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

Abstract

This invention relates to the therapeutic use of non-classical human major histocompatibility complex (MHC) molecules (also referred to as MHC class Ib molecules) in combination with peptide antigens for the treatment of Parkinson's disease. More specifically, this invention relates to recombinant polypeptides comprising a peptide antigen and one or more domains of a non-classical MHC class Ib molecule. This invention also relates to methods for producing such recombinant polypeptides, pharmaceutical compositions comprising them, and their use for the treatment of Parkinson's disease.

Description

[Technical Field]

[0001] This invention relates to the therapeutic use of non-classical human major histocompatibility complex (MHC) molecules (also known as MHC class Ib molecules) in combination with peptide antigens for the treatment of Parkinson's disease. More specifically, this invention relates to recombinant polypeptides comprising one or more domains of a peptide antigen and a non-classical MHC class Ib molecule. This invention also relates to methods for producing such recombinant polypeptides, pharmaceutical compositions comprising them, and their use for the treatment of Parkinson's disease. [Background technology]

[0002] Parkinson's disease (PD) is the most common neurodegenerative movement disorder, affecting more than 7 million people worldwide, and there is still no cure. Patients suffer from severe motor and non-motor symptoms, such as tremors, bradykinesia, rigidity, postural instability, and depression. The pathological features of the disease include the degeneration of dopaminergic neurons in the substantia nigra and the formation of α-synuclein-containing protein aggregates called Lewy bodies. While the physiological role of α-synuclein (aSyn) is indicated as being involved in the release of synaptic neurotransmitters and the amplification of immune responses (Alam et al., Cell Reports 38:110090. doi:10.1016 / j.celrep.2021.110090), the pathogenic role of insoluble α-synuclein remains a mystery. Observations that amplification and mutation of the SNCA gene encoding aSyn can induce genetically inherited forms of PD strongly link aSyn to the pathogenesis of PD.

[0003] Autopsies of human Parkinson's disease (PD) brains are characterized by microglial activation and T cell infiltration in the substantia nigra (Brochard V et al., 2009; McGeer PL et al., 1988). Pro-inflammatory cytokines within the nigrostriatal system, such as tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukins IL-1β and IL-6, further support the idea that PD progresses with significant neuroinflammation (Mogi M et al., 1994; Mogi M et al., 1994). An increased ratio of IFN-γ-producing T cells to IL-4-producing T cells in the peripheral blood of PD patients suggests a systemic shift to a pro-inflammatory environment (Baba Y et al., 2005). Recently, aSyn-specific T cell responses have been detected in peripheral blood cells of PD patients, and these responses have been found to precede motor symptoms, indicating that the immune system is actively and specifically involved in disease progression. Two aSyn-derived peptides have been shown to induce MHC-restricted cytokine release from peripheral blood mononuclear cells (Sulzer D et al., 2017). Currently, PD therapy is primarily based on compensating for SN neuron loss by administering L-dopamine or its derivatives. While this intervention initially improves disease symptoms, it does not prevent disease progression.

[0004] In a case-control study based on a population of approximately 48,000 PD patients, the risk of developing PD associated with immunosuppressant use was analyzed. Patients taking azathioprine had a significantly lower risk of PD (Racette BA et al., 2018). A recent cohort study further demonstrated that patients receiving anti-TNF-α therapy for inflammatory bowel disease had a reduced risk of developing PD (Peter I et al., 2018). The effects of immunomodulators, such as β2-adrenergic receptor agonists, or anti-IL-17 therapy have been explored in small, open-label clinical trials, yielding partially promising results (Magistrelli L and Comi C, 2020; Storelli E et al., 2019).

[0005] Given the often slow-progressing nature of this chronic disease, the side effects of strong immunosuppressants are a major obstacle in trials involving early-stage PD patients. However, in later-stage patients, irreversible neurodegeneration limits the achievable benefits by blocking further progression. In contrast, highly targeted immunomodulatory drugs enable early treatment, thereby maximizing therapeutic benefits.

[0006] For this purpose, a clinical trial (https: / / clinicaltrials.gov / ct2 / show / NCT03318523) was conducted using the aSyn-specific antibody cinpanemab (BIIB054). It was expected that sequestering free aSyn with cinpanemab would prevent further aSyn aggregation and thus prevent disease progression. Unfortunately, this study failed to meet its primary or secondary endpoints. Therefore, there is still an urgent need for targeted and effective disease-modifying therapies for PD.

[0007] The pathogenic role of T cells in Parkinson's disease (PD) has primarily been analyzed using toxin-based models, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 6-hydroxydopamine (6-OHDA) models. However, these models do not adequately reflect the molecular aspects of the human disease due to the lack of aSyn aggregation and Lewy body formation. This motivated Dr. Ip to develop a novel mouse model of PD that more faithfully reproduces the essential characteristics of the human disease. Human alpha-synuclein A53TUnilateral injection of the AAV1 / 2 vector encoding ("haSyn") into the substantia nigra, in a manner that induces contact taxis, induces behavioral defects, loss of dopaminergic substantia nigra neurons and striatal fibers, decreased striatal dopamine, and Lewy body formation (Ip CW et al., 2017). The high apparent validity of this model is achieved by injecting α-synuclein mutants, which are known to be disease-related in humans; therefore, the pathogenic significance of this model is also supported by the validity of its disease-related constructs. Furthermore, insoluble aSyn aggregates exhibiting S129 phosphorylation, as well as Lewy-like neurites and bodies, are detectable in this animal model (Ip CW et al., 2017). Thus, the haSyn PD model provides an important tool for advancing preclinical and translational research toward novel and better therapies for patients with PD. In a collaborative project, the inventors confirmed the broad pro-inflammatory profile of brain immune cells in this haSyn PD model and CD4 + and CD8 + We demonstrated the infiltration of an activated T cell subset into the brain of haSyn PD mice. These T cells induced neurodegeneration accompanied by PD-like symptoms. Genetic T cell deficiency (RAG-1 - / - While ) reduced dopaminergic neurodegeneration, T cell rearrangement exacerbated neuronal loss in vivo. T cells isolated from the brains of haSyn PD mice were cytotoxic to haSyn-expressing neurons in vitro (Karikari et al., Brain Behav Immun., March 2022, Vol. 101, pp. 194-210).

[0008] Immunosuppressive MHC class Ib molecules, such as HLA-G, are crucial for tolerance induction during pregnancy. These molecules exert immunosuppressive effects on various immune cells via immunosuppressive receptors, such as ILT2, ILT4, and Kir2DL4. International Publication 2018 / 215340 concerns combinations of MHC class Ib molecules and peptides for targeted therapeutic immunomodulation, but does not address the treatment of Parkinson's disease.

[0009] Therefore, there is an urgent need for targeted drugs that are effective in treating Parkinson's disease. [Overview of the project]

[0010] The inventors have found that human MHC class Ib molecules, such as HLA-G, have the ability to induce antigen-specific tolerance to presented peptide antigens. Thus, despite having a structure and sequence similar to classical human MHC class Ia molecules that induce antigen-peptide-specific immune responses, MHC class Ib molecules can be advantageously used according to the present invention to suppress immune responses in an antigen-specific manner. Furthermore, the inventors have found that molecules other than naturally occurring MHC class Ib molecules, particularly polypeptides comprising at least one domain of an MHC class Ib molecule, preferably only at least the [alpha]3 domain of an MHC class Ib molecule, can be used for suppressing immune responses according to the present invention: the [alpha]1 and [alpha]2 domains of a variable class Ia molecule can be combined with the [alpha]3 domain of a human MHC class Ib molecule to suppress immune responses to peptides presented by these antigens.

[0011] HLA-G molecules loaded with antigens can be unstable. Therefore, the inventors designed soluble recombinant polypeptides containing a peptide antigen, an MHC class Ib molecule such as HLA-G, and β2-microglobulin (b2m), and covalently linked these three components (e.g., via a covalent linker). Alternatively, the antigen-binding α1 and α2 domains of an MHC class Ib molecule such as HLA-G were replaced with the respective domains of other MHC molecules to enhance the flexibility and versatility of these recombinant polypeptides (see, for example, Figure 2). These alternative recombinant polypeptides can be designed using the antigen-binding domains of other human HLA molecules. (Mouse H2-K) bIt has been previously found that constructs containing the α1 and α2 domains can present the ovalbumin-derived peptide SIINFEKL (SEQ 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).

[0012] The literature teaches that Parkinson's disease is a neurodegenerative disease caused by the accumulation of pathogenic alpha-synuclein. The more recent concept that a crucial pathogenic event is due to an immune response to alpha-synuclein has not yet been translated into therapeutic drug development.

[0013] Surprisingly, the inventors have found that in a mouse model that faithfully mimics human Parkinson's disease, a therapeutic effect can be achieved in treating Parkinson's disease by using a substitute for the recombinant polypeptide of the present invention to suppress the immune response to alpha-synuclein. Therefore, according to the present invention, Parkinson's disease can be treated with the recombinant polypeptide of the present invention.

[0014] Furthermore, according to the present invention, the recombinant polypeptide of the present invention is expected to be extremely advantageous in immunotherapeutic treatment of Parkinson's disease because it causes little to no systemic immunosuppression. Importantly, patients in the early stages of Parkinson's disease should not be exposed to the strong side effects of systemic immunosuppression, as this can lead to opportunistic and potentially fatal infections. However, in patients in the later stages, neurodegeneration is often irreversible. Nevertheless, the induction of highly specific tolerance achieved by presenting the alpha-synuclein antigen on the recombinant polypeptide of the present invention means that tolerance is already present in the early stages of the disease, such as prodromal Parkinson's disease, which opens the way for novel therapies. [Means for solving the problem]

[0015] Therefore, the present invention relates to the following preferred embodiments. 1. Recombinant polypeptide capable of presenting a peptide antigen, wherein the order from the N-terminus to the C-terminus is: i) A peptide antigen presented by the recombinant polypeptide, which is a human alpha-synuclein peptide, ii) Linker arrangement depending on the case, iii) The sequence of human β2-microglobulin represented by Sequence ID No. 5, or the sequence of a human polypeptide domain containing an amino acid sequence that is at least 90% identical to the amino acid sequence of human β2-microglobulin, iv) Linker array depending on the case, v) The [alpha]1 domain of the MHC molecule, vi) The [alpha]2 domain of the MHC molecule, vii) A derivative of the [alpha]3 domain of an MHC class Ib molecule that can bind to ILT2 or ILT4, viii) Depending on the case, the protease cleavage site, ix) Depending on the case, spacer arrangement, and x) Affinity tags depending on the case Recombinant polypeptides containing [specific components].

[0016] The recombinant polypeptide according to item 1, wherein the peptide antigen described in 2.i) is 7 to 11 amino acids in length, preferably 8 to 11 amino acids in length.

[0017] 3.i) The recombinant polypeptide according to item 1 or 2, wherein the peptide antigen described in item 3.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.

[0018] 4.i) The recombinant polypeptide according to item 1 or 2, wherein the peptide antigen described in item 4.i) consists of the amino acid sequence of SEQ ID NO: 30.

[0019] 5.i) The recombinant polypeptide according to item 1 or 2, wherein the peptide antigen described in item 5.i) consists of the amino acid sequence of SEQ ID NO: 33.

[0020] 6.i) The recombinant polypeptide according to item 1 or 2, wherein the peptide antigen described in item 6.i) consists of the amino acid sequence of SEQ ID NO: 34.

[0021] 7.i) The peptide antigen described is a recombinant polypeptide as described in item 1 or 2, comprising the amino acid sequence of SEQ ID NO: 37.

[0022] 8.i) The recombinant polypeptide according to item 1 or 2, wherein the peptide antigen described in item 8.i) consists of the amino acid sequence of SEQ ID NO: 40.

[0023] 9.i) The recombinant polypeptide according to item 1 or 2, wherein the peptide antigen described in item 9.i) consists of the amino acid sequence of SEQ ID NO: 50.

[0024] 10. The recombinant polypeptide according to any one of the preceding items, wherein the [alpha]1 domain described in (v) and the [alpha]2 domain described in (vi) are derived from a human MHC class Ia molecule or a human MHC class Ib molecule.

[0025] 11. The recombinant polypeptide according to item 10, wherein the [alpha]1 domain described in (v) and the [alpha]2 domain described in (vi) are derived from a human MHC class Ib molecule, and the human MHC class Ib molecule is a human HLA-G molecule.

[0026] The recombinant polypeptide according to item 10, wherein the [alpha]1 domain described in (v) and the [alpha]2 domain described in (vi) are derived from a human MHC class Ia molecule, and the [alpha]1 domain described in (v) and the [alpha]2 domain described in (vi) are derived from a human HLA-A2 molecule, a human HLA-A1 molecule, or a human HLA-A11 molecule.

[0027] The recombinant polypeptide according to any one of the preceding items, wherein the [alpha]3 domain of the MHC class Ib molecule described in 13.(vii) is the [alpha]3 domain of human HLA-E, human HLA-F, or human HLA-G, preferably human HLA-G.

[0028] 14.(vii) The [alpha]3 domain or derivative described in 14.(vii) is identical to the [alpha]3 domain having the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 2, or SEQ ID NO: 51, 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, or is identical to the [alpha]3 domain having the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 2, or SEQ ID NO: 51, according to any one of the preceding items, recombinant polypeptide.

[0029] A recombinant polypeptide according to any one of items 1 to 14, wherein the [alpha]3 domain or derivative described in 15.(vii) is identical to the [alpha]3 domain having the amino acid sequence of SEQ ID NO: 9.

