). Subsequently, excess of unreacted GMBS is removed by either dialysis or ultrafiltration. The obtained activated CRM197 solution is subsequently incubated with AFFITOPE® peptides dissolved in phosphate buffer (pH 6.7). The free thiol group of the cysteine within the peptide reacts with the maleimido group forming the final AFFITOPE®-CRM197 product.

Application of the Immunogen

[0213] Vaccines are brought to ambient temperature, vortexed and applied subcutaneously (s.c.) in the flank of mice (200 μl) with an insulin 20 syringe with a G30-gauge (Omnican® 50, B. Braun Melsungen AG, Melsungen, Germany). Immunization is repeated three times in biweekly intervals.

Sample Collection

[0214] Blood was taken two weeks after each injection. Plasma was collected and stored at −20° C. until analysis. Blood collection and sacrifice of the animals was performed using FELASA-approved procedures.

Monoclonal Antibodies

[0215] In this study, two monoclonal Abs were used as controls: LB509 (Biolegend, CA, US) and 28A7. LB509 is a commercially available purified anti-aSyn, 115-121 Ab. The mouse mAb 28A7 (IgG1) was generated in-house with mouse B cell hybridomas (Mandler et al., 2014) against the AFFITOPE® PD01 which mimics the aSyn.sub.115-121 epitope.

Sample Collection

[0216] Blood was taken approximately two weeks after each injection, at least one day before the next injection. Plasma was collected and stored at −20° C. until analysis. Blood collection and sacrifice of the animals was performed using FELASA-approved procedures.

Titer Determination by ELISA

[0217] Titers against the immunizing peptides and against the recombinant human aSyn protein were analyzed. The presence of AFFIOTPE®-induced antibodies in plasma of immunized mice was determined by ELISA. 96-well plates (Nunc-Maxisorp) were coated with either recombinant human aSyn (1 μg/ml) or the injected peptide (BSA-conjugate; 1 μM). Titers were calculated as EC.sub.50-values with PRISM® 5.04 (GraphPad Inc, San Diego, Calif.) by non-linear regression analysis (four-parameter logistic fit function).

Immunohistochemistry (IHC)

[0218] Recognition of aSyn-positive inclusions (Lewy bodies) was performed on post-mortem brain sections from a frontal cortex biopsy of a DLB patient, (DLB patient, case No. X.sub.5631, Department of Neuroscience at UCSD, La Jolla, Calif.).

[0219] Plasma from AFFITOPE®-immunized mice was used to stain sections from frontal cortex brain biopsies from DLB patients. After tissue preparation (rehydration, deparaffinization, antigen retrieval, and blocking), sections were incubated with diluted mouse plasma for 2 h at RT or overnight at 4° C. Sections were incubated for 1 h at RT with undiluted Dako EnVision HRP labelled polymer (Agilent, Santa Clara, Calif.). For each IHC staining, counterstaining was done with Haematoxylin, and after this step, slides were de-hydrated and mounted in Entellan (Sigma-Aldrich). Slides were scanned in the brightfield mode using a Panoramic (Mirax) Scanner 150 (Carl Zeiss Microlmaging GmbH).

Surface Plasmon Resonance (Biacore) Analysis

[0220] All experiments were run on a Biacore T200 (GE Healthcare, Chicago, Ill.) using Biacore T200 Control Software 2.0.1. A CM5 chip was immobilized with an antibody from a commercially available mouse antibody capture kit (GE-Healthcare) according to manufacturer's instructions by amine coupling on flow-cells (Fc) 1 and Fc2 of the chip. Fc1 served as reference flow cell. The immobilization levels of the anti-mouse antibody for both Fc were comparable and resulted in approximately 11000 RU for Fc1 and Fc2.

[0221] To reach comparable levels of the captured antibodies, the injection time for each specific antibody was tested before the actual run and injection time was adjusted accordingly. This resulted in comparable capture levels for all tested antibodies including the unspecific antibodies captured on Fc1 in each cycle.

