MHC 1b-mediated alpha-synuclein-specific tolerance induction as a novel treatment for parkinson's disease

Abstract

The present invention relates to therapeutical uses of non-classical human major histocompatibility complex (MHC) molecules (also named MHC class Ib molecules) in combination with peptide antigens for the treatment of Parkinson's disease. The invention more specifically relates to recombinant polypeptides comprising peptide antigens and one or more domains of a non-classical MHC class Ib molecule. The invention also relates to methods of producing such recombinant polypeptides, pharmaceutical compositions comprising the same, as well as their uses for treating Parkinson's disease.

Description

[0001] MHC 1b-mediated alpha-Synuclein-specific tolerance induction as a novel treatment for Parkinson's disease

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to therapeutical uses of non-classical human major histocompatibility complex (MHO) molecules (also named MHO class lb molecules) in combination with peptide antigens for the treatment of Parkinson's disease. The invention more specifically relates to recombinant polypeptides comprising peptide antigens and one or more domains of a non-classical MHC class lb molecule. The invention also relates to methods of producing such recombinant polypeptides, pharmaceutical compositions comprising the same, as well as their uses for treating Parkinson's disease.

[0004] BACKGROUND

[0005] Parkinson's disease (PD) is the most common neurodegenerative movement disorder with over 7 million people affected worldwide and still no cure existing. Patients suffer from severe motor and non-motor symptoms such as tremor, bradykinesia, rigidity, postural instability, and depression. Pathologic disease hallmarks are degeneration of dopaminergic neurons in the substantia nigra and formation of o-Synuclein-containing protein aggregates called Lewy-bodies. While the physiological role of o-Synuclein (aSyn) points towards functions in synaptic transmitter release and amplification of immune responses (Alam et al, Cell Reports 38: 110090. doi: 10.1016 / j.celrep.2021.110090), the pathogenic role of insoluble o-Synuclein remains enigmatic. The observation that multiplications and mutations of the aSyn encoding SNCA gene can elicit genetically inherited forms of PD has robustly linked aSyn to PD pathogenesis.

[0006] Autopsies of human PD brains are characterized by microglia activation and T cell infiltration in the substantia nigra (Brochard V et al., 2009;McGeer PL et al., 1988). Pro-inflammatory cytokines such as tumor necrosis factor (TNF)-o, interferon (IFN)-y and interleukins IL-1 p and IL-6 in the nigrostriatal system further confirm that PD goes along with significant neuroinflammation (Mogi M et al., 1994; Mogi M et al., 1994). An increased ratio of IFN-y to IL-4 producing T cells in peripheral blood of PD patients indicates a systemic shift to a pro- inflammatory environment (Baba Y et al., 2005). Recently, aSyn-specific T cell responses were detected in peripheral blood cells of PD patients and found to precede motor symptoms, indicating an active and specific involvement of the immune system in disease progression. Two aSyn-derived peptides were shown to elicit MHC-restricted cytokine release from peripheral blood mononuclear cells (Sulzer D et al., 2017).

[0007] Currently, PD therapy mostly relies on compensating for SN neuron loss by administering L-Dopamine or derivative substances. This intervention initially improves disease symptoms, but does not prevent disease progression.

[0008] A population-based case-control study including approximately 48,000 PD patients analyzed the risk of developing PD with the intake of immunosuppressants. Patients medicated with azathioprine had a significantly lower risk for PD (Racette BA et al., 2018). A recent cohort study further demonstrated that patients who receive anti-TN F-a therapy for inflammatory bowel disease have a reduced risk for developing PD (Peter I et al., 2018). Effects of immunomodulating agents such as |32-Adrenoceptor agonists or anti-IL-17 therapy have been explored in small open label clinical trials that yielded partly encouraging results (Magistrelli L and Comi C, 2020; Storelli E et al., 2019).

[0009] Considering the often slowly progressing nature of this chronic disease, side effects of strong immunosuppressants represent a major obstacle for trials on early-stage PD patients. In late-stage patients, however, irreversible neurodegeneration limits the benefit achievable by blocking further progression. A highly targeted immunomodulatory drug would, in contrast, allow for early treatment, thereby maximizing therapeutic benefit.

[0010] To this end, clinical trials (https: / / clinicaltrials.gov / ct2 / show / NCT03318523) were conducted with the aSyn specific antibody cinpanemab (BIIB054). It had been hoped that sequestration of free aSyn by cinpanemab would prevent further aSyn aggregation and thus prevent disease progression. Unfortunately, this study failed to meet its primary or secondary endpoints. Thus, targeted and effective disease-modifying therapeutics for PD are still urgently needed.

[0011] The pathogenic role of T cells in PD has mostly been analyzed in toxin-based models such as the 1 -methyl-4- phenyl-1 ,2,3,6-tetrahydropyridine (MPTP) or the 6-hydroxydopamine (6-OHDA) models. However, given their lack of aSyn aggregation and Lewy body formation, these models poorly reflect the molecular aspects of the human disease. This has prompted Dr. Ip to generate a new mouse model for PD that recapitulates essential characteristics of the human disease more faithfully. Stereotaxis-guided, unilateral injection of an AAV1 / 2 vector encoding for human alpha-SynucleinA53T("haSyn”) into the substantia nigra induces behavioral deficits, loss of dopaminergic substantia nigra neurons and striatal fibers and reduction of striatal dopamine, as well as Lewy- body formation (Ip CW et al., 2017). As the high face validity of this model is achieved by injecting an a- Synuclein mutant that is known to be disease-associated in humans, the pathogenic relevance of this model is also supported by its disease-related construct validity. Furthermore, insoluble aSyn aggregates that show S129 phosphorylation and Lewy-like neurites and bodies are detectable in this animal model (Ip CW et al., 2017). The haSyn PD model thus provides an important tool to advance the preclinical and translational work towards new and better therapies for patients with PD. In a collaborative project, the inventors confirmed a broadly pro- inflammatory profile of brain immune cells in this haSyn PD model, and demonstrated infiltration of CD4+and CD8+activated T cell subsets into the brain of haSyn PD mice. These T cells induced neurodegeneration with PD-like symptoms. Genetic T cell deficiency (RAG-1 / ) reduced dopaminergic neurodegeneration, while T cell reconstitution aggravated neuronal loss in vivo. T cells isolated from brains of haSyn PD mice were cytotoxic towards haSyn-expressing neuronal cells in vitro (Karikari et al., Brain Behav Immun. 2022 Mar; 101 : 194-210.) Immunosuppressive MHC class lb molecule such as HLA-G are critical for tolerance induction during pregnancy. They exert immunosuppressive effects on various immune cells via immunosuppressive receptors such as ILT2, ILT4 and Kir2DL4. WO 2018 / 215340 relates to combinations of MHC class lb molecules and peptides for targeted therapeutic immunomodulation but remains silent on the treatment of Parkinson's disease.

[0012] Thus, there is an urgent need for targeted drugs which are effective for the treatment of Parkinson's disease.

[0013] DESCRIPTION OF THE INVENTION

[0014] The inventors have found that human MHC class lb molecules such as HLA-G possess the ability to induce antigen-specific tolerance towards presented peptide antigens. Thus, albeit being of similar structure and sequence as classical human MHC class la molecules which induce antigen peptide-specific immune responses, MHC class lb molecules can advantageously be used according to the invention to suppress immune responses in an antigen-specific manner. Additionally, the inventors have found that for the suppression of immune responses according to the invention, molecules other than naturally occurring MHC class lb molecules, and in particular polypeptides which only comprise at least one domain of an MHC class lb molecule, preferably at least an [alpha]3 domain of an MHC class lb molecule, can be used: The [alpha] 1 and [alpha]2 domains of variable class I a molecules can be combined with the [alpha]3 domain of a human MHC class lb molecule in order to suppress immune responses towards peptides presented by these antigens.

[0015] Antigen-loaded HLA-G molecules can be unstable. Thus, the inventors designed soluble recombinant polypeptides comprising a peptide antigen, an MHC class lb molecule such as HLA-G and p2-microglobulin (b2m), and connected these three components covalently (e.g., via covalent linkers). Alternatively, the antigenbinding a1 and a2 domains of an MHC class lb molecule such as HLA-G were exchanged by the respective domains of other MHC molecules to enhance the flexibility and versatility of these recombinant polypeptides (see, for instance, Figure 2). These alternative recombinant polypeptides can be designed with antigen-binding domains of other human HLA molecules. It was previously found that constructs comprising the a1 and a2 domains of murine H2-Kbcan present the ovalbumin-derived peptide SIINFEKL (SEC ID NO: 58) to OT-1 T cells. (OT-1 T cells express a transgenic T cell receptor that specifically recognizes this antigen) (WO 2018 / 215340). The literature teaches that Parkinson's disease is a neurodegenerative disease caused by pathogenic accumulation of alpha-Synuclein. The more recent concept that crucial pathogenic events are due to immune responses against alpha-Synuclein has not yet been translated towards therapeutics development.

[0016] Surprisingly, the inventors have found that by suppressing immune responses against alpha-Synuclein using surrogates of the recombinant polypeptides of the invention in a mouse model which faithfully mimics human Parkinson's disease, a therapeutic effect in the treatment of Parkinson's disease can be achieved. Thus, according to the invention, Parkinson's disease can be treated by the recombinant polypeptides of the invention.

[0017] Furthermore, according to the invention, the recombinant polypeptides of the invention are expected to be highly advantageous in the immunoterapeutic treatment of Parkinson's disease, because they cause hardly any systemic immunosuppression. Importantly, early-stage patients suffering from Parkinson's disease should not be exposed to the strong side effects of systemic immunosuppression, as this would likely result in opportunistic and potentially deadly infections. In late-stage patients, however, neurodegeneration is often irreversible. A highly specific tolerance induction achieved by presenting alpha-Synuclein antigens on the recombinant polypeptides of the invention would, however, be already tolerable at early stages of disease such as prodromal Parkinson's disease, which will open new therapeutic avenues.

[0018] Accordingly, the invention relates to the following preferred embodiments:

[0019] 1. A recombinant polypeptide capable of presenting a peptide antigen, the recombinant polypeptide comprising, in an N- to C-terminal order,

[0020] I) a peptide antigen presented by said recombinant polypeptide, wherein the peptide antigen is a peptide of human alpha-Synuclein; ii) optionally a linker sequence; ill) a sequence of a human polypeptide domain comprising a sequence of a human p2 microglobulin or an amino acid sequence at least 90% identical to the amino acid sequence of human p2 microglobulin represented by SEQ ID NO: 5; iv) optionally a linker sequence; v) an [alpha] 1 domain of an MHO molecule; vi) an [alpha] 2 domain of an MHO molecule; vii) an [alpha] 3 domain of an MHO class lb molecule or a derivative of an [alpha] 3 domain of an MHO class lb molecule, said derivative being capable of binding to ILT2 or ILT4; viii) optionally a protease cleavage site; lx) optionally a spacer sequence; and x) optionally an affinity tag. The recombinant polypeptide according to item 1, wherein said peptide antigen according to i) is 7 to 11 amino acids in length, preferably 8-11 amino acids in length. The recombinant polypeptide according to item 1 or 2, wherein said peptide antigen according to i) consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 28, 30, 31, 33, 34, 37, 40 and 50. The recombinant polypeptide according to item 1 or 2, wherein said peptide antigen according to i) consists of the amino acid sequence of SEQ ID NO: 30. The recombinant polypeptide according to item 1 or 2, wherein said peptide antigen according to i) consists of the amino acid sequence of SEQ ID NO: 33. The recombinant polypeptide according to item 1 or 2, wherein said peptide antigen according to i) consists of the amino acid sequence of SEQ ID NO: 34. The recombinant polypeptide according to item 1 or 2, wherein said peptide antigen according to i) consists of the amino acid sequence of SEQ ID NO: 37. The recombinant polypeptide according to item 1 or 2, wherein said peptide antigen according to i) consists of the amino acid sequence of SEQ ID NO: 40. The recombinant polypeptide according to item 1 or 2, wherein said peptide antigen according to i) consists of the amino acid sequence of SEQ ID NO: 50. The recombinant polypeptide according to any one of the preceding items, wherein said [alpha]1 domain according to (v) and said [alpha]2 domain according to (vi) are from a human MHC class la molecule or from a human MHC class lb molecule. The recombinant polypeptide according to item 10, wherein said [alpha]1 domain according to (v) and said [alpha]2 domain according to (vi) are from a human MHC class lb molecule, and wherein the human MHC class lb molecule is a human HLA-G molecule. The recombinant polypeptide according to item 10, wherein said [alpha]1 domain according to (v) and said [alpha]2 domain according to (vi) are from a human MHC class la molecule, and wherein said [alpha]1 domain according to (v) and said [alpha]2 domain according to (vi) are from a human HLA-A2 molecule, a human HLA-A1 molecule, or a human HLA-A11 molecule. The recombinant polypeptide according to any one of the preceding items, wherein the [alpha] 3 domain of the MHC class lb molecule according to (vii) is an [alpha] 3 domain of human HLA-E, human HLA-F or human HLA-G, preferably human HLA-G. The recombinant polypeptide according to any one of the preceding items, wherein the [alpha]3 domain or derivative according to (vii) is identical to or has at least 80% amino acid sequence identity, at least 90% amino acid sequence identity, at least 92% amino acid sequence identity, at least 94% amino acid sequence identity, at least 96% amino acid sequence identity, at least 98% amino acid sequence, or at least 99% amino acid sequence identity with the [alph a]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 2 or SEQ ID NO: 51 or is identical to the [alpha]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 2 or SEQ ID NO: 51 . The recombinant polypeptide according to any one of items 1-14, wherein the [alpha]3 domain or derivative according to (vii) is identical to the [alpha]3 domain having the amino acid sequence of SEQ ID NO: 9. The recombinant polypeptide according to any one of items 1-14, wherein the [alpha]3 domain or derivative according to (vii) is identical to the [alpha]3 domain having the amino acid sequence of SEQ ID NO: 2. The recombinant polypeptide according to any one of items 1-14, wherein the [alpha]3 domain or derivative according to (vii) is identical to the [alpha]3 domain having the amino acid sequence of SEQ ID NO: 51 . The recombinant polypeptide according to any one of the preceding items, wherein the linker sequence according to (ii) and / or the linker sequence according to (iv) comprises the amino acid sequence (GGGGS)n, wherein n is an integer equal to or higher than 1 , and wherein n is preferably an integer selected from the group consisting of 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10 and is preferably selected from the group consisting of 2, 3, 4 and 5. The recombinant polypeptide according to any one of the preceding items, wherein said sequence of a human polypeptide domain according to (ill) is at least 95% identical to the amino acid sequence of SEQ ID NO: 5, preferably at least 98% identical to the amino acid sequence of SEQ ID NO: 5 and more preferably identical to the amino acid sequence of SEQ ID NO: 5. The recombinant polypeptide according to any one of the preceding items, wherein said polypeptide is dimeric or multimeric. The recombinant polypeptide according to any one of the preceding items, wherein the polypeptide comprises or consists of all of the components I) to vii), does not comprise components viii) to x), or comprises or consists of all of the components I) to x). The recombinant polypeptide according to any one of the preceding items, further comprising an N-terminal secretion signal peptide sequence. The recombinant polypeptide according to any one of items 1-21 , wherein the recombinant polypeptide consists of an amino acid sequence consisting of the following ((a) and (b)) in an N- to C-terminal order:

[0021] (a) a peptide antigen consisting of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 28, 30, 31 , 33, 34, 37, 40 and 50; and

[0022] (b) the amino acid sequence of SEQ ID NO: 16 or SEQ ID NOs: 53-54. The recombinant polypeptide according to any one of the preceding items, wherein the recombinant polypeptide is soluble. A nucleic acid encoding one or more polypeptides according to any one of the preceding items, wherein the nucleic acid is preferably a vector. A pharmaceutical composition or kit comprising at least one nucleic acid according to item 25. A pharmaceutical composition or kit comprising at least one recombinant polypeptide according to any one of items 1-24. The pharmaceutical composition or kit according to item 27, wherein the pharmaceutical composition or kit comprises at least two different recombinant polypeptides according to any one of items 1-24, and wherein each of the different polypeptides comprises a different peptide antigen as defined in item 3. A pharmaceutical composition or kit according to any one of items 26-28, for use in the treatment of Parkinson's disease in a human patient. The pharmaceutical composition or kit for use according to item 29, wherein the Parkinson's disease is prodromal Parkinson's disease. The pharmaceutical composition or kit for use according to item 29 or 30, wherein the treatment is treatment of Parkinson's disease by immunotherapy, and wherein the treatment is preferably by inducing immunological tolerance against autoantigenic human alpha-Synuclein. A recombinant host cell comprising a nucleic acid or a vector according to item 25 and expressing the recombinant polypeptide according to any one of items 1-24. A method for obtaining pharmaceutical composition comprising a polypeptide according to any one of items 1-24, the method comprising the steps of (a) culturing the recombinant host cell of item 32 under conditions allowing expression of the recombinant polypeptide from the nucleic acid molecule, (b) recovering the recombinant polypeptide, (c) purifying the recombinant polypeptide, and (d) formulating the recombinant polypeptide into a pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Depiction of a peptide-loaded soluble MHO lb molecule suitable to achieve therapeutic antigenspecific immunomodulation.