[0030] A recombinant polypeptide according to any one of items 1 to 14, wherein the [alpha]3 domain or derivative described in 16.(vii) is identical to the [alpha]3 domain having the amino acid sequence of SEQ ID NO: 2.

[0031] A recombinant polypeptide according to any one of items 1 to 14, wherein the [alpha]3 domain or derivative described in 17.(vii) is identical to the [alpha]3 domain having the amino acid sequence of SEQ ID NO: 51.

[0032] 18. The recombinant polypeptide according to any one of the preceding items, wherein the linker sequence described in (ii) and / or the linker sequence described in (iv) comprises an amino acid sequence (GGGGS)n, where n is an integer of 1 or more, and n is preferably an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and preferably selected from the group consisting of 2, 3, 4, and 5.

[0033] The recombinant polypeptide according to any one of the preceding items, wherein the sequence of the human polypeptide domain described in 19.(iii) 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.

[0034] 20. The recombinant polypeptide described in any one of the preceding items, wherein the polypeptide is a dimer or a polymer.

[0035] 21. A recombinant polypeptide according to any one of the preceding items, wherein the polypeptide contains or consists of all of components i) to vii) and does not contain components vii) to x), or contains or consists of all of components i) to x).

[0036] 22. A recombinant polypeptide according to any one of the preceding items, further comprising an N-terminal secretory signal peptide sequence.

[0037] 23. From the N-terminus to the C-terminus, (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) Amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 53-54 A recombinant polypeptide according to any one of items 1 to 21, comprising an amino acid sequence consisting of the following.

[0038] 24. A soluble recombinant polypeptide as described in any one of the preceding items.

[0039] 25. A nucleic acid, preferably a vector, that encodes one or more polypeptides described in any of the preceding items.

[0040] 26. A pharmaceutical composition or kit comprising at least one nucleic acid as described in item 25.

[0041] 27. A pharmaceutical composition or kit comprising at least one recombinant polypeptide as described in any one of items 1 to 24.

[0042] 28. A pharmaceutical composition or kit according to item 27, comprising at least two different recombinant polypeptides as described in any one of items 1 to 24, each of which different polypeptides comprises a different peptide antigen as defined in item 3.

[0043] 29. A pharmaceutical composition or kit according to any one of items 26 to 28, for use in the treatment of Parkinson's disease in human patients.

[0044] 30. A pharmaceutical composition or kit for use as described in item 29, wherein Parkinson's disease is a prodromal Parkinson's disease.

[0045] 31. A pharmaceutical composition or kit for use as described in item 29 or 30, wherein the treatment is an immunotherapy treatment for Parkinson's disease, and the treatment preferably induces immune tolerance to the autoantigen human alpha-synuclein.

[0046] 32. Recombinant host cells comprising the nucleic acid or vector described in item 25 and expressing any one of the recombinant polypeptides described in items 1 to 24.

[0047] 33. A method for obtaining a pharmaceutical composition comprising a polypeptide described in any one of items 1 to 24, comprising the steps of (a) culturing recombinant host cells described in item 32 under conditions that enable the expression of recombinant polypeptide from nucleic acid molecules, (b) recovering recombinant polypeptide, (c) purifying recombinant polypeptide, and (d) formulating recombinant polypeptide into a pharmaceutical composition. [Brief explanation of the drawing]

[0048] [Figure 1] A depiction of a peptide-loaded soluble MHC Ib molecule suitable for achieving therapeutic antigen-specific immunomodulation. The presented peptide antigen is depicted as a dotted sphere, the HLA-G alpha 1-3 domains are sketched in light gray, and the beta-2 microglobulin domain is shown in dark gray. Any linkers connecting the antigenic peptide to the beta-2 microglobulin molecule are shown in gray stick style, and any disulfide traps are depicted as black spheres. This drawing was generated using Pymol and adapted from structures published in Clements et al., Proc Natl Acad Sci USA. 2005 Mar 1;102(9):3360-5 and Hansen et al., Trends Immunol. 2010 Oct;31(10):363-9. [Figure 2]Examples of constructs based on vectors encoding single-chain MHC Ib molecules suitable for therapeutic peptide-specific immunomodulation. HLA-G1 and HLA-G5 each consist of three [alpha] domains (shown here in black), a non-covalently bound beta-2-microglobulin subunit (shown here in dark gray), and an 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 can bind to receptors ILT2 (see Shiroishi et al., Proc Natl Acad Sci USA. 2003 July 22;100(15):8856-8861) and ILT4 (see Shiroishi et al., Proc Natl Acad Sci USA. 2006 Oct 31;103(44):16412-7) on immune cells. Physiologically, these sequences form a non-covalently linked MHC class 1 complex. To simplify the purification of the complex MHC Ib molecule, one or more protein tags (such as SpotTag, myc tag, and / or His(6x) tag) may be introduced. They can be introduced in a manner that allows for subsequent optional removal by cleavage using an optional factor Xa cleavage site. Furthermore, antigenic peptides, beta-2-microglobulin, and the MHC Ib[alpha] chain can be linked to increase stability. Vector maps were generated using Snapgene Viewer Software. [Figure 3]This figure shows the prediction and confirmation of antigenic aSyn peptides. Ten weeks after injection of EV or A53T vectors, mononuclear cells containing CD4+ and CD8+ T cells derived from the spleen of haSyn PD mice (gray) and control mice (black) were stimulated with the predicted peptides, and the results were obtained from subsequent IFN-g ELISpot analysis. Five aSyn peptides induced antigen-specific IFN-γ immune responses. Using NCBI protein BLAST, www.syfpeithi.de (Ver. 1.0) and netMHC4.0 (http: / / www.cbs.dtu.dk / services / NetMHC / ), haSyn protein sequences were aligned with mouse α-synuclein, and potential nascent antigens likely to be presented on MHC I (H-2Db and H-2Kb) and MHC II (I-Ab) molecules were screened. Three peptides (8-15 amino acids in length) with the highest scores for each MHC molecule were synthesized in aqua basic quality by Thermo Scientific. After stimulating lymph node or spleen-derived cells with each peptide, the ELISpot assay was performed. Splenocytes obtained from two initially unresponsive haSyn mice and two controls were cultured for 10 days in RPMI containing 10% FCS and 20 ng / ml IL-2 in the presence of all 10 peptides (1 μg / ml each). Multiscreen PVDF plates (MSIPS4510, Millipore) were activated with 35% ethanol, washed with sterile PBS, and coated overnight at 4°C with 2 μg / ml filtered anti-mouse IFN-γ antibody (AN18, Mabtech). Isolated single-cell suspensions from cervical lymph nodes and spleen were seeded in IMDM containing 7.5% FCS, with or without 5 μg / ml of the synthetic peptide. Next, IFN-γ spots were detected in PBS-T supplemented with 0.05% BSA using 2 μg / ml biotinylated anti-mouse IFN-γ antibody (R4-6A2, Mabtech), followed by horseradish peroxidase conjugate streptavidin (Cell Signaling).In the assay, a filtered, ready-made TMB substrate (Mabtech) was used for color development, and the results were analyzed using an Immunopost S6 Core Analyzer. [Figure 4-1] This figure shows that the aSyn-loaded AIM Bio surrogate molecule can prevent neuronal death and improve endurance in a PD model. Treatment with a peptide, here aSyn peptide, a polypeptide containing an MHC class 1 antigen-presenting domain and an HLA-G alpha 3 domain (AIM Bio), for 9 cycles at one dose per week induced significant dose-dependent neuroprotection of dopaminergic neurons in haSyn PD mice compared to medium-treated haSyn PD mice, and the number of tyrosine hydroxylase (TH)+ neurons was significantly higher in the substantia nigra of the treated group. Immunohistochemical staining was performed using 40 μm PFA-fixed frozen sections treated for unbiased stereology (TH) covering the entire SN. After 4% PFA fixation, sections were incubated with rabbit anti-mouse TH (1:1000) antibody, followed by biotinylated goat anti-rabbit secondary antibody (Vector Labs). Chromogenesis was performed using (DAB)-HCl-peroxidase (Vector Labs). The number of dopaminergic neurons in the substantia nigra was estimated using unbiased stereology with the Stereo Investigator software package (v11.07, MicroBrightField Biosciences). Durability of treated and untreated mice was assessed by comparing pre- and post-treatment latencies in a rotarod test, as described in (Ip CW et al., 2017). A: Image (scale bar = 100 micrometers); B: Quantitative. C: Latency compared to pre-treatment, assessed by rotarod test. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 4-3] This is a continuation of Figure 4-1. [Figure 5-1]This figure shows that in a PD model, aSyn-loaded AIM Bio surrogate molecule prevents the infiltration of CD11b+ myeloid cells (microglia) into the striatum but induces the infiltration of CD8+ T cells. Immunohistochemical staining of mouse tissue for CD11b+ myeloid cells and CD8+ T cells was performed using 10 μm fresh coronal frozen sections of the striatum. After 4% PFA fixation or acetone fixation, the sections were incubated with rat anti-mouse CD11b (1:100, Serotec) or rat anti-CD8 MCA609G 1:500 antibody, respectively, and then incubated with biotinylated rabbit anti-rat secondary antibody (Vector Labs). Chromogenesis was performed using (DAB)-HCl-peroxidase (Vector Labs). Microglia were quantified at 200x magnification on a BH2 light microscope (Olympus) in the SN and striatal regions represented by serial sections stained for TH. A: Image showing CD11b+ microglia cells in the striatum (scale bar = 50 micrometers for the entire image, 20 micrometers for the magnified area in the lower left corner); B: Quantitative analysis. C: Image showing CD8+ T cells in the striatum (scale bar = 100 micrometers for the entire image, 50 micrometers for the magnified area in the lower left corner). D: Quantitative analysis. [Figure 5-2] This is a continuation of Figure 5-1. [Figure 5-3] This is a continuation of Figure 5-1. [Figure 5-4] This is a continuation of Figure 5-1. [Figure 6]This figure shows that aSyn-loaded AIM Bio surrogate reduces aSyn accumulation in the substantia nigra in a PD model. Scale bar = 100 micrometers. Neurons were fixed with 4% PFA, blocked with 5% NGS and 0.3% Triton X100, and stained with rabbit anti-human α-synuclein (1:30,000, Sigma) and goat anti-rabbit Cy3 (1:300, Jackson Laboratories, cat # 111-165-003) secondary antibodies, as well as DAPI counterstaining. Lower intensity aSyn staining was observed in mice treated with aSyn-loaded AIM Bio surrogate. [Figure 7-1] This figure shows that aSyn-loaded AIM Bio protects tyrosine hydroxylase+ fibers and substantia nigra neurons in the striatum. See Figure 4 for methods. A significant decrease in tyrosine hydroxylase+ fibers was observed, particularly in the upper striatum, on the side injected with A53T aSyn AAV (A53T) (right side in this case). Injection of aSyn-loaded AIM Bio significantly reduced unilateral loss of tyrosine hydroxylase+ fibers in a dose-dependent manner. A: Image; scale bar = 1000 micrometers. B: Quantification. C: Repeated experiment of Figure 4, using alternative Nissl staining to show neurons. [Figure 7-2] This is a continuation of Figure 7-1. [Figure 7-3] This is a continuation of Figure 7-1. [Figure 8]This figure shows that aSyn-loaded AIM Bio induces CD8+CD122+ regulatory T cells. Significant induction of CD8+CD122+ regulatory T cells was observed in both the spleen and cervical lymph nodes in response to aSyn-loaded AIM Bio treatment. Frozen sections were fixed with acetone at -20°C for 10 minutes, washed, and blocked in PBS with 5% BSA, 5% NGS, and 0.2% Triton-X100 for 1 hour. CD122 was stained overnight in 1% BSA, 1% NGS, and 0.2% Triton-X100 with anti-CD122i rabbit polyclonal antibody (MyBiosource, 1:100) and anti-CD8i clone YTS191.1 rat (Biorad, 1:200). Secondary antibody staining was performed for 1 hour using anti-rabbit-Cy3 (Dianova, 1:300) and anti-rat-AF488 (Invitrogen, 1:300). [Figure 9-1] Stability of purified single-chain MHC Ib molecules. After purification, the stability of single-chain MHC Ib molecules was analyzed after 1 and 3 freeze-thaw cycles, storage at room temperature for 5 days, and heating to 50°C for 30 minutes. For this purpose, A) Coomas-Siegel staining of a 12% polyacrylamide gel using 2 μg of AIM Bio and B) aHLA-G Western blotting using 1 μg of protein with 2A12aHLA-G antibody (1:1000) were performed under non-reducing conditions. Both monomers and dimers are detectable. [Figure 9-2] This is a continuation of Figure 9-1. [Figure 10]Single-chain MHC Ib molecules are thermally stable. For the temperature shift assay (TSA), 3 μg of each single-chain MHC Ib molecule or motabizumab as a control molecule was diluted with PBS and 5x SYPRO Orange dye (stock 5000-fold, final concentration: 5-fold) to a volume of 25 μl. The melting curve program was set up on the StepOnePlus Instrument using StepOnePlus Software 2.3. Autofluorescence was measured in arbitrary units by increasing the temperature at a rate of 1°C per minute over 2 minutes, starting at 25°C for 1 minute, and then increasing the temperature to a final temperature of 95°C at a rate of 1°C per minute. The data were exported and the graphs were plotted in Prism V7.04. The Boltzman sigmoid function was used to determine the melting point (Tm). [Figure 11] Single-chain MHC Ib molecules induce Tregs in a dose-dependent manner. OT-I mice were intravenously injected with the indicated amounts of single-chain H2_Kb alpha 1+2 and HLA-G alpha 3 domain constructs containing human beta-2 microglobulin and the indicated peptide or carrier (PBS). Ova was the allogeneic peptide for OT-I TCRs in these mice, and Gp34 was an unrelated virus-derived control peptide. After 14 days, mice were sacrificed, and splenocytes were tested for IL-10 secreting cells in recall mouse IL-10 ELISpot (200,000 cells per well, MabTech mouse IL-10 ELISpot kit, supplemented with 5 μg / ml of the indicated peptide or PBS only, 48 h). Clear induction of IL-10 secreting cells reacting with the Ova peptide was observed upon injection of 50 and 500 μg of mouse-adapted Ova_KbG. [Figure 12]This figure shows that single-chain MHC Ib molecules inhibit T cell lysis in a dose-dependent manner. OT1 / BL6 mice were sacrificed, spleen cells were collected and washed once in RPMI 5% FCS. Red blood cells were removed for 3 minutes using 2 ml of 1× sterile RBC lysis buffer. Cells were cultured for 72 hours at high density (10⁷ cells / ml) in RPMI 10% FCS medium containing 20 ng / ml GMCSF, 20 ng / ml IL-2, and 10 ng / ml IL-4, as well as an increasing dose of Ova_KbG. Cells were then scraped from the plate, and CD8+ cells were purified via magnetic beads. Sterile 96-well white plates were used. Luciferase-expressing Panc02 target cells were loaded with 20 μg / ml Ova peptide (SIINFEKL) at 37°C for 60 minutes with shaking at 500 rpm. CD8+ effector T cells were added in a 50:1 ratio, along with luciferin. Luminescence was measured at 0, 24, and 48 hours. [Figure 13]Single-chain MHC Ib molecules induce IL-10 expression in EAE-ODC Ova mice. Serum cytokines obtained from EAE-ODC Ova mice were measured using the Th1 / Th2 10plex Flowcytomix kit (eBioscience) according to the manufacturer's instructions. The kit was used to simultaneously detect mouse granulocyte-macrophage colony-stimulating factor (GMCSF), interleukin-1 alpha (IL-1a), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-17 (IL-17), and tumor necrosis factor alpha (TNF-α) in a single sample. Beads coated with 10 different specific capture antibodies were mixed. Subsequently, 25 μL of mixed capture beads, 25 μL of an unknown serum sample or standard dilution, and 25 μL of phycoerythrin (PE) detection reagent were successively added to each well of a 96-V bottom-well plate and incubated in the dark at room temperature for 2 hours. The samples were washed with 1 mL of washing buffer for 5 minutes and centrifuged. After discarding the supernatant, the bead pellet was resuspended in 200 μL of buffer. The samples were measured on an Attune® NxT Flow Cytometer and analyzed using Attune Cytometric Software (Thermo Fisher Scientific). [Figure 14] This figure shows a graphic representation of the development process for a preferred polypeptide construct of the present invention. [Figure 15]This figure shows the thermal shift assay. In the thermal shift assay (TSA), 3 μg of each single-chain MHC Ib molecule was diluted in 25 μl volumes with PBS and 5× SYPRO Orange dye (stock 5000×, final concentration: 5×). A melting curve program was set up on a StepOnePlus instrument using StepOnePlus Software 2.3. The starting temperature was 25°C, held for 1 minute, then the temperature was increased by 1°C per minute, and the final temperature was 95°C, held for 2 minutes. Autofluorescence was measured throughout the entire process. The data was exported and graphed in Prism V7.04. The melting temperature (Tm) was determined via the Boltzmann sigmoid function. A high melting temperature suggests good protein stability for therapeutic use. [Figure 16-1] This figure shows the stability of the polypeptide construct of the present invention. The sc HLA-A2 or A11 alpha-1-2 HLA-G alpha-3 constructs containing the indicated peptides were purified via spot-cap purification according to the manufacturer's protocol and then dialyzed overnight. The constructs were then subjected to three freeze-thaw cycles and heated at 50°C for 30 minutes or held at room temperature for 6 days. The degradation was then analyzed on a Coomasie gel (12% gel, 4% stacking gel, 1 μg purified protein, non-reducing conditions, 15 μl sample + 5 μl 4×Laemmli). Roti Mark TRICOLOR XTRA (Roth, 2244) was used for Coomasie staining for 5-6 hours. Destaining was performed with 12.5 ml methanol + 37.5 ml distilled water for 5 minutes, followed by another round of destaining with distilled water. [Figure 16-2] This is a continuation of Figure 16-1. [Figure 17-1] This figure shows the results of ELISpot obtained from healthy donor PBMCs. It shows the induction of regulatory IL-10 secreting cells over 14 days in PBMCs obtained from healthy donors using the construct described in the present invention. [Figure 17-2] This is a continuation of Figure 17-1. [Figure 18-1]This figure shows the results of IL-10 ELISpot obtained from Parkinson's disease patients (A) and age-matched healthy individuals (B). It shows the demonstrated construct of the present invention and the induction of regulatory IL-10 secreting cells over 14 days through age-matched healthy donors (C1-C3) or normal healthy donors (B10, B12, B13). [Figure 18-2] This is a continuation of Figure 18-1. [Figure 19-1] Figure A shows the results of a Simple Western filtration, and Figure B shows the supernatant concentration measured by ELISA. The construct used in this figure is as specified in the "Sequence" section below and in Example 6 below. The Simple Western filtration was performed as described in Example 6, and the ELISA was performed as a sandwich ELISA as described in Example 6. [Figure 19-2] This is a continuation of Figure 19-1. [Figure 20-1] This figure shows ELISpot using patient PBMCs. A: Image of the wells in the assay. The number of counted spots is shown in the figure. B: Quantification. ELISpot was performed as described in Example 6. Compared to untreated PBMCs, IL-10 spots increased in 4 out of 5 NMO patient PBMCs treated with AIM Bio AQP65P65K_G. [Figure 20-2] This is a continuation of Figure 20-1. [Figure 21] This is a diagram showing the acceleration rotor rod test. [Figure 22] This figure shows the particle size exclusion chromatography (SEC) analysis of AIM Bio aSyn63-71_H2-KbG. [Figure 23] This figure shows the thermal shift analysis of aSyn63-71_H2-KbG. [Figure 24] This is a diagram showing the Jess Simple Western for aSyn63-71_H2-KbG. [Figure 25]This figure shows IL-10 secreting cells detected in the ELISpot assay. The bar graphs on the left and right show the gating rate (%) and cell number of CD8+ cells in HLA-A2-PE staining, respectively. The mean and standard deviation are shown for each donor PBMC. [Figure 26] This figure shows CD8+ T cell subpopulations in healthy controls and Parkinson's disease patients. Two types of α-Syn-specific AIM Bio (AIM_aSyn69-77_A2G and AIM_aSyn70-78_A2G) stimulated the CD8+ T cell response in PBMCs derived from Parkinson's disease patients. The panel compares four CD8+ T cell subpopulations, including CD8+CD103+, CD8+DexLowCD103+, and CD8+IL-10+ cells, in individuals with HLA-A2+ (middle panel). After incubation of the subpopulations with AIM Bio for 14 days, the expression of CD103 and IL-10 was analyzed. Induction of IL-10 and CD103 was shown in PBMCs obtained from patients with Parkinson's disease. P values ​​are shown where applicable. Please note that due to the small number of cells, stimulation of healthy donor PBMCs using AIM_aSyn70_A2G was omitted. [Modes for carrying out the invention]