[0222] The experimental setup for one cycle was as follows: [0223] 1. Capturing of unspecific antibody on reference Fc1 [0224] 2. Capturing of aSyn-specific antibody on Fc2 [0225] 3. Injection of sample (oligomeric aSyn, monomeric aSyn, buffer only) over Fc1 and Fc2 [0226] 4. Regeneration of Fc1 and Fc 2 with low pH; only anti-mouse antibody remains on the chip [0227] 5. Chip surface ready for next cycle

[0228] Preparation of oligomeric and monomeric aSyn: aSyn oligomers (SynAging, Vandceuvre-les-Nancy, FR) were thawed and diluted to 5 μg/ml in HBS shortly before injection. To remove the high molecular weight fraction from bona fide monomeric aSyn (rPeptide), aSyn was freshly dissolved, diluted to 5 μg/ml in HBS, and cut-off centrifuged for 10 min at 14.000×g using 50 kDa cut-off columns (Amicon Ultra 0.5 ml). As controls, two aSyn-specific antibodies were used, LB509 (BioLegend, San Diego, Calif.) and 28A7 (AFFiRiS AG).

Affinity Purification of AFFITOPE® Specific Antibodies Out of Immune Plasmas

[0229] Iodoacetyl magnetic beads (FG-106, Bioclone Inc., San Diego, Calif.) were coupled with the respective peptide (HPLC purified) for 1 h at RT and the remaining excess free sites were blocked with cysteine for an additional hour. After the blocking reaction, AFFITOPE®-coupled beads were incubated with 150 μl plasma of mice immunized with the corresponding AFFITOPE® candidate (for 2 h at RT). The AFFITOPE®-specific Abs were then eluted with Elution Buffer (Thermo Scientific). Afterwards the eluents were concentrated by Ultra Centrifugation (Millipore) tubes (30 kDa) to a volume of 150 μl (equal to the input volume).

Competition ELISA

[0230] Titrated amounts of different aSyn species, monomer (rPeptide), and aggregated forms including fibrils (Proteos Inc), or oligomers (Crossbeta Bicosciences), in concentrations ranging from 100 to 0.05 μg/ml (this corresponds to a range of 69 to 0.034 μM relating to monomeric aSyn) were pre-incubated with antibodies that were previously purified from end-plasma from AFFITOPE® candidate-immunized mice, as well as the control anti-aSyn antibody 28A7. Added aSyn species compete for the binding to plate-coated aSyn fibrils (Proteos Inc). IC.sub.50 values were calculated as the concentration of either monomeric, oligomeric, or fibrilic aSyn which was needed to quench half of the ELISA signal. IC.sub.50 values were calculated with PRISM® 5.04 (GraphPad Inc, San Diego, Calif.) by non-linear regression analysis (four-parameter logistic fit function).