[0024] The presented peptide antigen is depicted in dotted spheres, the HLA-G alpha1-3 domains are sketched in light-grey, and the beta2microglobulin domain is shown in dark grey. An optional linker connecting the antigenic peptide with the beta2microglobulin molecule is displayed in grey stick style, and an optional disulfide trap is depicted in black spheres. This figure was generated using Pymol and is adapted from structures published in Clements et al., Proc Natl Acad Sci U S A. 2005 Mar 1 ; 102 (9): 3360-5 and Hansen et al., Trends Immunol. 2010 Oct;31 (10):363-9.

[0025] Figure 2: Example for a vector-based construct encoding a single chain MHO lb molecule suitable for therapeutic peptide-specific immunomodulation.

[0026] HLA-G1 and HLA-G5 each consist of 3 [alpha] domains (here in black), a non-covalently associated beta 2- microglobulin subunit (here in dark grey) and the antigenic peptide presented on HLA-G (short black arrow). HLA-G1 further contains a transmembrane domain and a short intracellular chain (not shown here). As shown here, the [alpha]-3 domain is capable of binding to the receptors ILT2 (see Shiroishi et al., Proc Natl Acad Sci U S A. 2003 July 22; 100(15):8856-8861 ) and ILT4 (see Shiroishi et al., Proc Natl Acad Sci U S A. 2006 Oct 31; 103(44): 16412-7) on immune cells. Physiologically, these sequences form a non-covalently linked MHC class 1 complex. To simplify purification of the complex MHC lb molecule, one or more protein tags (such as SpotTag, myc tag and / or His(6x) tag) may be introduced. They may be introduced in such a way as to enable their later optional removal via cleavage using an optional Factor Xa cleavage site. Furthermore, the antigenic peptide, beta 2-microglobulin and MHC lb [al pha]chai n can be linked in order to increase the stability. The vector map was generated using Snapgene Viewer Software.

[0027] Figure 3: Prediction and confirmation of antigenic aSyn-peptides.

[0028] Results from IFN-y ELISpot analysis after stimulation of mononuclear cells containing CD4+, CD8+T cells from spleen of haSyn PD (grey) and control mice (black) with predicted peptides, 10 weeks after EV or A53T vector injection. 5 aSyn peptides elicit an antigen-specific IFN-y immune response. Using NCBI protein Blast, www.syfpeithi.de (Ver. 1.0) and netMHC4.0 (http: / / www.cbs.dtu.dk / services / NetMHC / ), the haSyn protein sequence was aligned with murine o-synuclein and screened for potential neo-antigens likely to be presented on MHC I (H-2Dband H-2 Kb) and MHC II (l-Ab) molecules. The three highest scoring peptides for each MHC molecule (8-15 amino acids long) were synthesized in aqua basic quality by Thermo Scientific. Each peptide was used to stimulate cells from lymph nodes or spleen followed by ELISpot assay. Splenocytes from two initially non-responsive haSyn mice and two controls were cultured for 10 days in the presence of all 10 peptides (1 pig / ml each) in RPMI with 10% FCS and 20 ng / ml IL-2. Multiscreen PVDF plates (MSIPS4510, Millipore) were activated with 35% ethanol, washed with sterile PBS and coated overnight with 2 pig / ml filtered anti-mouse IFN-y antibody (AN18, Mabtech) at 4 °C. Isolated single cell suspensions from cervical lymph nodes and spleen were plated with or without 5 pig / ml synthetic peptide in IMDM with 7.5% FCS. IFN-y spots were then detected with 2 pig / ml biotinylated anti-mouse IFN-y antibody (R4-6A2, Mabtech), followed by horseradish peroxidase- conjugated streptavidin (Cell Signaling) in PBS-T supplemented with 0.05% BSA. The assay was developed with filtered "ready to use” TMB substrate (Mabtech) and analyzed using an Immunopost S6 Core Analyzer

[0029] Figure 4: aSyn-loaded AIM Bio surrogate molecules prevent neuron death in PD model and may improve endurance.

[0030] Polypeptides comprising a peptide, here an aSyn peptide, MHC class 1 antigen presenting domains and HLA- G alpha3 domain (AIM Bio) treatment once a week for 9 cycles resulted in a significant, dose-dependent neuroprotection of dopaminergic neurons in haSyn PD mice compared to vehicle-treated haSyn PD mice with significantly higher numbers of tyrosine hydroxylase (TH)+neuron numbers in the substantia nigra of the treated groups. Immunohistochemical stainings were performed using 40 pm PFA-fixed cryosections processed for unbiased stereology (TH) covering the whole SN. After 4% PFA fixation, sections were incubated with rabbit anti-mouse TH (1 : 1000) antibodies followed by biotinylated goat anti-rabbit secondary antibodies (Vector Labs). Development was done using (DAB)-HCI-peroxidase (Vector Labs). Estimation of the number of dopaminergic neurons in the substantia nigra was done by unbiased stereology using the Stereo Investigator software package (v11 .07, MicroBrightField Biosciences).

[0031] Endurance of treated and untreated mice was tested by comparing pre-op and post-op latencies in a Rotarod test as described in (Ip CW et al., 2017).

[0032] A: images (scalebar=100 micrometers); B: quantification. C: Latencies relative to pre-op, assessed by the Rotarod test.

[0033] Figure 5: aSyn-loaded AIM Bio surrogate molecules prevent infiltration of CD11 b+myeloid cells (microglia) but induce infiltration of CD8+T cells into the striatum in PD model.

[0034] Immunohistochemical stainings of mouse tissue for CD11 b+myeloid cells and CD8+T cells were performed using 10 pirn fresh coronal cryo-sections of the striatum. After 4% PFA fixation or acetone fixation, respectively, sections were incubated with either rat anti-mouse CD11 b (1 :100, Serotec) or rat anti CD8 MCA609G 1 :500 antibodies, respectively, followed by biotinylated rabbit anti-rat secondary antibodies (Vector Labs). Development was done using (DAB)-HCI-peroxidase (Vector Labs). Microglia were quantified at a magnification of 200x in the region of the SN and striatum as depicted by consecutive sections stained for TH on a BH2 light microscope (Olympus).

[0035] A: images (scalebar=50 micrometers for the whole images and 20 micrometers for the enlarged area in the lower left corners, respectively) showing CD11 b+ microglia cells in the striatum; B: quantification. C: images (scalebar=100 micrometers for the whole images and 50 micrometers for the enlarged area in the lower left corners, respectively) showing CD8+ T cells in the striatum. D: quantification.

[0036] Figure 6: aSyn-loaded AIM Bio surrogate molecules reduce aSyn accumulation in Substantia Nigra in PD model. Scalebar=100 micrometers.

[0037] Neurons were fixed with 4% PFA and blocked with 5% NGS and 0.3% Triton X100 and stained with rabbit antihuman a-synuclein (1 :30,000, Sigma) and goat-anti rabbit Cy3 (1 :300, Jackson laboratories, cat # 111-165- 003) secondary antibodies and DAPI counterstaining. A less intense aSyn staining was observed in mice treated aSyn-loaded AIM Bio surrogate molecules.

[0038] Figure 7: aSyn-loaded AIM Bio protect Tyrosine Hydroxylase - fibers in the striatum and substantia nigra neurons

[0039] Methods see Figure 4.

[0040] A significant reduction of tyrosine hydroxylase4- fibers especially in the upper part of the striatum on the side in which A53T aSyn AAV (A53T) was injected (here: right side) was observed. Injection with aSyn-loaded AIM Bio significantly reduced the unilateral loss tyrosine hydroxylase4- fibers in a dose-dependent manner.

[0041] A: images; Scalebar=1000 micrometers. B: quantification. C: Repeat experiment of Figure 4 using an alternative Nissl staining to show neurons.

[0042] Figure 8: aSyn-loaded AIM Bio induce CD8+CD122+regulatory T cells

[0043] A significant induction of CD8+CD122+regulatory T cells both in the spleen and cervical lymph nodes was observed in response to aSyn-loaded AIM Bio treatment. Frozen sections were fixed with acetone for 10min at -20 °C, washed and blocked with 5% BSA; 5% NGS, 0.2% Triton-X100 in PBS for 1 h. CD122 was stained with antiCDI 221 rabbit polyclonal antibody (MyBiosource, 1 : 100) and anti-CD8i clone YTS191.1 rat (Biorad, 1 :200) in 1 % BSA; 1 % NGS, 0.2% Triton-X100 overnight. Secondary antibody staining used anti-rabbit-Cy3 (Dianova, 1 :300) and anti-rat-AF488 (Invitrogen, 1 :300) for 1 h.

[0044] Figure 9: Stability of purified single-chain MHC lb molecules. After purification of the single chain MHC lb molecules, their stability was analysed after 1 and 3 freeze-thawing cycles, storage for 5 days at room temperature and heating up to a temperature of 50°C for 30 min. For this, A) a Coomassie gel staining of a 12% polyacrylamide gel using 2 pig AIM Bio and B) an aHLA-G Western blot using the 2A12aHLA-G antibody (1 : 1000) blot using 1 pig protein was performed under non-reducing conditions. Both monomers and dimers are detectable.

[0045] Figure 10: Single-chain MHC lb molecules are thermally stable. For the Thermal Shift Assay (TSA), 3 pig of the respective single chain MHC lb molecule or Motavizumab as control molecule were diluted with PBS and 5x SYPRO Orange dye (stock 5000x, final concentration: 5x) to a volume of 25 pil. A melting curve program was set up on a StepOnePlus Instrument using the StepOnePlus Software 2.3. The start temperature was 25°C for one minute followed by a temperature increase of 1 °C per minute to a final temperature of 95°C for 2 min, thereby measuring the autofluorescence as arbitrary unit. Data were exported and graphs were drawn in Prism V7.04. For determination of the melting temperature (Tm), the Boltzman sigmoidal function was used.

[0046] Figure 11 : Single-chain MHC lb molecules induce Treg in a dose-dependent manner. OT-I mice were injected i.p. with indicated amounts of single-chain H2_Kbalphal +2 and HLA-G alpha3 domain constructs with human beta-2-microglobulin and the indicated peptide or carrier (PBS). Ova is the cognate peptide for the OT-I TOR in these mice, Gp34 is an irrelevant, virus derived control peptide. After 14 days, mice were sacrificed and splenocytes tested for IL10 secreting cells in a recall mouse IL-10 ELISpot (200,000 cells per well, MabTech mouse IL-10 ELISpot kit, 5 pig / ml of the indicated peptide or only PBS were added, 48h). A clear induction of IL-10 secreting cells reactive to Ova peptide was observed when 50 and 500 pig mouse adapted Ova_KbG were injected.

[0047] Figure 12: Single-chain MHC lb molecules inhibit T cell lysis in a dose-dependent manner. OT1 / BL6 Mice were sacrificed and splenocytes were collected and washed once in RPMI 5% FCS. Red blood cells were removed with 2ml 1x sterile RBC lysis buffer for 3 min. Cells were cultured in high density culture (10mio cells / ml) for 72h in RPMI 10% FCS medium with GMCSF 20 ng / mL, IL-2 20ng / ml and IL-4 10 ng / ml and increasing doses of Ova_KbG. Cells are then scraped from the plates, CD8+ cells are then purified via magnetic beads. Sterile 96-well white plates were used. Luciferase expressing Panc02 target cells were loaded with 20pig / ml Ova peptide (SIINFEKL) for 60 min at 37°C with 500 rpm shaking. CD8+ effector T cells were added in a 50:1 ratio, as well as luciferin. Luminescence was measured after Oh, 24h, 48h.

[0048] Figure 13: Single-chain MHO lb molecules induce expression of IL-10 in EAE-ODC Ova mice. Serum cytokines from EAE-ODC Ova mice were measured with Th 1 / Th2 10plex Flowcytomix Kit (eBioscience) according to the manufacturer's instruction. The kit was used for the simultaneous detection of mouse granulocyte-macrophage colony-stimulating factor (GMCSF), interleukin 1 alpha (IL-1 a), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), t interleukin-17 (IL-17), and tumor necrosis factor alpha (TNF-a) in a single sample. Beads coated with ten specific capture antibodies were mixed. Subsequently, 25 pL of the mixed captured beads, 25 pL of the unknown serum sample or standard dilutions, and 25 pL of phycoerythrin (PE) detection reagent were added consecutively to each well in 96-V bottom well plates and incubated for 2 h at room temperature in the dark. The samples were washed with 1 mL of wash buffer for 5 min and centrifuged. The bead pellet was resuspended in 200 pL buffer after discarding the supernatant. Samples were measured on the Attune™ NxT Flow Cytometer and analyzed Attune Cytometric Software (Thermo Fisher Scientific).

[0049] Figure 14: Graphic representation showing the development process of preferred polypeptide constructs of the invention.

[0050] Figure 15: Thermal Shift Assay. For the Thermal Shift Assay (TSA), 3 pig of the respective single chain MHC lb molecule were diluted with PBS and 5x SYPRO Orange dye (stock 5000x, final concentration: 5x) to a volume of 25 pil. A melting curve program was set up on a StepOnePlus Instrument using the StepOnePlus Software 2.3. The start temperature was 25°C for one minute followed by a temperature increase of 1 °C per minute to a final temperature of 95°C for 2 min with autofluorescence being measured throughout the process. Data were exported and graphs were drawn in Prism V7.04. The melting temperature (Tm) was determined via the Boltzmann sigmoidal function. The high melting temperatures indicate good protein stability for therapeutic use.

[0051] Figure 16: Stability of polypeptide constructs of the invention. sc HLA-A2 or A11 alpha1-2 HLA-G aplha3 constructs with indicated peptides were purified via spot-cap purification according to the manufacturers protocol, followed by ON dialysis. Constructs then underwent 3 freeze-thaw cycles, were heated to 50 °C for 30min or kept for 6 days at room temperature. Degradation was then analyzed on a Coomassie gel (12% gel, 4% stacking gel, 1 pig purified protein, non-reducing conditions, 15 pl sample +5 pl 4x Laemmli. Roti Mark TRICOLOR XTRA (Roth, 2244) was used for coomassie staining for 5-6 h. Destaining was done with 12.5 ml Methanol + 37.5 ml H2O dest for 5 min, another round of destaining with H2O dest

[0052] Figure 17: ELISpot results from healthy donor PBMCs. Induction of regulatory, IL-10 secreting cells through indicated constructs of the invention over a period of 14 days in PBMCs from healthy donors is shown

[0053] Figure 18: IL-10 ELISpot results from Parkinson patients (A) and healthy / age matched individuals (B). Induction of regulatory, IL-10 secreting cells through indicated constructs of the invention and age-matched healthy donors (C1-C3) or normal healthy donors (B10, B12, B13) over a period of 14 days is shown.

[0054] Figure 19: A: Simple Western results; B: Supernatant concentration measured in ELISA.

[0055] The constructs used in this Figure are as specified in the "sequences” section below and in Example 6 below. The Simple Western was performed as described in Example 6; the ELISA was carried out as a sandwich ELISA, as described in Example 6.

[0056] Figure 20: ELISpot with patient PBMCs. A: Pictures of wells in the assay. Numbers of the counted spots are indicated in the figure. B: Quantitation.

[0057] The ELISpot was carried out as described in Example 6.

[0058] IL-10 spots were increased in 4 out of 5 NMO patient PBMC treated with AIM Bio AQP65P65K_G compared to untreated PBMC.

[0059] Figure 21 : Accelerating rotarod test

[0060] Figure 22: Size Exclusion Chromatography (SEC) analysis of the AIM Bio aSyn63-71_H2-KbG

[0061] Figure 23: Thermal Shift analysis of aSyn6371_H2-KbG

[0062] Figure 24: Jess Simple Western for aSyn6371_H2-KbG Figure 25: IL-10 secreting cells detected in ELISpot assay. Left and right bar graphs respectively show % gated and cell counts of CD8+cells on HLA-A2’ PE staining. Mean and standard deviation is depicted in each donor PBMC.

[0063] Figure 26. CD8+T cell subpopulations in healthy controls and Parkinson's patients. Two x oSyn-specific AIM Bios (AIM_aSyn6977_A2G and AIM_aSyn7078_A2G) stimulated CD8+T cell responses in PBMC from Parkinson patients. The panels compare four CD8+T cell subpopulations, including CD8"CD103+, CD8+DexLowCD103+, and CD8+IL-10+cells, in HLA-A2+(middle row), individuals. The subpopulations were incubated with AIM Bios for 14 days, before being analyzed for expression of CD103 and IL-10. PBMC from patients with Parkinson's disease showed induction of IL-10 and CD103. P-values are indicated where appropriate. Please note that low cell numbers precluded stimulation of healthy donor PBMC with AIM_aSyn70_A2G.

[0064] DETAILED DESCRIPTION OF THE INVENTION

[0065] Definitions and General Techniques

[0066] Unless otherwise defined below, the terms used in the present invention shall be understood in accordance with their common meaning known to the person skilled in the art. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. Publications referred to herein may be cited either by specifying the full literature reference in the text, or by naming the first author and the publication date (e.g., "Brochard V et al., 2009”) the text, followed by the corresponding full literature reference in the "References” section of the present description.

[0067] All proteins in accordance with the invention, including the recombinant polypeptides of the invention, can be obtained by methods known in the art. Such methods include methods for the production of recombinant polypeptides. The recombinant polypeptides of the invention can be expressed in recombinant host cells according to the invention. Recombinant host cells of the invention are preferably mammalian cells such as CHO and HEK cells.