[0049] Definitions and General Techniques Unless otherwise defined below, terms used in this invention shall be understood in accordance with the general meaning known to those skilled in the art. All publications, patents, and patent applications cited herein, for any purpose whatsoever, are incorporated herein by reference in their entirety. Publications cited herein may be cited by providing a complete reference within the text, or by listing the first author and publication date (e.g., "Brochard V et al., 2009") within the text, followed by a corresponding complete reference in the "References" section of this specification.

[0050] All proteins according to the present invention, including the recombinant polypeptide of the present invention, can be obtained by methods known in the art. Such methods include methods for the production of recombinant polypeptides. The recombinant polypeptide of the present invention can be expressed in recombinant host cells according to the present invention. The recombinant host cells of the present invention are preferably mammalian cells such as CHO and HEK cells.

[0051] It will be understood that the recombinant polypeptide of the present invention may contain a secretion signal peptide sequence. Similarly, the recombinant polypeptide of the present invention may contain, for example, an affinity tag to facilitate purification, and an optional protease cleavage site between the tag and the polypeptide to facilitate removal of the tag by protease cleavage, for example.

[0052] Furthermore, references to amino acid sequences referred to herein include not only unmodified amino acid sequences but also typical post-translational modifications of these sequences that occur in cell expression systems known in the art, including mammalian cells such as CHO and HEK cells (e.g., glycosylation or deamidation of amino acids, clipping of specific amino acids, or other post-translational modifications).

[0053] Similarly, it will be understood that the recombinant polypeptides of the present invention may contain their respective propeptides.

[0054] It will also be understood that the recombinant polypeptides of the present invention may be in a soluble form or a membrane-bound form. Whether a recombinant polypeptide is "soluble" under these conditions can be determined by methods known in the art, for example, by measuring the turbidity of the recombinant polypeptide under the reference conditions shown above. As used herein, solubility means that at least 95% of the recombinant polypeptide is determined to be soluble under these reference conditions.

[0055] Single-chain MHC molecules can be stored, for example, in PBS at -80°C (with or without 0.1% human albumin as a carrier, depending on the protein concentration) or in 50% glycerol at -20°C.

[0056] According to the present invention, the MHC molecule is preferably a human MHC molecule.

[0057] The recombinant polypeptide of the present invention is preferably an isolated recombinant polypeptide.

[0058] A method for preparing recombinant polypeptides capable of binding to and presenting peptide antigens according to the present invention will be understood. For example, peptide antigen-binding domains such as the [alpha]1 and [alpha]2 domains are well known, and these domains can be modified. The ability of the polypeptides and peptide antigens according to the present invention to bind to MHC molecules can be determined by techniques known in the art, including, but not limited to, exploratory methods such as MHC peptide elution followed by mass spectrometry and in silico bioinformatics prediction, as well as confirmatory methods such as MHC peptide polymerization and stimulation assays.

[0059] According to the present invention, the recombinant polypeptide, pharmaceutical composition, and kit are preferably suitable for use in human patients.

[0060] According to the present invention, the recombinant polypeptide, pharmaceutical composition, and kit are preferably suitable for use in the treatment of Parkinson's disease in human patients.

[0061] According to the present invention, the recombinant polypeptide, pharmaceutical composition, and kit are preferably suitable for inducing immune tolerance to human alpha-synuclein in, for example, human patients.

[0062] According to the present invention, these recombinant polypeptides, pharmaceutical compositions, and kits of the present invention, wherein the [alpha]1 domain in (v) and the [alpha]2 domain in (vi) are derived from human HLA-A2 or HLA-A11 molecules, are preferably for use in the treatment of multiple sclerosis (MS), MOG antibody disease, or MOG antibody-positive neuromyelitis optica in human patients who are positive for the same HLA molecule (i.e., HLA-A2 and HLA-A11, respectively). This use is expected to be associated with a more favorable (lower immunogenicity) immunogenicity profile and a longer half-life (slower clearance) than in patients who are negative for HLA-A2 and HLA-A11, respectively.

[0063] In contrast, HLA-G does not exhibit the high levels of polymorphism observed with respect to classical HLA alleles. The HLA-G-based polypeptides of the present invention are expected to have a favorable immunogenicity profile and half-life across the human population.

[0064] According to the present invention, it is understood that the recombinant polypeptide, pharmaceutical composition, and kit of the present invention are stable.

[0065] In relation to peptide antigens used in accordance with the present invention, it will be understood that any length of these peptide antigens referred to herein (e.g., "7-11 amino acid length") refers to the length of the peptide antigen itself. Therefore, the length of the peptide antigen referred to herein does not include the length conferred by additional amino acids that are not part of the peptide antigen, such as additional amino acids derived from a possible linker sequence, etc.

[0066] According to the present invention, each occurrence of the term "contains" may be replaced with the term "consisting of".

[0067] Methods and Techniques Generally, unless otherwise defined herein, the methods used in the present invention (e.g., cloning methods or methods related to antibodies) are carried out in accordance with procedures known in the art, such as those described in Sambrook et al. ("Molecular Cloning: A Laboratory Manual," 2nd edition, 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 incorporated herein by reference).

[0068] Protein-protein binding, such as antibody binding to each of these target proteins, can be evaluated by methods known in the industry. Protein-protein binding is preferably evaluated by surface plasmon resonance spectroscopy.

[0069] For example, the binding of MHC class Ib molecules or recombinant polypeptides according to the present invention to their receptors, including ILT2 and ILT4, is preferably evaluated by surface plasmon resonance spectroscopy. More preferably, the binding of MHC class Ib molecules or recombinant polypeptides according to the present invention to their receptors is evaluated by surface plasmon resonance spectroscopy at 25°C. Suitable conditions for such surface plasmon resonance spectroscopy are described by Shiroishi et al., Proc Natl Acad Sci USA. 2003 July 22;100(15):8856-8861.