TABLE-US-00018 TABLE 18 AFFITOPERs and original sequence peptides tested Internal Sequence peptide ID no name Peptide sequence Additional Information  1 p4456 C-DQPVLPD aSyn115-121 (M116.fwdarw.Q, D11.fwdarw.L)  2 p4572 C-YDRPVQPDR aSyn114-122 (E114.fwdarw.Y, M116.fwdarw.R, D119.fwdarw.Q, N122.fwdarw.R)  3 p9964 C-EDMPVDPDNEAYE aSyn114-126  4 p9524 C-LEDMPVDPDNEA aSyn113-124  5 p9556- C-DMPVDPDNEA aSyn115-124  6 p9557 C-EDMPVDPDNE aSyn114-123  7 p9663 C-VDPDNEAYE aSyn118-126  8 p9988 C-AEDMPVDPDNEA aSyn113-124 (L113.fwdarw.A)  9 p9989 C-LADMPVDPDNEA aSyn113-124 (E114.fwdarw.A) 10 p9990 C-LEAMPVDPDNEA aSyn113-124 (D115.fwdarw.A) 11 p9991 C-LEDAPVDPDNEA aSyn113-124 (M116.fwdarw.A) 12 p9992 C-LEDMAVDPDNEA aSyn113-124 (P117.fwdarw.A) 13 p9993 C-LEDMPADPDNEA aSyn113-124 (V118.fwdarw.A) 14 p9994 C-LEDMPVAPDNEA aSyn113-124 (D119.fwdarw.A) 15 p9995 C-LEDMPVDADNEA aSyn113-124 (P120.fwdarw.A) 16 p9996 C-LEDMPVDPANEA aSyn113-124 (D121.fwdarw.A) 17 p9997 C-LEDMPVDPDAEA aSyn113-124 (N122.fwdarw.A) 18 p9998 C-LEDMPVDPDNAA aSyn113-124 (E123.fwdarw.A) 19 p9999 C-SEDMPVDPDNEA aSyn113-124 (L113.fwdarw.S) 20 p10000 C-LSDMPVDPDNEA aSyn113-124 (E114.fwdarw.S) 21 p10001 C-LESMPVDPDNEA aSyn113-124 (D115.fwdarw.S) 22 p10002 C-LEDSPVDPDNEA aSyn113-124 (M116.fwdarw.S) 23 p10003 C-LEDMSVDPDNEA aSyn113-124 (P117.fwdarw.S) 24 p10004 C-LEDMPSDPDNEA aSyn113-124 (V118.fwdarw.S) 25 p10005 C-LEDMPVSPDNEA aSyn113-124 (D119.fwdarw.S) 26 p10006 C-LEDMPVDSDNEA aSyn113-124 (P120.fwdarw.S) 27 p10007 C-LEDMPVDPSNEA aSyn113-124 (D121.fwdarw.S) 28 p10008 C-LEDMPVDPDSEA aSyn113-124 (N122.fwdarw.S) 29 p10009 C-LEDMPVDPDNSA aSyn113-124 (E123.fwdarw.S) 30 p10010 C-LEDMPVDPDNES aSyn113-124 (A124.fwdarw.S) 31 p10026 C-WEDMPVDPDNEA aSyn113-124 (L113.fwdarw.W) 32 p10027 C-IEDMPVDPDNEA aSyn113-124 (L113.fwdarw.I) 33 p10028 C-NEDMPVDPDNEA aSyn113-124 (L113.fwdarw.N) 34 p10029 C-KEDMPVDPDNEA aSyn113-124 (L113.fwdarw.K) 35 p10030 C-DEDMPVDPDNEA aSyn113-124 (L113.fwdarw.D) 36 p10031 C-LEWMPVDPDNEA aSyn113-124 (D115.fwdarw.W) 37 p10032 C-LEEMPVDPDNEA aSyn113-124 (D115.fwdarw.E) 38 p10033 C-LEKMPVDPDNEA aSyn113-124 (D115.fwdarw.K) 39 p10034 C-LELMPVDPDNEA aSyn113-124 (D115.fwdarw.L) 40 p10035 C-LENMPVDPDNEA aSyn113-124 (D115.fwdarw.N) 41 p10036 C-LEDWPVDPDNEA aSyn113-124 (M116.fwdarw.W) 42 p10037 C-LEDLPVDPDNEA aSyn113-124 (M116.fwdarw.L) 43 p10038 C-LEDNPVDPDNEA aSyn113-124 (M116.fwdarw.N) 44 p10039 C-LEDDPVDPDNEA aSyn113-124 (M116.fwdarw.D) 45 p10040 C-LEDKPVDPDNEA aSyn113-124 (M116.fwdarw.K) 46 p10041 C-LEDMPVDPDNEW aSyn113-124 (A124.fwdarw.W) 47 p10042 C-LEDMPVDPDNEV aSyn113-124 (A124.fwdarw.V) 48 p10043 C-LEDMPVDPDNEG aSyn113-124 (A124.fwdarw.G) 49 p10044 C-LEDMPVDPDNED aSyn113-124 (A124.fwdarw.D) 50 p10045 C-LEDMPVDPDNEK aSyn113-124 (A124.fwdarw.K) 51 p10074 C-LESMPVDPDNES aSyn113-124 (D115.fwdarw.S, A124.fwdarw.S) 52 p10075 C-SESMPVDPDNEA aSyn113-124 (L113.fwdarw.S, D115.fwdarw.S) 53 p10076 C-LESSPVDPDNEA aSyn113-124 (D115.fwdarw.S, M116.fwdarw.S) 54 p10077 C-SEDMPVDPDNES aSyn113-124 (L113.fwdarw.S, A124.fwdarw.S) 55 p10078 C-LEDSPVDPDNES aSyn113-124 (M116.fwdarw.S, A124.fwdarw.S) 56 p10079 C-SEDSPVDPDNEA aSyn113-124 (L113.fwdarw.S, M116.fwdarw.S) 57 p10114 CGG-KEDMPVDPDNEA aSyn111-124 (I112.fwdarw.G, D115.fwdarw.K) 58 p10115 C-LEKMPVDPDNES aSyn113-124 (D115.fwdarw.K, A124.fwdarw.S) 59 p10116 C-SEKMPVDPDNEA aSyn113-124 (L113.fwdarw.S, D115.fwdarw.K) 60 p10117 C-LEKMPVDPDNEK aSyn113-124 (D115.fwdarw.K, A124.fwdarw.K) 61 p10118 CGG-KESMPVDPDNEA aSyn111-124 (I112.fwdarw.G, D115.fwdarw.K, P117.fwdarw.S) 62 p10119 CGG-KESMPVDPDNEK aSyn111-124 (I112.fwdarw.G, D115.fwdarw.K, P117.fwdarw.S, A124.fwdarw.K) 63 p10166 C-LEKMPVDPDNE aSyn113-123 (D115.fwdarw.K) 64 p10167 CGG-KESMPVDPDNE aSyn111-123 (I112.fwdarw.G, D115.fwdarw.K, P117.fwdarw.S)