[0068] It will be understood that the recombinant polypeptides of the invention are meant to optionally include a secretion signal peptide sequence. Similarly, the recombinant polypeptides of the invention are meant to also optionally include affinity tags, e.g. in order to facilitate purification, and optional protease cleavage sites between the tag and the polypeptide, e.g. in order to facilitate removal of the tags by protease cleavage. It is also understood that any reference to amino acid sequences referred to herein is meant to encompass not only the unmodified amino acid sequence but also typical posttranslational modifications of these amino acid sequences (e.g., glycosylation or deamidation of amino acids, the clipping of particular amino acids or other posttranslational modifications) occurring in cellular expression systems known in the art, including mammalian cells such as CHO and HEK cells.

[0069] Likewise, it will be understood that the recombinant polypeptides of the invention are meant to optionally include the respective pro-peptides.

[0070] It will also be understood that the recombinant polypeptides of the invention can be in form of their soluble or their membrane-bound form. Whether a recombinant polypeptide is "soluble” under these conditions can be determined by methods known in the art, e.g., by measuring the turbidity of the recombinant polypeptide under the above-indicated reference conditions. As used herein, soluble means that at least 95% of the recombinant polypeptide is determined to be soluble under these reference conditions.

[0071] Single chain MHC molecules can be stored, for instance, in PBS at -80°C (with or without 0.1 % human albumin as carrier, depending on the protein concentration) or in 50% glycerol at -20°C.

[0072] According to the invention, MHC molecules are preferably human MHC molecules.

[0073] The recombinant polypeptides of the invention are preferably isolated recombinant polypeptides.

[0074] It will be understood how a recombinant polypeptide capable of binding and presenting an peptide antigen according to the invention can be prepared. For example, peptide antigen-binding domains such as [alpha]1 and [alpha]2 domains are well-known, and modifications of these domains can be made. The capability of a peptide antigen to bind to the polypeptides and MHC molecules according to the invention can be determined by techniques known in the art, including but not limited to explorative methods such as MHC peptide elution followed by Mass spectrometry and bio-informatic prediction in silico, and confirmative methods such as MHC peptide multimere binding methods and stimulation assays.

[0075] In accordance with the invention, the recombinant polypeptides, pharmaceutical compositions and kits of the invention are preferably suitable for use in a human patient.

[0076] In accordance with the invention, the recombinant polypeptides, pharmaceutical compositions and kits of the invention are preferably suitable for use in the treatment of Parkinson's disease in a human patient. In accordance with the invention, the recombinant polypeptides, pharmaceutical compositions and kits of the invention are preferably suitable for inducing immunological tolerance against human alpha-Synuclein, e.g., in a human patient.

[0077] In accordance with the invention, those recombinant polypeptides, pharmaceutical compositions and kits of the invention, wherein said [alpha] 1 domain according to (v) and said [alpha]2 domain according to (vi) are from a human HLA-A2 or HLA-A11 molecule, are preferably for use in the treatment of multiple sclerosis (MS), MOG antibody disease or MOG antibody positive neuromyelitis optica in a human patient who is positive for the same HLA molecule (i.e., HLA-A2 and HLA-A11, respectively). It is expected that this use will be associated with a more favorable (less immunogenic) immunogenicity profile and a longer half-life (slower clearance) than in patients who are negative for HLA-A2 and HLA-A11, respectively.

[0078] HLA-G, in contrast, does not show the high level of polymorphism observed with classical HLA alleles. It is expected that HLA-G-based polypeptides of the invention will have a favorable immunogenicity profile and halflife across the human population.

[0079] It is understood that in accordance with the invention, the recombinant polypeptides, pharmaceutical compositions and kits of the invention are stable.

[0080] It will be understood that in connection with the peptide antigens used in accordance with the invention, any lengths of these peptide antigens referred to herein (e.g. "7 to 11 amino acids in length”) are meant to refer to the length of the peptide antigens themselves. Thus, the lengths of peptide antigens referred to herein do not include the length conferred by additional amino acids which are not part of the peptide antigens such as additional amino acids from possible linker sequences etc.

[0081] In accordance with the present invention, each occurrence of the term "comprising” may optionally be substituted with the term "consisting of'.

[0082] Methods and Techniques

[0083] Generally, unless otherwise defined herein, the methods used in the present invention (e.g. cloning methods or methods relating to antibodies) are performed in accordance with procedures known in the art, e.g. the procedures described in Sambrook et al. ("Molecular Cloning: A Laboratory Manual.”, 2ndEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1989), Ausubel et al. ("Current Protocols in Molecular Biology.” Greene Publishing Associates and Wiley Interscience; New York 1992), and Harlow and Lane ("Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1988), all of which are incorporated herein by reference.

[0084] Protein-protein binding, such as binding of antibodies to their respective target proteins, can be assessed by methods known in the art. Protein-protein binding is preferably assessed by surface plasmon resonance spectroscopy measurements.

[0085] For instance, binding of MHC class lb molecules or recombinant polypeptides according to the invention to their receptors, including ILT2 and ILT4, is preferably assessed by surface plasmon resonance spectroscopy measurements. More preferably, binding of MHC class lb molecules or recombinant polypeptides according to the invention to their receptors is assessed by surface plasmon resonance measurements at 25°C. Appropriate conditions for such surface plasmon resonance measurements have been described by Shiroishi et al., Proc Natl Acad Sci U S A. 2003 July 22; 100(15):8856-8861.

[0086] Sequence Alignments of sequences according to the invention are performed by using the BLAST algorithm (see Altschul et al. (1990) "Basic local alignment search tool.” Journal of Molecular Biology 215. p. 403-410.; Altschul et al.: (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.). Appropriate parameters for sequence alignments of short peptides by the BLAST algorithm, which are suitable for peptide antigens in accordance with the invention, are known in the art. Most software tools using the BLAST algorithm automatically adjust the parameters for sequence alignments for a short input sequence. In one embodiment, the following parameters are used: Max target sequences 10; Word size 3; BLOSUM 62 matrix; gap costs: existence 11 , extension 1; conditional compositional score matrix adjustment. Thus, when used in connection with sequences, terms such as "identity” or "identical” preferably refer to the identity value obtained by using the BLAST algorithm.

[0087] Preparation of pharmaceutical compositions of the Invention

[0088] Pharmaceutical compositions of the present invention are prepared in accordance with known standards for the preparation of pharmaceutical compositions. For instance, the pharmaceutical compositions are prepared in a way that they can be stored and administered appropriately. The pharmaceutical compositions of the invention may therefore comprise pharmaceutically acceptable components such as carriers, excipients and / or stabilizers.

[0089] Such pharmaceutically acceptable components are not toxic in the amounts used when administering the pharmaceutical composition to a human patient. The pharmaceutical acceptable components added to the pharmaceutical compositions may depend on the chemical nature of the active ingredients present in the composition, the particular intended use of the pharmaceutical compositions and the route of administration.

[0090] In general, the pharmaceutically acceptable components used in connection with the present invention are used in accordance with knowledge available in the art, e.g. from Remington's Pharmaceutical Sciences, Ed. AR Gennaro, 20th edition, 2000, Williams & Wilkins, PA, USA. Pharmaceutical compositions comprising the nucleic acids of the invention (e.g., RNAs) may also be formulated in accordance with knowledge available in the art, e.g. using liposomal formulations targeting dendritic cells.

[0091] Peptide Antigens in Accordance with the Invention

[0092] The peptide antigens which can be used in accordance with the invention, including the peptide antigens as defined above, are not particularly limited other than by their ability to be presented on MHC molecules. It is understood that a "peptide antigen presented by said recombinant polypeptide” as referred to in relation to the invention is a peptide antigen that is presented by said recombinant polypeptide to human T cells, if such T cells are present, in a way that it binds to a T cell receptor on the human T-cells.

[0093] Peptides which are able to be presented on MHC molecules can be generated as known in the art (see, for instance, Rammensee, Bachmann, Emmerich, Bachor, Stevanovic. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics. 1999 Nov;50(3-4):213-9; Pearson et al. MHC class l-associated peptides derive from selective regions of the human genome. J Clin Invest. 2016 Dec 1;126(12):4690-4701 ; and Rock, Reits, Neefjes. Present Yourself! By MHC Class I and MHC Class II Molecules. Trends Immunol. 2016 Nov;37(1 1)724-737).

[0094] Peptide antigens are generally known in the art. Generally, the peptide antigens in accordance with the invention are capable of binding to MHC class I proteins. It will be understood by a person skilled in the art that for each MHC class lb molecule or polypeptide capable of presenting peptides in accordance with the invention, peptide antigens which are capable of binding to said MHC class lb molecule or recombinant polypeptide will preferably be used. These peptide antigens can be selected based on methods known in the art.

[0095] Binding of peptide antigens to MHC class lb molecules or to polypeptides capable of peptide antigen binding in accordance with the invention can be assessed by methods known in the art, e.g. the methods of: Rammensee, Bachmann, Emmerich, Bachor, Stevanovic. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics. 1999 Nov;50(3-4):213-9;

[0096] Pearson et al. MHC class l-associated peptides derive from selective regions of the human genome. J Clin Invest. 2016 Dec 1 ; 126(12):4690-4701 ; and

[0097] Rock, Reits, Neefjes. Present Yourself! By MHC Class I and MHC Class II Molecules. Trends Immunol. 2016 Nov;37(11):724-737.

[0098] Such methods include experimental methods and methods for the prediction of peptide antigen binding.

[0099] Anchor residues which serve to anchor the peptide antigen on the MHC class I molecule and to ensure binding of the peptide antigen to the MHC class I molecule are known in the art.

[0100] In a preferred embodiment in accordance with all embodiments of the invention, the peptide antigen used in accordance with the invention contain any of the anchor or preferred amino acid residues in the positions as predicted for MHC class I molecules.

[0101] Such predictions can preferably be made in as described in any one of the following publications:

[0102] - Rammensee et al, SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics (1999) 50: 213-219

[0103] - Nielsen et al, Protein Sci (2003) 12:1007-1017

[0104] - Neefjes et al. Nat Rev Immunol. 2011 Nov 11 ;11 (12):823-36

[0105] - Diehl et al. Curr Biol. 1996 Mar 1 ;6(3):305-14,

[0106] - Lee et al. Immunity. 1995 Nov;3(5):591-600.

[0107] - Desai & Kulkarni-Kale, T-cell epitope prediction methods: an overview. Methods Mol Biol. 2014;1184:333-64.

[0108] - Jumper, J., Evans, R., Pritzel, A. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583-589 (2021). https: / / doi.org / 10.1038 / s41586-021-03819-2

[0109] In the invention, the peptide antigen is from human alpha-Synuclein.

[0110] It is understood that the non-anchor amino acid residues of the peptide antigen of the invention may or may not contain conservative substitutions, preferably not more than two conservative substitutions, more preferably one conservative substitution with respect to the corresponding amino acid sequence of a peptide antigen from human alpha-Synuclein. Peptide antigens of the invention preferably consist of naturally occurring amino acids. However, non-naturally occurring amino acids such as modified amino acids can also be used. For instance, in one embodiment, a peptide antigen of the invention encompasses the peptidomimetic of the indicated peptide antigen amino acid sequence of human alpha-Synuclein.

[0111] Methods for the synthesis of peptide antigens, including peptide antigens in accordance with the invention, are well known in the art.

[0112] Sequences

[0113] Preferred amino acid sequences referred to in the present application can be independently selected from the following sequences. The sequences are represented in an N-terminal to C-terminal order; and they are represented in the one-letter amino acid code.

[0114] Exemplary sequences which are part of the recombinant polypeptides of the invention:

[0115] Leader Peptide (absent from the recombinant polypeptide due to processing during cellular expression): e.g. MSRSVALAVLALLSLSGLEA (SEQ ID NO: 1), encoded by the DNA sequence ATGAGCAGATCTGTGGCCCTGGCTGTTCTGGCTCTGCTGTCTCTGTCTGGCCTGGAAGCC (SEQ ID NO: 49)

[0116] Alternative leader peptides:

[0117] Mouse Ig Heavy peptide: MGWSCIILFLVATATGVHS (SEQ ID NO: 66)

[0118] Human OSM peptide: MGVLLTQRTLLSLVLALLFPSMASM (SEQ ID NO: 67)

[0119] VSV-G peptide: MKCLLYLAFLFIGVNC (SEQ ID NO: 68)

[0120] Mouse Ig Kappa peptide: METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 69)

[0121] BM40 peptide: MRAWIFFLLCLAGRALA (SEQ ID NO: 70)

[0122] Secrecon peptide: MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO: 71)

[0123] Human IgKVIll peptide: MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 72)

[0124] CD33 peptide: MPLLLLLPLLWAGALA (SEQ ID NO: 73) tPA peptide: MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 74)

[0125] Human Chymotrypsinogen peptide: MAFLWLLSCWALLGTTFG (SEQ ID NO: 75)

[0126] Human trypsinogen-2 peptide: MNLLLILTFVAAAVA (SEQ ID NO: 76)

[0127] Human IL-2 peptide: MYRMQLLSCIALSLALVTNS (SEQ ID NO: 77)

[0128] Gaussia luc peptide: MGVKVLFALICIAVAEA (SEQ ID NO: 78)

[0129] Albumin (HSA) peptide: MKWVTFISLLFSSAYS (SEQ ID NO: 79) Influenza Haemagglutinin peptide: MKTIIALSYIFCLVLG (SEQ ID NO: 80)

[0130] Human insulin peptide: MALWMRLLPLLALLALWGPDPAAA (SEQ ID NO: 81)

[0131] Silkworm Fibroin LC peptide: MKPIFLVLLWTSAYA (SEQ ID NO: 82)

[0132] Peptide antigen: any MHC class I peptide corresponding to MHC class I [alpha] 1&2 domains, e.g. GAPQEGIL (SEQ ID NO: 20), or any peptide antigen according to the invention, e.g.:

[0133] First linker: For instance GGGGSGGGGSGGGGS (SEQ ID NO: 3) or GCGASGGGGSGGGGS (SEQ ID NO: 4) beta 2 Microglobulin, for instance:

[0134] IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTE

[0135] KDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 5, human beta 2 Microglobulin)

[0136] Second Linker, for instance:

[0137] GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 6)

[0138] [Alpha] 1 & 2 domain derived either from human HLA-G or from any other MHC class I [alpha] 1 &2 domain suitable to present the selected antigenic peptide, Y84 may be C in DT variant e.g. [Alpha] 1 & 2 domain derived from human HLA-G: E.g.: GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAH

[0139] AQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAA

[0140] QISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRA (SEQ ID NO: 7)

[0141] Or: Human HLA-A2 [alpha]1 & 2 domain: E.g.: GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQ SQTDRVDLGTLRGCYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQ ITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRT (SEQ ID NO: 8)

[0142] Or: Human HLA-A11 [alpha]1 & 2 domain: E.g.: GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQ SQTDRVDLGTLRGCYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQ ITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRT (SEQ ID NO: 57)

[0143] Human HLA-G [alpha]3 domain (or any MHO lb [alpha]3 domain, such as HLA-F, which also interacts with ILT2 and ILT4 receptors), for instance: DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVWPSGE EQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL (SEQ ID NO: 9; sequence of HLA-G [alpha]3 with furin cleavage site in the C-terminal amino acid sequence from intron 4 of human HLA-G), or DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVWPSGE EQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESGSLSEDL (SEQ ID NO: 51 ; sequence of HLA-G [alpha]3 wherein the furin cleavage site in the C-terminal amino acid sequence from intron 4 of human HLA-G has been removed).

[0144] Note that the following underlined amino acids of this sequence are relevant for ILT2 or ILT4 receptor interaction: DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDyELVETRPAGDGTFQKWAAVyVPSGE EQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL, or DPPKTHVTHHPyFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVyVPSGE EQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESGSLSEDL Alternatively, a shorter form of a human HLA-G [alpha]3 domain may be used which lacks the optional C- terminal amino acid sequence from intron 4 (SKEGDGGIMSVRESRSLSEDL; SEQ ID NO: 47, or SKEGDGGIMSVRESGSLSEDL; SEQ ID NO: 52), i.e.: DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVWPSGE EQRYTCHVQHEGLPEPLMLRW (SEQ ID NO: 2).

[0145] The inventors found that the recombinant polypeptides can be produced using any one of the above-indicated sequences of HLA-G [alpha]3. The presence of the sequence of SEQ ID NO: 51 in the recombinant polypeptides, i.e., the presence of the sequence of HLA-G [alpha]3 wherein the furin cleavage site in the C- terminal amino acid sequence from intron 4 of human HLA-G has been removed, was found to advantageous for an improved purification of the recombinant polypeptides, e.g., for the purification of recombinant polypeptides containing C-terminal tags.