[0070] Sequence alignment of the sequence according to the present invention is performed 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 alignment of short peptides using the BLAST algorithm, suitable for the peptide antigen according to the present invention, are known in the art. Many software tools using the BLAST algorithm automatically adjust the parameters for sequence alignment for short input sequences. In one embodiment, the following parameters are used: maximum target sequence 10; word size 3; BLOSUM62 matrix; gap cost: present 11, elongated 1; conditional composition score matrix adjustment. Therefore, when used in relation to arrays, terms such as "identity" or "identical" preferably refer to identity values ​​obtained by using the BLAST algorithm.

[0071] Preparation of the pharmaceutical composition of the present invention The pharmaceutical composition of the present invention is prepared according to known standards for the preparation of pharmaceutical compositions.

[0072] For example, pharmaceutical compositions are prepared in a manner that allows them to be stored and administered appropriately. Accordingly, the pharmaceutical compositions of the present invention may contain pharmaceutically acceptable components such as carriers, excipients and / or stabilizers.

[0073] Such pharmaceutically acceptable components are not toxic at the amounts used when the pharmaceutical composition is administered to a human patient. The pharmaceutically acceptable components added to a pharmaceutical composition may depend on the chemical properties of the active ingredients present in the composition, the specific intended use of the pharmaceutical composition, and the route of administration.

[0074] Generally, pharmaceutically acceptable components used in connection with the present invention are used in accordance with the knowledge available in the industry, for example, from Remington's Pharmaceutical Sciences, Ed. AR Gennaro, 20th edition, 2000, Williams & Wilkins, PA, USA. Pharmaceutical compositions comprising nucleic acids (e.g., RNA) of the present invention can also be formulated in accordance with the knowledge available in the industry, for example, using liposomal formulations targeting dendritic cells.

[0075] The peptide antigen according to the present invention Peptide antigens that can be used in accordance with the present invention, including the peptide antigens defined above, are not particularly limited other than their ability to be presented on MHC molecules. The “peptide antigen presented by the recombinant polypeptide” as referred to in connection with the present invention is understood to be the peptide antigen presented to human T cells by the recombinant polypeptide, provided that such T cells are present such that the peptide antigen binds to T cell receptors on human T cells.

[0076] Peptides that can be presented on MHC molecules can be generated as is well known in this industry (see, for example, 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 I-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(11):724-737).

[0077] Peptide antigens are generally known in the art. Generally, the peptide antigens according to the present invention can bind to MHC class I proteins. For each MHC class Ib molecule or polypeptide capable of presenting the peptide according to the present invention, it will be understood that a peptide antigen capable of binding to the said MHC class Ib molecule or recombinant polypeptide is preferably used. These peptide antigens can be selected based on methods known in the art.

[0078] The attachment of a peptide antigen to an MHC class Ib molecule, or to a polypeptide capable of peptide antigen binding according to the present invention, can be done by methods known in the art, for example, 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 I-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(11):724-737 It can be evaluated by the following method.

[0079] Such methods include experimental methods and methods for predicting peptide antigen binding.

[0080] Anchor residues that are useful for immobilizing peptide antigens on MHC class I molecules and for ensuring the binding of peptide antigens to MHC class I molecules are well known in the art.

[0081] In preferred embodiments according to all embodiments of the present invention, the peptide antigen used in accordance with the present invention contains either an anchor or a preferred amino acid residue at a predicted position for an MHC class I molecule.

[0082] Such predictions can preferably be made as described in one of the following publications: - Rammensee et al., SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics (1999) 50: 213-219 - Nielsen et al., Protein Sci (2003) 12:1007-1017 - Neefjes et al., Nat Rev Immunol. 2011 Nov 11;11(12):823-36 - Diehl et al., Curr Biol. 1996 Mar 1;6(3):305-14 - Lee et al., Immunity. 1995 Nov;3(5):591-600 - Desai & Kulkarni-Kale, T-cell epitope prediction methods: an overview. Methods Mol Biol. 2014;1184:333-64 - 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.

[0083] In this invention, the peptide antigen is derived from human alpha-synuclein.

[0084] The non-anchored amino acid residues of the peptide antigen of the present invention may or may not contain conservative substitutions with respect to the corresponding amino acid sequence of the peptide antigen derived from human alpha-synuclein, but preferably contain two or fewer conservative substitutions, more preferably one conservative substitution.

[0085] The peptide antigen of the present invention preferably consists of naturally occurring amino acids. However, amino acids that do not exist in nature, such as modified amino acids, can also be used. For example, in one embodiment, the peptide antigen of the present invention comprises a peptide mimetic of the shown peptide antigen amino acid sequence of human alpha-synuclein.

[0086] Methods for synthesizing peptide antigens, including peptide antigens based on the present invention, are well known in the art.

[0087] array The preferred amino acid sequences referenced in this application may be independently selected from the following sequences. The sequences are presented in N-terminus to C-terminus and are represented by single-letter amino acid codes.

[0088] An example sequence that is part of the recombinant polypeptide of the present invention: Leader peptides (which are not present in recombinant polypeptides due to processing during cell expression): e.g., MSRSVALAVLALLSLSGLEA (SEQ ID NO: 1). This is encoded by the following DNA sequence ATGAGCAGATCTGTGGCCCTGGCTGTTCTGGCTCTGCTGTCTCTGTCTGGCCTGGAAGCC (SEQ ID NO: 49). Alternative leader peptides: Mouse Ig heavy chain peptide: MGWSCIILFLVATATGVHS (SEQ ID NO: 66) Human OSM peptide: MGVLLTQRTLLSLVLALLFPSMASM (SEQ ID NO: 67) VSV-G peptide: MKCLLYLAFLFIGVNC (SEQ ID NO: 68) Mouse Ig kappa peptide: METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 69) BM40 peptide: MRAWIFFLLCLAGRALA (SEQ ID NO: 70) Secrecon peptide: MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO: 71) Human IgKVIII peptide: MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 72) CD33 peptide: MPLLLLLPLLWAGALA (SEQ ID NO: 73) tPA peptide: MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 74) Human chymotrypsinogen peptide: MAFLWLLSCWALLGTTFG (SEQ ID NO: 75) Human trypsinogen 2 peptide: MNLLLILTFVAAAVA (SEQ ID NO: 76) Human IL-2 peptide: MYRMQLLSCIALSLALVTNS (SEQ ID NO: 77) Gaussia luc peptide: MGVKVLFALICIAVAEA (SEQ ID NO: 78) Albumin (HSA) peptide: MKWVTFISLLFSSAYS (SEQ ID NO: 79) Influenza hemagglutinin peptide: MKTIIALSYIFCLVLG (SEQ ID NO: 80) Human insulin peptide: MALWMRLLPLLALLALWGPDPAAA (SEQ ID NO: 81) Silkworm fibroin LC peptide: MKPIFLVLLVVTSAYA (SEQ ID NO: 82)

[0089] Peptide antigen: Any MHC class I peptide corresponding to the MHC class I [alpha]1 & 2 domains, e.g., GAPQEGIL (SEQ ID NO: 20), or any peptide antigen according to the present invention, e.g.:

[0090] [Table 1]

[0091] First linker: e.g., GGGGSGGGGSGGGGS (SEQ ID NO: 3) or GCGASGGGGSGGGGS (SEQ ID NO: 4) Beta-2 microglobulin, for example: IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (Sequence ID 5, Human Beta-2 Microglobulin) A second linker, for example: GGGGSGGGGSGGGGSGGGGS (Sequence 6)

[0092] The [alpha]1&2 domains are derived from human HLA-G or any other MHC class I [alpha]1&2 domains suitable for presenting a selected antigen peptide, and Y84 may be C in the DT variant. For example, derived from the [alpha]1&2 domain human HLA-G: for example: [ka]

[0093] Human HLA-G[alpha]3 domains (or any MHC Ib[alpha]3 domains, such as HLA-F, which also interacts with ILT2 and ILT4 receptors), e.g.: [ka]

[0094] Alternatively, a shorter human HLA-G[alpha]3 domain lacking an optional C-terminal amino acid sequence derived from intron 4 can be used (SKEGDGGIMSVRESRSLSEDL; SEQ ID NO: 47, or SKEGDGGIMSVRESGSLSEDL; SEQ ID NO: 52), i.e.: DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (Sequence ID 2).

[0095] The inventors have found that recombinant polypeptides can be produced using any one of the above sequences of HLA-G[alpha]3. The presence of the sequence of SEQ ID NO: 51 in the recombinant polypeptide, i.e., the presence of the HLA-G[alpha]3 sequence from which the furin cleavage site in the C-terminal amino acid sequence derived from intron 4 of human HLA-G has been removed, was found to be advantageous for improving the purification of recombinant polypeptides, for example, for the purification of recombinant polypeptides containing a C-terminal tag.

[0096] Factor Xa restriction site: IEGRTGTKLGP (SEQ ID NO: 10) SpotTag:PDRVRAVSHWSSC (Sequence ID 11) Myc tag:EQKLISEEDL(sequence number 12) His tag: HHHHHH * (Sequence ID 13) Spacer array: For example, NSAVD (sequence number 14) or GS.

[0097] Exemplary peptide antigens that may be part of the recombinant polypeptide of the present invention are as follows: GAPQEGIL (SEQ ID NO: 20), preferably used in recombinant polypeptides containing human HLA-G [alpha]1 and 2 domains. KTKEGVLYV (SEQ ID NO: 28), preferably used in recombinant polypeptides containing human HLA-A2 [alpha]1&2 domains. AVVTGVTAV (SEQ ID NO: 33), preferably used in recombinant polypeptides containing human HLA-A2 [alpha]1&2 domains. GVVHGVTTV (SEQ ID NO: 31), preferably used in recombinant polypeptides containing human HLA-A2 [alpha]1&2 domains. MDVFMKGLSK (SEQ ID NO: 22), preferably used in recombinant polypeptides containing human HLA-A11 [alpha]1&2 domains. GVVAAAEKTK (SEQ ID NO: 25), preferably used in recombinant polypeptides containing human HLA-A11 [alpha]1&2 domains. MPVDPDNEAY (SEQ ID NO: 21), preferably used in recombinant polypeptides containing human HLA-A1 [alpha]1&2 domains. PVDPDNEAY (SEQ ID NO: 44), preferably used in recombinant polypeptides containing human HLA-A1 [alpha]1&2 domains. SIAAATGFV (SEQ ID NO: 37), preferably used in recombinant polypeptides containing human HLA-A2 [alpha]1&2 domains. VVTGVTAVA (SEQ ID NO: 34), preferably used in recombinant polypeptides containing human HLA-A2 [alpha]1&2 domains. VVAAAEKTK (SEQ ID NO: 26), preferably used in recombinant polypeptides containing human HLA-A11 [alpha]1&2 domains. VFMKGLSKAK (SEQ ID NO: 24), preferably used in recombinant polypeptides containing human HLA-A11 [alpha]1&2 domains. AAATGFVKKD (SEQ ID NO: 42), preferably used in recombinant polypeptides containing human HLA-A11 [alpha]1&2 domains. AATGFVKK (SEQ ID NO: 43), preferably used in recombinant polypeptides containing human HLA-A11 [alpha]1&2 domains. SIAAATGFVKK (SEQ ID NO: 50), preferably used in recombinant polypeptides containing human HLA-A11 [alpha]1&2 domains.

[0098] Examples of recombinant polypeptides (excluding leader peptides): GAPQEGILGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQP KIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGC YNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDY EATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESLSLSEDLGSPDRVRAVSHWSSC* (Note: Sequence ID 15; the asterisk indicates the position of the stop codon.)

[0099] It should be noted that the sequence of the peptide antigen of the full-length protein described above (here: GAPQEGIL) can be replaced with any peptide antigen sequence according to the present invention, that is, any peptide antigen presented by the recombinant polypeptide, which is a peptide of human alpha-synuclein. In other words, the recombinant polypeptide of the present invention consists of a sequence of a peptide antigen that is a peptide of human alpha-synuclein (for example, any one of the peptide antigens of SEQ ID NOs. 20 to 46 or SEQ ID NO. 50 according to the present invention), followed by the following sequence GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWD RDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGCYNQS EDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEA TLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESLSLSEDLGSPDRVRAVSHWSSC* (Sequence ID 16; note that the asterisk indicates the position of the stop codon), or the following sequence that follows it GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWD RDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGCYNQS EDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEA TLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESGSLSEDLGSPDRVRAVSHWSSC* (Note that the asterisk in sequence number 53 indicates the position of the stop codon.) Alternatively, the following sequences that follow, lacking the C-terminal amino acid sequence derived from intron 4 of the HLA-G[alpha]3 domain, a variable cleavage site, and optionally a tag: It can consist of GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGCYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (Sequence ID 54).