REFERENCES

[0231] Dufty et al., Am J Pathol. 2007 May; 170(5):1725-38. [PubMed: 17456777] [0232] Bassil et al., Proc Natl Acad Sci USA. 2016 Aug. 23; 113(34): 9593-8. doi: 10.1073/pnas.1609291113 [PubMed: 27482103] [0233] Kosaka, J Neurol. 1990; 237:197-204. [PubMed: 2196340] [0234] Dickson et al., Acta Neuropathol. 1989; 78:572-584. [PubMed: 2683563] [0235] Braak et al., Acta Neuropathol. 2007; 114:231-241. [PubMed: 17576580] [0236] Lee et al., Int J Biochem Cell Biol. 2008; 40:1835-1849. [PubMed: 18291704] [0237] Bengoa-Vergniory et al., Acta Neuropathol. 2017; 134(6):819-838. doi: 10.1007/s00401-017-1755-1. [PubMed: 28803412] [0238] Bernis et al., Acta Neuropathol Commun. 2015 Nov. 26; 3:75. doi: 10.1186/s40478-015-0254-7. [PubMed: 26612754] [0239] Mandler et al., Acta Neuropathol. 2014; 127(6):861-79. doi: 10.1007/s00401-014-1256-4. [PubMed: 24525765] [0240] Spillantini et al., Nature. 1997 Aug. 28; 388(6645):839-40. (PubMed: 9278044] [0241] Lashuel, et al., Nat. Rev. Neurosci. 14, 38-48. doi: 10.1038/nrn3406 [0242] Volc, et al., Lancet Neurol. 19, 591-600. doi: 10.1016/S1474-4422(20)30136-8