[0146] Factor Xa restriction site: IEGRTGTKLGP (SEQ ID NO: 10)

[0147] SpotTag: PDRVRAVSHWSSC (SEQ ID NO: 11)

[0148] Myc tag: EQKLISEEDL (SEQ ID NO: 12)

[0149] His tag: HHHHHH* (SEQ ID NO: 13)

[0150] Spacer sequence: e.g. NSAVD (SEQ ID NO: 14) or GS

[0151] Exemplary peptide antigens which can be part of the recombinant polypeptides of the invention are as follows: GAPQEGIL (SEQ ID NO: 20), preferably used in recombinant polypeptides containing human HLA-G [alpha]1 & 2 domain

[0152] KTKEGVLYV (SEQ ID NO: 28), preferably used in recombinant polypeptides containing human HLA-A2 [alpha]1 & 2 domain

[0153] AWTGVTAV (SEQ ID NO: 33), preferably used in recombinant polypeptides containing human HLA-A2 [alpha]1 & 2 domain

[0154] GWHGVTTV (SEQ ID NO: 31), preferably used in recombinant polypeptides containing human HLA-A2 [alpha]1 & 2 domain

[0155] MDVFMKGLSK (SEQ ID NO: 22), preferably used in recombinant polypeptides containing human HLA-A11 [alpha]1 & 2 domain GWAAAEKTK (SEQ ID NO: 25), preferably used in recombinant polypeptides containing human HLA-A11 [alpha]1 & 2 domain

[0156] MPVDPDNEAY (SEQ ID NO: 21), preferably used in recombinant polypeptides containing human HLA-A1 [alpha]1 & 2 domain

[0157] PVDPDNEAY (SEQ ID NO: 44), preferably used in recombinant polypeptides containing human HLA-A1 [alpha]1 & 2 domain

[0158] SIAAATGFV (SEQ ID NO: 37), preferably used in recombinant polypeptides containing human HLA-A2 [alpha]1 & 2 domain

[0159] WTGVTAVA (SEQ ID NO: 34), preferably used in recombinant polypeptides containing human HLA-A2 [alpha]1 & 2 domain

[0160] WAAAEKTK (SEQ ID NO: 26), preferably used in recombinant polypeptides containing human HLA-A11 [alpha]1 & 2 domain

[0161] VFMKGLSKAK (SEQ ID NO: 24), preferably used in recombinant polypeptides containing human HLA-A11 [alpha]1 & 2 domain

[0162] AAATGFVKKD (SEQ ID NO: 42), preferably used in recombinant polypeptides containing human HLA-A11 [alpha]1 & 2 domain

[0163] AATGFVKK (SEQ ID NO: 43), preferably used in recombinant polypeptides containing human HLA-A11 [alpha]1 & 2 domain

[0164] SIAAATGFVKK (SEQ ID NO: 50), preferably used in recombinant polypeptides containing human HLA-A11 [alpha]1 & 2 domain

[0165] Example for a recombinant polypeptide (without the leader peptide):

[0166] GAPQEGILGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKV EHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGS GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQ SQTDRVDLGTLRGCYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQ ITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEI

[0167] ILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIM SVRESRSLSEDLGSPDRVRAVSHWSSC* (SEQ ID NO: 15; note that the asterisk denotes the position of the stop codon) Note that the sequence of the peptide antigen (here: GAPQEGIL) of the above full-length protein can be substituted by any peptide antigen sequence in accordance with the invention, i.e. by any peptide antigen presented by said recombinant polypeptide, wherein the peptide antigen is a peptide of human alpha-Synuclein. That is, recombinant polypeptides of the invention may consist of a sequence consisting of a peptide antigen which is a peptide of human alpha-Synuclein (e.g., any one of the peptide antigens of SEQ ID NOs: 20-46 or SEQ ID NO: 50 according to the invention), followed by the sequence of

[0168] GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFS KDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRY FYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVD LGTLRGCYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWE AAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRD GEDQTQDVELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGI MSVRESRS LSEDLGSPDRVRAVSHWSSC* (SEQ ID NO: 16; note that the asterisk denotes the position of the stop codon), or followed by the sequence of

[0169] GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFS KDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRY FYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVD LGTLRGCYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWE AAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRD GEDQTQDVELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGI MSVRESGS LSEDLGSPDRVRAVSHWSSC* (SEQ ID NO: 53; note that the asterisk denotes the position of the stop codon), or followed by the sequence without the C-terminal amino acid sequence from intron 4 of the sequence of the HLA-G [alpha]3 domain, the variable cleave site, and the optional tag of

[0170] GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFS KDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRY FYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVD LGTLRGCYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWE AAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRD

[0171] GEDQTQDVELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO: 54).

[0172] Examples of recombinant polypeptides according to the invention (shown without the optional leader peptide sequence, the C-terminal amino acid sequence from intron 4 of the sequence of the HLA-G [alpha]3 domain, the variable cleave site, and the optional tag): aSyn87-97_A11G_fs-_Spt (very good response rate in ELISPOT assays with, good stability)

[0173] Protein sequence:

[0174] SIAAATGFVKKGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIE KVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGG GSGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVK

[0175] AQSQTDRVDLGTLRGCYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMA AQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYP AEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO: 55)

[0176] Encoded by the DNA sequence (note that the following DNA sequence does not show the DNA sequence encoding the N-terminal leader peptide):

[0177] AGCATCGCCGCCGCCACCGGCTTCGTGAAGAAGGGATGCGGAGCTAGCGGCGGAGGCGGATCTGGTG GCGGAGGATCCATTCAGAGAACCCCTAAGATCCAGGTGTACAGCAGACACCCCGCCGAGAACGGCAAG AGCAACTTCCTGAACTGCTACGTGTCCGGCTTTCACCCCAGCGACATTGAGGTGGACCTGCTGAAGAAC GGCGAGCGGATCGAGAAGGTGGAACACAGCGATCTGAGCTTCAGCAAGGACTGGTCCTTCTACCTGCT GTACTACACCGAATTCACCCCTACCGAGAAGGACGAGTACGCCTGCAGAGTGAACCACGTGACACTGAG CCAGCCTAAGATCGTGAAGTGGGACAGAGACATGGGCGGAGGCGGATCTGGTGGCGGAGGAAGTGGC GGCGGAGGATCTGGCGGTGGTGGTTCTGGAAGCCACAGCATGCGGTACTTCTACACCAGCGTGTCCAG ACCTGGAAGAGGCGAGCCCAGATTCATTGCCGTGGGCTACGTGGACGACACCCAGTTCGTCAGATTCGA TTCCGATGCCGCCAGCCAGCGGATGGAACCTAGAGCACCTTGGATCGAGCAAGAGGGACCCGAGTACT GGGACCAAGAGACAAGAAACGTGAAGGCCCAGAGCCAGACCGACAGAGTGGATCTGGGAACCCTGAGA GGCTGCTACAACCAGAGCGAGGATGGCAGCCACACCATCCAGATTATGTACGGCTGCGACGTGGGACC CGACGGCAGATTTCTGAGGGGCTATAGACAGGACGCCTACGACGGCAAGGACTATATCGCCCTGAACG AGGACCTGCGGTCTTGGACAGCTGCCGATATGGCCGCTCAGATCACCAAGCGGAAATGGGAAGCCGCT CATGCCGCCGAACAGCAGAGAGCCTATCTGGAAGGCAGATGCGTGGAATGGCTGCGGAGATACCTGGA AAACGGCAAAGAGACACTGCAGCGGACCGATCCTCCTAAGACACATGTGACCCACCATCCTGTGTTCGA CTACGAGGCCACACTGAGATGTTGGGCCCTGGGCTTTTACCCTGCCGAGATCATCCTGACCTGGCAGAG

[0178] GGACGGCGAGGACCAGACCCAGGATGTGGAACTGGTGGAAACCAGACCTGCCGGCGACGGCACCTTC CAGAAATGGGCTGCTGTGGTGGTGCCCTCCGGCGAGGAACAGCGGTACACCTGTCACGTGCAGCACGA GGGACTGCCCGAGCCCCTGATGCTGCGGTGG (SEQ ID NO: 56)

[0179] Preferred A2G constructs: aSyn69 77_A2G (high response rate in ELISPOT assays with PBMC from healthy donors, excellent production yield and stability)

[0180] Protein sequence:

[0181] AyyTGyTAVGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEK VEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGG SGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKA

[0182] HSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMA AQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYP AEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID

[0183] NO: 61)

[0184] Encoded by the DNA sequence (note that the following DNA sequence does not show the DNA sequence encoding the N-terminal leader peptide):

[0185] GCTGTGGTCACAGGCGTGACAGCTGTTGGATGCGGAGCTAGCGGCGGAGGCGGATCTGGTGGCGGAG

[0186] GATCCATCCAGCGGACCCCTAAGATCCAGGTGTACAGCAGACACCCCGCCGAGAACGGCAAGAGCAAC

[0187] TTCCTGAACTGCTACGTGTCCGGCTTTCACCCCAGCGACATTGAGGTGGACCTGCTGAAGAACGGCGAG

[0188] CGGATCGAGAAGGTGGAACACAGCGATCTGAGCTTCAGCAAGGACTGGTCCTTCTACCTGCTGTACTAC

[0189] ACCGAATTCACCCCTACCGAGAAGGACGAGTACGCCTGTAGAGTGAACCACGTGACCCTGAGCCAGCCT

[0190] AAGATCGTGAAGTGGGACAGAGACATGGGAGGCGGAGGTAGTGGTGGTGGCGGTAGTGGCGGCGGTG GAAGCGGAGGTGGTGGATCTGGATCTCACAGCATGCGGTACTTTTTCACCAGCGTGTCCAGACCTGGCA GAGGCGAGCCTAGATTCATTGCCGTGGGCTACGTGGACGACACCCAGTTCGTCAGATTCGATTCCGATG

[0191] CCGCCAGCCAGCGGATGGAACCTAGAGCACCTTGGATCGAGCAAGAGGGCCCCGAGTATTGGGACGGC GAGACAAGAAAAGTGAAGGCCCACTCTCAGACCCACAGAGTGGACCTGGGAACCCTGAGAGGCTGCTA CAATCAGTCTGAGGCCGGCAGCCATACCGTGCAGAGGATGTATGGCTGTGACGTGGGCAGCGATTGGA GATTCCTGAGGGGCTACCACCAGTACGCCTACGACGGCAAGGACTATATCGCCCTGAAAGAGGACCTGC GGTCTTGGACAGCCGCCGATATGGCTGCTCAGACCACCAAGCACAAGTGGGAAGCCGCTCATGTGGCC GAACAGCTGAGAGCCTATCTGGAAGGCACCTGTGTGGAATGGCTGCGGAGATACCTGGAAAACGGCAA AGAGACACTGCAGCGGACCGATCCTCCTAAGACACATGTGACCCACCATCCTGTGTTCGACTACGAGGC

[0192] CACACTGAGATGTTGGGCCCTGGGCTTTTACCCTGCCGAGATCATCCTGACCTGGCAGAGGGACGGCG AGGACCAGACCCAGGATGTGGAACTGGTGGAAACCAGACCTGCCGGCGACGGCACCTTCCAGAAATGG GCTGCTGTGGTGGTGCCCTCCGGCGAGGAACAGCGGTACACCTGTCACGTGCAGCACGAGGGACTGCC

[0193] CGAGCCCCTGATGCTGCGGTGG (SEQ ID NO: 89) aSyn7078_A2G (high response rate in ELISPOT assays with PBMC from healthy donors, and PBMC from patients with Parkinson's disease, very good production yield and stability)

[0194] Protein sequence:

[0195] WTGVTAVAGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEK VEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGG SGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKA

[0196] HSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMA AQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYP AEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO: 62)

[0197] Encoded by the DNA sequence (note that the following DNA sequence does not show the DNA sequence encoding the N-terminal leader peptide):

[0198] GTGGTGACGGGTGTGACAGCAGTAGCCGGATGCGGAGCTAGCGGCGGAGGCGGATCTGGTGGCGGAG

[0199] GATCCATCCAGCGGACCCCTAAGATCCAGGTGTACAGCAGACACCCCGCCGAGAACGGCAAGAGCAAC

[0200] TTCCTGAACTGCTACGTGTCCGGCTTTCACCCCAGCGACATTGAGGTGGACCTGCTGAAGAACGGCGAG

[0201] CGGATCGAGAAGGTGGAACACAGCGATCTGAGCTTCAGCAAGGACTGGTCCTTCTACCTGCTGTACTAC ACCGAATTCACCCCTACCGAGAAGGACGAGTACGCCTGTAGAGTGAACCACGTGACCCTGAGCCAGCCT AAGATCGTGAAGTGGGACAGAGACATGGGAGGCGGAGGTAGTGGTGGTGGCGGTAGTGGCGGCGGTG

[0202] GAAGCGGAGGTGGTGGATCTGGATCTCACAGCATGCGGTACTTTTTCACCAGCGTGTCCAGACCTGGCA GAGGCGAGCCTAGATTCATTGCCGTGGGCTACGTGGACGACACCCAGTTCGTCAGATTCGATTCCGATG CCGCCAGCCAGCGGATGGAACCTAGAGCACCTTGGATCGAGCAAGAGGGCCCCGAGTATTGGGACGGC

[0203] GAGACAAGAAAAGTGAAGGCCCACTCTCAGACCCACAGAGTGGACCTGGGAACCCTGAGAGGCTGCTA CAATCAGTCTGAGGCCGGCAGCCATACCGTGCAGAGGATGTATGGCTGTGACGTGGGCAGCGATTGGA GATTCCTGAGGGGCTACCACCAGTACGCCTACGACGGCAAGGACTATATCGCCCTGAAAGAGGACCTGC GGTCTTGGACAGCCGCCGATATGGCTGCTCAGACCACCAAGCACAAGTGGGAAGCCGCTCATGTGGCC

[0204] GAACAGCTGAGAGCCTATCTGGAAGGCACCTGTGTGGAATGGCTGCGGAGATACCTGGAAAACGGCAA

[0205] AGAGACACTGCAGCGGACCGATCCTCCTAAGACACATGTGACCCACCATCCTGTGTTCGACTACGAGGC

[0206] CACACTGAGATGTTGGGCCCTGGGCTTTTACCCTGCCGAGATCATCCTGACCTGGCAGAGGGACGGCG AGGACCAGACCCAGGATGTGGAACTGGTGGAAACCAGACCTGCCGGCGACGGCACCTTCCAGAAATGG GCTGCTGTGGTGGTGCCCTCCGGCGAGGAACAGCGGTACACCTGTCACGTGCAGCACGAGGGACTGCC

[0207] CGAGCCCCTGATGCTGCGGTGG (SEQ ID NO: 90)

[0208] A preferred A11 G construct: aSyn37 45_A11G (good response rate in ELISPOT assays with PBMC from healthy donors and, in particular, with PBMC from patients with Parkinson's disease, very good production yield and stability)

[0209] Protein sequence:

[0210] VLYVGSKIKGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEK VEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGG SGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKA

[0211] QSQTDRVDLGTLRGCYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAA QITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPA EIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID

[0212] NO: 63)

[0213] Encoded by the DNA sequence (note that the following DNA sequence does not show the DNA sequence encoding the N-terminal leader peptide):

[0214] GTGCTGTACGTGGGCAGCAAGACAAAAGGATGCGGCGCTAGCGGCGGAGGCGGATCTGGTGGCGGAG GATCCATTCAGAGAACCCCTAAGATCCAGGTGTACAGCAGACACCCCGCCGAGAACGGCAAGAGCAACT TCCTGAACTGCTACGTGTCCGGCTTTCACCCCAGCGACATTGAGGTGGACCTGCTGAAGAACGGCGAGC

[0215] GGATCGAGAAGGTGGAACACAGCGATCTGAGCTTCAGCAAGGACTGGTCCTTCTACCTGCTGTACTACA

[0216] CCGAATTCACCCCTACCGAGAAGGACGAGTACGCCTGCAGAGTGAACCACGTGACACTGAGCCAGCCTA

[0217] AGATCGTGAAGTGGGACAGAGACATGGGCGGAGGCGGATCTGGTGGCGGAGGAAGTGGCGGCGGAGG ATCTGGCGGTGGTGGTTCTGGAAGCCACAGCATGCGGTACTTCTACACCAGCGTGTCCAGACCTGGAAG AGGCGAGCCCAGATTCATTGCCGTGGGCTACGTGGACGACACCCAGTTCGTCAGATTCGATTCCGATGC CGCCAGCCAGCGGATGGAACCTAGAGCACCTTGGATCGAGCAAGAGGGACCCGAGTACTGGGACCAAG AGACAAGAAACGTGAAGGCCCAGAGCCAGACCGACAGAGTGGATCTGGGAACCCTGAGAGGCTGCTAC

[0218] AACCAGAGCGAGGATGGCAGCCACACCATCCAGATTATGTACGGCTGCGACGTGGGACCCGACGGCAG

[0219] ATTTCTGAGGGGCTATAGACAGGACGCCTACGACGGCAAGGACTATATCGCCCTGAACGAGGACCTGCG

[0220] GTCTTGGACAGCTGCCGATATGGCCGCTCAGATCACCAAGCGGAAATGGGAAGCCGCTCATGCCGCCG

[0221] AACAGCAGAGAGCCTATCTGGAAGGCAGATGCGTGGAATGGCTGCGGAGATACCTGGAAAACGGCAAA GAGACACTGCAGCGGACCGATCCTCCTAAGACACATGTGACCCACCATCCTGTGTTCGACTACGAGGCC ACACTGAGATGTTGGGCCCTGGGCTTTTACCCTGCCGAGATCATCCTGACATGGCAGAGAGATGGCGAG

[0222] GATCAGACCCAGGACGTGGAACTGGTGGAAACAAGACCTGCCGGCGACGGCACCTTCCAGAAATGGGC TGCTGTGGTGGTTCCCAGCGGCGAGGAACAGAGATACACCTGTCACGTGCAGCACGAGGGACTGCCTG AACCTCTGATGCTGCGGTGG (SEQ ID NO: 91)