[0100] Examples of recombinant polypeptides according to the present invention (shown without optional leader peptide sequences, C-terminal amino acid sequences derived from intron 4 of the HLA-G[alpha]3 domain sequence, variable cleavage sites, and optional tags):

[0101] aSyn87-97_A11G_fs-_Sp (Exhibits excellent stability and a very good response rate in ELISP assays) Protein sequence: [ka]

[0102] The DNA sequence that codes for it (note that the DNA sequence below does not show the DNA sequence that codes for the N-terminal leader peptide): [ka] [ka]

[0103] Preferred A2G constructs: aSyn 69-77 _A2G (exhibits a high response rate in ELISPOT assays, excellent production yield and stability when using PBMCs derived from healthy donors) Protein sequence:

Chemical formula

[0104] DNA sequence encoding it (note that the DNA sequence encoding the N-terminal leader peptide is not shown in the following DNA sequence):

Chemical formula

Chemical formula

[0105] aSyn 70-78 _A2G (exhibits a high response rate in ELISPOT assays, very good production yield and stability when using PBMCs derived from healthy donors and PBMCs derived from patients with Parkinson's disease) Protein sequence:

Chemical formula

[0106] DNA sequence encoding it (note that the DNA sequence encoding the N-terminal leader peptide is not shown in the following DNA sequence):

Chemical formula

Chemical formula

[0107] Preferred A11G constructs: aSyn 37-45_A11G (When using PBMCs derived from healthy donors and especially from patients with Parkinson's disease, the ELISPOT assay shows a good response rate, very good production yield, and stability.) Protein sequence: [ka]

[0108] The DNA sequence that codes for it (note that the DNA sequence below does not show the DNA sequence that codes for the N-terminal leader peptide): [ka] [ka]

[0109] Further recombinant polypeptides of the present invention, tested in PBMCs from healthy donors and, optionally, from patients: aSyn 87-95 _A2G (When using PBMCs derived from healthy donors and patients with Parkinson's disease, it shows good response rates, very good stability, and productivity in the ELISPOT assay.) Protein sequence: [ka]

[0110] The DNA sequence that codes for it (note that the DNA sequence below does not show the DNA sequence that codes for the N-terminal leader peptide): [ka] [ka]

[0111] aSyn 86-96_A11G (When using PBMCs derived from healthy donors and PD patients, it shows very good response rates, very good stability, and productivity assessments in the ELISPOT assay.) Protein sequence: [ka]

[0112] The DNA sequence that codes for it (note that the DNA sequence below does not show the DNA sequence that codes for the N-terminal leader peptide): [ka]

[0113] The recombinant polypeptides of the present invention described above may optionally include, as illustrated above, a leader peptide, a C-terminal amino acid sequence derived from intron 4 of the HLA-G[alpha]3 domain sequence (e.g., SKEGDGGIMSVRESRSLSEDL; SEQ ID NO: 47, or SKEGDGGIMSVRESGSLSEDL; SEQ ID NO: 52), an optional protease cleavage site, an optional linker (e.g., GS), and / or an optional tag.

[0114] The following three general structures of the recombinant polypeptide according to the present invention are preferred: [Peptide antigen described in i) of the present invention]-[amino acid sequence] GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQ FVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEA TLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (Sequence ID 83, which includes the linker described in ii), the human β2 microglobulin sequence described in iii), the linker described in iv), the HLA-G[alpha]1 & 2 domains described in v) and vi), and the HLA-G[alpha]3 domain described in vii)))-[Optionally, the C-terminal amino acid sequence derived from intron 4 of the HLA-G[alpha]3 domain sequence (e.g., SKEGDGGIMSVRESRSLSEDL; Sequence ID 47, or SKEGDGGIMSVRESGSLSEDL; Sequence ID 52)]-[Optionally, a variable cleavage site]-[Optionally, a tag]

[0115] [Peptide antigen described in i) of the present invention]-[amino acid sequence] GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQF VRFDDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDDPKTHVTHHPVFDYEAT LRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (Sequence ID 84, which includes the linker described in ii), the human β2 microglobulin sequence described in iii), the linker described in iv), the HLA-A2 [alpha]1 & 2 domains described in v) and vi), and the HLA-G [alpha]3 domain described in vii)))-[Optionally, the C-terminal amino acid sequence derived from intron 4 of the HLA-G [alpha]3 domain sequence (e.g., SKEGDGGIMSVRESRSLSEDL; Sequence ID 47, or SKEGDGGIMSVRESGSLSEDL; Sequence ID 52)]-[Optionally, a variable cleavage site]-[Optionally, a tag]

[0116] [Peptide antigen described in i) of the present invention]-[amino acid sequence] GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGS GGGGSGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGCYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYI ALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO: 85, which includes the linker described in ii), the human β2 microglobulin sequence described in iii), the linker described in iv), the HLA-A11 [alpha]1 & 2 domains described in v) and vi), and the HLA-G [alpha]3 domain described in vii)] - [Optionally, the C-terminal amino acid sequence derived from intron 4 of the HLA-G [alpha]3 domain sequence (e.g., SKEGDGGIMSVRESRSLSEDL; SEQ ID NO: 47, or SKEGDGGIMSVRESGSLSEDL; SEQ ID NO: 52)] - [Optionally, a variable cleavage site] - [Optionally, a tag]

[0117] The receptors ILT2 (also known as LILRB1) and ILT4 (also known as LILRB2) are well known in the art. Preferred sequences of these receptors according to the present invention are as follows: ILT2: MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRL YREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTG AYIKPTLSAQPSPVVNSGGNVILQCDSQVAFDGFSLKEGEDEHPQCLNSQPHARGSSRA IFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEE TLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGA HNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKE GAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVS GPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLF LILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQ PEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMD TEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (Sequence ID 17).

[0118] ILT4: MTPIVTVLICLGLSLGPRTHVQTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRL YREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGA YPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEEEHPQCLNSQPHARGSSRAI FSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKKPSLSVQPGPVVAPGES LTLQCVSDVGYDRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAH NLSSECSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAG AADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPLELVVSG PSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVVLLLLLLLLLF LILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKDTQ PEDGVEMDTRAAASEAPQDVTYAQLHSLTLRRKATEPPPSQEREPPAEPSIYATLAIH(Sequence ID 18).

[0119] The sequences of human alpha-synuclein are known in the art. Preferred human alpha-synuclein sequences are as follows: MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA (Sequence ID 19)

[0120] Application of this invention to therapy: Parkinson's disease (PD), including prodromal Parkinson's disease (prodromal PD), and its classification are well known in the art.

[0121] Prodromal Parkinson's disease (PD) is a stage in which an individual does not meet the diagnostic criteria for PD (i.e., bradykinesia and at least one other motor sign), but exhibits signs and symptoms that suggest a higher-than-average risk of developing motor symptoms and being diagnosed with PD in the future. Most prodromal symptoms are non-motor and have a significant impact on the quality of life for both patients with prodromal PD and those whose disease progresses to motor PD. Therefore, early detection and treatment of such prodromal symptoms are essential for high-quality care. The most characteristic symptoms of prodromal PD include decreased olfaction, constipation, mood disturbances, and REM sleep behavior disorder (RBD).

[0122] The present invention is further illustrated by the following non-limiting embodiments: [Examples]

[0123] Reference example: Screening of target antigens in mice To screen for suitable target antigens, mice injected with haSyn or a control virus were sacrificed. Immune cells were isolated from the brain, peripheral lymph nodes, and spleen. These cells were then incubated with the A53T-aSyn peptide predicted in silico (e.g., via NetMHC). By screening for aSyn-specific immune responses in C57BL / 6 mice, we identified MHC I-restricted and MHC II-restricted haSyn peptides that induce IFN-γ release from T cells in affected mice (Figure 3). T cell reactivity to haSyn peptides was also elucidated in cervical lymph node and brain-derived T cells.

[0124] Example 1 (reference): Prevention of neuronal death in a PD in vivo model Given the crucial role of T cells in neurodegenerative processes (as revealed by depletion / reconstitution experiments), the inventors reasoned that this haSyn PD mouse model, which closely resembles human disease, is ideally suited for evaluating the effectiveness of immunomodulation against a background of PD-like aSyn-based pathology, and for assessing the suitability of the recombinant polypeptide of the present invention for treating PD.

[0125] The inventors further reasoned that inducing immune tolerance to aSyn would induce tissue-wide protection via local antigen-specific activation of Tregs. Having identified that the aSyn 68-78 peptide derived from A53T-aSyn is immunogenic in mice, the inventors identified this peptide antigen, which corresponds to the recombinant polypeptide of the present invention but is immunogenic in mice, as well as mouse H2-D b Recombinant polypeptides carrying alpha-1 and alpha-2 domains (hereinafter referred to as "mouse AIM Bio substitute molecule" or "AIM Bio") aSyn They developed (also known as "[AIM Bio]"). In pilot experiments, haSyn PD mice were given 0.5 μg / g AIM Bio starting on day 3 after AAV injection. aSyn , or 2 μg / g AIM Bio aSyn Either or an off-target, unrelated control peptide (gp34) was injected weekly. The Gp34 peptide is a well-characterized T-cell epitope derived from the lymphocytic choriomeningitis virus (LCMV) glycoprotein. This antigen was traditionally named Gp33, but H2-K b It was later discovered that the epitopes presented above only contain amino acids 34-41 (in contrast, the epitope starting with amino acid 33 is H2-K d (As presented above). Therefore, following the most recent recommendation, H2-K bThe epitope will be referred to as Gp34. In the literature, there is still ambiguous usage regarding the nomenclature of Gp33 and Gp34. The peptide sequence used here is AVYNFATM (SEQ ID NO: 48). Control mice were injected with carrier (PBS) only. Ten weeks after AAV delivery, we found that haSyn PD mice injected with PBS or Gp34_KbG lost approximately 45% of substantia nigra neurons compared to animals injected with a non-pathogenic empty vector control (EV). Surprisingly, this neuronal loss was observed with both low-dose (0.5 μg / g) and high-dose (2 μg / g) AIM Bio aSyn This was attenuated in a dose-dependent manner (Figure 4). Furthermore, the inventors found that the aSyn-loaded AIM Bio substitute molecule was attenuated in the striatum in the PD model by CD11b + It inhibits bone marrow cell infiltration (Figure 5), the aSyn-loaded AIM Bio substitute molecule reduces aSyn accumulation in the substantia nigra in a PD model (Figure 6), and aSyn-loaded AIM Bio affects tyrosine hydroxylase in the striatum. + We found that it protects the fibers (Figure 7).

[0126] Example 2: aSyn-specific MHC class I-based therapeutic agent for treating PD in human patients Our findings have shown that single-chain proteins containing the aSyn peptide antigen and the HLA-G alpha-3 domain can largely prevent neuronal death in animal models of Parkinson's disease, meaning they can be used to treat PD. Based on these findings, we have created an aSyn peptide-MHC class I fusion molecule for therapeutic use in human PD patients.

[0127] Target sequence (human alpha-synuclein): MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA (Sequence ID 19)

[0128] Exemplary peptides: GAPQEGIL (Sequence ID 20) MPVDPDNEAY (Sequence ID 21) MDVFMKGLSK (SEQ ID NO: 22) DVFMKGLSK (SEQ ID NO: 23) VFMKGLSKAK (Sequence ID 24) GVVAAAEKTK (Sequence ID 25) VVAAAEKTK (Sequence ID 26) GVAEAAGKTK (Sequence ID 27) KTKEGVLYV (Sequence ID 28) GVLYVGSKTK (Sequence ID 29) VLYVGSKTK (Sequence ID 30) GVVHGVTTV (SEQ ID NO: 31) (Based on the A53T mutant) GVTTVAEKTK (SEQ ID NO: 32) (Based on the A53T mutant) AVVTGVTAV (Sequence ID 33) VVTGVTAVA (Sequence ID 34) VTGVTAVAQK (Sequence ID 35) GSIAAATGF ​​(Sequence ID 36) SIAAATGFV (Sequence ID 37) SIAAATGFVK (Sequence ID 38) IAAATGFVK (Sequence ID 39) GSIAAATGFVK (Sequence ID 40) AAATGFVKK (Sequence ID 41) AAATGFVKKD (Sequence ID 42) AATGFVKK (Sequence ID 43) PVDPDNEAY (Sequence ID 44) MPSEEGYQDY (Sequence ID 45) PSEEGYQDY (Sequence ID 46) SIAAATGFV (Sequence ID 37) + HLA-A2 presented domain KTKEGVLYV (Sequence ID 28) + HLA-A2 presented domain AVVTGVTAV (SEQ ID NO: 33) + HLA-A2 presented domain GVVHGVTTV (SEQ ID NO: 31) + HLA-A2 presented domain VVTGVTAVA (SEQ ID NO: 34) + HLA-A2 presented domain GSIAAATGFVK (SEQ ID NO: 40) + HLA-A11 presented domain AAATGFVKK (Sequence ID 41) + HLA-A11 presented domain VLYVGSKTK (SEQ ID NO: 30) + HLA-A11 presented domain MPVDPDNEAY (SEQ ID NO: 21) + HLA-A1 presented domain PVDPDNEAY (SEQ ID NO: 44) + HLA-A1 presenting domain VVAAAEKTK (Sequence ID 26) + HLA-A11 presented domain VFMKGLSKAK (SEQ ID NO: 24) + HLA-A11 presented domain AAATGFVKKD (Sequence ID 42) + HLA-A11 presented domain AATGFVKK (SEQ ID NO: 43) + HLA-A11 presented domain MDVFMKGLSK (SEQ ID NO: 22) + HLA-A11 presented domain GVVAAAEKTK (Sequence ID 25) + HLA-A11 presented domain GAPQEGIL (SEQ ID NO: 20) + HLA-G presentation domain

[0129] Based on online prediction algorithms (Syfpeithi, NetMHC), a literature review on immunodominant regions, 3D protein folding prediction (AlphaFold2), protein expression data, and experimental results reported herein, the inventors have found that these epitopes are highly likely to induce tolerogenic effects in Parkinson's disease patients. Therefore, the most preferred peptide and preferred peptide shown above are suitable.

[0130] Example 3: Method used in the present invention Method for producing recombinant polypeptides according to the present invention - Expi-293F cells (Thermo Fisher) grown in Expi-293® expression medium (Thermo Fisher): 2.5 x 10⁻¹⁶ cells using the Expifectamine® 293 T transfection kit (Thermo Fisher) with Opti-MEM (Thermo Fisher) for complexation of DNA with Expifectamine. 6 Transfection of 1 μg of DNA into cells / ml, addition of enhancer according to protocol after 18-20 hours, 19 mm 2 The supernatant was collected after 4-6 days on an orbital shaker at 125 rpm (37°C, 8% CO2, humidified incubator). - Spot-tagged protein purification: Equilibration of Spot-Cap resin: Transfer the desired amount of slurry into a suitable tube, settle the beads by centrifugation (4°C, 4 min, 2500 g), remove and discard the supernatant, add 10 beds of PBS (cold) to the beads, mix and invert, settle the beads by centrifugation (4°C, 4 min, 2500 g), remove and discard the supernatant, repeat twice. - Add the required volume of beads to the supernatant, incubate on a rotator at 4°C, wash the beads by repeating centrifugation (4°C, 4 min, 2500 g), and remove the supernatant. - Prepare a 500 μM Spot-peptide solution in PBS, remove the supernatant, and incubate with 1 / 3 of the Spot-peptide solution for 5-10 minutes. The beads are settled by centrifugation. An Amicon Ultra-4 centrifugation filter (15kDa cutoff) is used for protein concentration, and spot peptides are removed using a 15kDa Amicon cutoff column. After rinsing the Amicon Ultra-4 centrifugation filter (15kDa cutoff) with PBS, wash it with 0.1N NaOH (4000g, centrifugation at 4°C) to remove trace amounts of glycerin.

[0131] ELISPOT: 1)Cell culture A) Isolation of PBMCs (under laminar flow hood) To isolate peripheral blood mononuclear cells (PBMCs), density centrifugation was performed using leukocytes from a leukocyte removal chamber and density gradient media (e.g., Ficoll or ROTI Sep 1077). After centrifugation at 1200xg for 20 min without using a brake, the interphase ring was washed with 1xPBS (5 min, 300xg). The PBMCs were frozen until further use.

[0132] B) Pulsing of PBMC (under laminar flow hood) The PBMCs were thawed one day before PBMC pulsing (d-1) and kept overnight in 5 ml of X-VIVO 15 medium containing 5% human AB serum in each well of a 6-well plate at 37°C.

[0133] The next day (d0), count the cells, 3 x 10 6 The cells were resuspended in X-VIVO 15 complete medium (5% hAB serum & cytokine cocktail: 20 ng / ml hIL-2, 20 ng / ml hGM-CSF, 10 ng / ml hIL-4 & 10 ng / ml hTGF-b1) at a cell density of cells / ml.

[0134] For the experiment, 3x10 6The cells were seeded in each well of a 12-well plate containing a final volume of 1000 μl of X-VIVO complete medium, along with a cytokine cocktail and 5 μg / ml of AIM Bio molecule (recombinant polypeptide of the present invention) or their respective controls.

[0135] On day 3, 1 ml of complete medium (containing cytokines) was added, and on day 6, a second pulse was performed using 5 μg / ml of the recombinant polypeptide of the present invention or its surrogate (collectively referred to as the "AIM Bio" molecule) (after removing the medium). On days 7, 10, and 12, 1 ml of complete medium (containing cytokines) was added. What is needed: X-VIVO 15 medium + 5% human AB serum X-VIVO 15 complete medium: X-VIVO 15 medium + cytokine cocktail: 2% human AB serum supplemented with 10 ng / ml TGF-b1, 10 ng / ml IL-4, 20 ng / ml IL-2, and 20 ng / ml GM-CSF. 6-well plate. 12-well plate.

[0136] 2) ELISPOT Laminar flow hood On day 13, ELISPOT plates were coated with anti-hIL10 (clone 9D-7, 1:500 dilution in sterile filtered PBS) and aIL10 (10G8-biotin), and on day 14, 200,000 cells per well, including negative control (cells + PBS) and positive control (e.g., LPS), were seeded onto ELISPOT plates in two replicates.

[0137] The PFDF membrane was activated with 50 μl / well EtOH (35% v / v) for 1 min, and then washed five times with 200 μl of distilled sterile water. The plate was coated with 100 μl / well antibody solution overnight at 4°C. The following day, unbound coating antibody was removed, and the plate was washed five times with 200 μl of PBS, 200 μl of blocking buffer (X-VIVO 15 5% hAb serum) was added, and the plate was incubated at room temperature for 30 min to 2 hours.

[0138] Each antigenic peptide or control DMSO was prepared in DMSO, and a final volume of 5 μg peptide / ml was added to the final volume of 100 μl / well. 150,000 cells per well were seeded in X-VIVO 15 medium containing 5% human AB serum. The blocking buffer (X-VIVO 15 medium + 5% hAB serum) was carefully removed, and medium containing PBS as a negative control and a stimulant (total volume of 5 μg / ml in each well) was added to the other wells, and incubated overnight at 37°C.

[0139] Outside of the laminar flow hood Secondary antibodies were prepared: a solution of 1 μg / ml aIL-10 biotinylated antibody in 0.5% BSA / 1x PBS (1:1000 dilution) and horseradish peroxidase-conjugated streptavidin (1:750 in 0.5% BSA / PBS) and tetramethylbenzidine was filtered using a 0.45 μm filter and stored at 4°C until use.

[0140] The cell supernatant was removed and washed five times with 100 μl of PBS. Finally, excess buffer was removed using a paper towel.

[0141] 25 μl of diluted HRP-streptavidin (1:750) was added to each well, incubated in a dark room at room temperature for 1 hour, and then washed five times with sterile 1x PBS.

[0142] 100 μL of filtered TMB substrate per well was added for 15-25 min until blue spots developed. The reaction was stopped by thoroughly washing the wells with tap water.

[0143] The plastic underdrain of the plate was removed, and the bottom and sides of the plate were washed with tap water and dried.

[0144] Plates were read using an ImmunoSpot S6 Ultra-V Analyzer (Cellular Technology Limited), analyzed in Excel, and graphing / statistics were performed in GraphPad Prism.

[0145] Required items: Capture antibodies: anti-hIL10 antibody (Clone: 9D-7, Mabtech #3430-3-250; diluted 1:500), anti-hIL10-biotinylated antibody (Mabtech, #3430-6-250) 1×PBS (sterile) 35% EtOH (v / v) Blocking buffer: X-vivo 5% hAB serum (sterile) [Blocking is performed in the same medium as the cell culture] Dilution buffer: 0.5% BSA in PBS Wash buffer: 1×PBS Medium: For T cells, X-VIVO 15 medium (Lonza) Filter syringe: Millex GV ELISPOT PVDF plate (#MSIP4510, Millipore) TMB substrate

[0146] Example 4: Further proof of principle regarding the stability and effect of the recombinant polypeptide of the present invention Additionally, to obtain further proof of principle that the recombinant polypeptides of the present invention and their substitute molecules are stable and effective, the inventors undertook to obtain and test recombinant polypeptides having the general structure of the recombinant polypeptides of the present invention but containing various different peptide antigens. As shown in Figures 9 and 10, respectively, the tested recombinant polypeptides were stable during freeze-thaw cycles and storage, and were thermally stable. Furthermore, the recombinant polypeptides induced Tregs in a dose-dependent manner (Figure 11) and inhibited T cell lysis in a dose-dependent manner (Figure 12). Figure 13 shows the effect of the recombinant polypeptides on the serum cytokine profile in EAE-ODC Ova mice. Induction of IL-10 and possibly IL-4 was observed, both of which are known as immunosuppressive cytokines that downregulate the immune response in an inflammatory environment. These require a congener peptide in addition to the HLA-G alpha-3 domain. IL-2 appears to be induced in response to the presentation of a congener peptide unrelated to the alpha-3 domain to cells. IL-2 is required for T cell activation and survival. Further evidence regarding the stability of the recombinant polypeptide of the present invention is also provided in Figures 15 and 16.

[0147] Example 5: Results of ELISpot obtained from healthy donor PBMCs (Figure 17), and results of IL-10 ELISpot obtained from Parkinson's disease patients (Figure 18A) and age-matched healthy individuals (Figure 18B). This experiment demonstrates that significant induction of immunosuppressive cells in response to the construct of the present invention can be observed in vitro as early as 14 days later in both healthy donor PBMCs and patient PBMCs.

[0148] Example 6: Expression and testing of a polypeptide ("AIM Bio") having the polypeptide structure defined in claim 1 and containing the peptide antigen or its surrogate antigen according to the present invention. Sandwich ELISA protocol Nunc 96-well microtiter plates (Thermo Fisher, 442404) were coated overnight with 50 μl of 1 μg / ml anti-HLA-G (2A12) capture antibody (MAI-10358, Thermo Fisher). The plates were washed three times with 200 μl of PBS / 0.05% Tween 20 (PBS / T), blocked at RT for 2 hours, blocked for 1 hour with 150 μl of 1% BSA (blocking buffer) in PBS, and then washed three times with 200 μl of PBS / T. Expi293F expression cultures were diluted with blocking buffer (1:10) and tested in two replicates. Biotinylated anti-β2 microglobulin antibody (MA1-19506, Thermo Fisher) was diluted in blocking buffer (1:1000) and 100 μl was added to each well over 1 hour. After three washes with 200 μl of PBS / T, 100 μl of horseradish peroxidase (HRP) (405210, Biolegend; diluted 1:100 in blocking buffer) conjugated with streptavidin was added for 20 minutes. The binding of the biotinylated anti-β2 microglobulin detection antibody was then visualized by washing three times for 10–15 minutes with 200 μl of PBS / T, followed by 100 μl of TMB substrate solution (BioLegend). The reaction was stopped with 100 μl of 1N H2SO4. Absorbance was measured at 450 nm using a Tecan Sunrise® ELISA plate reader, corrected at 620 nm, and the results were evaluated using Magellan® software.

[0149] Transfection protocol Expi-293F cells (Thermo Fisher) were grown in Expi-293® expression medium (Thermo Fisher) and transfected using the Expifectamine® 293 transfection kit (Thermo Fisher). Transfection was 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 the number was 4.5-5.7 x 10⁶.6 Transfected at a density of individual live cells / ml (over 99% viability).

[0150] For a final culture volume of approximately 12 ml, 3 x 10 7 1000 viable cells were diluted in 10 ml of Expi293® expression medium containing GlutaMAX (Gibco, A14351-01). 10 μg of DNA per 1 ml of total transfection volume was diluted in 600 μl of Opti-MEM (Thermo Fisher, 31985-047). 30 μl of Expifectamine® 293 reagent was diluted in 570 μl of Opti-MEM or Opti-Plex complexing buffer (Thermo Fisher). After 3–5 min, the DNA was added to the Expifectamine-293® / Opti-MEM® transfection mix and incubated at RT for 15–20 min for the formation of the DNA-Expifectamine-293F® reagent complex. Next, 1200 μl of DNA-Expifectamine-293® reagent complex was added dropwise to 50 ml bioreactors containing 30 million cells in 10 ml of culture medium. The cells were then shaken using an orbital shaker, 19 mm 2 The samples were incubated at a diameter of 225 rpm in a humidified incubator at 37°C and 8% CO2.

[0151] After 18-20 hours of incubation, cells were enhanced by adding 60 μl of Enhancer I and 600 μl of Enhancer II (ExpiFectamine® 293 Transfection Enhancer 1+2, Catalog A14524, part of the ExpiFectamine® 293 Transfection Kit) while shaking the bioreactor. The cells were spun down at 300 g for 5 minutes at room temperature, and then the supernatant was harvested on day 5 by centrifugation at 4000 g for 20 minutes at 4°C.

[0152] Simple Western Protocol Simple Western electrophoresis of Jess cells was performed under non-reducing conditions using EZ Standard Pack 1 (bio-techne, Ref PS-ST01E7) reagents, employing a 12-230 kDa cartridge (bio-techne, Ref SM W004-1), 2A12αHLA-G antibody as the primary antibody (Thermo Fisher, MAI-10358, 1:50 in bio-techne antibody dilution 2, part of the anti-mouse detection kit), and the anti-mouse detection kit (bio-techne Ref DM-002). The supernatant from transfected Expi293F cells was pre-diluted 1:10 with 1x PBS, then separately diluted 1:10 with 0.1x sample buffer. After further dilution 1:5 with 5x fluorescence master mix, 3 μl was applied to a single cartridge well with a molecular weight marker. 5 μl of protein ladder (EZ Standard Pack 1) was used for molecular weight determination in one well of the first column. In the next column, 10 μl of antibody dilution 2 (bio-techne, part of the anti-mouse detection kit) was loaded per well as a blocking reagent. In the third column, 10 μl of 1:50 diluted primary antibody 2A12αHLA-G was loaded. In the fourth column, 10 μl of mouse secondary antibody from the anti-mouse detection kit was loaded. In the fifth column, 15 μl of luminol-peroxide mix was loaded. In the last three columns, 500 μl of washing buffer was loaded per compartment. The plate was centrifuged at 1000 g for 5 mins in RT to remove air bubbles, and the remaining cover foil was removed. The cartridge and plate were then loaded into the Jess system (bio-techne) using Compass 6.2.0 software, and the instrument was started.