[0223] Further recombinant polypeptides of the invention that were tested in PBMC of healthy donors and in some cases in patients: aSyn87 95_A2G (good response rate in ELISPOT assays with PBMC from healthy donors and patients with Parkinson's disease, very good stability and producibility)

[0224] Protein sequence:

[0225] SLAAATGFVGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKV EHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGS GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAH

[0226] SQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAA QTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPA EIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID

[0227] NO: 64)

[0228] Encoded by the DNA sequence (note that the following DNA sequence does not show the DNA sequence encoding the N-terminal leader peptide):

[0229] AGCATCGCCGCCGCCACCGGCTTCGTGGGATGCGGAGCTAGCGGCGGAGGCGGATCTGGTGGCGGAG

[0230] GATCCATCCAGCGGACCCCTAAGATCCAGGTGTACAGCAGACACCCCGCCGAGAACGGCAAGAGCAAC TTCCTGAACTGCTACGTGTCCGGCTTTCACCCCAGCGACATTGAGGTGGACCTGCTGAAGAACGGCGAG CGGATCGAGAAGGTGGAACACAGCGATCTGAGCTTCAGCAAGGACTGGTCCTTCTACCTGCTGTACTAC

[0231] ACCGAATTCACCCCTACCGAGAAGGACGAGTACGCCTGTAGAGTGAACCACGTGACCCTGAGCCAGCCT

[0232] AAGATCGTGAAGTGGGACAGAGACATGGGAGGCGGAGGTAGTGGTGGTGGCGGTAGTGGCGGCGGTG

[0233] GAAGCGGAGGTGGTGGATCTGGATCTCACAGCATGCGGTACTTTTTCACCAGCGTGTCCAGACCTGGCA

[0234] GAGGCGAGCCTAGATTCATTGCCGTGGGCTACGTGGACGACACCCAGTTCGTCAGATTCGATTCCGATG

[0235] CCGCCAGCCAGCGGATGGAACCTAGAGCACCTTGGATCGAGCAAGAGGGCCCCGAGTATTGGGACGGC

[0236] GAGACAAGAAAAGTGAAGGCCCACTCTCAGACCCACAGAGTGGACCTGGGAACCCTGAGAGGCTGCTA

[0237] CAATCAGTCTGAGGCCGGCAGCCATACCGTGCAGAGGATGTATGGCTGTGACGTGGGCAGCGATTGGA

[0238] GATTCCTGAGGGGCTACCACCAGTACGCCTACGACGGCAAGGACTATATCGCCCTGAAAGAGGACCTGC

[0239] GGTCTTGGACAGCCGCCGATATGGCTGCTCAGACCACCAAGCACAAGTGGGAAGCCGCTCATGTGGCC

[0240] GAACAGCTGAGAGCCTATCTGGAAGGCACCTGTGTGGAATGGCTGCGGAGATACCTGGAAAACGGCAA

[0241] AGAGACACTGCAGCGGACCGATCCTCCTAAGACACATGTGACCCACCATCCTGTGTTCGACTACGAGGC

[0242] CACACTGAGATGTTGGGCCCTGGGCTTTTACCCTGCCGAGATCATCCTGACCTGGCAGAGGGACGGCG

[0243] AGGACCAGACCCAGGATGTGGAACTGGTGGAAACCAGACCTGCCGGCGACGGCACCTTCCAGAAATGG

[0244] GCTGCTGTGGTGGTGCCCTCCGGCGAGGAACAGCGGTACACCTGTCACGTGCAGCACGAGGGACTGCC CGAGCCCCTGATGCTGCGGTGG (SEQ ID NO: 92) aSyn86 96_A11 G (very good response rate in ELISPOT assays with PBMC from healthy donors and PD patients, very good stability and producibility assessment)

[0245] Protein sequence:

[0246] GSIAAATGFVKGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERI

[0247] EKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGG

[0248] GGSGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNV

[0249] KAQSQTDRVDLGTLRGCYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADM

[0250] AAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFY

[0251] PAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO: 65)

[0252] Encoded by the DNA sequence (note that the following DNA sequence does not show the DNA sequence encoding the N-terminal leader peptide): GGGAGCATTGCAGCAGCCACTGGCTTTGTCAAAGGATGCGGCGCTAGCGGCGGAGGCGGATCTGGTG GCGGAGGATCCATTCAGAGAACCCCTAAGATCCAGGTGTACAGCAGACACCCCGCCGAGAACGGCAAG AGCAACTTCCTGAACTGCTACGTGTCCGGCTTTCACCCCAGCGACATTGAGGTGGACCTGCTGAAGAAC GGCGAGCGGATCGAGAAGGTGGAACACAGCGATCTGAGCTTCAGCAAGGACTGGTCCTTCTACCTGCT GTACTACACCGAATTCACCCCTACCGAGAAGGACGAGTACGCCTGCAGAGTGAACCACGTGACACTGAG

[0253] CCAGCCTAAGATCGTGAAGTGGGACAGAGACATGGGCGGAGGCGGATCTGGTGGCGGAGGAAGTGGC GGCGGAGGATCTGGCGGTGGTGGTTCTGGAAGCCACAGCATGCGGTACTTCTACACCAGCGTGTCCAG ACCTGGAAGAGGCGAGCCCAGATTCATTGCCGTGGGCTACGTGGACGACACCCAGTTCGTCAGATTCGA TTCCGATGCCGCCAGCCAGCGGATGGAACCTAGAGCACCTTGGATCGAGCAAGAGGGACCCGAGTACT

[0254] GGGACCAAGAGACAAGAAACGTGAAGGCCCAGAGCCAGACCGACAGAGTGGATCTGGGAACCCTGAGA GGCTGCTACAACCAGAGCGAGGATGGCAGCCACACCATCCAGATTATGTACGGCTGCGACGTGGGACC CGACGGCAGATTTCTGAGGGGCTATAGACAGGACGCCTACGACGGCAAGGACTATATCGCCCTGAACG AGGACCTGCGGTCTTGGACAGCTGCCGATATGGCCGCTCAGATCACCAAGCGGAAATGGGAAGCCGCT

[0255] CATGCCGCCGAACAGCAGAGAGCCTATCTGGAAGGCAGATGCGTGGAATGGCTGCGGAGATACCTGGA AAACGGCAAAGAGACACTGCAGCGGACCGATCCTCCTAAGACACATGTGACCCACCATCCTGTGTTCGA CTACGAGGCCACACTGAGATGTTGGGCCCTGGGCTTTTACCCTGCCGAGATCATCCTGACATGGCAGAG AGATGGCGAGGATCAGACCCAGGACGTGGAACTGGTGGAAACAAGACCTGCCGGCGACGGCACCTTCC

[0256] AGAAATGGGCTGCTGTGGTGGTTCCCAGCGGCGAGGAACAGAGATACACCTGTCACGTGCAGCACGAG GGACTGCCTGAACCTCTGATGCTGCGGTGG (SEQ ID NO: 93)

[0257] The recombinant polypeptides of the invention shown above may also contain any one of the optional leader peptide, the C-terminal amino acid sequence from intron 4 of the sequence of the HLA-G [alpha]3 domain (e.g., SKEGDGGIMSVRESRSLSEDL; SEQ ID NO: 47, or SKEGDGGIMSVRESGSLSEDL; SEQ ID NO: 52), the optional protease cleavage site, the optional linker (e.g., GS) and / or the optional tag as exemplified above.

[0258] The following three general structures of recombinant polypeptides according to the invention are preferred:

[0259] [a peptide antigen i) according to the invention]— [the amino acid sequence GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFS

[0260] KDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRY FSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMN LQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCE AANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRD GEDQTQDVELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO: 83, which comprises a linker according to ii), a sequence of a human p2 microglobulin according to iii), a linker according to iv), HLA-G [alpha]1&2 domain according to v) and vi), and a HLA-G [alpha]3 domain according to vii))]— [optionally a C-terminal amino acid sequence from intron 4 of the sequence of the HLA-G [alpha]3 domain (e.g., SKEGDGGIMSVRESRSLSEDL; SEQ ID NO: 47, or SKEGDGGIMSVRESGSLSEDL; SEQ ID NO: 52)]- [optionally a variable cleavage site]— [optionally a tag]

[0261] [a peptide antigen i) according to the invention]— [the amino acid sequence GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFS KDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRY FFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDL GTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKW EAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQR DGEDQTQDVELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO: 84, which comprises a linker according to ii), a sequence of a human p2 microglobulin according to iii), a linker according to iv), HLA-A2 [alpha]1&2 domain according to v) and vi), and a HLA-G [alpha]3 domain according to vii))]— [optionally a C-terminal amino acid sequence from intron 4 of the sequence of the HLA-G [alpha]3 domain (e.g., SKEGDGGIMSVRESRSLSEDL; SEQ ID NO: 47, or SKEGDGGIMSVRESGSLSEDL; SEQ ID NO: 52)]- [optionally a variable cleavage site]— [optionally a tag]

[0262] [a peptide antigen i) according to the invention]— [the amino acid sequence GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFS KDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRY FYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVD LGTLRGCYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWE AAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRD GEDQTQDVELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO: 85, which comprises a linker according to ii), a sequence of a human p2 microglobulin according to iii), a linker according to iv), HLA-A11 [alpha]1&2 domain according to v) and vi), and a HLA-G [alpha]3 domain according to vii))]— [optionally a C-terminal amino acid sequence from intron 4 of the sequence of the HLA-G [alpha]3 domain (e.g., SKEGDGGIMSVRESRSLSEDL; SEQ ID NO: 47, or SKEGDGGIMSVRESGSLSEDL; SEQ ID NO: 52)]- [optionally a variable cleavage site]— [optionally a tag]

[0263] The receptors ILT2 (also known as LILRB1 ) and ILT4 (also known as LILRB2) are known in the art. Preferred sequences of these receptors in accordance with the invention are as follows:

[0264] ILT2:

[0265] MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRL

[0266] YREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELWTG

[0267] AYIKPTLSAQPSPWNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRA

[0268] IFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEE

[0269] TLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGA

[0270] HNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKE

[0271] GAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELWS

[0272] GPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGWIGILVAVILLLLLLLLLF

[0273] LILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQ

[0274] PEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMD

[0275] TEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO: 17)

[0276] ILT4:

[0277] MTPIVTVLICLGLSLGPRTHVQTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRL

[0278] YREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGA

[0279] YPKPTLSAQPSPWTSGGRVTLQCESQVAFGGFILCKEGEEEHPQCLNSQPHARGSSRAI

[0280] FSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKKPSLSVQPGPWAPGES

[0281] LTLQCVSDVGYDRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAH

[0282] NLSSECSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAG

[0283] AADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPLELWSG

[0284] PSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAWLLLLLLLLLF

[0285] LILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKDTQ

[0286] PEDGVEMDTRAAASEAPQDVTYAQLHSLTLRRKATEPPPSQEREPPAEPSIYATLAIH (SEQ ID NO: 18) The sequence of human alpha-Synuclein is known in the art. A preferred human alpha-Synuclein is as follows: MDVFMKGLSK AKEGWAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGWH GVATVAEKTK

[0287] EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP

[0288] DNEAYEMPSE EGYQDYEPEA (SEQ ID NO: 19)

[0289] Therapeutic applications of the invention:

[0290] Parkinson's disease (PD) including prodromal Parkinson's disease (prodromal-PD), and the classification thereof, are known in the art.

[0291] Prodromal-PD refers to the stage at which individuals do not fulfill diagnostic criteria for PD (ie, bradykinesia and at least 1 other motor sign) but do exhibit signs and symptoms that indicate a higher than average risk of developing motor symptoms and a diagnosis of PD in the future. Most prodromal symptoms are nonmotor and have a major impact on quality of life both for patients with prodromal-PD and for those whose disease stage has progressed to motor-PD. Thus, early detection and treatment of these prodromal symptoms is essential for high-quality care. The best-characterized symptoms of prodromal-PD include hyposmia, constipation, mood disorders, and REM sleep behavior disorder (RBD).

[0292] The present invention is further illustrated by the following non-limiting examples:

[0293] EXAMPLES

[0294] Reference Example: Screening for Target Antigens in Mice

[0295] To screen for suitable target antigens, mice injected with haSyn or control virus were sacrificed. Immune cells were isolated from brain, peripheral lymph nodes and spleen. Then, these cells were incubated with A53T-aSyn peptides predicted in silico (e.g. via NetMHC). By screening for aSyn-specific immune responses in C57BL / 6 mice, the inventors identified MHC I- and MHC Il-restricted haSyn peptides that induce IFN-y release from T cells of diseased mice (Fig. 3). T cell reactivity towards haSyn peptides was also shown for cervical lymph node and brain derived T cells.

[0296] Example 1 (reference): Prevention of Neuron Death in a PD in vivo Model The inventors reasoned that in view of the critical role for T cells in the neurodegenerative process (which was shown by depletion / reconstitution experiments), this haSyn PD mouse model, which closely resembles the human disease, is ideally suited to evaluate the efficacy of immune modulation on the background of a PD-like aSyn-based pathology, and to evaluate the suitability of recombinant polypeptides of the invention for the treatment of PD.

[0297] The inventors further reasoned that induction of immune tolerance towards aSyn will induce a tissue-wide protection via local antigen-specific activation of Treg. Having identified the A53T-aSyn derived aSyn 68-78 peptide as immunogenic in mice, the inventors developed a recombinant polypeptide, which corresponds to the recombinant polypeptides of the invention but carries this peptide antigen which is immunogenic in mice and murine H2-Dbalphal and 2 domains (this recombinant polypeptide is hereinafter also referred to as "mouse AIM Bio surrogate molecule” or "AIM Bioasyn”). In a pilot experiment, haSyn PD mice were injected weekly with either 0.5 pig / g AIM Bioasyn or 2 pig / g AIM Bioasyn or an off-target- irrelevant control peptide (gp34), starting 3 days after AAV injection. The Gp34 peptide is a well-characterized T cell epitope derived from Lymphocytic Choriomeningitis virus (LCMV) Glycoprotein. While this antigen was traditionally named Gp33, the epitope presented on H2-Kbwas later found to comprise just amino acids 34-41. (An epitope beginning at amino acid 33 is, in contrast, presented on H2-Kd.) Therefore, we call the H2-Kbepitope Gp34, which is in line with the most recent recommendations. Still, there is an ambiguous use of the Gp33 and Gp34 nomenclature in the literature. The sequence of the peptide used here is AVYNFATM (SEQ ID NO: 48). Control mice were injected with carrier (PBS) only. 10 weeks after AAV delivery the inventors found that PBS-injected or Gp34_KbG injected haSyn PD mice had lost about 45% of substantia nigra neurons compared to animals injected with a non-pathogenic empty vector control (EV). Surprisingly, this neuron loss was attenuated by both low-dose (0.5pig / g) and high dose (2 pig / g) AIM Bioasyn in a dose-dependent manner (Fig. 4). Furthermore, the inventors found that aSyn- loaded AIM Bio surrogate molecules prevent infiltration of CD11 b+myeloid cells into the striatum in PD model (Fig. 5), aSyn-loaded AIM Bio surrogate molecules reduce aSyn accumulation in Substantia Nigra in PD model (Fig. 6), and aSyn-loaded AIM Bio protect Tyrosine Hydroxylase - fibers in the striatum (Fig. 7).

[0298] Example 2: aSyn-specific MHC class l-based Therapeutics for the Treatment of PD in Human Patients

[0299] The inventors' findings show that single-chain proteins containing an aSyn peptide antigen and an HLA-G alpha 3 domain can largely prevent neuron death in an animal model for Parkinson's disease, i.e. that they can be used for the treatment of PD. Based on these findings, the inventors have generated aSyn-peptide and MHC class I fusion molecules for therapeutic use in human PD patients. Target sequence (human alpha-Synuclein):

[0300] MDVFMKGLSK AKEGWAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGWH GVATVAEKTK

[0301] EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP

[0302] DNEAYEMPSE EGYQDYEPEA (SEQ ID NO: 19)

[0303] Exemplary peptides:

[0304] GAPQEGIL (SEQ ID NO: 20) MPVDPDNEAY (SEQ ID NO: 21) MDVFMKGLSK (SEQ ID NO: 22) DVFMKGLSK (SEQ ID NO: 23) VFMKGLSKAK (SEQ ID NO: 24)

[0305] GWAAAEKTK (SEQ ID NO: 25)

[0306] WAAAEKTK (SEQ ID NO: 26)

[0307] GVAEAAGKTK (SEQ ID NO: 27)

[0308] KTKEGVLYV (SEQ ID NO: 28)

[0309] GVLYVGSKTK (SEQ ID NO: 29)

[0310] VLYVGSKTK (SEQ ID NO: 30)

[0311] GWHGVTTV (SEQ ID NO: 31) (based on A53T mutant)

[0312] GVTTVAEKTK (SEQ ID NO: 32) (based on A53T mutant)

[0313] AWTGVTAV (SEQ ID NO: 33)

[0314] WTGVTAVA (SEQ ID NO: 34)

[0315] VTGVTAVAQK (SEQ ID NO: 35)

[0316] GSIAAATGF (SEQ ID NO: 36)

[0317] SIAAATGFV (SEQ ID NO: 37)

[0318] SIAAATGFVK (SEQ ID NO: 38)

[0319] IAAATGFVK (SEQ ID NO: 39)

[0320] GSIAAATGFVK (SEQ ID NO: 40)

[0321] AAATGFVKK (SEQ ID NO: 41)

[0322] AAATGFVKKD (SEQ ID NO: 42)

[0323] AATGFVKK (SEQ ID NO: 43)

[0324] PVDPDNEAY (SEQ ID NO: 44)

[0325] MPSEEGYQDY (SEQ ID NO: 45) PSEEGYQDY (SEQ ID NO: 46)

[0326] SIAAATGFV (SEQ ID NO: 37) +HLA-A2 presenting domains

[0327] KTKEGVLYV (SEQ ID NO: 28) +HLA-A2 presenting domains

[0328] AWTGVTAV (SEQ ID NO: 33) +HLA-A2 presenting domains

[0329] GWHGVTTV (SEQ ID NO: 31) +HLA-A2 presenting domains

[0330] WTGVTAVA (SEQ ID NO: 34) +HLA-A2 presenting domains

[0331] GSIAAATGFVK (SEQ ID NO: 40) +HLA-A11 presenting domains

[0332] AAATGFVKK (SEQ ID NO: 41) +HLA-A11 presenting domains

[0333] VLYVGSKTK (SEQ ID NO: 30) +HLA-A11 presenting domains

[0334] MPVDPDNEAY (SEQ ID NO: 21) +HLA-A1 presenting domains

[0335] PVDPDNEAY (SEQ ID NO: 44) +HLA-A1 presenting domains

[0336] WAAAEKTK (SEQ ID NO: 26) +HLA-A11 presenting domains

[0337] VFMKGLSKAK (SEQ ID NO: 24) +HLA-A11 presenting domains

[0338] AAATGFVKKD (SEQ ID NO: 42) +HLA-A11 presenting domains

[0339] AATGFVKK (SEQ ID NO: 43) +HLA-A11 presenting domains

[0340] MDVFMKGLSK (SEQ ID NO: 22) +HLA-A11 presenting domains

[0341] GWAAAEKTK (SEQ ID NO: 25) +HLA-A11 presenting domains

[0342] GAPQEGIL (SEQ ID NO: 20) +HLA-G presenting domains

[0343] The above-indicated most preferred and preferred peptides are preferable, because the inventors found that based on online prediction algorithms (Syfpeithi, NetMHC), literature review with regards to immunodominant regions, 3D protein folding predictions (AlphaFold2) and protein expression data as well as the experimental results reported in the present specification, these epitopes are highly likely to induce tolerogenic effects in Parkinson patients.

[0344] Example 3: Methods used in the invention

[0345] Methods for producing recombinant polypeptides of the invention

[0346] Expi-293F cells (Thermo Fisher), grown in Expi-293™ expression medium (Thermo Fisher): transfection of 1 pig DNA into 2.5x106cells / ml using the Expifectamine™ 293 Transfection kit (Thermo Fisher) using Opti-MEM (Thermo Fisher) for complexation of DNA with Expifectamine, after 18-20 h, addition of enhancer according to the protocol, harvesting of the supernatant after 4-6 days (37°C, 8% CO2, humidified incubator), 19 mm2orbital shaker 125 rpm

[0347] Spot-tag protein purification: equilibration of Spot-Cap resin: transfer of desired slurry amount into an appropriate tube, sediment beads by centrifugation (4°C, 4 min, 2500 g), remove & discard supernatant, add 10 bed volumes PBS (cold) to beads, invert to mix, sediment beads by centrifugation (4°C, 4 min, 2500 g), remove & discard supernatant, repeat 2 times

[0348] Add required volume beads to supernatant, incubate ON, 4°C on a rotator, wash beads by repeated centrifugation (4°C, 4 min, 2500 g), and removal of supernatant

[0349] Prepare a 500 piM Spot-peptide solution in PBS, remove the supernatant, incubate with 1 / 3rd of the spot-peptide solution for 5-10 min

[0350] Sediment beads by centrifugation. Use Amicon Ultra-4 centrifugal filters (15 kDa cutoff) for Protein concentration and spot-peptide removal with 15 kDa Amicon cutoff columns

[0351] Rinse the Amicon Ultra-4 centrifugal filters (15 kDa cutoff) with PBS followed by 0.1 N NaOH (centrifugation at 4000 g, 4°C) to remove trace amounts of glycerine.

[0352] ELISPOT:

[0353] 1) Cell culture

[0354] A) PBMC isolation (under a laminar flow hood)

[0355] To isolate peripheral blood mononuclear cells (PBMC), a density centrifugation was performed with white blood cells from a leukocyte reduction chamber and density gradient medium (e.g. Ficoll, or ROTI Sep 1077). Cells were centrifuged for 20 min at 1200 x g without brake followed by collection of the interphase ring that was washed with 1x PBS (5 min, 300 x g). PBMC were frozen till further use.

[0356] B) PBMC pulsing (under a laminar flow hood)

[0357] PBMCs were thawed 1 day prior to PBMC pulsing (d-1) and kept overnight in 5 ml X-VIV0 15 medium containing 5% human AB serum in a well of a 6 well plate at 37°C.

[0358] On the next day (dO) cells were counted and resuspended in X-VIVO 15 complete medium (5% hAB serum & cytokine cocktail: 20 ng / ml hlL-2, 20 ng / ml hGM-CSF, 10 ng / ml hlL-4 & 10 ng / ml hTGF-b1) at a cell density of 3x106cells / ml.

[0359] For experiments, 3x106cells were seeded in the respective wells of a 12-well plate with a final volume of 1000 pl X-VIVO complete medium with cytokine cocktail and

[0360] 5 pjg / ml of an AIM Bio molecule (recombinant polypeptide of the invention) or the respective controls. On day 3, 1 ml complete medium (with cytokines) was added, on day 6, a second pulse with 5 pg / ml of a recombinant polypeptide of the invention or a surrogate thereof (collectively referred to as "AIM Bio” molecule) was performed (after removing medium). On days 7, 10 & 12, 1 ml complete medium (with cytokines) was added.

[0361] Required:

[0362] X-VIVO 15 medium + 5% human AB serum

[0363] X-VIV0 15 complete medium: X-VIVO 15 medium +2% human AB serum supplemented with cytokine cocktail: 10 ng / ml TGF-b1 , 10 ng / ml IL-4, 20 ng / ml IL-2, 20 ng / ml GM-CSF

[0364] 6 well plate

[0365] 12 well plate

[0366] 2) ELISPOT

[0367] Laminar flow hood

[0368] On day 13, ELISPOT plates were coated using anti-hlLW (clone 9D-7, 1:500 dilution in PBS, sterile filtered) and alL10 (10G8-biotin) and on day 14, 200,000 cells were seeded per well on the ELISPOT plates in duplicates, including negative controls (cells plus PBS) and a positive control (e.g. LPS).

[0369] The PFDF membrane was activated with 50 pl / well EtOH (35% v / v) for 1 min followed by 5x washing with 200 pl distilled sterile water. Plate was coated with 100 pl / well antibody solution at 4°C overnight. On the next day, unbound coating antibody was removed, 5 washing steps were performed with 200 pl PBS and 200 pl blocking buffer (X-VIVO 15 5% hAB serum) was added and the plate incubated for 30 min - 2 h at room temperature.

[0370] The respective antigenic peptide in DMSO or DMSO as a control were prepared, and a final amount of 5 pg peptide / ml was added to the final volume of 100 pl / well. 150,000 cells were seeded per well in X-VIVO 15 medium with 5% human AB serum. Blocking buffer (X VIVO 15 medum + 5% hAB serum) was carefully removed, and medium with PBS as negative control and stimulants (5 pg / ml total volume in each well) were added to the other wells and incubated at 37°C overnight.

[0371] Outside the laminar flow hood

[0372] Secondary antibody was prepared: 1 pg / ml alL-10-biotinylated antibody in 0.5% BSA / 1x PBS (1 :1000 dilution) and horseradish peroxidase-conjugated streptavidin (1 :750 in 0.5% BSA / PBS), tetramethylbenzidine solution was filtered using a 0.45 pm filter and stored at 4°C till use.

[0373] Cell supernatant was removed and 5x washed using 100 pl PBS. Last excess buffer was removed using paper towels. 25 l diluted HRP-streptavidin (1 :750) was added per well and incubated for 1 h at room temperature in the dark followed by 5 washing steps using sterile 1xPBS.

[0374] 100 pl of filtered TMB substrate was added per well for 15-25 min till blue sports developed. Reaction was stopped by washing the wells thoroughly with tapped water.

[0375] Plastic underdrains of the plates was removed and the bottom and sides of the plates were washed with tap water and dried.

[0376] Plates were read out using an ImmunoSpot S6 Ultra-V Analyzer (Cellular Technology Limited), analysed in Excel and graphs / statistics were done in Graphad Prism.

[0377] Required:

[0378] Capture antibodies: anti-hlL10 (Clone: 9D-7, Mabtech #3430-3-250; 1 :500 dilution), anti-hlL10-biotinylated (Mabtech, #3430-6-250)

[0379] 1x PBS (sterile) 35% EtOH (v / v)

[0380] Blocking buffer: X-vivo 5% hAB serum (sterile) [blocking is done in the same medium as cell culture]

[0381] Dilution buffer: 0.5% BAS in PBS

[0382] Washing buffer: 1x PBS

[0383] Medium: for T cells, X-VIVO 15 medium (Lonza)

[0384] Filter syringe: Millex GV

[0385] ELISPOT PVDF plate (#MSIP4510, Millipore)

[0386] TMB substrate

[0387] Example 4: Further proof-of-principle of stability and effects of the recombinant polypeptides of the invention. Additionally, the inventors set out to obtain and test recombinant polypeptides having the general structure of the recombinant polypeptides of the invention but containing various different peptide antigens, in order to obtain further proof-of-principle that recombinant polypeptides of the invention and surrogates thereof are stable and efficacious. As shown in Figures 9 and 10, respectively, the tested recombinant polypeptides are stable during freeze-thawing and storage and are thermally stable. Further, they induce Treg in a dose-dependent manner (Figure 11) and inhibit T cell lysis in a dose-dependent manner (Figure 12). Effects of the recombinant polypeptides on the serum cytokine profile in EAE-ODC Ova mice are shown in Figure 13. There is an induction of IL-10 and possibly IL-4, both known to be immunosuppressive cytokines downregulating immune responses in inflammatory settings. This requires an HLA-G alpha3 domain plus a cognate peptide. IL-2 seems to be induced in response to presenting the cells with a cognate peptide that is irrespective of the alpha3 domain. IL-

[0388] 2 is needed for T cell activation and survival.

[0389] Further proof for stability of recombinant polypeptides of the invention is also provided in Figures 15 and 16.

[0390] Example 5: ELISpot results from healthy donor PBMCs (Figure 17), and IL-10 ELISpot results from Parkinson patients (Figure 18 A) and healthy / age matched individuals (Figure 18 B). These experiments show that a significant induction of immunosuppressive cells in response to the constructs of the invention can be observed in vitro already after 14 days both in healthy donor PBMCs and patient PBMCs.

[0391] Example 6:

[0392] Expression and testing of polypeptides ("AIM Bio(s)”) having a polypeptide structure as defined in claim 1 , and containing peptide antigens according to the invention or surrogate antigens thereof.

[0393] Sandwich ELISA Protocol

[0394] Nunc 96-well microtiter plates (Thermo Fisher, 442404) were coated overnight with 50 pl 1 pg / ml of anti-HLA- G (2A12) capture antibody (MAI-10358, Thermo Fisher). The plates were washed 3 times with 200 pl PBS / 0.05% Tween 20 (PBS / T) and blocked for 1 h hour with 150 pl 1 % BSA in PBS (blocking buffer) for 2 h at RT followed by 3x washing with 200 pl PBS / T. Expi293F expression cultures were diluted (1 :10) with blocking buffer and tested in duplicates. Biotinylated anti-p2 microglobulin antibody (MA1 -19506, Thermo Fisher) was diluted in blocking buffer (1 : 1000) and 100 pl were added to the each well for 1 hour. After 3 washing steps with 200 pl PBS / T, binding of the biotinylated anti-p2 microglobulin detection antibody was visualized by adding 100 pl horseradish peroxidase (HRP) conjugated to streptavidin (405210, Biolegend; 1 :100 dilution in blocking buffer) for 20 minutes and 3x washing with 200 pl PBS / T, followed by 100 pl TMB substrate solution (BioLegend) for 10-15 minutes. The reaction was stopped with 100 pl of 1 N H2SO4. Absorbance was measured at 450 nm and a correction at 620 nm using a Tecan Sunrise™ ELISA plate reader and results were evaluated using Magellan™ software. Transfection protocol

[0395] Expi-293F cells (Thermo Fisher) grown in Expi-293™ expression medium (Thermo Fisher) and transfections were performed using the Expifectamine™ 293 Transfection kit (Thermo Fisher). Transfections were performed in 50 ml round bottom tubes (TPP, 91056) using the filter-caps of 50 ml bioreactor tubes (TPP, 87050). Expi293F cells were counted and transfected with a density of 4.5-57x106viable cells / ml (viability >99%).

[0396] For a ca. 12 ml final culture volume, 3x107viable cells were diluted in 10 ml Expi293™ Expression medium containing GlutaMAX (gibco, A14351 -01). 10 pig DNA / ml total transfection volume was diluted into 600 pl Opti- MEM (Thermo Fisher, 31985-047). 30 pl ExpifectamineTM293 reagent was diluted into 570 pl Opti-MEM or Opti-Plex complexation buffer (Thermo Fisher). After 3-5 min, DNA was added to the Expifectamine-293™ / Opti-MEM™ transfection mix and incubated for 15-20 min at RT for forming of DNA-Expifectamine-293F™ reagent complexes. Then, 1200 pl DNA-Expifectamine-293™ reagent complex was added dropwise to each 50 ml bioreactor containing 30 mio cells in 10 ml medium. Cells were incubated at 37°C, 8% CO2 in a humidified incubator on an orbital shaker, 19 mm2diameter, 225 rpm.

[0397] After 18-20 h incubation, cells were enhanced by dropwise addition of 60 pl enhancer I and 600 pl enhancer II (ExpiFectamine™293 Transfection Enhancer 1 +2, Cat. A14524, part of the ExpiFectamine™ 293 Transfection Kit) while shaking the bioreactor. The supernatants were harvested on day 5 by spinning down the cells at 300 g for 5 min at room temperature followed by 20 min centrifugation with 4000 g at 4°C.

[0398] Simple Western protocol

[0399] Jess simple Western runs were performed using the EZ Standard Pack 1 (bio-techne, Ref PS-ST01 E7) reagents under non-reducing conditions using 12-230 kDa cartridges (bio-techne, Ref SM W004-1), 2A12oHLA-G antibody (Thermo Fisher, MAI-10358, 1 :50 in bio-techne antibody diluent 2 part of the anti-mouse detection kit) as primary antibody and anti-mouse detection kit (bio-techne Ref DM-002). Supernatants from transfected Expi293F cells were pre-diluted 1 :10 with 1xPBS followed by another 1 : 10 dilution with O.lx sample buffer. After a further 1 :5 dilution with the 5x fluorescence master mix, 3 pl were subjected to the single cartridge well along with the molecular weight marker. 5 pl of the protein ladder (EZ Standard Pack 1) were used for molecular weight determination in one well of the first row. In the next row, 10 pl antibody diluent 2 (bio-techne, part of anti-mouse detection kit) were loaded per well as blocking reagent, in the 3rd row, 10 pl of the 1 :50 diluted primary antibody 2A12oHLA-G, in the 4th row, 10 pl mouse-secondary antibody from the anti-mouse detection kit, in the 5th row, 15 pl luminol-peroxide mix and in the last 3 rows, 500 pl wash buffer per compartment. Plate was centrifuged for 5 min at 1000 g at RT to remove air bubbles and the remaining cover foil was removed. Then, cartridge and plate were loaded into the Jess system (bio-techne) using Compass 6.2.0 software and machine was started.

[0400] Separation time was 25 min at 375 V followed by 5 min blocking time with antibody diluent (bio-techne), 30 min primary antibody incubation and 30 min secondary antibody incubation followed by detection.