[0153] The separation time was 25 minutes at 375V, followed by a 5-minute blocking time with antibody diluent (bio-techne), 30 minutes of primary antibody incubation, 30 minutes of secondary antibody incubation, and then detection.

[0154] ELISpot protocol ELISpot assay was performed according to the following protocol:

[0155] Phase 1: PBMC: AIM Bio culture Reagents · Human PBMC derived from patients with NMOSD (neuromyelitis optica spectrum disorder) · AIM Bio: AQP64_G, AQP65 P65K _G · Complete culture medium: X-vivo 15 (Lonza, Biozym 881026) supplemented with 5% human AB serum and human cytokines -rhIL-2 (20ng / ml); Immunotools, 11340027 -rhGM-CSF (20ng / ml); Immunotools, 11343128 -rhIL-4 (10ng / ml); Immunotools, 11340047 -rhTGFβ (10ng / ml); Immunotools, 11343160 · 24-well culture plate · PBS (sterile)

[0156] Thawing of PBMC (Day -1) · Thaw cryopreserved PBMC by rotating in a water bath for approximately 20~30s. · Immediately transfer the cells to a 15ml Falcon tube filled with PBS, and centrifuge at 300g for 5min at room temperature (RT). · Wash the cells with PBS for 2 additional times. · Resuspend the cells in 5ml X-vivo 15 medium supplemented with 5% hAB serum, and let the cells rest overnight at 37°C in a 6-well culture plate.

[0157] Induction of Treg: Culture PBMC with AIM Bio for 14 days (sterile conditions) · Test conditions: ○ Negative control (no AIM Bio treatment / untreated)

Chemical Formula

[0158] Phase 2: ELISpot assay (Perform the EliSpot assay according to the guidelines provided in the Human IL-10 ELISpot (Basic) Kit (Mabtech; 3430-2H))

[0159] reagent • ELISPOT PVDF plate (Millipore) • Human IL-10 EliSpot basic kit (Product code: 3430-2H) 35% ethanol • Blocking medium: X-vivo 5% hAB serum • Dilution buffer: 0.5% BSA in PBS or 0.5% FCS in PBS • EliSpot medium: X-vivo 15 (Lonza, Biozym 881026) supplemented with 5% human AB serum and hIL-2 (20 ng / ml) • Streptavidin-HRP for detection (Biolegend #405210; 1:750 dilution) • ELISpot substrate: TMB for HRP (Mabtech Code: 3651-10) ·PBS (sterile)

[0160] Cell coating (sterilization conditions) • Activate the PVDF wells with 25 μl of ethanol (35% v / v) for a maximum of 1 minute. Wash the plate five times with sterile water (200 μg / well). Dilute the coated antibody anti-hIL-10 (9D7) in sterile PBS to a final concentration of 2 μg / ml in sterile PBS. Add 50 μl of the diluted antibody per well (guideline: 15 μg / ml; 100 μl / well). Incubate overnight at 4-8°C.

[0161] Cell incubation in a plate (under sterile conditions) • Prepare the plate layout. The sample was tested in two replicates. ·sample: ○ Negative control: Untreated cells (no stimulation) ○Positive control: Untreated cells + 1 μg / ml α-CD3 / CD28 stimulating cocktail ○Test samples: AQP64_G treated cells and AQP65 P65K _G treated cells • Count PBMCs from 14D cultures. Calculate the required number of cells based on all conditions. • The minimum cell viability is greater than 50%. Otherwise, discard the sample. Remove any excess coated antibody under a hood and wash the plate five times with 200 μl of sterile PBS. Add 50 μl of blocking medium per well. Incubate at room temperature for 2 hours. ·1.5x10 in Elispot medium 6 Resuspend the PBMCs derived from all test conditions at the final concentration of / ml. • Resuspend the positive control cells at a higher concentration and supplement the volume with a stimulant cocktail. • Carefully remove excess blocking buffer under the hood. • Place 100 μl of PBMC (1.5 x 10) derived from each condition into each well. 5 Add the suspension of individual cells. Incubate at 37°C for 12-48 hours.

[0162] Spot detection Dilute the biotinylation detection antibody (12G8-biotin) to a concentration of 1 μg / ml (1:1000) in dilution buffer. Remove the cell supernatant and wash the plate five times in 200 μl of PBS. Remove the final excess buffer by pressing it onto a paper towel. Add 50 μl of detection antibody to each well. Incubate overnight at 4°C. Wash five times with 200 μl of PBS. Remove excess liquid by pressing onto a paper towel. Prepare HRP-conjugated streptavidin (1:750 in dilution buffer). Add 50 μl of streptavidin-HRP conjugate to each well. Incubate at room temperature for 1 hour. Wash five times with 200 μl of PBS. Remove excess liquid by pressing onto a paper towel. Add 50 μl of filtered TMB substrate to each well and incubate in a dark room for 10-20 minutes until blue spots appear. • Stop the reaction by rinsing the well with water (running tap water can be used). Remove the plastic underdrain from the plate and thoroughly rinse the bottom and sides of the plate with tap water. The analysis was performed using S6 Universal M2 readers (Immunospot, Cleaveland, Ohio).

[0163] result: Figure 19 shows that the AIM Bio of the present invention is expressed in Expi293F cells and that the monomer (approximately 50 kDa) is detectable. Expression was further confirmed and quantified in the supernatant via ELISA. This suggests that these molecules can be successfully produced.

[0164] Figure 20: ELISpot using patient PBMCs. A: Image of the wells in the assay, where double repeats are always present in the same row. The number of counted spots is shown in the figure. B: Quantification. ELISpot was implemented as described in Example 9, however, 1.2 × 10 6 Except for using only one PBMC, seeding the cells in a 24-well plate, and reducing all culture medium volumes accordingly. Compared to untreated PBMCs, AIM Bio AQP65 P65K In four of the five NMO patient PBMCs treated with _G, IL-10 spots increased. This suggests that AIM Bio can induce protective T cells in patient cells.

[0165] Furthermore, by using substitute constructs based on the claims, except for the inclusion of substitute peptides, the inventors have found that these constructs, which can be expressed at favorable expression levels, are functional, that is, they can induce antigen-specific immune tolerance. Therefore, in view of the favorable expression levels of the constructs of the present invention, it is expected that the constructs of the present invention are functional and can be used to treat the medical indications defined in the claims.

[0166] Example 7: In subsequent examples, the mouse AIM Bio used for the proof-of-concept experiment: Peptide sequence aSyn 63-71 : VTNVGGAVV (Sequence ID 86) Protein sequence aSyn 63-71 _H2-K b G [ka]

[0167] Peptide sequence gp 34-41 : AVYNFATM (Sequence ID 48) Protein sequence gp 34-41 _H2-K b G: [ka]

[0168] Example 8: Additional proof-of-concept data from in vivo studies: A53T / A30P Mouse Protein aggregation is a significant pathological event in the pathology of Parkinson's disease. A30P and A53T are two mutations in alpha-synuclein associated with increased aggregation and an increased risk of developing PD. Tg-hm2α-SYN-tg transgenic mice have these mutations and exhibit loss of SN neurons, terminals, and motor dysfunction. This model is described in 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., vol. 11(23):3770p., 2022, and 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 α-synuclein transgenic mice reveals upregulation of Sel1l., PLoS One. vol. 12(8):e0182092, 2017.

[0169] Accelerator rotor rod test Prior to preoperative measurements, Tg-hm2α-SYN-tg mice underwent a two-day training program. The mice were placed on a RotaRod Advanced (TSEsystems) and accelerated from 5 rpm to a maximum of 50 rpm over 300 seconds. Each day's training consisted of five 300-second runs. RotaRod measurements were recorded in asymptomatic 3-4 month old mice after the two-day training session. Subsequently, 9-10 mice per group were treated with AIM Bio as follows. experimental group 1. Wild type + PBS 2. A53T / A30P+PBS 3. A53T / A30P + 2μg / g gp 34-41 _H2-K b GAIM Bio (control) 4. A53T / A30P+2μg / g αSyn 63-71 _H2-K b G AIMio (Mouse-adapted α-Syn-specific AIM Bio)

[0170] At 5-6 months of age, the animals received three consecutive IP injections at 2-week intervals. Subsequently, the accelerated rotorodization test was repeated in mice at 7-8 months of age. aSyn 63-71 _H2-K b Mice treated with GAIM Bio showed almost no decrease in rotarod performance compared to healthy controls (Figure 21).

[0171] In summary, the results shown in Figures 4-8 demonstrate that several different aSyn peptide antigens, when combined with a suitable antigen-presenting domain, can induce immune tolerance in the recombinant polypeptide of the present invention.

[0172] Example 9: Testing of exemplary human recombinant polypeptides ("AIM Bio") according to the present invention: Candidate molecules tested in vitro using PBMCs derived from healthy donors or patients with Parkinson's disease (PD): aSyn 69-77 _A2G: Best results were obtained from healthy donors, with 21 responders out of 30 tested. In PD, when used at 5 μg / ml, 2 out of 9 PD patients responded; when used at 20 μg / ml, 4 out of 8 PD patients responded (and 3 out of 5 healthy donors responded). aSyn 70-78 _A2G: Best results were obtained in healthy donors, with 20 out of 29 cases tested. In PD, when used at 5 μg / ml, 5 out of 9 PD patients responded; when used at 20 μg / ml, 2 out of 5 PD patients responded. aSyn 37-45 _A11G: In healthy donors, 14 out of 29 tested cases were responders; in PD, when used at 5 μg / ml, 7 out of 9 PD samples were responders / / when used at 20 μg / ml, 2 out of 5 PD patients were responders (and 1 out of 2 healthy donors were responders). aSyn 87-95 _A2G: In healthy donors, 12 out of 32 cases tested were responders. In PD patients, when used at 5 μg / ml, 3 out of 9 PD patients were responders. aSyn86-96_A11G: In healthy donors, 18 out of 24 cases tested were responders. In PD patients, when used at 5 μg / ml, 5 out of 9 PD patients were responders. aSyn 47-55 _A2G: In the healthy donors tested, when used at 5 μg / ml, 3 out of 6 tested cases were responders, and PD was not tested. aSyn 32-41 _A2G: In healthy donors, when used at 5 μg / ml, 9 out of 12 cases tested were responders; PD was not tested.

[0173] Example 10: Characterization of the recombinant polypeptide ("AIM Bio") of the present invention Particle size exclusion chromatography (SEC) For characterizing purified proteins, size exclusion chromatography was performed at room temperature using an Akta FPLC (Amersham Biosciences) equipped with a Superdex 200 increase 10 / 300 column and Unicorn 5.31 software (GE Healthcare). Before operation, the buffer was replaced from 20% EtOH (storage solution) to 2 CV (column volume) of H2O. For analysis, the column was rinsed with 2 CV of PBS (Sigma, D8537-500 ml) at a flow rate of 0.5 ml / min. Subsequently, 10-20 μg of AIM Bio (volume 500 μl) was attached to a Superdex 200 increase 10 / 300 column (Cytiva), and the column was run at a flow rate of 0.5 ml / min for 1 CV to collect the protein fraction. The UV curve was plotted against time, and peaks were analyzed.

[0174] AIM Bio aSyn63-71_H2-K b SEC analysis of G indicates that purified AIM Bio is a monomeric protein (Figure 22).

[0175] Thermal shift assay aSyn 63-71 _H2-K b Thermal shift analysis of G shows that Tm is 44.84°C (Figure 23).

[0176] For the analysis of protein stability of purified AIM Bio, a thermal shift assay (TSA) was performed. In the TSA, 3 μg of AIM Bio was dissolved in 25 μl of PBS (Sigma, D8537-500 ml) containing SYPRO Orange dye (Merck, S5592, final dilution to 1:2000 in PBS). The melting curve was recorded using a StepOnePlus instrument (ThermoFisher) by starting at 25°C and increasing the temperature by 1°C per minute until the final temperature of 95°C was held for 2 minutes. The melting curve was analyzed using StepOnePlus 2.3 software. To determine Tm, a Boltzmann sigmoid curve approximation was performed and Tm was calculated accordingly.

[0177] Jess SimpleWestern Operation The Jess Simple Western blotting system was operated under non-reducing conditions using EZ Standard Pack 1 (bio-techne, Ref PS-ST01E7) reagents, with a 12-230 kDa cartridge (bio-techne, Ref SM W004-1), 2A12α HLA-G antibody (Thermo Fisher, MAI-10358, diluted 1:50 in bio-techne antibody diluent 2, part of the anti-mouse detection kit) as the primary antibody, and the anti-mouse detection kit (bio-techne, Ref DM-002). The supernatant was pre-diluted 1:10, and the purified protein was pre-diluted 1:100 with 1× PBS, then further diluted 1:10 with 0.1× sample buffer. After further dilution 1:5 with 5× fluorescent master mix, 3 μl was added to a single cartridge well along with a molecular weight marker. 5 μl of protein ladder (EZ Standard Pack 1) was used in one well in the first row for molecular weight determination. The next row contained 10 μl of antibody diluent 2 (bio-techne, part of the anti-mouse detection kit) per well as a blocking reagent; the third column contained 10 μl of 1:50 diluted primary antibody 2A12αHLA-G (2A12□HLA-G); the fourth column contained 10 μl of mouse secondary antibody from the anti-mouse detection kit; the fifth column contained 15 μl of luminol-peroxide mixture; and the last three columns contained 500 μl of washing buffer per compartment. The plate was centrifuged at 1000 g for 5 minutes at room temperature to remove air bubbles, and any remaining cover foil was removed. The cartridge and plate were then loaded into the Jess system (bio-techne) using Compass 6.2.0 software, and the machine was started. The separation time was 25 minutes at 375V, followed by a 5-minute blocking period using antibody diluent (bio-techne), a 30-minute primary antibody incubation, and a 30-minute secondary antibody incubation before detection.

[0178] aSyn 63-71 _H2-K b G is primarily a monomeric protein (Figure 24).

[0179] Example 11: Additional functional data using human constructs: method Cell culture for ELISpot assay In the ELISpot assay, PBMCs derived from PD patients were thawed and left to stand overnight in a 6-well plate (Greiner, #657160). The following day (day 0), the cells were counted, and the cell count was 1.5 × 10⁶. 6 Cells / well were placed 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 (Immunotools, #11340047), and 10 ng / ml h-TGFβ1 (BioLegend, #781804)), 3 × 10 cells / well. 6 Cells were seeded at a density of cells / ml in 12-well plates (Greiner, 665180). Cells were stimulated with 20 μg / ml aSyn-specific AIM Biologicals on days 0 and 6. An additional 500 μl of X-VIVO 15 complete medium was added to the cells on days 3, 6, 8, 10, and 13. 20 mM HEPES / PBS was added to the control wells. On day 14, cells were counted and used for ELISpot assay and dextramer staining.

[0180] ELISpot assay Cell seeding An ELISpot plate (Millipore, MSIPS4510) was activated for 1 minute with 50 μl of 35% EtOH per well, followed by five washes with 200 μl of PBS / well. The plate was coated with 2 μg / ml of anti-human IL-10 antibody (9D7, Mabtech, 3430-3-250) in PBS (overnight at 4°C). The following day, unbound antibody was removed, and the plate was washed five times with 200 μl of PBS / well, followed by blocking for 2 hours in 50 μl of X-VIVO 15 medium containing 5% hAB serum. The blocking medium was discarded. 100 μl of AIM Bio-treated cell suspension and control cell suspension were seeded onto the plate (cells 1.5 × 10⁶). 6 (Cells / ml). ImmunoCult® Human CD3 / CD28 T cell activator (25 μl / ml; Immunocult, #10971) was used as the positive stimulant. PBS was used as the negative stimulant. Plates were incubated for 48 hours in a humidified incubator containing 5% CO2.

[0181] IL-10 spot color development The cell suspension was discarded, and the plate was washed five times with 200 μl of PBS. Biotinylated anti-IL10 detection antibody (12G8-biotin, Mabtech, 3430-6-250) was diluted to a concentration of 1 μg / ml in PBS containing 0.5% BSA. 50 μl was added to each well and incubated at room temperature for 2 hours or overnight at 4°C. The plate was washed five times with 200 μl of PBS. 50 μl of streptavidin conjugate horseradish peroxidase (HRP) was added to each well and incubated for 1 hour. 50 μl / well of TMB substrate solution (Mabtech, 3651-10) was added to each well until a distinct spot appeared. The color development was stopped by thorough washing in deionized water. The underdrain was removed, and the bottom of the membrane was rinsed. The plates were dried, and the spots were counted using an ELISpot reader (Immunospot CTL machine and Immunospot SC Suite, CTL). The data was analyzed using Excel 365 (Microsoft), and graphs were generated using Prism 10.2 (GraphPad).

[0182] Figure 25 shows IL-10 secreting cells detected in the ELISpot assay. As shown in the figure, AIM_aSyn 70-78 _A2G stimulated IL-10 secretion in healthy PBMCs and Parkinson's disease patient PBMCs. IL-10 induction was linked to HLA-A2 + It is more potent in donor-derived PBMCs.

[0183] HLA-A2 + and HLA-A11 + CD8 in donors + Characterization of regulatory T cells Age-matched PBMC samples were collected from 8 PD patients and 5 healthy controls at University Hospital Würzburg. Informed consent was obtained from the patients, and the experiment was approved by the local ethics committee (approval number AZ54 / 15). Six donors (4 PD patients and 2 controls) were haplotyped as HLA-A2 positive. Following the protocol for the ELISpot assay, two types of α-synuclein (aSyn)-specific AIM Biological assays were performed, namely aSyn 69-77 _A2G and aSyn 70-78 Cells were stimulated with _A2G for 14 days. PBS was used as a negative stimulation control. Cells were then washed, counted, and further cultured in X-VIVO 15 complete medium. PE-labeled dextramers loaded with peptides (aSyn) were then used. 69-77 (AVVTGVTAV; Sequence ID 33) and aSyn 70-78 HLA-A2 dextramers loaded with (VVTGVTAVA; SEQ ID NO: 34) were prepared 48 hours prior using a modified Immudex protocol utilizing one-quarter of the standard reagent volume. Next, cells were treated with Brefeldin A at a 1:1000 dilution to inhibit protein secretion, and then incubated with the prepared dextramers at room temperature for 15 minutes. 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. Haplotyping was performed using HLA-A2 antibody (PE, BB7.2, BioLegend). After surface staining in the dark at 4°C for 1 hour, a washing step was performed to remove unbound antibody.

[0184] For intracellular staining, cells were first fixed for 20 minutes, and then permeabilized using FIX / PERM kit buffer according to the manufacturer's instructions (Thermo Fisher). The permeabilized cells were incubated in permeabilization buffer with antibodies targeting IL-10 (BV421, clone JES3 / 9D7, BioLegend), IFN-γ (PE-Cy7, clone B27, BioLegend), FoxP3 (APC, Miltenyi Biotec), and Granzyme B (PE Vio770, clone Qa16A02, BioLegend), diluted 1:75. This incubation was performed in the dark at room temperature for 30 minutes. After staining, the cells were washed, resuspended in FACS buffer, and analyzed using an Attune NxT flow cytometer autosampler (Thermo Fisher Scientific). Flow cytometry results were transferred to Microsoft Excel and analyzed using GraphPad Prism version 9 for visualization. Statistical analysis was performed using a chi-squared test with a mixed-effects model, and the results were compared to a PBS control.

[0185] Figure 26 shows CD8 levels in healthy controls and Parkinson's disease patients. + Shows a subpopulation of T cells. Two αSyn-specific AIM Bio(AIM_aSyn 69-77 _A2G and AIM_aSyn 70-78 _A2G) indicates that CD8 is present in PBMCs derived from Parkinson's disease patients. + The T cell response was stimulated. The panel was HLA-A2 + In the individual (middle row), CD8 + CD103 + CD8 + Dex Low CD103 + , and CD8 + IL-10 + Four CD8 cells +Compare T cell subpopulations. The subpopulations were incubated with AIM Bio for 14 days and then analyzed for the expression of CD103 and IL-10. PBMCs obtained from patients with Parkinson's disease showed induction of IL-10 and CD103. P values are indicated where applicable. Due to the low cell count, AIM_aSyn 70 It should be noted that stimulation of healthy donor PBMCs using _A2G was omitted.

[0186] AIM_aSyn 69-77 When co-cultured with _A2G, the proportion of CD8 in 7 donors (3 healthy subjects and 4 PD patients) + CD103 + , CD8 + IL-10 + , CD8 + dex low CD103 + , and CD8 + dex low IL-10 + populations increased. In this assay, no induction of FoxP3 + , IFNγ + , and granzyme B + cells was observed. When co-cultured with AIM_aSyn 70-78 _A2G, the increase of CD8 + Dex low CD103 + cells was smaller. AIM_aSyn 69-77 _A2G induced IL-10 secretion in 6 donors (2 healthy subjects and 4 PD patients). The induction of IL-10 was more potent in HLA-A2 - donors compared to HLA-A2 + donors.

[0187] Conclusion: AIM_aSyn 69-77 _A2G induces regulatory CD8 + T cells in PBMCs.

[0188] References: Ageno W, Steidl L, Marchesi C, Dentali F, Mera V, Squizzato A, Crowther MA, Venco A (2002), Selecting patients for home treatment of deep vein thrombosis: the problem of cancer. Haematologica 87:286-291. Baba Y, Kuroiwa A, Uitti RJ, Wszolek ZK, Yamada T (2005), Alterations of T-lymphocyte populations in Parkinson disease. Parkinsonism Relat Disord 11:493-498. 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. 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. Ip CW, Klaus LC, Karikari AA, Visanji NP, Brotchie JM, Lang AE, Volkmann J, Koprich JB (2017), AAV1 / 2-induced overexpression of A53T-alpha-synuclein in the substantia nigra results in degeneration of the nigrostriatal system with Lewy-like pathology and motor impairment: a new mouse model for Parkinson's disease. Acta Neuropathol Commun 5:11. 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. 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. 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. Mogi M, Harada M, Kondo T, Riederer P, Inagaki H, Minami M, Nagatsu T (1994), Interleukin-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. 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. 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. Peter I, Dubinsky M, Bressman S, Park A, Lu C, Chen N, Wang A (2018), Anti-Tumor Necrosis Factor Therapy and Incidence of Parkinson Disease Among Patients With Inflammatory Bowel Disease. JAMA Neurol 75:939-946. 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. 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. 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. 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. 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. Sulzer D, Alcalay RN, Garretti F, Cote L, Kanter E, Agin-Liebes J, Liong C, McMurtrey C, et al. (2017), T cells from patients with Parkinson's disease recognize alpha-synuclein peptides. Nature 546:656-661. [Industrial applicability]

[0189] The pharmaceutical compositions, polypeptides, nucleic acids, cells, and products used in this invention are industrially applicable. For example, they can be used in the manufacture of pharmaceuticals or as pharmaceuticals themselves.

Claims

1. A recombinant polypeptide capable of presenting a peptide antigen, wherein, in order from the N-terminus to the C-terminus, i) A peptide antigen presented by the recombinant polypeptide, which is a human alpha-synuclein peptide, ii) Linker arrangement depending on the case, iii) The sequence of human β2-microglobulin represented by Sequence ID No. 5, or the sequence of a human polypeptide domain containing an amino acid sequence that is at least 90% identical to the amino acid sequence of human β2-microglobulin, iv) Linker array depending on the case, v) The [alpha]1 domain of the MHC molecule, vi) The [alpha]2 domain of the MHC molecule, vii) A derivative of the [alpha]3 domain of an MHC class Ib molecule that can bind to ILT2 or ILT4, viii) Depending on the case, the protease cleavage site, ix) Depending on the case, spacer arrangement, and x) Affinity tags depending on the case Recombinant polypeptides containing [specific components].

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

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

30.

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

33.

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

34.

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

37.

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

40.

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

50.

10. The recombinant polypeptide according to any one of claims 1 to 9, wherein the [alpha]1 domain described in (v) and the [alpha]2 domain described in (vi) are derived from a human MHC class Ia molecule or a human MHC class Ib molecule.

11. The recombinant polypeptide according to claim 10, wherein the [alpha]1 domain described in (v) and the [alpha]2 domain described in (vi) are derived from a human MHC class Ib molecule, and the human MHC class Ib molecule is a human HLA-G molecule.

12. The recombinant polypeptide according to claim 10, wherein the [alpha]1 domain described in (v) and the [alpha]2 domain described in (vi) are derived from a human MHC class Ia molecule, and the [alpha]1 domain described in (v) and the [alpha]2 domain described in (vi) are derived 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 claims 1 to 12, wherein the [alpha]3 domain of the MHC class Ib molecule described in (vii) is the [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 claims 1 to 13, wherein the [alpha]3 domain or derivative described in (vii) is identical to the [alpha]3 domain having the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 2, or SEQ ID NO: 51, 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, or is identical to the [alpha]3 domain having the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 2, or SEQ ID NO:

51.

15. The recombinant polypeptide according to any one of claims 1 to 14, wherein the [alpha]3 domain or derivative described in (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 to 14, wherein the [alpha]3 domain or derivative described in (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 to 14, wherein the [alpha]3 domain or derivative described in (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 claims 1 to 17, wherein the linker sequence described in (ii) and / or the linker sequence described in (iv) comprises an amino acid sequence (GGGGS)n, where n is an integer of 1 or more, and n is preferably an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and preferably selected from the group consisting of 2, 3, 4, and 5.

19. The recombinant polypeptide according to any one of claims 1 to 18, wherein the sequence of the human polypeptide domain described in (iii) 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 claims 1 to 19, wherein the polypeptide is a dimer or a polymer.

21. The recombinant polypeptide according to any one of claims 1 to 20, wherein the polypeptide contains or consists of all of components i) to vii) and does not contain components vii) to x), or contains or consists of all of components i) to x).

22. A recombinant polypeptide according to any one of claims 1 to 21, further comprising an N-terminal secretory signal peptide sequence.

23. From the N-terminus to the C-terminus, (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) Amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 53-54 A recombinant polypeptide according to any one of claims 1 to 21, comprising an amino acid sequence consisting of the above.

24. A recombinant polypeptide according to any one of claims 1 to 23, which is soluble.

25. A nucleic acid encoding one or more polypeptides according to any one of claims 1 to 24, preferably a vector.

26. A pharmaceutical composition or kit comprising at least one nucleic acid as described in claim 25.

27. A pharmaceutical composition or kit comprising at least one recombinant polypeptide as described in any one of claims 1 to 24.

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

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

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

31. A pharmaceutical composition or kit for use according to claim 29 or 30, wherein the treatment is an immunotherapy treatment for Parkinson's disease, and the treatment preferably involves inducing immune tolerance to the autoantigen human alpha-synuclein.

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

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

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