[0401] ELISpot protocol

[0402] ELISpot analysis was performed according to the following protocol:

[0403] Phase 1: PBMC:AIM Bio culture

[0404] Reagents

[0405] • Human PBMC from patients with NMOSD {neuromyelitis optica spectrum disease)

[0406] • AIM Bios: AQP64_G, AQP65P65K_G

[0407] • Complete culture medium: X-vivo 15 (Lonza, Biozym 881026) supplemented with 5% human AB serum and human cytokines

[0408] -rhlL-2 (20ng / ml); Immunotools, 11340027

[0409] -rhGM-CSF (20ng / ml); Immunotools, 11343128

[0410] -rhlL-4 (10ng / ml); Immunotools, 11340047

[0411] -rhTGFp (10ng / ml); Immunotools, 11343160

[0412] • 24-well culture plates

[0413] • PBS (sterile)

[0414] Thawing the PBMCs (Day -1)

[0415] • Thaw the cryo-preserved PBMC by swirling in the water bath for around 20-30s

[0416] • Transfer the cells immediately into 15ml falcons filled with PBS and centrifuge: 300g, 5min, RT

[0417] • Wash the cell 2 more times with PBS

[0418] • Re-suspend the cells in 5 ml X-vivo 15 medium supplemented with 5% hAB serum and rest the cells overnight at 37°C in a 6-well culture plate

[0419] Treg Induction: 14d PBMC culture with AIM Bios (sterile conditions)

[0420] • Test conditions: o Negative control (No AIM Bio treatment / Untreated) o AQP64_G treatment (sequence AQP64_G:

[0421] MSRSVALAVLALLSLSGLEAKPLPVDMVLGCGASGGGGSGGGGS / QRPPK / QV'YSRHPA ENGKSNFLNCYYSGFHPSDIEYDLLKNGERIEKYEHSDLSFSKDWSFYLLYYFEFFPYEKD

[0422] EYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRXFSAAVS RPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAH AQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALN

[0423] EDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADP

[0424] PKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQK WAAWVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESGSLSEDLGSPDRV RAVSHWSSC*; SEQ ID NO: 59; note that the asterisk denotes the stop codon] o AQP65P65K_G treatment (sequence AQP65P65K:

[0425] MSRSVALAVLALLSLSGLEAKLPVDMVLGGGASGGGGSGGGGS / QRPPK / QV'YSRHPAE NGKSNFLNCYYSGFHPSDIEYDLLKNGERIEKYEHSDLSFSKDWSFYLLYYTEFTPTEKDE YACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGGGGSGSHSMRYFS

[0426] AAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRN TKAHAQTDRMNLQTLRGAYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDY LALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQ

[0427] RADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDG TFQKWAAVWPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESGSLSEDLGS PDRVRAVSHWSSC*; SEQ ID NO: 60; note that the asterisk denotes the stop codon)

[0428] • PBMC number: 1 .2x106in 400 l of complete culture medium

[0429] • Count the overnight rested cells and calculate the required number of cells for the assay.

[0430] • Wash the cells and resuspend them in complete culture medium at a final concentration of 3x106 / ml

[0431] • Add 400 pl (1.2x106cells) of cell suspension in each well in a 24-well plate

[0432] • Pulse the cells with 5 pg / ml AIM Bios AQP64_G and AQP65P65K_G (Day 0)

[0433] • Feed the cells with 400 pl of complete culture medium every 2 days.

[0434] • Pulse with 5 pg / ml AIM Bios AQP64_G and AQP65P65K_G on Day 6

[0435] • Seed the cells on ELISpot plate on day 14

[0436] Phase 2: ELISpot Assay

[0437] (EliSpot assay is performed according to the guidelines provided in human IL-10 ELISpot (Basic) kit (Mabtech;

[0438] 3430-2H)) Reagents

[0439] • ELISPOT PVDF plate (Millipore)

[0440] • Human IL-10 EliSpot basic Kit (Product code: 3430-2H)

[0441] • 35% Ethanol

[0442] • Blocking medium: X-vivo 5%hAB serum

[0443] • Dilution buffer. 0.5% BSA in PBS or 0.5% FCS in PBS

[0444] • EliSpot Medium: X-vivo 15 (Lonza, Biozym 881026) supplemented with 5% human AB serum and hlL-2 (20ng / ml)

[0445] • Streptavidin-HRP (Biolegend #405210; 1 :750 dilution) for detection

[0446] • ELISpot substrate: TMB for HRP (Mabtech Code: 3651-10)

[0447] • PBS (sterile)

[0448] Cell coating (sterile conditions)

[0449] • Activate PVDF wells with 25pl of Ethanol (35% v / v) for maximum 1 minute

[0450] • Wash the plate 5 times with sterile water (200 pl / well)

[0451] • Dilute coating antibody anti-hlL-10 (9D7) in sterile PBS to a final concentration of 2 pg / ml in sterile PBS. Add 50 pl of diluted antibody / well (Guidelines: 15 pig / ml; lOOpil / well)

[0452] • Incubate overnight at 4-8°C

[0453] Incubation of cells in the plate (under sterile conditions)

[0454] • Prepare a plate layout. Samples were tested in duplicates.

[0455] • Samples: o negative control: untreated cells (no stimulation) o Positive control: untreated cells + 1 pg / ml of O-CD3 / CD28 stimulation cocktail o Test samples: AQP64_G treated cells and AQP65P65K_G treated cells

[0456] • Count the PBMC from 14D culture. Calculate the required number of cells from every condition

[0457] • Minimum viability of the cells is >50%. Otherwise, discard the sample.

[0458] • Under the hood, remove excess coating antibody and wash the plate 5 times with 200 pl of sterile PBS

[0459] • Add 50 pl blocking medium per well. Incubate for 2h at room temperature

[0460] • Resuspend PBMC from every test condition at a final concentration of 1.5x106 / ml in Elispot medium

[0461] • Resuspend positive control cells at higher concentration and make up the volume with stimulation cocktail Under the hood, remove excess blocking buffer carefully

[0462] Add 100 l of PBMC (1 .5 x 105cells) suspension from each condition in respective wells

[0463] Incubate for 12-48h at 37°C.

[0464] Detection of Spots

[0465] • Dilute the biotinylated detection antibody (12G8-biotin) to a concentration of 1 pig / ml (1 :1000) in dilution buffer

[0466] • Remove the cell supernatant and wash the plate 5x in 200 l PBS. Remove last excess buffer by pressing onto paper towels.

[0467] • Add 50 l of detection antibody per well. Incubate overnight at 4° C.

[0468] • Wash 5x with 200 pl PBS. Remove excess liquid by pressing onto paper towels.

[0469] • Prepare HRP-conjugated streptavidin (1 :750 in dilution buffet)

[0470] • Add 50pl of streptavidin-HRP conjugate to each well. Incubate for 1 hour at room temperature.

[0471] • Wash 5x with 200 pl PBS. Remove excess liquid by pressing onto paper towels.

[0472] • Add 50pl of filtered TMB substrate per well and incubate for 10-20 minutes in dark until blue spots develop.

[0473] • Stop the reaction by washing wells with water (running tap water can be used).

[0474] • Remove the plastic underdrains of the plates and wash the bottom and sides of the plates well with tap water.

[0475] Analysis was performed using an S6 Universal M2 reader (Immunospot, Cleveland, Ohio).

[0476] Results:

[0477] Figure 19 shows that AIM Bios of this invention are expressed in Expi293F cells and that monomers (~50kDa) are detectable; Expression is furthermore confirmed and quantified in supernatants via ELISA. This suggests that these molecules can be successfully produced.

[0478] Figure 20: ELISpot with patient PBMCs. A: Pictures of wells in the assay, dublicates are always in a row. Numbers of the counted spots are indicated in the figure. B: Quantitation.

[0479] The ELISpot was carried out as described in Example 9, with the difference that only 1 .2 mio PBMC were used, cells were seeded in a 24 well plate, and all media volumes were reduced accordingly.

[0480] IL-10 spots were increased in 4 out of 5 NMO patient PBMC treated with AIM Bio AQP65P65K_G compared to untreated PBMC. This suggests that AIM Bios can induce protective T cells in patient cells. Further, by using surrogate constructs which are in accordance with the claims, except that they contain surrogate peptides, the inventors found that those constructs which can be expressed at good expression levels are functional, i.e., that they are able to induce antigen-specific immunological tolerance. In view of the good expression levels of the constructs of the invention, it is therefore expected that the constructs of the invention are functional and can be used to treat the medical indications defined by the claims.

[0481] Example 7:

[0482] Murine AIM Bios used for proof-of-concept experiments in the subsequent example:

[0483] Peptide sequence aSyn63 1: VTNVGGAW (SEQ ID NO: 86)

[0484] Protein sequence aSyn6371_H2-KbG yiNyGGAyyGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEK VEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGG SGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKG NEQSFRVDLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAAL ITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEII LTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO:

[0485] 87)J4_variable cleavage site and tag

[0486] Peptide sequence gp34'41: AVYNFATM (SEQ ID NO: 48)

[0487] Protein sequence gp34’41_H2-KbG:

[0488] AyYNFATMGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKV EHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGS GPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKG NEQSFRVDLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAAL ITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEII LTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO:

[0489] 88)_ I4_variable cleavage site and tag

[0490] Example 8:

[0491] Additional Proof-of-concept data in vivo:

[0492] A53T / A30P mice Protein aggregation is a key pathological event in Parkinson's disease pathology. A30P and A53T are two mutations in alpha-Synuclein associated with increased aggregation and an increased risk of PD development. Tg-hm2a-SYN-tg transgenic mice have these mutations and show a loss of SN neurons, terminals and motor deficits. The model has been described by Tiwari S, Singh A, Gupta P, Singh S. UBA52 Is Crucial in HSP90 Ubiquitylation and Neurodegenerative Signaling during Early Phase of Parkinson's Disease. Cells. 11 (23): 3770, 2022 and by Yan J, Zhang P, Jiao F, Wang Q, He F, Zhang Q, Zhang Z, Lv Z, Peng X, Cai H, Tian B. Quantitative proteomics in A30P*A53T o-synuclein transgenic mice reveals upregulation of Sell I. PLoS One. 12(8):e0182092, 2017.

[0493] Accelerating rotarod test

[0494] Tg-hm2a-SYN-tg mice were initially trained for two days prior to pre-operative measurements. They were placed on a rotarod (RotaRod Advanced, TSEsystems) with an accelerating speed from 5 rpm to a maximum of 50 rpm for the duration of 300 s. Each day of training consisted of five runs of 300 s each. Rotarod measurements were recorded in 3-4-month-old mice, which do not have symptoms after two-day training session. 9-10 mice per group were then treated with AIM Bios as follows: Experimental Groups

[0495] 1. Wildtype + PBS

[0496] 2. A53T / A30P + PBS

[0497] 3. A53T / A30P + 2 pg / g gp34'41_H2-KbG AIM Bio (control)

[0498] 4. A53T / A30P + 2 pg / g aSyn6371_H2-KbG AIMBio (mouse-adapted oSyn-specific AIM Bio)

[0499] At the age of 5-6 months, animals received 3 consecutive i.p. injections each 2 weeks apart.

[0500] The accelerating rotarod test was then repeated in 7-8 months old mice.

[0501] It was found that mice treated with aSyn6371_H2-KbG AIM Bios showed hardly any decline in rotarod performance compared to the healthy control (Figure 21).

[0502] In combination with the results of Figures 4-8, this experiment shows that different aSyn peptide antigens can induce immunotolerance in the recombinant polypeptides of the invention when combined with suitable antigen- presenting domains.

[0503] Example 9: Testing of exemplary human recombinant polypeptides according to the invention ("AIM Bios”): Candidate molecules tested in vitro with PBMC from healthy donors or patients with Parkinson's disease (PD): aSyn6977_A2G: best in healthy donors, 21 responders among 30 tested; PD: 2 responders among 9 PD patients, when used at 5 pig / ml II 4 responders among 8 PD patients (and 3 responders among 5 healthy donors) when used at 20 pig / ml aSyn7078_A2G: best in healthy donors, 20 among 29 tested; PD: 5 responders among 9 PD patients when used at 5 pig / ml II 2 responders among 5 PD when used at 20 pig / ml aSyn37 45_A11 G: in healthy donors, 14 responders among 29 tested; PD 7 responders among 9 PD samples when used at 5 pig / ml II 2 responders among 5 PD patients (and 1 responder among 2 healthy donors) when used at 20 pig / ml aSyn87 95_A2G: in healthy donors, 12 responders among 32 tested; PD: 3 responders among 9 PD patients when used at 5 pig / ml aSyn86-96_A11 G: in healthy donors, 18 responders among 24 tested; PD: 5 responders among 9 PD patients when used at 5 pig / ml aSyn47 55_A2G: in healthy donors tested 3 responders among 6 tested when used at 5 pig / ml, no PD tested aSyn32 41_A2G: in healthy donors, 9 responders among 12 tested when used at 5 pig / ml, no PD tested

[0504] Example 10: Characterization of recombinant polypeptides of the invention ("AIM Bios”)

[0505] Size Exclusion Chromatography (SEC) For protein characterization of purified proteins, a size exclusion chromatography run was performed at room temperature using a Superdex 200 increase 10 / 300 column on an Akta FPLC (Amersham Biosciences) and the Unicorn 5.31 software (GE Healthcare). Buffer was exchanged from 20% EtOH (storage solution) to 2 CV (column volumes) H2O prior to runs. For analysis, the column was rinsed with 2 CV PBS (Sigma, D8537-500 ml) at a flow rate of 0.5 ml / min. Thereafter, 10-20 pig AIM Bio (volume 500 pil) were subjected to a Superdex 200 increase 10 / 300 column (Cytiva) and column was run with a flow of 0.5 ml / min for 1 CV to collect the protein fractions. The UV curve was plotted against the time and peaks were analyzed.

[0506] The SEC analysis of the AIM Bio aSyn63-71_H2-KbG shows that the purified AIM Bio is a monomer protein (Figure 22).

[0507] Thermal Shift Assay

[0508] Thermal Shift of aSyn6371_H2-KbG analysis shows a Tm of 44.84°C (Figure 23).

[0509] For analysis of protein stability of the purified AIM Bios, a Thermal Shift Assay (TSA) was performed. For the TSA, 3 pig AIM Bio were dissolved in 25 pil PBS (Sigma, D8537-500 ml) containing SYPRO Orange dye (Merck, S5592, final dilution 1 :2000 in PBS). A melting curve was recorded in a StepOnePlus Instrument (ThermoFisher) by starting at 25°C and increasing the temperature by 1 °C per min to a final temperature of 95°C for 2 min. Melting curves were analysed using StepOnePlus 2.3 software. To determine the Tm, a Boltzmann sigmoidal curve fit was performed and the Tm was calculated accordingly.

[0510] Jess Simple Western Runs

[0511] Jess simple Western runs were performed using the EZ Standard Pack 1 (bio-techne, Ref PS-ST01 E7) reagents under non-reducing conditions using 12-230 kDa cartridges (bio-techne, Ref SM W004-1), 2A12oHLA-G antibody (Thermo Fisher, MAI-10358, 1 :50 in bio-techne antibody diluent 2 part of the anti-mouse detection kit) as primary antibody and anti-mouse detection kit (bio-techne Ref DM-002). Supernatants were prediluted 1 :10 and purified proteins were pre-diluted 1 :100 with 1xPBS followed by another 1 : 10 dilution with 0.1x sample buffer. After a further 1 :5 dilution with the 5x fluorescence master mix, 3 pi I were subjected to the single cartridge well along with the molecular weight marker. 5 pil of the protein ladder (EZ Standard Pack 1) were used for molecular weight determination in one well of the first row. In the next row, 10 pil antibody diluent 2 (bio-techne, part of anti-mouse detection kit) were loaded per well as blocking reagent, in the 3rd row, 10 pil of the 1 :50 diluted primary antibody 2A12DHLA-G, in the 4th row, 10 pl mouse-secondary antibody from the anti-mouse detection kit, in the 5th row, 15 pl luminol-peroxide mix and in the last 3 rows, 500 pl wash buffer per compartment. Plate was centrifuged for 5 min at 1000 g at RT to remove air bubbles and the remaining cover foil was removed. Then, cartridge and plate were loaded into the Jess system (bio-techne) using Compass 6.2.0 software and machine was started.

[0512] Separation time was 25 min at 375 V followed by 5 min blocking time with antibody diluent (bio-techne), 30 min primary antibody incubation and 30 min secondary antibody incubation followed by detection. aSyn6371_H2-KbG is a mainly monomeric protein (Figure 24).

[0513] Example 11 : Additional Functional Data with human constructs:

[0514] Methods

[0515] Cell culture for ELISpot Assay

[0516] For ELISpot assays, PBMC from PD patients were thawed and rested overnight in a 6-well-plate (Greiner, #657160). On the next day (dO), cells were counted and 1.5 mio cells / well were seeded in 12-well-plates (Greiner, 665180) at a density of 3x106cells / ml in X-VIVO 15 complete medium. (X-VIVO 15 medium (Lonza / Biozym #881026), 5% hAB Serum (HUABSE-0100, Bio&sell, Feucht, Germany), 20 ng / ml h-IL2 (Immunotools, Friesoythe, Germany, #11340027), 20 ng / ml h-GMCSF (Immunotools, #11343128), 10 ng / ml h- IL4 (Immunotool, #11340047) and 10 ng / ml h-TGF|31 (BioLegend, #781804)). Cells were stimulated with 20 pg / ml aSyn-specific AIM Biologicals on dO and d6. 500 pl additional X-VIVO 15 complete medium was added to the cells on d3, d6, d8, d10 and d13. 20 mM HEPES / PBS were added in control wells. On day 14, cells were counted and used for ELISpot assay and Dextramer staining.

[0517] ELISpot Assay

[0518] Seeding the cells

[0519] ELISpot plates (Millipore, MSIPS4510) were activated with 50 pl 35% EtOH per well for one minute, followed by 5 washing steps using 200 pl PBS / well. Plates were coated (overnight at 4°C) with anti-human IL-10 antibody (9D7, Mabtech, 3430-3-250) at a concentration of 2 pg / ml in PBS. On the next day, unbound antibody was removed and the plate was washed 5x with 200 pl PBS / well, followed by blocking for 2 h with 50 pl X-VIVO 15 medium containing 5% hAB Serum. The blocking medium was discarded. 100 pl of AIM Bio-treated and control cell suspensions were seeded on the plate (1.5x106cells / ml). ImmunoCult™ Human CD3 / CD28 T Cell Activator (25 pil / ml; Immunocult, #10971) was used as a positive stimulant. PBS was used as negative stimulant. The plate was incubated in a humified incubator with 5% CO2 for 48 h.

[0520] Development of IL-10 Spots

[0521] Cell suspension was discarded and the plate was washed 5 times with 200 l PBS. Biotinylated anti-IL10 detection antibody (12G8-biotin, Mabtech, 3430-6-250) was diluted to a concentration of 1 pig / ml in PBS containing 0.5% BSA. 50 l were added to each well and incubated for 2 h at room temperature or overnight at 4°C. The plate was washed 5 times with 200 pl PBS. 50 pl of Streptavidin-conjugated horse radish peroxidase (HRP) were added to each well and incubated for 1 hour. 50 pl / well TMB substrate solution (Mabtech, 3651 - 10) were added to each well until distinct spots emerged. Color development was stopped by extensive washing in deionized water. The underdrain was removed and the bottom side of the membrane was rinsed. The plate was allowed to dry and spots were counted using an ELISpot reader (Immunospot CTL machine and Immunospot SC Suite, CTL). Data were analyzed using Excel 365 (Microsoft) and graphs were generated using Prism 10.2 (GraphPad).

[0522] Figure 25 shows IL-10 secreting cells detected in ELISpot assay. As shown in the figure, AIM_aSyn7078_A2G stimulated IL10 secretion in healthy and Parkinson's patient PBMC. IL-10 induction is stronger with PBMC from HLA-A2+donors.

[0523] Characterization of CD8+regulatory T cells in HLA-A2+and HLA-A11+donors

[0524] Age-matched PBMC samples from 8 PD patients and 5 healthy controls were collected at the University Hospital Wurzburg. Patients gave informed consent and experiments were approved by the local ethics committee (vote no. AZ 54 / 15). 6 donors were haplotyped as HLA-A2 positive (4 of PD and 2 control each). Cells were stimulated for 14 days with two oSynuclein (aSyn)-specific AIM Biologicals, namely aSyn6977_A2G and aSyn7078_A2G, following the protocol described above for the ELISpot assays. PBS was used as negative stimulation control. Then, the cells were washed, counted, and further cultured in X-VIVO15 complete medium. Peptide-loaded, PE-labeled dextramers (HLA-A2 dextramer loaded with the aSyn6977(AWTGVTAV; SEQ ID NO: 33) and aSyn7078(WTGVTAVA; SEQ ID NO: 34) were prepared 48 hours in advance using a modified Immudex protocol, utilizing one-fourth of the standard reagent volumes. The cells were then treated with Brefeldin A at a 1 :1000 dilution to inhibit protein secretion, followed by incubation with the prepared dextramers for 15 minutes at room temperature. Surface staining was performed using BioLegend antibodies specific to CD3 (APC-Cy7, clone HIT3a), CD103 (FITC, clone Ber-ACT8), and CD8 (BV510, clone HIT8) at a 1 :50 dilution. For haplotyping, HLA-A2 antibody (PE, BB7.2, Biolegend) was used. This surface staining was carried out for 1 hour at 4°C in the dark, followed by washing steps to remove any unbound antibodies.

[0525] For intracellular staining, the cells were first fixed for 20 minutes and then permeabilized using the FIX / PERM kit buffer according to the manufacturer's instructions (Thermo Fisher). The permeabilized cells were incubated with antibodies targeting IL-10 (BV421 , clone JES3 / 9D7, BioLegend), IFN-y (PE-Cy7, clone B27, BioLegend), FoxP3 (APC, from Miltenyi Biotec), and Granzyme B (PE Vio770, clone Qa16A02, BioLegend at a 1 :75 dilution in the permeabilization buffer. This incubation was conducted at room temperature in the dark for 30 minutes. After staining, the cells were washed, resuspended in FACS buffer, and analyzed using the Attune NxT Flow Cytometer Autosampler (Thermo Fisher Scientific). The flow cytometry results were transferred to Microsoft Excel and analyzed using GraphPad Prism version 9 for visualization. Statistical analysis was performed using a Chi-Square test with a mixed-effect model, comparing the results to the PBS control.

[0526] Figure 26 shows CD8+T cell subpopulations in healthy controls and Parkinson's patients. Two x oSyn-specific AIM Bios (AIM_aSyn6977_A2G and AIM_aSyn7078_A2G) stimulated CD8+T cell responses in PBMC from Parkinson patients. The panels compare four CD8+T cell subpopulations, including CD8"CD103+, CD8+DexLowCD103+, and CD8+IL-10+cells, in HLA-A2+(middle row), individuals. The subpopulations were incubated with AIM Bios for 14 days, before being analyzed for expression of CD 103 and IL-10. PBMC from patients with Parkinson's disease showed induction of IL-10 and CD103. P-values are indicated where appropriate. Please note that low cell numbers precluded stimulation of healthy donor PBMC with AIM_aSyn70_A2G.

[0527] Co-culture with AIM aSyn69'77_A2G increased the proportion of CD8-CD103+, CD8-IL-10+, CD8+dexlowCD103+, and CD8+dexlowIL-10+populations in 7 donors (3 healthy and 4 PD patients). No induction of FoxP3+, IFNy+, and Granzyme B+cells was observed in this assay. Co-culture with AIM aSyn7078_A2G resulted in a lesser increase in CD8+DexlowCD103+cells. AIM aSyn6977_A2G induced IL10 secretion in 6 donors (2 healthy and 4 PD patients). IL10 induction was stronger in HLA-A2+donors compared to HLA-A2- donors.

[0528] Conclusion:

[0529] AIM aSyn6977_A2G induces regulatory CD8+T cells in PBMC.

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[0532] Bas J, Calopa M, Mestre M, Mollevi DG, Cutillas B, Ambrosio S, Buendia E (2001 ), Lymphocyte populations in Parkinson's disease and in rat models of parkinsonism. J Neuroimmunol 113:146-152.

[0533] Brochard V, Combadiere B, Prigent A, Laouar Y, Perrin A, Beray-Berthat V, Bonduelle O, Alvarez-Fischer D, et al. (2009), Infiltration of CD4+ lymphocytes into the brain contributes to neurodegeneration in a mouse model of Parkinson disease. J Clin Invest 119:182-192.

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[0535] Lacan G, Dang H, Middleton B, Horwitz MA, Tian J, Melega WP, Kaufman DL (2013), Bacillus Calmette-Guerin vaccine-mediated neuroprotection is associated with regulatory T-cell induction in the 1 -methyl-4-phenyl-1 ,2,3,6-tetrahydropyridine mouse model of Parkinson's disease. J Neurosci Res 91 :1292-1302.

[0536] Liu J, Gong N, Huang X, Reynolds AD, Mosley RL, Gendelman HE (2009), Neuromodulatory activities of CD4+CD25+ regulatory T cells in a murine model of HIV-1 -associated neurodegeneration. J Immunol 182:3855-3865.

[0537] Magistrelli L, Comi C (2020), Beta2-Adrenoceptor Agonists in Parkinson's Disease and Other Synucleinopathies. J Neuroimmune Pharmacol 15:74-81. McGeer PL, Itagaki S, Boyes BE, McGeer EG (1988), Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains. Neurology 38:1285-1291.

[0538] Mogi M, Harada M, Kondo T, Riederer P, Inagaki H, Minami M, Nagatsu T (1994), lnterleukin-1 beta, interleukin-6, epidermal growth factor and transforming growth factor-alpha are elevated in the brain from parkinsonian patients. Neurosci Lett 180:147-150.

[0539] Mogi M, Harada M, Riederer P, Narabayashi H, Fujita K, Nagatsu T (1994), Tumor necrosis factor-alpha (TNF-alpha) increases both in the brain and in the cerebrospinal fluid from parkinsonian patients. Neurosci Lett 165:208-210.

[0540] Musacchio T, Rebenstorff M, Fluri F, Brotchie JM, Volkmann J, Koprich JB, Ip CW (2017), Subthalamic nucleus deep brain stimulation is neuroprotective in the A53T alpha-synuclein Parkinson's disease rat model. Ann Neurol 81 :825-836.

[0541] Peter I, Dubinsky M, Bressman S, Park A, Lu C, Chen N, Wang A (2018), AntiTumor Necrosis Factor Therapy and Incidence of Parkinson Disease Among Patients With Inflammatory Bowel Disease. JAMA Neurol 75:939-946.

[0542] Racette BA, Gross A, Vouri SM, Camacho-Soto A, Willis AW, Searles Nielsen S (2018), Immunosuppressants and risk of Parkinson disease. Ann Clin Transl Neurol 5:870-875.

[0543] Reynolds AD, Banerjee R, Liu J, Gendelman HE, Mosley RL (2007), Neuroprotective activities of CD4+CD25+ regulatory T cells in an animal model of Parkinson's disease. J Leukoc Biol 82:1083-1094.

[0544] Reynolds AD, Stone DK, Hutter JA, Benner EJ, Mosley RL, Gendelman HE (2010), Regulatory T cells attenuate Th17 cell-mediated nigrostriatal dopaminergic neurodegeneration in a model of Parkinson's disease. J Immunol 184:2261-2271. Rosenkranz D, Weyer S, Tolosa E, Gaenslen A, Berg D, Leyhe T, Gasser T, Stoltze L (2007), Higher frequency of regulatory T cells in the elderly and increased suppressive activity in neurodegeneration. J Neuroimmunol 188:117-127.

[0545] Saunders JA, Estes KA, Kosloski LM, Allen HE, Dempsey KM, Torres-Russotto DR, Meza JL, Santamaria PM, et al. (2012), CD4+ regulatory and effector / memory T cell subsets profile motor dysfunction in Parkinson's disease. J Neuroimmune Pharmacol 7:927-938.

[0546] Storelli E, Cassina N, Rasini E, Marino F, Cosentino M (2019), Do Th17 Lymphocytes and IL-17 Contribute to Parkinson's Disease? A Systematic Review of Available Evidence. Front Neurol 10: 13.

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[0548] INDUSTRIAL APPLICABILITY

[0549] The pharmaceutical compositions, polypeptides, nucleic acids, cells, and products for use in the invention are industrially applicable. For example, they can be used in the manufacture of, or as, pharmaceutical products.

Claims

CLAIMS1. A recombinant polypeptide capable of presenting a peptide antigen, the recombinant polypeptide comprising, in an N- to C-terminal order, i) a peptide antigen presented by said recombinant polypeptide, wherein the peptide antigen is a peptide of human alpha-Synuclein; ii) optionally a linker sequence; iii) a sequence of a human polypeptide domain comprising a sequence of a human p2 microglobulin or an amino acid sequence at least 90% identical to the amino acid sequence of human p2 microglobulin represented by SEQ ID NO: 5; iv) optionally a linker sequence; v) an [alpha] 1 domain of an MHO molecule; vi) an [alpha] 2 domain of an MHO molecule; vii) an [alpha] 3 domain of an MHO class lb molecule or a derivative of an [alpha] 3 domain of an MHO class lb molecule, said derivative being capable of binding to ILT2 or ILT4; viii) optionally a protease cleavage site; ix) optionally a spacer sequence; and x) optionally an affinity tag.

2. The recombinant polypeptide according to claim 1 , wherein said peptide antigen according to i) is 7 to 11 amino acids in length, preferably 8-11 amino acids in length.

3. The recombinant polypeptide according to claim 1 or 2, wherein said peptide antigen according to i) consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 28, 30, 31 , 33, 34, 37, 40 and 50.

4. The recombinant polypeptide according to claim 1 or 2, wherein said peptide antigen according to i) consists of the amino acid sequence of SEQ ID NO: 30.

5. The recombinant polypeptide according to claim 1 or 2, wherein said peptide antigen according to i) consists of the amino acid sequence of SEQ ID NO: 33.

6. The recombinant polypeptide according to claim 1 or 2, wherein said peptide antigen according to i) consists of the amino acid sequence of SEQ ID NO: 34.

7. The recombinant polypeptide according to claim 1 or 2, wherein said peptide antigen according to i) consists of the amino acid sequence of SEQ ID NO: 37.

8. The recombinant polypeptide according to claim 1 or 2, wherein said peptide antigen according to i) consists of the amino acid sequence of SEQ ID NO: 40.

9. The recombinant polypeptide according to claim 1 or 2, wherein said peptide antigen according to I) consists of the amino acid sequence of SEQ ID NO: 50.

10. The recombinant polypeptide according to any one of the preceding claims, wherein said [alpha]1 domain according to (v) and said [alpha]2 domain according to (vi) are from a human MHO class la molecule or from a human MHO class lb molecule.11 . The recombinant polypeptide according to claim 10, wherein said [alpha] 1 domain according to (v) and said [alpha]2 domain according to (vi) are from a human MHC class lb molecule, and wherein the human MHC class lb molecule is a human HLA-G molecule.

12. The recombinant polypeptide according to claim 10, wherein said [alpha] 1 domain according to (v) and said [alpha]2 domain according to (vi) are from a human MHC class la molecule, and wherein said [alpha]1 domain according to (v) and said [alpha]2 domain according to (vi) are from a human HLA-A2 molecule, a human HLA-A1 molecule, or a human HLA-A11 molecule.

13. The recombinant polypeptide according to any one of the preceding claims, wherein the [alpha] 3 domain of the MHC class lb molecule according to (vii) is an [alpha] 3 domain of human HLA-E, human HLA-F or human HLA-G, preferably human HLA-G.

14. The recombinant polypeptide according to any one of the preceding claims, wherein the [alpha]3 domain or derivative according to (vii) is identical to or has at least 80% amino acid sequence identity, at least 90% amino acid sequence identity, at least 92% amino acid sequence identity, at least 94% amino acid sequence identity, at least 96% amino acid sequence identity, at least 98% amino acid sequence, or at least 99% amino acid sequence identity with the [alph a]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 2 or SEQ ID NO: 51 or is identical to the [alpha]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 2 or SEQ ID NO: 51 .

15. The recombinant polypeptide according to any one of claims 1-14, wherein the [alpha]3 domain or derivative according to (vii) is identical to the [alpha]3 domain having the amino acid sequence of SEQ ID NO: 9.

16. The recombinant polypeptide according to any one of claims 1-14, wherein the [alpha]3 domain or derivative according to (vii) is identical to the [alpha]3 domain having the amino acid sequence of SEQ ID NO: 2.

17. The recombinant polypeptide according to any one of claims 1-14, wherein the [alpha]3 domain or derivative according to (vii) is identical to the [alpha]3 domain having the amino acid sequence of SEQ ID NO: 51.

18. The recombinant polypeptide according to any one of the preceding claims, wherein the linker sequence according to (ii) and / or the linker sequence according to (iv) comprises the amino acid sequence (GGGGS)n, wherein n is an integer equal to or higher than 1 , and wherein n is preferably an integer selected from the group consisting of 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10 and is preferably selected from the group consisting of 2, 3, 4 and 5.

19. The recombinant polypeptide according to any one of the preceding claims, wherein said sequence of a human polypeptide domain according to (ill) is at least 95% identical to the amino acid sequence of SEQ ID NO: 5, preferably at least 98% identical to the amino acid sequence of SEQ ID NO: 5 and more preferably identical to the amino acid sequence of SEQ ID NO: 5.

20. The recombinant polypeptide according to any one of the preceding claims, wherein said polypeptide is dimeric or multimeric.

21. The recombinant polypeptide according to any one of the preceding claims, wherein the polypeptide comprises or consists of all of the components I) to vii), does not comprise components viii) to x), or comprises or consists of all of the components I) to x).

22. The recombinant polypeptide according to any one of the preceding claims, further comprising an N- terminal secretion signal peptide sequence.

23. The recombinant polypeptide according to any one of claims 1-21 , wherein the recombinant polypeptide consists of an amino acid sequence consisting of the following ((a) and (b)) in an N- to C-terminal order:(a) a peptide antigen consisting of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 28, 30, 31 , 33, 34, 37, 40 and 50; and(b) the amino acid sequence of SEQ ID NO: 16 or SEQ ID NOs: 53-54.

24. The recombinant polypeptide according to any one of the preceding claims, wherein the recombinant polypeptide is soluble.

25. A nucleic acid encoding one or more polypeptides according to any one of the preceding claims, wherein the nucleic acid is preferably a vector.

26. A pharmaceutical composition or kit comprising at least one nucleic acid according to claim 25.

27. A pharmaceutical composition or kit comprising at least one recombinant polypeptide according to any one of claims 1-24.

28. The pharmaceutical composition or kit according to claim 27, wherein the pharmaceutical composition or kit comprises at least two different recombinant polypeptides according to any one of claims 1-24, and wherein each of the different polypeptides comprises a different peptide antigen as defined in claim 3.

29. A pharmaceutical composition or kit according to any one of claims 26-28, for use in the treatment of Parkinson's disease in a human patient.

30. The pharmaceutical composition or kit for use according to claim 29, wherein the Parkinson's disease is prodromal Parkinson's disease.

31. The pharmaceutical composition or kit for use according to claim 29 or 30, wherein the treatment is treatment of Parkinson's disease by immunotherapy, and wherein the treatment is preferably by inducing immunological tolerance against autoantigenic human alpha-Synuclein.

32. A recombinant host cell comprising a nucleic acid or a vector according to claim 25 and expressing the recombinant polypeptide according to any one of claims 1-24.

33. A method for obtaining pharmaceutical composition comprising a polypeptide according to any one of claims 1-24, the method comprising the steps of (a) culturing the recombinant host cell of claim 32 under conditions allowing expression of the recombinant polypeptide from the nucleic acid molecule, (b) recovering the recombinant polypeptide, (c) purifying the recombinant polypeptide, and (d) formulating the recombinant polypeptide into a pharmaceutical composition.

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